Arnica montana subsp. atlantica: Really a subspecies?

Arnica montana subsp. atlantica: Really a subspecies? Genet Resour Crop Evol https://doi.org/10.1007/s10722-018-0653-2 RESEARCH ARTICLE . . . Corinna Schmiderer Paula Torres-Londono Andrea Lutz-Röder Virginia K. Duwe Johannes Novak Received: 22 January 2018 / Accepted: 8 May 2018 © The Author(s) 2018 Abstract In Arnica montana L. (Asteraceae) two corrected. Genetically, AMA separated very well subspecies are described, A. montana subsp. atlantica from AMM with a G between the subspecies of ST (AMA), present only on the Iberian Peninsula and A. 0.81, genetically justifying the subspecies concept of montana subsp. montana (AMM) with a very wide A. montana. Genetic variability in AMA (H =0.28) exp distribution area. The morphological differences was lower than in the AMM populations (H =0.70). exp between the two subspecies are small and variable. A somewhat higher fixation index of AMA (F = ST Therefore, this concept is sometimes questioned. To 0.17, compared to an F =0.08 for AMM) may ST establish the genetic background of the two sub- indicate that geneflow in AMA is a bit more restricted species, populations of AMA and AMM together than in alpine AMM. However, the fixation index of with herbarium samples and DNA Bank material of AMA is not deviating from Hardy–Weinberg equi- AMM were tested with 12 microsatellite markers. A. librium. No inbreeding was observed for AMA (F = IS montana propagates by seeds or by clonal propaga- 0.10) and AMM (F =0.08). IS tion of its rhizome. In AMA, clonality was frequent while in AMM only one case of clonality could be Keywords Arnica montana · identified. Therefore, further results were clone- Arnica montana subsp. atlantica · Arnica montana subsp. montana · Genetic analysis · Microsatellites C. Schmiderer · J. Novak (&) Institute for Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria Introduction e-mail: Johannes.Novak@vetmeduni.ac.at Arnica L. (Asteraceae, Heliantheae s.l.) is a circum- P. Torres-Londono Kra¨utermix GmbH, Wiesentheider Str. 4, 97355 boreal genus of about 30 species mostly of montane Abtswind, Germany habitats. Arnica montana L. (Asteraceae, mountain arnica, wolfs’ bane) is a perennial, facultative A. Lutz-Ro¨der apomictic species (Yankova-Tsvetkova et al. 2016), Kneipp GmbH, Winterha¨user Str. 85, 97084 Wu¨rzburg, predominantly self-incompatible, insect pollinated Germany which reproduces sexually with seeds and vegeta- V. K. Duwe tively with short rhizomes (Luijten et al. 1996, 2000). Botanischer Garten und Botanisches Museum Berlin- A. montana grows on acidic grass- and shrublands Dahlem, Freie Universita¨t Berlin, Ko¨nigin-Luise-Straße and is distributed from the Iberian Peninsula to the 6-8, 14195 Berlin, Germany 123 Genet Resour Crop Evol Ukraine (Maurice et al. 2012). Bolos y Vayreda scale genetic study of Duwe et al. (2017) on AMM. (1945) distinguished two subspecies, A. montana Furthermore, in recent decades a significant decline subsp. atlantica A. Bolos (AMA) and A. montana in the populations of arnica were also observed in subsp. montana (AMM), where AMA is present only Galicia (Lange 1998; Romero et al. 2011). in SW-France, N-Spain and Portugal. AMA is Cultivation of A. montana subsp. montana is morphologically different in its smaller height, thin- possible, but not without problems. The species need ner floral stems, lanceolate leaves and smaller flower a loose, well-aerated soil with an ample supply of heads with fewer bracts. This subspecies differs from water and a lime content of less than 1%. Otherwise, AMM also in its habitat preferences, it occurs the plant reacts immediately with chlorosis. Seed between 0 and 440 m (max. 1000 m) in areas of germination is another difficulty in cultivation (von oceanic climate while AMM occurs from 0 to Raison et al. 2000). 3000 m. This subspecies concept was recently Since lowland proveniences of AMA are signifi- questioned by the analysis of biometrical data from cantly different in their chemical composition from different populations in Galicia where the only AMM, it would be interesting to know if the genetic significant difference between low- and highland distance justifies the proposed division into two plants was found in plant height (Romero et al. 2011). subspecies. To address this question, individuals from The two major proveniences of arnica flowers for NW Spain and Central Europe were classified as subspecies AMA or AMM according to the criteria the pharmaceutical/cosmetics industry in Europe are the Romanian Carpathians and NW Spain (Galicia) defined by Bolos y Vayreda (1945) and compared (Vera et al. 2016). Mountain arnica is an old folk with a set of microsatellite markers recently pub- medicine still popular for the treatment of pain, lished by Duwe et al. (2015). swelling and bruises. Due to its topical application of flower extracts in gel or cream form it is regarded as ‘cosmeceutical’ (Baumann 2007). Sesquiterpene lac- Materials and methods tones (SL) are responsible for the anti-inflammatory activity of arnica (Wagner et al. 2004) with helenalin Sample material esters (H) showing higher anti-inflammatory activity than dihydrohelenalin esters (DH) (Klaas et al. 2002) In total a sample set of 89 individuals was analysed while DH are less allergenic than H (Lass et al. and classified according to Bolos y Vayreda (1945)as 2008). Lowland arnica (AMA) is a DH-chemotype, A. montana subsp. montana (33 samples, AMM) or A. while AMM possesses predominantly helenalin esters montana ssp. atlantica (55 samples, AMA). One (cf. for example chromatograms in the European sample of A. chamissonis Less. was used as outgroup Pharmacopoeia where the DH-chemotype is (Table 1). The samples were obtained from the described as the ‘Spanish type’, while the Hele- herbarium of the University of Vienna (WU), nalin-chemotype is called ‘East-European type’ collected from the wild in 2016 (aerial plant parts (EDQM 2014)). only) and were obtained from the DNA Bank of the In general, A. montana is so abundant in many Botanic Garden and Botanical Museum Berlin- countries that The IUCN Red List of Threatened Dahlem (BGBM) (see Table 1). All tissue samples Species classifies A. montana as “Least Concern”, from the BGBM and the underlying voucher speci- although monitoring population trends is suggested mens are deposited at the Botanic Garden and due to the decline in some countries (Falniowski et al. Botanical Museum Berlin and are available via the 2011). Following this suggestion, Luijten et al. Global Genome Biodiversity Network (GGBN) (1996, 2000) studied the effect of habitat fragmen- (Droege et al. 2016) and the Global Biodiversity tation on population structure of AMM in the Information Facility (GBIF). Specimens collected in Netherlands and found reduced levels of genetic Spain were deposited in the herbarium of Kra¨uter- variation and limited gene flow between the popula- Mix, specimens collected in Austria in WU. tions. A strong genetic differentiation and a suggested restricted gene flow with signs of genetic erosion in lower altitudes were also recently found in the large- 123 Genet Resour Crop Evol Table 1 Locality and specimen information of reference samples used in this study Pop. n ng Species Origin (location; elevation in m.s.m., GPS coordinates; collection date) ES01 10 8 AMA Spain, Galicia, 0.4 km NNE of Vilouris; 480 m, 43°12.30 N, 8°2.33′W; 2016-06-28 ES02 10 6 AMA Spain, Galicia, 2.6 km NW of Vilouris; 482 m, 43°12.70′N, 8°04.12′W; 2016-06-28 ES03 10 3 AMA Spain, Galicia, 1.4 km SW of Vilouris; 513 m, 43°11.56′N, 8°03.10′W; 2016-06-28 ES04 5 1 AMA Spain, Galicia, 3 km NNE of Xermade; 451 m, 43°22.97′N, 7°48.45′W; 2016-06-29 ES05 10 4 AMA Spain, Galicia, 3.3 km WNW of Susana; 705 m, 43°28.52′N, 7°40.40′W; 2016-06-29 ES06 10 6 AMA Spain, Galicia, 1.5 km NNE of Susana; 607 m, 43°28.86′N, 7°37.48′W; 2016-06-29 OG01 1 1 ACH WU: Austria, Vienna, cultivated at HBV; 1995-09-04 CE01 10 9 AMM Austria, Styria, Steinplan; 1640 m, 47°9.73′N, 14°54.09′E; 2016-07-21 CE02 10 10 AMM Austria, Carinthia, 6 km N of Millstatt, Hansbauerhutte; 1720 m, 46°51.47′N, 14°53.10′E; 2016-07-24 CE03 1 1 AMM WU: Switzerland, Grisons, NW Ravaisch; 2004-08-22 CE04 1 1 AMM WU: Italy, S-Tyrol, Central Alps, Passeier, Platt; 1995-07-10 CE05 1 1 AMM WU: Austria, E-Tyrol, Defereggen, Oberberg, N St. Jakob; 1987-08-04 CE06 1 1 AMM WU: Austria, Carinthia, Hohe Tauern, Hafnergruppe; 2003-07-07 CE07 2 2 AMM WU: Austria, Lower Austria, SW Waldviertel; 2009-06-05 AMM WU: Austria, Lower Austria, Waldviertel, Langsehschlag; 1913-06-05 CE08 1 1 AMM WU: Austria, Vienna, NW-Plateau of Sophienalpe; 1950 CE09 1 1 AMM BGBM (BGT 0008920): Germany, Saxony, Oelsen, Osterzgebirge; 632 m, 50°47′N, 13°56′E; 2013-06-03 CE10 1 1 AMM BGBM (BGT 0012009): Germany, Mecklenburg-Western Pomerania, Zarrendorf, Stralsund; 0 m, 54°14′N, 13°05′E; 2013-06-17 CE11 1 1 AMM BGBM (BGT 0013108): Germany, Brandenburg, Naturpark Niederlausitzer Heidelandschaft; 97 m, 51°30′ N, 13°46′E; 2013-06-20 CE12 1 1 AMM BGBM (BGT 0011921): Italy, S-Tyrol, 3 km ENE of Badia; 2062 m, 46°37′N, 11°56′E; 2013-06-29 CE13 1 1 AMM BGBM (BGT 0013144): Germany, Baden-Wu¨rttemberg, Black Forest; 1424 m, 47°52′N, 8°01′E CE14 1 1 AMM WU: France, E-Pyrenees, Superbolquere; 1750 m; 1944-07-17.-20 Pop. … population, n … number of sampled individuals per population, ng … number of individuals with different multilocus genotypes (genets), AMA … Arnica montana subsp. atlantica, AMM … A. montana subsp. montana, ACH … A. chamissonis, BGBM … Botanical Garden and Botanical Museum Berlin-Dahlem, Germany, HBV … Botanical Garden of the University of Vienna, Austria, WU … Herbarium of the University of Vienna, Austria Extract for HPLC analysis of sesquiterpene After shaking for 120 s the mixture was filtered lactones through a folded filter into a 100 mL flask. The filtrate was brought to dryness in vacuo and re-suspended in Extraction for the analysis of sesquiterpene lactones 3.0 mL of a mixture of equal volume parts of MeOH was performed using a modification of the European and H O. After filtration, the solution was used for Pharmacopoeia Monograph Arnica flower (EDQM HPLC analysis. 2014). In detail, dried flowers (approx. 5 g) were milled. 1.0 g of the powdered drug was weighed HPLC analysis exactly into a 250 mL flat bottom flask. After addition of 2.0 mL internal standard solution containing 1 mg/ HPLC analysis of sesquiterpene lactones were per- mL santonin in MeOH, immediately prepared prior to formed using an Alliance 2695 high pressure gradient use, and 50 mL MeOH, the mixture was extracted for system (Waters GmbH, Eschborn, Germany) 1 h using a reflux condenser. The cooled solution was equipped with a DAD detector. The following centrifuged for 15 min at 4500 rpm. To the super- parameters were applied: column, Merck Superspher natant, 7 g of neutral aluminium oxide was added. 100 RP 18e 12594mm (4 µm particle size); guard 123 Genet Resour Crop Evol column, Merck LiChrospher 100 RP 18e, 494mm For 15 μL PCR reactions, 10 ng of genomic DNA (5 µm particle size); mobile Phase A, H O; mobile was added to a master mix containing 19PCR buffer Phase B, MeOH; flow rate, 1.2 ml/min; injection B, 2 mM MgCl , 200 μM dNTPs (each), 0.6 U Taq volume, 20 µL; detection wavelength 225 nm; oven HOTFIREPol DNA Polymerase (all reagents from temperature, 20 °C; isocratic 0–3 min 38% B; linear Solis BioDyne, Tartu, Estonia), 200 nM fluorescent gradient 3–20 min 45% B; isocratic 20–30 min 45% labelled forward primers, 50 nM locus specific B; linear gradient 30–55 min 55% B; linear gradient forward primers and 250 nM locus specific reverse 55–57 min 100% B, 70 min stop. The assignment of primers. Samples with no or insufficient amplification chemotypes was deduced by comparing the chro- were repeated with different DNA amounts (0.25– matograms with the chromatograms in the European 10 ng) and 0.9 U polymerase. The PCR conditions Pharmacopoeia representing the two chemotypes included a denaturation step at 95 °C for 15 min, (‘Spanish Type’ and ‘East European Type’) (EDQM followed by 30 cycles at 95/58/72 °C for 30/45/45 s, 2014). 15 cycles at 95/53/72 °C for 30/45/45 s, and a final elongation step at 72 °C for 10 min. PCR products DNA extraction were checked on 2% agarose gels stained with PeqGreen (VWR International, Vienna, Austria; Genomic DNA was extracted from air dried speci- 4 µL/100 mL agarose solution; products including mens using a modified CTAB-protocol (Schmiderer 6-FAM) or without staining (PCR products including et al. 2013) based on Doyle and Doyle (1990). DNA ATTO dyes). Six amplified loci per sample (1 µL concentrations of the extracts were determined using PCR product each; Table 2) were mixed and diluted a NanoDrop ND-2000c (Peqlab Biotechnologie with 24 µL ddH O. The determination of the GmbH, Erlangen, Germany). DNA extracts were sequence lengths was performed by Microsynth diluted with Milli-Q water to 5 ng/µL. (Balgach, Switzerland) using GeneScan™ 500 LIZ™ dye size standard (Thermo Fisher Scientific, Microsatellite analysis Waltham, MA, USA). The obtained chromatogram files were edited using Peak Scanner 2.0 software Microsatellite markers and part of the primer (Applied Biosystems, Waltham, Massachusetts, sequences were adopted from Duwe et al. (2015). USA). Remade primers with an optimum melting tempera- ture ranging from 51 to 53 °C were designed using Statistical analysis Primer Express 2 (Applied Biosystems, Foster City, California, USA). Primer dimers were evaluated The number of multilocus genotypes (MLG) stan- using NetPrimer software (http://www.premier dardized by sample numbers, Stoddard and Taylors’ biosoft.com/netprimer). Multiplexing of different index of MLG diversity (Stoddart and Taylor 1988), loci was performed using Multiplex Manager Simpsons’ index (Simpson 1949) corrected by N/(N- (www.multiplexmanager.com). 1), Evenness, E.5 (Gru¨nwald et al. 2003), Nei’s PCR amplification was performed using tailed expected heterozygosity (H ), Nei’s genetic dis- Exp locus-specific forward primers, fluorescence labelled tances and a Neighbor-joining tree were calculated nested forward primers (5′ modified with 6-FAM, using R 3.3.0 (R Core Team 2016) with poppr 2.2.0 ATTO532, ATTO550 or ATTO565) binding to the (Kamvar et al. 2014, 2015). For more detailed forward primer extensions and “PIG-tailed” reverse population analysis, accessions with just one sample primers to reduce stutter bands (with 3–4 bp exten- (i.e., herbarium specimen) were excluded. Hence, sions to achieve a GTTT consensus sequence at the two Austrian AMM populations were compared to 5′-end, according to Brownstein et al. 1996). Unla- the six Spanish AMA populations. Putative clonality belled and 6-FAM labelled primers were obtained and AMOVA were calculated with Genalex 6.5 from Sigma-Aldrich (Vienna, Austria), all ATTO (Peakall and Smouse 2006, 2012). Putative clonality labelled primers were obtained from Microsynth was determined by multilocus genotypes (MLG). (Vienna, Austria). Individuals with the same MLG may either be parts of a clone (ramets of a genet) or—after sexual 123 Genet Resour Crop Evol Table 2 Primers used for analysis Primer name T Primer sequence Repeat motif* allele size Analysis range reaction Arm02(565) F 58* gaatcaccatcgtcgcatAACACACATCCACGTTTGGC TACA 2 Arm02 R 58* gtttAACCGTGCATCATTCTGTGG 190-274 Arm03(532) F2 52 tgtaaaacgtcggcgactCAAAAACCCTAATTCTCCATC TACA 2 Arm03 R2 52 gtttCTGCGCAATGGGTTTACT 109-145 Arm05(532) F 58* tgtaaaacgtcggcgactACTGTCACCTAGGGGTGTTC AACA 2 Arm05 R 58* gttTAAGCGGGGAGTCTTTCTGG 186-206 Arm06(550) F 58* ccaagtagggcggtatctTGTCGCCTCAATCCTTGGTG ACAT 1 Arm06 R 58* gtttGCTGAAGTCCTTCCTTGGAC 119-271 Arm07(M13) F 58* tgtaaaacgacggccagtACATGACGCAAAAAGCGTAG TATG 2 Arm07 R 58* gtttCCATGTTACCACCATGTCGC 211-251 Arm08(M13) F 58* tgtaaaacgacggccagtAGATGAGGTTCTTGCAGCATC TGTA 2 Arm08 R 58* gttTGCTTGCAGTTGAAGTAAAGGG 134-180 Arm09(565) F 58* gaatcaccatcgtcgcatTAGGCGTGAGTTTGTACTCG TATG 1 Arm09 R 58* gtttAAGCGTGTTAACTTCGTGAG 236-264 Arm10(565) F 58* gaatcaccatcgtcgcatACCAGCTGACTCTCTTTCCG CATA 1 Arm10 R 58* gtttCAAGGATGAACATCGGCCTC 147-207 Arm11(532) F2 52 tgtaaaacgtcggcgactGCACAAGGTATGTGTTGCA GT 1 Arm11 R 58* gttTCTTCGACCGAATGTTTTCACC 167-183 Arm12(550) F2 52 ccaagtagggcggtatctCTTGCTTCTTCTCTTTATAGATGTC AG 2 Arm12 R2 52 GGTTACCATTTTGGGTTCA 96-126 Arm13(532) F (= 58* tgtaaaacgtcggcgactGGTTTGAACACGAGATAGCG AT 1 Armo02 F*) Arm13 R (=Armo02 58* gtttACAAACTTCCTGTTGTCCCG 224-254 R*) Arm14(M13) F (= 58* tgtaaaacgacggccagtTCAAACAGTCACCAGCAACC ACCTGG 1 Armo03 F*) Arm14 R (=Armo03 58* gtttCAGAGGCTGCAACCCTAATG 213-241 R*) M13-FAM 53 [6FAM]-TGTAAAACGACGGCCAGT ATTO532 53 [ATTO532]-TGTAAAACGTCGGCGACT ATTO565 53 [ATTO565]-GAATCACCATCGTCGCAT ATTO550 51 [ATTO550]-CCAAGTAGGGCGGtATCT Capital letters of the primer sequences indicate the locus specific sequences; small form letters indicate artificial primer tails. The microsatellite repeat motif was published by Duwe et al. (2015). The allele size range was obtained with the used sample set including Arnica chamissonis *Allele specific primer sequence and T according to Duwe et al. (2015) Primer melting temperatures without asterisks were calculated with Primer Express 2, excluding primer tails reproduction—equal by chance. Briefly, the program separately for AMA and AMM with the Codom- estimates the probability (P ) of the occurrence of Allelic distance with 999 permutations. The division sex an MLG in a randomly mating population and gives allowed us a closer insight into the structure of the statistical significance levels based on observed allele two subspecies that would have been covered by the frequencies. Analysis of Molecular Variance high genetic distance between the two subspecies. (AMOVA) (Excoffier et al. 1992) was calculated 123 Genet Resour Crop Evol Results ramet of a genet or sexually reproduced and equal by chance. Of the 75 samples in the population sample In total 6 populations of lowland Arnica montana subset only 45 MLG could be detected. 10 MLG were from Spain with 5–10 individuals each were classi- present in multiple copies, all of them with a P - sex fied as A. montana ssp. atlantica (AMA) according value lower than 0.05 indicating that the probability the proposed criteria by Bolos y Vayreda (1945) and of equal MLG by sexual reproduction is rather low were compared to two populations of A. montana ssp. and clonality is more likely. Apart from one genet in montana (AMM) from Central Europe (Austria) with AMM with only two ramets all other 9 genets were 10 individuals each. This sample set was comple- found in AMA. Subsequently, only one ramet of a mented by a geographically wide range of individual genet was left in the sample set for further analysis samples from Germany to Northern Italy and the (Table 4). East-Pyrenees (France). All AMA plants belonged in their sesquiterpene lactone profile to the ‘Spanish Genetic difference between AMA and AMM type’, while AMM plants were of the ‘East-European type’ (EDQM 2014). The genetic study with 12 The results show a clear genetic distinction of microsatellite loci showed between 6 and 15 different Spanish AMA from Central European AMM indi- alleles, the expected heterozygosity H ranged from viduals (G =0.81, Table 3, Fig. 1). Although the exp ST 0.46 to 0.82 (mean=0.61) and the mean evenness genetic variability was much smaller in AMA, the ranged from 0.37 to 0.86 (mean=0.58) (Table 3). separation of populations within this group is far better supported than amongst AMM. Especially the Clonality population from Xermade (ES04) is distinctively different, but also the other two population groups Arnica montana has two propagation strategies, a from Susana (ES05) and Vilouris (ES03) are well sexual strategy (a facultative apomictic species with separated from each other, indicating limited gene predominant sexual reproduction (Yankova-Tsvet- flow between AMA populations. kova et al. 2016)) and an asexual strategy by AMM samples were only in some cases grouped rhizomes (Sugier et al. 2013). To avoid clonal by their geographic distance. Samples from Styria influence on the estimation of variability, probabil- (CE01) and Carinthia (CE02) are geographically ities of equal multilocus genotypes (MLG) were close, as well as from Vienna (CE08) and Lower estimated that an individual of a MLG was either a Austria (CE07) and from E-Tyrol (CE05) and Table 3 Characteristics of the microsatellites used in this study different alleles; G … proportion of genetic diversity that ST (Alleles … number of different alleles detected; H … resides among the two subspecies) Exp expected heterozygosity; evenness … distribution of the Locus Alleles H Evenness G between AMA and AMM Exp ST Arm14 8 0.53 0.67 0.993 Arm11 6 0.54 0.74 0.152 Arm13 9 0.64 0.53 0.829 Arm06 15 0.57 0.41 0.690 Arm10 15 0.58 0.37 0.939 Arm09 8 0.67 0.63 0.998 Arm08 10 0.60 0.62 0.343 Arm07 12 0.57 0.43 0.976 Arm03 7 0.46 0.60 0.898 Arm05 6 0.68 0.86 0.898 Arm12 14 0.82 0.68 0.983 Arm02 13 0.64 0.48 0.991 mean 10.25 0.61 0.58 0.808 123 Genet Resour Crop Evol Population structure Table 4 Characteristics of AMA and AMM (n … number of samples, eMLG … number of multilocus genotypes standard- ized by sample numbers, G … Stoddard and Taylors’ index of To compare population structures between AMA and MLG diversity, lambda … Simpsons’ index corrected by N/(N- AMM populations, populations with just one indi- 1), E.5 … Evenness, H … Nei’s expected heterozygosity) Exp vidual (herbarium samples) were excluded. So, two Subspecies n eMLG G lambda E.5 H exp AMM populations from Austria were compared to the six Spanish AMA populations. The linear Before clone correction distance of the two Austrian populations was AMA 55 13.2 13.7 0.736 0.726 0.249 107 km while the linear distance between the most AMM 20 19 18.2 0.986 0.973 0.657 distant AMA populations was 47 km. The number of Total 75 15.9 23.1 0.970 0.69 0.518 expected multilocus-genotypes (eMLG), the stan- After clone correction dardized MLG for unequal sample numbers, as well AMA 28 9.76 24.5 0.959 0.965 0.278 as Simpsons’ index and evenness were almost AMM 32 10 32 0.969 1 0.702 identical between AMA and AMM (Table 4). Total 60 9.95 57.2 0.983 0.982 0.704 Expected heterozygosity (gene diversity) was low in AMA (H =0.28) and high in AMM (H =0.70). exp exp S-Tyrol (CE04). The German populations (Saxony, These results were also reflected in AMOVA anal- Brandenburg, Mecklenburg, Baden-Wu¨rttemberg), ysis. Both subspecies, analysed separately, showed however, grouped separately with completely differ- here significant variation between populations ent geographical locations, but bootstrap support is (Table 5). However, AMA populations differed to a generally very weak in the AMM group (Table 1; higher degree from each other than the two AMM Fig. 1). Fig. 1 Neighbor joining tree based on Nei’s genetic distances of Arnica montana of Spanish (ES) AMA, and Central European (CE) AMM samples. Arnica chamissonis was used as outgroup (OG) 123 Genet Resour Crop Evol Table 5 Results of analysis of molecular variance (AMOVA), calculated separately for AMA and AMM Source df SS MS Est. Var. % F value P value AMA Among populations 4 18.721 4.680 0.294 17 F 0.171 0.001 ST Among individuals 22 34.742 1.579 0.151 9 F 0.105 0.074 IS Within individuals 27 34.500 1.278 1.278 74 F 0.258 0.002 IT AMM Among populations 1 10.050 10.050 0.312 8 F 0.075 0.001 ST Among individuals 17 70.450 4.144 0.322 8 F 0.084 0.020 IS Within individuals 19 66.500 3.500 3.500 85 F 0.153 0.001 IT populations did (17% of variation among populations two chloroplast markers. Genetic variability in Dutch in AMA compared to 8% in AMM, indicating a AMM populations (H =0.09) (Luijten et al. 2000) exp higher degree of panmixis in AMM). The degree of were even much lower than in AMA from this study inbreeding (variation among individuals) was almost (H =0.28). As in AMA (F =0.17), the Dutch exp ST equal for AMA and AMM (F =0.105 (P=0.07) and populations showed moderately significant popula- IS F =0.084 (P=0.02*) for AMA and AMM, tion differentiation (F =0.14). IS ST respectively). Clonality Discussion The elevated level of clonality in AMA is either an indication of negative influences on sexual reproduc- The subspecies concept tion or more favourable conditions for vegetative growth. Many reasons can negatively influence seed The subspecies concept in A. montana was recently propagation. Decreased pollination and seed devel- questioned by morphological analysis of an extensive opment, low seed longevity and poor possibilities for sample set (Romero et al. 2011) where the authors seeds to germinate in densely covered vegetation found that the defined criteria to distinguish AMA (competition) may be reasons linked to flower and from AMM were highly variable not allowing a clear seed biology. Attacks on and diseases of floral tissues distinction. However, AMA was genetically highly caused e.g., by herbivore slugs and fruit flies distinguishable from AMM in our microsatellite specialized on A. montana (Tephritis arnicae L., study. Vera et al. (2015) found also a phylogenetic Diptera, Tephritidae) (Sugier et al. 2013) which grouping of the two sesquiterpene lactone chemo- parasites in flower heads may lead to low seed yields. types by sequencing two polymorphic chloroplast Nutrient-rich (especially nitrogen-rich) soils are pro- markers (rps16 intron and ycf4-cemA). From the moting vegetative growth over flower and seed chloroplast data they could even deduce that the development. Grassland management (early cutting Spanish chemotype is ancestral to the Central-Euro- or grazing, intensity of use) has also influence on pean Chemotype and Galicia may be the source for successful propagation by seeds. Finally, flower the post-glacial colonization of A. montana in Europe collection intensity may also promote clonality. (Vera et al. 2015). Conservation Genetic diversity and population structure Applying a decision-making framework based on Although AMA showed a much lower expected genotyping developed for threatened species (Ot- heterozygosity compared to AMM (0.28 and 0.70, tewell et al. 2016), management for AMM should respectively) the lower genetic variability of AMA focus on habitat quality and maintaining large was also found by Vera et al. (2015) in sequencing populations rather than managing genetic diversity 123 Genet Resour Crop Evol Duwe VK, Ismail SA, Buser A, Sossai E, Borsch T, Muller (Duwe et al. 2017). For AMA, which shows higher LAH (2015) Fourteen polymorphic microsatellite markers genetic differentiation than AMM, lower genetic for the threatened Arnica montana (Asteraceae). Appl variability and no inbreeding, this framework pro- Plant Sci. https://doi.org/10.3732/apps.1400091 poses to increase artificially gene flow to increase Duwe VK, Muller LAH, Borsch T, Ismail SA (2017) Pervasive genetic differentiation among Central European popula- genetic diversity. Introduction of AMA cultivation in tions of the threatened Arnica montana L. and genetic the region collection could support gene flow by erosion at lower elevations. Perspect Plant Ecol Evol Syst bridging natural populations. In future, cultivating 27:45–56. https://doi.org/10.1016/j.ppees.2017.02.003 AMA could supplement wild collection. EDQM (2014) Arnicae tinctura. In: EDQM (ed) Ph. Eur., 8.0th edn., vol 1809 Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among Conclusion DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131:479–491 Falniowski A, Bazos I, Hoda´lova´ I, Lansdown R, Petrova A The recognition within Arnica montana of two (2011) Arnica montana. http://dx.doi.org/10.2305/IUCN. infraspecific taxa at subspecific rank, A. montana UK.2011-1.RLTS.T162327A5574104.en. Accessed 25 subsp. montana and A. montana subsp. atlantica,is April 2017 supported by the data presented in this paper. Gru¨nwald NJ, Goodwin SB, Milgroom MG, Fry WE (2003) Analysis of genotypic diversity data for populations of microorganisms. Phytopathology 93:738–746 Acknowledgements Open access funding was provided by Kamvar ZN, Tabima JF, Gr˙unwald NJ (2014) Poppr: an R the University of Veterinary Medicine Vienna. We cordially package for genetic analysis of populations with clonal, thank Remigius Chizzola, Brigitte Schmiderer and Reinhold partially clonal, and/or sexual reproduction. 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Plant Ecol 213:831–842 Mulcahy D, Nussbeck SY, O’Tuama E, Orrell T, Petersen Ottewell KM, Bickerton DC, Byrne M, Lowe AJ, Burridge C G, Robertson T, So¨hngen C, Whitacre J, Wieczorek J, (2016) Bridging the gap: a genetic assessment framework Yilmaz P, Zetzsche H, Zhang Y, Zhou X (2016) The for population-level threatened plant conservation priori- Global Genome Biodiversity Network (GGBN) Data tization and decision-making. Diversity Distrib 22:174– Standard specification. Database (Oxford). https://doi.org/ 188. https://doi.org/10.1111/ddi.12387 10.1093/database/baw125 123 Genet Resour Crop Evol Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in to reduce the pressure on endangered and high-valued Excel. Population genetic software for teaching and medicinal plant species. Sci World J 2013, Article ID research. Mol Ecol Notes 6:288–295 414363 Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Vera M, Romero R, Rodrı´guez-Guitia´n MA, Barros RM, Real Excel. 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Arnica montana subsp. atlantica: Really a subspecies?

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Abstract

Genet Resour Crop Evol https://doi.org/10.1007/s10722-018-0653-2 RESEARCH ARTICLE . . . Corinna Schmiderer Paula Torres-Londono Andrea Lutz-Röder Virginia K. Duwe Johannes Novak Received: 22 January 2018 / Accepted: 8 May 2018 © The Author(s) 2018 Abstract In Arnica montana L. (Asteraceae) two corrected. Genetically, AMA separated very well subspecies are described, A. montana subsp. atlantica from AMM with a G between the subspecies of ST (AMA), present only on the Iberian Peninsula and A. 0.81, genetically justifying the subspecies concept of montana subsp. montana (AMM) with a very wide A. montana. Genetic variability in AMA (H =0.28) exp distribution area. The morphological differences was lower than in the AMM populations (H =0.70). exp between the two subspecies are small and variable. A somewhat higher fixation index of AMA (F = ST Therefore, this concept is sometimes questioned. To 0.17, compared to an F =0.08 for AMM) may ST establish the genetic background of the two sub- indicate that geneflow in AMA is a bit more restricted species, populations of AMA and AMM together than in alpine AMM. However, the fixation index of with herbarium samples and DNA Bank material of AMA is not deviating from Hardy–Weinberg equi- AMM were tested with 12 microsatellite markers. A. librium. No inbreeding was observed for AMA (F = IS montana propagates by seeds or by clonal propaga- 0.10) and AMM (F =0.08). IS tion of its rhizome. In AMA, clonality was frequent while in AMM only one case of clonality could be Keywords Arnica montana · identified. Therefore, further results were clone- Arnica montana subsp. atlantica · Arnica montana subsp. montana · Genetic analysis · Microsatellites C. Schmiderer · J. Novak (&) Institute for Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria Introduction e-mail: Johannes.Novak@vetmeduni.ac.at Arnica L. (Asteraceae, Heliantheae s.l.) is a circum- P. Torres-Londono Kra¨utermix GmbH, Wiesentheider Str. 4, 97355 boreal genus of about 30 species mostly of montane Abtswind, Germany habitats. Arnica montana L. (Asteraceae, mountain arnica, wolfs’ bane) is a perennial, facultative A. Lutz-Ro¨der apomictic species (Yankova-Tsvetkova et al. 2016), Kneipp GmbH, Winterha¨user Str. 85, 97084 Wu¨rzburg, predominantly self-incompatible, insect pollinated Germany which reproduces sexually with seeds and vegeta- V. K. Duwe tively with short rhizomes (Luijten et al. 1996, 2000). Botanischer Garten und Botanisches Museum Berlin- A. montana grows on acidic grass- and shrublands Dahlem, Freie Universita¨t Berlin, Ko¨nigin-Luise-Straße and is distributed from the Iberian Peninsula to the 6-8, 14195 Berlin, Germany 123 Genet Resour Crop Evol Ukraine (Maurice et al. 2012). Bolos y Vayreda scale genetic study of Duwe et al. (2017) on AMM. (1945) distinguished two subspecies, A. montana Furthermore, in recent decades a significant decline subsp. atlantica A. Bolos (AMA) and A. montana in the populations of arnica were also observed in subsp. montana (AMM), where AMA is present only Galicia (Lange 1998; Romero et al. 2011). in SW-France, N-Spain and Portugal. AMA is Cultivation of A. montana subsp. montana is morphologically different in its smaller height, thin- possible, but not without problems. The species need ner floral stems, lanceolate leaves and smaller flower a loose, well-aerated soil with an ample supply of heads with fewer bracts. This subspecies differs from water and a lime content of less than 1%. Otherwise, AMM also in its habitat preferences, it occurs the plant reacts immediately with chlorosis. Seed between 0 and 440 m (max. 1000 m) in areas of germination is another difficulty in cultivation (von oceanic climate while AMM occurs from 0 to Raison et al. 2000). 3000 m. This subspecies concept was recently Since lowland proveniences of AMA are signifi- questioned by the analysis of biometrical data from cantly different in their chemical composition from different populations in Galicia where the only AMM, it would be interesting to know if the genetic significant difference between low- and highland distance justifies the proposed division into two plants was found in plant height (Romero et al. 2011). subspecies. To address this question, individuals from The two major proveniences of arnica flowers for NW Spain and Central Europe were classified as subspecies AMA or AMM according to the criteria the pharmaceutical/cosmetics industry in Europe are the Romanian Carpathians and NW Spain (Galicia) defined by Bolos y Vayreda (1945) and compared (Vera et al. 2016). Mountain arnica is an old folk with a set of microsatellite markers recently pub- medicine still popular for the treatment of pain, lished by Duwe et al. (2015). swelling and bruises. Due to its topical application of flower extracts in gel or cream form it is regarded as ‘cosmeceutical’ (Baumann 2007). Sesquiterpene lac- Materials and methods tones (SL) are responsible for the anti-inflammatory activity of arnica (Wagner et al. 2004) with helenalin Sample material esters (H) showing higher anti-inflammatory activity than dihydrohelenalin esters (DH) (Klaas et al. 2002) In total a sample set of 89 individuals was analysed while DH are less allergenic than H (Lass et al. and classified according to Bolos y Vayreda (1945)as 2008). Lowland arnica (AMA) is a DH-chemotype, A. montana subsp. montana (33 samples, AMM) or A. while AMM possesses predominantly helenalin esters montana ssp. atlantica (55 samples, AMA). One (cf. for example chromatograms in the European sample of A. chamissonis Less. was used as outgroup Pharmacopoeia where the DH-chemotype is (Table 1). The samples were obtained from the described as the ‘Spanish type’, while the Hele- herbarium of the University of Vienna (WU), nalin-chemotype is called ‘East-European type’ collected from the wild in 2016 (aerial plant parts (EDQM 2014)). only) and were obtained from the DNA Bank of the In general, A. montana is so abundant in many Botanic Garden and Botanical Museum Berlin- countries that The IUCN Red List of Threatened Dahlem (BGBM) (see Table 1). All tissue samples Species classifies A. montana as “Least Concern”, from the BGBM and the underlying voucher speci- although monitoring population trends is suggested mens are deposited at the Botanic Garden and due to the decline in some countries (Falniowski et al. Botanical Museum Berlin and are available via the 2011). Following this suggestion, Luijten et al. Global Genome Biodiversity Network (GGBN) (1996, 2000) studied the effect of habitat fragmen- (Droege et al. 2016) and the Global Biodiversity tation on population structure of AMM in the Information Facility (GBIF). Specimens collected in Netherlands and found reduced levels of genetic Spain were deposited in the herbarium of Kra¨uter- variation and limited gene flow between the popula- Mix, specimens collected in Austria in WU. tions. A strong genetic differentiation and a suggested restricted gene flow with signs of genetic erosion in lower altitudes were also recently found in the large- 123 Genet Resour Crop Evol Table 1 Locality and specimen information of reference samples used in this study Pop. n ng Species Origin (location; elevation in m.s.m., GPS coordinates; collection date) ES01 10 8 AMA Spain, Galicia, 0.4 km NNE of Vilouris; 480 m, 43°12.30 N, 8°2.33′W; 2016-06-28 ES02 10 6 AMA Spain, Galicia, 2.6 km NW of Vilouris; 482 m, 43°12.70′N, 8°04.12′W; 2016-06-28 ES03 10 3 AMA Spain, Galicia, 1.4 km SW of Vilouris; 513 m, 43°11.56′N, 8°03.10′W; 2016-06-28 ES04 5 1 AMA Spain, Galicia, 3 km NNE of Xermade; 451 m, 43°22.97′N, 7°48.45′W; 2016-06-29 ES05 10 4 AMA Spain, Galicia, 3.3 km WNW of Susana; 705 m, 43°28.52′N, 7°40.40′W; 2016-06-29 ES06 10 6 AMA Spain, Galicia, 1.5 km NNE of Susana; 607 m, 43°28.86′N, 7°37.48′W; 2016-06-29 OG01 1 1 ACH WU: Austria, Vienna, cultivated at HBV; 1995-09-04 CE01 10 9 AMM Austria, Styria, Steinplan; 1640 m, 47°9.73′N, 14°54.09′E; 2016-07-21 CE02 10 10 AMM Austria, Carinthia, 6 km N of Millstatt, Hansbauerhutte; 1720 m, 46°51.47′N, 14°53.10′E; 2016-07-24 CE03 1 1 AMM WU: Switzerland, Grisons, NW Ravaisch; 2004-08-22 CE04 1 1 AMM WU: Italy, S-Tyrol, Central Alps, Passeier, Platt; 1995-07-10 CE05 1 1 AMM WU: Austria, E-Tyrol, Defereggen, Oberberg, N St. Jakob; 1987-08-04 CE06 1 1 AMM WU: Austria, Carinthia, Hohe Tauern, Hafnergruppe; 2003-07-07 CE07 2 2 AMM WU: Austria, Lower Austria, SW Waldviertel; 2009-06-05 AMM WU: Austria, Lower Austria, Waldviertel, Langsehschlag; 1913-06-05 CE08 1 1 AMM WU: Austria, Vienna, NW-Plateau of Sophienalpe; 1950 CE09 1 1 AMM BGBM (BGT 0008920): Germany, Saxony, Oelsen, Osterzgebirge; 632 m, 50°47′N, 13°56′E; 2013-06-03 CE10 1 1 AMM BGBM (BGT 0012009): Germany, Mecklenburg-Western Pomerania, Zarrendorf, Stralsund; 0 m, 54°14′N, 13°05′E; 2013-06-17 CE11 1 1 AMM BGBM (BGT 0013108): Germany, Brandenburg, Naturpark Niederlausitzer Heidelandschaft; 97 m, 51°30′ N, 13°46′E; 2013-06-20 CE12 1 1 AMM BGBM (BGT 0011921): Italy, S-Tyrol, 3 km ENE of Badia; 2062 m, 46°37′N, 11°56′E; 2013-06-29 CE13 1 1 AMM BGBM (BGT 0013144): Germany, Baden-Wu¨rttemberg, Black Forest; 1424 m, 47°52′N, 8°01′E CE14 1 1 AMM WU: France, E-Pyrenees, Superbolquere; 1750 m; 1944-07-17.-20 Pop. … population, n … number of sampled individuals per population, ng … number of individuals with different multilocus genotypes (genets), AMA … Arnica montana subsp. atlantica, AMM … A. montana subsp. montana, ACH … A. chamissonis, BGBM … Botanical Garden and Botanical Museum Berlin-Dahlem, Germany, HBV … Botanical Garden of the University of Vienna, Austria, WU … Herbarium of the University of Vienna, Austria Extract for HPLC analysis of sesquiterpene After shaking for 120 s the mixture was filtered lactones through a folded filter into a 100 mL flask. The filtrate was brought to dryness in vacuo and re-suspended in Extraction for the analysis of sesquiterpene lactones 3.0 mL of a mixture of equal volume parts of MeOH was performed using a modification of the European and H O. After filtration, the solution was used for Pharmacopoeia Monograph Arnica flower (EDQM HPLC analysis. 2014). In detail, dried flowers (approx. 5 g) were milled. 1.0 g of the powdered drug was weighed HPLC analysis exactly into a 250 mL flat bottom flask. After addition of 2.0 mL internal standard solution containing 1 mg/ HPLC analysis of sesquiterpene lactones were per- mL santonin in MeOH, immediately prepared prior to formed using an Alliance 2695 high pressure gradient use, and 50 mL MeOH, the mixture was extracted for system (Waters GmbH, Eschborn, Germany) 1 h using a reflux condenser. The cooled solution was equipped with a DAD detector. The following centrifuged for 15 min at 4500 rpm. To the super- parameters were applied: column, Merck Superspher natant, 7 g of neutral aluminium oxide was added. 100 RP 18e 12594mm (4 µm particle size); guard 123 Genet Resour Crop Evol column, Merck LiChrospher 100 RP 18e, 494mm For 15 μL PCR reactions, 10 ng of genomic DNA (5 µm particle size); mobile Phase A, H O; mobile was added to a master mix containing 19PCR buffer Phase B, MeOH; flow rate, 1.2 ml/min; injection B, 2 mM MgCl , 200 μM dNTPs (each), 0.6 U Taq volume, 20 µL; detection wavelength 225 nm; oven HOTFIREPol DNA Polymerase (all reagents from temperature, 20 °C; isocratic 0–3 min 38% B; linear Solis BioDyne, Tartu, Estonia), 200 nM fluorescent gradient 3–20 min 45% B; isocratic 20–30 min 45% labelled forward primers, 50 nM locus specific B; linear gradient 30–55 min 55% B; linear gradient forward primers and 250 nM locus specific reverse 55–57 min 100% B, 70 min stop. The assignment of primers. Samples with no or insufficient amplification chemotypes was deduced by comparing the chro- were repeated with different DNA amounts (0.25– matograms with the chromatograms in the European 10 ng) and 0.9 U polymerase. The PCR conditions Pharmacopoeia representing the two chemotypes included a denaturation step at 95 °C for 15 min, (‘Spanish Type’ and ‘East European Type’) (EDQM followed by 30 cycles at 95/58/72 °C for 30/45/45 s, 2014). 15 cycles at 95/53/72 °C for 30/45/45 s, and a final elongation step at 72 °C for 10 min. PCR products DNA extraction were checked on 2% agarose gels stained with PeqGreen (VWR International, Vienna, Austria; Genomic DNA was extracted from air dried speci- 4 µL/100 mL agarose solution; products including mens using a modified CTAB-protocol (Schmiderer 6-FAM) or without staining (PCR products including et al. 2013) based on Doyle and Doyle (1990). DNA ATTO dyes). Six amplified loci per sample (1 µL concentrations of the extracts were determined using PCR product each; Table 2) were mixed and diluted a NanoDrop ND-2000c (Peqlab Biotechnologie with 24 µL ddH O. The determination of the GmbH, Erlangen, Germany). DNA extracts were sequence lengths was performed by Microsynth diluted with Milli-Q water to 5 ng/µL. (Balgach, Switzerland) using GeneScan™ 500 LIZ™ dye size standard (Thermo Fisher Scientific, Microsatellite analysis Waltham, MA, USA). The obtained chromatogram files were edited using Peak Scanner 2.0 software Microsatellite markers and part of the primer (Applied Biosystems, Waltham, Massachusetts, sequences were adopted from Duwe et al. (2015). USA). Remade primers with an optimum melting tempera- ture ranging from 51 to 53 °C were designed using Statistical analysis Primer Express 2 (Applied Biosystems, Foster City, California, USA). Primer dimers were evaluated The number of multilocus genotypes (MLG) stan- using NetPrimer software (http://www.premier dardized by sample numbers, Stoddard and Taylors’ biosoft.com/netprimer). Multiplexing of different index of MLG diversity (Stoddart and Taylor 1988), loci was performed using Multiplex Manager Simpsons’ index (Simpson 1949) corrected by N/(N- (www.multiplexmanager.com). 1), Evenness, E.5 (Gru¨nwald et al. 2003), Nei’s PCR amplification was performed using tailed expected heterozygosity (H ), Nei’s genetic dis- Exp locus-specific forward primers, fluorescence labelled tances and a Neighbor-joining tree were calculated nested forward primers (5′ modified with 6-FAM, using R 3.3.0 (R Core Team 2016) with poppr 2.2.0 ATTO532, ATTO550 or ATTO565) binding to the (Kamvar et al. 2014, 2015). For more detailed forward primer extensions and “PIG-tailed” reverse population analysis, accessions with just one sample primers to reduce stutter bands (with 3–4 bp exten- (i.e., herbarium specimen) were excluded. Hence, sions to achieve a GTTT consensus sequence at the two Austrian AMM populations were compared to 5′-end, according to Brownstein et al. 1996). Unla- the six Spanish AMA populations. Putative clonality belled and 6-FAM labelled primers were obtained and AMOVA were calculated with Genalex 6.5 from Sigma-Aldrich (Vienna, Austria), all ATTO (Peakall and Smouse 2006, 2012). Putative clonality labelled primers were obtained from Microsynth was determined by multilocus genotypes (MLG). (Vienna, Austria). Individuals with the same MLG may either be parts of a clone (ramets of a genet) or—after sexual 123 Genet Resour Crop Evol Table 2 Primers used for analysis Primer name T Primer sequence Repeat motif* allele size Analysis range reaction Arm02(565) F 58* gaatcaccatcgtcgcatAACACACATCCACGTTTGGC TACA 2 Arm02 R 58* gtttAACCGTGCATCATTCTGTGG 190-274 Arm03(532) F2 52 tgtaaaacgtcggcgactCAAAAACCCTAATTCTCCATC TACA 2 Arm03 R2 52 gtttCTGCGCAATGGGTTTACT 109-145 Arm05(532) F 58* tgtaaaacgtcggcgactACTGTCACCTAGGGGTGTTC AACA 2 Arm05 R 58* gttTAAGCGGGGAGTCTTTCTGG 186-206 Arm06(550) F 58* ccaagtagggcggtatctTGTCGCCTCAATCCTTGGTG ACAT 1 Arm06 R 58* gtttGCTGAAGTCCTTCCTTGGAC 119-271 Arm07(M13) F 58* tgtaaaacgacggccagtACATGACGCAAAAAGCGTAG TATG 2 Arm07 R 58* gtttCCATGTTACCACCATGTCGC 211-251 Arm08(M13) F 58* tgtaaaacgacggccagtAGATGAGGTTCTTGCAGCATC TGTA 2 Arm08 R 58* gttTGCTTGCAGTTGAAGTAAAGGG 134-180 Arm09(565) F 58* gaatcaccatcgtcgcatTAGGCGTGAGTTTGTACTCG TATG 1 Arm09 R 58* gtttAAGCGTGTTAACTTCGTGAG 236-264 Arm10(565) F 58* gaatcaccatcgtcgcatACCAGCTGACTCTCTTTCCG CATA 1 Arm10 R 58* gtttCAAGGATGAACATCGGCCTC 147-207 Arm11(532) F2 52 tgtaaaacgtcggcgactGCACAAGGTATGTGTTGCA GT 1 Arm11 R 58* gttTCTTCGACCGAATGTTTTCACC 167-183 Arm12(550) F2 52 ccaagtagggcggtatctCTTGCTTCTTCTCTTTATAGATGTC AG 2 Arm12 R2 52 GGTTACCATTTTGGGTTCA 96-126 Arm13(532) F (= 58* tgtaaaacgtcggcgactGGTTTGAACACGAGATAGCG AT 1 Armo02 F*) Arm13 R (=Armo02 58* gtttACAAACTTCCTGTTGTCCCG 224-254 R*) Arm14(M13) F (= 58* tgtaaaacgacggccagtTCAAACAGTCACCAGCAACC ACCTGG 1 Armo03 F*) Arm14 R (=Armo03 58* gtttCAGAGGCTGCAACCCTAATG 213-241 R*) M13-FAM 53 [6FAM]-TGTAAAACGACGGCCAGT ATTO532 53 [ATTO532]-TGTAAAACGTCGGCGACT ATTO565 53 [ATTO565]-GAATCACCATCGTCGCAT ATTO550 51 [ATTO550]-CCAAGTAGGGCGGtATCT Capital letters of the primer sequences indicate the locus specific sequences; small form letters indicate artificial primer tails. The microsatellite repeat motif was published by Duwe et al. (2015). The allele size range was obtained with the used sample set including Arnica chamissonis *Allele specific primer sequence and T according to Duwe et al. (2015) Primer melting temperatures without asterisks were calculated with Primer Express 2, excluding primer tails reproduction—equal by chance. Briefly, the program separately for AMA and AMM with the Codom- estimates the probability (P ) of the occurrence of Allelic distance with 999 permutations. The division sex an MLG in a randomly mating population and gives allowed us a closer insight into the structure of the statistical significance levels based on observed allele two subspecies that would have been covered by the frequencies. Analysis of Molecular Variance high genetic distance between the two subspecies. (AMOVA) (Excoffier et al. 1992) was calculated 123 Genet Resour Crop Evol Results ramet of a genet or sexually reproduced and equal by chance. Of the 75 samples in the population sample In total 6 populations of lowland Arnica montana subset only 45 MLG could be detected. 10 MLG were from Spain with 5–10 individuals each were classi- present in multiple copies, all of them with a P - sex fied as A. montana ssp. atlantica (AMA) according value lower than 0.05 indicating that the probability the proposed criteria by Bolos y Vayreda (1945) and of equal MLG by sexual reproduction is rather low were compared to two populations of A. montana ssp. and clonality is more likely. Apart from one genet in montana (AMM) from Central Europe (Austria) with AMM with only two ramets all other 9 genets were 10 individuals each. This sample set was comple- found in AMA. Subsequently, only one ramet of a mented by a geographically wide range of individual genet was left in the sample set for further analysis samples from Germany to Northern Italy and the (Table 4). East-Pyrenees (France). All AMA plants belonged in their sesquiterpene lactone profile to the ‘Spanish Genetic difference between AMA and AMM type’, while AMM plants were of the ‘East-European type’ (EDQM 2014). The genetic study with 12 The results show a clear genetic distinction of microsatellite loci showed between 6 and 15 different Spanish AMA from Central European AMM indi- alleles, the expected heterozygosity H ranged from viduals (G =0.81, Table 3, Fig. 1). Although the exp ST 0.46 to 0.82 (mean=0.61) and the mean evenness genetic variability was much smaller in AMA, the ranged from 0.37 to 0.86 (mean=0.58) (Table 3). separation of populations within this group is far better supported than amongst AMM. Especially the Clonality population from Xermade (ES04) is distinctively different, but also the other two population groups Arnica montana has two propagation strategies, a from Susana (ES05) and Vilouris (ES03) are well sexual strategy (a facultative apomictic species with separated from each other, indicating limited gene predominant sexual reproduction (Yankova-Tsvet- flow between AMA populations. kova et al. 2016)) and an asexual strategy by AMM samples were only in some cases grouped rhizomes (Sugier et al. 2013). To avoid clonal by their geographic distance. Samples from Styria influence on the estimation of variability, probabil- (CE01) and Carinthia (CE02) are geographically ities of equal multilocus genotypes (MLG) were close, as well as from Vienna (CE08) and Lower estimated that an individual of a MLG was either a Austria (CE07) and from E-Tyrol (CE05) and Table 3 Characteristics of the microsatellites used in this study different alleles; G … proportion of genetic diversity that ST (Alleles … number of different alleles detected; H … resides among the two subspecies) Exp expected heterozygosity; evenness … distribution of the Locus Alleles H Evenness G between AMA and AMM Exp ST Arm14 8 0.53 0.67 0.993 Arm11 6 0.54 0.74 0.152 Arm13 9 0.64 0.53 0.829 Arm06 15 0.57 0.41 0.690 Arm10 15 0.58 0.37 0.939 Arm09 8 0.67 0.63 0.998 Arm08 10 0.60 0.62 0.343 Arm07 12 0.57 0.43 0.976 Arm03 7 0.46 0.60 0.898 Arm05 6 0.68 0.86 0.898 Arm12 14 0.82 0.68 0.983 Arm02 13 0.64 0.48 0.991 mean 10.25 0.61 0.58 0.808 123 Genet Resour Crop Evol Population structure Table 4 Characteristics of AMA and AMM (n … number of samples, eMLG … number of multilocus genotypes standard- ized by sample numbers, G … Stoddard and Taylors’ index of To compare population structures between AMA and MLG diversity, lambda … Simpsons’ index corrected by N/(N- AMM populations, populations with just one indi- 1), E.5 … Evenness, H … Nei’s expected heterozygosity) Exp vidual (herbarium samples) were excluded. So, two Subspecies n eMLG G lambda E.5 H exp AMM populations from Austria were compared to the six Spanish AMA populations. The linear Before clone correction distance of the two Austrian populations was AMA 55 13.2 13.7 0.736 0.726 0.249 107 km while the linear distance between the most AMM 20 19 18.2 0.986 0.973 0.657 distant AMA populations was 47 km. The number of Total 75 15.9 23.1 0.970 0.69 0.518 expected multilocus-genotypes (eMLG), the stan- After clone correction dardized MLG for unequal sample numbers, as well AMA 28 9.76 24.5 0.959 0.965 0.278 as Simpsons’ index and evenness were almost AMM 32 10 32 0.969 1 0.702 identical between AMA and AMM (Table 4). Total 60 9.95 57.2 0.983 0.982 0.704 Expected heterozygosity (gene diversity) was low in AMA (H =0.28) and high in AMM (H =0.70). exp exp S-Tyrol (CE04). The German populations (Saxony, These results were also reflected in AMOVA anal- Brandenburg, Mecklenburg, Baden-Wu¨rttemberg), ysis. Both subspecies, analysed separately, showed however, grouped separately with completely differ- here significant variation between populations ent geographical locations, but bootstrap support is (Table 5). However, AMA populations differed to a generally very weak in the AMM group (Table 1; higher degree from each other than the two AMM Fig. 1). Fig. 1 Neighbor joining tree based on Nei’s genetic distances of Arnica montana of Spanish (ES) AMA, and Central European (CE) AMM samples. Arnica chamissonis was used as outgroup (OG) 123 Genet Resour Crop Evol Table 5 Results of analysis of molecular variance (AMOVA), calculated separately for AMA and AMM Source df SS MS Est. Var. % F value P value AMA Among populations 4 18.721 4.680 0.294 17 F 0.171 0.001 ST Among individuals 22 34.742 1.579 0.151 9 F 0.105 0.074 IS Within individuals 27 34.500 1.278 1.278 74 F 0.258 0.002 IT AMM Among populations 1 10.050 10.050 0.312 8 F 0.075 0.001 ST Among individuals 17 70.450 4.144 0.322 8 F 0.084 0.020 IS Within individuals 19 66.500 3.500 3.500 85 F 0.153 0.001 IT populations did (17% of variation among populations two chloroplast markers. Genetic variability in Dutch in AMA compared to 8% in AMM, indicating a AMM populations (H =0.09) (Luijten et al. 2000) exp higher degree of panmixis in AMM). The degree of were even much lower than in AMA from this study inbreeding (variation among individuals) was almost (H =0.28). As in AMA (F =0.17), the Dutch exp ST equal for AMA and AMM (F =0.105 (P=0.07) and populations showed moderately significant popula- IS F =0.084 (P=0.02*) for AMA and AMM, tion differentiation (F =0.14). IS ST respectively). Clonality Discussion The elevated level of clonality in AMA is either an indication of negative influences on sexual reproduc- The subspecies concept tion or more favourable conditions for vegetative growth. Many reasons can negatively influence seed The subspecies concept in A. montana was recently propagation. Decreased pollination and seed devel- questioned by morphological analysis of an extensive opment, low seed longevity and poor possibilities for sample set (Romero et al. 2011) where the authors seeds to germinate in densely covered vegetation found that the defined criteria to distinguish AMA (competition) may be reasons linked to flower and from AMM were highly variable not allowing a clear seed biology. Attacks on and diseases of floral tissues distinction. However, AMA was genetically highly caused e.g., by herbivore slugs and fruit flies distinguishable from AMM in our microsatellite specialized on A. montana (Tephritis arnicae L., study. Vera et al. (2015) found also a phylogenetic Diptera, Tephritidae) (Sugier et al. 2013) which grouping of the two sesquiterpene lactone chemo- parasites in flower heads may lead to low seed yields. types by sequencing two polymorphic chloroplast Nutrient-rich (especially nitrogen-rich) soils are pro- markers (rps16 intron and ycf4-cemA). From the moting vegetative growth over flower and seed chloroplast data they could even deduce that the development. Grassland management (early cutting Spanish chemotype is ancestral to the Central-Euro- or grazing, intensity of use) has also influence on pean Chemotype and Galicia may be the source for successful propagation by seeds. Finally, flower the post-glacial colonization of A. montana in Europe collection intensity may also promote clonality. (Vera et al. 2015). Conservation Genetic diversity and population structure Applying a decision-making framework based on Although AMA showed a much lower expected genotyping developed for threatened species (Ot- heterozygosity compared to AMM (0.28 and 0.70, tewell et al. 2016), management for AMM should respectively) the lower genetic variability of AMA focus on habitat quality and maintaining large was also found by Vera et al. (2015) in sequencing populations rather than managing genetic diversity 123 Genet Resour Crop Evol Duwe VK, Ismail SA, Buser A, Sossai E, Borsch T, Muller (Duwe et al. 2017). For AMA, which shows higher LAH (2015) Fourteen polymorphic microsatellite markers genetic differentiation than AMM, lower genetic for the threatened Arnica montana (Asteraceae). Appl variability and no inbreeding, this framework pro- Plant Sci. https://doi.org/10.3732/apps.1400091 poses to increase artificially gene flow to increase Duwe VK, Muller LAH, Borsch T, Ismail SA (2017) Pervasive genetic differentiation among Central European popula- genetic diversity. Introduction of AMA cultivation in tions of the threatened Arnica montana L. and genetic the region collection could support gene flow by erosion at lower elevations. Perspect Plant Ecol Evol Syst bridging natural populations. In future, cultivating 27:45–56. https://doi.org/10.1016/j.ppees.2017.02.003 AMA could supplement wild collection. EDQM (2014) Arnicae tinctura. In: EDQM (ed) Ph. Eur., 8.0th edn., vol 1809 Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among Conclusion DNA haplotypes: application to human mitochondrial DNA restriction data. 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Genetic Resources and Crop EvolutionSpringer Journals

Published: Jun 5, 2018

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