THE CONSEQUENCES AND IMPLICATIONS OF ECOLOGY ON BODY SIZE DISTRIBUTIONS IN SOCIAL INSECTS

BY

BILL DOUGLAS WILLS

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology with a concentration in Ecology, Ethology, and Evolution in the Graduate College of the University of Illinois at Urbana-Champaign, 2013

Urbana, Illinois

Doctoral Committee:

Professor Carla Cáceres, Chair Associate Professor Andrew Suarez, Director of Research Professor Ken Paige Research Associate Samuel Beshers

ABSTRACT

The study of life history traits is central to the fields of ecology, behavior, and evolution. Life history theory explores investment into key biological characteristics that figure directly into the reproductive success and survival of an organism (e.g. size at birth, age at maturity, and size at maturity). One of the major tenets of life history theory is that finite resources must be allocated to the development of traits associated with growth, defense, and reproduction. Consequently, investment into life history traits are subject to tradeoffs between resources allocated to each demand, where resources devoted to one function can no longer be devoted to another (offspring size versus offspring number). Relative to solitary species, the study of life history traits in eusocial organisms is complicated by their reproductive division of labor. In social insects, reproduction is dominated by a queen (reproductive) caste while the majority of other tasks within the colony are performed by a worker (non-reproductive) caste, often made up of sterile individuals. The separation of reproductive and non-reproductive individuals within a colony can influence tradeoffs that constrain the evolution of life history traits in solitary organisms. For example, workers no longer invest in morphological traits required for dispersal, mating, and reproduction (with few exceptions). Additionally, in approximately 13% of ant species worker variation is large enough that the worker caste can be subdivided into sub-castes based on size and shape. For these polymorphic species, body size is both an individual and a colony-level trait (distribution of worker body sizes). Ants are typically described as most diverse and successful organisms in terrestrial ecosystems. One explanation for their ecological dominance and success is that ant species are social allowing workers to display a high degree of morphological and behavioral specialization. All of which make ants a useful system to study in order to answer a variety of questions in ecology, behavior, and evolution. Investment into worker body size and number is thought to play a major role in determining colony growth, maintenance, and reproductive output (fitness). The investment into worker body size is integral to colony fitness because it impacts most every aspect of its existence. For example, size affects worker behavior, metabolism, thermal tolerance, locomotion, longevity, and food retrieval/storage. Consequently, body size plays an important role in determining how organisms interact with the biotic and abiotic components of their environment and is often under strong selection. However, variation in size often remains pronounced within populations. In addition to its biological importance, size is also easy to measure and a common metric for a variety of research areas.

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Variation within and between colony investment into worker size and number may result as a response to local conditions and within evolutionary constraints. For example, within a single colony, body size and body size distributions of workers is determined by evolutionary history, genetics, the social environment within the colony, abiotic factors, nutrition, and competitive environment. Evolutionary constraints, genetics and social environment generally act “within the colony” to influence worker body size distributions. In contrast, ecological influences, which include the abiotic environment, nutrition, and competitive environment, act “outside the colony.” It is important to note that these factors do not work singly, and the interactions between factors are also important in determining body sizes within a colony. Moreover, these factors may simultaneously impact worker body size from both outside and within a colony. In the following chapters, I explore how external (ecological) factors influence body size and body size distributions in ants. I also examine how variation in worker size and number influence the outcomes of competitive interactions. I begin in Chapter One with a general review of research discussing the determinates of body size in ant species with polymorphic workers. Chapter One explores the literature examining how ecology influences body size distributions in polymorphic ant species and how variability within a colony influences measures of colony fitness. The within-colony factors influencing body size (evolutionary constraints, genetics, and social environment) have been reviewed recently, thus I keep this section brief. In the review, I discuss the intrinsic (“within a colony”) and extrinsic (“outside a colony”) determinants of intra-specific variation in ant body size. Additionally, I review the literature on how variation in worker body size can promote division of labor by increasing worker efficiency and specialization. The review focuses on species with polymorphic workers and addresses the following two questions: 1. What factors influence the distribution of worker body sizes within a colony? and 2. How does variation in body size benefit the colony? Despite considerable research in these areas, I find few studies that directly link body size variation or caste ratios to components of colony fitness. I conclude with recommendations for future work, aimed at addressing current limitations in this field. This includes a need for experimental studies that explicitly relate body size variation to fitness in social insects. In the subsequent chapters I use comparative and experimental approaches to examine the ecological factors influencing, and consequences of, variation in worker body size in polymorphic invasive ant species. Introduced species are ideal for examining the role of ecological variation on investment into worker size as they often encounter vastly different ecological conditions throughout their introduced ranges (e.g. fewer competitors and access to additional resources). These differences in ecology likely impact colony investment in growth, reproduction, and defense. Moreover, the success of invasive species is thought to occur as a result of changes to competitive environment or behavioral

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dominance. In this way, invasive species provide an opportunity to investigate novel species interactions that would otherwise be unethical to do experimentally if these species were intentionally introduced into new environments. In Chapter Two, I quantify the variation in size and shape of workers among geographically distinct populations of the dimorphic (two distinct worker castes) big-headed ant (Pheidole megacephala) (Fabricius). This species has been introduced nearly worldwide including areas with species rich, competitively dominant ant fauna (e.g. Australia) and to islands that have no native ants (e.g. Hawaii). I utilize this variation in ant community species richness to investigate how P. megacephala species varies investment into soldier (major) worker size, shape, and number, depending on the competitive nature of the environment. As in other dimorphic species in this genus, minor workers are behavioral “generalists” within a colony. Minor workers are significantly smaller than major workers and complete the bulk of tasks within a colony. Majors are considerably larger and more energetically costly to produce. Majors play a more specialized role in colony defense, food retrieval, and food storage. As outlined in Chapter One different ecological conditions (e.g. nutrition, food resources, abiotic environment, and competition) among populations is hypothesized to alter colony investment into worker body size. I also use genetic data to determine if the populations of P. megacephala represent cryptic species or if morphological differences are attributable to changes following introduction. I find significant variation in worker mass among five populations. Both major and minor workers were largest in Australia, whereas minor workers were smallest in Hawaii and Mauritius. I also find differences in major and minor worker morphology among populations. Majors from Mauritius have significantly larger heads (width and length) than those from Hawaii and Florida. The length of the head and tibia of minor workers are greater in South Africa compared to samples from Australia, Hawaii, and Florida. I did not find differences in caste ratios among populations. The molecular data place all samples within the same clade, supporting that these morphologically different populations represent the same species. These results suggest that the variation in shape and morphology of major and minor workers may result from a rapid adaptation or plastic response to local conditions. In Chapter Three, I examine how access to plant-based carbohydrate resources (nutrition) influences colony investment into worker body size distributions in the continuously polymorphic, red imported fire ant (Solenopsis invicta). As holometabolous insects, differences in reproductive investment and feeding prior to pupation have dramatic effects on adult ant worker body size. An increase in the total amount and quality of food resources available to a colony can increase worker size, body size distributions, and colony size. Large colonies are themselves more likely to produce larger workers, further influencing worker body size variation. Moreover, an increase in access to plant-based

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carbohydrate resources is suspected of playing an important role in determining invasion success of ant species as carbohydrate resources are more commonly monopolized within introduced ranges of invasive ants than in their native ranges. In this chapter, I use multiple queen (polygyne) colonies from populations in Texas, split into four treatment colonies, to experimentally test how nutrition influences colony investment into worker number, body size, body size distributions, and fat content (worker quality). Experimental colonies were reared on a diet of insect protein and one of four treatment solutions (water, amino acids, carbohydrates, and amino acid and carbohydrates). Overall, colonies with access to carbohydrates (which mimic plant based resources) produce a higher biomass of workers after 60 days. The differences in worker biomass are attributable to changes in both worker number and mean size. Worker number and size generally increases in response to carbohydrate supplementation to their diet but there is no difference in worker fat content among treatments. There is a slight shift in body size distributions (more, medium sized workers) in treatments reared on diets of carbohydrates than those denied access to carbohydrates. Invasive species with access to additional carbohydrate resources may increase colony investment into worker number and medium sized workers and subsequently influence the outcomes of competitive interactions (Chapter Four). Variation in investment into worker number and body size can influence the outcomes of competitive interactions. This includes resource discovery, food retrieval, and resource defense. There is growing interest in co-opting Lanchester’s laws, originally designed for human warfare, to predict the outcomes of aggressive interactions in ants. In Chapter Four, I use a foraging experiment and field surveys to test the predictions of Lanchester’s laws in interference competition with the continuously polymorphic, S. invicta. I also use the foraging experiment to explore how varying worker number and size influences exploitation competition. Lanchester’s square law predicts that if a group is numerically superior then attacks should occur simultaneously against their opponent and is likely most effective in open environments (or above ground). If a group is numerically inferior but possess weapons of superior “quality”, fighting in a series of one-on-one duels is more advantageous. This strategy is potentially most effective in closed and confined spaces (or below ground). I find that for colonies of equal biomass but different worker size (e.g. different worker number), colonies with smaller workers typically discover baits more quickly than those with larger workers but both large and small worker colonies retrieve equal amounts of food resources. Colonies with smaller workers do not suffer fewer losses when outnumbering their opponents than when equally matched. I however find that when colonies with large bodied workers compete with numerically dominant opponents they suffer a greater number of losses in open arenas than closed arenas (e.g. support for the square law). In closed arenas, colonies with relatively few but large workers use their size advantage in

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confined spaces to defend against the numerically superior but smaller workers (e.g. support for the linear law). In the field, I find more small workers above ground than below ground which supports the predictions of the linear law because in narrow confined spaces larger body size is advantageous. Lanchester’s laws provide a framework to build predictions for outcomes of competitive interactions and in the future may help identify how competition influences community structure and species composition Body size is one of the most important life history traits as it directly affects an organism’s existence. Understanding determinates of variation in ant worker body size is no trivial task. The complexity arises because of reproductive division of labor and the presence of distinct worker castes. The relative importance of factor determining worker body size within a colony varies within and among different ant species (and in some cases between worker castes). This may explain why the vast majority of research examining body size has focused on solitary organisms while relatively little work is done using social insects. Given the ecological dominance of ants in many environments however, failing to examine the factors influencing within and between colonies often limits current and future understanding of insect ecology. There is a substantial amount of information regarding the factors influencing body size in ants, but there is much left to explore.

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To my mother Joyce K. Wills who has always been my adviser, coach, and teacher

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ACKNOWLEDGEMENTS

I would like to thank my advisor Andrew Suarez for his guidance, support, and patience. His positive attitude and calm demeanor guided me through the whole PhD process and perhaps more importantly, through major life events. Without his patience, the process would not have been as successful, rewarding, or enjoyable. I would also like to thank my committee members, Carla Cáceres, Ken Paige, and Samuel Beshers for their helpful comments and insightful criticism. Their commitment to education, student enrichment, and developing better scientists lies well above mean. Please know all of your efforts are appreciated. It has been and continues to be a privilege to learn from and work with you. I could not have been completed this work without the help of undergraduate assistants and lab mates. They have dealt with my anxiety, fears, and stubbornness. I have had the pleasure of working with a number of excellent undergraduate students who include, but are not limited to, Amanda Robinson, Cody Chong, Catherine Choi, Stephanie Tham, Zachary Buddle, Adam Skrzekut, Newton Hood, and Jennifer Ferchau. Each of whom has been exceedingly helpful in completing a variety of my projects. A thank you to my former labmates: Chris Smith, Moni Berg-Binder, Sarah Kantarovich, Joe Sapagna, and Ryan Thum, who helped me find projects, tolerated a young graduate student, and developed protocols which I have used throughout my dissertation. Additionally, I owe many thanks to Jo-anne Holley, Alex Wild, Adrian Smith, Dietrich Gotzek, Andrea Walker, and Selena Ruzi for their comments and advice. I also owe a special thanks to Fred Larabee for his tireless effort in the field, as a co-pilot and sounding board for research ideas. He has always been there to help me out of the holes, both literal and figurative, in which I often found myself. I learned how to collect, maintain, and use fire ants in research with the guidance of Edward LeBrun from the University of Texas Brackenridge Field Laboratory. His generosity saved a novice graduate student from countless logistical problems. Without him I would still be struggling to decipher them. He guided me through Argentina and his methodologies have helped me throughout my graduate career. My time at Brackenridge helped me survive working with an angry model organism. The funding which made this work possible came from the Department of Animal Biology, School of Integrative Biology, U. of I. Graduate College, Illinois Natural History Survey, Center for Latin American & Caribbean Studies, and Sigma Xi. In addition, this work has also been paid for with grants to my adviser from the National Science Foundation. Finally, I would like to thank my family, whose love and support provide the foundation for my success. I would especially like to thank my mother who taught me unrelenting perseverance.

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TABLE OF CONTENTS

CHAPTER 1: DETERMINANTS AND CONSEQUENCES OF WORKER POLYMORPHISM IN ANTS.…………………………………………………………………………………...………………….1

CHAPTER 2: BODY SIZE VARIATION IN GEOGRAPHICALLY DISTINCT POPULATIONS OF THE INVASIVE ANT, PHEIDOLE MEGACEPHALA…………………………………………………..19

CHAPTER 3: INFLUENCE OF NUTRITION ON INVESTMENT IN WORKER NUMBER, SIZE, AND CONDITION IN A POLYMORPHIC SOCIAL INSECT ………………………………………....37

CHAPTER 4: TESTING THE PREDICTIONS OF LANCHESTER’S LAWS OF COMBAT WITH A CONTINUOUSLY POLYMORPHIC ANT: TRADEOFFS BETWEEN WORKER NUMBER AND SIZE ………………………………………………………………………………………………………53

CHAPTER 5: SUMMARY AND CONCLUSIONS ……………………………………………………..77

REFERENCES……………………………………………………………………………………….……81

APPENDIX: SUPPLEMENTARY FIGURE AND TABLES.....………………………………………...99

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CHAPTER 1: DETERMINANTS AND CONSEQUENCES OF WORKER POLYMORPHISM IN ANTS

Abstract Body size is a key life history trait that influences the way organisms interact with each other and with their environment. Eusocial organisms, such as ants, provide an interesting model for ecological and evolutionary examinations of body size variation because of their reproductive division of labor, and the existence of worker polymorphism making body size both an individual and a colony-level trait (e.g. distribution of body sizes within a colony). In this perspective, I discuss the intrinsic and extrinsic determinants of intraspecific variation in ant body size. Additionally I review the literature on how variation in worker body size can promote division of labor by increasing worker efficiency and specialization. This review focuses on species with a polymorphic worker caste and addresses the following two questions: 1. What factors influence the distribution of worker body sizes within a colony? and 2. How does variation in body size benefit the colony? Despite considerable research in these areas, I found few studies that directly link body size variation or caste ratios to components of colony fitness. I conclude with recommendations for future work, aimed at addressing current limitations in this field. This includes a need for experimental studies that explicitly relate body size variation to fitness in social insects.

Introduction Body size is one of the most important life history traits influencing the reproduction and survival of an organism (Stearns 1992, Brown 1995, Chown and Gaston 2010). An animal’s body size affects its metabolism, thermoregulation, locomotion, reproduction, longevity, and diet (Peters 1983, Schmidt- Nielsen 1984, Calder 1984, Losos 1990, Douglas and Mathews 1992, Foote 1997, Kaspari and Weiser 1999, 2007). Consequently, body size plays an important role in determining how organisms interact with the biotic and abiotic components of their environment (Brown 1995). Body size is often under strong selection (Larsson et al. 1998, Kingsolver and Pfennig 2007), yet variation in body size is often pronounced in populations. Variation in body size may result from a response to constraints and is subject to tradeoffs, as is the case with life history traits generally (Zera and Harshman 2001). For example, a reproducing organism may have to choose between investing into individual offspring size versus total offspring number (Smith and Fretwell 1974). For many organisms body size is relatively easy to measure allowing for quick and robust data sets (Lindsey 1966, Peters 1983, Brown 1995). The causes and consequences of intraspecific variation in

1 body size are commonly discussed in studies of life history traits (Kozlowski et al. 2002, Stearns 1992, Fox and Czesak 2000), ecological physiology (Lindstedt and Calder 1981, Blanckenhorn 2000, Chown and Gaston 2010), and macroecology (Brown 1995, Chown and Gaston 2010). Subsequently, body size and its associated tradeoffs have been the focus of biologists for decades with the majority of this work focused on solitary organisms (Peters 1983, Stearns 1992, Brown 1995). Eusocial organisms, however, are often missing from studies that examine how ecological factors influence intraspecific variation in traits (Bolnick et al. 2003, Forster et al. 2012), even though the study of body size variation in social insects also has a long history (Wilson 1953, Wilson 1971, Oster and Wilson 1978, Cushman et al. 1993, Bourke and Franks 1995, McGlynn 1999, Kaspari 2005, Gouws et al. 2011). Relative to solitary species, the study of life history traits in eusocial organisms is complicated by their reproductive division of labor; reproduction is dominated by a queen caste while the majority of other tasks within the colony are performed by the worker caste that is often made up of functionally sterile individuals. This separation of reproductive and non-reproductive individuals within a colony may influence tradeoffs thought to constrain the evolution of life history traits such as size. For example, in most ants workers no longer invest in morphological characters related to dispersal, mating and reproduction. Additionally, ants often have dramatic variation in worker body size within colonies (Figure 1.1). In approximately 13% of ant species (~16% of ant genera), the worker caste can be subdivided based on variation in size and shape (Wilson 1953, Oster and Wilson 1978, Hölldobler and Wilson 1990, Fjerdingstad and Crozier 2006). For species with a polymorphic worker caste, body size is both an individual trait (e.g. average worker size) and a colony-level trait (distribution of worker body sizes). Body size and variation in body size are both thought to be under strong selection and may be important features influencing a colony’s fitness through colony maintenance, survival, and reproduction (Oster and Wilson 1978, Hölldobler and Wilson 1990, Bourke and Franks 1995, Billick 2002, Whitehouse and Jaffé 1996, Powell 2009). Inter- and intraspecific variation in body size can also influence ant community dynamics through foraging behavior and prey selection (Traniello 1987, Traniello 1989, Wetterer 1994a, 1994b), by influencing competitive interactions (Davidson 1977, 1978), and by mediating ant-plant mutualisms (Ness et al. 2004, Chamberlain and Holland 2009). In this perspective, I discuss the intrinsic and extrinsic determinants of intraspecific variation in ants with polymorphic workers. It is important to note that I do not comprehensively summarize the literature on variation in mean individual body sizes in species with monomorphic workers, or on variation in colony size (e.g. the number of workers) across populations (e.g. Kaspari and Vargo 1995). The review specifically addresses the following two questions: 1) What factors influence the distribution of body sizes as a colony-level trait? and 2) How does variation in body size benefit the colony? Theory predicts body size variation can increase the efficiency of division of labor through role specialization

2 (Oster and Wilson 1978). Consequently, worker polymorphism should influence colony efficiency, survival, and reproductive output (e.g. components of fitness). I therefore also explore the relationship between variation in worker body size and components of colony fitness. Finally, I end with recommendations for future work aimed at addressing current limitations in this field and ultimately ask: Why is worker polymorphism relatively rare in ants?

Body Size and Ants Ants are among the most diverse and successful organisms in terrestrial ecosystems (Hölldobler and Wilson 1990, Folgarait 1998). Their success is driven, in part, by a sophisticated division of labor (Hölldobler and Wilson 1990). Notably, nearly all ants have discrete reproductive (queen) and non- reproductive (worker) castes with remarkable morphological variation between castes (Hölldobler and Wilson 1990, Whitehouse and Jaffé 1996, Fjerdingstad and Crozier 2006). Ants also often have pronounced morphological variation within castes (Wilson 1953, Oster and Wilson 1978) adding complexity to discussions of body size because body size is both an individual trait and a colony-level trait. As holometabolous insects, ants are good models for examining tradeoffs in worker size and number because differences in parental investment and feeding prior to transition from larva to pupae results in dramatic effects on adult body size (Nijhout and Williams 1974, Wheeler 1991, Moczek 1998, Bezemer et al. 2005). The amount of variation observed in worker body size can broadly be categorized as monomorphic and polymorphic (Figure 1.1). Monomorphic workers are those that display isometric morphological variation, limited body size variation, or both (Wilson 1953, Oster and Wilson 1978). Polymorphic workers display a non-isometric increase in size occurring over a sufficient range of adult size variation to produce individuals of distinctly different proportions (Wilson 1953, Oster and Wilson 1978). Worker polymorphism can be further broken down in to subcategories (monophasic, diphasic, triphasic, and tetraphasic allometries) (Figure 1.1). Finally, some genera of ants (e.g. Pheidole) contain species that are completely dimorphic where colonies have two distinct worker body sizes (majors and minors) with no intermediately sized workers (Wilson 1958, Oster and Wilson 1978). In ants, a queen’s investment into offspring size or number is hypothesized to be important in determining the efficiency of a colony, and therefore colony success (Oster and Wilson 1978, Dornhaus et al. 2012). In species with distinct morphological castes in workers, one caste is often specialized for food retrieval, food processing, and/or colony defense (Hölldobler and Wilson 1990, Stapley 1999, Wilson 2003, Powell and Franks 2005, Powell 2008). Therefore, investment into these castes should influence colony survival, competitive ability, and reproduction (Detrain and Pasteels 1992, Beshers and Traniello 1994, Whitehouse and Jaffé 1996, Powell 2009).

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Intrinsic Determinates of Body Size In this section, I highlight a few intrinsic factors influencing body size within a colony as a general overview. I use the term intrinsic to describe largely proximate or mechanistic factors that influence body size “within the colony” including the role of genetics and development. In contrast, I use extrinsic to describe ecological influences on body size variation based on interactions between a colony and its environment. Some factors such as the role of nutrition and the social environment may be the result of a feedback between both intrinsic and extrinsic factors so I discuss these separately. It is important to note that the factors do not work singly and the interactions between factors are important in determining body sizes within a colony (Figure 1.2). As much of the research in this area has been reviewed recently (e.g. Anderson et al. 2008, Smith et al. 2008, Heinze et al. 2008, Schwander et al. 2010), I keep this section intentionally brief so I can focus the review on the ecological factors that may influence body size variation.

Genetic Factors In most ants, castes are determined by environmental cues, specifically by the amount and type of nutrition received during larval development (Wheeler 1986, 1991). However, recent studies on hybrid lineages in both Pogonomyrmex and Solenopsis, and a number of widely introduced “tramp” ant species (including Vollenhovia emeryi, Wasmannia auropunctata, Paratrachina longicornis and Anoplolepis gracilipes) have shown that caste determination can be largely, if not entirely, genetically based (Heinze 2008, Schwander et al. 2010, Pearcy et al. 2011). There is also growing evidence that a genetic influence on body size can result from patriline identity in species with multiply mated queens. For example, research on Pogonomyrmex (Rheindt et al. 2005, Smith et al. 2008), Acromrymex (Hughes et al. 2003, Hughes and Boomsma 2007), and Eciton (Jaffe et al. 2007) have found evidence that worker body size may be influenced by the genotype of the father. In multiple queen (polygyne) colonies different reproductive queens may also produce different sized workers (Schwander et al. 2005). Genotype may therefore influence a developing ant larva’s response to environmental stimuli, as observed in solitary organisms (Emlen and Nijhout 2000, Nijhout 2003), resulting in a genetic influence on size (but see Wiernasz and Cole 2010). Even in the absence of genetic caste determination, genetic factors (including maternal and other indirect genetic effects) can affect a larva’s response to environmental stimuli and therefore influence the development of body size variation within a colony (Hughes et al. 2003, Linksvayer and Wade 2005, Rheindt et al. 2005, Anderson et al. 2008). For example, larval begging behavior can influence the amount of food obtained from workers and theoretically will impact size and even caste at development

4 (Creemers et al. 2003, Kaptein et al. 2005, Anderson et al. 2008). The influence of genetics on body size through larval and worker behaviors may be pervasive (Andersen et al. 2008) although there is little experimental work in this area (but see Linksvayer et al. 2011 for an example with honey bees). Moreover, the relative contribution of genetic factors on worker body size is likely to vary depending on species (Schwander et al. 2010) and caste (reproductive, major, minor) within a colony (Smith et al. 2008).

Developmental Factors Wheeler’s (1986, 1991) and Nijhout’s (1999) reviews provide a thorough discussion of the developmental basis of worker body size variation. The relative amount and timing of hormone release can play an important role in determining worker body size (Wheeler 1986, Nijhout 1999). For example, in the dimorphic species Pheidole bicarinata, juvenile hormone plays an integral role in controlling the timing of developmental periods and the critical size at which metamorphosis occurs resulting in small “minor” workers and larger “major” workers (Nijhout and Wheeler 1981). Once present, majors can then influence the ratio of minors to majors in a colony by increasing the threshold level of juvenile hormone necessary for larvae to develop into majors through a contact pheromone (Wheeler 1986). If it is relatively easy to manipulate body size developmentally, why do only 16% of all ant species have a polymorphic worker caste? One of the explanations as to why polymorphism has not evolved more frequently is due to the developmental pathway of the queen (Wheeler 1986). In a comparative study of 35 ant species, Fjerdingstad and Crozier (2006) found that diversity in the worker caste is more likely to be observed when queen/worker developmental switches occur early during larval development. Caste determination at early developmental stages may improve the queen’s influence of the reproductive status of offspring within the colony (Schmid-Hempel 1993, Wheeler 1986, Fjerdingstad and Crozier 2006). A recent developmental study by Rajakumar and colleagues (2011) combined hormonal manipulation and gene expression to investigate how “super-majors” develop in the genus Pheidole. They were able to create workers much larger than typically found in a colony and conclude that it may be possible for species to generate extreme morphological specialization within the worker caste based on genetic accommodation though selection on genes that control the frequency and form of expression (through the development of additional juvenile hormone sensitive periods or developmental switch points). Ultimately, more developmental studies are needed to uncover how morphological diversity is generated within a colony, particularly to uncover how existing variation in developmental pathways between queens and workers can be co-opted to generate novel phenotypes (Molet et al. 2012).

5 Evolutionary Constraints As with other insects, morphological variation in ants varies within and between species (Gouws et al. 2011). While there has been no comprehensive phylogenetic study examining the evolution of body size or body size variation across the entire family Formicidae (but see Fjerdingstad and Crozier 2006), there is a considerable phylogenetic signal at the generic level. For example, workers of the two most diverse ant genera are almost entirely dimorphic (Pheidole) or polymorphic (Camponotus) (Wheeler 1986, Hölldobler and Wilson 1990). However, only 40 genera out of around 260 are known to have polymorphic worker castes (Oster and Wilson 1978). It is important to consider that worker polymorphism evolved multiple times in ants (Wilson 1971) and may be the result of different selection pressures. Body size variation in ants may therefore be largely driven by variation in other life history characters. For example, depending on the data set examined, the relationship between queen number (or mating frequency) and worker body size variation has been reported as both positive (Fjerdingstad and Crozier 2006) and negative (Frumhoff and Ward 1992). The evolution of worker body size variation between and within species is an area of research in need of further research.

Extrinsic (Ecological) Determinants of Body Size As mentioned above, caste and size variation within caste is largely environmentally determined through larval nutrition. The ecological environment experienced by a colony should therefore play a large role in determining how resources are acquired and then invested into offspring size and number. In this section, I explore research examining how extrinsic factors influence body size variation in workers within and between colonies. These include the abiotic environment, variation in food availability and quality, the competitive environment, and the social environment (including colony size). Ultimately, my goal is to understand how extrinsic factors influence body size independently and also through feedbacks with intrinsic factors such as genetics, development, and evolutionary history.

Physical Environment Temperature can strongly affect developmental processes influencing both the amount of time it takes larva to develop and the size of adults in holometabolous insects (Brian 1965, Calabi and Porter 1989, Wheeler 1991, Chown and Klock 2003, Davidowitz et al. 2004, Forster et al. 2012). Differences in worker body size within a colony can also vary seasonally due to differences in survival. Seasonal variation in body sizes within a colony may therefore occur both as a direct response to variation in temperature as well as a result of differences in resistance to environmental stress (Holldobler and Wilson 1990, Greenberg et al. 1992, Tschinkel 1993, Beshers and Traniello 1994, Goodisman et al. 2007). For example, workers sampled from summer months are expected to be smaller than those sampled in winter

6 because smaller workers suffer relatively higher mortality during the cold winter days (Tschinkel 1998). Seasonal variation in abiotic conditions also influences food availability and food storage within a colony. For example, the body mass of majors and minors of Pheidole morrisi, tended to be larger in North Carolina and New York than Florida (Yang et al. 2004). This pattern is observed because colonies in higher latitudes experience longer winters, and workers from higher latitudes generally store more fat and majors are used as replete stores (Yang 2006). Similarly, in Trachymyrmex septentrionalis worker body size distributions differ between populations (Florida vs. Long Island), possibly because larger workers are more likely to survive the longer northern winters subsequently influences colony fitness (Beshers and Traniello 1994). Latitude, a proxy for variation in climate, is proposed to play a significant role in determining of an individual body size (Peters 1983, Schmidt-Nielsen 1984, Brown 1995). Specifically, body size is predicted to increase with decreasing temperature and therefore with an increase in latitude (Bergman 1847). Studies examining variation over larger latitudinal gradients have generally shown that mean worker body size increases with increasing latitude (Heinze et al. 2003, Cushman et al. 1993, Kaspari 2005). However, a more recent study that examined collection data from eastern North America found no evidence to support Bergman’s rule (Gerarhty et al. 2007). When considering the overall patterns and impact of temperature on individual survival (Hölldobler and Wilson 1990, Tschinkel 1993) and larvae development (Wheeler 1991, Atkinson 1994), it appears reasonable to think that climate plays a significant role in determining worker body size. Body size may also influence foraging behavior in response to differences in habitat complexity. For example, smaller ants can more easily move through rather than over leaf litter and larger ants may move faster (Kaspari and Weiser 1999, Weiser and Kaspari 2009). Microclimate of foraging area may also influence competition by limiting the timing and location of foragers. For example, in a tropical rainforest in Costa Rica, smaller ant species tend to forage in moist microclimates, while species with large body size forage in a variety of microclimates (Kaspari 1993). Thus species with smaller workers potentially experience more interspecific interactions than species with larger workers (Kaspari 1993).

Competitive Environment The competitive environment in which a colony exists can have strong influence on the body size of ants within a colony. For example, in seven ant communities in Southern California and Arizona, Davidson (1978) found that variation in worker body size of the seed-harvesting ant Messor pergandei was inversely related to the amount of interspecific competition. In the absence of other seed-harvesting ant species, M. pergandei colonies exhibit more variation in body size compared to when other seed harvesters were present (Davidson 1978). Moreover, body size of individual workers was positively

7 correlated with the size of collected seeds suggesting diet breath may be widened by the degree of body size variation within a colony (Davidson 1978). The success of a colony’s discovery, dominance, and retrieval of food resources may be subject to tradeoffs between worker size and number. Generally, colonies with greater numbers of small workers discover food resources more quickly than colonies with fewer large workers (Fellers 1987, Holway 1999). However, in certain habitats with high complexity, larger ants may discover food faster because they typically have longer strides and can move over obstacles (Kaspari and Weiser 1999, Pearce-Duvet et al. 2011). Larger ants may also remove more food items faster than smaller bodied ants (Holldobler et al. 1978, Fellers 1987). Although it is generally thought behavioral dominance is not attributed to worker body size (Holldobler et al. 1978, Fellers 1987, Davidson 1998), in intraspecific interactions in Formica rufa, larger workers are more dominant than smaller workers (Batchelor and Briffa 2010, Batchelor et al. 2011, Batchelor and Briffa 2011). Continued research examining worker body size variation within and between species and its influence on the outcomes of inter- and intraspecific competition may provide valuable insights into community composition (Davidson 1977, Traniello 1989, Davidson 1998, LeBrun and Feener 2007). It has been proposed that the outcome of competitive interactions between social organisms can be predicted using Lanchester’s (1916) ‘linear’ and ‘square’ laws of combat based on individual (body size) and colony level (worker number) characteristics (Franks and Partridge 1993, Whitehouse and Jaffé 1996, McGlynn 2000, Adams and Mesterton-Gibbons 2003, Powell and Clark 2004, Plowes and Adams 2005). The ‘linear law’ assumes individuals engage in a series of one on one duels and the winner of the contest is determined by an individual’s fighting ability; in ants, fighting ability is likely correlated with body size. The ‘square law’ assumes larger groups of individuals can concentrate attacks on members of the smaller group and predicts the larger a group becomes relative to its enemy, the fewer casualties it will sustain. To date, examinations of Lanchester’s models suggest the ability to “win” a contest depends more on individual fighting ability than group size (Whitehouse and Jaffe 1996, Adams and Mesterton- Gibbons 2003, Powell and Clark 2004, Plowes and Adams 2005). For example, when leaf-cutting ants of the genus Atta, respond to an army ant predator (Nomamyrmex esenbeckii), they recruit larger workers (Powell and Clark 2004). Individual size also plays an important role in determining the outcome of ritualized aggressive displays observed in Myrmecocystus species, where smaller individuals are more likely to yield to larger ants (Hölldobler 1981). Large worker size is however, associated with numerically larger colonies and numerically larger colonies often overwhelm and subsequently raid smaller colonies (Hölldobler 1976, Hölldobler 1981). In addition to diet and foraging, the competitive environment may also influence body size through defense (Powell 2009). In the genus Cephalotes, variation in worker body size is an important

8 determinant of colony reproductive success and nest defense (Powell 2008, Powell 2009). Major workers in Cephalotes play an important role in preventing access to nest entrances of the colony (Powell 2008, 2009). A relationship between defensive behavior and body size has been observed in a variety of ant genera (McGlynn 2000) including Crematogaster (Stapley 1999), Atta (Whitehouse and Jaffe 1996, Powel and Clark 2004), Formica (Batchelor et al. 2012), Solenopsis (Plowes and Adams 2005, Haight 2010) and Pheidole (Detrain and Pasteels 1992, Huang 2010, Huang and Wheeler 2011). In Pheidole pallidula even the perceived threat of competition can lead to an increase in colony investment into the major caste (Passera et al. 1996). Yang and colleagues (2004) drew similar conclusions from field collections of Pheidole morrisi colonies from geographically distinct populations along the eastern coast of the United States. They attributed differences in worker body size and caste ratios among populations to differences in the competitive environment (Yang et al. 2004). Interspecific observations of Pheidole in Amazonian Ecuador found that species that dominated baits recruited higher proportions of major workers to baits, likely for resource defense (Mertl et al. 2010). The competitive environment is likely to influence the size and proportions of major workers with in Pheidole colonies because majors play an important role in nest and food resource defense and food processing (Detrain and Pasteels 1992, Wilson 2003, Mertl et al. 2010, Huang 2010, Huang and Wheeler 2011).

Feedback between intrinsic and extrinsic factors Nutrition Nutrition has long been considered an important factor determining body size of insects (Wheeler 1986, 1991). Larger bodied workers require a greater investment in biological building materials and in many species nutrition and development are linked (Wheeler 1986). Therefore one expects to find feedbacks between food availability, foraging success, and investment into larger workers. A test of this theory in food-supplemented forest plots found an increase in soldier pupae production (change in caste ratios) of Pheidole flavens colonies compared to forest plots not supplemented with food (McGlynn and Owen 2002). Body size is not only influenced by the overall amount of food received, but also by diet quality. The use of stable isotope analysis allows researchers to estimate the relative proportions of carbohydrate and protein investment into worker pupae. In Pogonomyrmex badius nitrogen to carbon ratios were higher for majors than minor workers and highest for queens (Smith and Suarez 2010). The ratio of carbohydrates and proteins may also affect individual worker survival and overall colony size (Dussutour and Simpson 2012) this in turn may have an impact on body sizes of workers within the nest (see Social Environment below).

9 The availability and sizes of food items may also influence worker body size within a colony. Changes in climate and temperature will likely influence the size and type of food resources available (Schoener and Janzen 1986, Blackburn et al. 2008). Habitats with larger food items may require larger individuals, and variation in food item size may contribute to the distribution of worker body size (Oster and Wilson 1978). In general however, body size in ants is less likely to limit the types of prey/food collected compared to other animals because of division of labor and cooperative foraging (Traniello 1987, reviewed in Traniello 1989, Traniello and Beshers 1991, Franks et al. 2001). Size matching has been observed to occur (to some degree) in a variety of seed eating ants (Davidson 1978, Rissing 1987, Kaspari 1996, Willott et al. 2000, Pfieffer et al. 2006), and leaf-cutting ants (Wilson 1980a, 1980b, Wilson 1983, Wetterer 2008). However, in most cases smaller workers can typically carry loads considerably more than their own weight and are not limited by mass of naturally occurring forage (reviewed in Traniello 1989). Matching of worker body size to foraging tasks may increase overall efficiency of colony foraging (Arnan et al. 2010) (Table 1.1). As mentioned above, highly seasonal environments with periods where foraging may be restricted, may select for large workers to act as repletes or “living food stores” (Rissing 1984, Beshers and Traniello 1994).

Social Environment Social environment within a colony can have large impact on the variation of body sizes within a colony. By social environment, I mean factors that are directly related to the colony’s traits and ontogeny as a whole. These include, but are not limited to, the age and size of a colony, the social form of the colony (e.g. monogyne versus polygyne), the worker caste distribution, and influences of pheromone control that are related to any of the aforementioned factors. For example, in many ants, the distribution of body size changes as the colony ages and grows. Specifically, it may take many years for long-lived colonies to start producing the largest workers (Tschinkel 1988, Tschinkel 1993, Billick and Carter 2007, Wilson 1983). In many species, recently founded queens initially produce nanitic workers, which are smaller than those produced later in life (Hölldobler and Wilson 1990). These workers are energetically less costly for the non-foraging queen to produce during the early founding stages of colony growth (Porter and Tschinkel 1986). Subsequently as colonies continue to grow and more workers are produced the production of larger workers (majors) increases (Tschinkel 1988, Tschinkel 1993, Araujo and Tschinkel 2010). This pattern has been well described in Solenopsis invicta (Tschinkel 1988), and is also seen in many species of Pheidole (Huang and Wheeler 2011), Pogonomyrmex (Johnson 2002), and the long-lived colonies of Atta (Wilson 1983). Queen number may be an important determinant of body size variation in ants. In the red imported fire ant, Solenopsis invicta, colonies can occur in two social forms, single queen (monogyne)

10 and multiple queen (polygyne) colonies. Generally, the distribution of worker sizes is greater in monogyne colonies than in polygyne colonies (Greenberg et al. 1985, Goodisman et al. 1999, Araujo and Tschinkel 2010). In addition, male body mass is smaller in polygyne colonies than in monogyne colonies (Goodisman et al. 1999). This variation in S. invicta’s social form is associated with allelic differences at the general protein-9 (GP-9) locus. Monogyne colonies have GP-9BB queens while polygyne queens are GP-9Bb (the genotype GP-9bb is largely lethal) (Ross 1997, Keller and Ross 1998). Like males, queens in monogyne colonies tend to be larger and more fecund than those in polygyne colonies (Porter et al. 1988, Vargo and Fletcher 1989, Keller and Ross1993, Keller and Ross 1999). Interestingly, during development monogyne and polygyne queens differ little in weight and fat content (Keller and Ross 1993). However, cross fostering experiments revealed that mature queens (post-metamorphosis) in monogyne colonies weighed more and had a greater fat content than those from polygyne colonies (Keller and Ross 1993). This exemplifies the synergistic nature of intrinsic influences on body size in ants. Here, the genotype at GP-9 (and its closely linked genes) plays an important role in determining queen phenotype and the number of queens in the colony. In turn, queen number influences the social environment and the body sizes of workers and new reproductives (both males and queens) produced by the colony. The current distribution of worker body sizes can also influence future investment into body size. For example in Pheidole species major workers tend to show a reduced efficacy in brood care (Mertl and Traniello 2009). The presence of majors in a colony can suppress the development of larvae into more majors by influencing developmental size thresholds (Wheeler and Nijhout 1984, Wheeler 1986). Experiments that artificially remove castes from lab colonies can lower the rate of worker production (Porter and Tschinkel 1985, Billick 2002) and the development of early and late instar larvae (Porter and Tschinkel 1985). The mechanism by which body sizes vary as a result of differences in social environment may be attributable to differences in food availability (but see Porter and Tschinkel 1985), queen pheromone control (Goodisman and Ross 1996), and the influence of cues that come directly from specific worker castes (Wheeler and Nijhout 1984). The strong influence of ecological factors such as temperature (Tschinkel 1993, Beshers and Traniello 1994) and competition (Herbers 1986, Passera et al. 1996, Oliveira et al. 2011) on the social environment within a colony highlights the interconnectedness of factors determining worker body size.

Division of Labor and Colony Fitness Division of labor (DOL) is a hallmark of eusocial insects and key to the success of cooperative groups generally (Robinson 1992, Beshers and Fewell 2001). One mechanism by which DOL can increase efficiency is by promoting specialization within groups. For example, workers of different sizes may specialize on different tasks such as brood rearing, foraging, and colony defense (Hölldobler and

11 Wilson 1990) (Table 1.1). Theory predicts that in order to optimize colony efficiency, a colony must allocate enough workers to complete each task (see Oster and Wilson 1978, Hasagawa 1997). It is therefore not surprising that many studies have aimed at identifying behavioral specialization and task specificity in workers based on variation in their size (Wilson 1968, Wilson 1976, 1978, 1980a, 1980b, 1984, Porter and Tschinkel 1985, Wheeler 1986, Detrain and Pasteels 1992, Hasegawa 1993, Wetterer 1999, Franks et al. 2001, Yang 2006, Powell 2009, Huang 2010, Muscedere and Traniello 2012). Many species, however, do not show evidence of size specific task matching (Schmid-Hempel 1992, Waser 1998, Schwander et al. 2005, Sempo and Detrain 2010). While colonies may be organized into physical castes, there is little evidence to support the prediction that the ratio of physical castes in workers plays a role in determining foraging efficiency, production of new reproductive individuals, or colony success (Schmid-Hempel 1992). Despite some success in predicting optimal caste ratios (e.g. Hasegawa 1997), few studies examine the relationship between caste ratios and fitness directly but instead use indirect measures such as worker growth as a surrogate for fitness (Wilson 1983, Rissing and Polock 1984, Walker and Stamps 1986, Calabi and Traniello 1989b, Waser 1998, Billick 2002, Arnan et al. 2011). As pointed out by Wilson (1980), efficiency is difficult to measure due to the unknown energetic costs associated with producing workers of different sizes. The energetic costs associated with building and maintaining a workforce may also influence task partitioning in a manner that is difficult to initially detect, but of significant importance (Porter and Tschinkel 1985). Finally, an absence of a relationship between caste ratio and fitness may be due to the high levels of behavioral plasticity seen in ants and other eusocial insects (Robinson 1992, Beshers and Fewell 2001 Johnson and Frost 2012). Experimental studies that change worker demography (e.g. removal of majors or minors) often show no effect on colony reproduction or foraging (Wilson 1983, Wilson 1984) as the remaining castes can complete the tasks once performed by the “specialists”. It is important to note that in each of the previously cited sources, few relate size-based task specificity directly to components of colony fitness (efficiency, survival, and reproductive output - but see Porter and Tschinkel 1985, 1986, Beshers and Traniello 1994, Powell 2008, 2009). Moreover, when components of fitness are measured, the same data are not consistently gathered across species, and multiple components of fitness are rarely measured for a single species. The relationship between body size variation and related task specificity to life-time reproduction and/or population growth remains unknown. Considering the variety of mating systems, life spans, and perennial nature of ant colonies, associating colony fitness with worker body size variation, caste ratios, and efficiency will be very difficult. This may be true even for species with distinct morphological castes whose specific roles are known. For example, majors in the genus Cephalotes play an essential role in defending nest entrances from intruders, an important behavior for colony survival (Powell 2008, 2009). The evolution of a

12 “soldier” caste in relation to nest defense is not restricted to the genus Cephalotes; highly specialized head shapes that serve to plug nest entrances have evolved multiple times in ants (Hölldobler and Wilson 1990).

Discussion and Future Directions A colony’s investment into offspring size and number is hypothesized to be related to colony efficiency and ultimately, colony success (Oster and Wilson 1978, Hölldobler and Wilson 1990, Powell 2008, 2009, Dornhaus et al. 2012). With the increasing availability of genetic resources for ants, including whole genomes for a wide variety of species (Gadau et al. 2012), is an exciting era for understanding the molecular and developmental basis for body size variation both within and among species. However, despite a growing understanding of the roles that specialized castes may play in many ant species, there still is very little known about how variation in body size both within and among colonies contributes to variation in fitness. The primary goal of this review was to examine the ecological factors that influence worker body size variation in ants. Here I summarize possible future research directions. The evolution of morphologically distinct castes in workers is an area that has received a considerable amount of attention. However, more quantitative data is needed, describing the amount of variation in worker body size within and between species (Gouws et al. 2011). This information will be useful for understanding the relative roles of phenotypic plasticity, developmental controls, and how body size changes as a response to nutrition, climate, and competition. The incorporation of phylogenic analyses on variation in worker body size will also be particularly important for testing hypotheses relating to the evolution and maintenance of worker polymorphism in ants.

Does body size variation generally lead to more efficient division of labor? In many species, morphologically distinct worker sub-castes appear specialized for defense, food processing, and/or food storage which can improve task efficiency (Oster and Wilson 1978, Hasegawa 1997, Hölldobler and Wilson 1990, Fjerdingstad and Crozier 2006). However, research supporting a general relationship between size variation and increased efficiency of division of labor is mixed (Table 1.1). The lack of resounding support may be explained because of evolutionary constraints on worker size (Wheeler 1986, Schmid-Hempel 1992). For example, morphological variation among workers may appear due to selection on different traits (such as queen-worker development, Wheeler 1986, Molet et al. 2012) or because behavioral plasticity may play a more important role in determining colony success than morphological variation (Calabi 1988, Beshers and Fewell 2001, Muscedere and Traniello 2012). Experimental studies that address these questions directly either by manipulating physical caste ratios for

13 long periods of time (e.g. many years) or by creating single cohort colonies to control for age related division of labor (as done with honey bees) are still needed. In doing so one could finally begin to empirically test the models for body size variation proposed decades ago by Oster and Wilson (1978). Although difficult, conducting these experiments under field conditions would be ideal to determine the fitness consequences (e.g. reproductive output) of such manipulations.

What are the evolutionary patterns of body size and body size variation in ants? E.O. Wilson (1976) asked if prevalence is associated with specific morphological or behavioral traits in ants. Although he found no general relationships, he noted that the two most specious genera, Camponotus and Pheidole, are both polymorphic (Wilson 1976). The recent development of robust phylogenies describing the relationships between most ant genera (Brady et al. 2006, Moreau et al. 2006) will allow for a careful examination of how body size and its variation relate to a wide variety of factors while controlling for evolutionary history. Using the comparative method, one can finally start examining if clades with polymorphic ants are more diverse, abundant, or ecologically dominant. For example, recently McGlynn et al. (2012) examined how 26 different species of Pheidole from Costa Rica invest in worker body size and caste ratios while controlling for phylogeny. They found that the greater the investment in majors (proportion of majors within a colony), the smaller the body sizes of both majors and minors (McGlynn et al. 2012).

How do intrinsic and extrinsic factors feedback to promote variation in body size? Most examinations of body size variation focus on either intrinsic or extrinsic factors. Even for ecological studies, there have been few holistic attempts to measure the relative contribution of many factors simultaneously. Experiments that co-vary temperature and nutrition to determine their relative impact on foraging behavior and caste differentiation would be a good start. Similarly, more research quantifying community ecological factors, such as the presence of conspecifics or the influence of the competitive environment generally, on body size are still needed. Ultimately, this will help bridge the gap between single species patterns, as in this review, and community ecological approaches that seek to explain how ant assemblages form in nature based on interactions between body size and climatic, metabolic or biogeographic factors (Kaspari 2005, Geraghty et al. 2007, King 2010, Hou et al. 2010). For single species approaches, future research should take advantage of ant species with polymorphic workers that live over a wide geographic range so natural variation in biotic and abiotic factors can be utilized. The black carpenter ant, Camponotus pennsylvanicus, provides a good model for this approach as it has a wide geographic distribution throughout North America (Hansen and Koltz 2005), is common in both urban and a wide variety of natural environments across a large range of

14 latitudes (Wheeler 1910, Buczkowski 2011), and is easy to maintain in the lab (Traniello 1977) allowing for common garden experiments. Similarly, research examining ecological correlates of body size variation among populations of widely introduced species that occur in a range of habitats may provide insight in ways not readily available within the native range of a typical ant species (McGlynn 1999). There are many introduced ants that could be used for this effort including Solenopsis geminata, S. invicta, Pheidole megacephala, P. fervens, Camponotus planatus, and C. sexguttatus.

Final Remarks Body size is one of the most important life history traits as it directly affects an organism’s metabolism, thermoregulation, locomotion, reproduction, abundance, longevity, and diet (Peters 1983, Schmidt-Nielsen 1984, Calder 1984, Losos 1990, Douglas and Mathews 1992, Foote 1997, Kaspari and Weiser 1999, 2007). Understanding determinates of body size variation in ants is no trivial task. The complexity arises because of division of labor and the presence of distinct worker castes in some species. This suggests that the relative importance of extrinsic and intrinsic factors on worker body size varies within and between species and in some cases between worker castes (Schwander et al. 2008 Gouvs et al. 2011). This may explain why the vast majority of research examining body size is focused on solitary organisms (Stearns 1992, Brown 1995) while relatively little work is done using social insects (Oster and Wilson 1978, Bourke and Franks 1995, Gouws et al. 2011). Given the ecological dominance of ants in many environments (Folgarait 1998, Farji-Brener 2010), failing to examine the factors influencing within/between colony body size variation would limit understanding of the role body size in insect ecology. There exists a substantial amount of information about the factors influencing body size in ants, but there is much left to discover and to include with what is already known. Here I reviewed the literature on variation in body size of ants with polymorphic worker castes. One of the goals of the review was to examine how body size variation influences colony fitness. However, I found few studies that have measured the fitness consequences of variation in worker body size. However, this limitation is not uncommon even in studies of solitary organisms (Kingsolver et al. 2001, Kinsolver and Pfennig 2007). While I focused on variation in body size within colonies, variation in body size among species is equally dramatic, and cannot truly be separated conceptually from variation within colonies. This would be an interesting topic for a future review from the perspectives of behavioral ecology, community ecology and phylogenetic constraints. Experimental work examining the relative roles of extrinsic and intrinsic factors on body size distributions within colonies is still needed. Given that multiple factors influence worker body size variation, experimental work that can address each independently and in concert will be particularly insightful for elucidating how intrinsic and extrinsic factors work together to determine body size variation in ants.

15 Chapter 1 Figures and Tables Figure 1.1. Allometric growth curves, body size distributions, and examples of variation in ant worker body size. A. Hypothetical examples of allometric growth curves (see Wilson 1953 and Oster and Wilson 1978). I.a. Monomorphism is represented by an isometric growth curve (slope = 1), while I.b. monophasic allometric growth is nonisometric (slope > or < 1). II. Diphasic allometry is represented by “breaks” with two segments with different slopes and intersect. III. Triphasic allometry is represented by two “breaks” with three segments with different slopes (minors, medias, and majors). IV. Complete dimorphism is represented by two distinct segments separated by a gap with no intermediates. B. Example body size distributions for ants with each allometric growth. I.a. Linepeithema humile, I.b. Camponotus pennsylvanicus (Fowler 1986), II. Atta sexdens (Wilson 1953), III. Oecophylla smaragdina (Wilson 1953), IV. Pheidole sp. (Wilson 1953). C. Photos of species with each allometric growth curve I.a. L. humile, I.b. C. pennsylvanicus. II. A. cephalotes, III. O. smaragdina, and IV. P. aberrans (Photos: © Alex Wild used with permission).

16 Figure 1.2. Worker body size and body size distributions within a colony are influenced by a variety of factors from within (“intrinsic”) and outside (“extrinsic) a colony. Intrinsic factors I discuss in the review include: evolutionary constraints, genetic factors, and the social environment. Extrinsic factors I discuss include: the physical environment, competition, and nutrition. These factors also can interact with one another and ultimately influence the development, survival, and longevity of workers within a colony.

17 Table 1.1 Summary of empirical examinations supporting or contradicting the prediction of task- matching based on ant body size. * Indicates division of labor based on body size associated with a component of fitness. This is a non-exhaustive list. Additional citations can be found in Traniello 1989, Beshers and Fewell 2001, Schimd-Hempel 1992, and references cited therein.

Support for DOL Species Predictions Citation based on body size Acromyrmex +/- (depends on Division of labor based on body size Muscedere et al. 2011 octospinosus access to forage) Atta Foraging efficiency based on body size – Wilson 1983 cephalotes Atta sexdens Foraging efficiency based on body size + Wilson 1980a,b Powell and Clark Atta spp. Nest defense efficiency based on body size + 2004 Camponotus Nest defense/ Brood production efficiency Hasegawa 1993*, + nipponicus based on body size 1997* Eciton Foraging efficiency based on body size + Franks et al. 2001 burchelli Powell and Franks Eciton spp. Foraging efficiency based on body size + 2005, 2006 Formica Body size distributions influence worker + Billick 2002* neorufibarbis production Formica Body size distributions influence worker Billick and Carter + obscuripes production 2007* Pheidole Nest defense efficiency based on body size + Wilson 1976a* dentata Pheidole Calabi and Traniello Task performance based on body size – dentata 1989a,b* Pheidole Caste ratios differ in response to Yang et al. 2004, + morrisi competitive /climatic differences Yang 2006 Pheidole Nest defense efficiency based on body size + Huang 2010 obtusopinosa Pheidole Detrain and Pasteels Nest defense efficiency based on body size + pallidula 1992. Pheidole Sempo and Detrain Task specialization based on body size – pallidula 2010 Pheidole Task specialization based on body size + Wilson 1984 species Solenopsis Efficiency of brood production based on Porter and Tschinkel + invicta body size 1985*,1986* Solenopsis invicta and S. Foraging efficiency based on body size + Wilson 1978 geminata Trachymyrmex Alate production efficiency based on body Beshers and Traniello + spetentrionalis size 1994* Veromessor Worker body size changes in response to + Davidson 1978 pergandei competition Veromessor Rissing and Pollock Foraging efficiency based on body size – pergandei 1984, Waser 1998 Formica Task specialization based on body size –/+ Schwander et al. 2005 selysi

18 CHAPTER 2: BODY SIZE VARIATION IN GEOGRAPHICALLY DISTINCT POPULATIONS OF THE INVASIVE ANT, PHEIDOLE MEGACEPHALA

Abstract Body size is an important life history trait that influences how individuals interact with each other and their environment. Invasive species often encounter vastly different ecological conditions throughout their introduced range that can influence relative investment into growth, reproduction, and defense among populations. In this study, I quantified variation in worker size, morphology, and caste ratios among five populations of a worldwide invasive species the big-headed ant, Pheidole megacephala (Fabricius). The sampled populations differed in ant community composition allowing us to examine if P. megacephala invests differently in the size and number of majors based on the local ant fauna. I also used genetic data to determine if these populations of P. megacephala represented cryptic species or if morphological differences could be attributed to change following introduction. I found significant variation in worker mass among the populations. Both major and minor workers were largest in Australia, where the ant fauna was most diverse, and minor workers were smallest in Hawaii and Mauritius, where P. megacpehala interacted with few to no other ants. I also found differences in major and minor worker morphology among populations. Majors from Mauritius had significantly larger heads (width and length) relative to whole body size than those from Hawaii and Florida. Minors had longer heads and tibias in South Africa compared to populations from Australia, Hawaii, and Florida. Caste ratios did not differ among populations suggesting that these populations may not be subject to tradeoffs in investment in major size versus number. The molecular data place all samples within the same clade supporting that these morphologically different populations represent the same species. These results suggest that the variation in shape and morphology of major and minor workers may therefore be the result of rapid adaptation or plastic responses to local conditions.

Introduction Inducible defenses are a primary strategy by which an organism can respond to immediate threats in its environment including predation, parasitism and competition (Harvell 1990, Agrawal 2001). Trans- generational induced defenses, or adaptive material effects, are a method by which parents can improve their direct and indirect fitness by modifying the phenotype of their offspring in response to current environmental conditions (Agrawal et al. 1999). Maternally induced defenses can include increases in chemical defensive compounds, the generation of specialized morphological structures (such as trichomes

19 in plants or helmet and spine formation in Daphnia), or a general increase in investment into the number or size of offspring (Harvell 1990, Repka and Pihlajamaa 1996, Boersma et al. 1998, Agrawal 1999, Agrawal et al. 1999, Mondor et al. 2005, Kaplan et al. 2008). Body size is a key life history trait that is closely associated with an organism’s physiology, behavior, reproduction, and survival. Size also influences how individuals interact with each other and their environment (Peters 1983, Calder 1984, Schmidt-Nielsen 1984, Losos 1990, Stearns 1992, Brown 1992, Brown 1995, Moczek and Emlen 2000, Chown and Gaston 2010, Gouws et al. 2011). Subsequently, body size is often measured in studies that examine trans-generational induced defenses (Passera et al. 1996, Boersma et al. 1998, Van Buskirk 2000, Relyea 2004, Yang et al. 2004). Moreover, tradeoffs exist in how limited resources are allocated into offspring size, number, and condition (Smith and Fretwell 1974), and these tradeoffs may explain when organisms induce defense strategies (Harvell 1990, Agrawal et al. 1999b, Van Buskirk 2000). While measuring investment into body size may be relatively straightforward in solitary organisms, in eusocial species, colonies partition investment into the number or quality of different castes. In ants, for example, a colony can invest into females by partitioning resources into discrete reproductive (queen) and non-reproductive (worker) castes. In species that have polymorphic workers, ant colonies can further differentially invest into worker castes that may be specialized for food storage, foraging or defense (e.g. minors versus majors or soldiers) (Oster and Wilson 1978, Hölldobler and Wilson 1990, Dornhaus and Powell 2010). Castes in ants are determined by the amount and type of nutrition received during larval development (Wheeler 1986, Hölldobler and Wilson 1990; but see Heinze 2008, Schwander et al. 2010 for exceptions). The same is true for body size variation within the worker caste (Wheeler 1991), although a number of recent studies have found evidence for a genetic influence on some size classes in polymorphic species (Hughes et al. 2003, Jaffe et al. 2007, Smith et al. 2008; but see Wiernasz and Cole 2010). An advantage to environmental caste determination is that colonies can rapidly alter investment into the number and size of workers in response to biotic and abiotic conditions to optimize colony growth, maintenance, foraging, and defense (Oster and Wilson 1978, Wilson 1984, Beshers and Traniello 1994, Yang et al. 2004). Both intra- and inter- specific competition, for example, may influence body size variation through foraging behavior and prey selection (Davison 1978, Traniello 1987, Traniello 1989, Wetterer 1994a). In the genus Pheidole, which has a dimorphic (and rarely trimorphic) worker caste consisting of majors and minors, colony investment into major production can be influenced by diet supplementation (McGlynn and Owen 2002) and in response to both intra- and inter-specific competition (Passera et al. 1996, Yang et al. 2004). For example, Yang et al. (2004) found a greater number of majors in colonies of Pheidole morrisi from populations that overlapped with the red imported fire ant

20 (Solenopsis invicta). However, the average size of these majors was smaller suggesting that tradeoffs existed between investment into major size or number. Shifts in caste ratio have also been observed in Pheidole pallidulla where the perceived threat of competitors increased the production of major workers within laboratory colonies (Passera et al. 1996). Differences in the competitive environment among populations may therefore induce shifts in investment into worker sub-castes that specialize on colony defense. In this study, I quantified variation in worker size, morphology, and caste ratios among five populations of a worldwide invasive species the big-headed ant, Pheidole megacephala (Fabricius 1793). Introduced species often encounter dramatically different ecological conditions within their invaded ranges and this variation in selective pressures can impact relative investment into growth, reproduction, and defense among introduced populations (e.g. Wolfe 2002, Zangerl and Berenbaum 2005, Berenbaum and Zangerl 2006). I chose populations that differed in ant community composition allowing us to examine if P. megacephala invests differently in the size and number of workers based on its interactions with the local ant fauna. Specifically, I use this data to test two hypotheses: 1) Populations introduced to areas with higher local ant diversity will produce either larger or a greater number of majors relative to populations introduced to areas with few or no resident ants. 2) Populations will exhibit a trade-off between investment into major size and number. Finally, to determine if morphological differences among populations could be due to the presence of more than one “cryptic” species, I use genetic data to examine the phylogenetic relationships among the populations relative to each other and to P. megacephala sequences available from the literature.

Methods Study System As with nearly all Pheidole species, P. megacephala has a dimorphic worker caste consisting of minors and majors (also referred to as soldiers) that are easily distinguished by differences in size and head shape. Major workers have larger heads relative to their body size, and specialize in nest defense and in food processing, retrieval, and storage (Wilson 1984). Minor workers typically undertake the remainder of tasks within the colony (Wilson 1984, Wilson 2003). I predicted that differences in ecological conditions among geographically distinct populations (particularly the competitive environment) will result in variation how colonies invest in worker body mass, morphology, and caste ratios. I chose P. megacephala because it has successfully invaded a wide range of geographic locations (Wetterer 2007, 2012) where different populations experience dramatically different ecological conditions. The native range of P. megacephala has not yet been determined although it is suspected to be from Africa (Ethiopian region) or Madagascar due to the richness of the megacephala species complex

21 from these regions (Wheeler 1922, Wetterer 2007, Fournier et al. 2012, Wetterer 2012, Fischer et al. 2012, Fischer and Fisher 2013). I collected workers of P. megacephala from five distinct populations that vary considerably in terms of species richness within the ant community: Northern Territory, Australia; Hawaii, USA; Florida, USA; Sabi Sands, South Africa; and Mauritius (Table 2.1). In each population, I sampled three to six nests and each nest was separated by between 200 m and up to 2 km. From each nest, I collected as many workers as possible by turning over a cover object that housed the nest, and aspirating as quickly as possible while also scooping ants into a container. All specimens were stored in 90% ethanol after collection. Local ant community composition surrounding each invaded population was estimated by visual sampling and from the literature. This allowed us to estimate a range of ant species that P. megacephala had historically, or is currently, interacting with. The higher estimate represents the total pool of species within the region and the lower estimate represents the local species pool. For example, Hawaii has no native ant species (Wilson and Taylor 1967), and the population of P. megacephala I sampled in Hawaii Volcanoes National Park had the potential to interact with between 2-10 species (Wetterer 1998, A.V. Suarez personal observation). In contrast, tropical Australia has one of the most species rich ant communities in the world. In the Howard Springs study area, 21-157 species have been recorded with the estimated number of ants declining as the invasion progressed (Hoffmann et al. 1999, Hoffmann and Parr 2008). The estimate range of ant species in South Africa (34-121 species) (Parr and Chown 2001, Parr 2008), Florida (60-100 species) (Deyrup 2003, M. Deyrup personal communication), and Mauritius (8-16 species) (Smith and Fischer 2009, A.V. Suarez personal observation) fall in between the estimated numbers for Hawaii and Australia. Since information on the biology of most individual ant species interacting with P. megacephala at each site is lacking (including their colony size, dominance, and ecological overlap), I instead examine if investment into worker size or major number is related to the overall diversity of resident ants at each site. Subsequently, I predict that investment into major production will be highest in Australia, intermediate in South Africa, Florida, and Mauritius, and lowest in Hawaii.

Phylogenetic analysis DNA Isolation Field collections were made in 90-95% EtOH and kept in the laboratory until the time of DNA extraction. Thirty-three new specimens of P. megacephala were sequenced for this study (Table 2.2). Total genomic DNA was isolated for one individual worker by first pulverizing with a tungsten carbide bead in a TissueLyser (Qiagen Inc., Valencia, CA), followed by purification using the DNeasy Tissue Kit (Qiagen Inc., Valencia, CA) following the manufacturer’s protocols.

22

Polymerase Chain Reaction (PCR) Amplification For most specimens, four fragments were amplified via PCR using specific primers for each gene region following the protocols of Moreau et al. (2006) and Moreau (2008): cytochrome oxidase I (COI) protein encoding mitochondrial marker, long-wavelength rhodopsin (LR) protein encoding nuclear marker, H3 histone (H3) protein encoding nuclear marker, and 12S mitochondrial ribosomal DNA marker, for a total of almost 2300 base pairs (bp) of aligned sequence.

Sequencing All sequencing was done using dye terminator cycle sequencing using BigDye terminator v3.1 and an ABI 3730 DNA analyzer (Life Technologies, Grand Island, NY). Primers used for amplification served as sequencing primers. All samples were sequenced in both directions again following the protocols of Moreau et al. (2006) and Moreau (2008).

Sequence alignment In addition to the new sequence data collected for 33 new specimens, I incorporated 56 additional relevant sequences generated for other studies (Moreau 2008, Smith and Fisher 2009, Fournier et al. 2012) for a total of 89 taxa included in this study (Supplementary Table 2). After sequence data was collected they were analyzed and initially aligned using the computer program Geneious v6.1.2 (Drummond et al. 2012). Inferred amino acid sequences were used for all protein-coding genes, allowing for comparatively uncomplicated alignment using Mesquite v2.75 (Maddison and Maddison 2011).

Phylogenetic analysis To infer relationships among the species of Pheidole included in this study, several model based phylogenetic analyses were performed on the CIPRES Science Gateway (Miller et al. 2010) using RAxML v7.3.2 (Stamatakis et al. 2005) and MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001). In order to evaluate the fit of the data, likelihood analyses were conducted using both the COI only data set (COI only) and the data partitioned by individual genes (partitioned). Modeltest 3.06 (Posada and Crandall 2001) was used to determine the most appropriate nucleotide substitution model. Two maximum likelihood searches were implemented in RAxML: 1) a single model of sequence evolution was assumed to underlie the mtDNA COI gene (COI only) with 500 bootstrap pseudoreplicates and 2) one that allowed each of the four gene regions to have a separate model of sequence evolution with parameters unlinked (partitioned) with 500 bootstrap pseudoreplicates.

23 Bayesian inference analyses were performed using MrBayes, with model parameters being estimated during the run, and using the default value of four Markov chains. A “temperature” parameter of 0.2 was implemented to produce incremental heating of each chain. The Markov chain Monte Carlo (MCMC) length was 25,000,000 generations, with the chain sampled every 1000 generations. Bayesian posterior probabilities (BPP) were estimated as the proportion of trees sampled after 10% burn-in that contained each of the observed bipartitions (Larget and Simon 1999; Rannala and Yang 1996). Again two analyses were preformed 1) a single model of sequence evolution was assumed to underlie the mtDNA COI gene (COI only) and 2) one that allowed each of the four gene regions to have a separate model of sequence evolution with parameters unlinked (partitioned). Independence of runs was insured by only accepting analyses where the average standard deviation of split frequencies was below 0.01.

Body Mass and Morphology I collected as many workers as possible from multiple nests within each population (see Table 2.1). From those collections, 25 minor workers and as many major workers as available (n=3-20) were dried in an oven at 50ºC for ~ 48 hours. After 48 hours, I placed workers into 1.5mL microcentrifuge tubes (to prevent re-hydration) and then weighed each individual using a UMX2 microbalance with 0.1 μg resolution (Mettler-Toledo, Columbus, OH). After weighing, each specimen was point mounted and head length (HL), head width (HW), pronotal width (PW), and tibia length (TL) were measured using a Semprex Micro-DRO digital stage micrometer (0.005 mm resolution, Semprex Corporation, San Diego, CA) connected to a Leica MZ 12.5 stereomicroscope. To examine how mean body mass and morphological measurements differed among populations; I first selected a model using R v2.15.1 (R Development Core Team 2012) (Crawley 2012). I compared the mean measurement per colony among populations with colonies as replicates with each worker as a subsample of colony. Majors and minors were identified a priori as workers are completely dimorphic and worker sub-caste was easily distinguished based on overall size and the shape of the head. For both sets of workers, and for all measurement data, the first model included population as a fixed effect and colony as random effect. Both were not significantly different than models including only population as a fixed effect (p > 0.99) thus I only used population for the following analysis. I compared among populations variation in body mass for each caste using an ANOVA and then used a post-hoc Tukey’s honesty significant difference (HSD) correction to determine which populations were different from each other. I compared morphological measurements among populations for each caste using a Principle Components Analysis (PCA) and compared differences between significant principle components with a post-hoc Tukey HSD.

24 Caste Ratios To compare caste ratios from each population, I counted the total number of majors and minors sampled from each nest, and calculated the proportion of majors within a colony. I compared proportions of majors among populations using a binomial distribution with GLM in R v2.15.1 (R Development Core Team 2012). The original model is over-dispersed and I refit the model with quasi-binomial to account of the over-dispersion (Crawely 2012).

Results Simple Sequence Statistics This study produced a final aligned 2,282 bp fragment with the following four gene regions: a fragment spanning the mitochondrial cytochrome oxidase I (COI) (1054 bp) gene, a fragment of the mitochondrial ribosomal DNA marker 12S (349 bp), a portion of the nuclear protein-encoding gene long- wavelength rhodopsin (LR) (555 bp), and a fragment of the nuclear protein-encoding gene H3 histone (H3) (324 bp). The aligned fragment contained 1351 constant sites (59.3%), 264 uninformative variable sites (11.5%) and 667 parsimoniously informative sites (29.2%). Most specimens were sequenced for all four genes with the following exceptions: COI missing for two taxa (AVS1813, SabiSands2); LR missing for 16 taxa (Table 2.2, and see Supplementary Table 1); 12S missing for two taxa (AVS1813, SabiSands2); and H3 missing for three taxa (AVS1812, AVS1814, AVS2717) (Table 2.2).

Phylogenetic Analyses The maximum likelihood topology for the COI only analysis is presented in Figure 2.1 (and Supplementary Figure 1) with maximum likelihood bootstrap (ML BS) support values for both the COI only and partitioned analyses, as well as the Bayesian PP (BPP) support values for both the COI only and partitioned analyses included. A GTR + G model of sequence evolution was found to be the best fit to the data for all partitions. All maximum likelihood and Bayesian inference tree topologies show strong support (100% ML BS COI only; 87% ML BS partitioned; 1.0 BPP COI only; 1.0 BPP partitioned) for the monophyly of the Pheidole megacephala samples from this study with previously sampled specimens from Moreau (2008), Smith and Fisher (2009), and Fournier et al. (2012).

Body Mass and Morphology

The body mass of majors differed significantly among populations (ANOVA F4,17 = 29.73, p < 0.0001) (Figure 2.2A). Majors from Australia were larger than those in other comparisons (Tukey HSD post-hoc p-adj < 0.001), but no other populations were different from one another. The body mass of the minors also differed significantly among populations (ANOVA F4,17 = 9.47, p < 0.0001) (Figure 2.2B).

25 The minors were larger in Australia compared to minors from the other four populations (Tukey HSD post-hoc p-adj < 0.001). Minors from South Afirca and Florida were also larger than minors from Mauritius and Hawaii (Tukey HSD post-hoc p-adj < 0.05). For majors, PC1 explained 79% of the variation in the morphological measurements and was primarily associated with head length and head width (Table 2.3). Populations differed with respect to PC

1 values (ANOVA F4,17 = 6.96, p < 0.01) with majors from Mauritius have longer and wider heads than majors from Florida and Hawaii (Tukey HSD post-hoc p-adj < 0.001) (Figure 2.3A). For minors, PC1 explained 53% and PC2 explained 17% of the variance in morphology (70% overall). As in majors, PC1 was associated with variation in head length and head width. PC2 also included variation in tibia length.

Unlike in majors, PC1 was not informative in distinguishing minors from any population (ANOVA F4,17 = 1.98, p = 0.14). However, there was a difference among populations in the shape of minors using PC2

(ANOVA F4,17 = 20.6, p < 0.0001). Minors from South Africa have longer heads and tibia length than those in Australia, Hawaii, and Mauritius (Tukey HSD post-hoc p-adj < 0.05) and minors from Australia were different than those from Florida and Hawaii (Tukey HSD post-hoc p-adj < 0.05) (Figure 2.3B).

Caste Ratios The proportion of majors within a colony was not significantly different among populations

(ANOVA F4,17 = 2.67, p = 0.0619) although there was a trend for nests in Florida to have fewer majors relative to the other four populations (Figure 2.4).

Discussion Ants are interesting model organisms for the study of inducible defenses as extensive overlap between generations allows queens to directly benefit from differential investment into offspring condition. Previous work has suggested that competition for resources (Davison 1978, Traniello 1987, Traniello 1989, Wetterer 1994a) and perceived threats (Passera et al. 1996, Yang et al. 2004) may influence colony investment into worker body size and caste ratios. Invasive species can encounter different competitive environments throughout their ranges (Holway et al. 2002, Wolfe et al. 2002, Zangerl and Berenbaum 2005, Berenbaum and Zangerl 2006) and may be particularly good models to examine how colonies invest into defense. I predicted that the invasive P. megacephala would increase investment into major size or number in populations introduced to areas with high local ant diversity relative to populations introduced to areas with few or no ant species. In addition, if a tradeoff exists between investment into the number and size of major workers, then I predict that populations that invest more into larger majors will also produce fewer of them.

26 I found substantial variation in how colonies invest into worker mass and morphology among five populations of the invasive ant, P. megacephala. Both major and minor workers were largest in Australia, and minor workers were smallest in Hawaii and Mauritius. Head shape also varied among populations in both worker sub-castes. The genetic results suggest that these populations are nominally all the same species. Subsequently, differences into investment among populations in the size of majors likely results from rapid adaptation or a plastic response to local conditions. While I only sampled ants from five different countries, these populations varied considerable in ecological conditions and I discuss the results in the context of this ecological variation. The preliminary phylogeny placed ants from all five populations within a clade that included most previously published sequences from populations identified as P. megacephala. However, recent molecular evidence (Moreau 2008, Fournier et al. 2012) suggests that the P. megacephala complex in Africa and Madagascar is taxonomically unresolved and that there may be more than one cryptic species introduced from these regions that is nominally called P. megacephala. The presence of cryptic species can present challenges to the study of invasions from a number of perspectives. First, they can hinder efforts to identify the source of introduction and effective biological control agents. Second, cryptic species can mislead researchers to believe large variation in ecological, behavioral, and genetic characteristics exist among populations of the same species when it is actually separate species that are being compared. This has been an issue with a number of ant species (e.g. Solenopsis invicta/S. richteri – Wilson 1951; Tetramorium caespitum/T. tsushimae - Steiner et al. 2006; Technomyrmex albipes/T. difficilis - Bolton 2007). In the case of P. megacephala, I need considerably more work examining genetic variation among introduced populations and also across its putative native range. Without this information, it will be impossible to identify the geographic origin of this widespread species and conclusively determine if multiple, related taxa may have been introduced. In principle, this can only be done with an extensive and careful taxonomic revision of this species group in Africa and Madagascar (sensu Fischer et al. 2012, Fischer and Fisher 2013). My results support one of the two predictions; that populations of P. megacephala introduced to areas with high native ant diversity would invest more into majors. Specifically, I found that both majors and minors were larger in Australia than in other populations. Although many studies have quantified body size variation among species (Cushman et al. 1993, Beshers and Traniello 1994, Kaspari 2005, Geraghty et al. 2007, McGlynn et al. 2012), few examine how variation differs across wide geographic locations within a single species. My results also correspond well with previous studies examining geographic variation in worker body size in Pheidole. In a study of three populations of Pheidole morrisi in the United States, Yang and colleagues (2004) found colonies from Florida produced significantly more, smaller majors in Florida than colonies from New York and New Jersey. Populations in New York

27 and New Jersey experience longer colder winters than colonies in Florida, whereas colonies in Florida encounter the aggressive red imported fire ant (Solenopsis invicta). In addition to differences in major size, both major and minor workers from New York and New Jersey stored more fat than those collected in Florida (Yang 2006). Unfortunately, I was unable to examine fat content in collected samples because the specimens were stored in 90% ethanol. Interestingly, I also found that colonies from Hawaii and Mauritius had the smallest minor workers. While I did not make any a priori predictions about minor worker size, this pattern warrants further investigation. Careful examination of how P. megacephala colonies invest into worker size, condition, at small spatial scales in relation to interactions with resident ant species will likely uncover patterns missed at larger scales as in this study. The relative shape of both majors and minor workers also varied among populations. Majors from Mauritius had significantly larger heads (width and length) than those from Hawaii and Florida. In minor workers, the length of the head and tibia were greater in South Africa relative to minors from Australia, Hawaii, and Florida. My results suggest that differences in body mass do not necessarily translate to predictable differences in morphology and shape. Most morphological variation among species of Pheidole can be attributed to size differences among species (Pie and Traniello 2007). However, variation in morphology between castes may reflect differences in selective pressures based on their function (Pie and Traniello 2007). Prior work has shown that the interaction between phylogenetic history and ecology induce interspecific variation in Pheidole species in the Neotropics, where interspecific variation in major worker size and morphology is tightly related to habitat type (ground- or twig-nesting), foraging strategy (discovery versus dominance), and major worker behavior (major worker recruitment to food resources) (Mertl et al. 2010). Variation in shape among populations in this study suggests a response to local ecological conditions separate from selection on overall size. For example, eco-mophological studies have shown that head shape and leg length can influence foraging behavior, trophic position, community assembly, and how ants can navigate complex topography (e.g. size-grain hypothesis) (Traniello 1989, Kaspari and Weiser 1999, Weiser and Kaspari 2006, Silva and Brandão 2010). The observed intraspecific variation in shape and morphology of major and minor workers may therefore be the result of rapid adaptation or plastic responses to widely varying local conditions. I did not find support for the second prediction that colony caste ratios would vary among populations. Worker body size may result from a tradeoff between colony investment into size and number of workers, so that a colony producing larger majors may produce fewer majors (Yang et al. 2004, Mertl and Traniello 2009, McGlynn et al. 2012). However, the presence of soldiers in a colony can inhibit further soldier production thereby creating stable minor/major ratios (Wheeler and Nijhout 1984). My results may reflect this general constraint although this seems unlikely given patterns seen in other Pheidole species where the number of majors increases in response to changes in diet or the competitive

28 environment (Passera et al. 1996, McGlynn and Owen 2002, Yang et al. 2004). It is possible the current sampling method was insufficient to accurately estimate caste ratios. To better estimate worker demography, greater effort is required to collect the entire colonies. However, P. megacephala colonies are difficult to collect in their entirety because they are extremely polydomous. Future efforts to estimate caste ratios should include quantifying the ratio of majors that respond to a specific stimulus to the colony such as access to a bait or threat for competitors, by counting the number of soldiers that show up to baits at fixed distances to the colony (Huang 2010, Mertl et al. 2010). In addition to competition, diet (McGlynn and Owen 2002) and climate (Yang et al. 2004) also influence colony investment into worker body size in Pheidole species. With the current sampling design, I was unable to explicitly test the contribution of each of these factors separately. Future work should sample more populations across a wider range of biotic and abiotic conditions. Furthermore, field surveys should be combined with common-garden experiments to explore the relative influence of each factor on the morphology of workers. Despite the limitations of the current study, my results serve as a foundation for the testing of specific hypotheses. For example, although I did not directly test the influence of competition on worker body size, I do see a pattern that suggests a relationship between competitive environment and major worker body size. Further work is needed to decipher the role of diet, climate, and competition on the observed variation in worker body sizes. The interplay of body size and invasion success has been investigated in a biogeographic context in a variety of taxa (plants: Crawley 1987, Thébaud and Simberloff 2001; vertebrates: Veltman et al. 1996, Jeschke and Strayer 2006, Blackburn et al. 2013; invertebrates: Lawton et al. 1986, McGlynn 1999, Roy et al. 2001, Miller et al. 2002, Roy et al. 2002). In fact, differential investment into defense, growth or reproduction between native and introduced populations forms the basis for many hypotheses as to why introduced species are so successful in invaded areas (Blossey and Notzhold 1995, Shea and Chesson 2002, Keane and Crawley 2002, Parker et al. 2013). Much research in this area examines whether species are larger in introduced populations relative to populations in their native range (Parker et al. 2013). While the data supporting the role of body size and invasion success is generally mixed, introduced species of insects are often smaller than their native counterparts (Lawton et al. 1986, McGlynn 1999). Additionally, some introduced ant species are known to smaller within their introduced range than in their native range (McGlynn 1999, Mikheyev and Mueller 2007). In social insects, tradeoffs between worker size and worker number may promote the success of invaders by either promoting larger individuals that will do well in one-on-one encounters, or by allowing species to obtain the high worker densities needed to displace resident species by outnumbering them with smaller individuals (Franks and Partridge 1993). Understanding how body size both responds and contributes to establishment success in new areas promises to continue to be an important area of research for invasions biology and ecology.

29 Chapter 2 Figures and Tables Figure 2.1. Phylogram of Pheidole megacephala as inferred through maximum likelihood analysis for the COI dataset. Collections of P. megacephala measured for size, morphology, and caste ratios as part of this study are noted by a star next to the taxa names. Branch lengths are proportional to substitution/site as indicated by the bottom legend inset. Clade support greater than 50% is denoted on branches as follows: Values above branches represent maximum likelihood bootstrap (ML BS) for the COI only dataset followed by the partitioned dataset and values below branches represent Bayesian posterior probabilities (BPP) for the COI only dataset followed by the partitioned dataset. Clade support of “--” denotes clades not supported in an individual analysis. Taxa names include taxonomic identity, state and country of collection site, and collector code (and GenBank accession number and citation to original publication if from a previous study).

30 Figure 2.2. Variation in mean body mass of majors and minors among populations of Pheidole megacephala. Majors and minors scale bars represent mean ± SE body mass per colony and letters indicate differences among populations (Tukey HSD post-hoc p-adj < 0.05). A. The body mass of majors differed significantly among populations (ANOVA F4,17 = 29.73, p < 0.0001). Majors from Australia were larger than those in other comparisons (Tukey HSD post-hoc p-adj < 0.001). B. The body mass of the minors differed significantly among populations (ANOVA F4,17 = 9.4662, p < 0.0001) and minors were larger in Australia compared to minors from Hawaii and Mauritius (Tukey HSD post-hoc p-adj < 0.001), and minors from Mauritius were larger than those collected in South Africa (Tukey HSD post-hoc p-adj < 0.05).

31 Figure 2.3. PCA of majors and minors among populations (Australia = green, A, Florida = red, F, Hawaii = black, H, Mauritius = blue, M, and South Africa = orange, SA) of Pheidole megacephala. A. For majors, PC1 explained 79% of the variation in the morphological measurements and was primarily associated with head length and head width. Populations differed with respect to PC1 values (ANOVA F4,17 = 6.96, p < 0.01) with majors from Mauritius being longer and wider than majors from Florida and Hawaii (Tukey HSD post-hoc p-adj < 0.001). B. For minors, PC1 explained 53.01% and PC2 explained 17% of the variance in morphology (70 % overall). As in majors, PC1 was associated with variation in head length and head width. PC2 also included variation in tibia length. For minors, PC1 was not informative in distinguishing minors from any population (ANOVA F4,17 = 1.98, p = 0.14). However, there was a difference among populations in the shape of minors using PC2 (ANOVA F4,17 = 20.6, p < 0.0001). Minors from South Africa have longer heads and tibia length than those from Australia, Hawaii, and Mauritius (Tukey HSD post-hoc p-adj < 0.05) and minors from Australia were different than those from Florida and Hawaii (Tukey HSD post-hoc p-adj < 0.05).

32 Figure 2.4. Mean (± SE) of caste distributions of Pheidole megacephala among populations. Caste ratios represent the number of majors divided by total number of ants collected. The proportion of majors within a colony was not significantly different among populations (ANOVA F4,17 = 2.67, p = 0.06).

33 Table 2.1. List of populations sampled for this study and characteristics for each population including the year P. megacephala was first reported, the number of colonies sampled, climate, location, and estimates of local ant community richness (native and introduced). Dates of first record from Wetterer (2012); except Mauritius, (taken from Forel’s (1905) surveys of the Seychelles and surrounding islands) and South Africa (taken from Hoffman et al. 1999). For Australia I provide two dates, one for the continent and a second for its first detection in Howard Springs (Reichel and Andersen 1996). South Africa is possibly within the native range of P. megacephala but see discussion in Wetterer (2012). Ant diversity estimate ranges are derived from personal observations and from the following published accounts. The high end represents the total pool of species within the region and local estimates and the lower end estimates of local species pool from Hawaii (Wetterer 1998, A.V. Suarez personal observation), Australia (Hoffmann et al. 1999, Hoffman and Parr 2008), Florida (Deyrup 2003, M. Deyrup personal communication), South Africa (Parr and Chown 2001, Parr 2008) and Mauritius (Smith and Fisher 2009, A.V. Suarez personal observation).

Population Coordinates First Record # Colonies Resident Ant Diversity Australia, Howard Springs 12.49S 131.04E 1887, 1996 3 21 – 157 South Africa, Sabi Sands 24.80S 31.50E 1905 4 34 – 121 Florida, Sarasota 27.35N 82.53W 1932 5 60 – 100 Mauritius 20.26S 57.55E 1905 4 8 – 16 Hawaii, Volcanoes N.P. 19.35N 155.47W 1879 6 2 – 10

34 Table 2.2. List of P. megacephala specimens, collection accession numbers, collection locality, and GenBank accession numbers.

Accession no. Locality mtDNA CO1 mtRNA 12s nDNA LR nDNA H3 AVS 1809 Hawaii submitted submitted X submitted AVS 1810 Hawaii submitted submitted X submitted AVS 1811 Hawaii submitted submitted X submitted AVS 1812 Hawaii submitted submitted X X AVS 1813 Hawaii X X X submitted AVS 1814 Hawaii submitted submitted X X AVS 1823 Hawaii submitted submitted X submitted AVS 1848 Hawaii submitted submitted X submitted AVS 2625 Mauritius submitted submitted submitted submitted AVS 2632 Mauritius submitted submitted X submitted AVS 2647 Mauritius submitted submitted submitted submitted AVS 2659 Mauritius submitted submitted submitted submitted AVS 2694 Mauritius submitted submitted submitted submitted AVS 2695 Mauritius submitted submitted submitted submitted AVS 2717 Mauritius submitted submitted X X BD 10 Missouri submitted submitted submitted submitted BD 3 Missouri submitted submitted submitted submitted BD 5 Missouri submitted submitted submitted submitted BD 9 Missouri submitted submitted submitted submitted BFL13 Florida submitted submitted X submitted BFL14 Florida submitted submitted submitted submitted BFL15 Florida submitted submitted submitted submitted BFL16 Florida submitted submitted X submitted BFL17 Florida submitted submitted X s ubmitted CSM1381 Florida Keys submitted submitted submitted submitted CSM1403 Florida Keys submitted submitted submitted submitted CSM2617 Uganda submitted submitted submitted submitted HS1 Australia submitted submitted submitted submitted HS2 Australia submitted submitted X submitted HS3 Australia submitted submitted submitted submitted HS4 Australia submitted submitted submitted submitted Sabi Sands 2 S. Africa X X X submitted Sabi Sands 3 S. Africa submitted submitted X submitted

35 Table 2.3. The eigenvectors calculated from the original morphological measurements and the percent contribution of components to observed variation in major and minor workers. Bolded values highlight morphological measurements are closely associated (>|0.5|) with principle components.

Principle Components PC 1 PC 2 PC 3 PC 4 Majors Head Length -0.7075 0.7002 -0.0237 -0.0926 Head Width -0.6329 -0.6822 -0.2627 -0.2549 Pronotal Width -0.2768 -0.1508 0.1492 0.9372 Tibia Length -0.1487 -0.1470 0.9530 -0.2193 Proportion of Variance 0.7873 0.1098 0.0569 0.0464 Cumulative Proportion 0.7873 0.8971 0.9536 1.0000 Minors Head Length 0.5632 0.5319 -0.4763 0.4160 Head Width 0.7127 0.0737 0.5416 -0.4394 Pronotal Width 0.1708 -0.3095 -0.6927 -0.6286 Tibia Length 0.3814 -0.7847 0.0001 0.4886 Proportion of Variance 0.5302 0.1733 0.1547 0.1409 Cumulative Proportion 0.5302 0.7074 0.8591 1.0000

36 CHAPTER 3: INFLUENCE OF NUTRITION ON INVESTMENT IN WORKER NUMBER, SIZE, AND CONDITION IN A POLYMORPHIC SOCIAL INSECT

Abstract In social insects, investment into worker size versus number is thought to play an important role in determining colony success. Additionally, colony investment into the worker force may shift in response to the availability of resources. Access to plant-based carbohydrates can influence colony growth and the monopolization of plant-based resources has been implicated in the ecological success of many groups of ants. I explored how access to carbohydrates and amino acids typical of plant-based resources influence colony investment into worker number, mean worker body size, worker size distributions, and individual worker quality (fat content) of a polymorphic ant species (Solenopsis invicta). Field collected colonies (n = 15) were divided into four experimental sub-colonies, each consisting of two queens, ~1192 workers, and ~50 brood. Each was reared on a diet of insect protein and one of four macronutrient treatment solutions (water, amino acids, carbohydrates, and amino acid & carbohydrates). Having access to carbohydrates increased the overall biomass of colonies after 60 days. This increase in biomass was a result of greater investment into worker number and size, but not an increase in the fat content of individual workers. Increased access to carbohydrate resources altered the body size distributions of colonies so that colonies produced more and larger “minor” workers but not more or larger “major” workers. These changes in colony investment shed insight into how limited resources are partitioned among a colony’s work force and may play an important role in determining invasion success of invasive ant species.

Introduction The study of life history traits is central to the fields of ecology, behavior, and evolution (Stearns 1992, Roff and Fairbairn 2007, Wolf et al. 2007). Life history theory explores investment into key biological characteristics that figure directly into the reproductive success and survival of an organism (e.g. size at birth, age at maturity, and size at maturity) (Reis 1989, Stearns 1992, Reznick et al. 2000). One of the major tenets of life history theory is that finite resources are differentially allocated to traits associated with growth, defense, and reproduction (Stearns and Fretwell 1978, Reznick 1985, Reiss 1989, Stearns 1992, Simmons and Emlen 2006). Consequently, investment into life history traits are subject to tradeoffs between resources allocated to each demand, where resources devoted to one function can no longer be devoted to another (offspring size versus offspring number) (Smith and Fretwell 1978, Stearns 1989, Stearns 1992, Roff 2000, Reznick et al. 2000). 37

Relative to solitary species, the study of life history traits in eusocial organisms is complicated by their reproductive division of labor. In social insects, reproduction is dominated by a queen caste while the majority of other tasks within the colony are performed by a worker caste often made up of sterile individuals. This separation of reproductive and non-reproductive individuals within a colony can influence tradeoffs that constrain the evolution of life history traits in solitary organisms. For example, ant workers no longer invest in morphological characters related to dispersal and mating, and rarely invest in reproduction. Additionally, ants often display considerable variation in worker body size even within a single colony. Ants within the worker caste can be subdivided based on variation in size and shape (Wilson 1953, Oster and Wilson 1978, Hölldobler and Wilson 1990, Fjerdingstad and Crozier 2006). For species with polymorphic workers, body size is both an individual trait and a colony-level trait (e.g. distribution of worker body sizes). Body size is often considered a key life history trait and is a common metric within the subdisciplines of biology (Peters 1983, Calder 1984, Schluter 1994, Brown 1995, Schoener 2011). Similarly, an ant colony’s investment into individual worker size, body size variation, and condition may influence colony fitness through colony maintenance, survival, and reproduction (Oster and Wilson 1978, Bourke and Franks 1995, Whitehouse and Jaffé 1996, Billick 2002, Powell 2008, 2009). For example, body size affects worker metabolism (Calabi and Porter 1989, Vogt and Appel 1999, Hou et al. 2010), thermal tolerance (Francke et al. 1985, Wiescher et al. 2012), locomotion (Kaspari and Weiser 1999, 2007), longevity (Porter and Calabi 1989), and foraging / prey selection (Rissing and Pollock 1984, Wetterer 1994a, 1994b, Powell and Franks 2005, Silva and Brandão 2010, Pirk and de Casenave 2011). Consequently, body size plays an important role in determining how organisms interact with their biotic and abiotic components of their environment (Davidson 1978, Traniello 1989, Silva and Brandão 2010, Wiescher et al. 2010). Variation within and between colonies in investment into the number, size and quality of workers may result from plastic responses to colony needs (e.g. defense) and the availability of food resources (Passera et al. 1996, Yang et al. 2004, Powell 2009). Furthermore, as holometabolous insects, differences in reproductive investment and feeding prior to pupation have dramatic effects on adult worker body size (Nijhout and Williams 1974, Wheeler 1991, Moczek 1998). An increase in the total amount of food resources available to a colony can increase worker size (Davidson 1978, Rissing and Pollock 1984, Davidson 1998, Smith and Suarez 2010), body size distributions (Beshers and Traniello 1994, Wheeler 1991, Tschinkel 1993), and colony size (Porter 1989, Wilder et al. 2011a). Colonies are more likely to produce larger workers as they increase in size and age, further influencing worker body size variation (Porter and Tschinkel 1985, Tschinkel 1988).

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Body size is not only influenced by the overall amount of food received, but also by diet quality. For example, in the seed-harvesting ant Pogonomyrmex badius, major workers have greater nitrogen to carbon ratios and are more enriched in δ15N compared to minor workers suggesting they assimilated more insect resources relative to seed resources than smaller workers during larval development (Smith and Suarez 2010). The relative amount of carbohydrates and proteins consumed may also affect individual worker survival and overall colony size (Dussutour and Simpson 2012). Colonies reared are carbohydrate rich diets have greater worker and brood production compared those deprived of carbohydrates (Porter 1989, Wilder et al. 2011a, Dussutour and Simpson 2012). Given that many ants get carbohydrates from plant-based resources such as honeydew excreting Homoptera, there has been a growing interest in linking ant access to such resources with their ecological success (Davidson et al. 2003, Styrsky and Eubanks 2007), particularly for invasive species (Davidson 1998, Grover et al. 2007, Tillberg et al. 2007, Helms et al. 2011, Wilder et al. 2011a). Invasive ant species such as the red imported fire ant (Solenopsis invicta) may monopolize plant-based resources within introduced ranges (Helms and Vinson 2002, Helms et al. 2011, Wilder et al. 2011b, Wilder et al. 2013). This in turn may increase colony growth and abundance within a community (Helms et al. 2011, Wilder et al. 2011a) that can determine the outcomes of direct and indirect competition (Franks and Partridge 1993, Traniello 1994, Davidson 1998). In this study, I manipulated diet in experimental colonies of Solenopsis invicta to examine how variation in macronutrient availability influences colony investment into worker number, and mean worker body size, condition (fat content). . Wilder and colleagues (2011) reported that access to additional carbohydrates resulted in a near doubling of dry biomass of workers over a 60 day period. Here I examine the relative contribution of worker number, body size, and fat content to the increased biomass. While previous work has examined the role of diet on worker production or biomass (Porter 1989, Wilder et al. 2011a, Dussutour and Simpson 2012), few studies examine multiple metrics (e.g. worker number, size, and quality) simultaneously. I also test whether additional macronutrients (carbohydrates, amino acids or both) change how colonies invest into the distribution of worker body sizes within a colony and if there are potential tradeoffs between investment into worker quality or number across treatments.

Methods Study System I chose to examine the effects of plant-based resources on colony investment into worker number, condition and size in the red imported fire ant (Solenopsis invicta), an invasive species native to northern Argentina and southern Brazil which was first introduced to Mobile, Alabama in the 1930’s and has successfully spread throughout the southeastern United States and elsewhere (Tschinkel 2006). These ants are continually polymorphic with a wide range of body sizes (head widths 0.45 mm – 1.50 mm) and a 39

distribution skewed towards smaller workers (Wood and Tschinkel 1981, Araujo Tschinkel 2010). Although it is largely omnivorous (Tennant and Porter 1991, Vogt et al. 2002, Wilder et al. 2011b), S. invicta frequently consumes plant-based resources particularly within its introduced range in the southeastern United States (Helms et al. 2011, Wilder et al. 2011b).

Colony Collections and Experimental Design I excavated colonies of polygyne S. invicta from the campus of Texas A&M University (College Station, Brazos County, Texas, USA) in the spring of 2008 and 2009 (Wilder et al. 2012). I slowly flooded the field-collected colonies with water to separate workers, brood, and queens from the soil. This material was used to make standardized laboratory colonies that each consisted of two queens, ~50 brood and 1 g wet mass of workers (0.3456 ± 0.009 g dry mass or 1192 ± 62 individuals) (Wilder et al. 2011a). Each field-collected colony was split into four lab colonies, one replicate for each treatment. Laboratory- colonies were housed in plastic containers (56 cm length x 40 cm width x14 cm height) lined with fluon, and provided with a vial of water and a darkened petri dish lined with plaster for a nest. The plaster substrate was moistened twice a week. I provided all colonies with two freshly killed crickets, Acheta domesticus, three times per week, which was ad libitum prey for colonies used in these experiments. Colonies were maintained on a 12:12 light:dark photoperiod with 40 – 70% humidity and a daily temperature cycle that included 8 hours during daylight at 32ºC and 16 hours at 24ºC. The experiments were run for 60 days after which the colonies were frozen. To test the effects of carbohydrates and amino acid components of artificial extrafloral nectar on investment into worker number and condition, I supplemented the cricket fed colonies with a 5 mL vial of one of four randomly assigned treatments: “water” (n = 12); “carbohydrate” - a solution containing only the carbohydrate component of extrafloral nectar (n = 13); “amino acid” - a solution containing only the amino acid component of extrafloral nectar (n=14); “nectar” -a solution containing both the carbohydrate and amino acid components of extrafloral nectar (n=15). The artificial nectar mimicked the chemical composition of extrafloral nectar of Passiflora sp. (Lanza 1991) and consisted of 1 L of water mixed with carbohydrates (108 g sucrose, 90 g glucose, 53 g fructose) and amino acids (0.0232 g aspartic acid, 0.512 g glutamine, 0.0404 g glutamic acid, 0.0194 g histidine, 0.0436 g isoleucine, 0.04 g leucine, 0.118 g phenylalanine, 0.368 g proline, 0.0704 g tryptophan, and 0.1122 g tyrosine). This artificial nectar recipe has carbohydrate (251 g/L) and amino acid (1.3 g/L) concentrations similar to a wide range of extrafloral nectars (carbohydrate, mean = 222, median = 183; amino acid, mean = 3.4, median = 1; Blüthgen et al. 2004). I only included carbohydrates and amino acids in the artificial extrafloral nectar as other components (such as volatiles to attract pollinators) are not likely needed for the nutritional demands of

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consumers (Blüthgen et al. 2004, Stadler and Dixon 2005). I replaced the vials with the experimental treatments twice each week.

Measurements At the end of the experiment, I counted the total number of workers and took head measurements from 200 randomly selected individuals per colony to determine mean size and body size distributions for each treatment. I mounted the heads of workers on paper cards with double-sided tape, and for each ant head length and width was measured using a Leica M205 C stereo microscope (467 nm resolution) attached to a five megapixel Leica DFC 425 digital microscope camera. Head length (HL) and head width (HW) were chosen as they are reliable and widely used indicators of overall body size (Porter 1983, Kaspari and Weiser 1999, Araujo and Tschinkel 2010).

Fat Content To estimate the average fat content of workers, I randomly chose ten workers from each replicate and dried specimens in an oven at 60ºC for 48 hours. After 48 hours, I placed workers into 1.5mL microcentrifuge tubes (to prevent re-hydration) and then weighed each individual using a UMX2 microbalance with 0.1 μg resolution (Mettler-Toledo, Columbus, OH). Specimens were then placed in gelatin capsules and arranged in a Soxhlet Extractor for 24 hours with ether. After 24 hours, the ants were again dried in an oven at 60ºC for several hours and were re-weighed on the microbalance. I estimated fat content as (dry mass – lean mass)/dry mass (after Smith and Tschinkel 2009).

Data Analyses Worker Number, Size and Fat content Worker number, size and percent fat content were compared between treatments in a strip-block mixed model ANOVA with treatments (amino acids and carbohydrates) as subplots and colony as a random blocking factor. Worker number and fat content were not normally distributed and were log10 and arcsine-root square transformed respectively. For both worker number and worker size, colony identity was significant in the model (p < 0.01) (see Supplementary Table 2). Therefore when analyzing worker number, head length, and head width, I only included replicates with all four treatments from the same field colony (n = 9) (see Supplementary Table 3). Analyses completed in SAS software, version 9.3 Copyright © 2002-2010, SAS Institute, Cary, NC, USA.

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Worker Body Size Distributions To compare body size distributions I used a G-test to compare the distributions of worker head widths, separated into two bins and four bins. Within a mature colony, the worker population consists to two distinct subpopulations (majors and minors) where major workers range in size between 0.75 – 1.6 mm (Tschinkel 1998) (Figure 3.5). The observed head measures ranged in size from 0.5 – 1.491 mm. I therefore created two groups to compare distributions of workers within a colony across treatments: minors 0.5 – 0.75 mm and majors 0.75 – 1.5 mm. To more comprehensively compare the distribution of workers within the two subpopulations, I divided the minors into two bins (0.5 – 0.675 mm and 0.675 – 0.75 mm) and the majors into two bins (0.75 – 1.125 mm and 1.125 – 1.5 mm). I used separate G-tests to compare the distributions of workers within each of the two subpopulations.

Tradeoffs: Worker Number, Size, and Fat Content I used correlation coefficients to examine if tradeoffs existed between total dry worker biomass of workers and colony investment into worker number of workers, body size, or fat content. For these analyses I pooled all replicates, regardless of treatment. Analysis completed with SAS software, version 9.3 Copyright © 2002-2010, SAS Institute, Cary, NC, USA.

Results Worker Number, Size and Fat Content Lab colonies supplemented with carbohydrates produced more workers than colonies not supplemented with carbohydrates (ANOVA F1,24 = 4.47, p = 0.045) (Figure 3.1). I found no effect of amino acid supplementation (ANOVA F1,24 = 0.03, p = 0.87) or an amino acid and carbohydrate interaction (ANOVA F1,24 = 0.50, p = 0.49) on worker number (Figure 3.1). Mean worker size (both HL and HW) was larger in colonies supplemented with carbohydrates than those of colonies not supplemented with carbohydrates (HL ANOVA F1,24 = 4.67, p = 0.041; HW

ANOVA F1,24 = 5.85, p = 0.024) (Figure 3.2). However, there was no effect of amino acid supplementation on worker size (HL ANOVA F1,24 = 0.01, p = 0.96; HW ANOVA F1,24 = 0.05, p = 0.82)

(Figure 3.2) or an amino acid and carbohydrate interaction (HL ANOVA F1,24 = 0.27, p = 0.608; HW

ANOVA F1,24 = 0.05, p = 0.83 ). Supplementation of carbohydrates (ANOVA F1,24 = 2.22, p = 0.149), amino acids (ANOVA F1,24 = 0.04, p = 0.84), or their interaction (ANOVA F1,24 = 0.25, p = 0.62) had a significant effect on worker fat content (Figure 3.3).

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Worker Body Size Distributions Investment into minor (0.5 – 0.75 mm) and major (0.75 – 1.5 mm) workers differed between treatments (G-test G =39.99, d.f. = 3, p < 0.0001) (Figure 3.4A). Further subdividing minors and majors each into two categories, I found differences in investment within the minor subpopulations (0.5 – 0.675 mm and 0.675 – 0.75 mm) (G-test G = 314.26, d.f. = 3, p < 0.0001) (Figure 3.4B) but not between the subpopulations of majors (0.75 – 1.125 mm and 1.125 – 1.5 mm) (G-test G = 2.24, d.f. = 3, p = 0.52) (Figure 3.4C, Figure 3.5). This pattern was driven by colonies with access to carbohydrates producing more minor workers with wider heads (0.675 – 0.75 mm) compared to without access to carbohydrates (G-test G = 191.42, d.f. = 1, p < 0.0001) (Figure 3.4B). There was no significant difference in the distribution of majors between fed carbohydrates versus not fed carbohydrates (G-test G = 0.20, d.f. = 1, p = 0.66) (Figure 3.4C).

Tradeoffs: Worker Number, Size, and Fat Content Across all lab colonies, increased worker biomass resulted in increased worker number within a colony (R2 = 0.7126, p < 0.001) (Figure 3.6A) and decreased mean worker size (R2 = 0.1269, p < 0.001) (Figure 3.6B). There was no relationship between biomass and percent worker fat content (R2 = 0.0015, p = 0.82) (Figure 3.6C). There was also significant negative correlation between worker number and mean worker size, (r = 39.32, p < 0.001) (Figure 3.6D).

Discussion I previously found that access to carbohydrates increased overall biomass in both field and lab colonies of S. invicta (Wilder et al. 2011a). In this study, I determined that differences in worker biomass of lab colonies results from colony investment into more workers and / or larger workers but not into individual worker condition (fat content). More specifically there are a greater number of “large minor” workers within the minor subpopulation (0.5 – 0.75 mm) in colonies with access to carbohydrate resources than those from colonies without access to carbohydrates. The observed differences in worker size therefore result from a shift in the mean body size of minor workers (the creation of more “medium” sized workers) rather than investment into more major workers. It is has been established that colonies reared on high carbohydrate (or relatively high carbohydrate to amino acid ratio) diets have higher dry biomass of workers, dry biomass of brood, and lower mortality then those reared on low carbohydrate (or relatively low carbohydrate to amino acid ratio) diets (Williams et al. 1980, Porter 1989, Wilder et al. 2011a, Dussutour and Simpson 2012). However, it was unclear if differences in worker biomass a difference in colony investment into work size, number or

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quality (fat content). In the present study, I found that differences in dry biomass of colonies supplemented carbohydrates (carbohydrates and carbohydrates & amino acid solutions) is a result of colonies investing more into more workers of greater mean size, rather than only into worker condition. Across all colonies, regardless of treatment, I found evidence for a tradeoff between investment into worker number and worker size. The pattern suggests as investment into worker number increases, the average size of workers decreases. This increase in the number of workers in the carbohydrate treatment is due to two factors, an increase in worker production and an increase in worker longevity (Wilder et al. 2010, Wilder et al. 2011a), which may also be related to worker size. Future work is still need to untangle how these factors are inter-related and contribute to the observed patterns. I found a significant effect of colony identity on measures of size and worker number. This is not surprising because as colonies age, the colony worker force grows both in size and number (Wilson 1983, Tschinkel 1988, Tschinkel 1993, Johnson 2002). Thus variation among field collected colonies in both age and size likely influenced initial worker metrics and these differences may have carried through the 60-day period of the experiment. Colony age in S. invicta is known to impact both the size and the distribution of worker size within a nest (Tschinkel 1988, 1993). Larger colonies typically produce larger workers (Tschinkel 1988, 1993, 2006). The initial colony size (1192 ± 62 individuals) is significantly smaller than mature S. invicta colonies of 2200,000 workers (producing 20,000 to 40,000 workers annually) (Tschinkel 1988, 1993). It is also important to note, that the initial worker number may have artificially constrained the size of workers produced during the duration of the experiment. If I started with larger laboratory colonies I potentially could have seen a shift in the size and number of workers within the major subpopulation (0.75 – 1.6 mm) (Tschinkel 1988). Despite being relatively small compared to natural colony sizes, the response of small colonies to access to carbohydrate resources is informative. As invasive ant species are likely introduced as small propagules, consisting of 10 – 1000 workers and a queen (Hee et al. 2001, Tsutsui and Suarez 2003). Finally, because I standardize initial lab colony size and account for colony identity in the statistical models, I am confident the observed differences reflect a response to diet. The importance of carbohydrate resources to worker size and growth, suggests that small colonies with access to carbohydrate resources potentially invest more into more workers and more “medium” sized workers. Within introduced populations, colonies of S. invicta are often at a lower estimated trophic position, (feeding more heavily on plant-based resources) than colonies in native populations (Wilder et al. 2011b). The importance of carbohydrate resources to colony investment into its worker force is likely a result of differences in digestive abilities of ant larvae and adult workers. Adult workers of S. invicta, for example, have a reduced digestive system compared to larvae and may carry solid foods to larvae for

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digestion (Vinson 1983, Tschinkel 2006). When only insect prey is available, larvae may divert resources away from their own growth to digest food to provide energy to adult workers. This dynamic could influence larval growth and development as well as limit the available energy to workers because larvae must balance nourishing themselves and feeding workers. Liquid carbohydrates, on the other hand, are relatively easy to digest by workers that allow workers to fully satisfy their own energetic requirements without diverting resources away from developing larvae. The availability of carbohydrate resources is known to play an important part in fueling worker activity and colony growth. It also helps explain why carbohydrate resources are tightly regulated by ant colonies and why mutualisms between ants and plants (or in some cases hemipterans that produce carbohydrate resources) have repeatedly evolved and are widespread in nature (Hölldobler and Wilson 1990, Dussutour and Simpson 2009, Cook et al. 2010). While access to carbohydrate resources increased colony biomass (Wilder et al. 2011a), and influenced body size distributions, all of the colonies were fed an ad libitum diet of crickets. The addition of crickets to colony diet likely reduced the impact of amino acid supplementation to worker number, size, and quality. If these colonies relied solely on the diet supplement treatments, it is likely that worker size, number, and overall worker quality would have also varied among individual treatments. Future experiments that include more extreme variation in diet, and run for longer periods of time, will presumably elucidate the importance of macronutrients to colony investment into worker number and size. Access to plant-based resources, in particular carbohydrate resources, plays an important role in determining colony investment into an important life history trait (worker body size and body size distribution). To a lesser extent, plant-based resources also influence colony investment in worker number and fat content. Nearly all studies of the role carbohydrates resources for ants have assumed the resources are used to fuel worker activity (Davidson 1997), aggression or foraging (Grover et al. 2007, Kay et al. 2010). However, carbohydrate resources are also essential to the production of larval workers (Helms and Vinson 2008, Dussutour and Simpson 2009, Wilder et al. 2011a, Dussutour and Simpson 2012). Larval development of ants and other insects is thought to be protein limited, but growing evidence suggest that energy is also needed growth, particularly in holometabolous insects (Fagan et al. 2002, Dussutour and Simpson 2009, Raubenheimer et al. 2009, Wilder and Eubanks 2010, Simpson and Raubenheimer 2012). My results suggests that the traditional paradigms of ant nutrition, including colony investment into worker production is limited by protein (amino acids) may need to be reevaluated. Ants require specific concentrations of nutrients in particular ratios to maintain worker activity, colony growth, and worker mortality (Dussutour and Simpson 2009, 2012). Finally, the results show the nutritional ecology can have important implications for the growth and development of invasive species like S. invicta.

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Monopolization of plant based resources changes may facilitate the invasion of new environments because a larger and more numerous worker forces can overpower competitors (Franks and Partridge 1993, Silva and Brandão 2010), withstand larger temperature fluctuations (Francke et al. 1985, Wiescher et al. 2012) and longer periods of food deprivation (Kaspari and Vargo 1995, Smith 2007), and overall live longer (Porter and Calabi 1989). Therefore, access to carbohydrate resources may significantly contribute to the establishment and subsequent spread of invasive ant species.

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Chapter 3 Figures and Tables Figure 3.1. Mean (± SE) worker number for colonies supplemented with amino acids (dashed lines) and/or carbohydrates (grey bars) to their diet. Worker number was greater for colonies supplemented with carbohydrates compared to colonies not supplemented with carbohydrates (ANOVA F1,24 = 4.47, p = 0.045). There was no affect of amino acid supplementation on worker number (ANOVA F1,24 = 0.03, p = 0.87).

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Figure 3.2. Affect of diet supplementation on mean (± SE) worker head length and width. A. Colonies supplemented with carbohydrates (gray bars) had larger head lengths than those not supplemented carbohydrates to their diets (ANOVA F1,24 = 4.67, p = 0.041) but there was no effect of amino acid (dashed lines) diet (ANOVA F1,24 = 0.01, p = 0.96) or interaction between carbohydrates and amino acid (ANOVA F1,24 = 0.01, p = 0.96) supplementation on worker head length. B. Similarly, head width was significantly larger in colonies supplemented carbohydrates than those not supplemented carbohydrates (ANOVA F1,24 = 5.85, p = 0.024) and there was no effect of amino acid (ANOVA F1,24 = 0.05, p = 0.82) or interaction between carbohydrates and amino acid (ANOVA F1,24 = 0.05, p = 0.83) supplementation on worker head width.

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Figure 3.3. The mean (± SE) percent fat content workers supplemented carbohydrates (gray bars) and/or amino acids (dashed lines) to their diet. There was no significant effect of carbohydrates (ANOVA F1,24 = 2.22, p = 0.15), amino acids (ANOVA F1,24 = 0.04, p = 0.84), or their interaction (ANOVA F1,24 = 0.25, p = 0.62) on worker fat content.

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Figure 3.4. The proportion of workers from two subpopulations (minors = 0 - 0.75 mm and majors = 0.75 – 1.6 mm) (see Tschinkel 1988). A. There proportion of workers is significantly different between diet supplements (G-test G =39.99, d.f. = 3, p < 0.0001). Specifically, there is a greater proportion of minor workers with wider HW from colonies reared with access to carbohydrates (gray bars) (G =34.2 d.f. = 1, p < 0.0001). B. The proportion of smallest minor workers (0 – 0.675 mm) is significantly different between treatments (G-test G = 314.26, d.f. = 3, p < 0.0001). Colonies with access to carbohydrates had significantly more workers with larger heads (G = 191.42, d.f. = 1, p < 0.0001). C. The proportion of the smallest major workers (0.75 – 1.125 mm) was not significantly different between treatments (G-test G = 2.24, d.f. = 3, p = 0.52). There is no significant difference in HW of major workers from colonies reared without access to carbohydrates and those with access to carbohydrates (G-test G = 0.2, d.f. = 1, p = 0.66).

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Figure 3.5. Worker body size distributions based on head width of experimental colonies of Solenopsis invicta after being reared for 60 days on diets that varied access to water (blue diamonds) carbohydrates (green triangles), amino acids (red squares), or both (purple asterisks).

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Figure 3.6. The relationships between: A. Worker biomass and worker number (R2 = 0.7126, p < 0.001). B. Worker biomass and the mean worker head width. (R2 = 0.1269, p < 0.001) C. Worker biomass and worker percent fat content. (R2 = 0.0015, p = 0.82) D. Workers number and mean worker head length (r = 39.32, p < 0.001). All colonies were pooled for these analyses.

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CHAPTER 4: TESTING THE PREDICTIONS OF LANCHESTER’S LAWS OF COMBAT WITH A CONTINUOUSLY POLYMORPHIC ANT: TRADEOFFS BETWEEN WORKER NUMBER AND SIZE

Abstract There is growing interest in co-opting mathematical models from operations research to form predictions of the outcomes of aggressive competitive interactions or interference competition of social groups. Lanchester’s laws of combat describe attrition rates during battles between groups and are potential tools to develop predictions to the outcome of aggressive competitive interactions in ants. Lanchester’s square law predicts that if a group is numerically superior then attackers should simultaneously engage their opponents. If a group is numerically inferior, fighting in a series of one-on- one duels would be advantageous. Using laboratory foraging experiment, I tested the predictions of Lanchester’s laws in interference competition and how varying worker number and body size influences exploitation competition with the continuously polymorphic, Solenopsis invicta. From the foraging experiments, I found that for colonies of equal biomass but different worker size (i.e. different worker number) colonies with smaller bodied workers typically discover baits more quickly than those with larger workers but both large and small worker colonies retrieve equal amounts of food resources. Colonies of smaller workers do not suffer fewer losses when outnumbering their opponents 4: 1 than when equally matched. However colonies of larger body size suffer greater losses when facing numerically superior opponents than when equally matched. More specifically when colonies of large bodied workers compete with numerically dominant opponents they suffer a greater number of losses in open arenas than closed arenas (e.g. “support for the square law”). In closed arenas, the relatively few but large workers can use their size advantage in the narrow, confined spaces to defend against the numerically superior but smaller workers (e.g. “support for the linear law”). In the field I found more small workers above ground than below ground which supports the predictions of the linear law because in narrow confined spaces larger size is advantageous. Lanchester’s laws provide context, however simple, to predict the outcomes of competitive interactions and in the future may help decipher how competition influences community structure and species composition.

Introduction Organisms can benefit from living in groups through a variety of mechanism including: reduced predation risk (Powell and Clark 2004, Lung and Childress 2007, Townsend et al. 2011), cooperative

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breeding (Wilson 1971, Cockburn 1998, Leadbeater et al. 2011, Brouwer et al. 2012), increased foraging success (Creel and Creel 1995, Fernández Campón 2007, Liker and Bokony 2009), and/or group defense (Jerome 1998, Wilson et al. 2001, Powell and Clark 2004). Specialization within a group can further enhance the benefits associated with sociality (Oster and Wilson 1978, Ridley and Raihani 2008, Settepani et al. 2013). For example, in eusocial organisms, reproduction is dominated by a queen caste while most remaining tasks are completed by a functionally sterile caste (Wilson 1971). Reproductive caste specialization reduces the relative importance of losing individual workers through risky behaviors like foraging, and can increase efficiency in worker production (Oster and Wilson 1978, Porter and Tschinkel 1985, Powell 2009). Additionally, by separating reproductive from non-reproductive individuals into separate castes, workers can be “released” from constraints associated with dispersal, mating, and reproduction, allowing for increased morphological variation. In approximately 13% of ant species worker variation is so great that the worker caste can be subdivided into sub-castes based on size and shape (Hölldobler and Wilson 1990). Increased morphological and behavioral specialization within groups may contribute to why many ants have achieved high abundance and ecological dominance in many terrestrial habitats (Hölldobler and Wilson 1990, Folgarait 1998, Lach et al. 2010). Variation in investment into worker number and body size, both within and between ant species, can influence the outcomes of competitive interactions (Davidson 1998, Kaspari and Weiser 1999, Sarty et al. 2006, Pearce-Duvet et al. 2011, Dornhaus et al. 2012). Specifically, colony investment into worker number and body size can affect resource discovery (Davidson 1977, Davidson 1998, Pearce-Duvet et al. 2011), food retrieval (Traniello 1989, Powell and Franks 2005, Schöning et al. 2005, Dornhaus et al. 2012), and resource defense (Adams 1990, 1994, Whitehouse and Jaffe 1996, Batchelor and Briffa 2012). The outcomes of competitive interactions between colonies of different worker body size and number are also context dependent (Traniello 1989, Franks and Partridge 1993, Davidson 1998). For example, in the context of direct competition, larger individuals may more easily kill smaller individuals (Adams 1994, McGlynn 2000, Powel and Clark 2004), while small workers in large numbers may be just as likely to overpower larger competitors (Plowes and Adams 2005, Holway and Suarez 2006). Given the general importance of competition in ecology (MacArthur 1958, Paine 1966, Schoener 1983, Tillman 1994), developing a predictive framework for the outcomes of competitive interactions between colonies based on investment into worker number and size will improve our general understanding of ants and other social species. There is a growing interest in co-opting mathematical models from operations research of human warfare, which provides sets of assumptions to specific context of conflict necessary to form predictions to the outcomes of aggressive, competitive interactions of social groups (Franks and Partridge 1993,

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McGlynn 2000, Wilson et al. 2001, Adams and Mesterton-Gibbons 2003, Plowes and Adams 2005). Partridge and Franks (1993) first suggested the utility of Lanchester’s (1916) laws of combat to predicting the outcome of aggressive competitive interactions among ants. The laws are based on attrition rates of two competing forces, where groups of combatants suffering fewer losses relative to their opponent “win” a contest (Lanchester 1916, Wallace 1968, Karr 1983, Franks and Partridge 1993, Adams and Mesterton- Gibbons 2003). Lanchester’s laws provide the framework to predict the outcomes of aggressive interactions solely based on two assumptions relative to worker number and body size. The “square law” assumes a larger group can simultaneously engage their opponent, concentrating their attacks. The likelihood of winning an interaction under the assumptions of the square law, is proportional to the square of group size (worker number), hence its name. The “linear law” assumes combat is a series of parallel one-on-one duels where individuals with greater fighting ability (worker body size) determine the outcome of duels. This law derives its name because likelihood of winning an engagement is proportional to group size and fighting ability. The prediction of Lanchester’s Laws for ants is largely based on predictions derived from Franks and Partridge (1993) who proposed that the outcome of competition between two groups will predominantly be determined by group size (McGlynn 2000, Wilson et al. 2001, Adams and Mesterton- Gibbons 2003, Plowes and Adams 2005). Franks and Partridge (1993) proposed that if the attacking side is numerically superior, the attackers should simultaneously engage each of their opponents. However, if the attacking side is numerically inferior, engaging in a series of one-on-one duels would be advantageous (Franks and Partridge 1993). These predictions have largely been supported in a few different systems (McGlynn 2000, Wilson et al. 2001, Adams and Mesterton-Gibbons 2003, Powell and Clark 2004, Plowes and Adams 2005). In army ants, for example, large-scale raids consisting of hundreds of thousands of relatively small workers (low individual fighting value) prey upon numerically inferior colonies of social insects (Franks 1982, Franks 1985). When army ants raid leaf-cutting ants (Atta), with worker forces in the millions (Fowler et al. 1986, Wirth et al. 2003), army ants recruit their largest workers (high individual fighting value) to engage the defending Atta workers (Powell and Clark 2004). Most prior examinations of Lanchester’s laws are based on interactions in open arenas that mimic above ground foraging (McGlynn 2000, Wilson et al. 2001, Adams and Mesterton-Gibbons 2003, Plowes and Adams 2005). However, not all species forage above ground; many ant species forage below ground (Porter and Tschinkel 1986, Berghoff et al. 2002, Wilkie et al. 2007) and interact with other ants in the confined spaces of tunnels or the opposing colony during raids (Alloway 1979, Tschinkel 1992, Powell and Clark 2004). The context within which an aggressive interactions occurs (above vs. below ground) may be important in determining its outcome. The relatively open spaces above ground may be more

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conducive to simultaneously engaging opponents, reduce the numerically larger group’s attrition rate, and favor groups with more individuals. In contrast, below ground interactions are more likely to occur in confined spaces and force combat into a series of one-on-one duels where the attrition rate of large bodied individuals may decrease and favors groups of larger size. In this study, I use a polymorphic ant species (Solenopsis invicta) to examine how tradeoffs in worker number versus worker size influences the outcomes of competitive interactions. I stage interactions between colonies of equal biomass but workers of different sizes thus different worker number. If worker number and body size influence attrition rate based on foraging arena (open vs. closed) then experimental colonies consisting of fewer large workers will suffer a lower attrition when competing for resources in confined spaces where combat can occur one-on-one. In contrast, I predict that colonies with a greater number of smaller workers will experience lower attrition in open arenas where they can surround their larger opponents. I complement the laboratory experiments with a survey of how S. invicta deploys their workers in the field to explore if the number and size of workers foraging above and below ground match the predictions of Lancaster’s square and linear laws. If worker number and body size influence attrition rate based on foraging location (above vs. below ground) then the number of foragers from above ground pitfall traps will be greater than below ground traps; and below ground foragers will be larger than above ground foragers. While Lanchester’s laws are useful for building predictions for aggressive interactions they do not account for indirect (exploitation) competitive interactions. Competitors can win a contest not only by causing attrition in an opposing force to gain access to resources but also by discovering and removing a food resource faster than competitors (Davidson 1998, Holway 1999). I therefore also use laboratory experiments to explore how tradeoffs between investing in worker size versus worker number influence food resource discovery, dominance, and retrieval under different foraging conditions. If investment into greater worker number or size affects discovery and dominance of food resources, I predict experimental colonies with more workers will discovery baits more quickly than colonies with fewer, larger workers. I also predict that colonies with larger workers will retain or usurp baits in confined foraging arenas, while colonies with more, smaller foragers will be more likely to monopolize resources in open arenas.

Methods Study System I test the predictions of Lanchester’s laws using the red imported fire ant (Solenopsis invicta), an aggressive invasive species native to northern Argentina and southern Brazil. Red imported fire ants were first introduced to Mobile, Alabama in the 1930’s and have since successfully spread throughout the

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southeastern United States and elsewhere (Tschinkel 2006). These ants are continually polymorphic with a wide range of body sizes (head widths 0.45 mm – 1.50 mm) with a distribution skewed towards smaller individuals (Wood and Tschinkel 1981, Araujo Tschinkel 2010). Fire ant colonies can contain tens of thousands of workers and they forage both above and below ground for resources (Porter and Tschinkel 1987, Tschinkel 2010). The monogyne (single queen) form is also very territorial; territory size in strongly influenced by the fighting ability of neighboring colonies and workers from different colonies will exhibit pronounced intra-specific aggression in laboratory assays (Adams 2003).

Foraging Experiment Colony Collections I excavated monogyne colonies of S. invicta in central Texas in the summer 2009. To separate workers, brood, and queens from the soil colonies were slowly flooded the field-collected colonies with water. Field-collected colonies were place in a fluon lined plastic containers (55 cm length x 40 cm wide x 14 cm height). A series of soil sieves (1.4 mm, 0.85 mm, 0.71 mm) was used to separate workers into three size classes (“small” “medium” “large”). Sieved-workers were housed in smaller fluon lined plastic containers (35 cm length x 20 cm width x 11 cm height). Worker populations were maintained on a sugar- water, water, and insect prey (Acheta domesticus) diet, feed three times a week, and provided a darkened petri dish lined with plaster for a nest moistened twice a week. To avoid measuring mortality induced by nest excavation and sieving during trials, I allowed colonies to acclimate for minimum of three days prior to sieving and trials. All colonies were maintained in a green house under natural temperature and daily light cycles from June - August 2009.

Experimental Design To test the predictions of Lanchester’s laws I created experimental colonies from the field- collected colonies. I first confirmed that workers in each colony pair were highly aggressive to one another using an assay consisting of adding ten workers from each field-collected colony in a fluon lined vial. High levels of aggression were defined as biting and stinging in the first minute. Each experimental colony received equal biomass (0.5 g wet weight of workers) and consisted of 250 ± 12 workers (mean ± SE) large worker or 840 ± 23 workers (mean ± SE) small workers. From the sieved colonies I used “large” workers (0.85 – 1.0 mm head width) and “small” workers (0.5 – 0.71 mm head width) size classes. Sieved worker groups were not fed 48 hours prior to being placed into experimental colonies. Each experimental colony was placed in a plastic container (17 cm x 17 cm x 6 cm) lined with fluon and provided a with a 5 ml water tube 24 hours prior to foraging experiment. Each field-collected colony pair

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was used to generate one replicate. Colonies were then connected to a common foraging arena that simulated either an open environment or a confined environment (see below) in the following size combinations: small vs. small, small vs. large, and large vs. large. As a negative control I used the same staged interactions for experimental colonies from the same field-collected colony (nestmates). For open foraging arenas, I used a fluon lined plastic container (80 cm x 40 cm x 14 cm). For the confined foraging arena, I simulated below ground foraging tunnels using clear plastic tubing (2 mm inner diameter) in a plastic container (80 cm x 40 cm x 14 cm). Plastic tubing (50 cm of clear plastic tubing x 6 mm inner diameter) connected all foraging arenas to experimental fractional colonies (Figure 4.1). Bait was placed in the center of each foraging arena at approximately 1 m from plastic containers of experimental colonies. For the confined arenas, I placed bait in a 1.5 mm flat bottom microcentrifuge tube connected to plastic tubing from either end. In the open arenas, I placed 0.5 g of tuna bait and for the closed arenas I used 0.25 g of tuna. The reduced amount of bait for closed arena was due to space restrictions of the microcentrifuge tube. I estimated worker mortality for interactions between experimental colonies with similar body size (large vs. large and small vs. small) by dividing the total number of deceased workers by half. Experimental colonies of equal worker number and body size are assumed to be equally matched. Otherwise worker size and identity was determined by distinct differences in worker head size. I took behavioral observations for four hours (8 am – 12 pm). During observation period I recorded the colony identity and size of foragers to locate the bait resource first and any turnover of bait ownership. After 24 hours I recorded the mass of bait post-trial and counted the number of deceased workers to estimate attrition for each colony.

Field Survey To examine the number and size of workers that foraged above or below ground, I sampled foragers from five locations in the southern United States (Corsicana, TX; Shreveport, LA; Monroe, LA; Macon GA; and Lake Placid, FL) during the summer 2010. Within each location I sampled foragers using pitfall traps surrounding five colonies with > 50 m between each colony. Eight pitfall traps circumscribed each colony and consisted of two sets of below and above ground pitfall traps. Traps of each type were placed around colonies in pairs, (in series) at 0.5 m and 1.0 m from nearest edge, and in the four cardinal directions. I used 50 mL tubes for both above and below ground pitfall traps. Below ground pitfall traps were built using 50 mL tubes with 16 holes (7 mm diameter) in two offset rows, around the circumference of the tube (1.5 – 4.0 cm from the top). Above ground pitfall traps were placed flush with the soil surface.

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Below ground pitfall traps were buried flush with the 50 mL cap. All pitfall traps were filled with salt and soap water solution and collected after 48 hours. For each colony I counted the number of foragers and measured forager body size (up to 100 workers per colony). To measure body size, I mounted heads of foragers on paper cards with double-sided tape. For each ant, head width was measured using a Leica M205 C stereo microscope (467 nm resolution) attached to a five megapixel Leica DFC 425 digital microscope camera. Head width (HW) was chosen as they are reliable and widely used indicators of overall body size (Porter 1983, Kaspari and Weiser 1999, Araujo and Tschinkel 2010).

Statistical Analysis Foraging Experiment Attrition To compare mean forager attrition and proportion of colony lost I used proc mixed and lsmeans with treatment, arena type, and size pairings as fixed effects and colony pairing as a random effect. Proportion workers lost was calculated by (number workers lost/mean number workers in 0.5 g workers) and was used to estimate colony biomass lost. The total number of dead small and large foragers was analyzed separately. For both size categories, experimental colonies foraging against nestmates (negative controls) suffered fewer losses than experimental colonies foraging against non-nestmates (small:

ANOVA F1,34 = 22.24, p = 0.001; large: ANOVA F1,34 = 6.87, p = 0.01).

Time to Discovery I examined the time to discovery of the bait resource using proc mixed and lsmeans using size pairing as a fixed effect and colony identity as a random effect. There was no effect of colonies foraging against competitors or nestmates on time to discovery (open: ANOVA F1,20 = 0.3, p = 0.93; closed:

ANOVA, F1,20 = 0.5, p = 0.82) therefore both sets of trials were included in each analysis. Additionally I separated the analysis of open and confined arenas because the tunnels of closed arenas tubes lead foraging worker directly to bait, requiring less “exploration”.

Resource Retrieval The proportion of resource retrieved was compared between interactions with proc mixed and lsmeans with treatment and size pairings as fixed effects and colony pairing as a random effect. Resource retrieval was analyzed separately for arena type because the microtubing in closed arenas forging limits traffic and load capacity not observed in open arenas.

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Field Survey To compare differences in forager number and body size from below and above ground pitfall traps from each colony, from five populations in the southern United States, I used a nested ANOVA.

Forager number was not normally distributed and was log10 transformed. The majority of the variation (> 90%) in forager number and body size is attributable to trap type. I therefore used a paired t-test the difference in mean forager number and size by trap type. All analyses were completed in SAS software, version 9.3 Copyright © 2002-2010, SAS Institute, Cary, NC, USA.

Results Foraging Experiment Attrition Overall mortality of experimental colonies paired with nestmates suffered the lowest mortality

(small: ANOVA F1,34 = 22.24, p = 0.001; large: ANOVA F1,34 = 6.87, p = 0.01). For small worker experimental colonies, there was no effect of arena type and size pairing on the mean number of deceased workers (ANOVA, p > 0.05) (Figure 4.2A). In contrast, experimental colonies of large worker suffered higher attrition when competing against smaller bodied colonies (F1,15 = 9.06, p > 0.01) and when in open arenas. Specifically, when competing in open arenas, more large workers die when competing against small worker experimental colonies than when competing against larger worker colonies (t-test, t = -3.46 p = 0.003). Similarly when larger worker colonies compete against smaller worker colonies, attrition of large workers is higher in open arenas then in closed arenas (t-test, t = -2.35, p = 0.03) (Figure 4.2B). The proportion of workers lost from each experimental colony, was not different between treatments, arena type, or size pairings (ANOVA, p > 0.05) (Figure 4.2C).

Time to Discovery There no effect of colonies foraging against competitors or nestmates on time to discovery (open:

ANOVA F1,20 = 0.3, p = 0.93; closed: ANOVA, F1,20 = 0.5, p = 0.82). From all trails where bait discovery occurred within the four hour observation period (n = 74), colonies of small workers arrived first in ~55% of staged interactions. In open arenas, foragers from small worker experimental colonies discovered baits in less time (38 ± 12 minutes) than larger worker colonies (87 ± 7 minutes; ANOVA, F1,28 = 13.46, p = 0.001) (Figure 4.3A). In closed arenas, small bodied experimental colonies also discovered baits more quickly (22 ± 7 minutes) than larger bodied colonies (38 ± 6 minutes; ANOVA, F1,18 = 8.36, p = 0.01)

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(Figure 4.3B). In all trials with potential for aggressive interactions (n =72) I observed no turnover of baits within the 4 hours of observation.

Resource Retrieval In open arenas, experimental colonies foraging against experimental colonies of nestmates retrieved more food resources than non-nestmate experimental colonies (ANOVA, F1,25 = 5.36, p = 0.03) but there was no effect of size of competitors on proportion bait removed (ANOVA, F1,25 = 0.30, p = 0.74) (Figure 4.4). In closed arenas there was no significant difference in resource retrieval when competing with nestmate or non-nestmates (ANOVA, F1,24 = 1.11, p = 0.30) or between different size pairing (ANOVA, F1,24 = 1.11, p < 0.35) (Figure 4.4).

Field Survey The mean number of foragers collected from above ground traps (170 ± 33 workers, n = 25) was not significantly different than those collected from below ground pitfall traps (544 ± 136 workers, n = 25; paired t-test, p = 0.66) (Figure 4.5A.) The mean HW of workers collected per colony from below ground traps (0.746 ± 0.01 mm, n = 25) were significantly larger than those collected above ground traps (0.79 ± 0.02 mm, n = 25; paired t-test, p = 0.04) (Figure 4.5B).

Discussion Variation in colony size and worker body sizes within and between ant species are important in determining niche use and foraging competition within a community (Davidson 1998, Kaspari and Weiser 1999, Sarty et al. 2006, Pearce-Duvet et al. 2011, Dornhaus et al. 2012). This occurs because worker number and body size can influence resource discovery (Fellers 1987, Davidson 1998, Pearce-Duvet et al. 2011), food retrieval (Traniello 1989, Powell and Franks 2005, Schöning et al. 2005, Dornhaus et al. 2012), and resource defense (Adams 1990, 1994, Whitehouse and Jaffe 1996, Batchelor and Briffa 2011). For polymorphic ant species, body size distributions of workers within a colony may shift in response to competition (Davison 1978, Traniello 1987, Traniello 1989, Wetterer 1994b) and perceived threat of competition (Passera et al. 1996). Recently mathematical models from operations research have be co- opted to form predictions about how worker size and number influence the outcomes of competitive interactions of social groups (Franks and Partridge 1993, McGlynn 2000, Wilson et al. 2001, Adams and Mesterton-Gibbons 2003, Plowes and Adams 2005). Partridge and Franks (1993) first suggested the utility of Lanchester’s (1916) laws of combat to predicting the outcome of competitive interactions in ants. For my purposes, I used Lanchester’s square

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and linear law to form and test predictions to the outcomes of aggressive interactions. Lanchester’s square law assumes larger groups simultaneously engage their opponents, concentrating their attacks and larger groups kill more workers than smaller groups. Lanchester’s linear law assumes interactions occur as a series of duels where individual’s body size determines the outcome of each duel. Previous examinations of Lanchester’s laws largely support the square law, but test the predictions of Lanchester’s laws in the context of above ground interactions (McGlynn 2000, Wilson et al. 2001, Adams and Mesterton-Gibbons 2003, Plowes and Adams 2005). However, not all aggressive interactions take place above ground (Porter and Tschinkel 1986, Berghoff et al. 2002, Wilkie et al. 2007). In the present study I assumed narrow, confined spaces (below ground foraging tunnels) force a series of one-on-one duels. In contrast, above- ground interactions are conducive to simultaneously engaging your opponents. Thus I predicted within foraging tunnels or confined spaces, larger workers suffer fewer losses than smaller workers (linear law) and in above ground, or open areas, workers in greater number suffer fewer losses than groups with relatively fewer workers (square law). I tested these predictions with the aggressive, polymorphic, and above/below ground foraging red imported fire ant (Solenopsis invicta). The results generally supported the predictions of Lanchester’s laws. I found that in open areas, it is potentially advantageous to invest in many, small bodied workers. Smaller workers require fewer resources (Calabi and Porter 1989) and take less time to produce (Wheeler 1991) allowing colonies to increase in size relatively quickly. Larger colonies can discovery food resources faster than colonies with fewer workers. In addition, if many small workers engage in aggressive interactions with few, large workers, the large opponents suffer higher attrition rates. There was no effect of size pairing or arena type on the number of small deceased foragers (Figure 4.2A). In open arenas, larger foragers suffered greater losses when facing smaller, more numerous competitors than when facing larger competitors of equal number. Experimental colonies of large (but relatively few) workers competing against small (but relatively more) competitors suffered greater losses in closed arenas than open arenas (Figure 4.2B). The attrition of larger foragers supports the predictions of the square law because competitors in greater number killed a larger number of foragers relative to an equal sized force, particularly in open arenas where competitors can simultaneously engage their opponents. There is also some support for the linear law in that despite being outnumbered, larger foragers in closed arenas can use their size effectively to oppose or overpower smaller opponents. There was no difference in the proportion of original worker number lost due to attrition between interactions between nestmates and non-nestmates, arena type, or size of competitors (Figure 4.2C). For example, in open arenas where colonies with larger workers lost more individuals when competed with colonies with smaller workers, the proportion of large foragers lost was not significantly different than

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small foragers. This suggests that the investment into worker number versus size could be equivocal from the perspective of relative fighting ability under the conditions of this study. While the biomass lost was not different across treatments, there are other costs to consider. The production of larger workers takes longer than smaller workers (Tschinkel 1988, Wheeler 1991) and in polymorphic ant species, larger workers also have additional “value” because they play important and specialized roles in nest defense, and food retrieval, processing, and storage (Wilson 2003, Schöning et al. 2005, Powell and Franks 2004, Powell 2009). Some ant species also assess colony strength during ritualized displays (Hölldobler 1976, Hölldobler 1981, Van Wilgenburg et al. 2005) or during territorial intrusions (Gordon 1992, Tschinkel et al. 1995, Adams 2003) based solely on worker number. Previous work also suggests colonies with greater worker number, outcompete numerically inferior colonies (Davidson 1977, Holway 1999, Dornhaus et al. 2012). Thus a slight increase in worker mortality of colonies with a few larger workers may suffer greater more than energy invested into worker biomass than colonies of many, small workers. The results are consistent with prior examinations of Lanchester’s laws (McGlynn 2000, Wilson et al. 2002, Powell and Clark 2004, Plowes and Adams 2005). For example, in the Neotropics army ants (Nomamyrmex esenbeckii) attack and kill mature colonies of leaf-cutting (Atta) (Powell and Clark 2004). Mature colonies of Atta consist of millions of workers (Fowler et al. 1986, Wirth et al. 2003) and army ant raids consist of hundreds of thousands of workers (Franks 1982, Franks 1985). Against the numerically superior force the success of raids is dependent on the speed and defensive response of Atta. Powell and Clark (2004) found, if N. esenbeckii engage Atta in near the nest entrance or entrance tunnels and recruit larger workers to the line of engagement, N. esenbeckii can overcome their numerical inferiority and successfully raid the Atta colony utilizing workers with high individual fighting potential (linear law). However, if Atta respond quickly recruiting a large defensive force with more workers and their own larger (major) workers they can defend their colony outside the entrance tunnels and utilize their numerical superiority (square law). Although Powell and Clark (2004) did not test the predictions of Lanchester’s laws explicitly they also found field evidence suggesting the outcomes of aggressive interactions are influenced by worker number, size, and foraging condition (open vs. confined spaces). A previous examination of Lanchester’s law in S. invicta found no support for the square law (Plowes and Adams 2005). A portion of the data supports their findings. In comparisons of small worker mortality and the proportion of workers lost, I also found no difference in mortality in small workers lost despite outnumbering their opponents ~4:1 (Figure 4.2A, 4.2C). I also observed that colonies of larger bodied workers suffered fewer losses in closed arenas (support for the linear law) but found when opponents can simultaneously engage larger foragers more large foragers are lost (support for the square law) (Figure 4.2B). This may reflect two completely different approaches to addressing Lanchester’s

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laws. Plowes and Adams (2005) tested Lanchester’s laws using Bayesian statistical inference to estimate θ (group fighting ability) based on experimental worker mortality of smaller groups (50 – 100 workers), of small, medium, and large workers in only an open arena (5 cm diameter cup). They estimated θ = 1.04, which supports the predictions of the linear law (θ = 2, would support the square law; Adams and Mesteron-Gibbons 2003, Plowes and Adams 2005). In both open and closed arenas the time till bait was discovered was more quickly by smaller workers than larger workers (Figure 4.3). Larger bodied workers of S. invicta spend less time in search of food than do smaller bodied workers (Wilson 1978, Porter and Tschinkel 1987, Tschinkel 2010) and more time as in active reserves (Mirenda and Vinson 1981). Smaller workers are more commonly foragers (Porter and Tschinkel 1987, Tschinkel 2006) and the smaller bodied experimental colonies also consisted of 3.5 fold as many workers as larger bodied colonies. In other ants species colonies consisting of many small are more diffuse within a habitat and are more likely to locate food resources (Fellers 1987, Davidson 1997, Holway 1999). This is particularly true in habitats with complex physical structures (ex. topical forest leaf litter) smaller ants recruit more quickly to bait resources than do larger bodied ants (Farji-Brener et al. 2004, Sarty et al. 2006). There was no effect of size pairing (small vs. small, small vs. large, or large vs. large) on the proportion of bait removed in open or closed arenas (Figure 4.4). I originally predicted more bait removal in trials with all small workers than trials with all large workers because of the small workers numerical advantage and propensity to forage. Instead I found that relatively small colonies (250-840 workers) are foraging for resources within 1 m of their nests may collect the same amount of food resources regardless of investing in a few, larger workers or many, small workers. Both the time to discovery and resource retrieval data reflect behavior that is observed in natural populations of S. invicta. In the field I found above ground foragers of S. invicta are typically smaller than below ground foragers, but it is established that when resources are discovered workers typically recruit additional and larger workers from nearby foraging tunnels (Wilson 1971, Markin et al. 1975, Porter and Tschinkel 1987). Larger workers help process large food items into smaller, more easily transported items but play and participate equally during recruitment to food items (Wilson 1978, Tschinkel 2006). Smaller workers may have a greater propensity of find resources but both sizes classes of workers are equally important and effective at removing food resources. I found no significant difference in the total number of foragers collected in above ground and below ground pitfall traps (Figure 4.1A). I did find support for the linear law, as foragers from below ground pitfall traps were larger than foragers collected from above ground pitfall traps (Figure 4.1B). Differences between body size of above and below ground foragers is not necessarily a response to

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competitive interactions. Forager body size and number in S. invicta is known to shift in response environmental conditions (Porter and Tschinkel 1987, Tschinkel 2010) and parasites (Phorid flies; Orr 1992, Morrison 1999). Smaller foragers above ground may represent expendable units that discover resources because foraging is high risk (Wilson 1971) and larger workers are energetically more costly to produce than smaller workers (Porter and Tschinkel 1985, Calabi and Porter 1989). When more and/or larger workers are required in then smaller bodied workers can recruit workers in reserve, often located nearby in below ground foraging tunnels (Markin et al. 1975, Porter and Tschinkel 1987). Thus below ground pitfalls may only sample the reserve worker force necessary for above ground recruitment rather than a response to below ground conflicts. The difference in forager size may also not reflect a response to aggressive interactions because aggression between groups of S. invicta foragers from mature colonies is uncommon. For example, territorial boundaries between S. invicta colonies are typically separated by zones devoid of S. invicta foragers (Tschinkel et al. 1995, Tschinkel 2010). Since territorial boundaries rarely overlap, it is also improbable that aggressive interactions with competing colonies of S. invicta occur within the sampling distance from the nest (1 m) because territories of mature colonies are significantly larger, extending several meters from nest center (Tschinkel et al. 1995, Tschinkel 2006). Though, there is evidence to suggest worker number is important for resource defense because when worker populations decrease territory size also decreases (Adams 2003, Tschinkel 2010). Foraging is dangerous (Wilson 1971) and maintaining foraging number of small foragers may serve as a cheap but effective indicator of resource holding potential to competitors (Gordon 1992, Batchelor and Briffa 2010). I also collected other ant species at traps from 0.5 and 1 meter from both above and below ground pitfall traps. Other ant species collected below ground were generally smaller than S. invicta foragers (personal observation). If an intraspecific aggression were to occur the larger S. invicta would have the size advantage. By utilizing small foragers above ground, S. invicta mitigate their energetic losses while maintaining an energetically inexpensive worker force (Calabi and Porter 1989) necessary to discovery resources quickly and also maintain foraging numbers to prevent territorial intrusion. The worker force in reserve, within the foraging tunnels, allows colonies to benefiting from a worker force with major workers important for resource defense, processing, retrieving, and storage (Wilson 1978, Calabi and Porter 1989, Tschinkel 2006, Tschinkel 2010) while also mitigating the loss of majors to exposure (Porter and Tschinkel 1987, Tschinkel 2010) and parasites (Orr 1992, Morrison 1999).

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Future Directions Competition has long been considered influential in determining the distribution and abundance of organisms within a community (MacArthur 1958, Paine 1966, Schoener 1983, Tillman 1994). Ants are typically described as most diverse and successful organisms in terrestrial ecosystems (Hölldobler and Wilson 1990, Folgarait 1998, Lach et al. 2010). One explanation for their ecological dominance and success is that ant species are social allowing workers to display a high degree of morphological variation and behavioral specialization (Wilson 1987, Hölldobler and Wilson 1990, Lach et al. 2010). Variation in worker number and size, within and between species, is believed to directly impact the outcomes of competitive interactions (Kaspari and Weiser 1999, Sarty et al. 2006, Dornhaus et al. 2012). Lanchester’s laws provide context, however simple, to predict the outcomes of competitive interactions. They are useful for explaining the amount of variation observed in ant species and potentially how competition can influences community structure and species composition. As noted by others testing the predictions of Lanchester’s laws, these models are not complete models of competition between social groups for a number of reasons (McGlynn 2000, Wilson et al. 2001, Adams and Mesterton-Gibbons 2003, Powell and Clark 2004, Plowes and Adams 2005). (1) Not all competitive interactions between groups are direct (interference) and measuring indirect (exploitation) competition, as I did, may be needed to understand differences in investment into workers size or number. (2) Many animals avoid physical combat, relaying on displays to assess outcome of combat without fighting (Parker 1974, Hölldobler 1976, Hölldobler 1981). (3) The models are based on the costs of conflict and not a group’s perceived value of a resource (Maynard Smith 1974, Mesterton-Gibbons and Adams 1998). (4) The mathematical models also assume each member of a group has the same propensity to fight. In ant species with workers of different sizes, it is likely size influences the probability of performing a variety of behaviors including aggressive interactions (Wilson 1976, Whitehouse and Jaffe 1996, Powell and Clark 2004). Although not perfect, Lanchester’s laws do provide a structure useful to predicting the outcomes of competitive interactions. Developing more realistic models could potentially add to current understanding of how competitive interactions influence tradeoffs in investment within colonies and potentially even community structure. I first however first suggest tests of the predictions of Lanchester’s laws in other ant species. The red imported fire ant (S. invicta), for example, fights with their mandibles a stinger which is effective only in close combat (Davidson 1998, Feener et al. 2008). Other ant species can spray chemicals to damage or immobilize competitors (Hölldobler and Wilson 1990). The methods outlined by Plowes and Adams (2005) to test the predictions of Lanchester’s laws may be useful in a community context. In a community of relatively few ant species one could compare θ within and

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between species. Such information would be useful to evaluate Lanchester’s laws utility in a broader ecological context.

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Chapter 4 Figures and Tables Figure 4.1. Size pairings (small x small, large x small, and large x large) and foraging arena (open and closed environment) for each field- collected colony pairing.

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Figure 4.2A. The attrition of small foragers lost from each experimental colony. For experimental colonies with smaller workers, there was no effect of arena type and size pairing on the mean number of deceased workers (ANOVA, p > 0.05).

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Figure 4.2B. The attrition of large foragers from each experimental colony. Experimental colonies with larger workers, suffered greater number of mean number of workers lost when competing against smaller bodied colonies (F1,15 = 9.06, p > 0.01). In open arenas, when large workers compete with experimental colonies of small workers a greater number of larger foragers are die than when competing against colonies with large workers (t-test, t = -3.46 p = 0.003). Similarly when larger bodied colonies compete against smaller bodied colonies, larger workers are more are likely to die in open arenas then closed arenas (t-test, t = -2.35, p = 0.03).

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Figure 4.2C. The proportion of workers lost (mean ± SE) from experimental colonies was not significantly different between treatments, arena type, or size pairings (ANOVA, p > 0.05).

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Figure 4.3A. The mean (± SE) time till bait was discovered, within a four hour observation period (n = 74), for both worker size classes in open arenas, foragers from experimental colonies with small workers discovered baits in less time (38 ± 12 minutes) than larger bodied colonies (87 ± 7 minutes; ANOVA, F1,28 = 13.46, p = 0.001).

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Figure 4.3B. The mean (± SE) time till bait was discovered, within a four hour observation period (n = 74), for both worker size classes in closed arenas, small bodied experimental colonies also discovered baits more quickly (22 ± 7 minutes) than larger bodied colonies (38 ± 6 minutes; ANOVA, F1,18 = 8.36, p = 0.01).

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Figure 4.4. The proportion of bait removed after 24 hours in open vs. closed arenas, for all size pairings, and colony pairings between nestmates and non-nestmate workers. In open arenas, experimental colonies foraging against experimental colonies of nestmates retrieved more food resources than when paired with non-nestmate colonies (ANOVA, F1,25 = 5.36, p = 0.03) but there was no effect of size of pairings on resources removal (ANOVA, F1,25 = 0.30, p = 0.74). In closed arenas there was no significant difference in resource retrieval when competing with nestmate or non-nestmates (ANOVA, F1,24 = 1.11, p = 0.30) or between different size pairing (ANOVA, F1,24 = 1.11, p < 0.35).

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Figure 4.5A. The mean number (± SE) of foragers (170 ± 33 workers, n = 25) collected above ground was not significantly different than those collected from below ground (544 ± 136 workers, n = 25; paired t-test, p = 0.66).

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Figure 4.5B. The mean (± SE) HW of workers collected from below ground (0.746 ± 0.01 mm, n = 25) was significantly larger than those collected above ground (0.79 ± 0.02 mm, n = 25; paired t-test, p = 0.04).

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CHAPTER 5: SUMMARY AND CONCLUSIONS

Body size is an important life history trait (Stearns 1992, Brown 1995, Chown and Gaston 2010) that impacts most, if not all, aspects of its existence including: metabolism, thermoregulation, locomotion, reproduction, longevity, and diet (Peters 1983, Schmidt-Nielsen 1984, Calder 1984, Losos 1990, Douglas and Mathews 1992, Kaspari and Weiser 1999, 2007). Moreover, variation in size has important ecological, evolutionary, and behavioral implications (Peters 1983, Calder 1984, Schmidt-Nielsen 1984, Traniello 1989, Brown 1995, Blanckenhorn 2000, Chown and Gaston 2010, Dornhaus et al. 2012). Variation in body size may result from a response to constraints and tradeoffs of reproductive investment, as is the case with life history traits generally (Smith and Fretwell 1974, Stearns 1992, Zera and Harshman 2001). Body size variation in social insects has a long history (Wilson 1953, Wilson 1971, Oster and Wilson 1978, Cushman et al. 1993, Bourke and Franks 1995, McGlynn 1999, Kaspari 2005, Gouws et al. 2011) but rarely is their examinations of the tradeoffs associated with investment into worker number and size (but see Davidson 1978, Fellers 1987, McGlynn et al. 2012). Body size and its variation are considered important features influencing a colony’s fitness through colony maintenance, survival, and reproduction (Oster and Wilson 1978, Hölldobler and Wilson 1990, Bourke and Franks 1995, Billick 2002, Whitehouse and Jaffé 1996, Powell 2009). In Chapter One, I reviewed the literature on the ecological factors influencing variation in worker size. This includes the abiotic environment, variation in diet, and the competitive environment. Overall, I found that despite a considerable amount of attention to the evolution of morphologically distinct castes in workers (Wilson 1953, Wilson 1971, Oster and Wilson 1978, Cushman et al. 1993, Bourke and Franks 1995, McGlynn 1999, Kaspari 2005, Gouws et al. 2011), more quantitative data describing the amount of variation in worker body size within and between species is needed (Gouws et al. 2011). This information will be useful to understand the how phenotypic plasticity and developmental controls change in response to nutrition, climate, and competition. In addition, despite decades of work describing the variation in worker body little is known about how this variation contributes to measures of colony fitness. Using both comparative and experimental approaches, I examined the ecological factors influencing variation, and consequences of variation, in worker body size in two polymorphic invasive ant species. Introduced species are ideal for examining the role of ecological variation on investment into worker size as they often encounter vastly different ecological conditions throughout their introduced ranges (e.g. fewer competitors and access to additional resources) (Vitousek et al. 1987). Differences in

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ecology likely impact colony investment in growth, reproduction, and defense (Oster and Wilson 1978, Passera et al. 1996, Yang et al. 2004, Powell 2009). In Chapter Two, I quantified the variation in size and shape of workers among geographically distinct populations of the dimorphic (two distinct worker castes) big-headed ant (Pheidole megacephala). I found a substantial amount of variation in how colonies invest into worker mass and morphology among five populations of the invasive ant, P. megacephala. Both major and minor workers were largest in Australia, and minor workers were smallest in Hawaii and Mauritius. Head shape also varied among populations in both worker castes. The genetic results suggest that these populations are nominally all the same species. Subsequently, differences into investment among populations in the size of majors may result from a response to local competitive environment. While I only sampled ants from five different countries, these populations varied considerable in ecological conditions (local and regional species richness). However, in addition to competition, diet (McGlynn and Owen 2002) and climate (Yang et al. 2004) also influence colony investment into worker body size in Pheidole species. With this sampling design, I was unable to test the contribution of each of these factors separately. Field surveys should be combined with common-garden experiments to explore the relative influence of each factor on the morphology of workers. Despite these limitations, my results serve as a foundation for the testing of specific hypotheses. More work is needed to decipher the role of diet, climate, and competition on the observed variation in worker body sizes. In Chapter Three, I examine how access to plant-based carbohydrate resources (diet) influences colony investment into worker body size distributions in the continuously polymorphic, red imported fire ant (Solenopsis invicta). Access to plant-based resources, in particular carbohydrate resources, plays an important role in determining colony investment into number and worker body size (Helms and Vinson 2008, Dussutour and Simpson 2009, Dussutour and Simpson 2012) and is suspected of playing an important role in determining invasion success of ant species (Helms and Vinson 2002, Helms et al. 2011, Wilder et al. 2011b, Wilder et al. 2013). I used multiple queen colonies from populations in Texas, split into four treatment colonies to experimentally test how diet influences colony investment into worker number, body size, body size distributions, and fat content. Differences in worker biomass are attributable to changes in both worker number and mean size. Worker number and size generally increased in response to carbohydrate supplementation but there is no difference in worker fat content among treatments. There is a slight shift in body size distributions in treatments reared on diets of carbohydrates than those denied access to carbohydrates. My results suggest that the traditional paradigms of ant nutrition, such as worker production is protein (amino acids) limited may need reevaluation. Finally, the results show the nutritional ecology can have important implications

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for the growth and development of invasive species like S. invicta. Monopolization of plant based resources may facilitate the invasion of new environments because a larger and more numerous worker forces can overpower competitors (Franks and Partridge 1993, Silva and Brandão 2010) and withstand larger temperature fluctuations (Francke et al. 1985, Wiescher et al. 2012), and longer periods of food deprivation (Kaspari and Vargo 1995, Smith 2007). Therefore, access to carbohydrate resources may significantly contribute to the establishment and subsequent spread of invasive ant species. An increase in resources available for investment into worker number and body size can influence the outcomes of competitive interactions (Davidson 1998, Kaspari and Weiser 1999, Sarty et al. 2006, Pearce-Duvet et al. 2011, Dornhaus et al. 2012). This includes resource discovery (Davidson 1977, Davidson 1998, Pearce-Duvet et al. 2011), food retrieval (Traniello 1989, Powell and Franks 2005, Schöning et al. 2005, Dornhaus et al. 2012), and resource defense (Adams 1990, 1994, Whitehouse and Jaffe 1996, Batchelor and Briffa 2012). There is growing interest in co-opting Lanchester’s laws, originally designed for human warfare, to predict the outcomes of aggressive interactions in ants (Franks and Partridge 1993, McGlynn 2000, Adams and Mesterton-Gibbons 2003, Plowes and Adams 2005). In Chapter Four, I used a foraging experiment and field surveys to test the predictions of Lanchester’s laws in interference competition with the continuously polymorphic, S. invicta. I also used the foraging experiment to explore tradeoffs in worker number and size in exploitation competition. I found colonies of equal biomass but different worker size (e.g. different worker number), colonies with smaller workers typically discover baits more quickly than those with larger workers but both large and small worker colonies retrieve equal amounts of food resources. Colonies with smaller workers do not suffer fewer losses when outnumbering their opponents than when equally matched. I however found that when colonies with large bodied workers compete with numerically dominant opponents they suffer a greater number of losses in open arenas than closed arenas (support for the square law). In closed arenas, colonies with relatively few but large workers use their size advantage in confined spaces to defend against the numerically superior but smaller workers (support for the linear law). In the field, I found more small workers above ground than below ground which supports the predictions of the linear law because in narrow confined spaces larger body size is advantageous. The production of larger workers takes longer than smaller workers (Tschinkel 1988, Wheeler 1991) and in polymorphic ant species, larger workers also have additional “value” because they play important and specialized roles in nest defense, and food retrieval, processing, and storage (Wilson 2003, Schöning et al. 2005, Powell and Franks 2004, Powell 2009). Some ant species also assess colony strength during ritualized displays (Hölldobler 1976, Hölldobler 1981, Van Wilgenburg et al. 2005) or during territorial intrusions (Gordon 1992, Tschinkel et al. 1995, Adams 2003) based solely on worker

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number. Previous work also suggests colonies with greater worker number, outcompete numerically inferior colonies (Davidson 1977, Holway 1999, Dornhaus et al. 2012). Thus a slight increase in worker mortality of colonies with a few larger workers may suffer greater more than energy invested into worker biomass than colonies of many, small workers. Foraging is dangerous (Wilson 1971) and maintaining foraging number of small foragers may serve as a cheap but effective indicator of resource holding potential to competitors (Gordon 1992, Batchelor and Briffa 2010). Ants are typically described as most diverse and successful organisms in terrestrial ecosystems (Hölldobler and Wilson 1990, Folgarait 1998, Lach et al. 2010). One explanation for their ecological dominance and success of ant species is that they are social allowing workers to display a high degree of morphological variation and behavioral specialization (Wilson 1987, Hölldobler and Wilson 1990, Lach et al. 2010). The amount of worker variation within a colony is influenced in part by competition (see Chapter Two and Four), nutrition (see Chapter Three), the abiotic environment, social environment, genetics, and evolutionary constraints (reviewed in Chapter One). The introduction of invasive ant species likely impacts a colony’s investment in growth, reproduction, and defense (Tillberg et al. 2007, Wilder et al. 2011a, Chapter Two, Chapter Three). This may occur as a result of changes in competition (Passera et al. 1996, Chapter Two) or increased access to food resources (McGlynn and Owen 2002, Wilder et al. 2011b, Chapter Three). Subsequently increases in worker number and size can improve their territory size (Adams 1994, Adams 2003), resource discovery and dominance (Holway 1999, Holway and Suarez 2006, Chapter Four), and reduce group losses during aggressive interactions (Tillberg et al. 2007, Helms et al. 2011, Wilder et al. 2011a, Chapter Four). Understanding determinates of variation in ant worker body size is no trivial task. The relative importance of factors determining worker body size is likely different within ant species, among ant species, and in some cases between worker castes. This complexity and amount of variation within a single species, colony, and sex and the remarkably plastic larval forms (Wheeler 1991) also make ants useful model organisms for exploring life history traits. The size and morphological specialization of ants also make them important tools to explore how morphological specialization within a group contributes to fitness of the colony (or group). Given the ecological dominance of ants in most terrestrial environments (Hölldobler and Wilson 1990), failing to examine the factors influencing within and between colonies limits our current understanding of insect ecology. There exists a substantial amount of information regarding the factors influencing ant body size, but is much left undiscovered.

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APPENDIX: SUPPLEMENTARY FIGURE AND TABLES

Appendix: Supplementary Figure 1. Full phylogram of Pheidole megacephala as inferred through maximum likelihood analysis for the COI dataset. Portion of phylogram presented in Figure 2.1 denoted in dashed box. Collections of P. megacephala measured for size, morphology, and caste ratios as part of this study are noted by a star next to the taxa names. Branch lengths are proportional to substitution/site as indicated by the bottom legend inset. Clade support greater than 50% is denoted on branches as follows: Values above branches represent maximum likelihood bootstrap (ML BS) for the COI only dataset followed by the partitioned dataset and values below branches represent Bayesian posterior probabilities (BPP) for the COI only dataset followed by the partitioned dataset. Clade support of “--” denotes clades not supported in an individual analysis. Taxa names include taxonomic identity, state and country of collection site, and collector code (and GenBank accession number and citation to original publication if from a previous study).

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Appendix: Supplementary Table 1. Pheidole Species Accession no. Locality mtDNA CO1 mtRNA 12s nDNA LR nDNA H3 megacephala AVS 1809 Hawaii submitted submitted X submitted megacephala AVS 1810 Hawaii submitted submitted X submitted megacephala AVS 1811 Hawaii submitted submitted X submitted megacephala AVS 1812 Hawaii submitted submitted X X megacephala AVS 1813 Hawaii X X X submitted megacephala AVS 1814 Hawaii submitted submitted X X megacephala AVS 1823 Hawaii submitted submitted X submitted megacephala AVS 1848 Hawaii submitted submitted X submitted megacephala AVS 2625 Mauritius submitted submitted submitted submitted megacephala AVS 2632 Mauritius submitted submitted X submitted megacephala AVS 2647 Mauritius submitted submitted submitted submitted megacephala AVS 2659 Mauritius submitted submitted submitted submitted megacephala AVS 2694 Mauritius submitted submitted submitted submitted megacephala AVS 2695 Mauritius submitted submitted submitted submitted megacephala AVS 2717 Mauritius submitted submitted X X megacephala BD 10 Missouri submitted submitted submitted submitted megacephala BD 3 Missouri submitted submitted submitted submitted megacephala BD 5 Missouri submitted submitted submitted submitted megacephala BD 9 Missouri submitted submitted submitted submitted megacephala BFL13 Florida submitted submitted X submitted megacephala BFL14 Florida submitted submitted submitted submitted megacephala BFL15 Florida submitted submitted submitted submitted megacephala BFL16 Florida submitted submitted X submitted megacephala BFL17 Florida submitted submitted X submitted megacephala CSM1381 Florida Keys submitted submitted submitted submitted megacephala CSM1403 Florida Keys submitted submitted submitted submitted megacephala CSM2617 Uganda submitted submitted submitted submitted megacephala HS1 Australia submitted submitted submitted submitted megacephala HS2 Australia submitted submitted X submitted megacephala HS3 Australia submitted submitted submitted submitted megacephala HS4 Australia submitted submitted submitted submitted megacephala Sabi Sands 2 S. Africa X X X submitted megacephala Sabi Sands 3 S. Africa submitted submitted X submitted

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Appendix: Supplementary Table 1. (cont.) megacephala Fournier2012_CH Australia ** X X X megacephala Fournier2012_HS Australia ** X X X megacephala Fournier2012_SP Australia ** X X X megacephala Fournier2012_WP Australia ** X X X megacephala RA0357 Australia EF518366.1 EF518664.1 X EF518833.1 megacephala Fournier2012_SC1 Cameroon ** X X X megacephala Fournier2012_SC2 Cameroon ** X X X megacephala Fournier2012_SC3 Cameroon ** X X X megacephala Fournier2012_SC4 Cameroon ** X X X megacephala CS0242 Madagascar EF518412.1 EF518713.1 EF519034.1 EF518881.1 megacephala HQ547393* Madagascar HQ547393.1 X X X megacephala Pouce750* Mauritius EF610013.1 X X X megacephala Cocotte700* Mauritius EF610016.1 X X X megacephala PieterBoth770* Mauritius EF610035.1 X X X megacephala Camizard375* Mauritius EF610039.1 X X X megacephala BlackRiver750* Mauritius EF610054.1 X X X megacephala Fournier2012 Mauritius ** X X X megacephala Fournier2012_Ds South Africa ** X X X megacephala Fournier2012_Sku South Africa ** X X X ampla RA0358 Australia EF518309.1 EF518603.1 EF518938.1 EF518774.1 noda RA0479 Vietnam EF518375.1 EF518673.1 EF518998.1 EF518842.1 oceanica RA0713 Palau EF518379.1 EF518677.1 EF519002.1 X pallidula RA0195 France EF518381.1 EF518680.1 EF519004.1 EF518848.1 plagiaria RA0482 Vietnam EF518385.1 EF518684.1 EF519008.1 EF518852.1 protea RA0478 Thailand EF518390.1 EF518689.1 X EF518857.1 quadrensis RA0320 Malaysia EF518392.1 EF518691.1 EF519014.1 EF518859.1 sexspinosa RA0712 Palau EF518404.1 EF518704.1 EF519026.1 EF518872.1 sp. RA0536 Africa EF518411.1 EF518711.1 EF519033.1 EF518879.1 sp. RA0538 Africa X EF518712.1 X EF518880.1 sp. RA0557 Africa EF518413.1 EF518714.1 EF519035.1 EF518882.1 sp. RA0558 Africa EF518417.1 EF518718.1 EF519039.1 EF518886.1 sp. RA0559 Africa EF518421.1 EF518722.1 EF519043.1 EF518890.1 sp. RA0563 Africa EF518424.1 EF518725.1 EF519045.1 EF518893.1 sp. RA0329 Australia EF518409.1 EF518709.1 EF519031.1 EF518877.1 101

Appendix: Supplementary Table 1. (cont.) sp. RA0422 Indonesia EF518414.1 EF518715.1 EF519036.1 EF518883.1 sp. RA0423 Indonesia EF518418.1 EF518719.1 EF519040.1 EF518887.1 sp. RA0492 Indonesia EF518410.1 EF518710.1 EF519032.1 EF518878.1 sp. RA0314 Madagascar EF518415.1 EF518716.1 EF519037.1 EF518884.1 sp. RA0315 Madagascar EF518419.1 EF518720.1 EF519041.1 EF518888.1 sp. RA0316 Madagascar EF518422.1 EF518723.1 EF519044.1 EF518891.1 sp. RA0317 Madagascar EF518425.1 EF518726.1 EF519046.1 EF518894.1 sp. RA0473 PNG EF518416.1 EF518717.1 EF519038.1 EF518885.1 sp. RA0516 PNG EF518420.1 EF518721.1 EF519042.1 EF518889.1 sp. RA0518 PNG EF518423.1 EF518724.1 X X sp. RA0519 PNG EF518426.1 EF518727.1 EF519047.1 EF518895.1 sp. RA0520 PNG EF518427.1 EF518728.1 EF519048.1 EF518896.1 comata RA0477 Malaysia EF518334.1 EF518629.1 EF518959.1 EF518799.1 dugasi RA0318 Thailand EF518343.1 EF518639.1 EF518969.1 EF518809.1 gatesi RA0319 Vietnam EF518350.1 EF518647.1 EF518976.1 EF518817.1 tandjongensis RA0481 Thailand EF518431.1 EF518732.1 EF519051.1 EF518900.1 variabilis RA0360 Australia EF518441.1 EF518742.1 EF519061.1 EF518908.1 pilifera RA0707 USA EF518384.1 EF518683.1 EF519007.1 EF518851.1 senex RA0462 USA EF518402.1 EF518702.1 EF519024.1 EF518870.1 tucsonica CS0224 USA EF518437.1 EF518738.1 EF519057.1 EF518905.1 tysoni RA0448 USA EF518438.1 EF518739.1 EF519058.1 EF518906.1 * Denotes SmithFisher2009 accession number. ** See Fournier et al. 2012. PNG denotes Papua New Gun

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Appendix: Supplementary Table 2. ANOVA table of all fixed interactions with colony identity. Worker Number Source SS df MS F p Carbohydrates 0.1052 1 0.1052 3.45 0.10 A. Acids 0.0006 1 0.0006 0.02 0.88 A. Acids & Carbohydrates 0.0118 1 0.0118 0.89 0.37 Colony 3.1605 8 0.3951 8.96 < 0.01 Colony*Carbohydrates 0.2444 8 0.0305 2.31 0.13 Colony*Amino Acids 0.2143 8 0.0268 2.02 0.17 Col*Carbohydrates*Amino Acids 0.1059 8 0.0132 1.19 e10 - Residual 3.94 e-15 0 1.11 e-12 - -

Head Length Source SS df MS F p Carbohydrates 0.0044 1 0.0044 3.46 0.10 A. Acids 2.74 e-6 1 2.74 e-6 0.00 0.96 A. Acids & Carbohydrates 0.0003 1 0.0003 0.60 0.46 Colony 0.0864 8 0.0108 5.48 0.01 Colony*Carbohydrates 0.0101 8 0.0013 3.02 0.07 Colony*Amino Acids 0.0090 8 0.0011 2.69 0.09 Col*Carbohydrates*Amino Acids 0.0033 8 0.0004 3.77 e8 - Residual 5.64 e-15 0 1.11e-12 - -

Head Width Source SS df MS F p Carbohydrates 0.0060 1 0.0060 4.00 0.08 A. Acids 5.21 e-5 1 5.21 e-5 0.05 0.83 A. Acids & Carbohydrates 4.65 e-5 1 4.65 e-5 0.09 0.77 Colony 0.0963 8 0.0120 5.93 0.01 Colony*Carbohydrates 0.0120 8 0.0015 2.84 0.08 Colony*Amino Acids 0.0084 8 0.0011 1.99 0.18 Col*Carbohydrates*Amino Acids 0.0042 8 0.0005 4.78 e8 - Residual 9.80 e-15 0 1.11e-12 - -

Fat Content Source SS df MS F p Carbohydrates 0.0369 1 0.0369 1.87 0.21 A. Acids 0.0011 1 0.0011 0.07 0.79 A. Acids & Carbohydrates 0.0059 1 0.0059 0.51 0.50 Colony 0.3432 8 0.0429 1.82 0.24 Colony*Carbohydrates 0.1580 8 0.0198 1.70 0.23 Colony*Amino Acids 0.1237 8 0.0155 1.33 0.35 Col*Carbohydrates*Amino Acids 0.0929 8 0.0116 1.05 e10 - Residual 1.02 e-14 0 1.11 e-12 - -

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Appendix: Supplementary Table 3. ANOVA table of all fixed effects and colony identity. Worker Number Source SS df MS F p Carbohydrates 0.1052 1 0.1052 4.47 0.045 A. Acids 0.0006 1 0.0006 0.03 0.87 A. Acids & Carbohydrates 0.0118 1 0.0118 0.50 0.49 Colony 3.1605 8 0.3951 16.79 0.0001 Residual 0.5646 24 0.0235 - -

Head Length Source SS df MS F p Carbohydrates 0.0044 1 0.0044 4.67 0.041 A. Acids 2.74 e-6 1 2.74 e-6 0.00 0.957 A. Acids & Carbohydrates 0.0003 1 0.0003 0.27 0.608 Colony 0.0864 8 0.0108 11.54 0.0001 Residual 0.0225 24 0.0009 - -

Head Width Source SS df MS F p Carbohydrates 0.0060 1 0.0060 5.85 0.0235 A. Acids 5.21 e-5 1 5.21 e-5 0.05 0.8240 A. Acids & Carbohydrates 4.65 e-5 1 4.65 e-5 0.05 0.8335 Colony 0.0963 8 0.0120 11.68 0.0001 Residual 0.0247 24 0.0010 - -

Fat Content Source SS df MS F p Carbohydrates 0.0257 1 0.0257 2.22 0.149 A. Acids 0.0005 1 0.0005 0.04 0.837 A. Acids & Carbohydrates 0.0029 1 0.0029 0.25 0.620 Colony 0.2510 8 0.0314 2.71 0.028 Residual 0.2777 24 0.0116 - -

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