Phylogenies and Secondary Chemistry in Arnica (Asteraceae)

Home , Achyrachaena, Adenothamnus, Amblyopappus, Arnica acaulis, Arnica cernua, Arnica chamissonis

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-κB 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-κB nuclear factor-κB 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-α 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 modified 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 chromosome 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 considered 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 d2 Anisocarpus (Raillardiopsis ) 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-κB (NF-κB) to DNA by alkylation of cysteine-38 in the p65/NF-κB 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 α,β- unsaturated carbonyl structures such as α-methylene-γ-lactones or α,β- 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-κB 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 polymorphic 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 angiosperm 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 universally 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 divergence 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 lessingii 40 AN 1 0.98 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 confirmed 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 archetypal major group and is placed nearest the base’’ in Arnica (directly descendent 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 polymorphic 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-κB to DNA.

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

60 % inhibition % 40

2 a

is a

6 2

1 2

iva

lat

arryi

tifolia

moll

inensis

gracil

fulgen

A. A.

M. sat M.

griscomii

A. A.

gus

discoide

A. A.

lanceo

montan

longifolia

A. A.

hamissonis hamissonis

A. A.

sachal

A. A.

L. hieracioid L.

A. A.

A. chamisson A. A. A.

Figure 22. Results of the inhibition of HNE release (induced by PAF and fMLP) and the inhibition NF-κB 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-κB 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-κB and the HNE release bioassays, the possibility that HNE release inhibition is mediated by inhibition of NF-κB or downstream events cannot be excluded. NF-κB 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-κB 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-κB 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-κB 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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57 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 392

Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology”.)

ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-8459 2008 Paper I

A Molecular Phylogenetic Study of Arnica (Asteraceae): Low Chloroplast DNA Variation and Problematic Subgeneric Classification

CATARINA EKENA¨ S,1,3 BRUCE G. BALDWIN,2 and KATARINA ANDREASEN1 1Department of Systematic Botany, Evolutionary Biology Center, Uppsala University, Norbyva¨gen 18D, SE-752 36 Uppsala, Sweden; 2Jepson Herbarium and Department of Integrative Biology, 1001 Valley Life Sciences Bldg, University of California, Berkeley, California 94720-2465 U.S.A. 3Author for correspondence ([email protected])

Communicating Editor: Matt Lavin

ABSTRACT. DNA sequences from five chloroplast DNA regions (the rpl16 and rps16 introns and the psbA–trnH, ycf4–cemA, and trnT–L spacers), and the nuclear ribosomal internal and external transcribed spacer (ITS and ETS) regions, were analyzed using maximum parsimony and Bayesian methods to explore the putatively complicated history of the mainly North American genus Arnica. The chloroplast regions were found to contain minimal variation in Arnica. Of 3710 nucleotides, only 119 were variable and 45 informative. However, combined with the ribosomal DNA data, the analysis yielded a number of well-supported clades. Strong support for the monophyly of Arnica was found in both the separate and combined analyses but none of the five currently recognized subgenera was resolved as monophyletic in any of the analyses. Arnica (Whitneya) dealbata and A. mallotopus (Mallotopus japonicus), two species that were previously placed outside Arnica, were confidently confirmed as members of the genus. The analyses revealed that A. nevadensis (subg. Austromontana) is most closely related to A. dealbata and that A. mallotopus forms a strongly supported clade with A. unalaschcensis (subg. Andropurpurea). Earlier biogeographical hypotheses that suggested an arctic origin and southward spread of the genus are not supported by our analyses. Hybridization, homoplasy, and rapid evolution are possible explanations for conflicts between the chloroplast and nuclear ribosomal data sets and for low support of the deeper nodes.

KEYWORDS: Arnica, Asteraceae, chloroplast, Compositae, dealbata, ETS, ITS, mallotopus, phylogeny.

Arnica L. is a circumboreal but mostly western Robinson 1981). In a discussion of the origin and North American genus of rhizomatous, perennial biogeographical history of the genus, Maguire herbs. Morphological features of the genus include proposed that Arnica is of arctic origin and spread simple, opposite leaves, epaleate receptacles, yel- southward along four major routes. Using ITS low (rarely whitish) to orange corollas, yellow or sequence data, Baldwin and Wessa (2000) found purple anthers, gray or brown to black (carbon- that Arnica (subtribe Arnicinae) is sister to the ized) cypselae, and usually a pappus of bristles. A tarweeds and silverswords (subtribe Madiinae) base chromosome number of x 5 19 is evident for and nested within an otherwise temperate western the genus, wherein hybridization and polyploidy, North American lineage (tribe Madieae sensu in association with agamospermy, have been pro- Baldwin; see Baldwin et al. 2002). Baldwin and posed to be common (e.g., Straley 1980; Wolf and Wessa suggested that the genus originated in Denford 1984a). Complex and diverse patterns of temperate western North America, rather than in morphological variation in Arnica are reflected by the arctic, as previously suggested by Maguire widely differing taxonomic treatments and close to (1943). In the analyses by Baldwin et al. (2002), two 100 described species. Currently, 32 minimal-rank species not included within the genus Arnica by taxa found in North America, including 26 species Maguire (1943), A. dealbata (A. Gray) B. G. Baldwin and six subspecies, are recognized by Wolf (2006). and A. mallotopus (Franch. & Sav.) Makino, nested Including the species endemic to Europe and Asia, well within Arnica. 29 Arnica species are currently recognized world- Since Maguire’s (1943) monograph, the subge- wide. nera Austromontana, Arctica, and Chamissonis have In the only revision of the entire genus (Maguire been revised separately (Wolf and Denford 1984a; 1943), Arnica was divided into five novel subge- Downie and Denford 1988a; Gruezo and Denford nera: Arctica, Andropurpurea, Austromontana, Cha- 1994). In addition, secondary metabolites of Arnica, missonis,andMontana (5subg. Arnica,which such as flavones, flavonoids, and sesquiterpene contains the type species, A. montana L.). At the lactones, have been studied in a systematic context time of Maguire’s work, Arnica was considered to (Downie and Denford 1986, Wolf and Whitkus belong within tribe Senecioneae, as proposed by 1987; Downie and Denford 1988b; Downie 1988; Bentham (1873), but was later moved to tribe Ebert et al. 1988; Gruezo and Denford 1995; Heliantheae sensu lato (Nordenstam 1977; Willuhn et al. 1995). However, until now no 917 918 SYSTEMATIC BOTANY [Volume 32 phylogenetic analysis of the genus has been were prepared according to recommendations from the published, apart from broader-scale efforts that manufacturers. For ITS and ETS sequences, the procedure followed the methods of Andreasen et al. (1999). In most included a few exemplar species (Baldwin et al. cases sequencing primers were the same as those used in the 2002). PCR reactions (Table 1). The main objective of this study was to use Forward and reverse sequences were reconciled using different lines of DNA sequence evidence to help Sequence Navigator 1.0.1 (Applied Biosystems). Sequences were aligned by eye, without ambiguity. Gaps of equal reveal the putatively complicated history of Arnica length and position shared by two or more taxa were and to evaluate the monophyly of the subgenera. considered homologous and binary coded as additional An additional objective was to determine the characters (see Simmons and Ochoterena 2000). Sequences positions of the two most morphologically anom- were submitted to EMBL (see Appendix 1 for accession alous and understudied species, A. dealbata and A. numbers) and the final matrix submitted to TreeBASE (study number 1859). mallotopus, within the genus. Maximum parsimony (MP) analyses of the combined sequence data as well as separate matrices of the cpDNA and ribosomal DNA regions were carried out using PAUP* MATERIAL AND METHODS version 4.0b10 (Swofford 2002). In the MP analyses, a heuris- Five non-coding chloroplast DNA (cpDNA) regions (the tic approach was used, with random addition of sequences rpl16 intron, the rps16 intron, the trnT–L spacer, the psbA– (100 replicates) and TBR branch swapping. In MP bootstrap trnH spacer, and the ycf4–cemA spacer) and the nuclear analyses (10 000 replicates), a heuristic approach was used ribosomal internal transcribed spacer (ITS; 18S–26S) and with random addition of sequences (100 replicates), TBR external transcribed spacer (ETS; upstream from the 18S branch swapping, and the MULTREES option off. subunit) regions were sequenced for 42 accessions of 28 Bayesian analyses were performed on the combined matrix species and four subspecies of Arnica (Appendix 1). Se- using MrBayes v.3.1 (Huelsenbeck and Ronquist 2001). The quences from all but one currently recognized Arnica species GTR model with gamma-distributed rate variation across (A. louiseana Farr) and two subspecies (A. lanceolata Nuttall sites and a proportion of invariable sites resulted in the subsp. lanceolata, and A. griscomii Fernald subsp. griscomii) lowest AIC scores, as calculated by Mr.Modeltest v.2 were examined. Multiple accessions of 12 of the taxa were (Nylander 2004). In two independent runs, the number of included. Eatonella nivea (D. C. Eaton) A. Gray, Hulsea algida MCMC chains was set to four and trees were sampled every A. Gray, Raillardella argentea (A. Gray) A. Gray, and Venegasia 100th generation. Analyses were run for 10.833106 genera- carpesioides DC., belonging to closely related lineages of tribe tions, until the standard deviation of the split frequencies of Madieae (Baldwin et al. 2002), were chosen as outgroup the two independent runs was below 0.01. The first quarter of species. the sampled trees was discarded as burnin. Fresh field-collected leaf material dried in silica was used MP analyses of cpDNA sequences for all accessions could for most DNA extractions. In addition, DNAs were extracted not be performed due to memory overflow, likely caused by from dry leaf material of herbarium specimens deposited in missing data and few potentially informative characters. the University and Jepson Herbaria (UC/JEPS), the Califor- Therefore, when analyzing the cpDNA data, all accessions nia Academy of Sciences herbarium (CAS), and the Swedish with one or more missing sequence region(s) were initially Museum of Natural History (S). Total DNA was isolated and excluded, and later these partial sequences were added one purified from leaf tissue using Mini-Beadbeater (BioSpec by one until the analysis could not be completed without Products, Bartlesville, Oklahoma) and the DNeasy mini kit memory overflow. This procedure resulted in the removal of (Qiagen, Hilden, Germany). For some specimens, DNA was nine accessions (including two outgroup taxa). In the extracted using the CTAB procedure (Saghai-Maroof et al. combined MP and Bayesian analyses of cpDNA and nuclear 1984; Doyle and Doyle 1987), with phenol extraction and rDNA data, and in the MP analysis of nuclear rDNA data, ethanol precipitation. accessions from all sampled taxa (Appendix 1) were in- PCR reactions were conducted on an Eppendorf Master- cluded except three ingroup samples, for which both ITS and cycler Gradient thermal cycler (Hamburg, Germany). Primers ETS data were missing. used for amplification and sequencing of cpDNA, ITS, and ETS regions are listed in Table 1. Typically, the PCR products RESULTS were amplified using an initial 95uC denaturation (5 min) and 35 cycles of 95uC denaturation (30 sec), 44uCto56uC A total of 3710 cpDNA nucleotides or less were annealing (1 min), and 72uC extension (2 min). The 25 ml sequenced for each of 42 accessions of Arnica and reactions consisted of 0.1 ml Taq DNA polymerase (Abgene, m four outgroup taxa. The cpDNA sequences (ex- Epsom, U.K.), 2.5 l each of reaction buffer, 25 mM MgCl2, 10 mM dNTP, and 0.1 M TMACL, 1.25 ml each of 10 mM cluding outgroup taxa) were found to contain forward and reverse primers (MWG Biotech, Ebenberg, minimal variation (119 variable and 45 parsimony Germany), and 12.5 ml of ten-fold diluted DNA. In some informative characters, including 15 coded indels) cases, AccuPower PCR Premix (Bioneer, Seoul, Korea) was relative to variation in the nuclear ribosomal DNA used to amplify the spacer or intron regions. The Phusion transcribed spacer regions (ITS and ETS). In the ITS enzyme (Finnzymes, Espoo, Finland) was used to amplify the long spacer region ycf4-cemA. Ethanol precipitation or and ETS, 302 variable and 174 parsimony in- Millipore plates were used to purify PCR products. formative characters were found among 1191 Sequencing reactions were conducted on a Gene Amp PCR nucleotides, excluding outgroup taxa. The level of system 9700 thermal cycler (Applied Biosystems, Foster City, sequence variation differs among the cpDNA California) and loaded on a MegaBACE 1000 DNA Analysis System (Amersham Biosciences, Buckinghamshire, U.K.) or regions, with the rpl16 intron, the rps16 intron, an ABI version 3.1 automated sequencer (Perkin-Elmer, and the psbA-trnH spacer being almost twice as Waltham, Massachusetts). Sequencing reaction mixtures variable as the ycf4-cemA spacer and the trnT-L 2007] EKENA¨ S ET AL.: PHYLOGENY OF ARNICA (ASTERACEAE) 919

w TABLE 1. Primers used for amplification and sequencing of chloroplast and ribosomal DNA regions in Arnica. Primer used for sequencing only.

Region Forward primer Reverse primer rpl16 intron 1067f (Asmussen pers. comm.) 18R (Asmussen 1999) and exon1r (Downie et al. 2000) rps16 intron rpsf (Oxelman et al. 1997) rps16r2 (Oxelman et al. 1997) trnT-L spacer TrnLA (Taberlet et al. 1991) and TrnLA1w TrnLD (Taberlet et al. 1991), TrnLBw (Taberlet et (Lantz and Bremer 2004) al. 1991), and TrnLIw (Bremer et al. 2002) psbA-trnH spacer psbAf (Sang et al. 1997) TrnHr (Tate and Simpson 2003) ycf4-cemA spacer rbcL/cemA-F5 (Erixon and Oxelman cemA-R (Erixon and Oxelman pers. comm.) pers. comm.) ITS (18S–26S) ITSI (Urbatsch et al. 2000), ITS3w ITS4 and ITS2w (White et al. 1990) (White et al. 1990), and ITS5w (Downie and Katz-Downie 1996) ETS (upstream from 18S) ETS-Hel1 (Baldwin and Markos 1998) 18S-E (Baldwin and Markos 1998)

spacer (see Table 2). The ribosomal ITS and ETS strong support (B: one accession of A. gracilis regions are about ten times as variable as the most Rydberg, subg. Austromontana, with A. rydbergii variable cpDNA regions. Greene, subg. Arctica;C:A. acaulis (Walter) Britton, Chloroplast DNA Analyses. MP analyses of Sterns & Poggenburg subg. Montana, with A. fulgens cpDNA sequences yielded 508 most parsimonious Pursh, subg. Arctica; D: the two accessions of A. trees. Bootstrap support values are indicated on the longifolia D. C. Eaton, subg. Chamissonis, with one strict consensus tree (Fig. 1). Three clades are well accession of A. lonchophylla Greene, subg. Arctica). supported: clade A, uniting all Arnica accessions, Clade E unites two accessions of different species of clade B, with two accessions of A. griscomii Fernald subg. Chamissonis (A. chamissonis and A. lanceolata subsp. frigida (C. A. Meyer ex Iljin) S. J. Wolf, and Nuttall subsp. prima (Maguire) Strother & S. J. Wolf) clade C, consisting of all three accessions of A. and clade F includes six accessions of subg. angustifolia Valhl subsp. angustifolia (subg. Arctica) Austromontana (A. spathulata Greene, A. discoidea and the sole accession of A. chamissonis Lessing Bentham, A. cordifolia Hooker, A. gracilis, and A. (subg. Chamissonis). cernua Howell). Clade G of both trees unites three Ribosomal DNA and Combined Analyses. The representatives of subg. Austromontana (two acces- analysis of ribosomal DNA data alone resulted in 16 sions of A. nevadensis A. Gray and one of A. viscosa most parsimonious trees and the combined analysis A. Gray) with A. (Whitneya) dealbata. Clade H of both of cpDNA and ribosomal DNA data resulted in 144 trees includes A. unalaschcensis Lessing (subg. most parsimonious trees. Bootstrap support values Andropurpurea) with A. mallotopus (Mallotopus japo- are indicated on the strict consensus trees of both nicus). In the ribosomal DNA tree, another well analyses (Figs. 2 and 3). Bayesian posterior proba- supported clade unites A. unalaschcensis and A. bilities are indicated on the strict consensus tree of mallotopus with A. lessingii (Torrey & A. Gray) the combined data (Fig. 3). Most of the well- Greene (subg. Andropurpurea). This clade is also supported clades are present in both trees. Clade strongly supported by Bayesian inference of the A of both trees unites all Arnica accessions. In clades combined dataset. Bayesian posterior probabilities B, C, and D of both trees, sequenced representatives for two basal clades, that are not present on the strict of different subgenera are grouped together with consensus trees of the MP analyses, are indicated as

TABLE 2. Length and variability for chloroplast and ribosomal DNA regions in Arnica. Characters used to denote insertions or deletions are listed in parenthesis.

Region Length Variable characters Informative characters Informative characters/length rpl16 intron 903 27 13 (5) 0.014 rps16 intron 717 32 11 (2) 0.015 trnT-L spacer 529 17 4 (1) 0.008 psbA-trnH spacer 521 21 8 (4) 0.015 ycf4-cemA spacer 1040 22 9 (3) 0.009 ITS (18S–26S) 686 174 98 (7) 0.143 ETS (upstream from 18S) 505 128 76 (4) 0.150 920 SYSTEMATIC BOTANY [Volume 32

FIG. 1. Strict consensus of 508 most-parsimonious trees based on cpDNA sequences (heuristic search, TBR, 100 replicates). Tree length 5 304 steps, CI (excluding uninformative characters) 5 0.68 and RI 5 0.73. 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 subgenus abbreviation (MO 5 subg. Montana,AR5 subg. Arctica,AU5 subg. Austromontana,CH 5 subg. Chamissonis, and AN 5 subg. Andropurpurea). *Outgroup taxa. Missing sequences are indicated by superscript numbers (1 5 rpl16 intron, 2 5 rps16 intron, 3 5 trnT-L spacer, 4 5 PsbA-trnH spacer, and 5 5 ycf4-cemA spacer). dotted lines on the strict consensus tree of the DISCUSSION combined analysis (Fig. 3). Both trees include other In all three trees, strong support for the clade well supported groups uniting pairs of accessions uniting all Arnica accessions reinforces a circum- of the same species or subspecies, such as A. scription of Arnica that includes A. dealbata (5 angustifolia subsp. angustifolia, A. griscomii subsp. Whitneya dealbata A. Gray) and A. mallotopus (5 frigida, A. lonchophylla, A. longifolia, A. montana L., A. Mallotopus japonicus Franch. & Sav.), which are nevadensis (combined tree), and A. spathulata discussed in more detail below. Most basal nodes (ribosomal tree). of the MP strict consensus trees are unresolved and 2007] EKENA¨ S ET AL.: PHYLOGENY OF ARNICA (ASTERACEAE) 921

FIG. 2. Strict consensus of 16 most-parsimonious trees based on ribosomal DNA (ITS and ETS) sequences (heuristic search, TBR, 100 replicates). Tree length 5 882 steps, CI (excluding uninformative characters) 5 0.52, and RI 5 0.66. 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 subgenus abbreviation (MO 5 subg. Montana,AR5 subg. Arctica,AU5 subg. Austromontana,CH5 subg. Chamissonis, and AN 5 subg. Andropurpurea). *Outgroup taxa. **ETS sequence of A. mallotopus is from voucher source 2. Missing sequences are indicated by superscript numbers (1 5 ITS and 2 5 ETS). bootstrap support values are low, whereas Bayes- Low cpDNA Variation. Although the basal ian inference analysis of the combined dataset nodes of the cpDNA strict consensus tree are resulted in moderate support for three basal nodes, better resolved than those of the ribosomal DNA as indicated on the strict consensus tree (Fig. 3). and combined data trees, only three cpDNA clades Generally in the tree, Bayesian posterior probabil- are well supported. Missing data is probably one ities are higher than bootstrap support values, explanation for the low bootstrap support in the which has also been shown for true clades in chloroplast analysis compared to the ribosomal simulation studies by Erixon et al. (2003). analysis. The latter data set is more complete; we 922 SYSTEMATIC BOTANY [Volume 32

FIG. 3. Strict consensus of 144 most parsimonious trees based on the combined data matrix of cpDNA and ribosomal DNA (ITS and ETS) sequences (heuristic search, TBR, 100 replicates). Tree length 5 1325 steps, CI (excluding uninformative characters) 5 0.52 and RI 5 0.63. Bootstrap values over 50% are indicated above branches and Bayesian posterior probabilities over 0.80 are indicated below branches. Major supported clades are marked with capital letters. Three groups supported by Bayesian posterior probabilities but collapsed in the strict MP consensus tree (and one group uniting A. lessingii with clade H) are indicated by the three vertical lines. Taxon samples are indicated by species and subspecies followed by population number and subgenus abbreviation (MO 5 subg. Montana,AR5 subg. Arctica,AU5 subg. Austromontana,CH5 subg. Chamissonis, and AN 5 subg. Andropurpurea). *Outgroup taxa. **ETS sequence of A. mallotopus is from voucher source 2. Missing sequences are indicated by superscript numbers (1 5 rpl16 intron, 2 5 rps16 intron, 3 5 trnT-L spacer, 4 5 PsbA-trnH spacer, 5 5 ycf4-cemA spacer, 6 5 ITS, and 7 5 ETS). were unable to amplify all cpDNA regions for most nucleotides were sequenced from five regions representatives (see Appendix 1). Another proba- spanning the chloroplast genome, a low percentage ble reason for this low support is the low number of parsimony informative cpDNA sites were of informative characters (45) in the cpDNA discovered for the sampled taxa within Arnica dataset. Even though as many as 3710 chloroplast (1.2%), compared to the percentage of such sites in 2007] EKENA¨ S ET AL.: PHYLOGENY OF ARNICA (ASTERACEAE) 923 ribosomal DNA (14.6%). Low cpDNA variation has japonicus) is grouped strongly (clade H) with A. been found in many other recently diversified taxa, unalaschcensis. The two species have an overlap- such as Gossypium L., Malvaceae (Small et al. 1998; ping geographical distribution in Japan and may Cronn et al. 2002), Lophocereus (A. Berger) Britton & represent an example of diversification there. Rose, Cactaceae (Hartmann et al. 2002), and Soja Arnica mallotopus, with white-lanate peduncles, Moench, Fabaceae (Xu et al. 2000). With addition of discoid heads, and yellow anthers, is morpholog- the ribosomal DNA data, the analysis yielded ically quite different from members of subg. a greater number of well-supported clades within Andropurpurea, including A. unalaschcensis,and Arnica, as anticipated from the addition of 174 was excluded from Arnica by Maguire (1943). In informative characters. Flora of Japan (Jeffrey and Yi-Ling 1995) A. Species Delimitations. For most taxa, accessions mallotopus is treated as a member of Arnica, but of the same species or subspecies are grouped classified in a separate, monotypic subgenus (subg. together in at least one of the analyses: A. acaulis Mallotopus). (cpDNA tree), A. angustifolia subsp. angustifolia, A. Arctica. Subgenus Arctica is exceptionally angustifolia Vahl subsp. tomentosa (Macoun) G. W. widespread, with a distribution extending Douglas & Ruyle-Douglas (cpDNA tree), A. grisco- throughout north temperate and arctic regions. mii subsp. frigida, A. lonchophylla (cpDNA tree), A. Seven species (A. angustifolia (with two currently longifolia, A. montana, A. nevadensis (combined tree), recognized subspecies, A. angustifolia subsp. angu- and A. spathulata (ribosomal DNA tree), in accord stifolia and A. angustifolia subsp. tomentosa), A. with taxonomic delimitations. Two exceptions dis- fulgens, A. griscomii, A. lonchophylla, A. louiseana, A. cussed in more detail below are A. ovata Greene and rydbergii, and A. sororia Greene) are recognized A. gracilis, each with two accessions that are widely in the latest revision by Downie and Denford separated from one another in all of the trees and (1988a). each of putative hybrid ancestry (Gruezo and As discussed in more detail below, subg. Arctica Denford 1994; Wolf and Denford 1984b). Also, the is not resolved as monophyletic in our analyses; two subspecies of A. angustifolia (subspp. angustifolia accessions of subg. Arctica are grouped strongly and tomentosa) do not constitute a clade in any of with accessions belonging to the subgenera Aus- the trees. tromontana, Chamissonis, and Montana. The sub- Whereas most accessions of the same species are genus was suggested by Maguire (1943) to be the grouped together, substantial conflict between the most ‘‘archetypal’’ subgenus in Arnica, derived tree topologies and the subgeneric classification of directly from an hypothesized ancestor, ‘‘Proto- Arnica is evident, as discussed below for each arnica’’. Plesiomorphy of the characteristics used to subgenus. delimit subg. Arctica, such as large, broadly hemi- Andropurpurea. Subgenus Andropurpurea, spheric to campanulate-turbinate involucres, which has not been revised since Maguire’s and white, barbellate pappus bristles, could con- (1943) treatment, is distributed from Alaska, across ceivably explain the evident polyphyly of the the Aleutian Islands, to Sachalin Island and Japan. group. The three species of the subgenus (A. lessingii, A. Austromontana. The taxa belonging to subg. sachalinensis (Regel) A. Gray, and A. unalaschcensis) Austromontana are distributed in montane to alpine are easily distinguished from the rest of the genus habitats from central Alaska to southern California. by having purple rather than yellow anthers. Nine species were recognized in the last revision of Sequences of two of the three species in the subgenus (Wolf and Denford 1984a), including Maguire’s (1943) subg. Andropurpurea, A. lessingii A. cernua, A. cordifolia, A. discoidea, A. gracilis, A. and A. unalaschcensis, are grouped together (along latifolia Bongard, A. nevadensis, A. spathulata, A. with A. mallotopus) with strong support in the MP venosa H. M. Hall, and A. viscosa. As in subg. ribosomal DNA analysis as well as in the Bayesian Arctica, most species within subg. Austromontana analysis of the combined data. That result supports are highly variable in morphology. Only the rare A. Maguire’s (1943) hypothesis that A. lessingii and A. cernua, A. venosa, and A. viscosa, restricted to the unalaschcensis, which overlap in distribution in the KlamathregionofsouthwesternOregonand Aleutian archipelago (but were each sampled northwestern California, are morphologically well elsewhere) are more closely related to one another defined (Wolf and Denford 1984a). Species within than to the other species of subg. Andropurpurea, A. subg. Austromontana are distinguished from mem- sachalinensis, which is of uncertain relationship bers of the other four subgenera by turbinate to based on the molecular trees. campanulate involucres, broad leaves, and white, In both the ribosomal and combined trees, the barbellate pappus bristles. Japanese endemic A. mallotopus (5 Mallotopus Two molecular clades include all but two 924 SYSTEMATIC BOTANY [Volume 32 representatives of subg. Austromontana.Sixof cypselae, A. dealbata was originally described as eleven accessions belonging to the subgenus (i.e., the sole member of the genus Whitneya A. Gray. the sole accessions of A. discoidea, A. cordifolia, and Morphological and cytological similarities to Arni- A. cernua, both accessions of A. spathulata, and one ca were noted (e.g, Ornduff et al. 1967; Turner and of two accessions of A. gracilis) constitute clade F in Powell 1977; Nordenstam 1977) prior to phyloge- the ribosomal and combined trees. Weak support netic analyses, which led to the transfer of Whitneya for placement of the major Austromontana clades in dealbata to Arnica by Baldwin (1999). A phyloge- the Arnica trees leaves open the possibility that netic position of A. dealbata within subg. Austro- most of the species in the subgenus constitute montana is consistent with the strong morpholog- a monophyletic group. ical similarity between that species and A. In the ribosomal tree, a clade with low support, nevadensis—the closest relative of A. dealbata based uniting A. discoidea and A. spathulata, upholds the on the ribosomal and combined data trees. earlier hypothesis based on morphology that they The morphologically anomalous Arnica viscosa, are closely related (Maguire 1943; Wolf and with discoid heads, cream-colored florets, and Denford 1984a). Wolf and Denford (1984a) sug- viscid foliage, which has been considered to be gested that the Klamath endemic A. spathulata one of the most distinctive species in Arnica (Wolf descended from A. discoidea by gradual adaptation and Denford 1984a), is also within this Austromon- and ultimately restriction to serpentine soils. These tana clade (G). This stands in contrast with earlier two species are morphologically very similar in suggestions of a closest relationship of that species leaf arrangement, number of heads, shape of to A. latifolia or A. venosa (Maguire 1943; Wolf and involucres, and disc floret morphology (both Denford 1984a), both of which are only weakly species lack ray florets). There are only a few placed in the molecular trees. Morphological differences such as leaf and petiole shape, degree similarities between A. viscosa and A. nevadensis of pubescence, and cypsela color. Also, the include stipitate-glandular leaves, dark gray stipi- flavonoid profile of A. spathulata represents a subset tate-glandular cypselae, and barbellate to subplu- of that in diploid A. discoidea (Wolf and Denford, mose pappus bristles. 1984a). Chamissonis. The distribution of subg. Chamis- Arnica gracilis has been suggested by Wolf and sonis is mostly confined to western North America, Denford (1984b) to be an apomictic hybrid of A. with a disjunct subspecies (A. lanceolata subsp. latifolia and A. cordifolia. The flavonoid profile of A. lanceolata) and some populations of A. chamissonis gracilis is the sum of that of A. latifolia and A. in eastern North America. In the most recent cordifolia and many morphological features of A. revision of subg. Chamissonis, Gruezo and Denford gracilis are intermediate between the two putative (1994) adopted the same circumscription of the parent species (Wolf and Denford 1984b). A hybrid group as proposed by Maguire (1943), but recog- origin of A. gracilis involving one of the two nized seven taxa (A. chamissonis, A. lanceolata putative parental lineages is consistent with the subsp. amplexicaulis (Nuttall) Gruezo, A. lanceolata molecular data. In the ribosomal tree analysis, one subsp. lanceolata, A. longifolia, A. mollis Hooker, A. of the accessions of A. gracilis is united with A. ovata, and A. parryi A. Gray) as opposed to the 12 cordifolia with low support. The other A. gracilis taxa treated by Maguire (1943). Arnica lanceolata accession is grouped with A. rydbergii (clade B), of subsp. amplexicaulis was subsequently changed to subg. Arctica, with very strong support in both the A. lanceolata subsp. prima (Maguire) Strother and ribosomal and combined trees. The sole accession Wolf (2006). As in subgenera Arctica and Austro- of A. latifolia, one of the putative parent species of montana, morphological variation is high in most A. gracilis, is well removed from both samples of A. species of subg. Chamissonis. The subgenus is gracilis and other members of subg. Austromontana distinguished from the other subgenera by posses- in all of the trees, but always with less than 50% sing stramineous, tawny to deep brown, and bootstrap support. In sum, the molecular data are subplumose to plumose pappus bristles, as well intriguing with regard to a possible hybrid origin as numerous pairs of cauline leaves. of A. gracilis, and this needs further attention. Two clades in the ribosomal tree unite all but one The second clade of subg. Austromontana (clade taxon (A. parryi) of subg. Chamissonis. A sister- G) accessions in the ribosomal and combined trees, group relationship between A. chamissonis and A. includes both accessions of A. nevadensis and A. lanceolata (clade E) in the ribosomal and combined viscosa, as well as the Californian endemic, A. DNA trees is consistent with the close relationship dealbata (5 Whitneya dealbata). With morphological of those taxa proposed by Maguire (1943). Exam- characteristics such as persistent ray corollas, ples of morphological similarities between A. staminate-functional disc florets, and epappose chamissonis and A. lanceolata include similar 2007] EKENA¨ S ET AL.: PHYLOGENY OF ARNICA (ASTERACEAE) 925 numbers of cauline leaf pairs (4–10), basal leaves A. fulgens, of western North America, are widely often withered at anthesis, similar numbers of separated. Therefore, the molecular results cannot heads per capitulescence (3–10), and short-stipi- be explained by recent hybridization between the tate-glandular cypselae. The cpDNA tree, in widely allopatric A. acaulis and A. fulgens.In contrast, includes the sole A. chamissonis accession addition to the molecular findings, the flavonoid in a well supported clade that otherwise consists of profiles of A. acaulis and A. montana are quite members of subg. Arctica. This incongruity be- different (Ebert et al. 1988). Morphological similar- tween chloroplast and nuclear data could be an ities between the two species of subg. Montana example of introgression and chloroplast capture noted by Maguire (1943) could be due to parallel in this specific lineage of A. chamissonis. evolution rather than a close relationship between Some weakly to moderately supported clades in the two species. the molecular trees unite members of subg. Hybridization, Homoplasy, and Rapid Evolu- Chamissonis with taxa of subg. Arctica, which was tion. Different collections of two taxa that have perceived by Maguire (1943) as directly ancestral to been suggested to be of hybrid origin, A. gracilis subg. Chamissonis. In the ribosomal and combined and A. ovata, were widely separated in the trees, sequences of A. angustifolia (subg. Arctica) are molecular trees, although not precisely in positions in a (weakly supported) clade with four taxa of that conform to previous hybridization hypotheses subg. Chamissonis (A. chamissonis, A. lanceolata (see above). An alternative hypothesis for the subsp. prima, A. mollis, and A. ovata). separated accessions of the same species could be In the sister clade, the two A. longifolia accessions lineage sorting as a result of disrupted concerted are united with one of the A. lonchophylla (subg. evolution. Other taxa were attached in unexpected Arctica) accessions. In the chloroplast tree, howev- positions in the trees (e.g., A. lonchophylla, A. er, the three accessions of A. lonchophylla are rydbergii,andA. parryi) and/or in conflicting grouped together (but with low support). In the positions in different molecular trees (e.g. A. ribosomal and combined analyses, A. parryi is chamissonis and A. lonchophylla). One possible placed in a moderately supported MP clade (but explanation for the odd positions of some acces- strongly supported Bayesian inference clade) with sions and conflict between the cpDNA and A. rydbergii (subg. Arctica) and A. gracilis (subg. ribosomal trees is hybridization, which has been Austromontana). proposed to be common within the genus. Low The two accessions of A. ovata are not grouped support for many relationships in cpDNA or together in any of the trees. The sequences from ribosomal trees does not allow for homoplasy to two accessions are different enough to exclude be dismissed as an alternative hypothesis for homoplasy alone as responsible for this diver- topological conflicts. Finally, the low support for gence. A. ovata has earlier been suggested to be of deep nodes in all three analyses is concordant with hybrid origin, involving A. amplexicaulis (5 A. short branch lengths of individual trees for those lanceolata prima)orA. mollis and A. latifolia or A. nodes (trees not shown), which may reflect rapid cordifolia of subg. Austromontana (Cronquist 1955; evolution without enough time between branching Ediger and Barkley 1978; Wolf 1980). Although events for mutations to accumulate. none of the A. ovata accessions were grouped Origin and Spread of Arnica. Maguire (1943) strongly with any of these species, hybridization hypothesized that Arnica originated in arctic North cannot be excluded as one possible reason for the America and spread south along four major routes, separated A. ovata accessions in the analyses. whereas Baldwin et al. (2002) suggested that Arnica Montana. The European A. montana and eastern originated in temperate western North America North American A. acaulis constitute subg. Mon- based on broad-scale relationships of the genus tana, which has not been revised since Maguire’s inferred from ITS sequence results and the geo- (1943) original treatment of the group. Maguire graphic distribution of sexual, diploid lineages. (1943) suggested that A. acaulis was derived Maguire further hypothesized that subg. Arctica is directly from A. montana, based on morphological ‘‘the closest archetypal major group and is placed similarities, such as trichome anatomy. nearest the base’’ in Arnica (directly descendent In the combined analysis, A. acaulis was strongly from his hypothetical ‘‘Protoarnica’’). Although united (clade C) with A. fulgens (subg. Arctica), of additional data will be necessary to ascertain the western North America, rather than with A. earliest diverging lineages in Arnica, none of the montana. Although weakly supported, the same trees provide any indication that members of subg. species were united in the separate cpDNA and Arctica constitute a deep grade or early diverging ribosomal DNA analyses as well. The distribution clade. Maguire’s alternative suggestion that areas of A. acaulis, of eastern North America, and the subgenera Austromontana, Chamissonis,and 926 SYSTEMATIC BOTANY [Volume 32

Montana arose from subg. Arctica perhaps comes hybridization may well have been important closest to our results, in the sense that taxa of subg. throughout the evolution of Arnica, as suggested Arctica are variously placed with members of those by Maguire (1943) and subsequent workers, e.g., three subgenera in the molecular trees. Wolf and Denford (1984b). More molecular data, Members of subg. Andropurpurea (including A. e.g., from low-copy nuclear gene regions, will be mallotopus) constitute early divergent clades in all necessary to clarify relationships and evolutionary of the MP and Baysian trees, which, although history of this complex genus. weakly supported, raises the intriguing possibility that an early lineage of modern arnicas may have ACKNOWLEDGEMENTS. The authors would like to thank occurred in the northwestern Pacific Rim, where Steven J. Wolf for examining voucher specimens, Henrik Lantz for help with laboratory work, and Per Erixon for help those taxa are endemic. That hypothesis is difficult with Bayesian analyses. The research was supported by to reconcile with the basally temperate, western grants to Katarina Andreasen from The Swedish Research North American distribution of the sister group of Council, The Royal Swedish Academy of Sciences, The Arnica, i.e., subtribe Madiinae, and the other Swedish King Carl XVI Gustaf Foundation, and The Lawrence R. Heckard Endowment Fund of the Jepson genera of tribe Madieae (e.g., Eatonella A. Gray, Herbarium, U.C. Berkeley. Hulsea Torr.&A.Gray,andVenegasia DC.), however, and the abundance of sexual, diploid LITERATURE CITED species of Arnica in the same region. More evidence will be required to examine the possibility that ANDREASEN,K.,B.G.BALDWIN,andB.BREMER. 1999. Phylogenetic utility of the nuclear rDNA ITS region in long-distance dispersal or migration from temper- the subfamily Ixoroideae (Rubiaceae): Comparisons ate America to boreal America or Asia occurred with cpDNA rbcL sequence data. Plant Systematics and very early in the history of the Arnica lineage. Evolution 217: 119–135. After divergence of subg. Andropurpurea, succes- ASMUSSEN, C. 1999. Toward a chloroplast DNA phylogeny of the tribe Geonomeae (Palmae). Memoirs of the New York sive nodes, moderately supported by Bayesian Botanical Garden 83: 121–129. posterior probabilities of the combined data (and BALDWIN, B. G. 1999. New combinations in Californian Arnica with low support in MP analyses), suggest di- and Monolopia (Compositae). Novon 9: 460–461. vergence of two lineages of subg. Austromontana ——— and S. MARKOS. 1998. Phylogenetic utility of the external transcribed spacer (ETS) of 18S–26S rDNA: and one lineage of subg. Montana. The two most Congruence of ETS and ITS trees of Calycadenia basally attached clades of subg. Austromontana (Compositae). Molecular Phylogenetics and Evolution 10: sequences suggest that these species are early 449–463. diverging lineages within Arnica, which is in line ——— and B. L. WESSA. 2000. Origin and relationships of the with Baldwin’s (2002) hypothesis of an origin of tarweed–silversword lineage (Compositae–Madiinae). American Journal of Botany 87: 1890–1908. Arnica in temperate western North America, ———, ———, and J. L. PANERO. 2002. Nuclear rDNA although more evidence is required to fully un- evidence for major lineages of helenioid Heliantheae derstand the early evolutionary history of the (Compositae). Systematic Botany 27: 161–198. genus. BENTHAM, G. 1873. Notes on the classification, history, and geographical distribution of Compositae. Journal of the To summarize, although analyses of five non- Linnean Society (Botany) London 13: 335–577. coding cpDNA regions alone did not generate BREMER,B.,K.BREMER,N.HEIDARI,P.ERIXON,R.G. enough resolution to yield much data on relation- OLMSTEDT,A.A.ANDERBERG,M.KA¨ LLERSJO¨ , and E. ships within Arnica, the combined analysis with BARKHORDARIAN. 2002. Phylogenetics of asterids based on 3 coding and 3 non-coding chloroplast DNA markers nuclear ribosomal data (ITS and ETS) and compar- and the utility of non-coding DNA at higher taxonomic isons between cpDNA and ribosomal DNA trees levels. Molecular Phylogenetics and Evolution 24: 274–301. provide new perspectives on phylogeny of the CRONN, R. C., R. L. SMALL,T.HASELKORN, and J. F. WENDEL. genus that should stimulate further systematic 2002. Rapid diversification of the cotton genus research. Based on these analyses, monophyly of (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast genes. American Journal of Botany any previously recognized subgenus is doubtful 89: 707–725. and warrants further study. Notwithstanding CRONQUIST, A. 1955. Compositae. In Vascular plants of the future changes in infrageneric classification, sub- Pacific Northwest. Part 5. Seattle: University of Washing- generic positions can now be proposed confidently ton Press. DOWNIE, S. R. 1988. Morphological, cytological, and flavonoid for the two species that were previously treated variability of the Arnica-angustifolia aggregate (Astera- outside Arnica; A. (Whitneya) dealbata belongs in the ceae). Canadian Journal of Botany 66: 24–39. same subgenus as its sister species, A. nevadensis ——— and K. E. DENFORD. 1986. The flavonoids of Arnica (subg. Austromontana) and A. mallotopus (Malloto- frigida and Arnica louiseana (Asteraceae). Canadian Journal of Botany 64: 2748–2752. pus japonicus) belongs in the same subgenus as its ——— and ———. 1988a. Taxonomy of Arnica (Asteraceae) sister-species, A. unalaschcensis (subg. Andropur- subgenus Arctica. Rhodora 90: 245–275. purea). Conflicts between the trees suggest that ——— and ———. 1988b. Flavonoid variation in Arnica 2007] EKENA¨ S ET AL.: PHYLOGENY OF ARNICA (ASTERACEAE) 927

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APPENDIX 1. Information on collection localities, voucher AM690619, AM690497, AM690575, AM690650, AM690722, sources, and accession numbers of taxa included in the AM690681. A. lonchophylla AR – 1 herb. Yukon Territory, analyses. Taxa are indicated by species and subspecies Canada, Cody 32481 (CAS); –, AM690624, AM690503, followed by subgenus abbreviation (MO 5 subg. Montana, AM690581, –, AM690728, AM690687. 2 herb. Nova Scotia, AR 5 subg. Arctica,AU5 subg. Austromontana,CH5 subg. Canada, Smith 6503 (S); AM690541, AM690627, –, AM690585, Chamissonis, and AN 5 subg. Andropurpurea). Numbers –, –, AM690691. 3 herb. Alberta, Canada, Breitung 13916 (S); before voucher citations indicate multiple samples analyzed AM690544, –, AM690507, AM690588, –, –, AM690693. A. per species and correspond to population numbers in Figs. 1, longifolia CH – 1 Washington, U.S.A., Baldwin 1101 (UC); –, 2, and 3. Samples were from herbarium specimens (‘‘herb.’’), AM690607, AM690485, AM690563, AM690640, AM690710, botanical gardens (‘‘cult.’’), or wild populations (collected by AM690669. 2 herb. Wyoming, U.S.A., Williams 1022 (S); the authors). Voucher information is listed as follows: AM690542, –, –, AM690586, AM690655, AM690731, collection locality, collector name and number (herbarium), AM690692. A. mallotopus – 1 herb. Japan, Mizushima 12615 EMBL accession numbers for the seven regions in the (S); –, –, –, AM690589, –, AM690732, –. 2 herb. Japan, Ono s.n. following order: rpl16 intron, rps16 intron, trnT-L spacer, (UC); –, –, –, –, –, –, AM696307. A. mollis CH – Oregon, U.S.A., PsbA-trnH spacer, ycf4-cemA spacer, ITS, and ETS; sequences Baldwin 1129 (UC); AM690528, AM690612, AM690490, not obtained are indicated by ‘‘–’’. AM690568, AM690644, AM690715, AM690674. A. montana MO – 1 Sweden, Andreasen 304 (UPS); AM690527, AM690611, A. acaulis MO – 1 herb. South Carolina, U.S.A., Wilbur 8964 AM690489, AM690567, AM690643, AM690714, AM690673. 2 (UC); AM690520, AM690603, –, AM690559, –, AM690706, herb. France, s.c. 500084 (DS); AM690540, AM690625, AM690665. 2 herb. North Carolina, U.S.A., Helms 1169 (S); AM690504, AM690582, AM690654, AM690729, AM690688. AM690543, AM690628, –, AM690587, –, –, –. A. angustifolia A. nevadensis AU – 1 herb. California, U.S.A., Straley 1938 subsp. angustifolia AR – 1 Sweden, Andreasen 312 (UPS); (UC); AM690519, –, –, AM690557, –, AM690704, AM690663. 2 AM690530, AM690615, AM690493, AM690571, AM690647, California, U.S.A., Baldwin 1093 (JEPS); AM690522, AM690718, AM690677. 2 herb. Quebec, Canada, Albe 3549 (S); AM690605, AM690483, AM690561, AM690639, AM690708, AM690545, AM690629, AM690508, AM690590, –, AM690733, AM690667. A. ovata CH – 1 Alaska, U.S.A., Baldwin 1340 AM690694. 3 herb. Greenland, Gelting 9396 (S); AM690546, –, (UC); AM690535, AM690620, AM690498, AM690576, –, AM690591, –, –, –. A. angustifolia subsp. tomentosa AR – 1 AM690651, AM690723, AM690682. 2 Washington, U.S.A., herb. Alberta, Canada, Breitung 13897 (S); AM690547, Baldwin 1104 (UC); AM690524, AM690608, AM690486, AM690630, AM690509, AM690592, AM690656, AM690734, AM690564, AM690641, AM690711, AM690670. A. parryi CH AM690695. 2 herb. Newfoundland, Canada, Fernald s.n. (S); –, – herb. Nevada, U.S.A., Howell et al. 50828 (CAS); AM690538, AM690631, AM690510, AM690593, –, –, –. A. cernua AU – –, AM690501, AM690579, –, AM690726, AM690685. A. herb. Oregon, U.S.A., Howell et al. 48823 (CAS); AM690539, rydbergii AR – Washington, U.S.A., Baldwin 1152 (UC); AM690623, AM690502, AM690580, –, AM690727, AM690686. AM690550, AM690634, AM690513, AM690596, AM690658, A. chamissonis CH – Alaska, U.S.A., Baldwin 1335 (UC); AM690736, AM690697. A. sachalinensis AN – cult. Uppsala AM690531, AM690616, AM690494, AM690572, AM690648, Bot. Garden (2316-1980), Andreasen 301 (UPS); AM690525, AM690719, AM690678. A. cordifolia AU– Washington, U.S.A., AM690609, AM690487, AM690565, –, AM690712, AM690671. Baldwin 1096 (UC); AM690523, AM690606, AM690684, A. sororia AR – herb. California, U.S.A., Schoolcraft 2082 (UC); AM690562, –, AM690709, AM690668. A. dealbata – California, AM690518, AM690601, AM690481, AM690555, AM690638, U.S.A., Baldwin 920 (JEPS); AM690549, AM690633, AM690702, AM690661. A. spathulata AU – 1 herb. Oregon, AM690512, AM690595, –, AF229307, AM690696. A. discoidea U.S.A., Howell et al. 48844 (CAS); –, AM690626, AM690505, AU – California, U.S.A., Baldwin 918 (JEPS); AM690548, AM690583, –, AM690730, AM690689. 2 herb. California, AM690632, AM690511, AM690594, AM690657, AM690735, –. U.S.A., Heckard et al. 6283 (JEPS); AM690521, AM690604, –, A. fulgens AR – cult. Uppsala Bot. Garden (1992-1230), AM690560, –, AM690707, AM690666. A. unalaschcensis AN – Andreasen 303 (UPS); AM690526, AM690610, AM690488, herb. Russia, Joneson 1094 (WTU); AM690529, AM690614, AM690566, AM690642, AM690713, AM690672. A. gracilis AM690492, AM690570, AM690646, AM690717, AM690676. A. AU – 1 herb. California, U.S.A., Cronquist 8102 (UC); –, venosa AU – herb. California, U.S.A., Taylor 9856 (UC); –, –, –, AM690602, AM690482, AM690558, –, AM690705, AM690664. AM690556, –, AM690703, AM690662. A. viscosa AU – Oregon, 2 herb. Utah, U.S.A., Bernett et al. 8431 (CAS); –, –, AM690506, U.S.A., Baldwin 1117 (UC); –, AM690613, AM690491, AM690584, –, –, AM690690. A. griscomii subsp. frigida AR – 1 AM690569, AM690645, AM690716, AM690675. Eatonella nivea Alaska, U.S.A., Baldwin 1341 (UC); AM690536, AM690621, – herb. Nevada, U.S.A., Taylor s.n. (JEPS); AM690554, AM690499, AM690577, AM690652, AM690724, AM690683. 2 AM690637, AM690517, AM690600, AM690660, AF229300, Alaska, U.S.A., Baldwin 1345 (UC); AM690537, AM690622, AM690701. Hulsea algida – California, U.S.A., Baldwin 678 AM690500, AM690578, AM690653, AM690725, AM690684. A. (JEPS); AM690552, –, AM690515, AM690598, AM690659, lanceolata subsp. prima CH – Alaska, U.S.A., Baldwin 1336 AM690738, AM690699. Raillardella argentea – California, (UC); AM690532, AM690617, AM690495, AM690573, –, U.S.A., Baldwin 625 (JEPS); AM690551, AM690635, AM690720, AM690679. A. latifolia AU – Alaska, U.S.A., AM690514, AM690597, –, AF229309, AM690698. Venegasia Baldwin 1337 (UC); AM690533, AM690618, AM690496, carpesioides – California, U.S.A., Baldwin 893 (JEPS); AM690574, AM690649, AM690721, AM690680. A. lessingii AM690553, AM690636, AM690516, AM690599, –, AF229301, AN – Alaska, U.S.A., Baldwin 1338 (UC); AM690534, AM690700. Paper II

Secondary chemistry and ribosomal DNA data congruencies in Arnica (Asteraceae)

C. Ekenäs 1. J. Rosén 2, S. Wagner 3, I. Merfort 3, A. Backlund 2, K. Andreasen 1

1 Department of Systematic Biology, Evolutionary Biology Center, Uppsala University, Uppsala, Sweden 2 Division of Pharmacognosy, Department of Medicinal Chemistry, BMC, Uppsala University, Uppsala, Sweden 3 Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-Universität, Freiburg, Germany

Abstract To investigate possible congruencies between DNA sequence data and secondary chemistry, we compared nuclear ribosomal DNA (nrDNA) sequence data, sesquiterpene lactone (STL) contents, and also cytometric data from 35 accessions of 16 Arnica (Asteraceae) species and two outgroup taxa (Layia hieracioides and Madia sativa), using phylogenetic inference, principal component analysis (PCA) and flow cytometry. Several groups supporting multiple accessions of the same species (of A. montana, A. longifolia, A. gracilis and A. chamissonis) are congruent between the phylogenetic trees based on nrDNA and STL data. Sesquiterpene lactone profiles were found to be highly consistent within multiple samples of A. montana and A. longifolia respectively. Moreover, sesquiterpene lactone data support subspecies classifications of A. chamissonis and A. parryi, with additional support from DNA sequence data and cytometric data. Morphology, STL data (PCA), cytometric data, and DNA sequence data suggest a hybrid origin of one accession (A. gracilis × longifolia). In A. gracilis, A. latifolia, and Layia hieracioides, previously not investigated for STLs, we found large amounts of xanthalongin derivatives. This is the first time STLs are reported from subtribe Madiinae.

Introduction The evolutionary success of many taxa within Asteraceae (23 000 species) is probably partly due to the elaborate secondary chemistry within the family (Cronquist, 1988; Bremer et al., 2003). Acting as defence against microorganisms, protection against UV radiation, or attractants of pollinators, secondary metabolites improves fitness and serve specific needs of the producing organism. These compounds tend to be taxon-specific and have therefore been applied as taxonomic markers long before DNA markers were introduced (e.g., Abbott, 1887). With the emergence of molecular biology, DNA sequence data has to a large extent replaced chemical and morphological data in phylogenetic studies and phylogenies based on DNA sequence data now frequently serve as a basis for interpretations of biosynthetic pathways of secondary metabolites (e.g., Wink and Mohamed, 2003; Windsor et al., 2005). Besides the use as chemosystematic markers, secondary metabolites are also of high interest in the search for pharmacologically interesting lead compounds (Müller-Kuhrt, 2003; Larsen et al., 2005; Larsson et al., 2007) and many pharmaceuticals of today originate from these types of compounds or derivatives of them (Newman and Cragg, 2007). Sesquiterpene lactones (STLs) constitute one of the largest groups of natural products known, with more than 5000 identified structures (Schmidt, 2006), of which most are found in Asteraceae. These compounds have been investigated for potential use as chemotaxonomic markers in the family as a whole, using cluster

1 analyses, principal component analyses (PCA), or other multivariate statistical methods (e.g., Alvarenga et al., 2001; Hristozov et al., 2007), and have also been investigated as a source of bioactive compounds (Schmidt and Heilmann, 2002; Kanashiro et al., 2006; Wagner et al., 2006; Scotti et al., 2007). Arnica L. (29 species; Wolf, 2006) is a genus in Asteraceae of which many species have a history of use in traditional medicine (e.g., Speck, 1937; Palmer, 1975; Turner, 1980; Turner, 1990; Strike, 1994; Alestine and Fehr, 2002). Numerous sesquiterpene lactones have been isolated and structurally elucidated from several Ar- nica species as summarized in Willuhn et al. (1995). Arnica has a circumpolar northern hemispheric distribution, with most species restricted to western North America. Hybridization, polyploidy, and agamospermy have been proposed to be common within Arnica, which is reflected in complex and diverse patterns of morphological variation (e.g., Straley, 1980; Wolf, 1980; Wolf and Denford, 1984). In the last revision of the genus by Maguire (1943), it was divided into five subgenera, of which Arctica, Austromontana, and Chamissonis have been separately revised since (Wolf and Denford, 1984; Downie and Denford, 1988; Gruezo and Denford, 1994). However, in a molecular study based on five chloroplast regions and the nuclear ribosomal DNA (nrDNA) regions ITS and ETS (Internal and External Tran- scribed Spacers), and in another study based on the second largest subunit of RNA polymerase II (RPB2), none of the five subgenera were found to be monophyletic (Ekenäs et al., 2007; Ekenäs et al., manuscript). Molecular phylogenetics based on DNA sequence data constitutes the primary basis for investigating evolutionary relationships among taxa. However in Arnica, analyses based on independent regions of DNA sequence data indicate complex evolutionary patterns (Ekenäs et al., 2007; Ekenäs, manuscript). In the search for additional phylogenetic information from independent data we here investigate possible congruencies between secondary chemistry, cytometric data, and DNA sequence data. Sesquiterpene lactone profiles, ITS and ETS sequence data, and flow cytometry data are investigated from 16 Arnica species (33 specimens) using phylogenetic inference and PCA. Other objectives were to explore consistency of STLs within species, and if phylogenetic information based on DNA sequence data could potentially be used as a predictive tool for secondary chemistry contents in closely related species.

Methods A total of 33 accessions from 16 Arnica species (with multiple representatives of six species), and two accessions of species from the sister group to Arnica (the subtribe Madiinae; Baldwin and Wessa, 2000), Layia hieracioides and Madia sativa, were cultivated outdoors under controlled conditions at Uppsala Botanical Garden, Swe- den (Table 1). Samples originated from seeds or rhizomes of wild specimens from Alaska, Alberta, California, Greenland, Quebec, and Wyoming, or cultivated specimens mainly from Uppsala Botanical Garden.

Ribosomal DNA sequence data DNA extraction and PCR reactions. The nuclear ribosomal internal transcribed spacer (ITS; 18S-26S) and external transcribed spacer (ETS; upstream from the 18S subunit) regions were sequenced for all 35 representatives. Leaf material (fresh or dried in silica) was used for DNA extractions. Total DNA was isolated and purified

2 from leaf tissue using Mini-Beadbeater™ (BioSpec Products, Bartlesville, OK) and the DNAeasy® mini kit (Qiagen, Hilden, Germany). ETS-Hel1 and 18S-E (Baldwin and Markos, 1998) were used as forward and reverse primers for ETS. For ITS, p16 (Popp and Oxelman, 2001) or Leu.1 (Andreasen et al., 1999) and ITS4 (White et al., 1990) were used as forward and reverse primers respectively. For ETS, PCR products of were amplified using an initial 95°C denaturation (2 min) and 40 cycles of 95°C denaturation (15 sec), 53°C annealing (45 sec), and 72°C extension (1 min). For ITS, PCR products were amplified using an initial 97°C denaturation (1 min) and 35 cycles of 97°C denaturation (10 sec), 55°C annealing (45 sec), and 72°C extension (1 min). The 40-50 μl reactions consisted of 0.15-0.25 μl Taq DNA polymerase (Abgene, Epsom, UK), 5 μl each of reaction buffer, 25 mM MgCl2, and10 mM dNTP, 0.5 μl each of 20 μM forward and reverse primers (MWG Biotech, Ebenberg, Germany), and 1-10 μl of ten- to fifty-fold diluted DNA. Millipore plates (Millipore, Billerica, MA) were used to purify PCR products. Sequencing reactions and alignment. Purified PCR products were sequenced by Macrogen (Seoul, Korea) under BigDyeTM terminator cycling conditions using the same primers as in the PCR reactions (see above). Forward and reverse sequences were reconciled using Sequencher version v.3.1.1 (Nishimura, 2000). Sequences were aligned by eye and gaps of equal length and position shared by two or more taxa were considered homologous and were binary coded as additional characters (see e.g. Simmons and Ochoterena, 2000). Cloning. When direct sequences contained polymorphisms, PCR products were cloned using TOPO TA cloning® Kit for Sequencing (Invitrogen, Carlsbad, CA). From each accession 10 clones were sequenced and aligned as described above.

Sesquiterpene lactone data Extraction methods. Isolation of an STL-rich extract mainly followed the procedure of Willuhn and Leven (1991). The material was harvested in two batches, in 2005 and 2006. Mature flower heads (20 g) were collected, weighed, and stored in dichloromethane for 24 hours. In some cases less than 20 g of flower heads were available, and the procedure below was scaled down accordingly. After vacuum filtration, the plant material was transferred to methanol, finely chopped in a mixer, extracted in methanol, agitated for 24 hours, and again filtrated. The methanol filtrate was combined with the dichloromethane filtrate and santonin (0.1 % in methanol; 3 ml) was added as an internal standard. After evaporation, the mixture was redissolved in methanol (20 ml) and Milli-Q water (80 ml), and extracted three times with ethyl acetate (200 ml) using a separatory funnel. The dried extract was dissolved in dichloromethane (40 ml), vacuum-filtered using a sterile nylon filter (0.45 μm, Cameo, USA), and the filter was washed with dichloromethane. The dried filtrate was dissolved in methanol and water (20 ml each), aluminium oxide (14 g) was added, and the mixture was agitated and centrifuged (5 minutes, 20°C and 2000 rpm) in a Sorvall RT 6000D centrifuge, followed by filtration evaporation of the filtrate.

3 Table 1. Source, voucher information and genome size of all accessions. Species Source and voucher Genome size Arnica angustifolia Vahl ssp. angustifolia 1 Quebec, Canada; Archambault 2005-MA-001 (MT), Ekenäs 45 (UPS) n. a. Arnica angustifolia Vahl ssp. angustifolia 2 Greenland; Ekenäs 49 (UPS) n. a. Arnica chamissonis Less. 1 California, USA; Ekenäs 35 (UPS) 1.00 Arnica chamissonis Less. 2 California, USA; Ekenäs 36 (UPS) 1.00 Arnica chamissonis Less. 3 Alberta, Canda; Andreasen & Ekenäs 413 (UPS) 0.85 Arnica chamissonis Less. 4 Alaska, USA; Baldwin 1335 (UC) 0.85 Arnica chamissonis Less. 5 cult. 1988:1710; Ekenäs 68 (UPS) 0.85 Arnica chamissonis Less. 6 cult. 1231:1992; Ekenäs 58 (UPS) 0.86 Arnica chamissonis Less. 7 cult.2; Ekenäs 69 (UPS) 0.85 Arnica cordifolia Hook. Alberta, Canada; Andreasen & Ekenäs 402 (UPS) 1.32 Arnica discoidea Benth. California, USA; Ekenäs 42 (UPS) 0.72 Arnica fulgens Pursh cult. 1230:1992; Ekenäs 59 (UPS) 0.70 Arnica gracilis Rydb. 1 Alberta, Canada; Andreasen & Ekenäs 416 (UPS) 1.00 Arnica gracilis Rydb. 2 Alberta, Canada; Andreasen & Ekenäs 418 (UPS) 1.00 Arnica gracilis Rydb. 3 Wyoming, USA; Baldwin 1393 (UC) 1.10 Arnica gracilis × longifolia Alberta, Canada; Andreasen & Ekenäs 417 (UPS) 1.26 Arnica griscomii Fernald ssp. frigida (Iljin) S. J. Wolf Alaska, USA; Baldwin 1341 (UC) 1.00 Arnica latifolia Bong. Alberta, Canada; Andreasen & Ekenäs 405 (UPS) 0.72 Arnica lanceolata Nutt. ssp. prima (Maguire) Strother & S. J. Wolf Alaska, USA; Baldwin 1336 (UC) 1.00 Arnica longifolia D. C. Eaton 1 cult.1 6640; Ekenäs 60 (UPS) 1.30 Arnica longifolia D. C. Eaton 2 cult. 1989:1451; Ekenäs 62 (UPS) 1.34 Arnica longifolia D. C. Eaton 3 cult. 1966:2035; Ekenäs 70 (UPS) 1.32 Arnica lonchophylla Greene Alberta, Canada; Andreasen & Ekenäs 412 (UPS) 1.00 Arnica mollis Hook. Alberta, Canada; Andreasen & Ekenäs 408 (UPS) 1.183 Arnica montana L. 1 Italy; Ekenäs 71 (UPS) 0.54 Arnica montana L. 2 Italy; Ekenäs 64 (UPS) 0.52 Arnica montana L. 3 cult. 1973:1109; Ekenäs 67 (UPS) 0.52 Arnica montana L. 4 Switzerland; Ekenäs 72 (UPS) 0.52 Arnica montana L. 5 Slovenia; Ekenäs 65 (UPS) 0.55 Arnica nevadensis A. Gray. California, USA; Ekenäs 66 (UPS) 1.00 Arnica parryi A. Gray. 1 cult. 1991:1856; Ekenäs 62 (UPS 1.00 Arnica parryi A. Gray. 2 California, USA; Ekenäs 37 (UPS) 1.22 Arnica sachalinensis A. Gray. cult. 2316:1980; Ekenäs 63 (UPS) 0.76 Layia hieracioides Hook. & Arn. California, USA; Baldwin s.n. (JEPS) n. a. Madia sativa Molina California, USA; Bainbridge s.n. (JEPS), Ekenäs 57 (UPS) n. a. Samples were collected in the field or when indicated by “cult.” from cultivated specimen in Uppsala Botanical Garden, Sweden. Herbarium acronyms are according to Holmgren et al. (1990). 1 collected in Bergius Botanic Garden, Stockholm. 2 collected in University of Columbia Botanical Garden. The genome size is relative to that of Lactuca sativa (internal standard 1.00; 2C = 5.47 pg). Not available: n. a. 3 diploid (chromosomes counted).

GC-MS analyses. The extracts were dissolved in methanol (1 ml) and GC analyses were carried out with an HP6890 series GC-system (Hewlett Packard, Wilming- ton, USA), using helium 5.0 as carrier gas. A fused silica capillary column (25 m × 0.25 mm I.D.) coated with 0.25 µm dimethylsiloxan was used (Optima 1, Machery- Nagel, Düren, Germany). The flow rate was set to 1.0 ml/min and the temperature profile started at 120°C followed by an increase rate of 10°C /min up to 270°C, where it was kept for 20 min. The injector and detector temperatures were 290°C and the injection volume was 1.0 µL using a split ratio of 1:10. An Agilent 5973 Network Mass Selective Detector (Agilent Technologies, Palo Alto, USA) at ioniza- tion energy 70 eV was used. Identification of STLs was achieved by holding the EI mass spectra between 40 and 400 m/z. Mass spectra of the same retention times were compared between species representatives using Peacock version 0.19 (http://peacock.johankool.nl/). The internal standard (santonin) was scaled to the same area for all representatives and the peak areas were adjusted accordingly as a measure of relative amounts. A few peaks that were clearly present in extracts of one batch and absent in extracts of the other batch were removed to avoid results biased towards differences between batches. The peak of the internal standard, santonin, was also removed from further analyses. Some STLs were identified by comparing with mass spectra and retention times of previously published and structurally determined STLs from Arnica species.

Phylogenetic analyses In all maximum parsimony analyses, a heuristic approach was used with a random addition of sequences (100 replicates) and TBR branch swapping with the MUL- TREES option on, using PAUP version 4.0b10 (Swofford, 2002). In MP bootstrap analyses (10 000 replicates), a heuristic approach was used with random addition of sequences (100 replicates for each bootstrap replicate), followed by TBR branch swapping, and with the MULTREES option off. Ribosomal DNA sequence data. Maximum parsimony analyses were performed on nrDNA sequence data (ITS and ETS, separately and combined). Cloned sequences from one representative at a time were added to all non-cloned representatives in separate analyses for ITS and ETS. For cloned sequences of the same accession that formed a monophyletic group, a single representative was included in the analysis. For cloned sequences that did not form a monophyletic group, multiple representative sequences were included (one from each well-supported clade). Al- most all sequences cloned from the same representatives ended up in congruent topological positions for ITS and ETS and the two DNA regions were therefore combined in the final analysis. Exceptions were cloned ETS sequences of five representatives of which no ITS sequences were detected in the same positions in the tree (marked in Figure 1A). In the combined analysis these were coded as unknown for the entire ITS region. STL data. Maximum parsimony analyses were also performed on the matrix of compounds resulting from the GC-MS analyses after transforming peak areas to the binary coded ”presence” or ”absence” of peak using the same program and settings as above.

PCA and OPLS analyses Principal component analysis. PCA (e.g., Eriksson et al., 2006) is a mathematical method used to reduce an n-descriptor space into a more manageable space of

5 smaller dimensions. Correlated variables are compressed into a reduced number of new uncorrelated variables, principal components (PCs), while as much information as possible is retained. PCA provides an overview of all data in a table, allowing for systematic variation and patterns like clusters, trends, and outliers to be detected. The results can be viewed in score and loading plots, where the scores are related to the objects (here the species representatives) and the loadings are related to the variables (here the peak areas). The relative distances between compounds in chemical space becomes a measure of their similarity with respect to the particular set of variables considered. PCA was performed on centered, un-scaled, and log transformed data and revealed clusters were investigated. Orthogonal partial least squares analyses. OPLS (Trygg and Wold, 2002) represents a modeling method for correlating two data tables (X and Y). In this study OPLS was used to relate peak presence and area as X matrix to certain groups of representatives (Y), with the aim to identify compounds important for the formation of certain groups based on chemical data.

Cytometric data With the aim to detect ploidy levels, flow cytometry was used to measure the genome size for most representatives (Table 1). In flow cytometry assays, the amount of stained nuclear DNA is measured by its florescence and correlated to an internal standard of which the DNA amount is known. Analyses were carried out by Plant Cytometry Services (Schijndel, Netherlands) using Lactuca sativa, (2x = 5.47 pg; Arumuganathan and Earle, 1991) as internal standard. Chromosomes were counted for one accessions (Arnica mollis) mainly following the same procedure as Baldwin (2007). Floral buds were fixed in chloroform, 95% ethanol, and glacial acetic acid (6:3:1) and chromosomes were counted from squashed microsporocytes at meiotic metaphase I in acetocarmine mixed with Hoyer´s solution (Beeks, 1955) under mi- croscope.

Results

DNA sequence data The final DNA sequence data matrix consists of 1052 characters (20% parsimony informative sites including outgroups and 16% excluding outgroups) of 46 ITS and 51 ETS sequences. Well-supported nodes are indicated with bootstrap values on the strict consensus tree of 439 most parsimonious trees (Fig. 1A). Clade A (100% bootstrap support; bs) strongly unites all Arnica representatives. Within clade A, cloned sequences of some accessions end up in two or three strongly supported groups, marked as paralogue I (83% bs), II (78% bs) and III (98% bs). Some cloned accessions are represented in all three paralogues: both representatives of A. parryi (1 and 2), A. gracilis 3, and the possible hybrid A. gracilis × longifolia. Arnica chamissonis 3-7, A. lanceolata, and A. angustifolia 2 are represented in two paralogues (I and II). Some accessions are represented in one paralogue (I: A. angustifolia 1, A. mollis, II: all A. longifolia representatives, A. chamissonis 1 and 2, and III: A. gracilis 1 and 2).

6 Secondary chemistry data Phylogenetic analyses. The matrix of 239 characters based on STL data includes 76% parsimony-informative sites excluding outgroups and 77% including outgroups. A maximum parsimony analysis yielded one single most parsimonious tree (Fig. 1B). Although basal branches are weakly supported, Arnica does not constitute a monophyletic group based on STL data. A few clades are well supported; all A. montana accessions are united in clade J (99% bs) and all A. longifolia accessions in clade K (100% bs). Arnica chamissonis 1 and 2 form clade L (99% bs) and with A. parryi 2 these form clade M (93% bs). The remaining A. chamissonis representatives are united in clade N (86% bs). PCA. The PCA performed on the matrix of peak areas for all species accessions resulted in a model with four principal components that explains 63% of the variation in the dataset. A few groups are well defined in the score plot (Fig. 2), e.g., all five A. montana representatives, all three A. longifolia representatives, and A. chamissonis 1 and 2. Identified STLs important for the clusterings of multiple sequences of A. montana, A. chamissonis 1-2, A. chamissonis 3-7, A. longifolia and A. gracilis accessions, as calculated by OPLS analyses, are indicated in Table 2. Identified STLs. We identified no less than 29 STLs of seven core structural types by comparing with known and published mass spectra and retention times of structurally known STLs (see Fig. 3), some of which are new to taxa. The distribution of these among all included species is summarized in Table 2.

Cytometric data Genome sizes as measured by flow cytometry are listed in Table 1. Chromosomes were counted for A. mollis, which was found to be diploid (2x= 38).

Discussion

Arnica - a complex evolutionary history Phylogenetic relationships in Arnica are complex, probably as a result of an evolutionary history involving hybridization, polyploidy and agamospermy. Cloned ITS and ETS sequences of A. angustifolia, A. chamissonis, A. gracilis, A. lanceolata, and A. parryi accessions are present in two or three paralogues in the phylogenetic tree (Fig. 1A). Polymorphism was also detected in the low-copy region RPB2 for the same taxa (Ekenäs et al. manuscript). Similar patterns of polymorphic ITS sequences have been found in a few taxa, e.g., Amelanchier (Rosaceae), where it was explained by hybridization in combination with reduced concerted evolution due to agamospermy (Campbell et al., 1997). It is possible that agamospermy has lead to a reduction in concerted evolution between different copies of the ITS and ETS region in Arnica and that this is causing the different paralogues to be maintained.

7 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 1.

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 2.

8 As expected, accessions of the same taxa end up close or in monophyletic groups in at least one of the paralogues (A. angustifolia, A. chamissonis, A. longifolia, A. gracilis, and A. parryi). However, some unexpected congruencies in interspecific phylogenetic relationships can be detected between paralogues, for example the close relationship that is suggested between the morphologically very different species A. gracilis and A. parryi in all three paralogues. A few moderately supported relationships, such as those between A. gracilis and A. parryi and between A. fulgens and A. lonchophylla, were also detected in a previous study based on ITS, ETS and chloroplast regions in Arnica. However, we did not identify the three different ITS and ETS paralogues in that study (Ekenäs et al., 2007).

STLs in Arnica PCA and phylogenetic analyses. Secondary metabolites (e.g., STLs and flavonoids) have been isolated and structurally identified for many species in Arnica (e.g., Wil- luhn et al., 1995 and references therein; Schmidt and Willuhn, 2000; Kos, 2005). Sesquiterpene lactones have been of special interest since these constitute the main anti-inflammatory compounds in A. montana (e.g., Hall et al., 1979; Lyss et al., 1997; Lyss et al., 1998; Siedle et al., 2003). However, although Willuhn et al. (1995) summarized STLs in Arnica, no cluster or phylogenetic analyses have been performed with the aim to present an overview of STLs in the genus as a whole. In the present study STL profiles were analysed in an evolutionary framework by phylogenetic inference which resulted in a resolved single most parsimonious tree (Fig. 1B). The data was also analysed with PCA (Fig. 2), allowing for relative amounts to be considered in addition to absence or presence of STLs. Generally, representatives forming strongly supported groups in the phylogenetic tree form distinct clusters in the fringes of the PCA score plot (A. montana, A. longifolia, and A. chamissonis 1-2 and 3-7). By comparing with mass spectra of already structurally determined STLs we identified the major compounds supporting these groups (Table 2 and Fig. 3). Sesquiterpene lactones of the helenalin and dihydrohelenalin types are mainly present in A. montana accessions and almost all are important for defining this group. The cluster of A. chamissonis (1 and 2), separated from the remaining accessions of the same species, is supported by STLs of the chamissonolide and dihydrohelenalin types and the cluster of three A. longifolia accessions is mainly supported by STLs of the xanthalongin type. A weakly supported group of acccessions in the phylogenetic tree forms a group close to the center of the PCA score plot and these generally contain fewer identified STLs.

Figure 1. Phylogenetic trees based on (A) nrDNA sequence data and (B) STL data for the 33 Arnica accessions and accessions of Layia hieracioides and Madia sativa (outgroup taxa). Accession 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 nrDNA (ITS and ETS) sequences (heuristic search, TBR, 100 replicates). CI (excluding uninformative characters) = 0.57 and RI = 0.80. Most cloned sequences 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). CI (excluding uninformative character) = 0.31 and RI = 0.61.

Figure 2. 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.

9 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 × 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. 3 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 (1-2) group, 4 the A. chamissonis (3-7) group, and 5 the A. gracilis group, 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 STL data in Fig. 1.

Identified STLs new to species. In of A. chamissonis we identified STLs of the xanthalongin type (e.g., 4H-xanthalongin, present in all seven A. chamissonis accessions). STLs of this type have previously not been reported in A, chamissonis. Moreover, large amounts of xanthalongin derivatives (particularly xanthalongin and dihydro-4H-xanthalongin) were found in accessions of A. gracilis and A. latifolia, neither of which have been investigated for STLs previously. Very few STLs were found in A. discoidea, A. fulgens, and A. griscomii, which are also species not earlier investigated for STLs to our knowledge. Two STLs of the xanthalongin type (xanthalongin and dihydroxanthalongin) were identified in Layia hieracioides, belonging to the sister group of Arnica (Madiinae; Baldwin and Wessa, 2000). Although diverse in other closely related groups (e.g., Gaillardiinae and Heliantheae; Ottosson 2003 and references cited therein), sesquiterpene lactones have previously not been identified in subtribe Madiinae.

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 3. Identified STLs in all accessions of Arnica and outgroup taxa (see Table 2).

Cytometric data Our results show that variation in DNA amounts between representatives within species to some extent correlates with differences in morphology, genetic, and chemical data (see below). However, as discussed by Suda et al. (2006), genome content measured by flow cytometry is not always correlated to ploidy level in species of the same genus. Even within species polyploidy is not always directly pro- portional to DNA content measured by flow cytometry. Indeed, in our study the

11 genome size of the reference sample, A. mollis (1.18), is almost double that of A. montana (0.52-0.54) although the chromosome number is the same in these two diploid representatives (no polyploidy has been reported in A. montana; Table 1). Still, our results show that differences in STL data and nrDNA sequence data between multiple accessions within species often is accompanied with differences in genome size, as discussed below.

Intraspecific congruencies Although basal nodes were weakly supported, phylogenetic analyses resulted in some strongly supported congruencies between STL data and nrDNA sequence data (Fig. 1). The congruent clades unite accessions of the same species: clades I (100% bs) and J (99% bs), uniting all A. montana accessions, clades 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 data tree, but with low support. Accessions within groups are also similar in genome size and the clusterings are also distinguished in the PCA score plot, except the one uniting A. gracilis accessions.

Support for subspecies classification In Maguire’s revision (1943) of the whole genus, A. chamissonis and A. parryi were both divided into subspecies (A. chamissonis ssp. chamissonis, ssp. foliosa and ssp. incana; A. parryi ssp. parryi and ssp. sonnei). However, no subspecies were recognized by Gruezo and Denford (1994). Our study show that multiple A. chamissonis and A. parryi representatives each form two groups in the analyses based on STLs that can be correlated to subspecies classification. Earlier taxonomic classifications and results from the present study are summarized in Table 3. Based on Maguire’s classification (1943), the two A. chamissonis accessions (1 and 2; originating from California) that are clearly separated from remaining A. chamissonis accessions based on STL data, are determined as A. chamissonis ssp. foliosa. The remaining representatives are classified as A. chamissonis ssp. chamissonis. This is also supported by earlier studies, e.g., Kresken (1984), in which subspecies of A. chamissonis are distinguished based on isolated STLs. Differences between the two groups are also found in genetic and cytometric data. In the nrDNA tree, A. chamissonis 1 and 2 are only detected in paralogue I, whereas remaining A. chamissonis accessions are detected in paralogues I and II. Cytometric data show a slightly larger genome size (1.00) in A. chamissonis 1 and 2 compared to that of A. chamissonis 3-7 (0.85-0.86). Similarly the two representatives of A. parryi (1 and 2; originating from Colorado and California would be classified as A. parryi ssp. parryi and A. parryi ssp. sonnei respectively based on Maguires classification (1943) and are separated from each other based on STL data. 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 low STL contents in A. parryi 1. In paralogue II of the tree based on nrDNA data, the representatives are separated with A. parryi 1 associated with A. lanceolata and A. gracilis 3 and A. parryi 2 associated with A. chamissonis representatives. In cytometric data, A. parryi 2 has a slightly larger genome size (1.22) compared to A. parryi 1 (1.00).

12 Table 3. Classifications of A. chamissonis and A. parryi according to different authors in comparison with the results of the present study.

Sequenced sample Taxonomic classification Present study Gruenzo & Maguire (1943) Jepson (1925) Cytometric data Chemical data (Fig. 1B) ITS and ETS sequence data Denford (1994) (Table 1) (Fig. 1A) A. chamissonis 1-2 A. chamissonis A. chamissonis ssp. foliosa (Nutt.) A. foliosa Nutt. 1.00 with A. parryi 2 paralogue II found Maguire A. chamissonis 3-7 A. chamissonis A. chamissonis ssp chamissonis A. chamissonis 0.85 - paralogues I and II found (as: ssp. genuina) Maguire A. parryi 1 A. parryi A. parryi ssp. parryi (as: ssp. genuina) A. parryi 1.00 - with A. lanceolata in paralogue II Maguire A. parryi 2 A. parryi A. parryi ssp. sonnei (Greene) Maguire A. foliosa Nutt. 1.22 with A. chamissonis 1-2 with A. chamissonis in paralogue II

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. 1) and contains many of the same STLs, but in smaller amounts (Table 3). 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 STL and nrDNA 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 Based on morphological characters one accession collected in Alberta, Canada, is suggested to be a hybrid between 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). Nuclear ribosomal DNA and STL data of the present study also suggest that it may be a hybrid of these two species. If so, directional concerted evolution has probably 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). Cytometric data shows that the genome size of the assumed A. gracilis × longifolia (1.26) is in between that of the A. gracilis accessions (1.00-1.10) and that of the A. longifolia accessions (1.30-1.34). In the tree based on chemical data, A gracilis × longifolia forms a weakly supported group with the rest of the A. gracilis representatives and in the PCA analysis it ends up alone in a position in between the groups consisting of A. gracilis and A. longifolia representatives. Maximo et al. (2006) propose the possibility of detecting hybrids in intermediate positions compared to the parent species in PCA based on secondary metabolite profiles (quinolizidine alkaloids) in Ulex (Fa- baceae), which is in line with these results. In addition, the accession has many STLs in common with each of its suggested parent species accessions (see Table 3).

STLs within species Whereas a group of secondary metabolites usually dominates within a taxon, there are usually differences not only between individual plants or populations, but also between different tissues of the same individual (Wink, 2003). 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. 1B and 2). 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). These can however be correlated to subspecies classification as discussed above. 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. The two accessions of A. angustifolia, in a basal clade of the tree and in the center of the PCA score plot, both contain fewer STLs. Based on our sampling, we can conclude that when abundant, STLs are consistent within species.

14 Congruencies between STL and nrDNA data One of our objectives was to investigate the degree of congruency between STL and nrDNA data and hence if phylogenetic studies based on nrDNA data can potentially be used to predict secondary chemistry in Arnica. More strongly supported basal nodes in both datasets would be necessary to as- sess congruencies between the two datasets regarding interspecific relationships. Still, our analyses show that no less than four clades, of which three are well supported, are congruent (or almost congruent) between analyses based on STL and nrDNA data, uniting multiple accessions of A. montana, A. longifolia, A. gracilis, and A. chamissonis accessions. 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.

Acknowledgements The authors thank B. G. Baldwin for sending seeds and rhizomes collected in Alaska and for providing seeds of Madia sativa and Layia hieracioides, A. Currier and M. Archambault for sending material from Quebec, R. Ejrnaes for sending material from Greenland, and S. Bal- dauf, P. Erixon, S. Larsson, B. Oxelman, and M. Thulin for valuable comments on the manuscript. The research was supported by grants to C. Ekenäs from Smålands nation and the Royal Swedish Academy of Sciences and by grants to K. Andreasen from the Swedish Re- search Council and the Royal Swedish Academy of Sciences.

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Paper III

Anti-inflammatory activity of extracts from Arnica (Asteraceae) species assessed by inhibition of NF-κB and release of human neutrophil elastase

Catarina Ekenäs 1,2, Anna Zebrowska 2, Barbara Schuler 3, Tobias Vrede 4, Katarina Andreasen 1, Anders Backlund 2, Irmgard Merfort 3*, Lars Bohlin 2*

Affiliation 1 Department of Systematic Biology, Evolutionary Biology Center, Uppsala University, Sweden 2 Division of Pharmacognosy, Department of Medicinal Chemistry, BMC, Uppsala University, Sweden 3 Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-Universität, Freiburg, Germany 4 Department of Ecology and Environmental Science, Umeå University, Sweden * Both authors contributed equally to the work

Correspondence Catarina Ekenäs, Department of Systematic Biology, Evolutionary Biology Center, Norbyvägen 18, 752 36 Uppsala, Sweden. E-mail: [email protected] Phone: +46 18 471 64 54 Fax: +46 18 471 64 57

Abstract Several species in the genus Arnica have been used in traditional medicine to treat inflammatory-related disorders. Extracts of twelve Arnica species and two closely related species (Layia hieracioides and Madia sativa) were investigated for anti-inflammatory activity, evaluating both the inhibition of human neutrophil elastase release and the inhibition of transcription factor NF-κB. Statistical analyses reveal significant differences in inhibitory capacities between extracts. Sesquiterpene lactones of the helenanolide type, of which some are known inhibitors of human neutrophil elastase release, and NF-κB, are present in large amounts in the very active extracts of A. montana and A. chamissonis. Furthermore, A. longifolia, which has previously not been investigated for anti-inflammatory properties, shows a high activity similar to that of A. montana and A. chamissonis in both bioassays. Sesquiterpene lactones of the xanthalongin type are present in large amounts in A. longifolia and other active extracts and would be of interest to evaluate for anti-inflammatory properties.

Key words sesquiterpene lactone; anti-inflammatory; neutrophil; elastase; NF-κB; xanthalongin; Arnica, Asteraceae

1 Abbreviations COX-2: cyclooxygenase 2 DMEM: Dulbecco´s modified Eagle medium EMSA: electrophoretic mobility shift assay fMLP: N-formyl-methionyl-leucyl-phenylalanine HaCaT: human keratinocyte HNE: human neutrophil elastase IκB: inhibitory subunit κB iNOS: inducible nitric oxide synthase NF-κB: nuclear factor κB PAF: platelet activating factor pNA: p-nitroanilide SAAVNA: N-succinyl-L-alanyl-l-alanyl-valine-L-p-nitroanilide TNF-α: tumor necrosis factor α STL: sesquiterpene lactone

2 Introduction Plant species closely related to those used for pharmacologically active compounds often express similar secondary chemistry and constitute potential sources for finding new active compounds of similar structures [1], [2]. Several of the 29 species in the genus Arnica have been used in traditional medicine by widely separated ethnic groups to treat inflammatory-related disorders (see Table 1). The best known member of the genus is Arnica montana, a native European medicinal plant used in pharmaceutical practice since the medieval ages. Flower heads of A. montana are still used as a tincture or oil to treat inflammatory-related topical disorders such as sprains, swellings, and bruises. Sesquiterpene lactones (STLs) have been found to constitute the major anti-inflammatory components of A. montana [9], [10], [11]. Although secondary metabolites have been characterized for many Arnica species, the anti-inflammatory properties of pharmaceutical preparations or extracts have only been evaluated for A. montana [12], [13] and A. mollis [14].

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 [3] infusion roots Catawba, North Carolina

A. angustifolia stomach problems [4] infusion - Gwich’in, Yukon and North- west Territories A. cordifolia sore eyes [5] - - Shuswap, British Columbia

A. cordifolia bruises, swellings and poultice whole plant Thompson, British Columbia and cuts [6] A. latifolia tuberculosis [6] infusion whole plant Thompson, British Columbia

aphrodisiac [7] - roots Okanagan-Colville, British Columbia A. discoidea wounds [8] - - Maidu, California

Anti-inflammatory mediators, for example STLs, intervene with the inflammatory process by targeting different parts of the inflammatory cascade. Neutrophil granu- locytes constitute a central component in the inflammatory response, with the ability to migrate to the actual site of inflammation and degrade foreign material by in situ secretion of degradative enzymes. One example of a degrading enzyme is human neutrophil elastase (HNE), which targets the connective tissue protein elastin as well as bacterial proteins. Activation of neutrophils is initiated by chemoattractants, such as the bacterial product N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the platelet activating factor (PAF). The inhibitory effects of compounds or extracts on HNE release by neutrophils can be studied in a multitarget bioassay first developed by Dewald et al. [15] and further modified by Johansson et al. [14]. These assays have been used to demonstrate that isolated STLs of the helenanolide type from A. montana and A. chamissonis inhibit the release of HNE from neutrophils [11]. Another step in the inflammatory pathway is the binding of transcription factors to promoter regions of genes coding for important mediators in the inflammatory process. Nuclear factor-κB (NF-κB) is one of the principal inducible transcription factors and has become a focal point in drug discovery. NF-κB regulates the transcription of over 400 target genes coding for cytokines, such as interleukins and tumor necrosis factor-α (TNF-α), as well as cell adhesion molecules and enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [16]. When activated by inflammatory conditions NF-κB is detached from its inhibitory

3 subunit (IκB) in the cytosol and translocated to the nucleus where it stimulates the transcription of target genes. Earlier studies showed that STLs prevent the binding of NF-κB to DNA by alkylation of cysteine-38 in the p65/NF-κB subunit [17]. There are strong indications that this is a general mechanism for STLs possessing α,β- unsaturated carbonyl structures such as α-methylene-γ-lactones or α,β-unsaturated cyclopentenones [18], [19]. The influence of STLs on the binding of NF-κB to DNA can be investigated by the electrophoretic mobility shift assay (EMSA). We have investigated anti-inflammatory activity of extracts from twelve Arnica species. Our aims were (1) to evaluate the anti-inflammatory potency of extracts from a wide range of Arnica species and (2) to compare the results of two bioassays testing anti-inflammatory activity to find out more about the mechanism of action of the active compounds. The bioassays test the capacity of extracts to inhibit HNE release from neutrophils and to inhibit transcription factor NF-κB binding to DNA. The results are discussed with regard to the STL composition of the extracts and documented traditional use of the species.

Materials and Methods

Plant material Seeds or rhizomes of 16 accessions from twelve Arnica species (A. angustifolia, A. chamissonis, A. discoidea, A. fulgens, A. gracilis, A. griscomii, A. lanceolata, A. longifolia, A. mollis, A. montana, A. parryi, and A. sachalinensis) were collected in the field, or obtained from plants cultivated in botanical gardens (see Table 2). In addition, representatives of two species, Layia hieracioides and Madia sativa, belonging to the group most closely related to Arnica [20], were also included. All plant material was cultivated outdoors at Uppsala Botanical Garden, Sweden.

Extraction and isolation of the STL-rich fraction The procedure for isolating STLs mainly followed the protocol of Willuhn and Leven [21]. Fresh flower heads were collected and weighed, and the material was first stored in dichloromethane (Merck). After a pre-extraction of 24 hrs the material was filtered and transferred to methanol (Merck), macerated in a mixer, agitated for 24 hrs, and vacuum filtered. The dried combined extract was dissolved in methanol so that 20 ml corresponded to 20 g of original plant material, and santonin (0.1 % in methanol; 3 ml) was added as an internal standard. Milli-Q water (80 ml) was added and the mixture was extracted three times with 200 ml ethylacetate (Labscan Ana- lytical Sciences). The dried extract was dissolved in 40 ml dichloromethane (Merck), filtered using a sterile nylon filter (0.45 μm, Cameo), dried, and redissolved in 40 ml of methanol and water (1:1 by volume). Aluminium oxide (14 g; GTF) was then added, and the mixture was agitated followed by centrifugation (5 minutes, 20°C and 2000 rpm) in a Sorvall RT 6000D centrifuge. Finally, the mixture was filtered again, and a stock solution was prepared (10 mg/ml in 10% ethanol).

4 Table 2. Source followed by voucher information for all Arnica species accessions. Species Source and voucher(s) Arnica angustifolia Vahl Quebec, Canada; Archambault 2005-MA-001 (MT), Ekenäs 45 (UPS) Arnica chamissonis Less. 1 California, USA; Ekenäs 36 (UPS) Arnica chamissonis Less. 2 Alaska, USA; Baldwin 1335 (UC) Arnica chamissonis Less. 3 cult.1231:1992; Ekenäs 58 (UPS) Arnica discoidea Benth. California, USA; Ekenäs 42 (UPS) Arnica fulgens Pursh cult.1230:1992; Ekenäs 59 (UPS) Arnica gracilis Rydb. Wyoming, USA; Baldwin 1393 (UC) Arnica griscomii Fernald Alaska, USA; Baldwin 1341 (UC) Arnica lanceolata Nutt. Alaska, USA; Baldwin 1336 (UC) Arnica longifolia D.C. Eaton 1 cult. 6630*; Ekenäs 60 (UPS) Arnica longifolia D.C. Eaton 2 cult. 1989:1451; Ekenäs 61 (UPS) Arnica mollis Hook. Alberta, Canada; Andreasen & Ekenäs 408 (UPS) Arnica montana L. 1 Italy; Ekenäs 64 (UPS) Arnica montana L. 2 Slovenia; Ekenäs 65 (UPS) Arnica parryi A. Gray. cult. 1991:1865; Ekenäs 62 (UPS) Arnica sachalinensis A. Gray. cult. 2316:1980; Ekenäs 63 (UPS) Layia hieracioides Hook. & Arn. California, USA; Baldwin s.n. (JEPS) Madia sativa Molina California, USA; Bainbridge s.n. (JEPS), Ekenäs 57 (UPS) Samples were collected in the field or when indicated by “cult.” from cultivated representatives at Upp- sala Botanical Garden, Uppsala, Sweden. *Collected in Bergius Botanical Garden, Stockholm, Sweden.

HNE release multitarget bioassay Each STL-rich extract was tested in duplicates in 4-8 independent experiments for inhibition of HNE release of PAF- and fMLP- activated neutrophils respectively. Extracts were tested at the concentration 100 μg/ml. Solutions of cytochalasin B (Sigma), the elastase substrate N-succinyl-L-alanyl-l-alanyl-valine-L-p-nitroanilide (SAAVNA; Bachem), fMLP (Sigma) and PAF (Bachem) were prepared from stock solutions, and neutrophils were isolated from heparanized blood of two healthy volunteers according to the method described by Johansson et al. [14]. The extract (10 mg/ml; 10 μl), cytochalasin B (5 μl), and neutrophil suspension (100 μl) were added to the SAAVNA solution (750 μl) and incubated for 5 minutes at 37°C. For the negative controls, ethanol (10%; 10 μl) was added instead of the extract. After incubation, the reaction was initiated by adding the inducer (100 μl of PAF or fMLP), followed by incubation at 37°C for 10 minutes. For the background samples (representing the spontaneous release of elastase without inducers), buffer was added instead of inducers. After incubation, the reaction was terminated by citric acid (2%; 250 μl), followed by centrifugation (1500 rpm; 20°C; 5 minutes). The substrate SAAVNA is cleaved by HNE, which leads to the formation of the colored product p-nitroanilide (pNA). The absorbance of each sample was recorded at 405 nm using a Shimadzu UV-160A UV visible recording spectrophotometer. For each extract, the inhibition of HNE release as a result of added inducer (fMLP or PAF), was calculated as the relative decrease in absorbance compared to the absorbance of the negative control:

⎡ ABSsample+inducer − ABSsample+buffer ⎤ %inhibition = 100 × ⎢1− ⎥ ⎣ ABSinducer − ABSbuffer ⎦

5 NF-κB bioassay Twelve extracts of eleven Arnica species and Madia sativa were tested for the ability to inhibit the transcription factor NF-κB. Each extract was tested in two concentrations of 66 and 33 μg/ml and repeated at least once. Before conducting the EMSA, HaCaT (human keratinocyte) cells were maintained in Dulbecco´s modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal calf serum (FCS; Sigma), 100 IU/ml penicillin and 100 µg/ml streptomycin (Roche Diagnostics). On the day prior to conducting the EMSA, cells were plated in small Petri dishes (BD Falcon) at a density of 7x105-1x106 cells in 5 ml of starved medium (DMEM with 100 IU/ml penicillin and 100 µg/ml streptomycin but without FCS). A lower basal stimulation is obtained under these starving conditions. Each extract was incubated

(37°C; 5% CO2) with the cell culture (5 ml) at the two concentrations on 60 mm Petri dishes for one hour. Activation of NF-κB was triggered by incubation with TNF-α (2.5 ng/ml; 5 μl) for another hour. Neither TNF-α nor extract was added to the negative control, and to the positive control TNF-α was added but no extract. Protein extraction and the EMSA were carried out as described in [18]. However, the [32P]-labeled oligonucleotide was replaced by a [33P]-labeled oligonucleotide. Signals were detected and quantified by densitometry using a PhosphoImager.

Statistical analysis Results were analysed with ANOVA using JMP TM 5.0.1a (SAS Institute) for Win- dows (see [22], for more details on ANOVA). The results of the inhibition of PAF- and fMLP-induced HNE release were separately compared for all extracts using the main effect factors Extract, which evaluates differences in inhibitory activities between extracts, Method, which evaluates differences between PAF- and fMLP- mediated inhibition, and the interaction effect factor Method × Extract (which evaluates if the difference between PAF- and fMLP-induced inhibition differs between extracts) as fixed effects. We also included the random effect batch nested within extract to find out if the results of inhibition of extracts were biased with regard to in which batch they were tested. In a second ANOVA, extracts and methods were compared for the subset of extracts that were also analysed for inhibition of NF-κB. For post hoc comparisons among treatments, Tukey honestly significant difference (HSD) tests were used with α = 0.05.

Results and discussion Large differences in anti-inflammatory activities were found between extracts from twelve Arnica species as measured by two bioassays. Ranges of inhibition were 10- 97% for PAF-induced HNE release, 1-100% for fMPL-induced HNE release, and 35-90% for inhibition of NF-κB (Table 3, Figure 1). Inhibitory capacities of extracts are discussed below with regard to STL content and earlier documented anti- inflammatory activities of isolated STLs. An ANOVA encompassing the results of the HNE bioassay for all extracts reveals that there is a highly significant difference (P <0.0001) in inhibitory activity among extracts of the different Arnica species tested (Table 4; part A). There is no significant difference (P = 0.74) between the PAF-induced and fMLP-induced inhibitory effects (discussed more below), but the interaction effect as measured by Method × Extract is significant (P = 0.0005). This indicates that the difference in

6 Table 3. Percentage of inhibition in the HNE release bioassay (mediated by PAF and fMLP) and the NF-κB bioassay, and presence or absence of STL core structures (from Ekenäs et al., manuscript) for all extracts (see Fig. 1 for STL core structures). Species* PAF-mediated HNE** fMLP-mediated HNE** NF-κB** STL core structure***

N Inhib. (%) CI N Inhib. (%) CI N Inhib. (%) CI He DHe Ar Cha Xa Ca Fl Eu

A. angustifolia 4 45 ± 32 5 26 ± 40 4 41 ± 8 - - - - (X) - - - A. chamissonis 1 6 97 ± 5 6 99 ± 1 3 86 ± 14 X X X X X (X) - - A. chamissonis 2 5 16 ± 14 5 28 ± 13 6 42 ± 8 (X) (X) X (X) X (X) - X A. chamissonis 3 6 82 ± 9 6 101 ± 10 - - - X (X) X (X) X (X) - X A. discoidea 5 10 ± 8 5 14 ± 21 6 46 ± 4 - - - - (X) - - - A. fulgens 5 38 ± 13 5 30 ± 18 6 50 ± 13 - (X) X - (X) (X) X - A. gracilis 6 46 ± 18 6 38 ± 15 4 71 ± 10 - (X) X - X - - - A. griscomii 6 69 ± 10 6 79 ± 9 2 35 ± 15 - - - - (X) (X) - - A. lanceolata 5 81 ± 9 5 69 ± 16 - - - (X) - X - X - - X A. longifolia 1 6 91 ± 10 6 97 ± 3 - - - X X X - X X X - A. longifolia 2 5 95 ± 4 5 95 ± 9 3 89 ± 10 X X X - X X X - A. mollis 5 26 ± 13 5 18 ± 6 6 44 ± 14 ------A. montana 1 6 88 ± 14 6 100 ± 5 3 90 ± 12 X X X - (X) (X) - - A. montana 2 6 92 ± 10 6 86 ± 12 - - - X X X - - X - - A. parryi 8 29 ± 16 7 39 ± 27 4 41 ± 6 - (X) (X) (X) (X) (X) - - A. sachalinensis 5 58 ± 13 5 79 ± 10 6 55 ± 13 ------L. hieracioides 5 68 ± 4 5 55 ± 23 ------X - - - M. sativa 5 13 ± 19 5 1 ± 19 2 60 ± 20 ------* A: Arnica, L: Layia, M: Madia. ** N: number of independent experiments, Inhib. (%): average percentage inhibition, CI: 95 % confidence interval. *** He: helenalin, DHe: dihydrohelenalin, Ar: arnifolin, Cha: chamissonolid, Xa: xanthalongin, Ca: Carabron, Fl: florilenalin, Eu: eudesmanolide, X: present, (X): present in trace amounts (area under peak < 5 % of internal standard

extracts. However, a Tukey HSD test (not shown) indicates no statistical difference between the methods (PAF- or fMLP-induced effects) for any single extract. The extracts were analysed in batches (each extract was run in duplicates in 4-8 batches) and our results show that there is variation associated with analysis batch (P <0.001). One reason could be individual variations caused by the state of the immune system of blood donors, which is discussed by Schorr et al. [23] as a possible reason for activated neutrophils and elevated basal stimulation. However, the sum of squares of analysis batch corresponds to only 26% of the sum of squares that relates to extract. Hence, the major part of the variation in inhibitory activity is due to differences between extracts.

Table 4. Results of two ANOVAs: A) ANOVA on the results of the HNE release bioassay (18 extracts of 14 species) and B) Two-way ANOVA on the results of the HNE release and NF-κB bioassays (13 extracts of 12 species).

Source of variation dfnum dfden SS F -2logLikelihood P

A) Method 1 271 28 0.11 0.74 Extract 17 82 134505 32.3 <0.0001 Method × Extract 17 271 11030 2.65 <0.0005 Analysis batch [Extract] 82 271 35357 3149 <0.001 Residual error 271 66411

B) Method 2 158 2692 4.94 0.008 Extract 12 158 116806 35.72 <0.0001 Method × Extract 24 158 20057 3.07 <0.0001 Residual error 158 44348 A) Main effects Method (differences between PAF- and fMLP- mediated HNE release), Extract (differences between extracts), the interaction effect Method × Extract, and the nested random effect Analysis batch (nested within extract) as factors, and % inhibition as dependent variable. Whole model 2 F117,271=14.8, P<0.0001, R =0.83. B) Main effects Method (differences between PAF- and fMLP- mediated HNE release and NF-κB) and Extract and the interaction effect Method × Extract as factors, and % 2 inhibition as dependent variable. Whole model F38,158=15.5, P<0.0001, R =0.79. dfnum: degrees of freedom in the numerator, dfden: degrees of freedom in the denominator, SS: sum of squares, F: test statistic for F test, -2logLikelihood: test statistic for χ2 test, P: probability that the effect is not different from 0, R2: coefficient of determination.

PAF- and fMLP-induced effects of the HNE release and the inhibition of NF-κB are compared in a second ANOVA for the subset of extracts that were tested for NF-κB inhibition (Table 4; part B). Similar to the previous ANOVA, inhibitory differences among extracts explain most of the variation. There is no significant difference in effect between the two concentrations tested for NF-κB inhibition (F13,32=1.34, P=0.24), and these were therefore combined and pooled. Since the effect of analysis batch was small in comparison to the main effect in the previous ANOVA, the inhibitory effects between PAF- and fMLP-induced HNE release differs between random effect batch was omitted.

Fig. 1. Results of the inhibition of HNE release (induced by PAF and fMLP) and the inhibition NF-κB for all extracts with 95% confidence intervals.

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. 1). Extracts of A. lanceolata, A. griscomii, A. sachalinensis, and Layia hieracioides in the HNE bioassay, and A. gracilis and Madia sativa in the NF-κB bioassay possess intermediate inhibition (60-80%). The tested extracts contain numerous STLs, of which some have been identified (Ekenäs et al. manuscript) and are classified according to core structure in Table 3. Core structures of STL skeletons identified in the extracts are illustrated in Figure 2. Accessions of species containing STLs of the helenanolide type (e.g., A. montana, A. chamissonis, and A. longifolia) possess strong inhibitory activity, which is in agreement with a previous study by Siedle et al. [11], in which two dihydrohelenalin derivatives from A. montana were active in the HNE release bioassay at micromolar concentrations. In Siedle et al. [17], it is also demonstrated that isolated helenalin and dihydrohelenalin derivatives inhibit NF-κB. However, in two extracts of intermediate activity in the HNE bioassay (A. griscomii and Layia hieracioides), only STLs of the xanthalongin structure could be identified. Xanthalongins are also the major expressed STLs in A. longifolia, and A. gracilis. This indicates that STLs of this type may also be potent inhibitors, which has previously not been shown.

Figure 2. Core structures of STLs identified in the extracts (Ekenäs et al. manuscript; see Table 3); helenalin (A), dihydrohelenalin (B), arnifolin (C), chamissonolid (D), xanthalongin (E), carabron (F), florilenalin (G), eudesmanolide (here exemplified by ivalin) (H).

Multiple representatives of a few species were included. Two representatives each of A. montana and A. longifolia, all consistently show very high inhibitory effects (86- 100%). However, two of the three A. chamissonis representatives included exhibit high effects (82-100%; A. chamissonis 1 and 3) and one low effect (16-42%; A. chamissonis 2). The representative with low effect originates from Alaska, whereas of the two exhibiting high effect, one originate from California and the other is of unknown origin. Perhaps the difference in chemistry originates in different ecological niches. Although most extracts of the same species are consistent based on our sampling, the example of A. chamissonis demonstrates that so is not always the case. The results of the NF-κB and HNE release bioassays are different with a statistical significance (P = 0.008). However, a Tukey HSD test (not shown) indicates that when comparing within extracts, the result is significantly different only for one single extract (between NF-κB and fMLP-induced HNE release for Madia sativa). The interaction Method × Extract is highly significant (P <0.0001), which is probably due to a difference in range between the bioassays. The NF-κB bioassay seems to be more sensitive at low concentrations of active compounds since all extracts possess at least 40% inhibition. Since the same STL-rich extracts possess strong inhibitory activity in both the NF-κB and the HNE release bioassays, the possibility that HNE release inhibition is mediated by inhibition of NF-κB or downstream events cannot be excluded. Within the neutrophil multitarget bioassay there is no significant difference between PAF- or fMLP-induced inhibitory activity, indicating that the mechanism of action is not correlated specifically to either of these stimulators. This is also in agreement with earlier studies of isolated STLs using the same bioassay [11], [23], [24]. Furthermore, Siedle et al. [11] tested the isolated STLs for inhibitory effects on

10 HNE with no significant effect, indicating that isolated HNE is not a direct target for STLs. Some accessions lack STLs but still possess intermediate inhibitory activity. Even though it has been shown that A. sachalinensis contains no STLs (probably because this species lacks the stipitate glandular trichomes containing these types of compounds) [25], the extract of A. sachalinensis exhibits significant inhibitory activity in both bioassays. Also Madia sativa exhibits inhibitory activity in the NF-κB bioassay, although we were not able to determine any STLs in this extract. Interest- ingly, Madia sativa has been used to treat rheumatoid disorders in South America, and for the same purpose by native populations of the Vancouver Islands [26]. Ma- jor compounds of A. sachalinensis and M. sativa responsible for this anti- inflammatory effect remain to be isolated and structurally determined. Comparing our findings with the traditional and present use of Arnica species, we can conclude that A. chamissonis, which has already been explored as a substitute to A. montana [27] exhibits very high anti-inflammatory activity. The extracts of some species that have been used in traditional medicine (e.g., A. discoidea and A. angustifolia) show low anti-inflammatory activity in our study. In contrast, extracts of A. longifolia show very high anti-inflammatory activity according to our results, but we could find no documented traditional use of this species. Furthermore, A. longifolia has to our knowledge not been tested for anti-inflammatory compounds. As discussed previously, xanthalongin derivatives may be partly responsible for the high anti-inflammatory activity of A. longifolia. This could also explain the moderate activity of A. lanceolata, A. gracilis and Layia hieracioides, which contain xanthalongin derivatives as major compounds. Extracts of these species as well as isolated xanthalongin derivatives will be further investigated in the search for anti- inflammatory agents.

Acknowledgements The authors thank B. G. Baldwin for sending seeds and rhizomes collected in Alaska and for providing seeds of Madia sativa and Layia hieracioides, A. Currier for sending material from Quebec, S. Ortlepp for instructions on the use of the HNE release bioassay, and S. Baldauf, M. Thulin, and S. Ortlepp for comments on the manuscript. The research was supported by grants to C. Ekenäs from Smålands nation and the Royal Swedish Academy of Sciences, by a grant to K. Andreasen from the Swedish Research Council, and by a FORMAS grant to L. Bohlin for the study of anti-inflammatory compounds.

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11 7 Turner N. Ethnobotany of the Okanagan-Colville Indians of British Columbia and Washington. Victoria British Columbia Provincial Museum; 1980. 8 Strike S. Ethnobotany of the California Indians. Aboriginal uses of California´s indigenous plants. Illinois Balogh Scientific Books; 1994. 9 Hall IH, Lee KH, Starnes CO, Sumida Y, Wu RY, Wadell TG et al. Anti- inflammatory activity of sesquiterpene lactones and related compounds. Journal of Pharmaceutical Sciences 1979; 68: 537-542. 10 Lyss G, Schmidt TJ, Merfort I, Pahl HL. Helenalin, an anti-inflammatory sesquiterpene lactone from Arnica, selectively inhibits transcription factor NF-κB. Biological Chemistry 1997; 378: 951-961. 11 Siedle B, Gustavsson L, Johansson S, Murillo R, Castro V, Bohlin L et al. The effect of sesquiterpene lactones on the release of human neutrophil elastase. Bio- chemical Pharmacology 2003; 65: 897-903. 12 Lyss G, Scmidt TJ, Pahl HL, Merfort I. Anti-inflammatory activity of Arnica tincture (DAB 1998) using the transcription factor NF-kappaB as molecular target. Pharmaceutical and Pharmacological Letters 1999; 9: 5-8. 13 Klaas CA, Wagner G, Laufer S, Sosa S, Loggia RD, Bomme U et al. Studies on the anti-inflammatory activity of phytopharmaceuticals prepared from Arnica flowers. Planta Medica 2002; 68: 385-391. 14 Johansson S, Göransson U, Luijendijk T, Backlund A, Claeson P, Bohlin L. A neutrophil multitarget functional bioassay to detect anti-inflammatory natural products. Journal of Natural Products 2002; 65: 32-41. 15 Dewald B, Baggiolini M. Evaluation of PAF antagonists using human neutrophils in a microtiter plate assay. Biochemical Pharmacology 1987; 36: 2505-2510. 16 Baeuerle PA, Henkel T. Function and activation of Nf-κB in the immune-system. Annual Review of Immunology 1994; 12: 141-179. 17 Siedle B, Garcia-Pineres AJ, Murillo R, Schulte-Mönting J, Castro V, Rungeler P et al. Quantitative structure - activity relationship of sesquiterpene lactones as inhibitors of the transcription factor NF-κB. Journal of Medicinal Chemistry 2004; 47: 6042-6054. 18 Garcia-Pineres AJ, Castro V, Mora G, Schmidt TJ, Strunck E, Pahl HL et al. Cys- teine 38 in p65/NF-κB plays a crucial role in DNA binding inhibition by sesquiterpene lactones. The Journal of Biological Chemistry 2001; 276: 39713-39720. 19 Lyss G, Knorre A, Schmidt TJ, Pahl HL, Merfort I. The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-κB by directly targeting p65. The Journal of Biological Chemistry 1998; 273: 33508-33516. 20 Baldwin BG, Wessa BL. Origin and relationships of the tarweed - silversword lineage (Compositae - Madiinae). American Journal of Botany 2000; 87: 1890-1908. 21 Willuhn G, Leven W. Zur qualitativen und quantitativen analyse der Sesquiter- penlactone von Arnikablüten DAB 9. Pharmzeutische Zeitung Wissenschaft 1991; 1: 32-39. 22 Zar J. H. 1984. Biostatistical analysis. 2nd ed. 1984; Prentice-Hall; pp 718. 23 Schorr K, Rott A, Da Costa FB, Merfort I. Optimization of a human neutrophil elastase assay and investigation of the effect of sesquiterpene lactones. Biologicals 2005; 33: 175-184. 24 Stefani R, Schorr K, Tureta JM, Vichnewski W, Merfort I, Da Costa FB. Ses- quiterpene lactones from Dimerostemma species (Asteraceae) and in vitro potential anti-inflammatory activities. Zeitschrift für Naturforschung 2006; 61: 647-652.

12 25 Junior I. Untersuchungen zur Inhaltsstoffürung der Blüten von Arnica chamissonis Less. ssp. genuina Maguire und Arnica sachalinensis (Regl.) A. Gray [dissertation]. Düsseldorf: Düsseldorf Universität; 1983. 26 Zardini E. Madia sativa Mol. (Asteraceae-Heliantheae-Madiinae): an ethno- botanical and geographical disjunkt. Economic Botany 1992; 46: 34-44. 27 Cassells AC, Walsh C, Berlin M, Cambornac M, Robin JR, Lubrano C. Estab- lishment of a plantation from micropropagated Arnica chamissonis: a pharmaceutical substitute for the endangered A. montana. Plant Cell, Tissue and Organ Culture 1999; 56: 139-144.

Paper IV

RPB2 phylogeny in Arnica (Asteraceae) reveals multiple d-paralogues

Catarina Ekenäs1, Nahid Heidari1, and Katarina Andreasen1

1Department of Systematic Biology, Evolutionary Biology Center, Uppsala University, Uppsala, Sweden.

Abstract The utility of the region coding for the second largest subunit of RNA polymerase II (RPB2) for resolving relationships in lower level taxa was explored by cloning and sequencing the region between exons 17 and 23 for 33 accessions of Arnica and four outgroup taxa. Three paralogues of the RPB2-d copy (RPB2- d A, B and C) were detected in Arnica and outgroup taxa, indicating that the duplications must have occurred before the divergence of Arnica. Low levels of homoplasy in combination with relatively high sequence variation indicates that the introns of the RPB2 region should be suitable for phylogenetic studies in recently diverged taxa. Parsimony and Bayesian analyses of separate alignments of the three copies reveal complex patterns in Arnica, likely reflecting a history of lineage sorting in combination with apomixis, polyploidization, and possibly hybridization. Cloned sequences of some taxa do not form monophyletic clades within paralogues, but form multiple strongly supported clades with sequences of other taxa. Some well supported groups are present in more than one paralogue and many groups are in line with earlier hypotheses regarding interspecific relationships within the genus.

Introduction In the search for more variable DNA regions for resolving phylogenetic relationships in recently diverged taxa, low copy nuclear DNA (lcnDNA) regions have been investigated (e.g., Small et al. 1998, Popp and Oxelman 2001, Lee et al. 2002, Pfeil et al. 2004, Joly et al. 2006). The majority of sequence data currently used in plant systematics originates from chloroplast DNA (cpDNA) or nuclear ribosomal DNA (nrDNA) data. However, in non-coding chloroplast regions there is often low sequence variation in recently diversified taxa, resulting in problems with low resolution in phylogenetic analyses (e.g., Small et al. 1998, Cronn et al. 2002, Hartman et al. 2002, Xu et al. 2000). Chloroplast DNA is mostly maternally inherited in flowering plants and since the cpDNA evolutionary history may differ from that of the organism due to e.g. hybridization, it is recommended to be used in combination with at least one region from the nuclear genome. Nuclear ribosomal DNA, such as the Internal Transcribed Spacer (ITS) region has the advantage of being more variable than any cp region and has proven successful, especially in combination with non-coding cpDNA, to resolve relationships in several young groups (Soltis et al. 1996, Campbell et al. 1997, Yang et al. 2002). However, ribosomal DNA exists in tandem repeat multiple copies, which are believed to be homogenized by concerted evolution. Low copy nuclear DNA regions exist in one or just a few copies, making it possible to trace paralogues in the genome to infer the evolutionary history without homogenizing processes like concerted evolution (Hillis et al. 1991). Each of the four classes of nuclear RNA polymerases present in green plants is composed of two large and several smaller subunits, of which the two large subunits tend to be present in a single or a few copies. The RPB2 region, coding for the second largest subunit of RNA polymerase II (Sawadogo and Sentenac 1990), has been

1 successfully used for resolving phylogenetic relationships at higher taxonomic levels (Denton et al. 1998, Oxelman and Bremer 2000, Oxelman et al. 2004, Ronçal et al. 2005) as well as at 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 angiosperm taxa which has been suggested to be the result of a gene duplication near the core eudicots. The RPB2-d copy is found almost universally in investigated angiosperms, while the RPB2-i copy is scattered within two major clades of eudicots (Oxelman et al. 2004, Luo et al. 2007). Arnica L. is a circumboreal genus of 29 species (Wolf 2006) with a distribution mostly confined to western North America. Complex and diverse patterns of morphological variation in Arnica are reflected by widely differing taxonomic treatments (Maguire 1943 and references therein). In the only revision of the entire genus by Maguire (1943), Arnica was divided into five subgenera: Arctica, Andropur- purea, Austromontana, Chamissonis, and Arnica (as: “Montana”; which contains the type species, A. montana L.). Three of the subgenera (Arctica, Austromontana, and Chamissonis) have been revised separately (Downie and Denford 1988, Wolf and Denford 1984a, Gruezo and Denford 1994). Hybridization and polyploidy in association with agamospermy have been proposed to be common in the genus (e.g., Afzelius 1936, Barker 1966, Straley 1980, Wolf and Denford 1984b) and we found complex patterns in earlier phylogenetic studies of the genus involving nrDNA, and cpDNA (Ekenäs et al. 2007, Ekenäs et al. manuscript). The aims of the present study were (1) to further investigate the utility of RPB2 for resolving phylogenetic relationships in low-level taxa and (2) to elucidate the interspecific relationships in Arnica, and (3) to compare the phylogeny based on RPB2 with earlier phylogenetic studies based on nrDNA and cpDNA regions in Arnica.

Material and Methods Plant material and PCR reactions. A total of 37 Arnica accessions, (including du- plicate representatives of seven taxa) and four outgroup taxa from other genera of the tribe Madieae (Eatonella nivea, Hulsea algida, Raillardella argentea, and Vene- gasia carpesioides) were included in the study. Samples were collected in the field, from cultivated populations in Uppsala Botanical Garden or from herbarium specimen (Table 1). Total DNA was isolated and purified from leaf tissue using Mini- Beadbeater™ (BioSpec Products, Bartlesville, OK) and the DNeasy® mini kit (Qiagen, Hilden, Germany). The region between exons 17–23 (including introns 17- 22, see Fig. 1) of the genome encoding for the second largest subunit RNA polymerase II (RPB2) was amplified using primers 7F (forward) and 10R (reverse; Ox- elman et al. 2004). Polymerase chain reactions were carried out using an initial 95°C denaturation (2 min) and 36-38 cycles of 95°C denaturation (30 sec), 58°C annealing (60 sec), and 72°C extension (2 min). The 50 μl reactions consisted of 0.25 μl Taq DNA polymerase (Abgene, Epsom, UK), 5 μl each of reaction buffer and 10 mM MgCl2, 4 μl of 10 mM dNTP, 1 μl each of 10 μM forward and reverse primers (MWG Biotech, Ebenberg, Germany), and 5 μl of 10-20-fold diluted DNA. Milli- pore plates (Millipore, Billerica, MA) were used to purify PCR products. The PCR products were further separated using gel electrophoresis, and bands of 1200-1800 bp were cut out under UV light and purified using the DNA and Gel Band Purifica- tion Kit (Amersham, Buckinghamshire, UK).

2 Table 1. Vouchers and accession numbers. Species Voucher information Arnica angustifolia Vahl. ssp. angustifolia Sweden, Andreasen 312 (UPS) Arnica angustifolia Vahl. ssp. tomentosa (Macoun) G. herb., Newfoundland, Fernald s.n. (S) W. Douglas & Ruyle-Douglas Arnica acaulis Britton, Sterns & Poggenb. herb., South Carolina, Wilbur 8964 (UC) Arnica cernua Howell herb., California, Fahver s.n (JEPS) Arnica chamissonis Less. 1 Alaska, Baldwin 1335 (UC) Arnica chamissonis Less. 2 California, Ekenäs 36 (UPS) Arnica cordifolia Hook. California, Baldwin 1096 (UC) Arnica dealbata (A. Gray) B. G. Baldwin California, Baldwin 920 (JEPS) Arnica discoidea Benth. California, Baldwin 918 (JEPS) Arnica fulgens Pursh cult., Andreasen 303 (UPS) Arnica gracilis Rydb. 1 herb., California, Cronquist 8102 (UC) Arnica gracilis Rydb. 2 Wyoming, Baldwin 1393, (UC) Arnica griscomii Fernald ssp. frigida (Iljin) S. J. Wolf Alaska, Baldwin 1345 (UC) Arnica lanceolata Nutt. ssp. prima (Maguire) Strother Alaska, Baldwin 1336 (UC) & S. J. Wolf Arnica latifolia Bong. Alaska, Baldwin 1337 (UC) Arnica lessingii Greene Alaska, Baldwin 1338 (UC) Arnica lessingii Greene Alaska, Stensvold 4260* Arnica lonchophylla Greene herb., Alberta Breitung 13916 (S) Arnica longifolia D. C. Eaton 1 Washington, Baldwin 1101 (UC) Arnica longifolia D. C. Eaton 2 cult., Ekenäs 70 (UPS) Arnica louiseana D. C. Eaton Alberta, Andreasen and Ekenäs 407 (UPS) Arnica mallotopus Makino 1 Japan, Ono s.n. (UC) Arnica mallotopus Makino 2 Japan, Mizushima 12615 (S) Arnica mollis Hook. Oregon, Baldwin 1129 (UC) Arnica montana L. Sweden, Andreasen 304 (UPS) Arnica nevadensis A. Gray California, Baldwin 1093 (JEPS) Arnica ovata Greene 1 Washington, Baldwin 1104 (UC) Arnica ovata Greene 2 Alaska, Baldwin 1340 (UC) Arnica parryi A. Gray herb., Nevada, Howell et al. 50828 (CAS) Arnica rydbergii Greene Washington, Baldwin 1152 (UC) Arnica sachalinensis A. Gray cult., Andreasen 301 (UPS) Arnica sororia Greene herb., Nevada, Tiehm 12876 (CAS) Arnica spathulata Greene herb., California, Heckard 6283 (JEPS) Arnica unalaschcensis Less. herb., Russia, Joneson 1094 (WTU) Arnica venosa Hall. herb., California, Taylor 9856 (UC) Arnica viscosa A. Gray Oregon, Baldwin 1117, (UC) Outgroups Eatonella nivea A. Gray herb., Nevada, Taylor s.n. (JEPS) Hulsea algida A. Gray California, Baldwin 678 (JEPS) Raillardella argentea A. Gray California, Baldwin 625 (JEPS) Venegasia carpesioides DC. California, Baldwin 893 (JEPS) Accronyms of herbaria are according to Holmgren et al. (1990). Specimen were collected in the field except when indicated by ”cult.”: collected in Uppsala Botanical Garden, Sweden, or ”herb.”: from herbarium specimens. * DNA was sent by Jenny DeWoody at USDA Forest Service, Placerville, CA.

Fig. 1. RPB2 structure in Arabidopsis thaliana (modified from Popp and Oxelman 2004) on chromosome 4. Boxes represent exons and lines represent introns. The arrows flank the region between exon 17 and 23, which is amplified and sequenced in the present study.

Cloning and sequencing. PCR products were cloned using TOPO TA cloning® Kit for Sequencing (Invitrogen, Carlsbad, CA) and 10-18 clones were sequenced by Macrogen (Seoul, Korea) under BigDyeTM terminator cycling conditions. Forward and reverse sequences were reconciled using Sequencher version 3.1.1 (Gene Codes Corporation, MI). Alignment and phylogenetic analyses. Sequences were aligned using ClustalW2 (Labarga et al. 2007). Gaps of equal length and position shared by two or more taxa were considered homologous and binary coded as additional characters using the “simple gap coding” of Simmons and Ochoterena (2000). A fast stepwise addition bootstrap analysis (10 000 replicates) was completed for the aligned matrix consisting of all sequences using PAUP version 4.0b10 (Swofford 2002), which resulted in a tree with four well-supported clades. By comparing with published RPB2 sequences, three of the clades were identified as RPB2-d copies and one as the RPB2-i copy. Each RPB2-d paralogue was aligned in a separate matrix. When cloned sequences of the same specimen formed a monophyletic group in the fast stepwise bootstrap analysis described above, a single representative was included. Regarding outgroups, one representative sequence for each taxon was included for each paralogue. The span of variation within each paralogue and between paralogues was estimated by calculating pairwise distances between all sequences, using a maximum-likelihood substitution model to correct for multiple hits. A maximum parsimony phylogenetic analysis was carried out for each paralogue using a heuristic approach with 100 replicates, a maximum of 1000 trees saved per replicate and TBR swapping. A full bootstrap analysis (10 000 replicates, 100 replicates for each bootstrap replicate, with a maximum of 1000 trees saved per replicate and TBR swapping) was completed for each paralogue and with the MULTREES option off. Bayesian analyses were also performed for each RPB2-d paralogue using MrBayes v.3.1 (Huelsenbeck and Ronquist 2001). For all three paralogues, the GTR model with gamma-distributed rate variation across sites and a proportion of invariable sites resulted in the lowest AIC scores, as calculated by Mr.Modeltest v.2 (Nylander 2004). In two independent runs, the number of MCMC chains was set to four and trees were sampled every 100th generation until the standard deviation of the split frequencies of the two independent runs was below 0.01 (RPB2-dA: 800 000 generations, RPB2-dB: 3 200 000 generations, and RPB2-dC: 200 000 generations). For all Bayesian analyses the first quarter of the sampled trees was discarded as burnin.

Results A fast stepwise addition bootstrap analysis (10 000 replicates) of the total matrix (426 sequences) resulted in four clearly separated clades (Fig. 2). Three of the clades consist of RPB2-d copy sequences (here denoted as RPB2-dA, RPB2-dB and RPB2- dC). In a fourth clade RPB2-i copy sequences were detected (Andreasen et al., unpublished data, see Oxelman and Bremer 2000, Oxelman et al. 2004). Within the

4 RPB2-d clade, RPB2-dB and RPB2-dC constitute a sister group to RPB2-dA. Out- group taxa are present in all paralogues. Results of the separate analyses of the three RPB2-d copies are illustrated in Figures 3 (RPB2-dA), 4 (RPB2-dB), and 5 (RPB2- dC) respectively, with a summary of parameter values of the three analyses in Table 2. Comparisons by pairwise distances indicate that variation within Arnica is 0.1- 5.4% within paralogues and 18-24% between paralogues (Table 3).

Table 2. Comparison of separate analyses of the three RPB2-d paralogues and taxa in major supported clades for all paralogues. Parameters RPB2-d RPB2-d RPB2-d A B C Number of sequences (excluding outgroup) 70 55 22 Number of taxa (excluding outgroup) 25 25 18 Informative characters (excluding outgroup) 173 177 76 Region length of aligned matrix 15271 1599 15292 Informative characters/ region length 0.11 0.11 0.05 Number of most parsimonious trees 100 000 4208 63 CI (excluding uninformative characters) 0.68 0.66 0.68 RI 0.93 0.87 0.79 Clades uniting taxa with major support (bootstrap values > 80%): A. mallotopus and A. chamissonis X - - A. mallotopus, A. chamissonis, and A. lanceolata X - - A. lonchophylla and A. angustifolia X X - A. lonchophylla and A. gracilis - X - A. lonchophylla and A. rydbergii X - - A. lonchophylla, A. gracilis, A. rydbergii, and A. angustifolia X - - A. latifolia and A. cordifolia X X - A. lessingii and A. latifolia X X - A. lessingii and A. ovata - X - A. lessingii and A. nevadensis - X - A. lessingii, A. nevadensis, and A. chamissonis X - - A. lessingii, A. chamissonis, and A. mollis - - X A. lessingi, A. chamissonis, A. mollis, A. parryi, A. gracilis, and A. ovata X - - A. lessingii, A. latifolia, A. discoidea, A. spathulata, and A. cernua X X - A. sachalinensis and A. unalaschcensis - X X A. cernua and A. spathulata - X X A. griscomii and A. louiseana - X - A. discoidea and A. dealbata - X - A. longifolia, and A. parryi - X - A. longifolia, A. chamissonis, and A. mollis - - X 1 excluding a 249 bp insertion in A. longifolia. 2 excluding a 338 bp insertion in A. discoidea. 3 A parsimony analysis of RPB2-dC in which all trees per replicate were saved resulted in the same 6 most parsimonious trees.

Table 3. Variation between and within paralogues among Arnica sequences calculated by pairwise differences. Variation within paralogues (%) Variation between paralogues (%) RPB2-dA RPB2-dB RPB2-dC RPB2-dA and RPB2-dA and RPB2-dB and RPB2-dB RPB2-dC RPB2-dC 0.1-5.4 0.2-4.5 0.2-4.6* 18.1-23.9 20.0-23.2 12.4-15.8 *9.0% if A. viscosa is included

The RPB2-dA copy (Fig. 3) consists of 70 sequences of 25 Arnica species, with multiple representatives of seven species and one outgroup taxon (Raillardella argentea). Some taxa (A. angustifolia ssp. tomentosa, A. lonchophylla, A. latifolia, A. gracilis, A. lessingii, and A. chamissonis) have sequences that are part of multiple well-supported clades within the RPB2-dA paralogue tree.

5 1

RPB2-d A

100

100 RPB2-d B

100

100 RPB2-d C RPB2-i 99

30 changes Figure 2. Parsimony phylogram of the total matrix (426 sequences) illustrating the interrelationships between the three RPB2-d paralogues A, B, and C, and the RPB2-i copy. Bootstrap support values (fast step addition, 10 000 replicates) are indicated for branches uniting paralogues. The tree is rooted with the outgroup taxa of the RPB2-i copy.

The RPB2-dB copy (Fig. 4) consists of 55 Arnica sequences of 25 species with multiple representatives of five species and one outgroup taxon (Venegasia carpesioides). Sequences of four taxa (A. lonchophylla, A. lessingii, A. latifolia, and A. discoidea) belong to multiple well-supported clades in the tree. The RPB2-dC copy (Fig. 5) constitutes a sister group to RPB2-dB and contains 26 Arnica sequences of 20 species with multiple representatives of two species. Sequences of all four outgroup taxa are included in the RPB2-dC copy. Within RPB2-dC, sequences of only a single taxon (A. mollis) belong to two well-supported clades. Arnica viscosa (which is not represented in RPB2-dA or RPB2-dB) is not nested within the Arnica clade in this paralogue but diverges before Raillardella argentea - one of the outgroups. Basal nodes are unresolved or weakly supported in the trees resulting from parsimony and Bayesian analyses of RPB2-dA (Fig. 3) and RPB2-dB (Fig. 4). In RPB2- dC (Fig. 5), basal nodes are slightly better resolved and some taxa (A. viscosa, A. dealbata, A. spathulata, A. cernua, and A. discoidea) successively diverge from the main Arnica lineage basally in the tree. Sequences of different taxa united by strongly supported nodes (> 80% bootstrap support) in one or two paralogues are indicated in Table 2. Out of 20 well-supported taxon groups, six (30%) are present in two paralogues. In paralogues RPB2-dA and RPB2-dB, close relationships between sequences of A. angustifolia and A. lonchophylla, A. lessingi and A. latifolia, and A. latifolia and A. cordifolia are strongly supported. In each of the paralogues there is also strong support for a larger clade uniting sequences of A. lessingi, A. latifolia, A. discoidea, A. spathulata, and A. cernua. Similarly in RPB2-dB and RPB2-dC there is support for a group consisting of A. cernua and A. spathulata as well as A. sachalinensis and A. unalaschcensis. Arnica sachalinensis and A. unalaschcensis are also united in the RPB2-dA paralogue but with lower bootstrap support (63%). Six strongly supported clades in

6 each of the paralogues RPB2-dA and RPB2-dB, and two clades in RPB2-dC, unite taxa that do not form a group in any of the other paralogues, as listed in Table 2.

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 3. 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). 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 strict consensus tree. Branches uniting clades with bootstrap support > 80% are bold. Some of the strongly supported clades are indicated (see Table 2).

7 100 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 4. One of 4 208 most parsimonious trees resulting from a maximum parsimony analysis of RPB2- d B (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). 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 strict consensus tree. Branches uniting clades with bootstrap support > 80% are bold. Some of the strongly supported clades are indicated (see Table 2).

8 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 5. One of 6 most parsimonious trees resulting from a maximum parsimony analysis of RPB2-d C (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). 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 strict consensus tree. Branches uniting clades with bootstrap support > 80% are bold. Some of the strongly supported clades are indicated (see Table 2).

Discussion Multiple RPB2 paralogues. The two major RPB2 copy types (RPB2-d and RPB2-i), of which the d-copy is found in all land plants and the i-copy in some eudicot taxa, are probably a result of a duplication event near the origin of the ”core” eudicots (Oxelman and Bremer 2000, Oxelman et al. 2004, Luo et al. 2007). Multiple paralogues of RPB2-d in Hibiscus and Sidalcea have been explained as a result of a more recent duplication event within Malvaceae (Pfeil et al. 2004, Stone and An- dreasen in prep.) and in Silene as result of polyploidization (Caryophyllaceae; Popp and Oxelman 2001). In Arnica and outgroups we detected three RPB2-d copies (RPB2-dA, RPB2-dB, and RPB2-dC; Figs. 3-5) and one RPB2-i copy (Andreasen et al., unpublished data). The variation between RPB2-d copies calculated by pairwise distances, is at least twice the variation within each copy (Table 3) which adds additional support for the interpretation of the three copies as paralogues. Since at least one of the outgroup taxa (Venegasia, Hulsea, Eatonella, and Raillardella) are nested within each of the RPB2-d copies (Figs. 3-5) the two duplication events within the d-copy must have occurred prior to the divergence of Venegasiinae, Hulseinae, Arnicinae, and Madiinae in Madieae. In a phylogenetic study of helenioid Heli- antheae, 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 outgroups. Utility of RPB2 at lower taxonomic levels. Compared with earlier studies involving ribosomal nuclear DNA and chloroplast sequence data in Arnica, the percentage of informative characters (excluding the outgroup) for the RPB2-d regions (5-11%) is in between that of chloroplast regions (1-2%) and ribosomal nuclear regions (14- 15%). However, the variation within introns of RPB2 are higher since the sequenced

9 region contains many exons with little variation. Retention indicies of the RPB2-d copies (0.79-0.93) are higher compared to that of earlier analyses of ITS and ETS (0.66) and chloroplast regions (0.73), implying that there is less homoplasy in RPB2-d compared to ribosomal and chloroplast regions in Arnica. Low levels of homoplasy (retention indices > 90) have also been found in other studies involving RPB2 (Popp and Oxelman 2001, Pfeil et al. 2004, Lou et al. 2006), which could indicate a history of less recombination in RPB2 compared to other regions such as ITS and ETS. The combination of low levels of homoplasy and relatively high sequence variation suggests that RPB2 introns are suitable for investigating interrelationships in low-level taxa. Polymorphism and incomplete lineage sorting. Basal nodes are generally weakly resolved in RPB2-d A and RPB2-d B, and slightly more resolved in RPB2-d C. Bayesian posterior probabilities are generally higher compared to bootstrap support values, which has also been shown for true clades in simulation studies by Erixon et al. (2003). Of the strongly supported groups, 30% are present in more than one RPB2-d paralogue. Sequences of some taxa are polymorphic and end up in more than one highly supported group in the trees (Figs. 3-5; A. lessingii, A. lonchophylla, A. latifolia, A. discoidea, A. gracilis, A. chamissonis, A. angustifolia, and A. mollis; Table 2). This has not previously been reported in studies of the RPB2 region for other species. A similar pattern of polymorphism with cloned sequences of a single taxon distributed in multiple well supported clades with different mixtures of other taxa was also found in nrDNA (Ekenäs et al. manuscript). Barker (1966) showed that there is a correlation between polyploidy and agamospermy in Arnica and he found that the strictly diploid species are all amphimictic. Similarly we detected no polymorphism in the strictly diploid species A. montana, A. acaulis, A. unalaschcensis, A. sachalinensis, A. fulgens, A. sororia, A. cernua, A. viscosa, and A. venosa included in our analyses. It is possible that polyploidy and agamospermy in combination with incomplete lineage sorting is the cause of polymorphism and the clustering of some accessions with different taxa within paralogues. Hybridization could also be a contributing factor to these complex patterns in Arnica. Congruencies with earlier phylogenetic studies of Arnica. The complex evolutionary history in Arnica is evident from the RPB2 results, as well as from earlier results of phylogenetic studies of the genus using ITS, ETS, and chloroplast regions (Ekenäs et al. 2007, Ekenäs et al. manuscript). Low resolution and weak support for basal branches in the RPB2 region is consistent with earlier studies and could be due to rapid evolution and diversification of these taxa in the early history of Arnica. A general trend that agrees with earlier studies is that sequences present in basal branches mostly constitute taxa of subgenera Andropurpurea and Austromontana. All basally diverged Arnica taxa in the RPB2-dC paralogue are Austromontana sequences, which supports Baldwin’s (2002) hypothesis that Arnica originated in temperate western North America where some Arnica species have a limited distribution and where subtribe Madiinae, sister to Arnica, occurs. Sequences of taxa belonging to subgenus Chamissonis are only present in branches diverging further up in the trees in all paralogues, which is also true for earlier studies based on cpDNA and nrDNA regions. Taxa of subgenus Chamissonis (e.g., A. chamissonis, A. parryi, A. longifolia, and A. lanceolata) possess more diverse sesquiterpene lactone chemistry compared to taxa of subgenera Andropurpurea, Arctica and Aus- tromontana (Willuhn et al. 1995, Ekenäs et al. manuscript), which can be interpreted as additional support for the circumscription of this subgenus.

10 Strongly supported groups correlated to earlier hypotheses. Close to half of the well supported clades in all paralogues (Table 2) unite taxa treated as closely related in earlier taxonomic treatments. Some taxa within subgenus Arctica (Maguire 1943, Downie and Denford 1988) are clustered with high support, e.g., A. lonchophylla and A. angustifolia (in paralogue RPB2-dA and RPB2-dB) and A. lonchophylla and A. rydbergi (in paralogue RPB2-dA). Arnica griscomii and A. louiseana, united in a well supported group in RPB2-dB, have previously been recognized as one species (A. louiseana ssp. louiseana, ssp. griscomii, and ssp. frigida (not included in this study); Downie and Denford 1986). Arnica latifolia and A. cordifolia (in RPB2-dA and RPB2-dB) have been suggested to be closely associated within Austromontana (Wolf and Denford 1984a). It has been proposed that A. gracilis is a hybrid of these two taxa (Wolf and Denford 1984b), but there is no support for this in the present study or in earlier studies based on cpDNA, nrDNA or secondary chemistry (Ekenäs et al. 2007, Ekenäs et al. manuscript). Also within Austromontana, A. spathulata and A. cernua (sisters in RPB2-dB and RPB2-dC) are both endemics of the Klamath region and found in serpentine habitats. Arnica discoidea and A. delabata (= Whit- neya dealbata; Baldwin 1999), also within subg. Austromontana, form a clade in RPB2-dB. Two clades of subg. Chamissonis sequences consist of A. longifolia, and A. parryi in RPB2-dB, and A. longifolia, A. chamissonis, and A. mollis in RPB2-dC. Finally, a clade of A. sachalinensis and A. unalaschcensis is present in all three paralogues (although moderately supported in RPB2-dA). These taxa belong within subg. Andropurpurea (as well as A. lessingii) and are easily recognized by having purple rather than yellow anthers. Whereas the groups described above unite sequences of the same earlier proposed subgenera that have been proposed to be closely related based on morphology, accessions of a few taxa (e.g., A. lessingii, A. gracilis, A. lonchophylla, A. chamissonis, and A. mallotopus) form groups with taxa presumed to be more distantly related. Interestingly, some of these taxa (A. gracilis, A. lonchophylla and A. chamissonis) were also retrieved in unexpected positions and/ or in conflicting positions in trees of earlier analyses based on chloroplast and ribosomal DNA data (Ekenäs et al. 2007). Again, this could be explained by incomplete lineage sorting in these taxa, possibly coupled to hybridization events. Arnica viscosa is not nested within Arnica in RPB2-dC. One taxon, Arnica viscosa, deserves special attention since it is not nested within Arnica in RPB2-dC (Fig. 5) and was not detected in any of the other two RPB2-d copies. A member of the tarweed genus, Raillardella argentea, diverges in a clade after A. viscosa, resulting in a non-monophyletic Arnica in the RPB2-dC tree. Bayesian analyses, more robust to rate heterogeneity, gave the same results as parsimony analyses, leading us to conclude that long branch attraction is not likely the cause of this pattern. In nrDNA analyses (Ekenäs et al. 2007), A. viscosa is clearly nested within Arnica, where it forms a moderately supported group with A. nevadensis and A. dealbata of subgenus Austromontana - the same subgenus in which it has been placed based on morphology (Maguire 1943, Wolf and Denford 1984a). 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 Raillardella. To summarize, although the evolutionary history of Arnica is complex, congruencies with topologies based on nrDNA and secondary chemisty in Arnica were

11 observed in the trees of the three RPB2-d paralogues. Whereas sequences of a few taxa are widely distributed in the tree, some well supported groups that are in agreement with earlier hypotheses of interrelationships in Arnica are also observed.

Acknowledgements We thank B. G. Baldwin and M. Stensvold for collecting and sending material, S. Baldauf B. Oxelman, L. Tibell, M. Thulin, and S. Aagaard for valuable comments on the manuscript, the Royal Academy of Sciences and the Swedish Research Council for grants to K. Andreasen.

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