Adamson, Jon Donald McPhail, Don Moerman, and Bill Neill, a great debt. First, thank you ..... history of southwestern British Columbia (Mathews et al. 1970 ...
COMPETITION A N D C H A R A C T E R DISPLACEMENT IN STICKLEBACKS by JOHN ROBERT PRITCHARD B . Sc. magna cum laude, University of Arizona, 1990
A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (DEPARTMENT OF ZOOLOGY)
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ABSTRACT This thesis investigates competition's effects on the evolution of sympatric threespine sticklebacks in small, post-glacial, coastal lakes of southwestern British Columbia. A few lakes contain two morphologically and ecologically distinct stickleback forms ("limnetics" and "benthics"), whereas most lakes contain a single "intermediate" form. These forms are believed to be derived from marine threespine sticklebacks that invaded lakes following the retreat of glaciers. I examine body shape differences of marine and freshwater stickleback forms and I test whether competition experienced by planktivorous marine sticklebacks in experimental ponds is consistent with the hypothesis that sympatric lake forms evolved, at least in part, in response to interspecific competition. A thin-plate spline geomorphometric analysis quantified differences in body shape among four stickleback forms: the marine [ancestral] and three [descendent] lake forms (limnetics, intermediates, and benthics). I found significant shape differences among all forms: marine and limnetic sticklebacks were most similar in body shape, while intermediates fell between them and benthics. This pattern matches the divergence observed in other characters among these forms. The main differences were in head size, distance between fin insertion points, and first dorsal spine location. Convergence in body shape among the freshwater sticklebacks may represent anti-predator adaptation, whereas divergence may represent adaptations for foraging. To test whether marines compete with intermediates, I placed juvenile marine sticklebacks either alone or with intermediate sticklebacks in experimental ponds. I also tested whether there is an adaptive shift in marine morphology between treatments due to natural
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selection, phenotypic plasticity, or both. Marine sticklebacks competed with intermediates; marines experienced both significant reduction in growth and increased consumption of zooplankton when intermediates were present. Marine morphology also showed an adaptive shift; however, this shift could not be unambiguously attributed to the presence of intermediates. I tested whether competition experienced by marines declines as the competitor becomes more divergent by pairing marines with either intermediates or benthics. Competition experienced by marine sticklebacks decreased when the competitor was more ecologically and morphologically differentiated; marines with intermediates grew significantly slower than marines with benthics. This research provides both a more complete description of morphological differentiation among sticklebacks and crucial evidence that interspecific competition is an important factor in evolution.
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T A B L E OF CONTENTS
ABSTRACT
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T A B L E OF CONTENTS
iv
LIST OF T A B L E S
vi
LIST OF FIGURES
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ACKNOWLEDGMENTS
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CHAPTER ONE: INTRODUCTION A N D OVERVIEW ECOLOGICAL CHARACTER DISPLACEMENT THREESPINE STICKLEBACKS THESIS O V E R V I E W CHAPTER TWO: SHAPE CHANGES IN FRESHWATER DIVERSIFICATION OF THREESPINE STICKLEBACKS KEYWORDS INTRODUCTION '. MATERIALS AND METHODS Fish populations Photography and digital scanning of stickleback slides Landmark selection and positions Overview of thin-plate spline (TPS) geomorphometric analysis Superimposition of specimens Uniform and non-uniform components of shape variation Shape divergence among sticklebacks Testing for growth and shape relationships RESULTS DISCUSSION
1 1 4 9
12 12 12 15 15 17 18 19 20 21 21 23 24 27
CHAPTER THREE: COMPETITION, SELECTION, A N D PHENOTYPIC PLASTICITY IN M A R I N E S T I C K L E B A C K S A F T E R C O L O N I Z A T I O N OF F R E S H W A T E R 40 KEYWORDS INTRODUCTION METHODS Fish populations Fish Collection and Rearing Experimental Design Data Acquisition Morphology
40 40 43 43 43 44 46 46 iv
Diet Intensity of Interspecific Competition Morphological Shifts Controlling for ontogenetic changes Controlling for standard length RESULTS Test of Interspecific Competition Natural Selection and Phenotypic Plasticity DISCUSSION Growth and Survival Diet Shifts Morphological Shifts Phenotypic Plasticity and Character Displacement CONCLUSION
47 49 50 51 52 53 53 54 56 57 60 61 63 64
CHAPTER FOUR: E X P E R I M E N T A L E V I D E N C E OF DECLINING INTERSPECIFIC COMPETITION DURING C H A R A C T E R DISPLACEMENT 75 KEYWORDS INTRODUCTION METHODS Fish Populations Fish Collection and Rearing Experimental Design Data Acquisition and Analysis Growth Survivorship Diet RESULTS Growth Survivorship Diet Shifts DISCUSSION
75 75 77 77 78 78 81 81 82 83 85 86 87 87 88
CHAPTER FIVE: CONCLUSION A N D RECOMMENDATIONS FOR F U T U R E RESEARCH
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REFERENCES
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LIST OF T A B L E S Table 2.1 Table 2.2 Table 3.1 Table 3.2 Table 4.1 Table 4.2 Table 4.3 Table 4.4
Summary of morphological differences among stickleback forms Centroid size and standard lengths of TPS specimens Summary of fish sampling Relationships with standard length Fish recovery and survivorship summary Mass and length of common prey items Diet analysis summary Summary of mean number of prey items consumed
39 39 74 74 98 98 99 100
LIST OF FIGURES Figure 1.1 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5
Character displacement model TIFF image of the stickleback species Stickleback images and landmark positions Stickleback consensus configuration Discriminant function analysis Relative warp analysis Deformations along first relative warp Transitions between marine and lacustrine configurations Experimental design and pond schematic Mean marine growth over 90 days Marine survivorship Mean proportion zooplankton in marine diets Natural selection on marine gill raker number Plasticity and/or selection in marine gill raker length Plasticity and/or selection in marine body depth Plasticity and/or selection in marine gape width Character displacement model and experimental treatments Experimental design and pond schematic Mean marine growth Absolute marine survivorship Diet analysis of marine sticklebacks
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11 32 33 34 35 36 37 38 66 67 68 69 70 71 72 73 93 94 95 96 97
ACKNOWLEDGMENTS Like the graduate students before me, and those surely to follow, I am a parasite. J. D. McPhail and his colleagues built the conceptual and logistical foundation upon which I based my research. They initially recognized and described the differences among populations of sticklebacks, and formulated hypotheses for how these populations evolved. I was fortunate to be able to utilize a study system with more than three decades of intensive research. A big thanks to those whose work I have used. I owe my supervisor, Dolph Schluter, and graduate research committee, Martin Adamson, Jon Donald McPhail, Don Moerman, and Bill Neill, a great debt. First, thank you for the opportunity and freedom to follow my own path. Thank you for having the courage and foresight to say "no" when I went astray. And thank you for sharing your individual perspectives and experiences so that we could reach a successful conclusion. Several people were especially helpful from the beginning of my graduate study. Lance Barrett-Leonard, Alistair Blanchford, Andrea Byrom, Dennis Chitty, Don Reid, Dave Ward, and Dave Westcott helped immensely with my thesis proposal. All of these people provided excellent suggestions, intellectual banter, and positive reinforcement. Dennis impressed upon me the importance of writing and speaking clearly. I hope this thesis reflects positively on his efforts. I had considerable help setting up and completing my experiments. In the beginning I either "borrowed" my supervisor's technicians (Nic Grabovac, Laurin Hummelbrunner, and Rees Kassen) or enlisted the help of my fellow lab mates. Once I had "proven" my ability to use assistance, I was granted my own field assistant. Except for dropping one of my socket wrenches into Marion Lake and letting "frog boy" escape, Matt McLeod was an ideal field
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assistant. Matt had a capacity to make our tasks fun and never complained about the long and erratic hours, bizarre working conditions, or redundant tasks. Several people assisted me with various stages of my research. Todd Hatfield showed me the ropes of stickleback biology, as well as provided the limnetic sticklebacks analyzed in Chapter Two. Gordon Haas always had his door open, and freely shared his knowledge, experience, and toys. Beren Robinson and Sharilynn Wardrop had the time and patience to teach me how to photograph and digitize sticklebacks. Dean Adams convinced me to tackle the challenge of geomorphometric shape analysis, and helped me through the labyrinth of jargon, analyses, and software bugs. Danusia Dolecki shared her knowledge of zooplankton and zoobenthos, and was always available to check my identification of "gobbled" critters. Michelle Roberge shared her time and enthusiasm. Sally Otto and "Ohta" were especially helpful in checking my bootstrapped survivorship estimates through numerical integration of various binomial probability functions. Rick Taylor kept me abreast of his work on stickleback phylogenetics, provided encouragement, and let me know when I made mistakes. Thank you. The students, post-docs and faculty from both "The Huts" and "The Big House" all contributed to this thesis and my development as a graduate student. My fellow graduate students and colleagues of U B C provided a rich intellectual and social environment. Dick Cannings, Charlie Krebs, Judy Myers, Jamie Smith, Rick Taylor, and Mike Whitlock all taught me things while we were teaching "the kids". Reuven Dukas, Stephen Heard, Mats Linden, Arne Mooers, and Beren Robinson provided stimulating discussions and grand adventures. My lab mates, Dick Repasky, Todd Hatfield, Durrell Kapan, Laurel Nagel, Troy Day, Steve Vamosi, Howard Rundle, and Elizabeth Clifford, taught me a lot about myself, the hunger for
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knowledge, and cooperation. Alistair Blanchford and Lance Bailey rescued me from numerous computer disasters, answered my UNIX, network, and S-plus questions, and otherwise kept me and our Bioscience Computing Unit "up and running". Elaine Humphrey showed me the finer points of imaging software and the capabilities of the E M Lab's hardware. Bruce, Granville, and Ken of the "Zoology Shop" were supportive of my need to "make things", and provided technical assistance, consulting, and "scrap bits" for building everything from small fish enclosures to bagpipes. I very much appreciate the help of the U B C Zoology Department office staff. Kathy Gorkoff and Diane Mellor both went out of their way to offer me assistance, advice, and support. Thanks so much to the hordes of people who came into the field to give me a hand! A few of my colleagues willingly subjected themselves to early versions of this thesis. While at times I questioned their sanity, I am truly grateful for their comments, criticisms, and perseverance. Dolph Schluter and Andrea Byrom did the "lion's share" improving countless drafts. My committe, Sally Otto, Beren Robinson, Karen Hodges, and Kathy Heise also provided useful suggestions and encouragement. My friends and family provided the support I needed to slay this beast. Mom, Dad, Ken, Grammy and Grampy, Grandma and Grandad, I've missed you. You've often been in my thoughts and I look forward to seeing more of you soon. Really... I'm "almost" done. Laurel, your love and tenacity never ceases to amaze me. Ma, Emerald and Earl, Perry and Jo, Brad and Warren, Angie and Travis, Tom and Aud and Ryan, thank you all for your encouragement, support, and "escapes" from Vancouver. Andrea and Andy, John and Darce, Lance and Kathy, Lenny and Katrina, Y i and Uwe, Mike, Doug and Colleen, thank you for adding a bit of balance to this affair. Jordan, thank you for sharing your passion for biology,
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woodworking, and pottery. My friends from Bujinkan Budo Taijutsu and Muso Jikiden Eishen Ryu Iaido helped me learn the meaning of both nin and kokoro. Thank you all for the tools to survive. Dolph, I can't thank you enough. You and NSERC provided funding for my research, food, and rent. But, you also did much more. You gave me the opportunity to complete this PhD. You let me go with my crazy ideas even when you suspected they might not "pan out", challenged me to think and work independly, and convinced me to keep going when I wanted to quit. Thank you. Your confidence and enthusiasm has meant a lot. I can't imagine anyone I'd rather have had as a supervisor. Keep at it. You have found your calling.
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CHAPTER ONE: INTRODUCTION A N D O V E R V I E W
Although reviews of field experiments show that competition is commonly found in nature (Connell 1983; Schoener 1983; Goldberg and Barton 1992; Gurevitch et al. 1992), there is little strong empirical evidence that competition results in coevolution among competitors (Taper and Case 1992a). Connell (1980) questioned the importance of competition as an evolutionary force, and demanded experimental evidence that competition can result in evolution. This thesis presents experimental evidence that interspecific competition played a role in the evolution of morphological divergence between sympatric limnetic and benthic threespine sticklebacks (Gasterosteus aculeatus). In addition, I describe the morphological divergence among freshwater sticklebacks with a morphological analysis of body shape.
E C O L O G I C A L C H A R A C T E R DISPLACEMENT The process of evolution in response to interspecific competition is "ecological character displacement" (Brown and Wilson 1956; Grant 1972; Dunham et al. 1979; Schluter and McPhail 1992; Taper and Case 1992a). In theory, interspecific competition can produce both evolutionary divergence and evolutionary convergence between competitors (Brown and Wilson 1956; Grant 1972; Rosenzweig 1978; Grant and Abbott 1980; Arthur 1982; Abrams 1986, 1987a, b, 1997; Schluter and McPhail 1992; Taper and Case 1985, 1992a, b). Thus, it is expected to produce a pattern in which two species occurring together (sympatry) are either more different from, or more similar to, one another than when they occur alone (allopatry) (Futuyma 1986).
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Divergence is the pattern most often described in the literature, and this thesis focuses on one such pattern. Convergence is possible when there is competition for non-substitutable resources, such as nutrients and light. Divergent character displacement is expected when resources are nutritionally substitutable, such as for many carnivores. If each species could reduce interspecific competition through resource partitioning and thereby increase its own fitness, this would result in divergent selection on the characters which confer efficiency in use of the resource. Thus, interspecific competition would drive the evolution of divergent characters which utilized opposite ends of the resource continuum (Futuyma 1986; Maynard Smith 1989). The pattern of greater morphological divergence between species, or morphs, in sympatry versus allopatry is common in a wide range of organisms. It has been observed in white clover (Turkington and Harper 1979), mud snails (Fenchel 1975; Fenchel and Kofoed 1976; Fenchel and Christiansen 1977), lizards (Schoener 1969, 1970; Losos 1990a, 1990b), charr (Skiilason et al. 1992; Magnan and Stevens 1993; Skiilason et al. 1993; Snorrason et al. 1994), sunfish (Robinson and Wilson 1994; Robinson et al. 1996), finches (Grant 1972; Schluter and Grant 1984; Schluter et al. 1985; Schluter 1988), honeyeaters (Diamond et al. 1989), bird-eating hawks (Schoener 1984), canids (Dayan et al. 1990, 1992; Van Valkenburgh and Wayne 1994; Kiesser 1995) and numerous other organisms (Malmquist 1985; Whitten and Williams 1992; Stol 1994; Reynolds andMayville 1994; Kawano 1995; Poeser 1995; Skiilason and Smith 1995; Chiba 1996; Smith and Skiilason 1996; Werdelin 1996). However, demonstration of this pattern alone is not sufficient to invoke an explanation of divergent ecological character displacement.
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Several items must be addressed before an example of greater divergence in sympatry than allopatry be attributed to character displacement. First, the pattern should occur multiple times and cannot be re-created by randomly sampling different allopatric populations (Grant 1972; Grant and Abbott 1980; Schluter and McPhail 1992,1993; Taper and Case 1992a; Schluter 1995b). Second, sites of sympatry and allopatry should be ecologically similar. Third, the phenotypic differences producing the pattern must have a genetic basis (Slatkin 1980) and the morphological differences between sites of sympatry and allopatry must represent evolutionary shifts. Fourth, the morphological differences should also reflect differences in resource use. Yet even if all these criteria were met, one more condition must be true. Similar phenotypes must compete for food. I focus on this last criterion through experimental demonstration of interspecific competition for food resources. Demonstration of interspecific competition is critical evidence of character displacement. Yet, most hypothesized instances of character displacement never go beyond describing morphological differences between sites of sympatry and allopatry. Only three "character displacement" systems have convincingly demonstrated interspecific competition: mud snails (Fenchel and Kofoed 1976; Gorbushin 1996), Anolis lizards (Pacala and Roughgarden 1982, 1985; Rummel and Roughgarden 1985; Losos 1990b, 1994; Losos et al. 1994), and sticklebacks (Schluter 1993, 1994, 1995a). These examples are not without their problems. In mud snails, no one has demonstrated that there is a genetic basis to the pattern of greater divergence in sympatry than allopatry. Indeed, recent evidence suggests that differences between sites of sympatry and allopatry have a non-genetic explanation (Salomeni 1993; Gorbushin 1996). In Caribbean Anolis lizards, one phylogenetic analysis of sympatric
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populations concluded that if character displacement occurred, it likely happened only once (Losos 1990a; 1994). The most convincing example of ecological character displacement to date comes from threespine sticklebacks (Gasterosteus aculeatus complex) inhabiting postPleistocene coastal lakes in southwestern British Columbia (Schluter and McPhail 1992, 1993; Schluter 1993, 1994, 1995a, 1996a,b; Taylor et al. 1997). The present thesis continues work on this system. Below, I describe the pattern and work done so far. I then describe my work in the following chapters.
THREESPINE STICKLEBACKS Threespine sticklebacks clearly exhibit greater morphological divergence in sympatry than in allopatry. Five (previously six) small, coastal lakes of southwestern British Columbia contain two genetically divergent forms of threespine sticklebacks (Schluter and McPhail 1992). One form, the "limnetic", is an open-water habitat specialist that forages primarily on zooplankton. The other form, the "benthic", is a bottom habitat specialist that forages primarily on littoral-dwelling invertebrates. Limnetics and benthics have distinctive body morphologies. Limnetic sticklebacks are smaller, and have longer, more numerous gill rakers, narrower gapes and thinner bodies than benthic sticklebacks. Most lakes in southwestern British Columbia contain a single stickleback form which can range in morphology and ecology from a limnetic-like form to a benthic-like form. However, the majority of small coastal lakes, similar to lakes with sympatric forms, contain an "intermediate" form, which utilizes both the open-water and littoral habitats and has a body morphology intermediate between the two sympatric forms (Bentzen and McPhail 1984; McPhail 1984, 1992, 1993,
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1994; Lavin and M c P h a i l 1985, 1986, 1987; Schluter and M c P h a i l 1992, 1993; Schluter 1993, 1994, 1995a). Although these sticklebacks are ecologically, morphologically, and reproductively differentiated, and maintain these differences in sympatry, they have not been formally described as "species". Nevertheless, for convenience, I refer the limnetic, benthic, intermediate, and marine populations used in this thesis as "species". Schluter and M c P h a i l (1992) proposed that divergent ecological character displacement may be responsible for the formation of sympatric stickleback populations. They proposed two alternative character displacement models: 1) one based on two invasions of lakes by marine sticklebacks (double invasion), and 2) one based on a single marine invasion followed by sympatric speciation. The double invasion model is as follows (Figure 1.1; also see McPhail 1993). Marine sticklebacks initially colonized or were trapped in newly formed lakes following the retreat of the glaciers and subsequent geological rebound of landmasses approximately 13,000 years ago (first invasion). These sticklebacks then evolved to an intermediate morphology which was adapted to lake environments. Many hundred (e.g. 250 2500) years later, a second temporary rise in sea level again allowed marine sticklebacks access to some lake populations (second invasion). In lakes where marine sticklebacks colonized but did not overwhelm freshwater populations, competition for food between marine and intermediate sticklebacks resulted in the evolution of the present-day sympatric stickleback species. The first marine invaders evolved into intermediate and then benthic sticklebacks, whereas the second marine invaders evolved into limnetic sticklebacks. The single invasion model also predicts character divergence between two morphologically and ecologically similar stickleback species, but these species derive from sympatric speciation of
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the intermediate sticklebacks (e.g. via competitive speciation; Rosenzweig 1978) rather than a second marine invasion. Currently, the double invasion ecological character displacement model appears to be more likely than the single marine invasion model. In this thesis, I used the two marine invasion model to test predictions of character displacement in sticklebacks. The geological history of southwestern British Columbia (Mathews et al. 1970, Clague 1983) and restricted distribution of the sympatric populations (McPhail 1993), lends support to the double invasion model, since conditions allowing a second invasion would likely be rare and restricted to a small geographic area. The model is further corroborated by the following evidence: allozyme data (Withler and McPhail 1985), salinity tolerance of stickleback eggs (Kassen et al. 1995), and microsatellite D N A data (E. B. Taylor, personal communication). Allozyme and microsatellite data both suggest two invasions of freshwater with limnetics as the most recent colonists. These data suggest each limnetic population is more closely related to marine sticklebacks than to either their sympatric benthic population or other limnetic populations. Salinity tolerance data also suggests limnetics were more recent colonists of freshwater than benthics (if salinity tolerance is assumed to decay with time). Mitochondrial D N A (mtDNA), however suggests each limnetic and benthic pair resulted from sympatric speciation, because limnetics and benthics of each population appear to be most closely related to one another (Taylor et al. 1997). However, the mtDNA results are likely contaminated by gene flow following secondary contact (Taylor et al. 1997; E. B. Taylor, personal communication). The marine threespine stickleback (Gasterosteus aculeatus) is primarily a pelagic planktivore that inhabits most of the northern hemisphere (Miinzing 1963; Bell 1976; Schluter and McPhail 1992; McPhail 1993). Marines are anadromous and breed along the coastline in
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freshwater drainages (Hay and McPhail 1975; McPhail and Hay 1983; Siamoto 1993). They have repeatedly colonized freshwater environments for at least ten million years (Bell 1977, 1994; McPhail 1994), and although freshwater populations often diverge markedly from their marine ancestor and each other, they do not persist over geological time. The marine stickleback has little morphological differentiation throughout its holoarctic range (Bell 1976; Francis et al. 1986; McPhail 1994), and little molecular differentiation among western North American populations (Withler and McPhail 1985; Withler et al. 1986; O'Reilly et al. 1992; Orti et al. 1994). Comparison between modern and fossil marine threespine sticklebacks indicates little morphological change since the late Miocene (several million years ago) (Bell 1977, 1994; Sychevskaya and Grechina 1981). This lack of differentiation among modem and Miocenic marine sticklebacks supports my assumption that the present-day marine species closely resembles, ecologically and morphologically, the ancestor of the lacustrine stickleback populations. Several other factors lend credibility to character displacement in the stickleback system. The pattern of greater morphological divergence in sympatry than allopatry is likely not a chance event as the displacement pattern occurred six times and cannot be generated by randomly sampling allopatric populations (Schluter and McPhail 1992). As well, the morphological traits differing among, and within, stickleback populations are heritable (Miinzing 1963; McPhail 1977, 1984, 1992; Hagen 1973; Hagen and Gilberston 1973; Baumgartner 1986; Schluter and McPhail 1992; Hatfield 1995, 1997). In a series of complementary laboratory and field experiments, Schluter (1993, 1995a) demonstrated the adaptive significance of the morphological differences among different forms of sticklebacks. Limnetics and benthics had higher feeding efficiency and growth in their primary habitats than
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in each others habitats, whereas sticklebacks of intermediate morphology had intermediate feeding efficiency and growth in both habitats. There also are other demonstrations that foraging success in sticklebacks depends on habitat and morphology (Bentzen and McPhail 1984; Lavin and McPhail 1986). Schluter (1994) tested the prediction from character displacement theory that interspecific competition on intermediate sticklebacks would result in selection favoring intermediate phenotypes most divergent from the competitor species. For his experiment, Schluter created a phenotypically-variable intermediate stickleback population by hybridizing Cranby Lake intermediates with Paxton Lake limnetic and benthic sticklebacks. His experimental design was based on the double invasion character displacement model (Figure 1.1). The modified intermediate stickleback population was present alone and in combination with limnetic sticklebacks on separate sides of two, divided experimental ponds. He found that in the presence of limnetic planktivores, the intermediate phenotypes most similar to limnetic sticklebacks suffered the greatest growth depression, an effect that decreased in magnitude with decreasing morphological resemblance between intermediate phenotypes and limnetic sticklebacks. In contrast, when intermediate sticklebacks were present alone, there was no detectable difference in growth among the phenotypes present. Thus, Schluter demonstrated interspecific competition between intermediate and limnetic sticklebacks resulted in divergent natural selection favoring more benthic-like phenotypes within the intermediate population.
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THESIS OVERVIEW Schluter's (1994) experiment provides one of the most compelling pieces of evidence that interspecific competition plays a significant role in the evolution of the sympatric stickleback forms. Yet, many questions concerning the role of character displacement in this system remained unanswered. This thesis addresses two of these questions. 1) Does the planktivore experience competition from intermediate sticklebacks? 2) If so, does the strength of competition experienced by the planktivore decrease as the other form becomes more divergent from it ecologically and morphologically? I used a novel approach to answer these questions by using the hypothesized ancestor, the marine threespine stickleback, as the target species. Juvenile marine sticklebacks were introduced into freshwater ponds either alone or together with juvenile freshwater sticklebacks. These manipulations were used to measure the intensity and effects of interspecific competition on the marine sticklebacks, and to test whether these results conformed to predictions from character displacement theory. The experiment presented in Chapter Three tested the prediction that marine sticklebacks would compete with intermediate sticklebacks. In addition, I tested whether this competition, or lack of it, would result in adaptive shifts in marine morphology. The experiment presented in Chapter Four tested the prediction that competition on marine sticklebacks would decline as the other species became more divergent from it. To test this prediction, I compared the intensity of competition on marines in the presence of intermediate sticklebacks with that on marines in the presence of benthic sticklebacks. An additional goal of this thesis is to provide a more complete description of the character displacement pattern in threespine sticklebacks. While marine, limnetic, benthic, and
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intermediate sticklebacks have been described in terms of gill raker number, gill raker length, gape width, and body depth and length, little is known about differences among these sticklebacks in body shape. In Chapter Two, I describe a morphological analysis of body shape for these sticklebacks. I tested whether body shape follows the same pattern of character divergence as other morphological traits. Furthermore, I describe the shape changes from the ancestral marine state to each of the descendant freshwater states.
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Ecological character displacement (ECD) model First marine invasion Intermediate form evolves
A. •*
CD
.5
Second marine invasion
A
Character displacement V
Modern species pair
Q #
Q
benthivore
planktivore
Ecological Morphology
Figure 1.1 Character displacement model Illustration of the hypothesized sequence of events leading to the morphological differentiation between contemporary sympatric stickleback forms: the ecological character displacement model assuming double invasion with allopatric speciation. Symbols indicate the mean position of a population along a resource-based morphological continuum. Positions in ecological morphology range from littoral resource specialist (i.e. benthivore) to zooplankton resource specialist (i.e. planktivore). The marine (ancestral) form is denoted by a star (*), whereas intermediates are a triangle (A), benthics a closed circle (•), and limnetics an open circle (O). The model begins when a lake environment is colonized by the marine ancestor (a planktivore). Marines evolve toward an intermediate phenotype which utilizes the entire resource continuum. Following a second marine invasion, competition for resources between marines and intermediates results in ecological character displacement. The intermediate form is displaced toward an increased benthivore morphology (i.e. benthic form), whereas the marine form is displaced toward a lake-adapted planktivore morphology (i.e. limnetic form). Figure modified from Taylor et al. (1997), after Schluter (1996a) and Schluter and McPhail (1993).
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C H A P T E R TWO: SHAPE CHANGES IN F R E S H W A T E R DIVERSIFICATION OF THREESPINE S T I C K L E B A C K S
John Robert Pritchard The Centre for Biodiversity Research, Department of Zoology, University of British Columbia 6270 University Blvd., Vancouver, BC V6T1Z4 Canada pritchrd® zoology, ubc. ca
KEYWORDS Body shape, evolution, fish, morphological divergence, geometric morphometries, landmark data, partial-warp scores, thin-plate splines, threespine sticklebacks (Gasterosteus spp.).
INTRODUCTION Planktivorous marine threespine sticklebacks (Gasterosteus aculeatus) have repeatedly colonized freshwater environments over the past ten million or more years (Bell 1977, 1994; McPhail 1994). Freshwater populations have diverged markedly in ecology and morphology from the marine ancestor and from each other (Bell and Foster 1994). Throughout their circumpolar range (Miinzing 1963; Hagen and McPhail 1970), marine sticklebacks exhibit little molecular (Withler and McPhail 1984; O'Reilly et al. 1992; Orti et al. 1994) or morphological (Heuts 1947; Miinzing 1963; Hagen 1967; Bell 1976) variation. Fossil evidence from the Late Miocene (approximately 10 million years ago) suggests marine sticklebacks have not changed in millions of years. Consequently, marines forming freshwater populations after the last glacial period (the Pleistocene) are presumed "identical", at least in morphology, to present day marines (Bell 1977, 1994; Bell and Foster 1994). In contrast,
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post-glacial freshwater populations of sticklebacks have radiated into an ecologically diverse species complex (Heuts 1947; Miinzing 1963; Hagen and McPhail 1970; Bell 1976; Moodie and Reimchen 1976; Francis et al. 1986; Schluter and McPhail 1992, 1993; Bell and Foster 1994; McPhail 1994). The present-day marine threespine stickleback is useful as a reference and tool for investigation of morphological and ecological changes that occur during freshwater diversification. In this paper, I use geometric shape analysis to investigate morphological shifts in a subset of freshwater species. McPhail (1994) summarizes a recent (less than 13,000 year old) adaptive radiation in threespine sticklebacks (Gasterosteus aculeatus complex) inhabiting small coastal lakes of southwestern British Columbia. All the populations formed when the marine stickleback colonized freshwater at the end of the Pleistocene. In five (previously six) small, coastal lakes, two stickleback species have formed, which partition the lakes' environment between the open-water and littoral/benthic habitats. The "limnetic" stickleback utilizes primarily openwater zooplankton and has a morphology specialized for this way of life: a thin, fusiform body; numerous, long gill rakers; and a narrow gape. The "benthic" stickleback utilizes primarily the littoral zone and deeper bottom substrates and has a morphology specialized for preying on benthic invertebrates: a deep, robust body; few, short gill rakers; and a wide gape. Most other small lakes contain a single species of stickleback, referred to as the "intermediate" or "solitary" stickleback, which is morphologically intermediate between limnetic and benthic sticklebacks, and utilizes both open-water and bottom habitats (Bentzen and McPhail 1984; McPhail 1984, 1992, 1993, 1994; Lavin and McPhail 1985, 1986, 1987; Baumgartner et al. 1988; Schluter and McPhail 1992, 1993; Schluter 1993,1994, 1995a).
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Schluter and McPhail (1992) summarized the size and shape differences among sticklebacks from these six sympatric lake populations, as well as ten solitary populations and a nearby marine population. Most variation among stickleback populations was accounted for in differences in body "size", defined as the first principal component axis of standard length, body depth, gape width, gill raker number and length: marine sticklebacks were described as the largest, while benthics, intermediates, and limnetics were successively smaller (Table 2.1). "Shape" was described in terms gill raker number and length, gape width, body depth, and body length when all were adjusted for differences in "size". Marines were nearly equivalent to limnetics in "shape", both were different from benthics, and solitary populations were intermediate. This approach provided a limited description of lateral shape differences among the species because the measurements describing fish profiles used solely body length and depth. However, these fish populations differ greatly in other aspects of profile that have never been quantified. Recent advances in morphometric methodology provide new approaches that are effective in capturing shape information of organisms (reviewed in Rohlf and Marcus 1993). These approaches (e.g. thin-plate spline analysis) are more powerful than previous techniques because they retain information about the geometric relationships among measurements, and permit graphical depiction of body shape changes among specimens and groups. This contrasts with other multivariate shape analyzes (e.g. PCA) which describe shape with numerous distance measurements between body structures, but retain no information that certain measurements share structures, and are not able to graphically display specimens from the distance measurements.
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In this paper, I use one of these new methods, thin-plate spline (TPS) geomorphometric analysis, to analyze body shape variation among four stickleback species: limnetic, benthic, intermediate, and marine (Figure 2.1). As a matter of opportunity, I was able to use sticklebacks which were all raised under common conditions and used in freshwater pond competition experiments; in contrast, Schluter and McPhail (1992) used marines returning to freshwater after growing primarily at sea. I will use the results of this analysis to answer three questions. First, can this method be used to discriminate completely between limnetics, benthics, intermediates and marines? Second, does geometric body shape variation among these stickleback species match the divergence patterns reported by Schluter and McPhail (1992)? More specifically, are limnetics and marines equivalent in shape, and do intermediates lie between them and benthics? Finally, what have been the main evolutionary trends in body shape from the ancestral marine state to descendent populations in small lakes?
MATERIALS A N D METHODS Fish populations I examined body shape differences among a marine population, and three lake stickleback species: a sympatric limnetic and benthic species pair (Paxton Lake), and an intermediate stickleback population (Cranby Lake). Marine sticklebacks were from the mouth of the Little Campbell River. All fish were hatched in the laboratory from wild-caught fertilized eggs, and transferred into freshwater ponds at the University of British Columbia experimental pond facility after approximately one month of growth. These four types of sticklebacks were subjects of two experiments carried out in the summer of 1995.
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As a matter opportunity and convenience the stickleback species were taken from two experiments. Marine, intermediate and benthic sticklebacks were sampled from one experiment, which consisted of two treatments paired within divided ponds (for details, see Chapter Four). Limnetic sticklebacks were sampled from a pond-half subjected to a second experiment (Schluter and Hatfield, unpublished experiment), where limnetics were together with benthics and limnetic-benthic hybrids. This experiment began on 12 June 1995 and ended after about thirteen weeks. The two experiments differed with respect to duration, frequency of species, and total fish density; therefore, growth rate and final size among species are not directly comparable. This prevented me from using size as a character to help distinguish the species, and required that I test the effect of this difference in size on my shape measurements. Both experiments were ended by adding 0.5kg of rotenone
(C23H22O6,
Syndel Laboratories,
Vancouver, BC) to each half-pond. After anesthetizing with MS-222 (tricaine methanesulfornate, Syndel Laboratories), the fish were fixed with 10% formalin (3.7% formaldehyde) for one to two weeks, then stained in alazarin red, and preserved finally in 37% isopropyl alcohol. In addition to convenience, other advantages of using these sticklebacks for shape comparisons is that I control for differences in shape arising from sea versus lake environment, and age structure within populations. There may be significant differences in the body shape of marine sticklebacks raised in freshwater versus saltwater. For instance, Honna and Tamura (1984) reported dwarfism in marine sticklebacks raised in freshwater. I have also observed decreased size in marine sticklebacks, relative to intermediate and benthic sticklebacks, when raised in freshwater (Chapters Three and Four). Since I am concerned with the evolution of freshwater stickleback species from marine sticklebacks invading freshwater, it was most
16
appropriate to compare marines raised in freshwater, versus the sea, to the each of the freshwater stickleback species. Furthermore, since each experimental population of stickleback species came from a single age class, I was able to control for any shape differences arising from age structure which would be present in wild populations. Thus, I was able to minimize shape differences within species due to growth-related (age-related) allometry. Previous shape comparisons using thin-plate spline (TPS) geomorphometric analyses suggest sample sizes per group of approximately twice the number of landmarks being analyzed to maintain statistical power (Rohlf et al. 1996; Adams and Funk 1997; Caldecutt and Adams 1997). I had twenty-eight fish representing each stickleback species and my shape analysis was based on fifteen landmarks per specimen. Photography and digital scanning of stickleback slides Fish were photographed in pairs with Ektachrome 160 Tungsten slide film under tungsten lighting conditions (F-stop: 22, Exposure: 1 second, Focus: by eye). Sticklebacks were placed in pairs on a paraffin wax mount with a Canadian dime, which was included in each picture as a standard size reference (diameter = 17.93 mm ± .004 mm). Slides were scanned at 1333 DPI as black and white images using a K O D A K Professional RFS 2035 Plus Film Scanner and Adobe Photoshop version 4.0. Each slide image was then trimmed to minimize unnecessary information, resized to a 200 DPI image, adjusted by eye for contrast and brightness, and saved as TIFF formatted file from Photoshop. Figure 2.2 illustrates the photographic arrangement and a facsimile of the resulting image.
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Landmark selection and positions I chose fifteen landmarks to define the major components of stickleback body shape (Figure 2.2). The landmarks, which formed a two-dimensional grid of discrete points for each stickleback, were chosen by two criteria: 1) all landmarks must be readily identifiable on all individuals and species, and 2) the set of landmarks would outline a stickleback shape. All of the landmarks chosen, with the exception of landmark 13, represent the meeting of two or more tissues; Bookstein (1990a) reports that these landmarks are the most informative and classifies them as Type I landmarks. Landmark 13 is a point of maximum curvature, which Bookstein (1990a) classifies as a Type II landmark. The position of landmark 14, at the base of the first dorsal spine, had to be estimated in Paxton Lake benthic sticklebacks because some individuals of this species lack this spine (13 of 28 benthics examined lacked the first spine). However, the spine when present is always associated with a clear indentation in the dorsal surface that is present also in benthics lacking the spine; hence, it was apparent where landmark 14 would be positioned. I had no landmarks between landmarks 7 and 8 because Paxton Lake benthics have no pelvic girdle or spines, and there was no readily identifiable position for a landmark in this region on benthics. The x,y coordinates of all landmarks were determined with the public domain NIH Image program version 1.61 (developed for Macintosh computers at the US National Institutes of Health; available on the Internet at http://rsb.info.nih.gov/nih-image). First, each TIFF image was calibrated by setting the diameter of the Canadian dime to 17.93 mm. The fifteen landmark positions were then marked on the sticklebacks as shown in Figure 2.2, and the x,y coordinates calculated by the NIH Image program were saved to a spreadsheet.
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Overview of thin-plate spline (TPS) geomorphometric analysis In this paper, I use thin-plate splines as a tool to analyze body shape. Consequently, I only provide an overview of the thin-plate spline methodology, and do not discuss the mechanics of the method in detail. Technical reviews of thin-plate splines and similar geomorphometric analyses are present by Rohlf and Bookstein (1990), and Rohlf et al. (1996). Zelditch et al. (1992), Zelditch and Fink (1995), and Fink and Zelditch (1997) provide detailed and intuitive descriptions of thin-plate spline analysis. I used thin-plate spline geomorphometric analysis to analyze stickleback body shape while controlling for body size. I completed this analysis using the landmark data of all 112 stickleback specimens and a series of programs developed by F. James Rohlf (1997a, b, c). The landmarks allowed calculation of a "mean shape", called the consensus configuration, which was used as a reference grid for comparison of shape among all specimens in the data set. I also calculated "mean shapes" for each of the different species, which I refer to as "species configurations". Shape differences due to geometric body size, or centroid size, were controlled by scaling all specimens to a centroid size of one; centroid size is defined later, but is analogous to the geometric area defined by the landmarks. Shape variation was then partitioned into two uniform components and twenty-four non-uniform components. Uniform shape variation is defined as the portion of variation accounted for by linear compression, stretching, or tilting of the consensus configuration in two dimensions (Adams and Funk 1997). The uniform components, ul and u2, are the two principal axes of this variation (Bookstein 1990b; Rohlf 1997a), and are calculated using Bookstein's (1996) linearized Procrustes estimate (Rohlf et al. 1996; Rohlf 1997a). Non-uniform shape variation
19
is defined as that requiring non-linear deformations of the grid of landmarks. There are (2k-6) non-uniform components of shape variation, where k= the number of landmarks. The nonuniform components are the partial-warp scores calculated using thin-plate splines (Bookstein 1990b; Rohlf and Marcus 1993; Rohlf et al. 1996; Rohlf 1997a; Adams and Funk 1997; Caldecutt and Adams 1997). Thin-plate splines are functions that describe the bending of a hypothetical, infinitely thin metal sheet (the "thin-plate"), which has been laid over the consensus configuration and bent, or "warped", so that each consensus configuration landmark lines up with the corresponding landmark on a target specimen. Thus, for every specimen there is a series of unique, non-uniform shape parameters, called partial-warp scores, which describe the thinplate spline function needed to bend the consensus configuration into that specimen's configuration. Superimposition of specimens Relative Warps version 1.09 (Rohlf 1997a) was used to superimpose all 112 stickleback specimens and calculate the consensus configuration. This program was also used to generate a species configuration for marine, limnetic, benthic and intermediate sticklebacks, by restricting the superimposition process to the 28 stickleback specimens representing each group. Relative Warps version 1.09 uses the generalized orthogonal least-squares (GLS) Procrustes method to superimpose specimens and generate a mean position for each landmark. While other superimposition methods exist (for a review, see Rohlf 1990), the GLS Procrustes method is required for thin-plate spline analysis (Rohlf and Bookstein 1990; Rohlf et al. 1996; Rohlf 1997a; Adams and Funk 1997).
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This superimposition process also removed all non-shape variation among sticklebacks by centering, scaling, and rotating each specimen. First, each specimen was centered by positioning its centroid, or "center of gravity" of its landmarks, at the origin of a twodimensional (x, y) grid. New x,y coordinates for each set of landmarks were then calculated for each specimen, based on the new position of its center at (0,0). Next, all specimens were scaled to a centroid size of one; where centroid size is defined as the square root of the sum of squared distances from each landmark to the centroid (Rohlf 1997a; Adams and Funk 1997). Finally, each specimen was rotated "to minimize the sum of squared deviations between homologous landmarks across all specimens" (Caldecutt and Adams 1997). Uniform and non-uniform components of shape variation Relative Warps version 1.09 also calculated the two uniform and twenty-four nonuniform components of shape variation. These shape components for each specimen were saved to a text file and imported into a statistical analysis program (S-plus version 3.3 for Windows; Mathsoft 1995). I used multiple analysis of variance (MANOVA) to test whether there were significant differences in means among marine, limnetic, benthic and intermediate sticklebacks in both uniform and non-uniform shape components. Shape divergence among sticklebacks I performed several analyses with the non-uniform components of shape variation, the partial-warp scores, to answer questions about shape divergence in sticklebacks. I also performed these analyses with the addition of the uniform components of shape variation, but the results were only slightly different and did not change my conclusions. Because of this and because some of the TPS software (Rohlf 1997b) only uses partial warp scores, I restricted most of my analysis to the non-uniform shape components. First, I performed a discriminant
21
function analysis of the variance-covariance matrix of partial-warp scores in order to separate marine, limnetic, intermediate and benthic sticklebacks into different groups. Second, I performed a principal component analysis of the partial-warp score variancecovariance matrix, also called a relative warp analysis (with a=0, as described by Rohlf and Marcus 1993; Rohlf et al. 1996; Rohlf 1997a; Caldecutt and Adams 1997), to summarize the nonlinear shape variation among specimens in as few dimensions as possible. The S-plus principal component analysis was performed on the partial-warp score matrix divided by the negative square root of the sample size (n=l 12). This "scaling" of the partial-warp scores was necessary to keep graphical outputs from Rohlf s (1997a) Relative Warps at the same scale as the principal component analysis. The first principal component axis, or first relative warp, accounts for the maximum amount of non-uniform shape variation. Similarly, subsequent principal component axes, which correspond to subsequent relative warps, account for successively smaller amounts of nonlinear shape variation (Rolhf and Bookstein 1990; Rohlf and Marcus 1993; Rohlf et al. 1996; Adams and Funk 1997). I also used the Relative Warps program to display graphical deformations of the consensus configuration at the extreme ends of the first relative warp. Finally, I used the species configurations of marines, limnetics, benthics and intermediates to describe the transition between the marine [ancestral] state and each of the lacustrine species. I used S-plus to plot the four stickleback species configurations, which were calculated by Relative Warps (Rohlf 1997a). For each lacustrine species configurations, I added vectors indicating the direction of movement for each landmark toward the marine configuration. All vectors were scaled by a factor of three in order to be visible. I also added an eye, and major fins and spines to each fish for illustrative purposes.
22
Testing for growth and shape relationships I calculated two measures of size, standard length and centroid size, for each specimen to test for size differences within and among species. Standard length (mm) was calculated from raw landmark coordinates as the distance between the base of the caudal peduncle (landmark 4) to the tip of the snout (landmark 10). Centroid size was calculated from the landmark positions of each specimen after the superimposition process. I used linear regression of centroid size on stickleback form, with species coded as a factor (i.e. species equaled marine, limnetic, intermediate or benthic), to test that the four species were different sizes. This test was also completed substituting standard length for centroid size. There were significant differences in size among the four stickleback species in both centroid size (R = 0.4518, F , i = 29.67, P= 4.5xl0" ) and standard length (R = 0.4348, 2
13
3
2
08
F3,108= 27.69, P= 2.3 x 10" ) (Table 2.2). This finding presented the possibility that any shape 13
differences observed among species could be explained by allometry; if growth affects shape, different species may have different shapes merely because they are different sizes and not because they are different species. However, there was no evidence of allometry, or growth-dependent shape changes, within species. Similarly, there was little evidence that shape changes along the gradients identified by the discriminant function analysis and the relative warp analysis were related to size. These conclusions were based on the following tests using centroid size as measure of fish size. These conclusions did not change when standard length was used as the measure of size. I tested whether centroid size could explain shape differences within each of the four stickleback species by performing separate linear regressions on each of the 24 non-uniform
23
shape components and the 2 uniform shape components by centroid size and form, with species coded as a factor. Since this involved 26 unplanned comparisons, I calculated a Bonferroni-corrected alpha-level of oc= 0.05/26 = 0.001923 for testing significance of each analysis (Sokal and Rohlf 1995). There was very little evidence of centroid size affecting shape within species; only one of the twenty-six shape components, the first partial-warp score, showed a significant (P< 0.00017) relationship between size and shape. Therefore, I did not correct for size differences within species. I also used linear regression, with species coded as a factor, to test whether centroid size could explain the shape differences described by either of the two discriminant function axes or either relative warp axes. There were no relationships between centroid size and either the first (P= 0.0669) or the second (P= 0.7113) discriminant function axes within species. Similarly, there were no relationships between centroid size and either the first (P = 0.0682) or the second (P= 0.5741) relative warp axes within species.
RESULTS The consensus configuration of all superimposed sticklebacks, with an outline formed by connecting the consensus coordinates of landmarks, is illustrated in Figure 2.3. The discriminant function analysis of the partial-warp scores revealed that it was possible to segregate the four stickleback species into nearly discrete, non-overlapping groups (Figure 2.4). There was no overlap between limnetic and benthic sticklebacks, while intermediates spanned the space separating these two stickleback species with minimal overlap. Marine sticklebacks occupied a unique area of space, separated completely from all of the freshwater stickleback species. There were significant amounts of variation among
24
marine, limnetic, benthic, and intermediate sticklebacks in both the two uniform ( M A N O V A , df= 3, Wilks' lambda = 0.3768, F
2
, 9 10
= 90.1257, P= 2.99xl0" ) and twenty-four non-uniform 24
( M A N O V A , df= .3, Wilks' lambda = 0.0.581, F
24
,87=
58.7753, P= 6.70xl0" ) components of 44
shape. Alternative M A N O V A test statistics (Pillai-Bartlett trace, Hotelling-Lawley trace, and Roy's largest eigenvalue) all yielded similar results. The principal components analysis of the non-uniform components of shape (partialwarp scores), or relative warp analysis, reveal the principal axes of shape variation among specimens (Figure 2.5). The position of each stickleback specimen, as well as the mean position of each stickleback form, was plotted relative to the first two relative warps in Figure 2.5. The first relative warp, or first principal component, explained 43.71% of the nonuniform shape variation among all 112 sticklebacks. The first three relative warps explained a total of 67.88% of this variation (13.73% from the second relative warp and 10.44% from the third relative warp). The mean positions of each stickleback species were arrayed in sequence along the first relative warp axis and showed little variation with respect to the second relative warp. Marine sticklebacks were at the lower end of the first relative warp, whereas benthics fell at the upper end. The first relative warp described non-uniform body shape variation among the four species that was consistent with the divergence patterns reported by Schluter and McPhail (1992): marine and limnetic sticklebacks had similar body shapes while intermediate and benthic sticklebacks had become increasingly different (Figure 2.5 and Table 2.1). Figure 2.6 illustrates the direction and magnitude of nonuniform shape changes from an extreme marine-like shape, denoted with the reference mark M at position (- 0.0045, 0.0) 1
in Figure 2.5, to an extreme benthic-like shape, denoted with the reference mark B at position 1
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(+ 0.0045, 0.0) on Figure 2.5. There were two major changes in non-uniform body shape associated with the transition from an extreme marine-like to an extreme benthic-like form: 1) the size of the head increases, and 2) the distance between the insertion points of both the dorsal and anal fins decreases. The increase in head size of benthic sticklebacks relative to marine sticklebacks is likely related to a morphological change in foraging specialization: benthics consume large prey items in the littoral habitat, whereas marines consume small zooplankton in the open-water habitat. Likewise, the decreased distance between anal and dorsal fin insertion points probably corresponds to decreased sizes of these two fins, which are important in performing "S-dart" swimming motions associated with plankton-foraging. Thus, movement along first relative warp corresponds with morphological changes that may be important in foraging specialization. The second relative warp accounted for few differences among the four populations, as the mean positions of each population all had approximately the same position relative to the second relative warp. However, the second relative warp accounted for mainly shape divergence within each stickleback species (Figure 2.5). The marine species configuration (Figure 2.7A) was plotted as a baseline and compared with each of the freshwater lake species configurations. Figures 2.7B, C, and D illustrate the non-linear shape changes required to move from limnetic, intermediate, and benthic configurations, respectively, toward the marine configuration. This series of comparisons illustrated that shape divergence from the ancestral state increases from the limnetic to the benthic configuration, with the intermediate configuration being intermediate. Few non-linear shape changes were required to obtain the limnetic configuration from the marine, whereas "large" shape changes were required to obtain the benthic configuration. The
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main body shape changes from the ancestral marine state to the limnetic species can be visualized as a "squeezing" of the marine configuration: the limnetic body is more fusiform and its mouth is moved slightly forward (Figure 2.1 A). In this respect, transition between the marine and limnetic stickleback species was primarily due to non-uniform reduction along the dorsal-ventral axis. In contrast, transition between marine and intermediate stickleback species was primarily due to compression of the trunk region along the anterior-posterior axis, and slight increase in size of the head region (Figure 2.7B). Transition between the marine and benthic stickleback species involved stretching of the body along the dorsal-ventral axis, and further increases size of the head region (Figure 2.7C). In all freshwater stickleback species, there was a steady migration of the first dorsal spine toward the caudal region.
DISCUSSION There were significant differences both uniform and nonuniform components of shape variation among marine, limnetic, intermediate and benthic stickleback populations. Uniform shape changes resulted from stretching, compression, or tilting of the consensus configuration in a particular direction (Rohlf et al. 1996), whereas nonuniform shape changes resulted from more complex deformations of the consensus configuration. This analysis focused on the differences among stickleback species due to differences in non-uniform components of shape variation. M y first objective was to see i f stickleback shape could distinguish marine, limnetic, benthic and intermediate sticklebacks. A discriminant function analysis of the partial-warp scores demonstrated clear separation among these species (Figure 2.4). Sympatric limnetic and benthic sticklebacks were completely separated in this analysis while intermediate
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sticklebacks lay directly between these two distributions. In contrast, the traditional morphometric techniques employed by Schluter and McPhail (1992) could not separate limnetics and benthics into two non-overlapping groups. One useful implication of this result is that thin-plate splines may be a useful tool for detecting interspecific hybrids between limnetic and benthic sticklebacks. This suggestion presumes that hybrids are intermediate in body shape between limnetic and benthic sticklebacks. This is likely because hybrids between these two stickleback species are intermediate in morphology, and resemble the phenotype of Cranby Lake intermediate sticklebacks in all characters measured (Schluter and McPhail 1992; Schluter 1994). To date, potential hybrids have been identified as individuals within the region of overlap between limnetics and benthics (McPhail 1984, 1992). Thin-plate spline analysis may be more powerful if it can identify individuals that fall between the sympatric species. Furthermore, the thin-plate spline method was much less labor intensive than the traditional method of detecting hybrids, which required dissection to count and measure gill rakers. In this respect, analysis of body shape with thin-plate splines may present a nondestructive method for distinguishing limnetics and benthics and possibly detecting hybrids. My second objective was to see if lateral body shape variation among marine, limnetic, benthic and intermediate stickleback species matched the divergence patterns reported by Schluter and McPhail (1992). Schluter and McPhail (1992) reported that marines and limnetics were nearly equivalent in shape; as measured by gill raker number and length, body depth and gape width (Table 2.1), while intermediate and benthic sticklebacks were increasingly more different. I found a similar pattern among the four species with respect to non-uniform shape differences. The relative warp analysis showed a gradation in body shape from marines to benthics with limnetics and intermediates between these extremes (Figure
28
2.5). While the first relative warp suggests a similar divergence pattern among the four species, it shows that marine and limnetic sticklebacks are not equivalent in shape. While the limnetic shape was most similar to the marine shape among the freshwater stickleback species, there was only minor overlap between marine and limnetic sticklebacks with respect to the first relative warp (Figure 2.5). The position of marine sticklebacks along the first relative warp indicates that they have smaller head regions, longer dorsal and anal fins, and more anterior first dorsal spine than any of the freshwater stickleback species. Such morphological differences are likely adaptations for foraging on zooplankton and/or anti-predator defense that change along the first relative warp. I discuss each of these possibilities in the subsequent section. My final objective was to describe the main body shape changes from the ancestral marine state to each of the freshwater species. There was a pattern of increased non-uniform shape changes between the marine species and the limnetic, intermediate, and benthic stickleback species, respectively (Figure 2.7). By deforming the marine species configuration into each of the freshwater species configurations I was able to visualize non-uniform shape changes not evident in the first relative warp. The deformation of the marine species into the limnetic species suggested minor shape differences, but the limnetic had narrower trunk and caudal regions than the marine state. Such decreases in body depth are often described as adaptations to foraging on zooplankton (Baumgartner et al. 1988; Walker 1997). The deformation of the marine species into the intermediate and benthic species showed that size of the head region continues to increase as seen with the first relative warp. Both the discriminant function analysis and the relative warp analysis suggest that there has been convergence in body shape among the freshwater stickleback species. This
29
may have resulted from the presence of a littoral-habitat in freshwater lakes that is lacking for pelagic zooplankton foragers (marine sticklebacks). The limnetic stickleback species is unique in the sense that it appears to have balanced the selection pressure of being a planktivore in a lake environment; it converged with intermediate and benthic sticklebacks with respect to shape described by the first relative warp, yet became more fusiform than marine sticklebacks in terms of non-uniform shape changes in body depth. Although this study did not test the adaptive significance of changes in body shape, previous studies of stickleback morphology and shape provide some clues to the potential importance of these changes. First, three key shape changes from marine to benthic sticklebacks along the first relative warp may reflect a transition from a zooplankton specialist to a zoobenthos specialist. First, head size increases. Second, caudal region length decreases. Third, the distance between the posterior and anterior insertion points of the dorsal fin and of the anal fin decreases. These changes in shape are consistent with predictions from hydromechanical models for increased maneuverability, which is considered adaptive for foraging on zoobenthos (Baumgartner et al. 1988; Walker 1997). Moreover, a comparison of limnetic and benthic sticklebacks (Baumgartner et al. 1988) suggested that the more fusiform bodies and longer pectoral fins of limnetic sticklebacks increased the potential for prolonged swimming, while the deeper body of benthics aided in zoobenthic foraging. In this respect, shape divergence among the sticklebacks may reflect adaptation to different modes of foraging. Freshwater sticklebacks may have converged on the ability to escape from predation events using "C-type fast starts" (a burst swimming maneuver). A recent comparison of swimming performance for limnetic and benthic sticklebacks found convergence in fast-start
30
ability (Law and Blake 1996). However, a previous study had found this ability lacking in anadromous marine sticklebacks (Taylor and McPhail 1986). Law and Blake (1996) suggested that the convergence in limnetic and benthic fast-start ability resulted from convergence in body shape of the caudal region. While they attributed this shape convergence to the larger size and depth of limnetic dorsal and anal fins, I found a similar convergence in the shape of the caudal region without looking at size of the dorsal and anal fins. Since, I found intermediates between limnetics and benthics in this convergence, they may also have this fast-start ability. In addition, freshwater sticklebacks converge in the posterior migration of the first dorsal spine also suggests body shape may have converged among freshwater sticklebacks in response to a particular predation regime. Thus, divergence and convergence of stickleback body shape in freshwater may reflect differing selection pressures due to resource competition and predation in freshwater lake environments.
31
Marine
Limnetic
Intermediate
Benthic
F i g u r e 2.1 T I F F image of the stickleback species Photographic image of all four stickleback species, which were raised in freshwater ponds. The species were scaled, with proportions constrained, to have roughly the same standard length.
32
Figure 2.2 Stickleback images and landmark positions Sticklebacks were photographed in pairs to produce 56 stickleback images, which all looked similar to the illustration above. A Canadian dime measuring 17.93 mm in diameter was included in each image as a standard size reference. The lower stickleback shows positions of the fifteen landmarks used in morphometric analysis.
33
Figure 2.3 Stickleback consensus configuration Stickleback consensus configuration from generalized orthogonal least-squares (GLS) Procrustes superimposition of all 112 stickleback specimens. The outline was formed by connecting lines between the mean (consensus) positions of each landmark. Caudal, dorsal, and anal fins are shown with respect to their surrounding landmarks. Head, trunk, and caudal regions of fish morphology are also shown for reference.
34
1st D i s c r i m i n a n t V a r i a b l e
Figure 2.4 Discriminant function analysis Discriminant Function Analysis of partial-warp scores for all 112 stickleback specimens. Marine (ancestral) specimens were denoted by stars (*), whereas intermediates were triangl (A), benthics closed circles (•), and limnetics open circles (O). The mean shape of each stickleback form is marked with a large letter (M= marine, L= limnetic, B= benthic, and 1= intermediate).
35
-0.004
-0.002
0.0
0.002
0.004
1st R e l a t i v e W a r p ( 4 3 . 7 1 % v a r i a t i o n )
Figure 2.5 Relative warp analysis Principal components analysis (PCA) of the partial-warp scores of all 112 stickleback specimens. Each PCA axis is referred to as a "relative warp". Marine (ancestral) specimens were denoted by stars (*), whereas intermediates were triangles (A), benthics closed circles (•), and limnetics open circles (O). The mean shape of each stickleback form is marked with a large letter (M= marine, L= limnetic, B= benthic, and 1= intermediate). The symbols M and B refer to the start (extreme marine-like shape) and end (extreme benthic-like shape) positions, respectively, for the changes in stickleback shape along the first relative warp axis, as depicted in Figure 2.6. 1
1
36
Figure 2.6 Deformations along first relative warp A graphical depiction of the deformation from an extreme marine-like shape to an extreme benthic-like shape along the first relative warp axis. The extreme benthic-like shape (position B on Figure 2.5) occurs at the ends of the dark lines (vectors), while the extreme marine-like shape (position M on Figure 2.5) occurs at the dark circles. Fish morphological characters such as fins and spines are for illustration purposes only. 1
1
37
A) Marine configuration
B) Limnetic configuration
C) Intermediate configuration
D) Benthic configuration
Figure 2.7 Transitions between marine and lacustrine configurations Illustration of each of the four stickleback form configurations. The lacustrine form configurations have vectors, which indicate the direction of change for each landmark position from the marine [ancestral] configuration. All vectors have been scaled by a factor of three to make direction and magnitude of transitions visible. Fins, eyes, spines and other aspects of fish morphology are included only for illustration purposes and do not reflect quantitative changes in characters.
38
Population
Lang Creek Paxton Lake Cranby Lake Paxton Lake
Form
Body Size (PC1)
Marine Limnetic Intermediate Benthic
4.30 3.50 3.96 3.94
Body Depth 2.30 2.20 2.30 2.38
Gape Width
Gill Gill Raker Raker Length Number
1.12 1.23 1.34 1.51
0.35 0.28 0.02 -0.37
22.33 22.73 19.91 17.49
Table 2.1 Summary of morphological differences among stickleback forms Mean differences in morphology among stickleback forms as reported by Schluter and McPhail (1992). Gill raker number is presented as untransformed counts. Body depth, gape width, and gill raker length are reported as PCI-size-corrected ln-transformed traits.
Population
Little Campbell River Paxton Lake Cranby Lake Paxton Lake
Form
Marine Limnetic Intermediate Benthic
Centroid Size (±SE) 0.99915 0.99947 0.99956 0.99903
±0.00005 ± 0.00004 ± 0.00004 ± 0.00006
Standard Length (mm) (±SE) 28.78 35.74 34.78 32.33
± ± ± ±
0.32 0.55 0.71 0.69
Experiment Duration (# days) 51 93 52 52
Table 2.2 Centroid size and standard lengths of TPS specimens Mean differences in centroid size and standard length among four stickleback forms used in thin-plate spline geomorphometric analysis of body shape.
39
C H A P T E R THREE: COMPETITION, SELECTION, A N D PHENOTYPIC PLASTICITY IN MARINE STICKLEBACKS A F T E R COLONIZATION OF F R E S H W A T E R
John Robert Pritchard The Centre for Biodiversity Research, Department of Zoology, University of British Columbia 6270 University Blvd., Vancouver, BC V6T1Z4 Canada pritchrd@ zoology, ubc. ca
KEYWORDS Ecological character displacement, ecological experiments, experimental ponds, evolution, Gasterosteus aculeatus, interspecific competition, morphological divergence, natural selection, phenotypic plasticity, sticklebacks
INTRODUCTION The threespine sticklebacks of southwestern British Columbia are a convincing example of ecological character displacement. One of the most compelling pieces of evidence suggesting competition had a role in the evolution of the limnetic and benthic sympatric forms was a competition experiment (Schluter 1994). He demonstrated that competition between intermediate and limnetic sticklebacks resulted in divergent natural selection favoring more benthic-like phenotypes within the intermediate population. While this experiment established that intermediate sticklebacks compete with planktivores, it left several other questions unanswered. I attempt to answer some of these questions in an experiment to be described below. First, does the planktivore experience competition? Second, does it experience selection? And finally, does competition induce phenotypic shifts? Schluter's experiment controlled for
40
effects of phenotypic plasticity, however it remains a possible mechanism for producing morphological divergence. Herein, I present results of an experiment completed during the same time period as Schluter's (1994) experiment, which used the same experimental pond facility and a comparable experimental design. My experimental design uses: 1) marine rather than limnetic sticklebacks as the surrogate for the ancestral planktivore; 2) marines as the target population and Cranby Lake intermediates as the competitors; and, 3) the "natural" rather than hybridization-"enhanced" distribution (sensu Schluter 1994) of marine stickleback phenotypes. It is justified to use marines rather than limnetics as the planktivore in this experiment. Marines are the hypothesized planktivore (Figure 1.1). Marine and limnetic sticklebacks are also similar in gill raker number, and body-size corrected measures of gill raker length, gape width and body depth (Schluter and McPhail 1992) and body shape (Chapter Two). My experiment is designed to test predictions of the character displacement hypothesis. First, the model predicts that marine and intermediate sticklebacks will compete for food. I used mean growth and survivorship of marine forms as two indices for measuring the intensity of competition experienced by marine sticklebacks in the presence of intermediate sticklebacks. Second, the model predicts a diet shift for marine sticklebacks in response to competition; marine sticklebacks in the presence of intermediate sticklebacks are predicted to consume a higher proportion of plankton than marine sticklebacks alone. A final goal of the experiment was to test for the presence of adaptive shifts in marine stickleback morphology between treatments. Specifically, I tested whether marine sticklebacks with intermediates had longer, more numerous gill rakers, narrower gapes, and slimmer bodies at the end of the experiment than marine sticklebacks that did not experience interspecific competition.
41
Such adaptive morphological differences in marine sticklebacks between treatments, if they exist, could arise by 1) divergence of marine morphology from intermediates, when interspecific competition was present, 2) convergence of marine morphology with intermediates, when interspecific competition was absent, or 3) both. This experiment can not distinguish between these alternatives. Two mechanisms could produce adaptive morphological differences in marine morphology between treatments over the course of a few weeks: 1) natural selection, and/or 2) phenotypic plasticity. Natural selection could produce morphological differences between treatments through differential mortality of different phenotypes. Diet-induced phenotypic plasticity could also produce adaptive morphological differences between treatments. To date, no stickleback competition experiments have detected differential mortality of different phenotypes within a species. However, a previous laboratory experiment I performed with Day and Schluter (Day et al. 1993) demonstrated that diet reversal decreases morphological differences between limnetic and benthic sticklebacks. Three traits associated strongly with foraging efficiency, gill raker length, gape width, and head depth, experienced the most change. Although these diet-induced changes could not remove the effect of species (e.g. turn a limnetic into a benthic), the phenotypic changes were adaptive. In this experiment, I used gill raker number, a non-plastic trait, to assess the presence of natural selection and gauge whether morphological differences should be attributed to phenotypic plasticity, natural selection, or both.
42
METHODS Fish populations I used intermediate sticklebacks from Cranby Lake, Texada Island, B C because this solitary population is intermediate in morphology and diet between the sympatric limnetic and benthic species (Schluter and McPhail 1992; McPhail 1993). This was the intermediate population also used by Schluter (1994). Marine (anadromous) sticklebacks from the mouth of the Little Campbell River, Semiahmoo First Nation Reserve, White Rock, B C were chosen because they breed at the same time as the Cranby Lake population. Fish Collection and Rearing All fish used were created by artificial fertilization of eggs from wild-caught sticklebacks. I collected adult marine and intermediate sticklebacks with minnow traps. Eggs from gravid female fish were fertilized in the field with the minced testes of male conspecific fish and then transported to the University of British Columbia. Sixteen batches of approximately 500 eggs each were assigned randomly to 16 100L aquariums. I suspended the eggs in mesh-bottomed cups along the side of each aquarium, and provided gentle aeration from an airstone beneath each cup. Cups were tended by removing unsuccessful (i.e. diseased, damaged or unfertilized) eggs, releasing hatchlings into the aquarium below and discarding empty egg cases. Baby fish were initially fed a combination of Liquid Small Fry baby fish food (Wardley Products Company, Secaucus, NJ), finely-ground flake food (Wardley's Basic Food Flakes for Tropical Fish, Wardley Products Company, Secaucus, NJ), and infusoria broth (the liquid resulting from soaking straw in stagnant, warmed water for several days). Three days after hatching, this diet was supplemented with brine shrimp nauplii (Artemia franciscana GSL, Supreme 99 Artemia, San Francisco Bay Brand, Inc., Newark, CA). All fish were fed to
43
satiation once per day. After approximately one month of rearing in the lab, the fish were added to the experimental ponds, marking the beginning of the experiment. Experimental Design This experiment was carried out in the summer of 1993 in two experimental ponds (Pond 9 and Pond 11) located on the south campus of the University of British Columbia. The ponds are square, measuring 24.4 meters per side and slope to a maximum depth of 3 meters (Figure 3.1). Each pond was divided in two with plastic barriers the day before starting the experiment. Barriers were constructed from a 30.5m x 4.25m sheet of 4-mil (0.1016 mm thick), UV-resistant polyethylene greenhouse film with a lm x 2m nitex (100|im mesh) window, and kept afloat with 4" diameter "Big-O" sewage pipe slipped into a sleeve at the top of the plastic sheet. Barriers were sealed against the bottom of each pond with a 30.5m steel chain and rocks. A scuba diver inspected the bottom seal of each barrier. The treatments were paired within ponds, and randomly assigned to a pond-side. The control treatment contained 970 marine sticklebacks and no intermediate sticklebacks, whereas the experimental treatment contained 970 marine sticklebacks and 1000 intermediate sticklebacks. This design results in differences in fish density between control and experimental treatments; however, such density differences are unavoidable if the experimenter controls for intraspecific competition. Since the goal of this experiment was to test specifically for interspecific competition, it was necessary to control for intraspecific competition, by keeping the number of intraspecific competitors (marines) constant, while varying interspecific competition, by adding interspecific competitors (intermediates) to the experiment treatment (for discussion, see Werner and Hall 1977; Maiorana 1977; and Hairston 1989). A consequence of this design is that I can say nothing about the relative competitive
44
abilities of marine and intermediate sticklebacks; however, this information is not germane to the questions being asked in this experiment. Ponds were stocked over two days (17 and 18 June 1993). Marines were added on the first day, intermediates on the second. Fish were allowed to grow throughout the summer and into the fall. The experiment ran for about thirteen weeks; it ended on 15 September 1993 when I began harvesting fish (Table 3.1). I harvested fish with minnow traps throughout September and October, but completed harvesting using rotenone. I added 0.5 kg of rotenone (C23H22O6, Syndel Laboratories, Vancouver, BC) to each half-pond and dipnetted fish as they swam to the surface. All fish were anesthetized with MS-222 (tricaine methanesulfonate, Syndel Laboratories) and then fixed in labeled jars of 10% formalin (3.7% formaldehyde) for at least two weeks. Minnow traps seemed biased to catching intermediate sticklebacks (Table 3.1), while rotenone removed any remaining fish. The frequency and/or density of sticklebacks in each treatment changed through the process of harvesting fish (Table 3.1). While the control and experimental treatments remained qualitatively unchanged (i.e. no interspecific competition versus interspecific competition), the harvesting process and duration had two probable effects on the experimental results: 1) fish continued to grow until harvested, and 2) differences in marine diet between treatments probably weakened through the harvesting process. I describe later how these two effects were taken into account when comparing marine diet and growth between treatments.
45
Data Acquisition I counted and recorded the number of marine and intermediate sticklebacks in each treatment. Marines were easily distinguished from intermediates by the number of lateral plates: marine sticklebacks have many (30-40) plates (Hay and McPhail 1975; McPhail and Hay 1983; personal observation) whereas intermediate sticklebacks only have a few (5-7) plates (also see Figure 2.1). The fish were stained in a 2% K O H and alizarin red solution for 12 hours and then preserved in 37.5% isopropyl alcohol. Contamination between control (marine sticklebacks only) and experimental (marine and intermediate sticklebacks) treatment pond-sides were assessed by looking for intermediate sticklebacks on control treatment pond-sides. There was no evidence of contamination in Pond 11. There was evidence of minor contamination in Pond 9; 2 of the 376 fish retrieved the control treatment pond-side were intermediate sticklebacks (Table 3.1). Since a low exchange rate of fish between sides of Pond 9 should weaken differences between pond-sides, the effect was conservative and I did not correct for it. Morphology I used fifty marine sticklebacks from each pond-side for morphological analysis. These fish were randomly sampled from jars of both minnow trapped and rotenone harvested sticklebacks. For each stickleback, I measured standard length, body depth and gape width. I also counted the number of gill rakers on the long arm of the first gill arch, and measured the length of the longest gill raker on the same arch. All measurements were taken from the left side of each fish. These measurements were completed as described in previous morphological analyses of sticklebacks (for examples see, Bentzen and McPhail 1984; Lavin and McPhail 1985, 1986, 1987; Schluter and McPhail 1992; Schluter 1993a, 1993b).
46
Diet I used fifteen marine sticklebacks from each pond-side for diet analysis. These fish were randomly sampled from jars of rotenone harvested sticklebacks. Sticklebacks sampled with minnow traps were not used for diet analysis because these fish often sat in traps for several hours before being killed and preserved, potentially obscuring their true foraging patterns through physical confinement and/or prey digestion. Since minnow traps were more effective in capturing intermediate sticklebacks than marine sticklebacks (Table 3.1), I assumed that interspecific competition weakened through the harvesting process. In this respect, the estimates of differences in marine diet between treatments will be conservative, because these observations are based on diet differences present at the end of the harvesting process. Pond identity and treatment was concealed during stomach content description. Stomach contents of each fish was placed in 10 mL scintillation vial with a randomly coded ID label and preserved with 70% ethanol. The corresponding fish was placed in another 10 mL scintillation vial with the identical ID label and a second label indicating its pond and treatment, and preserved with 37.5% isopropyl alcohol. Randomly coded ID labels allowed me to match individual stomachs with specific fish after their contents had been analyzed. I used a dissection microscope (WELD M3C, Wild Leitz Canada Ltd., Willowdale, Ontario) and an event recorder (TANDY 102 Portable Computer, Tandy Electronics Limited) to examine and record the contents of each stomach. I counted and identified whole prey items (Pennak 1978) as copepods (calanoids, cyclopoids or harpacticoids), cladocera (Bosmina, chydorids, Diaphanosoma, Ceriodaphnia or Simocephalus), ostracods, amphipods, snails, odonates, or other insects and insect larvae.
47
Prey types were grouped into two broad categories: planktonic prey (zooplankton) and benthic prey (zoobenthos). Calanoid copepods, Bosmina, Diaphanosoma, Ceriodaphnia and Simocephalus were classified as planktonic prey. Amphipods, snails, benthic copepods (cyclopoid or harpacticoid species), odonates, and other insects or insect larvae were classified as benthic prey. Ostracods and chydorids were excluded from the analysis because they can be classified as either plankton or benthos. These prey types are most frequently found in the water column a few centimeters above the bottom substrate. They are routinely ignored in both plankton and benthic sampling protocols because of their ambiguous status as zooplankton or zoobenthos (Pennak 1978; D. Dolecki, personal communication; Neill, personal communication). Although ostracods and chydorids are not found in zooplankton tows, an argument can be made to classify them as such, because of their small size (Pennak 1978; Chapter Four, Table 4.2) and the foraging mode fish would utilize to consume them. Five stomachs were excluded from the analysis because they contained only ostracods and/or chydorids. Four of the excluded stomachs came from the Pond 11 experimental treatment and the fifth excluded stomach came from the Pond 9 experimental treatment. If chydorids and ostracods were not excluded, and were classified as zooplankton, my results do not change. Diet composition was quantified for each fish as the proportion plankton in the diet, which was calculated as the ratio of zooplankton biomass (mg, dry-weight) to the summed biomass of zooplankton and zoobenthos in the stomach. For each fish, the biomass of zooplankton and zoobenthos was calculated by multiplying the total number of each prey type by its mean mass and summing the total mass of planktonic or benthic prey consumed, respectively. The mean mass of each prey type was estimated with a computer program
48
(Hopcroft 1991a or 1991b), which predicted prey mass from length measurements using length-weight regressions supplied for most zooplankton and zoobenthos prey types. Length measurements for various prey types were obtained by measuring the length of prey items consumed by sticklebacks in these ponds in 1995 (Chapter Four and Table 4.2). Intensity of Interspecific Competition Growth and survival of marine sticklebacks were used as indices of interspecific competition from intermediate sticklebacks. I also used the presence of marine diet shifts between treatments as a test of interspecific competition. The extended duration of harvesting following the end of the experiment allowed time for sticklebacks to continue to grow. This growth was controlled for with linear regression. When testing for differences between pond-sides, ln-transformed standard length was modeled as a function of days and treatment. I tested for treatment effects with a one-tailed, paired ttest (n= 2 ponds) on the changes in regression intercepts between control and experimental pond-sides. Mean growth of marine sticklebacks in each pond-side was presented as mean growth per 90 days, calculated from growth-rate (mm/day) of individual sticklebacks and scaled to growth per 90 days, the length of the experiment. Marine survivorship was compared between control and experimental pond-sides and treatments. I estimated survivorship of marine sticklebacks as the ratio of the number of marines retrieved to the number stocked. The standard error of marine survival was estimated from a binomial distribution represented as a proportion with rj = (pq / n ) ° , where p= 5
estimated marine survival, q= 1-p and n= 970 (Sokal and Rohlf 1995). Differences in marine survivorship between control and experimental pond-sides were tested with a Mantel-
49
Haenszel test (Sokal and Rohlf 1995). The difference between control and experimental treatments was tested with a one-tailed, paired t-test (n= 2 ponds) on arcsin, square root transformed marine survivorships. Mean proportion plankton consumed was calculated for marines in each pond-side as the average of the proportions of individual fish in the sample. I tested for differences between pond-sides with separate one-tailed, two-sample t-tests after applying an arcsin, square root transformation to the proportions. I tested for differences between treatments with a one-tailed, paired t-test (n= 2 ponds) on the mean of the transformed proportions. Morphological Shifts I tested whether there were morphological changes in marines between treatments because of natural selection, phenotypic plasticity, or a combination of these two mechanisms. Under the character displacement model, in the absence of interspecific competition, natural selection should favor marines with more benthic-like morphology: few, short gill rakers, deep bodies, and wide gapes; whereas in the presence of interspecific competition, natural selection should favor marines with more limnetic-like morphology: numerous, long gill rakers, slim bodies, and narrow gapes. I predict diet-induced adaptive phenotypic plasticity such that if interspecific competition results in a marine diet shift to increased proportion of zooplankton, as predicted under the character displacement model, marines in the presence of intermediates will have longer gill rakers, slimmer bodies, and narrower gapes than marines not in the presence of interspecific competition. I attempted to distinguish natural selection from phenotypic plasticity as causes of morphological shifts in the following way. First, I assumed that marine stickleback gill raker number is not phenotypically plastic in the face of diet changes. This assumption is supported
50
by evidence that neither limnetic or benthic stickleback gill raker numbers were phenotypically plastic with respect to diet (Day et al. 1993). Significant differences in gill raker number between treatments are therefore attributed to selection. Gill raker length, body depth and gape width experienced significant diet-induced phenotypic plasticity in limnetic sticklebacks (Day et al. 1993). If the same is true in marine sticklebacks, then shifts in these cannot be unambiguously attributed to selection or phenotypic plasticity. Nevertheless, the evidence of selection on gill raker number will provide a gauge for whether or not selection contributed to morphological shifts in gill raker length, gape width, or body depth. Controlling for ontogenetic changes I wanted to make certain any differences in morphology I observed between treatments were not due to ontogenetic changes. For example, if young (or slow growing) fish are more planktivorous in morphology than old (or fast growing) fish, I could observe my expected morphological shifts merely as a function of growth. To control for this possibility, I restricted morphological analysis to marines with overlapping standard length ranges between the pond-sides of each pond. This step was conservative. If I did not restrict my analysis to fish with overlapping standard lengths, my results did not change. In Pond 9, this restricted the analysis to fish between 37.3 mm and 48.3 mm in standard length (n=43 individuals from the control side, and n=35 individuals from the experimental side). In Pond 11, this restricted the analysis to fish between 36.8 mm and 48.0 mm in standard length (n=43 individuals from the control side, and n=30 individuals from the experimental side).
51
Controlling for standard length All data was normalized by transformation prior to analysis. Standard length, gill raker length, gape width, and body depth were all ln-transformed, while gill raker number was square root transformed. I used single factor analysis of variance to test whether there was a significant relationship between standard length and gill raker number, gill raker length, body depth, or gape width. If I found a significant relationship between standard length and a morphological trait, I included standard length as a covariate when testing for differences in the trait between pond-sides or treatments. I found significant relationships between standard length and gill raker length, body depth, and gape width, but no relationship between standard length and gill raker number (Table 3.2). I presented morphological shifts for each of the transformed traits in separate figures to show the direction and magnitude of the difference between pond-sides. Gill raker number was presented as the mean number of rakers ± one standard error in each pond-side. Gill; raker length, body depth, and gape width were presented for each pond-side as the mean residual ± one standard error from regressions within each pond with standard length as a covariate. For each pond, I used multiple analysis of variance (MANOVA) to test whether there were significant differences between control and experimental pond-sides in overall marine trophic morphology, as described by gill raker number, gill raker length, body depth, and gape width. Pond was nested within treatment as a random effect to test for overall differences between control and experimental pond-sides. A one-tailed, paired t-test (n= 2 ponds) on the differences in intercepts, resulting from the A N O V A model regression of the traits on standard
52
length and treatment, between control and experimental pond-sides was used to test for treatment effects. I also used analysis of variance (ANOVA) with pond-side as a factor to test for differences in gill raker number, gill raker length, body depth, and gape width between control and experimental pond-sides. Pond was again nested within treatment to test for overall differences. Standard length was included as a covariate when testing for differences in gill raker length, body depth, or gape width. I used one-tailed, paired t-tests (n= 2 ponds) on the differences in intercepts between control and experimental pond-sides to test for treatment effects.
RESULTS Test of Interspecific Competition As predicted, marine sticklebacks alone had significantly higher mean growth than marine sticklebacks with intermediate sticklebacks (Figure 3.2; One-tailed, paired t-test, t=513.5, df= 1, P=0.0006). There were significant differences in growth between control and experimental pond sides in Pond 9 (ANOVA, Fi, = 57.9003, P< 0.0001), Pond 11 (ANOVA, 95
F,,96=
84.6897, P< 0.0001), and overall (nested A N O V A , F , 1
193
= 136.2873, P< 0.0001).
These observations indicate greater than a ten percent decrease in mean growth of marine sticklebacks in the presence of intermediate sticklebacks (Figure 3.2). Overall, there was no significant difference in marine survival between the control and experimental treatments (Figure 3.3; One-tailed, paired t-test, t= 2.6389, df= 1, P= 0.8847). However, in both ponds, marine sticklebacks on control sides had significantly lower survival
53
than marine sticklebacks on experimental sides (Mantel-Haenszel test, X = 4.1945, df= 1, P= 2
0.0406). This pattern is the opposite of what I had predicted. Marine sticklebacks in the presence of intermediates ate significantly higher proportions of plankton than marine sticklebacks alone (Figure 3.4; One-tailed, paired t-test, t= 50.7432, df= 1, P= 0.0063). This finding was consistent with the prediction that marines in the presence of intermediate sticklebacks would eat more zooplankton and less zoobenthos than marine sticklebacks alone. There were also significant differences between control and experimental sides in both Pond 9 (t=3.1395, df= 27, P= 0.002) and Pond 11 (t= 2.0445, df= 25, P= 0.0258). Natural Selection and Phenotypic Plasticity In almost every case, the morphological shifts were in the predicted direction. There were trends for gill rakers to increase in number (Figure 3.5) and length (Figure 3.6), and body depth to decrease (Figure 3.7) between control and experimental treatments. Gape width narrowed between the experimental and control treatments in one pond, but there was a trend in the opposite direction in the second pond (Figure 3.8). Thus, seven of the eight comparisons were in the predicted direction. There was a consistent trend among the morphological characters which matched the predictions from natural selection and/or phenotypic plasticity. When gill raker number, gill raker length, body depth, gape width were examined together as a single variable describing marine trophic morphology, there were significant differences between control and experimental pond-sides in Pond 9 ( M A N O V A , df= 1, PillaiBartlett trace= 0.1750, trace= 0.1362, F
4
, 7= 6
F , 2= 4
7
3.8188, P= 0.0072), Pond 11 ( M A N O V A , df= 1, Pillai-Bartlett
2.6409, P= 0.0412) and overall (nested M A N O V A , df= 1, Pillai-Bartlett
54
trace= 0.1047, F i = 4.2095, P= 0.003). However, despite consistent significant differences 4j
44
between control and experimental pond-sides, I could not detect a significant treatment effect (One-tailed, paired t-test, t= 0.2667, df= 1, P= 0.417). I expected marines on experimental sides to have more gill rakers than those on control sides. The effect of treatment on gill raker number approached significance and was in the expected direction (Figure 3.5; One-tailed, paired t-test, t= 4.5556, df= 1, P=0.0688). However, there were no significant differences between experimental and control pond-sides in Pond 9 (ANOVA, Fi, = 0.0414, P= 0.8393), Pond 11 (ANOVA, F,, i= 0.0138, P= 76
0.9068), or overall (nested A N O V A , F
7
l j l 4 8
= 0.0488, P= 0.8255).
Since marines in the experimental treatments consumed significantly higher proportions of plankton than marines in control treatments, I expected phenotypic plasticity in trophic characters consistent with this diet shift. Specifically, I expected marines with intermediates on the experimental sides would have longer gill rakers, slimmer bodies and narrower gapes than marines alone on the control sides. Thus, marines alone on control sides would have shorter gill rakers, deeper bodies, and wider gapes than marines with intermediates. Indeed, marine sticklebacks with intermediates appeared to have longer gill rakers than marine sticklebacks alone (Figure 3.6). The effect of treatment on gill raker length approached significance (One-tailed, paired t-test, t= 4.7615, df= 1, P=0.0659) as did the overall difference between control and experimental pond-sides (nested A N O V A , F i i = i
47
3.7009, P= 0.0563). However, the pattern was not statistically significant in either Pond 9 (ANOVA, F
lj75
= 0.7171, P= 0.3998) or Pond 11 (ANOVA, F,, o= 2.8507, P= 0.0958). 7
55
Marine sticklebacks with intermediates also appeared to have slimmer bodies than marine sticklebacks alone (Figure 3.7), and there was an overall significant difference between control and experimental pond-sides (nested A N O V A , was highly significant in Pond 9 (ANOVA, F
u 5
F
U 4
7=
6.9735, P= 0.0092). This result
= 9.7230, P= 0.0026), but absent in Pond 11
(ANOVA, Fi = 0.8173, P= 0.3691). The effect of treatment was not significant (One-tailed, i70
paired t-test, t= 1.6269, df= 1, P=0.1754). Marine sticklebacks with intermediates had significantly narrower gape widths than marine sticklebacks alone in Pond 11 (Figure 3.8; A N O V A , Fi, o= 7.5014, P= 0.0078). 7
However, marine sticklebacks in Pond 9 showed a non-significant trend (ANOVA, F i = i75
0.2309, P= 0.6323) in the opposite direction (Figure 3.8). There was no significant overall difference between control and experimental pond-sides (nested A N O V A , F
U 4 7
= 2.5848, P=
0.1100), and the effect of treatment was also not significant (One-tailed, paired t-test, t=0.6828,df= 1,P=0.3093).
DISCUSSION Growth and survivorship have both been used as indices of competition, but differences in survivorship are less frequently observed. For instance, Pacala and Roughgarden (1984) did not detect a difference in survivorship for either lizard species when a second species was added. This was also true for mud snails (Fenchel 1975), sunfishes (Werner and Hall 1977), and sticklebacks (Schluter 1994). Yet, all of these studies detected significant differences in growth with competition. In this respect, growth may be a more sensitive index of competition intensity than survivorship.
56
Growth and survivorship can also be seen as effects of competition. In this respect, two species may compete even though survivorship of one or both species increases in the presence of the second. This was the case in a recent study of interspecific competition between two benthic stream fish; mottled sculpins had significantly higher survivorship, but reduced growth and body condition when in the presence of fantail darters (Resetarits 1997). In this example, survival, growth and condition were considered effects of competition, which all related to eventual reproductive output. Growth and Survival If the process of ecological character displacement was responsible, completely or in part, for the evolution of sympatric limnetic and benthic sticklebacks, then there has been selection for individuals with divergent character states which reduced competition between the two species. If present day sympatric species are the result of this continued selection, then, presumably, when these two species initially came into contact with one another the two species were 1) more similar to one another morphologically and ecologically than they are today, and 2) competition was more intense between the two species than it is today. I concluded that marine and intermediate sticklebacks compete. Marines were significantly smaller in the presence of intermediates. There was at least a ten percent decrease in mean marine body size when marines were in the presence of intermediate sticklebacks. This finding was significant between control and experimental pond-sides in both ponds, and overall. Furthermore, this pattern could be unambiguously attributed to treatment. Previous studies have also used growth to demonstrate competition. Pacala and Roughgarden (1984) observed decreased growth in one species of Anolis lizards when in the
57
presence of a second Anolis lizard species. In this example interspecific competition was asymmetric: one species of Anolis lizard was effected by the presence of the second, whereas the second species was unaffected by the presence of the first species. In mud snails, interspecific competition was also demonstrated by reduced growth of one species when in the presence of a second species (Fenchel 1975; Fenchel and Kofoed 1976; Fenchel and Christiansen 1977; Saloniemi 1993; Gorbushin 1996), and this competition was also asymmetric (Fenchel and Christiansen 1977; Gorbushin 1996). Werner and Hall (1977) observed decreased growth in bluegill sunfish when in the presence of pumkinseed sunfish, and Schluter (1994) used growth to demonstrate competition and selection in sticklebacks. Growth has also been frequently used to demonstrate competition in field experiments (for reviews, see Connell 1983; Schoener 1983; Gurevitch et al. 1992; Goldberg and Barton 1992). Decreased growth would likely have an influence on fitness via reductions in body size. Decreased body size has been shown to have negative fitness consequences in fishes including reduced overwinter survival (Bell and Foster 1994; Wootton 1984; Schluter and Hatfield, unpublished experiment), lower behavioral dominance (Bell and Foster 1994; Roberge, unpublished manuscript), and reduced fecundity (Rounsefell 1957). In contrast to the a priori survival predictions, there was a trend for marines in the presence of intermediates to have higher survivorship than marines alone. This pattern was significant between control and experimental pond-sides overall, but could not be attributed unambiguously to treatment. In this respect, survivorship was not useful as an index of competition intensity. This pattern of marine survivorship resulted in a negative relationship between final size and number of marines in each experimental unit; therefore, competition among marines could be an alternative explanation for the observed differences in marine
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growth between treatments. While this alternative explanation is possible, differences in survival only varied from 5-10% between treatments, whereas differences in marine growth varied from 10-12% between treatments. Although I could not attribute increased marine survivorship to interspecific competition, it is possible that this was the case. In hindsight, this unexpected result may be explained by other factors not accounted for by my design. In particular, I did not consider the potential role of predation. Reduced predation with competition, or apparent mutualism, could have produced increased survivorship with interspecific competition through predator satiation (Abrams and Matsuda 1996). The experimental treatments may have had so many fish that predation of marine sticklebacks decreased relative to predation in the control treatments. Thus, predator satiation could explain increased marine survivorship in the presence of intermediate sticklebacks. The experimental ponds had both avian and insect predators (Pritchard, personal observation), but no predatory fish. Predation events by backswimmers (Notonecta) were regularly observed during the introduction of juvenile fish. In addition, backswimmers were observed attacking adult sticklebacks after the introduction of rotenone. If most mortality resulted from predation by backswimmers during the introduction of juvenile fish, then my predator satiation explanation makes sense. Perhaps the risk of mortality is higher in the littoral habitat than the open water habitat, and marines in the presence of intermediates switch to open water habitat. The risk of mortality in the littoral habitat could be higher than the open water habitat via differences in predation, disease, and/or parasitism. Studies by Reimchen (1980) and Hay (1974) suggest littoral invertebrates are significant predators on sticklebacks. Sticklebacks are hosts to many
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littoral-specific parasites (Wootton 1976, 1984), which can produce both decreased fecundity and higher mortality. Diet Shifts The second goal of this experiment was to test the prediction of the character displacement model that marines in the presence of intermediate sticklebacks should consume higher proportions of zooplankton than marines in the absence of intermediate sticklebacks. This prediction was also confirmed with my experiment. In both ponds and overall, there was a significant increase in the proportion of zooplankton consumed by marine sticklebacks on the experimental pond-sides versus the control pond-sides. Indeed, there was approximately a thirty percent increase in the proportion of zooplankton consumed by marine sticklebacks between control and experimental sides in both ponds. Moreover, this diet shift could be attributed to the treatment. Diet shifts in response to competition have been observed in many temperate-zone fish species (Werner and Hall 1976; Schluter and McPhail 1993; Robinson et al. 1994). For instance, bluegill sunfish consumed greater amounts of zooplankton when in the presence of. pumpkinseed sunfish (Werner and Hall 1977). This has also been observed for brook charr in the presence of white suckers (Magnan and Stevens 1993), and arctic charr in the presence of sympatric charr morphs (Skiilason et al. 1992; Skiilason and Smith 1995). Such competitioninduced changes in resource use could present the necessary conditions for coevolution among competitors (Connell 1980). Diet and diet shifts are an integral component of ecological character displacement theory, in which there is selection for individuals that minimize interspecific competition by decreasing overlap in resource use. Genetically-controlled morphological characters which
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affect the efficiency of resource acquisition become the focus of this divergent natural selection. Hence, over time, two competing species evolve to more divergent morphologies through the displacement of characters related to resource acquisition. The stickleback character displacement model predicts that intermediate sticklebacks evolved into benthic sticklebacks, and the second invasion of marine sticklebacks evolved into limnetic sticklebacks (Figure 1.1). Schluter's (1994) demonstration that selection favors intermediates with more benthic-like phenotypes when planktivore competitors are added, provided support for intermediates evolving into benthic sticklebacks. The marine diet shifts demonstrated in the current experiment suggests that there would be selection on marine sticklebacks in the presence of intermediate competitors to evolve, or at least maintain, morphological characters which confer efficient use of zooplankton resources. Morphological Shifts A final goal of this experiment was to test for adaptive shifts in marine stickleback morphology between the two treatments. I expected marines from experimental treatments to have 1) more numerous gill rakers, 2) longer gill rakers, 3) slimmer bodies, and 4) narrower gapes than marines from control treatments. This combination of morphological characters was expected for two reasons. First, under the character displacement model, natural selection should favor this suite of characters for marine sticklebacks in the presence of intermediates. Second, if marines experience diet shifts as predicted, diet-induced phenotypic plasticity should produce changes in gill raker length, body depth, and gape width in an adaptive direction. Lastly, a morphological shift between treatments could arise by marines in the experimental treatments diverging from intermediate stickleback morphology, by marines in the control treatments converging with intermediate stickleback morphology, or both.
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There were significant changes in marine morphology as predicted, however these shifts could not be unambiguously attributed to treatment. In every case, except one, morphology changed as predicted. Gill raker number, gill raker length, and body depth showed trends in the predicted direction for both ponds (Figures 3.5, 3.6, and 3.7), whereas gape width only changed as predicted in one pond (Figure 3.8). For each of the morphological characters, one of the ponds showed a significant difference between control and experimental pond-sides in the expected direction; however, there was not a consistent shift for all morphological characters in any one pond replicate. Nevertheless, when I treated gill raker number, gill raker length, body depth, and gape width as a multivariate measure of marine morphology, I found significant differences between control and experimental pondsides in Pond 9, Pond 11, and overall. Although these differences could not be unambiguously attributed to treatment, it does suggest morphological shifts in these characters may result from interspecific competition (or lack of it). I can not distinguish between natural selection and phenotypic plasticity as the mechanism responsible for shifts observed in marine morphology between pond-sides. In fact, both mechanisms may have operated. I used gill raker number, which was not phenotypically plastic in either limnetic or benthic sticklebacks (Day et al. 1993), to test for the presence of selection on marine stickleback morphology between treatments. Although I may have observed selection in gill raker number in this experiment, previous stickleback competition experiments have not detected differential mortality among stickleback phenotypes even when designed to measure selection (Schluter 1994). In contrast, laboratory manipulation of diet has shown phenotypic plasticity in trophic morphology (i.e. gill raker length, body depth, and gape width) of limnetic and benthic sticklebacks (Day et al. 1993), which suggests
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competition-dependent changes in diet may produce similar plasticity and thereby exaggerate the morphological divergence between stickleback species utilizing different resources. Since there was no reproduction in this experiment, morphological shifts must have arisen by either differential mortality or phenotypic plasticity. Theory predicts that selection should act on the tails of the phenotypic distribution, which are rare. Consequently, it is difficult to have the statistical power to demonstrate selection even when it is present. An alternative experiment, which could test the direction and magnitude of selection, would be to produce an "enhanced" marine distribution through interspecific hybridization with limnetic, intermediate, and benthic sticklebacks. Growth of the different marine phenotypes could then be compared in the presence and absence of interspecific competition with either intermediate or benthic sticklebacks as an index of selection. This alternative experiment would be analogous, and complementary, to the selection experiment completed by Schluter (1994) on intermediate stickleback phenotypes. If there was no evidence of differential mortality among marine phenotypes, then morphological shifts could be attributed to plasticity. Phenotypic Plasticity and Character Displacement Phenotypic plasticity has received increasing amounts of attention over the past few years. Although most plasticity that has been described is behavioral (for reviews, see WestEberhard 1989; Gottard and Nylin 1995; Skulason and Smith 1995; Smith and Skulason 1996), there are also examples of morphological changes. However, the instances of morphological phenotypic plasticity have mostly been dependent on the presence of a predator (or prey) (see Bronmark and Miner 1992; Harvell 1992; Loeb et al. 1994; Reimer and Tedengren 1996). However, in a few cases interspecific competition has also been shown to
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produce morphological changes (Peterson and Andre 1980; Saloniemi 1993; Gorbushin
'
1996). In only two of the hypothesized instances of character displacement has phenotypic plasticity been reported: mud snails (Saloniemi 1993; Gorbushin 1996), and sticklebacks (Day et al. 1993). In the mud snail example, competition-induced phenotypic plasticity is presented as an alternative to evolutionary shifts for explaining the morphological divergence. Likewise, in character displacement studies in general, environmentally-induced differences are the default explanation for observations of morphological divergence. In sticklebacks, I demonstrated the presence of phenotypic plasticity, but that this plasticity could not produce the morphological divergence observed between sympatric limnetic and benthic sticklebacks (Day et al. 1993). In this respect, providing additional support for the genetic basis to the morphological divergence between sympatric stickleback species. While the shifts I observed may have been influenced, at least in part, by selection, it is also likely that diet-induced phenotypic plasticity played a role. In this respect, diet-induced plasticity may have increased the morphological divergence between competing marine and intermediate sticklebacks.
CONCLUSION This experiment provides evidence that marine and intermediate sticklebacks compete, and that marine sticklebacks change diet in the presence of intermediates. In this respect, two predictions of the stickleback character displacement model were confirmed. The experimental treatment of marine and intermediate sticklebacks recreates a hypothesized early stage of the stickleback character displacement series. If phenotypic plasticity increased the morphological divergence between these competing species, this may have prevented competitive exclusion early in the character displacement series and allowed time for
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coevolution between the species. Hence, competition-induced phenotypic plasticity may have aided the evolution of morphological divergence in the stickleback system.
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TOP VIEW of an Experimental Pond
Permeable Divider "Big-O" Sewage Pipe
INTERSPECIFIC COMPETITION CONTROL TREATMENT
INTERSPECIFIC COMPETITION EXPERIMENTAL TREATMENT
(970 marine sticklebacks)
(970 marine and 1000 intermediate sticklebacks)
24.4 m
12.2m
12.2m
SIDE VIEW of an Experimental Pond
Figure 3.1 Experimental design and pond schematic Experimental design. This experiment was replicated in two ponds with treatments paired in each pond. Each pond was divided in half with a permeable plastic divider, which allowed the exchange of water but not food resources (a lm x 2m, lOOjxrn mesh nitex window was placed in the middle of each divider). 970 marine stickleback were placed on both sides of each pond. I added an additional 1000 intermediate sticklebacks to one of the sides (chosen at random).
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00 CO
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Pond 9
CM CO
Pond 11 o
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No Competition
Competition Treatment
Figure 3.2 Mean marine growth over 90 days Mean growth per 90 days ± one standard error of marine sticklebacks in each experimental unit.
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Pond 11
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