Chapter 2: Mate Choice & Selection

The interaction between natural and sexual selection and the maintenance of biological diversity: From diversification to speciation

Amy K. Schwartz

Department of Biology McGill University, Montreal December 2009

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy

ABSTRACT Speciation involves the reduction of gene flow between populations resulting in reproductive isolation. Natural selection can be a strong promoter of this process when populations in different environments diverge in traits relevant for reproductive compatibility. If such traits are involved in mate choice or recognition, sexual selection is further implicated. Speciation is theoretically most likely when natural and sexual selection act in concert; but both the extent to which this occurs, and which factors mediate this interaction remain unclear. This thesis examines the relative roles of natural and sexual selection during adaptation, and their potential for eliciting reproductive isolation. A quantitative review shows that divergence in sexually selected traits between populations does not predict the magnitude or likelihood of reproductive isolation. Instead, variation in the strength of reproductive isolation depends on the opportunity for gene flow and the nature of selection acting on the traits. Similar results were found in an empirical test of this interaction in Trinidadian guppies (Poecilia reticulata). Populations from different environments do show mating isolation to some degree, but only against geographically proximate, and potentially maladapted, migrants. Together, this suggested that selection on mating preferences is not necessarily coupled with selection on mating traits. An introduction experiment that transplanted guppies into a new environment confirmed this intuition, as female preferences do not appear to respond predictably to broad-scale ecological differences. An opportunity to further examine phenotypic responses to environmental change was afforded with a human alteration of the forest surrounding guppy habitat. Clear-cutting likely influences the way visual signals are maintained, perceived and transmitted. However, spatial and temporal variation in male colour was shown to be independent of the light environment, and is instead likely to be maintained by subtle microhabitat and demographic fluctuations. In summary, this thesis shows that sexual and natural selection can be powerful promoters of diversification between populations, but whether or not this results in speciation depends largely

ii on the geographic context, the genetic covariance between male and female mating traits, and the strength of selection acting on each. This work further highlights the importance of considering feedbacks between evolutionary and ecological processes in order to understand the evolution of, and establish the correct targets for the preservation of biological diversity.

iii RÉSUMÉ Le processus de spéciation implique une réduction du flux génique entre des populations, et se complète quand le processus de reproduction les isole. La sélection naturelle peut être un important promoteur de ce processus quand les traits responsables de la compatibilité des populations pendant la reproduction divergent. Si ces traits sont impliqués dans le choix du partenaire, la sélection sexuelle est également impliquée. Théoriquement, la spéciation est plus probable quand les sélections naturelle et sexuelle agissent simultanément, mais la prévalence de ce processus et les éléments qui régissent cette interaction ne sont toujours pas clairs. Cette thèse examine les rôles respectifs de la sélection naturelle et la sélection sexuelle au cours de l'adaptation, et des conditions qui mènent à l'isolement reproductif. Une analyse quantitative montre que la divergence des traits sexuels secondaires entre des populations ne prédit ni la quantité, ni la probabilité d'isolement reproductif. Par contre, la variation de l'isolement reproductif entre les taxons dépend de la magnitude du flux génique et du genre de sélection qui agit sur les traits. Des résultats comparables ont été trouvés dans un test empirique de cette interaction avec les guppys de Trinidad (Poecilia reticulata). Les populations qui habitent dans des environnements différents démontrent un certain niveau d‟isolement pour l'accouplement, mais seulement contre les immigrants potentiellement mal adaptés qui proviennent des environs. Par conséquent, la sélection sur les préférences sexuelles qu‟expriment les femelles pour une série de traits chez les mâles n‟est pas nécessairement associée à la sélection sur les traits des mâles. Une expérience où les guppys étaient introduits dans un nouvel environnement a confirmé cette intuition: les préférences des femelles ne semblent pas répondre aux différences écologiques à grande échelle, ni aux traits des mâles. Une autre occasion d‟étudier les réponses phénotypiques aux changements environnementaux s‟est présentée lorsque la forêt aux environs de l‟habitat d‟une population de guppys a été altérée. La coupe à blanc peut influencer la façon dont les signaux visuels sont maintenus, perçus et transmis. Cependant, la variabilité spatiale et temporelle de la couleur des mâles est indépendante de l'environnement lumineux, mais elle est susceptible aux

iv fluctuations démographiques et des microhabitats. En résumé, cette thèse montre que la sélection sexuelle et la sélection naturelle peuvent être des promoteurs puissants de la diversification entre les populations. Que ce processus de diversification mène à la spéciation dépende largement du contexte géographique, de la covariance génétique entre les caractères des mâles et des femelles, et de la force de sélection agissant sur chacun d‟eux. Ce travail met également en évidence l'importance de considérer les boucles de rétroactions entre les processus évolutifs et écologiques afin de comprendre l'évolution et d'établir des objectifs appropriés pour préserver la diversité biologique.

v ACKNOWLEDGMENTS

Although specific contributions to the work are acknowledged at the end of each chapter, the whole is more than a sum of its parts and would not have been possible without the support, guidance and dedication of certain individuals. First, I thank my supervisor, Andrew Hendry for allowing his knowledge, his confidence, his humility, and his enthusiasm to be so contagious throughout my graduate career. I could not have asked for a better editor: one who can consistently read between my (sometimes) jumbled lines, and catch my (common) lack of visual perspective. I could not have asked for a better mentor: providing ample space to think and the freedom and confidence to follow through on my thoughts, while maintaining a firm and close whip to make sure I developed thought into action. It‟s been my privilege to grow as scientists together. Funding was gratiously provided by the Vineberg Family scholarship, a doctoral fellowship from the Fonds Québecois de Recherche sur la Nature et les Technologies (FQRNT) and Discovery grants from the Natural Sciences and Engineering Research Council (NSERC) to A.Hendry. I‟m extremely grateful to members of the Hendry lab and collaborators whose core has always been one of support. Particular thanks go to those in the formative years that helped shape my perspective: Erika Crispo, Nate Millar, Jean-Sébastien Moore, Katja Räsänen, and Dylan Weese. Thanks also to current and recent members for useful discussion, argument and new perspectives: Daniel Berner, Joey DiBattista, Cristian Correa, Luis deLeon, Renaud Kaeuffer, Joost Rayemaekers and Xavier Thibert-Plante. Although no-one in the Hendry lab can avoid feeding a fish at least once, the following individuals went above and beyond to ensure the survival and fecundity of hundreds of guppies. Special thanks to fellow graduate students Swanne Gordon, Ann McKellar and Maryse Boisjoly who shared the responsibility of the guppy lab. Thanks also to the many volunteers and undergraduate students, especially: Sara Elhajoui, Laura Easty, Zaki Jafry, Artur Kondrash‟ev, and Anil Patel. I‟m particularly grateful to the staff of McGill‟s

vi phytotron facility - Mark Romer, Claire Cooney and Frank Scopeletti, for welcoming fish into the greenhouse and responding to every bizarre whim or temperature adjustment I requested without hesitation. Thank you to the graduate students, faculty and staff of the Biology department and Redpath museum for fostering an environment of true collaboration, creativity and freedom for intellectual curiousity. I was also lucky to find true friends in those hallways that inspired me and comiserated with me when I needed it most: Jeremiah Busch, Rod Docking and Anneli Jokela. I am particularly grateful to Lisa Jones who started on this path and changed through it beside me, all along with great understanding and great strength. Finally, I had the good fortune of being able to do my PhD work surrounded by family and a family of friends. Their weekly traditions and regular distractions have kept me grounded, but their unconditional confidence in me have kept me focused on the prize. This thesis is dedicated to all of „my people‟. Thank you.

vii CONTRIBUTION OF AUTHORS

This thesis consists of versions four manuscripts that are in press, in review or in preparation for submission to journals for publication. As the primary author for all, I conceived of and executed the majority of the experimental design and analyses, and wrote the main draft of the manuscripts. Other than Chapter 1, for which I am sole author (in preparation for submission to Evolution), Chapters 2-4 greatly benefitted from collaboration with co-authors whose specific contributions are stated below.

Chapter 2: Interactions between ecology and geography influence mating isolation in guppies. Authors: Amy K. Schwartz, Dylan Weese, Paul Bentzen, Michael T. Kinnison, & Andrew P. Hendry

The field enclosure experiment and parentage assignment analyses were performed by A. Schwartz and D. Weese. P. Bentzen performed DNA extraction and sequencing for parentage assignment. A. Schwartz performed all laboratory mate choice experiments, fish rearing, colour analyses and further statistical analyses and wrote the main draft of the manuscript. M. Kinnison and A.P. Hendry assisted in experimental design, statistical analyses and improvement of the manuscript.

At the time of thesis submission, this manuscript has been submitted to Science.

Chapter 3: This is not déjà vu all over again. II. Female preferences for male guppy colour in a new experimental introduction. Authors: Amy K. Schwartz*, Laura Easty*, Swanne P. Gordon, and Andrew P. Hendry L. Easty performed all mate choice experiments, phenotypic measurements and ran preliminary statistical analyses and wrote the main draft of viii the manuscipt together with A.K. Schwartz. A.K. Schwartz conceived of the study and was responsible for the execution, experimental design and final statistical analyses. S.P. Gordon conducted field work (fish collections), was primarily responsible for fish population maintenance and rearing and helped improve the final manuscript. A.P. Hendry greatly assisted in experimental design, statistical analyses and improvement of the manuscript. *These authors contributed equally

At the time of thesis submission, this manuscript is resubmitted to the Journal of Evolutionary Biology, following a first round of generally positive reviews.

Chapter 4: Testing the influence of local forest canopy clearing on phenotypic variation in Trinidadian guppies Authors: Amy K. Schwartz and Andrew P. Hendry

A.K. Schwartz designed the study, performed all phenotypic measurements and statistical analyses and drafted the main manuscript. A.P. Hendry conceived of the study upon discovering the clear-cut, assisted with fish collection in Trinidad, and greatly improved the manuscript.

A version of this manuscript is in press at Functional Ecology

ix ANIMAL ETHICS

All laboratory experiments with fish were approved by and complied with animal care policies and protocols at McGill University (see Appendix III) and the Canadian Council for Animal Care (CCAC). Field work and collections were granted by the Department of Fisheries and Agriculture, Trinidad, W.I.

x TABLE OF CONTENTS

ABSTRACT...... ii RÉSUMÉ...... iv ACKNOWLEDGMENTS...... vi CONTRIBUTION OF AUTHORS ...... viii ANIMAL ETHICS...... x LIST OF TABLES...... xv LIST OF FIGURES...... xvii GENERAL INTRODUCTION Integrating Perspectives: Is there a unified model of speciation? ...... 1

CHAPTER 1 Divergence in sexually selected traits and assortative mating in natural populations...... 15 1.1. Abstract...... 16 1.2. Introduction...... 17 1.3. Methods...... 20 1.3.1. Data Collection...... 20 1.3.2. Phenotypic differences...... 20 1.3.3. Assortative mating...... 21 1.3.4. Other data extracted...... 23 1.3.5. Statistical analyses...... 23 1.4. Results...... 24 1.4.1. Divergence in sexual selection and assortative mating...... 24 1.4.2. What predicts the magnitude of trait divergence?...... 25 1.5. Discussion...... 26 1.5.1.Do sexual selection and sexual isolation form a continuum?....26

xi 1.5.2. Isolation asymmetry and female preference evolution….....…28 1.5.3. Estimating assortative mating: from the lab to the wild……....29 1.5.4. Summary and Implications……………………………………30 1.6. Acknowledgments…………………..………………………………...30

PREFACE TO CHAPTER 2 From general patterns to a natural model system...... 37

CHAPTER 2 Interactions between ecology and geography influence mating isolation in guppies………………………………………………………………………...... 38

2.1. Abstract………………………………………………………………..39 2.2. Introduction…………………………………………………………...40 2.2.1. Guppies as a natural model…………………………………...41 2.3. Methods……………………………………………………………….42 2.3.1. Laboratory mate choice experiment…………………………..42 2.3.2. Field enclosure experiment…………………………………...45 2.3.3. Genetic paternity analysis…………………………………….47 2.4. Results………………………………………………………………...48 2.4.1. Patterns of mating biases……………………………………...48 2.4.2. Male phenotype and mating success………………………….49 2.5. Summary and Implications……………………………………………50 2.6. Acknowledgments…………………..………………………………...51

PREFACE TO CHAPTER 3 Before our eyes: Contemporary evolution in nature……………………..58

xii CHAPTER 3 This is not déjà vu all over again. II. Female preferences for male guppy colour in a new experimental introduction...... 59 3.1. Abstract………………………………………………………………..60 3.2. Introduction…………………………………………………………...61 3.2.1. Natural selection, sexual selection and guppies……………....62 3.3. Methods……………………………………………………………….65 3.3.1. Experimental populations and laboratory rearing…………….65 3.3.2. Mate choice trials……………………………………………..65 3.3.3. Quantifying male colour and female preference……………...67 3.3.4. Statistical analysis…………………………………………….68 3.4. Results………………………………………………………………...69 3.4.1. Male colour…………………………………………………...69 3.4.2. Variation in female preference functions……………………..70 3.4.3. Results summary……………………………………………...71 3.5. Discussion……………………………………………………………..71 3.5.1. Why has evolution been so modest?...... 72 3.5.2. Implications…………………………………………………...74 3.6. Acknowledgments…………………………………………..………...76

PREFACE TO CHAPTER 4 Microhabitat variation and colour evolution...... 90

CHAPTER 4 Testing the influence of local forest canopy clearing on phenotypic variation in Trinidadian guppies………………………………...……..91 4.1. Abstract………………………………………………………………..92 4.2. Introduction………………………………………………………...…93 4.2.1. Phenotypic responses to environmental change………………93

xiii 4.2.2. Environmental variation and guppy colour…………………...95 4.3. Methods…………………………………………………………….....97 4.3.1. Study sites………………………………………………….....97 4.3.2. Guppy collection and trait measurement……………………...98 4.3.3. Statistical Analyses………………………………………….100 4.4. Results……………………………………………………………….101 4.4.1. Environmental change at the disturbed site………………….101 4.4.2. Patterns of phenotypic variation……………………………..102 4.5. Discussion……………………………………………………………103 4.5.1. Summary of results…………………………………………..103 4.5.2. Implications for mate choice and sexual selection…………..105 4.5.3. Future directions: Re-examining guppy colour……………...106 4.6. Acknowledgments……………………………………………..…….108

GENERAL SUMMARY AND CONCLUDING REMARKS Toward a unified model of speciation: Why do we care?...... 116

LITERATURE CITED...... 123

APPENDIX I Table of studies and references included in meta-analysis (Chapter 1)...... 158

APPENDIX II Study site locations and map of populations used in laboratory study (Chapter 2)...... 175

xiv LIST OF TABLES

Table 1.1. Factors affecting variation in the magnitude of assortative mating (index of isolation, IPSI) across studies...... 31

Table 2.1 Results of geneneralized linear mixed model for variation in female preference as a function of male ecology and geography...... 52

Table 2.S1 Phenotypic variation among males in the enclosure experiment...... 55

Table 2.S2. Indices of ecological and geographical mating isolation by female population....56

Table 3.1. Variation in relative area of male colour among the four populations examined (ancestral, natural colonized and introduced)...... 77

Table 3.2 Variation in female preferences for relative area of male colour within and between populations...... 78

Table 3.S1 Results of two-way ANOVA for variation in male colour and size...... 85

xv Table 3.S2 Variation in preference functions among populations when male traits are considered individually...... 86

Table 3.S3 Estimates of the strength and direction of sexual selection within populations from linear regression...... 87

Table 3.S4 Variation in preference functions among populations due to predation and river effects...... 88

Table 3.S5 Variation in preference functions among populations due to predation and river effects when male traits are considered individually...... 89

Table 4.1. Effects of space, time and disturbance on phenotypic variation...... 109

Table 4.2 Pairwise comparisons between sites before (2002-03) and after (2006-2007) the clear-cutting event at the disturbed site...... 110

Table 4.S1 Site locations ………………………...... 114

Table 4.S2 Temporal variations in canopy cover. Canopy openness measures are compared between years within sites with one-way analysis of variance...... 115

xvi LIST OF FIGURES

Figure i.i. Conceptual framework for the integration of factors during the evolution of reproductive isolation...... 9

Figure 1.1. Number of empirical studies from Web Of Science (ISI) search relating divergence in sexually selected traits and assortative mating since 1959...... 32

Figure 1.2. Investigation for publication bias in the dataset with respect to (a) statistically significant evidence for reproductive isolation and (b) the magnitude of divergence in sexually selected traits between populations...... 33

Figure 1.3.

Correlation between assortative mating (IPSI) and divergence in sexually selected traits (log SMD) between populations...... 34

Figure 1.4. Proportion of studies in the database showing statistical evidence for symmetric, assymetric and no assortative mating in (a) sympatry or parapatry and (b) allopatry...... 35

Figure 1.5. Average magnitude of standardized trait differences between populations as a function of signal type and taxa...... 36

xvii Figure 2.1. Mean ( s.e) female preference scores for males from environments of the same or different predation regime…………………………………………………….53

Figure 2.2 Relative mating success with high-predation females between high and low- predation males in field enclosures...... 54

Figure 2.S1 Fitted lines of logistic regression of male mating success (whether or not a male sired at least one offspring in the enclosure) on variation in area of orange (%) when high-predation males were competing against (a) parapatric low-predation males and (b) allopatric low-predation males...... 57

Figure 3.1 Map of the study sites in Trinidad used in the introduction experiment...... 79

Figure 3.2. Mean relative area of male colour and body size by population: Damier high- predation (DH), Damier low-predation (DL), Yarra high-predation (YH), and Yarra low-predation (YL)...... 80

Figure 3.3. Population-level female preference functions in home population mating trials...... 81

Figure 3.4. Population-level female preference functions in standard-male population (YH) mating trials...... 83

xviii

Figure 4.1. Means and standard errors for male and female size (standard length) in four years across sites varying in canopy cover...... 111

Figure 4.2. Means and standard errors of residuals (from body area) of the area of colour spots in four years across sites varying in canopy cover...... 112

Figure 4.S1. Map of Rivers in Trinidad‟s Northern Mountain Range. Inset shows locations of the three main study sites...... 113

xix

GENERAL INTRODUCTION Integrating perspectives: Toward a unified model of speciation

“Natural selection is, if not the only driving force in evolution, certainly the only known force capable of producing the illusion of purpose that so strikes all those who contemplate nature.” -Richard Dawkins in a public address at the 92nd Street YMCA, New York City, 2009

“The process [of speciation] is not easy to observe and measure in action, because it erases its own traces.” -Jonathan Weiner, The Beak of the Finch, p. 144

The overwhelming diversity of life forms has been the “mystery of mysteries” to observers of the natural world long before Charles Darwin borrowed the phrase in his introduction to The Origin of Species (1859). He was, however, the first to offer a mechanism of the origin and maintenance of this diversity: natural selection. Yet, despite over 150 years of research and study of natural selection‟s role in speciation, a mechanistic understanding of the process in nature still remains somewhat mysterious. The two quotes above articulate part of this difficulty. First, how do we go about making predictions of a process that has no foresight or purpose? And since speciation results in discontinuity or separation, how do we reconstruct it once it is already complete? The job of an evolutionary biologist is therefore to extend a timeline in both directions from the snapshot in which they find themselves and their object of study. Yet, in order to reconstruct history and develop general foundations for predicting the future, we first require a working definition of

species. Despite the title of his book, Darwin himself did not offer a definition for the actual point of origin of a species – instead resting on the view that since the same mechanism (natural selection) is responsible for variation at any scale, such a point was likely indefinable:

“...no clear line of demarcation has as yet been drawn between species and sub-species... or, again, between sub-species and well-marked varieties... These differences blend into each other in an insensible series; and a series impresses the mind with the idea of an actual passage” -Darwin, The Origin of Species p. 34

Today, mechanism-based definitions of species provide the working „lines of demarcation‟ that allow for hypothesis driven study (reviewed in Coyne & Orr 2004). According to the biological species concept, speciation involves the accumulation of reproductive isolation between populations. That is, individuals are biologically different species when they can no longer breed successfully, and thereby do not exchange genetic material (Mayr 1963). Functional definitions such as this provide a method for drawing the line between variation among populations (diversification), and discontinuities between them (speciation), yet the relative roles of the factors that exclude, promote, or maintain this line remain elusive. Thus, identifying the barriers to genetic exchange (gene flow) between differentiated, but not yet speciated, populations is crucial in the development of our understanding of the entire process of speciation. In this thesis, I examine how interactions between the main evolutionary processes (natural and sexual selection) within the environmental contexts in which they act (geography and ecology) influence the procession of reproductive isolation. One primary mechanism of gene flow reduction occurs when individuals no longer recognize each other as potential mates (i.e. “pre-mating isolation”). This may occur if environmental differences result in divergent selection on traits that are also signals for mate choice or discrimination. If such traits readily coevolve with mating preferences, then pre-mating isolation, and ultimately

speciation, should result as consequence of divergent adaptation to local environments (i.e. “ecological speciation”, Mayr 1947; Dobzhansky 1951; Schluter 2000). Mate choice, however, may not evolve at the same rate, or in the same direction as the traits that are under divergent natural selection. Consequently, sexual selection may act to oppose local adaptation by promoting gene flow between diverging populations (Felsenstein 1981; Schwartz & Hendry 2006). Recent research in speciation has focused on ecology‟s role, thus shifting the paradigm of speciation from pattern to process. Ecology is only one context however, and its effect on isolating mechanisms may vary depending on (i) the geographic context (opportunity for gene flow) and (ii) the nature of selection acting on mating preferences. Because these factors are often examined in isolation in populations where the process is complete or near-complete, it has been difficult to establish a general framework and identify the primary drivers to explain observed patterns of biological diversity. This thesis aims to integrate these factors into a common framework, specifically asking how the effects of natural and sexual selection on mating traits within populations translate into patterns of mate discrimination between populations. To address these questions, I conducted a quantitative review (Chapter 1) to identify the factors most likely to be responsible for the variation in pre-mating isolation across taxa. I then tested these questions empirically using laboratory and field experiments with Trinidadian guppies (Poecilia reticulata)  a natural model system of adaptive divergence and sexual selection (Chapters 2- 4). Because each chapter addresses particular questions related to this framework in detail and reviews the theoretical and empirical literature extensively, I use this introduction to present the historical link in the study of how natural selection, sexual selection and geographic overlap can individually drive speciation. I then illustrate how these factors may interact throughout the process of diversification. Finally, I describe the guppy system and demonstrate its appropriateness as a model for testing this framework.

Speciation, gene flow and selection: From pattern to process Because speciation involves barriers to genetic exchange, the first context in which it was best understood involved circumstances where such isolation was primarily extrinsic. That is, genetic divergence initially requires physical separation, rather than biological processes. This allopatric, or vicariant, speciation occurs when a population becomes physically fragmented, either passively (e.g. due to mountain range or island formation) or actively (e.g. due to migration of some individuals). As a consequence of a long period of physical isolation, genetic divergence between populations will result due to (i) chance effects (genetic drift), (ii) founder effects of a subset of colonizing individuals in a new location, and/or (iii) different ecological demands between locations. Once such differences develop between populations, reproductive isolating mechanisms will “inevitably evolve as an incidental by-product” (Mayr 1963, p. 581), so that if they come back into contact, they will no longer be reproductively compatible. Isolating mechanisms come in many forms. For example, individuals from different populations may (i) no longer be physically capable of mating (i.e. mechanical isolation), (ii) no longer recognize one another as potential mates (i.e. behavioural isolation), (iii) have developed different cues for when and/or where to breed (i.e. temporal and/or habitat isolation), or (iv) breed readily but their offpring are inviable or sterile (i.e. postzyogotic isolation). Regardless of the mechanism of reproductive isolation, geographic isolation was seen as a necessary starting condition. Intuitively this makes sense: if reproductive isolation is an incidental by-product of genetic divergence, then continued gene flow will prevent the diversification necessary to initiate the process of reproductive isolation. Although a number of theoretical studies in the 1960s and 1970s demonstrated the plausibility of speciation with gene flow - sympatric or parapatric - (e.g., Maynard-Smith 1966; Balkau & Feldman 1973), overall consensus was that it was not readily possible in nature and certainly was not common. Felsenstein (1981) formalized this argument in a seminal paper where he showed that species numbers are limited as a direct result of genetic constraints.

In particular, even if natural selection results in two different alleles (e.g. allele B produces a large body and allele b produces a small body) that are fixed in two populations, recombination will, in most circumstances, prevent absolute divergence between these alleles (e.g. variation in body size will be unimodal as opposed to bimodal). This influential paper, along with the general lack of empirical examples of sympatric - relative to allopatric - divergence, reinforced the popular view that gene flow prevents the opportunity for speciation. Although speciation has since been shown to occur with gene flow, this has not eliminated the controversy surrounding the likelihood and generality of sympatric speciation in nature (Diekmann & Doebeli 1999; Via 2001; Gavrilets 2003). However, recent focus has shifted from classifying speciation mechanisms geographically to studying the underlying mechanisms of divergence (selection and gene flow) and their relative impacts on isolating mechanisms. A second major conclusion of Felsenstein‟s (1981) model was that the limiting effects of recombination on speciation could be counter-acted by the effects of natural selection (e.g. if medium body sizes are particularly unfit or Bb is a lethal genetic combination). Recent theoretical advances have since incorportated the role of selection directly into the models. Rather than treating an environment of species coexistence as homogeneous, a deeper examination of ecological differences and/or habitat use within and between locations have shown several contexts in which speciation readily occurs without the requirement of physical isolation (e.g. Endler 1977; Lande & Kirkpatrick 1988; Doebeli & Diekmann 2003). Furthermore, increasing empirical evidence in the last two decades has shown that speciation with gene flow is not simply plausible, but perhaps even common. Nonetheless, sympatric speciation is most readily observed in cases where populations compete over limited resources, such as for food (e.g. Grant & Grant 2002; Nagel & Schluter 1998) or host-plants (Feder & Bush 1989; Via 1999; Funk 1998; Nosil et al. 2002). This suggests that ecological divergence may be a necessary prerequiste for speciation, regardless of the geographic context (Funk et al. 2006).

Speciation may also occur without natural selection, but instead due to sexual selection alone. For example, the over 500 species of haplochromide fishes of Lake Victoria are maintained by preferences for different colour morphs with no obvious ecological differentiation (Seehausen 1997). Sexual selection is generally defined as a change in phenotype or genotype frequencies as a result of variation in reproductive success among individuals in a population (Darwin 1871; Andersson 1994). Such variation may occur as a result of intrasexual interactions (e.g. male-male competition) or intersexual interactions (e.g. female choice). Regardless of the mechanism, particular traits will be associated with increased mating success (e.g. larger size is preferred by, or facilitates combats for access to, females). If such traits diverge between populations, then so might the direction of sexual selection for them. Early models of sexual selection commonly assumed that mating preferences were genetically linked to, and therefore co-evolved with, mating traits. For example, if two populations differ in body size (alleles B and b as above), sons of a B father will be large and daughters will have a preference for large males, whereas sons of a b father will be small and daughters will prefer to mate with smaller males. Such coupled inheritance will readily result in assortative mating by body size and hence result in speciation (Lande 1981, 1982; Diekmann & Doebeli 1999). The complication of sexual selection occurs when traits and preferences are not inherited together, but rather evolve independently, as in the Felsenstein (1981) scenario. If there is a second allele, C, that determines the strength of assortative mating (i.e. „mate with someone that looks like me‟), recombination between populations is more likely to break down the association between traits and preferences. Sexual selection may thus faciliate divergence and reproductive isolation, but reproductive isolation is not a necessary outcome of selection on sexual signals (Schwartz & Hendry 2006; for reviews see Ritchie 2008; Nosil et al. 2007 and Chapter 1). Thus, the interaction between the directions and strengths of each type of selection will likely modify the opportunity for gene flow between populations.

Integrating process and pattern Figure i.i illustrates the nature of this interaction. First, imagine an environment in which divergent natural selection results in a bimodal fitness function. For example, competition for space or resources results in exploitation of two types of habitats or food sources. Consequently, populations diverge in a trait that enhances optimal exploitation of this resource (e.g. small and large beaks to eat small and large seeds in Darwin‟s finches, Huber et al. 2007). In Figure i.i. this is illustrated by the filled and open stars, representing mean trait values in the two populations that correspond with two different fitness peaks, i.e. divergent local adaptation. If this trait is also a target of mate choice, two scenarios are possible. In scenario 1 of Fig. i.i., preferences are linked (either directly or indirectly) to selection on the trait. The mean trait values preferred in each population (filled and open stars) thus correspond with the average trait value in each population. Local adaptation therefore enhances both survival and reproductive success within each population. This will result in assortative mating where individuals mate within populations, thus reducing gene flow and lead to reproductive isolation (Scenario 2, Fig. i.i). In scenario 3, although natural selection favours two distinct trait values, sexual selection favours only one (shown here as an open-ended preference for larger trait values). This could occur, for example if females have a pre-existing preference or sensory bias for „largeness‟ (Ryan & Rand 1993), or if larger trait values confer a direct benefit to the female (for example, protection from predators, or territory and/or resource aquisition). Whatever the mechanism, in this circumstance gene flow will be maintained due to the high mating success of one male type, regardless of the costs to his survival in the „wrong‟ environment. This could ultimately prevent the evolution of reproductive isolation and local adaptation resulting in a genetically and phenotypically homogeneous population (4).

Alternatively, assuming additive inheritance on the trait subject to divergent natural selection, matings between females from the small trait value population (open stars) and males from the large trait value population (closed stars) will result in offspring with an intermediate phenotype (5). Such hybrids will find themselves in a fitness valley, and if natural selection is sufficiently strong (i.e. there is no intermediate environment, for example medium-sized seeds), these matings will be selected against. This „reinforcement‟ (Servedio & Noor 2003) can result in reproductive isolation due to direct selection against these hybrids (6) and/or indirect selection on mating preferences in the „small trait value‟ population (Servedio 2000, 2001), thus again producing a pattern of divergent sexual selection and assortative mating (7). In summary, the extent to which gene flow can be reduced in sympatry or upon secondary contact with respect to allopatric populations, will likely depend on (i) the extent of divergence in mating traits, (ii) the strength of selection against migrants, and (iii) the strength of selection against hybrids.

Figure i.i. Conceptual framework for the integration of factors during the evolution of reproductive isolation. Ecological differentiation results in divergent selection on a trait that is also important in mate choice. A population diverges into sub-populations with two mean trait values (open and closed stars). Shown are various scenarios for the evolution of reproductive isolation depending on (i) the mean value of the preferred trait in mate choice between the two populations (open and closed stars in second row) and (iii) the strength of selection against hybrids or intermediate trait values (gray star). See text (pp. 7-8) for detailed explanation.

Trinidadian guppies: a model for adaptation and (lack of?) speciation The rivers of Trinidad‟s Northern mountain range offer an excellent system to integrate evolutionary processess (natural and sexual selection) and environmental contexts (ecology and geography) in the study of speciation. In general, predation intensity varies along the upstream-downstream axis, with sharp changes occurring across waterfalls that prevent upstream colonization by predatory fishes (Haskins et al. 1961; Shaw et al. 1991). As a result, headwaters and tributaries are generally characterized by low predation, whereas downstream sections and the main channel areas are generally characterized by high predation. Specifically, guppies in downstream sections of rivers on the southern slope of the mountains coexist with the pike cichlid (Crenicichla alta) – a large piscivore, whereas upstream guppies coexist with only the killifish (Rivulus haarti) – a predator of mild to minimal effect. In contrast, the downstream reaches of rivers on the northern slopes of the mountains, which meet the Carribean sea, were colonized by piscivorous fish of marine origins, such as the mountain mullet (Agonostomus monticola) and the goby (Gobiomorus maculatum), whereas guppies in upstream sites again coexist with the R. haarti and another predator of minimal effect; freshwater prawns (Macrobrachium sp.). Regardless of the particular predator assemblage, high- and low-predation populations within a river show adaptive divergence in many traits, including male colour, behaviour, and life history (Seghers 1974; Endler 1980; Reznick & Endler1982; Reznick et al. 1996). This adaptive divergence has occurred independently in multiple drainages (i.e., parallel evolution), as inferred from patterns of geographical separation and genetic variation (Carvahlo et al. 1991; Reznick et al. 1996; Alexander et al. 2006). In this thesis, I focus on divergence in colour patterns between predation environments, as colour is a conspicuous target to predators and to potential mates. The evolution of traits that could potentially influence reproductive isolation in guppies is therefore simultaneously subject to both natural (predators) and sexual (female choice) selection. Divergence in mate signaling traits (male

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colour) therefore appears to be an example of parallel evolution, i.e. similar phentoypes are observed independently and repeatedly in response to similar environmental conditions. Furthermore, this parallel divergence is in a trait that forms the basis for female mate choice therefore providing a rare opportunity to study the interaction between natural and sexual selection in the evolution of premating isolation.

Mate choice in guppies The guppy system presents itself as a potential model for testing the theory of ecological speciation: divergent natural selection has repeatedly resulted in differentiation of a trait important for mating success. But is sexual selection also divergent? Indeed, high-predation females appear to discriminate against colourful males in some populations (Breden & Stoner 1987), and the strength of preferences co-evolves with colour variation in others (Houde & Endler 1990). Together, this would suggest that natural and sexual selection against migrants could effectively reduce gene flow and lead to reproductive isolation (Fig. i.i). Although the ingredients for isolation are there, gene flow appears to be high between predation environments (Crispo et al. 2006; Becher & Magurran 2000), and hybrids between population types are viable and fertile, leading many authors to conclude that guppies provide an example of where natural selection does not lead to reproductive isolation (reviewed in Magurran 2005). One possibility is that female choice does not have a strong influence on male mating success between populations, despite its role in sexual selection within populations. Guppies have a promiscuous mating system and mate throughout their lives. Most successful fertilizations occur as a result of female acceptance of a male following his display. Male displays vary among individuals, but for the most part consist of fanning out all fins, contorting the body into an „S‟ or sigmoid shape and then twitching around the female in a circle (Baerends et al. 1955; Liley 1966). A female in turn may ignore the male or consent to copulation by gliding toward him in a circle and tilting her gonopore up to allow fertilization (Houde 1997). Although there is no particular breeding season, females are only

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receptive to mating when virgins and for a brief period of time following parturition. Males therefore adopt another tactic for securing fertilizations: sneaky mating, in which a male will coerce copulation without a female‟s consent. A majority of these attempts are unsuccessful, however sneaking may contribute to up to 15% of matings in the wild (Matthews & Magurran 2000; Evans et al. 2003). Females are therefore constantly being harrassed by males, whether they are receptive to mating or not. The costs associated with avoiding this harrassment will likely be elevated in high-predation environments which may mediate the importance of female choice as the main determinant of male mating success between predation populations. Indeed, females in high-risk situations have been shown to modify their discrimination level (Gong 1997), school more often (Seghers 1974) and are likely to resort to mate choice copying rather than individual assessment (Dugatkin & Godin 1992) in order to remain less conspicuous. If females are less discriminating in high-predation conditions, low- predation migrants may succeed in passing on their genes, despite suffering low survival (Fig. i.i., scenario 3). This continued gene flow would prevent local adaptation and further divergence in mating traits and preferences (Fig. i.i. scenario 5). However, these „hybrid‟ offspring will inherit their father‟s colour pattern (colour is primarily a Y-linked trait, Houde 1992; Lindholm et al. 2004) and will in turn become high targets of predation. Such selection may act to reinforce female discrimination against migrants or migrant phenotypes (Fig. i.i. scenarios 6 & 7).

Mate choice vs. mating isolation in guppies: A direct test Despite much research on natural and sexual selection within guppy populations, the relative strengths of natural and sexual selection against migrants has not yet been directly tested. Selection studies have revealed that migrants between predation environments have reduced survival (Gordon et al. 2009; Weese et al., unpublished data) and that adaptation to new environments can evolve relatively quickly (Endler 1980; Reznick et al. 1997; Gordon et al. 2009).

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This suggests that natural selection against migrants should effectively reduce gene flow between populations. But do migrants also suffer a fitness compromise in terms of mating success? In a previous study, I quantified the amount of divergence in population preference functions (the relationship between male traits and mating probability) between high- and low-predation environments and found that preferences are generally evolving in parallel across replicate evolutionary lineages (Schwartz & Hendry 2007). In particular, high predation females discriminated against males with more colour, whereas low predation females showed very little discrimination based on colour. These results suggest that female preferences are evolving to increase matings with locally adapted males within their populations. Although male traits and female preferences thus appear to be coevolving to some extent, high variation in the strength of preferences suggests that (i) mate choice alone may not be enough to limit gene flow between these populations, (ii) female preferences and male traits may not evolve at the same rates or respond to predation differences in the same way, and (iii) variation in sexual selection and sexually selected traits may be influenced by ecological factors other than the dichotomous ecological contrast of „high‟ and „low‟ predation. To address these possibilities, I first looked for evidence of mating isolation between guppy populations adapted to different predation environments (Chapter 2). In order to further test for the influences of geography, ecology and mate preferences on the potential evolution of mating isolation, both laboratory and field experiments were employed using high- and low-predation population pairs, differing in colonization history and the potential for migration between them. Although the effects of predation regime on secondary sexual traits has been well studied (e.g. Haskins et al. 1961; Endler 1978, 1980, 1983; Millar et al. 2006), the nature of selection on female preferences has not received as much attention. Generally speaking, few studies have documented the contemporary evolution of female preferences in the wild. Chapter 3 tackles this question by looking for evidence of change in the direction and strength of female preferences following colonization of new predation environments.

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Finally, in Chapter 4, I consider alternative explanations for the extreme variation observed in male guppy colour. Although much of this variation can generally be attributed to predation regime, recent studies are finding that variation within and among rivers in a given predation regime can be just as substantial as that observed between predation regimes (e.g. Houde & Endler 1990; Brooks 2002; Karim et al. 2007; Kemp et al. 2008). Other environmental factors covary with predation which could also have an effect on the expression and maintenance of colour signals as well as preferences for them. Furthermore, low-predation guppies are thought to be more colourful simply due to the effects of relaxed selection from predators. However, this has never been directly tested and there may be other unmeasured sources of selection in “low-predation” that have contributed to divergence between upstream and downstream populations. Here, I investigate the impact of one such factor – canopy openness –to better understand the nuances of variation in sexually selected traits, and the potential for their response to environmental change. Overall, the guppy system therefore provides an excellent opportunity to study a species that is potentially at an early stage of the speciation process, where the particular factors that limit and promote progress toward local adaptation and the evolution of reproductive isolation can be investigated.

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CHAPTER ONE

Divergence in sexually selected traits and assortative mating in natural populations

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1.1 ABSTRACT

Sexual selection can, in theory, be a powerful promoter of population divergence and reproductive isolation. How much, how often, and under what circumstances this holds true is still a question of open debate. Most studies that have addressed these questions have done so from a large-scale comparative approach, relating proxies of sexual selection (e.g. sexual dimorphism or polyandry) to the number of species in a taxonomic group. Although results have been mixed, proxies for sexual selection are generally associated with higher speciation rates. However, the role that sexual selection plays in driving the process of divergence and speciation is still uncertain. If sexual selection is an important driver, greater divergence in sexually selected traits should be associated with greater levels of mating isolation. To address this prediction, I extracted data from 158 pairs of populations or incipient species from 69 study systems. I found no association between the magnitude of divergence in sexually selected traits and the strength of assortative mating. Instead, assortative mating was closely related to the degree of geographic overlap between populations and the degree of viability selection acting on the sexually selected trait. Together, these results suggest that although sexual selection can be important for speciation in some contexts, it probably often plays a role secondary to natural selection against hybridization.

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1.2. INTRODUCTION

According to the biological species concept, speciation involves the accumulation of reproductive isolation between populations (Mayr 1963). Identifying barriers to gene flow between diverging populations is therefore crucial to understanding speciation (Coyne and Orr 2004). Recognizing the primary mechanisms driving the evolution of reproductive barriers, however, can be difficult - because many factors can interact to increase or decrease gene flow during population divergence. Overall, the magnitude of reproductive isolation varies substantially across populations (Coyne and Orr 1989, 2004; Morjan and Rieseberg 2004; Funk et al. 2006; Nosil et al. 2009), but the particular factors that determine the amount of divergence remain uncertain. Here, I present results of a meta-analysis which considers the potential role of sexual selection in contributing to the maintenance of reproductive barriers. Sexual selection can, in theory, reduce gene flow and thus facilitate progress toward speciation. The reason is that sexual selection causes changes in traits important for mate recognition within populations, and so differences in sexual selection between populations can result in sexual isolation; i.e., a reduction of inter-population matings relative to intra-population matings (Dobzhansky 1937; Mayr 1963; Lande 1981; West-Eberhard 1983). I use the term „speciation‟ here to refer to the entire process, whose endpoint is complete reproductive incompatability. „Sexual isolation‟, on the other hand, refers to the degree to which such reproductive incompatability is evident. Whether or not sexual selection within populations and sexual isolation between populations are closely linked, however, is still a topic of some controversy (e.g. Paterson 1989; Etges 2002; Nosil et al. 2007). The reason is that sexual isolation can arise from a number of different mechanisms that influence species recognition (e.g. genetic incompatabilities, temporal or spatial differences in breeding, mechanical isolation, behavioural isolation), but sexual selection within populations relies on the relationship between traits and preferences for trait values, and thus evolves independently. Behavioural patterns of assortative mating within and between

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populations (i.e. „premating isolation‟) can therefore provide clues into the link between sexual selection and sexual isolation. Although many species are defined primarily by differences in traits that determine reproductive compatability, the general relationship between divergence in sexually selected traits and assortative mating is still uncertain (reviewed in Boake 2000). The potential connection between sexual selection and speciation was first proposed by Darwin (1871), but its role as a driver of diversification was long viewed as secondary to other isolating mechanisms (Dobzhansky 1937, Mayr 1963). This view began to change in the 1980s as a series of theoretical models demonstrated that speciation is readily initiated under a variety of scenarios when mating preferences coevolve with disruptive selection on sexually selected traits (Lande 1981,1982; Lande and Kirkpatrick 1988), or as a result of intrasexual competition for mates (West-Eberhard 1983). Further work has since expanded these earlier models and offerred (i) new insights regarding the limits and contexts in which speciation is most likely to occur via sexual selection (e.g. Schluter and Price 1993; Iwasa and Pomiankowski 1995; Payne and Krakauer 1997; Servedio 2004; Thibert-Plante and Hendry 2009), (ii) theoretical explorations of specific natural systems (e.g. Gavrilets et al. 2007; Gavrilets and Vose 2007), and (iii) new predictions for empirical testing (e.g. de Cara et al. 2008). Recent empirical work has supplemented theory by attempting to quantify the general relationship between sexual selection and speciation. A common approach has been a macro-evolutionary, or comparative, perspective within taxa (reviewed in Barrachlough et al. 1998; Coyne & Orr 2004; Ritchie 2007; Nosil et al. 2009), and the results have been variable. On the one hand, several studies have found positive correlations between speciation rates and the opportunity for sexual selection in birds (Barrachlough et al. 1995; Mitra et al. 1996; Moller & Cuervo 1998; Seddon et al. 2008). On the other hand, Morrow et al. (2003) did not find such a relationship based on a variety of estimates of sexual selection. Similarly, neither estimates of female choice or male-male competition correlate with species number in mammals, spiders and butterflies (Gage et al. 2002). In ray-finned fishes, sexual selection is correlated with diversification within clades

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in some studies (Mank 2007) but not in others (Ritchie et al. 2005). Overall, these studies indicate that sexual selection likely helps to keep species apart, but they cannot speak to its role throughout the process of speciation. That is, comparative analyses between species cannot clearly reveal whether sexual selection is a driving force of speciation or a secondary outcome of other evolutionary processes. If sexual selection is a driver of speciation, then greater levels of mate discrimination should result in greater divergence in mating traits, regardless of the factors responsible for the initial phenotypic divergence. For instance, traits such as body size or conspicuous colouration are commonly subject to both natural and sexual selection (Endler 1978; Andersson 1994). Hence, even if divergent natural selection is often the initiator of population differences, sexual selection may act as the tipping point in determining whether such differentiation will ultimately result in genetic discontinuity between populations, i.e. complete speciation. The study of speciation as a process is therefore best achieved by examining populations where speciation is not yet complete (Hendry 2009). Population divergence within species thus becomes an informative way to assess the relative contribution of sexual selection versus other factors to the evolution of mating isolation. In an earlier review of population divergence in sexually selected traits, Questiau (1999) found that the role of sexual selection in mating isolation was secondary to the apparent roles of genetic drift and natural selection. Data were limited, however, and the last ten years has seen an exponential increase in the number of studies relating sexual selection to premating isolation (Fig.1.1), making it an appropriate time to reassess the relative importance of sexual selection to speciation. This study applies a meta-analytic framework to determine how much, how often, and under what circumstances divergence in sexually selected traits is associated with premating isolation. The analysis is restricted to mating pairs at the early stages of divergence (populations, incipient species, or sister species) that show little to no post-zygotic isolation (viable hybrids can be formed in captivity). I further qualified and included studies representing a wide range of

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ecological and geographic contexts in order to provide the best opportunity to infer the relative importance of sexual selection during the evolution of premating isolation.

1.3 METHODS

1.3.1. Data Collection Web Of Science, and citations in the resulting papers, were searched for studies that quantified differences between populations, incipient species, or sister species in a trait used in mate choice in at least one of the populations. Search terms included combinations of “sexual selection”, “sexually selected traits”, “speciation”, “assortative mating”, and “reproductive isolation”. The data were further restricted to studies of animals from natural populations; i.e., experimental manipulations or artificial selection were not included which resulted in a total of 69 appropriate study systems (a particular population or species group in a particular location). The data for each comparison included mean values of a sexually selected trait in two populations (n = 437), and a quantitative estimate of assortative mating due to behavioural premating isolation (n = 145). A total of 66 species from eight taxa (Amphibians, n = 24; Birds, n = 28; Fish, n = 32; Insects, n = 69, Arthropods, n =1, Invertebrates, n = 2, Mammals, n =1; Reptiles, n =1) were represented (see Appendix I for the full data set and references).

1.3.2. Phenotypic differences Sexually selected traits were classified into five different signal types: acoustic, n = 65; behavioural, n = 10; chemical, n =3; colour, n = 40; and morphological, n = 40. For each trait, data on population means, standard deviations, and sample sizes were obtained directly from summary tables in papers wherever possible. If only graphical information was available, values

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were extracted from the images with digital imaging software (Image J, http://rsbweb.nih.gov/ij/). In each case, three repeat measures were taken and averaged. Trait differences between populations were calculcated as the standardized mean difference (SMD):

(X1 - X2) / p

where X represents the trait mean in each population, and p is the pooled standard deviation:

2 2 p = ((1 * (n1 – 1) + (2 * (n2 – 1)) / ((n1 – 1) + (n2 – 1))

Trait differences were calculated separately for each variable measured and the average was calculated in cases where multiple variables contribute to a single sexually selected trait for a total of 158 averaged comparisons. For example, in cases where „song‟ is generally known to influence mate choice, the SMD between all variables that contribute to song characteristics were averaged to yield one overall song metric. In systems where one particular element of many is known to be most important in sexual selection, (e.g. dominant frequency as opposed to interpulse rate), only this element was included. This was done since the main interest here is not to ask which traits contribute to assortative mating, but rather whether or not sexually selected traits do. SMD measures were log transformed to obtain normal distribution for use in linear models.

1.3.3. Assortative Mating As the main question of this study concerned how the evolution of sexually selected traits within populations affects patterns of mate recognition between populations, only data related to behavioural pre-mating isolation were included. Different studies estimate this type of isolation using a variety of metrics. Rather than using these values, I used the raw data to calculate a common

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index (IPSI - Rolan-Alvarez & Caballero 2000) for all studies. This index was deemed most appropriate since (i) it is commonly used in recent work; (ii) it is shown to have the least statistical bias, particularly for small sample sizes; and (iii) it incorporates information from all possible mating pair combinations, thus potentially revealing assymetries in isolation (Rolan-Alvarez & Caballero 2000; Perez-Figueroa et al. 2005).

IPSI compares the frequencies of homotypic copulations (or indicators of willingness to copulate) to the frequencies of both types of heterotypic pairings between two populations („A‟ and „B‟) as:

(PSIAA + PSIBB – PSIAB - PSIBA) / (PSIAA + PSIBB + PSIAB + PSIBA) where the first subscript letter indicates the population of the female of the pair, and PSI („pair sexual isolation) represents the number of observed mating pairs divided by the expected number of pairs, for each type of cross (AA, AB, BA, and BB). For example,

PSIAB = AB * (AA + AB + BA +BB) / (AA + AB) * (AB + BB)

The resulting data were classified as to whether or not they revealed statistically significant evidence for assortative mating, and whether the assortative mating was symmetric or not. Finally, the data were classified into the four common types of experimental methods employed: (1) natural behavioural observations (n = 19), which generally involve studies of census data of all individuals and the relative proportion of homotypic and heterotypic mating pairs observed, conducted in a contact zone between populations, (2) dichotomous choice (n = 50) laboratory experiments in which one individual of the choosy sex (often female) is given the choice between a mating partner of her own and the other population type, (3) multiple choice laboratory experiments (n = 23), in which a number of individuals of both sexes from both populations are combined and the proportion of homotypic and heterotypic copulations are noted, and (3) no choice

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laboratory experiments (n = 52) in which individuals are presented to individual members of the opposite sex sequentially and the probability of copulation in each cross type is calculated.

1.3.4. Other data extracted In order to qualify the relationship between divergence in sexually selected traits and assortative mating further, each population comparison was further categorized by (1) the extent of geographic overlap (sympatric, parapatric, allopatric) between them, (2) whether or not the trait is subject to divergent natural selection due to ecological differences (e.g., populations occupy different habitats or use different resources), (3) independent estimates of divergence time where available and (4) whether or not the populations are classified taxonomically as „true‟ species.

1.3.6. Statistical Analysis Publication bias might be present if studies with low sample sizes and low effect sizes are less likely to be submitted or accepted for publication. This potential bias was considered for phenotypic differences by examining the relationship between sample size and effect size, the latter being the difference between the natural logs of the population trait means divided by their pooled standard deviation. Potential publication bias was considered for premating isolation by examining the relationship between the number of mating pairs tested or observed and the statistical significance of assortative mating. No obvious biases were detected (Figure 1.2) making it appropriate to draw general conclusions from further analyses. Three main questions were addressed in the meta-analysis. (1) Does the magnitude of assortative mating (IPSI) increase with increasing divergence between populations in sexually selected traits? (2) Is this relationship influenced by the opportunity for gene flow between populatons, i.e the geographic context (sympatric, allopatric, parapatric)? (3) Is the relationship mediated by the influence of natural selection acting on the trait (i.e. ecological context)?

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Question 1 was addressed with a simple linear regression of the assortative mating index (IPSI) as the response variable, and the standardized mean trait difference (log SMD) as the predictor variable. Questions 2 and 3 were examined with analysis of covariance with log SMD as the covariate. Fixed factors included trait type, taxa, and the geographic and ecological contexts . A significant interaction between SMD and these factors would indicate that the effect of sexual selection is context-dependent. Furthermore, analysis of variance was used to determine the primary factors that influence phenotypic divergence and the magnitude of assortative mating independently from one another. Separate analyses were run with IPSI, and SMD as response variables. Factors included in each model were: taxa, trait type, geographic context, ecological context, and the method of estimation of assortative mating (for IPSI analyses only). Models were run with and without the inclusion of „population pair‟ as a random factor because, in some cases, multiple trait types are included for one measure of assortative mating. Results were similar with both models and therefore only models without the random factor are presented here. Finally, contingency analysis was used to relate the same fixed factors as above to the probability of detecting statistically positive, negative or asymmetric evidence for assortative mating.

1.4. RESULTS

1.4.1.Divergence in sexual selection and assortative mating Ignoring all other factors (simple linear regression), the magnitude of divergence in sexually selected traits was not associated with the degree of 2 assortative mating (F = 1.19, p = 0.27; R = 0.008). What then influences variation in the magnitude of the index of isolation (IPSI)? Neither taxa nor trait type influenced IPSI. Instead, the geographic (sympatric, parapatric, allopatric) and ecological contexts (whether or not the trait is subject to divergent natural selection) played a large role (Figure 1.3, Figure 1.4; Table 1.1). That is, for the same degree of divergence in sexually selected traits, populations that are not

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physically isolated show greater discrimination against heterospecifics (F2,144 = 8.37, p = 0.0004). Although the slopes of allopatric and non-allopatric lines are statistically the same (no interaction, Table 1.1), interestingly, their different effect on the magnitude of assortative mating was only evident for lower amounts of trait divergence, as the intercepts converge toward the maximum observed phenotypic difference between populations. In addition, IPSI was greatest in systems where the sexually selected trait is subject to divergent selection owing to differences in habitat and/or resource use (F = 5.39, p =0.02). Regardless of the magnitude of isolation, 49% (72/145) of studies reported statistically significant evidence for assortative mating. Of those that did not show assortative mating, 44% (32/73) showed that this was due to asymmetric isolation: i.e., one population/species prefers homotypic partners while the other prefers heterotypic partners. Contingency analysis confirmed that geography to some extent, and ecology to a large extent, are the most important determinants as to whether or not assortative mating is present, absent, or asymmetrical (Ecology 2 2  2,145 = 16.06, p = 0.013; Geography  4,145 = 18.82, p =0.0045). That is, assortative mating is most likely when the trait is under divergent natural selection and when geographic distributions overlap so that contact between individuals from the different populations is possible (Figure 1.4).

IPSI did not differ, on average, among the three different laboratory estimation methods (no choice, multiple choice, dichotomous choice). In contrast, estimates of IPSI conducted in nature were far greater (F3,140 = 8.23, p < 0.0001), suggesting that preference for conspecifics is underrepresented in captivity or common garden settings.

1.4.2. What predicts the magnitude of trait divergence? In contrast to the above results for assortative mating, the magnitude of divergence in sexually selected traits was not affected by the geographic or ecological context. Instead, variation in trait divergence was best explained by differences among taxa (F7,146 = 4.99, p < 0.0001) and trait types (F4,146 = 3.12, p

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= 0.02). Specifically, acoustic signals are more divergent than behaviour, morphology, or colour in fishes and in insects (Fig. 1.5).

1.5. DISCUSSION

Overall, the magnitude of divergence in sexually selected traits generally does not determine the magnitude of assortative mating between populations or closely-related species. Rather, the presence or absence of assortative mating and the magnitude of discrimination is associated with the degree of geographic overlap (allopatry, parapatry, sympatry) among populations, and to a lesser extent, with divergent natural selection acting on sexually selected traits. That is, heterospecific individuals are more likely to be discriminated against when they are encountered regularly (sympatry), and when sexually selected traits are subject to different strengths of viability selection in the different populations. Stated another way, even small differences in sexually selected traits will result in assortative mating between sympatric populations, but much larger trait differences are needed between allopatric populations. This result confirms previous intuition from models and quantitative analyses that (i) secondary contact promotes the evolution of mating isolation (reviewed in Servedio and Noor 2003), and (ii) sexual selection is most important combined with other evolutionary forces (Kirkpatrick and Lande 1988; Schluter and Price 1993; Questiau 1999).

1.5.1. Do sexual selection and sexual isolation form a continuum? If sexual selection within populations is the route to sexual isolation between populations, then we would expect greater divergence in sexually selected traits to lead to greater assortative mating. Given that this was not the case, our results suggest a decoupling between sexual selection within populations and sexual isolation between populations. Furthermore, it suggests that divergence in sexually selected traits does not always stem from or result in

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divergence in sexual selection due to mate choice (Svensson and Gosden 2007) since phenotypic divergence is likely a result of numerous other processes such as natural selection, genetic drift and founder effects. Studies of specific taxa have revealed variable results in this regard. In some cases, sexual selection within populations seems to contribute to sexual isolation, particularly when an important sexually selected trait is under strong divergent natural selection in sympatry. For example, body size is under divergent natural selection between ecomorphs of the rough periwinkle, Littorina saxatilis, and benthic/limnetic threespine stickleback, Gasterosteus aculeatus (Johannesson et al. 1993; Rolan-Alvarez et al. 1997; Nagel and Schluter 1998). In both cases, mating is assortative based on body size within populations, which translates to high degrees of sexual isolation between populations (Cruz et al. 2004; Boughman et al. 2005, but see Head et al. 2009). Studies of other systems suggest decoupling between sexual selection and sexual isolation. First, high rates of assortative mating are sometimes associated with little divergence in mating traits, such as in the grasshopper species C. brunneus/C. jacobsi (Bridle and Butlin 2002; Bailey et al. 2003) and song sparrows, Melospiza melodia (Patten et al. 2004). Second, low rates of assortative mating are sometimes present despite strong divergence in mating traits, such as between allopatric populations of the tungara frog, Physalaemus pustulosus (Ryan et al. 2007) and Trinidadian guppies, Poecilia reticulata (Endler and Houde 1995; Chapter 2). Third, several studies show evidence for both sexual isolation and trait divergence, but find no relationship between the two (e.gs. Colias butterflies, Ellers and Boggs 2003; Lake Malawi cichlids, Blais et al. 2009). Divergent sexual selection may therefore not be the main driver of sexual isolation between species, and instead might often first require correlations with other divergent evolutionary forces, particularly natural selection. Divergent natural selection clearly correlates with divergent sexual selection in some cases, but whether or not this is a necessary prerequisite for the evolution of assortative mating is uncertain. Due to a lack of within-population studies of sexual selection, this question could not be directly addressed here, however certain patterns

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emerging from the data suggest that the extent to which mating preferences diverge between populations will determine the limits to the completion of speciation.

1.5.2. Isolation asymmetry and female preference evolution The relatively high frequency of asymmetric premating isolation recorded here (22%) suggests that females often prefer trait values not found in their current population but rather found in other populations, often from other environments. Asymmetry has been well documented in multiple Drosophila species pairs (e.g. Coyne and Orr 1989; Price and Boake 1995; Yukilevich 2008), but is also common in other insects species and birds (see Appendix I). Such asymmetry may simply be a function of divergence time (Arnold et al. 1996), particularly if mating preferences take longer to respond to selection than phenotypes. That is, trait values might diverge quickly owing to divergent natural selection but ancestral preferences for those traits are retained for much longer. This is an unlikely explanation in the present dataset, however, because neither divergence time nor whether the compared populations are considered „true‟ species, influenced the likelihood of asymmetry (results not shown). A more likely driver of symmetry in mate discrimination concerns the degree of parallelism in natural and sexual selection (Boake 1991; Ryan & Rand 1993). Unlike the examples of Littorina and stickleback mentioned above, phenotypic divergence between populations often does not closely match the direction of sexual selection acting on that trait (Ryan and Rand, 1993). This mismatch might occur due to a pre-existing sensory bias for a trait that is shared across environments (e.g. Basolo 1995), or because a particular trait value is generally associated with high condition. For example, orange colouration can indicate vigor, and therefore genetic or phenotypic male quality, due to the positive effect of carotenoids on immune function (Hamilton and Zuk 1982). As long as orange, in this example, remains correlated with high condition, divergence in the trait owing to natural selection migh not lead to symmetric mating isolation because sexual selection for the trait remains conserved among

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the populations (Hill 1994; Grether 2000).

1.5.3. Estimating assortative mating: from the lab to the wild Even though ecological differences and geographic overlap appear to accelerate the evolution of assortative mating, premating isolation is rarely complete (Nosil 2009). In fact, out of the 158 population/species pairs compared here, only two showed evidence of complete assortative mating: Gambel‟s quail, Calliplela gambelli/C. californica (Gee 2003) and the wood white butterfly, Leptidea reali/L. sinapsis (Friberg et al. 2008). Although the explicit focus of this study was on the early stages of divergence and therefore isolation would not be expected to be complete, in some instances, post-zygotic isolation was strong despite little or no evidence of behavioural premating isolation. For example, offspring of crosses between pseudoscorpion (Cordylochernes scorpioides) populations from French Guiana and Panama have very low fitness, but 78.3% of inter-population mating trials resulted in copulation (Zeh and Zeh 2007). The discordance between different isolating barriers is further indicated by the higher, on average, estimates of assortative mating reported from field observations as opposed to any method of assessment performed in captivity. This is true across the data set and is also true within systems where both lab and field estimates are obtained. For example, the damselfly (Calopteryx) species pair, C. virgo and C. splendens have a heterospecific mating probability of 42.5% in no-choice experiments (IPSI = -0.47), but this is reduced to 0.03% (IPSI = 0.93) in their contact zone in the wild (Svensson et al. 2007; Tynkkynen et al. 2008). These results indicate that the environmental context of mate discrimination is likely just as important, or even more so, than the trait values independent of the environment. Such experiments are thus useful in revealing the genetic component of the evolution of premating isolation, but are not necessarily informative about the consequences of this evolution for population structure and speciation. Wherever possible, laboratory studies should therefore be complemented by observations (sympatric populations) or experimental manipulations (allopatric populations) in the wild.

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1.5.4. Summary and Implications Intuitively, sexual selection should result in assortative mating if the same criteria for mate selection apply both within and between populations. The present analysis suggests that this is likely not the case in general. That is, although sexual selection can be a promoter of population differentiation, it does not appear to be a consistent driver of premating isolation on its own. Rather, sexual selection seems to reinforce assortative mating only in contexts where making the „wrong‟ choice is more likely (geographic overlap) and more costly (divergent natural selection). The high incidence of asymmetric behavioural isolation in our dataset suggests that the magnitude of divergence between mating preferences may be a strong mediator of the likelihood of speciation. Even without knowing about the direction of mating preferences within and between populations, the lack of correlation between divergence in sexually selected characters and assortative mating suggests that species recognition may not rely on the same cues as mate choice. This does not necessarily rule out a dominant role for sexual selection, however, as mate choice is only one mechanism that results in variation in reproductive success among individuals. Intraspecific competition and modes of antagonistic sexual selection are also potential drivers of diversification (Arnqvist et al. 2000; Gavrilets 2000). Future research would benefit from comparing divergence between sexual and non-sexual characters in both sexes. The key to understanding the potential for sexual selection to drive premating isolation will therefore most likely emerge with more detailed studies on the direction and mode of inheritance of mating preferences within populations.

1.6. ACKNOWLEDGMENTS I am grateful to members of the Hendry and Chapman labs for useful discussion and inspiration. Andrew Hendry helped to greatly improve the manuscript.

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Table 1.1. Results of analysis of variance explaining variation in the magnitude of assortative mating (index of isolation, IPSI) across studies. Trait difference refers to the (standardized) difference in mean values for a trait that is the target of sexual selection within populations. Ecological context refers to whether or not the trait is subject to divergent natural selection, and geographic context refers to the degree of overlap between populations being compared (sympatric, parapatric or allopatric). Significant effects of the model are in bold.

Factor Df F P Trait difference 1, 144 1.50 0.22 (log SMD) Taxa 4, 144 0.73 0.65 Ecological 1, 144 5.34 0.02 Context Geographic 2, 144 8.37 0.0004 Context Trait type 4, 144 2.3 0.07 Taxa x SMD 4, 144 0.78 0.54 Ecological 1, 144 0.31 0.58 Context x SMD Geographic 2,144 0.2 0.82 Context x SMD Trait type x 4, 144 0.78 0.54 SMD

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Figure 1.1. Number of empirical studies from Web Of Science, investigating divergence in sexual selection, sexually selected traits, and/or assortative mating between populations from 1959 to the present.

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Figure 1.3. Linear regression of the index of assortative mating (IPSI) and the difference in a sexually selected trait between populations. Data points and lines are shown separately for populations that have overlapping geographic ranges (“non-allopatric”, open symbols, dashed line) and those that are physically isolated (“allopatric, closed symbols, solid line).

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Figure 1.4. Proportion of studies in the database showing statistical evidence for symmetric, assymetric and no assortative mating in (a) sympatry or parapatry and (b) allopatry. Solid bars indicate that the populations being compared are subject to divergent natural selection on a sexually selected trait.

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Figure 1.5. Average magnitude (and standard errors) of standardized trait differences between populations as a function of signal type and taxa.

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PREFACE TO CHAPTER 2: From general patterns to a natural model system

The results of Chapter 1 revealed that mating isolation is more likely to evolve and be maintained when traits that are under sexual selection within populations are further subject to divergent natural selection between populations. Furthermore, the geographic context, or the likelihood for encounters between individuals from diverging populations, will further determine the magnitude of mating isolation that can evolve. Although a meta-analysis is suggestive of interesting generalities common across taxa and study systems, it nonetheless provides only patterns of correlation, and cannot directly attest to the particular causal mechanisms responsible for such patterns. In the following three chapters, I use controlled laboratory and field experiments with Trinidadian guppies (Poecilia reticulata) to investigate the effects of ecological differences on the evolution of mating traits, mating preferences and mating isolation. In Chapter 2, I use laboratory and field experiments to ask (i) whether or not there is evidence for mating isolation between populations adapted to different predation environments and (ii) how the potential for gene flow, the nature and context of sexual selection, and the divergence in mate signaling traits may modify patterns of mating isolation.

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CHAPTER TWO

Interactions between ecology and geography influence mating isolation in guppies

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2.1. ABSTRACT

Mating isolation can be an important barrier to gene flow during the early stages of speciation, but the common mechanisms and contexts for the evolution of this isolation remain uncertain. For mechanisms, one possibility is adaptation to divergent environments; i.e., “ecological speciation”, in which the geographic context of divergence is largely irrelevant so long as traits under divergent selection influence mating success. Another possibility is that geography matters, with gene flow influencing the type and degree of mating isolation that evolves. In some cases, gene flow impedes mating isolation owing to recombination, whereas in other cases it may promote isolation through selection to avoid inter- population mating (i.e. reinforcement). We consider these possibilities in Trinidadian guppies (Poecilia reticulata), where populations in multiple rivers experience divergent natural selection owing to predation regime. Using laboratory mate choice experiments and genetic parentage assignment in field enclosures, we find evidence for mating isolation between predation regimes – but to an extent that is modified by geography. Specifically, low-predation females always discriminate against high-predation males, whereas high-predation females only discriminate against low-predation males from upstream in the same river. Ecologically driven mating isolation is thus reinforced by selection to avoid mating with maladapted immigrants from geographically proximal sources. This study thus confirms the theoretical intuition that the influence of divergent natural selection on early stages of speciation is likely modified by geography and shared history.

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2.2. INTRODUCTION

Renewed interest in speciation mechanisms has revealed that divergent natural selection can often be a powerful promoter of mating isolation, such that stronger barriers to gene flow exist between populations from more divergent environments („ecological speciation‟, reviewed in Schluter 2000; Coyne and Orr 2004; Funk et al. 2006; Nosil 2008a). When this result holds independent of the geographic relationships among source populations, then mating isolation is often inferred to be an incidental by-product of adaptation. But geographic correspondences might also be important. In particular, ecologically-based mating isolation may be strengthened by selection to avoid maladaptive mating between individuals from different environments – but this „reinforcement‟ can only take place where individuals from the two environments encounter each other (i.e., parapatry or sympatry). This selection to avoid maladaptive matings can be “indirect” through the reduced fitness of hybrid offspring (Dobhzanksy 1940; Butlin 1987) or “direct” through reductions in fitness owing to the actual act of between-type courtship or mating (Servedio 2001; Albert & Schluter 2004; Nosil & Crespi 2006). Fitting this possibility, several laboratory studies of ecological speciation have reported that mating isolation between ecologically differentiated groups is stronger for populations that actually interact in nature (Rundle & Schluter 1998; Nosil et al. 2003; Kay & Schemske 2008). These results extend the current interest in “divergence with gene flow” (Smith et al. 2005; Grant et al. 2005; Nosil 2008b) into the realm of “divergence-because-of-gene flow”, or reinforcement (Servedio & Noor 2003). We addressed this possibility through laboratory and field experiments designed to examine how ecology (adaptation to different environments) and geography (potential for dispersal) might interact to influence the evolution of mating isolation in Trinidadian guppies (Poecilia reticulata). Once reproductive isolation is complete or near complete, it is more difficult to determine how barriers to gene flow initially arose. Studying guppies in this context allowed us to

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focus on differentiated populations within a species in order to best make inferences about mechanisms involved in the early stages of divergence.

2.2.1. Guppies as a natural model Guppies are well known as a study system for documenting the role of ecology for evolutionary processes because, for the most part, they have independently colonized strongly contrasting environments  “high-predation” and “low-predation” sites within each of many rivers (Fajen and Breden 1992; Alexander et al. 2006). These divergent predation environments have led to the parallel evolution of adaptive differences in colour (Endler 1980; Millar et al. 2006), morphology (Hendry et al. 2006), life history (Reznick & Endler 1982), and behaviour (Magurran et al. 1992). Fitting with the predictions of ecological speciation (Schluter 2001), high- and low-predation guppies should preferentially mate with fish from a comparable predation regime. Although the role of predation in adaptive divergence is well established in guppies, its influence on assortative mating and reproductive isolation remains more uncertain (Magurran 1998, 2005), in contrast to patterns seen in closely related species (Langerhans et al. 2007). Natural and sexual selection for male size and colour could be potential mediators of any such assortative mating. In general, low-predation males are more colourful and larger than are high-predation males (Endler 1980; Millar et al. 2006), and laboratory mate choice studies have indicated that these traits influence female mate choice (e.g. Houde 1987; Brooks & Endler 2001a). Moreover, females in different predation environments appear to have evolved different mating preferences for these traits (Stoner & Breden 1988; Houde & Endler 1990; Schwartz & Hendry 2007), which might thus form a foundation for mating isolation. Either direct or indirect selection on high-predation females may account for their preference for male coloration (e.g., females mating with more colorful males might be more visible to predators or colourful male offspring might have lower survival).

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Guppies are also useful for studying the importance of interactions between ecology and geography. Dispersal frequently occurs between some populations inhabiting different predation regimes, but not others, as revealed by surveys of neutral genetic variation. For instance, high-predation populations receive migrants from directly upstream in the same river (Becher & Magurran 2000; Crispo et al. 2006), but essentially never from low-predation populations in other rivers (Alexander et al. 2006; Suk et al. 2009) or from geographically distant low-predation populations in the same rivers (Crispo et al. 2006). Moreover, low-predation populations rarely if ever experience dispersal from high-predation populations further downstream, because large waterfalls frequently separate the two environments. The guppy system thus allows us to test how the evolution of mating isolation in nature might be influenced by ecology (predation regimes) and geography (potential for dispersal).

2.3. METHODS

2.3.1. Laboratory mate choice experiment Our laboratory experiment employed a typical approach used in studies of parallel speciation. This approach involves the use of “no-choice” laboratory experiments to test whether or not females are more likely to mate with males from similar (as opposed to different) environment types, and whether this assortment is influenced by the geographic context of the source populations (Rundle et al. 2000; Nosil et al. 2002; McKinnon et al. 2004; Funk et al. 2006). We implemented this approach by using laboratory-reared guppies from paired high- and low-predation populations in each of three separate watersheds that represent distinct guppy lineages (see Appendix II for detailed information on site locations). At least twenty pregnant females were collected from each of the six sites. These fish and their descendants were then maintained in the laboratory following standard protocols (Schwartz and Hendry 2007) that kept populations separate and avoided mating between relatives. Fish used in the experiments were the first-

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generation lab-born offspring of wild-caught females. As juveniles, these offspring were raised together in family-specific tanks. As males began to mature they were removed and reared separately with untested females from their own population. Lab-born females therefore remained virgins, a state in which they are more receptive to mating. Mate choice trials involved placing single pairs of males and females into aquaria and recording their behavior. Female preference for a given male was scored following standard protocols for guppies (Houde 1987, 1990, 1997), in which the female responses to each male display (indicating her willingness to mate) are summed and then standardized for male display rate. This yields the “fractional intensity of response” (FIR) of a given female to a given male‟s displays (Schwartz & Hendry 2007; see Chapter 3 for more details on this index). A total of 196 trials were conducted with individual virgin females, each tested only once. Each male, however, was tested with four different female types, thus controlling for variation in individual male phenotype. Specifically, individual males from each of the six populations (high-predation and low- predation from the Aripo, Quare, and Yarra rivers) were paired sequentially and in random order with females from four populations - leading to four different “cross types:” (1) same predation and same river, (2) same predation and different river, (3) different predation and same river, and (4) different predation and different river. A predominant role for ecology would be indicated if males were consistently preferred by females of their own predation type, irrespective of the river from which the test individuals came. A predominant role for geography would be indicated if males were consistently preferred by females from the same river, irrespective of the predation environment from which the test individuals came. An interaction between ecology and geography would be indicated if the extent of mating isolation between predation regimes depended on whether males and females were from the same or different rivers. Ideally, all 36 possible mating combinations would have been performed, but due to the large number of fish required, each female population was tested with males from one of the two different rivers for cross types (3) and (4).

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Specifically, females from the Quare River were paired with males from the

Aripo River (High predation: Ncross type 3 = 6, Ncross type 4 = 8; Low predation: Ncross type 3 = 8, Ncross type 4 = 6), Aripo females were paired with Yarra males (High predation: Ncross type 3 = 8, Ncross type 4 = 9; Low predation: Ncross type 3 = 9, Ncross type 4 = 8), and Yarra females were paired with Quare males (N = 9 for all cross types). Trial order was randomized for each male; but, regardless, trial order did not affect male courtship behaviour (e.g., sneaking rate: repeated measures ANOVA, F = 0.2, p =0.90; display rate: F = 0.19, p =0.89). Once a male had been tested with all four females, he was anaesthetized and photographed with a digital camera under full spectrum lights. The sizes of his individual colour spots and of his body were then analyzed using standard protocols (Chapter 3-4; Endler 1980; Millar et al. 2006) implemented in digital image software (http://rsbweb.nih.gov/ij/). Variation in female preference (FIR) was analyzed with generalized linear mixed models (GLMM) that included male identity as a random factor. Fixed factors included female predation type (high or low), female river of origin (Yarra, Aripo, Quare), male river cross type (same or different), male predation cross type (same or different), and all possible interactions. Pure ecologically- based mating isolation would be indicated by a significant effect of male predation cross type that was consistent across all rivers; i.e., with a non- significant interaction between female river and cross type. We first ran models with all populations included. Finding some interesting interactions, we then broke the data down into subsets, including high-predation or low-predation females separately and cross within or between rivers separately. Significance was assessed using restricted maximum likelihood as implemented in SAS. The influence of male orange colouration on relative mating success was examined in two ways. First, male trait values were included as a factor in the GLMM above. Second, linear regressions were used within each female population to assess the relationship between female FIR for a given male and the difference between his phenotype from the average trait value in the female‟s population. A negative correlation would support the hypothesis that mating

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isolation is driven by divergent selection on male colour since foreign males with phenotypes closest to a female‟s local resident males would have the highest mating success. An index of mating isolation (IMI) between populations was calculated as the difference between the average preference (FIR) of females from a given population for their local males relative to foreign males of a given type (FIR own – FIR foreign/ FIR own + FIR foreign, as in Langerhans et al. 2007). Three types of indices where calculated based on the predation contrast and geographic context of the foreign male: different predation type from the same river, different predation type from a different river, and same predation type from a different river. This index ranges from -1 to +1 with 0 indicating no mating isolation and a positive value indicating preference for the native male.

2.3.2. Field-enclosure experiment Laboratory experiments of this sort can detect genetic differences in intrinsic female preferences (Houde 1997), but they might not reflect the full suite of mating biases in nature. In guppies, for instance, mating patterns can be modified by sneaky copulations (Evans et al. 2003), cryptic female choice (Pilastro et al. 2007), sperm competition (Evans & Rustein 2008), male-male interactions (Houde 1988; Price & Rodd 2006), mate choice copying (Briggs et al. 1996), and daily fluctuations in light environments (Gamble et al. 2003), none of which are apparent in no-choice laboratory mating trials. We therefore complemented our laboratory mate choice experiments with field experiments that allowed for all of these effects in the natural environment. Our field experiment used genetic parentage assignment to determine the relative mating success of different male types in stream enclosures in the Marianne River. Here we tried to mimic the type of between-population interactions that might occur in nature: i.e., low-predation males dispersing into a high-predation environment (rather than the reverse). Enclosures were constructed by isolating side-channel habitats in the source site of the high-predation experimental fish with chicken wire and mesh

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fabric, therefore excluding both local guppies and aquatic predators but allowing for natural habitat conditions and water flow. Low-predation males from two different source populations were used, each in competition with high-predation males for mating opportunities with virgin high-predation females (see Supplementary Information, Table 2.S1 for details on site locations). The first low-predation male source population (MLP) was located less than 1km upstream of the high-predation source population, and their pairing in an enclosure in the high-predation site thus represents an interaction that might occur in nature (i.e., “parapatric”). The other low-predation male source population (MLA) was located approximately 3.5km downstream of the high-predation site in a separate tributary. Given that guppy dispersal is primarily downstream (Becher & Magurran 2000; Suk & Neff 2009), this represents an interaction that would occur much less frequently in nature (i.e., effectively “allopatric”). Indeed, pairwise estimates of neutral genetic differentiation indicate that gene flow is likely higher from the upstream low-predation site (FstMH-MLP = 0.195; FstMH-MLA = 0.312, Crispo et al. 2006). A predominant role for ecology would be here indicated if local high-predation males had a mating advantage that was similarly strong over both parapatric and allopatric low-predation males. An interaction between ecology and geography would be indicated if the mating advantage of high- predation males differed when in competition with parapatric versus allopatric low-predation males. Juveniles were collected from a high-predation section (MH) of the Marianne River, and were held in our laboratory in Trinidad. As males matured, they were separated from the females. Forty-six of the resulting virgin females were split equally and randomly into two experimental enclosures in their home (MH) environment (25 females in Enclosure A and 21 females in Enclosure B). Local high-predation males (MH) and foreign low-predation males (MLA or

MLP) were collected from the wild (Enclosure A: NMH= 23, NMLA = 22;

Enclosure B: NMH=12, NMLP= 12) and held in the laboratory for one day before release into the enclosures where they were photographed (see below) and scales removed for DNA analysis. Males were then introduced into the field enclosures

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with the virgin females. After 48 hours in the enclosures, males were returned to their source population, and females were recaptured and returned to the laboratory where they were kept individually in tanks for three weeks. The females were then killed with an overdose of MS-222 and four offspring were dissected from each. These offspring and their mothers were then preserved in 95% ethanol for later DNA analysis.

2.3.3. Genetic paternity analysis Paternity was determined based on allele sharing at six tetra-nucleotide microsatellite markers: Pre8, Pre9, Pre15, Pre46 (Paterson et al. 2005), Pre32 and Pre53 (Watanabe et al. 2003). DNA was extracted from either scale samples (candidate parents) of whole tissue (embryos). Extraction and PCR protocols were as previously described (Paterson et al. 2005). Individual assignment of offspring to potential fathers in their enclosure was conducted with a likelihood-based exclusion analysis in the program CERVUS 3.0 (Marshall et al. 1998; Kalinowski et al. 2007) based on the difference in log-likelihood scores between candidate fathers. The power of assignment based on these markers was relatively low at the individual level but was very high at the source population level. Specifically, 93.67% of the offspring in Enclosure A and 88.3% of the offspring in Enclosure B could be unambiguously assigned to either local high-predation (MH) or foreign low-predation (MLA or MLP) males. Relative mating success of the two male ecotypes within each enclosure was statistically analyzed with contingency analysis. This analysis compared success at siring offspring relative to the random expectation (51% success of MH in enclosure A and 50% in enclosure B). For those offspring that were assigned to individual males with 95% confidence, we then used logistic regression to determine if individual male mating success was associated with colour or body size separately for each enclosure.

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2.4 RESULTS

2.4.1. Patterns of mating biases The laboratory study revealed evidence of ecological divergence in mate choice given a significant interaction (ANOVA F3,123= 7.34, p = 0.01) between female predation type (high or low) and cross type. We therefore subdivided the data set to determine the nature of this divergence. Overall we found that ecology largely determines mating patterns in low-predation females, whereas ecology and geography interact to determine mating patterns in high-predation females (Table 2.1; Fig.2.1). Low-predation females generally discriminated against high-predation males, despite the fact that they essentially never encounter such males in nature. Moreover, this mating isolation was present regardless of whether the high- predation males were from the same or different rivers. Ecology therefore largely overwhelms geography in mating preferences of low-predation females (note that, in this case, “geography” may be more relevant to historical gene flow during colonization than to contemporary gene flow). This is not to say, however, that geography is totally irrelevant. Specifically, low-predation females were less discriminating against high-predation males from the same river (index of mating isolation, IMI = 0.233  0.09 s.d.) than they were against high-predation males from a different river (IMI = 0.413  0.09 s.d.; Table 2.1; Fig. 2.1;Supplementary Information, Table 2.S2). Since low-predation populations were likely originally colonized from high-predation populations in the same river, this result suggests that some vestiges of ancestral preferences may persist for a considerable time following colonization of new environments. High-predation females, in contrast, discriminated against low-predation males from the same river (IMI = 0.395  0.04 s.d), but not against low-predation males from different rivers (IMI = -0.101  s.d; Table 2.1; Fig. 2.1; Supplementary Information, Table 2.S2). Lack of discrimination in the latter context is particularly clear given that high-predation females showed similar

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responses to high-predation males from different rivers (IMI = 0.12  0.2 s.d). The same general pattern was found in the field experiment (Fig. 2.2), where (1) high-predation males significantly out-competed parapatric low-predation males, siring 90.54% of the offspring (p = 0.001), whereas (2) high-predation males in competition with allopatric low-predation males produced only 65% of the offspring of high-predation females, which does not deviate from random mating (2 = 0.2, p = 0.65). This result therefore mirrors that of the no-choice laboratory mating trials, even with the potential for the full suite of aforementioned mating biases (as opposed to female preference alone) to operate. In short, both the laboratory and field experiments suggest that ecologically-based mating isolation against low-predation males can evolve in high-predation populations – but seemingly only when the specific populations are likely to interact in nature. We infer that mating isolation in high-predation populations does not evolve solely as a by-product of divergence in secondary sexual traits between predation environments, but also requires direct or indirect selection against mating with males of the other predation type in the same river. Importantly, this selection to avoid cross-type matings would likely be ecologically based since guppy populations from the same river do not show any intrinsic genetic incompatibilities (Ludlow & Magurran 2006).

2.4.2. Male phenotype and mating success To further investigate the basis of the observed patterns of mating isolation, we examined the influence of individual male phenotype on his mating success. If mating isolation were evolving as a consequence of divergence in sexually selected traits, then males more phenotypically divergent from the female‟s native population should be discriminated against most strongly, regardless of predation type (e.g. Boughman et al. 2005). The correlation between the extent of divergence in male trait values and relative mating success between predation types varied depending on context. In the field experiment, males from the „allopatric‟ low-predation population are substantially more orange (a trait subject to female mate choice in a number of populations – see

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Houde 1997; Magurran 2005 for reviews) than high-predation males (ANOVA

F1,44 = 9.32, p =0.004; Supplementary Information, Table 2.S1), but neither male predation type nor variation in orange influenced the likelihood of male mating success (logistic regression p = 0.95; Fig. 2, Fig. 2.S1). In contrast, in the „parapatric‟ enclosure, male mating success is positively related to orange colour (logistic regression p = 0.009), and the high-predation males who sired most of the offspring were indeed more orange on average than the low-predation males (ANOVA F1, 25 = 30.51, p < 0.001), (Fig. 2.2, Fig.2.S1). Finally, in the laboratory trials, there was no indication that variation in orange among males generally influenced the observed patterns of mate choice (High predation females F1,81 = 2.4, p = 0.12; Low-predation females: F1,95 = 0.76, p =0.4). These results suggest that even when natural and sexual selection target the same trait, sexual selection within populations and sexual isolation between them do not necessarily follow from each other. Few studies directly address this question empirically; therefore more information is required in order to know how often, and under what contexts mating preferences and mating isolation will be decoupled.

2.5. SUMMARY AND IMPLICATIONS Our results reinforce the importance of examining interactions between ecological and geographical factors in the early stages of divergence and speciation (Nosil 2008a). In fact, our results highlight the importance of additional complexities not normally considered even in speciation research that does examine such interactions. That is, not only did the effect of ecology depend on the geographic context but also the nature of this dependence varied among ecological types. On the one hand, low-predation females routinely discriminated against high-predation males, but more so when they were from a different river. On the other hand, high-predation females only discriminated against low- predation males when they were from the same river. Furthermore, these effects were strongest when males were in direct competition with each other, indicating

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that the social environment and intrasexual selection may be particularly important with respect to mate discrimination. Finally, we showed that colour divergence is not predictable based on predation environment alone, and although it influences mating success in some contexts, orange colour is not universally preferred under all circumstances. These sorts of complexities have not been partitioned out in previous guppy research, which may be why previous workers have argued that guppies do not show progress toward ecological speciation in nature (Magurran 2005; Magellan & Magurran 2007). Importantly, variable interactions of this sort also suggest that the development of mating isolation and speciation can be simultaneously repeatable, complex and asymmetrical due to the interactions of phylogenetic history, divergent ecological context and geography.

2.6. ACKNOWLEDGMENTS Fish collection, rearing and maintenance was greatly assisted by Ann McKellar, Jean-Sébastien Moore, Maryse Boisjoly, Sara Elhajoui, Swanne Gordon and Laura Easty. Zaki Jafry assisted with the field enclosure experiment. Lynn Anstey assisted with DNA extraction and sequencing. Funding was provided by a doctoral fellowship from the Natural Science and Technology Research Council of Quebec (AKS), Discovery grants from Natural Sciences and Engineering Research Council of Canada (APH, PB) and a the National Science Foundation (MTK).

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Table 2.1 Results of geneneralized linear mixed model for variation in female preference as a function of male ecology and geography

High Predation Females Low Predation Females (n = 98) ( n = 98) All Males1 Factor (fixed) DF F DF (num, den) F (num, den) (p -value) (p-value) Male predation 1, 43.41 0.41 1, 44.99 4.65 (same or different as female) (0.53) (0.04) Male river 1, 42.62 1.49 1, 43.86 0.005 (same or different as female) (0.23) (0.94) Male predation x Male river 1, 42.62 6.81 1, 44.02 0.71 (0.01) (0.40) Female river 2, 65.3 1.21 2, 70.09 0.52 (0.30) (0.6) Female river x Male predation 2, 73.19 0.40 2, 69.89 0.4 (0.67) (0.67) Female River x Male river 2, 74.61 0.45 2, 71.22 0.44 (0.64) (0.65) Parapatric Male predation 1, 43 8.79 1, 43 0.89 Males Only (same or different as female) (0.005) (0.35) Female River 2, 43 0.22 2, 43 0.38 (Aripo, Quare, Yarra) (0.80) (0.69) Male predation x Female river 2, 43 0.02 2, 43 0.47 (0.98) (0.63) Allopatric Male predation 1, 43 1.73 1, 43 5.49 Males Only (same or different as female) (0.20) (0.02) Female river 2, 43 1.95 2, 43 0.46 (Aripo, Quare, Yarra) (0.15) (0.63) Male predation x Female river 2, 43 0.67 2, 43 0.35 (0.52) (0.71)

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Figure 2.1. Mean ( s.e) female preference scores for males from the same or different predation regime. Data points represent least-square means of female preference from three rivers within a predation type: (a) high, (b) low. Closed symbols, connected with solid lines, represent males from the two environments in the female‟s own river; open symbols connected with dashed lines represent allopatric males. See Table 2.1 for statistical details

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Figure 2.2. Relative mating success in field enclosures for high-predation (solid bars) and low-predation (open bars) males with high-predation females. Results are shown for two enclosure types in which high-predation males competed for matings with low-predation males from an upstream population („parapatric‟) and an isolated population („allopatric‟) in the same river. Mating success is shown as the proportion of total offspring sired by males of each population

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Supplementary Information.

Table 2.S1. Phenotypic variation among males in the enclosure experiment.

Population N Latitude Longitude Mean Mean Body relative Area orange area (mm2) (%) High- 35 10˚46.071 N 061˚18.246 W 62.04a,b 7.45 (0.49)a Predation (1.46) (MH) „Parapatric‟ 20 10˚45.207 N 061˚18.707 W 66.44a 3.65 (0.62)b Low- (1.84) Predation (MLP) „Allopatric‟ 22 10˚46.378 N 061˚17.541 W 57.98b 11.31 (0.84)c Low- (2.49) predation (MLA) F-statistic 3.97 27.81 (p-value) (0.02) (<0.0001)

Least-square means, standard errors, and results of ANOVAs comparing variation in body area and relative orange area in males among the three populations used in the field enclosure experiment. Superscripts above means indicate homogeneous subsets from post-hoc Tukey tests.

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Table 2.S2. Indices of ecological and geographical mating isolation by female population. Female Population Parapatric Ecological Allopatric Ecological Geographic Mating Mating Isolation Mating Isolation Isolation

River Predation Aripo High 0.405 -0.443 0.386 Quare High 0.348 -0.026 -0.116 Yarra High 0.433 0.167 0.101 All High 0.395 -0.101 0.124 Aripo Low 0.133 0.307 -0.307 Quare Low 0.343 0.447 -0.566 Yarra Low 0.224 0.486 0.381 All Low 0.233 0.413 -0.164

Indices of mating isolation are estimated as the standardized difference in female response to a foreign or local male (see Methods). Because three types of foreign crosses were performed (see text), multiple indices can be calculated. The three shown here inform different hypotheses about the roles of ecology, geography and their interaction in mating isolation. “Parapatric ecological mating isolation” compares female preferences for a local male to a male from the same river but different predation type; allopatric ecological mating isolation compares local preferences to preferences for males from a different river and predation type and geographic mating isolation compares preferences within a predation type but from different rivers.

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Figure 2.S1. Fitted lines of logistic regression of male mating success (whether or not a male sired at least one offspring in the enclosure) on variation in area of orange (%) when high-predation males were competing against (a) parapatric low-predation males and (b) allopatric low-predation males.

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PREFACE TO CHAPTER 3 Before our eyes: Contemporary evolution in nature

In Chapter 1 we saw that the magnitude of reproductive isolation within many systems appeared to be dictated to a large extent by whether or not mate discrimination was symmetric. Specifically, despite large differences in male signaling traits, it is common to see heterospecific/typic preferences in one of the two female species/population types. The results of Chapter 2 somewhat mirrored this pattern as low-predation females always discriminate against high-predation males, but high-predation females only discriminate against the other type when they are geographically proximate. Furthermore, this study showed that both divergence in male traits and the nature of selection for these traits varies spatially across rivers and population pairs. Together, this work suggests that the potential for the evolution of mating preferences can mediate the extent of diversification and speciation possible. In order to address this question, we must first understand how and if female preferences respond to divergent natural selection. Very few studies have been able to establish the nature and rate of the evolution of female preferences in natural populations. Chapter 3 aims to do just that by taking advantage of a transplant experiment of a high-predation guppy population into a guppy-free low-predation river. Ten years later, after guppies had colonized the entire river (including a high-predation section downstream of the introduction site), we were able to ask whether the magnitude and direction of divergence in male colour and female preferences parallel (i) that observed in the ancestral river and (ii) patterns observed in other rivers throughout the range.

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CHAPTER THREE

This is not déjà vu all over again II. Female preferences for male guppy colour in a new experimental introduction.

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3.1. ABSTRACT

The contemporary evolution of female preferences in response to environmental changes has not been examined in natural populations. The introduction of guppies (Poecilia reticulata) from the Yarra River into the Damier River in Trinidad afforded an opportunity to do so. We here compare female preferences for aspects of male colour among four populations: ancestral Yarra high- predation, descendant Damier high- and low-predation, and Yarra low-predation. We employed two mate choice designs, one that paired males and females within populations and another that paired females from each population with ancestral (Yarra high predation) males. We found some evidence that female preference had evolved somewhat since the introduction (after 8 years or 13-26 guppy generations) for orange colour, but no indication that divergent natural selection has influenced patterns of mate choice for any male trait. In combination with previous work, these results suggest that the evolution of guppy colour and female preferences are influenced by factors in addition to predation, and that female preferences may take longer to evolve than other types of traits.

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3.2. INTRODUCTION

Many studies have documented adaptive changes in phenotypic traits for natural populations experiencing environmental change (reviews: Hendry & Kinnison, 1999; Reznick & Ghalambor, 2001; Stockwell et al., 2003). Interestingly, comparatively few of these studies have documented changes in traits subject to sexual selection (Svensson & Gosden, 2007), although some studies have found that such traits can indeed evolve on short time scales (e.gs. Seehausen et al. 1997; Candolin et. al., 2007). Svensson & Gosden (2007) suggested that this apparent deficit might reflect something fundamentally different about how the targets of sexual selection, as opposed to natural selection, respond to environmental changes. For example, secondary sexual traits might be stabilized by the often opposing nature of natural and sexual selection, and by differences in selection between the sexes that nevertheless share a genetic background (Tufvesson et al., 1999; Taylor et al., 2008). To date, however, too few studies have been conducted to be certain whether or not secondary sexual traits really do respond qualitatively differently to environmental change.

Environmental changes that can influence natural selection are well known (Endler 1986; Wade & Kalisz, 1990; Bell & Collins, 2008 ), and so we here concentrate on how environmental changes might influence sexual selection. First, the nature of sexual signals (visual, acoustic, olfactory) might be directly affected by the environment. For example, altered light properties can change how signals are transmitted and received (Seehausen,1997; Candolin et al.,2007). Second, individuals may respond to environmental changes through plastic alterations of their sexual signals or preferences for them. For example, birds near cities change their songs so as to be more distinctive from background noise (Slabbekoorn & Ripmeester, 2008), and female guppies are less choosy in the presence of predators (Gong, 1997). Changing environments might therefore directly influence sexual signals and preferences for them, which might then alter the influence of preference functions on overall fitness and lead to the evolution of traits and preferences.

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Alternatively, sexual selection need not always respond to environmental change. One obvious example comes from the asymmetric patterns of mating isolation seen in many taxa, where (for example) males of species A are preferred by females of both species A and B (e.g., Dettman et al., 2008; Nickel & Civetta, 2009). Such conservatism of mate preferences could occur, for example, through good genes benefits (Watt et al., 2001) or sensory biases (Rodd et al., 2002) - because these properties might sometimes be similar among populations, even in different environmental conditions. Another possibility is that traits and preferences would ultimately co-evolve in a reasonably stable environment, but dynamic conditions prevent this in contemporary populations.

Therefore, environmental change might or might not influence the evolution of sexually-selected traits and preferences for them, yet the particular circumstances resulting in either outcome remain elusive. The few relevant studies conducted so far suggest that spatial variation in secondary sexual traits is at least sometimes driven by spatial variation in sexual selection that is associated with the environment (Arnqvist, 1992; Kwiatkowski & Sullivan, 2002; Svensson et al., 2006). These observations suggest that female preferences can sometimes evolve in response to environmental change – but the generality of this result is unknown. As with studies of adaptation in general, a powerful approach is to experimentally alter the environment and then track evolutionary changes. In the laboratory, for example, female preferences evolve in response to altered natural selection (Rice & Hostert, 1993; Higgie et al., 2000; Rundle et al., 2005). What we ultimately care about, however, is how any such changes might take place in nature, where multiple environmental variables potentially interact. We explored these questions using experimentally-introduced guppies (Poecilia reticulata) in natural populations in Trinidad.

3.2.1. Natural Selection, Sexual Selection and Guppies Guppies provide one of the classic examples of interactions between natural and sexual selection. With respect to sexual selection, colourful males are

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often at a reproductive advantage because they are more often favoured by females (Kodric-Brown, 1985; Houde, 1987; Endler & Houde, 1995; Brooks & Endler, 2001b). With respect to natural selection, however, colourful males are thought to be at a survival disadvantage because they are more susceptible to predation (Endler, 1980). Spatial variation in natural selection is thus thought to cause the observed spatial variation in male colour, with males in low-predation locations generally being more colourful than those in high-predation locations (Endler, 1980; Magurran & Ramnarine, 2005; Millar et al.,2006). Endler (1980) elegantly tested this hypothesis through an experimental introduction in the Aripo River in Trinidad and found that high-predation males introduced into a low- predation site quickly evolved increased colour. We recently reported on the first attempt to replicate Endler‟s (1980) results (Karim et al., 2007). The Damier River, located on the north slope of the Northern Range Mountains of Trinidad, was guppy-free in the early 1990s. In 1996, D.N. Reznick introduced 200 fish from a high-predation site on the Yarra River into a low-predation site above a barrier waterfall in the Damier River (Fig. 1; Karim et al. 2007; Gordon et al. 2009). Qualitative surveys in the Damier a year later revealed that guppies had become established at the low-predation site and had also spread downstream over the barrier waterfall to colonize the high- predation site. In 2004, 8 years (13-26 guppy generations) after the introduction, we sampled guppies from the Damier and the Yarra and found that little genetic change in male colour had taken place (Karim et al., 2007). This striking contrast with the changes observed in Endler‟s (1980) study has motivated our ongoing investigation into the contemporary evolution of secondary sexual traits and preferences for them. This motivation has been strengthened by recent additional work showing that (1) guppy colour has actually changed little in other historical introductions, including Endler‟s, (2) the direction of evolution of colour between high- and low-predation males varies spatially and temporally (Kemp et al. 2008; Millar et al., unpublished data), and (3) that viability selection against colour is not dramatically stronger in high-predation populations than in low-predation populations (Weese et al. unpublished data).

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One hypothesis for variation in how guppy colour evolves is variation among populations in sexual selection owing to female preference. In this case, the evolution of male colour following an experimental introduction would depend on female preferences in the introduced fish, as well as the ability of female preference to evolve. Although female guppies from low-predation environments do often seem to converge on a preference for colourful males (Stoner & Breden, 1988; Houde & Endler, 1990; Godin & Biggs, 1996; Gong, 1997; Brooks & Endler, 2001b), geographic variation in female preferences is certainly prevalent even within a given predation regime (Houde & Endler, 1990; Endler & Houde, 1995; Schwartz & Hendry, 2007). Under this view, the lack of evolution of measured colour components in the Damier River would reflect (1) the lack of preference for high colour in the ancestral Yarra population and (2) relatively little evolution of female preference for high colour in the Damier following the introduction. Schwartz & Hendry (2007) confirmed the first prediction in that Yarra high-predation females do not seem to prefer more colourful males in their population. Here we test the second prediction by asking whether female preferences have evolved in the Damier. We do so by examining population-level female preference functions in the ancestral Yarra high-predation population, the derived Damier high- and low-predation populations, and a reference Yarra low-predation population (Fig. 1). Note that our interpretations of this comparison assume that female preferences have not evolved in the Yarra high-predation population since it was used for the Damier introduction. We have no direct evidence of this, but it seems reasonable given the lack of any obvious changes in environmental conditions (D.N. Reznick, pers. comm.).

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3.3. METHODS

3.3.1. Experimental populations and laboratory rearing In April of 2004, 15-20 pregnant females from each of the four study populations (Fig. 3.1) were captured in Trinidad and transported to our laboratory at McGill University. The offspring born of these females were then mated in a randomized design within each population – although always excluding brother- sister matings. These matings are the same as those described in more detail by Karim et al. (2007). Guppies in the resulting first-generation lab-reared offspring were then mated according to the same design. The resulting second-generation lab-reared offspring were raised in population-specific 5-gallon aquaria. These aquaria were checked daily for developing males, which were immediately removed, thus ensuring the females remained virgins. Virgins were desired because they are more likely to be receptive to males during mating trials (Bearends et al., 1955; Liley, 1966). The males were held in population-specific tanks with non-experimental females from their own population to ensure mating experience (Farr 1980; Price & Rodd 2006). Males were then individually isolated in the experimental aquarium for at least one hour prior to the trials described below.

3.3.2. Mate choice trials We employed a „no-choice‟ design that placed single males with single females to examine male-female interactions (e.g., Dugatkin, 1992; Houde, 1997; Nosil et al., 2002; Nosil et al., 2003; Rutstein et al., 2007; Schwartz & Hendry, 2007). Although a „no-choice‟ design might not represent typical conditions in the wild, it does reveal intrinsic female preferences while controlling for social interactions (Dugatkin & Godin, 1992). This study design seemed the most appropriate a priori because we were specifically interested in relative intrinsic female preferences (see Introduction), rather than absolute mating rates or intrasexual interactions.

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The trials took place in 2-gallon tanks covered on three sides with black paper and illuminated by an overhead full-spectrum fluorescent bulb (Vita-Lite 40W, which approximates the colour spectrum of full sunlight; Duro-Test Canada). After a female was introduced into a tank with the male, a given trial lasted a minimum of twenty minutes. Trials continued for longer (up to a total of 30 minutes) if the male continued to court the female. All trials were recorded with a video camera (Canon XL1-S) and then transferred to DVD for analysis. We used two experimental designs that allowed complementary inferences. In the first design („home population‟), males and females were paired within each of the four populations, so that each fish was used only once. Ten trials were performed for each population, generating a total of 40 trials that used a total of 40 males and 40 females. Females from the different populations thus interacted with males from different populations (i.e., their own population). As a consequence, the resulting population-level female preference functions were likely influenced by variation among populations in both female preferences and in male traits/behaviours. We consider this design useful for understanding the nature of sexual selection operating as a result of paired male-female interactions within populations. In the second experimental design („standard-male population‟), females from each of the four populations were presented individually and in random order to single males from a common test population (Yarra high-predation - the ancestral population). That is, a single Yarra high-predation male was tested in random order with each of four females: one Yarra low-predation female, one Yarra high-predation female, one Damier low-predation female, and one Damier high-predation female. This procedure was then repeated nine more times, each time with a new male and a new random sequence of females from the four populations. The result was a total of 40 trials that used a total of 10 males and 40 females. This design was useful in several respects. First, it yielded female preferences that were independent of any population-level variation among males (although variation in female preferences might still depend on the specific male

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population). Second, it allowed us to statistically control for individual variation among males – because each male was presented to each female type.

3.3.3. Quantifying male colour and female preference After the trials ended for a given male, he was anaesthetized with MS-222 and photographed (Nikon Coolpix F995) on a standard, grid-ruled background. The pictures were then analyzed (Scion Image Software: www.scioncorp.com) to determine the size of the male (surface area on the left side of the body) and the number and area of spots of different colours: black, orange, violet-blue, and green. These measurements excluded the tail, which is fragile and difficult to position in a standard and repeatable fashion. For a detailed description of these methods see Millar et al. (2006) and Karim et al. (2007). The total area of a given colour on a male depends on the size of the male, and so we also calculated the „relative area‟ of each colour as the total area of a given colour divided by the total area of the male. A female‟s response to a given male was estimated as the intensity of her responses to his displays. Guppy courtship usually involves a „sigmoidal display,‟ in which a male places himself in front of a female, arches his body, extends his fins, and performs several jerking vibrations (Baerends et al., 1955; Liley, 1996; Houde, 1997). Females may respond to a given male display in several ways: no response (score = 0), turning and orienting her body toward the male (1), gliding toward him (2), circling him (3), receiving an attempted copulation (4), or allowing full copulation (5) (Houde, 1997). Following full copulation, a male‟s body will jerk over an interval of several minutes, and he will closely guard the female (Baerends et al.1955; Pilastro et al., 2007). Using this scheme, we scored each response of each female to each male display over the course of each trial. The scores for female responses were combined in a given trial to yield an overall estimate of a given female‟s preference for a given male (Houde, 1997; Schwartz & Hendry, 2007). This was done by first calculating the total (cumulative) response (TR) for a given trial as the sum of the female‟s responses to all of the male‟s displays. TR was then standardized as a fraction of the

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maximum total response (MR) that a female could have shown given the number of male displays in that trial. MR was calculated assuming an attempted copulation (score = 4) after each male display followed by full copulation on the last display (score = 5): i.e., MR = ((Number of displays)-1)*4)+5. This standardization yielded a „fractional intensity of response‟ (FIR = TR/MR) for a given female to a given male over the course of a trial. Note that FIR controls for variation among trials in the frequency of male displays. After quantifying male traits and female FIR, female preference functions were calculated for each population in each type of trial (home population or standard-male population) as a linear relationship of FIR on each male trait (Houde & Endler, 1990; Endler & Houde, 1995; Brooks & Endler, 2001b; Gamble et al., 2003; Syriatowicz & Brooks, 2004; Schwartz & Hendry, 2007). We recognize that female preferences can be much more complicated multivariate surfaces (e.g. Blows et al., 2003) and can be frequency dependent (Kokko et al., 2007; Drullion & Dubois, 2008), but we were here interested specifically in trying to explain directional changes (or the lack thereof) in the overall amount of a given colour based on directional female preferences (or the lack thereof). In this sense, the slopes of linear relationships represented a sexual selection gradient appropriate for predicting evolutionary change in the relative amount of each colour.

3.3.4. Statistical analysis Variation in male colour was first analyzed in one-way ANCOVAs (populations as four levels of a single, fixed factor) considering the relative area of each colour category individually: black, orange, violet-blue, and green. Male body area was included as a covariate in all analyses so as to control for possible allometric effects of body size on the relative area of colour. Post-hoc Tukey tests were used to determine which of the four populations differed from each other following a significant result in (M)ANCOVA. We were also interested in the possibility of general river and predation regime effects, and so we repeated the

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above analyses as two-factor ANCOVAs (river and predation as fixed effects, plus their interaction) with the same response variables. Variation in female preference functions was first analyzed in ANCOVAs (populations as four levels of a single, fixed factor) considering each of the four colour categories both individually (as a single covariate per model) or together (all colour covariates in the same model). For both experimental designs (home population and standard-male population), the models included population (fixed), the relative area of each male colour category (covariate), and interactions between population and each male colour category. These models also included male size as a covariate – to control for possible allometry. Differences in female preference functions among populations would be revealed by significant population-by-trait interactions. The contemporary evolution of female preference would be indicated if preference functions differed between the Yarra high- predation (ancestral) population and the Damier (derived) populations. Natural selection would be inferred if divergence between predation regimes in the Damier was in the same direction as divergence between predation regimes in the Yarra. As was done with male colour (above), we also reran the above analyses in two-way ANCOVAs with river (fixed), predation regime (fixed), and their interaction, as separate factors. For the „standard-male population‟ trials, the models were run with male identity as a random factor, thus controlling for variation among males.

3.4. RESULTS

3.4.1. Male colour Males reared for two generations in a common-garden laboratory environment differed significantly among the four populations for two traits: the relative area of orange and body size (Table 3.1). Most notably, Yarra low- predation males had relatively more orange than did Yarra high-predation males (Fig. 3.2). Damier males were generally intermediate in orange between the two Yarra populations, although they were not significantly different from either

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(Table 3.1; Fig. 3.2). Similarly, Yarra low-predation males were larger than males from the other three populations, and significantly different from Damier-low predation males indicating no general genetic effect of predation regime on body size. Results were similar when variation in male colour was analyzed in a two- factor framework (river vs. predation regime – Table 3.S1). That is, predation regime did not have a consistent effect between the two rivers and so was not significant as a main effect. River effects, however, were evident in that Yarra males were larger than Damier males and Damier males had more violet-blue color than did Yarra males (Fig 3.2). One significant interaction was evident: that between predation and river for orange colour. This reflects the previously mentioned evidence of divergence between predation regimes in the Yarra but not the Damier (Fig. 3.2).

3.4.2. Variation in female preference functions In the home population trials (females tested with males from their own population), female preference functions for male colour were statistically similar among the four populations (i.e., no population-by-colour interactions) when traits were considered together (Table 3.2) or independently (Table 3.S2) in linear models. Further, few significant relationships between male colour and female responses were found (i.e., main effects of male colour covariates) indicating that the strength of sexual selection owing to intrinsic female preferences appears to be weak and/or variable among populations. The only exception here was that females generally preferred males with less violet-blue when traits were examined independently (Fig. 3.3; Table 3.S2). Results were generally similar in the standard-male population trials (females paired with males from the Yarra high-predation population). That is, preference functions were similar (i.e., no population-by-colour interactions) across the four female populations, when traits were considered together (Table 3.2) or independently (Table 3.S2). Moreover, none of the colour traits here showed main effects on female preference – again demonstrating that preference

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functions were generally quite flat and/or variable among populations (Fig.4). These results were confirmed by simple linear regressions for each colour in each population (Table 3.S3; Figs. 3.3 & 3.4). As in the one-factor models above, few significant differences in preference functions among female populations were detected in models which considered the effects of predation regime and river (Tables 3.S4 and 3.S5). The one exception was a significant effect of river for preferences for orange: females from both Damier populations preferred more orange males in their home populations, whereas Yarra females discriminated against the more orange males in their populations (Fig. 3.3; Table 3.S5).

3.4.3. Results Summary We found very few differences in female preference functions among populations. The only difference that we did find (preference for orange), was more strongly associated with river than with predation regime. This suggests that this evolution in female preferences since introduction to the Damier River did not occur because of the broad contrast between high- and low-predation environments. To confirm these conclusions, we removed the Yarra low- predation population from analyses in order to directly compare the female preferences of each Damier population to their Yarra high-predation ancestors. Results were similar to those reported above in that female preferences did not differ except for orange (results not shown).

3.5. DISCUSSION We have tested for contemporary evolution following the experimental introduction of high-predation guppies into the Damier River, where they colonized both high- and low-predation environments. In a previous paper (Karim et al.,2007), we showed that the introduced guppies have not experienced any noteworthy evolution with respect to the size and number colour spots, a result in contrast to the large changes documented in Endler‟s (1980) introduction experiment in another river. This difference between studies led us to consider the

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potential role of female preferences in constraining the evolution of male colour. We first found that contemporary representatives of the introduced Yarra high- predation population do not generally prefer more colourful males (see also Schwartz & Hendry, 2007). Assuming preferences have not changed in the Yarra since it was used for the introduction, any evolution of increased male colour in the Damier would therefore first require the evolution of female preference for colourful males. In this study we present evidence that while the evolution of female preference may have started in the Damier, it is relatively weak and not associated with differences in selection due to predation environment. That is, some analyses showed that females from both Damier populations currently prefer more orange males than do females from the Yarra high-predation source population. The observed change might reflect the fact that the Damier is a very different environment from the Yarra, being smaller and having a more closed canopy (Gordon et al. 2009). Ultimately however, we found no evidence of the evolution of preferences for other colours. We therefore first discuss potential reasons for why the evolution of female preferences has been so modest. We then discuss the implications of our results for understanding some key topics in evolutionary ecology.

3.5.1. Why has evolution been so modest? First, aspects of our experimental protocols may have misrepresented the evolution of female preferences as they would be expressed in nature. For instance, our experimental trials did not account for variation in light, water, and background conditions, which can influence male courtship and female choice (Long & Rosenqvist, 1998; Gamble et al., 2003). In addition, we used “no- choice” trials (single male:female pairs) that isolate intrinsic female preferences from the social interactions that can be important in nature. Examples of such interactions include mate choice copying (Godin et al., 2005), male-male competition (Houde, 1988; Jirotkul, 1999), and frequency dependence (O‟Donald & Majerus, 1988; Pemberton et al. 2003). Further, our inferences apply only to

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the aspects of male colour that we measured. For instance, evolution might have occurred with respect to the spectral characteristics of spots (sensu Kemp et al., 2008) or multivariate trait combinations (Blows et al.,2003). Second, female preferences might not evolve if the introduced population lacked appropriate genetic variation. For example, the original introduction may have been accompanied by founder effects or subsequent bottlenecks. We do not favour this explanation because (1) 200 introduced individuals would likely harbor considerable genetic variation, particularly given that multiple mating and sperm storage is typical of guppy females (Becher & Magurran, 2004; Kobayashi & Iwamatsu, 2002; Pitcher et al,. 2003); (2) the population was quite robust a year after the introduction (D.N. Reznick, pers. obs.); and (3) Damier guppies are indeed variable for male colour and female preference (Karim et al.,2007; present study). More generally, sexually selected traits tend to harbor considerable genetic variation (Svensson & Gosden, 2007; Ahuja & Singh, 2008), as is certainly the case for male guppy colour (Brooks & Endler, 2001a). For female preferences of guppies, however, the amount of genetic variation is less certain (Godin & Dugatkin, 1995; Brooks & Endler, 2001b; Brooks, 2002; Lindholm & Breden 2002; Hall et al. 2004). Third, selection might not strongly favour the evolution of new female preferences in the Damier. Although this explanation is certainly possible, we have no data to directly inform its plausibility. Indirectly, however, our results suggest that predation regime is not an overwhelmingly important determinant in these rivers. For example, females from both Yarra populations show similar preference functions despite dramatic differences in predation and in male traits. These results suggest that factors other than intrinsically-based female preferences for singly-encountered males might be contributing to the colour differences. Finally, selection and genetic variation may both be present but noteworthy evolution might just take more time. It is hard to evaluate this possibility in general because no study has examined the time course of evolution for sexually selected traits or female preferences in populations experiencing environmental change. In our study, the evidence in some analyses that Damier

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females are starting to evolve a changed preference for orange does seem to suggest that some subtle change has begun, though time will tell just how far it goes.

3.5.2. Implications Our results inform several topics in evolutionary ecology. Our focus is on the potential rate of evolution of sexually selected traits in natural populations – a topic of broad interest and importance but one that has received little empirical attention, making it difficult to generalize. The contemporary evolution of mate choice might influence the contemporary evolution of reproductive isolation (Ritchie, 2007). Furthermore, the contexts in which preferences are expected to evolve and how quickly this can be accomplished relative to secondary sexual traits can inform not only sexual selection theory, but enhance conservation strategies to predict how populations will respond to environmental change. We now consider each of these topics in turn. We have shown that male colour does not always increase when high- predation guppies are introduced into low-predation environments (Karim et al.,2007; Fig. 3.2). This result has been recently reinforced by other studies (see Introduction). Divergence in male colour between predation environments thus appears less consistent than divergence in other types of traits, such as life history and behaviour (Endler, 1995; Reznick et al.,1997; Magurran & Ramnarine, 2005, Gordon et al. 2009). Perhaps environmental change, at least in the present context, imposes stronger directional selection on traits that influence survival and fecundity than on those that influence mating success. This would not be surprising given that one must first survive before mating becomes relevant. It also suggests that the evolution of sexually selected traits might require the evolution of female preferences. The contemporary evolution of mate choice might influence the contemporary evolution of reproductive isolation (Ritchie, 2007). Divergent natural selection has been argued to drive the onset of pre-mating reproductive

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barriers (e.g., selection against migrants and hybrids) (Hendry, 2004; Hendry et al., 2007; Thibert-Plante & Hendry, 2009) and correspondingly, in this system Yarra fish now survive at lower rates in the Damier than do Damier residents (Gordon et al. 2009). The completion of reproductive isolation, however, might require the evolution of female preferences, which may take longer (Gavrilets & Boake, 1998; Dieckmann & Doebeli, 1999; Thibert-Plante & Hendry, 2009) but in some cases can drive rapid diversification (e.g., Maan et al. 2006). If mating isolation requires divergence in secondary sexual traits and preferences for them, then the lack of preference evolution we found here could suggest that in some cases slower preference evolution might limit the rate of reproductive isolation. This might also help to explain why reproductive isolation has not been found in other similar Trinidadian drainages (e.g. the Marianne, Crispo et al. 2006). Although some studies have documented the contemporary evolution of mate choice in the laboratory, none has tested for such evolution in nature. We found that female preferences did not evolve quickly when guppies were introduced into new environments. Although some evolutionary change was implicated (change in preference for orange), this change was modest and only detectable in some analyses. Perhaps more time will allow more change. Regardless, when contrasted with the repeated evidence for noteworthy evolution of a large suite of traits in introduced guppy populations (see above), the apparently small change in preference suggests constraints on the rapidity of preference evolution which, in turn, can influence the rate and magnitude of evolution of secondary sexual signals. Relative to the influence of natural selection, very little is known about how elements of sexual selection can respond to abrupt environmental changes (Candolin & Heuschele, 2008). The few empirical studies conducted so far show mixed results and focus on correlative patterns at macro-evolutionary scales (e.g., Morrow & Pitcher, 2003). Given that many populations are subject to increasing rates of human-induced environmental perturbations, there is a need for understanding evolutionary responses at a contemporary scale. The Damier introduction experiment afforded us a first opportunity to address this question and suggests that sexual selection may be less

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dramatically altered by environmental changes than is natural selection. Indeed, the only quantitative analysis to date of the strength of selection in nature suggested that sexual selection is only stronger than natural selection over long time scales (Hoekstra et. al., 2001). Determining the extent to which this potential discrepancy affects the overall adaptive potential of a population responding to environmental perturbations will clearly be the next challenge.

3.6. ACKNOWLEDGMENTS We are grateful to D. Reznick for introducing us to the Damier experimental introduction, and for helping in the field. Field work was also assisted by M. Kinnison, D. Weese and N. Millar. Laboratory fish rearing was assisted by M. Piette, Z. Jafry and N. Karim. Funding was provided by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant to APH, postgraduate scholarship to SG and USRA to L Easty) and by the Fonds Québecois de Recherche sur la Nature et les Technologies (postgraduate fellowship to AKS).

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Table 3.1. ANOVA results for differences in male colour (relative area) among the four populations, corresponding to the summaries shown in Figure 3.2. Significant differences (at are indicated in bold.

DF (F-statistic) P ANCOVAs Orange 3, 35 3.61 0.01 Black 3, 35 0.62 0.61 Green 3, 35 0.74 0.54 Violet-blue 3, 35 1.67 0.19 Body Size 3, 35 4.31 0.01

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Table 3.2. ANCOVA results for female preferences for male colour (relative area) when all colours were included in the same model. The „Home population‟ design paired individual females and males from the same populations. The „Standard-male population‟ design paired females from each population with the same males from the ancestral population (Yarra high-predation). Home Standard-male population population Term DF F P F P Population 3,39 0.31 0.82 0.68 0.58 Orange 1,39 0.12 0.74 2.23 0.20 Black 1,39 0.00 0.96 1.77 0.24 Green 1,39 0.09 0.77 0.10 0.76 Violet-blue 1,39 2.01 0.17 4.69 0.08 Body size 1,39 0.04 0.85 0.14 0.73 Population-by-Orange 3,39 0.59 0.63 1.71 0.21 Population-by-Black 3,39 0.33 0.80 1.01 0.42 Population-by-Green 3,39 0.20 0.89 0.96 0.43 Population-by-Violet-blue 3,39 0.05 0.99 1.10 0.38 Population-by-Body size 3,39 0.50 0.69 0.24 0.87

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Figure 3.1 Map of the study sites in Trinidad (after Karim et al.,2007). In 1996, 200 fish from the Yarra high-predation site were transplanted to the Damier low- predation site, from which the Damier high-predation site was then naturally colonized.

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Figure 3.2. Aspects of male colour and body size by population: Damier high- predation (DH), Damier low-predation (DL), Yarra high-predation (YH), and Yarra low-predation (YL). High-predation populations are indicated by solid bars; low-predation by open bars. The mean relative area of each colour (%) is shown for each population, along with standard errors of population means. Letters above the bars indicate homogenous subsets as revealed by Tukey tests.

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Figure 3.3. Population-level female preference functions in the home population trials as determined by linear regression of female preference score (FIR) on each male colourcategory (percent of colour on body). Preference functions for the two predation populations in each river are shown together, where the high-predation populations are indicated by solid symbols and lines and the low-predation populations are indicated by open symbols and dashed lines.

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Figure 3.3 continued

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Figure 3.4. Standard-male population female preference functions from linear regression of female preference score (FIR) on variation in male trait values for four colours (percent of colour on body). Preference functions are shown for each of the four female populations, in this case tested against the same ten males from the ancestral (Yarra high predation) population. Preference functions for the two predation populations in each river are shown together, where the high-predation populations are indicated by solid symbols and lines and the low-predation populations are indicated by open symbols and dashed lines.

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Figure 3.4. continued

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Supporting Information

Table 3.S1. Results of two-way ANOVAs examining variation in male colour (relative area) as a function of predation regime (high or low) and river of origin (Yarra or Damier). Significant differences (at are indicated in bold.

Predation River River-by-Predation Male Trait F P Orange 2.2 0.12 0.36 0.55 9.29 0.004 Black 0.002 0.97 1.32 0.26 0.75 0.39 Green 1.09 0.3 0.98 0.32 0.09 0.77 Violet-blue 0.22 0.65 4.31 0.04 0.97 0.33 Body size 0.1 0.76 9.78 0.004 3.04 0.09

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Table 3.S2. ANCOVA results for female preferences for male colour (relative area) when each colour was analyzed in a separate model. The „Home population‟ design paired individual females and males from the same populations. The „Standard-male population‟ design paired females from each population with the same males from the ancestral population (Yarra high-predation). Significant preference functions (at are indicated in bold.

Home Standard-male population population Male Trait Terms F P F P Orange Orange (main effect) 0.4 0.53 0.001 0.97 Population 0.18 0.91 0.72 0.55 Population-by-Orange 1.76 0.17 1.79 0.18 Black Black (main effect) 0.09 0.76 0.62 0.61 Population 0.56 0.65 0.02 0.88 Population-by-Black 0.51 0.68 0.34 0.79 Green Green (main effect) 0.36 0.55 0.04 0.85 Population 0.63 0.6 0.65 0.59 Population-by-Green 0.53 0.67 0.79 0.51 Violet-blue Violet-blue (main effect) 5.94 0.02 2.27 0.17 Population 1.14 0.34 0.82 0.5 Population-by-Violet-blue 0.59 0.62 0.51 0.68 Male size Body area (main effect) 0.18 0.67 1.34 0.28 Population 0.59 0.63 0.82 0.49 Population-by-Body Area 0.26 0.86 0.56 0.64

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Table 3.S3. Linear regressions of female preference (FIR) on male trait values in the „home population‟ trials, indicating the strength (r2) and direction (slope,  in parentheses within each, corresponding to summaries in Figures 3 and 4. Significant preference functions (at are indicated in bold.

Female Yarra Low Population Yarra predation Damier Damier Low High (control) High predation Predation Predation (colonized from (introduced) introduction site) Male Trait Orange 0.04 0.40 0.26 0.09 (-0.0294) (-0.0215) (0.0477) (0.0561) Black 0.00 0.02 0.13 0.05 (-0.003) (0.006) (-0.0451) (0.0232) Green 0.01 0.01 0.02 0.14 (0.006) (0.003) (-0.009) (-0.04) Violet-blue 0.02 0.26 0.35 0.31 (-0.018) (-0.016) (-0.054) (-0.042) Body Size 0.02 0.17 0.06 0.02 (-0.006) (-0.006) (-0.012) (0.011)

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Table 3.S4. Two-way ANCOVA results for female preferences for male colour (relative area) as a function of predation regime (high or low) and river of origin (Yarra or Damier) for all traits. The „Home population‟ design paired individual females and males from the same populations. The „Standard-male population‟ design paired females from each population with the same males from the ancestral population (Yarra high-predation). Term Home Standard-male Population population F P F P Predation 0.11 0.74 0.88 0.36 River 1.03 0.32 1.3 0.27 Orange 0.00 0.95 1.06 0.36 Predation-by-Orange 0.66 0.43 0.25 0.62 River-by-Orange 2.32 0.14 0.84 0.37 Black 0.05 0.82 1.43 0.3 Predation-by-Black 0.74 0.4 0.42 0.52 River-by-Black 0.01 0.91 1.55 0.23 Green 0.14 0.71 0.04 0.86 Predation-by-Green 0.35 0.56 0.56 0.47 River-by-Green 0.09 0.76 0.00 0.98 Violet-blue 2.39 0.14 2.63 0.18 Predation-by-Violet-blue 0.07 0.8 0.29 0.59 River-by-Violet-blue 0.41 0.53 1.11 0.31 Male size 0.1 0.76 0.14 0.73 Predation-by-Male size 0.66 0.43 0.00 0.95 River-by-Male size 0.8 0.38 0.56 0.46

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Table 3.S5. Two-way ANCOVA results for female preferences for male colour (relative area) as a function of predation regime (high or low) and river of origin (Yarra or Damier) when each colour was analyzed in a separate model. The „Home population‟ design paired individual females and males from the same populations. The „Standard-male population‟ design paired females from each population with the same males from the ancestral population (Yarra high- predation). Significant preference functions (at are indicated in bold.

Colour Terms Home Standard-male population population F P F P Orange Predation 0.00 0.94 0.98 0.33 Orange 0.17 0.68 0.01 0.95 River 0.71 0.40 0.15 0.24 Predation-by-Orange 0.05 0.83 0.14 0.71 River-by-Orange 5.25 0.03 0.22 0.64 Black Predation 1.14 0.29 0.84 0.37 Black 0.08 0.79 0.02 0.9 River 0.17 0.69 1.09 0.3 Predation-by-Black 1.16 0.29 0.08 0.78 River-by-Black 0.76 0.39 0.21 0.65 Green Predation 1.75 0.20 1.02 0.32 Green 0.63 0.43 0.04 0.85 River 0.46 0.50 1.5 0.24 Predation-by-Green 0.30 0.59 1.19 0.29 River-by-Green 1.25 0.27 0.17 0.68 Violet-blue Predation 1.6 0.22 0.98 0.33 Violet-blue 6.25 0.02 2.30 0.17 River 2.18 0.15 1.45 0.24 Predation-by-Violet-blue 0.09 0.77 0.01 0.92 River-by-Violet-Blue 1.61 0.21 0.27 0.61

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PREFACE TO CHAPTER 4: Microhabitat variation and colour evolution

Many studies have shown that differences in predation intensity result in repeated and predictable effects on male colour (e.g. Endler 1978, 1980; Millar et al. 2006) and female preference evolution (Stoner and Breden 1988; Houde and Endler 1990; Schwartz and Hendry 2007) in guppies. Although there is an intuitive and demonstrated explanation as to why guppies in high-predation environments should be less colourful (more conspicuous individuals are less likely to survive), explanations as to why guppies should evolve to be more colourful once this threat is removed are not as intuitive. The predominant hypothesis has been related to sexual selection for more colour, yet insight from Chapters 1 and 2 as well as recent re-evaluations of earlier studies (e.g. Karim et al. 2007; Kemp et al. 2008) show that neither predation or sexual selection alone are sufficient to explain the extreme polymorphism observed in guppy colour patterns. This suggests that selection likely varies both spatially and temporally due to habitat and/or biotic factors other than predation risk. In Chapter 4, I take advantage of a human modification of a guppy population‟s natural environment in order to isolate one particular variable  light availability  on the temporal and spatial variation in colour patterns.

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CHAPTER FOUR

Testing the influence of local forest canopy clearing on phenotypic variation in Trinidadian guppies

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4.1. ABSTRACT

The factors contributing to the maintenance of phenotypic variation in nature are often difficult to determine. Secondary sexual traits might be particularly interesting in this regard due to the interaction they experience between multiple selective agents. One way to examine such effects is to monitor populations experiencing environmental change. Human-caused changes might be particularly useful here because they often involve an abrupt and extreme alteration of specific habitat features. This alteration can then precipitate phenotypic plasticity, changes in adaptive landscapes, and modified evolutionary trajectories. The consequences of habitat manipulations on local populations can therefore improve our understanding of phenotypic variation in complex ecological systems. We took advantage of a human-caused environmental disturbance to examine factors influencing phenotypic variation in Trinidadian guppies (Poecilia reticulata). Differences in canopy cover along the stream have been hypothesized to explain some of this variation, but this has been hard to test directly. We here attempt a direct test of this hypothesis by monitoring changes in guppy size and colour following a dramatic decrease in canopy cover due to tree removal for agricultural activity. Although male and female body size increased following canopy clearing, little change was observed in the overall amount of melanin- based colours, carotenoid-based colours, and structural colours on males. We further compared phenotypes before and after canopy clearing at the disturbed site to those from two nearby reference sites that are at extreme ends of canopy cover. Overall, variation in colour was attributed to differences among sites, irrespective of canopy differences. We also found considerable temporal variation in some colour elements at a given site. Our results suggest that differences in canopy cover do not cause rapid and dramatic changes in guppy colour. The substantial unexplained variation must therefore be due to factors other than canopy (measured here) and predation regime (all sites were “low-predation”). Because of the multiple and complex interactions involved in the expression and maintenance of sexually selected traits, our study emphasizes the need for a better

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understanding of both the genetic and environmental sources of co-variation between sexual ornaments and preferences.

4.2. INTRODUCTION

4.2.1. Phenotypic Responses to Environmental Change The pace of adaptation in natural populations is an important question from an academic perspective (Hendry & Kinnison 1999; Kinnison & Hendry 2001; Estes & Arnold 2007; Fisk et al. 2007). More recently, its applied relevance has become increasingly apparent (Burger & Lynch 1995; Stockwell, Hendry & Kinnison 2003; Bell & Collins 2008). One common question is whether organisms are capable of responding adaptively so as to avoid population declines in the face of environmental change (Gomulkiewicz & Holt 1995; Willi, Buskirk & Hoffman 2006; Kinnison & Hairston 2007; Visser 2008). These concerns can be particularly relevant in the case of human-caused disturbances, because these often surpass the natural baseline of environmental perturbations (Vitousek et al. 1997; Palumbi 2001). If we are to understand the effects of human disturbance on evolutionary and ecological processes, we must first understand the main environmental drivers in a given situation. That is, although adaptive phenotypic changes have now been observed in many natural populations, it is more difficult to conclusively link specific environmental factors to specific phenotypic responses. These links may sometimes be more obvious in the case of human-caused environmental disturbances (Baker & Stebbins 1965; Reznick & Ghalambor 2001; Blondel 2008; Hendry, Farrugia & Kinnison 2008) in situations where one aspect of the habitat is manipulated. The present study considers one such possibility in Trinidadian guppies (Poecilia reticulata) that show extreme variation in colour patterns. Male guppy colour is a trait subject to both natural and sexual selection, yet polymorphism within and among populations remains

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high. Despite active research in this system, how particular environmental agents are responsible for the maintenance of this variation is not yet evident (Endler 1995; Brooks 2002). Generally, phenotypic variation and responses to environmental change may be particularly complicated with regards to secondary sexual traits. Complications arise here because such traits are sensitive to both natural and sexual selection (Darwin 1871; Andersson 1994), often involve multiple correlated traits (Brooks & Couldridge 1999; Candolin 2003), and can show phenotypic plasticity and context dependence (Griffiths & Sheldon 2001; Price 2006). Colour-based secondary sexual traits may be particularly sensitive to environmental change. For instance, colour is responsive to small scale variation in habitat and social interactions (Chunco, McKinnon & Servedio 2007; Gray & McKinnon 2007; Roulin & Bize 2007), and so even subtle variation in microhabitats can promote colour change. Larger-scale perturbations should have even larger effects, as suggested by the impact of urbanization, pollution and deforestation on bird plumage (e.g. Eeva, Lehikoinen & Ronka 1998; Horak et al. 2001; Smith et al. 2008). Other examples can be found in fishes. One is eutrophication of the Baltic Sea which resulted in accelerated algal growth, and a subsequent decrease in visibility for mate signaling. Consequently, the strength of sexual selection for red colouration in sticklebacks decreased; likely due to the increased costs of carrying (males) and choosing (females) bright red colour (Candolin, Salesto & Evers 2007; Engstrom-Ost & Candolin 2008). Similar effects seem to have occurred in the sand goby (Jarvenpaa & Lindstrom 2004) and in cichlid fishes (Seehausen, van Alphen & Witte 1997). Moreover, trait expression and selection can have cascading effects on each other. On the one hand, plastic changes in sexually selected signals can influence both natural and sexual selection, thus influencing their future evolution (West-Eberhard 2005; Price 2006; Svensson & Gosden 2007). On the other hand, sexual selection can change without direct alteration of traits, such as when environmental change alters signal reception and transmission (Endler 1992; Rosenthal 2007; Gray et al. 2008; Cockburn, Osmond & Double 2008). To date,

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however, the impacts of environmental change on components of sexual selection are rarely investigated, although the topic is gaining interest (Svensson & Gosden 2007; Candolin & Heuschele 2008; Cockburn et al. 2008). In this study we take advantage of a population of guppies that recently experienced a dramatic environmental disturbance in the form of canopy clearing around the stream. We had fortuitously sampled guppies for two years immediately prior to the canopy clearing and were able to sample guppies again for two years afterward. We thus consider the disturbance to be an unplanned “experiment” with before and after samples. We also compare samples from the same years at nearby sites that spanned the natural range of canopy cover, but remained relatively undisturbed during the same time period.

4.2.2. Environmental variation and guppy colour With respect to sexual selection in guppies, colourful males are often preferred by females (e.g., Houde 1987; Long & Houde 1989; Houde & Endler 1990; Endler & Houde 1995; Grether 2000; Brooks & Endler 2001a; Pilastro et. al. 2004). With respect to natural selection, colourful males may be more susceptible to predatory fishes, perhaps because they are more conspicuous (Endler 1978; Endler 1983; Godin & McDonough 2003). Spatial variation in male guppy colour should therefore reflect spatial variation in the relative strengths of opposing natural and sexual selection. Indeed, male guppies in low-predation sites are usually, although not always, more colourful than are male guppies in high- predation sites, the former often evolving larger and more numerous orange and structural (blues, greens, violets) spots (Haskins et al. 1961; Endler 1978; Endler 1980; Millar et al. 2006). Factors other than predation can also influence the evolution and expression of male colour  a fact evident by the extreme variation in male colour among sites within a given predation environment (Houde & Endler 1990; Endler & Houde 1995; Grether 2000; Grether, Hudon & Endler 2001a; Millar et al. 2006). It has been suggested that at least part of this variation may be due to

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differences in forest canopy cover (Endler 1995; Grether, Hudon & Millie 1999; Millar et al. 2006) and thus the amount of incident light reaching the stream. No previous study has looked at the effects of temporal changes in canopy cover. Some possible outcomes, however, might be suggested from among-site comparisons and from diet manipulations. First, adult guppies might be larger after canopy clearing. This prediction comes from studies showing that sites with more open canopies have greater primary productivity (Endler 1993; Grether et al. 1999; Reznick, Butler & Rodd 2001; McKellar, Turcotte & Hendry 2009) and therefore larger and faster-growing guppies (Grether et al. 2001b). In addition, increasing diet levels in the laboratory leads to faster growth and larger size at maturity (Arendt & Reznick 2005). Second, canopy clearing may change some aspects of guppy colour. In particular, sites with more open canopies in nature have males with less and smaller black spots (Millar et al. 2006). Moreover, increasing diet and carotenoid levels in the lab (both likely associated with more open canopies in nature) leads to males with increased saturation but reduced brightness of orange spots, larger yellow tail spots, and less black colouration (Kodric-Brown 1989; Grether 2000). Another potential influence of canopy cover is on the spectral properties of incident light, which can change how visual signals are transmitted and received. For example, lower light can cause guppies to court at shorter distances (Endler 1991; Long & Rosenqvist 1998), thus influencing the conspicuousness of colour spots and the responsiveness of females (Endler 1991; Gamble et al. 2003). Moreover, canopy cover alters the distribution of wavelengths reaching the stream and can therefore change how female guppies perceive a particular male colour pattern (Endler 1991; Endler 1993). Similar effects have been seen in many other taxa as well (e.g. Boughman 2001; Thery, Pincebourde, & Feer 2008; Schultz, Anderson, & Symes 2008). Any of the above effects may then alter selection and thus future evolutionary trajectories of male colour. Although the above comparative and laboratory studies lend some support to a role of canopy cover on colour variation, it has been difficult to isolate this factor from other co- varying environmental factors in nature. Comparisons within and among sites in

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relation to the clear-cut event thus provide an opportunity to assess the consequences of canopy clearing on the expression of male colour.

4.3. METHODS

4.3.1. Study Sites Sampling took place in the Marianne River, located on the north slope of Trinidad‟s Northern Mountain Range (see Figure 4.S1 in Supporting Information), at the end of the dry season (March/April) in each of four years (2002, 2003, 2006, 2007). Guppies at all sites coexist with only two potential aquatic predators: killifish (Rivulus hartii) and freshwater prawns (Macrobrachium spp.). Both killifish and prawns have relatively mild effects on guppy demography (Reznick et al. 1996), and our study sites are thus categorized as “low-predation” environments (Haskins et al. 1961; Reznick & Endler 1982). The focal site ("Disturbed") is a low-order stream that we first sampled in 2002 as part of our survey of variation in the Marianne (site “M16” in Crispo et al. 2006; Hendry et al. 2006; Millar et al. 2006).We did not visit the site in 2004, but in 2005 we found the forest canopy being cleared by farmers for a papaya plantation (it also had been a plantation at some unknown time in the past). Returning in 2006, we found that the canopy had been completely removed. We therefore sampled guppies from this site in both 2006 and 2007. This enababled a comparison of pre-disturbance samples (2002 and 2003) to post-disturbance samples (2006 and 2007). In each of the four years, we also sampled two “reference” sites of similar size and stream-order to the disturbed site. These sites represent the extremes of canopy cover among all our sampled low-predation sites in the Marianne and the canopy remained undisturbed throughout the sampling period. These reference sites are used to place temporal changes at the disturbed site in the context of (1) differences between sites with an extreme contrast in canopy cover, and (2) changes through time at sites without recent canopy clearing. See Table 4.S1 in

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Supporting Information for more details on the specific locations of the sites sampled. At each of the four sites in 2003 and 2007, we estimated canopy cover by use of a concave spherical densitometer. This method yields readings that are closely correlated with those from hemispherical photography (Englund, O‟Brien & Clark 2000) and with standing algal biomass (Grether et al. 2001b; McKellar et al. 2009). For these readings, each of the sample sites was first divided along its length into 3-6 evenly-spaced transect locations. Spacing between locations was constant at a site but varied among sites (from 5-20 m) to match the local areas from which guppies were sampled. At each location, four densiometer readings were taken (one in each cardinal direction) while standing in the middle of the wetted channel.

4.3.2. Guppy collection and trait measurement Handheld butterfly nets were used to collect at least twenty fish of each sex at each site in each year. These fish were transported live to our laboratory in Trinidad, held for 24-48 h, and then killed with an overdose (40mg/L) of tricaine methanosulfate (MS-222). We used MS-222 to immobilize fish for the photographs and also to standardize for behaviourally-plastic effects on colour variation, which are common in fishes. Although MS-222 may potentially influence the expression of colour elements, we have observed that for most colours the average size of spots on individual males is not affected by the anaesthetic (orange: t1, 29 = 0.05, p = 0.96; structural: t1, 29 = 0.48, p = 0.63). The only exception is that fish photographed under anaesthetic have larger black spots on average compared to unaesthetized fish (t1, 29 t= 3.09, p = 0.005) and similarly, brightness of all colour spots is reduced under MS-222. (A. Schwartz & N.P. Millar, unpublished data). These results are repeatable across years and consistent among the 13 populations included, and should not bias our results for the present comparative analysis. Nontheless, it is important to remember that colour quantified in this manner is useful for relative comparisons among groups but not

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for an absolute characterization of what guppies would look like in the field (quantifying colour on free-swimming guppies has not yet been attempted.) Each fish was photographed with a digital camera positioned at a standard height above a grid-ruled background. In 2002, the photographs were taken under natural light in the shade. In all other years, photographs were taken under full- spectrum fluorescent lights that approximate the colour spectrum of sunlight (Vita-Lite by Duro-Test Canada). A set of colour standards was placed in all photographs in 2006 and 2007. Ideally, had we pre-planned this “experiment,” we would have used common lighting conditions and colour standards throughout, and we would also have included more sophisticated analyses, such as spectrometry (Grether et al. 2001a; Grether et al. 2005; Kemp et al. 2008). This would have been useful because (1) populations can differ in the spectral properties of their colour spots even if they do not differ in the numbers and sizes of those spots (Kemp et al. 2008), and (2) diet can influence the spectral properties of spots (Grether 2000). Such data were not available, however, because our pre-disturbance samples were extracted from a large survey (34 sites in each of two years, Millar et al. 2006), for which time limitations did not allow spectrometry. Therefore, we rely on the simple and classic methods of counting and measuring the size and number of spots of different colour classes. This method may be biased toward human vision but it is the most broadly comparable to previous work, as nearly all studies report such data (e.g. Endler 1980; Endler & Houde 1995; Grether 2000; Brooks & Endler 2001b; Alexander & Breden 2004; Millar et al. 2006), even if they also report spectral properties (Grether 2005; Kemp et al. 2008). The digital images were analyzed in random order by a single individual (AKS) who was blind to each fish‟s site and year of collection. Scion ImageJ (http://rsb.info.nih.gov) was used to perform the following measurements. Standard length was measured from the tip of the snout to the end of the caudal peduncle. Body area was measured by tracing the fish‟s outline with the „free- hand‟ tool. Each colour spot on the body and the tail was then located and assigned to one of the nine colour categories used by Millar et al. (2006): black,

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fuzzy black, orange, red, blue, violet, silver, green, and yellow. The area of each spot was then measured with the free-hand tool, and these areas were summed to yield the total area of a fish covered by each colour category. For all colours except yellow, tail and body spot areas were summed. Repeatability estimates, based on multiple photographs of the same 60 fish, were high: e.g., r = 0.82 for orange area and r = 0.87 for black area. To simplify the analysis and presentation of so many possible response variables, some of which are correlated, we further combined the above nine categories in three more inclusive categories that have particular biological relevance (Endler 1980; Brooks & Endler 2001b; Blows, Brooks & Kraft 2003). These categories were black (includes black and fuzzy black), carotenoid- and pteridine-based (red, orange, yellow), and structural (blue, violet, silver & green). “Colour categories” in the rest of this paper refer to these three inclusive categories. We also analyzed yellow tail area separately because most yellow colour is found on the tail and this trait is very sensitive to diet manipulation in the laboratory (Grether 2000).

4.3.3. Statistical Analysis Our main goal was to determine if the disturbance event (canopy clearing) influenced phenotypic variation at the disturbed site. Phenotypic changes between sampling periods (2002-2003 vs. 2006-2007) at the disturbed site but not at the reference sites would suggest that some aspect of the disturbance was responsible. Furthermore, if changes at the disturbed site are in the direction of differences between the closed-canopy and open-canopy reference sites, then canopy cover could be implicated as the specific cause. We addressed these questions through analyses of female and male size (standard length) and the area of different colour categories on males. Analyses of colour area controlled for allometry by using the residuals of colour area on total body area. Each of the variables was analyzed individually (ANOVAs) and colour variables were analyzed jointly (MANOVA). We first analyzed variation among all our samples in two-way models with the following structure: sampling period (2002-2003 vs. 2006-2007), year

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(nested within sampling period), and site. We then analyzed pair-wise variation between sites before and after the clear-cut event. Statistical analyses were performed in JMP for Macintosh (v.7.02. SAS Institute, Cary, NC).

4.4. RESULTS

4.4.1. Environmental change at the disturbed site Canopy openness at the disturbed site was 22.19% (+/- 1.11 s.e) in 2003 and increased to 69.68% (+/- 7.54 ) in 2007. This change should increase incident light and therefore primary productivity and food availability (Grether et al. 2001b). We could not directly test for such cascading effects at the disturbed site because we did not sample algae before the disturbance. However, we have indirect evidence from samples of algal standing crop collected in 2007. In that year, the disturbed site had higher levels of chlorophyll a than did all 22 of the other Marianne and Paria sites (McKellar et al. 2009), wherein a strong positive correlation was found between canopy cover and productivity. We therefore suggest that the apparent increase in productivity at the disturbed site was the result of increased incident light. An alternative (agriculture fertilizers) was unlikely given the low agricultural activity intensity  it only continues for 50 m upstream of our study site. Over the same time period (2003 vs. 2007), the reference sites showed much less change in canopy cover. At the closed reference site, canopy openness remained essentially constant: 12.17% in 2003 and 7.37% in 2007. At the open reference site, canopy openness appeared to decrease from 87.05% in 2003 to 52.65% in 2007. This apparent increase may be illusory – being driven primarily by chance variation in the precise placement of canopy cover readings. That is, canopy cover is quite variable along this site (Table 4.S2) and slight variation in the locations of canopy readings taken 4 years apart may well lead to a difference in canopy cover. Regardless, the key point is that the two reference sites differed dramatically in canopy cover, and that the disturbed site changed through time along this same continuum.

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4.4.2. Patterns of phenotypic variation Both male and female body size increased from the first (2002–2003) to the second (2006–2007) sampling period at the disturbed site but not at the reference sites (Table 4.1, Fig. 4.1). As a qualification, however, note that only one of the two post-disturbance samples showed a substantial size difference from the pre-disturbance samples for each of the sexes (Fig. 4.1). Overall, fish were larger at the open reference site (females: F1, 248 = 16.63, p <0.0001; males: F1,168 = 4.74, p = 0.03), however the magnitude of this difference varied throughout the sampling period: no differences in size were detectable between reference sites for males within the first and second sampling periods, whereas females were significantly larger at the open reference site in the first, but not the second, sampling period (Table 4.2, Fig. 4.1). When comparing the disturbed and reference sites, guppies at the former were larger in most cases, although the differences were always largest after the disturbance (Table 4.2, Fig. 4.1). That is, although guppies were somewhat larger at the disturbed site before canopy clearing, they were considerably more so after canopy clearing. None of the colour variables showed consistent, statistically significant differences between the first and second sampling periods at any of the sites (Table 4.1, Table 4.2, Fig. 4.2). Instead, male colour varied considerably among sampling sites (Table 4.1, Fig. 4.2) but this was generally independent of both the disturbance event and differences in canopy cover among sites. Males at the two reference sites did differ somewhat in some aspects of colour; however, the closed reference site had males with more black in both sampling periods and less structural colour in the first sampling period (Table 4.2, Fig. 4.2). Males at the disturbed site also differed from males at the two reference sites: the disturbed site had males with more black compared to the open reference site, although only prior to canopy clearing. Moreover, males at the disturbed site had the least amount of orange colouration throughout the sampling period. Temporal variation in site differences were evident for the other aspects of colour: males in the disturbed site had more yellow on their tails than males at the closed site in the

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second sampling period and more black and structural colour than males at the open site in the first sampling period (Table 4.2, Fig.4.2).

4.5. DISCUSSION

4.5.1. Summary of Results We explored temporal and spatial variation in guppy body size and male colour in relation to (1) a recent disturbance event that dramatically reduced canopy cover at a single location (“disturbed” site) and (2) longer standing variation in canopy cover (“reference” sites). Some of our findings agree broadly with previous work. First, we found that canopy clearing generally led to larger guppies (Fig. 4.1; Table 4.2), consistent with the expectation that sites with more open canopies have more resources for guppies which then enhances their growth (Grether et al. 2001b; Karino & Hajima 2004; Arendt & Reznick 2005). Second, we found that more open canopies were, to some extent, associated with less black colour for a given male body size (Fig. 4.2; Table 4.2). That is, males had less black at the open canopy reference site than at the closed canopy reference site and males had less black at the disturbed site following canopy clearing. This trend further fits with expectations from previous work based on among-site comparisons in the Marianne River (Millar et al. 2006) and diet manipulations in the laboratory (Grether 2000). Overall, however, canopy cover was not a particularly strong determinant of phenotypic variation, particularly for colour when controlling for body size. A statistical reason may be relatively low power owing to only two years of samples before and after the disturbance – but the lack of consistent patterns in the two years within a given period suggests that this is not the primary cause. Instead, among-site variation independent of canopy cover proved very important (Table 4.1). Perhaps most striking, males at the disturbed site, both before and after the disturbance, had less orange and more structural colour, than did males at both reference sites (Fig. 4.2). One reason for this variation might be unmeasured environmental parameters, such as parasite loads (e.g., Houde and

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Torio 1992, but see Martin & Johnsen 2007) and the number of red-blind Macrobrachium prawns (Endler 1978; Kemp et al. 2008; NP Millar, unpublished data). Another reason may be among-site variation in female choice for male colour (e.g. Endler & Houde 1990; Endler & Houde 1995; Kwiatkowski & Sullivan 2002; Schwartz & Hendry 2007). Indeed, recent studies in several taxa are increasingly pointing to the importance of spatial variation in sexual selection, instead of just natural selection, as a driver of spatial variation in phenotypes (Roulin & Bize 2007; Gosden & Svensson 2008). Of course, spatial variation in sexual selection might be itself the result of spatial variation in environmental parameters.. For example, the direction of sexual selection on particular colours may be influenced by variation in the visual background (Endler 1978; 1991) or the presence/absence of various predators (Breden & Stoner 1987; Stoner & Breden 1987; Houde & Endler 1990; Schwartz & Hendry 2007). Alternatively, sexual selection may diverge among populations in arbitrary directions owing to founder effects (e.g. Carlson 1997; Gavrilets & Boake 1998), drift (Lande 1981), or population-specific sensory biases (reviewed in Endler & Basolo 1998). In addition to spatial variation, we documented considerable temporal variation at a given site, even when predation regime or canopy cover did not change appreciably. We do not have a clear explanation for this variation and we are not certain how common this variation is – because few guppy studies temporally replicate their samples. Possible reasons for temporal variation in colour are several, including frequency-dependent predation (Olendorf et al. 2006), frequency-dependent sexual selection (Eakley & Houde 2004; Zajitcheck & Brooks 2008), temporal variation in population size or sex ratio (Rodd & Sokolowski 1995; Magellan & Magurran 2007), gene flow from adjacent sites in different environments (Becher & Magurran 2000; Crispo et al. 2006), temporal variation in directional selection (Weese et al. In review), or small-scale variation in visual environments (Brooks 2002). In short, colour variation in guppies increasingly seems to be a question of multiple, interacting causal factors. Another factor contributing to colour variation in guppies may be the movement of fish between sites in a stream, given that a number of studies have

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shown guppies can move substantially over time (Croft et al. 2003; Barson, Cable & van Oosterhout 2009). Such movement may be passive, such as due to flooding, or active, such as to increase foraging or mating opportunities. In such cases, the increase in productivity at our focal site may, for example, have provided a better foraging opportunity for fish from other sites of lower quality. The resulting movement might have increased density at the disturbed site, thereby keeping the level of resource competition relatively consistent despite the increase in food availability. We cannot address this possibility in the current study but it would certainly be interesting to consider in future work.

4.5.2. Implications for mate choice and sexual selection Most colour pattern elements did not show consistent associations with canopy cover. This result might imply that canopy clearing will not influence sexual selection on male colour but this is not necessarily so. For example, the increase in fish size under open canopies may alter the visibility or attractiveness of particular colour patterns (even though the relative amounts of colour spots themselves do not change), thus influencing the degree of contrast with the background or other colour spots. In addition, canopy clearing changes the light environment and therefore influences the manner in which females perceive and respond to males (Gamble et al. 2003; Kolluru, Grether & Contreras 2007). As a result, the benefit to mate choice for both sexes (e.g., good genes or parasite infection levels) may change under different foraging environments. Although we cannot specifically address this hypothesis, it is worthwhile to provide an example of how it might be important – by reference to male orange colour. Female guppies often (although not always) prefer males with larger and more saturated orange spots (Houde 1987; Kodric-Brown 1989; Grether 2000; Brooks & Endler 2001a; Blows et al. 2003), perhaps because large areas of saturated orange reflect male quality through foraging ability (Houde and Torio 1992; Grether et al. 2004; Nicoletto 1991; Godin & Dugatkin 1996; Locatello et al. 2006)  the ability to saturate orange spots depends on the uptake of carotenoids and other pigments from the diet (Grether 2000). Sites with more

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open canopies have greater carotenoid availability and could allow even males of genetically poor quality to optimally saturate large areas of orange. If so, we might expect a reduction in the strength of female preferences for this trait, ultimately weakening the influence of sexual selection on this aspect of orange colour. Although we did not measure the saturation of orange spots, canopy- clearing events may provide an opportunity to test this aspect of the indicator hypothesis. In addition to possible effects of canopy clearing, temporal variation within sites likely has considerable implications for mate choice and sexual selection. Temporal variation in the strength and direction of sexual selection generally appears most common when the costs associated with sexually selected traits fluctuate due to seasonal variations in demographic or environmental components (e.g. Jann, Blackenhorn & Ward 2000; Lindstrom 2001; Hegyi et al. 2008). One possibility for the observed temporal variation in colour within sites in the present study may be related to common flooding of the Marianne River, which tends to move fish passively downstream. Such regular fluctuations in population demography may influence the strength of sexual selection for particular traits, even if the direction does not change (Endler 1992). This could occur due to changes in the visual environment (e.g. water turbidity), the overall size and condition of the population, or frequency of males carrying a preferred trait changing the indicator value of being choosy to a female. Furthermore, the strength and direction of female preferences are flexible depending, in part, on the social context in which mate choice is assessed (Godin & Dugatkin 1995; Brooks 1996; Godin & Briggs 1996). Determining the potential genetic and plastic contributions to female preferences, particularly in temporally heterogeneous versus homogenous populations, may help explain the maintenance of multiple traits and overall diversity in male guppies.

4.5.3. Future directions: Re-examining guppy colour Our baseline data provide the opportunity for an assessment of future evolutionary responses to canopy clearing. Our results indicate that differences in

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light levels do not appear to be causing changes in the heritable portion of variation in basic colour categories, but appear to influence some traits that are known to respond plastically to variations in resource levels (i.e. size and black colour). Such changes in phenotypes coupled with changes in the light environment may nonetheless influence the mechanisms and strengths of mate choice (as above). Little is known about the rate of evolution of female preferences in natural populations, but such traits might take more time to produce a phenotypic response in males. Our study can therefore be viewed as a stepping-stone for further work on the influence of human-caused perturbations on the expression and evolution of sexual signals. Will male colour evolve to a new state, as might be expected if natural or sexual selection has been altered? Will female preferences evolve and, if so, will they evolve in response to changes in canopy cover or to the male traits? What will happen if the canopy is allowed to grow back? Such work would be particularly informative in a general sense because few studies have examined the contemporary evolution of sexually selected traits (Svensson & Gosden 2007), although it is likely that their responses may be qualitatively different from traits subject only to natural selection (Kingsolver et al. 2001; Hoekstra et al. 2001). Trinidadian guppies may be an excellent model of such a circumstance. Despite the long history of research on colour in Trinidadian guppies, recent studies are increasingly pointing to the need for a closer look. Although life-history trait evolution has been unmistakably and repeatably attributed to differences in mortality rates due to predation (Reznick et al. 1997; Gordon et al. 2009) and differences in canopy cover (Grether et al. 2001b), the effect of the same broad predation differences on colour evolution is less consistent. The present study demonstrates considerable spatial and temporal variation in the colour of low-predation males in a single river that is not predictable based on differences in canopy. Due to the multiple agents interacting on the maintenance and perception of colour elements in both conspecific and heterospecific interactions, it appears that understanding the evolution of guppy colour will require a further quantification of habitat and demographic variation

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that goes beyond dichotomous (e.g. high vs. low predation; open vs. closed canopy) environmental parameters.

4.6. ACKNOWLEDGMENTS Erika Crispo, Michael Hendry, Michael Kinnison, Ann McKellar, Nathan Millar, David Reznick, Martin Turcotte, and Dylan Weese assisted with field collections and photography. Export permits were kindly granted by the Ministry of Agriculture, Land and Marine Resources – Fisheries Division of Trinidad & Tobago. Financial support was provided by the Natural Sciences and Engineering Council of Canada (APH) and the Fonds Québecois de Recherche sur la Nature et les Technologies (AKS).

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Table 4.1. Overall effects of sampling period (2002-2003 vs. 2006-2007), year (nested within sampling period), and site (Open, Closed, Disturbed) on phenotypic variation. Shown are F-ratios with significance indicated by asterisks. *p < 0.05; ** p < 0.01; *** p < 0. 001; **** p < 0.0001. Factors with the largest effects are shown in bold for each trait.

Factor Sampling Period Year Site Sampling Period x Site Df 1/243 2/243 2/243 2/243

Female length 4.65* 3.1* 15.01**** 11.41****

Male length 10.14* 3.1* 98.92**** 16.36****

Male colour 4.29** 2.5* 7.82**** 1.1 (MANOVA)

Orange area 2.23 0.05 12.0**** 0.35

Yellow tail area 2.67 2.1 3.73* 0.74

Black area 2.47 1.57 4.65* 1.34

Structural area 0.44 5.48** 7.27*** 1.72

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Table 4.2. Pairwise comparisons between sites before (2002-03) and after (2006-2007) the clear-cutting event at the disturbed site. Shown are F-ratios from one-way analysis of variance with significance indicated by asterisks. *p < 0.05; ** p < 0.01; *** p < 0. 001; **** p < 0.0001. The direction of the difference is indicated for significant differences (O = open; C = closed; D = disturbed).

Open vs. Closed Open vs. Disturbed Closed vs. Disturbed

Year 02-03 06-07 02-03 06-07 02-03 06-07 Df (females) 1/76 1/81 1/78 1/91 1/76 1/88 Df (males) 1/78 1/108 1/78 1/84 1/78 1/84 Female length 15.23*** 0.468 2.71 23.78**** 5.85* 28.76**** O > C D > O D > C D >C

Male length 3.18 1.63 16.33*** 122.62**** 30.18**** 133.93**** D > O D > O D > C D > C

Male colour 1.77 3.24 * 12.04**** 4.54* 6.11** 4.05* (MANOVA)

Orange area 0.25 0.17 9.89** 8.07** 11.56** 7.06** D < O D < O D < C D < C

Yellow tail 0.08 0.97 0.93 2.05 1.34 6.52* D > C

Black area 4.54* 6.72* 4.29* 0.44 0.10 2.94 O < C O < C D > O Structural Area 8.92** 0.19 16.24*** 1.43 2.51 2.11 O < C D > O

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Figure 4.1. Means and standard errors for male and female size (standard length). “Closed” = closed canopy reference site. “Open” = open canopy reference site. “Disturbed” = site where canopy clearing occurred between March of 2005 and March of 2006. Solid bars indicate closed-canopy samples and open bars indicate open-canopy samples. Letters below years indicate homogeneous subsets (post-hoc Tukey tests) comparing variation among years within each site.

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Figure 4.2. Means and standard errors of residuals (from body area) of the area of colour spots in different categories. See the caption for Figure 1 for labeling and other conventions

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Supporting Information

Figure 4.S1. Map of study sites in Trinidad‟s Northern Mountain Range

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Table 4.S1. Site locations. Grid references are from the Trinidad National Grid System 1: 25,000 map series. Site numbers here correspond to references in previous studies (Millar et al. 2006; Crispo et al. 2006; Hendry et al. 2006).

Site Latitude Longitude Grid Reference

Closed 10°46.378N 061°17.541W PS 868 914 (M10)

Disturbed 10°44.693 N 061°18.158 W PS 856 880 (M16)

Open 10°44.867 N 061°17.491W PS 867 885 (M4)

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Table 4.S2. Temporal variations in canopy cover. Canopy openness measures are compared between years within sites with one-way analysis of variance.

Site % Canopy Openness (2003) % Canopy Openness (2007) Analysis of Variance N Min- Mean N Min-max Mean max (+/- s.e) (+/- s.e) Closed 20 7-19 12.17 12 3-10 7.37 F = 0.09 (M10) (0.47) (0.72) P = 0.77 Disturbed 20 7-32 22.19 12 18-92 69.68 F = 61.24 (M16) (1.11) (7.54) P = 9.87 x 10-9 Open 20 39-96 87.05 20 11-96 52.65 F = 23.47 (M4) (1.83) (6.53) p = 1.76 x 10-5

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GENERAL SUMMARY & CONCLUDING REMARKS Toward a unified model of speciation: why do we care?

The study of evolution can be viewed as an exercise in working backwards: we see the forest, but not always the trees. We therefore start from a snapshot of history and attempt to resolve the steps that came before. This thesis was an exercise in identifying the main „trees‟ of nature: both extrinsic (environment and geography) and intrinsic (variation in survival and reproduction among individuals) that can help to explain the forest of biological diversity. This work has highlighted that although nature is messy, studying the components in an integrated perspective can form the foundations for the development of a clearer picture. Both academically and practically, this process serves the function of establishing generalities for understanding our relationship to our world. The hope is that in preventing ignorance of this history, we will in turn prevent the destruction of the future potential it carries with it.

Summary of Findings This thesis examined how the colonization of new environments, or environmental change in situ impacts divergence in characters important for mate discrimination (male signaling traits and female preferences), and ultimately speciation. Both a quantitative review of the literature (Chapter 1) and a test for the parallel evolution of mating isolation in guppies (Chapter 2) revealed that divergence in sexually selected phenotypes within populations does not necessarily predict patterns of mate discrimination between them. Specifically, context and history matter in determining whether or not sexual selection and sexual isolation will form a continuum. The review showed that the magnitude of divergence in sexually selected traits is not a result of sexual selection alone. In fact, many populations show directional preferences for particular trait values or types, even when these are rarely, if ever, encountered in their native environments. In other situations, despite large differences in a male signaling trait between populations, females do not rely on it as a target for mate

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discrimination at all. Reproductive isolation instead appears to evolve most quickly and/or easily when populations are ecologically differentiated and physically connected. This general result was also observed in Trinidadian guppies. Chapter 2 revealed that (i) the direction of sexual selection acting on male orange colour within populations does not always predict patterns of male mating success between populations that differ in the mean value of orange, and (ii) mating isolation in guppies is not a by-product of general adaptation to different predation environments. Specifically, high-predation females only discriminate against low-predation males when they are from the same river. Low-predation females on the other hand, always discriminate against high-predation males but also show more variation in their preferences. From a broad perspective it sometimes appears that male colour and female preferences are divergent between predation environments, in which low- predation males are more colourful, and low-predation females prefer more colourful males. However, Chapter 2 and the work of my Masters research (Schwartz & Hendry 2007) also showed that both the direction and the magnitude of this divergence varies within and across rivers, suggesting that differences in predation regime do not always lead to parallel evolutionary responses. From the perspective of male colour, this intuition was made more apparent by the results of a natural introduction experiment. Ten years after high- predation guppies were transplanted and established into high-and low-predation sections of a new river, no obvious divergence in colour was apparent (Karim et al. 2007). This result was in sharp contrast to the magnitude of divergence observed in the ancestral river, as well as to results of previous similar studies (e.g. Endler 1980). One possibility to explain the lack of parallel evolution in this new introduction could be a lack of time; however, in this same introduction, life history traits have diverged between predation populations to the same extent as that seen in the ancestral river (Gordon et al. 2009) and previously observed in other studies (e.g. Reznick et al. 1997). This suggested that there are potentially

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more constraints related to adaptation of sexually, rather than naturally, selected traits. Female preferences may be one such constraint, so that colour may only be able to evolve (once viability selection on it is relaxed) to the point that female preferences select for it. If females from the source population do not prefer colourful males initially, divergent adaptation, and therefore the opportunity for mating isolation, cannot evolve. Chapter 3 compared female preference functions in the ancestral and derived populations of this introduction and revealed that indeed, females generally do not prefer more colourful males in the ancestral population. This is not the full story, however, as we did not detect any evidence of divergence in preference functions between populations – even those that show divergence in colour. Instead, female preferences were more variable among individuals within populations than between them, suggesting that the context of mate choice is likely a stronger determinant of male mating success than intrinsic genetic preferences for trait values. The importance of the context of mate choice is also evident in considering the difference between the laboratory and field mate choice experiments in Chapter 2. Although in both cases high-predation males only have a mating advantage with their own females relative to upstream low-predation males, the strength of this effect was strongest when males were in direct competition for mating opportunities (field experiment). Although no-choice mating experiments showed that females‟ responses are, on average, higher toward their own males, copulation rates did not differ substantially across male types. In contrast, the field experiment showed that offspring production (and therefore the „currency‟ of gene flow and selection) is strongly biased toward local males when in competition with upstream migrants. This suggests that female choice alone is not sufficient to prevent gene flow, but can be reinforced by differences in male aggressiveness, sneaking rates, female mate choice copying, and/or postcopulatory mechanisms such as sperm competition. More direct experiments addressing each of these possibilities would be helpful in

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determining the relative importance of each mechanism of sexual selection for reducing gene flow. Although predation plays a strong role in explaining some aspects of variation in sexually selected traits and preferences, it clearly is not a sufficient explanation for the extreme variation observed in the guppy system. If divergent predation environments imposed consistently strong selection, then variation within populations would be reduced. The maintenance of genetic variation in the face of selection is a large topic of research beyond the scope of this thesis. However, I was able to examine the nature of this variation by considering the potential influence of one other habitat feature - light availability - due to tree removal at one of our long-term study sites (Chapter 4). Treating this disturbance as a natural experimental manipulation, I was able to place spatial and temporal phenotypic variation in context of the role of canopy openness. Although light availability has been shown to affect the maintenance and perception of colours in other species (Chuco et al. 2007; e.g. stickleback, Boughman 2001; bluefin killifish, Fuller & Travis 2004; haplochromide cichlids, Seehausen 1997), we found that spatial variation in guppy colour was independent of differences in the lighting environment. This result also opposes expectations for guppies as light affects courtship behaviours, growth rates, and the spectral properties (i.e. brightness and chroma) of orange spots (Grether 2000; Gamble et al. 2003; Arendt & Reznick 2005). Overall, this work suggests that demographic factors, biotic interactions, as well as spatial and temporal fluctuations in unmeasured habitat parameters, are likely responsible for the maintenance of colour polymorphism in guppies as opposed to broad environmental contrasts. In summary, it appears that the „lines of demarcation‟ between differentiated populations are best drawn by integrating information from various sources. Sexual selection is generally viewed as a „non-ecological‟ mode of speciation because these traits are not necessarily as affected by habitat differences as those linked directly to survival (e.g. life-history traits). Similarly, geographical and ecological perspectives on defining speciation have polarized, and at times, stagnated research in the field. The conclusions of this thesis show

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that a general framework will likely not be achieved by proving which of these factors is most important, but rather by instead focusing on how they interact.

Questions for Future Study A common question I hear when I tell people that I study evolution is: “What is there left to know?” I have difficulty answering this question as the tome of what I don‟t know would exceed the volume of what I think I now know. This is not to say that deeper study results in less understanding, but rather because the answers are more complicated than anticipated, new doors open for further investigation. Here, I focus on two major avenues of research that could shed light on some questions arising from this work. Although these questions were mainly inspired by my work with guppies, they are generalizable to all sexual animal populations. First, in reviewing the literature on sexual selection it was quickly evident how little is known about mating preferences relative to secondary sexual traits. Most theoretical models of sexual selection and speciation either assume a particular mode of inheritance of preferences (e.g. Lande 1982; Doebeli & Diekmann 2003), or reveal how different the outcome is depending on the mode assumed (e.g. Servedio 2000; Servedio & Saetre 2003). Yet, few studies have addressed selection on, and the inheritance of female preferences, particularly in wild populations (but see Haesler & Seehausen 2005; Saether et al. 2007). In guppies, male colour is Y-linked and therefore „hybrids‟ between high-and low- predation populations will inherit their fathers‟ colour pattern, rather than an intermediate phenotype. How and if female preferences are inherited or linked to colour traits has not yet been investigated, although it would shed light on the opportunity for co-evolution, divergent adaptation and reproductive isolation in this system. Second, one main conclusion of these studies is that sexual selection within populations often does not influence mating decisions between them, even when the same traits are in question (e.g. Head et al. 2009). Determining the contexts in which sexual selection and isolation are driven by the same

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mechanisms and when they are not, is therefore the next major challenge in speciation research.

General Implications A deeper understanding of the factors and contexts that limit and promote the evolution of reproductive isolation will surely contribute to our general knowledge of the ultimate evolutionary process – speciation. Furthermore, identification of the line between differentiation and speciation has applied consequences. A potential outcome of defining these boundaries will be a more clear definition of the units of biodiversity that should be of priority management concern. The results of this thesis emphasize the importance of the environmental context on maintaining diversity – a phenomenon mirrored in a number of recent cases of biodiversity loss. For example, eutrophication of waters impact resource availability and the visual environment, and have thus resulted in a breakdown of assortative mating where visual cues are important, for example in Lake Victoria cichlids (Seehausen et al. 2008) and both Canadian and European stickleback populations (Candolin et al. 2007; Taylor et al. 2006). Similarly, human introduction of new resources due to eco-tourism in the Galapagos appears to be resulting in a homogenization of finch populations (Hendry et al. 2006). Furthermore, the observation that pairs that will mate assortatively in the wild show less discrimination in the lab (Chapters 1 & 2) offers a warning for the implication of captive breeding programs: if the correct units of diversity aren‟t appreciated in their natural context, we run the risk of losing evolutionary potential. Similarly, the fact that contact zones appear to accelerate the evolution of assortative mating imply that development and land-use resulting in fragmentation should aim to preserve the potential for gene flow, even between seemingly disparate evolutionary units. Finally, attempts to preserve genetic diversity may fall short depending on how such diversity is defined. For example, I have here shown that species at early stages of divergence that show little to no neutral genetic differentiation can inter-breed despite high divergence in ecologically or sexually important traits.

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The complexities of the guppy system mirror the general complexities found in nature. Although clear and simple answers may be more readily interpretable, they do not open the door for new discovery. Since we have now accumulated a lot of evidence that natural selection is clearly a strong force in diversification (reviewed in Schluter 2000; Coyne & Orr 2004; Funk et al. 2006), further studies of the same prediction run the risk of being confirmatory and bringing us back into a time where the study of speciation was equal to taxonomic classification. In order to understand the mess of nature, we need to keep a broad perspective while paying attention to the trees. The route to a general model of speciation will therefore most likely emerge from multidisciplinary collaboration and approaches that do not presuppose a particular context, but rather incorporate the geographic and ecological circumstances in which evolutionary processes (e.g. natural and sexual selection) operate.

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APPENDIX I

Table of studies and references included in meta-analysis (Chapter 1). Species are listed by taxa and categorized according to the populations/sub-species compared, the type of sexually selected trait compared (A = acoustic; B = behaviour; C = colour; M = morphological), the nature of reproductive isolation observed (A = asymmetric; N = none; S = symmetric), whether or not populations are subject to divergent natural selection on the signaling trait (ecological difference, Y= yes; N = no), and the geographic context (A = allopatric; P = parapatric; S = sympatric

STUDY SYSTEM POPULATIONS/ TRAIT EVIDENCE ECOLOGICAL GEOGRAPHY REF. SPECIES COMPARED TYPE FOR R.I. DIFFERENCE? Amphibians Triturus T. vulgaris/T. M S N A 1-4 montadori Pseudacris P. ferarium/P. nigritat A S N A and S 5-6 Physalaemus Gamboa vs. 9 other A 6S: 4N N A 7-8 pustulosus locations

Physalaemus Yasuni/La Selva A S Y A 9 petersi

Dendrobates Isla Colon/ C N Y A 10 pumilio Bastimentos Orange

Dendrobates Solarte/ C N Y P 10 pumilio Bastimentos Orange Dendrobates Solarte/ C N Y P 10 pumilio Basimentos C. Dendrobates Isla Colon/ C N Y A 10 pumilio Bastimentos Green Dendrobates Bastimentos C./ C S Y S 10 pumilio Bastimentos Orange

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STUDY SYSTEM POPULATIONS/ TRAIT EVIDENCE ECOLOGICAL GEOGRAPHY REF. SPECIES COMPARED TYPE FOR R.I. DIFFERENCE? Dendrobates Solatrte/ C N Y P 10 pumilio Bastimentos Green Dendrobates Bastimentos C./ C S Y S 10 pumilio Bastimentos Orange Hyla sp. H. cinera/ H. gratiosa A S Y S 11-12 Eurycea bislineata E.wilderae C S Y P 13-15 /E.cirrigera

Rana sp. R.lessonae/ A A N S 16-18 R.esculenta

Arthropods Cordylochernes French Guiana/Panama M N Y A 19 scorpioides Birds Carpodacus Sonoran/Urban M & A - Y P 20 mexicanus Emberiza sp, E. schoeniclus/E. M & A - Y P 21-22 intermedi Geospiza fortis Large beak/small beak M & A S Y S 23-24 morphs Ficedula sp. Pied/collared flycatcher A S Y S & A 25-27

Myrmeciza Fragmented/ A S Y P 28-29 hemimelaena continous forest

Loxia curvirostra Type 2/Type 5 M & B S Y P 30-32

Loxia curvirostra Type 2/Type 9 M S Y P 31, 33

Loxia curvirostra Type 1/Type 2 M & A S Y P 34-35

Melospiza melodia M.mheermanni/M.m.fal C, M S Y S 36-37 lax & A

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STUDY SYSTEM POPULATIONS/ TRAIT EVIDENCE ECOLOGICAL GEOGRAPHY REF SPECIES COMPARED TYPE FOR R.I. DIFFERENCE? Poecile sp, black-capped/ M & A N N A 38-40 Carolina Uraeginthus Blue-breast/ M N N A 41 Red cheek Uraeginthus Blue-breast/ M N N S 41 Blue cap Uraeginthus Blue-cap/ M N N A 41 Red cheek Callipepla C.gambelli/ A A-wild N S 42-43 (Gambel's quail) C.californica S-lab Geospiza difficilis Genovesa/Wolf A S Y A 44-45

Geospiza difficilis Genovesa/ A N Y A 44-45 Darwin Luscinia svecica L.s.svetica/ C - N A 46-47 L.s.namnetum

Catharus ustulatus Coastal/ Inland A - Y P 48-49

Fishes Gasterosteus Misty Lake/ M A Y P 50, aculeatus Misty Inlet K.Rasanen, unpubl. Gasterosteus Misty Lake/ M N Y P 50, aculeatus Misty Outlet K.Rasanen, unpubl. Gasterosteus Misty Outlet/ M N N P 50, aculeatus Misty Inlet K.Rasanen, unpubl. Gasterosteus Lava/nitella habitats M S Y S 51-52 aculeatus (Iceland) Gasterosteus Benthic/ M & C S Y S 53-57 aculeatus Limnetic Campylomormyrus C. compressirostris/ A S N S 58-59 sp. C.rhyncophorus

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STUDY SYSTEM POPULATIONS/ TRAIT EVIDENCE ECOLOGICAL GEOGRAPHY REF. SPECIES COMPARED TYPE FOR R.I. DIFFERENCE? Campylomormyrus C. compressirostris/ A N N S 58-59 sp. C.tamandua

Gambusia huubsi High predation/ M Y Y A 60 Low predation Pseudomugil Willinga/Nelligan M N N P 61 signifer Pseudomugil Willinga/Ross M S N A 61 signifer Girardinichthys Five populations M Y N A 62 multiradiatus Poecilia reticulata Orupuche4/Paria C N Y A 63-64

Poecilia reticulata Orupuche 4/ C N Y A 63-64 Quare 6

Poecilia reticulata Quare 6/Paria C A N A 63-64

Poecilia reticulata Arima/Paria C N N A 63-64

Poecilia reticulata Arima/Quare 6 C N N A 63-64

Poecilia reticulata Arima/Marianne C A N A 63-64

Poecilia reticulata Marianne/Paria C N N A 63-64

Poecilia reticulata Oropuche 1/Paria C N Y A 63-64

Poecilia reticulata Oropuche 1/ C N Y A 63-64 Guanapo Poecilia reticulata Aripo, Quare, Yarra C See Chapter 2 Chapter 2 Insects Chorthippus sp. C. jacobsi/ A Y N S 65-66 C. brunneus

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STUDY SYSTEM POPULATIONS/ TRAIT EVIDENCE ECOLOGICAL GEOGRAPHY REF. SPECIES COMPARED TYPE FOR R.I. DIFFERENCE? Chorthippus Mull/Jersey A & M N N A 67 parallelus Chorthippus Mull/Pyrenees A & M S N A 67 parallelus Chorthippus Jersey/Pyrenees A & M N N A 67 parallelus Magicicada sp. M. tredecim/ C & A N N S 68-69 M. neotredecim Drosophila Baja/Mainland A A Y A 70-74 mojavensis Drosophila D. santomea/ A Y N S 75-76 melanogaster D. yakuba Drosophila N. American/ C & M A Y A 77-78 melanogaster African Drosophila Zimbabwe/ A S Y A 79-82 melanogaster Cosmopolitan Hawaiian D.silvestris/ A & M A N S 83-85 Drosophila sp. D.heterneura

Laupala sp. L.kohalensis/ A S N S 86-88 L.cerasina

Laupala sp. L.kohalensis/ A S N S 86-88 L.paranigra Anabrus simplex Solitary/ A - Y A 89-90 Gregarious Nasonia sp. N.vitripenis/ B & M A N A 91-93 N.longicornus Cytrodiopsis sp. C.whitei/ M S N A 94-95 C.dalmanni Papillonidae sp. P.g.glaucus/ C A Y P 96 P.g.candensis Colias sp. High elevation/ C A Y P 97-98 Low elevation

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STUDY SYSTEM POPULATIONS/ TRAIT EVIDENCE ECOLOGICAL GEOGRAPHY REF. SPECIES COMPARED TYPE FOR R.I. DIFFERENCE? Gryllus sp. G. rubens/ A S N A 99-101 G. texensis Habronattus Santa Rita/ A N N A 102-103 pugillis Griswold Antascosa

Chrysopidae N.American/Asian A N N A 104 adamsi

Chrysopidae Idaho/Connecticut A N N A 105-106 plorabunda.

Chrysopidae sp. C.plorabunda/ A S N S 106-107 C.downsei

Tetrix sp. T. subulata/ A A Y S 108-110 T. ceperoi Callosobruchus Asian/African M N N A 111 maculatus Chrysomelidae sp. C.colbatinus/ M A Y S 112-114 C.auratus Ephigger ephigger monosyllabic/ A S Y A 115-116 polysallabic Enchenopa Viburnum lentango host A 4S:1N Y S 117-118 binotata / 5 other host plants Orchelimum sp. O.nigrippes/ B A N S 119-122 O.pulchellum Teleogryllus sp. T.taiwanemma/ A S Y A 123 T.yezoemma Teleogryllus sp. T.taiwanemma/T.emma A S Y P 123 Luciola cruciata Ohtsu/Inuyama B N N A 124 Luciola cruciata Ohtsu/Sendai B S N A 124 Luciola cruciata Ohtsu/Aomori B S N A 124 Luciola cruciata Inuyama/Sendai B A N A 124 Luciola cruciata Inuyama/Aomori B A N A 124 Luciola cruciata Inuayam/Sendai B N N A 124

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STUDY SYSTEM POPULATIONS/ TRAIT EVIDENCE ECOLOGICAL GEOGRAPHY REF. SPECIES COMPARED TYPE FOR R.I. DIFFERENCE? Lycaenid sp. L.melissa/L. idas C S Y S 125-126 Leptidea sp. L.sinapsis/L.reali C S Y S 127 Invertebrates Littorina saxatilis Rough-banded/ M S Y S 128-132 Smooth-unbanded

Asellus aquaticus Chara/Reed M S Y P 133 Mammal Arctocephalus sp. A. gazella/A.tropicalis A A Y S 134-135

Reptile Sceloporus Oregon/California B S N A 136-137 graciosus

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APPENDIX II

Map and location of collection sites for laboratory mate choice experiment (Chapter 2) in Trinidad‟s Northern range mountains. Grid references are from Trinidad National Grid System 1:25, 000 map series.

Site Predation Location and Elevation Drainage Risk Upper Aripo tributary Low PS 933 818 Caroni (Naranjo river) Lower Aripo High PS 780 940 Caroni

Upper Quare tributary Low PS 810 970 Orupuche

Lower Quare High PS 792 975 Orupuche

Upper Yarra tributary Low PS 834 876 Northern (Limon river) (Carribean Sea) Lower Yarra High PS 802 940 Northern (Carribean Sea)

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