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

1234567 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 species1011 Kendra Cipollini, Kyle Titus, and Crystal Wagner1213 Kendra Cipollini1, Kyle Titus2, and Crystal Wagner3. Wilmington College, 187014 Quaker Way, Wilmington, OH 45177, USA15 1Corresponding author (e-mail: [email protected]). 16 2Current address: (e-mail: [email protected])17 3Current address: (e-mail: [email protected])1819 Phone: 937-382-6661 x367 (w), 937-532-6128 (cell)20 FAX: 937-383-85301 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 fewer29siliques 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 affected33germination 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.3940Key words: allelopathy, exotic species, germination inhibition, leaf extracts, 41phytotoxicity2 2 42Introduction43 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 (USDA832011) 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 taken116a 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 leaf133extract.6 6 134 Methods135Extract Preparation136 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 concentration148(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).150151Germination and reproduction of Arabidopsis in potting soil152 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 between187species 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).196197Reproduction of Arabidopsis in field soil198 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 invasive200species. 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. Pots205with 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.216217Germination of test species on paper218 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 frequently224used in allelopathy studies (McCarthy and Hanson 1998, Pisula and Meiners 2010). Four225replicates 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-way234ANOVA 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.243244Results245Germination and reproduction of Arabidopsis in potting soil246 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 = 0.10.263264Reproduction of Arabidopsis in field soil265 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).27012 12 271Germination of test species on paper272 All of the seeds of L. sativa and B. oleracea germinated in each of the four control273replicates. 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 the284strongest 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 had286stronger 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 species292responded 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).296297Discussion298 In our experiments, we confirmed the presence of allelopathy from leaves of three299invasive 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 the311two 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. thaliana326on 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 of329concentration 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 finding332a generalizable result from this study.333 While our study provides some interesting insights into the comparative effects of334allelopathy for these three species, there is still much research to be done to fully evaluate335the 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 seek339to 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 provides341important information on the relative allelopathic impact of each invasive species, as well342as 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).347348Acknowledgements349 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. 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Three-way Analysis of Variance (ANOVA) results for germination of three test484species - 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 p488Test Species 2 29.93 <0.001489Extract Species 2 11.63 <0.001490Extract Concentration 2 62.86 <0.001491Extract Species*Test Species 4 11.42 <0.001492Extract Species*Extract Concentration 4 3.50 0.011493Test Species*Extract Concentration 4 0.85 0.495494Extract Species* Test Species*Concentration 8 6.56 <0.001495Error 8149623 23 497Figure legends498Fig. 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 = 0.05.503504Fig. 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’s508test at α = 0.05. 509510Fig. 3. Mean days to flowering (± SE) of Arabidopsis thaliana for treatments containing 511no invasive species (control) and separate leaf extracts of three invasive species - Alliaria512petiolata, Lonicera maackii and Ranunculus ficaria. Treatments with different letters are513significantly different from each other using Tukey’s test at α = 0.10. 514515Fig. 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. 51924 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.524525Fig. 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).529530Fig. 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 = 0.05.53553653753825 25 10 d * e t a 8 a n i m a r e 6 * G s d a e 4 eS b f o2 0 g/mL r e b 0.1 g/mL b b 0.2 g/mL m 0 b u 0.3 g/mL N1 2 3 4 5 6 7539 Days26 26 900 p = 0.049 a a 800 s e u q i l ab i 700 S b f o r e 600 b m uN 500400 Control A. petiolata L. maackii R. ficaria540 Extract Type27 27 p = 0.071 28 ab ab b g n i r 26 e a w o l F o 24 t s y aD 2220 Control A. petiolata L. maackii R. ficaria541 Extract Type28 28 350 a s 300 e u b q b i l i S f 250 o r e b m u 200 N150 A. petiolata L. maackii R. ficaria542 Extract Type29 29 d A. petiolata e t 12 a L. maackii n i R. ficaria m 10 r e G 8 s d e e 6 S f o r 4 e b m 2 u N 0 B. oleracea L. sativa O. basilicum543 Test Species30 30 d 0.1 g/mL e t 12 a 0.2 g/mL n i 0.3 g/mL m 10 r e G 8 s d e e 6 S f o r 4 e b m 2 u N 0 A. petiolata L. maackii R. ficaria544 Extract Type31 31 10 *8 * * 64 A. petiolata * L. maackii 2 R. ficaria0 Brassica oleracea d e t a n 10 i * m * r 8 e G6 * s d * e 4 * e S2 f o r 0 Lactuca sativa e b m u 10 N8 *6 *4 * *2 *0 Ocimum basilicum0.1 0.2 0.3 Extract Concentration (in 545 g fresh leaf/mL distilled water)32 32

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

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