1, Kyle Titus

FAX: 937-383-8530

Invasive species pose a threat worldwide, negatively impacting biodiversity (Wilcove et al. 1998, McGeoch et al. 2010) and exerting significant economic costs (Pimentel et al. 2005). One focus in invasive species ecology is to determine factors that contribute to the success of invasive species (Sakai et al. 2001, Levine et al. 2003). These factors can range from life history traits (Kolar and Lodge 2001) to release from natural enemies (Keane and Crawley 2002). One hypothesis to explain invasive species success is the novel weapons hypothesis (Bais et al. 2003), whereby an invading species possesses a trait novel to the invaded ecosystem. The invasive species can then take advantage of this trait in its new ecosystem during interactions with native species that are evolutionarily-naïve to the trait. In plants, allelopathy can represent a novel weapon (Hierro and Callaway 2003, Callaway and Ridenour 2004). Allelopathy is simply the release of a chemical from the roots or leaves that affects germination, growth and/or reproduction of surrounding species (Rice 1974). Plants that exude these chemicals may be more likely to dominate in a new environment because their neighbors in their native range have evolved resistance or tolerance to their allelochemicals, while neighbors in their invaded range have not (Callaway and Aschehoug 2000). Allelopathy can have direct plant-to-plant effects, whereby allelochemicals directly impact another species (Dorning and Cipollini 2006). Alternatively, allelopathy may have indirect effects on other plants, such as through changing soil ecology or mutualisms (Stinson et al., 2006; Callaway et al. 2008, Zhang et al. 2009). Allelopathic effects may vary depending on target species (Cipollini et al. 2008a) or may vary by conditions such as life stage (Barto et al. 2010a) and nutrients (Cipollini et al. 2008a).

Allelopathy can be studied in a variety of experimental ways (Inderjit and Callaway 2003), with varying degrees of realism and control. Experiments with the greatest amount of experimental control, yet lowest amount of realism, are simple germination and growth experiments involving the application of specific chemicals or plant extracts with putative allelochemicals, usually in Petri dishes with a paper substrate (e.g., Dorning and Cipollini 2006, Cipollini et al. 2008b, McEwan et al. 2010). Other studies seek to increase the degree of realism at the cost of experimental control in greenhouse studies and field experiments, many times with the use of activated carbon as a manipulative tool (Ridenour and Callaway 2001, Cipollini et al. 2008, Cipollini and Schradin 2011). Field experiments show the greatest amount of realism and ecological relevance yet can be difficult in teasing out exact mechanism due to low amounts of experimental control. Generally, studies of allelopathy start with simple, controlled laboratory experiments before scaling up to field experiments.

Three important invasive species in forests and riparian areas in the Midwestern United States that have evidence of allelopathy are garlic mustard (Alliaria petiolata (Bieb.) Cavara & Grand – Brassicaceae), Amur honeysuckle (Lonicera maackii (Rupr.) Maxim - Caprifoliaceae) and lesser celandine (Ranunculus ficaria L. - Ranunculaceae). Lonicera maackii, native to Asia, in found in the eastern half of the United States (USDA 2011) and negatively affects trees and understory plants (Gould and Gorchov 2000, Collier et al. 2002, Hartman and McCarthy 2004). Leaf extracts of L. maackii inhibit germination of several test species in the laboratory (Dorning and Cipollini 2006, Cipollini et al. 2008b) and affect growth of Arabidopsis thaliana in the greenhouse (Cipollini et al. 2008a). Field soils collected from areas infested with L. maackii negatively impacted growth of A. thaliana (Cipollini and Dorning 2008). Cipollini et al. (2008) were unable to demonstrate any allelopathic effects of L. maackii on Impatiens capensis in the field with the use of activated carbon, though sample size issues limited the conclusions of the study.

While there is some evidence of allelopathy for all of these species, there is no research that investigates their comparative allelopathic effects. Other studies have taken a comparative approach to studying allelopathy and allelochemicals, either comparing a suite of invasive species (Pisula and Meiners 2010) or comparing an invasive species to co-occurring similar native species (Barto et al. 2010b, McEwan et al. 2010). Because allelopathic effects can vary with the species on which they are tested (Cipollini et al. 2008a, McEwan et al. 2010), we tested multiple species to have more generalizable results. A comparative approach using more than one test and invasive species would be useful in prioritizing restoration activities and possible use of mitigation treatments such as activated carbon (Kulmatiski and Beard 2006), particularly in areas invaded by more than one species. The purpose of our research was to compare allelopathic effects of the leaves of the three invasive species - A. petiolata, L. maackii and R. ficaria - on germination, growth, and/or reproduction of other test plant species, using three different leaf extract concentrations. We predicted that L. maackii would overall be the most allelopathic of the invasive species (e.g., Dorning and Cipollini, 2006, Cipollini et al. 2008a), followed by A. petiolata (e.g., McCarthy and Hanson 1998) and R. ficaria. We predicted that A. petiolata would have little to no impact on other species in the Brassicaceae, but have impacts on species in other plant families (e.g., Cipollini et al. 2008a). We also predicted that negative effects would increase with concentration of leaf extract.

During the spring, leaf extracts were made from locally-collected leaves of L. maackii, A. petiolata and R. ficaria. Leaves were soaked for 48 hours in distilled water and then filtered. The extracts were then diluted to three different concentrations: 0.1, 0.2, and 0.3 g fresh leaf tissue/mL distilled water. The two low concentrations used were similar to previous studies (Dorning and Cipollini 2006, Cipollini et al. 2008a). An additional higher concentration (0.3 g leaf/mL) was used in our current studies. While we have no information about natural concentrations of allelochemicals in the field, this high concentration represents approximately 30% of a mature L. maackii leaf in 1 mL of water (Dorning and Cipollini 2006), which is likely within field levels. Extracts were stored in the freezer until the start of an experiment and stored at 4ºC for the duration of the experiments. For all experiments, we used the fully factorial treatment combinations of extract type or species (A. petiolata, L. maackii or R. ficaria) and extract concentration (0.1, 0.2 or 0.3 g leaf/mL), for a total of 9 extract treatment combinations (3 species x 3 extract concentrations = 9 experimental treatment combinations).

In May of 2008, we planted 10 seeds of Arabidopsis thaliana into 100 mL pots containing potting soil (Pro-Mix BX, Premier Horticulture, Inc., Quakertown, PA) and 1mL of slow release fertilizer (Osmocote, The Scotts Company, Marysville, OH). Arabidopsis thaliana was chosen as a target species due to its sensitivity to allelochemicals (Pennacchio et al. 2005) and its successful use in previous allelopathy studies (Cipollini et al. 2008a, Cipollini and Dorning 2008). Four replicates were used for each treatment combination (3 species x 3 concentrations x 4 replicates = 36 experimental units). Additionally, there were also four replicate controls that received distilled water as a treatment, for a total of 40 pots in the experiment. Pots with seeds were immediately treated with 10mL of their specified extract (or control). The number of germinated plants in each pot was recorded every day for 2 weeks, at which time plants were thinned to one plant per pot. No plants germinated after 7 days. Each pot was treated with 10mL of extract every other week and water was given to the plants as needed. We performed the experiment in an air-conditioned growth room equipped with grow lights with high output fluorescent lights. Light levels were ~50 μmol/m²·s PAR and set on a timer for 15 h days and 9 h nights. We measured date of first flowering. After 13 weeks, we counted the number of siliques per plant and we collected 10 randomly selected siliques from each plant to assess seed mass per silique. Two plants died during the experiment and were therefore not included in the analysis of final measurements.

For the effect of extract concentration on germination over 7 days, we performed a Multivariate Analysis of Variance (MANOVA) for each species, using the number germinated as a separate variable in the model (Von Ende 1993). When significance was found in the MANOVA using Wilk’s λ, we ran separate univariate Analyses of Variance (ANOVAs) for each date, followed by Tukey’s test to determine significant differences between treatments. For the final response variables, due to constraints of the design we were unable to perform fully-crossed two-way ANOVAs for the two factors of species and extract concentration with the control treatments in the model. We first performed a series of three two-way ANOVAs with the factors of species and concentration and their interaction on the response variables of days to flowering, silique number and seed mass. There was a significant effect of species for the response variables of silique number and days to flowering (F_2,25 = 3.98, p = 0.031 and F_2,25 = 3.42, p = 0.049, respectively). There were no significant differences for the factor of concentration or the interaction between concentration and species for any response variable.

One major objective of this study was to statistically compare differences between species and the control. Since the effect of concentration was not significant for any response variable, we made a post hoc decision to remove the factor of concentration from the model. We then performed a MANOVA with the response variables of days to flowering, silique number and seed mass with the factor of extract type, either control or one of the three invasive species. When significance was found in the ANOVA using Wilk’s λ, we ran separate univariate ANOVAs for each response variable, followed by Tukey’s test to determine significance between means. We set α at 0.05 for all tests and used Type III sums of squares in this unbalanced design. Minitab was used for all statistical analyses (Ryan et al. 2005).

In August of 2009, we planted Arabidopsis thaliana (L.) Heynh (Brassicaceae) into 100 mL pots containing field soil, locally-collected in a woodlot area free of invasive species. Four replicates were used for each treatment combination (3 species x 3 concentrations x 4 replicates = 36 experimental units). Because we had found with previous treatments that A. petiolata extracts served as a negative control for A. thaliana (see results above and Cipollini et al. 2008a) and because of issues with data analysis, using a control with our design, we did not use a control of no extract for this study. Pots with seeds were immediately treated with 10mL of their specified extract. Plants were thinned to one plant per pot one week later. Each pot was treated with 10mL of extract every two weeks and water was given to the plants as needed. Ten mL of 0.4g/L fertilizer (Peters 20-20-20 N-P-K plus micronutrients; Grace-Sierra, Milpitas, CA) dissolved in distilled water were added approximately every other week. We performed the experiment in an air-conditioned growth room equipped with grow lights with high output fluorescent lights. Light levels were ~50 μmol μmol/m²·s PAR and set on a timer for 15 h days and 9 h nights. After 10 weeks, we counted the number of siliques per plant. We performed an ANOVA with the response variable of silique number with the fully-crossed factors of species and extract concentration, followed by Tukey’s test to determine significance between means. We set α at 0.05 for all tests.

The allelopathic potential on germination removing any soil effects was further explored by applying extracts to three agricultural species in three separate plant families: Brassica oleracea ‘Copenhagen Early Market' (Brassicaceae), Lactuca sativa ‘Grand Rapids, Tipburn Resistant’(Asteraceae) and Ocimum basilicum (Laminaceae). We chose these species since they were readily available, germinate easily and represent different plant families. Additionally, agricultural species such as lettuce and radish are frequently used in allelopathy studies (McCarthy and Hanson 1998, Pisula and Meiners 2010). Four replicates were used for each treatment combination (3 extract species x 3 concentrations x 3 test species x 4 replicates = 108 experimental units). Additionally, there were also four replicate controls per test species that received distilled water as a treatment, for a total of 120 experimental units in the experiment. Ten seeds of each appropriate species were placed on folded paper towels, which were watered with 10 ml of extract solution (or control). Paper towels were placed in plastic sandwich bags and placed under fluorescent lights with a daylength of 14 hours. Germination (measured as emergence of the radicle) was followed for 28 days. No additional seeds germinated after 14 days.

We analyzed the number germinated after 14 days using a fully-crossed three-way ANOVA with the factors of extract type (A. petiolata, L. maackii or R. ficaria), extract concentration (0.1, 0.2 or 0.3 g/mL) and test species (B. oleracea, L. sativa or O. basilicum). Data were transformed prior to analysis to meet model assumptions. We used Tukey’s test to determine significance between means. We set α at 0.05 for all tests. Because we could not use our control treatments directly in our full model and because we want to determine which extracts actually inhibit germination compared to the control, we performed a series of nine one-way ANOVAs for each test species and for each extract species separately with the factor of concentration (0, 0.1, 0.2 or 0.3 g leaf/mL) as the source of variation.
Results

Germination and reproduction of Arabidopsis in potting soil

For the germination over 7 days, there was a significant difference for L. maackii in the MANOVA (F_{21, 17} = 3.398, p = 0.007). In the univariate ANOVA, there was significant delay in germination for the first two days of the time course (F_3,12 = 9.13, p = 0.002 and F_3,12 = 13.80, p < 0.001). For the first day of germination, there were less seeds germinated in all extract treatments compared to the control (Fig. 1). For the second day of germination, there were less seeds germinated in the 0.2 g/mL and 0.3 g/mL concentrations compared to the control and the 0.1 g/mL concentration (Fig. 1). For the final response variables, there was a significant effect of extract type in the MANOVA (F_9,78 = 2.038, p = 0.046). In the ANOVA, there was significant effect of extract type for silique number (F_3,34 = 2.89, p = 0.049) and a near significant effect of extract type for flowering (F_3,34 = 2.56, p = 0.071). There were significantly less siliques in the L. maackii extract treatment compared to the control and A. petiolata extract treatments, with the R. ficaria extract treatment intermediate between the two groups (Fig. 2). Because the effect of species on days to flowering was significant in the first full ANOVA model, we present here the means for each extract treatment to investigate the nature of the effect (Fig. 3). Flowering in plants treated with R. ficaria extracts were slightly delayed compared to A. petiolata extract treatments at p = 0.10.

In the ANOVA, there was a significant effect of extract type on silique number (F_{2, 27} = 3.55, p = 0.043) and a near-significant effect of extract concentration on silique number (F_{2, 27} = 3.31, p = 0.052). There were more siliques produced by plants treated with the A. petiolata extracts compared to plants treated with the L. maackii or R. ficaria extracts (Fig. 4).

All of the seeds of L. sativa and B. oleracea germinated in each of the four control replicates. In the control for O. basilicum, nearly all germinated (mean ± SE = 9.3 ± 0.5). In the ANOVA, there was a significant effect of test species, extract species and extract concentration on germination (Table 1). Across all other treatments, B. oleracea (8.4 ± 0.4) and L. sativa (7.6 ± 0.4) and had higher germination than O. basilicum (6.0 ± 0.5). Across all other treatments, there was significantly lower germination in A. petiolata extract treatments (6.3 ± 0.5) compared to L. maackii and R. ficaria extract treatments (8.1 ± 0.3 and 7.6 ± 0.5, respectively). Across all other treatments, with each increase in concentration, there was a decrease in germination (9.1 ± 0.3, 7.5 ± 0.5, 5.5 ± 0.5 for 0.1 g/mL, 0.2 g/mL and 0.1 g/mL, respectively). There was a significant effect of the interaction of extract species with test species and with extract concentration (Table 1). The effect of extract species varied with test species, with A. petiolata extracts having the strongest effects on germination of L. sativa and O. basilicum and L. maackii extracts having strongest effects on germination of B. oleracea (Fig. 5). Extracts of R. ficaria had stronger effects than extracts of L. maackii on germination of O. basilicum and L. sativa. The effect of extract concentration varied with extract species, with greater inhibition of germination with increasing concentration in extracts of A. petiolata and R. ficaria compared to extracts of L. maackii, which had smaller changes with increasing extract concentration (Fig. 6). Additionally, there was a significant three way interaction of test species, extract concentration and extract species (Table 1). Essentially, each test species responded to increasing concentration of extracts of each species in different ways. For example, while increasing concentrations of L. maackii extract had strong effects on germination of B. oleracea, increasing concentration of L. maackii had little effects on germination of L. sativa and O. basilicum (Fig. 7).

In our experiments, we confirmed the presence of allelopathy from leaves of three invasive Midwestern species and, more importantly, provided information on the comparative effect of each. Pisula and Meiners (2010) similarly used standardized methods to compare a suite of 10 invasive species, but they did not use either L. maackii or R. ficaria in their study. Pisula and Meiners (2010) found A. petiolata to be one of the four highest inhibitory invasive species, though only one test species, radish, was used. Our comparative approach was enhanced by the use of multiple test species, as previous work shows that allelopathic effects vary with test species (Prati and Bossdorf 2004, Orr and Rudgers, 2005, McEwan et al. 2010).

Allelopathic effects of each invasive species varied with test species. Generally, effects of extracts of L. maackii were greatest on species from the Brassicaceae, while extracts of A. petiolata and R. ficaria had the highest inhibitory effect on species in other families (Asteraceae and Laminaceae). Extracts of A. petiolata did not strongly affect the two species in the Brassicaceae, as was found in previous work (Cipollini et al. 2008a). This is most likely caused by the similar chemical composition of plants in the same family, which makes A. thaliana and B. oleracea more resistant to the effects of these chemicals. Effects of extracts of R. ficaria were generally weaker though still had allelopathic effects, particularly at the highest concentration. Ranunculus ficaria had strongest effects on germination of L. sativa and O. basilicum.

Allelopathic effects of each invasive species also varied by experimental venue. Extracts of R. ficaria showed a trend to reduce reproduction and to delay flowering in A. thaliana in potting soil, while extracts of R. ficaria significantly inhibited silique production of A. thaliana in field soil. There was also higher seed production in potting soil compared to field soils, suggesting differing growing conditions, which may have influenced the differential response to allelopathy (Cipollini et al. 2008a, Cipollini and Dorning 2008). Interestingly, we found little long-term effect of extract of L. maackii on germination in A. thaliana in potting soil, as germination was only delayed by 2 days. This contrasts previous work, which showed 50% reduction of germination of A. thaliana on filter paper after one week (Cipollini and Dorning 2006). There was no significant effect of extract concentration on response variables in potting soil and only a near-significant effect in field soil, in comparison to previous work that found strong effects of concentration in similar experimental conditions (Cipollini et al. 2008a). In comparison, differing concentrations did affect germination on paper. Further, the concentration affect varied with extract species and with test species, increasing the difficulty in finding a generalizable result from this study.

While our study provides some interesting insights into the comparative effects of allelopathy for these three species, there is still much research to be done to fully evaluate the allelopathic potential of these species in the field. In order to evaluate whether the allelopathic effects truly represent novel weapons to native plants, a comparative approach using co-occurring native species should be used (Barto et al., 2010b, McEwan et al. 2010). Additionally, a combination of field and laboratory experiments should seek to identify allelopathic compounds and determine their bioactivity and persistence in situ (Inderjit and Callaway 2003, Barto and Cipollini 2009). Nevertheless, our study provides important information on the relative allelopathic impact of each invasive species, as well as illustrates the importance of using multiple test species and experimental conditions to incorporate consideration of differing sensitivities to and conditions for allelopathic effects. Finally, our study also importantly provides additional information about the allelopathic potential of R. ficaria, a species for which there is no published information despite increasing interest in its role as an invasive species (Axtell et al. 2010).
Acknowledgements

Doug Burks, Don Troike, Doug Woodmansee, and the students of BIO 440/441 provided valuable comments throughout the design and completion of this experiment. Don Cipollini also provided assistance and expertise when needed. We thank Wilmington College’s Instructional Development and Resources Committee for supporting a writing workshop during which this paper was produced. We thank Laura Struve and Michele Beery for creating and facilitating this workshop and all the participants for their support.

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Table 1. Three-way Analysis of Variance (ANOVA) results for germination of three test species - Brassica oleracea, Lactuca sativa and Ocimum basilicum - treated with extracts of three invasive species - Alliaria petiolata, Lonicera maackii and Ranunculus ficaria at three extract concentrations.

Source of variation Df F p

Test Species 2 29.93 <0.001

Extract Species 2 11.63 <0.001

Extract Concentration 2 62.86 <0.001

Extract Species*Test Species 4 11.42 <0.001

Extract Species*Extract Concentration 4 3.50 0.011

Test Species*Extract Concentration 4 0.85 0.495

Extract Species* Test Species*Concentration 8 6.56 <0.001

Error 81
Figure legends