published_or_final_version / Chinese Medicine / Master / Master of Philosophy

Public land management agencies, conservation organizations, and landowners are interested in expanding the diversity of plant species used in rangeland restoration seedings. While the restoration of native grasses and shrubs in the Great Basin has become increasingly successful, restoration of native forbs continues to be problematic. In the Great Basin, soil water availability and soil fungal pathogens are thought to limit to restoration success. During the course of two years, we conducted two field experiments at three sites in the Great Basin that spanned a latitudinal gradient encompassing different precipitation and temperature patterns. In the first experiment, we evaluated two treatments for enhancing native forb restoration – snow fences and N-sulate fabric. In addition, we tested whether multiple fungicide and hydrophobic seed coatings could reduce seed and seedling mortality from soil fungal pathogens. To quantify the effectiveness of treatments, we tracked the fate of sown seeds over four life stages: germination, seedling emergence, establishment, and second-year survival. We found that snow fences and N-sulate fabric had varying degrees of success for increasing seedling emergence or establishment but ultimately did not increase second-year survival. Seed coatings increased seedling emergence but did not increase establishment or second-year survival. In the second experiment, we replicated the first experiment and also measured soil water availability to better understand how snow fences and N-sulate fabric alter soil water availability, and if differences in soil water availability can explain restoration outcomes. While we found that our treatments can increase soil water availability, increased soil water did not consistently result in better restoration outcomes. Snow fences did not benefit any life stage at any site while N-sulate fabric had positive and negative effects on forb restoration depending on the site. Seed coatings increased seedling emergence and establishment at all sites, warranting further research with other forb species. Results from both experiments provide insights for developing new treatments and techniques that can improve native forb restoration in the Great Basin and similar semiarid systems.

Seed germination is pivotal in the life cycle of native plants in a restorative context because initiation of the metabolic processes critical to establishment is key to survival in such a competitive environment. Dormancy characteristics of some native plants including the subject species, Astragalus utahensis, have evolved mechanisms to control germination in order to maintain a seed bank and ensure germination at the right time under optimal conditions. In vitro germination studies confirm beneficial interactions between Alternaria and Aspergillus fungi and Astragalus utahensis seed. Inoculated seed trials (1.0 x 106 spores/mL) exhibited a highly significant difference in percent germination between the uninoculated control at 5.0 % germination and seeds inoculated with Alternaria and Aspergillus germinating at 95 % and 55 %, respectively. Germination trials conducted in the greenhouse revealed a beneficial relationship between fungal spore inoculation and seed germination. Control seeds germinated in soil at a rate of 16.0 %; three times as high as exhibited in vitro. Seed inoculated with either Alternaria or Aspergillus seeds germinated in soil at the same rate of 50.0 %. A seed germination trial conducted in the field demonstrated a beneficial response with Aspergillus inoculation. Fall plantings on two sites near Fountain Green and Nephi, Utah confirm this beneficial response to Aspergillus spore inoculation. These field trials indicated a highly significant response with the germination of scarified control seed at 14.7 % and the Aspergillus and Alternaria treated seed germinating at 29.3 and 19.3 %, respectively. Greenhouse germination trials with spore-inoculated seed indicated a 100% survival rate. Astragalus utahensis seeds germinated at an accelerated rate when inoculated with Aspergillus and Alternaria spores in-vitro. The beneficial germination response of fungal inoculated seeds indicates the efficacy of these treatments in dormancy contravention in hard-seeded species.

Astragalus ampullarioides and A. holmgreniorum are two rare endemics of southwestern Utah. Over two consecutive field seasons (2009-2010) we examined pre-emergent reproductive success, based on F/F and S/O ratios, from populations of both Astragalus ampullarioides and A. holmgreniorum, estimated the density of the soil seed bank of A. holmgreniorum as a measure of potential post-emergent reproductive success, and estimated seed persistence within the soil seed bank. Fruit/flower (F/F) ratios and seed/ovule (S/O) ratios varied significantly between populations and among years in both species, and showed low reproductive output in both taxa. In Astragalus ampullarioides F/F and S/O were 0.06±0.01 and 0.16±0.02, respectively (2009), and 0.14±0.01 and 0.41±0.02, respectively (2010). For Astragalus holmgreniorum F/F and S/O ratios were 0.11±0.01 and 0.38±0.02, respectively (2009), and 0.23±0.01 and 0.66±0.02, respectively (2010). Although Astragalus holmgreniorum exhibited a low soil seed bank density (4.3 seeds m-2), seed persistence data showed low a low percentage of seeds germinated during the first year in the soil seed bank. Seeds remaining in the seed bank maintained high percent viability. Soil seed persistence of Astragalus ampullarioides differed from A. holmgreniorum in that a high percentage of seeds germinated during the first year in the soil seed bank. A high percentage of viability in ungerminated seeds was also maintained in A. ampullarioides. Although these species differ in life histories and dependence on soil seed banks, an understanding of the strategies unique to each species will prove useful in management plans.

Le genre Astragalus L. (Fabaceae) est l'un des genres ayant le plus grand nombre de représentants parmi les angiospermes. Son centre d'origine et de diversité est situé dans les zones arides des montagnes de l'Asie centrale et sud-ouest. Au Liban, ce genre est aussi l'un des plus genres représentés dans la flore, avec 62 espèces et sous-espèces et 22 espèces endémiques identifiés. Les différents taxons de ce genre sont difficiles à identifier en se basant uniquement sur les caractères morphologiques et leur statut actuel de la distribution doit être évaluée. Les relations phylogénétiques, les variations dans la taille du génome et le rôle de la polyploïdie dans l'évolution du genre Astragalus dans les chaînes de montagnes du Liban ont été étudiés. Nos données confirment qu'un polymorphisme chromosomique interspécifique significatif existe dans le genre Astragalus du Liban et la polyploïdie et l'évolution subséquente du génome peuvent être d'importants moteurs de l'évolution de ce genre. Le processus de diversification du genre Astragalus qui a eu lieu au Liban a été analysé en utilisant des méthodes de datation phylogénétiques et moléculaires et des analyses des aires ancestrales. Nos résultats confirment que le Liban est le troisième centre de diversité pour les Astragales et doit être considéré comme un «berceau» de la biodiversité. Par conséquent, cette étude est une contribution à une meilleure compréhension de l'évolution et des processus biogéographiques à l'origine de la mise en place de la biodiversité au Liban, avec une finalité appliquée de conservation biogéographique. / The genus Astragalus L. (Fabaceae) is one of the genera with the largest number of representatives among the angiosperms. Its center of origin and diversity is located in the arid mountains of Central and Southwest Asia. In Lebanon, this genus is also one of the most represented genera in the flora, with 62 species and subspecies and 22 endemic species identified. The different taxa of this genus are difficult to identify based only on morphological characters and their current status of distribution must be evaluated. Phylogenetic relationships, changes in genome size and the role of polyploidy in the evolution of Astragalus genus in the Lebanese mountains range were studied. Our data confirm that a significant interspecific chromosomal polymorphism exists in the genus Astragalus of Lebanon and polyploidy and the subsequent evolution of the genome may be important drivers of the evolution of this genus. The diversification process of Astragalus genus that took place in Lebanon was analyzed using phylogenetic and molecular dating methods and analysis of ancestral areas. Our results confirm that Lebanon is the third center of diversity and should be considered as a "cradle" of biodiversity. Therefore, this study is a contribution to a better understanding of evolutionary and biogeographic processes behind the development of biodiversity in Lebanon, with an applied purpose of biogeographic conservation.

Law Sui-Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 99-109). / Abstracts in English and Chinese. / CONTENTS --- p.i / ABSTRACT --- p.v / 撮要 --- p.vii / ACKNOWLEDGEMENTS --- p.ix / LIST OF FIGURES & TABLES --- p.xi / ABBREVIATIONS --- p.xv / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- The Transition of Hyperplastic to Hypertrophic Growth During Heart Development --- p.1 / Chapter 1.2 --- The Controversial Capability of Heart Regeneration --- p.3 / Chapter 1.3 --- Challenges in Treating Heart Diseases --- p.5 / Chapter 1.4 --- A New Insight Behind Traditional Chinese Medicine (TCM) for Treating Heart Diseases --- p.7 / Chapter 1.5 --- The Potential Mitogenic TCMs on Cardiomyocytes --- p.10 / Chapter 1.5.1 --- Radix Astragali --- p.11 / Chapter 1.5.2 --- Radix Ophiopogonis --- p.12 / Chapter Chapter 2 --- MATERIALS & METHODS --- p.14 / Chapter 2.1 --- Materials --- p.14 / Chapter 2.2 --- Cell Culture --- p.16 / Chapter 2.2.1 --- Primary neonatal rat cardiomyocytes cell culture --- p.16 / Chapter 2.2.1.1 --- Mayer's hemalum-eosin staining --- p.17 / Chapter 2.2.2 --- Primary rat fibroblasts cell culture --- p.18 / Chapter 2.2.3 --- H9C2 cardiac cell culture --- p.18 / Chapter 2.3 --- TCMs Preparation and Treatment --- p.19 / Chapter 2.3.1 --- Preparation of TCMs powder from aqueous extracts --- p.19 / Chapter 2.3.2 --- Preparation of culture medium with TCMs powder --- p.19 / Chapter 2.3.3 --- Pre-treatment of undifferentiated and differentiated H9C2 cardiac cells with TCMs --- p.20 / Chapter 2.3.4 --- Post-treatment of differentiated H9C2 cardiac cells with TCMs --- p.20 / Chapter 2.4 --- Assessment of DNA Synthesis and Proliferation --- p.21 / Chapter 2.4.1 --- Tritiated thymidine incorporation assay --- p.21 / Chapter 2.4.2 --- 5-Bromo-2'-deoxy-uridine (BrdU) assay --- p.22 / Chapter 2.4.3 --- Cell counting --- p.23 / Chapter 2.4.4 --- Statistical analysis --- p.23 / Chapter 2.5 --- Screening of Differentially Expressed Genes in H9C2 Cells after TCM Treatment by cDNA Microarray --- p.25 / Chapter 2.5.1 --- Total RNA extraction --- p.25 / Chapter 2.5.2 --- RNA labeling --- p.26 / Chapter 2.5.2.1 --- Synthesis of fluorescence labeled probe --- p.26 / Chapter 2.5.2.2 --- Purification of fluorescence labeled probe --- p.27 / Chapter 2.5.3 --- Microarray hybridization --- p.28 / Chapter 2.5.3.1 --- Concentration of fluorescence labeled probe --- p.28 / Chapter 2.5.3.2 --- Hybridization --- p.28 / Chapter 2.5.3.3 --- Post-hybridization treatment --- p.29 / Chapter 2.5.4 --- Data collection --- p.29 / Chapter 2.5.4.1 --- Scanning of slide --- p.29 / Chapter 2.5.4.2 --- Image processing: spots finding and quantification --- p.30 / Chapter 2.5.5 --- Data normalization and analysis --- p.30 / Chapter 2.6 --- Confirmation of Differentially Expressed Genes in H9C2 Cells after TCM Treatment by RT-PCR --- p.32 / Chapter 2.6.1 --- DNase I digestion of total RNA sample --- p.32 / Chapter 2.6.2 --- First-strand cDNA synthesis --- p.32 / Chapter 2.6.3 --- RT-PCR of the candidate genes --- p.33 / Chapter Chapter 3 --- RESULTS --- p.36 / Chapter 3.1 --- Neonatal Primary Rat Cardiomyocytes --- p.36 / Chapter 3.1.1 --- Preparation of high-purity neonatal primary rat cardiomyocytes --- p.36 / Chapter 3.1.2 --- Neonatal primary rat cardiomyocytes ceased to undergo DNA replication after 6-day in vitro culturing --- p.38 / Chapter 3.1.3 --- Both MD and HQ promoted the growth of day 1 primary rat cardiomyocytes in dose- and time-dependent manners --- p.40 / Chapter 3.1.4 --- HQ is more potent than MD in promoting the growth of day 7 primary rat cardiomyocytes --- p.43 / Chapter 3.2 --- H9C2 Cardiac cells --- p.45 / Chapter 3.2.1 --- Proliferative effect of MD and HQ on undifferentiated H9C2 cardiac cells --- p.45 / Chapter 3.2.2 --- Pre-treatment of HQ on H9C2 cardiac cells during differentiation --- p.50 / Chapter 3.2.3 --- Pre-treatment of MD and HQ on differentiated H9C2 cardiac cells --- p.52 / Chapter 3.2.4 --- Post-treatment of MD on differentiated H9C2 cardiac cells…… --- p.55 / Chapter 3.3 --- Primary Rat Fibroblasts --- p.57 / Chapter 3.3.1 --- Proliferative effect of MD and HQ on primary rat fibroblasts --- p.58 / Chapter 3.4 --- Screening of Differentially Expressed Genes in H9C2 Cells after HQ Treatment by cDNA Microarray --- p.60 / Chapter 3.4.1 --- Differentially expressed genes in undifferentiated H9C2 cardiac cells after HQ treatment --- p.60 / Chapter 3.4.2 --- Differentially expressed genes in differentiated H9C2 cardiac cells after HQ treatment --- p.66 / Chapter 3.4.3 --- Comparison of differentially expressed genes in both undifferentiated and differentiated H9C2 cardiac cells after HQ treatment --- p.72 / Chapter 3.5 --- Confirmation of Differentially Expressed Genes in H9C2 Cells after HQ Treatment by RT-PCR --- p.73 / Chapter 3.5.1 --- "Preferential up-regulation of N-G, N-G-dimethylarginine dimethylaminohydrolase mRNA expression level in undifferentiated H9C2 cardiac cells after HQ treatment " --- p.74 / Chapter 3.5.2 --- Preferential up-regulation of heme oxygenase-3 mRNA expression level in undifferentiated H9C2 cardiac cells after HQ treatment --- p.75 / Chapter 3.5.3 --- Preferential up-regulation of cyclin B mRNA expression level in differentiated H9C2 cardiac cells after HQ treatment --- p.76 / Chapter Chapter 4 --- DISCUSSION --- p.77 / Chapter 4.1 --- HQ Being a More Effective Mitogenic TCM than MD on Cardiomyocytes Exerted its Effect in Dose- and Time Dependent --- p.79 / Chapter 4.2 --- Mitogenic Effect of Both MD and HQ might Possibly Due to the Regulation of Intrinsic Factors --- p.82 / Chapter 4.3 --- HQ Rather Than MD Showed a Higher Specificity in Promoting DNA Synthesis in Cardiomyocytes --- p.83 / Chapter 4.4 --- The Differentially Expressed Genes were Supported by The Clinical Functions of HQ --- p.85 / Chapter 4.5 --- Relating the Differentially Expressed Genes with Cardiac Growth and Development --- p.87 / Chapter 4.6 --- The Hypothetic Mechanisms of Action that HQ Exerted on Cardiac Growth and Development --- p.92 / Chapter 4.7 --- Future Prospect --- p.94 / Chapter 4.7.1 --- In vivo study of HQ on the proliferation of rat cardiomyocytes from neonatal to postnatal development --- p.94 / Chapter 4.7.2 --- The study of transgenic mice carrying the target gene regulated by HQ on cardiac growth and development --- p.96 / Chapter 4.7.3 --- The determination of active component of HQ on cardiac growth and development --- p.97 / REFERENCES --- p.99 / APPENDIX --- p.110

In the exploration into new therapeutic agents for human disease, medicinal herbs offer an enormous resource due to their wide range of biologically active components. However, because of these biologically active components, medicinal herbs can also have toxic side effects. The focus of this thesis is the effect of herbal therapies, both good and bad, on chronic kidney disease (CKD) and tubulointerstitial fibrosis. Tubulointerstitial fibrosis is considered one of the defining characteristics of CKD. In Chapter 1, the literature regarding the pathogenesis of tubulointerstitial fibrosis is reviewed, beginning with the mechanisms of its development, the main structural and functional features, and the molecular mediators. The structural features include activation of resident fibroblasts and transition of tubular epithelial cells into myofibroblasts, deposition of extracellular matrix proteins, increased apoptosis of normal cells of the renal nephron and development of tubular atrophy, increased renal oxidative stress, and hypoxia of renal tissues. Molecular mediators that are explored include angiotensin II, transforming growth factor-ß1 and numerous other cytokines and growth factors. Pharmacological manipulation of these features and their molecular mediators for regression of tubulointerstitial fibrosis is then discussed. Currently, the gold standard of therapy for people with CKD is blockade of the renin-angiotensin-aldosterone system with angiotensin converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers (ARBs). Because of the complexity of the pathogenesis of renal fibrosis and the multiple mechanisms by which ACEIs and ARBs work, this portion of the thesis focuses on the qualities that additional agents should have to complement their actions. These additional agents could work by decreasing oxidative damage, by decreasing fibroblast numbers through apoptosis, through the interruption of inflammatory, fibrotic mediators, by increasing anti-inflammatory mediators or through other mechanisms. The literature review therefore continues with a discussion of the historical use of medicinal herbs in genitourinary pathologies and the known contributions that medicinal herbs can make to the treatment or development of tubulointerstitial fibrosis and CKD. From this review, a large number of herbs were identified as having traditional use in renal disorders or as being of interest, currently, to researchers of renal pathologies. However, much information is lacking regarding the mechanisms by which the hypothesised benefits occur, making it impossible to assess which herb(s) may offer valuable additive support or alternative treatments to the therapies currently given to people with CKD. Furthermore, there is a lack of information regarding toxicity of these herbs; many herbs have never been assessed in cell culture or in animal toxicity studies. It was apparent that preliminary in vitro work was necessary before in vivo pharmacological work could be undertaken. This thesis, therefore, aimed to test the following hypotheses: (1) That medicinal herbs used currently for treatment of renal dysfunction have high anti-oxidant properties that can be further enhanced by specific extraction processes; (2) That the in vitro testing of selected extracts from medicinal herbs, identified in (1), will reveal some anti-oxidant benefits or indications of toxicity that need careful analysis in animal studies; (3) That careful in vivo testing of specific toxic medicinal herbs identified in these leadup studies will define specific pathological processes that predict an outcome of CKD; and (4) That careful in vivo testing of selected medicinal herbs, used in conjunction with more conventional medicines for CKD, will show an additive benefit when used to ameliorate development of CKD induced using an established animal model. The subsequent laboratory work was designed to test the validity of these hypotheses and the results are then presented in Chapters that each comprise a publication. The aim of Chapter 2 was to present a systematic analysis of the oxidant properties of 55 medicinal herbs that have been used traditionally to treat kidney and urinary disorders or have been of recent interest to researchers of renal disorders. Since different extraction processes yield different constituents, each of the herbs was sequentially extracted with three solvents of decreasing polarity. An assay was performed on each of the fractions to determine the oxygen radical absorbance capacity. The aim of Chapter 3 was to test the benefit or otherwise of each of the three extracts of the chosen herbs using an in vitro cell study. Each extract was tested for potential toxic, apoptotic, mutagenic and antioxidant activity on normal mammalian renal tubular epithelial cells (NRK-52E). The effect of the extracts on renal fibroblasts (NRK-49F) was also analysed. Several specific hypotheses arose from the combination of the systematic analyses and the literature review regarding benefits and toxicities of a number of the extracts. The subsequent in vivo work was designed to test the validity of two of these hypotheses. The aim of Chapter 4 was to test the hypotheses developed from the results of the previous Chapter. The herb Dioscorea villosa had demonstrated extreme cytotoxicity to mammalian renal epithelial cells and had caused transdifferentiation of epithelial cells into fibroblasts. An in vivo rodent model was used to test chronic dosage with this herb and its toxicity and predisposition for induction of CKD verified. The aim of Chapter 5 was to determine whether a herbal preparation (Angelica sinensis and Astragalus membranaceus) that had some support from the literature and the results from Chapters 2 and 3, could complement the actions of ACEIs in a rodent model of renal fibrosis (unilateral ureteral obstruction). The combination of herbal medicines and the ACEI was significantly more effective than the ACEI alone in ameliorating several characteristics of CKD development. To conclude the thesis, Chapter 6 provides an overview discussion of the results and a critical analysis of the methods used. Further, Chapter 6 looks towards future experiments that are planned to further resolve issues of concern about effects on renal health from use of medicinal herbs. .

Increased temperatures due to anthropogenic-induced climate change may raise the threat of extinction for taxa with sessile life histories (e.g., plants) in the near future. Linking climate change models to demographic models may provide useful insights into the potential effects of environmental changes on rare plants, and therefore aid in their current and future conservation. Population demographers generally agree that mechanistic models from a reductionist perspective are necessary to test assumptions in population drivers. For the first study, I assessed the climate vulnerability of a rare plant species, Pyrrocoma radiata, with a mechanistic model of four climatically-similar populations. I used environmentally-driven demographic models to estimate vital rates and population sizes from a nonlinear, nonparametric regression with local climate variables. I assessed the utility of this environmentally-correlated, stage-structured population matrix model compared to a stationary model of independent and identically-distributed environmental stochasticity. I then simulated future population projections based on climate conditions predicted by General Circulation Models (GCMs) under opposing emission scenarios. The second study hopes to answer population-level questions using a traditionally community-level method, non-metric multidimensional scaling, which considers correlation structure between response variables and can be used to find environmental correlates of the ordination axes. Demographic data on a threatened perennial, Astragalus tyghensis, were collected from five sites in the Tygh Valley, OR. I considered correlation structure between demographic vital rates to find environmental correlates of the ordination axes. The search for an environmental driver of population vital rates was successful for the two study species. Previous year dry dormant season precipitation likely affects the fertility rates a year later in P. radiata populations, and dry growing season reference evapotranspiration rates positively correlated with a growth gradient in A. tyghensis. Based on predicted precipitation, P. radiata is expected to rapidly decline by 2050, but this may be due to biases in the two GCMs and reliance on only one environmental factor. The NMS ordination adequately captured most of the variation in transition elements for the years and populations from A. tyghensis demographics. I provided support to the claim that model predictions can improve with the inclusion of mechanistic relationships. The inclusion of abiotic drivers in models used to predict population trends is supported by our study and may enhance predictive power in population viability assessments under changing climates. / Graduation date: 2013

University of Macau / Institute of Chinese Medical Sciences

Evolutionary relationships within and among three Astragalus sections (Incani DC., Hypoglottidei DC., and Dissitiflori DC.) that were native to Turkey were inferred from variations of nucleotide sequences of both chloroplast and nuclear genome regions. In the current study, Fifty-six species included in the three Astragalus sections were utilized to figure out phylogenetic relationships and estimate evolutionary divergence time based on DNA sequence of trnL intron (trnL5&rsquo / -L3&rsquo / ) , trnL3&rsquo / -F(GAA) (trnL-F intergenic spacer), trnV intron, matK (maturase kinase) cpDNA (chloroplast) and ITS (internal transcribed spacer) nDNA (nuclear) regions. Fifty-six Astragalus species with their replicas and one Cicer species as outgroup were analyzed by polymerase chain reaction amplification and DNA sequencing methods. Eleven unknown samples were also used in the current study to understand their section and species name. The results of the study indicated that unknown A35 and A52 samples could be named as A. dasycarpus, while unknown A65 and A66 samples as A. ovatus and lastly unknown A2 sample as A. nitens or A. aucheri. Section of unknown A3, A16, A20, A108, A109 and A110 samples were determined as Incani, but the exact species identification of these samples were not possible because of their close phylogenetic associations with more than one species. Highest genetic diversity was observed when the DNA sequences of ITS nrDNA (nuclear ribosomal) region comprising three subregions as ITS1, 5.8S and ITS2 was used, while the lowest one was calculated when DNA sequence of trnL-F cpDNA region was analyzed. The genetic divergence between Incani and Dissitiflori sections was highest whereas between Hypoglottidei and Dissitiflori was lowest based on all used regions. To figure out phylogenetic relationships among Astragalus species distributed in Turkey and in other regions of the World, DNA sequences of studied regions of foreign samples were collected from the NCBI database and were evaluated with DNA sequence of Turkish species used in the curent study. The Iranian samples either scattered in the phylogenetic tree or attached to our samples externally. South and North American samples (New World Astragalus or Neo Astragalus group) were nested within a different subcluster, which was located in the main cluster produced by samples of Old World Astragalus group (Turkish samples). With these results, we can say that New World Astragalus group is monophyletic and diverged from Old World Astragalus group. Evolutionary divergence time for Astragalus genus was estimated as about 12.5 - 14.5 million years (Ma), and that of New World Astragalus group as 5.0 - 4.0 Ma when rates of nucleotide substitutions of trnL intron and matK cpDNA regions were analyzed. In addition to evolutionary divergence time estimation for Astragalus and New World Astragalus group, divergence times among used three sections of the genus were also calculated by using DNA sequences of trnL, trnV intron and matK cpDNA regions and results indicated that Hypoglottidei and Dissitiflori sections diverged about 5.0-7.0 million years later than Incani section.