Oecologia
DOI 10.1007/s00442-007-0784-1
PLANT ANIMAL INTERACTIONS
Heritable variation in the foliar secondary metabolite
sideroxylonal in Eucalyptus confers cross-resistance to herbivores
Rose L. Andrew Æ Ian R. Wallis Æ Chris E. Harwood Æ
Michael Henson Æ William J. Foley
Received: 9 November 2006 / Accepted: 24 May 2007
Springer-Verlag 2007
Abstract Plants encounter a broad range of natural enemies and defend themselves in diverse ways. The cost of
defense can be reduced if a plant secondary metabolite
confers resistance to multiple herbivores. However, there
are few examples of positively correlated defenses in plants
against herbivores of different types. We present evidence
that a genetically variable chemical trait that acts as a strong
antifeedant to mammalian herbivores of Eucalyptus also
deters insect herbivores, suggesting a possible mechanism
for cross-resistance. We provide field confirmation that
sideroxylonal, an important antifeedant for mammalian
herbivores, also determines patterns of damage by Christmas beetles, a specialist insect herbivore of Eucalyptus. In a
genetic progeny trial of Eucalyptus tricarpa, we found
significant heritabilities of sideroxylonal concentration
(0.60), overall insect damage (0.34), and growth traits
(0.30–0.53). Population of origin also had a strong effect on
each trait. Negative phenotypic correlations were observed
between sideroxylonal and damage, and between damage
and growth. No relationship was observed between sideroxylonal concentration and any growth trait. Our results
Communicated by Julia Koricheva.
R. L. Andrew (&) I. R. Wallis W. J. Foley
School of Botany and Zoology,
The Australian National University,
Canberra, ACT 0200, Australia
e-mail: [email protected]
C. E. Harwood
Cooperative Research Centre for Forestry,
College Road, Sandy Bay, TAS 7005, Australia
M. Henson
Forests NSW, Northern Research Centre,
357 Harbour Drive, Coffs Harbour Jetty, NSW 2450, Australia
suggest that potential for evolution by natural selection of
sideroxylonal concentrations is not strongly constrained by
growth costs and that both growth and defense traits can be
successfully incorporated into breeding programs for plantation trees.
Keywords Eucalyptus tricarpa Formylated phloroglucinol compound (FPC) Anoplognathus montanus Heritability Growth-defense trade-off
Introduction
Plants have evolved a broad range of defenses against both
mammalian and invertebrate herbivores. If a plant secondary metabolite (PSM) confers resistance to multiple
herbivores, then the cost of defense can be reduced.
However, there are few examples of positively correlated
defenses in plants against herbivores of different types,
such as generalists and specialists (Leimu and Koricheva
2006) or mammalian and insect herbivores (Rousi et al.
1997; Mutikainen et al. 2002; Puustinen et al. 2004). Because the challenges of multiple herbivores can place
constraints on the evolution of plant defenses (Rausher
1996), the chemical and genetic basis of cross-resistance is
important.
Formylated phloroglucinol compounds (FPCs) may be
responsible for cross-resistance to mammalian and insect
herbivores of eucalypts (Eucalyptus l’Héritier and
Corymbia Hill and Johnson, Myrtaceae). These compounds
are important determinants of palatability to a range of
specialist and generalist mammalian herbivores, both captive and free-ranging (Lawler et al. 1998, 2000; Wallis
et al. 2002; Moore et al. 2004b; Scrivener et al. 2004;
123
Oecologia
Moore and Foley 2005). The physiological mechanism
underlying aversion to FPCs has also been elucidated,
making the system one of the best understood of plant–
mammal interactions (Lawler et al. 1998). A subgroup of
FPCs, known as sideroxylonals, also affects the feeding of
insect herbivores in the laboratory (Floyd and Foley 2001),
but evidence of deterrence in wild insects is needed to
support the hypothesis that FPCs contribute to crossresistance to mammalian and insect herbivores. We present
here the first field demonstration that concentrations of
sideroxylonals are related to food plant selection by wild
insects, in this case a species of Christmas beetle (Anoplognathus montanus Macleay; Coleoptera: Scarabaeidae),
which is a specialist herbivore of eucalypts.
There is substantial variation in the concentrations of
FPCs among eucalypt trees within populations (Lawler
et al. 2000; Wallis et al. 2002; Moore et al. 2004b). This
can create significant habitat patchiness at ecologically
relevant scales (Andrew et al. 2007), and understanding
how this variation arises requires comprehension of the
heritability of the trait. Narrow-sense heritability, the proportion of variation within populations that is due to
additive genetic causes, is of crucial importance because it
determines the ability of a species to evolve in response to
both natural and artificial selection. The heritability of foliar sideroxylonal concentration has been estimated in a
natural population of Eucalyptus melliodora using a marker-based method (Andrew et al. 2005), but comparisons
have not yet been made with traditional common-garden
estimates. Since genetic relatedness is not inferred from
molecular markers and sample sizes are less likely to be
limited by expense, genetic trial experiments in a common
environment (often called common-garden experiments)
can offer greater precision in estimates of quantitative
genetic parameters. Such estimates from genetic trial
experiments are more appropriate than those from natural
populations for predicting likely gains from breeding programs, which are aimed at genetic improvement of useful
traits in tree plantations. Common-garden experiments also
allow variation among populations to be studied without
the confounding effects of site differences (O’ReillyWapstra et al. 2002; Moore et al. 2004b). Phenotypic and
genetic correlations measured in progeny can also provide
insight into the evolutionary constraints on sideroxylonal
concentrations.
We addressed the relationship between foliar sideroxylonal concentrations and insect herbivory and studied
the quantitative genetics of sideroxylonal variation in
Eucalyptus tricarpa (LAS Johnson) LAS Johnson and K.D.
Hill. This was, until recently, a subspecies of E. sideroxylon (Hill and Johnson 1991), in which mammal feeding
studies have been conducted (Lawler et al. 2000). The two
species are chemically similar, with 0–23 mg g–1 DW
123
sideroxylonal (mean 10.9 mg g–1 DW) in E. sideroxylon
(Lawler et al. 2000; Floyd and Foley 2001) and 0–
47 mg g–1 DW sideroxylonal (mean 13.5 mg g–1 DW) in
E. tricarpa (this study). Since they also share mammalian
and insect herbivores, the influence of foliar defenses on
herbivory is likely to be sufficiently similar to provide a
reasonable grounds for inferring cross-resistance. Studying
a field trial testing open-pollinated progenies from a range
of natural provenances, we asked the following questions:
1.
2.
3.
4.
Do Christmas beetles preferentially attack E. tricarpa
trees that have low foliar concentrations of sideroxylonal?
Does significant narrow-sense heritability of variation
in foliar sideroxylonal occur in E. tricarpa? Are heritability estimates similar to marker-based estimates
from a wild E. melliodora population?
Do E. tricarpa populations display genetic differentiation in foliar sideroxylonal concentrations?
Do phenotypic and/or genetic correlations occur
among foliar sideroxylonal, growth traits, and insect
damage?
Materials and methods
Experimental design and sampling
Eucalyptus tricarpa is a widespread species occurring in
the low to medium rainfall zone in Victoria and New South
Wales (NSW), Australia (Brooker and Kleinig 1999). This
species is closely related to E. melliodora, in which sideroxylonal is highly heritable (Andrew et al. 2005). E.
tricarpa is being developed for forestry in areas of low
rainfall. The E. tricarpa provenance/progeny trial sampled
in this study was set up by Forests NSW, near Culcairn,
NSW (14656¢E, 3543¢S) in August 2000, as part of the
Australian Low Rainfall Tree Improvement Group (ALRTIG) program (Harwood et al. 2001). The trial tested 108
open-pollinated families, representing 15 natural populations in Victoria and NSW (Table 1). Eucalypts have
mixed mating systems, with typical outcrossing rates of
70% (Eldridge et al. 1993). Open pollination leads to
families consisting of mixtures of half-siblings and selfed
or outcrossed full siblings. Trees were arranged in four
replicates, with each family represented in each replicate
by one five-tree row plot. Trees were planted 1.8 m apart
with 4 m between rows and a buffer two trees deep on the
outside edges of the trial. The experimental area covered
1.8 ha, and an adjoining 0.6-ha area was planted with
surplus stock of some of the families. It was surrounded on
all sides by pasture, with the nearest Eucalyptus trees
approximately 200 m away.
Oecologia
Table 1 Geographical and climatic variables of populations represented in an E. tricarpa genetic experiment. Longitude, latitude, and
approximated altitude were obtained from seedlot collection reports, and climatic variables were predicted using WORLDCLIM data grids
Population
Region
Latitude
(S)
Longitude
(E)
Altitude
(m)
Annual mean
temperature
(C)
Minimum
temperature of
coldest month (C)
Lorne
Southern
3828¢
14403¢
105
13.9
5.7
Annual
precipitation
(mm)
691
Mt Bealiba
Goldfields
3648¢
14338¢
290
13.6
2.5
588
Clunes
Craigie
Goldfields
Goldfields
3716¢
3704¢
14344¢
14345¢
340
250
13.1
13.7
2.6
2.7
625
585
Tarnagulla
Goldfields
3645¢
14350¢
500
14.5
3.1
509
Heathcote
Goldfields
3659¢
14445¢
280
13.5
2.7
682
Mt Douglas Dam
Goldfields
3649¢
14449¢
240
14.3
2.9
610
Whroo
Goldfields
3642¢
14458¢
160
14.5
3
599
Christmas Hills
Central
3741¢
14523¢
14.5
4.6
801
Heyfield
Central
3756¢
14643¢
115
13.4
2.7
794
Mt Nowa Nowa
Gippsland
3742¢
14806¢
110
13.7
3.8
827
Tucker Box Track
Gippsland
3738¢
14815¢
100
13.6
3.4
877
Martin’s Ck
Gippsland
3728¢
14833¢
325
13.8
3.4
925
Bodalla
NSW
3603¢
14958¢
100
15.6
5.5
1,017
Narooma
NSW
3617¢
14958¢
160
14.8
4.7
1,026
Our sampling was conducted in November 2002, when
the plants were producing foliage that was intermediate
between juvenile and fully mature morphologies. The
transition from juvenile to adult foliage is not as dramatic
in E. tricarpa as in many other species of Eucalyptus. Leaf
shape differs only slightly and foliar chemistry is stable
across years (R. L. Andrew, unpublished observations). We
selected 9 populations covering the natural distribution of
the species and sampled from 3 to 9 families from each
population (9 families where possible), giving a total of 67
families selected. In each block, we sampled the two
westernmost trees of each plot planted for the selected
families. Where trees were stunted or dead, the next suitable tree in the plot was sampled. From the southern side of
each tree, approximately 50 g of leaf material was sampled, frozen, and stored at –20C. We sampled one replicate at a time, so that time of day was not confounded with
population or family. Height, diameter at 1.3 m (DBH) of
largest stem, and the sum of the squared diameters at 1.3 m
of all stems (D2) were measured for all trees by Forests
NSW in January 2004.
Insect damage
At the time of sampling, the experimental plants were
under attack by Anoplognathus montanus, a local, native
Christmas beetle. A simple presence/absence score was
used to assess Christmas beetle attack, as the pattern of
damage and beetle presence was very clear: a tree was
either heavily infested or free from damage. This is
consistent with previously observed patterns of damage
by Anoplognathus spp. (Edwards et al. 1993). Paired
comparisons were made to test the relationship between
beetle infestation and sideroxylonal concentrations. Each
pair consisted of one infested tree and one unaffected tree
from the same family and replicate (i.e., the same plot),
and sideroxylonal was measured using high-performance
liquid chromatography (HPLC) for both trees (see below).
The 23 pairs sampled were from 16 families, 12 of which
were among those included in the quantitative genetic
analysis.
In addition to Christmas beetles, defoliation and damage
to photosynthetic leaf area by other agents was also
apparent. A range of folivorous and sap-sucking insects,
such as chrysomelid leaf beetles (Paropsis spp.) and their
larvae, sawfly larvae (Perga sp.), leaf-blister sawflies
(Phylacteophaga sp.), and Eucalyptus weevils (Gonipterus
scutellatus), may have contributed to the observed damage.
Each tree that was sampled for chemical analysis was given
a score for overall insect damage. Trees were scored from
one to four for minimal defoliation, light defoliation,
moderate defoliation, and heavy defoliation, respectively.
Heavy defoliation (damage score 4) resulted in visible bare
branch ends over the whole of the canopy. Our damage
classes were similar to those employed by Raymond
(1995), but differed in the order of the ranking (from least
to most damage) and in the description of overall damage
(rather than of that produced by a single insect species).
The damage score affected by the presence of Christmas
beetle because of their contribution to overall leaf damage,
123
Oecologia
but also describes variation in leaf damage from other
sources, which were difficult to distinguish.
Chemical analysis
The leaves were freeze–dried and ground to pass through a
1-mm screen in a Tecator Cyclotec mill. A subset of 105
samples (including 46 paired trees selected for the Christmas beetle test) was assayed for sideroxylonal using the
HPLC technique described by Wallis et al. (2003). Sideroxylonal A and sideroxylonal C are found in constant
proportions in this solvent system (Moore et al. 2004a) and
their concentrations were combined to give the concentration of total sideroxylonals, which will be referred to as
sideroxylonal henceforth. We collected the visible and
near-infrared spectra (400–2,500 nm) of all samples from
dry, ground leaf following Moore et al. (2004b). Calibration equations were developed using modified partial least
squares regression using WINISI II (Infrasoft International,
Port Matilda, PA, USA) and following standard procedures
(American Society for Testing and Materials 1995). The
optimal model used the 1,100- to 2,498-nm region, standard normal variate and detrend scatter correction, and a
math treatment of 1881 (i.e., first derivative treatment, with
a gap width of 8 nm and smoothing over 8 data points; see
American Society for Testing and Materials 1995). The
model explained 97.8% of foliar sideroxylonal variation
(mean 17.9 mg g–1 dm; range 0.0–47.6 mg g–1 dm) in the
calibration set and had a standard error of cross-validation
of 2.4 mg g–1 dm.
Statistical analysis
Statistical analyses were conducted using GenStat version
8 (VSN International Ltd, Hemel Hempstead, UK). The
relationship between sideroxylonal and presence of
Christmas beetle was investigated by choosing paired
damaged and undamaged trees from the same plot. Beetledamaged trees were paired with the nearest available
undamaged tree within the same plot. This approach not
only avoided the large number of zero Christmas beetle
damage scores that would result if all trees assessed for
sideroxylonal had been included but also controlled for
genetic background (family) and location in the trial. The
difference in foliar sideroxylonal concentration between
the 23 affected trees and their paired unaffected trees was
tested using a two-sided, one-sample t-test.
Estimates of narrow-sense heritability were computed
for foliar sideroxylonal concentration, damage score, and
the growth traits using pooled variance components estimated by restricted maximum likelihood analysis (REML).
Variance components were estimated by fitting a mixed
123
model, with replicate and population as fixed effects.
Family within population was fitted as a random effect, as
well as plot within replicate, to account for the remaining
plot-level variance. Variance components estimated for
families within populations (r2f ), plots within replicates
(r2plot), and residual error (r2e ) were summed to give r2p, the
total phenotypic variance within populations and replicates
(Williams et al. 2002, p. 103). The use of a single pooled
family variance component leads to an estimate of the
average heritability among populations. Standard errors
were calculated from the family and residual variance
components using VFUNCTION (Payne 2005). All available trees were used for heritability estimation for each
trait. All 108 families represented in the trial were included
for height and DBH, which were measured for 1,772 and
1,692 trees, respectively. In contrast, only 67 families from
nine populations were available for estimation of heritability for sideroxylonal and damage (465 trees).
Additive genetic variance in phenotypic traits can be
estimated using the expected covariance of half-siblings
and adjusted to account for the outcrossing rates, according
to the common procedure used for trees with mixed mating
systems (Eldridge et al. 1993; Williams et al. 2002).
Within-population, narrow-sense heritability (h2) specific
to this environment was calculated using the formula,
h2 ¼
1= r2
f
r
;
r2p
where r is the mean relatedness within families, which is
typically assumed to be 0.4 in Eucalyptus (Eldridge et al.
1993). Instead of four times the family variance, additive
genetic variance is estimated as 2.5 times (or 1/0.4 times)
the family variance. As with all half-sibling designs, this
model assumes that inbreeding and epistasis are absent.
Maternal effects may be important in maternal half-sibling
families, but they are not commonly observed in Eucalyptus, based on controlled cross experiments (Lopez et al.
2003). In addition, this design cannot fully control for
dominance effects, which may inflate the within-family
covariance in plants with mixed mating systems.
Additive genetic correlations (subject to the same
assumptions as for heritability estimates) between pairs of
traits were estimated from plot means using multivariate
REML in GenStat 8 and tested using likelihood ratio tests
(Payne 2005). Replicate and population were fixed effects,
while family and plot were random effects in the model.
Standard errors of genetic correlations were estimated
using a standard Taylor expansion formula, implemented in
GenStat. Genetic correlations were estimated using only
populations for which both growth and sideroxylonal had
been measured.
Oecologia
The effects of geographic and climatic variables on
phenotypic traits were tested using the best linear unbiased
estimates (BLUEs) of population means obtained from the
REML mixed model analysis. Geographic variables considered were latitude, longitude, and approximate altitude.
Annual means of temperature and precipitation for each
population were obtained using the WORLDCLIM climatic grids (Hijmans et al. 2005) in ArcView GIS 3.3
(Environmental Systems Research Institute, Redlands, CA,
USA). Since minimum temperatures have been found to
be related to foliar sideroxylonal concentrations in Eucalyptus microcorys (Moore et al. 2004b), this variable was
also included. Univariate regression and Spearman’s rank
correlation were used to test the relationships between
geographic or climatic variables and the BLUEs for each
trait. Since multiple tests were carried out, sequential
Bonferroni corrections were used to adjust P values (Rice
1989).
Results
At the time of investigation, Christmas beetles occurred on
7.7% of trees sampled. The proportion of trees affected by
these beetles varied from 0% to 34% among populations
(Table 2), with 0–100% of trees within families under
Table 2 Population characteristics and traits measured in an E.
tricarpa genetic experiment. Best linear unbiased estimates (BLUEs)
of population effects for sideroxylonal, damage score, diameter at
1.3 m (DBH) and the sum of the squared diameters of all stems (D2)
were estimated using REML mixed model estimation, accounting for
attack. Four populations were unaffected by Christmas
beetles, including both NSW populations and the southernmost population, from Lorne (Fig. 1). Comparison of
paired affected infested with unaffected trees showed that
foliar sideroxylonal concentration was, on average,
10.4 mg g–1 dry weight lower in trees under attack
(t = 3.81 on 22 df; P < 0.001).
The mean concentrations of foliar sideroxylonal in
populations, predicted by the REML analysis, ranged from
12.8 to 33.8 mg g–1 dry weight (Table 2). Leaves from the
Clunes, Mt Nowa Nowa and Bodalla populations were all
consistently high in sideroxylonal, showing that high-sideroxylonal populations can be found throughout the range
of the species. Significant differences were also observed
among populations for damage score (P < 0.001), and
growth traits also varied among populations (P < 0.001).
Populations from Gippsland and NSW generally grew
faster than those from Central and Goldfields regions
(Table 2).
Genetic analysis revealed significant heritability for
each trait (Table 3). Sideroxylonal and damage demonstrated heritabilities of 0.60 and 0.34, respectively. The
heritabilities of height (0.44) and DBH (0.30) were high for
eucalypts and higher still when estimated only for the
populations in which sideroxylonal was measured (0.53
and 0.34, respectively; Table 3). Replicates differed sig-
replicate (fixed), plot within replicate (random) and family within
population (random). The grand mean and average standard error of
differences among population means were estimated using the same
model
Sideroxylonal
(mg g–1 DW)
Damage
score (1–4)
Height
(m)
DBH
(cm)
D2
(cm2)
5
27.2
2.55
3.21
2.96
3.48
9
24.6
2.11
4.15
4.61
4.89
Goldfields
10
32.8
2.36
4.42
5.07
5.41
Craigie
Goldfields
8
4.16
4.87
5.15
Tarnagulla
Goldfields
8
4.90
5.83
6.04
Heathcote
Mt Douglas Dam
Goldfields
Goldfields
10
3
3.57
1.83
4.31
2.61
4.61
2.84
Whroo
Goldfields
2
4.03
4.47
4.95
Christmas Hills
Central
8
4.00
4.21
4.75
Heyfield
Central
11
20.6
2.78
3.45
4.00
4.26
Martin’s Ck
Gippsland
11
12.8
2.69
4.74
5.32
5.71
Mt Nowa Nowa
Gippsland
11
33.8
1.84
Tucker Box Track
Gippsland
3
Population
Region
Lorne
Southern
Mt Bealiba
Goldfields
Clunes
Families
in trial
17.7
2.76
4.69
5.11
5.55
4.78
5.31
5.77
Bodalla
NSW
6
29.2
1.52
4.91
5.63
6.05
Narooma
NSW
3
26.6
1.75
5.17
5.97
6.43
25.0
2.26
4.14
4.68
5.06
2.8
0.24
0.36
0.52
0.55
Grand mean
S.E.D.
123
Oecologia
21
Lorne
Mt Bealiba
65
Clunes
70
Heathcote
69
Heyfield
60
Mt Nowa Nowa
66
Martin's Ck
68
Bodalla
44
Narooma
24
0
10
20
30
40
Trees attacked (%)
Fig. 1 Percentage of progeny from nine E. tricarpa populations
attacked by Christmas beetles during an outbreak in a common garden
experiment. Sample sizes (the number of trees from each population)
are shown next to each bar
nificantly for damage and growth traits, but not for sideroxylonal concentration. The two growth traits were highly
phenotypically correlated, and damage was negatively
correlated with both growth and foliar sideroxylonal concentrations (Table 4). However, there was no phenotypic
correlation of foliar sideroxylonal with either growth trait.
The additive genetic correlation of height and DBH was
high, but sideroxylonal concentration and damage score
were not significantly genetically correlated with any trait
(Table 4). Predicted population means were negatively
correlated for insect damage and plant height (r = –0.734,
P = 0.024). The correlation of predicted population means
for sideroxylonal and damage score was marginally significant (r = –0.659, P = 0.054).
When the effects of source population geographic or
climatic variables on traits were tested, no rank correlations
and only one univariate regression was significant at the
0.05 level prior to Bonferroni corrections. However, none
was significant or close to significance (P < 0.1) after
corrections were made for multiple comparisons.
Discussion
Cross-resistance of insect and mammalian herbivores
This study provides field evidence that cross-resistance to
insect and mammalian herbivores of Eucalyptus may be
conferred by foliar sideroxylonals at typical concentrations.
Trees with low concentrations of foliar sideroxylonals were
123
chosen by Christmas beetles over nearby, genetically related trees with higher concentrations. At concentrations
similar to those in the present study, sideroxylonals and
other FPCs have also been shown to reduce intake by a
range of mammalian herbivores (Lawler et al. 2000; Wallis
et al. 2002; Moore et al. 2005). The congruence of these
results with those demonstrating sideroxylonal-related
resistance to herbivory by common ringtail possums
(Pseudocheirus peregrinus) in a closely related and
chemically similar species (E. sideroxylon, Lawler et al.
2000) suggests that cross-resistance is likely in at least
these two eucalypt species, but probably also in others. The
variation observed in E. tricarpa foliar sideroxylonal
concentrations is most likely constitutive, as there is no
evidence of induced defenses in Eucalyptus (Henery 2006).
While previous tests of foliar defense induction may have
failed to detect a small response, a large change in concentration would be needed to produce the 10.4-mg g–1
difference between the trees affected and unaffected by
beetles in the present study.
In contrast to studies of annuals and herbaceous perennials, the chemical basis of defense has been identified in
few tree-herbivore systems and can be complex (Haukioja
2003). That the same chemicals are responsible for crossresistance to both mammalian and insect herbivores at
concentrations commonly found in leaves is ecologically
significant. Although broad classes of secondary chemicals,
such as phenolic glycosides and terpenes, are active against
both mammalian and arthropod herbivores, we are not
aware of any other examples of specific chemicals known
to confer resistance to both mammalian and insect herbivores. The strength of natural selection, both historical and
contemporary, on FPCs in eucalypts is still unknown, but
their evolution may have been favored by their effectiveness as defensive chemicals against diverse specialist and
generalist herbivores.
Adult Christmas beetles are specialist herbivores of
eucalypts and can cause severe damage to trees in plantations (Carne et al. 1974). They appear to have increased in
numbers in rural areas, probably because their larvae feed
on roots underground and can benefit from the increased
productivity of pasture and crops (Carne et al. 1974).
Christmas beetle numbers are extremely variable and severe outbreaks can result in nearly 100% defoliation of
some trees (Pryor 1953; Edwards et al. 1993). Laboratory
bioassays have shown that variation in sideroxylonal concentration in leaves can determine the feeding preferences
of Christmas beetles (Floyd and Foley 2001) and this study
provides evidence that wild Christmas beetles select trees
on the basis of foliar sideroxylonal concentration.
In the past, Christmas beetle herbivory has been related to
essential oil concentration and in particular the foliar concentration of 1,8-cineole (Edwards et al. 1993). However,
Oecologia
Table 3 Summary of quantitative genetic analysis using univariate REML
na
Sideroxylonal
Damage
Height
DBH
465
465
1,772
1,692
Random model
r2f (s.e.)b
2
v prob
c
17.68 (4.71)
0.082 (0.036)
0.272 (0.055)
<0.0001
0.478 (0.112)
<0.0001
0.0050
r2plot (s.e.)d
0.77 (3.77)
0.154 (0.042)
0.084 (0.032)
<0.0001
0.194 (0.089)
v2 probc
0.0606
0.0001
0.0032
0.0182
r2e (s.e.)e
55.33 (4.80)
0.368 (0.035)
1.178 (0.046)
3.318 (0.132)
Fixed model
Replicate
Wald statistic
0.89
dff
3
23.63
v2 probg
0.829
<0.001
Wald statistic
132.52
69.09
df
v2 probg
8
<0.001
8
<0.001
h2 (s.e.)h
0.60 (0.13)
19.91
3
3
<0.001
12.09
3
0.007
Population
131.75
14
<0.001
0.34 (0.14)
0.44 (0.08)
82.97
14
<0.001
0.30 (0.06)
Wald tests were used to test fixed effects by dropping individual terms from the model
a
n total number of trees included in REML analysis
b
r2f estimated variance component attributable to families within populations
c
v2 prob chi-square probability associated with change in deviance
d
r2plot residual plot-level variance component
e
r2e residual tree-level variance component
f
df degrees of freedom
g
v2 prob chi-square probability associated with Wald statistic
h
h2 estimated average narrow-sense heritability based on pooled family variance (r2f )
All heritability estimates are significant (P < 0.001) based on likelihood ratio tests
Table 4 Phenotypic (below diagonal) and additive genetic (above
diagonal, with standard errors in parentheses) correlations for defense
and growth traits in E. tricarpa. Average narrow-sense heritability
(with standard errors in parentheses), estimated using the same
Trait 1
Trait 2
Sideroxylonal
Sideroxylonal
Damage
provenances are shown italicized and on the diagonal. The genetic
correlation of height and DBH was estimated using Fisher optimisation to obtain model convergence
0.60* (0.13*)
–0.40*
Damage
Height
–0.39 (0.21)
0.34* (0.14*)
DBH
–0.07 (0.19)
0.00 (0.20)
–0.30 (0.20)
–0.34 (0.22)
Height
0.05
–0.27*
0.53* (0.08*)
0.96* (0.03)
DBH
0.02
–0.19*
0.84*
0.34* (0.06*)
* Significantly different from zero (P < 0.05)
resistant E. grandis clones were no higher in 1,8-cineole
concentration than susceptible clones (Johns et al. 2004).
Furthermore, experiments manipulating the concentration of
both 1,8-cineole and sideroxylonal in leaves have suggested
that FPCs have a larger effect on Christmas beetles (Floyd
and Foley 2001). Foliar concentrations of cineole and sideroxylonal tend to be correlated in natural populations
(Moore et al. 2004a; Andrew et al. 2005), which explains
why previous studies have identified cineole as the primary
deterrent. This association allows mammalian herbivores to
use cineole, which is volatile and has a strong odor, as a guide
to sideroxylonal concentrations (Lawler et al. 1999). It appears that terpenes may have a similar role as an olfactory cue
in feeding by Christmas beetles (Floyd and Foley 2001).
123
Oecologia
In addition to resistance, plants may employ alternative
or additional strategies to limit the costs of herbivory, such
as tolerance (Fornoni et al. 2004) or escaping herbivory
through faster growth (Kursar and Coley 2003). We found
some support for the latter in E. tricarpa, as overall insect
damage was related to both sideroxylonal concentration
and growth. However, the relationship of damage with
growth may also be due to the costs imposed by herbivore
damage, since the growth measurements were made a year
after the observed damage was done. A negative effect of
defoliation on subsequent growth rates was observed by
Raymond (1995) in trees damaged by chrysomelid leaf
beetles (Chrysoptharta bimaculata), which also chose
smaller trees. We were unable to separate these effects, as
growth measurements were made once only and insects
were not experimentally excluded.
Heritability of sideroxylonal and herbivore defense
Foliar sideroxylonal concentration is highly variable in E.
tricarpa, and our estimate of the narrow-sense heritability
of this trait is high. Previous progeny experiments in
Eucalyptus have significant genetic differentiation in FPCs
among populations, but within-population genetic variation
in sideroxylonal has not yet been shown (O’Reilly-Wapstra
et al. 2002, 2004). The present study supports the finding of
significant additive genetic variation of foliar sideroxylonal, with narrow-sense heritability estimates between 0.55
and 0.89, in E. melliodora using a genetic marker-based
approach (Andrew et al. 2005). Common-garden experiments are useful because they provide greater precision
than those in natural populations. However, the coefficient
of relationship of 0.4 among the individuals of open-pollinated families used for estimating heritability in this study
is only an approximation (Griffin and Cotterill 1988; Eldridge et al. 1993, p. 174). The degree of relatedness within
open-pollinated Eucalyptus families is affected by the
levels of selfing and correlated paternity. Studies of progeny arrays using molecular markers have shown that these
vary widely both among species (McDonald et al. 2003;
Jones et al. 2005) and within species (Burgess et al. 1996;
Butcher and Williams 2002). Direct estimates are not yet
available for E. tricarpa, and the relationships among
progeny within and among families may be influenced by
additional factors (Squillace 1974). The true level of
relatedness in field-grown progeny may therefore differ
substantially from the estimate we have used, but it is clear
that heritabilities of sideroxylonal concentration and insect
damage in our trial are moderate to high. Our estimates of
heritability, especially for growth traits, could be inflated
by inbreeding depression, since the open-pollinated design
does not control for differences in mating system parameters among families. The heritabilities of height and DBH
123
are surprisingly high, as values of about 0.2 for these traits
are more typical of Eucalyptus (Eldridge et al. 1993, p.
175). Estimates of heritability based on a single site may be
upwardly biased due to the inclusion of a genotype · environment interaction component in the estimate
of additive genetic variance (Tibbits and Hodge 1998;
Costa e Silva et al. 2006). There have been few studies on
chemical defenses carried out across sites (Bowers et al.
1992; Rosner and Hannrup 2004) and replicating this study
at multiple sites will be a high priority for future research
on FPCs.
The resistance of eucalypts to insect herbivores is often
highly heritable (Raymond 1995; Jones et al. 2002; Jordan et al. 2002; Rapley et al. 2004). Insect damage was
heritable in E. tricarpa and phenotypically correlated with
sideroxylonal concentration, although the genetic correlation was not significant. The lack of additive genetic
correlation between these traits may imply independence,
and that different genes are controlling the traits or may
have been due to insufficient statistical power. Genetic
variation in FPC production may also explain the difference in susceptibility to Christmas beetles observed between interspecific hybrids and pure E. grandis (Shepherd
2000).
Implications for coevolution and genetic improvement
The heritability of a trait is important for understanding the
evolutionary dynamics of the trait. While selection pressures are difficult to measure in long-lived trees, we now
have some evidence of the evolutionary potential of foliar
sideroxylonal concentrations in E. tricarpa. Our results
suggest that such concentrations may be capable of
responding to human-induced changes in herbivore pressures, such as increasing Christmas beetle or mammalian
herbivore abundance in agricultural landscapes. As yet, it is
unclear which groups of herbivores might act as the
strongest selective agent on sideroxylonal, because the
ability of plants to tolerate herbivory by different agents
may vary (Kotanen and Rosenthal 2000). The response to
breeding for increased concentrations of sideroxylonal will
depend on the heritability and phenotypic variation of this
trait, which our results suggest are high in E. tricarpa.
Increasing leaf sideroxylonal concentration through
breeding may increase the economic viability of plantation
forestry by reducing the impact of insect and mammalian
herbivores.
Population differentiation in heritable traits is of interest
because it may result from different selection regimes,
among other causes (Merila and Crnokrak 2001; McKay
and Latta 2002). High foliar sideroxylonal concentrations
and low variability would be expected in populations with
high herbivore pressure, but other environmental factors,
Oecologia
such as nutrient availability and rainfall, could also affect
the costs and benefits of defense. Moore et al. (2004b)
related differences in sideroxylonal concentrations among
natural populations of E. microcorys to temperature regimes, but could not separate the effects of genetic differentiation from those of environmental differences
between the sites, as experiments in controlled conditions
were lacking. E. tricarpa populations displayed genetic
differentiation in mean sideroxylonal concentrations, although there was no consistent pattern in differences
among geographical regions, suggesting that populationlevel processes are important in this species, rather than
broad-scale climatic variation. The lack of any strong
relationship between climatic variables at the source populations and sideroxylonal or damage in this species contrasts with those present in E. microcorys (Moore et al.
2004b). This may suggest that the effects observed in the
previous study may have been the result of plasticity rather
than genetic differentiation driven by environmental variation, although the results of the present study are based on
fewer populations and a smaller range of environmental
variation. However, our results are congruent with those of
Warren et al. (2005), who found only weak relationships
between physiological variables and seed-source rainfall in
E. tricarpa; the weak tendency of families from wetter
population locations.
The long-term stability of population differences in foliar sideroxylonal concentrations is unclear. Seedling
establishment and development to the sapling stage, which
is likely to occur following fire in most E. tricarpa
woodlands (Gill 1997), may be the stage in the life of a tree
during which herbivores can inflict the most damage.
Christmas beetle densities during recruitment may have a
strong influence on the sideroxylonal concentrations of the
next generation of E. tricarpa. The impact of Christmas
beetles on eucalypts varies considerably from year to year,
as populations fluctuate in response to rainfall and temperature patterns. Given the high heritability and phenotypic variance of sideroxylonal concentration, a population
may respond quickly to a transient change in herbivore
pressure and may not reflect the selection pressures facing
the trees at present or further in the past.
Additive genetic correlations may act as evolutionary
constraints, but are also important in animal and plant
breeding (Lynch and Walsh 1998; Williams et al. 2002). In
Eucalyptus, they could limit the potential benefits of
breeding for both growth and defense. The lack of correlation between sideroxylonal and growth suggests that
breeding for high sideroxylonal production need not
interfere with selection gains in growth traits under plantation conditions, although genetic analysis on additional
sites would be needed to draw this conclusion.
Physiological trade-offs may constrain the evolution of
defense traits in plants, although evidence for the costs of
plant defense has been contradictory (Koricheva 2002). No
attempts have yet been made to test this hypothesis for
foliar FPCs such as sideroxylonal. We found no relationship between foliar sideroxylonal concentrations and
growth, implying that the physiological cost of producing
sideroxylonal is not great or that the costs and benefits are
balanced under the conditions of the experiment. Experiments excluding herbivores are required to measure the
true physiological cost of sideroxylonal production, which
may be difficult for long-lived trees with many natural
enemies. Defenses have been shown to be costly in many
plants, but the expression of physiological costs can depend
heavily on the environmental conditions in which plants
are grown (Siemens et al. 2003). Care must be taken when
extrapolating from single common-garden trials such as
these to natural populations.
Conclusion
We have provided field confirmation that sideroxylonal is
related to resistance to an insect, as well as to mammalian
herbivores. Our results support the estimate of narrowsense heritability made for sideroxylonal using a markerbased method in a natural population of E. melliodora. We
found no evidence of a strong growth-defense trade-off in
E. tricarpa; however, this only highlights the need for
investigation of sideroxylonal production and growth in the
absence of herbivory, and of the ability of eucalypts to
escape or tolerate damage by different herbivores. We have
shown that differences in sideroxylonal concentration
among populations have a genetic basis, although the
importance of environmental factors should also be
investigated. Further understanding of the inheritance of
sideroxylonal could be gained from similar experiments in
a range of sites aimed at detecting interactions between
genotype and environment. These may be critical in
applying our results to the genetic improvement of the
insect resistance of eucalypts.
Acknowledgments The authors thank Emlyn Williams for statistical advice, and Nadine Scholtz and Junko Kondo for assistance in
field and laboratory work. We are grateful to Jim and Jane Rowe and
later Sharon and Lockie Altmeier for access to their property and to
ALRTIG for providing background information on the trial. We also
thank Ian Johnson and Hans Porada of Forests NSW for their support
and assistance. We thank Brian Baltunis and anonymous reviewers
for their comments on the manuscript. The work was funded by grants
from the Australian Research Council and the Rural Industries Research and Development Corporation (RIRDC) to W.J.F. and by a
scholarship from RIRDC and the Australian National University to
R.L.A. This study complies with the laws of the country in which it
was conducted, Australia.
123
Oecologia
References
American Society for Testing and Materials (1995) Standard practices
for infrared, multivariate, quantitative analysis. Designation:
E1655–94. West Conshohocken, PA
Andrew RL, Peakall R, Wallis IR, Wood JT, Knight EJ, Foley WJ
(2005) Can marker-based quantitative genetics in the wild be
successful? Heritability and genetic correlation of chemical
defences in Eucalyptus. Genetics 171:1989–1998
Andrew RL, Peakall R, Wallis IR, Foley W (2007) Spatial
distribution of defense chemicals and markers: implications for
the maintenance of variation. Ecology 87:716–728
Bowers MD, Collinge SK, Gamble SE, Schmitt J (1992) Effects of
genotype, habitat, and seasonal variation on iridoid glycoside
content of Plantago lanceolata (Plantaginaceae) and the implications for insect herbivores. Oecologia 91:201–207
Brooker MIH, Kleinig DA (1999) Field guide to eucalypts volume
1: South-Eastern Australia, 2nd edn. Bloomings Books,
Hawthorn
Burgess IP, Williams ER, Bell JC, Harwood CE, Owen JV (1996) The
effect of outcrossing rate on the growth of selected families of
Eucalyptus grandis. Silvae Genet 45:97–100
Butcher PA, Williams ER (2002) Variation in outcrossing rates and
growth in Eucalyptus camaldulensis from the Petford Region,
Queensland; evidence of outbreeding depression. Silvae Genet
51:6–12
Carne PB, Greaves RTG, McInnes RS (1974) Insect damage to
plantation-grown eucalypts in north coastal New South Wales,
with particular reference to Christmas beetles (Coleoptera:
Scarabaeidae). J Aust Entomol Soc 13:189–206
Costa e Silva J, Potts BM, Dutkowski GW (2006) Genotype by
environment interaction for growth of Eucalyptus globulus in
Australia. Tree Genet Genomes 2:61–75
Edwards PB, Wanjura WJ, Brown WV (1993) Selective herbivory by
Christmas beetles in response to intraspecific variation in
Eucalyptus terpenoids. Oecologia 95:551–557
Eldridge K, Davidson J, Harwood C, Van Wyk G (1993) Eucalypt
domestication and breeding. Clarendon Press, Oxford, UK
Floyd RB, Foley WJ (2001) Identifying pest-resistant eucalypts using
near-infrared spectroscopy. RIRDC Publication No 01/112.
Rural Industries Research and Development Corporation, Canberra, Australia
Fornoni J, Nunez-Farfan J, Valverde PL, Rausher MD (2004)
Evolution of mixed strategies of plant defense allocation against
natural enemies. Evolution 58:1685–1695
Gill AM (1997) Eucalypts and fires: interdependent or independent?
In: Williams J, Woinarski J (eds) Eucalypt ecology: individuals
to ecosystems. Cambridge University Press, Cambridge, pp 151–
167
Griffin AR, Cotterill PP (1988) Genetic variation in growth of
outcrossed, selfed and open-pollinated neighbourhood inbreeding progenies of Eucalyptus regnans and some implications for
breeding strategy. Silvae Genet 37:124–131
Harwood CE, Bulman P, Bush D, Mazanec R, Stackpole D (2001)
Australian low rainfall tree improvement group: compendium of
hardwood breeding strategies. Rural Industries Research and
Development Corporation, Canberra
Haukioja E (2003) Putting the insect into the birch-insect interaction.
Oecologia 136:161–168
Henery M (2006) Foliar secondary metabolites in Eucalyptus and
their role in resistance to defoliating insects. PhD Thesis, The
Australian National University, Canberra, Australia
Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A (2005) Very
high resolution interpolated climate surfaces for global land
areas. Int J Climatol 25:1965–1978
123
Hill KD, Johnson LAS (1991) Systematic studies in eucalypts 3. New
taxa and combinations in Eucalyptus (Myrtaceae). Telopea
4:223–267
Johns CV, Stone C, Hughes L (2004) Feeding preferences of the
Christmas beetle Anoplognathus chloropyrus (Coleoptera:
Scarabaeidae) and four paropsine species (Coleoptera: Chrysomelidae) on selected Eucalyptus grandis clonal foliage. Aust For
67:184–190
Jones TH, Potts BM, Vaillancourt RE, Davies NW (2002) Genetic
resistance of Eucalyptus globulus to autumn gum moth defoliation and the role of cuticular waxes. Can J For Res 32:1961–
1969
Jones RC, McKinnon GE, Potts BM, Vaillancourt RE (2005) Genetic
diversity and mating system of an endangered tree Eucalyptus
morrisbyi. Aust J Bot 53:11
Jordan GJ, Potts BM, Clarke AR (2002) Susceptibility of Eucalyptus
globulus ssp globulus to sawfly (Perga affinis ssp insularis)
attack and its potential impact on plantation productivity. For
Ecol Man 160:189–199
Koricheva J (2002) Meta-analysis of sources of variation in fitness
costs of plant antiherbivore defenses. Ecology 83:176–190
Kotanen PM, Rosenthal JP (2000) Tolerating herbivory: does the
plant care if the herbivore has a backbone? Evol Ecol 14:537–
549
Kursar TA, Coley PD (2003) Convergence in defense syndromes of
young leaves in tropical rainforests. Biochem Syst Ecol 31:929–
949
Lawler IR, Foley WJ, Pass GJ, Eschler BM (1998) Administration of
a 5HT3 receptor antagonist increases the intake of diets
containing Eucalyptus secondary metabolites by marsupials. J
Comp Physiol B 168:611–618
Lawler IR, Stapley J, Foley WJ, Eschler BM (1999) Ecological
example of conditioned flavor aversion in plant–herbivore
interactions: effect of terpenes of Eucalyptus leaves on feeding
by common ringtail and brushtail possums. J Chem Ecol 25:401–
415
Lawler IR, Foley WJ, Eschler BM (2000) Foliar concentration of a
single toxin creates habitat patchiness for a marsupial folivore.
Ecology 81:1327–1338
Leimu R, Koricheva J (2006) A meta-analysis of genetic correlations
between plant resistances to multiple enemies. Am Nat 168:E15–
E37
Lopez GA, Potts BM, Vaillancourt RE, Apiolaza LA (2003) Maternal
and carryover effects on early growth of Eucalyptus globulus.
Can J For Res 33:2108
Lynch M, Walsh B (1998) Genetics and analysis of quantitative traits.
Sinauer, Sunderland, Mass
McDonald MW, Rawlings M, Butcher PA, Bell JC (2003) Regional
divergence and inbreeding in Eucalyptus cladocalyx (Myrtaceae). Aust J Bot 51:393–403
McKay JK, Latta RG (2002) Adaptive population divergence:
markers, QTL and traits. Trends Ecol Evol 17:285–291
Merila J, Crnokrak P (2001) Comparison of genetic differentiation at
marker loci and quantitative traits. J Evol Biol 14:892–903
Moore BD, Foley WJ (2005) Tree use by koalas in a chemically
complex landscape. Nature 435:488–490
Moore BD, Wallis IR, Pala-Paul J, Brophy JJ, Willis RH, Foley WJ
(2004a) Antiherbivore chemistry of Eucalyptus—cues and
deterrents for marsupial folivores. J Chem Ecol 30:1743–1769
Moore BD, Wallis IR, Wood JT, Foley WJ (2004b) Foliar nutrition,
site quality, and temperature influence foliar chemistry of
tallowwood (Eucalyptus microcorys). Ecol Monogr 74:553–568
Moore BD, Foley WJ, Wallis IR, Cowling A, Handasyde KA (2005)
Eucalyptus foliar chemistry explains selective feeding by koalas.
Biol Lett 1:64–67
Oecologia
Mutikainen P, Walls M, Ovaska J, Keinänen M, Julkunen-Tiitto R,
Vapaavuori E (2002) Costs of herbivore resistance in clonal
saplings of Betula pendula. Oecologia 133:364–371
O’Reilly-Wapstra JM, McArthur C, Potts BM (2002) Genetic
variation in resistance of Eucalyptus globulus to marsupial
browsers. Oecologia 130:289–296
O’Reilly-Wapstra JM, McArthur C, Potts BM (2004) Linking plant
genotype, plant defensive chemistry and mammal browsing in a
Eucalyptus species. Funct Ecol 18:677–684
Payne RW (ed) (2005) The guide to genstat release 8 Part 2: statistics.
VSN International, Oxford, UK
Pryor LD (1953) Variable resistance to leaf-eating insect in some
eucalypts. Proc Linn Soc NSW 77:364–368
Puustinen S, Koskela T, Mutikainen P (2004) Direct and ecological
costs of resistance and tolerance in the stinging nettle. Oecologia
139:76
Rapley LP, Allen GR, Potts BM (2004) Genetic variation in
Eucalyptus globulus in relation to susceptibility from attack by
the southern eucalypt leaf beetle, Chrysophtharta agricola. Aust
J Bot 52:10
Rausher MD (1996) Genetic analysis of coevolution between plants
and their natural enemies. Trends Genet 12:212–217
Raymond CA (1995) Genetic variation in Eucalyptus regnans and
Eucalyptus nitens for levels of observed defoliation caused by
the eucalyptus leaf-beetle, Chrysophtharta bimaculata Olivier,
in Tasmania. For Ecol Man 72:21–29
Rice WR (1989) Analyzing tables of statistical tests. Evolution
43:223–225
Rosner S, Hannrup B (2004) Resin canal traits relevant for
constitutive resistance of Norway spruce against bark beetles:
environmental and genetic variability. For Ecol Man 200:77–87
Rousi M, Tahvanainen J, Henttonen H, Herms DA, Uotila I (1997)
Clonal variation in susceptibility of white birches (Betula spp.)
to mammalian and insect herbivores. For Sci 42:396–402
Scrivener NJ, Johnson CN, Wallis IR, Takasaki M, Foley WJ,
Krockenberger AK (2004) Which trees do wild common
brushtail possums (Trichosurus vulpecula) prefer? Problems
and solutions in scaling laboratory findings to diet selection in
the field. Evol Ecol Res 6:77–87
Shepherd M (2000) Variation and inheritance of resistance to
defoliation by Christmas beetles, Anoplognathus sp. (Leach) in
eucalypts. For Genet 7:57–64
Siemens DH, Lischke H, Maggiulli N, Schurch S, Roy BA (2003)
Cost of resistance and tolerance under competition: the defensestress benefit hypothesis. Evol Ecol 17:247–263
Squillace AE (1974) Average genetic correlations among offspring
from open-pollinated forest trees. Silvae Genet 23:149–156
Tibbits W, Hodge G (1998) Genetic parameters and breeding value
predictions for Eucalyptus nitens wood fiber production traits.
For Sci 44:587–598
Wallis IR, Watson ML, Foley WJ (2002) Secondary metabolites in
Eucalyptus melliodora: field distribution and laboratory feeding
choices by a generalist herbivore, the common brushtail possum.
Aust J Zool 50:507–519
Wallis IR, Herlt AJ, Eschler BM, Takasaki M, Foley WJ (2003)
Quantification of sideroxylonals in Eucalyptus foliage by highperformance liquid chromatography. Phytochem Anal 14:360–
365
Warren CR, Tausz M, Adams MA (2005) Does rainfall explain
variation in leaf morphology and physiology among populations
of red ironbark (Eucalyptus sideroxylon subsp tricarpa) grown in
a common garden? Tree Physiol 25:1369–1378
Williams ER, Matheson AC, Harwood CE (2002) Experimental
design and analysis for tree improvement, 2nd edn. CSIRO
Publishing, Collingwood
123