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Phd. Thesis Sana Jabeen.Pdf ECTOMYCORRHIZAL FUNGAL COMMUNITIES ASSOCIATED WITH HIMALAYAN CEDAR FROM PAKISTAN A dissertation submitted to the University of the Punjab in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in BOTANY by SANA JABEEN DEPARTMENT OF BOTANY UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN JUNE 2016 TABLE OF CONTENTS CONTENTS PAGE NO. Summary i Dedication iii Acknowledgements iv CHAPTER 1 Introduction 1 CHAPTER 2 Literature review 5 Aims and objectives 11 CHAPTER 3 Materials and methods 12 3.1. Sampling site description 12 3.2. Sampling strategy 14 3.3. Sampling of sporocarps 14 3.4. Sampling and preservation of fruit bodies 14 3.5. Morphological studies of fruit bodies 14 3.6. Sampling of morphotypes 15 3.7. Soil sampling and analysis 15 3.8. Cleaning, morphotyping and storage of ectomycorrhizae 15 3.9. Morphological studies of ectomycorrhizae 16 3.10. Molecular studies 16 3.10.1. DNA extraction 16 3.10.2. Polymerase chain reaction (PCR) 17 3.10.3. Sequence assembly and data mining 18 3.10.4. Multiple alignments and phylogenetic analysis 18 3.11. Climatic data collection 19 3.12. Statistical analysis 19 CHAPTER 4 Results 22 4.1. Characterization of above ground ectomycorrhizal fungi 22 4.2. Identification of ectomycorrhizal host 184 4.3. Characterization of non ectomycorrhizal fruit bodies 186 4.4. Characterization of saprobic fungi found from fruit bodies 188 4.5. Characterization of below ground ectomycorrhizal fungi 189 4.6. Characterization of below ground non ectomycorrhizal fungi 193 4.7. Identification of host taxa from ectomycorrhizal morphotypes 195 4.8. Soil analysis 198 4.9. Community analysis 199 4.9.1. Alpha diversity 199 4.9.2. Beta diversity 205 4.9.3. Gamma diversity 206 CHAPTER 5 Discussion 234 References 242 Annexure LIST OF FIGURES TITLE PAGE NO. Figure 1. Map of Pakistan showing sampling sites 13 Figure 2. Morphology of Amanita ahmadii 26 Figure 3. Anatomy of Amanita ahmadii 27 Figure 4. Molecular phylogenetic analysis of Amanita ahmadii based on ITS sequences 28 Figure 5. Molecular phylogenetic analysis of Amanita ahmadii based on LSU sequences 29 Figure 6. Morphology of Amanita brunneopantherina 32 Figure 7. Anatomy of Amanita brunneopantherina 33 Figure 8. Molecular phylogenetic analysis of Amanita brunneopantherina based on ITS sequences 34 Figure 9. Morphology of Amanita flavipes 37 Figure 10. Anatomy of Amanita flavipes 38 Figure 11. Molecular phylogenetic analysis of Amanita flavipes based on ITS sequences 39 Figure 12. Morphology and anatomy of Amanita glarea 42 Figure 13. Molecular phylogenetic analysis of Amanita glarea based on ITS sequences 43 Figure 14. Molecular phylogenetic analysis of Amanita glarea based on LSU sequences 44 Figure 15. Morphology of Amanita swatica 47 Figure 16. Anatomy of Amanita swatica 48 Figure 17. Molecular phylogenetic analysis of Amanita swatica based on ITS sequences 49 Figure 18. Morphology of Boletus himalayensis 52 Figure 19. Anatomy of Boletus himalayensis 53 Figure 20. Molecular phylogenetic analysis of Boletus himalayensis based on ITS sequences 54 Figure 21. Molecular phylogenetic analysis of Boletus himalayensis based on LSU sequences 55 Figure 22. Morphology and anatomy of Hortiboletus rubellus 58 Figure 23. Molecular phylogenetic analysis of Hortiboletus rubellus based on ITS sequences 59 Figure 24. Morphology and anatomy of Neoboletus luridiformis 62 Figure 25. Molecular phylogenetic analysis of Neoboletus luridiformis based on ITS sequences 63 Figure 26. Morphology of Xerocomellus rimosus 66 Figure 27. Anatomy of Xerocomellus rimosus 67 Figure 28. Molecular phylogenetic analysis of Xerocomellus rimosus based on ITS sequences 68 Figure 29. Molecular phylogenetic analysis of Xerocomellus rimosus based on LSU sequences 69 Figure 30. Morphology of Cortinarius corrosus 72 Figure 31. Anatomy of Cortinarius corrosus 73 Figure 32. Molecular phylogenetic analysis of Cortinarius corrosus based on ITS sequences 74 Figure 33. Morphology of Cortinarius longistipus 77 Figure 34. Anatomy of Cortinarius longistipus 78 Figure 35. Molecular phylogenetic analysis of Cortinarius longistipus based on ITS sequences 79 Figure 36. Morphology of Geastrum galiyensis 82 Figure 37. Anatomy of Geastrum galiyensis 83 Figure 38. Molecular phylogenetic analysis of Geastrum galiyensis based on ITS sequences 84 Figure 39. Morphology of Gomphidius flavostipus 87 Figure 40. Anatomy of Gomphidius flavostipus 88 Figure 41. Molecular phylogenetic analysis of Gomphidius flavostipus based on ITS sequences 89 Figure 42. Molecular phylogenetic analysis of Gomphidius flavostipus based on LSU sequences 90 Figure 43. Morphology of Hebeloma angustisporium 93 Figure 44. Anatomy of Hebeloma angustisporium 94 Figure 45. Molecular phylogenetic analysis of Hebeloma angustisporium. based on ITS sequences 95 Figure 46. Morphology of Inocybe alba 98 Figure 47. Anatomy of Inocybe alba 99 Figure 48. Morphology of Inocybe flavellorimosa 102 Figure 49. Anatomy of Inocybe flavellorimosa 103 Figure 50. Molecular phylogenetic analysis of Inocybe spp. based on ITS sequences 104 Figure 51. Morphology of Inocybe mimica 107 Figure 52. Anatomy of Inocybe mimica 108 Figure 53. Molecular phylogenetic analysis of Inocybe mimica based on ITS sequences 109 Figure 54. Morphology of Inocybe oblectabilis 112 Figure 55. Anatomy of Inocybe oblectabilis 113 Figure 56. Molecular phylogenetic analysis of Inocybe oblectabilis based on ITS sequences 114 Figure 57. Morphology and anatomy of Rhizopogon flavus 117 Figure 58. Molecular phylogenetic analysis of Rhizopogon flavus based on ITS sequences 118 Figure 59. Morphology of Russula amythistina 122 Figure 60. Anatomy of Russula amythystina 123 Figure 61. Ectomycorrhiza of Russula amythestina 124 Figure 62. Molecular phylogenetic analysis of Russula amethystina based on ITS sequences 125 Figure 63. Morphology of Russula delica 128 Figure 64. Anatomy of Russula delica 129 Figure 65. Molecular phylogenetic analysis of Russula delica based on ITS sequences 130 Figure 66. Morphology of Russula pakistanica 136 Figure 67. Scanning electron micrographs of basidiospores of Russula pakistanica 137 Figure 68. Anatomy of Russula pakistanica 138 Figure 69. Ectomycorrhiza of Russula pakistanica 139 Figure 70. Molecular phylogenetic analysis of Russula pakistanica based on ITS sequences 140 Figure 71. Molecular phylogenetic analysis of Russula pakistanica based on LSU sequences 141 Figure 72. Morphology of Russula rubecola 144 Figure 73. Anatomy of Russula rubecola 145 Figure 74. Molecular phylogenetic analysis of Russula rubecola based on ITS sequences 146 Figure 75. Morphology of Suillus convexatus 149 Figure 76. Anatomy of Suillus convexatus 150 Figure 77. Molecular phylogenetic analysis of Suillus convexatus based on ITS sequences151 Figure 78. Morphology of Tricholoma terreum 154 Figure 79. Anatomy of Tricholoma terreum 155 Figure 80. Molecular phylogenetic analysis of Tricholoma terreum based on ITS sequences 156 Figure. 81. Morphology of Gyromitra khanspurensis 159 Figure 82. Anatomy of Gyromitra khanspurensis 160 Figure 83. Molecular phylogenetic analysis of Gyromitra khanspurensis based on ITS sequences 161 Figure. 84. Morphology of Morchella pakistanica 164 Figure 85. Anatomy of Morchella pakistanica 165 Figure 86. Molecular phylogenetic analysis of Morchella pakistanica based on ITS sequences 166 Figure. 87. Morphology of Verpa asiatica 169 Figure 88. Anatomy of Verpa asiatica 170 Figure 89. Molecular phylogenetic analysis of Verpa asiatica based on ITS sequences 171 Figure 90. Molecular phylogenetic analysis of Verpa asiatica based on LSU sequences 172 Figure. 91. Morphology of Peziza khanspurensis 175 Figure 92. Anatomy of Peziza khanspurensis 176 Figure 93. Molecular phylogenetic analysis of Peziza khanspurensis based on ITS sequences 177 Figure. 94. Morphology of Geopora pinyonensis 181 Figure 95. Anatomy of Geopora pinyonensis 182 Figure 96. Molecular phylogenetic analysis of Geopora pinyonensis based on ITS sequences 183 Figure 97. Molecular phylogenetic analysis of ectomycorrhizal hosts based on ITS sequences 185 Figure 98. Graph showing absolute abundance of above ground ectomycorrhizal fungal taxa in stand 1 208 Figure 99. Pie chart showing relative abundance of above ground ectomycorrhizal fungal taxa in stand 1 208 Figure 100. Rank abundance curve of above ground ectomycorrhizal fungal taxa in stand 1 209 Figure 101. Graph showing absolute abundance of below ground ectomycorrhizal fungal taxa in stand 1 209 Figure 102. Pie chart showing relative abundance of below ground ectomycorrhizal fungal taxa in stand 1 210 Figure 103. Rank abundance curve of below ground ectomycorrhizal fungal taxa in stand 1 210 Figure 104. Graph showing absolute abundance of above ground ectomycorrhizal fungal taxa in stand 2 211 Figure 105. Pie chart showing relative abundance of above ground ectomycorrhizal fungal taxa in stand 2 211 Figure 106. Rank abundance curve of above ground ectomycorrhizal fungal taxa in stand 2 212 Figure 107. Graph showing absolute abundance of below ground ectomycorrhizal fungal taxa in stand 2 212 Figure 108. Pie chart showing relative abundance of below ground ectomycorrhizal fungal taxa in stand 2 213 Figure 109. Rank abundance curve of above ground ectomycorrhizal fungal taxa in stand 2 213 Figure 110. Graph showing absolute abundance of above ground ectomycorrhizal fungal taxa in stand 3 214 Figure 111. Pie chart showing relative abundance of above ground ectomycorrhizal fungal taxa in stand 3 214 Figure 112. Rank abundance curve of above ground
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