Comparative Allelopathy of Three Midwestern Invasive Species (Lonicera Maackii, Alliaria

7 Comparative allelopathic effects of three invasive species (Alliaria petiolata,

8 Lonicera maackii and Ranunculus ficaria) in the Midwestern United States:

9 Variation in response with experimental venue and target species

11 Kendra Cipollini, Kyle Titus, and Crystal Wagner

13 Kendra Cipollini1, Kyle Titus2, and Crystal Wagner3. Wilmington College, 1870

14 Quaker Way, Wilmington, OH 45177, USA

15 1Corresponding author (e-mail: [email protected]).

16 2Current address: (e-mail: [email protected])

17 3Current address: (e-mail: [email protected])

19 Phone: 937-382-6661 x367 (w), 937-532-6128 (cell)

20 FAX: 937-383-8530

1 1 21Abstract: Garlic mustard (Alliaria petiolata), Amur honeysuckle (Lonicera maackii)

22and lesser celandine (Ranunculus ficaria) are three species that invade Midwestern

23forests in the US and exhibit allelopathy. There is little known about their comparative

24allelopathic effects on multiple test species. In three experiments, the comparative

25allelopathy of these species was investigated by making leaf extracts of each species and

26measuring their effects on test species. In potting soil, there were fewer siliques of A.

27thaliana when plants were treated with extracts of L. maackii compared to plants treated

28with no extract and with A. petiolata extracts. In field soil, there were significantly fewer

29siliques in A. thaliana treated with extracts of L. maackii and R. ficaria compared to with

30extracts of A. petiolata. The effect of these treatment solutions on germination of three

31species (Broccoli - Brassica oleracea, Lettuce - Lactuca sativa, and Basil - Ocimum

32basilicum) was studied. Across all test species, R. ficaria and L. maackii extracts affected

33germination the least, while A. petiolata extracts affected germination the most.

34However, the extracts impacted species differentially; L. sativa and O. basilicum were

35more sensitive to A. petiolata and R. ficaria extracts and B. oleracea was more sensitive

36to L. maackii extracts. These results provide evidence of differential allelopathic effects

37of three invasive species, as well as the importance of experimental venue and test

38species.

40Key words: allelopathy, exotic species, germination inhibition, leaf extracts,

41phytotoxicity

2 2 42Introduction

43 Invasive species pose a threat worldwide, negatively impacting biodiversity

44(Wilcove et al. 1998, McGeoch et al. 2010) and exerting significant economic costs

45(Pimentel et al. 2005). One focus in invasive species ecology is to determine factors that

46contribute to the success of invasive species (Sakai et al. 2001, Levine et al. 2003).

47These factors can range from life history traits (Kolar and Lodge 2001) to release from

48natural enemies (Keane and Crawley 2002). One hypothesis to explain invasive species

49success is the novel weapons hypothesis (Bais et al. 2003), whereby an invading species

50possesses a trait novel to the invaded ecosystem. The invasive species can then take

51advantage of this trait in its new ecosystem during interactions with native species that

52are evolutionarily-naïve to the trait. In plants, allelopathy can represent a novel weapon

53(Hierro and Callaway 2003, Callaway and Ridenour 2004). Allelopathy is simply the

54release of a chemical from the roots or leaves that affects germination, growth and/or

55reproduction of surrounding species (Rice 1974). Plants that exude these chemicals may

56be more likely to dominate in a new environment because their neighbors in their native

57range have evolved resistance or tolerance to their allelochemicals, while neighbors in

58their invaded range have not (Callaway and Aschehoug 2000). Allelopathy can have

59direct plant-to-plant effects, whereby allelochemicals directly impact another species

60(Dorning and Cipollini 2006). Alternatively, allelopathy may have indirect effects on

61other plants, such as through changing soil ecology or mutualisms (Stinson et al., 2006;

62Callaway et al. 2008, Zhang et al. 2009). Allelopathic effects may vary depending on

63target species (Cipollini et al. 2008a) or may vary by conditions such as life stage (Barto

64et al. 2010a) and nutrients (Cipollini et al. 2008a).

3 3 65 Allelopathy can be studied in a variety of experimental ways (Inderjit and

66Callaway 2003), with varying degrees of realism and control. Experiments with the

67greatest amount of experimental control, yet lowest amount of realism, are simple

68germination and growth experiments involving the application of specific chemicals or

69plant extracts with putative allelochemicals, usually in Petri dishes with a paper substrate

70(e.g., Dorning and Cipollini 2006, Cipollini et al. 2008b, McEwan et al. 2010). Other

71studies seek to increase the degree of realism at the cost of experimental control in

72greenhouse studies and field experiments, many times with the use of activated carbon as

73a manipulative tool (Ridenour and Callaway 2001, Cipollini et al. 2008, Cipollini and

74Schradin 2011). Field experiments show the greatest amount of realism and ecological

75relevance yet can be difficult in teasing out exact mechanism due to low amounts of

76experimental control. Generally, studies of allelopathy start with simple, controlled

77laboratory experiments before scaling up to field experiments.

78 Three important invasive species in forests and riparian areas in the Midwestern

79United States that have evidence of allelopathy are garlic mustard (Alliaria petiolata

80(Bieb.) Cavara & Grand – Brassicaceae), Amur honeysuckle (Lonicera maackii (Rupr.)

81Maxim - Caprifoliaceae) and lesser celandine (Ranunculus ficaria L. - Ranunculaceae).

82Lonicera maackii, native to Asia, in found in the eastern half of the United States (USDA

832011) and negatively affects trees and understory plants (Gould and Gorchov 2000,

84Collier et al. 2002, Hartman and McCarthy 2004). Leaf extracts of L. maackii inhibit

85germination of several test species in the laboratory (Dorning and Cipollini 2006,

86Cipollini et al. 2008b) and affect growth of Arabidopsis thaliana in the greenhouse

87(Cipollini et al. 2008a). Field soils collected from areas infested with L. maackii

4 4 88negatively impacted growth of A. thaliana (Cipollini and Dorning 2008). Cipollini et al.

89(2008) were unable to demonstrate any allelopathic effects of L. maackii on Impatiens

90capensis in the field with the use of activated carbon, though sample size issues limited

91the conclusions of the study.

92 Alliaria petiolata, native to Europe, is found from coast-to-coast in the United

93States in nearly all but the southern-most states (USDA 2011). Alliaria petiolata

94negatively affects understory plants (McCarthy 1997, Meekins and McCarthy 1999,

95Carlson and Gorchov 2004). Alliaria petiolata has been shown to exhibit allelopathic

96effects on germination of Geum species (Prati and Bossdorf 2004), though at least one

97study has shown negligible effects (McCarthy and Hanson 1998) Garlic mustard has

98several candidate compounds that may be responsible for allelopathic effects (Vaughn

99and Berhow 1999, Cipollini et al. 2005, Cipollini and Gruner 2007), though exact

100compounds responsible have not been identified (Barto and Cipollini 2009). Indirect

101allelopathic effects mediated through mychorrhizae have been demonstrated in the

102greenhouse, (Stinson et al. 2008, Callaway et al. 2008), though the effect may vary with

103species or life stage (Barto et al. 2010a). Allelopathic effects of A. petiolata have been

104shown in the field (Cipollini et al. 2008a).

105 Ranunculus ficaria, native to Europe, is found in the Northeast, Midwest, and

106Pacific Northwest regions of the United States (USDA 2011). Ranunculus ficaria is

107considered an invasive species (Axtell et al. 2010), though there is only one published

108information confirming its negative impact except (Cipollini and Schradin 2011).

109Because R. ficaria has purported medicinal effects (Chevallier 1996), it likely exhibits

110allelopathy (Ehrenfeld 2006). Indeed, the allelopathic effects of R. ficaria on

5 5 111reproduction of I. capensis were demonstrated in the field (Cipollini and Schradin 2011),

112but clearly more information is necessary to fully evaluate the impact of R. ficaria as an

113invasive species, let alone the mechanism for its success.

114 While there is some evidence of allelopathy for all of these species, there is no

115research that investigates their comparative allelopathic effects. Other studies have taken

116a comparative approach to studying allelopathy and allelochemicals, either comparing a

117suite of invasive species (Pisula and Meiners 2010) or comparing an invasive species to

118co-occurring similar native species (Barto et al. 2010b, McEwan et al. 2010). Because

119allelopathic effects can vary with the species on which they are tested (Cipollini et al.

1202008a, McEwan et al. 2010), we tested multiple species to have more generalizable

121results. A comparative approach using more than one test and invasive species would be

122useful in prioritizing restoration activities and possible use of mitigation treatments such

123as activated carbon (Kulmatiski and Beard 2006), particularly in areas invaded by more

124than one species. The purpose of our research was to compare allelopathic effects of the

125leaves of the three invasive species - A. petiolata, L. maackii and R. ficaria - on

126germination, growth, and/or reproduction of other test plant species, using three different

127leaf extract concentrations. We predicted that L. maackii would overall be the most

128allelopathic of the invasive species (e.g., Dorning and Cipollini, 2006, Cipollini et al.

1292008a), followed by A. petiolata (e.g., McCarthy and Hanson 1998) and R. ficaria. We

130predicted that A. petiolata would have little to no impact on other species in the

131Brassicaceae, but have impacts on species in other plant families (e.g., Cipollini et al.

1322008a). We also predicted that negative effects would increase with concentration of leaf

133extract.

6 6 134 Methods

135Extract Preparation

136 During the spring, leaf extracts were made from locally-collected leaves of L.

137maackii, A. petiolata and R. ficaria. Leaves were soaked for 48 hours in distilled water

138and then filtered. The extracts were then diluted to three different concentrations: 0.1,

1390.2, and 0.3 g fresh leaf tissue/mL distilled water. The two low concentrations used were

140similar to previous studies (Dorning and Cipollini 2006, Cipollini et al. 2008a). An

141additional higher concentration (0.3 g leaf/mL) was used in our current studies. While

142we have no information about natural concentrations of allelochemicals in the field, this

143high concentration represents approximately 30% of a mature L. maackii leaf in 1 mL of

144water (Dorning and Cipollini 2006), which is likely within field levels. Extracts were

145stored in the freezer until the start of an experiment and stored at 4ºC for the duration of

146the experiments. For all experiments, we used the fully factorial treatment combinations

147of extract type or species (A. petiolata, L. maackii or R. ficaria) and extract concentration

148(0.1, 0.2 or 0.3 g leaf/mL), for a total of 9 extract treatment combinations (3 species x 3

149extract concentrations = 9 experimental treatment combinations).

151Germination and reproduction of Arabidopsis in potting soil

152 In May of 2008, we planted 10 seeds of Arabidopsis thaliana into 100 mL pots

153containing potting soil (Pro-Mix BX, Premier Horticulture, Inc., Quakertown, PA) and

1541mL of slow release fertilizer (Osmocote, The Scotts Company, Marysville, OH).

155Arabidopsis thaliana was chosen as a target species due to its sensitivity to

156allelochemicals (Pennacchio et al. 2005) and its successful use in previous allelopathy

7 7 157studies (Cipollini et al. 2008a, Cipollini and Dorning 2008). Four replicates were used

158for each treatment combination (3 species x 3 concentrations x 4 replicates = 36

159experimental units). Additionally, there were also four replicate controls that received

160distilled water as a treatment, for a total of 40 pots in the experiment. Pots with seeds

161were immediately treated with 10mL of their specified extract (or control). The number

162of germinated plants in each pot was recorded every day for 2 weeks, at which time

163plants were thinned to one plant per pot. No plants germinated after 7 days. Each pot

164was treated with 10mL of extract every other week and water was given to the plants as

165needed. We performed the experiment in an air-conditioned growth room equipped with

166grow lights with high output fluorescent lights. Light levels were ~50 μmol/m2·s PAR

167and set on a timer for 15 h days and 9 h nights. We measured date of first flowering.

168After 13 weeks, we counted the number of siliques per plant and we collected 10

169randomly selected siliques from each plant to assess seed mass per silique. Two plants

170died during the experiment and were therefore not included in the analysis of final

171measurements.

172 For the effect of extract concentration on germination over 7 days, we performed

173a Multivariate Analysis of Variance (MANOVA) for each species, using the number

174germinated as a separate variable in the model (Von Ende 1993). When significance was

175found in the MANOVA using Wilk’s λ, we ran separate univariate Analyses of Variance

176(ANOVAs) for each date, followed by Tukey’s test to determine significant differences

177between treatments. For the final response variables, due to constraints of the design we

178were unable to perform fully-crossed two-way ANOVAs for the two factors of species

179and extract concentration with the control treatments in the model. We first performed a

8 8 180series of three two-way ANOVAs with the factors of species and concentration and their

181interaction on the response variables of days to flowering, silique number and seed mass.

182There was a significant effect of species for the response variables of silique number and

183days to flowering (F2,25 = 3.98, p = 0.031 and F2,25 = 3.42, p = 0.049, respectively). There

184were no significant differences for the factor of concentration or the interaction between

185concentration and species for any response variable.

186 One major objective of this study was to statistically compare differences between

187species and the control. Since the effect of concentration was not significant for any

188response variable, we made a post hoc decision to remove the factor of concentration

189from the model. We then performed a MANOVA with the response variables of days to

190flowering, silique number and seed mass with the factor of extract type, either control or

191one of the three invasive species. When significance was found in the ANOVA using

192Wilk’s λ, we ran separate univariate ANOVAs for each response variable, followed by

193Tukey’s test to determine significance between means. We set α at 0.05 for all tests and

194used Type III sums of squares in this unbalanced design. Minitab was used for all

195statistical analyses (Ryan et al. 2005).

197Reproduction of Arabidopsis in field soil

198 In August of 2009, we planted Arabidopsis thaliana (L.) Heynh (Brassicaceae)

199into 100 mL pots containing field soil, locally-collected in a woodlot area free of invasive

200species. Four replicates were used for each treatment combination (3 species x 3

201concentrations x 4 replicates = 36 experimental units). Because we had found with

202previous treatments that A. petiolata extracts served as a negative control for A. thaliana

9 9 203(see results above and Cipollini et al. 2008a) and because of issues with data analysis,

204using a control with our design, we did not use a control of no extract for this study. Pots

205with seeds were immediately treated with 10mL of their specified extract. Plants were

206thinned to one plant per pot one week later. Each pot was treated with 10mL of extract

207every two weeks and water was given to the plants as needed. Ten mL of 0.4g/L

208fertilizer (Peters 20-20-20 N-P-K plus micronutrients; Grace-Sierra, Milpitas, CA)

209dissolved in distilled water were added approximately every other week. We performed

210the experiment in an air-conditioned growth room equipped with grow lights with high

211output fluorescent lights. Light levels were ~50 μmol μmol/m2·s PAR and set on a timer

212for 15 h days and 9 h nights. After 10 weeks, we counted the number of siliques per

213plant. We performed an ANOVA with the response variable of silique number with the

214fully-crossed factors of species and extract concentration, followed by Tukey’s test to

215determine significance between means. We set α at 0.05 for all tests.

217Germination of test species on paper

218 The allelopathic potential on germination removing any soil effects was further

219explored by applying extracts to three agricultural species in three separate plant families:

220Brassica oleracea ‘Copenhagen Early Market' (Brassicaceae), Lactuca sativa ‘Grand

221Rapids, Tipburn Resistant’(Asteraceae) and Ocimum basilicum (Laminaceae). We chose

222these species since they were readily available, germinate easily and represent different

223plant families. Additionally, agricultural species such as lettuce and radish are frequently

224used in allelopathy studies (McCarthy and Hanson 1998, Pisula and Meiners 2010). Four

225replicates were used for each treatment combination (3 extract species x 3 concentrations

10 10 226x 3 test species x 4 replicates = 108 experimental units). Additionally, there were also

227four replicate controls per test species that received distilled water as a treatment, for a

228total of 120 experimental units in the experiment. Ten seeds of each appropriate species

229were placed on folded paper towels, which were watered with 10 ml of extract solution

230(or control). Paper towels were placed in plastic sandwich bags and placed under

231fluorescent lights with a daylength of 14 hours. Germination (measured as emergence of

232the radicle) was followed for 28 days. No additional seeds germinated after 14 days.

233 We analyzed the number germinated after 14 days using a fully-crossed three-way

234ANOVA with the factors of extract type (A. petiolata, L. maackii or R. ficaria), extract

235concentration (0.1, 0.2 or 0.3 g/mL) and test species (B. oleracea, L. sativa or O.

236basilicum). Data were transformed prior to analysis to meet model assumptions. We

237used Tukey’s test to determine significance between means. We set α at 0.05 for all tests.

238Because we could not use our control treatments directly in our full model and because

239we want to determine which extracts actually inhibit germination compared to the

240control, we performed a series of nine one-way ANOVAs for each test species and for

241each extract species separately with the factor of concentration (0, 0.1, 0.2 or 0.3 g

242leaf/mL) as the source of variation.

244Results

245Germination and reproduction of Arabidopsis in potting soil

246 For the germination over 7 days, there was a significant difference for L. maackii

247in the MANOVA (F21, 17 = 3.398, p = 0.007). In the univariate ANOVA, there was

248significant delay in germination for the first two days of the time course (F3,12 = 9.13, p =

11 11 2490.002 and F3,12 = 13.80, p < 0.001). For the first day of germination, there were less

250seeds germinated in all extract treatments compared to the control (Fig. 1). For the

251second day of germination, there were less seeds germinated in the 0.2 g/mL and 0.3

252g/mL concentrations compared to the control and the 0.1 g/mL concentration (Fig. 1).

253For the final response variables, there was a significant effect of extract type in the

254MANOVA (F9,78 = 2.038, p = 0.046). In the ANOVA, there was significant effect of

255extract type for silique number (F3,34 = 2.89, p = 0.049) and a near significant effect of

256extract type for flowering (F3,34 = 2.56, p = 0.071). There were significantly less siliques

257in the L. maackii extract treatment compared to the control and A. petiolata extract

258treatments, with the R. ficaria extract treatment intermediate between the two groups

259(Fig. 2). Because the effect of species on days to flowering was significant in the first

260full ANOVA model, we present here the means for each extract treatment to investigate

261the nature of the effect (Fig. 3). Flowering in plants treated with R. ficaria extracts were

262slightly delayed compared to A. petiolata extract treatments at p = 0.10.

264Reproduction of Arabidopsis in field soil

265 In the ANOVA, there was a significant effect of extract type on silique number

266(F2, 27 = 3.55, p = 0.043) and a near-significant effect of extract concentration on silique

267number (F2, 27 = 3.31, p = 0.052). There were more siliques produced by plants treated

268with the A. petiolata extracts compared to plants treated with the L. maackii or R. ficaria

269extracts (Fig. 4).

12 12 271Germination of test species on paper

272 All of the seeds of L. sativa and B. oleracea germinated in each of the four control

273replicates. In the control for O. basilicum, nearly all germinated (mean ± SE = 9.3 ± 0.5).

274In the ANOVA, there was a significant effect of test species, extract species and extract

275concentration on germination (Table 1). Across all other treatments, B. oleracea (8.4 ±

2760.4) and L. sativa (7.6 ± 0.4) and had higher germination than O. basilicum (6.0 ± 0.5).

277Across all other treatments, there was significantly lower germination in A. petiolata

278extract treatments (6.3 ± 0.5) compared to L. maackii and R. ficaria extract treatments

279(8.1 ± 0.3 and 7.6 ± 0.5, respectively). Across all other treatments, with each increase in

280concentration, there was a decrease in germination (9.1 ± 0.3, 7.5 ± 0.5, 5.5 ± 0.5 for 0.1

281g/mL, 0.2 g/mL and 0.1 g/mL, respectively). There was a significant effect of the

282interaction of extract species with test species and with extract concentration (Table 1).

283The effect of extract species varied with test species, with A. petiolata extracts having the

284strongest effects on germination of L. sativa and O. basilicum and L. maackii extracts

285having strongest effects on germination of B. oleracea (Fig. 5). Extracts of R. ficaria had

286stronger effects than extracts of L. maackii on germination of O. basilicum and L. sativa.

287The effect of extract concentration varied with extract species, with greater inhibition of

288germination with increasing concentration in extracts of A. petiolata and R. ficaria

289compared to extracts of L. maackii, which had smaller changes with increasing extract

290concentration (Fig. 6). Additionally, there was a significant three way interaction of test

291species, extract concentration and extract species (Table 1). Essentially, each test species

292responded to increasing concentration of extracts of each species in different ways. For

293example, while increasing concentrations of L. maackii extract had strong effects on

13 13 294germination of B. oleracea, increasing concentration of L. maackii had little effects on

295germination of L. sativa and O. basilicum (Fig. 7).

297Discussion

298 In our experiments, we confirmed the presence of allelopathy from leaves of three

299invasive Midwestern species and, more importantly, provided information on the

300comparative effect of each. Pisula and Meiners (2010) similarly used standardized

301methods to compare a suite of 10 invasive species, but they did not use either L. maackii

302or R. ficaria in their study. Pisula and Meiners (2010) found A. petiolata to be one of the

303four highest inhibitory invasive species, though only one test species, radish, was used.

304Our comparative approach was enhanced by the use of multiple test species, as previous

305work shows that allelopathic effects vary with test species (Prati and Bossdorf 2004, Orr

306and Rudgers, 2005, McEwan et al. 2010).

307 Allelopathic effects of each invasive species varied with test species. Generally,

308effects of extracts of L. maackii were greatest on species from the Brassicaceae, while

309extracts of A. petiolata and R. ficaria had the highest inhibitory effect on species in other

310families (Asteraceae and Laminaceae). Extracts of A. petiolata did not strongly affect the

311two species in the Brassicaceae, as was found in previous work (Cipollini et al. 2008a).

312This is most likely caused by the similar chemical composition of plants in the same

313family, which makes A. thaliana and B. oleracea more resistant to the effects of these

314chemicals. Effects of extracts of R. ficaria were generally weaker though still had

315allelopathic effects, particularly at the highest concentration. Ranunculus ficaria had

316strongest effects on germination of L. sativa and O. basilicum.

14 14 317 Allelopathic effects of each invasive species also varied by experimental venue.

318Extracts of R. ficaria showed a trend to reduce reproduction and to delay flowering in A.

319thaliana in potting soil, while extracts of R. ficaria significantly inhibited silique

320production of A. thaliana in field soil. There was also higher seed production in potting

321soil compared to field soils, suggesting differing growing conditions, which may have

322influenced the differential response to allelopathy (Cipollini et al. 2008a, Cipollini and

323Dorning 2008). Interestingly, we found little long-term effect of extract of L. maackii on

324germination in A. thaliana in potting soil, as germination was only delayed by 2 days.

325This contrasts previous work, which showed 50% reduction of germination of A. thaliana

326on filter paper after one week (Cipollini and Dorning 2006). There was no significant

327effect of extract concentration on response variables in potting soil and only a near-

328significant effect in field soil, in comparison to previous work that found strong effects of

329concentration in similar experimental conditions (Cipollini et al. 2008a). In comparison,

330differing concentrations did affect germination on paper. Further, the concentration

331affect varied with extract species and with test species, increasing the difficulty in finding

332a generalizable result from this study.

333 While our study provides some interesting insights into the comparative effects of

334allelopathy for these three species, there is still much research to be done to fully evaluate

335the allelopathic potential of these species in the field. In order to evaluate whether the

336allelopathic effects truly represent novel weapons to native plants, a comparative

337approach using co-occurring native species should be used (Barto et al., 2010b, McEwan

338et al. 2010). Additionally, a combination of field and laboratory experiments should seek

339to identify allelopathic compounds and determine their bioactivity and persistence in situ

15 15 340(Inderjit and Callaway 2003, Barto and Cipollini 2009). Nevertheless, our study provides

341important information on the relative allelopathic impact of each invasive species, as well

342as illustrates the importance of using multiple test species and experimental conditions to

343incorporate consideration of differing sensitivities to and conditions for allelopathic

344effects. Finally, our study also importantly provides additional information about the

345allelopathic potential of R. ficaria, a species for which there is no published information

346despite increasing interest in its role as an invasive species (Axtell et al. 2010).

348Acknowledgements

349 Doug Burks, Don Troike, Doug Woodmansee, and the students of BIO 440/441

350provided valuable comments throughout the design and completion of this experiment.

351Don Cipollini also provided assistance and expertise when needed. We thank

352Wilmington College’s Instructional Development and Resources Committee for

353supporting a writing workshop during which this paper was produced. We thank Laura

354Struve and Michele Beery for creating and facilitating this workshop and all the

355participants for their support.

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22 22 483Table 1. Three-way Analysis of Variance (ANOVA) results for germination of three test

484species - Brassica oleracea, Lactuca sativa and Ocimum basilicum - treated with extracts

485of three invasive species - Alliaria petiolata, Lonicera maackii and Ranunculus ficaria at

486three extract concentrations.

487Source of variation Df F p

488Test Species 2 29.93 <0.001

489Extract Species 2 11.63 <0.001

490Extract Concentration 2 62.86 <0.001

491Extract Species*Test Species 4 11.42 <0.001

492Extract Species*Extract Concentration 4 3.50 0.011

493Test Species*Extract Concentration 4 0.85 0.495

494Extract Species* Test Species*Concentration 8 6.56 <0.001

495Error 81

23 23 497Figure legends

498Fig. 1. Mean number of seeds germinated (± SE) of Arabidopsis thaliana for control (0

499g/mL) and three concentrations (0.1, 0.2 and 0.3 g fresh leaf/mL distilled H2O) of

500Lonicera maackii leaf extracts. Asterisks indicate dates for which there were significant

501differences between treatments. Letters indicate significant differences within each date

502using Tukey’s test at α = 0.05.

504Fig. 2. Mean number of siliques (± SE) of Arabidopsis thaliana in potting soil for

505treatments containing no invasive species (control) and separate leaf extracts of three

506invasive species - Alliaria petiolata, Lonicera maackii and Ranunculus ficaria.

507Treatments with different letters are significantly different from each other using Tukey’s

508test at α = 0.05.

510Fig. 3. Mean days to flowering (± SE) of Arabidopsis thaliana for treatments containing

511no invasive species (control) and separate leaf extracts of three invasive species - Alliaria

512petiolata, Lonicera maackii and Ranunculus ficaria. Treatments with different letters are

513significantly different from each other using Tukey’s test at α = 0.10.

515Fig. 4. Mean number of siliques (± SE) of Arabidopsis thaliana in field soil for

516treatments containing separate leaf extracts of three invasive species - Alliaria petiolata,

517Lonicera maackii and Ranunculus ficaria. Treatments with different letters are

518significantly different from each other using Tukey’s test at α = 0.05.

24 24 520Fig. 5. Mean number of seeds germinated (± SE) of three test species - Brassica

521oleracea, Lactuca sativa and Ocimum basilicum - treated with leaf extracts of three

522invasive species - Alliaria petiolata, Lonicera maackii and Ranunculus ficaria across

523three extract concentration treatments.

525Fig. 6. Mean number of seeds germinated (± SE) across three test species treated with

526leaf extracts of three invasive species - Alliaria petiolata, Lonicera maackii and

527Ranunculus ficaria – at three leaf extract concentrations (0.1, 0.2 and 0.3 g fresh leaf/mL

528distilled H2O).

530Fig. 7. Mean number of seeds germinated (± SE) of three test species - Brassica

531oleracea, Lactuca sativa and Ocimum basilicum - treated with leaf extracts of three

532invasive species - Alliaria petiolata, Lonicera maackii and Ranunculus ficaria at three

533extract concentrations. Asterisks indicate significant difference within each extract

534species from the control in one-way ANOVAs using Tukey’s test at α = 0.05.

25 25 10

d * e t

a 8 a n i

e 6 * G

2 0 g/mL r

e b 0.1 g/mL b b 0.2 g/mL

u 0.3 g/mL N

1 2 3 4 5 6 7

539 Days

26 26 900 p = 0.049 a a 800 s e u q i

l ab i 700 S

e 600 b m u

400 Control A. petiolata L. maackii R. ficaria

540 Extract Type

27 27 p = 0.071 28 ab ab b g n i

r 26 e a w o l F

o 24 t

20 Control A. petiolata L. maackii R. ficaria

541 Extract Type

28 28 350 a

s 300 e

u b q b i l i S

f 250 o

r e b m

u 200 N

150 A. petiolata L. maackii R. ficaria

542 Extract Type

29 29 d A. petiolata e

a L. maackii n

i R. ficaria

m 10 r e G 8 s d e

r 4 e b

m 2 u N 0 B. oleracea L. sativa O. basilicum

543 Test Species

30 30 d 0.1 g/mL e

a 0.2 g/mL n i 0.3 g/mL

m 10 r e G 8 s d e

r 4 e b

m 2 u N 0 A. petiolata L. maackii R. ficaria

544 Extract Type

31 31 10 *

8 * * 6

4 A. petiolata * L. maackii 2 R. ficaria

0 Brassica oleracea d e t a

n 10 i * m *

r 8 e G

6 * s d *

e 4 * e S

r 0 Lactuca sativa e b m

u 10 N

0 Ocimum basilicum

0.1 0.2 0.3 Extract Concentration (in 545 g fresh leaf/mL distilled water)