Herbivore release drives parallel patterns of evolutionary divergence in invasive plant phenotypes

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1 Journal of Ecology 216, 14, doi: / Herbivore release drives parallel patterns of evolutionary divergence in invasive plant phenotypes Akane Uesugi 1 and Andre Kessler 2 1 School of Biological Sciences, Monash University, Building 18, Melbourne, Vic. 38, Australia; and 2 Department of Ecology & Evolutionary Biology, Cornell University, Ithaca, NY 1485, USA Summary 1. Herbivory can drive rapid evolution of plant chemical traits mediating defensive and competitive ability. At a geographic scale, plant populations that escape selection from their ancestral herbivores may evolve decreased defence and increased competitiveness. While contrasts between native and invasive populations of plants lend support to this hypothesis, such experiments cannot establish causal links between herbivory and evolved invasive phenotypes. 2. Here, we conducted geographic contrasts, and coupled these with long-term selection experiments that directly test for evolutionary responses to herbivore exclusion. In common gardens, we contrasted Solidago altissima genotypes that were historically exposed or protected from herbivory across two experimental time-scales: (i) a natural experiment where plant populations evolved either with native herbivory (in Minnesota and New York) or evolved relatively free from herbivory for ~1 years in Japan, and (ii) a 12-year manipulative experiment where plants were either exposed to ambient herbivory or treated with insecticide. 3. In both experiments, plant populations responded to herbivore release by evolving increased production of root allelochemicals and interspecific competitive ability against Poa pratensis. While plant resistance to a beetle herbivore did not diverge between plant origins, we still observed parallel evolutionary shifts in leaf secondary metabolite and protease inhibitor production, which may confer resistance to diverse herbivore species. 4. Synthesis. Observed evolutionary convergence for multiple plant traits, between the natural and manipulative experiments, emphasizes the role of insect herbivores as key drivers of plant adaptation and geographic differentiation. Key-words: allelopathy, artificial selection experiment, evolution of increased competitive ability, invasion ecology, plant resistance, plant herbivore interactions, secondary metabolites Introduction Insect herbivory has been hypothesized as a major selective force driving plant adaptation, due to its ubiquitous nature and its significant impacts on plant fitness (Mauricio & Rausher 1997; Strauss et al. 22; Carmona, Lajeunesse & Johnson 211). Recent long-term manipulative experiments demonstrated that herbivory can drive rapid evolution of plant defence-related traits (Agrawal et al. 212; Bode & Kessler 212; Z ust et al. 212), as well as competitive ability (Agrawal et al. 212; Uesugi & Kessler 213) that are in trade-off with defence (Coley, Bryant & Chapin 1985; Herms & Mattson 1992; Lind et al. 213). At a global scale, a geographic variation in herbivore communities is predicted to result in divergent plant traits among plant populations Correspondence author: akane.uesugi@monash.edu (Thompson 25; Zangerl, Stanley & Berenbaum 28; Z ust et al. 212). Numerous studies have tested this idea by taking advantage of natural experiments where exotic plants are introduced into a novel environment that often lacks their specialist herbivores (reviewed in Orians & Ward 21). For example, the evolution of increased competitive ability (EICA) hypothesis predicts that a release from herbivory drives evolution of increased competitiveness and reduced herbivore defence in introduced ranges (Blossey & N otzold 1995). Furthermore, the Novel Weapons hypothesis (NWH) suggests that some invasive plants gain competitive advantage by producing allelopathic compounds that native competitors are not adapted to, and such allelopathy could be selected for in invasive ranges (Callaway & Ridenour 24). These hypotheses, which were tested by contrasting plants from native and invasive ranges in a common garden, have gained some support (e.g. Wolfe, Elzinga & Biere 24; Zheng et al. 215), but 216 The Authors. Journal of Ecology 216 British Ecological Society

2 Convergent evolution of plant invasiveness 877 overall support has been equivocal (reviewed in Bossdorf et al. 25; Colautti, Maron & Barrett 29; Felker-Quinn, Schweitzer & Bailey 213). Furthermore, the causal relationship between herbivory and evolution of invasive phenotypes has not been established in most of the natural experiments, because trait shifts observed in invasive plant populations could result from founder effects and/or adaptation to other environmental variables (Bossdorf et al. 25; Colautti, Maron & Barrett 29). Here, we propose that combining natural experiments with long-term manipulative experiments, where evolutionary response to herbivore exclusion is directly measured, provide a powerful tool for elucidating the direct role of herbivory on plant adaptation at a global scale (Z ust et al. 212). If herbivory is a major driver of plant adaptation in invasive ranges, we would expect a convergent evolutionary pattern of trait shifts in invasive plant populations and in populations in which herbivory has been experimentally removed. The tall goldenrod, Solidago altissima L., allows for such multiscale contrasts because we could compare plant genotypes that originated from two historical experiments: (i) a natural experiment, where native North American populations were introduced to Japan and evolved relatively free from herbivores for approximately 1 years (see Methods) and (ii) a 12-year manipulative experiment, where half of the experimental plots were treated with insecticide to remove above-ground insect herbivores (H plots), and the rest were exposed to ambient herbivory (H+ plots, see Uesugi & Kessler 213), We previously reported that the manipulative herbivore exclusion in S. altissima resulted in an evolutionary increase in interspecific competitive ability against Poa pratensis, which was likely mediated by increased allelochemical compound production in roots (Uesugi & Kessler 213). Bode & Kessler (212), in turn, showed an evolutionary decrease in resistance to a specialist herbivore, Trirhabda virgata (Chrysomelid, Coleoptera). Here, under the same common garden conditions as in Uesugi & Kessler (213), we contrasted S. altissima genotypes that originated from both the natural and manipulative experiments. Anti-nutritive secondary compounds and anti-digestive proteins can mediate plant resistance to herbivory, while allelopathic compounds released from roots may provide competitive advantages to the plant (Uesugi & Kessler 213). To understand the chemical mechanisms underlying the evolution of plant resistance and competitiveness, we particularly focused on evolutionary changes in plant leaf and root secondary metabolite production as the traits mediating the ecological interactions observed previously (Bode & Kessler 212; Uesugi & Kessler 213). Assuming that a rapid adaptive evolution occurs in Japanese S. altissima populations (see Methods), our goal here was to test whether geographic variation in plant traits observed in nature is compatible with the herbivore release scenario, as predicted by the EICA hypothesis. We did this by contrasting natural geographic divergence between native and invasive regions with the pattern of divergence observed under experimental evolution. We predicted convergent evolutionary shifts in Japanese and H populations that were historically protected from herbivory (hereafter called protected genotypes ) relative to those from North America and H+ populations that were historically exposed to herbivory ( exposed genotypes ). Our results strongly validate these predictions and provide experimental support for herbivore release as a critical driver of adaptive divergence in natural invasive plant populations. Materials and methods STUDY SYSTEM The tall goldenrod, S. altissima L. (Asteraceae), is a dominant perennial species of old-field plant communities native to eastern North America (Werner, Bradbury & Gross 198). It reproduces both sexually and asexually: wind-dispersed seeds are predominantly responsible for colonization of an abandoned field, while the plants spread clonally via rhizomes within a field (Cain 199). Solidago altissima was introduced to Japan more than 1 years ago, but its distribution only exploded in late 195s (Fukuda 1982). The native source populations that were introduced into Japan are currently unknown, but previous studies showed that there is an equivalent level of molecular genetic (AFLP) diversity between Japanese and North American populations (Y. Ando, unpublished data). Japanese populations also exhibit genotypic variation in plant resistance to recently introduced herbivores, Uroleucon nigrotuberculatum (introduced in 199s, Ando, Utsumi & Ohgushi 21) and Corythucha marmorata (introduced in 2s, Sakata et al. 214) and show rapid adaptation in response to a current change in herbivore environment (Sakata et al. 214), suggesting that they are highly evolutionary labile. Finally, its invasion history with a prolonged lag time between the initial introduction and the spread (Fukuda 1982) could potentially suggest a high propagule pressure and adaptive evolution within Japan immediately prior to its geographic spread there (Sakai et al. 21). Herbivore communities on Japanese S. altissima drastically differ from those in its native range. For example, native NY populations host more than 1 insect herbivores, many of which are Solidago specialists (Root & Cappuccino 1992). The community is dominated by chewing and leafmining beetles, T. virgata and Microrhopala vittata, respectively (Chrysomelid: Root & Cappuccino 1992). In outbreak years, a single stem may harbour close to 9 beetle larvae (Root & Cappuccino 1992), which can defoliate entire plants (A. Uesugi, personal observations). Herbivory by these insects also reduces competitive advantages of S. altissima over understorey forbs and grasses, such as P. pratensis (Carson & Root 2). By contrast, the current herbivore community on Japanese S. altissima is dominated by exotic sap feeders, which were introduced from North America in the 199s (Ando, Utsumi & Ohgushi 21; Sakata et al. 214). Ando, Utsumi & Ohgushi (21) found 18 other herbivore species on the Japanese plants, whose per plant density was approximately 1/3 of NY populations during their non-outbreaking years (Root 1996). Thus, prior to 199s, the Japanese S. altissima populations presumably enjoyed drastically reduced herbivore loads compared to its native populations. SOURCE OF SOLIDAGO ALTISSIMA GENOTYPES Genotypes from the natural experiment were collected from each of the three geographic regions, including two native North American regions in New York (NY) and Minnesota (MN), and one region in the introduced range in Japan (JP: see Table S1 in Supporting Infor-

3 878 A. Uesugi & A. Kessler mation). In the native range, we sampled from widely geographically diverged populations (across the major longitudinal axis of the native species range) in an attempt to capture the potentially broad spectrum of phenotypic diversity that is characteristic of S. altissima within its native range. In each region, rhizomes from 1 genotypes were collected from distant fields to capture variations within each region. Genotypes from the manipulative experiment resulted from longterm selection plots that were established in 1995 in a mid-successional old field at Whipple Farm, Tompkins Co., NY. The details of the manipulative experiment were described in Bode & Kessler (212) and Uesugi & Kessler (213), but briefly, twelve plots (5 m 9 5 m) were established, of which six were exposed to ambient insect herbivory (H+). The remaining six were treated with pyrethroid insecticide, fenvalerate (ORTHO â Group, Marysville, OH, USA), biweekly during the growing season (H ). Fenvalerate reduces herbivore damage to < 1% of leaf area, while having no phytotoxic or soil fertilization effects and minimal effects on soil microbes and belowground herbivory (Carson & Root 2). In 28, 16 haphazardly chosen plants from each of the twelve plots (192 plants total) were used to initiate clone libraries at Cornell University, Ithaca, NY. We randomly selected 3 H and 29 H+ genotypes from the collection for the subsequent characterization of plant traits in a common garden. COMMON GARDEN EXPERIMENTS Genotypes of S. altissima from both manipulative and natural experiments were propagated clonally from rhizome cuttings in the greenhouse for two growing cycles prior to the common-garden experiment to minimize maternal effects. A common-garden experiment contrasting genotypes from the manipulative experiment was conducted in summer 21, and the results on competitive ability and allelopathic compound production were reported in Uesugi & Kessler (213). Genotypes from the natural experiment were examined in summer 211 under the same condition. Because the focus of this study was to test parallel pattern of evolution in natural and manipulative experiments, we used an identical common garden environment where standard competition and herbivory treatments were applied in both experiments to make trait estimates comparable. In early spring, we propagated six plants from each genotype (hereafter called target plants ) from rhizome cuttings, and newly sprouted target plants were transplanted into 2 cm azalea pots containing 1: 3 ratio of sand (Bonsal American, Charlotte, NC, USA) and potting soil (Sun-Gro, Bellevue, Washington, DC, USA) and placed on the rooftop growing space of Corson Hall, Cornell University on 13 May 21 and 9 May 211. Plants were subsequently put under three levels of competition treatment and two levels of damage treatment in a full factorial manner. Competition treatments included (i) a control, in which the target plant was growing by itself, (ii) interspecific competition when growing with P. pratensis and (iii) intraspecific competition when growing with another S. altissima plant. Competitive ability was assessed using the additive design by estimating the importance of competition on target plant growth relative to other factors that could affect its growth (Goldberg & Fleetwood 1987). We chose to use a standard model species (or genotype in case of intraspecific competition) as a competitor in each of the interspecific and intraspecific competition to make our results for the native and manipulative experiments comparable and tractable. In the interspecific competition treatment, approximately 2 seeds of P. pratensis (obtained from Seeds Trust, Inc. Cornville, AZ, USA) were sprinkled evenly on the soil surface around the target plant. Poa pratensis is an ecologically relevant heterospecific competitor of S. altissima in both the native and invasive ranges, and it should therefore provide a good index of interspecific competitive ability in both ranges. In the native range, it is one of the common understorey plants found in old fields dominated by S. altissima (Carson & Root 2). It is also widespread in Japan and elsewhere (Holm et al. 1979) and thus is likely to compete with S. altissima in invasive ranges. The relevance is particularly strong in the context of allelopathy. We previously found that cis-dehydromatricaria ester from S. altissima roots strongly inhibited the growth of P. pratensis (Uesugi & Kessler 213). While its allelopathic effects on other plant species are also known (Kobayashi et al. 198; Johnson, Halitschke & Kessler 21), grass species, in general, seems to be more vulnerable to Solidago s allelopathy than forbs and legumes (Yang et al. 27). Because Japanese S. altissima often co-occurs with grass species in disturbed habitats (Fukuda 1982), allelopathy may be an effective mode of competition against common heterospecific competitors in the invasive range. In the intraspecific competition treatment, each target genotype competed against a single reference genotype of S. altissima (hereafter called, the neighbour genotype ), which was selected from the clone libraries of the manipulative experiment on the basis of its intermediate (and therefore representative) growth and allelopathic compound production (A. Uesugi, unpublished). Of those genotypes that originated from the natural experiment, the neighbour genotype was also placed within the second quartile for those traits, and thus, we avoided using an extreme genotype as an intraspecific competitor. The common assumption of the approach using a single reference neighbour genotype is that the identity of neighbour does not alter how the target genotypes compete, such that the relative rank of competitiveness among the target genotypes is transitive. While it is possible that target and neighbour genotypes interact in a non-transitive manner (i.e. there is a G 9 G interaction mediating the rank order of competitive outcomes), no such relationships were found in relative plant growth measures in another study using S. altissima (Genung, Bailey & Schweitzer 212). Damage treatment was applied 1 days after plants were transplanted. All target plants were bagged with mesh sleeves (Palm Tree Packaging, Apopka, FL, USA), and four field-collected T. virgata larvae per plant were added for 7 days in the damage treatment, while no larvae were added in the control. Damage and competition treatments were fully crossed. Plant resistance against T. virgata was measured on plants in the damaged plants by measuring mean initial and final larval mass. We selected T. virgata for the bioassay of plant resistance because it is the most common, specialist herbivore in New York. Previous work showed that the species can significantly reduce plant fitness during outbreak years (Root & Cappuccino 1992) and may shape resistance traits in S. altissima (Bode & Kessler 212). After 1 weeks from the initial transplant, the number of newly produced ramets was counted as an indicator of plant growth in the early growing season. Ramet production is an appropriate estimate of S. altissima fitness, as increased ramet production will enhance plant s ability to shade out other plants and increase competitive dominance in the field during the early growing season (McBrien, Harmsen & Crowder 1983; Bode & Kessler 212; Heath et al. 214). CHEMICAL ANALYSES Root samples were collected after 1 weeks of plant growth by removing approximately 2 mg of lateral roots from living target

4 Convergent evolution of plant invasiveness 879 plants. We quantified allelopathic polyacetylenes, which are known to inhibit seed germination and growth of heterospecific competitors (Kobayashi et al. 198; Johnson, Halitschke & Kessler 21; Uesugi & Kessler 213), as well as phenolics and diterpene acids. Leaf samples were collected for secondary metabolite analyses following the damage treatment to capture the chemical variations that T. virgata larvae experienced in bioassays. We targeted two classes of anti-nutritive compounds, phenolics and diterpene acids, that are commonly found in S. altissima (Uesugi, Poelman & Kessler 213), and serine protease inhibitor (SPI), which is an anti-digestive protein that inhibits herbivore gut protease (Green & Ryan 1972; Bode, Halitschke & Kessler 213). Root and leaf tissues were flash frozen in liquid nitrogen and stored at 8 C until chemical extraction and analysis. ANALYSIS OF PHENOLICS, DITERPENE ACIDS AND POLYACETYLENES WITH HPLC Approximately 2 mg of leaf or root tissue per sample was extracted in 1 ml 9% methanol using FastPrep â tissue homogenizer (MP Biomedicals â, Solon, OH, USA) at 6 m s 1 for 6 s with.9 g grinding beads (Biospec â, Zirconia/Silica 2.3 mm). Fifteen microlitres of the supernatant was analysed for secondary metabolites by highperformance liquid chromatography (HPLC) on an Agilent â 11 series HPLC equipped with a Gemini C18 reverse-phase column (3 lm, mm, Phenomenex, Torrance, CA, USA) using a standard method targeted at phenolic compounds (Keinanen, Oldham & Baldwin 21) with a slight modification to quantify phenolics, polyacetylenes and diterpene acids simultaneously. Our elution system consisted of solvents (A).25% H3PO4 in water (ph 2.2) and (B) acetonitrile was 5 min, 2% of B; 5 35 min, 2 95% of B and min, 95% of B, with the flow rate of.7 ml min 1. Peaks of phenolics (caffeic and coumaric acid derivatives, and flavonoids), polyacetylenes and diterpene acids were identified to compound classes using UV spectra information and quantified at 32 nm (caffeic and coumaric acid derivatives), 36 nm (flavonoids), 254 nm (polyacetylenes) and 21 nm (diterpene acids). For each of five compound classes, we generated a standard curve for a representative compound (i.e. caffeic acid, coumaric acid, rutin, pimaric acid and cis-dehydromatricariaester, respectively) and expressed sampled compounds as the equivalents of the respective standard (Tsao & Yang 23). N-benzoyl-dl-arginine-b-naphthylamide (BANA) in DMSO was added to each sample in the microplate and incubated for another 2 min at 37.5 C. The reaction was terminated by adding 1 ll of2%hcl in ethanol, and the absorbance at 54 nm was measured as a background reading with microplate reader. Dye reaction with 1 ll of.6 % p-dimethylaminocinnamaldehyde in ethanol lasted for 3 min at room temperature before the total absorbance was measured again at 54 nm. A standard curve using soya bean trypsin inhibitor standards was used to calculate SPI concentration of each sample. The SPI concentration was expressed as mg SPI per mg total protein. STATISTICAL ANALYSIS All statistical analyses were done separately for the natural and manipulative experiments using R (R version 2.8.1; R Foundation for Statistical Computing, Vienna). In the natural experiment, two localities within a native range (i.e. MN and NY) were nested within a continent (or plant origin ). Root and leaf secondary metabolites were first analysed using a MANOVA to test for the effect of plant origin on overall composition of compounds (Table 1). For root compound production, we first calculated genotype mean of each compound concentration and log transformed prior to a MANOVA analysis. For leaf tissues, genotype means for leaf secondary metabolites were calculated separately for undamaged and damaged leaves to examine constitutive and induced levels of compound production. Where overall MANOVA showed a significant origin effect, we conducted an univariate ANOVA of each compound (Figs 1 and 2). Logtransformed SPI production in leaf tissue (SPI/protein (mg/mg)) was also tested for plant origin separately for the undamaged and damaged leaves using an ANOVA. For each secondary metabolite and SPIs, per cent increase in compound production in protected populations (JP and H ) compared to that in exposed populations (native and H+) was calculated as: (c prot c exp )/c exp, where c prot is the mean concentration in protected populations, and c exp is that in exposed populations (Figs 1 and 2). Inducibility of leaf secondary metabolites in response to T. virgata damage was compared among plant origins. First, inducibility index of each compound was calculated as: c damage c control, where c damage was the concentration in damaged treatment, and c control was the concentration in undamaged control (Morris, Traw & Bergelson 26). SERINE PROTEASE INHIBITOR (SPI) ANALYSIS SPI analysis was conducted following methods by Bode, Halitschke & Kessler (213). Briefly, about 2 mg of fresh leaf samples was extracted in 1 ml of extraction buffer that contained 15 mm sodium chloride, 2. mm EDTA, 2 mg ml 1 phenylthiourea, 5 mg ml 1 diethyldithiocarbamate and 5 mg ml 1 polyvinylpolypyrrolidone (PVPP). Samples were homogenized using FastPrep â, centrifuged, and supernatant was transferred to a sample vial. Protein was quantified using the Bradford assay (Bradford 1976) with sample extracts diluted 1:1 in.1 M TRIS. Ten microlitres of each diluted sample was combined with 2 ll of Bradford reagent, incubated at 25 C for 1 min, and absorbance at 595 nm was measured using a Synergy HT multi-detection microplate reader (Bio-Tek, Winooski, VT, USA). A calibration curve was prepared with a dilution series of immunoglobulin standards. SPI activity was measured by combining 2 ll of reaction buffer (.1 M TRIS, ph 7.6), 1 ll of.25 mg ml 1 trypsin in.1 M TRIS, and 2 ll sample, and incubating at 37.5 C for 5 min. Then, 2 ll of 3.1 mg ml 1 Table 1. Results of MANOVA showing the effect of plant origin on root and leaf (undamaged control and damaged leaves) secondary metabolite composition analysed separately for the natural and manipulative experiments. Inducibility of secondary metabolites was calculated as (c damage c control ), where c damage was the concentration in damaged treatment, and c control was the concentration in the undamaged control. Numbers in bold indicate statistical significance Pillai s trace F (d.f. num, d.f. den ) P Natural experiment Root (19, 9).13 Undamaged leaf (15, 14) <.1 Damaged leaf (15,14) <.1 Leaf inducibility (15, 14).84 Manipulative experiment Root (19, 39).38 Undamaged leaf (15, 41).23 Damaged leaf (15, 41).79 Leaf inducibility (15, 39).96

5 88 A. Uesugi & A. Kessler 4 (a) Natural experiment % increase in root compound production (protected exposed)exposed (b) Phenolics Manipulative experiment Phenolics Polyacetylenes Polyacetylenes Diterpene acids Diterpene acids Fig. 1. Per cent increase in root secondary metabolites (phenolics, polyacetylenes, & diterpene acids) in (a) JP compared to native MN and NY genotypes in the natural experiment, and (b) H compared to H+ genotypes in manipulative experiment. Numbers near the x-axis indicate compound identity (see Table S2). Asterisks show significance in univariate analyses: <.5, <.1, <.1. 2 (a) Natural experiment % increase in leaf compound production (protected exposed)exposed Phenolics 2 (b) Manipulative experiment Phenolics Diterpene acids Diterpene acids SPI SPI Fig. 2. Per cent increase in leaf secondary metabolites (phenolics & diterpene acids) and SPIs in (a) JP compared to native MN and NY genotypes in the natural experiment, and (b) H compared to H+ genotypes in manipulative experiment. Grey bars represent constitutive difference in undamaged control, and black bars represent difference at damage-induced state. Asterisks show a significant effect of plant origin on compound concentrations: <.5, <.1, <.1. Crosses indicate significant plant origin effect on inducibility of compound production: <.5, <.1. Using the inducibility indexes, the overall inducibility of compound blends was tested for the plant origin effect using a MANOVA, followed by univariate ANOVA (Table 1). To test for potential trade-offs between inducibility and constitutive compound production, we further tested the genotypic correlations using a regression analysis for those compounds that showed differences in inducibility between plant origins. Competitive ability was examined by testing the number of new ramets against fixed effects of plant origin, competition treatment, damage treatment and a random factor of plant genotype in a generalized linear mixed model (GLMM) with Poisson error distribution with glmer function in lme4 package in R. Due to smaller mean number of ramets per treatment groups, we used a Laplace approximation to estimate parameters, and overdispersion was tested using overdisp_fun (Bolker et al. 29). Finally, a model selection was conducted using a likelihood ratio test following Bolker et al. (29). The resistance to T. virgata larvae was estimated for each plant by first calculating relative growth rate of larvae (RGR) as: [(final mass initial mass)/initial mass] and then converting to resistance index as: [1 (RGR/RGR max )] where RGR max is the maximum RGR observed in each experiment. The initial model for resistance was examined using a linear mixed model with lme function in nlme package of R and included plant origin, competition treatment and their interaction as fixed effect, and plant genotype as a random

6 Convergent evolution of plant invasiveness 881 factor. A model selection was conducted using a likelihood ratio test (Bolker et al. 29). Genotypic correlations of secondary metabolite production were tested using a linear regression analysis. First, genotype means of total phenolics, total diterpene acids and total polyacetylenes (only in roots) were calculated for root and leaf tissues separately. Correlation analyses were conducted between compound classes within a tissue type, and within a compound class between tissue types using corr.test function with multiple comparisons adjusted with the Holm Bonferroni method. To test whether root secondary metabolite production was associated plant competitive ability, we conducted a multiple regression analysis with GLMM with Poisson error distribution. Here, inter- and intraspecific competitive ability was estimated as ramet growth under P. pratensis and conspecific S. altissima competition, respectively. Total phenolics, diterpene acids and polyacetylenes were used as explanatory variables. Similarly, leaf secondary metabolites and SPIs were tested for their effects on plant resistance to T. virgata using a linear mixed model. inducibility of the four phenolic compounds (t < 1.43, P >.16), whereas that of the two diterpene acids showed positive correlations (t > 4.3, P <.2). Inducibility of SPIs did not differ by plant origin (F 1,23 = 1.54, P =.23). In the manipulative experiment, overall leaf secondary metabolite blends did not differ between H+ and H genotypes constitutively, but significantly varied with origin when leaves were damaged (Table 1). In the univariate analyses of damage-induced leaves, two phenolic compounds showed higher concentrations in H than H+ genotypes, while one diterpene acid showed a lower concentration in H than H+ genotypes (Fig. 2b). Induced level of SPI production tended to be lower in H compared to H+, but the difference was not significant (F 1,55 = 2.57, P =.11, Fig. 2b). Inducibility of overall secondary metabolites and SPIs did not vary with plant origin (Table 1). Results ROOT SECONDARY METABOLITES Overall root secondary metabolite composition significantly varied with plant origin in the natural experiment (MAN- OVA, Table 1). Univariate analyses of each compound showed that the invasive JP genotypes had a higher production of all five polyacetylenes, whereas they produced lower amounts of all three diterpene acids compared to those observed in native genotypes (Fig. 1a). Similarly, in the manipulative experiment, the overall metabolite composition significantly varied with plant origin (Table 1). Univariate analyses revealed that H genotypes had higher production of four of the five polyacetylenes, as previously reported in Uesugi & Kessler (213), and lower production of two diterpene acids, compared to H+ genotypes (Fig. 1b). LEAF SECONDARY METABOLITES AND SPIS Overall leaf secondary metabolite production varied with plant origin in the natural experiment at both constitutive (undamaged) and induced (damaged) levels (Table 1). In the univariate analyses of constitutive compound production, we found that the concentrations of two phenolic compounds were higher in JP compared to native genotypes, whereas one phenolic and three diterpene acid were lower in JP compared to the native populations (Fig. 2a). The difference between plant origins became more prominent in the damage-induced state, where the production of five phenolic compounds increased, and that of one phenolic, three diterpene acids and SPIs decreased in JP compared to native genotypes (Fig. 2a). Ten out of 15 compounds we examined were inducible by T. virgata damage, and overall inducibility of compounds differed among plant origins with MANOVA (Table 1). Univariate analyses of inducibility revealed that, compared to native genotypes, JP genotypes exhibited greater induction of four phenolic compounds, but lower induction of two diterpene acids. No correlation was found between constitutive production and COMPETITIVE ABILITY The ramet number in the natural experiment was Poisson distributed with no sign of overdispersion (v 2 = 134., d.f. = 168, P =.97). A GLMM model in the absence of interactions with herbivory provided the best fit (AIC = 613.3, with DAIC < 1.4 when the interaction terms were removed). Interestingly, plant origin interacted with interspecific competition with P. pratensis (GLMM: z = 2.87, P =.41), but not with intraspecific competition with the other S. altissima (z =.89, P =.37). Competition from P. pratensis decreased the rate of ramet production in S. altissima plants from MN and NY, whereas it did not affect ramet production in JP genotypes (Fig. 3), suggesting the evolution of Competition Control With Poa With Solidago Number of ramets H H+ JP MN NY Fig. 3. Vegetative reproduction (measured as number of ramets produced) under three competition treatments: non-competitive control (white bars), interspecific competition with Poa pratensis (grey bars) and intraspecific competition with Solidago altissima (black bars). The main panel shows the results of the natural experiment, and the subpanel on upper right shows the result from the manipulative experiment reported in Uesugi & Kessler (213) for a comparison. Interactions between plant origin and competition with P. pratensis in both the natural (P =.41) and manipulative (P =.36, Uesugi & Kessler 213) experiments suggest evolution of increased interspecific competitive ability. No origin x intraspecific competition interactions were found. Error bars represent standard errors.

7 882 A. Uesugi & A. Kessler increased competitiveness against P. pratensis in JP plants. Corresponding results for the manipulative experiment showing parallel patterns of competitiveness evolution are reported in Uesugi & Kessler (213), and shown in a subpanel of Fig. 3 for comparison. RESISTANCE TO TRIRHABDA VIRGATA Resistance to T. virgata in both natural and manipulative experiments was best fit by a linear mixed model without plant competition; thus, the final model included only plant origin as a fixed effect. Plant resistance to T. virgata in JP plants was lower than MN plants (t = 2.63, P =.14), but did not differ from NY plants (t = 1.13, P =.26: Fig. 4a). In the manipulative experiment, there was no difference in resistance to T. virgata between H and H+ genotypes (t =.29, P =.78, Fig. 4b). GENOTYPIC CORRELATIONS AMONG TRAITS In the natural experiment, total root polyacetylenes negatively correlated with root and leaf diterpene acids (root: coef =.75, P adj <.1; leaf: coef =.71, P adj <.1, see Fig. S1). Incidentally, root and leaf diterpenes were positively correlated (coef =.72, P adj <.1). In the manipulative experiment, leaf phenolics positively correlated with leaf diterpenes (coef =.58, P adj <.1, Fig. S1). 1.5 (a) Natural experiment NS None of the root secondary metabolite classes explained the variation in interspecific or intraspecific competitive ability in the natural experiment, while in the manipulative experiment, total polyacetylenes was positively correlated with interspecific competitiveness (coef =.47, z = 2.54, P =.11). Plant resistance to T. virgata was marginally positively correlated with SPIs in the natural experiment (coef = 2.23, t = 2.8, P =.5), but negatively in the manipulative experiments (coef = 2.18, t = 1.89, P =.65). Finally, competitiveness against P. pratensis did not correlate with plant resistance against T. virgata in either the natural (t = 1.7, P =.1) or manipulative experiments (t = 1.6, P =.11). Intraspecific competitiveness and resistance did not correlate in the natural experiment (t =.9, P =.39), while they were positively correlated in the manipulative experiment (t = 2.3, R 2 adj =.72, P =.25). Discussion Herbivory can drive rapid evolution of plant chemical traits that mediate defence and competitive interactions (Zangerl, Stanley & Berenbaum 28; Agrawal et al. 212; Z ust et al. 212; Uesugi & Kessler 213). Because geographic variation in herbivore communities can generate a selection mosaic among populations (the Geographic Mosaic Theory of Coevolution; Thompson 25), we would expect plant traits under herbivore selection to also diverge in space. If, in fact, herbivory drives plant adaptation at a geographic scale (Thompson 25; Zangerl, Stanley & Berenbaum 28; Z ust et al. 212), we would expect convergent evolution between the manipulative and the natural experiments. Here, we compared representative invasive and native populations of S. altissima and tested the hypothesis that escape from herbivory in invasive ranges would result in the evolution of plant chemical traits as predicted in the manipulative experiment. Resistance (1 RGR/RGR max ) 1.5 JP MN NY (b) Manipulative experiment NS H H+ Fig. 4. Plant resistance to Trirhabda virgata larvae calculated as [1 (RGR/RGR max )] in the (a) natural and (b) manipulative experiments. Error bars represent standard errors. Asterisks show significance: <.5. EVOLUTION OF ALLELOPATHY AND COMPETITIVENESS In S. altissima, we previously showed that evolution of increased competitiveness against P. pratensis coincided with evolutionary increase in the production of polyacetylenes in roots (Uesugi & Kessler 213). Our current analysis also demonstrated that total polyacetylenes positively influenced plant s interspecific competitiveness in the manipulative experiments. Moreover, one of the major polyacetylenes, cisdehydromatericaria ester (DME), inhibited the growth of P. pratensis but not S. altissima, suggesting its function in interspecific, but not in intraspecific, competitive ability (Kobayashi et al. 198; Johnson, Halitschke & Kessler 21; Uesugi & Kessler 213). In this study, we found remarkably congruent shifts in polyacetylene production between the natural and manipulative experiments, where all polyacetylene compounds were produced at higher levels in herbivore-protected (i.e. JP and H ) than exposed (i.e. native and H+) genotypes. This observed convergence suggests that evolution of allelopathy

8 Convergent evolution of plant invasiveness 883 was primarily driven by herbivore release. Parallel to our previous results showing increased interspecific competitive ability in H plants (Uesugi & Kessler 213), we also found that JP plants better competed against P. pratensis than native Minnesota (MN) and New York (NY) genotypes, while they competed equally against conspecifics. Because grass species such as P. pratensis, which are particularly vulnerable to Solidago allelochemicals (Yang et al. 27), are common competitors of S. altissima in Japan (Fukuda 1982), the observed trait shifts further suggest that evolution of polyacetylenes mediates the evolution of invasiveness in Japanese S. altissima populations. While the direction of the shift in polyacetylene production was surprisingly consistent between the natural and manipulative experiments, the magnitude of increase was much greater in the natural experiment. The difference in magnitude could be a result of their different evolutionary time-scales: ~1 years in the natural experiment, relative to 12 years in the manipulative experiment. Alternatively, selection for allelopathy may be stronger in introduced ranges where invasive plants encounter competitors with no prior exposure or evolved resistance to the exotic compounds, as suggested by the novel weapons hypothesis (Callaway & Aschehoug 2; Qin et al. 213; Gruntman, Zieger & Tielb orger 215; Zheng et al. 215). Evolutionary increase in polyacetylene production coincided with reduced production of root diterpene acids. While the exact functions of diterpene acids in S. altissima roots is unknown, studies from other systems indicate that diterpenes could be anti-feedants for root herbivores and bacterial pathogens (Seo et al. 212; Vaughan et al. 213). The observed chemical shifts may therefore be driven by relaxed selection from below-ground herbivores in invasive plant populations, although the effect of the fenvalerate insecticide used in the manipulative experiments on below-ground communities is thought to be minimal (Root 1996; pers. comm. R. Johnson). Alternatively, the shifts in root diterpene acids could be driven by selection on genetically correlated secondary metabolites (see Fig. S1). A negative trade-off between diterpene acids and polyacetylenes within roots suggests that strong selection for increased allelopathy could inadvertently reduce diterpene acids in roots. Because total diterpene acid concentrations in leaf and root tissues are positively correlated, relaxed above-ground herbivory may also indirectly result in a reduction of root diterpene acid production. EVOLUTION OF PLANT DEFENCE-RELATED COMPOUNDS A relaxation of above-ground herbivore pressure should select for reduced production of defence-related compounds if their cost of production is high (Coley, Bryant & Chapin 1985; Herms & Mattson 1992). Leaf secondary metabolites and serine proteinase inhibitors (SPIs) showed convergent evolutionary shifts between natural and manipulative experiments, suggesting that herbivory could drive evolution of plant defence chemistry. In general, S. altissima genotypes from protected populations showed higher production of several phenolic compounds, while expressing lower production of diterpene acids and SPIs than plants from herbivory-exposed populations. Similar patterns of secondary metabolite evolution have been implied in other geographic comparisons, where phenolic compounds increased in invasive populations (Triadica sebifera: Wang et al. 212; Solidago canadensis: Yuan et al. 212) and mono- and diterpenes decreased in invasive populations (Solidago gigantea: Johnson, Hull-Sanders & Meyer 27) compared to native populations. These studies, along with ours, suggest that release from herbivory could lead to predictable shifts in the production of certain functional classes of defence-related compounds. Furthermore, the pattern could indicate greater metabolic costs of constitutive production of diterpenes and SPIs than phenolics (Zavala et al. 24; Sampedro, Moreira & Zas 211), or a selective advantage of increased phenolic production in invasive populations due to altered abiotic conditions (Lavola 1998). While we observed clear shifts in leaf chemistry between protected and exposed genotypes, we currently have weak evidence for their biotic consequences. In the natural experiment, resistance to T. virgata was lower in JP compared to MN plants, but did not differ from NY plants. In the manipulative experiments, we found no difference in plant resistance to T. virgata between H and H+ genotypes, while Bode & Kessler (212) found a significant reduction in resistance in H compared to H+ genotypes using the same plant populations. This inconsistency could result from variation in experimental conditions, such as the specific source of test insects, which could differ in their tolerance to plant resistance (Uesugi and Kessler pers. obs), or it may simply indicate a relatively small effect of herbivore exclusion on the evolution of resistance to T. virgata. Because native S. altissima populations face diverse array of herbivores, and different blends of secondary metabolites are known to affect herbivore species differentially (Hull-Sanders et al. 27; Uesugi, Poelman & Kessler 213), plant resistance to T. virgata alone may not necessarily capture the potential evolution of plant resistance in this system. In fact, the resistance to T. virgata was only marginally correlated with SPI levels, whose effects were inconsistent across experiments. Additional contrasts using multiple herbivore species are necessary to determine how the observed evolutionary shifts in leaf chemistry influence plant resistance to the herbivore community as a whole. In extension of the EICA hypothesis, plant defence theory predicts that introduced plant populations should evolve increased inducibility when costs of constitutive defence are high and frequencies of herbivory are low (Karban & Baldwin 1997; Beaton et al. 211). We found that, compared to native genotypes, JP genotypes had increased inducibility of several phenolic compounds, but decreased inducibility of diterpene acids. We found no trade-offs between inducibility and constitutive production of these compounds in our study, suggesting that observed differences in inducibility cannot be attributed to the cost-saving mechanism hypothesized above.

9 884 A. Uesugi & A. Kessler Moreover, we found no evidence of inducibility evolution in the manipulative experiment, suggesting that differences in inducibility we observed between JP and native populations are not likely to be the result of adaptation to herbivore release. The lack of divergence in inducibility of secondary metabolites between invasive and native populations has been observed elsewhere (Cipollini et al. 25; Eigenbrode et al. 28). These patterns are consistent with the hypothesis that due to infrequent herbivory in invasive populations, plants rarely express induced resistance, which then reduces opportunities for selection to act against induction of costly defences (Eigenbrode et al. 28; Lahti et al. 29). Alternatively, maintaining inducibility is advantageous in both native and invasive populations, perhaps for different ecological reasons, if inducibility has multiple functions (reviewed in Kessler 215). Conclusion The two approaches used in this study not only demonstrated rapid evolution of plant traits on an ecological time-scale, but also showed convergent evolutionary patterns, suggesting that release from herbivory is a major driver of plant adaptation in invasive ranges. Alternative to the evolutionary scenario, the observed trait differences between Japanese and North American populations could potentially result from a founder event. However, the presence of molecular and quantitative genetic diversity within Japanese S. altissima populations (Sakata et al. 214; Y. Ando, unpublished data) suggests that the Japanese population has accumulated ample levels of genetic variation for a rapid adaptation to take place (Sakata et al. 214). Although the number of sampled regions was limited here, we also found that the JP population showed consistently divergent patterns in multiple traits from both MN and NY populations, which were sampled from geographically diverged populations in the native range. Future studies will contrast plant phenotypes across multiple populations of S. altissima in multiple independent invasion events to assess the predictability of invasive phenotype evolution at a global scale. A combination of natural and manipulative experiments used here provides a powerful tool to directly link geographic patterns of trait variations to evolutionary mechanisms and drivers of natural selection, yet studies employing such an approach are rare (Z ust et al. 212). Z ust et al. (212) showed in laboratory artificial selection experiments that Arabidopsis thaliana populations facing differential selection from two aphid species diverged in defence chemistry associated with aphid resistance, a pattern matching the geographic variations in the aphid abundance and plant chemotype frequency. While they found that pairwise evolution with a single dominant herbivore species can drive a geographic pattern of plant defence traits, plant-herbivore interactions are often more complex. In our manipulative experiment, we removed a suite of herbivore species with insecticide treatments, which allowed us to examine selection imposed by an entire aboveground herbivore community. A major assumption of the EICA hypothesis is that evolution of increased competitiveness is an indirect consequence of selection against the maintenance of costly defence, which genetically trade-off with plant competitiveness (Blossey & N otzold 1995). Surprisingly, we did not find such negative correlation between plant resistance to T. virgata and plant competitive ability. However, relatively weaker shifts in plant resistance traits compared to competitive traits suggest that the above assumption of trade-off is not required for competitiveness to evolve in response to escape from herbivory. Rather, a relaxation of herbivory could modify ecological interactions (Carson & Root 2; Lahti et al. 29; Agrawal et al. 212), such that major selection pressures on plant traits might shift in magnitude from herbivore- to competitiondominated, and this shift in their relative strengths of selection may underlie the predominant evolutionary shift in competitiveness. Acknowledgements We thank Yoshino Ando and Tim Craig for providing us with S. altissima plants for the natural experiment, Anurag Agrawal and Tim Connallon for helpful discussions and suggestions on earlier drafts of this study, and Rayko Halitschke and Robert Bode for help with chemical analyses. The study was supported with funds from the National Science Foundation (USA, NSF-IOS 95225) and Cornell University. Data accessibility Data associated with this paper are deposited in the Dryad repository (doi: 1.561/dryad.s492 m) (Uesugi & Kessler 216). References Agrawal, A.A., Hastings, A.P., Johnson, M.T.J., Maron, J.L. & Salminen, J.P. (212) Insect herbivores drive real-time ecological and evolutionary change in plant populations. Science, 338, Ando, Y., Utsumi, S. & Ohgushi, T. (21) Community structure of insect herbivores on introduced and native Solidago plants in Japan. Entomologia Experimentalis et Applicata, 136, Beaton, L.L., Van Zandt, P.A., Esselman, E.J. & Knight, T.M. (211) Comparison of the herbivore defense and competitive ability of ancestral and modern genotypes of an invasive plant, Lespedeza cuneata. Oikos, 12, Blossey, B. & N otzold, R. (1995) Evolution of increased competitive ability in invasive nonindiginous plants- a hypothesis. Journal of Ecology, 83, Bode, R.F., Halitschke, R. & Kessler, A. (213) Herbivore damage-induced production and specific anti-digestive function of serine and cysteine protease inhibitors in tall goldenrod, Solidago altissima L. (Asteraceae). Planta, 237, Bode, R.F. & Kessler, A. (212) Herbivore pressure on goldenrod (Solidago altissima L., Asteraceae): its effects on herbivore resistance and vegetative reproduction. Journal of Ecology, 1, Bolker, B.M., Brooks, M.E., Clark, C.J., Geange, S.W., Poulsen, J.R., Stevens, M.H.H. & White, J.S.S. (29) Generalized linear mixed models: a practical guide for ecology and evolution. Trends in Ecology & Evolution, 24, Bossdorf, O., Auge, H., Lafuma, L., Rogers, W., Siemann, E. & Prati, D. (25) Phenotypic and genetic differentiation between native and introduced plant populations. Oecologia, 144, Bradford, M.M. (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Analytical Biochemistry, 72, Cain, M.L. (199) Patterns of Solidago altissima ramet growth and mortality - the role of below-ground ramet connections. Oecologia, 82,