Comparison of foliar terpenes between native and invasive Solidago gigantea

Biochemical Systematics and Ecology 35 (2007) 821e830 www.elsevier.com/locate/biochemsyseco

Comparison of foliar terpenes between native and invasive Solidago gigantea Robert H. Johnson a,*, Helen M. Hull-Sanders b, Gretchen A. Meyer c a

Medaille College, Mathematics and Science, 18 Agassiz Circle, Buffalo, NY 14214, USA b Canisius College, 2001 Main Street, Buffalo, NY 14208, USA c University of Wisconsin e Milwaukee Field Station, 3095 Blue Goose Road, Saukville, WI 53080, USA Received 27 December 2006; accepted 16 June 2007

Abstract To test a defensive chemistry prediction of the Evolution of Increased Competitive Ability (EICA) hypothesis, Solidago gigantea plants from North American and European (invasive) populations were grown in a screen-enclosed garden. Terpenes from 80 seed grown (dried leaves) and 320 rhizome propagated (moist leaves) individuals were confirmed by GC/MS and quantified by GCFID. Native seed grown plants were found to have significantly greater diterpene concentrations than their European counterparts; foliar sesquiterpenes did not differ. The occurrence of specific sesquiterpenes and diterpenes was homogeneous across the two seed sources suggesting these biochemical pathways remain unchanged. Leaves from native rhizome propagated plants also had significantly greater monoterpene and diterpene concentrations; again sesquiterpene levels did not differ. Rhizome propagated plants exhibited significant population differences in monoterpene and diterpene concentrations. These data support the defensive chemistry predictions of the EICA hypotheses but cannot discount the role of possible founder effects in the invasive range. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: EICA; Invasive species; Introduced species; Monoterpenes; Sesquiterpenes; Diterpenes; Chemical defenses; Solidago gigantea

1. Introduction The Evolution of Increased Competitive Ability (EICA) hypothesis advanced by Blossey and No¨tzold (1995) predicts that introduced plant species, which have escaped their natural specialist herbivores, should evolve to decrease their investment in anti-herbivore chemical defenses. Resources no longer needed for defense could be reallocated to characteristics that provide a selective advantage in the novel habitat. The newly evolved invasive phenotypes should thus exhibit increased growth and fecundity relative to native conspecifics, but lower resistance to herbivores, particularly specialists from their native range (Blossey and No¨tzold, 1995; Wolfe, 2002; Maron et al., 2004). The EICA hypothesis was formulated to provide a mechanism to explain invasive plant vigor and success in general and in particular the observation that invasive purple loosestrife (Lythrum salicaria L.) produced greater plant biomass and hosted larger specialist herbivore larvae than conspecifics in the native range. Blossey and No¨tzold (1995) did not * Corresponding author. Tel.: þ1 716 880 2238; fax: þ1 716 884 0291. E-mail address: [email protected] (R.H. Johnson). 0305-1978/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2007.06.005

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measure plant’s secondary chemistry but did assume that differences in insect growth were a result of altered patterns of plant defenses. Recent modifications to the EICA hypothesis propose that invasive genotypes may still experience moderate attack by local generalist herbivores (Muller-Scharer et al., 2004). In some cases selection may favor a reduction in quantitative chemical defenses (effective against specialists but metabolically expensive) and an increase in the concentrations of less costly qualitative defenses that may be more toxic to generalists (Joshi and Vrieling, 2005; Stastny et al., 2005). The Solidago system offers multiple advantages for testing the biochemical predictions made by the EICA hypothesis. First, the genus Solidago is indigenous to North America, although a single species, Solidago virgaurea, is native to Europe (Gleason and Cronquist, 1991; Voss, 1996). Two species (Solidago altissima and Solidago gigantea) were introduced to Europe in the mid 18th century, quickly naturalized, and began to spread during the mid 19th century (Weber, 1998). Both species are now considered among the most aggressive plant invaders in Europe (Weber and Schmid, 1998). Second, native goldenrod hosts a number of specialist and generalist insect herbivores known to strongly impact populations (Root and Cappuccino, 1992; Abrahamson and Weis, 1997; Meyer, 2000), whereas few insects have been observed feeding on the plant in Europe (Jobin et al., 1996). Third, a comparison of S. gigantea populations growing in the US and Europe showed that stem density is about twice as high in Europe as in the US based on sampling from three widely separated areas within each continent (Jakobs et al., 2004). Finally, biochemical studies indicate that the genus Solidago exhibits a diverse terpenoid profile, the accumulation of which can impose substantial metabolic costs (Gershenzon, 1994; Zangerl and Bazzaz, 1992). For example, S. gigantea foliar tissue was found to contain 95 different mono and sesquiterpenes (Kalemba et al., 2001). S. gigantea rhizomes were found to contain solidagoic acids A and B (Anthonsen et al., 1973), and eight furan-containing cis-clerodane diterpenes (Henderson et al., 1973). S. virgaurea (European goldenrod) foliar tissue has also been found to contain a number of cis-clerodane lactones (Goswami et al., 1984), but no information on the concentrations of S. virgaurea foliar cis-clerodanes was found. Solidago diterpenes are known to influence feeding preference of Trirhabda canadensis, a chrysomelid specialist on goldenrod (Le Quesne et al., 1986) as well as a broad range of generalist insect herbivores (Cooper-Driver and Le Quesne, 1987; Maddox and Root, 1987, 1990). When fed fresh S. gigantea leaves, growth of Spodoptera exigua larvae was found to decline with increasing host leaf cis-clerodane diterpene concentrations; however, growth of Trirhabda virgata larvae (another chrysomelid specialist on goldenrod) was not affected (Hull-Sanders et al., 2007). This same study found that neither S. exigua nor T. virgata was affected by sesquiterpene levels. Meyer et al. (2005) found that European seed grown S. gigantea plants hosted greater insect biomasses than seed grown natives and that both seed and rhizome propagated European invasives were more susceptible to rust fungus and Xanthomonas leaf blight infections than native conspecifics. In the study reported here, we looked for a biochemical mechanism for the differential susceptibility to insects and pathogens reported by Meyer et al. (2005) between seed and rhizome propagated native and invasive goldenrods. Specifically, we tested the prediction that invasive goldenrods, in the absence of damage, should exhibit reduced levels of foliar terpenoids relative to native conspecifics. The reduction could come about in several ways: (1) by the loss of specific constitutive pathways that would be reflected in differential compound occurrence, (2) by a simple reduction in the leaf terpene concentration among the invasive individuals, or by some combination of (1) and (2).

2. Materials and methods 2.1. Common garden conditions All plants were grown in a 12  12 m aluminum window screen enclosure to protect them from insect herbivores located at the University of Wisconsin e Milwaukee Field Station, Saukville, WI, where they were randomly arranged and watered as needed. Within the screenhouse, plants were cultivated in pots in a commercial potting mix (Farfard Grow Mix #2, Conrad Farfard Inc., Agawam, Massachusetts). To further protect against pathogen or insect damage, plants were sprayed with a mixture of fungicide (mancozeb) and insecticide (carbaryl) at 1e3 week intervals as needed. Light levels in the screenhouse were approximately 70% of full sun, and height, phenology and appearance of goldenrod in the screenhouse were similar to plants grown outside (Meyer, personal observation).

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2.1.1. Seed grown plants Seeds from 22 invasive European and 10 native USA S. gigantea populations were collected in 1999. Within each population, infructescences of individual ramets were bagged separately. Ramets from which seeds were collected were well separated in space to ensure that they came from separate clones. Some seeds were germinated in 2000 for the experiment reported in Meyer et al. (2005); all remaining seeds were stored under cool, dry conditions. In early April 2003, the stored seeds were sown into separate cups containing a commercial potting mix, with each cup containing the seeds from one maternal parent. There were 110 European maternal parents (5 per population) and 92 US maternal parents (2e14 per population) randomly arranged within the greenhouse at the Field Station. Germination in the cups was scored in late April and again in June. The presence of at least one seedling in a cup was counted as successful germination. Germination of European seeds was much better than that of US seeds (see Section 3), resulting in many more European seedlings than US seedlings. In early July, one seedling per maternal parent was transplanted to a 6.6 L pot and moved to the screen enclosure maintained at the Field Station. All US cups with seedlings were used (n ¼ 32, representing eight populations), but there were many more European seedlings than US, thus seedlings from only 53 of the European maternal parents were transplanted. European seedlings were chosen so that 19 populations were represented, with the number of replicates within populations as balanced as possible. Because of the need to allow these plants to develop sufficiently large rhizomes for future propagation, leaf harvesting was limited to five leaves per plant. This restriction precluded our obtaining additional matched control leaves (for wetedry mass conversion), and necessitated dry leaf analysis. On September 10, five fully expanded leaves from the upper third to upper half of each plant were harvested and immediately placed into a circulating air oven (60  C) at the Field Station and dried for 48 h. Dried leaves were subsequently express shipped to Buffalo, NY; upon arrival, leaves were maintained in sealed silica-gel desiccators until processed. Leaves of each plant were weighed just prior to chemical analysis to obtain mean leaf mass. Population locations and final sample sizes for chemical analyses are shown in Table 1. 2.1.2. Rhizome propagated plants Seeds from the same seed collections as those used for the seed grown plant experiment were germinated in spring 2000 and grown in field plots at the Field Station for the experiment reported in Meyer et al. (2005). This experiment included 20 European populations and 10 US populations. At the conclusion of this experiment in October 2001, rhizomes of all plants were harvested from the common garden and over-wintered in a refrigerator in closed plastic bags. Thereafter, plants were propagated each year by planting rhizomes each spring in pots and harvesting new rhizomes from each plant at the end of the growing season for storage over-winter in the refrigerator. These methods allowed the same genotypes to be propagated over time for use in multiple experiments. In May 2004, the rhizomes were planted, grown and used for leaf chemistry measures in the experiments described in Hull-Sanders et al. (2007). This experiment included two clones of each genotype: one was left undamaged and the other was damaged by Trirhabda beetles. At the end of the 2004 growing season, the rhizomes were again harvested and stored under refrigeration. In May 2005, two rhizomes from each of 80 US and 80 EU genotypes were cut to 7e15 cm lengths and weighed to ensure homogeneous planting masses. One rhizome of each pair was undamaged in the previous experiment and the other was previously damaged. Rhizomes were planted in individual 4 L pots and grown under the screenhouse common garden conditions as described above. In August 2005, 10 fully expanded leaves from the upper third of each stem were harvested and placed in sealed plastic bags in a cooler. Five leaves were blindly selected from each bag, weighed, dried in a forced air oven and reweighed. These control leaves were used to determine wetedry mass conversions so that terpenoid concentrations determined from wet leaves could be standardized to dry leaf mass. The remaining bagged fresh leaves were express shipped to Buffalo, NY in an insulated container with commercial ice-packs. Upon arrival, leaves were immediately placed in a 80  C freezer and stored frozen until processing. Thawed leaves were weighed just prior to chemical analysis to obtain moist leaf mass (see Table 1). 2.2. Chemical analysis Five dried (seed grown) or freshly frozen (rhizome propagated) moist leaves from each experimental plant were placed into 15 ml of 70% hexanee30% ethyl acetate to which 0.26 mg of nonadecane (internal standard) was added. Leaves were then disrupted using a Polytron tissue homogenizer; the resulting slurry was centrifuged (10 min at 3000 rpm) and the supernatant decanted into 20 ml glass scintillation vials. Vial contents were concentrated under a fume hood to a final volume of approximately 1.5 ml and analyzed using a Varian 3900 GC with a flame ionization

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Table 1 Original seed collection localities, population code (used in Fig. 2) coefficients of variation (% diterpene concentration variability) and population sample sizes for seed grown (dry leaf analysis) and rhizome propagated (moist leaf analysis) Site

Pop code

Lat

European populations Austria (Imst) France (Rosenau) France (Kembs) France (Strasbourg) France (Erstein) France (L’Isle sur le Doubs) Germany (Rosdorf) Germany (Celle) Germany (Do¨rnten) Germany (Neumunster) Germany (Karlsruhe) Hungary (Devecser) Hungary (Mosonmagyarovar) Italy (Albate) Italy (Fino-Mornascio) Switzerland (Tenero) Switzerland (Cadepezzo) Switzerland (Arlesheim) Switzerland (Sihlbrugg) Switzerland (Cadenazzo)

EU22 EU1 EU4 EU5 EU6 EU8 EU10 EU13 EU14 EU16 EU17 EU11 EU12 EU18 EU19 EU27 EU2 EU3 EU7 EU20

47.18 47.65 47.68 48.58 48.43 47.45 53.94 52.37 51.40 47.95 49.01 47.10 47.86 45.40 45.76 46.18 46.12 47.51 47.21 46.13

N N N N N N N N N N N N N N N N N N N N

10.77 7.53 7.50 7.77 7.69 6.58 9.89 10.05 6.58 11.78 8.39 17.51 17.21 9.16 9.07 8.95 8.94 7.66 8.53 8.96

US populations Iowa (Zimmerman Field) Iowa (Cone Marsh) Michigan (Ann Arbor) New Hampshire (N. Haverhill) New Hampshire (Claremont) New York (Turkey Hill) New York (Trumansberg) North Dakota (Oakville Prairie) Vermont (Green Mtn Audubon) Wisconsin (Saukville)

US1 US2 US8 US4 US5 US9 US10 US6 US3 US7

41.66 41.49 42.28 44.07 43.35 42.45 42.53 47.89 44.36 43.22

N N N N N N N N N N

91.54 91.43 83.73 72.03 72.36 76.50 76.67 97.30 73.01 88.01

CV (%) seed

N

CV (%) rhizome

E E E E E E E E E E E E E E E E E E E E

64.7 64.2 55.5 45.9 34.3 18.6 66.2 49.8 68.7 e 22.3 58.6 72.3 80.3 90.0 62.9 69.9 10.5 41.5 e

3 3 3 2 2 3 3 2 2 e 3 3 3 3 2 3 3 3 3 (1)

130.8 45.2 47.8 53.3 22.3 39.6 55.4 31.3 52.7 26.7 55.5 55.5 57.3 82.2 49.7 38.8 76.2 53.8 52.9 45.0

7 6 8 8 7 5 6 7 8 6 8 9 8 8 7 7 8 6 8 5

W W W W W W W W W W

e 58.5 e 89.4 61.8 e e 58.1 59.3 70.1

(1) 3 (1) 3 6 e e 4 5 7

55.6 59.3 37.6 43.8 48.3 65.9 46.3 44.2 60.8 74.5

18 18 15 14 16 8 4 19 19 18

Long

N

Populations represented by a single individual (n ¼ 1) were dropped from analysis; localities given in decimal degrees latitude and longitude.

detector (FID). Compounds were separated using a RTx-5ms column (30 m  0.25 mm id); injector temperature was maintained at 250  C and the FID at 300  C. Dry leaf chemical analysis used an initial oven temperature of 80  C (3 min) increasing to 280  C at 5  C/min. Moist leaf analysis used an initial oven temperature of 30  C (3 min) increasing to 280  C at 5  C/min. Monoterpenes were not integrated for the seed grown plants as the leaf drying process could volatilize these lower molecular weight terpenes. Terpene FID peaks were grouped by retention times and integrated separately to differentiate monoterpene, sesquiterpene and diterpene concentrations. Prior to quantitative GC analysis, leaf samples from both the seed grown (dried) and rhizome propagated (freshly frozen) plants from US and European populations were extracted and concentrated separately as described above. Identities of the sample peaks were confirmed with an Agilent 6850 GCe5973 MSD using a similar RTx-5ms column and the same thermal elution program used for the quantitative runs. The resulting mass spectral ion patterns for each peak were compared to the NIST2000 software database and literature spectra (Anthonsen et al., 1973; Henderson et al., 1973) for compound confirmations. 2.3. Statistical analysis Data were analyzed using SPSS version 13.0 for Windows. All data were tested for normality and equality of variance (Levene’s test); all terpene concentrations were log transformed to meet test assumptions. The effects of continent of origin on monoterpene (moist leaves only), sesquiterpene and diterpene concentrations were analyzed

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using nested GLM-univariate analysis. Continental origin was treated as a fixed effect and population of origin was included in the model as a nested, random effect. In experiment 1 (seed grown plants) one EU and two US populations were dropped from the GLM nested analysis because each was represented by a single individual and offered no intrapopulation replication. To look for possible carry-over effects of leaf loss from the previous year (2004; reported in Hull-Sanders et al., 2007), previously damaged plants were compared with previously undamaged plants using a one-way ANOVA. No differences were found in monoterpene, sesquiterpene or diterpene concentrations between the two treatments (all p values  .5), thus carry-over effects of leaf loss were discounted. Independent chi-square tests of association were conducted to determine if associations existed between seed source and the presence of three sesquiterpene hydrocarbons, an oxygenated sesquiterpene, three diterpenes and a linear hydrocarbon (octadecane) that were selected to represent the diverse chemical groups spanning the GC thermal elution gradient found in these plants. A chi-square test of association was also conducted between seed source and germination frequency. Correlation analysis was conducted to test for a relationship between sesquiterpene and diterpene concentrations of seed grown plants. 3. Results 3.1. Seed grown plants Ten sesquiterpene hydrocarbons (Mþ 204) and one sesquiterpene ketone (Mþ 218) were confirmed from both the US and European populations based on relative peak retention times, parent ion mass and mass/abundance ratios of major ion fragments. Of the 11 confirmed sesquiterpenes, spectral comparisons of individual peaks with the software database allowed the tentative identification of six specific compounds (Table 2). Eight diterpenes were confirmed based on relative retention times, parent ion mass and mass/abundance ratios of major ions and spectral comparisons with software and literature. Of these, two were confirmed as kaurenes, four as cis-clerodanes and two as diterpene acids. The presence of ions 67, 81 and 95 m/z indicated the attachment of a furan ring which provides additional confirmation that the oxygenated diterpenes are furan-containing dicyclic clerodanes and acids (Table 2). (þ)-epiBicyclosesquiphellandrene was the dominant sesquiterpene peak exhibited by all individuals from both seed sources and with the exception of three US individuals, and was the dominant terpenoid overall. Visual comparison of quantitative chromatograms suggested that the occurrence of major sesquiterpene and diterpene peaks was similar between US and European plants. Statistical testing of this observation likewise found no differences in the frequencies of the eight selected compounds that spanned the GC thermal gradient (see Table 2 for selected compounds) between European and US plants (significance values for all eight c2 tests: p  .585). Mean dry leaf diterpene concentration from plants of US seed origin was significantly greater than the mean concentration from their European counterparts (Fig. 1A; F ¼ 6.054, df ¼ 1, 17; p ¼ .025). Population diterpene means were not seen to differ (F ¼ 1.089, df ¼ 22, 53; p ¼ .387, nested ANOVA); however, individual diterpene concentrations were generally a variable characteristic within populations of either seed source resulting in large standard deviations (Fig. 2A) and coefficients of variation (Table 1). In addition, plants of US seed origin exhibited greater overall variance in diterpene concentration than did EU plants (Levene’s test: s2 ratio ¼ 2.59, F ¼ 7.34; p ¼ .008). In contrast Table 2 Solidago foliar terpenes identified by GC/MS Monoterpenes

Sesqiterpenes

Diterpenes

a-Pinene Terpenine Camphene Sabinene Myrcene Phellandrene Limonene Ocimene

Cycloprop[e]azulene Germacrene-D (two isomers)a (þ)-epi-Bicyclosesquiphellandrenea Germacrene-B Sesquiterpene hydrocarbon (Mþ m/e 204)a Isolongifolen-5-onea

Kaur-16-ene (Mþ m/e 272)a Kaurene isomer (Mþ m/e 272) Methyl cis-clerodane (Mþ m/e 298) cis-Clerodane aldehyde (Mþ m/e 300) cis-Clerodane g-lactone (Mþ m/e 314)a cis-Clerodane dialdehyde (Mþ m/e 314) Solidagoic acid A (Mþ m/e 316)a Solidagoic acid isomer (Mþ m/e 316)

Only a single representative from each isomeric series was used; octadecane (Mþ m/e 254)a was included in the chi-square analysis. a Indicates terpenes used in chi-square tests of association.

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A

B 4.0

Sesquiterpenes mg/g dry leaf

Diterpenes mg/g dry leaf

2.50 2.00 1.50 1.00 0.50

3.0

2.0

1.0

0.0

0.00

EU

US

EU

US

Fig. 1. Mean foliar terpenes  1 SE of seed grown S. gigantea plants from European (n ¼ 50) and US (n ¼ 30) seed origins.

A

Diterpenes mg/g dry leaf

6

5

4

3

2

1

0 E E E E E E E E E E E E E E E E E E U U U U U U U U U U U U U U U U U U U U U U U U S S S S S S 1 10 11 12 13 14 17 18 19 2 21 27 3 4 5 6 7 8 2 3 4 5 6 7

Populations (from seeds)

B Diterpenes mg/g dry leaf

12 10 8 6 4 2 0 E E E E E E E E E E E E E E E E E E E E U U U U U U U U UU U U U U U U U U U U U U U U U U U U U U S S S S S S S S SS 1 10 11 12 13 14 16 17 18 19 2 20 22 27 3 4 5 6 7 8 1 10 2 3 4 5 6 7 8 9

Populations (from rhizomes) Fig. 2. Mean foliar diterpene concentrations (1 SD) of EU and US S. gigantea populations from seed grown (A) and rhizome propagated (B) plants. Population sample sizes and coefficients of variation (%) are given in Table 1.

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to diterpenes, mean sesquiterpene concentration did not differ between plants of US versus EU origin (Fig. 1B; F ¼ .166, df ¼ 1, 20; p ¼ .688). Likewise, sesquiterpene concentration variance was found to be homogeneous between plants of US versus EU origin (Levene’s test: p > .1). Sesquiterpene concentration did vary significantly between populations (F ¼ 2.365, df ¼ 22, 53; p ¼ .005, nested ANOVA). Although a sesquiterpene pattern was not observed with seed origin, an overall positive relationship did exist between leaf sesquiterpene and diterpene concentrations (r ¼ .336, df ¼ 78, p ¼ .002). Greater germination success of European plants was observed (c2 ¼ 26.17, df ¼ 1, p < .001) and was a factor in the limited replication of US populations. For example, at least one seedling was produced from 71.8% (1 SE  7.6%) of the European maternal parents, while only 29.78% (1 SE  7.64%) of the US maternal parents had at least one germinated seedling. 3.2. Rhizome propagated plants The 10 sesquiterpene hydrocarbons, sesquiterpene ketone and eight diterpenes previously described from the seed grown plants were also detected in the rhizome propagated leaves. Extraction of freshly frozen leaves from these plants allowed the additional analysis of monoterpenes. Eight monoterpene hydrocarbons (Mþ 136) were confirmed from both the US and European populations based on relative peak retention times, parent ion mass and mass/abundance ratios of major ion fragments and comparison with the NIST2000 database (Table 2). (þ)-epi-Bicyclosesquiphellandrene was again the dominant sesquiterpene peak exhibited by all individuals from both continents of origin and remained the dominant terpenoid overall. Rhizome propagated plants of US origin had significantly greater mean leaf diterpene concentrations than their European counterparts (Fig. 3A; F ¼ 7.75, df ¼ 1, 28; p ¼ .01). Population diterpene means of these plants were also found to differ (F ¼ 2.396, df ¼ 28, 261; p < .001, nested ANOVA). Individual diterpene concentrations were generally a variable characteristic within populations of either origin (Fig. 2B) with correspondingly high coefficients of variability (Table 1). Plants of US origin exhibited greater overall variance in diterpene

B Sesquiterpenes mg/g dry leaf

Diterpenes mg/g dry leaf

A 6.0 5.0 4.0 3.0 2.0 1.0 0.0 EU

US

EU

US

12.5 10.0 7.5 5.0 2.5 0.0 EU

US

Monoterpenes mg/g dry leaf

C 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Fig. 3. Mean foliar terpenes  1 SE of rhizome propagated S. gigantea plants from European (n ¼ 142) and US (n ¼ 149) seed origins.

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concentration than did EU plants (Levene’s test: s2 ratio ¼ 1.96, F ¼ 11.234; p ¼ .001). In contrast to diterpenes, mean sesquiterpene concentration did not differ between plants of US versus EU origin (Fig. 3B; F ¼ .664, df ¼ 1, 28; p ¼ .422); however, sesquiterpene concentration was found to differ significantly between populations (F ¼ 4.772, df ¼ 28, 260; p < .001, nested ANOVA). Sesquiterpene concentration variance was found to be homogeneous between plants of US versus EU origin (Levene’s test; p > .5). The sesquiterpene ketone isolongifolen-5-one was identified in only 42 plants overall; 19 of which were from US origin and 23 from EU origin. Monoterpene concentrations generally mirrored the diterpene pattern (Fig. 3C), with plants of US origin exhibiting greater mean levels than EU plants (F ¼ 20.178, df ¼ 1, 28; p < .001) and exhibiting significant population means differences (F ¼ 2.80, df ¼ 28, 261; p < .001). 4. Discussion The foliar cis-clerodane diterpenes confirmed from these US and European S. gigantea populations correspond to clerodane diterpenes previously reported from S. gigantea rhizomes (Anthonsen et al., 1973; Henderson et al., 1973). S. virgaurea, the only species of Solidago native to Europe, is also reported to contain a number of foliar cisclerodanes (Goswami et al., 1984), thus compositional similarities in chemical defenses appear to exist between invasive S. gigantea and the native S. virgaurea. The presence of clerodane diterpenes in S. virgaurea suggests that these compounds may confer a selective advantage by presumably limiting damage from potential European generalist herbivores. Invasive goldenrod species, if exposed to similar generalist herbivore pressure, could also be expected to maintain clerodane-producing pathways. Within S. gigantea, the homogeneous occurrence of three sesquiterpene hydrocarbons, a sesquiterpene ketone, a diterpene hydrocarbon and two oxygenated diterpenes across both US and EU seed source plants suggests that genes involved in these specific terpene synthetic pathways remain active between the native and invasive ranges. The sesquiterpene ketone isolongifolen-5-one occurred with relatively low frequency across all individuals (14.4% overall) but was found in at least one individual in eight of the 10 US populations. Among the EU populations, isolongifolen-5-one occurred in at least one individual in eight of the 20 EU populations. A German population (EU14) exhibited the highest frequency of occurrence: three out of four genotypes tested. In total, over 81% of all EU isolongifolen-5-one containing genotypes were found within German, Swiss and Austrian populations. Isolongifolen-5-one was not detected in any of the four French populations sampled. If the genetic basis of isolongifolen-5-one production is established, the molecule could provide a rapid and inexpensive biochemical marker for future studies of Solidago invasion biology and evolution. Because generalists can be highly sensitive to qualitative chemical defenses, there may be less pressure to maintain these chemicals at high constitutive levels in the invasive range particularly if there are evolutionary tradeoffs. Although terpene compositional profiles were similar between the native and invasive ranges, diterpene concentration of native seed grown leaves was 32.6% higher than those of the invasive EU plants. This pattern was repeated in our second experiment; diterpene concentrations from rhizome propagated leaves of native US origin were 27.3% higher than EU levels. The lower diterpene levels may explain why Meyer et al. (2005) found higher insect biomasses on seed grown European plants and that European plants were also more heavily attacked by fungal and bacterial pathogens. Plants used in our experiments and Meyer et al. (2005) were derived from the same seed collection. Our second experiment also found that native US rhizome propagated plants exhibited 36% greater mean monoterpene concentrations than plants of EU origin. Monoterpenes are a relatively common foliar constituent and at high levels are known to inhibit a range of generalist herbivores (Lincoln and Couvet, 1989; Zangerl and Bazzaz, 1992); however, we found no research that investigated their possible role in influencing Trirhabda feeding on goldenrod. Our findings of reduced diterpene levels from invasive goldenrods are in contrast to the higher pyrrolizidine alkaloid concentrations reported among invasive Senecio jacobaea (Joshi and Vrieling, 2005; Stastny et al., 2005). Joshi and Vrieling (2005) and Stastny et al. (2005) argue that the higher levels of alkaloids found in invasive Senecio result from evolutionary pressure to reduce costly quantitative defenses (e.g. tannins) most effective against adapted specialists (not present in the invasive range) and to increase less costly qualitative defenses that are most effective against generalists. The argument assumes that qualitative-type chemical defenses are less costly because they are not maintained at very high constitutive levels, but may not take into full account the substantial metabolic costs of complex biosynthetic pathways and storage structures that terpenoids often require (Gershenzon, 1994). On the other hand, if European generalists remain deterred by qualitative allelochemicals (e.g. cis-clerodane diterpenes) at low levels, the EICA hypothesis still predicts overall reductions in the concentrations of these defensive chemicals relative to native

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plants as long as lower concentrations incur lower metabolic costs. Cooper-Driver and Le Quesne (1987) found that adapted Trirhabda beetles were stimulated to feed by the diterpene kaur-16-en-19-oic acid but that high levels of three other kauranoid diterpenes inhibited both larval and adult feeding. Thus in the absence of Trirhabda, invasive S. gigantea may escape an important evolutionary pressure that might otherwise act to maintain higher levels of these defensive diterpenes. The level of Solidago diterpene tolerance by non-adapted generalists in the invasive range could then become a factor in determining potential thresholds in the evolution of lower foliar diterpene concentrations. Within both continents of origin we did find a high degree of diterpene variability within populations (CV% for both seed and rhizome plants) and concentration differences between populations (rhizome plants); however, EU plants did exhibit lower overall variance. When looking at the diterpene variability (CV%) of seed grown plants, US populations ranged from 58.5 to 89.4% and EU populations ranged from 10.5 to 90%; US rhizome propagated plant populations ranged from 37.6 to 74.5% and EU populations ranged from 22.3 to 130.8%. This level of variability in allelochemical concentration is not uncommon in native plant populations, although less information exists for invasive plant populations. Joshi and Vrieling (2005) did report that the variability of the pyrrolizidine alkaloid jacobine from S. jacobaea populations ranged from 4 to 78% within the native range and from approximately 35 to 160% in the introduced range, with only one of two chemotypes being present in the introduced range. Our study suggests that even though monoterpene and diterpene concentrations are lower in the introduced range, generalist herbivores will still encounter a very heterogeneous foliar terpene landscape. In contrast to monoterpenes and diterpenes, sesquiterpenes were not seen to differ between native and introduced ranges in either seed or rhizome propagated plants. The lack of difference within the seed grown plants could have been an artifact of leaf drying prior to chemical analysis. Sesquiterpene hydrocarbons are semi-volatile and extraction from dried leaves may not provide quantitative yields; however, our second experiment with rhizome propagated plants used freshly frozen moist leaves and still found no difference between continents of origin. Within the seed grown plants we did find a positive relationship between sesquiterpene and diterpene concentrations suggesting sufficient resolution of sesquiterpene variability to detect a biochemical relationship with diterpenes. Sesquiterpenes and diterpenes of the rhizome propagated plants exhibited mean concentrations that were over twice those of the seed grown leaves. These differences could have resulted from a number of factors including leaf processing (dry versus moist) and/or developmental differences (seed versus rhizome propagation). However, the proportion of mean sesquiterpene and diterpene concentrations in the total terpenoid profile of EU and US plants remained similar between both seed and rhizome propagated plants and provides additional evidence that sesquiterpenes were not differentially lost from our dry leaf analysis. In conclusion, this study offers partial support to the biochemical predictions of the EICA hypothesis, namely that foliar monoterpene and diterpene but not sesquiterpene concentrations were reduced in plants originating from invasive populations relative to natives grown in a common garden. Because terpene profile composition remained similar between native and invasive ranges, the lower concentrations would most likely have come about by reduced production and/or accumulation and not by the loss of synthetic pathways. Alternative hypotheses do exist that can also explain our observed differences. For instance, there could have been chance introductions of several low diterpene chemotypes across the invasive range that was coupled with vegetative spread and limited gene flow. In either case, these findings suggest that use of introduced Trirhabda beetles as potential control agents may yield different levels of success based on the plant phenotypes present in different regions. Our study did not address the growth predictions of the EICA hypothesis; however, other studies have examined the growth of native and invasive S. gigantea in more detail, with mixed results. Using garden experiments in Europe, Jakobs (2004) found that invasive genotypes had significantly greater shoot mass, leaf mass, infructescence mass, and rhizome mass than native genotypes, in accord with the EICA hypothesis. However, other studies have found infructescence mass of invasive S. gigantea plants to be generally equal to or significantly less than that of native genotypes (Meyer and Hull-Sanders, in press). These studies suggest that decreases in chemical defenses in invasive plants, as demonstrated here, may not necessarily translate into increased plant performance. Acknowledgement This project was funded by National Science Foundation grant DEB-035127 to Johnson and DEB-0315430 to Meyer. Special thanks are given to R. Grebenok (Canisius College, Buffalo, NY) for GC/MS use and D. Snieszko, M. Bucheker and T. Tinti (Medaille College) for laboratory assistance. We also thank B. Young and anonymous reviewers for insightful comments on previous versions of the manuscript.

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