VOLKSMED Database: A Source for Forgotten Wound Healing Plants in Austrian Folk Medicine.

The global increase in the incidence of wounds is concerning and fuels the search for new treatment options. The use of traditional medicinal plants in wound healing represents an appreciated available therapeutic possibility. This work introduces the VOLKSMED database, which contains plants and other materials used in Austrian folk medicine, either as monographs or mixtures. This work focuses on the monographs of the database. Concerning wound healing, Hypericum sp., Arnica montana, Calendula officinalis, Plantago sp., and Malva sp. are the most commonly used plants. The focus of this paper is set on selected lesser-known plants (Abies alba, Anthyllis vulneraria, Brassica sp., Gentiana sp., Larix decidua, Picea abies, Sambucus sp., Sanicula europaea) and their status quo in literature concerning wound healing. A systematic search using the databases SciFinder, SCOPUS, and PubMed yielded substantial evidence for the wound healing potential of Brassica sp., Gentiana sp., the Pinaceae A. abies, L. decidua, and P. abies, as well as Sambucus nigra. In vivo and clinical studies substantiate their use in Austrian folk medicine. According to the literature, especially A. vulneraria, Sambucus racemosa, and S. europaea would be worth investigating in-depth since data concerning their wound healing effects - even though scarce - are convincing. In conclusion, the VOLKSMED database contains promising opportunities for further treatment options in the field of wound healing. Future research should consider the listed plants to support their traditional use in Austrian folk medicine and possibly promote the implementation of old knowledge in modern medicine.

Norway Spruce Balm: Phytochemical Composition and Ability to Enhance Re-epithelialization In Vitro.

The balm of the Norway spruce (Picea abies) is a well-known traditional herbal medicine used to cure wounds. Even though clinical trials have confirmed its empirical use, the active constituents, their mode of action, and the exact composition of this natural product are still unknown. In this study, the balm was subjected to fractionated extraction and further purified employing flash chromatography, HPLC-PDA-ELSD, preparative and analytical TLC. Hydroxycinnamic acids ( 1: - 3: ), the lignan pinoresinol ( 4: ), four hydroxylated derivatives of dehydroabietic acid (DHAA) ( 5:  -  8: ), and dehydroabietic acid ( 9: ) were isolated. Their structures were elucidated by LC-MS, 1D- and 2D-NMR. Four extracts, two commercially available resin acids-pimaric acid ( 10: ) and isopimaric acid ( 11: )-and the isolated compounds were tested for increased re-epithelialization of cell-free areas in a human adult low calcium high temperature keratinocytes monolayer. Lysophosphatidic acid (10 µM) served as positive control and ranged between 100% and 150% rise in cell-covered area related to the vehicle control. Two extracts containing carboxylic acids and non-acidic apolar constituents, respectively, boosted wound closure by 47% and 36% at 10 and 3 µg/mL, respectively. Pinoresinol, DHAA, three of its hydroxylated derivatives, and pimaric and isopimaric acid as well as defined combinations of the hydroxylated DHAA derivatives led to a significantly enhanced wound closure by up to 90% at concentrations between 1 and 10 µM. Overall, lignans and diterpene resin acids, main constituents of Norway spruce balm, are able to increase migration or proliferation of keratinocytes in vitro. The presented data link the phytochemistry of this natural wound healing agent with boosted re-epithelialization.

Apparent penetration depth in attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy of Allium cepa L. epidermis and cuticle.

Over the past decades, ATR-FTIR has emerged as promising tool for the identification of plants at the genus and (sub-) species level through surface measurements of intact leaves. Theoretical considerations regarding the penetration depth of the evanescent wave into the sample and the thickness of plant leaf cuticles suggest that the structure and composition of the cuticle represent universal taxonomic markers. However, experimental evidence for this hypothesis is scarce. In the current contribution, we present results of a series of simple experiments on epidermal monolayers derived from the bulbs of Allium cepa L. (Amaryllidaceae) as a model system to study the effect of an IR active probe located beyond the theoretical penetration depth of the evanescent wave. We found that this probe had a significant influence on the ATR-FTIR spectra for up to 4 epidermal layers stacked on top of each other corresponding to a total thickness of around 60 µm, exceeding the theoretical penetration depth of the evanescent wave by a factor of around 20. Altogether, our data indicate a major discrepancy between theory and practice in ATR-FTIR spectroscopy in general and provide strong evidence that in general plant leaf spectra cannot be fully explained by the structure and composition of the cuticle alone.

Quality Control of Valerianae Radix by Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy.

(Acetoxy-)valerenic acid and total essential oil content are important quality attributes of pharmacy grade valerian root (Valerianae radix). Traditional analysis of these quantities is time-consuming and necessitates (harmful) solvents. Here we investigated an application of attenuated total reflection Fourier transform infrared spectroscopy for extractionless analysis of these quality attributes on a representative sample comprising 260 wild-crafted individuals covering the Central European taxonomic diversity of the Valeriana officinalis L. s. l. species aggregate with its three major ploidy cytotypes (i.e., di-, tetra- and octoploid). Calibration models were built by orthogonal partial least squares regression for quantitative analysis of (acetoxy-)valerenic acid and total essential oil content. For the latter, we propose a simplistic protocol involving apolar extraction followed by gas chromatography as a reference method for multivariate calibration in order to handle the analysis of samples taken from individual plants. We found good predictive ability of chemometric models for quantification of valerenic acid, acetoxyvalerenic acid, total sesquiterpenoid acid, and essential oil content with a root mean squared error of cross-validation of 0.064, 0.043, and 0.09 and root mean squared error of prediction of 0.066, 0.057, and 0.09 (% content), respectively. Orthogonal partial least squares discriminant analysis revealed good discriminability between the most productive phenotype (i.e., the octoploid cytotype) in terms of sesquiterpenoid acids, and the less productive ones (i.e., di- and tetraploid). All in all, our results demonstrate the application of attenuated total reflection Fourier transform infrared spectroscopy for rapid, extractionless estimation of the most important quality attributes of valerian root and minimally invasive identification of the most productive phenotype in terms of sesquiterpenoid acids.

Suitability of Root and Rhizome Anatomy for Taxonomic Classification and Reconstruction of Phylogenetic Relationships in the Tribes Cardueae and Cichorieae (Asteraceae).

The value of root and rhizome anatomy for the taxonomic characterisation of 59 species classified into 34 genera and 12 subtribes from the Asteraceae tribes Cardueae and Cichorieae was assessed. In addition, the evolutionary history of anatomical characters was reconstructed using a nuclear ribosomal DNA sequence-based phylogeny of the Cichorieae. Taxa were selected with a focus on pharmaceutically relevant species. A binary decision tree was constructed and discriminant function analyses were performed to extract taxonomically relevant anatomical characters and to infer the separability of infratribal taxa, respectively. The binary decision tree distinguished 33 species and two subspecies, but only five of the genera (sampled for at least two species) by a unique combination of hierarchically arranged characters. Accessions were discriminated-except for one sample worthy of discussion-according to their subtribal affiliation in the discriminant function analyses (DFA). However, constantly expressed subtribe-specific characters were almost missing and even in combination, did not discriminate the subtribes. Most anatomical characters showed at least some degree of homoplasious evolution limiting their suitability as phylogenetically informative characters.

Underestimated diversity in one of the world's best studied mountain ranges: The polyploid complex of Senecio carniolicus (Asteraceae) contains four species in the European Alps.

Senecio carniolicus (Asteraceae) is an intricate polyploid complex distributed in the European Alps (di-, tetra- and hexaploids) and Carpathians (hexaploids only). Molecular genetic, ecological, and crossing data allowed four evolutionary groups within S. carniolicus to be identified. Here, we establish that these four groups (two vicariant diploid lineages, tetraploids and hexaploids) are also morphologically differentiated. As a consequence, we draw taxonomic conclusions by characterizing four species, including the more narrowly circumscribed S. carniolicus (lectotypified here), the taxonomically elevated S. insubricus comb. nov. (lectotypified here), and the two newly described species S. disjunctus and S. noricus.

Ethnopharmacological in vitro studies on Austria's folk medicine--an unexplored lore in vitro anti-inflammatory activities of 71 Austrian traditional herbal drugs.

ETHNOPHARMACOLOGICAL RELEVANCE: In Austria, like in most Western countries, knowledge about traditional medicinal plants is becoming scarce. Searching the literature concerning Austria's ethnomedicine reveals its scant scientific exploration. Aiming to substantiate the potential of medicinal plants traditionally used in Austria, 63 plant species or genera with claimed anti-inflammatory properties listed in the VOLKSMED database were assessed for their in vitro anti-inflammatory activity. MATERIAL AND METHODS: 71 herbal drugs from 63 plant species or genera were extracted using solvents of varying polarities and subsequently depleted from the bulk constituents, chlorophylls and tannins to avoid possible interferences with the assays. The obtained 257 extracts were assessed for their in vitro anti-inflammatory activity. The expression of the inflammatory mediators E-selectin and interleukin-8 (IL-8), induced by the inflammatory stimuli tumor necrosis factor alpha (TNF-α) and the bacterial product lipopolysaccharide (LPS) was measured in endothelial cells. The potential of the extracts to activate the nuclear factors PPARα and PPARγ and to inhibit TNF-α-induced activation of the nuclear factor-kappa B (NF-κB) in HEK293 cells was determined by luciferase reporter gene assays. RESULTS: In total, extracts from 67 of the 71 assessed herbal drugs revealed anti-inflammatory activity in the applied in vitro test systems. Thereby, 30 could downregulate E-selectin or IL-8 gene expression, 28 were strong activators of PPARα or PPARγ (inducing activation of more than 2-fold at a concentration of 10µg/mL) and 21 evoked a strong inhibition of NF-κB (inhibition of more than 80% at 10µg/mL). CONCLUSION: Our research supports the efficacy of herbal drugs reported in Austrian folk medicine used for ailments associated with inflammatory processes. Hence, an ethnopharmacological screening approach is a useful tool for the discovery of new drug leads.

Sex-specific-differences in cardiometabolic risk in type 1 diabetes: a cross-sectional study.

BACKGROUND: Little is known about the impact of sex-specific differences in the management of type 1 diabetes (T1DM). Thus, we evaluated the influence of gender on risk factors, complications, clinical care and adherence in patients with T1DM. METHODS: In a cross-sectional study, sex-specific disparities in glycaemic control, cardiovascular risk factors, diabetic complications, concomitant medication use and adherence to treatment recommendations were evaluated in 225 consecutive patients (45.3% women) who were comparable with respect to age, diabetes duration, and body mass index. RESULTS: Although women with T1DM had a higher total cholesterol than men, triglycerides were higher in obese men and males with HbA1c>7% than in their female counterparts. No sex differences were observed in glycaemic control and in micro- or macrovascular complications. However, the subgroup analysis showed that nephropathy was more common in obese men, hyperlipidaemic women and all hypertensive patients, whereas peripheral neuropathy was more common in hyperlipidaemic women. Retinopathy was found more frequently in women with HbA1c>7%, obese men and in both sexes with a long duration of diabetes. The multivariate analysis revealed that microvascular complications were associated with the duration of disease and BMI in both sexes and with hyperlipidaemia in males. The overall adherence to interventions according to the guidelines was higher in men than in women. This adherence was concerned particularly with co-medication in patients diagnosed with hypertension, aspirin prescription in elderly patients and the achievement of target lipid levels following the prescription of statins. CONCLUSIONS: Our data showed sex differences in lipids and overweight in patients with T1DM. Although glycaemic control and the frequency of diabetic complications were comparable between the sexes, the overall adherence to guidelines, particularly with respect to the prescription of statins and aspirin, was lower in women than in men.

Identification of ostruthin from Peucedanum ostruthium rhizomes as an inhibitor of vascular smooth muscle cell proliferation.

Inhibition of vascular smooth muscle cell (VSMC) proliferation is of substantial interest in combating cardiovascular disease. A dichloromethane extract from the rhizomes of Peucedanum ostruthium, a traditionally used Austrian medicinal plant with anti-inflammatory properties, was examined for a putative antiproliferative activity in rat aortic VSMC. This extract inhibited serum (10%)-induced VSMC proliferation concentration dependently. Further identification and biological testing of its major constituents revealed that the coumarin ostruthin (7) is the major antiproliferative substance. In summary, a new bioactivity of P. ostruthium rhizomes is described, and 7 has been identified as the responsible compound.

Anatomy of subterranean organs of medicinally used cardueae and related species and its value for discrimination.

Numerous species of the Asteraceae, the composites, are famous for their use in both traditional and conventional medicine. Reliable anatomical descriptions of these plants and of possible adulterations provide a basis for fast identification and cheap purity controls of respective medicinal drugs by means of light microscopy. Nevertheless, detailed comparative studies on root and rhizome anatomy of valuable as well as related inconsiderable composite plants are largely missing yet. The presented study aims to narrow this gap by performing anatomical analyses of roots and rhizomes of 16 species belonging to the tribe Cardueae, of formerly and currently used drugs as well as their near relatives as potential adulterations (Carlina acaulis L., Carlina vulgaris L., Arctium lappa L., Arctium tomentosum Mill., Carduus defloratus L., Carduus personata (L.) Jacq, Cirsium arvense (L.) Scop., Cirsium vulgare (Savi) Ten., Cirsium erisithales (Jacq.) Scop., Onopordum acanthium L., Silybum marianum (L.) Gaertn., Rhaponticum scariosum Lam., Centaurea jacea L., Centaurea scabiosa L., Centaurea cyanus L., Cnicus benedictus L.). A detailed verbal and graphical survey of the analysed anatomical features is provided. Several characters were finally extracted which allow for discrimination of the examined species and may be effectively used for drug quality controls.

Microscopical discrimination of the subterranean organs of medicinally used plants of the Cichorieae and their relatives.

CONTEXT: Light microscopy is in most cases a quick method for the identification and discrimination of medicinally used plant drugs; moreover, this technique is very inexpensive. Reliable descriptions of the anatomy of plants and their adulterations are prerequisites for necessary purity controls. OBJECTIVE: The anatomy of the subterranean organs of 18 pharmaceutically useful as well as related but inconsiderable Asteraceae species from nine genera (Taraxacum F. H. Wigg., Leontodon L., Scorzoneroides Moench, Hypochaeris L., Crepis L., Aposeris Neck., Cichorium L., Scorzonera L., and Tragopogon L.; tribe Cichorieae, Asteraceae) is described in detail and graphically illustrated. Features characterizing and discriminating the studied taxa are presented and discussed. MATERIALS AND METHODS: The roots/rhizomes of various species were examined by means of light microscopy. RESULTS: Useful anatomical characters were found for the discrimination between the species, and some of them were examined for the first time. DISCUSSION AND CONCLUSION: Discrimination of most genera and species investigated was possibly based on the anatomy of their underground parts. The identified characters may be effectively used for quality control of commercial drugs and the identification of adulterations.

Identification and quantification of coumarins in Peucedanum ostruthium (L.) Koch by HPLC-DAD and HPLC-DAD-MS.

The rhizomes of Peucedanum ostruthium (L.) Koch (masterwort) are traditionally used in the alpine region as ingredient of liqueurs and bitters, and as a herbal drug. A sensitive and specific high-performance liquid chromatography-diode-array detection-mass spectrometry (HPLC-DAD-MS) method has been developed for the simultaneous identification and quantification of its main coumarins, oxypeucedanin hydrate, oxypeucedanin, ostruthol, imperatorin, osthole, isoimperatorin, and ostruthin. Fast HPLC separation could be achieved on an Acclaim C18 column (150 mm × 2.1 mm i.d., 3 µm) using a mobile phase gradient of acetonitrile-water modified with 0.01% acetic acid. The quantification by HPLC-DAD was performed with imperatorin as external standard and validated to demonstrate selectivity, linearity, precision, and accuracy. The content of the main coumarins was quantitated in various batches of commercial and field-collected rhizomes of Peucedanum ostruthium, as well as in beverages prepared thereof.

Ethnobotanical survey of traditionally used plants in human therapy of east, north and north-east Bosnia and Herzegovina.

AIM OF THE STUDY: The study aims to provide a systematical revision of the traditional use of wild and cultivated plants in north-eastern Bosnia and Herzegovina (Western Balkan Peninsula; Southeast Europe). Thereby, it will extend and complement a recent previous study carried out in middle, southern and western Bosnia and Herzegovina. MATERIAL AND METHODS: Information was collected by performing so called open ethnobotanical interviews. The following data were recorded and systematically assembled in a database: name, age and occupation of the interviewed person; the geographic locality and date of the interview; the name of the used plant; plant parts used; prescription background and preparation procedure as well as indication. Plants mentioned to be used by the informants were collected during field trips done together with the informants and taxonomically determined. The corresponding material was finally deposited in the herbarium of the Department of Pharmacognosy of the University of Vienna for the purpose of documentation. RESULTS: In total, 45 places including villages and mountain areas were visited and 84 persons questioned. 254 wild and cultivated species and 1655 different preparations for the use in traditional human therapy were recorded. The most frequently mentioned indications were disorders of the gastrointestinal tract, respiratory system, urogenital tract, skin, blood system, cardiovascular system, nervous system as well as rheumatism. Infusions were the most frequently prepared formulation. Other applied preparations mentioned with decreasing frequency were decocts, sirups, tinctures, collars, direct application of plants without prior preparation, ointments, freshly pressed juices, oils, powders, fluid unctions, macerations and finally suppositories. Special preparations, typical only for the area of Bosnia and Herzegovina were "mehlems" and some kind of sirup called "dulbe secer" (eng. dulbe sugar). While "mehlems" were already recognized and accordingly discussed for the central, southern and western parts of Bosnia and Herzegovina, "dulbe secer" seems to be known in north-eastern region only. The main compounds of this preparation are sugar or honey, lemon and flowers of one particular species of Rosa (with very small flowers), in Bosnia called "dulbe" rose or "sugar" rose. Prescriptions were verbally delivered for up to more than six generations, traditionally from mother to daughter. For the objective of further analyses and comparisons, the recorded data were inserted in the "VOLKSMED" data base of Austrian prescriptions. CONCLUSIONS: The study showed that there exist considerable similarities in medicinal plant use including indications and type of preparations between the different regions and ethnicities of Bosnia and Herzegovina. Interestingly, there were also only little differences in frequencies of medicinal plant use, indications and preparations between middle, western and southern part Bosnia and Herzegovina on one hand and the eastern, northern and north-eastern part of this county on the other hand. The results also demonstrated the high importance of medicinal plants for the physical health of Bosnian people. 70 of the species reported by Bosnian people were also used in official pharmacy. In addition, a variety of less known plants has been used since ages in traditional therapy of this country and hence may be potential sources for new therapies. Therefore, further pharmaceutical research into this particular and scientifically still underexplored proportion of Bosnian plant biodiversity appears promising and is recommended by the authors.

Traditional medicine in the pristine village of ProkoÅko Lake on Vranica Mountain, Bosnia and Herzegovina.

The results of an ethnobotanical study conducted in the pristine village of ProkoÅko Lake (Vranica Mountain, Bosnia and Herzegovina) in summer 2007 is presented. Informal interviews involving 12 informants known as âtraditional healersâ provided data from 43 plants used in 82 prescriptions. The applied plants were used for a broad spectrum of indications. The most frequent were gastro-intestinal tract ailments, blood system disorders, skin ailments, respiratory tract ailments and urinary-genital tract ailments. The most frequent preparation was an infusion. Other often used preparations were ointments or balms and decocts. The special Bosnian balms known as âmehlemsâ were prepared from freshly chopped or freshly pressed herbal parts of various plant species. Warmed resins from Abies or Picea species, raw cow or pig lard, olive oil and honey served as basis. The traditional doctors, who usually worked as a team, enjoyed such a good reputation that people from all over the country were visiting in search of alternative ways to cure their ailments and diseases. The practical techniques applied by the healers and some of their attitudes and values are reported.

Ethnobotanical study on medicinal use of wild and cultivated plants in middle, south and west Bosnia and Herzegovina.

AIM OF THE STUDY: The results of an ethnobotanical study on the use of wild and cultivated plants in middle, west and south Bosnia and Herzegovina (Western Balkan Peninsula; Southeast Europe) carried out in the years 2006-2009 are presented. Despite the country's extraordinary high plant diversity with about 3600 known species of vascular plants, plant usage in traditional medicine remained largely unexplored in the past. The purpose of this study was the systematic collection of information about usage of wild and cultivated plants in human therapy from Bosnia and Herzegovina. MATERIALS AND METHODS: Information was gathered by performing so called open ethnobotanical interviews by which data on the name, age and occupation of the interviewed person; the geographic locality and date of the interview; the name of the used plant; part of the plant used; the prescription background and preparation procedure as well as the indication was systematically collected. Plants mentioned to be used by the informants were collected, taxonomically determined and corresponding material was deposited in the herbarium of the Department of Pharmacognosy of the University of Vienna. RESULTS: In total, 34 places including villages and mountain areas were visited and 51 people, known as 'traditional healers' by the rest of the inhabitants, questioned. 228 wild and cultivated species and 730 different preparations for the use in human therapy were recorded. Species of the genera Achillea, Hypericum, Mentha, Teucrium, Thymus, and Urtica were particularly highly recommended by the majority of the informants. The most frequently mentioned indications were urogenital tract disorders, respiratory system disorders, gastrointestinal tract disorders, skin ailments, blood system disorders, nervous system disorders, cardiovascular system disorders, and rheumatism. Infusions were the most frequently prepared formulation. Other applied preparations mentioned with decreasing frequency were decocts, ointments, direct application of plants without prior preparation, fluid unctions, sirups and tinctures or collars, freshly pressed juices, powders, and finally macerations. Balms known as "mehlems" were special to Bosnia and were prepared from freshly chopped or freshly pressed herbal parts of various plants. Warmed resins from Abies or Picea species, bees wax, raw cow or pig lard, olive oil and honey were used as additives in the mehlem formulations. Representatives of the genera Arctium, Carlina, Euphrasia, Hypericum, Plantago, Teucrium, and Urtica were most frequently used in these balms. Prescriptions were verbally delivered, usually from mother to daughter and for even more than six generations. For the purpose of further analyses and comparisons, the collected data were inserted in the so called "VOLKSMED" data base of Austrian prescriptions. CONCLUSIONS: The identified broad variety of indications and their frequent applications suggests that traditional plant use is of high importance and still crucial for the medicinal accommodation of Bosnian people. Fifty-seven of the species reported by Bosnian people were used in official pharmacy. In addition, a variety of less known plants has been used since ages in traditional therapy of this country and hence may be potential sources for new therapies. Therefore, further pharmaceutical research into this particular and scientifically still underexplored proportion of Bosnian plant biodiversity appears promising and is recommended by the authors.

Distribution of phenolic compounds in Middleeuropean taxa of the Achillea millefolium L. aggregate.

Achillea millefolium L. s.l. is a cytogenetically, morphologically, and chemically polymorphic aggregate. Besides the sesquiterpenes that possess chemotaxonomic relevance and mediate the antiphlogistic activity, the plant contains phenolic compounds such as dicaffeoylquinic acids and flavonoids causing choleretic and spasmolytic effects. To evaluate their contribution to the chemotaxonomy of European taxa of the A. millefolium group, we developed a SPE-HPLC/UV method that allows quantification of the phenolic constituents in the different taxa. The investigated species displayed differences in the quantitative and qualitative composition of phenolic acids and flavonoids. Hence, they seem to be of chemotaxonomic significance, especially for the distinction of the diploid taxa. Combining the obtained results with the data of the sesquiterpene analyses gives a comprehensive insight into the distribution of those pharmacologically relevant plant constituents in the A. millefolium group.

AFLP analyses demonstrate genetic divergence, hybridization, and multiple polyploidization in the evolution of Achillea (Asteraceae-Anthemideae).

Achillea, a temperate genus of herbaceous allogamous perennials, is a model for evolutionary radiation through hybridization and polyploidization. AFLP analyses were performed on 300 individuals of 66 populations and 27 taxa/cytotypes, mainly from the polyploid A. millefolium aggregate and its suspected hybrid links with other clades of the genus. The mosaic genetic structure of hybrids and polyploids is revealed by specific AFLP bands shared with their assumed parents. In E Asia, A. alpina-4x and A. wilsoniana-4x are allotetraploids between A. acuminata-2x (sect. Ptarmica) and A. asiatica-2x (sect. Achillea-A. millefolium agg.). A. virescens-4x is a hybrid species linking A. nobilis agg. and A. millefolium agg. in S Europe. The hybrid swarm A. clypeolata-2x yen A. collina-4x recently formed in Bulgaria shows no AFLP bands additive to its parents; by contrast, other more ancient allopolyploids exhibit genetic innovations. Relationships within A. millefolium agg. are complex. Five 2x-taxa, mostly well separated and regressive, are limited to Eurasia; seven 4x- and 6x-taxa are intimately linked by hybridization, are expansive, and through A. asiatica-2x/4x have formed the N American polyploids. All these results from AFLPs correspond well to other evidence, and indicate a long history of reticulate evolution in Achillea.