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Ecology and evolutionary biology of phenotypic plasticity in the threespine stickleback (Gasterosteus… Day, Troy Michael Charles 1994

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ECOLOGY AND EVOLUTIONARY BIOLOGY OF PHENOTYPIC PLASTICITY INTHE THREESPINE STICKLEBACK (GASTEROSTEUS)byTROY MICHAEL CHARLES DAYB.Sc., The University of British Columbia, 1990A THESIS SUBMflTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THEDEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Zoology)We acceuJis thesis as conforming1,A6 the re ed standardTHE UNIVERSITY OF BRITISH COLUMBIAJune 1994© Troy Michael Charles DayIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)____________________________Department of________________The University of British ColumbVancouver, CanadaDate 22)DE-6 (2/88)11ABSTRACTThis work addresses questions concerning the evolution of diet-induced plasticityof trophic morphology in two species of freshwater threespine stickleback (Gasterosteuspp.). In chapter one I describe an experiment designed to answer the following questionsabout diet-induced morphological plasticity in these fish: (1) do the study species exhibitdiet-induced morphological plasticity, and is this plasticity likely to be adaptive, (2) aredifferent selective regimes associated with different degrees of plasticity, and (3) is theregenetic variation for phenotypic plasticity in contemporary populations. The experimentrevealed that these species exhibit plasticity that appears to be adaptive, and that anassociation exists between diet variability and the degree of diet-induced morphologicalplasticity as predicted by theory. It also revealed that genetic variation for morphologicalplasticity exists in both species.Chapter two presents a second experiment designed to further explore thepossibility that diet variability can drive the evolution of morphological plasticity. Thisexperiment also had three objectives: (1) to quantify the time scale of morphologicalchange to determine if it is compatible with that of natural diet variability, (2) to explicitlyexamine the adaptive significance of diet-induced morphological plasticity by measuring itseffect on foraging efficiency, and (3) to examine the effect that short-term learning(behavioural plasticity) has on foraging efficiency and compare its importance to that ofmorphological plasticity. This second experiment revealed that the time scale of plasticchange is roughly compatible with that of diet variability and that diet-inducedmorphological changes result in changes in foraging efficiency. It also revealed thatbehavioural plasticity affects foraging efficiency but that it affects a different component ofthe prey ingestion process than does morphological plasticity.111TABLE OF CONTENTSABSTRACT.iiTABLE OF CONTENTS iiiLIST OF TABLES vLIST OF FIGURES viACKNOWLEDGMENTS viiGENERAL INTRODUCTION 1Chapter One: A COMPARISON OF DIET-INDUCED MORPHOLOGICALPLASTICITY BETWEEN TWO SPECIES OF THREESPINESTICKLEBACK 3Introduction 3Materials and Methods 8Crossing technique and rearing program 8Measurements 10Analysis 11Multiple significance tests 12Effect of diet 13Interspecific comparison 14Family x diet interaction 14Results 16Effect of diet 16Interspecific comparison 17Family x diet interaction 17Discussion 18Evolutionary implications 23Chapter Two: THE ECOLOGICAL SIGNIFICANCE OF MORPHOLOGICAL ANDBEHAVIOURAL PLASTICITY 31Introduction 31ivMorphological and behavioural plasticity 32Plasticity and sticklebacks 34Materials and Methods 35Diet treatment 36Foraging trials 37Measures of foraging efficiency and morphology 37Analysis 39Time scale 40Effect of morphological plasticity and learning 41Results 42Time scale 42Effect of morphological plasticity 43Effect of learning 44Discussion 45Evolution of morphological plasticity in sticklebacks 48GENERAL DISCUSSION 58LITERATURE CITED 60VLIST OF TABLESTable one. The percentage of the ‘morphological gap’ between benthics and limnetics thatis removed by diet reversal 25Table two. The extent of diet-induced morphological plasticity in benthics and limneticsand results of the interspecific comparison 26Table three. Results of the analysis for genetic variation of morphological plasticity 27Table four. The magnitude of diet-induced morphological change for limnetics and resultsof the analysis of the time scale of morphological change 51Table five. Results of the analysis for the effect of morphological plasticity and the effectof behavioural plasticity (learning) on foraging efficiency 52Table six. Results of the multiple regression analyses for the effect of morphologicalplasticity on foraging efficiency 53viLIST OF FIGURESFigure one. The expected pattern of diet-induced morphological plasticity under theproposed evolutionary hypothesis 29Figure two. Diet-induced morphological plasticity in benthics and limnetics for fourmorphological traits 30Figure three. Diet switching experimental design 55Figure four. Magnitude of diet-induced morphological change across the three sets 56Figure five. The effect of morphological plasticity and behavioural plasticity on the fourforaging efficiency variables 57viiACKNOWLEDGMENTSI would like to thank my supervisor, J. D. McPhail for allowing me the time topursue my sometimes whimsical interests and permitting me the freedom to conduct mythesis research on a subject of my choice. Without his support and knowledge ofsticklebacks, none of this research would have been possible. I also wish to thank D.Schiuter for his assistance with all parts of this thesis, from providing lab space andequipment for part of the project, to providing an enthusiastic ear that was always eager tolisten to (and sometimes redirect) my ramblings. Bill Neill also provided much appreciatedboosts of enthusiasm and clarified many thoughts by helping me ground them in reality.I thank my parents for their continued support, sometimes financial and sometimesemotional, even though I’m sure they often had valid wonders about what I was doing andwhy.I thank my friends for providing much needed and appreciated sarcastic (cynical)commentaries on life in general and life as a gradual student in particular.Finally, I wish to thank Laura Nagel for helping me conduct the experiments, forbeing my best friend, and for tolerating many a mood swing both on Texada Island andelsewhere.1GENERAL INTRODUCTIONPhenotypic plasticity is defined as repeatable environmentally-induced phenotypicchange which occurs within an organism’s lifetime (Bradshaw 1965; Steams 1989;Schemer 1993a). The functional relationship between the environmental factor of interestand the phenotype is described by a ‘norm of reaction’ (Suzuki et al. 1989). Although thesignificance of phenotypic plasticity was recognized years ago (Wright 1931;Schmalhausen 1949; Bradshaw 1965), phenotypic plasticity was not formally incorporatedinto evolutionary theory until quite recently (Levins 1968; Via and Lande 1985;Gomullciewicz and Kirkpatrick 1992; Leon 1993; Gavrilets and Schemer 1993a, 1993b).Consequently, empirical research in this field has undergone a recent increase as well(Schlicting and Levin 1986; Dodson 1989; van Noordwijk 1989; Witte et al. 1990;Wimberger 1991, 1992).This thesis addresses questions concerning the evolution of diet-induced plasticityof the trophic morphology of two sympatric species of threespine stickleback(Gasterosteus The first chapter introduces the two study species and briefly reviewssome relevant natural history. Here, I also describe an experiment designed to answersome fundamental questions regarding diet-induced morphological plasticity insticklebacks. These include: (1) do these species exhibit diet-induced morphologicalplasticity, and if so, is the plasticity likely to be adaptive, (2) do the two species exhibitdifferent degrees of plasticity as predicted by theory based on differences in their dietvariability, and (3) is there genetic variation for phenotypic plasticity present incontemporary populations? As a subsidiary question I also asked: what proportion of the2morphological difference between the species might result from phenotypic plasticity? Toanswer these questions I compared the diet-induced morphological plasticity of these tworecently diverged stickleback species.This experiment is the first step towards testing theory concerning the evolution ofmorphological plasticity in wild populations. if diet-induced morphological changes areadaptive and if the species that normally experiences high diet variability also exhibits agreater degree of plasticity, then this would suggest that diet variability may be importantin the evolution of trophic morphological plasticity. The presence of genetic variability formorphological plasticity would strengthen this claim. Such results would also lend supportto recent theoretical studies of the evolution of phenotypic plasticity in general.Chapter two presents a second experiment designed to further explore thepossibility that diet variability can drive the evolution of morphological plasticity. With thisexperiment I had three objectives: (1) to quantify the time scale of morphological changein order to determine if it is compatible with that of natural diet variability, (2) to explicitlyexamine the adaptive significance of diet-induced morphological plasticity by measuring itseffect on foraging efficiency, and (3) to examine the effect of short-term learning(behavioural plasticity) on foraging efficiency and to compare its importance to that ofmorphological plasticity. Results of this experiment were intended to supplement those ofthe first experiment and increase our understanding of the factors influencing the evolutionof morphological plasticity in sticklebacks. In addition, this experiment allowed me toassess the importance of different types of phenotypic plasticity with regard to foragingefficiency. The second experiment focused solely on the species that exhibited the greatestdiet-induced morphological change in the first experiment (the limnetic).3CHAPTER ONEA COMPARISON OF DIET-INDUCED MORPHOLOGICAL PLASTICITYBETWEEN TWO SPECIES OFTHREESP1NE STICKLEBACK (GASTEROSTEUS .)INTRODUCTIONPhenotypic plasticity is environmentally-induced phenotypic change that occurswithin an organism’s lifetime (Bradshaw 1965; Stearns 1989). A resurgence of empiricaland theoretical interest in this phenomenon has brought about a re-evaluation of itsecological and evolutionary significance (West-Eberhard 1989). At one time phenotypicplasticity was thought to result from developmental accidents (West-Eberhard 1989), butnew evidence suggests that much environmentally-induced phenotypic variation may beselectively advantageous (Steams 1983; Bernays 1986; Greene 1989; Spitze 1992;Thompson 1992). This has led some to view plasticity as a trait, subject to evolutionarypressures just as any other phenotypic character (Schlicting and Levin 1986; Schemer1993b). The discovery of widespread genotype x environment interaction in naturalpopulations (genetic variation for phenotypic plasticity) further suggests that phenotypicplasticity is an evolutionarily labile character.Here, I present an empirical study that addresses four fundamental questionsconcerning the ecological and evolutionary significance of phenotypic plasticity. These are4(1) do my study organisms exhibit plasticity in an adaptive direction; (2) how much of the‘match’ between the morphology of a species and its environment is a result of plasticityand, in particular, how much of the morphological difference between species inhabitingdifferent environments is a result of plasticity; (3) have different selective regimes resultedin the evolution of different degrees of plasticity; and (4) is genetic variation forphenotypic plasticity present in contemporary populations? While a variety of studies haveaddressed (1) and (4) (Lindsey 1962; Bernays 1986; Thompson 1992), few havedetermined the degree to which species differences in morphology are the result ofplasticity (i.e. (2)). Additionally, few studies have compared the degree of plasticity acrossspecies for which selection on plasticity is likely to differ (but see Wimberger 1991,1992).I studied two sympatric species of threespine stickleback (Gasterosteus pp.) fromPaxton Lake, British Columbia, Canada (McPhail 1992; Schluter and McPhail 1992).These species are not formally described but are referred to as the ‘benthic’ species and the‘limnetic’ species after the regions of the lake in which they usually forage (University ofBritish Columbia Fish Museum Catalogue #83-351). The benthic species is specialized forlittoral foraging and possesses a suite of traits suited to this. Individuals are deep-bodied,possess a small number of short gill rakers and have a wide and terminal gape (fig. 1). Thelimnetic species is more slender-bodied, with numerous, long gill rakers and a narrow,upturned gape (fig. 1). It is more planktivorous but it also exhibits a seasonal shift inhabitat use. Individuals forage in the littoral zone in spring during the breeding season, andthen switch to foraging in the water column in summer and fall (Schiuter and McPhail1992; Schluter 1993). Morphological differences between species are largely heritable5(McPhail 1992; Schiuter unpubl. data) and strongly affect feeding efficiency and growthrates in different habitats (Bentzen and McPhail 1984; Lavin and McPhail 1986; Schiuter1993, Schiuter, unpubi. data).The benthic and limnetic species are extremely closely related (Nei’s geneticdistance 0.0 18; McPhail 1992) and they are probably both descended from the marinethreespine sticicleback which colonized the lake on two separate occasions (Schiuter andMcPhail 1992). Comparative evidence suggests that their present morphological andhabitat differences are the result of competition-induced character displacement but atpresent the exact sequence of morphological stages that occurred during their evolution isnot resolved. Schluter and McPhail (1992) provide the evidence for characterdisplacement and a discussion of the two main competing phylogenetic hypotheses. Thepost-glacial history of this region of British Columbia indicates that these two species havecoexisted for no more than 13,000 years (Mathews et al. 1970; Clague et al. 1982; Clague1983). Thus, these species are exceptionally well suited for study because much of theirhistories is shared, their differences have evolved extremely recently, and this evolutionhas likely taken place under ecological conditions that are still experienced by the twospecies.I addressed the four questions mentioned above in the following way. First, Iexamined whether plasticity of trophic morphology is adaptive by reversing the ‘natural’diets of the two species, and asking whether they become morphologically more similar. Iconclude that phenotypic plasticity is adaptive for either or both species if themorphological distinction between them is reduced when they are diet-reversed (fig 1).6Such inference is reasonable because of the effect of trophic morphology on efficiency ofprey capture in the two habitats (Bentzen and McPhail 1984; Lavin and McPhail 1986;Schiuter 1993). Thus, an individual whose morphology grew to resemble that of the otherspecies when raised on a diet that is characteristic of the other species would likely enjoyan increase in foraging efficiency. An explicit test of the adaptive significance of thismorphological plasticity is presented in chapter two.Second, by comparing morphological differences of the species when diet-reversedwith the differences when fed their natural diets, I was able to determine how much of thenatural morphological difference between these two species is a direct result of diet. Therehas been considerable debate over how much of the morphological variation presentamong some populations is a result of plasticity, especially in systems in which large scaleadaptive radiation has occurred (Witte 1984; Meyer 1987; Wimberger 1991,1992). Theexample most frequently cited is probably that of the cichlid radiation in the African riftlakes but other impressive examples exist (Skulason et al. 1989; Snorrason et al. 1989).Significant adaptive radiation of the Gasterosteus species complex has occurred throughcolonization of freshwater. The freshwater species have adapted to a variety of conditionspresent throughout the northern hemisphere (Hagen and McPhail 1970; Bell 1976; Lavinand McPhail 1985; McPhail 1993). My study lends insight into the possible importance ofmorphological plasticity in these instances.Third, I compared the degree of plasticity between the two species, one of which isspecialized for feeding in the littoral zone (the benthic), and the other of which exploitsboth habitats seasonally (the limnetic). The limnetic species exploits the littoral habitat in7the spring during reproduction (April - June). Limnetic males build and defend nests in thelittoral habitat, and gravid limnetic females use the habitat when searching for prospectivemates. Stomach samples indicate that littoral and planktonic prey items are nearly equallyrepresented in the diet of limnetic fish during this life history stage (Schiuter and McPhail1992; Schiuter 1993). Once reproduction has ceased, surviving limnetic fish move backinto the water column where they feed on zooplankton during the summer and fall(Schluter 1993). In contrast, fish of the benthic species forage in the littoral habitat yearround.The conventional wisdom is that plastic trophic morphology would be beneficial iforganisms are faced with significant variability of resource use on the appropriate temporalscale (Gomulkiewicz and Kirkpatrick 1992). Wimberger (1991,1992) compared twocongeneric species of cichlids (Geophagus brasiliensis and steindachneri) and predictedthat differences of diet variability would select for different degrees of morphologicalplasticity. His results did not bear out this prediction of an interspecific difference. Mystudy addresses the same issue by using a similar type of interspecific comparison.Because individuals of the limnetic species experience significantly greater variability ofresource use over the course of their lifetime than do individuals of the benthic species, Iexpected that limnetics would be more plastic than benthics.Finally, I employed a full-sib design in my experiment in order to estimatequantitative genetic parameters of phenotypic plasticity. Theoretical results demonstratethat optimal levels of phenotypic plasticity can evolve given the appropriate type ofgenotype x environment interaction (Via 1987). While some studies were able to quantify8additive genetic vanance for phenotypic plasticity (Via 1984a), my design only allowed thecharacterization of family x diet interaction which is a measure of broad sense heritabilityof phenotypic plasticity. This parameter is still useful however, because it reveals whethergenetic variation for phenotypic plasticity exists. Such a crude partitioning of the geneticvariance is usually the norm when dealing with organisms that require extensive laboratoryfacilities for rearing.MATERIALS AND METHODSCrossing Technique and Rearing ProgramFish of both species were raised from artificially fertilized eggs. Eggs from gravidfemales were stripped into a petri dish by applying gentle pressure to the abdomen in ananterior to posterior direction. Males were sacrificed in MS-222, rinsed, and their testeswere removed and minced with forceps in a sterile aqueous saline solution (15% salt byvolume). This solution was poured over the egg mass and then left for 3-5 minutes untilfertilization had taken place. The egg mass was then rinsed and 30 fertilized eggs wereselected randomly. This procedure was repeated to yield 12 full-sib broods of 30 eggsfrom both the benthic and limnetic species.Each 30-egg brood was split into a pair of 15-egg half-broods, one being assignedto a live brine shrimp diet (Artemia salina), and the other being assigned to a diet of liveblackworms (iiibifex) and frozen bloodworms (Diptera spp.). These two diet treatmentswere representative of the planktonic and littoral habitats respectively. Consequently, my9experiment allowed a comparison of the two species when raised on their ‘naturaF dietswith the two species when their diets were reversed.Fertilized eggs were placed in plastic cups with mesh bottoms that were suspendedin an aquarium of continuously aerated water. After approximately seven days the eggshatched and all 15 fish of each half-brood were released into one side of a partitioned 102L aquarium. There were a total of 24 partitioned aquaria and each aquarium contained ahalf-brood of both species. Thus the half-broods of each family were raised in differentand randomly determined aquaria. Diet treatment was assigned randomly to each aquariumwith all aquaria having the same diet treatment on each side of the partition.Not all fertilizations were performed at the same time due to the sporadicavailability of adult fish. The time span between the first fertilization and the lastfertilization was approximately one month. The experiment was initiated in the spring of1992 and terminated in the fall of 1992.During the first month of life, fish in the littoral treatment were too small to be feda diet of blackworms. Consequently, all fish were fed a diet of brine shrimp nauplii duringthis period. After the first month had elapsed, the two experimental diet treatments wereused. Fish assigned to the littoral treatment were fed chopped frozen bloodworms for anadditional three weeks and then were fed live blackworms for the remainder of theexperiment. All fish were fed to satiation on their assigned diet treatment each day.Blackworms (the littoral prey) were administered by depositing the worms into a sandfilled petri dish at the bottom of the aquaria. Brine shrimp (the planktonic prey) werereleased into the water column. These two methods of prey deployment mimic the natural10feeding habitats of the two species. Brine shrimp were cultured in the laboratory andblackworms were purchased weekly from a local pet store. Photo-period was held at aconstant 16 L: 8 D cycle and temperature was maintained between 1 7C and 20C.MeasurementsThe experiment was terminated in November 1992 when fish had been fed thedifferent diet treatments for approximately 4 months. By this time both species hadreached a mean size of approximately 40 mm. This is the adult body size of the limneticspecies but the benthic species typically attains sexual maturity at 50mm or larger. All fishwere sacrificed in MS-222 and fixed in a solution of 10% formalin for one week. Afterfixation, fish were stained in a solution of Alizarin Red and 10% KOH in order to rendercalcified tissue more visible. The fish were then permanently stored in a solution of 37%isopropyl alcohol.Due to mortality, not all half-aquaria contained the same number of fish when theexperiment was terminated. This could potentially confound the comparison between diettreatments if mortality was non-random with respect to diet (Lindsey and Harrington1972). Unfortunately a comparison between the morphology of surviving and dying fishwas not possible because most of the mortality occurred within a few weeks of hatching.Therefore, to rule out natural selection as a cause of morphological differences betweengroups, I performed a two-way ANOVA with diet and species as factors and mortalitylevel as the dependent variable. Neither factor alone nor their interaction was significant(diet, 0.2 <P < 0.3; species, 0.05 <P < 0.1; interaction, 0.7 <P < 0.8) and there were no11obvious trends in the data to suggest that mortality was biased with respect tomorphology. Consequently I concluded that mortality was random with respect to theexperimental treatments.To balance the design for analysis, three fish from each half-aquarium wereselected randomly for measurement. This yielded a total of 6 fish per family per species(69 limnetic fish and 70 benthic fish). The number of fish of each species was slightlylower than 72 because five half-aquaria had only 2 surviving fish.Six characters were measured on each fish: (1) standard length, (2) gape width,(3) gill raker length, (4) gill raker number, (5) head depth, and (6) snout length. Traits(2) through (6) were chosen priori since they are correlates of foraging efficiency(Bentzen and McPhail 1984; Lavin and McPhail 1986; Schluter 1993). Standard lengthwas used as an overall size measure of each fish. These six traits are also among the mostvariable between sympathc benthic and limnetic species as well as among allopatricpopulations of threespine sticklebacks (Hagen and Gilbertson 1972; Gross and Anderson1984; McPhail 1993; Schluter and McPhail 1992).All dimensions were measured using an ocular micrometer on a Wild M3Cdissecting microscope, except standard length, which was measured using Verniercalipers.AnalysisAll traits except gill raker number were correlated with body size, andconsequently a covariate was needed to examine diet-induced morphological changes. To12simplify interpretation, I used standard length as a covariate rather than a compositevariable such as a pooled first principal component (PCi). The results were unchanged,however, when PC 1 was used instead. Additionally, I use untransformed data in allanalyses because this resulted in homogeneous variances between species.I examined all traits for size x diet interaction by using analysis of covariance. Nointeraction was evident in either species, implying that the effect of diet was independentof an individual’s size (limnetics, 0.05 <P < 0.6 for all traits; benthics, 0.1 <P < 0.9 for alltraits). Consequently I size-adjusted all traits by least squares regression against standardlength using a common slope between treatments for each species. These size-adjustedvariables are used in all subsequent analyses.MULTIPLE SIGNIFICANCE TESTS- Because five traits were examined for phenotypicplasticity, and a variety of comparisons were carried out using these traits, there was adanger that Type I error rates would escalate. I attempted to minimize the number ofstatistical tests by first carrying out a multivariate test of a given hypothesis using all fivetraits simultaneously. If the result was statistically significant (P < 0.05), I then attemptedto decompose the multivariate result into univariate measures to determine the relativerole of each trait in the significant multivariate result. There is no single establishedprocedure for performing such a test and Wilkinson (1975) suggests four alternatives. Iuse univariate tests and employ a sequential Bonferroni procedure (at 1 = 0.05) to guardagainst Type-I error (Rice 1989). It should be noted however, that it is primarily therelative magnitude of the P-values of the traits that are of interest in such an analysis rather13than their absolute values because the multivariate test establishes an a priori table-widesignificance level of cx. = 0.05.EFFECT OF DIET- If diet-induced phenotypic plasticity is adaptive, then I expected thatthe benthic and limnetic species would become more similar to one another when theirdiets were reversed (y2 <yi in fig 1). Testing this is equivalent to testing whether thesum of effects ‘U and ‘B’ in figure 1 is greater than zero. An individual fish does notrepresent the experimental unit in the breeding design because full sibs are not statisticallyindependent. Consequently, I calculated a mean diet-induced change for each of thetwelve families of both species for each of the five traits.Let B and L represent two matrices where b (or l) is the mean diet-induceddifference for the ith family and the jth trait of the benthic and limnetic speciesrespectively. I denote the five dimensional (co)variance matrices for B and L by SB andSL, and the five dimensional vectors of mean diet-induced differences among family meansby b and I. I let the vector t = b + I whose (co)variance matrix is then S1 = SB + S andthen tested whether t> 0 using Hotelling’s T2 statistic T2 = n(t — 0)TS1 (t —0) where 0 isthe five dimensional zero vector and where n =12 is the sample size. This statistic isdistributed as [(n—l)p/ (n — p)]F,_ where p is the number of dependent variables, anddenotes the F-distribution with p and n—p degrees of freedom (Johnson andWichern 1982).The above multivariate analysis revealed whether the overall ‘morphological gap’between the two species was narrowed. Subsequently, I carried out univariate t-tests to14determine if the sum of ?L? and ‘B’ in figure 1 was greater than zero using the sequentialBonferroni procedure mentioned above. This revealed the nature of the multivariatedifference.INTERSPECIFIC COMPARISON.-The above analysis does not reveal whether diet-induced changes were exhibited equally by both species or if one species was primarilyresponsible for the ‘narrowing of the gap? when both were diet-reversed. My expectationwas that the gap would be narrowed primarily by diet-induced change in the limneticspecies (i.e. L> B in fig 1), thus reflecting greater adaptive phenotypic plasticity in thespecies with the more variable natural diet.To test this expectation I performed a one-way multivariate analysis of variance(Johnson and Wichem 1982) comparing the mean diet-induced change of the twelvebenthic families with the mean diet-induced change of the twelve limnetic families for allfive traits. A significant Wific’s lambda indicates that there is a significant overall differencebetween species in phenotypic plasticity. The multivariate result was then decomposedusing a sequential Bonferroni procedure (Rice 1989) on univariate ANOVA results foreach trait.FAMILY x DIET INTERACTION-For phenotypic plasticity to evolve, genetic variationfor plasticity must exist in the population (Via 1987). My experimental design allowed meto estimate family x diet interaction. This is a measure of broad sense genetic variation forphenotypic plasticity and thus reflects whether plasticity has a genetic component. I15estimated the significance of this interaction using a two-way mixed model, multivariateanalysis of variance (Johnson and Wichern 1982). Separate MANOVAs were carried outfor each species. Family was considered as a random factor in this analysis and diet wasconsidered as a fixed factor.A potential complication in calculating family x diet interaction arises from theconfounding effects of micro-environment. Each of the 15 fish from a half-brood that wereraised on the same diet, were also raised in the same half-aquarium, and thus they are notstrictly independent. All of these fish experienced the same micro-environmental(aquarium) effects during their growth. Because I assumed that sibs are independent whencalculating the family x diet interaction term, my findings must be regarded as tentative. Aprevious quantitative genetic analysis of sticklebacks by Lavin and McPhail (1987)demonstrated, however, that aquarium effects on size-corrected measurements arenegligible, lending support to my assumption.There is considerable difficulty in interpreting the relationship between thedependent variables and the factors in a two-way MANOVA when there is a significantinteraction effect (Morrison 1976; Johnson and Wichern 1982). My purpose with this testwas mainly to determine whether there was significant overall genetic variation forplasticity and consequently I did not attempt to examine the effect of diet or family in eachspecies using this procedure. To probe the nature of the genetic variation for plasticity Icalculated two-way univariate ANOVATsfor each trait of both species.All analyses were carried out using Systat 5.01 on an IBM-compatiblemicrocomputer (Wilkinson et al. 1992).16RESULTSEffect of DietAll diet-induced changes were in a direction that is suggestive of adaptivephenotypic plasticity. Limnetic fish raised on a littoral diet developed a morphology thatwas displaced toward that of the benthic species relative to control fish raised on aplanktonic diet. Benthic fish raised on a planktonic diet developed a morphology that wasdisplaced toward that of the limnetic species relative to control fish raised on a littoraldiet. This trend was exhibited by all morphological characters (fig. 2) except gill rakernumber which displayed no trend of change.The results of the multivariate analysis reveal that the ‘morphological gap’ betweenthe benthic and limnetic species was significantly reduced by diet-reversal (Hotelling’s T2 =45.14, df = 5 and 7, 0.01 <P < 0.05). Thus, when considering all traits simultaneously,diet affects the degree to which these species differ morphologically. This suggests thatdifferences in diet may contribute to the morphological difference between the two speciesin the wild.Table 1 presents the percent reduction of the morphological gap between the twospecies caused by diet-reversal for each of the five traits. Percent reductions ranged from -1 percent for gill raker number to 58 percent for head depth. Also presented in table 1 arethe results of the univariate t-tests for each trait. Gill raker length was the only trait thatwas significant using the sequential Bonferroni procedure. It is informative, however, thatall traits except gill raker number exhibited a diet-induced change in the same direction.17Additionally, head depth had a P-value less than 0.05 which suggests that it alsocontributed to the multivariate significance.Interspecific ComparisonFigure 2 and table 2 suggest that the limnetic species tends to be more plastic thanthe benthic species in all traits except snout length and gill raker number (which showedvirtually no plasticity). MANOVA confirmed that the two species differed in their amountof plasticity, although statistical significance is marginal (P = 0.05). Univariate ANOVAs(table 2) revealed that only gill raker length displayed a significant difference in themagnitude of plasticity between the two species. All other traits had P-values at least anorder of magnitude larger than gill raker length. Thus, the degree of plasticity of the twospecies appears to be more distinct in some traits than in others, suggesting that thebenthic and the limnetic species differ not only in the magnitude of plasticity but in thepattern of plasticity among these morphological characters as well.Family x Diet InteractionMultivariate analysis revealed the presence of significant family x diet interaction inboth species (P < 0.001) indicating that there were differences among families in the extentand/or direction of diet-induced change. This is a measure of broad sense heritability ofphenotypic plasticity. Thus, there is genetic variability for morphological plasticity presentin both species’ populations.18Results of the univariate analysis are presented in table 3. Family x diet interactionin the benthic species is exhibited primarily by gape width, although gill raker length andsnout length are nearly significant. Family x diet interaction in the limnetic species isexhibited primarily by gape width. This suggests that morphological plasticity hasevolutionary potential and could respond to natural selection in both species ofsticklebacks.DISCUSSIONThe ecology of morphological plasticity is not well understood and is justbeginning to receive empirical attention (Meyer 1990; Witte et al. 1990; Wimberger199 1,1992). The ecological conditions that select for morphological plasticity are stillunclear. Yet this knowledge is necessary to understand the interplay between selectiveregimes, plasticity, and morphological evolution. The wider effects of morphologicalplasticity on niche partitioning, speciation and adaptive radiation are also poorlyunderstood. The extent to which plasticity accounts for morphological variation withinand between species would lend some insight to this problem.This study addressed four important issues of morphological plasticity using twosympatric species of stickleback. First, I demonstrated that when the two species are dietreversed, their morphologies become more similar overall. This suggests that either orboth species exhibits adaptive morphological plasticity. The individual traits thatcontributed most to this overall morphological change were gill raker length and perhaps19head depth. Both of these traits are probably important with respect to feeding efficiency(Bentzen and McPhail 1984; Lavin and McPhail 1986; Schiuter 1993).The second issue I addressed was how much of the interspecific difference inmorphology of these species can be attributed to plasticity. The percentage of the‘morphological gap’ between the two species that was closed by diet-reversal ranged from-1 percent for gill raker number to 58 percent for head depth. Thus a considerable amountof the morphological difference between the two species in the wild might be attributableto diet-induced morphological change.Third, I compared degree of plasticity between the two species, on the expectationthat greater natural diet-variability in the limnetic species would drive the evolution ofgreater plasticity in that species. The two species did exhibit a significant difference inoverall level of phenotypic plasticity and this difference was primarily attributable to agreater plasticity of gill raker length in the limnetic than in the benthic species. Gill rakerlength has been strongly implicated as a determinant of foraging efficiency in sticklebacks(Bentzen and McPhail 1984; Lavin and McPhail 1986; Schiuter 1993) and therefore theseresults lend support to the hypothesis that diet variability can select for morphologicalplasticity. Head depth and gape width also tended to be more plastic in the limnetic than inthe benthic species but these differences were not statistically significant (fig 2).Lastly, I addressed whether contemporary populations of benthics and limneticsmaintain genetic variation for morphological plasticity. Theoretical work suggests thatoptimal levels of phenotypic plasticity can evolve given appropriate genotype xenvironment interaction (Via and Lande 1985; Via 1987). The family x diet interaction I20demonstrate reveals a broad sense heritability of plasticity (Via 1984a, 1984b). Thisheritability includes variation due to dominance and epistatic interactions as well asadditive genetic variance (Falconer 1989). While this does not completely satisfy theconditions required for the evolution of phenotypic plasticity, it does reveal that plasticityis genetically determined.When making comparisons between species it is often difficult to distinguish theeffect of phylogeny from the effect of recent natural selection (Lauder 1982). Because thebenthic and limnetic species have both recently evolved from a common marine ancestor(McPhail 1993), and because both are relatively ‘young’ species, I can be reasonablyconfident that phylogenetic effects are not responsible for their differences in plasticity.Additionally, the wealth of natural history information of these two species ofstickleback and the well documented glacial history of this region, suggest that theenvironmental context in which these two species have evolved is relatively well-understood. Consequently, the adaptive nature of all diet-induced morphological changes,the greater degree of plasticity in the limnetic species, and the presence of geneticvariation for plasticity, together provide compelling evidence to support the hypothesisthat morphological plasticity has evolved as a result of diet variability. It has often beensuggested that diet variability might drive the evolution of trophic morphological plasticitybut there has been little such evidence.At this point however, two alternative hypotheses for the observed results deservemention. One involves the possibility of differences between the two species in their abilityto ingest prey. Because benthic fish are generally larger than limnetic fish in most21dimensions, it may be easier for a benthic fish to ingest a planktonic prey than for alimnetic fish to ingest a littoral prey. Assuming that diet-induced morphological changeresults from mechanical stress (as discussed below), limnetic fish would likely exhibitgreater morphological plasticity than benthic fish. However, if this were the case, onewould also expect there to be an overall effect of body size on the degree of plasticity. Nointeraction between size and the effect of diet was evident in the data and consequently Ido not believe this to be the explanation for my results.The second alternative basis for the interspecific difference in plasticity may lie indifferences in the way benthic fish and limnetic fish are constructed. Since their divergencefrom a common ancestor, the limnetic species has evolved a less robust morphology thanthe benthic species. For example, gill rakers of the limnetic species are not only longer andmore numerous than those of the benthic species, but they are more slender as well. Thus,if slender-built morphology is more susceptible to stress-induced change, this wouldexplain how the difference in plasticity between the two species is determined.Conceivably, such differences in robustness have evolved merely as an incidentalbyproduct of evolutionary divergence in body form, in which case interspecific differencesin plasticity are simply a (non adaptive) coffelated response to selection on the mean valueof each trait. If, however, robustness of morphology can evolve independently of the meanvalues of morphological traits, then such structural differences may be the proximatemechanism by which adaptive evolution of plasticity is realized.Diet-induced morphological plasticity could result from either nutritionaldifferences between diets or from differences in the mechanics of prey ingestion.22Nutritional effects are confounded with mechanical effects in my experiment. Nutrition canbe an important determinant of fish morphology (Halver 1984; Wimberger 1993). I feel,however, that differences in the mechanics of prey ingestion are more important in thisstudy. If nutritional differences were important, diet-induced morphological changeswould likely have exhibited a more random pattern (Wimberger 1993). The pattern of dietinduced change demonstrated is consistent with the difference in morphology observedbetween many littoral foraging and plankton foraging species (Lavin and McPhail 1985,1986; Schluter and McPhail 1993). Littoral and planktonic ecomorphs likely result fromthe mechanical requirements of foraging in these habitats rather than from nutritionaleffects.I suspect that mechanical stress is the cause of observed morphological plasticity inthe traits I examined, because these structures are composed of either cartilage or bone. Ithas long been realized that structures made of bone remodel and change shape dependingon the stresses imposed upon them (Lanyon 1984; Lanyon and Rubin 1985). The lack ofdiet-induced change in gill raker number is consistent with this hypothesis, becausewhereas mechanical stress can change the shape of particular structures it can not easilyalter their number.Behavioural plasticity also likely plays an important role in adaptation to resourcevariability. Changes in foraging behaviour have large effects on foraging efficiency (Dill1983; Ehlinger 1989a, 1989b) and behaviour is probably amenable to more rapid changethan morphology. For example, very different modes of foraging are used to exploitplankton and benthos, and individual fish switch rapidly between them when moving23between habitats (Schiuter 1993). An experiment which tests the adaptive significance ofbehavioural plasticity versus morphological plasticity is presented in chapter two.Evolutionary ImplicationsWhether phenotypic plasticity retards or enhances evolution is still a matter ofsome debate (West-Eberhard 1989; Stearns 1989). The distinction may be particularlyimportant when considering the Gasterosteus species complex. Invasion of freshwater bymarine aculeatus is pervasive throughout the holarctic region (Wootton 1976).Colonization of freshwater occurred as the Pleistocene glaciation ended and dramaticadaptive radiation ensued (Hagen and McPhail 1970; Bell 1976; McPhail 1993). Evenslight differences among bodies of freshwater in the same drainage basin have led to finescale adaptation in resident sticklebacks (Lavin and McPhail 1985). Trophic morphologymaps remarkably well onto lake ecology in all populations examined (Lavin and McPhail1985; Schluter and McPhail 1992). Heritability of trophic morphology also providescompelling evidence that this diversity is the result of evolutionary change.What has not been known is the extent to which this radiation owes its diversity tophenotypic plasticity. A comparison of several populations of sticklebacks in BritishColumbia (Schluter and McPhail 1992) has shown that gill raker length, gape width andgill raker number are among the most variable traits among sticldeback populations. Giventhat gill raker length has exhibited considerable phenotypic plasticity, it is possible thatsome of this interpopulation variability is environmentally-induced.24The effects of phenotypic plasticity on adaptive radiation and speciation inGasterosteus is not clear. It is evident that not all sticklebacks are equally phenotypicallyplastic, but the extent to which plasticity plays a role in the evolution of species pairs suchas the benthic and the limnetic is unknown. Given that trophic character displacement is animportant component of evolution and speciation in sticklebacks (Schluter and McPhail1992), it is possible that plasticity of trophic traits plays a very important role as well.25Table 1. Absolute magnitude of the ‘morphological gap’ between species when raised ontheir ‘natural’ diets and when diet-reversed. Values are calculated using the mean of thetwelve family means from each species for each trait. Units are in millimeters except forgill raker number. Asterisks indicate the results of univariate t-tests for a reduction of themorphological gap between species.Trait Natural Diet Diet-Reversed Percent ReductionGape Width 0.5 85 0.339 42Gill Raker Length 0.456 0.283 38***Gill Raker Number 5.29 1 5.3 19 -1Head Depth 0.477 0.20 1 58*Snout Length 0.665 0.469 29***p<o.oo1 *0.01 <P<0.0526Table 2. Percentage difference in each morphological character under diet-reversal. Eachvalue was calculated by dividing the absolute value of mean diet-induced change by themean value of that character when the species was raised on its natural diet. Asterisksindicate a significant difference between species as tested using univariate ANOVAs.Benthics LimneticsCharacter (% difference) (% difference)Gape Width 1.8 7.1Gill Raker Length** 5.1 11.0Gill Raker Number 1.4 1.0Head Depth 0.8 2.5Snout Length 2.6 2.8**p< 0.0127Table 3. F-ratios from univariate ANOVAs for family x diet interaction (F x D df =11,Error df =46 for all values).SpeciesTrait Benthic LimneticGape Width 734*** 13.76***Gill Raker Length 2.29* 0.68Gill Raker Number 1.58 1.99Head Depth 1.55 1.44Snout Length 2.18* 1.52***p<fl *OO1<p,<00528Fig 1. The benthic species (left) and the limnetic species (right). Also the expected patternof diet-induced morphological change. The four points represent the mean value of a traitfor the four species x diet combinations. The diet-induced change of the limnetic species(L) is predicted to be greater than the diet-induced change of the benthic species (B) ifdiet-variability has driven the evolution of morphological plasticity. The differencebetween the two species when raised on their ‘natural’ diets (y 1) is expected to be greaterthan the difference between the two species when they are diet-reversed (y2) ifmorphological plasticity is adaptive.Fig 2. Plots of the four species x diet combinations for all traits except gill raker number(which exhibited no plasticity). Plots are the mean of the 12 family means in eachcombination (i.e. N=12 for each of the means for each trait) and the 95 percent confidenceintervals. Units are in millimeters.29TLG) %j/20- 1B •Benthic Benthic Limnetic LimneticWorms P’ankton Worms PlanktonFigure 13.6ft’33.4•23.2ci) 0 (U CD3. U (U U) a) -J1.0CU a: =0.9( C)C Cl)3.6-J 4-.0 c (.1)3.2BenthicBenthicLimnoticLimneticWormsPlanktonWormsPlankton.fBenthicBenthicLimneticLirnneticWomisPlanktonWoimsPlankton-BonthicLiriinohcLitunolicPintiIcIn iW,iiitFi;i,)i( I)IflonihicBonthicLimneticLimnolicBenihicVVi,ip;IIIItkt()I)‘i.’()IIIISIIII1t(I(IIl031CHAPTER TWOTHE ECOLOGICAL SIGNIFICANCE OFMORPHOLOGICAL AND BEHAVIOURAL PLASTICITYINTRODUCTIONRecently, interest in the evolution of phenotypic plasticity has increased (Stearns1989; West-Eberhard 1989; Schemer 1993a) and, although theoretical research hasadvanced significantly (Levins 1968; Via and Lande 1985; Gomulkiewicz and Kirkpatrick1992; Leon 1993; Gavrilets and Schemer 1993a, 1993b), empirical research, especially onnatural populations, has lagged behind. While there have been several examinations ofphenotypic plasticity in natural populations (Lindsey 1962, 1972; Schlicting and Levin1986; Dodson 1989; van Noordwijk 1989; Witte et al. 1990; Wimberger 1991, 1992), themajority of empirical tests of theory on the evolution of phenotypic plasticity have comefrom laboratory studies of Drosophila (Schemer 1 993a). These studies contributesubstantially to our understanding of how phenotypic plasticity evolves but, ultimately, wewant to know if theoretical predictions are borne out in natural populations.In this chapter I extend the results of chapter one that suggested diet variability candrive the evolution of plastic trophic morphology. In chapter one I demonstrated anassociation between diet variability and the degree of diet-induced morphological plasticityin two sympatric species of threespine stickleback. Additionally, I documented theheritability of morphological plasticity and provided evidence that suggests diet-induced32morphological change is adaptive. The present chapter extends these results in three ways.First, I examine the time scale over which diet-induced morphological change occurs todetermine if it is compatible with the time scale of natural diet variability. Second, Iexamine the adaptive significance of diet-induced morphological plasticity by measuring itseffect on foraging efficiency. Third, I examine the effect of short-term learning(behavioural plasticity) on foraging efficiency and compare its importance to that ofmorphological plasticity.Theoretical work suggests that the degree of plasticity that evolves depends uponthe rate of occurrence of the plastic response to environmental cues (Gomulkiewicz andKirkpatrick 1992; Leon 1993). Traits are usually classified as labile or non-labile in suchtheoretical analyses. Labile traits are those whose plastic response to environmental cues iseffectively immediate, whereas non-labile traits are those whose expression is plastic butwhose value is fixed at some point during development. In reality it is possible for traits tobe anywhere between these two extremes. While many studies examine morphologicalplasticity, few determine whether the time scale of the plastic response is compatible withthat of the environmental variability for which the plasticity is suspected to be anadaptation.Morphological and Behavioural PlasticityStudies of the adaptive significance of diet-induced morphological plasticity arerare, and in those that exist (e.g. Thompson 1992), few consider of the potential effect ofdiet-induced behavioural change. Yet, behavioural plasticity can have a large impact on33foraging efficiency (Werner et al. 1981; Dill 1983; Ehlinger 1989a, 1989b, 1990; Croy andHughes 1991). Consequently, where diet-induced morphological change is accompaniedby a change in foraging efficiency, the effects of behavioural and morphological plasticityoften are confounded.My experiment is designed explicitly to examine both types of phenotypicplasticity. I define learning (behavioural plasticity) as behavioural changes that take placeon a time scale short enough to preclude most morphological change. The effect oflearning is measured as the changes in foraging efficiency that accrue from short-termexperience with a prey type.Morphological plasticity is relatively easy to measure but its effect on foragingefficiency is more difficult to quantify. Long-term exposure to a particular prey may alternot only trophic morphology but also neural morphology and thus result in indirectbehavioural changes (Krebs 1990; Healy et al. 1994 and references therein). Also, learning(as defined above) may be morphology-dependent. That is, its effects on foragingefficiency may change as morphology changes (an interaction). Consequently, it is notpossible to completely disentangle the effects of morphological and behavioural plasticity.Because of this problem I define the effect of morphological plasticity as the usual effectof diet-induced changes in trophic morphology plus any effect of changes in behaviour thatresult from long-term exposure to prey. This effect is measured by documenting changesin foraging efficiency that take place with long-term exposure to prey.34Plasticity and SticidebacksMy study organism is the planktivorous stickleback (Gasterosteus p) found in sixlakes of coastal British Columbia, Canada (McPhail 1993). The species is not formallydescribed but is termed the ‘limnetic’ species because it forages predominantly on calanoidcopepods (Diaptomus in the ‘limnetic’ (water column) habitat of lakes. The limneticspecies coexists with a ‘benthic’ species which forages predominantly on invertebrates(gammarids) in the littoral region of the lakes. The two species are morphologicallydistinct, and differences in morphology have important effects on foraging efficiency andgrowth in the two habitats (Bentzen and McPhail 1984; Schluter 1993, 1994).The limneticspecies has a long snout, long gillrakers, a slender head and a narrow gape relative to thebenthic species (McPhail 1992). These morphological differences are largely heritable(McPhail 1992; chapter one) and are thought to have evolved as a result of competitionfor resources and character displacement (Schluter and McPhail 1992, 1993).In chapter one I demonstrated that the limnetic species exhibits greater diet-induced morphological plasticity than the benthic species. Because limnetics forage in thebenthic habitat during their breeding season (Schluter and McPhail 1992; Schluter 1993),they experience substantial resource variability. In contrast, benthic fish forage in thebenthic habitat throughout their lives and therefore have a relatively monotonous diet.Therefore, in chapter one, I suggested that greater diet-induced morphological plasticity inthe limnetic species may have evolved to cope with this high degree of resource variability.35In this chapter I document the time scale over which morphology changes bykeeping limnetics on a diet of either calanoid copepods (limnetic prey) or gammarids(benthic prey) and sampling fish from these two treatments over time. A comparison ofthis time scale with that of the natural resource variability arising from the breeding cycleallows me to determine if the two are compatible. Also, by conducting foraging thals ongammarid prey items with fish from the two diet groups, I can quantify the effect ofmorphological plasticity and learning. If diet-induced morphological plasticity has evolvedas a result of diet variability, then limnetics with gammarid-induced morphology should bemore efficient foragers on gammarids than limnetics with calanoid-induced morphology(all else being equal).MATERIALS AND METHODSAll fish were taken from Paxton Lake, British Columbia, Canada in mid-April 1993using dipnets. Each individual (25mm - 30mm standard length) was randomly assigned toa diet of either calanoid copepods (Diaptornus spp) or gammarid amphipods (Gammaruslacustris). These diet treatments represent prey commonly found in the natural foraginghabitats of the limnetic species (Schluter and McPhail 1992). Fish were kept in eight 102 Laquaria (four per diet treatment) at initial densities of approximately 30 individuals peraquarium. These densities declined throughout the summer as a result of mortality andsampling for foraging trials. All fish that died during the course of the summer were keptfor comparison with surviving individuals. This allowed me to check that morphologicaldifferences between diet treatments were the result of diet-induced plasticity rather than36differential mortality. Photoperiod was constant (16L:8D cycle) for the entire period andtemperature varied with ambient temperature (between 13 and 19 degrees Celsius).Diet TreatmentI used a diet-switching experimental design (fig. 3). The pre-switch period waslongest and was meant to induce an effect of both learning and morphological plasticity onforaging efficiency. The shorter post-switch period was meant to induce only a learningeffect. There were three sets of foraging trials, each with a different duration of pre-switchdiet exposure (sets 1, 2 and 3 had 18, 39 and 72 days of exposure respectively). Thepurpose was to estimate the time scale over which morphology changes, and to determinethe effect of different degrees of morphological change on foraging efficiency. The post-switch period was ten days for all three sets. Although the duration of the post-switchperiod is somewhat arbitrary, ten days probably is sufficient to produce efficiency changesthrough learning (Werner et al. 1981; Ehlinger 1989a; Schiuter 1993) yet short enough topreclude most morphological change.Switching involved selecting a random sample of fish (18, 24 and 24 fish for sets 1,2 and 3 respectively) from each of the two diet treatments. Half the fish were kept on thesame diet, and the remaining half were given a diet opposite to what they had experiencedduring the pre-switch period. This design provided three sets of four experimentaltreatments: calanoid pre-switch/calanoid post-switch (C/C), calanoid pre-switch/gammaridpost-switch (CIG), gammarid pre-switch/calanoid post-switch (0/C), and gammarid preswitch/gammarid post-switch (GIG) (fig. 3).37Foraging TrialsThese trials were to determine how experience with gammarids or copepodsaffects foraging efficiency on gammarids. In each trial a single fish was placed in a 102 Laquarium containing ten gammarids (mean length = 3.47 [SD = 0.53], mean width = 1.06[SD = 0.181) on a sand substrate. I recorded the time at which each of the followingevents occurred: orientation towards substrate or prey item, prey attacked on substrate,prey attacked in water column, prey spat out, end of orientation towards substrate or preyitem, prey swallowed, ‘empty’ strike on substrate and ‘empty’ strike in water column.Empty strikes are instances where the fish appeared to strike at nothing, or at debris in theaquarium other than gammarids. Because foraging efficiency varies with satiation level,trials ended either after ten minutes or after the consumption of two prey items, whichevercame first.Measures of Foraging Efficiency and Moho1ogvThe information recorded during each foraging trial allowed me to calculate fourmeasures of foraging efficiency: two for searching efficiency and two for handlingefficiency.The searching efficiency measures are (1) time from fish introduction to first attack(henceforth termed ‘latency time’) and (2) search time per prey item. Search time/preywas calculated by dividing the total search time by the number of prey items consumedplus one. One was added to the denominator to prevent division by zero in instances38where a fish searched for, but did not consume, prey items. Both variables were logtransformed to normalize their distributions.The handling efficiency measures are (3) handling time per prey item consumedand (4) number of attacks required per prey item consumed. Both were calculated bydividing total handling time, or number of attacks, by the number of prey consumed plusone. Both measures were log transformed to normalize their distributions, and both weresize-corrected by least-squares regression against standard length because they werecorrelated with body size. This regression was carried out on combined data from thethree different pre-switch durations (the three sets).Five morphological characters were measured on each fish: (1) gillraker length, (2)head depth, (3) gape width, (4) snout length, and (5) standard length. Characters (1)through (4) were chosen because chapter one demonstrated they have a plastic responseto diet, and character (5) was used as a covariate for size-correction of variousmeasurements. All were log transformed to equalize their variances. Although othermorphological characters probably exhibit diet-induced plasticity, these characters providean index of overall trophic morphology. Characters (1) through (4) were measured usingan ocular micrometer on a Wild M3C microscope at 6.4x - 16x magnification and standardlength was measured using Vernier calipers.Size-corrected morphological characters were used in all analyses except for thatof time scale. Size-correction was carried out by least-squares regression of each characteragainst standard length using combined data from all three sets. The size-correctedvariables were adjusted to the mean standard length of the combined data.39AnalysisMost of the analyses are one-tailed. The analysis for diet-induced morphologicalchange is one-tailed because chapter one demonstrated a consistent direction to all diet-induced change. Likewise, the analyses for changes in foraging efficiency are one-tailedbecause morphological and behavioural experience with gammarids is never expected todecrease foraging efficiency on gammarids. Analyses are univariate in all cases where adirectional alternative hypothesis is appropriate because it is difficult to incorporatedirectional hypotheses into multivariate statistical procedures. In these instances, I correctfor table-wide significance levels using the sequential Bonferroni technique (Rice 1989).All statistical analyses are performed using Systat 5.02 for Windows (Wilkinson et al.1992).To rule out differential mortality (natural selection) as a cause of observedmorphological change, I conducted a one-way MANOVA (Johnson and Wichern 1982)on the fish that died. I used the pre-switch diet treatment as the independent variable andthe morphological characters that exhibited change as the dependent variables. Ifdifferential mortality caused the observed pattern of morphological change, thenmorphology should differ between diet groups in a direction opposite to the difference inmorphology of surviving fish. The effect of pre-switch diet was not significant (F2,80=2.32, P = 0.11) and the direction of difference between diet treatments was the same inboth dead and surviving fish. This indicates that diet-induced plasticity occurred in both40the fish that died and those that survived. Therefore, the morphological changes are notthe result of biased mortality.TIME SCALE. - An assumption in the experimental design is that most diet-inducedmorphological change occurs on a time scale longer than ten days. This assumption wastested by combining data from the three pre-switch durations (sets) for treatments C/C andCIG and then comparing the two treatments using a one-way MANOVA on the fourmorphological variables. The same procedure was conducted with groups G/C and GIG.The results of these tests confirm that ten days of diet treatment is not sufficient to inducea detectable morphological change (C/C vs. CIG, F461 = 0.568, P = 0.69; G/C vs. GIG,F4,61 = 0.589, P = 0.67).To determine the time scale over which morphology does change I compared themorphology of treatments CIC and CIG combined with treatments G/C and GIG combinedacross the three pre-switch durations (sets). Thus I had two ‘morphology’ groups fromeach pre-switch duration (set) to compare (the groups were determined by pre-switch diettreatment, fig. 3). These groups were compared using a univariate one-tailed ANCOVA(Neter and Wasserman 1974) for each morphological character for each pre-switchduration. Standard length was the covariate. The pre-switch duration at which the twogroups are significantly different is an estimate of the time necessary to inducemorphological change.41EFFECT OF MORPHOLOGICAL PLASTICITY AND LEARNING. - Treatments C/C,C/G and GIG (fig. 3) were used to estimate the effect of morphological plasticity andlearning. If diet-induced morphological change affects foraging efficiency, then divergencein morphology between the two pre-switch diet treatments from set 1 to 2 to 3 should beparalleled by divergence in foraging efficiency between groups [C/C + C/G] and [GIG]because these two groups differ in pre-switch diet. Testing for such divergence tests forthe effect of morphological plasticity. The effect of learning was tested by comparinggroup [C/Cl with [C/G + GIG] because these two groups differ in post-switch diet.I used the following regression model to test for both effects simultaneously:Y = a + /5. duration + pre )‘we duivtion2+ Spost 7postY is the measure of foraging efficiency and duration is the length of pre-switch diettreatment in days. 6pre and st are indicator variables specifying the type of pre- or post-switch diet: pre or st equals 1 if the diet treatment is calanoids and -1 if the diettreatment is gammarids. This model assumes that foraging efficiency is equal in treatmentsC/G and G/G (Y = a) at zero pre-switch duration and that efficiency of the two pre-switchdiet groups ([C/C and C/G] versus [GIG]) diverges non-linearly with time (represented bythe term, Ypre pre duration2);the quadratic was a good first approximation to this nonlinear divergence (fig. 3). Because there was no significant interaction between the effectof post-switch diet type and pre-switch duration for any foraging efficiency variable (F1,5642= 2.161, 0.106, 0.037, 0.038 for attacks/prey, handling time/prey, search time/prey,latency time), the effect of learning is represented by a constant displacement (y) of C/Cfrom C/G for all pre-switch durations. The model also specifies a common linear term forall three treatments (fi . duration). If Ypre or ‘Yst are significantly different from zero thenforaging efficiency was affected by morphological plasticity or learning, respectively.As an additional analysis for the effect of morphological plasticity I conducted aunivariate multiple regression for each (un-corrected) efficiency variable using those (sizecorrected) morphological variables that exhibited diet-induced change as independentvariables. If the characters that exhibited a diet-induced change (or any characterscorrelated with them) actually do affect foraging efficiency, the efficiency variables shoulddepend on them. This analysis was carried out on data from all three pre-switch durationscombined.RESULTSTime ScaleHead depth was the only morphological character that exhibited significant dietinduced plasticity, and then only in the longest pre-switch duration (table 4). None of thecharacters exhibited statistically significant plasticity before this set although the trend ingape width appeared to plateau (or even decline) after the second set (fig. 4). Combiningsets 2 and 3 for gape width, however, results in a statistically significant differencebetween the two diet groups (F1,92 = 4.00, P 0.024). This suggests that small effect sizeand/or high variability result in the need for larger sample sizes. There was no consistent43pattern of diet-induced plasticity in giliraker length or snout length across the three preswitch durations (sets). These results differ from those in chapter one which demonstratesubstantial plasticity in all four morphological characters. Fish were kept on different dietsfrom an earlier age in the previous study, however, and this suggests that some traitsmight exhibit non-labile plasticity. Perhaps, differences in diet and/or growth rate betweenthe present and previous experiments accounts for the difference in plasticity as well.Effect of Morphological PlasticityForaging efficiency on gammarids continually improved as an individual’sexperience with gammarids increased up to 72 days; all measures of forging efficiency,except latency time, significantly diverged between the two pre-switch diet treatments (i.e.Ypre is significantly different from zero, table 5; c/c [and C/G] diverge from GIG as preswitch duration increases, figure 5). This increase in foraging efficiency parallels, and isroughly on the same time scale, as diet-induced morphological change (fig. 4). Thissuggests that part of the change in efficiency may be due to plasticity in the charactersexamined.In general, the pattern exhibited by the foraging efficiency variables suggests thatdivergence in efficiency between treatments /C (or C/G) and GIG over time is due toboth the increased efficiency of treatment GIG and the decreased efficiency of treatmentcic (or c/G). Such an interpretation should be treated cautiously, however, because atrend of decreased efficiency irrespective of treatment group across the three pre-switchdurations would produce this same pattern.44Multiple regression analyses of specific morphological characters reveal that onlyhead depth is significantly related to handling time/prey (table 6). Apparently gape width isnot related to any of the efficiency variables. Note, however, that the R2 values are low(handling time/prey, 0.049; attacks/prey, 0.032; search time/prey, 0.0 16; latency time,0.007), and this implies large variations in foraging efficiency that are not explained byvariation in either morphological character. Although measurement error was undoubtedlypartly responsible for these low R2 values, other additional factors (e.g., unmeasuredtraits) probably also play important roles in determining foraging efficiency.Effect of LearningBoth searching efficiency variables were improved by post-switch experience withgammarids (y for latency time, P = 0.02; search time/prey, P = 0.007; table 5) but, usingthe sequential Bonferroni technique, only search time/prey was significant. This variabledisplayed both an effect of morphological plasticity and an effect of learning (table 5; fig.5[iiij). Lack of a significant effect of learning on latency time might be the result of lowstatistical power since latency time was consistently highest in treatment C/C at each ofthe three pre-switch durations (fig. 5[ivj). Neither handling efficiency variable exhibited asignificant effect of learning nor did they display any consistent pattern at each pre-switchduration.A comparison of the elevations of the three regression lines at 72 days of preswitch duration allows an estimate of the relative effects of morphology plasticity andlearning on foraging efficiency (fig. 5). The magnitude of the effect of learning on handling45efficiency (C/C versus C/G at duration = 72, fig.5[i-ii]) is much smaller than that ofmorphological plasticity (CIG versus GIG at duration = 72, fig. 5[i-iij), suggesting thathandling efficiency is predominantly effected by morphological plasticity. In contrast,learning has a large effect on both searching efficiency variables. Its effect appears roughlyequal to that of morphological plasticity for search time/prey and only learning producedan effect on latency time (fig. 5[iii-iv]).DISCUSSIONRecent theoretical explorations of the evolution of phenotypic plasticity revealsome ecological conditions under which plasticity should evolve (Via and Lande 1985;Gomulkiewicz and Kirkpatrick 1992; Gavrilets and Schemer 1993a, 1993b), and a fewempirical studies have attempted to correlate ecological factors and degree of plasticity(Wimberger 1991,1992). Some of these studies suggest that phenotypic plasticity can beadaptive; however, studies that quantify the adaptive significance of phenotypic plasticityare rare (Thompson 1992). Yet, such evidence is crucial to understanding the evolution ofphenotypic plasticity. Further, even in instances where the adaptive significance ofmorphological plasticity has been measured, other possible explanations such as changes inbehaviour usually have not been considered.In my experiment with limnetic sticklebacks it took between 39 and 72 days of diettreatment to induce a detectable morphological change. The only morphological characterthat exhibited significant change was head depth, although gape width exhibited aconsistent pattern of change when the duration of the diet treatment increased.46Comparison of the present results with those in chapter one suggest that diet-inducedmorphological changes may not be reversible but, instead, are fixed at some point indevelopment (i.e., they are non-labile). Thus, the marginal diet-induced changes in onlytwo of the four traits measured in this study may be a result of using wild fish that werealready approximately 25mm - 30mm in standard length. Other differences between thetwo experiments, however, include the use of different prey items as well as a differenttotal amount of growth achieved by fish in each experiment. Thus, an unequivocalexplanation of the discrepancy cannot be given.Diet-induced morphological changes probably result from plastic responses ineither muscle or bone. Both substances can change size and shape under prolonged novelstresses (Lanyon 1984; Lanyon & Rubin 1985), especially if the stresses are imposedduring critical periods in ontogeny (Wainwright et al. 1991; see Bertram & Swartz 1991for discussion). In chapter one I suggested why mechanical stress is the probableproximate cause of plasticity rather than an alternative such as nutritional differencesbetween diets.Diet-induced changes in morphology were paralleled by changes in foragingefficiency (table 5; fig. 5). Additionally, head depth was significantly related to one of thehandling efficiency variables (table 6). Although the functional utility of head depth is notknown, this character probably is correlated with other, unmeasured morphologicalcharacters. Also, a large proportion of the variation in the efficiency variables was notexplained by variation in the morphological characters measured. This implies that changesin unmeasured characters also must cause some of the divergence in efficiency. One47possible example is diet-induced change in neural morphology and the consequent effectson behaviour (Masai and Sato 1965; Krebs 1990; Healy et al. 1994). If neural morphologyis more labile than the morphological characters measured, then low R2 values for gapewidth and head depth may not represent the total effect of diet-induced morphologicalchange.The morphological characters chosen for study are the important (plastic)correlates of foraging efficiency in sticklebacks (Bentzen and McPhail 1984; Lavin andMcPhail 1986; Schiuter 1993) and therefore are a logical part of the anatomy to quantify.Unquestionably, however, other attributes of a fish’s morphology also contribute tosuccessful foraging. Thus, the divergence in foraging efficiency that I attribute tomorphological plasticity probably is not solely due to diet-induced change in the charactersexamined. Rather, plasticity in these characters should be considered as an index of overallmorphological plasticity.Learning has a significant effect on search time/prey and appears to result in aconsistent, though non-significant, effect on latency time. In contrast, learning does nothave any discernible effect on either of the handling efficiency variables. This differs fromthe results of previous studies where learning affects both searching and handlingefficiency (Werner et al. 1981; Dill 1983; Ehlinger 1989a, 1989b; Croy and Hughes 1991).The reason for this discrepancy between my data and earlier studies is not known.Morphological Plasticity versus Learning. - The efficiency of the prey ingestion process isseparable into two distinct components: searching efficiency, and handling efficiency. Myresults suggest that the effects of morphological plasticity and learning parallel this48division of foraging efficiency. The magnitude of the effect of learning on search time/preyis roughly equal to that of morphological plasticity and it appears to have had the onlyeffect on latency time (fig. 5 [iii-iv], at duration = 72 days). In contrast, the magnitude ofthe effect of morphological plasticity on both handling efficiency variables is substantiallygreater than that of learning (fig. 5{i-iij, at duration = 72 days). Several studies havedemonstrated that learning affects both components of foraging efficiency but the presentresults are the first to my knowledge that compares the effects of learning with those ofmorphological plasticity.Evolution Of Morphological Plasticity in SticklebacksIn chapter one I demonstrated an association between diet variability and thedegree of trophic morphological plasticity in two species of sticklebacks. Here I havedemonstrated that this diet-induced morphological change is adaptive in that it increasesforaging efficiency. Together these results provide some of the best evidence from naturalpopulations to date suggesting that diet variability can drive the evolution of plastictrophic morphology. However, a few complicating factors remain to be addressed.if morphological plasticity has evolved as a result of diet variability then it isimportant that the time scale of diet-induced morphological change is appropriatelymatched to the time scale of natural diet variability. I have shown that morphologicalchanges take place on a time scale of 39 to 72 days. Observations suggest that thebreeding season for the entire limnetic species’ population in Paxton Lake is roughly 100to 120 days (Schiuter and McPhail 1992; pers. ohs.). Although this is likely an49overestimate for the amount of time an individual spends breeding, it is a reasonable firstapproximation because most fish do not survive the winter to breed again the followingyear. Alternatively, plastic changes may occur more rapidly in the wild because fish areexposed to other environmental cues in addition to a change of diet (e.g., temperature,pH).A potential objection to the above evolutionary hypothesis stems from the lack ofsubstantial diet-induced morphological change in this study as compared to results ofchapter one. The present results on diet-induced morphological change suggest that someof the traits considered might exhibit non-labile plasticity. if this is true, then the argumentof intra-generation diet variability driving the evolution of morphological plasticity inthese traits is not plausible (Gomulkiewicz and Kirkpatrick 1992; Schemer 1993a). It isthen curious why the limnetic species exhibits greater plasticity in these traits. A potentialexplanation resides in the differences in lifespan of the two species. Because individuals ofthe limnetic species have a shorter lifespan (approx. 1-2 years) than individuals of thebenthic species (approx. 3+ years), the limnetic species may experience greater inter-generation resource variability than the benthic species. Non-labile plasticity could then bean adaptation to uncertain resource levels that are present in any particular generation.Yoshimura and Clark (1991), Gomullciewicz and Kirkpatrick (1992), Schemer (1993a)and Leon (1993) all provide discussions of adaptation to inter-generation variability. If thisis true, then the effect of morphological plasticity on foraging efficiency that I havedemonstrated may well be an underestimate. The full effect of morphological plasticitycould only be demonstrated by administering the diet treatments early in ontogeny.50A fmal alternative hypothesis for the difference in degree of plasticity between thetwo species was suggested in chapter one. The limnetic species is morphologically lessrobust than the benthic species. For example, limnetic giirakers are not only longer andmore numerous than those of the benthic species, but are more slender as well. If lessrobust morphology is more susceptible to stress-induced change, this would explain howthe interspecific difference in plasticity is realized. It is possible that differences inrobustness have evolved as an incidental byproduct of evolutionary divergence in bodyform, and thus interspecific differences in plasticity are simply a (non adaptive) correlatedresponse to selection on the mean value of each trait. At present it is not possible todistinguish between these alternatives.51Table 4. Results of the one-tailed univariate ANCOVAs for a diet-induced change inmorphology and also the proportional changes calculated as percent = 100. (trait -traitcai) / t1aitcai. Only results from the third set are presented.Morphological Character F1, (Diet) Percent ChangeHead Depth 6.10** 1.4Gape Width 1.95# 2.2Giliraker Length 0.423 8.3Snout Length 0.030 0.0** P<O.O1,#O.05 <P<O.1(“IU,Table5.Resultsoftheanalysisforaneffectofmorphologicalplasticityandlearningonforagingefficiency.Statisticalmodel:Y=+f3duration+ö.(Iuratson2+•yO.Indicatorvariablesandö 1 ,equalI(-I)ifdiettreatmentiscalanoids(gammauids).P—valuesarcnotBonferronicorrected.F 1 , 87______________________________________Estimate___________________EfficiencyVatiable‘postXf3(.i0-)Ypre(.l0)Handlingtime/Prey0.88812.9***0.1972.65-3.980.1110.0466Attacks/Prey0.10413.2***0.7310.701.330.1100.0876Searchtime/Prey0.9444•49*6.40**1.585.880.09480.379Latencytime0.5160.1994.40*2.665.340.02400.388**P<O.O01,**P<O.0l,*P<0.0553Table 6. Results of the multiple regression analyses for the effect of morphologicalplasticity on foraging efficiency. N= 124 for each._______________t-statistic______________Efficiency Variable Head Depth Gape WidthHandling time/Prey 2.49** 0.61Attacks/Prey 1.68* -0.51Search time/Prey -1.27 -0.22Latency time -0.62 0.39** P<O.O1, * P<O.0554Fig. 3. Design of the diet treatments. The pre-switch period is meant to inducemorphological change and the post-switch period is meant to induce short-termbehavioural change.Fig. 4. Magnitude of the diet-induced change across the three sets plus/minus SE. Bothmorphological characters are size-corrected to a fish of 32.95 mm in standard length.Fig. 5. The foraging efficiency variables across the three sets with regression linescalculated using estimates of table 2 as Y = a + fi . duration + . . duration2+y. Dashed (CIG) and solid lines (GIG) depict divergence of efficiency resulting frommorphological plasticity and dotted line (C/C) reveals the change in efficiency resultingfrom learning relative to group CIG. Symbols are means of each group plus/minus SE;A=C/C, A=C/G, and •=G/G.55Calanoids C21flOidS. Grnmarus[ C1iojdsGainmarus I G2T—Pre-switch Post-switchDiet DietTimeC/Cdo0/CGIGFFigure 3-Q(PC’)C)CDC-‘0C!)Diet-InducedChangeHeadDepthIn(mm),0099>-LS.— C)a)L>>‘Lt).5.-’-__5(I)ci) Eo0)________________cadUIa) a a)a)(0U)-.9>-(1)(1)Eci ci)>-.EC)C ci)-c C) (‘I ci)____________________(I)02040602060Pre-SwitchDietDuration(days)(i) 0204060LI) C’)(üi)C’)0204060(iv)to ciC’)040(315.458GENERAL DISCUSSIONIn chapter one I demonstrated an association between diet variability and thedegree of trophic morphological plasticity in two species of stickleback. I also providedevidence suggesting that morphological plasticity is adaptive and demonstrated thatmorphological plasticity is heritable. These results are some of the best evidence from anatural population to suggest that diet variability can drive the evolution of plastic trophicmorphology.In chapter two I extended these results by demonstrating that diet-inducedmorphological change is adaptive in that it increases foraging efficiency. I also providedevidence revealing that the time scale of morphological change is roughly compatible withthe time scale of natural resource variability. Together, the results from both chaptersprovide strong support for the proposed evolutionary hypothesis. However, it is importantto emphasize the complications discussed in chapters one and two. The alternativehypotheses mentioned there require a more in depth treatment before they can becompletely dismissed. Additionally, it would be useful to have a clear understanding of theproximate mechanism whereby diet-induced changes are realized.In addition to addressing issues regarding the evolution of morphologicalplasticity, the results of these two experiments suggest ways in which individual fish adaptto resource variability. Short-term conditioning on a particular prey largely results inbehavioural change which increases searching efficiency. Long-term conditioning on aparticular prey results in behavioural and morphological change which increases both59searching and handling efficiency. These mechanisms result in adaptation to environmentalconditions on time scales shorter than those usually envisaged for evolutionary change.Additionally, the present results reveal that substantial adaptation of a population can takeplace without any genetic change.60LITERATURE CITEDBell, M. A. 1976. 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