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\begin{document}

\title{Intensity of herbivory correlates with stronger constitutive and weaker
induced defenses for non-native plant species -- another mechanism for
EICA?}



\author[1]{Xiao Sun}%
\author[2]{Yumei Sun}%
\author[2]{Xueyao Cao}%
\author[2]{Xincong Zhai}%
\author[3]{Ray Callaway}%
\author[4]{Jinlong Wan}%
\author[5]{S. Luke Flory}%
\author[6]{Wei Huang}%
\author[1]{Jianqing Ding}%
\affil[1]{Henan University}%
\affil[2]{Affiliation not available}%
\affil[3]{University of Montana}%
\affil[4]{Wuhan Botanical Garden Chinese Academy of Sciences}%
\affil[5]{University of Florida}%
\affil[6]{Key Laboratory of Aquatic Botany and Watershed Ecology}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Non-native plants are typically released from specialist enemies in new
ranges, but continue to be attacked by generalists, but whether they
shift relative allocation to constitutive or induced defenses is
unknown. We compared herbivory on co-occurring native and non-native
species and also constitutive and induced defenses. Non-natives suffered
less damage than natives and constitutive defenses of non-natives was
lower than that of native congeners, whereas induced defense was the
opposite. The strength of constitutive defenses for a species was
correlated with the intensity of herbivory experienced, for non-natives,
whereas induced defenses showed the reverse. The defenses of natives
were not related to herbivory pressure. Finally, the strength of induced
defenses correlated positively with growth, suggesting a novel mechanism
for the evolution of increased competitive ability. These results expand
our understanding of fundamental tradeoffs in constitutive and induced
defenses and provide novel insight into how herbivory pressure affects
defense allocation.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Non-native plant invasions have provided remarkable insights into rapid
adaptation in defense allocation (Callaway \& Maron, 2006; Waller et
al., 2020). In part, this is because non-native plant species are
typically released from specialist enemies in introduced ranges but
continue to be attacked by generalists - the Enemy Release Hypothesis
(Keane \& Crawley, 2002). Thus, the most predictable change in herbivore
communities in non-native ranges, at least until biological control
agents are introduced, is the loss of specialists and disproportionate
exposure to naive generalists. Generalist-dominated communities in
non-native ranges commonly correspond with exotic species developing
greater qualitative chemical defenses against generalists, relative to
conspecifics in the native range - the Shifting Defense Hypothesis
(Joshi \& Vrieling, 2005; Zhang et al., 2018). These qualitative
defenses may be constitutive or induced, but little is known about
selection on constitutive versus induced defenses in non-native ranges.
Theory and hypotheses that specifically focus on how constitutive and
induced defenses might respond to generalists in native and non-native
ranges have not led to clear predictions (Orians \& Ward, 2010), which
is barrier to understanding successful invasion.

Plants are thought to maximize their fitness by balancing resource
allocation to defense with other functions (Rotter \& Holeski, 2018;
Mertens et al., 2021), and this allocation can be affected by herbivore
pressure (Stamp, 2003). Constitutive defenses protect plants from attack
without a time lag, but constitutive defenses are thought to be costly
in the absence of herbivores (Wittstock \& Gershenzon, 2002; Ali \&
Agrawal, 2012; Aljbory \& Chen, 2018). Thus, when herbivore pressure is
low, plant fitness might be maximized by investing less in constitutive
defenses. In contrast, induced defenses appear to be cost-saving
strategies where defenses are expressed only in response to herbivore
pressure (Gatehouse, 2002; Agrawal \& Hastings, 2019).

From the ``quagmire'' (Stamp, 2003) of hypotheses for defense allocation
in plants, a few possibilities emerge for how induced and constitutive
defenses might evolve in plant species in their non-native ranges. In a
review of invasive plants, Orians \& Ward (2010) suggested that induced
defense should evolve to be more prevalent in non-native ranges where
the cost of defense is high and plants are attacked infrequently
(Bixenmann et al., 2016; Agrawal \& Karban, 1999). Different non-native
species often experience very different degrees of generalist herbivore
pressure (Zou, 2008; Sims-Chilton et al., 2009), providing opportunities
to test Orian \& Ward's idea. For example, a common garden survey found
that different non-native species suffered from 0\% to more than 40\%
leaf herbivory from generalists (Agrawal \& Kotanen, 2003). Variation in
herbivore pressure should lead to variation in defense traits (Coverdale
\& Agrawal, 2022; Wan et al., 2022) and less intense and less frequent
herbivory is theorized to select for allocation to induced defenses
(Bixenmann et al., 2016). However, how this variation in generalist
herbivore pressure affects any potential tradeoffs in
constitutive\emph{vs.} induced defensive strategies among non-native
plant species is unknown.

Growth-defense tradeoffs appear to derive from plasticity or adaptive
variation in allocation that maintains fitness in a variable environment
(Monson et al., 2022). Increased investment in defense may result in
reduced growth (Hahn et al., 2021), leading to differential investment
in growth or defense by species based on their evolutionary responses to
environmental conditions - the Resource Availability Hypothesis (Coley
et al., 1985). Most studies have focused on how variation in herbivore
pressure across geographical clines shapes evolution of plant defense
(Woods et al., 2012; Moreira et al., 2018). However, these studies have
not generally considered how biogeographical shifts in defense
strategies might constrain growth.

Non-native species provide a good system for studying induced and
constitutive defenses. If non-native species are attacked less by
generalists than native species, we can test the prediction that (i)
non-natives increase expression of cheaper induced defenses and decrease
expression of expensive constitutive defenses (Figure S1A and S1B). If
non-native species are attacked disproportionately by generalists, then
this disentangling of generalist and specialist effects might permit a
cleaner opportunity to test another prediction (ii), that the herbivore
pressure a species experiences will increase expression of constitutive
defenses and decrease induced defenses (Figure S1A and S1B). We can then
test the prediction (iii) that allocation to less expensive induced
defense allow allocation to greater growth (Figure S1C).

To explore these predictions, we first carried out large spatial scale
measurements of herbivore intensity on 27 non-native and 59 co-occurring
native species in a field survey in northern China (Table S1, S2 and
S3). We then quantified herbivore intensity on 12 pairs of non-native
species and native congeners in a common garden. Finally, we conducted
measurements of constitutive and induced defenses as well as plant
growth on these pairs of common garden species (Table S4).

\textbf{Materials and Methods}

Herbivore pressure in the field

To compare herbivore pressures on different non-native and native plant
species under natural conditions, we conducted a field survey in
abandoned agricultural fields at 28 sites, ranging from 34\selectlanguage{ngerman}°00' to 38°54'
N and 112°55' to 119°44' E in northern China in 2018 (Table S1).

At each site, we selected the three tallest plants of each non-native
species and co-occurring native species in a 5 x 10 m plot. To determine
herbivore pressure, we calculated the percentage of damaged leaves by
counting 10-50 leaves starting from the top of each plant and recording
the proportion of damaged leaves (Table S2 and S3).

To determine whether herbivore biomass correlated with leaf damage, we
recorded the number of each herbivore species on each non-native plant.
Native plants were not measured as these measurements were simply to
correlate damage with insect biomass. To obtain the mean biomass of each
herbivore category, we carried out a field survey at eight sites in
2019. We collected all herbivores on non-native species and determined
the fresh weight of individual insects to the nearest 0.01g as described
in Supplementary Methods 1.

Herbivore pressure in the common garden

We conducted a common garden experiment at Henan University, Kaifeng,
China in 2020 (34°30' N, 114°10' E). We selected the 12 most common
non-native herbaceous plant species in the field survey and compared
herbivory on these species and 12 co-planted native congeners (Table
S4). Specialist herbivores were absent in our system. Thus, these
species provided an explicit test of how non-native plant species cope
with generalist herbivores (see Müller-Schärer et al., 2004; Joshi \&
Vrieling, 2005).

In the field survey, we found no significant effect of site on leaf
damage for most non-native species or native congeners (Table S2 and
S3). Therefore, for controlled experiments we collected seeds from 2 to
3 individuals of each non-native species and their native congeners at
each site and mixed them. We sowed seeds in trays (20×30 cm) and placed
them in the greenhouse. Similar sized seedlings of all species (about 5
cm in height) were used for the following experiments.

We removed standing vegetation by hand from an agricultural field at
Henan University and established 168 1×1 m plots grouped into 6 blocks.
Plots within each block were separated by 1.5 m and blocks were
separated by 3 m. Then, the 28 plots within each block were randomly
assigned to one of 14 pairs of non-native species and their native
congeners. We transplanted four seedlings into each plot spaced 25 cm
apart. All plants were exposed to natural herbivores.

Three months after planting, we recorded herbivore abundance and
measured herbivore biomass as described above and also evaluated the
percentage of damaged leaf area instead of the percentage of damaged
leaves for all plants. We clipped 20 leaves from each plant and
calculated damaged leaf area and total leaf area using Image Proexpress
V.6.0 (Media Cybernetics, Inc., Bethesda, MD, USA). We used the average
of herbivore biomass and percentage of damaged leaf area of the four
plants in each plot for analysis.

Constitutive and induced bioassay defense

We used the same 12 pairs of non-native species and their native
congeners for assessment of constitutive and induced defenses. We sowed
the seeds of each species separately into 96-cell trays and then
transplanted similar sized plants of each species individually into pots
(15 cm diameter, 18 cm height) filled with 50\% potting media
(Pindstrup, Denmark) and 50\% topsoil collected from the common garden.
Greenhouse conditions were as described above.

When plants had 20 leaves, we started the herbivory treatment and
bioassay. As the non-native plant species were mainly attacked by
Lepidoptera in the field survey (Figure S2A) and common garden
experiment (Figure S2B), thus we applied \emph{Spodoptera
littoralis}(Lepidoptera: Noctuidae), a species that feeds on more than
40 plant families (Kempel et al., 2011), as the generalist herbivore. We
obtained eggs of \emph{S. littoralis} from Keyun Biological Control Co.,
Ltd., China and fed them on bean-based artificial diet. The newly
emerged\emph{S. littoralis} larvae from the next generation were used to
quantify the constitutive defense of pairs of non-native species and
their native congeners. We measured larval weight gain on leaves
harvested from healthy undamaged plants. We clipped one fully expanded
leaf from the top of plants and placed it on a Petri dish (9 cm
diameter) with moist filter paper. Then, we added a newly emerged
pre--weighed larva (weight\textsubscript{1}) into the Petri dish. We
replaced each leaf with a fresh one from another healthy plant every
day. After four days we re-weighed larva and calculated weight gain for
10 replicates.

To quantify induced defenses of pairs of non-native species and native
congeners, we measured larval weight gain on leaves that were harvested
from plants previously damaged by herbivores. The initial damage level
was at roughly 10\%, which was consistent with the percentage of leaf
damage in common garden experiment (11.5±1.2 \%). We then added 2-6
second instar larvae to the two leaves in the middle of each plant,
depending on the size of the different plant species, and covered them
with a mesh bag (0.8 mm openings). After two days we removed larvae.
Plants were grown 4-8 days to produce new leaves and then we followed
the same protocol as above with 10 replicates.

Constitutive and induced chemical defense

To test the contribution of secondary metabolites to constitutive and
induced defenses, we measured chemicals in leaves that were harvested
from previously healthy and herbivore-damaged plants. Terpenoids and
phenols are important secondary metabolites for defense against a wide
range of herbivores (Mithöfer \& Boland, 2012). Thus, we used the same
non-native species and their native congeners used in the bioassay to
measure total phenolics and total triterpenoids in leaves harvested from
healthy and herbivore-damaged plants.

Leaves were flash frozen in liquid nitrogen and stored at -80°C for
analysis. Total phenolic concentration was calculated as described in
Supplementary Methods 2.

Growth rates

To evaluate the relationship between the strength of induced defenses
and plant growth, we measured the relative growth rate of non-native and
native plants. Two weeks after transplanting, we harvested ten
similar-sized plants for each species and measured total dry biomass
(weight\textsubscript{1}). After six more weeks, we harvested
conspecifics and determined total dry biomass (weight\textsubscript{2}).
We calculated relative growth rate (RGR) of each species as: RGR =
(weight2 - weight1) / 42 days. There were 12 replicates for each
combination of weight\textsubscript{1} plants and
weight\textsubscript{2} plants for each non-native species and native
congeners.

Statistical analysis

Herbivore pressure - To test for difference in herbivore pressure
(binary data of undamaged \emph{vs.} damaged leaves in a cbind matrix)
among the non-native plant species in the field survey, we used a Wald
Chi-square test applied on a Generalized Linear Mixed Model (GLMM) with
a binomial distribution. Site was a random effect. We used a bootstrap
method to test whether results differed when the number of native plant
species was equal to the number of non-native species. We tested for
difference in herbivore pressure (logit-transformed percentage of
damaged leaf area) among the non-native plant species in the common
garden experiment using a Wald Chi-square test applied on a Linear Mixed
Model (LMM) with blocks as random effects. We conducted same analyses
for native species. To test for differences in herbivore pressure
(binary data of undamaged\emph{vs.} damaged leaves in a cbind matrix)
between the non-natives and natives in the field survey, we used a Wald
Chi-square test applied on a GLMM with a binomial distribution. Site and
species nested in origin as random effects. We also tested for
differences in herbivore pressure (logit-transformed percentage of
damaged leaf area) in the common garden experiment using LMM with blocks
and species nested in origin as random effects. Furthermore, we used
GLMM with binomial distribution that included random terms for sites and
species to test whether percentage of damaged leaves (binary data of
undamaged \emph{vs.} damaged leaves in a cbind matrix) depended on
herbivore biomass in the field survey and used LMM that included random
terms for block and species to test whether percentage of damaged leaf
area (logit-transformed) depended on herbivore biomass in the common
garden experiment. Finally, for non-native species and their native
congeners, in both the field survey and common garden experiment, we
used LM to examine the relationship between leaf damage and herbivore
biomass for the two experiments using mean values for species.

Constitutive and induced defenses - We assessed constitutive and induced
defenses using larval weight gain and chemical contents for each
species. For constitutive defense, we used larval weight gain on the
leaves of plants that had not been previously attacked. For induced
defense, we calculated the larval weight gain on the leaves of
previously attacked plants minus the mean of larval weight gain on the
leaves of un-attacked plants. Constitutive and induced defenses
expressed by chemicals were evaluated using the same methods. We used
the percentage of herbivore-damaged leaf area for each species in the
common garden experiment as herbivore pressure. To evaluate
relationships among constitutive defense, induced defense and herbivore
pressure, we carried out Pearson correlations in which we multiplied
larval weight with -1 since higher larval weight gain indicates lower
defense. Mean values per species were used for above analyses and
non-native and native species were analyzed separately. To test
constitutive and induced defense between non-native and native species
we used LMM with species as a random effect. Finally, to test whether
changes in chemicals might underly changes in herbivore growth, we
conducted Pearson correlations across both herbivory treatments and all
species to examine the dependence of larval weight gain on phenolics or
triterpenoids using mean values per species.

Strength of growth and induced defense - To test for differences in
relative growth rate between non-native plant species and native
congeners, we used a LMM with species nested within origin as random
effects. Furthermore, we calculated difference in induced defense in
term of larval weight gain and difference in plant growth rate between
non-native species and corresponding native congeners. We then used
Pearson correlation to evaluate relationship between difference in
induced defense and difference in plant growth rate.

Homogeneity of variances and normality of distributions of data were
checked before data analysis and P-values were corrected by False
Discovery Rate (FDR) (Benjamini \& Hochberg, 1995). All statistics were
carried out using R (version 4.0.5) with the `car', `lme4', and
`RVAideMemoire' packages (Bates, 2014).

\textbf{Results}

Herbivore pressure on native and non-native plant species

In the field survey of 28 sites, 396 individuals of 27 non-native plant
species and 678 individuals of 59 native plant species were evaluated.
There were large differences among non-native plant species (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 3744,
P \textless{} 0.001, Figure 1A) and among native species (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 3025, P
\textless{} 0.001, Figure 1B) in herbivore damage, with non-native plant
species damaged 46.2\% less than natives (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 22.63, P\textless{}0.001,
Figure 1C).

In the common garden experiment, herbivore damage varied by over an
order of magnitude among non-native species (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 198.43, P \textless{}
0.001, Figure 1D) and among native species (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 71.22, P \textless{}
0.001, Figure 1E). Herbivore damage on non-native species was 41.5\%
less than on natives (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 6.53, P = 0.011, Figure 1F).

Non-native plant species with higher herbivore biomass on them received
more leaf damage in the field survey (r = 0.97, P \textless{} 0.001,
Figure S3A) and in the common garden (r = 0.38, P \textless{} 0.001,
Figure S3B). Furthermore, there was a strong positive linear
relationship between herbivore damage on particular species in the field
survey and those species in the common garden experiment (r = 0.88, P
\textless{} 0.001, Figure S4).

Herbivore pressure and constitutive and induced defense

Assessed by \emph{S. littoralis} larval weight gain, constitutive
defense (-1 × larval weight gain on healthy plants) was positively
correlated with herbivore pressure (assessed as described above) across
all 12 non-native plant species (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 0.00, P = 0.020), whereas there
was no correlation between constitutive defense and herbivore pressure
across the 12 native congeners (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 0.32, P = 0.306, Figure 2A).
Induced defense {[}-1 × (larval weight gain on previously damaged plants
- larval weight gain on healthy plants){]} was negatively correlated
with herbivore pressure for non-native plant species (\selectlanguage{greek}χ\selectlanguage{ngerman}² = -0.69, P =
0.014), whereas there was no correlation between induced defense and
herbivore pressure for native congeners (\selectlanguage{greek}χ\selectlanguage{ngerman}² = -0.49, P = 0.106) (Figure
2B). Overall, constitutive defense of non-native plant species was
38.7\% lower than that of native congeners (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 4.28, P = 0.039)
(Figure 2A), while induced defense of non-native plant species was
55.3\% higher than that of native congeners (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 5.56, P = 0.018)
(Figure 2B).

For chemical defenses assessed by phenolics, results were similar to
that assessed by larval weight gain. Constitutive defense (phenolics in
healthy plants) (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 0.76, P = 0.004) and induced defense (phenolics in
previously attacked plants - phenolics in non-attacked plants) (\selectlanguage{greek}χ\selectlanguage{ngerman}² =
-0.84, P \textless{} 0.001) were positively and negatively correlated
with herbivore pressure, respectively, across all 12 non-native plant
species (Figure 2C and 2D). Whereas, for native congeners, defense
assessed by phenolic content showed no correlation between constitutive
defense and herbivore pressure (\selectlanguage{greek}χ\selectlanguage{ngerman}² = -0.20, P = 0.525) or between
induced defense and herbivore pressure (\selectlanguage{greek}χ\selectlanguage{ngerman}² = -0.18, P = 0.571, Figure 2C
and 2D). Overall, constitutive defense of non-native plant species,
measured as phenolic content, was equal to that of native congeners (\selectlanguage{greek}χ\selectlanguage{ngerman}²
= 0.02, P = 0.885, Figure 2C), whereas induced defense was higher than
that of native congeners (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 163.20, P \textless{} 0.001, Figure 2D).
However, for defense assessed by triterpenoids, there was no
relationship between constitutive defense and herbivore pressure
(non-natives, \selectlanguage{greek}χ\selectlanguage{ngerman}² = -0.08, P = 0.807; natives, \selectlanguage{greek}χ\selectlanguage{ngerman}² = -0.20, P = 0.527), or
between induced defense and herbivore pressure (non-natives, \selectlanguage{greek}χ\selectlanguage{ngerman}² = -0.07,
P = 0.820; natives, \selectlanguage{greek}χ\selectlanguage{ngerman}² = 0.01, P = 0.970) for non-native plant species
and native plant species (Figure 2E and 2F). Constitutive defense (\selectlanguage{greek}χ\selectlanguage{ngerman}² =
0.03, P = 0.852) and induced defense (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 2.15, P = 0.143) of
non-native plant species, measured as triterpenoids, was equal to that
of native congeners (Figure 2E and 2F).

Across all herbivory treatments (leaves from un-attacked and attacked
plants) and plant species (non-natives and natives), larval weight gain
was negatively correlated with phenolic content (r = -0.29, P = 0.047,
Figure 3A) and with triterpenoid content (r = -0.29, P = 0.044, Figure
3B).

Tradeoff between constitutive and induced defense

For defense assessed by larval weight gain, plant species that had
higher constitutive defense had lower induced defense, resulting in a
negative correlation across the 12 non-native plant species (r = -0.58,
P = 0.048) and the native congeners combined (r = -0.85, P \textless{}
0.001) (Figure 4A). This was similar for non-native plant species when
defense was assessed by phenolic content (r = -0.76, P = 0.004), but not
for native congeners (r = 0.05, P = 0.881) (Figure 4B).

Strength of induced defense and growth

For the 12 pairs of non-native plant species and native congeners, RGR
of non-native plant species was 1.6 times higher than that of native
congeners (\selectlanguage{greek}χ\selectlanguage{ngerman}² = 19.08, P \textless{} 0.001) (Figure 5A, Figure S5). This
variation in RGR was strongly positively correlated with variation in
the strength of induced defense as measured by larval weight gain (r =
0.72, P = 0.019) (Figure 5B).

\textbf{Discussion}

Our results make two key conceptual contributions to understanding
allocation of plant defenses. First, we showed that non-native species,
which presumably have escaped from specialist natural enemies, had lower
constitutive defenses and higher induced defenses, which correlated with
higher growth rates. Second, non-native plant species, ostensibly
exposed primarily to generalists, showed a strong positive relationship
between herbivore pressure and constitutive defense, and a strong
negative relationship between herbivore pressure experienced by a
species and the intensity of induced defenses exhibited by that species.
These findings expand our understanding of constitutive vs. induced
defense allocation associated with herbivore pressure on non-native
plants.

Integrating these results suggests that reduced herbivore attack (Figure
1C and 1F; i.e., escape from enemies) favored allocation to induced
defenses (Figure 2B) instead of constitutive defenses (Figure 2A). Our
results also suggest that induced defenses are a ``cheaper'' overall
strategy which favors higher growth rates (Figure 5). It appears that
the generalist-dominated herbivore community that attacked non-native
plant species drove a strong positive relationship between the intensity
of herbivore pressure and allocation to constitutive defense among our
target species (Figure 2A and 2C). This supports long-held theory that
has had minimal empirical support - intense or consistent herbivory
should select for more expensive constitutive defenses over cheaper
induced defenses (Ito \& Sakai, 2009).

Generalist herbivore pressure

Native generalist herbivores often avoid non-native plant species or do
not suppress them (Schaffner et al., 2011; Sedio et al., 2020). For
example, Cappuccino \& Carpenter found that non-native invasive plants
experienced 96\% less leaf damage than non-native species that were not
classified as invasive (Cappuccino \& Carpenter, 2005). One hypothesis
for this pattern is the novelty of biochemicals, in their new ranges,
produced by non-native plants (Schaffner et al., 2011; Callaway \&
Aschehoug, 2000). Cappuccino \& Arnason (2006) found that a large suite
of invasive species had more unique secondary chemicals than exotic
plants, relative to native species. These, and other studies (see review
by Inderjit et al. 2021), indicate that unusual or novel biochemicals of
some non-native species may be why native herbivores avoid them
(Inderjit et al., 2021).

Defense strategies

Intense and predictable herbivory is thought to select for constitutive
plant defenses (Kalske \& Kessler, 2020). Induced defenses appear to be
adaptive to less intense and infrequent herbivory (Agrawal \& Karban,
1999; Ito \& Sakai, 2009) as a cost-saving alternative to constitutive
defense (Detto \& Xu, 2020). Relevant literature on invasives is
minimal, but the invasive genotype\emph{Lespedeza cuneata} exhibited
greater induced defense but lower constitutive defense than ancestral
native genotypes (Beaton et al., 2011) as might be predicted by reduced
or more sporadic herbivory in the non-native range. In contrast,
non-native genotypes of\emph{Alternanthera philoxeroides} have higher
constitutive defenses and lower induced defenses (Liu et al., 2020).
Clearly, the literature on evolutionary changes in constitutive or
induced defenses by exotic plants is limited.

Other recent results indicate that secondary metabolism plays key roles
in the interactions between non-native plants and herbivores (Tian et
al., 2021; Yu et al., 2022). Phenolics inhibit the digestion of protein
(Rehman et al., 2012) and have been central in many studies of
plant-defense mechanisms (e.g., Kumar et al., 2020). We showed that
weight gain of a generalist herbivore was negatively correlated with
phenolic concentration across all species and herbivory treatments,
suggesting that defense might derive from variation in constitutive and
induced phenolic concentrations. Triterpenes are terpenoids, also
abundant in plants (Kumar et al., 2020), and larval herbivore weight
gain was negatively correlated with triterpenoid content across our
native species. However, triterpenoid content did not correspond with
herbivore pressure. Triterpenes have many other functions
(González-Coloma et al., 2011), and the functional diversification of
triterpenes may have masked simple responses to herbivores.

Cost-benefit allocation

Optimal defense theory assumes that organisms are under strong natural
selection to allocate resources to optimize their cost-benefit ratio in
terms of fitness (Stamp, 2003; Alba et al., 2012). Much of this research
has focused on a proposed evolutionary tradeoff where invasive plants
experience relaxed selection on herbivore defense and evolve greater
allocation to growth and competitive ability - the Evolution of
Increased Competitive Ability hypothesis (EICA) (Zhang et al., 2020;
Callaway et al., 2022). Generalist herbivory can mediate defensive
strategies and resource allocation (Müller-Schärer et al., 2004). Thus,
we suggest that the costs and benefits of induced defenses should differ
in low-versus high-damage risk environments. Such damage
pressure-dependent defensive strategies could drive strong selection on
developmental defensive strategies (Maron et al., 2019) in ways that
optimize plant defense and maximize plant fitness components for each
non-native species. In this context, constitutive defense was lower
whereas induced defense was higher for non-native species than native
congeners, respectively. The striking positive relationship between
herbivory experienced by different non-native species in the field and
constitutive defense, and negative relationship between herbivory
experienced by non-native species and induced defense imply that
introduced plants may adopt a cost saving strategy between constitutive
defense and induced defense in response to generalist pressure.

Plant species commonly show a strong trade-off between defense and
growth (Lazzarin et al., 2021), but herbivore-driven changes in
defensive strategy might alter the cost-benefit ratios of defense and
growth allocation. Maintaining constitutive defense at high levels
appears to require plants to invest substantial resources, potentially
increasing the total cost of chemical defenses. If induced defenses are
less costly than constitutive defenses, perhaps relaxed selection on
constitutive defense could allow greater growth, and such a tradeoff
might contribute to the dominance of some non-native species. To our
knowledge, no study of EICA has experimentally integrated potential
increased growth tradeoffs of non-native plants derived from a shift
from constitutive to induced defenses. Our results support the defense
strategies that have proposed such tradeoffs between constitutive
defense and induced defense where cheaper induced defenses allow greater
growth and reproduction (Mumm \& Hilker, 2006; Mauch-Mani et al., 2017).
Altogether, the results suggest that the defense strategy of stronger
protection against generalist herbivores is a factor contributing to
invasion success.

An important caveat is that we only used one generalist
herbivore,\emph{S. littoralis} , to explore the defensive strategy of
non-native plants. Other studies indicate that generalist preferences
and impacts can vary a great deal among herbivore species (Inderjit et
al., 2021). Our non-native plants were occupied by many species of
generalists in the field, thus, to better understand patterns of defense
strategies when non-native plants face generalist herbivore pressure,
several generalist herbivore species should be included in future
testing of plant defenses. We emphasize that our binary approach to
herbivore diet (generalist vs. specialist) was heuristic, and thus too
simplistic (Hardy et al., 2020), but appeared to have allowed us to
detect some fundamental ecological phenomena. Finally, the best tests of
hypotheses such as ours compare the same species in native and
non-native ranges (Sheng et al., 2022), and it should be noted that
studying defense-related tradeoffs among native and non-native species
in the same non-native range provides strong, but not the best, evidence
for defense adaptations (Van Kleunen et al., 2010). However, including
many non-native and native congeners increases the strength of our
results.

Integrating herbivore-related hypotheses for non-native species invasion

Interactions between non-native plants and herbivores have improved our
understanding of plant-herbivore interactions, underlying mechanisms,
and rapid evolution of these dynamics (Müller-Schärer et al., 2004; Lin
et al., 2021). Two key hypotheses derived from non-native invasions are
the Enemy Release Hypothesis - that non-native species escape much of
the herbivory experienced in their native ranges, primarily by
specialists, and the related Shifting Defense Hypothesis - that
non-native invaders increase defenses, chiefly qualitative, against
generalists. However, we do not know how constitutive and induced
defenses might respond to generalist-dominated herbivore communities or
the intensity of attack by these communities. Our results show a shift
to induced defenses by non-native plants, and that as herbivore pressure
intensifies on non-native species, so does the strength of their
constitutive defenses. Finally, our results suggest new ways to consider
the Evolution of Increased Competitive Ability Hypothesis - perhaps the
``cheaper'' induced strategy adopted by non-natives allows more
allocation to growth and competitive ability. Thus, our results suggest
that all three hypotheses may be surprisingly integrated (Figure 6).
These results expand our understanding of fundamental tradeoffs in
constitutive and induced defenses and provide novel insight into how
variation in herbivore communities might affect defense allocation in
plants.

\textbf{Acknowledgments} : We thank Hongwei Yu and Sichong Chen for data
analysis. We thank the assistance for secondary metabolism from Shanghai
BIOTREE Biological Technology Co., Ltd., Shanghai, China. This work was
supported by National Natural Science Foundation of China (U21A20190,
32071658, 32071660, 31822007, 32001239), Henan Province University
Innovation Talents Support Program (22HASTIT039), Young Talent Support
Project of Henan Province (2021HYTP034). RMC was supported by National
Science Foundation EPSCoR Cooperative Agreement OIA-1757351.

\textbf{Competing interest statement} : The authors have declared that
there is no conflict of interests.

\textbf{References}

Agrawal, A.A. \& Hastings, A.P. (2019) Trade--offs constrain the
evolution of an inducible defense within but not between plant
species.\emph{Ecology} , 100, e02857.

Agrawal, A.A. \& Karban, R. (1999) Why induced defenses may be favored
over constitutive strategies in plants. In: \emph{The Ecology and
Evolution of Inducible Defenses} (eds Tollrian, R. \& Harvell, C.D.).
Princeton University Press, Princeton, NJ, pp. 45--61.

Agrawal, A.A. \& Kotanen, P.M. (2003) Herbivores and the success of
exotic plants: a phylogenetically controlled experiment. \emph{Ecol}
.\emph{Lett} ., 6, 712--715.

Alba, C., Bowers, M.D. \& Hufbauer, R. (2012) Combining optimal defense
theory and the evolutionary dilemma model to refine predictions
regarding plant invasion. \emph{Ecology} , 93, 1912--1921.

Ali, J.G. \& Agrawal, A.A. (2012) Specialist versus generalist insect
herbivores and plant defense. \emph{Trends Plant Sci} ., 17, 293--302.

Aljbory, Z. \& Chen, M.S. (2018). Indirect plant defense against insect
herbivores: a review. \emph{Insect Sci} ., 25, 2--23.

Bates, D., Mäechler, M., Bolker, B.M. \& Walker, S.C. (2014) Fitting
linear mixed-effects models using Lme4. \emph{J} . \emph{Stat} .
\emph{Softw} ., 67, 1--48.

Beaton, L.L., Van Zandt, P.A., Esselman, E.J. \& Knight, T.M. (2011)
Comparison of the herbivore defense and competitive ability of ancestral
and modern genotypes of an invasive plant, \emph{Lespedeza cuneata} .
\emph{Oikos} , 120, 1413--1419.

Benjamini, Y. \& Hochberg, Y. (1995) Controlling the false discovery
rate: a practical and powerful approach to multiple testing. \emph{J}
.\emph{R} . \emph{Stat} . \emph{Soc} . \emph{B} ., 57, 289--300.

Bixenmann, R.J., Coley, P.D., Weinhold, A. \& Kursar, T.A. (2016) High
herbivore pressure favors constitutive over induced defense. \emph{Ecol}
. \emph{Evol} ., 6, 6037--6049.

Callaway, R.M. \& Aschehoug, E.T. (2000) Invasive plants versus their
new and old neighbors: a mechanism for exotic invasion. \emph{Science} ,
290, 521--523.

Callaway, R.M. \& Maron J.L. (2006) What have exotic plant invasions
taught us over the past 20 years? \emph{Trends Ecol. Evol.} , 21,
369--374.

Callaway, R.M., Lucero, J.E., Hierro, J.L. \& Lortie, C.J. (2022) The
EICA is dead? Long live the EICA! \emph{Ecol} . \emph{Lett} ., 25,
2289--2302.

Cappuccino, N. \& Carpenter, D. (2005) Invasive exotic plants suffer
less herbivory than non--invasive exotic plants. \emph{Biol} .
\emph{Lett} ., 1, 435--438.

Cappuccino, N. \& Arnason, J.T. (2006) Novel chemistry of invasive
exotic plants. \emph{Biol} . \emph{Lett} ., 2, 189--193.

Coley, P.D., Bryant, J.P. \& Chapin III, F.S. (1985) Resource
availability and plant antiherbivore defense. \emph{Science} , 230,
895--899.

Coverdale, T.C. \& Agrawal, A.A. (2022) Experimental insect suppression
causes loss of induced, but not constitutive, resistance in\emph{Solanum
carolinense} . \emph{Ecology} , 103, e3786.

Detto, M. \& Xu, X. (2020) Optimal leaf life strategies determine
V\textsubscript{c,max} dynamic during ontogeny. \emph{New Phytol} .,
228, 361--375.

Gatehouse, J.A. (2002) Plant resistance towards insect herbivores: a
dynamic interaction. \emph{New Phytol} ., 156, 145--169.

González-Coloma, A., López-Balboa, C., Santana, O. \& Fraga, R. (2011)
Triterpene-based plant defenses.\emph{Phytochem} . \emph{Rev} ., 10,
245--260.

Hahn, P.G., Keefover\selectlanguage{english}-Ring, K., Nguyen, L. \& Maron, J.L. (2021)
Intraspecific correlations between growth and defense vary with resource
availability and differ within and among populations. \emph{Funct} .
\emph{Ecol} ., 35, 387--396.

Hardy, N.B., Kaczvinsky, C., Bird, G. \& Normark, B.B. (2020) What we
don't know about diet-breadth evolution in herbivorous insects.
\emph{Annu} . \emph{Rev} .\emph{Ecol} . \emph{Evol} . \emph{S} ., 51,
103--122.

Ito, K. \& Sakai, S. (2009) Optimal defense strategy against herbivory
in plants: Conditions selecting for induced defense, constitutive
defense, and no--defense. \emph{J} .\emph{Theor} . \emph{Biol} ., 260,
453--459.

Joshi, J. \& Vrieling, K. (2005) The enemy release and EICA hypothesis
revisited: incorporating the fundamental difference between specialist
and generalist herbivores.\emph{Ecol} . \emph{Lett} ., 8, 704--714.

Kalske, A. \& Kessler, A. (2020) Population--wide shifts in herbivore
resistance strategies over succession. \emph{Ecology} , 101, e03157.

Keane, R.M. \& Crawley, M.J. (2002) Exotic plant invasions and the enemy
release hypothesis. \emph{Trends Ecol} . \emph{Evol} ., 17, 164--170.

Kempel, A., Schaedler, M., Chrobock, T., Fischer, M. \& van Kleunen, M.
(2011) Tradeoffs associated with constitutive and induced plant
resistance against herbivory.\emph{P} . \emph{Natl} . \emph{Acad}
.\emph{Sci} . \emph{USA} , 108, 5685--5689.

Kumar, S., Abedin, M., Singh, A. K. \& Das, S. (2020) Role of phenolic
compounds in plant-defensive mechanisms. In: \emph{Plant Phenolics in
Sustainable Agriculture Singapore} (eds Lone, R., Shuab, R., Kamili,
A.N.). Springer, Singapore, pp. 517--532.

Lazzarin, M., Meisenburg, M Meijer, D., Van Ieperen, W., Marcelis,
L.F.M., Kappers, I.F. \emph{et al} . (2021) LEDs make it resilient:
effects on plant growth and defense. \emph{Trends Plant Sci} ., 26,
496--508.

Lin, T., Vrieling, K., Laplanche, D., Klinkhamer, P.G., Lou, Y. Bekooy,
L. \emph{et al} . (2021). Evolutionary changes in an invasive plant
support the defensive role of plant volatiles. \emph{Curr} .\emph{Biol}
., 31, 3450--3456.

Liu, M., Pan, X., Zhang, Z., Kleunen, M.V. \& Li, B. (2020) Testing the
shifting defense hypothesis for constitutive and induced resistance and
tolerance.\emph{J} . \emph{Pest Sci} ., 93, 264--355.

Maron, J.L., Agrawal, A.A. \& Schemske, D.W. (2019) Plant--herbivore
coevolution and plant speciation. \emph{Ecology} , 100, e02704.

Mauch-Mani, B., Baccelli, I., Luna, E. \& Flors, V. (2017) Defense
priming: an adaptive part of induced resistance. \emph{Annu} .
\emph{Rev} . \emph{Plant Biol} ., 68, 485--512.

Mertens, D., Fernandez de Bobadilla, M., Rusman, Q., Bloem, J., Douma,
J.C. \& Poelman, E.H. (2021) Plant defence to sequential attack is
adapted to prevalent herbivores.\emph{Nat} . \emph{Plants} , 7,
1347--1353.

Mithofer, A. \& Boland, W. (2012) Plant defense against herbivores:
chemical aspects. \emph{Annu} . \emph{Rev} . \emph{Ecol} . \emph{Evol}
.\emph{S} ., 63, 431--450.

Monson, R.K., Trowbridge, A.M. Lindroth, R.L. \& Lerdau, M.T. (2022)
Coordinated resource allocation to plant growth--defense tradeoffs.
\emph{New Phytol} ., 233, 1051--1066.

Moreira, X., Petry, W.K., Mooney, K.A., Rasmann, S. \& Abdala-Roberts,
L. (2018) Elevational gradients in plant defences and insect herbivory:
recent advances in the field and prospects for future research.
\emph{Ecography} , 41, 1485--1496.

Muller-Scharer, H., Schaffner, U. \& Steinger, T. (2004) Evolution in
invasive plants: implications for biological control. \emph{Trends Ecol}
. \emph{Evol} . 19, 417--422.

Mumm, R. \& Hilker, M. (2006) Direct and indirect chemical defence of
pine against folivorous insects.\emph{Trends Plant Sci} ., 11, 351--358.

Orians, C.M. \& Ward, D. (2010) Evolution of plant defenses in
nonindigenous environments. \emph{Annu} . \emph{Rev} . \emph{Entomol} .,
55, 439--459.

Rehman, F., Khan, F.A. \& Badruddin, S. M.A. (2012) Role of phenolics in
plant defense against insect herbivory. In: \emph{Chemistry of
phytopotentials: health, energy and environmental perspectives} (eds
Khemani, L.D., Srivastava, M.M., Srivastava, S.). Springer, Berlin,
Heidelberg, pp. 309--313.

Rotter, M.C. \& Holeski, L.M. (2018) A meta--analysis of the evolution
of increased competitive ability hypothesis: genetic-based trait
variation and herbivory resistance trade-offs. \emph{Biol}
.\emph{Invasions} , 20, 2647--2660.

Schaffner, U., Ridenour, W.M., Wolf, V.C., Bassett, T., Muller, C.,
Muller-Scharer, H. \emph{et al} . (2011) Plant invasions, generalist
herbivores, and novel defense weapons. \emph{Ecology} , 92, 829--835.

Sedio, B.E., Devaney, J.L., Pullen, J., Parker, G.G., Wright, S.J. \&
Parker, J.D. (2020) Chemical novelty facilitates herbivore resistance
and biological invasions in some introduced plant species. \emph{Ecol} .
\emph{Evol} ., 10, 8770--8792.

Sheng, M., Rosche, C., Al-Gharaibeh, M., Bullington, L.S., Callaway,
R.M., Clark, T. \emph{et al} . (2022). Acquisition and evolution of
enhanced mutualism--an underappreciated mechanism for invasive
success?\emph{ISME J} ., 16, 2467--2478.

Simberloff, D., Kaur, H., Kalisz, S. \& Bezemer, T.M. (2021) Novel
chemicals engender myriad invasion mechanisms. \emph{New Phytol} ., 232,
1184--1200.

Sims-Chilton, N.M., Zalucki, M.P. \& Buckley, Y.M. (2009) Patchy
herbivore and pathogen damage throughout the introduced Australian range
of groundsel bush,\emph{Baccharis halimifolia} , is influenced by
rainfall, elevation, temperature, plant density and size. \emph{Biol} .
\emph{Control} , 50, 13--20.

Stamp, N. (2003) Out of the quagmire of plant defense hypotheses.
\emph{Q} . \emph{Rev} . \emph{Biol} ., 78, 23--55.

Tian, B, Pei, Y., Huang, W., Ding, J. \& Siemann, E. (2021) Increasing
flavonoid concentrations in root exudates enhance associations between
arbuscular mycorrhizal fungi and an invasive plant. \emph{ISME J} ., 15,
1919-1930.

Van Kleunen, M., Dawson, W., Schlaepfer, D., Jeschke, J.M. \& Fischer,
M. (2010). Are invaders different? A conceptual framework of comparative
approaches for assessing determinants of invasiveness. \emph{Ecol} .
\emph{Lett} ., 13, 947--958.

Waller, L.P., Allen, W.J., Barratt, B.I.P., Condron, L.M., Franca, F.M.,
Hunt, J. E. \emph{et al} . (2020) Biotic interactions drive ecosystem
responses to exotic plant invaders. \emph{Science} , 368, 967--972.

Wan, J., Yi, J., Tao, Z., Ren, Z., Otieno, E. O., Tian, B. \emph{et al}
. (2022). Species\selectlanguage{english}-specific plant\selectlanguage{english}-mediated effects between herbivores
converge at high damage intensity. \emph{Ecology} , 103, e3647.

Wittstock, U. \& Gershenzon, J. (2002) Constitutive plant toxins and
their role in defense against herbivores and pathogens. \emph{Curr}
.\emph{Opin} . \emph{Plant} . \emph{Biol} ., 5, 300--307.

Woods, E.C., Hastings, A.P., Turley, N.E., Heard, S.B. \& Agrawal, A.A.
(2012) Adaptive geographical clines in the growth and defense of a
native plant. \emph{Ecol} .\emph{Monogr} ., 82, 149--168.

Yu, H., He, Y., Zhang, W., Chen, L., Zhang, J., Zhang, X. \emph{et al} .
(2022) Greater chemical signaling in root exudates enhances soil
mutualistic associations in invasive plants compared to natives.
\emph{New Phytol} ., 236, 1140-1153.

Zhang, Z., Liu, Y., Yuan, L., Weber, E. \& Kleunen, M.V. (2020) Effect
of allelopathy on plant performance: a meta--analysis. \emph{Ecol} .
\emph{Lett} ., 24, 348--362.

Zhang, Z., Pan, X., Blumenthal, D., Van Kleunen, M., Liu, M. \& Li, B.
(2018) Contrasting effects of specialist and generalist herbivores on
resistance evolution in invasive plants.\emph{Ecology} , 99, 866--875.

Zou, J., Siemann, E., Rogers, W. E. \& DeWalt, S.J. (2008) Decreased
resistance and increased tolerance to native herbivores of the invasive
plant \emph{Sapium sebiferum} . \emph{Ecography} , 31, 663--671.

\textbf{Figures}

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/571956/articles/617053-intensity-of-herbivory-correlates-with-stronger-constitutive-and-weaker-induced-defenses-for-non-native-plant-species-another-mechanism-for-eica}

\textbf{Figure 1.} Herbivore pressure on non-native and native plant
species. Percentage of damaged leaves on non-native (A) and co-occurring
native (B) species in the field survey, and percentage of damaged leaf
area on non-native species (D) and native congeners (E) in the common
garden experiment. Differences in the percentage of damaged leaves on
non-native and co-occurring native species in the field (C) and in the
percentage of damaged leaf area on non-native species and native
congeners in the common garden (F). Boxplots represent the interquartile
range and median, and points represent outliers. There were 27
non-native plant species measured (n = 3 to 66 individuals per species
depending on occurrence in the field) and 59 non-native plant species
measured (n = 3 to 54 individuals per species depending on occurrence)
in the field survey across 28 sites. The most common 12 non-native plant
species and their native congeners were used in the common garden
experiment (n = 6 per species). * indicate \emph{p} \textless{}0.05 and
*** indicate \emph{p} \textless{}0.001.

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/571956/articles/617053-intensity-of-herbivory-correlates-with-stronger-constitutive-and-weaker-induced-defenses-for-non-native-plant-species-another-mechanism-for-eica}

\textbf{Figure 2.} Correlations between constitutive or induced defenses
and herbivore pressure across 12 non-native plant species and 12 native
plant species. Relationships between the percentage of damaged leaf area
in the common garden experiment and constitutive defense assessed by
generalist \emph{Spodoptera littoralis} larval weight gain on the leaves
of healthy plants (A) and assessed by phenolics (C) and triterpenoids
(E) contents in the leaves of healthy plants. Relationships between the
percentage of damaged leaf area in the common garden experiment and
induced defense assessed by difference in \emph{S. littoralis} larval
weight gain on the leaves between healthy and damaged plants {[}-1 x
(larval weight gain on the leaves of damaged plant -- average of larval
weight gain on the leaves of healthy plant), B{]}, and assessed by
differences in phenolics in the leaves between healthy and damaged
plants (content in the leaves of damaged plant -- average of content in
the leaves of healthy plant, D) and difference in triterpenoids on the
leaves between healthy and damaged plants (content in the leaves of
damaged plant - average of content in the leaves of healthy plant, F).
Each point represents individual non-native species (red points) and
native congeners (blue points). Lines indicate significant linear
relationships. The embedding boxplots represents the comparison for each
group of species overall. * indicate \emph{p} \textless{}0.05 and ***
indicate \emph{p} \textless{}0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Relationships between the growth of \emph{Spodoptera
littoralis} larvae and leaf secondary chemicals across all species (12
pairs of non-native plants and native congeners) and herbivory
treatments (healthy plants and larvae-damaged plants). Relationship
between larval weight gain and leaf phenolic content (A) and leaf
triterpenoid content (B). Data points represent individual non-natives
and native species. Lines indicate significant linear relationships.

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/571956/articles/617053-intensity-of-herbivory-correlates-with-stronger-constitutive-and-weaker-induced-defenses-for-non-native-plant-species-another-mechanism-for-eica}

\textbf{Figure 4.} Trade-off between constitutive defense and induced
defense across 12 pairs of non-native plants and native congeners.
Relationship between constitutive defense (-1 \selectlanguage{ngerman}× larval weight gain on
the leaves of healthy plant) and induced defense {[}-1 × (larva weight
gain on the leaves of damaged plant -- average of larval weight gain on
the leaves of healthy plant){]} assessed by generalist \emph{Spodoptera
littoralis} larval growth (A). Relationship between constitutive defense
(content in the leaves of healthy plant) and induced defense (content in
the leaves of damaged plant - average of content in the leaves of
healthy plant) assessed by phenolics (B). Blue points represent
individual non-native species, and red points represent individual
native species. Lines indicate significant linear relationships.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} Relative growth rates of non-native species and
native congeners (A). Relationship between the difference in the
relative growth rate between non-native species and its corresponding
native congener and the difference in the induced defense between
non-native species and its corresponding native congener (B). Induced
defense was assessed by difference in \emph{Spodoptera littoralis}larval
weight gain on the leaves between healthy and damaged plants {[}-1 \selectlanguage{ngerman}×
(larval weight gain on the leaves of damaged plant -- average of larval
weight gain on the leaves of healthy plant){]}. Each point represents a
non-native species and its native congener. Lines indicate significant
linear relationships. *** indicates\emph{p} \textless{}0.001.

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/571956/articles/617053-intensity-of-herbivory-correlates-with-stronger-constitutive-and-weaker-induced-defenses-for-non-native-plant-species-another-mechanism-for-eica}

\textbf{Figure 6.} Appropriation of ancient Chinese folk mythology to
illustrate integration of the ERH, SDH, and EICA hypotheses with
variation in defense allocation. Sun Wukong, the monkey king
(representing a plant species), possesses inherent strength that is
limited by the incantation of the golden hoop (A) - specialist
herbivores - which keeps him under control. However, when Sun Wukong
undertakes a pilgrimage to the west (B) - non-native introduction - the
golden hoop is no longer required, allowing a rapid increase in defense
capacity (C) - the golden cudgel he acquired on his journey - against
the now predominant generalist herbivores. Simultaneously, removing the
golden hoop allows a strong and consistent response to \emph{variation}
in generalist herbivore pressure, resulting in a positive correlation
between herbivore pressure and allocation to cheaper induced defense
(D). This reallocation leads to greater growth consistent with the
Evolution of Increased Competitive Ability Hypothesis (E).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}

: Sun Wukong. : The golden hoop. : Generalist herbivore. : specialist
herbivore. : Golden cudgel, plant defense. ERH: Enemy Release
Hypothesis. SDH: Shifting Defense Hypothesis. EICA: Evolution of
Increased Competitive Ability Hypothesis.

\textbf{Supporting Information}

\textbf{Herbivore pressure correlates with stronger constitutive and
weaker induced defenses for non-native plant species}

Xiao Sun\textsuperscript{1}, Yumei Sun\textsuperscript{1}, Xueyao
Cao\textsuperscript{1}, Xincong Zhai\textsuperscript{1}, Ragan M.
Callaway\textsuperscript{2}, Jinlong Wan\textsuperscript{3,4}, S. Luke
Flory\textsuperscript{5}, Wei Huang\textsuperscript{3,4}*, Jianqing
Ding\textsuperscript{1}

\textsuperscript{1} State Key Laboratory of Crop Stress Adaptation and
Improvement, School of Life Sciences, Henan University, Kaifeng 475004,
China

\textsuperscript{2} Division of Biological Sciences and Institute on
Ecosystems, University of Montana, Missoula 59812, USA

\textsuperscript{3} Key Laboratory of Aquatic Botany and Watershed
Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan
430074, China

\textsuperscript{4} Core Botanical Garden, Chinese Academy of Sciences,
Wuhan 430074, China

\textsuperscript{5} Agronomy Department, University of Florida,
Gainesville 32611, USA

*Correspondence: Wei Huang, huangwei0519@wbgcas.cn

This file contains the following Supplementary Information:

\begin{itemize}
\tightlist
\item
  Supplementary Methods: Appendix Methods 1-2
\item
  Supplementary Figures: Appendix Figure S1-S5
\item
  Supplementary Tables: Appendix Table S1-S4
\end{itemize}

\emph{Supplementary Methods 1: Evaluate herbivore biomass in the field
survey}

We used herbivore biomass to represent herbivore pressure (Allen et al.,
2021) as higher herbivore abundance may not always translate to greater
herbivore damage if feeding by particular herbivore species is
relatively minor (e.g., aphids and beetles) or extensive (e.g., larvae).
To determine whether the herbivore biomass correlated with leaf damage,
we checked all leaves and recorded the number of each herbivore species
on each non-native plant. We classified observed herbivores into five
orders (nine categories), including Lepidoptera (larvae), Orthopter,
Thysanoptera, Hemiptera (leafhoppers, stinkbugs, whiteflies, and
aphids), and Coleoptera (beetles and herbivorous ladybirds) since some
larvae were difficult to identify to species. A total of 20,723 insects
on non-native plants were counted (Native plants were not measured as
these measurements were primarily to correlate damage to insect
biomass). Then, we evaluated herbivore biomass on each plant by
multiplying abundance and mean herbivore biomass of each category and
then summing herbivore biomass of all categories. To obtain the mean
biomass of each herbivore category, we carried out a field survey at
eight sites in 2019. We collected all herbivores on non-native species,
stored them in plastic tubes with leaves of host plants and determined
the fresh weight of individual insects to the nearest 0.01g in the lab.
The mean biomass of each herbivore category was as follows: Lepidoptera,
0.25 g/ individual; Orthoptera, 0.45 g/ individual; Thysanoptera,
0.01g/30 individuals; leafhopper, 0.03g/ individual; stinkbug, 0.19 g/
individual; whitefly, 0.01 g/30 individuals; aphid, 0.01 g/5
individuals; leaf beetle, 0.05 g/ individual; ladybird, 0.04 g/
individual.

Supplementary Methods 2: Chemical analysis of total phenolics and total
triterpenoid

Leaves were flash frozen in liquid nitrogen and stored at -80\selectlanguage{ngerman}°C for
analysis. Total phenolic concentration was calculated with the modified
Folin-Ciocalteau method (Singleton \& Rossi, 1964). Briefly, an aliquot
of 1 mL of free phenolic acid extract was added to 5 mL of water, 1.0 mL
Folin-Ciocalteau reagent, and 3 ml of saturated sodium carbonate
solution (7.5\%) and mixed in a screw-top test tube. The absorbance was
measured at 765 nm with a spectrophotometer, after incubation for 2 h at
room temperature. Quantification was based on the standard curve,
established with 0, 50, 100, 150, 200 and 250 mg/L of gallic acid, and
the results were expressed as gallic acid equivalent in milligrams per
gram dry weight (mg GAE/g DW). Total triterpenoid concentration was
determined via vanillin-glacial acetic acid-perchloric acid
spectrophotometry using oleanolic acid as the standard (Fan \& He,
2006). Briefly, total triterpenoids were extracted by ultrasonic
extraction using anhydrous ethanol as a solvent. Oleanolic acid was used
as the reference, 5\% vanillin-acetic acid glacial solution and
perchlorate acid were used as the chromogenic agent, followed by water
bath at 70°C for 15min, and the absorbance was measured at 560 nm.
Because the amount of one leaf was not sufficient for each chemical
analysis, we gathered leaves from six plants as a biological
replication. There were 12 replicates for each combination of health
plant and herbivory treatment for each non-native species or their
native congeners.

\textbf{References}

Allen, W.J., Waller, L.P., Barratt, B.I., Dickie, I.A. \& Tylianakis,
J.M. (2021) Exotic plants accumulate and share herbivores yet dominate
communities via rapid growth. \emph{Nat. Commun} ., 12, 1--12.

Singleton, V. \& Rossi, J.A. (1964) Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. \emph{Am} . \emph{J}
.\emph{Enol} . \emph{Viticult} ., 16, 144--158.

Fan, J.P. \& He, C.H. (2006) Simultaneous quantification of three major
bioactive triterpene acids in the leaves of \emph{Diospyros kaki} by
high--performance liquid chromatography method. \emph{J}
.\emph{Pharmaceut} . \emph{Biomed} ., 41, 950--956

\textbf{Hosted file}

\verb`image12.emf`  available at \url{https://authorea.com/users/571956/articles/617053-intensity-of-herbivory-correlates-with-stronger-constitutive-and-weaker-induced-defenses-for-non-native-plant-species-another-mechanism-for-eica}

\textbf{Figure S1.} Predictions for how herbivore pressure affects plant
defense and growth traits. Plant species under higher herbivore pressure
(putatively natives, blue line) are predicted to have higher mean
constitutive defense and lower mean induced defense than plants under
lower herbivore pressure (putatively non-natives, red line). Also,
differences in herbivore pressure among species should correlate with
increasing constitutive defense and decreasing induced defenses for all
species, illustrated by the slopes of the lines (A and B). These defense
responses should lead to growth-defense trade-offs in which greater
allocation to less expensive induced defense allows allocation to
greater growth (C).

\textbf{Hosted file}

\verb`image13.emf`  available at \url{https://authorea.com/users/571956/articles/617053-intensity-of-herbivory-correlates-with-stronger-constitutive-and-weaker-induced-defenses-for-non-native-plant-species-another-mechanism-for-eica}

\textbf{Figure S2.} Total herbivore biomass collected on the non-native
plants in the field survey (A) and common garden experiment (B). We
classified herbivores in the field survey and common garden experiment
into five orders (nine categories), including larva Lepidoptera,
Orthopter, Thysanoptera, Hemiptera (leafhopper, stinkbug, whitefly, and
aphid), and Coleoptera (beetle and ladybird) since larvae of some
species were difficult to identify to species. The values are the total
herbivore biomass in each category.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}

\textbf{Figure S3.} Relationship between herbivore biomass and leaf
damage for non-native plant species in the field survey (A) and in the
common garden experiment (B). Data points represent individual plants (n
= 396 in the field survey, n = 84 in the common garden experiment).
Lines through points indicate significant linear relationships.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}

\textbf{Figure S4.} Relationship between percentage of damaged leaves in
the field survey and percentage of damaged leaf area in the common
garden experiment for non-native species. Each points represent
individual non-native species. The line through points indicates a
significant linear relationship.

\textbf{Hosted file}

\verb`image16.emf`  available at \url{https://authorea.com/users/571956/articles/617053-intensity-of-herbivory-correlates-with-stronger-constitutive-and-weaker-induced-defenses-for-non-native-plant-species-another-mechanism-for-eica}

\textbf{Figure S5.} Pairs of non-native plants and native congeners at
the end of experiment. Left, native plants; right, non-native plants.
a,\emph{Amaranthus tricolor} \emph{vs.} \emph{Amaranthus retroflexus} ;
b,\emph{Xanthium sibiricum} \emph{vs. Xanthium italicum} ; c,
\emph{Bidens tripartite} \emph{vs.} \emph{Bidens frondose} ; d,
\emph{Solidago decurrens} \emph{vs} . \emph{Solidago canadensis} ; e,
\emph{Rhus chinensis} \emph{vs} . \emph{Rhus typhina} ; f,
\emph{Phytolacca acinosevs} . \emph{Phytolacca americana} ; g,
\emph{Amaranthus blitumvs} . \emph{Amaranthus spinosus} .

\textbf{Table S1.} Locations of 28 sites for the field survey in
northern China. Codes of the sites with asterisks indicate those
surveyed again for evaluation of herbivore biomass.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Number & Code & Site & Province & Longitude & Latitude\tabularnewline
\midrule
\endhead
1 & CZ1 & Cangzhou & Hebei & 38\selectlanguage{ngerman}°11\selectlanguage{english}'N & 116\selectlanguage{ngerman}°43\selectlanguage{english}'E\tabularnewline
2 & CZ2 & Cangzhou & Hebei & 38\selectlanguage{ngerman}°54\selectlanguage{english}'N & 116\selectlanguage{ngerman}°09\selectlanguage{english}'E\tabularnewline
3 & HD & Handan & Hebei & 36\selectlanguage{ngerman}°13\selectlanguage{english}'N & 114\selectlanguage{ngerman}°23\selectlanguage{english}'E\tabularnewline
4 & HS1* & Hengshui & Hebei & 37\selectlanguage{ngerman}°51\selectlanguage{english}'N & 115\selectlanguage{ngerman}°41\selectlanguage{english}'E\tabularnewline
5 & HS2 & Hengshui & Hebei & 37\selectlanguage{ngerman}°35\selectlanguage{english}'N & 115\selectlanguage{ngerman}°37\selectlanguage{english}'E\tabularnewline
6 & XT1 & Xingtai & Hebei & 36\selectlanguage{ngerman}°54\selectlanguage{english}'N & 114\selectlanguage{ngerman}°29\selectlanguage{english}'E\tabularnewline
7 & XT2* & Xingtai & Hebei & 36\selectlanguage{ngerman}°52\selectlanguage{english}'N & 114\selectlanguage{ngerman}°24\selectlanguage{english}'E\tabularnewline
8 & XT3 & Xingtai & Hebei & 36\selectlanguage{ngerman}°54\selectlanguage{english}'N & 114\selectlanguage{ngerman}°24\selectlanguage{english}'E\tabularnewline
9 & DF1 & Dengfeng & Henan & 34\selectlanguage{ngerman}°27\selectlanguage{english}'N & 112\selectlanguage{ngerman}°58\selectlanguage{english}'E\tabularnewline
10 & DF2 & Dengfeng & Henan & 34\selectlanguage{ngerman}°32\selectlanguage{english}'N & 112\selectlanguage{ngerman}°55\selectlanguage{english}'E\tabularnewline
11 & GY* & Gongyi & Henan & 34\selectlanguage{ngerman}°48\selectlanguage{english}'N & 113\selectlanguage{ngerman}°01\selectlanguage{english}'E\tabularnewline
12 & KF1* & Kaifeng & Henan & 34\selectlanguage{ngerman}°52\selectlanguage{english}'N & 114\selectlanguage{ngerman}°18\selectlanguage{english}'E\tabularnewline
13 & KF2 & Kaifeng & Henan & 34\selectlanguage{ngerman}°49\selectlanguage{english}'N & 114\selectlanguage{ngerman}°16\selectlanguage{english}'E\tabularnewline
14 & SQ* & Shangqiu & Henan & 34\selectlanguage{ngerman}°08\selectlanguage{english}'N & 115\selectlanguage{ngerman}°25\selectlanguage{english}'E\tabularnewline
15 & XX* & Xinxiang & Henan & 35\selectlanguage{ngerman}°06\selectlanguage{english}'N & 114\selectlanguage{ngerman}°16\selectlanguage{english}'E\tabularnewline
16 & ZK* & Zhoukou & Henan & 34\selectlanguage{ngerman}°00\selectlanguage{english}'N & 114\selectlanguage{ngerman}°24\selectlanguage{english}'E\tabularnewline
17 & XZ1 & Xuzhou & Jiangsu & 34\selectlanguage{ngerman}°26\selectlanguage{english}'N & 117\selectlanguage{ngerman}°08\selectlanguage{english}'E\tabularnewline
18 & XZ2 & Xuzhou & Jiangsu & 34\selectlanguage{ngerman}°29\selectlanguage{english}'N & 117\selectlanguage{ngerman}°08\selectlanguage{english}'E\tabularnewline
19 & BZ* & Binzhou & Shandong & 36\selectlanguage{ngerman}°58\selectlanguage{english}'N & 117\selectlanguage{ngerman}°38\selectlanguage{english}'E\tabularnewline
20 & DZ & Dezhou & Shandong & 36\selectlanguage{ngerman}°50\selectlanguage{english}'N & 116\selectlanguage{ngerman}°41\selectlanguage{english}'E\tabularnewline
21 & JN & Jinan & Shandong & 36\selectlanguage{ngerman}°53\selectlanguage{english}'N & 117\selectlanguage{ngerman}°30\selectlanguage{english}'E\tabularnewline
22 & JN1 & Jining & Shandong & 34\selectlanguage{ngerman}°45\selectlanguage{english}'N & 117\selectlanguage{ngerman}°08\selectlanguage{english}'E\tabularnewline
23 & JN2 & Jining & Shandong & 35\selectlanguage{ngerman}°39\selectlanguage{english}'N & 116\selectlanguage{ngerman}°57\selectlanguage{english}'E\tabularnewline
24 & JN3 & Jining & Shandong & 35\selectlanguage{ngerman}°47\selectlanguage{english}'N & 117\selectlanguage{ngerman}°04\selectlanguage{english}'E\tabularnewline
25 & LW & Laiwu & Shandong & 36\selectlanguage{ngerman}°13\selectlanguage{english}'N & 117\selectlanguage{ngerman}°27\selectlanguage{english}'E\tabularnewline
26 & LY & Linyi & Shandong & 36\selectlanguage{ngerman}°05\selectlanguage{english}'N & 118\selectlanguage{ngerman}°38\selectlanguage{english}'E\tabularnewline
27 & WF & Weifang & Shandong & 36\selectlanguage{ngerman}°33\selectlanguage{english}'N & 119\selectlanguage{ngerman}°44\selectlanguage{english}'E\tabularnewline
28 & ZB & Zibo & Shandong & 36\selectlanguage{ngerman}°51\selectlanguage{english}'N & 118\selectlanguage{ngerman}°19\selectlanguage{english}'E\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Table S2.} Occurrence and herbivore pressure of 27 non-native
plant species in 28 sites in field survey across North China. Species
with asterisks were used in the common garden experiment, bioassay and
chemical analysis. The difference in the percentage of damaged leaf
(binary data for undamaged \emph{vs.} damaged leaves in a cbind matrix)
among sites was tested using a Wald Chi-square test applied on a
Generalized Linear Model (GLM) with a binomial distribution. Each
non-native species was analyzed separately.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
Non-native species & Number of measured individuals & Frequency of
occurrence (\%) & Number of insects & Difference in herbivore pressure
(binary data of undamaged vs. damaged leaves in a cbind matrix) &
Difference in herbivore pressure (binary data of undamaged vs. damaged
leaves in a cbind matrix) & Difference in herbivore pressure (binary
data of undamaged vs. damaged leaves in a cbind matrix)\tabularnewline
\midrule
\endhead
& & & & Df & Chisq & P\tabularnewline
\emph{Erigeron canadensis}* & 66 & 78.6 & 4131 & 21 & 9.88 &
0.980\tabularnewline
\emph{Ipomoea cairica}* & 36 & 42.9 & 355 & 11 & 1.43 &
1.000\tabularnewline
\emph{Bidens frondosa}* & 30 & 35.7 & 1747 & 9 & 8.76 &
0.460\tabularnewline
\emph{Amaranthus spinosus}* & 27 & 32.1 & 1396 & 8 & 19.61 &
0.012\tabularnewline
\emph{Bidens pilosa}* & 27 & 32.1 & 1708 & 8 & 6.16 &
0.629\tabularnewline
\emph{Amaranthus retroflexus}* & 18 & 21.4 & 696 & 5 & 26.96 &
0.000\tabularnewline
\emph{Aster subulatus}* & 15 & 17.9 & 19 & 4 & 1.38 &
0.848\tabularnewline
\emph{Datura stramonium}* & 15 & 17.9 & 1098 & 4 & 100.21 &
0.000\tabularnewline
\emph{Hibiscus trionum}* & 15 & 17.9 & 1105 & 4 & 13.21 &
0.010\tabularnewline
\emph{Abutilon theophrasti}* & 12 & 14.3 & 3066 & 3 & 6.34 &
0.096\tabularnewline
\emph{Phytolacca americana}* & 12 & 14.3 & 1329 & 3 & 20.57 &
0.000\tabularnewline
\emph{Rhus typhina}* & 12 & 14.3 & 2 & 3 & 3.65 & 0.302\tabularnewline
\emph{Solidago canadensis}* & 12 & 14.3 & 1122 & 3 & 0.52 &
0.914\tabularnewline
\emph{Xanthium italicum}* & 12 & 14.3 & 18 & 3 & 1.02 &
0.798\tabularnewline
\emph{Cyperus rotundus} & 9 & 10.7 & 8 & & &\tabularnewline
\emph{Erigeron bonariensis} & 9 & 10.7 & 15 & & &\tabularnewline
\emph{Flaveria bidentis} & 9 & 10.7 & 12 & & &\tabularnewline
\emph{Veronica didyma} & 9 & 10.7 & 0 & & &\tabularnewline
\emph{Veronica persica} & 9 & 10.7 & 0 & & &\tabularnewline
\emph{Ambrosia artemisiifolia} & 6 & 7.1 & 1370 & & &\tabularnewline
\emph{Cosmos bipinnata} & 6 & 7.1 & 173 & & &\tabularnewline
\emph{Daucus carota} & 6 & 7.1 & 4 & & &\tabularnewline
\emph{Helianthus tuberosus} & 6 & 7.1 & 243 & & &\tabularnewline
\emph{Lactuca seriola} & 6 & 7.1 & 648 & & &\tabularnewline
\emph{Solanum aculeatissimum} & 6 & 7.1 & 5 & & &\tabularnewline
\emph{Erigeron annuus} & 3 & 3.6 & 320 & & &\tabularnewline
\emph{Sonchus asper} & 3 & 3.6 & 133 & & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S3.} Number and frequency of occurrence 59 native plant
species in 28 sites in a field survey across North China. Species with
asterisks were used in the common garden experiment, bioassay and
chemical analysis. The difference in the percentage of damaged leaf
(binary data of undamaged \emph{vs.} damaged leaves in a cbind matrix)
among sites was tested using a Wald Chi-square test applied on a
Generalized Linear Model (GLM) with a binomial distribution. Each
non-native species was analyzed separately. Each native species was
analyzed separately.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Non-native species & Number of measured individuals & Frequency of
occurrence (\%) & Difference in herbivore pressure (binary data of
undamaged vs. damaged leaves in a cbind matrix) & Difference in
herbivore pressure (binary data of undamaged vs. damaged leaves in a
cbind matrix) & Difference in herbivore pressure (binary data of
undamaged vs. damaged leaves in a cbind matrix)\tabularnewline
\midrule
\endhead
& & & Df & Chisq & P\tabularnewline
\emph{Xanthium sibiricum} & 54 & 64.3 & 17 & 107.97 & \textless{}
0.001\tabularnewline
\emph{Bidens biternata} & 45 & 53.6 & 14 & 69.79 & \textless{}
0.001\tabularnewline
\emph{Amaranthus blitum} & 39 & 46.4 & 12 & 7.19 & 0.845\tabularnewline
\emph{Amaranthus tricolor} & 33 & 39.3 & 10 & 16.41 &
0.088\tabularnewline
\emph{Abutilon indicum} & 30 & 35.7 & 9 & 6.06 & 0.734\tabularnewline
\emph{Phytolacca acinosa} & 27 & 32.1 & 8 & 8.22 & 0.412\tabularnewline
\emph{Datura metel} & 15 & 17.9 & 4 & 12.14 & 0.016\tabularnewline
\emph{Erigeron acer} & 12 & 14.3 & 3 & 0.25 & 0.969\tabularnewline
\emph{Rhus chinensis} & 12 & 14.3 & 3 & 1.11 & 0.775\tabularnewline
\emph{Solidago decurrens} & 12 & 14.3 & 3 & 1.21 & 0.752\tabularnewline
\emph{Aster indicus} & 9 & 10.7 & 2 & 5.57 & 0.062\tabularnewline
\emph{Bidens tripartita} & 9 & 10.7 & 2 & 0.15 & 0.929\tabularnewline
\emph{Hibiscus mutabilis} & 9 & 10.7 & 2 & 1.46 & 0.482\tabularnewline
\emph{Ipomoea aquatica} & 9 & 10.7 & 2 & 0.60 & 0.741\tabularnewline
\emph{Setaria viridis} & 45 & 53.6 & & &\tabularnewline
\emph{Cirsium arvense} & 42 & 50.0 & & &\tabularnewline
\emph{Taraxacum mongolicum} & 42 & 50.0 & & &\tabularnewline
\emph{Artemisia argyi} & 27 & 32.1 & & &\tabularnewline
\emph{Eclipta prostrata} & 15 & 17.9 & & &\tabularnewline
\emph{Sonchus wightianus} & 12 & 14.3 & & &\tabularnewline
\emph{Artemisia sacrorum} & 9 & 10.7 & & &\tabularnewline
\emph{Aster ageratoides} & 9 & 10.7 & & &\tabularnewline
\emph{Cirsium japonicum} & 9 & 10.7 & & &\tabularnewline
\emph{Lactuca indica} & 9 & 10.7 & & &\tabularnewline
\emph{Alternanthera sessilis} & 6 & 7.1 & & &\tabularnewline
\emph{Chloris virgata} & 6 & 7.1 & & &\tabularnewline
\emph{Digitaria sanguinalis} & 6 & 7.1 & & &\tabularnewline
\emph{Eupatorium chinense} & 6 & 7.1 & & &\tabularnewline
\emph{Eupatorium fortunei} & 6 & 7.1 & & &\tabularnewline
\emph{Imperata cylindrica} & 6 & 7.1 & & &\tabularnewline
\emph{Ixeris chinensis} & 6 & 7.1 & & &\tabularnewline
\emph{Plantago asiatica} & 6 & 7.1 & & &\tabularnewline
\emph{Portulaca oleracea} & 6 & 7.1 & & &\tabularnewline
\emph{Pulsatilla chinensis} & 6 & 7.1 & & &\tabularnewline
\emph{Solanum nigrum} & 6 & 7.1 & & &\tabularnewline
\emph{Sonchus oleraceus} & 6 & 7.1 & & &\tabularnewline
\emph{Cynodon dactylon} & 6 & 7.1 & & &\tabularnewline
\emph{Artemisia carvifolia} & 3 & 3.6 & & &\tabularnewline
\emph{Artemisia scoparia} & 3 & 3.6 & & &\tabularnewline
\emph{Bolboschoenus yagara} & 3 & 3.6 & & &\tabularnewline
\emph{Bothriospermum chinense} & 3 & 3.6 & & &\tabularnewline
\emph{Carduus nutans} & 3 & 3.6 & & &\tabularnewline
\emph{Chrysanthemum lavandulifolium} & 3 & 3.6 & & &\tabularnewline
\emph{Coreopsis basalis} & 3 & 3.6 & & &\tabularnewline
\emph{Cyperus rotundus} & 3 & 3.6 & & &\tabularnewline
\emph{Elephantopus scaber} & 3 & 3.6 & & &\tabularnewline
\emph{Euphorbia thymifolia} & 3 & 3.6 & & &\tabularnewline
\emph{Gaillardia pulchella} & 3 & 3.6 & & &\tabularnewline
\emph{Helianthus annuus} & 3 & 3.6 & & &\tabularnewline
\emph{Hydrocotyle sibthorpioides} & 3 & 3.6 & & &\tabularnewline
\emph{Ixeris japonica} & 3 & 3.6 & & &\tabularnewline
\emph{Lactuca raddeana} & 3 & 3.6 & & &\tabularnewline
\emph{Rehmannia glutinosa} & 3 & 3.6 & & &\tabularnewline
\emph{Salvia plebeia} & 3 & 3.6 & & &\tabularnewline
\emph{Silene conoidea} & 3 & 3.6 & & &\tabularnewline
\emph{Torilis scabra} & 3 & 3.6 & & &\tabularnewline
\emph{Tribulus terrestris} & 3 & 3.6 & & &\tabularnewline
\emph{Trifolium repens} & 3 & 3.6 & & &\tabularnewline
\emph{Trigonotis peduncularis} & 3 & 3.6 & & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S4.} List of the 12 non-native plant species and their
native congeners used in the common garden experiment, bioassay and
chemical analysis.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Number\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Family\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Genus\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Native\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Growth type\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Life cycle\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Non-native\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Growth type\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Life cycle\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Origin\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Asteraceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Aster\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Aster ageratoides}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Aster subulatus}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
North America\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Asteraceae\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Bidens\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Bidens tripartite}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{Bidens frondosa}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
North America \emph{Aster subulatus}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Asteraceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Bidens\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Bidens biternata}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Bidens pilosa}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
America\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Asteraceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Erigeron\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Erigeron acer}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Erigeron canadensis}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
North America\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Asteraceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Solidago\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Solidago decurrens}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Solidago canadensis}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
North America\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Asteraceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Xanthium\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Xanthium sibiricum}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Xanthium italicum}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
America/Europe\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Amaranthaceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Amaranthus\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Amaranthus tricolor}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Amaranthus retroflexus}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
America\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Amaranthaceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Amaranthus\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Amaranthus blitum}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Amaranthus spinosus}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
South America\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Solanaceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Datura\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Datura metel}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Datura stramonium}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
South Asia\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
10\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Convolvulaceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Ipomoea\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Ipomoea aquatica}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Ipomoea cairica}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Asia/Africa\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
11\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Phytolaecaceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Phytolacca\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Phytolacca acinosa}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Perennial\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Phytolacca americana}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
perennial\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
North America\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
12\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Malvaceae\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Abutilon\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Abutilon indicum}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Abutilon theophrasti}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
herbaceous\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
annual\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
South Asia\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

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\end{document}

