SOIL AND LITTER LEGACY EFFECTS OF INVASIVE FLOWERING RUSH (BUTOMUS UMBELLATUS) ON LAKE ERIE WETLAND RESTORATION

Alyssa Dietz

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2015

Committee:

Helen Michaels, Advisor

Enrique Gomezdelcampo

C. Eric Hellquist

Jeffrey Miner ii ABSTRACT

Helen Michaels, Advisor

The Great Lakes region has been impacted by the invasion of over 180 different alien

species. As invasives have reduced wetland habitat availability and altered community structure,

managers have developed extensive eradication programs. However, even following the removal

of dense monocultures, invasive plants can influence native ecosystems through long-term

chemical and biological changes, known as legacy effects. My research investigates the potential

for these legacy effects following the removal of Flowering Rush (Butomus umbellatus), an

understudied emergent in Lake Erie wetlands. This research focuses on how legacy effects of B.

umbellatus may influence restoration of native communities and investigates whether the

presence of remnant stands of B. umbellatus propagules or litter alters the success of native

reestablishment.

A seed mix of 25 native species was sown into flats with soil from either native

dominated soils, areas with formerly moderate invasions, or areas with persistent Butomus

monocultures. These sown plant communities were then subjected to single and combined

treatments of living B. umbellatus vegetative propagules and litter alongside unsown flats that examined the response of seedbank communities.

The presence of propagules greatly reduced the growth of native seedlings developing in experimental plantings by 69%. While there was no difference in biomass between native seedlings grown in univaded soils and those from areas of B. umbellatus monocultures, native

diversity and community evenness were lower. Contrary to initial predictions, B. umbellatus litter increased native biomass and taxon diversity. Planted propagules also reduced applied iii invasive litter decomposition. Nutrient analysis of soils from sites of monocultures had elevated levels of phosphorus and nitrogen release.

This work documents specific negative impacts of this understudied invasive emergent plant on Great Lakes wetland communities. My results demonstrate the importance of controlling vegetative propagules and emphasizes their potential role in inhibiting restoration efforts that are costly and time consuming endeavors for management partners. This work also suggests that there may be possible changes to microbial communities and related ecosystem nutrient cycles once monocultures have developed. My research suggests the presence of legacy effects of B. umbellatus that alter community composition and soil conditions that warrant further investigation. iv ACKNOWLEDGMENTS

First and foremost I would like to thank my advisor Dr. Helen Michaels for her help and guidance on my thesis. I would also like to thank my committee members Dr. Jeffrey

Miner, Dr. Enrique Gomezdelcampo, and Dr. C. Eric Hellquist for their help and willingness to answer endless questions throughout my project. I also thank Ron and Kathy Huffman of US

Fish and Wildlife who provided key background information about ONWR and whose discussions played a central part in the creation of my thesis.

Many thanks go to my labmates Paige Arnold and Jacob Sublett for not only their help in hours upon hours of harvesting but also their friendship and support every day in the office. I’m also thankful for the help that I received from the graduated members of the lab Mike Plenzler,

Jennifer Shimola, and Ryan Walsh in their guidance and advice in the development of my thesis.

My research could have never been accomplished without the help of multiple people in the field. I am extremely grateful for the help I received from the Miner lab (Jake Miller, Dani

McNeil, Jamie Russell, Kevin Bland, Jordan Pharris) for their help in collecting soils and the setup of my mesocosms. I’m also thankful for the help of Rachel Schirra, without whom the native species mix may have never been finished. I am eternally grateful for the help of Dylan

Jacobs, who not only labored away at the collection of my field soils and harvest but who also served as a great friend and supporter throughout my masters. Last, and certainly not least I would also like to thank my family and friends for their kind words and support throughout not only my research but my whole college career. I want to thank my parents for encouraging me to take on projects that challenged me and for helping me in my times of need including hours spent looking at the radar. To my friends Ashlee Thomas, Mike Fashinpaur, Addie Bagford,

Jessica LaHurd, and Meredith Barnes, thank you for being the much needed escape from the lab and for always getting me through even the toughest times. v

TABLE OF CONTENTS

Page

INTRODUCTION...... 1

MATERIALS AND METHODS ...... 5

Site Description ...... 5

Experimental Design ...... 5

Environmental Factors ...... 9

Soil, Leaf Tissue, and Litter Collection ...... 10

Statistical Analyses ...... 11

Environmental Variables ...... 11

Native Seedling Response Measures ...... 12

B. umbellatus Growth and Litter Decomposition ...... 12

RESULTS ...... 14

Environmental Conditions ...... 14

Seed Bank Community ...... 15

Native Monocultures ...... 16

Taxon Richness of Seeded Treatments ...... 16

Total Biomass ...... 17

Community Attributes: Diversity, Evenness, and Similarity ...... 18

B. umbellatus Responses ...... 19

Litter Decomposition and Tissue Quality ...... 19

DISCUSSION ...... 21

Implications for Managers and Further Areas of Research ...... 26 vi

REFERENCES ...... 28

APPENDIX A. STATISTICAL TABLES ...... 49 vii

LIST OF TABLES

Table Page

1 Applied Seed Mix Composition ...... 33

2 Average Recorded Nutrients of Soils ...... 34

3 Common Emerged Species and Responses to Applied B. umbellatus Treatments ... 35

4 Ungerminated Applied Seed Species ...... 36 viii

LIST OF FIGURES

Figure Page

1 National Distribution of B. umbellatus ...... 37

2 Map of Ottawa National Wildlife Refuge ...... 38

3 Butomus umbellatus nodules ...... 39

4 Vegetation of Common Seedbank Taxa ...... 40

5 Effect of Soil Invasion History on Native Response Measures ...... 42

6 Effect of Applied Nodules and Litter on Native Response Measures ...... 44

7 B. umbellatus Litter and Nodule Responses to Soil Invasion History ...... 45

8 Effect of Nodules on the Decomposition of Litter...... 46

9 Effect of Soil and Nodule Interactions on the Decomposition of Litter ...... 47

10 B. umbellatus Tissue Nutrients ...... 48 1

INTRODUCTION

Although wetlands provide nearly 40% of renewable ecosystem services (Costanza et al.

1997, Zedler 2000), the United States is annually losing 60,000 acres (24,281 hectares) of these important ecosystems (EPA 2015). Given that less than 5% of the original wetlands of some

Great Lakes states still remain (Zedler 2003), understanding major causes of destruction and hindrances to their successful restoration is critical. While major losses of wetland habitat are

from agricultural development and urban spread, invasive species are also viewed as a substantive cause of wetland destruction (Drexler and Bedford 2002, Zedler 2006, Frieswyk et al. 2007, Zedler et al. 2012). Over $100 million is spent annually repairing the damage caused by invasive species (Rosaen et al. 2012) in the Great Lakes region. Most regional attention and funding has been placed on the management of many exotic fish and mussel species. However, invasive wetland plants such as common reed (Phragmites australis), narrow leaved cattail and

its hybrid (Typha angustifolia and Typha x glauca respectively), as well as reed canarygrass

(Phalaris arundinacea) (Holdredge and Bertness 2011, Larkin et al. 2012, Kaproth et al. 2013)

have also had major impacts on wetland ecosystems. These well studied invasives alter

community structure, change dissolved oxygen (DO) levels, increase hypoxia and allelopathic

chemical concentrations, and create habitat changes that can decrease native seedling success and reduce native biomass (Grman and Suding 2010, Vilà et al. 2011, Shultz and Dibble 2012).

Such changes to natural wetland ecosystems not only allow invaders to outcompete natives, but lead to positive feedback loops that further benefit past and even new invaders (Parkinson et al.

2010, Kaproth et al. 2013).

Given the impact of these common wetland invasive plants, managers have initiated many wetland restoration projects in the Great Lakes region, including $4.6 million spent on the 2 management of Phragmites annually (Martin and Blossey 2013). Because most invasive species are thought to outcompete natives through better adaptations for acquiring resources (Farrer and

Goldberg 2009, Meisner et al. 2012), restoration plans often focus on the removal or eradication of large established monotypic stands of invasives. However, following removal of invasives, restoration efforts have often failed to achieve desired community composition due to more indirect effects, known as legacy effects. These legacy effects, which are long lasting ecosystem changes, can alter native soil chemistry due to remaining decaying plant matter (Grman and

Suding 2010, Holdrege and Bertness 2011, Larkin et al. 2012). For example, leaf litter remaining in Typha hybrid infested wetlands has been found to increase soil ammonium and nitrogen (N) mineralization, as well as decrease native diversity more so than dense stands of living Typha plants (Farrer and Goldberg 2009). In addition, a study conducted by Elgersma et al. (2011) found that soil microbial communities are determined by previous invasive species rather than current natives, and restoration of original microbial communities occurs slowly over time.

Observations and studies such as these suggest that the impacts of invasives can extend well beyond removal of invasive stands, greatly altering the time frame and success of restoration projects.

Although the impacts and legacy effects of major wetland invasive plants on Great Lakes wetlands have been well documented, relatively little is known about flowering rush (Butomus umbellatus, Butomaceae). Originally introduced from Europe through the St. Lawrence Seaway in the early 1900s, B. umbellatus is an emergent species known for its characteristic tall triangular leaves and pink flowers (Brown and Eckert 2005, Parkinson et al. 2010). Even though

B. umbellatus has been found in Great Lakes regional wetlands for over a century (Brown and

Eckert 2005, Kliber and Eckert 2005, Parkinson et al. 2010), there are still substantial knowledge 3

gaps regarding its impacts on native wetland communities. Some of the few documented

ecosystem impacts of B. umbellatus invasions have been described from ongoing management

efforts in the Flathead Lake region of Montana. In these wetlands, B. umbellatus has altered

native habitat structure by forming stands in areas that were formerly open water. These newly

vegetated areas reduce habitat for native fish species that prefer vegetated waters, and lead to

increased spread of invasive fish species such as Northern Pike and Largemouth Bass that use

the dense stands as cover in ambush hunting of prey (Parkinson et al. 2010).

Like many wetland invasives, B. umbellatus is capable of rapid spread both sexually

through seed as well as asexually through rhizomes (Brown and Eckert 2005). In addition, B.

umbellatus can disperse asexually through the production of vegetative propagules or “nodules”

(Figure 3, Brown and Eckert 2005, Parkinson et al. 2010). Nodules are propagules or buds that

originate from a parental rhizome and readily break off the parent plant, forming roots and leaves while floating in the water column. During low water levels in spring and early summer, nodules are able to quickly develop into small plants, giving B. umbellatus a size advantage over native seeds still emerging from dormancy. A two cm rhizome fragment can produce an average of 50 nodules per plant under experimental conditions (range 7-200 nodules/ plant, Dietz and

Michaels, personal observation). The ability of B. umbellatus to reproduce rapidly and become readily established via seeds, nodules, and rhizomatous growth provide significant challenges to managers who do not fully understand the impacts of this species. With the further spread of B. umbellatus into wetlands in Montana and Washington from the Great Lakes region, ecological knowledge of B. umbellatus will be critical for effective management (Figure 1, Parkinson et al.

2010). 4

Here I examine the potential legacy effects of B. umbellatus on restoration of local Lake

Erie wetlands by quantifying the effects of B. umbellatus litter and juvenile plants that develop from vegetative propagules or nodules on the emergence of native seeds and the growth of seedlings. Specifically I ask the following two questions:

1) Is there a legacy effect in soils with a history of B. umbellatus invasion?

2) Does the presence of B. umbellatus vegetative propagules or litter alter the success of

native reestablishment?

5

MATERIALS AND METHODS

Site Description

In the Great Lakes region, extensive areas of B. umbellatus infestation occur at Ottawa

National Wildlife Refuge (ONWR) along the southwestern shore of Lake Erie in Oak Harbor,

Ohio (Figure 2). ONWR is comprised of approximately 6,500 acres (2,631 hectares) of diked,

managed wetlands along the Northwest Ohio coast of Lake Erie (ONWR 2014). Originally part

of the Great Black Swamp, this area is now surrounded by agricultural land and managed for use

by migratory waterfowl, including the federally listed Bald Eagle. Water levels in these diked

wetland systems are carefully monitored by ONWR staff in the early spring and summer during

major bird migrations (ONWR 2014). Given the extensive invasion of B. umbellatus in these

wetlands, ONWR managers have employed several different eradication methods over the past

decade (Ron and Kathy Huffman, personal communication). In areas of heavy infestation,

managers have altered hydrology, used aerial application of herbicides, and disked mature stands

of B. umbellatus beneath the soil with limited success since 2005. In addition, given the

extensive agricultural activity surrounding the refuge, nutrients such as phosphorus and nitrogen

maybe be locally high from fertilizer runoff originating in nearby farm fields. As found in studies

of invasive Typha stands (Farrer and Goldberg 2009), these areas of nonpoint source pollution

may be influencing the distribution and spread of B. umbellatus across the refuge.

Experimental Design

To examine the legacy effects of B. umbellatus on native germination through the

influences of litter and living plants of B. umbellatus, native seeds were sown in shallow (8 cm

deep) mesocosms filled with one of three soil types collected from four managed Lake Erie

wetlands at ONWR in June 2014 (Figure 2). Soils with stands of native emergent species and no 6

history of B. umbellatus invasion were collected from two ONWR wetlands. Soil samples were

collected from two native wetland management units (MS8a, MS8b) with no previous

observations of B. umbellatus stands at the time of soil collection. Two types of invaded soils

with different invasion levels of B. umbellatus were also collected in June of 2014 from two

managed ONWR wetland sites once unusually high water levels allowed for site access. Soils

were collected from a site with a moderate history of B. umbellatus invasion (Entrance Pool). In

fall 2012, this site was dominated by a B. umbellatus monoculture. However, at the time of

collection (June 2014), stands of B. umbellatus were limited to shoreline areas possibly due to

increased water levels and muskrat activity (Dietz, personal observation). The last site of soil

collection (MS7) was a heavily invaded wetland with a history of persistent large Butomus

monocultures. These stands have returned despite several years of extensive management

including aerial herbicide application, disking, and permanent drawdowns of water levels,

(Kathy Huffman personal communication). Soils were collected haphazardly within each site

from areas of similar hydrology (0-0.5 m water depth) by removing the top 15 cm of the soil

profile so as to maximize collection of soil with the most recent impacts.

Following collection, soils with similar invasion history were homogenized and sifted

through a 1 cm mesh sieve to remove rocks and large rhizomes. In addition, care was taken to

remove any dormant B. umbellatus rhizome bud propagules (nodules) present in invaded soils.

Following sieving, I filled mesh bottomed standard 10 x 20 in. greenhouse flats (25.4 x 50.8 cm,

Dillen Products, Middlefield, OH, model A10200G18D050) with a single soil type. After random assignment of a single soil type to each of 24 five ft. diameter (1.5 m) plastic pools

(General Foam, Norfolk, VA, GV24DAC), I placed 5 trays within each pool, with eight

replicate pools for each soil type (120 trays total, 8 pools x 5 trays/pool x 3 soils). Each tray 7

within a pool was randomly assigned one of five specific treatments: 1) an unseeded treatment to

determine seedbank vegetation composition of each soil type, 2) seeded with a native mix

applied to the soil surface, 3) seeded treatments applied alongside planted B. umbellatus rhizome

bud propagules, 4) seeded trays with dried B. umbellatus litter placed on top of sown seed, and

5) seeded trays with a combined treatment of both living B. umbellatus rhizome bud propagules

and dried litter. Because of space limitations, parallel treatments to examine direct effects of applied B. umbellatus propagules and litter on the unseeded native seedbank were not included in this study.

Unseeded flats received no further experimental treatments beyond the designated soil

type so as to assess the natural diversity of native and invaded seedbanks in each of the three

selected soil histories. In addition, seedbank trays were left unpruned, while seeded treatments

were pruned once before sowing to reduce competition during germination with applied seed.

Flats designated for seeding were sown with a selected native seed mix comprised of 25 wetland

species (Table 1) with similar ecohydrological distributions to B. umbellatus in July 2014 at

BGSU’s Ecology-Ethology Research Station. Seed mix composition and sowing density were

designed based on Lake Erie marsh vegetation literature (Lowden 1969, Thiet 2004) and

recommendations of ONWR managers (Kathy Huffman, personal communication). Individual

proportions of seeds (purchased from Cardno JF New, Ann Arbor, MI) for each of the 25 species

were weighed according to desired mixed composition (Table 1) in April 2014 and distributed

into twelve plastic containers between layers of filter paper and sphagnum moss to provide a

moist, cold stratification of 60 days at 4˚C before application to trays and light mixing into the

soil surface in July 2014. In June 2014 prior to planting, I also sowed monocultures of each of

the 25 native seeds used to create the native wetland seed mix to aid in identification of young or 8

non-reproductive individuals at the time of harvest. The native monocultures were grown in standard greenhouse trays containing a 1:1:1 soil medium of vermiculite, sand, and peat moss in

the BGSU greenhouse under natural growing season photoperiods until October 2014.

Naturally senesced and overwintered leaf litter was collected off mature emergent B.

umbellatus plants from a single wetland unit at ONWR separate from soil collection sites in late

May 2014. Litter was washed, air dried, and weighed (63.0 ± 0.36 g/flat) before application on

top of seeds immediately after sowing. Application weights were determined from observed

percent litter cover at the site of collection at ONWR based on average mass of litter found

within thirty 0.25 m2 haphazardly sampled quadrats. Litter was placed directly on top of seeds

and was laid flat against the soil surface similar to litter conditions found in the field (Dietz,

personal observation). Litter was held in place using 1 cm mesh screen secured with pins. The 1

cm mesh was selected so as to minimize reduction of light to germinating seeds beyond that from applied litter. Floating vegetative propagules with young leaves and roots were collected from

ONWR in June 2014 and weighed in groups of 10 propagules applied per tray for use as a covariate. B. umbellatus propagules were allowed to establish for one week before seed sowing

and were replaced as needed for one month following planting to ensure maintenance of this

treatment. Propagules that escaped screening of invaded soils were also removed for one month

following planting. Pools were filled to the height of flats with municipal dechlorinated water

for thermoregulation during summer heat and to mimic the saturated soil conditions of early

summer mudflats of local wetlands. To help with variations in tray height created by uneven

ground under mesocosm pools, clean sand (Quickcrete Premium Play Sand, Atlanta, GA) was

applied to the bottom of each pool to create a uniform water level across all trays. Water levels

were maintained so as to keep soils saturated and carefully monitored for the first month to avoid 9

drying of the soil and around rain events to prevent flooding that could wash away applied seeds.

Water was dechlorinated for two days prior to use and also changed approximately every two

weeks.

In early September after thirteen weeks of growth, I identified plants to genus and to

species when possible and recorded the total number of emerged taxa in each tray to document

total taxon richness prior to harvest. For collection of biomass and to later determine Shannon

Weiner Diversity, aboveground biomass was harvested from October 4-19, following initial fall frost. After harvesting, biomass was dried at 60˚C until constant weight (at least 48-72 hours).

Dried tissue samples were stored until weighing to the nearest 0.0001gram (g). Applied B. umbellatus propagules and litter were also collected from trays at harvest, dried, and weighed to determine effects of treatments on the growth of planted propagules and decay of litter.

To test similarities of vegetation communities I calculated Jaccard index coefficients (SJ)

based on the presence and absence of taxa among litter and nodule treatments within all three

soil types and five seeding treatments using the equation:

SJ = C/ (A+B+C)

where C represents the shared number of species amongst both treatments while A and B represent species or taxa unique to each (Real and Vargas 1996). Differences in Jaccard values were calculated using methods described in Real and Vargas (1996).

Environmental Factors

Throughout the duration of the experiment, environmental variables of the mesocoms were monitored to ensure uniform conditions across established treatments and for comparison to natural conditions observed at ONWR taken at the time of soil collection. Soil temperatures taken in the spring at ONWR may represent an underestimate of the maximum surface 10

temperatures of mudflats in the later summer, but still allowed natural comparisons to

experimental mesocosms. Water and soil temperature were monitored daily throughout the summer using a soil thermometer (Oakton TempTest®IR, Vernon Hills, IL) while DO, and pH levels were measured over the month of July with a sensION multiparameter probe (Hach, Inc.,

Loveland, CO) just before biweekly water changes.

Soil, Leaf Tissue, and Litter Collection

Newly senesced leaves were collected from the base of emerged plants in November

2014, dried, and cut for use in a litter decomposition study. B. umbellatus litter was collected from the same wetlands at ONWR from which I collected the applied litter used in the mesocosm experiment. Following collection, the litter was air dried for one week, weighed, and placed into

11 x 19 cm nylon mesh (2 x 2 mm) bags. Litter bags were placed in the field mounted on 6 ft.

(1.8 m) steel posts driven into the soil at three locations placed 15 m apart along a 45 m transect through B. umbellatus stands in the MS5 wetland unit of ONWR (chosen for accessibility during hunting season) on November 25, 2014. A total of nine bags were placed (3 bags/location) on the soil surface submerged approximately 0.30 m underwater. Litter bags were secured with zip ties and also weighted down with two small pebbles (32.2-36.6 g) taken from the field to keep bags submerged.

After 23weeks, decomposition bags were collected on May 7, 2015. Litter bags were

washed, and dried at 60˚C before nutrient analysis. Changes in mass were used to determine

decomposition rate (k) of B. umbellatus tissues using the equation:

-kt Mo = Mte

where Mo and Mt represent original litter mass and mass of the litter at time t respectively

(Olson 1963, Allison and Vitousek 2004b). To acquire preliminary overview of potential 11 variation in soil chemistry, I conducted a limited soil survey. At the time of litter bag collection, soil samples were also taken for nutrient analysis of the soils used in mesocosms. Five 15 cm samples were collected using a soil corer every 15 m along a 75 m transect within Entrance Pool and MS7. I also took three soil samples 15 m apart in two locations of MS8a and one location within MS8b from the same areas where soils were originally collected for the experiment.

Samples were dried at 70˚C prior to nutrient analysis by Brookeside Laboratories (New Bremen,

OH). Soils were tested for the following nutrients (ppm): phosphorus, calcium, magnesium, potassium, sodium, boron, iron, manganese, copper, zinc, aluminum, ammonium, and soluble sulfur. Percent organic matter, pH, estimated nitrogen release (kg/ha), and total ion exchange capacity were also measured for each sample. Three tissue samples of newly emerged B. umbellatus leaves (collected May 2015) along with previously collected litter samples, were also analyzed by Brookeside Laboratories for tissue nutrient content.

Statistical Analyses

Environmental Variables

All statistical analyses were performed using JMP version 12.0 (2007, SAS Institute Inc.,

Cary, NC) and for each analysis I used α ≤ 0.05 to determine significance. Environmental variables (soil and water temperature, DO, and water pH) in the mesocosms were averaged across the season. I used one-way ANOVAs to test the influence of soil invasion history on water temperature, DO, and pH. To test for differences in soil temperatures in the pools, I used a least squares regression of the five seeding treatments and three soil types. I used multiple nonparametric Wilcoxon tests to determine differences among soil histories in nutrient levels. 12

Native Seedling Response Measures

Seeding treatment types of applied nodules and litter were coded for presence and absence to directly test the influence of these variables in my models of native response

measures (taxon richness, total biomass, Shannon-Weiner diversity, and community evenness). I

used a generalized linear model to assess the need for and effects of the application of a

restoration seed mix on the total number of species harvested (native or the number of observed

invasive species) under a Poisson distribution with effects tests estimated as Maximum Log

Likelihood ratios. I also used a generalized linear model comparing the total number of harvested

species among seeded treatments under the influence of the three soil types and the presence of

B. umbellatus nodules and litter.

I ran least squares regression to test the main effects of soil history and seeding

treatments (nodules, litter) on total biomass, Shannon-Weiner diversity, and community

evenness. All response variables were tested for meeting assumptions of parametric tests

(normality, heterogeneity of variance) and Ln(x) transformations were used when needed (total

biomass). Because pairwise interaction effects among treatments of litter, soil, and nodules were

not significant, they were dropped from subsequent models. Although initial weight of planted

nodules were included in preliminary analyses as a covariate, this variable had no explanatory

power and was dropped from subsequent models.

B. umbellatus Growth and Litter Decomposition

A least squares regression was used to test for effects of soil type and the

presence/absence of applied litter on the growth of B. umbellatus propagules. To examine effects

on the decomposition of applied litter amongst treatment types in mesocosms, I used a least

squares regression to test the influence of the presence of nodules and soil invasion history on 13 changes in litter weight. In addition, I also used a repeated measures MANOVA to assess differences in tissue nutrient concentrations among leaves from three leaf conditions (green, recently senesced, overwintered) among collection times across seasons.

14

RESULTS

Environmental Conditions

Water and soil temperatures varied as expected across the growing season of the

experiment. From July to October 2014, water temperatures ranged from 4.5 - 38˚C with an overall seasonal average of 18.4˚C across all pools. Seasonal average water temperature did not exceed the maximum water temperatures (24˚C) taken at the time of soil collection in early summer at ONWR. In addition, water temperature did not significantly differ among pools of varying soil histories (Table A1, F2, 23 = 1.11, p = 0.35).

Similar to water temperature, soil temperatures of mesocosms were quite variable over

the growing season. Temperatures from July-September ranged from 9.5-33˚C with a seasonal

average of 22.3˚C amongst all trays, lower than the maximum value of 31˚C found in exposed

shoreline mudflats at ONWR in May 2014. Soil temperature of the trays did not vary with soil

invasion history (F2, 61.9 = 1.40, p = 0.25), or the presence of nodules (F1, 87 = 0.000, p = 1.00) or

litter (F1, 87 = 0.001, p = 0.98) in seeded treatments. Interactions between applied treatments such as soil x litter, and soil x nodules also did not influence soil temperatures (Table A2).

Over July when data were recorded, dissolved oxygen (DO) ranged from 7.05-9.23 mg/L across all soil types. In addition, DO was not significantly influenced by soil invasion history, but soils with a history of monocultures tended to have the lowest average DO (Table A1, mean

= 6.87, SD = 0.24, F2, 23 = 2.87, p = 0.08). Water pH varied from 7.4-8 for the month of July,

and like DO, was not influenced by soil invasion history (Table A1, F2, 23 = 0.49, p = 0.62).

Nonparametric tests of Wilcoxon/ Kruskal-Wallis rank sums (Table A3) revealed that soil histories differed only in estimated nitrogen release (ChiSquared = 8.88, DF = 2, p = 0.01), soluble sulfur (ChiSquared = 11.20, DF = 2, p = 0.004), phosphorus (ChiSquared = 7.33, DF = 2, 15

p = 0.03), sodium (ChiSquared = 15.53, DF = 2, p = 0.0004), potassium (ChiSquared = 9.02, DF

= 2, p = 0.01), manganese (ChiSquared = 6.89, DF = 2, p = 0.03) and zinc (ChiSquared =12.29,

DF = 2, p = 0.002). Soils of moderate invasion had the lowest recorded averages for soluble

sulfur, estimated nitrogen release, phosphorus, sodium, manganese, and zinc (Table 2). Native

soils had the lowest amounts of potassium, while moderately invaded soils had the next lowest.

In addition, though ammonium (NH4) and organic matter levels did not significantly differ among soils (most likely due to small sample sizes), levels of these important soil nutrients were found to be the lowest in areas of moderate invasion. Also, for 10 of the 17 measured variables, monoculture soils have the highest levels observed. (Table 2).

Seed Bank Community

A total of 25 taxa emerged from the dormant seedbank of native, moderately invaded, and monoculture soils. Though all three soils types had 18 taxa emerge, vegetation community composition varied among the soil types (Figure 4). Some taxa such as Bidens spp., and

Potomogeton spp. emerged from the seedbank of only one soil type (moderately invaded). Of the

total 25 taxa observed, 11 comprised eighty percent of the taxonomic diversity found across the three soil types (Table 3). The eleven most common taxa found in unseeded soils were

Eleocharis spp. (Spikerushes), Ammannia robusta (Grandredstem), Leersia oryzoides (Rice cutgrass), Polygonum spp. (Knotweeds), Typha spp. (Cattails), Cyperus spp. C (Umbrella sedges), Echinochloa crus-galli (Barnyard Grass), Lindernia dubia (Yellowseed False

Pimpernel), and Ludwigia spp. A (Primrose-Willow), Alisma subcordatum (American Water

Plantain), and Lythrum salicaria (Purple Loosestrife).

Taxon richness (S) of plants emerging from the dormant seedbank in unseeded trays ranged from 7-15 taxa (mean = 10.5, SD = 2.30), similar to the seeded trays with 8-15 taxa 16

(mean = 12.0, SD = 2.26). As expected, the application of seed significantly increased the total number of taxa produced (Table A4, ChiSquared = 9.91, DF = 1, p = 0.002). Although the number of taxa ranged from 7-19 across the three soil types, it was not significantly changed by soil invasion history (ChiSquared = 0.06, DF = 2, p = 0.97). The number of invasive species that emerged from trays ranged from 0-3 species with an average of 1.77 species (SD = 0.90).

Individuals of Lythrum salicaria (purple loosestrife), Echinochloa crus-galli (barnyard grass), and Typha angustifolia (narrowleaf cattail) were found growing in all field soils. Soil history did not influence the number of invasive species that emerged from the seedbank (Table A4,

ChiSquared = 3.57, DF = 2, p = 0.17).

Native Monocultures

Successful germination of applied seed mix species was evaluated with established greenhouse native monocultures. Out of a mix of 25 species, five species failed to germinate

(Table 4) suggesting either inadequate stratification periods or germination conditions or, most likely, unviable seed. These species were also never observed germinating in the field soils of mesocosms. In addition, some species such as Asclepias incarnata (swamp milkweed) showed low germination rates, with only a small number of individuals growing (<10) in greenhouse conditions and were never observed in field soils (Table 4).

Taxon Richness of Seeded Treatments

For trays that received the applied seed mix, soil invasion history did not affect the total number of taxa that grew (Table A4, ChiSquared = 0.31, DF =2, p = 0.86). Although I predicted that taxon richness would be lower in trays planted with B. umbellatus nodules (mean = 12.5, SD

= 2.69), I did not see this difference in the number of taxa that grew in trays with versus without nodules (mean = 13.4, SD = 2.52, ChiSquared = 0.97, DF = 1, p = 0.33). However, taxon 17

richness increased in the presence of B. umbellatus litter (Figure 6C, ChiSquared = 7.62, DF = 1,

p = 0.01), as communities exposed to litter produced an average of three more taxa.

Total Biomass

The total biomass of seedbank trays (mean = 20.5 g, SD = 7.38) was greater than that of

seeded trays (mean = 13.8 g, SD = 4.27), most likely due to the extra two weeks seedlings in these trays grew before seeding and because emerged plants in seedbank trays were not removed

at the time seeded trays were planted (Table A5, F1, 47 = 16.5, p = 0.0002). As such, seedbank

treatments were not included in subsequent analyses of total biomass. The combined biomass

harvested from planted trays ranged from 3.61-27.6 g across all soil types, while the average

biomass of moderately invaded soils (mean = 9.63 g, SD = 3.11) was 66% lower than for native

(mean = 14.6 g, SD = 5.54) and monoculture soils (mean = 14.6 g, SD = 4.18). In addition, in

trays with applied seeds, this decrease in biomass in moderately invaded soils was consistent across all seeding treatments of applied nodules and litter (Figure 5A, Table A6, F2, 66.2 = 10.3, p

= 0.0001). When the vegetative propagules of the invasive were present, total biomass produced

was significantly lower (mean = 10.63 g, SD = 3.88) than when nodules were absent (Figure 6A,

mean = 15.2 g, SD = 4.84, F1, 67.0= 24.8, p < 0.0001). For trays exposed to B. umbellatus litter,

productivity increased as these treatments had significantly greater biomass (mean = 14.20 g, SD

= 5.02) compared to trays with no litter (Figure 6A, mean = 11.7 g, SD = 4.56, F1, 67.0 = 5.5, p =

0.02). There was no interaction effect of combined litter and nodule treatments on the final harvested biomass.

Following analyses of total biomass, I also conducted posthoc nonparametric analyses

(Table A7) on taxon-specific growth responses to the applied seeding treatments of B. umbellatus nodule and litter addition for the 11 most common taxa (Table 3) because not all taxa 18

occurred in all experimental combinations, limiting effective analysis. Responses of these taxa to

nodules and litter differed among species. Only one of these species, Echinochloa crus-galli, was

reduced in both treatments of nodules and litter. Also, two native taxa, Eleocharis spp. and

Alisma subcordatum, showed negative responses to the presence of nodules. No other species

had significant positive responses to the presence of added nodules, while Lindernia dubia and

Ludwigia spp. showed positive responses to litter.

Community Attributes: Diversity, Evenness, and Similarity

Shannon-Weiner Diversity averaged 0.69 (SD = 0.13) (range for H’ = 0.36-1.05) across

all soil types and seeding treatments. Diversity decreased where soils had a longer history of

invasion (Figure 5B, Table A6, F2, 20.1 = 9.30, p = 0.0014). In contrast to their effect on biomass

production, the presence of nodules did not reduce diversity (Figure 6B, mean = 0.68, SD = 0.12,

F1, 70.1 = 1.61, p = 0.21). However, litter treatment did increase the diversity of vegetation within

treatments (Figure 6B, F1, 70.1 = 19.1, p < 0.0001). Pairwise interaction effects (nodules x litter,

soil x nodules, litter x soil) did not affect diversity.

Community Evenness ranged from J’= 0.15-0.40 with an average of 0.27 (SD = 0.04).

Soils from the site with B. umbellatus monocultures had the lowest evenness of the three soil

types (Figure 5C, Table A6, mean = 0.25, SD = 0.04, F2, 20.6 = 12.9, p = 0.0002), showing a shift in vegetation composition towards increased dominance of a few species. Neither litter addition

(F1, 70.9 = 2.67, p = 0.11) nor nodules (F1, 70.9 = 0.06, p = 0.81) significantly altered species

richness in seeded trays. Also, no interactions between variables significantly affected species

evenness.

High Jaccard coefficients amongst treatments in all three soil types showed strong

similarities amongst vegetation communities observed between applied treatments of nodules 19

and litter. When comparing Jaccard Indices for addition of nodules or litter within soil types, SJ values were never significantly different. This lack of significant difference demonstrates a

strong similarity of the relative evenness of taxa within all seeded communities of the three soils

types.

B. umbellatus responses

At planting, nodule mass ranged from 3.1-6.1 g (mean = 4.35 g, SD = 0.77). At the time of harvest, biomass ranged 6.30-19.39 g with an average of 11.14 g (SD = 2.83). Although

biomass of B. umbellatus was unaffected by the presence of litter (Table A6, F1, 25.5 = 0.47, p =

0.50), the growth of nodules differed among the three soil types (Figure 7A, F2, 42 = 3.44, p =

0.04). Nodules had the greatest biomass when grown in native soils (average mass of 8.96 g, SD

= 3.61), while growth was lowest in soils with a history of moderate invasion (mean = 5.00, SD

= 1.49). Interactions between litter and soil did not influence the biomass of planted nodules.

Litter Decomposition and Tissue Quality

Changes in the weight of applied litter were used to determine decomposition over the

growing season. Across all trays with applied litter, initial weights of litter decreased by 36.9-

56.7 g (mean = 47.7 g, SD = 4.70). Litter in native and moderately invaded soils decomposed by similar amounts in the presence and absence of nodules, but the presence of nodules greatly reduced decomposition in monoculture soils. Litter decomposed the least in soils with no history

of B. umbellatus invasion (mean = 3.55 g/week, SD = 0.42), while litter decayed the most (78% of biomass lost) in monoculture soils (Figure 7B, Table A6, mean = 3.78 g/week, SD = 0.36, F2,

39.1 = 6.26, p = 0.004). Also, when nodules were present (mean = 45.7 g, SD = 4.04), litter

decomposition was consistently reduced (Figure 8, mean= 3.82 g/week, SD = 0.35, F1, 21.5 = 17.8,

p = 0.0004). Interactions between soil and nodules also significantly influenced the 20

decomposition of litter (Figure 9, F2, 21.4 = 4.92, p = 0.02). The average calculated decomposition

constant of B. umbellatus litter (k) of retrieved litter bags was 1.22 (yr.)-1.

I observed a significant seasonal change in all nutrient concentrations in the different ages of B. umbellatus tissues (Table A8, F2, 6 = 10.1, p = 0.001). Nutrient concentrations for

nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), and sulfur (S), were all highest

in newly emerged leaf tissues and decreased as leaf tissues began to senesce into the fall (Figure

10). Nitrogen and phosphorus levels of new tissues collected in early spring 2015 were 3.52 and

0.79% respectively. Calcium (Ca) levels were the only nutrient to not be highest in newly

emerged tissues. As expected, nutrient levels tended to be lowest in decaying overwintered

tissues.

21

DISCUSSION

Since the introduction of B. umbellatus to the Great Lakes around the turn of the

twentieth century, its direct impacts on local ecosystems has received little attention. The observed 69% decrease in biomass of native wetland communities exposed to growing B.

umbellatus nodules compared to those without nodules is the first documented impact of this

invasive on native wetland restoration. These reductions in seedling growth due to direct

competition from live stands of B. umbellatus are similar to the effects of other exotic grass and

forb species shown to impact forested and prairie ecosystems (Jordan et al. 2008, Grman and

Suding 2010, Grove et al. 2012).

However, I did not find the expected increase in growth of B. umbellatus in soils from the

longest invaded sites. This result contrasts with studies of other invasive plants in which self-

generated positive feedback loops have been suggested (Jordan et al. 2008, Farrer and Goldberg

2009, Vaccaro et al. 2009). Also, in this study, total biomass of species was the lowest in soils

from areas of moderate invasion. While this pattern might suggest that this is due to underlying

changes in soil composition, similarities in the biomass of native taxa of historically native and

monoculture soils suggest that this observation is from other site differences and not legacy

effects. The site of moderate invasion had higher levels of water throughout the year and was

prone to elevated Lake Erie water levels in higher water years, unlike the water levels of the

other selected pools that were much more heavily regulated and controlled. Though high water

levels suggest that higher levels of nutrients should be correlated with increased anaerobic

conditions from high water (Lyon et al. 1986), the more frequent movement of water into and

out of the areas of this soil collection may have reduced the accumulation of nutrients and litter 22

in sediments as evident in the lower percentage of organic matter and significantly lower amounts of estimated nitrogen release in the moderately invaded soils of the Entrance Pool.

Contrary to the influence of planted nodules and original predictions, applied B.

umbellatus litter had a positive influence on total biomass. Similar studies conducted on another

Great Lakes invader, Typha angustifolia, found that the presence of litter decreased native stem

density and also increased rates of N mineralization (Farrer and Goldberg 2009), while in

another study Typha litter increased survival and growth of Lythrum salicaria (Hager 2004).

Though litter may reduce light availability to emerging seedlings and provide physical

challenges to young plants establishing roots (Vacarro et al. 2009), increases in available

nitrogen from quickly decaying leaf tissues of larger established invasive plants can positively influence the growth of natives (Ehrenfeld 2003). Estimated decomposition rates from this initial study of B. umbellatus tissues revealed that these leaf tissues decay much more rapidly than found for other invaders such as Phragmites or Typha (Windham 2001, Vaccaro et al. 2009).

These data suggest that release of nutrients from B. umbellatus litter may influence wetland nutrient cycles differently than as documented for other common wetland invasives.

Invasives may gain competitive advantages over natives through a number of physiological and reproductive methods (Ehrenfeld et al. 2001). Low leaf construction costs but high photosynthetic rates have led many invasive species to contain high levels of foliar N and P

(Baruch and Goldstein 1999). These high concentrations of tissue nutrients coupled with faster decomposition rates of invasives (Ehrenfeld et al. 2001 and 2003, Allison and Vitousek 2004b,

Ashton et al. 2005) may provide soils in areas of invasion with higher levels of available nutrients. Positive feedbacks of this increased nutrient release are also further encouraged by increased microbial enzymatic activity in areas of increased N and P (Sinsabaugh and 23

Moorehead 1994, Allison and Vitousek 2004a) leading nutrient rich soils to become even richer

over time. Also, because I did not observe any harmful effects of the presence of litter, but only

beneficial effects on biomass and taxon diversity, it is unlikely that B. umbellatus tissues contain

allelopathic chemicals as seen in other invasive species (Grove et al. 2012).

Species specific growth responses to the application of litter may also result from

invasion history differences in the litter decomposition rates amongst the three different soil

types that may have arisen through chemical and biological changes that occurred following

invasion. Applied B. umbellatus litter decayed less in treatments with native soils and

decomposed the most in soils of historical B. umbellatus monocultures. These results correspond

to findings by a similar study (Strickland et al. 2009) in which litter decomposed faster in soils

with prior species history due to changes in soil microbial communities. Microbial community

composition and ecosystem processes are influenced not only by current vegetation community

composition (Strickland et al. 2009), but more importantly by past or removed species (Elgersma

et al. 2011). Native soils, with no history of B. umbellatus occurrence, may have microbial

communities that are not well-suited to decompose B. umbellatus tissue. Such a scenario would

be consistent with the significantly reduced litter decomposition observed in the mesocosms with

native soils and suggests that microbial communities may change following B. umbellatus invasion.

Alternatively, altered decomposition rates among the different soil types also could have been influenced by inherent nutrient differences in soils. B. umbellatus litter rich in nutrients may have accumulated in areas of high infestation and in turn led to locally high nutrient levels in these sites. Increased soil nutrient levels could have in turn influenced the rate of decomposition in these high nutrient sites (Sinsabaugh and Moorehead 1994, Allison and 24

Vitousek 2004a) even without a direct change to the microbial community of the soil. Also,

already locally high nutrients, possibly due to runoff from landscape inputs of surrounding

agriculture, would have supported higher levels of growth, even before the addition of nutrients

acquired through decomposition. I observed significantly higher rates of nitrogen release,

sodium, and potassium in soils of former monocultures consistent with increased nitrogen

mineralization levels found in stands of Typha (Farrer and Goldberg 2009). These increased

levels may be due to the higher percentages of N and P found in both fresh and overwintered

tissues of B. umbellatus that are even greater than levels in fresh tissues of Typha and Phalaris

arundinacea (Kao et al. 2003, Vaccaro et al. 2009). In addition, the presence of nodules also

reduced the decomposition of litter across all three soil invasion histories. This suggests that as

invasions of B. umbellatus build, denser stands of nodules may slow or decrease nutrient cycling

through the decomposition of litter, reducing the resources available for native communities.

Although nutrient inputs of litter decomposition and agricultural runoff may begin to explain elevated levels of nutrients in monocultures sites, this increase may have also been from a third source of inputs into these wetland systems. The increased nutrient levels observed in monoculture soils may also be evidence of management practices that left chemical legacies following the herbicide application. The addition of nitrogen and phosphorus rich herbicides like glyphosate could have increased N and C mineralization (Haney et al. 2002, Saxton et al. 2011)

of monoculture soils independent of microbial community changes related to living B.

umbellatus or its litter inputs.

Similar to its effects on biomass, litter also increased taxon richness and Shannon-Weiner

diversity. Meisner et al. (2012) found that soil respiration rates were higher in areas of invasive

litter application. Such increased levels of root respiration could have allowed additional species 25

to germinate in trays with B. umbellatus litter compared to those without. Also, litter application can create the establishment of favorable conditions for seedlings on small scales (Molofsky and

Augspurger 1992). Throughout the summer, soil surfaces tended to dry quickly between watering times, but more slowly in trays with applied litter due to moisture that remained in the litter tissues (Dietz personal observation). This may have promoted greater establishment of seeds exposed on the soil surface similar to Eckstein and Donath (2005) who found that litter prevented humidity and soil temperature reaching levels that would otherwise decrease seedling germination. Lastly, in the week following seed sowing, one large rain storm washed small amounts of seed from trays through splashing by heavy rainfall. Washout may have been less severe in trays with applied litter due to the litter holding seed in place and could have led to higher diversity in these trays though the majority of applied seed remained in all trays.

Although the total number of species emerging from seeded trays did not significantly differ among soils with differing B. umbellatus invasion histories, this was most likely due to the presence of a substantial viable dormant seedbank. This result suggests that seeding may not be needed to restore these wetlands. However, the absence of differences in taxon richness could be the result of six species failing to germinate from the applied seed mix in field soils in this short time span. In my treatments the soil was kept moist, not inundated, for long periods of time, possibly reducing germination in some species like Pontederia cordata (Gettys and Dumeroese

2009). Also, the germination requirements of other species such as Sparganium americanum and

S. eurycarpum are not well understood and successful germination of wild seedlings is poor

(Belyakov and Laprirov 2015).

26

Implications for Managers and Further Areas of Research

Data documenting decreased growth of natives exposed to stands of living B. umbellatus propagules supports the continuation of eradication programs to remove dense stands of B. umbellatus from wetlands of the Great Lakes. Although other studies (Dietz and Michaels, unpublished) suggest cutting can reduce production of the nodules (rhizome buds), the persistence of these buds in the infested soils and their propensity to float, sprout, and disperse makes these propagules a critical factor to consider when planning the restoration of previously invaded sites even long after dense stands are removed. Furthermore, even though total taxon diversity and community evenness were not significantly influenced by the presence of B. umbellatus nodules, both were reduced in soils with long histories of invasion, leading to shifts in community structure of natives establishing following the removal of B. umbellatus stands.

Decreased growth of two important native wetland taxa, Eleocharis spp. and Alisma subcordatum warrant caution for managers interested in achieving a prescribed community composition in wetlands after restoration. Preliminary studies conducted at ONWR comparing macroinvertebrate communities in B. umbellatus stands to areas of native pinkweed (Persicaria pensylvanicum) and American water plantain (Alisma subcordatum) found that family richness was lower in areas of B. umbellatus (Dietz and Michaels, unpublished). Therefore, subsequent ecosystem cascades involving food chains associated with these natives that support wildlife are possible and in need of future study.

This work also suggests that wetland microbial communities maybe altered by the presence of B. umbellatus leading to changes in nutrient cycling. However, these changes may also be from responses of the microbial community to wetland nutrient fluxes and not direct changes to the microbial community composition. My mesocosms are a highly simplified model 27

of natural conditions found in Lake Erie wetlands. Further research that examines differences in

wetland water and soil chemistry in relation to natural fluctuations in hydrology and the

microbial communities of native and B. umbellatus soils is essential to better understand the influences of this invasive on Great Lakes wetland ecosystems.

28

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33

TABLES AND FIGURES Table 1. Applied Seed Mix Composition. Proportions of all individual species are given by weight in grams relative to the overall weight of the total seed mix. Choice of individual species was determined from information taken from available Lake Erie vegetation composition literature, commercially available wetland emergent seed mixes, and recommendations of wildlife managers at Ottawa National Wildlife Refuge (ONWR) in Oak Harbor, OH. Species Common Proportion Name Name (by weight) Acorus calamus Sweet Flag 0.36 Alisma subcordatum American Water Plantain 1.89 Asclepias incarnata Swamp Milkweed 1.60 Carex comosa Longhair Sedge 2.47 Carex cristatella Crested Sedge 0.44 Carex lurida Shallow Sedge 4.15 Carex vulpinoidea Fox Sedge 5.82 Eleocharis palustris Common Spike Rush 1.38 Eupatorium maculatum Spotted Joe Pye Weed 2.26 Hibiscus laevis Halberdleaf Rosemallow 8.88 Juncus effusus Common Rush 1.53 Leersia oryzoides Rice Cut Grass 3.28 Lycopus americanus American Water 0.91 Horehound Mimulus ringens Allegheny 1.24 Monkeyflower Peltandra virginica Green Arrow Arum 15.36 Penthorum sedoides Ditch Stonecrop 0.51 Polygonum Pennsylvania Smartweed 0.91 pensylvanicum Pontederia cordata Pickerelweed 19.07 Sagittaria latifolia Broadleaf Arrowhead 1.89 Scirpus acutus Hardstem Bulrush 2.77 Scirpus atrovirens Green Bulrush 4.15 Scirpus validus Softstem Bulrush 7.79 Sparganium American bur-reed 1.31 americanum Sparganium Simple stem bur-reed 3.86 eurycarpum Verbena hastata Swamp Verbena 2.55 34

Table 2. Average Recorded Nutrients of Soils. The average values of recorded nutrient variables are listed below. Highlighted variables show significant differences amongst soil types for that variable based on the results of Nonparemetric Wilcoxon/Kruskal-Wallis analyses. Superscripts represent results of Tukey-Kramer comparison of means.

Soil Type pH N Exchange Humus NH4 P K S Ca release Capacity (%) (ppm) (ppm) (ppm) (ppm) (ppm (kg/ha) (ME/100g) Native 6.39 467a 23.5 4.24 24.7 28.7ab 157b 69.9a 2815 (N=9) Moderately 6.58 265b 22.2 2.67 16.68 20.2b 195a 23.0b 2726 Invaded (N=5) Monoculture 5.98 515a 24.9 4.51 22.9 38.8a 196a 28.0b 2734 (N=5) Soil Type Mg Na Fe Cu Al Zn B Mn (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)

Native 645 41.3b 499 2.02 670 1.53b 0.92 110a (N=9) Moderately 653 32.0c 490 2.75 773 1.25b 0.85 75.6b Invaded (N=5) Monoculture 596 69.8a 514 2.85 770 2.39a 0.81 101ab (N=5)

35

Table 3. Common Emerged Species and Responses to Applied B. umbellatus Treatments. Eleven species comprised 80% of the total species harvested from seedbank treatments. Species are listed in descending order with the most frequent species being listed first. Biomass of individual species responded differently to planted nodules and applied litter treatments. Responses are indicated if there was a significant change in biomass to that treatment. Positive signs represent an increase in the biomass of that species under the presence of that experimental condition, while negative sings represent decreases in biomass based on nonparametric Wilcoxon Tests. Species Name Common Name (+)Nodules (+)Litter Eleocharis spp. Spikerushes (-) 0 Ammannia robusta Grandredstem 0 0 Leersia oryzoides Rice Cutgrass 0 (-) Polygonum spp. Knotweeds 0 0 Typha spp. Cattails (-) 0 Cyperus spp. A Umbrella Sedges 0 0 Echinochloa crus-galli Barnyard Grass (-) (-) Lindernia dubia Yellowseed False Pimpernel 0 (+) Ludwigia spp. A Primrose-Willow or Water- 0 (+) Primrose Alisma subcordatum American Water Plantain (-) 0 Lythrum salicaria Purple Loosestrife 0 0

36

Table 4. Ungerminated Applied Seed Species. Six species included in our applied seed mix did not germinate in experimental treatments and greenhouse conditions. The last column provides the soil conditions under which a species did not germinate. Species Name Common Name Soil Type Asclepias incarnata Swamp Milkweed Field Soil Mimulus ringens Allegheny Monkeyflower Field Soil and Greenhouse Penthorum sedoides Ditch Stonecrop Field Soil and Greenhouse Pontederia cordata Pickerelweed Field Soil and Greenhouse Sparganium americanum American bur-reed Field Soil and Greenhouse Sparganium eurycarpum Simple stem bur-reed Field Soil and Greenhouse 37

Figure 1. National Distribution of Butomus umbellatus. Gray areas represent known invasive populations of B. umbellatus as of the taking of this map in 2012. Additional known populations can be found in Washington State and Louisiana. Map available at: http://plants.usda.gov/core/profile?symbol=BUUM

38

Figure 2. Map of Ottawa National Wildlife Refuge. Highlighted areas show pool locations of collected soils with native (MS8a, MS8b), moderate invasion (Entrance Pool), and monocultures of B. umbellatus histories within our field site of ONWR. Individual soil collection sites occurred within multiple locations of each designated pool type. GIS data is courtesy of ONWR and US Fish and Wildlife Staff. Three soil samples were taken every 15 m along 45 m transects starting at N 41˚37.012 W 083˚12.690, N 41°36.884 W 083°12.363, and N 41˚36.731 W 083˚12.248 for native soils. Five samples were taken every 15 m along 75 m transects starting at N 41˚36.325 W 083˚12.101 for moderately invaded soils, and N 41˚36.787 W 083˚14.272 for monoculture soils.

39

A B Figure 3. Butomus umbellatus nodules. A. Nodules attached to the parental rhizome. Nodules, or vegetative propagules form on the rhizome of parental plants before breaking off. B. Vegetative nodule in water column. While floating in the water column, nodules are able to form leaf and root tissues. 40

Vegetation Composition of Common Seedbank Taxa Eleocharis spp. Ammannia robusta Leersia oryzoides Polygonum spp. Typha spp. Cyperus spp. A Echinochloa crus-galli Lindernia dubia Ludwigia spp. A Alisma subcordatum Lythrum salicaria 1 0.9 0.8 0.7 0.6

Proportion 0.5 0.4 0.3 0.2 0.1 0 Native Moderate Invasion Monocultures Soil History

Figure 4. Vegetation Composition of Common Seedbank Taxa. Though each soil type had a total of 18 species germinate from the seedbank, the relative abundance of the 11 most common species differed across soils. While emerged species abundance was more evenly dispersed in native soils, monoculture soils were heavily dominated by Ammania robusta and Eleocharis spp. 41

18 A A A 16

14 B 12

10

8 Biomass (g) 6

4

2

0

B A A 0.7 B

0.6

0.5

0.4 Weiner DiversityWeiner - 0.3

0.2 Shannon 0.1

0

C 0.35 A A 0.3 B

0.25

0.2

0.15

Community Evenness Community 0.1

0.05

0 Native Moderate Invasion Monocultures Soil Invasion History

42

Figure 5. Effect of Soil Invasion History on Native Response Measures. A. Effects on Total Biomass. Soils with a history of moderate B. umbellatus invasion significantly lowered the biomass of species harvested from seeded treatments (mean = 9.63, SD = 3.11, F2, 66.2 = 10.3, p = 0.0001). Native soils (mean = 14.6, SD = 5.54) and soils with a history of B. umbellatus monocultures (mean = 14.6, SD = 4.18) were not significantly different from one another. B. Effects on Diversity. Monocultures had lower taxon diversity (mean = 0.63, SD = 0.09) than soils of native (mean = 0.73, SD = 0.14) and soils of moderate B. umbellatus invasion (mean = 0.71, SD = 0.12, F2, 20.1 = 9.30, p = 0.001). C. Effects on Community Evenness. Species Evenness was lowest in soils with a history of B. umbellatus monocultures (mean = 0.25, SD = 0.04, F2, 20.2 = 12.9, p = 0.0002). Bars connected with the same letter are not significantly different from one another. Least square means are shown for all response measures. All error bars represent standard errors. 43

A 18 16 14 12 10 8 Biomass Biomass (g) 6 4 2 0

B 0.8

0.7

0.6

0.5

0.4

0.3

0.2 - WeinerShannon Diversity 0.1

0

C 16 14

12

10

8

6

4 Species Number 2

0 (-) Nodules, (-) (+) Nodules, (-) (-) Nodules, (+) (+) Nodules, (+) Litter Litter Litter Litter

Presence/Absence of Nodules and Litter 44

Figure 6. Effect of Applied Nodules and Litter on Native Response Measures. A. Effects on Total Biomass. Biomass of native seeds sown alongside B. umbellatus nodules (mean =10.63, SD =3.88) was significantly lower than plants seeded alone (mean = 15.2, SD =4.84, F1, 67.0 = 24.8, p < 0.0001). In contrast, exposure to B. umbellatus litter (mean = 14.20, SD = 5.02) increased the biomass of harvested species over trays without litter (mean = 11.7, SD = 4.56, F1, 67.0 = 5.5, p = 0.02). B. Effects on Taxon Diversity. The presence of planted B. umbellatus nodules (mean = 0.68, SD = 0.12) did not significantly alter the species diversity compared to trays without nodules (mean = 0.70, SD = 0.13, F1, 70.1 = 1.61, p = 0.21). However, the presence (mean = 0.75, SD = 0.12) of B. umbellatus litter significantly increased species diversity over trays without litter (mean = 0.65, SD = 0.12, F1, 70.1 = 19.1, p < 0.0001). C. Effects on Taxon Richness. Species richness was lower in treatments with planted B. umbellatus nodules (mean = 12.5, SD = 2.69), but was not significantly lower than trays without (mean = 13.4, SD = 2.52, ChiSquared = 0.97, DF = 1, p = 0.33). The presence of litter (mean = 14.40, SD = 2.08) significantly increased the total number of species harvested compared to trays without litter (mean = 11.48, SD = 2.32, ChiSquared = 7.62, DF = 1, p = 0.01). Means are shown for richness. Least square means are shown for biomass and diversity. All error bars represent standard error. 45

A 10 A 9

8 AB 7 B 6

5

4

3 Biomass Biomass of Nodules (g) 2

1

0

B 5 A 4.5 AB B 4 3.5 3 2.5 2 1.5 1

Loss of Biomass (g/week) Biomass of Loss 0.5 0 Native Moderate Invasion Monocultures Soil Invasion History

Figure 7. B. umbellatus Litter and Nodule Responses to Soil Invasion History. A. Growth of Nodules. Nodules in moderately invaded sites had the lowest biomass of all three soil types (mean = 5.00, SD = 1.49). Nodules grew the most in soils of native history (mean = 8.96, SD = 3.61, F2,42 = 3.44, p = 0.04). B. Decomposition of Litter. Decomposition of B. umbellatus litter was the lowest in soils of native history (mean = 3.55 g/week, SD = 0.42) while litter decreased more in soils of former monocultures (mean = 3.78 g/week, SD = 0.36, F2,39.1 = 6.26, p = 0.004). Bars connected by the same letter are not significantly different from one another. Least square means are shown. Error bars represent standard errors. 46

Effect of Nodules on the Decomposition of Litter 5 4.5 A B 4 3.5 3 2.5 2 1.5 1

Loss of Biomass (g/week) Biomass of Loss 0.5 0 (-) Nodules (+) Nodules Presence of Nodules

Figure 8. Effect of Nodules on the Decomposition of Litter. The presence of nodules (mean = 3.51 g/week, SD = 0.31) greatly decreased the decomposition of applied litter in trays (mean = 3.82 g/week, SD = 0.35, F1,21.5 = 17.8, p = 0.0004). Bars connected by the same letter are not significantly different from one another. Least square means are shown. Error bars represent standard errors. 47

Effect of Soil and Nodule Interactions on the Decompostion of Litter 5

4.5 A AB 4 B B B B 3.5 3 2.5 2 1.5 1

Loss of Biomass (g/week) Biomass of Loss 0.5 0 (-) Nodules (+) Nodules (-) Nodules (+) Nodules (-) Nodules (+) Nodules Native Moderatley Invaded Monocultures Presence/Absence of Litter Amongst Soil Histories

Figure 9. Effect of Soil and Nodule Interactions on the Decomposition of Litter. Interactions between planted nodules and soil influenced the decomposition of litter (F120, 21.4 = 4.92, p = 0.02). Across all soil types the presence of nodules decreased the amount of decomposition. However, in native soils, the decrease in decomposition amounts were not significant like those found in moderately invaded soils and monocultures. Bars connected by the same letter are not significantly different from one another. Least square means are shown. Error bars represent standard error.

48

B. umbellatus Tissue Nutrients 4.5

4

3.5 Fresh

3 Newly Senesced

2.5 Overwintered

2

1.5 Concentration (%) Concentration 1

0.5

0 N P K Ca Mg S Nutrient

Figure 10. B. umbellatus Tissue Nutrients. Leaf nutrient concentration significantly differed amongst tissues collected during different seasons (F2,6=10.1, p =0.001). Newly emerged litter (fresh) had the highest concentrations of all nutrients except for calcium levels. Overwintered litter tended to have the lowest concentrations. Least square means are shown. Error bars represent standard error. 49

APPENDIX A. STATISTICAL TABLES Table1. Effect of Soil Invasion History on Mesocosm Water Environmental Variables. The effect of B. umbellatus invasion history of soils on water environmental variables was conducted using one way analysis of variance (ANOVA) tests. Only influences of soil history were included in these analyses. Source DF SS MS F Prob > R2 Ratio F Water Soil History 2 0.12 0.06 1.11 0.35 0.10 Temperature N=24 Error 21 1.18 0.06

Total 23 1.18

Dissolved Soil History 2 0.43 0.21 2.87 0.08 0.21 Oxygen N=24 Error 21 1.56 0.07

Total 23 2.00

pH Soil History 2 0.05 0.02 0.49 0.62 0.04 N=24 Error 21 0.95 0.05

Total 23 1.02

Table 2. Effects of Soil Invasion History and Seeding Treatments on Soil Temperature of Mesocosms. I ran a least squares regression to test the effect of soil invasion history and seeding 50 treatments (application of a 25 spp. native seed mix as well as B. umbellatus nodules and litter) on the temperature of soils in established mesocosm trays through the growing season. N=120, R2=0.36. Source Nparm DF DFDen F Ratio Prob > F

Soil History 2 2 61.9 1.40 0.25

Nodules 1 1 87 0.00 1.00

Litter 1 1 87 0.001 0.98

Nodules x Litter 1 1 87 0.82 0.37

Nodules x Litter x Soil 2 2 87 1.29 0.28

Soil x Nodules 2 2 87 0.47 0.63

Soil x Litter 2 2 87 2.45 0.09

Table 3.Nonparametric Wilcoxon Tables for Significant Differences in Soil Nutrients. I used Nonparametric Wilcoxon analyses to test the effects of soil invasion history on the nutrients of 51 collected soil samples from ONWR in the spring of 2015. Only nutrient levels that showed significant differences are shown in the table below. For all tests N=19. ChiSquare DF Prob > ChiSq

Potassium 9.02 2 0.01

Zinc 12.29 2 0.002

Estimated Nitrogen 8.88 2 0.01 Release Sodium 15.53 2 0.0004

Soluble Sulfur 11.20 2 0.004

Phosphorus 7.33 2 0.03

Manganese 6.89 2 0.03

Table 4. Results of Generalized Linear Models For Taxon Richness. I used generalized linear models to test the effects of seed application and soil history on the number of total and invasive 52

taxa that emerged. I also used a generalized linear model to test the impacts of soil invasion history and seeding treatments of applied nodules and litter on total taxon richness. Nonsignificant interaction effects were removed from the model for seeded trays. All models were run assuming a Poisson distribution. Significant values are bolded. Source DF ChiSquare Prob>ChiSq Seebank vs Soil History 2 0.06 0.97 Seeded Trays N= 120 Seed 1 9.91 0.002

Soil x Seed 2 0.04 0.98

Invasive Species Seed 1 1.49 0.22 N=120 Soil History 2 3.57 0.17

Soil x Seed 2 0.84 0.66

Seeded Trays Soil History 2 0.31 0.86 N=96 Nodules 1 0.97 0.33

Litter 1 7.62 0.01

Table 5. Effect of Seed Application on Biomass. I used a one way analysis of variance (ANOVA) to test the effects of seed application on the biomass of harvested taxa. Comparison of 53 means was only used to look at differences between seedbank and seeded trays without the presence of applied nodules and litter. N= 48 R2= 0.26. Source DF SS MS F Ratio Prob > F Model 1 1.76 1.76 16.5 0.0002

Error 46 4.92 0.11

Total 47 6.68

Table 6. Effect of Seeding Treatments and Soil Invasion History on Native and B. umbellatus Response Measures. I used least squares regression to test the impact of soil invasion history and 54

applied B. umbellatus litter and nodules on native response measures of biomass, Shannon- Weiner Diversity, and Community Evenness. Least squares regression was also used to determine effects of applied nodules and litter treatments on B. umbellatus nodule growth and litter decomposition. Nonsignificant interaction effects were dropped from all models. Significant values are bolded. Source DF DFDen F Ratio Prob > F R2 Total Biomass Soil History 2 66.2 10.3 0.0001 0.54 N=96 Nodules 1 67.0 24.8 <0.0001

Litter 1 67.0 5.5 0.02

Shannon- Soil History 2 20.1 9.30 0.001 0.32 Weiner Diversity Nodules 1 70.1 1.61 0.21 N=96 Litter 1 70.1 19.1 <0.0001

Community Soil History 2 20.6 12.9 0.0002 0.14 Evenness N=96 Nodules 1 70.9 0.06 0.81

Litter 1 70.9 2.67 0.11

Planted Soil History 2 42.0 3.44 0.04 0.34 Nodules N=48 Litter 1 25.5 0.47 0.50

Litter Soil History 2 39.1 6.26 0.004 0.63 Decomposition N=48 Nodules 1 21.5 17.8 0.0004

Soil x Nodules 2 21.4 4.92 0.02

Table 7. Nonparametric Wilcoxon/Kruskal-Wallis Rank Sums for Treatments on Common Taxa. Nonparametric analyses were used to test the effects of applied B. umbellatus nodules and litter on the harvested biomass of the 11 most common harvest taxa. Significant values are bolded. 55

Species Model ChiSquare DF Prob > ChiSq Alisma subcordatum Nodules 6.39 1 0.01

N=80 Litter 0.16 1 0.69

Echinochloa crus-galli Nodules 13.39 1 0.0002

N=50 Litter 6.28 1 0.01

Eleocharis spp. Nodules 15.67 1 <0.0001

N=120 Litter 2.19 1 0.14

Ammannia robusta Nodules 3.60 1 0.06

N=111 Litter 2.81 1 0.09

Leersia oryzoides Nodules 0.73 1 0.39

N=90 Litter 5.18 1 0.02

Lindernia dubia Nodules 0.42 1 0.51

N=84 Litter 6.58 1 0.01

Lythrum salicaria Nodules 0.64 1 0.42

N=65 Litter 1.57 1 0.21

Polygonum spp. Nodules 3.50 1 0.06

N=87 Litter 0.33 1 0.56

Typha spp. Nodules 4.12 1 0.04

N=80 Litter 0.00 1 1.00

Cyperus spp. C Nodules 0.12 1 0.73

N=105 Litter 0.03 1 0.86

Ludwigia spp. Nodules 0.36 1 0.55

N=86 Litter 15.07 1 0.0001

Table 8 Effect of Age of Tissues on B. umbellatus Tissue Nutrients. To test for differences in the nutrient concentration of B. umbellatus tissues during different seasons on fresh (green), recently senesced, and overwintered leaves, I used a repeated measures MANOVA. N= 9. 56

Test Value Exact F NumDF DenDF Prob > F F-Test 10.1 30.4 2 6 0.001