Limnetic Zooplankton Structure and the Impact of Invasion by an Exotic Cladoceran, Daphnia Lumholtzi

Limnetic Zooplankton Structure and The Impact of Invasion by an Exotic Cladoceran, Daphnia lumholtzi

A dissertation submitted To Kent State University in partial Fulfillment of the requirements for the Degree of Doctor of Philosophy

Susan R. Pasko

August 2009

Dissertation written by Susan R. Pasko B.S., Richard Stockton College, 2000 Ph.D., Kent State University, 2009

Approved by

______, Chair, Doctoral Dissertation Committee Dr. Robert E. Carlson ______, Member, Doctoral Dissertation Committee Dr. Mark W. Kershner ______, Member, Doctoral Dissertation Committee Dr. James L. Blank ______, Member, Doctoral Dissertation Committee Dr. Alison J. Smith

Accepted by

______, Chair, Department of Biological Sciences Dr. James L. Blank

______, Dean, College of Arts and Sciences Dr. John R. D. Stalvey

TABLE OF CONTENTS

LIST OF FIGURES ...... vii

LIST OF TABLES ...... ix

BACKGROUND ...... 1

OBJECTIVES OF THE STUDY...... 6

CHAPTER 1: CONTEMPORARY SURVEY

INTRODUCTION ...... 8

Dispersal ...... 8

Seasonal Influences in Invasion Success ...... 11

Biotic Factors ...... 12

Objectives of the Study ...... 14 METHODS

Contemporary Survey ...... 15

Zooplankton Measurement Study ...... 17

Zooplankton Diversity Study ...... 18 RESULTS

Contemporary Survey ...... 20

Zooplankton Measurement Study...... 24

Zooplankton Diversity Study ...... 27 DISCUSSION

Range Expansion ...... 28

Abiotic Factors ...... 30

Community Interactions ...... 36

Conclusions...... 41

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CHAPTER 2: PALEOLIMNOLOGICAL STUDY

INTRODUCTION ...... 43

Paleolimnology: A Review ...... 45

Sedimentary Pigments and Trophic State ...... 46

Objectives of the Study ...... 47

METHODS ...... 48

Lake Profiles ...... 50

Coring Techniques...... 54

Analytical Techniques ...... 54 RESULTS

Percent Dry Weight...... 59 Organic Matter ...... 60

Sedimentary pigments ...... 62

Zooplankton Microfossil Measurements ...... 69

Carapace Measurements...... 80

Head Shield Measurements ...... 81

Daphnid Claw Measurements ...... 82

Mucro to Carapace Ratio ...... 83

Zooplankton Microfossil Percent Abundance ...... 84

Diversity Indices ...... 94

Community Compositions ...... 96 DISSCUSSION

Dry Weight ...... 98

Organic Matter ...... 99

Sedimentary Pigments ...... 100

Species Diversity ...... 101

Species Composition ...... 104

Influence of Predation ...... 107

Conclusions ...... 108 CHAPTER 3 – RESOURCE OVERLAP STUDY

INTRODUCTION ...... 111

Objectives of the Study ...... 114

METHODS ...... 116 RESULTS

Grazing Rates...... 120

Alpha Index - Correlation Coefficient Matrix ...... 122

Patterns within Species ...... 122

Alpha Index - Principle Component Analysis ...... 128

DISSCUSSION ...... 131

Evidence of Food Resource Overlap ...... 132

Thermal Influences ...... 135

Depth and Horizontal Segregation ...... 137

Conclusions ...... 138 CHAPTER 4 – EXPERIMENTAL MESOCOSM STUDY

INTRODUCTION ...... 140

Hypotheses of Competitive Interactions ...... 140

Influences of Predation ...... 144

Objectives of the Study ...... 145

METHODS ...... 147

RESULTS

Intrinsic rate of increase ...... 150

Biomass...... 152

DISSCUSSION...... 155

Zooplankton Age Structure ...... 158

Influence of Predation...... 159

Influence of Season Succession...... 160

Paradox of the Plankton...... 162

Overlapping Generations Resulting from Diapause...... 163

Conclusions ...... 164

FINAL DISCUSSION...... 166

LITERATURE CITED...... 173 Appendix A: Lakes included in the contemporary survey ...... 188 Appendix B: Lakes included in the zooplankton measurement study ...... 190 Appendix C: Lakes included in the zooplankton diversity study ...... 191

LIST OF FIGURES

1. Range of Daphnia lumholtzi.in the United States ...... 3

2. Comparison of the morphology of Daphnia lumholtzi to common native Daphnia species...... 5

3. Location of the lakes and reservoirs included in the contemporary survey ...... 16

4. Location of the Invaded and Non-Invaded localities used in the Zooplankton Measurement Study...... 18

5. Location of the Invaded and Non-Invaded localities used in the Zooplankton Diversity Study...... 19

6. Distribution of the invasive zooplankton species, Daphnia lumholtzi and Eubosmina coregoni, in the state of Ohio ...... 21

7. Cluster analysis from the contemporary survey ...... 22

8. Location of the lakes used in the paleolimnological study...... 49

9. The percentage of dry weight and organic matter from the sediments cores ...... 61

10. The concentration of chlorophyll derivatives from the sediment cores...... 65

11. The percent native chlorophyll from the sediment cores ...... 66

12. Concentration of oscillaxanthin from the sediment cores ...... 70

13. Concentration of myxoxanthrophyll from the sediment cores ...... 71

14. Oscillaxanthin to myxoxanthophyll ratio from the sediment cores ...... 72

15. Cladoceran microfossil carapace lengths ...... 73

16. Cladoceran microfossil carapace lengths ...... 74

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17. Cladoceran microfossil head shield. lengths ...... 75

18. Cladoceran microfossil head shield lengths ...... 76

19. Daphnid microfossil claw lengths ...... 77

20. The mucro to carapace length ratios for Bosmina longirostris...... 78

21. Cladoceran Microfossil percent abundance data from Lake Berlin...... 87

22. Cladoceran Microfossil percent abundance data from Lake Milton...... 88

23. Cladoceran Microfossil percent abundance data from Mosquito Creek Lake ...... 89

24. Cladoceran Microfossil percent abundance data from LaDue Reservoir...... 90

25. Cladoceran Microfossil percent abundance data from Lake Punderson...... 91

26. Cladoceran Microfossil percent abundance data from East Branch Reservoir ...... 92

27. Comparison of diversity indices between the cladoceran communities...... 95

28. Comparison of littoral cladoceran diversity indices ...... 95

29. DCA results using the percent zooplankton species abundance...... 97

30. DCA results using the percent species abundance of the sediment core layers ...... 97

31. Box plot representing the overlap of the grazing rates between the species...... 121

32. Alpha Index values for the eight microsphere classes ...... 127

33. PCA results using zooplankton feeding preferences...... 130

34. Hypothesized relationship of the successful invasion of D. lumholtzi...... 145

35. Final Biomass of the native cladoceran species ...... 153

36. Final Biomass of D. lumholtzi from the invaded community treatments...... 154

37. Comparison of the relative biomass between treatments ...... 154

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LIST OF TABLES

1. Comparison of the abiotic characteristics of invaded lakes...... 23

2. Comparison of mean cladoceran length and width values...... 25

3. Comparison of individual cladoceran species mean length and width values ...... 26

4. Comparison of diversity values ...... 27

5. Summary of environmental factors which may influence invasion ...... 34

6. Average percent of dry weight and organic matter from the surface sediments ...... 61

7. Average values of sedimentary pigments from the sediment cores ...... 64

8. Median microfossil length values...... 79

9. Average microfossil length from the surface sediments ...... 80

10. Median percent species abundance values ...... 93

11. Pearson correlation coefficient matrix using Alpha Index values...... 128

12. Intrinsic rate of increase for the cladoceran species ...... 151

BACKGROUND

The invasion of aquatic habitats by non-indigenous species is a cause for concern as these exotic organisms continuously threaten the integrity of freshwater ecosystems. Consequences of invasion at the species level range from extinction of native species through predation or competitive exclusion to hybridization. At the ecosystem level, consequences may consist of loss of biodiversity and the alteration of ecosystem function (Work and Gophen 1999b, Lennon et. al. 2001). Although the majority of introduced species have had little or no effect on native communities (Williamson and Fitter 1996), some have produced dramatic effects. One familiar example is the zebra mussel,

Drissena polymorpha, whose invasion led to increases in water clarity and nutrient recycling rates in

North American freshwater systems (Dzialowski et. al. 2000). The predacious cladoceran,

Bythotrephes cederstroemi, invaded the Great Lakes in the early 1980’s and has since been linked to the decline of native zooplankton including Leptodora kindii, Daphnia retrocurva and D. pulicaria

(Havel et. al. 1995). The severe impacts of invading species on native ecosystems are reason for a global concern.

Invasive species grant opportunities to examine the ecology of aquatic communities because these invaders may restructure the existing community and modify its development. Little is understood about which species will or will not be able to successfully establish in new environments or what factors at the community level facilitate or resist invasions. Species detected in an early stage of invasion may provide new information pertaining to the

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characteristics of habitats that are vulnerable to invasion, traits of the non-native species that allows them to be successful, as well as the impact that the new species may have on the native habitat

(Havel et. al. 1995, Dzialowski et. al. 2000, Miller et. al. 2002). Current research has focused on attempts to answer these questions. These studies may be beneficial to identify patterns of invasions, allowing prediction and possibly prevention of future invasions.

One of the most recent freshwater invaders to North America is the daphnid, Daphnia lumholtzi.

This cladoceran is native to east Africa, south-west Asia, and northern and eastern Australia (Havel et. al. 1995, Hiskey 1996, Work and Gophen 1999a/b, Dzialowski et. al. 2000). First reported within a lake in eastern Texas in 1991, D. lumholtzi has since been detected in at least 225 lakes and reservoirs and 22 states, spreading as far east as Florida, west to California, and north into the Great

Lakes (Figure 1). In addition, numerous studies have indicated that the range of this species is continuing to expand across North America (Havel and Herbert 1993, Work and Gophen 1999a, East et. al. 1999, Dzialowski et. al. 2000, Muzinic 2000, Lennon et. al. 2003).

D. lumholtzi, as described by G.O. Sars in 1885, has a number of morphological characteristics which make it distinct from native Daphnia species (Figure 2). These features include a large pointed helmet, long tail spine with numerous spines, distinctive pointed lateral fornices, ten to fourteen anal spines, and approximately ten prominent spines along the ventral carapace margin

(Havel and Herbert 1993, Havel et. al. 1995, Hiskey 1996). The length of the head spine ranges from one half to slightly more than the body length (Muzinic 2000). D. lumholtzi is greater in total body length then most native daphnids; for example, measurements of the colonists in Lake Erie revealed total lengths ranging from 5.0 to 5.7 mm within females and 4.9 to 5.3 mm within males. Other studies have reported smaller total body lengths; for instance Illinois lakes reported lengths between

2.0-2.6 mm in Lake Chantauqua (Lemke et. al. 2003) and 2.31-2.89 mm in Lake Springfield (Kolar et. al. 1997). In the Ohio River, adults average about 1.1 mm long (Jack and Thorp 1995). The differences in size may be the result of cyclomorphism, since D. lumholtzi has demonstrated the ability to change the length of its head spine in response to the environment (Dzialowski et. al.

2003).

Figure 1: Lakes within the United Stated which have reported introductions of Daphnia lumholtzi. Locations were complied from Havel and Shurin 2004 as well as zooplankton samples supplied by the OEPA and identified as a part of this present study.

Cladocerans are important to freshwater systems as species in this group are herbivores that are important dietary items for fish (Gillooly and Dodson 2000). Daphnids have been commonly referred to as keystone herbivores as they act as a vital step in the transfer of energy from lower trophic levels, such as bacteria and algae, to higher levels which includes planktivorous insects and fish (Dzialowski et. al. 2000). Consequently, a change in the zooplankton community could lead to changes in both the lower and upper trophic levels. Ecological theory suggests that larger zooplankton may be better competitors because of greater effectiveness of food collection and should be able to displace those smaller in size (Brooks and Dodson 1965). Further, the elongated spines of D. lumholtzi may create handling difficulties for predators. Consequently, if D. lumholtzi were to become a dominant member of the zooplankton community, displacement of native zooplankton, alteration of the phytoplankton community, and effects on higher tropic levels may possibly result (East et. al. 1999, Stoeckel and Charlebois 2004). However, the potential of D. lumholtzi to impact freshwater systems in any significant way has been questioned because the abundance of any of the introduced populations has been characteristically low thus far (Johnson and

Havel 2001, Lennon et. al. 2003). Further, D. lumholtzi reaches its highest densities during the late summer when most other zooplankton are observed in low numbers, suggesting that the interaction of this species with other native species is likely to be low and the effects of the invader on the new habitat minimal (Work and Gophen 1999b). However, the effect of the species on native zooplankton communities may be yet to be observed since the invasion may be too recent to detect any ecological impact (Johnson and Havel 2001).

Figure 2: Comparison of the morphology of Daphnia lumholtzi to common native Daphnia species.

Large Bodied Cladocerans are defined as those with an average body length greater than 1.0 mm. A: Daphnia magna B: Daphnia pulex C: Daphnia galeata D: D. lumholtzi

Small Bodied Cladocerans are defined as those with an average body length less than 1.0 mm E: Daphnia retrocurva F: Daphnia parvula G: Daphnia ambigua H: Bosmina longirostris I: Eubosmina coregoni J: Chydorus sphaericus.

The illustration of D. lumholtzi was taken from Havel and Herbert (1993), E. coregoni from Balcer et. al. (1984), all others from Pennak (1989).

Objectives of the Study

The overall goal of this research is to better understand the success rates and consequences of D. lumholtzi on lake ecosystems. To achieve this goal, this study used a combination of contemporary surveys, paleolimnological techniques, and laboratory experiments. Each of these approaches has been organized into separate chapters and is briefly introduced below.

Chapter 1: Contemporary Survey: This study utilized archived samples from the Ohio

Environmental Protection Agency (OEPA) and surveys of Ohio lakes to investigate the current distribution of D. lumholtzi as well as to identify zooplankton community structures associated with this non-indigenous species.

Chapter 2: Paleolimnological Study: In this investigation sediment cores were processed for cladoceran remains and sedimentary plant pigments in order to determine community types that are vulnerable to invasion and the potential impact that D. lumholtzi may have on native communities.

Chapter 3: Resource Overlap Study: In order to identify the amount of food resource overlap, and hence competition, between zooplankton species this study utilized fluorescent microspheres to determine the grazing efficiency and range of particle sizes able to be ingested for D. lumholtzi as well as other native zooplankton species.

Chapter 4: Experimental Mesocosm Study: Experimental manipulations using laboratory mesocosms were performed in order to verify that the patterns found from the sediment core analysis occurred as a result of biological interactions. D. lumholtzi was introduced into different community types identified by the paleolimnological study. The zooplankton were observed over an eight week period in order to determine in which communities D. lumholtzi would be able to persist.

Each of the studies were performed to identify the conditions in freshwater communities that may facilitate the establishment of D. lumholtzi and to evaluate the potential impact that this species may have on native communities. This information may be beneficial to identify patterns of invasions, thus constructing mechanisms to possibly prevent future invasions.

CHAPTER 1: CONTEMPORARY SURVEY

INTRODUCTION

In order for an invasive species to be successful within a novel environment, it must be have the means to disperse outside its native range. Upon arrival at a new location, the species must be able to tolerate the environmental conditions of the new environment. Finally, in order to persist in a non-native system, the non-indigenous species must have defenses against new predation pressures and be able to out-compete native species for resources, or occupy an empty niche (Miller et. al.

2002). The ability of D. lumholtzi to meet these criteria is discussed below.

Dispersal

The distribution of cladocerans is controlled by their capability to disperse, as well as their ability to tolerate physical, chemical, and biological pressures in new environments (Smol et. al.

2001). Dispersal of individuals refers to the ability of the zooplankton to move by their own means or be transported by other agents including physical, animal, or human vectors. The dispersal of non- indigenous species has often been the consequence of human activities, either deliberate or unintentional. Several vectors have been identified in the transport of invasive zooplankton into aquatic ecosystems including the release of ballast water from ships and accidental or planned release of aquatic life.

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The consequences of these actions are poorly understood, especially in plankton systems

(Dzialowski et. al. 2000, Bollens et. al. 2002). Past research on freshwater invasive species has focused on those which are transported through ballast water, resulting in a rapid establishment into large bodies of water, such as inland bays and the Great Lakes. D. lumholtzi provides the opportunity to study the invasion of a small number of individuals released into smaller bodies of water (Havel and Herbert 1993).

The invasion of D. lumholtzi is unique for several reasons. For example, intercontinental dispersal of cladocerans is rare with most species being limited to only one continent. This indicates that either long distance dispersal is ineffective or integrating into new habitats is difficult for zooplankton (Havel et. al. 1995). The invasion of D. lumholtzi is also distinctive because the vector responsible for the invasion is unknown. Other non-native freshwater species, such as the bivalves,

Drissena polymorpha and Corbicula fluminea, and cladocerans, Eubosmina coregoni and

Bythotrephes cederstroemi, have likely been introduced through ballast water from large ships into the Great Lakes. The inland distribution of D. lumholtzi and its late detection in the Great Lakes suggest that ballast water of large boats was not the agent of introduction (Havel and Herbert 1993,

Havel et. al. 1995, Dzialowski et. al. 2000). Another hypothesis proposes that storms dispersed dry sediments along with ephippia into the atmosphere, resulting in the deposition of the eggs into North

American waters. In a test of zooplankton dispersal rates, Jenkins and Underwood (1998) demonstrated that wind and rain did not play a significant role in the dispersal of zooplankton. The results of that study along with the fact that no other species has ever demonstrated an introduction through this mechanism (Havel et. al. 1995) indicates that wind as a method of transport for D. lumholtzi is doubtful. A more likely vector may be the release of exotic fish or plants imported by

the aquarium trade. The Nile perch seems to be a possible suspect since at least one of the lakes initially stocked with the fish has reported populations of D. lumholtzi. Furthermore, the perch were obtained from their native locations where D. lumholtzi is a dominant member of the zooplankton community. If the species was indeed introduced along with the Nile perch, this would imply that D. lumholtzi was able to expand its range from a few locations in Texas to over 22 states in less than a decade (Havel and Herbert 1993, Havel et. al. 1995). The rapid rate of dispersal and colonization success suggests that D. lumholtzi could become a common member of the zooplankton communities throughout the United States. The rapid rate at which the species is expanding its range emphasizes the need to determine lake habitats that may be vulnerable to invasion as well as potential impacts that this species may create.

The rapid rate of expansion of D. lumholtzi may lead to the impression that dispersal may be relatively simple for zooplankton. However, once a new habitat is reached by an individual, the abiotic conditions and biological interactions will determine whether or not the species will persist

(Shurin and Havel 2002). Other invasive species of zooplankton have similar means of dispersal as

D. lumholtzi, yet most have not significantly expanded their range. For example, Eubosmina coregoni was introduced into the Great Lakes in the 1960’s and Bythotrephes cederstroemi invaded

North America in the early 1980’s, however, both species remain limited to locations near their point of introduction (Havel and Herbert 1993). It is possible that widespread dispersal has occurred among these cladocerans, yet their inability to persist in new freshwater communities has allowed their dispersal to go undetected.

Seasonal Influences in Invasion Success

Seasonality affects the temperature and stratification of lakes, ultimately resulting in a change in nutrient and phytoplankton composition, thereby changing the quality and quantity of food resources available to the zooplankton. These changes may lead to resource competition among the species, with a shift in competitive advantage whenever the environment changes (Johnson and Havel 2001).

Late summer phytoplankton communities in lakes and reservoirs are often dominated by cyanobacteria, which are often associated with handling difficulties, toxins, and low nutrient content for the zooplankton. During the warmer months concentrations of edible algae may become limiting for zooplankton, leading to resource competition (Lennon et. al. 2001, Pattinson et. al. 2003). In the tropical lakes of D. lumholtzi’s native range, cyanobacteria are present throughout most of the year, indicating that this species may have a greater tolerance to cyanobacteria (Pattinson et. al. 2003).

Furthermore, the large size of D. lumholtzi may allow it to filter more efficiently and consume a larger range of particles than smaller zooplankton, thus allowing higher survival and reproduction rates at lower food concentrations than smaller species (Johnson and Havel 2001).

Limnetic thermal variation that results from seasonal shifts may be among the most important abiotic characteristics for the success or failure of invasive plankton. It has been suggested that the timing of zooplankton peak abundance is not associated with seasons, but rather with specific ranges of temperature (Gillooly and Dodson 2000). In zooplankton populations, an increase in temperature will result in increased locomotion, filtering rates, ingestion rates, and assimilation rates; however, higher energy costs also result as elevated temperatures cause an increase in respiration.

Consequently, increased temperatures are often not optimal for survival and typically limit numbers and growth rates in zooplankton communities.

In North American reservoirs, D. lumholtzi populations reach maximum numbers during the mid- to late summer months. Increased temperatures during this time usually result in the decline of

Daphnia populations. This decline may encourage invasion by D. lumholtzi as the non-indigenous species may have more success establishing during periods of lower diversity (Elton 1958). Further, this period of increased temperatures and decreased competition may form an open thermal niche that D. lumholtzi is able to fill due to its tolerance to warm water conditions and low quality food resources (Havel et. al. 1995, Pattinson et. al. 2003).

Biotic Factors

In addition to seasonality, several hypotheses have been put forth to support the idea that invasion may be influenced by biological interactions that occur among community members. For example, the Biotic Resistance Hypothesis (Miller et. al. 2002) predicts that successful invasion is negatively related to species richness. Greater species diversity is often the result of numerous trophic levels, which may lead to a high strength of interaction. These interactions may produce strong competitors, well-defined prey, and efficient predators within the community, ultimately resulting in fewer open niches. The foundation of this model dates back to Elton’s (1958) hypothesis that species-rich communities are more resistant to invasion due to a lower number of available niches which results from increased resource use, competition, and predation. Accordingly, the rate of establishment of non-native species should decrease with time.

The Invasion Meltdown Theory (Simberloff and Von Holle 1999) was proposed as an alternative to the Biotic Resistance model. This hypothesis was based on the observation that once an invasive species has been established, it may have the ability to alter the surrounding environment and create

favorable conditions for other non-native species. In these situations, native community structure may be disrupted following each invasion, allowing for subsequent establishment of other exotic species. This hypothesis implies that establishment of an invasive species may occur if the community has been invaded several times. The latter theory appears to have more credibility in regards to North American freshwater systems. For example, the Great Lakes have long been subjected to repeated invasion yet, perhaps due to greater boating traffic, the number of successful invasions have substantially increased over the last few decades (Ricciardi 2001). The prior history of invasion within the Great Lakes and their recent invasion by the cladocerans, Bythotrephes cederstroemi and Cercopagis pengoi, suggest that this habitat is vulnerable to invasion by D. lumholtzi.

Establishment of non-indigenous species may also be dependent of individual characteristics of the non-indigenous species themselves. Successful invasive species often share similar characteristics including wide physiological tolerances, short generation times, high dispersal rates, and they are commonly generalists with regards to food resources and habitat (Havel et. al. 1995,

Work and Gophen 1999b). D. lumholtzi shares many of these properties; hence, it is an ideal species to study invasion models. The success of this cladoceran may be further enhanced by its ability to reproduce parthenogenically, allowing for quick reproduction as well as a reduction in the number of individuals that must be transported for colonization. It is theoretically possible that a single female could initiate a successful invasion (Havel et. al. 1995, Work and Gophen 1999a).

Objectives of the Study

The broad physiological tolerances, ease of dispersal, and the potential predation deterrence of D. lumholtzi suggest that this species could successfully establish within every lake in the United Stated into which it is introduced. This species was first detected in an eastern Texas lake in 1989 (Work and Gophen 1999a); by 1993 the species was reported in Ohio waterways (Hiskey 1996). In spite of this rapid expansion, little investigation has taken place to investigate what factors may facilitate the establishment of D. lumholtzi or to identify characteristics of habitats which are vulnerable to invasion. To investigate these questions archived zooplankton samples provided by the Ohio EPA as well as samples collected during the summers of 2005 and 2006 were used to determine the distribution of D. lumholtzi across the state of Ohio as well as to identify zooplankton community structures associated with the non-indigenous species. This survey also attempted to determine abiotic characteristics of the lakes that may facilitate the invasion by D. lumholtzi. Examination of the achieved and collected samples provided information of the dispersal of D. lumholtzi into Ohio lakes as well as species compositions associated with invasion by this exotic zooplankton.

Additionally, the samples were analyzed for mean zooplankton body size, zooplankton species diversity, as well as abiotic characteristics from invaded and non-invaded lakes and reservoirs.

Comparison of these samples provided insight into the conditions of invasion for D. lumholtzi by revealing the abiotic and biotic factors that are associated with invasion events.

METHODS

Contemporary Survey

Archived zooplankton samples were provided by The Ohio Environmental Protection Agency

(OEPA) for this survey. The agency conducted zooplankton surveys for 87 public lakes and reservoirs across the state of Ohio between the years 1989 and 2005 (Figure 3). These samples were collected using a vertical tow beginning at twice the Secchi depth with a 63 µm, 20 cm diameter zooplankton net (Davic, personal communication). All samples were preserved in 5% formalin.

In addition to these samples, 26 Ohio lakes and reservoirs were surveyed during the summer months of 2005 and 2006 (Figure 3). Six of the locations were not included in the OEPA surveys.

The samples were taken using a vertical tow beginning at the lake bottom using a 153 µm, 20 cm diameter zooplankton net. To prevent ballooning, zooplankton were preserved in a sucrose formalin solution (Haney and Hall 1973). Cladocerans were identified to species while copepods were identified to suborder following Balcer et. al. (1984), Pennak (1989) and Ward and Whipple (1959).

To identify zooplankton communities that were associated with the presence of D. lumholtzi, cluster analysis using Jaccard’s Coefficient was preformed on the presence / absence data using MVSP Plus

(version 3.13). Jaccard’s Coefficient is preferred in numerical taxonomy as it measures similarity in habitats in respect to the species present in the sample and ignoring the impact of species that are absent. By excluding variables in which neither habitat contains the species, similarity is only measured by common attributes (Jobson 1991).

Water chemistry and nutrient data for locations in the survey was provided by the 1996 OEPA

Ohio Water Resource Inventory Report (Davic et. al. 1997). This data was available 76 of the 93

lakes and reservoirs in this survey. Paired Student’s t-tests were applied to compare abiotic factors between the invaded and non-invaded localities. Invaded and Non-Invaded locations were distinguished by the presence or absence of D. lumholtzi as determined by the analysis described above.

Figure 3: Location of the 93 Ohio lakes and reservoirs included in the

contemporary survey.

Zooplankton Measurement Study

From the contemporary survey samples, 20 samples were selected for the zooplankton measurement study; 10 representing invaded locations, and 10 representing the non-invaded (Figure

4). The samples were chosen based on the quality of preservation; such that cladocerans could be identified to species and an adequate number of cladocerans could be counted. The invaded samples each included D. lumholtzi, the non-invaded lakes did not. Two of the non-invaded samples, Alum

Creek Reservoir and Hargus Lake, were chosen as they were collected prior to invasion. The remaining non-invaded lakes were selected as the samples were taken during the late summer months, the time when D. lumholtzi reaches its highest peak numbers; thereby the absence of D. lumholtzi indicates that the species has not yet been introduced. The preserved zooplankton were identified and counted until at least 100 cladocerans were tallied or the entire sample volume, whichever came first. Identifications followed Balcer et. al. (1984), Pennak (1989) and Ward and

Whipple (1959). To avoid bias from the length of head and tail spines, carapace length measurements were made from the top of the eyespot to the base of the tail spine. Width measurements were taken at the widest section of the body. Paired Student’s t-tests were used to compare mean cladoceran length and values between invaded and non-invaded localities.

Figure 4: Location of the Invaded and Non-Invaded localities used in the zooplankton measurement study.

Zooplankton Diversity Study

Seventeen samples from the contemporary survey were used to represent invaded locations in this study (Figure 5). This included every sample in which D. lumholtzi was present, provided that it had adequate preservation such that cladocerans could be identified to species and a sufficient number of zooplankton could be counted. All samples from the contemporary survey that were collected between the summer months of July and September and did not contain D. lumholtzi were used to represent the non-invaded localities, totaling 34 samples. The preserved zooplankton were

identified and counted until at least 200 individuals, or the entire sample contents, were tallied.

Identifications followed Balcer et. al. (1984), Pennak (1989) and Ward and Whipple (1959).

Cladocerans were identified to the lowest taxonomic level possible while copepods were identified to suborder. Shannon Weiner Diversity, Maximum Diversity, Species Evenness, and Species

Richness were calculated for each the invaded and non-invaded samples. Paired Student’s t-tests were used to compare the diversity indices between the lakes.

Figure 5: Location of the Invaded and Non-Invaded localities used in the zooplankton diversity study.

RESULTS

Contemporary Survey

Zooplankton surveys of 93 Ohio lakes and reservoirs identified the presence of D. lumholtzi in

19 locations (Figure 6). Cluster analysis of the zooplankton species composition (Figure 7) revealed that D. lumholtzi is closely associated with smaller sized zooplankton such as D. ambigua and

Ceriodaphnia species. D. lumholtzi did not demonstrate a close association with larger sized zooplankton including D. galeata and D. pulex. The exotic cladoceran was also not closely associated with species that maintain high numbers during the summer months such as Bosmina longirostris and Diaphanosoma birgei nor was it ever found together with the invasive Eubosmina coregoni.

Comparison of invaded to non-invaded localities showed that those with D. lumholtzi present in their water column tended to be significantly larger in surface area (p > 0.05, df = 75). The surface area of the invaded lakes averaged 15.5 (+ 0.45) km2; however, there was a considerable range of surface area among these lakes. The smallest invaded locations were New Concord Reservoir and

Lake Choctaw, with surface areas of 0.04 and 1.0 km2 (Davic et. al. 1997). The largest water bodies where D. lumholtzi was detected was Whitewater Reservoir and Grand Lake St. Mary’s with surface areas of 105.4 and 51.4 km2. Furthermore, the range of surface areas belonging to the non-invaded lakes, between 0.02 and 14.4 km2, falls within the range of the invaded lakes. The other limnological characteristics also demonstrated extensive overlap between the two categories; accordingly, no other significant differences between invaded and non-invaded lakes were found

(Table 1).

Figure 6: Distribution of the invasive zooplankton species, Daphnia lumholtzi and Eubosmina coregoni, in the state of Ohio. Points were generated by data from OEPA samples as well as contemporary surveys taken during the summers of 2005 and 2006.

Figure 7: Cluster analysis using Jaccard’s Coefficient representing presence / absence data from all lakes surveyed in the contemporary survey.

Table 1: Comparison of mean (+ standard error) and ranges of the abiotic characteristics of invaded lakes and reservoirs (n=16) and non-invaded localities (n=61). As values were taken from the 1996 OEPA Ohio Water Resource Inventory Report (Davic et. al. 1997), not all samples in the survey are accounted for in this analysis. Significant differences derived for t-tests are denoted with * (p>0.05).

Non-Invaded Invaded Limnological Characteristic Mean Range Mean Range * Surface Area (km2) 2.1 + 6.8 0.02 – 14.4 15.5 + 0.45 0.04 – 105.4 Temperature (°C) 22.6 + 0.5 11.8 - 29.2 25.4 + 0.9 23.5 - 27.1 Trophic State (TSI ) 57.7 + 2.6 40 - 72 58.7 + 1.2 44 - 71 Secchi Depth (m) 1.3 + 0.3 0.2 - 4.8 1.2 + 0.1 0.2 - 5.0 1.5 - Chlorophyll a (µg / l) 25.8 + 3.7 116.11 36.2 + 0.5 2.4 - 119.6 10.0 - Total Phosphorus (µg / l) 72.1 + 6.4 660.0 43.3 + 13.8 11.0 - 90.0 Total Nitrogen (µg / l) 0.8 + 0.1 0.2 - 6.0 0.5 + 0.2 0.3 - 0.9 Nitrite (µg / l) 0.03 + 0.0 0.02 - 0.13 0.02 + 0.0 0.02 - 0.04 Nitrate / Nitrite (µg /l ) 0.7 + 0.0 0.1 - 6.9 0.1 + 0.3 0.1 - 0.2 pH 8.2 + 0.2 6.9 - 10.0 8.3 + 0.1 7.6 - 9.3 D.O (mg / L) 8.1 + 2.8 5.3 - 14.2 10.8 + 0.3 3.5 - 26.7 77.0 - Conductivity (µS / cm) 373.8 + 58.9 945.0 356.1 + 28.9 215.0 - 650.0 17.0 - Alkalinity (µeq / l) 103.5 +14.7 260.0 79.9 + 10.4 50.0 - 165.0 * Significant (p>0.05)

Zooplankton Measurement Study

Twenty of the contemporary samples were analyzed to determine if the mean zooplankton size differed between non-invaded and invaded locations (Table 2). With D. lumholtzi removed from the analysis, the cladoceran measurements from the invaded and non-invaded lakes were compared using a paired Student’s t-test. The invaded locations showed a significantly lower mean body length

(p > 0.05, df = 18). When D. lumholtzi was included in the analysis, the mean length in the invaded lakes rose from 0.44 (+ 0.01) mm to 0.48 (+ 0.01) mm and a significant difference was no longer found between the two lake categories. However, the addition of D. lumholtzi to the analysis produced a mean body width of 0.263 (+ 0.003) in the invaded communities, significantly higher than the mean width of the non-invaded lakes (p > 0.05, df = 18). Finally, the length: width ratios of the invaded communities were significantly lower when D. lumholtzi was included in the analysis as well as when it was removed (p > 0.05, df =1958, 1709).

Mean zooplankton size was also calculated for each individual species from the invaded and non-invaded lakes (Table 3). Bosmina longirostris demonstrated a significantly lower mean body width in the invaded lakes (p > 0.05, df = 303), whereas Diaphanosoma birgei was significantly smaller in the invaded lakes for both mean body length and width (p > 0.05, df = 777). Ceriodaphnia was the only species group to exhibit a significantly higher mean body width in the invaded lakes (p

> 0.05, df = 126). B. longirostris, D. galeata, D. parvula, and Ceriodaphnia species each exhibited significantly lower length: width ratios in the invaded lakes (p > 0.05, df = 303, 345, 22, and 126).

With the exception of Diaphanosoma birgei; none of the species exhibited a significant difference in mean body length between the invaded and non-invaded lakes.

The core body length of D. lumholtzi, measured from the top of the eyespot to the base of the tail spine, averaged 0.59 (+ 0.05) mm. This measurement demonstrates that when the elongated head and tail spines are excluded, the core body length of D. lumholtzi is similar, although slightly larger, to other native daphnids. For example, D. galeata exhibited a core body length of 0.58 (+

0.01) mm and D. parvula showed a mean core length of 0.47 (+ 0.05) mm.

Table 2: Comparison of mean (+ standard error) cladoceran length and width values between invaded and non-invaded localities. All measurements are in millimeters. Analysis was performed with and without D. lumholtzi included in the analysis. The range for each variable is reported below the mean. Variables with * denote a significant difference from the non-invaded localities as revealed by a t-test (p > 0.05). Invaded Localities Non-Invaded Localities Length:Width Length Width Length Length:Width Ratio Width Ratio Without 0.47 + 0.44 + 0.01 * 0.24 + 0.01 1.87 + 0.02 * D. lumholtzi 0.01 0.24 + 0.00 2.00 + 0.02 (0.18 - (0.15 - 1.20) (0.08 - 0.64) (0.85 - 3.77) 1.00) (0.11 - 0.56) (0.85 - 4.06) With 0.48 + 0.01 0.26 + 0.00 * 1.82 + 0.02 * D. lumholtzi (0.15 - 1.20) (0.08 - 0.84) (0.85 - 3.77)

Table 3: Comparison of individual cladoceran species mean (+ standard error) length and width values between invaded and non-invaded localities. All measurements are in millimeters. The range for each variable is reported below the mean. Variables with * denote a significant difference from the non-invaded localities as revealed by a t-test (p > 0.05). Species Invaded Localities Non-Invaded Localities

Length:Width Length:Width Length Width Length Width Ratio Ratio Bosmina 0.27 + 0.00 0.24 + 0.00 * 1.15 + 0.01 * 0.27 + 0.01 0.22 + 0.00 1.28 + 0.02 longirostris (0.19 - 0.41) (0.14 - 0.39) (0.85 - 2.93) (0.18 - 0.57) (0.13 - 0.33) (0.85 - 1.74) Chydorus 0.23 + 0.01 0.24 + 0.01 0.99 + 0.05 0.25 + 0.00 0.22 + 0.00 1.09 + 0.00 sphaericus (0.19 - 0.27) (0.20 - 0.23) (0.88 - 1.14) (0.25 - 0.25) (0.22 - 0.22) (1.09 - 1.09) Diaphanosoma 0.46 + 0.01 * 0.21 + 0.00 * 2.29 + 0.04 0.51 + 0.00 0.22 + 0.00 2.35 + 0.02 birgei (0.15 - 1.04) (0.08 - 0.50) (1.15 - 3.77) (0.22 - 1.00) (0.11 - 0.48) (0.96 - 4.06) Ceriodaphnia 0.40 + 0.01 0.26 + 0.01 * 1.59 + 0.03 * 0.37 + 0.01 0.22 + 0.01 1.69 + 0.03 spp. (0.21 - 0.74) (0.11 - 0.62) 1.00 - 2.67) (0.22 - 0.55) (0.14 - 0.38) 1.25 - 2.47) Daphnia 0.32 + 0.03 0.20 + 0.03 1.65 + 0.06 0.49 + 0.17 0.28 + 0.05 1.68 + 0.31 ambigua (0.29 - 0.40) (0.16 - 0.26) (1.56 - 1.75) (0.28 - 0.82) (0.21 - 0.38) (1.13 - 2.19) Daphnia 0.47 + 0.05 0.28 + 0.04 1.70 + 0.06 * 0.58 + 0.06 0.28 + 0.03 2.09 + 0.04 parvula (0.29 - 0.85) (0.15 - 0.62) (1.36 - 2.04) (0.31 - 0.99) (0.15 - 0.41) (1.78 - 2.43) Daphnia 0.58 + 0.01 0.30 + 0.01 1.98 + 0.03 * 0.62 + 0.02 0.30 + 0.01 2.16 + 0.03 galeata (0.28 - 1.20) (0.13 - 0.64) (1.00 - 3.57) (0.35 - 1.00) (0.12 - 0.56) (1.40 - 3.44) Eubosmina na na na 0.36 0.01 + 0.31 + 0.01 1.18 + 0.02 coregoni (0.21 - 0.71) (0.14 - 0.46) (0.85 - 1.94) Daphnia 0.59 + 0.05 0.31 + 0.03 1.00 + 0.09 na na na lumholtzi (0.31 - 1.20) (0.14 - 0.84) (1.19 - 2.91)

Zooplankton Diversity Study

In effort to determine if the invaded and non-invaded locations differed in zooplankton species diversity, the zooplankton of 51 samples were identified and counted. Shannon-Weiner diversity, maximum diversity, species evenness, and richness were each calculated for the samples in the non- invaded lakes and the invaded lakes. Values were calculated with and without D. lumholtzi included in the analysis. The diversity indices performed on this data revealed remarkably similar results between the lake categories (Table 4); consequently, no significant differences were found (p > 0.05, df = 50).

Table 4: Comparison of mean (+ standard error) diversity values between invaded (n=17) and non- invaded localities (n = 40). A t-test revealed no significant differences between any of the variables (p > 0.05). Invaded Localities

Invaded (D. lumholtzi removed

Localities from analysis ) Non-Invaded Localities

Shannon-Weiner 1.04 + 0.09 0.83 + 0.09 0.87 + 0.06

Maximum Diversity 1.59 + 0.07 0.091 + 0.08 1.44 + 0.06

Evenness 0.65 + 0.04 0.51 + 0.05 0.60 + 0.03

Richness 0.81 + 0.06 0.57 + 0.06 0.72 + 0.05

DISCUSSION

Range Expansion

The contemporary survey revealed that the range D. lumholtzi is rapidly expanding across the state of Ohio. The species was first reported in Ohio in 1993 in Grand Lake St. Mary’s (Hiskey

1996). This study identified the presence of D. lumholtzi in 19 of the 93 Ohio lakes and reservoirs surveyed. Throughout North America D. lumholtzi has rapidly colonized a variety of habitats including reservoirs, flood lake plains, large rivers, and the Great Lakes (Pattinson et. al. 2003). The rapid spread of the species is likely facilitated by flowing water connecting the lakes and reservoirs.

As the species has shown the ability to maintain populations in large rivers, it suggests that flowing water may be an important vector for the dispersal of the species (Havel et. al. 1995). An investigation performed by Shurin and Havel (2002) supported this idea by demonstrating that lakes and reservoirs located downstream from source populations were more likely to be invaded then those upstream, indicating the importance of the movement of surface water in dispersal. If D. lumholtzi does actively use flowing water as a means of dispersal it suggests that the recent detection of the species in the Illinois River provides the potential for the further extension of its range into

Lake Michigan, it if has not done so already (Stoeckel and Charlebois 2000).

The study performed by Shurin and Havel (2002) also revealed the occurrence of invasion in lakes with no potential upstream source, implying the presence of dispersal mechanisms other than flowing water. The existence of numerous dispersal vectors is supported by an investigation performed by Dzialowski et. al. (2000) in which 40 Kansas ponds were surveyed to reveal that D. lumholtzi was absent from each site. As the species inhabits a variety of habitats within its native

range including small pools, lakes, and reservoirs, it seems unlikely that local environmental conditions would have prohibited the species from establishing in these ponds. Lack of colonization may be explained by the small area of the ponds and the fact that they were inaccessible to boating traffic, suggesting that humans play an essential role in the dispersal of the species.

In stressful conditions daphnids produce ephippia that are resistant to harsh environmental conditions, including digestion and desiccation. The diapausing eggs of D. lumholtzi are well adapted to dispersal as they include a long point on each end and several small hairs along the dorsal edge. These morphological features serve as hooks, attaching to boats and other material which is transported to different locations. Consequently, the epihippia produced by daphnids offer the ability of the species to be transported into new areas as well as the ability to reestablish in the same habitat at a later time (Shurin 2000).

Many of the non-invaded localities in the contemporary survey have heavy boating traffic or are downstream from invaded locations. This indicates that these non-invaded lakes have strong potential for the introduction of D. lumholtzi, yet have been able to resist invasion. It has been suggested that abiotic factors may determine the successful establishment of a non-indigenous species, thus these factors are an important consideration in investigations regarding the distribution and spread of invasive species. Comparison of invaded to non-invaded localities in this study showed that those with D. lumholtzi present in their water column tended to be significantly larger in surface area (p > 0.05, df = 75). Although other studies have also reported this trend (Havel et. al.

1995, Dzialowski 1996), lack of colonization within smaller lakes may be explained by reduced boating traffic within these water bodies. Consistent with MacArthur and Wilson’s (1967) theory of island biogeography, larger water bodies offer a bigger target for invasion as an exotic species

expands its range; therefore, these lakes are potentially subjected to a higher rate of immigration.

This study demonstrated that smaller water bodies are able to support populations of D. lumholtzi as the species was detected in New Concord Reservoir, which, with a surface area of 0.04 km2, was one of the smallest water bodies in the survey. Further the surface areas of the non-invaded localities ranged from 0.02 to 14.4 km2. These values strongly overlapped the invaded lakes’ surface area range of 0.04 – 105.34 km2, indicating that surface area was not a factor preventing the invasion of

D. lumholtzi.

Abiotic Factors

A common theory behind biological invasions states that species adapted to disturbed habitats will have a greater ease persisting in a novel environment then those native to stable environments.

Fluctuating water levels, turbidity, and deliberate manipulations of food webs are typical in reservoirs, qualifying these lakes as habitats that are vulnerable to the colonization of exotic species

(Havel et. al. 1995). As D. lumholtzi has been observed in reservoirs and temporary lakes and ponds within its native habitat, it can be considered a prime candidate for successful invasion of North

American reservoirs. Vulnerable habitats may also include those with a similar habitat to that of the invaders. Maximum summer temperatures in temperate waterbodies often mimic the temperatures found in the native environment of D. lumholtzi, thus similar conditions may enhance the establishment of D. lumholtzi (Work and Gophen 1999 b).

Thermal variation of aquatic ecosystems has been previously recognized as a major factor contributing to the coexistence of zooplankton species. Past studies have suggested the idea of an open thermal niche (Havel et. al. 1995, Pattinson et. al. 2003), characterized by changes in

temperature, along with fluctuating phytoplankton levels and increased predation pressures during the warmer summer months. These conditions usually result in the decline of Daphnia populations and may allow for the successful establishment of D. lumholtzi as this period of increased temperatures, increased food, and decreased competition may form an empty niche that D. lumholtzi is able to fill due to its tolerance to warm water conditions and the possible predation protection offered by its elongated spines. This study and others (Dzialowski et. al. 2000) failed to find support for this theory as no significant differences in temperature between invaded and non-invaded lakes were found. Although D. lumholtzi populations reach maximum numbers during the mid- to late summer months, these results indicate that temperature may not be the only factor affecting the seasonal densities of this species.

D. lumholtzi has demonstrated a high reproductive rate, short life cycle duration, early maturity, and fast development within higher temperature ranges; all of which are traits of a successful invader

(Work and Gophen 1999a, Lennon et. al. 2001). However, discrepancies have been reported regarding to the thermal range for the species. For example, laboratory experiments have demonstrated that the species is sensitive to temperatures below 10°C, exhibiting no reproduction and reduced survivorship (Lennon et. al. 2001); however, reproduction at low temperatures may be possible as individuals carrying eggs have been collected within the Illinois River in temperatures from 3 to10°C (Stoeckel and Charlebois 2004). Further, high densities of D. lumholtzi have been observed at temperatures outside 28.7 to 29.2° C, the optimal range determined by laboratory experiments (Havens et. al. 2000). Zooplankton samples from Lake James, North Carolina found that D. lumholtzi populations increased in late summer when temperatures reach about 30°C and persisted until temperatures dropped to approximately 15°C (Celik et. al. 2003). Comparisons of D.

lumholtzi numbers to native Daphnia in Lake Okeechobee (East et. al. 1999) found that the invasive species was present within the lake year-round. Densities of D. lumholtzi were highest within the lake, comprising 60 to 70% of the daphnids assemblage, when the water temperature was greater than 27°C, yet consisted of 10% of the species composition when temperatures dropped below this range. The fact that D. lumholtzi was able to survive year round within lakes is indication that the species is able to tolerate and persist in lower water temperatures. Thus, temperature may not be the sole cause of the seasonal density changes of D. lumholtzi.

This study failed to find any significant differences in the abiotic or biotic characteristics between invaded and non-invaded lakes. Thus, it seems unlikely that local environmental conditions would prohibit D. lumholtzi from establishing in additional lakes. This finding is supported by other investigations that found conflicting results regarding factors that may facilitate invasion of D. lumholtzi. For example, in a survey of Louisiana water bodies, Davidson and Kelso (1997) found that the abundance of D. lumholtzi was positively correlated to specific conductivity, yet it was noted that specific conductivity also correlated to surface water temperature. Soon after, experimental manipulations by Work and Gophen (1999a) found changes in conductivity yielded no significant effects on invasion resistance as increases in conductivity did not effect the reproduction, molting, or survivorship rates of D. lumholtzi. Dzialowski et. al. (2000) found that lakes and reservoirs which supported introduced populations of D. lumholtzi demonstrated lower levels of nitrogen, phosphorus, chlorophyll a, as well as higher Secchi disk depths. However, no pre-invasion data was recorded on the water bodies in this study, thus it remains uncertain whether the differences in characteristics were the cause or effect of the invasion. Significant differences were reported in the study by

Dzialowski et. al. (2000) between the invaded and non-invaded locations, yet the values from the

locations with D. lumholtzi had a wide range and overlapped with the values from the reservoirs without the species. For example, the total nitrogen concentration from invaded lakes in this study was 0.24 to 0.86 mg/L, whereas the non-invaded reservoirs ranged from 0.32 to 2.16 mg/L. These ranges suggest that D. lumholtzi should be capable of surviving in the non-invaded locations if given the opportunity. Moreover, the results from the study by Dzialowski et. al. (2000) conflict with a previous study performed by Havel et. al. (1995), in which significantly higher levels of total nitrogen were found within invaded reservoirs and no significant differences for most limnological characteristics including Secchi depth, phosphorus, or chlorophyll a values. The study by

Dzialowski et. al. (2000) also reported significant differences between invaded and non-invaded locations for mean water temperature, nitrate, and lake surface area; however, for each of these characteristics the data values from the reservoirs with D. lumholtzi overlapped those in the non- invaded reservoirs. This overlap suggests that there may not be a difference in the vulnerability of invasion among the lakes.

The above investigations reported conflicting results; however, they do illustrate that D. lumholtzi has broad physiological tolerances and can persist in a wide range of environmental conditions. Past research as well as this study demonstrates that D. lumholtzi is able to survive, grow, and reproduce under a wide range of conditions of turbidity, salinity, conductivity, and temperature. Studies have also reported that cyanobacteria levels and nutrient levels are not a likely cause of the season succession of D. lumholtzi (Work and Gophen 1999, Lennon et. al. 2001,

Pattinson et. al. 2003). The results of this preceding research as well as the conclusions from this study are summarized in Table 5.

Table 5: Summary of results investigating environmental factors which may influence the invasion of Daphnia lumholtzi.

Limnological Result Source Characteristic Invaded reservoirs tend to be larger in area. Ranging from 0.2 – 595.5 km2 for Havel et. al. 1995 Area invaded reservoirs * Dzialowski et. al. Not significant (p>0.05) * 2000 Invaded localities were significantly larger is size (p>0.05). Range of invaded locations was 0.04 – 105.4 km2, overlapping the range of non-invaded lakes This study which was 0.02 – 14.4 km2. * Invaded reservoirs significantly different (p<0.01) Ranges overlap: 23.1-27.2° Havel et. al. 1995 Water Temperature invaded, 11.6-28.1°C for non-invaded reservoirs. * No significant difference between invaded and non-invaded reservoirs Dzialowski et al.

(p>0.05) * 2000 Individuals in 15 and 22° treatments survived longer that those in 29°C. Fast Work and Gophen

maturation and epihippia production with increasing temperature ** 1999a No offspring between 5-10°; 15° lowest positive intrinsic rate of increase. Population growth increase curvilinearly up to 25°. declined at 30°. Lennon et. al. 2001 Survivorship highest between 10-20°C ** Optimum temperature 28.7-29.2°C ** Havens et. al. 2000 Highest densities above 27°, densities declined below 27°C * East et. al. 1999 Not significant (p>0.05) * This study Invaded reservoirs significantly different (p<0.05) Ranges overlap: Havel et. al. 1995 Conductivity 196-716 uS/cm invaded, 31-935uS/cm for non-invaded reservoirs. * No effect on reproduction, yet survival highest in moderate treatments Work and Gophen

(1250 uS/cm) ** 1999a Densities positively correlated with specific conductivity. Specific Davidson and Kelso

conductivity positively correlated with surface water temperature * 1997 Dzialowski et. al. Not significant (p>0.05) * 2000 Not significant (p>0.05) * This study Work and Gophen Increasing levels lowered reproduction, no significant effect on survival ** Turbidity 1999a Secchi Depth Not significant (p>0.05) * Havel et. al. 1995 Invaded reservoirs significantly different (p<0.039) Ranges overlap: 30-250 Dzialowski et. al.

cm invaded, 30-190 cm for non-invaded reservoirs. * 2000 Not significant (p>0.05) * This study Total Nitrogen Not significant (p>0.05) * Havel et. al. 1995 Invaded reservoirs significantly different (p<0.038) Ranges overlap: 0.24- Dzialowski et. al.

.86mg/L invaded, 0.32-2.16mg/L for non-invaded reservoirs. * 2000 Not significant (p>0.05) * This study Dissolved nitrogen Not significant (p>0.05) * Havel et. al. 1995

Invaded reservoirs significantly different (p<0.001) Ranges overlap: 4-543 µg Nitrate/Nitrite Havel et. al. 1995 /l invaded, 1-768 µg /L for non-invaded reservoirs. * Not significant (p>0.05) * Havel et. al. 1995 Not significant (p>0.05) * This study Ammonium Not significant (p>0.05) * Havel et. al. 1995

Total Phosphorus Not significant (p>0.05) * Havel et. al. 1995

Invaded reservoirs significantly different (p<0.048) Ranges overlap: 0.014- Dzialowski et. al.

0.123 mg/L invaded, 0.017-0.214mg/L for non-invaded reservoirs. * 2000

Not significant (p>0.05) * This study Total Dissolved Not significant (p>0.05) * Havel et. al. 1995 Phosphorus Dzialowski et. al. pH Not significant (p>0.05) * 2000 Not significant (p>0.05) * This study

Si2 Not significant (p>0.05) * Havel et. al. 1995 Nonvolatile Not significant (p>0.05) * Havel et. al. 1995 suspended solids

Volatile suspended Not significant (p>0.05) * Havel et. al. 1995 solids

Ca++ Not significant (p>0.05) * Havel et. al. 1995 Mg++ Not significant (p>0.05) * Havel et. al. 1995 Na+ Not significant (p>0.05) * Havel et. al. 1995 K+ Not significant (p>0.05) * Havel et. al. 1995 Chlorophyll a Not significant (p>0.05) * Havel et. al. 1995 Dzialowski et. al. Not significant (p>0.05) * 2000 Not significant (p>0.05) * This study Reduced survivorship, reproduction, and intrinsic rate of increase as level Cyanobacteria Pattinson et. al. 2003 increase. **

* Results are based on surveys between invaded and non-invaded water bodies.

** Results are based on experimental manipulations within laboratory mesocosms.

Community Interactions

The failure to find any significant differences among the abiotic characteristics in this study implies that it is unlikely that local environmental conditions prohibit D. lumholtzi from establishing in additional lakes. This suggests that interactions with the native species may be important in controlling the establishment of D. lumholtzi.

The Biotic Resistance model states that higher species diversity may lead to a greater invasion resistance as a result of high strength of interaction within the community and fewer resources available for utilization. Past research involving invasive species has only mildly supported this hypothesis. Some studies have demonstrated that species richness has caused a decrease in the probability of invasion (Tilman 1997); other investigations have shown the opposite effects

(Robinson et. al. 1995). Still other research has found no significant effect in either direction (Miller et. al. 2002). The Biotic Resistance hypothesis was also unsupported in this study as several diversity indices failed to reveal any significant differences between the invaded and non-invaded localities.

The Biotic Resistance hypothesis also suggests that more diverse communities may resist invasion as fewer resources will be available. This idea has also not received strong support in previous studies as increasing food resources through nutrient enrichment does not appear to facilitate the establishment of D. lumholtzi . For example, Lennon et. al. (2003) used experimental mesocosms to investigate the invasion success of the species under different trophic conditions. That study concluded that nutrient enrichment increased zooplankton biomass and reduced species diversity. D lumholtzi was most commonly found in communities with high species diversity that

were dominated by calanoid copepods and Bosmina. The invasive species was only rarely found in communities that exhibited lower species diversity and high numbers of Chydorus and

Simocephalus. Further, the highest peak relative abundance of D. lumholtzi occurred in the medium level nutrient treatment; however, the highest relative abundance was found within the low nutrient treatment. Trophic state indicators, chlorophyll a, zooplankton biomass, or diversity could not explain these conflicting results. However, the fact that the species was always rare in high nutrient conditions and in low diversity communities suggests that relaxing resource limitation does not facilitate the invasion success of D. lumholtzi.

Although no differences in species diversity were found between invaded and non-invaded localities, the present study did find a significantly lower mean zooplankton body length within the invaded lakes. This difference was no longer found when D. lumholtzi was included in the analysis; however this can be explained as D. lumholtzi is larger than many of the native cladoceran species, thus its presence in the water column will result in an increase mean length and width of the zooplankton community. Further, several species including D. parvula, D. galeata, B. longirostris, and Ceriodaphnia species exhibited significantly lower length: width ratios in the invaded communities. The lower mean body size within the invaded communities found in this study is consistent with surveys of Lake Springfield, Illinois taken from 1992 to 1994 (Kolar et. al. 1997).

Zooplankton samples from that lake reported that increases in the abundance of D. lumholtzi correlated with changes in body size of the native zooplankton populations. As the densities of D. lumholtzi increased from 1992 to 1994, the mean length of the native zooplankton shifted from

0.41+ 0.04 mm to 0.18 + 0.01 mm. The presence of a smaller-sized community may suggest higher levels of vertebrate predation in invaded communities, as these predators have been shown to

selectively feed on larger zooplankton individuals (Brooks and Dodson 1965). The elongated spines of D. lumholtzi may aid in defense, allowing the invasive species to persist in vertebrate predator- dominated communities.

The presence of D. lumholtzi in communities with smaller mean body size could also be explained by competitive interactions. Because of its large size, D. lumholtzi may have a higher grazing efficiency than smaller individuals. A greater effectiveness of food collection may allow the invasive species to displace the native species and successfully establish. Alternatively, larger sized individuals may not become as easily displaced since, compared to D. lumholtzi, they may be equal or greater competitors for food resources; hence, the presence of large bodied zooplankton may result in a failed invasion. D. lumholtzi is routinely reported as greater in length than native Daphnia species (Havel and Herbert 1993, Hiskey 1996, Kolar et. al. 1997, Muzinic 2000, Lemke et. al.

2003). This study demonstrated that the core body length of D. lumholtzi, measured from the top of the eyespot to the base of the tail spine, averaged 0.59 (+ 0.05) mm. Therefore the length of D. lumholtzi, once the head and tail spines are excluded, is similar to that of other large sized daphnids.

For example, the core body length of D. galeata was 0.58 (+ 0.01) mm. Feeding efficiency and the maximum size particle capable of being consumed have shown a positive relationship with cladoceran body size (Brooks and Dodson 1965, Burns 1965, Smith and Cooper 1982). Thereby, the measurements from this study provide evidence that D. lumholtzi should perform more similarly to larger sized daphnids than previously assumed.

This study utilized cluster analysis to determine the zooplankton community composition most closely associated with D. lumholtzi. D. lumholtzi is closely associated with smaller sized zooplankton such as D. ambigua and Ceriodaphnia species. The species was not closely associated

with larger sized zooplankton including D. galeata and D. pulex. If larger bodied zooplankton are more efficient at collecting food particles than D. lumholtzi, the absence of D. lumholtzi from larger sized communities may be explained by competitive interactions. However, communities composed of large sized zooplankton are often subjected to high levels of invertebrate predation. It seems reasonable to assume that the large size and pronounced spines of D. lumholtzi would create handling difficulties for invertebrate predators, thus the invasive species would be selected against.

However, Dzailowski et. al. (2003) found that D. lumholtzi exhibited shorter tail spine and core body lengths when subjected to invertebrate kairomones. This contradiction to zooplankton life history theory, warrants further research into the role of invertebrate predation in resisting the invasion by

D. lumholtzi within lake communities

Alternatively, if D. lumholtzi is more efficient at collecting particles than smaller species, the presence of the exotic cladoceran in communities dominated by small bodied species suggests that it should be a stronger competitor than the smaller zooplankton. However, communities comprised of small bodied species are often characterized by high vertebrate predation levels that reduce the densities of larger sized species. The survival of D. lumholtzi in such predator- dominated communities may be explained by the elongated spines of the species. Exotic species often lack natural predators in invaded habitats or possess mechanisms for predation resistance

(Johnson and Havel 2001). D. lumholtzi supports a long helmet and post-abdominal spine, which possibly aids in defense against predators and allows for selective advantage over other Daphnia species.

The persistence of the pronounced spines of D. lumholtzi in non-native habitats suggest invertebrate and vertebrate predation pressures may have a strong influence in determining which

lakes may be vulnerable to invasion. Production of the elongated spines is energetically costly to D. lumholtzi, possibly resulting in longer maturation times and smaller broods. Given these high costs and the fact that the size of the species may be enough to deter predation, the reason why the species produces such elaborate spines is questioned. One explanation for such extreme morphology may be related to high predation pressure in its native environment (Kolar and Wahl 1998). Although the defense provided by the spines may facilitate the invasion of D. lumholtzi, the high energetic demands associated with this defense may also hinder the population growth rates of the species; a disadvantage which may explain why not all introductions result in successful establishment.

D. lumholtzi has been reported to reach peak numbers in the late summer months when other daphnids are rare or absent, suggesting that the species may be taking advantage of an open niche within these environments. However, this idea does not rule out that D. lumholtzi may be interacting with other taxa that have customarily filled this thermal niche. Dzialowski et. al. (2000) observed that the abundance of Diaphanosoma was higher in reservoirs where D. lumholtzi was undetected.

The cluster analysis in this study also revealed that D. lumholtzi was not closely associated

Diaphanosoma birgei as well as Bosmina longirostris and Eubosmina coregoni. Each of these species maintain high numbers during the warmer months. High densities of these native species during the summer may result in a suppression of D. lumholtzi numbers at a time when the exotic species typically reaches its peak numbers. This suppression may prevent the successful establishment of D. lumholtzi.

Conclusion.

The contemporary survey of 93 Ohio lakes and reservoirs identified the presence of D. lumholtzi in 19 locations. Many of the non-invaded localities are in close proximity or are downstream from invaded locations. For example, 22 non-invaded lakes are within the same county as an invaded location. Further, two of the main tributaries of the Ohio River are the Muskingum

River, in southeastern Ohio and the Sciotio River, which begins in west-central Ohio and flows southward. Each of these rivers are fed by waterbodies that contain reservoirs that have been invaded by D. lumholtzi. Studies have suggested that flowing water may be an important vector for the dispersal of D. lumholtzi (Havel et. al. 1995, Shurin and Havel 2002), however, the watersheds of these Muskingum and Sciotio Rivers still contain several lakes and reservoirs that have remained non-invaded. This indicates that several non-invaded lakes have strong potential for the introduction of D. lumholtzi, yet have been able to resist invasion thus far.

Invaded localities tended to be significantly larger in surface area than non-invaded lakes.

Invasion in larger lakes may be explained by increased boating traffic in these waterbodies. The limnological data of the lakes revealed no other significant differences between invaded and non- invaded lakes; it seems unlikely that local environmental conditions would prohibit D. lumholtzi from establishing in additional lakes.

This survey of Ohio lakes and reservoirs failed to show any significant differences in species diversity between the invaded and non-invaded locations. Analysis of the zooplankton species composition revealed that D. lumholtzi is closely associated with smaller sized zooplankton, but not with larger sized zooplankton or species that maintain high numbers during the summer months.

Predation, either by invertebrate or vertebrate predators, may have a strong influence in determining

in which lakes D. lumholtzi can successfully invaded. The elongated spines and large size of the species may act as a deterrent to predation, allowing the species to persist in communities characterized by high levels of predation. These results also suggest that larger bodied zooplankton may inhibit the establishment of D. lumholtzi, although the mechanism used to exclude the invasive species is unknown.

This study finds that it may be possible that invasion resistance in habitats is influenced by identities or traits of a particular species and not the overall diversity. In these situations, species identity may be more important than richness, thus high diversity is not needed to resist invasion.

The contemporary study was able to determine the communities compositions commonly associated with D. lumholtzi; however, more research is needed to identify the mechanisms responsible for making these communities vulnerable to the invasion of D. lumholtzi.

CHAPTER 2: PALEOLIMNOLOGICAL STUDY

INTRODUCTION

Studying the dispersal of exotic zooplankton into new environments offers the potential to estimate the rate at which species colonize and establish into new habitats. The results of the contemporary survey in the previous chapter suggested that failure to invade a particular habitat may be the result of interactions with the native species rather than abiotic characteristics. Other investigations of the invasion of D. lumholtzi suggest that the morphological features of the species may cause handing difficulties for several invertebrate and vertebrate predators, resulting in prey selection against the exotic species (Dzialowski et. al. 2000, Muzinic 2000, Johnson and Havel 2001, Celik et. al. 2002). Studies have also suggested that the presence of one particular zooplankton species may be enough to prevent establishment of D. lumholtzi (Work and Gophen 1999b, Havens et. al. 2000, Dzialowski et. al. 2007); accordingly, if no strong competitors are present at the time of introduction, D. lumholtzi may be able to establish.

The lakes and reservoirs in the contemporary survey were rarely assessed in continuous years; several of the locations were surveyed only once within the 16 year span that the OEPA collected samples. Consequently, this survey may not necessarily represent the changes in zooplankton composition over time or the changes in species abundance that follow the

43 44

invasion of D. lumholtzi. Further, samples were collected only from the pelagic region of the lakes between April and September, thus the littoral species may be underestimated and changes in zooplankton abundance which resulted from seasonal succession cannot be accounted for. In order to provide a continuous record of zooplankton species abundance and to examine changes in the species composition that may occur from the invasion of D. lumholtzi, a paleolimnological investigation was performed.

One source of information to address the questions regarding past and present zooplankton populations and community dynamics lies within the lake sediments, as sediments store the histories of community dynamics of many organisms (Hairston et. al. 1999, Duffy et. al. 2004). A paleolimnological study can be valuable in the aspect that this field of ecology is capable of adding the perspective of time to understand the changes that take place in lake systems. Through examination of sediment cores it is possible to reconstruct the environmental history of a lake (Brugam 1983). While the frequency of a failed invasion is hard to detect, the failure of an invasive species to persist after it becomes established is even less commonly reported. Further, paleolimnological investigations have the advantage that they provide a complete successional history of any lake, include each habitat, and avoid the problems associated with seasonality and patchiness (Warner 1983). Previous studies of zooplankton remains have been used to determine the response of lake systems to impacts such as eutrophication, pesticides, and acidification (Fritz and Carlson 1982, Binford et. al. 1983,

Duigan and Birks 2000).

Paleolimnology: A Review

Cladocerans are a common member of the zooplankton community where they occur both in the open water as well as the littoral zone. Typically, the families of Daphniidae and

Bosminidae are established offshore and others such as Chydoridae densely populate the littoral flora and substrates (Duigan and Birks 2000). In most situations, investigation of cladoceran remains are based on sediment profiles collected from the deepest part of the basin.

This methodology is based on the idea that the remains of the species from littoral habitats are transported offshore and mixed with those of the pelagic species before they are both incorporated into the sediments. Thereby, the sediments in the deepest region of the lake are thought to be composed of a spatially and temporally-integrated sample of the communities that lived in different habitats within the lake (Warner 1983). Recent studies have demonstrated that samples from other offshore locations yield the same proportion of remains as those from the deepest location, suggesting that the cladocerans species are well represented in all samples from the pelagic regions of any particular lake (Smol et. al. 2001).

Sediments contain a variety of inorganic and organic remains that may be used to reconstruct the history of a lake. As zooplankton grow, molt, and die, their exoskeletons become a part of the lake sediments (Duigan and Birks 2000). The chitinous skeletal structure of the cladocerans preserve better then those of copepods, usually resulting in a shift from copepod-dominated water to cladoceran microfossil-dominated sediments. Chitin degrades slowly, yet not all exoskeleton remains degrade at the same rate. Those with certain crystalline structures in their chitin polymers are more hydrated and thus become more preserved (Smol et. al. 2001). A major constraint is that, while members of the families Chydoridae and

Bosminidae are well preserved, the skeletal components from the remaining nine families of cladocerans preserve selectively. For example, daphnids are weakly preserved as their exoskeleton is too fragile to resist decomposition and attack by fungi. Consequently, only certain parts of their exoskeleton may be represented within the sediments. The remains left by well-preserved cladoceran taxa include head shields, carapaces, post abdominal claws, and epihippia (Whiteside and Swindoll 1988, Smol et. al. 2001). Among the Daphnidae only the post-abdominal claws, mandibles, and epihippia occur within the sediments (Frey 1960).

However, due to unique differences in morphology of epihippia and post-abdominal claws, most daphnid exoskeletons and remains are identifiable to species (Shumate et. al. 2002).

Sedimentary Pigments and Trophic State

The accumulation rate of sedimentary pigments has demonstrated a positive correlation with the primary productivity of a lake at the time of deposition (Swain 1985). As photosynthetic material dies in the water column, chlorophyll a is degraded to chlorophyll derivatives which include pheophytin, pheophorbide, chlorophyllide, as well as their isomers and allomers. Degradation of the pigments occurs though bacterial activity, photoxidation, and grazing. The percent native chlorophyll is the portion of chlorophyll that has yet to be decomposed, thus it can be used as an indication of preservation for other sedimentary pigments. High values of percent native chlorophyll may indicate increased algal biomass, whereas high values may also signify decreased oxygen concentration in the hypolimnion since degradation of sedimentary pigments will decline in anoxic conditions. (Brown 1968, Smol et. al. 2001).

Pigments within the sediment profile have been used to determine which types of algae were present since some pigments are specific to certain algae types. For example, myxoxanthophyll and oscillaxanthin are unique to Cyanobacteria. While myxoxanthophyll is common in many cyanobacteria species, oscillaxanthin is found in only two genera:

Oscillatoria and Arhrospira. The presence of cyanobacteria is often related to eutrophic conditions in a water body, thus shifts in these pigments may indicate a change in trophic status. Further, the accumulation rate of sedimentary pigments may also exhibit a positive correlation with primary productivity at the time of deposition (Swain 1985). Cyanobacterial pigment data can be simplified using the oscillaxanthin to myxoxanthophyll ratio, which provides a measure of the relative productivity of these pigments independent of preservation effects. A ratio value greater than 1.0 indicates higher levels of algae containing oscillaxanthin, suggesting a phytoplankton assemblage dominated by Oscillatoria species and, hence, eutrophic conditions (Swain 1985).

Objectives of the Study

The specific objectives of this paleolimnological study were to utilize the cladoceran remains from the sediment cores to distinguish the community structure prior to invasion and to identify particular species, or species compositions, which either facilitated or resisted the establishment of D. lumholtzi. The microfossils were also used to identify changes in species abundance that followed the invasion of D. lumholtzi and to determine the impact that the species had on zooplankton community structure. Finally, changes in sedimentary pigments

were examined to determine if the presence of D. lumholtzi in the water column had the ability to alter the phytoplankton composition or the rate of primary productivity.

METHODS

Sediment cores were taken from six Ohio reservoirs: East Branch Reservoir, Lake

Punderson, LaDue Reservoir, Mosquito Creek Reservoir, Lake Berlin, and Lake Milton. All locations are located in the northeastern corner of Ohio (Figure 8). The sampling was limited to locations in one area of the state in order to minimize differences in zooplankton composition that could have been attributed to differences in land use practices, topography, or climate.

The reservoirs represented three different D. lumholtzi categories: non-invaded, failed invasion, and invaded. These distinctions were made based on the data collected from the contemporary study. Lake Punderson and East Branch Reservoir were classified as the non-invaded lakes; D. lumholtzi was not detected within any samples from the contemporary survey. The failed invasion category, or lakes where D. lumholtzi was unable to successfully establish, included

LaDue Reservoir and Mosquito Creek Reservoir. Finally, Lake Milton and Lake Berlin were classified as the invaded lakes as high levels of D. lumholtzi were found in these lakes from

2005 to 2007.

Figure 8: Location of the lakes used in the paleolimnological study.

Lake Profiles

Non-Invaded Lakes:

East Branch Reservoir:

East Branch Reservoir is located in eastern Geauga County, near Chardon, Ohio. The reservoir is created by East Branch Reservoir Dam on the East Branch of the Cuyahoga River.

East Branch Reservoir Dam was constructed in 1939 by the City of Akron in order to create a water body to be used for drinking water and recreation purposes. At normal levels it has a surface area of 1.6 km2 with 12 km of shoreline (Find Lakes, nd).

The contemporary survey revealed that this lake has high numbers of Daphnia retrocurva and Diaphanosoma birgei. Although this reservoir is a public lake used for recreational purposes, this lake did not have D. lumholtzi. Seventeen lakes in the contemporary survey were located in Geauga County or in the Cuyahoga River watershed; none were invaded by D. lumholtzi. However, East Branch Reservoir is in close proximity to lakes in which D. lumholtzi has been reported. For example, the reservoir is only 32 km away from

Mosquito Creek Reservoir, 43 km from Lake Milton, and 52 km from Lake Berlin.

Additionally, each of these lakes are used for recreational purposes and exhibit heavy boating traffic; thereby, the proximity of East Branch Reservoir to these invaded lakes suggests a high probability for invasion.

Lake Punderson

Lake Punderson is the largest and deepest kettle lake in Ohio, with a maximum depth of

20 m and covering 0.4 km2 with 4 km of shoreline. Located in Newbury, Ohio, Geauga

County, this lake is situated in the glaciated plateau region of Ohio. The last glacier to enter

Ohio's boundaries, the Wisconsinan, receded about 12,000 years ago resulting in the formation of Lake Punderson. (Ohio Department of Natural Resources a, nd)

Zooplankton species in high abundance in Lake Punderson include Bosmina longirostris and Chydorus sphaericus, as shown in the contemporary survey. Although D. lumholtzi was not found in any water bodies with Geauga County; Lake Punderson is only 39 km from Mosquito Creek Reservoir, 42 km from Lake Milton, and 49 km from Lake Berlin.

Each of these lakes, along with Lake Punderson, are public state lakes; thus it is reasonable to assume there is a high level of traffic among the lakes for recreational purposes, which may increase the risk of invasion by D. lumholtzi into Lake Punderson.

Failed Invasion Lakes

LaDue Reservoir

LaDue Reservoir is located in Geauga County near Mantua, Ohio. The reservoir was constructed in 1963 on a tributary of the Cuyahoga River to provide an additional water supply to the city Akron. At normal levels, the lake covers 6 km2 with 33 km of shoreline (Ohio

Department of Natural Resources b, nd)

The contemporary survey determined that D. lumholtzi was present in LaDue Reservoir in 1997; but by 2005, the invasive species was no longer detected within the lake. The disappearance of D. lumholtzi from LaDue Reservoir suggests that this species was unable to successfully establish in this water body.

Mosquito Creek Lake

Mosquito Creek Lake is situated in Trumbull County, near the town of Cortland, Ohio.

With a surface area of 32 km2 and a shoreline length of 64 km, it is the second largest inland lake in the state. The reservoir has an average depth of 3 m, while the maximum depth is 13 m.

Completed in 1944, the Mosquito Creek Reservoir Dam stands 14 m high and 1722 m long. At the north end of the lake is an uncontrolled natural spillway, designed to reverse the flow of water when levels reach 275 m above sea level, such that the water flows north through the spillway into a tributary of the Grand River, eventually flowing into Lake Erie (Ohio

Department of Natural Resources c, nd)

D. lumholtzi was found in Mosquito Creek Lake in 2005, but was not found in samples collected from 2006 to 2008, thus it was assumed that the invasive daphnid was unsuccessful at establishing a population in this reservoir. From 2006 to 2008 high numbers of the invasive,

Eubosmina coregoni, were found within this lake. This species was not detected during the time when D. lumholtzi was present in Mosquito Creek Lake.

Invaded Lakes

Lake Berlin

Lake Berlin is located on the Mahoning River, upstream from Warren, Ohio. The reservoir crosses the county boundaries of Mahoning, Portage and Stark Counties. Along with

Mosquito Creek Lake, Lake Berlin is one of sixteen flood control projects by the U.S. Army

Corps of Engineers, Pittsburgh District. Lake Berlin covers 15 km2 and has a shoreline length of 30 km (US Army Corp of Engineers, nd). Lake Berlin has a mean depth of 7 m and a maximum depth of 17 m; although annual water level fluctuations can be as much as 6 m.

(Ohio Department of Natural Resources d, nd).

The contemporary survey revealed that D. lumholtzi has been present in this lake from at least 2005. Other zooplankton species found within the lake include Bosmina longirostris,

Diaphanosoma birgei, D. galeata, and D. parvula.

Lake Milton

Lake Milton is located in Milton Township, Ohio, in Mahoning County. Lake Milton dam, which impounds the Mahoning River experienced structural problems during the 1970’s.

Consequently, the reservoir was drained from 1986 to 1988 is order to repair the dam.

Presently, Lake Milton covers 7 km2 and has a shoreline length of 37 km. (Ohio Department of

Natural Resources b, nd). Zooplankton species found this lake, as detected by the contemporary survey, include Bosmina longirostris, Diaphanosoma birgei, Ceriodaphnia species, D. galeata, D. parvula, as well as D. lumholtzi.

Coring Techniques

The sediment cores were taken using a WildCo drop corer; Model # 2404 A14, fitted with a 5 cm diameter plastic tube. One core was taken from each lake or reservoir, near the deepest area. The length of the cores ranged from 23 to 34 cm, determined by the maximum depth of which the corer could penetrate the sediment. Each core was sectioned into one centimeter sections and placed into individual plastic vials. As the core was extruded, the outer portion of each section was discarded in order to prevent contamination from other layers. The vials were stored at approximately 8 °C in the dark to prevent degradation.

Analytical Techniques

The following techniques were applied to each one centimeter layer of the sediment core. a. Dry weight: Sediment density was measured by placing five cm3 of sediment from each

layer in tared porcelain crucibles. Each sample was dried at 105 °C for 36 hours, allowed to

cool to room temperature in a desiccant jar, and reweighed. Percent dry weight was

calculated using the formula:

Percent Dry Weight = (Dry Weight / Wet weight) x 100 b. Organic matter: The dried samples used to determine sediment density and dry weight were

ignited at 550 °C for four hours, allowed to cool to room temperature in a desiccant jar, and

reweighed. The weight loss on ignition was considered to be the result of the loss of

organic matter. Percent organic matter was calculated using the formula:

Percent organic matter = ((Dry Weight – Weight after ignition at 550 °C)/Dry Weight) x 100

c. Sedimentary pigments: Sediment pigments were extracted to determine the percent native

chlorophyll, chlorophyll derivatives, and two pigments distinctive of Cyanobacteria from

each one centimeter section of the cores. The procedure followed Swain (1985). To avoid

further degradation of the pigments, the procedure was performed under near dark

conditions. Two replicates were analyzed from each core layer.

Two 2.5 cm3 samples of sediment from each layer were placed into 50 ml centrifuge

tubes and five ml of 90% ethanol was added. The tubes were than mixed and centrifuged

for 10 minutes. The extract was brought up to a final volume of 25 ml with the addition of

90% ethanol.

The concentrations of the pigments were measured colorimetricaly using a Bausch

and Lomb Spectronic 88 Spectrophotometer. Chlorophyll derivates were read at 665 nm.

Swain (1985) expressed the amount of pigment as absorbance per gram of organic matter;

where 1 absorbance unit is equal to 1 absorbance in a 10 centimeter cell when dissolved in

100 ml solvent. However, a 1 centimeter cell was used in this study; thus, the results were

multiplied by 10. Percent native chlorophyll was measured by adding 0.1 ml of 10%

hydrochloric acid to the sample. Ninety seconds after adding the acid, the extract was read

again at 665 nm. Percent native chlorophyll was calculated using the following formula:

Percent native chlorophyll = (665 nm before acidification – 665 nm after acidification)

0.7 (665 after acidification) x 100

The cyanobacterial pigments, myxoxanthrophyll and oscillaxanthin, were measured by pouring 18 ml of the extract into a separation funnel. As the funnel was swirled, 40 ml of petroleum ether was added. The hypophase was removed from the funnel and transferred into test tubes. The tubes were allowed to dry overnight under air jets. Once dry, the pigments were redissolved in 5 ml of absolute ethanol. Myxoxanthrophyll was read at 504 nm and oscillaxanthin was read at 529 nm on the spectrophotometer. Phorbin, a pigment complex which is needed to calculate the final absorbance of the pigments, was read at 412 nm. The following formulas were used to calculate the concentration of the pigments:

Net absorbance:

O 529 = (1.266 x A529) - (0.219 x A504) - (0.081 x A412)

M 504 = (1.358 x A504) - (1.308 x A529) - (0.031 x A412)

Absorbance at peaks:

O 495 = 1.27 (O529)

M 473 = 1.20 (M504)

Pigment concentration = 10,000 (V) (A) / (E) (P)(g org)

Where:

V = Volume of extract (% ml in this study)

A = Absorbance at peak

E = Extinction coefficient (1450 for oscillaxanthin, 2100 for myxoxanthrophyll

P = Proportion of the extract used (0.6 in this study)

g org = Weight of organic matter from the extracted sediment.

d. Cladoceran microfossils. The technique for processing the sediment for cladoceran remains

follows Smol et. al. (2001). This procedure is recommended for all sediment types and

provides a gentle method for the removal of inorganic material and carbonates while

reducing the fragmentation and degradation of the zooplankton remains.

Four cm3 of sediment was added at 150 ml of 10% potassium hydroxide (KOH)

solution. The solution was heated to 70 to 80 ° for 30 minutes, with a magnetic stirrer

providing constant stirring at a low speed. The solution was poured onto a 35 µm sieve and

rinsed using tap water until the water came through clear. Examination of the rinse water

determined that all cladoceran remains were retained on the sieve. The residue retained on

the sieve was transferred into plastic vials. Three drops of safranin glycerin solution and 1

ml of 90% ethanol was added to the vial. The safranin glycerin solution was added to stain

the microfossil remains, whereas the ethanol was used as a preservative.

Slides were prepared by pipeting the sample onto a glass slide and covering it with a

cover slide. The slide was scanned at 100x magnification. At least 200 remains were

counted from each one centimeter section of each core, which is the minimum number

recommended for counting (Smol et. al. 2001). All cladoceran remains were tabulated

separately, but only the most frequent body part (i.e. head shields, shells, post abdomens,

claws) from each taxon was be used as an estimate of species abundance. Identification of

the remains were made using Balcer, Korda, and Dodson (1984), Brooks (1957), Deevey

and Deevey (1971), Dodson and Frey (1991), Frey (1959, 1965), Pennak (1978), as well as

comparison to preserved zooplankton from the contemporary survey.

Daphnids were identified to the lowest possible taxonomic level, either species or species-type. The identifications were determined by the size and position of the teeth within the pectens from each post-abdominal claw. D. galeata – type claws are distinguished by small teeth that are equal size on all three pectens. Native zooplankton found in Ohio that exhibit this morphology include D. galeata and D. ambigua. Teeth found in the middle and proximal pecten that are slightly larger than those found on the distal pecten are characteristic of species with D. parvula – type claws. In addition to D. parvula, this claw type is also found in D. retrocurva. Teeth of the middle pecten that are distinctly larger than either the middle or distal pecten are distinctive of D. pulex-type claws. Although four species of Daphnia share this morphology, D. pulex is the only daphnid found in Ohio that exhibits this claw type. The post- abdominal claw of D. lumholtzi is distinct from native zooplankton species found in Ohio. The claw of this invasive species is characterized by teeth of the proximal claw that are slightly larger than the teeth found on the middle or proximal pectens.

All zooplankton microfossil carapaces, head shields, and post-abdominal claws were measured for length. A linear correlation between claw length and body length has been found in Daphnia species (Dodson 1970), thus the length of all daphnid claws were measured to estimate the mean body size with the daphnid community. It has also been suggested that bosminids produce longer mucro lengths to reduce the risk of predation; hence, mucro length may be correlated to the presence of invertebrate predators (Kerfoot 1981, Post et. al. 1995,

Hellsten et. al. 1999). For that reason, the mucro of Bosmina microfossils was measured independently of the carapace.

Statistical analysis on the zooplankton remains was performed using MVSP Plus (version

3.13). Species data was analyzed using detrended correspondence analysis (DCA) to identify particular species, or species compositions associated with D. lumholtzi. MVSP Plus (version

3.13) was also used to calculate Shannon Weiner diversity, species evenness, and species richness. The Shannon Weiner diversity index was chosen to represent the diversity of the zooplankton communities as it minimizes the effects of the large number of rare species

(Pielou 1966). ANOVA and post hoc tests used to analyze the differences among the diversity indices were performed using BioStat 2008.

RESULTS

Percent Dry Weight.

The percent dry weight values in Lake Punderson remained relatively constant throughout the entire core, having a median value of 10.0%. The values from the uppermost sections, layers 0 to 2 cm, were averaged to provide a value representing the most recent conditions within the lake (Table 6). Lake Punderson had a percent dry weight surface average of 8.6%, which was the lowest value of the lakes used in this study. The percent dry weight from the LaDue Reservoir core decreased from the bottom of the core to core surface, with one sharp increase at the depth of 2 cm. The increase occurred just after the appearance of D. lumholtzi, which was detected at a depth of 4 cm. The percent dry weight surface average for this lake was 51.9% and the median of the core was 62.3%, the highest value in the lakes in this study. East Branch Reservoir, Lake Milton, Lake Berlin, and Mosquito Creek Reservoir

also had trends of decreasing percent dry weight from the bottom to core surface, although these trends were less exaggerated in these cores. No notable changes occurred after the detection of D. lumholtzi within the cores from the invaded or failed invaded lakes. (Figure 9)

Organic Matter

The highest percent of organic matter was found in Lake Punderson, as this lake demonstrated a median value of 36.7% and an average surface value of 36.5% (Table 6). This lake also exhibited the greatest amount of fluctuation for this variable, as the percent values ranged from 34.0 to 40.5%. LaDue Reservoir had increasing percents of organic matter throughout the core. The percent of organic matter in deepest section of this core was 3.3%, yet the amount of organic material increased to 5.4% at the core surface. Despite the increasing values, LaDue Reservoir exhibited the lowest values in percent organic matter, as the median value of the core was 4.0% and the surface average was 5.1%. The percent organic matter in

East Branch Reservoir, Lake Milton, and Lake Berlin remained fairly constant through the core, yet each lake exhibited a slight increase near the core surface. As the slight increase in values is found only near the surface of the cores, no correlation can be made with the detection of D. lumholtzi within the core. Mosquito Creek Reservoir exhibited relatively constant levels from the bottom of the core until a depth of 4 cm, where a slight fluctuation was seen. The fluctuation occurred as the percent of organic matter decreased from 8.0% to 5.8% between layers 5 to 4 cm. D. lumholtzi was detected within this core at a depth of 10 cm; however, the highest numbers of the invasive species was found at the same depth that this fluctuation occurred.

Table 6: The average percent of dry weight and organic matter from the surface sediments (core layers 0 to 2 cm). Median values were calculated using the entire cores taken from each of the lakes used in the paleolimnolgical study.

Organic Matter Dry Weight % Dry Weight % Organic Matter % Surface Surface Average Median % Median Average East Branch Reservoir 19.6 34.0 10.7 9.3 Lake Punderson 8.6 10.0 36.5 36.7 LaDue Reservoir 51.9 62.3 5.1 4.0 Mosquito Creek Lake 26.7 37.8 6.6 7.5 Lake Berlin 28.4 40.0 6.9 6.8 Lake Milton 37.0 34.7 8.8 7.9

0 0

5 5

10 10

15 15

20 20 Depth (cm) Depth Depth (cm) Depth

25 25

30 30

35 35 0 20406080100 0 1020304050 Percent Dry Weight Percent Organic Matter

East Branch Reservoir Lake Punderson East Branch Reservoir Lake Punderson LaDue Reservoir Mosquito Creek Lake LaDue Reservoir Mosquito Creek Lake Lake Berlin Lake Milton Lake Berlin Lake Milton

Figure 9: The percentage of dry weight and organic matter from the six sediments cores in this study.

Sedimentary pigments

Chlorophyll

The pigment analysis revealed numerous fluctuations in both the concentration of chlorophyll derivatives and percent native chlorophyll throughout each of the six sediment cores. None of the fluctuations correlated with the appearance of D. lumholtzi in the invaded or failed invasion lakes. In effort to facilitate viewing any trends that may occur in the cores, a 5 point moving average was calculated for the pigments of each core (Figures 10 and 11).

The concentration of chlorophyll derivatives in East Branch Reservoir demonstrated a period of decreasing values near the core bottom and an increase, followed by a slight decrease, near the core surface. East Branch Reservoir exhibited the lowest median chlorophyll derivatives value from the lakes at 0.22 (absorbance units/ g organic matter). However, the surface sediment average from this reservoir was 0.36 (absorbance units/ g organic matter), which was higher than Lake Punderson, Mosquito Creek Lake, and Lake Berlin (Table 7).

Lake Punderson demonstrated nearly an opposite trend from East Branch Reservoir as concentrations of chlorophyll derivatives peaked in depths 9 to 18 cm. LaDue Reservoir demonstrated continuously increasing values of chlorophyll derivatives throughout the core.

The moving average also indicated an increasing trend of chlorophyll derivatives concentrations in Lake Milton, although the actual data from the reservoir demonstrated numerous fluctuations throughout the core. Lake Milton also exhibited the highest values of concentrations of chlorophyll derivatives as the core revealed a median value of 0.56

(absorbance units/ g organic matter) and an average value of 1.07 (absorbance units/ g organic

matter) at the surface. Lake Berlin and Mosquito Creek Reservoir exhibited fairly constant values throughout the cores; however, in the more recent sediments Lake Berlin showed an increase in concentration of chlorophyll derivatives, while Mosquito Creek Reservoir exhibited a decrease. As a result of this decrease Mosquito Creek Lake had the lowest surface average for concentration of chlorophyll derivates with a value of 0.18 (absorbance units/ g organic matter).

East Branch Reservoir exhibited increasing percentages of native chlorophyll from the core bottom to a depth of 28 cm. Depths from 27 to 13 cm showed decreasing percents; however, values began to increase again in recent sediments. East Branch Reservoir demonstrated the lowest median values of percent native chlorophyll with a value of 33.23%

(Table 7). However, as a result of the increase near the top of the core, the surface average value increased to 54.09%. Lake Punderson revealed a similar trend to East Branch Reservoir as percent native chlorophyll values decreased from the bottom of the core to a depth of 10 cm, after which values began to increase, Lake Punderson exhibited the highest value of percent native chlorophyll as the core revealed a median value of 68.94% and an average value of

68.63% at the surface Lake Milton exhibited the lowest average surface value at 34.17% native chlorophyll. This reservoir, along with Lake Berlin, Mosquito Creek Lake, and LaDue

Reservoir demonstrated a fluctuating pattern similar to the pervious lakes. Each of these lakes exhibited decreasing values near the bottom of the core, yet percent native chlorophyll values began to increase in the most recent sediment layers.

Table 7: The average chlorophyll derivatives (CH Der) (absorbance units/ g organic matter), percent native chlorophyll (& Na CH), oscillaxanthin (OSC) concentration (µg pigment / g organic matter), myxoxanthophyll (MYX) concentration (µg pigment / g organic matter), and oscillaxanthin to myxoxanthophyll ratio (OSC/MYX) from the surface sediments (core layers 0 to 2 cm) and median values (Med.) from entire cores taken from each of the lakes used in the paleolimnolgical study. The study lakes are as follows: East Branch Reservoir (EB), Lake Punderson (LP), LaDue Reservoir (LD), Mosquito Creek Lake (MC) Lake Berlin (LB), and Lake Milton (LM). % Na OSC/ CH Der CH CH % Na OSC MYX MXY OSC/ Surface Der Surface CH Surface OSC Surface MYX Surface MYX Average Med. Average Med. Average Med. Average Med. Average Med.

EB 0.36 0.22 54.09 33.23 6.46 2.67 98.19 18.32 0.06 0.11

LP 0.27 0.27 68.63 68.94 7.88 21.51 62.70 100.21 0.22 0.32

LD 0.60 0.24 43.90 40.21 5.47 1.27 2.65 3.54 1.67 0.50

MC 0.18 0.24 58.79 48.94 3.85 6.73 8.28 5.68 0.81 1.46

LB 0.34 0.29 66.90 38.24 4.37 3.74 25.46 15.16 0.26 0.24

LM 1.07 0.56 34.17 35.79 1.72 6.01 9.78 13.29 0.16 0.66

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 5 10 10 10 15 15 20 Depth (cm) Depth Depth (cm) Depth (cm) Depth 15 20 25

20 30 25

35 30 25 0.0 0.2 0.4 0.6 0.8 1.0 0.00.20.40.60.81.0 0.0 0.2 0.4 0.6 0.8 1.0 Concentrartion of chlorophyll Concentrartion of chlorophyll derivatives (absorbance units/ Concentrartion of chlorophyll derivatives (absorbance units/ g organic matter) derivatives (absorbance units/ g organic matter) g organic matter)

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5 10 10 10 15 15 20 Depth (cm) Depth (cm) Depth (cm) Depth 15 20 25

20 25 30

30 35 25 0.0 0.2 0.4 0.6 0.8 1.0 0.00.20.40.60.81.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Concentrartion of chlorophyll Concentrartion of chlorophyll Concentrartion of chlorophyll derivatives (absorbance units/ g derivatives (absorbance units/ g derivatives (absorbance units/ g organic matter) organic matter) organic matter)

Figure 10: The concentration of chlorophyll derivatives from the six sediment cores of this study. The black trend lines represent the moving average of each five consecutive layers. The arrow represents the point where D. lumholtzi was detected in the core.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 5

10 10 10 15 15 Depth (cm) 20 Depth (cm) 15 Depth (cm) Depth 20 25 20 25 30

35 30 25 0 20406080100 0 20406080100 0 20406080100

Percent Native Chlorophyll Percent Native Chlorophyll Percent Native Chlorophyll

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5

10 10 10 15 15

Depth (cm) Depth 20 Depth (cm) Depth Depth (cm) Depth 15 20 25 25 20 30

30 35 25 0 20406080100 0 20406080100 0 20 40 60 80 100 Percent Native Chlorophyll Percent Native Chlorophyll Percent Native Chlorophyll

Figure 11: The percent native chlorophyll from the six sediment cores of this study. The black trend lines represent the moving average of each five consecutive layers. The arrow represents the point where D. lumholtzi was detected in the core.

Cyanobacterial Pigments

The concentrations of the cyanobacterial pigments, oscillaxanthin and myxoxanthophyll, exhibited considerable variation in each of the sediment cores (Figures 11 to 13). Since fluctuating patterns continued near the top of the cores, no correlation can be made with the detection of D. lumholtzi in the invaded and failed invasion lakes. The cyanobacteria pigment data can be simplified using the oscillaxanthin to myxoxanthophyll ratio. A ratio value greater than 1.0 indicates higher levels of algae containing oscillaxanthin, indicating a phytoplankton assemblage dominated by Oscillatoria species, thus eutrophic conditions (Swain 1985).

Concentrations of both oscillaxanthin and myxoxanthophyll gradually increased throughout the core from East Branch Reservoir. The ratio of the two pigments remained relatively stable throughout the core, reaching the highest values from depths 8 to 15 cm; yet the values of the ratio never reached 1.0. Oscillaxanthin was also never the dominant pigment in Lake Punderson, as the oscillaxanthin to myxoxanthophyll ratio never exceeded 1.0. Within this core, the pigment ratio steadily increased from the core bottom to a depth of 15 cm; however, the highest ratio obtained was 0.73. In a contrast from East Branch Reservoir, the concentrations of oscillaxanthin and myxoxanthophyll in Lake Punderson decreased near the top of the core. Although the oscillaxanthin to myxoxanthophyll ratios from East Branch

Reservoir and Lake Punderson, were among the lowest in the study; surface average concentrations of oscillaxanthin and myxoxanthophyll from these non-invaded lakes were higher than those from invaded or failed invasion lakes (Table 7)

Concentrations of oscillaxanthin remained relatively constant throughout the entire core from LaDue Reservoir, although levels did increase near the top of the core. Myxoxanthophyll

concentrations increased from the bottom of the core until a depth of 6 cm, afterwards a decreases was seen. The oscillaxanthin to myxoxanthophyll ratios remained above 1.0 in the bottom layers of the core. Following the depth of 16 cm, the ratio value dropped below 1.0.

However the ratio sharply increased at the core surface as a result of the abrupt increase in oscillaxanthin in the top layers of the core. LaDue Reservoir exhibited the lowest median concentrations of oscillaxanthin and myxoxanthophyll, with values of 1.27 and 3.54 (µg pigment / g organic matter). However, with a value of 1.67, this reservoir maintained the highest average surface sediment value for the oscillaxanthin to myxoxanthophyll ratio. The other failed invasion lake, Mosquito Creek Lake, exhibited the highest median cyanobacterial pigment ratio. The ratio values for this core remained above 1.0 for nearly the entire core. The ratio between the two pigments fell below 1.0 only in the top layers of the core. The recent sediments of Mosquito Creek Lake also demonstrated increasing levels of myxoxanthophyll, while oscillaxanthin concentrations decreased.

Oscillaxanthin to myxoxanthophyll ratio values higher than 1.0 were seen in the bottom sediments of the Lake Milton core; however above a depth of 16 cm, the ratio decreased and remained below 1.0 to the core surface. The concentrations of oscillaxanthin increased from the bottom to a depth of 13 cm. Levels then decreased until 8 cm, followed by a slight increase.

Concentrations of oscillaxanthin decreased again at the top of the core, resulting in the lowest surface average found from the lakes within this study. Myxoxanthophyll concentrations also demonstrated this same fluctuating pattern in the Lake Milton core. Highest myxoxanthophyll concentrations were seen at depths of 5 and 15 cm. High amounts of variability in cyanobacterial pigments were also found throughout the core from Lake Berlin. The recent

sediments of Lake Berlin demonstrated a decrease in the oscillaxanthin to myxoxanthophyll ratio near the top of the core, although the ratio of the two pigments remained below 1.0 throughout the entire core.

Zooplankton Microfossil Measurements

The mean length measurements of the carapaces, head shields, and claws are represented in

Figures 15, 17, and 19. Interactions between D. lumholtzi and the littoral community members are expected to be low; thus Figures 16 and 18, are presented to examine the changes in carapace and head shield length among only the pelagic species. Since D. lumholtzi is considered a large bodied daphnid, its presence in the community will likely result in larger mean body size of the community; hence, mean claw length was calculated using only the native Daphnia species as well as with the invasive species (Figure 19). To examine the length of the mucro in proportion to the size of the carapace, the mucro to carapace ratio was calculated for Bosmina longirostris, shown in Figure 20. Median microfossil length throughout each core and the average microfossil length from the surface sediments are shown in Tables 9 and 10.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 5

10 10 10 15 15

20 Depth (cm) Depth Depth (cm) Depth (cm) 15 20 25 25 20 30

30 35 25 0 20 40 60 80 100 024681012 0 5 10 15 20

Concentration of oscillaxanthin Concentration of oscillaxanthin Concentration of oscillaxanthin (ug pigment / g organic matter) (ug pigment / g organic matter) (ug pigment / g organic matter)

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5 10 10 10 15 15 20

Depth (cm) 15 Depth (cm) Depth Depth (cm) 20 25

20 25 30

25 30 35 0 5 10 15 20 0 5 10 15 0 10203040 Concentration of oscillaxanthin Concentration of oscillaxanthin Concentration of oscillaxanthin (ug pigment / g organic matter) (ug pigment / g organic matter) (ug pigment / g organic matter)

Figure 12: Concentration of oscillaxanthin (µg pigment / g organic matter)from the six sediment cores of this study. The black trend lines represent the moving average of each five consecutive layers. The arrow represents the point where D. lumholtzi was detected in the core.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 5 10 10

15 15 10

20 20 Depth (cm) Depth Depth (cm) (cm) Depth 15 25 25

20 30 30

35 35 0 50 100 150 0 50 100 150 200 250 25 0246810 Concentration of myxoxanthrphyll Concentration of myxoxanthrphyll Concentration of myxoxanthrphyll (ug pigment / g organic matter) (ug pigment / g organic matter) (ug pigment / g organic matter)

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5

10 10 10 15

20 Depth (cm) Depth Depth (cm) Depth (cm) Depth 15 20 25

20 25 30

30 35 25 0 5 10 15 0 1020304050 0 1020304050 Concentration of myxoxanthrphyll Concentration of myxoxanthrphyll Concentration of myxoxanthrphyll (ug pigment / g organic matter) (ug pigment / g organic matter) (ug pigment / g organic matter)

Figure 13: Concentration of myxoxanthrophyll (µg pigment / g organic matter)from the six sediment cores of this study. The black trend lines represent the moving average of each five consecutive layers. The arrow represents the point where D. lumholtzi was detected in the core.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 10

15 10 Depth Depth Depth (cm) 15 (cm) (cm) 20

20 25

20 30 25

35 30 0.0 0.2 0.4 0.6 0.8 1.0 1.2 25 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Oscillaxanthin to Oscillaxanthin to Oscillaxanthin to Myxoxanthophyll Ratio Myxoxanthophyll Ratio Myxoxanthophyll Ratio

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5

10 10 10

15 Depth Depth Depth (cm) 15 15 (cm) (cm) 20

20 20 25

25 25 30

30 35 30 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0.00.20.40.60.81.0 0.0 0.5 1.0 1.5 2.0 2.5 Oscillaxanthi to Oscillaxanthin to Oscillaxanthin to Myxoxanthophyll Ratio Myxoxanthophyll Ratio Myxoxanthophyll Ratio

Figure 14: Oscillaxanthin to myxoxanthophyll ratio from the six sediment cores of this study. The black trend lines represent the moving average of each five consecutive layers. The arrow represents the point where D. lumholtzi was detected in the core.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 5 10 10 15 10 15 20 15 Depth (cm) Depth Depth (cm)

20 (cm) Depth 25

25 20 30

35 30 25 00.51 0.0 0.5 1.0 1.5 00.51 Carapace Length (mm) Carapace Length (mm) Carapace Length (mm)

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0 5 5 5 10 10 10 15 15 20 15 Depth (cm) Depth Depth (cm) Depth Depth (cm) Depth 20 25

20 25 30

35 30 25 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 Carapace Length (mm) Carapace Length (mm) Carapace Length (mm)

Figure 15: Cladoceran microfossil carapace lengths from each of the lakes within the paleolimnological study. Carapace lengths in these figures include all species, pelagic and littoral, found within the cores. The arrow represents the point where D. lumholtzi was detected in the core.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 5 10 10 15 10 15 20 15 Depth (cm) Depth Depth (cm) Depth 20 (cm) Depth 25

25 20 30

30 35 25 0.0 0.5 1.0 00.51 00.51 Pelagic Carapace Length Pelagic Carapace Length Pelagic Carapace Length (mm) (mm) (mm)

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5 10 10 10 15 m) 15 20 15 Depth (cm) Depth Depth (c Depth 20 (cm) Depth 25 20 25 30

30 35 25 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 Pelagic Carapace Length Pelagic Carapace Length Pelagic Carapace Length (mm) (mm) (mm) Figure 16: Cladoceran microfossil carapace lengths from each of the lakes within the paleolimnological study. Carapace lengths are from the pelagic species found in the core, Bosmina longirostris, Eubosmina coregoni, and Chydorus sphaericus. No Daphnia carapaces were found in this study, accordingly daphnids are not represented in these figures. The arrow represents the point where D. lumholtzi was detected in the core.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 5 10 10 10 15 15 20 15 Depth (cm) Depth (cm) Depth

20 (cm) Depth 25

20 25 30

35 30 25 00.511.5 0.01.02.03.0 00.511.5 Headshield Length (mm) Headshield Length (mm) Headshield Length (mm)

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5

10 10 10 15 15 20 15 Depth (cm) Depth Depth (cm) Depth 20 (cm) Depth 25 25 20 30

30 35 25 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 Headshield Length (mm) Headshield Length (mm) Headshield Length (mm)

Figure 17: Cladoceran microfossil head shield lengths from each of the lakes within the paleolimnological study. Head shield lengths in these figures include all species, pelagic and littoral, found within the cores. The arrow represents the point where D. lumholtzi was detected in the core.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 5 10 10 15 10 15 20 15 Depth (cm) Depth (cm) Depth 20 Depth (cm) 25

25 20 30

35 30 25 00.511.5 0.01.02.03.0 00.511.5 Pelagic Headshield Pelagic Headshield Pelagic Headshield Length (mm) Length (mm) Length (mm)

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5 10 10 10 15 15 20 15 Depth (cm) Depth (cm) 20 Depth (cm) 25

25 20 30

30 35 25 0.0 1.0 2.0 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 Pelagic Headshield Pelagic Headshield Pelagic Headshield Length (mm) Length (mm) Length (mm)

Figure 18: Cladoceran microfossil head shield lengths from each of the lakes within the paleolimnological study. Head shield lengths are from the pelagic species found in the core, Bosmina longirostris, Eubosmina coregoni, and Chydorus sphaericus. No Daphnia head shield were found in this study, accordingly daphnids are not represented in these figures. The arrow represents the point where D. lumholtzi was detected in the core.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0 5 5 5

10 10 10 15 15

20 15 th (c th 20 Depth (cm) Depth p m) Depth (cm) Depth

De 25 25 20

30 30 25 35 35 00.51 00.51 00.51 Claw Length (mm) Claw Length (mm) Claw Length (mm) Claw Length - Native Daphnids Claw Length - Native Daphnids Claw Length - Native Daphnids Claw Length - All Daphnids

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5

10 10 10

m) 15 15 20

pth ( pth 15 Depth (cm) Depth

De20 c Depth (cm) Depth 25 20 25 30

30 35 25 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 Claw Length (mm) Claw Length (mm) Claw Length (mm)

Claw Length - Native Daphnids Claw Length - Native Daphnids Claw Length - Native Daphnids Claw Length - All Daphnids Claw Length - All Daphnids Claw Length - All Daphnids

Figure 19: Daphnid microfossil claw lengths from each of the lakes within the paleolimnological study. The “native daphnid claw lengths” includes claws from the D. galeata - type, D. parvula - type, and D, pulex - type species groups. The “all daphnid claw length category” includes these three Daphnia species groups as well as D. lumholtzi. In the non-invaded lakes this group this value was not calculated as D. lumholtzi was not detected within these lakes. The arrow represents the point where D. lumholtzi was detected in the core.

East Branch Reservoir Lake Punderson LaDue Reservoir 0 0 0

5 5 5 10 10

10 15 15 20 15 Depth (cm) Depth Depth (cm) Depth 20 (cm) Depth 25 20 25 30

35 30 25 0 0.1 0.2 0.3 0 0.1 0.2 0.3 0 0.1 0.2 0.3 Mucro to Carapacre Ratio Mucro to Carapacre Ratio Mucro to Carapacre Ratio

Mosquito Creek Lake Lake Berlin Lake Milton 0 0 0

5 5 5

10 10 10 15 15 20 15 Depth (cm) Depth Depth (cm) Depth Depth (cm) Depth 20 25 20 25 30

30 35 25 0.0 0.2 0.4 0.0 0.2 0.4 0.0 0.2 0.4 Mucro to Carapacre Ratio Mucro to Carapacre Ratio Mucro to Carapacre Ratio Figure 20: The mucro to carapace length ratios for Bosmina longirostris. This ratio was calculated for each of the lakes within the paleolimnological study. The arrow represents the point where D. lumholtzi was detected in the core.

Table 8: The median microfossil length values from entire cores taken from each of the lakes used in the paleolimnological study. All length measurements are in millimeters. The carapace length grouping includes all carapace microfossils found in the core layer. Pelagic carapace length includes microfossils from Bosmina longirostris, Eubosmina coregoni, and Chydorus sphaericus (no daphnid carapaces were found in this study). Head shield length grouping includes all head shield microfossils found in the core layer. Pelagic head shield length includes microfossils from Bosmina longirostris, Eubosmina coregoni, and Chydorus sphaericus (no daphnid head shields were found in this study). The native daphnid claw lengths includes claws from the D. galeata - type, D. parvula - type, and D, pulex - type species groups. The all daphnid claw length category includes these three Daphnia species groups as well as D. lumholtzi. In the non-invaded lakes this group this value was not calculated as D. lumholtzi was not detected within these lakes. The study lakes are as follows: East Branch Reservoir (EB), Lake Punderson (LP), LaDue Reservoir (LD), Mosquito Creek Lake (MC) Lake Berlin (LB), and Lake Milton (LM). Pelagic Native All Mucro to Pelagic Head Head Daphnid Daphnid Carapace Carapace Carapace shield shield Claw Claw Length Length Length Length Length Length Length Ratio

EB 0.72 0.58 0.88 0.98 0.46 na 0.19

LP 0.75 0.65 0.80 0.89 0.59 na 0.23

LD 0.73 0.62 0.93 1.00 0.68 0.68 0.22

MC 0.72 0.60 0.92 0.94 0.60 0.61 0.28

LB 0.74 0.64 0.93 1.10 0.51 0.52 0.24

LM 0.68 0.58 0.84 0.95 0.49 0.49 0.27

Table 9 : The average microfossil length from the surface sediments (core layers 0 to 2 cm) from each of the lakes used in the paleolimnological study. All length measurements are in millimeters. Measurement categories are as described in Table 8. The study lakes are as follows: East Branch Reservoir (EB), Lake Punderson (LP), LaDue Reservoir (LD), Mosquito Creek Lake (MC) Lake Berlin (LB), and Lake Milton (LM).

Pelagic All Pelagic Head Head Native Daphnid Mucro to Carapace Carapace shield shield Daphnid Claw Carapace Length Length Length Length Claw Length Length Length Ratio

EB 0.68 0.58 0.85 0.90 0.47 na 0.20

LP 0.72 0.61 1.07 1.21 0.52 na 0.23

LD 0.70 0.60 0.89 1.01 0.60 0.62 0.21

MC 0.68 0.67 0.88 0.91 0.61 0.64 0.27

LB 0.71 0.60 0.91 1.00 0.46 0.52 0.25

LM 0.64 0.58 0.66 0.82 0.30 0.33 0.31

Carapace Measurements

The carapace lengths of Lake Milton remained relatively constant throughout the core, at approximately 0.68, until a depth of 5 cm, at which the carapace lengths within the core began to decrease until the top of the core. This decrease was among the littoral carapaces; data representing the carapaces from only the pelagic species shows an increase in length near the top of the core. Lake Berlin exhibited a similar trend as the carapace lengths of this core show mild fluctuations in the bottom of the core, yet lengths began to decrease in the most recent sediments. No decrease was found in the data from the pelagic species. Lake Milton, along with East Branch Reservoir, demonstrated the lowest median and surface average values for

overall and pelagic carapace lengths. However, the values in these categories were among the highest in Lake Berlin.

A slight decrease was seen in the carapace lengths of the pelagic species within the recent sediments of Mosquito Creek Lake. However, slight variations of length were found throughout the core in both the lengths including all carapace microfossils as well as only the pelagic species. LaDue Reservoir, the other failed invasion lake, also exhibited fluctuations of carapace length throughout the entire core. These changes are expected to be related to the abundance of littoral species since the carapace lengths of the pelagic species of remained very consistent throughout the length of the sediment core.

East Branch Reservoir, a non-invaded lake, exhibited patterns similar to that of LaDue

Reservoir as the carapace lengths varied slightly throughout the core, yet lengths including only the pelagic species remained consistent, at approximately 0.58 mm. The lengths from

Lake Punderson were relatively stable over time, both in the pelagic as well as the lengths including all carapace microfossils found in the core layers. The pelagic species in Lake

Punderson did show a slight peak at a depth of 8 cm, where mean carapace length increased to

1.05 mm.

Head shield Measurements

The head shield lengths of Lake Berlin varied throughout the core. Only slight variation was found in the pelagic species from depths 26 to 12 cm, where head shield lengths of the pelagic species averaged 0.95 mm. From depth 12 cm, the depth at which D. lumholtzi was detected in the core, until the top of the core; the head shield lengths began to fluctuate as

values varied between 0.84 and 1.05 mm. The mean head shield length measurements of Lake

Milton showed strong variation throughout the sediment core. Lake Milton exhibited the lowest values for head shield length at sediment surface.

The head shield lengths from Mosquito Creek Lake demonstrated variations throughout the core. However, the head shield lengths including all microfossils as well as those from the pelagic species began to decrease from a depth of 8 cm to the top of the core. The other failed invasion lake, LaDue Reservoir, showed mild fluctuations, yet no noticeable shift in head shield length cold be identified.

The non-invaded lakes, East Branch Reservoir and Lake Punderson, demonstrated only minor variations of head shield length throughout the length of the sediment core. However, a slight peak in head shield length was found at a depth of 1 cm in the sediment core from Lake

Punderson. As a result of the increase, Lake Punderson exhibited the highest values of head shield length within the surface sediment; however, this lake exhibited the lowest median value for head shield length among the pelagic species.

Daphnid Claw Measurements

The native Daphnia claw length measurements of Lake Milton varied throughout the core; however, a decrease in claw length was noticed at a depth of 8 cm until the top of the core. When the claw lengths of D. lumholtzi were included in the analysis the same pattern was found, although mean claw lengths were slightly higher. Claw length decreased after the detection of D. lumholtzi in the Lake Milton core, but no noticeable shift was found in Lake

Berlin. Some mild fluctuations were found at the bottom of the Lake Berlin core, yet the claw

lengths of were consistent near the top of the core. The mean claw lengths at the sediment surface varied among the invaded lakes; however Lake Milton and Lake Berlin exhibited the lowest average value of all the lakes included in this study.

Mosquito Creek Lake exhibited strong variations in claw length, particularly in the top half of the sediment core. From depths 10 to 6 cm, claw length of the native daphnids increased to 0.60 mm. Claw lengths then decreased to 0.42 mm by the top of the core. This same pattern was seen when D. lumholtzi was included in the analysis; however, mean claw length was higher, particularly from depths 10 to 6 cm. LaDue Reservoir also exhibited a fluctuation within recent sediment that was stronger than any variation seen in the remainder of the core.

The failed invasion lake showed a decrease in native daphnid claws from depths 4 to 2 cm, at which D. lumholtzi achieved its highest percent abundance within this lake. As the abundance of the invasive species began to lessen at the top of the core, the native daphnid claw lengths in

LaDue Reservoir began to increase.

Daphnid claws were rare in the non-invaded lakes, East Branch Reservoir and Lake

Punderson, thus claw lengths is unrepresented in many of the core layers. The native claw lengths that are presented exhibit strong fluctuations throughout the entirety of the core, such that no patterns in claw length are visible.

Mucro to Carapace Ratio

The mucro to carapace ratio remained very consistent throughout the core from Lake

Milton, although a slight increase is seen in recent sediments. Although also very consistent through the core, the ratio values of Lake Berlin began to fluctuate at a depth of 14 cm and

continued until the top of the core. The invaded lakes, along with Mosquito Creek Lake, exhibited the highest median ratio values as well as the highest values at the sediment surface.

Only minor variations were founding the failed invaded lake, Mosquito Creek Lake and

LaDue Reservoir, and the non-invaded lakes, East Branch Reservoir and lake Punderson; such that no notable shift in mucro to carapace ratios could be identified.

Zooplankton Microfossil Percent Abundance

The cladoceran community of Lake Berlin was largely dominated by Bosmina longirostris. The percent abundance of this species had a median value of 86.7% (Table 10), and never fell below 76% within the sediment core. D. lumholtzi was first detected at a depth of 12 cm, yet by the top of the core the percent abundance of this species had risen to 6.0% of the cladoceran remains. D. galeata – type and D. parvula – type post-abdominal claws were also found within this core. The highest levels of these species types were found at depths 21 to

23 cm. Percent abundances of these daphnids decreased to 3.5% prior to the introduction of D. lumholtzi, yet began to increase again near the core surface (Figure 21).

The cladoceran community of Lake Milton was composed almost entirely of B. longirostris; however, the lower half of this core was also showed high percent abundances of

Eubosmina coregoni (Figure 22). The abundance of E. coregoni continuously declined throughout the core The percent abundance of this species was only 0.5% of the community at

7 cm, which was the depth D. lumholtzi was detected in the core. The percentage of E. coregoni remained low as the percent abundance of D. lumholtzi increased, reaching 5.2% in the most recent sediments. D. galeata – type and D. parvula – type claws were also detected in

the Lake Milton core. D, parvula – type remained at low numbers, but demonstrated consistent levels throughout the core. D. galeata-type reached its highest percent abundance between depths of 14 to 19 cm; however, percentages declined prior to the introduction of D. lumholtzi and continued to remain at low levels for the remainder of the core.

Mosquito Creek Reservoir also exhibited a heavy dominance by B. longirostris, although the median percent abundance value for this species, 54.0%, was lower than that found in the invaded lakes. E. coregoni was also present in the core; however, unlike Lake Milton and Lake

Berlin, this species exhibited high numbers throughout the entire core (Figure 23). The median percent abundance value of E. coregoni was 14.9% in Mosquito Creek Lake, whereas it was only 3.8% in Lake Milton and 0.0% in Lake Berlin. High percent abundances were also seen in

Chydorus sphaericus, Alona species, D. galeata and parvula – types. Additionally, D. pulex – type claws were found; this species group was absent in both invaded locations. D. lumholtzi was first seen at a depth of 7 cm; it reached its highest percent abundance of 3.5% at 4 cm, then declined in abundance near the core surface.

Unlike the previous lakes, LaDue Reservoir was not dominated by one species, rather had high percent abundances of B. longirostris, E. coregoni, and C. sphaericus. The daphnid species types D. galeata and D. parvula were found consistently throughout the core, but never achieved percent abundances higher than 1.0% (Figure 24). D. lumholtzi was detected at depths

3 to 4 cm, yet it only composed 1.5% of the total species abundance at this depth. The invasive species was not found in the most recent sediments of this core.

The sediment core from Lake Punderson exhibited high percent abundances of C. sphaericus and B. longirostris as well as littoral species of the genera Alona and Alonella

(Figure 25). E. coregoni was also present throughout the core, but only retained a median

abundance of 0.5%. D. galeata and D. parvula species were represented by very low percent

abundance, not exceeding 1.3%, but these species types were found throughout the entire core.

East Branch Reservoir also demonstrated high percent abundances of C. sphaericus, E. coregoni, and B. longirostris. The abundance of E. coregoni decreased in the upper half of the core, while percentage abundance of B. longirostris increased (Figure 26). D. galeata and D. pulex species types were found, yet each never exceeded a percent abundance greater than 0.5% of the cladoceran community. D. parvula - type was in greater abundance than the other daphnids. This species group increased in abundance near the top of the core, reaching a percent abundance of 2.5

% at the depth of 3 cm.

Bosmina longirostris Eubosmina coregoni Daphnia lumholtzi 0 0 0

5 5 5

10 10 10

15 15 15

20 20

Depth (cm) 20 Depth (cm) Depth (cm)

25 25 25

30 30 30

35 35 35 0% 20% 40% 60% 80% 100% 0% 5% 10% 0% 5% 10% Percent Abundance Percent Abundance Percent Abundance

Small Bodied Daphnids Other Pelagic Species Littoral Species Large Bodied Daphnids 0 0 0

5 5 5

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20 20 20 Depth (cm) Depth (cm) Depth (cm)

25 25 25

30 30 30

35 35 35 0% 10% 20% 0% 5% 10% 0% 10% 20% 30% Percent Abundance Percent Abundance Percent Abundance

Figure 21: Cladoceran Microfossil percent abundance data from Lake Berlin. List of species in the categories, small bodied daphnids, large bodied daphnids, other pelagic species, and littoral species are as listed in Table 10.

Bosmina longirostris Eubosmina coregoni Daphnia lumholtzi 0 0 0

5 5 5

10 10 10

Depth (cm) Depth

15 Depth (cm) Depth (cm) Depth 15 15

20 20 20

25 25 25 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 0% 5% 10% Percent Abundance Percent Abundance Percent Abundance

Small Bodied Daphnids Other Pelagic Species Littoral Species Large Bodied Daphnids 0 0 0

5 5 5

10 10 10

Depth (cm) 15 Depth (cm) 15 Depth (cm) Depth 15

20 20 20

25 25 25 0% 5% 10% 0% 5% 10% 0% 10% 20% Percent Abundance Percent Abundance Percent Abundance

Figure 22: Cladoceran Microfossil percent abundance data from Lake Milton. List of species in the categories, small bodied daphnids, large bodied daphnids, other pelagic species, and littoral species are as listed in Table 10.

Bosmina longirostris Eubosmina coregoni Daphnia lumholtzi 0 0 0

5 5 5

10 10 10

15 15 15 Depth (cm) Depth Depth (cm) Depth (cm) 20 20 20

25 25 25

30 30 30 0% 20% 40% 60% 80% 100% 0% 20% 40% 0% 5% 10% Percent Abundance Percent Abundance Percent Abundance

Small Bodied Daphnids Other Pelagic Species Littoral Species Large Bodied Daphnids 0 0 0

5 5 5

10 10 10

15 15 15 Depth (cm) (cm) Depth (cm) Depth 20 20 20

25 25 25

30 30 30 0% 10% 20% 30% 0% 5% 10% 0% 10% 20% 30% Percent Abundance Percent Abundance Percent Abundance

Figure 23: Cladoceran Microfossil percent abundance data from Mosquito Creek Lake. List of species in the categories, small bodied daphnids, large bodied daphnids, other pelagic species, and littoral species are as listed in Table 10.

Bosmina longirostris Eubosmina coregoni Daphnia lumholtzi

0 0 0

5 5 5

10 10 10

Depth (cm) 15 Depth (cm) 15 Depth (cm) 15

20 20 20

25 25 25 0% 5% 10% 0% 20% 40% 60% 80% 0% 20% 40% 60% 80%

Percent Abundance Percent Abundance Percent Abundance

Small Bodied Daphnids Other Pelagic Species Littoral Species Large Bodied Daphnids

0 0 0

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Depth (cm) Depth (cm) 15 15 Depth (cm) 15

20 20 20

25 25 25 0% 5% 10% 0% 10% 20% 30% 40% 0% 10% 20% 30%

Percent Abundance Percent Abundance Percent Abundance

Figure 24: Cladoceran Microfossil percent abundance data from LaDue Reservoir. List of species in the categories, small bodied daphnids, large bodied daphnids, other pelagic species, and littoral species are as listed in Table 10.

Bosmina longirostris Eubosmina coregoni Small Bodied Daphnids Large Bodied Daphnids 0 0 0

5 5 5

10 10 10

15 15 15 Depth (cm) Depth Depth (cm) Depth Depth (cm) 20 20 20

25 25 25

30 30 0% 5% 10% 30 0% 20% 40% 60% 0% 5% 10% Percent Abundance Percent Abundance Percent Abundance

Other Pelagic Species Littoral Species

0 0

5 5

10 10

15 15 Depth (cm) Depth (cm) 20 20

25 25

30 30 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% Percent Abundance Percent Abundance

Figure 25: Cladoceran Microfossil percent abundance data from Lake Punderson. List of species in the categories, small bodied daphnids, large bodied daphnids, other pelagic species, and littoral species are as listed in Table 10.

Bosmina longirostris Eubosmina coregoni Small Bodied Daphnids Large Bodied Daphnids 0 0 0

5 5 5

10 10 10

15 15 15

20 20 Depth (cm) 20 Depth (cm) Depth (cm)

25 25 25

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35 35 0% 20% 40% 60% 80% 35 0% 20% 40% 60% 80% 0% 5% 10% Percent Abundance Percent Abundance Percent Abundance

Other Pelagic Species Littoral Species

0 0

5 5

10 10

15 15

20 20 Depth (cm) Depth Depth (cm) 25 25

30 30

35 35 0% 20% 40% 60% 0% 10% 20%

Percent Abundance Percent Abundance

Figure 26: Cladoceran Microfossil percent abundance data from East Branch Reservoir. List of species in the categories, small bodied daphnids, large bodied daphnids, other pelagic species, and littoral species are as listed in Table 10.

Table 10: Median percent species abundance values for each lake in this study. Species listed under the species category are as represented in Figures 21 to 26.

East Mosquito Branch Lake LaDue Creek Lake Lake Species Group Reservoir Punderson Reservoir Lake Berlin Milton Bosmina longirostris 49.0% 20.8% 22.0% 54.0% 86.7% 77.7% Eubosmina coregoni 21.7% 0.5% 41.8% 14.9% 0.0% 3.8% Daphnia lumholtzi 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Small Bodied Daphnids Daphnia parvula - type 0.5% 0.0% 0.0% 2.0% 3.0% 1.0% Large Bodied Daphnids Daphnia galeata -type 0.0% 0.5% 0.0% 8.9% 2.0% 1.0% Daphnia pulex - type 0.0% 0.0% 0.0% 0.5% 0.0% 0.0% Other Pelagic Species Chydorus sphaericus 21.2% 40.0% 21.7% 3.5% 1.5% 2.5% Diaphanosoma birgei 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Leptodora kindii 0.5% 0.0% 0.0% 1.5% 2.0% 1.7% Littoral Species Acroperus 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Alona 4.4% 22.3% 9.7% 10.2% 2.2% 5.0% Alonella 0.0% 5.1% 0.0% 0.0% 0.0% 0.0% Anchistropus 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Camptocercus rectirostris 0.5% 1.2% 0.5% 1.0% 0.0% 1.0% Eurycercus 0.0% 0.9% 0.0% 0.0% 0.0% 0.0% Graptoberis testudinaria 0.0% 3.4% 0.0% 0.0% 0.0% 0.0% Leydigia 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Monospilus 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Percantha 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Pleuoxus 0.4% 2.4% 0.0% 0.0% 0.0% 0.0% Scapholeberis 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Sida 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Simocephalus 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%

Diversity Indices

Shannon Weiner diversity index as well as species evenness and richness indices were performed for each 1 cm layer in the cores (Figure 27). ANOVA test results showed a significant difference between the diversity indices among the non-invaded. failed invasion, and invaded lakes (p<0.05). In order to test which of the specific species pairs were significantly different, Fisher’s least significant difference (LSD) test was utilized. The test revealed that the communities before invasion, defined as the layers prior to the detection of D. lumholtzi, in Lake Milton and Lake Berlin had a significantly lower Shannon diversity, evenness, and richness than the communities before invasion of the failed invasion lakes and the communities of the non-invaded lakes..

Following the introduction of D. lumholtzi into Lake Milton and Lake Berlin, higher percent abundances were found in the littoral species, particularly those from the genera

Acroperus, Alona, Alonella, and Camptocercus. Shannon diversity, evenness, and richness indices were performed only on the littoral species of the invaded lakes (Figure 28). A paired

Student t-test revealed that the littoral community of Lake Milton was significantly higher following invasion in Shannon diversity and species richness (p >0.05, df = 21). Lake Berlin revealed a significantly higher Shannon diversity and species evenness values in the littoral community following the invasion of D. lumholtzi (p >0.05, df = 31).

1.6 1.4 Invaded: 1.2 Before Invasion 1

0.8 Failed: 0.6 Before 0.4 Invasion

Value Index Diversity 0.2 Non - 0 Invaded Shannon Species Species Diversity Evenness Richness

Figure 27: Comparison of diversity indices between the cladoceran communities prior to detection of D. lumholtzi of the invaded and failed invasion lakes as well as the communities of the non-invaded lakes.

Lake Berlin Lake Milton 1.2 1.2 Shannon 1 1 Diversity 0.8 0.8 Species 0.6 0.6 Evenness 0.4 0.4 Diversity Index Value Index Diversity Diversity Index Value Diversity 0.2 Specie Richness 0.2 0 0 Before Invasion After Invasion Before Invasion After Invasion

Figure 28: Comparison of littoral cladoceran diversity indices between the cladoceran communities before and after the detection of D. lumholtzi within the invaded lakes.

Community Composition

Detrended correspondence analysis (DCA) was used to determine which species were associated with the presence of D. lumholtzi (Figure 29) For this analysis, D. galeata- type and

D. parvula-type claws were assumed to be represented by D. galeata and D. parvula as these daphnids were detected in the lakes during the contemporary survey. D. pulex - type is assumed to be represented by D. pulex, as this is the only species found in Ohio that is characterized by the post-abdominal claw morphology that defines this species group. When using the percent abundances from each of the six sediment cores, the DCA results revealed that the larger daphnid species, D. galeata and D. pulex cluster together in the upper portion of the scatterplot. E. coregoni and C. sphaericus were found together in the far right. The littoral species clustered near the center of the graph. D. lumholtzi was found to the left of the graph grouping with B. longirostris, D. parvula, Diaphanosoma birgei, and the planktivorus

Leptodora kindii. Percent species abundance of the sediment core layers were overlaid onto the

DCA graph (Figure 30). The resulting scatterplot revealed that the core layers from Mosquito

Creek Reservoir clustered towards the top of the graph near the D. galeata and D. pulex groupings. The core sections from other failed invasion lake, LaDue Reservoir, were found near the right of the graph, indicating they are characterized by high abundances of E. coregoni and C. sphaericus. Finally the sediment layers from the invaded lakes, Lake Milton and Lake

Berlin, were found at the lower left portion of the scatterplot. This location is associated with the presence of D. lumholtzi, B. longirostris, D. parvula, and Diaphanosoma birgei.

Figure 29: DCA results from the percent species abundance from the failed invasion and invaded lakes.

Figure 30: DCA results using the percent species abundances of the individual sediment core layers from the failed invasion and invaded lakes.

DISSCUSSION

Dry Weight

The sediment cores were sliced into 1 cm segments; a consistent sedimentation rate over time was assumed. However, sediment deposition often fluctuates over time, influenced by soil erosion, productivity within the lake, and lake flushing rates (Smol et. al. 2001). The sediment cores were not dated, nor were sedimentation rates calculated. These procedures were deemed not necessary as this study was focused on changes occurring before and after invasion rather than specific dates in the lakes’ history.

Each of the lakes, except Lake Milton, exhibited higher percent dry weight in the lower portion of the core. These higher percents of dry weight are expected as the lower sections of the cores are subjected to compaction (Smol et. al. 2001). Fluctuations in dry weight may represent an influx of erosion material as well as variations of the water-retentive ability of the sediments. Since reservoirs are frequently disturbed habitats, characterized by fluctuating water levels. The changing water levels may be a contributing factor to changes in erosion rates, which could explain why the percentages of dry weight in Lake Milton remained consistent throughout the core rather than demonstrate signs compaction. Further, Lake Milton has been periodically drained over the years. The periods of time when the reservoir was empty may have reduced the amount of compaction within the sediments.

Organic Matter

The highest percents of organic matter were found in Lake Punderson. This lake is the largest and deepest kettle lake in Ohio with a maximum depth of approximately 20 m, thus this water body may be more vulnerable to oxygen depletion than the reservoirs. As oxygen concentrations decline, microbial activity and, consequently, organic matter breakdown is reduced. In this situation organic material tends to be preserved, thus increased concentrations of organic matter may be indicative of hypolimnetic anoxia as well as increased productivity

(Smol et. al. 2001).

Changes in organic matter of the sediment record has been traditionally regarded as evidence for changes in past productivity (Whiteside 1983); however, this correlation may be less valid for reservoirs as a result of the fluctuating water levels in these water bodies. In this study the non-invaded lakes exhibited the highest average percentage of organic matter from the surface sediments as well as median values from entire cores, whereas the failed invasion lakes exhibited the lowest values. If the percent of organic material does correlate with the amount of productivity in the reservoirs, than the median position of the invaded lakes suggests that productivity may not be a determining factor for the successful invasion of D. lumholtzi.

However, these results may also indicate that this median trophic status provides optimal conditions for D. lumholtzi. Supporting evidence for this hypothesis is given by experimental mesocosms used to investigate the invasion success of the D. lumholtzi under different trophic conditions (Lennon et. al. 2003). This investigation found that highest peak relative abundance of D. lumholtzi occurred in the medium level nutrient treatment. It may be possible that habitats characterized by low productivity do not have enough resources to support additional

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species, whereas lakes with high productivity often exhibit low diversity; dominated by a few species that are strong competitors. Environments with an intermediate level of productivity may be more vulnerable to invasion as there are enough resources to support the invasive species, yet the native species are not strong enough to competitively exclude these species.

Sedimentary Pigments

Chlorophyll

The sedimentary pigments from each lake demonstrated considerable variation throughout the cores. No noticeable shifts occurred in the pigments following the detection of

D. lumholtzi with the sediments. Thereby, there is no evidence that the presence of D. lumholtzi in the water column was related to phytoplankton composition or the rate of primary productivity.

The invaded lakes were higher in chlorophyll derivatives than the non-invaded or failed invasion lakes, but were lower in percent native chlorophyll. The high percentages of the chlorophyll derivatives suggests increased algal biomass within the invaded lakes while the percent native chlorophyll represents the portion of that has not been decomposed to its derivatives and is used as an indicator of preservation (Swain 1985). The low values of native chlorophyll along with the high percentages of the chlorophyll derivatives within Lake Milton and Lake Berlin suggest that pigment degradation is enhanced in these lakes. Degradation of the pigments occurs though bacterial activity, photoxidation, and grazing. It is uncertain which is dominant process; however, each of these processes requires ample oxygen. Thereby, the

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chlorophyll values found in the invaded lakes may indicate dual effects of increased algal biomass and sufficient oxygen concentrations within the hypolimnion.

Cyanobacterial Pigments

Cyanobacteria pigment data can be simplified using the oscillaxanthin to myxoxanthophyll ratio. The ratio is a better estimate of the relative production of the two pigments as it is degradation-independent. A ratio value greater than 1.0 indicates the higher levels of algae containing oscillaxanthin, indicating a phytoplankton assemblage dominated by

Oscillatoria species, and thus eutrophic conditions (Swain 1985). The oscillaxanthin to myxoxanthophyll ratio of Lake Berlin remained below 1.0 throughout the entire core. Ratio values higher than 1.0 were seen in the bottom sediments of the Lake Milton; however, above a depth of 16 cm, the ratio decreased and remained below 1.0 to the core surface. The other lakes, except Mosquito Creek Lake, also exhibited consistent ratio values below 1.0; such that the cyanobacterial pigments data does not sufficiently explain why D. lumholtzi was unable to establish in the other lakes. However, the success of the species in Lake Berlin and Lake

Milton does imply that this species invasion may be negatively correlated with phytoplankton assemblages dominated by Oscillatoria species.

Species Diversity

The invaded lakes exhibited lower species diversity, richness, and evenness values than either the non-invaded or failed invaded lakes. This result provides evidence for the Biotic

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Resistance model (Elton 1958, Miller et. al. 2002) which states that higher species diversity may lead to a greater invasion resistance due to high strength of interaction within the community and fewer resources available for utilization. Hence, competitive interactions between native zooplankton species and D. lumholtzi may contribute to preventing the invasive species from establishing. Little is known about the feeding preferences of D. lumholtzi and its functional redundancy with native species, thus more research is warranted before implications can be made regarding the competitive abilities of D. lumholtzi.

Lake Milton and Lake Berlin also experienced an increase in the species diversity of the littoral cladocerans following the invasion of D. lumholtzi. The invasive daphnid is a pelagic species; thereby its presence in a lake system should not influence the littoral zone.

Nonetheless, daphnids are keystone herbivores that are capable of influencing lower and upper trophic levels. The changes in the littoral community may be explained by a decrease in the trophic state of the lake through selective grazing. A decrease in lake trophic status may increase the boundaries of the littoral zone, creating more niches and supporting a higher number of littoral species. However, the sedimentary pigment data from this study does not offer any evidence that the presence of D. lumholtzi in the water column is capable of producing alterations to the trophic state.

The increased diversity in the littoral community may also be the result of decreased predation pressure in this habitat. The late summer peak of D. lumholtzi may offer planktivorous fish an additional food source at a time when they normally move inshore to prey on the littoral zooplankton (Kolar and Wahl 1998). The benefit of an additional food source by the invasion of a large cladoceran has been documented by the invasion of

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Bythotrephes into the Great Lakes. Consumption of zooplankton by yellow perch in Lake Erie nearly doubled after the invasion of this exotic species (Mills et. al.2004). Although further investigation is needed, a shift in feeding preferences of vertebrate predators may offer explanation for the alterations of littoral communities following the invasion of D. lumholtzi.

Smol (1981) noted that differences in species diversity found within sediment cores may not necessarily provide information on the biology of the species involved, rather these differences may be artifacts of the sedimentation rate. Sedimentation rates change throughout the lake’s history, thus it can be difficult to compare species diversity overtime as the length of the core will represent different time periods of sediment accumulation. Although the indices used in this study may not estimate diversity over equal time intervals, the values are still useful in determining patterns of invasion. Changes in diversity throughout the depth of the core are used in this study to examine the shifts that occurred within the littoral community of

Lake Milton and Lake Berlin. The invasion of D. lumholtzi into these lakes occurred within the last decade, thus reducing the possibility that a drastic shift is sediment accumulation rate took place within this limited time period. Further, Smol (1981) predicted that species diversity should be highest at the bottom of the core as a result of compaction of the sediments.

However, examination of percent abundances of the species in this study clearly shows an increase in the number of species near the top of the core. Although the exact date that these species appeared may be questioned, it is certain that this shift occurred following the detection of D. lumholtzi within the cores.

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Species Composition

The sediment cores from Lake Berlin and Lake Milton were characterized by high percent abundances of smaller daphnids and B. longirostris. The non-invaded lakes, Lake

Punderson and East Branch Reservoir, exhibited high numbers of B. longirostris, E. coregoni,

C. sphaericus, and several littoral species. Mosquito Creek Lake and LaDue Reservoir represented lakes where D. lumholtzi was unable to successfully establish. Mosquito Creek

Lake contained high percent abundances of the large-bodied daphnids, D. galeata and D. pulex; whereas LaDue Reservoir was characterized by high numbers of E. coregoni and C. sphaericus . These results indicate that the success of D. lumholtzi is facilitated in lakes composed of smaller sized zooplankton species. Additional evidence for this hypothesis is given by the measurements of the microfossils. The measurements showed that the invaded lakes had median carapace and head shield lengths that were among the smallest in the study.

The data suggest that lakes characterized by a small sized zooplankton community may be more vulnerable to invasion by D. lumholtzi, whereas the presence of larger sized daphnids, such as D. pulex and D. galeata, may be capable of resisting establishment of D. lumholtzi. The invaded and failed invasion lakes provide evidence that interactions with one or more native species may prevent the establishment of non-indigenous species; however, the non-invaded lakes, Lake Punderson and East Branch Reservoir, suggest that other factors must also be considered. These lakes have been able to resist invasion thus far, yet are largely composed of

E. coregoni and C. sphaericus, which are among the smallest of the pelagic cladocerans. It may be possible that D. lumholtzi has not been given the opportunity to disperse into these water bodies, although this seems unlikely given the high amount of recreational activity on these

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lakes and their close proximity to invaded localities. Lennon et. al. (2003) suggested that that

C. sphaericus was able to prevent the establishment of D. lumholtzi, while communities dominated by Bosmina longirostris were more vulnerable to invasion. As these observations were also found in this study, there is strong implication that species composition of the community may determine the outcome of invasion. However, factors other than species size may be important in making this determination.

C. sphaericus is typically found in clumps of filamentous algae and only becomes pelagic in eutrophic systems (Pennak 1989). Consequently, the presence of this species in the pelagic region of the lake may be an indicator of eutrophic conditions. The disassociation between D. lumholtzi and C. sphaericus may be related to the trophic status of the lake and not species interactions. The invaded lakes each exhibited oscillaxanthin to myxoxanthophyll ratio less than 1.0 at the time of invasion, indicating lower levels of Oscillatoria and, possibly, less eutrophic conditions. Mosquito Creek lakes exhibited cyanobacterial pigment ratios above 1.0 throughout the entire core; whereas the other failed invasion lake, LaDue Reservoir demonstrated a ratio above 1.0 in the top layers of the core. This suggests that high levels of

Oscillatoria species may prevent the establishment of D. lumholtzi within a water body. Larger bodied daphnids, such as D. lumholtzi, are often more affected by the inhibitory effects of filamentous algae, thus when cyanobacteria is present in high numbers smaller sized zooplankton should be expected to dominate (East et. al. 1999) An investigation by Pattinson et. al. (2003) also supports the hypothesis that the invasion of D. lumholtzi is resisted by increased levels of cyanobacteria. In the Pattinson et. al. (2003) study enclosures were utilized to examine the diet, survival, and fecundity of daphnids under conditions of good and poor

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food quality by using different concentrations of cyanobacteria. D. lumholtzi exhibited reduced survivorship, fecundity, and intrinsic rate of increase as concentrations of cyanobacteria increased, suggesting that the exotic species does not have a greater tolerance of filamentous algae than native species.

The failed invasion and non-invaded lakes demonstrated a consistent presence of E. coregoni throughout the entire length of the sediment core. E. coregoni was also present in

Lake Milton and Lake Berlin; however, the percent abundance of the species declined to near zero prior to the detection of D. lumholtzi. These results parallel those from the contemporary survey, as D. lumholtzi and E. coregoni were never found in the same lake at the same time in any of the surveyed locations. E. coregoni is a European species that was first detected in the

Great Lakes watershed within the 1960’s. Little is known about the ecology of this invasive species, thus it remains uncertain why E. coregoni and D. lumholtzi are not found together, even in lakes where there is evidence that both have invaded. In Mosquito Creek Lake and

LaDue Reservoir, E. coregoni continued to persist in high numbers as D. lumholtzi declined in abundance. This suggests that E. coregoni exclude D. lumholtzi from the water column.

However, in Lake Milton percent abundances of E. coregoni declined to near zero prior to the arrival of D. lumholtzi. It seems probable that the two invasive species differ in their optimal requirements for survival, thus the environmental conditions of a lake may favor only one of the species at a particular time.

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Influence of Predation

The mucro to carapace length ratios of B. longirostris from each lake maintained relatively consistent values throughout the length of the cores. Although mild fluctuations in

Mosquito Creek Lake and LaDue Reservoir were seen, these variations could not be correlated to the appearance of D. lumholtzi. Changes in the mucro to carapace ratio were found in the invaded lakes, Lake Milton and Lake Berlin, and included a slight increase in recent sediments.

The mucro to carapace length ratios were found to be higher in the invaded lakes than in either the failed invasion or non-invaded communities. A longer mucro in relation to body size offers a stronger defense against invertebrate predators (Kerfoot 1981 Post et. al. 1995, Hellsten et. al. 1999). This suggests that invaded lakes may have higher predation pressures by invertebrate predators; however, the large size and elongated spines of D. lumholtzi may allow the invasive species to persist in invertebrate predator dominated communities.

If the invasive species is selected against by invertebrate predators, it may increase the predation pressure on other zooplankton species. If these predators prey on larger, less protected species; selective predation will likely results in a reduction in the mean body size of the zooplankton community as well as an increase in the mucro length of B. longirostris. These results have been found in this study as well as past investigations (Kolar et. al. 1997,

Dzialowski et. al. 2003). For example, the mucro to carapace ratio increased within the recent sediments of Lake Milton and Lake Berlin. Additionally, the microfossil length measurements of the native cladocerans began to decrease or show strong fluctuations following the detection of D. lumholtzi in the invaded lakes. The contemporary survey (Chapter 1) as well as investigations of Lake Springfield, Illinois (Kolar et. al. 1997) also reported a decrease in

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zooplankton body size following the invasion of D. lumholtzi. The changes in zooplankton size and morphology indicate that the invasion of D. lumholtzi is capable of altering the zooplankton community structure, although further examination is required to identify the underlying mechanisms of the community changes.

Conclusions

1. Comparison of the percent organic matter values of the invaded lakes to the non-invaded and

failed invasion lakes suggests that environments with an intermediate level of productivity may

be more vulnerable to invasion.

2. The low oscillaxanthin to myxoxanthophyll ratio values of the invaded lakes and the

disassociation of D. lumholtzi with Chydorus sphaericus indicate that high levels of Oscillatoria

algal species may prevent the establishment of D. lumholtzi within a water body.

3. Lower species diversity in lakes may make them vulnerable to invasion by D. lumholtzi. Low

diversity may result is more open niches and low interaction strengths between the members of

the community, possibly facilitating the establishment of exotic species.

4. Results from this study indicate that the species diversity of littoral cladocerans increases

following the invasion of D. lumholtzi. This trend may occur as selective grazing by D.

lumholtzi clears the water column, increasing the area of the littoral zone. However, this study

does not offer any evidence to support this hypothesis. D. lumholtzi may also provide an

additional food source for planktivorous fish in the late summer months; thus allowing these

predators to remain in the pelagic zone at a time when daphnids are usually absent. A shift in

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feeding preferences of vertebrate predators following the invasion of D. lumholtzi may explain

the changes found in the littoral communities, yet further investigation is required

5. Lakes characterized by a small sized zooplankton community may be more vulnerable to

invasion by D. lumholtzi, whereas the presence of larger sized daphnids, such as D. pulex and D.

galeata, may be capable of resisting establishment of D. lumholtzi. These results suggest that

interactions with one or more native species may be an important factor is determining the

outcome of invasion.

6. D. lumholtzi failed to establish in communities containing E. coregoni; however, the reason is

unknown. It may be possible that E. coregoni is able to exclude D. lumholtzi through

competition. It is also possible that the two species have different optimal environmental

preferences, thus the limnological characteristics of a lake make it difficult for the species to co-

exist. As little is known about the ecology of E. coregoni, the interactions between these two

invasive species requires further investigation to determine why these species fail to co-exist.

7. The successful establishment of D. lumholtzi has been shown to correlate with a reduction of the

mean body size in the zooplankton community. This pattern has also been seen in past studies

(Lennon et. al. 2001) as well as the contemporary survey (Chapter 1) of this study; however, the

mechanism behind this trend is uncertain. It may the result of increased predation pressures on

the native zooplankton, while it is also possible that D. lumholtzi is able to suppress the native

species through competitive interactions. Further investigation is necessary as a change in the

mean body size of the zooplankton community may indicate a change in zooplankton species

composition which may lead to changes in both the lower and upper trophic levels.

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8. The higher mucro to carapace length ratios found in the invaded lakes indicates that lakes with

higher levels of invertebrate predation may be more vulnerable to the establishment of D.

lumholtzi. Increases in the ratio within the recent sediments of the invaded lakes suggest that

invertebrate predation pressure may increase on native zooplankton species after the invasion of

D. lumholtzi.

CHAPTER 3 – RESOURCE OVERLAP STUDY

INTRODUCTION

Zooplankton live in dilute environments; large lakes may represent a limitless supply of food, but this resource may be greatly dispersed such that zooplankton starvation is still possible. Therefore, zooplankton must be efficient in nutrient uptake and utilization (Conover

1968). Ecological theory suggests that when two zooplankton species compete for a single limiting resource, under stable conditions and in the absence of predation, the species with the greatest feeding efficiency should be the superior competitor (Gliwicz 1990).

Studies of feeding efficiency have shown that food collecting surfaces are proportional to the square of the body length of the species. For example, the body length of Daphnia is approximately four times as great as Bosmina, such that the filtering area of daphnids will be about sixteen times larger than bosminids (Brooks and Dodson 1965). Further, Burns (1968) developed a study that examined the relationship between cladoceran body size and the maximum size particle that is capable of being consumed. The author demonstrated that a positive relationship exists between cladoceran body size and the size of particles able to be ingested. These results indicate that the size of particles ingested by zooplankton are related to body length. Species

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similar in size will overlap their utilization of food resources and may compete (Smith and

Cooper 1982)

Larger individuals not only ingest larger particles, but they can also capture very small particles. Accordingly, the food used by small cladocerans is completely overlapped by that of larger species (DeMott 1982). This suggests that in the absence of other biological interactions, large sized zooplankton may be stronger competitors than the small sized zooplankton species.

The relationship between body size and competitive ability among zooplankton assumes that the size of an individual will determine its rate of ingestion, assimilation, and respiration.

However, feeding efficiency will also be determined by the morphology or feeding behavior of the zooplankton. Naumann (1918) identified three types of cladoceran filter feeders: Daphnia type, Sida type, and Bosmina type. The Daphnia type (including Daphnia and Ceriodaphnia) and the Sida type (Sida, Diaphanosoma, and Holopedium) generally feed in a stationary position or using a hop-and-sink motion. They use their thoracic limbs to separate out particles and draw them from the water into the carapace opening where fine filter plates collect the particles. Observations of zooplankton feeding methods revealed that rejection is the major mechanism for selective feeding in Daphnia species (Burns1968; DeMott and Kerfoot 1982,

Porter 1983). Particles which clog the filtering appendages are rejected with the post- abdominal claw, while filaments or excess food can be removed at the mouth. This method may be inefficient since rejection causes frequent interruptions in feeding and edible food may be rejected along with the unacceptable particles.

The Bosmina type (Bosmina and Eubosmina) was described as swimming horizontally to collect food particles by Naumann (1918). The continuous swimming of Bosmina may be a

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behavioral adaptation to increase the encounter rate with food particles (DeMott and Kerfoot

1982). This group exhibits a morphology that differs from other cladocerans. Unlike the fixed filtering surfaces characteristic of Daphnia, species within the genera Bosmina, Eubosmina,

(DeMott 1982) and some species within Chydorus (Fryer 1965), have feeding appendages modified for seizing and manipulating individual particles. Individuals can also reject or capture particles by adjusting the space between the last pair of thoracic limbs. Increasing this space may increase water flow, resulting in a higher feeding rate on larger food items.

Additionally, the tooth-bearing spines and filter plates on the second trunk limbs of these species can be used to rake large particles into the food groove (DeMott and Kerfoot 1982).

The ability to capture a wide range of food particles may allow individuals in this group to ingest high quality food without expending energy sorting and rejecting undesirable particles.

Zooplankton species have been traditionally regarded as taxonomically similar and thus are included within the same functional feeding group. However, the descriptions of morphology and feeding behavior suggest that zooplankton species may differ in grazing selection and efficiency. Observations of feeding modes in zooplankton suggest that differences between

Daphnia species may be minimal; consequently, having a larger body size may be optimal for daphnids (DeMott 1982). This may not be the case for small cladoceran species. Some studies have found that Bosmina species selectively ingest smaller particles (Burns 1968, Gliwicz

1969), consistent with a positive correlation between body size and feeding efficiency.

However, other experiments have revealed differences in food gathering by Daphnia and

Bosmina. DeMott and Kerfoot (1982) labeled food particles using radioisotopes to demonstrate that bosminids are able to selectively consume specific flagellate species and continually

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switch preferences over time as food abundance changes. Feeding manipulations by Porter et. al. (1983) found a positive correlation between body size and filtering rate among Daphnia species, but concluded that this relationship was no longer significant when Bosmina was included in the analysis. These observations suggest that factors other than body length may influence feeding in smaller cladocerans.

Objectives of the Study

Zooplankton communities are usually not dominated by one superior competitor, rather are composed of several species that coexist. It can be hypothesized that resource partitioning may be an important factor in structuring of zooplankton communities. Daphnids have demonstrated the ability to feed on a wide variety of sources, including phytoplankton, organic matter, and bacteria, suggesting that this group may be generalists and non-selective in their feeding behavior. Currently it is unknown how much food resource overlap exists between D. lumholtzi and native zooplankton species, thus it is impossible to predict how the invasion of this daphnid may impact community structure through competitive interactions.

Analysis of zooplankton community compositions in the contemporary survey (Chapter 1) and the paleolimnological study (Chapter 2) found that D. lumholtzi is closely associated with smaller sized zooplankton species. The invasive species was not closely associated with larger sized daphnids in either study. Competitive exclusion may be one explanation for these findings. D. lumholtzi may have a higher grazing efficiency than smaller individuals because of its large size. A greater effectiveness of food collection may allow the invasive species to displace the smaller native species and successfully establish. Alternatively, larger sized

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individuals may not become as easily displaced since, compared to D. lumholtzi, they may be equal or greater competitors for food resources. If competition among zooplankton species is a contributing factor to the successful invasion of D. lumholtzi; it would be expected that the invasive species will exhibit a higher grazing rate and ability to ingest larger particles than small bodied individuals.

The contemporary survey (Chapter 1) also determined that the core body length of D. lumholtzi, which excludes the length of the head and tail spines, is similar to that of other large sized daphnids. If feeding efficiency and the maximum size particle capable of being consumed does have a positive relationship with cladoceran body size than D. lumholtzi may perform more similarly to larger sized daphnids than previously assumed.

In order to investigate the amount of resource overlap, and hence competition, between zooplankton, this study utilized fluorescent microspheres to determine the range of particle sizes able to be ingested and feeding rate of D. lumholtzi as well as other native zooplankton species. Fluorescent microspheres have become a popular method of evaluating zooplankton grazing interactions. The microspheres are advantageous as they are easily counted and are well-defined. Unlike techniques involving radiotracers or glass or plastic particles, fluorescent microspheres allow distinction between particles in the gut and those in the food groove or attached to the animal. The microspheres also make it easier to control the gut passage time to make certain that particles are not defecated, which may result in an underestimation of feeding rates (Wiedner and Vareschi 1995). Studies have shown that the level of taste discrimination varies among zooplankton taxa (DeMott 1982, Porter 1983), thus it is possible that certain zooplankton species will be unwilling to ingest the fluorescent microspheres. However, the

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microspheres are often used in the medical field for antigen-antibody agglutination test, thus have a proven ability to absorb proteins and other organic compounds. Accordingly, the microspheres are also able to absorb flavors from algal cultures. DeMott (1986) demonstrated that Daphnia and Bosmina species could not discriminate between algae and the “flavored” microspheres.

METHODS

Resource overlap experiments were performed for the following species, each categorized into groups dependent on body size: Large Daphnids: D. pulex, and D. magna,

Mid-sized Daphnids: D. retrocurva, D. galeata, and D. lumholtzi, Small Daphnids: D. parvula and Ceriodaphnia dubia, and Small Cladocerans: Chydorus sphaericus, Bosmina longirostris,

Eubosmina coregoni. The groupings were assigned based on core body length, measured from the top of the eyespot to the base of the tail or mucro for all Daphnia species and B. longirostris. For Chydorus sphaericus and Eubosmina coregoni, species which lake tail spines and mucro, the core body length was measured from the top of the eyespot to the most posterior point of the carapace. Large Daphnid group consists of species whose average core body length is greater than 1.5 mm whereas those in the Mid-sized Daphnid groups have an average core body length between 1.0 – 1.5 mm. Species included in the Small Daphnid and

Small Cladoceran have an average core body length less than 1.0 mm.

Individuals from the species Ceriodaphnia dubia, D. pulex, and D. magna were obtained from cultures at Sachs Systems Aquaculture (St. Augustine, Florida). The remaining

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species were collected from Ohio lakes and were cultured in the laboratory. Chydorus sphaericus, Bosmina longirostris, Eubosmina coregoni D. parvula, D. retrocurva were collected from East Twin Lake, D. galeata from Mosquito Creek Reservoir, and D. lumholtzi from Lake Milton. Each species culture was maintained in artificial lake water and fed a mixture of Selenestrum and powdered plankton feed (Sachs Systems Aquaculture). The cultures were kept in 16:8 light dark photoperiod using fluorescent fixtures with Nutri-Grow full spectrum bulbs.

A stock solution of florescent microspheres (Fluoresbrites, Polysciences Inc.) was prepared using 0.53, 0.78, 1.01, 2.02, 4.16, 7.32, 15.02, and 27.01 µm at concentrations of approximately 6.62 x 106, 2.08 x 106, 9.56 x 105, 1.20 x 105, 1.37 x 104, 2.51 x 103, 2.91 x 102, and 50 microsphere / mL. These concentrations were chosen such that each microsphere size was represented by equal particle volume in the solution. Microspheres of 0.53, 0.78, and 1.01

µm corresponded to sizes of bacteria, whereas the other diameters represented algal sizes.

Accordingly, the total concentration of microspheres in the stock solution represented natural lake concentrations. Flavored microspheres were prepared following DeMott (1986) by incubating 25 ml of the microsphere stock solution for 12 -24 hours in 10 ml of a Selenestrum algal suspension (5 x 105 cell / liter). A magnetic stirrer was used to keep the microspheres in suspension during this time.

In a 100 ml beaker, 15 ml of the Selenestrum algal suspension was added to the flavored spheres to form a 1:1 mixture of microspheres and algae. This mixture was brought up to 25°C, the optimal temperature for D. lumholtzi (Lennon et. al. 2001). Ten zooplankton individuals were randomly collected from the cultures and immediately placed into the beaker. The

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animals were allowed to feed for two minutes. To avoid underestimation of filtration rates, it was essential that feeding times were shorter than gut passage times; thus the feeding time was two minutes following the recommendations of Wiedner and Vareschi (1995). After the two minute feeding time the zooplankton were killed with hot water (60° C), filtered on 53 µm

Nitex mesh, and rinsed with tap water. This procedure was repeated for each species in the experiment.

After being rinsed, the zooplankton were measured for core body length as described above. After being measured each individual was placed into a vial with 1 ml of distilled water and sonicated for 15 minutes. This process causes a breakdown of animal tissue and disperses the gut contents into suspension. The suspension was then filtered on black 0.2 µm polycarbonate filters. The filters were examined under an epifluorescent microscope such that each microsphere could be counted and measured.

Grazing Rate (um3 /ind / min) for the total volume of microspheres was calculated using for each species using the following formula (Edmonson and Winberg 1971):

GR = (V)(ln C) / (tn) where V the volume of water in the beaker (50 ml in this study), C is the volume of microspheres (um3) ingested in time t, t is the total incubation time (2 min in this study), and n is the zooplankton number used for the experiment (n = 10 in this study).

One way analysis of variance (ANOVA) was used to establish the statistical probability of a significant difference between the grazing rates of each species on the different microsphere sizes. Fisher’s least significant difference (LSD) test was used to determine significant differences among specific species pairs.

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Zooplankton feeding selectivity was calculated for each microsphere size using the Alpha

Index developed by Chesson (1978). This index derived from a stochastic model involving probabilities of encounter and ingestion upon encounter independent of prey availability, and is given by:

αi = (ri / pi) / Σ (ri / pi)

where ri is the relative abundance of a prey in a predator's diet, represented by the number of microspheres found in the gut. pi is the prey's relative abundance in the ecosystem, or the number of microspheres available in the beaker of water. The index results in values between 0 and 1, with α = 0 representing avoidance and α = 1 representing exclusive feeding.

Pearson correlation coefficients were calculated from the Alpha Index values using

BioStat 2008 to determine the level of size selection similarity for each possible pair of the zooplankton species used in this study. Multi-Variant Statistics Program (MVSP) Plus (version

3.13). was used to perform Principle component analysis (PCA) on the correlation coefficient matrix values. ANOVA and post hoc tests used to determine significant differences between the grazing rates of the species were performed using BioStat 2008.

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RESULTS

Grazing Rates

Grazing rate (um3 / ind / min) was calculated for each species used in this study (Figure

31). Median grazing rates ranged from 7.8 (um3 / ind / min), demonstrated by Chydorus sphaericus to 17.7 um3 / ind / min, reported from D. pulex. D. lumholtzi was found to have a grazing rate of 14.3 um3 / ind / min, which was exceeded by 4 other species D. galeata, D. pulex, D. magna, and D. retrocurva.

ANOVA test results showed a significant difference between the grazing rates (p<0.05).

In order to test which of the specific species pairs were significantly different, Fisher’s least significant difference (LSD) test was utilized. The test revealed that the grazing rate of

Chydorus sphaericus was significantly different from D. retrocurva, D. galeata, D. pulex, D. magna, and D. lumholtzi (p<0.05). B. longirostris and E. coregoni were significantly different from D. retrocurva,, D. pulex, and D. magna (p<0.05). Ceriodaphnia dubia had a significantly different rate from D. magna (p<0.05). Lastly, D. parvula showed a significant difference from

D. retrocurva and D. magna (p<0.05).

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Chydorus

Bosmina

Eubosmina

Ceriodaphnia

D. parvula

D. retrocurva

D. lumholtzi

D. galeata

D. pulex

D. magna

0 5 10 15 20 25

Grazing Rate - um3 /individual / min

Figure 31: Box plot representing the overlap of the grazing rates between the species. Grazing rates were calculated for each microsphere size ingested, the median is represented by • . The box represents the 25 and 75 quartiles. The whiskers represent the maximum and minimum data points.

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Alpha Index - Correlation Coefficient Matrix

Alpha Index values (Chesson 1978) were calculated for each microsphere size to determine feeding selectivity of the zooplankton species (Figure 32). Pearson correlation coefficients were calculated from these values to provide a numerical description of the feeding similarities between the species pairs. The correlation matrix is shown in Table 11. The correlation coefficients can range between -1 and +1 and measures the degree of similarity in feeding preference. A positive value for the correlation implies a high level of similarity between the species involved, indicating they are consuming similar amounts of microspheres within the same size range. A negative value for the correlation implies the species do not share similar feeding preferences and are not ingesting microspheres of similar sizes. Details of the feeding preferences for each of the species used in this study are described below.

Patterns within Species

Chydorus sphaericus

C. sphaericus demonstrated a strong preference for the smallest microsphere size classes, 0.53 and 0.78 µm, demonstrated by Alpha Index values of 0.46 and 0.41. The microspheres ingested by these two size classes were 97.4% of the total microspheres consumed. The remainder of the microspheres ingested where within the 1.01 and 2.02 µm class size. C. sphaericus individuals did not ingest microspheres greater than 2.02 µm. The correlation coefficient matrix revealed negative values, ranging from -0.120 to -0.512, for all

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Ceriodaphnia and Daphnia species. Positive correlation coefficient values of 0.722 and

0.561 were from for Bosmina longirostris and Eubosmina coregoni.

Bosmina longirostris

B. longirostris exhibited the highest Alpha Index values, 0.45, for microspheres within the 0.78 µm class size, followed by values of 0.26 and 0.15 for those within the 1.01 and 0.53

µm class sizes. Microspheres from the 2.02 and 4.16 µm class size consisted of 1.1% of the total microspheres ingested. No microspheres larger than 4.16 µm were selected by this species. B. longirostris demonstrated a strong similarity in food selectivity to Eubosmina coregoni, as shown by the correlation coefficient of 0.955. Correlation coefficients were very low or negative between the other cladocerans used in this study.

Eubosmina coregoni

The feeding preferences of E. coregoni were similar to that of B. longirostris. The largest preference was also for microspheres within the 0.78 µm class size, as shown by an Alpha Index value of 0.38. However, this species ingested slightly more microspheres from the 1.01 µm class size (Alpha Index values of 0.36 as opposed to 0.26) and fewer from the 0.53 µm class size (0.12 to 0.15). Comparable to B. longirostris, this bosminid species ingested 1.0% microspheres from the

2.02 and 4.16 µm class size and did not selected any microspheres larger than 4.16 µm.

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Ceriodaphnia dubia

C. dubia was capable of ingesting microspheres from 0.53 to 7.32 µm, however, the Alpha

Index value, 0.51, for this species indicates a high preference for microspheres within the 2.02 µm class size. Correlation coefficients values were highest with D. parvula and D. galeata, whereas the values where low or negative with the smallest cladoceran species, Chydorus sphaericus, B. longirostris, and E. coregoni.

Daphnia parvula

The food selectivity of D. parvula is very similar to that of C. dubia, as shown by both the

Alpha Index values and correlation coefficient matrix. Both species have shown a strong preference for the 2.02 µm class size. Comparable to C. dubia, D. parvula also selected microspheres from 0.53 to 4.16 µm; however, no individuals from this species ingested microspheres larger than 4.16 µm. Correlation coefficients were very high between C. dubia as well as D. retrocurva, D. galeata, and D. lumholtzi, with values ranging from 0.818 to 0.918.

Daphnia retrocurva

D. retrocurva showed the highest feeding selectivity for microspheres with the 2.02 and 4.16

µm class size, as revealed by Alpha Index values of 0.42 and 0.36. This daphnid ingested microspheres from 0.53 to 27.01 µm, although no microspheres within the 15.02 µm class size were selected. Correlation coefficient values were high, ranging from 0.811 to 0.986, between all other daphnid species used in this study.

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Daphnia galeata

D. galeata demonstrated feeding selectivity patterns very similar to those of D. parvula.

It also demonstrated a high preference for microspheres within the 2.02 µm class size, yielding an Alpha Index value of 0.52. D. galeata selected microspheres the 0.53 to 7.32 µm; however, unlike D. parvula this species did not ingest any microspheres larger than 7.32 µm. Correlation coefficient values indicated that D. galeata has similar feeding preferences to the Daphnia species as well as Ceriodaphnia dubia.

Daphnia pulex

D. pulex showed a strong preference for microspheres from the 2.02 to 15.12 µm class sizes, demonstrated by Alpha Index values from 0.37 to 0.16. This study found that the daphnid species was capable to ingesting smaller particles, although did not actively select them. Ingestion of microspheres from the size classes of 0.53, 0.78, and 1.01 µm resulted in

Alpha Index values of 0.01, 0.03, and 0.03. The feeding selectivity of this species was similar to other daphnid species, including D. retrocurva, D. galeata, D. magna, and D. lumholtzi, as shown by the high correlation coefficient values within the matrix.

Daphnia magna

This study demonstrated the D. magna was capable of ingesting microspheres from all eight microsphere size classes. Alpha Index values ranged from 0.05 to 0.26, with the highest selectivity found for the 2.02 and 4.16 µm class sizes. Correlation coefficient values were high

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between the species and D. retrocurva, D. galeata, D. magna, and D. lumholtzi. Negative values were found in the matrix between Chydorus sphaericus, B. longirostris, and E. coregoni.

Daphnia lumholtzi

D. lumholtzi exhibited a high feeding preference for microspheres of the 2.02 and 4.16

µm class sizes, as Alpha Index values for the classes were 0.46 and 0.45. Although much lower amounts were selected, this species also demonstrated that it was capable of ingesting microsphere from 0.53 to 7.32 µm. Microspheres from the 15.02 or 27.01 µm classes were not found in the gut of any of the individuals from this species. High correlation coefficient values from the matrix suggest that the feeding preferences of D. lumholtzi may be similar to those of the other daphnid species used in this study. These values ranged from 0.748 between D. pulex to 0.986 found between D. retrocurva. Correlation coefficients were negative between this species and Chydorus sphaericus, B. longirostris, and E. coregoni.

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Chydorus sphaericus Daphnia retrocurva 1 1

x 0.8

0.6 0.6 0.4 0.4 Alpha Inde 0.2 Alpha Inde 0.2 0 0 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 Bead Diameter (u) Bead Diameter (u)

Bosmina longirostris Daphnia lumholtzi 1 1

x 0.8 0.8 x 0.6 0.6 0.4 0.4 Alpha Inde Alpha

0.2 Alpha Inde 0.2 0 0 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 Bead Diameter (u) Bead Diameter (u)

Eubosmina coregoni Daphnia galeata 1 1

0.8 x 0.8 x 0.6 0.6 0.4 0.4

Alpha Inde Alpha Inde 0.2 0.2 0 0 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 Bead Diameter (u) Bead Diameter (u)

Ceriodaphnia dubia Daphnia pulex 1 1 x x 0.8 0.8 0.6 0.6 0.4 0.4 Alpha Inde

0.2 Alpha Inde 0.2 0 0 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 Bead Diameter (u) Bead Diameter (u)

Daphnia parvula Daphnia magna 1 1 x

x 0.8 0.8 0.6 0.6 0.4 0.4 Alpha Inde 0.2 Alpha Inde 0.2 0 0 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 0.53 0.78 1.01 2.02 4.16 7.32 15.02 27.01 Bead Diameter (u) Bead Diameter (u)

Figure 32: Alpha Index values for the eight microsphere classes offered to the cladoceran species.

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Table 11: Pearson correlation coefficient matrix, where each coefficient quantifies the similarity of the Alpha Index values of two cladoceran species. The Alpha Index describes the selectivity for a particular microsphere size class. Cladoceran species are: Chydorus sphaericus (CHY), Bosmina longirostris (BOS), Eubosmina coregoni (EUB), Ceriodaphnia dubia (CER), Daphnia parvula (D PA), Daphnia retrocurva (DR), Daphnia galeata (DG), Daphnia pulex (D PU), Daphnia magna (DM), and Daphnia lumholtzi (DL). Bold number indicate a significant correlation coefficient value (p > 0.05), indicative of a similarity in food selection between the two species. CHY BOS EUB CER D PA DR DG D PU DM DL

CHY 1 BOS 0.722 1 EUB 0.561 0.955 1 CER -0.120 0.151 0.188 1 D PA -0.184 0.091 0.140 0.985 1 DR -0.415 -0.273 -0.268 0.767 0.825 1 DG -0.331 -0.154 -0.137 0.921 0.918 0.913 1 D PU -0.512 -0.385 -0.376 0.731 0.732 0.811 0.898 1 DM -0.485 -0.389 -0.421 0.638 0.666 0.930 0.876 0.900 1 DL -0.354 -0.219 -0.216 0.748 0.818 0.986 0.880 0.793 0.906 1

Alpha Index - Principle Component Analysis

Differences between one zooplankton species and the feeding selectivity of the other species are described by a single row of the correlation matrix; whereas differences among the ten species used in this study are described by the entire matrix. As these differences can be more challenging to recognize than those between two specific species, principle component analysis (PCA) was used to visualize and quantify the differences in feeding selectivity (Figure

33). Principle component analysis collapses the data to form an “n” dimensional shape, yet maintains much of the similarity and dissimilarity information between the species. The

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dimensions consist of the principle components, which are linear combinations produced from the original variables (in this study, the Alpha Index values from each microsphere class size).

Each one of the principle components explains some of the variation seen in the correlation coefficient matrix (Miracle 1974).

The analysis represents the similarities in food selectivity between the ten zooplankton species described in the correlation coefficient matrix. The first three components of the PCA explained 75.3%, 11.8%, and 6.4% of the variation with the correlation coefficient matrix, roughly 93.5% of the total variation. Component 1 is largely driven by consumption of microspheres in the 4.16 µm size class, whereas the loading scores of component 2 were highest for microspheres in the 1.01 µm size class. Chydorus sphaericus is isolated in the top left corner of the PCA. This position is not unexpected as this species exhibited feeding preferences dissimilar from the other zooplankton species, represented by negative values on the matrix. B. longirostris, and E. coregoni, exhibited similar feeding patterns and are closely grouped together on the PCA as a result. D. parvula and Ceriodaphnia dubia occupy nearly identical positions on the PCA. These two species have very similar feeding preferences, represented by a correlation coefficient of .985. The largest of the daphnids, D. magna and D. pulex are clustered together on the PCA. This grouping was anticipated as these two species exhibited a strong similarity in feeding selectivity. D. retrocurva and D. lumholtzi also demonstrated a strong preference for the same microsphere size classes, thus resulting in a high correlation coefficient, 0.986, between the species. This similarity resulted in these two daphnids being positioned close together on the PCA.

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D. galeata is positioned closest to the other mid-sized daphnids, D. retrocurva and D. lumholtzi as these species each share similar feeding preferences. However, D. galeata also has feeding preferences similar to D. parvula and Ceriodaphnia dubia. Consequently, D. galeata is positioned on the PCA between the mid-sized and small-sized daphnids.

-0.3

-0.4 D. magna Chydorus -0.5 D. pulex -0.6 -0.7 D. retrocurva -0.8 D. lumholtzi -0.9

-1 Bosmin a D. galeata -1.1 Eubosmina

-1.2

-1.3 D. parvula Ceriodaphnia -1 0 1 2 3

Component 1 Figure 33: Principle component analysis showing the relative position of the ten zooplankton species based on feeding preferences. Circles represent groupings based in core body length. Large Daphnids: D. pulex, and D. magna, Mid-sized Daphnids: D. retrocurva, D. galeata ,and D. lumholtzi, Small Daphnids: D. parvula and Ceriodaphnia dubia, and Small Cladocerans: Bosmina longirostris and Eubosmina coregoni

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DISSCUSSION

This study utilized eight different microsphere sizes to determine the ability of a zooplankton to ingest a wide variety of different size particles. The information gained was used to describe and compare the food resource overlap of the zooplankton species.

The correlation coefficient matrix provides a numerical description of any similarities or dissimilarities in feeding preference. The usage of the correlation coefficient has been used for illustrative purposes in past zooplankton resource studies (Bodgan and Gilbert 1987) as the authors felt that the results provided a quantitative comparison of the abilities of zooplankton to utilize different size cells. As the value of the coefficient from any of the zooplankton pairs increases (from -1 to 1), so does the similarity between the feeding preferences of the species involved. The principle component analysis (PCA) was used to visualize and quantify the feeding differences among the ten species used in this study.

Species that exhibit high similarity in feeding presences exhibit the potential for exploitative competition, which occurs as the consumption of a resource by one species makes that resource unavailable for consumption by another. Further, as the number of species with similar requirments increases, the occurance of diffuse competiton becomes more likely

(Pianka 1974). In this situation, weak competitive interactions accumulate and may potenially inhibit many species. In order to avoid exclusion, each species must have ecological requirments that differ by at least some minimum amount from the others. The zooplankton in this study exhibited differences in their resources use as the amount and size of microspheres ingested varied by at least some small amount among the ten species. These minute differences

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may be important for the species to avoid competitive exclusion in zooplankton communities.

Ecological theory suggests that a community with a great amount of resource overlap should support more species than those where each species has a discrete niche. However, diffuse competition suggests that there is a limit to the amount of resource overlap that can occur within a community (Pianka 1974). This study suggests that zooplankton species are cabable of resource partitioning, yet compeitive interactions may be present that limit the amount of species diversity present within the community.

Evidence of Food Resource Overlap

In this study, considerable overlap in feeding selectivity was found between several species pairs. Among the small cladoceran group, B. longirostris, and E. coregoni demonstrated similar feeding requirements while the preferences of Chydorus sphaericus were unique. The small daphnids, D. parvula and Ceriodaphnia dubia, showed strong similarities in feeding preferences and, as a result, were positioned closely together on the PCA. Similarities were also found within the largest daphnids: D. magna and D. pulex. These similarities and the size of microspheres ingested by the species support the hypothesis of a positive correlation between body size and feeding efficiency (Gophen and Geller 1984, Burns 1968, DeMott 1986,

Borsheim and Anderson 1987).

D. lumholtzi exhibited similar feeding preferences with the other daphnid species used in this study, as shown by the high correlation coefficients within the matrix. The invasive species exhibited the highest similarity to D. retrocurva, with a correlation coefficient of 0.986. The scatter plot of the PCA also positioned D. lumholtzi near D. retrocurva.

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The high level of similarity in feeding preferences between D. lumholtzi and D. retrocurva found in this study may explain why these species were never found concurrently in the contemporary survey (Chapter 1). This study provides evidence that the food resources of the two species overlap, thus suggesting the potential for competition. D. retrocurva exhibited a higher grazing rate than D. lumholtzi which indicates that D. retrocurva has a greater effectiveness of food collection. This higher feeding efficiency may allow D. retrocurva to effectively exclude D. lumholtzi from the community, thus preventing the species from successfully establishing. In the paleolimnological study (Chapter 2) D. parvula - type post- abdominal claws were found within the sediment cores of the failed invasion lakes. It was assumed that these microfossils represented D. parvula, since this species was found in the lakes during the contemporary survey. However, the claws may also represent D. retrocurva, as this species shares the same post-abdominal morphology as D. parvula. Thereby, it is possible that D. retrocurva was present at the time of introduction into these lakes and may have contributed to the failure of D. lumholtzi to establish.

The contemporary survey and paleolimnological study (Chapters 1 and 2) also reported that D. lumholtzi and E. coregoni were never found in the same lake at the same time in any of the surveyed locations. The negative correlation coefficient value between the feeding preferences of these two species implies that these species do not share similar feeding preferences and are not ingesting microspheres of similar sizes. The lack of resource overlap between D. lumholtzi and E. coregoni suggests that these two species do not compete for food; thus other factors are likely responsible for the failure of these two species to co-exist. The contemporary survey revealed a strong association between D. retrocurva and E. coregoni, as

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these two species were commonly found together within lakes in northern Ohio. Accordingly, the presence of D. retrocurva and its potential ability to exclude D. lumholtzi, may be the reason why D. lumholtzi failed to establish in communities containing E. coregoni.

In this study D. lumholtzi exhibited the highest selectivity for microspheres in the 2.02 and 4.16 µm class sizes and failed to ingest any microspheres larger than 7.32 µm. The large- sized daphnids in the study, D. pulex, and D. magna, as well as the mid-sized D. galeata showed a higher preference for the microspheres within the 7.32 µm size class and exhibited larger grazing rates than D. lumholtzi. Further, D. pulex and D. magna were able to ingest the microspheres in the 15.02 and 27.01 µm class sizes. These results lend support to the hypothesis that larger body size results in higher feeding efficiency. If feeding efficiency estimates the competitive ability among zooplankton, then D. galeata, D. pulex, and D. magna may be superior competitors to D. lumholtzi since they are able to feed from a wider range of particle sizes are more efficient at collecting these particles. This supports the hypothesis brought forth by the contemporary survey and paleolimnological study that larger sized daphnids may prevent establishment of D. lumholtzi through direct competition for food.

The small- sized cladoceran species in the study: Chydorus sphaericus, B. longirostris, E. coregoni, Ceriodaphnia dubia, and D. parvula each exhibited maximum feeding selectivity values for microsphere size classes smaller than those selected most by D. lumholtzi. These results, along with the low correlation coefficient values between D. lumholtzi and the small cladocerans, suggest that there is little food resource overlap between the invasive species and small bodied cladocerans. Additionally, D. lumholtzi demonstrated higher grazing rates than each of these species. These results suggest that D. lumholtzi is a more efficient feeder, and possibly a stronger

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competitor, than the smaller species. Accordingly, D. lumholtzi should be able to successfully

establish in communities where smaller-sized zooplankton are present. The ability of D. lumholtzi

to successfully invade such communities may be further aided by the presence of vertebrate

predation. Temperate lake communities dominated by small sized communities are usually

characterized by high levels of predation, as vertebrate predators eliminate the larger species

allowing smaller species that escape predation to become more common. The elongated spines of

D. lumholtzi may be an effective deterrent to vertebrate predation, thus allowing the species to

survive in communities where predators are high in number. Thus, the low level of food resource

overlap between D. lumholtzi and small bodied cladocerans as well as predatory interactions may

be responsible for the success of this invasive species

Thermal Influences

Although this study supports the hypothesis that feeding efficiency is positively correlated to body length, feeding behavior of zooplankton may also be determined by factors other than body size. For example, temperature influences affect the feeding efficiency of zooplankton as metabolic demands may increase at higher temperatures, making it difficult to meet growth and reproduction energy requirements (Kolar et. al. 1997). Investigations of the feeding efficiency of a large cladoceran, D. pulex, at different temperatures revealed that maximum feeding efficiency at different temperatures was obtained at approximately 2 mm

(Lynch 1977). Feeding efficiency decreased with larger body sizes as the respiration rate increased more rapidly with body size than did feeding rate. These results imply that the largest individuals are not necessarily the most efficient feeders. However, a body size of 2 mm is larger

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then that of small species of zooplankton; predicting that even at a size of 2 mm, D. pulex should maintain a higher feeding efficiency, and possibly be able to out-compete the smaller species.

Consequently, the largest individuals of the population may not always have the highest feeding efficiency; however, the largest species may still be able to maintain the highest efficiency within the community, resulting in the potential to displace smaller species as a result of competitive interactions.

As a tropical species, D, lumholtzi is well-adapted to warmer temperatures which may explain why the species reaches peak numbers during the mid- to late summer months. It is possible that during this time period D. lumholtzi achieves its maximum feeding efficiency as the warm temperatures of the summer months mimic this species’ natural habitat. Further, investigations of the life history characteristics of D. lumholtzi under different temperature regimes (Lennon et. al. 2001) determined that 15°C was the lowest temperature at which D. lumholtzi exhibited a positive intrinsic rate of increase. The highest intrinsic rate of increase (r =

0.364) for the species occurred at 25° C, followed closely by 20 °C (r = 0.345). No eggs were produced below 10° C (r = 0). At elevated temperatures, D. lumholtzi exhibited a higher reproductive rate, short life cycles and early age of maturation. These characteristics are typical of a successful invader; therefore higher temperatures may a contributing factor to the success invasion of D. lumholtzi. In this study each feeding trial was preformed at 25° C, the optimal temperature for D. lumholtzi. The results found in this investigation are assumed to represent the warm summer period when D. lumholtzi reaches its peak numbers. However, if the feeding efficiency of D. lumholtzi is influenced by temperature, then different results may be found if the feeding trials were performed at lower temperatures. Repeating the experiment at different

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temperature regimes may be necessary to understand why D. lumholtzi fails to achieve high numbers throughout the year.

Depth and Horizontal Segregation

Zooplankton may be capable of partitioning the environment in other ways besides the division of food resources. The limnetic environment is represented by simple gradients of light, temperature, and chemicals and is relatively uniform in comparison to the littoral habitat.

Horizontal segregation of zooplankton is made difficult by wind driven circulation. However, zooplankton are able to control their vertical position, allowing for depth segregation. Five discrete depth habitats may be recognized; the upper and lower epilimnion, metalimnion, upper and lower hypolimnion. Each of these locations is characterized distinctive temperature and light profiles as well as food conditions (Hall et. al. 1976). Diel vertical migration in zooplankton is generally assumed to be a predator avoidance behavior; however, this activity may also reduce competition for food resources among the species.

Several studies have provided evidence of zooplankton segregating through depth. For example, Milbrink et. al. (2003) reported differences in the behavior of daphnids in experimental mesocosms. D. longispina spent most of the time near the water surface and D. magna remained in the middle depth of the enclosure. D. pulex and D. obtusa both spent most of the time on the bottom of the enclosure. Further, lake observations demonstrated that Diaphanosoma has a preference for warm surface water while Bosmina prefers greater depths (DeMott and Kerfoot

1982). These observations suggest that different foraging strategies exist between the species, possibly as a result of interspecific competition. The preferred position in the water column is

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currently unknown for D. lumholtzi. Thus, it remains possible that D. lumholtzi may be able to avoid interactions with native species, resulting in a successful invasion, through depth segregation as well as food resource partitioning.

Conclusions

The evidence that zooplankton exhibit different feeding preferences and are able to partition available resources suggest that competition may be an important influence that structures zooplankton communities. The ability of an invasive species to impact a zooplankton community would partially depend on the food resources it uses and whether those resources differ from those of the native species. D. lumholtzi demonstrated considerable food resource overlap with

D. retrocurva, which may explain why these two species were not found concurrently in either the contemporary survey or paleolimnlogical study.

D. lumholtzi demonstrated higher grazing rates and an ability to consume larger particles than the small- sized cladoceran species in the study. These results suggest that D. lumholtzi is a more efficient feeder, and possibly a stronger competitor, than the smaller species. However, the paleolimnological study in Chapter 2 failed to show any changes in the abundance of pelagic species following invasion. Thus, there is no evidence that D. lumholtzi is able to competitively exclude native zooplankton species. However, the feeding selectivity indices also revealed that there is little food resource overlap between D. lumholtzi and the small bodied zooplankton.

These results indicate that D. lumholtzi may not be in direct competition with the small bodied zooplankton species. Communities dominated by small sized zooplankton species are usually characterized by high levels of predation. In these communities larger sized species are typically

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eliminated by vertebrate predators, thus the larger food particles consumed by these organisms may become available for utilization. The elongated spines of D. lumholtzi may allow the species to persist in high predation environments. Thus, the predatory deterrence of D. lumholtzi and its ability to consume the larger particles may allow the invasive species to co-exist with these small bodied cladocerans.

The large-sized daphnids in the study, D. pulex, and D. magna, as well as the mid-sized D. galeata demonstrated an ability to consume the largest microsphere sizes in this study and also exhibited grazing rates that exceeded that of D. lumholtzi. These results suggest that these species may be superior competitors to D. lumholtzi since they are able to feed from a wider range of particle sizes are more efficient at collecting these particles. These results lend support to the hypothesis that larger sized daphnids may prevent establishment of D. lumholtzi in lake communities through direct competition for food.

CHAPTER 4 – EXPERIMENTAL MESOCOSM STUDY

INTRODUCTION

Competition is assumed to be one of the dominant factors responsible for community organization. Invasive species may have specific traits that allow them to outcompete native species. Consequently, non-indigenous species may change the species composition of native habitats resulting in an alteration of ecosystem function (Johnson and Havel 2001). In zooplankton communities; resource levels, such as food or space, are important factors to the biological interactions that occur among the species (Dzialowski et al. 2000). To successfully establish into such communities, an invader must be able to utilize these resources, which requires the species to displace the current user, find an unexploited resource, or coexist with the native species through resource partitioning (Miller et al. 2002).

Hypotheses of Competitive Interactions

Several hypotheses for the factors that lead to competitive dominance among cladoceran species have been suggested. These hypotheses, described below, include the R Hypothesis put forth by Tilman (1982), the Low Food Efficiency Hypothesis

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(Romanovsky 1984), the Small Body Size Hypothesis (Neill 1975, Hall 1976), the R-max

Hypothesis (Goulden 1978), as well as Brooks and Dodson’s (1965) Size Efficiency

Hypothesis.

The R Hypothesis (Tilman 1982) proposes that the species capable of surviving at the lowest resource concentration will be the strongest competitor. This suggests that differences in zooplankton behavior and morphology may affect competitive outcomes at lower food concentrations. Gliwicz (1990) attempted to find support for this hypothesis by comparing the filtering ability of eight daphnid species under steady state conditions. This study found that at low resource concentrations, the growth rate of larger species was higher than that of smaller- sized species. In another study, it was demonstrated that larger zooplankton species have a better ability than smaller species to store fat and use it as an energy reserve during periods of low food concentrations (Milbrink et al. 2003). These results suggest that larger body size results in a decreased threshold food level, defined as the amount of food needed for assimilation to balance metabolic losses (Gliwicz 1990), and indicates that larger zooplankton species may have a competitive advantage over smaller bodied species in a resource limited environment.

Low Food Efficiency Hypothesis (Romanovsky 1984) is similar to the R Hypothesis as it suggests that species with the lowest resource threshold for reproduction are the best competitors. Romanovsky (1984) found that the ability of a species to survive at low food concentrations is related to individual growth rate and slow-growing individuals have the most competitive advantage. He also stated that the outcome of competition among cladoceran species in the absence of predation depends on the trophic status of the water body. In

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oligotrophic and mesotrophic water bodies, small, slow-growing cladocerans are the stronger competitors. In eutrophic waters, large rapid-growing species dominate and can outcompete the smaller cladocerans.

Romanovsky‘s (1984) predictions of competitive dominance in oligotrophic waters parallels that of the Small Body Size Hypothesis (Neill 1975, Hall 1976). This hypothesis suggests that smaller sized species may be able to filter smaller particles more efficiently, thus the optimal body size may decrease with decreasing food concentration. Thus, in some environments smaller species may be better competitors than larger species.

The R-max hypothesis (Goulden et al. 1978) proposes that species with the highest intrinsic rate of increase are superior competitors. Goulden et al. (1978) suggested that in the absence of predation, large cladoceran species dominate because of greater reproductive rates, measured by the intrinsic rate of natural increase. The major factors that determine the intrinsic rate of increase are time of first birth and fecundity. Although Bosmina species matures earlier than most Daphnia species, higher fecundities of the larger daphnids result in higher R-max; hence, body length was found to positively correlate with intrinsic rate of increase.

Finally, the Size-Efficiency Hypothesis (Brooks and Dodson 1965) holds that when smaller species utilize food particles that are a subset of those used by larger species then the larger species should dominate during periods of low predation intensity. This may occur since the larger species are hypothesized to be better competitors as a result of greater effectiveness of food collection and a reduced metabolic demand. However, size selection by vertebrate predators will likely eliminate the larger species and smaller species that escape predation will become more common. Coexistence may occur when predation is of moderate intensity. In this

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situation it is suggested that the larger zooplankton will be retained to low densities such that the competing smaller species will be able to persist.

In a test of the Size Efficiency Hypothesis, Dodson (1974b) found that invertebrate predation typically selects smaller cladocerans. Since invertebrate predators are the preferred food items for fish, their occurrence at high densities is often connected with the absence or low number of vertebrate predators. It is generally believed that invertebrate predators can play a significant role in the structuring of zooplankton communities only if the density of planktivorous fish is low (Dodson 1974b). Accordingly, the Size Efficiency Hypothesis suggests that the mean size of the dominant zooplankton present within the water column are determined by the influences of competition as well as vertebrate and invertebrate predation.

The above hypotheses suggest that competitive ability can be the result of higher filtration rates, higher intrinsic rate of increase, tolerance of low food concentrations, or susceptibility to predation. Which factor, if any, is the dominant characteristic that leads to competitive dominance among cladoceran species remains unclear. The R Hypothesis, R-max

Hypothesis , and Size Efficiency Hypothesis each indicate that larger bodied cladoceran species are stronger competitors than smaller species. The Low Food Efficiency Hypothesis and Small Body Size Hypothesis state that larger cladocerans will be able to outcompete smaller sized species only during eutrophic conditions or when food resources are high in abundance. The lakes and reservoirs included in the contemporary survey and paleolimnological study (Chapters 1 and 2) have been characterized as eutrophic (Davic et al.

1997); consequently, each of the hypotheses described above indicates that the large size of D. lumholtzi should facilitate invasion into these environments.

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Influences of Predation

The Size Efficiency Hypothesis recognizes predation is an important influence structuring communities. Further, the Low Food Efficiency Hypothesis and R-max Hypothesis each state that the competition outcome described occurs only in the absence of predation.

Thus, susceptibility to predation is most likely positively related to the competitive ability of zooplankton species. As a result, predation influences on interspecific competition may be one of the most important combination of factors that structure zooplankton communities (Allan

1974, Lynch 1977, Kerfoot 1981).

D. lumholtzi produces two morphs (Green 1967). One form has a short rounded head, while the other has an elongated head spine. The latter is almost always found in Ohio lakes.

The spined form is thought to be less visible and suffers less predation, appearing when planktivorous fish are present in the water column. D. galeata and D. retrocurva share a similar core body size as D. lumholtzi, a measurement that excludes the size of the head and tail spines by measuring from the top of the eyespot to the base of the tail. These species also exhibit cyclomorphism of the helmet at high predation levels (Dodson 1974, Sorensen and

Sterner 1992); however, neither of the species is capable of producing head spines comparable to the exaggerated form seen in D. lumholtzi. Accordingly, the elongated spines of D. lumholtzi may facilitate invasion by allowing the species to avoid predation in new environments.

However, in order to establish the species must also be able to acquire food resources either through resource portioning with the native species or by exploiting unused resources.

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Objectives of the Study

The results from the previous chapters suggest that the successful invasion by D. lumholtzi may be a balance between a partitioning of food resources and an ability to avoid predation

(Figure 34). At low predation levels competition is expected to be high between D. lumholtzi and the large-sized zooplankton that are typically present in communities lacking vertebrate predators. The resource overlap study determined that large-sized daphnids are able to feed from a wider range of particles than D. lumholtzi are may be more efficient at collecting these particles. These findings suggest that larger sized zooplankton may exclude D. lumholtzi from lake communities through direct competition for food.

Figure 34: Hypothesized relationship between competition and predation on the successful invasion of D. lumholtzi. The invasive species is expected to be excluded from communities with high levels of predation, presumably because of resource competition. D. lumholtzi survives at low levels of predation because of its ability to partition food resources and resist vertebrate predation.

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The resources overlap study also found that D. lumholtzi exhibits minimal food resource overlap with the small bodied zooplankton species. Communities dominated by small bodied zooplankton species are usually characterized by high levels of predation that typically reduces the number of larger sized zooplankton species. However, the elongated spines of D. lumholtzi may allow the species to persist in high predation environments. According, the predatory deterrence of D. lumholtzi and its difference in feeding preferences may minimize competition, allowing the invasive species to co-exist with small bodied cladocerans.

The final section of this study will explore if interactions with native zooplankton species can prevent the invasion of D. lumholtzi. This exotic cladoceran has shown to have strong dispersal capabilities, broad physiological tolerances and its elongated spines appear to deter predation; all which contribute to making this species a successful invader. In spite of these characteristics, there are still locations in which the species has not yet been able to invade, thereby investigations of the competitive abilities of this species become essential.

This study will test the community resistance hypothesis developed from previous chapters, which suggests that communities dominated by large sized zooplankton will resist the establishment of D. lumholtzi, yet the invasive species will be able to persist in communities where small-sized zooplankton species are present. Experimental mesocosms were constructed to represent the different zooplankton communities found in the paleolimnological study: a large bodied community represented by large daphnids, D. galeata and D. pulex, a small bodied community composed of Bosmina longirostris and Ceriodaphnia dubia, and a small bodied community consisting of Eubosmina coregoni and C. dubia. D. lumholtzi will be introduced into each of these communities. Observations the communities will provide

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information regarding the role of competition in preventing invasion in situations where predation is absent and food resources are not limiting.

METHODS

Eighteen 38 L aquaria were used as experimental mesocosms for this study. Each aquarium was filled with 20 L of artificial lake water. The mesocosms were covered in thin plastic sheeting to prevent water loss. Each aquaria were illuminated in a 16:8 light dark cycle using fluorescent fixtures with Nutri Grow full-spectrum bulbs. The containers were also maintained at approximately 24 °C, well within the optimal temperature range for D. lumholtzi

(Lennon et. al. 2001). To each aquarium 250 mL of a Selenestrum algal suspension (5 x 105 cell/liter) was added. After the addition of the algae, the mesocosms were left undisturbed for two weeks prior to the addition of zooplankton. This allowed for an adequate food source to establish for the zooplankton.

Six community treatments were created in the mesocosms, each of which was replicated in triplicate. All zooplankton were obtained and cultured as described in the resource overlap study (Chapter 3). The communities, with number of individuals added at the beginning of the experiment are represented in the diagram below:

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Non – Invaded Treatments: Invaded Treatments:

Without D. lumholtzi With D. lumholtzi 43 Bosmina longirostrius Small bodied community 43 Bosmina longirostrius 41 Ceriodaphnia dubia with Bosmina longirostrius 41 Ceriodaphnia dubia 15 Daphnia lumholtzi 42 Eubosmina coregoni Small bodied community 42 Eubosmina coregoni 41 Ceriodaphnia dubia with Eubosmina coregoni 41 Ceriodaphnia dubia 15 Daphnia lumholtzi 32 Daphnia galeata 32 Daphnia galeata Large bodied community 15 Daphnia pulex 15 Daphnia pulex 15 Daphnia lumholtzi

The number of native zooplankton added to each aquarium was determined to account for the differences in body size between each species. Length-width regressions (Durmont et. al.

1975, Rosen 1981) were used to determine biomass, such that each treatment demonstrated equal biomass at the beginning of the experiment. To the invaded treatments, 15 individuals of

D. lumholtzi were added. This number was chosen to represent an accidental introduction rather than equal biomass. All native zooplankton were added to the appropriate mesocosm on the same date and were maintained for two weeks in order to establish stable populations. D. lumholtzi was added to each of the invasion treatments two weeks later, representation of an invasion event.

Each mesocosm was sampled weekly after the addition of the native zooplankton.

Zooplankton were sampled by gently stirring each aquarium and withdrawing 1 L of water from the center of each aquarium with a graduated pitcher. The sample was then filtered through 53 µm Nitex mesh. The zooplankton retained on the mesh were preserved in a sucrose

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formalin solution (Haney and Hall 1973) until identified and counted under a dissecting

microscope. In order to maintain the starting volume of 20 L and to prevent nutrient depletion,

1 L of artificial lake water was added each mesocosm following weekly sampling. The

mesocosms were sampled over an eight week period. Following the last sampling period, each

aquarium was drained and filtered through 53 µm Nitex mesh. The zooplankton retained on the

mesh were preserved such that the sample could later by scanned for the presence of D.

lumholtzi. This practice ensured that any D. lumholtzi individuals that were present, but below

detection, would be accounted for.

Weekly zooplankton counts from each aquaria were used to calculate the intrinsic rate of

increase and biomass of each species. Intrinsic Rate of Increase (ind / day), or r, was calculated

using the formula:

r = (lnNt – lnN0)/t

Where No = population size at time zero, Nt = population size at time t, and t is the number of days since the individuals were added to the aquaria. Biomass was calculated using

published length width regressions (Durmont et. al. 1975, Rosen 1981). Paired Student’s t-tests were utilized to compare the intrinsic rate of increase and biomass of each species between the

non-invaded and invaded treatments.

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RESULTS

Intrinsic rate of increase

Intrinsic rate of increase (ind / day) was calculated for each species after the last sampling period (Table 12) The intrinsic rate of increase was slightly lower in the invaded treatments for each of the native species, yet paired Student’s t-tests failed to find any significant differences between the non-invaded and invaded treatments (p>0.05, df = 4).

Intrinsic rate of increase was also calculated for D. lumholtzi in the two small bodied communities. The rate of increase for the invasive species was somewhat lower in the small bodied community with E. coregoni; however, no significant differences between the two small bodied treatments were found (p>0.05, df = 4).Intrinsic rate of increase for D. lumholtzi was not calculated for the large bodied community as no individuals were present in this treatment on the final day of sampling. One week after D. lumholtzi was introduced to the invaded treatment, the species was found in all 9 aquaria of the invaded treatments. However, the following week D. lumholtzi was found in only one mesocosm belonging to the large bodied, invaded treatment; whereas numbers of D. lumholtzi had increased in all aquaria belonging to the small bodied treatments. One week later D. lumholtzi was not found in any of the large bodied communities, yet numbers of this species continued to increase in both the small bodied community with B. longirostrius, invaded and small bodied community with E. coregoni, invaded treatments.

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Table 12: Intrinsic rate of increase (ind / day) for the cladoceran species grown in different community treatments Intrinsic rate of increase for D. lumholtzi was not calculated for the large bodied community as no individuals were present in this treatment on the final day of sampling.

Without Daphnia lumholtzi With Daphnia lumholtzi

Small bodied community with Bosmina Bosmina longirostrius 0.091 + 0.001 0.090 + 0.003 Ceriodaphnia dubia 0.092 + 0.003 0.089 + 0.005 Daphnia lumholtzi na 0.076 + 0.003 Small bodied community with Eubosmina Eubosmina coregoni 0.089 + 0.003 0.086 + 0.000 Ceriodaphnia dubia 0.096 + 0.001 0.089 + 0.004 Daphnia lumholtzi na 0.066 + 0.005

Large bodied community

Daphnia galeata 0.091 + 0.001 0.088 + 0.001 Daphnia pulex 0.093 + 0.001 0.091 + 0.003 Daphnia lumholtzi na na

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Biomass

Biomass (µg) for each zooplankton species was also calculated after the last sampling period. Figure 35 shows the final biomass for each of the native cladoceran species in the non- invaded and invaded treatments. All of the species demonstrated a slight decrease in biomass in the invaded treatments, except for D. pulex which showed a slight increase. Paired Student’s t- test revealed a significant difference in the biomass of E. coregoni between the two treatments

(p>0.05, df = 4). No other significant differences were found.

D. lumholtzi exhibited a lower biomass in the small bodied community with E. coregoni treatment than that with B. longirostrius (Figure 36); however no significant difference was found between the two treatments (p>0.05, df = 4). D. lumholtzi was not present any of the large bodied communities at the end of this study.

The percent relative biomass for each species was also calculated for each of the 6 treatments (Figure 37) Although, D. lumholtzi was able to persist in the small bodied community with B. longirostrius, the relative biomass of the native species were not significantly altered (p>0.05, df = 4). B. longirostrius exhibited 47.0% of the relative biomass in both the invaded and non-invaded treatments. Relative biomass of C. dubia decreased only slightly in the invaded treatment, from 53.0% to 45.9%. In the small bodied community with E. coregoni treatments, both C. dubia and E. coregoni exhibited a decrease of relative biomass in the invaded treatment. While the decrease in C. dubia was not significant, E. coregoni demonstrated a significant decrease (p>0.05, df = 4) between the non-invaded and invaded treatments as its relative biomass decreased from 46.0% to 41.5% with the presence of D. lumholtzi.

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D. lumholtzi was not present in the final sampling of the large bodied treatments, however the relative biomass of the large biomass was altered between the two treatments. Although no significant differences were found (p>0.05, df = 4), D. pulex did experience an increase in relative biomass, from 70.8% to 75.3%, within the invaded treatment. As a result, the relative biomass of D. galeata was lowered, from 29.2% to 24.7 %, in the large bodied, invaded treatment.

2500

2000

1500

1000

Final Biomass (ug) Biomass Final 500

0 BOS EUB CER DG DP Non-Invaded Invaded

Figure 35: Final Biomass (µg) of the native cladoceran species in the non-invaded and invaded treatments. Paired student’s t-tests revealed a significant different in the biomass values of E. coregoni. (p>0.05, df = 4) No other significant differences were found. Native Cladoceran species are: Bosmina longirostris (BOS), Eubosmina coregoni (EUB), Ceriodaphnia dubia (CER), Daphnia galeata (DG), Daphnia pulex (DP).

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100

20 Final Biomass (ug) Biomass Final

Small w/ Small w/ Large BOS EUB

Figure 36: Final Biomass (µg) of D. lumholtzi from each of the invaded community treatments. D. lumholtzi was not present in any of the large bodied communities by the end of the experiment. Paired student’s t-tests did not find any significant differences (p>0.05, df = 4) between the small-bodied community treatments

100% 100% 100% ss 80% 80% 80%

60% 60% 60% e Bio e 40% 40% 40% tiv ma Relative Biomass Relative 20% Relative Biomass 20% Rela 20%

0% 0% 0% Non-Invaded Invaded Non-Invaded Invaded Non-Invaded Invaded BOS CER DL EUB CER DL DG DP DL

Figure 37: Comparison of the relative biomass between the non-invaded and invaded treatments for the Small bodied community with Bosmina longirostrius (left), Small bodied community with Eubosmina coregoni (center), and Large bodied community (right) treatments. Native Cladoceran species are: Bosmina longirostris (BOS), Eubosmina coregoni (EUB), Ceriodaphnia dubia (CER), Daphnia galeata

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DISSCUSSION

This study investigated community resistance toward D. lumholtzi by introducing the invasive species into three community types: Small bodied community with Bosmina longirostrius, Small bodied community with Eubosmina coregoni, and Large bodied community. This investigation was necessary in order to determine if a particular species or species group can prevent invasion.

In the present study, D. lumholtzi did not persist in the large bodied community. The invasive species was found in low numbers in the first and second week after it was introduced into the mesocosms, but it was no longer detected in any of the large bodied communities by week three. These results support the contemporary survey and paleolimnological study in that

D. lumholtzi is not able to persist in communities dominated by large daphnid species. In the mesocosms no predation was present and environmental conditions were optimal for the survival of D. lumholtzi, hence, the inability of the invasive species to survive in the large bodied community supports the hypothesis of competition for the same size food particles between D. lumholtzi and the large Daphnia species. The resource overlap study in Chapter 3 found that D. pulex and D. galeata exhibit a higher preference for larger sized beads and a higher feeding efficiency than D. lumholtzi. According to the Size Efficiency Hypothesis

(Brooks and Dodson 1965), these characteristics may allow D. pulex and D. galeata to become stronger competitors, thus resulting in D. lumholtzi being excluded from the community. D. pulex exhibited a higher intrinsic rate of increase and relative biomass than the smaller sized D.

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galeata in both the non-invaded and invaded treatments; giving further support for the hypothesis that larger sized cladocerans are stronger competitors.

D. pulex showed a higher final biomass in the invaded treatment while the other native species exhibited higher values in the non-invaded treatments. The food resource overlap study of Chapter 3 demonstrated that the food preferences of D. lumholtzi more closely resemble D. galeata than D. pulex. Thus, competition may have been strongest between D. lumholtzi and D. galeata in the large bodied treatment. If the D. galeata population was even slightly suppressed by D. lumholtzi, it may have allowed D. pulex to gain an even larger advantage within this community.

Both the contemporary survey and paleolimnological study suggest that communities dominated by small-bodied cladocerans are vulnerable to invasion. This conclusion was supported in the present study since D. lumholtzi was able to establish in the small bodied community with Bosmina longirostrius. The invasive species was also successful in the small bodied community with Eubosmina coregoni although the previous investigations (Chapter 1 and 2) failed to show D. lumholtzi and E. coregoni existing concurrently. The presence of D. lumholtzi in the small bodied community with Eubosmina coregoni treatment resulted in a significant decrease in biomass and, although not significant, a lower intrinsic rate of increase for E. coregoni. These results suggest that not only is D. lumholtzi capable of surviving in a community comprised of E. coregoni, but D. lumholtzi may be the stronger competitor.

Alternative explanations for the lack of coexistence of these two species in the natural environment must be explored. It may be possible, as suggested in Chapter 3, that other species associated with E. coregoni may exclude D. lumholtzi from the community.

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Environmental conditions may also be responsible, as there may be a particular limnetic condition that favors one species over the other.

Although D. lumholtzi was able to persist in the small bodied communities, there was little alteration to the community structure of these treatments. No significant differences in biomass or intrinsic rate of increase were found between the invaded and non-invaded treatments for B. longirostrius or C. dubia. Further, the relative biomass of the small cladoceran species, including E. coregoni, changed very little with the addition of D. lumholtzi to the community.

It is commonly accepted that for an invasive species to be successful, it must be able to displace the current holder or find an empty niche (Miller et al. 2002). In this study, the latter appears to be true. The resource overlap study (Chapter 3) concluded that B. longirostrius and

E. coregoni have a high preference for particles roughly 1.01 µm in size while C. dubia has the highest feeding selectivity for particles within the 2.02 µm class size. D. lumholtzi is capable of ingesting particles much larger than those that the small-sized species frequently consume, thus it appears likely that D. lumholtzi is able to exploit unused food resources. As small sized communities are usually characterized by high levels of predation, the native larger sized zooplankton species that would typically consume the larger sized particle are removed from the community by size selective predation. However, it is possible that the elongated spines of

D. lumholtzi protect the species from vertebrate predators, allowing the invasive species to coexist with small sized zooplankton species.

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Zooplankton Age Structure

Although body size and use of food resources can be used to predict the strongest competitor in laboratory experiments; other influences are present that may influence zooplankton community structure within the natural environment. This may be why experimental evidence regarding zooplankton communities has not been supportive of the Size

Efficiency Hypothesis and contributed to the general non-acceptance of the hypothesis that competitive interactions influence zooplankton community composition (Work and Gophen

1999a). One reason for the dispute may be that the Size-Efficiency Hypothesis does not consider the age classes of the species involved. If the adults of the smaller species share the same resources as the offspring of the larger species, lessened viability of the neonates may occur; ultimately resulting in a displacement of the larger species if the young fail to survive into adulthood. For example, Lynch (1978) was able to demonstrate that Ceriodaphnia may be a superior competitor to Daphnia species because the juvenile daphnids were sensitive to resource depression.

In enclosure experiments investigating the level of competition between D. lumholtzi and

D. parvula in McDaniel Lake, Missouri (Johnson and Havel 2001), it was expected that the larger body size of D. lumholtzi would be advantageous and allow the species to become competitively superior over D. parvula. It had been demonstrated that D. lumholtzi was able to suppress the numbers of D. parvula when the two species were in direct competition in the enclosures; however, D. lumholtzi was never found in any significant numbers in the lake following introduction. The experiment showed that competition with D. parvula was not limiting the growth of D. lumholtzi and an alternative hypothesis was needed to explain the low

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occurrences of the invasive species within the lake. As shown in past studies, including the paleolimnological study in Chapter 2, higher diversity appears be associated with invasion resistance. Thus, it may be that other cladocerans within McDaniel Lake, including

Diaphanosoma birgei, D. retrocurva, and D. ambigua, also contributed to the suppression of

D. lumholtzi numbers by limiting the number of available feeding niches.

Influence of Predation

In addition to competition, predation could also influence zooplankton community structure as well as determine the outcome of invasion. The large size of D. lumholtzi as well as the elongated head and tail spines may result in handing difficulties for several invertebrate and vertebrate predators (Celik et al. 2002). Thus, prey selection against the exotic species may grant D. lumholtzi a competitive advantage (Dzialowski et al. 2000, Muzinic 2000). For example, in Lake Okeechobee, Florida, D. ambigua was found in low densities in the northern sections of the lake where planktivores were in high abundance, yet in this location D. lumholtzi was found in high numbers (East et al. 1999). This suggests that D. lumholtzi is able to survive in areas of high vertebrate predation.

Examinations of the extent to which the morphology of D. lumholtzi obstructs fish predation demonstrated that bluegill less than 50mm in length selected against the species both in the field and laboratory (Kolar and Wahl 1998). In most cases these juvenile fish attacked D. lumholtzi readily in the beginning of the study; however, as the bluegill gained experience of the challenges associated with capturing and ingesting the zooplankton they stopped attacking it. Larger bluegill, 52-80 mm, did not select against D. lumholtzi, although predation on the

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daphnid was associated with lower ingestion rates and longer handling times when compared to foraging times on native D. pulex (Kolar and Wahl 1998). In an investigation of the feeding preferences of silversides in Lake Texoma, Texas (Lienesch and Gophen 2005) it was found that fish as small as 22 mm can ingest D. lumholtzi but select smaller prey when available.

These feeding investigations support the hypothesis that D. lumholtzi may be able to persist in communities dominated by smaller sized zooplankton because the morphology of this species deters the high level of fish predation that may be responsible for keeping the native large sized zooplankton species in low abundance.

Large bodied zooplankton communities are often characterized by high levels of invertebrate predation, as invertebrates usually prey on zooplankton smaller than 2 mm

(Dodson 1974b). The large size and elongated spines of D. lumholtzi appear to effectively deter predation from invertebrates (Swaffer and O’Brien 1996). Celik et. al (2003) found a positive relationship between D. lumholtzi and Chaoborus in a North Carolina reservoir; hence, competitive interactions, rather than invertebrate predation, are likely responsible for the absence D. lumholtzi from large sized zooplankton communities.

Influence of Season Succession

In addition to the biological interactions of competition and predation, seasonal influences could also have a dramatic impact on the zooplankton community. In heterogeneous environments, such as most lakes and reservoirs, changing environmental conditions result in a change of the phytoplankton composition. This fluctuation in food conditions may influence the outcome of competition as certain species may be able to utilize short pulses of high food

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concentrations, have a lower food threshold, or more efficient at consuming grazing-resistant algae; each of which may allow the species to become favored within the community (Kreutzer and Lampert 1999). In addition, it is unlikely that any two species will have identical feeding rates in natural situations. Should one species be more efficient at high food concentrations and the other low, coexistence may occur. Seasonal succession within zooplankton populations as a result of resource partitioning has been supported by numerous field studies. In a Michigan lake it was observed that D. galeata had higher birth rates, death rates, and rates of increase when placed in direct competition with D. pulicaria (Johnson and Havel 2001). The numbers of D. galeata increased in mid-summer as this species was able to exploit low quality resources more effectively than D. pulicaria. In spring, the situation reversed as D. pulicaria becomes more efficient at utilizing higher quality resources and reached a peak in its population density.

Lake enclosure experiments have also shown that D. rosea had a competitive advantage in the spring, but its numbers rapidly declined by late summer as a direct result of competition with

D. pulicaria (Johnson and Havel 2001).

In the United States, D. lumholtzi populations have been recorded to reach maximum numbers during the mid- to late summer months. During this period, most native zooplankton species decline which may result in decreased competition. The paleolimnological study in

Chapter 2 indicated that lower species diversity within lakes may make these habitats vulnerable to invasion by D. lumholtzi. The lowest diversity within a lake typically occurs during the summer months; hence, conditions at this time of the year become ideal for the invasive species. Additionally, phytoplankton communities in the late summer months are often dominated by cyanobacteria, which are associated with handling difficulties, toxins, and

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low nutrient content for zooplankton. In the tropical lakes of D. lumholtzi’s native range, cyanobacteria are present throughout most of the year, indicating that this species may have a greater tolerance for cyanobacteria (Pattinson et al. 2003). Thus, seasonal succession of the zooplankton species and a tolerance to grazing-resistant algae may also facilitate invasion of D. lumholtzi.

Paradox of the Plankton

Bengtsson (1987) conducted a literature review of cladoceran competitive interactions.

An examination of 37 species combinations in 20 studies found that the size-efficiency hypothesis was only supported in 60% of the investigations. The R-max hypothesis was supported in 68% of the studies, while the efficiency at low food levels found support in 36%.

Lastly, small species were the superior competitors in only 17% of the experiments. Of the experiments that were conducted under different conditions in the laboratory or during more than one experimental period in the field, 76% showed different outcomes in different treatments. This suggests that competitive ability in cladocerans may vary with environmental conditions, which may result in non-equilibrium coexistence in many zooplankton communities.

Environmental conditions vary both temporally and spatially within lakes, thus trade offs between predation, resource levels, and abiotic conditions may result in different combinations of species favored under different environmental conditions. The situation of several potential competitors present in an open water community was described by Hutchinson (1961) as the

“paradox of the plankton”. Originally this concept was applied to phytoplankton, but later was

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expanded to zooplankton communities (Herbert and Crease 1980). This concept suggests that limiting the amount of ecological segregation necessary to permit coexistence may imply the existence of competitive equilibrium which may not apply to zooplankton communities.

Hutchinson (1961) stated that non-equilibrium conditions, created by season transitions and environmental disturbances might lead to the greater than expected diversity. Furthermore, plankton may never reach equilibrium, as multi-species competition may lead to oscillations and chaos, contributing greater diversity within the water column (Rohde 2005). The rapid shifts in competitive advantage as a result of this non-equilibrium may allow species which would eventually be competitively excluded to persist longer than expected.

Overlapping Generations Resulting from Diapause

Competition is further complicated in zooplankton communities as many species produce diapausing eggs, creating overlapping generations. Even if competition is able to completely exclude another species from the water column, these eggs provide a colonization source for future years. The overlapping generations provided by the eggs along with fluctuating environmental conditions common may allow the coexistence of species as well as maintain high species diversity in zooplankton communities (Caceres 1998). Further, if conditions in an introduced habitat are unsuitable for D. lumholtzi to persist or if the species is competitively excluded, the ephippia left behind will guarantee D. lumholtzi an addition chance to invade, perhaps in a different set of conditions that will facilitate establishment.

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Conclusions

This study provides evidence that resource partitioning among zooplankton species is a contributing factor to facilitating the invasion of D. lumholtzi. The invasive species demonstrated a high feeding overlap with other large size daphnids, indicating the potential for resource competition which may prevent invasion. D. lumholtzi has been shown to consume larger particles than small sized cladoceran species, thus it may be able to fulfill a vacant niche and persist in communities dominated by small sized zooplankton species. These hypotheses were supported in this study as D. lumholtzi was able to successfully establish in both communities dominated by small sized cladocerans; however the invasive species failed to persist in the mesocosms where large daphnids were present.

Two main conclusions can be drawn from past studies of zooplankton competition. First, zooplankton commonly demonstrate resource overlap; and secondly, competition is hard to demonstrate in nature as the outcome can be unpredictable, rarely resulting in competitive exclusion (Hesson 1990). The absence of competitive exclusion in zooplankton communities could be explained by size selective predation, age structure, or seasonal succession. As food quality increases, intraspecific competition may also become more important in reducing the likelihood of exclusion. Competitive exclusion may also be avoided by the zooplankters’ ability to produce resting eggs. The epihippia may act as a measure of fitness of the preceding generations as well as create a link between the seasons and generations.

For these reasons, the influence of competition still remains controversial despite numerous examples of potential competition and resource partitioning. Given that zooplankton competition experiments often take place under a variety of conditions, it is not surprising that

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the experiments often have different outcomes. A reliable indicator of competition has yet to be determined as the outcome of competition may be dependent on a wide range of circumstances including temperature, body size, feeding modes, reproductive strategies, as well as food composition and availability (Caceres 1998, Milbrink et al.2003). Thus, it may be unlikely that complex ecological interactions, such as competition, can be predicted from the evaluation of a single trait. However, the main conclusions found in this study, were also found in the contemporary survey, paleolimnological investigation, and resource overlap study. Although competition can be difficult to confirm using only the overlap of food resources, parallel conclusions from multiple methods lend support. Future work is warranted regarding the predator deterrence as well as the depth and algal preferences of D. lumholtzi. However, the results of this investigation of the community resistance toward the invasive D. lumholtzi is consistent with the hypothesis that invasion by D. lumholtzi may be resisted by larger sized species, while communities dominated by small sized cladoceran species may be vulnerable to invasion. More importantly, this study is a reminder that community structure is an important factor to consider while identifying patterns of invasions as the identities or traits of a particular species may be capable of preventing invasion.

FINAL DISCUSSION

Successful invasion is likely dependent on numerous factors, including resource availability, species interactions, dispersal abilities, and the life history characteristics of the non-native species. Efforts to fully understand the success rates and consequences of exotic species on communities has brought forth numerous investigations concentrating on identifying the qualities of invasions, the invaders, as well as vulnerable habitats. This study used a combination of techniques including a contemporary survey, paleolimnological study, and laboratory experiments to explore conditions of freshwater communities that may facilitate the establishment of D. lumholtzi and to evaluate the potential impact that this species may have on native environments.

Past research involving D. lumholtzi has focused on the abiotic conditions (Havel et al. 1995,

Work and Gophen 1999a/b, Dzialowski et al. 2000, Lennon et al. 2001) or species diversity

(Lennon et al. 2003, Dzialowski et al. 2007) to describe patterns of invasion There have not been any investigations that have considered the influence of the identity or trait of a particular species on the invasion of D. lumholtzi. Consequently, this study was performed in order to assess the role of biological interactions in either facilitating or preventing the invasion by this exotic cladoceran. Understanding the mechanism for successful invasion is warranted because of the rapid rate in which D. lumholtzi is expanding its range, which suggests that the species will become a permanent and common member of lake ecosystems in North America.

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Prior to this investigation, the presence of D. lumholtzi was only recorded in 3 lakes within the state of Ohio (Havel and Shurin 2004). However, D. lumholtzi was detected in 19 of the 93 lakes and reservoirs included in the contemporary survey, demonstrating that the spread of this species has been underestimated. Many of the non-invaded localities are in close proximity or are downstream from invaded locations. This indicates that several non-invaded lakes have strong potential for the introduction of D. lumholtzi, yet have been able to resist invasion thus far.

It seems unlikely that local abiotic conditions prohibit D. lumholtzi from establishing in additional lakes, as the limnological data failed to find any significant differences in the abiotic factors or productivity levels between the invaded and non-invaded lakes. Further, D. lumholtzi has shown to have strong dispersal capabilities, broad physiological tolerances and elongated spines which may deter predation; all which contribute to making this species a successful invader. However, in spite of these characteristics, there are still several locations in which the species has not yet been able to invade. The remainder of this study focused on the biological interactions between D. lumholtzi and native zooplankton species in order to explain the apparent paradox in which a species cannot persist in a particular location despite the presence of physiological tolerances adapted for the environmental conditions.

Past hypotheses that suggested that invasion may be influenced by biological interactions among community members have focused on species diversity (Elton 1958, Simberloff and Von

Holle 1999, Ricciardi 2001, Miller et al. 2002). Accordingly, the biodiversity of the invaded locations were considered in this study. The contemporary survey did not find any differences in

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species diversity between the invaded and non-invaded localities. The paleolimnological study determined that that invaded lakes were characterized by lower species diversity, providing support for the Biotic Resistance model (Elton 1958, Miller et. al. 2002) This difference may not have been seen in the contemporary survey since it only consisted of samples collected from the pelagic region of the lakes between April and September, thereby data from the littoral species and changes in zooplankton abundance resulting from seasonal succession were not accounted for. These limitations in contemporary sampling emphasize the value of paleolimnological techniques in ecological studies. Paleolimnological investigations provide a complete successional history of any lake, include each habitat, and avoid the problems associated with seasonality and patchiness. The lake sediments represent the changes in zooplankton composition over time as well as changes in species abundance that follow an invasion. Further, paleolimnological studies were able to detect the failure of an invasive species to persist after it becomes introduced into a new habitat. Cladoceran microfossils store the histories of community dynamics, including the date of their arrival. The potential of paleolimnology in investigations regarding biological invasion as been greatly underestimated and warrant more attention.

The paleolimnological study in this investigation was used to analyze the zooplankton community compositions associated with D. lumholtzi. The results of this analysis paralleled those found in the contemporary survey. Both investigations found that D. lumholtzi is closely associated with smaller sized zooplankton species, but not found with larger sized daphnids. It is recognized that many factors may influence the structuring of zooplankton communities, including predation, food resources, as well as numerous abiotic conditions. Experimental mesocosms were used explore the role of competition on the invasion of D. lumholtzi in the

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absence of other factors. It was found that D. lumholtzi was unable to persist in the mesocosms comprised of large bodied daphnids, while the invasive species was able to establish in the mesocosms with smaller bodied species. These results coincide with those found in the contemporary and paleolimnological study, supporting the idea that the presence of a particular species or species group may be capable of preventing invasion.

The resource overlap study investigated the utilization of food resources among different cladoceran species and its role in the facilitation or resistance of biological invasion. In this study the mid and large-sized daphnids demonstrated an ability to consume the largest particle sizes offered and exhibited grazing rates that exceeded that of D. lumholtzi. The mid-sized D. retrocurva exhibited the highest food resource overlap with D. lumholtzi, which may explain why these species were never found concurrently in the contemporary survey. D. lumholtzi and

E. coregoni were also never found in the same lake at the same time in any of the surveyed locations; although in the experimental mesocosm study, D. lumholtzi was able to persist in treatments where E. coregoni was present. Since the contemporary survey revealed a strong association between D. retrocurva and E. coregoni, the presence of D. retrocurva and its potential ability to exclude D. lumholtzi, may be the reason why D. lumholtzi failed to establish in communities containing E. coregoni.

The resource overlap study further revealed that D. lumholtzi consumes larger particles than the small- sized cladoceran species. This implies that there is minimal food resource overlap between D. lumholtzi and the small bodied zooplankton and that D. lumholtzi may not be in direct competition with these species. Lack of competition suggests that D. lumholtzi cannot displace these native cladocerans and should not impact the existing zooplankton community

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composition following invasion. Analysis of the sediment cores in the paleolimnological study did not reveal any changes in the abundance of pelagic species following invasion. Further, the experimental mesocosms showed little alteration to the community structure of the small bodied treatments following the introduction of D. lumholtzi. These results support the hypothesis that

D. lumholtzi is unable to competitively exclude native zooplankton species and changes in zooplankton abundance following invasion are minimal.

The conclusions from each of the investigations used in this study suggest that communities with larger sized species may inhibit the establishment of D. lumholtzi as a result of the high food resource overlap with D. lumholtzi. The invasive cladoceran does not appear to be in direct competition with small sized zooplankton species; however, communities dominated by these species may be more susceptible to invasion. This conclusion can be explained by considering the influence of predation on zooplankton community structure. Communities with smaller sized species are typically characterized by high levels of vertebrate predation. Vertebrate predators usually select larger prey items, thus reducing the number of the larger sized zooplankton species that would typically consume the larger sized particles. It is possible that the elongated spines of

D. lumholtzi protect the species from vertebrate predators, allowing the invasive species to coexist with small sized zooplankton species and exploit the larger particles typically consumed by the larger bodies species that may be eliminated by vertebrate predators. Thus, both competition and predation may influence the outcome of invasion by D. lumholtzi. This study focused on the competitive interactions between D. lumholtzi and native cladocerans, yet further research is needed to understand the role of vertebrate predation on invasion success

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This investigation has also prompted other questions related to the invasion of D. lumholtzi.

For example, the paleolimnological study found higher Bosmina mucro to carapace length ratios in the sediment cores from the invaded lakes, indication of higher invertebrate predation levels in these locations. Currently it is unknown if invertebrate predators are able to consume D. lumholtzi or if the elongated spines of this species are able to deter predation. Additional research is needed to determine if lakes with higher levels of invertebrate predation are more vulnerable to the establishment of D. lumholtzi.

Results from the paleolimnological study also revealed that the species diversity of littoral cladocerans increased following the invasion of D. lumholtzi. This trend may occur as selective grazing by D. lumholtzi clears the water column, increasing the area of the littoral zone; however this study does not offer any evidence to support this hypothesis. D. lumholtzi may also provide an additional food source for planktivorous fish in the late summer months; thus allowing these predators to remain in the pelagic zone at a time when daphnids are usually absent. A shift in feeding preferences of vertebrate predators following the invasion of D. lumholtzi may explain the changes found in the littoral communities. Additional research is needed to determine if the increased diversity within the littoral community was related to the invasion D. lumholtzi and if this exotic species has the ability to impact upper or lower trophic levels.

In conclusion, the results of this study suggests that the identities or traits of a particular species may resist biological invasion. The outcome of competitive interactions may depend on a wide range of circumstances including temperature, body size, feeding modes, reproductive strategies, as well as food composition and availability. Consequently, competition can be difficult to document and as a result its role in preventing biological invasions may be

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underestimated. D. lumholtzi was found to be associated with smaller sized zooplankton species and excluded from communities dominated by larger species in the contemporary survey, paleolimnological investigation, resource overlap study, and experimental mesocosm study.

Competition cannot be confirmed in any of the individual studies; however the similar conclusions found from each studies strongly imply that biological interactions are capable of determining the outcome of invasion. This study concludes that identities or traits of a particular species do shape the outcome of invasion; thus it is important to consider the structure of a community while investigating patterns of invasion.

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Appendix A: All lakes included in the Contemporary Survey (Chapter 1)

Lake Samples provided by Lakes sampled in between Lakes sampled by the OEPA and the OEPA 2205 and 2006 in 2005 and 2006 Acton Lake Atwood Reservoir Barberton Reservoir Aldrich Pond Lake Milton Charles Mill Reservoir Alum Creek Lake Lake Berlin Chippewa Reservoir Amann Reservoir Leesville Reservoir Clendening Reservoir Barberton Reservoir Seneca Reservoir Deleware Reservoir Belmont Lake Tappan Reservoir East Branch Reservoir Blue Limestone Lake Hoover Reservoir Bowling Green Reservoir LaDue Reservoir Bucyrus Reservoir Mogadore Reservoir Caesar Creek Reservoir Mosquito Creek Reservoir Caldwell Lake Nimisila Reservoir Cambridge Reservoir O'Shaughnessy Reservoir Charles Mill Reservoir Piedmont Lake Chippewa Reservoir Pymatuning Reservoir CJ Brown Reservoir Salt Fork Reservoir Clendening Reservoir Turkey Foot Lake Crystal Lake Twin Lake, East Deer Creek Lake Twin Lake, West Delaware Reservoir West Branch Reservoir Delco Lake Wills Creek Reservoir Deleware Reservoir East Reservoir East Branch Reservoir Echo Lake Evans Lake Evergreen Lake Grand Lake St. Mary's Grand River Wildlife Lake Grant Lake Guildford Lake Hargus Lake Harrison Lake Hoover Reservoir Huffman Reservoir Indian Lake Jefferson Reservoir LaDue Reservoir Lake Choctaw Lake Isabella Lake Lavere Lake Loretta

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Lake Romona Lake Rupert Lake Snowden Lake Sue Lampson Reservoir Madison Lake Maysville Reservoir Meander Creek Reservoir Mogadore Reservoir Mosquito Creek Reservoir Mount Gilead Mount Orab Reservoir Nettle Lake New Concord Reservoir New Lexington Reservoir Nimisila Reservoir North Baltimore Reservoir Opossum Creek Lake O'Shaughnessy Reservoir Piedmont Lake Possumack Reserve Pond Pymatuning Reservoir Raccooon Creek Reservoir Richwood Lake Rio Grande Reservior Roaming Rock Shores Rocky Fork Lake Rush Run Lake Salt Fork Reservoir Sardinia Reservoir Schrock Lake Sharon Woods Lake St. Clairsville Reservoir Sunny Lake Thoreau Pond Turkey Foot Lake Twin Lake, East Twin Lake, West West Branch Reservoir (MJ Kirwin) Westerville Reservoir Whitewater Lake William Harsha Lake (East Fork) Wills Creek Reservoir Wolf Run Lake

Appendix B: List of Lakes and Reservoir that were included in the Zooplankton Measurement Study (Chapter 1, Contemporary Survey) Non-Invaded Localities Invaded Localities

Lake or Reservoir Date Sampled Lake or Reservoir Date Sampled Alum Creek Reservoir 8/23/1989 Alum Creek Reservoir 7/21/2005 Deer Creek Reservoir 8/20/1990 Grand Lake St. Mary's 5/20/1993 Hargus Lake 8/23/1990 Hargus Lake 8/21/1997 Highlandtown Lake 9/14/2005 Indian Lake 5/24/1993 LaDue Reservoir 7/20/2005 Lake Milton 8/31/2005 Mosquito Creek Reservoir 9/3/2006 Maysville Reservoir 8/9/1994 Sippo Lake 7/25/2002 New Concord Reservoir 8/9/1994 West Branch Reservoir 7/20/2005 Salt Fork 8/8/1994 Wills Creek Reservoir 8/9/2005 Tappan Lake 8/9/2005 Wolf Run Reservoir 8/13/1996 Whitewater Lake 9/24/1996

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Appendix C: List of Lakes and Reservoir that were included in the Diversity Study Non-Invaded Invaded Lake or Reservoir Date Sampled Lake or Reservoir Date Sampled Alum Creek Reservoir 8/23/1989 Alum Creek Reservoir 8/2/2005 Barberton Reservoir 8/1/2005 Atwood Reservoir 8/15/2005 Charles Mill Reservoir 9/11/1990 Berlin Lake 8/31/2005 Chippewa Lake 8/1/2005 Deer Creek Reservoir 8/21/1997 Clendenington Reservoir 8/13/2005 Deleware Reservoir 8/2/2005 Crystal Lake 8/24/1994 Hargus Lake 8/21/1997 Deer Creek Reservoir 8/20/1990 Hoover Reservoir 8/2/2005 Deer Creek Reservoir 8/22/1994 Lake Choctaw 8/22/1997 East Branch Reservoir 8/1/2005 Lake Milton 8/31/2005 Mosquito Creek Findley Reservoir 7/23/2001 Reservoir 8/31/2005 Findley Reservoir 9/24/1997 New Concord Reservoir 8/9/1994 Findley Reservoir 8/5/1997 O'Shagonessy Reservoir 8/2/2005 Hargus Lake 8/23/1990 Salt Fork Lake 8/8/1994 Hargus Lake 7/17/1997 Salt Fork Lake 8/13/2005 Highlandtown Lake 9/14/2005 Seneca Lake 8/13/2005 Jefferson Reservoir 8/24/1995 Tappan Reservoir 8/13/2005 LaDue Reservoir 8/1/2005 Whitewater Lake 9/24/1996 Lake Sue 8/14/1997 Leesville Reservoir 8/15/2005 Long Lake 8/19/1998 Lost Creek Reservoir 8/22/1996 McComb Reservoir 8/22/1996 Mosquito Creek Reservoir 9/3/2006 Morgadore Lake 8/19/1996 Morgadore Lake 8/1/2005 Nettle Lake 8/13/1997 New Lexington Reservoir 9/11/1995 Nimisilla Reservoir 8/1/2005 PiedmontC 8/15/05 9/10/1998 Pleasant Hill Lake 8/22/1996 Pymatuning Reservoir 8/1/2005 Sebalo Pond 8/15/1995 Shrock Lake 10/21/1996 Sippo Lake 10/7/2002 Summit Lake 8/20/1996 Turkeyfoot Lake 9/9/1997 Turkeyfoot Lake 8/1/2005 West Branch 8/1/2005 Westerville Lake 7/20/1997 Wills Creek Reservoir 8/13/2005 Wills Creek Reservoir 8/13/2005

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