MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF SELECTED MACROFUNGI IN NORTHERN AREAS OF KHYBER PAKHTUNKHWA, PAKISTAN PhD (Botany) Junaid Khan CENTER FOR PLANT SCIENCES AND BIODIVERSITY UNIVERSITY OF SWAT 2018 MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF SELECTED MACROFUNGI IN NORTHERN AREAS OF KHYBER PAKHTUNKHWA, PAKISTAN PhD (Botany) Submitted by Junaid Khan Roll No. 120851 Supervised by Dr. Hassan Sher Co supervisor Dr. Abdul Nasir Khalid CENTER FOR PLANT SCIENCES AND BIODIVERSITY UNIVERSITY OF SWAT 2018 TABLE OF CONTENTS CONTENTS Page Number Acknowledgements i Abstract ii Chapter 1. Introduction 1 Aims and Objectives 08 2. Materials and Methods 10 Study area 10 Field visits and collection of fruiting bodies 12 Morphological and anatomical characterization 13 Specimen identification and deposition 14 Molecular characterization 14 DNA extraction 14 PCR amplification, Visualization and sequencing 15 Editing of Sequences and BLAST Analysis of ITS sequences 16 3. Results 18 4. Discussion 382 5. Conclusions and Recommendations 419 6. References 421 7. Annexure-A 451 LIST OF FIGURES No. TITLE Page No. 1. Map of the study area……………………………………………………... 11 2. Macroscopic and microscopic characters of Chlorophyllum hortense…… 22 3. Molecular Phylogenetic analysis by of Chlorophyllum hortense and associated taxa inferred from nITS data by using Maximum Likelihood method……………………………………………………………………… 23 4. Macroscopic and microscopic characters of Echinoderma asperum………. 26 5. Macroscopic and microscopic structures of Lepiota cristata………………. 29 6. Molecular Phylogenetic analysis of Lepiota cristata and associated taxa by 30 Maximum Likelihood method inferred from nITS data using MEGA6…….. 7. Basidiomata of Leucoagaricus campestris………………………………… 34 8. Microscopic structures of Leucoagaricus campestris……………………… 35 9. Molecular Phylogenetic analysis of Leucoagaricus campestris and associated 36 taxa by Maximum Likelihood method using MEGA6 software……………. 10. Macroscopic and microscopic characteristics of Lycoperdon microsporon…. 39 11. Molecular Phylogenetic analysis of Lycoperdon microsporon and associated 40 taxa inferred from nITS data by Maximum Likelihood method………….. 12. Macroscopic and microscopic features of Lycoperdon perlatum………….. 42 13. Macroscopic and microscopic features of Macrolepiota excoriata………… 45 14. Molecular Phylogenetic analysis of Macrolepiota excoriata and associated taxa inferred from nITS data using Maximum Likelihood method in 46 MEGA6……………………………………………………………………... 15. Morphological characters of Albatrellus flettiodes…………………………. 50 16. Morphological characters of Albatrellus roseus……………………………. 54 17. Phylogenetic analysis of Albatrellopsis, Albatrellus and associated genera 56 based on nITS molecular data………………………………………………. 18. Morphology of Jahnoporus oreinus………………………………………… 59 19. Molecular Phylogenetic analysis of Jahnoporus oreinus and associated taxa 60 by Maximum Likelihood method using nITS data…………………………. 20. Basidiomata of Amanita ahmadii…………………………………………… 64 21. Microscopic structures of Amanita ahmadii………………………………… 65 22. ITS based Molecular Phylogenetic analysis of Amanita ahmadii and 66 associated species by Maximum Likelihood method……………………….. 23. Basidiomata of Amanita cinerea…………………………………………… 69 24. Microscopic structures of Amanita cinerea………………………………... 70 25. Molecular Phylogenetic analysis of Amanita cinerea by Maximum 71 Likelihood method inferred from nITS data………………………………… 26. Colored photographs of different collections of Amanita cinnamomescens 74 27. Microscopic structures of Amanita cinnamomescens……………………….. 75 28. Morphology of Amanita fusca………………………………………………. 79 29. nITS based Molecular Phylogenetic analysis of Amanita fusca and associated 80 taxa……………………………………………………………… 30. Morphology of Amanita pakistanica……………………………………….. 83 31. Morphology of Amanita pallidorosea………………………………………. 86 32. Molecular Phylogenetic analysis by Maximum Likelihood method of Amanita pallidorosea and associated taxa inferred from nITS data using 87 MEGA6……………………………………………………………………... 33. Morphology of Amanita pantherina………………………………………… 90 34. Molecular Phylogenetic analysis of Amanita pantherina and associated taxa 91 by Maximum Likelihood method based on the Jukes-Cantor model. 35. Basidiomata of Descolea quercina………………………………………….. 96 36. Microscopic structures of Descolea quercina………………………………. 97 37. Molecular Phylogenetic analysis of Descolea quercina and associated taxa 98 inferred form nITS data……………………………………………………... 38. Basidiomata of Butyriboletus pakistanicus …………………………………. 102 39. Microscopic structures of Butyriboletus pakistanicus ………………………. 103 40. Molecular Phylogenetic analysis by Maximum Likelihood method of 104 Butyriboletus pakistanicus and associated taxa by using nITS data ……….. 41. Basidiomata of Caloboletus flavoporus …………………………………….. 106 42. Microstructures of Caloboletus flavoporus ………………………………… 108 43. Molecular Phylogenetic analysis of Caloboletus flavoporus and associated 109 taxa by Maximum Likelihood method using nITS data. ………………….. 44. Basidiomata of Lanmaoa angustispora …………………………………… 112 45. Microscopic structures of Lanmaoa angustispora ………………………… 113 46. Molecular Phylogenetic analysis by Maximum Likelihood method of 114 Lanmaoa angustispora and associated taxa by using nITS data…………… 47. Morphology of Strobilomyces strobilaceus………………………………… 117 48. Morphology of Tylopilus porphyrosporus………………………………….. 120 49. Morphology of Veloporphyrellus purpureus……………………………….. 123 50. RaxML tree of Veloporphyrellus purpureus and associated taxa inferred from 124 ITS data……………………………………………………………….. 51. Different collections of Clavariadelphus elongatus…………………….. 129 52. Microscopic structures of Clavariadelphus elongatus………………….. 130 53. Molecular Phylogenetic analysis of Clavariadelphus elongatus and 131 associated taxa by Maximum Likelihood method…………………………... 54. Basidiomata of Cortinarius clavatus………………………………………... 136 55. Microscopic structures of Cortinarius clavatus…………………………….. 137 56. Basidiomata of Cortinarius longistipes……………………………………... 141 57. Microscopic structures of Cortinarius longistipes………………………….. 142 58. Morphology of Cortinarius striatus………………………………………… 145 59. Maximum likelihood tree of Cortinarius sp. reported during this study and 146 associated ones inferred from ITS data……………………………………… 60. Morphology of Calocera viscosa…………………………………………… 149 61. Morphology of Astraeus hygrometricus……………………………………. 154 62. Molecular Phylogenetic analysis of Astraeus hygrometricus and associated 155 taxa by Maximum Likelihood method inferred from nITS data……………. 63. Morphology of Rhodocybe luteus…………………………………………... 160 64. Molecular Phylogenetic analysis of Rhodocybe luteus and associated taxa by 161 Maximum Likelihood method…………………………………………… 65. Morphology of Coriolopsis gallica…………………………………………. 164 66. Morphology of Gomphus clavatus………………………………………….. 167 67. Morphology of Turbinellus floccosus………………………………………. 170 68. Morphology of Hericium cirrhatum………………………………………… 174 69. Molecular phylogenetic analysis of Hericium cirrhatum and related taxa by 175 maximum likelihood method……………………………………………….. 70. Morphology of Laccaria tortilis…………………………………………….. 178 71. Morphology of Coltircia abieticola………………………………………… 182 72. Microscopic structures of Coltricia abieticola……………………………… 183 73. Molecular Phylogenetic analysis of Coltricia abieticola and associated taxa 184 inferred from nITS data……………………………………………………... 74. Morphology of Galerina marginata………………………………………… 188 75. Morphology of Gymnopilus penetrans……………………………………… 191 76. Basidiomata of Gymnopilus swaticus……………………………………….. 195 77. Microscopic characters of Gymnopilus swaticus……………………………. 196 78. Molecular Phylogenetic analysis of Gymnopilus taxa collected during present 197 study and associated taxa by Maximum Likelihood method………... 79. Basidiomata of Hebeloma rostratum……………………………………….. 200 80. Microscopic structures of Hebeloma rostratum…………………………….. 201 81. Molecular Phylogenetic analysis of Hebeloma rostratum and associated taxa 203 by Maximum Likelihood method…………………………………………… 82. Basidiomata of Phaeocollybia varicolor…………………………………… 206 83. Microscopic structures of Phaeocollybia varicolor………………………… 207 84. Molecular Phylogenetic analysis of Phaeocollybia varicolor and associated 208 taxa by Maximum Likelihood method inferred from nITS data……………. 85. Morphology of Incoybe pallidorimosa……………………………………… 213 86. Molecular Phylogenetic of Inocybe pallidorimosa and associated taxa by 214 Maximum Likelihood method………………………………………………. 87. Morphology of Inocybe putilla……………………………………………… 217 88. Molecular Phylogenetic analysis of Inocybe putilla and associated taxa 218 inferred from nITS data……………………………………………………... 89. Morphology of Inocybe salicetum…………………………………………... 221 90. Molecular Phylogenetic analysis of Inocybe salicetum and associated taxa 222 using nITS data Maximum Likelihood method…………………………….. 91. Morphology of Gymnopus dysodes………………………………………… 226 92. Microscopic structures of Gymnopus dysodes……………………………… 227 93. Molecular Phylogenetic analysis of Gymnopus dysodes and associated taxa 228 by Maximum Likelihood method…………………………………………… 94. Basidiomata of Oudemansiella sparslamellata……………………………... 233 95. Microscopic structures of Oudemansiella sparslamellata………………….. 234 96. Molecular Phylogenetic analysis of ITS dataset of Oudemansiella 235 sparslamellata and associated taxa by Maximum Likelihood method……… 97. Basidiomata of Pluteus populicola………………………………………….. 239 98. Microscopic structures of Pluteus populicola……………………………….