J Chem Ecol (2013) 39:516–524
DOI 10.1007/s10886-013-0266-3
Performance of an Herbivorous Leaf Beetle
(Phratora vulgatissima) on Salix F2 Hybrids:
the Importance of Phenolics
Mikaela Torp & Anna Lehrman & Johan A. Stenberg &
Riitta Julkunen-Tiitto & Christer Björkman
Received: 18 September 2012 / Revised: 30 January 2013 / Accepted: 31 January 2013 / Published online: 1 March 2013
# Springer Science+Business Media New York 2013
Abstract The genotype of the plant determines, through the
expression of the phenotype, how well it is suited as food for
herbivores. Since hybridization often results in profound genomic alterations with subsequent changes in phenotypic
traits, it has the potential to significantly affect plantherbivore interactions. In this study, we used a population of
F2 hybrids that originated from a cross between a Salix
viminalis and a Salix dasyclados genotype, which differed in
both phenolic content and resistance to the herbivorous leaf
beetle Phratora vulgatissima. We screened for plants that
showed a great variability in leaf beetle performance (i.e.,
oviposition and survival). By correlating leaf phenolics to
the response of the herbivores, we evaluated the importance
of different phenolic compounds for Salix resistance to the
targeted insect species. The performance of P. vulgatissima
varied among the F2 hybrids, and two patterns of resistance
emerged: leaf beetle oviposition was intermediate on the F2
hybrids compared to the parental genotypes, whereas leaf
beetle survival demonstrated similarities to one of the parents.
The findings indicate that these life history traits are controlled
by different resistance mechanisms that are inherited differently in the hybrids. Salicylates and a methylated luteolin
derivative seem to play major roles in hybrid resistance
M. Torp (*) : J. A. Stenberg : C. Björkman
Department of Ecology, Swedish University of Agricultural
Sciences, P.O. Box 7044, 750 07 Uppsala, Sweden
e-mail: [email protected]
A. Lehrman
Department of Crop Production Ecology, Swedish University
of Agricultural Sciences, P.O. Box 7043, 750 07 Uppsala, Sweden
R. Julkunen-Tiitto
Natural Product Research Laboratory, Department of Biology,
University of Eastern Finland, P.O. Box 111,
801 01 Joensuu, Finland
to Phratora vulgatissima. Synergistic effects of these compounds, as well as potential threshold concentrations, are
plausible. In addition, we found considerable variation in both
distributions and concentrations of different phenolics in the
F2 hybrids. The phenolic profiles of parental genotypes and
F2 hybrids differed significantly (e.g., novel compounds
appeared in the hybrids) suggesting genomic alterations with
subsequent changes in biosynthetic pathways in the hybrids.
Keywords Hybridization . Phenolics . Luteolin .
Salicylates . Phratora vulgatissima performance .
Resistance . Salix
Introduction
Hybridization between species is common in nature (Arnold,
1997) and can result in substantial changes in various plant
characteristics. Parental species and hybrids often differ in
resistance to herbivores. This variation is probably mediated
by modifications of plant chemistry, morphology, or phenology
in hybrid plants compared to their parents (Fritz et al., 1999;
Cheng et al., 2011). Patterns of hybrid resistance are diverse:
hybrids can express intermediate (Fritz et al., 1998; Czesak et
al., 2004; Hochwender and Fritz, 2004), equal (Orians et al.,
1997; Fritz et al., 1998; Hallgren et al., 2003), increased (Fritz,
1999; Hjältén and Hallgren, 2002; Hochwender and Fritz,
2004), or decreased (Whitham, 1989; Orians et al., 1997;
Hjältén, 1998; Czesak et al., 2004) resistance compared to
their parents. This is not surprising since different traits that
affect herbivore responses, e.g., plant secondary chemistry
(Tahvanainen et al., 1985) and nutrient content (Mattson,
1980), exhibit varying and often complex inheritance patterns
(Rieseberg and Ellstrand, 1993; Cheng et al., 2011). The resistance of parental species and hybrids depends partially on
J Chem Ecol (2013) 39:516–524
environmental conditions (Hochwender et al., 2000). In
addition, different herbivore species respond differently
to variation in plant traits (Matsuki and MacLean, 1994;
Orians and Fritz, 1996).
The importance of plant chemistry for the outcome of
plant-herbivore interactions and plant resistance to herbivore attacks has been thoroughly addressed (e.g., Fraenkel,
1959; Haukioja, 1980; Harborne, 2001; Treutter, 2006;
Barbehenn and Constabel, 2011). However, much is still
unknown about genetic or regulatory aspects of the biosynthesis of many of these defensive chemical compounds.
Since small genetic changes can cause large shifts in the
chemical properties of both plant individuals and species
(Nyman and Julkunen-Tiitto, 2005), hybridization should
further increase the variability in plant chemical profiles.
According to classic hybridization theory, a trait, whether it
is expressed by one or several genes, is expected to show
greater variance in the F2 hybrids compared to either
the parental species or the F1 hybrids (Hochwender et al.,
2000). This is a result of recombination between chromosomes during the production of F2 hybrids (Hochwender
and Fritz, 2004). Orians (2000) showed that F2 hybrid plants
demonstrated greater variance in secondary chemistry
than F1 and parental plants. The variability in plant
chemical content expressed in the F2 generation can be used
to evaluate the effect of plant chemistry on herbivore responses and to identify the mechanisms behind such plantherbivore interactions.
Willows (Salix spp.) are excellent for investigating the
effects of hybridization on insect herbivore responses. Most
of the species of this genus are cross-fertile, and many
hybrids occur naturally. There is a diverse insect community
that feeds on willows; in particular, chrysomelid beetles
(Coleoptera) may cause severe damage to the plants.
Willows generally can be divided into two groups based
on their content of phenolics: some species or genotypes
contain high levels of phenolic glycosides (e.g., salicylates)
and low levels of condensed tannins, whereas other species
or genotypes demonstrate high levels of condensed tannins
but no phenolic glycosides (Julkunen-Tiitto, 1986, 1989;
Baum et al., 2009). Insect herbivores vary in their ability
to cope with these compounds; specialists often prefer to
feed on willows with high levels of phenolic glycosides,
while generalists usually avoid these plants (Rowell-Rahier,
1984; Tahvanainen et al., 1985; Kelly and Curry, 1991;
Orians et al., 1997; Boeckler et al., 2011).
Salix hybrids previously have been used as a model
system in studies that try to correlate the response of herbivorous insects and plant chemical composition. Some of
these studies have focused on parental species and F1 hybrids only (Orians et al., 1997; Hallgren, 2003), whereas
others have included F2 hybrids (Hallgren et al., 2003;
Glynn et al., 2004) and back-crosses (Hallgren et al.,
517
2003). To understand the effect of hybridization from an
ecological point of view, it is important to include the more
complex hybrid categories (Arnold, 1997). One way to
elucidate how hybridization influences plant-herbivore interactions is to study how plant characteristics that affect
herbivore responses (e.g., plant chemistry) are inherited
from parental species to hybrids (Orians et al., 2000).
Furthermore, by correlating variation in plant traits to the
response of herbivores, it might be possible to gain knowledge about the complex interactions among genes, traits,
and patterns on a broader ecological scale.
In this study, we used hybridization between Salix
viminalis, which is susceptible to the chrysomelid leaf beetle
Phratora vulgatissima, and S. dasyclados, which exhibits a
high degree of resistance to this insect pest. Crossing of two
F1 hybrids created a F2 generation that gave us an opportunity
to screen for plants that showed variability in the performance
of P. vulgatissima. Moreover, by analyzing the phenolic profiles of the F2 hybrids, we aimed to find chemical clues that
explained the varied performance of the targeted insect species. This study was done as a part of a Salix molecular
breeding program with the long-term goal of developing
methods for selecting plants with increased resistance to insect
pests and pathogens.
Methods and Materials
The Study System Willows (Salix spp.) are deciduous trees
or shrubs that are commonly cross-fertile and hybridize
easily. Cuttings root readily making cultivation and cloning
simple. The foliage of Salix species often is rich in carbonbased phenolics, and generally there is a significant genetic
effect on the phenolic metabolite content (Julkunen-Tiitto,
1986; Baum et al., 2009). Phenolic compounds play a major
role in Salix resistance to herbivory (e.g., Tahvanainen et al.,
1985). The two willow species used in this study vary in their
phenolic profiles; the foliage of S. viminalis is high in condensed tannins but does not contain any salicylates, whereas S.
dasyclados leaves contain high levels of salicylates and small
amounts of condensed tannins (Julkunen-Tiitto, 1986). The
variation in phenolic content has been proposed to be responsible for the different resistance of the two species for the
herbivorous leaf beetle P. vulgatissima; S. viminalis is susceptible while S. dasyclados demonstrates a high degree of resistance (Glynn et al., 2004; Lehrman et al., 2012).
The blue willow leaf beetle (P. vulgatissima) is a common
herbivore on many species of Salix, including short rotation
coppice crops such as S. viminalis. The beetles are univoltine
in Sweden and they overwinter as adults. In early spring, they
start feeding on their host plants and in late spring or early
summer, the beetles mate and start laying eggs. The eggs are
laid in clusters on the leaves, and egg hatching takes place
518
after approximately 2 wk (Kendall et al., 1996). After passing
through three instar stages, the larvae pupate in the soil. The
beetles, and especially their larvae, can cause severe damage
to Salix plants (Björkman et al., 2000).
Plants A female S. viminalis (cv. ‘Jorunn’) was crossed with
a male S. dasyclados (cv. ‘SW901290’), and two of the F1
progenies were further crossed to produce an F2 generation.
Twelve of these F2 hybrids were selected for testing of resistance against P. vulgatissima. To enable as much genetic
variation as possible among the selected F2 hybrids, the
selection was done based on differences in morphology, such
as leaf shape and branching, between the F2 hybrids. Winterdormant cuttings from annual shoots of the two parental
genotypes and the selected F2 hybrids were harvested and
stored at 4 °C. About six weeks prior to when the plants were
to be used in the experiments, 10 cm cuttings were planted in
pots (diam=11 cm) containing Hasselfors™ planting soil.
Pots were transferred to a greenhouse where the cuttings were
grown in natural light with supplementary illumination at
300 μmolm2 s-1 and temperatures ranging from 20 °C to
26 °C. Plants were watered regularly and fertilized (N:P:K=
51:10:43). New cuttings were continuously planted in order to
provide fresh leaves from plants of the same age throughout
the whole experimental period.
Bioassay In March, adult P. vulgatissima were collected in
the field from their overwintering sites in the vicinity of
Uppsala, central Sweden. To avoid disruption of their hibernation period, the beetles were stored at -4 °C until the start of
the bioassay. At the start, P. vulgatissima beetles were transferred from storage to room temperature, and their sexes were
determined.
In a first bioassay, where beetle performance on the parental
genotypes ‘Jorunn’ (S. viminalis) and ‘SW901290’ (S.
dasyclados) was tested (see Lehrman et al., 2012), individual
beetles were kept isolated in 30 ml plastic jars (26×67 mm) with
perforated lids. Each jar contained 1 cm3 moistened oasis floral
foam and one mature leaf from the mid-section of the genotype
to be tested. The jars were placed in a climate room at 20 °C
with a diurnal rhythm of 18:6, L:D. Beetles were provided with
fresh leaves every secondday and at the same time, the jars were
cleaned, the oasis moistened, and beetle survival monitored.
After 10 d, females and males were paired in the jars for 24 h
to mate. After mating, males were removed and only females
remained in the bioassay until they died. Every second day, eggs
were counted and removed from the jars. The bioassay was
finished when the last beetle had laid no eggs for 4 d; the best
performing beetles continued their oviposition for about 3 mo.
Results from the bioassay showed that the P. vulgatissima
oviposition rate levelled out about 6 d after mating.
In the current study, we used the F2 hybrids in a bioassay
similar to the one described briefly above and in more detail
J Chem Ecol (2013) 39:516–524
in Lehrman et al. (2012). In this F2 hybrid bioassay, 24
females and 16 males were caged together, in order to feed
and mate, for 20 d. Cages contained three potted plants of
one of the twelve F2 hybrids (i.e. 12 cages, each containing
3 plants of respective hybrid genotype). After 20 d, the
female beetles were transferred to separate jars and transferred to a climate room as described above. Each jar
contained one mature leaf from the mid-section of the appropriate F2 hybrid, and as in the parental bioassay, beetles
were provided with fresh leaves every secondday when
eggs were counted and removed from the jars. The F2
hybrid bioassay was terminated after 14 d. About 20 females
per F2 hybrid were included in the bioassay but the final
number varied due to high mortality on some of the hybrids
in the mating cages.
Chemical Analyses Six F2 hybrids were selected for screening of phenolics that might be related to beetle egg production
and survival (Fig. 1). These F2 hybrids were chosen due to
their variable effects on beetle performance. Five leaves from
6-wk-old plants were sampled from the mid-section of 5
individual plants of each genotype. The leaves were dried in
a greenhouse (20–26 °C, natural light) for 3 d and then frozen
at −20 °C in sealed plastic bags. Prior to extraction, composite
leaf samples were milled using a ball mill. Phenolics were
extracted from 6 to 8 mg leaf material with the Precellys 24homogenizer (Betrin Technologies, France) for 30 s at
4,780 × g with 600 μl of 100 % MeOH in 2 ml vials.
Samples were kept in an ice bath for 15 min with subsequent
centrifugation for 5 min at 11,300 × g at 4 °C. Supernatants
were transferred to 6 ml tubes, and the extraction procedure
was repeated for three additional 5 min extractions. Methanol
from the combined extracts (2.4 ml) was evaporated to dryness
under nitrogen. Dried samples were re-dissolved in 300 μl
100 % MeOH and 300 μl distilled H2O. Samples were analyzed with a high-performance liquid chromatography, HPLC
(Agilent, Series 1100, Germany), equipped with a diode array
detector, DAD (G1315B). A reverse-phase (RP) C18 column
(Agilent Technologies, USA) was used for the separation of
the compounds using conditions described in Julkunen-Tiitto
and Sorsa (2001). The compounds were detected at 270 nm
and 320 nm based on their UV-spectra and retention times.
Concentrations were calculated using the following standards:
salicin for salicin; salicortin for salicortin and HCH-salicortin;
hyperin for hyperin and quercetin derivatives; luteolin 7glucoside for luteolin derivatives; (+)-catechin for (+)-catechin; kaempferol 3-glucoside for kaempferol-diglycoside and
dicoumaroyl-astragalin; chlorogenic acid for chlorogenic acid
derivatives (i.e., compounds expressing chlorogenic acid spectrum); and cinnamic acid for cinnamic acid derivatives (i.e.,
compounds expressing p-OH-cinnamic acid spectrum).
Condensed tannins (methanol soluble and insoluble) were
quantified by oxidative depolymerization to anthocyanidins
J Chem Ecol (2013) 39:516–524
519
Fig. 1 Phratora vulgatissima a
oviposition (eggs/day; means±
S.E.) and b survival
(number of days: means±S.E.)
during a 14 d bioassay in which
the beetles were feeding on
leaves from either one of the
parental genotypes or one of
twelve F2 hybrids originating
from a crossing between Salix
viminalis (‘Jorunn’) and S.
dasyclados (‘SW901290’).
The percentages given in b
describe the number of beetles
that survived during the 14 d
bioassay (e.g., 100 % implies
that all beetles survived the
14 d trial while 25 % denotes
that only 1/4 of the beetles
survived for 14 d). The
highlighted hybrids were
selected for phenolic analysis
in acid buthanol as described by Porter et al. (1986) using
purified Betula nana leaf tannin as the standard.
Statistics Data were analyzed using SAS 9.2. The nonparametric Kruskal-Wallis test was performed on beetle
oviposition and survival on the parental generation since
the data were not normally distributed. One-way ANOVAs
were performed on beetle oviposition and survival on the F2
generation, since these data met the criteria for adopting
parametric tests showing normal distribution. Regression
analyses were performed on the leaf concentration of different phenolic compounds vs. the mean number of eggs
produced per day and beetle survival in order to find phenolic metabolites that were correlated to these key life
history traits of the beetles.
Results
In a previous study in which the host effect on P. vulgatissima
life history traits was examined, Lehrman et al. (2012) showed
that a two week oviposition period is a good indicator of the
suitability of the host plant. In the same study, P. vulgatissima
oviposition and survival on the two parental genotypes
‘Jorunn’ (S. viminalis) and ‘SW901290’ (S. dasyclados) were
evaluated. In our current study, we used the data from
Lehrman et al. (2012) on P. vulgatissima oviposition and
survival on ‘Jorunn’ and ‘SW901290’ during the first 14 days
following mating (i.e., day 12 to 26 in their study). Since
beetle egg laying on the two parental genotypes was relatively
stable during the selected time period, we argue that beetle
oviposition during those two weeks should be compatible and
comparable to the results of the current study.
Leaf Beetle Performance on Parental Species and F2
Hybrids There was a significant difference between the two
parental Salix genotypes in P. vulgatissima oviposition (H=
12.22, P<0.001; Fig. 1a) and survival (H=5.03, P=0.025;
Fig. 1b) with females feeding on the S. viminalis genotype
‘Jorunn’ performing better compared to females provided with
leaves from the S. dasyclados genotype ‘SW901290’. There
was also a significant difference in oviposition (number of
eggs/day) among the beetles fed on the F2 hybrid genotypes
(F11,230 =44.16, P<0.001; Fig. 1a). The highest egg production
was found for beetles on hybrid 312 while those on hybrid 310
exhibited the lowest egg production. Beetle oviposition on the
F2 hybrids seemed to be intermediate between the two parental
genotypes, and none of the hybrids yielded egg production as
high as that for beetles on the S. viminalis genotype ‘Jorunn’.
On the other hand, beetle egg production was low on several of
the F2 hybrids, resembling the oviposition on the S. dasyclados
genotype ‘SW901290’. Beetle survival (number of days) on the
520
J Chem Ecol (2013) 39:516–524
F2 hybrids varied significantly between the F2 genotypes
(F11,230 =3.90, P<0.001; Fig. 1b). Survival rate generally was
high with the exception of the beetles feeding on hybrid 310,
which exhibited high mortality with only 25 % surviving the
two week bioassay period. Beetle survival on hybrid 310 was
even lower than on the parental S. dasyclados genotype
‘SW901290’.
(Fig. 2), and the total concentration of these other phenolics
in the leaves varied among the hybrids (F5,29 =6.00, P=
0.001). The concentration and distribution of the individual
substances varied among the hybrids, e.g., chlorogenic acid,
hyperin, and luteolin derivatives were present in all hybrids
whereas (+)-catechin, quercetin 3-glucoside, and some other
flavonoids showed a more scattered distribution (Table 1).
Phenolic Profiles of F2 Hybrids The phenolic profiles of the
F2 hybrids varied considerably (Table 1). Salicylates were
present in all the studied F2 hybrids, but the total concentration of salicylates varied significantly between the hybrids (F5,29 =94.77, P<0.001) with hybrid 312 exhibiting
the lowest concentrations and hybrid 310 the highest
(Fig. 2). All hybrids also contained condensed tannins, and
the condensed tannin concentrations differed between the
hybrids (F5,29 = 6.75, P < 0.001). The hybrids with high
levels of salicylates generally had low levels of condensed
tannins and vice versa (Fig. 2). In addition, a wide variety of
other phenolic compounds were found in the F2 hybrids
Relationships Between Leaf Beetle Performance and F2
Hybrid Plant Chemistry When beetle oviposition was compared to the individual phenolic compounds found in the F2
hybrids, some noteworthy relationships appeared (Fig. 3).
Phratora vulgatissima oviposition was negatively related to
the leaf concentrations of both salicin and salicortin (R2 =0.72,
P=0.032 and R2 =0.56, P=0.085, respectively) as well as to the
total concentration of leaf salicylates (R2 =0.58, P=0.080;
Fig. 3a). Hybrid 416, however, can be seen as an outlier;
despite the relatively low salicylate concentration, oviposition
on this hybrid was extremely low. Negative relationships were
found between beetle egg production and the leaf concentration
Table 1 Concentrations (means±S.E. mg/g d.w.) of different phenolic substances in the leaves of six F2 hybrids originating from a cross between
Salix viminalis (‘Jorunn’) and Salix dasyclados (‘SW901290’)
Chemical compound
F2 hybrids
Phenolics
312
169
386
396
416
310
Salicin
Salicortin
HCH-Salicortin
Chlorogenic acid
Neochlorogenic acid
Hyperin
p-OH-cinnamic acid derivative 1
p-OH-cinnamic acid derivative 2
p-OH-cinnamic acid derivative 3
p-OH-cinnamic acid derivative 4
(+)-catechin
Quercetin diglycoside 1
Quercetin diglycoside 2
Quercetin 3-glucoside
Quercetin glycoside+Kaempferol 3-glucoside
0.91±0.12
4.45±0.50
5.26±0.94
0.22±0.02
0.07±0.01
0.25±0.05
0.46±0.09
1.04±0.13
0.87±0.11
0.09±0.01
0.83±0.02
0.50±0.02
0.79±0.04
5.88±0.26
4.50±0.27
0.96±0.10
0.14±0.01
1.50±0.16
0.34±0.02
1.70±0.13
1.36±0.09
0.22±0.14
0.80±0.07
0.19±0.01
0.35±0.03
2.29±0.21
20.35±1.24
4.48±0.54
2.30±0.65
0.06±0.01
0.27±0.03
0.80±0.25
1.42±0.37
0.50±0.05
0.39±0.02
2.75±0.13
28.71±1.12
2.03±0.26
5.90±1.44
0.83±0.07
0.13±0.01
0.48±0.02
0.56±0.05
1.01±0.08
0.78±0.07
1.28±0.18
0.08±0.01
0.15±0.01
0.35±0.03
1.71±0.08
9.11±0.68
2.14±0.11
2.04±0.59
1.04±0.08
0.10±0.01
0.63±0.07
0.41±0.04
1.84±0.27
1.52±0.24
2.36±0.43
0.09±0.01
0.40±0.02
0.16±0.02
0.39±0.04
2.74±0.19
29.12±2.51
3.93±0.47
2.55±0.29
0.19±0.01
0.26±0.03
0.73±0.03
1.33±0.33
0.18±0.01
0.19±0.01
0.37±0.06
Kaempferol diglycoside
Quercitrin
Methyl-apigenin-glycoside
Methyl-lureolin-glycoside 1
Methyl-lureolin-glycoside 2
Methyl-lureolin-glycoside 3
Dicoumaroyl-astragalin
Condensed tannins
Methanol soluble
Methanol insoluble
0.19±0.01
2.52±0.27
0.93±0.05
0.33±0.01
2.18±0.20
0.09±0.01
0.30±0.04
0.15±0.01
3.13±0.16
1.20±0.07
0.76±0.04
1.88±0.24
1.63±0.10
1.30±0.06
0.41±0.04
2.10±0.30
3.48±0.07
1.60±0.07
0.95±0.05
1.99±0.13
1.72±0.16
2.01±0.13
0.94±0.06
1.73±0.13
3.20±0.24
1.87±0.12
0.59±0.04
0.56±0.04
43.57±6.43
40.50±2.33
45.13±3.99
43.85±0.50
42.29±10.35
28.90±1.08
18.84±4.87
25.26±1.87
33.91±7.80
28.65±1.80
15.26±2.33
25.50±2.57
J Chem Ecol (2013) 39:516–524
521
P=0.019; Fig. 3d). No significant relationships were found
between beetle survival and leaf phenolic content.
Discussion
Fig. 2 The sum concentration (means±S.E.) of a salicylates (salicin,
salicortin, and HCH-salicortin), b condensed tannins (methanol soluble
and methanol insoluble), and c other phenolic compounds (phenolic
acids, phenolic glycosides, and flavonoids) found in the leaves of the
F2 hybrids. The hybrids are ranked according to Phratora vulgatissima
oviposition with beetles feeding on hybrid 312 exhibiting the
highest egg production, while beetles feeding on hybrid 310 completely
failed in their oviposition. Means capped with different letters are significantly different
of a luteolin derivative (R2 =0.86, P=0.008; Fig. 3b) and (+)catechin (R2 =0.81, P=0.015; Fig. 3c). Moreover, beetle oviposition and the concentration of condensed tannins in the
leaves demonstrated a positive relationship (R 2 = 0.79,
Host plant suitability is influenced by the genotype of the
plant and accordingly, plant genetic variation can be projected
upward onto higher trophic levels (Hochwender and Fritz,
2004). Two different patterns of Salix resistance to P.
vulgatissima emerged from the results of this study. We found
that leaf beetle oviposition was intermediate on the F2 hybrids
compared to the parental genotypes, suggesting an additive
inheritance of resistance traits (Fritz et al., 1998). On the other
hand, beetle survival on the F2 hybrids demonstrated more
similarities to one of the parents, the S. viminalis genotype ‘Jorunn’, proposing dominant inheritance (Fritz et
al., 1998). These findings indicate that there are different resistance mechanisms controlling oviposition and survival, and that these resistance mechanisms are inherited
differently in hybrids.
When considering plant chemistry, Orians (2000) stated
that while the most common pattern is that hybrids are intermediate compared to their parental species (e.g., Orians et al.,
2000; Hallgren et al., 2003; Yarnes et al., 2008; Cheng et al.,
2011), other patterns of expression have been observed; the
concentration of chemical compounds in hybrids can be similar to one of the parents (Fahselt and Ownbey, 1968; Court et
al., 1992; Yarnes et al., 2008), higher than any of the parents
(Spring and Schilling, 1989; Court et al., 1992; Hallgren et al.,
2003; Nahrung et al., 2009; Kirk et al., 2010; Cheng et al.,
2011), or lower than any of the parents (Court et al., 1992;
Yarnes et al., 2008; Cheng et al., 2011). Hybrids also can lack
chemical substances that are present in the parental species or
exhibit compounds that are not produced by the parents
(Rieseberg and Ellstrand, 1993; Orians, 2000; Kirk et al.,
2010; Cheng et al., 2011). When comparing the total concentration of all phenolic compounds found in the leaves of our
parental genotypes and F2 hybrids, the hybrids showed phenolic profiles that were intermediate compared to the parents
(for data on the chemical content of the S. viminalis genotype
‘Jorunn’ and the S. dasyclados genotype ‘SW901290’, see
Lehrman et al., 2012). An additive inheritance for all phenolic
substances could thus be assumed. However, when evaluating
individual phenolic compounds or phenolic groups, other
patterns appeared. Salix viminalis does not contain any salicylates, while this substance group is present in S. dasyclados
(Julkunen-Tiitto, 1986). According to the study of Lehrman et
al. (2012), ‘SW901290’ contain about 4.5 mg salicylates per g
d.w. leaf material. The salicylate concentration in the F2
hybrids in this study varied between approximately 5 –
35 mg/g d.w., which is higher than the salicylate concentration
of both parental genotypes. In the F2 hybrids, condensed
522
J Chem Ecol (2013) 39:516–524
Fig. 3 Relationships between
Phratora vulgatissima
oviposition (eggs/day) and leaf
concentrations (mg/g d.w.) of a
salicylates, b methyl-luteolin
glycoside 2, c (+)-catechin and
d condensed tannins in willow
F2 hybrids. Numbers in the
figure correspond to the
different hybrids
tannins demonstrated concentrations that were intermediate or
lower than the parental genotypes, while the group of other
phenolic compounds showed equal or higher concentrations
in the F2 hybrids compared to the parents. In addition, we
found some individual phenolics that were present in one of
the parents (‘Jorunn’) but were lacking in the hybrids (e.g.,
some quercetin, myricetin, kaempferol, and rhamnetin derivatives) and some new compounds that were present in the
hybrids but not in either of the parents (e.g., some quercetin,
apigenin, and luteolin derivatives). Loss of parental chemical
compounds is common in hybrids and the appearance of novel
hybrid chemicals increases in later generation hybrids
(Rieseberg and Ellstrand, 1993). This is due primarily to i)
inhibition of biochemical pathways, which results in an accumulation of precursor chemicals and a simultaneous loss of
end-products, ii) re-direction of biochemical pathways, which
results in new combinations of compound skeleton and side
chains where one part of the substance generally originates
from one parent and the other part is derived from the other
parent, iii) disruption of regulatory genes that alter gene expression and lead to changes in where the chemicals are
produced, and iv) further segregation of alleles at multiple
loci (Orians, 2000).
An intriguing result of this study is the characteristics of the
F2 hybrids 416 and 310. Beetle oviposition was extremely
low on these two hybrids; 416 demonstrated an egg production of 0.09 eggs/day, while there was no egg production at all
on 310. As a comparison, oviposition on the resistant parent
‘SW901290’ was 0.11 eggs/day. In addition, beetles feeding
on 310 demonstrated high mortality with only 25 % surviving
the two week bioassay period (compared to a survival rate of
43 % on the resistant parent ‘SW901290’). Beetle survival on
416 was, however, almost as high as on the susceptible parent
‘Jorunn’. These findings suggest that, in the process of hybridization, 310 and 416 gained modifications that were not
present in the parental genotypes, making them less susceptible to the leaf beetle P. vulgatissima. Examining the phenolic
profiles of these two hybrids shows that 416 contains fairly
low amounts of salicylates, while the salicylate concentration
in 310 is high. Salicylates previously have been reported as
important compounds in willow resistance (Rowell-Rahier,
1984; Kelly and Curry, 1991; Tahvanainen et al., 1985;
Lehrman et al., 2012). The high levels of salicylates in 310
could, therefore, be a plausible explanation for the lack of
oviposition and high mortality of beetles feeding on this
hybrid. However, there was low egg production in 416, despite the rather low concentrations of salicylates found in this
hybrid, leading us to propose that there are resistance components other than salicylates. A comparison of the concentration of a methyl-luteolin glycoside in 310 and 416 reveals
high amounts of this compound in both hybrids. In a previous
study that included the parental genotypes ‘Jorunn’ and
‘SW901290’, we showed potential negative effects of both
salicylates and a luteolin derivative on P. vulgatissima adult
and larval performance (Lehrman et al., 2012). The effect of
the luteolin derivative was, however, non-linear, and there was
a tendency towards a possible critical threshold concentration
above which beetle performance was reduced. Noteworthy is
the fact that the luteolin derivative discussed in Lehrman et al.
(2012) was not present in any of the F2 hybrids in this study;
instead novel methylated luteolin derivatives were found.
Methylated flavonoids have higher insect and mammalian
toxicity than the corresponding non-methylated ones
(Seigler, 1998). We hypothesize that the performance of the
beetles feeding on the 310 and 416 hybrids probably is a
consequence of synergistic effects between the concentrations
J Chem Ecol (2013) 39:516–524
of salicylates and the methylated luteolin derivatives. The
presence of potential threshold values, above which either
compound group alone or a combination of both compound
groups together results in toxic or even lethal effects, is
plausible.
Every hybridization event within a group of potentially
interbreeding plant genotypes will result in a unique variation
in the progeny. If the extreme variation in plant quality that
was correlated to the performance of herbivorous beetles in
this study is representative for any cross between these two
taxa, it could be argued that the results presented here are
conservative estimates of the existing variation. The fact that
we found such large variation in beetle performance and plant
chemistry highlights the potentially strong impact of plant
traits on beetle performance in willows. Although we were
able to capture a high degree of chemical variation within the
F2 population by analyzing a very limited number of plant
individuals, the design did not allow us to determine which
chemical traits actually were responsible for the observed
bioassay patterns. We can only conclude that beetle oviposition rate was negatively associated with salicylates, positively
associated with condensed tannins, and that high concentrations of a methylated luteolin derivative may contribute to the
poor beetle performance observed on some of the F2 hybrids.
Further studies including a larger set of plant individuals is
needed to identify the mechanistic connections in this study
system.
A benefit of using F2 hybrids in experimental studies is that
the plants have been subjected to recombination, which results
in a break-up of previous gene combinations and dissociation
of one trait from other traits, thus providing a greater ability to
determine the independent contribution of different traits to
overall plant resistance (Hochwender et al., 2000). Moreover,
a broad range of chemical phenotypes enables screening for
favorable individuals, which can be used to further expand our
understanding of the intricate mechanisms behind plantherbivore interactions. In this study, we found significant
differences in the phenolic profiles of parental genotypes
and F2 hybrids; compounds present in the parents were absent
in the hybrids, novel compounds appeared in the hybrids, and
the ratios between different compounds changed. The divergence in chemical phenotypes of the F2 hybrids indicates
substantial genetic alterations in these plants and changes of
biosynthetic pathways. In addition to genomic alterations,
hybridization can result in profound changes to gene expression (Hegarty et al., 2008). New patterns of gene expression
may arise by several mechanisms involving changes to upstream regulators and mutations of non-coding regulatory
DNA sequences (e.g., enhancers) of a gene (Rebeiz et al.,
2011). Genetic recombination during hybridization may increase such alterations and result in activation of previously
latent regulatory sequences. Recent transcriptomic studies in
several plant hybrid systems (e.g., maize, Stupar et al., 2007;
523
Arabidopsis, Wang et al., 2006; and Senecio, Hegarty et al.,
2008) have revealed significant changes to hybrid gene expression patterns compared to the expression patterns found in
the parental species. These findings, together with the results
presented in our study, motivates further research that penetrate deeper into the biochemical, genetic, and molecular
mechanisms behind the observed patterns. We would not find
it surprising if such research leads to the detection of hitherto
unknown mechanisms that may explain anomalies in plant
resistance studies.
Acknowledgements We thank Elena Ahlbin, Marie Melander, Sussi
Andersson Björkman, and Karin Eklund for assistance with insect
bioassays and plant nursing, and Ann-Christin Rönnberg-Wästljung
for advice and for providing plant material. This study was done as a part
of the Salix Molecular Breeding Activities (SAMBA) project financed by
the Swedish Energy Agency, the Faculty of Natural Resources and
Agricultural Sciences at the Swedish University of Agricultural Sciences
and Lantmännen Agroenergi AB.
References
ARNOLD, M. L. 1997. Natural Hybridization and Evolution. Oxford
University Press, New York.
BARBEHENN, R. V. and CONSTABEL, C. P. 2011. Tannins in plantherbivore interactions. Phytochemistry 72:1551–1565.
BAUM, C., TOLJANDER, Y. K., ECKHARDT, K.-U., and WEIH, M. 2009.
The significance of host-fungus combinations in ectomycorrhizal
symbioses for the chemical quality of willow foliage. Plant Soil
323:213–224.
BJÖRKMAN, C., HÖGLUND, S., EKLUND, K., and LARSSON, S. 2000.
Effects of leaf beetle damage on stem wood production in coppicing willow. Agr. Forest Entomol. 2:131–139.
BOECKLER, G. A., GERSHENZON, J., and UNSICKER, S. B. 2011.
Phenolic glycosides of the Salicaceae and their role as antiherbivore defences. Phytochemistry 72:1497–1509.
CHENG, D., VRIELING, K., and KLINKHAMER, P. G. L. 2011. The effect
of hybridization on secondary metabolites and herbivore resistance: implications for the evolution of chemical diversity in
plants. Phytochem. Rev. 10:107–117.
COURT, W. A., BRANDLE, J. E., POCS, R., and HENDEL, J. G. 1992. The
chemical composition of somatic hybrids between Nicotiana
tabacum and N. debneyi. Can. J. Plant Sci. 72:209–225.
CZESAK, M. E., KNEE, M. J., GALE, R. G., BODACH, S. D., and FRITZ,
R. S. 2004. Genetic architecture of resistance to aphids and mites
in a willow hybrid system. Heredity 93:619–626.
FAHSELT, D. and OWNBEY, M. 1968. Chromatographic comparison of
Dicentra species and hybrids. Am. J. Bot. 55:334–345.
FRAENKEL, G. S. 1959. The raison d’être of secondary plant substances. Science 129:1466–1470.
FRITZ, R. S. 1999. Resistance of hybrid plants to herbivores: genes,
environment, or both? Ecology 80:382–391.
FRITZ, R. S., ROCHE, B. M., and BRUNSFELD, S. J. 1998. Genetic
variation in resistance of hybrid willows to herbivores. Oikos
83:117–128.
FRITZ, R. S., MOULIA, C., and NEWCOMBE, G. 1999. Resistance of
hybrid plants and animals to herbivores, pathogens and parasites.
Annu. Rev. Ecol. Syst. 30:565–91.
GLYNN, C., RÖNNBERG-WÄSTLJUNG, A.-C., JULKUNEN-TIITTO, R.,
and WEIH, M. 2004. Willow genotype, but not drought treatment,
524
affects foliar phenolic concentrations and leaf-beetle resistance.
Entomol. Exp. Appl. 113:1–14.
HALLGREN, P. 2003. Effects of willow hybridization and simulated
browsing on the development and survival of the leaf beetle
Phratora vitellinae. BMC Ecol. 3:5.
HALLGREN, P., IKONEN, A., HJÄLTÉN, J., and ROININEN, H. 2003.
Inheritance patterns of phenolics in F1, F2, and back-cross hybrids of willows: implications for herbivore responses to hybrid
plants. J. Chem. Ecol. 29:1143–1158.
HARBORNE, J. B. 2001. Twenty-five years of chemical ecology. Nat.
Prod. Rep. 18:361–379.
HAUKIOJA, E. 1980. On the role of plant defences in the fluctuation of
herbivore populations. Oikos 35:202–213.
HEGARTY, M. J., BARKER, G. L., BRENNAN, A. C., EDWARDS, K. J.,
ABBOTT, R. J., and HISCOCK, S. J. 2008. Changes to gene expression associated with hybrid speciation in plants: further insights
from transcriptomic studies in Senecio. Philos. T. Roy. Soc. B
363:3055–3069.
HJÄLTÉN, J. 1998. An experimental test of hybrid resistance to insects
and pathogens using Salix caprea, S. repens and their F1 hybrids.
Oecologia 117:127–132.
HJÄLTÉN, J. and HALLGREN, P. 2002. The resistance of hybrid willows
to specialist and generalist herbivores and pathogens: the potential
role of secondary chemistry and parent host plant status, pp. 153–
168, in M. R. Wagner, K. M. Clancy, F. Lieutier, and T. D. Paine
(eds.), Mechanisms and Deployment of Resistance in Trees to
Insects. Kluwer Academic Publishers, New York.
HOCHWENDER, C. G. and FRITZ, R. S. 2004. Plant genetic differences
influence herbivore community structure: evidence from a hybrid
willow system. Oecologia 138:547–557.
HOCHWENDER, C. G., FRITZ, R. S., and ORIANS, C. M. 2000. Using
hybrid systems to explore the evolution of tolerance to damage.
Evol. Ecol. 14:509–521.
JULKUNEN-TIITTO, R. 1986. A chemotaxonomic survey of phenolics in
leaves of northern Salicaceae species. Phytochemistry 25:663–
667.
JULKUNEN-TIITTO, R. 1989. Phenolic constituents of Salix: a chemotaxonomic survey of further Finnish species. Phytochemistry
28:2115–2125.
JULKUNEN-TIITTO, R. and SORSA, S. 2001. Testing the effects of drying
methods on willow flavonoids, tannins, and salicylates. J. Chem.
Ecol. 27:779–789.
KELLY, M. T. and CURRY, J. P. 1991. The influence of phenolic
compounds on the suitability of three Salix species as hosts
for the willow beetle Phratora vulgatissima. Entomol. Exp.
Appl. 61:25–32.
KENDALL, D. A., HUNTER, T., ARNOLD, G. M., LIGGITT, J., MORRIS,
T., and WILTSHIRE, C. W. 1996. Susceptibility of willow clones
(Salix spp.) to herbivory by Phyllodecta vulgatissima (L.) and
Galerucella lineola (Fab.) (Coleoptera, Chrysomelidae). Ann.
Appl. Biol. 129:379–390.
KIRK, H., VRIELING, K., VAN DER MEIJDEN, E., and KLINKHAMER, P.
G. L. 2010. Species by environment interactions affect
pyrrolizidine alkaloid expression in Senecio jacobaea, Senecio
aquaticus, and their hybrids. J. Chem. Ecol. 36:378–387.
LEHRMAN, A., TORP, M., STENBERG, J. A., JULKUNEN-TIITTO, R., and
BJÖRKMAN, C. 2012. Estimating direct resistance in willows
against a major insect pest (Phratora vulgatissima) comparing
life history traits. Entomol. Exp. Appl. 144:93–100.
J Chem Ecol (2013) 39:516–524
MATSUKI, M. and MACLEAN JR., S. F. 1994. Effects of different leaf
traits on growth rates of insect herbivores on willows. Oecologia
100:141–152.
MATTSON, W. J. 1980. Herbivory in relation to plant nitrogen content.
Annu. Rev. Ecol. Syst. 11:119–161.
NAHRUNG, H. F., WAUGH, R., and HAYES, R. A. 2009. Corymbia
species and hybrids: chemical and physical foliar attributes and
implications for herbivory. J. Chem. Ecol. 35:1043–1053.
NYMAN, T. and JULKUNEN-TIITTO, R. 2005. Chemical variation within and
among six northern willow species. Phytochemistry 66:2836–2843.
ORIANS, C. M. 2000. The effects of hybridization in plants on
secondary chemistry: implications for the ecology and evolution of plant-herbivore interactions. Am. J. Bot. 87:1749–1756.
ORIANS, C. M. and FRITZ, R. S. 1996. Genetic and soil-nutrient effects on
the abundance of herbivores on willows. Oecologia 105:388–396.
ORIANS, C. M., HUANG, C. H., WILD, A., DORFMAN, K. A., ZEE, P.,
DAO, M. T. T., and FRITZ, R. S. 1997. Willow hybridization
differentially affects preference and performance of herbivorous
beetles. Entomol. Exp. Appl. 83:285–294.
ORIANS, C. M., GRIFFITHS, M., ROCHE, B. M., and FRITZ, R. S. 2000.
Phenolic glycosides and condensed tannins in Salix sericea, S.
eriocephala and their F1 hybrids: not all hybrids are created
equal. Biochem. Syst. Ecol. 28:619–623.
PORTER, L. J., HRSTICH, L. N., and CHAN, B. G. 1986. The conversation of proanthocyanidins and prodelohinidins to cyanidins and
delphinidins. Phytochemistry 25:223–230.
REBEIZ, M., JIKOMES, N., KASSNER, V. A., and CARROLL, S. B. 2011.
Evolutionary origin of a novel gene expression pattern through
co-option of the latent activities of existing regulatory sequences.
Proc. Natl. Acad. Sci. USA 108:10036–10043.
RIESEBERG, L. H. and ELLSTRAND, N. C. 1993. What can molecular
and morphological markers tell us about plant hybridization? Crit.
Rev. Plant Sci. 12:213–241.
ROWELL-RAHIER, M. 1984. The presence or absence of phenolglycosides
in Salix (Salicaceae) leaves and the level of dietary specialization
of some of their herbivorous insects. Oecologia 62:26–30.
SEIGLER, D. S. 1998. Plant Secondary Metabolism. Kluwer Academic
Publishers, USA.
SPRING, O. and SCHILLING, E. E. 1989. Chemosystematic investigation
of the annual species of Helianthus (Asteraceae). Biochem. Syst.
Ecol. 17:519–528.
STUPAR, R. M., HERMANSON, P. J., and SPRINGER, N. M. 2007.
Nonadditive expression and parent-of-origin effects identified by
microarray and allele-specific expression profiling of maize endosperm. Plant Physiol. 145:411–425.
TAHVANAINEN, J., JULKUNEN-TIITTO, R., and KETTUNEN, J. 1985.
Phenolic glucosides govern the food selection pattern of willow
feeding leaf beetles. Oecologia 67:52–56.
TREUTTER, D. 2006. Significance of flavonoids in plant resistance: a
review. Environ. Chem. Lett. 4:147–157.
WANG, J., TIAN, L., LEE, H.-S., WEI, N. E., JIANG, H., WATSON, B.,
MADLUNG, A., OSBORN, T. C., DOERGE, R. W., COMAI, L., and
CHEN, Z. J. 2006. Genomewide nonadditive gene regulation in
Arabidopsis allotetraploids. Genetics 172:507–517.
WHITHAM, T. G. 1989. Plant hybrid zones as sinks for pests. Science
244:1490–1493.
YARNES, C. T., BOECKLEN, W. J., AND SALMINEN, J. P. 2008. No
simple sum: seasonal variation in tannin phenotypes and leafminers in hybrid oaks. Chemoecology 18:39–51.