Intraspecific Variation of Three Phenotypic Morphs of Daphnia Pulicaria in the Presence of a Strong Environmnetal Gradient

INTRASPECIFIC VARIATION OF THREE PHENOTYPIC MORPHS OF DAPHNIA PULICARIA IN THE PRESENCE OF A STRONG ENVIRONMNETAL GRADIENT

Ariel Shaleka Gittens

A thesis submitted to the Department of Biology

In conformity with the requirements for

the degree of Masters of Science

Queen’s University

Kingston, Ontario, Canada

(April, 2014)

Freshwater lake ecosystems often exhibit strong oxygen, and temperature gradients across which many zooplankton species live. Daphnia sp. vary in their ability to up-regulate hemoglobin in response to low oxygen environments. However; the role that hemoglobin up-regulation plays in diel vertical migration, and how it might mediate coexistence of Daphnia within lakes is still unclear. Using an oligotrophic lake in

Ontario, I studied three distinct phenotypes of Daphnia pulicaria, which differed in the ability to up-regulate hemoglobin (classified as red, pink, and pale). Twenty-four hour surveys were conducted during the fall of 2012 and samples were drawn at 1m intervals to monitor changes in diel vertical migration. At each 1m interval Daphnia were color indexed, photographed, and preserved for genetic analysis using cellulose acetate electrophoresis. Red and pink Daphnia showed little change in distribution over the water column through time, suggesting individuals experienced little vertical migration. Pale individuals showed strong changes in vertical distribution through time suggesting vertical migration. The phenotypes are strongly correlated with multi-locus genotypes, suggesting genetic differences in migration behavior. Mesocosm experiments were used to manipulate migration over heterogeneous environments to test the hypothesis that vertical migration impacts genetic and phenotypic diversity in Daphnia pulicaria. The first mesocosm experiment contained two treatment groups; a migrating and non- migrating treatment containing the three phenotypes. The migrating treatment permitted unrestricted movement throughout the water column, and the non-migrating treatment restricted Daphnia to discrete 1m intervals. The second mesocosm experiment comprised two non-migrating treatments; red non-migrating and pale non-migrating. Results from ii the first set of mesocosm experiments indicate decreased genetic and phenotypic diversity in the migrating treatment. Shifts in hemoglobin up-regulation between pales and reds in the second mesocosm experiments suggest hemoglobin up-regulation is plastic, whereby pale, pink, and red individuals have the ability to up and down regulate hemoglobin. The differences in Daphnia migration patterns and the plastic response in hemoglobin up-regulation permits migrating genotypes to withstand low oxygen conditions. Overall implications of this study suggest that migration over a strong environmental gradient plays a key role in fostering phenotypic plasticity and genetic diversity in organisms living in heterogeneous environments.

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Acknowledgements

This thesis was competed thanks to the support of many people, so if I forget to mention you, you know who you are. First and foremost I would like to thank my supervisor Bill; it takes a very special person to handle my particular brand of crazy. Your guidance and wisdom have been invaluable to this research and my development as a scientist. I could not have wished for a more kind and patient, emphasis on the word patient, mentor.

I am thankful for the support of Shirley French, you were my field mentor. Where would

I be without your particular brand of funny and kind words of encouragement? I have to thank

Clay Cressler and his wife Sam Cressler. Clay, you are a much smarter, adult, male version of myself; I hope to be like you in my next life. Sam, what would I do without you feeding me? I would be dead in a ditch somewhere. Thank you for opening up your home and family to me. My lab mates, Stefan Bengstan and Leslie Holmes. Leslie, I literally could rule the world from your bedroom and Stefan, remember when I almost lost you in the Minneapolis airport?

I give my thanks to the graduate students who were my village, because it takes a village to raise a child. To all of the graduate students that went out into the field with me, I could not have completed my mesocosm field experiments without you. My field assistant Ayo

Adurogbanga, you were the best thing besides my data that came out of my summer field season.

I will never forget your hard work and dedication to my project; I thank you for all of your hard work.

Thank you to my committee, Dr. Shelley Arnott, Dr. Chris Eckert, and Dr. Paul Grogan for their helpful suggestions and experience. Your insights have provided me with direction and support throughout this project.

Last but not least, I have to thank my family and my mother Lisa Gittens. Thank you for your encouragement; I could not have completed this degree without you. You answered every phone call and told me when I needed to take a nap. iv

Statement of Originality

I hereby certify that all of the work described within this thesis is the original work of the author.

Any published (or unpublished) ideas and/or techniques from the work of others are fully acknowledged in accordance with the standard referencing practices.

(Ariel Gittens)

(September, 2014)

Table of Contents

Abstract ...... ii Acknowledgements ...... iv Statement of Originality ...... v List of Figures ...... viii List of Tables ...... x List of Abbreviations ...... xi Chapter 1 General Introduction ...... 1 1.1 Spatial and Temporal Environmental Heterogeneity ...... 2 1.2 Diel Migration in Zooplankton ...... 4 1.3 Daphnia ...... 7 1.4 Hemoglobin in Daphnia ...... 9 1.5 Study Site ...... 10 1.6 Thesis Objectives ...... 10 Chapter 2 To migrate, or not to migrate: two co-existing strategies for how Daphnia use environmental gradients ...... 13 2.1 Introduction ...... 13 2.2 Methods ...... 16 2.3 Results ...... 25 2.4 Discussion ...... 27 2.5 Figures ...... 36 Chapter 3 Manipulating migration over a heterogeneous environment: how the ability to migrate influences genotypic and phenotypic diversity in Daphnia pulicaria ...... 47 3.1 Introduction ...... 47 3.2 Methods ...... 50 3.3 Statistical Analyses ...... 55 3.4 Results ...... 56 3.5 Discussion ...... 59 3.6 Figures ...... 69 Chapter 4 General Discussion ...... 83 References ...... 85 Appendix A ...... 106 Appendix B ...... 109

Appendix C ...... 112

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List of Figures

Figure 1.1 A diagram that depicts the sexual and asexual parthenogeneic life cycle of Daphnia (Ebert 2005)...... 12 Figure 2.1 Mean vertical temperature (°C ) profiles ...... 36 Figure 2.2 Mean vertical oxygen (mg/L) profiles ...... 40 Figure 2.3 Cholorophyll-a (μg /L) profiles ...... 41 Figure 2.4 Particulate ogranic carbon (μg /L) profiles ...... 42 Figure 2.5 Light Levels (lux) ...... 43 Figure 2.6 Relative abundances of the three phenotypes during the summer of 2012 ...... 44 Figure 2.7 24h diel vertical migration patterns ...... 45 Figure 2.8 12h diel vertical migration patterns ...... 46 Figure 2.9 Total hemoglobin mass (μg /mL) of individual Daphnia from the three phenotypes .. 47 Figure 2.10 Total hemoglobin concentration (mg.g/ dry weight) of individual Daphnia from the three phenotypes ...... 48 Figure 2.11 Hue values of individual Daphnia from the three phenotypes ...... 49 Figure 2.12 Principal coordinate analysis of the 4pm and 4am samplings combined ...... 50 Figure 2.13 Principal coordinate analysis of the 4pm sampling ...... 51 Figure 2.14 Principal coordinate analysis of the 4am sampling ...... 52 Figure 3.1 Mesocosm Experiment 1 Setup ...... 77 Figure 3.2 Mesocosm Experiment 2 Setup ...... 78 Figure 3.3 Genotype absolute abundances: lake sampling, non-migrating, migrating ...... 79 Figure 3.4 Rank abundance curves: lake sampling, non-migrating, migrating...... 80 Figure 3.5 Genotype richness, Shannon-Wiener, and Simpson genetic diversity for the non- migrating and migrating treatments ...... 81 Figure 3.6 Density and Shannon-Wiener genetic diversity, Simpson genetic diversity, and genotype richness for the migrating and non-migrating treatments ...... 82 Figure 3.7 Absolute phenotype abundances: lake sampling, non-migrating, migrating ...... 83 Figure 3.8 Phenotype Shannon-Wiener and Simpson diversity indexes for the non-migrating and migrating treatment groups ...... 84 Figure 3.9 Phenotype abundances by depth non-migrating treatment mesocosm experiment 1 .. 85 Figure 3.10 Phenotype abundances by replicate migrating treatment mesocosm experiment 1 ... 86 Figure 3.11 Phenotype abundances red non-migrating treatment mesocosm experiment 2 ...... 87

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Figure 3.12 Phenotype abundances pale non-migrating treatment mesocosm experiment 2 ...... 88 Figure 3.13 Shannon-Wiener, and Simpson phenotypic diversity indexes for the red, and pale non-migrating treatments mesocosm experiment 2 ...... 89

List of Tables

Table 2.1 Summary of one factor ANOVA values for total hemoglobin mass per animal (μg /L)...... 36 Table 2.2 Summary of Tukey HSD comparisons for total hemoglobin mass per animal (μg /L). 36 Table 2.3 Summary of one factor ANOVA values for total hemoglobin concentration (mg/g dry weight) per animal...... 37 Table 2.4 Summary of Tukey HSD comparisons for total hemoglobinconcentration (mg/g dry weight) per animal...... 37 Table 2.5 Summary of one factor ANOVA values for hue values for individuals analyzed spectrophotometrically...... 38 Table 2.6 Summary of Tukey HSD comparisons for hue values ...... 38 Table 3.1 Genotype richness, Shannon-Wiener diversity and Simpson diversity indexes for the non-migrating and migrating treatments...... 72 Table 3.2a Phenotype Shannon-Wiener diversity index for the non-migrating and migrating treatments used in the first mesocosm experiment ...... 73-74 Table 3.2b Phenotype Simpson diversity index for the non-migrating and migrating treatments used in the first mesocosm expriment ...... 73-74 Table 3.3a Phenotype Shannon-Wiener diversity index for the red non-migrating treatments used in the second mesocosm experiments ...... 75 Table 3.3b Phenotype Simpson diversity index for the red non-migrating treatments used in the second mesocosm experiments ...... 75 Table 3.4a Phenotype Shannon-Wiener diversity index for the pale non-migrating treatments used in the second mesocosm experiments ...... 76 Table 3.4b Phenotype Simpson diversity index for the pale non-migrating treatments used in the second mesocosm experiments ...... 76

List of Abbreviations

ANOVA: Analysis of Variance

ANCOVA: Analysis of Co-Variance

Tukey HSD: Tukey Honest Significant Difference

χ2: Chi Squared

Chl-a: Chlorophyll ɑ

POC: Particulate Organic Carbon

Chapter 1

General Introduction

One of the main goals of community ecology is to understand the patterns of distribution and abundance of ecological communities. Ecological communities contain a wide range of species diversity, and ecologists have long questioned how this diversity is maintained (Chesson

2000, Cadotte and Carscadden 2011,Ricklefs 1987). This has motivated the proposition of niche based hypotheses to help explain the maintenance of species diversity. Grinnell (1917) first coined the term ‘niche’ in his seminal paper “The niche relationship of the California Thrasher”, which states that the niche of a species is the habitat requirements that allow a species to persist and produce offspring. Elton’s (1927) definition of a niche proposed that the niche was the

“ecological position a species occupies within its community” and encompasses the idea that the niche is a role a species plays in its community rather than habitat. Gause (1934) proposed the competitive exclusion principal, which simply states that two complete competitors cannot co- exist, and the stable co-existence of organisms requires individuals to differ in their niches

(Hardin 1960). Hutchinson (1957) took the niche concept one step further by introducing the fundamental versus realized niche. Modern ecological definitions of a niche highlight the importance of heterogeneous environments in restricting the potential area and resources an organism is capable of acquiring (Levins 1963, Rainey and Travisano 1998).

Abiotic and biotic factors that contribute to heterogeneous environments include but are not limited to spatial heterogeneity (Holt 1984), interspecific and intraspecific competition

(Wilson and Keddy 1986), predation (Wellborn et al. 1996), and productivity (Abrams

1988). Abiotic factors such as temperature (Hare et al. 2007, Skirnisdottir et al. 2000), and 1 geological features (Pianka 1966) can create physiological constraints on a species, influencing their distribution and altering community level properties such as species richness. Biotic factors such as predation help regulate community assemblages through mortality (Menge and

Sutherland 1976). Environments can vary spatially and temporally in the composition of abiotic and biotic factors which can influence species distribution and community assemblages.

Additionally species vary in their responses to environmental heterogeneity which may include changes in behavior and physiology in response to spatial and temporal changes in abiotic and biotic factors. However these changes in behavior and physiology involve trade-offs in some other component of fitness. Trade-offs in fitness (i.e. growth, survivorship, and reproduction) promotes stable co-existence because organisms differ in their responses to environmental heterogeneity, which helps to promote species diversity (Levins 1962, Macpeek 1996).

1.1 Spatial and Temporal Environmental Heterogeneity

Environments can vary across space or in time, and the spatial variation can be fine grained or coarse grained. Fine grained variation occurs when a mobile organism experiences more than one set of environmental conditions (Pigliucci 2001). Coarse grained variation occurs when mobile organisms experience fairly homogenous environmental conditions, but the environment varies between subspecies occupying different spatial locations, or across generations (Gilespie 1974). An example of fine grained variation occurs in lakes, where oxygen and temperature gradients vary spatially throughout the water column (Wetzel 2001). In terrestrial systems, coarse grained variation occurs in the coat colors of beach mice, which inhabit diverse habitats in the Southern US, mouse coat color varies spatially in response to owl predation (Steiner et al. 2007). Differences in spatial variation whether fine or coarse grained,

2 influences the assemblage of species present within a habitat, and species are spatially distributing differently in response to environmental heterogeneity.

Temporal heterogeneity occurs in response to complex abiotic and biotic interactions through time (Menge and Sutherland 1976). For example, temporal oscillations in resource availability allows for species coexistence because no two species are limited by the same resource at the same time (Armstrong and MacGehee 1980, Hutchinson 1961, Huisman et al.

1999). Organisms can adopt different responses to both spatial and temporal heterogeneity. For example an organism’s ability to respond to temporal heterogeneity may depend on generation time, while its ability to respond to spatial heterogeneity may depend on the distance over which it can disperse (Townsend and Hildrew 1994).

In response to environmental heterogeneity individuals can adopt behavioral responses such as migration (Cagnacci et al. 2011), and differences in habitat selection (Karr and Freemark

1983) in reaction to changes in their environment. Behavioral responses to environmental heterogeneity can be interspecific or intraspecific and can change depending on the life stage of the organism (Cheng et al. 2013, Weins 1976). A common change in behavior occurs when organisms undergo migration across heterogeneous environments (Brown and Pavolic 1992). For example, ungulates move seasonally between geographic locations that produce differences in terms of predation risk and food quality (Holdo et al. 2009). Migration strategies over heterogeneous environments differ within and between species and involve differences in migratory timing (Woodrey and Chandler 1997), and distance migrated (Nolan and Ketterson

1990). An example of this occurs in the American Shad (Alosa sapidissima), an organisms whose spawning migrations vary with latitude, with northern population spawning earlier than southern populations (Leggett and Whitney 1972, Quinn and Adams 1996). Migration is an

3 important determinant of the spatial distribution of populations in heterogeneous landscapes, and there are costs and benefits associated with migration. Differences in migratory strategy involves trade-offs in fitness (i.e. growth, reproduction, and survivorship), and organisms select habitat that may optimize a component of fitness such as growth, and be detrimental to another such as reproduction. For example, in response to predation risk year of the young fish spend more time in the littoral zone, which provides protection from predators but decreases feeding rates

(Baystrom et al. 2003). Several strategies related to migration can occur in the same species, due to environmental fluctuations and individual differences in the cost of migration (Smith and

Nisson 1987). Migration strategies differ inter and intraspecifically in species of plankton, in response to fluctuations in environmental heterogeneity.

Plankton are a diverse group of organisms that inhabit waterways worldwide, there are two types of plankton: holoplankton and meroplankton. Holoplankton spend their entire life cycle as plankton (e.g. krill, and copeopods), and by contrast meroplankton are only planktonic for a part of their lives (e.g. sea urchins, and sea stars) (Wetzel 2001). Zooplankton, are a type of holoplankton and are present in oceans, seas, and lakes; they are herbaceous grazers that feed on phytoplankton present in the water column. Zooplankton can be classified by size, and/ or developmental stage (Wetzel 2001). Species of zooplankton have a wide range of genetic diversity and phenotypic plasticity, and occupy an essential position in many aquatic foodwebs.

In response to spatial and temporal changes in environmental heterogeneity within lakes, zooplankton undergo a type of behavioral response known as diel migration.

1.2 Diel Migration in Zooplankton

During the summer, primary production and growth of phytoplankton in stratified lakes takes place in the warmer waters of the epilimnion (Wetzel 2001). In deeper thermally stratified lakes

4 the epilimnion is the top-most layer occurring above the deeper hypolimnion; typically the epilimnion has warmer water temperatures and higher dissolved oxygen concentrations (Wetzel

2001). The hypolimnion is the dense bottom layer in a thermally-stratified lake, and is the layer of water that lies below the thermocline. Usually the hypolimnion is the coldest layer of a lake, has lower dissolved oxygen profiles, and receives less light for photosynthesis (Wetzel 2001).

In zooplankton, diel migration occurs in diverse animal phyla across widespread geographical locations in both marine and freshwater habitats (Elser and Hassett 1994, Havel and

Shurin 2004). Diel migration encompasses the movement of individuals over a 24h period, and light is the proximate factor believed to control migration (Haney and Hall 1975, Forward 1988).

However predation risk is thought to be the ultimate factor that controls migration strategies in zooplankton (Hall et al. 1979). Migrations can vary temporally and spatially among developmental stages of species, and between co-existing species (Bohrer 1980, George 1983).

There are three types of diel migrations that commonly occur in species of zooplankton: horizontal, vertical, and reversed vertical migration.

Diel horizontal migration in zooplankton is characteristic of shallow lakes that do not stratify, where light penetrates throughout the entire water column (Lauridsen and Buenk et al. 1996,

Lauridsen et al. 1996). In diel horizontal migration, zooplankton migrate into the littoral zone during the day where submerged macrophytes provide a refuge from visual predators, and migrate into the pelagic zone at night to feed on phytoplankton (Burks et al. 2002). Horizontal migration is advantageous in zooplankton that inhabit shallow lakes that are well-oxygenated throughout the water column, and do not have a hypolimnion that can act as a prey refuge from visual predators (Iglesias et al. 2007, Merhoff et al. 2007).

Diel vertical migration is the vertical movement of individuals into the hypolimnion during the day and back up into the epilimnion at night (Hays 2003), this is the most common form of diel migration in zooplankton. Migrating animals spend the day in the deep cold waters of the hypolimnion, which is frequently oxygen limited, and migrate back up into the warm well oxygenated waters of the epilimnion at night to feed on phytoplankton (Enright and Honegger

1977). This migration is typical of zooplankton that inhabit deeper lakes, seas and oceans.

Predation risk from visual predators is thought to be the ultimate factor that controls vertical migration patterns in zooplankton. Visual predators, such as planktivores and first year juvenile fish, feed most efficiently at high light intensities (Vineyard and O’Brien 1979), and the low light conditions in the hypolimnion during the day can provide some protection from visual predators for larger bodied zooplankton species. There are costs associated with remaining in the cold, food poor hypolimnion during the day; for example zooplankton experience slower growth rates in colder water (Huntley and Lopez 1992) and those costs are usually balanced by avoiding predation mortality.

Another form of vertical migration, reversed vertical migration, has also been documented in a variety of aquatic systems, and involves zooplankton present in the epilimnion during the day, with a migration into the hypolimnion at night (Carey et al. 2011, Wojtal-Frankiewicz et al.

2010). Reversed migration is typical of fishless lakes, where aquatic invertebrate predators are dominant (Ohman et al. 1983). However this migration pattern is less common in species of zooplankton compared to vertical migration.

Predation risk is not the only determinant of vertical migration patterns in zooplankton. Food availability, temperature and oxygen can be just as important in modifying vertical migration patterns, and the resulting distribution of zooplankton in the water column is influenced by a

6 combination of predation risk, food availability, temperature and oxygen gradients (Larrson and

Lampert 2012). Such that food availability, and temperature may be a stronger determinant of vertical migration in zooplankton, compared to predation risk. Work by Giske et al. (1997) and

Lampert (2005) suggests that zooplankton are distributing in the water column according to the ideal free distribution with costs hypothesis. This hypothesis states that zooplankton should be selecting habitats that are best suited to their survival and reproduction while decreasing mortality risk and the resulting choice of habitat is dependent on a combination of food availability, predation risk, and temperature and oxygen gradients (Guisande et al. 1991, Lampert and Grey 2003). An example of this occurs in Daphnia that inhabit ponds with intensive predation risk in the epilimnion, in response to the increased predation risk Daphnia will not undergo vertical migration into the food poor hypolimnion, and remain permanently in the epilimnion (Huntley and Brooks 1982, Johnson and Jakobsen 1987). In species of Daphnia there are well-documented examples of differences in reversed, horizontal, and vertical migration, in response to factors such as predation risk, food availability, and oxygen and temperature gradients (Lampert et al. 2003).

1.3 Daphnia

Daphnia are a genus of zooplankton that live in a variety of aquatic environments ranging from freshwater lakes and ponds to oceans, streams and rivers. They are a member of the order

Cladocera and there are over 50 species of Daphnia distributed worldwide (Hebert 1978).

Daphnia spp. largely reproduce asexually for part of the year (cyclic parthenogens), with each daughter offspring being genetically identical to the mother (Stross 1966). However, in response to changes in the environment (e.g. changes in temperature, food availability, and photoperiod), or temporary habitats (e.g. ephemeral ponds), some Daphnia spp. can undergo sexual

7 reproduction and females can produce male offspring, and haploid eggs known as ephipphia

(Figure 1.1, Hebert and Ward 1972). These sexual eggs are resistant to desiccation, and have the ability to persist in the environment, until an environmental cue such as a change in temperature, or photoperiod causes them to hatch (Ebert 2005). Additionally, some Daphnia spp. reproduce asexually year round (obligate parthenogens) and can also asexually produce viable ephippial eggs in response to changes in the environment, or in the presence of ephemeral habitats (Figure

1.1, Hebert 1981). In lakes abiotic and biotic factors can vary throughout a season, leading to fluctuations in resource availability (Tilman et al. 1982) and predation (Carpenter et al. 1985).

Spatial and temporal variation in an environment can promote genetic diversity in Daphnia if the genotypes have the correct tradeoffs (Ebert 1993, Weider 1989). Populations of Daphnia also reveal many different types of phenotypic morphs in heterogeneous environments (Kussell and

Leibler 2005, Stiber and Luning 1994). Phenotypic plasticity is influenced by both environmental and genetic factors, and allows for adaptations to variable environments (e.g. amphibians accelerate growth rate in ephemeral ponds: Newman 1988). Plastic responses can be illustrated using reaction norms, a visual representation of phenotypic traits of a genotype across a range of environmental conditions (Schlichting and Pigliucci 1998). Species of Daphnia also have various levels phenotypic plasticity, such that some species of Daphnia can undergo drastic morphological changes in response to kairomone cues released by predators (e.g. development of helmets:(Hebert and Grewe 1985, Hanazato 1990), neck teeth: (Dodson 1989), or alter their physiology with the up-regulation of hemoglobin in low oxygen environments (Fox 1948 ). In some lakes oxygen can be a strong determinant constraining habitat selection in species of

Daphnia. Hemoglobin up-regulation is an adaptive response to low oxygen conditions, and can influence vertical migration patterns in Daphnia.

1.4 Hemoglobin in Daphnia

Spatial and temporal differences in oxygen can act as a strong selective force, influencing the genotypic composition in natural zooplankton populations (LaBerge and Hann 1990).

Oxygen levels vary with depth in stratified lakes and in the presence of low oxygen, Daphnia populations can possess substantial variation in their ability to up-regulate hemoglobin (Duffy

2010). The synthesis of hemoglobin has been shown to improve physiological performance of

Daphnia in low oxygen conditions (Paul et al. 2004). There are eleven genes involved in the up- regulation of hemoglobin in Daphnia pulex, and eight genes involved in hemoglobin up- regulation in Daphnia magna (Colbourne et al.2011). In Daphnia pulex there are over 25 hemoglobin subunits expressed in hemoglobin synthesis, and there are shifts in which hemoglobin subunits are expressed depending on temperature and oxygen concentrations present in the water column (Lamekeyer 2003). Daphnia that up-regulate hemoglobin have a distinct pink or red color present in the hemolymph due to the oxy-hemoglobin present in solution in the blood. (Ebert 2005). In maternally reproducing females that up-regulate hemoglobin, oxy- hemoglobin is found in solution in the plasma as well as present in the parthenogenic eggs (Fox

1948). The production of hemoglobin can provide an ecological advantage in predator avoidance and foraging behavior in the oxygen-poor hypolimnion because hemoglobin up-regulation allows individuals to tolerate the lower oxygen conditions of the hypolimnion (Pirow et al. 2001).

Hemoglobin up-regulation differs intraspecifically, and intraspecific variation in hemoglobin synthesis can lead to differences in fitness and fecundity promoting phenotypic and genetic diversity in heterogeneous environments.

1.5 Study Site

Round Lake is a small 15 hectare (ha) oligotrophic lake located in Southern Ontario near

Queen’s University Biological Station (Chaffey’s Locks, Ontario). The lake has a mean depth of

12m and a maximum depth of 30m, and is connected to Garter Lake by a wetland. Oligotrophic lakes are classified as low-production lakes, associated with low phosphorous and nitrogen inputs; they are often very deep clear water lakes. Wetzel (2001) characterizes oligotrophic lakes as lakes with chlorophyll a and total phosphorous levels that range from 0.3-3.0 μg /L and 1-5.0

μg /L respectively, Round Lake’s chlorophyll a levels range from 0.236 μg /L to 3.86 μg /L.

Summer total phosphorous levels in Round Lake range from 3.0 μg /L to 4.0 μg /L (Reavie et al.

2006). Turnover stops in Round Lake at 17m, meaning that the nutrients below 17m are not re- introduced back into the water column. A strong temperature and oxygen gradient exists in

Round Lake year-round due to the lack of turnover indicated by the temperature gradient in the fall (Appendix A, Figure 1A). A large zooplankton community exists in Round Lake, and

Daphnia pulicaria is the dominant Cladoceran in the pelagic zone. There are over thirteen species of fish present in Round Lake, and Chaoborus flavicans is the dominant invertebrate predator of Daphnia in the pelagic zone (Gittens personal observation). Three phenotypic morphs of Daphnia pulicaria exist that differ visually in the amount of hemoglobin visible in the thorax, classified as pales, pink, and reds.

1.6 Thesis Objectives

The overall aim of my thesis is to examine differences in hemoglobin up-regulation between three distinct phenotypes of Daphnia pulicaria, and how these differences correlate with migration patterns among the three morphs. The differences in migration patterns may be a

10 mechanism allowing for intraspecific co-existence between the three phenotypes. My main thesis objectives were:

(1) To determine if differences in hemoglobin up-regulation were correlated with

differences in diel vertical migration patterns, and to understand if there was a genetic

basis to difference in hemoglobin up-regulation between the three morphs (Chapter

2).

(2) To determine the importance of migration over a strong environmental gradient in

maintaining genetic and phenotypic diversity in Round Lake (Chapter 3).

Figure 1.1 A diagram that depicts the sexual and asexual parthenogeneic life cycle of Daphnia (Ebert 2005

Chapter 2

To migrate, or not to migrate: two co-existing strategies for how Daphnia use

environmental gradients

2.1 Introduction Habitat selection is an important component in the ecology of a species, and will be defined here as a place where organisms spend a disproportionate amount of time to help promote the survival and fitness of individuals (Morris 2003). Habitat selection is influenced by a variety of factors such as resource quantity (MacArthur 1964), resource quality (Forsman et al.

2008), competition for resources from both conspecific and interspecific individuals (Fretwell and Carvel 1969, Muller et al. 1997), predation risk (Brown 1991), and environmental and abiotic stressors (Martin 2001, Quan and Pechenik 1998). Resource availability within a habitat can be patchy, with individuals dispersing between patches (Barton et al. 1992), or exist along a habitat gradient (Karr and Freemark 1983) and the qualities of resources available within a habitat contribute to components of fitness through growth, reproduction, and survivorship

(Rozenweig 1991). In addition, individuals can modify their habitat selection based on fitness.

For example, in the Black Legged Kittiwakes (Rissa tridactyla) a cliff nesting seabird, the breeding success of the previous year is influential in determining whether or not the nesting pairs will emigrate and select a different, more productive cliff face (Danchin et al. 1998).

Similarly, the choice in habitat may be modified in terms of the costs and benefits perceived by the animal (Lima and Dill 1990), and individuals select habitats that maximize their overall fitness. This may include selecting habitat that may be detrimental in maximizing growth and reproduction, but may maximize some other component of fitness such as survivorship. For example, Mayfly larvae (Callibaetis montamus), will spend more time in low oxygen conditions 13 in the presence of fish, which increases survivorship but decreases growth rates (Rahel and Kolar

1990). However, habitat selection itself is not static, and individuals can adopt different strategies to deal with temporal changes in resource availability, predation pressures, and density in the number of animals present. These strategies involve modifications in behavior (Nelson et al. 2004), morphology (Dodson 1989) and physiology (Creel and Winnie 2005, Scheuerlein et al.

2001).

Changes in behavior, morphology, and physiology that are likely to increase survival often come at a cost of other fitness-related life-history traits. In response to increased predation risk, ungulates such as elk, moose, and deer avoid high-risk, food rich meadow habitats and switch to low-risk, food poor forest habitats in the presence of wolves and coyotes (Creel et al.

2005, Dussalt et al. 2005, Godvik et al. 2009). Changes in habitat selection often occur along an environmental gradient, whereby abiotic interactions such as temperature and oxygen provide a constraint on habitat selection. For example, in zooplankton, habitat selection is modified as a result of a strong environmental gradient in the water column due to the presence of variable light, oxygen, and temperature gradients (Hays 2003).

Zooplankton have the ability to alter their habitat selection in response to a variety of biotic and abiotic factors by modifying their behavior, for example, diel vertical migration. This vertical migration is characterized by the movement of individuals into the cold, poorly oxygenated hypolimnion during the day and back up into the warm, well oxygenated epilimnion at night (Lampert 1989). Zooplankton can also undergo reversed migration by remaining in the epilimnion during the day and migrating down into the hypolimnion at night (Ohman et al.

1983). The strength of vertical migrations can vary seasonally, as well as among and between zooplankton species. According to a number of studies, predation risk from both fish and

14 aquatic invertebrate predators are thought to be the primary control in the diel vertical migration patterns of various zooplankton species (Dini and Carpenter 1991, Dodson 1990, O’Brien 1979).

For example, zooplankton may use the oxygen and light diminished hypolimnion as a refuge from visual predators such as planktivorous fish (Hanazato and Hosomi 1989).

Vertical migration in zooplankton can be modified by food availability (Winder et al.

2004), UV radiation (Fischer et al. 2006, Rhode et al. 2001) and temperature (Loose and

Dawidowicz 1994). Specifically, these environmental factors are important for fitness in

Daphnia, such that higher food concentrations increase clutch sizes (Guisande and Gliwicz

1992), and warmer water temperatures increase developmental rates (Stich and Lampert 1984).

Different combinations of food, temperature, and predation pressures may also cause a large variability in vertical migration patterns. Using a large indoor mesocosm, Larsson and Lampert

(2012) showed that Daphnia pulicaria did not undergo diel vertical migration in the presence of predators when food concentrations in the hypolimnion were increased. However complete exploitation of the hypolimnion was constrained by the presence of low oxygen and colder water temperatures (Larsson and Lampert 2012).

In lakes with a deep water food maximum, which is defined as the presence of chlorophyll-a (chl-a) located below the thermocline (Cullen 1982), Daphnia are faced with the presence of an alternative food source located in a low oxygen environment. To help tolerate low oxygen conditions, some species of Daphnia have the ability to alter their physiology and up- regulate hemoglobin. In Daphnia, the up-regulation of hemoglobin is stimulated by exposure and acclimation to low oxygen conditions (Fox et al. 1951). However, there are costs to the production of hemoglobin in Daphnia. Warmer water temperatures increase metabolic demands making the up-regulation of hemoglobin energetically costly (Fox and Phaer 1953, Lamkemeyer

15 et al. 2003), and resources used in the synthesis of hemoglobin are no longer available for growth and reproduction (Fox 1955, Pirow et al. 2001). In addition, Daphnia that up-regulate hemoglobin are more visible and susceptible to predation, due to the red color of the hemoglobin in the thorax (Heisey and Porter 1977). Despite the costs, increases in hemoglobin concentrations lead to a higher oxygen transport capacity, improving the physiological performance of Daphnia in low oxygen conditions (Kobayashi and Gonoi 1985).

Interspecific variation in habitat selection is well studied in populations of Daphnia (De

Meester 1994, Hu and Tessier 1995, Tessier and Leibold 1997). However, few studies have looked at the correlation between the differences in hemoglobin up-regulation related to differences in diel vertical migration. It is unclear how differences in hemoglobin up-regulation correlate with differences in diel vertical migration strategies in Daphnia. I will look to see if differences in vertical migration in Daphnia are correlated with differences in hemoglobin up- regulation. I posited that Daphnia that up-regulate hemoglobin may remain permanently in the hypolimnion, which may act as a permanent refuge from predation risk. I used a relatively deep oligotrophic lake in Ontario, to study the differences in diel vertical migration patterns among three distinct phenotypes of D. pulicaria, classified as reds, pinks, and pales, which differ visually in hemoglobin up-regulation. The three phenotypes of D. pulicaria in Round Lake are thought to be obligate asexuals during the fall due to observations of Daphnia producing ephippia eggs asexually throughout the fall sampling seasons (Gittens personal observation).

2.1 Methods The study site is a deep lake located near Queen’s University Biological Station

(44°34'2.64"N, -76°19'28"W) known as Round Lake. The lake is oligotrophic with a mean depth of

12m and a maximum depth of 30m. Spring and fall turnover do not extend deeper than 17m

16 based on the seasonal changes in temperature profiles, which means that the deepest 13m do not mix with high oxygen water surface waters at any point during the year (Appendix A). As a result, the deep water is low in oxygen year round, providing a strong temperature and oxygen gradient over the water column. The copepod community comprises cyclopoid copepods, and calanoid copepods (Gittens personal observation). D. pulicaria is the most abundant Cladoceran species in the pelagic zone. The fish community is composed of thirteen species of fish including: blunt nose minnows, Pimephales notatus, black chin shiners Notropis heterodon, black nose shiners Notropis heterolepis, blanded killifish Fundulus diaphanous, northern pike

Esox lucius, central mudminnow Umbra limi, largemouth bass Micropterus salmoides, pumpkinseed Lepomis gibbosus, bluegill Lepomis macrochirus, rock bass Ambloplites rupestris, yellow perch Perca flavescens, yellow bullheads Ameiurus natalis, and brown bullheads

Ameiurus nebulosus (Opinicon Natural History). The phantom midge larvae Chaoborus flavicans is the dominant invertebrate predator of Daphnia in the pelagic zone (Gittens personal observation).

Physical parameters such as changes in the chlorophyll ɑ (chl-a), particulate organic carbon (POC), dissolved oxygen (mg/L) and temperature (°C) gradients were monitored throughout the summer in 2012. Dissolved oxygen and temperature profiles were taken in the pelagic zone using an YSI probe (model 560A) starting at 0m and continuing at 1m intervals to a maximum depth of 20m. Chl-a samples were collected on three sampling dates: July 17, 2012,

August 11, 2012, November 11, 2012. Water samples were collected and stored in brown 1L

Nalgene bottles using a water pump, starting at 1m and continued sequentially at 1m intervals to a maximum depth of 25m. POC samples were collected using the aforementioned automated water pump on August 11, 2012, starting at 1m and continued sequentially at 1m intervals to a

17 maximum depth of 25m, and water was stored in clear 1L nalgene bottles. For chl-a samples,

200mL of water was filtered onto 25mm GF/F filters and then frozen. Chl-a samples were extracted overnight in 25ml of methanol and analyzed using a TD-700 flurometer. To measure

POC samples, 400mL of water was filtered onto 25mm GF/F filters that were pre-combusted at

400°C for four hours to burn off any carbon present. POC filters were analyzed using a Thermo

Fisher Scientific Elemental Analyzer (FlashEA 1112), using a combustion method to measure carbon levels captured on the filter. Light levels were measured using a LI-COR underwater radiation sensor (model LI-1400) starting at 1m and continued sequentially at 1m intervals to a maximum depth of 25m on August 12, 2008, August 19, 2008, and May 4, 2009.

To quantify changes in the genotypic and phenotypic composition of D. pulicaria populations, zooplankton samples were taken on September 5, 2011 and November 29, 2011, and weekly during the summer of 2012 starting June 18, 2012, and ending on September 29,

2012. Samples were collected by taking integrated hauls of the water column using an 80μm mesh zooplankton net, starting at 25m ending at 1m. Daphnia were randomly sorted using a zooplankton sorter and 200 adults were size classed using four pre-measured counting circles

(Size 1:1.0mm-1.4mm, Size 2:1.4mm-2.0mm, Size 3:2.0mm-2.5mm, Size 4: 2.5mm and up, color indexed using a color card (Appendix A, Figure 2A). Individuals were assigned a color by assigning a number from 0-6 based on the degree of redness seen in the body, and frozen in a -80 freezer.

To track differences in the diel vertical migration patterns of pale, pink, and red D. pulicaria a 24h vertical migration study was conducted on September 28, 2012. Starting at 8am, two replicate samples were collected every 4h for 24h. Samples were collected using a 19.7 L plexiglass Schindler trap with an 80μm mesh, starting at 1m and continued sequentially at 1m

18 intervals to a maximum depth of 25m. Due to the large number of individuals collected during the 24h vertical migration survey, the first replicate of live animals was counted, size classed, and color indexed in the field under a dissecting microscope. The second replicate of live animals was collected and transported back to the lab in insulated coolers held at 4-8°C to minimize mortality. Daphnia in the second replicate were size classed and color indexed using a

Lecia M80 camera scope. All live individuals from each 1m interval were recorded. Samples in the second replicate were processed over a week and mortality of individuals, although not recorded, was only seen on the last day of processing.

To track possible differences between genotypic and phenotypic vertical structure in D. pulicaria two additional 12h surveys were conducted. The surveys were done at 4pm and 4am on

October 20, 2012 and November 11, 2012. These two time periods were selected based on results of the vertical migration survey that indicated the greatest difference in vertical migration between color phenotypes existed between 4am and 4pm. Samples were collected at 1m intervals, starting at 1m and ending at 25m using a 19.7 L plexiglass Schindler trap with an 80μm mesh. Each 12h survey consisted of two replicates for each of the 4am and 4pm time periods.

Animals were collected live and transported to the lab in insulated coolers as described above.

Individuals were color indexed and the length of each daphnid was measured from the top of the eye to the base of the tail spine using a Lecia M80 camera scope. Daphnids were photographed on a plexiglass petri dish with a white piece of paper taped to the bottom to help increase contrast at the 32 X magnification. Daphnia were stored individually in 0.5mL vials with a number written on the cap that corresponded to the depth the Daphnia was captured at, before being stored in a -80°C freezer for electrophoresis. All live individuals from each 1m depth interval were recorded and frozen, (mortality of individuals was only seen on the last day of processing).

Hemoglobin analysis

In order to quantitatively assess hemoglobin content between individual Daphnia, hemoglobin analysis was conducted using a spectrophotometer machine. Daphnia were collected on December 13, 2012 by taking integrated hauls of the water column using an 80μm zooplankton net starting at 25m and ending at 1m. A total of 80 adult Daphnia were processed spectrophotmeterically. Animals were first measured and then photographed at 32X magnification on a Lecia M80 camera scope. The individuals were then crushed and placed in

450mL of M4 media (Elendt and Bias 1990). Phenylmethylsulfonylfluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA) (for a final volume of 1mmol-L of each), were added to the M4 media to help prevent proteolysis of the homogenates (Schwerin et al. 2010). Following

Schwerin et al. (2010), Daphnia were centrifuged at 12,000 rpms at 4°C for 15min, and 200µL of the supernatant was analyzed spectrophotometrically. The absorbance spectra for oxygenated hemoglobin concentrations were recorded at a wavelength of 442nm. To quantify hemoglobin content in Daphnia, a stock solution of 150mg of lyophilized powdered human hemoglobin

(Sigma Aldrich H7379) was diluted in 15mL of M4 media. From this stock solution a series of

15 standards were created, each standard had a final volume of 1mL (including a blank comprised solely of 1mL of M4 media). The lowest standard concentration contained 5µL of the hemoglobin stock solution in 995µL of M4 media, the highest standard concentration contained

125µL of hemoglobin stock solution in 875µL of M4 media. All samples were analyzed using the FIAlab-2500 flow injection system that includes a UV/VIS spectrometer (USB2+F00122) based detector. The FIAlab-2500 has a 50cm path length, which helps to increase the sensitivity of the detection limit in low volumes of liquid, this coupled with the flow cell (LS2-1) allowed for the detection of hemoglobin in individual Daphnia.

Since both hemoglobin and electrophoresis analysis are destructive techniques, we used image analysis to estimate the hemoglobin content of animals analyzed spectrophotometrically.

All Daphnia collected for spectrophotometric analysis were photographed on a petri dish with a white piece of paper taped to the bottom at the highest lighting intensity using the Lecia M80 camera scope to help standardize lighting conditions. Images were analyzed in Adobe Photoshop

(version CS6); the thorax of each Daphnia was outlined (Appendix A Figure 3A), to capture the redness of animals from the flow of hemoglobin through the body cavity. Daphnia photographs were analyzed in order to determine differences in the degree of coloration in Daphnia in response to hemoglobin content using the red green blue (RGB) color space derived from the

RGB color model (Hill and McGraw 2006). I used the RBG color model and calculated the distance from pure red in order to relate total hemoglobin content to visual redness. The histogram tool was used to display one median red, blue, and green value for each photograph.

Red, blue, and green values were recorded and used to calculate the distance from pure red.

Cellulose acetate electrophoresis

To evaluate the relationship between the observed phenotype and the genotypic identity of individual Daphnia, I scored allozymes at five loci of individuals collected during the second replicate of the 4am and 4pm samplings using the October 20, 2012 time period. Adult Daphnia were color indexed, photographed, measured (from the top of the eye to the base of the tail spine), and stored in a -80°C freezer. Frozen individuals were analyzed using cellulose acetate electrophoresis at five loci: aldehyde oxidase (ao), lactate dehydrogenase (ldh), phosphoglucomutase (pgm), malate dehydrogenase (me), and glucose-6-phosphate isomerase

(pgi). D. pulicaria were prepped for electrophoresis by placing individuals in 6µL of distilled water in Super Z applicator 12 well plate (Helena Laboratories), and ground with a glass cover

21 slip. All frozen individuals were compared against two controls of known laboratory genotypes.

The controls were either a heterozygote or homozygote at each of the five loci tested; this was done to ensure accurate genotyping of the unknown individuals. The homogenates were then applied to cellulose acetate electrophoresis gel plates (76mm x 76mm, Helena Laboratories) and ran at a 200V for 20min using a VWR power supply. All electrophoresis chemicals buffers and stains follow Hebert and Beaton (1989). Pgm and ao were co-stained, staining individuals for pgm first and ao second (Hebert and Beaton 1989).

Statistical Analyses Cluster Analysis To quantify whether Daphnia phenotypes had any inherent grouping based on hemoglobin concentrations (mgHb/ g dry weight), a k-means cluster analysis was conducted that statistically groups individuals based on similarities. Objects (in this case individuals) are assigned to a cluster based on a similarity matrix, and the first step in k-means partitioning method measures similarity by assigning objects to a number of clusters that minimize within cluster variation (Quinn and Keogh 2002). The objects are then successively reassigned to clusters to help minimize the within-cluster variation; this allows the ability of cluster affiliations to change during the course of the analysis. The cut off point for the number of clusters used in the k-means analysis was determined by calculating the within-group sum of squares for different numbers of clusters. The within group sum of squares decreases as the number of clusters increases, and often forms an “elbow” that indicates the transition from substantial decreases to marked flattening of the variation (Tibshirani et al. 2000). The location of the

“elbow” indicates the appropriate number of clusters present (Tibshirani et al. 2000). To provide additional support for the number of cluster present, we calculated average silhouette widths.

The average silhouette widths provide a visual representation, and quantitative metric of the average distance between clusters. Specifically, the distance of points within a group is compared to the distance between points in the nearest group (Rousseeuw 1986). The optimal number of clusters to select is indicated by the largest silhouette coefficient (Rousseeuw 1986).

24hr Vertical migration survey To evaluate the trends in migration patterns over a 24h period between pale, pink, and red

Daphnia I conducted a two way ANOVA comparing changes in mean depth between pale, pink, and red Daphnia. The numerical variable was changes in mean depth, with color (whether individuals were pale, pink, and red), and time (one of the time periods during the 24hr survey) as categorical variables. To better understand the pattern of results obtained in the two way

ANOVA; a contrast analysis was performed post hoc. The contrast analysis was estimated from the regression coefficients from the ANOVA; and compared the changes changes in the mean depths throughout the 24hr survey between pale versus pink Daphnia, pink versus red Daphnia, and red versus pale Daphnia.

Hemoglobin content and color analysis

To understand the relationship between total hemoglobin content per animal (ug of Hb) and the visual redness of individual Daphnia, each individual assayed for total hemoglobin concentration was first photographed in order to analyze the amount of redness. The distance from pure red was determined by calculating Euclidian distances of how far individuals were from RGB= (255, 0,0), which is the color of pure red. Specifically, the distance from pure red was calculated using the following equation:

Linear regressions were performed on the raw, and log transformed distance from pure red, and total hemoglobin content. To understand which model was a better fit for the data, I compared the goodness of fit in the raw and log transformed linear regression models using Akaike information criterion values (AIC’s) (Wagenmakers and Farrell 2004).

Genetic Analysis

A principal coordinate analysis was performed on the data of all the individuals combined for the 4 am and 4 pm time samplings, and individuals of each time period separately.

The presence (1) or absence (0) of alleles were recorded at each of the five loci for 345 individuals (248 individuals in the 4am sampling, and 97 individuals in the

4pm sampling) and a genetic dissimilarity matrix was created using classical Euclidean distances following Rodgers (1972). The analysis of the combined 4am and 4pm data was constrained to the first seven dimensions, because the first seven dimensions explained 99.7% of the variance seen in the Euclidian distance matrix. The analysis of the 4am data was constrained to six dimensions, which explained 95.7% of the variance seen in the Euclidian distance matrix. The analysis of the 4pm data was constrained to six dimensions, which explained 98.4% of the variance seen in the Euclidian distance matrix. All graphs and statistical analyses were generated in R 2.15.1 2012 (R Core Development Team 2013), the genetic distance matrix was generated using the ‘ade4’ package (Chessel et al. 2013). A G-test for goodness of fit was conducted on individuals in the 4am and 4pm surveys, both combined and separately to see if the genotype was independent of the recorded phenotype within and between the two time periods (reds, pinks, and pales); the G-test was generated using the ‘vegan’ package (Oksanen et al. 2013). In the 4 am, and 4 pm time samplings, separately and combined, rare genotypes were classified as genotypes that had one individual or less of each phenotype. Genotypes that were classified as

24 rare were pooled to create one large rare genotype in order to satisfy the assumptions of a G-test.

The 4 am sampling had 57 genotypes total, 18 genotypes were classified as rare, and in the four pm sampling 9 out of the 25 genotypes were classified as rare.

2.2 Results Physical parameters

A strong temperature and oxygen gradient exists year round in Round Lake, with decreases in both temperature and oxygen below 14m (Figure 2.1). In July and August chl-a levels were highest around the thermocline (7-13m), low again below 18m, and high near the bottom (Figure

2.2 a). Particulate organic carbon levels start to increase at 14m and peak at 24m (Figure 2.2 b).

Below 10m, light levels are less than 1.50 lux, and at 13m, light levels fall below 1.0 lux (Figure

2.3).

Changes in phenotype abundances through time

Phenotypic relative abundances between pales, pinks and reds remained relatively constant throughout the summer 2012 sampling season, such that, half of the individuals collected were pale, while the other half were pink or red (Figure 2.4).

24h Vertical migration

D. pulicaria that up-regulated hemoglobin (pinks and reds) showed little change in their vertical distribution through the water column in the 24h survey and 12h survey; suggesting that these individuals remained low in the water column with little vertical migration (Figure 2.5 and

Figure 2.6). In contrast individuals classified as pales showed strong changes in their vertical distribution through time suggesting vertical migration (Figure 2.5). Strong overlap and normal diel vertical migration patterns exist between the three phenotypes until the 8pm sampling period during the 24h survey. After the 8pm time period pales migrate back up in the water column, and

25 are found at mean depths ranging from 13-10 m (Figure 2.6), while pinks and reds remain low in the water column throughout the 24h time samplings and are found at mean depths below 16m

(Figure 2.6). The 12h samplings display strong overlap in pales, pinks and reds during the 4pm time period (Figure 2.6). The results of the ANOVA indicate that there is a significant difference in mean depth between pale, pink, and red Daphnia over a 24 hr. period (F10,18=6.741, p<0.001).

The results of the contrast analysis suggest that there is is a significant difference in the mean depth between red and pale individuals in the 8pm (df=18, t=3.80, p<0.001), 12am (df=18, t=4.15, p<0.001), and 4am (df=18, t=8.74, p<0.001) time samplings, indicating that there is a strong difference in migratory patterns between red, and pale Daphnia in those time periods

(Figure 2.6, Table 2.2a). However there is no significant difference in mean depth between red and pink Daphnia during the 8pm (df=18, t=-0.60, p=0.556), 12am (df=18, t=-0.30, p=0.766), and 4am (df=18, t=-0.16, p=0.877) time periods, suggesting that there is no difference in migratory patterns during red and pink Daphnia (Figure 2.6, Table 2.2c).

Cluster Analysis: A histogram of total hemoglobin concentration (mgHb/g dry weight) suggests there are multiple modes in the data (Figure 2.7 a). Visually there is little change in the within sum squares between the third and fourth cluster suggesting that three clusters is a good cutoff point

(Figure 2.7 b). The silhouette coefficient is highest at 0.66, when k=3, providing additional support that the optimal number of cluster present is 3 (Figure 2.7 c) (Rousseeuw 1986). Based on this, for the remaining analyses I compare the results among the three phenotypes groups: pales, pinks, and reds.

Hemoglobin content and color analysis:

There is a strong relationship between total hemoglobin content and the degree redness in individual Daphnia (Figure 2.8). The fit of the line of the linear and exponential distance from 26 pure red and total hemoglobin content is y=209.448-0.6249x, and log(y) =5.46-0.0696*log(x) respectively. The linear and exponential models had AIC values of 607.54, and -239.265 respectively with k=2 (Table 2.1). The smaller AIC value of the exponential model suggests that the log transformed linear regression model is the best model.

Genetic Analysis:

Genotype is not independent of phenotype in the 4pm and 4am sampling combined

(G=76.8515, df=50, p=0.008) (Figure 2.9), the 4 pm sampling (G=47.5869, df=32, p=0.037) (Figure 2.10), and the 4 am sampling separately (G=64.6153, df=42, p=0.014) (Figure 2.11). There are some genotypes that are comprised of individuals from all three phenotypes (i.e an even distribution of pales, pinks, and reds), other genotypes are dominated by individuals of one phenotype (i.e. mostly pales with some reds and pinks present and vice versa), and some genotypes are comprised of individuals from one phenotype (i.e. all pale, pink, or red) (Figure 2.9, Figure 2.10, and Figure 2.11).

2.3 Discussion In Round Lake, the three phenotypes of D. pulicaria, pales, pinks and reds, differ in how far they migrate vertically in the water column. The differences in vertical migration among the three morphs reflect differences in habitat selection, which may be a mechanism that helps mediate intraspecific co-existence. The lack of independence between the observed phenotype and the underlying genotype indicates that there is a genetic basis for differences in hemoglobin up-regulation, which may suggest genetic differences in migratory strategy. The strong environmental gradient present in Round Lake plays a role in differences in hemoglobin up- regulation and habitat selection between the three phenotypic morphs of D. pulicaria. In response to environmental variability Daphnia can alter their migratory behavior and select

27 habitat that helps to maximize fitness through growth, survivorship, and reproduction (Hays

2003). However trade-offs exist in habitat selection whereby individuals are selecting habitat that may optimize a component of fitness such as growth, yet may be detrimental to survivorship and reproduction (Stich and Lampert 1981). Changes in habitat selection in Daphnia can occur in response to a variety of factors such as predation risk, food availability, and oxygen, and temperature gradients

Selective feeding by year of young and planktivorous fish can alter the species composition, size distribution and vertical migration strategies in zooplankton communities (Brooks and

Dodson 1965, Gliwicz 1986, Gliwicz and Pijanowska 1989). Light levels are an important factor mediating the size selectivity of planktivores by regulating prey visibility, with larger prey more visible at higher light levels. Planktivorous fish are vertically distributed in the water column at depths, with light levels that help to maximize capture rates of prey (Bohl 1982, Diel 1988).

During the 8am time sampling, Daphnia are found below 11m and migrate below 14m during the 12pm time period. Daytime light levels on August 2012 at 11m and 14m range from 1.73 to

0.648 lux. Light intensities at these depths would likely not be limiting to the feeding behavior of planktivorous fish. Given that during the day all three phenotypes stay below 14m, which is likely a response to fish predation, we can postulate that some other environmental factor is limiting the distribution of fish in the water column. While we have no direct observation of this, the oxygen concentrations we find in the lake are likely to restrict the vertical distribution of planktivorous fish. Most fish (both freshwater and marine) cannot tolerate oxygen environments below 2mg L-1 (Ludsin et al. 2009, Vanderploeg et al. 2009). Oxygen levels are routinely less than 2mg L-1 at 14m and below in Round Lake, indicating that 14m and below may act as a prey refuge for Daphnia. Chaoborus flavicans, an aquatic invertebrate predator of D. pulicaria is a

28 tactile non-visual predator that is tolerant to low oxygen environments. Larvae are gape limited and only third and fourth instars of C. flavicans have the ability to feed on adult Daphnia

(Hoback and Stanley 2001). Since Chaoborus also undergoes vertical migration in response to predation pressures from visual predators, it is likely there is strong overlap during the daytime of both C. flavicans and D. pulicaria in the hyplomnion. The preferred sizes of Daphnids suitable for third and fourth instars of C. flavicans include small and intermediate sized individuals ranging from 0.78mm to 1.40mm (Kajak and Rybak 1979). The mean size of red, pink, and pale adults collected during the 4pm time period of the 12h survey were 2.61mm,

2.55mm, and 2.20mm, respectively. Suggesting that red, pink and pale adult Daphnia located in the hypolimnion during the day are on average too large to be consumed by third and fourth instars of C. flavicans larvae, suggesting that fish predation risks may play a larger role in the vertical distribution of Daphnia than invertebrate predators. Predation risk is not the only determinant of the vertical migration patterns in species of Daphnia, the distribution of phytoplankton in the water column is also important in the vertical distribution of Daphnia.

Food availability fluctuates in both quantity and quality in stratified lakes (Johnsen and

Jakobson 1987, Pijanowska and Dawidowicz 1987), with generally higher quality and higher food concentrations present in the epilimnion and lower quality and lower food concentrations present in the hypolimnion. Overall, lakes with a deep water food maximum provide an alternative food source in the hypolimnion for invertebrates. In Round Lake the thermocline starts at 7m and ends at 13m (Gittens personal observation), chl-a abundances increases around

10m and peaks at 14m in July and August. Particulate organic carbon (POC) levels, which includes the presence of phytoplankton, bacteria, as well as detritus increase sharply at 14m and peaks at 24m. The increase in POC may indicate the presence of bacteria located below 14m and

29 the effect of food raining down from above. Bacteria provide another possible food source for

Daphnia. The increase in POC and chl-a below 13m suggests that there is more food available in the hypolimnion compared to the epilimnion in Round Lake. Red and pink individuals did not undergo vertical migration throughout the 24h survey and were roughly distributed in the water column between 14m and 21m. The lack of vertical migration in reds and pinks may allow individuals that up-regulate hemoglobin the ability to access the chl-a and/or bacteria present below 14m. In addition to an increase of food availability in the hypolimnion, individuals that up-regulate hemoglobin have the added benefit of being able to permanently stay in the oxygen poor predation refuge. In a lake with high food concentrations in the hypolimnion and high predation pressure in the epilimnion, Gilwicz and Pijanowska (1988) showed that Daphnia did not undergo vertical migration and remained in the hypolimnion day and night. In Round Lake pale Daphnia undergo vertical migration even though there is more food located below 14m suggesting that food is not the only component influencing the vertical distribution of Daphnia in

Round Lake. In lakes with increased food abundances in the hypolimnion, low oxygen concentrations in the hypolimnion, constrain Daphnia’s ability to access food located at those depths (Kessler and Lampert 2004).

Hemoglobin up-regulation in Daphnia allows for increased binding affinity of oxygen in low oxygen conditions and the increase in oxygen supports higher rates of survival, feeding, and respiration (Hanazato and Dodson 1995, Kobayashi and Gonoi 1985, Pirow et al. 2001). The amount of dissolved ambient oxygen present in water is inversely related to the amount of hemoglobin found in Daphnia. Red and pink Daphnia have higher hemoglobin concentrations

(mg/g dry weight), compared to pale Daphnia. The increase in hemoglobin content increases the tolerance to the low oxygen conditions of the hypolimnion, and is correlated with a lack of

30 vertical migration in red and pink Daphnia. Pale Daphnia have lower hemoglobin concentrations

(mg/g dry weight), and may not be able to tolerate the low oxygen conditions present in the hypolimnion, which may be a factor influencing vertical migration back up into the epilimnion at night. A disadvantage to up-regulating hemoglobin is that pink and red Daphnia are redder in appearance and are more visible to visual predators such as fish. However, that wouldn’t generate a cost to migrating up during the night since fish predation is likely limited by light during these time periods. Both temperature and oxygen play important roles in influencing hemoglobin up-regulation in Daphnia.

Temperature plays an important role in maximizing reproductive components, including age at maturity and brood duration. Work by Lampert et al. (2003) in a large mesocosm experiment showed that in the absence of predators and in the presence of food in the hypolimnion, Daphnia still underwent vertical migration back up into the epilimnion at night. This result suggests that temperature may play an important role in maximizing fitness. Even though the food quality in the hypolimnion is better than in the epilimnion, at night the warmer temperatures in the epilimnion may help to increase growth rates and egg production in pale Daphnia. However, pale Daphnia that migrate out of the hypolimnion and up into the epilmnion at night are exposed to a rapidly changing temperature and oxygen gradient. Pale Daphnia migrate up into the epilimnion and are found at depths ranging from 1-11m during the 12am and 4am time periods.

Temperature may have a significant effect on juvenile development rates, which help to reduce generation times, which helps to increase age at first reproduction and brood duration time in

Daphnia (Orcutt and Porter 1984). However, the effect of temperature and the influences it may have on generation time between reds, pinks, and pales was not looked at in this study. The lack of vertical migration in reds and pinks at night may be due to the energetic costs of up-regulating

31 hemoglobin at the warmer water temperatures of the epilimnion, and hemoglobin degrades quickly at warmer temperatures (Landon and Stasiak 1983). The differences I observed in migratory behavior between the three phenotypic morphs of D. pulicaria are indicative of two different strategies in spatial habitat selection between the three phenotypes. In the first strategy, pinks and reds up-regulate hemoglobin and remain in the cold water, oxygen poor hypolimnion.

Daphnia that remain in the hypolimnion have access to food located below 14m, and the light diminished waters of the hypolimnion provide a prey refuge from visual predators. In the second strategy, pales do not up-regulate hemoglobin and undergo vertical migration back up into the warm waters of the epilimnion at night. The benefits of migrating include warmer water temperatures in the epilimnion, which helps to increase age at maturation, and shorten brood duration times. However Daphnia that migrate, do so over rapidly changing temperature and oxygen gradients, and still face some predation risk from visual predators in the epilimnion. For non-migrators, the colder waters of the hypolimnion provide a permanent refuge from fish and access to food present in the hypolimnion; but Daphnia that remain low in the water column may experience slower growth rates and longer brood duration times. Each strategy likely enhances a different component of fitness, and Daphnia are employing a strategy that helps maximize individual fitness. In Round Lake hemoglobin up-regulation is indicative of behavior (whether or not individuals migrate) and the difference in habitat selection between reds, pinks, and pales is modified by predation pressures, food availability, oxygen and temperature gradients. The proportion of pale, pink, and red Daphnia present during the summer 2012 sampling season remains constant, suggesting that differences in vertical migration strategy co-exists over multiple asexual generations.

In conclusion, in lakes with food present in the hypolimnion, Daphnia are faced with a trade-off between low temperatures and high feeding opportunities in the hypolimnion, and high temperatures but low feeding opportunity in the epilimnion. This tradeoff suggests that there are factors other than food influencing the upward migration and habitat selection in Daphnia, and differences in migration strategy helps to maximize different components fitness such as growth and reproduction. There is a genetic component to the behavioral differences in migration strategy between the three phenotypes in Round Lake. In other systems where hemoglobin up- regulation is not present, intraspecific variation in vertical migration strategy may help to mediate co-existence in Daphnia genotypes. Overall this study extends the growing body of literature on the importance of intraspecific variation in mediating species co-existence, and highlights the importance of hemoglobin up-regulation in vertical migration strategies in

Daphnia.

Table 2.1 A summary of AIC values used to assess the goodness of fit between the exponential (A) and linear models (B) of the distance from pure red and total hemoglobin content.

Model Df AIC K ΔAIC A 3 -239.65 2 0 B 3 607.54 2 -846.80

Table 2.2 a) A summary of the contrast tables from the final pairwise comparisons of differences in mean depths for pale versus pink Daphnia collected during the 24hr sampling survey.

Time Contrast S.E. Lower Upper t df p-value 4am 8.58 1.05 6.37 10.80 8.14 18 0 8am 2.10 1.05 -0.10 4.32 2.00 18 0.06 12pm -0.62 1.05 -2.8 1.58 -0.59 18 0.56 4pm 0.27 1.05 -1.94 2.48 0.26 18 0.79 8pm 3.69 1.05 1.47 5.90 3.50 18 0.01 12am 4.21 1.05 1.99 6.42 4.00 18 0.01 b) A summary of the contrast tables from the final pairwise comparisons of differences in mean depths for pink versus red Daphnia collected during the 24hr sampling survey. Time Contrast S.E. Lower Upper t df p-value 4am -0.63 1.05 -2.84 1.58 -0.60 18 0.55 8am -0.36 1.05 -2.57 1.85 -0.34 18 0.732 12pm -0.15 1.05 -2.37 2.05 -0.15 18 0.88 4pm -0.63 1.05 -2.85 1.57 -0.60 18 0.55 8pm -0.31 1.05 -2.53 1.89 -0.30 18 0.76 12am -0.16 1.05 -2.37 2.05 -0.16 18 0.87 c) A summary of the contrast tables from the final pairwise comparisons of differences in mean depths for red versus pale Daphnia collected during the 24hr sampling survey. Time Contrast S.E. Lower Upper t df p-value 4am 9.21 1.05 7.00 11.43 8.74 18 0 8am 2.46 1.05 0.25 4.68 2.34 18 0.03 12pm -0.46 1.05 -2.68 1.74 -0.44 18 0.66 4pm 0.91 1.05 -1.30 3.12 0.86 18 0.39 8pm 4.01 1.05 1.79 6.22 3.80 18 0.01 12am 4.37 1.05 2.16 6.59 4.15 18 0.01

2.4 Figures

A B

Depth (m) Depth

Temperature (°C) Oxygen (mg/L)

Figure 2.1 A) Mean temperature (°C) profiles taken from the pelagic zone in Round Lake during the summer of 2012. B) Mean oxygen (mg/L) profiles taken from the pelagic zone in Round Lake during the summer and fall of 2012.

A B

(m)

Depth

Chl-a (μg /L) Particulate Organic Carbon (μg /L)

Figure 2.2 Vertical profiles of chl-a (μg /L) (A) taken from the pelagic zone in Round Lake during the summer and fall of 2012 (points). To smooth the data, a three-point moving average was calculated represented by the solid black, blue, and yellow lines. Vertical profiles of particulate organic carbon (μg /L) (B) taken in the pelagic zone of Round Lake on August 11, 2012 (points). To smooth the data, a three- point moving average was calculated represented by the solid black line.

Depth (m) Depth

Light Values (lux)

Figure 2.3 Vertical light profiles taken from the pelagic zone in Round Lake during the summer of 2008, and 2009 (points). To smooth the data, a three point moving average was calculated represented by the solid lines.

Figure 2.4 Phenotypic relative abundances of D. pulicaria samples collected twice in the fall of 2011 on September 5, 2011 and November 29, 2011 and collected weekly during the summer of 2012 starting on June 18, 2012 ending September 29, 2012. The grey, pink, and red colors represent pale, pink, and red Daphnia respectively.

Depth (m) Depth

Relative Abundance of D. pulicaria (#/19.7L) Figure 2.5 Changes in the diel vertical migration patterns between the three phenotypes pales, pinks, and reds over a 24hr period. The grey, pink, and red colors represent pale, pink, and red Daphnia respectively. Individuals captured in the first and second replicates are represented using solid circles and asterisk respectively. The solid and dashed lines represent a three point moving average for the first and second replicates.

Figure 2.6 Changes in the mean depths for pale, pink, and red Daphnia collected during the 24 hr vertical migration survey. Individuals collected during the first and second replicates at each time period are represented by the grey, pink, and red dots which correspond with mean depths for pale, pink, and red Daphnia respectively. An asterick denotes a significant difference in mean depth between the three phenotypes, and the words NS indicates no-significant differences in changes in the mean depths.

A B C

Frequency

Averagewidth silhouette Within groups sum of sum Withinsquares of groups

Hemoglobin (mgHb/g dry weight) Number of clusters Number of clusters

Figure 2.7 a) The frequency of hemoglobin concentration (mgHb/g dry weight) of Daphnia from a lake sample collected by taking integrated hauls of the water column starting at 1m and ending at 25m on December 13, 2012. Clustering analysis suggests three modes in the data, which are depicted here as grey, pink, and red colors corresponding to hemoglobin concentrations (mg/g dry weight). The dotted lines on the histogram represent the centers 72.33, 182.58, and 299.17 estimated by the k-means analysis. b) Within group sum of squares suggest that the hemoglobin content of individuals clusters into three groups and c) the average silhouette coefficient peaks at the third cluster.

A B

Distance fromRed Pure Distance from Pure Red (log) Red fromPure Distance

Total Hemoglobin Content (ug Hb per animal) Total Hemoglobin Content (log ug Hb per animal) Figure 2.8 a) The relationship between the raw distance from pure red, and total hemoglobin content data of Daphnia shown in Figure 2.5. Linear regressions are shown with solid lines. b) The relationship between log transformed distance from pure red, and total hemoglobin content. The relationship is better the best fit line is log(y)=5.46-0.0696*log(x) with an R2 value is 0.56.

Figure 2.9 Principal coordinate analysis of the combined 4 am and 4 pm data for 345 individuals, the analysis was constrained to the first seven dimensions, which explained 99.7% of the variance seen in the Euclidian distance matrix. Each pie chart represents particular genotype; and the size of the pie chart corresponds to the number of individuals in that genotype. The color of the pie chart corresponds to the recorded phenotype of that individual.

Figure 2.10 Principal coordinate analysis of the 4 pm data for 97 individuals, the analysis was constrained to the first six dimensions, which explained 95.7% of the variance seen in the Euclidian distance matrix Each pie chart represents particular genotype; and the size of the pie chart corresponds to the number of individuals in that genotype. The color of the pie chart corresponds to the recorded phenotype of that individual.

Figure 2.11 Principal coordinate analysis of the combined 4 am data, for 248 individuals. The analysis was constrained to the first seven dimensions, which explained 98.4% of the variance seen in the Euclidian distance matrix. Each pie chart represents particular genotype; and the size of the pie chart corresponds to the number of individuals in that genotype. The color of the pie chart corresponds to the recorded phenotype of that individual.

Chapter 3

Manipulating migration over a heterogeneous environment: how the ability to

migrate influences genotypic and phenotypic diversity in Daphnia pulicaria

3.1 Introduction Natural environments are rarely homogeneous, often varying in biotic and abiotic factors.

These factors can include changes in resource availability (Huisman and Weissing 1999), predation risk (Lecomte et al. 2008), elevation (Kerr and Packer 1997), or thermal gradients

(Shade et al. 2008). The previously mentioned factors help contribute to the heterogeneity present within an environment and play an important role in maintaining species diversity

(Menge and Sutherland 1987). For example, the variability in rainfall in arid environments allows for ecological differentiation in plant communities due differences in water acquisition in individual plants, which helps to promote species co-existence (Chesson et al. 2004).

Environmental heterogeneity may occur along an environmental gradient or in discrete patches and species are distributed along this gradient or between patches on the basis of their own environmental requirements (Laiolo 2013). Environments fluctuate spatially and temporally in abiotic and biotic factors, and species vary in their responses to this heterogeneity. A common response to environmental heterogeneity is migration, and species of zooplankton in response to spatial and temporal heterogeneity undergo a phenomenon known as diel vertical migration.

Zooplankton are small planktonic crustaceans that can alter their behavior and undergo diel vertical migration in response to changes in predation risk (Williamson et al. 1989), food availability (Williamson et al. 1996) and temperature and oxygen gradients (MacLern 1964,

Svetlichny et al. 2000). Diel vertical migration in zooplankton is characterized by the movement

47 of individuals into the hypolimnion during the day, and back up into the epilimnion at night

(Hays 2003). Interspecific and intraspecific variation in zooplankton migration differs between species and clonal genotypes in response to a variety of factors such as predation risk (Gilwicz and Pijanowska 1989, Vuorinen 1987), ultra violet (uv) radiation (Cooke et al. 2008, Williamson et al. 1993), temperature (Orcutt and Porter 1983), and food availability (Lampert 2012). Diel vertical migration is thought to be strongly influenced by predation risk from visual predators

(Tollorian and Harvel 1999 , Zaret and Suffern 1976), and may be a compromise between predator avoidance and foraging opportunities (Enright 1977, Lampert 1989). In response to environmental heterogeneity, Daphnia clones also display a wide range of phenotypic plasticity, which includes morphological changes (Luning 1992, Tollrian 1995), and in physiology

(Kobayashi and Gonoi 1984). For example the induction of the hemoglobin protein in species of

Daphnia is extremely plastic in response to environmental changes in oxygen concentration and temperature (Green 1956, Zeis et al. 2009). The differences in spatial heterogeneity within lakes are an important process that impacts genetic diversity in species of zooplankton (Weider 1985).

Spatial heterogeneity impacts genetic and species diversity in zooplankton within lakes in response to changes in a variety of abiotic and biotic factors as individuals migrate throughout the water column In response to changes in spatial heterogeneity zooplankton have the ability to alter their behavior, and alter their diel vertical migration strategies. The variation in vertical migration strategies in zooplankton are thought to promote genetic and species diversity due to the fact that Daphnia genotypes and species differ in vertical migration strategies (Larsson and

Lampert 2012, Weider 1984). The differences in vertical migration strategy can be genetic and

Daphnia genotypes differ in how far they vertically migrate in response to the previously mentioned factors (De Meester 1993, King and Miracle 1995). It is well documented that

Daphnia can differ intraspecifically and interspecifically in their vertical migration strategies in response to spatial and temporal environmental heterogeneity (Weider 1989). However no studies have manipulated Daphnia’s ability to migrate throughout the water column over a strong environmental gradient that varies spatially in predation risk, temperature, and oxygen.

The mesocosm experiments from this chapter will manipulate the ability to migrate over a heterogeneous environment, to elucidate the importance of migration in maintaining the possible genetic and phenotypic diversity in Daphnia.

Three phenotypic morphs of D. pulicaria exist in Round Lake that differ visually in the amount of hemoglobin present in the throax, and are classified as pales, pinks, and reds (Chapter

2). Hemoglobin production in the hemolymph is stimulated by exposure to low oxygen environments. Elevated levels of hemoglobin prolong survival, and are necessary for egg development under low oxygen conditions (Engle 1985). Differences in hemoglobin up- regulation were correlated with differences in the vertical distribution of pale, pink, and red

Daphnia in the water column (Chapter 2). I will use Daphnia as a model system to answer the question of how vertical migration over a heterogeneous environment maintains genetic and phenotypic diversity in D. pulicaria. I hypothesize that the differences in vertical migration in reds, pinks and pales may allow for the intraspecific maintenance of phenotypic and genetic diversity in D. pulicaria. Traditionally, most experiments that examine clonal existence in

Daphnia populations focus primarily on phenotypic diversity (Colbourne 2011, Scoville and

Pfrender 2010), or primarily on genetic diversity (Hebert and Crease 1980, Hebert et al. 1988).

However, migration over a heterogeneous environment may be a factor that helps influence genetic and phenotypic compositions in natural Daphnia populations. My study used mesocosm experiments to manipulate migration over an environmental gradient to test the hypothesis that

49 migration has an impact on genetic and phenotypic diversity. The first mesocosm experiment was composed of two treatment groups: a migrating and non-migrating treatment comprised of individuals from all three phenotypes. The migrating treatment allowed Daphnia to move unrestricted throughout the water column, and the non-migrating treatment restricted Daphnia to discrete 1m intervals and provided a test of the main hypothesis. The second mesocosm experiment was composed of two non-migrating treatment groups: a red non-migrating and pale non-migrating treatment. This second mesocosm experiment was intended to test the shifts in genetic and phenotypic diversity when migration over a heterogeneous environment was manipulated.

3.2 Methods Round Lake is a relatively deep lake located near Queen’s University Biological Station

(QUBS). The lake is oligotrophic with a mean depth of 12m and a maximum depth of 30m.

Spring and fall turnover do not extend deeper than 17m at the deepest point based on the seasonal changes in temperature profiles, meaning, the deepest 13m remain low in oxygen year round, providing a strong temperature and oxygen gradient. There is a large zooplankton community and Daphnia are the dominant herbaceous zooplankton, with D. pulicaria as the dominant Cladoceran species in the pelagic zone. The fish community is comprised of thirteen species of fish. The phantom midge larvae Chaoborus flavicans (Gittens personal observation) is the dominant invertebrate predator of Daphnia in the pelagic zone. More detailed information about Round Lake can be found in the second chapter of this thesis.

Physical parameters:

Physical parameters such as changes in the chlorophyll a (chl-a), particulate organic carbon (POC), dissolved oxygen (mg/L) and temperature (°C) gradients were monitored

50 throughout the summer 2012 sampling season. Dissolved oxygen and temperature profiles were taken in the pelagic zone using an YSI probe (model 560A); starting at 0m and continued sequentially at 1m intervals to a maximum depth of 25m. Chl-a samples were collected during two separate sampling dates in the summer: July 17, 2012, August 11, 2012, and one sampling date in the fall: November 11, 2012. Samples were collected and preserved in brown 1L

Nalgene bottles using 25PSI automated water system pump, starting at 1m and continued sequentially at 1m intervals to a maximum depth of 25m. POC samples were collected using the aforementioned water pump on August 11, 2012, starting at 1m and continued sequentially at 1m intervals to a maximum depth of 25m, and water was preserved in clear 1L nalgene bottles. For chl-a samples, 200mL of water was filtered onto 25mm GF/F filters. To measure POC samples

400mL of water was filtered onto 25mm GF/F filters that were pre-combusted at 400°C for 4h to burn off any carbon present. POC filters were analyzed using a Thermo Fisher Scientific

Elemental Analyzer (FlashEA 1112).

Mesocosm Design:

The mesocosms used in this study were 1m long nytex/polycarbonate plastic tubes with a mesh near the bottom of the tube, which allowed for water to flow through the mesocosm. The mesocosms were suspended off of a floating dock located over the deepest part of Round Lake

(30m). Each 1m section of the mesocosm held 20.43L of water and had two flaps, one flap located at the top and one located at the bottom of the section. The flaps were either kept closed, or remained open to create the two treatment groups. In the non-migrating treatment group the top and bottom flap for each 1m section were kept closed, sealed with a thin layer of silicone.

The same was done in the migrating treatment, but both the top and bottom flap were left open.

Each 1m section of the mesocosm was physically connected to form a long mesocosm that

51 spanned 10m in the water column. The individual 1m sections of the mesocosms had their own aquarium pumps to actively circulate water from the depth they were suspended at in the water column, capturing the natural environmental gradient present. In order to power the aquarium pumps I used two 12V DC deep cycle Natulis Marine batteries, connected to a 1000W

Eliminator inverter that converted power from 12V DC to120V AC. Connected to the inverter was a 200W transformer that transformed the power from 120V AC to 12VAC. The transformer was necessary because the aquarium pumps were 12VAC, and would not be able to handle

120VAC. The aquarium pumps were attached to a NOMA timer that came on for two minutes every two hours, each pump circulated 1.22L of water a minute (2.44L of water per 2 minutes).

Over a 24h period the pumps circulated 29.28L of water through each 1m mesocosm section, this allowed for water to be fully replenished in the mesocosms once a day.

Mesocosm Experiments:

The first mesocosm experiment had two treatment groups: migrating and non-migrating.

The second mesocosm experiment had two non-migrating treatment groups. Daphnia were collected the day prior to the start of the first and second mesocosm experiments. Using an 80μm mesh zooplankton closing net, integrated hauls of the water column were taken starting at 25m ending at 1m. Individuals were stored in 1L plastic water bottles and transported back to the lab in insulated coolers kept between 4-8 C° to help minimize mortality in Daphnia. A random sub- sampling of 200 individuals were size classed (Size 1:1.0mm-1.4mm, Size 2:1.4mm-2.0mm,

Size 3:2.0mm-2.5mm, Size 4: 2.5mm and up), color indexed (by assigning a number from 0-6 based on the degree of redness seen in the thorax), and stored in 0.5ml vials before being frozen in the -80 freezer. Hereafter, the random sub-sampling will be referred to as the lake sampling for all statistical analyses, results, and discussion points. Only adult Daphnia were stored in the

0.5ml vials and frozen; adult Daphnia were defined as any individuals that were size class 3

(2.0mm-2.5mm) and up. Thirty animals (10 of each phenotype), were size classed and adult

Daphnia were sorted and placed in 500ml plastic water bottles filled with filtered lake water from 20m and below. Water temperatures at depth below 20m were cooler and helped to reduce mortality rates in Daphnia. The water bottles were kept at temperatures between 4-8 C° in insulated coolers to help prevent mortality in Daphnia before being placed in the mesocosm experiments.

The first mesocom experiment ran for a total of 4 weeks and started on July 4, 2012. The migrating treatment allowed the Daphnia to move unrestricted throughout the mesocosm and the non-migrating treatment restricted the Daphnia to discrete 1m intervals (Figure 3.1). The mescosms spanned 10m in the water column, each section started at 5m and ended at 15m. The two treatment groups had 5 replicates each and a replicate consisted of 10 connected 1m mesocosm sections, for a total of 50 1m sections in each of the 5 replicates. In both the migrating and non-migrating treatments, 10 individuals of each phenotype, for a total of 30 individuals, were placed in each 1m section of the mesocosms. A total of 1500 individuals were placed in both treatments, with 300 individuals per replicate (where a replicate contained a set of 10 connected 1m mesocosms).

The second mesocom experiment ran for a total of 4 weeks and started on August 4,

2012. In the second experiment both treatment groups were non-migrating. The first treatment group contained only pale Daphnia, and the second treatment group was comprised of only red

Daphnia (Figure 3.2). The mesocosms spanned 10m in the water column, with the first section starting at 5m and the last section ending at 15m. The two treatment groups had 5 replicates each and each replicate consisted of 10 connected 1m mesocosm sections, for a total of 50, 1m

53 sections in the 5 replicates. Daphnia were collected by taking integrated hauls of the water column using an 80μm mesh zooplankton closing net, and sorted following the same protocol mentioned in the first mesocosm experiment. In the pale non-migrating treatment group 30 pale individuals were placed in each 1m section for a total of 300 pale individuals per replicate, or

1500 pale individuals total across all 5 replicates. In the red non-migrating treatment group 30 red individuals were placed in each 1m section for a total of 300 red individuals per replicate, or

1500 red individuals across all 5 replicates.

Cellulose Acetate Electrophoresis

I analyzed cellulose acetate electrophoresis results at five loci of individuals collected in the first and second mesocosm experiments. At the end of the first and second mesocosm experiments, each 1m section was drained and Daphnia were placed in 1L plastic bottles which corresponded to the depth and replicate they were collected. The 1L bottles were stored in insulated coolers and transported back to the lab, all individuals were size classed, and color indexed. Adult Daphnia were placed in 0.5ml vials with a number written on the cap that corresponded to the depth and replicate associated with that individual and frozen in the -80 freezer. Frozen individuals were analyzed using cellulose acetate electrophoresis at five loci: aldehyde oxidase (ao), lactate dehydrogenase (ldh), phosphoglucomutase (pgm), malate dehydrogenase (me), and glucose-6-phosphate isomerase (pgi). D pulicaria were prepped for electrophoresis by placing individuals in 6µl of distilled water in Super Z applicator 12 well plate

(Helena Laboratories), and ground with the corner of a glass slide. All frozen individuals were compared against two controls of known laboratory genotypes. The controls were either a heterozygote or homozygote at each of the five loci tested; this was done to ensure accurate genotyping of the unknown individuals. The homogenates were then applied to cellulose acetate

54 electrophoresis gel plates (76mm x 76 mm Helena Laboratories) and ran at a 200V for 20 min using a VWR power supply. All electrophoresis chemicals buffers and stains follow Hebert and

Beaton 1989. Pgm and ao were co-stained, staining individuals for pgm first and ao second

(Hebert and Beaton 1989). In the first mesocosm experiment, 521 and 169 individuals were processed across all five replicates of the non-migrating and migrating treatments respectively.

In the second mesocosm experiment there were only 50 animals alive in the red non-migrating replicates and 48 animals alive in the pale non-migrating replicates. None of the animals collected from the second mesocosm experiments were analyzed electrophoretically.

3.3 Statistical Analyses Mesocosm Experiment 1

To visualize evenness and abundance of genotypes present rank abundance curves were calculated for the lake sampling, non-migrating, and migrating treatments. To examine the overall differences in genotypic and phenotype diversity between the non-migrating and migrating treatment groups, I calculated Shannon-Wiener, Simpson diversity indexes and richness. The Shannon-Wiener diversity index was calculated as

H= -∑(p_i*(ln)(p_i)), where p_i represents the proportional abundance of individuals belonging to the ith species and ln is the natural logarithm. The Simpson diversity index was calculated as D= 1- ∑( p_i2) where p_i represents the proportional abundance of individuals belonging to the ith species.

Using a two sample t-test, I compared the genotypic and phenotypic diversity indexes, as well as richness between the two treatment groups. An analysis of covariance (ANCOVA) was conducted to see if density had an effect on genotypic or phenotypic richness within and between the migrating and non-migrating treatments. The response variable was either genotype or

55 phenotype richness, the categorical variable was treatment (migrating or non-migrating), with density as the covariate. I conducted the same analysis to examine if density had an effect on the two forms of diversity, for both genotype and phenotype in the migrating and non-migrating treatments.

Mesocosm Experiment 2

Due to high mortality rates in the second mesocosm experiment the individuals collected were not analyzed using cellulose acetate electrophoresis. Shannon-Wiener and Simpson diversity indexes were calculated to estimate phenotypic diversity for the red non-migrating, and pale non-migrating mesocosm experiments using the equations mentioned above. The Shannon-

Wiener and Simpson diversity indexes were compared using a two sample t-test.

All analyses were completed in R 2.15.1 (R Development Core Team 2013), the

Shannon-Wiener and Simpson diversity indexes were calculated using the ‘vegan’ package

(Oksanen et al. 2013), rank abundance curves were calculated using the ‘biodiversity’ package

(Kindt 2013).

3.4 Results Mesocosm Experiment 1 Overall Genotype Differences There were 32 distinct genotypes (Appendix C) collected in lake sampling, migrating, and non-migrating treatments (Figure 3.3). Non-migrating had the greatest diversity and was most even, the lake sample had less diversity and was less even that either treatments, and the migrating treatment had the lowest diversity and intermediate levels of evenness (Figure 3.3,

Figure 3.4). In the non-migrating treatment, genotypes L, G, D and N are the most abundant and genotypes present in the lake sampling, migrating, and non-migrating treatments (Figure 3.3,

Figure 3.4). Genotype L is the dominant genotype in the lake sampling, migrating, and non- 56 migrating treatments (Figure 3.3). There were fewer individuals in the migrating treatment

(Figure 3.3) compared to the non-migrating treatment (Figure 3.3). Genotype richness, Shannon-

Wiener, and Simpson diversity indexes, were calculated for the non-migrating and migrating treatment groups (Table 3.1, Figure 3.5). Genotype richness was significantly different between the migrating and non-migrating treatment groups (t=3.888, p=0.004, Figure 3.5). There was a significant difference in genetic diversity between the migrating and non-migrating treatment groups using the Shannon-Wiener diversity index (t=2.721, p=0.026, Figure 3.5); however, there was no significant difference in the genetic diversity using the Simpsons Diversity index between the migrating and non-migrating treatment groups (t=0.006, p=0.996, Figure 3.5). As density increases, there is an increase in genetic diversity, and genotype richness in the non- migrating and migrating treatment groups (Figure 3.6). There was no significant interaction between density and genetic diversity in the Shannon-Wiener, and Simpson diversity index between the migrating and non-migrating treatments (Shannon-Wiener: F3,6=4.094, p=0.578,

Simpson:F3,6=0.351, p=0.344 ). Additionally there was no interaction between density and genotype richness in the migrating and non-migrating treatment groups (F3,6=12.51, p=0.498).

Overall Phenotype Differences:

There were few red individuals present in the genotypes collected in the migrating and lake sampling (Figure 3.7). In the lake sampling there were more genotypes present that had both pales and pinks present compared to the migrating or non-migrating treatment groups (Figure

3.7). There is a significant difference in the Simpson diversity index of red Daphnia between the migrating and non-migrating treatments (t=1.67, p<0.001, Figure 3.8 a, b), however there were non-significant differences in phenotypic diversity of pale and pink individuals between the two treatments (pale: t=1.67, p=0.133, pink: t=0.748, p=0.475, Figure 3.8 a, b). There were

57 significant differences in the Shannon-Wiener diversity indexes in pale and red individuals between the migrating and non-migrating treatments (pale: t=2.316, p=0.04, red: t=8.407, p<0.001. Figure 3.8 c, d), the differences in pink individuals between the two treatments were non-significant (t=12.28, p=0.828).

Phenotype changes by depth

Mesocosm Experiment 1:

There are more reds and pinks present below 10m in the non-migrating replicates, and very few reds and pinks present at 5, 6, and 7m (Figure 3.9a, b, c, d, e). There are fewer red individuals present in the non-migrating replicates compared to pink individuals (Figure 3.9a, b, c, d, e). In the migrating treatment pale individuals dominate the phenotypic composition of all five replicates (Figure 3.10a, b, c, d, e). Red individuals are only present in in the first migrating replicate (Figure 3.10 a), however pink individuals are present all of the migrating replicates

(Figure 3.10 a, b, c, d, e). A breakdown of phenotype abundances by genotype for

Mesocosm Experiment 2:

In the red non-migrating replicates there are pinks and reds present below 12m, and pales present at 11m and above (Figure 3.11). There are more pales above 11m, and more reds below

12m, and 14m in red non-migrating replicate 1 (Figure 3.11), there are more pinks and reds present below 11m in red non-migrating (Figure 3.11). There is an increase in pinks below 13m in red non-migrating replicate (Figure 3.11). In the pale non-migrating treatment, there is an increase of reds and pinks below 11m (Figure 3.12). There are more pinks than pales at 10 and

11m, and an increase in reds below 11m in the pale non-migrating treatment (Table 3.3, Table

3.4, Figure 3.12). There is an increase in Shannon-Wiener and Simpson diversity indexes in pink and red Daphnia compared to pale, in the red non-migrating treatment (Table3.3, Table 3.4,

Figure 3.13 a, c). In the pale non-migrating treatment there is increases Shannon-Wiener and

Simpson diversity indexes in pale Daphnia compared to pink and red (Table. Figure 3.13 b, d)

3.5 Discussion I conducted two sets of mesocosm experiments that manipulated the ability of Daphnia to migrate in the water column. The first mesocosm experiment had two treatment groups: a migrating group and a non-migrating group each comprised of individuals from all three phenotypes. The migrating treatment showed decreased genetic diversity compared to the non- migrating treatment; the increase in genetic diversity seen in the non-migrating treatment also coincides with an increase in density (the total number of individuals per replicate). The analysis of the correlation with density suggests that the differences in density between the two treatments are driving the increases in genetic and phenotypic diversity in the non-migrating treatment compared to the migrating. The results from the second mesocosm experiment indicate that hemoglobin up-regulation is plastic, whereby both red and pale Daphnia have the ability to up- regulate and down-regulate hemoglobin in response to environmental conditions.

There is an increase in genetic diversity in the migrating treatment compared to the non- migrating, and the genetic diversity in the non-migrating treatment closely resembles the genetic diversity present in the lake sampling. The shifts in genetic diversity in the migrating and non- migrating treatments compared to the lake sampling may exist due to competitive processes among the three morphs. Intraspecific variation in habitat selection maintains genetic diversity because Daphnia genotypes segregate spatially in the water column (Leibold 1991, Weider and

Lampert 1985). In chapter 2 of my thesis, I correlated migration strategies with differences in hemoglobin up-regulation, and the differences in hemoglobin up-regulation were related to genetic differences in behavior. Red and pink individuals remain in the hypolimnion indicating

59 no vertical migration compared to pale individuals that migrate back up into the epilimnion at night.

The increased genetic diversity in the non-migrating treatment compared to the migrating may be a result of competitive release of non-migrators compared to migrators. When migrating

Daphnia are restricted to 1m intervals, their competitive advantage over non-migrating Daphna is reduced, which may help to explain the increase in genetic diversity in the non-migrating treatment, however this hypothesis is purely speculative. The rare genotypes are comprised mostly of pink and red individuals suggesting that they are genotypes that do not undergo vertical migration and remain in the hypolimnion. In Round Lake, pale Daphnia that migrate may have a competitive advantage over non-migrating individuals. In general Daphnia that undergo vertical migration may have a thermal advantage over non-migrators due to increased growth rates in the warmer water temperatures of the epilimnion, which may help to increase individual fitness in Daphnia (McLaren 1963, Orcutt and Porter 1983).

The differences in genetic diversity between the two treatments may be due to differences in density. There is no significant difference in the Shannon-Wiener and Simpson diversity indexes when differences in density between the two treatment groups are accounted for. This result suggests that density is an important factor influencing genetic diversity between the migrating and non-migrating treatment in the first mesocosm experiment, or that the differences in diversity are the result of a sampling effect through differences in density. Additionally, the density of animals in the non-migrating treatment was closer to the density of animals collected in the lake sampling, and the genotypes present in the non-migrating treatment were closer to the genotypes present in the lake sampling. There may have been differences in mortality rates and survivorship between the two treatment groups, however that was not examined in this study.

Phenotypic diversity increased in the non-migrating treatment compared to the migrating treatment in the first mesocosm experiment. The differences in phenotypic diversity between the two treatments may occur in response to differences in temperature and oxygen concentrations.

In the first mesocosm experiment, there were more pinks and reds present in the non-migrating treatment compared to the migrating treatment. In the non-migrating treatment, there was an increase in reds and pinks below 11m suggesting that individuals that up-regulate hemoglobin may have increased survivorship in low oxygen conditions compared to Daphnia that do not up- regulate hemoglobin. Oxygen concentrations in Round Lake at 11m and below range from 11 mg/L-1.26 mg/L, which may help stimulate and maintain hemoglobin production in red and pink

Daphnia. In Daphnia, reduced oxygen concentrations and colder temperatures stimulate the up- regulation and production of hemoglobin (Heisey and Porter 1977, Lamekeyer 2003). In turn hemoglobin up-regulation increases tolerance and survivorship to environmental hypoxia (Gorr et al. 2004). The increase in pale Daphnia above 11m may suggest that migrating Daphnia may have better survivorship at warmer water temperatures. In the migrating treatment red and pink

Daphnia were present in lower abundances compared to pale Daphnia. Shifts in the phenotypic diversity in the two treatment groups may occur in response to differences in survivorship, however, mortality rates between the individuals from each of the three phenotypes were not examined in this study. Additionally, shifts in phenotypic diversity in the migrating and non- migrating treatment groups may be due to the apparent plasticity of hemoglobin up-regulation.

Daphnia display a wide range of phenotypic plasticity in response to environmental heterogeneity including the ability to up-regulate hemoglobin in response to low oxygen conditions (Fox 1951). The results from the second mesocosm experiment indicate that hemoglobin up-regulation in D. pulicaria is plastic in response to consistent exposure to anoxic

61 environments. Previous studies have found that in D. pulex and D. magna, hemoglobin up- regulation is plastic and inducible in response to low oxygen environments (Zeis et al. 2003,

Pinkhaus et al. 2007). In second mesocosm experiment, there were shifts towards a dominance of pale individuals at 11m and above in the red non-migrating treatment, suggesting that red

Daphnia are down-regulating hemoglobin. In Round Lake depths above 11m have warmer water temperatures and are well oxygenated. Similarly in the pale non-migrating treatment there was a shift towards red individuals below 11m, since depths below 11m in Round Lake have lower oxygen concentrations and colder water temperatures.

Both temperature and oxygen play a role in hemoglobin up-regulation in Daphnia. To maintain hemoglobin up-regulation, species of Daphnia require consistent exposure to low oxygen conditions; in the absence of exposure to low oxygen conditions, individuals shift towards down-regulation of hemoglobin (Sell 1998). The shift towards hemoglobin down- regulation in red individuals at warmer temperatures provides evidence that hemoglobin up- regulation may be energetically costly at warmer temperatures, and that both temperature and oxygen play a role in hemoglobin up-regulation. In D. pulex it takes between three to four weeks to fully up-regulate hemoglobin under anoxic conditions (Zeis et al. 2009). D. pulex is a similar species to D. pulicaria and it took up to four weeks for individuals in the second mesocosm experiment to up-regulate hemoglobin.

The differences in migration strategy over a strong environmental gradient among the three morphs are a mechanism that helps to maintain the phenotypic and genetic diversity in Round

Lake. This is due to the fact that there are differences in habitat selection between the three morphs. The behavior; whether or not individuals underwent vertical migration, and the strong environmental gradient are important in stimulating and maintaining hemoglobin up-regulation

62 in Daphnia. The results from the second mesocosm experiment indicate that hemoglobin up- regulation is plastic, and whether or not hemoglobin is up-regulated depends on the temperature and oxygen concentrations of where individuals reside at in the water column. The vertical partitioning of Daphnia in the water column reduces the competitive interactions among the three phenotypes. The non-migrators remain low in the water column and do not undergo vertical migration; as a consequence, they are exposed to lower temperature and oxygen concentrations which stimulate hemoglobin up-regulation. Migrators undergo vertical migration, do not up-regulate hemoglobin due to exposure of the higher temperature and oxygen concentrations of the epilimnion. When Daphnia are restricted to 1m intervals, there is an increase in the genetic and phenotypic diversity. The increase in rare genotypes in the non- migrating treatment compared with the migrating, suggests that individuals that up-regulate hemoglobin are inferior competitors compared to pale Daphnia. However, the differences in density between the two treatments may have had an effect on the shifts in genetic and phenotypic diversity among the three phenotypes.

In conclusion, hemoglobin up-regulation is plastic and is stimulated by behavioral differences between the three phenotypic morphs. The difference in migratory strategy between the three phenotypic morphs promotes habitat segregation between red, pink, and pale Daphnia.

The separation of red and pink individuals in the water column compared to pale individuals helps to reduce competitive processes between the three morphs. There was a decrease in genetic diversity in the migrating treatment compared to the non-migrating treatment and this may be due to density differences between the two treatments.

Table 3.1 Genotype richness, Shannon-Wiener diversity, and Simpson diversity indexes for all five replicates of the non-migrating and migrating treatments.

Treatment Shannon-Wiener Simpson Diversity Density (absolute # of Diversity Index individuals from each Index replicate) Non-Migrating 1.437 0.606 75 Replicate 1 Non-Migrating 1.567 0.596 77 Replicate 2 Non-Migrating 1.704 0.632 175 Replicate 3 Non-Migrating 1.390 0.533 170 Replicate 4 Non-Migrating 1.388 0.591 70 Replicate 5 Migrating Replicate 1 1.560 0.673 38 Migrating Replicate 2 1.122 0.481 55 Migrating Replicate 3 1.299 0.598 58 Migrating Replicate 4 1.039 0.625 2 Migrating Replicate 5 0.990 0.580 33

Table 3.2 Phenotype, Shannon-Wiener (a), and Simpson diversity (b) indexes for all five replicates of the non-migrating and migrating treatments. a) Treatment Pale Pink Red Non-Migrating 1.35557 1.116367 0.9556999 Replicate 1 Non-Migrating 1.053373 1.648847 1.3296613 Replicate 2 Non-Migrating 1.507511 1.57325 1.2798542 Replicate 3 Non-Migrating 1.777509 1.998681 1.0397208 Replicate 4 Non-Migrating 1.706126 1.294545 0.6365142 Replicate 5 Migrating Replicate 1 3.4011974 0 0 Migrating Replicate 2 3.4339872 3.091042 0 Migrating Replicate 3 3.6109179 3.044522 0 Migrating Replicate 4 0.6931472 0 0 Migrating Replicate 5 2.7080502 2.302585 0 b) Treatment Pale Pink Red Non-Migrating 0.6394558 0.6020408 0.5714286 Replicate 1 Non-Migrating 0.5032556 0.7857143 0.7222222 Replicate 2 Non-Migrating 0.7024691 0.7382920 0.7000000 Replicate 3 Non-Migrating 0.7980008 0.8338615 0.6250000 Replicate 4 Non-Migrating 0.7990398 0.717438 0.4444444 Replicate 5 Migrating Replicate 1 0.9666667 0 0 Migrating Replicate 2 0.9677419 0.9545455 0

Migrating Replicate 3 0.9729730 0.9523810 0 Migrating Replicate 4 0.5000000 0 0 Migrating Replicate 5 0.9333333 0.9000000 0

Table 3.3 Phenotype, Shannon-Wiener (a) and Simpson diversity (b), for all five replicates of the red non-migrating treatments in the second mesocosm experiment. a) Treatment Pale Pink Red Red Non-Migrating 0.812 0.636 0 Replicate 1 Red Non-Migrating 0 0 0 Replicate 2 Red Non-Migrating 0 0.693 0.890 Replicate 3 Red Non-Migrating 0 0 0 Replicate 4 Red Non-Migrating 0 0 0.893 Replicate 5

Treatment Pale Pink Red Red Non-migrating 0.884 0.351 0 Replicate 1 Red Non-migrating 0 0 0 Replicate 2 Red Non-migrating 0 0.444 0.790 Replicate 3 Red Non-migrating 0 0 0 Replicate 4 Red Non-migrating 0 0 1.00 Replicate 5

Table 3 4.Phenotype, Shannon-Wiener (c) and Simpson diversity (d), for all five replicates of the pale non-migrating treatments in the second mesocosm experiment. c) Treatment Pale Pink Red Pale Non-Migrating 0 0 0 Replicate 1 Pale Non-Migrating 0.825 0 0.275 Replicate 2 Pale Non-Migrating 0.346 0.693 0 Replicate 3 Pale Non-Migrating 0 0 0 Replicate 4 Pale Non-Migrating 0.293 0.500 0.870 Replicate 5

d) Treatment Pale Pink Red Pale Non-Migrating 0.692 0 0.243 Replicate 1 Pale Non-Migrating 0 0 0 Replicate 2 Pale Non-Migrating 0.302 0.769 0 Replicate 3 Pale Non-Migrating 0 0 0 Replicate 4 Pale Non-Migrating 0.431 0.790 0.899 Replicate 5

3.6 Figures

Figure 3.1 A schematic of the two treatment groups: migrating and non-migrating used in the first set of mesocosm experiments. Individuals in the migrating treatment were allowed to move un-restricted over the water column, whereas individuals in the non-migrating treatment were restricted to one meter intervals. Each treatment had five replicates and was made up of 10 1m mesocosm sections. The mesocosm sections spanned 10m in the water column and were suspended starting at 5m and ending at 15m.

Figure 3.2 A schematic of the two treatment groups: pale non-migrating, and red non-migrating, used in the second set of mesocosm experiments. Each treatment had five replicates and was made up of 10 1m mesocosm sections. The mesocosm sections spanned 10m in the water column and were suspended starting at 5m and ending at 15m.

Figure 3.3 Absolute abundance of each genotype for the lake sampling, non-migrating, and migrating, treatment groups and each letter on the x-axis corresponds to a distinct genotype.

Figure 3.4 Ranked genotype abundance curves for the lake sampling (a), non-migrating (b), and migrating treatments (c).

Figure 3.5 Shannon-Wiener diversity, Simpson genetic diversity indexes, and Genotype richness for the migrating and non-migrating treatments from mesocosm experiment 1.

Figure 3.6 Density (number of individuals per replicate) and the Shannon-Wiener genetic diversity index(a), Simpson genetic diversity indexes (b), and genotype richness (c), for all five replicates in the migrating and non-migrating treatment in the first mesocosm experiment.

Absolute Abundance Absolute

Lake Sampling

Absolute Abundance Absolute

Non-Migrating

Absolute Abundance Absolute

Migrating

Figure 3.7 Absolute phenotype abundances for the lake sampling (a), non-migrating (b), and migrating

(c) treatment groups. On the x-axis, each letter represents a distinct genotype and across all three graphs the letters represent the same genotype.

Figure 3.8 Phenotype Shannon-Wiener and Simpson diversity indexes for the migrating and non- migrating treatment groups from mesocosm experiment 1. The treatments are represented as migrating

Shannon-Wiener (a), non-migrating Shannon-Wiener (b), migrating Simpson (c), non-migrating Simpson

(d).

A B C

D E

Figure 3.9 Phenotype abundances by depth from mesocosm experiment 1. Non -migrating replicate 1 (a), non- migrating replicate 2 (b), non-migrating replicate 3 (c), non-migrating replicate 4 (d), and non-migrating replicate

5 (e). Pale, pink, and red Daphnia are represented by the colors grey, pink, and red respectively.

A B C

D E Absolute Absolute Abundance

Figure 3.10 Phenotype abundances by replicate from mesocosm experiment 1 for migrating replicate 1 (a), migrating replicate 2 (b), migrating replicate 3 (c), migrating replicate 4 (d), and migrating replicate 5 (e). Pale, pink, and red Daphnia are represented by the colors grey, pink, and red respectively.

Figure 3.11 Absolute phenotype abundances from mesocosm experiment 2 by depth for non-migrating red replicate 1 (a), non-migrating red replicate 3 (b), and non-migrating red replicate 5 (c) collected in the second set of mesocosm experiments. Individuals collected were not analyzed electrophoretically, thus only abundance and phenotype are depicted. Pale, pink, and red Daphnia are represented by the colors grey, pink, and red respectively.

Figure 3.12 Absolute phenotype abundances from mesocosm experiment 2 by depth for non-migrating pale replicate 2 (a), non-migrating pale replicate 3 (b), and non-migrating pale replicate 5 (c) collected in the second set of mesocosm experiments. Individuals collected were not analyzed electrophoretically, thus only abundance and phenotype are depicted. Pale, pink, and red Daphnia are represented by the colors grey, pink, and red respectively.

Pale Pink Red Pale Pink Red Pale Pink Red Pale Pink Red

Figure 3.13 Phenotypic Shannon Wiener and Simpson diversity indexes for the second mesocosm experiment red and pale non-migrating replicates. Red non-migrating Shannon-Wiener diversity (a), pale non-migrating Shannon-Wiener diversity (b), red non-migrating Simpson diversity (c), pale non- migrating Simpson diversity (d).

Chapter 4

General Discussion

The niche is a central concept in community ecology and seeks to provide insights into factors that help promote species diversity. Environments can vary spatially and temporally in abiotic and biotic factors, which can act as limiting factors that constrain the breadth of an organism’s niche. Species can respond to the variance in abiotic and biotic factors behaviorally within their environment, however, this change in behavior often results in tradeoffs in fitness related traits (Creel et al. 2005, Quinn and Adams 1996, Rahel and Kolar 1990). Phenotypic plasticity and genetic diversity in the presence of a strong environmental gradient influences both population dynamics and community assemblages (Menge and Sutherland 1987, Hebert and

Crease 1980). However, there is still a lot to learn about what role intraspecific variation plays in the maintenance of species diversity, specifically the importance of heterogeneous environments in promoting genetic diversity and phenotypic plasticity.

In chapter 2, I discuss a set of 24h and 12h surveys conducted at Round Lake to track changes in diel vertical migration patterns over a strong environmental gradient among three distinct phenotypes of D. pulicaria (pales, pinks, and reds), that differed in hemoglobin up- regulation. My results indicated there is a genetic component to differences in hemoglobin up- regulation between red, pink, and pale Daphnia, suggesting that the differences in migration strategies are genetic. Whereby individuals that up-regulated hemoglobin, reds and pinks remained low in the water column indicating no vertical migration, versus individuals that did not up-regulate hemoglobin, pales which undergo vertical migration. The differences in habitat selection among the three morphs promote genetic and phenotypic diversity. The three 83 phenotypes co-exist throughout a summer season, and the proportion of pale, pink, and red

Daphnia present during the summer 2012 sampling season remains constant. This suggests that the differences in migration patterns are not temporary and co-exist over multiple asexual seasons during the summer.

The ability to migrate over a heterogeneous environment is an important component in maintaining the phenotypic and genetic diversity in the three morphs of D. pulicaria. Therefore in chapter 3, I conducted two sets of mesocosm experiments that limited migratory ability, to understand how the ability to migrate influenced genotypic and phenotypic diversity. In the first set of mesocosm experiments there was an increase in genetic and phenotypic diversity in the non-migrating treatment compared to the migrating treatment. However, the increase in diversity was attributed to differences in density between the two treatments.

An interesting result from the second set of mesocosm experiments were the shifts in hemoglobin up and down regulation in red and pale Daphnia; suggesting that both the environment and genetics plays a role in hemoglobin up-regulation in Daphnia. Pale individuals have the ability to up-regulate hemoglobin, and red individuals have the ability to down regulate hemoglobin but whether or not hemoglobin is up-regulated is dependent on the migratory strategy. The results indicate that hemoglobin up-regulation is plastic, and all individuals from the three phenotypes have the ability to up-regulate, or down-regulate hemoglobin. Due to the plastic nature of hemoglobin up-regulation, in Round Lake it is likely the behavior that influences whether or not the production of hemoglobin is stimulated. The plastic nature of the production of the hemoglobin protein in some species of Daphnia may be adaptive, and occurs in response to consistent exposure to low oxygen environments. In lakes and ponds oxygen

84 concentrations vary spatially and temporally and the plastic nature of hemoglobin up-regulation in Daphnia may allow individuals to withstand anoxic conditions (Duffy 2010, Engle 1985).

The difference in habitat selection between reds, pinks, and pales is modified by predation pressures, food availability, oxygen and thermal temperature gradients. These results add to the growing body of literature that predation risk is not the ultimate factor that controls diel vertical migration strategies in zooplankton. In Round Lake the environmental variation in food availability, predation risk, and temperature and oxygen gradients helps to promote differences in behavioral vertical migration patterns in Daphnia. In general the role of plasticity in promoting novel phenotypes and divergence in populations is largely overlooked (Pfenning et al. 2010). In

Round Lake hemoglobin up-regulation is plastic, and stimulated in response to consistent exposure to low oxygen environments. The differences in migratory strategy between the three morphs in the face of a strong oxygen gradient selected for a change in physiology in Daphnia that remain low in the water column. This change in physiology allowed individuals that up- regulate hemoglobin to exploit a new environment, promoting ecological differentiation. In other systems that do not have such the obvious marker of hemoglobin up-regulation, it may be easy to overlook the role that phenotypic plasticity plays in maintaining intraspecific variation.

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Appendix A

Figure 1A. Mean temperature profiles were recorded using an YSI probe (model 560 A) at one meter intervals starting at 1m ending at 20m. Temperature profiles were recorded once a week during the months of September, October, November, and December. The temperature profile was only taken once in the month February on February 24. 2012.

106

Figure 2A A picture of the color scale used to classify the three phenotypes. Daphnia classified as 0 were pales, animals classified as a 1, 2, or 3 were pinks, and animals classified as 4, 5, or 6 were reds

107

Figure 3A A picture of the outline of the thorax used to analyze the hue values of Daphnia collected during the 12h 4am and 4pm sampling on October 20, 2012 and animals analyzed for hemoglobin content. Images were analyzed using Adobe photoshop (CS6).

108

Appendix B

Table B1 A table of the 28 distinct genotypes analyzed using cellulose acetate electrophoresis from the 4am sampling replicate 2 collected October 20, 2012. Frozen individuals were analyzed looking for polymorphisms at five loci: aldehyde oxidase (AO), lactate dehydrogenase (LDH), phosphoglucomutase (PGM), malate dehydrogenase (ME), and glucose-6-phosphate isomerase (PGI). Homozygotes and heterozygotes were recorded as medium medium (mm), and medium fast (mf) respectively for me, ldh, and pgi. There were two possible heterozygotes for the pgm loci, slow medium (sm), or medium fast (mf),and three possible homozygotes at the ao loci, slow slow (ss), medium medium (mm), and fast fast (ff).

Genotype Me Ldh Pgi Pgm Ao

A mm mf mm mm mm

C mm mf mm sm mm

D mm mf mm mf mm

E mm mf mm mm ss

F mm mf mm mm mm

G mm mf mm mm ff

H mm mm mf sm mm

I mm mm mf sm mm

J mm mm mf mf ss

K mm mm mf mf mm

L mm mm sm mm mm

M mm mm mm sm ss

N mm mm mm sm mm

P mm mm mm mf ss

Q mm mm mm mf mm

109

R mm mm mm mf ff

S mm mm mm mm ss

T mm mm mm mm mm

U mm mm mm mm ff

V mf mf mm sm ss

W mf mf mm mm ff

X mf mm mm sm ss

Y mf mm mm sm mm

Z mf mm mm sm ff

A1 mf mm mm mm mm

B1 mf mm mm mm ff

B3 mm mm mm sm mm

110

Table B2 A table of the 19 distinct genotypes analyzed using cellulose acetate electrophoresis from the 4pm sampling replicate 2 October 20, 2012. Frozen individuals were analyzed looking for polymorphisms at five loci: aldehyde oxidase (AO), lactate dehydrogenase (LDH), phosphoglucomutase (PGM), malate dehydrogenase (ME), and glucose-6-phosphate isomerase (PGI). Homozygotes and heterozygotes were recorded as medium medium (mm), and medium fast (mf) respectively for me, ldh, and pgi. There were two possible heterozygotes for the pgm loci, slow medium (sm), or medium fast (mf),and three possible homozygotes at the ao loci, slow slow (ss), medium medium (mm), and fast fast (ff).

Gentoype Me Ldh Pgi Pgm Ao A mm mf mm mm mm D mm mf mf mf mm H mm mm mf mm mm I mm mm mf sm mm K mm mm mf mf mm M mm mm mf sm ss N mm mm mm sm mm P mm mm mm mf ss Q mm mm mm mf mm R mm mm mm mf ff S mm mm mm mm ss T mm mm mm mm mm U mm mm mm mm ff A1 mf mm mm mm mm B3 mm mm mf sm mm C1 mf mf mf sm mm D1 mf mm mm sm mm E1 mf mm mm mf mm F1 mm mm mm sm ff

111

Appendix C

Table C1 A table of the 32 distinct genotypes analyzed using cellulose acetate electrophoresis from the lake sampling, non-migrating, and migrating treatments. Frozen individuals were analyzed looking for polymorphisms at five loci: aldehyde oxidase (AO), lactate dehydrogenase

(LDH), phosphoglucomutase (PGM), malate dehydrogenase (ME), and glucose-6-phosphate isomerase (PGI). Homozygotes and heterozygotes were recorded as medium medium (mm), and medium fast (mf) respectively for me, ldh, and pgi. There were two possible heterozygotes for the pgm loci, slow medium (sm), or medium fast (mf),and three possible homozygotes at the ao loci, slow slow (ss), medium medium (mm), and fast fast (ff).

Genotype Me Ldh Pgi Pgm Ao

A mf mm mm mm mm

A1 mf mm mm mm ss

B mf mm mm sm mm

B2 mf mm mm mf mm

C mm mf mf mm mm

C3 mf mm mm mf mm

D mm mf mm mm mm

D4 mf mm mf sm mm

E mm mf mm sm mm

E5 mf mm mf mf mm

F mm mm mf mf mm

G mm mm mf mm mm

G6 mf mf mm sm ss

H mm mm mf sm mm

112

H7 mf mf mm mm mm

I mm mm mm mf mm

I8 mf mf mm mf mm

J mm mm mm mf ss

K mm mm mm mm ff

L mm mm mm mm mm

M mm mm mm mm ss

N mm mm mm sm mm

P mm mm mm sm ss

Q mm mm sm mm mm

R mm mm mm sm ff

S mm mm mf mm ss

T mm mf mm mm ss

W mm mf mm mm ff

X mm mf mm mf mm

Y mm mf mf sm mm

Z mm mf mf mf ff

Z12 mf mf mm sm mm

113