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To Gustaf and Vilda, who by their mere existence gave me

new perspectives on what is truly important in life.

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I E. Rova, and M. Björklund. Can preference for oviposi-tion sites initiate reproductive isolation in Callosobru-chus maculatus? Submitted manuscript.

II E. Rova, and M. Björklund (2010). Competitive envi-

ronments induce shifts in host fidelity. Journal of Evolu-tionary Biology, 23: 1657-1663.

III E. Rova, and M. Björklund. The influence of migration

on the maintenance of assortative mating. Submitted ma-nuscript.

IV E. Rova, and M. Björklund. An experimental study on

the interaction between inbreeding and assortative mating in the process of speciation. Submitted manuscript.

V M. Björklund, and E. Rova. Assortative mating and the

cost of inbreeding. - A simulation approach. Submitted manuscript.

Reprints were made with permission from the respective publishers.

Contents

Introduction ..................................................................................................... 9 General Background ................................................................................... 9 Methods .................................................................................................... 10 Study Species ........................................................................................... 11

Ecology and life history ....................................................................... 11 Callosobruchus maculatus as a model organism ................................. 11

Aims of the thesis.......................................................................................... 12 Main questions addressed; ....................................................................... 12

Results and Discussion ................................................................................. 13 The role of assortative mating in reproductive isolation with free dispersal between hosts (Paper I) ............................................................................ 13 Frequency dependent selection based on host preference underlying shift in host fidelity (Paper II) .......................................................................... 14 Different rates of migration between adjacent populations utilizing different host plants, and its effects on the persistence of assortative mating (Paper III) ..................................................................................... 16 An experimental study on the interaction between inbreeding and assortative mating and how it affects the process of speciation (Paper IV) .................................................................................................................. 21 An alternative approach to study the interaction between inbreeding and assortative mating, and it effects on divergence, using computer simulations (Paper V) ............................................................................... 22

Conclusions and future challenges ................................................................ 23

Sammanfattning på svenska .......................................................................... 25 Bakgrund .................................................................................................. 25

Fröbaggen som modellorganism .......................................................... 25 Syfte ..................................................................................................... 26 Resultat ................................................................................................ 26

Acknowledgements ....................................................................................... 28

References ..................................................................................................... 31

9

Introduction

General Background No aspect of speciation has been as vigorously disputed as the view that new species can arise in the presence of gene flow. The question of whether the first stages of divergence absolutely require complete geographic isolation, as in the case of allopatric speciation, emerged in the nineteenth century and ever since allopatric, versus parapatric, versus sympatric speciation has been a topic of intense discussion (Berlocher 1988). Indeed, divergence-with-gene-flow remains to be a subject of no consensus. It is a matter of great interest still and has gained both theoretical and empirical support as a con-sequence (Bolnick and Fitzpatrick 2007). However, empirical data support-ing the theory of speciation with gene flow, though growing, is still scarce and badly needed.

Theory has identified a variety of evolutionary processes that under cer-tain ecological conditions can lead to speciation despite the presence of gene flow (Maynard-Smith 1966; Kondrashow and Mina 1986; Doebeli 1996; Dieckmann and Doebeli 1999; Kondrashow and Kondrashow 1999; Doebeli and Dieckmann 2000; Via 2001; Gavrilets 2003, 2004). One particular set of models where speciation with gene flow is considered likely is when a shift occurs in host preference in phytophagous insects, and particularly when mating takes place on the host plant (Schluter 1998, Feder 1988; Menken and Roessingh 1988). Many speciation models, and especially sympatric such, have been motivated by the observation of host races and involve eco-logically driven reproductive isolation associated with adaptation to alterna-tive and discrete resources or habitats (Via 2001; Dres and Mallet 2002; Berlocher and Feder 2002; Gavrilets 2003, 2004). In general, these models predict that when a host shift occurs, and mating takes place on the host plant, sympatric speciation can be facilitated if assortative mating linked to host choice develops as a by-product of selection for enhanced performance on the new host. In such a scenario, rapid speciation is theoretically plausi-ble. Experimental evidence of rapid shifts in host plant utilization has been provided by Wasserman and Futyama (1981), and Bolnick and Fitzpatrick (2007).

Ecological contact does not necessarily have to be a hindrance to specia-tion, but can instead be a prerequisite and even, in special cases, a driving force, as gene flow may enhance speciation through reinforcement (Doebeli

10

and Dieckmann 2004). In fact, it has been shown earlier that evolutionary divergence is feasible in the face of gene flow if the evolution of assortative mating is allowed for (Dieckmann and Doebeli 1999). Thus, the develop-ment of a mating system based on positive assortative mating seems to be crucial for the initiation and continuation of ecological diversification and speciation in sympatric and parapatric populations.

Methods The empirical approach used in this study has been to assess the develop-ment of resource and mating preferences and to quantify costs and benefits related to a system of assortative mating. By employing artificial selection for colour (black and brown) in conjunction with natural selection for two alternative resources, I aimed to link colour to resource. The colour dimor-phism was then used as a neutral marker of mating preference when studying mating patterns between the two morphs.

To assess development of host preferences, female host-choice pheno-types of the two colour morphs were tested, and artefacts due to larval condi-tioning and maternal effects were controlled for (Paper I). Assortative mat-ing was measured as a deficit of heterozygotes compared to Hardy-Weinberg equilibrium in the case of random mating in the F1 generation after crossing the two color morphs (Paper I, II and III). In a system based on positive as-sortative mating, homozygotes prefer to mate with individuals of the same color, producing an excess of homozygotes and a deficit of heterozygotes. Viability inequalities of the different morphs on the two resources, i.e. selec-tion against heterozygotes, would have generated the same pattern. There-fore, the viability of all three morphs was tested on both resources.

An alternative method also used in this study to measure assortative mat-ing, and to see whether it was linked to resource use, was mating trials (Pa-per II). Virgin females were introduced to virgin males from both resource types and allowed to choose which male they preferred to mate with. To discern which type of male the female chose to copulate with, one male in each trial was sterilized before mating. Females mated to sterile males lay unfertilised transparent eggs whereas females mated to fertile males lay fertilised white eggs. Female preference for type of male could then easily be determined. Investigating female mating propensity is yet another way of assessing assortative mating (Paper II). Here, time to mating and duration of mating were used to elucidate whether females were more prone to engage in mating, and if they were willing to mate for a longer time, with males of their own kind. In paper (IV) inbreeding depression was measured in terms of reduced female fecundity (the actual number of eggs laid), decreased juvenile hatching success, changed hatched offspring sex ratio and, popula-tion extinction. In paper (V) computer simulations were used to explore the

11

joint effects of inbreeding and assortative mating on population divergence, including costs and benefits.

Study Species Ecology and life history The seed beetle Callosobruchus maculatus (Fabr.), belonging to the family of Bruchidae, with approximately 1400 species, is a cosmopolitan pest of cultivated and wild legumes (Fabaceae) throughout the tropics. It particu-larly infests beans of the genus Vigna (Fox et al. 2004) both in storage and in the field leading to considerable losses of food in third world countries. The life cycle of this species is fairly simple, starting out with females cementing their eggs onto the surface of host beans. The larvae then burrow their way into the bean, feeding from it during their complete development into adults. When fully grown, adults emerge through holes in the surface of the beans (Southgate 1979) and start to reproduce shortly after emergence. The species has non-overlapping generations, and a generation time of approximately 28 days when maintained in climate chambers at 26° C and 55 % RH.

Callosobruchus maculatus as a model organism The genus Callosobruchus consists of about 20 species (Borowiec 1987), many of which over the last two decades have been kept in various labs, frequently used as model organisms in studying sexual conflict, male-female interactions, and sperm competition (Fox 1993a, 1993b; Arnqvist et al. 2004; Eady et al. 2007; Edvardsson 2007; Fricke & Arnqvist 2007; Harano 2007; Rönn et al. 2007). There are many advantages in working with seed beetles as model organ-isms. They are easy to rear in laboratory environments and the adults readily reproduce and survive for several weeks without access to food and water (Wightman 1978; Savalli and Fox 1998). Also, two colour morphs occur naturally in this species, even in the same populations; one brown and one black. When performing reciprocal crosses between the black and brown morphs in C. maculatus, it was found that all resulting offspring were of the intermediate type and that F1 crosses yielded a distribution of morphs in concordance with expectations from Hardy-Weinberg (Eady 1991), suggest-ing that the genetic background of the colour dimorphism is controlled by a single locus with two alleles. This forms an ideal base for studying mating patterns using the dimorphism as a neutral marker of mating preferences. The seed beetle population used in the work behind this thesis, originated in Brazil, and was provided from the lab of Paul Eady, where it had been cul-tured for about 20 years.

12

Aims of the thesis

The main aim of this thesis was to investigate the role of gene-flow and as-sortative mating in the initial stages of speciation, to better understand the different ways in which new species can evolve, and to analyze under which ecological conditions, and through which mechanisms, speciation with gene flow is likely to occur.

Main questions addressed; • Is assortative mating, based on host preference, alone suffi-

cient to initiate and maintain reproductive isolation when dispersal is free between hosts? (Paper I)

• Can frequency-dependent selection induced through compe-tition, and based on host preference lead to a shift in host fi-delity and initiate divergence within sympatric populations through the development of assortative mating? (Paper II)

• At which migration rates can assortative mating, based on preference and not performance, persist in a population, making divergence possible? Is it really as strict as the theory predicts? (Paper III)

• How does assortative mating and inbreeding interact and af-fect the process of speciation? - An experimental approach. (Paper IV)

• How does assortative mating and inbreeding interact and af-fect the process of speciation? - A simulation approach. (Pa-per V)

13

Results and Discussion

The role of assortative mating in reproductive isolation with free dispersal between hosts (Paper I) The aim of paper I was to evaluate the role of assortative mating, based on host plant choice, in maintaining partial reproductive isolation between pop-ulations of the seed beetle Callosobruchus maculatus, when dispersal was free between hosts.

According to host shift theory, the two main requirements for sympatric speciation to occur are disruptive selection for a certain trait and subsequent assortative mating based on that particular trait (Maynard Smith 1966; Udovic 1980; Felsenstein 1981). In Paper I, I investigated whether selection for different resources in allopatry could cause reproductive isolation in sec-ondary contact through linkage disequilibrium between mating- and host preference. The experiment showed that after only ten generations of selec-tion on different resources/hosts and for different colour morphs, strains of the Cowpea weevil Callosobruchus maculatus develop new host preferences and show resource-dependent assortative mating when given the possibility to choose mates and hosts. The newly evolved host preference was shown as a change in ovipositional preference (Fig. 1) and was followed by assortative mating (revealed as fewer hybrids than expected in the F1 generation when comparing the distribution of morphs with Hardy Weinberg equilibrium). There seemed to be no larval conditioning or maternal effects operating to obscure the interpretation of the results, thus the conclusion can be made that the degree of assortative mating developed in this system was due to a change in resource preference alone. The consequential reduced gene flow between the different strains stayed intact for two generations before finally resulting in random mating. Since no viability differences could be detected between the different morphs on the two resources, no selection against het-erozygotes seem to have been operating, suggesting that no fitness differ-ences have evolved between individuals on the two hosts. In the lack of se-lection against intermediate phenotypes, assortative mating is difficult to maintain as there are no costs to recombination.

14

Resource chosen: Black-eye Resource chosen: Mung

Mung Black-eyed

Origin

0,30

0,35

0,40

0,45

0,50

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0,65

0,70P

ropo

rtion

egg

s

Figure 1. The proportion of eggs (± 95 % confidence interval) laid by females in the different treatments. Origin refers to the bean type females were raised on for ten generations, and resource chosen refers to the bean type they chose to lay their eggs on when offered a mixture of the two bean types as oviposition substrate.

Based on the results of this study, I conclude that a reduction of gene flow can evolve in a population due to a link between host preference and assorta-tive mating. However, consistent with models of speciation, assortative mat-ing alone is not sufficient to maintain reproductive isolation when dispersal is free between hosts. The evolution of reproductive isolation in this system cannot proceed without selection against hybrids (Gavrilets 2004). Thus, it is required that assortative mating and disruptive selection work in concert to hinder gene flow between diverging populations. In other words, the estab-lishment and maintenance of linkage disequilibrium between pre- and post zygotic isolation is decisive for speciation to proceed to completion.

Frequency dependent selection based on host preference underlying shift in host fidelity (Paper II) The aim of paper II was to assess whether frequency-dependent selection based on host preference could be induced by strong competition for re-sources and lead to a shift in host plant utilization, by the development of assortative mating linked to resource use.

15

The evolution of disruptive selection and the subsequent development of assortative mating are the two key challenges to the theory of sympatric speciation (Dieckmann et al. 2004; Fitzpatrick et al. 2009). A set of models dealing with these issues allow for sympatric speciation to evolve as a result of disruptive selection for resource use, where extreme phenotypes suffer less from competition, mate assortatively and produce offspring with the same benefits of reduced competition (Doebeli 1996; Dieckmann and Doebeli 1999; Doebeli and Dieckmann 2000; Drossel and McKane 2000).

Paper II investigates whether a change in host preference and subsequent assortative mating can be obtained in a case of total sympatry based on fre-quency dependent selection. In this experiment, a highly competitive envi-ronment was created to force less competitive females to relax their host fidelity and instead utilize a novel resource. I examined whether a shift in host fidelity could be induced through disruptive selection in sympatry, and if a linkage between mating- and food preference i.e. assortative mating would consequently evolve.

As disruptive selection acts against intermediate phenotypes, individuals that mate assortatively are favoured by natural selection. Once assortative mating based on the trait under disruptive selection has evolved, reproduc-tive isolation automatically develop as the ecological trait diverges, followed by evolutionary branching as a natural consequence (Metz et al. 1996; Geritz et al. 1998). The evolution of assortative mating is crucial here, as evolution-ary branching cannot come about without it (Doebeli and Dieckmann 2003).

The results gained show that after only four generations on a mixture of resources/hosts, strains of the seed beetle, Callosobruchus maculatus, start to develop new host preferences leading to a sympatric relaxation of their host fidelity (Fig. 2). One explanation for the change in host fidelity could be the act of disruptive selection resulting from high competition leading to a shift in host preference. A second explanation could be the evolution of a more generalistic behaviour in resource utilisation among females. To elucidate which one of the two explanations were true, I studied female laying prefer-ence over time in an uncrowded environment with an excess of resources at hand. I show that under high competition on a mixture of resources/hosts, strains of the Seed beetle, Callosobruchus maculatus, change their host fi-delity and evolve a more generalistic behaviour in resource utilisation among females. The change in host fidelity did not result in disruptive selection and was not followed by assortative mating. This means that only one out of two fundamental prerequisites for sympatric speciation evolved as a result of the frequency-dependent selection. I conclude that for this process to work, a shift to a novel food resource as a result of selection must also lead to a loss of preference for the original source such that individuals are only able to use either one of the two.

16

1 2 3 4 5 6 7 8 9 10

Generation

0,25

0,30

0,35

0,40

0,45

0,50

0,55

0,60

Prop

ortio

n of

egg

s on

mun

g

Figure 2. The proportion of fertilized eggs laid on mung beans over generations under high competition. There is a significant positive trend in the proportion of eggs laid on mung beans over generations. Vertical bars denote 95 confidence intervals.

Different rates of migration between adjacent populations utilizing different host plants, and its effects on the persistence of assortative mating (Paper III) The aim of paper III was to investigate the effects of different levels of mi-gration between two populations, utilizing different host plants, on the main-tenance of assortative mating within each population.

Most scenarios of speciation underline the importance of the formation and maintenance of geographical variations as a necessary step for diver-gence to occur (Jakoby and Whigham 2005). However, many researchers have suggested that rapid and extensive speciation is in fact possible without complete geographic isolation (Endler 1977; Rice & Hostert 1993; Gavrilets et al. 1998; Gavrilets et al. 2000). Traditional parapatric models often as-sume that some form of geographic differentiation is induced by discontinui-ties in the external environment, and that speciation is driven by divergent local adaptation or genetic drift in spatially distant locations and hindered by gene flow because of ecological contact (Gavrilets 1999; Turelli et al. 2001). However, ecological contact is not necessarily a hindrance to speciation, but, instead, can be a prerequisite and a driving force, as gene flow may enhance speciation through the process of reinforcement (Doebeli and Dieckmann

rs = 0.58

17

2004). In such a situation, the mating system influences whether evolution-ary branching, and hence parapatric speciation is feasible, as it has been shown earlier that evolutionary branching in well-mixed sexual populations is possible if the evolution of assortative mating is allowed for (Dieckmann and Doebeli 1999).

Standard population genetic theory states that very weak migration, on the order of one individual exchange per two populations per generation, is ade-quate to prevent genetic differentiation, making speciation impossible (for basic model see e.g. Templeton 2006), a conclusion however only shown for neutral alleles (Slatkin 1987). In simulations done by Gavrilets et al. (2000) speciation was observed even when the migration rate was on the order of several individuals per subpopulation per generation. In paper III, I examine the effects of migration on the possibility of divergence and speciation by introducing different levels of migration between populations of the seed beetle Callosobruchus maculatus, inhabiting two different resources, and constituting two different colour morphs. By monitoring mating patterns in the populations exposed to immigration over several generations, and com-paring them to Hardy Weinberg equilibrium as a test statistic for assortative mating, I can obtain an idea of the possibilities for speciation in the presence of gene flow.

I show that assortative mating can be upheld for several generations in populations experiencing immigration rates of up to eight percent, or 13-15 immigrants per generation, despite the lack of adaptive divergence and trade-offs between the exchanging populations (Fig. 3). Since assortative mating vanishes after some generations of extensive gene flow without selection against hybrids, I conclude that selection is likely to be an important factor in speciation in the face of gene flow.

18

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< 0.001 < 0.001 < 0.001 0.99 0.027 0.99

Figure 3. The index of assortative mating over generations, Index in the BE popula-tions (first three) and in the M populations (last three). P-values from a combined probability test over replicates are given in the figure. Each open circle refers to a single replicate, while the filled circles refer to the mean values.

21

An experimental study on the interaction between inbreeding and assortative mating and how it affects the process of speciation (Paper IV) The aim of paper IV was to experimentally explore the joint effects of in-breeding and assortative mating on population divergence, and to highlight the importance of taking both of these two factors into account when study-ing speciation processes.

One of the key factors in the process of speciation is assortative mating i.e. when individuals select mates more phenotypically similar to themselves than expected under random mating (Kirkpatrick and Servedio 1998). Assor-tative mating can be beneficial when preventing co-adapted gene complexes from being broken up, which could be an important aspect in populations well adapted to their respective habitats. A case where assortative mating should be adaptive is when sympatric host races of phytophagous insects mate assortatively based on host plant choice (Via 2001; Berlocher and Fed-er 2002; Dres and Mallet 2002). To mate assortatively, is however costly, not only in terms of time and effort spent searching for the right mates (Verzijden et al. 2005), but there is also the risk of inbreeding to be consid-ered, a cost mostly overlooked and therefore only modestly discussed earlier in the literature (Ödeen and Florin 2000). When assortative mating works through phenotype-matching the individuals most similar to one another are often the ones of closest kin. This in turn would increase the risk of assimi-lating deleterious mutations within small populations, subjecting them to an elevated risk of extinction.

In models of sympatric and parapatric speciation dealing with cases where assortative mating should be adaptive, for example those where assortative mating starts on an island on the basis of an ecological trait (Kirkpatrick and Servedio 1998), or as mentioned above, within a group of phytophagous insects encountering and utilizing a new host plant (Via 2001; Berlocher and Feder 2002; Dres and Mallet 2002), population sizes are most likely fairly small given the ecological settings.

My study experimentally explores the interaction of inbreeding and assor-tative mating in the process of speciation. I show that in small populations of the seed beetle, Callosobruchus maculatus, signs of inbreeding depression, such as reduced female fecundity, decreased juvenile hatching success, changed hatched offspring sex ratio, and extinction develop in relation to assortative mating. I also show that in these small, inbred populations, the mating system changes in terms of a reduced tendency to mate assortatively. Based on these results, I advocate for the importance of considering the joint effects of assortative mating and inbreeding when dealing with theories of speciation with gene flow.

22

An alternative approach to study the interaction between inbreeding and assortative mating, and it effects on divergence, using computer simulations (Paper V) The aim of paper V was to appraise the importance of the interaction of as-sortative mating and inbreeding in the initial stages of speciation using com-puter simulations.

Models of speciation frequently deal with small founder populations often with mating preferences based on ecological traits or habitat preferences (Kirkpatrick and Servedio 1998; Via 2001; Berlocher and Feder 2002; Dres and Mallet 2002). Small populations, on the other hand might suffer from inbreeding, however, few studies have explored the combined effects of assortative mating and inbreeding in such populations.

The possible outcomes of inbreeding are difficult to predict since they ul-timately depend on the actual deleterious mutations accumulated in each population, and of population history (Theodorou and Couvet 2006). In small populations it also depends on the stochastic effects of drift, and the effectiveness of purging (Crnokrak & Barret 2002). Since the effects of in-breeding are highly stochastic, experiments can only capture a small part of the potential problem. An alternative approach is to use computer simula-tions. In this study I have, using computer simulations, contrasted two sets of populations selected to move to a new adaptive peak through a change in an ecological trait. With this simulation I show that assortative mating based on similarities increases the possibility for change in a population, as long as the population does not suffer from inbreeding depression. Inbred populations seem not to be able to cope with strong assortative mating, as this is likely to elevate the level of inbreeding, increasing the risks of inbreeding depression and as a result decreasing population mean fitness. This in turn hinders the possibility of change, and instead might drive the population to extinction.

23

Conclusions and future challenges

According to the results in paper I, divergence can evolve fast in a popula-tion due to a link between resource preferences and assortative mating. This promotes reproductive isolation as a by-product of habitat choice and opens for the possibility of speciation in sympatry. However, assortative mating alone does not seem to be sufficient enough to maintain the divergence when there is unlimited dispersal between hosts. Selection against intermediate morphs is needed to counteract the process of recombination and keep the two diverging morphs apart. The findings of paper I, in terms of a swift change in resource preference, is supported by the results in Paper II, in which I show that frequency dependent selection in the form of high compe-tition leads to a change in host fidelity as females evolve a more generalistic behaviour in their resource utilisation. The change in host fidelity did not however result in disruptive selection and was not followed by assortative mating. This means that only one out of two fundamental prerequisites for sympatric speciation evolved as a result of the frequency-dependent selec-tion. I therefore conclude that for this process to work, a shift to a novel host as a result of selection must also lead to a loss of preference for the original host such that individuals are only able to use either one of the two.

In paper III, I show that assortative mating to some degree can be upheld for several generations in populations experiencing immigration rates of up to eight percent, or 13-15 immigrants per generation, despite the lack of adaptive divergence and trade-offs between the exchanging populations. Since assortative mating vanishes after some generations of extensive gene flow without selection against hybrids, I conclude that disruptive selection is likely to be of crucial importance for speciation to occur in the face of gene flow.

Paper IV experimentally investigates the interaction of inbreeding and as-sortative mating in the process of speciation. The results show that small populations often show signs of inbreeding depression in relation to assorta-tive mating, and that within these populations the mating system changes from strong to weak assortative mating. I draw the conclusion that the change in mating system is a consequence of inbreeding, and a strategy to avoid further development of inbreeding depression. I also strongly advocate for the importance of considering the joint effects of assortative mating and inbreeding when dealing with theories of speciation in the face of gene flow, and conclude that further investigation are needed for a deeper under-

24

standing of these effects and to elucidate their role and possible conse-quences in the initial stages of sympatric and parapatric speciation.

Paper V uses a computer simulation approach to investigate the impor-tance of the interaction of assortative mating and inbreeding in the initial stages of speciation. The main findings of paper V supported the experimen-tal results of paper IV, in showing that assortative mating leads to decreased mean fitness and a decreased probability of change in mean value as a result of the presence of deleterious mutations, in populations of limited size. Based on these results I conclude that the risk of inbreeding, and as a result inbreeding depression, needs to be considered seriously when assortative mating based on phenotype matching is invoked in models of speciation.

The evolution of behavioural isolation is the most critical step in modelling sympatric and parapatric speciation and therefore further examination of the ecological conditions under which such isolation could evolve is needed. A large amount of researce remains to be done on the subject, and an interesting future experimnet would be to empirically test the assumptions of the model of van Doorn et al. (2009) on sexual selection on good genes. My model system meets the assumptions of van Doorn's model, and is therefore highly suitable for empirically testing the hypothesis that sexual selection on condition dependent signals creates reproductive isola-tion. With my system this can be done in a very simple and straight forward way. Using my ecologically diverged populations, I wish to test whether the mating success of males from each population is larger when performing in their own environment than in the alternate environment, even though they are exposed to the choice of the same kind of female. It would indeed be interesting to explore this type of sexual selection further, and see if it could perhaps maintain a split and lead to reproductive isolation between sympa-tric populations where my earlier research has failed to do so.

25

Sammanfattning på svenska

Bakgrund Artbildning utgör idag ett av de större forskningsfälten inom den moderna evolutionära biologin. Teorierna kring hur nya arter utvecklas från redan existerande arter kan dateras ända tillbaka till Darwin och hans verk om ar-ternas uppkomst, i vilket han bland annat utvecklade sina idéer om sympat-risk (och paraptrisk) artbildning, dvs. när artbildning sker inom samma geo-grafiska område trots fortsatt genutbyte mellan de olika grupperna. Anled-ningen till att artbildning faktiskt kan ske trots pågående genflöde mellan grupper har att göra med ekologiska omständigheter som minskar integration mellan dem. Artbildning kan då ske genom disruptiv selektion och på grund av fördelen med att para sig med individer av samma karaktär, eftersom avkomma mellan individer med olika karaktärer ofta har sämre fitness.

Under senare decennier har arbildningsteorin erhållit ett förnyat intresse då Darwins kontroversiella teorier om artbildning med genflöde dammats av, till viss del förnyats, och studerats i detalj. Ingen annan aspekt inom den moderna evolutionära biologin har förmodligen någonsin skapat så stora meningsskiljaktigheter och vildsinta diskussioner som just sympatrisk och parapatrisk artbildning. Artbildningsprocessen fortsätter att fascinera forska-re, och stora luckor återstår att fylla i kunskapen om hur livets diversitet uppstått.

Mitt doktorandprojekt har främst inriktats på att experimentellt undersöka de grundläggande förutsättningarna för den sympatriska och den parapatris-ka artbildningens ekologi, samt utvärdera vikten av att para sig med indivi-der av samma karaktär som en själv i stävjandet av integrationen mellan divergerande grupper. I små populationer kan valet att para sig inom samma sort leda till inavel med svår inavelsdepression som följd. Detta kan ställa till med problem för den sympatriska och parpatriska artbildningen, som ofta initieras i just sådana små populationer.

Fröbaggen som modellorganism I den här avhandlingen använder jag mig av fröbaggen av släktet Callosob-ruchus. Familjen Bruchide, som fröbaggen tillhör, består av omkring 1 400 arter, varav ett tjugotal utgörs av just släktet Callosobruchus. Under de se-naste 20 åren har fröbaggar utvecklats till ett väletablerat system för studiet

26

av frågor som rör interaktioner mellan hanar och honor, spermiekonkurrens, sexuella konflikter, och artbildning. Fördelarna med fröbaggen som modell-organism är många. Bland annat är de lätta att föda upp på laboratorium, kräver lite i form av skötsel, och anses vara oerhört robusta vad gäller tem-peratur, fuktighet, och hantering. Fröbaggarna är en av världens största ska-deinsekter, och förekommer naturligt kring hela ekvatorn. Livscykeln är ca 4 veckor lång, och påbörjas då en hona cementerar fast sina befruktade ägg på ytan till olika typer av bönor. Larven kläcks sedan och äter sig in i bönan varpå den spenderar hela sin utvecklingstid inuti bönan och äter av dess in-nanmäte. Som fullvuxen individ äter sig fröbaggen sedan ut ur bönan, letar upp en partner att para sig med, och så är en ny livscykel påbörjad.

Syfte Syftet med mitt avhandlingsarbete har varit att utforska rollerna av genflöde och parningspreferens i artbildningens startskede. Detta för att bättre förstå de olika sätt på vilka nya arter kan uppstå, samt att analysera under vilka ekologiska förutsättningar, och genom vilka mekanismer som sympatrisk och parapatrisk artbildning kan ske.

Resultat I artikel I undersökte jag rollen av partnerval, baserat på val av värdväxt, i upprätthållandet av reproduktiv isolation under fri spridning av individer mellan olika värdväxter. Resultaten visar på att skillnader kan utvecklas fort mellan grupper kopplade till olika värdväxter, om partnerpreferens och part-nerval är länkat till just val av värdväxt. Detta främjar i sin tur utvecklingen av reproduktiv isolation som en biprodukt av resursval, och öppnar upp för möjligheten till sympatrisk artbildning. Den reproduktiva isolationen kan dock inte upprätthållas av egen kraft under fri spridning mellan värdväxter, utan kräver selektion mot hybrider, dvs. att avkomma mellan individer med olika karaktärer har sämre fitness än avkomma mellan individer med samma karaktärer.

Resultaten i artikel I stödds av resultaten i artikel II, vilka visar på att se-lektion skapad genom kraftig konkurren kan leda till en förändring i värd-växtpreferens hos sympatriska grupper. Med förändringen följde dock inte en utveckling av partnerval kopplad till val av värdväxt, ett förbehåll för att den sympatriska artbildningen de facto ska kunna ske.

Artikel III undersökte migrationens påverkan på artbildning, och jämförde olika nivåer av genflöden mellan populationer. Resultaten visade att partner-val baserat på val av värdväxt kan upprätthållas i flera generationer med migrationsflöden på omkring 13-15 individer per generation, utan hjälp av någon som helst selektion. Detta är mycket högre migrationsflöden än de traditionella parapatriska teorierna tillåter.

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Artikel IV och V undersökte båda interaktionen mellan inavel och partnerval och dess påverkan på sympatrisk och parapatrisk artbildning, artikel IV ex-perimentellt, och artikel V via datorsimuleringar. Resultaten visade på att små populationer ofta lider av inavel och inavelsdepression som ett direkt resultat av ackumulation av mutationer. I dessa populationer kan valet att para sig inom samma sort öka graden av inavel, med svår inavelsdepression, eller utdöende som följd. Då sympatrisk och parpatrisk artbildning ofta initi-eras i justs sådana små populationer anser jag att risken för inavel, och in-avelsdepression, bör tas i beaktande i de modeller där partnerval baseras på likheter, och i och med det även släktskap mellan individer.

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Acknowledgements

I owe most of my gratitude to my supervisor Mats Björklund for taking the time to teach me everything I know about science. It took a few more years than any of us had expected. Thank you for all the knowledge, support, time, understanding, and patience that you devoted. Not only did you help me achieve my goal of obtaining my PhD-degree, you also helped me fulfill a dream that was born in the head of a 12 year old girl 20 years ago, the dream of becoming a scientist. I would also like to thank my second supervisor Göran Arnqvist, who all this time lingered in the background, as a safety backup. When at times I had doubts in Mats (which of course did not happen more than once or twice), I always knew I could turn to you for a second opinion. Thank you for always keeping your office door open, for answering the questions I had, and for sharing with me your immense knowledge and great ideas. Also, thank you for your great advice of keeping quiet rather than revealing a lack of knowledge. I will keep that in mind during my dis-sertation, and when needed later in life.

Marnie, what in the world would I have done without you? For sure I would not have finished my thesis in time. Thank you for all the numerous fun days in the field, and for each and every one of our early morning talks, dealing with everything ranging from our children, to life in general, and the annoying creatures called husbands and men (and for that I am ever so sorry Niklas, but for what it is worth, Marnie and I never counted you as one of them). Thank you, Marnie, for being my dictionary, for knowing the answer to every little one of my numerous questions, and for being such a great friend. Johanna, thank you for taking over where Marnie left off, it did not pass one week before I got the first SMS with the query of where I was at and why I was not at work. Also, thank you for always being cheerful, al-ways updating me on the latest department gossip (gosh, I would have been totally ignorant if it had not been for you), and for popping in every morning for a highly unscientific chat. Also, thank you for all the times that you have helped me out in the lab, for all the good ideas that you have provided me with, and for being a great friend! Nina, you were the one that got me into all of this in the first place. Thank you for that! To me you went from being the scariest person imaginable to meet in early mornings before coffee, to the most lovable of friends and co-workers. One of the best periods in my life, were the months in Africa with you! Thank you for always letting me have my time in that god awful looking sofa in your office. And just so you know,

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fika just wasn’t worth attending once you had left. We should start hanging out more often once you get back home. I miss you tons! Mårten and Chris, my first office mates, I have both of you to thank for helping me out with the software in the first couple of years, and for a lot of fun office hours. Chris, we share some great travel memories together. With you I barely survived in a hurricane on a small boat filled with vomiting Chinese tourists. I almost drowned on the Great Barrier Reef. I was pretty much sucked dry on blood by leaches when lost in the rainforest, and I was very close to receiving a full body exam and being shot on the airport when entering the states, just be-cause you found it funny to joke about me bringing drugs into the country, and then suggesting that we should try to make a run for it. Despite living on the edge when travelling with you, the vacations I have had ever since, just doesn´t measure up! Sara and Niklas, you have both been great office mates and good friends. Niklas, I do applaud you for enduring mine and Marnies morning chats, and I also appreciate your tendency to always agreeing with us. Amber, never will I forget that pearling laughter of yours echoing from down the hall every now and again. Also, thank you for all the life saving, super yummy cookies and cakes you baked. Evy, thank you for your many encouraging words, and for sharing with me your immense experience of life! Irene, thank you for splendidly guiding me through my inner chaos, for helping me set up intermediate goals, and for always giving me a brighter view of life. Claudia and Sussie, you have both been an immense help in the lab, Claudia in educating me, and Sussie in caring for my beetles during the year I was in England. I would also like to take the opportunity to thank Rei-ja for all the help in ordering and fixing things to the lab, and Ingela for tak-ing care of office stuff, and always taking time to ask about how things are going. I also wish to send thanks to many old and special friends, for shap-ing me into the one that I am today, and for staying in contact despite my much too infrequent efforts to keep in touch. You know who you are.

Jeanette, what would life have been like if it had not been for that first day in college? You are by far my very best friend in life. Always there, supporting, loving, soothing. Thank you for being my wake-up calls those last mornings before handing in my thesis. Thank you for calling every day, several times a day, those last weeks of writing, encouraging me to continue working. Thank you for always making me believe that I could do what at times seemed to be the impossible. Being a single parent of two little trolls, working full time in the last year of her PhD, seriously, I could never have done it without you!

I owe great thanks to all members of my family, my parents, my brothers and sisters. Indeed I feel truly blessed to belong to such a big family. Mum and dad, despite the fact that none of you have had the least idea of what I have been working with these last seven years, both of you did a great job equipping me early in life with the skills needed to get to where I am today. Thank you for being great parents, and even greater grandparents! I know I

30

don´t say it very often, but I do love you. Gustaf, my baby brother, you and I have always been two of the same kind. Thank you for taking time and step-ping in during the last weeks before the handing in of my thesis, offering to read through my articles and helping me improving them despite the fact that you really did not have the time to do it. I truly appreciate that! Love u bro! We have now come to the end of what is most probably the only part of a thesis that is thoroughly red by everyone, and I have only one more person to acknowledge, Paula. How can I ever explain to you what you mean to me? Never have I ever met a person more caring, loving, kindhearted, fun, stub-born, ingenious, romantic, secure, handy, loud when angry, cuddly, unsel-fish, warm, and good looking. I am completely and utterly in love with you, and my children absolutely adore you! Thank you for making us a whole family again. You are my happy place, and I love you. / Emma

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