Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 660

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Bottlenecks and Blowflies Speciation, Reproduction and Morphological Variation in Lucilia

BY

ANN-BRITT FLORIN

ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001 Dissertation for the Degree of Doctor of Philosophy in Animal Ecology presented at Uppsala University in 2001 Abstract Florin, A.-B. 2001. Bottlenecks and blowflies. Speciation, reproduction and morphological variation in Lucilia. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations 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 the process of speciation. First, the effect of population size on speciation is studied using several meta-analyses of published laboratory experiments. Second, the effect of population size on behaviour is studied using a laboratory population of the blowfly Lucilia sericata. Third, the effect of population size on morphological and genetic variation is studied using wings and microsatellites from wild populations of L. illustris as well as experimentally bottlenecked populations of L. sericata. The meta-analyses showed that the result of many previous laboratory experiments on sympatric and parapatric speciation may have been biased by too small population sizes. Reduced interbreeding was less likely to develop in small populations where the selection against hybridisation often seemed to have been opposed by inbreeding depression or loss of genetic variation. In allopatric speciation experiments, no general consistent effect of population size was observed. There was no support for speciation through founder events. In fact, significant assortative mating was only found in vicariance experiments where derived populations was 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 only as long as the female was in the company of at least one other female. Female mate choice and a positive effect of number of eggs on larval survival are suggested to be the underlying factors. No historic bottlenecks could be detected in the fly populations, but strong genetic indications suggest a fine grained genetic population structure of wild Lucilia flies. Bottlenecks had unpredictable effects on wing morphology as well as on genetic variation and fitness in a laboratory stock of L. sericata. Thus a bottlenecked population will not necessarily have a higher chance of evolving morphological novelties than one which has not undergone a bottleneck. However, among many bottlenecked populations there is a good chance that in at least one of them the conditions will be conducive to morphological change and evolution. In this statistical sense, thus, strong population fluctuations may enhance the probability of speciation events. Key words: Population size, speciation, Lucilia sericata, morphological variation, Lucilia illustris, bottlenecks. Ann-Britt Florin, Department of Animal Ecology, Evolutionary Biology Centre, Norbyv. 18D, SE-752 36 Uppsala, Sweden ([email protected]) © Ann-Britt Florin 2001 ISSN 1104-232X ISBN 91-554-5133-0 This thesis is based on the following papers, which will be referred to in the text by the roman numerals I-VI

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

II Florin, A.-B. & Ödeen, A. Laboratory environments are not conducive for allopatric speciation. J. Evol. Biol. In press.

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

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

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

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

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

4 INTRODUCTION

Why are there so many species? This is a question that has been asked by mankind since the beginning of time. At the same time the preservation of form, morphologic stasis, is also apparent (Wake et al., 1983; Larson, 1989; Björklund, 1991, 1996; Benton & Pearson, 2001). Some traits differ greatly even in closely related species while other traits seem to stay unaltered through million of years (Gould & Eldredge, 1977; Turner, 1986). This paradox of stasis and yet a biological richness has led to the concepts of limiting adaptive gene complexes and founder effect which could break these complexes and allow for evolution of morphological novelties and even 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 property of the whole genome rather than of the constituent genes, and hence further evolution may require a thorough rebuilding of the genotype. Several lines of 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 male courtship behaviour (Kaneshiro, 1976); genetic transilience theory (Templeton, 1980); variance induced peak shifts (Whitlock, 1995)) offer alternative proposals how these complexes may break up and allow evolution to continue. Common for these models, however, are the stress on the stochastic effects of inbreeding and genetic drift associated with small population size.

Speciation Mayr (1942) defines a species as a set of "groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups". I will use this Biological Species Concept throughout the thesis. Allopatric speciation, where new species arise from geographically isolated populations of the same ancestral species, is the most widely accepted of all current speciation models (Mayr, 1942, 1963; Lynch, 1989; Coyne, 1992; Rice & Hostert, 1993). It can be divided into vicariance 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,

5 giving rise to two or more large, isolated sub-populations. With time the sub-populations are thought to evolve reproductive isolation as a by- product of divergent selection pressures and/or genetic drift. In peripatric speciation (Mayr, 1954) (also commonly known as founder speciation) a small peripheral portion of the population becomes isolated and may undergo one or several (Carson, 1975) bottlenecks. Genetic drift caused by low population size during the bottlenecks together with relaxed selection pressure under the following flush phase, when the population rapidly increases in size, allows the formation of new gene combinations that would not have survived in the original population. Reproductive isolation is then believed to evolve either as a by-product of the genetic changes (Mayr, 1954; Carson, 1975; Templeton, 1980) or as a consequence of relaxation 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 without geographic isolation. Several hypotheses called “divergence-with-gene- flow speciation” (see Rice & Hostert, 1993) assume that populations become genetically isolated because traits for positive assortative mating coevolve with other traits under disruptive selection. Either selection against interbreeding is thought to build non-random associations between the genes responsible for the traits (linkage disequilibrium), or the traits represents different phenotypes deriving from the same gene (pleiotropy). The theory of “reinforcement” (Dobzhansky, 1940) implies that a feedback process between selection against hybrids and positive assortative mating reinforces reproductive isolation until the interbreeding populations have become genetically isolated. More theories of speciation are reviewed in Turelli et al. (2001).

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

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

The study species Lucilia illustris and Lucilia sericata are two of the many, widely distributed, shiny, green blowflies in the Lucilia genus (Zumpt, 1965; Dear, 1985, Fig. 1). The adults feed on flowering plants, although females also require 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 the egg-laying substrate, like in the closely related L. cuprina where males place themselves on the vegetation downwind from the egg-laying substrate (Bartell et al., 1969; Kitching, 1977; Barton Browne et al., 1987). Apart from visual cues the males also respond to pheromones released by females (Bartell et al., 1969). Overall sex-ratio probably is equal, but close to an egg-laying substrate it is highly female biased and the egg-laying females can come in hundreds (Hall et al., 1995). An adult female lays on average 200 eggs per batch (Wall, 1993) and can produce a maximum of eight to ten batches in her three week long life given benign conditions (Zumpt, 1965; Hayes et al., 1998). The median lifespan of wild L. sericata (and

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

The larva requires a moist environment and a food resource. The female lays her eggs primarily on carcasses, but occasionally also on open wounds or the soiled and wet fleece of sheep, causing a skin disease known as myiasis (Zumpt, 1965; Hall & Wall, 1995). L. sericata acts as a primary agent 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 the eggs hatch within 24 hours and the larvae feed on organic matter while passing through three larval stages in approximately one week. As they enter the prepupal stage they leave the feeding substrate and dig into the soil to pupate. In summer the prepupal period lasts from three days to several weeks, depending on temperature, but in autumn the larvae enter diapause and the prepupae remain inactive until spring (Zumpt, 1965; Ring, 1967a-b). With increasing latitude local populations tend to become univoltine (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 the conditions and amount of food obtained by the larvae (Coyler, 1951; Hanski, 1976a-b). Prinkkilä and Hanski (1995) showed that larvae compete through scramble competition and that larval survival is highly density dependent. Nutritional stress also results in the production of many small adults instead of a few large ones (Daniels et al., 1991, personal observation). The two sexes are essentially monomorphic, although females are generally larger than males and their eyes are further apart and smaller than males (Rognes, 1991). The eyes cover a larger part of the head in males than in females, a pattern that is common in many species of flies (Thornhill & Alcock, 1983) and reflects the males’ strategy of watching out for 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 reared flies in Uppsala at 25 oC and 70% humidity under a 16:8 hour light:dark cycle. Adults received pig liver as a protein source on their third day of life and on subsequent occasions as a substrate for egg-laying. Flies had free access to water and sugar. Wild L. illustris flies were collected during three consecutive years (1996-1998) from five different localities in Sweden. Three of the localities were approximately 100 km apart, ranging from Uppsala in the south via Gävle to Söderhamn in the north (Fig. 2). Two

8 localities, only one kilometre apart, were sampled in Uppsala (Zootis and Polack). Flies were collected repeatedly during June to August using funnel traps baited with pig liver.

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

G96: 23,0 G98: 22,22 Gävle

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

Po97: 14,14 50 km

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 are wings followed by microsatellites.

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

Size was measured as centroid size, the square root of the sum of squared differences from a set of landmarks to their centroid (Bookstein, 1991). To separate shape from size, the X and Y coordinates were transformed into Bookstein Coordinates (Bookstein, 1991; Dryden & Mardia, 1998) by scaling them to the baseline size (distance between landmarks No. 10 and No. 11, Fig. 3). The resulting 24 variables (X and Y coordinates) can be used as independent shape variables in ordinary multivariate statistics (Bookstein, 1991).

9 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 morphological components in development and function will be directly related to their morphological integration. The dependence between landmarks within wings were studied in two ways. First, by comparing the observed to the expected variance of eigenvalues in correlation matrices (Cheverud et al., 1989), it is possible to test whether or not the wings are integrated and to compare the strength of integration between different populations. A descriptive measure of morphological integration is the effective number of landmarks, Meff, i.e. the total number of independent effective characters in a multivariate data set (Cheverud, 2000). Second, by comparing covariance matrices in a Flury analysis (Phillips & Arnold, 1999) it is possible to test which model best describes the relationship between different phenotypic variance-covariance matrices ranging from totally unrelated, via different numbers of common principal components and proportional principal components to identical matrices.

10 Genetic data

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

Three different measurements of population genetic variation were obtained from the microsatellite data: average heterozygosity, Ho, Fisher’s inbreeding coefficient, F, and θ. The latter is a function of effective population size and mutation rate (Nielsen, 1997). For a measurement of individual genetic variation I used the mean d2, which is the squared difference in repeat units between alleles at a microsatellite locus averaged over all loci at which an individual is typed (Coulson et al., 1998). This is a more informative measurement of degree of inbreeding than average heterozygosity since it also takes the distance between alleles into account and a larger difference in number of repeats reflects a higher degree of ancestral outbreeding (Pemberton et al., 1999).

Microsatellites can be very helpful in detecting the occurrence of bottlenecks. When a population is reduced in size the allelic diversity is reduced faster than heterozygosity (Nei et al., 1975). This means that the observed number of alleles in a sample from a bottlenecked population is less than the number of alleles expected from the observed heterozygosity assuming mutation drift equilibrium (Cornuet & Luikart, 1996) or, in other words, the population exhibits a heterozygosity excess. The test statistic T2 (Cornuet & Luikart, 1996) is used to determine whether heterozygosity excess, characteristic of a bottleneck, is statistically significant.

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

11 RESULTS AND DISCUSSION Population size and sympatric and parapatric speciation Speciation in sympatry or parapatry is an intriguing possibility, which has attracted a great deal of scientific attention over the last 50 years. Important criticism against the sympatric and parapatric speciation models comes from 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 sympatric and parapatric speciation. However, positive assortative mating can lead to inbreeding and hence to inbreeding depression, in which case natural selection will favour outbreeding. The rate of inbreeding (∆F) will be higher the smaller the population, as described by ∆F = 1/2Ne (Wright, 1931), hence population size can have a profound effect on the outcome of such experiments. In paper I we review the published records of sympatric and parapatric speciation experiments to test the relative importance of selection intensity applied (i), duration of experiment (ttot), and effective population size (Ne).

The basic design in these experiments consisted of two sub-populations that were allowed to interbreed to different degrees. A selective penalty was then applied on hybridisation, either directly or indirectly. To quantitatively determine to what extent a certain experiment should be considered successful, we calculated the hybrid frequency change, ∆H, as the difference in percentage of hybrids between the beginning (H0) and the end (H1) of the experiment relative to the total percentage of hybrids in the beginning:

H − H ∆H = 100* 1 0 H 0 A negative value of ∆H means that the frequency of hybrids has declined during the experiment, and indicate that reproductive isolation has beginning to evolve. Successful experiments, i.e. those resulting in significant positive assortative mating, on average comprised a larger effective population size (Mann-Whitney U-test: n = 63, P < 0.01), and there was a negative correlation between ∆H and Ne (Spearman’s rank correlation: n = 63, ρ = -0.37, P < 0.01). One explanation for the influence of the population size could be that as selection proceeds genetic variation becomes depleted more rapidly in smaller populations. An alternative possibility is that development of inbreeding depression prevents the evolution of reproductive isolation. Selection against outbreeding must be

12 stronger than the selection against inbreeding that will develop in all finite populations for any positive assortative mating to evolve. In most cases, the experimentally applied selection against outbreeding was kept at a constant level over time. In contrast, natural selection against inbreeding will continually increase as more and more detrimental alleles become fixed. Should the two selection intensities come to balance each other, neither traits for inbreeding nor traits for outbreeding will be favoured, and the speciation process will grind to a halt.

There was a positive correlation between i and ∆H among small experiments, Ne<107 (Spearman’s rank correlation: n = 39, ρ = 0.33, P < 0.05) and a negative correlation among large experiments (Spearman’s rank correlation: n = 20, ρ = - 0.50, P < 0.05; Fig. 4). Intense selection will produce an earlier but smaller response than will weak selection (Robertson 1970 b) as a result of the depletion of genetic variation, which will be more significant in smaller populations. Furthermore, in a smaller population, inbreeding depression is more likely to arise rapidly enough for selection against inbreeding to overwhelm the applied selection. Therefore, we will expect to see a negative correlation between hybrid frequency change and selection intensity only among large experiments and a positive rather than a negative correlation among small experiments. This is the pattern we found on either side of Ne = 107 (Fig. 4).

The duration of the experiments was highly positively correlated with effective population size and to determine which had the highest impact on the outcome of an experiment we performed a partial correlation test using Spearman’s ρ. Keeping ttot constant resulted in a significant negative effect of Ne on ∆H (n = 63, ρ = - 0.27, P < 0.05) while keeping Ne constant showed 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 in Rice & Hostert (1993). C = divergent selection with hybrid inviability; D = divergent selection with hybrid viability; E = divergent selection with hybrid viability in which assortative mating was intended to evolve as a pleiotropic effect of selection on other traits, for example breeding-habitat choice. The experiment types differed in hybrid frequency change, ∆H 2 (Kruskal-Wallis: n = 63, χ = 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 the largest and, together with the C-group, the most successful experiments (Fig. 5).

13 200

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-80 Large -120 0,0 0,4 0,8 1,2 1,6 2,0 2,4 2,8 Small i Figure 4. Relationship between hybrid frequency change (∆H) and selection intensity (i) in laboratory sympatric and allopatric speciation experiments with effective population size larger or smaller than 107.

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

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

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

Population size and allopatric speciation The vicariance model of allopatric speciation has been repeatedly confirmed empirically, while peripatric speciation has suffered severe criticism 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 have yielded equivocal results (Rice & Hostert, 1993; Ödeen & Florin, 2000), implying that there are certain conditions required for speciation but not fulfilled in some of these studies.

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

15 importance of multifarious divergent selection, as opposed to parallel or no selection, as a promoter of reproductive isolation. Likewise, in our review of sympatric and parapatric speciation experiments (Ödeen & Florin, 2000), selection intensity was found to have a positive effect on reproductive isolation, but only in large populations.

We surveyed the literature from the 1950’s to the present day for laboratory experiments on allopatric speciation and found 25 papers, most of which are discussed in Rice and Hostert (1993). The basic design in these experiments consists of spatially isolated populations of Drosophila and Musca flies, taken from the same, often outbred, stock. After a number of generations in isolation, mating tests are performed to assess the amount of reproductive isolation achieved between experimental populations or to the original stock. First, we separated the experiments according to the two types of isolation tests made: Derived — two derived populations tested against each other, Origin — a derived population tested against the original, unselected, unbottlenecked stock. Second, we divided the experiments according to the allopatric speciation model tested: Vicariance or Peripatric. Third, we divided these groups with respect to selection regime: experimental populations where no selection, or parallel selection, was applied were classified as Drift, and experiments with divergent selection, for example for high and low bristle number, were classified as Selected. 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 was used (Bishop et al., 1975). Y can assume values between –1 (negative assortative mating) and 1 (positive assortative mating). A value of zero means 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), that peripatric speciation through founder-flush events does not occur under laboratory conditions. More surprisingly, in paper II we were able to show that experiments of the more widely accepted vicariance model, with or without selection, have also failed to produce significant reproductive isolation against a control population. With the exception of vicariance speciation experiments where derived populations were tested for reproductive isolation against each other, laboratory allopatric speciation experiments thus have had little if any effect on reproductive isolation. The means weighted by sample size were not significantly different from the null hypothesis Y = 0, except in Derived tested Vicariance experiments (t = 3.50, d.f. = 36, P = 0.001, Fig 6a and b).

16 Fig 6a 0.9

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

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

17 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 allopatric speciation 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 types there 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 the peripatric model remains unsupported—not the least since neither the bottleneck size nor the number of bottlenecks affected the amount of reproductive isolation achieved . In Derived tested Peripatric experiments neither the number of bottlenecks nor the length of the experiment was correlated 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 the contrary, the length of the experiment was positively correlated to the development of reproductive isolation while the number of bottlenecks had no 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 bottlenecks had 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 Y and length of experiment in Origin tests but a non-significant negative trend in 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 enough time. The experiments might simply have been too short (cf. Hostert, 1997; Mooers et al., 1999).

In Derived tested experiments Y differed between subgroups; Selected selection regimes produced higher Y than did Drift and Vicariance experiments 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 that divergently selected vicariance experiment (Vicariance-Selected) have been more successful than simple drift and peripatric experiments. However, this was true only in mating tests where two experimentally derived populations had been tested against each other (Derived tests), suggesting that reproductive isolation more easily evolves between two daughter populations than between a mother and daughter population.

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

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

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Figure 7. Reproductive isolation (Y) between derived populations in experiments testing different 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 size and composition of the group can have a profound effect on the behaviour of the individuals comprising it. In paper III I have conducted four experiments on the effect of group size and sex ratio on reproduction in the blowfly, Lucilia sericata. In particular I focused on the following questions: Is there a critical minimum group size for reproduction? Do higher densities increase reproduction? Does the sex ratio affect the egg-laying propensity of females?

In the first experiment I varied the number of blowflies and discovered a positive effect of group size. There was a clear difference in the egg-laying propensity between treatments with differing numbers of flies (Cochran Q test 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 much lower than at higher (3-5 pairs) fly densities and also lower than expected if all female had the same egg-laying propensity as the control females from

19 large base population (Table 1). In experiment 2 I used a constant number of 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 experiment 3 I had a constant number of flies (six) but varied the sex-ratio. Egg-laying differed 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-laying frequency increased, except for the most male-biased ratio (5:1), where the egg-laying frequency was extremely low (Table 1). To determine if the low egg-laying propensity among single females was due to male harassment I conducted a fourth experiment in which I kept the sex-ratio equally biased but varied the number of flies. The egg-laying frequencies of females in the 5:1 treatment were significantly lower than expected (Binomialtest: 0.20 compared to 0.75; P = 0.0004) and significantly lower compared to that of the 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 in experiment 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-laying propensity of 0.75. The P-values are from an binomialtest of observed versus expected frequencies. 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 0.025-0.44 2-pair 0.40 0.94 0.000010 0.19-0.74 3-pair 0.90 0.98 0.14 0.69-1.0 4-pair 0.80 0.996 0.00067 0.55-0.97 5-pair 0.70 0.999 1.1 * 10-7 0.44-0.93 14-20pair 1.00 1 1 - 1:5 0.20 0.999 3.7*10-23 0.067-0.56 2:4 0.50 0.996 2.2*10-10 0.26-0.81 3:3 0.70 0.98 0.00042 0.44-0.93 4:2 0.80 0.94 0.12 0.55-0.97 5:1 0.10 0.75 0.000030 0.025-0.44

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

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

My experiments have shown that successful reproduction requires more than 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 number of males (n) to assure her of good male quality. The inflexibility of this strategy, which in the experiment imposes a drastic fitness cost, probably is adaptive in nature if males normally occur in great numbers in places easily located by the females, as in the vicinity of a carrion. In that case females can benefit from this behaviour since it encourages male competition and improves the female’s chance of mating with a superior male.

Although larval density has been found to adversely affect survival and adult size (Hanski, 1976a-b; Prinkkilä & Hanski, 1995; Prinkkilä, 1996), blowflies often lay eggs on the same spot as previously used by other females (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 if they exceed a certain number, preferably of the same species (cf. group feeding Hanski, 1990). It is important to be among the first to lay eggs on a new carcass, thus granting the offspring a head start in the fierce scramble competition (Hanski, 1976a; Hanski & Kuusela, 1977). This means that the benefits of postponing egg-laying until another female arrives must be substantial. If females obtain a great fitness gain by laying eggs on a carcass together with one or more other females, it could pay to wait until another 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 Induced Peak Shifts the stochastic effects of bottlenecks and inbreeding could break up limiting coadapted gene complexes and allow evolutionary morphologic novelties to develop. In paper IV the genetic variation (using microsatellite

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

The distribution of carcasses is both patchy and highly unpredictable in time and space. This makes it probable that sometimes the offspring of only a few females can monopolise an entire carcass and thus represent the next generation of blowflies in that area. The genetic consequences of this life style is dependent on the distribution of resources in relation to dispersal ability. On one hand, if migration between patches is substantial the population could be considered essentially panmictic, with no local differentiation, but due to the high stochasticity of the resource, in both time and space, the risk of the entire population going through a bottleneck could be considerable. On the other hand, if dispersal is insufficient, there will be local differentiation and high levels of inbreeding in a highly structured system, perhaps comparable to a meta-population. The first scenario predicts signs of bottlenecks in the genetic pattern of blowflies while the second scenario suggests high levels of inbreeding. Regardless of which scenario is true, the colonisation of an ephemeral habitat, a founding event, could be argued to act to break up gene complexes limiting morphologic evolution. If this is happening there should be multivariate phenotypic differences among populations of this fly and a negative relationship between the amount of genetic variation and morphological integration 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 of M: 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, the populations may have gone through repeated number of bottlenecks and hence become highly inbred, but these bottlenecks will not be detected in the traditional tests since they presume that the population is outbreeding and panmictic before the bottleneck.

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

22 & Zaklan, 2000; Guerra et al., 1997; Pezzoli et al., 1997). There was no difference 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 almost significant and year turned out to have a slight effect on wing shape (Nested Anova, Wilks test, places within years: F88,484 = 1.28, p = 0.05; between years F44,244 = 1.45, p = 0.04). The limited variation in wing shape could be due to stabilising selection on wings which may typify traits whose function is hampered by even slight deviations. It is also possible that opportunistic species, living on ephemeral resources, have become adapted to cope with inbreeding and bottlenecks and hence are less prone to change morphologically due to these factors as compared to species with a great degree of panmixis.

Table 2. Genetic variation represented by observed heterozygosity (H0), Fisher’s coefficient of inbreeding (F), the outbreeding coefficient (d2 ) and a function of effective population size (θ) in wild populations of Lucilia illustris. The last two columns are test variables from bottleneck tests, where a value of M much lower than 1 and a high positive value of T2 would indicate a bottleneck. 2 2 Population n H0 F θ Mean d ΜΤ S96 24 0.29 0.46* 2.21 1.41 1 -1.28 Z96 11 0.34 0.44# 3.72 0.87 0.87 -0.67 S97 25 0.60 0.21 3.09 1.76 0.93 -1.43 Z97 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 -1.49 S98 7 0.32 0.49# 3.42 1.16 0.90 0.54 Z98 25 0.42 0.38* 6.13 0.72 0.91 -1.46 G98 22 0.26 0.56** 2.45 1.69 0.92 -0.29 F and Τ2 sign. diff. from 0: *P<0.05, ** P<0.01, # could not be tested due to low sample size

Table 3. Morphological integration of eleven wing landmarks in different populations. Observed (Vo) and expected (Ve) variance of eigenvalues of correlation matrices with confidence interval of the observed values given and effective number of landmarks (Meff).

Pop nVo Ve 95 % C. I. Meff S96 17 4.12 0.59 2.59-6.06 7.2 G96 11 2.75 0.91 2.31-4.45 8.5 S97 18 3.05 0.56 2.36-4.18 8.2 Z97 13 4.46 0.77 3.66-6.5 6.9 Po97 11 3.36 0.91 2.62-5.40 7.9 Z98 14 3.40 0.71 2.59-5.27 7.9 G98 15 4.47 0.67 2.99-6.53 6.93

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

Place or year did not have any effect on mean d2 (Nested Anova: places within 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 differentiation low (FIS = 0.37; FST = 0.05), but there was a significant difference in genotypic 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 are not low. Assuming a mutation rate between 1*10-3 and 9*10-6 (range of mutation rates in insect microsatellites (Ellegren, 2000)) gives a value between 500 and 4 500 000 individuals for the population with the smallest value of θ (S96). The estimated frequency of potential null alleles, which can 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 substantial values of F will still result (above 0.20 in four populations). A third possible reason for the high values of F could be that samples from separate populations in fact consist of samples pooled from several distinct populations, suggesting a fine-grained genetic structuring of L. illustris.

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

Bottlenecks and morphological and genetic variation in L. sericata The disruptive effect of founder events, bottlenecks and inbreeding on the genetic system could have an effect on morphological differentiation. Thus, there is some evidence suggesting that bottlenecks can induce morphological changes (Jablonski et al., 1983; Templeton, 1986; Bryant & Meffert, 1988, 1990, 1991, 1992, 1996a-b, 1998; Meffert, 2000). In paper V I subjected a laboratory stock population of Lucilia sericata to artificial bottlenecks. In experiment 1, experimental populations were subjected to a single 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

24 wings and microsatellite variation. If bottlenecks facilitate the evolution of morphologic variation, morphological integration should be lower in bottlenecked populations, while variation in size and shape should be higher. Furthermore, genetic variation should be negatively correlated to morphological integration of wings. Since bottlenecks and the concomitant inbreeding can result in inbreeding depression and lowered fitness (Saccheri et al., 2001), I also investigated the effects of these factors on a fitness 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 between three and six in experiment 1 and between four and six in experiment 2. No general difference in integration between bottlenecked and control populations was observed. The number of bottlenecks had no effect on the effective number of landmarks (rs = 0.082, n = 11, p = 0.81). This suggests that bottlenecks do not reduce overall morphological integration and hence they do not increase the probability of evolutionary change. The pattern of phenotypic correlation, as detected in covariance matrices, however, was found to change over time as well as differ between different populations. In experiment 1, one generation after the bottleneck, two of the bottlenecked populations turned out to have four principal components in common with the control population while two other bottlenecked populations shared only one principal component with the control. Three generations after the bottleneck, two of the tested populations shared all principle components with the control, while one bottlenecked population shared only three or four principal components with the control and none with the rest of the bottlenecked populations. Finally, at the end of experiment 1, all remaining populations had all principal components in common. This suggests that the effects of bottlenecks had been eroded over time as populations had become more similar to the control. In experiment 2, all experimental populations shared all nine principal components with the controls, except the twice bottlenecked population 3A2 which was different from both the controls (3 common principal components) and the other repeatedly bottlenecked populations (2 common principal components). This shows that bottlenecks do not necessarily lead to changes in morphological integration although they probably have the potential 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 of the experiment. When comparing wing size in bottlenecked populations and control populations sampled at the same time it was evident that differences

25 between populations were present during the whole experiment (One-Way Anova, 1 generation after bottleneck: F5,46 = 3.25, p = 0.014; 3 generations after 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 between bottlenecked and control populations.

The wing shape in the control populations changed over time in experiment 1(One-way Anova, Wilks test: F54,242 = 5.66, p < 0.0001). It also differed between 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 of experiment: F36,28 = 2.42, p = 0.009). While all populations were different from each other at one and three generations after the bottleneck, only two of the remaining three populations were different from each other at the end of the experiment, once again pointing to the transient effect of a bottleneck. In experiment 2, the once bottlenecked populations had different overall shape compared to control populations (One-Way Anova, Wilks test: F18,117 = 2.69, p = 0.0008), and a closer inspection of pairwise relationship revealed that all bottlenecked populations were different from each other but the control populations did not differ from each other. The same pattern was found in repeatedly bottlenecked populations, where experimental populations differed from control populations (One-Way Anova, Wilks test: F36,178 = 5.73, p < 0.0001) and populations differed from all others except for the control populations (Fig. 8). This suggests that bottlenecks do have a potential to cause differentiation in wing shape among L. sericata, although changes probably need to be positively selected to achieve permanency, otherwise they will drift back to “normal” again.

A comparison of control and bottlenecked populations revealed that genetic variation declined markedly during the course of the experiment even though 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 was confirmed in experiment 2 where number of bottlenecks were neither correlated 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 genetic variation between control and bottlenecked populations in both experiments suggests that bottlenecks do not always lead to loss of heterozygosity.

26 7

6

5

4

3

2

1

0

Root 2 2A2 -1 3A2 -2 1B3 -3 3B3 -4 K1 -5 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 K2 Root 1

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 from discriminant analysis.

5.0

4.0

2 3.0 d

2.0 mean

1.0

0 01314

Time

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 errors shown.

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

27 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 remove the genetic restrictions for evolution of morphological novelties.

Hatching success was high throughout and in experiment 1 no correlation was observed between hatching success and F (rs = -0.27, n = 15, p = 0.34) 2 or Ho (rs = 0.38, n = 15, p = 0.16) or mean d (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 of heterozygosity and outbreeding on hatching success be detected (n = 11, 2 Ho: rs = 0.055, p = 0.87; F: rs = -0.24, p = 0.48, mean d : rs = -0.23, p = 0.50). Yet, half of the bottlenecked populations went extinct during the course of the experiment. This strongly suggests that bottlenecks and inbreeding elevate the risk of extinction. The lack of correlation between genetic variation and fitness in paper V may be a result of most detrimental effect on fitness already having been purged from the genome at the time of sampling (Bryant & Meffert, 1991; Saccheri et al., 1996; Miller & Hedrick, 2001).

CONCLUSIONS

Our results suggest that population size can affect the likelihood of speciation. Previous laboratory experiments designed to elucidate the mechanisms of sympatric and parapatric speciation in many cases may have been handicapped by too small population sizes. Reduced interbreeding and reproductive isolation was less likely to develop in small populations where the selection process often seemed to have been opposed by inbreeding depression or loss of genetic variation. This means that, according to our results, the experimental evidence frequently used as an argument against sympatric and parapatric speciation models is not as strong as previously believed. On the contrary, there was no general effect of population size on the amount of positive assortative mating achieved in published allopatric speciation experiments. Moreover, our meta-analysis showed no support for speciation through founder events (peripatric speciation) in laboratory experiments. In fact, the only experiments leading to significant positive assortative mating were the simulations of vicariance speciation which had been tested against other derived populations. However, none of the reviewed experiments, regardless of population size or selection regime, has succeeded in generating pre-mating isolation towards a control population. This shows that the method of testing is at least as important as the speciation model.

28 Population size and composition were found to have an important influence on reproductive behaviour. In Lucilia sericata the number and sex of the surrounding individuals had a profound effect on egg-laying. There was a positive effect of increasing number of males but only as long as the female was in the company of at least one other female. The mechanisms behind these behaviours are not fully understood, but I suggest that female mate choice and a positive effect of number of eggs on larval survival could be the underlying factors. This shows the importance of looking at individual reproductive behaviour in the context of the social environment in which it lives and not treating the behaviour of individuals as independent of each other.

The effect of population size on genetic and morphologic variation in my experiments was not straightforward but rather stochastic. Wings of both wild L. illustris and laboratory L. sericata were found to be a highly morphological integrated structure. Still there was variation in shape, but not size, in wings between wild populations separated in space and/or time. No historic bottlenecks could be detected. There was a slight temporal and spatial genetic differentiation, but the most striking feature was the high values of inbreeding in most populations. One possible reason for this could be a fine grained genetic population structure of wild Lucilia flies. Bottlenecks were shown to have unpredictable effects on wing size and shape as well as on the genetic variation in experimental populations of L. sericata. While it is clear that bottlenecks can facilitate morphological change, the changes probably need to be subjected to selection to become fixed and lead to some substantial difference in morphology. This means that bottlenecks do not inevitably increase the probability for evolutionary changes in morphology. In fact, the effects of bottlenecks seem to be quite unpredictable. The same seems to be true regarding the negative effects of bottlenecks. Half of the bottlenecked populations went extinct during my experiments while among the surviving populations there was no trace of lowered fitness. This stochasticity means that bottlenecked populations do not have a higher chance of evolving morphological novelties as compared to a population not having undergone a bottleneck. However, if many populations are bottlenecked one could argue that there is, statistically speaking, a good chance that in at least one of them the conditions will be right to facilitate morphological change and evolution. As Mayr (1963) put it :"Most peripheral isolates do not evolve into new species, but when a new species evolves, it is almost invariably from a peripheral isolate" .

29 ACKNOWLEDGEMENTS

I like to thank my supervisor Mats Björklund for always being optimistic and always taking himself time to discuss my ideas and Staffan Ulfstrand for his help with improving many scientific writings, including this thesis. I am 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 as well as the former department of Zoology. Thank you all people for a good working atmosphere, lots of fun spexes, and putting up with the less pleasing side-effects of blowfly rearing. I also like to thank everybody who has been part of this work: collected flies, reared flies, mounted wings, measured wings, extracted DNA, helped in the laboratory, revealed the secrets of cloning, helped with statistics, helped with computers, helped with proof-reading and participated in scientific discussions. I specially want to thank my mother and father and sister for supporting me through ups and downs and collecting a substantial part of my flies. Without you this thesis never would have been written. Thank you also all friends for not giving up on me when I always work, and thank you for not letting me totally disappear into the magic world of biology. Last, but not least, I give my thanks to “The Bad Boys of Biology” for a lot of fun in what for the last summer has been my home away from home. Financial support for this work was provided by the Royal Swedish Academy of Science, the Ax:son- Johnson foundation, Lennanders foundation, the Zoological foundation and HM Carl XVI Gustav 50 years foundation.

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37 SVENSK POPULÄRVETENSKAPLIG SAMMANFATTNING

Hur uppstår nya arter och hur uppstår morfologiska (kroppsliga) förändringar? Det finns teorier som hävdar att ny variation uppstår när en population (grupp av individer) drastiskt minskar i storlek för att sedan öka igen, d.v.s. när den går genom en ”flaskhals”. När en population är mycket liten spelar slumpen en stor roll för vilka gener som kombineras i avkomman. När sedan populationen ökar i storlek igen kommer den naturliga selektionen att vara låg. Detta innebär att även individer med mindre lyckade nya genkombinationer som aldrig skulle ha överlevt i den ursprungliga populationen kan ge upphov till nya fördelaktiga genkombinationer i efterföljande generationer. Detta kan leda till morfologiska nyheter och i extremfallet till en ny art. Flaskhalsar är dock inte bara positiva, den ökade risken för inavel kan få fatala konsekvenser för populationen. Därför är populationsstorlekar intressanta även i naturvårdssamanhang.

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

Artbildning En art är, enligt det biologiska artbegreppet, en grupp verkligt eller potentiellt reproducerande (fortplantande) naturliga populationer, som är reproduktivt isolerade från andra sådana grupper. Det vill säga fortplantning sker inom arten men inte mellan olika arter. Det finns två huvudgrupper av modeller för hur artbildning går till: 1) allopatrisk artbildning – då en art delas i två genom en geografiskt barriär, t.ex. en bergskedja; 2) Sympatrisk och parapatrisk artbildning – då nya arter uppstår utan någon geografisk åtskillnad från ursprungsarten. Genom att analysera resultat från andra författares publicerade artbildningsexperiment kunde jag och min medförfattare dra slutsatsen att populationsstorleken spelade en stor roll hos sympatriska och parapatriska artbildningsexperiment. Den här typen av experiment avsåg att förhindra parningar mellan två populationer genom att döda en andel av de hybrider som bildades. På så sätt gynnas de individer som väljer att para sig inom sin egen population. Vi upptäckte att ju större experimentpopulation som använts desto närmare artbildning hade experimentet nått, d.v.s. desto färre hybrider bildades. Det kan bero på att de små experimentpopulationerna led av inavel och att det därför fanns en naturlig selektion för att para sig med någon så olik sig själv som möjligt, d.v.s. någon från en annan population. Denna naturliga selektion

38 motverkade forskarnas försök att förhindra hybridisering och ledde till att försöken misslyckades.

I allopatriska experiment har istället två populationer totalt isolerats ifrån varandra och efter en tid i isolering har parningsförsök gjorts för att se om individer föredrar att para sig inom eller mellan populationerna. I vår litteraturundersökning kunde vi inte hitta något tydligt samband mellan populationsstorlek och hur stark reproduktiv isolering som experimenten uppnått. Däremot hade förvånansvärt många experiment misslyckats att uppnå någon som helst isolering, utan individerna parade sig slumpmässigt. Vi kunde också visa att reproduktiv isolering hade en större chans att bildas mellan två dotterpopulationer än mellan en moder- och en dotterpopulation.

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

Fortplantning Jag gjorde fyra olika försök med en laboratoriepopulation av L. sericata för att utröna vilken effekt sammansättningen och storleken av en grupp hade för fortplantningen. Det visade sig att antalet hannar påverkade honornas villighet att lägga ägg: ju fler hannar desto större chans att honorna lade ägg. Om en hona bara blev tilldelad en slumpmässig hanne så lade hon bara ägg i 20 % av fallen, jämfört med 80% av fallen om hon hade fyra hannar att 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 av minst en annan hona var mycket mer benägna att lägga ägg än ensamma

39 honor. Det kan bero på att larvernas överlevnad påverkas positivt av antalet larver, men bara till en viss gräns. Det är en hård konkurrens mellan larverna om den begränsade födan och det är därför viktigt för en spyflugehona att vara bland de första att lägga sina ägg och ge sin avkomma ett försprång. Om vinsten, i form av ökad larvöverlevndad, av att lägga ägg tillsammans är tillräckligt stor kan en hona tjäna på att vänta tills en annan hona är närvarande innan hon lägger sina ägg.

Morfologisk och genetisk variation Levnadssättet hos de gröna spyflugorna kan innebära att de regelbundet går genom populationsflaskhalsar. Man kan tänka sig att det i vissa fall bara är avkomman från ett fåtal honor som lägger beslag på ett helt kadaver och ger upphov till nästa generation av spyflugor. Jag studerade morfologisk variation genom att jämföra storlek och form på vingarna hos vilda populationer av L.illustris i Sverige. Det visade sig att formen på flugornas vingar skilde sig något mellan de olika platserna och också något mellan de olika åren. Däremot fanns inget samband mellan genetisk variation och variation i vingform. Det fanns heller inga genetiska tecken på att populationerna hade genomgått flaskhalsar.

Genom att låta laboratoriepopulationer av L. sericata gå igenom experimentella flaskhalsar, kunde jag visa att effekterna av en flaskhals är oförutsägbara. I vissa fall verkade morfologisk variation underlättas i andra inte. Den genetiska variationen var konstigt nog inte lägre i populationer som gått genom flaskhalsar än i kontrollpopulationer. Inte heller kunde några negativa effekter av flaskhalsar på kläckning av puppor upptäckas. Ändå dog flera av experimentpopulationerna ut. Det tyder på att i vissa fall innebar flaskhalsen att populationen helt dog ut medan i andra fall fick flaskhalsen inga negativa effekter alls.

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

40