Factors Affecting the Restoration of Vallisneria Americana in the Caloosahatchee River

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Factors affecting the restoration of Vallisneria americana in the Caloosahatchee River

A Thesis

Presented to

The Faculty of the College of Arts and Sciences

Florida Gulf Coast University

In Partial Fulfillment

of the Requirement for the Degree of

Master of Science

K. Michael Ross 2015

APPROVAL SHEET

This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science

______

Kory Michael Ross

______

Edwin M. Everham, III, Ph.D. Committee Chair

______

David W. Ceilley, M.S. Committee Member

______

James G. Douglass, Ph.D. Committee Member

The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline

ii Acknowledgements

My research would not have been possible without the guidance of my major advisor, Edwin M. Everham III, and my committee. Thank you Win for your guidance and patience throughout this journey. I would also like to express my sincerest gratitude to my committee members, David W. Ceilley and James G. Douglass.

In addition, I would like to thank the South Florida Water Management District for funding this research. Specifically, I’d like to thank Beth Orlando, Peter Doering, and

Theresa Coley. Dr. Lynn Gettys at the UF/IFAS Center for Aquatic and Invasive Plants thank you for your help investigating the donor Vallisneria genetics. Also, I’d like to thank the staff at Sun Splash Family Waterpark for direct access to Lake Kennedy. I would like to thank the Lake Trafford Marina for unlimited boat ramp access and providing additional boats. I would especially like to thank John Ferlita and Geoff

Rosenaw for their numerous days dedicated to this project. They helped harvest and plant

V. americana transplants, build and deploy exclosures, and collect water quality. Jamie

McDonald, thank you for your instruction and support with the sediment analysis. Alex

Leynse, although the sediment nutrient data didn’t make into the final print, thank you for your time and guidance in prepping the sediments for the AutoAnalyzer.

I also wish to thank my mother Kim, my grandmother Laina, and my grandfather

Ed. They have provided unconditional support and love throughout my life. To Nicole, my significant other, thank you for your patience, love, and support throughout this endeavor.

iii Table of Contents

List of Figures………………………………………………………………………………...…vii

List of Appendices………………………………………………………………………………..ix

Abstract……………………………………………………………………………………x

Chapter 1- Introduction…………………………………………………………………1

Global Threats to Coastal Waterways…………………………………………………….1

Importance of Submerged Aquatic Vegetation in Estuaries………………………………1

Caloosahatchee River and Estuary………………………………………………………...2

Ecology of Vallisneria americana………………………………………………………...4

Morphological description………………………………………………………...5

Life cycle and reproduction……………………………………………………….6

Habitat requirements………………………………………………………………7

V. americana in the Caloosahatchee River and Estuary …………...... 7

Research Objectives……………………………………………………………………….9

Chapter 2- Methods…………………………………………………………………….11

Overview of Study Design……………………………………………………………….11

Study Location…………………………………………………………………………...12

General Procedures………………………………………………………………………14

Donor V. americana……………………………………………………………...14

Exclosures and planting method…………………………………………………16

Sediments………………………………………………………………………………...16

Soil sample collection……………………………………………………………16

Total organic content……………………………………………………………16

Sediment grain size analysis…………………………………………………….17

Water Quality……………………………………………………………………………18

Phase I-Small Exclosures………………………………………………………………..18

Exclosure design, installation, and planting…………………………………….18

Quantitative plant sampling……………………………………………………..21

Monitoring………………………………………………………………………22

Data analysis……………………………………………………………………22

Phase II- Large Exclosures……………………………………………………………...23

Exclosure design, installation, and planting……………………………………23

Monitoring………………………………………………………………………25

Snail monitoring……………………………………………………………...…26

Data analysis……………………………………………………………………26

Chapter 3- Results……………………………………………………………………..27

Sediment Characteristics………………………………………………………………..27

Total organic content and grain size analysis……………………………….…27

Water Quality…………………………………………………………………...29

Objective 1: Effect of two planting densities on the growth of V. americana.....31

Objective 2: Difference of growth between two genetic strains of V. americana..32

Objective 3: Effect of site on growth……………………………………………34

Phase II…………………………………………………………...... 34

Objective 1: Effect of site on growth of V. americana………………………….38

Objective 2: Effect of strain on V. americana growth………………………….39

Chapter 4- Discussion…………………………………………………………………..41

Limitations of the Study……………………………………………………………….…41

Effect of exclosure size………………………………………………………………….43

Effect of two planting densities………………………………………………………….45

Growth between two genetic strains……………………………………………………..46

Effect of sediment characteristics………………………………………………………..48

Future Considerations……………………………………………………………………50

Conclusions and Recommendations……………………………………………………..51

References………………………………………………………………………………53

vi List of Figures

Figure Page

1. Map of Caloosahatchee River and Estuary watershed with S-77, S-78 and S-79 control structures……………………………………...... 3

2. Map depicting Vallisneria americana distribution………………...... 5

3. Map depicting the Caloosahatchee River and Estuary with Franklin Lock and Dam (S-79). …………………………………………………………. 12

4. Map for Phase I depicting sites 1, 2, and 3…………………………...... 13

5. Map for Phase II illustrating sites 1, 2, 3, 4, 5, 6, and…………………… 13

6. Map of V. americana donor sites……………...………………………… 15

7. Photograph of Florida Gulf Coast University vessel used to transport V. americana……………………………………………………………...... 15

8. Diagram of the 1-m2 exclosure design for Phase I……………………… 19

9. Diagram of the experimental design of Phase I………………………….. 20

10. Photograph of site two exclosure installation……………………………. 21

11. Diagram of the exclosure coordinate system…………………………….. 22

12. Photograph of a large exclosure used in Phase II of the study…………... 24

13. Diagram of Phase II large exclosure and planting methodology………… 24

14. Graph of sediment organic content among donor and transplant sites………………………………………………….……………………. 27 15. Graph of percent sand, clay/silt, and gravel among donor and transplant sites……………………………………………………………………….. 28

16. Graph of percent silt/clay among donor and restoration sites……………. 28

17. Graph of Water quality results for sites 1-7, including temperature (°C), dissolved oxygen (mg/L), conductivity (μS), salinity (ppt), pH, and Secchi depth (m)…………………………………………………………. 31

18. Graph of calculated light attenuation coefficients, K (m-1), for each site through the sampling period……………………………………………... 31

vii

19. Graph of planting density effect on mean biomass (g)………………….. 32

20. Graph comparing between the two strains in Phase I……………………. 33

21. Graph comparing between the two strains in Phase I……………………. 33

22. Graph of planting site effect on mean biomass (g)………………………. 34

23. Graph of Lake Kennedy V. americana transplant mean percent frequency through time at sites 4, 5, 6 and 7…………………………….. 35

24. Graph of Lake Trafford V. americana transplant mean percent frequency through time at sites 4, 5, 6 and 7………………………………………... 35

25. Graph of Lake Kennedy mean number of rosettes (shoots) through time at sites 4, 5, 6 and 7………………………………………………………. 36

26. Graph of Lake Trafford mean number of rosettes through time at sites 4, 5, 6 and 7……………………………………………………………...... 36

27. Graph of final V. americana transplant mean percent frequency of occurrence through time among sites in Phase II………………………... 37

28. Graph of final mean number of rosettes through time among sites in Phase II…………………………………………………………………… 37

29. Graph of average blade length comparison among sites in Phase II…….. 38

30. Graph of average leaf area comparison among sites in Phase II………… 39

31. Graph of average blade length comparison between strains in Phase II…. 39

32. Graph of average blade width comparison between strains in Phase II…. 40

33. Graph of average leaf area comparison between strains in Phase II……... 40

viii

List of Appendices

Appendix Page

A. Graph of Phase I above and below ground biomass…………. 67

B. Graph of Phase I percent coverage through time graphs…….. 68

C. Table of LSD post-hoc tests for total organic content and silt/clay averages among sites in Phase II…………………… 70

D. ANOVA tables for Phase I analysis………………………… 72

E. ANOVA tables for Phase II analysis………………………... 75

F. Table of LSD post-hoc test for final mean number of rosettes… 77

G. Table of LSD post-hoc tests for average blade length and area among sites in Phase II………………………………………… 78

Abstract

Globally, rivers and estuaries have been exposed to heavy exploitation, pollution, and landscape alterations, leading to losses of habitat and ecological functions. In particular, submerged aquatic vegetation (SAV) habitats have declined globally, impacting the ecological functions they contribute. The Caloosahatchee River and Estuary are located on the southwest coast of Florida. Development and hydrologic alteration in the

Caloosahatchee watershed have drastically altered the ecology of the river and estuary.

This has led to loss of SAV, which serves as an indicator of the overall health of the system. The aim of this project was to examine factors affecting the growth of restoration plantings of the freshwater SAV Vallisneria americana in the Caloosahatchee River.

Factors examined included sediment characteristics, planting densities, herbivore exclosure size, and genetic strain of donor plants. Response variables included the number of rosettes and blades, blade morphology, and total plant biomass. Significant differences were found between the two strains in terms of mean number of rosettes and blades. No significant differences were found between the two sites used in Phase I. In comparing the mean final percent frequency of occurrence among sites in Phase II, a significant difference was found with strain. In comparing the final number of rosettes among sites in Phase II, a significant difference was found with site. Significant differences were found with average blade length and average leaf area among the sites in

Phase II. Average blade length, width, and leaf area were significantly different when compared between strains in Phase II. Determining the difference of small versus large exclosures was strictly a qualitative assessment. Differences and similarities were observed when examining transportation and deployment, planting and sampling effort,

x wave action, and herbivory. When examining planting density the results indicate the initial planting density had no effect on the growth of V. americana. Although planting density lacks a vital role in the growth and establishment of V. americana, genetics may account for observed differences. Preparation for a SAV restoration that includes an investigation of the donor plants genetic makeup should be standard practice. Restoration of any species to an extirpated site will involve attempting to find appropriate genetic material often from geographically separated populations. In addition to genetics the site selection is another critical factor when considering a SAV restoration attempt.

xi Chapter 1

Introduction

Global threats to coastal waterways. People have inhabited coastal regions and river shorelines for thousands of years (Conley et al. 2000), benefitting from the food, transportation, energy, and other resources that aquatic environments provided. Today coastlines rank among the most densely populated areas globally (Courrat et al. 2009;

González-Ortegón et al. 2010). These areas have been exposed to heavy exploitation, pollution, and landscape alterations, which threatens their ecological integrity and their ability to continue to provide resources for humanity (Lotze et al. 2006, Barbier et al.

2011).

Importance of submerged aquatic vegetation in coastal waterways. Shallow aquatic ecosystems may include diverse, biogenic-structured benthic habitats (Courrat et al. 2000) such as seagrass meadows, mangrove forests, marshes and oyster reefs. Such habitats are valued for their contributions to biodiversity and ecosystem functions. In the last few decades seagrasses and freshwater submerged aquatic vegetation (SAV) have declined globally (Orth and Moore 1983; Walker and McComb 1992; Short and Wyllie

1996; Kemp et al. 2004) impacting the ecological functions they contribute. SAV beds provide nurseries for multiple fish and invertebrate species (Heck and Thoman 1984;

Bertelli and Unsworth 2014; Unsworth et al. 2014), food and habitat for water fowl

(Rybicki and Landwehr 2007; Perry et al. 2007;) and other animals facilitating a diverse ecosystem (Lubbers et al. 1990; Heck et al. 1995; Kemp et al. 2004; Patrick et al. 2014).

SAV takes up dissolved nutrients (Wigand et al 2001; Kemp et al. 2004; Patrick et al.

2014), oxygenates the water column, and captures suspended sediments helping to

1 control erosion and reduce turbidity (Lubber et al. 1990; Hemminga and Duarte 2000;

Patrick et al. 2014). In addition to its ecological importance, SAV supports both commercial and recreational fisheries, providing substantial economic benefits (McBride and Bartleson, 2010). In Florida’s Caloosahatchee River and Estuary, SAV is an integral component of the system (Kraemer et al., 1999; Bartleson, 2014).

Caloosahatchee River and Estuary. The Caloosahatchee River and Estuary are located on the southwest coast of Florida (Figure 1). The river originally had its headwaters near Lake Hicpochee and a tidal influence as far as the town of La Belle

(Barnes, 2005). The once meandering river was channelized and made into a canal (C-43) to aid in flood control, land reclamation, and navigation (Barnes, 2005). The canal extended the river east to Lake Okeechobee, creating a new outlet for the lake. Due to these anthropogenic alterations the Caloosahatchee River now has two main sources of freshwater: its historic watershed, and Lake Okeechobee (Doering and Chamberlain,

1999). Discharges from Lake Okeechobee are required for flood control for lands south of the lake, and freshwater supply for Lee County (Doering and Chamberlain, 1999).

These sources of freshwater affect the total discharge, quality and quantity of water released into the river and estuary (Doering and Chamberlain, 1999). Water quality, quantity, and movement are further affected by a total of three lock and dam structures,

S-77, S-78 & S-79, built along the C-43 canal to control flow and stage height (Barnes,

2005) (Figure 1).

Figure 1: Map of Caloosahatchee River and watershed showing major basins and water management structures. (SFWMD, 2014).

Completed in 1966, S-79, also known as Franklin Lock and Dam, is the furthest downstream structure and serves primarily as a salinity barrier to the river (Doering et al.,

2002; Barnes, 2005). Annual freshwater discharge from S-79 to the estuary is enough to fill the entire volume of the estuary over eight times per year (Doering and Chamberlain,

1999).

Currently the Caloosahatchee River extends 67 km from Lake Okeechobee to the

Franklin Lock and Dam (S-79) where the estuary begins and continues roughly 40 km ending at Shell Point (Doering et al. 2002). The freshwater portion varies in width from

50 to 130 m with depths ranging from 6 to 9 m (Barnes, 2005). Remnants of the river’s natural meandering path exist as oxbows (Barnes, 2005). The upper portion of the estuary has a width of 160 m and widens to 2500 m downstream with an average depth of 6 m and 1.5 m, respectively (Scarlatos, 1988; Barnes, 2005). Land and canal development in

3 the Caloosahatchee watershed coupled with water management policy have changed the natural hydrology and negatively affected the ecology of the estuary (Barnes, 2005).

These ecological stressors include altered hydrology, altered estuarine salinity, physical alterations to the estuary, boating and fishing pressure, and elevated levels of nutrients, toxins, and dissolved organics (Barnes, 2005).

SAV in the Caloosahatchee River and Estuary serves as an indicator of the overall health of the system due to its sensitivity to major changes in long-term water quality

(McBride and Bartleson, 2010). Historically, the lower portion of the estuary was dominated by the seagrasses Thalassia testudinum and Halodule wrightii, and the middle and upper portions were comprised of the euryhaline SAV Ruppia maritima (widgeon grass) and the freshwater SAV Vallisneria americana (tape grass) (Chamberlain and

Doering, 1999; Kraemer et al., 1999). Prior to construction of the S-79 lock and dam, estuarine and riverine SAV would have mingled along a gradual gradient. Now, however, they are disjointed, i.e., the conditions experienced by SAV above the lock and dam can be very different from those in the estuary.

Ecology of Vallisneria americana. Vallisneria americana Michaux (American wild celery, tape grass), a native to eastern North America, can be found from southern

Canada south to Texas and Florida (Catling et al., 1994; McFarland, 2006). The USDA

NRCS (2014) recognizes V. americana’s presence in forty-two of the lower forty-eight states (Figure 2). Additionally, V. americana has been documented in Cuba, Mexico,

Guatemala, Honduras, east and southeast Asia, Oceania, and Australia (Catling et al.

1994; McFarland 2006).

Figure 2: Map depicting Vallisneria americana distribution. ("Plants Profile for Vallisneria americana (American eelgrass)", 2016)

Morphological description. V. americana is a dioecious, perennial, submerged aquatic macrophyte, found in fresh and slightly brackish environments (Lovett-Doust and

LaPorte, 1991; Catling et al., 1994). The ribbon-like leaves can grow in excess of two meters depending on the lotic properties and depth of the water (Lovett-Doust and

LaPorte, 1991; Catling et al., 1994; McFarland, 2006). The leaves are toothed along the margins and heavily veined with a distinct mid longitudinal stripe, and blunt round tips.

They form rosettes from short vertical stems that send out rhizomes and stolons (Catling et al., 1994; McFarland, 2006). The roots form fibrous unbranched clusters underneath the stolons (Catling et al., 1994; McFarland, 2006).

5 Life cycle and reproduction. V. americana reproduces sexually but is capable of extensive clonal growth through the formation of stolons (Lovett-Doust and LaPorte,

1991; Doering et al., 2001). The importance of vegetative reproduction is evidenced in the variety of asexual propagules created (McFarland, 2006). Northern populations rely on the development of a dormant winter bud to survive the winter (Titus and Hoover,

1991; Doering et al., 2001). However, in South Florida, populations of V. americana do not completely die back in winter and growth may be observed year round (Dawes and

Lawrence, 1989; Doering et al., 2001). If favorable conditions are met V. americana exhibits a seasonal pattern of growth with the greatest biomass occurring in late summer, flowering in the late summer-early fall, and a decline in biomass during the winter

(Bortone and Trupin, 2000; Doering et al., 2001). Although most individuals in a population appear to remain vegetative, an elaborate sexual process occurs in this species

(Lovett-Doust and LaPorte, 1991).

Sexual reproduction occurs in the late summer-early fall in the Caloosahatchee estuary (Doering et al., 2001). Once the pistillate flowers have formed within their spathe they elongate to the water’s surface via a peduncle. On a shorter peduncle, attached near the base of the male plant, as many as 2000 staminate flowers fit into a spathe

(McFarland, 2006). The staminate flowers are released via an abscission zone, float to the water’s surface, and enter a depression created by the pistillate flower where the transfer of pollen to the stigma occurs (Cox, 1993; Catling et al., 1994; McFarland, 2006). After pollination the stalk of the pistillate flower coils drawing the cylindrical fruit downwards

(McFarland, 2006). The cylindrical capsule ranges between five and fifteen centimeters and contains hundreds of seeds nested in a gelatinous medium (McFarland, 2006).

6 Habitat requirements. V. americana can survive under broad environmental conditions and various water chemistry regimes (Korschgen and Green, 1988). These conditions include high turbidity (Davis and Brinson, 1980), low light levels (Titus and

Adams, 1979), a pH range of 5.1-7.2 (Titus and Hoover, 1991; Catling et al., 1994), and salinities between 3-5ppt (Steenis, 1970; Catling et al., 1994) and up to 8ppt but with limited growth (Boustany et al., 2010). Hunt (1963) found that V. americana thrives best in silty clay but can establish in various substrates that allow the roots to penetrate and anchor. Catling et al. (1994) cited several studies (Meyer et al., 1943; Hunt, 1963;

Wilkinson, 1963; Stodola, 1967; Titus and Adams, 1979; Barko et al., 1981) indicating a temperature range of V. americana typically between 18 and 50 ̊C.

V. americana in the Caloosahatchee River and Estuary

Distribution and Status of V. americana in the Caloosahatchee River and

Estuary. Hoffacker (1994) estimated V. americana coverage at 1000 acres with 60 percent between Beautiful Island and the Fort Myers Bridge. In 1998, when SAV monitoring of the Caloosahatchee River and Estuary began, V. americana was abundant

(Mazzotti et al., 2007; Douglass, 2014). Dense beds of V. americana were documented from Fort Myers to Beautiful Island and east to the S-79 lock and dam. A drought spanning 1999-2001 coupled with high salinity levels depleted V. americana from the estuary (Bartelson, 2014; Douglass, 2014). A partial recovery was documented in 2004 followed by another high salinity event and die off in 2006. V. americana was absent throughout 2007-2009 with a mini reoccurrence in 2010 followed by another decimation in 2011 (Douglass, 2014).

7 V. americana studies in the Caloosahatchee River and Estuary (CRE). The

South Florida Water Management District (SFWMD) has determined V. americana to be a “valued ecosystem component” (VEC) and is used to determine its MFL (minimum flows and levels) regime to support V. americana in the upper estuary ( SFWMD 2001).

McBride and Bartleson (2010) identified a high water column light attenuation coefficient, and surface water run off (causing algal blooms and further light attenuation) as impediments to SAV recovery. Furthermore, several studies (Kraemer et al., 1999;

Doering et al., 2001; Doering et al., 2002; French et al., 2003; Boustany et al., 2010;

Bartleson et al., 2014) indicate a delicate relationship between salinity, temperature, and subsequent success of V. americana americana transplants. The varying flow regimes found in the Caloosahatchee River and Estuary are the direct result of large influxes of freshwater from the watershed and Lake Okeechobee (McBride and Bartleson, 2010).

These releases, or lack of releases, cause the light and salinity to fluctuate and ultimately combine to compress or eliminate the oligohaline environment favored by V. americana

(Bartleson, 2014). The expansion or contraction of viable habitat for V. americana below

S-79 occurs at different temporal and spatial scales; both intra-annually (dry/wet season) and interannually (dry/wet years). By comparison, the freshwater V. americana habitat above S-79 is relatively stable. Successful establishment of V. americana above S-79 has the potential to create an upriver seed source for the estuarine population and to serve as a possible local source of donor plant material for future restoration efforts in the estuary.

Although abiotic factors are important to the success of SAV restoration efforts

(Rybicki and Carter, 1986; Kraemer et al., 1999; French and Moore, 2003; Kemp et al.,

2004; McFarland and Shafer, 2008; Moore et al., 2010), herbivory is also important

8 (Ceilley et al., 2003, 2009, 2013; Hauxwell et al., 2004; Moore et al., 2010). In 2002

Ceilley and Bortone identified herbivory as an impediment to SAV recovery in the CRE

(personal communication). Ceilley et al. (2003) documented the successful use of exclosures to aid in the restoration of V. americana. Similarly, Hauxwell et al. (2004) demonstrated that recently transplanted, unprotected V. americana transplants in the

Crystal River were decimated by grazing manatees. Adjacent plots protected by herbivory exclosures survived, increased in biomass, flowered, and reproduced clonally

(Hauxwell et al., 2004). These exclosures allow newly transplanted SAV to establish without the added pressure of grazing. Manatees, turtles, muskrats, and waterfowl are documented as negatively affecting transplants in other studies (Carter and Rybicki,

1985; Hauxwell et al., 2004).

Research Objectives

Although the freshwater and estuarine environments of the Caloosahatchee River and Estuary are separated by the S-79 Lock and Dam, some connectivity still exists.

Therefore it is the aim of this project to focus restoration efforts in the freshwater portion of the Caloosahatchee and determine relevant factors affecting successful establishment.

The successful establishment of V. americana transplants in the freshwater portion of the

Caloosahatchee may provide an upriver seed source and local source of donor material for future restoration efforts in the estuary. Lastly, the methods used in this study may provide guidance for future restoration projects in the estuary.

9 Objective 1. Determine the effect of exclosure size (1 m2 vs. 12 m2) on growth of

V. americana. This objective aims to investigate the ability of fewer large exclosures versus several smaller exclosures to establish beds of V. americana. Additionally, the objective will explore differences in transportation and deployment, ability to plant and sample V. americana transplants, resiliency to wave action, and efficiency in excluding herbivores.

Objective 2. Determine the effect of two planting densities on the growth of V. americana. Manual methods of restoration are time consuming and labor intensive. To minimize cost and maximize efficiency this objective aimed to determine if planting two densities, ten and twenty rosettes, would yield the same desired outcome.

Objective 3. Determine the difference of growth between plants from two donor sites, possibly reflecting two genetic strains of V. americana. To increase the likelihood of a successful restoration attempt the genetics of donor plants should be considered. A greater genetic variation among donor plants may increase the capacity to prove resilient to dynamic changes in both the short-term and long-term.

Objective 4. Determine the effect of sediment characteristics (organic content and particle size) on growth of V. americana. The sediment characteristics of donor and restoration sites may differ. Understanding the similarities and differences of each could provide insight towards selecting potential restoration locations.

10 Chapter 2

Methods

Overview of Study Design. Vallisneria americana was planted at several restoration sites in the Caloosahatchee River, testing the effects of different genetic strains, planting techniques, sediment types, and herbivore exclusion methods on establishment success. Phase I of the project focused on examining the effects of smaller exclosures, different planting densities and donor strains, and sites on the establishment of Vallisneria americana in the Caloosahatchee River. Phase I was meant to serve as a pilot study for future projects and subsequent funding. After the Phase I results were analyzed a second Phase was implemented to investigate the use of larger exclosures, uniform densities, and different genetic strains at four new locations in the river.

11 Study location. The Caloosahatchee River and Estuary are located on the

Southwest coast of Florida (Figure 3). All V. americana transplant sites were located in the freshwater portion of the Caloosahatchee, east of the Franklin Lock and Dam (S-79),

26.7217° N, 81.6939° W. (Figures 4 and 5, respectively).

Figure 3: Site map depicting the Caloosahatchee River and estuary. The Franklin Lock and Dam (S-79) denotes the beginning of the estuary.

12 Figure 4: Site map for Phase I depicting sites 1, 2, and 3. Sites 1 and 2 are the locations for Vallisneria transplants. Site 3 is a reference water quality site.

Figure 5: Site map for Phase II illustrating sites 1, 2, 3, 4, 5, 6, and 7. Sites 1 and 2 are from Phase I and were monitored throughout Phase II. Sites 4, 5, 6, and 7 are the Phase II V. americana transplant sites. Site 3 from Phase I remained a water quality reference site throughout Phase II.

13 General procedures.

Donor V. americana. The V. americana for Phase I and II was collected locally from Lake Kennedy in Cape Coral Florida and Lake Trafford in Collier County, Florida

(Figure 6). The donor sites offered significantly different genetic strains (Lyn Gettys, personal communication) with Lake Kennedy being dominated by a possible clonal population. After a multi-year restoration project V. americana began to establish throughout Lake Trafford providing both male and female plants (Ceilley et al. 2013, unpublished). V. americana was harvested from both sites three days prior to transplanting. Transplants were stored in coolers and five gallon buckets with ambient water and transported to Florida Gulf Coast University (Figure 7). Transplants were then processed to remove non-native snails, clams, and invasive plants. The V. americana was stored outside for no longer than 72 hours before being planted. During this time all containers remained filled with ambient water and covered with a shade awning to lessen the exposure to direct sunlight.

Figure 6: V. americana donor sites Lake Kennedy, Cape Coral, Florida (Top right) and Lake Trafford, Collier County, Florida (Bottom right).

Figure 7: Florida Gulf Coast University vessel with coolers and five gallon buckets used to transport V. americana.

15 Exclosures and planting method. The use of exclosures has been documented in several SAV restoration efforts (Sheridan et al., 1998; Tatu, 2006; Burkholder et al.,

2013). Multiple V. americana restoration projects have incorporated exclosures (Carter and Rybicki, 1985; Ceilley et al., 2003, 2009, 2013; Hauxwell et al., 2004; Moore and

Jarvis, 2007; McBride and Bartleson, 2010; Moore and Jarvis, 2010). The exclosures implemented in this project were made of PVC and built at two different dimensions. The specifics of the exclosure dimension and materials used are detailed below. By taking readings every 10 cm. Transplants for both phases consisted of mature rosettes with a robust root structure. Rosettes were planted bare root in a small depression in the sediment by hand or in some cases a hand trowel (Smart et al., 1998; Smiley and Dibble,

2006; Moore and Jarvis, 2010). As part of the study Phase I and II implemented different exclosure designs and planting methods and therefore will be addressed separately.

Sediments.

Soil sample collection and processing. Sediment samples from the six transplant sites and two donor sites were collected. At each site three randomly selected sediment samples were collected using a 20 cm long hand soil auger for a total of twenty-four samples. The sediment sample was placed in a Ziploc bag and stored in a cooler until processed. These samples were analyzed for total organic content and sediment grain size.

Total organic content (TOC). Methods used to determine TOC were adopted from (Schumacher, 2002). To prevent any altering of the samples and preserve the organics the samples were stored in a freezer at -10 ̊C until analysis could be completed.

16 Between fifteen and nineteen grams of each sample were homogenized and placed in a drying oven at 105 ̊C for at least 16h or until no further change in mass was observed.

They were then removed, placed in a desiccator to cool, and the mass was recorded. The moisture content was calculated using the following formula:

Moisture Content, percent = [(A – B) x 100]/B

where:

A = as-received test specimen, g, and B = mass of the oven-dried specimen, g.

The sample used to determine the moisture content was then used to determine the ash content. The sample was placed in a muffle furnace with a gradual increase in temperature to 440 ̊C until the sample was completely ashed. The sample was covered, cooled in a desiccator and weighed. The ash content was calculated as follows:

Ash Content, percent = (C x 100)/B

where:

C = ash, g, and B = oven-dried sample, g.

The amount of organic matter was determined by:

Organic matter, percent = 100.0 – D

where:

D = ash content, percent.

The sediments from all sites were compared for mean TOC using ANOVA.

Sediment grain size analysis.

17 To examine the potential differences in sediment composition among the sites a sub sample of roughly 50 grams was measured from each of the twenty-four samples.

Samples were processed using a W.S. Tyler Ro-Tap RX-812 Coarse Sieve Shaker with

30 cm diameter brass sieves: specifically a No. 10, No. 230, and a pan to determine gravel, coarse sand, and the fraction of silt/clay, respectively. Each sample was subjected to a sieving interval of ten minutes at 280 oscillations per minute. The sediment samples were compared for mean gravel, coarse sand, and silt/clay percentages using ANOVA.

Water quality. Water quality parameters were measured using a Yellow Spring

Instruments, Inc. (YSI, Inc., Yellow Spring, Ohio) model 85 water quality sonde and included temperature (̊C), dissolved oxygen (mg 1-1), specific conductance (μS) and

-1 salinity (psu). Secchi depth and light attenuation, or Kd (m ) were measured using a

Secchi disc and Li-Cor Model 1400 with 2 pi and 4 pi radiometers. Water quality parameters during Phase I were measured weekly, June 9th, 2011 through August 25,

2011, when possible. During Phase II water quality parameters for sites 1 through 7 were collected approximately weekly from July 20, 2012 through November 21, 2012.

Phase I. This phase included the planting of two strains of Vallisneria americana in replicated small exclosures at two locations and at two planting densities. A total of twenty-four 1-m2 exclosures were assembled, deployed, and planted.

Exclosure design, installation, and planting. Each 1 m2 exclosure was made of

Tenax™ poultry fence with 1 inch hexagonal mesh attached to four 1.5 meter long ¾’’ diameter schedule 40 PVC poles (Figure 8). The poles were attached with elbows and a square PVC frame top. Tenax™ mesh measured 1.25 meters in height and attached to

18 each PVC pole with cable ties. One half meter of each pole was pushed down into the substrate along with the mesh to prevent potential herbivory. To prevent birds and turtles from entering the exclosure from above, a 1-m2 piece of thinner weight Vexar mesh was attached to the top of the exclosure. A metal tag was attached to each exclosure for identification, along with a permanent marker identifying the treatment. To identify the restoration boundaries additional PVC pipe with reflective tape was installed along the perimeter of the study site. Exclosures were partially constructed on land prior to deployment and finished on site before being planted with V. americana.

Figure 8: Diagram of the 1 m2 exclosure design for Phase I (not drawn to scale).

19 Plants of each genetic strain (Lake Trafford strain and Lake Kennedy strain) were established at two densities (10 and 20 rosettes per exclosure), at each of two sites (site 1 and site 2), for a total of eight unique treatments (Figure 9). Each treatment had three replicates, so there were a total of 24 planting plots for Phase I. Plants were hand planted inside a 0.09 m2 frame using a hand trowel. Sediment was pressed back around the roots as the trowel was removed to secure each plant’s roots in the sediment. Exclosure cages were then place over the outside of the planting square (to cover all plants inside the grid) and then the planting square was removed carefully. The exclosure cage was then pushed down until the mesh was flush with the sediment surface and then pounded with a rubber mallet on each leg into the sediment. To help ensure exclusion of herbivores, the mesh area between each leg of the cage was then secured to the bottom with three-hole bricks and heavy-duty plastic cable ties (UV resistant). Planting of Site 1 was completed on

June 1, and Site 2 was planted on June 2, 2011 (Figure 11).

Figure 9: Graphic depicting the experimental design used in Phase I. Pictured are sites one and two. At each site two densities were planted, 20 rosettes and 10 rosettes for each donor source (Lake Trafford and Lake Kennedy) in replicates of three.

Figure 10: Site two installment. Quantitative plant sampling. On August 25th, 2011 quantitative plant sampling was conducted at site 1 and 2. To capture a representative sample the exclosures were numbered 1-12 and sectioned into nine, 0.33 meter quadrats and assigned a random (x, y) coordinate (Figure 11). Once the coordinate was determined, a 0.11 m² PVC quadrat was placed over that section of the grid and secured on the bottom using metal stakes. All V. americana rosettes within the quadrat were harvested, bagged, and stored in a cooler.

Above and below ground biomass were separated and dried at 60 ̊C until a constant mass was achieved (Appendix A). Any plant material originating outside the quadrat was excluded from the sample. On August 26th, 2011 all V. americana samples were processed at Florida Gulf Coast University. Within each sample the number of rosettes

21 and blades were enumerated and the five longest blades from each sample were measured for length and width.

Figure 11: Depicted is an example of the exclosure coordinate system. The X marks a sampling coordinate of (2, 2). Monitoring. During Phase II, we continued to monitor the small exclosures from

Phase I on a monthly basis. On August 20, September 24 and again on November 21,

2012 the small exclosures were inspected by removing the top mesh and then visually and tactilely assessing percent coverage by V. americana. Cover was estimated and recorded into one of four categories based on approximate cover where; 0 = none present,

1 = 1-33 percent cover, 2 = 34-66 percent cover and 3 = 67-100 percent cover. The graphs can be found in (Appendix B). The small exclosures were also searched for herbivores including turtles and snails.

Data analysis. Treatment effects (strain, density, and site) of each measured variable (number of rosettes, number of blades, biomass, blade length, blade width, and leaf area) were determined using a Three-Way Analysis of Variance (ANOVA; SPSS

Version 22 for Windows, IBM).

22 Phase II.

Exclosure design, installation, and planting. Phase II incorporated a larger exclosure design (Figure 12). Each exclosure was again made of Tenax™ poultry fence with 1 inch hexagonal mesh attached to four 1.5 meter long ¾’’ diameter schedule 40

PVC poles. One half meter of each pole was pushed down into the substrate along with the mesh to prevent potential herbivory. On May 22, 2012 and May 24, 2012 four large exclosures, measuring 6 m x 2 m, were deployed at each site (4, 5, 6 and 7) in the

Caloosahatchee River. Each exclosure was planted with Vallisneria americana from

Lake Trafford and Lake Kennedy which had been identified by Dr. Lyn Gettys at the

University of Florida Center for Aquatic Plants as different genetic strains. For each strain, 60 plants were planted (per cage) using a meter-squared quadrat (ten plants per square meter/low density planting from Phase I) to ensure an even planting effort (Figure

13). On June 8th, 2012 the four large exclosures were repaired with additional schedule

40 PVC supports and 3/8” rebar attached to the bottom of the Vexar™ mesh.

Figure 12: A large exclosure used in Phase II of the study.

Figure 13: An overhead diagram depicting the phase II large exclosure and planting methodology (not drawn to scale).

24 Monitoring. On August 20, September 24, November 21, and December 20, 2012 the large cages were inspected for herbivores and for assessing percent cover by V. americana in each cage. The large cages were opened to allow a snorkeler into the cage.

Visibility and photographic documentation was problematic due to poor water clarity and occasional boat wakes that stirred up sediments on the bottom. Therefore we decided to use a tactile and visual method for assessing percent cover and growth. Using a one- meter square quadrat, subdivided into nine subplots with nylon line, the quadrat was placed on the river bottom beginning at the upper left corner of the cage and moving back and forth from one end of the cage to the other until the entire cage bottom was assessed.

When the quadrat was placed on the river bottom each subplot was inspected for the presence/absence of V. americana rosettes. Frequency of occurrence (x/9) of V. americana rosettes from the subplots was reported verbally by the diver to the recorder outside the cage. Then a random number between 1 and 9 was given to the diver for counting the total number of rosettes within the randomly assigned subplot. In this manner, an estimate of percent cover for each square meter of the cage was recorded along with a subsample of shoot density for each quadrat (Smiley and Dibble, 2006). This method allowed us to obtain a non-destructive but quantitative measurement of both the percent coverage and shoot density for each strain (half of each cage). Additionally, at the end of Phase II each site was sampled to collect plants of each strain. A random sampling effort was used to collect V. americana rosettes. Rosettes ranged from 10 to fifteen per sample. For each rosette the number of blades, blade lengths, blade widths, and leaf areas were measured.

25 Snail monitoring. At the same time the quadrats were being assessed for presence/absence of rosettes, the diver searched by hand for non-native apple snails,

Pomacea insularum. In addition, the exclosure itself was searched for presence of apple snail egg clusters. The total number of adult apple snails and egg clusters was recorded at each location and cage. Egg clusters were removed and discarded into the river. Adult snails were thrown as far as possible onto land to minimize likelihood of survival.

Data analysis. Treatment effects (strain and site) of each variable (number of rosettes, number of blades, biomass, blade length, blade width, and leaf area) were determined using a Two-Way Analysis of Variance (ANOVA; SPSS Version 22 for

Windows, IBM).

26 Chapter 3 Results Sediment characteristics. Total organic content and grain size analysis. A total of twenty-four sediment samples were collected from the six planting sites and the two donor sites, Lake Kennedy and Lake Trafford. The mean percent of total organic content (TOC) of the samples is shown in Fig 15. The mean percent total organic content among the planting and donor sites were significantly different (Figure 14, p =

0.001). The average percent of sand, clay/silt, and gravel at the donor and restoration sites are depicted in Figure 15. A significant difference was found among sites when comparing the mean percent silt/clay fraction (Figure 16, p=0.025).

20.00

15.00 a

c c c,b 10.00 c,b c,b

b c,b Content

5.00 Mean % OrganicTotal

0.00 Lake Kennedy Lake Trafford Site 1 Site 2 Site 4 Site 5 Site 6 Site 7 Figure 14: Comparison of mean percent total organic content among donor and transplant sites. Error bars represent standard deviation. A significant difference was found (p = 0.001). Identical letters indicate no significant difference (p > 0.05) from LSD post-hoc test (Appendix C).

27 Average % Sand Average % Clay/Silt Average % Gravel 100 98 96 94 92 90

88 Average% 86 84 82 80 Lake Lake 1 2 4 5 6 7 Kennedy Trafford Site

Figure 15: Graph depicting average percent sand, clay/silt, and gravel for the donor and restoration sites.

10 a 8 a,c 6 a,b,c

Percent Silt/Clay b,c 4 b,c b,c b 2 b 0 Lake Kennedy Lake Trafford Site 1 Site 2 Site 4 Site 5 Site 6 Site 7 Figure 16: Comparison of mean percent silt/clay among donor and restoration sites. Error bars represent standard deviation. A significant difference was found (p= 0.025). Identical letters indicate no significant difference (p > .05) from LSD post-hoc test (Appendix C).

28 Water quality. Water quality results measured for each sampling period at each planting site are shown in (Figure 17). In general, water quality followed similar patterns among the sites throughout the sampling period. In addition, the light attenuation (K m-1) was calculated for each site during the sampling effort (Figure 18).

A Site 1 Site 2 Site 3 (Reference) Site 4 Site 5 Site 6 Site 7 34

32 C) ° 30 28 26 24 Temperature Temperature ( 22 20 3/6/2012 4/6/2012 5/6/2012 6/6/2012 7/6/2012 8/6/2012 Date

B Site 1 Site 2 Site 3 (Reference) Site 4 Site 5 Site 6 Site 7 10 9 8 7 6 5

Dissolved Oxygen (mg/L) 4 3/6/2012 4/6/2012 5/6/2012 6/6/2012 7/6/2012 8/6/2012 Date

29 C Site 1 Site 2 Site 3 (Reference) Site 4 Site 5 Site 6 Site 7 1000

) 900 μS 800

700

600 Conductance (

500 3/6/2012 4/6/2012 5/6/2012 6/6/2012 7/6/2012 8/6/2012 Date

D Site 1 Site 2 Site 3 (Reference) Site 4 Site 5 Site 6 Site 7 0.55 0.5 0.45 0.4

0.35 Salinity (ppt) 0.3 0.25 3/6/2012 4/6/2012 5/6/2012 6/6/2012 7/6/2012 8/6/2012 Date

E Site 1 Site 2 Site 3 (Reference) Site 4 Site 5 Site 6 Site 7 9 8.5 8

7.5 pH 7 6.5 6 3/6/2012 4/6/2012 5/6/2012 6/6/2012 7/6/2012 8/6/2012 Date

30 F Site 1 Site 2 Site 3 (Reference) Site 4 Site 5 Site 6 Site 7 2

1.5

0.5Secchi Depth(m) 3/6/2012 4/6/2012 5/6/2012 6/6/2012 7/6/2012 8/6/2012 Date Figure 17: Water quality results for sites 1-7. Included in the figure are measurements for A. temperature (°C), B. dissolved oxygen (mg/L), C. conductance (μS), D. salinity (ppt), E. pH, and F. Secchi depth (m).

Site #1 Site #2 Site #3 (Reference Site) Site #4 Site #5 Site #6 Site #7

5.0

) 1 - 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

Light Light AttenuationK (m 0.5

Date

Figure 18: Calculated K (m-1) values for each site through the sampling period.

Objective 1: Effect of two planting densities on the growth of V. americana. In comparing the two planting densities in Phase I, no significant differences were found with the final mean number of rosettes, mean number of blades, mean blade length, mean blade width, mean leaf area, or total biomass (Figure 19). ANOVA tables for each analysis can be found in (Appendix D).

Low (10) High (20) 16.00

14.00

12.00

10.00

8.00

6.00 Mean Biomass MeanBiomass (g) 4.00

2.00

0.00 Figure 19: Planting density effect on mean biomass (g). Error bars represent standard deviation. No significant difference was found (ANOVA, p = .811). Objective 2: Difference of growth between two genetic strains of V. americana.

Significant differences were found between the two strains in Phase I with mean number of rosettes (Figure 20, p = .022) and mean number of blades (Figure 21, p = .018). No significant differences were found with mean blade length, mean blade width, mean leaf area, or total biomass. ANOVA tables for each analysis can be found in (Appendix D).

Lake Kennedy Lake Trafford 12.0

10.0

8.0

6.0

4.0 Mean Number MeanNumber Rosettesof 2.0

0.0 Figure 20: Comparison between the two strains in Phase I. Error bars represent standard deviation. A significant difference was found with mean number of rosettes (ANOVA, p = 0.022).

Lake Kennedy Lake Trafford 100

30 Mean Number MeanNumber Bladesof 20

0 Figure 21: Comparison between the two strains in Phase I. Error bars represent standard deviation. A significant difference was found with mean number of blades (ANOVA, p = 0.018).

33 Objective 3: Effect of site on the growth of V. americana. No significant differences were found between the two sites used in Phase I for the mean number of rosettes, mean number of blades, mean blade length, mean blade width, mean leaf area, or total biomass (Figure 22). ANOVA tables for each analysis can be found in (Appendix

D).

Site 1 Site 2 18.00 16.00 14.00 12.00 10.00 8.00 6.00

Mean Biomass MeanBiomass (g) 4.00 2.00 0.00

Figure 22: Planting site effect on mean biomass (g). No significant difference was found (ANOVA, p = .505). Phase II. For Phase II of this study we monitored the status of the planting three times. The trends through time for mean percent frequency of occurrence

(presence/absence within each subplot) and mean number of rosettes are shown in

Figures 23, 24, 25, and 26. The mean percent frequency of Lake Kennedy at sites 6 and 7 declined while Lake Kennedy material at sites 4 and 5 remained constant. In general, the

Lake Trafford plants at sites 4, 5 and 7 declined and at site 6 remained constant. The mean number of Lake Kennedy rosettes decreased at sites 5, 6 and 7. An increase in the mean number of rosettes was observed at site 4. Lake Trafford rosettes at sites 4, 6 and 7 increased through time while rosettes at site 5 displayed a decrease.

34 Site 4 Lake Kennedy Site 5 Lake Kennedy Site 6 Lake Kennedy Site 7 Lake Kennedy 130% 120% 110% 100% 90% 80%

Mean % Frequency 70% 60% Sample 1 Sample 2 Final Sampling Event

Figure 23: Lake Kennedy V. americana transplant mean percent frequency (presence/absence in the subplots) through time at sites 4, 5, 6 and 7.

Site 4 Lake Trafford Site 5 Lake Trafford Site 6 Lake Trafford Site 7 Lake Trafford 110% 105% 100% 95% 90% 85% 80%

Mean % Frequency 75% 70% 65% Sample 1 Sample 2 Final Sampling Event

Figure 24: Lake Trafford V. americana transplant mean percent frequency (presence/absence in the subplots) through time at sites 4, 5, 6 and 7.

35 Site 4 Lake Kennedy Site 5 Lake Kennedy Site 6 Lake Kennedy Site 7 Lake Kennedy 40 30 20 10 0 Sample 1 Sample 2 Final -10 Sampling Event

Mean Number MeanNumber Rosettesof Figure 25: Lake Kennedy mean number of rosettes per 0.11m2 sample through time at sites 4, 5, 6 and 7. Initial planting density was 1.1 rosettes per subplot.

Site 4 Lake Trafford Site 5 Lake Trafford Site 6 Lake Trafford Site 7 Lake Trafford 25 20 15 10 5 0 Sample 1 Sample 2 Final

Mean Number MeanNumber Rosettesof -5 Sampling Event

Figure 26: Lake Trafford mean number of rosettes per 0.11m2 through time at sites 4, 5, 6 and 7. Initial planting density was 1.1 rosettes per subplot. All statistical analysis were performed on the data from the final monitoring. In comparing the mean final percent frequency of occurrence among sites in Phase II, a significant difference was found with strain (Figure 27, p = 0.022). No significant difference was found among sites. In comparing the final number of rosettes among sites in Phase II, a significant difference was found with site (Figure 28, p = 0.003). No significant difference was found with strain. ANOVA tables for each analysis can be found in (Appendix E).

120

100

MeanPercent Frequency Occurrenceof 20

0 Lake Kennedy Lake Trafford

Figure 27: Final V. americana transplant mean percent frequency of occurrence through time among sites in Phase II. Error bars represent standard deviation. A significance was found between the strains (ANOVA, p = 0.022).

120

100

FinalAverage Rosettesof 20

0 Site 4 Site 5 Site 6 Site 7

Figure 28: Final mean number of rosettes through time among sites in Phase II. Error bars represent standard deviation. Sites were significantly different (ANOVA, p = 0.003).

37 Identical letters indicate no significant difference (p > .05) from LSD post-hoc test (Appendix F).

Objective 1: Effect of site on growth of V. americana. In comparing among the sites in Phase II, significant differences were found with average blade length (Figure 29, p = 0.001) and average leaf area (Figure 30, p = 0.006).

40 a a,b 30 b b 20

AverageBlade Length(cm) 10

0 Site 4 Site 5 Site 6 Site 7

Figure 29: Comparison among sites in Phase II. Error bars represent standard deviation. A significant difference was found with average blade length (ANOVA, p = 0.001). Identical letters indicate no significant difference (p > .05) from LSD post-hoc test (Appendix, H).

90 80 70 60 50 a a,b

40 a,b 30 b

20 AverageLeaf (cm2) Area 10 0 Site 4 Site 5 Site 6 Site 7

Figure 30: Comparison among sites in Phase II. Error bars represent standard deviation. A significant difference was found with average leaf area (ANOVA, p = 0.006). Identical letters indicate no significant difference (p > .05) from LSD post-hoc test (Appendix G).

Objective 2: Effect of strain on V. americana growth. Significant differences were found with average blade length (p = 0.023), average blade width (p = 0.034) and average leaf area (p = 0.018), in comparing between strains for Phase II (Figures 31, 32, and 33, respectively).

AverageBlade Length(cm) 10

0 Lake Kennedy Lake Trafford

39 Figure 31: Comparison between strains in Phase II. Error bars represent standard deviation. A significant difference was found with average blade length (ANOVA, p = 0.023).

1.8 1.6 1.4 1.2 1 0.8 0.6

0.4 AverageBlade (cm) Width 0.2 0 Lake Kennedy Lake Trafford

Figure 32: Comparison between strains in Phase II. Error bars represent standard deviation. A significant difference was found with average blade width (ANOVA, p = 0.034).

20 AverageLeaf (cm2) Area 10

0 Lake Kennedy Lake Trafford

Figure 33: Comparison between strains in Phase II. Error bars represent standard deviation. A significant difference was found with average leaf area (ANOVA, p = 0.018).

40 Chapter 4 Discussion

Limitations of the study.

Seasonal and inter-annual variability. The time frame in which this study was conducted was not long enough to fully capture seasonal variation or inter-annual variability. To gain a better understanding of the V. americana transplants and the ecosystem as a whole the study should have spanned a year at minimum. Monitoring the transplants over the course of a year would have provided insight into the seasonality of the ecosystem. The Caloosahatchee River and Estuary are subject to varying environmental conditions during the year depending on the season. During winter months and or droughts the oligohaline portion of the estuary may shrink or become non-existent all together as it depends on regular releases from S-79 to maintain previously established minimum flow levels. During the summer months releases from

Lake Okeechobee and run-off from the watershed increase both flows and nutrient loading to the river and estuary. This study would also have benefited from a more complete understanding of the dynamics associated with inter-annual variability. Moore et al. (2010) investigated the role of habitat and herbivory on the restoration of freshwater angiosperms that spanned seven years and noted the importance of annual differences.

Additionally, regardless of the time in which the study was conducted, the exclosures used to protect the V. americana transplants should have remained in the field and additional monitoring implemented. Instead the study concluded and the exclosures were removed. The use of mesh exclosures to protect the plants from herbivory is critical and long-term protection may be necessary to establish founder colonies to withstand

41 initial grazing pressures (Moore et al., 2010). Lastly, after the cages were removed continued monitoring could have provided important data regarding the resilience of the newly established V. americana beds to herbivory.

Site selection. When restoring a damaged or lost ecosystem several factors may affect the success of the project. The most important aspect of a restoration project is site selection (Fonseca et al., 1998; Shafer and Bergstrom, 2008). Often the underlying reason(s) for a failed attempt at restoration stems from selection of sites that are unsuitable for the growth and survival of the target species (Fonseca et al., 1998; Shafer and Bergstrom, 2008). Aquatic ecologists have introduced key criteria to aid in the selection of sites based on the probability of SAV restoration success: the historic presence/absence of SAV, water depth and light availability, water quality and water column nutrient concentrations, sediment quality, and wave exposure (Fonseca et al.,

1998; Shafer and Bergstrom, 2008). The water quality parameters typically investigated in aquatic restoration projects include: water clarity, total suspended solids, concentrations of chlorophyll a, dissolved inorganic nitrogen and dissolved inorganic phosphorous (Shafer and Bergstrom, 2008). Eventually light requirements were included to incorporate the demands of epiphytic growth on SAV leaves. These five factors were developed by seagrass ecologists in the Chesapeake Bay but can be applied to aquatic ecosystems globally. This study was not intended to examine site factors. Instead a qualitative approach was used to determine habitat suitability mostly defined by sediment quality (solid enough to anchor a bare root transplant) and depth. In hindsight the above- mentioned criteria should have been incorporated to quantitatively rather than qualitatively to better define/develop site standards in the Caloosahatchee River. Shafer

42 and Bergstrom (2008) also point out that newly planted sites may require a more narrow and strict set of site conditions than those found within an existing SAV bed, specifically in areas unvegetated for decades. Evidence supporting historically established beds of V. americana within the study area is minimal and at best anecdotal. Given the above limitations, the following interpretations of the four objectives of this study are explored below.

Objective 1.

Determine the effect of exclosure size (1 m2 vs. 12 m2) on growth of V. americana. The idea to investigate exclosure size on the growth of V. americana stemmed originally from the question, “Is it more feasible to establish V. americana beds with multiple small exclosures or fewer large exclosures?” The objective posed here is difficult to address because the different exclosure sizes were used at different locations.

To definitively compare the exclosure size on growth the small and large exclosures would need to occupy the same site. Future studies could investigate, quantitatively, the application and feasibility of small exclosures versus large exclosures. All sites and both exclosure sizes exhibited success. Throughout the course of the study similarities and differences in both exclosures were apparent.

Transportation and deployment. The twenty-four 1 m2 small exclosures occupied more space than the four 2 x 6 m large exclosures. This was critical when assessing and planning the transportation of the exclosures to ensure staff, vehicle, and boat availability. A team of two could deploy and install the small exclosures while the large exclosures required additional staff.

43 Planting and sampling. Although the method of planting the two exclosure sizes differed slightly each method was unique and necessary for its respective phase. The most notable difference when sampling the small and large exclosures was the accessibility to the plants. The large exclosures could incorporate a single diver. When diving the visibility was minimal but ample to visually count rosettes, apple snails, etc.

The small exclosures’ limited access from the top required relying on touch rather than sight.

Wave action. Although both exclosure sizes were susceptible to wave action, the small exclosures were more resilient. Within a week of deployment half of the large exclosures had suffered considerable damage. To prevent additional damage the large exclosures were reinforced with larger diameter PVC to serve as anchors. Despite the attempt to reinforce the large exclosures, weekly maintenance was needed to uphold the exclosures’ integrity.

Herbivory. Regardless of the sizes used the role of the exclosure was to exclude potential herbivory and facilitate the growth and establishment of V. americana.

Although both exclosure sizes were successful the feasibility of using small versus large exclosures for scaled-up restoration efforts is less clear. In other SAV restoration projects exclosure sizes include 1.5 m2 (Hauxwell et al., 2004), 2 m2 (Doyle et al., 1997), and 4 x

10 m (Moore et al., 2010) with success. Success, in most cases, was limited to within the protected areas. Hauxwell et al. (2004) observed unprotected transplants were completely consumed within one month of transplanting. In addition, allowing plants to become established five months before being subjected to grazing pressure did not affect their ability to survive (Hauxwell et al., 2004). A common thread among SAV-herbivory

44 restoration projects is the need to establish large enough founder colonies. Doyle et al.

(1997) conducted test plantings to develop founder colonies of freshwater SAV species including V. americana; Expansion outside of the exclosures occurred only when herbivore pressure was low. The size of the exclosure and ultimately the amount of SAV transplanted appears to vary case by case. The presence of surrounding SAV may diffuse grazing pressure (Doyle et al., 1997). In areas of sparse or zero SAV the amount of SAV required to successfully establish sustaining beds increases. To expand beyond herbivore exclosures large founder colonies may need to be established to withstand grazing pressures (Moore et al., 2010). The lack of SAV in the study area of the Caloosahatchee may indicate the need for larger scale restoration efforts to establish founder beds. Lastly,

Moore et al. (2010) states at least three years are needed for complete bed development to buffer the impact of grazing pressures.

Objective 2.

Determine the effect of two planting densities on the growth of V. americana.

In this study the lower planting density (10 rosettes-m2) resulted in no significant difference in final biomass compared to double the planting density. Manual methods of restoration are time consuming and labor intensive involving harvesting, processing, storing, and planting. Although this study investigated a limited variation of plant densities on a very short time scale the findings could have a significant impact on larger restoration projects. SAV restoration projects range in scale from small, totaling tens or hundreds of square meters (Fishman et al., 2004; Orth et al., 2006) to several acres

(Busch et. al., 2010; Shafer and Bergstrom, 2010). Larger projects typically involve mechanized methods for harvesting and planting seeds and/or plants. Conversely, manual

45 methods of collection and planting are used for but not limited to the smaller scale. The use of seeds coupled with the development of mechanical methods provides a cost- effective means to restore larger acreages of SAV (Shafer and Bergstrom, 2010.)

However, not all projects are equal in size and require different methods. Moore et al.

(2010) found that V. americana beds can be established using seeds, seed pods, and whole rosettes. Restoration methods using whole rosettes proved to be the most successful in terms of establishment (Moore et al., 2010).

Objective 3.

Determine growth between two genetic strains. The spatial scale of a restoration project either large or small must prove resilient to dynamic changes in both the short- term and long-term (Lloyd et al., 2012). To ensure success at the short-term the genetics of donor plants must be considered (Lloyd et al., 2012) but restored populations often have too little genetic variation to prove resilient over time. The subsequent consequences of too few individuals (genetic variation) are (1) directly impact fitness due to inbreeding (Dudash, 1990; Gigord et al., 1998; Keller and Waller, 2002; Lloyd et al.,

2012) and (2) low allelic diversity and the potential to evolve through increased rates of genetic drift (Whitlock, 2000; Hartl and Clark, 2007; Lloyd et al., 2012). Low diversity also inhibits a population’s ability to rebound from environmental pressures, including grazing, heat shock, or nitrogen loading (Lloyd et al., 2012).

To increase the likelihood of a successful restoration attempt two genetic strains of V. americana donor plants were collected at two locations. Dr. Lyn Gettys, Ph.D.

(University of Florida) identified Lake Trafford and Lake Kennedy as genetically

46 different strains of donor V. americana. During Phase I a significant statistical difference was observed in the growth pattern between the two strains. The mean number of rosettes and blades were greater in Lake Kennedy. The results indicate initially that Lake

Kennedy would be more suitable to restoration in the Caloosahatchee River. However, other parameters, may also be important. While morphological characteristics and total biomass did not differ among strains in the Phase I experiment, the results from Phase II indicate that Lake Trafford donor material averaged greater blade length, width, and leaf area. Additionally, the donor material originating from Lake Trafford had a higher final mean percent frequency of occurrence. The results from Phase II indicate that Lake

Trafford stock is more appropriate for SAV restoration in the Caloosahatchee River.

Despite statistically significant differences in growth patterns (Phase I) and morphological characteristics/frequency of occurrence (Phase II) the results indicate either donor strain as a suitable candidate for restoration in the Caloosahatchee River.

However, sites in the river vary so the characteristics of donor suitability should be considered carefully along the river. Lloyd et al. (2011) outline three main approaches for selecting material for re-vegetation efforts: (1) select a few particularly well performing genotypes for a particular set of criteria and propagate; (2) select propagules to ensure restored populations reflect genetic diversity of local populations; and (3) use large numbers of propagules of diverse origin and let natural selection sort out appropriate genotypes for a particular site.

Understanding how donor genetics contribute to restoration outcome is critical.

Preparation for a SAV restoration project that includes an investigation of the donor plants genetic makeup should be standard practice. This consideration creates the

47 potential to begin the study with multiple genotypes and increase the likelihood of a successful restoration. Furthermore the genetics of the plants after a restoration project should be considered, thus allowing the researcher to clearly identify the specific genotype(s) that thrive at a particular restoration site hopefully leading toward a better understanding of plant/environment ecology. Any effort to understand genotype and habitat characteristic ‘fit’ must include a clear articulation of what defines ‘success’. This is not clear-cut and could include, but is not limited to: total biomass, number of plants, plant density, morphology, survivability, and sexual reproduction. SAV researchers might incorporate these and other factors to define a standard definition of restoration success.

The importance of genetics is not limited to SAV restoration projects and therefore should be considered in any restoration effort. Restoration of any species to an extirpated site will involve attempting to find appropriate genetic material often from geographically separated populations. When the cause of extirpation is unknown, or unconnected, a genetic ‘fit’ might be doomed to similar end. Given the rapidly changing environmental conditions of most or all ecosystems, perhaps the best guideline is Lloyd et al.’s (2012) third approach using a maximum of propagules with a maximum of diversity to create most resilient system.

Objective 4.

Determine the effect of sediment characteristics on growth of V. americana.

The sediment characteristics of donor or “natural” sites often differ from planned restoration sites and may inhibit long-term establishment (Gettys and Haller, 2013).

Therefore, in addition to environmental parameters and genetic variability the sediment

48 characteristics of proposed restoration sites should be considered. The majority of aquatic plants utilize nutrients stored within the sediment instead of those in the water column

(Barko and Smart, 1981). V. americana tolerates a wide range of sediment fertility

(Gettys and Haller, 2013) but Anderson and Kalff (1986) found V. americana maximized biomass production when cultured with low levels of N, P, and K. Hunt (1963) found V. americana on substrate ranging from gravel to hard clay but grew best in silty sand.

Furthermore, Hunt (1963) concluded only an impervious or overly soft substrate prevents anchoring and subsequent establishment of V. americana.

The total organic content (TOC) analysis of the donor and restoration sites yielded significant differences among the two donor sites and restoration sites (Figure 15). Lake

Kennedy measured the greatest mean percent TOC at 13.1% and differed significantly from all remaining sites. Lake Trafford and the restoration sites ranged from 7.10% to

9.83% TOC but did not exhibit a significant difference among sites (Figure 15). In comparing the two donor strains (sites) in Phase I Lake Kennedy had a higher number of rosettes and blades (Figures 21 and 22, respectively). This is different than we would expect considering donor plants collected at Lake Kennedy thrived in a greater percent

TOC. Based on TOC the plants originating from Lake Trafford should have had better success than Lake Kennedy as they were relocated to sites with similar TOC. In Phase II significant differences were established between the two donor strains. The mean percent frequency of occurrence in Lake Trafford was greater than Lake Kennedy (Figure 28).

Additionally, significant differences were found in average blade length, width, and leaf area (Figure 32, 33, and 34, respectively). All of which were greater in Lake Trafford.

These results differ from Phase I and indicate that Lake Trafford was a more suitable

49 donor plant. This variation between donor site and restoration success may indicate a complex interaction of factors or possibly a critical factor that has not yet been identified.

The grain size analysis indicates no significant difference among sites when measuring average percent gravel and sand (p = 0.126 and p = 0.167, respectively).

Gravel percentages ranged from .09% to 8.3% of the total composition while average percent sand among the sites ranged from 87% to 99% comprising the bulk of the sediment. This would indicate that the source and targeted restoration sites are similar in sediment composition. Despite the observed similarities it is important to note that the silt/clay fraction was significantly different (p = 0.025). The percentages of silt/clay ranged from 0.7% in Lake Trafford to 8.0% in Lake Kennedy. In Phase II Lake Trafford appeared to outcompete Lake Kennedy with a greater mean percent frequency of occurrence, blade length, blade width, and leaf area. The results of the grain size analysis do not provide conclusive evidence to support this observation. Instead the results indicate that either of the sources would be acceptable donors given the similarities in sediment composition among the sites. It is possible that the sediment composition (TOC and grain size) are not driving the observed differences. The fertility of the soils (N, P, and K) among the sites may be a crucial factor contributing to the different growth responses.

Future Considerations.

 The time frame in SAV restoration should be long enough to capture seasonal and

inter-annual variability.

 Site selection may be the most critical factor when considering a SAV restoration

project.

50  Although multiple small exclosures appear more resilient in the field the

researcher(s) must take into consideration the feasibility of: transportation and

deployment, planting and sampling, and wave action when considering exclosure

size.

 The initial planting density of a SAV restoration project should be determined on

a case by case basis. Ultimately the researcher(s) would maximize the number of

initial transplants.

 To create the most resilient system it is important to investigate the genetic stock

of the donor source(s) to maximize genotypic diversity.

 A thorough investigation of the sediment composition and fertility at potential

donor and restoration sites should be conducted to maximize transplant success.

 When measuring success of transplanted SAV it may be necessary to measure

survivability, sexual reproduction, total biomass, number of plants, and plant

density.

Conclusions and Recommendations

V. americana restoration efforts should continue in both the estuary and freshwater portion of the Caloosahatchee. The freshwater portion of the CRE provides a unique area to investigate V. americana restoration methodology within a relatively stable environment. Future research should focus on using additional sources of V. americana in an effort to maximize genotypic diversity. The best approach to site selection would be to plant anywhere and everywhere the sediment will allow transplants to anchor. Additionally, the use of exclosures to protect the newly transplanted V. americana is necessary but the size and number needed are dependent on the scale and

51 individual needs of the project. To repair our estuaries will require fixing both the timing and quality of the water inundating the CRE. Restoring native SAV populations will be critical to this effort.

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66 APPENDIX A

Qualitative plant sampling from Phase I. This graph depicts the mean above and below ground biomass for sites 1 and 2. The error bars represent standard deviation.

67 APPENDIX B

Lake Kennedy Mean Percent Cover through Time

Site 1 Lake Kennedy (L) Site 1 Lake Kennedy (H) Site 2 Lake Kennedy (L) Site 2 Lake Kennedy (H)

66%, -

2.5 33%,2=34% - 2

1.5 3=67%100%) 1

0.5

Mean % Cover Thru Time Mean % Cover Thru Time (0=0%, 1=1% 0 Sample 1 Sample 2 Final Sample

During the course of Phase II the small exclosures were monitored on a monthly basis. Cover was estimated and recorded into one of four categories based on approximate cover where; 0 = none present, 1 = 1-33 percent cover, 2 = 34-66 percent cover and 3 = 67-100 percent cover. The graph depicts high and low densities of Lake Kennedy at sites 1 and 2.

68 Lake Trafford Mean Percent Cover through Time

Site 1 Lake Trafford (L) Site 1 Lake Trafford (H) Site 2 Lake Trafford (L) Site 2 Lake Trafford (H)

100%) -

2.5

66%,3=67% -

33%,2=34% - 1.5

0.5 Mean % Cover Thru Time Mean % Cover Thru Time (0=0%, 1=1% 0 Sample 1 Sample 2 Final Sample

69 APPENDIX C

Table showing the LSD Post Hoc test for Total organic content.

Table depicting the LSD Post Hoc Test for the average Silt-Clay fraction.

71 APPENDIX D

The above ANOVA tables are for Objective 1-3 from Phase I investigating the different response variables. Including the number of rosettes, the number of blades. Blade length, blade width, leaf area, and biomass.

74 APPENDIX E

Phase II ANOVA table with the mean V. americana transplant final percent cover.

Phase II ANOVA table with the mean V. americana final number of rosettes.

Phase II ANOVA table that includes the four response variables and their interaction with site and strain.

76 APPENDIX F

Phase II LSD Post Hoc test for final number of rosettes.

77 APPENDIX G

LSD Post Hoc test for Phase II response variables and their interaction with site