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Influence of soil characteristics on the spatial distribution of Fallopia japonica (Japanese Knotweed) David Tian1, Christopher Neill2 Department of Biology1 Swarthmore College Swarthmore, PA 19081 The Ecosystems Center2 The Marine Biological Laboratory Woods Hole, MA 02543 SES Fall Semester 2015 Abstract Invasive species pose a serious threat to biodiversity and cause upwards of $120 billion worth of damage and maintenance costs annually. Very little is known about what controls the spatial distribution of invasive species. In the Woods Hole and Falmouth areas, Japanese knotweed is an aggressive and successful invasive species that grows along roadsides. It typically does not grow within oak-pine forests, which are characterized by native species. This stark contrast is the driving force behind my study. I sought to understand why Japanese knotweed does not grow in the oak-pine forest, as a means to investigate what determines the spatial distribution of invasive species. I tested the hypothesis that a lack of seed rain prevented Japanese knotweed from growing in the Oak-pine forest. Furthermore, I compared soil characteristics between Japanese knotweed and oak-pine forest soils to investigate if there were significant differences in organic matter, pH, extractable nitrogen, and salinity. Lastly, I conducted a germination experiment to investigate whether differences in soil characteristics and pH could influence germination rates of Japanese knotweed seeds. Seed traps indicate that Japanese knotweed seeds enter the oak-pine forest. Japanese knotweed soils tend to have lower percentages of organic matter, but have significantly higher pH, higher concentrations of extractable nitrogen in the forms of NH4+ and NO3-, as well as higher cation concentrations in terms of Na+, Ca+2, K+, and Mg+2. The germination experiment indicated that Japanese knotweed seeds germinated roughly half as well in natural oak-pine soils, compared to natural Japanese knotweed soils. Furthermore, pH has a significant effect on germination rates. Finally, a significantly higher number of seedlings sprouted from rhizomes planted in Japanese knotweed seed, as opposed to oak-pine soil. Japanese knotweed illustrates that soil characteristics, in particular pH, play an instrumental role in determining the spatial distribution of invasive species. Key words: Fallopia japonica, Japanese knotweed, oak-pine forest, soil characteristics, germination Introduction Invasive species pose a serious threat to biodiversity across the country. It has been estimated that 57% of federally listed imperiled species are affected by invasive species 1 (Wilcove et al. pg. 242). Fallopia japonica, commonly known as Japanese knotweed, is an invasive species that has spread across the majority of the United States and has been designated by the World Conservation Union as one of the worst invasive species in the world. Today, Japanese knotweed can be found in roughly three quarters of North America, in locations as remote as Alaska (Lowe et al. 2000). Overall, invasive species are a problem all over the world, but especially in the United States where invasive species cost the US economy roughly 120 billion dollars annually (Pimentel, Zuniga, and Morrison, 2005). Japanese knotweed was originally introduced to the United States from East Asia in the th 19 century as an ornamental plant, or a plant to stabilize soil in coastal areas. Today, it causes many infrastructure problems such as damage to concrete, roads, and walls, as well as a reduction in capacity of flood defense channels to carry water (Stone 2010). Due to all of these reasons, the control of the spread of invasive species is of utmost importance to conservation science (Kareiva and Marvier, 2010). The success of Japanese knotweed is due in large part to its rapid growth rate as well as its ability to form thick and dense colonies that completely crowd out native species, as well as its ability to block sunlight to shorter plants. Furthermore, part of the knotweed’s success is due to the ability of its rhizomes to extend 3 meters deep and 7 meters horizontally (Stone 2010). Currently, there is no direct way to effectively cull Japanese knotweed populations. Herbicides have yielded mixed results and are often not an effective solution, although trials in Haida Gwaii, British Colombia have shown that spraying sea water on Japanese knotweed increases salinity and reduces populations (Mclean and Eastham, 2009). Up until this point, there has been relatively little research conducted on the spatial distribution of invasive species and why they are able to invade some habitats, but not others. What research has been done has not focused on soil characteristics. Instead it has focused on creating spatial logistic regression models to predict the geographical targets of spreading invasive species (Zhu et al., 2007; Liu et al., 2011, and Thomas, 2013). My approach focuses on the effect of soil characteristics on the spatial distribution of invasive species. In the Woods Hole and Falmouth areas of MA, Japanese knotweed is an aggressive and successful invasive species that grows along roadsides, but not within oak-pine forests. Oak-pine forests are characterized by native species including pitch pine and black oak, plus an understory of huckleberry, wintergreen, and blueberry. Based on this observation, I used Japanese knotweed 2 as a model system to investigate the soil characteristics that influence the spatial distribution of invasive species. My goal was to better understand their invasion mechanism and gain knowledge that can be put towards improved management practices. My investigation focused on three questions: does seed rain from Japanese knotweed reach the oak-pine forest, do soil characteristics differ between Japanese knotweed and oak-pine forest soils, and if the aforementioned questions are affirmed, how do soil characteristic differences affect the germination of Japanese knotweed? I hypothesize that Japanese knotweed seeds are able to enter the oak-pine forest due to wind, and that soil characteristics between the two treatments will differ greatly, particularly in terms of pH and extractable nitrogen. Additionally, I hypothesize that germination rates will differ in the two soil treatment types due to pH. Methods Collecting Soils for Treatments In the Woods Hole and Falmouth vicinities, I took twelve soil samples for each treatment, from Japanese knotweed sites along roadsides and oak-pine forest sites with no Japanese knotweed present (Figure 1). Out of these 24 sites, there were four sampling pairs where the oakpine site was directly behind the Japanese knotweed site. At each of these sites, I took two 15 cm soil cores and placed them in plastic bags. One was used for analyzing soil characteristics in lab, while the other was combined with other samples of the same treatment to form a conglomerate soil for a germination experiment. Furthermore, at each site I took LAI readings using an LAI LiCOR 6000. In addition, a 50 cm x 50 cm quadrat was randomly placed on a representative portion of the litter, and I collected litter within the quadrat into a plastic bag. Finally, for each Japanese knotweed site I laid a transect through a representative portion of the patch. At each meter along the transect, I measured and recorded the height of the nearest Japanese knotweed using a meter stick. I also placed two 50 cm x 50 cm quadrats within the patch, to measure the number of Japanese knotweeds within each quadrat, and these two values were averaged and scaled up to a full square meter to determine the density of Japanese knotweed within each patch. Upon returning to lab, I stored the soil samples in a refrigerator until further analysis, and I dried litter in a 60 °C oven before determining the litter mass of each site. Seed Rain 3 I used a total of three seed traps to measure whether or not seed rain from Japanese knotweed reaches the oak-pine forest. Each set of seed traps consisted of four separate pieces of mesh window screen that were one square meter each (Figure 2). I laid the seed traps at one meter intervals from the edge of the Japanese knotweed patch into the oak-pine forest, and weighted them down with logs or rocks to prevent the wind from blowing the traps away. I placed two at the MBL Devil’s Lane parking lot (JK3 in Figure 1) site; the first one leading from the Japanese knotweed patch into the oak-pine forest, and the second one leading towards the edge of the road, where two colonies were visible on the other side of the road. I placed the third trap leading from the Japanese knotweed patch to the oak-pine forest at the Algonquin Ave. – Leisure Green Drive (JK4 in Figure 1) site. I collected all three traps after 20 days in the field. Comparing Soil Characteristics I calculated the wet:dry weight ratio by weighing out roughly 10 g of wet soil, and subsequently drying the soil to measure the amount of water mass lost. Percent organic matter was measured based on loss on ignition. I weighed out 3 g of soil into tins and placed them in a furnace at 450°C for 4 hours. Afterwards, I recorded the weight again to calculate the amount of mass lost in the form of organic matter being burned off. The wet:dry ratio was incorporated into this calculation. I measured pH by placing a 10 g sample of wet soil into a small beaker with 50 ml of DI water. I then stirred the soil water slurry before lowering a pH electrode into the sample. After waiting for the measurement to stabilize, pH was recorded to the nearest tenth. I determined NH4+and NO3- concentrations using a modification of the Wood et al. (1967) and Strickland (1972) methods, respectively. I weighed 15 g of de-rooted and homogenized soil for each sample into a plastic cup. 100 ml of 1M KCl was then poured into each cup before the cups were capped and agitated on a shaker table for 1 hour to extract NH4+and NO3-. Afterwards, I filtered 20 ml of sample through an ashed GF/F filter into a scintillation vial for further analysis. I measured nitrate concentrations using an automated flow injection analyzer (Lachat QuickChem® 8500). For ammonium, I added 0.12 ml of phenol solution, 0.12 ml of sodium nitroprusside solution and 0.3 ml of oxidizing solution to a 3 ml sample of supernatant. Samples sat in the dark for an hour before colorimetric analysis. I constructed a standard curve by diluting known concentrations of ammonium and using colorimetric analysis. 4 I measured cation concentrations of Na+, Ca+2, K+, and Mg+2 by first obtaining a 10 g of de-rooted, homogenized dry soil for each sample. Cations were extracted from the soil using 100 ml of 1M NH4Cl. After I shook the soil samples for 30 min at 50 RPM, I refrigerated them for 24 hours. Afterwards, I thawed the samples before gravity filtering them into 20 ml scintiallation vials using a funnel and a 110 mm Whatman filter paper. Finally, I analyzed samples on the Perkins Elmer AAnalyst 400® using atomic absorption spectrophotometry. Dilutions were made as necessary for samples of higher concentrations. Germination I de-rooted and homogenized the conglomerate soils for both the oak-pine forest and Japanese knotweed treatments. A total of four treatments were created: natural oak-pine soil, natural Japanese knotweed soil, manipulated oak-pine soil with a pH increased by one, and a manipulated Japanese knotweed soil with a pH decreased by one. These will be referred to as treatments 1, 2, 3, and 4, respectively. Each pot contained roughly 50 g of soil. For each treatment, there were four pots of 25 Japanese knotweed seeds each, for a total of 100 seeds per treatment. Beforehand, I had frozen the seeds for one month to allow for proper germination. I placed my germination pots in bins containing two tins of water, covered in plastic wrap in order to prevent moisture loss (Figure 3). I germinated the seeds in a 25°C growth chamber that received 770 PAR for 12 hours per day. I watered my pots twice per day, or as needed to prevent desiccation. Additionally, I planted five rhizomes each in pots of natural oak-pine forest soil and natural Japanese knotweed soil. Seeds and rhizomes were germinated for 20 days. Manipulations I created treatments 3 and 4 by adding lime and sulfur, respectively, based on Neill et al. (2015). The amount added was based online gardening recommendations for loamy soil. Recommendations entailed raising pH by one using 80 lbs of lime and decreasing pH by one using 20 lbs of sulfur per 1,000 ft2. I converted the amounts of lime and sulfur needed into grams, and scaled down the recommended amount from 1,000 ft2, to the surface area of the pot (31.7 cm2). I mixed the lime and sulfur into the top 1 cm of the soil and sprayed the pot with water to assist in the integration into soil. Statistical Analyses 5 I used Microsoft Office 365 Excel® to calculate means and standard errors, along with independent t-tests and one-way ANOVA tests. I used an online Tukey HSD program from Vassar College to conduct the Tukey HSD test. Results The average litter masses within each quadrat for the oak-pine forest and Japanese knotweed sites varied little and did not show statistical significance (Figure 4) (t = 0.182, df = 22, p = 0.86). Data on the growth vigor of the Japanese knotweed sites in terms of both average height and density showed no significant correlations to any of the Japanese knotweed soil characteristics. LAI data were not used considered because the sampling period occurred in late fall/early winter and most leaves had already fallen, thus rendering the data inaccurate. Seed Rain The seed trap set at the Algonquin Ave. – Leisure Green Drive (JK4) site yielded no seeds. Furthermore, the second trap set at the MBL Devil’s Lane parking lot (JK3) site next to the road also did not contain any seeds. However, the first seed trap at this location leading from the Japanese knotweed to the oak-pine forest contained many seeds. At the intervals of 1 m, 2 m, 3 m, and 4 m into the oak-pine forest, there were 92, 14, 49, and 56 seeds per m2, respectively (Figure 5). Soil Characteristics Analyses of soil characteristics showed many significant differences between oak-pine forest soils and Japanese knotweed soils. The oak-pine forest soil was composed of 28.6% organic matter while the Japanese knotweed soil was composed of 16.5% organic matter on average (Figure 6). This was not a significant difference (t = 1.40, df = 21, p = 0.18). Furthermore, there was a significant difference in the average pH the two treatments (Figure 7) as the oak-pine forest soil had a pH of 4.7, compared to 5.4 for the Japanese knotweed soil (t = 7.54, df = 22, p = 1.6E-07). In terms of extractable nitrogen, there were significant differences for both ammonium and nitrate. For ammonium, the oak-pine forest soil contained 0.301 µg N/g dry soil on average, while the Japanese knotweed soil contained 2.07 µg N/g dry soil on average (Figure 8). These differences were significant (t = -3.66, df = 21, p = 0.001). Meanwhile, for nitrate the oak-pine forest soil contained 0.09 µg N/g dry soil on average, while the Japanese knotweed soil contained 4.21 µg N/g dry soil on average (Figure 9). Once again, these 6 differences in nitrogen between the two treatments were significant (t = -4.08, df = 21, p = 0.0005). On average, the oak-pine soil contained lower concentrations of the cations Na+, Ca+2, K+, Mg+2 in comparison to Japanese knotweed soil (Figure 10). For Na+, the oak-pine forest soil contained 0.13 mEq per 100 g dry soil on average while the Japanese knotweed soil contained 0.48 mEq per 100 g dry soil, which was not statistically significantly different (t = -1.93, df = 21, p = 0.06). As for Ca+2, the oak-pine forest contained 0.48 mEq per 100 g dry soil on average, while the Japanese knotweed soil contained 3.67 mEq per 100 g dry soil on average, which was a significant difference (t = -3.74, df = 21, p = 0.001). Differences between the two treatment soils in K+ and Mg+2 were not significant. For K+, the oak-pine forest soil contained 0.33 mEq per 100 g dry soil while the Japanese knotweed soil contained 0.42 mEq per 100 g dry soil. This was a not a significant difference (t = -1.21, df = 21, p = 0.24). For Mg+2, the oak-pine forest soil contained 0.64 mEq per 100 g dry soil on average while Japanese knotweed soil contained 0.76 mEq per 100 g dry soil on average. This was not a significant difference (t = -0.47, df = 21, p = 0.64). Germinations On average, there were 7.5 germinations per pot in treatment 2, and 7.75 germinations treatment 3. Treatment 1 averaged 4 germinations per pot, while treatment 4 averaged 5.5 greminations per pot. Among these four treatments, there was statistically significant variation among the means based on an one-way ANOVA (F(3, 12) = 3.53, p = 0.048). A post-hoc Tukey test revealed that the treatment 1 was statistically significantly different from both Treatments 2 and 3. Moreover, treatments 2 and 3 were not statistically significant from each other. However, treatment 4 was not statistically significantly different from any of the other three treatments (Figure 11). The heights of seedlings within treatments 2 and 3 averaged 1.08 cm and 1.19 cm, respectively. Seedlings in treatment 1 had an average height of 0.55 cm. Meanwhile, seedlings in treatment 4 had an average height of 0.66 cm. Among these four treatments, there was also statistically significant variation among the means based on an one-way ANOVA (F(3, 12) = 5.43, p = 0.013). Once again, a post-hoc Tukey test revealed that the treatment 1 was statistically significantly different from both treatments 2 and 3. Moreover, treatments 2 and 3 were not 7 statistically significant from each other. However, treatment 4 was not statistically significantly different from any of the other three treatments (Figure 12). Seven seedlings sprouted from Japanese knotweed rhizomes grown in natural Japanese knotweed soil, while no seedlings sprouted from rhizomes grown in natural oak-pine forest soil (Figure 13). Discussion Seed Traps The second seed trap leading towards the road at the MBL Devil’s Lane parking lot site did not catch any seeds. This is likely due to the fact that laying traps near roads, instead of within the forest, allows for wind to blow seeds away easily. In addition, the third seed trap at the Algonquin Ave – Leisure Green Drive did not catch any seeds either. This is likely due to the topographical variation of the site, where the seed traps had to be set atop a small mound, in between the Japanese knotweed and the oak-pine forest. However, it is still very clear that seeds are able to enter the oak-pine forest based on the data from the first seed trap at the Devil’s Lane. Even at a distance of 4 m in the oak-pine forest, there are over 50 seeds per m2 (Figure 5). I would expect that there may be a gradient of a decreasing number of seeds as the distance into the oak-pine forest increases, but due to topographical variations, there is no gradient (Figures 2 and 5). The low number of seeds found at 2 m into the oak-pine forest is likely due to many of them falling down into the traps set a 3 m and 4 m. Even if no seeds had been found within the seed traps, it would not have meant that no seeds were reaching the oak-pine forest. Although I cannot account for variability in terms of wind and weather, I believe that seed traps are an accurate method of measuring seed rain as long as the topography is level. For future investigations, I hope to place additional seed traps farther into the forest until no seeds are caught, to understand the extent of how far seed rain is able to travel. Soil Characteristics The higher percentage of organic matter in oak-pine forests in comparison to Japanese knotweed soils can be attributed to the fact that there tends to be more litter within forests. Additionally, along road sides where Japanese knotweed grows, organic matter tends to be striped away. The difference in pH of 0.7 is significant considering pH is measured on a log scale. The lower pH in the oak-pine forest soil can likely be attributed to the acidic pine needles and other organic acids in the forest soils (Nehar et al, 2013). 8 In terms of extractable nitrogen, there were significantly higher concentrations of NH4+ and NO3- in the Japanese knotweed soil, compared to the oak-pine forest soil (Figure 8 and 9). This is likely due to the fact that the lower pH in the oak-pine forest inhibits nitrification (Neill et al, 2007). Of the nitrogen that is present in these oak-pine forest soils, there is a higher concentration of NO3-, compared to NH4+ which may be due to the trend that plants preferentially take up NH4+, since it is easier to metabolize and costs the plant less energy. Cation concentrations of Na+ and Ca+2 were significantly higher in Japanese knotweed soils, compared to oak-pine forest soils (Figure 10). This can be attributed to the winter practice of salting roads in the Falmouth area. The road salt that the Department of Public Works uses consists of NaCl, mixed with a Safemelt 40/60 blend of forestry byproduct and CaCl2 (Mineral Safety Data Sheet, 2012). Since the Japanese knotweed grow along roadsides, they live in conditions of high salinity. However, it is important to note that it is not likely that Japanese knotweed grow better in conditions of high salinity. Most plants are negatively affected by salinity since the uptake of water becomes more difficult. Rather, the higher salinity along roadsides likely put additional stresses on the former native species that used to grow along roadsides, and made them more susceptible to invasion by Japanese knotweed. Differences between the two types of soils were not significant in terms of K+ and Mg+2 since they are not included in road salt and are not added through human influence along road sides. For both cations, they are mainly present in soils due to weathering and throughfall. Overall, Japanese knotweed soils were characterized by higher levels of extractable nitrogen, cations, and a higher pH, but a lower amount of organic matter. Germination Germination was roughly twice as likely in natural Japanese knotweed soils, compared to natural oak-pine soils. It is possible that this is due to higher levels of nitrogen and cations and lower levels of organic matter within Japanese knotweed soil, but it is clear that pH greatly affects germination. The manipulated oak-pine soil that had a pH increased by one to a level consistent with natural Japanese knotweed soil contained similar rates of germinations per pot, indicating that pH has a major affect. Allelopathy from pine needles was definitely considered as a potential driving force behind lower rates of germination in the natural oak-pine forest soil, but the manipulated treatments indicate that pH is the true driver of whether or not Japanese knotweed seeds are able to germinate. Interestingly, a pH decrease of 1 to a level consistent with 9 natural oak-pine forest soil in the manipulated Japanese knotweed sample did not yield an average germination rate per pot in between the two natural treatments. Data on the average seedling height closely matched onto the germination rate results. The differences in soil characteristics between natural oak-pine and Japanese knotweed soils along with the factor of pH affect not only germination rates, but also seedling growth (Figure 12). It is important to note that germination of Japanese knotweed soils was still possible in natural oak-pine forest soils. However, matched with the observation that there are no Japanese knotweed plants within the oak-pine forest, I conclude that the mortality rate of Japanese knotweed seedlings may be very high. If the germination experiment had been extended, it is possible that seedlings within the oak-pine soil treatment could have died. It seems that reproduction through seed dispersal is rather rare for the Japanese knotweed. Instead Japanese knotweed tends to reproduce and spread from its rhizomes, as it slowly invades the forest. This way, the plant is able to slowly change and alter its soil conditions to make them more advantageous to itself, and less advantageous to native species in its sites. Furthermore, although it is possible that seeds of other species germinated, many seedlings retained the three winged calyx in which the seed was originally incased (Figure 14). Seedlings of questionable origin were compared to seedlings that had the three winged calyx and were without a doubt Japanese knotweed (Figure 14). Interestingly, germination was possible within roughly 7-10 days, while a previous study indicated that germination could take anywhere from 5-55 days, which is much wider range (Stone 2010). Variations in the time until germination are likely due to variations in temperature and the amount of time that the seeds were frozen. The results from the rhizome experiment further support the conclusion that differences in soil characteristics between Japanese knotweed and oak-pine forest soils affect germination and seedling growth (Figure 13). However, the sample size for this portion of the study was not high enough and I was unable to test the effect of pH on sprouting seedlings due to not enough rhizomes. In the future, it would be interesting to see how well the seedlings grow into actual full grown Japanese knotweed plants, and to conduct the germination experiment in the field, instead of the lab. Previous studies have shown that when plants are germinated in the presence of an aqueous extract of pitch pine needles, plants that lived with pitch pine had relatively higher germination rates, while species that did not normally grow with pitch pine suffered lower 10 germination rates due to allelopathy (Rizvi pg. 217-219). Additionally, it would be very interesting to study the carbon and nitrogen contents through CHN analysis of the litter within oak-pine forest and Japanese knotweed patches. Perhaps differences in litter composition could help to explain differences in organic matter and extractable nitrogen levels. Finally, previous literature has listed soil moisture, light intensity, and plant species richness as determining factors regarding the spatial distribution of invasive species (Liu et al. 2011). For future studies I would like to include the three aforementioned factors to my study to better understand the conditions in which Japanese knotweed are able to invade or not. Looking forward, invasive species will continue to pose a threat to biodiversity and cause extensive damage. In the past, the eradication of invasive species has been the primary goal of ecological management groups, but this has rarely been done successfully on a large scale (Kettenring and Adams, 2011). Recent studies have indicated that top down controls of grazing by goats are able to effectively and economically cull populations of phragmites, a notorious marsh invasive species in the Northeast (Silliman et al. 2014). Japanese knotweed is also considered grazing edible by animals and perhaps grazing could be an effective method of controlling Japanese knotweed populations. However, changing soil conditions such as pH could be an additional cheap and easy method to control invasive species populations, from a bottom up point of view. Conclusion Previous literature has focused on building models to predict the spatial distribution of invasive species or other factors, but there has been little to no work done on soil conditions and characteristics. My study indicates that there are stark differences in soil characteristics between Japanese knotweed soils and native oak-pine forest soils, particularly in terms of pH, nitrogen levels, and salinity. These differences, especially pH, likely affect the spatial distribution of invasive species by effecting where seeds are able to germinate and grow effectively. 11 Acknowledgements I would like to thank my mentor Chris Neill for helping me to design my project and guiding me through the data analysis. Moreover, I would like to thank Anne Giblin for guiding me in the usage of the atomic absorption spectrophotometer. Finally, I would like to thank Rich McHorney and the TAs Hannah Kuhns, Aliza Ray, and Brecia Douglas for helping me with lab analyses and answering every possible question that I could have. Literature Cited Kareiva P, Marvier M. 2010. Conservation science: balancing the needs of people and nature. Greenwood Village, Colorado: Roberts Publishers. Kettenring KM, Adams CR. 2011. Lessons learned from invasive plant control experiments: a systematic review and meta-analysis. Journal of Applied Ecology 48:970–979 Liu, D., Jiang, H., Zhang, R., and K. He. 2011. Predicting the spatial distribution of Lonicera japonica, based on species occurrence data from two watersheds in Western Kentucky and Tennessee. Proceedings of the 17th Central Hardwood Forest Conference. 418-424. Lowe S., Browne M., Boudjelas S. and M. De Poorter. 2000. 100 of the World’s Worst Invasive Alien Species A selection from the Global Invasive Species Database. Published by The Invasive Species Specialist Group (ISSG) a specialist group of the Species Survival Commission (SSC) of the World Conservation Union (IUCN). Mclean, D. and A. Eastham. 2009. Northwest Invasive Plant Council (NWIPC) 2009 Strategic Plan and Plant Profiles. British Colombia Ministry of Agriculture Food and Fisheries. Mineral Safety Data Sheet. 2012. Eastern Minerals, Inc. Nehar, D., Asmussen, D. and S. Lovell. 2013. Roads in northern hardwood forests affect adjacent plant communities and soil chemistry in proportion to the maintained roadside area. Sci Total Environment 449:320-327. Neill, C., Von Holle, B., Kleese, K., Ivy, K., Colllins, A., Treat, C., and M. Dean. 2007. Historical influences on the vegetation and soils of the Martha’s Vineyard, Massachusetts coastal sandplain: Implications for conservation and restoration. Biological Conservation 136: 17-32. 12 Neill, C., Wheeler, M., Loucks, E., Weiler, A., Von Halle, B., Pelikan, M. and T. Chase. 2015. Influence of soil properties on coastal sandplain grassland establishment on former agricultural fields. Restoration Ecology 23: 531-538. Pimentel D, Zuniga R, Morrison D. 2005. Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecological Economics 52:273–288 Rizvi, S.J. 1992. Allelopathy: basic and applied aspects. Springer Science & Business Media, Netherlands. Shyam, Thomas. 2013. Predicting the spatial distribution of an invasive plant species and modeling tolerance to herbivory using Lythrum salicaria L. as a model system" Graduate thesis, University of Iowa, Iowa City, Iowa, USA. Silliman, B., Mozder, T., Angelini, C., Brundage, J., Esselink, P., Bakker, J., Gedan, K., van de Koppel, J., and A. Baldwin. 2014. Livestock as a potential biological control agent for an invasive wetland plant. PeerJ: 1-19. Stone, Katharine R. 2010. Polygonum sachalinense, P. cuspidatum, P. × bohemicum. In: Fire Effects Information System. U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station, Fire Sciences Laboratory. Strickland, J.D.H. and T.R. Parsons A practical handbook of Seawater Analysis 1972 Ottawa, Fisheries Research Board of Canada 2nd. Ed. Wilcove, David S., et al. "Leading Threats to Biodiversity: What's Imperiling U.S. Species." Precious Heritage. Ed. Bruce A. Stein, Lynn S. Kutner, and Jonathan S. Adams. New York: Oxford University Press, 2000. 239-55. Print Wood ED, Armstrong FAJ, and Richards FA. (1967) Determination of nitrate in seawater by cadmium-copper reduction to nitrate. Journal of the Marine Biological Association of the United Kingdom 47: 23-31. Zhu, L., Sun, O., Sang, W., Li, Z., and K. Ma. 2007. Predicting the spatial distribution of an invasive plant species (Eupatorium adenophorum) in China. Landscape Ecology 22: 1143-1154. 13 Figures Figure 1. Map of sampling sites in the Woods Hole and Falmouth vicinity. Red dots represent oak-pine forests sites, while yellow dots represent Japanese knotweed sites. Paired sites include OP1 and JK3, OP4 and JK4, OP6 and JK5, and OP9 and JK12. Figure 2. A photograph of the experimental setup of Trap 1 at the MBL Devil’s Lane parking lot site (JK3). Traps lead from the edge of the Japanese knotweed patch at the top, down into the oak-pine forest. Figure 3. The experimental setup of the germination experiment. The plastic wrap is not shown. Figure 4. Mean litter mass (g) ± SE per m2 of natural oak-pine forest and Japanese knotweed soils. Figure 5. Number of seeds per m2 entering the oak-pine forest at the MBL Devil’s Lane parking lot site. Data from the additional two traps located towards the road at Devil’s Lane and at the Algonquin Ave. – Leisure Green Drive site were not included in this figure. Figure 6. Mean percent organic matter ± SE of natural oak-pine forest and Japanese knotweed soils. Figure 7. Mean pH ± SE of natural oak-pine forest and Japanese knotweed soils. Figure 8. Mean concentrations of NH4+ (µg N/g dry soil) ± SE of natural oak-pine forest and Japanese knotweed soils. Figure 9. Mean concentrations of NO3- (µg N/g dry soil) ± SE of natural oak-pine forest and Japanese knotweed soils. Figure 10. Mean cations concentrations of Na+, Ca+2, K+, and Mg+2 (mEq/100 g dry soil) ± SE of oak-pine forest and Japanese knotweed soils. Figure 11. Average number of germinations ± SE per pot grown in all four germination treatments, including natural and pH manipulated oak-pine forest and Japanese knotweed soils. Letters represent results of the Tukey HSD test. Figure 12. Average height (cm) ± SE of seedlings grown in all four germination treatments, including natural and pH manipulated oak-pine forest and Japanese knotweed soils. Letters represent results of the Tukey HSD test. Figure 13. Number of sprouted seedlings from rhizomes in natural oak-pine forest and Japanese knotweed soils. Figure 14. A close up image of one of the germination pots. Heights among seedlings vary. Many of the seedlings still include the three winged calyx in which the Japanese knotweed seed is naturally contained. 14 Figure 1. Map of sampling sites in the Woods Hole and Falmouth vicinity. Red dots represent oak-pine forests sites, while yellow dots represent Japanese knotweed sites. Paired sites include OP1 and JK3, OP4 and JK4, OP6 and JK5, and OP9 and JK12. 15 Figure 2. A photograph of the experimental setup of Trap 1 at the MBL Devil’s Lane parking lot site (JK3). Traps lead from the edge of the Japanese knotweed patch at the top, down into the oak-pine forest. 16 Figure 3. The experimental setup of the germination experiment. The plastic wrap is not shown. 17 700 LitterMassPerSquareMeter 600 500 400 300 200 100 0 Oak-PineForest JapaneseKnotweed Figure 4. Mean litter mass (g) ± SE per m2 of natural oak-pine forest and Japanese knotweed soils. 18 100 SeedsperSquareMeter 90 80 70 60 50 40 30 20 10 0 0 1 2 3 MetersintoOak-PineForest 4 Figure 5. Number of seeds per m2 entering the oak-pine forest at the MBL Devil’s Lane parking lot site. Data from the additional two traps located towards the road at Devil’s Lane and at the Algonquin Ave. – Leisure Green Drive site were not included in this figure. 19 40 35 PercentOrganicMatter 30 25 20 15 10 5 0 Oak-PineForest JapaneseKnotweed Figure 6. Mean percent organic matter ± SE of natural oak-pine forest and Japanese knotweed soils. 20 6.0 5.8 5.6 5.4 pH 5.2 5.0 4.8 4.6 4.4 4.2 4.0 Oak-PineForest JapaneseKnotweed Figure 7. Mean pH ± SE of natural oak-pine forest and Japanese knotweed soils. 21 3 2.5 µgNpergdrysoil 2 1.5 1 0.5 0 Oak-PineForest JapaneseKnotweed Figure 8. Mean concentrations of NH4+ (µg N/g dry soil) ± SE of natural oak-pine forest and Japanese knotweed soils. 22 6 5 µgNpergdrysoil 4 3 2 1 0 Oak-PineForest JapaneseKnotweed Figure 9. Mean concentrations of NO3- (µg N/g dry soil) ± SE of natural oak-pine forest and Japanese knotweed soils. 23 Na+ Ca+2 0.8 5 0.7 4.5 4 mEq per 100 g Dry Soil 0.6 3.5 0.5 3 0.4 2.5 0.3 2 1.5 0.2 1 0.1 0.5 0 0 K+ Mg+2 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Oak-PineForest Japanese Knotweed Oak-PineForest Japanese Knotweed Figure 10. Mean cations concentrations of Na+, Ca+2, K+, and Mg+2 (mEq/100 g dry soil) ± SE of oak-pine forest and Japanese knotweed soils. 24 9 NumberofGerminationsPerPot 8 7 B 6 B 5 AB 4 3 2 A 1 0 NaturalOak-Pine NaturalJapanese Oak-Pine:pH+1 Japanese Knotweed Knotweed:pH- 1 Figure 11. Average number of germinations ± SE per pot grown in all four germination treatments, including natural and pH manipulated oak-pine forest and Japanese knotweed soils. Letters represent results of the Tukey HSD test. 25 1.6 1.4 AverageHeight(cm) 1.2 1.0 0.8 B B 0.6 0.4 A AB 0.2 0.0 Japanese NaturalOak-Pine NaturalJapanese Oak-Pine:pH+1 Knotweed Knotweed:pH- 1 Figure 12. Average height (cm) ± SE of seedlings grown in all four germination treatments, including natural and pH manipulated oak-pine forest and Japanese knotweed soils. Letters represent results of the Tukey HSD test. 26 8 NumberofSproutedSeedlings 7 6 5 4 3 2 1 0 Oak-PineForest JapaneseKnotweed Figure 13. Number of sprouted seedlings from rhizomes in natural oak-pine forest and Japanese knotweed soils. 27 Figure 14. A close up image of one of the germination pots. Heights among seedlings vary. Many of the seedlings still include the three winged calyx in which the Japanese knotweed seed is naturally contained. 28