Population genetic structure and trait associations in forest savory using molecular,morphological and phytochemical markers

In this investigation, morphological, phytochemical and ISSR markers were used to estimate the relationships among and within seven populations of white savory (Satureja mutica), belonging to four provinces in Iran. The individuals were phenotypically diverse, which stamen length, corolla length, corolla diameter, calyx length, bract length, inflorescence length, calyx length and bracteole width were characteristics with the highest variation. Leaf dimensions were in significant correlation with flower and inflorescence characteristics. Chemical compounds of essential oils were found variable in various individuals and all samples were principally composed of phenolic constituents (carvacrol and/or thymol). As a consequence, the plants were classified into two major chemotypes including carvacrol and thymol. A total of 197 band positions were produced by 14 ISSR primers, of which 176 were found polymorphic with 88.91% polymorphism. ISSR genetic similarity values among individuals ranged between 0.45 and 0.94 which was indicative of a high level of genetic variation. Multiple regression analysis (MRA) revealed that phytochemical compositions as dependent variable, showed statistically significant correlation and in association with leaf and flower traits as independent variable, indicating a main role of leaf and flower on production of these compounds. Also, several ISSR fragments were found associated with some morphological traits and phytochemical compositions. The high diversity within and among populations of S. mutica according to different data systems could provide useful information for conservation and selection of cross-parents in breeding programs.     

Differentiating Thymus vulgaris chemotypes with ISSR molecular markers

Common thyme (Thymus vulgaris L.) is a Mediterranean evergreen shrub of the Lamiaceae that shows chemical polymorphism in its natural populations. In Mediterranean habitats six basic chemotypes have been described according to the main component in its essential oil: thymol, carvacrol, linalool, geraniol, α-terpineol, thuyanol-4, of which the thymol chemotype is widely used in cultivation and as a spice. In this study ten selected clones were analysed in terms of chemical composition of the volatile oil and genetic fingerprint. The study question was whether individual chemotypes can be distinguished by molecular markers. Chemical composition was analysed by GC-MS, and for assessing the genetic polymorphism ISSR marker system was chosen as it is reliable and easy to use even in case of less studied species. The studied T. vulgaris clones represented five different chemotypes and could be distinguished using 12 ISSR primers. Clones belonging to the thymol chemotype showed the most distinct separation. The study shows the potential of using molecular markers in breeding and selection of T. vulgaris, being able to differentiate different chemotypes.     

Comparative analysis of essential oil compositions in seven populations of Bakhtiarian savory in natural and field conditions

Bakhtiarian savory (Satureja bachtiarica Bunge) is an endemic herb growing wild in Iran with interesting pharmacological and biological properties. In this research, the variability of essential oil content and phytochemical compositions among seven populations of this species was studied in both natural and field conditions. There was a wide interpopulation variability in phytochemical variation of studied populations under both natural and field conditions, indicating the existence of different chemotypes. Six chemical compositions showed high value in studied plants so that carvacrol and thymol were known as major compounds and confirmed by principal component analysis. Ilam population had the highest thymol in both field (77.10 %) and natural (42.32 %) conditions and was known as a thymol chemotype. Beside, Yazd and Shahrekord populations, respectively, showed the highest carvacrol percentages in both field (84.83 and 63.81 %) and natural (72.50 and 58.05 %) conditions and were known as carvacrol chemotypes. In other populations, carvacrol and thymol were the main components and showed partly equal percentage and these populations were known as carvacrol/thymol chemotypes. According to Pearson correlation analysis, carvacrol was negatively correlated with p-cymene, γ-terpinene and thymol (r = ?0.61, ?0.70 and ?0.98, respectively), indicating very high reverse correlation between carvacrol and thymol. In conclusion, the high value of the essential oil compositions in studied S. bachtiarica populations could provide useful information for conservation and selection of cross parents in breeding programs to develop improved cultivars.     

Chemical and genetic variability of selected Turkish oregano (Origanum onites L.) clones

Turkish oregano (Origanum onites L.) is a major aromatic plant that belongs to Labiatae family. In this work fourteen Turkish oregano clones have been characterized chemically and genetically. Essential oil obtained by hydro-distillation was characterized by gas chromatography. Genetic variation was determined by use of random amplified polymorphic DNA (RAPD) markers. Variation of essential oils in the selected clones was subjected to cluster analysis, and two chemotypes, carvacrol and thymol, were identified. Carvacrol was found to be the main component in all clones except clone-661, the main component of which was thymol. The oregano clones were divided into three main groups by clustering on the basis of RAPD markers. Genetic similarity values among the oregano clones ranged between 0.49 and 0.73 which was indicative of a low level of genetic variation. Clones originating from locations close to each other had similar RAPD markers. Correlation analysis of the genetic distance matrix and the Euclidian distance matrix revealed no significant correlation between them. The results also indicated that there is no relationship between genetic structure of the selected clones and essential oil composition.     

Essential oil chemical diversity of twenty Iranian Origanum vulgare L. subsp. viride populations

Origanum vulgare L. belonging to the Lamiaceae family, commonly known as oregano, is one of the most traded and consumed medicinal plants worldwide. It is a rich natural source of specialized metabolites including essential oils. The present study is the first comprehensive account of the essential oil profile of wild O. vulgare subsp. viride plants in Iran. Twenty native populations from four provinces of northern Iran were investigated for the content and composition of essential oils. Essential oil contents were found to vary from 0.1 to 0.5% (v/w) based on dry weight. Analysis of these oils by GC-FID and GC-MS resulted in a total of forty-nine constituents, representing 96.2–99.7% of the total compositions. The subsequent classification of the studied populations by multivariate statistical procedures characterized four chemotypes: i) γ-terpinene/p-cymene; ii) linalool/α-phellandrene; iii) sabinene; and iv) carvacrol/trans-caryophyllene. These data may be useful in the future selection of populations comprising specific chemical characteristics for industrial purposes, and furthermore contribute beneficial information for developing conservation strategies, domestication and breeding programs of Iranian O. vulgare subsp. viride germplasm.     

Genetic,phenotypic, and phytochemical polymorphism in Eastern European populations of <Emphasis Type="Italic">Mentha arvensis</Emphasis> L.

Variability of M. arvensis from five geographically distanced populations was examined using morphological traits and phytochemical composition of essential oil and with the help of DNA fingerprinting using ISSR markers. The population differentiation based on morphological traits was weak. Analysis of the essential oil composition provided the subdivision of the sample into three groups and, on the basis of the composition of ISSR amplicons, into four groups of specimens. A high degree of genetic polymorphism of M. arvensis and substantial, though incomplete, population differentiation were identified. It was demonstrated that the population of M. arvensis from the Komi Republic was the most genetically isolated, while the populations from Moscow and Penza provinces were weakly differentiated from each other. The population from the Republic of Belarus (near Grodno) was genetically and phytochemically considerably different from the other studied populations, although morphologically indistinguishable from them. We argue that the differentiation was caused not only by the isolation by distance but also owing to the formation of three different ecotypes adapted to different climatic conditions.     

Influence of environmental and genetic factors on the stability of essential oil composition of Thymus pulegioides

Thymus pulegioides plants were collected from various natural habitats of Lithuania and transferred into a new uniform environment. The plants were cloned annually at controlled conditions and their essential oil composition was monitored by capillary GC and GC/MS. The geraniol/geranial/neral (G/G/N), thymol (T), linalool (L), carvacrol/γ-terpinene/p-cymene (C/γT/pC) and thymol/carvacrol/γ-terpinene/p-cymene (T/C/γT/pC) chemotypes of T. pulegioides were studied. It was found that according to the stability of essential oil composition on a sudden change of environmental conditions two types of T. pulegioides can be distinguished: (1) plants which preserve their chemical composition of the essential oils; (2) plants, which considerably change their chemical composition of the essential oils.     

Genetic variability and structure of Quercus brantii assessed by ISSR,IRAP and SCoT markers

Persian oak (Quercus brantii Lindl.) is one of the most important woody species of the Zagros forests in Iran. Three molecular marker techniques: start codon targeted (SCoT), inter-simple sequence repeat (ISSR) and inter-retrotransposon amplified polymorphism (IRAP) markers were compared for fingerprinting of 125 individuals of this species collected from different geographical locations of north-west of Iran. A total of 233 bands were amplified by 18 ISSR primers, of which 224 (96.10%) were polymorphic, and 126 polymorphic bands (97.65%) were observed in 129 bands amplified by 10 IRAP primers. Besides, 118 bands were observed for all 10 SCoT primers, of which 113 were polymorphic (95.71%). Average polymorphism information content (PIC) for ISSR, IRAP and SCoT markers was 0.30, 0.32 and 0.38, respectively, and this revealed that SCoT markers were more informative than IRAP and ISSR for the assessment of diversity among individuals. Based on the three different molecular types, cluster analysis revealed that 125 individuals taken for the analysis can be divided into three distinct clusters. The Jaccard's genetic similarity based on the combined data ranged from 0.23 to 0.76. These results suggest that efficiency of SCoT, IRAP and ISSR markers was relatively the same in fingerprinting of individuals. All molecular marker types revealed a low genetic differentiation among populations, indicating the possibility of gene flow between the studied populations. These results have an important implication for Persian oak (Q. brantii) germplasm characterization, improvement, and conservation.     

Chemical composition, antioxidant and antimicrobial activities of essential oil of Thymus algeriensis wild-growing in Libya

The composition of essential oil isolated from Thymus algeriensis growing wild in Libya was analyzed by GC and GC-MS. The essential oil was characterized with thymol (38.50%) as the major component. The oil was screened for antioxidant activity using DPPH assay, and compared to thymol and carvacrol. Antioxidant activity was high, with the IC₅₀ of 0.299 mg/ml, compared to 0.403 and 0.105 mg/ml for thymol and carvacrol, and 0.0717 mg/ml for BHA. In addition, antimicrobial activity was tested against eight bacteria and eight fungi. T. algeriensis oil showed inhibitory activity against tested bacteria at 0.001–0.05 mg/ml, while bactericidal activity (MBC) was achieved at 0.0025–0.05 mg/ml. For antifungal activity MICs ranged 0.0005–0.025 mg/ml and MFC 0.001–0.05 mg/ml. High antimicrobial activity against the fungi in particular suggests that the essential oil of Thymus algeriensis could have a useful practical application.     

Acaricidal property of the essential oil from <Emphasis Type="Italic">Lippia gracilis</Emphasis> against <Emphasis Type="Italic">Tetranychus urticae</Emphasis> and a natural enemy, <Emphasis Type="Italic">Neoseiulus californicus</Emphasis>, under greenhouse conditions

The essential oil from the leaves of Lippia gracilis was investigated for fumigant and residual activity against Tetranychus urticae (Acari: Tetranychidae) and Neoseiulus californicus (Acari: Phytoseiidae). The results were compared to eugenol, Ortus^® and Azamax^®, as positive controls. Gas chromatography (GC) and GC/mass spectrometry analysis enabled the identification of 28 compounds, accounting for 99.1?±?0.6% of the essential oil. The major constituents were carvacrol (61%), p-cymene (11%) and thymol (11%). Mites were more susceptible to the oil in fumigant tests than in residual tests. Among the components, thymol and β-caryophyllene had the greatest fumigant and residual toxicity against T. urticae, respectively. The role of selected constituents (carvacrol, p-cymene, thymol, limonene, β-pinene, 1,8-cineole, terpinolene and β-caryophyllene) in the acaricidal properties of the L. gracilis essential oil is also discussed. Fumigant and residual effects of Lippia oil were more selective than eugenol with regard to a natural enemy of T. urticae, Neoseiulus californicus. Experiments under greenhouse conditions demonstrated greater toxicity of the Lippia oil in comparison to the positive control at 24, 48 and 72 h after treatment. The results suggest that Lippia oil is a good candidate for the formulation of a botanical acaricide for the integrated management of T. urticae.     

Environmental and seasonal impacts on the chemical composition of Satureja horvatii Šilić (Lamiaceae) essential oils

The chemical composition of the essential oils of Satureja horvatii Šilić from two natural habitats (Mt. Orjen and Mt. Lovćen in Montenegro) and from cultivated plants (Belgrade, Serbia) were characterized. For the latter, plants from the locus classicus, i.e., Orjenske Lokve (Mt. Orjen), were transferred to Belgrade and, after three years of cultivation, the chemical composition of their essential oils at different phenological stages was analyzed. The essential oils were obtained from the aerial parts of the plants by hydrodistillation and analyzed by GC and GC/MS. The yields and chemical compositions of the S. horvatii oils showed significant differences between the plants collected in the natural habitats and those from cultivation, as well as between the plants at different phenological stages. In the populations from the natural habitats, growing in Mediterranean conditions, the most abundant oil constituents were the phenols thymol (63.7% in the samples from Mt. Orjen) or carvacrol (68.1% in the samples from Mt. Lovćen), while the oils from the cultivated plants (Belgrade), growing in continental conditions, were dominated by linalool (up to 65.8 and 55.9% in average). The basic characteristics of the essential oil from plants at the early phenological stage (before flowering) were high percentages of linalool (37.4%), thymol (27.3%), and carvacrol (12.2%). At the stage of flowering, the percentage of linalool (56.6–57.5%) increased, while those of thymol (15.5–15.8%) and carvacrol (1.4–1.5%) significantly decreased. The essential oil of plants in the full stage of fruiting was characterized by the domination of linalool (58.4 and 65.8%) and lower percentages of thymol (7.6 and 1.3%) and carvacrol (0.7 and 0.1%). In conclusion, the oil composition of S. horvatii was found to depend on the pedoclimatic conditions of the habitat and the phenological stage of the plants.     

Variation of Essential Oil Content and Composition,Phenolics, and Yield Related Traits Using Different Pollination Systems in Populations of Thymus Species

The production of self-pollinated plants could be important for improving medicinal plants secondary metabolites. In this study, 11 Thymus populations from eight species were evaluated to determine the effect of self and open pollination on agro-morphological characteristics, total phenolic content (TPC), essential oil (EO) content, and EO components. Inbreeding led to some positive effects of above mentioned traits in most of the studied populations. Total phenolic content ranged from 7.07 to 52.69 mg tannic acid equivalents (TAE) g⁻¹ dry weight (DW) in open pollinated derived populations, while it varied from 1.2 to 55.03 mg TAE g⁻¹ DW in self-pollinated ones. Under open and self-pollination condition, the highest EO content was obtained in T. trautvetteri (3.37 %) and T. pubescens (1.96 %), respectively. Gas chromatography-mass spectrometry (GC/MS) identified 42 compounds including thymol, carvacrol, linalool, p-cymene, γ-terpinene, terpinen-4-ol, and α-terpineol as the main compounds. In most cases, selfed plants compared to open pollinated ones, revealed higher thymol content. T. daenensis-1 showed a significant increase in thymol content (from 25.22 % to 74.3 %) due to self-pollination. Moreover, self-pollination led to emergence of some new compounds. Carvacrol methyl ether was the constituents of Thymus EO that are being reported in self-pollinated populations. Finally, inbreeding in Thymus might be suggested as a useful tool to increase genetic homogeneity for the selection of superior plants for improving secondary metabolite.     

Phytochemical Diversity of the Essential Oils of Mexican Oregano (Lippia graveolens Kunth) Populations along an Edapho‐Climatic Gradient

Mexican oregano (Lippia graveolens) is an important aromatic plant, mainly used as flavoring and usually harvested from non‐cultivated populations. Mexican oregano essential oil showed important variation in the essential‐oil yield and composition. The composition of the essential oils extracted by hydrodistillation from 14 wild populations of L. graveolens growing along an edaphoclimatic gradient was evaluated. Characterization of the oils by GC‐FID and GC/MS analyses allowed the identification of 70 components, which accounted for 89–99% of the total oil composition. Principal component and hierarchical cluster analyses divided the essential oils into three distinct groups with contrasting oil compositions, viz., two phenolic chemotypes, with either carvacrol (C) or thymol (T) as dominant compounds (contents >75% of the total oil composition), and a non‐phenolic chemotype (S) dominated by oxygenated sesquiterpenes. While Chemotype C was associated with semi‐arid climate and shallower and rockier soils, Chemotype T was found for plants growing under less arid conditions and in deeper soils. The plants showing Chemotype S were more abundant in subhumid climate. High‐oil‐yield individuals (>3%) were identified, which additionally presented high percentages of either carvacrol or thymol; these individuals are of interest, as they could be used as parental material for scientific and commercial breeding programs.     

DNA typing and genetic relations among populations of Kelussia odoratissima using ISSR and SRAP markers

Kelussia odoratissima is well known for its medicinal importance. It has been announced as an endangered species. Thus, examining the genetic variation and conservation of this plant is necessary. In the present study, inter simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) molecular markers were employed for the first time to access the genetic diversity and relationships of 77 wild individual plants of K. odoratissima collected from seven populations in Central Zagros region of Iran. A total of 146 bands were amplified by 12 ISSR primers, of which 129 (87.80 %) were polymorphic, while 69 polymorphic bands (83.30 %) were observed among 86 bands amplified by 11 SRAP primers. Polymorphic information content (PIC = 0.32), resolving power (Rp = 7.80), and marker informativeness (MI = 3.48) generated by ISSR primers were higher than that of SRAP analysis (PIC = 0.30, Rp = 5.61, and MI = 1.88). The study indicated that ISSR were more effective than SRAP markers for assessing the degree of genetic variation of K. odoratissima. In both UPGMA dendrograms of ISSR and SRAP, in most cases, individuals from each population were clustered in various groups without clear separation, which demonstrates the high variability of this germplasm in Iran. UPGMA cluster analysis revealed inconsistencies in the clustering patterns, as the Mantel’s test between the dendrograms for ISSR and SRAP data indicated a poor fit for the ISSR and SRAP data types (r = 0.10). Besides, principal coordinate analysis results showed that the first three principal coordinates account for 65.57 % of the total variation and studied seven populations were separated from each other and placed into five groups. These results have an important implication for K. odoratissima germplasm characterization, improvement, and conservation.     

Salicylic acid affects growth,essential oil and chemical compositions of thyme (Thymus daenensis Celak.) under reduced irrigation

Salicylic acid (SA) may reduce the negative impact of water deficit on growth and metabolite yield of Thymus daenensis Celak subsp. daenensis Celak. The effect of foliar application of SA and reduced irrigation on growth, oil yield, chemical components, and antibacterial and antioxidant activities of T. daenensis in field condition were investigated. Treatments comprised 0.0, 1.5 and 3.0 M SA applied to plants under normal irrigation and stressed conditions. Results indicated that irrigation regime had a significant effect on growing degree days (GDD) required to reach early and full flowering. Foliar application of SA influenced GDD from early growing stage to 50 % and full flowering, minimum radius and canopy diameter. The highest values of oil content (3.2 % v/w) and yield (14.9 g m^?2) were obtained from application of 3.0 M SA. Percentage of some chemical constituents in the essential oil extracted from the plants under stress was higher than non-stressed plants. Thymol content was significantly reduced under stressed conditions. Foliar application of SA significantly improved carvacrol, α-thujene, α-pinene and p-cymene contents in the oils, but reduced thymol and, β-caryophyllene amounts. Our results showed that foliar application of SA reduced the negative effect of water deficit on thymol content in the essential oil of T. daenensis. The essential oils of T. daenensis exhibited antioxidant and antibacterial activities when plants were sprayed with 1.5 and 3.0 M SA, respectively.     

Essential oil production in shoot cultures versus field-grown plants of Thymus caespititius

Thymus caespititius Brot. is an important aromatic species, due to synthesis and production of essential oils for the pharmaceutical and food industries. In the present study, levels of essential oils from two chemotypes, including carvacrol/thymol (CT) and sabinene/carvacrol (SC), were evaluated in proliferating shoot cultures (6–12 subcultures following establishment) and compared to those from field-grown plants. The essential oils were isolated by hydrodistillation and analysed by gas chromatography (GC) and GC–mass spectrometry (GC–MS). Cultures grown under in vitro culture conditions, evaluated over six subcultures, were found to maintain stable composition of essential oils. For the CT chemotype, carvacrol (42 %) and thymol (23 %) were the main essential oil components detected in field-grown plants; in proliferating shoot cultures the levels detected attained 17–25 % in the case of carvacrol and 18–23 % in that of thymol, closely followed by carvacryl acetate (15–23 %) and thymyl acetate (11–15 %). For the SC chemotype, carvacrol (13–28 %), sabinene (18–45 %), and thymol (9–12 %) were the main essential oil components detected in both field-grown and proliferating shoot cultures. Our experiments showed that the essential oil composition in proliferating shoot cultures was not only stable, but also qualitatively similar to that of field-grown plants, notwithstanding minor quantitative differences.     

Genotypic diversity and structure of Satureja mutica revealed by inter-simple sequence repeat markers

Satureja mutica (Lamiaceae) is an herbaceous medicinal plant which grows in Iran. The objective of the study was to obtain an overview of the genetic relatedness among and within seven populations of this species using inter-simple sequence repeat (ISSR). Fourteen ISSR primers amplified a total of 197 DNA fragments of which 176 (88.91%) were polymorphic. All ISSR primers were highly effective in discriminating among the populations. Genetic similarity coefficients ranged from 0.45 to 0.94, indicating considerable distance and diversity in the germplasm and were confirmed by clustering analysis. The dendrogram showed a clear clustering pattern of plants indicating a significant association between genetic similarity and geographical distance. Analysis of molecular variance revealed that a greater proportion of total genetic variation existed within populations (75%) rather than among populations (25%). The study indicated that ISSR markers were effective and reliable for assessing the degree of genetic variation of S. mutica. These findings can support future research on the selection of S. mutica for breeding and medicinal plant development.     

<Superscript>1</Superscript>H NMR-based metabolic profiling and target analysis: a combined approach for the quality control of <Emphasis Type="Italic">Thymus vulgaris</Emphasis>

The characterization of T. vulgaris plant material for quality control purposes was performed by NMR-based methods. Direct extraction of 141 T. vulgaris samples with DMSO-d ₆ enabled the obtainment of crude extracts with a representative composition in terms of both volatile and non-volatile constituents. The acquisition of 600 MHz ¹H NMR spectra resulted in a dataset which was analyzed by a combination of metabolic profiling and target analysis approaches. Preliminary analysis of the ¹H NMR spectra was performed by principal component analysis, which revealed sample discrimination on a chemotype basis (thymol, carvacrol and linalool chemotypes). Further minor discriminative constituents were identified as p-cymene, γ-terpinene, rosmarinic acid, and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl. Metabolite identification was accomplished by 1D and 2D NMR techniques and supported by spiking experiments. Fast dereplication of constituents not available as reference compounds was performed by HPLC–SPE–NMR experiments. A targeted approach based on qHNMR was validated for quantification of the identified secondary metabolites. Validation was performed in terms of precision (intra-day RSD ≤ 4.51%, inter-day RSD ≤ 4.18%), repeatability (RSD ≤ 2.30%), accuracy (recovery rates within 93.4 and 103.4%), linearity (correlation coefficients ≥ 0.9990), robustness, and stability. The amount of the dominant monoterpene in thymol, carvacrol, and linalool chemotypes was respectively found to be within 0.4–2.6, 0.7–2.3, and 1.1–3.6% (w/w). Variable amounts of the precursors p-cymene and γ-terpinene were found in thymol and carvacrol chemotypes. The highest amount of rosmarinic acid and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl in the analyzed samples was respectively 4.6 and 0.4% (w/w). Since quantification is performed on a weight basis, the essential oil content can be estimated based on the sum of the quantified monoterpenes. The NMR-based analysis of T. vulgaris represents a more comprehensive approach in comparison to traditional chromatographic methods such as GC and LC, respectively employed for the analysis of volatile and non-volatile constituents. Further advantages lie in the simple sample preparation, rapidity and reproducibility of the NMR analysis.     

Highly differentiated populations of the narrow endemic and endangered species Primula cicutariifolia in China,revealed by ISSR and SSR

The perennial herb Primula cicutariifolia Pax is an endangered and endemic species with narrow distribution in eastern Anhui and Zhejiang provinces of China. In this study, the levels of genetic variation and the pattern of genetic structure in five natural populations of P. cicutariifolia were assessed by inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) markers. Both markers revealed that there was remarkably low genetic variation within populations (e.g., H_e = 0.19 and 0.18, for ISSR and SSR respectively) and high differentiation among populations (G_ST = 0.714 and 0.611; Φ_ST = 0.698 and 0.599, for ISSR and SSR respectively). The level of population genetic diversity was correlated to population size only detected by ISSR markers. The genetic structure of P. cicutariifolia may be explained by limited gene flow that was caused by habitat fragmentation and limited seeds and pollen dispersal ability, self breeding system and biennial life form. To protect and avoid disappearance of P. cicutariifolia, much more attentions should be paid to protect all the remnant populations and their habitats, and three management units, i.e. Tianmushan, Damodao, and Panan units, were proposed.