Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 660

_____________________________ _____________________________

Bottlenecks and BlowfliesSpeciation, Reproduction andMorphological Variation in

Lucilia

BY

ANN-BRITT FLORIN

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

Dissertation for the Degree of Doctor of Philosophy in Animal Ecology presented atUppsala University in 2001

AbstractFlorin, A.-B. 2001. Bottlenecks and blowflies. Speciation, reproduction and morphologicalvariation in Lucilia. Acta Universitatis Upsaliensis. Comprehensive Summaries of UppsalaDissertations from the Faculty of Science and Technology 660. 40 pp. Uppsala. ISBN 91-554-5133-0.

This thesis attempts to improve our understanding of the role of population size for theprocess of speciation. First, the effect of population size on speciation is studied usingseveral meta-analyses of published laboratory experiments. Second, the effect ofpopulation size on behaviour is studied using a laboratory population of the blowflyLucilia sericata. Third, the effect of population size on morphological and geneticvariation is studied using wings and microsatellites from wild populations of L. illustris aswell as experimentally bottlenecked populations of L. sericata. The meta-analyses showedthat the result of many previous laboratory experiments on sympatric and parapatricspeciation may have been biased by too small population sizes. Reduced interbreeding wasless likely to develop in small populations where the selection against hybridisation oftenseemed to have been opposed by inbreeding depression or loss of genetic variation. Inallopatric speciation experiments, no general consistent effect of population size wasobserved. There was no support for speciation through founder events. In fact, significantassortative mating was only found in vicariance experiments where derived populationswas tested against each other. Population size influenced reproductive behaviour in L.sericata. There was a positive effect of increasing number of males on egg-laying but onlyas long as the female was in the company of at least one other female. Female mate choiceand a positive effect of number of eggs on larval survival are suggested to be theunderlying factors. No historic bottlenecks could be detected in the fly populations, butstrong genetic indications suggest a fine grained genetic population structure of wildLucilia flies. Bottlenecks had unpredictable effects on wing morphology as well as ongenetic variation and fitness in a laboratory stock of L. sericata. Thus a bottleneckedpopulation will not necessarily have a higher chance of evolving morphological noveltiesthan one which has not undergone a bottleneck. However, among many bottleneckedpopulations there is a good chance that in at least one of them the conditions will beconducive to morphological change and evolution. In this statistical sense, thus, strongpopulation fluctuations may enhance the probability of speciation events.

Key words: Population size, speciation, Lucilia sericata, morphological variation, Luciliaillustris, bottlenecks.

Ann-Britt Florin, Department of Animal Ecology, Evolutionary Biology Centre, Norbyv.18D, SE-752 36 Uppsala, Sweden (Ann-Britt.Florin@ebc.uu.se)

© Ann-Britt Florin 2001

ISSN 1104-232XISBN 91-554-5133-0

This thesis is based on the following papers, which will be referred to in thetext by the roman numerals I-VI

I Ödeen, A. & Florin, A.-B. 2000. Effective population sizemay limit the power of laboratory experiments to demonstratesympatric and parapatric speciation. Proc. R. Soc. Lond. B.267, 601-606.

II Florin, A.-B. & Ödeen, A. Laboratory environments are notconducive for allopatric speciation. J. Evol. Biol. In press.

III Florin, A.-B. Group size and composition affects reproductionin blowflies. Manuscript.

IV Florin, A.-B. Morphological and genetic variation in wildpopulations of Lucilia illustris (Diptera: Calliphoridae).Manuscript.

V Florin, A.-B. The effects of bottlenecks on morphological andgenetic variation in Lucilia sericata (Diptera: Calliphoridae).Manuscript.

VI Florin, A.-B. & Gyllenstrand, N. Isolation andcharacterization of polymorphic microsatellite markers in theblowflies Lucilia illustris and Lucilia sericata. Manuscript.

Reprints were made by permission from the publisher. In paper I and IIboth authors contributed equally to the work. In paper VI the order of theauthors reflects their involvement in the paper. Cover illustration andblowfly photo p. 7, copyright Mark C. Cassino.

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Contents

INTRODUCTION 5

Speciation 5

The study species 7

GENERAL METHODS 9

Morphological data 9

Morphological integration 10

Genetic data 11

RESULTS AND DISCUSSION 12

Population size and sympatric and parapatric speciation 12

Population size and allopatric speciation 15

Population size and reproduction in blowflies 19

Genetic and morphological variation in wild L. illustris 21

Bottlenecks and morphological and genetic variation in

L. sericata 24

CONCLUSIONS 28

ACKNOWLEDGEMENT 30

REFERENCES 30

SVENSK POPULÄRVETENSKAPLIG SAMMANFATTNING/

SWEDISH SUMMARY 38

5

INTRODUCTION

Why are there so many species? This is a question that has been asked bymankind since the beginning of time. At the same time the preservation ofform, morphologic stasis, is also apparent (Wake et al., 1983; Larson, 1989;Björklund, 1991, 1996; Benton & Pearson, 2001). Some traits differ greatlyeven in closely related species while other traits seem to stay unalteredthrough million of years (Gould & Eldredge, 1977; Turner, 1986). Thisparadox of stasis and yet a biological richness has led to the concepts oflimiting adaptive gene complexes and founder effect which could breakthese complexes and allow for evolution of morphological novelties andeven lead to speciation.

Dobzhansky (1937) coined the term “coadaptation of the gene pool”meaning that the fitness of a gene is dependent on its genetic environment.Because genes are integrated, the adaptive value of a genotype is a propertyof the whole genome rather than of the constituent genes, and hence furtherevolution may require a thorough rebuilding of the genotype. Several linesof evidence suggest that such coadapted gene complexes exist (Cavener &Clegg, 1981; Carson & Templeton, 1984; Service & Rose, 1985; Stephan &Kirby, 1993; Clarke, 1997). Different models (peripatric speciation (Mayr,1954); founder-flush speciation (Carson, 1975); accidental loss of malecourtship behaviour (Kaneshiro, 1976); genetic transilience theory(Templeton, 1980); variance induced peak shifts (Whitlock, 1995)) offeralternative proposals how these complexes may break up and allowevolution to continue. Common for these models, however, are the stress onthe stochastic effects of inbreeding and genetic drift associated with smallpopulation size.

Speciation

Mayr (1942) defines a species as a set of "groups of actually or potentiallyinterbreeding natural populations, which are reproductively isolated fromother such groups". I will use this Biological Species Concept throughoutthe thesis. Allopatric speciation, where new species arise fromgeographically isolated populations of the same ancestral species, is themost widely accepted of all current speciation models (Mayr, 1942, 1963;Lynch, 1989; Coyne, 1992; Rice & Hostert, 1993). It can be divided intovicariance and peripatric (or “peripheral isolates”) speciation (Lynch, 1989)depending on the location of the geographical split and the size of the sub-populations. In vicariance speciation (“the dumbbell model” in Mayr(1982)) a continuous population is split in the centre of its distribution,

6

giving rise to two or more large, isolated sub-populations. With time thesub-populations are thought to evolve reproductive isolation as a by-product of divergent selection pressures and/or genetic drift. In peripatricspeciation (Mayr, 1954) (also commonly known as founder speciation) asmall peripheral portion of the population becomes isolated and mayundergo one or several (Carson, 1975) bottlenecks. Genetic drift caused bylow population size during the bottlenecks together with relaxed selectionpressure under the following flush phase, when the population rapidlyincreases in size, allows the formation of new gene combinations thatwould not have survived in the original population. Reproductive isolationis then believed to evolve either as a by-product of the genetic changes(Mayr, 1954; Carson, 1975; Templeton, 1980) or as a consequence ofrelaxation of mating preferences in bottlenecks (Kaneshiro, 1989).

A more disputable model, although, recently more widely accepted (Via,2001), is sympatric or parapatric speciation, that is, speciation withoutgeographic isolation. Several hypotheses called “divergence-with-gene-flow speciation” (see Rice & Hostert, 1993) assume that populationsbecome genetically isolated because traits for positive assortative matingcoevolve with other traits under disruptive selection. Either selectionagainst interbreeding is thought to build non-random associations betweenthe genes responsible for the traits (linkage disequilibrium), or the traitsrepresents different phenotypes deriving from the same gene (pleiotropy).The theory of “reinforcement” (Dobzhansky, 1940) implies that a feedbackprocess between selection against hybrids and positive assortative matingreinforces reproductive isolation until the interbreeding populations havebecome genetically isolated. More theories of speciation are reviewed inTurelli et al. (2001).

With respect to allopatric speciation, the evolutionary role of populationsize has fuelled a hot debate for the last 70 years. Disciples of Wright(1931) and Mayr (1963) have argued that since small population size isassociated with increased genetic drift and the subsequent breaking up oflimiting coadapted gene complexes, it may allow a population to evolveonto new peaks in the adaptive landscape, which could be inaccessible tolarge populations. Therefore, small population size will facilitate speciation.In contrast, followers of Fisher (1930) have maintained that, since smallpopulations are characterised by low genetic diversity, high risk ofinbreeding depression, and relatively inefficient selection (Robertson,1970), evolutionary changes are more likely to lead to speciation in a largepopulation.

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This thesis concerns the effect of population size on speciation (Paper I andII), the effect of population size on behaviour (Paper III), and the effect ofpopulation size on morphological variation (Paper IV and V).

The study species

Lucilia illustris and Lucilia sericata are two of the many, widelydistributed, shiny, green blowflies in the Lucilia genus (Zumpt, 1965; Dear,1985, Fig. 1). The adults feed on flowering plants, although females alsorequire a protein-source to mature the eggs (Zumpt, 1965; Daniels et al.,1991) and to become sexually receptive (Bartell et al., 1969; Heath, 1985).

Figure 1. A blowfly, Lucilia sp. © M. C. Cassino

Under natural conditions mating is likely to take place in the vicinity of theegg-laying substrate, like in the closely related L. cuprina where malesplace themselves on the vegetation downwind from the egg-laying substrate(Bartell et al., 1969; Kitching, 1977; Barton Browne et al., 1987). Apartfrom visual cues the males also respond to pheromones released by females(Bartell et al., 1969). Overall sex-ratio probably is equal, but close to anegg-laying substrate it is highly female biased and the egg-laying femalescan come in hundreds (Hall et al., 1995). An adult female lays on average200 eggs per batch (Wall, 1993) and can produce a maximum of eight toten batches in her three week long life given benign conditions (Zumpt,1965; Hayes et al., 1998). The median lifespan of wild L. sericata (and

8

presumably also L. illustris) is, however, only 3-6 days (Wall, 1993), andthe high reproductive potential is therefore seldom realised.

The larva requires a moist environment and a food resource. The femalelays her eggs primarily on carcasses, but occasionally also on open woundsor the soiled and wet fleece of sheep, causing a skin disease known asmyiasis (Zumpt, 1965; Hall & Wall, 1995). L. sericata acts as a primaryagent of myiasis, while L. illustris more seldom is attracted to living tissue.The development time in blowflies is highly dependent on temperature(Hanski, 1976b; Greenberg & George, 1985; Singh, 1985). At 25 oC theeggs hatch within 24 hours and the larvae feed on organic matter whilepassing through three larval stages in approximately one week. As theyenter the prepupal stage they leave the feeding substrate and dig into thesoil to pupate. In summer the prepupal period lasts from three days toseveral weeks, depending on temperature, but in autumn the larvae enterdiapause and the prepupae remain inactive until spring (Zumpt, 1965; Ring,1967a-b). With increasing latitude local populations tend to becomeunivoltine (Ring, 1971). The pupal period lasts for about a week at 25 oC,after which the adults emerge.

All Lucilia species are extremely variable in size depending on theconditions and amount of food obtained by the larvae (Coyler, 1951;Hanski, 1976a-b). Prinkkilä and Hanski (1995) showed that larvae competethrough scramble competition and that larval survival is highly densitydependent. Nutritional stress also results in the production of many smalladults instead of a few large ones (Daniels et al., 1991, personalobservation). The two sexes are essentially monomorphic, although femalesare generally larger than males and their eyes are further apart and smallerthan males (Rognes, 1991). The eyes cover a larger part of the head inmales than in females, a pattern that is common in many species of flies(Thornhill & Alcock, 1983) and reflects the males’ strategy of watching outfor passing females.

In all my experiments I used a laboratory population of L. sericata,obtained from a stock culture held at the University of Bristol, UK. I rearedflies in Uppsala at 25 oC and 70% humidity under a 16:8 hour light:darkcycle. Adults received pig liver as a protein source on their third day of lifeand on subsequent occasions as a substrate for egg-laying. Flies had freeaccess to water and sugar. Wild L. illustris flies were collected during threeconsecutive years (1996-1998) from five different localities in Sweden.Three of the localities were approximately 100 km apart, ranging fromUppsala in the south via Gävle to Söderhamn in the north (Fig. 2). Two

9

localities, only one kilometre apart, were sampled in Uppsala (Zootis andPolack). Flies were collected repeatedly during June to August using funneltraps baited with pig liver.

Figure 2. Number of individuals analysed from five localities: Söderhamn (S), Gävle (G),Zootis (Z) and Polack (Po), during three consecutive years: 1996-98. Numbers given arewings followed by microsatellites.

GENERAL METHODS

Morphological data

For the analysis presented in paper IV and V wings from dead flies wereremoved and mounted between microscopic slides and photographed. Xand Y coordinates for 14 morphological landmarks (Fig. 3) were scored.All landmarks are at the intersection of wing veins, except No. 7, which isat the maximum curvature of the media wing vein. To assess measurementerror a subsample of wings were measured twice.

Size was measured as centroid size, the square root of the sum of squareddifferences from a set of landmarks to their centroid (Bookstein, 1991). Toseparate shape from size, the X and Y coordinates were transformed intoBookstein Coordinates (Bookstein, 1991; Dryden & Mardia, 1998) byscaling them to the baseline size (distance between landmarks No. 10 andNo. 11, Fig. 3). The resulting 24 variables (X and Y coordinates) can beused as independent shape variables in ordinary multivariate statistics(Bookstein, 1991).

G96: 23,0 G98: 22,22

S96: 23,24 S97: 25,25S98: 7,7

Z96: 7,11 Z97: 19,20 Z98: 24,25

Po97: 14,14

Uppsala

Gävle

Söderhamn

50 km

10

Figure 3. Wing of Lucilia illustris with the 14 landmarks shown.

Morphological integration

Morphological integration is “the summation of the totality of characters,which, in their interdependency of form, produce an organism” (Olson &Miller, 1958). The degree of interdependency of morphologicalcomponents in development and function will be directly related to theirmorphological integration. The dependence between landmarks withinwings were studied in two ways. First, by comparing the observed to theexpected variance of eigenvalues in correlation matrices (Cheverud et al.,1989), it is possible to test whether or not the wings are integrated and tocompare the strength of integration between different populations. Adescriptive measure of morphological integration is the effective number oflandmarks, Meff, i.e. the total number of independent effective characters ina multivariate data set (Cheverud, 2000). Second, by comparing covariancematrices in a Flury analysis (Phillips & Arnold, 1999) it is possible to testwhich model best describes the relationship between different phenotypicvariance-covariance matrices ranging from totally unrelated, via differentnumbers of common principal components and proportional principalcomponents to identical matrices.

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Genetic data

Genetic variation was studied with the use of microsatellites. Paper VIdescribes the isolation and characterisation of the 13 loci used. DNA wasobtained from thoracic muscle tissue using Chelex extraction (Walsh et al.,1991). Primers were marked with fluorescent dye, and after multiplex PCRthe size of fragments were determined using ABI prism 310 geneticanalyzer. Six loci were screened for L. illustris and nine for L. sericata.

Three different measurements of population genetic variation were obtainedfrom the microsatellite data: average heterozygosity, Ho, Fisher’sinbreeding coefficient, F, and θ. The latter is a function of effectivepopulation size and mutation rate (Nielsen, 1997). For a measurement ofindividual genetic variation I used the mean d2, which is the squareddifference in repeat units between alleles at a microsatellite locus averagedover all loci at which an individual is typed (Coulson et al., 1998). This is amore informative measurement of degree of inbreeding than averageheterozygosity since it also takes the distance between alleles into accountand a larger difference in number of repeats reflects a higher degree ofancestral outbreeding (Pemberton et al., 1999).

Microsatellites can be very helpful in detecting the occurrence ofbottlenecks. When a population is reduced in size the allelic diversity isreduced faster than heterozygosity (Nei et al., 1975). This means that theobserved number of alleles in a sample from a bottlenecked population isless than the number of alleles expected from the observed heterozygosityassuming mutation drift equilibrium (Cornuet & Luikart, 1996) or, in otherwords, the population exhibits a heterozygosity excess. The test statistic T2

(Cornuet & Luikart, 1996) is used to determine whether heterozygosityexcess, characteristic of a bottleneck, is statistically significant.

Bottlenecks can also be detected using M, the mean ratio of the number ofalleles (k) to the range in allele size (r) (Garza & Williamson, 2001). Areduction in population size will lead to loss of alleles, but because the lossof any allele will lead to a reduction in k, but only the loss of the largest orsmallest allele will result in a reduction in r, the latter will be reduced moreslowly. This means that the ratio M will be smaller in recently bottleneckedpopulations than in equilibrium populations.

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RESULTS AND DISCUSSION

Population size and sympatric and parapatric speciation

Speciation in sympatry or parapatry is an intriguing possibility, which hasattracted a great deal of scientific attention over the last 50 years. Importantcriticism against the sympatric and parapatric speciation models comesfrom experimental evidence (Scharloo, 1971; Rice & Hostert, 1993).Positive assortative mating is a key ingredient in a speciation process(Kirkpatrick & Servedio, 1998) and is especially important in sympatricand parapatric speciation. However, positive assortative mating can lead toinbreeding and hence to inbreeding depression, in which case naturalselection will favour outbreeding. The rate of inbreeding (∆F) will behigher the smaller the population, as described by ∆F = 1/2Ne (Wright,1931), hence population size can have a profound effect on the outcome ofsuch experiments. In paper I we review the published records of sympatricand parapatric speciation experiments to test the relative importance ofselection intensity applied (i), duration of experiment (ttot), and effectivepopulation size (Ne).

The basic design in these experiments consisted of two sub-populations thatwere allowed to interbreed to different degrees. A selective penalty wasthen applied on hybridisation, either directly or indirectly. To quantitativelydetermine to what extent a certain experiment should be consideredsuccessful, we calculated the hybrid frequency change, ∆H, as thedifference in percentage of hybrids between the beginning (H0) and the end(H1) of the experiment relative to the total percentage of hybrids in thebeginning:

A negative value of ∆H means that the frequency of hybrids has declinedduring the experiment, and indicate that reproductive isolation hasbeginning to evolve. Successful experiments, i.e. those resulting insignificant positive assortative mating, on average comprised a largereffective population size (Mann-Whitney U-test: n = 63, P < 0.01), andthere was a negative correlation between ∆H and Ne (Spearman’s rankcorrelation: n = 63, ρ = -0.37, P < 0.01). One explanation for the influenceof the population size could be that as selection proceeds genetic variationbecomes depleted more rapidly in smaller populations. An alternativepossibility is that development of inbreeding depression prevents theevolution of reproductive isolation. Selection against outbreeding must be

0

01*100H

HHH

−=∆

13

stronger than the selection against inbreeding that will develop in all finitepopulations for any positive assortative mating to evolve. In most cases, theexperimentally applied selection against outbreeding was kept at a constantlevel over time. In contrast, natural selection against inbreeding willcontinually increase as more and more detrimental alleles become fixed.Should the two selection intensities come to balance each other, neithertraits for inbreeding nor traits for outbreeding will be favoured, and thespeciation process will grind to a halt.

There was a positive correlation between i and ∆H among smallexperiments, Ne<107 (Spearman’s rank correlation: n = 39, ρ = 0.33, P <0.05) and a negative correlation among large experiments (Spearman’s rankcorrelation: n = 20, ρ = - 0.50, P < 0.05; Fig. 4). Intense selection willproduce an earlier but smaller response than will weak selection (Robertson1970 b) as a result of the depletion of genetic variation, which will be moresignificant in smaller populations. Furthermore, in a smaller population,inbreeding depression is more likely to arise rapidly enough for selectionagainst inbreeding to overwhelm the applied selection. Therefore, we willexpect to see a negative correlation between hybrid frequency change andselection intensity only among large experiments and a positive rather thana negative correlation among small experiments. This is the pattern wefound on either side of Ne = 107 (Fig. 4).

The duration of the experiments was highly positively correlated witheffective population size and to determine which had the highest impact onthe outcome of an experiment we performed a partial correlation test usingSpearman’s ρ. Keeping ttot constant resulted in a significant negative effectof Ne on ∆H (n = 63, ρ = - 0.27, P < 0.05) while keeping Ne constantshowed that the duration of the experiment per se had no effect on ∆H (n =63, ρ = - 0.15, P > 0.05).

We classified the experiments from C to E, according to the notation inRice & Hostert (1993). C = divergent selection with hybrid inviability; D =divergent selection with hybrid viability; E = divergent selection withhybrid viability in which assortative mating was intended to evolve as apleiotropic effect of selection on other traits, for example breeding-habitatchoice. The experiment types differed in hybrid frequency change, ∆H(Kruskal-Wallis: n = 63, χ2 = 29.0, P < 0.0001) but also in Ne (Kruskal-Wallis: n = 63, χ2 = 15.9, P < 0.001) with the E-group containing both thelargest and, together with the C-group, the most successful experiments(Fig. 5).

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Large

Small

i

DH

-120

-80

-40

0

40

80

120

160

200

0,0 0,4 0,8 1,2 1,6 2,0 2,4 2,8

Figure 4. Relationship between hybrid frequency change (∆H) and selection intensity (i)in laboratory sympatric and allopatric speciation experiments with effective populationsize larger or smaller than 107.

Experiment type

-50

0

50

100

150

200

250

300

350

c d e

Figure 5. Difference in effective population size (squares) and the negative sign of hybridfrequency change (triangles) between experiments of different types: C, divergent selectionwith hybrid inviability(n = 19); D, divergent selection with hybrid viability (n = 32); E,divergent selection with hybrid viability and pleiotropy (n = 12). Medians are showntogether with upper and lower quartiles.

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Sympatric and parapatric speciation hypotheses have been widely criticisedand seem to require a narrow set of conditions. From the invariable successof E-type experiments Rice and Hostert (1993) concluded that strong,discontinuous, divergent selection, acting on multiple traits, with apleiotropic effect on assortative mating is required for reproductiveisolation to arise in sympatry or parapatry. However, it is necessary to takegreat care when interpreting such experimental results as these, becauseexperiments are highly susceptible to inadequacies in design. The successof the E-experiments could be explained at least as well by these particularexperiments having the largest population sizes.

In conclusion, our analysis does not support the sympatric and parapatricspeciation models, but reveals that the experimental evidence frequentlyused against them is not as strong as previously believed.

Population size and allopatric speciation

The vicariance model of allopatric speciation has been repeatedlyconfirmed empirically, while peripatric speciation has suffered severecriticism for being both implausible and empirically unsupported (Barton &Charlesworth, 1984; Barton, 1989, 1996; Coyne, 1992, 1994; Rice &Hostert, 1993; Moya et al., 1995; Rundle et al., 1998; Mooers et al., 1999).The published records on laboratory experiments on speciation haveyielded equivocal results (Rice & Hostert, 1993; Ödeen & Florin, 2000),implying that there are certain conditions required for speciation but notfulfilled in some of these studies.

In paper II we review the published records of allopatric speciationexperiments in the light of the BSC, using meta-analytic tools. Our purposewas to determine 1) the true effect size of the reproductive isolation thatexperimenters have achieved, 2) whether or not the isolation is permanent,and 3) the importance of different factors for the evolution of reproductiveisolation. Apart from effective population size and bottleneck size, thenumber of bottlenecks (founder-flush-crash cycles: Rundle et al., 1998), thenumber of generations that sister populations are kept in isolation (Hostert,1997; Mooers et al., 1999), and selection intensity (Schluter, 1996) arepotentially important to speciation. Peripatric speciation can be comparedto a lottery with great winnings (speciation) but also very high stakes(extinction). Each new bottleneck is then another ticket in the speciationlottery. Time should have a positive effect on the accumulation of genecombinations that could induce reproductive isolation. The review by Riceand Hostert (1993) of laboratory speciation experiments stresses the

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importance of multifarious divergent selection, as opposed to parallel or noselection, as a promoter of reproductive isolation. Likewise, in our reviewof sympatric and parapatric speciation experiments (Ödeen & Florin, 2000),selection intensity was found to have a positive effect on reproductiveisolation, but only in large populations.

We surveyed the literature from the 1950’s to the present day for laboratoryexperiments on allopatric speciation and found 25 papers, most of whichare discussed in Rice and Hostert (1993). The basic design in theseexperiments consists of spatially isolated populations of Drosophila andMusca flies, taken from the same, often outbred, stock. After a number ofgenerations in isolation, mating tests are performed to assess the amount ofreproductive isolation achieved between experimental populations or to theoriginal stock. First, we separated the experiments according to the twotypes of isolation tests made: Derived — two derived populations testedagainst each other, Origin — a derived population tested against theoriginal, unselected, unbottlenecked stock. Second, we divided theexperiments according to the allopatric speciation model tested: Vicarianceor Peripatric. Third, we divided these groups with respect to selectionregime: experimental populations where no selection, or parallel selection,was applied were classified as Drift, and experiments with divergentselection, for example for high and low bristle number, were classified asSelected. This subdivision resulted in four well-defined categories:Vicariance-Drift, Vicariance-Selected, Peripatric-Drift, and Peripatric-Selected. As a measurement of isolation Bishop’s isolation index Y wasused (Bishop et al., 1975). Y can assume values between –1 (negativeassortative mating) and 1 (positive assortative mating). A value of zeromeans that mating is random.

This meta-analysis shows, like many studies before it (Rice & Hostert,1993; Moya et al., 1995; Rundle et al., 1998; Mooers et al., 1999), thatperipatric speciation through founder-flush events does not occur underlaboratory conditions. More surprisingly, in paper II we were able to showthat experiments of the more widely accepted vicariance model, with orwithout selection, have also failed to produce significant reproductiveisolation against a control population. With the exception of vicariancespeciation experiments where derived populations were tested forreproductive isolation against each other, laboratory allopatric speciationexperiments thus have had little if any effect on reproductive isolation. Themeans weighted by sample size were not significantly different from thenull hypothesis Y = 0, except in Derived tested Vicariance experiments (t =3.50, d.f. = 36, P = 0.001, Fig 6a and b).

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PeripatricVicariance

Fig 6a

Sample size

Y

-0.3

-0.1

0.1

0.3

0.5

0.7

0.9

10 50 100 500

PeripatricVicariance

Fig 6b

Sample size

Y

-0.5

-0.3

-0.1

0.1

0.3

0.5

0.7

10 50 100 500

Figure 6. Reproductive isolation (Y) in published laboratory allopatric speciationexperiments with different number of matings observed (Sample size) to determine Y inmating tests between a) two derived populations (Derived) and b) one derived populationand the original population (Origin). Peripatric speciation experiments (Peripatric) areshown with filled symbols and vicariance speciation experiments (Vicariance) with opensymbols. The solid line denotes the null-hypothesis, namely random mating. The dashedline shows the weighted mean of Y for Vicariance and the dotted line for Peripatricexperiments. In b), weighted mean Y for Vicariance and Peripatric experiments are tooclose to Y = 0 to be shown.

The failure of the allopatric experiments manifests itself further in the poorconsistency of reproductive isolation. Isolation at the end of the experiment(Y) correlated with isolation in the penultimate mating test (Yp) only in

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Derived types of tests (Derived: r = 0.63, n = 18, P = 0.005; Origin: r =0.20, n = 21, P = 0.40).

There was no general effect of Ne on the likelihood of laboratory allopatricspeciation in this study (Derived: r = -0,084, n = 43, P = 0.59; Origin: r =0.12, n = 168, P = 0.13), however, in one of the tested experiment typesthere was a positive correlation with Y (Origin: rVicariance-Selected = 0.35, n =35, P = 0.04). This gives some support for the vicariance model, while theperipatric model remains unsupported—not the least since neither thebottleneck size nor the number of bottlenecks affected the amount ofreproductive isolation achieved . In Derived tested Peripatric experimentsneither the number of bottlenecks nor the length of the experiment wascorrelated with Y (number of cycles: rpartial = -0.004, n = 12, P = 0.99;duration of experiment: rpartial = 0.58, n = 14, P = 0.12). In Origin, on thecontrary, the length of the experiment was positively correlated to thedevelopment of reproductive isolation while the number of bottlenecks hadno effect per se (duration of experiment: rpartial = 0.42, n = 118, P = 0.002;number of cycles: rpartial = -0.13, n = 116, P = 0.33). The size of bottleneckshad no effect on the development of reproductive isolation (N = 2-32)(Derived: r = -0.16, n = 14, P = 0.58; Origin: r = -0.02, n = 116, P = 0.79).Among Vicariance experiments there was a positive correlation between Yand length of experiment in Origin tests but a non-significant negative trendin Derived tests (Origin: r = 0.31, n = 52, P = 0.02; Derived: r = -0.27, n =37, P = 0.10). This hints that speciation might have occurred given enoughtime. The experiments might simply have been too short (cf. Hostert, 1997;Mooers et al., 1999).

In Derived tested experiments Y differed between subgroups; Selectedselection regimes produced higher Y than did Drift and Vicarianceexperiments were more successful than Peripatric (F2, 48 = 3.74, P = 0.03,Fig. 7), but no such differences could be found in Origin tested experiments(F3, 165 = 0.48, P = 0.70). Like Rice & Hostert (1993), we found thatdivergently selected vicariance experiment (Vicariance-Selected) have beenmore successful than simple drift and peripatric experiments. However, thiswas true only in mating tests where two experimentally derived populationshad been tested against each other (Derived tests), suggesting thatreproductive isolation more easily evolves between two daughterpopulations than between a mother and daughter population.

The result from paper II implicates some suggestions for future design ofspeciation experiment: first, to discern true speciation from randomfluctuations in mating behaviour, it is necessary to use large sample sizes in

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mating tests and to test reproductive isolation in more than one generation;second, the experiments must be run for long enough; third, strongdivergent selection should be applied on large populations.

Y

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

Peri-Dri Vic-Dri Vic-Sel

Figure 7. Reproductive isolation (Y) between derived populations in experiments testingdifferent speciation models: vicariance speciation under divergent selection (Vic-Sel),peripatric speciation under drift (Peri-Dri), and vicariance speciation under drift (Vic-Dri).Means are shown together with standard errors (boxes) and standard deviation (whiskers).

Population size and Reproduction in blowflies

Aggregations of conspecific individuals often occur in nature, and the sizeand composition of the group can have a profound effect on the behaviourof the individuals comprising it. In paper III I have conducted fourexperiments on the effect of group size and sex ratio on reproduction in theblowfly, Lucilia sericata. In particular I focused on the following questions:Is there a critical minimum group size for reproduction? Do higher densitiesincrease reproduction? Does the sex ratio affect the egg-laying propensityof females?

In the first experiment I varied the number of blowflies and discovered apositive effect of group size. There was a clear difference in the egg-layingpropensity between treatments with differing numbers of flies (Cochran Qtest 1-5 pairs, Q = 17.8, df = 4, P = 0.0013; 1-20 pairs, Q = 13.1, df = 5, P =0.023). At the lowest density (1-pair) the egg-laying frequency was muchlower than at higher (3-5 pairs) fly densities and also lower than expected ifall female had the same egg-laying propensity as the control females from

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large base population (Table 1). In experiment 2 I used a constant numberof flies (six) but different sized cages to create different densities of flies.There were no differences in egg-laying frequency at the different fly-densities (Egg-laying propensity: low density, 1.0; medium density, 0.90;high density, 0.90; Cochran Q test: Q = 1.0, df = 2, P = 0.61). In experiment3 I had a constant number of flies (six) but varied the sex-ratio. Egg-layingdiffered significantly across the various sex ratios (Cochran Q test: Q=13.3,df = 4, P = 0.01). As the sex ratio became more male-biased the egg-layingfrequency increased, except for the most male-biased ratio (5:1), where theegg-laying frequency was extremely low (Table 1). To determine if the lowegg-laying propensity among single females was due to male harassment Iconducted a fourth experiment in which I kept the sex-ratio equally biasedbut varied the number of flies. The egg-laying frequencies of females in the5:1 treatment were significantly lower than expected (Binomialtest: 0.20compared to 0.75; P = 0.0004) and significantly lower compared to that ofthe 10:2 treatment groups (5:1, 0.20, 95% C.I. 0.067-0.56; 10:2, 0.9, 95%C.I. 0.69-1.0).

Table 1. The egg-laying propensity in treatments with different number of pairs of flies inexperiment 1 and in treatments with different sex ratios (Males:Females) in experiment 3.The expected frequencies are calculated using an assumption of binomial distribution(n=10 in all cases except 14-20 pair where n=5), and an individual female egg-layingpropensity of 0.75. The P-values are from an binomialtest of observed versus expectedfrequencies. The last column is a 95% confidence interval of the observed values.

Treatment Observed freq. Expected freq. P-values 95 % C.I.1-pair 0.10 0.75 0.000030 0.025-0.442-pair 0.40 0.94 0.000010 0.19-0.743-pair 0.90 0.98 0.14 0.69-1.04-pair 0.80 0.996 0.00067 0.55-0.975-pair 0.70 0.999 1.1 * 10-7 0.44-0.9314-20pair 1.00 1 1 -1:5 0.20 0.999 3.7*10-23 0.067-0.562:4 0.50 0.996 2.2*10-10 0.26-0.813:3 0.70 0.98 0.00042 0.44-0.934:2 0.80 0.94 0.12 0.55-0.975:1 0.10 0.75 0.000030 0.025-0.44

These experiments show that there is a positive effect of group size that isdue to the number of flies and not the density of flies. The number of maleshas the greatest influence; the more males a female has access too, thehigher is her egg-laying propensity. However, this is only true as long asthe female is in the company of at least one other female.

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The positive effects of increasing numbers of males can have many possiblecauses; females may gain direct or indirect benefits through multiplematings (Yasui, 1998; Newcomer et al., 1999; Arnqvist & Nilsson, 2000) orfrom mate choice (Bateson, 1983), or a combination of these (Eberhard,1996). If the low frequency of remating is true also for L. sericata, assuggested by Pollock (1971), given the low life expectancy of a blowfly(Wall, 1993), most females probably mate only once. Thus spermcompetition is unlikely, instead suggesting the importance of direct femalechoice.

My experiments have shown that successful reproduction requires morethan one male to be present. This suggests that females may use a best-of-n-males strategy (Gibson & Langen, 1996) but with a fixed threshold numberof males (n) to assure her of good male quality. The inflexibility of thisstrategy, which in the experiment imposes a drastic fitness cost, probably isadaptive in nature if males normally occur in great numbers in places easilylocated by the females, as in the vicinity of a carrion. In that case femalescan benefit from this behaviour since it encourages male competition andimproves the female’s chance of mating with a superior male.

Although larval density has been found to adversely affect survival andadult size (Hanski, 1976a-b; Prinkkilä & Hanski, 1995; Prinkkilä, 1996),blowflies often lay eggs on the same spot as previously used by otherfemales (Barton Browne et al., 1969; Hanski, 1987a-b). Moreover, an intra-specific aggregation at carcasses has been found in blowfly communities(Kouki & Hanski, 1995). This could be understood if larvae do better ifthey exceed a certain number, preferably of the same species (cf. groupfeeding Hanski, 1990). It is important to be among the first to lay eggs on anew carcass, thus granting the offspring a head start in the fierce scramblecompetition (Hanski, 1976a; Hanski & Kuusela, 1977). This means that thebenefits of postponing egg-laying until another female arrives must besubstantial. If females obtain a great fitness gain by laying eggs on acarcass together with one or more other females, it could pay to wait untilanother female arrives and then race to be the first to lay.

Genetic and morphological variation in wild L. illustris

According to models of founder effect speciation and Variance InducedPeak Shifts the stochastic effects of bottlenecks and inbreeding could breakup limiting coadapted gene complexes and allow evolutionary morphologicnovelties to develop. In paper IV the genetic variation (using microsatellite

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markers) and morphologic variation, size and shape of wings, werecompared in wild populations of Lucilia illustris in Sweden.

The distribution of carcasses is both patchy and highly unpredictable intime and space. This makes it probable that sometimes the offspring of onlya few females can monopolise an entire carcass and thus represent the nextgeneration of blowflies in that area. The genetic consequences of this lifestyle is dependent on the distribution of resources in relation to dispersalability. On one hand, if migration between patches is substantial thepopulation could be considered essentially panmictic, with no localdifferentiation, but due to the high stochasticity of the resource, in bothtime and space, the risk of the entire population going through a bottleneckcould be considerable. On the other hand, if dispersal is insufficient, therewill be local differentiation and high levels of inbreeding in a highlystructured system, perhaps comparable to a meta-population. The firstscenario predicts signs of bottlenecks in the genetic pattern of blowflieswhile the second scenario suggests high levels of inbreeding. Regardless ofwhich scenario is true, the colonisation of an ephemeral habitat, a foundingevent, could be argued to act to break up gene complexes limitingmorphologic evolution. If this is happening there should be multivariatephenotypic differences among populations of this fly and a negativerelationship between the amount of genetic variation and morphologicalintegration of wings.

No indications of prior bottlenecks were detected in any of the samples(Wilcoxon test of heterozygote excess in T2, p > 0.5; Randomisation test ofM: p>0.28 in all cases, Tab. 2). This can mean one of three things. First,none of the populations may have gone through bottlenecks or, second,tests have too low power to detect bottlenecks even if they exist. Third, thepopulations may have gone through repeated number of bottlenecks andhence become highly inbred, but these bottlenecks will not be detected inthe traditional tests since they presume that the population is outbreedingand panmictic before the bottleneck.

Wings were highly integrated structures, and the effective number oflandmarks was reduced from eleven to between seven and eight (Tab.3).The strength of the integration did not differ between populations. Thelarge number of shared principal components (eight of eleven, Fluryanalysis) of phenotypic covariance matrices of wing shape for flies fromdifferent places and different years underlines that wings are a highlyintegrated structure in Lucilia illustris. This also seems to be the case inother insects (bumblebees: Klingenberg et al., 2001; fruitflies: Klingenberg

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& Zaklan, 2000; Guerra et al., 1997; Pezzoli et al., 1997). There was nodifference in wing size between places within years or between years(Nested Anova, places within years: F4,121 = 1.55, p = 0.19; between years:F2,121 = 0.56, p = 0.57). The difference between places was almostsignificant and year turned out to have a slight effect on wing shape (NestedAnova, Wilks test, places within years: F88,484 = 1.28, p = 0.05; betweenyears F44,244 = 1.45, p = 0.04). The limited variation in wing shape could bedue to stabilising selection on wings which may typify traits whose functionis hampered by even slight deviations. It is also possible that opportunisticspecies, living on ephemeral resources, have become adapted to cope withinbreeding and bottlenecks and hence are less prone to changemorphologically due to these factors as compared to species with a greatdegree of panmixis.

Table 2. Genetic variation represented by observed heterozygosity (H0), Fisher’scoefficient of inbreeding (F), the outbreeding coefficient (d2 ) and a function of effectivepopulation size (θ) in wild populations of Lucilia illustris. The last two columns are testvariables from bottleneck tests, where a value of M much lower than 1 and a high positivevalue of T2 would indicate a bottleneck.Population n H0 F θ Mean d2 Μ Τ2

S96 24 0.29 0.46* 2.21 1.41 1 -1.28Z96 11 0.34 0.44# 3.72 0.87 0.87 -0.67S97 25 0.60 0.21 3.09 1.76 0.93 -1.43Z97 20 0.32 0.31 3.08 1.41 0.86 -1.86*Po97 14 0.34 0.16 3.60 1.52 0.89 -1.49S98 7 0.32 0.49# 3.42 1.16 0.90 0.54Z98 25 0.42 0.38* 6.13 0.72 0.91 -1.46G98 22 0.26 0.56** 2.45 1.69 0.92 -0.29F and Τ2 sign. diff. from 0: *P<0.05, ** P<0.01, # could not be tested due to low samplesize

Table 3. Morphological integration of eleven wing landmarks in different populations.Observed (Vo) and expected (Ve) variance of eigenvalues of correlation matrices withconfidence interval of the observed values given and effective number of landmarks (Meff).Pop n Vo Ve 95 % C. I. Meff

S96 17 4.12 0.59 2.59-6.06 7.2G96 11 2.75 0.91 2.31-4.45 8.5S97 18 3.05 0.56 2.36-4.18 8.2Z97 13 4.46 0.77 3.66-6.5 6.9Po97 11 3.36 0.91 2.62-5.40 7.9Z98 14 3.40 0.71 2.59-5.27 7.9G98 15 4.47 0.67 2.99-6.53 6.93

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The only detectable effect of inbreeding on morphology was the positivecorrelation between heterozygosity and effective number of landmarks (rs =0.81, n = 6, p = 0.05), suggesting that, if anything, inbreeding strengthensthe morphological integration and hence reduces the probability of peakshifts.

Place or year did not have any effect on mean d2 (Nested Anova: placeswithin years, F5,113 = 1.01, p = 0.42; between years, F2,113 = 0.96, p = 0.39).In the total sample, inbreeding was very high and genetic differentiationlow (FIS = 0.37; FST = 0.05), but there was a significant difference ingenotypic distribution among populations (G-like test, df = 12, χ2 = infinity,p<0.0001). Interestingly, the value of F is strikingly high in all populations(Tab. 2), suggesting that inbreeding occurs frequently in Lucilia illustris.The values of θ, however, indicate that the effective population sizes arenot low. Assuming a mutation rate between 1*10-3 and 9*10-6 (range ofmutation rates in insect microsatellites (Ellegren, 2000)) gives a valuebetween 500 and 4 500 000 individuals for the population with the smallestvalue of θ (S96). The estimated frequency of potential null alleles, whichcan inflate the estimate of F, was quite high for half of the loci (0.27-0.39)but also after removing these loci from the estimation of F substantialvalues of F will still result (above 0.20 in four populations). A thirdpossible reason for the high values of F could be that samples from separatepopulations in fact consist of samples pooled from several distinctpopulations, suggesting a fine-grained genetic structuring of L. illustris.

In conclusion, my data show no evidence for bottlenecks in the past, whilethe frequency of inbreeding that does exist seems to be insufficient to causeany VIPS in this species. Stabilising selection on wing shape probablyexplains the narrow morphological variation.

Bottlenecks and morphological and genetic variation in L. sericata

The disruptive effect of founder events, bottlenecks and inbreeding on thegenetic system could have an effect on morphological differentiation. Thus,there is some evidence suggesting that bottlenecks can inducemorphological changes (Jablonski et al., 1983; Templeton, 1986; Bryant &Meffert, 1988, 1990, 1991, 1992, 1996a-b, 1998; Meffert, 2000). In paperV I subjected a laboratory stock population of Lucilia sericata to artificialbottlenecks. In experiment 1, experimental populations were subjected to asingle founder-flush event and monitored for 13 subsequent generations,while in experiment 2 populations were subjected to repeated bottlenecks.The traits under focus are the same as in paper IV, i.e. shape and size of

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wings and microsatellite variation. If bottlenecks facilitate the evolution ofmorphologic variation, morphological integration should be lower inbottlenecked populations, while variation in size and shape should behigher. Furthermore, genetic variation should be negatively correlated tomorphological integration of wings. Since bottlenecks and the concomitantinbreeding can result in inbreeding depression and lowered fitness(Saccheri et al., 2001), I also investigated the effects of these factors on afitness related character, viz. the proportion of pupae hatching.

The nine landmarks in the wings were morphologically strongly integrated,and the number of effective landmarks was reduced from nine to betweenthree and six in experiment 1 and between four and six in experiment 2. Nogeneral difference in integration between bottlenecked and controlpopulations was observed. The number of bottlenecks had no effect on theeffective number of landmarks (rs = 0.082, n = 11, p = 0.81). This suggeststhat bottlenecks do not reduce overall morphological integration and hencethey do not increase the probability of evolutionary change. The pattern ofphenotypic correlation, as detected in covariance matrices, however, wasfound to change over time as well as differ between different populations.In experiment 1, one generation after the bottleneck, two of thebottlenecked populations turned out to have four principal components incommon with the control population while two other bottleneckedpopulations shared only one principal component with the control. Threegenerations after the bottleneck, two of the tested populations shared allprinciple components with the control, while one bottlenecked populationshared only three or four principal components with the control and nonewith the rest of the bottlenecked populations. Finally, at the end ofexperiment 1, all remaining populations had all principal components incommon. This suggests that the effects of bottlenecks had been eroded overtime as populations had become more similar to the control. In experiment2, all experimental populations shared all nine principal components withthe controls, except the twice bottlenecked population 3A2 which wasdifferent from both the controls (3 common principal components) and theother repeatedly bottlenecked populations (2 common principalcomponents). This shows that bottlenecks do not necessarily lead tochanges in morphological integration although they probably have thepotential to do so.

Wing size in control populations varied over time in experiment 1(One-Way Anova: F3,49 = 24.61, p < 0.0001) and was smallest at the middle ofthe experiment. When comparing wing size in bottlenecked populations andcontrol populations sampled at the same time it was evident that differences

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between populations were present during the whole experiment (One-WayAnova, 1 generation after bottleneck: F5,46 = 3.25, p = 0.014; 3 generationsafter bottleneck: F4,38 = 6.07, p = 0.0007; end of experiment: F2,29 = 19.59,p < 0.0001). There was however no consistent differences in size betweenbottlenecked and control populations.

The wing shape in the control populations changed over time in experiment1(One-way Anova, Wilks test: F54,242 = 5.66, p < 0.0001). It also differedbetween bottlenecked and control populations measured at the same time(One-way Anova, Wilks test: 1 generation after bottleneck: F90,300 = 4.98, p< 0.0001; 3 generations after bottleneck: F72,214 = 4.86, p < 0.0001; end ofexperiment: F36,28 = 2.42, p = 0.009). While all populations were differentfrom each other at one and three generations after the bottleneck, only twoof the remaining three populations were different from each other at the endof the experiment, once again pointing to the transient effect of abottleneck. In experiment 2, the once bottlenecked populations haddifferent overall shape compared to control populations (One-Way Anova,Wilks test: F18,117 = 2.69, p = 0.0008), and a closer inspection of pairwiserelationship revealed that all bottlenecked populations were different fromeach other but the control populations did not differ from each other. Thesame pattern was found in repeatedly bottlenecked populations, whereexperimental populations differed from control populations (One-WayAnova, Wilks test: F36,178 = 5.73, p < 0.0001) and populations differed fromall others except for the control populations (Fig. 8). This suggests thatbottlenecks do have a potential to cause differentiation in wing shapeamong L. sericata, although changes probably need to be positivelyselected to achieve permanency, otherwise they will drift back to “normal”again.

A comparison of control and bottlenecked populations revealed that geneticvariation declined markedly during the course of the experiment eventhough bottlenecks per se had no effect (Main-effect Anova, Time, F3,263 =32.2, p<0.0001; Bottlenecks, F1,263 = 0.56, p = 0.45, Fig. 9). This result wasconfirmed in experiment 2 where number of bottlenecks were neithercorrelated to inbreeding (n = 11, rs = 0.29, p = 0.39) nor to heterozygosity(n = 11, rs = -0.24, p = 0.48). The lack of a general difference in geneticvariation between control and bottlenecked populations in both experimentssuggests that bottlenecks do not always lead to loss of heterozygosity.

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2A2

3A2

1B3

3B3

K1

K2

Root 1

Root 2

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

7

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5

Figure 8. Wing shape in repeatedly bottlenecked (2A2, 3A2, 1B3 and 3B3) and control(K1 and K2) populations of L. sericata in experiment 2. Axes represent roots fromdiscriminant analysis.

Time

mean

d2

0

1.0

2.0

3.0

4.0

5.0

0 1 3 14

Figure 9. Genetic variation, expressed as mean d2, as a function of time in pooled L.sericata populations from bottleneck experiment 1. Means together with standard errorsshown.

There was no correlation between the strength of morphological integrationin wing shape and any of the estimates of genetic variation in neither of theexperiments (Exp1: F: n=14, rs = 0.28, p = 0.33; Ho: n = 14, rs = -0.098, p =

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0.74; θ: n=13, rs = -0.08, p = 0.98; Exp 2: F: n=11, rs = 0.086, p = 0.80; Ho:n = 11, rs = 0.066, p = 0.85). This suggests that inbreeding does not removethe genetic restrictions for evolution of morphological novelties.

Hatching success was high throughout and in experiment 1 no correlationwas observed between hatching success and F (rs = -0.27, n = 15, p = 0.34)or Ho (rs = 0.38, n = 15, p = 0.16) or mean d2 (rs = 0.29, n = 15, p = 0.29)but a significant positive correlation with θ (rs = 0.57, n = 14, p = 0.03).Nor in experiment 2 could any detrimental effects of inbreeding or lack ofheterozygosity and outbreeding on hatching success be detected (n = 11,Ho: rs = 0.055, p = 0.87; F: rs = -0.24, p = 0.48, mean d2: rs = -0.23, p =0.50). Yet, half of the bottlenecked populations went extinct during thecourse of the experiment. This strongly suggests that bottlenecks andinbreeding elevate the risk of extinction. The lack of correlation betweengenetic variation and fitness in paper V may be a result of most detrimentaleffect on fitness already having been purged from the genome at the time ofsampling (Bryant & Meffert, 1991; Saccheri et al., 1996; Miller & Hedrick,2001).

CONCLUSIONS

Our results suggest that population size can affect the likelihood ofspeciation. Previous laboratory experiments designed to elucidate themechanisms of sympatric and parapatric speciation in many cases may havebeen handicapped by too small population sizes. Reduced interbreeding andreproductive isolation was less likely to develop in small populations wherethe selection process often seemed to have been opposed by inbreedingdepression or loss of genetic variation. This means that, according to ourresults, the experimental evidence frequently used as an argument againstsympatric and parapatric speciation models is not as strong as previouslybelieved. On the contrary, there was no general effect of population size onthe amount of positive assortative mating achieved in published allopatricspeciation experiments. Moreover, our meta-analysis showed no support forspeciation through founder events (peripatric speciation) in laboratoryexperiments. In fact, the only experiments leading to significant positiveassortative mating were the simulations of vicariance speciation which hadbeen tested against other derived populations. However, none of thereviewed experiments, regardless of population size or selection regime,has succeeded in generating pre-mating isolation towards a controlpopulation. This shows that the method of testing is at least as important asthe speciation model.

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Population size and composition were found to have an important influenceon reproductive behaviour. In Lucilia sericata the number and sex of thesurrounding individuals had a profound effect on egg-laying. There was apositive effect of increasing number of males but only as long as the femalewas in the company of at least one other female. The mechanisms behindthese behaviours are not fully understood, but I suggest that female matechoice and a positive effect of number of eggs on larval survival could bethe underlying factors. This shows the importance of looking at individualreproductive behaviour in the context of the social environment in which itlives and not treating the behaviour of individuals as independent of eachother.

The effect of population size on genetic and morphologic variation in myexperiments was not straightforward but rather stochastic. Wings of bothwild L. illustris and laboratory L. sericata were found to be a highlymorphological integrated structure. Still there was variation in shape, butnot size, in wings between wild populations separated in space and/or time.No historic bottlenecks could be detected. There was a slight temporal andspatial genetic differentiation, but the most striking feature was the highvalues of inbreeding in most populations. One possible reason for this couldbe a fine grained genetic population structure of wild Lucilia flies.Bottlenecks were shown to have unpredictable effects on wing size andshape as well as on the genetic variation in experimental populations of L.sericata. While it is clear that bottlenecks can facilitate morphologicalchange, the changes probably need to be subjected to selection to becomefixed and lead to some substantial difference in morphology. This meansthat bottlenecks do not inevitably increase the probability for evolutionarychanges in morphology. In fact, the effects of bottlenecks seem to be quiteunpredictable. The same seems to be true regarding the negative effects ofbottlenecks. Half of the bottlenecked populations went extinct during myexperiments while among the surviving populations there was no trace oflowered fitness. This stochasticity means that bottlenecked populations donot have a higher chance of evolving morphological novelties as comparedto a population not having undergone a bottleneck. However, if manypopulations are bottlenecked one could argue that there is, statisticallyspeaking, a good chance that in at least one of them the conditions will beright to facilitate morphological change and evolution. As Mayr (1963) putit :"Most peripheral isolates do not evolve into new species, but when a newspecies evolves, it is almost invariably from a peripheral isolate" .

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ACKNOWLEDGEMENTS

I like to thank my supervisor Mats Björklund for always being optimisticand always taking himself time to discuss my ideas and Staffan Ulfstrandfor his help with improving many scientific writings, including this thesis. Iam glad I got the chance of enjoying you both as head of my department.It has been rewarding being a part of the department of Animal Ecology aswell as the former department of Zoology. Thank you all people for a goodworking atmosphere, lots of fun spexes, and putting up with the lesspleasing side-effects of blowfly rearing. I also like to thank everybody whohas been part of this work: collected flies, reared flies, mounted wings,measured wings, extracted DNA, helped in the laboratory, revealed thesecrets of cloning, helped with statistics, helped with computers, helpedwith proof-reading and participated in scientific discussions. I speciallywant to thank my mother and father and sister for supporting me throughups and downs and collecting a substantial part of my flies. Without youthis thesis never would have been written. Thank you also all friends for notgiving up on me when I always work, and thank you for not letting metotally disappear into the magic world of biology. Last, but not least, I givemy thanks to “The Bad Boys of Biology” for a lot of fun in what for the lastsummer has been my home away from home. Financial support for thiswork was provided by the Royal Swedish Academy of Science, the Ax:son-Johnson foundation, Lennanders foundation, the Zoological foundation andHM Carl XVI Gustav 50 years foundation.

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SVENSK POPULÄRVETENSKAPLIG SAMMANFATTNING

Hur uppstår nya arter och hur uppstår morfologiska (kroppsliga)förändringar? Det finns teorier som hävdar att ny variation uppstår när enpopulation (grupp av individer) drastiskt minskar i storlek för att sedan ökaigen, d.v.s. när den går genom en ”flaskhals”. När en population är mycketliten spelar slumpen en stor roll för vilka gener som kombineras iavkomman. När sedan populationen ökar i storlek igen kommer dennaturliga selektionen att vara låg. Detta innebär att även individer medmindre lyckade nya genkombinationer som aldrig skulle ha överlevt i denursprungliga populationen kan ge upphov till nya fördelaktigagenkombinationer i efterföljande generationer. Detta kan leda tillmorfologiska nyheter och i extremfallet till en ny art. Flaskhalsar är dockinte bara positiva, den ökade risken för inavel kan få fatala konsekvenserför populationen. Därför är populationsstorlekar intressanta även inaturvårdssamanhang.

I min avhandling har jag undersökt effekterna av populationsstorlek påartbildning (uppsats I och II), på fortplantning (uppsats III) och påmorfologisk och genetisk variation (uppsats IV och V).

ArtbildningEn art är, enligt det biologiska artbegreppet, en grupp verkligt ellerpotentiellt reproducerande (fortplantande) naturliga populationer, som ärreproduktivt isolerade från andra sådana grupper. Det vill sägafortplantning sker inom arten men inte mellan olika arter. Det finns tvåhuvudgrupper av modeller för hur artbildning går till: 1) allopatriskartbildning – då en art delas i två genom en geografiskt barriär, t.ex. enbergskedja; 2) Sympatrisk och parapatrisk artbildning – då nya arter uppstårutan någon geografisk åtskillnad från ursprungsarten. Genom att analyseraresultat från andra författares publicerade artbildningsexperiment kunde jagoch min medförfattare dra slutsatsen att populationsstorleken spelade enstor roll hos sympatriska och parapatriska artbildningsexperiment. Den härtypen av experiment avsåg att förhindra parningar mellan två populationergenom att döda en andel av de hybrider som bildades. På så sätt gynnas deindivider som väljer att para sig inom sin egen population. Vi upptäckte attju större experimentpopulation som använts desto närmare artbildning hadeexperimentet nått, d.v.s. desto färre hybrider bildades. Det kan bero på attde små experimentpopulationerna led av inavel och att det därför fanns ennaturlig selektion för att para sig med någon så olik sig själv som möjligt,d.v.s. någon från en annan population. Denna naturliga selektion

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motverkade forskarnas försök att förhindra hybridisering och ledde till attförsöken misslyckades.

I allopatriska experiment har istället två populationer totalt isolerats ifrånvarandra och efter en tid i isolering har parningsförsök gjorts för att se omindivider föredrar att para sig inom eller mellan populationerna. I vårlitteraturundersökning kunde vi inte hitta något tydligt samband mellanpopulationsstorlek och hur stark reproduktiv isolering som experimentenuppnått. Däremot hade förvånansvärt många experiment misslyckats attuppnå någon som helst isolering, utan individerna parade sig slumpmässigt.Vi kunde också visa att reproduktiv isolering hade en större chans att bildasmellan två dotterpopulationer än mellan en moder- och en dotterpopulation.

Den gröna spyfluganI uppsats III, IV och V har jag studerat två arter av den gröna spyflugan,Lucilia illustris och Lucilia sericata. Båda arterna är vanligt förekommandeöver större delan av världen och är kanske mest ökända för honornas vanaatt lägg ägg i ruttnande kött. De vuxna individerna lever dock påblomnektar, även om honorna behöver ett proteinrikt mål mat innan de kanlägga sina upp till 200 ägg. Parningen äger förmodligen rum i närheten avlämpligt äggläggningssubstrat, t.ex. ett kadaver. Larvernas utveckling ärmycket temperaturberoende men vid ca 25oC tar det ca en vecka innanlarverna gräver ned sig i marken och förpuppas och sedan ytterligare envecka innan pupporna kläcks. Den sista sommargenerationen blir dock intepuppor direkt utan övervintrar som larver och förpuppas först nästföljandevår. En vuxen spyfluga kan maximalt bli runt en månad gammal menmedellivslängden är inte mer än en knapp vecka. De båda könen är nästanidentiska men hannen är något mindre och har större ögon. Det senare ärvanligt hos många flugarter och man tror att det speglar hannarnas strategiatt sitta på marken och spana efter förbiflygande honor.

FortplantningJag gjorde fyra olika försök med en laboratoriepopulation av L. sericata föratt utröna vilken effekt sammansättningen och storleken av en grupp hadeför fortplantningen. Det visade sig att antalet hannar påverkade honornasvillighet att lägga ägg: ju fler hannar desto större chans att honorna ladeägg. Om en hona bara blev tilldelad en slumpmässig hanne så lade hon baraägg i 20 % av fallen, jämfört med 80% av fallen om hon hade fyra hannaratt välja på. Det tyder på att partnerval kan vara viktigt hos den här arten.Ett annat intressant fenomen som upptäcktes var att honor i sällskap avminst en annan hona var mycket mer benägna att lägga ägg än ensamma

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honor. Det kan bero på att larvernas överlevnad påverkas positivt av antaletlarver, men bara till en viss gräns. Det är en hård konkurrens mellanlarverna om den begränsade födan och det är därför viktigt för enspyflugehona att vara bland de första att lägga sina ägg och ge sinavkomma ett försprång. Om vinsten, i form av ökad larvöverlevndad, av attlägga ägg tillsammans är tillräckligt stor kan en hona tjäna på att vänta tillsen annan hona är närvarande innan hon lägger sina ägg.

Morfologisk och genetisk variationLevnadssättet hos de gröna spyflugorna kan innebära att de regelbundet gårgenom populationsflaskhalsar. Man kan tänka sig att det i vissa fall bara äravkomman från ett fåtal honor som lägger beslag på ett helt kadaver och gerupphov till nästa generation av spyflugor. Jag studerade morfologiskvariation genom att jämföra storlek och form på vingarna hos vildapopulationer av L.illustris i Sverige. Det visade sig att formen på flugornasvingar skilde sig något mellan de olika platserna och också något mellan deolika åren. Däremot fanns inget samband mellan genetisk variation ochvariation i vingform. Det fanns heller inga genetiska tecken på attpopulationerna hade genomgått flaskhalsar.

Genom att låta laboratoriepopulationer av L. sericata gå igenomexperimentella flaskhalsar, kunde jag visa att effekterna av en flaskhals äroförutsägbara. I vissa fall verkade morfologisk variation underlättas i andrainte. Den genetiska variationen var konstigt nog inte lägre i populationersom gått genom flaskhalsar än i kontrollpopulationer. Inte heller kundenågra negativa effekter av flaskhalsar på kläckning av puppor upptäckas.Ändå dog flera av experimentpopulationerna ut. Det tyder på att i vissa fallinnebar flaskhalsen att populationen helt dog ut medan i andra fall fickflaskhalsen inga negativa effekter alls.

Denna slumpmässighet betyder att populationer som går genom flaskhalsarinte nödvändigtvis har en större chans att utveckla nya morfologiska drageller bilda nya arter än populationer som inte gått genom flaskhalsar. Men,om många populationer går genom flaskhalsar finns det, statistiskt sett, engod chans för att det i åtminstone någon av populationerna ska finnas derätta förhållandena för morfologisk utveckling och artbildning.