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The region coding for the second largest subunit of RNA polymerase II (RPB2) was explored for resolving interspecific relationships in Arnica and lower level taxa in general. The region between exons 17 and 23 was cloned and sequenced for 33 accessions of Arnica and four outgroup taxa. Three paralogues of the RPB2-d copy (RPB2-dA, B and C) were detected in Arnica and outgroup taxa, indicating that the duplications must have occurred before the divergence of Arnica. 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. Low levels of homoplasy in combination with relatively high sequence variation indicates that the introns of the RPB2 region could be suitable for phylogenetic studies in low level taxonomy.

To investigate possible congruencies between DNA sequence data and secondary chemistry, we compared nuclear ribosomal DNA (nrDNA) sequence data, sesquiterpene lactone (STL) contents, and cytometric data from 35 accessions of 16 Arnica (Asteraceae) species and two outgroup taxa (Layia hieracioides and Madia sativa), using phylogenetic inference and principal component analysis (PCA). 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 have been reported from subtribe Madiinae.

Several species in the genus Arnica have been used in traditional medicine to treat inflammatory-related disorders. Extracts of twelve Arnica species and two species closely related to Arnica (Layia hieracioides and Madia sativa) were investigated for inhibition of human neutrophil elastase release and 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, 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 interesting to evaluate further.

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.