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A possible ontogenetic trade-off between defenseand tolerance in response to simulated herbivoryin seedlings and saplings of Araucaria angustifolia
Fernanda da Silva Alabarce •
Lucia Rebello Dillenburg
Received: 19 February 2014 / Accepted: 30 May 2014 / Published online: 22 June 2014
� Brazilian Society of Plant Physiology 2014
Abstract When facing herbivory, plants can defend
themselves and/or tolerate the inflicted damage. Unlike
defense, tolerance does not prevent herbivory, but
enables plants to compensate for damage. Resource
allocation theories assume that plants have a limited pool
of resources and that those allocated to one function or
structure cannot be used by another. Also, due to their
limited photosynthetic area and root biomass, seedlings
are expected to invest less in defenses than later plant
stages. Seedlings of the conifer species Araucaria
angustifolia (Brazilian pine) were shown to be quite
tolerant to shoot damage, but their ability to defend
themselves from herbivores is not yet known. In the
present study we tested whether the species is equally
tolerant to shoot damage at the seedling and early sapling
stages. We also looked for a possible ontogenetic trade-
off between tolerance and defense along early plant
ontogeny, and compared the allocation of starch reserves
between those two developmental stages in response to
the inflicted damage. To simulate herbivory, we severed
the shoots from seedlings and saplings (*80 % of shoot
mass removed). Shoot replacement by sprouting was
observed in both groups, although the number of sprouts
produced in saplings was greater than in seedlings.
Damage resulted in increased mobilization of seed starch
in seedlings. In saplings, underground reserves were
apparently deployed in response to simulated herbivory.
While seedlings had a greater ability to compensate for
tissue loss, saplings were more able to chemically defend
themselves, suggesting a possible ontogenetic trade-off
between tolerance and defense.
Keywords Brazilian pine � Chemical defense �Resource allocation � Tissue damage
1 Introduction
Herbivory is a biotic interaction that can potentially
generate negative impacts on plant fitness by causing
tissue loss, and these impacts represent an important
selective force that favors the evolution of resistance
mechanisms that enable plants to cope with their
consumers (Crawley 1997). Escape, defense and
tolerance are three resistance mechanisms that plants
express to reduce the negative impacts of their
interactions with herbivores (Boege and Marquis
2005). While escape mechanisms aim on hiding plants
from herbivores, defense and tolerance allow plants to
successfully deal with their presence. These two last
mechanisms will be the focus of the present study.
Most studies that investigated plant resistance
focused on defensive traits, which mostly prevent
events of herbivory (Berenbaum et al. 1986). Based on
the assumption that plant fitness is usually reduced by
F. da Silva Alabarce � L. R. Dillenburg (&)
Laboratorio de Ecofisiologia Vegetal, Departamento de
Botanica, Universidade Federal do Rio Grande do Sul,
91501-970 Porto Alegre, Brazil
e-mail: [email protected]
123
Theor. Exp. Plant Physiol. (2014) 26:147–156
DOI 10.1007/s40626-014-0014-2
natural enemies, such defense traits (e.g. repellent
secondary metabolites, spines and thorns) should
benefit plants (Marquis 1984). However, many studies
have questioned such assumption and suggested that
some plants may have the ability to resist herbivory by
developing an alternative strategy: tolerance to her-
bivory damage (McNaughton 1983; Paige and Whit-
man 1987; Mauricio et al. 1993). Tolerance of plants
to herbivory has been defined as the degree to which a
plant can regrow and reproduce after damage from
herbivores (Strauss and Agrawall 1999), or as the
ability of plants to maintain fitness through growth and
reproduction after experiencing herbivore damage
(Boege and Marquis 2005). Unlike defense, tolerance
does not prevent herbivory, but enables plants to
reduce the negative effects on plant fitness through
compensatory growth, meristem activation, increased
photosynthesis and resource reallocation (McNaugh-
ton 1983; Boege and Marquis 2005).
Resource allocation theories assume that plants
have a limited pool of resources, and that those
allocated to a function or structure cannot be used by
another, promoting trade-offs that determine resource
allocation constraints. During plant ontogeny, alloca-
tion of the available resources will be influenced by the
inherent increase in plant size, and by changes in
functional priorities (Weiner 2004). These priorities
may include resisting the attack by herbivores, and
both defending itself from this attack and tolerating the
inflicted damage involve costs that are imposed on
plant biochemical and physiological processes
(Strauss et al. 2002; Zhang and Jiang 2002).
The impacts of herbivory are expected to vary as
plants develop from very young seedlings to mature
plants. Therefore, the amount and type of resistance
mechanisms are also expected to change with plant
ontogeny (Farnsworth 2004), and such changes may
not follow a linear fashion (Boege and Marquis, 2005).
Indeed, this pattern of variation along plant ontogeny
seem to depend on many factors, including plant life
form, type of herbivore (mammal, insect, mollusk),
and type of resistance trait (Barton and Korsheva
2010). By using meta-analysis, these authors were able
to uncover complex and nonlinear patterns in the
ontogeny of plant resistance to herbivory. According
to their analysis, the ability of plants to tolerate
damage does not exhibit a significant variation
through plant ontogeny. In contrast, a clear pattern
of increase in constitutive levels of all classes of
chemical defenses through the seedling stage was
found, implying that plants would reach the early
sapling stage with a greater amount of chemical
defenses than young seedlings.
The longevity of the sapling stage of woody plants
has been suggested to play an important role in
discriminating ontogenetic patterns of resource alloca-
tion to resistance. This happens because woody plants
often survive for decades as saplings before light gaps
allow them to grow, and also because of their easier
accessibility compared to mature individuals (Feeny
1976). Chemical defense at the sapling stage is then
considered to be very important for survival and is likely
to lead to higher levels of defense at this stage compared
to adulthood (Barton and Korsheva 2010). Because
large-seeded woody species will tend to generate large
seedlings (Leishman et al. 2000; Westoby et al. 2002),
which may remain at the seedling stage for a longer
period than those coming from small seeds, we also see
the potential for the longevity of the seedling stage to
significantly affect the way species will defend them-
selves against and/or tolerate herbivore damage.
Araucaria angustifolia (Bertol.) Kuntze (Araucar-
iaceae), also known as Brazilian pine, is one of the few
native conifer species growing in South America. It is
a dominant and emergent tree species of the montane
forests of southern Brazil (Hueck 1972), and, in the
southernmost state of the country, these forests are
currently expanding over the adjacent grasslands
(Duarte et al. 2006). Its seeds are one of the largest
among conifers (Eckenwalder 2009), and its saplings
form long-lasting and slow-growing ‘plant banks’ in
the forest understory (Carvalho 1994; Duarte et al.
2002), making the species an interesting system for
studying seedling and sapling herbivore resistance.
The species also colonizes open grasslands, where it
acts as an important forest nucleation species (Duarte
et al. 2006, 2010; Dos Santos et al. 2011). In open
environments, adult individuals sometimes carry more
than one main stem, and such behavior has been
associated to the action of herbivores at early stages of
plant development (Mattos 1994). Zandavalli (2006),
for example, has observed Brazilian pine seedlings
carrying numerous sprouts, after being severely dam-
aged by ants (Acromyrmex crassispinus and Atta sp.).
Species of Araucariaceae have unique undifferen-
tiated axillary meristems, which are able to develop
into buds when released from apical dominance,
giving these conifers the ability to resprout after
148 Theor. Exp. Plant Physiol. (2014) 26:147–156
123
damage (Burrows 1987, 1989). Alabarce and Dillen-
burg (2012) reported that the ability of seedlings to
resprout in response to severe shoot damage persisted
even when seedlings were disconnected from their
large supporting seeds, but the availability of seed
reserves allowed for a more intense sprouting
response. Mass accumulation in the underground
hypocotyl was a very distinct initial response to
damage, but, on the long run, damaged seedlings were
able to re-establish a biomass allocation pattern which
was very similar to the undamaged plants. If, on one
hand, we already know that this species is capable of
tolerating damage by a compensatory growth response
at the seedling stage, on the other, no information is
yet available regarding such response at the sapling
stage. Also, there is no information on the possible
chemical defenses against herbivory exhibited by this
conifer species at any developmental stage. Flavo-
noids, for example, were shown to play an important
protective role against DNA UV-induced damage in
our target species (Yamagushy et al. 2009), but other
ecological roles of these compounds, such as chemical
defense against herbivory, are yet to be investigated.
The aims of this study were to compare tolerance to
simulated shoot damage in A. angustifolia at the
seedling and sapling stages, to compare their alloca-
tion of mass and starch reserves in response to such
damage, to investigate and compare the expression of
chemical defenses (flavonoid accumulation) at these
two ontogenetic stages, and to examine a possible
trade-off between tolerance and defense. A two-stage
experiment was then designed in order to answer the
following questions: (1) Is the species equally tolerant
to shoot damage at the seedling and early sapling
stages? (2) How does damage to seedlings and
saplings affect growth allocation and the mobilization
and allocation of starch reserves? (3) At which of these
two ontogenetic stages is the species more able to
chemically defend itself against herbivory? (4) Is there
any indirect evidence for a possible trade-off between
tolerance and defense along early plant ontogeny?
2 Materials and methods
2.1 Plant material and growth conditions
The study was conducted in an experimental area located
in the Federal University of Rio Grande do Sul, Brazil
(30�010S and 51�130W), between October 2010 and
December 2011. Brazilian pine seeds were collected in
June 2010 in the National Forest of Sao Francisco de
Paula, Rio Grande do Sul, Brazil (29�230S and 50�230W),
immersed in water to discard the floating ones, and
disinfested with a 2 % sodium hypochlorite solution.
They were then stored in a refrigerator at 4 �C. In August
2010, they were sowed in trays containing wet vermic-
ulite, and pre-germinated seeds were planted in 2.0-L
PET bottles, containing medium-sized sand. A modified
10 % Hoagland solution (Lowe and Dillenburg 2011)
was applied every two weeks. The volume provided
corresponded to the full water capacity of the bottles
(320 mL). Plants were grown under a light shade cloth,
with minimum and maximum values of recorded irradi-
ances (taken with a quantum sensor LI-190S-1, Li-Cor
Inc., Lincoln, NE, USA in two different cloudless days at
noon time) ranging from 400 to 810 lmol m-2 s-1.
2.2 Experimental design
Herbivory damage was simulated by severing, close to
the base, the main shoot of seedlings and saplings.
Such kind of damage was observed to be imposed by
ants in nature, particularly at the seedling stage. A total
of forty-eight plants were used in the experiment, and
they were initially separated in two groups: those to be
damaged two and 11 months after sowing. According
to Lowe and Dillenburg (2011), seed reserves are
mostly exhausted between 80 and 120 days after seed
sowing. Those two groups then represent plants at the
seedling (SD) and sapling (SP) stages, respectively,
each represented by twenty-four plants. Half of the
plants in each group were damaged (D), and the other
half remained undamaged (U). Shoot damage of
seedlings and saplings was imposed with a single cut
using a pair of scissors, with *80 % of the shoot mass
being removed in both cases. The whole experiment
was then composed by a factorial arrangement of two
ontogenetic stages and two levels of damage, totaling
four treatments, each with 12 experimental units: (1)
undamaged seedlings (USD); (2) damaged seedlings
(DSD); (3) undamaged saplings (USP); and (4)
damaged saplings (DSP).
2.3 Growth responses to shoot damage
Growth evaluations were made at two different times
during the course of the experiment: at day 0, when
Theor. Exp. Plant Physiol. (2014) 26:147–156 149
123
damage was imposed, and 60 days later (day 60). In
both occasions, six experimental units were harvested
for each of the four treatments. At each harvest, plants
were separated into shoot, shoot sprouts (at day 60, in
the case of damaged plants), root, underground
hypocotyl and seed. These plant parts were oven dried
at 60 �C until constant weight, their mass recorded,
and the percentage of mass contributions of shoot, root
and hypocotyl to the overall plant mass was calcu-
lated. At day 0, the removed tissue from damaged
plants was also dried and weighed to measure the
percentage of lost tissue. At day 60, the number and
length of shoot sprouts emitted from damaged plants
were also recorded.
The degree of plant tolerance to the imposed
damage was estimated by growth compensation for
the loss of photosynthetic shoot tissue. Such compen-
sation was evaluated at day 60, through the ratio
between shoot mass of damaged plants and shoot mass
of undamaged plants, with a full compensation
equivalent to 1. This approach is based on the estimate
of compensation of Strauss and Agrawall (1999). In
order to aid the interpretation of this compensatory
response, we also calculated the relative shoot growth
by dividing the difference between the final (day 60)
and initial (day 0) values of shoot mass and length by
the initial ones. Because shoot evaluations were not
made in the same plants in both days, these growth
rates were based on a single mean value for each
treatment on day 0 and individual plant values on day
60.
2.4 Starch concentration and mobilization
Starch concentration of underground storage organs
(hypocotyl and main root), as well as of seeds in the
case of seedlings, was quantified at day 60. Starch was
extracted from the dry material (in the case of seeds, it
only included the starchy megagametophyte) with
52 % perchloric acid, and the total starch concentra-
tion was estimated by the anthrone-sulphuric acid
method and absorbance readings in a spectrophotom-
eter at 630 nm (McCready et al. 1950).
2.5 Chemical defense
Plant chemical defense after damage (day 60) was
evaluated by measuring the concentration of flavo-
noids in leaves of six plants of each treatment. All
mature leaves and sprouts from seedlings, and *10
mature leaves and sprouts from saplings were used for
this analysis. Extraction of the dry material was made
in 95 % ethanol and the quantification followed the
aluminum chloride complex method using spectro-
photometric readings at 510 nm (Zhishen et al. 1999).
Flavonoid concentration was expressed in milligrams
per gram of dry leaf tissue.
2.6 Data analysis
The effects of ontogeny and damage on starch,
flavonoid and relative growth data were analyzed
using two-way ANOVA, followed by Tukey’s proce-
dure for mean comparisons. Ontogenetic effects on
growth compensation and number of sprouts, as well
as damage effects on seed starch of seedlings, were
analyzed using a two-sample t test. In order to tell
whether plant biomass differed between the three
major parts (root, hypocotyl and shoot), a one-way
ANOVA was run for each of the four treatments, using
plant part as the main variance factor, followed by
Tukey’s mean separation procedure. All analyses were
run with the statistical package Statistix 8.0 (Analyt-
ical Software), using P B 0.05.
3 Results
3.1 Growth responses to shoot damage
All plants (damaged or not) survived throughout the
experiment, and all damaged seedlings and saplings
exhibited stem sprouting on day 60. The number of
sprouts produced per plant was significantly higher in
saplings (4.25 ± 0.25) than in seedlings (2.5 ± 0.28)
(t = 21.0; P = 0.004). While in saplings there was no
dominance of one sprout in relation to the others, at
least 60 days after damage took place, a dominant
sprout (one sprout significantly taller than the others)
could be easily distinguished in 85 % of the damaged
seedlings (Fig. 1).
The shoot mass and shoot length relative growths
were affected by damage, stage of development and by
the interaction between these two factors (Table 1).
While the shoots of seedlings grew much more than
those of saplings, shoots of damaged plants grew more
than those of undamaged controls during the sixty-day
period. As attested by the significant interactions, such
150 Theor. Exp. Plant Physiol. (2014) 26:147–156
123
damage effects were much more pronounced for
seedlings than for saplings (for this later stage, only
shoot length was affected by damage) (Table 2).
Finally, growth compensation in response to damage
was much greater in seedlings than in saplings
(t = 81.5; P = 0.0003) (Fig. 2a).
On day 60, seedlings had a greater proportion of
their dry mass in shoot than in root structures as
compared to saplings, regardless of whether they had
suffered damage or not. Shoots and roots of undam-
aged saplings had similar contributions to the overall
plant mass, but shoot contribution turned smaller than
root contribution in damaged plants. Dry mass allo-
cation to the underground hypocotyl was lower than
the other two plant parts in all cases (Fig. 3; Table 3).
3.2 Starch concentration
The starch concentration remaining in seeds on day 60
was significantly reduced by the shoot damage
imposed to seedlings (t = 215; P = 0.0000). These
also responded to damage by holding a greater
Fig. 1 General aspect of representative damaged and control
plants of Araucaria angustifolia 60 days after treatments were
imposed: in a, sprouts in a damaged seedling (left), and a control
seedling (right); in b, sprouts in a damaged sapling (left), and a
control sapling (right). The black vertical bars indicate the
length and position of the remaining original shoot (stump) after
damage took place
Table 1 Results from two-way ANOVA for different plant
parameters of Araucaria angustifolia
Variable/sources df F P
Shoot mass relative growth
Stage 1 4,209.92 0.0000
Damage 1 355.56 0.0000
Stage 9 Damage 1 295.03 0.0000
Shoot length relative growth
Stage 1 82.44 0.0000
Damage 1 56.80 0.0001
Stage 9 Damage 1 32.86 0.0004
Hypocotyl starch
Stage 1 0.21 0.6584
Damage 1 15.07 0.0047
Stage 9 Damage 1 163.78 0.0000
Main root starch
Stage 1 28.24 0.0007
Damage 1 5.33 0.0499
Stage 9 Damage 1 83.71 0.0000
Flavonoid
Stage 1 920.63 0.0000
Damage 1 0.00 0.9921
Stage 9 Damage 1 228.89 0.0000
Significant P values (P B 0.05) are shown in boldface
Table 2 Means (standard error) of relative shoot growth
(biomass and length) of damaged and undamaged seedlings
and saplings of Araucaria angustifolia
Treatment Shoot biomass
relative growth
(g g-1)
Shoot length relative
growth (cm cm-1)
N
USD 3.97 (0.4) b 2.69 (0.36) b 6
DSD 6.83 (0.22) a 11.83 (1.3) a 6
USP 0.2 (0.03) c 0.38 (0.04) c 6
DSP 0.32 (0.05) c 1.62 (0.28) b 6
Different letters indicate significant differences between
treatments
USD undamaged seedlings, DSD damaged seedlings, USP
undamaged saplings, DSP damaged saplings (n = 6;
P B 0.05)
Theor. Exp. Plant Physiol. (2014) 26:147–156 151
123
concentration of starch in the hypocotyl and main root.
Damage to saplings, on the other hand, led to lower
concentrations of starch in both hypocotyl and main
root (Fig. 4; Table 1).
3.2.1 Chemical defense
Damage had no significant effect on leaf flavonoid
concentration. However, this concentration was much
greater in damaged saplings than in damaged
seedlings (Fig. 2b; Table 1) with a similar difference
also being present in control plants (data not shown).
4 Discussion
Seedlings replaced about 50 % of the lost tissue in two
months, while saplings replaced only about 10 %
during the same period. Besides, the relative shoot
growth rate, intrinsically higher in seedlings,
expressed an even greater difference between the
two life stages when plants were damaged. We cannot
predict whether such initial differences in compensa-
tory regrowth will also result in greater fitness (and
thus greater tolerance) of damaged seedlings com-
pared to damaged saplings. However, these results
strongly suggest that seedlings are more tolerant to
severe shoot damage than young saplings, considering
Fig. 2 Ontogenetic effects
on a tolerance to herbivory
(compensation) and
b defense against herbivory
(flavonoid concentration) in
damaged plants of
Araucaria angustifolia.
Asterisks indicate significant
differences between
ontogenetic stages (n = 6;
P B 0.05). Compensation is
the ratio between shoot mass
of damaged plants and shoot
mass of undamaged plants,
with a full compensation
equivalent to 1
Fig. 3 Ontogenetic and damage effects on biomass partitioning
of plants of Araucaria angustifolia at the end of the experiment.
USD = undamaged seedlings; DSD = damaged seedlings;
USP = undamaged saplings; and DSP = damaged saplings.
Different letters indicate significant differences between plant
parts at each treatment (n = 6; P B 0.05)
Table 3 Results from one-way ANOVA for testing the effects
of plant part on the percentage of mass contribution to total
plant mass
Source/variable df F P
% mass contribution USD 2 13,393 0.0000
% mass contribution DSD 2 15,016 0.0000
% mass contribution USP 2 15,675 0.0000
% mass contribution DSP 2 38,413 0.0000
USD undamaged seedlings, DSD damaged seedlings, USP
undamaged saplings, DSP damaged saplings
152 Theor. Exp. Plant Physiol. (2014) 26:147–156
123
that growth is an important estimate of fitness (Strauss
and Agrawall 1999). As such, it is a particularly useful
estimate in perennial plants (Steven et al. 2008).
Even though tolerance to herbivory is expected to
increase through ontogeny because of limitations on
resource acquisition, storage and bud bank in young
plants, this simple prediction finds much more com-
plex patterns in nature (Boege et al. 2011). Araucaria
angustifolia seems to be one of the species to go
against this general rule by producing seedlings that
can more fully compensate for shoot damage than
young saplings. Seedling versus sapling comparisons
for herbivory tolerance in woody plants are very
scarce. Weltzin et al. (1998) found that older seedlings
of Prosopis glandulosa were less tolerant to shoot
herbivory than younger seedlings, but tolerance was
judged by plant survival When shoot growth rates in
response to damage were compared, seedlings and
saplings were very much alike. In fact, the meta-
analysis presented by Barton and Korsheva (2010)
found that tolerance showed no ontogenetic pattern in
herbaceous and woody plants. The pattern we pre-
sented here is restricted to a very narrow region of the
whole ontogenetic sequence of the species, but it does
agree with what is commonly expected when seed-
lings at the cotyledonary stage are compared to older
seedlings or young saplings (Boege and Marquis
2005): when still relying on seed reserves, seedlings
would be more capable of resisting an herbivore attack
than after becoming more autonomous. In the case of
A. angustifolia, the role played by the cotyledons of
many angiosperm trees is replaced by the very well
developed and starchy megagametophyte, which, as
briefly discussed below, allows for a not so common
mass allocation pattern at this very young stage.
Woody species have an overall pattern of a
continuous decrease in shoot:root ratio during devel-
opment (Wilson 1988). Indeed, undamaged seedlings
of A. angustifolia allocated more mass to their shoots
than saplings. However, the mass allocation pattern
within the seedling stage presented by Dillenburg et al.
(2010) shows that, at least up to *160 days after seed
germination, shoot investment increases continuously
and is mostly supported by the abundant seed reserves
and those stored in the underground hypocotyl. This
major investment on shoot growth during the seedling
stage, at the expenses of seed reserves, may have
rendered the plants more tolerant to severe shoot
damage than saplings. Devoid of these initial reserves,
young saplings showed lower ability to replace tissues
when severely damaged, despite being able to produce
more sprouts. Shoot sprouts in Araucariaceae arises
from poorly differentiated, quiescent leaf axil meris-
tems, which develop into buds when released from
Fig. 4 Ontogenetic and damage effects on starch concentration
in seed, hypocotyl and main root of plants of Araucaria
angustifolia at the end of the experiment. USD = undamaged
seedlings; DSD = damaged seedlings; USP = undamaged
saplings; and DSP = damaged saplings. Different letters
indicate significant differences between treatments (n = 6;
P B 0.05)
Theor. Exp. Plant Physiol. (2014) 26:147–156 153
123
apical dominance (Burrows 1987, 1989). These mer-
istems increase in size as plants grow larger (Burrows
1990), which might explain the greater number of
sprouts in saplings than in seedlings of A. angustifolia.
In seedlings, shoot damage resulted in lower starch
concentration in the seeds and to a greater starch
accumulation in the underground hypocotyl and main
root. In saplings, on the other hand, starch concentra-
tion in these underground organs was reduced in
response to damage. These results allow us to suggest
some patterns of storage use in response to damage.
The greatest reduction in seed starch concentration in
damaged seedlings is a clear indication that shoot
removal led to a greater mobilization of this important
carbon reserve. It is reasonable to believe that much of
this carbon sustained shoot regrowth. Not relying on
maternal reserves to regrow anymore, it is not
surprising that starch concentration in the hypocotyl
and main root of saplings reduced in response to
damage in saplings. Very similar results were reported
for Gustavia superba (Barberis and Dalling 2008), in
which resprouting was supported by root reserves after
the establishment period.
The importance of non-structural carbohydrates
stored in roots after a defoliation event has been
commonly reported for young trees (Rodgers et al.
1995; Boege 2005; Kabeya and Sakai 2005). In A.
angustifolia, the hypocotyl joins the main root in
playing this role, and such underground storage seems
to play a critical role in plant tolerance to tissue
damage and removal. The greater starch concentration
in the main root and hypocotyl in damaged seedlings
may increase the chances of a new regrowth in case a
new damage event takes place, but the possibility of
seed resource sequestration should also be considered
(Orians et al. 2011). Because most of the shoot was
removed in both seedlings and saplings, only the
former had a significant source of carbon (supporting
seed) to be reallocated for underground storage.
Besides saving some of the seed resources for a new
damage event, increased hypocotyl storage may also
help hide these valuable seed reserves from herbi-
vores. Alabarce and Dillenburg (2012) had previously
reported the importance of the amount of available
seed reserves on the sprouting intensity of A. angust-
ifolia seedlings: when prematurely deprived from the
seeds, shoot growth compensation after damage was
much smaller. The present study demonstrates that
these abundant maternal reserves render the seedlings
more tolerant to shoot damage than young saplings, by
providing a ‘stabilizing effect’’ of the negative impacts
of herbivory. Such tolerance assumes paramount
importance, since seeds and seedlings are expected
to be the most vulnerable stages to herbivore attack.
Damage did not lead to an increased flavonoid-
based chemical defense in either seedlings or saplings.
However, flavonoid evaluations were made sixty days
after simulated injury took place. We should then
strongly consider the possibility that this was a long-
enough time for chemical defenses to go back to their
constitutive levels. Future studies should evaluate a
time course response of such chemical defenses. The
developmental stage, on the other hand, did have an
effect on leaf flavonoid concentration, with contrast-
ing results when compared to plant tolerance. While
saplings showed less tolerance to shoot damage than
seedlings, the former were potentially more able to
chemically defend their shoot tissues from herbivores
than the latter, as attested by the much greater
concentration of flavonoids in their shoot tissues.
The meta-analysis presented by Barton and Korsheva
(2010) revealed a clear pattern of increase in the levels
of constitutive chemical defenses through the seedling
stage. We only evaluated seedlings at one point along
their development, but the fact that young saplings had
higher levels of flavonoids than seedlings is a strong
indication that this concentration might increase as
seedlings grow older. It would be interesting to follow
flavonoid concentration in the shoots of A. angustifolia
from the time they arise until seedlings are no longer
seedlings.
A trade-off between the ability of different plant
genotypes to tolerate and to defend themselves against
herbivores has long been suggested, based on the fact
that both strategies serve the same function (Mauricio
et al. 1997). When tested, however, it has generated
contrasting results (e.g., Fineblum and Rausher 1995;
Mauricio et al. 1997; Leimu and Koricheva 2006).
From an ontogenetic perspective, this trade-off has
been observed in different woody species, such as
Pinus ponderosa (Wagner 1988), Nectandra ambigens
(Sanchez-Hidalgo et al. 1999), and Casearia nitida
(Boege et al. 2011). Our results corroborate these
previous studies by providing indications of a possible
ontogenetic trade-off between these two strategies
(tolerance and defense) in A. angustifolia as predicted
by Boege and Marquis (2005): as plants developed
from the seedling to the sapling stage, their ability to
154 Theor. Exp. Plant Physiol. (2014) 26:147–156
123
tolerate damage decreased, while their capacity to
chemically defend their shoot tissues increased.
According to Bryant et al. (1992) and Bryant and
Julkunen-Tiito (1995), there is a trade-off in allocation
of carbon to production of immobile chemical
defenses versus storage reserves, such that, at the
seedling stage, the allocation of photosynthates to
substances such as chemical defenses (that do not
support growth) would be selectively disadvanta-
geous. With these scenarios in mind, we suggest that
seedlings mobilized their reserves to the reconstruc-
tion of lost photosynthetic tissue at the expenses of low
flavonoid levels, while saplings had higher levels of
chemical defense at the expenses of a lower rate of
tissue replacement.
Acknowledgments We thank the Agronomy School of
Federal University of Rio Grande do Sul for the greenhouse
space and the Plant Physiology Laboratory for supporting the
chemical analysis. We also thank the Brazilian Council for
Scientific and Technological Development (CNPq/Brazil) for
fellowships awarded to the authors. This study is part of the
master thesis of the first author, which was developed in the
Postgraduate Program in Botany of the Federal University of
Rio Grande do Sul, Brazil, under the supervision of the
corresponding author.
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