Multiple Approaches for Taxonomic Study of Selected Artemisia L
Total Page:16
File Type:pdf, Size:1020Kb
MULTIPLE APPROACHES FOR TAXONOMIC STUDY OF SELECTED ARTEMISIA L. SPECIES FROM PAKISTAN By Muhammad Qasim Hayat Department of Plant Sciences Quaid-i-Azam University Islamabad, Pakistan 2011 MULTIPLE APPROACHES FOR TAXONOMIC STUDY OF SELECTED ARTEMISIA L. SPECIES FROM PAKISTAN A MANUSCRIPT PRESENTED TO THE QUAID-I-AZAM UNIVERSITY, ISLAMABAD IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN PLANT SCIENCES By Muhammad Qasim Hayat Department of Plant Sciences Quaid-i-Azam University Islamabad, Pakistan 2011 FOREIGN EVALUATORS OF THE THESIS 1. DR. SHAHINA A. GHAZANFAR Head of Temperate Regional Team Royal Botanical Garden, Kew Richmond, Surrey London TW9 3AB United Kingdom Email: [email protected] 2. DR. LORNA DAWSON Macaulay Institute Craigiebuckler Aberdeen AB15 United Kingdom Email: [email protected] 3. DR. PEI SHENGJI Professor Kunming Institute of Botany Chinese Academy of Sciences, Heilongtan Kunming 650204 China Email: [email protected] CERTIFICATION This thesis by Mr. Muhammad Qasim Hayat is accepted in its present form by the Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan as satisfying the thesis requirement for the degree of Doctor of Philosophy in Plant Sciences. Supervisor Dr. MUHAMMAD ASHRAF Professor of Cell Biology NUST Centre of Virology & Immunology National University of Sciences & Technology H-12 Islamabad, Pakistan External Examiner I Dr. M. K. LEGHARI Director Botanical Science Division Pakistan Museum of Natural History Garden Avenue, Shakarparian Islamabad, Pakistan External Examiner II Dr. MUQARRAB SHAH Professor & Dean Department of Microbiology Faculty of Health Sciences Hazara University Mansehra, Pakistan Chairperson Dr. ASGHARY BANO Professor & Chairman Department of Plant Sciences Quaid-i-Azam University Islamabad, Pakistan Date May, 24, 2011 i DEDICATION I DEDICATED MY THESIS TO MY SPIRITUAL MASTER “HAZOOR SUFI IQBAL MUHAMMAD” ii TABLE OF CONTENTS Chapter Title Page CERTIFICATION i DEDICATION ii TABLE OF CONTENTS iii LIST OF TABLES vi LIST OF PLATES vii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi ACKNOWLEDGEMENT xii LIST OF PUBLICATIONS xiii ABSTRACT xiv 1 INTRODUCTION 1-25 1.1. Origin, development and diversification of Artemisia 1 in the geological past 1.2. Evolutionary relationships with other groups in the 1 family Asteraceae 1.3. Pollen Evolution 2 1.4. Kariology 3 1.5. Ecology, center of diversity and geographical 3 distribution 1.6. Life form and morphology 5 1.7. Phenology and reproductive biology 5 1.8. Historical developments in classification 5 1.9. Molecular phylogeny 8 1.10. Generic delimitation 9 1.10.1. Infrageneric classification 10 1.11. Economic importance 12 1.11.1. Medicinal importance 12 1.11.2. Insecticidal, repellent and insect antifeedent 14 properties 1.11.3. Ecological importance 14 1.11.4. Other uses 15 1.12. Perspectives in the study of Artemisia L. 15 1.13. Introduction to the study area 16 1.13.1. Location 16 1.13.2. Geology 16 1.13.3. Climate 17 1.13.4. Phytogeographic regions 17 1.14. Artemisia in Flora of Pakistan 19 1.15. Artemisia in Pakistani - Economic potentials 19 1.15. Justification of Present work 22 1.16. Objectives of Present Study 24 2 REVIEW OF LITERATURE 26-38 2.1. Ethnotaxonomy 26 2.2. Morphology 26 2.3. Leaf epidermal anatomy 31 2.4. Palynology 33 iii 2.5. Phytogeography 35 2.6. Molecular phylogeny 35 2.7. Chemotaxonomy based on Elemental Contents 38 3 MATERIALS AND METHODS 39-66 3.1. Ethnotaxonomy 39 3.2. Morphology 39 3.3. Leaf epidermal anatomy 44 3.4. Palynology 54 3.5. Phytogeography 56 3.6. Molecular phylogeny 58 3.6.1. DNA Extraction 58 3.6.1.1. CTAB method 58 3.6.1.2. DNeasy kit (QIAGEN) method 59 3.6.2. PCR 59 3.6.3. PCR products Purification 59 3.6.4. Sequencing PCR 59 3.6.5. Sequencing 59 3.6.6. Sequence processing 64 3.6.7. Phylogenetic Analysis 64 3.6.7. Bootstrapping 64 3.6.7. Maximum parsimonious analysis 64 3.6.7. Bayesian inference 64 3.6.7. Neighbor joining trees 65 3.6.8. Maximum Like-Hood Phylogenetic trees 65 3.7. Diversity in elemental contents 65 3.7.1. Sample preparation 65 3.7.2. Digestion 65 3.7.3. Atomic absorption Spectrophotometery 66 3.7.3. Statistical analysis 66 3.7.3.1 Principal Component Analysis (PCA 66 3.7.3.2 Cluster Analysis 66 4 RESULTS 67-164 4.1. Ethnotaxonomy 67 4.2. Morphology 88 4.3. Leaf epidermal anatomy 100 4.3.1. Leaf epidermal cells and stomata 100 4.3.2. Foliar trichomes 100 4.3.3. Phylogenetic relationships based on distribution of foliar trichomes in the genus Artemisia 102 4.4. Palynology 128 4.4.1 Phylogenetic analysis of Artemisia l. (Asteraceae) based on micromorphological traits of pollen grains 128 4.5. Phytogeography 139 4.6. Molecular phylogeny 144 4.6.1. Neighbor joining consensus trees 144 4.6.2. Phylogenetic tree with posterior probabilities 144 4.6.3. Maximum likehood phylogenetic tree 144 4.6.4 Maximum parsimonious tree 144 4.7. Chemotaxonomy based on elemental diversity 158 4.7.1 Cluster analysis 158 iv 4.7.2 Principal component analysis 158 5 DISCUSSION 165-180 5.1. Ethnotaxonomy 165 5.2. Morphology 166 5.3. Leaf epidermal anatomy 168 5.4. Palynology 171 5.5. Phytogeography 173 5.6. Molecular phylogeny 173 5.7. Diversity in elemental contents 175 5.8. Conclusions 179 5.9. Recommendations 180 REFERENCES 182-203 v LIST OF TABLES Sr. Title Page 1.1. Comparison among different infrageneric classifications of Artemisia 7 1.2. Phytogeographical Regions in Pakistan 20 2.1. Ethnobotanically identified Artemisia species in the world 27 2.2. Ethnobotanically identified Artemisia species in Pakistan 28 3.1. List of taxa studied and their herbarium vouchers 41-43 3.2. Morphological characters and character states of Artemisia for the phylogenetic analysis. 45-46 3.3. Data matrix used in phylogenetic analysis of Artemisia based on morphological features. 47-50 3.4. Character and character states of foliar trichomes for the Phylogenetic analysis of Artemisia 52 3.5. Data matrix used in phylogenetic analysis of Artemisia based on foliar trichomes 53 3.6. Characters and character states of pollen grains for the phylogenetic analysis of Artemisia. 55 3.7. Character state matrix used in phylogenetic analysis of Artemisia based on pollen features. 57 3.8. Composition of PCR reaction mixture per sample 61 3.9. PCR conditions for genomic amplification 61 3.10. Details of primers used in this study 62 3.11. Reaction mixture composition for sequencing PCR per Sample 63 3.12. Conditions for sequencing PCR 63 4.1. Stomatal characteristics in Artemisia species 103-104 4.2. Guard cells characteristics in Artemisia species 105-106 4.3. Stomatal comples characteristics in Artemisia species 107-108 4.4. Foliar epidermal cells characteristics in Artemisia species 109-110 4.5. Quantitative characteristics of glandular foliar trichomes of Artemisia 123 4.6. Quantitative characteristics of nonglandular foliar trichomes of Artemisia 124-125 4.7. Pollen quantitative characteristics of the studied taxa 129-130 4.8. Chromosome numbers (2n), basic numbers (X) and ploidy levels (PL) of the Pakistani Artemisia 140-141 4.9. Average concentration (ppm) (n=5) along with standard deviation of the elements in studied Artemisia species. 159-160 vi LIST OF PLATES Sr. Title Page 4.1. Artemisia dubia 76 4.2. A. moorcraftiana 76 4.3. A. vulgaris (A: young plants, B: Inflorescence) 77 4.4. A. maritima 77 4.5. A. scoparia 78 4.6. A. santolinifolia 79 4.7. A. herba-alba 80 4.8. A. dubia (A: Young plants, B: Flowers) 80 4.9. A. dracunculus 81 4.10. A. annua (A: Young plants, B: Flowers) 81 4.11. A. absinthium (A: Young plants, B: Flowers) 82 4.12. A. absinthium dry plant 82 4.13. S. brevifolium Population 83 4.14. S. brevifolium 83 4.15. A. roxburghiana 84 4.16. A. roxburghiana 84 4.17. A. tournifortiana 85 4.18. A. biannus inflorescence 86 4.19. A. japonica 87 4.20. A. japonica Leaves 87 4.21. Stomatal variations in Artemisia by means of SEM. 111 4.22. Stomatal variations in Artemisia by means of LM. 112 4.23. Adaxial leaf epidermal cells in Artemisia tangutica by means of SEM 113 4.24. Abaxial foliar surface variations in Artemisia by means of LM 114 4.25. Adaxial foliar surface variations in Artemisia by means of LM 115 4.26. Types of glandular foliar trichomes in Artemisia by means of LM 116 4.26. Types of glandular and nonglandular foliar trichomes in Artemisia by means of LM 117 4. 28. Types of glandular foliar trichomes in Artemisia by means of SEM 118 4.29. Types of nonglandular foliar trichomes in Artemisia by means of SEM 119 4.30. Types of glandular and nonglandular foliar trichomes in Artemisia by means of LM 120 4.31. Types of glandular and nonglandular foliar trichomes in Artemisia by means of SEM 121 vii 4.32. Types of glandular foliar trichomes in Artemisia by means of LM 121 4.33. Scanning electron micrographs showing the equatorial view of pollens of studied taxa 131 4.34. Scanning electron micrographs showing the polar view of pollens of studied taxa 132 4.35. Scanning electron micrographs showing the exine sculpture of studied Taxa 133 4.36. Light micrographs of pollen grains 135 4.37. Scanning electron micrographs of the taxa studied 137 4.38. Scanning electron micrographs of the taxa studied 138 4.39. Gel image of ETS PCR products of studied Artemisia Species 145 viii LIST OF FIGURES Sr.