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MOLECULAR CHARACTERIZATION AND IDENTIFICATION OF GILLED FUNGI FROM HIMALAYAN MOIST TEMPERATE FORESTS OF PAKISTAN USING INTERNAL TRANSCRIBED SPACERS (ITS) OF rDNA A THESIS SUBMITTED TO THE UNIVERSITY OF THE PUNJAB IN FULFILLMENT OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANY BY ABDUL RAZAQ DEPARTMENT OF BOTANY UNIVERSITY OF THE PUNJAB, LAHORE. JANUARY, 2013 Dedication This dissertation is dedicated to my beloved brother Munawer Ali who always encouraged me to pursue my dreams. Table of Contents Acknowledgements viii 1. Summary 1 2. Introduction 3 3. Aims and Objectives 11 4. Materials and Methods 12 4.1. Sampling site 12 4.2. Sampling strategy 12 4.3. Morpho anatomical characterization 12 4.4. Molecular characterization 13 i. DNA isolation 13 a) General CTAB protocol 13 b) Extract-N-Amp Protocol 14 ii. PCR Amplification of target DNA 14 iii. PCR products purification 15 iv. Sequencing 15 i. Sequencing PCR 15 ii. Cleaning of sequencing PCR products 16 v. Sequence assemblage and BLAST analysis 16 vi. Alignments and phylogenetic analysis 17 5. Results 19 5.1. Morpho anatomical and molecular characterization 20 5.2. Phylogenetic Analysis 279 6. Discussion 346 7. References 370 i List of taxa 1. AGARICACEAE 1.1. Agaricus 1.1.1. Agaricus albus 21 1.1.2. Agaricus augustus 23 1.1.3. Agaricus calvatus 25 1.1.4. Agaricus pakistanicus 27 1.1.5. Agaricus rhodofloridus 29 1.1.6. Agaricus sp. SR101 31 1.2. Cystodermella 1.2.1. Cystodermella cinnabrina 33 1.3. Lepiota 1.3.1. Lepiota acutesquamosa 35 1.3.2. Lepiota brunneoincarnata 37 1.3.3. Lepiota cristata 40 1.3.4. Lepiota himalayensis 42 1.3.5. Lepiota subincarnata 45 2. AMANITACEAE 2.1. Amanita 2.1.1 Amanita flavoconiodes 49 2.1.2 Amanita khanspurensis 52 2.1.3 Amanita pakistanica 55 2.1.4 Amanita pantherina 57 2.1.5 Amanita pseudovaginata 60 2.1.6 Amanita rubescence 62 2.1.7 Amanita vaginatoides 64 3. BOLBITACEAE 3.1. Panaeolus 3.1.1. Panaeolus deosaiensis 69 3.1.2. Panaeolus sphinctrinus 71 4. COPRINACEAE 4.1. Coprinus 4.1.1. Coprinus comatus 75 5. CORTINARIACEAE 5.1. Corntinarius 5.1.1. Corntinarius brunneocarpus 79 5.1.2. Corntinarius hyacinthofolius 82 5.1.3. Corntinarius nigricans 85 5.1.4. Corntinarius olivaceopictus 88 5.1.5. Corntinarius virgafomus 90 6. ENTOLOMATACEAE 6.1. Entoloma 6.1.1. Entoloma pakistanica 94 ii 7. GLOEOPHYLLACEAE 7.1. Neolentinus 7.1.1. Neolentinus Lepideus 99 8. GOMPHIDIACEAE 8.1. Chroogomphus 8.1.1. Chroogomphus roseolus 103 9. HYGROPHORACEAE 9.1. Hygrophorus 9.1.1. Hygrophorus chrysodon 108 10. HYMENOGASTERACEAE 10.1. Hebeloma 10.1.1. Hebeloma mesophaeum 113 10.1.2. Hebeloma theobrominum 115 10.2. Timgrovea 10.2.1. Timgrovea descoleus 118 11. INOCYBACEAE 11.1. Inocybe 11.1.1. Inocybe genticus 122 11.1.2. Inocybe impurus 125 11.1.3. Inocybe leucoblema 128 11.1.4. Inocybe nitidiuscula 131 11.1.5. Inocybe obsoleta 134 11.1.6. Inocybe pakistanica 137 11.1.7. Inocybe rimosa 141 11.1.8. Inocybe squamata 144 11.1.9. Inocybe sp.SR.19 147 12. MARASMIACEAE 12.1. Gymnopus 12.1.1. Gymnopus sp. K-36 151 12.1.2. Gymnopus sp. K-38 153 12.1.3. Gymnopus sp. KP-32 155 12.2. Marasmius 12.2.1. Marasmius albimyceliosus 157 12.2.2. Marasmius oreades 160 12.3. Microphale 12.3.1. Microphale brassicolens 163 13. Mycenaceae 13.1. Mycena 13.1.1. Mycena pura 167 14. PHYSALACRIACEAE 14.1. Xerula 14.1.1. Xerula hispida 170 15. PLEUROTACEAE 15.1. Hohenbuehelia 15.1.1. Hohenbuehelia petaloides 174 iii 16. PLUTEACEAE 16.1. Pluteus 16.1.1. Pluteus pouzarianus 177 16.1.2. Pluteus subcervinus 182 17. RUSSULACEAE 17.1. Lactarius 17.1.1. Lactarius ahmedii 188 17.1.2. Lactarius sanguifluus 191 17.2. Russula 17.2.1. Russula acrifolia 194 17.2.2. Russula aurata 196 17.2.3. Russula densifolia 198 17.2.4. Russula foetenoides 200 17.2.5. Russula himalayca 204 17.2.6. Russula integriformis 207 17.2.7. Russula khanspurensis 209 17.2.8. Russula laricina 212 17.2.9. Russula laricina var. ahmedii 214 17.2.10. Russula pectinatoides 216 17.2.11. Russula pectinatoides var. pakistanica 219 18. STROPHARIACEAE 18.1. Gymmopilus 18.1.1. Gymnopilus acortinus 222 18.2. Hypholoma 18.2.1. Hypholoma xanthofolia 225 18.3. Pholiota 18.3.1. Pholiota cylindricus 229 18.3.2. Pholiota flavipes 233 19. TRICHOLOMATACEAE 19.1. Armilleria 19.1.1. Armillaria sp KP-48 237 19.2. Clitocybe 19.2.1. Clitocybe abundans 239 19.2.2. Clitocybe alkaliviolascens 242 19.2.3. Clitocybe bresadolina 245 19.2.4. Clitocybe obscuritus 249 19.2.5. Clitocybe sp 1 252 19.2.6. Clitocybe vibecina 254 19.3. Clitocybulla 19.3.1. Clitocybula himayalaca 257 19.4. Laccaria 19.4.1. Laccaria ahmedii 260 19.5. Lyophyllum 19.5.1. Lyophyllum connatum 264 iv 19.5.2. Lyophyllum microsporus 266 19.6. Melanoleuca 19.6.1. Melanoleuca angelesiana 268 19.7. Tricholoma 19.7.1. Tricholoma myomyces 270 19.8. Tricholomopsis 19.8.1. Tricholomopsis flammula 272 19.8.2. Tricholomopsis rutilans 274 v List of Phylogenetic Analysis of Taxa 1. Phylogenetic analysis of Agaricus 279 2. Phylogenetic analysis of Cystodermella 281 3. Phylogenetic analysis of Lepiota 282 4. Phylogenetic analysis of Amanita 284 5. Phylogenetic analysis of Panaeolus 286 6. Phylogenetic analysis of Coprinus 287 7. Phylogenetic analysis of Cortinarius 289 8. Phylogenetic analysis of Entoloma 291 9. Phylogenetic analysis of Neolentinus 293 10. Phylogenetic analysis of Chroogomphus 295 11. Phylogenetic analysis of Hygrophorus 297 12. Phylogenetic analysis of Hebeloma 299 13. Phylogenetic analysis of Timgrovea 301 14. Phylogenetic analysis of Inocybe 303 15. Phylogenetic analysis of Gymnopus 307 16. Phylogenetic analysis of Marasmius 309 17. Phylogenetic analysis of Microphale 311 18. Phylogenetic analysis of Mycena 313 19. Phylogenetic analysis of Xerula 314 20. Phylogenetic analysis of Hoenobuhelia 316 21. Phylogenetic Analysis of Pluteus 318 22. Phylogenetic analysis of Lactarius 320 23. Phylogenetic analysis of Russula 322 24. Phylogenetic analysis of Gymnopilus 325 25. Phylogenetic analysis of Hypholoma 327 26. Phylogenetic analysis of Pholiota 329 27. Phylogenetic analysis of Armellaria 331 28. Phylogenetic analysis of Clitocybula 332 29. Phylogenetic analysis of Laccaria 334 vi 30. Phylogenetic analysis of Melanoleuca 336 31. Phylogenetic analysis of Tricholoma 338 32. Phylogenetic analysis of Tricholomopsis 340 33. Phylogenetic analysis of Clitocybe and Lyophyllum 342 vii ACKNOWLEDGEMENTS There are many people that were integral to this process. I would like to thank all those who have contributed during five years of PhD. I would like to express my deepest gratitude to my supervisor, Dr. Abdul Nasir Khalid, associate professor, department of Botany, University of the Punjab, Lahore for his excellent guidance, care and patience. I would never have been able to finish my dissertation without him. I am also thankful to Prof. Dr. Khan Rass Masood, Chairman, Department of Botany, University of the Punjab, Lahore for providing such an excellent environment for research and field tours. I would like to thank Dr. Abdul Rehman Khan Niazi, Dr. Muhammad Shahid and Dr. Muhammad Ilyas who intoduced me in the field of Molecular Biology. I am really wordless to express my thanks to Prof. Dr. Tanveer Akhtar, Department of Zoology, University of the Punjab, Lahore for her moral courage and academic guidance. I am grateful to Higher Education Commission, Pakistan for funding under Indigenous fellowship batch 4 and International Research Support Initiative Programm (IRSIP). Amongst my fellows, my utmost gratitude to Ms. Nousheen Yousaf, whose sincerity and cooperation, I shall never forget. I am also thankful to my all lab fellows for providing creative environment for research and Dr. Muhammad Hanif, Mr. Bilal Chaudry, Mr. Akbar Ali Shah for accompanying me in field tours. I am obliged to my nephew, Ghulam Qadir for helping in finalizing my thesis. I extend my gratitude to my wife, Sobia Ilyas for her infinite support and encouragement to do better always. I would never forget to thank my foreign collaborators Kabir Peay, Jason Hoeksema, Else Vellinga, Takahito Kobayashi and Zhu Liang Yang for their insightful comments and most importantly Tom Bruns who privileged me with an opportunity to work in Bruns lab, Plant and Microbial Biology Department, University of California, Berkeley, USA. That was most memorable and informative part of whole period. Above all I am thankful to my brothers, sisters, nephews and nieces for supporting me and encouraging me with their best wishes. At the end I am also thankful to my son, Muhammad Ahmad, for his beautiful smiles while my stay abroad. ABDUL RAZAQ viii SUMMARY This work deals with the morpho-anatomical and molecular characterization and identification of those fungi which posses gills or slit like structures below their caps, from some sites of the Himalayan moist temperate forests of Pakistan‘ part Khyber Pakhtunkhwa Province (Khanspur-Ayubia, Kuzagali, Khairagali, Nathiagali, Kaghan Valley; Sheran) and northern part of Pakistan (Fairy meadows). Internal transcribed spacers (ITS) region of rNDA of fungi under investigation has been amplified, collected during monsoon 2009-2011. During this investigation, 85 species of fungi belonging to 36 genera of 19 families were collected growing either in association with plants or from decaying logs. All these taxa have been described in detail on morphological, anatomical basis and ITS- rDNA molecular marker data. The co-relationship among phenotypes and genotypes has been documented in the form of phylogeny. The treated taxa include six (6) species of Agaricus (Agaricus albus nom. prov., A. augustus, A. calvatus nom. prov., A. pakistanicus nom.
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    THE CHECKLIST OF SHROPSHIRE FUNGI 2011 Contents Page Introduction 2 Name changes 3 Taxonomic Arrangement (with page numbers) 19 Checklist 25 Indicator species 229 Rare and endangered fungi in /Shropshire (Excluding BAP species) 230 Important sites for fungi in Shropshire 232 A List of BAP species and their status in Shropshire 233 Acknowledgements and References 234 1 CHECKLIST OF SHROPSHIRE FUNGI Introduction The county of Shropshire (VC40) is large and landlocked and contains all major habitats, apart from coast and dune. These include the uplands of the Clees, Stiperstones and Long Mynd with their associated heath land, forested land such as the Forest of Wyre and the Mortimer Forest, the lowland bogs and meres in the north of the county, and agricultural land scattered with small woodlands and copses. This diversity makes Shropshire unique. The Shropshire Fungus Group has been in existence for 18 years. (Inaugural meeting 6th December 1992. The aim was to produce a fungus flora for the county. This aim has not yet been realised for a number of reasons, chief amongst these are manpower and cost. The group has however collected many records by trawling the archives, contributions from interested individuals/groups, and by field meetings. It is these records that are published here. The first Shropshire checklist was published in 1997. Many more records have now been added and nearly 40,000 of these have now been added to the national British Mycological Society’s database, the Fungus Record Database for Britain and Ireland (FRDBI). During this ten year period molecular biology, i.e. DNA analysis has been applied to fungal classification.
  • Toxic Fungi of Western North America

    Toxic Fungi of Western North America

    Toxic Fungi of Western North America by Thomas J. Duffy, MD Published by MykoWeb (www.mykoweb.com) March, 2008 (Web) August, 2008 (PDF) 2 Toxic Fungi of Western North America Copyright © 2008 by Thomas J. Duffy & Michael G. Wood Toxic Fungi of Western North America 3 Contents Introductory Material ........................................................................................... 7 Dedication ............................................................................................................... 7 Preface .................................................................................................................... 7 Acknowledgements ................................................................................................. 7 An Introduction to Mushrooms & Mushroom Poisoning .............................. 9 Introduction and collection of specimens .............................................................. 9 General overview of mushroom poisonings ......................................................... 10 Ecology and general anatomy of fungi ................................................................ 11 Description and habitat of Amanita phalloides and Amanita ocreata .............. 14 History of Amanita ocreata and Amanita phalloides in the West ..................... 18 The classical history of Amanita phalloides and related species ....................... 20 Mushroom poisoning case registry ...................................................................... 21 “Look-Alike” mushrooms .....................................................................................