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Home , Cyperaceae, Solidago ohioensis, Typha, Typha angustifolia

Final Report – angustifolia and T. x glauca suppression at Mukwonago River Floodplain (TNC Lulu Lake Preserve)

Walworth County, WI

∳Integrated Restorations, LLC Ecological Restoration & Land Management Services Integrating Ecological Theory and Research with Restoration Practices

Final Report – and T. x glauca suppression at Mukwonago River Floodplain (TNC Lulu Lake Preserve)

By Craig A. Annen Integrated Restorations, LLC June 2015 228 South Park Street Belleville, WI 53508 (608) 424-6997 (office) (608) 547-1713 (mobile) [email protected]

Deliverable final report for contract number 60112-2012028 between The Nature Conservancy, Inc. and Integrated Restorations, LLC

Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 2

Summary

1. A well-established population of Typha angustifolia was present in the Mukwonago River and was found to be spreading at an accelerated rate. This expansion was of immediate management concern and an effective suppression method needed to be developed and empirically tested to protect the biological and ecological integrity of the wetland basin. 2. Both Typha angustifolia and T. x glauca were present in the Mukwonago River floodplain wetland. 3. T. angustifolia covered 4.83 acres of the wetland basin in 2010 (prior to management intervention), equal to 10.9% of the wetland area. T. x glauca occurred sporadically as individual culms throughout the wetland basin. 4. Cumulative richness of the wetland basin (from 2012 through 2014) was S = 159, with a high proportion of conservative species. Cumulative Floristic Quality (FQI) was estimated at 67.2. 5. Three Special Concern species were detected during the three-year botanical survey: ohioensis ( goldenrod), Triglochin maritima (bog arrow-grass), and Thelypteris simulata (bog fern). The wetland also supports a small population of Platanthera huronensis (Huron pale green orchid). 6. The Mukwonago River wetland basin has high beta (habitat) diversity, with floristic elements of shrub-carr, southern sedge meadow, bog, fen, wet prairie, aquatic, and riparian emergent wetland types present. 7. The Mukwonago River wetland basin has exceptional natural area quality, and is a high quality natural resource in the Mukwonago River watershed. 8. A single directed cut surface application of imazapyr to T. angustifolia and T. x glauca reduced stem density > 90% for three consecutive growing seasons compared to untreated controls. 9. species density and diversity were similar between treated and non-treated areas, indicating that the suppression technique was methodically selective, even though a non- selective herbicide was used. 10. A variety of sensitive wildlife species were documented within the project area, indicating that the treatment protocol did not elicit any adverse effects to these organisms. 11. Imazapyr application to 1 in 4 cut Typha stems was effective in suppressing the majority of the stand via intra-clonal translocation, although limited follow up application will be required to suppress immature Typha that survived treatments because they were not connected to the rhizome network of the main clone at the time of treatment. 12. This selective approach was effective at reversing the T. angustifolia invasion in this wetland basin. 13. Although absent from control plots, increased in abundance in treated plots following a two-year lag time. Biocontrol should be concurrent with Typha suppression management in the of the Mukwonago Watershed.

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Site Description

The 40.3-acre project area is bisected by the Mukwonago River, which flows in a west-east direction and empties into Lulu Lake (map 1). Aquatic and emergent wetland vegetation coenoclines occupy the channel and banks of the Mukwonago River, and the wetland also supports diverse assemblages of sedge meadow, fen, bog, and wet prairie communities within the topographical depressions of the river’s floodplain (map 1). The wetland consists of 29.5 acres of remnant sedge meadow overlying a Marsh (Mf) soil series, with an additional 10.8 acres of the floodplain basin supporting a diverse wet prairie community atop Houghton muck (Ht) soil series (USDA NRCS 2012, Appendix 1). Both the Houghton muck and Marsh organic soils are derived from the decay of herbaceous organic plant material within the wetland and have similar profiles. Both soil types are very poorly drained, occur on slopes of 0 – 2%, have high water holding capacities, intersect the water table at the soil surface, and are prone to frequent ponding. The Houghton muck differs from the Marsh series in that the former is rarely flooded whereas the latter is frequently flooded. The wetland has a history of prescribed burning as a part of ongoing restoration and management within the Lulu Lake Nature Preserve. When this project was initiated, a population of Typha angustifolia had invaded the sedge meadow portion of this wetland and was rapidly expanding; this expansion was of immediate management concern and suppressive intervention was deemed necessary before this species could establish a monoculture to the exclusion of native wetland vegetation. Project Goals 1. Determine the extent of the Typha angustifolia infestation in the wetland basin. 2. Ascertain if the model derived by Boers and Zedler (2008) accurately predicted this species' rate of spread. 3. Determine if individuals of both narrow-leaved cattail (Typha angustifolia) and cattail (T. x glauca) were present in the wetland. 4. Develop a selective and effective Typha suppression technique for use in areas where Typha is commingled with desirable non-target native species. 5. Reduce the distribution and abundance of established Typha angustifolia and T. x glauca stands within the sedge meadow without causing excessive collateral damage to non-target sedge meadow species or disrupting the ecological integrity of the site. 6. Empirically determine if the suppression technique was effective at reducing Typha densities without causing excessive collateral damage to non-target sedge meadow species. 7. Conduct a detailed botanical inventory and Floristic Quality Assessment of the native vegetation communities present within the wetland. Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 4

Map 1: Vegetation communities of the Mukwonago River wetland basin.

Extent of Typha angustifolia invasion In a 2010 baseline survey, we mapped the extent of Typha angustifolia invasion in the Mukwonago River wetland. Where it was abundant enough to be discernible on aerial photos, T. angustifolia covered 4.35 acres of the wetland basin. The majority of T. angustifolia clones were located to the south of the Mukwonago River (map 2). T. angustifolia was intermixed with a diverse assortment of native sedge meadow species within the wetland basin. Boers and Zedler (2008) used a time-series of aerial photographs from 1963 – 2000 to construct a predictive model for T. angustifolia’s rate of spread within the watershed. Boers and Zedler (2008) reported that T. angustifolia dominated 5.3% of the Mukwonago River wetland in 2000, and based upon an analysis of its rate of spread during the 37-year time period they predicted a finite rate of population increase of r = 0.14 per year. Although these authors did not include a linear regression equation of their population growth model in the article, we were able to interpolate an expected value for the extent of T. angustifolia in 2010, the year of our baseline survey, using the linear regression figures they provided. The linear Boers and Zedler model predicted that T. angustifolia would dominate approximately 6.7% of the wetland by 2010. However,

Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 5 our baseline survey (using aerial photos produced in 2010) showed that T. angustifolia actually dominated 10.9% of the wetland, corresponding to a finite rate of increase of r = 0.51 per year, higher than the population growth rate predicted by the Boers and Zedler model. A higher than expected rate of expansion prompted us to consider the possibility that a linear model might not be the best long-term predictor for T. angustifolia population dynamics in the Mukwonago River wetland. State and transition models of community dynamics predict that species invasions increase linearly until they reach a critical mass, or threshold population density, beyond which invasion progresses at an accelerated, nonlinear rate due to internal system feedbacks (Kot 2001; Hobbs and Suding 2009) that can send communities on unpredictable trajectories of change. Accumulation of Typha litter and its secondary effects (such as mulching competing species) is likely one feedback that induces an accelerated expansion rate. Another probable feedback involves the effects of nitrogen and loading on biomass production and litter production. Moreover, and perhaps more importantly, the actual life expectancy and duration of reproductive fecundity (under field conditions) of clonal perennial monocots are not well known (Stüfer et al. 2002), and these variables are strong predictors of population dynamics. Although thresholds are difficult to pinpoint empirically and predictive trends involving nonlinear population dynamics are not yet completely understood, we felt there was sufficient evidence to conclude that this population had crossed a threshold and was expanding at an accelerated rate in the absence of management intervention. In summary, Typha angustifolia is capable of a geometric rate of spread once established, and T. angustifolia invasion pose a serious threat to remnant-condition wetlands such as the Mukwonago River floodplain basin. Both Typha angustifolia and T. x glauca were present in the sedge meadow Although Boers and Zedler (2008) documented only Typha angustifolia within the wetland in 2000, a small number of individuals of T. x glauca were observed during the July 2010 baseline survey, and again in July 2011 and August 2012 (Fig. 1). Both parent species (T. angustifolia and T. latifolia) were present and sympatric in the wetland basin, and with hybridization could have readily occurred in the decade between the Boers and Zedler survey and our baseline survey. At that time, the number of T. x glauca individuals was less than 1% of the total number of Typha stems.

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Map 2: Typha angustifolia distribution in 2010.

Figure 1: Typha x glauca (2012)

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Empirical Methods Development of Herbicide-Additive System and Application Method Choice of Herbicide for Typha suppression Reversing the Typha invasion within the Mukwonago River wetland posed a challenge from a tactical weed suppression standpoint. The target species (Typha angustifolia and T. x glauca) were comingled with desired non-target endpoint species, several of which were either classified as Wisconsin-Special Concern species or could be assigned high coefficient of conservatism values. Unfortunately, there are no commercially-available herbicide formulations that can be used to selectively target Typha without inflicting collateral damage to non-target species. However, we reasoned that a non-selective herbicide formulation could be a viable option for this abatement project provided that the herbicide was applied judiciously and with proper additives and carefully-directed application techniques. In other words, we needed to develop and empirically test a practical and cost-effective method of deploying a non- selective herbicide selectively. The choice of herbicide was between an aquatic formulation of glyphosate or the aquatic-approved ALS herbicide imazapyr. During the initial reconnaissance survey in 2010, we dug up Typha angustifolia phytomers and examined their . Morphologically, Typha rhizomes were similar to grass rhizomes, possessing an elongated apical bud, numerous non-elongated lateral buds at the rhizome nodes covered by cataphylls and appressed to the main rhizome axis, and a nearly complete lack of extensive rhizome branching (Fig. 2). We reasoned that, similar to grass rhizomes, Typha rhizomes were constrained by a system of rhizome apical dominance that could affect herbicide translocation patterns and contribute to post-application resurgence capacity when non- persistent herbicides (such as glyphosate formulations) were applied (Annen 2010). We postulated that glyphosate application would only provide one growing season worth of topkill, after which the Typha would re-sprout from dormant lateral rhizome buds and resurge to its pretreatment density. Figure 2: Typha angustifolia rhizome.

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Imazapyr (trade name Polaris®) is an ALS herbicide of the imidazolinone chemical family. ALS herbicides are noncompetitive inhibitors that attach to the regulatory binding site of acetolactate synthase, an allosteric enzyme that catalyzes biosynthesis of the branched-chain amino acids leucine, isoleucine, and valine (Subramanian et al. 1991). Imazapyr is a systemic, post-emergent, non-selective herbicide formulated as a water-soluble isoproplyamine salt. Imazapyr systemically translocates in both xylem and phloem and is persistent within plant tissues (weed suppression efficacy can continue for three months to two years after application), yet it has a low tendency to bioaccumulate in animal fatty tissue (Octanol-Water Partition Coefficient, KOW = 1.3) (WSSA 1994) and is approved for use in aquatic ecosystems, provided applicators possess certification in the Aquatic & Mosquito Application category (WDATCP commercial category 5.0). This herbicide’s persistence offers the potential for enhanced long-term control and suppression of regrowth compared to glyphosate formulations. Unlike glyphosate, which rapidly degrades once the dominant apical bud is killed, imazapyr remains chemically active after apical bud necrosis, and is able to affect resprouting lateral buds once they have been released from apical dominance and begin to receive assimilate and nutrients from the main rhizome axis. Thus, we postulated that this herbicide’s long-lasting phytotoxic action would diminish etiolated regrowth potential and resurgence capacity from the dormant rhizome bud bank, thereby enhancing Typha suppression. Herbicide Additives Imazapyr is formulated as an isoproplyamine (IPA) salt. Although this formulation makes the herbicide highly soluble in water, any calcium cations dissolved in hard mix water will rapidly bind with the dissociated weak acid conjugate of imazapyr and sequester the herbicide’s active moiety; calcium complexes are not loaded into plant phloem sieve tube elements and are not translocated within the plant body (Epstein 1973). Stochiometrically, each divalent calcium cation can sequester two imazapyr molecules. This problem was overcome by adding a water conditioning agent containing ammonium sulfate to the mix water. ReQuest® conditioning agent (Helena Chemical Company, Memphis, TN) was added to tank mixtures at a rate of 2% (v/v) (equivalent to 75 mL per gallon) prior to herbicide dilution. The sulfate anions in the conditioning agent are stronger ligands than the weak acid conjugate of imazapyr and are preferentially bound to calcium ions since they form insoluble calcium precipitates. This reaction is favored thermodynamically since it increases the entropy of the system, and ensures that the active herbicide moiety is chemically preserved:

- + 3- 2+ - + + H2O + SO4(NH4)3 + RCOO IPA -----> 2SO4 (aq) + 3Ca (aq) -----> Ca3(SO4)2 (s) + RCOO IPA (aq) + NH4 AMS + imazapyr salt calcium sulfate precipitate + active imazapyr

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In addition, 20N-20P-20K fertilizer (Miracle Grow®, Scott’s Company, Marysville, OH) was added to tank mixtures at a rate of 7.0 g/gallon. Fertilizers are widely used as herbicide tank-mix partners because they encourage active growth, which enhances herbicide uptake and translocation. A biodegradable sticking agent and pH buffering agent approved for use in aquatic ecosystems (Induce pH®, Helena Chemical Company, Memphis, TN) was added to imazapyr mixtures at a rate of 4% (v/v) (equivalent to 5 fluid ounces per gallon). This additive contains fatty acids that cause applied herbicide to physically adhere to treated surfaces, and also prevents herbicide drift and runoff from treated surfaces, stabilizes tank mixture pH, and curtails both evaporation and wash-off from rewetting of applied herbicide from treated surfaces. Spray pattern indicator (SPI) dye was also added to the herbicide mixture at a rate of one fluid ounce per gallon to prevent duplicate treatments of Typha cut surfaces and allow applicators to monitor overspray. Application Protocol A primary disadvantage of ALS herbicides is that they are almost too persistent; applications often create a kill zone lasting two or three growing seasons, even at low concentrations (in our experience, even rinsate from imazapyr can elicit phytotoxic effects). Foliar applications of imazapyr were deemed to be out of the question within this diverse natural wetland due to its persistence and non-selective mode of action. Aboveground stems of Typha angustifolia and T. x glauca were trimmed approximately 10 – 15 cm from the plant/soil or plant/water interface with a sharp bypass shear (Fig. 3). Imazapyr was applied to cut surfaces at a rate of 3.85% (v/v) (equivalent to 5 fluid ounces per gallon) with a small- capacity compression sprayer (APE Enterprises, Warsaw, Poland) at low output pressure. To prevent overspray, sprayer nozzles were fitted with a polyethylene cone-shaped drip/drift guard attachment (Fig. 4). Suppression treatments took place during the months of July and August in the 2011, 2012, 2013, and 2014 growing seasons, well after full leaf elongation and when Typha rhizome reserves were at a minimum due to drains for inflorescence development and flowering (and etiolated regrowth potential was minimal). To further curtail the possibility of inflicting collateral damage to non- target species and creating a large dead zone in the existing vegetation matrix, and to increase the cost- effectiveness of this suppression approach, we only cut and treated approximately one out of every four Typha stems. Once absorbed by the treated plant, imazapyr is rapidly translocated within the xylem and phloem throughout the entire plant body (WSSA 1994), and we reasoned that intra-clonal translocation might kill the remaining untreated tillers, provided they were connected to the main rhizome axis shared by multiple tillers within the Typha phytomers. This strategy posed a bit of a risk since the degree of clonal integration within perennial monocots is not well understood, despite several calls for more research in the pertinent literature (c.f. Stüfer et al. 2002). Different tillers connected to the same

Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 10 rhizome system might function independently after they reach maturity, or they may continue to support each other through material transfers and clonal subsidies. Figure 3: Imazapyr was applied to approximately one in four Typha cut stems.

Figure 4: Compression sprayer fitted with a polyethylene drip guard.

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By 2013 we had enough treatment data to conclude that the suppression approach and herbicide- additive mixture were both effective and selective, and we modified the cutting and treating procedures to enhance the efficiency and productivity of this technique. Typha stems were mowed to a stubble height of 10 – 15 cm with a STIHL 450 clearing saw (STIHL USA, Virginia Beach, VA) equipped with a circular cutting blade and herbicide was applied to cut surfaces with low-pressure gravity flow from a four-gallon backpack sprayer (Solo USA, Newport News, VA) modified with a polypropylene drift guard attachment (Fig. 5 – 8). Figures 5 & 6: Four-gallon backpack sprayer fitted with a polyethylene drip guard.

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Figures 7 & 8: Mowing was found to be more a productive treatment approach than hand-clipping.

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Quantitative Methods Botanical Inventory Botanical inventory of the marsh was conducted throughout the growing season from 2012 through 2014. The survey method consisted of a set of perpendicular passes through the wetland with each surveyor separated by ca. two meters. Botanical nomenclature follows the University of Wisconsin- Madison Herbarium (Wetter et al. 2001). Coefficient of conservatism (C) values (values assigned from the Wisconsin DNR, Bernthal 2003) were assigned to all species recorded in the botanical inventory. C values could not be assigned to non-vascular plants (e.g., bryophytes and mosses) since they were not listed in either Bernthal (2003) or in Swink and Wilhelm (1994). A Floristic Quality Index (FQI) was calculated as FQI = mean C√푆, where S = the native species richness of the site (Swink and Wilhelm 1994). Vegetation Sampling and Data Analysis The effects of imazapyr treatment on Typha angustifolia were measured in a stratified sampling design with two treatments and a single replication: (1) clipping followed by imazapyr application, and (2) no treatment (control). In 2014, we additionally measured the effects of (3) mowing followed by imazapyr application so that we could determine if the two cut surface treatments (clipping vs. mowing) elicited similar effects. Treatment responses were measured on 9 July 2012, 10 July 2013, and 14 July 2014. To assess the long-term suppression of Typha angustifolia resurgence capacity, we collected data from random samples from the same treatment and control areas during all sampling intervals. Plant stem density was measured in ten 0.125 m2 rectangular quadrats per treatment (quadrats were 0.5 m on the long axis and 0.25 m on the short axis). All species present within each quadrat were sampled. Quadrat shape and size were appropriate for this type of vegetation (Krebs 1989; Brummer et al. 1994), and rectangular quadrats were employed because they provide more detailed estimates of vegetation diversity than square quadrats (Krebs 1989). Estimates of Typha angustifolia stem density, species density, species diversity, and floristic quality (expressed as the mean coefficient of conservatism of species present) between treated and non-treated areas were used as indicators of treatment effectiveness. Species density (Magurran 1988) was determined within each quadrat subsample as the number of taxonomically distinct species per 0.125 m2. Plant species diversity was estimated with

th Shannon’s Entropy (H' = -Σ pi ln pi, where pi corresponds to the proportional abundance of the i species). Stem density was used as an indicator of vegetation abundance for diversity estimates.

Diversity estimates were transformed to the species density scale with a Hill Series formula (where N1 = eH') (Hill 1973). Hill Series transformation converts H' values from the esoteric bits of information per individual to the number of equally common species per unit area (Magurran 1988), which is more

Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 14 intuitive and ecologically meaningful. Since the experimental design only consisted of a single replication (Hurlbert 1984), statistical similarity between the treatment and control was estimated by measuring the degree of overlap in the 95% confidence interval for each response (Tacha et al. 1982; Ott and Longnecker 2001). When computing standard errors for the 95% CI estimates for Shannon’s Entropy, unbiased variance estimates for H' were calculated following methods outlined in Magurran (1988). Treatment effect sizes were calculated as [(control mean – treatment mean)/control mean]*100.

Results and Discussion Botanical Inventory and Floristic Quality of the Mukwonago River Floodplain Basin Botanical inventories detected the presence of S = 159 native plant species inhabiting the Mukwonago River floodplain marsh basin (Appendix A), including populations of three Wisconsin-Special Concern species Solidago ohioensis (Ohio goldenrod), Triglochin maritima (bog arrow-grass), and Thelypteris simulata (bog fern) (map 3). These 159 species belonged to 50 botanical families (Appendix A). 54% of the total taxonomic richness of the marsh belonged to only seven botanical families: (Compositae) comprised the majority of species (18% of total species richness), followed by (15%), (Graminae) (6%), (4%), (Labiatae) (4%), Apiaceae (Umbelliferae) (4%), and Salicaceae (3%). The mean coefficient of conservatism for all species present was 5.3 {range 1 – 10}, mode = 6, and the cumulative FQI of the site was 67.2. The marsh contained an unusually high proportion of conservative species: 29% of species had a coefficient of conservatism ≥ 7 and 13% had a coefficient ≥ 8. These results establish that the Mukwonago River floodplain marsh has exceptional high natural area potential (Swink and Wilhelm 1994) and that this wetland is a high quality natural resource (USFWS 2011). Suppression of Typha angustifolia and T. x glauca Treatment responses are summarized in Tables 1 through 3 and in Figures 9 - 15. In 2012, one year after initial treatment, Typha angustifolia stem density was 90% lower in the treated (clipped) areas of the marsh compared to the untreated areas (Table 1). Two individual Typha x glauca culms were cut and treated in 2011, and none were observed within the treated area in 2012, 2013, or 2014. In 2013, two years after initial treatment, Typha stem density remained suppressed in treated plots relative to the untreated control; stem density in treated (clipped) plots was 99% lower than in the control areas (Table 2). In 2014, Typha suppression was similar in both clipped and mowed plots, with treated plots possessing 96% and 97% less Typha stems than untreated control plots (Table 3). 2014 Typha suppression results suggest that mowing was as effective as clipping in terms of preparing Typha stems

Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 15 for herbicide application, and was a faster and more efficient approach to deploy in the field setting. This result also demonstrated that a backpack sprayer was an equally effective herbicide delivery method to use of a small capacity compression sprayer.

Map 3: Distribution of Wisconsin-Special Concern species Solidago ohioensis, Triglochin maritima, and Thelypteris simulata, and of Platanthera huronensis within the Mukwonago River floodplain wetland.

Herbicide application to approximately 1 in 4 cut Typha angustifolia stems was effective in suppressing the majority of the stand, presumably through intra-clonal rhizome translocation (although a molecular-tracer study would be required to confirm this suspicion). However, a limited number of surviving T. angustifolia plants were sampled in 2011 clipped plots in 2012, and also within 2013 mowed plots in 2014 (Tables 1 and 3). The majority of these T. angustifolia stems were flowering in the 2012 and 2014 sampling periods, indicating that they were mature plants that were cold-temperature vernalized the previous winter and thus not recruited from the seed bank following the spring 2012 burn. We hypothesized that these individuals were immature non-tillering individuals that were recruited from seed prior to when the 2011 treatments were carried out and were possibly not connected to the larger phytomer rhizome network laid down by the more well-established and fecund

Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 16 individuals within the population. We further hypothesized that the surviving plants were not clipped and treated, nor were they affected by imazapyr applications to other clipped Typha stems because they were not connected to the larger tiller network. We therefore conclude that a limited amount of follow- up suppression efforts will be necessary when this protocol for Typha suppression is used on established populations consisting of both mature and immature plants. Selectivity of Suppression Protocol In 2012, species density, H' diversity, and floristic quality were similar (Table 1), preliminary evidence that the treatment method was selective for Typha and minimized collateral damage to non-target endpoint species. In 2013, however, while species density and floristic quality remained similar in both treated and control plots, H' diversity was 26% higher in control plots (Table 2). There are three possible explanations for the measured decrease in Shannon’s entropy in treated plots in 2013: 1. The treatment protocol wasn’t entirely selective for T. angustifolia and some unintentional collateral damage occurred, 2. Random sampling error due to variability in species composition and abundance in this wetland basin during 2013 sampling, and/or 3. Suppression treatments that reduced Typha angustifolia and T x glauca stem density in turn lowered the output value of the Shannon's entropy estimate (which is a composite index of both species richness and abundance). To determine if the treatment protocol was indeed selective for Typha, we removed Typha stem density from the data set for the control plots and then re- calculated the degree of overlap in the 95% confidence interval. We reasoned that this analysis would indicate if the additional abundance scores provided by the presence of Typha had an impact on the value of Shannon’s entropy (H'). In the absence of the Typha data subset, control plots showed 81% overlap in the 95% confidence interval for species density, indicating that the differences measured in species density were largely an artifact of how the diversity estimate is calculated rather than the result of collateral damage to non-target species. In 2014, mowed plots had higher species density and H' diversity than clipped plots (Table 3). This result could be explained in terms of the intermediate disturbance hypothesis, which states that diversity is maximized along a disturbance gradient where disturbance is neither too rare nor too frequent. Mowing cattail culms to expose cut surfaces for herbicide treatment acted as a disturbance that opened up the lower canopy to light and other resources, which led to higher species density and diversity in mowed plots. Both suppression treatments (clipping and mowing) had higher species density and diversity than untreated control plots (Table 3), showing that the presence of Typha angustifolia exhibited a negative effect on native species density and diversity. Floristic quality was similar among all treatments in 2013 and 2014 (Tables 2 and 3).

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The overall patterns we measured among species density, diversity, and floristic quality between treated and non-treated areas indicated that the application method developed in this study was selective, even though a non-selective herbicide formulation was employed for Typha suppression (Fig. 9). Additionally, both flowering and non-flowering Platanthera huronensis (Huron pale green orchid) were detected within the 2011 clipped and 2013 mowed treatment areas during our 2012 and 2014 sampling surveys, a further indication that the suppression method was predominantly selective since P. huronensis is a disturbance-sensitive species that we would expect to be more negatively affected by off-target herbicide applications than more common matrix species within the mixed vegetation stand (Fig. 10). Similarly, we also observed several disturbance-sensitive wildlife species within treatment areas during the 2013 and 2014 field seasons, often within only a couple of hours or days after treatments were administered (Figs. 11 – 13). Another noteworthy observation occurred in 2014, when we observed a pair of Sandhill cranes and their colt foraging within the mowed treatment area immediately following cattail removal treatments. While working in the marsh, we noted that these cranes tended to avoid cattail-dominated areas and seemed to prefer open sedge meadow and wet prairie habitat. Previous studies on reed canarygrass (Phalaris arundinacea L.) demonstrated that reversing invasions not only promoted greater species diversity but also altered vegetation-height structure, with implications for habitat quality (Annen 2011). The effect of Typha angustifolia invasions on habitat quality and vegetation-height structure for wildlife merits additional study. Competitive Release of Lythrum salicaria Following Typha angustifolia Suppression Treatments A weed shift occurs when one gets replaced by another after treatments are administered. In the final year of this study we observed that L. salicaria (purple loosestrife) had appeared in both clipped and mowed Typha-suppression plots, yet was absent in control plots (Table 3). A two-year lag time was required before this effect was detected by quantitative quadrat sampling, but the implications are clear. We postulate that L. salicaria is a weaker competitor than T. angustifolia, and is normally absent or rare when Typha dominates, then subsequently increases in abundance once released from competition when Typha invasions are reversed. We therefore recommend that L. salicaria biocontrol agents be released concurrently with Typha management. Summary The Mukwonago River floodplain wetland is an exceptionally diverse wetland resource that contains elements of wet prairie, southern sedge meadow, bog, fen, shrub-carr, aquatic, and riparian emergent wetland coenoclines. Reversing the Typha angustifolia invasion will ensure that the biological and ecological integrity of this valuable reference site is preserved for future generations. Results from this experiment demonstrate that the Typha suppression protocols developed in this study were effective at

Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 18 suppressing Typha angustifolia and largely selective for the target species when carried out within a diverse mixed vegetation stand. The protocols also appeared to have no adverse effects on several disturbance-sensitive wildlife species. This suppression strategy will be expanded to other Typha angustifolia invasions within the TNC Nature Preserves of the Mukwonago Watershed during the 2015 – 2018 growing seasons. Figure 9: The suppression method was selective, even though a non-selective herbicide formulation was used. Non-target species and dead Typha stems within the treatment area (summer 2012 (l) and summer 2014 (r)).

Figure 10: Flowering Platanthera huronensis within cattail-treated area (2014).

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Figures 11 – 13: Examples of sensitive wildlife species that were observed within cattail-treated area, often within hours after treatments were administered.

Table 1: Summary of treatment effects (2012 growing season). Response Mean (SE) 95% Confidence Interval Population Separation T. angustifolia stem density Treated (Clipped) 0.6 (1.6) (0.0 – 3.5) CONTROL > CLIPPED Untreated 5.8 (2.0) (2.1 – 9.5) Species Density Treated (Clipped) 5.6 (1.8) (3.5 – 8.4) CLIPPED = CONTROL Untreated 6.0 (1.3) (2.3 – 8.9) Pooled Mean 5.8 (1.5) H' Diversity [eH'] Treated (Clipped) 1.291 [3.6] (0.111) (1.233 – 1.487) CLIPPED = CONTROL Untreated 1.360 [3.9] (0.127) (1.180 – 1.402) Pooled Mean 1.326 [3.8] (0.119) Floristic Quality (mean c) Treated (Clipped) 5.2 (0.8) (3.7 – 6.6) CLIPPED = CONTROL Untreated 4.4 (0.6) (3.2 – 5.5) Pooled Mean 4.8 (0.8)

Table 2: Summary of treatment effects (2013 growing season).

Response Mean (SE) 95% Confidence Interval Population Separation T. angustifolia stem density Treated (Clipped) 0.1 (0.3) (0.0 – 0.7) CONTROL > CLIPPED Untreated 7.1 (3.6) (0.3 – 14.0) Species Density Treated (Clipped) 7.5 (1.8) (4.1 – 10.9) CLIPPED = CONTROL Untreated 9.2 (2.3) (5.1 – 13.3) Pooled Mean 8.4 (4.8) H' Diversity [eH'] Treated (Clipped) 1.290 [3.6] (0.075) (1.215 – 1.365) CONTROL > CLIPPED Untreated 1.733 [5.7] (0.092) (1.641 – 1.825) Floristic Quality (mean c) Treated (Clipped) 4.8 (0.5) (3.8 – 5.7) CLIPPED = CONTROL Untreated 4.8 (0.7) (3.5 – 6.1) Pooled Mean 4.8 (0.6)

Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 20

Table 3: Summary of treatment effects (2014 growing season). Response Mean (SE) 95% Confidence Interval Population Separation T. angustifolia stem density Treated (Clipped) 0.5 (1.3) (0.0 – 1.8) Treated (Mowed) 0.4 (0.5) (0.0 – 0.9) CLIPPED = MOWED > CONTROL Untreated 12.6 (3.6) (0.3 – 24.9) L. salicaria stem density Treated (Clipped) 2.4 (3.5) (0.0 – 5.9) Treated (Mowed) 3.1 (3.6) (0.0 – 9.6) CLIPPED = MOWED > CONTROL Untreated 0.0 (0.0) (0.0 – 0.0) Species Density Treated (Clipped) 7.1 (1.2) (5.9 – 8.3) Treated (Mowed) 11.5 (2.0) (7.9 – 15.1) MOWED > CLIPPED > CONTROL Untreated 5.4 (2.0) (1.8 – 9.0) H' Diversity [eH'] Treated (Clipped) 1.381 [4.0] (0.082) (1.299 – 1.463) Treated (Mowed) 1.883 [6.6] (0.093) (1.791 – 1.976) MOWED > CLIPPED > CONTROL Untreated 0.816 [2.3] (0.094) (0.722 – 0.910) Floristic Quality (mean c) Treated (Clipped) 5.5 (0.7) (4.8 – 6.2) Treated (Mowed) 5.9 (0.7) (4.6 – 7.1) CLIPPED = MOWED = CONTROL Untreated 5.4 (0.7) (4.2 – 6.6) Pooled Mean 5.6 (0.7)

Figures 14 & 15: Visual comparison of treated areas (foreground) with untreated areas (background). Note the differences in vegetation height-structure between the treated and untreated vegetation stands.

Final Report – Typha angustifolia suppression at Lulu Lake Preserve Page 21

Vegetation Inventory of Mukwonago River Wetland Complex (2012-2015)

Observation dates: 7/9; 8/19; 9/3/2012 7/10; 7/24/2013 Observers: C. Annen, A. Budyak, S. 7/14; 7/22/2014 7/10/2015 Longabaugh, D. Cordray, C. Kregel, Victoria Lubner, S. Mayer, LEAF interns, TNC interns Botanical Name Common Name C S Botanical Family Acorus americanus Sweet Flag 7 1 Acoraceae Alisma subcordatum Water Plantain 3 2 Alismataceae Alnus incana subsp. rugosa Speckled Alder 4 3 Betulaceae Andropogon gerardii Big Bluestem 4 4 Poaceae (Graminae) Angelica atropurpurea Purple-Headed Angelica 6 5 Apiaceae Asclepias incarnata Swamp Milkweed 5 6 Asclepiadaceae Aster boreale Bog Aster 10 7 Asteraceae (Compositae) Aster firmus Shiny-Leaved Aster 6 8 Asteraceae (Compositae) Aster novae-angliae New England Aster 3 9 Asteraceae (Compositae) Aster lanceolatus var. simplex White Panicle Aster 4 10 Asteraceae (Compositae) Aster pilosus Frost Aster 1 11 Asteraceae (Compositae) Aster puniceus Purple-Stem Aster 6 12 Asteraceae (Compositae) Aster umbellatus Flat-Topped Aster 6 13 Asteraceae (Compositae) Bidens sp. Tickweed NA 14 Asteraceae (Compositae) Brasenia schreberi Water Shield 6 15 Cabomaceae Bromus ciliatus Fringed Brome 7 16 Poaceae (Graminae) Calamagrostis canadensis Blue-Joint Grass 5 17 Poaceae (Graminae) Caltha palustris Marsh Marigold 6 18 Ranunculaceae Campanula aparinoides Marsh Bellflower 7 19 Campanulaceae aquatilis Aquatic Sedge 7 20 Cyperaceae Carex conoidea Prairie Star Sedge 8 21 Cyperaceae Small Yellow Sedge 8 22 Cyperaceae Porcupine Sedge 3 23 Cyperaceae Inland Star Sedge 7 24 Cyperaceae Lake Sedge 6 25 Cyperaceae Hop Sedge 6 26 Cyperaceae Bog Sedge 10 27 Cyperaceae Broad-Leaved Wholly Sedge 4 28 Cyperaceae var. stipata Owlfruit Fox Sedge 2 29 Cyperaceae Tussock Sedge 7 30 Cyperaceae Hairy-Fruited Sedge 7 31 Cyperaceae Tuckerman's Bladder Sedge 8 32 Cyperaceae Yellow Lake Sedge 7 33 Cyperaceae Brown Fox Sedge 2 34 Cyperaceae Cerastium nutans Nodding Chickweed 4 35 Caryophyllaceae Ceratophyllum demersum Coon Tail 3 36 Ceratophyllaceae Chara vulgaris Musk Grass NA 37 Characeae Cicuta bulbifera Bulblet Water Hemlock 7 38 Apiaceae Cicuta maculata Spotted Water Hemlock 6 39 Apiaceae Cirsium muticum Swamp Thistle 8 40 Asteraceae (Compositae) Cornus amomun Silky Dogowood 4 41 Cornaceae Cornus racemosa Grey Dogwood 2 42 Cornaceae Cornus stolonifera Red Osier Dogwood 3 43 Cornaceae Cuscuta gronovii var. gronovii Swamp Dodder 4 44 Cuscutaceae strigosus Yellow Nut Sedge 1 45 Cyperaceae Danthonia spicata Fool's 4 46 Poaceae (Graminae) Dryopteris cristata Marsh Shield Fern 7 47 Dryopteridaceae Elodea canadensis Elodea 3 48 Hydrocharitaceae acicularis Needle Spike Rush 5 49 Cyperaceae Eleocharis erythropoda Bald Spike Rush 3 50 Cyperaceae Spike Rush 3 51 Cyperaceae Common Marsh Spike Rush 6 52 Cyperaceae Epilobium leptophyllum Marsh Willow-Herb 3 53 Onagraceae Eupatorium maculatum Spotted Joe-Pye Weed 4 54 Asteraceae (Compositae) Eupatorium perfoliatum Boneset 6 55 Asteraceae (Compositae) Euthamia graminifolia Grass-Leaved Goldenrod 4 56 Asteraceae (Compositae) Galium circaezans Licorice Bedstraw 7 57 Rubiaceae Galium labradoricum Marsh Bedstraw 10 58 Rubiaceae Galium tinctorium Southern Three-Lobed Bedstraw 5 59 Rubiaceae Glyceria grandis Reed Manna Grass 6 60 Poaceae (Graminae) Glyceria striata Fowl Manna Grass 4 61 Poaceae (Graminae) Helenium autumnale Sneezeweed 4 62 Asteraceae (Compositae) Helianthus giganteus Swamp Sunflower 4 63 Asteraceae (Compositae) Helianthus grosseserratus Sawtooth Sunflower 2 64 Asteraceae (Compositae) Heteranthera [Zosterella] dubia Water Star Grass 6 65 Pontederiaceae Impatiens capensis Orange Jewelweed 2 66 Balsaminaceae Impatiens pallida Pale Jewelweed 6 67 Balsaminaceae Iris versicolor Northern Blue Flag Iris 5 68 Iridaceae dudleyi Dudley's Rush 4 69 Juncus nodosus Jointed Rush 6 70 Juncaceae Juncus tenuis Path Rush 1 71 Juncaceae Krigia biflora Dwarf Two-Flowered Cynthia 4 72 Asteraceae (Compositae) Lathyrus palustris Marsh Pea 7 73 Fabaceae (Leguminosae) Leersia oryzoides Cut Grass 3 74 Poaceae (Graminae) Lemna minor Duckweed 4 75 Araceae Lemna trisulca Star Duckweed 6 76 Araceae Liatris linguistilis Showy Blazing-Star 7 77 Asteraceae (Compositae) Liatris pycnostachya Prairie Blazing-Star 7 78 Asteraceae (Compositae) Lilium michiganense Turk's Cap Lily 6 79 Liliaceae Lobelia siphilitica Great Blue Lobelia 5 80 Lobeliaceae Lonicera dioica var. dioica Red Vine Honeysuckle 7 81 Caprifoliaceae Lycopus americanus Water Horehound 4 82 Lamiaceae (Labiatae) Lycopus uniflorus Bugleweed 4 83 Lamiaceae (Labiatae) Lycopus virginicus Virginia Water Horehound 8 84 Lamiaceae (Labiatae) Botanical Name Common Name C S Botanical Family Lysimachia lanceolata Lance-Leaved Loosestrife 6 85 Primulaceae Lysimachia quadriflora Narrow-Leaved Smooth Loosestrife 9 86 Primulaceae Lysimachia thyrsifolia Swamp Loosestrife 7 87 Primulaceae Lythrum alatum var. alatum* Prairie Loosestrife 6 88 Lythraceae Maianthemum stellatum Starry False Soloman's Seal 5 89 Liliaceae Marchantia polymorpha Black-Lined Liverwort NA 90 Marchantiaceae Mimulus ringens Monkeyflower 6 91 Scrophulariaceae Nuphar variegata Yellow Pond Lily 6 92 Nymphaceae Nymphaea odorata White Water Lily 6 93 Nymphaceae Onoclea sensiblis Sensitive Fern 5 94 Dryopteridaceae Oxypolis rigidor Stiff Cowbane 6 95 Apiaceae Parietaria pensylvanica Pellitory 2 96 Urticaceae Pedicularis lanceolata Swamp Betony 8 97 Scrophulariaceae Poa palustris Marsh Bluegrass 5 98 Poaceae (Graminae) Polygonatum biflorum Soloman's Seal 4 99 Liliaceae Polygonum amphibium Water Smartweed 5 100 Polygonaceae Polygonum hydropiperoides Water Pepper 6 101 Polygonaceae Polygonum pensylvanicum Pennsylvania Knotweed 1 102 Polygonaceae Polygonum punctatum Dotted Smartweed 5 103 Polygonaceae Polygonum scandens Climbing Smartweed 3 104 Polygonaceae Polygonum sp. Smartweed NA 105 Polygonaceae Populus tremuloides Aspen 2 106 Salicaceae Potamogeton natans Floating-Leaved Pondweed 5 107 Potamogetonaceae Potamogeton pectinatus [Stuckenia pectinata] Sago Pondweed 3 108 Potamogetonaceae Pentaphylloides floribunda [Potentilla fruticosa] Shrubby Cinquefoil 9 109 Rosaceae Platanthera huronensis Huron Green Orchid 7 110 Orchidaceae Prunus virginiana var. virginiana Chokecherry 3 111 Rosaceae Pycnanthemum virginianum Virginia Mountain Mint 6 112 Lamiaceae (Labiatae) Ranunculus sceleratus Cursed Crowfoot 3 113 Ranunculaceae Ribes cynosbati Dogberry 3 114 Rosaceae Rudbeckia hirta Black-Eyed Susan 4 115 Asteraceae (Compositae) Rudbeckia laciniata Cut-Leaved Coneflower 6 116 Asteraceae (Compositae) Rudbeckia subtomentosa Sweet Black-Eyed Susan 7 117 Asteraceae (Compositae) Rumex orbiculatus Great Water Dock 8 118 Polygonaceae Salix bebbiana Bebb's Willow 7 119 Salicaceae Salix candida Sage Willow 10 120 Salicaceae Salix humilis Prairie Willow 6 121 Salicaceae Salix nigra Black Willow 4 122 Salicaceae Sagittaria latifolia Arrowhead 3 123 Alismataceae Sagittaria rigida Stiff Arrowhead 8 124 Alismataceae atrovirens Green Bulrush 3 125 Cyperaceae Rufous Bulrush 4 126 Cyperaceae acutus Hard-Stem Bulrush 6 127 Cyperaceae Three-Square Rush 4 128 Cyperaceae Schoenoplectus tabernaemontani [Scirpus validus] Soft-Stem Bulrush 4 129 Cyperaceae Scutellaria galericulata Marsh Skullcap 5 130 Lamiaceae (Labiatae) Silphium integrifolium var. deamii Rosinweed 6 131 Asteraceae (Compositae) Silphium perfoliatum Cup Plant 4 132 Asteraceae (Compositae) Silphium terebinthinaceum Prairie Dock 7 133 Asteraceae (Compositae) Sium suave Water Parsnip 5 134 Apiaceae Solidago gigantea Tall Goldenrod 3 135 Asteraceae (Compositae) Solidago ohioensis Ohio Goldenrod (Special Concern) 9 136 Asteraceae (Compositae) Solidago patula Rough-Leaved Swamp Goldenrod 8 137 Asteraceae (Compositae) Solidago riddellii Riddell's Goldenrod 7 138 Asteraceae (Compositae) emersum Narrow-Leaved Burr-Reed 8 139 Sparganium eurycarpum Burr-Reed 5 140 Sparganiaceae Sparganium natans Small Bur-Reed 9 141 Sparganiaceae Spartina pectinata Prairie Cord Grass 5 142 Poaceae (Graminae) Sphagnum sp. Sphagnum Moss NA 143 Sphagnaceae Spiraea alba Meadowsweet 4 144 Rosaceae Spirodela polyrrhiza Big Duckweed 5 145 Araceae Stachys palustris Marsh Hedge-Nettle 5 146 Lamiaceae (Labiatae) Stuckenia [Potamogeton] pectinata Sago Pondweed 3 147 Potamogetonaceae Symplocarpus foetidus Skunk Cabbage 8 148 Araceae Teucrium canadense American Germander 4 149 Lamiaceae (Labiatae) Thalicrum dioicum Meadow Rue 7 150 Ranunculaceae Thelypteris palustris var. pubescens Eastern Marsh Fern 7 151 Thelypteridaceae Thelypteris simulata Bog Fern (Special Concern) 10 152 Thelypteridaceae Triglochin maritima Bog Arrow-Grass (Special Concern) 10 153 Juncaginaceae Cattail 1 154 Utricularia vulgaris Common Bladderwort 7 155 Lentibulariaceae Vallisneria americana Wild Celery 6 156 Hydrocharitaceae Verbena hastata Blue Marsh Vervain 3 157 Verbenaceae Veronia fasciculata Ironweed 5 158 Asteraceae (Compositae) Viola sororia Common Wood Violet 3 159 Violaceae Zizia aurea Golden Alexander 7 160 Apiaceae Alga sp. Filamentous algae NA 161 Chlorophyta

50 Botanical Families Present Proportion of species with (C ≥ 8) = 13%; S = 21 Species Richness 161 Most Abundant Families S % of total S Proportion of species with (C ≥ 7) = 29%; S = 46 Mean C value 5.3 Asteraceae (Compositae) 29 18% Number of at-risk plant species = 3 FQA 67.5 Cyperaceae 25 16% Modal C value 6 Poaceae (Graminae) 9 6% Polygonaceae 7 4% Lamiaceae (Labiatae) 7 4% Apiaceae 6 4% Salicaceae 5 3% 88 55% References

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