Taylor University Pillars at Taylor University

Master of Environmental Science Thesis Collection

2012 Distribution of Biological Control Agents (Galerucella spp., Hylobius transversovittatus, Nanophyes marmoratus) and their impact on Lythrum salicaria (purple loosestrife) in Indiana Joshua S. Britton

Follow this and additional works at: https://pillars.taylor.edu/mes Part of the Environmental Sciences Commons

Recommended Citation Britton, Joshua S., "Distribution of Biological Control Agents (Galerucella spp., Hylobius transversovittatus, Nanophyes marmoratus) and their impact on Lythrum salicaria (purple loosestrife) in Indiana" (2012). Master of Environmental Science Thesis Collection. 5. https://pillars.taylor.edu/mes/5

This Thesis is brought to you for free and open access by Pillars at Taylor University. It has been accepted for inclusion in Master of Environmental Science Thesis Collection by an authorized administrator of Pillars at Taylor University. For more information, please contact [email protected]. Distribution of biological control agents (Galerucella spp.,

Hylobius transversovittatus, Nanophyes marmoratus) and their

impact on Lythrum salicaria (purple loosestrife) in Indiana

Joshua S. Britton

A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Environmental Science Taylor University

May 17, 2012

Appoved by:

Paul E. Rothrock, PhD Robert T. Reber, MS Commitee Chair, Interim MES Director Commitee Member Professor of Environmental Science Associate Professor of Environmental Science Taylor University Taylor University

Rich Dunbar Connie Lightfoot, EdD Committee Member Dean Regional Ecologist, Northeast Region School Professional and Graduate Studies Indiana Department of Natural Resources Taylor University Acknowledgements

This thesis would not have been possible without a number of people. The work on which this thesis is based was completed by the Indiana Department of Natural Resources, Division of Nature Preserves, so I extend my thanks to those who have contributed to the data collection over the year. The Indiana Department of Natural Resources also provided the funding for this work. Additionally, I would like to thank Rich Dunbar, for his time and assistance as a part of my thesis committee and for his role in coordinating the work with the Indiana Department of Natural Resources. Likewise, I would like to thank Rob Reber, another member of my committee, for his support and guidance throughout the project, particularly with the statistical analysis. I must also thank Paul Rothrock, my thesis advisor, for his directive and assistance to all aspects of this project. My studies at Taylor University have been funded by the scholarship fund provided by Leland Boren, so my thanks to him for his support of the Environmental Science program. Most importantly, I must thank my family and friends for their continued support. My parents, John and Barb, who travelled the country with me and my sister, Brianne. These miles and years of exploring the natural wonders led me to my passion for ecology and the environment. Finally, I must thank my wonderful wife, Bridget, for her love, support, and encouragement throughout the many stressful months.

ii Contents

Acknowledgements...... ii List of Tables...... iv List of Figures...... v

1 Impacts of Galerucella calmariensis and G. pusilla on Lythrum salicaria in Indiana1 1.1 Introduction...... 2 1.1.1 Impacts of Lythrum salicaria ...... 2 1.1.2 Biological Control...... 5 1.1.3 Objectives...... 7 1.2 Methods...... 8 1.2.1 Sites and Transects...... 8 1.2.2 Sampling...... 8 1.2.3 Data Analysis...... 9 1.3 Results...... 10 1.4 Discussion...... 12 1.4.1 Conclusions...... 14

2 Dispersal and Distribution of Biological Control Agents for Lythrum sali- caria in Indiana 16 2.1 Introduction...... 17 2.1.1 Impacts of Lythrum salicaria ...... 17 2.1.2 Control...... 18 2.1.3 Brief Insect Life Histories...... 18 2.1.4 History of Indiana Biological Control...... 19

iii 2.1.5 Project Goals...... 20 2.2 Methods...... 20 2.3 Results and Discussion...... 22

References 37

Appendices 40

iv List of Tables

1 Galerucella abundance categories...... 24 2 Midpoints used for percent cover and percent damage variables...... 24 3 Spearman rank-sum correlations between year and number of stems, percent cover by L. salicaria, number of inflorescence, or species richness. Spearman’s ρ followed by an asterisk (*) are significant...... 25 4 All Galerucella spp. locations with selected attributes. Last two columns show distances used for buffer radius in Figure 10...... 41 5 All Hylobius transversovittatus locations with selected attributes...... 47 6 All Nanophyes marmoratus locations with selected attributes...... 48

v List of Figures

1 Galerucella spp. transect analysis sites...... 26 2 Number of Lythrum salicaria stems per quadrat over time...... 27 3 Lythrum salicaria percent cover class over time. Jitter has been added to keep points from overlapping, since both variables are essentially categorical.... 28 4 Total number of Lythrum salicaria inflorescences over time...... 29 5 Total fall species richness over time...... 30 6 Galerucella abundance over time. Abundance is calculated as the mean cate- gorical value for the three life stages, as described in Chapter 1 and jitter has been added to keep points from overlapping...... 31 7 Linear regression of the relationship between density of L. salicaria and plant species richness by site...... 32 8 Hylobius transversovittatus sites in Indiana. All sites are release sites..... 33 9 Nanophyes marmoratus release sites (?) and non-release sites (•) in Indiana 34 10 Galerucella spp. release sites (?) and non-release sites (•) in Indiana with buffers showing potential area currently occupied by Galerucella spp. The small buffer area is based on 491 m/yr and the larger on 1822 m/yr...... 35 11 Locations where Galerucella spp. have been found in Indiana...... 36 12 Galerucella spp. map produced for the IDNR...... 49 13 Galerucella spp. population size map produced for the IDNR...... 50 14 Galerucella spp. distribution estimate map produced for the IDNR..... 51 15 Galerucella spp. statewide map produced for the IDNR...... 52 16 Nanophyes marmoratus map produced for the IDNR...... 53 17 Hylobius transversovittatus map produced for the IDNR...... 54

vi 1 Impacts of Galerucella calmariensis and G. pusilla

on Lythrum salicaria in Indiana

Abstract

Lythrum salicaria is an invasive wetland hydrophyte native to Eurasia. Since 1814, it has been spreading throughout North America and has been in Indiana since 1900. A wide vari- ety of negative ecosystem impacts have been documented but some authors have questioned these findings. Galerucella spp. have been utilized as biological control agents for Lythrum salicaria in Indiana since 1994, but this is the first publication to examine insect control in the state. Studies from other states have generally been shorter time frames than the eight to ten years of this study. This study examined the impacts of Galerucella spp. at four Indiana wetlands by considering changes in beetle abundance, plant species richness, Lythrum salicaria cover, flowering, and stem densities over time. These relationships were analyzed using Spearman’s rank-sum correlations. Galerucella spp. abundance was signif- icantly correlated at two sites, species richness and percent cover were both significantly correlated at three sites, and number of inflorescence and stem density were significant at all four sites. The number of inflorescence show the strongest correlation, with ρ’s between -0.4575 and -0.7761. Overall, it was determined that following the introduction of Galeru- cella spp. Lythrum salicaria had a significant negative response at each of the wetlands. Variation was observed between sites, but their impacts at these wetlands strongly suggest that Galerucella spp. can play a major role in controlling Lythrum salicaria. Keywords: Galerucella, Lythrum salicaria, biological control, invasive species, Spearman’s correlation

1 1.1 Introduction

Lythrum salicaria is a wetland hydrophyte which is native to Eurasia. It is considered an invasive species in North America. The slightly square stems grow in clumps of 30–50 from a single taproot (Mal et al. 1992). The lanceolate to ovate leaves are opposite or whorled on the lower portions of the stem, while upper leaves are alternate. Lythrum salicaria produces a terminal spike of tightly clustered flowers which may be over 1 meter in length (Mal et al. 1992). A single plant can produce over 2.5 million seeds in a single growing season (Malecki et al. 1993). Meanwhile, the plant may reach total heights as great at 2.7 meters (Mal et al. 1992). Thompson et al.(1987) summarized the global distribution of L. salicaria and Hult´en (1971) identified both an Asian and European center for distribution. Historically, the first observations of L. salicaria in North America occurred in New England, in 1814 (Mal et al. 1992). Introductions of L. salicaria likely came from several sources including inadvertent transport in shipping ballast and on imported wool. Intentional introduction also occurred by immigrants, who used the plant as a medicinal herb (Thompson et al. 1987). Following its initial introduction into the Western hemisphere, humans contributed to the spread of L. salicaria into the Midwest. Thompson et al.(1987) reported that the development and use of canals was key in spreading L. salicaria. The canals provided a disturbed habitat which would have been easily invaded and the heavy use of the canals for transport could have easily carried seed westward. Further intentional spread may have been caused by use of the plant as an ornamental and as a nectar plant for beekeeping. In Indiana the earliest record is from 1900 and Stuckey(1980) notes very little spread through 1940. Today, L. salicaria is found throughout Indiana, though it is most common in the northern counties. Additionally, L. salicaria occurs in 47 of the contiguous states (it is absent from Florida) (Blossey et al. 2001).

2 1.1.1 Impacts of Lythrum salicaria

Lythrum salicaria can form extensive stands which many authors have characterized as monotypic (Malecki et al. 1993). However, some evidence suggests that while L. salicaria becomes the dominate vegetative species, invasion does not reduce overall species richness (Farnsworth and Ellis 2001, Hager and Vinebrooke 2004). In addition to potential reductions in plant biodiversity, the documented ecosystem impacts of L. salicaria include reducing bird habitat quality, interference with pollination of native species, and alterations to wetland function(Blossey et al. 2001). One reason for the invasive nature of L. salicaria is the prolific seed production which results in domination of the seed bank, permitting high recruitment of seedlings even after removal of adult plants (Welling and Becker 1990). In addition to possessing a competitive advantage in seedling recruitment, adult plants also are highly competitive(Gaudet and Keddy 1995). Weiher et al.(1996) studied 120 microsom containers simulating 24 different water level treatments and found that L. salicaria was especially competitive in treatments that simulated non-flooded conditions. This competitive behavior also is demonstrated by Weihe and Neely(1997), where Typha latifolia was negatively affected by L. salicaria and analysis showed that over time L. salicaria would completely replace Typha latifolia. Schooler and McEvoy(2006) demonstrated a negative association between density of L. salicaria and plant species richness. Lythrum salicaria biomass was found to be negatively correlated with the biomass of other plant species (Farnsworth and Ellis 2001). Other studies have demonstrated that removal of L. salicaria results in an increase in other plant species density, including grasses and sedges (Gabor et al. 1996) and Typha angustifolia (Mal et al. 1997). Lythrum salicaria also can reduce the seed set of the native species L. alatum through competition for pollinators (Brown et al. 2002). The invasive L. salicaria produces a much larger and showier floral display than L. alatum, which led to reduced pollinator visitation to L. alatum. Brown and Mitchell(2001) reported that pollination with mixed pollen of L.

3 salicaria and L. alatum reduced seed set by 28% versus pollination with L. alatum pollen alone. Similarly, when Decodon verticillatus, another member of the Lythraceae family, were hand pollinated with mixed pollen from L. salicaria and D. verticillatus the seed set was reduced by 33.3% compared to hand pollination with only D. verticillatus pollen (Da Silva and Sargent 2011). In wetlands where L. salicaria replaces Typha spp. it can have a notable impact on the wetland function. Most leaves drop from L. salicaria in the autumn while Typha loses relatively few leaves in the fall (B¨arlocher and Biddiscombe 1996). In addition to this temporal shift in decomposition, decomposition rates and nutrient leaching vary between the species. Phosphorus concentration of L. salicaria leaves is twice that of Typha plants (Emery and Perry 1996). These changes could increase the nutrient input into wetlands and cause eutrophication. Furthermore, Grout et al.(1997) compared the decomposition of L. salicaria and Carex lyngbyei, finding that L. salicaria has significantly higher decay rate, causing detritus from L. salicaria to only be available during the autumn while C. lynbyei remains available through the spring. Changes in porewater chemistry were observed by Templer et al.(1996); specifically, L. salicaria communities had lower phosphate in the sediment porewater than Typha an- gustifolia or Phragmites austalis communities. As L. salicaria has high concentrations of phosphorus in the leaves, it is likely that the low concentrations in porewater are due to increased uptake by L. salicaria. Whitt et al.(1999) found that avian species diversity decreased in wetlands where L. salicaria was a dominant species. Muskrat (Agelaius phoeniceus) use and long-billed marsh wren (Cistothorus palustris) nesting were shown be lower in L. salicaria stands than adja- cent Typha spp. (cattail) stands (Rawinski 1982, Rawinski and Malecki 1984). Lor(2000) examined the use of L. salicaria by many avian species for feeding and nesting. Virginia rail (Rallus limicola), sora (Porazana coaolina), least bittern (Ixobrychus exilis), American bittern (Botaurus lentiginosus) and pied-billed grebe (Podilymbus podicpes) were found to

4 avoid L. salicaria stands. Meanwhile, nearby areas with Typha spp. provided habitat for many of these species. While many studies have identified negative effects of L. salicaria, there remains some debate as to whether it is causing damage to native wetland communities. Hager and Vine- brooke(2004) studied six Minnesota wetlands and found that species richness and diversity was significantly greater in wetlands patches which had been invaded by L. salicaria com- pared to uninvaded Typha angustifolia wetlands. Whether this association is a result of L. salicaria invasion or L. salicaria preferentially invades areas with high richness is unclear. A review of the relationship of L. salicaria with native flora and fauna by Anderson(1995) de- termined that, from the literature existing at the time of his review, the affect of L. salicaria on wetland ecosystems and native species was unclear. Additionally, Farnsworth and Ellis (2001) found no significant effect between density of L. salicaria stems and species richness.

1.1.2 Biological Control

Classical biological control involves the introduction of natural plant enemies in attempts to control the introduced plant species (Hight and Drea 1991). In its native European range, L. salicaria does not form large monotypic or dominant stands as it does in North America. Differences in growth patterns of native vs. invasive seed has been demonstrated (Chun et al. 2010). When stands form in its native range, they do not persist due to control of reproduction and growth that is provided by native insect herbivores (Blossey et al. 1994b). Attempts to control L. salicaria began in the 1950’s, but initial efforts were largely unsuc- cessful (Skinner et al. 1994). Flooding, cutting, and burning were found to be inadequate methods of controlling L. salicaria (Blossey et al. 2001, Skinner et al. 1994). Hand pulling the plants was the most successful of the early control methods, but requires the entire rootstock be pulled, which is highly labor intensive (Skinner et al. 1994). Chemical control has pri- marily utilized glyphosate, 2,4-D, or triclopyr, however, because of large and long-persisting seedbanks (Welling and Becker 1990), spraying must be repeated often (Blossey et al. 2001,

5 Skinner et al. 1994) perhaps every two to three years(Gabor et al. 1996). Additionally, the non-selective nature of chemical control reduced populations of sedges, grasses, cattails, and other native wetland plants (Skinner et al. 1994, Gabor et al. 1996). Due to the lack of effective control methods and the continued spread of L. salicaria, a biological control program for L. salicaria was established in North America (Malecki et al. 1993, Blossey et al. 2001, Hight and Drea 1991). This involved identifying natural insect herbivores and selecting those best suited for use as biological control agents. (Malecki et al. 1993, Blossey et al. 2001, Hight and Drea 1991). In the end, four insect species were approved for release, Hylobius transversovittatus Goeze (a root-mining weevil), Nanophyes marmoratus Goeze (a flower feeding weevil), Galerucella calmariensis L., and G. pusilla Duft (two leaf-beetles) (Blossey et al. 2001).

Brief Galerucella spp. Life History

Adult Galerucella spp. emerge in the spring (April or May) from the soil or plant litter (Blossey et al. 1994b). At this time, beetles begin feeding on meristematic tissue of L. salicaria. Oviposition begins after about one week of feeding and last approximately two months, peaking in May and June but continuing into July (Blossey et al. 1994b). Clusters of two to ten eggs are laid in the leaf axils or on the plant stems (Malecki et al. 1993). Early instars of the emerged larvae feed preferentially on the developing buds. Older larvae will feed on any portion of the plant tissue (Blossey et al. 1994b). They typically skeletonize the leaves of L. salicaria. Once larvae are mature, they will pupate in the soil or leaf litter (Blossey et al. 1994b). Adults emerge in July, and in some cases, will complete another oviposition period prior to overwintering (Landis 2003). This second generation will feed on L. salicaria regrowth and very severe impacts on L. salicaria have been observed in these cases. All adults are short-lived and die soon after oviposition (Malecki et al. 1993). In areas where live L. salicaria is lacking, Galerucella spp. adults may disperse to locations where L. salicaria is readily available. Dispersing insects are strongly attracted by conspecifics

6 (Grevstad and Herzig 1997), due to pheromone production by males feeding on L. salicaria (Bartelt et al. 2008, Hamb¨ack 2010).

Impacts of Biological Control

Following the selection of biological control agents, insect releases began in the United States in 1992; with these releases monitoring of insect impacts on L. salicaria and its associated wetland community (Blossey et al. 2001). Results of theses releases have been published from across the United States, from two to ten years post-release (Piper 1996, Grevstad 2006, Dech and Nosko 2002, Landis 2003, Stamm-Katovich et al. 1999). One of the earliest publications describing the results of biological control of L. salicaria occurred in Washington State Piper(1996). In central Washington state, Galerucella spp. were released to eight sites in 1992 and 1993. Surveys conducted in 1993 and 1994 found that Galerucella spp. had become established at all eight release sites. In another study (Dech and Nosko 2002) found that, in Ontario, establishment of Galerucella spp. was slow and the populations remained small throughout the three year study period. The G. pusilla popula- tion crashed in the second year after release, and only minor feeding damage was observed by Galerucella spp. over this short time period. Landis(2003) found 100% establishment of 24 releases made in Michigan between 1994 and 1999. Within 4–5 years, large populations were observed at all three 1999 release sites. In potted L. salicaria, Stamm-Katovich et al.(1999) found that L. salicaria biomass was reduced after a single growing season when Galerucella spp. were present. However, other studies suggest that in a natural setting, several years are required for the impacts of Galerucella spp. to be observable (Landis 2003, Dech and Nosko 2002, Piper 1996). Major effects of Galerucella spp. were observed by Landis(2003) and included suppression of flowering, up to 98% reduction in stem numbers, and an increase in site species richness. However, these results did not occur at all sites or in a uniform time. Rather, the results differed greatly between sites. A Minnesota wetland showed a 95% reduc- tion in flowering, and a 50% reduction in stem height four years after release (Blossey and

7 Skinner 2000). At the same site, the number of stems per square meter remained constant throughout the sampling period. Finally, Grevstad(2006) examined the ten-year impacts of Galerucella spp. in New York State and found that 24 of 36 release sites had Galerucella spp. present after ten years. Also, a single site had no persisting L. salicaria after ten years as an apparent result of Galerucella spp. Stem height was reduced significantly between 1994 and 2004, declining by 25.8%, and reduced flowering was observed at sites with moderate to high plant damage.

1.1.3 Objectives

In 1994 the Indiana Department of Natural Resources (IDNR), Division of Nature Preserves began releasing Galerucella spp. to various wetlands. Additional releases continue as deemed necessary. In order to evaluate the impact of biological control agents in Indiana, the IDNR began monitoring at one release sites in 1996, two additional sites in 1997, and a fourth site in 1999. Monitoring was conducted following the protocol of Blossey(1994). The main objective of this study is to examine the data gathered from these sites to provide insight into the impacts of Galerucella spp. in Indiana. Additionally, the data was also analyzed to examine the impact of L. salicaria on species richness.

1.2 Methods

1.2.1 Sites and Transects

The releases described in this study occurred in 1996 at three sites and 1999 at the fourth site. The 1996 releases were at Fish Creek, Wilson Wetland, and Bonneyville Mills and the 1999 release site was at Chapman Lake (Figure1). At all sites except Bonneyville Mills, two transects were oriented parallel to one another and one meter square quadrats were placed

8 every five meters for a total of ten quadrats. Spacing between the transects varied based on site size, shape, and distribution of L. salicaria within the wetland. At Fish Creek, the transects extended from a path to the southwest into the wetland. Wilson Wetland was a constructed wetland, which was created by adding sinuosity to the stream channel. The area inhabited by L. salicaria was restricted to the previous stream banks with one transect on each bank. This resulted in more spacing between the transects than Fish Creek or Chapman Lake. Chapman Lake was the driest of the sites and also had a strong Typha sp. population. Transects were established approximately parallel to the lake shore, with 5 meters between transect lines. At Bonneyville Mills the site was exceptionally small, which restricted the number and positioning of the quadrats. Five quadrats were arranged along two transects. The transects were perpendicular to one another with one quadrat at the intersection and the remaining quadrats place three meters from the intersection in either direction along the transects.

1.2.2 Sampling

Monitoring was conducted following the protocol outlined by Blossey(1994). This involved sampling each site in the spring and fall, from through 2007. Spring sampling was conducted between May 18th and June 19th and included insect and vegetative sampling. Each quadrat was surveyed to determine: the estimated abundance of Galerucella spp. in each life stage (eggs, larvae, adults), percent damage to L. salicaria, percent cover of L. salicaria, number of L. salicaria stems, percent cover of Typha spp., number of Typha spp. stems. Additionally, in each quadrat the height of the five tallest L. salicaria stems and the five tallest Typha spp. stems were recorded. Each quadrat also was surveyed to list all present plant species. Galerucella abundances for each life stage were recorded as a categorical variable as shown in Table1. Percent damage and percent cover were assigned to categories as shown in Table2. Fall sampling occurred from September 1st to 22nd. At this time L. salicaria has finished blooming and Galerucella spp. activity had ended. Since insect activity had ceased, no

9 measure of beetle abundance or damage was made. The same measures of L. salicaria and Typha spp. were made as during the spring sampling. The five tallest L. salicaria plants in each quadrat were surveyed for the following: number of inflorescence, length of the terminal inflorescence, number of flower buds in 5 cm of inflorescence. The total number of inflorescence in each quadrat also was recorded. Finally, a list of plant species was made for each quadrat.

1.2.3 Data Analysis

Field data were transferred to spreadsheets. Data obtained from the five tallest plants in each quadrat were averaged to obtain a single value for each quadrat per year. Galerucella spp. abundances presented some challenge. The numbers of each life stage were estimates and were assigned to a category (Table1). Additionally, the categories had unequal ranges, some of which were quite large; because of this using midpoints as estimates seemed misleading. Therefore, to analyze Galerucella spp. abundance the three categorical values were averaged for each quadrat in each year. These calculated abundances are of little use in describing the actual number of beetles (except for an abundance of 1, which means Galerucella spp. were not present); rather, they provide an indication of the relative size of the population. Percent cover and percent damage values were defined as the midpoint of the category range (Table2). Release timing (Chapman Lake) and replication (Bonneyville Mills) varied between sites, preventing analysis across sites. Therefore, each site was analyzed separately. Statistical analysis and graph plotting was done using R 2.14.2 (R Development Core Team 2011) and the lattice package (Sarkar 2008). Fall data were used to assess L. salicaria response over time. Data were not normally distributed, and therefore required the use of non-parametric methods. Spearman’s rank-sum correlations (ρ) were calculated to examine the relationship between year of observation and the following variables: species richness, number of stems, percent cover of L. salicaria, and number of inflorescence. Additionally, best fit lines were

10 drawn through scatterplots of the same data using the smooth option of the xyplot command which can be found in the lattice package. This utilizes loess smoothing which fits the line to the scatterplot using local polynomial regression, providing a graphical view of general trends in the data (Figures2,3,4,5). In order to provide some comparision to Landis(2003) and Farnsworth and Ellis(2001) the relationship between L. salicaria stem density and species richness also was examined. These data were normally distributed at all sites, so a simple linear regression was used to examine the data. The number of L. salicaria stems per quadrat (stem density) was used as the independent variable and species richness was the dependent variable.

1.3 Results

Spearman’s ρ showed significant positive correlation between species richness and year at Chapman Lake (p<0.001), Fish Creek (p<0.001), and Wilson Wetland (p<0.001), but not at Bonneyville Mills (p=0.2515). Fish Creek had a ρ of 0.689, the highest of the sites. Correlations at Chapman Lake and Wilson Wetland were noticeably lower with ρ’s of 0.4314 and 0.3912 respectively (Table3). All sites showed a significant correlation between year and number of stems per square meter. Spearman’s ρ was highest at Fish Creek (ρ=-0.6813) followed closely by Bonneyville Mills (ρ=-0.6407). At Chapman Lake ρ was -0.3842 and at Wilson Wetland ρ was -0.3912 (Table3). Percent cover of L. salicaria was significantly correlated to year (p<0.001) at all sites except Chapman Lake (p=.07951). Fish Creek again showed the highest correlation (ρ=- 0.6428). Spearman’s ρ was -0.5710 at Bonneyville Mills and -0.3715 at Wilson Wetland (Table3). Correlations between year and number of inflorescence per quadrat was significant at all sites (p<0.001). The highest correlation was at Wilson Wetland, where ρ was -0.7761.

11 Spearman’s ρ was -0.7431 at Fish Creek, -0.5180 at Chapman Lake, and -0.4575 at Bon- neyville Mills (Table3). In comparison to other metrics, the number of inflorescence showed a consistently high ρ. The abundance of Galerucella spp. was significantly correlated with time at Fish Creek (p=<0.0001 and Wilson Wetland (p=0.0281). It was very near the α=0.05 (at Chapman Lake (p=0.0502) and was insignificant at Bonneyville Mills (p=0.1803). Spearman’s corre- lations were consistently low: 0.3989 at Fish Creek, 0.2094 at Wilson Wetland, and 0.2071 at Chapman Lake. Species richness showed a significant relationship to L. salicaria stem density at Chap- man Lake(p=0.0133), Fish Creek (p<0.0001), and Wilson Wetland (p=0.0221), but was not significant at Bonneyville Mills (p=0.793). When analyzed across all four sites collectively the relationship was highly significant (p<0.0001).

1.4 Discussion

The response of L. salicaria to Galerucella spp. varied between sites, with some sites showing a very strong correlation between plant characteristics and time, while others showed only weak correlations. While all sites showed a negative correlation between number of stems and year, this relationship was very strong at Fish Creek and Bonneyville Mills, but weaker at Chapman Lake, and nearly insignificant at Wilson Wetland. One potential explanation for this is that heavy feeding by Galerucella spp. may reduce plant growth early in the season. Following this reduction in height and flowering, plants have been shown to respond by producing new stems (Blossey and Skinner 2000). Regardless, it is clear that in the years following release of Galerucella spp., the number of stems decreased at all sites. Blossey and Skinner(2000) observed similar reduction in stem densities at one site.

12 Percent cover by L. salicaria showed significant negative correlation with time at three sites (Bonneyville Mills, Wilson Wetland, Fish Creek), but was not significant at Chapman Lake. This could be caused by several factors. Possibly, this illustrates a recovery of L. salicaria at the Chapman Lake site; this seems unlikely because Chapman Lake is the newest of the sites. Other measures of L. salicaria health did not show similar trends. Alternatively, these minor changes that are appearing may be due to the categorical estimates over cover class or differences in personnel making these observations in subsequent years. Meanwhile, the other three sites demonstrate a significant decrease in cover of L. salicaria over time. All four sites had significant correlation between the number of L. salicaria inflorescence and year. Consistent reductions in flowering were observed three to five years after release by (Landis 2003). Flowering was essentially terminated at some point for all sites and in the final year (2007) remained extremely low. The prevention or reduction of flowering might indicate major long term impacts on L. salicaria populations. Seed accumulation in these wetlands is likely high, but if the flowering remains very low, the existing seed bank could become depleted, leading to further reductions in L. salicaria in the future. Species richness was not significantly correlated with year at Bonneyville Mills, but was significant at the other three sites. At Bonneyville Mills, species richness increase steadily for the first 3–5 years, but decreased over the next five years. During the same time period, all measures of L. salicaria health continued to decrease. The drop in species richness occurs at the same time as a decline in Galerucella spp. numbers at Bonneyville Mills (Figure5 and6). It is perhaps more likely, however, that the small number of quadrats at Bonneyville Mills may have impacted the accuracy of the species richness data. The significant correlation identified between L. salicaria stem density and species rich- ness at three of the four sites supports the findings of Landis(2003). However, Farnsworth and Ellis(2001) found no significant relationship one year after release of Galerucella spp. This suggests that a longer period of time is needed to observe these impacts. Also, the relationship of stem density and species richness was not significant at Bonneyville Mills.

13 This may have been due to natural species richness within that wetland, particularly since it was such a small area. The smaller number of quadrats within the site may have caused any relationship which did occur to remain unnoticeable. The correlation of Galerucella spp. abundance with time was low, even at sites where it was significant, with the highest correlation being 39.9% at Fish Creek. Examination of the scatter plots and best-fit lines (Figure6) better demonstrates the temporal patterns. A single year with high Galerucella spp. abundances occurred at each of the sites except Wilson Wetland. These one-year spikes occurred in 2001 at Fish Creek, 2000 at Bonneyville Mills, and 2002 at Chapman Lake. Following these increases in Galerucella spp. population, the population declined over the next several years. Past studies have shown that large populations of Galerucella spp. can decimate L. salicaria in an area (Landis 2003). Once this happens, Galerucella spp. are forced to disperse to new locations in order to find additional L. salicaria plants. This is likely occuring at these sites; several years after release the number of Galerucella spp. is sufficiently high to provide substantial control of L. salicaria but this control forces the population to disperse to a new area.

1.4.1 Conclusions

Considering the changes at each of the four sites several conclusions can be drawn. First, not all sites show the same response to Galerucella spp. over the 8–10 year time frame. These variations are likely due to subtle differences between sites, which may be biotic, abiotic, or anthropomorphic differences. Despite these notable differences, the overall trends remain. Species richness generally increased each year after Galerucella spp. were released. Similarly, L. salicaria stem density, percent cover, and number of inflorescence decreased over time. The general trends suggest that the impact of Galerucella spp. is significant, though the level of control and the impacts of the beetles may be variable between sites. The one impact which was significant at all sites was a reduction in flowering. This also is the most visible change, as the absence of large purple spike inflorescence is a dramatic

14 change from uncontrolled populations. Early studies of biological control insects suggested that a combination of several insects may best control L. salicaria; one of the approved biological control agents is Nanophyes marmoratus, a flower weevil. Considering the impact Galerucella spp. had on flower production, significant populations of both insects may not be sustainable within a wetland. In conclusion, following the introduction of Galerucella spp. Lythrum salicaria had a significant negative response at each of the wetlands in this study. However, L. salicaria was more inhibited at some sites than others, with Fish Creek showing the response most clearly. In addition to reducing the health of L. salicaria, species richness generally increased. This also adds to the existing evidence of the negative effects of L. salicaria. While this study did not examine the community diversity beyond simple richness, species were recorded. Examination of these data in the future may provide greater insight into the impacts of L. salicaria on species richness. Over time, additional impacts may arise, particularly a further decrease in L. salicaria stems as a result of the sudden decline in seed production. Whether the release of Galerucella spp. will provide the desired level of long-term control remains unclear, but their impacts at these wetlands strongly suggest that Galerucella spp. can play a major role in controlling L. salicaria. Other studies have observed similar changes, and this study adds a longer time frame than most past studies. This further demonstrates that Galerucella spp. can be an effective means for controlling populations of L. salicaria.

15 2 Dispersal and Distribution of Biological Control

Agents for Lythrum salicaria in Indiana

Abstract

The invasive wetland perennial, Lythrum salicaria has spread throughout Indiana wetlands since 1900. Four insect species were eventually approved for release. These species in- cluded: Hylobius transversovittatus, Nanophyes marmoratus, Galerucella calmariensis, and G. pusilla. The distribution of these beetles has been monitored by the Indiana Department of Natural Resources since 1994. This project aimed to expand the existing data on locations of these control agents and to estimate their distribution throughout the state. Nanophyes marmoratus and Hylobius transversovittatus have spread slowly between wetlands. Addi- tionally, the nocturnal behavior of Hylobius transversovittatus has increased the difficulty in locating populations. Geospatial analyses of Galerucella spp. suggests that they have become widely distributed in the northern region of the state. By calculating distances and date of initial observation between sites, it was estimated that Galerucella spp. spread at a rate of least 491 meter per year with a maximum rate of 1822. This is simplified calculation of dispersal rates allowed for visualization of areas for potential future releases. Additionally, it demonstrated that Galerucella spp. have become widely established through northern In- diana. Keywords: Lythrum salicaria, Galerucella, Nanophyes marmoratus, Hylobius transverso- vittatus, biological control, invasive species, geospatial distribution

16 2.1 Introduction

Lythrum salicaria is an invasive wetland perennial in North America(Thompson et al. 1987). It form clumps of 30–50 stem, arising from one taproot (Mal et al. 1992). A terminal spike of tightly clustered flowers is produced and may be over 1 meter in length. Additional lateral spikes arise on lower branches of the stem (Mal et al. 1992) and a single plant can produce upwards of 2.5 million seeds each year (Malecki et al. 1993). Lythrum salicaria may reach heights as great at 2.7 meters (Mal et al. 1992). The distribution of L. salicaria has been documented by Thompson et al.(1987) and Hult´en(1971). The earliest record of L. salicaria in North America were in 1814 from New England (Mal et al. 1992). Introductions of L. salicaria likely occurred in a variety of manners, including inadvertent transport in shipping ballast and on imported wool, and intentional introduction from immigrants, who used the plant as a medicinal herb (Thompson et al. 1987). Human affects contributed to the continued spread of L. salicaria into the Midwest. The development and use of canals provided a disturbed habitat, ideal for an invasive like L. salicaria Thompson et al.(1987). Heavy use of the canals for transport would have allowed for seed to be carried westward. Other use of the plant, both as an ornamental and as a nectar plant for beekeeping, resulted in intentional introductions as well Thompson et al.(1987). In Indiana the earliest record is from 1900, and Stuckey(1980) notes very little spread through 1940. Lythrum salicaria is distributed throughout Indiana and is most common in the northern counties.

2.1.1 Impacts of Lythrum salicaria

L. salicaria can form monotypic stands (Malecki et al. 1993) and a number of ecological impacts have been documented. These impacts included: domination of the seed bank (Welling and Becker 1990), reducing wildlife habitat quality (Whitt et al. 1999, Rawinski 1982, Lor 2000), interference with pollination of native species (Brown et al. 2002, Da Silva

17 and Sargent 2011, Templer et al. 1996), and alterations to wetland function (Emery and Perry 1996, B¨arlocher and Biddiscombe 1996,). Lythrum salicaria also has been shown to outcompete native plants in a variety of wetlands (Gaudet and Keddy 1995, Weihe and Neely 1997, Gabor et al. 1996, Mal et al. 1997).

2.1.2 Control

Control efforts against L. salicaria began in the 1950’s, and included attempts at flooding, cutting, and burning (Skinner et al. 1994). Initial efforts were largely unsuccessful for all but the smallest patches of L. salicaria (Blossey et al. 2001, Skinner et al. 1994). Glyphosate, 2,4-D, or triclopyr have been used in chemical control but, because of extensive seed banks (Welling and Becker 1990), spraying must be repeated often (Blossey et al. 2001, Skinner et al. 1994). The non-selective nature of chemical control also reduced populations of sedges, grasses, cattails, and other native wetland plants (Skinner et al. 1994, Gabor et al. 1996). The lack of effective methods of control and continued dispersal of L. salicaria resulted in the formation of a program to establish a biological control program for L. salicaria in North America. An overview of this process is provided by Malecki et al.(1993), while Blossey et al.(2001), and Hight and Drea(1991) examine the process in more detail. Four insect species eventually were approved for release. These species included: Hylobius transversovit- tatus Goeze (a root-mining weevil), Nanophyes marmoratus Goeze (a flower feeding weevil), Galerucella calmariensis L., and G. pusilla Duft (two leaf-beetles) (Blossey et al. 2001).

2.1.3 Brief Insect Life Histories

Galerucella calmariensis and G. pusilla

Adult Galerucella spp. emerge from the leaf litter or soil in the spring (April or May)(Blossey et al. 1994b). Beetles begin feeding on meristematic tissue of L. salicaria and oviposition begins after about one week of feeding. The oviposition period last around two months,

18 peaking in May and June but continuing into July (Blossey et al. 1994b). Eggs are laid in the leaf axils or on the plant stems in clusters of two to ten eggs and are covered with a layer of frass (Malecki et al. 1993). Early instars of the emerged larvae feed preferentially on the developing buds while older larvae will feed on any portion of the plant tissue (Blossey et al. 1994b). Intense larval feeding results can result in the leaves of L. salicaria being completely skeletonized. Once larvae are mature they pupate in the soil or leaf litter (Blossey et al. 1994b). Adults emerge in July and, in some cases, will carry out a second oviposition period prior to overwintering (Landis 2003). This second generation will feed on L. salicaria regrowth and in these cases very severe impacts on L. salicaria have been observed. Following intense feeding on L. salicaria, the lack of L. salicaria may cause Galerucella spp. adults to disperse into other areas where L. salicaria is readily available. Dispersing insects are strongly attracted by conspecifics (Grevstad and Herzig 1997), due to pheromone production by males feeding on L. salicaria (Bartelt et al. 2008, Hamb¨ack 2010).

Hylobius transversovittatus

Adult Hylobius transversovittatus emerge in the spring and begin feeding nocturnally on young L. salicaria leaves (Malecki et al. 1993). Oviposition lasts two to three months and occurs into the soil or into the plant stem (Malecki et al. 1993, Blossey et al. 1994a). Young larvae feed on roothairs or the stem pith, depending on where eggs were oviposited (Blossey et al. 1994a). Larvae continue feeding on the root tissue by mining into the root, eventually forming pupation chambers in the upper parts of the root. Adults may emerge in late summer or the following spring and live several years.

Nanophyes marmoratus

Nanophyes marmoratus adults emerge from the leaf litter in May or June and begin feeding on L. salicaria leaves. Once flower buds have developed they begin feeding on the buds and mate. Eggs are laid into the flower buds and larvae feed on the developing flower, preventing

19 flowering. Larvae pupate in the bud and adults emerge in July and August and continue feeding on leaf tissue before overwintering in the leaf litter (Wilson et al. 2001).

2.1.4 History of Indiana Biological Control

Following the approval of Galerucella spp., N. marmoratus, and H. transversovittatus for release, all three were released at sites in Indiana by the Indiana Department of Natural Re- sources (IDNR), Division of Nature Preserves. Galerucella spp. were the first to be approved, and a protocol for raising captive populations was quickly established. Both N. marmoratus and H. transversovittatus proved to be more difficult to raise. Galerucella spp. were released to a greater number of sites because of they were approved first and were easily raised. Releases of Galerucella spp. continued, and as populations accumulated, most releases were simply transfers of individuals collected from existing populations to a site where they were previously absent.

2.1.5 Project Goals

The aim of this project was to develop a geodatabase containing files to track the distri- bution of the biological control agents across the state. It was also a goal of this project to understand the current spread and patterns of dispersal by field checking numerous sites throughout northern Indiana.

2.2 Methods

The primary files in the geodatabase are point feature classes of locations where beetle populations have been located. To maintain these records, any new release site is added to the files and time each spring is spent checking locations where the beetles are likely to have spread. This surveying involves driving to areas around known release or dispersal sites and identifying areas infested by L. salicaria. Once L. salicaria is found, the area is

20 checked for presence of beetles and their abundance is ranked according to Table1. During the summer of 2011, additional efforts were made to survey a number of sites within spatial gaps in the current data. In order to check a large number of sites over a short period of time most of the sites examined were boat ramps. In addition to ease of checking for L. salicaria and biological control agents, boat ramps are disturbed habitats, often are in full sunlight (favored by L. salicaria, and boats and trailers provide a means of spreading loosestrife seed. As a result of this additional work, 103 sites were examined, with nearly all of them being boat ramps. Geospatial analysis, processing, and map construction was performed using ArcGIS Desk- top 10 (ESRI 2011). In addition, the Geospatial Monitoring Environment (Beyer 2012) was used where noted. The initial task was to restructure existing data to facilitate the updating of these files over time. A new geodatabase was created, containing all the required files. Three empty point feature classes were created, one for each beetle species. Appropriate fields were added to the feature class and wherever possible domains were created to limit the input options. These fields included Release, County, and Abundance. The original data were arranged in three shapefiles, one for each beetle species. The ”Add XY” tool was used in ArcMap to add the X,Y locations for each point to the shapefiles and the shapefiles were exported to text format. After ensuring all data were correct, the text files were imported to the new feature classes using the ”Add XY Data” tool. Following the creation of the feature classes, additional sites surveyed during 2011 were added to these files. A series of maps were created to show the dispersal of L. salicaria biological control insects in Indiana. numbers of known H. transversovittatus and N. marmoratus sites remain fairly low, therefore, only a single map was produced for each of these species (Figures8 and9). However, Galerucella spp. has dispersed quite well and are relativity easy to locate, resulting in 156 confirmed sites. Therefore, several maps were constructed to depict their distribution (Figures 10 and 11).

21 Finally, the geospatial data were analyzed to determine the likely dispersal rate of Galeru- cella spp. Non-release sites were joined to each release site manually, using data exported to spreadsheets. Using the convert.tabletolines command in the Geospatial Modelling Envi- ronment (Beyer 2012), the distance between each set of points was determined and added to the table. For each pair of sites the number of years between the release of Galerucella spp. at the release site and the first observation at the non-release site was determined. The distance between sites was divided by the years between these dates to calculate the average distance traveled per year. The assumption was made that the smallest distance travel per year was the most likely source of the beetles at the new site, and other pairs of points were removed. One record remained for each non-release site. The maximum distance per year (1822 m/yr) and the mean distance per year (491 m/yr) from this list were then used to draw buffers around known Galerucella spp. locations in ArcMap. The calculated dispersal distance per year was multiplied by the number of years since the first observation (or release date) at each site. The buffer was then drawn at this distance. The buffer distances around a newer site are smaller, while that of an old site is much larger (Figure 10).

2.3 Results and Discussion

Locating H. transversovittatus has been very difficult (Ferrarese and Garono 2010). The adult beetles are nocturnal (Blossey et al. 1994a), so surveying is usually conducted for beetles in the larval stage. However, this stage is completed below ground, making surveying labor intensive and requiring uprooting the plants for close examination (Malecki et al. 1993). Therefore, it is difficult to draw conclusions as to their distribution within Indiana. They have not been located at new sites and only occasionally at past release sites, this is possibly due to their secretive life cycle. Nanophyes marmoratus have been found at some non-release sites, but all of these have been located near to a release site, suggesting that their dispersal has been limited. However,

22 their dispersal also may be limited by the success of Galerucella spp. Large populations of Galerucella spp. have can reduce the flowering of L. salicaria (Blossey and Skinner 2000). These flowers are necessary to the life cycle of N. marmoratus. A drastic reduction in flow- ering could limit the success and dispersal of N. marmoratus. Additionally, N. marmoratus have been release at far fewer sites in Indiana than Galerucella spp., so the potential for locating new sites is less for N. marmoratus. Galerucella spp. have spread quickly and efficiently across the state The mean annual dispersal distance of Galerucella spp. was calculated at 491 meters per year and the maximum was 1822 meters per year. The buffered areas in Figure 10 allow for an approximation of the potential area to which Galerucella spp. may have dispersed. These areas cover a total of 5,294 km2 for the dispersal of 491 meter per year and 33,348 km2 based on 1822 meters per year. Using the National Wetland Inventory as a basis for L. salicaria habitat, the potential Galerucella spp. habitat is severely reduced to 747 km2 (491 m/yr) and 2,799 km2 (1822m/yr). When compared to previous studies, these dispersal estimations fall within the wide range of values observed. Albright et al.(2004) found Galerucella spp. 9 km from the nearest release site after 4 years, though they hypothesized that the total distance was travelled in one year. Even if the dispersal is estimated over four years, the distance of 2,250 meters per year is slightly larger than the 1822 meters per year found in this study. However, Dech and Nosko(2002) suggest a very limited dispersal rate of both Galerucella species. After four years Galerucella spp. were found a maximum of approximately 50 meters from the release location. Based on additional observations made during the surveying for Galerucella spp. it ap- pears that the beetles are well-distributed to potential habitat within the 491 meters per year buffer area. Dispersal throughout the larger buffer area is more sporadic, but a large number of sites do occur beyond this buffered area (Figure 10). While the assumption was made that Galerucella spp. dispersal occurs in a uniform fashion over time (x meters per

23 year) this is almost certainly not the case. This assumption allows for an approximation of the potential area inhabited by Galerucella spp., but their dispersal patterns are more complex. Bartelt et al.(2008), Hamb¨ack(2010), Grevstad and Herzig(1997) have shown the importance of pheromones released by Galerucella spp. and the resulting aggregation behavior. Additionally, when large Galerucella spp. populations accumulate they can defoli- ate nearly all L. salicaria in the area (Landis 2003). Following this defoliation, beetles have been observed to disperse in large numbers to new sites, further complicating the dynamics and patterns of dispersal. While the area defined in Figure 10 are potential areas, the provide an approximation to work from. Using these maps, the areas least likely to be currently occupied by Galerucella spp. can be identified and additional releases can be performed in these areas.

24 Table 1: Galerucella abundance categories. Abundance Number of egg masses, Category larvae, or adults 1 0 2 1–9 3 10–49 4 50–99 5 100–499 6 500–1000 7 <1000

Table 2: Midpoints used for percent cover and percent damage variables. Coded Category Category Value Midpoint Range A 0% 0% B 3% 1–5% C 15% 5–25% D 37.5% 25–50% E 62.5% 50–75% F 87.5% 75–100%

25 Table 3: Spearman rank-sum correlations between year and number of stems, percent cover by L. salicaria, number of inflorescence, or species richness. Spearman’s ρ followed by an asterisk (*) are significant. Site Variable Used Spearman’s ρ p-value Bonneyville Species Richness -0.1573 0.2515 Mills Stems -0.6407* <0.0001 % Cover -0.5710* <0.0001 Inflorescence -0.4575* 0.0006 Galerucella spp. Abundance 0.0981 0.4803 Chapman Species Richness 0.4314* <0.0001 Lake Stems -0.3242* 0.0018 % Cover -0.1858 0.0795 Inflorescence -0.5181* <0.0001 Galerucella spp. Abundance 0.2071 0.0502 Fish Species Richness 0.6895* <0.0001 Creek Stems -0.6813* <0.0001 % Cover -0.6427* <0.0001 Inflorescence -0.7431* <0.0001 Galerucella spp. Abundance 0.3989* <0.0001 Wilson Species Richness 0.3911* <0.0001 Wetland Stems -0.2100* 0.0277 % Cover -0.3715* <0.0001 Inflorescence -0.7761* <0.0001 Galerucella spp. Abundance 0.2094* 0.0281

26 Figure 1: Galerucella spp. transect analysis sites.

27 1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Fish Wilson

100 100

50 50 2

0 0

Bonneyville Chapman stems per meter

100 100 L. salicaria

50 50

0 0

1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Year

Figure 2: Number of Lythrum salicaria stems per quadrat over time.

28 1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Fish Wilson

80 80

60 60

40 40

20 20

0 0 percent cover Bonneyville Chapman

80 80 L. salicaria 60 60

40 40

20 20

0 0

1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Year

Figure 3: Lythrum salicaria percent cover class over time. Jitter has been added to keep points from overlapping, since both variables are essentially categorical.

29 1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Fish Wilson

500 500

400 400

300 300 2 200 200

100 100

0 0

Bonneyville Chapman inflorescence per meter 500 500

400 400

L. salicaria 300 300

200 200

100 100

0 0

1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Year

Figure 4: Total number of Lythrum salicaria inflorescences over time.

30 1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Fish Wilson 30 30

25 25

20 20

15 15

10 10

2 5 5

Bonneyville Chapman 30 30

species per meter 25 25

20 20

15 15

10 10

5 5

1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Year

Figure 5: Total fall species richness over time.

31 1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Fish Wilson

3.5 3.5

3.0 3.0

2.5 2.5

2.0 2.0

1.5 1.5

1.0 1.0

Bonneyville Chapman abundance catagoryabundance

3.5 3.5

3.0 3.0

Galerucella 2.5 2.5

2.0 2.0

1.5 1.5

1.0 1.0

1997 1999 2001 2003 2005 2007 1997 1999 2001 2003 2005 2007 Year

Figure 6: Galerucella abundance over time. Abundance is calculated as the mean categorical value for the three life stages, as described in Chapter 1 and jitter has been added to keep points from overlapping.

32 0 50 100 0 50 100 Fish Wilson 30 30

25 25

20 20

15 15

10 10

5 5

Bonneyville Chapman 30 30

pce Richness Species 25 25

20 20

15 15

10 10

5 5

0 50 100 0 50 100 L. salicaria stems per meter2

Figure 7: Linear regression of the relationship between density of L. salicaria and plant species richness by site.

33 Figure 8: Hylobius transversovittatus sites in Indiana. All sites are release sites.

34 Figure 9: Nanophyes marmoratus release sites (?) and non-release sites (•) in Indiana

35 Figure 10: Galerucella spp. release sites (?) and non-release sites (•) in Indiana with buffers showing potential area currently occupied by Galerucella spp. The small buffer area is based on 491 m/yr and the larger on 1822 m/yr.

36 Figure 11: Locations where Galerucella spp. have been found in Indiana.

37 References

Albright MF, Harman WN, Fickbohm SS, Meehan H, Groff S, and Austin T. 2004. Recovery of native flora and behavioral responses by Galerucella spp. following biocontrol of purple loosestrife. American Midland Naturalist 152:248–254.

Anderson MG. 1995. Interactions between Lythrum salicaria and native organisms: A critical review. Environmental Management 19:225–231.

B¨arlocher F and Biddiscombe NR. 1996. Geratology and decomposition of Typha latifolia and Lythrum salicaria in a freshwater marsh. Archiv fu¨rHydrobiologie 136:309–325.

Bartelt RJ, Coss´eAA, Zilkowski BW, Wiedenmann RN, and Raghu S. 2008. Early-summer pheromone biology of Galerucella calmariensis and relationship to dispersal and coloniza- tion. Biological Control 46:409–416.

Beyer H. 2012. Geospatial Modelling Environment. Version 0.6.0.0.

Blossey B. 1994. Purple loosestrife monitoring protocol. Departent of Natural Resources, Cornell University. Unpublished.

Blossey B, Schroeder D, Hight SD, and Malecki RA. 1994a. Host specificity and environmen- tal impact of the weevil Hylobius transversovittatus, a biological control agent of purple loosestrife (Lythrum salicaria). Weed Science 42:128–133.

Blossey B, Schroeder D, Hight SD, and Malecki RA. 1994b. Host specificity and environ- mental impact of two leaf beetles (Galerucella calmariensis and G. pusilla) for biological control of purple loosestrife (Lythrum salicaria). Weed Science 42:134–140.

Blossey B and Skinner L. 2000. Design and importance of post-release monitoring. In: Spencer NR, editor. Proceedings of the X International Symposium on Biological Control of Weeds, p 693–706.

Blossey B, Skinner LC, and Taylor J. 2001. Impact and management of purple loosestrife (Lythrum salicaria). Biodiversity and Conservation 10:1787–1807.

Brown B and Mitchell R. 2001. Competition for pollination: effects of pollen of an invasive plant on seed set of a native congener. Oecologia 129:43–49.

Brown BJ, Mitchell RJ, and Graham SA. 2002. Competition for pollination between and invasive species (purple loosestrife) and a native congener. Ecology 83:2328–2336.

Chun YJ, Kim CG, and Moloney KA. 2010. Comparison of life history traits between invasive and native populations of purple loosestrife (Lythrum salicaria) using nonlinear mixed effects model. Aquatic Botany 93:221–226.

38 Da Silva EM and Sargent RD. 2011. The effect of invasives Lythrum salicaria pollen depo- sition on seed set in the native species Decodon verticillatus. Botany 89:141–146.

Dech JP and Nosko P. 2002. Population establishment, dispersal, and impact of Galerucella pusilla and G. calmariensis, introduced to control purple loosestrife in Central Ontario. Biological Control 23:228–236.

Emery SL and Perry JA. 1996. Decomposition rates and phosphorus concentrations of purple loosestrife Lythrum salicaria and cattail Typha spp. Hydrobiologia 323:129–138.

ESRI. 2011. ArcGIS Desktop 10. Environmental Systems Research Institute, Redlands, CA.

Farnsworth EJ and Ellis DR. 2001. Is purple loosestrife (Lythrum salicaria) and invasive threat to freshwater wetlands? Conflicting evidence from several ecological metrics. Wet- lands 21:199–209.

Ferrarese E and Garono RJ. 2010. Dispersal of Galerucella pusilla and G. calmariensis via passive water transport in the Columbia River Estuary. Biological Control 52:115–122.

Gabor TS, Haagsma T, and Murkin HR. 1996. Wetland plant responses to varying degrees of purple loosestrife removal in southeastern Ontario, Canada. Wetlands 16:995–98.

Gaudet CL and Keddy PA. 1995. Competitive performance and species distribution in shoreline plant communities: A comparative approach. Ecology 76:280–291.

Grevstad FS. 2006. Ten-year impacts of biological control agents Galerucella pusilla and G. calmariensis (Coleoptera: Chrysomelidae) on purple loosestrife (Lythrum salicaria) in Central New York State. Biological Control 39:1–8.

Grevstad FS and Herzig AL. 1997. Quantifying the effects of distance and conspecifics on colonization: Experiments and models using the loosestrife leaf beetle, Galerucella calmariensis. Oecologia 110:60–68.

Grout JA, Levings CD, and Richardson JS. 1997. Decomposition rates of purple looses- trife (Lythrum salicaria) and Lyngbyei’s Sedge (Carex lyngbyei) in Fraser River Estuary. Estuaries and Coasts 20:96–102.

Hager HA and Vinebrooke RD. 2004. Positive relationships between invasive purple looses- trife (Lythrum salicaria) and plant species diversity and abundance in Minnesota wetlands. Canadian Journal of Botany 82:763–773.

Hamb¨ack P. 2010. Density-dependent processes in leaf beetles feeding on purple loosestrife: Aggregative behavior affecting individual growth rates. Bulletin of Entomological Research 100:605–611.

Hight SD and Drea JJ. 1991. Prospects for a classical biological control project against purple loosestrife (Lythrum salicaria L.). Natural Areas Journal 11:151–157.

Hult´enE. 1971. The circumpolar plants: 2. Almqvist & Wiksell, Stockholm. 39 Landis DA. 2003. Establishment and impact of Galerucella calmariensis L. (Coleoptera: Chrysomelidae) on Lythrum salicaria L. and associated plant communities in Michigan. Biological Control 28:78–91. Lor S. 2000. Population status and breeding biology of marsh birds in Western New York. [thesis], Ithaca (NY):Cornell University. Mal TK, Lovet-Doust J, and Lovet-Doust L. 1997. Time-dependent competitive displacement of Typha angustifolia by Lythrum salicaria. Oikos 79:26–33. Mal TK, Lovett-Doust J, Lovett-Doust L, and Mulligan G. 1992. The biology of Canadian weeds. 100. Lythrum salicaria. Canadian Journal of Plant Science 72:1305–1330. Malecki RA, Blossey B, Hight SD, Schroeder D, Kok LT, and Coulson JR. 1993. Biological control of purple loosestrife. BioScience 43:680–686. Piper G. 1996. Biological control of the wetlands weed puple loosestrife (Lythrum salicaria in the Pacific northwestern United States. Hydrobiologia 340:291–294. R Development Core Team. 2011. R: A Language and Environment for Statistical Comput- ing. R Foundation for Statistical Computing, Vienna, Austria. Rawinski TJ. 1982. The ecology and management of purple loosestrife (Lythrum salicaria L.) in central new york. [thesis], Cornell University. Rawinski TJ and Malecki RA. 1984. Ecological relationships among purple loosestrife, cattail and wildlife at the Montezuma National Wildlife Refuge. New York Fish and Game Journal 31:81–87. Sarkar D. 2008. Lattice: Multivariate Data Visualization with R. Springer, New York. Schooler SS and McEvoy PB. 2006. Relationship between insect density and plant damage for the golden loosestrife beetle, Galerucella pusilla, on purple loosestrife (Lythrum salicaria). Biological Control 36:100–105. Skinner LC, Rendal WJ, and Fuge EL. 1994. Minnesota’s purple loosestrife program: History, findings, and management recomendations. Special publication 145, Minnesota Depart- ment of Natural Resources. Stamm-Katovich EJ, Becker, Roger L, and Ragsdale DW. 1999. Effect of Galerucella spp. on survival of purple loosestrife (Lythrum salicaria) roots and crowns. Weed Science 47:360–365. Stuckey RL. 1980. Distributional history of Lythrum salicaria (purple loosestrife) in North America. Bartonia 47:3–20. Templer P, Findaly S, and Wigand C. 1996. Sediment chemistry associated with native and non-emergent macrophytes of Hudson River marsh ecosystem, p 1–32. In: Waldman J, Nieder W, and Blair E, editors. Final Reports of the Tibor T. Polgar Fellowship Program, 1995. 40 Thompson DQ, Stucky RL, and Thompson EB. 1987. Spread, impact, and control of purple loosestrife (Lythrum salicaria) in North American wetlands. Technical report, U.S. Fish and Wildlife Service.

Weihe PE and Neely RK. 1997. The effects of shading on competition between purple loosestrife and broad-leafed cattail. Aquatic Botany 59:127–138.

Weiher E, Wisheu IC, Keddy PA, and Moore DR. 1996. Establishment, persistence, and management implications of experimental wetland plant communities. Wetlands 16:208– 218.

Welling CH and Becker RL. 1990. Seed bank dynamics of Lythrum salicaria L.: Implications for control of this species in North America. Aquatic Botany 38:303–309.

Whitt MB, Prince HH, and Cox, Jr. RR. 1999. Avian use of purple loosestrife dominated habitat relative to other vegetation types in a Lake Huron wetland complex. The Wilson Bulletin 11:105–114.

Wilson LM, Schwarzlaender M, Blossey B, and Randal CB. 2001. Biology and Biological Control of Purple Loosestrife. USDA Forest Service / UNL Faculty Publications.

41 Appendix A: GIS Tables

Table 4: All Galerucella spp. locations with selected attributes. Last two columns show distances

used for buffer radius in Figure 10.

Location Name County Released First Obs. Abundance Latest Obs. 491 m/yr 1822 m/yr Ball Wetlands KOSCIUSKO Y 7/15/1997 D 6/16/2010 7365 27330 Ball Wetlands KOSCIUSKO Y 1/1/2003 E 6/16/2010 4419 16398 Big Otter Lake STEUBEN Y 7/1/1996 E 6/1/2009 7856 29152 Bonneyville Mills ELKHART Y 7/3/1996 C 1/1/2003 7856 29152 Boot Lake ELKHART Y 1/1/2002 E 7/1/2010 4910 18220 Calumet Prairie LAKE Y 6/17/2008 D 7/15/2010 1964 7288 42 Calumet River C LAKE Y 1/1/2004 D 5/19/2008 3928 14576 Calumet River D LAKE Y 1/1/2004 D 5/19/2008 3928 14576 Calumet Trail PORTER Y 7/16/1997 B 6/5/2001 7365 27330 Cedarville ALLEN Y 8/1/1996 E 6/19/2007 7856 29152 Chapman KOSCIUSKO Y 7/15/1997 F 7/6/2011 7365 27330 Clark and Pine LAKE Y 1/1/2000 C 7/28/2010 5892 21864 Cline Ave NP LAKE Y 1/1/2006 C 7/12/2010 2946 10932 Cole CASS Y 1/1/2003 F 5/23/2011 4419 16398 Cook Lake MARSHALL Y 6/1/2009 C 6/12/2006 1473 5466 Crooked Lake WHITLEY Y 7/3/1996 C 7/11/2011 7856 29152 Dixon MARSHALL Y 7/15/1997 D 6/17/2010 7365 27330 Dixon MARSHALL Y 7/15/1997 D 6/17/2010 7365 27330 Dupont LAKE Y 1/1/2000 C 7/6/2010 5892 21864 Fawn River STEUBEN Y 7/15/1997 E 7/6/2011 7365 27330 Fish Creek Fen LAPORTE Y 1/1/1996 F 5/14/2009 7856 29152 Location Name County Released First Obs. Abundance Latest Obs. 491m/yr 1822 m/yr Flat Lake MARSHALL Y 7/15/1997 E 6/17/2010 7365 27330 Gibson LAKE Y 6/26/1997 C 7/14/2010 7365 27330 Goshen Millpond ELKHART Y 1/1/2005 E 6/20/2011 3437 12754 Grassy Lake LAGRANGE Y 7/8/1996 C 6/16/2003 7856 29152 Green Lake STEUBEN Y 1/1/1997 D 1/1/2009 7365 27330 Hardy Lake SCOTT Y 6/19/1997 C 6/13/2004 7365 27330 I-70 Little Point N MORGAN Y 6/2/2011 B 6/2/2011 491 1822 I-70 Little Point S MORGAN Y 6/2/2011 B 6/2/2011 491 1822 I-70 Mile 57 HENDRICKS Y 6/2/2011 B 6/2/2011 491 1822 Indy Quick Mart ditch MARION Y 6/2/2011 B 6/2/2011 491 1822 Indy Quick Mart pond MARION Y 6/2/2011 B 6/2/2011 491 1822 Kankakee (Ten Mile Road) STARKE Y 6/15/1997 B 7/7/2011 7365 27330 LaSalle 5 NEWTON Y 1/1/2006 B 6/28/2011 2946 10932

43 Latonka MARSHALL Y 1/1/1996 F 1/1/2009 7856 29152 Latonka - Theiling MARSHALL Y 1/1/1994 F 1/1/2009 8838 32796 Loon Lake STEUBEN Y 7/15/1997 C 5/22/2007 7365 27330 Manitou KOSCIUSKO Y 1/1/2001 C 6/17/2010 5401 20042 Manitou FULTON Y 1/1/1996 E 6/28/2011 7856 29152 Manitou - Moose FULTON Y 1/1/1996 F 1/1/2011 7856 29152 Manitou - off island FULTON Y D 1/1/2009 0 0 Maxinkuckee MARSHALL Y 1/1/1996 D 6/17/2010 7856 29152 Mississinewa WABASH Y 1/1/1996 B 6/10/2011 7856 29152 Oak Ridge LAKE Y 7/16/1997 C 8/24/2004 7365 27330 Olin Lake LAGRANGE Y 5/31/2007 C 6/22/2010 2455 9110 Oxbow ELKHART Y 6/15/1997 C 6/20/2000 7365 27330 Pine Station (Bongi) LAKE Y 7/16/1997 C 7/28/2010 7365 27330 Pleasant Lake ST. JOSEPH Y 6/30/1996 E 6/28/2011 7856 29152 Potawatomi - beaver dam STEUBEN Y 1/1/1997 D 6/1/2005 7365 27330 Potawatomi - Lonidaw STEUBEN Y 1/1/2000 D 6/1/2005 5892 21864 Location Name County Released First Obs. Abundance Latest Obs. 491m/yr 1822 m/yr Potawatomi - yellow jack STEUBEN Y 1/1/1997 E 5/24/2007 7365 27330 Putterbaugh Creek ELKHART Y 5/26/2004 E 5/27/2011 3928 14576 Seidner Dune and Swale LAKE Y 1/1/2001 C 7/14/2010 5401 20042 Seven Sisters STEUBEN Y 1/1/1996 F 1/1/2008 7856 29152 Seven Sisters - Seidler STEUBEN Y 1/1/1994 F 1/1/2008 8838 32796 Springville LAPORTE Y 7/2/1996 C 6/14/2004 7856 29152 Stone Lake LAGRANGE Y 1/1/1999 B 7/14/2003 6383 23686 Terry Lake DEKALB Y 7/1/1996 D 6/13/2005 7856 29152 Tolleston (Gibson) LAKE Y 7/16/1997 D 7/12/2010 7365 27330 Wawasee KOSCIUSKO Y 9/20/1996 E 6/19/2009 7856 29152 Wawasee Boat Co. KOSCIUSKO Y 1/1/2003 E 6/19/2009 4419 16398 West Beach LAKE Y 1/1/1994 D 5/18/2010 8838 32796 West Beach LAKE Y 1/1/1999 D 5/18/2010 6383 23686

44 West Beach LAKE Y 1/1/1999 D 5/18/2010 6383 23686 West Beach LAKE Y 1/1/1999 D 5/18/2010 6383 23686 Wilson Ditch MARSHALL Y 7/6/1996 E 6/17/2010 7856 29152 Yellowwood BROWN Y 6/26/1997 F 7/25/2011 7365 27330 Gibson LAKE N 5/28/1998 C 5/28/1998 6874 25508 Koontz Lake MARSHALL N 6/12/2001 D 6/12/2011 5401 20042 Stone Lake LAGRANGE N 7/14/2003 D 6/3/2010 4419 16398 Westpoint mitigation LAKE N 7/13/2005 B 7/13/2005 3437 12754 Cook Millpond MARSHALL N 6/12/2006 C 6/12/2006 2946 10932 Chapman Big NP KOSCIUSKO N 5/22/2007 C 6/19/2010 2455 9110 Rochester Ramp MARSHALL N 5/22/2007 E 6/17/2010 2455 9110 Fish Creek Fen TNC LAPORTE N 7/9/2008 C 5/14/2009 1964 7288 Ropchan Memorial STEUBEN N 7/15/2008 C 7/15/2008 1964 7288 Cole CASS N 10/3/2008 E 5/23/2011 1964 7288 Potato Creek ST JOSEPH N 1/1/2009 C 1/1/2009 1473 5466 Ligonier NOBLE N 5/1/2009 D 5/1/2009 1473 5466 Location Name County Released First Obs. Abundance Latest Obs. 491m/yr 1822 m/yr North Chain Lake ST JOSEPH N 5/1/2009 C 5/1/2009 1473 5466 Houghton Lake MARSHALL N 5/14/2009 C 5/14/2009 1473 5466 Conklin Bay KOSCIUSKO N 6/19/2009 C 6/19/2009 1473 5466 Pretty Lake MARSHALL N 7/1/2009 C 7/1/2009 1473 5466 Story Lake DEKALB N 5/12/2010 C 5/12/2010 982 3644 Rogers Park LAGRANGE N 5/25/2010 B 5/25/2010 982 3644 Shriner Lake WHITLEY N 5/28/2010 C 5/28/2010 982 3644 Pigeon Lake LAGRANGE N 6/3/2010 C 6/3/2010 982 3644 Syracuse DQ KOSCIUSKO N 6/3/2010 C 6/3/2010 982 3644 Ball Wetlands KOSCIUSKO N 6/16/2010 C 6/21/2011 982 3644 Goshen Dam ELKHART N 6/16/2010 C 6/16/2010 982 3644 Orland Park LAGRANGE N 6/16/2010 C 6/16/2010 982 3644 Manitou KOSCIUSKO N 6/17/2010 C 6/17/2010 982 3644

45 McClosky Savanna LAKE N 1/1/2011 F 1/1/2011 491 1822 Adj. to CR 200 N CASS N 5/23/2011 C 5/23/2011 491 1822 CR 200 N and CR 275 E CASS N 5/23/2011 C 5/23/2011 491 1822 CR 200 N and Rt 17 CASS N 5/23/2011 D 5/23/2011 491 1822 CR 275 N and CR 400 E CASS N 5/23/2011 D 5/23/2011 491 1822 Leases Corner at Rt 17 CASS N 5/23/2011 D 5/23/2011 491 1822 Rt 17 south of CR500N CASS N 5/23/2011 D 5/23/2011 491 1822 South Mud Lake FULTON N 5/23/2011 B 5/23/2011 491 1822 Bachelor Rd West STEUBEN N 5/24/2011 D 5/24/2011 491 1822 Big Otter Lake Boat Ramp STEUBEN N 5/24/2011 E 5/24/2011 491 1822 Cedar Swamp STEUBEN N 5/24/2011 C 5/24/2011 491 1822 Clear Lake Boat Ramp STEUBEN N 5/24/2011 D 5/24/2011 491 1822 CR 675 W, East of Tamarack Lake STEUBEN N 5/24/2011 C 5/24/2011 491 1822 Crooked Creek STEUBEN N 5/24/2011 D 5/24/2011 491 1822 Crooked Creek, East of Tamarck Lake STEUBEN N 5/24/2011 D 5/24/2011 491 1822 Jimmerson Lake Outlet STEUBEN N 5/24/2011 B 5/24/2011 491 1822 Location Name County Released First Obs. Abundance Latest Obs. 491m/yr 1822 m/yr Kendallville GMC NOBLE N 5/24/2011 C 5/24/2011 491 1822 Lake James Inlet STEUBEN N 5/24/2011 C 5/24/2011 491 1822 Orland Cemetary STEUBEN N 5/24/2011 C 5/24/2011 491 1822 Outlet Mall STEUBEN N 5/24/2011 E 5/24/2011 491 1822 Pokagon Medows Srague Additon STEUBEN N 5/24/2011 C 5/24/2011 491 1822 Warner Lake STEUBEN N 5/24/2011 C 5/24/2011 491 1822 Sparta Lake Boat Ramp NOBLE N 5/25/2011 E 5/25/2011 491 1822 West Otter Lake Boat Ramp STEUBEN N 5/26/2011 D 5/26/2011 491 1822 CR 17 Mitigation B+C ELKHART N 5/27/2011 B 5/27/2011 491 1822 Harold’s Landing at Blue Lake WHITLEY N 5/27/2011 C 5/27/2011 491 1822 Round Lake Boat Ramp WHITLEY N 5/27/2011 D 5/27/2011 491 1822 Adj. to Rt 8, 0.5mi W of County Line STARKE N 5/31/2011 B 5/31/2011 491 1822 Lawrence Lake Boat Ramp MARSHALL N 5/31/2011 C 5/31/2011 491 1822

46 Maxinkuckee Boat Ramp MARSHALL N 5/31/2011 C 5/31/2011 491 1822 Maxinkuckee Wetland Area Public Access MARSHALL N 5/31/2011 C 5/31/2011 491 1822 Yellow River, adj. to West 14th Rd MARSHALL N 5/31/2011 C 5/31/2011 491 1822 Youche Country Club LAKE N 6/1/2011 B 6/1/2011 491 1822 Chapman Lake Boat Ramp KOSCIUSKO N 6/1/2011 D 6/1/2011 491 1822 Dewart Lake Boat Ramp KOSCIUSKO N 6/1/2011 D 6/1/2011 491 1822 Goshen Dam Pond at Kercher Rd ELKHART N 6/1/2011 D 6/1/2011 491 1822 Kuhn Lake Boat Ramp KOSCIUSKO N 6/1/2011 D 6/1/2011 491 1822 Lake Wawasee Boat Ramp KOSCIUSKO N 6/1/2011 C 6/1/2011 491 1822 Syracuse Lake Boat Ramp KOSCIUSKO N 6/1/2011 E 6/1/2011 491 1822 Wabee Lake Boat Ramp KOSCIUSKO N 6/1/2011 C 6/1/2011 491 1822 Edgewater Park Boat Ramp ELKHART N 6/2/2011 E 6/2/2011 491 1822 Elkhart River at Jackson Blvd Bridge ELKHART N 6/2/2011 F 6/2/2011 491 1822 Leppers Park, South Bend ST JOSEPH N 6/2/2011 D 6/2/2011 491 1822 Veterans Memorial Park Boat Ramp ST JOSEPH N 6/2/2011 E 6/2/2011 491 1822 Woodlawn Park, South Bend ST JOSEPH N 6/2/2011 D 6/2/2011 491 1822 Location Name County Released First Obs. Abundance Latest Obs. 491m/yr 1822 m/yr Adj to St Rt 110, West 1/2 mile of St Rt 331 MARSHALL N 6/3/2011 E 6/3/2011 491 1822 Palestine Lake Boat Ramp FULTON N 6/3/2011 D 6/3/2011 491 1822 West of Palestine Lake, adj to Rt 25 FULTON N 6/3/2011 C 6/3/2011 491 1822 Kingsbury LAPORTE N 6/11/2011 D 6/11/2011 491 1822 Yellow Creek Lake ELKHART N 6/20/2011 D 6/20/2011 491 1822 Ball Wetlands KOSCIUSKO N 6/21/2011 F 6/21/2011 491 1822 Engle Lake NOBLE N 6/21/2011 C 6/21/2011 491 1822 Grassy Creek boat ramp KOSCIUSKO N 6/21/2011 C 6/21/2011 491 1822 Manitou FULTON N 6/22/2011 B 6/22/2011 491 1822 Manitou boat ramp FULTON N 6/22/2011 C 6/22/2011 491 1822 LaSalle Black Oak Bayou NEWTON N 6/28/2011 B 6/28/2011 491 1822 Crooked Creek STEUBEN N 7/6/2011 B 7/6/2011 491 1822 Crooked Creek STEUBEN N 7/6/2011 B 7/6/2011 491 1822

47 Delong FULTON N 7/7/2011 C 7/20/2011 491 1822 Price Lake Tri-County KOSCIUSKO N 7/7/2011 C 7/7/2011 491 1822 CR 800N and 600 W NOBLE N 7/27/2011 D 7/27/2011 491 1822 Galena Marsh LAPORTE N 8/4/2011 E 8/4/2011 491 1822 Ivanhoe South LAKE N 8/4/2011 D 8/4/2011 491 1822 Grand Prairie LAKE N 8/25/2011 C 8/25/2011 491 1822 Table 5: All Hylobius transversovittatus locations with selected attributes.

Location Name County Released First Obs. Abundance Latest Obs. Life Stage Seven Sisters STEUBEN Y 1/1/1998 A — Adult and Eggs Springfield Fen LAPORTE — 1/1/2001 A — Adult and Eggs Fish Creek Marsh LAPORTE Y 1/1/1997 A — Adult Manitou - beaver lodge FULTON Y 1/1/2003 F 1/1/2008 Adult Tolleston Ridges LAKE Y 1/1/2001 F 1/1/2006 Adult Theiling MARSHALL Y 1/1/2001 F 1/1/2006 Adult Calumet Prairie 1 LAKE Y 5/18/2010 — — Adult Calumet Prairie 2 LAKE Y 5/18/2010 — — Adult Springfield NP LAPORTE Y 10/7/2009 — — Adult Kakakee (Ten Mile Road) STARKE Y 6/24/2004 — — Adult and Eggs Missisinewa WABASH Y 6/24/2004 — — Adult 48 Calumet River C LAKE Y — F 5/19/2008 Adult Calumet River D LAKE Y — B 5/19/2008 Adult Ivanhoe South LAKE Y 1/1/2001 — — Adult Crooked Lake WHITLEY Y 10/7/2009 — — — Table 6: All Nanophyes marmoratus locations with selected attributes.

Location Name County Released First Obs. Abundance Latest Obs. Manitou - off island FULTON N 1/1/2007 C 1/1/2009 Manitou - dome FULTON Y 1/1/2005 C 1/1/2009 Big Otter Lk - Pokagon Sprague STEUBEN Y 1/1/2002 B 5/24/2011 Crooked Creek STEUBEN Y 1/1/2002 C 5/24/2011 Crooked Lake WHITLEY Y 8/3/2010 C 7/11/2011 Ball Wetlands KOSCIUSKO Y 1/1/2002 C 6/16/2010 Ivanhoe South LAKE Y 1/1/2002 A 8/4/2011 Manitou boat ramp FULTON N 6/22/2011 B 6/22/2011 Kankakee Y5 STARKE Y 7/7/2011 B 7/7/2011 Ligonier NOBLE Y 6/11/2011 B 6/11/2011 Union Church MARSHALL Y 6/10/2011 B 6/10/2011 49 Missisinewa Grant Creek WABASH Y 6/10/2011 B 6/10/2011 Manitou FULTON N 6/22/2011 B 6/22/2011 Warner Lake Boat Ramp STEUBEN N 5/24/2011 B 5/24/2011 Bachelor Rd 1/4 mi W 675 STEUBEN N 5/24/2011 C 5/24/2011 Adj 675 .5 mi N Bachlor R STEUBEN N 5/24/2011 B 5/24/2011 Crooked Creek, CR 600 W STEUBEN N 5/24/2011 A 5/24/2011 Jimmerson Lake Outlet STEUBEN N 5/24/2011 A 5/24/2011 Lake James Inlet STEUBEN N 5/24/2011 A 5/24/2011 Outlet Mall STEUBEN N 5/24/2011 A 5/24/2011 Big Otter Lk STEUBEN N 5/24/2011 A 5/24/2011 Appendix B: Indiana DNR Maps

Distribution of Galerucella spp. in Northern Indiana !( !( !( ^_ ^_!( ^_!(!(!(!(!(^_^_!(^_!(^_ !( !( !( !( ^_ ^_ !( ^_ !( ^_^_^_^_ ^_!( !(! !(!( ^_ South !(Bend !( ^_ !( ^_ ^_ !( ^_^_!( ^_^_! ^_^_ ^_!( ^_ ^_!( ^_ !( Gary ^_!( !(!( ^_ ^_ !( !( !( !( ^_ !( !( !( !(!( !( !( !(!( !( !(!(^_^_ !( !( !( !( !( !(!( !( ^_!(^_ !(^_!( !( !(^_!( ^_!( ^_ !(^_!(^_!( !(^_!( !( ^_!(!( !( !(!( !( ^_ !( !( ^_ !( !( ^_ ^_!( !( !(!( ^_ ^_ !( !( ! Fort Wayne ^_ ^_!(^_!( !( ^_!(^_ !(

!( !( ^_!( !(!(

^_ ¯ 1 inch equals 20 miles Miles 0 25 50 100

Legend Known Galerucella Sites ^_ Release Site !( Non-release Site

Coordinate System: NAD 1983 UTM Zone 16N Projection: Transverse Mercator Datum: North American 1983 Credits: Indiana DNR, Division of Nature Preserves; J. Britton; indianamap.org Date: 12/28/2011

Figure 12: Galerucella spp. map produced for the IDNR

50 Populations Sizes of Galerucella spp. in Northern Indiana

South Bend

Gary

Fort Wayne

1 inch equals 20 miles ¯ Miles 0 25 50 100 Legend Abundance of Galerucella at Sites !( B- Hard to Find (! C- Easy to Find (! D- Moderate Damage (! E- Patchy Severe Damage (! F- Widespread Severe Damage

Coordinate System: NAD 1983 UTM Zone 16N Projection: Transverse Mercator Datum: North American 1983 Credits: Indiana DNR, Division of Nature Preserves; J. Britton; indianamap.org Date: 12/28/2011

Figure 13: Galerucella spp. population size map produced for the IDNR

51 Estimated Distribution Area of Galerucella spp. in Northern Indiana

!( !( !(!(!(!(!( !(!(!( !(!( !( !( !( !(!( !( !(!( !( !( !( !( !( !(!( !( !( !(!( !( !( !( !( South Bend !( !( !( !(!( !( !(!(!(!( !( !(!( !( ! !( !( !( !( Gary !( !( !( !( !( !( !( !( !( !( !( !( !(!( !(!( !( !( !( !( !( !( !( !( !( !( !( !(!(!( !(!( !( !(!( !( !(!( !(!( !( !( !( !( !(!( !( !( !( !( !( !( !( !( !(!( !( !( ! Fort Wayne !( !(!(!( !(

!( !( !( !(!(

!( ¯ Legend !( Known Galerucella Sites Area within 5 miles of known site Area within 10 miles of known site

1 inch equals 20 miles Miles 0 25 50 100 Coordinate System: NAD 1983 UTM Zone 16N Projection: Transverse Mercator Datum: North American 1983 Credits: Indiana DNR, Division of Nature Preserves; J. Britton; indianamap.org Date: 12/28/2011

Figure 14: Galerucella spp. distribution estimate map produced for the IDNR

52 !( !(!( !( !( !( !(!(!(!(!(!(!(!(!( !( !( !( !(!( !(!( !( !( !( !( !( !( !( !(!(!(!(!(!( !( !( !( !( !( !( !( !(!( !(Ga!(ry !( !( !( !( !( !( !(!( !( !( !( !(!(!( !( !( !( !( !(!(!( !( !( !(!(!(!(!( !(!( !( !( !(!( !(!(!( !( !(!( !( !( Distribution of !(!( !( Fort Wayne !(!( !( !( Galerucella spp. !( !(!(!( !( in Indiana

Legend !( !( Known locations of Galerucella populations Indianapolis

53 Major Cities !( !(

!(

1 in = 40 miles !( Miles 0 12.5 25 50 75 100

Coordinate System: NAD 1983 UTM Zone 16N Projection: Transverse Mercator Evansville Datum: North American 1983 ¯ Credits: Indiana DNR, Division of Nature Preserves; J. Britton; indianamap.org Date: 12/28/2011

Figure 15: Galerucella spp. statewide map produced for the IDNR Distribution of Nanophyes marmoratus in Indiana ^_!(!( ^_ ! South Bend

! Gary ^_ ^_ ^_ ^_ ^_

! Fort Wayne ^_!(!(

^_

Legend ¯ Known Nanophyes Locations ^_ Release Location !( Non-Release Location

1 inch equals 20 miles Miles 0 25 50 100

Coordinate System: NAD 1983 UTM Zone 16N Projection: Transverse Mercator Datum: North American 1983 Credits: Indiana DNR, Division of Nature Preserves; J. Britton; indianamap.org Date: 12/28/2011

Figure 16: Nanophyes marmoratus map produced for the IDNR

54 Distribution of Hylobius transversovittatus in Indiana

! South Bend

!( !(! Gary

!(

! Fort Wayne !(

Legend ¯ !( Known Hylobius Locations

1 inch equals 20 miles Miles 0 25 50 100

Coordinate System: NAD 1983 UTM Zone 16N Projection: Transverse Mercator Datum: North American 1983 Credits: Indiana DNR, Division of Nature Preserves; J. Britton; indianamap.org Date: 12/28/2011

Figure 17: Hylobius transversovittatus map produced for the IDNR

55