Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 924

Disentangling the ReticulateHistory of Polyploids in Silene

(Caryophyllaceae)

BY

MAGNUS POPP

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004

Therefore, one should know that Perfect Understanding is a great mantra, is the highest mantra, is the unequalled mantra, the destroyer of all suffering, the incorruptible truth. A mantra of Prajñaparamita should therefore be proclaimed. This is the mantra:

“Gate gate paragate parasamgate bodhi swaha”

(The Prajñaparamita Heart Sutra)

List of Papers

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

I Popp, M. and B. Oxelman. 2001. Inferring the History of the Polyploid Silene aegaea (Caryophyllaceae) Using Plastid and Homoeologous Nuclear DNA Sequences. Molecular Phylogenetics and Evolution 20 (3): 474–481.

II Popp, M. and B. Oxelman. Evolution of a RNA Polymerase Gene Family in Silene (Caryophyllaceae) –Incomplete Concerted Evolution and Topological Congruence among Paralogues. Submitted to Systematic Biology.

III Popp, M., P. Erixon, F. Eggens and B. Oxelman. Origin and Evolution of a Circumpolar Polyploid Species Complex in Silene(Caryophyllaceae). Manuscript.

IV Popp, M. and B. Oxelman. Origin and Evolution of North American Polyploid Silene (Caryophyllaceae). Manuscript.

The first paper is reproduced with the publisher’s kind permission.

All papers included in this thesis are written by the first author, with comments and suggestions given by the co-authors. The studies were planned in cooperation with the co-authors. The first author is responsible for all analyses and the major part of the laboratory work. The second author of the third paper produced the psbE-petL dataset.

Contents

Introduction.....................................................................................................1

Polyploidy – a brief history.............................................................................3

RNAP introns – a useful history .....................................................................5

Silene L. – a complex history..........................................................................6

Polyploidy in Silene L. – a reticulate history ..................................................8Objectives...................................................................................................8Materials and Methods ...............................................................................8Summary of results.....................................................................................9

Paper I....................................................................................................9Paper II ................................................................................................10Paper III ...............................................................................................10Paper IV...............................................................................................12

Conclusions ..............................................................................................14

Sammanfattning (Swedish summary) ...........................................................15

Acknowledgements.......................................................................................17

References.....................................................................................................19

1

Introduction

The significance of polyploidization, i.e., the multiplication of entire chromosomal complements (Stebbins, 1971), as an evolutionary process has been widely acknowledged (e.g., Stebbins, 1971; Grant, 1981; Masterson, 1994). Due to its hybrid origin, an allopolyploid contains two or more different nuclear genomes. This often leads to differences between species phylogenies and gene phylogenies as depicted in Fig. 1. There are, however, several other evolutionary important processes causing multiple variants in a sequence region. Allele variation, gene duplication, recombination and lateral gene transfer are examples of such processes (reviewed in e.g., Avise, 1989; Doyle, 1997; Wendel and Doyle, 1998). By analyzing several independent biparentally inherited DNA regions, it is possible to distinguish the effects of allopolyploidization from other processes.

Figure 1 Taxon B originates after hybridization between taxon A and C as depicted in (a). Biparentally inherited nuclear DNA reflects the history of both parental lineages (b). In a phylogenetic analysis, the B1 genome in taxon B obtained from taxon A is revealed as most closely related to taxon A, whereas the B2 genome obtained from taxon C is most closely related to taxon C.

The genus Silene L. comprises ca. 650 species (Oxelman et al., 2001), and is most diverse in the Mediterranean and Middle East. The incidence of polyploidy in Silene is generally low, but e.g., Silene aegaea (endemic to Greece), a number of Arctic and subarctic taxa and the vast majority of taxa endemic to North America, are polyploids (Kruckeberg, 1961; Oxelman, 1995, Elven et al., in prep.). Analyses of several putatively unlinked biparentally inherited low copy nuclear DNA regions, in combination with the ITS region and chloroplast regions, made it feasible to study the origin and evolution of some of these polyploids and their close relatives. The low

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copy regions are intron regions in four genes, RPA2, RPB2, RPD2a and RPD2b, encoding the second largest subunit of three different RNA polymerases. A duplication of RPD2 detected in (but not necessarily restricted to) the tribe Sileneae is inferred to account for the paralogy.

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Polyploidy – a brief history

During the work with vegetative grafts and chimeras of Solanum, Winkler (1916) discovered that some of the plants thus obtained had twice the number of chromosomes compared to the parent plant (Grant, 1981). To describe organisms with more than two complete sets of chromosomes, Winkler introduced the term “polyploid”. Preceding Winkler with almost a decade, Lutz and other workers demonstrated similar “sudden chromosome doublings in Oenothera mutants” (in Digby, 1912).

The polyploid condition had been known for some years, though, and a classical example of polyploidy is Primula kewensis (Digby, 1912). Primulakewensis was discovered 1899 as a spontaneous diploid hybrid between the closely related species P. floribunda and P. verticillata growing next to each other in the Royal Botanic Gardens, Kew, UK. The hybrid was highly sterile and thus propagated by cuttings. Half a decade later, one of the P. kewensisplants set seed after the pollination of a single pin flower with pollen from a thrum flower (pin and thrum referring to alternative stamen and style arrangements in the flowers). The progeny had both pin and thrum flowers and was fully fertile. Working with both sterile and fertile P. kewensis,Digby (1912) discovered that the sterile plants had the same number of chromosomes as the parental species, i.e., 2n = 2x = 18, whereas the fertile plants had twice that number, i.e., 2n = 4x = 36.

A number of definitions and criteria have been used to distinguish different types of polyploidy. Using the terminology introduced by Kihara and Ono (1926), the Oenothera and Solanum polyploids presented above are referred to as autopolyploids, whereas the Primula polyploid is an allopolyploid. Since the introduction of the terms auto- and allopolyploidy, the terminology has been extended to cope with the different meiotic behaviors and/or taxonomical ranks often used to describe the type of polyploidy and infer the mode of origin of polyploid organisms (e.g., Stebbins, 1971; Grant, 1981). In this thesis, the terms auto- and allopolyploidy are used in a strictly phylogenetic context, disregarding chromosomal behavior and taxonomical ranks.

The modes of origin of polyploids are mere hypotheses that can be tested in a phylogenetic context. If H0 = “the polyploid is an autopolyploid” then H1 = “the polyploid is an allopolyploid”. Autopolyploidy is the null-hypothesis, and as such cannot be “confirmed” even though the analysis may reveal a sistergroup relationship of the paralogues. If the analysis reveal a

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non-sistergroup relationship of the paralogues, then autopolyploidy is rejected in favor of allopolyploidy. However, duplication of a DNA region followed by lineage sorting is a potential source of Type I error, i.e., H0(autopolyploidy) is wrongly rejected in favour of H1 (allopolyploidy). Type I errors are minimized by analysing several independent biparentally inherited DNA regions, because it is unlikely that the same pattern of lineage sorting will be displayed in unlinked regions. Type II errors, i.e. cases where H0(autopolyploidy) is not rejected when, in fact, H1 (allopolyploidy) is true, are best adressed by carefully choosing DNA regions containing a phylogenetic signal strong enough to separate the lineages included in the analysis.

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RNAP introns – a useful history

The RNA polymerase family (RNAP) consists of three large nuclear DNA dependent RNA polymerase holoenzymes in most eukaryotes. RNAP I and III transcribe structural RNA such as rRNA and tRNA, respectively, whereas RNAP II mainly transcribes mRNA. However, a fourth member, RNAP IV, is found only in plants and its function is yet to be elucidated (The Arabidopsis Genome Initiative, 2000). In Arabidopsis thaliana, three of the genes (RPA2, RPB2, and RPC2), encoding the second largest subunits of these holoenzymes, are single-copy and are located on chromosomes 1, 4, and 5, respectively, whereas the fourth (RPD2) is present in two, presumably recently diverged paralogues located on chromosome 3 (The Arabidopsis Genome Initiative, 2000).

Two highly conserved amino acid motifs (GDK and GEMERD, Fig. 2) are present in all five genes, including the two RPD2 paralogues. By targeting these amino acid motifs with degenerated primers in a nested PCR approach, it is possible to amplify and separate the DNA regions between GDK and GEMERD in all four subunits.

Figure 2 Structure of second largest subunits of the RNAP gene family in Arabidopsis thaliana. Boxes represent exons and lines represent introns. Lengths are proportional to scale bar. Arrows indicate the highly conserved amino acid regions GDK and GEMERD, and also approximate primer sites for RNAP10F, RNAP10FF, RNAP11R, and RNAP11bR used in paper II. Note that the two paralogous RPD2sequences in A. thaliana are not orthologous to the two paralogues in Sileneae(paper II).

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Silene L. – a complex history

The genus Silene L. (Caryophyllaceae) is distributed mainly in the Northern hemisphere and is most diverse in the Mediterranean and Middle East. Together with the closely related Agrostemma, Cucubalus and Lychnis,Silene was included by Linnaeus (1753) in Species Plantarum. Based on the number of styles, Silene and Cucubalus (having three styles) were discerned from Agrostemma and Lychnis (with five styles). Traditionally, most post-Linnaean taxonomists have separated Cucubalus from Silene by fruit type (a fruit with a fleshy mesocarp vs. a capsule), and Agrostemma from Lychnisby its hairy styles and carpels alternating with the calyx segments (e.g., Chowdhuri, 1957). In 1812, Melandrium Röhl. was erected and discerned from Lychnis by its usually inflated calyces and capsule teeth twice the number of carpels, and from Silene by partial septation of the capsule. Melandrium was recognized by both the major 19th century monographers of Silene, Rohrbach (1869) and Williams (1896). Since Linnaeus, several additional genera have been distinguished by different authors (see Oxelman and Lidén, 1995, and Oxelman et al., 2001, for a more thorough review). The following text discusses some of the groups where polyploidy is known to occur.

In a revision of North American species of Silene, Hitchcock and Maguire (1947) noted that the characters used to separate Melandrium from Sileneand Lychnis were too variable among American species to be of any taxonomical value at the generic level. They chose to include the majority of North American representatives of Melandrium in Silene, the revision thus encompassing 54 species in total.

Chowdhuri (1957) dismissed the use of subgenera in his worldwide revision of Silene, and instead erected 44 sections that he considered natural groups. The native North American species, of which the majority was, or had been, included in Melandrium, were included in Silene and dispersed among several sections. Most of the species were included in the sections Occidentales Chowdhuri, Graminifoliae Chowdhuri, and QuadrilobataeChowdhuri. Chowdhuri also brought back S. menziesii and its close allies (=Anotites Greene) and placed them in section Rupifraga Otth in DC.

A number of Melandrium species not considered by Hitchcock and Maguire (1947) were included in Silene section Physolychnis (Benth.) Bocquet (=S. section Gastrolychnis (Fenzl.) Chowdhuri) by Bocquet (1969). The taxonomy of some of the groups in S. section Physolychnis is somewhat

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confused despite the revision by Bocquet (1969). The Arctic/subarctic taxa have been referred to as Gastrolychnis (e.g., Cherepanov et al., 2000), Lychnis (e.g., Polunin, 1959), Melandrium (e.g., Hultén, 1968; Komarov, 1970), or, more appropriately, included in Silene (e.g., Oxelman et al., 2001; Elven et al., in prep.). Furthermore, recent phylogenetic studies (e.g., Oxelman and Liden, 1995; Oxelman et al., 1997; Oxelman et al., 2001) based on molecular data have identified a well supported clade of representatives from S. section Physolychnis, section Occidentales, section Odontopetalae, the S. ajanensis (=Lychnis ajanensis) group, and several other species.

Silene sedoides and its close allies have previously been classified in S.section Dichasiosilene ser. Rigidulae by Rohrbach (1869) and in S. section Atocion subsection Divaricatae by Chowdhuri (1957). Both morphological and molecular data reject these groups as monophyletic (Oxelman, 1995; Oxelman and Liden, 1995; Oxelman, 1996), and instead, Silene section Sedoideae Oxelman and Greuter was erected (Oxelman, 1995). The tetraploid S. aegaea was hypothesized by Oxelman (1995) to be an allopolyploid, closely related to S. sedoides and S. pentelica.

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Polyploidy in Silene L. – a reticulate history

Objectives The objectives of paper I are to: 1) test the hypothesis of an allopolyploid origin of Silene aegaea, 2) investigate whether evolutionary processes have homogenized the putative paralogous DNA regions under study in S.aegaea, and 3) evaluate whether the combination of RPB2, ITS, and rps16sequence data may be a useful system to study the evolution of polyploids.

The objectives of paper II are to: 1) test the phylogenetic hypothesis based on ITS and rps16 data in Sileneae (Oxelman et al., 2001), 2) provide future studies of Sileneae with backbone information from several, presumably unlinked regions, thus facilitating inferences of gene duplications and allopolyploidizations, and 3) investigate the topological congruence among the datasets.

The objectives of paper III are to: 1) test hypotheses of the origin and evolution of a circumpolar polyploid species complex in Silene.

The objectives of paper IV are to: 1) test if the Silene native to North America included in the study form a monophyletic group. 2) infer the mode of origin (auto- or allopolyploid origin) and the relationships of the polyploid taxa, and 3) investigate the relationship of S. section Physolychnis and the native North American Silene.

Materials and Methods All plant materials used are listed with voucher data in paper I – IV.

The approach here taken for studying the origin and evolution of polyploid taxa in Silene, is using gene phylogenies inferred from biparentally inherited, putatively independent, DNA sequences combined with chloroplast DNA regions. The inferred gene phylogenies can explain the phylogenetic network responsible for the historical relationships among allopolyploids and their parental lineages.

In the polyploids, the biparentally inherited DNA regions are isolated and characterized by cloning PCR products and sequencing secondary PCR products obtained from individual clones. The chloroplast DNA is

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sequenced from primary PCR products, as are the nuclear regions for the majority of the diploids.

To make inferences of phylogenetic relationships, PAUP* (Swofford, 2002) is used. For the different optimality criteria and search strategies employed, see paper I – IV.

Summary of results

Paper I Phylogenetic analyses reveal two distinct types of biparentally inherited nuclear RPB2 and ITS sequences in the two accessions of tetraploid (2n = 4x = 48) Silene aegaea investigated. From the two datasets, respectively, one sequence type is found in a clade formed by diploid (2n = 2x = 24) S. pentelica and one in the clade formed by diploid (2n = 2x = 24) S. sedoides.Analysis of the chloroplast rps16 dataset recovers both S. aegaea accessions in the S. pentelica clade. Using the gene phylogenies of RPB2, ITS, and rps16 to infer the history of tetraploid S. aegaea, there is strong support for an allopolyploid origin of S. aegaea, with the maternal ancestor from the S.pentelica lineage, and the paternal contributor from the S. sedoides lineage (Fig 3).

Figure 3 The species phylogeny inferred from the gene phylogenies obtained from analysis of RPB2, ITS and rps16 (paper I). Silene aegaea is inferred to be an allopolyploid, originating from the S. sedoides and S. pentelica lineages.

Three of the 17 clones obtained from the ITS regions in S. aegaea were inferred to be recombinants, i.e., chimerical sequences partly of sedoidestype and partly of pentelica type. A PCR and cloning experiment with S.sedoides/S. pentelica template mixture demonstrates that under the

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prevailing PCR conditions, PCR recombination cannot be ruled out as an explanation for the recombinant S. aegaea clones. The conclusion is therefore that no convincing sign of ongoing homogenization of homoeologous DNA regions, such as in the ITS regions of Gossypium(Wendel et al., 1995) and Saxifraga (Brochmann et al., 1996), is detected in S. aegaea.

Paper II A low stringency nested PCR approach is used to amplify the region between the highly conserved amino acid regions GDK and GEMERD in the low copy nuclear DNA genes RPA2, RPB2, RPD2a, and RPD2b (Fig. 2). Used in concert with the ITS region and the rps16 intron from the chloroplast, they resolve previously poorly known major relationships in Sileneae (Fig. 4). Maximum parsimony analyses of the separate datasets result in largely congruent phylogenetic trees for all regions.

Overall model congruence is tested in a likelihood context using the software PLATO. It is found that ITS, RPA2, and RPB2 deviates from the maximum likelihood model for the combined data. The topology parameter is isolated and topological congruence assessed by non-parametric bootstrapping. No strong topological incongruence is found. Two paralogues of RPD2 are found in –but not necessarily restricted to– Sileneae. Several independent losses and incomplete concerted evolution are inferred in the RPD2 gene phylogeny.

Paper III Phylogenetic analyses of two chloroplast DNA regions and five putatively unlinked nuclear DNA regions are used to explore the relationship of a circumpolar Arctic/subarctic polyploid species complex in Silene. Gene phylogenies inferred from introns in the low copy nuclear genes RPA2,RPB2, RPD2a and RPD2b, and the ITS region from the nuclear ribosomal DNA region, indicate two consecutive hybridization events. The inferred species phylogeny is depicted in Fig. 5.

In general, two paralogous sequences are identified from the tetraploids and three paralogues from the hexaploids in the low copy nuclear genes. Interlocus concerted evolution appears to have homogenized the ITS regions, and only the sequences inferred to correspond to the paternal lineages were recovered in the polyploids.

Paralogous RPA2 introns sequences obtained from the diploid S.ajanensis group were inferred to have originated by gene duplication followed by extinction in the polyploid lineages, or by lineage sorting of an ancient allele pool.

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Figure 4 One of two most parsimonious trees from the analysis of the combined datasets (paper II). Branch lengths are proportional to number of changes. Numbers associated with nodes indicate parsimony bootstrap percentages. Nodes without numbers have bootstrap percentages < 50.

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Figure 5 Inferred species phylogeny of the polyploids S. involucrata (2n = 4x = 48) and of S. sorensenis and S. ostenfeldii (2n = 6x = 72; paper III). The first hybridization event (a) involved the diploid S. uralensis (2n = 2x = 24) lineage as the cytoplasmic donor, and the diploid S. ajanensis (2n = 2x = 24) lineage as pollen donor. The hybridization and polyploidization resulted in the tetraploid S.involucrata lineage. A second hybridization and polyploidization with the S. ajanensis lineage as pollen donor, and the tetraploid S. involucrata lineage as cytoplasmic donor, resulted in the hexaploid lineages of S. sorensenis and S.ostenfeldii.

Paper IV DNA sequences from the rps16 intron and the psbE-petL spacer from the chloroplast genome, combined with the ITS region and introns from the low copy nuclear genes RPA2 and RPB2, are used to infer origins and phylogenetic relationships of North American polyploid Silene and its close relatives. Although the vast majority of North American Silene are polyploid (2n = 4x, 6x, 8x), which contrasts to the diploid condition dominating in other parts of the world, the phylogenetic analyses reject a single, monophyletic origin of the North American polyploids. Two separate North American lineages are revealed (Fig. 6). One of the lineages consists of tetraploid Silene menziesii (2n = 4x = 48) and its diploid allies. The second lineage leads to a clade consisting of Arctic, European and Asian taxa in addition to the majority of the North American polyploids.

The tetraploid S. californica (2n = 4x = 48) and the hexaploid (2n = 6x = 72) S. hookeri are derived from separate allopolyploidization events between the two main lineages. Despite extensive cloning, no trace of paralogy indicating an allopolyploid origin is found in S. menziesii.

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Figure 6 One of >32100 MP trees found in the RPB2 dataset analysis (paper IV). Branches drawn in thin lines are collapsed in the strict consensus tree. Numbers associated with nodes indicate maximum parsimony bootstrap frequencies. Branch lengths are proportional to number of changes. Numbers of sequenced clones are given after the taxon names. Arrows in bold indicate the positions of sequences recovered from hexaploid S. hookeri. Groups with North American taxa are in square brackets.

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Due to sampling differences between the datasets, its it difficult to distinguish patterns of allopolyploidy from e.g., lineage sorting in several other taxa. There are, however, several more putatively separate allopolyploid origins indicated by the failure of multiple sequences obtained from individual accessions from both the RPA2 and the RPB2 intron to form monophyletic groups.

Conclusions Polyploidy in general, and allopolyploidy in particular, has played, and most likely, still plays an important role in the evolution of Silene. It occurs rather isolated in the allotetraploid Silene aegaea, only known from a steep slope, overlooking a stony beach on the Aegean island of Ikaria, Greece. It also occurs in a complex pattern of reticulations, connecting Asia, Northern Scandinavia, the Arctic and North America down to South America, and gives the impression of gaining momentum from both diploid and polyploid genomes.

The DNA regions used to infer the phylogenetic relationships all have their share of advantages and disadvantages. Chloroplast regions, such as the rps16 intron and the psbE-petL spacer, are usually easy to amplify and sequence. They are useful for discerning the cytoplasmic lineage in a phylogenetic network. Since the chloroplast is uniparentally inherited, it cannot reveal reticulations.

The nuclear ribosomal regions, such as the ITS region, are biparentally inherited and easy to amplify, too. The homogenizing effect of interlocus concerted evolution of the paralogues, however, can virtually hide the history of one or more parental genome in allopolyploids if standard PCR methods are used. This is not always the case, though, as seen in S. aegaea.

Low copy nuclear DNA (lcnDNA), a group to which the RNA polymerase gene family RPA2, RPB2 and RPD2 belongs, is the most promising candidate in the toolbox. It is assumed to be less vulnerable to interlocus concerted evolution than the ITS region. Furthermore, the number of potentially useful low copy regions in the nucleus is practically inexhaustible. The evolution of lcnDNA regions is largely unknown, but seem to be rather dynamic with fluctuating copy numbers, differences in chromosomal locations, and recombination events. Another drawback is that few, if any, protocols for lcnDNA regions can be applied to a given plant group without optimization and redesign of PCR primers and of PCR parameters. However, by using degenerated primers and nested PCR, and an increasing understanding of the dynamics of lcnDNA regions, they may prove very well suited for inferring polyploid evolution.

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Sammanfattning (Swedish summary)

När genmodifierade grödors vara eller icke vara diskuteras, är det sällan naturens egen “genmodifiering” nämns. Som alltid är naturen mer storslagen. Där människan nöjer sig med att ändra en egenskap här och en där, slår naturen till och fördubblar nästan hela arvsmassan, cellkärnans kromosomer, i ett slag! Ofta sker fördubblingen i samband med en hybridisering, det vill säga en korsning mellan två olika arter. Kromosomtalsfördubblingen gör ofta den vanligtvis sterila hybriden fertil, samtidigt som den innebär en barriär mot återkorsningar med de båda föräldraarterna. På så sätt har en ny utvecklingslinje, eller en ny art om man så vill, bildats.

Kromsomtalsfördubblade hybrider är både vanliga och viktiga. Vanliga på så sätt att uppskattningsvis 35 – 50% av alla nu levande blomväxtarter är kromosomtalsfördubblade och av dem är troligen en stor del av hybridursprung. I ett mer historiskt perspektiv kan man säga att de flesta nulevande arter är härstammar från kromsomtalsfördubblade hybridföräldrar. Viktiga är de så tillvida att mycket av det vi äter varje dag, är naturligt kromsomtalsfördubblade. Potatis, majs, ris, vete, kaffe, äpple, banan, sockerrör, sojabönor, de flesta av våra kålsorter – listan kan göras väldigt lång! Två andra ekonomiskt viktiga kromosomtalsfördubblade växter är tobak och bomull.

Genom att “läsa av” arvsmassan hos olika arter går det att analysera släktskapsförhållandena mellan dem. Kromomsomtalsfördubblade hybrider har kvar en stor del av sitt genetiska arv från de olika föräldra-arterna. Om man “delar upp” arvsmassan kan man genom en släktskapsanalys se var de olika delarnas närmaste släktingar finns, och sedan göra tolkningar om föräldra-arterna utifrån det släktträdet.

Jag har studerat uppkomst och utveckling av kromsomtalsfördubblade hybrider hos släktet Silene, bläror och glimmar, i nejlikfamiljen. Släktet omfattar ca 650 arter och finns nästan uteslutande på norra halvklotet. Arbetet har dels rört en liten, väl avgränsad grupp i medelhavsområdet, och dels en betydligt större och mer komplex grupp som verkar sträcka sig över Asien, norra Skandinavien, Arktis, Nordamerika och Sydamerika.

I det första arbetet (paper I) visar jag att den mycket sällsynta grekiska Silene aegaea från ön Ikaria är en kromsomtalsfördubblad hybrid mellan de närbesläktade arterna S. sedoides och S. pentelica som båda finns på Ikaria. Det andra arbetet (paper II) innebär en utveckling av de metoder jag använde

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i det det första arbetet. Fler delar av arvsmassan används för att få mer tillförlitliga resultat i släktskapsanalyserna. Jag tar också fram ett mer omfattande släktskapsträd för släktet Silene och några närbesläktade växtsläkten. Det tredje arbetet (paper III) visar att den arktiska S. involucrata(polarblära) uppkommit som en kromsomtalsfördubblad hybrid mellan förfäder till en grupp växter (representerad av S. ajanensis och S. linnaeana)från Sibirien och nordöstra Asien och en annan arktisk grupp (representerad av S. uralensis, fjällblära). Genom en återkorsning och ytterligare en kromosomtalsfördubbling mellan S. involucrata och S. ajanensis-gruppenuppkom sedan bland annat den grönländska S. sorensenis. Det fjärde och sista arbetet (paper IV) visar att de nordamerikanska kromsomtals-fördubblade hybriderna inom Silene inte bildar en isolerad grupp, utan ingår i en större grupp där växter från Asien, Europa (studerade också i paper II och III) och Sydamerika också ingår. De olika geografiskt vitt skilda växtarterna hålls samman av ett komplext nätverk av hybridiseringar och kromsomtalsfördubblingar.

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Acknowledgements

Det har varit lite pyssel att få ihop den här avhandlingen... Den hjälp jag fått av er, mina vänner, har inte varit en tillgång utan själva förutsättningen för att lyckas. Jag hoppas att ni förstår det.

En som kanske tycker att det här har varit lika pyssligt som jag, är min huvudhandledare Bengt Oxelman. Bengan, du har dragit ett enormt lass, framförallt nu när slutet närmat sig med överraskande snabba steg. Jag fick en känsla av samförstånd redan när du var i Seattle i början av min doktorandperiod. Jag skulle köpa en dator och mailade ett förslag till dig. Ditt motförslag var att skippa datorn och i stället investera de ca. 20000 kronorna i Single Malt av god kvalitet. Du erbjöd dig till och med att ansvara för lejonparten av inköpen under din hemresa från Seattle! Det erbjudandet, tillsammans med din förmåga att lyssna, diskutera och engagera, har gjort att jag förstått att ingen rimligtvis kan önska sig en bättre handledare. Tack Bengt.

Min biträdande handledare, Magnus Lidén, var den som fick mig intresserad av botanik från början. Visserligen lurade du mig på fältarbete i Kina, men det kompenserades mer än väl genom att vi åkte till Iran där vi samlade Dionysia. Det var också i Iran som du gav mig tipset att söka den här doktorandtjänsten. Magnus, jag har mycket att tacka dig för. Inte att undra över att jag alltid blir väldigt glad av att se dig!

Ett stort tack vill jag ge till Birgitta Bremer, Kåre Bremer, Leif Tibell och Mats Thulin, dels för stöd och uppmuntran under åren som gått, och dels för värdefulla kommentarer av manuskript och avhandling nu mot slutet. Sylvain Razafimandimbison have spent quite some time discussing the peculiarities of ITS with me, and I greatly acknowledge you for that, and also for your valuable comments on the manuscripts. Jag vill också tacka Ulla Hedenquist, inte för det administrativa arbetet du utför och som jag tackar dig för i tid och otid. Nej, istället vill jag tacka dig för dina oerhört träffsäkra cynismer, dräpande kommentarer och små underfundigheter med vilka du förgyllt min vardag under fem år. Utan Reija Dufva, Inga Hallin och Nahid Heidari hade det inte blivit mycket till avhandling. Ett stort tack till er för allt arbete med mina sekvenser. Ett speciellt tack till Nahid för ditt stora personliga engagemang, alla uppmuntrande ord och allt godis!

Ett stort tack vill jag ge Per Kornhall, min rumskamrat sedan fem år. Jag har trivts oerhört bra i ditt sällskap och du vet att jag inte skulle vilja byta dig mot någon annan! Eller förresten, kanske mot Raveena Tandon, men annars

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ingen. Eller kanske mot någon som vattnar sina blommor själv. Helst Raveena dock. Jag vill också passa på att tacka för illustrationen som pryder omslaget. Tack till Per Erixon för att du alltid sett till att jag verkligen fått valuta för min arbetstid. Genom dig har jag alltid varit ordentligt uppdaterad vad det gäller din senaste PCR, dina konstiga sekvenser och hur det gick för dina studenter på förra tentan. Utan dig skulle doktorandtiden känts mycket längre –fast i verkligheten skulle den antagligen ha varit mycket kortare! Få har på samma sätt som Frida Eggens fått mig att känna fysisk och psykisk smärta. Fysisk smärta genom att i svåra stunder med osviklig precision pricka mig med jordnötter i pannan, och psykisk smärta genom att i andra svåra stunder (men med samma osvikliga precision) annonsera sina fältarbeten på Grönland och i Tibet. Johan Nylander har, bland mycket annat, lärt mig att timing är allt. En av många konsekvenser av det är att man måste kunna prata tydligt med mat i munnen. Alla vet (Kristina Articus inte minst!) att jag är väldigt svag för Hege Vårdal och Annika Vinnersten. Utan er skulle mitt liv vara väldigt torftigt. Ord kan inte beskriva hur glad jag är att jag har er!

Det finns många fler att tacka på avdelningarna för systematisk botanik och systematisk zoologi och även en dryg handfull människor på andra avdelningar inom EBC, men jag kan inte få tiden att räcka till att klä mina tack i ord och nöjer mig därför med ett stort och varmt kollektivt tack till er alla –nya, gamla och blivande doktorander, personalen på Fytoteket, i bibliotek och Botaniska trädgården– ja, alla. Tack så mycket!

Många och varma tack till Petra Korall, Jenny Smedmark och Torsten Eriksson för allt stöd och all vänskap. Min bana började på Botan i Göteborg och där finns en hoper människor som förtjänar många tack. Ett stort och kärleksfullt tack går till Hanne Hegre Grundt i Oslo –jag hoppas du kommer över till den rätta sidan (den som har växter med både morfologisk och molekylär variation) och att Bengan välkomnar dig till Uppsala igen nu när du har en egen korkskruv. Bernard Pfeil, thank you for your guidance with the title and also the good company!

Så har turen kommit till mina familjer. Familj av blod och ingifte –jag vill bara säga tack för att ni funnits medan jag varit borta! Och du far, inte gick det väl så illa som du trodde, när jag åkte tillbaka till Indien istället för att börja läsa kemi? Mitt glas har alltid fyllts på hos Harriet och Börje och jag har alltid givits en rättvis chans att slåss om en filt eller en varm plats vid kaminen. Tack också till Ulrika. Jag har alltid känt mig välkommen hos Benny och Christina. Anders och Johanna, Sari och Strokarn –ord är otillräckliga. Tack. Pelle –du har alltid haft en tumme ledig att peta mig i ögat med. Ord hade varit tillräckligt! Jonsson och Krille, tack för att ni alltid har ett mer än tillräckligt ont ord till övers för en vän i nöd. Algot, i alla former och tider, i alla skepnader –med dig som måttstock kommer jag alltid att framstå i mycket god dager. Det är väl min smala lycka.

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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 91-554-5845-9

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”.)