Dispersal and Invasion Dynamics of the Round Goby, Neogobius Melanostomus, Facilitating Colonization of Great Lakes Tributaries

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2010

Dispersal and invasion dynamics of the round goby, Neogobius melanostomus, facilitating colonization of Great Lakes tributaries

Jennifer Ellen Bronnenhuber University of Windsor

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Recommended Citation Bronnenhuber, Jennifer Ellen, "Dispersal and invasion dynamics of the round goby, Neogobius melanostomus, facilitating colonization of Great Lakes tributaries" (2010). Electronic Theses and Dissertations. 8105. https://scholar.uwindsor.ca/etd/8105

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Jennifer Ellen Bronnenhuber

A Thesis Submitted to the Faculty of Graduate Studies through Environmental Science in Partial Fulfillment of the Requirements for the Degree of Master of Science at the University of Windsor

Windsor, Ontario, Canada

2010

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1+1 Canada DECLARATION OF CO-AUTHORSHIP

I hereby declare that this thesis incorporates material that is result of joint research, as follows:

This thesis also incorporates the outcome of a joint research undertaken in collaboration with Brad Dufour under the supervision of Dr. Daniel Heath. The collaboration is covered in Chapter 2 of the thesis. In all cases, the key ideas, primary contributions, experimental designs, data analysis and interpretation, were performed by the author, and the contribution of co-author was 1) providing the genotype data for 32 round goby lake populations from the Great Lakes region used as reference populations for genotype exclusion and assignment analyses, and 2) providing genotype data for round goby collected from Maitland River sites (2005-2006).

I am aware of the University of Windsor Senate Policy on Authorship and I certify that I have properly acknowledged the contribution of other researchers to my thesis, and have obtained written permission from each of the co-author(s) to include the above material(s) in my thesis.

I certify that, with the above qualification, this thesis, and the research to which it refers, is the product of my own work.

in ABSTRACT

The recent upstream colonization of Great Lakes tributaries by round goby,

Neogobius melanostomus, provides an opportunity to identify dispersal mechanisms and examine invasion dynamics associated with successful colonization of a novel aquatic environment. Genetic analyses identified a stratified dispersal strategy associated with upstream colonization. Genetic diversity was maintained during colonization by natural dispersal suggesting founder effects may be mitigated and adaptation may facilitate river colonization. Swimming performance analyses indicates that the uni-directional and high flow of rivers does not limit range expansion. However, morphological differences between lake and river populations suggest that dispersal is not a random diffusion offish from the lake. Continued expansion and population persistence will increase the impact of this highly invasive species. Future research should monitor river invasion front populations for changes in dispersal and rate of colonization.

IV DEDICATION

This thesis is dedicated to Jon for teaching me that a dream is just the first step. It is also

dedicated to my 2 families for their endless faith, patience, and support. ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my two supervisors Dr. Daniel

Heath and Dr. Dennis Higgs for the opportunity they provided me with, and the guidance and support throughout this project. I would also like to thank my committee Dr.

Melania Cristescu and Dr. Lynda Corkum for their comments and suggestions.

A special thanks to J. Ens for his unparalleled assistance in the field collecting samples and keeping us safe; long weeks, dirty fingers, and lots of fresh air.

Additionally, I would like to thank C. Beneteau for introducing me to the great world of field work. Thanks to L.Barthe for participating in an internship for this project, and

L.Helou for collecting samples. I am grateful to K. Tierney and the staff at the Technical

Support Centre, University of Windsor for design and construction of the swimming flume. Special thanks to A. Barkley for fish care and round goby illustration.

Finally I would like to thank Jon, Jon's family, and my family for their unending support and faith in me.

Funding for this project was provided by the Canadian Aquatic Invasive Species

Network (CAISN), the Natural Science and Engineering Research Council (NSERC), the

Ontario Ministry of Natural Resources (OMNR), and the Ontario Federation of Anglers and Hunters (OFAH).

VI TABLE OF CONTENTS

DECLARATION OF CO-AUTHORSHIP Hi

ABSTRACT iv

DEDICATION v

ACKNOWLEDGEMENTS vi

LIST OF TABLES ix

LIST OF FIGURES xi

LIST OF APPENDICES xiv

1.0 INTRODUCTION 1

1.1 BIOLOGICAL INVASIONS 1

1.2 DISPERSAL 3

1.3 ROUND GOBY {NEOGOBIUS MELANOSTOMUS) 5

1.4 CHAPTER 2 OBJECTIVES 8

1.5 CHAPTER 3 OBJECTIVES 9

1.6 REFERENCES 10

2.0 DISPERSAL STRATEGIES, SECONDARY RANGE EXPANSION, AND

INVASION GENETICS OF THE ROUND GOBY {NEOGOBIUS

MELANOSTOMUS) IN GREAT LAKES TRIBUTARIES 16

2.1 INTRODUCTION 16

2.2 MATERIALS AND METHODS 20 vii 2.3 RESULTS 30

2.4 DISCUSSION 41

2.5 REFERENCES 48

3.0 DIFFERENCES IN DISPERSAL ABILITY BETWEEN EXPANDING AND

NON-EXPANDING POPULATIONS OF ROUND GOBY

{NEOGOBIUS MELANOSTOMUS): A COMPARISON OF SWIMMING

PERFORMANCE AND MORPHOLOGY 60

3.1 INTRODUCTION 60

3.2 MATERIALS AND METHODS 65

3.3 STATISTICAL ANALYSIS 73

3.4 RESULTS 77

3.5 DISCUSSION 85

3.6 REFERENCES 95

4.0 GENERAL DISCUSSION 106

4.1 REFERENCES 113

VITAAUCTORIS 115

viii LIST OF TABLES

Table 2.1 Watershed and location, site description, site code, GPS coordinates, distance to river mouth (km), and number of samples per site (N) per year 22

Table 3.1 Expected differences in morphology between expanding river (R) and non- expanding lake populations (L) of round goby, Neogobius melanostomus, in the Great

Lakes region. Round goby have recently initiated upstream colonization of river systems and it is expected that an adaptive response in dispersal ability to high flow is facilitating successful upstream expansion. Eight morphological traits (plus standard length) known to be functionally related to swimming performance, station holding ability, and habitat use in other fishes are used for population level morphological comparisons, and as predictors of swimming performance (UCRU, UQAIT, UBURST)- Trait - performance combinations not expected to have a functional relationship are indicated by a dashed line 73

Table 3.2 Sample sizes of round goby, Neogobius melanostomus, from lake and river populations at 3 Great Lakes tributaries used in swimming performance trials and morphometric comparisons 78

Table 3.3 MANOVA results for body shape comparison between lake and river populations of round goby, Neogobius melanostomus, at 3 Great Lakes tributaries. Traits were chosen from the literature based on the potential for a functional relationship with swimming ability in different hydrodynamic regimes. Residual traits were grouped

ix according to expected functional relationship with 1) swimming endurance, 2) station holding to the substrate, and 3) thrust production. Bold type and underlined indicates significance after Bonferroni correction 82

Table 3.4 Significant stepwise multiple regression results testing the effect of residual morphological traits (including standard length, SL) on swimming performance of round goby {Neogobius melanostomus) collected from lake and river populations at 3 Great

Lakes tributaries. Morphological traits were grouped according to an expected association with 3 swimming performance measures (swimming endurance, UCRIT; station holding ability, UGAIT; maximum thrust potential, UBURST)- Regional data

(Maitland, Grand, Saugeen) were tested independently and pooled (All) 85

x LIST OF FIGURES

Figure 2.1 Map of the round goby sampling sites in 2 watersheds of south western

Ontario. Site abbreviations are as shown in Table 2.1. Sites where no round gobies were captured are indicated by an asterisk (*) 23

Figure 2.2 Dispersal distance frequency of round goby {Neogobius melanostomus) river migrants from the Maitland River (2005 - 2009), Grand River (2008 - 2009), and

Saugeen River (2009) that successfully assigned (19 out of 65 fish) to a source population within the Great Lakes basin (n = 40). Genotype assignment (Rannala and Mountain

1997) was performed in Geneclass 2.0. First generation migrants were successfully assigned if the score for the first ranked putative source population was 4 times greater than the score for the second ranked source population 28

Figure 2.3 Number of round goby {Neogobius melanostomus) first generation migrants in the Maitland (2005-2009), Grand (2008-2009), and Saugeen (2009) lake and river populations (river populations as determined by cluster analysis) identified by genotype exclusion (Rannala and Mountain 1997) in Geneclass 2.0. Downstream to upstream sites within each river are positioned from left to right. Migrants were identified in 4 out of 10 lake populations (ML1 (2007-2009) and GL1 (2009 only)). River migrants were identified within all sites in the Maitland River with the exception of MR9. River migrants in the Saugeen River were captured at the lowermost and uppermost river sites.

xi River migrants in the Grand River were restricted to the 2 downstream sites in the lower reach of the river. Refer to Table 2.1 for site abbreviations and sample sizes 34

Figure 2.4 Genetic diversity, as measured by A) mean allelic richness (A), after rarefaction to 12 individuals to account for sample size differences, B) expected heterozygosity (HE), and C) mean relatedness (r), in round goby {Neogobius melanostomus) populations in the Maitland, Grand, and Saugeen Rivers. River population values represent "without migrants" (black bars) and "with migrants" (light bars; A and HE only). Mean (solid line) and 95% confidence interval (dashed lines) represents expected values as calculated from 40 baseline lake populations. Asterisk (*) indicates a significant difference between the river and generalized lake population. Site abbreviations are as indicated in Table 2.1 39

Figure 3.1 Morphological variables measured from digital photographs of round gobies

{Neogobius melanostomus) collected from lake and river sites located at 3 Great Lakes tributaries in 2009. Maximum body depth (BDMAX: deepest body region from lateral view), maximum body width (BWMAX: widest body region from dorsal view; not shown), caudal fin area (CA: caudal fin fully extended), caudal fin height (CH: dorsal to ventral tip of caudal fin when fully extended), pectoral fin area (PC: left (right was used if left fin damaged) pectoral fin fully extended), minimum peduncle depth (PDMIN: narrowest vertical depth of caudal peduncle), and pelvic fin area (PL: pelvic fin fully extended) All measurements were made to the nearest 0.01mm 72

xii Figure 3.2 Comparison of mean (± SE) residual swim performance values as measured by critical station holding speed (UCRIT), gait transition speed (UGAIT), and maximum burst swimming speed (UBURST), of round gobies, Neogobius melanostomus, from lake

(dark bars) and river (light bars) populations at 3 Great Lakes tributaries (Maitland,

Grand, Saugeen). All population comparisons at each tributary were non-significant

(p>0.05) 80

(light bars) populations of round goby {Neogobius melanostomus) collected at 3 Great

Lakes tributaries. Morphological traits were chosen based on their functional relationship with swimming ability in other fishes. Asterisk (*) denotes significance according to univariate ANOVA results comparing estimated marginal means of residual traits between lake and river populations 83

xiii LIST OF APPENDICES

Appendix 2.1 Pairwise comparisons of Weir and Cockerham's (1984) FST among round goby, Neogobius melanostomus, sampling sites at the Maitland (2005-2009), Grand

(2008-2009), and Saugeen (2009) rivers. Bold type and underlined indicates significant pairwise FST comparisons after Bonferroni correction. Site abbreviations are as in Table

2.1 and Figure 2.1 58

Appendix 3.1 Mean standard length (±SE) of round gobies, Neogobius melanostomus, from lake and river populations at the Maitland, Grand, and Saugeen Rivers in 2009

(n = 98). Minimum and maximum values are listed for each lake and river population 106

Appendix 3.2 Frequency distribution of standard length (mm) of round goby, Neogobius melanostomus, males (black bars) and females (grey bars) collected from lake and river populations at the Maitland, Grand, and Saugeen Rivers in 2009. Fish whose sex was undetermined by visual inspection of external papillae are represented by white bars

(Maitland and Saugeen river only). Total sample size is 98 107

1 Appendix 3.3 Absolute (cm s" ) critical station holding speed (UCRIT), gait transition speed (UGAIT), and maximum burst swimming speed (UBURST) for round gobies,

Neogobius melanostomus, sampled from lake and river populations at the Maitland,

Grand, and Saugeen Rivers in 2009. Total sample size is 58 108

xiv 1.0 INTRODUCTION

1.1 BIOLOGICAL INVASIONS

Species ranges are naturally dynamic (Caughley et al. 1988). The distribution of a species reflects their ability to disperse, colonize and persist in new areas with the colonization process typically involving 4 stages: dispersal from a native region, introduction to a recipient region, establishment of a reproductive population, and demographic spread. Many contemporary colonization events are influenced by human activities that facilitate long distance introductions beyond natural dispersal barriers and biogeographic regions (Wilson et al. 2009). The consequences of human-mediated introductions may be negligible if the species fails to establish a reproductive population.

Establishment success depends on the species ability to pass through three forms of ecological resistance that can act as a filtering mechanism throughout the invasion process: demography (e.g. age structure, effective population size), biota (e.g. competition, predation), and/or environment (e.g. temperature, salinity). On the other hand, human-mediated introductions can often be compounded by high propagule pressure where a large size of inoculation and/or high frequency of introduction events increase establishment success (Kolar and Lodge 2001, Wilson et al. 2009). Large-scale negative ecological and economic consequences have accompanied some successful introductions which have proliferated into highly invasive species (sea lamprey

{Petromyzon marinus; Mills et al. 1993), zebra mussel {Dreissena polymorpha; Mills et al. 1993), cane toad {Bufo marinus; Alford et al. 2009), water hyacinth {Eichhornia crassipes; Villamagna and Murphy 2010), and emerald ash borer {Agrilus planipennis;

Gandhi and Herms 2010).

1 The Laurentian Great Lakes have been subject to many adverse colonization events in recent decades due to human activity (Mills et al. 1993, Mack et al. 2000).

Since the opening of the St. Lawrence Seaway in 1959, human activity in the Great Lakes region increased and was paralleled by an increase in deliberate release of organisms, unintentional introductions through cultivation and aquarium trade, canal construction, habitat modification, and bait release which have contributed to the growing number of exotic species that have successfully established populations in the Great Lakes watershed (Mills et al. 1993). One of the largest contributors of introduced species to the

Great Lakes in recent decades is transoceanic shipping (Mills et al. 1993, Ricciardi and

Maclsaac 2000, Ricciardi 2006). By 2000, it was estimated that 75% of non-indigenous species that have been introduced to the Great Lakes regions since the early 1970's are due to ballast water releases (Ricciardi and Maclsaac 2000) with the majority of introductions (55%) originating from Eurasia (Mills et al. 1993). The substantial contribution of Eurasian donor regions is due to the intensity of commercial traffic relative to other regions of the world (Ruiz et al. 1997, Ricciardi 2006). Additionally, the history of environmental changes in the Ponto-Caspian region (i.e. water level fluctuations and salinity gradients; see Ricciardi and Maclsaac 2000) is a suggested factor contributing to long distance dispersal success of Eurasian species. Many of the successfully introduced aquatic fauna are tolerant to environmental changes, or have life history stages able to survive harsh conditions during transport including mid-ocean ballast water exchanges, which is expected to reduce survival of freshwater organisms due to increased salinity in the ballast tanks (but see Gray and Maclsaac 2010). Despite ballast water exchange legislation, euryhaline species continue to establish and spread

2 throughout the Great Lakes (Ricciardi and Maclsaac 2000), which has led to additional ballast regulations (i.e. salt water flushing) to further reduce introductions to the Great

Lakes (see Briski et al. 2010). Human dispersal vectors have also been implicated in the rapid, post-establishment spread of introduced species (Johnson and Carlton 1996, Brown and Stepien 2009). Ballast water exchanges among Great Lakes ports, recreational boating transfers through surface, hull, and equipment fouling, and bait bucket transfer are mechanisms facilitating regional movement of successful invaders (Johnson and

Carlton 1996, Brown and Stepien 2009). Identifying and reducing human-mediated dispersal pathways is an effective way to decrease the rate and range of expansion of introduced species since once an invader is established at detectable levels, eradication is unlikely (Dimond et al. 2010). However, natural dispersal following establishment in the newly invaded region can also be a potent source of colonization on a local scale which can lead to region-wide expansion at an accelerating rate (Alford et al. 2009).

1.2 DISPERSAL

Range expansion by natural dispersal, while expected to be generally less effective at generating a rapid regional distribution compared to human mediated dispersal vectors, can facilitate colonization into regions inaccessible to anthropogenic dispersal vectors. Natural dispersal can occur by regional diffusion (contiguous dispersal) where organisms move into adjacent territories at a rate relative to their population growth multiplied by a diffusion coefficient (Shigesada et al. 1995). Range expansion can also occur by a stratified dispersal strategy where long distance jump events accompany diffusion dispersal and lead to complex species distribution patterns

3 and accelerating rates of spread (Shigesada et al 1995). Colonization events are ideal for rapid evolutionary change due to population growth within a new biotic and abiotic environment (Reznick and Ghalambor 2001). Natural dispersal in the new region can be under strong selective forces and can result in colonizing populations with the ability to adapt to novel environments leading to extra range dispersal within their new region

(Phillips et al. 2006, Lombaert 2010). The evolutionary features of natural dispersal include the propensity to disperse where behaviour and motivation to disperse may be affected by population dynamics, life stage, or environmental cues (Duckworth and

Badyaev 2007). The evolution of dispersal can also be correlated to individual traits that affect the capacity to disperse (Phillips et al. 2006). For example, a re-colonization event of the speckled wood butterfly {Pararge aegeria) in the United Kingdom was led by individuals with greater dispersal ability, through increased investment in flight (wing morphology and thorax size), compared to continuously occupied sites that had greater investment in reproduction (e.g. abdomen size; Hill et al. 1999). When a species is expanding its range into a novel environment, it is expected that the invasion front will be dominated by a dispersal phenotype associated with successful colonization.

Colonization can lead to negative effects on native biodiversity through secondary range expansion, especially into areas with at risk species (see Poos et al. 2010), and sensitive areas such as nursery habitats (Cooper et al. 2007). When natural dispersal facilitates secondary range expansion into a new region, it can be expected that the source population density is high enough to trigger expansion, and the expanding population consists of dispersive individuals able to adapt to and successfully colonize new territories.

4 Contemporary colonization events create natural experiments to test theory and identify mechanisms facilitating successful introduction and establishment in a novel environment. This is especially important when the ecological and economic impact of the species increases as range expansion proceeds. The invasion front of an expanding population can therefore yield valuable information into the mechanisms facilitating colonization success.

1.3 ROUND GOBY {NEOGOBIUS MELANOSTOMUS)

One of the most speciose families of fishes is the Gobiidae family, in the Order:

Perciformes. The Gobiidae family consists of 5 subfamilies, 210 genera, and approximately 1950 known species (Corkum 2010). Species included in the Gobiidae family are generally small (10-50 cm), carnivorous, benthic fishes that have distributions in marine, brackish, and freshwater systems throughout the world (Fuller et al. 1999). Gobies are found in a variety of habitats including estuaries, reefs, algal beds, inland lakes and rivers, lagoons, kelp beds, mangrove swamps, and bays (Allen et al.

2006, Corkum 2010). Latitudinal distribution of gobies extends from the tropics to the subarctic region (Corkum 2010). A typical morphological feature that distinguishes most gobies from other fishes is a fused pelvic fin on the ventral surface of their body which can be used as a suction disk in high, turbulent flow or while ascending inland waters.

Due to their small size, gobies are often prey for larger piscivores while other goby species have developed symbiotic relationships with fishes and crustaceans (Randall et al.

2005). Facilitated by human introductions, some goby species (e.g. round: Neogobius melanostomus, tubenose: Proterorhinus semilunaris, shokihaze: Tridentiger barbatus,

5 shimofuri: Tridentiger bifasciatus, chameleon: Tridentiger trigonocephalus) have successfully invaded aquatic environments far beyond their native region and may constitute a large percentage of catch where they are introduced, while other goby species are listed as endangered in their native range (tidewater: Eucyclogobius newberryi; Fuller etal. 1999, Allen etal. 2006).

The round goby, Neogobius melanostomus, is a member of the subfamily:

Gobiinae. Round gobies are native to the Black Sea, Caspian Sea, and the Sea of Azov and typically occupy nearshore rocky habitat in brackish and fresh water during the warmer season and migrate to deeper water for the winter season (Werner 2004). In addition to their preferred rocky habitat, round gobies also occupy mud, silt, sand, gravel, and cobble substrates along with anthropogenic debris including shipwrecks (Corkum et al. 1998, Werner 2004). The diet of adult round gobies mainly consists of molluscs, which is facilitated by their pharyngeal teeth able to crush the hard shells of larger mollusc prey (Werner 2004). Round gobies also consume insects, zooplankton, gammarids, snails, worms, fish eggs, and smaller fish including juvenile round goby

(French and Jude 2001, Simonovic et al. 2001, Roseman et al. 2006). Juvenile round gobies typically refrain from feeding on molluscs until they grow to a length of approximately 60 mm (French and Jude 2001).

Round gobies can live up to 5 years and may grow up to 25 cm maximum total length. Round gobies mature at approximately 2-3 years old but age-at-maturity differs between sex and throughout their global distribution (see Corkum et al. 1998). The spawning season of the round goby extends from May to October in the Great Lakes region and is dependent on a water temperature in the range of 9 - 26 °C (Maclnnis and

6 Corkum 2000). The long spawning season allows for multiple spawning events estimated at 4 - 6 times per season, depending on water temperature (see Corkum et al.

1998). Male-male competition leads to individual male territories in which parental males, displaying secondary sexual characteristics (i.e. large size, black coloration, swollen cheeks, aggression), court females, fertilize and aerate eggs of multiple females, and protect eggs and young of year (Corkum et al. 1998, Werner 2004). The number of eggs is positively correlated to body length of females and ranges from approximately

252 - 1818 eggs in the Great Lakes region (Maclnnis and Corkum 2001), a relatively low number compared to females in the native range (328 - 5221 eggs; Kovtun 1978 in

Maclnnis and Corkum 2000). Eggs are usually deposited in a single layer on hard surfaces in the male's nest cavity. Parental care and the relatively large size of round goby eggs (3.2 mm) is suggested to increase hatching success and offspring survival (see

Maclnnis and Corkum 2000). In addition to parental male round gobies, an alternative male reproductive morphology has been observed. The alternative male morphology is smaller, lacks secondary sexual characteristics and matures earlier than parental males

(Corkum et al. 1998, L'Avrinkova and Kovac 2007, Marentette et al. 2009).

Round goby are a highly successful invader of the Laurentian Great Lakes.

Round gobies were first detected in the St. Clair River in 1990 (Jude et al. 1992), arriving from their native Ponto-Caspian region presumably by ballast water discharge (Hensler and Jude 1997). Within 5 years of detection, round gobies were distributed throughout all 5 Great Lakes and have had major ecological and economic impacts suggested to be due to their high population densities (Johnson et al. 2005), and competitive superiority

(Dubs and Corkum 1996, Janssen and Jude 2001, Balshine et al. 2005). Round goby

7 have been implicated in the reduced recruitment of mottled sculpin (Janssen and Jude

2001), decreased abundance of logperch populations (Balshine et al. 2005), and have disrupted food webs, creating a potential link for contaminant transfer from zebra mussels to larger piscivores therefore threatening human health (see Corkum et al. 2004). Round gobies also prey on eggs of economically important sports fish including walleye, smallmouth bass, and sturgeon (see Corkum et al. 2004, Roseman et al. 2006). Round goby recently initiated upstream expansion into river systems in their introduced North

American regions as well as river systems in their European distribution (Phillips et al.

2003, Wiesner 2005, Carmen et al. 2006, Poos et al. 2010). River expansion threatens native biodiversity and at-risk species (Poos et al. 2010), through establishment in canals, connected waterways, and inland lakes. Upstream river expansion provides an ideal opportunity to evaluate dispersal mechanisms, dispersal strategy, and invasion dynamics associated with colonization of a novel aquatic environment.

1.4 CHAPTER 2 OBJECTIVES

The main objective of chapter 2 is to use genetic methods to evaluate local dispersal and colonization dynamics associated with round goby river colonization.

Genetic structure was characterized within each river using highly polymorphic microsatellite markers to identify genetic subdivisions between sites. Genotype assignment methods were used to distinguish among contiguous and long distance dispersers within each river system. Genetic diversity was compared between lake and river populations to evaluate the effect of colonization on genetic diversity, including mean relatedness within populations to identify a dispersal genotype. Temporal stability

8 in spatial distribution and genetic structure was examined across two years and five years at two independent tributaries.

1.5 CHAPTER 3 OBJECTIVES

The main objective of chapter 3 is to characterize the dispersal ability of round gobies from lake and river populations at 3 tributaries to identify a non-random dispersal phenotype associated with river colonization. Using a subset of round gobies from the lake and river populations, I quantified 3 measures of swimming ability that represent aerobic endurance, behaviour, and maximum anaerobic ability in a closed system swimming flume. Measures of swim performance were tested for repeatability.

Morphological traits were measured from digital photographs and tested for population level differences among lake and river fish. Morphological variables were related to swimming ability in other fishes and therefore correlations with performance values were examined to identify a functional relationship between morphology and performance in river colonizers.

9 1.6 REFERENCES

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15 2.0 DISPERSAL STRATEGIES, SECONDARY RANGE EXPANSION, AND

INVASION GENETICS OF THE ROUND GOBY {NEOGOBIUS MELANOSTOMUS)

IN GREAT LAKES TRIBUTARIES

This chapter also incorporates the outcome of ajoint research undertaken in collaboration with Brad Dufour under the supervision of Dr Daniel Heath In all cases, the key ideas, primary contributions, experimental designs, data analysis and interpretation, were performed by the author, and the contribution of the co-author was 1) providing the genotype data for 32 round goby lake populations from the Great Lakes region used as reference populations for genotype exclusion and assignment analyses, and 2) providing genotype data for round goby collected from Maitland River sites (2005-2006)

2.1 INTRODUCTION

Successful colonization of habitat beyond a species current range requires the appropriate dispersal strategy to facilitate the initial introduction. Dispersal into new environments can occur by regional diffusion — where the population expands into adjacent habitats as a function of population growth rate and a diffusion coefficient leading to a stepping stone model of expansion (Shigesada et al. 1995, Thibault et al.

2009) — or by long distance dispersal where dispersal is not limited to nearby patches, but instead can lead to disjunct populations located far from the species range core; an island model of dispersal where each habitat patch has the same probability of being colonized (Kareiva et al. 1990). It is now recognized that multiple dispersal mechanisms such as short-distance diffusion combined with long-distance jump events, collectively termed stratified dispersal, contribute to the range expansion of many contemporary colonizing species (Muirhead et al. 2006, Worthington Wilmer et al. 2008, Darling and

Folino-Rorem 2009, Huebner 2010). Stratified dispersal can lead to complex species distribution patterns and increasing rates of expansion when a species is colonizing a new area (Shigesada et al.1995, Henne et al. 2007).

16 Species expanding their range into new environments are faced with selective pressures which will challenge their dispersal strategy and potentially limit their expansion. Selective forces include abiotic factors such as different climate, different chemical composition, and physical barriers to dispersal (Moyle and Light 1996,

Ricciardi and Maclsaac 2000, Kestrup and Ricciardi 2009), and biotic factors such as new predators, parasites, and community dynamics (Moyle and Light 1996,

Sakai et al. 2001, Chun et al. 2010). Dispersal is expected to be favoured in peripheral and expanding populations. Critical factors that affect a species' dispersal strategy include dispersal ability (Hochkirch and Damerau 2009), the location of the disperser within the species range (Gros et al. 2006, Dytham 2008), and its ability to respond to environmental heterogeneity (Kestrup and Ricciardi 2009). For example, an adaptation for increased dispersal capacity at the invasion front of the prolific cane toad in Australia has led to an accelerating rate of expansion into territories that were not expected to be colonized by cane toads based on models derived from their native region

(Phillips et al. 2007). Population genetic theory states that adaptive potential depends on genetic diversity, that is, populations with higher genetic diversity will have a greater opportunity to resist detrimental founder effects and adapt to different environments

(Lee 2002). At the same time, genetic drift and selective forces associated with colonizing new environments will act to deplete a population's genetic diversity

(Ciosi et al. 2008, Peacock et al. 2009).

Recent research shows that genetic diversity may be maintained in colonization events and ongoing invasions, despite theoretical expectations for a loss of genetic diversity through founder effects and genetic bottlenecks (Colautti et al. 2005,

17 Stepien et al. 2005, Stepien and Tumeo 2006, Hochkirch and Damerau 2009). Suggested mechanisms that may account for this anomaly include high propagule pressure

(Lockwood et al. 2005), and gene flow among independently colonized populations

(Kolbe et al. 2004, Stepien et al. 2005). Human-mediated introductions are known to have high propagule numbers including multiple introduction events from genetically distinct source populations (Kolbe et al. 2004, Therriault et al. 2005, Simon-Bouhet et al.

2006, Stepien and Tumeo 2006, Roman and Darling 2007, Zalewski et al. 2010).

Human-mediated dispersal within new environments will also promote gene flow among newly established populations therefore increasing genetic diversity and the potential to produce novel genotypes within these new populations (Lombaert et al. 2010).

Colonization patterns associated with human-mediated dispersal are different from that created by natural dispersal and thus if regional diffusion and long distance dispersal events are both contributing to range expansion (i.e. "stratified dispersal") the rate of expansion is expected to increase (Shigesada et al. 1995). Stratified dispersal is increasingly being accepted as a mechanism that can also maintain genetic diversity at the invasion front of an expanding population (Simon-Bouhet et al. 2006, Tobin and

Blackburn 2008). Furthermore, it has been suggested that a high level of genetic diversity at the invasion front will increase the probability of an introduction becoming a successful invasion by providing genetic variation for phenotypic traits associated with colonizing ability on which selection can act (Roman 2006, Parisod and Bonvin 2008).

The round goby, Neogobius melanostomus, is a highly successful invader originating from the Ponto-Caspian region. Round gobies were first detected in the

Laurentian Great Lakes in 1990 in the St. Clair River (Jude et al 1992). By 1995, round

18 gobies were reported in all 5 Great Lakes and continue to be detected in new areas throughout the Great Lakes watershed (Jude et al. 1995, Phillips et al. 2003,

Schaeffer et al. 2005, Pennuto et al. 2010, Poos et al. 2010). The rapid spread of round goby populations throughout the Great Lakes is attributed to ballast water transfer among

Great Lakes ports post-introduction (Hensler and Jude 2007). Successful establishment of round goby populations has been also facilitated by their high environmental tolerance, rapid population growth rate, and superior competitive ability (Dubs and Corkum 1996,

Charlebois et al. 2001). Despite their rapid spread, round goby populations were expected to remain limited to the Great Lakes basin within preferred rocky habitat

(Clapp et al. 2001, Ray and Corkum 2001). However, round gobies have recently expanded their range upstream into river systems in their introduced North American region as well as within their introduced range in Europe (Sapota and Skora 2005,

Phillips et al. 2003, Wiesner 2005, Pennuto et al. 2010, Poos et al. 2010). River expansion is occurring in areas void of ballast water vectors and therefore suggests that alternate dispersal mechanisms are facilitating upstream spread. Paradoxically, round gobies are expected to have poor natural dispersal ability, especially upstream, due to their benthic morphology and small home range (Wolfe and Marsden 1998,

Ray and Corkum 2001; but see Hensler and Jude 2007).

The ability of round goby to disperse and colonize river systems creates new dispersal corridors for their continued expansion into connected waterways and inland lakes which would increase their ecological and economic impact (Ricciardi and

Maclsaac 2000). It is critical to identify factors governing secondary spread into tributary watersheds in order to accurately predict the ultimate distribution and potential

19 impacts that may result from the round goby invasion. Here I examine the invasion genetics of the round goby river invasion front in three selected Great Lakes tributaries.

Specifically, I test two hypotheses: 1) river colonization occurs by contiguous diffusion or long distance dispersal, and 2) river dispersal results in a change in genetic diversity within a colonizing population relative to non-expanding lake population. Genetic characterization of the colonization dynamics and dispersal mechanisms associated with successful invasions will provide useful results for accurate risk assessment and effective management strategies. Additionally, the current study will contribute to the growing knowledge of how species expand their range into new environments.

2.2 MATERIALS AND METHODS

Sample collection

A total of 1288 round gobies were collected from May to October in 2005 to 2009

(Table 2.1). Round gobies were collected from the Maitland River, Goderich, Ontario,

Canada from 4 river sites (n = 44) and 1 lake site in 2005, and 5 river sites (n = 113) and

1 lake site in 2006 as part of another study (Dufour 2007). Samples collected from the

Maitland River lake site in 2005 and 2006 were combined to attain a reasonable sample size (n = 31) after testing for genetic similarity. Round gobies were collected from the

Maitland River at 6 river sites (n = 116) and 1 lake site (n = 44) in 2007, 8 river sites

(n = 197) and 1 lake site (n = 84) in 2008, and 9 river sites (n = 190) and 1 lake site

(n = 64) in 2009 (Figure 2.1, Table 2.1). I collected 297 round gobies from the Grand

River, from Port Maitland to Brantford, Ontario, Canada in 2 years (2008: 8 river sites n

= 59, 2 lake sites n = 50; 2009: 7 river sites n = 112, 2 lake sites n = 76). In 2009,1

20 collected round gobies from 3 river sites (n = 79) and 1 lake site (n = 23) in the Saugeen

River that drains into Lake Huron at Southampton, Ontario, Canada (Table 2.1, Figure

2.1). All fish were collected by a combination of techniques including a single pass with

Smith-Root LR-24 backpack electrofisher (Smith-Root Inc. Vancouver, WA, USA), three to six 100 m seine hauls with a 30 m beach seine with 6.35 mm mesh, 4 or more minnow traps baited with luncheon meat, and/or hook and line baited with worms. Round gobies were considered absent at a site when no specimens were captured with a minimum of

1200 electroshocking seconds, three 100 m seine hauls, or 4 angling hours plus 4 baited minnow traps depending on suitability of each sampling technique at each site. Samples were collected at the river mouth, in the lake close to the river mouth, and at upstream intervals until the invasion fronts were identified (Figure 2.1). The invasion fronts in each river were determined as the farthest upstream site where round gobies were captured when round gobies were absent at a minimum of two consecutive upstream sites. Additional random sites upstream from the invasion fronts (Maitland: 11.4 river km, Grand: 21.2 river km, Saugeen: 4.8, 6.6, and 7.8 river km upstream of the invasion front) were sampled to check for possible disjunct round goby populations (Figure 2.1).

All UTM (Universal Transverse Mercator) coordinates were recorded with a portable

GPS unit (Table 2.1). All round gobies were humanely euthanized with an overdose of clove oil (25 ppm), and fin clip samples were fixed in 95% ethanol and stored at room temperature until DNA extraction.

21 Table 2.1 Watershed and location, site description, site code, GPS coordinates, distance to river mouth (km), and number of samples per site (N) per year.

Site Distance to river Watershed and Site Code Latitude Longitude 2005 2006 2007 2008 2009 Description mouth (km) location

Lake Huron

Maitland River lake MLl 43°44'40 57"N 8!°43,176r,vV 09 31" 31* 46 84 65

mouth MRI 43°44,54 37"N 8]°43'26 69"W 03 17 31 0 52 9

river MR2 43°45'6 48"N 8I°42'52 32"W 1 1 16 24 16 61 48

river MR3 43°45'11 34"N 8I°42'40 02"W 1 5 4 29 45 26 35

river MR4 43°45'15 7S"N 81°42'22 98"W 1 9 7 17 43 28 15

river MR5 43°45'12 00"N 81°42'5 70"W 23 0 12 1 4 17

river MR6 43°45'l 98"N 8r42'0 36"W 27 0 0 2 14 22

river MR7 43°44'57 66"N 81°4r56 40"W 29 0 0 12 10 35 river MR8 43°44'43 86"N 81°41'52 74"W 34 - 0 0 2 6 river MR9 43°44,34 92"N 81=4139 06"W 39 • 0 0 3 Lake Erie Grand River lake GL1 42°51 183'N 79 °34 948'VV 07 - - 13 36 lake GL2 42°5I 12I'N 79° 34 785'W 05 - 37 40 mouth GRI 42°51'24 72"N 79°34'40 53"W 0 1 - 26 42 river GR2 42°54'2 09"N 79°37'7 45'W 79 - - - 10 35 river GR3 42°55'37 38"N 79°42'4I 40"W 83 4 5 river GR4 42°56'56 28"N 79°51'37 08"W 32 7 - 3 ? river GRS 43°4'26 73"N 79°57'29 5I"W 51 1 - 1 1 1 river GR6 43° 431 98"N 79°57'52 32"W 51 8 - - 2 0 river GR7 43°4'4I 70"N 79°58'I6 86"W 52 9 10 13 river GR8 43°5'38 15"N 80° r54 02"W 58 9 - 1 4 Lake Huron Saugeen River lake SL1 44° 30 OWN 81°22'522"W 0 1 - - 23 mouth SRI 44°30'4 08"N 8r22'l8 20"W 02 - - 49 river SR2 44°30'17 34"N 81°20'53 64"W 24 - - 17 river SR3 44°30,]5 39"N 8I°19'58 69"W 43 - - - 13 * genotype data from samples collected in 2005 and 2006 at site MLl were combined in a previous study to increase sample size after confirming no difference in allele frequency distribution Total sample size is 31.

22 Figure 2.1 Map of round goby sampling sites in 3 rivers of south western Ontario Site abbreviations are as shown in Table 2 1 Sites where no round gobies were captured are indicated by an asterisk (*)

23 DNA extraction

A 0.5 - 10.0 mg clip of caudal fin tissue was pressed and air dried to remove all ethanol from the tissue sample prior to proteinase K digestion. I extracted DNA following an automated silica plate-based extraction protocol (Elphinstone et al. 2003) and re-suspended DNA in lOOuL Tris-EDTA buffer (pH = 8.0). Samples were genotyped at 8 polymorphic nuclear microsatellite loci; Nme2, Nme3, Nme4, Nme5,

Nme6, Nme7, Nme9, and NmelO (Dufour et al. 2007). Polymerase chain reactions

(PCR) contained approximately 50 ng DNA, 1.0 X PCR reaction buffer (Applied

Biosystems), primer specific concentrations of MgCb (Nme 2 = 2.1 mM; Nme4, Nme9 &

NmelO = 2.4 mM; Nme 1, Nme3 & Nme5 = 2.7 mM; Nme6 & Nme7 = 2.8 mM), 0.19 mM of each dNTP (Promega), 0.057 uM reverse primer (Sigma Genosys), 0.038 uM

IR700 or 800 dye-labelled forward primer (MWG Biotech AG), and 0.05 U Taq polymerase (5U/uL, Applied Biosystems) with ddF^O to a final reaction volume of 11 uL. Polymerase chain reactions followed the protocol of: 95°C initial denaturation for

120 s; 35 cycles of 95°C for 15 s, optimized primer-specific annealing temperature

(Nme3, Nme5, Nme7 & NmelO = 48°C; Nme2 & Nme4 = 55°C; Nme9 = 58°C; Nme6 =

59°C) for 15 s, 72°C extension for 30 s; followed by 72°C final extension for 120 s and a

4°C holding temperature. The PCR products were run on a LiCor DNA analyzer for visualization. Amplicons were manually analyzed and scored to size with 1 base pair resolution using GenelmaglR 4.05 software (Scanalytics, Inc).

24 Genetic analyses

Microsatellite markers were tested for Hardy-Weinberg equilibrium and linkage disequilibrium within each sampling site using an exact probability test in Genepop

4.0.10 (Raymond and Roussett 1995). Significance was adjusted for multiple comparisons using sequential Bonferroni correction.

Genetic structure

Regional level genetic structure has been previously reported in the Great Lakes round goby distribution (Stepien et al. 2005, Stepien and Tumeo 2006, Dufour 2007) and therefore I focus my analyses on a local scale (i.e. each river independently). Population genetic structure within each river was inferred using a cluster based method in Structure

2.2 (Pritchard et al. 2000) within each sampling year. Results were generated with the admixture model using 100 000 burn-ins and 1 000 000 iterations of a Markov chain

Monte Carlo simulation. Genotype clustering uses all individuals as units simultaneously with no prior knowledge of the spatial distribution of sampling sites (Pritchard et al.

2000). The number of genetic clusters (K) was determined by the highest negative

In probability (-In P) of data of all possible K values, when K < the number of sampling sites within each river. Genetic structure results were used to cluster river sites for subsequent analyses.

I also estimated genetic divergence among river sites, and river versus lake sites within each sampling year, by calculating pairwise FST in Arlequin 3.11 (10 000 permutations; Excoffier et al. 2005). Significance was corrected for multiple comparisons following the sequential Bonferroni method.

25 Upstream dispersal

Individual and population-level genotype assignment and exclusion were performed in Geneclass 2.0 (Piry et al. 2004) to infer population connectivity and geneflow patterns. More specifically, I wanted to: 1) identify individual fish whose genotype is excluded from the lake or river population in which they were captured (e.g. first generation migrants), with the river populations as previously determined by cluster analyses (see above), 2) identify the most likely source population of each first generation migrant, and 3) identify the most likely source population for each river population (with migrants excluded) using group genotype assignment. Genotype assignment has been shown to be a more powerful approach to detect spatial patterns of dispersal compared to population level approaches that are based on the relationship between genetic differentiation and geographic distance (i.e. isolation by distance; Castric and Bernatchez

2004).

First, I used individual genotype exclusion (Rannala and Mountain 1997) with

Monte Carlo resampling of 10 000 simulations (Paetku et al. 2004) to identify migrant fish whose genotype is excluded from the lake or river population in which they were captured and thus are likely first generation immigrants to that population. Migrants were identified as fish that were excluded from the population based on a 5% type 1 error threshold. The assignment exclusion method by Rannala and Mountain (1997) is suitable to identify recent immigrants to a population when the source and sink populations have relatively low genetic divergence among them, and this approach is also robust to population genetic dis-equilibrium (Rannala and Mountain 1997). I performed a cross

26 tabulation analysis in Systat 7.0 to test for differences in number of migrants in the lake versus the river.

Second, I performed Bayesian rank-based genotype assignment (Rannala and

Mountain 1997) of migrants to identify a putative source population for these fish. I used genotype data from 32 baseline round goby populations (n = 1958) from the lower Great

Lakes region that were sampled and genotyped in a previous study (Dufour 2007) as reference populations for assignment. The rank-based method calculates the likelihood of each reference population being the putative source for each migrant fish. Successful assignment was achieved if the score for the most likely source population was a minimum of 4 times greater than the score for the second ranked population (Beneteau et al. 2009).

Lastly, I performed Bayesian rank-based group assignment (Rannala and

Mountain 1997) to identify the most likely source population for each river population

(with migrants removed). Criteria for successful assignment to a source population was as in step 2; the score for the most likely source population must be a minimum of 4 times greater than the score for the second ranked population (Beneteau et al. 2009). I identified three possible outcomes: 1) river populations that were assigned to their associated lake population are colonization events via contiguous diffusion dispersal,

2) river populations that were not assigned to the associated lake population though were successfully assigned to a lake population located within 150 km from the site of capture

(as measured by shortest water distance in Google Earth) are short-distance colonization, or "regional diffusion", and 3) river populations assigned to a lake population located more than 150 km away are long distance colonizations. The 150 km threshold used to

27 distinguish among contiguous and long distance dispersal events was determined by examining the peaks in the frequency distribution of dispersal distances travelled by migrant fish (as identified in step 2; Figure 2.2). The first peak consisted of dispersal distances ranging from 0 - 150 km which most likely represents natural dispersal and thus

I used 150 km as a threshold distance to distinguish among contiguous and long distance dispersal events. River populations were defined by genetic structure analyses described above.

7 a) CD ^H Maitland X! 6 • Grand E Regional Long distance c 5 <- -> II Saugeen -§—c» CO 4

"E 3

o 2 c CD 13 CX 1 CD 0 ill 1 o o o o o O o o o o o § o

Figure 2.2 Dispersal distance frequency of round goby {Neogobius melanostomus) river migrants from the Maitland River (2005 - 2009), Grand River (2008 - 2009), and Saugeen River (2009) that successfully assigned (19 out of 65 fish) to a source population within the Great Lakes basin (n = 40). Genotype assignment (Rannala and Mountain 1997) was performed in Geneclass 2.0. First generation migrants were successfully assigned if the score for the first ranked putative source population was 4 times greater than the score for the second ranked source population.

28 Genetic diversity

To evaluate genetic diversity differences among lake and river populations (river populations as defined by genetic structure analysis) I calculated allelic richness (A), with values corrected for unequal samples sizes by rarefaction to 12 individuals, in FSTAT v.2.9.3 (Goudet 2001) and expected heterozygosity (HE) in Arlequin 3.11 (Excoffier et al.

2005). I calculated mean A and HE and 95% confidence intervals for all lake populations combined (n=40) including 32 baseline populations from a previous study (Dufour 2007).

I tested for differences in genetic diversity (A and HE independently) between the river populations and the combined lake populations by identifying river population values that fell outside of the 95% confidence limits generated by the lake values. Genetic diversity

(A and HE) was estimated in river populations with and without long distance migrants included to determine the effect that long distance dispersal has on genetic diversity during colonization. Low genetic diversity can result from founder and population bottleneck events and is expected to be the result of recent colonization.

Relatedness was estimated within lake and river populations (with migrants excluded) to determine if there is a family effect associated with upstream dispersal (e.g. dispersal genotype). Pairwise relatedness (Queller and Goodnight 1989) among individuals was estimated within each lake (n = 40) and river population (n = 10) in

Genalex 6.1 (99 permutations, 99 bootstraps; Peakall and Smouse 2006). Within population mean relatedness (r) was calculated from pairwise relatedness among individuals. I tested for differences in relatedness between lake and river populations

(with migrants excluded) by examining if point estimates of r within river populations are within the 95% confidence interval (CI) derived from the lake values. River estimates

29 outside of the 95% CI would be considered significantly different based on a 5% error threshold.

Temporal stability

Since the round goby river invasion process is both recent and highly dynamic I expected the river populations to show temporal instability in spatial distribution and genetic structure in the two rivers for which I had temporal replicates (Maitland and

Grand Rivers). To examine temporal stability in spatial distribution, GPS coordinates were recorded at each sampling site within each river. The shortest water distance separating the river mouth and the uppermost invasion front site was measured in Google

Earth and compared among years in the Maitland River (2005 - 2009) and Grand River

(2008 - 2009). Temporal stability in allele frequency distribution was tested by performing exact tests within river populations (Maitland 2005 - 2009, Grand River

2008 - 2009) across adjacent years in TFPGA (Miller 1997). Significance was Bonferroni corrected to account for multiple comparisons.

2.3 RESULTS

A total of 1262 round gobies were genotyped (2005: 44; 2006: 144; 2007: 160,

2008: 390; 2009: 524). All 8 microsatellite markers were polymorphic with 4 to 18 alleles per locus. Departures from Hardy-Weinberg equilibrium were found in 21 of the

464 loci by sampling site comparisons. The majority of departures were found within

Maitland River sites (19 / 21: 90%), and the remaining 2 departures were found in

2009-GR1, 2009-SL1 (Site abbreviations are as shown in Table 2.1). The majority of

30 Hardy-Weinberg deviations were due to heterozygote deficiency (12/20: 60%).

Significant linkage disequilibrium was observed between 2 locus pairs with overall significance driven by one significant population comparison each (Nme3-Nmel0:

2007-MR3; Nme2-Nme9: 2009-ML1). I recovered 76 alleles from all river sites and 65 alleles from lake sites (lake sites associated with the sampled rivers) with an overall mean of 10 alleles per locus. Lake and river sites share 63 alleles with 2 out of 65 (3%) lake alleles only found in lake sites and 13 out of 76 (17%) river alleles only found within river sites. The number of private alleles identified ranged from 0 to 1 in the lake sites, and 0 to 2 in river sites.

Genetic structure

The highest negative In probability values (-In P) for individual genotype clustering within each river in each year inferred one cluster in the Maitland River in each sampling year (2005 - 2009), and one cluster in the Saugeen River (2009). Therefore I combined all of the Maitland and Saugeen river sites into a single "river population" (MR and SR, respectively) within each year for subsequent analyses. Two clusters were identified in the Grand River in each sampling year (2008 and 2009). The spatial distribution of Grand River clusters were similar across years and correspond to an upper and lower river distribution. River sites GR1 and GR2 were combined into a lower

Grand River population (GRLOW) in each year. River sites GR4 to GR8 were combined to make the upper Grand River population (GRUP) in each year. Fish from river site GR3 had a mixed membership among GRLow and GRyp in each year and were assigned

31 accordingly. In 2008, 2 fish from GR3 clustered with GRLOW and 2 fish clustered with

GRup. In 2009, 3 fish from GR3 clustered with GRLow, and 2 fish clustered with GRup.

Pairwise FST values for genetic divergence among sampling sites within a sampling year ranged from -0.16 to 0.25 (Appendix 2.1). All pairwise FST estimates among sites in the Maitland and Saugeen River system were non-significant (and sometimes negative). The Grand River sites had 23 out of 81 (28.4%) significant pairwise comparisons after Bonferroni correction (Appendix 2.1). Significant FST comparisons generally correspond to differentiation among upper and lower Grand River sites in 2009, mirroring our genotype clustering results.

Upstream dispersal

The results of the individual genotype exclusion identified 7 out of 366 (1.9%) lake fish and 65 out of 896 (7.3%) river fish that were excluded from the lake or river population (river populations defined by genetic structure analyses; see above) in which they were captured and therefore were likely first generation migrants to that population

(Figure 2.3). Six migrants were identified in the Maitland lake population in 2007-2009

(MLl: 2007 n = 1, 2008 n = 3, 2009 n = 2; Figure 2.3). One migrant was identified in one Grand River lake population (GL1: 2009). No migrants were identified in the

Saugeen lake population (SL1). River migrants were identified at all sampling sites in the Maitland River population (MR) with the exception of the uppermost upstream site

(MR9-2009; Figure 2.3). The frequency of migrants in the Maitland River within each year ranged from 2.3% (2005: n = 1) to 9.6% (2008: n = 19). Migrants were identified at

2 out of 3 sites (SRI: n = 4, SR3: n = 1) in the Saugeen River population (SR) and made

32 up 7.4% of all samples collected from the Saugeen River. The lower Grand River population (GRLOW) was composed of 6.8% (n = 4) migrants in 2008 and 5.6% (n = 7) migrants in 2009 and were collected from sites GR1 and GR2 in each year (Figure 2.3).

Interestingly, no migrants were detected in the upper Grand River population (GRUP) in either sampling year (2008 and 2009). The cross tabulation analysis revealed a significant difference between migrant loads in lake and river populations at the Maitland

River (p = 0.010, x2= 1-02, df = 1), and the Grand River (p = 0.002, %2= 9.87, df = 1). In both rivers the migrant load was higher than in the corresponding lake population

(Maitland: 49 / 612 (8.0%) river migrants, 6 / 221 (2.7%) lake migrants; Grand: 11 / 104

(10.6%) lower river (GRLOW) migrants, 1/126 (0.8%) lake migrants). No significant difference in migrant load was detected between the lake and river population in the

Saugeen River, though my sample size was small (n = 91; lake: n = 23, river: n = 68).

33 1 0 E I I 2005 I I 2006

!• 2007

2008 c 2009 CO E

r~ >~ rv o ~~i Q: Q: o: OOOOOOOOOO « CO (O S) Site

Figure 2.3 Number of round goby {Neogobius melanostomus) first generation migrants in the Maitland (2005-2009), Grand (2008-2009), and Saugeen (2009) lake and river populations (river populations as determined by cluster analysis) identified by genotype exclusion (Rannala and Mountain 1997) in Geneclass 2.0. Downstream to upstream sites within each river are positioned from left to right. Migrants were identified in 4 out of 10 lake populations (MLl (2007-2009) and GL1 (2009 only)). River migrants were identified within all sites in the Maitland River with the exception of MR9. River migrants in the Saugeen River were captured at the lowermost and uppermost river sites. River migrants in the Grand River were collected from the 2 downstream sites in the lower reach of the river. Refer to Table 2.1 for site abbreviations and sample sizes.

Successful assignment was achieved for 3 out of 7 (42.9%) lake migrants, and 19 out of 65 (29.2%) river migrants based on the assignment criteria (the score for the first ranked population is 4 times greater than the score for the second ranked population).

Genotype assignment indicated that 14 migrants (lake: n = 1, river: n = 13) assigned to source populations located within 150 km of their site of capture; 3 migrants (lake: n = 2,

34 river: n = 1) assigned to a source approximately 205 - 270 km away, 5 river migrants assigned to source populations located over 500 km away by shortest water distance

(Figure 2.2). Migrants from the Maitland populations (lake and river) that successfully assigned to a source lake population (n = 17: lake n = 3, river n = 14) were assigned to populations in the western basin of Lake Erie (n = 4: lake n = 2, river n = 2), eastern Lake

Erie (river n = 3), eastern Lake Ontario (river n = 1) and Lake Huron (n = 9: lake n = 1, river n = 8). Successful assignment of the Grand River migrants to a lake population

(n = 3) assigned to populations in eastern Lake Erie (n = 2), and Lake Huron (n = 1).

Migrants to the Saugeen River that successfully assigned to a source population (n=2) assigned to a population in Lake Huron close to the mouth of the Maitland River (n = 2).

Assignment results should be viewed with caution as 4 out of 7 (57.1%) lake migrants and 46 out of 65 (70.8%) river migrants did not assign to any potential source population based on my assignment criteria. Failed assignment most likely occurred because the source population was not sampled. Round goby populations may have significant genetic structure at a smaller spatial scale than the 32 source populations were sampled

(Stepien and Tumeo 2006, Dufour 2007, Brown and Stepien 2009). Additionally, the 32 putative source populations (Dufour 2007) primarily represent the mid to lower Canadian regions of the Great Lakes (Lake Huron, Lake Erie, and Lake Ontario; Dufour 2007).

Seven out often river populations (with long distance dispersers excluded) successfully assigned to their associated lake population and therefore were most likely established by contiguous dispersal from the lake. River populations that assigned to their nearby lake population included all Maitland River populations (2005 - 2009), and the 2 lower Grand River populations (GRLOW: 2008 and 2009). Group assignment scores

35 were high and ranged from 97.2% (GRLOw 2009) to 99.9 % (MR 2005, 2006, 2008). Of the remaining 3 river populations, one was assigned to a lake population within 150 km

(SR 2009: assignment score 99.9%), one was assigned to a lake population located over

150 km (shortest water distance) from the river mouth (GRUP 2008: assignment score

99.9%), and one river population failed to assign to any of the available source populations (GRup 2009). The Saugeen River population (SR 2009) successfully assigned to a Lake Huron lake population located near the mouth of the Maitland River

(89 km shortest water distance) and therefore was considered established by regional diffusion. The 2008 upper Grand River population (GRUP) also assigned to the Lake

Huron population near the mouth of the Maitland River. The shortest water distance separating these 2 populations (GRup and its putative source population in Lake Huron) is approximately 561 km suggesting the upper Grand River population (2008 only) was established by a long distance dispersal event. The most likely source population for the

2009 upper Grand River population (GRup) was also the Lake Huron population near the mouth of the Maitland River, though the assignment score was relatively low (59.6%) and was not 4 times higher than the second ranked source population (40.4%; located in the western basin of Lake Erie near Colchester, Ontario).

Genetic diversity

Mean allelic richness (A) within lake and river populations (river populations as defined by genetic structure analysis) ranged from 2.84 to 5.08 with a global mean A of

4.14 (Figure 2.4A). Expected values, as determined by the mean A (4.14) of the lake populations (n=40) bounded by the 95% confidence interval ranged from 4.00 to 4.29.

36 Mean allelic richness (A) within river populations (with migrants removed) was 4.03 and ranged from 3.25 (GRUP2009) to 4.36 (MR 2008). When migrants were included mean

A within river populations was 4.14 and ranged from 3.25 (GRup2009) to 4.51 (MR

2008). One out of 10 river populations (GRLOW 2008) had significantly higher A than the lake values when migrants were included and one river population (GRup 2009) had significantly lower A than the lake values (Figure 2.4A).

Mean expected heterozygosity (HE) in the lake and river populations ranged from

0.4.9 to 0.64 (global mean: 0.57). Mean HE for the lake populations (n = 40) was 0.57 and

95% confidence interval ranged from 0.56 to 0.59. Mean HE in river populations with migrants removed was 0.56 and ranged from 0.49 (GRUP 2008) to 0.61 (GRLOw 2008).

When long distance migrants were included in river populations mean HE was 0.57 and ranged from 0.49 (GRUP2008) to 0.63 (GRLow 2008). One out of 10 river populations

(GRLOW 2008) had significantly higher HE and one river population (GRup 2008) had significantly lower HE compared to the lake populations when migrants were included

(Figure 2.4B). Genetic diversity (A and HE) was elevated in all river populations when migrants were included compared to when migrants were removed from the analyses

(with the exception of GRup which had no long distance migrants), though the difference was not statistically significant.

Relatedness

Mean within population relatedness (r) values ranged from -0.05 to 0.33 in the lake and -0.01 to 0.30 in the river populations (with migrants removed). All river population estimates were outside the 95% confidence interval for the lake populations

37 (Figure 2.4C). Mean relatedness was significantly higher in 2 out of 10 river populations

(GRup 2008-2009) when compared to the generalized lake population. The remaining 8 out of 10 river populations (MR 2005-2009, GRLow 2008-2009, SR 2009) had significantly lower within population mean relatedness compared to lake values.

38 a)

o TO rz CO CD 5

oen >D), N O

CO sz -a £o CD Q. X LU

C. 04

(/) 03 cCD -a CD « 02 CD C co o 1 CD i__at

-0 1 03 05

03 £ £ f f £ or or or or o o

A and HE only). Mean (solid line) and 95% confidence interval (dashed lines) represents expected values as calculated from 40 baseline lake populations. Asterisk (*) indicates a significant difference between the river and generalized lake population. Site abbreviations are as indicated in Table 2.1.

39 Temporal stability

The temporal stability in spatial distribution of round goby river populations that were colonized from the lake (i.e. contiguous dispersal) was examined across 5 years

(2005 - 2009) in the Maitland River and 2 years (2008 - 2009) in the Grand River. In

2005 the uppermost river site (MR4) in the Maitland River that was colonized by round gobies from the lake (i.e. contiguous dispersal) was located 2.0 km from the river mouth at Goderich, ON (Table 2.1; Figure 2.1). By 2009 river colonization by contiguous dispersal had advanced upstream to site MR9 which is located approximately 4 km from the river mouth. Round goby colonization of the Maitland River expanded at a rate of approximately 500 m / year. No disjunct populations were identified in the Maitland

River upstream from the natural invasion front in any sampling year. The uppermost river site in the Grand River that was colonized by contiguous dispersers from the lake was located approximately 8.3 km (GR3) from the river mouth at Port Maitland, ON in

2008 (Table 2.1; Figure 2.1). Contiguous dispersers were not identified upstream from site GR3 in 2009 suggesting that the round goby population is relatively stable or moving at a rate undetectable by my sampling regime.

Exact tests of temporal change in allele frequency distribution within the Maitland

River population across subsequent years returned one significant pairwise comparison

(MR 2008 - 2009; p = 0.00, %2 = 56.77, df = 16). Pairwise comparisons of allele frequency distribution in the Grand River populations (GRLOW and GRUP) between the two sample years (2008 to 2009) were non-significant.

40 2.4 DISCUSSION

Colonization events provide unique opportunities to examine invasion dynamics associated with expanding populations. An expanding population can therefore yield valuable insight into mechanisms associated with dispersal, colonization success and hence range expansion. Genetic methods have allowed researchers to characterize mechanisms of dispersal during colonization which has led to an increase in studies reporting stratified dispersal as a mechanism facilitating secondary range expansion

(Colautti et al. 2005, Parisod and Bonvin 2008, Darling and Forino-Rorem 2009).

Additionally, modeling approaches have emphasized the importance of stratified dispersal in secondary spread (e.g. Muirhead et al. 2006). The results of my study suggest that round goby river colonization proceeds by a stratified dispersal strategy; contiguous and long-distance dispersal both operating together.

Contiguous dispersal moves individuals into an adjacent territory at a rate of spread equal to the population growth rate multiplied by a diffusion coefficient

(Shigesada et al. 1995). Genetic panmixia and non-equilibrium processes are typical in the early stages of colonization by contiguous dispersal with a stepping stone pattern of genetic structure developing over time as population expansion proceeds. I identified contiguous dispersal from the lake as the primary mechanism of upstream expansion on a local scale. A temporally unstable spatial distribution led to a constant rate of spread across years. Despite spatial instability the genetic structure of the colonizing populations were temporally stable. Genetic stability during colonization was unexpected but may be explained by expansion dominated by fish from the lake population. Range expansion by contiguous dispersal resulted in little to no genetic structure among the river

41 sampling sites in these colonizing populations providing further support that river colonization is recent and ongoing. These results suggest that there is a fundamental difference between round goby dispersal patterns reported for the Great Lakes (Brown and Stepien 2009) and dispersal on a local scale associated with river colonization.

Substantial genetic structure and genetic differentiation among Great Lakes round goby populations was reported early in their invasion of the Great Lakes (Stepien and Tumeo

2006) suggesting human activity was responsible for their rapid spread and regional distribution (see Wolfe and Marsden 1998) followed by limited gene flow post- establishment. In the current study, population expansion by contiguous dispersal suggests round gobies are undergoing secondary range expansion naturally. Dispersal from the range margin is expected to be under strong selective forces (Dyfham 2008) which can act to limit range expansion due to unsuitable habitat and physical barriers to dispersal. Rivers act as dispersal barriers for species limited to passive movement as downstream flow prevents upstream migration. The hydrodynamic flow regime of rivers can also act to limit dispersal among species employing an active dispersal strategy as sufficient swimming ability is required to hold their position and to move within high flow stochastic environments (Langerhans 2008). The recent colonization of river systems by round gobies through natural dispersal suggests that the costs associated with dispersing through such dispersal barriers have been reduced (Bonte et al 2010), although the mechanism of that reduction is not clear.

A higher migrant load was observed in river populations associated with contiguous dispersal compared to the lake population at two tributaries. Assignment of river migrants resulted in a wide range of dispersal distances, suggesting a combination

42 of short- and long-distance dispersal vectors. Peaks in the frequency distribution of migrant dispersal distance likely represent a combination of natural dispersal (shorter dispersal distance), and human activity (longer dispersal distance; Figure 2.2); however distinguishing between dispersal vectors remains speculative at this point. Darling and

Forino-Rorem (2009) proposed stratified dispersal as the mechanism responsible for the genetic structure patterns observed in the invasive hydrozoan, Cordylophora, in the Great

Lakes. It was suggested that natural current-driven dispersal was responsible for local scale colonization while long-distance dispersal was human mediated due to vessel movement through the Great Lakes. On the other hand, a combination of multiple natural dispersal mechanisms (i.e. pollen and seed dispersal) in the European wildflower,

Biscutella laevigata, has resulted in short- and long-distance dispersal into marginal populations (Parisod and Bonvin 2008). Although the current study focuses on dispersal at a local scale, river colonization by a dual dispersal strategy suggests that founder effects may be mitigated by gene flow from distinct source populations (Parisod and

Bonvin 2008).

A colonizing population is expected to have lower genetic diversity compared to its source population due to a limited number of founders (Ciosi et al. 2008, Darling and

Folino-Rorem 2009, Peacock et al. 2009, Ramakrishnan et al. 2010). Genetic diversity is expected to recover over time as additional dispersers migrate into the new population and gene flow increases (Ibrahim 1996, Brown and Stepien 2008, Parisod and Bonvin

2008, Darling and Folino-Rorem 2009). River habitats are located at the periphery of the round goby Great Lakes distribution and therefore were expected to have low genetic diversity as a result of limited gene flow due to population fragmentation, and drift

43 effects associated with small founding populations. Indeed, lower genetic diversity was observed in one river population (GRUP) however, this can be attributed to long distance founding event that established a relatively isolated upstream population. Population isolation of GRup is also supported by significantly higher relatedness compared to the generalized lake population. In the remaining river populations (those associated with contiguous dispersal) there was no reduction in genetic diversity relative to expected values from the generalized lake population. In fact, one river population (GRLOW 2008) had significantly higher genetic diversity relative to the mean lake values when long distance migrants were included in the analyses. In all cases when long distance migrants were included in the analyses genetic diversity was elevated, as expected, which resulted in 17% of river alleles only recovered from river populations (i.e. private alleles). High genetic diversity in peripheral, expanding populations has previously been attributed to stratified dispersal in which long distance dispersal facilitates gene flow from genetically distinct populations (Roman and Darling 2007, Parisod and Bonvin 2008, Brown and

Stepien 2009). In a study investigating local scale colonization dynamics, a cline in genetic diversity was observed within a core region with decreasing diversity towards the edge, as expected from multiple founding events during post-glacial re-colonization

(Parisod and Bonvin 2008). However, when marginal regions were compared to the core region, no difference in genetic diversity was observed. Genetic structure among recently established populations in peripheral regions, combined with evidence for short- and long- distance dispersal, suggests the maintenance of genetic diversity at the periphery is due to stratified dispersal and population admixture processes during expansion.

Therefore despite the potential diversity reducing effects of dispersal and colonization,

44 stratified dispersal can replenish genetic diversity in peripheral populations through direct migration into the new population, and indirectly through population coalescence following establishment. Lower relatedness in the river populations makes it unlikely that the observed variation in genetic diversity is due to family dispersal biases. Long distance dispersers make up a substantial component of river populations and thus have probably been supplementing river genetic diversity as migrants become naturalized into the population. Gene flow from long distance dispersal may provide the genetic diversity necessary to overcome bottlenecks and facilitate rapid colonization of a presumably non- ideal habitat (Kolbe et al. 2004, Stepien et al. 2005).

I identified two genetic clusters in the Grand River that coincided with the spatial distribution offish in upper and lower portions of the river. Group assignment to a source population over 500 km away indicates that the upper population (GRUP) likely originated from a separate, long distance introduction. Interestingly, migrants were not detected in GRUP in either year. The absence of recent migrants, high relatedness, and low diversity suggest that GRUP is a founding population that remains relatively isolated from the downstream river and lake populations. Geographic distance and physical barriers to dispersal (i.e. river weirs) may facilitate further isolation of GRUP but are not expected to prevent dispersal as fishways connect the lower, middle, and upper reaches of our Grand River study region. Indeed, genetic clustering results revealed that site GR3 had a mixed membership from the upper and lower Grand River populations. The location of GR3 is immediately upstream from the first potential dispersal barrier, the

Dunnville dam, a weir equipped with a Denil fishway. Admixture offish from GRLOW and GRUP at site GR3 suggest that the Dunnville dam is a permeable barrier which may

45 eventually lead to further population coalescence. Reid et al. (2008) reported low genetic divergence among populations of the black redhorse {Moxostoma duquesnei) separated by weirs in the Grand River. Unlike black redhorse, round goby dispersal capacity is expected to be low (Wolfe and Marsden 1998, Ray and Corkum 2001), yet is sufficient to allow population connectivity across this potential dispersal barrier. It should be noted that if round goby populations can penetrate rivers beyond physical barriers such as weirs, the Saugeen River population is poised at Denny's dam (SR3) for further upstream expansion.

Multiple introductions have been proposed as a mechanism to facilitate the successful establishment of introduced species (Colautti et al. 2005, Stepien et al. 2005,

Stepien and Tumeo 2006, Darling and Folino-Rorem 2009, Bjorklund and Almqvist

2010). Similarly, multiple dispersal vectors into a recipient population will increase propagule pressure during colonization as well as supplement genetic diversity through gene flow from genetically distinct source populations. In the current study, I demonstrate that a multiple dispersal strategy can facilitate colonization of new territories and can supplement genetic diversity in expanding populations. Contiguous diffusion dispersal acts to expand a species range on a local scale and can be an important mechanism of range expansion into new territory, while long-distance dispersal can increase, or at least maintain, genetic diversity in an expanding population through gene flow from distant source populations. I predict that round goby populations will continue to spread upstream in river environments possibly at an increasing pace. Physical dispersal barriers within river corridors are expected to slow upstream range expansion yet long-distance introductions as well as gradual diffusion dispersal across such barriers

46 can occur. I have established baseline information of the round goby river invasion front in three Great Lakes tributaries and identified contiguous dispersal as the predominant mechanism of upstream expansion. River colonization is recent and dominated by fish from the lake. Though complete eradication of round goby populations is not possible, a rapid response strategy incorporating population reduction near the river mouth and physical barriers to upstream dispersal may be suitable to slow the spread in select tributaries (Dimond et al. 2010).

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57 Appendix 2.1 Pairwise comparisons of Weir and Cockerham's (1984) FST among round goby, Neogobius melanostomus, sampling sites at the Maitland (2005-2009), Grand (2008-2009), and Saugeen (2009) rivers. Bold type and underlined indicates significant pairwise FST comparisons after Bonferroni correction. Site abbreviations are as in Table 2.1 and Figure 2.1.

Mailland 2005 MRI MR2 MR3 MR4

ML 0 006 0 005 -0 017 -0 002

MRI 0 009 -0 014 0 003

MR2 -0 027 0 006

MR3 -0 037

MR4

Maitland 2006 MRI MR2 MR3 MR4 MR5

MLl -0 016 -0 004 0011 0 003 0011

MRI -0 004 0011 0 003 0011

MR2 0 004 0018 0 001

MR3 0015 0011

MR4 0 042

MR5

Maitland 2007 MR2 MR3 MR4 MR5 MR6 MR7

MLl 0 009 0 004 0 009 0 107 -0 011 -0 006

MR2 0 011 0018 0 228 0 052 0 025

MR3 0 005 0 133 0 006 0 005

MR4 0 095 -0 020 0011

MRS 0017 0 072

MR6 0 002

MR7

Maitland 2008 MRI MR2 MR3 MR4 MRS MR6 MR7 MR8

MLl 0 002 0 001 0 005 0 004 -0018 0 015 0 008 0 045

MRI 0 000 0 003 -0 002 -0 023 0014 0 009 0 033

MR2 0 006 0 006 -0 006 0 021 0 007 0 078

MR! 0 003 0 006 0 006 -0 005 0 053

MR4 -0 026 0 003 -0 004 0 005

MRS 0 024 -0 015 0 004

MR6 0 001 -0019

MRI 0011

MR8

Maitland 2009 MRI MR2 MR3 MR4 MRS MR6 MR7 MR8 MR9

MLl 0 033 0 007 0 006 0 003 -0 002 0 000 0 004 0 006 0 068

MRI -0 013 0 009 0 024 -0 020 0017 -0 007 0010 0 027

MR2 0010 0017 -0 006 0011 0 006 0 009 0 031

MR3 0 003 -0 012 0013 0 00] 0 001 0 067

MR4 -0 006 0 003 0015 0 026 0 060

MR5 0 001 -0012 -0 014 0 021

MR6 0 008 -0013 0 057

58 MR7 -0 011 0 051

MR8 0 049

MR9

Grand 2008 GL1 GL2 GRI GR2 GR3 GR4 GR5 GR6 GR7 GR8

GLI 0008 -0013 0018 0 022 0.137 0019 0 088 0.123 -0 027

GL2 0 003 0 030 0 039 0.127 0 036 0 097 0.125 -0 014

GRI 0017 0015 0.105 0 027 0 081 0.112 -0 044

GR2 0 062 0 129 0 077 0 170 0.170 0 129

GR3 0 091 -0 052 0 054 0 063 0 001

GR4 -0017 -0 030 0 080 0 023

GR5 -0 121 -0 077 -0 051

GR6 -0 024 -0 165

GR7 0 082

GR8

Grand 2009 GLI GL2 GRI GR2 GR3 GR4 GR5 GR7 GR8

GLI 0011 0 002 0 009 0.153 0 105 0.161 0.246 0.092

GL2 0 008 0.028 0.105 0 067 0.091 0.184 0 049

GRI 0.015 0.092 0 036 0.109 0.197 0 058

GR2 0.124 0 072 0.152 0.224 0.114

GR3 -0 052 0 026 0 107 0 077

GR4 -0 006 0 092 0 100

GR5 0 050 0 040

GR7 0.182

GR8

Saugeen 2009 SLI SRI SR2 SR3

SL1 0011 -0 010 0 009

SRI 0 000 0O10

SR2 -0 011

SR3

59 3.0 DIFFERENCES IN DISPERSAL ABILITY BETWEEN EXPANDrNG AND

NON-EXPANDING POPULATIONS OF ROUND GOBY

{NEOGOBIUS MELANOSTOMUS): A COMPARISON OF SWIMMING

PERFORMANCE AND MORPHOLOGY

3.1 INTRODUCTION

The spatial distribution of a species reflects its ability to disperse to, and adapt to, different environments. A stable range limit suggests that population density within the species range is not high enough to trigger range expansion or the probability of survival and reproductive success beyond the range margin is drastically reduced (Thomas et al.

1986). When a species is expanding its range and colonizing a novel environment it is expected that a fundamental shift in its external environment (e.g. climate; Thomas et al.

2001) or an intrinsic change (e.g. adaptation; Lavergne and Molofsky 2007) has occurred

In contemporary colonization events, a lag time from establishment to spread is often observed (Johnson and Carlton 1996, Crooks 2005, Goudswaard et al. 2008, Lombaert et al. 2010) which has been suggested to be due to population dynamics limitations (e.g. exponential population growth), or the time required for an adaptive response to the new environment (Reznick and Ghalambor 2001, Lavergne and Molofsky 2007, Lombaert et al. 2010). Adaptive modifications not only allow a species to persist in a novel environment but, when coupled with colonization, can provide a mechanism for colonization through what was once a barrier to dispersal (Urban et al. 2007). Therefore, a species distribution reflects a combination of its dispersal capacity as well as the ecological conditions it is adapted to within a heterogeneous landscape.

60 Dispersal and traits associated with dispersal are usually strongly correlated with colonization success and have been shown to have undergone rapid evolutionary change in many contemporary species invasions (Cwynar and MacDonald 1987, Simmons and

Thomas 2004, Phillips et al. 2006, Alford et al. 2009). Habitat suitability and availability across a species' range generally follows a gradient from range core to range margin.

Typically the range core is a densely populated region with high connectivity and optimal habitat. Habitat towards the range margin becomes suboptimal and patchily distributed with lower density populations (Brown 1984, Lawton 1993). Strong selection on dispersal is expected at the range margin where there is an increased risk of failing to locate suitable habitat and resources (Hughes et al. 2007, Dytham 2008). During range expansion, suitable habitat beyond the range margin may be available and therefore dispersal can be favoured due to benefits associated with accessing habitats with lower intra-specific competition (Cwynar and MacDonald 1987). It has been shown that expanding range margins and newly established populations can be dominated by a dispersal phenotype (Thomas et al. 2001, Simmons and Thomas 2004, Phillips et al.

2006, Duckworth and Badyaev 2007, Hughes et al. 2007). For example, the coupling of aggression and propensity to disperse in the western bluebird {Sialia mexicana) has led to a cline where time since colonization was negatively related to aggressive behaviour

(Duckworth and Badyaev 2007). As range expansion continues, previously established populations may become less dispersive, often due to an energetic trade-off between fecundity and costly dispersal-associated traits (Zera and Denno 1997, Simmons and

Thomas 2004, Hughes et al. 2007). Variation in dispersal can also occur at the individual level where the invasion front is dominated by individuals with greater dispersal capacity

61 (Simmons and Thomas 2004, Phillips et al. 2006, Alford et al. 2009). Variation in dispersal associated traits can lead to an evolved shift in dispersal ability at the range margin as invasion front individuals mate, and produce dispersive offspring with high dispersal capacity, leading to an accelerating rate of colonization (Alford et al. 2009).

Spatial sorting of dispersal ability during colonization has been shown in plants (Cwynar and MacDonald 1987), insects (Thomas et al. 2001, Hughes et al. 2007), birds

(Duckworth and Badyaev 2007), and anurans (Phillips et al. 2006) but few studies have investigated non-random dispersal facilitating colonization in aquatic environments (but see Bonn et al. 2004). As dispersal and colonization of aquatic environments is greatly affected by abiotic conditions (e.g. water flow; Moyle and Light 1996), many aquatic organisms have developed modifications in their morphology, physiology and behavior to maximize efficiency in response to their hydrodynamic environment (McLaughlin and

Grant 1994, Sagnes et al. 1997, Langerhans 2008, Leavy and Bonner 2009). Therefore when a species is dispersing into a novel aquatic environment (i.e. low to high flow) it is expected that the colonizing population will have better swimming ability and morphological adaptations that facilitate a successful introduction.

In recent decades human-mediated dispersal vectors have contributed greatly to the number of successful aquatic introductions that have resulted in the establishment of highly invasive populations (Mills et al. 1993). Despite the importance of identifying dispersal pathways and vectors facilitating the introduction of non-indigenous species, the mechanisms facilitating range expansion in the introduced region also contribute to successful invasions. Natural dispersal in the introduced range can contribute substantially to invasiveness and secondary spread into areas that do not normally receive

62 anthropogenic introductions (Schaeffer et al. 2005). Round goby, Neogobius melanostomus, were first detected in the Laurentian Great Lakes in the St. Clair River in

1990 (Jude et al. 1992) and rapidly spread throughout all five Great Lakes within five years of detection establishing extremely high population densities (Johnson et al. 2005).

Ballast water transfer has been cited as the most likely vector responsible for the introduction of round gobies to the Great Lakes as well as their regional spread post- introduction (Jude et al. 1992, Hensler and Jude 1997, Brown and Stepien 2009).

Successful establishment of round goby populations throughout the Great Lakes is attributed to their superior competitive ability (Dubs and Corkum 1996, Janssen and Jude

2001), high reproductive capacity (Maclnnis and Corkum 2000), and broad environmental tolerance. During their Great Lakes invasion, round goby have established populations outside of their preferred shallow rocky habitat (Lapointe and Corkum 2007) and can occupy mud, silt, sand, and gravel habitats in water depths up to 73 m (Ray and

Corkum 2001, Schaeffer et al. 2005, Young et al. 2010). Currently, round goby populations are undergoing secondary range expansion from low flow lake environments into higher flow river tributaries in North America, as well as in introduced regions in

Europe (Simonovic et al 2001, Phillips et al. 2003, Wiesner 2005, Carmen et al. 2006,

Pennuto et al. 2010, Poos et al. 2010). Upstream river invasion in the Great Lakes tributaries is occurring by a stratified dispersal strategy in which natural dispersal is the primary mechanism facilitating upstream expansion (Chapter 2). Paradoxically, the natural dispersal ability of round gobies is expected to be greatly limited. Round goby have a benthic morphology, which suggests poor sustained swimming ability (Fisher et al. 2005), and lack a swim bladder for buoyancy. Adult round goby have been shown to

63 be philopatric within the breeding season, displaying small home ranges (5 ± 1.2 m2 Ray and Corkum 2001; Wolfe and Marsden 1998, but see Cookingham and Ruetz 2008).

Precocious young round goby have a benthic morphology at hatching and lack a pelagic larval phase. Early juvenile round goby vertically migrate into the nocturnal pelagic zone to forage which could facilitate passive dispersal within the lakes and downstream in rivers (Hensler and Jude 1997, Hayden and Miner 2009). However, the recent upstream colonization of river systems suggests that active, directed dispersal is being employed.

Recent dispersal and colonization of uni-directional, high flow environments suggests that natural dispersal ability has changed in the invaded regions, or there is a dispersive life stage undetected in previous studies (but see Ray and Corkum 2001; Schaeffer et al.

2005). Range expansion against water flow suggests that dispersal ability, in this case swimming ability, should be a limiting factor in the successful upstream dispersal into high flow stochastic environments. By examining the dispersal ability (swimming performance and morphology) of round gobies at the river invasion front I can test the hypothesis that the river colonizing round goby are adapted for upstream range expansion through a dispersal strategy different from that of the lake-dwelling fish.

In the current study I perform a comparison of round goby from expanding river populations to those from the lake population in close proximity to the river mouth to test for a dispersal phenotype associated with natural upstream dispersal. More specifically, I test for differences in swimming performance and morphological traits functionally related to swimming ability in other fishes, between recently colonized river populations and downstream lake populations. The ecological and economic impact of an invasive species will increase with its spread and distribution in the new region. A dispersal

64 phenotype associated with upstream river colonization threatens successful containment of round goby from expanding into connecting waterways and inland lakes.

3.2 MATERIALS AND METHODS

Sample collection

I followed a paired comparison design in which round goby collected from each river were compared to round goby captured from the lake population in close proximity to the river mouth. By comparing fish from adjacent locations in the same watershed I targeted populations expanding by contiguous dispersal (as identified in Chapter 2) and minimized abiotic and ecological differences that may have an impact on the phenotype offish associated with different watersheds. As part of the larger sampling regime

(Chapter 2), I collected round goby from May to October in 2009 from 3 Great Lakes tributaries; the Maitland River that drains into Lake Huron at Goderich, Ontario, the

Grand River that drains into Lake Erie at Port Maitland, Ontario, and the Saugeen River that drains into Lake Huron at Southampton, Ontario. Briefly, round gobies were collected from the lake, the river mouth, and at upstream intervals until the invasion front was reached (refer to Chapter 2 for details). Fish (n = 192) from predetermined sites

(lake, down-, mid-, and up- river sites) were kept alive in site specific containers which were filled and replenished with fresh river water every 2 days. Fish were kept in natural light conditions and fed TetraMin flake food to excess daily until transported back to the lab (Department of Biological Sciences, University of Windsor, Windsor, ON) for swim performance trials. In the lab, round gobies were kept in aerated aquariums, kept on a

14:10 hour light cycle and remained on the same feeding schedule as in the field.

65 Aquariums were filled with de-chlorinated tap water treated with Prime (SeaChem Inc.), with water temperatures ranging from 18-23°C All other round gobies (n = 381) captured were euthanized in the field with an overdose of clove oil (25ppm), and photographed from the dorsal and left lateral side using a Nikon Coolpix 4500 digital camera. Pelvic, caudal, and left pectoral fins (right pectoral fin was used when the left fin was damaged) were removed and expanded on a piece of paper, and photographed. Sex was determined by visual inspection of the external genital papillae. All digital photographs included an individual fish ID and a ruler for measurement calibration.

Swimming performance

Swimming performance trials were conducted in a modified 140L Brett-type swim tunnel (Brett 1964).The swim tunnel is a 1.0 m long transparent square tube with an internal dimension of 10 x 10 cm. Transparent box-shape holding chambers (25 x 25 x

25 cm) were connected to each end of the tunnel with a transition slope from tunnel to chambers of greater than 25cm horizontal distance. A return pathway for water recirculation connected both holding chambers. Water flow was propelled by a 14cm diameter ducted propeller (Bombardier, Montreal, QE) and flow speed was controlled with a 2.24 kW digitally controlled electric motor (Marathon motor with a Lenze

SMVector controller; Moncur Electric, Windsor, ON). The swim tunnel was isolated from the holding chambers at each end by a removable stainless steel wire gate located at each junction between tunnel and transition slopes. The downstream gate was equipped with connectors to which a slight electrical current could be applied to promote upstream swimming when contacted. Black garbage bags were hung from the ceiling surrounding

66 the flume to block direct fluorescent lighting from the swim tunnel test area and to block the view of the researcher from the fish. Two video cameras (EverSecure; Mateo, QE) were positioned directly below the swim tunnel to record the trials and allow live viewing of the fish during each trial.

Individual fish were starved for 24 hours prior to the initiation of the first swim trial to ensure a post-absorptive state (Beamish 1978). A minimum of 1 hour acclimation period (minimum of 30 minutes in a temporary holding tank filled with water from the flume and a minimum of 30 minutes in the swim tunnel with no flow) was provided prior to each trial. Three performance tests were conducted in 2 different swim trial periods.

The first swim trial was an incremental velocity test (critical station holding test, UCRIT),

to determine critical station holding endurance. The UCRIT test (Brett 1964) predominantly measures aerobically fuelled activity (but see Nelson et al. 1994). The

UCRIT test is a widely used and accepted method to measure steady swimming (cruising) speed and station holding endurance of fishes (Taylor and Foote 1991, Plaut 2001,

Nelson et al. 2002, Fisher et al. 2005, Peake 2008). Round gobies respond to increased water flow by station holding to the substrate (Hoover et al. 2003) and switch gait to a burst and coast style swimming at faster water flow. I used the UCRIT test to measure station holding endurance against increasing water flow. Following the acclimation period, the trial began with water flow initiated at 20cm-s"' (the minimum flow velocity attainable for the flume) for 10 minutes and increased by 10 cm-s"1 (Un) every 10 minutes

(600 seconds: Tn) until the fish was exhausted. Exhaustion was determined by a lack of response to the downstream electrical grid after which water flow was terminated and the

67 fish was removed and placed in an individual tank for a 24 hour recovery period. Critical station holding speed (UCRIT) was calculated as:

1 1 UCRIT (cm-s" ) = U, (cm-s"')+ (T, (s) / T„ (s) x U„ (cm-s" )

where Ui is the penultimate speed (cm-s"1) before exhaustion in which the fish swam or remained in station holding position for the full time interval (Tu: 600 s), and Ti (s) is the time at fatigue in the final velocity interval. Velocities did not need to be corrected for the solid blocking effect as fish occupied less than 10% of the cross-sectional area of the swim tunnel (see Plaut 2001 and references therein).

Next, I measured gait transition speed (UGAIT) as a complimentary measure of aerobic capacity (Drucker 1996, Peake 2008). Gait transition speed has been shown to reflect UCRIT speed but with less unexplained variance which is expected to be due to behaviour and motivation in the latter (Peake 2008). By measuring UGAIT, there is greater control over variance due to differences in relative activity level from body size and habitat and therefore is a physiologically equivalent measure of performance for comparison (Drucker 1996). In round gobies, the initial gait (at low flow velocity) is predominantly station holding to the substrate followed by a transition to burst and coast swimming at a higher flow velocity for the second gait. In this sense, the threshold velocity at the gait transition is essentially a "lift-off speed and represents the speed in which a switch from aerobic station-holding endurance to anaerobic unsteady swimming occurs. In the current study, gait transition speed was measured during the UCRIT test

(Peake 2008). Briefly, time was recorded when a fish lifted-off from its station holding

68 position within each time interval. The UGAIT value represents the first speed in which over 50% of the time interval was spent burst and coast swimming. My method of measuring gait transition is slightly modified from the gait transition speed (UsTmax) in other studies which report the speed of the last time interval used in full for steady swimming (Peake 2008, Tudorache et al. 2010). In contrast, I chose to use the speed of the first time interval in which a gait transition occurred (for a minimum of 50% of the time interval; Webb 2001) to have a direct measurement of flow velocity that initiates the gait change. Fish were given a 24 hour recovery period in individually labelled tanks prior to the initiation of swim trial 2 (UBURST; described below).

The second swim trial experiment was a maximum acceleration test, the UBURST test, to determine the maximum flow velocity that each fish can move against. The

UBURST test measures anaerobically fuelled activity and is a complimentary measure of locomotor performance to the UCRIT test as it targets unsteady locomotion, otherwise known as "burst and coast" swimming (Beamish 1978, Reidy et al. 2000). Burst and coast swimming is a gait used by fishes with a body-caudal fin style of swimming when experiencing high flow velocities (Pon et al. 2007) and is typical of round goby locomotion (personal observation). Following the acclimation period, water flow was initiated at the specific UCRIT speed for each fish for 30 seconds and increased by 10 cm-s"1 every 30 seconds until the fish was exhausted (i.e. no response to the downstream electrical grid). Burst and coast swimming occurred within the first time interval in all trials and was the predominant mode of swimming throughout the trial. The water velocity at the penultimate interval (i.e. the last time interval where the fish swam for the complete interval) was considered the UBURST speed for each fish and represents the

69 maximum flow velocity in which the fish could swim against. After the trials, the fish were humanely euthanized with an overdose of clove oil (25ppm) and digital photographs were taken as described above for morphological analyses.

All measures of swimming performance were tested for repeatability with a subset offish from the 3 watersheds (UCRIT, UGAIT n=l 1; UBURST n=9). Repeated trials were performed 28-32 days after the initial set of trials. The average of the 2 trials for each fish was used for analyses. Following repeatability trials, each round goby was euthanized and photographed (as described above) for morphological analyses.

Morphology

Swimming capacity in fishes is often correlated to their body morphology and therefore variation in specific morphological traits can affect the activity level and hydrodynamic stress which an organism experiences (Webb 1984, Taylor and Foote

1991, Sagnes et al. 1997, Boily and Magnan 2002, Blake 2004, Fisher et al. 2005,

Hanson et al. 2007). I selected eight morphological traits known to influence swimming performance and habitat use in other fishes, through thrust, drag, and station holding effects (Sambilay 1990, Blake 2004, Fisher and Hogan 2007; Figure 3.1, Table 3.1).

Swimming endurance is enhanced by minimizing drag forces with a streamlined body shape (Beamish 1978, Webb 1984, Boily and Magnan 2002) that has a high fineness ratio

(FR = standard length (SL) / maximum body depth (BDMAX)), small fin size, and a high caudal aspect ratio (AR = caudal fin height squared (CH) / caudal fin area(CA); Sambilay

1990, Boily and Magnan 2002). Thrust production is potentially affected by body size,

fin area, and caudal peduncle depth factor (CP = minimum peduncle depth (PDMIN) /

70 maximum body depth (BDMAX); Blake 2004). Round gobies respond to increased flow by clinging to the substrate with their fused pelvic fin - a defining morphological trait of the Gobiidae family (Corkum 2010) and therefore I included pelvic fin area (PL) as an adaptation for enhanced clinging. The fused pelvic fin acts as a suction-like appendage by some goby species to cling to the substrate and ascend into upstream environments

(Blob et al. 2006). It is expected that round gobies with a larger pelvic fin area will have better ability to remain attached to the substrate in high flow conditions and therefore I expect pelvic fin area to be larger in upstream dispersers (Table 3.1). Station holding ability can be enhanced by larger pectoral fins that are able to press against the substrate maintaining a downward force from the water flow (Webb 1994, Adams et al.

1999).Additionally, I measured flatness index (FI) as an adaptive trait for lotic organisms where flatness index (FI) = maximum body width (BWMAX) / maximum body depth

(BDMAX) (Pon et al. 2007). Flatness index is a measure of lateral compression and can be lower in fishes (lower body width to depth ratio) in high flow tributaries as greater body depth can enhance manoeuvrability(Webb 1984, Pon et al. 2007). On the other hand, flatness index is also a measure of dorso-ventral flattening and is expected to be greater in organisms associated with the benthos especially those that cling to the substrate in fast, uni-directional flow environments (Eastman and DeVries 1985, Webb 1994). I expect traits that maximize thrust, endurance, and/or station holding ability will be greater in riverine round goby compared to the lake population as an adaptive response to high flow

(Langerhans 2008: Table 3.1). Morphological traits were measured to the nearest 0.01 mm from enlarged digital photographs of the dorsal and left lateral view (Figure 3.1), and

71 3 fully extended fins (pelvic, pectoral, and caudal) using Northern Eclipse software

(Empix Imaging, Inc., Mississauga, ON).

Figure 3.1 Morphological variables measured from digital photographs of round gobies {Neogobius melanostomus) collected from lake and river sites located at 3 Great Lakes tributaries in 2009. Maximum body depth (BDMAX; deepest body region from lateral view), maximum body width (BWMAX' widest body region from dorsal view; not shown), caudal fin area (CA; caudal fin fully extended), caudal fin height (CH; dorsal to ventral tip of caudal fin when fully extended), pectoral fin area (PC; left (right was used if left fin damaged) pectoral fin fully extended), minimum peduncle depth (PDMrN: narrowest vertical depth of caudal peduncle), and pelvic fin area (PL; pelvic fin fully extended) All measurements were made to the nearest 0.01mm.

72 Table 3.1 Expected differences in morphology between expanding river (R) and non-expanding lake populations (L) of round goby, Neogobius melanostomus, in the Great Lakes region. Round goby have recently initiated upstream colonization of river systems and it is expected that an adaptive response in dispersal ability to high flow is facilitating successful upstream expansion. Eight morphological traits (plus standard length) known to be functionally related to swimming performance, station holding ability, and habitat use in other fishes are used for population level morphological comparisons, and as predictors of swimming performance (UCRIT, UGAIT, UBURST)- Trait - performance combinations not expected to have a functional relationship are indicated by a dashed line.

TRAIT THRUST ENDURANCE STATION HOLDING

AR = caudal fin height squared (CH) /caudal fin area CA R > L CA= caudal fin area R > L CP2 = min. peduncle depth PDMIN / max. body depth BDMAX R > L FI3 = max body width BWMAX / max. body depth BDMAX R = 1 R>L FR4 = standard length SL / max. body depth BDMAX R > L PC = pectoral fin area R > L R < L R>L PL = pelvic fin area R>L rWT = residual body weight R > L R < L ' AR caudal aspect ratio 2 CP caudal peduncle depth factor 1 FI flatness index 4FR fineness ratio

3.3 STATISTICAL ANALYSIS

Round gobies that were identified as long distance migrants using genotype assignment methods (Chapter 2) were removed from the performance analyses as their dispersal may have been human-mediated and therefore may not represent natural dispersal activity. Parametric assumptions of normality and equal variance were tested with a Shapiro-Wilk's test and Levene's test, respectively. All analyses were conducted in SPSS (IBM Corporation, Somers, NY) and results were assessed for significance at p = 0.05. When multiple comparisons were performed, significance was adjusted by

Bonferroni correction to account for an inflated repeated comparison error rate.

73 Swim performance

Pearson's correlation was performed to test for a correlation between swimming

performance and standard length. Gait transition speed was negatively correlated to

standard length (r = - 0.33, p < 0.05) therefore residuals from the regression of absolute

speeds (UCRIT, UGAIT, UBURST) on standard length were used for analyses to remove the

effect of body length on swimming. I performed two sample t-tests on residual swim

speeds among males and females for each of the 3 swim performance measures (rUcRiT,

IUGAIT, TUBURST)- There was no significant difference among males and females in

performance speeds and therefore data were pooled for subsequent analysis. I compared

residual swimming performance values (rUcRiT, TUGAIT, IUBURST) among lake and river

populations at each of the 3 tributaries (Maitland, Grand, Saugeen) by conducting two

sample t-tests with Bonferroni correction to account for multiple comparisons. Pearson's

correlation was used to determine the strength of the relationship among pairwise residual

performance measures (i.e. TUCRIT - rUGAiT, rUCRiT - IUBURST, IUGAIT - TUBURST).

Repeatability in each performance measure was examined using Pearson's correlation

coefficient between trial 1 and trial 2 of a subset offish (UCRIT, UGAIT: n = 11, UBURST:

n = 9; Hanson et al. 2007).

Morphology

Morphology was examined on swim trial fish (n = 58) plus an additional 40 fish

(Maitland: n=15, Grand: n=6, Saugeen: n=19) to increase sample size and include river

sites under-represented in swim trials. Two sample t-tests were used to test if the added fish differed in standard length and residual body weight (rWT: residual from regression

74 of body weight on standard length to correct for body length differences) from the swim trial fish of the same population. The additional fish did not differ in standard length from the swim trial fish (Maitland lake: t = 2.26, df = 8, p > 0.05; Maitland river: t =

0.07, df = 26, p > 0.05; Grand lake: t = 0.27, df = 8, p > 0.05; Grand river: t = 0.10, df =

8, p > 0.05; Saugeen lake: t = 1.80, df = 8, p > 0.05, Saugeen river: t = 1.10, df = 28, p >

0.05). Residual body weight (rWT) was significantly lower in the river fish in the Grand

River used in swim trials (n = 7: mean = -0.68 ± 0.34) compared to the additional river fish (n = 3: mean = 1.60 ± 0.29) used in morphological analyses (t = 4.04, df = 8, p <

0.01).

Linear regression of morphological traits on standard length was used to quantify the effect of standard length on morphology. Flatness index (FI), fineness ratio (FR), and fin area (PC, PL, CA) were significantly correlated with standard length (p < 0.01).

Residuals from the regression of each morphological trait on standard length were used for subsequent analyses to remove any effect of body length on morphology. Residual morphometric ratios (rAR, rCP, rFI, rFR), residual body weight (rWT), and residual fin area (rCA, rPC, rPL) were tested between males and females using two-sample t-tests.

No difference was observed between sexes and data was pooled for subsequent analyses.

Two-sample t-tests were used to test for differences in standard length (SL) and residual body weight (rWT) between lake and river fish at each tributary. I tested for body shape differences among lake and river populations at each tributary using 3 independent

MANOVA's to test the following 3 hypotheses:

75 1) River dispersers have morphological traits associated with enhanced swimming endurance compared to the lake population (i.e. rAR, rFI, rFR, rPC, rWT: Table 3.1).

2) Residual traits associated with enhanced thrust production (i.e. rCA, rCP, rPC, rWT) will be greater in river dispersers compared to the lake population (Table. 3.1).

3) Residual traits expected to increase station holding ability (i.e. rFI, rPC, rPL) will be greater in river dispersers compared to the lake population (Table 3.1).

Univariate ANOVA's were performed on all dependent variables to identify which variables differentiate lake and river populations at each tributary.

Swimming and morphology

To examine the functional relationship between swimming performance and morphology, I used a stepwise multiple linear regression with swimming values

(dependent variable) and morphological traits (including standard length) as independent variables. The stepwise approach was used as no a priori assumptions of the relative importance of each variable on swimming performance were made. Swimming performance and morphological variables were grouped according to the expected functional association between trait and performance (i.e. endurance, thrust, station holding; Table 3.1). Forward and backward multiple regressions were performed to verify stepwise results.

76 3.4 RESULTS

A total of 573 round gobies were collected from lake and river sites at 3 tributaries. Samples collected from the lake and river populations were male biased at each tributary (Maitland lake: male n = 60, female n = 4, river: male n = 103, female n = 28; Grand lake: male n = 62. female n = 14, river: male n = 83, female n = 19;

Saugeen lake: male n = 17, female n = 3, river: male n = 31, female n = 5) and sex was undetermined for 144 river fish (Maitland river: n = 59; Saugeen river: n = 85).

Reproductive males and females were collected from all lake and river populations, with reproductive status assessed based on a gonadosomatic index of 1% for males and 8% for females (Marentette and Corkum 2008). Eighteen fish (Grand: n = 16, Saugeen: n = 2) were removed from the swimming and morphological comparisons as they were previously identified as long distance dispersers through genetic analyses (Chapter 2) and may not represent natural dispersal events.

Swimming performance

Fifty eight round gobies (not including long distance dispersers) representing lake and river populations at 3 tributaries (Maitland: n = 23, Grand: n = 14, Saugeen: n = 21:

Table 3.2) were tested for swim performance. Three fish died (Maitland: n = 2, Saugeen: n = 1) following the first swim trial (incremental velocity trial; UCRIT, UGAIT) and therefore 55 fish were measured for performance in the second trial (constant acceleration test; UBURST).

77 Table 3.2 Sample sizes of round goby, Neogobius melanostomus, from lake and river populations at 3 Great Lakes tributaries used in swimming performance trials and morphometric comparisons

Region Site Swimming Morphometric

Maitland lake 1 "To

river 16 28

Grand lake 7 10

river 7 10

Saugeen lake 7 10

river 14 30

TOTAL 58 98

78 Critical station holding speeds (UCRIT) were highly variable. Mean absolute UCRIT

for lake fish was 104.6± 3.2 cm-s"'and river fish was 106.4 ± 4.8 cm-s"1 (Appendix 3.3).

There were no differences in mean residual UCRIT between lake and river populations at

1 any tributary (Figure 3.2). Mean gait transition speed (UGAIT) was 101.2 ± 4.5 cm-s" in

lake fish andl03.7± 5.3 cms" in river fish (Appendix 3.3). No differences in mean

residual UGAIT were observed between lake and river populations at any tributary (Figure

1 3.2). Mean maximum burst swimming speed (UBURST) was 147.1 ± 6.0 cm-s" in lake fish

and 143.2± 4.0 cm-s"1 in river fish (Appendix 3.3). No significant differences in mean

residual maximum burst speed were observed between lake and river populations at any

of the three tributaries (Figure 3.2). As expected, UBURST was significantly greater than

UCRIT and UGAIT in all populations (p < 0.0001). There was a significant and strong

= positive correlation between TUCRIT and TUGAIT (r 0.860, p < 0.0001) indicating these

reflect similar measures of endurance. Finally, there was a significant positive

correlation between TUCRIT and rUsuRSTO" = 0.472, p < 0.0001), and TUGAIT and TUBURST

(r = 0.332, p < 0.05) indicating that there is no trade-off between speed and endurance in

round gobies. Pearson's correlation coefficient for repeated trials indicated that UCRIT results were highly repeatable after approximately 1 month (r = 0.791, p < 0.01) which

suggests that a hierarchal rank among individual swimming performance likely reflects true variation in their natural environment, though longer time periods between trials may produce different results (Oufiero and Garland 2009). There was no significant correlation in UGAIT values (r = -0.195, p > 0.05) or UBURST values (r = 0.471, p > 0.05) between trial 1 and trial 2.

79 20 0

100

00 T -100 T

-20 0 | Lake 20 0 • River 100 < o 00 c P3 o M= -100

-20 0

20 0

100 CD X 00 mr 100

-20 0 Maitland Grand Saugeen

Figure 3.2 Comparison of mean (± SE) residual swim performance values as measured by critical station holding speed (UCRJT), gait transition speed (UGAIT), and maximum burst swimming speed (UBURST), of round gobies, Neogobius melanostomus, from lake (dark bars) and river (light bars) populations at 3 Great Lakes tributaries (Maitland, Grand, Saugeen). All population comparisons at each tributary were non significant (p > 0.05).

80 Morphology

Lake fish had greater mean standard length (SL) than river fish at 2 out of 3 tributaries (Maitland: t= 7.84, df=36, p < 0.0001; Saugeen: t = 10.17, df =38; Figure 3.3,

Appendix 3.1). Residual body weight was similar between lake and river fish at all tributaries. Multivariate ANOVA (MANOVA) results indicate that the lake and river population at the Maitland differed in overall body shape with respect to thrust production (Table 3.3). Univariate ANOVA's for the Maitland yielded one out of 4 significant comparisons between lake and river for variation in thrust producing variables. A larger caudal fin area was expected in river fish to enhance thrust production while burst and coast swimming. Maitland river fish had a significantly smaller caudal fin area compared to the lake fish (F = 6.13, df = 1, p < 0.05; Figure 3.3). Multivariate

ANOVA including morphological variables expected to influence station holding ability were non-significant between lake and river populations across all tributaries (Table 3.3).

However, univariate ANOVA results indicated that pelvic fin area differed among lake and river populations at the Grand River (F = 5.80, df = 1, p < 0.05; Figure 3.3). A larger pelvic fin area was expected to enhance station holding to the substrate in high flow environments. Despite expectations, the river fish at the Grand River had a significantly smaller pelvic fin area compared to the lake population (F = 6.13, df = 1, p < 0.05). The discord between MANOVA and ANOVA results is most likely due to small and disparate sample sizes (Willig et al. 1986).

81 Table 3.3 MANOVA results for body shape comparison between lake and river populations of round goby, Neogobius melanostomus, at 3 Great Lakes tributaries. Traits were chosen from the literature based on the potential for a functional relationship with swimming ability in different hydrodynamic regimes. Residual traits were grouped according to expected functional relationship with 1) swimming endurance, 2) station holding to the substrate, and 3) thrust production. Bold type and underlined indicates significance after Bonferroni correction.

Hypothesis Traits Region df Sig

1) Endurance traits rAR, rFI, rFR, rPC, rWT Maitland 34 0215 5,28 0 953

Grand 19 0 624 5, 13 0 684

Saugeen 36 0 735 5,30 0 603

2) Station holding traits rFI, rPC, rPL Maitland 37 1 349 3,33 0 275

Grand 19 1 847 3, 15 0 182

Saugeen 36 0 402 3,32 0 752

3) Thrust producing traits rCA, rCP, rPL, rWT Maitland 34 4 572 4,29 0.006

Grand 19 0 899 4, 14 0 491

Saugeen 36 0 779 4,31 0 547

Hotelling's Trace

82 X T—T^ f

_L_ -^^L

.r1. T, T I "1" 1 - T

Y^r Maitland Grand Saugeen

r * *

Maitland Grand Saugeen

Figure 3.3 Comparison of mean (± SE) residual morphometric ratios (rAR: residual caudal aspect ratio, rCP: residual caudal peduncle depth factor, rFI: residual flatness index, rFR: residual fineness ratio), residual fin area (rCA: residual caudal fin area, rPC: residual pectoral fin area, rPL: residual pelvic fin area), and body size variables (SL: standard length (mm), rWT: residual body weight) between lake (dark bars) and river (light bars) populations of round goby {Neogobius melanostomus) collected at 3 Great Lakes tributaries. Morphological traits were chosen based on their functional relationship with swimming ability in other fishes. Asterisk (*) denotes significance according to univariate ANOVA results comparing estimated marginal means of residual traits between lake and river population.

83 Swimming and morphology

Stepwise multiple regressions were performed for each region (Maitland, Grand,

Saugeen) and with all regions pooled. Individual regions were used to account for ecological differences that may impact the phenotype of fish associated with different watersheds. One out of 5 residual morphological variables was included in the best fit multiple regression model explaining inter-individual variation in maximum burst swimming speed (UBURST)- Residual caudal peduncle depth factor (rCP) was positively correlated to UBURST and explained 38% of the variation in UBURST when regional data were pooled. When regions were analyzed independently, rCP was included in the best fit model for the Maitland River and explained 19% of the variation in UBURST

(Table 3.4). One residual morphological trait was included in the best fit multiple regression model explaining inter-individual variation in critical station holding endurance (UCRIT)- Residual pectoral fin area (rPC) was positively correlated to UCRIT at the Grand River and explained 31% of the variation in UCRIT when regions were analyzed separately (Table 3.4). When regional data were pooled, all residual morphological variables were excluded from the best fit model. No residual morphological traits were included in the best fit model explaining variation in UGAIT when regional data were held independent or pooled.

84 Table 3.4 Significant stepwise multiple regression results testing the effect of residual morphological traits (including standard length, SL) on swimming performance of round goby {Neogobius melanostomus) collected from lake and river populations at 3 Great Lakes tributaries. Morphological traits were grouped according to an expected association with 3 swimming performance measures (swimming endurance,

UCRIT; station holding ability, UGAIT; maximum thrust potential, UBURST)- Regional data (Maitland, Grand, Saugeen) were tested independently and pooled (All).

DEPENDENT VARIABLE MODEL REGION R R2 Adj. R2 F df P

Endurance (UCRIT)1 rPC Grand 0 557 0310 0 247 4 940 1 0 048

Station holding ability (UGAIT)2

Maximum burst speed (thrust, UBURST)1 rCP All 0 440 0 193 0 175 10 551 1 0002

Maitland 0 621 0 385 0 347 10 027 1 0 006

1 excluded variables rAR, rCF, rFI, rFR, SL 2 excluded variables rFI, rPC, rPL, SL 1 excluded variables rCA, rCF, rPC, SL

3.5 DISCUSSION

The outcome of dispersal into a new aquatic environment depends on the ability of the organism to persist in a novel hydrodynamic regime (Moyle and Light 1996). The upstream invasion by round gobies from the Great Lakes is a recent and ongoing colonization event (Chapter 2) following a typical time lag from establishment to spread similar to other successful invasions (Crooks 2005). Round goby were detected at industrial harbours and piers located at the mouths of Great Lakes tributaries early in their invasion. However, despite high population densities resulting from rapid population growth round goby remained absent, or at least undetected, in adjacent tributaries with the exception of some larger rivers (e.g. Detroit River, Vistula River;

Corkum et al. 2004). Detection of round gobies in new areas throughout the Great Lakes region recently included many rivers (Poos et al. 2010) prompting concern surrounding the dispersal mechanism(s) facilitating secondary spread, which was identified to be

85 dominated by natural contiguous dispersal (Chapter 2).Upstream invasion by natural dispersal provides a unique opportunity to study dispersal and invasion dynamics into a novel aquatic environment as a product of secondary spread.

I reject my initial hypothesis that maximum swimming speed and station holding endurance is greater for river fish. First, river fish were not better swimmers than the lake fish. Maximum speed and endurance were similar across lake and river populations at each tributary which indicates that the uni-directional and higher flow of rivers is not strong enough to act as a dispersal barrier and drive selection on swimming performance during river colonization by natural dispersal. High inter-individual variation in swimming performance within each population indicates that strong directional selection for maximum swimming ability or spatial sorting by dispersal capacity is not occurring.

Secondly, river fish were not specialized for one swimming mode (i.e. speed versus endurance) as has been observed in other fishes (Webb 1984, Reidy et al. 2000,

Ojanguren and Brana 2003). The energetic cost of station holding in higher flow is lower for fishes that use the substrate for station holding rather than swimming. However, it is expected that the energetic cost of station holding in rivers depletes energy reserves that can be used towards maximizing unsteady (i.e. burst and coast) swimming (Langerhans

2008). Given equivalent energy stores it can be expected that lake fish would utilize only a portion of their energy reserves towards endurance leaving more energy resources for specialization on manoeuvrability and fast-start burst responses if a trade-off was occurring (Langerhans 2008). The positive relationship between endurance (UCRIT,

UQAIT) and speed (UBURST) suggests that a trade-off is not occurring in round goby which is most likely due to a decoupling of traits used in each gait. Finally, there was no

86 difference in 'lift-off speed (gait transition speed: UGAIT) between lake and river populations indicating that motivation to swim or behavioural differences in response to higher flow did not affect propensity to swim. Gait transition speed may also be affected by morphology especially in benthic fishes. A higher flatness index (the ratio of body width to body depth) has the potential to increase the lift-drag ratio which would facilitate lower (and therefore earlier) lift-off speeds (Webb 1994, Webb 1996). Since flatness index and gait transition speed did not differ between lake and river populations, it is difficult to determine the effect of behaviour versus morphology on gait transition from substrate to swimming in round goby. In a similar study, the slip speed (the first water speed that removed a fish from the substrate) of mottled sculpin {Cottus bairdi) from

Michigan streams was substantially lower (mean: 16.2 ± 1.2 cm-s"1: Webb et al. 1996) than the mean gait transition speed of river round gobies measured in this study (mean:

103.38 ± 5.2 cm-s"1). The difference in results was most likely affected by methodology as I did not measure the first speed that caused the goby to slip from the substrate but rather recorded the water speed that caused the round goby to remain burst and coast swimming for over 50% of the time interval. A comparison of slip speeds and body form between mottled sculpin and other benthic fishes led the authors to suggest that behavioural modification of forces in high flow (i.e. positioning of pectoral fins) may play a larger role in station holding to the substrate compared to hydrodynamic properties of body shape (Webb 1996, Taft et al. 2008). It is likely that gait transition speed in this study measured a combination of slip speed and aerobic endurance, similar to UCRIT, which was confirmed by a strong and positive correlation between the 2 performance measures (Peake 2008). The swimming flume and performance measures used may not

87 have reflected natural conditions that are ecologically relevant for round gobies.

Maximum endurance is unlikely to be used in every day routine activities. Instead, daily activities are most likely performed using only a proportion of the maximum effort possible. If this is the case then other measures of swimming performance (i.e. sustained station holding ability) may be more useful to identify swimming differences affecting dispersal and habitat selection in round goby, though the time required to conduct sustained swimming trials (> 200 minutes per fish) make it a less desirable performance measure.

Dispersal in high flow aquatic environments is energetically costly and therefore it was expected that river colonizing populations would display morphological adaptations that minimize drag and maximize thrust to facilitate successful dispersal and establishment in rivers. Caudal fin area was smaller in river fish at all tributaries but only one lake-river comparison was significant. The caudal fin of round gobies is rounded, with a low aspect ratio that maximizes surface area (Langerhans 2008). A large rounded caudal fin is associated with a deep caudal peduncle and fast-start, burst swimming

(Webb 1994, Blake 2004). In contrast, a caudal fin with a smaller fin area, wider span and small chord length (i.e. a high aspect ratio) is often associated with drag reduction in fish that undergo sustained steady swimming to reduce energetic losses from recoil effects, and in fish that inhabit or migrate in high flow environments (McLaughlin and

Grant 1994, Boily and Magnan 2002, Blake 2004, Morinville and Rasmussen 2008). I expected river fish to have a larger caudal fin area to enhance burst swimming ability, especially since the 3 swimming measures were positively correlated. However, a larger caudal fin area may be associated with other robust features similar among lake fish (i.e.

88 deeper caudal peduncle), and suggest that lake fish may be more specialized at acceleration and fast start manoeuvres, though no difference in maximum burst swimming ability was detected. Variation in morphology explained variation in swimming performance in two comparisons though this did not include morphological variables that differentiated lake and river populations. Variation in station holding endurance (UCRIT) was explained by variation in residual pectoral fin area. All lake populations had a larger residual pectoral fin area compared to the river fish though individual comparisons were non-significant, most likely due to small sample size. This is consistent with other studies that have observed an association of smaller pectoral fin area in fishes occupying high flow environments and was suggested to be due to drag effects associated with a larger fin area (Brinsmead and Fox 2002, Rouleau 2010).

However, it was expected that if a larger pectoral fin area contributes to drag, then UCRIT would be negatively related to fin area (Rouleau et al. 2010). There are two possible explanations to account for this relationship. The UCRIT test targets aerobically fuelled activity and leads to a lengthy swim trial (i.e. over 90 minutes) that can incorporate more than one gait (including anaerobic activity: Nelson et al. 1994) due to increasing flow velocity. Initially, round gobies respond to the water flow by clinging to the bottom of the swim tunnel. It is possible that round goby use their large, flexible pectoral fins to maintain position in water flow. Juvenile pallid sturgeon {Scaphirhynchus albus) is benthic and also clings to the substrate in high water flow. It has been suggested that pallid sturgeon position their pectoral fins at an angle that provides a downward force from the water flow therefore enhancing their ability to cling to the substrate and maintain position (Adams et al. 1999). Larger pectoral fin area is also expected to help

89 maintain position at higher flow speeds in other benthic fishes (Webb 1994, Webb 1996,

Taft et al. 2008). During the UCRIT test most round gobies switched to burst and coast swimming at higher flow velocities. In this case, maximum UCRIT would reflect performance ability in both gaits (station holding and burst and coast swimming). Burst and coast swimming may include the use of pectoral fins for propulsion in round goby

(but see Taft et al. 2008) therefore the positive relationship between pectoral fin area and

UCRIT may also be explained by thrust produced by the pectoral fins during unsteady swimming.

A robust caudal peduncle was associated with greater burst swimming ability.

Fish specialized in accelerating (i.e. anaerobic burst swimming) can exhibit poor sustained swimming ability due to robust morphological features including a deep caudal peduncle that enhances fast-starts, turns, and manoeuvres (Webb 1984, Webb 1994,

Langerhans 2008). The complex benthic habitats used by round gobies, as well as their aggressive, territorial behavior may provide an ecologically relevant explanation for the functional relationship between peduncle depth and maximum burst speeds. Round gobies are prey for many larger piscivores and therefore must generate fast movements to escape predation (Young et al. 2010). Caudal peduncle depth factor was greater in lake fish compared to the river fish across all tributaries though the difference was non significant. It is unlikely that a trend towards a smaller caudal peduncle in riverine round gobies is an adaptation for drag reduction as river gobies did not spend more time swimming compared to station holding at lower flow velocities (i.e. no difference in

UGAIT)- The trend of a larger peduncle area in lake fish may reflect an adaptive response favouring faster escape response especially if predation pressure is higher in lakes.

90 Interestingly, three out of four morphological variables that were either significantly correlated with swimming ability or significantly different between lake and river populations (i.e. pectoral fin area, caudal peduncle depth factor, caudal fin area) were larger in the lake fish across all tributaries, though non-significant, and represent three out of four variables expected to enhance thrust production. Small sample sizes and large variation in traits most likely contributed to the low power of statistical comparisons between lake and river populations.

Round goby body length was significantly smaller in 2 rivers compared to the lake population. It is possible that body size differences associated with river colonization represents early life stage dispersal. In a study that investigated round goby site affinity, a greater relative abundance of small round gobies was observed in more recently colonized habitats compared to longer established populations leading the authors to speculate that juveniles may be more dispersive than adults (Ray and Corkum

2001). Additionally, body size has been shown to predict the outcome of competition among non-reproductive males (when body size difference exceeded 3%) with larger round goby residents outcompeting smaller round gobies for shelter suggesting smaller gobies are more easily displaced (Stammler and Corkum 2005). Indeed the smaller body size observed at the river invasion fronts in two tributaries suggest that younger, smaller round gobies are more dispersive and more likely to colonize river systems, although I did not measure age. However, body length differences among lake and river populations may not be explained solely by juvenile dispersal. First, no ontogenetic niche shift has been reported in round gobies and ontogenetic changes in morphology have not been observed (L'Avrinkova et al. 2005). If river dispersal was a common juvenile strategy in

91 round gobies, river colonization would have occurred earlier in their Great Lakes invasion, especially due to the close proximity of rivers to the initial sites of introduction

(i.e. industrial harbours) throughout the Great Lakes. As well, size differences would be expected between lake and river populations at all tributaries, which was not observed. I collected reproductive males and females from each river. The typical parental male (i.e. large body size, black coloration, swollen cheeks) was collected from the Grand River but not from the Maitland and Saugeen Rivers despite exhaustive effort. Life history variation was observed early in their Great Lakes invasion with a trend towards a generalist life history (Maclnnis and Corkum 2000). The increasing evidence in the literature of life history variation in the invasive round goby (L'Avrinkova and Kovac

2007, French and Black 2009, Marentette et al. 2009, Young et al. 2010) suggests that round goby may be equipped to respond to changes in their environment at a rapid pace, and this may facilitate colonization of a novel environment (see Bohn et al. 2004), though this remains speculative at this stage.

The different results for body size comparisons that I observed between the Grand

River and the Maitland and Saugeen Rivers may be the result of habitat characteristics and regional location. Round goby populations in the Maitland and Saugeen Rivers occupy clear water, with cobbles and pebbles on top of a bedrock substrate. Despite flow modification in the upper portions of the rivers, habitat in the lower reaches where round gobies are present is dominated by shallow runs and riffles with occasional pools. The

Grand River is a large river that drains an agricultural region of south western Ontario.

The lower Grand River is turbid with soft, silty substrate deposited among allochthonous debris. Flow modification has resulted in slow moving reservoirs and backwaters, making

92 the river more suitable to invasion by lentic species. Additionally, the turbid water of the lower Grand River would help protect larger fish from visual predators and subsequently may facilitate the persistence of larger round gobies especially when predation pressure by piscivores seems to be increasing (Young et al. 2010). Additionally, the soft substrate in the Grand River may contribute to the significantly smaller pelvic fin size observed in river fish. It is possible that a burrowing behavior may replace clinging to a hard surface in areas with finer substrate particles leading to a reduction in pelvic fin size, though a more detailed study would be required to verify such a relationship.

The results of this study suggest that colonization of a new aquatic environment by round gobies is not limited by natural dispersal ability (i.e. swimming ability) into a uni-directional, higher flow environment. However, body shape and size differences were observed between the expanding river populations and non-expanding lake population suggesting that river colonization is not a random diffusion of individuals. I observed a pattern of larger fins and deeper caudal peduncle in lake fish with some correlation with burst swimming ability. The smaller, less robust river fish may remain station holding in areas of flow refuge rather than initiating short term burst and coast swimming at high flow velocity. It is curious that I observed morphological differences with minimal effect on swimming ability; however, a larger sample size, and a more ecologically relevant swimming measure (i.e. sustained station holding) may yet reveal differences in dispersal ability promoting river colonization. The ability of round goby to disperse and colonize high flow river environments will increase their ecological impact.

The high flow of river systems is not a strong dispersal barrier to round goby colonization. Future studies examining colonization and invasion front dynamics should

93 incorporate life history variation and quantify density dependent responses during colonization for a comprehensive understanding of dispersal potential and secondary spread in successful aquatic invasions.

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105 Appendix 3.1 Mean standard length (±SE) of round gobies, Neogobius melanostomus, from lake and river populations at the Maitland, Grand, and Saugeen Rivers in 2009 (n = 98). Minimum and maximum values are listed for each lake and river population.

Region Population n M ean (mm) SE M in (mm) Max (mm) Maitland lake 10 90.2 3.8 69.8 121.5

river 28 54.8 2.0 32.5 75.4

Grand lake 10 83.1 2.6 71.7 97.2

river 10 84.7 1.9 75.0 94.4

Saugeen lake 10 84.3 3.0 70.5 103.4

river 30 45.1 1.9 31.9 65.6

106 Appendix 3.2 Frequency distribution of standard length (mm) of round goby, Neogobius melanostomus, males (black bars) and females (grey bars) collected from lake and river populations at the Maitland, Grand, and Saugeen Rivers in 2009. Fish that sex was undetermined by visual inspection of external papillae are represented by white bars (Maitland and Saugeen river only). Total sample size is 98.

I^^H Maitland lake males Maitland river males Maitland river females I I Maitland lake females I I Maitland nver sex undetermined

HIM I

^HB Grand lake males ^^H Grand river males

\ 1 Grand lake females I ] Grand river females

^^H Saugeen lake males ^BB Saugeen river males j 1 Saugeen nver females I I Saugeen lake females I I Saugeen nver sex undetermined

11 v> (p

107 Appendix 3.3 Absolute (cm s"') critical station holding speed (UCRIT)> gait transition speed (UGAIT). and r maximum burst swimming speed (UBURST) f° round gobies, Neogobius melanostomus, sampled from lake and river populations at the Maitland, Grand, and Saugeen Rivers in 2009. Total sample size is 58.

Swimming test Region Population n Mean (cms") SE Min. Max

Ur Maitland lake 7 104.7 6.4 81.5 124.1

river 16 93.4 9.0 43.1 170.9

Grand lake 7 112.9 6.8 86.7 132.8

river 7 98.7 5.5 75.1 122.4

Saugeen lake 7 109.2 9.5 80.9 152.6

river 16 124.0 6.2 83.4 166.3 u,GAI T Maitland lake 7 87.1 2.7 80.0 99.4 river 16 93.2 8.7 60.5 180.2

Grand lake 7 95.7 4.3 80.0 139.8

river 7 95.0 3.7 70.6 99.4

Saugeen lake 7 113.4 9.4 89.3 160.0

river 16 123.9 8.2 70.6 170.2

U BURST Maitland lake 7 149.1 11.1 92.2 184.6

river 14 137.3 6.8 102.3 199.0

Grand lake 7 153.7 11.9 113.9 196.1

river 7 159.64 2.7 152.8 170.2

Saugeen lake 7 138.7 8.8 109.55 174.5

river 15 144.2 6.0 108.1 193.2

108 4.0 GENERAL DISCUSSION

A species distribution reflects a dynamic interplay between population growth, community dynamics, adaptive potential, and dispersal capacity. An element of stochasticity (e.g. human mediated dispersal) has the potential to affect the interactions among those factors and can cause a species range to change drastically as a result.

Range expansion is the dispersal and colonization of a species from a source region into a new region and can be accompanied by rapid evolutionary change affecting life history and dispersal dynamics (Reznick and Ghalambor 2001). Contemporary changes in species distributions are often associated with human activities that provide a mechanism for a subset of a population to move into a new region. The founding population is expected to have reduced genetic diversity relative to their source population and is faced with a new selective regime which can result in rapid evolutionary changes if population establishment and growth is achieved (Reznick and Ghalambor 2001). Likewise, evolutionary changes to the founding population may trigger secondary range expansion through modifications that facilitate colonization in the new territory (Cwynar and

MacDonald 1987, Hill et al. 1999, Thomas et al. 2001, Phillips et al. 2008, Alford et al.

2009). The recent invasion of Great Lakes tributaries by the highly invasive round goby,

Neogobius melanostomus, provides a unique opportunity to examine dispersal mechanisms and invasion dynamics associated with colonization of a novel aquatic environment.

In the current study, I observed a stratified dispersal strategy (contiguous and long distance dispersal) associated with round goby river colonization suggesting a combination of natural and human mediated dispersal vectors. Contiguous dispersal of

109 fish from the lake to the river was identified as the dominant mechanism of dispersal in the 3 tributaries examined, with evidence for ongoing expansion (approximately 500m / year) in one of two temporal replicates (Chapter 2). Genetic diversity was maintained during river colonization suggesting that founder effects may be mitigated and adaptation may play an important role during invasion of a new territory. River colonization by natural dispersal was dominated by smaller fish compared to the lake (Chapter 3). I observed a pattern of greater relative fin area (caudal and pectoral) and caudal peduncle depth in lake fish at all tributaries which are morphological traits associated with greater anaerobic ability (e.g. fast-starts, acceleration, manoeuvres) in other fishes (Webb 1994,

Blake 2004); two of these traits (relative pectoral fin area and caudal peduncle depth) were positively related to round goby swimming performance. Despite morphological differences, lake and river populations did not differ in maximum speed or endurance which indicates that river colonization is not selecting for fish with maximum dispersal ability. The river flow in sampled rivers may not be high enough to serve as an effective dispersal barrier. The lag time between establishment and secondary spread into rivers may reflect a density dependent response where river colonization has occurred due to increasing population density in the preferred lake environment. However, morphological differences in fin and body size between lake and river populations suggest that river colonization is not driven by entirely random dispersal of lake fish. I speculate that life history variation in round goby (L'Avrinkova and Kovac 2007,

Marentette et al. 2009) is associated with river colonization and stress the need for temporal studies to examine density dependent responses during colonization. Future studies should examine swimming behavior and sustained station holding endurance

110 associated with differences in substrate type and particle size. Evolutionary changes in natural dispersal and colonization at the invasion front may promote increased invasiveness and therefore invasion front populations should be monitored for changes in genetic diversity and rates of colonization.

Long distance dispersal was also associated with river colonization in two ways.

Recent immigrants were identified in all river populations associated with contiguous dispersal. The wide range of dispersal distances suggests that natural and human mediated dispersal is facilitating geneflow into rivers from different source populations distributed widely in the Great Lakes. I also identified a long distance dispersal event that established a disjunct river population upstream from the natural expanding front.

This disjunct population remains relatively isolated from the downstream and lake populations; however, evidence for connectivity indicates that population coalescence is likely if population growth and expansion continue. Long distance dispersal likely helps maintain genetic diversity in expanding river populations, an unwanted situation if diversity increases invasiveness and population persistence. I observed a higher migrant load in 2 out of 3 rivers compared to the lake population indicating that new migrants are targeting rivers. Despite round goby possession and transportation bans, the practice of using round gobies as bait may still occur and therefore bait bucket transfer into rivers would explain the higher migrant load observed in rivers. Alternatively, natural dispersal vectors such as river associated organisms (e.g. avian piscivores) may contribute to additional migrants into the river population, though dispersal from only nearby populations would be expected in this case. Natural long distance dispersal by round gobies may also contribute to the migrant load in rivers as a previous mark recapture

111 study identified an outlier that dispersed considerably farther than the majority of the population, though recapture success was low (Wolfe and Marsden 1998). As there is an indication that human activity may be involved in river colonization through long distance dispersal, management efforts stressing public awareness, stewardship, and compliance to regulations should be pursued immediately to reduce unwanted introductions into new river areas and to mitigate geneflow among established populations. Future genetic studies should quantify migrant load during colonization, and identify the source populations contributing long distance migrants into rivers using genetic assignment methods.

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114 VITA AUCTORIS

Name: Jennifer Ellen Bronnenhuber

Place of birth: London, Ontario

Year of birth: 1974

Education: Medway High School, Arva, Ontario

1988-1992

John Paul II Secondary School, London, Ontario

1993

University of Windsor, Windsor, Ontario

1993-1994, 2003-2008: B.Sc.[H]

University of Windsor (GLIER), Windsor, Ontario

2008 - 2010: M.Sc. Environmental Science

115