Effects of the social environment on the behaviour and fitness of a territorial squirrel

by

Erin Rose Siracusa

A Thesis presented to The University of Guelph

In partial fulfillment of requirements for the degree of Doctor of Philosophy in Integrative Biology

Guelph, Ontario, Canada

© Erin Rose Siracusa, September 2018 ABSTRACT

EFFECTS OF THE SOCIAL ENVIRONMENT ON THE BEHAVIOUR AND FITNESS OF A TERRITORIAL SQUIRREL

Erin Rose Siracusa Advisor: University of Guelph, 2018 Dr. Andrew G. McAdam

Most organisms interact with conspecifics and therefore have a social environment. While it is understood that variation in the composition of this social environment can have important consequences for gregarious species, solitary, territorial species may also live in socially complex environments where the composition of neighbouring conspecifics can directly influence time and energy spent on territory defence. The importance of the social environment for solitary species is, however, poorly understood. In this thesis, I combined behavioural observations, field experiments, and long-term data analysis from a population of North American red squirrels (Tamiasciurus hudsonicus) to explore the effects of the social environment on the territorial dynamics, behaviour and fitness of an ‘asocial’ species. In particular, I looked at two aspects of the social environment that were likely to have important effects on territorial species: familiarity and relatedness with neighbours. In my first chapter, I established that squirrels living in social neighbourhoods with high average familiarity faced reduced risk of intrusion and cache pilferage from conspecifics, providing evidence of the ‘dear- enemy’ phenomenon in red squirrels. In Chapter 2, I experimentally demonstrated that red squirrel ‘rattle’ vocalizations serve an important territorial defence function by deterring conspecifics from intruding. In Chapter 3, I found that red squirrels respond to changes in their social environment by adjusting their behaviour in a manner that reduces the costs of territoriality: in familiar social neighbourhoods red squirrels reduced their rattling rates and increased the proportion of time spent in nest. Finally, in Chapter 4, I used 21 years of data to show that living near familiar neighbours has substantial fitness benefits, increasing annual reproductive success and survival in both male and female squirrels. Collectively my thesis

contributes to our broader understanding of the importance of the social environment for ‘asocial’ species and provides evidence that interactions between territorial individuals are not strictly competitive but can also be cooperative in nature. In particular, mutualistic interactions, rather than kin-selection, were important in mitigating conflict and enhancing fitness. Studying social interactions in asocial animals may, therefore, provide important insights into the mechanisms driving the evolution of social systems.

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DEDICATION

To my parents, Jeanne Beno and Alan Siracusa, who first taught me the names of wild things, who gave me acres of woodland to romp, who let me wade in the mud after salamanders and tadpoles, and who pulled me from my bed at unreasonable hours of the morning to see the sun rise. To my parents, who taught me a deep respect for all living things and instilled in me an unsurpassed love for the natural world. It is because of you that I am a biologist today.

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ACKNOWLEDGEMENTS

My thanks must first and foremost go to my advisor, Andrew McAdam, and to the members of the McAdam lab, Julia Kilgour, Simon Denomme-Brown, Jack Robertson, David Fisher, Maggie Bain, Shelby Bohn and Sarah Guindre-Parker, who have been my academic family for the past 5 years. I consider myself extraordinarily lucky to have landed in a graduate program where I have had the opportunity to work with such supportive, caring, intelligent and creative colleagues on a daily basis. You have each been an inspiration to me, and have taught me not only how to be a better scientist but also a better human being. It is my most sincere hope that every graduate student has the opportunity to work with such a tremendous advisor and lab group.

To my incredibly hard-working and dedicated field assistants – Jack Robertson, Sam Sonnega, Marta Thorpe, and Yuqing Sun – without you none of this research would have been possible. Thank you for your quick thinking, your laughter, and your unending supply of wit that got me through even the toughest days in the field. I am indebted to you for helping make me a better mentor.

To my KRSP family, who helped to make the fifteen months that I spent at Squirrel Camp some of the best of my life, especially Lisa Bidinosti, Naomi Louchouarn, Sarah Nason, Alec Robetaille, Sarah Westrick and Marina Morandini. A very special thanks to Emily Studd, who first taught me the art of squirrelling and who has been an indomitable source of friendship and laughter ever since.

I am grateful to all of my collaborators and mentors, including my committee members, Ryan Norris, Elena Choleris, and Beren Robinson; the KRSP principle investigators, Stan Boutin, Murray Humphries, Ben Dantzer, Jeffrey Lane; and our bioacoustics expert, Dave Wilson. These folks have devoted their time and energy to providing feedback on experimental design, assisting with data analysis, listening to presentations, and commenting on manuscripts. Their efforts have enhanced the quality of my work and made me a better scientist. I am humbled and grateful to have had the opportunity to work with such an esteemed group.

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I owe a special thank you to Erika Barthelmess who changed my life and first set me down this path when she agreed to be my undergraduate advisor. When I couldn’t decide if I wanted to be a writer or a biologist, Erika encouraged me to be both, and I am a better scientist for it. Her teaching and mentorship have shaped the professor that I hope to one day become.

Finally, and most importantly, I am deeply thankful for my partner, Josh LeBlanc, who, despite completing a PhD of his own and taking on extensive teaching responsibilities in the course of his degree, has never once wavered in his support. In the course of 5 years together we have overcome three surgeries and a major concussion, we have thoroughly confused Canadian border agents by swapping out our vehicle at least four times, and somehow managed to wrangle a queen-sized mattress into fitting a full-size bed frame. You have dealt with my prolonged absences in the field, have seen me through tears of loneliness, sorrow, and frustration and somehow, through all of it, have remained patient, supportive, and kind. I am deeply indebted to you for your love and support throughout this process.

This research would not have been possible without the support of the Champagne and Aishihik First Nations, especially Agnes MacDonald and her family, who have allowed us for the past 30 years to study red squirrels on their traditional territory. My research was generously supported by a variety of funding sources including an Ontario Trillium Scholarship, Grants-in-Aid of Research from the American Society of Mammalogists and the Arctic Institute of North America, an Elgin Card Scholarship in Terrestrial Zoology from the University of Guelph, and the Natural Sciences and Engineering Research Council of Canada.

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TABLE OF CONTENTS

Abstract ...... ii Dedication ...... iv Acknowledgements ...... v Table of Contents ...... vii List of Tables ...... ix List of Figures ...... x Prologue ...... xi Chapter Publications and Author Contributions ...... xvi Chapter 1: Familiarity With Neighbours Affects Intrusion Risk In Territorial Red Squirrels ...... 1

Abstract ...... 1 Introduction ...... 1 Methods...... 6 Results ...... 13 Discussion ...... 14 Tables ...... 21 Figures...... 23

Chapter 2: Red Squirrel Territorial Vocalizations Deter Intrusions By Conspecific Rivals ...... 25

Abstract ...... 25 Introduction ...... 25 Methods...... 29 Results ...... 32 Discussion ...... 32 Figures...... 36

Chapter 3: Red Squirrels Mitigate Costs Of Territory Defence Through Social Plasticity ...... 37

Abstract ...... 37 Introduction ...... 37 Methods...... 41 Results ...... 51 Discussion ...... 54 Tables ...... 60 Figures...... 64

Chapter 4: Stable Social Relationships with Territory Neighbours Affect Reproductive Success and Survival in an ‘Asocial’ Squirrel...... 66

Abstract ...... 66 Introduction ...... 66 Methods...... 70

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Results ...... 74 Discussion ...... 77 Tables ...... 83 Figures...... 86

Epilogue ...... 88 Supplementary Material: Chapter 1 ...... 94 Supplementary Material: Chapter 3 ...... 97 References ...... 99

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LIST OF TABLES

Table 1.1. Cox proportional hazard mixed effects models and AICc results predicting risk of intrusion based on the average characteristics of the neighbourhood ...... 21

Table 1.2. Cox proportional hazard mixed effects models and AIC results predicting risk of intrusion based on individual squirrel characteristics from all intruders ...... 22

Table 3.1. Fixed effects from generalized linear mixed-effects (GLMM) and Beta-Binomial (BB) models, showing effects of average neighbourhood familiarity, local density, and focal squirrel’s age on rattling rate, nest use and vigilance behaviour ...... 60

Table 3.2. Random effects from all generalized linear mixed-effects (GLMM) and Beta-Binomial (BB) models, showing effects of average neighbourhood familiarity, local density, and focal squirrel’s age on rattling rate, nest use and vigilance ...... 62

Table 3.3. Fixed effects from exploratory post-hoc models including a within-individual

(FamiliarityW) and among-individual (FamiliarityA) effect of familiarity on rattling rate, nest use and vigliance ...... 63

Table 4.1. Fixed effects from annual survival, male ARS, and female ARS generalized linear mixed-effects models ...... 83

Table 4.2. Random effects from annual survival, male ARS, and female ARS generalized linear mixed-effects models ...... 85

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LIST OF FIGURES

Figure 1.1. Effects of average familiarity and season on risk of intrusion...... 23

Figure 1.2. Effects of relative distance, relative relatedness and relative familiarity on the risk of a neighbouring squirrel intruding ...... 24

Figure 2.1. Latency to intrusion following the temporary removal of a territory owner during trials with and without a speaker replacement ...... 36

Figure 3.1. Effects of average familiarity on rattling rate and proportion of time spent in nest. . 64

Figure 3.2. Three scenarios for how variation in mean rattling rate (random intercepts) in combination with variation in data sampling structure might change our ability to detect among- individual effects when individuals have the same slope ...... 65

Figure 4.1. Results from full models showing effects of average neighbourhood familiarity on annual survival, number of pups sired, and number of pups recruited ...... 86

Figure 4.2. Results from senescent models showing effects of average neighbourhood familiarity on annual survival, number of pups sired, and number of pups recruited ...... 87

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Prologue

For most organisms, interactions with conspecifics are a ubiquitous part of daily life. This social environment can play a critical role in individual behaviour, influencing where individuals forage, how they move between habitats, or with whom they mate. In the broadest sense, the number of interacting conspecifics (i.e. density or group size) can affect rates of agonistic interactions (Cubaynes et al., 2014) or mediate dispersal (Matthysen, 2005). But it is also understood that the characteristics of group members can vary widely, and that variation in the composition of the social environment can have important consequences for individual behaviour and fitness, beyond the effects of coarser-grained features such as group size (Farine, Montiglio, & Spiegel, 2015). This has been well demonstrated in gregarious species. The presence of kin has been shown to increase the likelihood of alarm calling (Cheney & Seyfarth, 1985; Sherman, 1977), while variation in sex ratio can result in individuals altering their levels of aggression and activity (Andino, Reus, Cappa, Campos, & Giannoni, 2011; Montiglio, Wey, Chang, Fogarty, & Sih, 2017). In groups with a mix of behavioural types, frequency-dependent payoffs can lead to switching of behavioural tactics (Morand-Ferron, Wu, & Giraldeau, 2011).

Although it is taken for granted that social interactions are important for group-living species, the possibility that the social environment might also be relevant for solitary, territorial species is somewhat incongruous with the idea that such species generally have limited social contact. However, the notion of territoriality as a social phenomenon has been prevalent since at least the 1950s (Allee, 1951; Darling, 1952; Fisher, 1954). The existence of a territory is dependent on having conspecifics that occupy adjoining habitat. An organism that exists alone merely occupies space (Darling, 1952); it does not meet the definition of ‘territorial’ in that it does not actively defend an area from conspecifics (Brown & Orians, 1970) or occupy a fixed area where it has priority of access to an important resource (Kaufmann, 1983). Territoriality therefore cannot exist without some form of competition or cooperation among conspecifics that allows for the establishment of territory boundaries and thereby exclusive access to a given resource. Even in circumstances where defence of resources does not involve physical contact with conspecifics, regular interactions among territorial neighbours can occur through auditory signaling or olfactory signatures. Since the term ‘social’ in the broadest sense can encompass any

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situation where there are reoccurring interactions between organisms (Danchin, Giraldeau, & Cezilly, 2008), territoriality can be understood as a fundamentally social phenomenon.

Importantly, while our understanding of social interactions among territory neighbours has classically been defined by competition and aggression, it is also possible that territorial species may benefit from the presence of neighbouring conspecifics. This idea was first formalized by Stamps (1988), who demonstrated that in the absence of variation in resource quality, juvenile lizards (Anolis aeneus) were more likely to settle near established territorial residents, suggesting some benefit of ‘grouping’ with conspecifics even for species whose interactions were fundamentally competitive. Interestingly, this means that territorial neighbours may provide benefits similar to those provided by the presence of conspecifics for gregarious species (Smith, Lacey, & Hayes, 2017). For instance, it has been suggested that territorial clusters of males might attract more females than isolated individuals, thereby improving access to mates (Allee, 1951; Lack, 1948), or that the presence of neighbouring conspecifics may provide some degree of protection against risk of predation through alarm calling (Beletsky, Higgins, & Orians, 1986). Additionally, neighbours may cooperate in expelling intruders, thereby reducing the costs of territory defence (Getty, 1987). The implication then, is that social interactions among territorial conspecifics are not exclusively limited to competitive interactions but may also be shaped by cooperative or mutually beneficial exchanges. This alludes to the possibility that ‘asocial’ species may experience a much more nuanced social environment than has typically been appreciated.

Solitary, territorial species, like their social counterparts, can live in socially complex environments where neighbouring conspecifics can vary in personality, sex, age, relatedness, or familiarity, among other possibilities (Beletsky & Orians, 1989; Booksmythe, Hayes, Jennions, & Backwell, 2012; Brown & Brown, 1996). Territoriality, as an adaptive strategy, relies on a basic cost/benefit ratio, whereby the benefits of resource acquisition must outweigh the costs of resource defence (Brown, 1964; Schoener, 1987). Variation in the social environment (i.e. territorial neighbours) might therefore have important consequences by directly influencing the time and energy spent on territorial defence, and thus affecting fitness outcomes. Relatedness and familiarity with neighbours are two important aspects of the social environment with

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potential to affect interactions among territorial individuals. Interactions with kin might be expected to facilitate cooperation among conspecifics due to inclusive fitness benefits (Hamilton, 1964a; 1964b). Specifically, kin selection might favour the evolution of reduced aggression among related neighbours (Brown & Brown, 1993; Viblanc, Pasquaretta, Sueur, Boonstra, & Dobson, 2016) thereby allowing more time spent on self-maintenance behaviours (e.g. foraging; Brown & Brown, 1996) and potentially enhancing fitness (Viblanc et al., 2016; Viblanc, Arnaud, Dobson, & Murie, 2010). On the other hand, long-term neighbours are also known to engage in a cooperative agreement of reduced aggression and territory defence called the ‘dear enemy’ phenomenon (Fisher, 1954; Temeles, 1994). This is assumed to occur either because role- mistakes become less likely with increased familiarity (Ydenberg, Giraldeau, & Falls, 1988) or because long-term neighbours pose less of a threat to resources (Temeles, 1994). Familiarity with neighbours therefore has potential to mitigate conflict between interactants (Bebbington et al., 2017) and by reducing the costs of territoriality, potentially increase fitness of territory holders (Beletsky & Orians, 1989; Grabowska-Zhang, Wilkin, & Sheldon, 2012b). The significance of these social relationships for territorial species under natural conditions is, however, poorly understood. Importantly, the roles of direct and indirect benefits in resolving conflict among conspecifics remains one of the enigmas of evolutionary biology (Clutton-Brock, 2009; Sachs, Mueller, Wilcox, & Bull, 2004). Understanding the relative roles of kin selection and mutualistic interactions in reducing conflict and enhancing fitness among territorial neighbours might offer insight as to the importance of these mechanisms in the evolution of social systems.

In this thesis, I address the question of how variation in the composition of the social environment can affect the territorial dynamics, behaviour, and fitness of an ‘asocial’ species, using the North American red squirrel (Tamiasciurus hudsonicus) as a model system. My research was conducted as part of the Kluane Red Squirrel Project, which has been monitoring the same population of red squirrels in the southwest Yukon since 1987 (Berteaux & Boutin, 2000; McAdam, Boutin, Sykes, & Humphries, 2007). In the boreal forest, red squirrels defend exclusive territories centered around a larder hoard of spruce cones called a ‘midden’ (Fletcher et al., 2010). There is good reason to expect that the composition of neighbours around an

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individual’s territory is likely to be variable in this system. Relatedness with neighbouring conspecifics has potential to be high, as mothers are known to bequeath part or all of their territory to offspring (Price 1992; Price & Boutin 1993; Berteaux & Boutin 2000). The presence of food resources appears to be a likely factor affecting post-breeding behaviour of mothers, with females displaying reluctance to share or bequeath their territories when food stores are high and current food production is low, and an increased likelihood to disperse when current food production is high and food stores are low (Berteaux & Boutin 2000). Additionally, while juveniles have been shown to disperse upwards of 500 m, average dispersal distance of successful juveniles is 90 m, suggesting that the advantages of philopatry, including familiarity with the environment, access to parental resources, or proximity to close relatives, drive individuals to remain much closer to home (Berteaux & Boutin 2000). This variability in juvenile philopatry and post-breeding dispersal of adult females lends itself to an environment in which neighbours have potential to vary in relatedness. Furthermore, while red squirrels typically show limited propensity to relocate to a vacant midden (Larsen & Boutin, 1995), turnover of middens can occur through the death of a territory owner or occasionally through bequeathal, setting the stage for an environment with variability in neighbour familiarity. Adult overwinter survival can be as high as 80% for two-year old squirrels but declines with age, and average adult longevity is 3.5 years (McAdam et al. 2007). Importantly, fluctuations in available food resources may also be an important driver of variation in neighbourhood familiarity. During ‘mast’ years in the Yukon white spruce trees (Picea glauca) synchronize their cone production (LaMontagne & Boutin, 2007). Since white spruce cones are the primary food resource for red squirrels in this ecosystem, this influx of cones leads to high recruitment of juveniles (McAdam & Boutin, 2003), resulting in increases in density (Dantzer et al. 2012), and reduced neighbourhood familiarity as a result (E. Siracusa unpublished data).

For red squirrels, maintaining a midden is crucial for overwinter survival and thus these territories are heavily defended, primarily through vocalizations called ‘rattles’ (Lair, 1990; Siracusa, Morandini, et al., 2017b; Smith, 1978). Squirrels use these vocalizations to obtain information about their social environment and have been demonstrated to adjust their behaviour (Dantzer, Boutin, Humphries, & McAdam, 2012) and life history (Dantzer et al., 2013) in

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response to auditory cues of changes in local density. Given that rattles are also individually unique (Wilson et al., 2015), I wanted to understand whether squirrels could use these vocalizations to obtain and respond to more nuanced information about their social environment, specifically information about relatedness and familiarity with neighbours. I set out to answer three main questions in my thesis:

1) In what social environments do squirrels face the greatest risk of territory intrusion? 2) Can squirrels adjust their behaviour in response to the social environment to minimize costs of territoriality? 3) Does the social environment have important fitness consequences for squirrels?

I used a combination of long-term data analysis, behavioural observations, and field experiments to answer these questions. In Chapter 1, I conducted an experimental removal of territory owners to assess under which social environments and from which individuals red squirrels faced the greatest risk of territory intrusion and cache pilferage (Siracusa, Boutin, et al., 2017a). In Chapter 2, I used a speaker occupation experiment to test whether red squirrel ‘rattle’ vocalizations actually function to deter intruders and are therefore an important component of territory defence (Siracusa, Morandini, et al., 2017b). In Chapter 3, I combined several different behavioural measures, including focal animal observation, audio recorders and accelerometers to assess whether squirrels could adjust their behaviour in response to changing social conditions to minimize time spent on territory defence (Siracusa et al. in review). In Chapter 4, I analyzed 21 years of long-term data to quantify the effects of the social environment on annual survival and reproductive success of male and female squirrels. Collectively my thesis assesses the importance of the social environment for the behaviour and fitness of a solitary, territorial species, providing insight into the ways in which social interactions can affect the stability of territorial systems and the extent to which ‘asocial’ species can assess and respond to nuanced changes in the composition of their neighbouring conspecifics.

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Chapter Publications and Author Contributions

All four of my chapters incorporate data from a long-term study of North American red squirrels (the Kluane Red Squirrel Project- KRSP) located in the southwest Yukon, Canada near Kluane National Park (61° N, 138° W). Stan Boutin initiated the long-term study in 1987 and has maintained the study until present day with support from Murray M. Humphries, Andrew G. McAdam, Ben Dantzer and Jeffrey Lane as principle investigators on the project. The pedigree has been maintained by David W. Coltman and Jamieson C. Gorrell. All of the long-term data is a result of the hard work of many dedicated technicians over the past 30 years.

Chapter 1 is published in Animal Behaviour (Siracusa, Boutin, et al., 2017a) and was coauthored by Stan Boutin, Murray M. Humphries, Jamieson C. Gorrell, David W. Coltman, Ben Dantzer, Jeffrey E. Lane and Andrew G. McAdam. ERS and AGM conceived of and designed the study. ERS conducted the fieldwork (removal experiment). ERS conducted the statistical analyses and wrote the manuscript with support from AGM. All authors provided comments and feedback on the manuscript.

Chapter 2 is published in Behaviour (Siracusa, Morandini, et al., 2017b) and was coauthored by Marina Morandini, Stan Boutin, Murray M. Humphries, Ben Dantzer, Jeffrey E. Lane and Andrew G. McAdam. ERS and AGM conceived of and designed the study. ERS and MM conducted the fieldwork (speaker occupation experiment). ERS and MM analyzed the data and wrote the manuscript, with comments and feedback from all authors.

Chapter 3 is in review at Animal Behaviour (ANBEH-D-18-00328) and was coauthored by David R. Wilson, Emily K. Studd, Stan Boutin, Murray M. Humphries, Ben Dantzer, Jeffrey E. Lane and Andrew G. McAdam. ERS and AGM conceived of and designed the study. ERS conducted the fieldwork (focal observations, deployment of audio recorders and accelerometer collars). EKS developed the accelerometer calibration to allow for the characterization of different red squirrel behaviours. DRW developed the discriminant function analysis to isolate red squirrel rattle vocalizations. ERS conducted statistical analyses with support from AGM,

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EKS, and DRW. ERS wrote the manuscript with support from AGM. All authors provided comments and feedback on the manuscript.

Chapter 4 is not yet out for review but will be coauthored by Stan Boutin, Murray M. Humphries, Ben Dantzer, Jeffrey E. Lane, Dave W. Coltman, and Andrew G. McAdam. ERS and AGM conceived of and designed the study. ERS conducted statistical analyses and wrote the manuscript with support from AGM.

Chapter 1: Familiarity With Neighbours Affects Intrusion Risk In Territorial Red Squirrels

Abstract

Interactions with conspecifics are an important aspect of an individual’s environment. Although it is well known that the presence of conspecifics can have important effects on behaviour, in general it is also now acknowledged that the composition of the social environment can vary, and that this variation may have profound effects on individual behaviour and fitness. Using a wild population of North American red squirrels, we investigated the importance of the composition of the social environment in a territorial species by assessing whether the risk of intrusion faced by territory owners varied with the degree of relatedness and familiarity in their social neighbourhoods. To test this, we conducted temporary removals of territory owners and observed the time until intrusion and the identity of intruding individuals. We found that individuals in neighbourhoods with low average familiarity faced a higher risk of intrusion and that unfamiliar neighbours were more likely to intrude. Surprisingly, we found that related neighbours also posed a higher risk of intrusion. The results from our study suggest that familiarity with neighbours may be an ecologically and evolutionarily relevant measure of the social environment, even in a species considered to be ‘asocial’. Future studies should consider the potential importance of the social environment, which has heretofore been mostly overlooked, as a relevant selective pressure in asocial, territorial species.

Introduction

An important aspect of an organism’s daily life consists of interactions with conspecifics, comprising what we consider to be an individual’s social environment. Such interactions with social partners are increasingly recognized to be important to individual behaviour and fitness (Montiglio, Ferrari, & Réale, 2013; Moore, Brodie, & Wolf, 1997; Saltz, 2013; Wolf, Brodie, & Moore, 1999). In the broadest sense, the presence of conspecifics may cause individuals to change the frequency with which they engage in certain behaviours (reviewed by Beauchamp, 2003), or may modulate the behavioural tactics that individuals use (Fischer, Schwartz, Hoke, & 1

Soares, 2015; Hellmann & Hamilton, 2014). However, variation in the composition of social groups can have effects on behaviour and fitness above and beyond the mere presence or number of conspecifics in an individual’s environment. For instance, the presence of kin in social groups can reduce aggressive interactions and vigilance behaviour while increasing proportion of time spent foraging or feeding (Brown & Brown, 1993; 1996; Davis, 1984; Viblanc et al., 2016) and may positively influence survivorship and reproductive success (Hokit & Blaustein, 1997). Social units with stable composition (familiar associations among individuals) benefit from undisrupted dominant and subordinate roles, which results in increased tolerance and resource sharing for all group members (Senar, Camerino, & Metcalfe, 1990). In degus (Octodon degus) group stability has been demonstrated to have a modulating effect on the relationship between group size and fitness (Ebensperger et al., 2016). Social instability (number of changed neighbours), on the other hand, has been demonstrated to result in increased aggressive signaling in Betta splendens (Matessi, Matos, Peake, McGregor, & Dabelsteen, 2010).

For territorial species the social environment is often defined by agonistic interactions. A variety of definitions for territoriality have been given, including but not limited to, the exclusive use of a fixed area (‘ecological’ territoriality; Pitelka, 1959), the active defence of an area from conspecifics (‘behavioural’ territoriality; Brown & Orians, 1970), or a fixed area where an individual has priority of access to an important resource (Kaufmann, 1983). All definitions, however, necessitate that territoriality involve social interactions, via defence of one’s territory, mutual avoidance, or cooperation among neighbours. Most territorial species live in socially complex environments with a composition of neighbours that vary in relatedness (Brown & Brown, 1993), familiarity (Beletsky & Orians, 1989), sex (Eckenweber & Knörnschild, 2013), body condition (e.g. size; Booksmythe et al., 2012), aggression (Hyman & Hughes, 2006) or resource holding potential (Parker, 1974), among other possibilities. Given that territoriality is not adaptive unless the benefits of increased access to resources outweigh the expenditure of time and energy in defending one’s territory (Brown, 1964; Brown & Orians, 1970), it should be expected that the composition of neighbours around an individual’s territory matters, as those individuals will directly influence the frequency, and therefore cost, of territorial defence. However, the role of neighbours remains contentious and appears to vary based both on the 2

conditions and system of study under observation (Carpenter, 1987).

One of the most widely documented patterns of differential behaviour among neighbouring individuals is the reduced aggression of territory owners toward familiar individuals relative to unfamiliar individuals (Fisher, 1954; reviewed in Temeles, 1994). Coined by Fisher in 1954, the ‘dear enemy effect’ is a cooperative phenomenon of reduced aggression among long-term neighbours, which has been hypothesized to occur either because long-term neighbours pose less of a threat to resources than unfamiliar neighbours or strangers (Temeles, 1994) or because the chances of role mistakes becomes less likely with increased familiarity (‘asymmetric war of attrition’; Ydenberg et al., 1988). In either case, the function of the dear enemy effect is believed to be reduced engagement in expensive conflicts, which allows for increased time devoted to reproduction and growth. Familiarity with neighbouring individuals has been demonstrated to increase reproductive success in red-winged black birds (Agelaius phoeniceus; Beletsky & Orians, 1989) and great tits (Parus major; Grabowska-Zhang, Wilkin, & Sheldon, 2012b), confer immediate fitness benefits through faster predator-evasion responses in brown trout (Griffiths, Brockmark, Hojesjo, & Johnsson, 2004), enhance survival and body condition in Arctic char (Salvelinus alpinus; Seppä, Laurila, Peuhkuri, Piironen, & Lower, 2001), as well as improve the survival and reproductive success of kangaroo rats (Dipodomys stephensi) following translocation (Shier & Swaisgood 2012). However, neighbours have also been demonstrated to be important territorial intruders (Paton & Carpenter, 1984; Smith & Ivins, 1986), and in cases where competition between territory owners is particularly intense, individuals display increased aggression toward neighbours (‘nasty neighbour effect’- Müller & Manser, 2007; Newey, Robson, & Crozier, 2010; Yoon, Sillett, Morrison, & Ghalambor, 2012).

Despite evidence that the composition of individuals in the social neighbourhood does matter for behaviour and fitness, our understanding of the effects of social composition on territorial interactions remains limited. For many species, territoriality is maintained through vocalizations, displays, and scent markings, as a means to establish borders and exclude potential competitors. However, territorial borders are not impenetrable and competitors may intrude as a means to increase access to resources. Hinsch & Komdeur (2010) have shown mathematically 3

that the evolutionary stability of territoriality depends on the rate of intrusions from territorial neighbours. Since territoriality is an energetic trade off between access to, and defence of, resources, intrusion by neighbours has significant potential to undercut the benefits of maintaining a territory (as a result of resource theft) while at the same time increasing the costs of defence (Hinsch & Komdeur, 2010; Hixon, 1980; Schoener, 1987). Understanding the social conditions under which territory owners face higher intrusion risk when defensive signaling cues are eliminated may provide insight into how territory owners should be expected to allocate defensive time and energy expenditure in response to their social environment in order to minimize costly intrusions.

North American red squirrels (Tamiasciurus hudsonicus) in the northern boreal forest harvest and cache newly matured white spruce (Picea glauca) cones in a larder hoard at the centre of their territory, called a ‘midden’ (Fletcher et al., 2010). Red squirrels depend heavily on these cached cones for overwinter survival and defend exclusive, non-overlapping territories surrounding their midden (Smith, 1968). Defence occurs primarily through the use of territorial vocalizations called ‘rattles’(Smith, 1978). Rattles are an important mechanism for communication between neighbours and function to deter intruders (Siracusa, Morandini, et al., 2017b), thus minimizing potential costly physical interactions (Lair, 1990; Smith, 1978). Although physical interactions between individuals occur infrequently (Dantzer et al., 2012; Gorrell, McAdam, Coltman, Humphries, & Boutin, 2010), red squirrels obtain information about their social environment through the acoustic information contained within neighbouring rattles (Dantzer et al., 2012). A squirrel’s social environment, therefore, is defined by the 130 m radius at which rattles can be detected (Smith, 1978). Within this acoustic social neighbourhood red squirrels use rattles to assess local population density and adjust their behaviour (Dantzer et al., 2012) and life history (Dantzer et al., 2013) accordingly. Playback experiments have demonstrated that red squirrels are able to recognize kin through rattles (Wilson et al., 2015) and that squirrels may also distinguish between neighbour and stranger vocalizations (Price, Boutin, & Ydenberg, 1990). The use of acoustic signaling, therefore, allows for the establishment of a rich social network, one that has so far been demonstrated to affect both behaviour and life history in a species typically considered to be ‘asocial’. 4

While the importance of the quantity of individuals in the social environment has been demonstrated in red squirrels (Dantzer et al., 2012; 2013), the composition of red squirrel social neighbourhoods are also likely to vary in two important ways: relatedness and familiarity (length of time as neighbours). Juveniles will sometimes settle next to their mother, as breeding females are known to sometimes bequeath part or all of their territory to offspring (Berteaux & Boutin, 2000; Price, 1992; Price & Boutin, 1993). However, it is also common for juveniles to disperse from their natal territories and settle next to siblings, their father, or in many cases mostly unrelated neighbours (Berteaux & Boutin, 2000), creating variation in relatedness among neighbourhoods. In addition, overturn of middens can occur through the death of a territory owner or occasionally through bequeathal. As a result some squirrels may occupy different territories each year, setting the stage for an environment with variation in familiarity (i.e. duration of tenure as neighbours).

The purpose of this study was to examine under what social conditions and from which individuals red squirrels face increased risk of territorial intrusion in the absence of territory defence. By measuring intrusion risk, we aimed to better understand how red squirrels should be expected to adjust territorial behaviour in response to the composition of their social environment. Removal experiments of territorial owners have demonstrated that red squirrels quickly recognize and will pilfer cones from vacated territories (Donald & Boutin, 2011). Most pilferers have been shown to be juvenile squirrels from adjacent middens (i.e. “neighbours”; Donald & Boutin, 2011), but the relationship (e.g. relatedness, familiarity) of the pilferer to the territory owner has not yet been explored. We tested two alternative, non-mutually exclusive hypotheses regarding risk of intrusion. Kin selection theory postulates that individuals might act altruistically toward relatives if this enhances their inclusive fitness (Hamilton, 1964a; 1964b). Since an intrusion is likely a costly event for the territory owner, we predicted that red squirrels should face greater risk of intrusion from non-kin since there is no indirect fitness cost to these individuals of pilfering or attempting to steal a territory from an unrelated neighbour. Alternatively, the dear enemy phenomenon suggests that long-term neighbours enter into a cooperative relationship of reduced vigilance and aggression along a shared territorial border 5

(Fisher, 1954). Long-term neighbours should, therefore, avoid intruding and breaking this cooperative relationship since it is likely to be defensively costly. We predicted that red squirrels should face greater risk of intrusion from new neighbours since levels of aggression are likely to be high as territory boundaries are negotiated, imposing little additional cost on the interactants when intrusions do occur.

To test these ‘kin selection’ and ‘dear enemy’ hypotheses, we temporarily removed territory owners to assess the risk of intrusion from neighbouring squirrels. We made the following predictions for each hypothesis: If kin-selection influences risk of intrusion we predicted that 1) the probability of intrusion would be higher and the latency to intrusion would be lower for territory owners in neighbourhoods with low average relatedness, 2) unrelated neighbours would be more likely to intrude, and would intrude faster than more closely related neighbours. If familiarity with neighbours affects risk of intrusion we predicted that 1) the probability of intrusion would be higher and the latency to intrusion would be lower for territory owners in neighbourhoods with low average familiarity (a high proportion of new neighbours), and 2) less familiar neighbours would be more likely to intrude and would intrude faster than more familiar neighbours.

Methods

Study Site and Population Between April and September 2015, we studied a population of free-ranging North American red squirrels, monitored since 1987 as part of the Kluane Red Squirrel Project (KRSP; see Berteaux & Boutin, 2000; McAdam et al., 2007 for a detailed description of study sites and project protocols). The study sites are located in the southwest Yukon, Canada near Kluane National Park (61N, 138W), and are characterized as open boreal forest, dominated by white spruce (Picea glauca; Krebs, Boutin, & Boonstra, 2001). Each year we enumerated all individuals in the population (identifiable via permanent alphanumeric ear tags) and threaded the ear tags with a unique combination of coloured wires. The use of coloured wires allowed us to identify individuals and record behaviour from a distance. We monitored the squirrels on the study sites from March until August each year and used a combination of live-trapping procedures and 6

behavioural observations to track reproduction and identify territory ownership.

We conducted our experiment on two study sites. Both sites were located along the Alaska Highway within 4.5 km of each other. Each site was flagged and staked at 30 m intervals to provide spatial coordinates for trapping and behavioural observations. Using these stakes, spatial data could be recorded to a resolution of 3 m. One study site was maintained as a control (40 ha), while the other site was provided with supplemental food beginning in 2004 as part of a larger ongoing study (45 ha). Briefly, between October and May, each individual squirrel was provided with 1 kg of all-natural peanut butter at six-week intervals. The peanut butter was placed in a suspended bucket at the centre of each individual’s territory. At the end of May the peanut butter was removed from all individuals except females that were pregnant or lactating. This food supplementation has been demonstrated to lead to an increase in population density compared to the average population density on control sites (Dantzer et al., 2012). However, 2014 was a year of high spruce cone production (mast year; LaMontagne & Boutin, 2007). The increased survival of juveniles following a mast year (McAdam & Boutin, 2003) led to high population densities on both the food-supplemented and control sites in 2015. At the start of this study, density on the control site was 3.55 squirrels/ha and on the food-add site was 4.74 squirrels/ha (average densities in the Yukon are 1.75 squirrels/ha; Dantzer et al., 2012).

Assessing Relatedness and Familiarity of Neighbourhoods For all territory owners in our study sites, we defined an individual’s ‘neighbourhood’ to be all conspecifics within a 130 m radius, since this is the known audible range of red squirrel rattles (Smith, 1978). We assessed how the composition of red squirrel neighbourhoods varied in relatedness and familiarity (length of time as neighbours).

Relatedness We collected DNA and permanently tagged red squirrel pups while still in their natal nest, so we could assign maternity with certainty. We assigned paternity starting in 2003 by genotyping all individuals at 16 microsatellite loci (Gunn et al., 2005) and assigning paternity with 99% confidence using CERVUS 3.0 (Kalinowski, Taper, & Marshall, 2007). Using the established 7

multigenerational pedigree, currently spanning 29 years and over 9,000 individuals, (Lane, Boutin, Gunn, Slate, & Coltman, 2008; McFarlane et al., 2015) we calculated the coefficient of relatedness between a territory owner and each of its neighbours. Among the neighbourhoods used in this study, 20% of neighbours were yearlings on the food-supplemented site in 2015 and were not included in the latest version of the pedigree (created in autumn 2014). As a result, these individuals were not genotyped and so relatedness to the territory owner was unknown. Averaging coefficients of relatedness across all neighbours provided a measure of average relatedness for each individual’s social neighbourhood.

Familiarity Every May and August we completely censused the population by using a combination of live- trapping and behavioural observations to determine the owner of every territory on each study site. For each territory owner, we determined the familiarity of neighbours using existing census data to assess the length of time that each pair of squirrels had been neighbours (i.e. occupied their current territories within 130 m of one another). An individual’s average familiarity was calculated as the average number of days that the territory owner had been a neighbour with each of the individuals in its social neighbourhood. As a result of the biannual census, familiarity is updated twice each year.

Temporary Removal Experiment To assess variation in the risk of territory intrusion within and among social neighbourhoods, we conducted a temporary experimental removal of male midden owners. Although only males were used in this study, both males and females defend exclusive food-based territories and exhibit territorial behaviour that is the same in both form and function (Smith, 1981; Smith, 1968). We did not remove females because of the potential animal welfare implications associated with temporarily removing of a female with dependent offspring. Furthermore, intrusion on territories owned by females could also be affected to some degree by the risk of infanticide of dependent offspring. Here we were only interested in intrusion risk associated with the defence of food or spatial resources.

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The focal individuals for this study were chosen in order to capture neighbourhoods with a range of relatedness and familiarity values. All focal individuals chosen for removals were 60 m apart, at minimum. The first set of experimental removals (N = 53) was conducted between 5 April and 20 June 2015 (hereafter, ‘summer’), which followed the mating season. We were careful not to conduct removals while mating was still ongoing to avoid intrusions by male squirrels in search for females in estrus. We conducted 28 of these removals on the control site and 25 on the experimental site. A second set of experimental removals was conducted on 50 of the same individuals and 1 new individual between 2 August and 8 October 2015 (hereafter, ‘autumn’), which followed juvenile settlement (N = 51, two of our original individuals died over the summer, the third lost both ear tags and we were unable to confirm the individual’s identity so we replaced this individual with a new squirrel). By conducting a second set of removals post- juvenile settlement our hope was to assess important changes in the composition of the social neighbourhood resulting from the settlement of new neighbours. Twenty-seven of these removals were conducted on the control site and 24 on the food-add site.

Methods for the temporary removals were adapted from Donald & Boutin (2011). Squirrels were live-trapped using Tomahawk traps (Tomahawk Live Trap Co., Tomahawk, Wisconsin) baited with peanut butter. Once trapped, we transferred the squirrel to a modified box (41 cm x 17.5 cm x 19 cm), which helped to keep it calm and quiet for the duration of the removal. We then moved the squirrel a distance of 20-30 m away from its midden. Care was taken not to place the captive squirrel on another squirrel’s midden. From the start of the removal an observer monitored the captive squirrel’s midden for a total of two hours, from a distance of no less than 5 m from the midden centre. Red squirrels rattle on average once every seven minutes (Dantzer et al., 2012). Therefore, a two-hour removal provided a reasonable time frame for neighbouring squirrels to notice and capitalize on the lack of territorial defence, as demonstrated by a previous study in this system (Donald & Boutin, 2011).

During the two-hour removal period we recorded between zero and four different intruders per trial (Fig. S1.1). A territory intrusion was defined as occurring when a squirrel moved onto the larder hoard of spruce cones (i.e. the ‘midden’) located near the centre of the 9

owner’s territory. The edges of the midden were defined by the extent of discarded cone scales on the surface of the ground. When an intrusion was witnessed, the observer recorded the time of intrusion and the identity of the intruding squirrel. At the end of the two hours we released the captive squirrel at the site of capture. This research was approved by the University of Guelph Animal Care Committee (AUP number 1807) and is in compliance with the ASAB/ABS Guidelines for the Use of Animals in Research.

Statistical Analysis For all statistical analyses, we used Cox proportional hazard mixed effects models (PHMM) in the R package coxme (version 2.2-5: Therneau, 2015). Cox proportional hazard models take into account both the probability of intrusion (1 = intrusion observed, 0 = no intrusion observed) as well as the time to intrusion, combining these into a single response measure called the hazard of intrusion. For the purposes of interpretability the response variable hereafter will be referred to as the ‘risk of intrusion’. A high risk of intrusion in all cases is therefore equivalent to a high probability of intrusion and a fast time to intrusion.

Assessing risk of intrusion among red squirrel social neighbourhoods We were first interested in identifying the characteristics of red squirrel social neighbourhoods affecting risk of intrusion. For this analysis, each removal was assigned a value for latency to intrusion based on the time of first intrusion. Trials with no intruders were given censored values, indicating that these trials may have resulted in intrusions given a longer removal time. We excluded all trials in which the first intruder was from greater than 130 m away, or had no known home (22 intruders; 21% of trials), since these intrusions were interpreted as risk extrinsic to the social neighbourhood. All models (excluding the intercept model) included season as a categorical variable, since removals were conducted in both summer (post-breeding) and autumn (post-juvenile settlement). We found that average age and average familiarity were positively correlated (Pearson’s product-moment correlation: r = 0.35, N = 82, P = 0.001). This was expected since when a territory is first established the neighbourhood may be composed of neighbours of a variety of ages, however, as average familiarity increases so too does average age of the neighbourhood. However, average age was not found to be a significant predictor of 10

risk of intrusion ( = 0.005 ± 0.17, z = 0.03, P = 0.98) and so was excluded from further analysis. The kinship model included average relatedness as a continuous covariate to test the hypothesis that red squirrels face higher risk of intrusion in neighbourhoods with low average relatedness. The dear enemy model included average familiarity as a continuous predictor to test the hypothesis that red squirrels face higher risk of intrusion in neighbourhoods with low average familiarity. Average familiarity was transformed using a natural logarithm transformation prior to analysis to account for a nonlinear relationship between the response and predictor variable.

We also tested for effects of neighbour proximity and neighbour density on risk of intrusion. The proximity model included average distance of neighbours from the focal midden as a continuous covariate and the density model included the density of squirrels in the social neighbourhood as a continuous predictor, measured as the number of squirrels per hectare within 130 m. We were initially interested in testing for an interaction between each continuous covariate and season to determine if the importance of the aforementioned predictors to risk of intrusion was affected by season. For each model above we fitted a separate model including an interaction between season and the primary covariate. None of these interactions were significant, however, and so were excluded from this analysis. The global model included all of the aforementioned continuous covariates, as well as a categorical effect of season. In all of these mixed effects models we included a random intercept term for individual identity of the squirrel (‘squirrel ID’) to account for inter-individual variance due to the repeated removals of the same squirrels. We also fitted a null model that included only the intercept. The fit of these seven a priori models were compared and evaluated based on differences (i) in Akaike’s Information Criterion corrected for small sample sizes (AICc; corrected for small sample sizes; Burnham & Anderson, 2002).

Assessing individual risk of intrusion within red squirrel social neighbourhoods Each territory owner that was temporarily removed had between 8 and 33 neighbours within their 130 m auditory neighbourhood. We were therefore interested in breaking down average neighbourhood effects to assess which factors affected variation in risk within the social neighbourhood. For this analysis we included trials that had intruders from greater than 130 m 11

away, or with no known home, but removed these individual intruders from the analysis (30 intruders), since these intrusions were interpreted as risk extrinsic to the social neighbourhood. At the end of the 120-minute removal, all intruding neighbours were given a value for latency to intrusion and all non-intruding neighbours were given censored values, indicating that these individuals may have intruded given a longer removal time. In all models we included a random intercept term for squirrel ID to account for both inter-individual variance due to the repeated removals of the same focal squirrel, as well as the fact that each focal squirrel had many neighbours in its social neighbourhood, each of which was scored for their risk of intrusion. For all continuous covariates, relative values were calculated by subtracting the average value of the neighbourhood from the individual value, giving a value for each covariate that was relative to all other squirrels in the neighbourhood. The following models include all intruders within 130 m of the focal midden. We completed an additional analysis using only the first intruders, but found very similar results (see Table S1.1).

As above, we found relative age and relative familiarity to be positively correlated (Pearson’s product-moment correlation: r = 0.41, N = 2353, P < 0.001). Age was not a significant predictor of an individual’s risk of intruding ( = -0.19 ± 0.13, z = -1.49, P = 0.14) and so was excluded from further analysis. We also included sex of the intruder in our initial analyses. In some models males tended to be more likely to intrude, but this effect was never significant and so was not explored further. The relatedness model included relative relatedness as a continuous covariate to account for our initial hypothesis that less related neighbours pose a higher risk of intrusion. The familiarity model included relative familiarity as a continuous covariate to account for our initial hypothesis that less familiar neighbours pose a higher risk of intrusion. We also included a distance model to assess if closer neighbours would be more likely to intrude. To determine whether the identity of the intruder was affected by season we fit four separate models that included an interaction between season and each of the aforementioned covariates. None of these interactions were significant and so were excluded from further analysis. The global model included all of the aforementioned continuous predictors. We also fitted a null model containing only the intercept. The fit of these six a priori models were compared and evaluated based on differences (i) in Akaike’s Information Criterion. 12

All Cox proportional hazard mixed effect models were found to adequately meet the proportional hazards assumption (Cox, 1972). We used dfbeta residuals to ensure that there were no outlying observations with undue influence. We used martingale residuals fitted by local linear regression to test for the presence of significant non-linearities between our response and predictor variables. All continuous variables were standardized to a mean of zero and unit variance prior to statistical analysis, and all neighbours missing from the pedigree were assigned a standardized relatedness value of zero. For the following results, we present all means ± SE, unless otherwise stated, and considered differences statistically significant at  < 0.05. All analyses were performed using R version 3.2.3 (R Core Team, 2015).

Results

During the 104 removal trials we observed 77 cases (74% of trials) of intrusion on the focal territories within the two-hour observation period. On average, first intruders arrived at the midden after 55.89 minutes (range: 0-120 min; Fig. S1.2). Seventy-one percent of these first intruders came from within the focal squirrel’s auditory social neighbourhood (with middens < 130 m away). The mean distance of neighbouring middens was 86 meters (range: 9-130 m) from the focal midden. Neighbouring squirrels were related by 0.05 on average (range: 0-0.55) and the average familiarity (length of time as neighbours) between focal individuals and their surrounding neighbours was 227.3 days (range: 0-1590 days).

Risk of intrusion among red squirrel social neighbourhoods

Model selection using AICc revealed the dear enemy model to be the most parsimonious for predicting risk of intrusion by average neighbourhood characteristics. All other models had substantially less support (i values were between 5.59 and 9.54; Table 1.1). Risk of intrusion was significantly higher for individuals in neighbourhoods with low average familiarity ( = - 0.44 ± 0.13, z = -3.28, P = 0.001; Fig. 1.1). Seasonality was also a significant predictor of risk of intrusion (2 = 7.27, df = 1, P = 0.007). Specifically, intrusion risk was significantly higher in the autumn (79% of trials had an intruder) relative to the summer (57% of trials had an intruder;  = 0.79 ± 0.29, z = 2.71, P = 0.007; Fig. 1.1). Risk of intrusion was not affected by average density

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of squirrels in the neighbourhood, average relatedness of the neighbourhood, or average distance of neighbours from the focal midden (all |z| < 0.73, all P > 0.46).

Individual probability of intrusion within red squirrel social neighbourhoods

In assessing the factors affecting variation in intrusion risk within the social neighbourhood, model selection using AIC revealed that there was substantial support for the global model, with considerably less support for the distance model (i = 7.73), and no support for all other models

(i values were between 123.79 and 145.91; Table 1.2). Based on the global model, neighbours that were closer to the focal midden were significantly more likely to intrude ( = -1.35 ± 0.14, z = -9.53, P < 0.001; Fig. 1.2) and less familiar neighbours (newer) were also more likely to intrude ( = -0.33 ± 0.15, z = -2.23, P = 0.03; Fig. 1.2). Surprisingly, neighbours that were more related to the focal individual were also significantly more likely to intrude ( = 0.22 ± 0.08, z = 2.80, P = 0.005; Fig. 1.2). Thirteen of the 79 intrusions were by neighbours related by at least 0.5 to the territory owner. Of these 13 closely related intruders 6 were mothers that intruded on their offspring’s territory, 4 were siblings, and 3 were daughters who intruded on their father’s territory.

Discussion

Conspecific intruders can represent a significant threat to the stability of territorial systems by simultaneously decreasing the value of the resource being defended (via pilfering) while increasing the cost of defence (Hinsch & Komdeur, 2010). The goal of this study was to measure risk of intrusion in the absence of territory defence in order to better understand how squirrels might be expected to adjust their territorial behaviour in response to social conditions to mitigate threat of intrusion while minimizing costs of defence. We used only male squirrels in this study, however both male and female red squirrels defend exclusive territories based around a central cache of food resources and exhibit territorial behaviour that does not differ between the sexes.

We set out to test two a priori hypotheses about the aspects of the social environment affecting risk of intrusion in a territorial tree squirrel. Given the presence of variation in the

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composition of red squirrel social neighbourhoods, we had hypothesized that intrusion risk would be affected by the presence of kin (kin-selection hypothesis) or familiarity with neighbours (dear-enemy hypothesis). The results from our study supported all of the predictions derived from the dear-enemy hypothesis: 1) among territories, individuals in neighbourhoods with low average familiarity faced a higher risk of intrusion (i.e. a higher probability of intrusion and a faster rate of first intrusion; Fig. 1.1) and, 2) within neighbourhoods, less familiar individuals were more likely to be the ones that intruded and had a faster rate of intrusion (Fig. 1.2). We found no effect of average relatedness on the overall risk of intrusion, but contrary to our prediction, more related neighbours posed a higher risk of intrusion than less related neighbours (Fig. 1.2).

We included distance in the AIC models to account for the null expectation that closer neighbours would be more likely to intrude, and found that distance was a significant predictor of intrusion risk. The average distance of neighbours did not affect intrusion risk, but unsurprisingly, within neighbourhoods, closer neighbours posed a higher risk of intrusion (Fig. 1.2). While squirrels can detect rattles up to 130 m away (Smith, 1978), we expected that closer neighbours would more easily detect the absence of neighbouring rattles and thus would be more likely to intrude onto the vacated territory. We also included density in the AIC models to account for our previous finding that squirrels rattle more frequently when surrounded by a higher density of conspecifics (Dantzer et al., 2012; Shonfield, Taylor, Boutin, Humphries, & McAdam, 2012), suggesting that higher density conditions might signal higher intruder pressure. However, like Dantzer et al. (2012), we found that density did not have an effect on intrusion risk.

Effects of Familiarity In many species, long-term neighbours reduce vigilance and aggression along a shared territorial border. This type of neighbour-stranger discrimination is a cooperative phenomenon known as the dear-enemy effect (Temeles, 1994). The results from our study demonstrate that red squirrels faced reduced risk of intrusion when they were more familiar with their neighbours, which suggests that this type of cooperative phenomenon is occurring in red squirrels. Our results 15

support those of Price et al. (1990), which demonstrated that red squirrels respond more intensely to territorial rattle playbacks from non-neighbours than from neighbours. However, more recently Wilson et al. (2015) found that while red squirrels discriminated between kin and non- kin, they did not respond differentially to the rattles of neighbours and non-neighbours. One possible explanation for this discrepancy is that Wilson et al. (2015) did not control for the location from which neighbour and non-neighbour rattles were broadcast. It is possible that squirrels differentiate between neighbours and non-neighbours based on both the identity and location of their calls. If some component of associative learning is involved in identification, a territory owner may no longer recognize a neighbour’s vocalization when disassociated from the location where that neighbour usually calls, or, alternatively, a neighbour calling from a new location may still be individually identifiable but may be recognized as an increased threat to territory ownership (Falls & Brooks, 1975; Godard, 1991). Either possibility could result in the similarity of response to neighbours and non-neighbours observed in Wilson et al. (2015).

Most demonstrations of the dear-enemy effect have attempted a straightforward test of the phenomenon by observing a territory owner’s response to a dyadic interaction with a familiar or unfamiliar conspecific. Here we have approached the dear enemy phenomenon from a different perspective, by observing the response of neighbours rather than territory owners. Arguably this allows for a deeper understanding of the implications of the dear enemy effect, beyond a simple demonstration of the phenomenon itself. Given the potential for intrusions to undermine the stability of the territorial system, due to associated costs of defensive time and energy and the loss of resources that may affect fitness (Hinsch & Komdeur, 2010), our study suggests that familiarity may be an ecologically and evolutionarily relevant measure of the composition of the social environment in red squirrels. While we found the average familiarity of the social neighbourhood to be a strong indicator of intrusion risk among territories, the effect was somewhat weaker within territories, and relatedness and distance were also found to be significant predictors of relative intrusion risk among neighbours. It is possible that in neighbourhoods with low average familiarity (lots of new neighbours) territory owners may have to spend disproportionate amounts of time defending their midden, allowing other typically “less risky” neighbours to take advantage of the occupant’s diverted attention to steal food resources. 16

Our findings imply that familiarity is a salient metric of the social environment in a species considered to be ‘asocial’ and may have important, and previously underappreciated, effects on behaviour and fitness.

The observed effect of familiarity on territorial intrusion risk may also help explain the variation in hoarding tactics seen in red squirrels across North America. Throughout most of their range, red squirrels hoard food items in a central location, however, in the eastern portion of their range red squirrels are known to frequently scatter-hoard food items (Dempsey & Keppie, 1993; Layne, 1954). Several hypotheses have been proposed to explain this variation in hoarding behaviour, including increased predation risk in western populations and the importance of a central, concentrated food source to minimize energy expenditure in conditions of extreme cold (Dempsey & Keppie, 1993). However, it is also possible that the risk of intrusion in eastern populations is such that it leads to the breakdown of a larder-hoarding system. If the demography (mortality, immigration, emigration) of these eastern populations is such that territory owners never become familiar enough with their neighbours to develop dear enemy benefits, it is possible that intrusion risk may remain too high for central caches to be economical (Clarke & Kramer 1994).

Effects of Relatedness Contrary to our predictions from the kin-selection hypothesis we found that, within neighbourhoods, more related individuals posed a higher risk of intrusion (Fig. 1.2), although there was no effect of the average relatedness of the neighbourhood on intrusion risk. We had predicted that more related individuals would be less likely to intrude since intruding and stealing food resources poses a cost to the victim, which would then be incurred by the intruder as an indirect fitness cost if that individual shares genes with the victim (Hamilton, 1964a; 1964b). However, it is also possible that squirrels are more tolerant of intrusions from related individuals if access to food resources enhances the intruder’s survival and thus the inclusive fitness of the territory owner (Hamilton, 1964a; 1964b). Previous research on this squirrel population has demonstrated that squirrels are able to recognize and discriminate between kin and non-kin. Red squirrels have been shown to adopt closely related orphaned juveniles (Gorrell 17

et al., 2010), and in cold temperatures will engage in communal nesting with kin (Williams et al., 2013), suggesting that under certain conditions red squirrels tolerate relatives on their territory. Red squirrels have also been demonstrated to rattle less frequently toward playbacks of vocalizations from kin vs. non-kin (Wilson et al., 2015). While it might be presupposed that red squirrels rattle less frequently because kin pose a reduced risk of intrusion, our data indicate instead that more related neighbours pose an increased risk of intrusion, meaning that territory owners may rattle less toward kin because they are more tolerant of intrusions by these individuals.

A similar exploitation strategy has been observed in zebra finches (Taeniopygia guttata) whereby social foragers tolerate higher levels of exploitation of foraging discoveries in kin groups (Mathot & Giraldeau, 2010). Mathot and Giraldeau (2010) demonstrated that this behaviour could arise because of the inclusive fitness benefits conferred by being scrounged by kin rather than non-kin. Other interactions among asocial or territorial species may be modulated by relatedness. For example, juvenile Atlantic salmon (Salmo salar) have been demonstrated to share high quality feeding territories for twice as long with kin as with non-kin (Griffiths & Armstrong, 2002). Kin groups result in reduced aggressive interactions among juvenile Atlantic salmon and rainbow trout (Oncorhynchus mykiss) and more foraging opportunities as a result (Brown & Brown, 1993; 1996). In raccoons (Procyon lotor), dyadic encounters occur more frequently among kin and the degree of home range overlap is greater among related females, which, during the winter, suggests an important role for kin in social thermoregulation (Robert, Garant, Vander Wal, & Pelletier, 2013; but see Hirsch, Prange, Hauver, & Gehrt, 2013). Southern flying squirrels (Glaucomys volans) also preferentially aggregate with highly related individuals during the winter months (Thorington & Weigl, 2011a; 2011b; Thorington, Metheny, Kalcounis-Rueppell, & Weigl, 2010; but see Garroway, Bowman, & Wilson, 2013; Winterrowd, Gergits, Laves, & Weigl, 2005).

Red squirrels may stand to gain similar, or additional, benefits from neighbouring kin. If red squirrels are more tolerant of reciprocal territorial intrusions among kin, then territory owners surrounded by closely related individuals might benefit from reduced territorial defence, 18

increased access to food resources, or shared warmth on cold nights. Given that six of the closely related intruders were mothers who intruded on their offspring’s territory (four of which were bequeathed territories) it may be an added benefit for red squirrel mothers to bequeath a portion of their own midden (Berteaux & Boutin, 2000; Price & Boutin, 1993), or a nearby secondary midden (Boutin, Larsen, & Berteaux, 2000) to their offspring in return for increased access to resources. It is important to note, however, that despite the increased intrusion risk by relatives, there was no effect of average relatedness on intrusion risk. For individuals in such neighbourhoods, it is possible that the higher risk of intrusion by relatives is offset if the presence of relatives in the neighbourhood helps to reduce the risk of unrelated squirrels intruding. While red squirrels clearly defend exclusive individual territories, there are nuances in our results that suggest more subtle interactions among red squirrel neighbours than previously appreciated.

Effects of Seasonality We conducted our removal experiment in both the summer and autumn of 2015 to assess if intrusion risk was consistent across seasons. We found that the overall risk of intrusion was higher in the autumn than in the summer (Fig. 1.1) but the characteristics of neighbouring individuals and neighbourhoods that predicted risk of intrusion did not differ between seasons. There are several potential explanations for why we might see this difference in overall intrusion risk between seasons. Our removals in the autumn were intentionally conducted post-juvenile settlement. Given the importance of familiarity in predicting risk of intrusion it is likely that the increased intrusion risk post-juvenile settlement reflects the risk posed by the influx of new neighbours. Alternatively, autumn is a particularly crucial time for squirrels to obtain and cache spruce cones and other food stores for the winter (Fletcher et al., 2010). Given that cache size is an important predictor of overwinter survival (LaMontagne et al., 2013), squirrels may be more willing to risk an intrusion in the autumn in an attempt to pilfer food stores. This may be particularly true in 2015 given that spruce trees produced no cones on our study sites, but cone stores in middens were still high from the previous mast year in 2014.

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Conclusion There is increasing interest in understanding the role of group composition as an agent of selection shaping individual fitness as well as the evolutionary trajectory of populations (Farine et al., 2015). While the social environment has typically been measured at a coarse scale such as “density”, there is now increased recognition of the importance of individual variation in characteristics including age, familiarity, and relatedness. This study demonstrated that social neighbourhoods with low average familiarity (lots of new neighbours) significantly increased intrusion risk for a territorial squirrel that is highly dependent on cached food resources for overwinter survival. Additionally, related neighbours posed a higher risk of intrusion than originally predicted, suggesting that there may be more subtle interactions among kin than previously detected in this study system. Although underappreciated, asocial species, similar to their social counterparts, have to manage relationships with neighbouring individuals while still allocating adequate time and energy to gather resources and reproduce successfully. The effects of neighbourhood familiarity on intrusion risk suggest that it is a relevant measure of the social environment that may have important consequences for behavioural time budgets and therefore fitness in red squirrels. Prior to now the relevance of the social environment as a potentially important selective pressure in ‘asocial’ species has primarily been overlooked. Our findings suggest that familiarity among neighbours may be an important metric to consider in both social and asocial species in order to understand how the composition of the social environment can impact the ecological and evolutionary dynamics of populations. Few studies have been able to directly quantify the effects of familiarity on fitness (cf. Beletsky & Orians, 1989; Grabowska- Zhang, Wilkin, & Sheldon, 2012b) and additional work is needed in this regard to further our understanding of the role of group composition as an agent of selection.

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Tables

Table 1.1. Cox proportional hazard mixed effects models and AICc results predicting risk of intrusion based on the average characteristics of the neighbourhood.*

Model K Log-likelihood AICc i wi Rank Dear Enemy Model 4 -205.06 427.83 0.00 0.89 1 ~ fam + season Global Model 7 -205.44 433.31 5.49 0.06 2 ~ r + fam + dis + density + season Density Model 4 -201.34 436.00 8.17 0.02 3 ~ density + season Proximity Model 4 -201.61 436.41 8.58 0.01 4 ~ dis + season Kinship Model 4 -202.13 436.52 8.69 0.01 5 ~ r + season Intercept Model 2 -208.14 437.37 9.54 0.01 6 ~ 1

Number of parameters (model complexity) is represented by K. Models were evaluated based on differences between AICc scores (i) and AICc weights (wi). i is the difference between the observed model (i) and the best model as determined by the lowest AICc. Model rank is based on differences among AICc scores.

*The significance of the random intercept term for squirrel identity was assessed using the Global Model (variance = 0.10; 2 = 0.14, df = 1, P = 0.70).

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Table 1.2. Cox proportional hazard mixed effects models and AIC results predicting risk of intrusion based on individual squirrel characteristics from all intruders.*

Model K Log- AIC i wi Rank likelihood Global Model 5 -530.89 1078.07 0.00 0.98 1 ~ dis + r + fam Distance Model 3 -536.86 1085.80 7.73 0.02 2 ~ dis Relatedness Model 3 -599.90 1201.86 123.79 1.29e-27 3 ~ r Familiarity Model 3 -607.75 1217.57 139.49 5.01e-31 4 ~ fam Intercept Model 2 -611.96 1223.98 145.91 2.02e-32 5 ~ 1

Number of parameters (model complexity) is represented by K. Models were evaluated based on differences between AIC scores (i) and AIC weights (wi). i is the difference between the observed model (i) and the best model as determined by the lowest AIC. Model rank is based on differences among AIC scores.

*The significance of the random intercept term for squirrel identity was assessed using the Global Model (variance = 0.08; 2 = 0.32, df = 1, P = 0.57).

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Figures

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Figure 1.1. Plots are based on the dear enemy model and show the effect of (a) average familiarity and (b) season (S = summer, A = autumn) on the risk of intrusion (N = 82). Values on the X axis are in standardized units. Partial residuals from the Cox proportional hazard mixed effects model are shown on the Y axis and do not include the random effect of squirrel ID.

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Figure 1.2. Plots are based on the global model and show the effect of (a) relative distance, (b) relative relatedness and (c) relative familiarity on the risk of a neighbouring squirrel intruding (N = 2353). Values on the X axis are in standardized units. Partial residuals from the Cox proportional hazard mixed effects model are shown on the Y axis and do not include the random effect of squirrel ID.

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Chapter 2: Red Squirrel Territorial Vocalizations Deter Intrusions By Conspecific Rivals

Abstract

In many species, territory advertisement is thought to be one of the primary functions of acoustic communication. North American red squirrels are a territorial species in which ‘rattles’ have long been thought to be the principal signal communicating territory ownership. These vocalizations have been assumed to deter intruders, thus reducing energy costs and the risk of injury associated with direct aggressive interactions. However, this hypothesis has not been directly tested. Here we used a speaker occupation experiment to test whether red squirrel rattles function to deter conspecific rivals. We studied 29 male squirrels and removed each individual from his territory twice in a paired design. During the experimental treatment we simulated the owner’s presence after its removal by broadcasting the owner’s rattle from a loudspeaker at the centre of the territory once every seven minutes. During the control treatment the territory was left in silence after the temporary removal of the owner. We found that the presence of a speaker replacement reduced the probability of intrusion by 34% and increased the latency to first intrusion by 7%, providing support for the hypothesis that rattles play an active role in reducing intrusion risk. However, intrusions were not completely averted by the speaker replacement, indicating that for some individuals vocalizations alone are not a sufficient deterrent without other cues of the territory owner.

Introduction

Vocal communication is thought to have several principal functions, including territory advertisement (Bradbury & Vehrencamp, 2011; Catchpole, 1982; Catchpole & Slater, 2008). While the role of vocalizations in repelling conspecific rivals is typically well accepted, this function has rarely been directly demonstrated. Evidence supporting the functionality of vocalizations as a deterrent for intruders has come mostly through indirect means, via observational and correlational studies in the field, rather than direct experimental tests of functionality. For example, vocalizations commonly observed in association with intrusion 25

events or aggressive interactions among individuals (Catchpole, 1983; Kramer & Lemon, 1983; Sharpe & Goldingay, 2009; Smith, 1978), containing characteristics such as low frequency and high intensity (believed to be associated with aggression; Anderson & Barclay, 1995; Morton, 1977), or seasonally associated with times of important territorial defence (Catchpole, 1973; Penteriani, 2002) have typically been ascribed a territorial function. However, while suggestive, these correlative studies lack the causal evidence to support the putative defensive functionality of vocalizations.

The use of experimental playbacks is one technique that has been employed to study the role of vocalizations in territory defence. Playbacks have been used to simulate the intrusion of a rival individual by broadcasting a vocalization from a loudspeaker placed on a focal territory (Weeden & Falls, 1959). The aggressive reaction of territory owners in response to simulated intrusions has been used as evidence to support the conclusion that vocalizations play a role in territory defence against conspecific rivals. This type of aggressive response has been observed in a variety of taxa including anurans (Bastos, Alcantara, & Morais, 2011; Morais, Siqueira, & Bastos, 2015; Wells, 1977), fishes (Bass & McKibben, 2003), birds (Brumm, Robertson, & Nemeth, 2011; Cain & Langmore, 2015; Odom & Mennill, 2010) and mammals (Barlow & Jones, 1997; Darden & Dabelsteen, 2008; Hayes et al., 2004; Reby, Cargnelutti, & Hewison, 1999). By inducing an aggressive reaction in territory owners, the use of playbacks can effectively demonstrate that vocalizations function in immediate territorial confrontations. However, by measuring the response of owners, rather than intruders, these studies fail to clarify whether vocalizations induce avoidance and so function to keep conspecifics off the territory, even when confrontations are not imminent.

Muting and speaker occupation are two experimental designs that have been used in songbirds to test the hypothesis that acoustic signals function to deter territory intrusions. In some muting experiments, territory owners have been rendered silent via devocalizing surgical procedures (Peek, 1972). These experiments have provided empirical evidence for the territorial function of song by demonstrating that muted males experience higher intrusion rates and increased territory loss relative to controls whose ability to sing is left intact (McDonald, 1989; 26

Peek, 1972; Smith, 1979; Westcott, 1992). In speaker occupation experiments, territory residents are removed and replaced by speakers broadcasting the owner’s song (Falls, 1988; Krebs, Ashcroft, & Webber, 1978; Nowicki, Hughes, & Searcy, 1998; Yasukawa, 1990). In most cases, territories with a speaker replacement remain unoccupied longer and experience lower rates of intrusion than territories that are left silent, suggesting that song may be important in helping to repel intruders (Falls, 1988; Krebs et al., 1978; Nowicki et al., 1998).

While birdsong is one of the most well studied phenomena in animal communication, fewer studies have attempted to experimentally demonstrate the territorial function of vocalizations in other taxa. Speaker occupation experiments have been used in bicolor damselfish (Pomacentrus partitus; Myrberg, 1997) and painted gobies (Pomatoschistus pictus; Pereira et al., 2013), and muting experiments more recently in Lusitanian toadfish (Halobatrachus didactylus; Conti, Fonseca, Picciulin, & Amorim, 2015) to show that vocalizations serve as a “keep-out” signal to other conspecifics. Due to the limitations of finding species amenable to such experimental designs our understanding of the territorial function of vocalizations in mammals has been limited to observational field studies or playback experiments that induce an aggressive response in the territory holder (Barlow & Jones, 1997; Grinnell & McComb, 2001; Grinnell, Packer, & Pusey, 1995; Reby et al., 1999; Sharpe & Goldingay, 2009; Smith, 1978). Harrington and Mech (1979) did demonstrate that simulated howling resulted in retreat or avoidance by neighbouring wolf (Canis lupus) packs, suggesting that howling serves to deter intruders and maintain territorial boundaries without direct aggression. Similarly, Waser (1975) demonstrated that playbacks of ‘whoopgobble’ vocalizations from mangabeys (Cercocebus albigena) served to mediate intergroup avoidance.

To experimentally test the function of vocalizations for territorial defence in a mammalian species we used a territorial tree squirrel (Tamiasciurus hudsonicus). North American red squirrels are small, arboreal squirrels in which both sexes defend exclusive, individual territories throughout the year. The core of each territory is a larder hoard of food resources called a “midden” (Smith, 1968). Red squirrels produce several vocalizations, of which the “rattle” is believed to be the most important for territorial defence (Smith, 1978). Rattles, 27

unlike the vocalizations of songbirds, are not known to be associated with mating and are used by both sexes. Rattles are known to have a repeatable acoustic structure that allows for individual identification and discrimination by conspecifics (Digweed, Rendall, & Imbeau, 2012; Wilson et al., 2015). Smith (1978) observed that red squirrels produce rattles when another squirrel enters its territory, but also periodically when there is no apparent threat. Rattles were also observed to elicit fleeing behaviour from the intruder (Smith, 1978). This suggests that rattles may function as an advertisement of occupancy and help to maintain the spacing of individuals while minimizing direct aggressive interactions (Lair, 1990; Smith, 1978). By enabling the avoidance of aggressive interactions such as chases or fights, rattles may reduce energy costs and risk of injury (Wilson, 1975). The use of playback experiments has demonstrated that red squirrels can differentiate between neighbours and strangers as well as kin and non-kin using rattles, and that they respond more aggressively toward simulated intrusions from strangers (Price et al., 1990) and non-kin (Wilson et al., 2015). In another study, increased population density was found to increase vigilance and rattling rates in red squirrels (Dantzer et al., 2012; Shonfield et al., 2012). However, intruder pressure at high densities did not increase, which suggests that increasing rattling rates may be effective in deterring territorial intrusions (Dantzer et al., 2012). These observations support the idea that in red squirrels, rattles serve to advertise the owner’s presence to other conspecifics, maintain territory boundaries, and deter intruders. However, to date there is no direct experimental evidence to support this perceived functionality.

The aim of our study was to experimentally test whether red squirrel rattling functions to deter intruders. To assess this we employed a speaker occupation experiment and temporarily removed 29 squirrels from their territories in a paired design. During the control treatment the territory was left in silence, while during the experimental treatment we simulated the owner’s presence by broadcasting the owner’s rattle from a loudspeaker at the centre of the territory. We predicted that if rattles function to deter intruders in the absence of the territory owner then, compared to territories left in silence, territories with a speaker replacement would have: 1) a lower probability of intrusion and, 2) a longer latency to intrusion.

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Methods

Population and Study Area We studied a wild population of North American red squirrels (Tamiasciurus hudsonicus) in the southwest Yukon, Canada (61° N, 138° W), near Kluane National Park. The habitat of the study area is open boreal forest dominated by white spruce (Picea glauca; Berteaux & Boutin, 2000; Humphries & Boutin, 2000). This population has been monitored continuously since 1987 as part of the Kluane Red Squirrel Project (McAdam et al., 2007), on up to six study sites. We conducted our experiment on two study sites; one site was maintained as a control (40 ha) while the other study site (45 ha) was an experimental food-add site that has been supplemented with peanut butter between October and May every year since 2004 as part of a larger on-going study (Dantzer et al., 2012).

Each year in May and August we enumerated all individuals in the population and determined territory ownership using live-trapping methods and behavioural observations. We permanently tagged squirrels with uniquely numbered metal ear tags (Monel #1; National Band and Tag, Newport, KY, U.S.A) around 25 days old in their natal nest. Each squirrel was also given a unique combination of coloured wires that were threaded through the ear tags to allow individuals to be identified from a distance (Berteaux & Boutin, 2000; McAdam et al., 2007).

Rattle Recordings Between June and August 2015 we recorded 240 rattles from 29 male squirrels (minimum 4-5 rattles each squirrel) using a Marantz® Professional Solid State Recorder (model PMD660; 44.1 kHz sampling rate; 16-bit accuracy; WAVE format) with a Sennheiser® shotgun microphone (model ME66 with K6 power supply; 40-20000 Hz frequency response (± 2.5 dB); super- cardioid polar pattern; Shonfield et al., 2016; Wilson et al., 2015). We collected all rattles opportunistically in the morning, between 0730 and 1100 hours. Squirrels were followed at a distance when attempting to collect a rattle and were not stimulated with a playback or otherwise provoked during rattle collection. Although we cannot exclude the possibility that the observer’s presence elicited the rattles, squirrels on our study sites were well habituated to human observers.

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We edited the recorded rattles using Avisoft-SASLab Pro (Avisoft Bioacustics). To preserve rattle characteristics, we imported the recorded calls into Avisoft as uncompressed 16-bit .wav files. For each squirrel, we chose three rattles with minimum background noise. Because recordings were collected at various distances from the calling squirrel, we adjusted rattles to ensure that all recordings had similar power (dB) by adjusting the amplitude in Avisoft-SASLab Pro. We stored the three rattles, interspersed with 7-minute intervals of silence, as .wav files.

Speaker Occupation Experiment Between August and September 2015, we conducted temporary removals of 29 male squirrels from their territories, 15 from the control site and 14 from the experimental food addition site (N = 29). We trapped squirrels with Tomahawk live traps (Tomahawk Live Trap Co., Tomahawk, Wisconsin), and removed each squirrel from his territory twice, once as a treatment and once as a control (order of treatment and control was randomly assigned). Trials were conducted no less than 3 days apart (range: 4-41 days). Once trapped, we placed the squirrel in a modified box (41 x 17.5 x 19 cm) to help keep the squirrel quiet and calm for the duration of the removal (Donald & Boutin, 2011). A small amount of peanut butter and some spruce cones were also provided in the modified box. We then moved the squirrel 20-30 m away from his midden, and placed the individual in a shady location. Care was taken not to place the removed individual on another squirrel’s territory.

Each trial commenced immediately following the removal of the territory owner. When removed as a control, we left the territory owner’s midden silent. We used a silent control because many biologically relevant control sounds, such as bird song, are likely to convey additional information (e.g. predator risk; Soard & Ritchison, 2009; Templeton, Greene, & Davis, 2005), which may be used by heterospecifics in decision-making (Huang, Sieving, & Mary, 2012; Seyfarth & Cheney, 1990). The speaker was not present on the midden during the control removals. When removed as a treatment, we placed a Saul Mineroff SME-AFS field speaker with a playback range of 0.1-22.5 kHz face-up on the ground at the centre of the squirrel’s midden. We then broadcast the owner’s rattle from this speaker at a sound pressure level (SPL) between 65-75 dB (Shonfield et al., 2016) measured using a digital sound level 30

metre (RadioShack model 33-2055A) at 2 m from the upwards-facing speaker. We broadcast the owner’s rattle at 7-minute intervals (Dantzer et al., 2012) for the duration of the removal.

During each removal an observer monitored the midden from a distance of no less than 5 m away from the midden centre. If an intrusion was witnessed the observer recorded the time of intrusion and the identity of the intruding squirrel. An intrusion was defined as occurring when a squirrel moved onto the pile of cone scales, which is considered to define the edges of a squirrel’s midden. Speaker replacement trials lasted 120 minutes or until an intruder arrived, whichever came first. Control trials lasted the full 120 minutes, but we only used data from the first intruder for analyses presented in this paper. After trials were completed we released the captive squirrel at the site of capture. This research was approved by the University of Guelph Animal Care Committee (AUP number 1807).

Statistical analysis To assess whether the presence of a speaker replacement reduced the probability of a squirrel intruding and the latency to intrusion we used a Cox proportional hazard mixed effects model (PHMM) in the R package coxme (version 2.2-5: Therneau, 2015). We used a survival analysis approach because removals in which no intruder was observed in the 120 min time-period had censored values for time to intrusion. The response variable for our Cox proportional hazard models was a single measure called the hazard of intrusion. This response variable takes into account both the probability of intrusion (1 = intrusion observed, 0 = no intrusion observed) as well as the time to intrusion (measured in minutes). For the purposes of interpretability, hereafter we will refer to the outcome of this model as the ‘risk of intrusion’. In all cases, a high risk of intrusion is equivalent to a high probability of intrusion and a fast time to intrusion. Although some control trials had multiple intrusion events, for this analysis we included all first intruders only. Our model included speaker treatment (speaker or no speaker) and study site as categorical predictors. We also included squirrel identity as a random intercept term to account for our matched pairs design.

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Our PHMM met the proportional hazards assumption (Cox, 1972; Cox & Oakes, 1984). We examined dfbeta residuals to ensure that there were no influential observations. All analyses were performed using R software 3.3.2 (R Core Team, 2016). We considered differences statistically significant at =0.05 and we reported all means ± SE, unless otherwise stated.

Results

Risk of intrusion was significantly lower in the presence of the speaker replacement ( = -0.92 ± 0.33, z = -2.77, P = 0.01). There were no differences in the risk of intrusion between the two study sites ( = -0.24 ± 0.32, z = -0.75, P = 0.45). For each treatment we conducted 29 temporary removals of territory owners. Twenty-five intrusions occurred when no speaker replacement was present (86% of removals) and only 15 intrusions were observed when a speaker was used to broadcast the rattle of the territory owner (52% of removals). The proportion of intrusions with the speaker replacement was significantly lower than when the territory was left silent (McNemar’s Chi-squared test; 2 = 5.79, df = 1, P = 0.02). The average time to first intrusion on empty territories was 52.20 minutes (SD: ± 36.84 min; range: 0-111 min) and on speaker- occupied territories was 60.13 minutes (SD: ± 38.62 min; range: 15-114 min), which were not significantly different (Welch Two Sample t-test; t = -0.64, df = 28.50, P = 0.74; Fig. 2.1). Thus, over a 120-minute period the presence of a rattle vocalization reduced the probability of intrusion by 34% but only reduced the time to intrusion by 7%, suggesting that the reduced risk of intrusion was primarily due to a reduced probability of intrusion. Risk of intrusion was not affected by either the order of treatment or the number of days between trials (all |z| < 0.88; all P > 0.38).

Discussion

We investigated the territorial function of vocalizations in the North American red squirrel. Specifically, we tested the role of rattles in discouraging conspecific intruders. The results from this study confirmed our hypothesis that red squirrel rattles reduce intrusion risk by demonstrating that territories occupied by a speaker replacement had a significantly lower probability of intrusion and a delayed time to first intrusion (although not significant) compared to territories that were left empty. Without an appropriate control sound it is difficult to 32

demonstrate unequivocally that it was the broadcast rattle rather than the sound of the speaker that deterred squirrels from intruding. However, previous studies have demonstrated that squirrels in this population respond to rattles broadcast from a speaker in a behaviourally appropriate manner, by rattling more towards non-kin or unfamiliar individuals (Price et al., 1990; Wilson et al., 2015; but see Shonfield et al., 2016). These studies would suggest that rattles broadcast through a speaker do not represent a novel sound but, a biologically relevant cue to which squirrels were responding appropriately. Additionally, because the speaker was not present on the midden during control trials we cannot rule out the possibility that the physical presence of the speaker served as a deterrent to intruders. However, since intruding neighbours came from within 12-176 m (mean: 56 m) of the focal midden, the primary deterrent to an intrusion is likely to be auditory rather than visual. Had the visual cue of the speaker been an important deterrent we would have seen more squirrels approach the territory during the speaker replacement trials but fail to actually intrude, which was not the case. We observed two squirrels approach within visual range of the observer (~15 m) but fail to intrude during the control trials and none during the speaker replacement trials.

Our findings support the conclusions of previous speaker occupation studies in both songbirds and territorial fishes that have demonstrated the importance of acoustic signalling for territory defence and retention. Experimental tests of the role of vocalizations in actively deterring conspecific rivals in mammals have been less commonly performed (but see Harrington & Mech, 1979; Waser, 1975). In red squirrels, the number of intrusions was clearly reduced by the rattle playback, but the speaker replacement was not 100% effective in deterring intrusions. Of the 29 territories which received a speaker playback, 15 experienced a territorial intrusion, which is just over half of the territories (52%), suggesting that some squirrels may only be deterred by visual cues, or other stimuli from the territory owner. This is consistent with previous speaker occupation studies which demonstrate lower intrusion rates or delayed intrusions in the presence of playbacks, but do not preclude all individuals from intruding (Falls, 1988; Myrberg, 1997; Nowicki et al., 1998). Falls (1988) suggested that the effectiveness of the speaker occupation design may depend in part on the conspicuousness of the territorial species that is being studied. Speaker replacement should be most effective in species that are visually 33

inconspicuous because it allows the façade of occupancy to be maintained for a longer period of time. Red squirrels are usually fairly visible to their neighbours as they are diurnal and spend a significant portion of their time foraging and feeding on or around their territory. Red squirrels will also actively chase intruders off their midden if necessary, although such direct acts of aggression are usually rare (Dantzer et al., 2012; Gorrell et al., 2010).

Given the potential importance of the physical presence of the territory owner, it is not surprising that the speaker replacement experiments were not wholly effective in deterring red squirrel intruders. In fact, in some studies, speaker replacements initially induced avoidance of ‘occupied territories’, but had unexpected effects on intrusion rates. For example, in red-winged blackbirds there were reduced rates of fly-through, but not neighbour trespass, on speaker replacement territories, suggesting that visual displays may be crucial for territory retention (Yasukawa, 1981). In painted gobies the presence of a playback ultimately led to higher rates of territory intrusion than in territories that were left silent. Painted gobies approached ‘occupied’ nests more frequently than silent nests but then demonstrated avoidance after approaching the agonistic calls. However, when the gobies were unable to associate the sound with the physical presence of a territory holder they proceeded to intrude on the ‘occupied’ territory (Pereira et al., 2013). Anecdotally, we observed similar patterns of behaviour in red squirrels. Potential intruders that appeared at the edge of the territory initially appeared to be deterred by the broadcast rattle, however, after remaining on the territory for several minutes without sighting the territory owner, these loitering individuals entered the midden and, when not chased off by a resident squirrel, proceeded to pilfer food resources. Without the visual presence of the territory owner, potential intruders appear to become habituated to a repetitive acoustic stimulus. While we have confirmed the significance of rattles in red squirrel territory defence, altogether these results suggest that, similar to other acoustic signals, rattles only serve as a temporary deterrent to territorial intrusions until the absence of the territory owner is confirmed. This underscores the importance of the combined effects of visual and acoustic cues for deterring conspecific rivals.

A more precise understanding of how rattles function in territory defence is still lacking. Red squirrel rattles are known to have unique signatures that allow for individual identification 34

by other conspecifics (Wilson et al., 2015), but it is unclear if rattles will indiscriminately discourage intruders regardless of whether that rattle is from a resident or non-resident conspecific. Yasukawa (1981) suggested that one reason the speaker replacement did not deter neighbour trespass in red-winged blackbirds is because song types were used which were unfamiliar to neighbouring conspecifics. Yasukawa speculated that neighbouring males interpreted this as an attempted territory establishment by a new individual and therefore responded by either attempting to re-negotiate boundaries or claim the abandoned territory before the new male had time to fully settle (Yasukawa, 1981). Here we used only the calls of the territory owners in our speaker replacements. Future studies should employ the use of speaker occupation experiments using calls from different signallers to better understand if red squirrels are able gather and respond to more complex information about their social environment.

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Figures

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0 No Speaker Speaker Treatment Figure 2.1. Latency to intrusion following the temporary removal of a territory owner during trials with (N = 15) and without (N = 25) a speaker replacement. Centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Percentages above each boxplot indicate the percentage of trials in which an intrusion occurred.

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Chapter 3: Red Squirrels Mitigate Costs Of Territory Defence Through Social Plasticity

Abstract

For territorial species, the ability to be behaviourally plastic in response to changes in their social environment may be beneficial by allowing individuals to mitigate conflict with conspecifics and reduce the costs of territoriality. Here we investigated whether North American red squirrels (Tamiasciurus hudsonicus) were able to minimize costs of territory defence by adjusting behaviour in response to the familiarity of neighbouring conspecifics. Since red squirrels living in familiar neighbourhoods face reduced risk of territory intrusion, we predicted that increasing familiarity among territorial neighbours would allow squirrels to spend less time on territorial defence and more time in the nest. Long-term behavioural data (1995-2004) collected from the same squirrels across several different social environments indicated that red squirrels reduced rates of territorial vocalizations and increased nest use in response to increasing neighbourhood familiarity. In contrast, cross-sectional data (2015-2016), which provided observations from each individual in a single social environment, did not provide evidence of this plasticity. Post-hoc analyses revealed that evidence of social plasticity in this system was primarily due to within- individual changes in behaviour, which we were unable to estimate in the cross-sectional data. Our results demonstrate that red squirrels respond to changes in their social environment by adjusting their behaviour in a manner that reduces the costs of territoriality. However, our results also suggest that estimating plasticity by comparing behaviour among individuals (i.e. cross- sectional analyses) may not always be reliable. Our ability to detect these effects may therefore depend on having data with multiple observations from the same individuals across different social environments.

Introduction

Phenotypic plasticity can broadly be defined as the ability of a single genotype to express multiple phenotypes in response to different environmental conditions (Pigliucci, 2001). Indeed, classic studies of phenotypic plasticity have typically focused on changes in non-reversible traits 37

(e.g. morphology) that are expressed within a single genotype (Greene, 1989; Hebert & Grewe, 1985; Lively, 1986). However, traits that are expressed repeatedly over the course of an organism’s lifetime (e.g. timing of reproduction) can be subject to reversible within-individual plasticity (Nussey, Wilson, & Brommer, 2007; Piersma & Drent, 2003). This ‘reversible plasticity’ (Gabriel, Luttbeg, Sih, & Tollrian, 2005), also referred to as ‘phenotypic flexibility’ (Piersma & Drent, 2003), or ‘responsiveness’ (Wolf, Van Doorn, & Weissing, 2008), is a powerful mechanism for adapting to changing and unpredictable environmental conditions. Behavioural traits, in particular, show capacity for substantial phenotypic lability in response to changing environmental conditions within an organism’s lifetime and can facilitate an organism’s ability to cope with both predictable and unpredictable variation in the environment (Ghalambor, Angeloni, & Carroll, 2010).

The social environment is arguably one of the most dynamic and variable realms of an individual’s environment, since high levels of unpredictability are inherent when interacting with other agents that can also exhibit plasticity in behaviour. Examples of social plasticity (changes in behaviour in response to changing social conditions; Montiglio et al., 2017; Sih, Chang, & Wey, 2014) are prevalent. For instance, individuals adjust their level of aggression according to the perceived level of threat imposed by neighbours versus strangers (Temeles, 1994). Interacting individuals change their signaling behaviour in response to bystanders ('audience effect' – Doutrelant, McGregor, & Oliveira, 2001; Pinto, Oates, Grutter, & Bshary, 2011). Behaviour may also be affected by previous social experiences such as ‘winner-loser effects’ (Hsu, Earley, & Wolf, 2006; Rutte, Taborsky, & Brinkhof, 2006), as well as by ‘eavesdropping’, in which bystanders extract information from interacting individuals (Earley, 2010; Mennill, Ratcliffe, & Boag, 2002).

The ability to adjust behaviour in response to social context should allow individuals to avoid costly interactions while appropriately engaging in other social interactions that might enhance fitness (Taborsky & Oliveira, 2012). This ability to show adaptive adjustments in social behaviour has been termed ‘social skill’ (Sih & Bell, 2008) or ‘social competence’ (Taborsky & Oliveira, 2012), although few studies have directly demonstrated fitness benefits of social 38

plasticity (Han & Brooks, 2015; Montiglio et al., 2017; Patricelli, Uy, Walsh, & Borgia, 2002). Given the substantial number of social interactions that group-living species must navigate, the benefits of social plasticity are expected to be high in such species (Taborsky, Arnold, Junker, & Tschopp, 2012). However, solitary, territorial species may also benefit from appropriate adjustments in social behaviour, as being socially plastic may allow individuals to mitigate conflict with conspecifics and reduce the costs of territoriality. For example, gladiator frogs (Hypsiboas rosenbergi) adjust the timing of vocalizations in response to changing levels of conspecific competition. By reducing calling rates in response to changing social conditions, individuals can help reduce an energetically costly behaviour (Höbel, 2015).

Solitary, territorial species, like their social counterparts, face variation in their social environments through their interactions with territorial neighbours. A well-described example of this variation is differences in familiarity with neighbours (Bebbington et al., 2017; Beletsky & Orians, 1989; Eason & Hannon, 1994; Grabowska-Zhang, Wilkin, & Sheldon, 2012b). Long- term social relationships with neighbours have been presumed to be advantageous by minimizing renegotiation of territory boundaries and therefore reducing aggression as well as time and energy spent on territory defence (‘dear-enemy effect’; Fisher, 1954). However, most evidence in support of this cooperative phenomenon comes from experimental studies where individuals are exposed to a familiar and unfamiliar stimulus and a behavioural response is recorded (Temeles, 1994). We know less about how territorial behaviour is affected by familiar social relationships under natural conditions when individuals may have to navigate territorial dynamics with multiple neighbours (but see Bebbington et al., 2017; Eason & Hannon, 1994).

In this study, we used a longitudinal dataset spanning eight years and cross-sectional data from two years to examine whether territorial North American red squirrels (Tamiasciurus hudsonicus, hereafter ‘red squirrels’) adjust their behaviour in response to their familiarity with their local social environment. Our long-term dataset contained multiple observations of the same individuals across different social environments, allowing us to measure within-individual changes in behaviour. In contrast, our cross-sectional data represented an intensive snapshot of a large number of individuals at a single point in time (i.e. a single social environment for each 39

individual), allowing us to compare across individuals to assess differences in behaviour. Although behavioural plasticity is fundamentally a within-individual phenomenon, it can be approximated by comparing among individuals in different environments (Legagneux & Ducatez, 2013; Slabbekoorn & Peet, 2003). While this among-individual approach is a useful tool (particularly where it is challenging or time-consuming to collect data on many individuals over several environments) it relies on the critical assumption that the among-individual relationship is an accurate representation of within-individual changes in behaviour.

Red squirrels are territorial rodents that defend year-round exclusive territories (Smith, 1968). In the Yukon, red squirrels cache white spruce cones (Picea glauca) in a larder hoard called a ‘midden’ at the centre of their territory (Fletcher et al., 2010). This food cache is important for overwinter survival (Kemp & Keith, 1970; LaMontagne et al., 2013) and both sexes heavily defend these resources from conspecifics, primarily through territorial vocalizations called ‘rattles’ (Smith, 1978). Rattles function to deter intruders (Siracusa, Morandini, et al., 2017b) but are also individually unique (Digweed et al., 2012; Wilson et al., 2015). Rattles therefore carry important information about the local social environment, such as the identity or density of neighbouring conspecifics. Squirrels have been shown to use this acoustic information to increase rattling rates and vigilance and decrease nest use in response to increasing local density (Dantzer et al., 2012), providing some evidence of functional plasticity in territorial behaviour. Additionally, there is evidence that local social conditions are temporally variable in this system. Overturn of middens can occur through the death of a territory owner or through bequeathal. As a result, some squirrels may occupy different territories each year, leading to variation in neighbour familiarity (i.e. duration of tenure as neighbours). This can affect local territory conditions. Familiarity with territory neighbours has been shown to have direct effects on territory intrusion risk. Specifically, individuals living in neighbourhoods with higher average familiarity faced reduced intrusion risk (Siracusa, Boutin, et al., 2017a), consistent with the dear enemy phenomenon (Fisher, 1954).

Given temporal heterogeneity in territorial neighbours and variation in signaling behaviour, we predicted that increasing familiarity with territorial neighbours would allow for 40

changes in other aspects of behaviour, specifically decreased time spent on territorial defence as evidenced by (1) decreasing rattling rates and (2) reducing time spent vigilant for conspecifics. We also predicted that, as neighbourhood familiarity increased, squirrels would increase the proportion of time spent in nest, as a proxy for time spent on offspring-care or self-maintenance. Changes in behaviour, as predicted above, would allow individuals to minimize aggression and reduce the time and energy allocation to territory defence under social conditions associated with reduced risk of territorial intrusion, and thus would be indicative of social competence in this territorial species.

Methods

We studied a natural population of North American red squirrels located in the southwest Yukon near Kluane National Park (61° N, 138° W) that has been monitored continuously since 1987 as part of the Kluane Red Squirrel Project (KRSP; McAdam et al., 2007). To assess functional social plasticity in red squirrels, we measured behaviour of individuals on three study grids characterized by open boreal forest where white spruce is the dominant tree species (Krebs et al., 2001).

Using the longitudinal dataset, we analyzed long-term focal animal observations (Altmann, 1974) of 41 red squirrels across eight years (between 1995 and 2004), collected on one unmanipulated control grid (Sulphur: SU; 40 ha). We also examined a cross-section of data by collecting and analyzing focal observations of 108 squirrels in one year (2016) on two unmanipulated control grids (Kloo: KL and SU; 40 ha each) and one food supplemented grid (Agnes: AG; 45 ha; see Dantzer et al., 2012 for a description of the food supplementation experiment). Since accurately capturing adjustments in behaviour is often challenging, even with the intensive use of focal observations, we also measured the behaviour of squirrels by deploying accelerometers in 2016 to assess nest use, and audio recorders in 2015 and 2016 to measure rattling rates. Details on these approaches are provided below.

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Measuring familiarity In each year, we enumerated all squirrels living on our study areas and monitored individuals from March until August. We used a combination of live-trapping procedures and behavioural observations to track reproduction, identify territory ownership, and determine offspring recruitment from the previous year (Berteaux & Boutin, 2000; McAdam et al., 2007 for a complete description of core project protocols). All study grids were staked and flagged at 30 m intervals, which allowed us to record the spatial location of all squirrel territories to the nearest 3 m. In this study system, territory locations were denoted based on the location of an individual’s midden, which approximates the centre of a squirrel’s territory. We did not explicitly map territory boundaries for all individuals.

We trapped squirrels using Tomahawk live traps (Tomahawk Live Trap Co., Tomahawk, Wisconsin, USA) baited with all natural peanut butter. If previously tagged, the identities of the squirrels were determined from their unique alphanumeric metal ear tags (Monel #1; National Band and Tag, Newport, KY, USA), which they received in their natal nest at around 25 days of age. During the first capture of the season, we marked each squirrel by threading coloured wires through each ear tag, which allowed for individual identification of squirrels during behavioural observations. We censused the population twice annually (in mid-May and mid-August) and determined territory ownership through a combination of consistent live-captures of the same individual at the same midden and behavioural observations of territorial ‘rattle’ vocalizations (Smith, 1978).

For each territory owner we defined the social neighbourhood to be all conspecifics whose middens were within a 130 m radius of the owner’s midden. One hundred and thirty meters is the furthest distance that red squirrel rattles are known to carry (Smith, 1978) and is similar to the 150 m distance found by (Dantzer et al., 2012) to be most relevant for local density effects of the social environment. We measured pairwise familiarity between the territory owner and each neighbour as the number of days that both individuals were within the same acoustic neighbourhood (i.e. occupied their current neighbouring territories). We then averaged pairwise measures of familiarity between the focal individual and each of the neighbours to provide a 42

measure of the average familiarity of each individual with its entire acoustic neighbourhood (Siracusa, Boutin, et al., 2017a). We used this measure of average familiarity for all subsequent analyses to assess how familiarity with neighbours affected red squirrel behaviour.

Focal observations Red squirrels are an ideal species for behavioural studies because they are diurnal, easy to locate visually or through acoustic cues, and habituate readily to the presence of humans. As part of the KRSP, we have recorded the behaviour of red squirrels through focal sampling of radio-collared individuals (model PD-2C, 4 g, Holohil Systems Ltd., Ontario, Canada) since 1994, although the sampling protocol has varied slightly across this period. For this study, we chose to use a subset of long-term behavioural data where focal observations were collected in a consistent manner by instantaneous sampling at 30-sec intervals for ten continuous minutes (Altmann, 1974) on a single control grid (SU; N = 487 10-min sessions over 41 individuals). We excluded any focals where the squirrel was out of sight for more than half the observation session (N = 8). These behavioural observations were available for female squirrels in 1995 (N = 41), 1996 (N = 10), 1997 (N = 25), 1999 (N = 34), 2001 (N = 70), 2002 (N = 110), 2003 (N = 120), and 2004 (N = 77) and were recorded by 38 different observers. On average, data were available for two different social environments per individual (range 1-8). We considered any change in average familiarity during the population census to be a change in the social environment. Since the population was censused twice annually (in mid-May and mid-August) individuals could experience up to two different social environments per year.

Between 7 May 2016 and 31 August 2016, we used focal animal sampling as described above for seven continuous minutes, rather than ten, to record red squirrel behaviour (N = 1060 7 min sessions over 108 individuals). In this cross-sectional data we only had observations from each individual in a single social environment. Since rattling is a rare behaviour and is often missed using instantaneous sampling, in 2016 we recorded all occurrences (Altmann, 1974) of rattle vocalizations emitted by the focal squirrel, including those which fell outside the 30-sec sampling interval (i.e ‘critical incidents’). We used all of these data, including critical incidents, to assess how familiarity affected rattling rates in 2016. Four observers collected behavioural 43

data on both male (N = 76) and female (N = 32) squirrels across two control grids (KL and SU) and one food-supplemented grid (AG). We monitored each individual for 2-10 days consecutively (barring inclement weather; mean = 4 days) and collected an average of 10 focals per individual (range: 2-29). In instances where multiple focal observations were collected for the same squirrel in a single day, observations were kept 30 minutes apart at minimum. Because an observer was in regular attendance at these territories we could be confident that there was no turnover in the social environment during the sampling period for any of these individuals. Territory turnovers in this system are accompanied by substantial rattling and chasing and are therefore easy to detect. The two squirrels for which we observed a disturbance in the local social environment during the sampling period were excluded from this analysis.

For all focal sampling, we recorded and classified red squirrel behaviours in a similar way to previous studies of squirrel behaviour in this system (Anderson & Boutin, 2002; Dantzer et al., 2012; Stuart-Smith & Boutin, 1994). We classified behaviours according to the following categories: vocalizing (“barking” or “rattling”; Smith, 1978), feeding, foraging, traveling, caching food items, interacting with conspecifics, grooming, resting, vigilant, in nest, or out of sight (unknown behaviour). Vigilance could be distinguished from resting by the alert posture of the squirrel; vigilant squirrels typically had their head up and appeared observant, sometimes standing on their hind limbs, while resting squirrels often had their head tucked down or lay stretched out.

Audio recording and acoustic analysis Between 23 June – 25 September 2015 and 8 May – 1 September 2016, we deployed Zoom H2n audio recorders (Zoom Corporation, Tokyo, Japan) to determine rattling rates of squirrels. We attached recorders with windscreens to 1.5 m stakes and placed a single recorder in the centre of each squirrel’s midden. Since Zoom H2n recorders are not weatherproof, we placed an umbrella approximately 30 cm above each audio recorder to protect it from rain and snow. Each morning, we deployed audio recorders between 0500-0600 h (just before squirrels typically became active). We set audio recorders to record in 44.1kHz/16bit WAVE format, and recorded in 2- channel surround mode. We allowed audio recorders to run for a full 24 hours, but in this study 44

we only use data collected between 0700-1300 h, which is the period during which squirrels are typically most active between early summer and early autumn (Studd, Boutin, McAdam, & Humphries, 2016; Williams et al., 2014). We deployed audio recorders for 137 squirrels (N = 109 males and N = 28 females) and recorded each squirrel for 5 consecutive days on average (range: 1-13 days; N = 714 days or 4284 hours over 137 individuals). Because we collected audio data over 2 years, we had observations from 2-3 different social environments for 28 of these individuals. Due to the large volume of recordings, we detected rattle vocalizations from recordings automatically using Kaleidoscope software (version 4.3.2; Wildlife Acoustics, Inc., Maynard, MA, USA). Detection settings included a frequency range of 200013000 Hz, a signal duration of 0.415 s, a maximum intersyllable silence of 0.5 s, a fast Fourier transform size of 512 points (corresponding to a frequency resolution of 86 Hz and a temporal resolution of 6.33 ms), and a distance setting of 2 (this value ensures that all detections are retained).

The purpose of using audio recorders was to provide a more accurate estimate of individual rattling rates. A challenge, however, is that the recorders also recorded vocalizations from neighbouring squirrels. Neighbours should be farther away from the recorder. Because sound degrades and attenuates predictably with distance, it should be possible to distinguish between the rattles of focal and neighbour squirrels on the basis of rattle acoustic structure. We tested this by conducting hour-long calibrations on 48 focal individuals between 13 September and 14 October 2015. During these calibrations, audio recorders were set up as described above. A single observer standing near the midden kept the territory owner in sight and recorded whether each rattle belonged to the territory owner or a neighbouring individual.

We detected rattle vocalizations from the calibration recordings using Kaleidoscope software (same settings as above). Based on a comparison with the observer's notes, the software detected 100% of the focal squirrel rattles. We then developed a procedure for distinguishing focal squirrel rattles from other types of detections, including neighbour rattles and non-rattles. First, we automatically measured the acoustic structure of every detection using the software package 'Seewave' (version 2.0.5; Sueur, Aubin, & Simonis, 2008) in R (see details of structural measures below). Second, we used the structural measurements in a discriminant function 45

analysis in SPSS (software, version 24; IBM Corporation, Armonk, New York, USA) to develop a predictive model for assigning detections to groups (i.e. focal rattle, neighbour rattle, non- rattle). We developed the model using detections from half of the 1-hour calibration files (selected at random), and then tested it for accuracy by applying it to the detections from the remaining half. The model correctly assigned 80.6% of the focal rattles to the 'focal rattle' group, meaning we missed 19.4% of focal rattles (i.e. false negatives). Some non-rattle detections were also assigned to the 'focal rattle' group, but we removed these by reviewing the spectrograms of all detections categorized as ‘focal rattles’. After removing non-rattle detections, 16.0% of the detections remaining in the 'focal rattle' group were false positives, meaning they were actually from the neighbour instead of the focal squirrel. We then applied the predictive model to the main set of audio files, and reviewed all detections labeled as ‘focal rattle’ in Kaleidoscope to remove the non-rattle detections.

The structural measures included in the discriminant function analysis were: (1) duration, (2) root-mean-square amplitude, (3) pulse rate, (4) duty cycle, and five variables that measured the distribution of energy in the frequency domain, including (5) peak frequency, (6) first energy quartile, (7) skewness, (8) centroid, and (9) and spectral flatness. Duration, root-mean-square amplitude, pulse rate, and duty cycle were measured from a waveform. Pulse rate is the number of pulses in the rattle minus one, divided by the period of time between the beginning of the first pulse and the beginning of the last (Wilson et al., 2015). Duty cycle is the proportion of the rattle when a pulse is being produced. For pulse rate and duty cycle, individual pulses were identified using the 'timer' function in seewave (50% amplitude threshold; 200-point smoothing window with 90% overlap). The five energy distribution variables were obtained using the 'specprop' function in seewave, and were based on a mean frequency spectrum (512-point fast Fourier transform, hanning window, 0% overlap). Peak frequency is the frequency of maximum amplitude. First energy quartile is the frequency below which 25% of the energy is found. Skewness, centroid, and kurtosis describe the shape of the power spectrum (detailed definitions can be found in Sueur et al., 2008).

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Accelerometers An accelerometer is an instrument that measures the acceleration of the body along three axes: anterior-posterior (surge), lateral (sway), dorso-ventral (heave) and records temperature, allowing for the characterization of different behavioural patterns. Between 4 May and 1 September 2016, we deployed AXY-3 accelerometers (Technosmart Europe srl., Rome, Italy) on 94 squirrels (N = 66 males and N = 28 females). Accelerometers were deployed in combination with radio transmitters (model PD-2C, Holohil Systems Ltd., Ontario, Canada). We deployed accelerometers on 94 individuals for an average of 9 days per individual (range: 4-17; N = 873 days over 94 individuals) at a sampling rate of 1 Hz. Accelerometers recorded data constantly while deployed, but for this study we only use data between 06:00-21:00 h to estimate time spent in nest during active hours of the day (Williams et al., 2014).

Raw accelerometer data were classified into 5 behavioural categories using threshold values of summary statistics according to the decision tree developed for red squirrel accelerometers and temperature data loggers by Studd et al. (in review). Following methods proposed by (Collins et al., 2015), the decision tree was created using 83.8 hours of direct behavioural observations on 67 free-ranging squirrels and had an overall accuracy of correctly classifying known behaviours of 94.9% (Studd et al. in review). Briefly, warm stable temperatures were used to identify when the animal was in the nest with the additional constraint that the individual must not be moving for the majority of each nest bout. Low acceleration values were associated with not moving, moderate acceleration values denoted feeding, and high acceleration corresponded to travelling. Travelling was further categorized as running when the peak acceleration value of the surge axis was above a threshold of 1.15 G.

Ethical note This study required trapping individuals in order to attach radio transmitters and accelerometers. Traps were checked every 60-90 minutes and squirrels were never left in a trap for longer than 120 minutes maximum. Radio transmitters and accelerometers were attached as a single collar around the squirrel’s neck using plastic zip ties covered with heat shrink to minimize irritation to the skin. Total package weight for collars with both accelerometers and radio transmitters 47

(including battery, packaging, and bonding material) was 9.6 g on average. For a 200-250 g red squirrel (Steele, 1998) this collar weight was less than the recommended 5-10% of the animal’s body weight (Wilson, Cole, Nichols, Rudran, & Foster, 1996). Because all squirrels were continuously monitored through behavioural observations we could check for irritation caused by the collars. If any irritation was detected (missing fur, red or raw skin around the neck) the squirrel was immediately trapped and the collar removed. Instances of irritation caused by the collar were extremely rare and no squirrels suffered any long-term consequences as a result of the collars. All behavioural observations were conducted at least 5 m away from the focal squirrel to minimize any effects of squirrel behaviour. Behavioural observations had no detectable negative impact on individuals. This research was approved by the University of Guelph Animal Care Committee (AUP number 1807) and is in compliance with the ASAB/ABS Guidelines for the Use of Animals in Research.

Statistical analyses Given that previous work in this study system (Dantzer et al., 2012) allowed us to make specific predictions about how squirrels should adjust rattling rates, vigilance and nest use in response to their social environment, here we used univariate models to test for the effects of familiarity on each of these behaviours explicitly. For all models we included local density, measured as the number of squirrels per hectare within 130 m, as a continuous predictor, to account for the fact that previous work in the study system has found local density to be an important predictor of behavioural time budgets. We also included age as a fixed effect in all rattling rate models since we expected that the vigor of territory defence might decline with physical deterioration, but we did not have specific predictions as to how age might affect nest use or vigilance. However, it is important to note that since young squirrels are inherently unfamiliar with their neighbours and familiarity increases with age, age and familiarity were correlated (Pearson’s correlation coefficient ranged between 0.42 and 0.58 for these analyses) although variance inflation factors were low (< 3; Zuur, Ieno, & Elphick, 2009). Fixed effects and random effects of all models are summarized in Tables 3.1 and 3.2, respectively.

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Focal data We analyzed the longitudinal (N = 487 10-min sessions) and cross-sectional (N = 1060 7-min sessions) focal data separately to account for the structural differences in our data sets. While we had multiple observations of the same individuals across different social environments in the longitudinal data, we only had observations from a single social environment for each of our individuals in the cross-sectional data. In the long-term data, there was a single data point where the number of rattles recorded was 25 times greater than the mean. This outlier was likely an error in data entry and was removed (see Fig. S3.1). We analyzed the effects of neighbourhood familiarity on (1) the frequency of territorial vocalizations (rattles), (2) the proportion of time spent vigilant, and (3) the proportion of time spent in nest. We modeled the frequency of territorial vocalizations using a generalized linear mixed-effect model (GLMM) with a BOBYQA optimizer and a Poisson error distribution (log-link) where the response variable was the number of rattles emitted during the focal session. For both the proportion of time spent in nest and the proportion of time spent vigilant, we fitted a Beta-Binomial model to account for overdispersion in the data (Harrison, 2015). Using the ‘cbind’ function, we defined the response variable as a 2-column matrix composed of the number of observations of the given behaviour (in nest or vigilant) and the number of observations of all other behaviours (not including observations when the squirrel was out of sight). In all models we included average familiarity and local density as continuous predictors, and for the rattling rate models we included age as a continuous fixed effect. We included grid, sex and observer identity as categorical fixed effects for the 2016 focal data (it was not necessary to include grid or sex for the long-term data as all data were collected on females on a single grid). For both datasets, we included a random intercept term for squirrel identity (squirrel ID) to account for repeated observations of the same squirrels. We also included a random effect of year and observer identity for the long-term dataset to account for inter-individual differences in behavioural scoring.

Audio recorder data To assess the effects of familiarity on rattling rates derived from the audio recorder data, we fit a GLMM with a Poisson error distribution (log-link). Our response variable was the number of rattles emitted between 0700 – 13:00 h (i.e. number of ‘focal rattles’, unadjusted for false 49

positive or false negative error rates; N = 714 days of recordings). We included average familiarity, local density, age, grid, and sex as covariates in the model, as well as a random intercept term for squirrel ID, and an observation-level random effect (OLRE) to account for overdispersion in the model.

Accelerometer data Using accelerometer data, we assessed the effect of neighbourhood familiarity on the proportion of time spent in nest between 06:00 – 21:00 h using a Beta-Binomial model (N = 873 days). Our response variable was defined as above, using a two-column matrix that included the number of nest observations and the number of observations of all other behaviours. We included average familiarity, local density, grid, and sex as fixed effects in the model, and included a random effect for squirrel ID and accelerometer collar.

Exploratory post-hoc analysis Upon finding evidence of behavioural plasticity in the long-term data but not the cross-sectional data (see results below), we conducted an exploratory post-hoc analysis in an attempt to understand the inconsistencies in our results. While the longitudinal data provided multiple measures of the same individuals across different social environments, allowing us to estimate within-individual relationships, we could only estimate among-individual relationships in the cross-sectional data. To assess whether our results might be driven by a within-individual effect, thus limiting our ability to detect behavioural plasticity in the cross-sectional data, we re-fit our rattling rate and nest use models from the long-term data using a within-subject mean centering approach. Following the methodology of van de Pol & Wright (2009), we split our familiarity term in the longitudinal data into an among-individual effect of familiarity (i.e. the mean familiarity score for an individual across all observations) and a within-individual effect of familiarity (i.e. the deviation in each familiarity observation for each individual from their mean score). We applied the same approach to the 2015 and 2016 audio recorder data for which we had observations from individuals across multiple social environments (Table 3.3).

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We conducted analyses using R version 3.4.1 (R Core Team, 2017) and fitted all GLMMs using the lme4 package (version 1.1-13; Bates, Maechler, Bolker, & Walker, 2015). For all analyses, we fitted generalized additive models to confirm that there were no significant non- linearities between our predictor and response variables. We checked for overdispersion by comparing the ratio of the sum of the squared Pearson residuals to the residual degrees of freedom in each model (Zuur et al., 2009) and assessing whether the sum of squared Pearson residuals approximated a Chi-squared distribution with N-P degrees of freedom (Bolker et al., 2009). As stated above, we accounted for overdispersion in Poisson models by including an observation-level random effect (OLRE; Harrison, 2014). For models with binomial data, we accounted for overdispersion using Beta-Binomial models, which have been demonstrated to better cope with overdispersion in binomial data (Harrison, 2015). We fitted all Beta-Binomial models using the package glmmADMB (version 0.8.3.3; Harrison, 2015; Skaug, Fournier, Nielsen, Magnusson, & Bolker, 2018). We standardized all continuous fixed effects to a mean of zero and unit variance. For the following results we present all means ± SE, unless otherwise stated, and consider differences statistically significant at P < 0.05.

Results

Heterogeneity in mean neighbourhood density and familiarity were very similar between the longitudinal and cross-sectional data sets. Among the years in which we analyzed long-term focal data (1995-2004), variation in average neighbourhood familiarity ranged from 0 (corresponding to when a squirrel first established its territory) to 813 days (mean: 229 ± 9 days) and variation in local density ranged from 0.57 to 5.84 squirrels/hectare (mean: 1.93 ± 0.05 squirrels/hectare). In our 2015 and 2016 cross-sectional data, there was a nearly equivalent amount of variation in average neighbourhood familiarity and local density. Neighbourhood familiarity ranged from 0 to 855 days (mean: 296 ± 5 days) and local density ranged from 1.13 to 6.03 (mean: 3.34 ± 0.03 squirrels/hectare). Below we discuss the effects of familiarity and age on behavioural patterns. Results for other fixed effects in the models can be found in Table 3.1.

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Longitudinal data Territorial defence During the long-term focal observations, red squirrels emitted an average of 0.37 ± 0.04 rattles per 10-min observation session (range: 0-4), which is equivalent to one rattle every 27.06 minutes. Red squirrels in the longitudinal dataset adjusted their behaviour in response to increasing average neighbourhood familiarity by emitting significantly fewer rattles ( = -0.29 ± 0.12, z = -2.48, P = 0.01; Fig. 3.1). This corresponds to a predicted three-fold decrease in rattling rates: in neighbourhoods with the lowest familiarity, squirrels were predicted to rattle once every 24.76 minutes and in neighbourhoods with the highest familiarity, only once every 79.75 minutes. The effect of age on rattling rates was marginally non-significant ( = -0.20 ± 0.11, z = -1.85, P = 0.06; Table 3.1). On average, squirrels spent 6.0 ± 0.7% of their time vigilant, but did not show changes in vigilance behaviour in response to changing neighbourhood familiarity ( = 0.02 ± 0.13, z = 0.15, P = 0.88; Table 3.1).

Nest use Based on the long-term data, red squirrels spent, on average, 31.0 ± 2.0% of their time in nest. Red squirrels responded to changing social conditions by increasing nest use in response to increasing familiarity ( = 0.26 ± 0.12, z = 2.31, P = 0.02; Fig. 3.1). This is equivalent to a predicted 23% increase in nest use: squirrels in neighbourhoods with the lowest familiarity were predicted to spend only 22% of their time in nest compared to 45% in neighbourhoods with the highest familiarity.

Cross-sectional data Territorial defence During focal observations in 2016, red squirrels emitted 0.71 ± 0.03 rattles per 7-min observation session (range: 0-6), which equates to approximately one rattle every 9.80 minutes. Data from audio recorders in 2015 and 2016 provided very similar estimates of rattling rates. We captured, on average, 33.96 ± 0.72 rattles per 6-hours of recording (range: 3-123), which, after correcting for the error rates in our discriminant function analysis, is equivalent to one rattle every 9.81

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minutes. Rattling rates were much higher than in the longitudinal data due to differences in behavioural sampling protocol. In 2016, all occurrences of rattling were recorded as ‘critical incidents’, while in the long-term data rattles were only recorded if they fell on a 30-second sampling interval. When critical incidents of rattling were removed from the 2016 data, rattling rates dropped to one rattle every 40.55 minutes. Based on both cross-sectional focal observations and audio recorder data, neither average familiarity of the social neighbourhood (all |z| < 1.25, all P > 0.21) nor age (all |z| < 1.87, all P ≥ 0.06) were significant predictors of rattling rate (Table 3.1). Focal observations indicated that red squirrels spent 7.0% ± 0.5% of their time vigilant, on average, but did not adjust vigilance behaviour in response to changing neighbourhood familiarity ( = 0.05 ± 0.07, z = 0.69, P = 0.49; Table 3.1).

Nest use Based on focal observations in 2016, on average red squirrels spent an average of 36.0% ± 1.0% of their time in nest. Accelerometer data from 2016 provided similar estimates of average proportion of time spent in nest during daylight hours (36.0% ± 0.4%). Both focal observations and accelerometer data indicated that squirrels did not adjust their nest use in response to neighbourhood familiarity (all |z| < 1.22, all P > 0.22; Table 3.1).

Exploratory post-hoc analysis In our post-hoc analyses we found evidence to suggest that effects of familiarity on rattling rates were primarily due to within-individual changes in behaviour rather than among-individual differences. In the long-term data, increasing familiarity led to a significant decrease in rattling rates within ( = -0.21 ± 0.08, z = -2.51, P = 0.01), but not among individuals ( = -0.18 ± 0.12, z = -1.50, P = 0.13; Table 3.3). There was a positive within and among-individual effect of familiarity on nest use, but neither of these effects were significant (all |z| < 1.69, all P > 0.08; Table 3.3). Audio recorder data from 2015 and 2016 also revealed a significant negative within- individual effect ( = -0.03 ± 0.01, z = -2.55, P = 0.01), but not among-individual effect of familiarity on rattling rates ( = 0.02 ± 0.05, z = 0.34, P = 0.74; Table 3.3). Results from the audio data should be interpreted with caution as the inclusion of year in the model affected these

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results (see Table S3.1).

Discussion

For territorial species, the ability to be responsive to changes in the social environment may convey a fitness advantage by allowing individuals to reduce time and energy investment in costly behaviours (Höbel, 2015; Krobath, Römer, & Hartbauer, 2017; Ydenberg et al., 1988). In this study, we used multiple types of behavioural data, as well as a longitudinal and cross- sectional dataset, to test a single overarching hypothesis: that red squirrels show behavioural plasticity in response to the familiarity of their social neighbourhood. Our results provide evidence that an ‘asocial’ species, the North American red squirrel, can respond to changes in the composition of its social environment, and that red squirrels do so under natural conditions and in a manner that is consistent with our expectations for adaptive behavioural change in this species. Although our evidence for social plasticity comes exclusively from female squirrels, both male and female red squirrels defend exclusive territories based around a central cache of food resources and emit territorial vocalizations that are the same in both form and function (Smith 1968; Smith 1981). Given this, and the fact that we found no evidence of an interaction between familiarity and sex in the cross-sectional analyses (data not shown), we have no reason to expect that social plasticity differs between male and female red squirrels.

Previous work in this study system has demonstrated that red squirrels face reduced intrusion risk in social neighbourhoods with high average familiarity (Siracusa, Boutin, et al., 2017a). As such, we predicted that red squirrels would show appropriate social plasticity by reducing territorial defence behaviours and increasing time and energy spent on self-maintenance behaviours when in familiar social neighbourhoods. Results from behavioural observations across eight years provided support for these predictions, indicating that red squirrels demonstrated social plasticity by reducing rattling rates and increasing the proportion of time spent in nest in social neighbourhoods with high average familiarity (Fig. 3.1). Such changes in behaviour not only minimize the time spent on territory defence but might also reduce associated costs of territoriality. For example, territorial vocalizations may attract the attention of predators (Abbey-Lee, Kaiser, Mouchet, & Dingemanse, 2016) and red squirrel rattles are loud, broadband 54

signals which should be easy to localize (Marler, 1955). By reducing rattling rates under less risky social conditions, squirrels may also benefit from reduced predation risk. Additionally, spending more time in nest when familiarity with neighbours is high also presumably reduces the risk of being detected by a predator.

We did not, however, find effects of neighbourhood familiarity on vigilance behaviour. This could be due to vigilance for conspecifics being easily confounded with vigilance for predators. In contrast, Dantzer et al. (2012) found significant effects of local density on vigilance using behavioural data collected over a similar time frame, indicating that conspecific rather than heterospecific effects on vigilance are detectable in this system. While we included local density as a covariate in all of our models to account for the potential effects of density on behaviour (Dantzer et al., 2012), our goal was not to directly estimate effects of density, and our results therefore are not a clean representation of density effects. In several cases density was correlated with other variables in the model, such as grid, leading to substantial changes in the parameter estimates for density. As a result, the effects of density on behaviour that we have reported here cannot be compared directly to previous studies of these effects in this population (e.g. Dantzer et al., 2012; Shonfield et al., 2012) and we do not discuss the effects of density further.

Results from the cross-sectional data in 2015 and 2016 did not corroborate our long-term results showing behavioural responses to familiarity. Findings from the behavioural observations, audio recorders, and accelerometers indicated that when using among-individual relationships to estimate the effects of the social environment on behaviour, there was no effect of familiarity on territorial behaviours (rattling rates, vigilance) or self-maintenance (nest use; Table 3.1). In light of this inconsistency with the longitudinal data, we conducted an exploratory post-hoc analysis to assess whether using longitudinal data (where we can estimate within- individual changes in behaviour) rather than cross-sectional data (where the analysis is largely among individuals) might account for our different findings. Since behavioural plasticity is functionally a within-individual phenomenon, using among-individual differences in behaviour to estimate these effects relies on the assumption that the among-individual relationship is an accurate representation of the within-individual relationship. Here we used a within-subject 55

centering approach (van de Pol & Wright, 2009) and found that the within and among-individual effects were not equivalent. In the long-term data, we found that individuals adjusted rattling rates in response to changes within their own social environment (i.e. a significant within- individual effect) but did not observe significant differences in rattling rates when comparing among individuals (Table 3.3). Similarly, for the audio recorder data (the only cross-sectional data for which we had observations of individuals across multiple social environments) we found evidence of a significant within-individual, but not among-individual, effect (Table 3.3). Thus, while we clearly see evidence of plasticity when considering changes in individual behaviour across different social environments, in this study system it appears that we cannot estimate these effects by comparing behaviour among individuals.

One potential explanation for this discrepancy is that the among-individual effect is masked by individual variation in plasticity, whereby substantially different individual ‘slopes’ result in a ‘mean slope’ of zero (i.e. the absence of a significant population-level response to the environment; Nussey, Wilson, & Brommer, 2007). We were unable to test this hypothesis as we lacked the statistical power to include a random slope term in our models (Martin, Nussey, Wilson, & Réale, 2011). Furthermore, even if all individuals demonstrate negative reaction norms (i.e. reduced rattling rate in response to increasing familiarity), there are still several reasons we might fail to detect differences among individuals. First, it seems unlikely that squirrels can assess their absolute familiarity, meaning that behavioural adjustments are dependent on the relative social environments individuals experience rather than absolute changes in familiarity. Nor were we able to precisely measure absolute familiarity since measures of the social environment were based on semi-annual census data, which may have added some noise to the data. Additionally, variation in individual mean ratting rates (i.e. random intercepts) due to differences in sex, age, personality, stress, among other possibilities, might mask an among-individual effect of familiarity. These factors, combined with variation in the range of social environments sampled for a given individual, mean that, even when all individuals show negative reaction norms, it is possible to measure a lack of, or even a positive among-individual effect (Fig. 3.2). Importantly, this means that studies that attempt to assess behavioural plasticity by comparing behaviour among individuals should interpret their results 56

with caution, as the estimated among-individual effect may not be an accurate reflection of within-individual changes in behaviour.

Although we have provided an explanation for the differences in our long-term and cross- sectional findings, there are a couple reasons why it is important to interpret our findings demonstrating effects of familiarity on red squirrel behaviour with caution. First, there is potential for changes in rattling rates to be driven by effects of age rather than familiarity if the strength of territory defence declines with physical deterioration. We have done our best to account for this possibility in our analyses but given that these variables are strongly correlated an experimental approach would prove useful in disentangling these effects, as they are difficult to tease apart statistically. Second, it is worth addressing our use of multiple univariate analyses to test a single overarching hypothesis. Previous research in this study system has detected effects of the social environment on vigilance and nest use using a multivariate analysis (Dantzer et al., 2012), allowing us to make specific predictions about how squirrels should adjust patterns of nest use and vigilance in response to neighbourhood familiarity. Given this, we felt that analyzing the effects of familiarity on each of these behaviours individually provided a more elegant test of our hypothesis. However, it is important to be aware that our use of univariate analyses increases our chances of committing a Type I error by attributing variance as unique to a single response variable when it may in fact be shared (Huberty & Morris, 1989).

Despite these limitations, we believe that the results from our study, in particular the data for which we can estimate within-individual changes in behaviour, provide evidence that red squirrels are socially plastic. Furthermore, although we have not directly tested the fitness consequences of social plasticity, red squirrels reduced rattling rates, thereby spending less time on territory defence and potentially minimizing risk of detection by predators, under social conditions where intrusion risk was low (Siracusa, Boutin, et al., 2017a). This suggests that ‘asocial’ species may be socially competent as well as socially responsive (Taborsky & Oliveira, 2013; 2012). Familiarity with conspecifics has long been assumed to be beneficial by allowing individuals to minimize energy expended on territory contests and increase time devoted to reproduction and growth (Getty, 1987; Temeles, 1994; Ydenberg et al., 1988). Evidence for 57

reduced aggression toward familiar conspecifics is taxonomically widespread (Temeles, 1994). However, these studies have typically been focused on documenting behavioural changes on short timescales through exposure to an experimental stimulus. Our study is one of few to demonstrate that natural variation in neighbourhood familiarity has direct consequences for behavioural time budgets by allowing individuals with familiar neighbours to reduce territory defence and increase time spent in nest. A handful of previous studies have demonstrated similar patterns in wild populations under natural social conditions. Willow ptarmigan (Lagopus lagopus) males were found to spend significantly more time engaged in territorial border disputes when they had more new neighbours (Eason & Hannon, 1994). In Seychelles warblers (Acrocephalus sechellensis), living near familiar individuals provided important benefits by reducing immediate energetic costs through fewer physical fights (Bebbington et al., 2017).

Additionally, recent research has increasingly noted the importance of group composition in shaping individual behaviour (Farine et al., 2015). For example, nutmeg mannikins (Lonchura punctulata) have been shown to forego consistent individual differences in scrounger-forager tactics when flock composition changes, and to adjust their social strategy according to frequency-dependent pay-offs (Morand-Ferron et al., 2011). Water striders (Aquarius remigis) also show plasticity in aggression and activity in response to the presence of hyperaggressive individuals in the group (Sih et al., 2014) or changes in male-male competition (Montiglio et al., 2017). Although territorial species do not act in clearly defined, discrete units, we have demonstrated that red squirrels show similar social plasticity in response to the composition of neighbouring territory holders at the scale of the acoustic social environment (i.e. 130 m radius). Our results emphasize that the composition of neighbouring conspecifics, in addition to quantity of individuals in the social environment (Dantzer et al., 2012), can shape the behaviour of territorial species.

Conclusion The advantage of territoriality is contingent on the benefits of resource acquisition outweighing the costs of defending those resources from competitors (Brown, 1964; Schoener, 1987). Although the dear-enemy effect has been a well-recognized phenomenon in territorial species for 58

several decades (Temeles, 1994), the relative importance of social interactions for maintaining this cost-benefit ratio under natural conditions has rarely been explored. Here we show that red squirrels adjust their behaviour in response to the familiarity of their social environment and that squirrels living in unfamiliar social neighbourhoods pay a cost in time by increasing rattling rates three-fold and reducing nest use by approximately 25% in order maintain their territory under conditions of high intrusion risk. Importantly, our results suggest that behavioural plasticity in this species cannot be estimated by comparing differences in behaviour among-individuals, emphasizing the need to have observations from the same individuals across multiple social environments in order to detect these behavioural patterns.

Taken altogether, these results provide evidence that territorial species have the capacity to assess and respond to nuanced changes in their social environment, despite not typically being considered to engage in important social interactions. Social relationships may therefore be more important than previously appreciated for apparently ‘asocial’ species. Several studies have demonstrated that familiarity with territory neighbours can have fitness benefits, including increased growth rate and survival (Höjesjö, Johnsson, Petersson, & Järvi, 1998; Seppä et al., 2001), reduced telomere attrition (Bebbington et al., 2017) and increased reproductive success (Beletsky & Orians, 1989; Grabowska-Zhang, Wilkin, & Sheldon, 2012b). Given that these long-term social relationships have been demonstrated to affect both intrusion risk (Siracusa, Boutin, et al., 2017a) and behavioural time budgets (this study) in red squirrels, it seems likely that familiarity with territory neighbours could also have important fitness consequences for this species, but this has yet to be explored.

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Tables

Table 3.1. Fixed effects from all generalized linear mixed-effects (GLMM) and Beta-Binomial (BB) models, showing effects of average neighbourhood familiarity, local density, and focal squirrel’s age on rattling rate, nest use and vigilance behaviour. Regression coefficients for familiarity, age and density are standardized. Significant effects are indicated in bold.

Method of data Years N Model Fixed effect Parameter +/- z P collection SE Longitudinal 1995-2004 487 focals Rattle rate Familiarity -0.29 ± 0.12 -2.48 0.01 (GLMM) Age -0.20 ± 0.11 -1.85 0.06 Density -0.17 ± 0.13 -1.38 0.17 Vigilance Familiarity 0.02 ± 0.13 0.15 0.88 (BB) Density 0.05 ± 0.26 0.19 0.85 Nest Use Familiarity 0.26 ± 0.12 2.31 0.02 (BB) Density -0.37 ± 0.15 -2.53 0.01 Cross-sectional 2016 1060 focals Rattle rate Familiarity 0.07 ± 0.07 1.09 0.27 (GLMM) Age -0.02 ± 0.07 -0.25 0.80 Density -0.12 ± 0.06 -2.01 0.04 Sex-M + -0.13 ± 0.13 -1.02 0.31 Grid-KL * -0.15 ± 0.13 -1.11 0.27 Grid-SU * -0.52 ± 0.17 -3.09 0.002 Obs- JR † 0.14 ± 0.25 0.58 0.56 Obs- MT † -0.34 ± 0.09 -3.87 <0.001 Obs- YS † -0.47 ± 0.10 -4.92 <0.001 Vigilance Familiarity 0.05 ± 0.07 0.69 0.49 (BB) Density -0.01 ± 0.09 -0.07 0.95 Sex-M + 0.26 ± 0.18 1.48 0.14 Grid-KL * -0.42 ± 0.17 -2.48 0.01 Grid-SU * -0.36 ± 0.22 -1.63 0.10 Obs- JR † 0.66 ± 0.38 1.73 0.08 Obs- MT † -0.86 ± 0.16 -5.44 <0.001 Obs- YS † 0.44 ± 0.14 3.24 0.001 Nest Use Familiarity -0.11 ± 0.09 -1.21 0.23 (BB) Density 0.08 ± 0.10 0.79 0.43 Sex-M + 0.11 ± 0.21 0.52 0.60 Grid-KL * 0.15 ± 0.22 0.70 0.48

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Grid-SU * 0.50 ± 0.27 1.85 0.06 Obs- JR † -0.59 ± 0.57 -1.02 0.31 Obs- MT † 0.41 ± 0.16 2.62 0.009 Obs- YS † 0.37 ± 0.16 2.24 0.02 Audio 2015-2016 714 recordings Rattle rate Familiarity -0.05 ± 0.04 -1.24 0.21 (GLMM) Age -0.09 ± 0.05 -1.86 0.06 Density 0.003 ± 0.04 0.08 0.94 Sex-M + 0.01 ± 0.10 0.13 0.90 Grid-KL * -0.28 ± 0.09 -3.13 0.001 Grid-SU * -0.73 ± 0.12 -6.10 <0.001 Accelerometers 2016 922 Nest Use Familiarity -0.0005 ± 0.04 -0.01 0.99 (BB) Density -0.07 ± 0.04 -1.51 0.13 Sex-M + 0.04 ± 0.08 0.53 0.60 Grid-KL * 0.13 ± 0.09 1.36 0.17 Grid-SU * 0.01 ± 0.11 0.11 0.91

+ Female taken as the reference * AG (food supplemented grid) taken as the reference † Observer ES taken as the reference

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Table 3.2. Random effects from all generalized linear mixed-effects (GLMM) and Beta- Binomial (BB) models. Significance assessed using a log-likelihood ratio test (LRT) with one degree of freedom to compare models with and without the listed random effect. Significant effects are indicated in bold.

Method of data Years Model Random effect Variance 2 df P collection Longitudinal 1995-2004 focals Rattle rate Squirrel ID 0.09 2.19 1 0.14 (GLMM) Year <0.01 <0.01 1 >0.999 Observer 0.48 18.55 1 <0.001 Vigilance Squirrel ID 0.08 0.56 1 0.45 (BB) Year 1.11 5.03 1 0.02 Observer 0.62 10.53 1 0.001 Nest Use Squirrel ID <0.01 <0.01 1 >0.999 (BB) Year 0.10 1.20 1 0.27 Observer 0.10 1.80 1 0.18 Cross-sectional 2016 focals Rattle rate Squirrel ID 0.14 37.53 1 <0.001 (GLMM) Vigilance Squirrel ID 0.14 8.16 1 0.004 (BB) Nest Use Squirrel ID 0.32 19.20 1 <0.001 (BB) Audio 2015-2016 recordings Rattle rate Squirrel ID 0.18 422.38 1 <0.001 (GLMM) OLRE 0.06 558.11 1 <0.001 Accelerometers 2016 Nest Use Squirrel ID 0.10 157.58 1 <0.001 (BB) AXY No. <0.01 <0.01 1 >0.999

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Table 3.3. Fixed effects from exploratory post-hoc models including a within-individual

(FamiliarityW) and among-individual (FamiliarityA) effect of familiarity. Regression coefficients for familiarity, age and density are standardized. Significant effects are indicated in bold.

Method of Years N Model Fixed effect Parameter z P data collection +/- SE Longitudinal 1995-2004 487 focals Rattle rate FamiliarityW -0.21 ± 0.08 -2.51 0.01 (GLMM) FamiliarityA -0.18 ± 0.12 -1.50 0.13 Age -0.22 ± 0.11 -2.02 0.04 Density -0.18 ± 0.13 -1.42 0.15 Nest Use FamiliarityW 0.17 ± 0.10 1.68 0.09 (BB) FamiliarityA 0.19 ± 0.13 1.53 0.13 Density -0.37 ± 0.15 -2.50 0.01 Audio 2015-2016 714 recordings Rattle rate FamiliarityW -0.03 ± 0.01 -2.55 0.01 (GLMM) FamiliarityA 0.02 ± 0.05 0.34 0.74 Age -0.10 ± 0.05 -2.09 0.04 Density -0.02 ± 0.04 -0.56 0.58 Sex-M + 0.01 ± 0.10 0.12 0.90 Grid-KL * -0.28 ± 0.09 -3.16 0.002 Grid-SU * -0.81 ± 0.12 -6.58 <0.001

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Figures

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Figure 3.1. Red squirrels adjust a) rattling rate and b) proportion of time spent in nest in response to the average familiarity of their social neighbourhood (N = 487). Results are based on 10-min behavioural observations of squirrels between 1995-2004. Values on x-axis are standardized measures of average familiarity. Points indicate raw data with a small amount of jitter introduced to show overlapping points.

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a. Negative Among−Individual Effect b. No Among−Individual Effect c. Positive Among−Individual Effect

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Figure 3.2. Three different scenarios for how variation in mean rattling rate (random intercepts) in combination with variation in data sampling structure might change our ability to detect among-individual effects when individuals have the same slope. We schematically depict the within-individual slopes (solid grey lines) of seven subjects (j = 1 to j = 7). The solid grey lines indicate the range over which each individual was sampled. Dotted lines provide an extension of these slopes to the edge of the figure. The among-subject slope (solid black line) is based on the association between x̄ j and ȳj as denoted by the filled black circles.

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Chapter 4: Stable Social Relationships with Territory Neighbours Affect Reproductive Success and Survival in an ‘Asocial’ Squirrel

Abstract

An important question in evolutionary biology is how conflict between interacting conspecifics can be resolved, allowing the evolution of various forms of stable social organization. In group- living species, stable social bonds among group members help to mitigate conflict and enhance fitness. Similarly, long-term social relationships in solitary, territorial species may help to minimize conflict between neighbours (and thus costs of territoriality), though the fitness benefits of these social relationships are poorly understood. In this study, we used 21 years of data from a population of territorial North American red squirrels (Tamiasciurus hudsonicus) to assess how familiarity with neighbours affected the reproductive success and survival of individuals. We also modeled how kinship among neighbours influenced fitness, to determine the importance of mutualistic interactions relative to kin-selection. While relatedness among territorial neighbours was not an important driver of survival or reproductive success, we found that familiarity with neighbours had substantial benefits for survival, and increased both the number of pups sired and the number of pups recruited annually. These effects were particularly pronounced in the ‘senescent’ period (i.e. squirrels aged 4 and older) suggesting that familiarity may help buffer squirrels against effects of age-related fitness declines. This study is one of few to show that familiar relationships among territorial neighbours can have direct fitness benefits. Mutually beneficial interactions among unrelated individuals may, therefore, play an important role in the evolution of social systems.

Introduction

Most organisms interact with conspecifics and, therefore, have a social environment. Conflict is a ubiquitous part of this social environment because the survival of an organism’s genes is dependent on pursuing selfish interests. Despite this, we know that cooperation regularly occurs in nature at all levels of biological organization (Maynard Smith & Szathmary, 1995). Understanding how this conflict can be mitigated, therefore, is central to our understanding of 66

how group living and sociality can evolve. One of the outstanding questions in evolutionary biology is the extent to which mutualistic benefits and kin-selection can mitigate conflict between interacting organisms (Clutton-Brock, 2009; Sachs et al., 2004). The indirect fitness benefits gained from associating with kin is well established as an important pathway to conflict resolution (Hamilton, 1964a; 1964b). However, for group living organisms it is also understood that stable social bonds among conspecifics can play an important role in mitigating intragroup conflict and enhancing fitness. The importance of social relationships has been well demonstrated in primates, whereby stable social bonds have been demonstrated to influence overall health and stress levels (Crockford, Wittig, Whitten, Seyfarth, & Cheney, 2008; Engh, Beehner, Bergman, Whitten, Hoffmeier, Seyfarth, & Cheney, 2006a; 2006b; Kaplan et al., 1991; Wittig et al., 2008), enhance longevity (Silk et al., 2010), and increase offspring production and survival (McFarland et al., 2017; Schülke, Bhagavatula, Vigilant, & Ostner, 2010; Silk, 2003; Silk et al., 2009). Female feral horses (Equus callabus) obtain similar benefits from social bonds with unrelated mares, including increased birth rates and foal survival, potentially resulting from reduced male harassment (Cameron, Setsaas, & Linklater, 2009). In female bighorn sheep, increased social centrality (a measure of the degree to which individuals in a group are well- connected to other group members) has also been demonstrated to lead to increased lamb production and survival (Vander Wal, Festa-Bianchet, Reale, Coltman, & Pelletier, 2015).

In addition to mitigating conflict among group-living species, there is substantial potential for stable social relationships to play an important role in helping to resolve conflict over territorial space. Territoriality, as a strategy, is only adaptive if the benefits of increased access to resources outweigh the time and energy spent defending those resources from competing conspecifics (Brown, 1964; Brown & Orians, 1970). One way that individuals can act to minimize the costs associated with territoriality is by maintaining relatively stable territories or home ranges over their lives (Beletsky & Orians, 1987; McDougall & Kramer, 2007; Part, 1995; Stamps, 1987). The benefits of this territory fidelity may include increased information about the local physical environment, such as resource availability or predation risk. However, maintaining a stable territory also ensures well-established territory boundaries and provides important information about neighbouring conspecifics, including dominance status and 67

competitive ability. Stable social relationships with territorial neighbours can therefore lead to a cooperative agreement of reduced aggression and territory defence, a phenomenon more commonly known as the ‘dear enemy’ effect (Fisher 1954). This kind of mutualistic relationship among long-term neighbours may help to alleviate the costs of territoriality.

Previous studies have demonstrated that stable social relationships among neighbours have important benefits for a variety of traits in territorial species. Some of these benefits include faster growth rate (Höjesjö et al., 1998; Liebgold & Cabe, 2008), faster predator evasion responses (Griffiths et al., 2004), enhanced juvenile body condition and survival (Seppä et al., 2001) and reduced telomere attrition (Bebbington et al., 2017). There has even been some evidence that familiar relationships among territorial neighbours can lead to the formation of defensive coalitions, as originally theorized by Getty (1987). Fiddler crabs (Uca spp.) were found to come to the aid of familiar neighbours when threatened by a potential usurper (Detto, Jennions, & Backwell, 2010), familiar rock pipits (Anthus petrosus) engaged in coordinated territory defence and evictions of intruders (Elfstrom, 1997) and great tits (Parus major) have been demonstrated to be more likely to engage in joint predator mobbing with familiar neighbours (Grabowska-Zhang, Sheldon, & Hinde, 2012a). While most of these findings have provided evidence of indirect fitness benefits, fewer studies have been able to demonstrate direct fitness consequences of stable social relationships among neighbours. The earliest evidence of this comes from male red-winged blackbirds (Agelaius phoeniceus), which were found to attract more females and fledge significantly more offspring when sharing a border with at least one familiar neighbour (Beletsky & Orians, 1989). More recently, great tits have been found to lay larger clutches and have an increased chance of successfully fledging offspring when surrounded by more familiar neighbours (Grabowska-Zhang, Wilkin, & Sheldon, 2012b).

Taken together, there is growing evidence to suggest that long-term social relationships may mitigate conflict and enhance fitness in both group-living and territorial species, thus potentially playing a role in the evolution of sociality. However, in many of the study systems that have investigated the importance of stable social relationships, social interactions typically take place between related individuals (Silk et al., 2009; Silk, Alberts, & Altmann, 2006a; Silk, 68

Altmann, & Alberts, 2006b; Silk, Cheney, & Seyfarth, 1999) obscuring our ability to understand the relative evolutionary roles of kin-selection and mutualistic interactions in reducing conflict and facilitating cooperation. Studies that have more clearly been able to parse out effects of relatedness indicate that social bonds among conspecifics can be adaptive even in the absence of kinship (Cameron et al., 2009), or suggest that familiarity may interact in complex ways with relatedness or other aspects of the social environment (Bebbington et al., 2017).

Here we used a long-term study of North American red squirrels (Tamiasciurus hudsonicus) to assess the importance of familiarity with neighbours on the reproductive success and survival of a territorial species. With a pedigree spanning 27 years and over 9000 individuals, this study system provided a unique opportunity to assess the importance of stable social relationships among territorial neighbours while controlling for effects of kinship. Red squirrels are arboreal rodents in which both sexes defend exclusive territories based around a central cache of food resources called a ‘midden’ (Smith, 1968). These resources are important for overwinter survival (Kemp & Keith, 1970; LaMontagne et al., 2013) and are defended primarily through territorial vocalizations called ‘rattles’ (Lair, 1990; Siracusa, Morandini, et al., 2017b). Previous work in this study system has demonstrated that once individuals settle on a territory they rarely show propensity to relocate to a vacant midden (Larsen & Boutin, 1995), thus allowing for the establishment of long-term familiarity among neighbouring individuals. For red squirrels, relationships with territory neighbours are primarily defined by antagonistic interactions, since individuals are in direct competition for limited resources. However, living near familiar neighbours has been shown to provide important benefits, including significantly reducing the risk of territory intrusion from conspecifics (Siracusa, Boutin, et al., 2017a) as well as the time spent on territory defence. Red squirrels emitted fewer territorial vocalizations in response to rattle playbacks from familiar conspecifics compared to unfamiliar conspecifics (Price et al., 1990), and similar results were found when observing squirrels under naturally occurring social conditions. Territory owners reduced rattling rates and allocated more time to nest-use when average familiarity with neighbouring conspecifics was high (Siracusa et al. in review).

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Given that familiarity with neighbouring conspecifics can reduce risk of pilferage and minimize time spent on territory defence, we hypothesized that stable social relationships with neighbours would have direct fitness benefits for red squirrels. To test this hypothesis we used 21 years of long-term data from a wild population of red squirrels. We predicted that red squirrels whose average familiarity with neighbouring conspecifics was high would have increased annual reproductive success and a higher probability of surviving to the following year.

Methods

Study population We studied a wild population of North American red squirrels located in the southwest Yukon, Canada near Kluane National Park (61° N, 138° W). This population has been monitored since 1987 on two 40-ha study grids separated by the Alaska Highway. The study grids are characterized by open boreal forest with white spruce (Picea glauca) as the dominant canopy species (Krebs et al., 2001). We monitored squirrels annually from March until August and used a combination of live-trapping and behavioural observations to assess territory ownership, track reproduction and survival, and determine offspring recruitment. Squirrels were trapped using Tomahawk traps (Tomahawk Live Trap Co., Tomahawk, Wisconsin, USA) baited with natural peanut butter. During their single-day oestrous, female red squirrels mate with multiple males and produce multiply-sired litters (Lane et al., 2008), with an average of three pups per litter (Humphries & Boutin, 2000). We monitored the reproductive statuses and parturition dates of females through palpation, mass changes, and evidence of lactation. After parturition, we used radio telemetry to follow females to their nests. We fitted pups with unique alphanumeric metal ear tags (Monel #1; National Band and Tag, Newport, KY, USA) at 25 days old, allowing us to follow individuals throughout their lifetimes. A detailed description of project protocols can be found in Berteaux & Boutin 2000 and McAdam et al. 2007.

Measuring familiarity We completed a full census of the population twice annually in May and August and determined territory ownership by using a combination of live-trapping and behavioural observations. We defined each squirrel’s social neighbourhood to be all conspecifics within a 130 m radius of the 70

focal squirrel’s midden (i.e. the centre of the territory). Red squirrel territorial vocalizations (‘rattles’) typically carry up to 130 m (Smith, 1978), so this radius defines the acoustic social environment that an individual experiences. Furthermore, 130 meters is similar to the distance at which red squirrels have been demonstrated to assess and respond to changes in density (150 m; Dantzer et al., 2012) or neighbour familiarity (130 m; Siracusa et al. in review) in their local environment. We measured the pairwise familiarity between the territory owner and each neighbour as the number of days that both individuals had occupied their current territories within 130 m of each other. We then averaged all pairwise measures of familiarity to obtain a measure of each individual’s average familiarity with its surrounding neighbours. We used this average familiarity term to assess for effects stable social relationships on fitness in the analyses described below.

Measuring relatedness We temporarily removed juveniles from their natal nest immediately following parturition to weigh, sex, and take ear tissue samples. Observing pups in the natal nest also allowed us to assign maternity with certainty. Starting in 2003, we determined paternity by genotyping all individuals at 16 microsatellite loci (Gunn et al., 2005) and assigned paternity with 99% confidence using CERVUS 3.0 (Kalinowski et al., 2007). Genotyping error based on known mother-offspring pairs was 2%. Additional details on the genetic methods can be found in Lane et al. 2008. Using the established multigenerational pedigree, we calculated the coefficient of relatedness between the territory owner and each neighbour. We averaged all pairwise measures of relatedness to provide a measure of the average relatedness between each squirrel and all of its neighbours.

Fitness measures We measured annual survival of 1009 squirrels between 1994 and 2015 (N = 2346 records). Given that long-term monitoring of individuals in this population first began in 1987, we excluded data prior to 1994 to ensure accurate measurement of familiarity between neighbours. Annual survival was defined as whether or not an individual survived until January 1st of the following year. An individual was considered to have survived if trapped or observed at any 71

point during or after January 1st. Our ability to successfully redetect adults in the population on a yearly basis is estimated to be 1.0 (Descamps, Boutin, McAdam, Berteaux, & Gaillard, 2009).

Female annual reproductive success (ARS) was measured as the number of pups recruited each year. Juveniles that survived overwinter were considered to have recruited. We included data from 451 breeding females between 1994 and 2015 (N = 982 records). All females with a known parturition date were considered to have bred; all other non-breeding females were excluded from analysis. Since red squirrel males do not contribute to the raising of pups and the male’s social environment is therefore unlikely to affect pup recruitment, we defined male ARS as the total number of pups sired each year. We used data from 207 males between 2003 and 2014 (N = 412 records). We excluded data prior to 2003 and after 2014 because pedigree data was incomplete. For all analyses we only used data for adults (≥ 1 year old) whose age could be assigned with certainty (i.e. individuals tagged in nest as juveniles).

Although our ability to accurately assess survival and recruitment has the potential to be confounded by dispersal beyond the borders of our study grids, this does not appear to be a problem in our study system. Juveniles typically disperse less than 100 m (Berteaux & Boutin, 2000; Cooper et al., 2017), and estimates of juvenile survival do not appear to differ between the centre and edge of grid, which might be expected if dispersing juveniles were mistakenly considered to have died (McAdam et al., 2007).

Statistical analyses To assess the effects of familiarity on ARS and survival we used generalized linear mixed effects models (GLMMs) with a BOBYQA optimizer. For survival models we fitted models with a binomial distribution (logit-link) where the response variable was a binary response (1 = yes, the squirrel survived to the next year; 0 = no, the squirrel did not survive to the next year). We fitted separate models for male and female ARS using GLMMs with a Poisson distribution (log-link). For females, the response variable was the number of pups recruited, for males the number of pups sired. For all models we fitted the squirrel’s average familiarity with its neighbours as a continuous predictor. To account for age-dependence of both survival and reproductive success 72

in this population (Descamps, Boutin, Berteaux, & Gaillard, 2008; McAdam et al., 2007) we included both age and quadratic term for age as continuous covariates in all models. We included grid density and the squirrel’s average relatedness with its neighbours as continuous predictors to account for the effects of density and kinship, respectively, on survival. We also included grid as a fixed effect. For the survival models we checked for an effect of sex, but sex was not significant and so was not considered further. To account for repeated measures of individuals and the fact that reproductive success might be confounded by differences in individual quality, we included a random effect of squirrel ID. We also included a random effect of year to account for temporal variation in resource availability.

Importantly, we wanted to account for inherent spatial structure in our data that could be related to familiarity. Although we discuss red squirrel neighbourhoods as if they were discreet units, in actuality individuals were distributed fairly continuously across our study grids, meaning that there was spatial overlap in individually defined neighbourhoods. We wanted to ensure that our observed effects on fitness were not driven by spatial variation in resource availability or predation risk. In particular, rather than familiarity influencing survival, areas with low survival might have high turnover and therefore low familiarity. To assess this, we grouped squirrels into 150 m squares (within each grid and within each year) based on their known spatial locations and included this ‘spatial ID’ as a random effect in all of our models.

Beyond assessing the overall effect of familiarity on annual reproductive success and survival in red squirrels, we were interested in understanding whether familiarity with neighbouring conspecifics could help to explain early life increases in reproductive success or might buffer against the effects of senescence (Descamps et al., 2008; McAdam et al., 2007). To do this we split our data into a ‘pre-senescent’ period (≤ 3 years old) and a ‘senescent’ period (≥ 4 years old), in addition to running models with all the data (i.e. ‘full models’). We divided the data in this way because age four has been demonstrated to correspond to either the peak or senescent period for most reproductive traits in the population (Descamps et al., 2008; McAdam et al., 2007). Furthermore, an understanding of the biological relationship between age and familiarity lends support for splitting the data in this manner. Young squirrels are inherently 73

unfamiliar with their neighbours and familiarity increases with age, to a point. Up to three years of age familiarity is strongly correlated with age (Pearson r = 0.60) but after four years old familiarity is more strongly driven by other demographic factors in the population. This decoupling of age and familiarity in the ‘senescent’ period (Pearson r = -0.04) enabled us to better assess the independent effects of familiarity and age on fitness. Models for the pre- senescent and senescent period included the same covariates as the full models described above, except that these models only included a linear effect of age.

We conducted statistical analyses using R version 3.4.1 (R Core Team, 2017) and fitted GLMMs using the lme4 package (version 1.1-13: Bates et al., 2015). For all models, we checked for significant non-linearities between the predictor and response variables by fitting generalized additive models. We checked for overdispersion by assessing whether the sum of squared Pearson residuals approximated a Chi-squared distribution with N-P degrees of freedom (Bolker et al., 2009). Of the 1013 squirrels used in this analysis 12 individuals were not in the pedigree, meaning that there were 25 neighbourhoods for which we could not calculate relatedness with neighbours. We assigned these neighbourhoods the mean average neighbourhood relatedness (r = 0.05). To facilitate direct comparison of effect sizes (Schielzeth, 2010) we standardized all continuous predictors to a mean of zero and unit variance. We present all means ± SE, unless otherwise stated, and consider all differences statistically significant at  < 0.05.

Results

Survival We analyzed 2346 records of annual survival from 1009 squirrels. Over the 11-year period in which we analyzed survival (1994-2015), average familiarity ranged between 0 and 1186 days (mean = 191 ± 4 days). Average relatedness ranged between 0 and 0.5 (mean = 0.05 ± 0.001) and average density ranged between 0.53 and 4.31 squirrels/hectare (mean = 2.10 ± 0.02 squirrels/hectare). Based on the full model with all data, increased familiarity with neighbouring individuals increased the probability of a squirrel surviving to the next year by approximately 10% ( = 0.18 ± 0.06, z = 2.78, P = 0.005; Fig. 4.1). There was a significant negative linear ( =

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-0.27 ± 0.09, z = -2.94, P = 0.003) and quadratic effect of age ( = -0.11 ± 0.04, z = -2.57, P = 0.01; Table 4.1).

The factors affecting survival also depended on life stage. In the pre-senescent period (≤ 3 years old; N = 1834 records over 996 individuals) neither age nor familiarity was a significant predictor of annual survival (all |z| < 0.51, all P > 0.61; Table 4.1) presumably because survival was fairly consistent in early life. In the senescent period (≥ 4 years old; N = 512 records over 288 individuals) familiarity increased the probability of survival by 40% (50% chance of survival when familiarity with neighbours was lowest to a 90% chance of survival when familiarity with neighbours was highest;  = 0.45 ± 0.11, z = 3.40, P <0.001; Fig. 4.2). As expected, there was also a negative linear effect of age on survival in the senescent period ( = - 0.36 ± 0.10, z = -3.42, P <0.001; Table 4.1).

Average relatedness, density, and grid were not significant predictors of annual survival in any of the models (all |z| < 1.68, all P > 0.09; Table 4.1), nor was the random grouping term of ‘spatial ID’ significant, indicating that there was no substantial spatial variation in annual survival at the scale of 150 m2 (all variance < 0.28, all 2 <1.25, all P > 0.25; Table 4.2).

Male ARS We analyzed 412 records of 207 males between 2003 and 2014. Over this timespan, average familiarity ranged between 0 and 1186 days (mean = 166 ± 9 days). Average relatedness ranged between 0 and 0.33 (mean = 0.04 ± 0.003) and density ranged between 0.53 and 2.04 squirrels/hectare (mean = 1.56 ± 0.02 squirrels/hectare). Full models including all data indicated that familiarity with neighbouring conspecifics had a significant positive effect on number of pups sired: males were predicted to sire one pup per year when familiarity with neighbours was lowest and nine pups per year when familiarity with neighbours was highest ( = 0.18 ± 0.09, z = 2.04, P =0.04; Fig. 4.1). There was a positive linear ( = 0.68 ± 0.14, z = 4.75, P <0.001) and negative quadratic effect of age on male ARS ( = -0.41 ± 0.08, z = -5.32, P <0.001; Table 4.1).

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Predictors of male ARS varied with life stage. In the pre-senescent period (≤ 3 years old; N = 334 records over 182 individuals) there was no effect of familiarity ( = 0.20 ± 0.10, z = 1.95, P = 0.05), but there was a positive linear effect of age on number of pups sired ( = 0.50 ± 0.11, z = 4.58, P <0.001; Table 4.1). In the senescent period (≥ 4 years old; N = 78 records over 60 individuals) number of pups sired increased six-fold with increasing familiarity ( = 0.39 ± 0.15, z = 2.67, P = 0.008; Fig. 4.2), and declined linearly with increasing age ( = -0.39 ± 0.19, z = -2.01, P = 0.04; Table 4.1).

Average relatedness, density and grid were not significant predictors in any of the models (all |z| < 1.47, all P > 0.14; Table 4.1). The random grouping term of ‘spatial ID’ was significant in all male ARS models, indicating that there were some areas of grid with substantially higher or lower male ARS (all variance > 0.36, all 2 > 4.49, all P < 0.04; Table 4.2).

Female ARS We analyzed 982 records of 451 breeding females between 1994 and 2015. Over this period, average familiarity ranged between 0 and 892 days (mean = 212 ± 6 days). Average relatedness ranged between 0 and 0.5 (mean = 0.07 ± 0.002) and density ranged between 0.53 and 4.31 squirrels/hectare (mean = 1.98 ± 0.03 squirrels/hectare). In the full model, there was no effect of familiarity on number of pups recruited ( = 0.06 ± 0.05, z = 1.38, P =0.17; Fig. 4.1). There was no linear effect of age ( = 0.08 ± 0.05, z = 1.44, P =0.15) but there was a negative quadratic effect of age on ARS ( = -0.10 ± 0.04, z = -2.72, P =0.006), and a negative linear effect of density ( = -0.37 ± 0.13, z = -2.77, P =0.006; Table 4.1).

Predictors of female ARS were also dependent on life stage. In the pre-senescent model (≤ 3 years old; N = 714 records over 438 individuals) there was no effect of familiarity ( = 0.01 ± 0.05, z = 0.28, P = 0.78) or age ( = 0.06 ± 0.05, z = 1.13, P =0.26), but density had a significant negative effect on female ARS ( = -0.48 ± 0.14, z = -3.54, P <0.001; Table 4.1). In the senescent period (≥ 4 years old; N = 268 over 170 individuals), familiarity with neighbours more than tripled the predicted number of pups recruited: when familiarity with neighbours was 76

lowest, females were predicted to recruit 0.5 pups annually, compared to the 1.8 pups recruited by females when familiarity with neighbours was highest ( = 0.23 ± 0.09, z = 2.52, P =0.01; Fig. 4.2). Number of pups recruited declined linearly with increasing age ( = -0.29 ± 0.10, z = - 2.79, P =0.005) and density also had negative effect on annual recruitment ( = -0.38 ± 0.17, z = -2.20, P =0.03; Table 4.1).

Average relatedness and grid were not significant predictors of female ARS in any of the models (all |z| < 1.86, all P > 0.06; Table 4.1). The random grouping term of ‘spatial ID’ was not significant in any of the models, indicating that there were was no substantial spatial variation in female ARS at the scale of 150 m2 (all variance < 0.11, all 2 <1.32, all P > 0.24; Table 4.2).

Discussion

Stable social relationships among group-living species are known to play an important role in mediating intragroup conflict and enhancing fitness (Cameron et al., 2009; Silk, 2003; Vander Wal et al., 2015). For territorial species, maintaining territorial boundaries can be a costly endeavor and long-term associations among neighbours may serve an equally important role in helping to alleviate the negative effects of competition between neighbours (Fisher, 1954; Temeles, 1994). Here we used 21 years of data from a population of territorial North American red squirrels to test the direct fitness effects of long-term familiarity with neighbouring conspecifics. We tested for these effects while controlling for kinship to help elucidate the importance of mutualistic interactions relative to inclusive fitness benefits. Our findings demonstrate that stable social relationships with neighbouring competitors has substantial benefits for survival and reproductive success in ‘asocial’ red squirrels, while relatedness with neighbours does not. In particular, while familiarity was not responsible for early life increases in reproductive success, familiarity was an important predictor of reproductive success and survival for both males and females in the senescent period (Fig. 4.2).

While the benefits of familiarity of territorial neighbours have long been posited (i.e. the ‘dear-enemy’ phenomenon; Fisher, 1954; Temeles, 1994) our study is one of few to empirically

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demonstrate direct fitness consequences of familiarity. Important benefits for reproductive success have also been found in red-winged blackbirds (Beletsky & Orians, 1989) and great tits (Grabowska-Zhang, Wilkin, & Sheldon, 2012b). While findings have been similar across these studies, the measures of familiarity used to assess these effects have been quite different. For instance, some studies have used the presence (or lack) of a single familiar neighbour to assess affects on fitness (Beletsky & Orians, 1989), while in other cases the familiarity status of the nearest neighbour or the total number of familiar neighbours has been used (Grabowska-Zhang, Wilkin, & Sheldon, 2012b). In red squirrels, average familiarity of the social neighbourhood has also been demonstrated to affect intrusion risk (Siracusa, Boutin, et al., 2017a) and behavioural plasticity (Siracusa et al. in review), providing good evidence to suggest that this is a relevant measure of familiarity in this study system. However, such variation in methodology raises interesting questions about what measure of familiarity is most salient, and whether this measure might vary among study species or even among fitness components. Further investigation into this question might shed light on the underlying mechanisms driving these effects of familiarity.

Cofounding Effects of Age/Implications for Senescence Previous work in this study system has demonstrated that red squirrels show evidence of both reproductive and actuarial senescence (Descamps et al., 2008; McAdam et al., 2007). To account for this, we included age, or a quadratic term for age, as a fixed effect in all of our models. However, a challenge associated with this is that age and familiarity are strongly correlated early in life. Young squirrels are inherently unfamiliar with their neighbours and familiarity typically increases with age, making it difficult to statistically tease apart the independent effects of these covariates. Evidence from our full models indicated that the effects of familiarity on survival and male ARS were robust to the inclusion of age. Increased familiarity with neighbours increased survival by up to 10% for both males and females and resulted in a 9-fold increase in number of pups sired (Fig. 4.1). We did not, however, find an effect of familiarity on female ARS in the full models. To help account for the potential confounding correlation between age and familiarity, and also to better understand how familiarity was contributing to fitness, we split our analysis into a pre-senescent (≤ 3 years old) and a senescent (≥ 4 years old) period.

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These partial models revealed the familiarity was not an important driver of early life reproductive success or survival, however neighbourhood familiarity had a substantial benefit for all fitness measures in the senescent period. For squirrels aged 4 and older, relative to living in neighbourhoods with low familiarity, living in neighbourhoods with high familiarity increased the probability of annual survival by 40%, increased the number of pups sired 6-fold, and more than tripled the number of pups recruited (Fig. 4.2). Importantly, the magnitudes of these effects of familiarity in the senescent period were equal to or greater than the effects of age (Table 4.1). While the decline in reproductive output and survival with age is a common phenomenon in many vertebrate species (Gaillard, Loison, Festa-Bianchet, Yoccoz, & Solberg, 2003; Reznick, Ghalambor, & Nunney, 2002), finding effects of familiarity that are similar in magnitude (and opposite in direction) to these well-established effects of age is somewhat astonishing given the lack of attention that such social relationships have been given. These results suggest that there is potential for familiarity to play an important role in buffering red squirrels against the effects of age-related fitness declines. By reducing the decline in reproductive success and survival in later life, stable social relationships may help to intensify selection against senescence. Therefore, if variation in the extent of established social relationships exists across populations (e.g. due to differences in demography) this may have implications for the evolution of senescence.

Mutualistic vs. Kinship Benefits Studies that have examined the benefits of social bonds have often been unable to differentiate between the evolutionary importance of mutualistic pathways relative to kin selection in reducing conflict and enhancing fitness (c.f. Bebbington et al., 2017; Cameron et al., 2009). In this study we had a unique opportunity to use a multigenerational pedigree to control for effects of kinship while testing our hypothesis that long-term social relationships would have direct fitness benefits for red squirrels. Our results provided substantial evidence that familiarity with territorial neighbours increased reproductive success and survival in red squirrels while average relatedness of the social neighbourhood had no effect on these fitness measures. Although we do not have direct evidence for the mechanism by which familiarity with neighbouring conspecifics might lead to increased reproductive success, previous work in this this study system has shown that increased familiarity with neighbours results in reduced intrusion risk (Siracusa, Boutin, et 79

al., 2017a) as well as reduced time spent on territory defence and increased time spent in nest (Siracusa et al. in review). Conceivably then, these direct fitness benefits may result from cooperative agreements of non-intrusion among long-term neighbours, allowing individuals to minimize aggressive interactions and spend more time and energy on growth and reproduction.

These findings suggest that mutualistic pathways may facilitate reduced conflict among conspecifics, with resulting fitness benefits, even in the absence of inclusive fitness benefits. Similar benefits of non-kin association have been demonstrated in female feral horses that live in long-term social groups with unrelated members. Increased social integration (measured through allogrooming and spatial relationships) among unrelated females was found to lead to increased birth rates and foal survival (Cameron et al., 2009). In Seychelles warblers (Acrocephalus sechellensis), the importance of social familiarity was also prevalent but shown to depend in complex ways on other aspects of the social environment such as kinship and density. For example, high numbers of new neighbours were associated with increased telomere shortening, but only when those neighbours were also unrelated. Relatedness among neighbours therefore mitigated the effects of unfamiliarity on somatic condition (Bebbington et al., 2017). While we looked for interactions between relatedness and familiarity in our models we found no evidence to support the possibility that relatedness might moderate the consequences of living with unfamiliar neighbours on survival and reproductive success (results not shown).

Spatial Confounds An alternative explanation for our findings is that spatial variation in resource availability or predation risk might instead affect probability of reproductive success or survival. In particular, areas with low survival would have high turnover and hence low average familiarity locally. If this kind of spatial autocorrelation in survival were present, low neighbourhood familiarity might be linked to low survival in the absence of a causal relationship. While this is a relevant concern that warrants consideration, there are several reasons why we do not think this is likely to be the case. First, there is no evidence of consistent spatial variation in spruce cone production (the primary food source for red squirrels; Boutin et al., 2006; Fletcher et al., 2010) across years in this study system (LaMontagne et al., 2013). Therefore, there shouldn’t be locations that are 80

consistently rich or poor in food resources. Second, all effects of familiarity we observed were robust to the inclusion of a spatial random effect at the scale of a 150 m2 (‘spatial ID’; Table 4.1). The spatial random effect was only significant in the male ARS models (Table 4.2). Given that we would expect spatial variation in resource availability or predation risk to affect males and females similarly, this result would suggest that some additional spatial factor, unique to males, might be at play. It is possible that if there are spatial clusters of females, males that are located near those clusters are particularly successful at breeding with multiple females and thus siring many pups.

Conclusion Together with previous findings (Bebbington et al., 2017; Beletsky & Orians, 1989; Grabowska- Zhang, Wilkin, & Sheldon, 2012b), our results provide growing evidence that, even in species whose primary social interactions are defined by competition for limited resources, stable social relationships with neighbouring conspecifics can have direct fitness benefits. The strength of these effects was particularly surprising, in some models familiarity increased annual survival by up to 40%, and more than tripled annual reproductive success, suggesting that stable social relationships may be underappreciated selective pressure in solitary, territorial species. Given the importance of maintaining long-term relationships with conspecifics, it would be interesting to investigate whether squirrels might play an active role in helping to ensure the survival of familiar neighbours. The idea that territorial neighbours might engage in cooperative behaviours to prevent neighbourhood turnover was originally postulated by Getty (1987). Since then, evidence of territorial neighbours forming defensive coalitions in response to both conspecific (Detto et al., 2010; Elfstrom, 1997) and heterospecific intruders (Grabowska-Zhang, Sheldon, & Hinde, 2012a) has been demonstrated in several species. Although it is important to remember that red squirrels defend exclusive territories and the interactions among neighbours are largely agonistic, our results suggest that, for a squirrel, the competitor you know is better than the competitor you don’t. The possibility that squirrels might help ensure the survival of long-term neighbours or engage in cooperative defence to prevent usurpers from taking over a neighbouring midden seems plausible given that the benefits of maintaining familiar neighbours might well outweigh the costs of such cooperative behaviours. 81

The relative role of mutualistic interactions versus kin selection in resolving conflict among conspecifics remains one of the enigmas of evolutionary biology. Our results indicate that intraspecific conflict can be mitigated through mutualistic pathways, with resulting fitness benefits, in the absence of kinship effects (see also Bebbington et al., 2017; Cameron et al., 2009). Inclusive fitness benefits, therefore, are not a necessary prerequisite to facilitate cooperation between social partners. Consequently, stable social relationships among neighbours are an important and often overlooked contributor to reproductive success and survival in territorial species, and may have implications for the evolution of social systems.

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Tables

Table 4.1. Fixed effects from annual survival, male ARS, and female ARS generalized linear mixed-effects models. Models based on the full, pre-senescent (≤ 3 years old), and senescent (≥ 4 years old) datasets are shown with significant effects indicated in bold. Regression coefficients are standardized.

Data set N Model Fixed effect Parameter ± SE z P Full data 2346 Survival Familiarity 0.18 ± 0.06 2.78 0.005 Age -0.27 ± 0.09 -2.94 0.003 Age2 -0.11 ± 0.04 -2.57 0.01 Relatedness 0.008 ± 0.05 0.15 0.88 Density 0.10 ± 0.11 0.92 0.36 Grid-SU 0.17 ± 0.10 1.67 0.10 412 Male ARS Familiarity 0.18 ± 0.09 2.04 0.04 Age 0.68 ± 0.14 4.75 <0.001 Age2 -0.41 ± 0.08 -5.32 <0.001 Relatedness -0.11 ± 0.08 -1.47 0.14 Density 0.04 ± 0.12 0.37 0.71 Grid-SU -0.07 ± 0.19 -0.38 0.70 982 Female ARS Familiarity 0.06 ± 0.05 1.38 0.17 Age 0.08 ± 0.05 1.44 0.15 Age2 -0.10 ± 0.04 -2.72 0.006 Relatedness 0.02 ± 0.04 0.63 0.53 Density -0.37 ± 0.13 -2.77 0.006 Grid-SU -0.06 ± 0.08 -0.79 0.43 Pre-senescent data 1834 Survival Familiarity 0.04 ± 0.07 0.50 0.62 Age 0.01 ± 0.07 0.16 0.87 Relatedness -0.01 ± 0.06 -0.20 0.84 Density 0.11 ± 0.11 0.94 0.35 Grid-SU 0.16 ± 0.12 1.40 0.16 334 Male ARS Familiarity 0.20 ± 0.10 1.95 0.05 Age 0.50 ± 0.11 4.58 <0.001 Relatedness -0.13 ± 0.09 -1.46 0.14 Density 0.004 ± 0.14 0.03 0.98 Grid-SU -0.19 ± 0.22 -0.89 0.37 714 Female ARS Familiarity 0.01 ± 0.05 0.28 0.78 Age 0.06 ± 0.05 1.13 0.26 Relatedness 0.03 ± 0.05 0.65 0.52 Density -0.48 ± 0.14 -3.54 <0.001 Grid-SU -0.01 ± 0.09 -0.15 0.88 83

Senescent data 512 Survival Familiarity 0.45 ± 0.11 3.40 <0.001 Age -0.36 ± 0.10 -3.42 <0.001 Relatedness 0.13 ± 0.11 1.20 0.23 Density 0.15 ± 0.11 1.33 0.18 Grid-SU 0.14 ± 0.21 0.68 0.49 78 Male ARS Familiarity 0.39 ± 0.15 2.67 0.008 Age -0.39 ± 0.19 -2.01 0.04 Relatedness 0.05 ± 0.14 0.37 0.72 Density 0.11 ± 0.19 0.60 0.55 Grid-SU 0.26 ± 0.32 0.82 0.41 268 Female ARS Familiarity 0.23 ± 0.09 2.52 0.01 Age -0.28 ± 0.10 -2.79 0.005 Relatedness 0.06 ± 0.08 0.79 0.43 Density -0.38 ± 0.17 -2.20 0.03 Grid-SU -0.32 ± 0.17 -1.85 0.07

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Table 4.2. Random effects from all generalized linear mixed-effects models. Significance assessed using a log-likelihood ratio test (LRT) with one degree of freedom to compare models with and without the listed random effect. Significant effects are indicated in bold.

Data set Model Random effect Variance 2 df P Full data Survival Squirrel ID 0.007 0.002 1 0.96 Year 0.18 29.00 1 <0.001 Spatial ID 0.07 0.55 1 0.46 Male ARS Squirrel ID 0.47 35.60 1 <0.001 Year 0.08 6.19 1 0.01 Spatial ID 0.44 39.28 1 <0.001 Female ARS Squirrel ID 0.03 0.69 1 0.40 Year 0.35 128.20 1 <0.001 Spatial ID 0.01 0.08 1 0.78 Pre-senescent data Survival Squirrel ID <0.01 <0.01 1 >0.999 Year 0.20 21.71 1 <0.001 Spatial ID 0.02 0.04 1 0.85 Male ARS Squirrel ID 0.35 32.68 1 <0.001 Year 0.14 8.10 1 0.004 Spatial ID 0.59 17.12 1 <0.001 Female ARS Squirrel ID <0.01 <0.01 1 >0.999 Year 0.31 89.64 1 <0.001 Spatial ID 0.01 0.04 1 0.84 Senescent data Survival Squirrel ID <0.01 <0.01 1 >0.999 Year 0.06 0.60 1 0.44 Spatial ID 0.27 1.24 1 0.26 Male ARS Squirrel ID 0.45 5.96 1 0.01 Year <0.01 <0.01 1 >0.999 Spatial ID 0.37 4.50 1 0.03 Female ARS Squirrel ID <0.01 <0.01 1 >0.999 Year 0.30 18.84 1 <0.001 Spatial ID 0.10 1.31 1 0.25

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Figure 4.2. Results from senescent models (≥ 4 years old) showing effects of average neighbourhood familiarity on annual survival (N = 512), male ARS (number of pups sired; N = 78), and female ARS (number of pups recruited; N = 268). Shaded grey bars indicate 95% confidence intervals. Values on x-axis are standardized measures of average familiarity. Points indicate raw data with a small amount of jitter introduced to show overlapping points.

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Epilogue

Prior to now, the idea that the social environment might contribute importantly to the behaviour and fitness of solitary species has been underappreciated, even though ‘asocial’ species, like their social counterparts, must manage relationships with neighbouring individuals while still allocating adequate time and energy to reproduction. By combining longitudinal data from a long-term study of red squirrels in the Yukon with observations and experiments in the field, I found clear evidence that the social environment affects intrusion risk and behavioural time budgets in a territorial species and also has direct fitness consequences. More specifically, the behaviour and fitness of red squirrels was affected by familiarity, rather than by relatedness with neighbours. Long-term relationships with neighbouring conspecifics led to reduced intrusion risk, allowing individuals to spend less time on territory defence and ultimately enhancing survival and annual reproductive success. These results indicate that, even among species whose interactions are fundamentally competitive, long-term social relationships can provide important benefits, suggesting a potentially important role for mutualistic interactions among unrelated individuals in the evolution of social systems.

Given that kin selection is widely accepted as a ubiquitous phenomenon facilitating cooperation among conspecifics, it is somewhat surprising that we didn’t find evidence that inclusive fitness benefits were an important mechanism mitigating conflict among red squirrel neighbours. In other territorial squirrels (Urocitellus columbianus) clustering with kin was found to reduce the costs of aggression with resulting fitness benefits (Viblanc et al., 2016). Although red squirrels are known to be able to differentiate between kin and non-kin (Gorrell et al., 2010; Wilson et al., 2015), the role of kin in territorial interactions remains ambiguous. In Chapter 1, I found that related neighbours were more likely to intrude on territory owners (Siracusa, Boutin, et al., 2017a). One potential explanation for this is that red squirrels tolerate intrusions by relatives due to associated indirect fitness benefits of allowing kin to share food resources. However, in Chapter 4 I did not find evidence that squirrels in neighbourhoods with high average relatedness had increased reproductive success or survival, as might be expected if kin provided increased access to food. Unlike Columbian ground squirrels, where there is clear philopatry

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(Viblanc et al., 2016), the average neighbourhood relatedness for red squirrels is quite low, typically averaging around 0.05 (see Chapter 4). Although red squirrel mothers occasionally bequeath a full or partial midden to offspring (Price, 1992; Price & Boutin, 1993), rates of bequeathal are typically low (Berteaux & Boutin, 2000). It may be that dispersal in this population is such that relatedness among red squirrel neighbours is never high enough for inclusive fitness benefits to play a substantial role in moderating territorial interactions and mitigating conflict. Alternatively, if the primary benefits of kinship come from sharing food resources (as implicated in Chapter 1), relatedness of nearest neighbours may be more relevant measure to assess whether red squirrels obtain social benefits from kin.

These considerations raise a more general and important point about the approaches that I have used in this thesis to measure the effects of the social environment. While I have provided good evidence to suggest that the average familiarity, measured at the scale of 130 m, is an appropriate and relevant way of measuring the effects of the social environment for red squirrels, this is only one of several possible ways of assessing the importance of social relationships for a territorial species. Other studies have looked for effects of familiarity by noting the presence or absence of a single familiar individual in the social environment (Beletsky & Orians 1989), counting the number of familiar individuals (Grabowska-Zhang, Wilkin, & Sheldon, 2012b), or assessing the familiarity of the nearest neighbour (Grabowska-Zhang, Wilkin, & Sheldon, 2012b). By focusing in on the social environment at the scale of 130 m it is possible that my thesis has failed to capture other meaningful patterns of social interaction which may occur at more localized scales. For example, as alluded to above, my findings do not mean that neighbour relatedness has no biologically meaningful effects on red squirrel behaviour or fitness, but simply that the scale at which I attempted to measure these effects did not detect any influence of kinship. Future work should attempt to use other measures of the social environment by, for example, explicitly mapping territory boundaries and assessing the relatedness and familiarity of nearest neighbours. Voronoi diagrams may also prove a useful tool for creating nearest- neighbour polygons where information on territory boundaries is not available. Exploring alternative approaches for measuring the social environment might offer additional insight as to the relative importance of kin-selection in this system and shed light on the underlying 89

mechanisms driving the effects of familiarity.

Regardless, the results from my thesis provide exciting evidence to support the idea that inclusive fitness benefits are not a necessary prerequisite to facilitate cooperation. Instead, long- term social relationships can lead to the formation of a cooperative agreement of non-intrusion among red squirrel neighbours, reducing time spent on territory defence and increasing fitness. A question that remains to be answered is the nature of the functional mechanisms that might maintain cooperation between unrelated individuals. Given that red squirrels engage in reoccurring interactions over the course of their lifetime, and familiar neighbours appear to pay a short-term cost by refraining from intruding (i.e. relinquishing possible benefits of territorial expansion or increased access to food stores), interactions among territory holders can be thought of as a form of iterated Prisoner’s Dilemma (Axelrod & Hamilton, 1981). Future research should therefore explore whether conditional reciprocity might play an important role in stabilizing cooperative relationships in this species. Specifically, non-intrusion among familiar neighbours might be reinforced if territory owners punish intruders by increasing aggression and thus forcing the defecting individual to allocate increased time and energy to territory defence.

While density-dependent fecundity and survival are well-recognized and well-supported mechanisms of population regulation (Danchin et al., 2008), the direct benefits of neighbour familiarity on reproductive success and survival suggests that the composition of the social environment, in addition to the quantity of interacting conspecifics, has potential to affect population level outcomes. In particular, periods of high density are likely to be coupled with low familiarity because of the influx of new individuals into the population. This means that high density and low familiarity might be contributing synergistically to limiting reproductive success and survival. If this is the case, then models that fail to account for the effects of familiarity might actually be over estimating the effects of density. Where possible, quantifying the effects of both density and familiarity on reproductive success and survival would help elucidate the relative importance of these different social mechanisms for regulating populations.

Although red squirrels do not interact in clearly defined groups, my thesis has provided 90

substantial evidence to suggest that apparently solitary species can interact in biologically relevant units (e.g. ‘acoustic neighbourhoods’) the composition of which can have important consequences for behaviour and fitness. Understanding how group composition affects selection acting on individual phenotypes and the resulting evolutionary implications has been a point of considerable interest in recent years (Farine et al., 2015). My research suggests that our investigation of the importance of group composition should not only encompass social species or organisms that act in discreet groups but should also extend to solitary species where interactions between individuals may still be localized. The idea that non-discrete units can be the basis for selection is not new (Nunney, 1985), and recent work in this system has provided evidence of selection on red squirrel growth rate and parturition date at the level of the acoustic social neighbourhood (i.e. being born earlier and growing faster than nearby litters is important for juvenile survival; Fisher et al., 2017).

By recognizing that solitary organisms can still interact in localized social groups, we can begin to appreciate that the social environment may be a potentially important selective pressure in ‘asocial’ species that has previously been mostly unacknowledged. Future research should investigate both the selective consequences and the evolutionary implications of the social environment for solitary, territorial species. Selection for aggression (Yoon et al., 2012), growth rate (McAdam & Boutin, 2003), or personality traits (Dingemanse, Both, Drent, & Tinbergen, 2004) that has been shown to be density dependent might also depend on the composition of the social environment. For example, in fruit flies (Drosophila melanogaster) selection for aggression has been shown to depend not only on the density of the social environment but also the relative frequency of aggressive phenotypes (Kilgour, McAdam, Betini, & Norris, 2018). Social heterogeneity might therefore impact how selection acts on individuals and contribute to the maintenance of phenotypic variation. Additionally, it is increasingly recognized that the genes of interacting partners can affect the phenotype of the focal individual (i.e. indirect genetic effects; Moore et al., 1997). Characteristics of interacting conspecifics may therefore influence evolutionary responses to selection, either by accelerating evolution or changing the evolutionary trajectory in a direction opposite to that favoured by selection. For example, in red squirrels, competition with neighbouring conspecifics has been shown to slow down the expected response 91

to selection for earlier parturition dates under high-density conditions (Fisher et al. unpublished). However, this study represents one of few studies of the evolutionary implications of social interactions in a wild, solitary species, emphasizing the need for further research. Understanding the capacity of wild organisms to respond to a changing environment is becoming an increasingly important issue. If social interactions can play a substantial role in limiting the evolutionary potential of populations it is important that we begin to take these interactions into account in order to make informed conservation and management decisions.

Ultimately, understanding the selective consequences of the social environment might also advance our understanding of the evolution of territorial systems. For instance, the benefits of familiar neighbours might help to explain why individuals of many migratory species return to the same territories in consecutive years (Austin, 1949; Beletsky & Orians, 1987) or why individuals of other more sedentary species maintain relatively stable territories and home ranges over their lifetimes (Greenwood, 1980; Harvey, Greenwood, & Perrins, 1979; Larsen & Boutin, 1995). The benefits of stable social relationships might mean that bequeathal behaviours are particularly costly for territorial species since mothers are not only giving up access to current food resources but must renegotiate social relationships, and might help to explain why such behaviours are relatively rare (Berteaux & Boutin, 2000). Additionally, in red squirrels, mothers were found to be more likely to disperse when food levels in their midden were low but current spruce cone production was high (Berteaux & Boutin, 2000). While this suggests that squirrels minimize costs with respect to food resources when deciding to bequeath, it might also indicate that squirrels minimize costs with respect to the social environment. During years of high cone production (i.e. ‘mast years’; LaMontagne & Boutin, 2007) there is typically high recruitment of juveniles (McAdam & Boutin, 2003), leading to the settlement of many new neighbours and reduced average neighbourhood familiarity as a result (unpublished data). Therefore, in such years, females may face no substantially greater social cost by bequeathing their current territory. Finally, I’ve demonstrated in this thesis that the social environment can undermine the stability of territorial systems by decreasing resources and increasing time spent on defence. The social environment might, therefore, have implications for when territoriality will evolve (i.e. when the benefits of exclusive access to a resource outweigh the costs of defence; Brown, 1964; Brown & 92

Orians, 1970) or what shape territorial systems might take. As discussed briefly in Chapter 1, in systems where stable social relationships with neighbours are never established it may be uneconomical to defend a larder hoard of resources due to the high rates of cache pilferage.

Final Remarks Although red squirrels rarely come into physical contact with one another, there is mounting evidence to suggest that squirrels none-the-less interact with each other in important ways that have behavioural, ecological, and evolutionary consequences (Fisher et al., 2017; Siracusa, Boutin, et al., 2017a; Siracusa, Morandini, et al., 2017b). An important take-home message from this work is that competition and cooperation are two sides of the same coin that operate in tension with, but not in exclusion of, one another. Although interactions among individuals in territorial species are fundamentally defined by competitive interactions for food, mates, or other resources, I have shown that social relationships can limit these competitive interactions and provide important benefits that might feed back to maintain strong affiliative relationships among territorial neighbours. Ultimately, territorial organisms do not exist in isolation and the implications of the social environment for apparently ‘asocial’ species should be more widely studied and understood. We are currently in an exciting time where the advent of new technologies (e.g. proximity collars) and statistical techniques (e.g. social network analysis) are moving us beyond ‘density’ as the sole metric of the social environment and providing us with a richer understanding of how organisms interact with one another and how these interactions shape individual fitness as well as the collective outcomes of groups. Future research should look ahead to how we can apply these techniques to better understanding competitive and cooperative dynamics in solitary, territorial animals.

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Supplementary Material: Chapter 1

Table S1.1. Cox proportional hazard mixed effects models and AIC results predicting risk of intrusion based on individual squirrel characteristics from first intruders only.*

Model K Log AIC i wi Rank likelihood Global model 5 -370.26 746.57 0.00 0.98 1 ~ dis + r + fam Distance model 3 -378.38 758.80 12.23 2.20e-03 2 ~ dis Relatedness model 3 -410.28 822.57 76.00 3.13e-17 3 ~ r Familiarity model 3 -422.60 847.22 100.65 1.39e-22 4 ~ fam Intercept model 2 -425.78 851.57 105.00 1.58e-23 5 ~ 1 Number of parameters (model complexity) is represented by K. Models were evaluated based on differences between AIC scores (i) and AIC weights (wi). i is the difference between the observed model (i) and the best model as determined by the lowest AIC. Model rank is based on differences among AIC scores.

*The significance of the random intercept term for squirrel identity was assessed using the global 2 model (variance = 0.0004;  1 = 0.005, P = 0.94).

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Supplementary Material: Chapter 3

Table S3.1. Results from the audio recorder data showing effects of within-individual

(FamiliarityW) and among-individual (FamiliarityA) familiarity on rattling rate when including year as a fixed effect. The inclusion of year helps to account for structure in our data, however, we had no a priori hypothesis for why year itself might affect behavioural patterns. Our expectation was that changes in rattling rates would be driven by changes in density or familiarity between years. Given this, and the fact that year was correlated with familiarity (r = 0.34) and density (r = -0.43), we excluded year from our primary analysis. We have included year here to be transparent about its effects in the model.

Model Fixed effect Parameter +/- SE z P

Rattle rate FamiliarityW 0.02 ± 0.02 1.30 0.19 (GLMM) FamiliarityA 0.05 ± 0.04 1.17 0.24 Age -0.09 ± 0.04 -1.93 0.05 Density -0.09 ± 0.04 -2.25 0.02 Sex-M + -0.15 ± 0.10 -1.53 0.13 Grid-KL * -0.23 ± 0.08 -2.76 0.006 Grid-SU * -0.75 ± 0.12 -6.40 <0.001 Year-2016 -0.40 ± 0.08 -5.13 <0.001 + Female is taken as the reference * AG (food supplemented grid) is taken as the reference

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Figure S3.1. Distribution of number of rattles emitted per 10-min focal observation. Grey bars show raw data (N = 488 observations); black outline indicates a theoretical Poisson distribution simulated using (N = 500 data points). The single outlier where 9 rattles were recorded in a single focal observation was removed before data analysis, as it appears to be a data entry error.

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