Phylogenies and Secondary Chemistry in Arnica (Asteraceae)

Home , Anisocarpus, Arnica, Arnica angustifolia, Arnica cernua, Arnica chamissonis, Arnica gracilis

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 392

CATARINA EKENÄS

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

I Ekenäs, C., B. G. Baldwin, and K. Andreasen. 2007. A molecular phylogenetic study of Arnica (Asteraceae): Low chloroplast DNA variation and problematic subgeneric classification. Sys- tematic Botany 32 (4): 917-928.

II Ekenäs, C., J. Rosén, S. Wagner, I. Merfort, A. Backlund, and K. Andreasen. Secondary chemistry and ribosomal DNA data congruencies in Arnica (Asteraceae). Submitted.

III Ekenäs, C., A. Zebrowska, B. Schuler, T. Vrede, K. Andreasen, A. Backlund, I. Merfort, and L. Bohlin. Anti-inflammatory activity of extracts from Arnica (Asteraceae) species assessed by inhibition of NF-B and release of human neutrophil elastase. Submitted.

IV Ekenäs, C., N. Heidari, and K. Andreasen. RPB2 phylogeny in Arnica (Asteraceae) reveals multiple d-paralogues. Manuscript.

Paper I is reprinted with the kind permission of the publisher.

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 laboratory work and analyses were conducted by CE with the exceptions that follow. In paper I KA did the nuclear ribosomal DNA laboratory work, in paper II JR did the PCA, in paper III AZ and BS did part of the laboratory work and TV the ANOVA, and in paper IV NH did most of the laboratory work.

Contents

Introduction...... 7 Arnica...... 13 Aims...... 19 Materials and Methods...... 20 Phylogenetic studies of Arnica (I, II, IV)...... 23 Sesquiterpene lactones in Arnica (II) ...... 33 Anti-inflammatory activity of Arnica (III)...... 40 Conclusions...... 43 Svensk sammanfattning ...... 45 Acknowledgements - Tack! ...... 48 Literature...... 51

Abbreviations

ANOVA analysis of variance cpDNA chloroplast DNA ETS external transcribed spacer fMLP N-formyl-methionyl-leucyl-phenylalanine GC-MS gas chromatography-mass spectrometry HNE human neutrophil elastase ITS internal transcribed spacer lcnDNA low-copy nuclear DNA NF-B nuclear factor-B nrDNA nuclear ribosomal DNA PAF platelet activating factor PCA principal component analysis PCR polymerase chain reaction RPB2 second largest subunit of RNA polymerase II SM secondary metabolite STL sesquiterpene lactone TBR tree bisection-reconnection TNF- tumor necrosis factor alpha

Introduction

It has always been of interest to mankind to learn more about the world we live in – curiosity is the basis of science. Historically, natural sciences such as botany, medicine and chemistry were all included as one field of science, comprehensive enough for one person to grasp in a lifetime. Only a few cen- turies ago, a physician like Linneaus was also a botanist, zoologist, and a geologist. With more knowledge accumulating, fields in science naturally get more specialized. The role of interdisciplinary studies is to link various fields of research and find patterns that would otherwise not be detected.

Plant systematics and phylogenetics Systematics deals with the relationships and classification of organisms in groups. Classification of plants and animals based on appearance and other characteristics such as utility has probably existed in most traditional cul- tures worldwide. However, with Darwin’s theories of evolution as a base, new aspects of ancestry, relatedness and formation of new species were introduced (Darwin 1859). Phylogenetic studies were initially based mainly on morphology (e.g. Haeckel 1866, Fig. 1). With the emergence of molecular biology in the 20th century, hypotheses of relationships between taxa based on morphology could be tested by inferring phylogenetic relationships based on DNA sequence data. Since the mid-1990´s, molecular systematics has often been applied in attempts to resolve relationships at higher taxonomic levels, e.g. between tribes or families, as well as lower levels, e.g. between species within a genus.

Figure 1. One of the first published phylogenetic trees (Haeckel 1866).

Useful DNA regions The majority of sequence data currently used in plant systematics originates from chloroplast DNA (cpDNA) or nuclear ribosomal DNA (nrDNA) data. Chloroplast DNA is mostly maternally inherited in flowering plants. Since the evolutionary history of the organism may differ from the gene based phylogenies due to e.g. hybridization (see below), it is recommended to be used in combination with at least one region from the nuclear genome. Non- coding chloroplast regions evolve relatively slowly in comparison to nuclear regions, resulting in low sequence variation in recently diverged groups and problems with lack of resolution in phylogenetic analyses (e.g., Small et al. 1998, Cronn et al. 2002, Hartman et al. 2002, Xu et al. 2000). Nuclear ribosomal DNA, such as the Internal Transcribed Spacer (ITS) region has the advantage of being more variable than any non-coding cp region and has proven successful, especially in combination with cpDNA, to resolve relationships in several young groups (e.g., Soltis et al. 1996, Camp- bell et al. 1997, Yang et al. 2002). Nulear ribosomal DNA exists in tandem repeat multiple copies, which are believed to be homogenized by concerted evolution (Hillis et al. 1991).

8 In the search for other DNA regions for resolving phylogenetic relationships in recently diverged taxa, low copy nuclear DNA (lcnDNA) regions have been much investigated lately (e.g., Small et al. 1998, Popp and Oxel- man 2001, Pfeil et al. 2004, Joly et al. 2006). Low copy nuclear DNA regions exist in just a few copies, making it possible to trace paralogues in the genome to infer the evolutionary history without homogenizing processes like concerted evolution.

Hybridization and lineage sorting In low-level taxonomy, when relationships among recently diversified taxa are investigated, the influence of hybridization and lineage sorting as recent processes can complicate the picture. Hybridization, the interbreeding of lineages representing different taxa, has been proposed to be frequently oc- curring among plants (Ellstrand et al. 1996). When resulting in the formation of a new lineage, hybridization is often accompanied with a polyploidization event (i.e. a multiplication of the entire genome; allopolyploidization). Poly- ploidization as a result of a multiplication of the genome of a single species on the other hand (autopolyploidization) often results in sterility and propa- gation via agamospermy (asexual reproduction). In multi-copy regions prone to concerted evolution, such as in nrDNA, asexual reproduction can result in disrupted concerted evolution and polymorphisms between copies (e.g. Campbell et al. 1997). Although hybridization can be reflected in gene phylogenies, other processes such as gene duplication and incomplete lineage sorting can result in similar patterns (Fig. 2).

AB1 B2 C A B1 B2 C

Figure 2. Incomplete lineage sorting (left) and hybridization (right), are two under- lying processes that can result in the same pattern.

Where polymorphism occurs (i.e. multiple paralogues caused for example by gene duplication or polyploidy), lineage sorting can lead to extinction of all multiple paralogues within lineages, or a few (incomplete lineage sorting), which may lead to inference of wrong organismal relationships (see Doyle 1992). By investigating several independent gene regions, it may be possible to distinguish between these processes. If the same typological pattern is evident from different regions of the genome, hybridization may be more likely

9 to be the cause, whereas if the pattern is detected in one region only, it is more likely to be caused by incomplete lineage sorting or gene duplication/loss.

Secondary metabolites in chemotaxonomy Whereas primary metabolites are essential for cell survival, secondary metabolites (SMs) can be generally defined as compounds improving the fitness of the producing organism. These compounds can act for example as defence against microorganisms, protection against UV radiation, or as an attractant of pollinators. Secondary metabolites tend to be taxon-specific and various groups of SMs have been applied as taxonomic markers long before DNA markers were introduced (e.g., Abbott 1887). DNA sequence data is now more frequently used in phylogenetic studies, and phylogenies based on DNA sequence data can serve as a basis for interpretations of biosynthetic pathways of secondary metabolites (e.g., Wink and Mohamed 2003, Win- dsor et al. 2005).

Pharmacognosy Secondary metabolites lead us to the field of pharmacognosy, which deals with the discovery, characterization, and evaluation of bioactive compounds or extracts originating from natural sources. Pharmacognosy is an interdisciplinary field of science that spans a wide range of fields, from ethnopharmacology to natural products chemistry. A model was recently proposed, sum- marizing interactions among aspects of the field of pharmacognosy today (Larsson 2007).

Figure 3. Model illustrating aspects of pharmacognosy (from Larsson 2007).

10 Secondary metabolites as bioactive compounds Acting as a chemical defence in the producing organism, secondary metabolites possess properties that are of high interest in the search for pharmacologically interesting lead compounds (Müller-Kuhrt 2003, Larsen et al. 2005, and Larsson et al. 2007). Historically all drugs were based on natural products. The World Health Organization estimates that 25% of pharmaceuticals currently on the market still originate from traditional medicine (WHO 2003), and more than 45% originate from compounds isolated or derived from natural products (Cragg et al. 1997). So, a large percentage of modern pharmaceuticals originate from compounds that would also be classified as secondary metabolites. Although synthetic chemistry has enabled the production of large amounts of compounds to be screened in a relatively short time, it is unlikely that a complex molecule such as paclitaxel (Fig. 4) would have been synthesized in a chemistry lab. Natural products have the potential to serve as leads that, in combination with synthetic chemistry, can be modi- fied and optimized.

O CH3 O OH O

O NH O H H

O H OH O CH3 OH O O O Figure 4. Paclitaxel, isolated from Taxus brevifolia (Taxaceae) and used in cancer chemotherapy (Wani et al. 1971, Cragg et al. 1993).

Selection of organisms With an estimation of several million species on earth (including the species- rich kingdoms of bacteria and fungi), how does one decide where to start looking for bioactive compounds? Random screening programs tend to be costly and time consuming and it is therefore advantageous to use an initial target approach when selecting objects for further investigations. A classical approach used in ethnobotany or ethnopharmacology is to investigate species that are or have been used in traditional medicine. A species that has been selected and used over long periods in human history to treat a certain disor- der is more likely to contain bioactive compounds compared to randomly selected species.

11 A third approach that may be combined with the two above is to examine close relatives to species already known to synthesize pharmacologically interesting compounds. Theoretically, if SMs can be used as taxonomic markers (as discussed above), the opposite should also be true: there should be a higher chance of finding similar types of SMs in groups that are closely related, as inferred by a phylogeny based on DNA markers. As relationships between species are resolved by phylogenetic inference based on DNA markers, this can be tested by comparing a phylogeny based on DNA with a phylogeny based on chemical markers of the same species.

12 Arnica

Arnica L. (Asteraceae) is a circumboreal genus of rhizomatous, perennial herbs with a center of diversity in western North America. Morphological features of the genus include simple, opposite leaves, epaleate receptacles, yellow (rarely whitish) to orange corollas, yellow or purple anthers, gray or brown to black cypselae, and usually a pappus of bristles. The base chromo- some number for the genus is x = 19. In North America 32 minimal-rank taxa, including 26 species and six subspecies, were recognized by Wolf (2006). Including the species endemic to Europe (A. montana) and Asia (A. mallotopus and A. sachalinensis), 29 Arnica species are currently recognized worldwide.

Figure 5. Distribution of Arnica (from Maguire 1943).

Figure 6. Jasper National Park, Alberta, Canada. Photo by the author.

Taxonomic history and phylogenetic position The name Arnica was mentioned for the first time in Kräeuterbuch by Jaco- bus Theodorus Tabernaemontanus (1625), where a woodcut of A. montana is displayed under the name Caltha alpina (Fig. 7), with the the phrase “… aber von den Medicis, Arnica” added as a synonym. In the same reference,

14 Ptarmica, from the Greek word ptaro (= I sneeze), was also mentioned as a synonym, and could possibly be the origin of the name arnica (Mayer and Czygan, 2000). In the 17th-18th century, the European representatives of Ar- nica were commonly referred to under the name Doronicum (e.g by Lin- neaus in Flora Lapponica, 1737). However, in Species plantarum (1753), Linnaeus adopts the name Arnica, although of the six species he described only A. montana is maintained within the genus as recognized today.

Figure 7. The first appearance of A. montana as a woodcut (Tabernaemontanus, 1625).

Complex and diverse patterns of morphological variation in Arnica are reflected by widely differing taxonomic treatments. In a revision by Rydberg (1927) over 100 species were recognized as belonging in the genus. Poly- ploidy is widespread in many species and Barker (1966) established that there is a strong connection between polyploidy and agamospermy in Ar- nica. A hybrid origin of some species has also been proposed (e.g., Wolf and Denford 1984a). In the latest revision of the whole genus Maguire (1943) recognized 32 species and divided the genus into five subgenera; Andropur- purea, Arctica, Arnica (as: Montana), Austromontana, and Chamissonis. As a result of later revisions of subgenera Arctica, Austromontana and Chamis- sonis, the total number of species was reduced to 27 (Downie and Denford 1988, Wolf and Denford 1984b, Gruezo and Denford 1994). Later, in analyses by Baldwin et al. (2002), two species not included within the genus by

15 Maguire (1943), A. dealbata (A. Gray) B. G. Baldwin and A. mallotopus (Franch. & Sav.) Makino were nested within Arnica. At the time of Maguire’s revision of the genus (1943), Arnica was con- sidered to belong within tribe Senecioneae, subfamily Asteroideae, as proposed by Bentham (1873), but was later moved to tribe Heliantheae sensu lato (Nordenstam 1977, Robinson 1981) based on chemistry and morphology. In a discussion of the origin and biogeographical history of the genus, Maguire proposed that Arnica is of arctic origin and that it spread southward along four major routes. Using ITS sequence data, Baldwin and Wessa (2000) found that Arnica (subtribe Arnicinae) is sister to the tarweeds and silverswords (subtribe Madiinae) and nested within an otherwise temperate western North American lineage (tribe Madieae sensu Baldwin; see Baldwin et al. 2002; Fig. 8). Baldwin and Wessa suggested that the genus originated in temperate western North America, rather than in the arctic, as previously suggested by Maguire.

100 Eriophyllum lanatum 100 d8 Eriophyllum staechadifolium 69 d8 98 Pseudobahia bahiifolia d1 d6 Pseudobahia peirsonii 100 Syntrichopappus fremontii

d10 Baeriinae Eriophyllum congdonii 95 Lasthenia californica 97 d5 Lasthenia burkei * 56 d8 100 Baeriopsis guadalupensis d2 d21 Amblyopappus pusillus 83 Monolopia gracilens d4 100 Monolopia major d14 Monolopia (Lembertia ) congdonii 100 Constancea (Eriophyllum )nevinii San Clemente Island d19 Constancea (Eriophyllum )nevinii Santa Catalina Island 76 Venegasia carpesioides Venegasiinae d2 100 Hulsea algida x = 19 taxa 93 d15 Hulsea californica 81 Hulseinae d6 Hulsea vestita d2 Eatonella nivea

Arnica mollis Arnicinae 100 Arnica cernua Clone 1 86 Arnica cernua Clone 2 d5 d3 Arnica cernua Clone 3 100 Arnica (Whitneya) dealbata d10 d1 100 Arnica unalaschensis d6 Arnica mallotopus (Mallotopus japonicus ) Arnica longifolia Raillardella argentea Adenothamnus validus 97 Hemizonella (Madia) minima d1 d7 Kyhosia (Madia) bolanderi 52 Raillardiopsis d2 Anisocarpus ( ) scabridus d1 83 99 Carlquistia (Raillardiopsis ) muirii d2 100 d11 Madia sativa d10 100 Argyroxiphium sandwicense d13 Wilkesia gymnoxiphium d1 Dubautia plantaginea Madiinae Deinandra (Hemizonia ) fasciculata d1 Holocarpha virgata Centromadia (Hemizonia ) perennis 53 Calycadenia truncata d1 Osmadenia tenella 56 Hemizonia congesta d1 Blepharizonia plumosa 94 100 Lagophylla ramosissima d6 d11 Lagophylla minor d1 Holozonia filipes d1 55 Layia heterotricha 61 Layia munzii d2 Layia gaillardioides Blepharipappus scaber d1 Achyrachaena mollis

Figure 8. Placement of Arnica as subtribe Arnicinae, sister to subtribe Madiinae within tribe Madieae (from Baldwin and Wessa 2000, with permission from the authors).

16 Traditional and modern use Documented medicinal use of the European representative, A. montana, stretches back at least to the 14th century when it was mentioned in an ency- clopedia by Matthaeus Sylvaticus (1317) for menstrual pains. From the 16th century it is widely mentioned as a “wound-remedy” to treat external injuries (Mayer and Czygan 2000). Arnica was also mentioned by Linnaeus as a substitute for tobacco in Flora Suecica (1755). The North American species A. acaulis, A. angustifolia, A. cordifolia, A. latifolia, and A. discoidea have also been used in traditional medicine according to a number of ethnobotani- cal surveys as summarized in Table 1. Interestingly, widely separated groups of people have used Arnica species to treat disorders that could be coupled to inflammation.

Figure 9. Cultivation of A. montana (with permission from Weleda).

Flower heads of A. montana are still used today, applied externally as a tincture or oil to treat inflammatory-related topical disorders such as sprains, swellings, and bruises. The more easily cultivated A. chamissonis has been proposed as a substitute to A. montana (Cassells et al. 1999). In a randomised double-blinded study of 204 patients, the short-term use of Arnica gel was compared to ibuprofen gel (5%) in the treatment of hand osteoarthritis. The result was no significant perceived difference between the two treatments (Widrig et al. 2007).

17 Table 1. North American Arnica species for which there is documented traditional use. Species Use Preparation Plant part Ethnic group and location A. acaulis back pain (Speck 1937) infusion roots Catawba, North Carolina A. angustifolia stomach problems (Alestine and infusion - Gwich’in, Yukon and NWT Fehr 2002) A. cordifolia sore eyes (Palmer 1975) - - Shuswap, BC A. cordifolia and bruises, swellings and cuts (Turner poultice whole plant Thompson, BC A. latifolia 1990) tuberculosis (Turner 1990) infusion whole plant Thompson, BC aphrodisiac (Turner 1980) - roots Okanagan-Colville, BC A. discoidea wounds (Strike 1994) - - Maidu, California NWT: Northwest Territories, BC: British Columbia

Sesquiterpene lactones With more than 5000 known structures, STLs constitute one of the largest groups of natural products known (Schmidt 2006). Most of these compounds are isolated from species in Asteraceae and are believed to be responsible for at least part of the evolutionary success of this large group of taxa. These compounds have been investigated for potential use as chemotaxonomic markers for the family as a whole, using cluster analysis, principal component analysis (PCA), or other multivariate statistical methods (e.g., Al- varenga et al. 2001, Hristozov et al. 2007), and as a source of bioactive compounds (Schmidt and Heilmann 2002, Kanashiro et al. 2006, Wagner et al. 2006, Scotti et al. 2007). Numerous STLs have been isolated and identified from Arnica species as summarized by Willuhn and co-workers (1995). Especially A. montana has been thoroughly investigated for STL contents and the anti-inflammatory effect of these compounds. Sesquiterpene lactones of the helenanolide type from A. montana and A. chamissonis have been shown to inhibit the release of human neutrophil elastase from neutrophils, and thereby intervene with the inflammatory process (Siedle at al. 2003). It has also been demonstrated that STLs prevent the binding of nuclear factor-B (NF-B) to DNA by alkylation of cysteine-38 in the p65/NF-B subunit (Siedle at al. 2004), thereby preventing the transcription of inflammatory mediators. There are strong indications that this is a general mechanism for STLs possessing ,- unsaturated carbonyl structures such as -methylene--lactones or ,- unsaturated cyclopentenones (Lyss et al. 1998, Garcia-Pineres et al. 2001).

18 Aims

The work presented in this thesis was conducted at the Department of Evolu- tion, Genetics and Systematics, Subdepartment of Systematic Biology, and at the Department of Medicinal Chemistry, Division of Pharmacognosy.

The specific aims of this thesis were to:

Investigate phylogenetic relationships in Arnica using several independent DNA regions from cpDNA, nrDNA, and lcnDNA (I, II, and IV).

Investigate the STL chemistry in Arnica and closely related taxa (II).

Investigate possible congruencies between ribosomal DNA and STL data in Arnica (II).

Evaluate anti-inflammatory activity of several species of Arnica using two bioassays and correlate the activity to STL content (III).

19 Materials and Methods

Taxon sampling At least one representative of all species (29) belonging to Arnica are included in (IV), and in (I) all are included except A. louiseana. Four outgroup taxa; Eatonella nivea, Hulsea algida, Raillardella argentea, and Venegasia carpesioides, belonging to closely related lineages in tribe Madieae were included. Samples originated from herbarium specimens or from fresh specimens collected in the field, or in botanical gardens.

Figure 10. Cultivation of Arnica species in Uppsala Botanical Garden. Photo by the author.

All specimens included in (II) and (III) were cultivated outdoors in Upp- sala Botanical Garden, Sweden. We collected seeds and rhizomes during

20 field trips to Alberta and California. Seeds were also obtained from specimens collected by others in the field and from botanical gardens. In (II) and (III), representatives of 16 and 12 Arnica species were included respectively. Two outgroup taxa, Layia hieracioides and Madia sativa, from the subtribe Madiinae (sister to Arnica), were included in both studies.

Sequenced DNA regions and cloning The internal and external transcribed spacers (ITS and ETS) of the nuclear ribosomal nrDNA, were sequenced in (I) and (II). When polymorphism was detected, PCR-products were cloned (10 clones) and sequenced. In (I), five cpDNA regions (the rpl16 and rps16 introns and the psbA---trnH, ycf4---cemA, and trnT---L spacers) were amplified and sequenced. In (IV) the region between exons 17 and 23 in the nuclear low-copy gene encoding the second largest subunit of RNA polymeras II (RPB2) was amplified, cloned (10-18 clones) and sequenced.

Isolation of STL-rich fraction For (II) and (III), flowerheads were collected from cultivated specimen in two batches (2005 and 2006). Extraction was followed by isolation of STL- rich fractions (see fractionation protocol, Fig. 11). These were subjected to gas chromatography and mass spectrometry (GC-MS) analyses in (II) and mass spectra and retention times of the resulting compounds were compared to those of already known STLs in Arnica.

CH2Cl2 and MeOH extracts filtration, evaporation

EtOAc H2O and MeOH evaporation

CH2Cl2 filtration, evaporation

MeOH

Al2O3, filtration, evaporation

STL-rich extract

Figure 11. Fractionation scheme for isolation of a STL-rich fraction (II and III).

21 Phylogenetic analyses and PCA In (I), the two matrices of cp and nrDNA regions were subjected to separate and combined parsimony heuristic tree searches and bootstrap analyses. The combined matrix was also subjected to phylogenetic analysis using Bayesian inference. Three RPB2-d paralogues were detected in (IV) and each was aligned separately and subjected to separate parsimony heuristic tree searches and bootstrap analyses as well as Bayesian analyses. In (II), nrDNA sequences and a matrix of STL data (in which peaks were coded as absent or present) were separately subjected to parsimony analyses and the matrix of STL data (peak areas) was also subjected to PCA.

Bioassay studies In (III), the STL-rich fractions were tested for anti-inflammatory activity using two bioassays, one testing the capacity of extracts to inhibit the release of human neutrophil elastase (HNE) from neutrophils, and one testing the capacity to inhibit the binding of transcription factor NF-B to DNA. The results were subjected to analysis of variance (ANOVA), comparing results between extracts as well as between results of the two bioassays.

22 Phylogenetic studies of Arnica (I, II, IV)

Phylogenetic investigations of plants are usually based on cpDNA, nrDNA, and/or lcnDNA regions. In this thesis we have sequenced DNA originating from all three types.

Chloroplast regions – low variation (I) Variation in non-coding chloroplast regions of Arnica was found to be low leading to weakly supported basal nodes (Fig. 12). Five chloroplast regions (3710 bp) were sequenced which resulted in and average of 12 parsimony- informative characters. Similarly low variation in chloroplast DNA has been found in many recently diversified taxa, such as Gossypium, Malvaceae (Small et al. 1998, Cronn et al. 2002), Lophocereus, Cactaceae (Hartmann et al. 2002), and Soja, Fabaceae (Xu et al. 2000).

23 sororia AR nevadensis 1 AU 2, 3, 5 nevadensis 2 AU cordifolia AU 5 lanceolata prima CH 5 ovata 1 CH parryi CH 2, 5 rydbergii AR lonchophylla 1 AR 1, 5 lonchophylla 2 AR 3, 5 lonchophylla 3 AR 2, 5 65 angustifolia tomentosa 1 AR angustifolia tomentosa 2 AR 1, 5 57 gracilis 1 AU 1, 5 spathulata 1 AU 1, 5 60 mollis CH cernua AU 5 dealbata 5 ovata 2 CH B 100 griscomii frigida 1 AR griscomii frigida 2 AR montana 1 MO latifolia AU lessingii AN angustifolia angustifolia 1 AR C 86 chamissonis CH angustifolia angustifolia 2 AR 5 angustifolia angustifolia 3 AR 2, 3, 5 67 montana 2 MO 52 acaulis 1 MO 3, 5 acaulis 2 MO 3, 5 100 A fulgens AR sachalinensis AN 5 discoidea AU unalaschcensis AN Venegasia carpesioides* 5 Eatonella nivea*

Figure 12. Strict consensus of 508 most-parsimonious trees based on cpDNA sequences (heuristic search, TBR, 100 replicates). Bootstrap values over 50% are indicated and major supported clades are marked with capital letters. Taxon samples are indicated by species and subspecies followed by population number and the first two letters of the subgenus in which it was placed by Maguire (MO: subg. Montana (now Arnica), AR: subg. Arctica, AU: subg. Austromontana, CH: subg. Chamissonis, and AN: subg. Andro- purpurea). *Outgroup taxa. Missing sequences are indicated by superscript numbers (1: rpl16 intron, 2: rps16 intron, 3: trnT-L spacer, 4: PsbA-trnH spacer, and 5: ycf4-cemA spacer).

Polymorphism in nrDNA regions (I and II) The nuclear ribosomal regions ITS and ETS were used in two studies. In (I), the aim was to compare phylogenies based on ribosomal DNA data (Fig. 13) and chloroplast data. In (II) ITS and ETS were sequenced for cultivated accessions of Arnica with the aim to compare the phylogeny (Fig. 19) with STL data of the same accessions. The two datasets are based on different accessions, with a more complete taxon sampling in (I). In (II) we detected three paralogues in the dataset for some species. Similar patterns of poly- morphic ITS sequences have been found in a few taxa e.g., Amelanchier (Rosaceae), where it was explained by hybridization in combination with

24 reduced concerted evolution due to agamospermy (Campbell et al., 1997). It is likely that agamospermy in Arnica is responsible for a similar reduction in concerted evolution between different ITS and ETS copies and that this is causing the divergent paralogues to be maintained.

sororia AR 100 griscomii frigida 1 AR griscomii frigida 2 AR B 96 gracilis 1 AU rydbergii AR parryi CH sachalinensis AN C 53 acaulis 1 MO 64 fulgens AR 94 lonchophylla 2 AR 1 lonchophylla 3 AR 1 100 longifolia 1 CH D 83 longifolia 2 CH lonchophylla 1 AR ovata 2 CH mollis CH E 98 chamissonis CH lanceolata prima CH ovata 1 CH 58 72 angustifolia angustifolia 1 AR angustifolia angustifolia 2 AR angustifolia tomentosa 1 AR latifolia AU 100 montana 1 MO montana 2 MO 98 spathulata 2 AU 62 spathulata 1 AU A 100 discoidea AU 2 F 70 66 cordifolia AU gracilis 2 AU 1 cernua AU venosa AU nevadensis 1 AU 100 78 nevadensis 2 AU G 73 dealbata viscosa AU H 97 unalaschcensis AN 93 mallotopus ** lessingii AN Raillardella argentea* 100 Hulsea algida* 100 Eatonella nivea* Venegasia carpesioides*

Figure 13. Strict consensus of 16 most-parsimonious trees based on nuclear ribosomal DNA (ITS and ETS) sequences (heuristic search, TBR, 100 replicates). Bootstrap values over 50% are indicated and clades discussed in (I) are marked with capital letters. Taxon samples are indicated by species and followed by population number and the first two letters of the subgenus in which it was placed by Maguire (MO: subg. Montana (now: Arnica), AR: subg. Arctica, AU: subg. Austromontana, CH: subg. Chamis- sonis, and AN: subg. Andropurpurea). *Outgroup taxa. **ETS sequence of A. mallotopus is from voucher source 2. Missing sequences are indicated by superscript numbers (1: ITS and 2: ETS).

RPB2 – three d-paralogues (IV) The low-copy nuclear gene coding for the second largest subunit of RNA polymerase II (RPB2; Sawadogo and Sentenac 1990), has been successfully

25 used for resolving phylogenetic relationships at higher taxonomic levels (Denton et al. 1998, Oxelman and Bremer 2000, Oxelman et al. 2004, Ron- çal et al. 2005) as well as lower levels (e.g., Popp and Oxelman 2001, Pfeil et al. 2004, Goetsch et al. 2005, Thomas et al. 2006, Loo et al. 2006, Sun et al. 2007, Kool et al. 2007). Two copies of RPB2 are present in some angio- sperm taxa, which has been suggested to be the result of a gene duplication near the origin of core eudicots. The RPB2-d copy is found almost univer- sally in investigated angiosperms, while RPB2-i is scattered within two major clades of eudicots (Oxelman et al. 2004, Luo et al. 2007). In Arnica, three RPB2-d paralogues were detected (RPB2-dA, RPB2-dB, and RPB2-dC; Fig. 14-16). Since the outgroup taxa (Venegasia, Hulsea, Eatonella, and Raillardella) are nested within the RPB2-d copies, the two duplication events within the d-copy must have occured prior to the diver- gence of Venegasiinae, Hulseinae, Arnicinae, and Madiinae in Madieae. In a phylogenetic study of helenioid Heliantheae, Baldwin et al. (2002) suggest a polyploidization event at the base of the Heliantheae s.l. and Eupatorieae, which could possibly be coupled to at least one of the duplication events detected in RPB2 in Arnica and the outgroups. Multiple paralogues of RPB2- d in Hibiscus and Sidalcea have been explained as a result of a more recent duplication event within in Malvaceae (Pfeil et al. 2004, Stone and An- dreasen, in prep.). Multiple RPB2-d copies have been found in Silene as result of polyploidization (Caryophyllaceae; Popp and Oxelman 2001). As in ITS and ETS, polymorphism was also detected within the three paralogues. Although the tree topologies of the three paralogues are not completely congruent, 30% of the strongly supported clades uniting different species are present in trees of more than one paralogue.

26 mallotopus 7 AN 2 86 A. mallotopus and mallotopus 1 AN 2 1.00 A. chamissonis chamissonis 3 CH 2 chamissonis 7 CH 2 84 lanceolata 1 CH 1.00 A. mallotopus, A. chamissonis lanceolata 7 CH and A. lanceolata chamissonis 1 CH 2 100 chamissonis 10 CH 2 1.00 chamissonis 4 CH 2 lanceolata 2 CH chamissonis 6 CH 2 0.66 51 fulgens AR 0.90 sororia AR gracilis 8 AU 2 0.98 gracilis 10 AU 1 gracilis 5 AU 1 76 gracilis 4 AU 1 67 1.00 gracilis 3 AU 1 mollis 7 CH A. lessingii, A. chamissonis, A. mollis, mollis 9 CH 0.95 58 0.98 A. parryi, A. gracilis, and A. ovata parryi 5 CH 98 ovata 5 CH 2 mollis 4 CH 1.00 0.98 lessingii 40 AN 1 77 chamissonis 8 CH 1 ovata 7 CH 1 0.95 lanceolata 8 CH longifolia b6 CH ovata 6 CH 2 0.98 63 sachalinensis 1 AN unalaschcensis 6 AN 0.99 1.00 57 nevadensis 1 AU louiseana 8 AR 69 lessingii 2 AN 2 1.00 nevadensis 10 AU 0.98 52 90 A. lessingii, A. nevadensis lessingii 6 AN 2 1.00 and A. chamissonis 96 lessingii 5 AN 2 chamissonis 9 CH 1 1.00 mallotopus 7 AN 1 louiseana 1 AR montana 5 MO angustifolia ssp. tomentosa 12 AR 100 A. lonchophylla and angustifolia ssp. tomentosa 13 AR 1.00 lonchophylla 10 AR A. angustifolia latifolia 10 AU 1 82 latifolia 2 AU 1 A. lessingii and A. latifolia 1.00 lessingii 4 AN 1 lessingii 9 AN 1 discoidea f AU A. lessingii, A. latifolia, A. discoidea, 72 0.80 lessingii 2 AN 1 0.99 lessingii 10 AN 1 A. spathulata, and A. cernua 59 100 lessingii 3 AN 1 discoidea c AU 1.00 0.98 spathulata 3 AU A. lonchophylla and cernua 4 AU 99 gracilis 4 AU 2 A. rydbergi gracilis 11 AU 1 67 1.00 86 rydbergii g AR 0.78 77 1.00 lonchophylla 38 AR 100 0.97 100 gracilis 10 AU 2 A. lonchophylla, A. gracilis, gracilis 9 AU 1 1.00 1.00 angustifolia ssp. tomentosa 6 AR A. rydbergi, and A. angustifolia 93 angustifolia ssp. angustifolia 1 AR 100 1.00 angustifolia ssp. angustifolia 1 AR 67 lonchophylla 6 AR 1.00 angustifolia ssp. angustifolia 8 AR A. lonchophylla and 99 latifolia 4 AU 2 A. angustifolia latifolia 7 AU 1 A. latifolia and A. cordifolia 1.00 67 cordifolia 8 AU 1.00 griscomii 13 AR Raillardella argentea n 5 changes Figure 14. One of 100 000 most parsimonious trees resulting from a maximum parsimony analysis of RPB2-dA (100 replicates with a maximum of 1000 trees saved per replicate). Species epithets are followed by clone denotation and the first two letters of the subgenus in which it was placed by Maguire (1943; MO: subg. Montana (now: Arnica), AR: subg. Arctica, AU: subg. Austromontana, CH: subg. Chamissonis, and AN: subg. Andropurpurea). This is followed by an accession number in cases of multiple representatives of the same species. Bootstrap support values > 50% are indicated above branches and Bayesian posterior probabilities >0.50 are indicated below branches. Dashed branches are not present in the parsimonious strict consensus tree. Branches uniting clades with bootstrap support > 80% are bold.

27 100 angustifolia ssp. tomentosa 9 AR A. lonchophylla and 0.99 1.00 lonchophylla 7 AR A. angustifolia parryi 1 CH 77 72 parryi 4 CH 0.98 64 mollis 14 CH 0.98 chamissonis 5 CH 1 56 0.98 gracilis 7 AU 2 51 gracilis 5 AU 2 0.98 ovata 12 CH 1 93 louiseana 9 AR A. griscomii and 97 1.00 griscomii 12AR A. louiseana 0.59 1.00 louiseana c11 AR montana 1 MO 0.68 acaulis a5 MO gracilis 3 AU 2 50 lanceolata 10 CH 0.80 griscomii 14 AR 92 lessingii 7 AN 2 A. lessingii and A. ovata 0.99 ovata 3 CH 2 gracilis 11 AU 2 95 gracilis 2 AU 1 1.00 gracilis 12 AU 1 gracilis 9 AU 2 A. lonchophylla and A. gracilis 89 60 gracilis 6 AU 1 1.00 gracilis 7 AU 1 0.81 0.99 lonchophylla 1 AR lonchophylla 4 AR longifolia 3 CH 2 longifolia b3 CH 1 parryi 6 CH 100 85 longifolia b4 CH 1 A. longifolia and A. parryi 1.00 1.00 longifolia a4 CH 1 longifolia 1 CH 2 longifolia b9 CH 1 0.97 64 fulgens 8 AR 97 sachalinensis 5 AN 61 A. sachalinensis and A. unalaschcensis 62 1.00 unalaschcensis 5 AN 1.00 0.92 100 latifolia 6 AU 2 A. latifolia and A. cordifolia 1.00 cordifolia 3 AU 97 cernua 2 AU 1.00 spathulata 2 AU A. cernua and A. spathulata 82 latifolia 8 AU 1 100 64 1.00 lessingii 1 AN 1 A. lessingii, A. latifolia, A. discoidea, 1.00 discoidea e AU 0.96 97 latifolia 5 AU 1 A. spathulata, and A. cernua 1.00 lessingii 8 AN 1 0.94 latifolia 4 AU 1 A. lessingii and A. latifolia 100 dealbata b AU 100 1.00 discoidea d AU A. dealbata and A. discoidea 1.00 dealbata d AU 63 lessingii 3 AN 2 62 0.98 nevadensis 15 AU A. lessingii and 100 0.90 lessingii 12 AN 2 A. nevadensis 60 1.00 lessingii 4 AN 2 1.00 venosa 1 AU Venegasia carpesioides b 5 changes Figure 15. One of 4 208 most parsimonious trees resulting from a maximum parsimony analysis of RPB2-dB (100 replicates with a maximum of 1000 trees saved per replicate). Species epithets are followed by clone denotation and the first two letters of the subgenus in which it was placed by Maguire (1943; MO: subg. Montana (now: Arnica), AR: subg. Arctica, AU: subg. Austromontana, CH: subg. Chamissonis, and AN: subg. Andropurpurea). This is followed by an accession number in cases of multiple representatives of the same species. Bootstrap support values > 50% are indicated above branches and Bayesian posterior probabilities >0.50 are indicated below branches. Dashed branches are not present in the parsimonious strict consensus tree. Branches uniting clades with bootstrap support > 80% are bold.

mollis 8 CH lessingii 6 AN 1 87 mollis 5 CH A. lessingi, A. chamissonis 1.00 mollis 15 CH and A. mollis 98 mollis 1 CH 1.00 chamissonis 4 CH 1 mollis 1 CH 77 100 A. longifolia, A. chamissonis 97 chamissonis 8 CH 2 1.00 longifoia 7 CH 2 and A. mollis sororia 1 AR griscomii 5 AR 1.00 63 lanceolata 6 CH 100 ovata 10 CH 2 1.00 83 sachalinensis 10 AN A. sachalinensis and 1.00 75 unalaschcensis 10 AN 81 1.00 A. unalaschcensis lessingii 1 AN 2 77 1.00 0.81 0.95 latifolia 1 AU 2 86 discoidea B AU 100 1.00 100 cernua 9 AU A. cernua and A. spathulata 1.00 1.00 spathulata 1 AU 87 dealbata h AU 1.00 90 Raillardella argentea h 1.00 viscosa 3 AU Venegasia carpesioides j Eatonella nivea 7 Hulsea algida e 10 changes Figure 16. One of 6 most parsimonious trees resulting from a maximum parsimony analysis of RPB2-dC (100 replicates with a maximum of 1000 trees saved per replicate). Species epithets are followed by clone denotation and the first two letters of the subgenus in which it was placed by Maguire (1943; MO: subg. Montana (now: Arnica), AR: subg. Arctica, AU: subg. Austromontana, CH: subg. Chamissonis, and AN: subg. Andropurpurea). This is followed by an accession number in cases of multiple representatives of the same species. Bootstrap support values > 50% are indicated above branches and Bayesian posterior probabilities >0.50 are indicated below branches. Dashed branches are not present in the parsimonious strict consensus tree. Branches uniting clades with bootstrap support > 80% are bold.

Complex evolutionary patterns Evolutionary relationships in Arnica are difficult to track, as expected in a young genus of recently diversified taxa, and in which processes such as polyplodization, agamospermy, and possibly hybridization are present. The phylogenetic trees resulting from analyses of the independent datasets of RPB2-d paralogue, nrDNA, and cpDNA regions, have few strongly supported groups in common. Polymorphism within nrDNA and RPB2-d paralogues makes it difficult to concatenate matrices based on the different regions. It was therefore not possible to produce an analysis on the combined data matrix. However, a few groups are supported in trees of more than one region, such as the close (although weakly supported) relationship between A. acaulis and A. fulgens based on nrDNA, cpDNA, and RPB2-dA regions. These species are not regarded as close and are classified into separated subgenera: A. acaulis in Arnica and A. fulgens in Arctica (Maguire 1943). Ar- nica sachalinensis, A. unalaschcensis, and A. lessingi have been proposed to be closely related and share the synapomorphy of purple rather than yellow anthers. In our phylogenetic analyses, A. sachalinensis and A. unalaschcen-

29 sis are sisters according to all RPB2-d paralogues, whereas in the tree based on ITS and ETS (Fig. 13), A. unalaschcensis, A. mallotopus and A. lessingi are found in a strongly supported group diverged from A. sachalinensis (although weakly supported). Arnica lanceolata and A. chamissonis have also been proposed to be closely related and end up close according to phylogenetic analyses based on rnDNA data and RPB2-d paralogues A and C. Barker (1966) noted a strong correlation between agamospermy and polyploidy in Arnica. He found all strictly diploid species in Arnica (A. montana, A. acaulis, A. unalaschcensis, A. sachalinensis, A. fulgens, A. sororia, A. cernua, A. viscosa, and A. venosa) to be also strictly amphimictic (i.e. only reproducing sexually) in all representatives investigated. Our results support the findings of Barker, since there is no polymorphism in any accessions of the species mentioned above in any of the regions we investigated. Polymorhism in both nrDNA and RPB2-d paralogues were found in accessions of A. angustifolia, A. chamissonis, A. gracilis, A. lanceolata, A. mollis, and A. parryi. It is likely that polymorphism observed in these accessions is connected to polyploidy, and that incomplete lineage sorting result in the clustering of these with different taxa in different regions or paralogues. This would explain the observed incongruencies between DNA regions and paralogues within regions.

Phylogeny and origin Although patterns are complex, some trends can be identified from all datasets. In phylogenetic analyses resulting from nrDNA and cpDNA regions, there are strong support values for clades uniting all Arnica sequences. Re- garding RPB2-dA and RPB2-dB, only one outgroup taxon was detected in each of the paralogues, and therefore we can draw no conclusions regarding monophyly of Arnica based on these paralogues. However, in the RPB2-dC paralogue, A. viscosa is not nested within Arnica. A member of the tarweed genus, Raillardella, diverges in a clade after A. viscosa, making Arnica non- monophyletic in the RPB2-dC tree. Interestingly, the anomalous morphology of A. viscosa, noted by Maguire (1943), is superficially like that of Rail- lardella, as reflected by R. paniculata Greene, a synonym of A. viscosa. One explanation for this pattern could be introgression between A. viscosa and a taxon outside Arnica, e.g., in Raillardella or the lineage leading to Rail- lardella. Two species previously placed outside Arnica - A. mallotopus (Mallotopus japonicus) and A. (Whitneya) dealbata are confidently con- firmed as members of Arnica in all analyses in which these are included.

Figure 17. Arnica viscosa. Photo by the author.

Based on analyses of all regions, none of the subgenera proposed by Maguire (1943) constitute a monophyletic group. However, in all analyses, all members of subgenus Chamissonis are only present in branches diverging further up in the trees. Another trend is that basally diverging branches mostly consist of members of subgenus Andropurpurea (nrDNA, and cpDNA regions in I), Austromontana (RPB2-dC) or both (RPB2-dA and RPB2-dB). Maguire hypothesized that subg. Arctica is ‘‘the closest arche- typal major group and is placed nearest the base’’ in Arnica (directly de- scendent from his hypothetical ‘‘Protoarnica’’). Although additional data will be necessary to ascertain the earliest diverging lineages in Arnica, none of the trees provide any indications that members of subg. Arctica constitute a deep grade or early diverging clade. Maguire’s proposal of an arctic origin of Arnica is not supported by our analyses. Instead, the hypothesis suggested by Baldwin (2002) of an origin in temperate western North America, which coincides with the distribution of the sister group to Arnica, is more in agreement with our results. As discussed above, members of subgenus Austromontana are present in basally diverged clades and many of these are typically or strictly diploid species of restricted distribution in California and Oregon (e.g., the successively basally diverging taxa in RPB2-dC: A. viscosa, A. dealbata, A. cernua, and A. spathulata). Members of subgenus Andropurpurea constitute early diverging clades in all of the trees based on cpDNA and rnDNA (in I), which, although weakly supported, raises the intriguing possibility that an early lineage of Arnica

31 may have occurred in the northwestern Pacific Rim, where those taxa are endemic. As discussed by Wolf and Denford (1984a), the East-West disjunct distribution of some species, and the circumboreal distribution of A. angustifolia, suggests that Arnica was part of the Arcto-Tertiary flora. Raven and Axelrod (1978) included Arnica in a group of genera that are well developed in Cali- fornia and at the same time widespread, suggesting that the spreading aridity from Upper Tertiary times which culminated in a full Mediterranean climate in the later Quaternary has affected these taxa. Our result of low support for basal branches, combined with short branch lengths, suggests that the spread of Arnica took place under rapid evolution, which may have coincided with this change in climate.

32 Sesquiterpene lactones in Arnica (II)

Identified STLs Sesquiterpene lactones constitute one of the largest groups of structurally identified natural products and many STLs have been identified in Arnica species (e.g., Willuhn et al. 1995 and references therein, Schmidt and Wil- luhn 2000, Kos 2005). The total matrix of 239 peaks resulting from GC-MS data was subjected to phylogenetic analysis and PCA. Some peaks that were important for the formation of clusters were examined closer we were able to identify 29 STLs of seven core structures (Fig. 18, Table 2) by comparing with retention times and mass spectra of already structurally determined STLs.

helenalin derivatives: R = R = 1 6-O-acetyl-helenalin 1: acetyl 8: H 2 6-O-isobutyryl-helenalin 2: isobutyryl 9: acetyl 3 6-O-methacryloyl-helenalin O 3: methacryloyl O 10: isobutyryl 4 6-O-methylbutyryl-helenalin 4: methylbutyryl 11: methacryloyl 5 6-O-isovaleryl-helenalin O 5: O 12: methacrylbutyryl O isovaleryl 6 6-O-angelicoyl-helenalin OR 6: angelicoyl O OR 13: isovaleryl 7 6-O-tigloyl-helenalin 7: tigloyl 14: angelicoyl 15: tigloyl dihydrohelenalin derivatives: 16: senecioyl 8 11,13-dihydrohelenalin 17 R1 = methyl, R2 = H 9 6-O-acetyl-11,13-dihydrohelenalin OH 18 R1 = H, R2 = methyl O 19 R1 = H, R2 = H 10 6-O-isobutyryl-11,13-dihydrohelenalin 20 R1 = acetyl, R2 = H 11 6-O-methacryloyl-11,13-dihydrohelenalin O O 21 R1 = acetyl, R2 = acetyl 12 6-O-2-methylbutyryl-11,13-dihydrohelenalin 13 6-O-isovaleryl-11,13-dihydrohelenalin O O 14 6-O-angelicoyl-11,13-dihydrohelenalin O O 15 6-O-tigloyl-11,13-dihydrohelenalin R1 O 16 6-O-senecioyl-11,13-dihydrohelenalin OR2 OR1 O R2 arnifolin derivatives: 23 17 11,13-dihydroarnifolin 18 flexuosin B 24 22 O chamissonolide derivatives: O O O O 19 chamissonolide OH O 20 6-O-acetylchamissonolide O O 21 4,6-Di-O-acetylchamissonolide 25 26 carabrone dervative: 22 carabrone O O xanthalongin d erivatives: O O O OH O 23 xanthalongin 24 4H-xanthalongin 25 11,13-dihydroxanthalongin O 26 11,13-dihydro-4H-xanthalongin 27 28 29 HO O HO O florilenalin derivative: O O O 27 2-O-acetylflorilenalin HO O eudesmanolide derivative: 28 ivalin 29 11,13-dihydroivalin Figure 18. Identified STLs in GC-MS profiles of Arnica and outgroup taxa.

33 Table 2. Distribution of the 29 identified STLs in accessions of Arnica and outgroup taxa. Accession Identified STLs He DHe Ar Cha Ca Xa Fl Eu 11 21 31 41 51 61 71,4 8 91 101 111 123 131 143 153 161,3 17 18 193 203 213 222 232,5 243,4 255 262 272 28 29 A. montana 1 X X X X X X X (x) X X X - X - X X X - - - - X ------A. montana 2 X X X X X X X (x) X X X - X - (x) X - - - - - (x) - - (x) - - - - A. montana 3 X X X X X X X (x) X X X - X - - X (x) ------A. montana 4 X X X X X X X (x) X X X - X - - X X - - - - (x) ------A. montana 5 X X X X X X X (x) X X X - X - (x) X (x) - - - - X ------A. longifolia 1 X - - - - (x) X (x) X - - - - (x) - (x) - - - - - X X X X X X - - A. longifolia 2 X - - - - (x) (x) (x) X - - - - (x) - (x) - - - - - X X X (x) X X - - A. longifolia 3 X - - - - (x) (x) (x) X - - - - (x) - (x) - - - - - X X X (x) X X - - A. cham. 1 - - - (x) - - X X X X - X X X X X - X X X X (x) X X - (x) - - - A. cham. 2 - - - (x) - - X X X X - X X X X X X X X X X (x) X X - (x) - - - A. parryi 2 ------(x) (x) (x) (x) - - - (x) (x) X (x) (x) (x) (x) (x) (x) - X (x) (x) - - - A. cham. 7 - (x) - (x) - - X (x) - (x) - - (x) (x) - (x) X (x) - - - (x) - X - (x) - - - A. cham. 5 ------X (x) - - - - - (x) - X (x) - X (x) - X - X - (x) - - - A. cham. 4 ------(x) (x) - - - - - (x) - (x) - - - - - (x) - X - (x) - - - A. cham. 6 ------X (x) - - - - - (x) - (x) - (x) - - - (x) - X - (x) - - - A. cham. 3 ------X ------(x) ------(x) X - - - - - A. parryi 1 ------(x) ------(x) - - - - - (x) - (x) (x) (x) - - - A. lanceolata (x) ------X - (x) X - - - A. grac x long X ------(x) - - - - - (x) ------X X - X X - X X A. gracilis 1 ------(x) ------X - X X - - - A. gracilis 2 ------(x) ------X (x) (x) X - - - A. gracilis 3 ------(x) ------X X X X - - - A. mollis ------A. cordifolia ------(x) ------A. lonch. ------(x) ------A. ang. 2 ------A. discoidea ------(x) ------A. ang. 1 ------(x) ------A. latifolia ------X - - X - - - A. nevadensis ------(x) - - - - X X A. fulgens ------(x) (x) - (x) - - - - - (x) (x) ------A. griscomii ------(x) (x) ------A. sach. ------M. sativa ------L. hieracioides ------X X - - - - - See Fig. 18 for names of identified STLs. Superscript numbers indicate STL important for the cluster of 1 the A. montana group, 2 the A. longifolia group, 3 the A. chamissonis (1 and 2) group, 4 the A. chamissonis (3-7) group, and 5 the A. gracilis group. This is also indicated by shaded areas. STLs are listed as derivatives of helenalin (He), dihydrohelenalin (DHe), arnifolin (Ar), chamissonolide (Cha), carabron (Ca), xanthanolide (Xa), florilenalin (Fl), and eudesmanolide (Eu). X: present, (x): present in trace amount (area under curve <5 % of internal standard), X: present in large amount (area under curve >150 % of internal standard) - :not detected. Accessions are listed according to the order they appear in the tree based on secondary chemistry in Fig. 19.

STLs new to Arnica species and Madiinae Arnica gracilis, A. latifolia, A. discoidea, A. fulgens, and A. griscomii were investigated for STL content for the first time. Xanthalongin derivatives (particularly xanthalongin and dihydro-4H-xanthalongin) were found in accessions of A. gracilis and A. latifolia, whereas very few STLs were found in A. discoidea, A. fulgens, and A. griscomii. Although A. chamissonis has been investigated for STLs, we found many of the xanthalongin type (e.g. 4H-xanthalongin, present in all A. chamissonis accessions). This type of STLs has previously not reported in this species. Two STLs of the xanthalongin type (xanthalongin and dihydroxanthalongin) were identified in Layia hieracioides, belonging to the sister subtribe to Arnicinae (Madiinae; Baldwin and Wessa 2000). Although diverse in other closely related groups (e.g., Gaillardiinae and Heliantheae; Ottosson 2003 and references cited therein), STLs have previously not been reported from subtribe Madiinae.

STL chemistry within species As discussed by Wink (2003), whereas a group of SMs usually dominates within a taxon, the pattern of SMs is complex and there are differences not only between individual plants or populations, but also between different tissues of the same individual. Still, STL chemistry was found to be very consistent within multiple samples of A. montana (five accessions) and A. longifolia (three accessions), each forming a distinct cluster in the PCA score plot and a strongly supported group in the phylogenetic tree (Fig. 19B and 20). The STL chemistry is somewhat less consistent in multiple accessions of A. chamissonis (seven accessions forming two groups) and A. parryi (two accessions diverged from each other; see below). The three A. gracilis accessions form a group of low support in the phylogenetic tree but do not form a distinct group in the PCA score plot. Major STLs supporting the for- mations of these clusters are indicated in Table 2. The two accessions of A. angustifolia, diverged in a basal clade of the tree and in the center of the PCA score plot, both contain less STLs. Based on our sampling, we can conclude that STLs can act as chemotaxonomic markers within Arnica species in which STLs are abundant.

35 A lanceolata 98 chamissonis 4 D 100 chamissonis 5 62 chamissonis 3 63 chamissonis 6 chamissonis 7 gracilis x longifolia* parryi 1 79 parryi 2 C 99 mollis I 83 gracilis 3* B 98 angustifolia 1 angustifolia 2 montana 1 B montana 2 I 100 montana 3 montana 1 52 montana 4 montana 2 montana 5 montana 3 J 99 parryi 2 montana 4 chamissonis 1 montana 5 chamissonis 2 longifolia 1 K 100 chamissonis 3 longifolia 2 65 E 97 chamissonis 4 longifolia 3 chamissonis 5 chamissonis 1 L 99 90 chamissonis 6 M 93 II 78 chamissonis 2 chamissonis 7 parryi 2 angustifolia 1 chamissonis 7 A 100 58 longifolia 1 chamissonis 5 51 F 99 longifolia 2 chamissonis 4 N 86 longifolia 3 66 chamissonis 6 gracilis x longifolia chamissonis 3 63 parryi 1* parryi 1 61 lanceolata* lanceolata gracilis 3* gracilis x longifolia G 100 parryi 1 gracilis 1 68 parryi 2 III 98 gracilis 3 gracilis x longifolia gracilis 2 H 94 gracilis 3 mollis gracilis 1 72 gracilis 2 cordifolia mollis lonchophylla angustifolia 2 68 fulgens lonchophylla discoidea griscomii angustifolia 1 68 cordifolia latifolia discoidea nevadensis latifolia fulgens nevadensis griscomii 75 sachalinensis sachalinensis 59 Layia hieracioides Madia sativa Madia sativa Layia hieracioides

Figure 19.

4 A. chamissonis 1 - 2

2 A. chamissonis 3 - 7 A. parryi 2

1 A. parryi 1 t[4] 0 A. gracilis A. montana -1

A. gracilis x longifolia -2 A. longifolia

-3

-5 -4 -3 -2 -1 0 1 2 3 4 5 6 t[2]

Figure 20.

36 Congruencies between DNA data and STL chemistry Complex evolutionary patterns, as are evident from phylogenetic studies based on multiple regions in the genome of Arnica, motivate the search for additional phylogenetically useful information in other types of data, such as secondary chemistry. Ribosomal DNA sequence data and STL profiles were analysed for Arnica species, using phylogenetic inference and PCA (Fig. 19 and 20). Phylogenetic analyses resulted in the following strongly supported congruencies between STL data and nrDNA: clades I (100% bs) and J (99% bs), uniting all A. montana accessions, F (99% bs) and K (100% bs), uniting all A. longifolia accessions (although A. gracilis x longifolia is included in F), and D (100%) and N (86%), uniting five of the A. chamissonis accessions (although A. lanceolata is included in D). Clade H (94% bs), uniting all A. gracilis accessions with A. gracilis x longifolia, is also present in the STL chemistry tree, but with low support. Another congruency between the datasets is that representatives present in the three paralogues of the nrDNA strict consensus tree (A. angustifolia, A. chamissonis, A. gracilis, A. lanceolata, A. longifolia, A. mollis, and A. parryi) also form a clade in the tree based on STL data (although also weakly supported and with A. montana included and A. angustifolia and A. mollis excluded). Hence, it seems as taxa containing more diverse STL profiles (see Table 2), also are more closely associated based on nrDNA data. Moreover, of the taxa mentioned above, A. chamissonis, A. lanceolata, A. longifolia, and A. parryi belong to subg. Chamissonis, of which sequences are only present in branches diverging further up in the trees in studies based on cpDNA and nrDNA and lcnDNA regions. According to identified STLs of the present study (see Table 2), taxa of this subgenus generally possess more diverse sesquiterpene lactone chemistry compared to taxa of subgenera Andropurpurea, Arctica and Austromontana (also shown by Willuhn et al. 1995), which can be interpreted as additional support for the circumscription of this subgenus.

Figure 19. Phylogenetic trees based on (A) DNA sequence data and (B) secondary chemistry data for the 33 Arnica accessions and accessions of Layia hieracioides and Madia sativa (outgroup taxa). Numbers are assigned to multiple accessions of the same species. Multiple accessions of A. chamissonis, A. gracilis, A. longifolia, A. montana, and A. parryi are marked by colour. Bootstrap values over 50% are indicated and branches with more than 75% bootstrap support are marked as bold. Major clades discussed are marked with capital letters. A. Strict consensus of 432 most parsimonious trees based on nuclear ribosomal DNA (ITS and ETS) sequences (heuristic search, TBR, 100 replicates). Sequences of polymor- phic taxa form three groups (I, II, III). *No ITS sequences were found in these positions. B. The single most parsimonious trees based on a matrix of 239 peaks (compounds) of STL-rich extracts (heuristic search, TBR, 100 replicates).

Figure 20. Score plot of principal component 2 vs component 4 of the PCA. Multiple accessions of A. chamissonis, A. gracilis, A. longifolia, A. montana, and A. parryi are marked by colour.

Figure 21. Arnica longifolia. Photo by the author.

Support for subspecies classification Although not recognised in the latest revision of subgenus Chamissonis (Gruezo and Denford 1994), A. chamissonis and A. parryi have each been classified into subspecies, based on morphology. Based on Maguire’s classification (1943), the two A. chamissonis accessions (1 and 2; originating from California) that are clearly separated from the remaining A. chamissonis accessions based on secondary chemistry data, are determined as A. chamissonis ssp. foliosa. The remaining representatives would be classified as A. chamissonis ssp. chamissonis (as: genuina). This is also supported by earlier studies, e.g., Kresken (1984) where subspecies of A. chamissonis have been distinguished based on isolated STLs. We also find differences between the two groups in genetic and cytometric data. In the ribosomal DNA tree, A. chamissonis 1 and 2 are only detected in paralogue I, whereas the remaining A. chamissonis accessions are detected in paralogues I and II. Similarly the two representatives of A. parryi (1 and 2; originating from Colorado and California), are differentiated based on secondary chemistry data and are determined as A. parryi ssp. parryi (as: genuina) and A. parryi ssp. sonnei respectively based on Maguires classification (1943). In a study by Gruezo and Denford (1995), relatively few flavonoids were found in A. parryi ssp. parryi, which is in line with our findings of a low secondary metabolite content in A. parryi 1. Additional support for the separation of the A. parryi accessions is found in ribosomal DNA data. In paralogue II the repre-

38 sentatives are separated with A. parryi 1 associated with A. lanceolata, and A. gracilis 3 and A. parryi 2 associated with A. chamissonis representatives. Arnica parryi 2 (= A. parryi ssp. sonnei) is nested within a strongly supported group with A. chamissonis 1 and 2 (= A. chamissonis ssp. foliosa; Fig. 19B) and contains many of the same STLs, but in smaller amounts (Table 2). These findings are consistent with a study by Merfort et al. (1986) in which a high number of flavonoids were detected in A. parryi ssp. sonnei, with a profile very similar to that of A. chamissonis ssp. foliosa. Jepson (1925) transferred A. parryi ssp. sonnei to A. foliosa (= A. chamissonis ssp. foliosa) based on morphological similarities. A close connection between A. chamissonis ssp. foliosa and A. parryi ssp. sonnei, is evident based on chemistry and ribosomal DNA results, and supported by earlier studies. Perhaps hybridization events between these two geographically overlapping taxa (both taxa have a restricted distribution in California) have resulted in chemical and morphological similarities. Alternatively, the taxonomy of these two taxa is in need of revision.

A possible hybrid Similarly, based on morphological characters one accession collected in Alberta, Canada, is suggested to be a hybrid of A. gracilis and A. longifolia (A. gracilis longifolia). It would be classified as A. gracilis except for leaf morphology, which is similar to A. longifolia (e.g., leaves are sessile and lanceolate rather than petiolate and ovate, as in A. gracilis). Genetic and chemical data of the present study also suggest that it may be a hybrid of these two species. If so, ribosomal DNA data indicate that directional concerted evolution has caused the ITS and ETS copies to converge towards each of the two parental types in paralogues I and III, while the affinity in paralogue II remains uncertain (A. longifolia is not represented in this clade). In the tree based on chemical data, A. gracilis longifolia, forms a weakly supported group with the rest of the A. gracilis representatives. The accession also has many STLs in common with each of its suggested parent species. In the PCA score plot it ends up alone in a position in between the groups consisting of A. gracilis and A. longifolia representatives. Using PCA based on secondary metabolite profiles (quinolizidine alkaloids) in Ulex (Fabaceae), Maximo et al. (2006) propose the possibility of detecting hybrids in intermediate positions between the parent species.

39 Anti-inflammatory activity of Arnica (III)

Several Arnica species have been used in traditional medicine. Extracts of A. montana – used in herbal medicine today – have been thoroughly investigated for anti-inflammatory properties, but most species in the genus have not been investigated. We tested extacts of several Arnica species for anti- inflammatory properties by evaluating inhibitory effects on release of human neutrophil elastase HNE by neutrophils and inhibitory effects on the binding of transcription factor NF-B to DNA.

100 PAF - mediated HNE fMLP - mediated HNE NF kappa B 80

60 % inhibition % 40

a 2

6 2

1 2

iva

A. parryi A.

A. mollis A.

M. sat M.

A. gracilis A.

gustifolia

A. fulgens A.

A. griscomii A.

A. discoide A.

hamissonis hamissonis

A. lanceolata A.

A. montana A.

A. longifolia A.

L. hieracioides L.

A. an A.

A. sachalinensis A.

A. c A.

A. chamissonis A. A. c A.

Figure 22. Results of the inhibition of HNE release (induced by PAF and fMLP) and the inhibition NF-B for all extracts with 95% confidence intervals. Accession numbers correspond to those in Table 2 and Figs. 19 and 20.

Comparing the inhibitory capacities of the tested extracts, Arnica montana, A. longifolia and two of the A. chamissonis accessions show high inhibitory effects in both bioassays (80-100%; Fig. 22). Extracts of A. lanceolata, A. griscomii, A. sachalinensis, and Layia hieracioides in the HNE bioassay, and A. gracilis and Madia sativa in the NF-B bioassay possess intermediate inhibition (60-80%). This can be correlated to the PCA score plot (Fig. 20; based on the total matrix of compounds), where the more active accessions

40 (of A. montana, A. chamissonis and A. longifolia) are present in the fringes of the PCA score plot as these are more diverse in STLs. Since the same STL-rich extracts possess strong inhibitory activity in both the NF-B and the HNE release bioassays, the possibility that HNE release inhibition is mediated by inhibition of NF-B or downstream events cannot be excluded. NF-B is involved in the transcription of Interleukin-8 (IL-8), which is also known to be involved in neutrophil activation (Fig. 23), and there may be other inflammatory mediators linking the two assays. The NF-B bioassay seems to be more sensitive at low concentrations of active compounds since all extracts possess at least 40% inhibition even though extracts were tested in lower concentrations in this assay.

fMLP PAF + + elastase 1 iNOS transcription + factor + inflammatory respons NF-kappaB IL-8

Neutrophil COX-2 2

TNFalfa

Figure 23. Possible connection between the two bioassays. The inhibition of HNE release from neutrophils (1) may be linked to the inhibition of NF-B binding to DNA (2).

Correlations between activity and STL content

Accessions of species containing STLs of the helenanolide type (e.g., A. montana, A. chamissonis, and A. longifolia) were found to possess strong inhibitory activity in both bioassays (Table 2, Fig. 22). This is in agreement with a previous study by Siedle et al. (2003) in which two dihydrohelenalin derivatives from A. montana were active in the HNE release bioassay at micromolar concentrations. In Siedle et al. (2004), it was also demonstrated that isolated helenalin and dihydrohelenalin derivatives inhibit NF-B binding to DNA. However, in two extracts of intermediate activity in the HNE bioassay (A. griscomii and Layia hieracioides), only STLs of the xanthalongin core structure could be identified. Xanthalongins are also the major expressed STLs in A. longifolia, and A. gracilis. This suggests that STLs of this type may also be potent inhibitors.

41 Activity within species Inhibitory capacity is very high in multiple accessions of A. montana and A. longifolia. As discussed above, the STL chemistry within these two species is also very homogenous. Two of the A. chamissonis representatives (2, collected in California and 6, cultivated and from unknown origin) also possess very high inhibitory activity, whereas the third (collected in Alaska) is much less active. According to subspecies classification within A. chamissonis discussed previously, A. chamissonis 1 belongs to A. chamissonis ssp. foliosa whereas the remaining two would be classified as A. chamissonis ssp. chamissonis. Therefore the difference in effect can not be correlated with subspecies classification. The PCA score plot (Fig. 20) reveals that the STL chemistry of A. chamissonis 4 (which is the blue spot closest to the center of the plot) is removed from the remaining A. chamissonis accessions, which shows that the difference in activity is accompanied with a difference in STL chemistry. As previously discussed, although a group of secondary metabolites dominates within a taxon, contents between populations and individuals vary depending on the origin and ecological niche of the plant. This has also been shown in studies comparing extracts of different origins from A. montana (e.g. Klaas et al 2002).

42 Conclusions

Phylogenetic studies of Arnica Complex evolutionary patterns in Arnica are evident from phylogenetic analyses of chloroplast, nuclear ribosomal and low-copy DNA regions. Polymorphism was detected in both the nuclear ribosomal and low-copy regions in accessions of A. angustifolia, A. chamissonis, A. gracilis, A. lanceolata, A. mollis, and A. parryi, probably caused by polyploidy, agamospermy and possibly hybridization. No polymorphism was detected for any of the diploid and strictly amphimictic species (A. montana, A. acaulis, A. unalaschcensis, A. sachalinensis, A. fulgens, A. sororia, A. cernua, A. viscosa, and A. venosa). Incongru- encies between topologies of the different regions may be due to processes such as lineage sorting. Taxa belonging to subgenus Chamissonis are not present in any early diverging branches in the phylogenetic trees of any investigated DNA region, which suggests that these species diverged later in the evolutionary history of Arnica. Species of subgenera Austromontana (and Andropurpurea) constitute early diverging lineages in all regions, which is in agreement with the hypothesis of an origin of Arnica in temperate Western North America.

Sesquiterpene lactones in Arnica Several STLs of the xanthalongin type were found in A. chamissonis, A. gracilis and A. latifolia, previously not reported for these species. For the first time STLs are also reported from subtribe Madiinae, since xanthalongin and 4H-xanthalongin were also found in large amounts in Layia hieracioides. It would be of interest to examine additional taxa from Madiinae in order to further evaluate STL chemistry of the subtribe.

Biological activity of Arnica species Two bioassays evaluating anti-inflammatory activity by measuring interactions with the inflammatory pathway show that accessions of A. chamissonis and A. longifolia are equally active as A. montana. Arnica longifolia has

43 previously not been investigated for biological activity and would be interesting to further evaluate for STL contents.

Correlations: phylogeny – STLs – biological activity Generally, species rich in STLs (A. montana, A. longifolia, A. chamissonis, and to some degree A. lanceolata and A. gracilis) also possess the strongest inhibitory activities in two bioassays measuring interactions with the inflammatory pathway. It is therefore likely that mainly STLs are responsible for the anti-inflammatory activity as measured by these bioassays in these species of Arnica. According to the phylogenetic analyses, clades supported by both STL chemistry and DNA sequence data consist of multiple accessions of the same species (A. montana, A. chamissonis, A. longifolia, and A. gracilis). These species are therefore well defined genetically and chemically, based on our sampling. Support for subspecies classification of A. chamissonis and A. parryi was found in STL data and differences between subspecies could also be detected in cytometry data and nrDNA data.

44 Svensk sammanfattning

För att kunna relatera till sin omgivning har människan alltid haft ett behov av att klassificera och sätta namn på det som finns i naturen. Växtsystematik handlar om att klassificera men även om att utreda släktskapet mellan väx- tarter, vilket numera ligger till grund för klassifikationer. Tidigare jämförde man morfologiska karaktärer, det vill säga växternas utseende, för att på så sätt komma fram till en hypotes om släktskap. Man upptäckte även att vissa typer av ämnen som växter använder som försvar, sekundärmetaboliter, ofta är specifika för en viss växtgrupp och kan användas för klassificering (kemosystematik). Senare, efter molekylärbiologins intåg, har det blivit möjligt att jämföra DNA mellan organismer och på så sätt få fram släktträd som kan jämföras med hypoteser baserade på morfologi eller kemi. Men det är inte så enkelt som det låter. Genregionerna har ibland sin egen utveckling och det är inte alltid ett släktträd baserat på en genregion visar det verkliga släktskapet mellan arterna. Om däremot flera släktträd baserade på olika genregioner visar på samma mönster är det ett starkt stöd för att detta också visar det verkliga släktskapet mellan arterna. Arnica är ett växtsläkte som består av 29 arter och hör till familjen As- teraceae, korgblommiga växter. Arnica har delats in i fem undersläkten: Arc- tica, Arnica, Andropurpurea, Austromontana och Chamissonis. Slåttergubbe (Arnica montana) och fjällarnika (Arnica angustifolia) är de svenska namnen på våra två representanter av släktet. Slåttergubbe finner man i delar av Eu- ropa och fjällarnikan över de nordliga delarna av hela norra halvklotet, men de flesta Arnica-arter förekommer enbart i västra Nordamerika. Vissa av arterna är väldigt variabla morfologiskt vilket resulterat i att släktet ibland delats in i fler än 100 arter! Vi undersökte flera oberoende DNA-regioner för att få fram en hypotes om hur släktet har utvecklats och fann komplexa mönster. I flera regioner hittade vi polymorfismer, vilket betyder att det verkar finnas flera olika kopior av en och samma genregion. Men de olika kopiorna visar inte på samma släktskapsmönster i de olika genregionerna, vilket troligen är resul- tatet av en process som gör att kopior slumpvis försvinner. Inget av de fem undersläktena som vanligen nämns inom Arnica bildar en enhetlig grupp i någon av våra analyser. Arter från undersläktet Austromontana utgör flera basala grupper i vissa av analyserna. Det stämmer överens med en hypotes om att hela släktet skulle ha uppstått i tempererade västra Nordamerika där

45 de närmaste släktingarna till Arnica finns och där en del av arterna inom Austromontana har en begränsad utbredning. Förr utgjorde naturen den enda källan till läkemedel och än idag här- stammar mer än 45% av våra moderna läkemedel från ämnen som kommer från naturen, många av dessa från traditionellt använda växter. Unika kemi- ska föreningar i växter, så kallade sekundärmetaboliter, som används som markörer inom kemosystematik är också vanligen de ämnen som är biolo- giskt aktiva. Arnica montana har sedan länge använts som medicinalväxt inom Europeisk folkmedicin och används fortfarande utvärtes som tinktur för att behandla exempelvis sträckningar, stukningar och blåmärken. Det finns även dokumenterad traditionell användning av flera av de nordameri- kanska arterna i släktet, t ex mot ryggsmärtor, blåmärken, svullnader och för sårläkning. Man har undersökt Arnica montana med avseende på dess verk- samma ämnen och kommit fram till att en grupp sekundärmetaboliter, så kallade seskviterpenlaktoner, står för den huvudsakliga antiinflammatoriska aktiviteten. Vi ville ta reda på om fler arter inom släktet uppvisar antiinflammatorisk aktivitet och undersöktes därför extrakt från 12 arter, som odlats i botaniska trädgården i Uppsala. För att testa antiinflammatorisk aktivitet använde vi oss av två olika biologiska testmodeller. Dels undersökte vi i vilken grad utsöndringen av det inflammatoriska enzymet elastas från neutrofiler (en sorts celler som förekommer i blod hos människor) hämmas och dels i vilken grad avläsning av gener som kodar för viktiga signalmolekyler i inflamma- tionsprocessen hämmas. Våra resultat visar att förutom den i Europa välkända medicinalväxten Arnica montana, uppvisar även de två nordameri- kanska arterna Arnica chamissonis och Arnica longifolia mycket hög aktivitet enligt båda testmodellerna. Vidare undersökte vi innehållet av seskviterpenlaktoner i alla extrakt och fann att de arter i släktet Arnica som uppvisar hög antiinflammatorisk aktivitet även innehåller mycket sesquiterpenlaktoner. Vi fann flera seskviterpenlaktoner av xanthalongin-typen i Ar- nica chamissonis, Arnica gracilis och Arnica latifolia, vilka tidigare inte rapporterats från dessa arter. Dessutom fann vi samma ämnen i Layia hieracioides, en art från gruppen Madiinae som utgör de närmaste släktingarna till Arnica. Seskviterpenlaktoner har inte heller tidigare rapporteras från Madii- nae. För att närmare studera om sekundärmetaboliter kan hjälpa till att utreda släktskap i växter som uppvisar komplexa evolutionära mönster, och om man kan använda ett släktträd baserat på DNA för att förutsäga förekomsten av seskviterpenlaktoner, jämförde vi dessa två typer av data för 16 arter av Arnica. I kemi-data kunde vi bland annat se stöd för tidigare underartsklassi- ficeringar av Arnica chamissonis och Arnica parryi. Vi fann även stöd i morfologi, DNA och kemi för att en av de prover som insamlats var en korsning (hybrid) mellan de två arterna Arnica gracilis och Arnica longifolia. Vissa grupperingar av flera exemplar av samma art (Arnica montana, Arnica longi-

46 folia, Arnica chamissonis och Arnica gracilis) sammanfaller mellan släktträdet baserat på DNA och släktträdet baserat på sesquiterpenlaktoner, vilket betyder att dessa är väl definierade såväl kemiskt som genetiskt, baserat på vårt urval.

Figure 24. The author collecting A. viscosa at Mt Shasta, California. Photo: Mike Park.

47 Acknowledgements - Tack!

Det här har varit fem innehållsrika och väldigt roliga år som jag har många att tacka för!

Först och främst ett jättetack till mina handledare som har bidragit stort till denna avhandling:

Katarina Andreasen, du har alltid tagit dig tid och funnits till hands när jag undrat över något, uppmuntrat och hittat lösningar på problem som dykt upp. Du har så många kvalitéer och är en förebild för mig både som pedagog och forskare – vilken tur jag hade som fick dig som handledare!

Anders Backlund, med din kreativitet och förmåga att hitta nya infalls- vinklar har du möjligjort en spännande vinkling till mitt projekt. Tack för ditt engagemang under de här åren, och för länken till farmakognosin!

I would like to thank everyone at the Department of Systematic Biology: Sandra Baldauf (for comments on all manscripts), Inga Hedberg (för din sommarkurs i etnobotanik som gjorde att jag hamnade på avdelningen), Mats Thulin (för kommentarer på alla manuskript), Leif Tibell (för kommentarer manuskript), Bengt Oxelman (för diskussioner och kommentarer på manuskript, och för att du uppmuntrade mig att söka doktorandtjänsten), Lars Björk (för diskussioner och tips på artiklar), Thomas Jaenson, Niklas Wikström, Magnus Lidén, Mariette Manktelow, Katinka Pålsson, Johanna Fehling, Åsa Kruys, Henrik Lantz, Per Erixon (för kommentarer på II!), Christina Wedén (för uppiggande halstabletter och för att du stått ut med alla utspridda papper i vårt rum den senaste tiden), Nahid Hei- dari (för alla uppmuntrande ord, hjälp på lab och medförfattarskap i IV!) Afsaneh Ahmadzadeh, Ulla Hedenquist och Agneta Brandtberg- Falkman.

Avdelningen skulle inte vara densamma utan andra doktorander! Tack till Sunniva Aagaard (för kommentarer på IV och kappan!), Hugo DeBoer och Anneleen Kool (för inspirerande etnobotanik-möten!), Anja Rautenberg (för all hjälp med Mac-frågor!), Donata Tibuwha, Il-Chan Oh, Mattias Forshage, Andreas Wallberg, Karolina Larsson (för kompanjon mot målet!), Samira Garboui, Fawzia Elmhalli, Chanda Vongsombath, Vi-

48 chith Lamxay, Ping Ajawatanawong, Ding He, Anders Larsson, och före detta doktorander: Sanja Savic (för alla resor tillsammans, och för Martin!), Bozo Frajman (för guidning i Slovenien!), Rikke Naesborg, Cajsa Andersson, Maria Backlund, Frida Eggens, Kristina Articus, Dick Andersson, Björn-Axel Beier, Jesper Kårehed, Elisabeth Långström, Maria Carlota Monroy, Johannes Lundberg, Magnus Popp, Annika Vinnersten, Hege Vårdal, Johan Nylander och Yonas Tekle. Tack även till före detta examensarbetare Marie Melander och Anna Petri, före detta post-doc Doug Stone, nydisputerade Hans-Henrik Fuxelius, och Katie St Onge (for inviting us for dinner in Edmonton!).

Jag har haft del av mitt forskningsarbete förlagd på avdelningen för farma- kognosi, institutionen för läkemedelskemi. Stort tack till Lars Bohlin för att jag har fått utföra en del av projektet på avdelningen, för medförfattarskap i III och för förtroendet att ha fått planera undervisningen för kursen Global farmaci under den här tiden, postumt till Premila Perera (för handledarskap och mycket inspiration), Ulf Göransson, Kerstin Ståhlberg (för tålamod med krångliga reseräkningar!), Maj Blad (för hjälp med det praktiska under kursplaneringar!), Hesham el-Seedi (for your amazing engagement in the Global pharmacy course in Egypt -06), Josefin Rosén (för medförfattarskap i II!), Sonny Larsson (för kommentarer på II!), Jenny Pettersson (för Global farmaci-resor till Sri Lanka -06 och Taiwan -07), Robert Burman (för Global farmaci-resa till Egypten -06), Sofia Ortlepp (för att du visade oss neutrofil-assayen och för kommentarer på III!), Erik Hedner, Teshome Aboye, Mariam Yeshak, Anders Hermann, Martin Sjögren, Erika Svangård och Petra Lindholm. Tack även till Lina Rådmark för Global farmaci-resa till Sri Lanka -05.

Ett stort tack till till min före detta student Anna Zebrowska för labarbete, Tobias Vrede för statistiska analyser, och båda för medförfattarskap i III.

Many thanks to Irmgard Merfort for the very nice and productive times at your lab at Albert-Ludwigs-Universität in Freiburg, and for coauthorship in II and III. Thanks also to everyone at the lab for making me feel so welcome, especially Steffen Wagner (for coauthorship in II), Barbara Schuler (for coauthorship in III), Agnés Millet (for many hiking trips in the Black For- est), Christoph Jäger, Claudia Kern, and Andrea Hrenn.

Also many thanks to Bruce Baldwin for the very nice time at your lab at UC, Berkeley, for sending many seeds and rhizomes, and for coauthorship in I. Special thanks to Bridget Wessa (for taking me to nice lunches and week- end trips) and to Mike Park (for an unforgetable fieldtrip in California with three flat tires and an encounter with the entire police force of Mt Shasta).

49 Tack även till Vänner och släkt, som jag håller av mycket trots att jag inte hört av mig så ofta den senaste tiden!

Stort tack till mina föräldrar Ulf och Anna-Karin som alltid finns till hands, min farmor Margareta, min bror Håkan och hans fru Maho.

Sist men inte minst, Martin, som lagat god mat, skött markservicen och som jag knappast hade klarat detta utan under de sista veckorna ;) – tack för att du finns i mitt liv och ständigt påminnner mig om vad som är viktigt!

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