Comparison of foliar terpenes between native and invasive Solidago gigantea

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www.elsevier.com/locate/biochemsysecoBiochemical Systematics and Ecology xx (2007) 1e10

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Comparison of foliar terpenes between native and invasiveSolidago 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, USAb 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 gigan-tea plants from North American and European (invasive) populations were grown in a screen-enclosed garden. Terpenes from 80seed grown (dried leaves) and 320 rhizome propagated (moist leaves) individuals were confirmed by GC/MS and quantified by GC-FID. 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 seedsources suggesting these biochemical pathways remain unchanged. Leaves from native rhizome propagated plants also had signif-icantly greater monoterpene and diterpene concentrations; again sesquiterpene levels did not differ. Rhizome propagated plantsexhibited significant population differences in monoterpene and diterpene concentrations. These data support the defensive chem-istry predictions of the EICA hypotheses but cannot discount the role of possible founder effects in the invasive range.� 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 Notzold (1995) pre-dicts that introduced plant species, which have escaped their natural specialist herbivores, should evolve to decreasetheir investment in anti-herbivore chemical defenses. Resources no longer needed for defense could be reallocated tocharacteristics that provide a selective advantage in the novel habitat. The newly evolved invasive phenotypes shouldthus exhibit increased growth and fecundity relative to native conspecifics, but lower resistance to herbivores, partic-ularly specialists from their native range (Blossey and Notzold, 1995; Wolfe, 2002; Maron et al., 2004). The EICAhypothesis was formulated to provide a mechanism to explain invasive plant vigor and success in general and in par-ticular the observation that invasive purple loosestrife (Lythrum salicaria L.) produced greater plant biomass andhosted larger specialist herbivore larvae than conspecifics in the native range. Blossey and Notzold (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 � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.bse.2007.06.005

Please cite this article in press as: Robert H. Johnson et al., Comparison of foliar terpenes between native and invasive Solidago gigantea, Bio-

chem. Syst. Ecol. (2007), doi:10.1016/j.bse.2007.06.005

mailto:[email protected]

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2 R.H. Johnson et al. / Biochemical Systematics and Ecology xx (2007) 1e10

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measure plant’s secondary chemistry but did assume that differences in insect growth were a result of altered patternsof plant defenses. Recent modifications to the EICA hypothesis propose that invasive genotypes may still experiencemoderate attack by local generalist herbivores (Muller-Scharer et al., 2004). In some cases selection may favor a re-duction in quantitative chemical defenses (effective against specialists but metabolically expensive) and an increase inthe 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 EICAhypothesis. 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 gi-gantea) were introduced to Europe in the mid 18th century, quickly naturalized, and began to spread during the mid19th 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 herbivoresknown 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 comparisonof S. gigantea populations growing in the US and Europe showed that stem density is about twice as high in Europeas 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 accumulationof 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. gigantearhizomes were found to contain solidagoic acids A and B (Anthonsen et al., 1973), and eight furan-containingcis-clerodane diterpenes (Henderson et al., 1973). S. virgaurea (European goldenrod) foliar tissue has also beenfound to contain a number of cis-clerodane lactones (Goswami et al., 1984), but no information on the concentra-tions of S. virgaurea foliar cis-clerodanes was found. Solidago diterpenes are known to influence feeding preferenceof Trirhabda canadensis, a chrysomelid specialist on goldenrod (Le Quesne et al., 1986) as well as a broad range ofgeneralist insect herbivores (Cooper-Driver and Le Quesne, 1987; Maddox and Root, 1987, 1990). When fed freshS. gigantea leaves, growth of Spodoptera exigua larvae was found to decline with increasing host leaf cis-clerodanediterpene concentrations; however, growth of Trirhabda virgata larvae (another chrysomelid specialist on golden-rod) was not affected (Hull-Sanders et al., 2007). This same study found that neither S. exigua nor T. virgata wasaffected by sesquiterpene levels.

Meyer et al. (2005) found that European seed grown S. gigantea plants hosted greater insect biomasses than seedgrown natives and that both seed and rhizome propagated European invasives were more susceptible to rust fungus andXanthomonas leaf blight infections than native conspecifics. In the study reported here, we looked for a biochemicalmechanism for the differential susceptibility to insects and pathogens reported by Meyer et al. (2005) between seedand rhizome propagated native and invasive goldenrods. Specifically, we tested the prediction that invasive golden-rods, 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 bereflected in differential compound occurrence, (2) by a simple reduction in the leaf terpene concentration amongthe 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 herbivoreslocated at the University of Wisconsin e Milwaukee Field Station, Saukville, WI, where they were randomlyarranged and watered as needed. Within the screenhouse, plants were cultivated in pots in a commercial pottingmix (Farfard Grow Mix #2, Conrad Farfard Inc., Agawam, Massachusetts). To further protect against pathogenor insect damage, plants were sprayed with a mixture of fungicide (mancozeb) and insecticide (carbaryl) at 1e3week intervals as needed. Light levels in the screenhouse were approximately 70% of full sun, and height, phenol-ogy and appearance of goldenrod in the screenhouse were similar to plants grown outside (Meyer, personalobservation).



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2.1.1. Seed grown plantsSeeds 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 collectedwere well separated in space to ensure that they came from separate clones. Some seeds were germinated in 2000for the experiment reported in Meyer et al. (2005); all remaining seeds were stored under cool, dry conditions. In earlyApril 2003, the stored seeds were sown into separate cups containing a commercial potting mix, with each cup con-taining the seeds from one maternal parent. There were 110 European maternal parents (5 per population) and 92 USmaternal parents (2e14 per population) randomly arranged within the greenhouse at the Field Station. Germination inthe cups was scored in late April and again in June. The presence of at least one seedling in a cup was counted assuccessful germination. Germination of European seeds was much better than that of US seeds (see Section 3), result-ing in many more European seedlings than US seedlings. In early July, one seedling per maternal parent was trans-planted to a 6.6 L pot and moved to the screen enclosure maintained at the Field Station. All US cups with seedlingswere used (n¼ 32, representing eight populations), but there were many more European seedlings than US, thus seed-lings from only 53 of the European maternal parents were transplanted. European seedlings were chosen so that 19populations were represented, with the number of replicates within populations as balanced as possible. Because ofthe need to allow these plants to develop sufficiently large rhizomes for future propagation, leaf harvesting was limitedto five leaves per plant. This restriction precluded our obtaining additional matched control leaves (for wetedry massconversion), and necessitated dry leaf analysis. On September 10, five fully expanded leaves from the upper third toupper half of each plant were harvested and immediately placed into a circulating air oven (60 �C) at the Field Stationand dried for 48 h. Dried leaves were subsequently express shipped to Buffalo, NY; upon arrival, leaves were main-tained in sealed silica-gel desiccators until processed. Leaves of each plant were weighed just prior to chemical anal-ysis to obtain mean leaf mass. Population locations and final sample sizes for chemical analyses are shown in Table 1.

2.1.2. Rhizome propagated plantsSeeds 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 experimentincluded 20 European populations and 10 US populations. At the conclusion of this experiment in October 2001, rhi-zomes 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 rhizomesfrom each plant at the end of the growing season for storage over-winter in the refrigerator. These methods allowed thesame 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 exper-iment included two clones of each genotype: one was left undamaged and the other was damaged by Trirhabda bee-tles. At the end of the 2004 growing season, the rhizomes were again harvested and stored under refrigeration. In May2005, two rhizomes from each of 80 US and 80 EU genotypes were cut to 7e15 cm lengths and weighed to ensurehomogeneous planting masses. One rhizome of each pair was undamaged in the previous experiment and the otherwas previously damaged. Rhizomes were planted in individual 4 L pots and grown under the screenhouse commongarden conditions as described above. In August 2005, 10 fully expanded leaves from the upper third of each stemwere 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 sothat terpenoid concentrations determined from wet leaves could be standardized to dry leaf mass. The remainingbagged 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 leaveswere 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 wereplaced 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 at3000 rpm) and the supernatant decanted into 20 ml glass scintillation vials. Vial contents were concentrated undera 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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detector (FID). Compounds were separated using a RTx-5ms column (30 m� 0.25 mm id); injector temperature wasmaintained 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) in-creasing to 280 �C at 5 �C/min. Monoterpenes were not integrated for the seed grown plants as the leaf dryingprocess could volatilize these lower molecular weight terpenes. Terpene FID peaks were grouped by retention timesand integrated separately to differentiate monoterpene, sesquiterpene and diterpene concentrations. Prior to quantita-tive GC analysis, leaf samples from both the seed grown (dried) and rhizome propagated (freshly frozen) plants fromUS and European populations were extracted and concentrated separately as described above. Identities of the samplepeaks were confirmed with an Agilent 6850 GCe5973 MSD using a similar RTx-5ms column and the same thermalelution program used for the quantitative runs. The resulting mass spectral ion patterns for each peak were comparedto the NIST2000 software database and literature spectra (Anthonsen et al., 1973; Henderson et al., 1973) for com-pound confirmations.

2.3. Statistical analysis

Data were analyzed using SPSS version 13.0 for Windows. All data were tested for normality and equality ofvariance (Levene’s test); all terpene concentrations were log transformed to meet test assumptions. The effects ofcontinent of origin on monoterpene (moist leaves only), sesquiterpene and diterpene concentrations were analyzed

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 Long CV (%) seed N CV (%) rhizome N

European populations

Austria (Imst) EU22 47.18 N 10.77 E 64.7 3 130.8 7

France (Rosenau) EU1 47.65 N 7.53 E 64.2 3 45.2 6

France (Kembs) EU4 47.68 N 7.50 E 55.5 3 47.8 8

France (Strasbourg) EU5 48.58 N 7.77 E 45.9 2 53.3 8

France (Erstein) EU6 48.43 N 7.69 E 34.3 2 22.3 7

France (L’Isle sur le Doubs) EU8 47.45 N 6.58 E 18.6 3 39.6 5

Germany (Rosdorf) EU10 53.94 N 9.89 E 66.2 3 55.4 6

Germany (Celle) EU13 52.37 N 10.05 E 49.8 2 31.3 7

Germany (Dornten) EU14 51.40 N 6.58 E 68.7 2 52.7 8

Germany (Neumunster) EU16 47.95 N 11.78 E e e 26.7 6

Germany (Karlsruhe) EU17 49.01 N 8.39 E 22.3 3 55.5 8

Hungary (Devecser) EU11 47.10 N 17.51 E 58.6 3 55.5 9

Hungary (Mosonmagyarovar) EU12 47.86 N 17.21 E 72.3 3 57.3 8

Italy (Albate) EU18 45.40 N 9.16 E 80.3 3 82.2 8

Italy (Fino-Mornascio) EU19 45.76 N 9.07 E 90.0 2 49.7 7

Switzerland (Tenero) EU27 46.18 N 8.95 E 62.9 3 38.8 7

Switzerland (Cadepezzo) EU2 46.12 N 8.94 E 69.9 3 76.2 8

Switzerland (Arlesheim) EU3 47.51 N 7.66 E 10.5 3 53.8 6

Switzerland (Sihlbrugg) EU7 47.21 N 8.53 E 41.5 3 52.9 8

Switzerland (Cadenazzo) EU20 46.13 N 8.96 E e (1) 45.0 5

US populations

Iowa (Zimmerman Field) US1 41.66 N 91.54 W e (1) 55.6 18

Iowa (Cone Marsh) US2 41.49 N 91.43 W 58.5 3 59.3 18

Michigan (Ann Arbor) US8 42.28 N 83.73 W e (1) 37.6 15

New Hampshire (N. Haverhill) US4 44.07 N 72.03 W 89.4 3 43.8 14

New Hampshire (Claremont) US5 43.35 N 72.36 W 61.8 6 48.3 16

New York (Turkey Hill) US9 42.45 N 76.50 W e e 65.9 8

New York (Trumansberg) US10 42.53 N 76.67 W e e 46.3 4

North Dakota (Oakville Prairie) US6 47.89 N 97.30 W 58.1 4 44.2 19

Vermont (Green Mtn Audubon) US3 44.36 N 73.01 W 59.3 5 60.8 19

Wisconsin (Saukville) US7 43.22 N 88.01 W 70.1 7 74.5 18

Populations represented by a single individual (n¼ 1) were dropped from analysis; localities given in decimal degrees latitude and longitude.



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using nested GLM-univariate analysis. Continental origin was treated as a fixed effect and population of origin wasincluded in the model as a nested, random effect. In experiment 1 (seed grown plants) one EU and two US populationswere dropped from the GLM nested analysis because each was represented by a single individual and offered nointrapopulation replication. To look for possible carry-over effects of leaf loss from the previous year (2004; reportedin Hull-Sanders et al., 2007), previously damaged plants were compared with previously undamaged plants usinga one-way ANOVA. No differences were found in monoterpene, sesquiterpene or diterpene concentrations betweenthe two treatments (all p values� .5), thus carry-over effects of leaf loss were discounted. Independent chi-square testsof association were conducted to determine if associations existed between seed source and the presence of three ses-quiterpene hydrocarbons, an oxygenated sesquiterpene, three diterpenes and a linear hydrocarbon (octadecane) thatwere 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. Correlationanalysis was conducted to test for a relationship between sesquiterpene and diterpene concentrations of seed grownplants.

3. Results

3.1. Seed grown plants

Ten sesquiterpene hydrocarbons (Mþ 204) and one sesquiterpene ketone (Mþ 218) were confirmed from both theUS and European populations based on relative peak retention times, parent ion mass and mass/abundance ratios ofmajor ion fragments. Of the 11 confirmed sesquiterpenes, spectral comparisons of individual peaks with the softwaredatabase allowed the tentative identification of six specific compounds (Table 2). Eight diterpenes were confirmedbased on relative retention times, parent ion mass and mass/abundance ratios of major ions and spectral comparisonswith software and literature. Of these, two were confirmed as kaurenes, four as cis-clerodanes and two as diterpeneacids. The presence of ions 67, 81 and 95 m/z indicated the attachment of a furan ring which provides additional con-firmation that the oxygenated diterpenes are furan-containing dicyclic clerodanes and acids (Table 2). (þ)-epi-Bicyclosesquiphellandrene was the dominant sesquiterpene peak exhibited by all individuals from both seed sourcesand with the exception of three US individuals, and was the dominant terpenoid overall. Visual comparison of quan-titative chromatograms suggested that the occurrence of major sesquiterpene and diterpene peaks was similar betweenUS and European plants. Statistical testing of this observation likewise found no differences in the frequencies of theeight selected compounds that spanned the GC thermal gradient (see Table 2 for selected compounds) betweenEuropean 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 con-centration from their European counterparts (Fig. 1A; F¼ 6.054, df¼ 1, 17; p¼ .025). Population diterpene meanswere not seen to differ (F¼ 1.089, df¼ 22, 53; p¼ .387, nested ANOVA); however, individual diterpene concentra-tions were generally a variable characteristic within populations of either seed source resulting in large standard de-viations (Fig. 2A) and coefficients of variation (Table 1). In addition, plants of US seed origin exhibited greater overallvariance in diterpene concentration than did EU plants (Levene’s test: s2 ratio¼ 2.59, F¼ 7.34; p¼ .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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USEU

2.50

2.00

1.50

1.00

0.50

0.00

Dite

rpen

es m

g/g

dry

leaf

A

USEU

4.0

3.0

2.0

1.0

0.0

Sesq

uite

rpen

es m

g/g

dry

leaf

B

Fig. 1. Mean foliar terpenes� 1 SE of seed grown S. gigantea plants from European (n¼ 50) and US (n¼ 30) seed origins.

US7

US6

US5

US4

US3

US2

EU8

EU7

EU6

EU5

EU4

EU3

EU27

EU21

EU2

EU19

EU18

EU17

EU14

EU13

EU12

EU11

EU10

EU1

Populations (from seeds)

US7

US8

US9

US6

US5

US4

US3

US2

US10

US1

EU8

EU7

EU6

EU5

EU4

EU3

EU27

EU22

EU20

EU2

EU19

EU18

EU17

EU16

EU14

EU13

EU12

EU11

EU10

EU1

Populations (from rhizomes)

6

5

4

3

2

1

0

Dite

rpen

es m

g/g

dry

leaf

A

12

10

8

6

4

2

0

Dite

rpen

es m

g/g

dry

leaf

B

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;F¼ .166, df¼ 1, 20; p¼ .688). Likewise, sesquiterpene concentration variance was found to be homogeneousbetween plants of US versus EU origin (Levene’s test: p> .1). Sesquiterpene concentration did vary significantlybetween populations (F¼ 2.365, df¼ 22, 53; p¼ .005, nested ANOVA). Although a sesquiterpene pattern was notobserved with seed origin, an overall positive relationship did exist between leaf sesquiterpene and diterpene concen-trations (r¼ .336, df¼ 78, p¼ .002). Greater germination success of European plants was observed (c2¼ 26.17,df¼ 1, p< .001) and was a factor in the limited replication of US populations. For example, at least one seedlingwas produced from 71.8% (1 SE� 7.6%) of the European maternal parents, while only 29.78% (1 SE� 7.64%) ofthe 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 seedgrown plants were also detected in the rhizome propagated leaves. Extraction of freshly frozen leaves from these plantsallowed the additional analysis of monoterpenes. Eight monoterpene hydrocarbons (Mþ 136) were confirmed from boththe US and European populations based on relative peak retention times, parent ion mass and mass/abundance ratios ofmajor ion fragments and comparison with the NIST2000 database (Table 2). (þ)-epi-Bicyclosesquiphellandrene wasagain the dominant sesquiterpene peak exhibited by all individuals from both continents of origin and remained thedominant terpenoid overall. Rhizome propagated plants of US origin had significantly greater mean leaf diterpene con-centrations than their European counterparts (Fig. 3A; F¼ 7.75, df¼ 1, 28; p¼ .01). Population diterpene means ofthese plants were also found to differ (F¼ 2.396, df¼ 28, 261; p< .001, nested ANOVA). Individual diterpene con-centrations were generally a variable characteristic within populations of either origin (Fig. 2B) with correspondinglyhigh coefficients of variability (Table 1). Plants of US origin exhibited greater overall variance in diterpene

USEU

6.0

5.0

4.0

3.0

2.0

1.0

0.0

Dite

rpen

es m

g/g

dry

leaf

A

USEU

12.5

10.0

7.5

5.0

2.5

0.0Sesq

uite

rpen

es m

g/g

dry

leaf

B

USEU

1.2

1.0

0.8

0.6

0.4

0.2

0.0Mon

oter

pene

s m

g/g

dry

leaf

C

Fig. 3. Mean foliar terpenes� 1 SE of rhizome propagated S. gigantea plants from European (n¼ 142) and US (n¼ 149) seed origins.



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concentration than did EU plants (Levene’s test: s2 ratio¼ 1.96, F¼ 11.234; p¼ .001). In contrast to diterpenes, meansesquiterpene concentration did not differ between plants of US versus EU origin (Fig. 3B; F¼ .664, df¼ 1, 28;p¼ .422); however, sesquiterpene concentration was found to differ significantly between populations (F¼ 4.772,df¼ 28, 260; p< .001, nested ANOVA). Sesquiterpene concentration variance was found to be homogeneous betweenplants of US versus EU origin (Levene’s test; p> .5). The sesquiterpene ketone isolongifolen-5-one was identified inonly 42 plants overall; 19 of which were from US origin and 23 from EU origin. Monoterpene concentrations generallymirrored the diterpene pattern (Fig. 3C), with plants of US origin exhibiting greater mean levels than EU plants(F¼ 20.178, df¼ 1, 28; p< .001) and exhibiting significant population means differences (F¼ 2.80, df¼ 28, 261;p< .001).

4. Discussion

The foliar cis-clerodane diterpenes confirmed from these US and European S. gigantea populations correspond toclerodane 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 cis-clerodanes (Goswami et al., 1984), thus compositional similarities in chemical defenses appear to exist betweeninvasive S. gigantea and the native S. virgaurea. The presence of clerodane diterpenes in S. virgaurea suggeststhat these compounds may confer a selective advantage by presumably limiting damage from potential European gen-eralist herbivores. Invasive goldenrod species, if exposed to similar generalist herbivore pressure, could also be ex-pected to maintain clerodane-producing pathways. Within S. gigantea, the homogeneous occurrence of threesesquiterpene hydrocarbons, a sesquiterpene ketone, a diterpene hydrocarbon and two oxygenated diterpenes acrossboth US and EU seed source plants suggests that genes involved in these specific terpene synthetic pathways remainactive between the native and invasive ranges. The sesquiterpene ketone isolongifolen-5-one occurred with relativelylow frequency across all individuals (14.4% overall) but was found in at least one individual in eight of the 10 USpopulations. Among the EU populations, isolongifolen-5-one occurred in at least one individual in eight of the 20EU populations. A German population (EU14) exhibited the highest frequency of occurrence: three out of four geno-types tested. In total, over 81% of all EU isolongifolen-5-one containing genotypes were found within German, Swissand Austrian populations. Isolongifolen-5-one was not detected in any of the four French populations sampled. If thegenetic basis of isolongifolen-5-one production is established, the molecule could provide a rapid and inexpensivebiochemical 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 maintainthese chemicals at high constitutive levels in the invasive range particularly if there are evolutionary tradeoffs. Al-though terpene compositional profiles were similar between the native and invasive ranges, diterpene concentrationof native seed grown leaves was 32.6% higher than those of the invasive EU plants. This pattern was repeated in oursecond experiment; diterpene concentrations from rhizome propagated leaves of native US origin were 27.3% higherthan EU levels. The lower diterpene levels may explain why Meyer et al. (2005) found higher insect biomasses on seedgrown 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 ex-periment also found that native US rhizome propagated plants exhibited 36% greater mean monoterpene concentra-tions than plants of EU origin. Monoterpenes are a relatively common foliar constituent and at high levels are knownto inhibit a range of generalist herbivores (Lincoln and Couvet, 1989; Zangerl and Bazzaz, 1992); however, we foundno 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 alka-loid concentrations reported among invasive Senecio jacobaea (Joshi and Vrieling, 2005; Stastny et al., 2005). Joshiand Vrieling (2005) and Stastny et al. (2005) argue that the higher levels of alkaloids found in invasive Senecio resultfrom evolutionary pressure to reduce costly quantitative defenses (e.g. tannins) most effective against adapted special-ists (not present in the invasive range) and to increase less costly qualitative defenses that are most effective againstgeneralists. The argument assumes that qualitative-type chemical defenses are less costly because they are not main-tained at very high constitutive levels, but may not take into full account the substantial metabolic costs of complexbiosynthetic pathways and storage structures that terpenoids often require (Gershenzon, 1994). On the other hand, ifEuropean generalists remain deterred by qualitative allelochemicals (e.g. cis-clerodane diterpenes) at low levels, theEICA 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 thatadapted Trirhabda beetles were stimulated to feed by the diterpene kaur-16-en-19-oic acid but that high levels of threeother kauranoid diterpenes inhibited both larval and adult feeding. Thus in the absence of Trirhabda, invasiveS. gigantea may escape an important evolutionary pressure that might otherwise act to maintain higher levels of thesedefensive diterpenes. The level of Solidago diterpene tolerance by non-adapted generalists in the invasive range couldthen 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 bothseed and rhizome plants) and concentration differences between populations (rhizome plants); however, EU plants didexhibit lower overall variance. When looking at the diterpene variability (CV%) of seed grown plants, US populationsranged from 58.5 to 89.4% and EU populations ranged from 10.5 to 90%; US rhizome propagated plant populationsranged from 37.6 to 74.5% and EU populations ranged from 22.3 to 130.8%. This level of variability in allelochemicalconcentration is not uncommon in native plant populations, although less information exists for invasive plant popula-tions. Joshi and Vrieling (2005) did report that the variability of the pyrrolizidine alkaloid jacobine from S. jacobaeapopulations 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 monoter-pene and diterpene concentrations are lower in the introduced range, generalist herbivores will still encounter a veryheterogeneous foliar terpene landscape.

In contrast to monoterpenes and diterpenes, sesquiterpenes were not seen to differ between native and introducedranges in either seed or rhizome propagated plants. The lack of difference within the seed grown plants could havebeen an artifact of leaf drying prior to chemical analysis. Sesquiterpene hydrocarbons are semi-volatile and extractionfrom dried leaves may not provide quantitative yields; however, our second experiment with rhizome propagatedplants used freshly frozen moist leaves and still found no difference between continents of origin. Within the seedgrown plants we did find a positive relationship between sesquiterpene and diterpene concentrations suggesting suf-ficient resolution of sesquiterpene variability to detect a biochemical relationship with diterpenes. Sesquiterpenes andditerpenes of the rhizome propagated plants exhibited mean concentrations that were over twice those of the seedgrown leaves. These differences could have resulted from a number of factors including leaf processing (dry versusmoist) and/or developmental differences (seed versus rhizome propagation). However, the proportion of mean sesqui-terpene and diterpene concentrations in the total terpenoid profile of EU and US plants remained similar between bothseed and rhizome propagated plants and provides additional evidence that sesquiterpenes were not differentially lostfrom our dry leaf analysis.

In conclusion, this study offers partial support to the biochemical predictions of the EICA hypothesis, namely that foliarmonoterpene and diterpene but not sesquiterpene concentrations were reduced in plants originating from invasive popula-tions relative to natives grown in a common garden. Because terpene profile composition remained similar between nativeand invasive ranges, the lower concentrations would most likely have come about by reduced production and/or accumu-lation and not by the loss of synthetic pathways. Alternative hypotheses do exist that can also explain our observed differ-ences. For instance, there could have been chance introductions of several low diterpene chemotypes across the invasiverange that was coupled with vegetative spread and limited gene flow. In either case, these findings suggest that use of in-troduced Trirhabda beetles as potential control agents may yield different levels of success based on the plant phenotypespresent in different regions. Our study did not address the growth predictions of the EICA hypothesis; however, other studieshave examined the growth of native and invasive S. gigantea in more detail, with mixed results. Using garden experiments inEurope, 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 infruc-tescence mass of invasive S. gigantea plants to be generally equal to or significantly less than that of native genotypes (Meyerand Hull-Sanders, in press). These studies suggest that decreases in chemical defenses in invasive plants, as demonstratedhere, 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 toMeyer. 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 re-viewers for insightful comments on previous versions of the manuscript.



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Comparison of foliar terpenes between native and invasive Solidago gigantea

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