Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 925

Genetics and the Origin of TwoFlycatcher Species

BY

THOMAS BORGE

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004

To Camilla, Benjamin, Martin & Caroline

List of Papers

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

I Sætre G-P, Borge T, Lindell J, Moum T, Primmer CR, Sheldon BC, Haavie J, Johnsen A & Ellegren H. 2001. Speciation, introgressive hybridization and nonlinear rate of molecular evolution in flycatchers. Molecular Ecology,10:737-749.

II Borge T, Webster MT, Andersson G & Sætre G-P. Recurrent selective sweeps on the Z chromosome characterize the evolutionary history of two closely-related flycatcher species (Ficedula hypoleuca and F. albicollis). Manuscript.

III Sætre G-P, Borge T, Lindroos K, Haavie J, Sheldon BC, Primmer C & Syvänen A-C. 2003. Sex chromosome evolution and speciation in Ficedula flycatchers. Proceedings of the Royal Society of London: Biological Sciences,270:53-59.

IV Borge T, Haavie J, Sæther SA, Bures S & Sætre G-P. Paternally determined species recognition in female flycatcher hybrids. Manuscript.

V Borge T, Lindroos K, Nádvorník P, Syvänen A-C & Sætre G-P. Rate of introgression in island versus clinal hybrid zones of Ficedula flycatchers are consistent with regional differences in hybrid fertility. Manuscript.

VI Veen T, Borge T, Griffith SC, Sætre G-P, Bures S, Gustafsson L & Sheldon BC. 2001. Hybridization and adaptive mate choice in flycatchers. Nature,411:45-50.

Article I (© Blackwell Publishing Ltd), III (© The Royal Society) and VI (© Macmillan Publishers Ltd) were printed with permission from the publishers.

Contents

Introduction.....................................................................................................1The origin of genetics, genomics and speciation........................................1Speciation ...................................................................................................2Evolution of prezygotic isolation ...............................................................4

The theory of Reinforcement.................................................................4On the genetics of prezygotic isolation .................................................6

Evolution of postzygotic isolation..............................................................7The Dobzhansky-Muller model of speciation .......................................7Haldane’s Rule ......................................................................................8On the genetics of postzygotic isolation ..............................................10

Genetics and the origin of bird species ....................................................10Prezygotic isolation: ............................................................................11Postzygotic isolation:...........................................................................12Sex-chromosomes................................................................................12

General research aims ...................................................................................14

Methods ........................................................................................................15Study species and populations..................................................................15

The pied flycatcher ..............................................................................15The collared flycatcher ........................................................................16Hybrids between the collared and the pied flycatcher .........................16The semicollared flycatcher.................................................................16The Atlas flycatcher.............................................................................17Study areas...........................................................................................17

Field methods ...........................................................................................18Laboratory methods..................................................................................19

DNA preparation .................................................................................19Sequencing...........................................................................................19mtDNA ................................................................................................20Microsatellites .....................................................................................20

Single Nucleotide Polymorphisms (SNPs) ..........................................21SNP identification................................................................................22SNP genotyping...................................................................................22

Results and discussion ..................................................................................25Summary paper I..................................................................................26Summary paper II ................................................................................28Summary paper III ...............................................................................31Summary paper IV...............................................................................33Summary paper V................................................................................35Summary paper VI...............................................................................37

The small pieces and the great puzzle...........................................................39

Future prospects ............................................................................................43

Acknowledgements.......................................................................................44

References.....................................................................................................46

Abbreviations

SNP Single nucleotide polymorphism bp Base pair(s) DNA Deoxyribose nucleic acid PCR Polymerase chain reaction mtDNA Mitochondrial DNA SSCP Single-strand conformation polymorphism RFLP Restriction fragment length polymorphism DHPLC Denaturing high performance liquid chromatography CR Control region

Micro, or Mutation rate N Population size Ne Effective population size

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Introduction

Man has always been fascinated by the great diversity of organisms which live in the world around him. Many attempts have been made to understand the meaning of this diversity and the causes that bring it about. To many minds this problem possesses an irresistible aesthetic appeal. Inasmuch as scientific inquiry is a form of aesthetic endeavor, biology owes its existence in part to this appeal.

Theodosius Dobzhansky

The origin of genetics, genomics and speciation When CHARLES DARWIN wrote On the Origin of Species (1859) he had no knowledge of the mechanisms of heredity. JEAN-BAPTISTE LAMARCK’s theory of inheritance (LAMARCK 1809), which held that that traits acquired (or diminished) during the lifetime of an organism could be passed to its offspring, was at that time a perfectly reasonable hypothesis. The missing link in Darwin’s argument came with the rediscoveries of GREGORMENDEL’s thesis (1866) in the beginning of the last century.

MENDEL’s work on peas led to a new theory of evolution known as mutationism (or Mendelianism) led by HUGO DE VRIES (1900). According to this theory, mutations could produce large modifications of organisms and give rise to new species. Mutationism was opposed by naturalists, such as the biometricians led by KARL PEARSON (1897), who defended DARWIN’sview of natural selection, but without taking into account MENDEL’s laws of inheritance.

The mutationism fell in the1920s and 1930s with the theoretical work of several geneticists including RONALD AYLMER FISHER (1930), SEWALL WRIGHT (1931) and JOHN BURDON SANDERSON HALDANE (1932). However, these men’s influence on most of their contemporary biologists was limited because the theories were written in a difficult mathematical language and the empirical examples were few.

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When THEODOSIUS DOBZHANSKY wrote the book Genetics and the Origin of Species (1937) he refashioned the mathematical formulations, dressed the equations with natural history and experimental population genetics, and extended the synthesis to speciation and other important problems omitted by the mathematicians. It had an enormous impact on naturalists and experimental biologists, who rapidly embraced the new understanding of the evolutionary process as one of genetic change in populations. Together with ERNST MAYR (1942), JULIAN HUXLEY (1942), GEORGE G. SIMPSON (1944)and GEORGE LEDYARD STEBBINS (1950), DOBZHANSKY contributed to the Modern Synthetic Theory of Evolution.

Biology has undergone a fundamental change in the last 50 years due to a series of discoveries. We know that DNA rather than proteins carry the genetic information (HERSHEY and CHASE 1952), the basic structure of DNA (WATSON and CRICK 1953), how DNA functions to be transcribed to RNA and translated into proteins (CRICK 1966), how DNA replicates (MESELSON and STAHL 1958), how gene expression can be regulated (PARDEE et al. 1959), that each amino acid is coded by three nucleotides (CRICK et al. 1961) and the key to these triplet codes (NIRENBERG et al. 1966). Inventions such as the polymerase chain reaction (PCR) (SAIKI et al. 1985) and automated sequencing (SMITH et al. 1986) lead to the first completely sequenced genome of a self replicating, free-living organism- the bacteria Haemoplius influenzae Rd. (FLEISCHMANN et al. 1995). This has recently been followed by the Drosophila genome (MYERS et al. 2000), the first genome sequence of a plant (THE ARABIDOPSIS GENOME INITIATIVE2000), the human genome (LANDER et al. 2001; VENTER et al. 2001), the mouse genome (WATERSTON et al. 2002) and the dog genome (KIRKNESS et al. 2003). Several additional genomes are now either actively being sequenced or are being strongly considered for sequencing. These include several microbes, fungi, rat, bee, tomato and other crops, several livestock species, chicken, rhesus and chimp. All this information, combined with automated technology and bioinformatics, has opened a new field in biology called genomics - the study of entire genomes. The recent advancements in genetics and genomics have brought us closer to elucidating "the mystery of mysteries" i.e. how species are formed.

SpeciationSpeciation processes give rise to the vast amount of diversity of organisms on earth. Accordingly, the study of speciation has been at the heart of evolutionary biology ever since Darwin introduced the concept in On the Origin of Species (DARWIN 1859).

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Despite the seemingly limitless number of examples of speciation that nature provides, little is known about the underlying biological mechanisms of the phenomenon that, in terms of the biological species concept, can be defined as: ”The separation of populations of plants and animals, originally able to interbreed, into independent evolutionary units which can interbreed no longer owing to accumulated genetic differences” ("SPECIATION" 1999). This splitting of a single evolutionary lineage into completely reproductively isolated groups normally requires thousands to millions of years. A large number of demonstrable factors contribute to the speciation process, the most important once being geographical isolation, sexual selection and natural selection of which relative importance is difficult to disentangle. However, since the accumulated genetic differences are the very essence of speciation, it is important to focus on the barriers to the exchange of genes.

It is well known that physical barriers such as topography, water (or land), or unfavourable habitats can separate populations and keep them separated until reproductive barriers based on biological attributes of species, makes interbreeding impossible if the populations meet again. The speciation event is successful only if ecological requirements have diverged/do diverge to the extent that competition will not drive one or the other of the new species extinct (e.g. BEGON et al. 1996). In fact, all evolutionary biologists today agree that this speciation model, known as allopatric speciation, occurs and many even hold that it is the most common mode of speciation in animals (MAYR 1963; FUTUYMA and MAYER 1980; COYNE 1992). However, separated populations often come into secondary contact before reproductive isolation is completed (e.g. ARNOLD 1997). The outcome will then depend on the degree of hybridization and the fitness of the hybrids. The reproductive barriers that limit gene flow can loosely be divided into prezygotic barriers – acting to prevent hybridization – and postzygotic barriers – acting to reduce hybrid fitness (for more detailed classification, see MAYR 1963; TEMPLETON 1989). If these barriers are ineffective, introgression (gene flow across a partial postzygotic barrier) will wipe out the genetic differences that accumulated in allopatry.

Because speciation is complete when reproductive isolation prevents gene flow in sympatry (areas where divergent lineages live together), the “genetic” part of speciation properly involves the study of only those isolating mechanisms evolving up to that moment. For some organisms it is possible to artificially cross individuals from different taxa or from different allopatric populations (e.g. LIJTMAER et al. 2003). Such experiments can tell us if these organisms have developed postzygotic barriers or not. However, since the setting is artificial and because multiple barriers typically isolate species, such experiments cannot point out which isolation barrier(s) are important in preventing gene flow in incipient speciation processes in nature.

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These active barriers can best be studied in species that already are in secondary contact. Hence, hybrid zones (areas where divergent lineages come into contact and hybridize) are often seen as natural laboratories in which to study evolutionary processes involved in speciation (ARNOLD1997).

Evolution of prezygotic isolation The gene pools of two sympatric populations will not be mixed if individuals of the two populations breed at different time (temporal isolation), in different types of habitats (ecological isolation), does not fancy each other (behavioral isolation) or are not physically able to interbreed (mechanical isolation). Prezygotic isolation is by far the most critical factor keeping populations separate. Cases where species remain distinct only because of postzygotic isolation are extremely rare, if they exist at all (KIRKPATRICKand RAVIGNE 2002).

MAYR (1942; 1963) held that isolating factors only were a by-product of natural selection and/or genetic drift in allopatric populations. According to this view, merging of the populations in sympatry is a likely outcome unless isolation is complete or nearly so. In contrast, MULLER (1940; 1942) and DOBZHANSKY (1935) believed that prezygotic isolation could evolve also in sympatry. DOBZHANSKY introduced the term “isolating mechanisms” as a replacement of reproductive barriers because he meant prezygotic barriers could evolve actively by natural selection to avoid maladaptive hybridization (DOBZHANSKY 1935). In his important, but controversial theory of reinforcement of prezygotic isolation, he described how speciation can be completed in sympatry in spite of hybridization and gene flow (DOBZHANSKY 1940).

The theory of ReinforcementThe theory suggests that natural selection against the production of unfit hybrids can induce reinforcement of prezygotic isolation that eventually completes the speciation process (DOBZHANSKY 1940). The theory was developed in a scenario where two partly genetically isolated populations met in secondary contact as described above, but it can also be a key component in models of speciation that do not involve extrinsic barriers to gene exchange. In these models, the initial divergence results from adaptation to different environments that may be spatially separated – parapatric speciation (ENDLER 1977; GAVRILETS et al. 2000) or intermingled – sympatric speciation (MAYNARD SMITH 1966; BUSH 1994). Reinforcement

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may operate on any trait that increases isolation, but signals and responses involved in mate recognition are the most obvious candidates.

The theory had wide popularity until several critiques arose in the 1980s (FELSENSTEIN 1981; TEMPLETON 1981; SPENCER et al. 1986; BUTLIN 1989; SANDERSON 1989). The most important arguments were that: (1) gene flow from allopatric populations would work against adaptations in sympatry; (2) if the fitness of F1 hybrids is reduced by many heterozygous loci, each with a small effect, backcrossing might produce a relatively wide hybrid zone with relatively fit backcross individuals, so selection for prezygotic isolation will be weakened; and (3) recombination between the isolating locus and the fitness-reducing locus counteracts natural selection for reinforcement. After this massive attack, biologists considered reinforcement to be, at best, a process that rarely occurs in nature. However, empirical reviews that documented patterns of reproductive isolation consistent with the occurrence of reinforcement (COYNE and ORR 1989a; HOWARD 1993; NOOR 1999), several mathematical models (SERVEDIO and KIRKPATRICK 1997; KIRKPATRICK and SERVEDIO 1999; SERVEDIO 2000; reviewed in TURELLIet al. 2001; KIRKPATRICK and RAVIGNE 2002) and publications of several well analyzed case studies which pointed out the occurrence of reinforcement in nature (COYNE 1992; NOOR 1995a; SÆTRE et al. 1997; RUNDLE and SCHULTER 1998; HIGGIE et al. 2000; JIGGINS et al. 2001) have strengthened the interest in reinforcement. Today, the question of whether reinforcement can or does happen no longer applies. The challenge now is to determine the frequency and importance of reinforcement relative to other means of speciation (SERVEDIO and NOOR 2003).

How frequent reinforcement is in nature is not known. HOWARD’S (1993) many more examples of possible reinforcement could equally well be what BUTLIN (1989) calls reproductive character displacement (Figure 1): selection against hybridization when postzygotic isolation is complete and gene flow never has occurred. Theory suggests that reproductive character displacement is a more plausible hypothesis, mainly because the process cannot be opposed by recombination. The difference between reproductive character displacement and reinforcement is a cause/effect relation to isolation. Reinforcement is the only mechanism by which character divergence is causing isolation and not just being the effect; one of the few mechanisms where natural selection has a direct role in speciation.

BUTLIN (1989) suggested that a convincing example of reinforcement would show that (1) gene exchange between populations occurs, or did occur in the past, (2) traits associated with mating have diverged in the area of contact, (3) the divergence alters the pattern of mating in a way that reduces the

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production of unfit hybrid genotypes, and (4) the divergence is not an incidental consequence of other selection pressures.

Figure 1 Reproductive character displacement. Two lines have diverged more in secondary sexual characters in sympatry than in allopatry.

My opinion in this regard is that BUTLIN’S (1989) distinction between reinforcement and reproductive character displacement is a bit arbitrary and misleading. In some cases of reinforcement you might find much introgression between the species, other species may have nearly complete postzygotic barriers (i.e. almost no gene flow). To separate no gene flow from almost none would be virtually impossible in the field. Moreover, displacement of sexual characteristics is not a necessary consequence of reinforcement (NOOR 1995b). Thus, I suggest that reinforcement should be the term for the process in which natural selection favours adaptations against the production of hybrids, irrespective of degree of postzygotic isolation between the taxa (and whether or not any morphological character shift occurs), and character displacement the term for a character divergence in sympatry whether it is caused by reinforcement or other factors. With these definitions reproductive character displacement is a possible but not necessary consequence of reinforcement.

On the genetics of prezygotic isolation Compared to postmating isolation (see below), little concerted effort has been spent on analyzing the genetic causes of premating isolation. Studies that have examined the genetic basis of prezygotic isolation appear

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inconsistent with the traditional view of gradual adaptive differentiation involving many small changes over long time. There are clear examples in nature where adaptation has occurred rapidly, suggesting that just a few genes have been involved (GRANT and GRANT 1997a; HENDRY et al. 2000; HIGGIE et al. 2000; HAAG and TRUE 2001; ORR 2001; VIA 2001). Even complex phenotypic shifts can sometimes be accounted for by a few mutations with major effects (WOODRUFF and THOMPSON 2002 and references therein).

Evolution of postzygotic isolation Species that produce sterile or inviable hybrids can be pictured, in terms of WRIGHT'S adaptive landscape (WRIGHT 1932), as residing on two adaptivepeaks, with the hybrid genotype residing in a valley between the parental species. The problem of speciation was for a long time how two species could come to reside on different peaks without either lineage passing trough the adaptive valley. This fundamental problem was first (as noted by ORR1996) solved by WILLIAM BATESON (1909), then independently by DOBZHANSKY (1937) and MULLER (1940; 1942).

The Dobzhansky-Muller model of speciation The model describes how two or more loci that have evolved together within each species will act poorly if they are introduced to a new genetic background (Figure 2). The ancestral population of species one and species two has loci homozygous for a and b respectively. During the divergence of the two species, species 1 evolve from genotype aa to AA. Both Aa and AA are harmless together with the bb locus. Similarly species 2 evolve from bb to BB without any problem. However, hybrids between the two species, heterozygous at both loci may find themselves in an adaptive valley if the A and B alleles does not function together. Genes showing this kind of deleterious epistatic interaction are known as “complementary genes”. The “Dobzhansky-Muller” model of speciation forms the basis of all modern work on the genetics of hybrid sterility and inviability.

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Figure 2 The Dobzhansky-Muller model of speciation. Species 1 and species 2 evolve from aabb to AAbb and aaBB, respectively. Whereas one mutation (a A or b B) function properly in their “normal” genetic backgrounds, they may not function together when they are brought together in a common genome (hybrids between species 1 and 2 will all have AaBb).

Hybrid sterility is not an all-or-none phenomenon. In the very beginning of a speciation process hybrid sterility is only partial, so that some fertile offspring are produced. This attracted the attention of biologists for centuries and in 1922 HALDANE formulated an observation known as Haldane’s rule (HALDANE 1922).

Haldane’s Rule

“When in the F1 [hybrids] offspring of two different animal races [or species], one sex is absent, rare, or sterile, that sex is the heterozygous [heterogametic] sex”

HALDANE 1922, p 101

HALDANE showed that this rule is obeyed in several animal groups. Later surveys suggest that it holds in most animals known to have sex chromosomes (reviewed in COYNE 1992; WU and DAVIS 1993; LAURIE1997). Evidence suggests that the majority of incipient species evolve through a phase in which the fitness of heterogametic hybrids is reduced before they evolve full reproductive isolation (COYNE and ORR 1997). The rule therefore tells us something about general genetic events that occur

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when a new species begins to evolve. The fact that Haldane’s rule holds in organisms where females are the heterogametic sex (e.g. Lepidoptera PRESGRAVES 2002; and birds PRICE and BOUVIER 2002; LIJTMAER et al. 2003) as well as in organisms where males are the heterogametic sex (e.g. mammals (WU and DAVIS 1993) and Drosophila (BOCK 1984)) implies that it may be unrelated to sex per se and is most probably caused by the difference between heterogametic vs. homogametic genotypes.

Of the many hypotheses that have been suggested to explain Haldane’s rule, only three – the dominance theory, the “faster-male” theory, and the “faster-X” theory–remain viable (ORR 1997).

The Dominance Theory The dominance theory posits that genes involved in reproductive isolation act as partial recessives in hybrids. Under this hypothesis, the XY hybrids suffer more than the pure XX individuals because an X-linked allele incompatible with another allele (e.g. autosomal) would have greater negative effects when hemizygous than when heterozygous. This is because the X-linked incompatibility allele is not masked by dominance by the allele on the homologous chromosome (ORR 1993; TURELLI and ORR 1995; TURELLI and ORR 2000).

The “Faster-Male” Theory Haldane’s rule would be the outcome if genes expressed only in males evolve faster (due to e.g. sexual selection) than genes expressed in females, or if male reproductive characteristics (for example, spermatogenesis) are more sensitive than female reproductive characteristics to perturbation in hybrids. Evidence for the “faster-male” theory has been found in Drosophila(HOLLOCHER and WU 1996; TRUE et al. 1996), but only for hybrid fertility. However, the theory cannot explain Haldane’s rule in taxa with female heterogamety as in these taxa the female hybrids should be the sterile sex (ORR and TURELLI 1996).

The “Faster-X” Theory The faster X theory is based on the “large X-effect”. The large X-effect theory holds that genes affecting reproductive isolation are typically located on the X chromosome (COYNE and ORR 1989b). The faster-X theory holds that these X-linked genes evolve faster than autosomal ones. The relative rate of evolution on the sex chromosome genes may exceed that of autosomal genes if new advantageous mutations are typically recessive (CHARLESWORTH et al. 1987).

How important intrinsic postzygotic barriers - problems in hybrid development - are in speciation is unknown. Many species coexist in

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sympatry without strong intrinsic postzygotic isolation, which suggests that other barriers to gene exchange are important, that is, prezygotic barriers and extrinsic hybrid problems of reduced sexual and ecological fitness. In Drosophila prezygotic isolation apparently evolves faster than postzygotic isolation in sympatry (COYNE and ORR 1989a; COYNE and ORR 1997), a pattern consistent with the theory of reinforcement. However, how often postzygotic barriers, both intrinsic and extrinsic, stimulate this process is unknown.

On the genetics of postzygotic isolation In animals, two types of genetic effects seem most likely to reduce hybrid fitness: chromosome rearrangements and genetic incompatibility. As the once so popular “chromosomal speciation” theory (WHITE 1969) is suffering from theoretical, as well as empirical difficulties (COYNE and ORR 1998), more and more studies support the genetic incompatibility model (but see RIESEBERG 2001). The increase of intrinsic postzygotic isolation with time is consistent with most theories of speciation in which isolation is the by-product of gradual genetic distance. Studies of the time-course of sex-specific inviability show that the heterogametic sex becomes less fertile and viable with time since divergence (genetic distance). Later, sterility and inviability also appear in the homogametic sex, completing speciation (COYNE and ORR 1989a; COYNE and ORR 1997; SASA et al. 1998; PRESGRAVES 2002; LIJTMAER et al. 2003; RUSSELL 2003). The common pattern is that many genes are involved (TRUE et al. 1996; NAVEIRA and MASIDE 1998; WU and HOLLOCHER 1998). However, identification of “speciation genes” has shown that reduced hybrid fitness can behave as a simple two-locus Dobzhansky-Muller incompatibility (WITTBRODT et al. 1989; BARBASH et al. 2003; PRESGRAVES et al. 2003). Identification and exploration of such genes reveals the main function of the loci, and can allow one to assess whether divergence was promoted by natural or sexual selection, or by random genetic drift (TING et al. 1998).

Genetics and the origin of bird species In this thesis I am looking at the genetics, genomics, and the process of speciation in two bird species, the pied flycatcher (Ficedula hypoleuca) and the collared flycatcher (F. albicollis). These birds are some of the best-studied bird species in Europe, subject to extensive studies on a variety of fields within biology since the 1930s (see e.g. LUNDBERG and ALATALO1992). Recently, the pied and the collared flycatcher have been used as a model system for studying speciation by reinforcement (SÆTRE et al. 1997).

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However, almost nothing was known about the genetics of the pre- and postzygotic isolation mechanisms between these two bird species.

So far, Drosophila species have been main model systems for investigating the evolution of reproductive isolation (POWELL 1997), and the most important progress has been made in these systems in understanding both prezygotic isolation (KELLY and NOOR 1996; NOOR et al. 2001a; SERVEDIOand NOOR 2003) and postzygotic isolation (ORR and PRESGRAVES 2000; PRESGRAVES 2003; PRESGRAVES et al. 2003). It is not only the origin of flycatchers that suffers from lack of genetic knowledge but also the origin of all other bird species. GRANT and GRANT (1997a) go as far as saying: “…the genetics of speciation are the genetics of other organisms [than birds], mainly Drosophila”. Obviously birds differ from Drosophila and many other organisms in many ways (see below) that can have major implications for the likelihood and rate of speciation. Accordingly, more information from birds should enable us to learn more about speciation in general.

Prezygotic isolation:DARWIN’s theory of sexual selection can explain why males, and not females, often evolve elaborate secondary sexual traits (DARWIN 1874). Abundant evidence indicates that male weapons (such as antlers or extreme canines) evolve through contest over females, while male ornaments (such as the Peacock’s tail) evolve through female mate choice (ANDERSSON 1994). Darwin noted, “Secondary sexual characters are more diversified and conspicuous in birds… than in many other class of animals” (DARWIN 1874). Why should birds be especially predisposed to sexual selection? REEVE and PFENNIG (2003) suggest that differences in sex chromosome systems will lead to taxonomic biases for showy (ornamented) males. They present theory, simulations and empirical data suggesting that male ornaments and female preferences for those traits are more likely to evolve in systems with ZZ/ZW (male homogamety) than in XY/XX (male heterogamety) systems. As sexual selection on male signals (such as song, behavior, morphology or plumage traits) may play a significant role in prezygotic isolation (SÆTRE 2000), speciation models developed on XY systems may not be accurate for birds and other organisms with male homogamety (snakes and Lepidoptera).

In birds, female preference for a conspecific partner often involves learning from the parents early in life (TEN CATE et al. 1993), a process called sexual imprinting (BATESON 1966; CLAYTON 1993). Moreover, song, which usually is an important species-specific male signal, often has a culturally inherited component (KROODSMA 1982). The genetic basis of mate recognition systems is contentious. So is the role of imprinting vs. genetic inheritance of these characters in sexual selection, and the role of sexual

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selection in speciation (OWENS et al. 1999a; OWENS et al. 1999b; PIZZARI1999).

Postzygotic isolation:One of the mechanisms suggested behind Haldane’s rule, the “faster male” hypothesis, is not supported in organisms where females are the heterogametic sex. If the “faster male” effect plays a large role in Drosophila, other effects must be more important in organisms with female heterogamety. In Lepidoptera, Haldane’s rule for sterility evolves as fast (if not faster) as in Drosophila (PRESGRAVES 2002). This author argues that a theory comprising dominance and faster sex-evolution (sex-related genes of both sexes evolve fast) can explain Haldane’s rule and the faster evolution of hybrid sterility versus inviability in this taxon. In addition, taxon-specific factors such as different gene composition of the sex chromosomes can explain the “large Z-effect” in Lepidoptera. PRICE AND BOUVIER (2002) and LIJTMAER (2003) report gradual evolution of postzygotic isolation and Haldane’s rule in birds and refer to PRESGRAVES’ (2002) arguments of how this is possible in a ZW system. In birds however, hybrid problems seems to evolve slowly compared to Drosophila and Lepidoptera. Often, but not always, hybrid sterility and inviability arise after the speciation process is essentially complete. Another difference between birds and the two insect taxa might be the order in which the hybrids of heterogametic and homogametic sexes become sterile and inviable. In contrast to Drosophilaand Lepidoptera, where the heterogametic sex are both sterile and inviable before the homogametic sex is sterile, both sexes seem to evolve sterility before the heterogametic sex is inviable in birds (but see LIJTMAER et al. 2003). PRICE AND BOUVIER (2002) suggest that taxon specific factors may facilitate structural changes in chromosomes and cause problems with chromosomal pairing in birds.

Sex-chromosomes A common denominator for many differences between birds and Drosophilain factors causing pre- and postzygotic barriers seems to be the different sex chromosome system. Differences between males and females in their characteristics cause different selection pressures on each sex, and because each sex is part of the environment of the other sex some alleles may be beneficial to one sex and disadvantageous to the other. Such sexually antagonistic traits open for a sexual conflict (PARKER and PARTRIDGE 1998). Both theory (RICE 1984) and experimental data (GIBSON et al. 2002) point out the macro-sex chromosome (X/Z) to be a hotspot for sexually antagonistic fitness variation. This means that in contrast to the XY males, that only pass their Xs (with the male beneficial allele) to daughters, ZZ

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males pass their Zs both to their daughters and sons which should substantially improve the heritability of fitness between fathers and their sons, and thereby facilitate the sexual selection process (GIBSON et al. 2002).

Another factor that affects genes on sex chromosomes differently in the XY and ZW systems is the phenomenon called male driven evolution (MIYATAet al. 1987). The theory says that a chromosome passed to the next generation from a male is expected to have more mutations than a chromosome passed from a female because it undergoes many more replication steps due to more cell divisions in the male germ-line than the female germ-line (HALDANE 1935). Since the Z chromosome spends twice as much time in males compared to the X (the Z spends 2/3 and the X chromosome spends 1/3 of its time in the male germline), more mutations are expected on Z than X relative to the autosomes in the respective genomes. Such effects will also affect the micro sex chromosomes (the Y and W).

Because all organisms that have sex chromosomes do not form a monophyletic group, it appears that sex chromosomes have evolved independently many times from different sets of autosomes (MARSHALL GRAVES and SHETTY 2001). This means that the sex chromosome size, density of genes, levels of dosage compensation and number of genes involved in reproductive isolation are expected to be different within and between the two systems. A consequence of this is that nucleotide compositions and recombination rates may vary between the systems. All these factors are likely to influence the evolution of the sexes and reproductive isolation in various taxa differently.

In this thesis I will examine theories on speciation made by DOBZHANSKYand his contemporaries in the light of modern DNA techniques. By using the flycatcher model system I hope I can contribute with information on the genetics of speciation in birds.

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General research aims

To reconstruct the most likely phylogeographic scenario for the black-and-white Ficedula flycatcher species complex.

To develop molecular markers suitable for detecting interspecific gene flow caused by hybridization between the pied and the collared flycatcher.

Investigate the genetic architecture of traits involved in prezygotic isolation (plumage characteristics and mate preferences).

Investigate genetic events in the evolution of reproductive isolation, factors influencing whether or not reinforcement can evolve and the genetics of breakdown of pre- and postzygotic isolation.

Investigate the patterns of hybridization and species-assortative mating, and estimate the fitness costs of hybridization.

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Methods

Study species and populations The European black-and-white flycatchers of the genus Ficedula are small passerine birds (12-13 g) belonging to the family Muscicapidae. They are long-distance migrants, breeding in forested areas in Northern Africa and in Europe during spring and summer, while spending the rest of the year in tropical habitats in Africa. Almost all studies on these birds are carried out on their breeding ground where they are easy to study because they prefer nest-boxes over natural holes for breeding. It is possible to attract almost the whole breeding population to nest-boxes (LUNDBERG and ALATALO 1992). Hence, within local populations it is possible to obtain good estimates of important parameters such as patterns of hybridization and consequences of hybridization on fitness. Further, because of their relative tameness, handling of the parents or nestlings for taking blood-samples and phenotypic measures does not seem to have any negative effect on breeding activity.

Phylogeographic evidence suggests that the ancestor of the black-and-white flycatchers in Europe split into four geographically isolated populations around the Mediterranean Sea during the Pleistocene, giving rise to the four species know today (Paper I, SÆTRE et al. 2001). These are the pied flycatcher (Ficedula hypoleuca), the collared flycatcher (F. albicollis), the semicollared flycatcher (F. semitorquata) and the Atlas flycatcher (F. speculigera).

The pied flycatcher The pied flycatcher is one of the most studied wild bird species in Europe (see e.g. LUNDBERG and ALATALO 1992). 548 documents matched the search on “Ficedula hypoleuca” in the ISI Web of Science database. Their breeding area ranges from Spain across Europe to Russia in the northeast (Figure 3). This area overlaps with the collared flycatchers breeding area in a cline in Central-Europe and on two islands in the Baltic Sea (Öland and Gotland). The Spanish population, which previously was considered a subspecies of the pied flycatcher (LUNDBERG and ALATALO 1992), is now

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regarded as the population from which the other European pied flycatchers have originated (SÆTRE et al. 2001). Male plumage colour varies from brown or greyish-brown to black. In areas where the species occur in sympatry with the collared flycatcher, greyish-brown males are most frequent (RØSKAFT and JÄRVI 1992; SÆTRE et al. 1993; SÆTRE et al. 1997). The white forehead patch is small but varies in form and size. Females are greyish-brown and similar to the extreme greyish-brown males.

The collared flycatcher The collared flycatcher is also a well-studied bird species. 126 documents matched the search on “Ficedula albicollis” in the ISI Web of Science database. The collared flycatchers breeding in Italy are assumed to be the population from which the other European collared flycatchers originated (parallel to the Spanish pied flycatchers) (Paper I). The collared flycatcher males are generally grey-black to black with a large white forehead badge and larger white wing patches than the pied flycatchers. The males living in Italy are more variable in their plumage characters than the ones living in sympatry with the pied flycatchers (Paper III). Other allopatric populations are found south of the sympatric areas in Central Europe (Figure 3).

Hybrids between the collared and the pied flycatcher Hybrids between the collared and pied flycatchers are found in the overlapping breeding areas of the two species in central Europe and on two islands in the Baltic Sea. Both hybrid males and females are intermediate to the pure species in plumage characteristics, which is more easily seen in males. Female hybrids are most easily recognized in the field by having alarm calls from both species but a trained eye can identify them by plumage. In addition to a mixed alarm call, male hybrids also sing a mixed song (GELTER 1987). Male hybrids have reduced fertility whereas females are apparently completely sterile (ALATALO et al. 1990; GELTER et al.; SÆTRE et al. 1999).

The semicollared flycatcherThis is a little studied species (only one document found by performing the ISI search: RØSKAFT and JÄRVI (1992)). This study, earlier field reports, and descriptions in ornithological field guides often mix this species with other Ficedula species (especially the collared flycatcher) because of previous phylogeny based on plumage colour and morphology. The phenotype of this species is in many respects intermediate between the pied and the collared flycatcher, e.g. a medium-sized forehead-patch and a partial neck collar.

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The Atlas flycatcherThis species is also very little studied and no matching ISI documents were found when searching for F. speculigera. The species was previously described as a sub-species of the pied flycatcher but is now recognized as a distinct species (SÆTRE et al. 2001). Males of this species resemble the allopatric collared flycatchers breeding in Italy phenotypically, but usually lack the characteristic neck collar of the latter species (SÆTRE et al. 2001).

Study areas

Figure 3 Present geographical distributions of four species of Ficedula flycatchers. The map is based on different ornithological field-guides. The accurate distribution of the semicollared flycatcher is somewhat uncertain, and is therefore marked with “?”. Numbers refers to sampling areas given in Table 1.

Table 1: Sympatric and allopatric populations of pied and collared flycatchers

In fig. 1 Sampling area % pied flycatchers% collared flycatchers Study

1 Madrid, Spain 100 0 I, II, III, V 2 Oslo, Norway 100 0 I, III, V 3 Lingen, Germany 100 0 I. III, V 4 Öland, Sweden 30 70 I, III, IV, V 5 Jesenik Mts., Czech Rep. 15 85 I, III, IV, V, VI 6 Gotland, Sweden 5 95 I, III, V, VI 7 Budapest, Hungary 1 99 I, V 8 Breclav, Czech Rep. 0 100 I, III, V 9 Abruzzo, Italy 0 100 II, III, V

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Field methods

TrappingBoth sexes of the flycatchers are most easily trapped in nest-boxes during the time when they are feeding their offspring. Males are also relatively easy to catch by mist-net or in the nest-box at other times during the breeding season because they can easily be induced to respond appropriately to “intruding males” or “prospecting females.” By simulating these situations with playback recordings and/or by presenting a caged bird in a male’s territory, it is easy to catch him with a mist-net or a nest-box trap. These latter methods were used to catch males early in the breeding seasons and were the only means of catching in areas without nest-boxes (e.g. Atlas flycatchers, semicollared flycatchers, and Italian collared flycatchers). When a bird is caught it is kept in a light proof bag where it stays calm until measuring.

Collection of data For DNA sampling, a blood sample was taken from the birds. A brachial vein was punctured with a sterile syringe and about 25µl blood taken with a capillary tube. The blood was preserved in Queens lysis buffer (SEUTIN et al. 1991) until DNA was extracted in the lab. Each individual was ring-marked with an individually numbered ring to be recognized in later breeding seasons or in other locations (e.g. on migration), and a unique combination of coloured rings for individual recognition. In addition to a number of different morphological measurements (Table 2), sex and age was determined.

Table 2: Morphological measurements taken in the field

Morphology Measured to the nearest

Body mass 0.1 g Wing length 0.5mm Tarsus length 0.1 mm White patch of primaries 3-7 0.1 mm White on tail 0.1 mm Beak length 0.1 mm

Males only: Plumage colour Drost scalea

Forehead patch (height and width) 0.1 mm Neck collar Collar index 1-5b

a Plumage colour can be categorized using the Drost scale (DROST 1936), from jet black (I) to fully brown (VII). In addition to Drost’s score we have also estimated the surface area on the back and head covered by non-black feathers (from 0 to 100% using 5% intervals). b Neck collar index: 0=none; 1=some whitish feathers; 2=broken (ideal hybrid); 3=partly broken; 4=grey-spotted; and 5=complete white.

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Age determination was based on plumage characteristics. First-year male collared flycatchers have brown primaries/secondaries whereas older individuals have black. Females of the two species as well as male pied flycatchers, can be aged by the wear of tail, wing and secondary covert (young have worn, unmolted feathers) and the colour of the “upper mouth-roof” (pink in young birds, black in older birds) (SVENSSON 1984).

Arrival date of males and females, the day of nest building, laying date of the first egg, and hatching date was recorded for each nest-box. Data on breeding success includes number of eggs, number of hatchlings, number of fledglings and recruitments to the breeding population.

Laboratory methods

DNA preparation The traditional phenol/chloroform method was used to extract DNA from nearly all flycatcher samples preserved in Queens lysis buffer. The Chelex 100 (WALSH et al. 1991), which is a much less time consuming method, produced many extractions with too low DNA concentrations for the samples preserved in Queens lysis buffer. However, for some reason Chelex 100 extractions from whole blood fixed in ethanol worked very well (LISLEVAND et al. MS). The DNeasy® Tissue Kit (QIAGEN) was also successfully used for DNA purification from tissue samples fixed in ethanol (LISLEVAND et al. MS) and from blood preserved in Queens lysis buffer (Paper IV). DNA concentrations were measured with a spectrophotometer and diluted to a standard concentration (10ng/ l). This was particularly important for the phenol/chloroform extractions as the DNA concentrations differed extremely between the samples.

SequencingDuring this work I have used three different sequencing methods. Whereas the core technology follows the SANGER’s basic 3´-dideoxynucleoside 5´-triphosphate (“dideoxy”) protocol (SANGER et al. 1977) in all three methods, almost everything else has successively been modified to make sequencing faster, safer, easier, and more accurate. My first sequencing experience was using radio labeled nucleotides, electrophoresis, and autoradiography (some of the sequences in Paper I). In later studies (Papers I-V) this time-consuming method of manual sequencing was replaced by automated sequencing on an ABI 377 (Perkin-Elmer). In this method, the radioactive nucleotides are replaced with fluorescently labeled ones, allowing all four

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chain-termination reactions to be performed in one tube and separated in one gel lane. The traditional slab gel-based units have recently been challenged by capillary electrophoresis systems (SMITH and HINSON-SMITH 2001). Most of the sequencing in paper II is carried out on a MegaBACE 1000 capillary instrument (Amersham Biosciences). This method is faster than the previous (no gels to pour) and the risk of contamination are reduced since there is no sample spillover from one lane to the next.

mtDNASince the first analysis of mitochondrial DNA (mtDNA) variation in nature (probably BROWN AND WRIGHT’s (1975) brief paper on parthenogenetic lizards [AVISE 2000] ) a series of studies spanning over two decades document the power of mtDNA analyses in deciphering the evolutionary origins and ages of numerous vertebrate taxa (reviewed by AVISE et al. 1992). Research in the 1970s and early 1980s uncovered the major molecular and transmission-genetic features of mtDNA that instill this molecule with special value as a micro-evolutionary phylogenetic marker. These are maternal inheritance (typically without intermolecular genetic recombination), rapid evolution at the nucleotide sequence level, and extensive intraspecific polymorphism (AVISE 2000). I used mtDNA in phylogenetic analyses in Paper I. A control region (CR) segment that differs in length by 32 bp between the pied and the collared flycatchers (SÆTRE and MOUM 2000) is used for molecular species identification and as a marker in the introgression studies (Papers I, III, V, and VI)

MicrosatellitesMicrosatellites are tandemly repeated non-coding DNA (1-6bp in length). Soon after the discovery of the PCR reaction (SAIKI et al. 1985; SAIKI et al. 1988), microsatellites were genotyped by electrophoresing the PCR products on polyacrylamide gels (LITT and LUTY 1989; TAUTZ 1989; WEBER and MAY 1989). Traditionally the microsatellite markers were used in genetic mapping and diagnostics (ELLEGREN 1993 and references therein) and in forensic science (e.g. WALSH et al. 1991). During the last ten years, microsatellites have been extensively used in analysis of structures of natural populations, both for studies of sub-populations within a single species and to elucidate the evolutionary relationship between species (GOLDSTEIN and SCHLÖTTERER 1999). Recently, microsatellites have been used for detecting selection and different demographic effects that causes deviation from the variation expected under neutrality (WIEHE 1998; SCHLÖTTERER 2002). In this thesis I used microsatellites to identify extra-pair parents by using a combination of four microsatellites (Paper V and VI). One of the microsatellites was included in studies of introgression between the pied and

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the collared flycatchers (Papers I, III and V). The microsatellites were genotyped by electrophoresis on an acrylamide gel and visualized by a standard silverstaining protocol.

Even though there are numerous advantages of microsatellites in the study of natural populations (e.g. SCHLÖTTERER and PEMBERTON 1994), I chose not to continue with microsatellites in the other studies for several reasons. Population studies and “selective sweep” studies (see e.g. SCHLÖTTERER2002) require many microsatellite markers. Since bird genomes generally tend to show low frequencies of microsatellites (PRIMMER et al. 1997), they are hard to find. Furthermore, microsatellites found in chicken (the genomic model organism among birds) are not likely to be present in flycatchers. There are many different ways of screening organisms for microsatellites, all of them are practically complex and expensive and may yield only a small number of potential microsatellite loci (ZANE et al. 2002). The microsatellites obtained by these methods can be located anywhere in the genome. It is hard to find markers that have the useful amount of variation. Microsatellites are often too variable to be used as interspecific markers.

Single Nucleotide Polymorphisms (SNPs) A single polymorphic base position in the genomic DNA is considered as a SNP if the least frequent allele has a frequency of 1% or greater. SNPs are usually bi-allelic because the mutation rate is normally so low that the probability for two or more independent mutation events at the same site is very low. Although SNPs are only bi-allelic and thus may be considered less informative than hyper-variable microsatellites they are getting increasingly popular as genetic markers (reviewed by VIGNAL et al. 2002). There are four main reasons for this:

1. SNPs have been found to occur every 100-300 bp in humans, so they are far more prevalent in the genome than microsatellites, providing large sets of markers near or in any locus of interest. In flycatchers they are even more common than in humans (PRIMMER et al. 2002).

2. Some of the SNPs located in genes can be expected to directly affect protein structure or expression levels, and these may therefore represent candidate alterations for genetic mechanisms of biological relevance.

3. While repetitive sequences exhibit some instability. That is, mutations occasionally alter the size of an allele complicating analysis of the inheritance, SNPs are much more stably inherited. Almost all of them are bi-allelic and inherited in a Mendelian fashion.

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4. Increasingly efficient SNP typing systems are becoming available with very large throughputs, offering sufficient power in genetic analyses.

I primarily wanted to use SNPs (in paper III and V) because they are relatively easy to find compared to microsatellites. The way I am using the acronym SNP may be a little confusing. A fixed difference between the species should be called an “interspecific” SNP, but for simplicity I use SNP for both within- and between species polymorphisms. However, I may have been somewhat inconsequent in that regard.

SNP identification Candidate avian intron sequences were extracted from GenBank using the criteria that they were about 400–600 bp in length, i.e. to enable single read sequencing of nearly the entire PCR products, and that there was sufficient exon sequence on either side of the intron for primer design. Candidate sequences were then subjected to a Blast homology search (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Sequences which were members of closely related gene families, or for which pseudo-genes had been identified, were excluded. PCR primers were designed using the program Primer ExpressTM 1.5 (Applied Biosystems). The primers that allowed successful PCR amplification were used to sequence a few individuals from allopatric populations of each species in order to see any potential fixed differences. Such loci were sequenced, or genotyped with other methods (see below), for several more individuals to “confirm” interspecific SNPs. The whole SNP identification strategy is described in more detail in PRIMMER et al. (2002).

SNP genotyping A handful of molecular strategies using various assay formats, are in use for SNP analysis (reviewed by LANDGREN et al. 1998; SYVÄNEN 1999). I have used four of them (RFLP, SSCP, DHPLC and microarray technology).

Restriction Fragment Length Polymorphism (RFLP) analysis RFLP was one of the first marker systems developed and it was the marker of choice before the microsatellite revolution (DNA fingerprinting). The concept is based on using restriction enzymes, which cleave DNA at highly specific sites. I used the method to confirm the presence of sequence polymorphisms at specific sites by using a restriction enzyme cutting at the site of interest (PRIMMER et al. 2002). The method is very reliable and was considered a possible method for large scale screening of SNPs. However, it was hard to find restriction enzymes that gave products different enough to

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allow separation on agarose-gels. Because I only managed to find enzymes for half of the loci, other large-scale screening methods were evaluated.

Single-Strand Conformation Polymorphism (SSCP) analysis The SSCP method (first described by ORITA et al. 1989) serves as an effective tool to separate heterozygous alleles on polyacrylamide gels. Whereas the mobility of double-stranded DNA in gel electrophoresis depends on the length and not on the particular nucleotide sequence, the mobility of single strands is noticeably affected by very small changes in the sequence (possibly one changed nucleotide out of several hundred). In the absence of a complementary strand, the single strand may experience intrastrand base pairing, resulting in loops and folds that give the single strand a unique 3D structure. A single nucleotide change is likely to give a different structure affecting the strand's mobility through a gel. The method was used to screen for variation in paper I and to double-check genotypes in paper III.

Denaturing High Performance Liquid Chromatography (DHPLC) analysisA more recently developed technique (OEFNER and UNDERHILL 1995), commonly referred to as DHPLC, allows automated detection of single base substitutions as well as small insertions and deletions (reviewed by XIAO and OEFNER 2001). For detecting mutations, the essence of DHPLC is heteroduplex (double-stranded DNA in which the two DNA strands do not show perfect base complementarity) analysis. Heteroduplexes differ from homoduplexes in electrophoretic mobility and melting properties that can be detected under controlled experimental analysis. Because the method does only reveal the presence of a mismatch, its location and chemical nature has to be established by sequencing. Initially, I considered DHPLC as a promising method for large-scale genotyping of SNPs, as it is faster and more accurate then SSCP (JONES et al. 1999). However, it is only possible to see the difference between a homoduplex and a heteroduplex and not between two different homoduplexes. Moreover, frequent technical problems with the DHPLC instrument (Varian ProStar) made me look for alternative methods for SNP genotyping.

Microarray technology We genotyped the flycatcher SNPs with a tag-array-based mini-sequencing assay (PASTINEN et al. 2000). The concept of the minisequencing primer extension assay is to anneal a detection primer to the nucleic acid sequence immediately 3´of the nucleotide position to be analyzed and to extend this primer with a single labeled nucleotide complementary to the nucleotide to be detected using a DNA polymerase (SYVÄNEN et al. 1990). The elongated primers (“tags”) were hybridized to complementary primers (“anti-tags”)

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that are immobilized covalently through their 5´-ends on a glass support (PASTINEN et al. 1997). The fluorescence signals were detected and quantified using a Scan Array 5000 instrument. The method is used in Papers III and V.

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Results and discussion

Once upon a time, in a place called the Old World, there lived a flycatcher species dressed in black and white. This flycatcher species was about to be the mother of other flycatcher species we find in Europe today. The beginning of the story is obvious. The preliminary end of the story can be seen today. Different versions of the in-between story have previously been told based on interpretations of morphology (VON HAARTMAN 1949; LUNDBERG and ALATALO 1992). Paper I and II tell a different story, a story written in A, C, G and T(U). Paper I is on the evolutionary history of Ficedula flycatchers read in mitochondrial and nuclear DNA sequence. Paper II is a more in-depth analysis on the history of the pied and the collared flycatchers that still are isolated in the respective areas where they initially originated. By focusing on the sequence variation on the Z-chromosome and the autosomes I evaluate different selective and demographic forces these allopatric populations have been exposed to.

An important and evolutionary interesting chapter of the flycatcher history is the sympatric interactions between the pied and the collared flycatcher.SÆTRE et al. (1997) showed that mixed pairs are less frequent than expected from random, indicating species-assortative mating. They showed that assortative mating is caused by female preference for conspecific males with sympatric colouring over those with allopatric colouring. They also presented a phylogeny showing that the diverged plumage characters and the female preferences in sympatry have evolved in situ. The authors held that assortative mating has evolved as an adaptation to avoid unfit hybridization in accordance with the reinforcement theory. Paper III show directional differences in introgression between the species as well as differences between the autosomes and the Z chromosome. Paper III and IV suggest that the Z chromosome have disproportionate effects on mate choice, male colouring and hybrid fitness. I argue that Z-linkage of these three components together with marginal introgression on the Z chromosome enhance the probability for speciation by reinforcement.

As time went by, the two flycatchers also met on two islands in the Baltic Sea (about 150 years ago). Paper V focus on differences in introgression rate between this hybrid zone and the (presumably) older hybrid zone in Central

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Europe where hybrids have lower fitness and the process of reinforcement apparently is more pronounced.

Despite the apparently strong barriers against hybridization, females of the two species still occasionally choose a heterospecific partner. The “happy ending” when speciation is completed has not been reached since the hybrids still have some fitness and genes do cross the species barriers. Paper VI gives a possible explanation for why hybridization still occurs, indicating that the last line in the evolutionary history of these flycatchers is “to be continued” rather than “… and they lived happily ever after in reproductive isolation.”

Summary paper I

Speciation, introgressive hybridization and nonlinear rate of molecular evolution in flycatchers The phylogeographic studies of a range of animal and plant species has shown that many closely related species and subspecies in Europe have their origin from areas around the Mediterranean Sea where they were isolated from each other during the Pleistocene (HEWITT 1993; HEWITT 1996; AVISEand WALKER 1998; AVISE et al. 1998; COMES and KADEREIT 1998; TABERLET et al. 1998). It is possible that birds would be less affected by geographical barriers, such as mountains and seas, compared to other taxa because of their greater dispersal abilities.

According to Haldane’s rule (HALDANE 1922), hybridization in birds may result in sex-biased introgression: exchange of nuclear DNA through fertile male hybrids (homogametic sex) but absence of mitochondrial introgression due to female (heterogametic) sterility (TEGELSTRÖM and GELTER 1990).

MtDNA and nuclear sequences was used to reconstruct phylogenetic trees of the four Ficedula flycatchers. Together with nuclear markers and a species-specific mtDNA marker they were used to infer the amount of introgression and to see if the introgression rate differed between the two genomes and between two different hybrid zones. However, the resolution of the markers available in this rather early study was not sufficient for strong inferences about introgression rates (but see paper III and V).

The phylogeny based on mtDNA sequences (ND6, cytochrome b, and three partial tRNA genes) gave a relatively high support for the semicollared flycatcher being the first of the ingroup species to split off. The three other ingroup species seems to have diverged relatively simultaneously (Figure 4).

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Figure 4 Phylogenetic relation among Ficedula flycatchers based on mtDNA sequences. A neighbour-joining tree is presented with genetic distances drawn to scale. The scale refers to Kimura 2-parameter distances (%). Numbers at the nodes refers to bootstrap replication scores based on 1000 resamplings: 1=1000, 2=760, 3= 403, 4=1000 and 5=1000. The spotted flycatcher (Muscicapa striata) was used for outgroup rooting.

The topology of the phylogeny inferred from the nuclear sequences was similar to that based on mtDNA with respect to the order at which the species split off. Genetic distances were overall smaller at nuclear compared to mitochondrial sequences reflecting a slower rate of evolution of the former sequences. However, the relative genetic distances differed between the genomes. Compared to the mtDNA sequences the ingroup species appeared more closely related at the nuclear DNA sequences, relative to the outgroup species. This would imply either that the divergence of the nuclear DNA sequences has increased with time or that divergence of the mtDNA has leveled off with time.

The first possibility might be expected from sex-biased introgression according to Haldane’s rule. However, even if fitness data suggests that Haldane’s rule is evident among hybrids in the sympatric areas (SÆTRE et al. 1999), genetic distances were not lower between sympatric than between allopatric populations of pied and collared flycatchers. Moreover, a comparison of genetic distance between these flycatchers and an outgroup species suggests that four microsatellite loci, exhibiting allele overlap, have diverged at a similar rate as the mitochondrial ND6 gene in these three species. Thus, even if the data indicate a slight sexual bias in rate of introgression (estimated mtDNA introgression 0.4% and nuclear introgression 0.88%), we suggest that sex-biased introgression only has a minor effect in equilibrating nuclear DNA at a higher rate than mtDNA in the present hybrid zones.

We consider it more likely that the divergence of the mtDNA has leveled off with time. First, because the mtDNA analyzed was coding sequences, and

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most of the nuclear DNA was non-coding, the evolution of the former would be more constrained and could deviate more from linear evolution with time. Second, the mutation rate is much higher on mtDNA than on nuclear DNA. Finally, there is a much stronger mutational bias at mtDNA, transitions being much more frequent than transversions. Accordingly, mutational saturation would occur faster on the mtDNA genome, particularly on the evolutionary constrained nucleotide positions (primarily the first and second codon positions). When we compared the rate of divergence between the nuclear sequences and the mtDNA at third positions only (and transversions only), one of the mtDNA genes (ND6) showed a linear relationship relative to the nuclear data while the other (cyt b) approached linearity. Accordingly, we consider it likely that the observed deviation from linear evolution at the nuclear and mitochondrial sequences is mainly a result of more rapid mutational saturation on the latter genome.

Because the southernmost distribution of allopatric flycatcher populations are the same as the areas forested during the Pleistocene glaciations, we consider it likely that these are the areas where the flycatchers were isolated during the ice ages. These areas are Iberia (the pied flycatcher), north-west Africa (the Atlas flycatcher), Italy (the collared flycatcher) and Caucasus (the semicollared flycatcher). Evolutionary conservative migration patterns could explain why the flycatchers were restricted to their specific refuges. The pied and the collared flycatchers probably followed the northward expansion of deciduous forests from the southern refuges after the last glaciation, as supported by the close genetic similarity between populations of these species from different geographical regions (HAAVIE et al. 2000). The Atlas flycatcher and the semi-collared flycatcher have apparently not expanded their breeding ranges from their presumed Pleistocene refuges. With reference to the leading edge theory (HEWITT 1993; HEWITT 1996), these two species may not have had any edge to new breeding habitats that reopened after the retardation of the ice, for instance due to competition with the two other species in areas north of their present distribution.

Summary paper II

Recurrent selective sweeps on the Z chromosome characterize the evolutionary history of two closely related flycatcher species (Ficedulahypoleuca and F. albicollis)

In this study we try to get a grip on the different forces that have been/are acting on the pied and the collared flycatcher before they encountered each other by examining the genetic variation in allopatric populations (Spanish pied and Italian collared flycatchers).

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When comparing closely related species, the number of shared and fixed polymorphisms is expected to follow a fixed pattern under neutrality (WAKELEY and HEY 1997), and the levels of polymorphism and divergence across loci are expected to be correlated (HUDSON et al. 1987). Whereas selection and gene flow will give locus specific deviation from the pattern of sequence variation expected under the neutral theory, demographic events will have an effect on the whole genome (reviewed by NURMINSKY 2001). These effects can be separated by multi-locus DNA sequence analyses (HEYand MACHADO 2003), an approach which has been effectively implemented to analyze regions associated with reproductive isolation in closely related Drosophila species (MACHADO et al. 2002).

Since other papers in the thesis (III and IV) points out the Z chromosome as an important piece in the speciation-puzzle of the pied and the collared flycatchers, we were especially interested the sequence variation on the Z chromosome compared with autosomes in the two species.

By analyzing patterns of polymorphisms and divergence at several unlinked loci on the Z chromosome and the autosomes (Table 3) we found (1) average variation on the Z chromosome to be greatly reduced compared to neutral expectations based on autosomal diversity in both species; (2) significant heterogeneity between patterns of polymorphism and divergence at Z-linked loci; (3) a relative absence of polymorphisms that are shared by the two species on the Z chromosome compared to the autosomes (Table 3); that the ancestral population was probably much larger than the present populations investigated; and (4) a slightly higher nucleotide diversity in the collared than in the pied flycatcher both on Z-linked and autosomal loci (Figure 5).

Table 3 Sample size, sequence variation and polymorphisms

Z-linked genes Autosomal genes

#Genes L W SFixed SShared #Genes L W SFixed SSharedPied 9 9657 0.0012 23 11538 0.0029

Collared 9 9655 0.0014 21 2

23 11539 0.00396 51

L – total sequence length in bp W – Watterson’s estimate of per site

S – number of segregating sites that are fixed or shared between the two species

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Figure 5 Total nucleotide diversity ( W based on silent sites, presented in %) on Z-linked and autosomal loci in pied and collared flycatchers.

We used an isolation model of speciation (WANG et al. 1997) and found that the ancestral population must have been much larger than the present populations of the two species. The estimated time since divergence was found to be much longer on the Z chromosome than on autosomes, probably reflecting the accelerated loss of shared polymorphisms on the former. Even though the results fit a simulated model (WANG et al. 1997), we have already presented evidence for factors (selection) that violate assumptions of the isolation speciation model. The results from the model must therefore be interpreted accordingly.

As the introns in this study are generally too short to allow reliable comparisons of linkage disequilibrium (LD) in shared and non-shared polymorphisms it was not possible to perform the LD test of gene flow as described by (MACHADO and HEY 2003). However, introgression between the two studied populations is not expected because (1) the populations are geographically isolated; (2) there are only two shared polymorphisms found on Z; and (3) the shared haplotypes found are in general the ancestral haplotype or their close derivatives.

The reduction in polymorphism found on Z is too great to be caused by demographic effects, such as a lower male than female effective population size, and indicates the action of recurrent selective sweeps on the Z chromosome during the evolution of the two species. This hypothesis is supported by the polymorphism/divergence heterogeneity found on the Z

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chromosome as well as the low level of shared polymorphisms on this chromosome.

Because the macro sex chromosomes are pointed out as hotspots for sexual antagonistic traits (RICE 1984; GIBSON et al. 2002), are found to harbor disproportional amounts of genes involved in sex-specific traits in various organisms (SPERLING 1994; PROWELL 1998; REINHOLD 1998; RITCHIE and PHILLIPS 1998; CIVETTA and SINGH 1999; WANG et al. 2001b; GIBSON et al. 2002; LERCHER et al. 2003; SÆTRE et al. 2003), and that many of these genes often evolve rapidly (CIVETTA and SINGH 1995; CIVETTA and SINGH1998a; CIVETTA and SINGH 1998b; CIVETTA and SINGH 1999; SWANSON et al. 2001a; SWANSON et al. 2001b; SWANSON and AQUADRO 2002; MEIKLEJOHN et al. 2003; TORGERSON and SINGH 2003), we suggest that the patterns observed in this study for a large part may be explained by sexual selection acting on Z-linked genes.

With the knowledge from the other papers presented in this thesis, the substantial divergence on the Z chromosome is likely to have important implications for speciation of the pied and collared flycatcher.

Summary paper III

Sex chromosome evolution and speciation in Ficedula flycatchers

Comparative evidence suggests that the genes controlling sex and reproduction-related traits are disproportionally abundant on the macro sex chromosome (X or Z) in various organisms (REINHOLD 1998; HURST and RANDERSON 1999; SAIFI and CHANDRA 1999; RITCHIE 2000; NOOR et al. 2001b; WANG et al. 2001a). Such traits are important in the development of post- and prezygotic barriers, a fact that has often been neglected in discussions of the reinforcement hypothesis (but see NOOR et al. 2001b). Here we investigate the significance of sex-chromosome evolution on the development of post and prezygotic isolation in the pied and collared flycatchers.

With a tag-array-based mini-sequencing assay to genotype single nucleotide polymorphisms (SNPs) and interspecific substitutions, we investigate the pattern of backcrossing and introgression among sympatric flycatchers. Specifically, we look for reduced rates of introgression at Z-linked genes. We also investigate whether male plumage traits involved in reinforcing prezygotic isolation are sex-linked by comparing the phenotypes and genotypes of individuals from sympatry.

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We used 10 autosomal and 10 Z-linked nucleotide sites for the large-scale genotyping. In addition we used one species-specific microsatellite locus and a mitochondrial control region fragment that harbors a species-specific indel (SÆTRE and MOUM 2000); Paper I. We investigated hybridization and backcrossing by comparing genotypes of sympatric birds (n=302) with those of allopatric pied flycatchers (n=106) and collared flycatchers (n=51) applying the model-based clustering method of PRITCHARD et al. (2000).

The two flycatcher species differ in the ratio of inter- and intra-specific polymorphism between Z-and autosomal linked loci. Moreover, they appear more differentiated than expected at the four Z-linked genes compared with phylogenies estimated using mitochondrial and autosomal gene sequences. We suggest that a likely explanation for the observed pattern is sweeps of selection in one or both species at Z-linked genes (see also paper II).

Among sympatric birds, only pure or non-recombinant genotypes were observed at Z. We find no cases where a Z-chromosome has collared alleles at some loci but pied alleles at others (i.e. no interspecific recombination on Z). We do find evidence for intraspecific recombination on Z. Since some males with a hybrid sex chromosome genotype apparently are fertile there are opportunities for interspecific recombination. It might be that some mechanism (e.g. chromosomal rearrangements) prohibits interspecific recombination, or that recombinant genotypes are efficiently removed from the populations by selection. Whether there is interspecific recombination or not, genes on the Z chromosome have a major influence on hybrid fertility.

Whereas autosomal alleles of collared flycatcher origin were almost completely absent among pied flycatcher, many of the collared flycatchers carried at least one allele of pied flycatcher origin (Figure 6). This asymmetric introgression may be due to a difference in incompatibility between the species (i.e. pied flycatcher genome is more sensitive to foreign genes than the collared genome), the pied flycatcher females are more selective than collared females (not supported by the empirical data in paper VI), that there are a higher influx of pied flycatchers to the hybrid zones (paper V) or the probability of back-crossing to the collared flycatcher is greater because of the much higher frequency of this species (paper VI).

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Figure 6 Pattern of introgression in sympatric pied and collared flycatchers. Whereas introgression occurs on autosomal loci (A) especially from pied to collared flycatchers, it is basically absent at Z-linked loci (Z). This suggests that Z-linked genes are involved in reproductive isolation.

Correlative evidence suggests an influence of Z-linked genes on the expression of three male-specific plumage traits (white forehead patch, plumage colour and neck collar). These traits have previously been shown to be involved in reinforcement of prezygotic isolation (SÆTRE et al. 1997).

Z-linkage of genes affecting prezygotic barriers (male plumage traits) as well as postzygotic barriers (hybrid fertility) can explain why reinforcement can operate despite rather extensive introgression on the autosomes. From this conclusion we suggest that divergent selection on Z-linked, sex-limited traits may affect hybrid fertility. The effects of such a causal link between pre- and postzygotic isolation has been modeled by SERVEDIO AND SÆTRE(2003). Their model show that selection on Z-linked traits involved in prezygotic isolation can lead to hitchhiking of linked genes involved in postzygotic isolation. Divergence of the latter increases the postzygotic barriers. Furthermore, a stronger postzygotic barrier fuels the reinforcement of prezygotic barriers by increasing the selection pressure. The process goes into a positive feedback loop.

Summary paper IV

Paternally determined species recognition in female flycatcher hybrids

In paper III we showed a Z-linked association between male plumage characters and hybrid fitness which facilitate reinforcement because one of the problems with reinforcement, the breakdown of linkage disequilibrium between genes affecting pre- and postzygotic isolation (FELSENSTEIN 1981), is greatly reduced. In this paper I am examining the inheritance of the

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complementary trait involved in prezygotic isolation between the pied and collared flycatchers: female mate preferences.

With species-specific genetic markers on the Z chromosome and the mitochondria we could identify the maternal and paternal species identity of female hybrids and compare this with the species identity of the male she had chosen. Females with a pied flycatcher father predominantly mated with a pied flycatcher male, whereas females with a collared flycatcher father mated predominantly with a collared flycatcher male (Figure 7).

The preference can be due to sexual imprinting on the father or be genetically determined and linked to the Z chromosome inherited from the father. However, whether imprinted or genetically determined female mate preferences the result would be a strong association between the female preference and the Z-linked male traits.

Together with the findings in paper III that link pre- and postzygotic barriers to the sex chromosomes, the findings presented in this paper not only link male and female components of prezygotic isolation, but show (effectively) Z-linkage of all three components of isolation (male signals, female preferences and hybrid fitness).

Figure 7 Hybrid females predominately mate with males of the paternal species.

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Summary paper V Rate of introgression in island versus clinal hybrid zones of Ficedulaflycatchers are consistent with regional differences in hybrid fertility

Prezygotic and postzygotic barriers to gene exchange are not fixed properties of a hybridizing taxon but are subjected to evolutionary change due to processes such as selection, drift and gene flow. As we have seen, the penalty from producing unfit hybrids can induce reinforcement of prezygotic isolation. Such reinforcement selection may have correlated effects on postzygotic isolation (SERVEDIO and SÆTRE 2003). When genes controlling pre- and postzygotic isolation are linked on the sex chromosome, reinforcement of prezygotic isolation (selection) can increase postzygotic isolation through hitchhiking. On the other hand, introgression may over time lead to a reduced penalty from hybridization. At high levels of introgression a “hybrid swarm” would result; that is, an array of different backcrossed and recombined genotypes. Mating between members of such backcrossed populations may result in offspring that are more fit than F1-hybrids of “pure” parental types. Hence, both pre- and postzygotic isolation may increase or decrease with time in a hybrid zone pending on circumstances.

The pied and the collared flycatcher meet in two main hybrid zones; a large cline in Central and Eastern Europe and on the Swedish island of Gotland and Öland in the Baltic Sea. Although there are many similarities between the two zones there are also some interesting differences. Thanks to historical notes from Swedish naturalists we know quite precisely when the two species came into secondary contact on the Baltic Isles. The collared flycatcher was first observed on these islands around 1850. We do not know the exact age of the Central European contact zone but it would necessarily have to be older considering the re-colonization routes the birds must have taken from their Pleistocene refugia after the last glaciation period. Accordingly, the two species would have been affected by interspecific interactions for a longer time in Central Europe than on the Swedish islands. The divergence in secondary sexual traits resulting from reinforcement is much more pronounced in Central Europe compared to on the islands. Moreover, data on hatching success suggest that hybrids are more often sterile in Central Europe compared to the islands (SÆTRE et al. 1999). In both hybrid zones female hybrids appears to be sterile but hatching failure of pairs with a male hybrid is higher in Central Europe. Thus, there is some evidence suggesting that both pre- and postzygotic isolation may be stronger in Central Europe.

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Direct estimates of fertility of male hybrids using molecular markers to determine the pattern of paternity, is somewhat compromised by the rarity of hybrids. GELTER et al. (1992) used DNA-fingerprinting to investigate pattern of paternity in 7 pairs with a male hybrid from Gotland, Sweden. The hybrid sired offspring in 6 of the 7 nests. In this paper I test paternity in 4 nests with a male hybrid from Central Europe using microsatellite analysis. Of the 20 nestlings produced only one had a genotype consistent with being sired by the male hybrid. Hence, although the sample sizes are small the data are consistent with data on hatching success suggesting regional differences in hybrid fertility.

Applying a tag-array-based minisequencing assay (as in paper III) we demonstrate that the rate of introgression is much higher on the Swedish islands than in Central Europe, despite the presumed younger age of the former hybrid zone. The findings in paper III; no signs of introgression of Z-linked markers and the apparent one-way introgression pattern from the pied to the collared flycatcher, are still valid with the larger sample size used here. Apparently therefore, rather extensive introgression from pied to collared flycatchers of autosomal genes through fertile male hybrids occurs in the Swedish island hybrid zones, whereas introgression is rare in Central Europe due to stronger pre- and postzygotic barriers to gene exchange.

I suggest that two processes may contribute to these differences between the hybrid zones. First, the pattern of gene flow from allopatry is more asymmetric on the islands and this may oppose reinforcement (SERVEDIOand KIRKPATRICK 1997; SÆTRE et al. 1999). The collared flycatchers on the islands are apparently isolated from any continental population whereas there would be influx of pied flycatchers (without any adaptation to avoid hybridization) from the surrounding mainland. Consequently, the collared flycatchers receive a constant influx of pied flycatcher alleles. The resulting backcrossed collared flycatcher population may accordingly produce “hybrids” with the pied immigrants that are more fertile than F1-hybrids of pure pied and collared flycatchers. This breakdown of postzygotic isolation would further decrease the selection potential for reinforcement. In Central Europe, on the other hand, the necessary symmetry in gene flow for reinforcement to occur may be present and no breakdown of postzygotic isolation would occur. The second process that can help to explain the differences between the hybrid zones is the loop effect explained above (SERVEDIO & SÆTRE 2003). Reinforcement-selection in Central Europe may have increased postzygotic isolation through hitchhiking. Sex-linkage of genes affecting pre- and postzygotic isolation (paper III and IV) suggest that the necessary conditions for the loop effect to occur are present in the flycatchers. Based on the data available we cannot determine the relative importance of the two processes.

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A final interesting result in this study is that we find unexpectedly high rates of introgression at one marker in both hybrid zones. I discuss the possibility that the introgressed marker may be linked to a gene under positive selection in the novel genetic background. That is, that the collared flycatcher may have inherited an adaptive allelic state from the pied flycatcher through introgressive hybridization.

Summary paper VI Hybridization and adaptive mate choice in flycatchers

Theory says that it is often females of the less frequent species that is involved in hybridization because there are no or few unpaired conspecific males available (HUBBS 1955; GRANT and GRANT 1997b). This can explain why female pied flycatchers can be forced to hybridize, since pied flycatchers are rare in the sympatric areas. However, it is more difficult to understand why a female collared flycatcher should choose to pair with a male pied flycatcher, given the pre- and postzygotic barriers described in paper III and V.

Here we look at rates and consequences of hybrid pairing and we dissect the fitness consequences of different pairing and mating events by using life history data from the Czech and the Swedish hybrid zones.

Female collared flycatchers pairing with a male pied flycatcher breed later than females in pure collared pairs. Since flycatchers are selected to breed as early as possible (WIGGINS et al. 1994), the hybridization could be due to time constraints. However, because the hybrids have very low fitness, the relative loss in fitness for a female that pairs with a heterospecific male will be substantial. It is therefore difficult to understand how time constraints can reduce fitness more than hybridization.

Field-observations indicated more extra-pair paternity in mixed pairs where the female is a collared flycatcher than in mixed pairs with a female pied flycatcher. Paternity testing with microsatellites confirmed this pattern; the mean rate of extra pair paternity is four times higher in mixed pairs including a collared female than the other three possible pair combinations. Extra pair copulations with male conspecifics may be a way of reducing a female’s fitness loss of heterospecific pairing.

All extra-pair offspring in mixed pairs with a collared female, was sired by a collared male (reflecting an active extra-pair copulation strategy or a collared flycatcher superiority in sperm-competition (PRICE 1997; HOWARD1999). In mixed pairs with a pied female very few extra-pair offspring were

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found, but the few cases found again always involved a extra-pair male conspecific with the female.

By using a Z/W-linked genetic marker-pair for molecular sexing of nestlings we found more male nestlings in PFmale X CFfemale broods than in CF X CF broods. This can be explained by increased mortality of females at any time after fertilization, in accordance with Haldane’s rule (HALDANE 1922). An alternative hypothesis is that females in PFmale X CFfemale pairs adjust the sex ratio of eggs they ovulate in favour of males, since birds may have some degree of control over their offspring sex-ratio (ELLEGREN et al. 1996; KOMDEUR et al. 1997; NAGER et al. 1999; SHELDON et al. 1999). Whatever reason, this represent a second means by which the cost of heterospecific pairing are lower than they appear at first.

The fitness effect of hybridizing is better seen in number of grand-offspring recruited. Observations from the life history data was tested against fitness predictions based on paternity and sex ratio data, for all three pair types including a collared flycatcher. The loss in fitness for a female collared flycatcher pairing with a heterospecific male compared to pairing with a conspecific is still apparently substantial despite the cost-reducing mechanisms.

However, it is possible to predict the point at which pairing with a hetrospecific male yields the same, or greater, fitness return as pairing with a conspecific male, by taking into account the fact that collared flycatchers involved in mixed pairs are later breeders than those in pure collared pairs; that the former pairs fledge a greater proportion of eggs than do the latter (for unknown reasons), and the negative standardized selection gradient on laying date.

The findings suggest that hybrid pairing sometimes can represent adaptive mate choice. It also shows that interactions between species in hybrid zones can be more complex than previously recognized. Predictions from reinforcement, or sympatric speciation by sexual selection may be too simple to capture the full diversity of possible interactions in hybrid zones.

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The small pieces and the great puzzle

When I started this journey I had the benefit of standing on the shoulders of giants. The scientific field of evolutionary biology has had a long and fruitful history and a rich forest of theoretical insights and empirical evidence has seen the light of dawn. These are exciting days in the history of evolutionary biology. New molecular techniques are constantly being developed and refined, enabling us to look into old controversies and once speculative ideas at depths our forerunners could only dream of. I feel privileged to have had this opportunity to contribute in the search for possible answers to one of the deepest philosophical questions ever asked: where do we come from we living creatures of planet Earth? In that regard I hope I may have added some small pieces to the great puzzle. Below I summarize some of my main findings and try to nest up some apparent loose ends.

The European black-and-white Ficedula flycatchers constitute four closely related species (or incipient species), which most likely originated from a large ancestral population that was subdivided in geographically separated areas around the Mediterranean Sea during Pleistocene (Paper I).

The population isolated in Spain and the one isolated in Italy have evolved into the pied and the collared flycatchers respectively. During the time they were separated, natural selection, different demographic events and random drift have driven the populations in different directions (genetically and phenotypically) (Paper II). So far this is in accordance with the traditional allopatric mode of speciation (MAYR, MULLER and DOBZHANSKY) where isolating factors are by-products of adaptations and drift in allopatry. However, I show that selection has particularly influenced the genetic diversity on the sex chromosomes. I suggest that much of this may be related to sexual selection that drive the sex chromosomes of the two species more apart than the rest of their genomes. This emphasis on differences between the sex-specific gene pool and the autosomal gene pool might (as highlighted in paper III and IV) give different prerequisites for the interactions in secondary contact that has been under discussion ever since the beginning on the Modern Synthetic Theory of Evolution.

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Today the two species meet each other in two different hybrid zones in Europe. The differences that accumulated in allopatry cause low fitness in hybrids between the two species. Female hybrids are less fertile than male hybrids, which is in accordance with Haldane’s rule (HALDANE 1922). According to this rule we would expect sex-biased introgression of nuclear genes. This pattern was not significant in Paper I. However, with the greater resolution provided by more markers in paper III and V, I was able to demonstrate significant effects of sex-biased introgression. The development from the initial attempt to estimate introgression (paper I) to the more refined analyses (paper III and V) also demonstrates that one may easily jump to wrong conclusions when data are insufficient. For instance, we erroneously concluded that some introgression of mtDNA occurs in paper I. With the higher resolution provided by more markers in the later papers we found out that these individuals which appeared to have mtDNA from the “wrong” species were actually mis-identified hybrids or first generation backcrosses. Hence, I will not exclude the possibility that some of my other conclusions may have to be revised when more data becomes available.

The observation that male fertility is affected before female inviability fit PRICE and BOUVIERS (2002) observations in other bird species. This observation can be explained by a “faster Z effect”. Intrinsic postzygotic reproductive isolation (inviability and sterility) is positively correlated with increasing genetic divergence between species in Drosophila. If the genetic divergence is higher on the Z chromosome (as indicated in paper II) and if this chromosome harbours more genes affecting hybrid fertility than viability, incompatibilities are expected to arise between fertility genes before any effects on viability evolves. The “faster Z effect” might be faster than the “faster X effect” if fertility-related genes are more sensitive to incompatibilities in birds and/or if the Z chromosome diverges faster than the X chromosome (relative to autosomes). Additionally, as discussed in paper V, selection on Z-linked genes due to the effect of reinforcement might under some circumstances increase the relative divergence of Z to autosomes even further (SERVEDIO and SÆTRE 2003).

SÆTRE et al. (1997) showed that reinforcement operates in flycatcher hybrid zones. In this thesis I have provided evidence suggesting that the process of reinforcement can be better understood when we take into account the genetic architecture of the traits affecting pre- and postzygotic isolation (papers III, IV and V). My analyses suggest a significant effect of Z-linked genes in controlling both pre- and postzygotic isolation. Accordingly, the two components of reproductive isolation would not evolve independently. One of the greatest theoretical obstacles against reinforcement is recombination between genes affecting pre- and postzygotic isolation (FELSENSTEIN 1981). Hybridization and backcrossing would result in a

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random combination of the two classes of genes in individuals of mixed ancestry unless there is strong linkage disequilibrium. The linkage of these genes to the Z chromosome can therefore help to explain why reinforcement can operate in these birds despite the occurrence of introgression of autosomal genes (Paper III and IV).

However, this enhancement of the potential for reinforcement to occur may not be sufficient in all circumstances (Paper V). Demographic effects such as asymmetric gene flow from allopatry may oppose reinforcement as possibly seen on the hybrid zone of the Swedish islands of Gotland and Öland in the Baltic Sea. I find a higher rate of introgression on the hybrid zone of the Baltic Isles than in Central Europe consistent with regional differences in hybrid fertility. Further studies are clearly needed to unravel the underlying forces causing these apparent differences in strength of pre- and postzygotic isolation in the different hybrid zones.

Despite the presence of effective prezygotic barriers, apparently driven by reinforcement, females from both species might be regarded as choosing a heterospecific partner surprisingly often. One explanation is suggested in paper VI. Female collared flycatchers may actively seek a heterospecific male as social partner and reduce hybridization costs by seeking extra pair copulation (EPC) with conspecific males; and to the extent that hybrids are produced, to preferentially produce offspring of the more fertile sex (males).

However, how adaptive this strategy is can be questioned in the light of the discussion in paper IV and the results in paper V. First, if female choice is imprinted on the father rather than genetically inherited through the Z chromosome, extra-pair females having a heterospecific social father will be imprinted on the wrong species. These females will thus be prone to hybridize. Second, the fitness calculations of paper VI were based on an implicit assumption that male hybrids father a similar proportion of the hatchlings in their nests as pure males in pure pairs. Paper V demonstrates that other males may sire a significant proportion of the nestlings in the nests of male hybrids. Accordingly, the suggestion in paper VI that heterospecific pairing may be adaptive appears less likely in light of the new information obtained in these later studies. Moreover, observations from the field indicate that females can be confused in species recognition (e.g. HAAVIE et al. in press). However, this does not compromise the important finding of paper VI that females evidently reduce the costs of heterospecific pairing. I think heterospecific pairing can best be explained by females doing “the best of a bad situation,” and that the extra pair paternity and sex ratio patterns revealed (paper VI) are factors that partly compensate for these costs.

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In conclusion, my results suggest that the sex chromosomes play a major role in the speciation process in the examined flycatcher species. To some extent it feels almost embarrassing these attempts of mine to unravel the genetic architecture of pre- and postzygotic isolation when I read about the work they are able to do on Drosophila. Clearly, the depth and sophistication of analysis and molecular details in the Drosophila model systems is beyond comparison to my work. Nevertheless, I think I have shown that it is possible to conduct fairly large-scale genetic analysis also in non-model organisms and that important progress can be made. I have specifically emphasized the role of the Z chromosome in prezygotic isolation, a phenomenon not studied in any other bird species. My hope is that this work can inspire other researchers to explore the generality of these results.

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Future prospects

During the work on this project I recognize that we are just seeing the tip of the genetic complexity that is floating around in a deep soup of interfering environmental factors. However, some more complexity could definitely be melted in the cocktail of flycatcher species.

By using more SNP markers we could investigate the introgression pattern at a finer scale. To be able to link the genetic markers to phenotypes that are directly involved in isolation mechanisms we will need to know more about accurate position of the genes on the different chromosomes. Such physical mapping could be done with linkage mapping analysis. This would be interesting also with respect to study the molecular evolution of chromosomes. Could genes have been rearranged in the two species? How is the cytological architecture of these passerines compared to the chicken? To be able to link genotypes and phenotypes to a more precise location on the Z chromosome, more hybrids and backcrosses will be needed.

Almost all the primers I have used are designed from chicken. They will probably fit many other bird species. It would be interesting to do nucleotide variation studies similar to paper II in other species-complexes (with e.g. different levels of sexual selection) to find out more about how general some of the findings in this thesis are.

As the chicken genome soon will be published, new genes will become available for use as templates for genetic analysis of flycatchers and other bird species.

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Acknowledgements

To be a PhD student and father to three small children involves a lot of hard work. Being mother to three small children whose father is a PhD student is harder. Much harder. Camilla, you are amazing! Thank you!!!!!!!!!!!!!!!!!

I would also like to thank:

Glenn for excellent supervising (again) and for being a very supportive friend. You were always there when I needed you.

Jon for being like a brother.

Stein Are for support and help during the last hectically weeks of writing up this thesis.

Katja for being such a wonderful person. Thanks a bunch for all help!

Annika for making our office shine (despite the extreme chaos in my corner).

Matt for all help with data analysis and writing. You have taught me a lot!

Gunilla: You are just one happy human being! Thank you for all the help. I really hope we will have the opportunity to work together in the future.

Henrik & Øystein, my two main sources of inspiration. You showed me that it is possible to carry out seemingly impossible amounts of lab work (Henrik) and to combine a PhD with a stock of children (Øystein).

Hans, Craig, Ann-Christine for letting me play in your labs, and Craig, Simon and Katarina for improving my lab skills.

All fieldworkers that have contributed with samples and Erik, Hannah, Lori, and Sofia for primers.

Gunilla K, Anneli and Gunilla A for conducting some of the sequencing.

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Hans, and the rest of the people at the department of evolutionary biology for making it a very nice place to work at.

All you other people I know at EBC

All coauthors: Ann-Christine, Arild, Ben, Glenn, Gunilla, Hans, Johan, Jon, Katarina, Lars, Matthew, Petr, Stein Are, Simon, Thor, Truls and Stanislav for the cooperation.

Nisse, for all the nest-boxes and Rose-Marie for taking care of business.

Special thanks to mom, dad, Kjersti, Vidar and to other family members visiting and helping us here in Uppsala.

My work was supported by Oscar & Lili Lamms Memorial Foundation.

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Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

from the Faculty of Science and TechnologyEditor: The Dean of the Faculty of Science and Technology

Distribution:Uppsala University Library

Box 510, SE-751 20 Uppsala, Swedenwww.uu.se, acta@ub.uu.se

ISSN 1104-232XISBN 91-554-5846-7

A doctoral dissertation from the Faculty of Science and Technology, UppsalaUniversity, is usually a summary of a number of papers. A few copies of thecomplete dissertation are kept at major Swedish research libraries, while thesummary alone is distributed internationally through the series ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Science and Technology.(Prior to October, 1993, the series was published under the title “ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Science”.)