Phylogenetic Relationships of Silene Sect. Melandrium and Allied Taxa - DocsLib

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Home , Agrostemma, Caryophyllaceae, Silene, Silene conica, Silene dioica, Silene latifolia

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Cover illustrations: Barry Breckling, Sara Gold, Kjell Lännerholm, Anja Rautenberg and Wiebke Rautenberg.

List of Papers

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

I Rautenberg, A., Filatov, D., Svennblad, B., Heidari, N., Oxel- man, B. (2008) Conflicting phylogenetic signals in the SlX1/Y1 gene in Silene. BMC Evolutionary Biology, 8:299 II Rautenberg, A., Hathaway, L., Oxelman, B., Prentice, H. C. Phylogenetic relationships of Silene section Elisanthe (Caryo- phyllaceae) as inferred from chloroplast and nuclear DNA sequences. Manuscript. III Rautenberg, A., Hathaway, L., Prentice, H. C., Oxelman, B. Phylogenetic relationships and optimal genealogical species delimitations of Silene section Melandrium (Caryophyllaceae). Manuscript. IV Sloan, D. B., Oxelman, B., Rautenberg, A., Taylor, D. R. Phy- logenetic analysis of mitochondrial mutation rate variation in the angiosperm tribe Sileneae. Submitted. V Rautenberg, A., Sloan, D. B., Aldén, V., Oxelman B. Phyloge- netic relationships of Silene multinervia and Silene section Conoimorpha (Caryophyllaceae). Manuscript.

AR was responsible for the writing of all papers, except IV, with help of the co-authors. AR was responsible for most of the lab work in I and III, parts of the lab work in II and V, and all analyses in papers I–III and V. The defen- dant’s contribution to paper IV is restricted to providing DNA material and comments to the analyses and the text.

Contents

Introduction...... 9 Gene trees and species trees ...... 9 On the existence of species...... 11 Study species...... 11 Aims ...... 14 Materials and Methods...... 15 Paper I (Conflicting phylogenetic signals in the SlX1/Y1/SlXY1 genes)...... 17 Paper II and III (Relationships of Silene section Melandrium)...... 20 Paper IV (Mitochondrial mutation rate variation) ...... 25 Paper V (Relationships of S. multinervia and S. section Conoimorpha) ...... 27 Conclusions...... 31 Summary in Swedish (Sammanfattning på svenska)...... 32 Fylogenetiskt släktskap bland Silene sektion Melandrium och närbesläktade grupper ...... 32 Acknowledgements – Tack – Danke – ...... 35 References...... 38

Abbreviations

bp base pairs cpDNA chloroplast DNA BEAST Bayesian Evolutionary Analysis Sampling Trees BEST Bayesian Estimation of Species Trees HPD highest posterior density MRCA most recent common ancestor mtDNA mitochondrial DNA mya/kya million/thousand years ago RS absolute synonymous substitution rates

Introduction

“A man with one clock always knows the time. A man with two clocks is never sure” could for this study be translated to something like “A phylogeneticist with one tree always (thinks she) knows the true relationships. A phylogeneticist with two trees is never sure”. Systematic biology deals with evolutionary relationships among organisms, often depicted by phylogenetic trees. These trees are nowadays usually based on the DNA sequences of different organisms and use mathematical models to infer a plausible graph over the organisms’ relationships. A challenge in phylogenetic research is that the relationships between organism lineages are not always straightforward processes that are reflected in every part of the organisms’ genomes. The observation that phylogenies sometimes tell us conflicting stories is, however, also one of the advantages with using molecular data: by investigating the unexpected patterns we find, we can learn more about the evolutionary processes that act on us and the world around us.

Gene trees and species trees Different genes within an organism can have different evolutionary histories, because of e.g. mode of inheritance, events of sexual reproduction (hybridization), or incomplete lineage sorting. In most flowering plants, chloroplasts and mitochondria are maternally inherited (through the egg cells). As a result, the genetic material present in chloroplasts and mitochondria will re- flect the evolutionary history of the maternal lineage only. The nuclear genome, on the other hand, will be inherited from both parents, and thus both parental lineages will be reflected in the nuclear genome. Chloroplasts and mitochondria are therefore generally expected to depict the seed dispersal, whereas nuclear DNA is expected to depict both seed and pollen dispersal patterns. A successful hybridization event leads to mixing of genes from two independent lineages into the progeny. When a lineage splits into two, ancestral alleles might be randomly sorted in the new lineages (incomplete lineage sorting), which also will result in conflicting gene trees. When different gene trees give different topologies, one would often like to find a tree that summarizes the species’ history (Figure 1). There are different ways to infer species trees from gene trees. One way is to concatenate

9 the data from different genes and use all information to produce a single tree. This method is appropriate if all the included genes have the same history, but if they have not, the result might be misleading (Kubatko and Degnan 2007). An alternative is to compare the separate gene trees and select the topology that occurs most frequently as the species tree. A problem with this approach is that the gene tree topology that is most likely to evolve from the species tree can differ from the species tree topology (Degnan and Rosenberg 2006). A third approach is to take into account the possibility of random differential survival of ancestral alleles in the daughter lineages in a coalescent framework (Maddison 1997; Maddison and Knowles 2006; Ed- wards et al. 2007). In coalescent theory, gene copies in a current population are traced back to their most recent common ancestor, while taking into account population size and population structure. AB

C A } B } } }

time

Figure 1. One way to view species is to imagine them as “tubes”, in which the evolution operates on the genes in individuals and populations. Here are three different gene phylogenies included in a “species” tree, where the outer thick, black lines represent organism lineages (taxa). The thin, black lines represent genes that are reciprocally monophyletic within taxa. The light grey lines represent gene copies that are more similar across the taxa A and B than within the taxa, because of incomplete lineage sorting (deep coalescence). The thin, dark grey lines represent a gene split that has occurred after the divergence of A and B, best explained by hybridization.

10 On the existence of species When referring to “species trees” we assume that there is such a thing as species. In reality, when working with closely related species, it is not always easy to draw the limits between species. There is no clear-cut distinc- tion between various taxonomic levels like varieties, races, subspecies, and species. With an open mind on species definitions, it can be interesting to explore what impact different a priori taxon delimitations have on the interpretations of conflicting gene trees. Imagine a case when the sampled gene trees are incongruent and all genes are not reciprocally monophyletic within taxa (Figure 1). It can here be reasonable to explore whether the sampled gene trees favour separate A and B lineages, or whether the genetic subdivision between A and B is so low that they can be considered one species. By choosing classifications that maximize the likelihood of the observed gene trees, we have a way to optimize species delimitations.

Study species The plant genus Silene L. (Caryophyllaceae) consists of around 650 mostly herbaceous species, with a centre of diversity in the Mediterranean and Southwest Asia. Silene and the closely related smaller genera Agrostemma, Atocion, Eudianthe, Heliosperma, Lychnis, Petrocoptis, and Viscaria are often referred to as Sileneae (Oxelman et al. 2001). The studies presented here focus on some groups within one of two large subgenera of Silene: Si- lene subgenus Behenantha (Otth) Endl. (=S. subgenus Behen (Dumort.) Rohrb.). The phylogenetic studies published so far, have failed to sort out the relationships among the major lineages of the subgenus. The members of Silene section Melandrium (Röhl.) Rabeler have received special interest, since they are dioecious (with male and female organs on separate individuals). They have sex chromosomes (X and Y), in a system similar to that in e.g. humans. Most other flowering plants have male and female organs in the same flower, or male and female flowers on the same individual. Many other dioecious plants lack sex chromosomes, but instead have other means of sex determination. Researchers interested in sex chromosome evolution have found it useful to compare the sex chromosomes of the section Melandrium species with the corresponding autosomes of closely related hermaphrodite species (e.g. Atanassov et al. 2001; Filatov and Charlesworth 2002; Filatov 2004; Filatov 2005). Most authors accept the taxonomy of the five European section Melan- drium species as delimited by Chater et al. (1993). Two of the species of section Melandrium are widespread in Europe: Silene latifolia Poir. (=S. alba (Mill.) E.H.L.Krause, white campion; Figure 2) and S. dioica (L.)

11 Clairv. (red campion). They are usually ecologically separated but hybridize frequently when they come in contact. Silene latifolia has a wider distribution range, extending into the temperate parts of Asia. The three other species in the section have more limited distributions: S. diclinis (Lag.) M.Laínz grows only near Valencia in Spain. The white-flowered S. heuffelii Soó is found in Romania and on the Balkan Peninsula, and S. marizii Samp. has a disjunct distribution in Portugal and in Spain. The endemic species are not known to form natural hybrids with S. latifolia or S. dioica, but they are in- terfertile under experimental conditions (Baker 1947; Prentice 1978).

Figure 2. Female (left) and male (right) individuals of S. latifolia (photo: Anja Rau- tenberg).

The dioecious species of section Melandrium were previously classified together with S. noctiflora L. in Silene section Elisanthe (Fenzl ex Endl.) Ledeb. Silene noctiflora is a hermaphrodite species with white night- flowering flowers (Figure 3). It has been shown to have extremely elevated synonymous substitution rates in the mitochondrial genome (Mower et al. 2007; Sloan et al. 2008).

Figure 3. S. noctiflora (photo: Anja Rautenberg), and S. multinervia (photo: Barry Breckling, used with kind permission).

12 The species in Silene section Conoimorpha Otth (Figure 4) are mainly recognized by their peculiar calyx nerves: they have 15–60 prominent parallel, unbranched nerves, whereas all other Silene species have either fewer nerves, or if they are several, they always show a branching pattern on the calyx. Section Conoimorpha also has a basic chromosome number of x=10 chromosomes (or possibly 11 in S. lydia Boiss.; Greuter 1995), whereas the other Silene species have x=12 (Chater et al. 1993). All species in the section are annuals, with a centre of diversity in the Eastern Mediterranean.

Figure 4. S. conoidea (photo: Anja Rautenberg), and S. coniflora (photo: Sara Gold, used with kind permission from http://www.wildflowers.co.il).

The Californian S. multinervia S.Watson (Figure 3) has been classified as a member of section Conoimorpha, and later been synonymized to the South West/Central Asian species S. coniflora Nees ex Otth (Morton 2005; Figure 4). Silene multinervia is an annual plant that grows on newly burnt lands. The calyx with 20 parallel, unbranched nerves has strong resemblance to that of Silene section Conoimorpha, but S. multinervia does not form a monophyletic group with the rest of section Conoimorpha (Popp and Oxelman 2007)

Figure 5. Silene pendula and S. uniflora (photo: Anja Rautenberg).

13 Silene pendula L. (Figure 5) is a popular garden plant, with original distribution in Italy (Jalas and Suominen 1986). Previous studies indicate that it is related to S. vulgaris (Moench) Garcke (Desfeux and Lejeune 1996; Oxel- man et al. 1997). Silene vulgaris, and its close relatives, among them S. uniflora Roth (Figure 5), are recognized by extremely inflated calyces with several nerves in an anastomosing pattern.

Aims The general aim of the studies included in this thesis is to use gene phylogenies to investigate evolutionary relationships among the taxa classified in or near the Silene sections Melandrium and Conoimorpha. In paper I we explore a case where different parts of the gene SlX1/SlY1 give rise to conflicting phylogenies within Silene subgenus Behenantha. To investigate whether gene duplication/loss may be a plausible explanation we present a novel probabilistic PCR approach to determine the number of sequence variants present in an individual. In paper II and III we investigate the evolutionary relationships among the dioecious members of section Elisanthe/Melandrium, and their closest relatives. We use an extensive sampling of several outgroup taxa that have previously been classified together with the dioecious species. In paper III we use a hierarchical Bayesian coalescent method to test the species delimitations in section Melandrium. Paper IV is a survey of mitochondrial DNA mutation rate variation in Si- leneae species, with special emphasis on Silene subgenus Behenantha, to see whether any more species experience as high substitution rates as those observed in S. noctiflora. In paper V we investigate S. multinervia, S. coniflora and other section Conoimorpha species in a phylogenetic and morphological study to see whether the peculiar morphological feature shared by S. multinervia and section Conoimorpha (several prominent, parallel, unbranched calyx nerves) has a common origin, or whether it has evolved separately.

14 Materials and Methods

The work presented here relies almost exclusively on DNA sequence data, obtained from live plant material, from herbarium specimens or GenBank sequences from other studies. In paper I, we use sequence data from the SlX1/SlY1 genes from the sex chromosomes of species in Silene section Melandrium, and the corresponding genes from the autosomes in hermaphrodite Silene species. The DNA regions used in studies II–V are several chloroplast DNA (cpDNA) regions (the matK and rpoC1 genes, the psbA- trnH spacer region, the rps16 intron, and the trnL-trnL/F intron/spacer region), four mitochondrial DNA (mtDNA) regions (the atp1, atp9, cox3, and nad9 genes), the internal transcribed spacer (ITS) region from the nuclear ribosomal DNA, and four low-copy number nuclear regions from the RNA polymerase gene family (non-coding parts of the genes RPA2, RPB2, RPD2a, and RPD2b). Most sequences have been obtained by direct sequencing of purified PCR products, but for paper I we also sequenced some cloned PCR products. In several cases, we have designed sequence-specific primers to sort out sequence copies in polymorphic individuals. The sequence data have been used to infer phylogenies, using several strategies. Maximum parsimony phylogenies, with bootstrap support values, have been constructed using PAUP* (Swofford 2002). A more sophisticated way is to take into account the probabilities of different mutation events in the DNA molecule, e.g. as implemented in the software MrBayes (Huelsen- beck and Ronquist 2001; Ronquist and Huelsenbeck 2003). When the data set contains conflicting phylogenetic signals, it can be informative to visual- ize the relationships by networks, as in the software SplitsTree (Huson and Bryant 2006). In cases where there is incongruence between trees obtained from different genes, dated phylogenies may help to distinguish between different causes of incongruence (Sang and Zhong 2000; B. Frajman, F. Eggens and B. Oxelman, submitted manuscript). We have used the software BEAST (Drummond and Rambaut 2007) to perform relative dating of our phylogenies. In paper I we use a genetic algorithm for recombination detection (GARD; Kosakovsky Pond et al. 2006), to find recombination breakpoints in a DNA sequence data set.

15 Dated phylogenies are also important tools when comparing substitution rates between lineages. In paper IV we use a cpDNA phylogeny with dated nodes to obtain absolute mtDNA substitution rates. In paper III and V we use coalescent methods implemented in the software BEST (Liu et al. 2008) to infer gene trees (one for each gene included), and at the same time a species tree that is compatible with the gene trees. BEST can deal with incomplete lineage sorting/deep coalescence, but if hybridization has been going on between the taxa, the assumptions of the model are violated. On the other hand, this limitation could also be used to test the species delimitations. We expect that hybridization and deep coalescence will cause the likelihood of the gene trees to be lower than if the in- volved taxa are classified as one species in a coalescent model. By giving BEST different input files where the species are delimited in different ways, and comparing the likelihoods of the gene trees given the species tree, we explore the relationships between closely related taxa in paper III. Likelihood values cannot simply be compared between two analyses with different models (different species delimitations can be seen as different models, because the number of distinct branches in the species tree differ), since different models with different numbers of parameters automatically will result in different likelihoods. The likelihood values can, however, be used to compute Bayes Factors (e.g. Kass and Raftery 1995), which can say something about how likely different models are, given the data. In paper III we compare different BEST runs with different a priori species delimitations, by calculating Bayes Factors for the marginal likelihood values. One possible cause of incongruence between different gene phylogenies is incomplete sampling of paralogues, i.e. some phylogenies would not give as “odd” relationships as they do, if all copies of the studied gene region had been found and included in the phylogeny. In paper I we develop an approach to calculate how much PCR screening that has to be done before one can be 95 % sure that there are no more existing undetected copies.

16 Paper I (Conflicting phylogenetic signals in the SlX1/Y1/SlXY1 genes)

In paper I, we planned to use the genes SlX1/SlY1 from the sex chromosomes of Silene section Melandrium and the homologous gene SlXY1 from the autosomes of related hermaphrodites species to infer phylogenies among the members of the section and its closest relatives. However, we soon realized that the region (collectively referred to as XY1) gave conflicting results re- garding the positions of some outgroup species, and we started to investigate the causes of this. A recombination detection test indicates recombination breakpoints approximately in the middle of the sequenced part of the gene (Figure 6). Different parts of the same gene give distinct topologies when included in separate phylogenetic analyses. Silene vulgaris and section Conoimorpha change places when comparing the 5' and the 3' end of the gene (clades C and V in Figure 7a and b).

a) Breakpoint placement support using c-AIC 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 Model averaged support 0.1 0 0 500 1000 1500 2000 2500 3000 3500 4000 Nucleotide S_nutans_A

b) c) S_acaulis_B S_nutans_A S_latifoliaY_AJ31065

S_acaulis_B S_dioicaX_AY084044 S_dioicaX_AY084044 S_latifoliaX_AJ31065 S_latifoliaX_AJ31065

S_dioicaY_AY084045 S_conica

S_latifoliaY_AJ31065 S_dioicaY_AY084045

S_vulgaris_C S_vulgaris_C S_conica S_pendula S_pendula 0.02 0.02 Figure 6. Results from the recombination detection analysis. a) Support for the sug- gested positions of breakpoints in the GARD analysis using the HKY85 nucleotide substitution model and beta-gamma rate distribution with 5 rate classes on the re- duced 9-sequence XY1 matrix (4045 bp), Neighbor-Joining trees for the 5' (b) and 3' non-recombinant partitions (c).

17 1/99 S_acaulis_B S_acaulis_A 1/- a) 1/100 S_nutans_B 1/100 S_nutans_C S_nutans_A L_flosjovis_AY084042 * S_latifoliaX_AY084036 S_latifoliaX_AJ310656 1/99 S_dioicaX_AY084044 2.0 S_dioicaY_AY084045 dE 1/92 S_latifoliaY_AJ310655 0.96/70 II 1/100 S_latifoliaY_AY084037 7.6 S_noctiflora_AJ631222 N 0.91/67 III 1/69 S_vulgaris_B 5.1 S_vulgaris_A 0.89/67 I 1/94 I V 9.2 2.3 S_vulgaris_AY084040 0.97/69 S_vulgaris_C S_pendula P 0.98/78 0.99/63 S_conica_AY082378 8.0 1/79 I S_conica C 4.2 S_conoidea

2.0

S_nutans_B b) 1/100 1/100 S_nutans_C 1/- S_nutans_A 1/100 S_acaulis_B S_acaulis_A L_flosjovis_AY084042 * 1/100 S_latifoliaY_AY084037 1/56 S_latifoliaY_AJ310655 0.99/73 S_latifoliaX_AY084036 S_dioicaY_AY084045 dE 1/100 1/95 3.5 S_dioicaX_AY084044 1/84 1/91 II S_latifoliaX_AJ310656 5.8 S_noctiflora_AJ631222 0.75/64 III N S_conoidea 4.4 1/99 I I 0.99/84 2.5 1/97 S_conica C 8.6 S_conica_AY082378 S_pendula P 1/100 I S_vulgaris_AY084040 5.0 1/100 I 1/94 S_vulgaris_A 3.1 S_vulgaris_B V 0.88/- S_vulgaris_C 2.0 Figure 7. Bayesian consensus chronograms for the 5' (a) and 3' parts (b) of the 6416 bp XY1 matrix. Values below branches are median ages in million years. Numbers above branches are posterior probabilities (PP)/Maximum parsimony bootstrap (MPB) support values for the same data set but with additional indel characters (values of PP < 0.70 and MPB < 60% are not shown). Hyphens (-) indicate nodes with high posterior probabilities that were not present in the MPB trees. Horizontal bars represent 95% HPD age intervals. Roman numerals are used to label nodes mentioned in paper I. Note how the positions of the groups section Conoimorpha (C) and S. vulgaris (V) change in the different partitions.

18 We present an approach to perform several independent PCR reactions and sequence them to detect all potential sequence copies, and to calculate the probability that there are no undetected copies. We show that it is very unlikely that the incongruence is caused by gene duplications/losses or artefacts, such as in vitro recombination during PCR. The observed phylogenetic incongruence may have been caused by recombination of two divergent alleles following horizontal gene transfer, inter- specific hybridization or incomplete lineage sorting.

19 Paper II and III (Relationships of Silene section Melandrium)

In papers II and III, we conclude that the dioecious species of section Elisan- the do indeed form a monophyletic group, and that S. noctiflora, which is the type of the section Elisanthe (Greuter 1995), is not closely related. Because the type species of the section no longer is included with the other species, the section name of the dioecious species should instead be Silene section Melandrium (Röhl.) Rabeler. The other hermaphrodites previously included in section Melandrium/Elisanthe are scattered among other Silene groups. Within section Melandrium, there is a correlation of the chloroplast phylogeny with geographic origin, rather than with traditional taxonomy. Most specimens from Southern/Western Europe group together (Figure 8; node d in Figure 9), nested in a larger clade, also containing specimens from Geor- gia and Iran (node c in Figure 9). Most specimens from Northern/Eastern Europe form a group (node f in Figure 9). Two Turkish specimens have a highly deviating DNA (node g in Figure 9). The origin of these deviating cpDNA sequences is unknown, but the observed pattern may be caused by introgression of cpDNA from a species not included in our study.

Figure 8. Origin of the section Melandrium specimens. Map: Anders Larsson.

20 A rupestre E laeta P pyrenaica

1/100 morpha Conoi- 1/100 ammophila 1/100 conica macrodonta conoidea 1/55 DIC_1_ES DIC_7_ES 0.72/ D118_1_FR L121_9_FR l) 1/77 D130_1_FR d)1/ L166_30_FR SW D184_24_FI L123_5_PT S L126_19_IT 0.95/62 M4_1_PT c) 0.94/ M4_7_PT 0.99/56 L_12851_GE 1/55 L_14771_GE e) L_14584_IR

1/97 L170_10_IT Melandrium dioecious M5_5_PT D3_1_SK D3_3_SK D16_1_DE D72_1_PL k) 0.89/67 D169_9_FI L175_3_FI L5_4_SE 0.77/77 L80_12_SK NE Elisanthe/ L81_9_SE f)0.99/ L138_27_PL L178_2_DE 0.75/ H4_2_RO H4_6_RO H5_3_RO 0.9/ b)1/100 H5_5_RO L200_2_ES 1/87 M5_4_PT D198_4_SE H_12642_GR g)1/100 L140_13_TR L140_19_TR 1/100 multinervia 1 multinervia 2 1/99 pendula 1/100 uniflora vulgaris samia elisabethae Silene littorea akinfievii subg. aprica 0.7/ davidii 1/98 Behenantha a) 1/100 sorensenis 1/75 fedtschenkoana Physolychnis h) 1/100 quadriloba viscosa zawadzkii 0.73/ 1/85 noctiflora 1 i) 1/100 noctiflora 2 0.99/71 turkestanica 1 turkestanica 2 Lychnis /74 Ly chalcedonica 1/91 1/100 Ly coronaria Ly floscuculi 1/100 fruticosa 0.61/51 schwarzenbergeri j) 1/79 pseudoatocion 1/100 hoefftiana 1 0.99/76 hoefftiana 2 Silene pygmaea

subg. 0.0050 Silene

Figure 9. Phylogram for the concatenated chloroplast sequences. Values above branches are Bayesian posterior probabilities/Maximum parsimony bootstrap percentages. Only support values higher than 0.60/50 are shown. Branch lengths represent expected number of changes. A= Atocion, D= Silene dioica, DIC= S. diclinis, E= Eudianthe, H= S. heuffelii, L= S. latifolia, Ly= Lychnis, M= S. marizii, P= Pet- rocoptis. Two-letter abbreviations at the end of sequence names correspond to coun- try codes according to ISO 3166. Names in bold represent members of the “Southern clade”, whereas names in bold italics represent members of the “South Western clade”. Names in grey represent members of the “North Eastern clade”.

The nuclear DNA regions we have sequenced correspond better to taxonomic affiliation (Figure 10), but there are also several signs that the species are not perfectly separated (Figure 11). The gene trees suggest that S. heuffelii and S. diclinis are distinct from the other section Melandrium species, while our sample of S. dioica, S. latifolia, and S. marizii contains sequences that apparently have a mixed origin. The two Turkish S. latifolia specimens with a deviating cpDNA are clearly placed within section Melandrium in the nuclear phylogenies (e.g. Figure 10).

21 The reticulate pattern between the dioecious section Melandrium species could be caused by e.g. incomplete sorting of common ancestral alleles, or by hybridization (present or past).

E laeta P pyrenaica Lychnis 1/100 Ly floscuculi Ly floscuculi 1/100 littorea littorea integripetala b 1/98 DIC1_ES DIC7_ES D31_SK D33A_SK 1/87 D33B_SK e 0.97/62 D1984_SE D161A_DE D721_PL D1301_FR 1/84 D1843A_FI 0.98/60 D18424a_FI D1699a_FI 0.99/63 D18424g_FI D1181_FR 0.90/65 D1699g_FI L1701_IT c M41_PT 0.97/65 M47_PT Melandrium 1/88 M54_PT 1/95 D161B_DE D1843B_FI L14013_TR sL1753B_FI Silene L12851_GE 1/79 L14584_IR subg. L14019A_TR Behenantha 0.91/66 L14019B_TR 1/100 L14771a_GE L14771g_GE L54c_SE 1/- L54t_SE L8012_SK L819_SE L1219_FR L1231_PT 0.99/63 Physolychnis + Conoimorpha + ... f 0.96/63 L17010_IT hoefftiana a L12619_IT -/56 littorea 1/99 sL1753A_FI Ly coronaria + flosjovis 1782_DE -/61 L2002_ES 1/100 Melandrium + integripetala M55_PT Ly floscuculi H12642a_GR h H12642c_GR 1/100 H42A_RO H42B_RO H46c_RO 1/64 H46t_RO 0.93/56 H53_RO H55a_RO H55d_RO 1/100 hoefftiana 1 hoefftiana 2 0.55/63 aprica 0.80/- caroliniana stellata 1 davidii involucrata 0.9/74 1/100 viscosa 1 1/98 viscosa 2 Physolychnis nigrescens sorensenis 1/89 uralensis viscosa 3 0.7/50 zawadzkii stellata 2 0.88/- 0.82/81 fabaria 1/89 1/100 uniflora vulgaris 0.98/84 samia 0.88/75 noctiflora 1 1/100 noctiflora 1 turkestanica 0.99/63 pendula 0.99/96 ammophila 0.96/- 1/100 conica Conoimorpha macrodonta conoidea 1/100 multinervia 1 0.89/- multinervia 2 Silene subg. Behenantha 1/98 acaulis -/53 -/60 fruticosa Silene subg. Silene 1/100 schafta pseudoatocion Lychnis 1/100 Ly coronaria Ly flosjovis

0.02 Figure 10. Phylogram for the RPB2 region. Values above branches are Bayesian posterior probabilities/Maximum parsimony bootstrap percentages. Values to the right of nodes are estimated median heights (in million years). Branch lengths correspond to expected number of changes. D = Silene dioica, DIC = S. diclinis, E = Eudianthe, H = S. heuffelii, L = S. latifolia, M = S. marizii, P = Petrocoptis. The smaller, partial tree represents nodes in the maximum parsimony tree that are not present in the MrBayes tree.

22 L200_2_ES L81_9_SE 59 L175_3_FI L138_27_PL L80_12_SK H5_3_RO 56 L12851_GE L166_30_FR O 52 62 H4_6_R H55_RO 78 H4_2_RO S. heuffelii L_14771_GE S. marizii 64 L12850_GE M5_4_PT

H_12642_GR M4_1_PT 74 78 M4_7_PT L140_19_TR M5_5_PT L140_13_TR

S. latifolia

100 S. diclinis L170_10_IT

DIC_1_ES DIC_7_ES 88 D3_3_SK D72_1_PL D3_1_SK 60 D184_24_FI 60 L170_1_IT

99 60 D169_9_FI

62 62 L_14584_IR 97 70 L123_1_PT 65 S. dioica D184_3_FI L123_5_PT D130_2_FR

84

72 73 D130_1_FR 0.0010 D118_1_FR

Figure 11. NeighborNet network of ITS sequences, based on uncorrected p dis- tances, and handling ambiguous states with the “Average States” option in Splits- Tree 4 (Huson and Bryant 2006). Numbers on the edges are bootstrap values. D = Silene dioica, DIC = S. diclinis, H = S. heuffelii, L = S. latifolia, M = S. marizii. Names in bold represent members of the “Southern clade” in the cpDNA phylogeny, whereas names in bold italics represent members of the cpDNA “South Western clade”. Names in grey represent members of the “North Eastern clade” in the cpDNA phylogeny.

In order to test if the traditional taxa in section Melandrium are supported by a genealogical approach to gene trees we use a Bayesian hierarchical method in paper III, as employed in the software BEST (Liu et al. 2008). We test three different combinations as priors for the species trees: all taxa treated as separate species; all taxa treated as one pooled species “Melandrium”; and as 3 species (S. diclinis and S. heuffelii as separate species, and S. dioica + S. latifolia + S. marizii as a pooled species). Silene zawadzkii was used as outgroup for these analyses. By calculating Bayes Factors for the marginal likelihoods of the different runs on the nuclear data set, we conclude that the optimal genealogical species setup given our data is separate S. diclinis and S. heuffelii species, and a joint species including S. dioica, S. latifolia, and S. marizii.

23 a) b) zawadzkii H12642 zawadzkii H4_6 H5_5B H5_3A diolatmar b 1 DIC_7t 1 DIC_7g 1 D118_1 1 diclinis D169_9A D130_2 heuffelii D169_9B D184_3 0.0030 L121_9 L123_5c L123_5t 0.61 L138_27c 0.74 L138_27t L175_3t 1 L166_30 L140_19B L200_2 0.87 D184_24L L140_19A D184_24S 0.84 L81_9A L175_3c L81_9B c L170_1 0.84 M4_1B M4_7 0.0050 d 0.9 L170_10 c) M5_5L

zawadzkii h H4_2A 1 0.62 H4_2B 0.66 H4_6 b H5_5A d) 1 DIC_1 DIC_7 D184_3A e D184_24B zawadzkii 0.99 D118_1 H12642 D16_1A h 1 1 H4_6 0.99 D3_3B H5_3 D3_3A H5_5 L1701 b 1 DIC_1 c 0.99 M4_1A DIC_7B M47 D3_3 D184_3B D16_1 L81_9A 0.98 D130_1 e 0.94 L121_9 D130_2 f 0.94 L123_1 D169_9 L170_10 D184_3 L12851 D18424 1 L5_4 0.97 L14584 0.98 L140_19A L5_4B L140_19B L80_12A L14771A L80_12B L14771G L81_9A L140_13 1 L121_9 L175_3c 0.90 L123_1 L178_2 1 L123_5A 0.0050 L200_2 L123_5B M5_5 L126_19 L140_19 L170_1A c L170_1B 1 M4_1A M4_7A M5_4B 0.0050 L170_10A L178_2

Figure 12. Species tree (a) and gene trees for RPA2 (b), RPB2 (c), and RPD2b (d) for the 3-species set-up (S. heuffelii and S. diclinis treated as separate species, with S. dioica, S. latifolia, and S. marizii treated as a pooled species, “diolatmar”). Values above branches are Bayesian posterior probabilities. D = Silene dioica, DIC = S. diclinis, H = S. heuffelii, L = S. latifolia, M = S. marizii.

24 Paper IV (Mitochondrial mutation rate variation)

In paper IV, we show that the extremely elevated mitochondrial substitution rates previously reported for S. noctiflora (Mower et al. 2007; Sloan et al. 2008) is shared with the very closely related S. turkestanica Regel, and with Silene section Conoimorpha. atp1 atp9

dN dS dN dS Beta vulgaris Beta vulgaris Agrostemma githago Agrostemma githago Petrocoptis pyrenaica Petrocoptis pyrenaica Heliosperma pusillum Heliosperma pusillum Eudianthe laeta Eudianthe laeta Viscaria alpina Viscaria alpina Viscaria vulgaris Atocion lerchenfeldianum Atocion lerchenfeldianum Silene delicatula Silene delicatula Silene cordifolia Silene cordifolia Silene sordida Silene sordida Silene odontopetala Silene odontopetala Lychnis coronaria Lychnis coronaria Silene noctiflora Silene noctiflora Silene turkestanica Silene turkestanica Silene zawadzkii Silene zawadzkii Silene seoulensis Silene seoulensis Silene douglasii Silene samojedora Silene stellata Silene douglasii Silene davidii Silene stellata Silene sachalinensis Silene davidii Silene involucrata Silene sachalinensis Silene argentina Silene sorensenis Silene laciniata Silene involucrata Silene conica Silene argentina Silene ammophila Silene laciniata Silene macrodonta Silene conoidea Silene samia Silene conica Silene khasyana Silene ammophila Silene akinfievii Silene macrodonta Silene latifolia Silene samia Silene hookeri Silene khasyana Silene menziesii Silene akinfievii Silene acutifolia Silene latifolia Silene auriculata Silene hookeri Silene integripetala Silene menziesii Silene lacera Silene acutifolia Silene dichotoma Silene auriculata Silene littorea Silene integripetala Silene nana Silene lacera Silene vulgaris Silene dichotoma Silene uniflora Silene littorea Silene caesia Silene nana Silene repens Silene vulgaris Silene gallinyi Silene uniflora Silene schafta Silene pendula Silene pygmaea Silene caesia Silene caryophylloides Silene repens Silene nicaeensis Silene gallinyi Silene succulenta Silene moorcroftiana Silene imbricata Silene schafta Silene muscipula Silene pygmaea Silene antirrhina Silene caryophylloides Silene vittata Silene ciliata Silene tunicoides Silene nicaeensis Silene acaulis Silene succulenta Silene multicaulis Silene imbricata Silene paucifolia Silene gallica Silene paradoxa Silene bellidifolia Silene fruticosa Silene muscipula Silene yemensis Silene antirrhina Silene flavescens Silene vittata Silene tunicoides Silene armena Silene otites Silene acaulis Silene nutans Silene schwarzenbergeri Silene multicaulis Silene paradoxa Silene fruticosa Silene paucifolia Silene gracilicaulis Silene yemensis 0.05 Silene flavescens

Figure 13. dN and dS trees for mitochondrial genes atp1 and atp9. Branch lengths are in terms of non-synonymous (dN) or synonymous (dS) substitutions per site as estimated by PAML under a constrained topology. The scale is the same for both trees.

25 The four genes investigated experience extremely elevated synonymous (those substitutions that do not alter the amino acid sequence) substitution rates in S. noctiflora+S. turkestanica and S. section Conoimorpha, whereas the non-synonymous substitution rates are low in all taxa (Figure 13). We also show that there are large differences in the genus Silene in mutation rate between the four included mtDNA regions. Synonymous substitution rate in atp9 is extreme and highly variable throughout most of Silene (Figure 13). The absolute synonymous substitution rates (RS) for all branches are shown in Figure 14. Silene noctiflora and S. turkestanica show a reduction in RS in their terminal branches. In contrast, section Conoimorpha has high RS values also in terminal branches.

0.25+/-0.09 Beta_vulgaris 0.20+/-0.15 Agrostemma_githago 0 Petrocoptis_pyrenaica 1.15+/-0.41 0.12+/-0.09 Heliosperma_pusillum 0.25+/-0.15 Eudianthe_laeta 0.78+/-0.71 0 0 Viscaria_alpina 1.42+/-1.02 0 Viscaria_vulgaris 0.32+/-0.25 0 Atocion_lerchenfeldianum 0.14+/-0.14 Silene_delicatula 0.15+/-0.10 Silene_cordifolia 1.38+/-0.79 0 Silene_sordida 0 0 Silene_odontopetala 3.09+/-0.89 Lychnis_coronaria 18.6+/-10.4 54.3+/-13.3 Silene_noctiflora 8.63+/-6.18 Silene_turkestanica 0.95+/-0.45 Silene_zawadzkii 21.6+/-8.6 2.75+/-2.56 Silene_seoulensis 1.52+/-3.81 0 5.13+/-2.38 Silene_samojedora 0 0 Silene_douglasii 0.71+/-0.73 Silene_stellata 0 Silene_davidii 2.07+/-3.32 0 0 Silene_sachalinensis 0 Silene_involucrata 0 Silene_argentina 0.71+/-0.54 Silene_laciniata 33.3+/-11.1 Silene_conoidea 55.3+/-19.3 16.0+/-7.2 0 Silene_macrodonta 70.1+/-50.7 29.4+/-14.5 42.5+/-36.4 Silene_ammophila 53.3+/-25.4 Silene_conica 1.15+/-0.46 Silene_samia 0.46+/-0.22 Silene_khasyana 0.69+/-0.37 Silene_akinfievii 0.23+/-0.23 0 Silene_latifolia 0 0.52+/-0.38 Silene_hookeri 1.98+/-2.25 Silene_menziesii 0 Silene_acutifolia 0.69+/-0.37 Silene_auriculata 7.15+/-1.67 Silene_integripetala 0.23+/-0.17 Silene_lacera 0.46+/-0.22 Silene_dichotoma 1.61+/-0.68 Silene_littorea 0.46+/-0.24 Silene_nana 0 0.29+/-0.28 Silene_vulgaris 0 1.17+/-0.93 Silene_uniflora 1.53+/-0.94 0 Silene_pendula 5.67+/-2.26 Silene_caesia 1.78+/-0.64 Silene_repens 1.78+/-0.60 0.75+/-0.29 Silene_gallinyi 13.9+/-6.5 Silene_moorcroftiana 4.14+/-2.23 0 Silene_schafta 1.38+/-0.87 Silene_pygmaea 2.76+/-2.22 Silene_caryophylloides 0 0 Silene_ciliata 0 11.4+/-3.0 Silene_nicaeensis 9.26+/-8.32 7.22+/-2.29 Silene_succulenta 6.7+/-11.2 1.04+/-0.78 Silene_imbricata 6.93+/-8.82 4.34+/-2.00 0 Silene_gallica 1.6+/-3.3 4.97+/-2.04 Silene_bellidifolia 7.34+/-2.83 2.16+/-1.62 Silene_muscipula 18.7+/-6.4 Silene_antirrhina 2.03+/-1.00 Silene_vittata 0.94+/-1.21 0.82+/-0.87 3.81+/-5.47 1.42+/-1.97 Silene_tunicoides 0.82+/-0.55 Silene_armena 14.3+/-4.83 Silene_otites 1.32+/-1.56 6.62+/-2.53 Silene_acaulis 41.1+/-12.2 Silene_nutans 15.1+/-5.0 Silene_schwarzenbergeri 5.17+/-2.26 3.96+/-2.85 Silene_multicaulis 12.8+/-4.4 Silene_paradoxa 14.8+/-5.0 Silene_fruticosa 18.0+/-5.7 Silene_paucifolia 15.1+/-4.9 Silene_gracilicaulis 0 15.9+/-7.2 Silene_yemensis 0 Silene_flavescens Figure 14. Absolute synonymous substitution rates (RS) and approximate standard errors based on concatenation of nad9, cox3, and atp1.

26 Paper V (Relationships of S. multinervia and S. section Conoimorpha)

None of our gene phylogenies in paper V gives any support at all to the hy- pothesis that S. multinervia in California is a recent introduction of the Eura- sian species S. coniflora to America. The two species are clearly separated in all phylogenies. Morphological comparison of the two taxa also reveals ob- vious differences (different leaf morphology, absence/presence of coronal scales; Figure 3–Figure 4). We present a chromosome count of 2n=24 in S. multinervia, which deviates from 2n=20 in section Conoimorpha (Greuter 1995). Silene multinervia is not grouped together with the Eurasian samples of section Conoimorpha in any of our phylogenies, but on the other hand, we also do not have strong support to reject them from being sister groups based on the gene trees. Different gene phylogenies give different patterns, and the relationships between the major groups within subgenus Behenantha are largely unresolved (Figure 15–Figure 16). We used the software BEST to infer a species tree that takes the information from all gene trees into account (Figure 17). Based on the nuclear data set, there is some support for a monophyletic group consisting of section Conoimorpha and S. noctiflora+S. turkestanica, which would indicate a single origin of the extremely elevated mitochondrial substitution rates observed in S. noctiflora+S. turkestanica and the Eurasian members of section Conoimorpha (paper IV). In the present analysis, the elevated substitution rate can be treated as a character in itself, and so can be used to support the non-sistergroup relationship of S. multinervia and section Conoimorpha. The calyx nervature, which is a potential synapomorphy for S. multinervia and section Conoimorpha, may be explained either by parallelism or by sorting effects.

27 A rupestre E laeta P pyrenaica 0.55/76 L chalcedonica 1/100 L coronaria L floscuculi 0.62/- akinfievii 0.68/- littorea elisabethae 1/98 pendula 1/100 uniflora vulgaris samia diclinis_7 0.94/- 0.71/- 1/- dioica_184_24 0.92/- latifolia_121_9 marizii_4_1 latifolia_170_10 1/100 heuffelii_4_6 1/99 1/86 latifolia_81_9 latifolia_200_2 0.94/- 1/100 ammophila 1/100 conica subconica macrodonta 1/97 coniflora_1 1/89 coniflora_5 0.65/- C 1/96 coniflora_3 1/100 coniflora_4 1/94 conoidea lydia_1 1/100 lydia_2 lydia_3 1/100 multinervia_7722 multinervia_13489 M 1/100 multinervia_14609 0.7/- aprica 1/98 davidii sorensenis 1/70 fedtschenkoana 1/100 quadriloba viscosa zawadzkii_2447 1/87 noctiflora_2 1/100 noctiflora_1 turkestanica_1 0.98/70 N turkestanica_2 0.95/87 fruticosa 1/94 pseudoatocion schafta

0.0060

Figure 15. Phylogram for the concatenated chloroplast sequences. Values above branches are Bayesian posterior probabilities/Maximum parsimony bootstrap values. Posterior probabilities/bootstrap values lower than 0.60/50 are not indicated. Branch lengths represent estimated number of changes per site.

28 E_laeta L_chalcedonicaA1 1/100 1/100 L_coronaria L_flosjovisA1 0.79/93 1/100 L_floscuculiA1 L_floscuculiA2 aprica fedtschenkoana 0.99/90 involucrata_P 0.89/- involucrata_E nigrescens 1/100 1/100 quadriloba viscosa sorensenis 0.92/75 zawadskii multinervia_1 1/100 multinervia_2 M 1/100 0.77/56 multinervia_3 pendula 0.85/76 0.98/89 uniflora 1/100 elisabethaeA1 elisabethaeA2 1/100 noctiflora 1/88 turkestanica N ammophila 0.96/83 conica 1/76 subconica 0.99/92 conoidea 1/95 coniflora_1 C 1/99 coniflora_3 1/94 coniflora_4 0.99/61 coniflora_5 1/97 macrodonta lydia_3 integripetala

0.99/- schafta_2 schafta_1 0.67/- littorea 0.99/74 pseudoatocion fruticosa 1/100 P_pyrenaicaA1 P_pyrenaicaA2 0.85/76 schafta_2 1/97 0.99/74 schafta_1 fruticosa 0.02 -/60 littorea pseudoatocion integripetala Figure 16. Phylogram for RPD2a. Values above branches are Bayesian posterior probabilities/Maximum parsimony bootstrap values. Posterior probabilities/bootstrap values lower than 0.60/50 are not indicated. Branch lengths represent estimated number of changes per site. The smaller, partial tree represents nodes in the maximum parsimony tree that are not present in the MrBayes tree.

29 a) Lychnis coronaria diclinis

1 noctiflora turkestanica

0.97 zawadzkii multinervia uniflora

1 ammophila 1 conica 1 macrodonta 1 coniflora 1 1 conoidea lydia 0.0060 elisabethae b) Lychnis coronaria diclinis 0.79 uniflora multinervia 1 zawadzkii 1 noctiflora 1 turkestanica ammophila 0.98 0.66 1 conica

1 macrodonta coniflora 1 0.0070 conoidea

Figure 17. BEST analyses. a) Species trees for the data set with cpDNA partition. b) Species trees for the data set without cpDNA partition and without S. lydia and S. coniflora. Values above branches are Bayesian posterior probabilities.

30 Conclusions

The studies included in this thesis clearly demonstrate that different parts of the genome may tell us different stories and stress the importance of using multiple genes in reconstruction of taxonomic relationships. We show that a combination of traditional phylogenetic methods and coalescent methods is a promising venue for the development of systematics and taxonomy. Our novel probabilistic PCR approach, in combination with phylogenetic methods, provides a way to discriminate between different paralogue types and to determine the number of outparalogues in a genome, when the entire genomic sequence is not known (paper I). The dioecious species S. diclinis, S. dioica, S. heuffelii, S. latifolia, and S. marizii form a strongly supported monophyletic group, Silene section Melandrium, distinct from the hermaphrodite species S. noctiflora. Hybridi- zation has been influencing the relationships within section Melandrium (papers II and III). The Californian species S. multinervia is not synonymous to the Eurasian S. coniflora. There is no molecular support for S. multinervia being the closest relative of the Eurasian section Conoimorpha species. Instead, there are indications that section Conoimorpha and S. noctiflora+S. turkestanica are close relatives. A sister-group relationship of S. noctiflora+S. turkestanica and section Conoimorpha would imply that the extremely elevated mitochondrial synonymous substitution rates in the two latter groups has a single origin (papers IV and V). The basal relationships among the major lineages in Silene subgenus Be- henantha remain unresolved, even if some groups have high support and are well resolved within them (papers II, III, V). Our phylogenies support a sce- nario of rapid radiation early in the evolution of subgenus Behenantha, as proposed by Erixon and Oxelman (2008).

A phylogeneticist with one tree always (thinks she) knows the true relationships. A phylogeneticist with several trees has a better chance to find the truth (if there is one).

31 Summary in Swedish (Sammanfattning på svenska)

Fylogenetiskt släktskap bland Silene sektion Melandrium och närbesläktade grupper Systematik handlar om att reda ut hur olika organismer är släkt med varandra. För att få tillräckligt mycket information för att kunna göra detta brukar vi systematiker nu för tiden läsa av bitar (sekvenser) av DNA- molekylerna från de organismer vi är intresserade av. Dessa DNA-sekvenser använder vi sedan för att med hjälp av matematiska modeller räkna ut det mest troliga släktskapsträdet (fylogenin). Ofta brukar de mönster vi ser stämma ungefär med teorier från tidigare studier, baserade på morfologiska karaktärer. Ibland stämmer det dock inte alls, och genom att undersöka de oväntade mönster vi hittar kan vi lära oss mer om de evolutionära processer som påverkar oss och världen omkring oss. Olika delar av arvsmassan kan återspegla olika släktskap. Det kan bero på att olika gener nedärvs på olika sätt. Exempelvis får de flesta blomväxter sina kloroplast- och mitokondriegener bara från modern, medan kärn-DNAt kommer från båda föräldrarna. Hybridisering mellan olika släktskapslinjer och slumpvisa populationsgenetiska processer är andra faktorer som kan orsaka motstridiga signaler i släktskapsträden. Konstiga mönster kan naturligtvis också helt enkelt bero på felhantering av material och analyser. Många gener finns i flera liknande kopior i arvsmassan. De olika kopiorna har ofta olika historisk bakgrund, och då är det viktigt att jämföra rätt kopior med varandra vid de fylogenetiska analyserna. I artikel I upptäcker vi att två olika ändar av en och samma gen ger upphov till olika släktskapsträd. Vi visar att det oväntade mönstret inte beror på att vi gjort fel i våra analyser, utan kommer fram till att hybridisering mellan olika linjer, och efterföljande omflyttning av DNA-sekvenser är en troligare orsak. DNA-sekvenserna hjälper oss att få fram fylogenier för de olika generna, men i själva verket är vi ofta mer intresserade av släktskapet mellan de organismer som generna finns i. Vi brukar därför prata om artträd, även om det inte alltid är så lätt att definiera vad som är en art. Det finns ett flertal olika artbegrepp (till exempel att arter är grupper där medlemmarna kan reprodu- cera sig med varandra, eller att arter är grupper som bildar avskilda grenar i släktskapsträd), men inget som är användbart på alla sorters organismer. Ett

32 sätt att se på arter är att betrakta dem som ”rör”, som evolutionära processer kan verka i (Figur 1). Jag har arbetat med några grupper inom nejlikväxtfamiljen (Caryophylla- ceae). Släktet Silene består av ca 650 arter, varav de flesta finns kring Me- delhavet och i sydvästra Asien. Jag har fokuserat på några grupper inom undersläktet Behenantha. Sektionen Melandrium är i Sverige representerad av vitblära (S. latifolia; Figur 1) och rödblära (S. dioica). Arterna i Meland- rium är speciella eftersom deras individer är enkönade, medan de flesta andra växter har tvåkönade individer (med han- och honorgan i samma blomma, eller med separata han- och honblommor på samma individ). Me- landrium-arterna är vidare speciella genom att individernas kön bestäms av X- och Y-kromosomer, på motsvarande sätt som hos till exempel däggdju- ren. I artikel II och III studerar vi släktskap inom sektionen Melandrium samt försöker hitta deras närmaste släktingar utan könskromosomer. Efter- som de olika genträden tyder på olika släktskapsförhållanden, använder vi oss i artikel III av en metod som räknar ut artträd och genträd samtidigt. Vi testar också att mata programmet med data där vi i förväg delat in individer- na i ”arter” på olika sätt, och ser vilka artkombinationer som stämmer bäst med genträden. Det visar sig att en kombination av separata (traditionella) och sammanslagna arter stämmer bäst, givet våra data. I artikel IV undersöker vi genetisk variation i mitokondrie-DNA i flera arter inom släktet Silene. Variationen inom mitokondrie-DNA är generellt väldigt låg i växter, även om det finns några undantag – bland andra nattglim (S. noctiflora; Figur 2). Vi visar att förutom nattglim har även den närbe- släktade arten S. turkestanica och medlemmar av sektionen Conoimorpha (bland andra sandglim, S. conica) ovanligt variabelt mitokondrie-DNA. Mängden icke-synonyma substitutioner (utbyte av DNA-byggstenar som leder till ändring av aminosyrasekvensen i proteinet som genen kodar för) är låg, medan mängden synonyma substitutioner (som inte påverkar aminosyrasekvensen) är hög. Den höga mängden synonyma substitutioner tyder på att sektionen Conoimorpha och S. noctiflora+turkestanica har en förhöjd muta- tionstakt i sina mitokondrier. Vi visar också att det är stor skillnad i substitu- tionshastighet mellan olika delar av mitokondriegenomet. I artikel V undersöker vi sektionen Conoimorpha närmare. I Kalifornien finns en art (S. multinervia; Figur 3) som påminner väldigt mycket om de europeiska och asiatiska medlemmarna av sektionen. Den har till och med blivit avfärdad som att vara S. coniflora (Figur 4) som spritts från Asien med hjälp av människan. Tidigare studier har inte lyckats visa något nära släkt- skap mellan S. multinervia och de andra arterna i sektionen Conoimorpha. Vi visar att S. multinervia och S. coniflora definitivt inte är samma art. När det gäller hur S. multinervia och sektionen Conoimorpha i stort är släkt, får vi däremot motstridiga besked från olika genträd. Även om inget träd stödjer att S. multinervia skulle vara den närmaste släktingen till sektion Conoi- morpha, finns det heller inte starka bevis som säger motsatsen. Vi får dock

33 indikationer på att S. noctiflora+S. turkestanica och sektion Conoimorpha kan vara varandras närmaste släktingar. I så fall kanske den extremt höga mutationshastigheten i dessa två linjer har samma ursprung. Om uppkomsten av nya arter går snabbt, genom att befintliga utvecklings- linjer delas upp flera gånger inom en relativt kort tidsrymd, blir grenarna i släktträdet korta. Då ökar också sannolikheten att olika genträd visar olika mönster på grund av olika slumpvisa populationsgenetiska processer. Vi tror att det är det som hänt inom undersläktet Behenantha. Sammantaget visar arbetena i den här avhandlingen hur viktigt det är att studera flera oberoende regioner i arvsmassan vid molekylära släktskapsana- lyser.

34 Acknowledgements – Tack – Danke –

Jag förstår inte riktigt hur det gick till. Bengt gav mig betalt för att sitta i solen i Botaniska trädgården och fota och pressa växter, och nu ska jag plöts- ligt lämna in min avhandling till tryckeriet? Tack till Bengt Oxelman för att du tjatade på mig att komma till ”rätt” avdelning, och via den lättare drogen ”assistenttjänstgöring” lurade in mig på tunga grejer som ”doktorandtjänst”, för din tillgänglighet och lagom blandning av uppmuntran och piska, samt för alla lärorika samtal om botanik i allmänhet och Silene i synnerhet! När Bengt försvann iväg till Göteborg har det känts tryggt att ha de biträdande handledarna Magnus Lidén och Mikael Thollesson på plats, ofta så nära som på armlängds avstånd vid lunchbordet. Extra tack till Magnus för kloka (om än ibland förvirrande) kommentarer på manuskript och kappa. It has been really fun to work with my co-authors from various parts of the world: Bodil Svennblad i Uppsala för hjälp med ”dra-bollar-ur-en-skål”. Dmitry Filatov in UK for sharing interesting sequence data with us, and for good contributions to the manuscript. Thanks to Honor C. Prentice and Louise Hathaway in Lund for letting me join the Elisanthe/Melandrium phylogeny project, and for your contributions to the manuscripts. Thanks to Daniel Sloan in USA for hard work with the mitochondrial sequences. Jag har haft stor nytta av material som nuvarande och tidigare medlemmar i Bengts Silene-grupp har arbetat fram, samt för de gruppmöten vi haft. Tack alltså till Frida Eggens, Per Erixon (för humorfulla svar på mina tusen frågor om analyser och labbprocedurer), Božo Frajman (for the Slovenia trip), Inga Hallin, Nahid Heidari (för ständig uppmuntran), Anders Larsson, Elisabeth Långström, Anna Petri (för våra spännande Rysslandsresor), Mikael Thol- lesson, Maria Ullbors och de tre Magnusarna (Lidén, Lundberg och Popp). Nahid Heidari och Afsaneh Ahmadzadeh har alltid varit redo att förklara mystiska labbprocedurer och beställa saker som behövs till labbet. Nahid och Vivian Aldén har omsorgsfullt producerat mängder av preppar och sekvenser. Jag är också enormt glad över att Vivian lyckades gro Silene multinervia, få den stackars plantan att överleva ända till våren, för att slutligen lyck- as räkna kromosomerna. Efter mina egna försök att räkna Silene-kromosomer åt Poppen ska jag erkänna att jag inte hade så mycket hopp om att de här rackarna över huvud taget har kromosomer...

35 Andreas Wallberg och Mats Töpel förtjänar ett varmt tack för idogt arbete med systematik-datorklustren i Uppsala och Göteborg, och närmast oändligt tålamod med de taffliga användarna (mig t ex). Övriga kollegor på avdelningen har bidragit till en både vetenskapligt och socialt trevlig arbetsmiljö. Tack till Agneta (en perfekt rumsgranne), Anders (för hjälp med kartor, gott samarbete med undervisning, utlåning av ergo- nomi-musen och trevliga utflykter), Anneleen, Cajsa (för matning, styling, fritidsaktiviteter och tysk humor), Frida (hade inte du börjat som doktorand före mig, skulle jag inte ha hamnat här. Tack också för gott sällskap under studietiden, och många spännande utflykter med Jesper H och Kajsa-Karin), Hobbe, Hugo, Jesper, Johanna (ich vermisse dich!), Katarina, Mats (tack för svar på alla taxonomiska och botaniska frågor), Sandie (for comments on manuscripts), Sunniva (for alle vitenskapelige og unscientific diskusjoner, godt samarbeid med undervisning, og andre aktiviteter. Jeg er også veldig glad for at du rydder vei ved å gå gjennom alle prosedyrer med avhandlingen jobbe noen uker før meg [Google translate, 2009]), Sylvain (for the great Madagascar trip!), Ulla, and to all other present and former colleagues. Fikarumskollegorna (molevol, fysbot och intendenturen) har bidragit till trevliga lunch- och kafferaster. Stefan, Ola och Håkan har hjälpt till med alla möjliga relevanta och irrelevanta datorfrågor (det är till exempel schysst att medan man tillbringar dagarna med att snöa in på konstiga biologiska processer, också då och då kunna komma hem med praktiska kunskaper, som hur man klämmer ihop sin egen ip-kabel). Extra tack till Stefan för oändligt tålamod med vår labbserver och dess (och dess administratörers) egenheter.

The curators and other staff have been really helpful during my visits to the herbaria GB, LE, MAG, MW, S, UPS, VLA and WU. Thanks to people who provided material: Doug Stone and the CAS herbarium, Jake Ruygt, Božo Frajman, Peter Schönswetter, and Georgy Lazkov, and to all the herbaria who have provided Bengt with material. Sara Gold (http://www.wildflowers.co.il) and Barry Breckling provided nice photographs of plants I had only seen as pressed, dry herbarium specimens. Ett varmt tack går också till Botaniska trädgården i Uppsala (främst till Christina Apell och hennes kollegor för omsorgsfullt omhändertagande av massor av Silene-plantor, men även för den otroliga servicen med material till undervisning).

, V ! Jag har också haft väldigt trevligt tillsammans med doktorander och andra kollegor från Göteborg, Stockholm och andra ställen, på olika kurser, resor och konferenser.

This PhD project was supported by grants from Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and The Swedish Research Council (VR) to Bengt Oxelman, and by finan-

36 cial support from The Royal Physiographic Society in Lund, Helge Ax:son Johnssons stiftelse, Royal Swedish Academy of Sciences (KVA), GBIF Sverige, Liljewalchs resestipendium, Linné-stipendiestiftelsen, V. Ekmans stipendiefond and Wallenbergstiftelsen to Anja Rautenberg. Jag är uppriktigt tacksam för bidragen till dator, pipetter, resor och labbkostnader! Part of this work was carried out using the resources of the Computational Biology Ser- vice Unit from Cornell University, which is partially funded by Microsoft Corporation.

Arkenfolket inklusive hang-arounds har i hög grad bidragit till trevlig av- koppling från biologin: Anja och Ronny med Aron, David och Noomi med Leo, Hasse-Mats, Kristina, PNK, Theres, Tobias, samt övriga korridorare, HemIT- och AAF-medlemmar, innebandyfolk och andra vänner. KinoRurik har bidragit till en arbetsbörda som Bengt nog skulle ha prote- sterat mot om han hade vetat (men även en viss övning i att sista-minuten- korrekturläsa texter, som kommer väl till pass nu), och desto mer inneburit trevligt samarbete med Anna, Magnus, Zhenja, Prune, familjen Lakstigal, Alexander och alla de andra. ! Aikidovännerna från Budohu- set har visserligen inte sett mig på mattan de senaste åren, men tiden där har ändå bidragit till grundkondition, samt ledarkurser och -erfarenhet som var nyttiga när jag började undervisa. Domo arigato gozaimashita! Det var för övrigt i samband med ett aikidoläger som jag första gången besökte herbariet i St Petersburg, så för mig hör aikido och herbariearbete ihop. Tack också till alla andra vänner och släktingar! Herzlichen Dank an mei- ne Deutschen Verwandte und Freunde, die immer sehr gastfreundlich sind! Ett extra varmt tack går till Inger, för kommentarer på kappan samt trevlig samvaro och generös support de senaste drygt 30 åren! Ulrik har bidragit med en del markservice, och desto mer med uppmuntran, glatt humör, galna upptåg, och en aldrig sinande ström av konspirations- teorier och dåliga skämt från obskyra internetforum, lägesrapporter från da- torspelsvärlden, detaljerade referat av din senast besökta föreläsning i håll- fasthetslära, och hela din trevliga familj! Och så till slut ett stort tack till mamma och pappa, för ständig uppmuntran, hjälp med bilder, och lagning av (nästan) allt som jag råkar ha sönder. Snart har jag äntligen tid att komma och pyssla om er och huset!

Och naturligtvis också tack till alla som inte står med ovan men ändå förkla- rat, matat, skjutsat, hjälpt till, lånat ut saker och uppmuntrat under åren!

37 References

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