Morphological and Molecular Characterization of Selected Macrofungi in Northern Areas of Khyber Pakhtunkhwa, Pakistan

Home , Albatrellus flettii, Amanita beckeri, Amanita crocea, Amanita flavella, Amanita flavoconia, Amanita franchetii

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………………………………. 240

99. Basidiomata of Volvariella hypopithys……………………………………... 242

100. Microscopic structures of Volvariella hypopithys…………………………... 243

101. Basidiomata of Volvariella bombycina……………………………………... 246

102. Microscopic structures of Volvariella bombycina………………………….. 247

103. Basidiomata of Volvolpluteus earlei……………………………………….. 250

104. Microscopic structures of Volvopluteus earlei……………………………… 251

105. Molecular Phylogenetic analysis of the taxa of Pluteacea of the present study 252 and associated taxa by Maximum Likelihood method………………………. 106. Morphology of Cerioporus squamosus……………………………………... 255

107. Basisiomata of Panus rudis…………………………………………………. 257

108. Microscopic structures of Panus rudis……………………………………… 258

109. Macro and Micro-morphological characters of Pterula bisporitica………… 262

110. Molecular Phylogenetic analysis of Pterula bisporitica and associated taxa by 263 Maximum Likelihood method inferred from ITS data…………………… 111. Morphological characters of Lactarius furcatlamellatus…………………….. 268

112. Molecular Phylogenetic analysis by Maximum Likelihood method of Lactarius furcatlamellatus and associated taxa by Maximum likelihood 269 analysis……… 113. Morphology of Lactarius mairieoides ………………………………… 273

114. Molecular phylogenetic analysis of Lactarius mairieoides ……………. 274

115. Morphological characters of Lactarius mediterraneensis ………………… 277

116. Molecular Phylogenetic analysis by Maximum Likelihood method of 278 Lactarius mediterraneensis and associated taxa using nrITS data………... 117. Morphology of Lactarius pterosporus………………………………… 281

118. Molecular Phylogenetic analysis of Lactarius pterosporus inferred from 282 nrITS sequence data…………………………………………………… 119. Morphology of Lactarius sanguifluus………………………………………. 285

120. Molecular Phylogenetic analysis of Lactarius sanguifluus and associated taxa 286 inferred from ITS data………………………………………………….. 121. Morphology of Lactifluus versiformis………………………………………. 289

122. Molecular Phylogenetic analysis of Lactifluus versifromis and associated taxa 290 from nITS data by using the Maximum Likelihood method…………… 123. Morphology of Russula amethystina………………………………………... 293

124. Molecular Phylogenetic analysis of Russula amethystina and associated taxa 294 inferred from nrITS data……………………………………………………. 125. Basidiomata of Russula kangchenjungae…………………………………… 298

126. Microscopic structures of Russula kangchenjungae………………………… 299

127. Molecular Phylogenetic analysis of Russula kangchenjungae and associated 300 taxa using nr ITS data……………………………………………………….. 128. Basidiomata of Russula olivacea…………………………………………… 303

129. Microscopic features of Russula olivacea…………………………………... 304

130. Molecular Phylogenetic analysis of Russula olivacea inferred from nITS 305 data…………………………………………………………………………... 131. Basidiomata of Russula postiana……………………………………………. 308

132. Microscopic features of Russula postiana…………………………………… 309

133. Molecular Phylogenetic analysis of Russula postiana and associated taxa 310

134. Morphology of Russula similis……………………………………………… 313

135. Molecular Phylogenetic analysis of Russula similis inferred form nITS data 314 using the Maximum Likelihood method…………………………………….. 136. Morphology of Agrocybe elatella…………………………………………… 317

137. Different views of Basidiomata of Pholiota microcarpa……………………. 321

138. Microscopic structures of Pholiota microcarpa…………………………….. 322

139. Molecular Phylogenetic analysis of Pholiota microcarpa and associated taxa 323 using nITS data by Maximum Likelihood method…………………………... 140. Macro and Micro-morphological characters of Lepista nuda……………….. 327

141. Basidiomes of different collections of Lepista panaeolus…………………… 330

142. Microscopic structures of Lepista panaeolus………………………………... 331

143. Molecular Phylogenetic analysis of Lepista panaeolus and associated taxa by 332 Maximum Likelihood method using nITS data…………………………... 144. Morphology of Leucopaxillus giganteus…………………………………….. 335

145. Molecular Phylogenetic analysis of Leucopaxillus giganteus and associated 336 taxa using nITS data by Maximum Likelihood method……………………… 146. Different views of the basidiomes of Melanoleuca cinereifolia…………….. 339

147. Microscopic structures of Melanoleuca cinereifolia………………………… 340

148. Molecular Phylogenetic analysis of Melanoleuca cinereifolia by Maximum 341 Likelihood method inferred from nrITS data using MEGA6……………….. 149. Basidiome of Tricholoma conicosulphureum………………………………… 344

150. Microscopic structures of Tricholoma conicosulphureum…………………… 345

151. Molecular Phylogenetic analysis of Tricholoma conicosulphureum and 346 associated taxa based of nITS data by Maximum Likelihood method………. 152. Morphology of Tricholomopsis robustus …………………………………. 351

153. Morphology of Tricholomopsis umbonatus …………………………………. 354

154. Molecular Phylogenetic analysis of Tricholomopsis taxa reported during 355 present investigation and associated taxa by Maximum Likelihood method… 155. Morphology of Caloscypha fulgens…………………………………………. 356

156. Morphology of Helvella crispa……………………………………………… 359

157. Morphology of Helvella elastica……………………………………………. 361

158. Macroscopic and microscopic features of Helvella leucopus………………. 363

159. Macroscopic and microscopic features of Helvella macropus……………… 365

160. Macroscopic and microscopic features of Helvella maculata………………. 367

161. Macroscopic and microscopic features of Helvella solitaria……………….. 369

162. Macroscopic and microscopic features of Verpa conica……………………. 372

163. Macroscopic and microscopic features of Otidea leporine…………………. 375

164. Ascomata of Xylaria polymorpha (A & D) and X. hypoxylon (B & C)…….. 379

165. Microscopic structures of Xylaria polymorpha and X. hypoxylon…………... 380

166. ITS based Molecular Phylogenetic analysis of Xylaria polymorpha and 381 associated taxa by Maximum Likelihood method…………………………... LIST OF CHARTS

No. TITLE Page No.

1. Pie chart showing details of the reported species.………………………….... 18

ACKNOWLEDGMENTS I would like to express my sincere thanks and deepest gratitude for my supervisor Dr. Hassan Sher (SAARC gold medalist), Associate Professor, Center for Plant Sciences and Biodiversity, University of Swat, Pakistan, for his guidance, encouragements, and invaluable comments for shaping this thesis. I am indebted to him for his kind attitude and keen interest in my work. I am also highly thankful to my co-supervisor Dr. Abdul Nasir Khalid, Professor Department of Botany, University of the Punjab Lahore, for his sincere efforts in shaping this dissertation, loving and caring attitude, moral support, providing lab and hostel facility, and encouragements throughout this journey. I would also like to express my deepest gratitude to Dr. Haider Ali, Dr. Zahidullah, Dr. Ahmad Ali (Assistant Professors, Center for Plant Sciences and Biodiversity, University of Swat) and Dr. Abdul Rehman Khan Niazi (Assistant Professor, Department of Botany, University of the Punjab Lahore) for their supportive role, friendly attitude, guidance and encouragements. My special thanks to Dr. Shah Hussain (Assistant professor, CPS & B, University of Swat, Pakistan) for his guidance and support in every part of this PhD process. I am grateful to my lab fellows, Ms. Hira Bashir, Ms. Munazza Kiran, Ms. Ayesha Farooqi, Ms. Amna Imran, Ms. Afshan Wahab, Mr. Zia Ullah, Mr. Sadiqullah, Ms. Aamna Ishaq, Dr. Sana Jabeen, Dr. Malka Saba, Ms. Tayyaba Qasim, Ms. Aamna Imran, Ms. Memoona Khan and Mr. Muhammad Usman Mughal for their help and providing friendly environment. I am also thankful to the Lab Assistants Mr. Israr ul Haq, Hussain Ahmad, Abbas Khan, Irfan Khan, and Lab attendant Mr. Azizullah for providing conducive environments in the laboratory. Thanks to Kabir Khan, Ihtisham and Rahim Bahadar for their friendly attitude during all the PhD period. I am also thankful to my friends and roommates Dr. Amjad Ali and Mr. Noman Fazal for giving me a nice company during my stay at Hostel (University of the Punjab), Lahore. I am indebted to all my family members especially my parents, brothers and sisters for their prayers and invaluable moral and financial support. Junaid Khan

ABSTRACT

Present study dealt with morphological and molecular characterization of macrofungi in northern parts of Khyber Pakhtunkhwa, Pakistan. This region represents floristically a rich part of Pakistan comprising well-known mountainous ranges of Himalaya and Hindu Kush, but a very few macrofungal surveys have been conducted so far. The present study was therefore endeavored to report some macrofungal species occurring in woodland and forest ecosystems of Northern parts of Khyber Pakhtunkhwa, Pakistan. Field surveys and collections of specimens were carried out at different sites of the study area during the years 2013‒2016. For collections, drying, preservation, micro-morphological and molecular characterization of the collected specimens, standard protocols were used.

A total of 88 species belonging to 30 families and 57 genera are being reported and described in detail, of which 75 species belong to Basidiomycota and 11 to Ascomycota.

Identity of 59 species was confirmed through molecular data inferred from nITS region.

Out of the described species more than thirty percent showed their novelty. Albatrellus roseus, Amanita griseofusca, Clavariadelphus elongates, Descolea quercina, Gymnopilus swaticus and Phaeocollybia pakistanica have been published as new species and 25 other species are proposed as new to science including: Albatrellopsis flettiodes nom.prov.,

Amanita ahmadii nom.prov., A. cinerea nom.prov., Butyriboletus pakistanicus nom.prov.,

Caloboletus alpinus nom.prov., Cortinarius clavatus nom.prov., C. longistipes nom.prov.,

C. striatus nom.prov., Inocybe pallidorimosa nom.prov., I. salicetum nom.prov., Lactarius furcatilamellatus nom.prov., L. maireiodes nom.prov., Leucoagaricus campestris nom.prov., Lycoperdon microsporon nom.prov., Oudemansiella sparslamellata nom.prov., Phaeocollybia varicolor nom.prov., Pholiota microcarpa nom.prov., Pluteus

ii populicola nom.prov., Pterula bisporitica nom.prov., Rhodocybe luteus nom.prov.,

Russula similis nom.prov., Tricholoma conicosulphureum nom.prov., Tricholomopsis robustus nom.prov., T. umbonatus nom.prov. and Veloporphyrellus purpureus nom.prov.

Furthermore, 28 species are new records for Pakistani mycobiota viz., Agrocybe elatella,

Amanita pallidorosea, Caloscypha fulgens, Coltricia abieticola, Coriolopsis gallica,

Gymnopilus penetrans, Gymnopus dysodes, Hebeloma rostratum, Helvella leucopus, H. macropus, H. maculata, H. solitaria, Hericium cirrhatum, Inocybe putilla, Jahnoporus oreinus, Lactarius mediterraneensis, L. pterosporus, Lactifluus versiformis, L. volemus,

Lanmaoa angustispora., Lepista panaeolus, Leucopaxillus giganteus, Russula kangchenjungae, R. olivacea, R. postiana, Verpa conica, Volvariella hypopithys and

Volvopluteus earlei. Of these Amanita pallidorosea and lactifluus volemus have been published.

Genera including Albatrellopsis, Albatrellus, Butyriboletus, Caloboletus, Caloscypha,

Jahnoprus, Lanmaoa, Phaeocollybia, Pterula, Veloporphyrellus and Volvopluteus are addition to the fungi of Pakistan. The most abundant and diverse family was Russulaceae with 11 species distributed among 3 genera, followed by Agaricacea, Amanitaceae and

Tricholomataceae with 7 species each. Boletaceae, Helvellaceae and Hymenogastraceae are represented by 6 species each.

Results of the study reveals that northern parts of Khyber Pakhtunkhwa are rich in terms of macrofungi and there may be many other species awaiting documentation. This study therefore recommends further and in-depth surveys on exploration of macrofungi in woodland and forest ecosystems of the Northern parts of Khyber Pakhtunkhwa in particular

iii and other parts of Pakistan in general. Published work form present PhD project is listed as annexure-A.

CHAPTER 1

INTRODUCTION

Macrofungi is an assemblage of all the fungi producing a fruiting body larger than 1 cm

(Redhead, 1997; Kaya, 2015). It is an artificial but a convenient group, includes well known fungi presented by common names viz. morels, truffles, puffballs, gilled fungi, cup fungi, boletes and bracket fungi (Food and Agriculture Organization [FAO], 1996).

Macrofungi are among the earliest form of fungi known to mankind (Okhuoya et al., 2010) but, defining their exact number has always remained a point of discussion for mycologists (Schmit & Mueller, 2007). Many studies concentrated on enumerating mushrooms diversity, which mostly are based on relating the number of mushrooms to the number of higher plants (Hawksworth, 1991; Schmit & Mueller, 2007). Estimates based on molecular data suggest as many as 5.1 million fungi (O’Brien et al., 2005; Blackwell,

2011), however, the estimate of 1.5 millions (Hawksworth, 2001a; Yang, 2011) seems convincing. In contrast to the expected large number of fungi, we roughly have the knowledge of 0.08 to 0.12 million species (Hawksworth, 2001b; Kirk et al., 2008). This huge difference between the estimated and known fungi seems related to the fact that, unfortunately there have been insufficient or inadequate samplings of fungi in many parts of the world (Kendrick, 2000; Blackwell, 2011). It is also evident from the account of

Hawksworth (2001a), that numerous potential fungal habitats and localities still remain understudied that may harbor large number of undiscovered fungi.

Macrofungi are perhaps the most important component of any ecosystem, performing variety of undeniable roles in ecosystem dynamics and stability (Mueller et al.,

2004). These like other members of kingdom fungi are heterotrophic, depending upon other

1 sources for their nutrition, living either as saprotrophic, symbiotic or parasitic partners of higher plants (Mueller et al., 2007). Saprotrophic macrofungi are vital for the breakdown and recycling of organic matter particularly woody tissues and help in the recycling of both natural and industrial forest waste and dead wood (Redhead, 1997). Symbiotic macrofungi form friendly association with majority of plants primarily flowering plants and conifers and in some cases with bryophytes and ferns (Pressel et al., 2010). Redhead (1997) elucidated that excepting tree species in the families of Cupressaceae (Chamaecyparis

Spach, Calocedrus Kurz, Thuja L.) and Aceraceae, all major timber trees and many ornamental plants are symbiotically dependent on ectomycorrhizal fungi (mostly macrofungi). Elimination of ectomycorrhizal fungi or an ample drop in their numbers can lead to loss or decline in number of the host trees, have a serious effect on the timber industry, and lead to desertification.

Macrofungi are also useful in the bioremediation of industrial waste and the accumulation of heavy metals from the environment (Kalac, 2012). It has been known that several macrofungal species, such as Trametes versicolor (L.) Lloyd, serve as decomposers of persistent organic pollutants (POPs) and other complex polymers (lignin and cellulose).

Some others are capable to decontaminate soil or groundwater of some types of pollutants

(Redhead, 1997).

The importance of macrofungi as diet has been well established since ancient times

(Chelela et al., 2014) and in many cases associated with potentates and royalty because of their pleasant taste and flavor (Da Silva, 2005). Edible mushrooms are considered as functional food (Jonathan et al., 2012) not only for texture and flavor (Tibuhwa, 2013) but also for their chemical and nutritional characteristics, which can provide health benefits

2 beyond the traditional nutrients they contain (Cheung, 2008). Most of the edible mushrooms are low in calories and rich in nutrients like proteins, vitamins, minerals, fibers and trace elements compared to the best local legumes (Mendil et al., 2004; Kalac, 2013).

The protein content of edible mushrooms ranges from 1.75 to 3.63% of their fresh weight

(Deepalakshmi & Mirunalini, 2014). This means that the protein content of edible mushrooms, in general, is about twice as that of Asparagus and cabbage, and 4 times of apples and 12 times those of oranges. On a dry weight basis, mushrooms normally contain

30‒40 % proteins (Sharma & Madan, 1993), as compared to 39.1% in soybean, 25.2% in milk, 13.2% in wheat and 7.3% in rice (Cheung, 2008). Therefore, in amount of crude protein, mushrooms rank well above most plants foods, including milk, which is an animal product (Prasad & Sethi, 2013). Beside proteins, carbohydrates also form a prevailing component of mushrooms, which is about 50‒60 % of dried mushrooms (Kalac, 2012).

The above account makes it clear that, mushrooms has the potential to combat food security, especially in the developing countries of the world (Sanchez et al., 2002) as well as key to proteins shortage and human wellbeing.

Mushrooms are also reported as therapeutic foods, beneficial in preventing diseases such as cancer, hypertension and hypercholesterolemia (Bobek & Galbavy 1999; Aina et al., 2012; Nithya et al., 2014). Studies also have shown antitumor, antiviral, antithrombotic and immune-modulating effects of mushrooms (Halpern & Miller, 2002; Wasser, 2002).

Lignicolous macrofungi such as Ganoderma lucidum (Curtis) P. Karst, have secondary metabolites which expressed significant effects such as antibacterial activity (Oluranti et al., 2012). These functional characteristics are mainly due to the presence of dietary fibers and in particular chitin and beta glucans (Manzi et al., 2001).

Macrofungi are among the most important non-timber forest products (Adekunle

& Ajao, 2005). Of the many non‒timber forest products collected commercially, wild mushrooms are one of the most economically significant (Alexander et al., 2002; Sher &

Shah, 2014). Mushrooms, such as chanterelles (Chantharellus spp.), king boletes (Boletus edulis Bull.), morels (Morchella spp.), false morels (Gyromitra spp.) and pine mushrooms

(Tricholoma magnivelare (Peck) Redhead), are commercially harvested directly from natural habitats, supporting a multimillion-dollar industry (Redhead, 1997) and can serve as an extra income generator (Sher et al., 2015a). In 1991, the global market value of medicinal mushrooms and their derivatives was 1.2 billion US $ (Wasser et al., 2000), in

2013 it was US $29.42792 billion and is estimated to grow at a compound annual growth rate (CAGR) of 9.5% from 2014 to reach US $50.03412 million by 2019

(Marketsandmarketscom, 2015). Knowing the huge agro-industrial, medicinal and commercial benefits, mushroom resources have been exploited in most developed

(Okhuoya et al., 2010) and developing countries of the world (Chang, 1997). Mushrooms cultivation and the processing of mushroom products have been beneficial to millions of people across Pakistan (Sher et al., 2011), China, India, Tanzania and other developing countries in terms of financial, social, and health improvement (Chelela et al., 2014). In addition, cultivation and development of mushroom industries positively generate economic growth and have an economic impact at national and regional levels. This is particularly evident from the findings of Sher and Shah (2014), that total harvested quantity of morels raised to 14000 kg in year 2012 in Palas valley, Pakistan, with pickers receiving a revenue of US$343,000 per annum. Supporting such a huge agro-economic industry, mushrooms can serve as an extra income generator as well (Tibuhwa, 2013).

Aside from the benefits of human consumption, mushrooms form a part of the diet for native animals as well (Zotti et al., 2013). Fungi like Truffles, are widely consumed by many animals which also serve as their obligate dispersal agents. Others, through wood decay, create essential habitats for a variety of animals either by causing cavitation of trees or logs, or preparing the wood for colonization by insects and, indirectly, by larger animals

(Redhead, 1997).

Review of the literature reveals that macrofungi are useful for man and his environment in number of ways, but, have received little attention worldwide (Mueller et al., 2004; Schmit & Mueller, 2007). This negligence has been particularly attributed to the higher interest in plants and animals as sources of food and medicine for man (Osemwegie

& Okhuoya, 2009). Moreover, macrofungi is considered as an odd group of organisms, poorly understood and difficult to study due to their cryptic morphologies and hidden, short lived fruiting bodies (Schmit & Mueller, 2007). Fungal taxonomy has also proved to be one of the most difficult task for taxonomists and has remained in a state of constant flux for many decades. Recently, taxonomy of fungi was revised classifying it into one kingdom, one subkingdom, seven phyla, ten subphyla, 35 classes, 12 subclasses, and 129 orders (Hibbett et al., 2007).

Accurate identification and delimitation of species is also important for the understanding our natural world. Errors in this process can have impacts on biodiversity assessment, ecological studies, and management planning (Bortolus, 2008).

Conventionally, macro-morphological traits were used for identification and classification of fungi. However, phenotypic plasticity causes difference in different environments which can lead to misidentification (Zhao et al., 2011). Therefore, a need of more reliable and

5 straightforward method for identification and classification of fungi was felt, that can be used for the recognition of mushrooms.

Fortunately during recent decades, molecular tools have been developed to describe the diversity of fungi in more practical and superior way (Taylor, 2008). Compared to conventional methods, molecular methods are more reliable and have made the process of species identification and classification in fungi more reliable and faster. In general, one specific short DNA fragment called the Internal Transcribed Spacer (ITS) is used for identification of fungi at the species level (Nilsson et al., 2009). The ITS region is composed of two non-coding polymorphic regions, nested within the nuclear ribosomal

DNA repeat between small subunit (18S), 5.8S and large subunit (28S). This region has intraspecific conserved but interspecific variable sequences, has a high copy number, conserved priming sites, a shorter size (650‒900bp) and enormous amount of sequence data in GenBank for comparison. These properties has made this region more powerful tool for species identification in fungi (Gardes & Bruns, 1993) and has been recommended as the universal fungal barcode sequence (Badotti et al., 2017; Schoch et al., 2012).

Although additional markers other than ITS, enhances the data, but ITS alone is enough for species delimitation in fungi including Boletaceae. For example Sarwar et al., (2018;

2016) published Suillus himalayensis, Neoboletus luridiformis and Hortiboletus rubellus

(members of Boletaceae) using ITS marker alone.

The phylogenetic analysis of fungi is carried out by assessing the ITS sequence of a specific species of fungi with those deposited to the GenBank and other databases (Neves et al., 2012). For most of the species a threshold value of 97% similarity is set for considering novelty of a species, however a ratio of more than 98% for Agaricus and even

6 some base differences in Cortinarius is considered as significant (Niskanen et al., 2008;

Gao & Yang, 2010).

Pakistan is among the biodiversity rich countries of the world blessed with varied agro-climatic regions. The history of fungal exploration of Pakistan dates back to Butler and Bisby (1931) and Mundkur (1938) when this region was a part of British India. Later on, fungal diversity of Pakistan was explored in detail by Dr. Sultan Ahmad, from 1939 to

1983. During this period, he produced many research article describing more than 2500 species of different groups of fungi including basidiomycetes, ascomycetes, pezizales, uredinales and ustilagonales (1952; 1955; 1956a;1956b; 1956c;1969b; 1972b).

The Japanese scientists (Izumi et al., 1992; Ono, 1992; Ono & Kakishima, 1992;

Shibata, 1992; Ono & Kakishima, 1993; Kakishima et al., 1993; Murakami, 1993), during their cryptogamic expedition to the Northern areas of Pakistan also made a significant contribution to fungi of Pakistan.

During 1990s, all the described fungi of Pakistan were assembled into “Fungi of

Pakistan” (Ahmad et al., 1997). Sultana et al., (2011) worked on Agarics of Kaghan valley and produced a checklist of mushrooms comprising of 110 species of aphyllophorales and

228 species of basidiomycetes. Gardezi (1998) and Gardezi and Ayub (2003) reported some valuable mushrooms from Azad Jammu and Kashmir. Other notable contributions include Sultan et al., (2001), Sabir et al., (2001) Tulloss et al., (2001), Burni et al., (2006) and Niazi et al., (2006) from different regions of Pakistan. So far, approximately 900 different species of macrofungi are reported from different ecoregions of Pakistan (Niazi,

2008; Niazi et al., 2010; Sarwar & Khalid, 2012; Razaq et al., 2013; Razaq, 2013; Fiaz,

2013, Nawaz et al., 2013; Hanif et al., 2014; Sher et al., 2014; Thongklang et al., 2014;

Hussain et al., 2015a, 2015b; Qasim et al., 2015; Sarwar et al., 2015, Hussain et al., 2016).

The above accounts clearly depict that current knowledge of the macrofungi of

Pakistan in general and Northern parts of Khyber Pakhtunkhwa in particular is insufficient that can be used for a thorough analysis desirable for biodiversity conservation. There are a very few published monographs, checklists and identification keys for the bulk of macrofungi of Pakistan. The present data is also inadequate for a comparison among different ecoregions of the country or other regions of the world. In fact, there is deficiency of information and the available data is too fragmentary and incomplete, that can be used for further studies in the region. Moreover, new species of macrofungi are constantly reported in every new area being surveyed, further clarifying the condition. In the light of these facts, it is hypothesized, that most of the macrofungi of Northern parts of Khyber

Pakhtunkhwa await documentation and there is a dire need for a detailed analysis of macrofungi in the forest and woodland ecosystem of Northern parts of Khyber

Pakhtunkhwa, Pakistan. The present planned study is, therefore, designed to explore the macrofungal diversity in the forest and woodland ecosystems of the northern parts of

Khyber Pakhtunkhwa with the following objectives.

Aims and Objectives

The objectives of the research are:

 To collect and record maximum possible number of species of macrofungi found

in the study area.

 To confirm the identity of the collected macrofungal species using morphological

and molecular techniques where required.

 To study the phylogenetic relationship of the collected macrofungi using DNA

sequence data from the ITS region.

CHAPTER 2

MATERIALS AND METHODS

Study area

Northern parts of Khyber Pakhtunkhwa are among the biodiversity rich parts of Pakistan, covering the districts of Swat, Shangla, Buner, Dir upper, Dir lower and Chitral (Fig. 1).

Topography of the area is dominated by mountains including well-known ranges of Hindu

Kush and Himalaya. Among these the Hindu Kush Range begin in the North in Gilgit-

Baltistan and extend into Chitral in the West. “Tirich Mir” towering more than 7690 meters is the highest peak of Hindu Kush range in Pakistan is also located in distrct Chitral.

To the south of the Hindu Kush lies three subparallel ranges of Kohistan, Swat and

Dir, occupying the areas of Malakand Diviosn and Kohistan District. All these three ranges run in a north-south direction and are separated from each other by rivers. The most easterly is the Kohistan range which is bounded by the Indus River in the East and Swat River in the West. Swat Range which is in the middle is bounded by Swat River on the East and by

Panjkora River on the west. The westerly range “Dir Range” is bounded on the east by

River Panjkora and River Kunar on the west. These ranges vary greatly in altitude as they move north to south with their elevation reaching 5,000‒6,000 meter in the north and as low as 200 meters to the south.

Fig. 1: Map of the study area

The study area is quite rich in terms of floristic biodiversity (Sher et al., 2015) and can be classified into moist temperate, dry temperate and subtropical regions. Mountains are characterized by evergreen forests including variety of conifers while low lying areas are characterized by deciduous vegetation. Climatically the area is characterized by moderate summers and extremely cold and snowy winters. December and January are the coldest while July and August are the hottest months of the year. Winter season starting from October extend to the mid of the April. Snowfall is common in the mountainous areas, occurring from December to March.

Field visits and Collection of fruiting bodies

The study was conducted from March 2013 to October 2016. Prior to field visits, necessary data about vegetation, climate, weather and geography of the visiting sites was obtained following Lodge et al., (2004). Important materials including pair of sharp knives, cutter, paper bags, tags, basket for carrying samples, digital camera and field notebooks were arranged. Accordingly frequent field visits were arranged to the collecting sites in all months of the year especially the monsoon during 2013‒2016. Specimens with range of all growing stages were preferred for collection and minimum of three specimens were collected for each species. The specimen present singly were considered only if were in good condition. Substrate other than soil especially wood was also collected along-with fruiting bodies. Every specimens was assigned a unique tag number and photographed accordingly in their natural habitat. To note the size of the specimen a centimeter scale was photographed alongside of the specimen. For DNA studies, parts of the lamellae and pores of boletes and polypores were preserved in 2 % Cetyl trimethyl ammonium Bromide

(CTAB) solution in autoclaved Eppendorf tubes.

A fan heater was used for drying of the specimens with temperature adjusted between 38‒50°C. Small specimens were dried as a whole, whereas large agarics and other fleshy specimens especially boletes were cut into half or quarter to aid in drying process.

This also prevent decaying of the interior and eating activity of the larvae of insects.

Properly dried specimens were put into separate zip-lock bags with small quantity of packed silica gel to absorb any moisture left during the drying procedure.

Morphological and anatomical characterization

Morphological characters viz. color, size, shape, surface features, cap margins, odor and taste, context (Flesh) characteristics, presence or absence of volva, annulus, gills attachment, stipe ornamentation, texture and bruising reaction were noted on the spot using a field notebook. Bruising reaction especially for boletes was checked by rubbing the pileus, hymenium and stipe with sharp knife and color changes were noted. To check the interior of the specimens, at least one specimen was sliced longitudinally and important characters were noted. Color codes follow Munsell color charts (1975).

Microscopic examination were carried out in the laboratory of Center for plant sciences and biodiversity, University of Swat, using light microscope (BOECO, Model BM

120). Slides were prepared using distilled water or 5 % Potassium Hydroxide solution

(KOH) as a mounting medium. Congo red and Phloxine were used for staining purposes where required. Amyloidy of the spores and hyphae in some genera were checked by using

Melzer’s reagent. Important microscopic characters were noted from all the available basidiomes and measurements were recorded using a calibrated computer based software

“Piximetre” version 5.9 (downloaded from http://ach.log.free.fr/Piximetre/ ). Total of 20 random readings were obtained for spores, basidia, cystidia and other micro‒structures.

For measurements “Q” represents the length / breadth ratio of individual spores, “Qe” represent average length / breadth ratio of all the spores from all basidiomes whereas “Me” is the mean of length and breadth of spores. Measurements of spores are without hilar appendages while those of basidia are without sterigmata.

Specimen identification and deposition

Based on morpho-anatomic characters, specimens were identified using the available literature (Ahmad, 1972b; 1980; Hanif, 2011; Sarwar & Khalid, 2012; Sharif, 2012; Ilyas,

2013; Razaq, 2013a; Razaq, 2013b; Fiaz, 2013; Yousaf, 2014; Hussain, 2016; Saba, 2016;

Sana, 2016). Properly dried and identified specimens are deposited to the Swat University

Herbarium (SWAT) and in duplicates to Herbarium of the department of Botany,

University of the Punjab, Lahore (LAH).

Molecular Characterization

The specimens which were unable to be identified using morpho-anatomic data, were subjected to molecular characterization for proper identification using the following procedures.

DNA extraction

Total genomic DNA extraction from fresh or dried specimen was done by using CTAB method (Porebski et al., 1997). The steps involved in the process are as follows.

• About 50 mg of tissue from the gills or pore surface was taken in an autoclaved

Eppendorf tube containing 300 µl of 2 % CTAB solution.

• The CTAB added tissues were subjected thrice to thaw and freeze by placing the

tube in a freezer and water bath alternatively.

• The tissues were crushed in the same Eppendorf tube using autoclaved micro-pestle

until a homogenous mixture was obtained.

• The homogenate was incubated for 30‒50 minutes in water bath adjusted at 65º C

with inverting the tube after every five minute interval.

• After incubation, 300 µl of Choloroform-Isoamyl alcohol (24:1) was added to the

tube and was vortexed for one minute to get a milky appearance.

• The tubes were centrifuged again for 20 minutes at 13200 rpm at 4º C.

• The supernatant was taken into another autoclaved Eppendorf tube with care

avoiding touching the debris layer.

• To the supernatant, 2/3rd or 133.33 µl of ice cold Iso-Propanol was added and kept

for overnight in a freezer.

• Next day, the tubes were centrifuged at the same rate and temperature and the

supernatant was discarded leaving the DNA pellets at the bottom of the tube.

• The DNA pellets were washed twice by adding 200 µl of ice cold ethanol and

centrifuged at the same rate for few minutes.

• After washing, the DNA pellets were kept for drying in the laminar flow for

minimum of 1 hour.

• DNA pellets were diluted by adding 50 µl of double distilled water (ddH2O) and

stored at ‒20 °C for further use.

PCR Amplification, Visualization and Sequencing

The extracted DNA was subjected to Polymerase Chain Reaction (PCR) to amplify the nuclear internal transcribed spacer (ITS1, 5.8 S and ITS2 hereafter referred as nITS) region with ITS1F as forward (Gardes & Bruns, 1993) and ITS4 as reverse primer (White et al.,

1990). Polymerase chain reaction (PCR) were performed in 25 µl volume per reaction. The

PCR master mix contained 2.5 µl of 10 X Taq Buffer with (NH4)2SO4, 2.5 µl of dNTP

Mix (Thermo Scientific #R0181), 0.5 µl forward primer (ITS1F), 0.5 µl reverse primer

(ITS4), 2 µl of 25 mM Mgcl2, 1–2 µl of template DNA and 0.125 µl Taq DNA polymerase

(Thermo Scientific #EP0402). PCR procedure consisted of initial 4 min denaturation at

94°C, 40 cycles of 1 minute at 94°C, 1 min at 55°C, 1 min at 72°C and a final extension of

10 minutes at 72°C.

Visualization of PCR products were accomplished using 1 % agarose gel added with 3 µl ethidium bromide and a UV illuminator.

Sequencing of the amplified products was accomplished through outsourcing.

Editing of Sequences and BLAST analyses of ITS sequences

The generated sequences were compared with other sequences in the GenBank using Basic

Local Alignment Search Tool (BLAST) at National Center for Biotechnology Information

(NCBI) and Unite database. Closely matching sequences, especially the published ones were downloaded for phylogenetic analysis. DNA sequences were aligned using BioEdit version 7.2.5 (Hall, 1999) and CLUSTAL X 2.1 (Larkin et al., 2007). Phylogenetic analyses were performed by using Maximum likelihood (ML) or Maximum parsimony

(MP) analyses. Maximum likelihood trees were generated with MEGA6 (Tamura et al.,

2013) by selecting the best model for analysis or with RAxML-HPC v.8 on XSEDE implemented on the CIPRES Science Gateway v. 3.3 (Miller et al., 2010). For the bootstrapping phase, GTRGAMMA model was used with one thousand rapid bootstrap replicates. A bootstrap proportion greater than 70% was considered as significant.

Maximum parsimony (MP) analyses was performed using PAUP* Version4 (Swofford,

2004), with all characters of type unordered and equally weighted. Gaps were treated as missing data. Heuristic searches were performed with 1000 replicate searches with random

16 taxon addition. MAXTREES was set to 5000 with MulTrees option in effect and TBR branch swapping. All characters were treated as of type 'unord' and equally weighted.

CHAPTER 3

RESULTS

Present study dealt with the morphological and molecular characterization of selected macrofungi in northern parts of Khyber Pakhtunkhwa, Pakistan.

A total of 88 species of macrofungi belonging to 30 families and 57 genera are being reported and described in detail, of which 75 species belong to Basidiomycota and

11 to Ascomycota. Of these, 31 species (35%) showed their novelty, 28 species (32%) are new records for Pakistani mybiota while 21 species (24%) are new records for current study area (Chart-1). The details of all species treated during current investigation are given below.

Chart 1: Pie chart showing details of the reported species.

AGARICACEAE

a) Chlorophyllum

1. C. hortense

b) Echinoderma

1. E. asperum

c) Lepiota

1. L. cristata

d) Leucoagaricus

1. L. campestris nom.prov

e) Lycoperdon

1. L. microsporon nom.prov.

2. L. perlatum

f) Macrolepiota

1. M. excoriata

CHLOROPHYLLUM Massee

Chlorophyllum hortense (Murrill) Vellinga, Mycotaxon 83: 416 (2002) Fig. 2

Pileus 60‒90 mm across, conical to conico-convex at first, later on palno-convex with a central umbo, pure white to cream colored in young specimens, with moderate pinkish brown colors (7.5YR 9/4) in mature specimens especially at the center, surface dry, shiny, smooth at first, cuticle breaking later on giving the pileus a velvety appearance, pileus edge striated, margin appendiculate, cracking with age, interior fleshy, white, turning red (7.5R

3/14) upon cutting, thicker at the disc (5‒7mm), less than 1mm at the edges. Lamellae free, broader at mid point (5‒7mm), narrowing towards edges, close, cream colored with light pinkish tone, olivaceous green in mature and Herbarium specimens, lamellulae present, in single tier, of variable lengths, lamellar edge even. Stipe 80‒10 × 6‒9 mm in dimensions, central, terete, thickening downward with a slightly bulbous base, bulb 10‒13 mm across, white, turning red (7.5R 3/14) upon bruising and handling, surface smooth, glabrous, context white, reddening (7.5R 3/14) upon cutting especially at the base, centrally hollow, annulus present, double, superior, subperonate, persistent.

Basidiospores (8.0) 8.5‒10.0 (11) × (5.5) 6.0‒7.0 (8.0) μm, Q = (1.23) 1.31‒1.50 (1.57),

Me= 9.0 × 6.4, Qe = 1.4, broadly ellipsoid, thick walled (≤4 µm), apiculus present, germpore absent. Basidia 20‒25 × 6‒7.5 µm, clavate, mostly 2-sterigmated, rarely 3- sterigated, sterigmata ≤4.5 µm. Cheilocystidia 40–60 × 5–8.5, cylindrical to narrowly clavate, rarely mucronate. Squamules composed of long-clavate to cylindro-clavate elements, terminal cells 60–100 × 10–15 µm, thin walled. Stipitipellis composed of fusoid hyphae, 100‒15 µm long.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Takhta Band village, 950 m a.s.l., on humus rich soil among grasses,

Junaid Khan, 05th September, 2015, TB-16 (SWAT000101).

Molecular Characterization Fig. 3

A 744 base pairs (bp) fragment of the ITS region of the TB-16 showed 99% similarity with

Chlorophyllum hortense (AY243611; AY243612) from USA upon BLAST search.

For phylogenetic reconstruction of the Pakistani collection of TB-16, a total of 22 nucleotide sequences were used with Lepiota cristata (Bolton) P. Kumm (AF391050;

AF391051) as outgroup taxon. The maximum likelihood analysis using MEGA6 software, clustered the 20 in-group sequences into 5 different clades viz. hortense/lusitanicum, agaricoides/medioflavoides, molybdites/globosum, neomastoidae and brunneum/rhacodes.

The Pakistani collection clustered within the hortense/lusitanicum clade along-with C. hortense with a strong bootstrap value of 100%. Phylogenetically, the Pakistani species of

C. hortense is close to the species from USA.

Comments: Chlorophyllum hortense is previously reported from Pakistan as

Leucoagaricus hortensis (Murrill) Pegler from Botanical Garden, University of the Punjab,

Lahore Pakistan (Nawaz, 2012; Qasim, 2013), with no other records. This is the third report of this taxon from Pakistan and first from Khyber Pakhtunkhwa.

Figure 2. A–H. Macroscopic and microscopic characters of Chlorophyllum hortense (TB-16). A‒C: Basidiomata. A: Basidiome in natural habitat. B: View of the lamellae. C: color change upon cutting. D‒H. Microscopic structures. D: Basidiospores. E: Basidia. F: Squamules on the pileus surface. G: Cheilocystidia. H: Stipitipellis hyphae. Bars: 10mm for A‒C, 5 µm for D, E & H, 14 µm for F, 3.8 µm for G.

AY243612.1 Chlorophyllum hortense 65 AY243611.1 Chlorophyllum hortense 100 Chlorophyllum hortense (TB-016)

70 Chlorophyllum hortense (TB-016) hortense/lusitanicum

KR233490.1 Chlorophyllum arizonicum

KR233482.1 Chlorophyllum lusitanicum 43 98 KR233483.1 Chlorophyllum lusitanicum

AY243616.1 Chlorophyllum agaricoides

DQ200928.1 Chlorophyllum agaricoides agaricoides/medioflavoides 28 96 89 GQ329055.1 Leucoagaricus medioflavoides

KJ524559.1 Chlorophyllum molybdites 100

60 KJ524557.1 Chlorophyllum molybdites molybdites/globosum 99 KU499923.1 Chlorophyllum globosum

99 KP229776.1 Chlorophyllum globosum

KF551251.1 Macrolepiota neomastoidea neomastoidea

AY243609.1 Chlorophyllum brunneum 100 AY083206.1 Chlorophyllum brunneum

96 JQ683124.1 Chlorophyllum rhacodes brunneum/rhacodes

AY081237.1 Chlorophyllum rhacodes 100 AY081240.1 Chlorophyllum rhacodes

AF391050.1 Lepiota cristata outgroup 100 AF391051.1 Lepiota cristata

0.02 Figure 3. Molecular Phylogenetic analysis by of Chlorophyllum hortense and associated taxa inferred from nITS data by using Maximum Likelihood method. The evolutionary history was inferred by using Maximum Likelihood method based on the Jukes-Cantor model using MEGA6. The tree with the highest log likelihood (-2124.0550) is shown.

ECHINODERMA (Locq. ex Bon) Bon

Echinoderma asperum (Pers.) Bon, Docums Mycol. 21(no. 82): 62 (1991) Fig. 4

Pileus 40‒60 mm across, convex in young stages, plane to plano-convex with a central umbo and seceding margin in mature stages, pink (2.5YR 7/4) to pinkish brown (5YR 7/4), surface dry, dull, fibrillose when young, granulose with a fibrillose background with aging especially at the disc, pileus margin appendiculate, often eroded, context white, 4‒5 mm at the disc and 1‒1.5 mm at the margin, soft to spongy. Lamellae remotely free, close, whitish with or without pinkish tone (7.5YR 9/2), developing brownish to pinkish spots upon maturity, broader at the center, 3‒4 mm, lamellar edge even, lamellulae present, mostly in single tier. Stipe central, 50‒80 × 5‒8 mm, terete, equal or slightly thicker at the base, concolorous with the pileus or slightly whitish in the upper part, fibrillose with whitish background, annulus cottony or cortinate, leaving a ring zone on the pileus, texture rigid, context solid, stuffed with a spongy pith, wet, grayish white below the surface.

Basidiospores (6.2‒) 6.3‒7.1 (‒7.9) × (2.2‒) 3.1‒3.4 (‒3.6) µm, Q = (1.8‒) 1.9‒2.3 (‒2.9),

Me = 6.7 × 3.2 µm, Qe = 2.2, ellipsoid to cylindrical, apiculate, smooth. Basidia 30‒45 ×

9‒13 µm in the widest part, clavate, clamped at the base, 4-spored. Cheilocystidia 20‒35

× 7.5‒15 µm, sphaeropedunculate to widely clavate, some septate. Pileipellis a cutis, hyphae 5‒8 µm wide, interwoven, clamp connections present. Squamules on the pileus surface composed of subglobose to more or less ellipsoidal cell, 10‒30 µm in diameter.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., among decomposing needles under Abies

pindrow (Royle ex D. Don) Royle, Junaid Khan, 14th August, 2014, MM-43,

(SWAT000102).

Comments: This taxon represent first report from the study area and second form Pakistan, first being from Sharan as Lepiota aspera (Pers.) Quél by Murakami (1993). By adopting the new name, the genus Echinoderma is introduced for the first time to the fungi of

Pakistan.

Figure 4: A–H. Macroscopic and microscopic characters of Echinoderma asperum (MM-43). A‒C: Basidiomata. A: Natural habitat. B: granulose pileus surface. C: View of the sliced fruiting body. D‒G. Microscopic structures. D: Basidiospores. E: Basidia. F: Cheilocystidia. G: Pileipellis. H: Squamules on the pileus surface. Bars: 12mm for A‒C, 4.2 µm for D, 8 µm for E, G & H, 10.6 for F. LEPIOTA (Pers.) Gray

Lepiota cristata (Bolton: Fr.) P. Kumm., Fu¨hrer in die Pilzkunde:137. 1871 Fig.5

Pileus 15‒25 mm across, convex to conical when young, turning plano-convex to plane by maturity, rarely uplifted, umbonate, pinkish brown with a darker disc (10R 5/6), background cream colored, surface dry, dull, scaly, scales radiating from disc to the margin, cap margin crenate, context thin, pinkish to pinkish red (2.5YR 6/6) below the umbo otherwise cream colored, thicker at the center (2‒2.5mm) than margin (up-to 1mm), dry to slightly moist in some specimens, fragile, mostly unchanging upon bruising or cutting, rarely turning slightly reddish later on. Lamellae free, broad, 3‒4 mm at the center, close, cream colored with a slight pinkish tone in mature specimens, lamellar edge even to slightly serrulate, lamellulae present, in single tier. Stipe 30‒40 × 2‒2.5 mm in dimensions, central, terete, equal in most of the specimens, slightly tapering in others, without any bulb or volva, reddish (10R 3/8) in young stages, reddish (10R 5/8) to cream colored later on, lightly pruinose above the ring, pruinose to smooth below, annulus present, cream colored, membranous, disappearing, may or may not be present in mature specimens, texture fibrous, interior hollow in the center, reddish with a darker upper part, unchanging or slightly reddening upon cutting, fibrous in the center, fibers whitish, appears wet.

Basidiospores (5.3‒) 5.5–7.0 (‒7.2) × (3‒) 3.2–4.5 (‒4.7) µm, Q= (1.6) 1.7–1.8, Me = 6.3

× 3.9 µm, Qe = 1.7, bullet shaped, smooth walled, hyaline to shiny in KOH. Basidia 22–

30 × 10–11 µm, clavate, 4-sterigmated, sterigmata ≤ 5 µm, clavate, thin walled, hyaline in

KOH. Cheilocystidia 22–45 × 10–15µm, clavate to broadly clavate, hyaline to pale yellow in 5% KOH solution, thin walled. Squamules on the pileus surface composed of clavate, narrowly clavate to cylindrical terminal elements, 25–50 × 8–15 µm.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Ingaro Dherai village, 950 m a.s.l., on soil with nearby Salix trees, Junaid

Khan, 22nd July, 2014, ING-16, (SWAT000103). Same location, Junaid Khan, 30th

July, 2015, ING-27, (LAH31328).

Molecular characterization Fig. 6

The ITS region of ING-16 and ING-27 upon amplification with fungal specific primers

(ITS1F and ITS4) yielded a 740 and 754bp fragments respectively. Both the query sequences matched 99% with Lepiota cristata (KJ873871) from Pakistan.

For phylogenetic reconstruction, a total of 26 nucleotide sequences were used that included

15 in-group and 02 outgroup taxa (Macrolepiota velosa Vellinga & Zhu L. Yang

NR119459; Macrolepiota turbinata T. Label JF495078). In the aligned dataset there were

719 sites out of which 452 were conserved, 261 were variable with 210 parsimony informative sites.

Upon maximum likelihood analysis, the sequences of ING-16 and ING-27 clustered with sequences of L. cristata, confirming the identity of present collections as L. cristata.

Comments: this taxon is previously reported twice from Khanspur, Sharan and Dungagali by Iqbal and Khalid and Razaq et al., (2013). This is the third report of this species from

Pakistan and first from the present study area.

Figure 5. A‒F: Macroscopic and microscopic structures of Lepiota cristata (ING-16). A & B: Basidiomata in natural habitat. C: Basidiospores. D: Cheilocystidia. E: Elements of squamules. F: Basidia. Bars: 8 mm for A & B, 6 µm for C, 4 µm for D–F.

99 AF391064.1 Lepiota castaneidisca

30 AF391065.1 Lepiota castaneidisca AF391053.1 Lepiota sp. Vellinga 2542

27 99 AF391052.1 Lepiota sp. Vellinga 2515

70 AY176363.1 Lepiota cristatoides AY176462.1 Lepiota apatelia 32 GQ375546.1 Lepiota neophana 57 99 GQ375547.1 Lepiota neophana KJ873871.1 Lepiota cristata Lepiota cristata (ING16) 76 KY950454.1 Lepiota cristata 96 AF391051.1 Lepiota cristata Lepiota cristata (ING27) NR 119449.1 Lepiota scaberula 99 GQ141550.1 Cystolepiota cystophora KC900376.1 Lepiota coloratipes 64 AF391066.1 Lepiota rufipes 100 KC900377.1 Lepiota coloratipes KC819622.1 Lepiota coloratipes KX711967.1 Lepiota aureofulvella 55 89 AF391025.1 Lepiota boudieri

57 EF080868.1 Lepiota tomentella

76 FJ998395.1 Lepiota brunneoincarnata 100 FJ481017.1 Lepiota brunneoincarnata NR 119459.1 Macrolepiota velosa 97 JF495078.1 Macrolepiota turbinata voucher P87

0.01 Figure 6. Molecular Phylogenetic analysis of Lepiota cristata and associated taxa by Maximum Likelihood method inferred from nITS data using MEGA6. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes-Cantor model. The tree with the highest log likelihood (-2067.5347) is shown.

LEUCOAGARICUS Locq. ex Singer

Leucoagaricus campestris nom.prov. Figs.7 & 8

Etymology: the epithet “campestris” refers to growing in open fields or plains, the habitat of present species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Ingaro Dherai Village,

920 m a.s.l., in humus rich soil in open fields with nearby trees of Populus nigra L., Junaid

Khan, 01st November, 2015, ING-1558 (Holotype), (SWAT000104).

Diagnosis: fruiting body medium to large sized, campanulate to plane with inrolled

margin, grayish brown to yellowish brown, pileus appressed scaly with

appendiculate margin, ellepsoid to amygdaliform basidiospores, large number of

different kinds of cheilocystidia.

Pileus 50‒90 mm across, campanulate with inrolled margin in young stages, plane with uplifted margin and somewhat depressed center in mature specimens, grayish yellowish brown (10YR 5/2) when young, moderate yellowish brown (10YR 5/4) when mature, unchanging upon bruising, surface dry, dull, appressed scaly, areolate in mature specimens, cap margin appendiculate, interior white to creamy white, unchanging upon cutting, thicker from disc to almost hafway of the total pileus diameter (5‒7mm), narrowing outward and less than 1mm at the edge. Lamellae free, broader at the edge and there (5‒7mm), close, cream colored, lamellar edge serrulate, lamellulae present, in 1‒3 tiers, usually one long and two very short. Stipe 40‒80 × 8‒10 mm (in the upper part) and 15‒18 mm at the base, central, terete, clavate, color cream colored in young speciemens, moderate grayish brown

(10YR 5/4) in mature specimens, bruising very light yellowish brown (7.5YR 9/8), very lightly hairy, annulus present, fibrillose, single, white with somewhat grayish brownish

31 zones on the under surface, white to creamy white above and there striate, non-pendent, interior fleshy with a hollow center, flesh white, fibrillose, at the base turning light orange yellow (10YR 9/8) upon cutting otherwise unchanging.

Basidiospores (6.1‒) 6.7‒8.6 (‒11.8) × (4.6‒) 4.8‒6 (‒6.8) µm,Q = (1.3‒) 1.4‒1.6 (‒1.7),

Me = 8 × 5.5 µm, Qe = 1.5, variable in size and shape, ellipsoid in side view, amygdaliform to long ovoid in front view, apiculus present, germ pore present, smooth, thick walled (0.4‒

0.55 µm). Basidia 22‒30 × 7‒9 µm, clavate, 4-spored, sterigmata up to 5 µm long.

Cheilocystidia 45‒60 × 8‒12 µm, variously shaped, narrowly utriform, flexuous, clavate with or without capitate to sub-capitate heads, some irregular with horn like structures.

Pleurocystidia none observed. Squamules on the pileus surface composed of clavate to narrowly clavate cells with septate hyphae, 7‒12 µm in diam.

Molecular characterization Fig.9

The ITS based search using BLAST, matched the query sequences (618bp) of ING-1558 with Leucoagaricus leucothites (Fr.) Singer (KT002154; HQ436119; GQ329038;

EU416308; GQ329058) and L. subcretaceus Bon. (GQ329063; GQ329052.1) with 99% identity.

A total of 22 nucleotide sequences were used in phylogenetic analysis including 08 in- group taxa and one outgroup taxon (Chlorophyllum molybdites KU049476; KU049675).

Upon maximum likelihood analysis, sequences of the Pakistani collection (ING-1558) formed a sister clade with L. leucothites with a strong bootstrap value of 77%, supporting its independent position. The two sequences of L. subcretaceus (GQ329063; GQ329052.1) also clustered with L. leucothites and needs confirmation.

Comments: based on morphological and molecular evidence, the collection ING-1558 is proposed as new to science as L. campestris nom.prov. herein.

Figure 7. A–F. Basidiomata of Leucoagaricus campestris (ING-1558). A: Full basidiome in natural habitat. B: View of the pileus surface. C: View of the appendiculate pileus margin. D: View of the underside. E: View of the annulus. F: View of the context. Bars: 10mm for A‒F.

Figure 8: A – C. Microscopic structures of Leucoagaricus campestris (ING-1558). A: Basidiospores. B: Cheilocystidia. C: Squamules on the pileus surface. Bars: 3.93 µm for A, 7 µm for B & C.

EU416308.1 Leucoagaricus leucothites

GQ329063.1 Leucoagaricus subcretaceus

GQ329038.1 Leucoagaricus leucothites

KF410815.1 Leucoagaricus subcretaceus 77 GQ329058.1 Leucoagaricus leucothites

GQ329052.1 Leucoagaricus subcretaceus

GQ329050.1 Leucoagaricus leucothites 37 Leucoagaricus campestris (ING-1558)

99 Leucoagaricus campestris (ING-1558)

61 KP013004.1 Leucocoprinus fragilissimus

100 U85324.1 Leucoagaricus fragilissimus

AF482868.1 Leucoagaricus nympharum

89 EU416310.1 Leucoagaricus nympharum voucher HMAS 99343 100

GU139790.1 Leucoagaricus hesperius 100

99 GU139789.1 Leucoagaricus hesperius

AY243642.1 Leucoagaricus sp. Vellinga 2587

KU886320.1 Leucocoprinus cepistipes 34 99

U85321.1 Leucoagaricus cepaestipes

83 AY176408.1 Leucoagaricus cinerascens

100 AY176410.1 Leucoagaricus cinerascens

KU049676.1 Chlorophyllum molybdites strain DMSC10488

KU049675.1 Chlorophyllum molybdites strain DMSC13590

0.02 Figure 9. Molecular Phylogenetic analysis of Leucoagaricus campestris and associated taxa by Maximum Likelihood method using MEGA6 software. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes-Cantor model. The tree with the highest log likelihood (-2360.9773) is shown.

LYCOPERDON Pers.

Lycoperdon microsporon nom.prov. Fig. 10

Etymology: the epithet “microsporon” refers to the comparatively small basidiospores of the present taxon among matching taxa

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Malam Jabba valley, 2000 m a.s.l., on soil with under Quercus incana Roxb., Junaid Khan, 25th July, 2015, MJ-1582,

(SWAT000105).

Diagnosis: pyriform fruiting body that ranges in color from pure white to cream

with a pinkish tone, flaking exoperidium, and smaller basidiospores (3.2‒3.6 × 2.9‒

3.29 µm) with a short pedicel (7-10 µm) compared to the L. mammiforme, the

closely matching taxon

Fruiting body 35‒55 mm tall, 25‒45 mm across in the upper part, 10‒12 mm in the basal part, pyriform, exoperidium pure white to cream colored with somewhat pinkish tone (5Y

9/4), smooth, flaking by maturity especially in the upper parts, those at the base are more persistent, often leaving a ring like zone at the junction of pileus and stipe, endoperidium white, smooth to finely granulose/spinulose, opening by an apical pore, gleba white and fleshy at first, powdery and olive brown by maturity. Stipe 8‒15 mm across, white to cream colored, tapering downward, interior white and fleshy at first, spongy and brownish by maturity, does not turn into a powdery mass.

Basidiospores (3.1‒) 3.2‒3.6 (‒3.7) × (2.7‒) 2.9‒3.29 (‒3.3) µm, Q = 1‒1.2 (1.3), Me =

3.4 × 3.1 µm, Qe = 1.1, globose to slightly sub-globose, thick walled (≤0.3 µm), warted,

37 warts (≤0.4 µm), pedicellate, some with pedicel, pedicel 7‒10 µm long, brown to chocolate brown in KOH. Capiliitial hyphae branched, simple without septae, golden brown in KOH.

Elements of the exoperidium 25‒35 × 11‒18 µm, broadly clavate to sphaeropedunculate, pedicel 3‒4 µm in diameter, hyaline in KOH solution. Elements of the peridium 15‒25 ×

13‒20 µm, oval to round in shape.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Malam

Jabba valley, 2000 m a.s.l., on soil with under Quercus incana Roxb., Junaid Khan,

30th July, 2015, MJ-15190, (LAH31329).

Molecular characterization Fig. 11

The ITS region of MJ-1582 and MJ-15190 resulted in a 729 and 734bp fragments, respectively. Upon BLAST search both the query sequences matched 99% with

Lycoperdon mammiforme (AJ237621; DQ112567) from USA and Korea respectively. For phylogenetic reconstruction, a total of 26 closely matching sequences were used in the maximum likelihood analysis with Bovista tomentosa (Vittad.) De Toni (KT958936;

KT958934) as outgroup taxon. The maximum likelihhod analysis clustered the Lycoperdon species into 7 different clades. The Pakistani sequences of L. mammiforme clustered with included sequences of L. mammiforme within the molle/mammiforme clade, forming a separate clade with a strong bootstrap values confirming its novel position.

Comments: based on concrete morphology and molecular evidence, L. microsporon is proposed as new to science.

Figure 10. A–F. Macroscopic and microscopic characteristics of Lycoperdon microsporon (MJ-1582). A: Basidiomes in natural habitat. B: View of the context. C: Basidiospores. D: Capillitial hyphae. E: Elements of the peridium. F: Elements of the exodoperidium. Bars: 6 mm for A & B, 16.2 µm for C & D, 23 µm for E, 9 µm for F.

Lycoperdon microsporon 94 Lycoperdon microsporon 87 Lycoperdon microsporon DQ112567.1 Lycoperdon mammiforme 65 molle/mammiforme 97 AJ237621.1 Lycoperdon mammiforme EU833664.1 Lycoperdon rimulatum DQ112566.1 Lycoperdon molle 38 68 88 EU833662.1 Lycoperdon molle

80 KT875054.1 Lycoperdon excipuliforme KP340186.1 Lycoperdon excipuliforme 44 excipuliforme/altimontanum 60 DQ112589.1 Lycoperdon altimontanum 79 DQ112588.1 Lycoperdon altimontanum DQ112575.1 Lycoperdon lambinonii 26 DQ112576.1 Lycoperdon lambinonii 32 lambinoii/umbrinum 55 DQ112591.1 Lycoperdon umbrinum 39 89 DQ112592.1 Lycoperdon umbrinum DQ112578.1 Lycoperdon echinatum echinatum

62 KU507392.1 Lycoperdon ericaeum ericaeum 99 KU507385.1 Lycoperdon ericaeum JN572908.1 Lycoperdon subcretaceum

subcretaceum/nigrescens 39 KU507387.1 Lycoperdon nigrescens 99 KU507384.1 Lycoperdon nigrescens JN572903.1 Lycoperdon rupicola rupicola 99 JN572901.1 Lycoperdon rupicola KT958936.1 Bovista tomentosa outgroup 99 KT958934.1 Bovista tomentosa

0.005

Figure 11. Molecular Phylogenetic analysis of Lycoperdon microsporon and associated taxa inferred from nITS data by Maximum Likelihood method. The tree with the highest log likelihood (-1465.7594) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. There were a total of 597 positions in the final dataset.

Lycoperdon perlatum Pers., Observ. mycol. (Lipsiae) 1: 4 (1796) Fig. 12

Fruiting body 50‒70 × 40‒60 mm across, mostly pear shaped with a distinct stipe, rarely clavate, stipe 20‒35 × 8‒15 mm in dimensions, often distorted, surface warty in the upper part, granular in the lower, warts longer 1‒1.5mm, pyramidal, white in young specimens, then with a brownish to pinkish tones (5Y 9/4) by maturity, warts fragile often senescing by maturity leaving a netted peridium, opening by an apical pore. Gleba fleshy and white at first, later on powdery and olivaceous brown, finally drying dark brown (10YR 4/4).

Basidiospores (4.5‒) 4.8‒5.2 × (4.3‒) 4.7‒5 (‒5.2) µm, Q = 1‒1.06 (‒1.1), Me = 5 × 4.8

µm, Qe = 1, globose, warted, warts (≤0.5 µm), blackish brown in KOH solution, thick walled (≤0.5 µm). Peridial cells 12‒20 × 9‒12 µm, sub-globose to clavate, hyaline in KOH solution, walls ≤0.5 µm.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Malam

Jabba valley, 2000 m a.s.l., on soil under Pinus wallichiana, Junaid Khan, 10th

August, 2014, MJ-51, (SWAT000106). PAKISTAN, KHYBER

PAKHTUNKHWA, Miandam valley, 2200 m a.s.l., on soil under Pinus

wallichiana, Junaid Khan, 14th August, 2014, MM-53, (LAH31330).

Comments: Lycoperdon perlatum is previously reported from different localities in

Pakistan including Swat district (Ahmad, 1952; 1956). This is the second report of this species from present study area.

Figure 12. A – F. Macroscopic and microscopic features of Lycoperdon perlatum. A: Fruiting body. View of the surface. C: sliced fruiting body showing internal gleba. D: Basidiospores. E: fruiting Body. E: Peridial elements. Bars: 5 mm for A‒C & F, 9.75 µm for D, 12 µm for F. MACROLEPIOTA Singer

Macrolepiota excoriata (Schaeff.) Wasser, Ukr. bot. Zh. 35(5): 516 (1978) Fig.13

Pileus 80‒100mm across, parabolic when young, turning plano-convex with age, light greenish yellow (10Y 9/6), surface dry, dull, appressed scaly, margin appendiculate, interior fleshy, cream colored, unchanging upon cutting, thicker at the disc(3-6mm), thinner at the margins (≤1mm). Lamellae free, broader at the middle (3‒6mm), close to crowded, cream to pinkish cream colored (7.5Y 9/4) in young specimens, olive brown

(2.5Y 5/4) in mature, lamellar edge even. Stipe 70‒110 × 15‒18 mm, central, terete, thickening downward with a slightly swollen base (20mm), cream colored with a yellowish to olivaceous tone, bruising yellow at the base only, surface covered with annular remnants below, scaly to scaly-fibrillose above, interior more or less concolorous to the exterior, turning yellow at the base upon cutting, centrally hollow except for the extreme apex and base, flesh thick and brittle.

Basidiospores (8.4‒) 8.7‒11.8 (‒12.6) × (4.8‒) 5.5‒6.6 (‒7.1) µm, Q = (1.4‒) 1.6‒1.8 (‒

1.9), Me = 10 × 5.9 µm, Qe = 1.7, ellipsoid to boat shaped with one broader and other slightly narrower end, smooth, apiculus present, germ pore present, thick walled (≤0.5 µm), guttulate, guttule one to many. Basidia 25‒30 × 6‒8 µm, clavate, mostly 2-sterigmated, rarely 4-sterigmated, sterigmata ≤0.4 µm, basal clamps absent. Cheilocystidia 15–45 × 8–

15 µm, clavate, sub cylindrical to lanceloate. Squamules on the pileus surface made of branched filamentous to cylindrical hyphae, 3–15 μm in diam, terminal elements 30–150

× 5–12 μm. Pileipellis hyphae composed of simple interwoven, branched septate hyphae,

3-6 µm in diam.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2200 m a.s.l., on Soil under Pinus wallichiana, Junaid

Khan, 27th July, 2014, MJ-30, (SWAT000107).

Molecular characterization Fig. 14

The ITS region of the Pakistani collection (MJ-30) produced 741bp sequences. Upon

BLAST search, the query sequences matched 99% with Macrolepiota excoriata

(HQ412660; JQ683100; JQ683089) from USA and Israel.

To infer the phylogenetic relationship of Pakistani collection of Macrolepiota excoriata, a total of 31 nITS nucleotide sequences were used with Leucoagaricus barssii (Zeller)

Vellinga (GQ329062) and Chlorophyllum brunneum (Farl. & Burt) Vellinga (AY081227;

AY081231) as outgroup taxa. There were a total of 736 characters in the final aligned dataset out of which 465 characters were conserved, 251 variable and 171 parsimony informative.

Maximum likelihood analysis clustered the included sequences into three clades viz.

Macrolepiota, Macrosporae and Volvatae. These clades are in conformity with the findings of Ge et al., (2010) except the M. clelandii Grgur., occupying a separate position in contrast to their finding. The Pakistani sequences of M. excoriata clustered with other sequences of the same species from USA (HQ412660) and Israel (JQ683100; JQ683089) within the

Macrosporae clade with a strong bootstrap value, thus confirming its identity as

Macrolepiota excoriate.

Comments: this is the second report of this taxon from Pakistan (first By Fiaz, 2013 form

Mansehra district) while first from the present study area.

Figure 13. A–G. Macroscopic and microscopic features of Macrolepiota excoriata (MJ-30). A‒C: Different views of basidiomata. D‒G. Miacroscopic structures. D: Basidiospores. E: Basidia. F: Cheilocystidia. G: Squamules on the pileus surface. Bars: refer to scale for A‒C, 4 µm for D & E, 6 µm for F, 17 µm for G.

HM246503.1 Macrolepiota permixta 70 HQ412661.1 Macrolepiota permixta 98 JQ683107.1 Macrolepiota procera JQ683106.1 Macrolepiota procera 43 100 AF482839.1 Macrolepiota dolichaula HM125522.1 Macrolepiota dolichaula 98 KR362911.1 Macrolepiota detersa 38 Macrolepiota 99 KJ524560.1 Macrolepiota detersa AY243598.1 Macrolepiota fuliginosa

63 AY243597.1 Macrolepiota fuliginosa 99 AY243597.1 Macrolepiota fuliginosa AY243596.1 Macrolepiota rhodosperma KF937346.1 Macrolepiota colombiana 98 U85311.1 Macrolepiota colombiana 26 JQ683089.1 Macrolepiota excoriata HQ412660.1 Macrolepiota excoriata 82 JQ683100.1 Macrolepiota excoriata Macrolepiota excoriata (MJ-30) 68 Macrolepiota excoriata (MJ-30) Macrosporae 68 98 72 AY243600.1 Macrolepiota psammophila JQ683125.1 Macrolepiota konradii AF482844.1 Macrolepiota mastoidea 99 HM125530.1 Macrolepiota mastoidea AY243605.1 Macrolepiota subsquarrosa NR 119459.1 Macrolepiota velosa Volvatae 100 KJ524569.1 Macrolepiota velosa

100 AF482838.1 Macrolepiota clelandii AY083203.1 Macrolepiota clelandii GQ329062.1 Leucoagaricus barssii

Outgroup 96 AY081227.1 Chlorophyllum brunneum 100 AY081231.1 Chlorophyllum brunneum

0.01 Figure 14. Molecular Phylogenetic analysis of Macrolepiota excoriata and associated taxa inferred from nrITS data using Maximum Likelihood method in MEGA6. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes-Cantor model. The tree with the highest log likelihood (-1815.3508) is shown. The analysis involved 31 nucleotide sequences. There were a total of 448 positions in the final dataset.

ALBATRELLACEAE

a) Albatrellopsis

1. A. flettiodes nom.prov.

b) Albatrellus

1. A. roseus nom.prov.

c) Jahnoporus

1. J. oreinus

ALBATRELLOPSIS Teixeira.

Albatrellopsis flettiodes nom.prov. Fig. 15

Etymology: The name “flettiodes” is derived from its closely matching Albatrellopsis flettii

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Miandam valley, 2250 m a.s.l., on Soil under Pinus wallichiana, Junaid Khan, 23rd August, 2014, MM-72

(Holotype), (SWAT000108).

Diagnosis: Fruiting body terrestrial, pileus infundibuliform to irregular, more or

less circular, shiny, grey to rusty greyish brown with a sterile margin beyond the

tube layer and whitish grey decurrent pore surface with small angular pores,

basidiospores sub-globose to broadly ellipsoid, inamyloid.

Fruiting body annual, terrestrial, single, without any odor. Pileus up-to 100mm across, infundibuliform, more or less circular in outline, sticky when wet, shiny, grey (10Y6/2) at first then developing rusty greyish brown (7.5YR7/6) stains with age, does not turn bluish upon handling or bruising, shape of margin arched, exceeding the pore surface, cap surface smooth to finely velutinous especially on edge, center finely areolate with age, context dry, firm, yellowish red (7.5YR6/8) just above the pore surface, turning paler towards pileal surface (10YR9/2), up to 5 mm at margins and up to 8 mm at the center, continuous with the stipe. Pore surface whitish grey (7.5GY7/2) with a darker (7.5GY4/2) marginal area, descending to the stipe up to the base, pores angular, very small, up to 7 per mm, does not bruise or slightly turning greyish (7.5GY5/2), tube layer thin, ≤ 1mm. Stipe up to 7 cm long and 3.5 cm in diameter, central, terete, unequal, tapering towards top, narrowing in the underground part, concolorous with the pore surface, pores present, shape of the pores irregular compared to the pore surface, sometimes, furfuraceous in appearance, grey

(7.5GY4/2), inside solid, context thick, fleshy, yellowish red to rusty brown (7.5YR6/8) just beneath the stipitipellis, creamy yellowish red (2.5YR9/2) in the center. Reaction with

KOH: no reaction or slightly turning lilaceous.

Basidiospores (4.6‒) 4.2‒5 (‒5.4) × (3.8‒) 3.4‒4 (‒4.4 µm), Q = 1.1‒1.3 (‒1.35), Me =

4.8 × 3.9 µm, Qe = 1.2, sub-globose to oval in face view, broadly ellipsoid in side view, with single guttule, thick walled (≤0.5 µm), hyaline in KOH solution, inamyloid, apiculate.

Basidia 15‒20 × 5‒7 µm, clavate to oblong or irregular, 4-spored, guttulate. Tramal hyphae 2‒3 µm in diameter, thin walled, branched, clamp connections frequent, hyaline to slightly brownish in KOH. Gloeopleurous hyphae scattered, rare, golden brown in

KOH, 5‒7 µm in diameter. Pileipellis simple septate, hyphae 4‒6 µm in diameter.

Contextual hyphae thin walled, branched, with frequent clamps, hyaline in KOH, 5‒7 µm in diameter.

Distribution: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Miandam valley.

Comments: based on discrete morphological and molecular data (nITS), the collection

MM-72 is being proposed as new to science as Albatrellopsis flettiodes herein.

Figure 15: A – I. Morphological characters of Albatrellopsis flettiodes (MM-72). A‒D: Different views of basidiome. E‒I. Microscopic structures. E: Basidiospores. F: Basidia. G: Tramal hyphae. H: Contextual hyphae. I: Pileipellis. Bars: 7mm for A & D, 10mm for B & C, 5 µm for E & F, 7 µm for G, 9.33 µm for H & I.

ALBATRELLUS Gray.

Albatrellus roseus J. Khan, Sher & Khalid Fig. 16

Etymology: the specific epithet “roseus” refers to the rose colored pileus of the present taxon

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat district, Gabin Jabba valley, on ground under Picea smithiana (Wall.) Boiss, Junaid Khan, 30th August 2015, GJ-1511

(Holotype) (SWAT0001009).

Diagnosis: Fruiting body annual, terrestrial in small scattered groups to gregarious

with overlapping pilei. Pileus yellowish brown to pinkish red squamulose, pore

surface decurrent, white to creamy white that turn yellow upon handling and

pinkish red upon aging and reaction with KOH, stipe white to creamy white turning

yellowish orange upon maturity, ovoid to ellipsoid, amyloid basidiospores,

contextual hyphae contorted.

Fruiting body annual, terrestrial, in small scattered groups, sometimes gregarious with overlapping pilei, usually single rarely fused. Pileus 40‒70 mm across, loosely convex to umbilicate with a slight central depression, circular to kidney shaped or sometimes irregular in shape, dry, smooth at first then cracking with age giving areolate appearance especially in dry conditions, patches radiating from center to margins, yellowish brown

(10YR8/8) then turning pinkish brown (7.5R4/4) at the margin and lighter reddish at the center (7.5R6/6), background creamy (5Y0/2) to whitish yellow (5Y9/4), color of the basidiomes turn dull brown to blackish brown upon aging and drying, cap margin even in young specimens then cracking with age, the edge turning yellow and then greenish with age, flesh white, not changing upon cutting, ≤ to 1mm at the margins, ≤ to 5mm at the

51 center. Pore surface white to creamy white, drying olivaceous, descending the stem up to the base, pores irregular ranging in shape from round, angular to elongated, small especially at the margins, gradually getting larger toward center and then from stipe top to base, 2‒5 per mm, bruising yellow (10Y8/10) especially in young stages, then drying pinkish red (5R5/10). Stipe usually central or slightly eccentric, rarely lateral, terete, ≤ 50

× 10 mm, usually equal or slightly swollen at the base in some specimens, with up‒to 20 mm long underground base that tapers gradually towards the base, with white rhizoidal cluster, white to creamy white at first then yellowish orange (5YR6/10) at maturity and drying blackish brown, aerial portion covered with pores with a smooth underground portion, pore size larger as compared to the pore surface of the cap, 2‒3 per mm, mostly angular becoming irregular with age, context white in the aerial portion while yellowish brown at the base, does not changing upon cutting or slightly turning yellowish (5GY9/8).

Odor none or mushroomy when young then with an unpleasant smell upon drying and aging. Reaction with KOH: The pileus surface at first turns yellow (10Y9/8), then olive green (10Y6/6) and within seconds turns pink to pinkish red (10RP8/14) which gets darker

(10RP8/16) with time. The pore surface and the stipe also turn pinkish red (10RP4/12) very quickly upon reaction with KOH.

Basidiodpores (4.8‒) 4.9‒6 (‒6.4) × (3.9‒) 4‒4.58 (‒4.6) µm, Q = 1.2‒1.38 (‒1.4), Me =

5.5 × 4.3 µm, Qe = 1.3, ellipsoid to ovoid, hyaline in KOH solution, amyloid in Melzer’s reagent, with single guttule, apiculate. Basidia 15‒20 × 4‒6 µm club shaped, simple septate at the base, hyaline in KOH. Cystidia none observed. Hyphal system monomitic. Tramal hyphae thin walled, hyaline to slightly golden in KOH, unbranched, with clavate terminal ends, 3‒4 µm in diameter. Pileipellis hyphae unbranched, simple septate, light golden in

KOH solution, mostly 4 µm in diameter. Contextual hyphae simple, thin walled, contorted with irregular constrictions and expansions, rarely branched, 5‒15 µm in diameter.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat district, Gabin Jabba valley, on ground under Picea

smithiana, Junaid Khan, 22nd September, 2016, (GJ-1621).

Distribution: Gabin Jabba valley, Swat District, Khyber Pakhtunkhwa Province.

Comments: based on morphological and molecular evidence, this species is reported as new to science and published in Mycoscience (Annexure-A).

Figure 16: A‒I. Morphological characters of Albatrellus roseus. A‒C: Holotype collection. D: GJ-1621. E‒I: Microscopic structures. E: Basidiospores. F: Basidia. G: Tramal hyphae. H: Pileipellis. I: Contextual hyphae. Bars: 12mm for A‒D, 3.5 µm for E &F, 7.5 µm for G & H, 8 µm for I. Molecular characterization Fig. 17

The target region (ITS1, 5.8S and ITS2) GJ-1511 and GJ-1621 yielded a 638bp long sequences which upon BLAST search showed 95% similarity with A. avellaneus Pouzar

(EU837239; EU669394) from USA. Similarly, the target region of MM-72 upon sequencing generated a 680bp long sequence which upon BLAST search matched 89 % with Albatrellus flettii Morse ex. Pouzar (AY621802; AY061738) from USA.

To reconstruct the phylogeny of Pakistani collections (GJ-1511;GJ-1621 & MM-72), closely matching sequences were downloaded and used in Maximum likelihood analysis.

Upon phylogenetic analysis, the included species clustered within different clades as mentioned by Audet (2010). Pakistani collections (GJ-1511 and GJ-1621) clustered within genus Albatrellus forming a sister clade with A. avellaneus with strong bootstrap value of

96%, supporting its independent position. The second Pakistani collection (MM-72) clustered within the genus Albatrellopsis forming a sister clade with A. flettii with a strong bootstrap value of 93% also supporting its unique nature.

Figure 17: Phylogenetic analysis of Albatrellopsis, Albatrellus and associated species based on nITS molecular data.

JAHNOPORUS Nuss.

Jahnoporus oreinus V. Spirin, J. Vlasák & O. Miettinen, Cryptog. Mycol. 36: 414 (2015)

Fig. 18

Fruiting body annual, fleshy when fresh, woody when mature or dry, several fused pilei arising from common stipe. Pileus individual pileus 30‒50 mm across, overall fruiting body 120 mm wide, semicircular to oval in shape, pale orange brown (7.5 YR 8/4) with a dark brownish center (5YR 3/4), surface dry, shiny, bumpy, hispid, pileus margin even to wavy, slightly incurved in some areas. Pore surface slightly decurrent, cream‒colored, drying brownish orange (5YR 5/8), pores mostly angular, some irregular, 2‒3 per mm, tubes 2‒3 mm deep at the center, 1mm at the edge, concolorous to the pore surface. Stipe

40‒50 × 8‒12 mm, lateral, hispid, hairs denser and longer than pileus, yellowish brown

(7.5YR 6/8) upward, strong brown (7.5YR 3/6), interior solid, white. Odor and Taste foul.

Basidiospores (12‒) 12.4‒12.9 (‒13.6) × (4.7‒) 5.1‒5.6 (‒5.9) µm, Q = (2‒) 2.2‒2.5 (‒

2.7), Me = 12.7 × 5.4 µm, Qe = 2.3, fusiod to navicular, smooth, mostly with single oil droplet at the center. Basidia 40‒50 × 11‒13 µm, narrowly clavate, mostly 2‒sterigmated, rarely 4‒sterigmated, sterigmata 6‒8 µm long, thin walled, hyaline in KOH, with oily droplet, clamped at bases. Cystidia 35‒45 × 3‒5 µm, narrowly clavate, thin walled to thick walled. Pileipellis hyphae, parallel with somewhat wavy walls, 3‒3.5 µm in diameter, clamped. Tramal hyphae elongated, septate, clamped, 3‒4 µm in diameter. Contextual hyphae composed of parallel hyphae which are thick walled, 3‒5 µm in diameter, mixed with gloeocystidia, gloecystidia 70‒100 × 10‒15 µm, thick walled (≤3µm), irregular.

Hyphal system monomitic.

Materials Examined: PAKISTAN, KHYBER PAKHTUNKHWA, District Swat,

Gabin Jabba valley, on ground under Picea smithiana, Junaid Khan 30th August

2015, GJ-1511, (SWAT000110).

Molecular characterization Fig. 19

The target region (ITS) yielded a 630bp fragment which showed 99 % similarity with J. oreinus (Cooke) Nuss (KU165786; KU165785) followed by J. brachiatus (KU165781) with 99 % similarity and J. hirtus (Cooke) Nuss (KU165782; KU165783; KU165784) with

97 % similarity.

A total of 11 nucleotide sequences were used in the phylogenetic analysis containing 9 in- group taxa and two (Albatrellus citrinus NR132801; A. piceiphilus DQ789396) outgroup taxa. The maximum likelihood analysis clustered Pakistani collection with J. oreinus confirming the identity as J. oreinus.

Comments: Jahnoporus orenius represent the first record of the genus and species for

Pakistani mycobiota and probably second from the Asia.

Figure 18: A–G. Morphology of Jahnoporus oreinus (GJ-1513). A & B: Basidiomata. C‒G: Microscopic structures. C: Basidiospores. D: Basidia. E: cystidia. F: Contextual hyphae with Gloeopleurous hyphae. G: Pileipellis. Bars: 10mm for A & B, 5.5 µm for C ‒ E, 12 µm for F, 5 µm for G.

Jahnoporus oreinus (Pakistani collection)

95 Jahnoporus oreinus (Pakistani collection) KU165786 Jahnoporus oreinus 72 KU165785 Jahnoporus oreinus

53 KU165781 Jahnoporus brachiatus KU165783 Jahnoporus hirtus 100 DQ911605 Jahnoporus hirtus 97 KU165784 Jahnoporus hirtus FJ439517 Jahnoporus hirtus NR 132801.1 Albatrellus citrinus DQ789396.1 Albatrellus piceiphilus

0.05

Figure 19. Molecular Phylogenetic analysis of Jahnoporus oreinus and associated taxa by Maximum Likelihood method using nrITS data. The tree with the highest log likelihood (‒1235.8588) is shown.

Family AMANITACEAE

a) Amanita

1. A. ahmadii nom.prov

2. A. cinerea nom.prov.

3. A. cinnamomescens

4. A. griseofusca

5. A. pakistanica

6. A. pallidorosea

7. A. pantherina

AMANITA Pers.

Amanita ahmadii nom.prov. Figs. 20 & 21

Etymology: “Ahmadii” in the honor of pioneer mycologist of Pakistan Dr. Sultan Ahmad.

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Gabin Jabba valley,

2300 m a.s.l., under Picea smithiana, Junaid Khan, 31st August, 2015, GJ-1507 (Holotype),

(SWAT000111).

Diagnosis: fruiting body medium sized, convex to plano-convex, dark grey to

brownish grey, pileus shiny with more or less regularly arranged pyramidal warts,

margin appendiculate, lamellae close to sub-distant, concolorous bulbous stipe,

reddening at the base, annulus superior with striated upper surface, pendent, sub-

globose to oval basidiospores.

Pileus 40‒70 mm across, convex when young, maturing plano-convex to plane with uplifted margin, dark grey (10Y 5/2) to brownish grey (10YR 5/2) in some specimens, surface dry, shiny, warty, warts pyramidal, more or less regularly arranged especially in young specimens, some specimens have very few warts concentrated especially at the margin, pileus margin appendiculate with hanging annular remnants, cracking in mature specimens, interior solid, flesh white to cream colored, slowly turning reddish upon breaking, thicker at the center (3‒4mm). Lamellae adnexed, appear slightly decurrent, broader at the center (2.5‒3.5mm), lamellar edge even to slightly serrulate, close to sub‒ distant, cream colored with or without a grayish (10Y 5/2) edge especially in young stages, unchanging, lamellulae present, mostly in single tier. Stipe 60‒90 × 9‒15 mm in dimensions, central, terete, straight in some specimens flexuous in others, thickening

62 downward with a bulbous base, color same as pileus to slightly light gray (10Y 6/2), base turning reddish orange (10R 5/12) to reddish brown (2.5YR 2/6), striated above the ring and there finally breaking to form horizontal zones, fibrillose to felty below and breaking there forming a cracked appearance, interior solid, flesh cream colored, turning reddish brown (2.5YR 2/6) at the base otherwise unchanging, annulus present, superior, membranous, pendant, striated above, felty below, concolorous with the pileus.

Basidiospores (6.7‒) 7.2‒8.6 (‒9.4) × (5.9‒) 6.3‒7.6 (‒8) µm, Q = (1‒) 1.1‒1.3, Me = 7.9

× 6.8 µm, Qe = 1.2, sub-globose to oval, hyaline in KOH solution, amyloid in Melzer’s reagent, uniguttulate to granular in KOH, with prominent apiculus. Basidia 35‒42 × 8.5‒

10.5 µm, clavate to narrowly clavate, 4-sterigmated, sterigmata 4‒5 µm, guttulate in Congo red, basal clamps absent. Cystidia same size as basidia, clavate. Pileipellis composed of globose to sub-globose elements, 30‒40 × 15‒20 µm, annulus composed of filamentous hyphae with sub‒globose to elongated terminal ends, terminal ends 40‒50 × 13‒17 µm in dimensions.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Gabin Jabba valley, 2300 m a.s.l., under Picea smithiana, Junaid Khan,

04th September, 2015, GJ-1508, (LAH31389).

Molecular characterization Fig. 22

The ITS region generated a 707 base pairs long fragment upon sequencing which showed

98 % similarity with an unknown species of Amanita (KJ466372; KJ466373) from China.

For phylogenetic reconstruction, maximum likelihood analysis was used including 26 nucleotide sequences with A. griseofolia Zhu L. Yang (NR119498; KF017934) as outgroup

63 taxon. The query sequences clustered with other species of section validae of subgenus

Lepidella along-with Amanita aff. fritillaria, A. aspera and A. franchetii.

Comments: Based on the distinctive morphology and supportive molecular analysis the collections (GJ-1507 and GJ-1508) are proposed as new to science.

Figure 20. A – D. Basidiomata of Amanita ahmadii. A: Basidioma in natural habitat (GJ-1507). B: View of the lamellae. C: View of full basidiomes (GJ-1508). D: Basidiome in natural habitat. Bars: 8mm for A‒D.

Figure 21. A – E. Microscopic structures of Amanita ahmadii. A: Basidiospores. B: Annular elements. C: Basidia. D: Cystidia. E: Pileipellis. Bars: 9 µm for A, 40 µm for B&E, 15.5 µm for C, 18 µm for D.

96 KF937300 Amanita flavoconia 64 KF937301 Amanita flavoconia AB015696 Amanita flavipes 75 JN020967 Amanita flavoconia KJ638281 Amanita flavoconia 56 82 JF313655 Amanita flavoconia KF245911 Amanita flavipes KR919762 Amanita morrisii 48 51 KR919760 Amanita morrisii 97 KT213441 Amanita morrisii

98 JF313650 Amanita flavorubescens GQ166902 Amanita flavorubescens KP711839 Amanita sp cruetilemuru 80 21 KC152067 Amanita novinupta 98 KC152066 Amanita novinupta KP711831 Amanita sp. cruetilemur

94 JX515561 Amanita franchetii AF085485 Amanita aspera 40 94 Amanita ahmadii (GJ-1507) 32 Amanita ahmadii (GJ-1508) 83 KJ466372 Amanita aff. fritillari 97 KJ466373 Amanita aff. fritillari KJ638284 Amanita rubescens EF493271 Amanita spissa NR119498 Amanita griseofolia 100 KF017934.1 Amanita griseofolia

0.05 Figure 22: ITS based Molecular Phylogenetic analysis of Amanita ahmadii and associated species by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes‒Cantor model. There were a total of 451 positions in the final dataset.

Amanita cinerea nom.prov. Figs. 23 & 24

Etymology: the epithet “cinerea” refers to the ash gray color and texture of the pileus

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

1950 m a.s.l., under Quercus dilatata Royle, in Quercus‒coniferous mixed forest, Junaid

Khan, 08th August, 2015, MJ-15108 (Holotype), (SWAT000112).

Diagnosis: pileus slate gray in color, surface ashy-felty especially in young stages,

with or without warts at mature stages, margin very slightly striated, stipe bulbous

with volval remnants arranged in rings, fibrillose often forming horizontal rings,

centrally hollow, basidiospores broadly ellipsoid, cystidia sphaeropedunculate.

Pileus 80‒130 mm across, hemispherical when young then turning plano-convex with age, silvery to slate gray (5GY 9/2) at first then slate gray with a brownish to pinkish tone (10Y

8/2) at maturity, covered with gray (5GY 5/2) ash-felty volval remnants which diminishes with age exposing the surface, some specimens with concentrically arranged warts, slightly striated at the extreme margins, pileus margin smooth, usually non-appendiculate, occasionally with a few volval remnants, context white, thicker at the center (2‒3mm) than margins (≤1mm), unchanging upon cutting. Lamellae free, broader at the center (3‒5 mm) crowded, white, cream colored, somewhat pruinose, lamellulae present, frequent, of diverse lengths, unchanging upon bruising. Stipe 100‒150 × 15‒20 mm, central, terete, bulbous at the base and there covered with remnants forming rings, white with light gray

(10Y 8/2) fibrils, fibrils more prominent downward, irregularly arranged at first, forming horizontal rings upon maturity, especially at the apex and base, color darkening (2.5GY

5/2) upon bruising except at the base where turning reddish (5R 4/10), annulate, annulus

67 membranous, pendulant, fragile, upper surface concolorous with the pileus and there striated, while under surface dark grey (5GY 5/2) and smooth to felty, volva absent, flesh white, centrally hollow, unchanging upon cutting.

Basidiospores: (6‒) 6.5‒8 (‒8.5) × (4.5‒) 5‒5.5 (‒6) µm, Q= (1.3‒) 1.4–1.45 (‒1.46) µm,

Me = 7.25 × 5.25, Qe = 1.3, broadly ellipsoid, contents appear granular in Congo red, rarely smooth with a single central oil granule, apiculus present. Basidia 30‒35 × 6‒8 µm, clavate, 4-strigmated, sterigmata 3‒4 µm long, guttulate, basal clamps absent. Cystidia

30‒35 × 12‒20 µm, sphaeropedunculate. Pileipellis composed of globose to sub-globose elements, 30‒40 × 15‒20 µm, with scattered simple septate and branched hyphae.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 1950 m a.s.l., under Quercus dilatata, Junaid Khan,

16th August, 2015, MJ-1557 (LAH31390). Same locality, Junaid Khan, 31st July,

2016, MJ-1613, (LAH31391).

Molecular characterization Fig. 25

A 685bp sequence was generated by amplifying the nITS region of MJ-15108 and MJ-

1557, which showed 97% identity with Amanita spissacea S. Imai (KM052543;

KM052546; KM052550) upon BLAST search. Unexpectedly the query results had some sequences of Amanita griseofolia Zhu L. Yang (KF017934; KF017936; NR119498) and based on their high similarity these were also included in the subsequent phylogenetic analysis.

The maximum likelihood analysis involved 26 nucleotide sequences of Amanita species with Amanita zangii (Zhu L. Yang, T.H. Li & X.L. Wu) Vizzini & Contu (KJ466433) as

68 outgroup taxon. The Pakistani sequences of MJ-15108 and MJ-1557 formed a separate clade along-with Amanita spissacea and Amanita australis Stev., with a strong bootstrap value of 98%. The included Amanita griseoflia sequences formed a separate clade. The analysis clearly support the independent position of Pakistani collections (MJ-15108 and

MJ-1557).

Comments: Morphological novelty together with molecular evidence supports the independent position of Amanita collection (MJ-15108 and MJ-1557) which is being proposed as new to science here as A. cinerea nom.prov.

Figure 23: A‒F. Basidiomata of Amanita cinerea. A: Holotype Collection in natural habitat showing different stages and ornamentation. B: View of the lamellae. C: Young stage (MJ-1613). D: View of the stipe and annulus. D: View of the felty pileus. E: Collection MJ-1557 in natural habitat. Bars: 5mm for A‒F.

Figure 24: A – D. Microscopic structures of Amanita cinerea (MJ-15108). A: Basidiospores. B: Basidia. C: Cystidia. D: Pileipellis. Bars: 6.5 µm for A, 12 µm for B&C, 21 µm for D.

KP711836 Amanita sp. cruetilemur 95 KP711839 Amanita sp. cruetilemur

KC152065 Amanita novinupta

KF245918 Amanita rubescens

95 KF245919 Amanita rubescens

JF313650 Amanita flavorubescens

GQ166902 Amanita flavorubescens 95

50 KP711842 Amanita novinupta 99 KR338836 Amanita novinupta

EF493271 Amanita spissa 100 EF493270 Amanita spissa

AY436453 Amanita excelsa

KR919762 Amanita morrisii 100 99 58 KR919761 Amanita morrisii

KF245911 Amanita flavipes

99 KC581322 Amanita franchetii

GU222314 Amanita australis

Amanita cinereus (MJ-15108) 99 Amanita cinereus (MJ-1557)

98 KM052546 Amanita spissacea KM052543 Amanita spissacea 99 KM052550 Amanita spissacea

KF017936 Amanita griseofolia

100 NR 119498 Amanita griseofolia 90 KF017934 Amanita griseofolia

KJ466433 Amanita zangii

0.02 Figure 25: Molecular Phylogenetic analysis of Amanita cinerea by Maximum Likelihood method inferred from nITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes‒Cantor model. All positions containing gaps and missing data were eliminated.

Amanita cinnamomescence Tulloss, S. H. Iqbal, A. N. Khalid & Bhandary Figs. 26 &

Pileus 100‒160 mm across, ovoid to conic at first, plano‒convex with an umbo later on, sometimes upturned with reduced umbo, in young specimens the color is strong orange

(5YR 6/12) with a deep orange (2.5YR 5/12) disc, in mature stages deep orange yellow

(10YR 6/12) with a strong brown (7.5YR 5/8) disc, surface dry, shiny, dull in some specimens (MM-1541), texture smooth with a striated margin, striation one third of the total pileus diameter, disc slightly velutinous otherwise glabrous, with or without volval remnants, when present mostly as a single central patch, cap margin entire to crenate, cracking in some specimens, interior solid, context white, unchanging or slightly getting brownish upon cutting. Lamellae free to adnexed, close, broad (≤7mm at the center), gill edge entire, ≤ 0.8 mm, yellowish white (5Y 9/4) in young specimens then aging cream colored, lamellulae present, of diverse lengths. Stipe 200‒300 × 15‒25 mm, central, terete, thickening downward, brilliant yellow (5Y 9/10) to yellowish white (5Y 9/6), fibrillose to fibrillose squamulose below the annulus, striated above the annulus, context white, breaking with a snap, annulus present, superior, membranous, pendent, yellow (5Y 7/10) above and striated there, smooth below, volva saccate, membranous, persistent, 40‒55 ×

30‒40 mm, white, unchanging.

Basidiospores (7.1‒) 8.3‒10.3 (‒11) × (6.5‒) 6.7‒8.7 (‒9) µm, Q = (1.1‒) 1.15‒1.3 (‒1.4),

Me = 9.3 × 7.7 µm, Qe = 1.2, mostly ellipsoid, rarely ovoid in face view, hyaline in KOH, thin walled, with a large central guttule, apiculus present. Basidia 40‒50 × 11‒14 µm, clavate, thin walled, hyaline in KOH, smooth to guttulate, clamped at the base. Cystidia same length as basidia, 15‒20 µm wide, broadly clavate, contents granular to guttulate at

72 the bases in Congo red. Pielipellis composed of thin walled, clamped, elongated hyphae of size 2‒3 µm in diameter. Volva composed of sphaerocytes of size 30‒50 × 20‒35 µm.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2150 m a.s.l., among decomposing needle litter under

Abies pindrow, Junaid Khan, 01st August, 2014, MM-06, (SWAT000113). Same

valley, 2250 m. a.s.l., in decomposing needle litter under Abies pindrow, Junaid

Khan, 09th August, 2015, MM‒1541, (LAH31392).

Comments: During our present venture, we collected several collections of this species form Miandam and Malam Jabba valleys repeatedly. Morphological and anatomical characters of the collections (MM-06; MM-1541) closely matches with Amanita cinnamomescens at www.amanitaceae.org and are treated as Amanita cinnamomescens.

This is one of the common and widespread Amanita species found in coniferous forests of the study area and can be easily recognized in the field by larger size, orange yellow coloration, striated pileus margin and yellowish annulate and volvate stipe.

Figure 26: Colored photographs of different collections of Amanita cinnamomescens. A: MM-06 in natural habitat. B: Pileus surface showing coloration, central umbo and striation. C: View of the striated annulus and stipe there. D: Juvenile stage showing different coloration (MM-1541).

Figure 27: A – E. Microscopic structures of Amanita cinnamomescens. A: Basidiospores. B: Basidia. C: Cystidia. D: Pileipellis. E: Volval elements. Bars: 10.5 µm for A, 14 µm for B&E, 20 µm for C, 7.5 µm for D.

Amanita griseofusca J. Khan & M. Kiran Fig. 28

Etymology: the epithet “griseofusca” refers to brown color with a grayish tinge, characterizing the species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

1950 m a.s.l., under Quercus dilatata in mixed coniferous forest, Junaid Khan, 16th August,

2015, MJ-1558 (Holotype), (SWAT000114).

Diagnosis: greyish brown pileus with white to beige, membranous volval remnants

present as one (large) to a few (small) warts, striations one third of the total pileus

radius, close, cream lamellae with a pink tone, fibrillose stipe with a white loose

and saccate volva and broadly ellipsoidal to ellipsoidal inamyloid basidiospores.

Pileus 50‒80mm across, hemispherical to elliptical when young, flat when mature, may have a slightly depressed center especially in mature specimens, grayish brown (10YR 5/2) with a dark grayish brown (10YR 3/2) disc, striated almost one third from the margin, smooth at the disc, warts present in some specimens absent in others, when present in many small to medium sized or few large patches, white at first then with a grayish (2.5Y 7/4) tone later on, soft, spongy, thick, cap margin entire at first then cracking with age, context creamy white, fragile, unchanging upon cutting, thicker at the center (2‒3mm). Lamellae free, crowded, pruinose especially in young specimens, broader (5‒7 mm) at the center, lamellar edge even, creamy at first the pale yellow with a pinkish tone (2.5Y 9/4) in mature stages, fragile, lamellulae rare and short. Stipe 80‒100 × 8‒12 mm, equal or slightly thickening downward, central, terete, creamy white to pale yellowish tan (2.5Y 9/4), fibrillose especially at the bottom, fibrils whitish to greyish white in young specimens

76 which turn brown (10YR 5/6) with age, the base may or may not have volval remnants, volva saccate, non-coherent in the upper part, breaking, whitish, reduced in mature specimens, unchanging, interior whitish to cream colored, unchanging, fragile.

Basidiospores (9.9‒) 10.1‒11.9 (‒12.9) × (8‒) 8.3‒9.3 (‒9.9) µm, Q = (1.1‒) 1.2‒1.3 (‒

1.5), Me = 11.2 × 8.9 µm, Q = 1.3, broadly ellipsoid to ellipsoid. Basidia 40‒47 × 12‒15

µm, club shaped, 4-spored, simple septate at the bases. Pileipellis with more or less cylindrical-fusoid cells, 20‒30 µm in diameter.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 1950 m a.s.l., under

Quercus dilatata, Junaid Khan, 10th August, 2016, MJ‒30 (LAH31393). Same

locality, 31st August, 2016, MJ1-664 (LAH31394).

Molecular characterization Fig. 30

The ITS region of Pakistani collections (MJ1558, MJ30 and MJ1664) resulted in a 610,

590 and 580bp sequences respectively. All the query ITS sequences were subjected to

BLAST search, and showed 97% similarity with Amanita beckeri Huijsman (JF907758) and Amanita oblongospora Contu (JF907762) followed by Amanita crocea (Quél.) Singer

(JQ912665) with 95 % identity.

For phylogenetic analysis, a total of 59 nucleotide sequences mostly comprising of species of section Vaginatae alongwith two species of section Caesareae (A. loosii Beeli and A. yuaniana Yang) and three species section Amanita (A. altipes Yang, M. Weiss & Oberw,

A. sinensis Yang (1997: 23), and A. subfrostiana Yang) were used. Limacella pitereka

Grgur (KX443412) and L. subillinita Guzmán (KR919759) were used as outgroup taxa.

The three Pakistani sequences of putative new species (A. griseofusca sp.nov.) formed a sister clade with a clade comprising of A. beckeri, A.crocea, and A. oblongospora.

Comments: Based on morphological characters and molecular support, this species is published as new t science as Amanita griseofusca.

Figure 28. A‒D: Morphology of Amanita fusca. A & B: Basidiomata (Holotype). C ‒ E. Microscopic structures. C: Basidiospores. D: Basidia. E: Pileipellis. Bars: 5mm for A & B, 4.8 µm for A: 10.5 µm for B. 11.25 µm for C.

Figure 29. nITS based Molecular Phylogenetic analysis of Amanita fusca and associated taxa. Evolutionary history was inferred using the Maximum Likelihood method based on the Jukes‒Cantor model. There were a total of 439 positions in the final dataset.

Amanita pakistanica Tulloss, Iqbal & Khalid. Mycotaxon, 2001 Fig. 30

Pileus 50‒70 mm across, convex when young, plane by maturity, light brownish white

(10Y 9/4) when young, turning whitish with a brownish disc (7.5YR 9/8) and white margins, viscid, shiny, smooth or slightly bumpy at the disc with a striated margin, striation almost 2/3rd of the total pileus diameter, warts present, rare, concentrated especially at the margin, rarely present on the disc, white, spongy, context white, thicker at the disc (≤

3mm), very thin at the edge (≤ 1mm), texture fragile, flesh unchanging upon cutting or bruising or slightly turning yellowish with time. Lamellae closely free to adnate, broad (3‒

4 mm at the center), thickness ≤ 1mm, close, cream colored to whitish with a pinkish tone

(10Y 9/4) when young then pure white at maturity, slightly pruinose, lamellar edge even, dry, lamellulae present, of diverse lengths. Stipe 120‒150 × 10‒15 mm, central, terete, thickening towards base, squamulose-fibrillose below, smooth above, texture firm, breaking with a snap, context white, slightly turning yellowish upon cutting, annulus present, membranous, superior, hanging, white above, below cream colored with few warts, volva saccate to slightly napiform, membranous, 20‒25 × 15‒20 mm, inseparable, friable leaving few irregular patches over the stipe.

Basidiospores (9.3‒) 9.7‒10.7 (‒10.9) × (6.6‒) 6.9‒7.8 (‒8.1) µm, Q = 1.3‒1.4 (‒1.6), Me

= 10.2 × 7.4 µm, Qe = 1.4, broadly ellipsoid, smooth, hyaline in KOH solution, uniguttulate, apiculus present. Basidia 35‒50 × 10‒14 µm, clavate, hyaline in KOH, without guttules. Cystidia 40‒50 × 12‒15 µm, narrowly utriform to narrowly clavate with somewhat irregular walls, hyaline in KOH, darky staining in Congo red. Pileipellis hyphae suspended, 3‒4 µm in diameter, simple septate.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2200 m a.s.l., under Pinus wallichiana, Junaid Khan,

08th August, 2015, MJ-15118 (SWAT000115).

Comments: this appears the second report of Amanita pakistanica, first being from Tulloss et al., (2011).

Figure 30. Morphology of Amanita pakistanica (MJ-15118). A‒C. Basidiomata A: Basidiomes in natural habitat. B: View of the pileus. C: View of the stipe and volva. D‒G. Microscopic structures. D: Basidiospores. E: Basidia. F: Pileipellis. G: Cystidia. Bars: 10mm for A-C, 11.5 µm for D, 16.5 µm for E&G, 10 µm for F.

Amanita pallidorosea P. Zhang & Zhu L. Yang, in Zhang, Chen, Xiao, Bau, Bao & Yang,

Fungal Diversity 42: 125 (2010) Fig.31

Pileus 40‒60 mm across, convex to plane with a slight umbo, shape of margin is straight, reddish (2.5YR 8/8) on the disc, whitish to cream colored towards margins, the reddish color may fade to whitish in some specimens upon aging or in dry conditions, unchanging upon cutting or bruising, surface smooth, dry, dull to silky, cap margin smooth, splitting with age or in dry conditions, context of the pileus dry, whitish or slightly paler than pileus,

2‒3 mm at the center and less than 1 mm at the margins, texture soft, unchanging upon cutting. Lamellae free, broad (2‒3mm at the center), spacing close, whitish at first then turning creamy to yellowish cream at maturity, mostly straight, rarely forking, lamellar edge entire, blunt, dry, lamellulae present, of diverse lengths. Stipe 80‒120 × 6‒12 mm, central, terete, sub‒cylindrical or thickening downward, white to creamy above the annulus and creamy white with a yellowish to reddish tone (10Y 9/8) below, volva present, saccate to sub‒globose, white, permanent, breaking in some specimens leaving fragments on the stipe that give a banded appearance to the stipe, annulus present, membranous, permanent, yellow at first then fades to light yellowish with age. Interior is solid with a central hollow portion that does not extend beyond the upper half, texture fragile, context whitish, unchanging upon cutting or bruising. Odor negligible, taste not recorded.

Basidiospores (6.8–) 7.1–8.5(–9.1) × (6.5–) 7.0–8.5(–9.0) µm, Q = 0.99–1.08, Me = 7.8 ×

7.75, Qe = 1.006, globose to sub-globose, apiculus prominent (1.5µm), guttulate, amyloid.

Basidia 30 – 40 × 8.0 – 10 µm, clavate, tetra-sterigmated, basal clamps absent. Pileipellis hyphae ≤ 7.5 µm, filamentous, hyaline, simple septate. Volval cells subglobose to oval (50

× 70 μm), hyphae 5–7 μm in diam. Volval remnants on the pileus sub-globose to globose,

20-30 μm in diam.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 1950m a.s.l., under Quercus dilatata., Junaid Khan,

16th August, 2015, MJ‒1554, (SWAT000116). Same locality, Junaid Khan, 31st

July, 2016, MJ‒1619 (LAH31394).

Molecular characterization Fig.32

For phylogenetic reconstruction of Amanita pallidorosea and associated species, a total of

59 sequences were used of which 58 sequences were of Amanita species, and one sequence of Limacella glioderma (Fr.) Maire (JF478086), was used for rooting purpose. In the aligned dataset there were a total of 891 sites out of which 338 sites were conserved, 490 variable and 345 were parsimony informative. The phylogenetic tree obtained from

Maximum likelihood analysis distributed the included sequences into four well supported clades corresponding to the four sections (sect. Phalloideae, sect. Validae, sect. Lepidella and sect. Amanita) within the genus Amanita. Sequences obtained from Pakistani collections (basidiocarps and morphotypes) nested with A. pallidorosea in sect.

Phalloideae. This lineage is consistent with the morphological identification.

Comments: there are no previous records of this taxon from Pakistan and is an addition to the fungi of Pakistan.

Figure 31: A‒G: Morphology of Amanita pallidorosea. A‒C: Basidiomes in natural

86 habitat. D‒F. Microscopic structure (MJ-1554). D: Basidiospores. E: Basidia. F: Volva. G: Volval remnants from Pileus. Bars: 10mm for A‒C, 10 µm for C, D & F, 15 µm for G.

Figure 32. Molecular Phylogenetic analysis by Maximum Likelihood method of Amanita pallidorosea and associated taxa inferred from nITS data using MEGA6. The tree with the highest log likelihood (-1352.9446) is shown. There were a total of 455 positions in the final dataset.

Amanita pantherina (DC.) Krombh., Naturgetr. Abbild. Beschr. Schwämme (Prague): 29

(1846) Fig.33

Pileus 30‒70 mm across, ovoid when young, turning convex and finally plane by maturity, pale yellow brown (5Y 9/4) with a light olive brown (2.5Y 5/4) disc, moist to viscid, shiny especially in wet conditions, warts present, white, soft, regular to irregular in arrangement, pileus margin striated almost halfway from the margin with a smooth disc, pileus margin even to slightly crenated, context white, thinner at the margin (≤1 mm) thicker at the disc

(2‒3 mm), soft, unchanging upon cutting. Lamellae close to remotely free, broad (3‒4mm at the center), close, whitish to creamy in young specimens then creamy with a pale yellowish to pinkish tone (5Y 9/4) in mature specimens, somewhat pruinose, lamellar edge entire to slightly serrulate, dry, lamellulae present, rare, in 1‒2 tiers. Stipe central, terete,

100‒120 × 5‒10 mm, equal or slightly swollen towards the base, white, punctate to squamulose below the ring, hairy above, annulate, annulus pendent, wooly, margin often with hanging warts, upper surface concolorous with the stipe, under surface yellow (10Y

9/10), volva saccate, membranous, inseparable, friable, leaving one or more wooly rings, concolorous with the stipe, texture fibrous, context white, centrally hollow, unchanging upon cutting or bruising.

Basidiospores (8.8‒) 9.2‒11.3 (‒11.5) × (6.2‒) 6.9‒8.5 (‒8.6) µm, Q = 1.3‒1.4 (‒1.6), Me

= 10.3 × 7.6 µm, Qe = 1.4. ellipsoid to broadly ellipsoid, smooth, hyaline in KOH, somewhat granular in Congo red. Basidia 35‒45 × 9‒12 µm, clavate, 4-sterigmated,

88 sterigmata 3‒5 µm long, basal clamps absent. Pileipellis a cutis, hyphae with obtuse terminal ends, 5‒9 µm in diameter.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 09th

August, 2015, MM-1543 (SWAT000117). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley 2250 m. a.s.l., in

decomposing needle litter under Abies pindrow, Junaid Khan, 10th August, 2014,

MJ-64 (LAH31395).

Molecular characterization Fig. 34

The target region of MM-1543 resulted in a 655bp long sequence which showed 99% similarity with Amanita pantherina (DC.) Krombh. (KF017942; AB080974) from Korea and Japan, respectively. A total of 23 closely matching sequences were used in the phylogenetic analysis with Amanita griseofolia Zhu. L. Yang (NR_119498; KF017934) as outgroup taxon.

Upon maximum likelihood analysis, the sequence of Pakistani collections (MM-1543) clustered with other species of A. pantherina included in the analysis, confirming the identity of our collections (MJ-1554 and MJ-1619) as A. pantherina.

Comments: Amanita pantherina is previously reported from Muzaffarabad by Shibata

(1992), Murakami (1993), Iqbal & Khalid (1996) with no records from present study area.

Figure 33: A ‒ E: Morphology of Amanita pantherina. A & B: Basidiome showing different stages of development and characters. C‒E. microscopic structures (MM-1543). C: Basidiospores. D: Basidia. E: Pileipellis. Bars: 10mm for A&B, 9 µm for C: 13.5 µm for D, 23 µm for E.

Amanita pantherina MM1543

KF017942.1 Amanita pantherina voucher KA12-0932

64 KJ609156.1 Amanita pantherina voucher SFC20120821-82

AB080974.1 Amanita pantherina

KF017944.1 Amanita pantherina voucher KA12-1393

100 KU248106.1 Amanita subglobosa voucher RET 410-2

KX810031.1 Amanita subglobosa voucher RET 717-5

KF651009.1 Amanita parvipantherina isolate Ap-LQ

KF651008.1 Amanita parvipantherina isolate Ap-WD 82 KF651000.1 Amanita parvipantherina isolate Ap-DQ

KX365198.1 Amanita pakistanica voucher RET 317-6

96 KX061525.1 Amanita pakistanica voucher RET 352-1

KU248129.1 Amanita albocreata voucher RET 634-8

KU248126.1 Amanita albocreata voucher RET 417-4

KP313583.1 Amanita frostiana voucher RET 588-6 100 KP313582.1 Amanita frostiana voucher RET 546-2

86 AB080779.1 Amanita muscaria

100 KP866193.1 Amanita muscaria voucher RET 514-2

HQ604823.1 Amanita gemmata voucher UBC:F19752

87 HQ604824.1 Amanita gemmata isolate BD 34 voucher UBC:F19764

KJ535439.1 Amanita breckonii voucher NY 00066695

NR 119498.1 Amanita griseofolia

100 KF017934 Amanita griseofolia

0.05 Figure 34. Molecular Phylogenetic analysis of Amanita pantherina and associated taxa by Maximum Likelihood method based on the Jukes-Cantor model. The tree with the highest log likelihood (‒1990.5325) is shown. There were a total of 606 positions in the final dataset.

Family BOLBITIACEAE

a) Descolea

1. Descolea quercina nom. prov.

Descolea quercina J. Khan & A. Naseer Figs. 35 & 36

Etymology: the epithet “quercina” refers to the association of the species with Quercus incana Roxb.

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

1950 m a.s.l., on Soil under Quercus incana, Junaid Khan, 25th July, 2015, MJ-1590

(Holotype), (SWAT000118).

Diagnosis: Fruiting body medium sized, agaricoid, convex to cenvexo-

campanulate, light yellowish brown to deep yellowish brown with or without

olivaceous tint, pileus squamose to squamose-granulose, lamellae close and light

brown, stipe yellowish brown with a pendent annulus, annulus strongly striated on

the upper surface, basidiospores limoniform, verrucose with a prominent apiculus.

Pileus 40‒70 mm across, convex to convexo-campanulate when young, plane with an uplifted margin when mature, light yellowish brown (7.5YR 7/4) to deep yellowish brown

(10YR 3/8), surface dry, dull, somewhat hygrophanous getting darker after rain, texture scaly, squamose to squamose-granulose at the disc, cap margin striated, interior strong yellowish brown (10YR 5/8), moist, thicker at the center (2‒3 mm) than the margins (not more than 1mm), unchanging upon cutting. Lamellae adnexed, forking rarely, broader at the center (4‒7mm), close, light brown (7.5YR 6/4) in young specimens and light yellowish brown in mature specimens (7.5YR 7/4), gill edge even, lamellulae present, mostly in 3 tiers, two short and the middle extending beyond the mid-point and often crisped at the end. Stipe 50‒70 × 8‒12 mm in dimensions, central, terete, thickening towards base, light yellowish brown (7.5 YR 7/4) to strong yellowish brown (10YR 4/8) above the ring and

93 there smooth, yellowish brown (10YR 5/6) below and fibrillose there, annulus superior, membranous, concolorous with the lamellae, strongly striated on the upper surface, smooth to slightly scaly below, margin appendiculate, texture fibrous, interior hollow at the center, flesh whitish above the ring, yellowish brown (10YR 5/6) below, center wet.

Basidiospores (9.9‒) 11.4‒12.9 (‒13.9) × (6.6‒) 6.64‒8.6 (‒9.1) µm, Q = 1.4‒1.7 (‒1.9),

Me = 12 × 7.9 µm, Qe = 1.5, limoniform, verrucose, verrucae partly joined together, germpore absent, with prominent apiculus. Basidia 25‒40 × 8‒12 µm, clavate, mostly 3- sterigmated, some 2‒sterigmated as well, sterigmata 3‒5 µm long, content granular in

Congo red. Cheilocystidia equal in size to basidia or slightly longer, 40‒45 × 10‒15 µm, clavate with acute apices and a 4‒6 µm long appendix, interior granular in Congo red.

Pleurcystidia same shape and size as cheilocystidia but with a longer (6‒8 µm) appendix.

Elements from pileal scales 20‒25 × 10‒20 µm, variously shaped, spherical, broadly clavate, ellipsoid, fusiform to narrowly utriform, thin walled, golden brown in KOH.

Pileipellis a hymeniform layer, consisting of broadly clavate, clavate to fusiform elements,

20–25 × 10–20 μm, strongly encrusted with golden brown pigment. Hyphae of the universal veil thin walled, cylindrical, 3–6 μm in diameter, strongly encrusted with golden brown pigment, clamp connections present. Stipitipellis hyphae 10‒12 µm in dimension, rarely clamped, tips, pinkish brown in KOH.

Molecular characterization Fig. 37

Three sequences were generated from three different basidiomes of collection MJ-1590, which yielded a 725bp fragments. The query sequences were subjected to BLAST search which matched 88% Descolea phlebophora (HQ533035) and D. recedens (JX178628).

The query sequences also matches with species of Descomyces, Setchelliogaster and

Timgrovea, and based on high similarity, the species of these genera were also included in the phylogenetic analysis. There were a total of 27 sequences with Hebeloma fastibile and

H. circinans used as outgroup taxa.

Maximum likelihood analysis was performed using MEGA6 while maximum parsimony analysis was performed using PAUP. Software. The maximum parsimony phylogram (not shown) was congruent with the maximum likelihood phylogram. The Pakistani collection formed a separate clade along with Descolea maculata, D. recedens and D. gunnii with strong bootstrap values of 87/61% (ML/MP). The phylogenetic analysis clearly support the independent position of our collection (MJ-1590).

Comments: based on morphological novelty and molecular data, the collection MJ-1590 is being proposed as new to science as Descolea quercina.

Figure 35: A‒D: Basidiomata of Descolea quercina (MJ-1590). A: Natural habitat. B: View of longitudinally sliced basidiome. C: view of the lamellae, under surface of annulus and fibrous nature of the stipe. D: view of young and mature basidiomes showing difference in colors of the stipe and lamellae.

Figure 36: A‒F: Microscopic structures of Descolea quercina. A: Basidiospores. B: Cheilocystidia. C: Elements of the Pileal scales. D: Basidia. E: Pileipellis. F: Pleurocystidia. Bars: 7 µm for a: 14.5 µm for b: 12 µm for c & d: 9 µm for E: 20 µm for F.

Figure 37. Molecular Phylogenetic analysis of Descolea species and associated taxa inferred form nITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3‒parameter model. The tree with the highest log likelihood (‒2193.4011) is shown. There were a total of 529 positions in the final dataset.

Family BOLETACEAE

a) Butyriboletus

1. B. pakistanicus nom.prov.

b) Caloboletus

1. C. alpinus nom.prov.

c) Lanmaoa

1. L. angustispora

d) Strobilomyces

1. S. strobilaceus

e) Tylopilus

1. T. porphyrosporus

f) Veloporphyrellus

1. V. purpureus nom.prov.

BUTYRIBOLETUS Arora & J.L. Frank

Butyriboletus pakistanicus nom.prov. Figs. 38 & 39

Etymology: the epithet “pakistanicus” refers to Pakistan, the country of collection of this species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

1950 m a.s.l., on Soil under Quercus incana, Junaid Khan, 31st July, 2015, MJ-1612

(Holotype), (SWAT000121).

Diagnosis: fruiting bodies medium to large sized, margin exceeding the tube layer,

pileus yellowish brown to pale brown, velutinous, pore surface greenish yellow,

stipe almost double in length to the pileus diameter, reticulate in the upper parts,

with whitish mycelium.

Pileus 40‒60 mm across, convex to plano–convex with a margin exceeding the tube layer, color light yellowish brown (10YR 7/8), to pale brownish (10YR 7/8), bruising dark brownish (10YR 4/4), surface dry, velutinous, texture smooth to bumpy, context solid, light yellowish 7.5Y 9/6), bruising yellowish green (10GY 7/4), thicker at the disc (≤

5mm). Pore surface short decurrent, greenish yellow (10Y 9/10), bruising grayish yellow green (5GY 3/2), pore small, 4‒5 per mm, round, tubes 3–4 mm deep, concolorous with the pore surface. Stipe 90‒120 × 12‒25 mm in dimensions, central, terete, same colored as pore surface in the upper one third and there reticulated, browning downward and there smooth to lightly fibrillose, turning brownish upon handling and bruising, base with pale whitish yellow mycelium, interior solid, more or less same colored as exterior, slowly turning yellowish red (10YR 7/8) upon cutting.

100

Basidiospores (8.8–) 9.5‒12.4 (–13.4) × (3.5–) 4‒4.7 (–4.9) µm, Q = (2–) 2.1‒3 (–3.1),

Me = 11 × 4.3 µm, Qe = 2.5, cylindrical, smooth walled, mostly with one large central oil granule. Basidia 25‒30 × 7‒10 µm, clavate, thin walled, 4-sterigmated, without basal clamps. Cystidia 15‒30 × 5‒7 µm, clavate-fusoid, flexuous, rare. Pileipellis a cutis, hyphae 3‒5.5 µm in diameter, terminal elements cylindrical. Hyphae in the stipe base 2‒

5 µm in diameter, simple septate, thin to moderately thick walled, without cystidia, with more or less cylindrical terminal elements, sometimes forking. Hyphae in the stipe upper surface 4‒6 µm in diameter, simple septate, with broadly clavate terminal elements.

Molecular characterization Fig. 40

The ITS region of two different basidiomata of MJ-1612 amplified with ITS1F and ITS4 primers yielded a 725 and 790bp fragments. Both the query sequences matched 94 % with

Butyriboletus sanicibus D. Arora & J.L. Frank (NR137795; KC184469) from USA and

90% with Boletus fechtneri (Velen.) Arora & J.L. Frank (KJ419930; KJ419929) from

Czech Republic.

For phylogenetic reconstruction, a total of 34 nITS nucleotide sequences were used in the maximum likelihood analysis using MEGA6 with Caloboletus calopus (Pers.) Vizzini

(KJ605655) selected as outgroup taxon. The aligned dataset was composed of 720 sites out of which 481 sites were conserved, 218 variable with 131 parsimony informative. The maximum likelihood analysis clustered the Pakistani sequences of MJ-1612 within a separate branch along-with Butyriboletus sanicibus and B. fechtneri. The strong bootstrap value of 96% confirm the unique position of Pakistani collection (MJ-1612).

101

Comments: based on distinctive character and molecular support, this species is proposed as new to science as Butyriboletus pakistanicus nom.prov.

Figure 38. A‒E. Basidiomata of Butyriboletus pakistanicus (MJ-1612). A: Basidiomes in natural habitat. B: View of the pore surface showing bruising reaction and reticulated stipe apex. C: Basidiomes with scale for size. D. View of the upper surface of pileus. E: View of the color change of context. Bars: 10mm for A ‒ E.

102

Figure 39. Microscopic structures of Butyriboletus pakistanicus (MJ-1612). A: Basidiospores. B: Basidia. C: Cystidia. D & E: Hyphae from the Stipe’s upper parts. F: Pileipellis. G & H: Hyphae from the stipe’s basal parts. Bars: 4.3 µm for A, 4 µm for B & C, 12 µm for D & E, 8 µm for F, 10 µm for G, 6.6 µm for H.

103

87 KC184453.1 Butyriboletus primiregius voucher JLF1973 KC184447.1 Butyriboletus primiregius voucher AHS69419 96 KC184428.1 Butyriboletus autumniregius voucher JDS289 KC184431.1 Butyriboletus autumniregius KC184461 Butyriboletus querciregius KC184419.1 Butyriboletus abieticola voucher OSC67698 91 KC184412.1 Butyriboletus abieticola

99 KC184446.1 Butyriboletus persolidus KC184444.1 Butyriboletus persolidus KC584789.1 Butyriboletus regius 99 KC184462.1 Butyriboletus regius KJ909519.1 Butyriboletus roseoflavus strain HKAS54099 99 KJ909518.1 Butyriboletus roseoflavus strain HKAS63609

99 KJ605668.1 Butyriboletus appendiculatus HM347641 Butyriboletus appendiculatus KC184471.1 Butyriboletus brunneus voucher Arora11221 99 KT002600 Butyriboletus brunneus HM347653.1 Butyriboletus subappendiculatus voucher JAM0189 99 JN903700.1 Boletus subappendiculatus voucher Wu 19720 KJ909522.1 Butyriboletus yicibus strain HKAS57668

97 MH234472 Butyriboletus taughannockensis MH236100 Butyriboletus taughannockensis KJ909521.1 Butyriboletus yicibus

51 KT002604.1 Boletus pulchriceps strain R. Chapman 0945 98 KT002604 Boletus pulchriceps EU569285 Boletus frostii KU317752 Butyriboletus peckii

99 KJ419930.1 Boletus fechtneri KJ419929 Boletus fechtneri

97 NR 137795.1 Butyriboletus sanicibus 86 KC184469.1 Butyriboletus sanicibus 88 Butyriboletus pakistanicus 99 Butyriboletus pakistanicus KJ605655.1 Caloboletus calopus

0.01 Figure 40. Molecular Phylogenetic analysis by Maximum Likelihood method of Butyriboletus pakistanicus and associated taxa by using nITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes–Cantor model. The analysis involved 34 nucleotide sequences. All positions containing gaps and missing data were eliminated.

104

CALOBOLETUS Vizzini.

Caloboletus alpinus nom.prov. Fig. 41 & 42

Etymology: the epithet “alpinus” refers to to alpine altitude where the species is growing

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Gabin Jabba valley,

2400 m a.s.l., on Soil under Picea smithiana, Junaid Khan, 30th August, 2015, GJ-1502 &

GJ-1503 (Holotype), (SWAT000119).

Diagnosis: fruiting body medium sized, pileus plano-convex, yellowish brown,

surface scaly-areolate, context greenish yellow, bruising bluish, pores surface

yellow to yellowish brown, bruising bluish, stipe surface reticulate, red in color,

basidiospores cylindrical, smooth, cystidia cylindrical, clavate or flexuous.

Pileus 50‒90 mm across, plano-convex, color moderate yellowish brown (10YR 7/8), surface dry, scaly-areolate forming small patches resembling granules, unchanging upon bruising, interior solid, greenish yellow (10Y 9/12), bruising blue. Pore surface adnexed, pale yellow (5Y 9/10) in young stages, drying yellowish brown (10YR 5/6), bruising bluish green (5GB 4/10), pores small, 4‒5 per mm, tubes 3‒4 mm deep. Stipe 50‒70 × 9‒12 mm, central, terete, equal or thickening downward, red (2.5R 4/12) in young stages, brown

(10YR 7/8) by maturity, bruising blue (7.5B 6/10), surface reticulated up-to the base, base with whitish mycelium.

Basidiospores (9.2–) 10.4‒14.4 (–15.1) × 5.4‒5.9 (–6.2) µm, Q = (1.7) 1.9‒2.5 (–2.7), Me

= 12.7 × 5.7 µm, Qe = 2.2, cylindrical, with apiculus, smooth, thick walled (≤ 0.4 µm), mostly with single large granule, rarely with few small, brownish in KOH. Basidia 25‒30

× 6‒10 µm, clavate, 4-sterigmated, granular-guttulate, basal clamps absent. Cystidia 22‒

105

30 × 6‒7 µm, cylindrical, lageniform, clavate to flexuous. Tubes hyphae 3‒5 µm, with clamps. Pileipellis a cutis, hyphae 6‒8 µm in diameter, septate without clamps, terminal elements cylindrical. Caulocystidia 30-45 × 3-6 µm, narrowly cylindrical, flexuous somewhat lanceolate.

Molecular characterization Fig. 43

The 645bp long ITS nucleotide sequence of GJ-1502 and GJ-1503 matched 97% with

Caloboletus panniformis (Taneyama & Har. Takah.) Vizzini (KU317758.1; KJ605658.1) from China upon BLAST search.

For phylogenetic reconstruction, a total of 23 matching nucleotide sequences were used with Phylloporus pumilus Naves & Halling (JQ003627) and Xerocomus ferrugineus

(Schaeff.) Alessio (KR019867) as outgroup taxa. There were a total of 858 sites in the aligned dataset containing 255 conserved and 589 variable with 467 parsimony informative sites. The maximum likelihood analysis using MEGA6 clustered the included taxa into five well supported clades. The Pakistani sequences of GJ-1502 and GJ-1503 clustered with

Caloboletus panniformis forming a sister clade, supporting its independent position.

Comments: based on morphological and molecular evidence, collection GJ-1502 and GJ-

1503 are proposed as new to science as Caloboletus alpinus nom.prov.

106

Figure 41. A‒F. Basidiomata of Caloboletus alpinus. A: Basidiome in natural habitat (GJ-1503). B: View of the pileus surface (GJ-1503). C: View of the pileus surface (GJ- 1502). D: View of the reticulated stipe. E: GJ-1502 in natural habitat. F. View of the pores and bruising reaction. Bars= 10mm for A ‒ F.

107

Figure 42. A‒E. Microscopic structures of Caloboletus alpinus (GJ-1502). A: Basidiospores. B: Cystidia. C: Basidia. D: Caulocystidia. E: Pileipellis. Bars: 4.6 µm for A, 5.6 µm for B‒D, 12 µm for E.

108

97 Caloboletus alpinus GJ1502 Caloboletus alpinus GJ1503 68 KJ605658 Caloboletus panniformis 73 KJ605667 Caloboletus panniformis KJ605657 Caloboletus inedulis 63 KJ605662 Caloboletus radicans KJ605656 Caloboletus firmus KJ605663 Caloboletus yunnanensis 100 KJ605664Caloboletus yunnanensis KJ605655 Caloboletus calopus 90 DQ679806 Caloboletus calopus FJ235148 Boletus amygdalinus 92 DQ974705 Boletus amygdalinus KR782307 Suillellus luridus KJ605668 Butyriboletus appendiculatus KJ605666 Boletus sinicus JF907783 Xerocomus impolitus 79 KJ729489 Pulchroboletus roseoalbidus 98 KJ729486 Pulchroboletus roseoalbidus EU231983 Boletus edulis 100 EU231984 Boletus edulis KR019867 Xerocomus ferrugineus JQ003627 Phylloporus pumilus

0.01 Figure 43. Molecular Phylogenetic analysis of Caloboletus alpinus and associated taxa by Maximum Likelihood method using nrITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2–parameter model. The tree with the highest log likelihood (–1033.4192) is shown.

109

LANMAOA G. Wu & Zhu L. Yang

Lanmaoa angustispora G. Wu & Zhu L. Yang Figs. 44 & 45

Pileus 60‒100 mm, pulvinate to plano-convex, color yellowish brown (5YR 6/8) to grayish red (10R 5/6), surface more or less sticky, shiny, smooth to lightly roughened especially with age, shiny. Pore surface adnexed, yellowish red to yellowish orange (2.5YR 7/12) in young stages, yellow (5Y 9/10) in mature, bruising bluish green (10G 3/4), pores small, 3‒

4 per mm, angular, tubes 4‒6 mm deep, yellowish (5Y 9/10) in color. Stipe 60‒80 × 12‒

15 mm, more or less equal, central, terete, color vivid yellow (5Y 8/16) near the pileus, reddening downward with a yellowish red (2.5YR 7/8) to reddish (10R 6/8) base, bruising bluish (5GB 8/6) in the upper part, yellowish red (2.5YR 7/8) in the basal part, base covered with whitish mycelium.

Basidiospores (8.1–) 8.2‒11.2 (–11.8) × 3.7‒4.3 (–4.8) µm, Q = (2.1–) 2.2‒2.78 (–2.8),

Me = 10.1 × 4 µm, Qe = 2.5, cylindrical, smooth walled, with apiculus. Basidia 30‒35 ×

6.9‒7.3 µm, cylindrical to narrowly clavate, 4-sterigmated, without basal clamps. Cystidia

20‒35 × 4.0‒10 µm, fusiform, thin walled. Pileipellis an ixocutis, hyphae 4‒9 µm in diameter, terminal elements cylindrical to narrowly clavate. Stipe base composed of moderately thick-walled hyphae, parallel, rarely branched, septate without clamps, 2‒3 µm in diameter. Hyphae from the stipe upper parts composed of suspended hyphae, 3‒5 µm in diameter, with more or less cylindrical terminal elements.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 1950 m a.s.l., on Soil under Quercus incana, Junaid

Khan, 30th July, 2015, MJ-1550 (LAH31395). PAKISTAN, KHYBER

110

PAKHTUNKHWA, Swat District, Malam Jabba valley, 1950 m a.s.l., on Soil

under Quercus incana, Junaid Khan, 31st July, 2016, MJ-1622, (SWAT000120).

Molecular characterization Fig. 46

The ITS regions of MJ-1622 upon sequencing yielded a 730bp fragments. Upon BLAST search, the query sequences matched 99% with Lanmaoa angustispora (NG_059537) followed by L. flavorubra (Halling & M. Mata) G. Wu, Halling & Zhu L. Yang (JQ924339) with 97% similarity from China.

A total of 24 nucleotide sequences were used in the maximum likelihood analysis using

MEGA6 with Boletus edulis (HQ161848; AF291300) as out-group taxon. There were 796 sites in the aligned dataset out of which 365 sites were conserved, 399 variable and 322 parsimony informative. The Pakistani sequences of MJ-1622 clustered with Lanmaoa angustispora from China, thus confirmed the identity of MJ-1622 as Lanmaoa angustispora.

Comments: the genus Lanmaoa is a newly erected genus in the Boletaceae by Wu et al.,

(2015). Both the genus and species (L. angustispora) are new records for Pakistan. The

Pakistani collections (MJ-1622 and MJ-1554) are well in conformity with the original description of L. angstispora from China which is also supported by molecular phylogentic analysis (Figure. 46) and are treated as L. angustispora herein.

111

Figure 44. A‒F. Basidiomata of Lanmaoa angustispora. A: MJ-1550 in natural habitat. B, E & F: view of pore surface of young and mature stages and bruising reaction. C: MJ- 1622 in natural habitat. D: Bruising reaction at the stipe base. Bars: 15mm for A ‒ F.

112

Figure 45. A‒G. Microstructures of Lanmaoa angustispora. A: Basidiospores. B: Basidia. C: Cystidia. D: Hyphae of the stipe base. E: Hyphae of the stipe upper parts. F: Pileipellis. Bars: 3.8 µm for A, 7 µm for B, 6 µm for C, 3.6 for D & E. 10.5 µm for F.

113

99 NG 059537 Lanmaoa angustispora 49 Lanmaoa angustispora (Pakistani collection) Lanmaoa

13 JQ924339 Lanmaoa flavorubra MH244204 Boletus subvelutipes

Neoboletus 15 29 MH236217 Boletus cf subvelutipes 99 MH234861 Neoboletus sp 13 KF030305 Boletus pallidoroseus 100 JQ327001 Lanmaoa carminipes 30 MH257548 Boletus fagicola KU160161 Neoboletus sp vividivelutinus Neoboletus

52 99 KP871802 Chalciporus radiatus Chalciporus 61 KP871800 Chalciporus radiatus 31 KY418892 Butyriboletus roseoflavus Butyriboletus MH371020 Baorangia aff bicolor Baorangia 12 54 KY418896 Rubroboletus sinicus HKAS:68620

90 KF030256 Rubroboletus pulcherrimus Rubroboletus 65 MH203877 Rubroboletus eastwoodiae 91 KF030252 Rubroboletus rhodosanguineus AF514825 Xerocomus pruinatus Xerocomus 87 JQ924322 Xerocomellus cisalpinus Xerocomellus EF183543 Octaviania columellifera Octaviana 99 EF183541 Octaviania columellifera HQ161848 Boletus edulis Boletus 100 AF291300 Boletus edulis

0.01 Figure 46. Molecular Phylogenetic analysis of Lanmaoa angustispora and associated taxa by Maximum Likelihood method using nITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes–Cantor model. The tree with the highest log likelihood (–1248.3432) is shown. There were a total of 316 positions in the final dataset.

114

STROBILOMYCES Berk.

Strobilomyces strobilaceus (Scop.) Berk., Hooker's J. Bot. Kew Gard. Misc. 3: 78 (1851)

Fig. 47

Pileus 30‒50 mm across, ovoid to parabolic in young stages, convex to plano-convex with inrolled margin in mature specimens, black to blackish grey when young, blackish to greyish with a brownish to pinkish tone by maturity, surface recurved scaly, scales soft, wooly, margin scaly, interior whitish, fragile, turning pinkish when cut and blackish later on. Pore surface cream colored in young specimens, grey to greyish black in mature specimens, pores 2‒3 per mm, angular, tubes 5‒10 mm deep, spongy, same color as pores surface, turning pink to pinkish red upon cutting. Stipe 80‒110 × 12‒18 mm in dimensions, central, terete, thicker at the base, concolorous to the pileus, scaly, scales recurved, often with a ring zone, soft, greyish black in color in upper part, with more pinkish to brownish tone at the base, interior solid, context creamy to whitish, turning pinkish upon cutting.

Basidiospores (9.4–) 9.6‒11.9 (–12) × (7.8–) 8.6‒10 (–10.2) µm, Q = (1.1–) 1.2 (–1.3),

Me = 11.1 × 9.3 µm, Qe = 1.2, sub–globose to ovoid, ornamented, ornamentation up-to 1

µm, ridges forming a complete reticulum, blackish in KOH as well Congo red. Basidia

30‒60 × 10‒18 µm, clavate. Cystidia 40–50 × 9–12 µm, fusoid-ventricose, blackish brown in KOH with darker upper parts. Peleipellis a cutis, composed of 35–55 × 10–15 µm wide cylindrical cells with rounded tips, blackish brown in KOH. Stipitipellis hyphae simple septate, shorter (20 ‒ 30 µm) and broader (15 ‒ 20 µm) than pileipellis hyphae.

115

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., on Soil under Pinus wallichiana, Junaid

Khan, 09th August, 2015, MM-1535, (SWAT000121).

Comments: Strobilomyces strobilaceus is the only reported member of this genus from

Pakistan. This is the second report of this fungus from Pakistan and first from the study area, first being from Malakundi (Shibata, 1992).

116

Figure 47. A‒I. Morphology of Strobilomyces strobilaceus (MM-1535). A: different stages of development. B: View of the pore surface. C: Pileipellis. D,E & H: Cystidia. F: Basidiospores. G: Stipitipellis. I: Basidia. Bars:15 mm for A & B, 36 µm for C, 12 µm for D,E & H, 15 µm for F, 80 µm for G, 18 µm for I.

117

TYLOPILUS P. Karst.

Tylopilus porphyrosporus (Fr. & Hök) A.H. Sm. & Thiers, Boletes of Michigan (Ann

Arbor): 98 (1971) Figs. 48

Pileus 40‒70 mm across, hemispherical at first then turning convex with age and finally plano-convex, surface dark brown to blackish brown especially when wet, dry, dull, velvety, smooth to bumpy, cracking with age especially at the margin, interior solid, firm, white, turning slowly bluish upon cutting, thicker at the center (4‒6 mm) than at the margin

(1‒1.5 mm). Pore surface adnexed, greyish to reddish brown in young stages then turning chocolate brown to yellowish brown later on, pores 2‒3 per mm, tubes concolorous to the pore surface, broad (6‒9 mm at the center), bluing slowly upon cutting. Stipe 50‒100 ×

10‒15 mm in dimensions, central, terete, equal to slightly enlarged at the base, concolorous with the pileus or slightly yellowish in some specimens, whitish in the basal part, fibrillose to fibrillose-striate, bruising darker, interior solid, fleshy, context white, turn blue upon cutting.

Basidiospores (15.1–) 15.2‒18.4 (–18.8) × (6.4–) 6.5‒8.4 (–8.5) µm, Q = (2–) 2.1‒2.4 (–

2.8), Me = 16.6 × 7.3 µm, Qe = 2.3, ellipsoid, smooth walled, yellowish brown in KOH, with single guttule. Basidia 40‒50 × 10‒15 µm, clavate, 2‒4 sterigmated. Cystidia 60‒80

× 17‒30 µm, fusiform with or without a long neck. Pileipellis hyphae 8‒12 µm wide, smooth, hyaline to yellow–brown in KOH, darkly staining in Congo red, terminal elements cylindrical with rounded apices. Stipitipellis hyphae simple septate, 7 ‒ 10 µm in diameter, more or less anastomosing, with more or less clavate, cylindrical ot flexuous terminal elements.

118

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2200 m a.s.l., on Soil under Pinus wallichiana, Junaid

Khan, 27th July, 2014, MJ-30, (SWAT000123). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Miandam valley, 2200 m a.s.l., on Soil under

Pinus wallichiana, Junaid Khan, 14th August, 2014, MM-02, (LAH31396).

PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Gabin Jabba valley,

2380 m a.s.l., on Soil under Picea smithiana, Junaid Khan, 09th July, 2016, GJ-1613

(LAH31397).

Comments: this is the second report of this taxon from Pakistan while first from the present study area. Previously it was only known from Shogran (Murakami, 1993). During present investigation, this species was encountered repeatedly from different localities of the study area and should be considered as a common find.

119

Figure 48. A‒E. Morphology of Tylopilus porphyrosporus. A & B: Basidiomata. A: Natural habitat. B: longitudinally sliced fruiting body showing interior and color change. C‒G: Microscopic structures. C: Basidiospores. D: Cystidia. E: Basidia. F: Cells of the stipe. G: Pileipellis terminal elements. Bars: 8mm for A & B, 7 µm for C, 9.6 µm for D, 7.5 µm for E, 36 µm for F, 12.8 µm for G.

120

VELOPORPHYRELLUS L.D. Gómez & Singer

Veloporphyrellus purpureus nom. prov. Fig. 49

Etymology: the epithet “purureus” refers to the warm red color, characterizing the species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2200 m a.s.l., on Soil under Pinus wallichiana, Junaid Khan, 31st July, 2016, MJ-1678

(Holotype), (SWAT000124).

Diagnosis: pileus convex to plano-convex, deep red to reddish pink, pores large

(2‒3 per mm), pink in color, stipe concolorous with the pileus, finely fibrillose,

basidiospores 12.6 × 6 µm, cylindrical, cystidia clavate with acute apices, some in

chains, pilepellis with fusiform hyphae.

Pileus 60‒80 mm across, convex when young, aging plano-convex, deep red (5R 3/10) at first, reddish pink (7.5R 6/6) with age, unchanging upon bruising, surface dry, roughened when young, then almost granulose by maturity, context cream colored, unchaining upon cutting, thicker at the disc (3‒4 mm). Pores surface whitish in young stages, then reddish pink (10R 6/6) by maturity, bruising darker reddish pink (7.5R 6/6), pores 1‒3 per mm, angular, tubes concolorous to the pore surface, 3–5 mm deep, unchanging upon cutting. Stipe 70‒110 × 10‒15 mm, central, terete, thickening downward, concolorous with the pileus, surface very finely fibrillose, interior solid, context white, unchanging upon cutting, base with white mycelium.

Basidiospores (9–) 11‒13.7 (–14.8) × (5.2–) 5.6‒6.5 (–6.9) µm, Q = (–1.7) 2‒2.2 (–2.3),

Me = 12.6 × 6 µm, Qe = 2.1, cylindrical, mostly smooth, some roughened, mono to bi- guttulate, moderately thick walled, apiculus present but not much prominent. Basidia 30‒

40 × 10‒12 µm, clavate, 4-sterigmated, without basal clamps. Cystidia 30‒55 × 10‒12

121

µm, clavate with acute apices, often in chains. Pileipellis hyphae 50‒90 × 15‒20 µm, fusiform with cylindrical to necked terminal.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2200 m a.s.l., on Soil

under Pinus wallichiana. Junaid Khan, 14th July, 2015, MJ-1546, (LAH31397).

Molecular characterization Fig. 50

The ITS region of MJ-1678 and MJ-1546 upon amplification generated a 675 and 637bp long fragments, respectively. The query sequences matched with taxa from different lineages of Boletaceae and based on high similarity species of different genera were included in the subsequent phylogentic analysis.

There were a total of 28 nucleotide sequences with Phylloporus pumillus M.A. Neves &

Halling (NR120023; JQ003826) as outgroup taxon. Maximum likelihood analysis was conducted through online CIPRES portal with GTRGAMMA model in effect. The

Pakistani sequences clustered within the Veloporphyrellus L.D. Gómez & Singer genus forming a separate clade with a robust bootstrap value of 100%, supporting its unique position.

Comments: based on unique morphology and supporting molecular data, the collections

MJ-1687 and MJ-1546 are proposed as new to science as Veloporphyrellus purpureus.

122

Figure 49. A‒G: Morphology of Veloporphyrellus purpureus. A&B: Basidiomata. C: Basidiospores (smooth ones). D: Pileipellis. E: Basidiospores (roughened). F&G: Cystidia. Bars: 25 mm for A & B, 8.5 µm for A & C, 20 µm for D, F & G.

123

Figure 50: RaxML tree of Veloporphyrellus purpureus and associated taxa inferred from ITS data. Names of the taxa are according to GenBank data.

124

Family CLAVARIADELPHACEAE

a) Clavariadelphus

1. C. elongatus nom.prov.

125

CLAVARIADELPHUS Donk.

Clavariadelphus elongatus nom. Prov. Fig. 52 & 53

Etymology: “elongatus” refers to the elongate larger fruiting body of present species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2250 m a.s.l., Junaid Khan, 28th August, 2016, MJ-16126 (Holotype), (SWAT000125).

Diagnosis: fruiting body (≤230 mm), sub-cylindrical with obtuse apices, color light

yellowish pink to moderate grayish reddish orange, basidiospores ellipsoidal and

cylindrical, narrowly clavate or flexuous cystidia.

Fruiting body 170–230 (including the underground part) × 8–12 mm in dimensions, sub- cylindrical, tapering upward with an obtuse tip, somewhat flattened at the middle, base may or may not be tapering, surface smooth and terete in in young specimens, rugose to rugulose by maturity and drying, body straight at first then somewhat flexuous with the tip bending downward, color of the above ground part light yellowish pink (5YR 9/4) in young specimens, moderate reddish orange (10R 5/8) to grayish reddish orange (10R 5/6) in mature specimens, light orange yellow (7.5YR 9/8) in below ground part, texture pliable in mature specimens, breaking with a snap in young stages, interior solid, cream colored, unchanging.

Basidiospores (8–) 8.4 – 9.9 (–11.2) × (5.9–) 6.2 – 7.3 (–8.1) µm, Q = (1.2–) 1.22 – 1.4 (–

1.6), Me = 9.2 × 6.9 µm, Qe = 1.3, ovoid in face view, ellipsoid in side view, with a prominent apiculus, content granular, hyaline in KOH solution, lightly staining in Congo red, inamyloid, smooth walled. Basidia 55–85 × 7–10 µm, narrowly clavate with a narrow basal part, mostly 4-sterigamted, rarely 2-sterigmated, sterigmata ≤ 10 µm, hyaline in KOH

126 solution except for the upper part where light golden brown, content granular, with one to many guttules concentrated especially at the basal parts, basal clamps present. Cystidia

45–75 × 5–10 µm, mostly cylindrical to somewhat flexuous, rarely narrowly clavate, some branched other with sub-capitate heads, some with hooked tips, frequent, mixed with basidia, contents granular, some guttulate, basal clamps none observed. Contextual hyphae 5–9 µm in diameter, parallel, septate, clamped, mostly normal at the septa, rarely inflated, inflation 10–15 µm in diameter, hyaline in KOH solution, smooth walled.

Pseudorhizal hyphae 5–9 µm in diameter, parallel, septate, clamped, rarely inflated, inflations ≤ 12 µm, hyaline in KOH, smooth walled.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2050 m a.s.l., on soil under Abies pindrow, Junaid

Khan, 25th July, 2015, MJ-1595 (LAH31397). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2200 m a.s.l., on soil under

Abies pindrow, Junaid Khan, 15th September, 2016, MJ-16107 (LAH31398).

Molecular characterization Fig. 53

The ITS region of MJ-1595, MJ-16107 and MJ-16126 yielded 500–530bp fragments which upon BLAST analysis matched 93% with C. occidentalis Methven (EU846242) from USA.

Another sequence of C. occidentalis (EU834202) from USA matched 91 % with the query sequence and was included in the analysis.

A total of 20 nITS nucleotide sequences were used in the phylogenetic analysis with

Lentaria byssiseda Corner (FJ596788) selected as outgroup taxon. There were a total of

127

697 sites in the aligned dataset with 395 conserved, and 268 variable sites containing 194 parsimony informative characters.

The maximum likelihood analysis clustered the in-group taxa into three well supported clades. The Pakistani sequences clustered within clade I with C. occidentalis (EU846242) forming a sister clade thus confirming its unique position. Unexpectedly the other sequence of C. occidentalis (EU834202, Voucher OSC 114250) clustered within clade II with C. subfastigatus V.L. Wells & Kempton (EU669206) and needs confirmation. Similarly the included sequences of C. subfastigatus occupied two different positions within clade I and

II and should be confirmed.

Comments: morphological uniqueness and molecular data support the unique nature of our collections (MJ-1595, MJ-16107 and MJ-16126) which is being proposed here as new to science as Clavariadelphus elongatus.

128

Figure 51. A ‒ D. Different collections of Clavariadelphus elongatus. A: MJ-16126 (Holotype) showing complete fruiting body. B: MJ-1595. C: MJ-16107. D: natural habitat (MJ-1595). Bars: 10 mm.

129

Figure 52: A – E. Microscopic structures of Clavariadelphus elongatus (MJ-16126, Holotype). A: Basidiospores. B: Contextual hyphae. C: Basidia. D: Cystidia. E: Pseudorhizal hyphae. Bars: 6.2 µm for A, 20 µm for B & E, 9.7 µm for C, 9.23 µm for D.

130

EU669207.1 Clavariadelphus subfastigiatus 86 EU697241.1 Clavariadelphus subfastigiatus

JX275756.1 Clavariadelphus subfastigiatus 85 EU526003.1 Clavariadelphus ligula voucher OSC 1064245

87 EU526000.1 Clavariadelphus mucronatus voucher OSC 1064138 65 KM248917.1 Clavariadelphus pistillaris clade I

86 KT874979.1 Clavariadelphus pistillaris voucher AR09762

EU846242.1 Clavariadelphus occidentalis voucher OSC 114281

Clavariadelphus elongatus 56

100 100 Clavariadelphus elongatus 83 Clavariadelphus elongatus

HQ650728.1 Clavariadelphus truncatus

EU669206.1 Clavariadelphus subfastigiatus 96 100 EU834202.1 Clavariadelphus occidentalis voucher OSC 114250 clade II

65 KT874983.1 Clavariadelphus truncatus voucher CB08379 HQ379937.1 Clavariadelphus pakistanicus

KM248918.1 Clavariadelphus ligula

61 EU834196.1 Clavariadelphus sachalinensis voucher OSC 96213 clade III 99 EU624411.1 Clavariadelphus sachalinensis

FJ596788.1 Lentaria byssiseda outgroup

0.02

Figure 53. Molecular Phylogenetic analysis of Clavariadelphus elongatus and associated taxa by Maximum Likelihood method by applying Neighbor–Join and BioNJ algorithms inferred from nrITS data.

131

Family CORTINARIACEAE

a) Cortinarius

1. C. clavatus nom.prov.

2. C. longistipes nom.prov.

3. C. striatus nom.prov.

132

CORTINARIUS (Pers.) Gray.

Cortinarius clavatus nom.prov. Figs. 54 & 55

Etymology: the epithet “clavatus” refers to club shaped stipe of the present species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Miandam valley, 2250 m a.s.l., on soil under Abies pindrow, Junaid Khan, 01st August, 2014, MM-20 (Holotype),

(SWAT000126).

Diagnosis: pileus convex to plano-convex with decurved margin and central umbo,

light brown to strong brown, hygrophanous, fibrillose to fibrillose-scaly, lamellae

with decurrent tooth, brown, stipe clavate to lightly thickened, fibrillose,

basidiospores broadly ellipsoid to sub-amygdaliform, ornamented, with suraphilar

depression, 10.9 × 7.1 µm, pleurocystidia present.

Pileus 25‒70 mm across, convex when young, plano-convex with decurved margin and central umbo later on and finally aging palne with uplifted margin and shallowly depressed center, when dry light orange yellow (10YR 9/8) with a moderate yellow orange (10YR

7/8) central part and strong brown (5YR 4/10) umbo, when wet light brown (7.5YR 5/4) with a dark reddish brown (10R 4/8) disc, hygrophanous, surface dry, shiny to dull, fibrillose when young, fibrillose-scaly when mature, cap margin even to eroded with remnants of veil in many cases, context concolorous with the exterior, unchanging upon cutting, thicker at the disc (1.5‒3mm), thinning towards margins and there ≤1mm.

Lamellae sinuate with a decurrent tooth, broader near stipe (3‒4mm), distant, yellowish brown (5YR 5/12), brownish orange (2.5YR 5/8) in mature specimens, develop rusty brown spots at maturity, lamellar edge even, acute, lamellulae present, mostly in single

133 tier, rarely in 3 tiers, crisped at apices in some specimens, some lamellae forking near the stipe. Stipe 40‒60 × 5‒10 mm, central, terete, clavate in young specimens, equal to slightly thickened downward later on, color reddish brown (2.5YR 5/6) to brownish (2.5YR 6/8), fibrillose overall, slightly striated at the extreme apex, annulus present, cortinate, superior, leaving a ring zone on the stipe, interior corky, concolorous with the exterior, unchanging upon cutting.

Basidiospores (9.2‒) 9.9‒11.6 (‒12.5) × (6.1‒) 6.7‒7.7 (‒8.1) µm, Q = (1.4‒) 1.44‒1.6 (‒

1.7), Me = 10.9 × 7.1 µm, Qe = 1.5, broadly ellipsoid to sub-amygdaliform, densely ornamented, ornamentation 0.4‒0.5 µm high, plage absent, suprahilar depression present, light yellowish brown to dark brown in KOH, reddish brown in Melzer’s reagent. Basidia

25‒35 × 7‒8 µm, clavate, 4-sterigamted, sterigmata ≤5 µm, clamped at the bases, hyaline in KOH, darkly staining in Congo red. Cheilocystidia none observed. Pleurocystidia 30‒

35 × 5‒8 µm, cylindrical to narrowly utriform, clamped at the bases. Pileipellis a cutis, individual hyphae 3‒4 µm in diameter, clamped at the septa, terminal ends clavate to cylindrical. Stipitipellis hyphae cylindrical, more or less parallel, 3‒4 µm in diameter, clamped, thin walled, ending in cylindrical terminals.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Miandam valley, 2250 m a.s.l., on soil under

Abies pindrow, 2250 m. a.s.l., Junaid Khan, 07th August, 2016, MM-1609

(LAH31398).

134

Molecular characterization Fig. 59

The target region (ITS) of MM-20 and MM-1609 upon amplification with ITS1F and ITS4 primer pair yielded 536 and 538bp long fragments respectively. The generated sequences were subjected to BLAST search using NCBI database. Both the query sequences matched

96% with Cortinarius hinnuleoarmillatus Reumaux (NR_131790; DQ499461;

DQ499464) from USA and Finland, respectively. It also showed 95% similarity with C. badioflavidus Ammirati, Beug, Niskanen, Liimat. & Bojantchev (KU041732; KU041723;

KU041733) form Finland.

For phylogentic reconstruction of Cortinarius species reported during present investigation, a combined tree was generated by including 125 nucleotide sequences.

Hebeloma alpinum was used for rooting purpose. The maximum likelihood phylogram was generated by using MEGA6 software and applying Jukes and Cantor model. The Pakistani sequences of MM-20 and MM-1609 clustered within Hinnulei section forming a separate clade with C. garciae (Figure. 59).

Comments: the morphological characters and molecular data, fits the species in section

Hinnulei Melot of Cortinarius and also support its independent position. Based on discrete morphological characters and molecular evidence, the collections (MM-20 and MM-1609) are proposed as new to science as Cortinarius clavatus nom.prov.

135

Figure 542: A‒F. Basidiomata of Cortinarius clavatus. A: MM-20 in natural habtat. B: MM-1609. C: View of lamellae and Cortina in young specimen (MM-20). D: Different developmental stages. E: pileus surface view. F: Lamellae and stipe view. Bars: 10 mm.

136

Figure 55: A – E. Microscopic structures of Cortinarius clavatus. A: Basidiospores. B: Pileipellis. C: Basidia. D: Pleurocystidia. E: Stipitipellis. Bars: 4.3 µm for A, 6.5 µm for B, 5.99 µm for C, 5.41 µm for D, 8.6 µm for E.

137

Cortinarius longistipes nom.prov. Figs. 56 & 57

Etymology: the epithet “longistipes” refers to the long stipe characterizing the present species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

1950 m a.s.l., on soil in mixed coniferous (Quercus-Pinus) forest, Junaid Khan, 08th

August, 2015, MJ-15107 (Holotype), (SWAT000127).

Diagnosis: pileus convex to plano-convex with decurved margin, with purplish

tone in young stages, velutinous, lamellae with decurrent tooth, stipe much longer

than pileus diameter, basidiospores broadly ellipsoid to sub-amygdaliform, 9.8 ×

6.2 µm, with suprahilar depression, cheilocystidia and pleurocystidia present.

Pileus 40‒60 mm across, convex when young, maturing plano-convex with a decurved margin, purplish pink (2.5R 7/2) with a brownish purple (7.5R 4/4) disc in young specimens, aging light brown (7.5YR 5/4) and drying dark brown (5YR 2/4), surface dry, dull, smooth, light velvety with scattered small appressed scales, disc streaked at the maturity otherwise smooth, cap margin lightly appendiculate at first, cracking with age.

Lamellae uncinated with a decurrent tooth, broader near the stipe (3‒5mm), narrowing towards edge and there1‒2 mm in diameter, distant, deep purple (7.5P 3/8) at the edge and purple (7.5P 7/12) inward in young specimens, maturing reddish brown (2.5YR 4/4) and drying dark blackish brown (7.5R 1/2), lamellar edge even, acute, lamellulae present, mostly in single tier, rarely in 3, of diverse lengths, some lamellae intervenose. Stipe 90‒

150 × 10‒15 mm, central, terete, flexuous, more or less clavate, in young specimens purplish blue (7.5PB 6/6) above the annular zone, grayish purplish pink below (2.5RP 7/4), pale orange yellow (10YR 8/4) to moderate brown (7.5YR 4/4), bruising brown (7.5YR

138

3/4), base with purplish to whitish mycelium, fibrillose overall, somewhat striated above the annular zone, annulus cortinate, leaving a ring zone on the stipe.

Basidiospores (8.9‒) 9‒11.1 (‒11.4) × (5.3‒) 5.4‒7.2 (‒7.3) µm, Q = 1.5‒1.8 (‒1.9), Me

= 9.8 × 6.2 µm, Qe = 1.6, broadly ellipsoid to sub-amygdaliform, heavily ornamented, ornamentation 0.5‒0.8 µm high, dark brown to reddish brown in KOH solution, dextrinoid in Melzer’s reagent, apiculate, suprahilar depression present, plage absent. Basidia 25‒40

× 8‒10 µm, clavate to narrowly clavate, 4-sterigmated, sterigmata ≤7 µm long, clamped at the bases. Cheilocystidia 20‒30 × 6‒9 µm, fusoid ventricose, mucronate, narrowly utriform, some spoon shaped, some hyaline in KOH solution, others brownish.

Pleurocystidia 30‒40 × 8‒10 µm, narrowly utriform, narrowly cylindrical, rare, hyaline in KOH solution. Pileipellis a cutis, hyphae 5‒10 µm in diameter, clamped, rarely branched. Stipitipellis composed of cylindrical hyphae, 6‒9 µm in diameter, mostly 6 µm, clamped, rarely branched, caulocystidia scattered, narrowly clavate to cylindrical, 6‒9 µm in diameter.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 1950 m a.s.l., on soil in

mixed coniferous (Quercus-Pinus) forest, Junaid Khan, 31st July, 2016, MJ‒1611

(LAH31399).

Molecular characterization Fig. 59

The ITS region of MJ-15107 and MJ-1611 yielded 595 and 604bp fragments respectively which matched 96% with Cortinarius torvus (Fr.) Fr. (AJ889977; AY669668) from

Germany followed by C. venustus P. Karst. (FJ039572; FJ039571) from Canada.

139

The phylogentic tree was generated by including 125 nucleotide sequences with Hebeloma alpinum as outgroup taxon. Maximum likelihood analysis clustered the Pakistani sequences of MJ-15107 and MJ-1611 with Cortinarius torvus and C. aff. multicolor forming a separate branch with a strong bootstrap value of 76% supporting its unique nature.

Comments: based on the molecular and morphological evidences, this species is being proposed as new to science as Cortinarius longistipes nom.prov.

140

Figure 56. A – F. Basidiomata of Cortinarius longistipes. For size refer to scale.

141

Figure 57: A – F. Microscopic structures of Cortinarius longistipes. A: Basidiospores. B: Cheilocystidia. C: Pleurocystidia. D: Basidia. E: Stipitipellis. F: Pileipellis. Bars: 6.5 µm for A, 5.1 µm for B, 8.9 µm for C, 10.3 µm for D, 17 µm for E & F.

142

Cortinarius striatus nom.prov. Fig. 58

Etymology: the epithet “striatus” refers to the straite pileus characterizing this species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Shangla District, Yakh tangay 1950 m a.s.l., on soil in mixed coniferous (Quercus incana and Pinus wallichiana) forest, Junaid

Khan, 08th August, 2015, MJ-15200 (Holotype), (SWAT000128).

Diagnosis: fruiting body small (≤30mm), convex to plano-convex with a central

umbo and decurved margin, yellow to reddish brown, hygrophanous, surface

striated at the margin, lamellae sinuate, sub-distant, concolorous stipe,

basidiospores moderately ornamented, broadly ellipsoid to sub-amygdaliform with

suprahilar depression, cheilocystidia and pleurocystidia present.

Pileus 20‒30 mm across, convex to plano-convex with a central umbo and somewhat decurved margin, color yellow (5Y 9/10) to reddish brown (5YR 6/8) with yellowish spots, probably hygrophanous, surface dry, dull, smooth at the disc, rugulose at the margin and there striated, striations almost halfway to the center, pileus margin crenate. Lamellae sinuate with a slight decurrent tooth, broad near the stipe (almost 3mm), narrowing towards margins and there almost 1mm, subdistant, color moderate orange yellow (10YR 7/8), lamellar edge even, lamellulae present, short, mostly in single tier. Stipe 25‒40 × 3‒5 mm, central, terete, equal, mostly curved, same colored as pileus with a somewhat whitish base, fibrillose, longitudinally striated especially in the upper part.

Basidiospores (7.3‒) 8.3‒9.7 (‒10) × (4.5‒) 4.7‒5.3 (‒5.9) µm, Q = 1.6‒1.9 (‒2.1), Me =

8.8 × 5.1 µm, Qe = 1.7, ellipsoid to sub-amygdaliform, moderately ornamented, ornamentation 0.6‒0.8 µm high, plage absent, suprahilar depression present, dark brown

143 in KOH, reddish brown in Melzer (dextrinoid). Basidia 20‒30 × 6‒8 µm, narrowly clavate, thin walled, hyaline in KOH, 4-sterigamted, sterigmata 3‒4 µm long, clamped at the bases.

Cheilocystidia 20‒30 × 7‒9 µm, narrowly clavtae, with or without capitate heads, some with prongs, rare, thin walled, hyaline in KOH. Pleurocystidia 20‒25 × 5‒8 µm, narrowly utriform to clavate, some with acute apices, some pronged. Pileipellis a cutis, individual hyphae 8‒11 µm in diameter, with cylindrical terminals, branched and rarely clamped.

Stipitipellis composed of parallel hyphae, 3‒5 µm in dimater, rarely clamped and branched. Caulocystidia 20‒30 × 3‒4 µm, cylindrical.

Molecular characterization Fig. 59

A 526bp generated from ITS region of MJ-15200 showed 95% similarity with Cortinarius annae‒maritae Bendiksen & Brandrud (KT591589) from Finland and Cortinarius candelaris Fr. (GQ159883) from Canada.

Upon maximum likelihood analysis, the sequences of SU561 clustered with C. velenovskyi

Rob. Henry (GQ159907) and C. candelaris Fr. forming a separate clade with a well- supported bootstrap value of 72%, thus evidently supporting its unique position.

Comments: unique morphological and molecular data favors independent position of the present Pakistani collection and is proposed as new to science as Cortinarius striatus.

144

Figure 58. Morphology of Cortinarius striatus (MJ-15200). A & B: Basidiomata. C‒H: Microscopic structures. C: Basidiospores. D: Cheilocystidia. E: Pileipellis. F: Pleurocystidia. E: Basidia. F: Stipitipellis. Bars: 4.8 µm for A & D, 6.3 for B & E, 11.25 µm for C, 12.8 µm for F.

145

91 KU041732.1 Cortinarius badioflavidus KU041723.1 Cortinarius badioflavidus KU041732.1 Cortinarius badioflavidus AY669667.1 Cortinarius helvolus 22 DQ117926.1 Cortinarius hinnuleus 86 AY669667 Cortinarius helvolus DQ499461.1 Cortinarius hinnuleoarmillatus DQ499463.1 Cortinarius hinnuleoarmillatus 88 DQ499461 Cortinarius hinnuleoarmillatus NR131790 Cortinarius hinnuleoarmillatus 96 KX234739.1 Cortinarius garciae 27 57 KX234738.1 Cortinarius garciae Cortinarius clavatus 99 Cortinarius clavatus 33 AY083183.1 Cortinarius hinnuleus strain IB19960139 AY083184.1 Cortinarius hinnuleus strain IB19930113 90 AY083184 Cortinarius hinnuleus AY083183 Cortinarius hinnuleus 7 KJ206485.1 Cortinarius fulvoisabellinus voucher RH1891 (PC) DQ117927 Cortinarius brunneus JF907949 Cortinarius saturatus 100 Cortinarius striatus 51 Cortinarius striatus GQ159907.1 Cortinarius velenovskyi 72 GQ159883 Cortinarius candelaris 99 GQ159849.1 Cortinarius scandens GQ159847.1 Cortinarius ceraceus 11 93 NR131868 Cortinarius roseivelatus KP114464 Cortinarius roseivelatus NR 131868.1 Cortinarius roseivelatus 72 GQ159898.1 Cortinarius laetissimus 14 AY669673.1 Cortinarius rubricosus KT591589 Cortinarius annaemaritae FJ039549 Cortinarius saturninus GQ159819 Cortinarius helvolus FJ039551 Cortinarius saturninus GQ159842 Cortinarius sertipes 13 GQ159848 Cortinarius casimiri GQ159853 Cortinarius casimiri HQ604707 Cortinarius subsertipes HQ604715 Cortinarius subsertipes FJ039547.1 Cortinarius saturninus voucher OC37 FJ039546.1 Cortinarius subsertipes voucher OC36 94 HQ604718.1 Cortinarius saturninus isolate 2081121-010 voucher UBC:F19597 FJ039552.1 Cortinarius subsertipes voucher OC13 FJ039550.1 Cortinarius subsertipes voucher OC27 GQ159819.1 Cortinarius helvolus voucher UBC F17231 OC159 HQ604719.1 Cortinarius casimiri isolate 2081105-024 voucher UBC:F19583(2) HQ604717.1 Cortinarius casimiri isolate 2081121-011 voucher UBC:F19598(2) HQ604715.1 Cortinarius subsertipes isolate 2081105-025 voucher UBC:F19584 HQ604707.1 Cortinarius subsertipes isolate 2071029-017 voucher UBC:F19458 1 FJ039547 Cortinarius saturninus 99 KP165545 Cortinarius uraceisporus KP165544 Cortinarius uraceisporus NR131878 Cortinarius uraceisporus KJ146705 Cortinarius boulderensis DQ499466 Cortinarius boulderensis NR121207 Cortinarius boulderensis 85 EU837216.1 Cortinarius boulderensis voucher OSC 109690 EU837218.1 Cortinarius boulderensis voucher OSC 109646 FJ039558.1 Cortinarius boulderensis voucher SMIA09 DQ499466.1 Cortinarius boulderensis voucher AHS17461 (MICH) 72 EU652364.1 Cortinarius boulderensis voucher OSC 115139 NR 121207.1 Cortinarius boulderensis MICH 10323 ITS region from TYPE material DQ497190 Cortinarius rubrovioleipes DQ497191 Cortinarius rubrovioleipes 18 86 DQ497193 Cortinarius rubrovioleipes DQ497194 Cortinarius rubrovioleipes AF430251.1 Cortinarius traganus DQ499465.1 Cortinarius pseudobovinus 97 DQ499465 Cortinarius pseudobovinus KU041741.1 Cortinarius roseobasilis 100 KU041740.1 Cortinarius roseobasilis 99 NR131829 Cortinarius politus KC608588 Cortinarius politus 9 98 KT591596 Cortinarius fuscescens 57 KP165548 Cortinarius fuscescens 8 DQ117925 Cortinarius armeniacus 99 KT591609 Cortinarius impennoides KT591604 Cortinarius impennoides 8 96 Cortinarius longistipes Cortinarius longistipes Cortinarius longistipes 29 GQ159889 Cortinarius aff multicolor 94 JX407337 Cortinarius torvus KM576383 Cortinarius sp LM5522 AY669668 Cortinarius torvus 76 91 KT591601 Cortinarius niveotraganus 43 NR131842 Cortinarius niveotraganus 12 EU433388 Cortinarius agathosmus 60 KP406560 Cortinarius calopus GQ159921 Cortinarius venustus 39 FJ039571 Cortinarius venustus 52 KC608583 Cortinarius fructuodorus KC608581 Cortinarius fructuodorus KC608582 Cortinarius fructuodorus 54 NR131827 Cortinarius fructuodorus 96 KX886282.1 Cortinarius biriensis KX831119.1 Cortinarius biriensis 49 KX831126.1 Cortinarius intempestivus 69 KX831125.1 Cortinarius intempestivus 97 KX831121.1 Cortinarius cristatosporus KX831120.1 Cortinarius intempestivus DQ367900 Cortinarius traganus KJ705152 Cortinarius traganus 92 DQ367900.1 Cortinarius traganus 54 EU486459.1 Cortinarius traganus HQ604720.1 Cortinarius leucopus HQ604721 Cortinarius leucopus GQ159816 Cortinarius vernus 86 FJ039539 Cortinarius vernus HQ604721.1 Cortinarius leucopus FJ039539.1 Cortinarius vernus KF048132.1 Cortinarius pseudofallax KF048131.1 Cortinarius pseudofallax 99 JQ724016.1 Cortinarius parvannulatus DQ114744 Cortinarius armillatus KM390762.1 Hebeloma alpinum 100 KF309411.1 Hebeloma alpinum

0.01 Figure 59. Maximum likelihood tree of Cortinarius sp. reported during this study and associated ones inferred from ITS data.

146

Family DACRYMYCETACEAE

a) Calocera

1. C. viscosa

147

CALOCERA (Fr.) Fr.

Calocera viscosa (Pers. ex Fr) Fr. (MJ‒82) Fig. 60

Fruiting body 40‒70 × 1‒1.5 mm, lignicolous, coralloid, branched, branches erect, forked resembling antlers, bright yellow (7.5YR 8/18) to orangish yellow(7.5YR 7/18) in color, shiny, greasy-slimy to touch especially in wet conditions, smooth, buried portion whitish yellow.

Basidiospores (8.7‒) 9.2‒11.5 (‒11.8) × (4‒) 4.1‒5.1 (‒5.2) µm, Q = (1.9‒) 1.95‒2.8 (‒

2.9), Me = 10.2 × 4.7 µm, Qe = 2.2, ellipsoidal to more or less phaseoliforme, smooth walled, with apiculus, with two central guttules, mostly aseptate, rarely with a single septum, hyaline in KOH solution. Basidia 20‒25 × 3‒5 µm, bi-sterigmated, shape resembling tuning fork, hyaline in KOH solution. Contextual hyphae 4‒6 µm, septate, inflated at some places, often with clavate to cylindrical terminal elements.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., on decomposing coniferous log, Junaid

Khan, 01st August, 2014, MM-22 (SWAT000129). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2150 m a.s.l., on same

substrate, Junaid Khan, 10th August, 2014, MJ-82, (LAH31400).

Comments: This species is previously reported from Changla gali and Kaghan valleys

(Ahmad, 1956b; 1972a). This is third report of this species from Pakistan and first from the study area.

148

Figure 60. A – E. Morphology of Calocera viscosa. A: Natural habitat. B: View of the harvested bodies. C: Basidiospores. D: Forked basidia. E: contextual hyphae. Bars:10 mm for A & B, 11 µm for C, D & E.

149

DIPLOCYSTIDIACEAE

a) Astraeus

1. A. hygrometricus

150

ASTRAEUS Morgan

Astraeus hygrometricus (Pers.) Morgan, J. Cincinnati Soc. Nat. Hist. 12: 20 (1889) Fig.

Fruiting body 25-35 mm, more or less globose shaped in young stages, exoperidium cracking later on forming 7‒12 rays resulting in a star shaped body, rays pointed, outer surface cream colored to light brown (2.5YR 7/2) and somewhat smooth at first, later on cracked, interior grayish brown (7.5YR 6/2). Spore case 10‒20 mm high and in width, more or less spherical, opening through an apical pore, rays refolding in dry conditions.

Interior white at first and fleshy at that time, chocolate brown to reddish brown (5YR 3/4) later on and powdery then, spores released through the apical pore.

Basidiospores (7.8 ‒) 7.8‒9.5 (‒9.6) × (6.8‒) 7.8‒9.1 (‒9.5) µm, Q = 1‒1.1 (‒1.2), Me =

8.9 × 8.6 µm, Qe = 1, globose, spiny, spines ≤ 1 µm high, blackish to reddish brown in

KOH solution, thick walled. Exoperidial cells, 5‒6 µm, elongated, clamped. Elaters present, ladder like.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Marghazar valley, 1100 m a.s.l., under Quercus dilatata Royle, Junaid

Khan, 31st July, 2014, MZ-22 (SWAT000130). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Miandam valley, 1500 m a.s.l., under Quercus

dilatata, Junaid Khan, 12th August, 2016, MZ-1610 (LAH31401).

151

Molecular characterization Fig. 62

The ITS region of MZ-22 and MZ-1610 yielded a 720 and 724bp fragments, respectively.

Upon BLAST search both the query sequences showed 99% similarity with Astraeus hygrometricus (AJ629406; HG000287). Unexpectedly, there were many other sequences of A. hygrometricus matching 90% to 95% with the query sequences which were also included in the subsequent analysis.

Accordingly, 15 nucleotide sequences of different Astraeus species were downloaded and used in the subsequent phylogenetic analysis using maximum likelihood analysis.

Geastrum pseudostriatus Hollós (NR_132884) and G. papinuttii J.C. Zamora

(NR_137855) were used as outgroup taxa.

The maximum likelihood analysis clustered the included in-group taxa into three different clades labeled as Clade A, Clade B, and Clade C. The Pakistani sequences of MZ-22 and

MZ-1610 clustered with sequences of A. hygrometricus (NR132884; NR137855), confirming their identity as A. hygrometricus. One of the sequence of A. hygrometricus

(FJ710188) clustered with A. pteridis (Shear) Zeller and needs confirmation. One other sequence of A. hygrometricus (FJ710187) clustered with A. telleriae and is supposed to be deposited with a wrong identity.

Comments: based on the close resemblance with description of A. htgrometricus (Phosri et al., 2007; Desjardin, 2015), the present collections of (MZ-22 & MZ-1610) are treated as A. hygrometricus. Molecular data of nITS also confirm the present collection as A. hygrometricus.

152

Morphologically, Astraeus hygrometricus is closely resembling to H. telleriae which has been recently raised as an independent taxon from A. hygrometricus complex by Phosri et al., (2013). Among these, A. telleriae is however easily differentiable by its very pubescent/wooly inner layer of outer peridium, which is without wooly hairs in H. hygrometricus.

This species is previously reported from Murree, Kaghan and Kalam Swat by Ahmad

(1956b) as Geastrum hygrometricum Pers. and this appears the fourth record of the species from Pakistan. The genus name “Astraeus” is introduced for the first time to the fungi of

Pakistan.

153

Figure 61. A – F. Morphology of Astraeus hygrometricus. A: Fruiting body in natural habitat. B: Immature stage. C: Full fruiting body. D & E: Excipular cells. F: basidiospores. Bars: 10mm for A‒C, 30 µm for D‒F.

154

Astraeus hygrometricus MZ1610 72

Astraeus hygrometricus MZ22

77 HG000287.1 Astraeus hygrometricus

AJ629406.1 Astraeus hygrometricus

HE681775.1 Astraeus sirindhorniae Clade A HG000286.1 Astraeus telleriae 99

FJ710187.1 Astraeus hygrometricus

FJ710188.1 Astraeus hygrometricus 63 HG000292.1 Astraeus pteridis 92

HG000291.1 Astraeus pteridis

JQ292819.1 Astraeus odoratus Clade B 100 AJ629882.1 Astraeus odoratus

AJ629383.1 Astraeus asiaticus Clade C 97 AB507404.1 Astraeus asiaticus

NR 132884.1 Geastrum pseudostriatus Outgroup 100 NR 137855.1 Geastrum papinuttii

0.05 Figure 62. Molecular Phylogenetic analysis of Astraeus hygrometricus and associated taxa by Maximum Likelihood method inferred from nITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2‒ parameter model. The tree with the highest log likelihood (‒1721.9774) is shown. All positions containing gaps and missing data were eliminated.

155

Family ENTOLOMATACEAE

a) Rhodocybe

Rhodocybe luteus nom.prov.

156

RHODOCYBE Maire,

Rhodocybe luteus nom.prov. Fig. 63

Etymology: “luteus” Lat. refers to the yellow color, characterizing the present taxon

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Gabin Jabba valley,

2250 m a.s.l., among decomposing needles under Pinus wallichiana, Junaid Khan, 30th

August, 2015, GJ-1525 (Holotype), (SWAT000131).

Diagnosis: pileus infundibuliform with arched margin, dull brownish grey to pale

orange yellow, surface dry, rugose to rugulose, lamellae decurrent, concolorous

with the pileus, stipe hirsute, with a white rhizomorph, basidiospores ellipsoid to

somewhat irregular, cheilocystidia and pleurocystidia present.

Pileus 60‒80 mm across, infundibuliforme with arched margin, pale yellow to orange yellow (5Y 9/18) when young, dull brownish grey (10Y 9/2) with a more brownish (10YR

8/4) disc by maturity, surface dry, dull, glabrous, rugose to rugulose with concentrically arranged wrinkles, smoother at the extreme pileus edge, cap margin even. Context dry, whitish in color, thicker at the center (2‒2.5 mm), thickness at margin not more than 1 mm in thickness, firm, unchanging upon cutting or exposure. Lamellae decurrent, long, narrow

(1‒2mm), average in thickness, close, concolorous with the pileus or more brownish (5Y

9/4), dry, gill edge entire, lamellulae present, frequent, of diverse lengths. Stipe 20‒40 ×

3‒5 mm, central, terete, equal or slightly wider in the upper part, color same as pileus or slightly paler (10Y 9/4), hirsute, hairs getting denser downward, attached to the soil

157 through white rhizomorph, texture firm, interior solid when young then corky in mature specimens, whitish than exterior (5GY 9/4), without any annulus.

Basidiospores (4.4‒) 4.9‒5.5 (‒6.5) × (3.8‒) 3.81‒4.7 (‒5.4) µm, Q = 1.2‒1.3 (‒1.4), Me

= 5.2 × 4.3 µm, Qe = 1.2, ellipsoidal to somewhat irregular, sub-globose to oval, appears smooth, hyaline both in KOH solution and Congo red, with a central guttule, apiculus present. Basidia 25‒35 × 5‒7 µm, clavate, 4-sterigamated, rarely 2-sterigmated, sterigmata

3‒4 µm long, hyaline in KOH solution, staining in Congo red. Cheilocystidia 18‒20 × 4‒

5 µm, clavate to narrowly utriform, lamellar edge sterile. Pleurocystidia 25‒35 × 5‒7 µm, cylindrical to narrowly clavate with or without flexuous bases. Pileipellis cutis, individual hyphae 3‒4 µm in diameter, thick walled (0.3‒4.5 µm), without clamps. Stipitipellis composed of parallel to somewhat anastomosing elongated hyphae with diameter not more than 3 µm, clamps absent, thin walled.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Gabin Jabba valley, 2250 m a.s.l., among

decomposing needles under Pinus wallichiana A. B. Jack.among decomposing

needles under Pinus wallichiana, Junaid Khan, 31st August, 2015, GJ-1515

(LAH31401).

Molecular characterization Fig. 64

The target region (ITS) of GJ-1525 and GJ-1515 yielded 610 and 615bp fragments, respectively, which on BLAST search at UNITE and NCBI databases matched 83 % similarity with Rhodocybe popinalis (Fr.) Singer, (UDB017726; UDB017718; FJ770400).

158

A total of 25 nucleotide sequences were used in the phylogenetic reconstruction of the

Rhodocybe species with Tricholoma joachimii Bon & A. Riva (HM590876) and T. equestre (L.) P. Kumm. (HM590872) as outgroup taxa. The maximum likelihood analysis showed that the species of Rhodocybe and Clitopilus form two well supported clades. The

Pakistani species of Rhodocybe (GJ-1515 and GJ-1525) clustered within the Rhodocybe-

Clitopilus complex with a strong bootstrap value of 99% forming a sister clade with different R. popinalis species. The present molecular position, support independent position of our collections.

Comments: Based on morphological uniqueness and data from molecular analysis, this taxon is being proposed as new to science as Rhodocybe luteus nom.prov within the

Rhodocybe popinalis complex.

159

Figure 63. A–F. Morphology of Rhodocybe luteus. A: Holotype collection (GJ-1525). B: Basidiomes in natural collection (GJ-1515). Basidiospores. B: Basidia. C: Stipitipellis. D: Pleurocystidia. E: Cheilocystidia. F: Pileipellis. Bars: 10mm for A & B, 3.33 µm for C, 7.7 µm for D, 8.57 µm for E, 6.6 µm for F, 4.34 µm for G, 8 µm for H.

160

FJ770409 Clitopilus passeckerianus 96 EU754988 Clitopilus

FJ770403 Clitopilus

FJ770385 Clitopilus hobsonii 55 FJ770388 Clitopilus passeckerianus

96 FJ770386 Clitopilus passeckerianus 94 Clitopilus FJ770407 Clitopilus prunulus

98 FJ770398 Clitopilus hobsonii

EF413030 Clitopilus giovanellae

UDB024702 Clitopilus scyphoides 85 FJ770401 Clitopilus scyphoides 99 UDB023462 Clitopilus scyphoides

AF357018.2 Rhodocybe fallax

99 AF357017.2 Rhodocybe fallax

KC885963 Clitopilus amarus

UDB011645 Clitocella popinalis 100 99 FJ770397 Clitopilus popinalis 92 100 DQ494694.1 Rhodocybe mundula Clitocella

FJ770400 Clitopilus popinalis

Rhodocybe luteus(GJ-1525) 94 100 Rhodocybe luteus (GJ-1515)

99 UDB017718 Rhodocybe popinalis

100 UDB017726 Clitopilus popinalis

HM590876.1 Tricholoma joachimii Outgroup 100 HM590872.1 Tricholoma equestre

0.02 Figure 64. Molecular Phylogenetic analysis of Rhodocybe luteus and associated taxa by Maximum Likelihood method. The tree with the highest log likelihood (‒2601.3464) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. There were a total of 458 positions in the final dataset.

161

FOMITOPSIDACEAE

a) Coriolopsis

1. C. gallica

162

CORIOLOPSIS Murrill 1905

Coriolopsis gallica (Fr.) Ryvarden, Norw. Jl Bot. 19: 230 (1973) Fig. 65

Fruiting body broadly attached, semi‒circular or bracket shaped, mostly imbricate, sometimes applanate to applanate with an umbo, 70‒100 mm across, grayish to brownish

(10YR 7/4), hairy, hairs brown to yellowish brown (7.5YR 6/8), margin turning bald with age, mostly without zones, may develop zones with age, context brown turning whitish with age, fibrillose, 3 ‒ 5 mm deep becoming narrow towards margin. Pore Surface brownish white (2.5Y 9/6) when young then turning brown to reddish brown (7.5YR 7/4) on maturity, pores round to angular, 2 ‒ 3 pores per mm, tubes up to 8 mm deep, two toned at maturity with brown outer surface and dark brown inner surface.

Basidiospores (10.7‒) 10.73 ‒ 13.1 × (‒3.8) 3.81 ‒ 4.8 µm, Q = (2.7‒) 2.73 ‒ 3.1, Me =

12.1 × 4.2 µm, Qe = 2.9, cylindrical; smooth, hyaline in KOH. Basidia 20 – 25 × Hyphal system trimitic, generative hyphae 3-5 µm in diameter, thick walled (0.3 to 0.5 µm), clamped at the septa, binding hypha 3 ‒ 5 µm in diameter, moderately thick walled (≤0.3

µm), simple septate, skeletal hyphae 3 ‒ 6 µm in diameter, simple septate.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Ingaro Dherai village, 1000 m a.s.l., on decomposing Populus stump, 30th

August, 2015, ING-25 (Lectotype), (SWAT XXX).

Comments: this taxon has no previous records from Pakistan (Ahmad et al., 1997), and is being reported for the first time.

163

Figure 65. A – F. Morphology of Coriolopsis gallica. A – C: Different views of basidiomes. D: Basidiospore. E: Basidia. Binding hyphae. Bars:10 mm for A ‒ C, 12 µm for D & E, 45 µm for F.

164

Family GOMPHACEAE

a) Gomphus

1. G. clavatus

b) Turbinellus

1. T. floccosus

165

GOMPHUS Pers.

Gomphus clavatus (Pers.) Gray, Nat. Arr. Brit. Pl. (London) 1: 638 (1821) Fig. 66

Fruiting body 100‒180 mm across and 100‒200 mm long, vase shaped, centrally depressed, composed of two to three overlapping caps arising from the same stipe. Upper surface brown (10YR 6/8), yellowish brown (10YR 4/8) to lilac brown (7.5YR 7/4), granulose especially at the margins and somewhat smoother at the disc, margin lobed to cracked in mature specimens, context white to cream colored, does not changing upon cutting, 1‒2 mm in diameter. Under surface fertile, folded to form false gills, false gills blunt, decurrent, reticulated or veined, lilac with a reddish brown margin. Stipe 10‒20 ×

5‒15 mm in dimensions, terete, tapering towards base, lilac (10R 7/2) in the above ground portion and there slightly wrinkled, white in the underground and smooth there, interior white, solid, unchanging upon cutting.

Basidiospores (11.2‒) 11.3‒14.4 (‒15.6) × (4.5‒) 5‒6.2 (‒6.4) µm, Q = (1.9‒) 2.2‒2.6 (‒

2.8), Me = 13.7 × 5.7 µm, Qe = 2.4, ellipsoidal, verrucose, hyaline in KOH solution.

Basidia 45‒55 × 8‒10 µm, clavte, simple septate at the bases. Cystidia 60‒70 × 4‒6 µm, cylindrical to somewhat flexuous.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Gabin Jabba valley, 2250 m a.s.l., on soil under Picea smithiana, Junaid

Khan, 30th August, 2015, GJ-1525 (SWAT000133).

Comments: Gomphus clavatus is previously reported from Murree by Ahmad (1956b;

1972a). This is third record of this taxon from Pakistan and first from Khyber

Pakhtunkhwa.

166

Figure 66. A – F. Morphology of Gomphus clavatus. A: Basidiome in natural habitat. B: View of the under surface. C: Basidiospores. D: View of the upper surface. E: Cystidia. F: Basidia. Bars: 10 mm for A, B & D, 13 µm for C, 3.1 µm for E, 11 µm for F.

167

TURBINELLUS Earle

Turbinellus floccosus (Schwein.) Earle ex Giachini & Castellano, Mycotaxon 115: 196

(2011) Fig. 67

Fruiting body 40‒120 mm across, 60‒140 in height, cylindrical at first turning vase shaped later on, centrally depressed, margin wavy, often with a shallow cleft on one side, depression deep rarely shallow. Upper surface scaly, scales appressed, yellowish orange

(2.5 YR 6/18) when young, turning bright orange (2.5YR 6/16) later on, finally aging with a reddish color (7.5R 5/20), margin yellowish, interior concolorous just below the surface otherwise white, unchanging upon cutting, 1‒2 mm at the edge 2‒4 mm at the base, moist.

Under surface wrinkled forming false gills, decurrent up to the beginning of the stipe, ridges inter-venose, blunt, creamy yellowish at first then aging light orange (2.5YR 8/8) to rosy brown (2.5YR 7/8). Stipe 10‒30 × 10‒20 mm in dimensions, short, concolorous with the under surface or with a slight brownish tint, whitish at the extreme base, interior white, unchanging, wet, fibrous.

Basidiospores (13.9‒) 14.3‒18 (‒18.1) × (5.9‒) 6.8‒10 (‒10.5) µm, Q = (1.6‒) 1.8‒2.4 (‒

2.5), Me = 16.4 × 8.3 µm, Qe = 2, ellipsoidal, apiculus present, finely verrucose, hyaline in KOH solution, not staining in Congo red as well, inamyloid. Basidia 70‒120 × 7‒12

µm, narrowly clavate, guttulate in mature stages, 4-spored, sterigmata 5‒7 µm long, simple septate. Cystidia 100‒120 × 7‒10 µm, cylindrical, moliniform to narrowly clavate with pointed apices. Pileipellis composed of cylindrical to oblong hyphae, 60‒100 × 20‒25 µm in diameter, septate and there constricted.

168

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2150 m a.s.l., in decomposing needle litter under Abies

pindrow, Junaid Khan, 01st August, 2014, MM-07 (SWAT000134). Same valley,

2250 m. a.s.l., in decomposing needle litter under Abies pindrow, Junaid Khan, 09th

August, 2015, MM-1550 (LAH31402). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2200 m. a.s.l., under Pinus

wallichiana, Junaid Khan, 08th August, 2016, MJ-16150 (LAH31403).

Comments: Turbinellus floccosus is already reported from Dungagali, Chandragali by

Ahmad (1956b; 1972a) and Murakami (1973) as Gomphus floccosus (Schwein.) Singer.

This is the third record of this species from Khyber Pakhtunkhwa and first from the present study area. This species is rare but widespread, and was collected from different location in the study area.

The genus name “Turbinellus” is a new addition to the fungi of Pakistan. This is also the first detailed description of the species from Pakistan.

169

Figure 67. A – F. Morphology of Turbinellus floccosus. A & B: Basidiomes in natural habitat. C: View of the pileus surface. D: Basidiospores. E: Basidia and Cystidia.F: Pileipellis elements. Bars: 14 mm for A‒C, 33.6 µm for D, 48 µm for E, 70 µm for F.

170

Family HERICIACEAE

a) Hericium

1. H. cirrhatum

171

HERICIUM Pers.

Hericium cirrhatum (Pers.) Nikol., Acta Inst. Bot. Acad. Sci. USSR Plant. Crypt., Ser. II

6: 343 (1950) Fig. 68

Fruiting body 70‒100 mm across, shelf like, mostly in tiers attached at the base laterally, semicircular to irregular, spongy when fresh, harder upon drying, pure white at first, later turning cream colored and finally drying brown (10YR 6/4). Upper surface granulose to echinulate with small sterile spines, under surface spiny, spines 10‒15 mm long, tapering downward with acute pointed terminals, close, white to creamy at first, turning pinkish and finally drying brownish (10YR 6/4). Context same colored as exterior, dry to moist, fibrous, 20‒30 mm thick at the center and 5‒15 mm at the edge. Stipe absent.

Basisdiospores (3‒) 3.1‒3.7 (‒3.9) × (2.2‒) 2.3‒2.9 (‒3.2) µm, Q = 1.2‒1.4 (‒1.5), Me =

3.4 × 2.7 µm, Qe = 1.3, ellipsoid in side view, subglobose to ovoid in face view, smooth or slightly warted, with apiculus, amyloid, hyaline in KOH solution. Basidia 4-spored, 30‒

40 × 4‒6 µm, slender, smooth, clamped at bases, hyaline in KOH solution. Contextual hyphae up to 5 µm in diameter, thick walled, with clamp connections. Gloeoplerous hyphae 4‒9 µm in diameter, frequent, present in trama, as well as in context.

Gloeocystidia 75‒110 × 6‒9 µm, scattered, sinuous.

Material Examined: Pakistan, Khyber Pakhtunkhwa, Swat District, Ingaro Dherai

village, 920 m a.s.l., saprobic on decomposing Populus stump, 30th April, 2015,

Junaid Khan, ING-26 (SWAT000135). Ingaro Dherai, 925 m a.s.l., on decaying

populous stump, 07th August 2015, Junaid Khan, ING-33 (LAH31404).

172

Molecular characterization Fig. 69

The amplified (ITS) region of ING-33 upon sequencing generated a 662bp fragment which on BLAST search showed 99 % similarity with Creolophus cirrhatus (Pers.) P. Karst

(synonym of Hericium cirrhatum) GenBank EU784261 and AF506385. All the sequences in the Blast showed significant match with the query sequence and belong to the genus

Hericium.

A total of 24 nucleotide sequences were used in the maximum likelihood analysis. There were a total of 672 sites in the final dataset, out of which 372 were conserved, 211 variable and 58 parsimony informative. Phylogenetic analysis nested Pakistani collection of

Hericium with Creolophus cirrhatus (EU784261) from Britain and Sweden (Accession #

AF506385) confirming the identity.

Comments: this taxon is reported for the first time from Pakistan.

173

Figure 68. A‒G. Morphology of Hericium cirrhatum. A: ING-33. B: Natural habitat (ING-26). C: Basidiospores. D: Basidia. E: Gloeopleurous hyphae. F: Contextual hyphae. G: Gloeocystidia. Bars: 5mm for A & B, 2 µm for C, 10 µm for D, 8 µm for E & F, 15 µm for G.

174

Figure 69: Molecular phylogenetic analysis of Hericium cirrhatum and related taxa by maximum likelihood method. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

175

Family HYDNANGIACEAE

a) Laccaria

1. L. tortilis

176

LACCARIA

Laccaria tortilis (Bolton) Cooke, Grevillea 12(no. 63): 70 (1884) Fig. 70

Pileus 10‒15 mm across, convex with a depressed center at first then plane to upturned with age with a depressed center and finally cracking becoming irregular, pinkish brown

(2.5YR 6/8) in color to reddish brown (10R 4/12) with a darker disc, moist to dry, surface smooth with a sulcate marginal portion, margin crenated or weary, context concolorous to the pileus, thin not more than 0.5 mm, equal or slightly thinner at the margins. Lamellae sinuate, ventricose, 3‒4 mm broad at the center, distant, concolorous with the pileus or slightly paler, lamellar edge serrullate, cracking later on, lamellulae present, in 1‒3 tiers.

Stipe 10‒20 × 1‒2 mm in dimensions, central, terete, equal to slightly enlarged at the base, straight or slightly flexuous, concolorous with pileus, smooth overall, otherwise slightly hairy at the base, interior same color, hollow at the center, wet, unchanging upon cutting or bruising.

Basidiospores (11.1‒) 11.4‒13.2 × (10.4‒) 10.9‒12.6 (‒13.2) µm, Q = 1‒1.06 (‒1.1), Me

= 12.3 × 11.9 µm, Qe = 1, globose, warty, warts 1‒2 µm, apiculus present, light bluish in

KOH solution. Basidia 35‒40 ×8‒12 µm, clavate to fusiform, strictly 2-spored, sterigmata up-to 8 µm long. Pileipellis a cutis composed of parallel clamped hyphae, 4‒6 µm in diameter.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Ingaro Dherai village, 1000 m. a.s.l., on the wet and shady banks of

streams, Junaid Khan, 05th November, 2014, ING-002 (SWAT000136). Same

location, Junaid Khan, 06th November, 2016, ING‒1690, (LAH31405).

177

Comments: This species is reported from Kaghan valley by Sultana et al., (2011) with no other records from Pakistan. This is the second report of the species from Pakistan and first from the study area.

Figure 70. A – F. Morphology of Laccaria tortilis. A: Basidiomes in natural habitat. B: View of basidomes from different angles. C: View of the lamellae. D: 2‒spored basidia. E: Pileipellis. F: basidiospores. Bars: 7 mm for A‒C, 30 µm for D, 12 µm for E & F. Family HYMENOCHAETACEAE

178 a) Coltricia

1. C. abieticola

179

COLTRICIA Gray

Coltricia abieticola Y.C. Dai, Fungal Diversity 45: 138 (2010) Figs. 71 & 72

Fruiting body fan shaped, annual, stipitate, soft and fibrous when young, drying corky.

Pileus 25‒35 mm across, more or less infundibuliform, circular in outline with more or less lobed margin, fibrillose to velutinate, dark reddish brown (10R 3/10) with a yellowish brown (10YR 7/8) margin, shiny, concentrically zoned, context dark reddish (7.5R 3/6) in color, thicker at the center (3 –4mm), thinning outward and 1mm at the extreme edge, Pore surface dark reddish (7.5R 3/4) in color with lighter margins, pores round to angular, small

1–3 per mm, tube layer shallow, thicker at the center (≤1mm) thinning on both sides therefrom, concolorous to the pore surface, fragile when young, corky when dried. Stipe

30‒40 × 3‒5 mm, central, terete, dark reddish black, to grayish red, surface finely scaly, texture corky.

Basidiospores (7.5–) 7.8‒10 (–10.2) × (5.3–) 5.5–6.5 (–7) μm, Q=1.45–1.53, Me= 8.96 ×

5.95 μm, Qe= 1.49, broadly ellipsoid, thick-walled, smooth, hyaline to pale yellow in KOH solution. Basidia 20‒25 × 6‒8 μm, clavate, basidioles same shaped without sterigmata.

Hyphal system monomitic, hyphae 5‒10 μm in diameter, lilaceous-cinnamon colored in

KOH solution, thick walled, branched, simple septate.

Molecular characterization Fig. 73

The target region (ITS) resulted in a 963bp sequence which showed 99% similarity with

Coltricia abieticola Y.C. Dai (KU360673; KU360674) from China.

A total of 26 nucleotide sequences were used in the final analysis containing 12 in-group taxa. Trichaptum fuscoviolaceum (Ehrenb.) Ryvarden (KJ668436) and Inonotus

180 pachyphloeus (Pat.) T. Wagner & M. Fisch. (KJ654381; KM030575) were used as outgroup taxa. Maximum likelihood analysis using MEGA6 resulted in phylogram clustering the Coltricia species into 5 well supported clades. The Pakistani sequences of

MM-29 and MJ-16120 clustered with Coltricia abieticola along-with C. weii Y.C. Dai and

C. pyriphila (Wakef.) Ryvarden in clade A thus confirming the identity of our present collections as C. abieticola.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2250 m a.s.l., on humus rich soil under Abies pindrow,

Junaid Khan, 14th August, 2014, MM-29 (SWAT000137). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2200 m. a.s.l., under Abies

pindrow, Junaid Khan, 08th August, 2015, MJ-16120 (LAH31406).

Comments: this is the first record of Coltricia abieticola from Pakistan.

181

Figure 71. A‒F. Morphology of Coltircia abieticola. A: View of the shiny and concentrically zoned pileus (MJ-16120). B-E : MM-29. B: Basidioma in natural habitat. C: View of the pore surface. D: View of the pileus and pore surface. E: View of the context. F: Lilaceous color of hyphae in KOH.

182

Figure 72. A‒D. Microscopic structures of Coltricia abieticola (MM-29). A: Basidiospores. B: Basidia. C: Tramal hyphae. D: Contextual hyphae. Bars: 11 μm for A & B, 16 μm for C & D.

183

KU360699.1 Coltricia weii 99 KU360698.1 Coltricia weii

KU360689.1 Coltricia pyrophila

36 98 KU360691.1 Coltricia pyrophila Clade A KU360674.1 Coltricia abieticola

97 Coltricia abieticola (Pakistan)

72 Coltricia abieticola (Pakistan)

98 KU360673.1 Coltricia abieticola

KU360682.1 Coltricia minima 99 KU360683.1 Coltricia minima

93 Clade B 73 KU360694.1 Coltricia verrucata

54 KU360693.1 Coltricia strigosipes

100 KU360692.1 Coltricia strigosipes 100 KU360679.1 Coltricia crassa Clade C 100 KU360678.1 Coltricia crassa

KU360681.1 Coltricia macropora Clade D 99 KU360680.1 Coltricia macropora

KU360670.1 Coltricia austrosinensis

KU360684.1 Coltricia minor 97 KU360687.1 Coltricia perennis 99 86 E KU360686.1 Coltricia perennis

89 KC152085.1 Coltricia confluens

95 KC152084.1 Coltricia confluens

KJ668436.1 Trichaptum fusco-violaceum

100 KJ654381.1 Inonotus pachyphloeus Outgroup taxa

100 KM030575.1 Inonotus pachyphloeus

0.1 Figure 73. Molecular Phylogenetic analysis of Coltricia abieticola and associated taxa inferred from nITS data.The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3–parameter model. The tree with the highest log likelihood (–2622.0184) is shown.

184

Family HYMENOGASTRACEAE

a) Galerina

1. G. marginata

b) Gymnopilus

1. G. penetrans

2. G. swaticus nom.prov.

c) Hebeloma

1. H. rostratum

d) Phaeocollybia

1. P. pakistanica sp.nov.

2. P. variacolora nom.prov

185

GALERINA Earle

Galerina marginata (Batsch) Kühner, Encyclop. Mycol. 7: 225 (1935) Fig. 74

Pileus 10‒15 mm across, conical at first the convex with age, brown to yellowish brown

(10Yr 7/8) with a darker disc, surface dry to slightly sticky, especially in wet conditions, dull or shiny when wet, smooth with striated margin that extend almost 2/3rd of the total pileus diameter, cap margin even to striate, context dark brown (10YR 3/8) , yellowish brown below the surface, less than 1 mm at the margin, 1.5‒2.5 mm at the disc, texture firm, wet, unchanging upon cutting. Lamellae slightly decurrent, 1‒2 mm broad, sub- distant, light brown to brown (10YR 7/8), slightly pruinose in some specimens, lamellar edge even or slightly serrulate, lamellulae present, in 3‒6 tiers. Stipe 20‒30 × 1‒2 mm, central, terete, equal or slightly thicker towards base, flexuous in most of the specimens, light brown above (10YR 9/8), turning darker downward, surface pruinose to punctate above fibrillose below, annulus present, membranous, pendant, superior, brown to dark brown especially in mature specimens, disappearing in some specimens, context, solid, whitish brown in the upper part, turning dark brown to reddish brown downward, wet, not changing upon cutting or bruising.

Basidiospores (7.6‒) 7.7‒8.45 (‒8.5) × (4.8‒) 4.9‒5.7 (‒5.8) µm, Q = (1.4) 1.43‒1.6 (‒

1.7), Me = 8.1 × 5.3 µm, Qe = 1.5, ellipsoidal in side view, amygdaliform in face view, smooth to slightly verrucose, apiculus present, with or without germpore. Basidia 20‒30

× 6‒8 µm, club shaped, 4-sterigamted, sterigmata 5‒6 µm long. Cheilocystidia narrowly lageniform, 30‒35 µm long. Pleurocystidia same size and shape as cheilocystidia.

Pileipellis a cutis, hyphae 5‒8 µm in diameter, clamp connections present. Stipitipellis hyphae 3‒5 µm in diameter, clamped at septa, more or less parallel.

186

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., on decomposing woodchips under Abies

pindrow, Junaid Khan, 22nd August, 2014, MM-64 (SWAT000138).

Comments: the collection MM-64 was identified as Galerina marginata by referencing it to the literature of (Gulden, 2010). It is the only species in the Galerina genus from

Pakistan, which is previously reported from Khanspur (Iqbal & Khalid, 1996). This report of Galerina marginata, is the second from Pakistan and first from present study area.

187

Figure 74: A – G. Morphology of Galerina marginata (MM-64). A: Basidioma in natural habitat. B: View of the interior. C: Basidiospores. D: Basidia. E: Cystidia. F: Pileipellis. G: Stipitipellis. Bars: 7 mm for A & B, 3.71 µm for C, 6.17 µm for D, 7.6 µm for E, 8 µm for F, 8.75 µm for G.

188

GYMNOPILUS P. Karst.

Gymnopilus penetrans (Fr.) Murrill, Mycologia 4(5): 254 (1912) Fig. 75

Pileus 45‒55 mm across, convex to plano-convex when young turning hemispherical to plano-convex to slightly concave at maturity with inflexed to involute margin, yellow- ochre to rusty brown (2.5YR7/8‒2.5YR6/8), surface smooth, context pale yellow

(2.5Y8/8), thicker at the center. Lamellae crowded, ventricose but sometimes also triangular, near stipe emarginate and decurrent with a small tooth or broadly adnate to slightly decurrent, yellow‒rusty (5YR6/6) to deep rusty brown (5YR5/6), edge concolorous or somewhat paler, irregularly serrulate. Stipe 60‒95 × 7 ̶ 13 mm, central to slightly off centric, stipe pale ochre (5YR8/2), brownish to rusty brown (5YR8/6), covered with remnants of velum which are white and tomentose-fibrillose, base white, tomentose with white mycelial cords, cylindrical or gradually thickening towards base, slightly bulbous in some cases.

Basidiospore (5.10 ̶ ) 6.97 ̶ 8.52 (̶ 8.68) × (3.87 ̶ ) 3.97 ̶ 5.05 (̶ 5.14) µm, Me= 7.86 × 4.57

µm, Q= 1.21 ̶ 2.03, Qe=1.72, rusty yellow in KOH solution, wall darker, rusty brown, ornamentation moderately developed, verrucose to rugulose-verrucose, mature spores distinctly red-brown in Melzer’s reagent (dextrinoid). Basidia 20 ̶ 30 × 5 ̶ 7 µm, tetra- sterigmate, sterigmata ≤4.6 µm, broadly cylindrical with median constriction and attenuated basal part. Cheilocystidia 20–30 × 6–8 μm, rarely intermixed with basidia at edge, variable in shape, cylindrical, narrowly fusiform-cylindrical, narrowly utriform to lecythiform, apex with more or less pronounced globose head 3.5–7 μm in diameter but sometimes not capitate. Pleurocystidia rarely present, lageniform with globose head or utriform. Pileipellis cells cylindrical, covered with nests of parallel to interwoven hyphae

189 forming the scales which are more intensely colored, rusty brown, of cylindrical to narrowly fusiform cells 4 – 10 μm broad, special terminal elements or pileocystidia absent.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Dir

Upper District, Kumrat valley, 2350 m a.s.l., under Pinus wallichiana, Junaid

Khan, 02nd September, 2015, MM-68 (SWAT000139).

Molecular characterization Fig. 78

A 597bp fragment was obtained by amplification of the ITS region of MJ-68. The query sequence upon BLAST analysis matched 99% with G. penetrans (AY281000; FJ481026;

AY281001).

Upon maximum likelihood analysis, the query sequences clustered with other sequences of G. penetrans, confirming the identity.

Comments: Gymnopilus penetrans is reported for the first time from Pakistan.

190

Figure 75: A‒G. Morphology of Gymnopilus penetrans (MM-68). A & B: Basidiomata. C: Cheilocystidia. D. Basidiospores. E. Basidia and pleurocystidia. F. Stipitipellis. G. Pileipellis. Scale bar = 5.4 μm for A; 4.75 μm for B; 9.5 μm for C; 1.9 μm for D; 1.6 μm for E.

191

Gymnopilus swaticus J. Khan, Sher & Khalid Figs. 76 & 77

Etymology: the specific epithet “swaticus” refers to the district of collection “Swat”.

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat district, Gabin Jabba Lalkoo,

2483 m a.s.l., on Picea smithiana, 31st August 2015, Junaid Khan (GJ-1518), (Holotype)

(SWAT000140).

Diagnosis: basidiomata yellowish orange to light orange. Pileus ≤ 70 mm across,

campanulate to plano-convex with inrolled margin in young stages, velutinous to

lightly tomentose. Lamellae sinuate to emarginate, sub-distant to close. Stipe

central to slightly eccentric, velutinous-fibrillose, longitudinally striated.

Basidiospores verrucose, ellipsoid to amygdaliform, 8.6–10.0 × 4.5–5.6 µm.

Pileus 50‒70 mm across, campanulate to hemispherical or convex when young then turning plano‒convex to plane at maturity, margin incurved to inrolled at first then turning straight with age, yellowish orange (2.5 YR 8/4) to light orange (2.5 YR 4/6) at first then rusty yellowish orange (2.5 YR 5/12 to 2.5 YR 6/ 12) at maturity, dry, dull, pruinose to velutinose when young then velutinous to pubescent when mature or lightly tomentose in some specimens, covered with whitish silky to pale yellowish hairs that finally turn shorter with age, appressed scaly when mature, cap margin entire when young then turning even to widely sulcate at maturity, context moist, solid, very thick at the center compared to the margins, concolorous just beneath the pileipellis otherwise paler with intermixed whitish and orangish portions, continuous with the stipe, compact, does not changing upon cutting or slightly turning darker later on, appears hygropanous especially in older stages, odor farinaceous, taste not observed. Lamellae sinuate to emarginate, broad, thickness average, sub‒distant to close, Sulphur yellow (2.5 YR 5/12 to 2.5 YR 6/ 12) when young then

192 turning rusty brown (2.5 YR 5/12 to 2.5 YR 6/ 12) with age with a paler margin, edge entire or slightly serrulate, dry unequal with many short lamellulae in between. Stipe central to slightly off centric especially in mature specimens or in growing from the side of the stumps, 60‒70 × 7‒10 mm, terete, equal, usually flexuous, concolorous with the cap (2.5

YR 8/4 to 2.5 YR 4/6), velutinous to fibrillose; covered with whitish to light yellowish small whitish hairs, longitudinally striated, base covered with whitish mycelium especially in young stages, texture firm, interior solid, flesh concolorous with the stipe or slightly darker especially at the center.

Basidiospores (8.5‒) 8.6‒10 (‒10.1) × (4.5‒) 4.5‒5.6 (‒5.7) µm, Q = (1.6‒) 1.7‒2.1 (‒2.3),

Me = 9.1 × 5.1 µm, Qe = 1.8, mostly ellipsoid, amygdaliforme, rarely ovoid, verrucose, apiculate, without germ pore. Basidia mostly tetra-sterigmated, rarely bi-sterigmated, up- to 35 × 8 µm in dimensions, narrowly clavate, sterigmata ≤5 µm, clamped at the bases.

Cheilocystidia scattered, 30‒40 × 8‒10 µm, narrowly utriforme with sub-capitate tips, hyaline to slightly golden brown in KOH solution. Pleurocystidia similar in shape and size to Cheilocystidia, slightly emergent. Pileipellis a ctuis with anastomosing hyphae, individual hyphae up‒to 6 µm in diameter, clamped, hyaline to slightly brown in KOH, with cylindrical terminal elements. Sub-hymenial cells up to 20 µm in diameter, broader and larger compared to pellicular cells, with clamps, hyaline in KOH solution.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat district, Gabin Jabba valley Lalkoo, 2483 m a.s.l., on

Picea smithiana (Wall.) Bioss, 15th September 2015, Junaid Khan (GJ-1519)

(LAH31406). Same location, 08th July 2016, Junaid Khan (GJ-1612) (LAH31407).

Same location, 04th September 2016, Junaid Khan (GJ-1640).

193

Molecular characterization Fig. 78

A 597bp long ITS fragment was obtained after trimming the motifs from the generated sequences of GJ-1518, GJ-1612 and GJ-1640. BLAST search of the query sequence showed that Pakistani collections are 97% similar to G. decipiens (Sacc.) P.D. Orton

(AF325669), G. odini (Fr.) Bon & P. Roux (AF325663) and G. turficola M.M. Moser &

H. Ladurner (AF325669).

Upon maximum likelihood analysis, the Pakistani sequences of GJ-1518, GJ-1612 and GJ-

1640 clustered along-with G. turficola, G.odini, G. decepiens forming a separate beanch supported by a bootstrap value of 86%. The current analysis supports independent position of these collections.

Comments: Gymnopilus swaticus sp. nov., is being proposed as new to science.

194

Figure 76. A‒D. Basidiomata of Gymnopilus swaticus. A: Holotype collection (GJ-1518) showing gills and surface features (Mature stage) B: GJ-1519. C: natural habitat (GJ-1640) D: young stage showing pruinose to pubescent appearance (GJ-1612). Bars: 10 mm for A‒ D.

195

Figure 77. A‒D. Microscopic characters of Gymnopilus swaticus (GJ-). A. Cheilocystidia. B. Basidiospores. C. Basidia and pleurocystidia. D. Pileipellis. Scale bar = 5.7 μm for A; 7.8 μm for B; 7 μm for C: 12.5 μm for D.

196

HG969652 Gymnopilus suberis 58 HG969654 Gymnopilus suberis KT368689 Gymnopilus suberis HG969653 Gymnopilus suberis 88 AY280975 Gymnopilus aeruginosus AY280992 Gymnopilus luteofolius AY280976 Gymnopilus aeruginosus AY281016 Gymnopilus subpurpuratu 92 AY280974 Gymnopilus aeruginosus KJ654557 Gymnopilus sp 88 KJ654555 Gymnopilus sp HG969655 Gymnopilus igniculus 84 EU401711 Gymnopilus cyanopalmico 80 EU401712 Gymnopilus purpureosqua 66 AY280998 Gymnopilus purpureosqua 44 AY280979 Gymnopilus purpureosqua DQ311084 Gymnopilus subearlei EU401710 Gymnopilus purpuratus KT368682 Gymnopilus dilepis 90 KT368680 Gymnopilus dilepis KT368681 Gymnopilus dilepis 54 KX035108 Gymnopilus lepidotus 44 AY280989 Gymnopilus lepidotus AY280991 Gymnopilus lepidotus 58 AY280994 Gymnopilus medius AY281013 Gymnopilus subearlei 70 EU401709 Gymnopilus ochraceus AY280978 Gymnopilus cerasinus 100 EU518419 Gymnopilus sp EU518418 Gymnopilus sp 100 KF727401 Gymnopilus allantopus AF501542 Gymnopilus allantopus 36 100 AF501548 Gymnopilus hybridus KJ146708 Gymnopilus penetrans 28 100 EU518421 Gymnopilus arenophilus EU518422 Gymnopilus arenophilus 92 FJ481026 Gymnopilus penetrans 82 86 Gymnopilus penetrans F292 Gymnopilus penetrans SJ1082

100 AY925212 Gymnopilus penetrans 84 KR011987 Gymnopilus penetrans AY281007 Gymnopilus sapineus AY925213 Gymnopilus penetrans JF340274 Gymnopilus penetrans 98 KX773872 Gymnopilus sapineus KX773873 Gymnopilus sapineus JF908048 Gymnopilus hybridus AY281000 Gymnopilus penetrans AY280999 Gymnopilus penetrans AY281002 Gymnopilus penetrans KT368685 Gymnopilus penetrans AF501551 Gymnopilus moabus 92 Gymnopilus swaticus GJ1612 100 Gymnopilus swaticus GJ1640 42 Gymnopilus swaticus GJ1518

86 AF325659 Gymnopilus odini AF325660 Gymnopilus decipiens 92 AF325669 Gymnopilus turficola 72 AF325663 Gymnopilus penetrans AY281017 Gymnopilus underwoodii AY280981 Gymnopilus cf. flavidel 100 AY281018 Gymnopilus validipes KF309422 Hebeloma pusillum Out group 100 AF325643 Hebeloma fastibile

0.02 Figure 78. Molecular Phylogenetic analysis of Gymnopilus taxa collected during present study and associated taxa by Maximum Likelihood method. The analysis involved 73 nucleotide sequences. There were a total of 653 positions in the final dataset.

197

HEBELOMA (Fr.) P. Kumm.

Hebeloma rostratum Beker, Vesterh. & U. Eberh., in Beker, Eberhardt, Vesterholt &

Schütz, Fungal Biology 120(1): 96 (2016) Figs. 79 & 80

Pileus 30–50 mm across, convex, often irregular due to overlapping pilei, light orangish brown (7.5YR 7/4) to moderate yellowish brown (10YR 5/4), surface smooth, sticky, glabrous to very finely fibrillose, cap margin crenate, interior solid, flesh cream colored, unchanging, moist, thicker at the disc (5‒7mm) than margins (≤ 1mm). Lamellae sinuate, broad (4‒5 mm at the broadest point), close to sub-distant, color light yellowish brown

(7.5YR 7/4) to strong yellowish brown (10YR 5/10), darkening with age, lamellar edge serrate to moderately eroded, lamellulae present, mostly in 3 tiers, of diverse lengths. Stipe

25‒40 × 5‒7 mm, central, terete, equal or slightly thickening downward, base lightly enlarged in some specimens, color pale yellowish pink (5Y 9/4), bruising brownish, surface glabrous to lightly pruinose, interior solid, flesh moist, light brownish beneath the stipe cuticle, whitish in the center, unchanging.

Basidiospores (11‒) 11.3‒13.3 (‒13.8) × (6.8‒) 6.9‒7.7 (‒7.9) µm, Q = (1.6‒) 1.64‒1.85

(‒1.9), Me = 12.5 × 7.2 µm, Qe = 1.7, amygdaliform to broadly ellipsoidal, ornamented, ornamentation resembling spines, thick walled (≤ 0.5 µm), apiculus present, germ-pore present, content granular, guttulate with single oily drop, dextrinoid. Basidia 20‒30 × 7‒

9 µm, clavate, tetra-sterigmated. Cheilocystidia 30‒45 × 5‒7.5 µm, narrowly clavate to narrowly mucronate, thin walled, in clusters. Pleurocystidia 25‒35 × 8‒10 µm, narrowly utriforme. Pileipellis an ixocutis with embedded hyphae, 70‒120 × 6‒10 µm, smooth walled, hyaline in KOH solution, clamped at the septa, terminal elements cylindrical.

198

MATERIALS STUDIED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Takhta Band village, 1000 m a.s.l., under Populus nigra L., 04th

November, 2014, TB-014 (SWAT000141). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Ingaro Dherai village, 1000 m a.s.l., under

Populus nigra L., 22nd November, 2015, ING-1559. (LAH31408).

Molecular characterization Fig. 81

The ITS region of ING-1559 and TB-014, by amplification with fungal specific primers, yielded a 640 and 681bp fragments, respectively. Both the query sequences were subjected to BLAST search which showed 100% similarity with Hebeloma rostratum Beker,

Vesterh. & U. Eberh (KT217532; KT217358; KT217472) from Germany.

For phylogenetic analysis of Pakistani collections, 32 nucleotide sequences were downloaded from GenBank comprising of 30 in-group taxa with Hebeloma mediorufum

Soop., (GU591656; GU591655) used as outgroup taxon.

The aligned dataset was composed of 663 characters out of which 584 characters were conserved, 79 variable characters were with 70 parsimony informative characters. After removal of the ambiguous characters there were a total of 556 positions in the final dataset.

The analysis clusters the Pakistani sequences of ING-1559 and TB-014 with the sequences of Hebeloma rostratum from Germany, thus confirming the identity as H. rostratum Beker,

Vesterh. & U. Eberh.

Comments: this is the first report of this taxon from Pakistan and probably from outside

Europe.

199

Figure 79: A – D. Basidiomata of Hebeloma rostratum. A: Basidiomes in natural habitat (ING‒1559). B: Collection TB‒014. C: View of the lamellae. D: View of the full basidiomes (ING‒1559). E: View of the full basidiomes (TB‒014). E: View of the context. Bars: 10 mm for A‒F.

200

Figure 80: A – D. Microscopic structures of Hebeloma rostratum. A: Basidiospores. B: Pleurocystidia. C: Cheilocystidia. D: Pileipellis. Bars: 8.1 µm for A, 5.94 µm for B, 7.1 µm for C, 11 µm for D.

201

KT071016.1 Hebeloma alpinum KX657845.1 Hebeloma aanenii KX657866.1 Hebeloma geminatum KX657857.1 Hebeloma eburneum KX657862.1 Hebeloma geminatum KX657852.1 Hebeloma eburneum

63 JN943879.1 Hebeloma lutense 72 72 JN943855.1 Hebeloma lutense KF309411.1 Hebeloma alpinum KX657861.1 Hebeloma geminatum

94 KF309415.1 Hebeloma crustuliniforme KF309413.1 Hebeloma crustuliniforme 41 GQ869511.1 Hebeloma hiemale 77 90 GQ869486.1 Hebeloma hiemale KX657844.1 Hebeloma aanenii KX687221.1 Hebeloma pseudofragilipes

88 KT217496.1 Hebeloma ingratum KX687220.1 Hebeloma pseudofragilipes 42 KX687206.1 Hebeloma fragilipes 97 KX687226.1 Hebeloma vaccinum KX687223.1 Hebeloma vaccinum 66 EU570163.1 Hebeloma cavipes KX687191.1 Hebeloma cavipes KT217548.1 Hebeloma echinosporum

89 KX687217.1 Hebeloma populinum 89 KT217560.1 Hebeloma populinum KT217472.1 Hebeloma rostratum KT217358.1 Hebeloma rostratum 93 Hebeloma rostratum Hebeloma rostratum GU591656.1 Hebeloma mediorufum 99 GU591655.1 Hebeloma mediorufum

0.01 Figure 81. Molecular Phylogenetic analysis of Hebeloma rostratum and associated taxa by Maximum Likelihood method using MEGA6. The tree with the highest log likelihood (‒1211.5006) is shown.

202

PHAEOCOLLYBIA R. Heim.

Phaeocollybia varicolor nom.prov. Figs. 83 & 84

Etymology: the epithet “varicolor” refers to the variable color of the species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2100 m a.s.l., under Abies pindrow, Junaid Khan, 16th August 2015, MJ-1561 (Holotype),

(SWAT000142).

Diagnosis: fruiting body medium sized (≤50mm), conical to conico-campanulate with an acute umbo, color variable, from reddish orange to reddish brown, surface viscid and shiny, glabrous to finely fibrillose, lamellae adnexed, stipe with pseudorhiza, base dark red, basidiospores amygdaliform to broadly ellipsoidal, densely ornamented with plage present, cheilocystidia and pleurocystidia present, clamp connection frequent, tibiiform diverticula present in rhiz0pellis only.

Pileus 30‒50 mm across, conical in young specimens then aging conico-campanulate with a prominent acute umbo, margin upturned in some specimens, color deep reddish orange

(10R 5/12) in young specimen, in mature specimens yellowish orange (5YR 6/12) with a dark reddish orange (10R 5/12) umbo, drying reddish brown (10R 4/8), viscid in young specimens, dry in mature stages, surface smooth to lightly wrinkled at the disc, glabrous to very lightly and sparsely fibrillose in young specimens, cap margin even to eroded, finally cracking in some specimens, interior solid, context thin, ≤ 2mm at the disc and up to the halfway, then narrowing immediately to less than 1mm at the edges, cream colored with a reddish tone, unchanging. Lamellae adnexed, narrow near the stipe and edges, 2‒2.5 mm at the center, close, moderate reddish orange (10R 5/10), developing strong reddish (10R

3/14) spots by maturity, lamellar edge even at first then eroded by maturity, lamellulae present, frequent, of diverse lengths. Stipe 50‒70 × 5‒7 mm above, pseudorhiza 30‒40 mm, tapering, central, terete, color vivid orange (2.5YR 6/18) to light orange brown (5YR

203

8/8) in the upper parts, base dark reddish (7.5R 4/10), darkening downward and with a dark red (10R 2/6) pseudorhiza, surface smooth to somewhat fuzzy near the base, texture pliable, interior fibrillose, hollow, context paler or more or less same colored as exterior, unchanging upon cutting, cavity covered with cream colored fibrils.

Basidiospores (9.1‒) 9.5‒11 (‒11.4) × (5.2‒) 5.4‒6.6 (‒6.9) µm, Q = 1.6‒1.9 (‒2.1), Me

= 10.3 × 6 µm, Qe = 1.7, amygdaliform to broadly ellipsoidal, densely ornamented, ornamentation up to 0.4 µm, plage present, reddish brown in KOH, non-amyloid. Basidia

25‒30 × 8‒9 µm, clavate, 4-sterigmated, sterigmata 3‒4 µm high. Cheilocystidia 40‒55 ×

3‒7 µm, cylindrical with or without capitate to sub-capitate heads. Pleurocystidia 25‒30

× 6‒8 µm, narrowly clavate to narrowly utriform, some with acute apices. Pileipellis a cutis, hyphae 2‒6 µm in diameter, some smooth others roughened, septate and branched, septa thick and staining brownish with KOH, simple septate. Stipitpellis hyphae 3‒6 µm, more or less parallel, clamped, clamps frequent almost at every septum,golden brown in

KOH solution. Tibiiform diverticula present in the rhizopellis. Contextual hyphae sarcodimitic.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2100 m a.s.l., under Abies

pindrow, Junaid Khan, 09th April 2015, MJ-1634 (LAH31409). PAKISTAN,

KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley, 2100 m a.s.l.,

under Abies pindrow, Junaid Khan, 28th August, 2016, MJ‒16114 (LAH31410).

204

Molecular characterization Fig. 84

The ITS regions of MJ-1561 and MJ-1634 resulted in a 618 and 665bp fragments, respectively. The query sequences upon BLAST search matched 93% with Phaeocollybia ammiratii Norvell, (KJ450919; KJ450914) from USA. Based on the outcome closely matching sequences were downloaded for the subsequent phylogenetic analysis.

The maximum likelihood analysis comprising of 32 nucleotide sequences, clustered the

Phaeocolybia species into different well supported clades. The Pakistani sequences of MJ-

1561 and MJ-1634, formed a separate branch along-with P. olivacea A.H. Sm., P. gregaria

A.H. Sm. & Trappe, and P. fallax A.H. Sm.

205

Figure 82. A – F. Basidiomata of Phaeocollybia varicolor. A‒C: MJ-1634. D‒E: MJ- 1561 (holotype). Bars: 10 mm.

206

Figure 83. A – F. Microscopic structures of Phaeocollybia varicolor. A: Cheilocystidia. B: rhizopellis with tibiiform diverticula. C: Sarcodimitic tissues of context. D: Basidiospores. E: Basidia. F: Roughened hyphae of the pileipellis. Bars: 40 µm for A, 20 µm for B, 50 µm for C, 9.9 µm for D, 12.2 µm for E, 13.5 µm for F.

207

GQ165653 Phaeocollybia gregaria GQ165650 Phaeocollybia gregaria JN102520 Phaeocollybia gregaria 63 EU846286 Phaeocollybia fallax EU846281 Phaeocollybia fallax

78 JN102530 Phaeocollybia olivacea 96 GQ165679 Phaeocollybia olivacea Phaeocollybia varicolor 99 Phaeocollybia varicolor

99 JN102495 Phaeocollybia ammiratii GQ165629 Phaeocollybia ammiratii N102545 Phaeocollybia redheadii 54 JN102541 Phaeocollybia redheadii JN102546 Phaeocollybia redheadii 99 98 GQ165685 Phaeocollybia oregonensis GQ165684 Phaeocollybia oregonensis 95 KJ450916 Phaeocollybia benzokauffmanii 99 KJ450910 Phaeocollybia benzokauffmanii

100 JN102515 Phaeocollybia fallax 98 JN102512 Phaeocollybia fallax JN102499 Phaeocollybia attenuata 99 JN102500 Phaeocollybia attenuata

100 KF219601 Phaeocollybia phaeogaleroides KF219600 Phaeocollybia phaeogaleroides

99 KF219569 Phaeocollybia dissiliens 66 KF219568 Phaeocollybia dissiliens

99 JN102548 Phaeocollybia sipei 97 EU644706 Phaeocollybia sipei 76 Phaeocollybia pakistanica 100 Phaeocollybia pakistanica EF091828.1 Squamanita odorata EF184305.1 Squamanita umbonata

0.02 Figure. Molecular Phylogenetic analysis of Phaeocollybia pakistanica varicolor and associated taxa by Maximum Likelihood method inferred from nITS data.

208

Family INOCYBACEAE

a) Inocybe

1. I. pallidorimosa nom.prov

2. I. putilla.

3. I. salicetum nom.prov.

209

INOCYBE (Fr.) Fr.,

Inocybe pallidorimosa nom.prov. Fig. 85

Etymology: the epithet “pallidorimosa” refer to the pallid color of the present species belonging to Rimosae section

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2050 m a.s.l., under Pinus wallichiana, Junaid Khan, 27th July, 2014, MJ-1430 (Holotype),

(SWAT000143).

Diagnosis: fruiting body small to medium sized, pileus conical, surface fibrillose

to sericeous, pale cream colored, lamellae sinuate to emarginate, stipe bulbous,

concolorous or slightly paler than pileus, basidiospores phaseoliform to oblong,

rarely broadly ellipsoid, both cheilocystidia and pleurocystidia present.

Pileus 20‒30 mm across, ovoid to conical when young, conical by maturity with a smooth margin, pinkish brownish (7.5YR 8/4) dry, dull, fibrillose to sericeous, cap margin entire to slightly appendiculate, context dry to moist, pale cream colored, thicker at the center with maximum thickness of 2.5 mm, firm, reddening upon cutting, odor spermatic, taste mushroomy and mild. Lamellae attached to the stipe, sinuate to emarginate, narrowly broad, average in thickness, subdistant to close, paler than pileus, cream colored with a pinkish tone, margin entire, acute to obtuse, dry, unequal with frequent short gills. Stipe central, terete, bulbous, 30‒40 mm × 5‒7 mm except at the base, where bulbous and 9‒12 mm in thickness, concolorous with the gills or slightly paler, smooth to velvety in some specimens, without any annulus, interior solid with somewhat conolorous flesh, faintly reddening upon cutting.

210

Basidiospores (9.6‒) 10.2‒12.4 (‒13.2) × (4.8‒) 5.2‒6.3 (‒6.6) µm, Q = (1.5‒) 1.8‒2.4 (‒

2.6), Me = 11.4 × 5.7 µm, Qe = 2, phaseoliform to oblong, rarely broadly ellipsoidal, smooth, thick walled (0.4‒0.5 µm), with apiculus, germ-pore absent, mostly with a single central guttule. Basidia 25‒30 × 5‒7 µm, clavate, tetra-sterigated. Cheilocystidia 20‒30 ×

5‒7 µm, narrowly clavate to clavate, some with capitate to sub-capitate heads, some septate above the bases. Pleurocystidia 20‒28 × 6‒9 µm, narrowly clavate, thin walled. Pileipellis a cutis, hyphae 9‒12 µm, parallel, clamped, terminal ends narrowing. Stipitipellis composed of more or less parallel hyphae, 9‒12 µm in diameter, clamped, terminal ends narrowing but not acute.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2050 m a.s.l., under Pinus

wallichiana, Junaid Khan, 31st July 2015, MJ-1566 (LAH31410).

Molecular characterization Fig. 86

The 585bp fragment (ITS) generated by the Pakistani Inocybe species (MJ-1430 & MJ-

1566) matched 94 % with Inocybe rimosa (Bull.) P. Kumm. (FJ904147; FJ904146) from

Sweden upon BLAST analysis. A total of 29 sequences were used in the phylogenetic analysis with 26 sequences from section Rimosae and 3 outgroup sequences. Maximum likelihood analysis clustered the 26 sequences of species of Section Rimosae into 4 sub- clades. The Pakistani collection clustered within sub-clade A, along-with I. rimosa, I. melliolens Kühner, I. sororia Kauffman, I. bulbosissima (Kühner) Bon, I. arenicola (R.

Heim) Bon and I. guttulifer Kühner. This clade is well supported compared to the other sub-clades. Sub-clade B is represented by I. squamata J.E. Lange, I. hygrophorus Kühner,

211

I. flavella P. Karst. and I. spuria Jacobsson & E. Larss. Sub-clade C and D are represented by single species viz. I. dulcamaroides Kühner and I. mimica Massee, respectively.

Comments: based on the morphological and molecular evidence, this species is proposed as new to science herein.

212

Figure 85. A – F. Morphology of Incoybe pallidorimosa (MJ-1430). A: Basidioma in natural habitat. B: View of the gills. C: Basidiospores. D. Cheilocystidia. E: Basidia with Pleurocystidium to the left. F. Pileipellis. Bars: 10 mm for A & B, 5.28 µm for C, 4.55 µm for D, 5.5 µm for E, 11 µm for F.

213

99 Incoybe pallidorimosa (MJ-1566) Inocybe pallidorimosa (MJ-1430) FJ904147.1 Inocybe rimosa 94 FJ904146.1 Inocybe rimosa 55 FJ904148.1 Inocybe melliolens

4871 FJ904149.1 Inocybe melliolens HQ604602.1 Inocybe sororia JQ408780.1 Inocybe sororia 99 HQ604620.1 Inocybe praetervisa 43 A 100 HQ604610.1 Inocybe sororia JQ408748.1 Inocybe arenicola 100 JF908110.1 Inocybe arenicola

100 FJ904160.1 Inocybe bulbosissima Rimosae FJ904159.1 Inocybe bulbosissima FJ904165.1 Inocybe umbrinella 44 52 FJ904164.1 Inocybe umbrinella 97 JF908233.1 Inocybe guttulifer 100 JF908100.1 Inocybe guttulifer

99 FJ904136.1 Inocybe squamata 42 FJ904132.1 Inocybe squamata

40 FJ904137.1 Inocybe hygrophorus 59 B AM882782.2 Inocybe flavella 58 FJ904139.1 Inocybe spuria 99 FJ904138.1 Inocybe spuria FJ904127.1 Inocybe dulcamaroides C FJ904124.1 Inocybe mimica D KJ399958.1 Inocybe phaeoleuca

100 KJ399904.1 Inocybe leiocephala 100 KJ399893.1 Inocybe leiocephala

0.02 Figure 86. Molecular Phylogenetic of Inocybe pallidorimosa and associated taxa by Maximum Likelihood method.

214

Inocybe putilla Bres., Fung. Trident. 1(4‒5): 81 (1884) Fig. 87

Pileus 15‒20 mm across, convex to plano-convex with an umbo, light brown to pinkish brown (7.5YR 8/4) at the edge with a dark brown to reddish brown (5YR 3/4) disc, background light brown, dry, dull to slightly shiny, fibrillose, fibrils radiating from disc to margin, disc lightly wrinkled, cap margin eroded to slightly appendiculate, context moist, concolorous with the pileal surface, thicker at the disc (2‒3 mm) thinner at the edge (0.8‒

1.2mm), texture soft, unchanging upon cutting. Lamellae adnate, ascending, broad (3‒

4mm at the disc), subdistant, light brown (7.5YR 8/4) to pinkish brown (7.5YR 7/4), lamellar edge even or slightly serrulate and whitish in color, lamellulae present, rare, in one tier, dry. Stipe 30‒50 × 3‒4 mm, central, terete, equal or slightly tapering towards base, straight or flexuous in some specimens, pruinose to punctate especially in the upper part, pruinose to slightly fibrillose in the lower part, whitish in the upper and lower parts, pale brown (2.5YR 9/4) in the middle, background light brown, interior solid, white beneath the umbo and base, dark brown in between, moist, unchanging upon cutting or bruising.

Basidiospores (7.9‒) 8.1‒9.2 (‒10.1) × (4.9‒) 5.3‒6.6 (‒6.7) µm, Q = (1.3‒) 1.34‒1.6 (‒

1.7), Me = 8.7 × 5.9 µm, Qe = 1.5, nodulose, golden brown to dark brown in KOH. Basidia

25‒30 × 8‒10 µm, clavate, 4-sterigmated. Cheilocystidia 55‒75 × 13‒20 µm, narrowly utriform with a long neck, with crystalliferous tips, thick walled (≤ 2µm), brownish in KOH solution. Pleurocystidia same size as cheilocystidia, narrowly utriform to narrowly lageniform with comparatively shorter neck, tips crystalliferous or not. Pileipellis a cutis or euhymeniderm, hyphae 7‒10 µm wide and clamped. Stipitipellis hyphae 4‒6 µm wide, elongated, clamped. Caulocystidia none observed.

215

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2050 m a.s.l., under Pinus wallichiana, Junaid Khan,

27th July, 2014, MJ-23 (SWAT000144).

Molecular characterization Fig. 88

Upon sequencing, the ITS region of MJ-23 resulted in a 596bp fragment which on BLAST query matched 99% with two voucher specimens (AM882752; JF908228) of Inocybe putilla Bres. from Sweden and USA, respectively.

A total of 26 matching nucleotide sequences (ITS) were retrieved form GenBank and used in the phylogenetic analysis. After removal of the ambiguous position from the final dataset, there were 645 sites comprising of 375 conserved, 260 variable and 222 parsimony informative sites. The Pakistani collections (MJ-23 & MJ-24) clustered with Inocybe putilla confirming the identity as Inocybe putilla.

Comments: this taxon is a new record for Pakistan.

216

Figure 87. A – G. Morphology of Inocybe putilla (MJ-23). A & B: Basidiomata in natural habitat. C: Basidiospores. D: Basidia. E: Cheilocystidia. F: Pleurocystidia. G: Pileipellis. Bars: 6mm for A & B, 3.3 µm for C, 3.8 µm for D, 7.8 µm for E, 7.5 µm for F, 6.25 for G.

217

HQ604230 Inocybe subcarpta HQ604232 Inocybe nitidiuscula HQ604235 Inocybe subcarpta HQ604223 Inocybe stellatospora 93 HQ604227 Inocybe stellatospora HQ604220 Inocybe lanuginosa HQ604222 Inocybe lanuginosa var HQ604221 Inocybe lanuginosa 52 JN580878 Inocybe pseudoteraturgu 97 JN580880 Inocybe pseudoteraturgu HQ604245 Inocybe nitidiuscula JN580825 Inocybe bufonia 99 JN580824 Inocybe bufonia 68 59 HQ604236 Inocybe acuta HQ604243 Inocybe cicatricata 51 HQ604237 Inocybe pseudoasterospo 67 HQ604239 Inocybe striata GU234104 Inocybe boltonii JN580814 Inocybe borealis 100 JN580820 Inocybe borealis FN550882 Inocybe putilla Inocybe putilla (MJ-23) 99 59 JF908228 Inocybe putilla 68 AM882752 Inocybe putilla GU234071.1 Mallocybe dulcamara 100 GU234082.1 Mallocybe dulcamara

0.02 Figure 88. Molecular Phylogenetic analysis of Inocybe putilla and associated taxa inferred from nITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes-Cantor model. The tree with the highest log likelihood (‒1484.0800) is shown.

218

Inocybe salicetum nom.prov. Fig. 89

Etymology: the epithet “salicetum” refers to a habitat dominated by Salix trees,

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Takhta band Village,

1000 m a.s.l., on a sandy soil under Salix babylonica L., Junaid Khan, 09th August 2014,

TB-04 (Holotype), (SWAT000145).

Diagnosis: fruiting body small to medium sized, growing in sandy soils under Salix babylonica, pileus hemispherical to convex with or without a short umbo, surface squamulose, brown, lamellae unicnate with a short decurrent tooth, fibrillose to fibrillose-pruinose stipe, broadly ellipsoidal to phaseoliform basidiospores, presence of pleurocystidia and cheilocystidia. Pileus 20‒35 mm across, hemispheric to convex with somewhat decurved margin and short umbo in young stages, turning plane or saucer shaped with a depressed pileus except the umbo, in some specimens turning plane with an uplifted margin later on, surface dry, brown

(7.5YR 6/8) with a deep brown (2.5YR 2/6) disc, squamulose, squamules pyramidal, radiating from disc to the margins, interior solid, context wet, concolorous with the pileus or slightly darker, unchanging upon cutting. Lamellae uncinated with a short decurrent tooth, moderately broad (2‒3mm) at the center, concolorous with the pileus, lamellar edge eve to crenate, lamellulae present, in 1‒3 tiers, short, seldom extending beyond the center.

Stipe 25‒35 × 3‒5 mm, central, terete, equal to slightly thickening upward, concolorous with the pileus with a whitish upper part, fibrillose to fibrillose-pruinose, cuticle cracking in some mature specimens.

Basidiospores (8.6‒) 8.8‒10.2 (‒11.2) × (5.1‒) 5.3‒6.1 (‒6.2) µm, Q = (1.4‒) 1.5‒1.9 (‒

2), Me = 9.6 × 5.7 µm, Qe = 1.7, broadly ellipsoidal to phaseoliform, smooth, thick walled

(≤0.5 µm), with single central guttule, apiculus present, not prominent. Basidia 20‒30 ×

219

6‒8 µm, clavate to narrowly utriform, 4-sterigmated, sterigmata 3‒3.5 µm in length, clamped at the bases. Cheilocystidia 23‒30.3 × 16‒18 µm, ovoid to obovoid, often in chains. Pleurocystidia 25‒30 × 7‒8 µm, narrowly clavtae to narrowly utiform, some with basal spur, without basal clamps. Pileipellis cutis, hyphae 7‒10 µm in diameter, septate and clamped, with clavtae to cylindrical terminal elements (≤20 µm in diameter), some smooth walled and light golden brown in KOH solution, other roughened and dark golden brown.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Takhta band Village, 1000 m a.s.l., on a sandy

soil under Salix babylonica L., Junaid Khan, 09th April 2015, TB-09 (LAH31411).

Same locality, Junaid Khan, 21st April, 2015, ING-1511 (LAH31412).

Molecular characterization Fig. 90

The ITS region of TB-04, TB-09 and ING-1511 gave 700, 702 and 725bp fragments respectively, which matched 85% with Inocybe leucoloma Kühner (GU980623;

KJ810600) from Sweden and China respectively.

A total of 17 closely matching nucleotide sequences from the BLAST results and those mentioned in Vaurus and Larsson (2011) were downloaded and used in the phylogenetic analysis. The maximum likelihood analysis clustered the Pakistani sequences with I. malenconii R. Heim, forming a sister clade. Current phylogenetic results supports independent position of our collections.

Comments: based on unique morphology and supporting molecular data, the collections

TB-04, TB-09 and ING-1511 are proposed here as new to science.

220

Figure 89. A – H. Morphology of Inocybe salicetum. A‒C: Different views of Basidiomata (Holotype). D – H. Microscopic structures. D: Basidiospores. E: Basidia. F: Cheilocystidia. G: Pleurocystidia. H: Pileipellis. Bars: 5mm for A ‒ C, 4.3 µm for D, 5 µm for E, 2.3 µm for F, 5.54 µm for G, 10 µm for H.

221

HM209794.1 Mallocybe sp. JV5968

HM209793.1 Mallocybe sp. JV19678 99 KX602262.1 Inocybe myriadophylla

62 KX602263.1 Inocybe myriadophylla

GU234129.1 Mallocybe dulcamara

GU234082.1 Mallocybe dulcamara

KJ810600.1 Inocybe leucoblema 60 GU980623.1 Inocybe leucoloma

HM209787.1 Inocybe malenconii 94 KX602259.1 Inocybe malenconii

44 Inocybe salicetum (TB-04)

Inocybe salicetum (TB-09) 94 Inocybe salicetum (ING-1511)

GU980653.1 Inocybe arthrocystis

99 GU980654.1 Inocybe arthrocystis

AY380365.1 Mallocybe agardhii

100 FJ904123.1 Mallocybe agardhii

0.05 Figure 90. Molecular Phylogenetic analysis of Inocybe salicetum and associated taxa using nITS data Maximum Likelihood method. The tree with the highest log likelihood (‒1342.2713) is shown. TAll positions containing gaps and missing data were eliminated.

222

Family OMPHALOTACEAE

a) Gymnopus

1. G. dysodes

223

GYMNOPUS (Pers.) Gray

Gymnopus dysodes (Halling) Halling, in Antonín, Halling & Noordeloos, Mycotaxon 63:

364 (1997) Figs. 91 & 92

Pileus 15‒30 mm across, conical at first, later on convex to plano-convex with a central umbo, finally drying irregular, color light brown (5YR 6/4) to dark brown (5YR 4/4), drying dark brown (7.5YR 1/2), surface dry, dull, disc smooth otherwise striated, pileus margin crenate to eroded in some specimens, context thin, 1‒2 mm at the disc, ≤1mm at the margins, flesh pinkish brown (7.5YR 9/4), unchanging. Lamellae adnexed, seceding, narrow (1.5‒2.5 mm) in diameter, distant, creamy pinkish (5Y 9/4) at first the light brown

(7.5YR 7/4) to dark brown (5Y 3/4) later on, lamellulae present, rare, short, mostly in single tier, lamellae intervenes. Stipe 10‒20 × 4‒7 mm, round in young specimens, flattened in mature specimens, equal or slightly thickening downward, pinkish brown (7.5 YR 9/4) lightly scaly to almost upward, scaly downward with hirsute base, texture pliable, interior hollow at the center, pith covered with whitish fibrils, interior same colored as exterior, unchanging upon cutting.

Basidiospores 4.8‒5.6 (‒6.2) × (1.7‒) 2.8‒3.6 (‒3.7) µm, Q = (1.5‒) 1.52‒1.9 (‒3), Me =

5.3 × 2.9 µm, Qe = 1.9, narrowly ellipsoid to oblong, apiculus present, smooth. Basidia

22‒25 × 4‒6 µm, mostly clavate, rarely cylindrical, clamped at bases, hyaline in KOH.

Cheilocystidia 25‒35 × 5‒6.5 µm, cylindrical to flexuous, thin walled, clamped at the bases, hyaline in KOH solution. Pleurocystidia 25‒35 × 5‒7 µm, variously shaped, cylindrical, narrowly utriform, flexuous, bottle shaped, horned, thin walled, clamped at

224 bases. Pileipellis a cutis, individual hyphae 5‒10 µm, terminals mostly acute, some cylindrical, others with irregular terminals, thin walled.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Ingaro Dherai Village, 920 m a.s.l., under Populus nigra L., Junaid Khan,

23rd July, 2014, ING‒21 (SWAT000146). Same locality, 950 m a.s.l., under

Populus nigra, Junaid Khan, 02nd August, 2015, ING‒1511a (LAH31411).

Molecular characterization Fig. 93

The ITS region of ING-21 and ING-1511a yielded a 737bp long sequence which matched

99% with Gymnopus dysodes (KY026666; AF505778; DQ449987; KR673491) upon

BLAST search.

Based on the outcome, 45 closely matching nucleotide sequences were downloaded and used in the subsequent phylogenetic analysis. Marasmius mbalmayoensis (EF160091;

EF160092) was used as outgroup taxon. The maximum likelihood analysis clustered the

Pakistani sequences of ING-21 and ING-1511a with the sequences of Gynopus dysodes confirming the identity.

Comments: this taxon is being reported as a new record for fungi of Pakistan.

225

Figure 91: A – D: Morphology of Gymnopus dysodes. A: Basidiomes in natural habitat (ING-21). View of the gills (ING-1511a). C: View of the context. D: Basidiomes showing different views of the Basidiomes. Bars: 10mm for A‒D.

226

Figure 92: Microscopic structures of Gymnopus dysodes. A: Basidiospores. B: Basidia. C: Cheilocystidia. D: Pleurocystidia. E: Pileipellis. Bars: 2.4 µm for A, 5.6 µm for B, 6.6 µm for C & D, 12.1 µm for E.

227

89 JX536162.1 Gymnopus ocior JX536161.1 Gymnopus ocior

32 JX536172.1 Gymnopus aquosus JX536169.1 Gymnopus aquosus 57 AY263449.1 Gymnopus sepiiconicus 15 DQ480112.1 Gymnopus alkalivirens 96 KR673434.1 Gymnopus aquosus 90 KR673415.1 Gymnopus aquosus AY263429.1 Gymnopus vitellinipes 81 KJ416261.1 Gymnopus ceraceicola KC248389.1 Gymnopus ceraceicola

76 KJ416260.1 Gymnopus ceraceicola 99 KJ416262.1 Gymnopus ceraceicola

8 NR 137797.1 Gymnopus imbricatus

96 NR 137798.1 Gymnopus hakaroa 85 KC248407.1 Gymnopus hakaroa

100 FJ596894.1 Gymnopus polyphyllus FJ596895.1 Gymnopus polyphyllus KJ416269.1 Gymnopus barbipes 16 35 KT271754.1 Gymnopus trabzonensis 99 LT594120.1 Gymnopus impudicus 85 LT594119.1 Gymnopus impudicus

86 KT222652.1 Gymnopus montagnei KT222653.1 Gymnopus montagnei AY263447.1 Gymnopus salakensis 21 9 KX926133.1 Gymnopus variicolor 31 LT594121.1 Gymnopus sp. 1 MT-2016 KX926134.1 Gymnopus variicolor 99 DQ449986.1 Gymnopus iocephalus DQ449984.1 Gymnopus iocephalus 96 DQ449985.1 Gymnopus iocephalus AF505779.1 Gymnopus impudicus Gymnopus dysodes (ING-21) 73 Gymnopus dysodes (ING-21)

99 DQ449987.1 Gymnopus dysodes KR673491.1 Gymnopus dysodes 62 KY026666.1 Gymnopus dysodes AF505778.1 Gymnopus dysodes

95 KT222659.1 Gymnopus atlanticus KT222654.1 Gymnopus atlanticus

85 KT222655.1 Gymnopus talisiae KT222657.1 Gymnopus talisiae 97 KT222656.1 Gymnopus talisiae EF160091.1 Marasmius mbalmayoensis 100 EF160092.1 Marasmius mbalmayoensis

0.05 Figure 93. Molecular Phylogenetic analysis of Gymnopus dysodes and associated taxa by Maximum Likelihood method. The tree with the highest log likelihood (‒2711.0685) is shown. There were a total of 426 positions in the final dataset.

228

Family PHYSALACRIACEAE

a) Oudemansiella

1. O. sparslamellata nom.prov.

229

OUDEMANSIELLA Speg.

Oudemansiella sparslamellata nom.prov. Fig. 94 & 95

Etymology the specific epithet “sparslamellata” refers to distant lamellae characterizing the species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat district, Malam Jabba

(Kishawra), 2050 m a.s.l. single specimen, on soil under Quercus dilatata, 16th August,

2015, Junaid Khan, MJ-1559 (Holotype) (SWAT000147).

Diagnosis: Basidiomata up to 50 mm across. Pileus cream to whitish grey with a darker center, glabrous to rugulose. Stipe scaly-flocculose and radicating. Lamellae uncinated and distant. Basidiospores, globose to sub-globose and smooth. Cheilocystidia capitate to sub-capitate, oblong, fusiform, thin walled. Pleurocystidia widely cylindric, oblong to narrowly ellipsoid, thin walled. Pileipellis an ixo-hymeniderm. Stipitipellis composed of filamentous hyphae with scattered elongated, sub-capitate to capitate caulocystidia and narrowly conical to setiform caulosetae.

Pileus 40‒50 mm across, convex when young turning plane with age and finally drying convex, umbonate in young stages only then plane with age and finally undulating without umbo, light brown (7.5YR 6/2) with a greyish hue all over and a darker disc in young stages, turning cream to whitish cream (5Y 9/4) with a brownish disc and slightly greyish margin by maturity and after rain, drying grey, surface dry, dull, unchanging upon bruising, glabrous to slightly rugulose especially towards margin when fresh and rugose to rugulose when dry, one specimen lightly reticulate near margin; context cream‒colored to creamy white, dry, 1‒2 mm thick at the disc and 1‒1.5 mm at the margin, firm. Lamellae adnate- uncinate, some lamellae with decurrent tooth and others slightly decurrent, ventricose to

230 sub-ventricose, 2‒4 mm broad at the center, ≤ 1mm thick, distant, white to creamy white, developing brownish spots with age, drying orangish brown; edge even, dry; lemellulae present, arranged in three to six tiers. Stipe central, 70‒90 × 3‒5 mm to the ground line, terete, more or less cylindrical, swollen at the base, whitish in the apical part and there longitudinally striated, greyish brown (10YR 5/2) to grey below with gradual darkening downward, punctate at first then lacerate with age, transversely zonate in some specimens, pilose at the base, dry, firm, becoming hollow by maturity; context creamy white in the upper part, greyish towards base, not changing in the upper part upon cutting, turning greyish in the lower part. Pseudorhiza 20‒35 mm, greyish brown to blackish brown, tapering downward.

Basidiospores (13.3‒) 13.8‒15.3 (‒16) × (12.1‒) 13‒14.3 (‒14.7) µm, Q = 1‒1.1 (‒1.2),

Me = 14.6 × 13.5 µm ; Qe = 1.1, globose to sub-globose or short ovoid, thin walled, with one to many guttules, hyaline in KOH, with prominent hilar appendage. Basidia 50‒70 ×

20‒25 µm, clavate to cylindrical in shape, mostly with 4 sterigmata rarely with 2, sterigmata 8‒10 µm long, guttulate, clamped at the base; basidioles with same shape and size as basidia or slightly smaller, lacking sterigmata, without guttules, smooth, hyaline, thin‒walled. Cheilocystidia scattered, 70‒90 × 20‒25 µm, oblong, fusiform to cylindrical, hyaline, thin‒walled. Pleurocystidia scattered, 50‒90 × 20‒30 µm, polymorphic, widely cylindric, oblong, narrowly ellipsoid, flexuous to cylindrical with capitulate to sub‒ capitulate apices, hyaline, thin‒walled. Pileipellis ixohymenidermal, composed of two elements; pileus margin composed of coralloid branched hyphae which mostly end in round, ovoid to subovoid pedicellate to sphaeropedunculate pileocystidia, 15 – 25 µm wide in the widest part with a 3 – 5 µm wide lower pedicel; rarely or obscurely clamped, those

231 of disc composed of clampless hyphae with scattered elongated sphaeropedunculate pileocystidia with comparatively longer pedicel, 20‒30 µm wide. Stipitipellis composed of clamped filamentous hyphae, 3‒4 µm wide with scattered caulocystidia. Caulocystidia sub-capitate to capitate, 80‒100 × 5‒6 µm, thin‒walled. Caulosetae present, narrowly fusiform to setiform, 150‒200 × 5‒6 µm, thick‒walled, golden brown in KOH solution.

ADDITIONAL MATERIALS EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat district, Malam Jabba (Kishawra), 2050 m a.s.l. single

specimen, on soil under Oak, 31st July, 2016, Junaid Khan, MJ-1633 (LAH31412)

Molecular characterization Fig. 96

The ITS region of nrDNA upon sequencing yielded 832bp long sequence which on

BLAST matches 92% with Oudemansiella furfuracea (Peck) Zhu L. Yang, et al.,,

(GQ913365, FJ596855 and GQ913367) followed by 92% similarity with O. radicata

(Relh. ex Fr.) Singer (GQ913391, GQ913389 and GQ913393). Other closely matching taxa included O. chiangmaiae Zhu L. Yang, et al., (GU980132) and O. raphanipes (Berk.)

Pegler & T.W.K. Young (GU980130). Using the ITS dataset, species of Oudemansiella resolved into four clades with present collections clustering within Radicatae clade between O. raphanipes, O. chinagmaie and O. radicata. The clades conform to the morphological sections of Oudemansiella suggested by Yang et al., (2009). Strong bootstrap values were observed among the species of Dactylosporina, Mucidula and

Oudemansiella with low bootstrap values among the members of Radicatae.

Comments: based on novel morphology and molecular support, Oudemansiella sparslamellata is proposed as new to science taxon.

232

Figure 94. A‒D. Basidiomata of Oudemansiella sparslamellata. A: Basidiome in natural habitat. B: View of distant lamellae. C: Holotype collection in natural habitat. D: MJ-1633. Bars: 10 mm for A‒D.

233

Figure 95. A‒K. Microscopic structures of Oudemansiella sparslamellata (Holotype). A: Basidiospores. B: Basidia. C: Cheilocystidia. D: Pleurocystidia. E: Caulosetae. F: Caulocystidia. G: fruiting body. H: Pileocystidia from disc region. I: Pileocystidia from pileus margin. J: Hyphal arrangement in the disc region. K: Hyphae in the margin. Bars: 6 µm for A, 15.2 µm for B, 17 µm for C & D, 23.3 µm for E, 15 µm for F, 18 mm for G, 25 µm for H & I, 12 µm for J & K.

234

Figure 96. Molecular Phylogenetic analysis of ITS dataset of Oudemansiella sparslamellata and associated taxa by Maximum Likelihood method. The tree with the highest log likelihood (2532.2361) is shown. Names are taken from Genbank. Family PLUTEACEAE

235 a) Pluteus

1. Pluteus populicola nom.prov b) Volvariella

1. V. bombycina

2. V. hypopithys c) Vlovopluteus

1. V. earlei

236

PLUTEUS Fr.

Pluteus populicola nom.prov. Figs. 97 & 98

Etymology: “poulicola” mean dwelling on Populus, the habitat of the present taxon

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Ingaro Dherai village,

1000 m. a.s.l., on decomposing stump of Populus nigra L., Junaid Khan, 07th August, 2014,

ING-32 (Holotype), (SWAT000148).

Diagnosis: fruiting body medium sized, pileus pulvinate to plano-convex with a central umbo, light orange yellow to dark yellow, glabrous to lightly velvety, lamellae free, pink in color, stipe longitudinally striate to pruinose, basidiospores mostly sub-globose, rarely oval to broadly ellipsoidal, smooth, cheilocystidia utriform. Pileus 50‒70 mm across, pulvinate to plano-convex with a central umbo, light orange yellow (10YR 9/8) to dark yellow (2.5Y 9/12), disc darker (2.5Y 7/12), surface smooth, glabrous to lightly velvety, cap margin crenate, interior concolorous with the pileus just beneath the cuticle otherwise cream colored, unchanging upon cutting, thin, 2‒3 mm at the disc, less than 1mm at the margin. Lamellae free, broad (≤6mm at the center), close, pale pink (7.5YR 9/4) in young stages, pink (7.5R 9/4) in mature specimens, lamellar edge even, lamellulae present, mostly in single tier, rarely in two tiers, short to long extending beyond the middle of lamellae. Stipe 50‒80 × 5‒7 mm in dimensions, central, terete, equal to very slightly thickening downward, pinkish (7.5YR 8/4), surface more or less pruinose, longitudinally striate in mature specimens, breaking with a snap, interior concolorous with the exterior or light yellowish brownish (10YR 6/4), unchanging upon cutting, flesh fibrous, center hollow, covered with loosely arranged whitish fibrils.

237

Basidiospores (5.1‒) 5.5‒6.3 (‒6.8) × (4.4‒) 4.8‒5.8 (‒5.9) µm, Q = 1.1‒1.19 (‒1.2), Me

= 5.9 × 5.1 µm, Qe = 1.1, mostly sub-globose, rarely oval to broadly ellipsoidal, smooth, thick walled (2‒3 µm), mostly with one central guttule, rarely with many small guttules.

Basidia 20‒25 × 6‒7 µm, cylindrical to broadly oblong, with 4 sterigmata, sterigmata 2‒

2.5 µm long. Cheilocystidia 45‒ 50 × 9‒15 µm, utriform. Pleurocystidia none observed.

Pileipellis ixocutis, composed of branched hyphae suspended in gelatinous matrix, individual hypha 5‒8 µm in diameter, terminals round. Stipitipellis tissues sarcodimitic, composed of narrow branched hyphae (7‒9 µm in diameter) intermixed with large (200‒

300 × 12‒18 µm) fusoid hyphae.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Ingaro Dherai village, 1000 m. a.s.l., on decomposing stump of Populus

nigra L., Junaid Khan, 27th July, 2015, ING-1507 (LAH31413).

Molecular characterization Fig. 105

The ITS region of both collections (ING-32 & ING-1507) produced a 730bp fragment which showed 95 % similarity with Pluteus leoninus (Schaeff.) P. Kumm., P. variabilicolor Babos and P. castroae Justo & E.F. Malysheva (KF668295; KP192912;

NR119871, respectively) upon BLAST search. Accordingly, different sequences of the matching taxa and those mentioned in (Lezzi et al., 2014) were downloaded and used in the subsequent phylogenetic analysis. The maximum likelihood analysis clustered the

Pakistani collections within the leoninus clade, forming a sister clade with P. variabilicolor. The strong bootstrap value confirms the independent position of our present collections.

238

Comments: this species is proposed as new to science herein.

Figure 97. Basidiomata of Pluteus populicola. A & B: Holotype collection (ING-32). C‒ E: ING-1507. Bars: 10mm for A‒E.

239

Figure 98. A – D. Microscopic structures of Pluteus populicola. A: Basidiospores. B: Cheilocystidia. C: Stipitipellis. D: Pileipellis. Bars: 2.7 µm for A, 5 µm for B, 6.35 µm for C, 9.6 µm for D.

240

VOLVARIELLA Speg.

Volvariella hypopithys (Fr.) Shaffer, Mycologia 49: 572 (1957) Figs. 99 & 100

Pileus 30‒38 mm across, convex with a slight central umbo, surface tomentose covered with silky white hairs, cap margin straight and tomentose, interior solid, cream colored unchanging upon cutting, thin, ≤3mm at the disc, narrowing quickly towards margins and there less than 1mm. Lamellae free, 3‒4 mm at the broader site, lamellar edge even, lamellulae present, in 1‒2 tiers, mostly long, extending beyond middle of the lamellae, rarely short, close, color pink (10R 9/6), lamellar edge even. Stipe 50‒60 × 4‒6 mm in dimension, central, terete, equal or very slightly thickened downward, pure white, surface velvety to canescent, hairs silky white, base with small volva (8‒10mm high), volva thin, membranous, white, free in the upper part, attached at the base, interior of the stipe cream colored, solid, turning yellowish brown (10YR 9/8) in the extreme base upon cutting, otherwise unchanging, flesh moist.

Basidiospores (6.2‒) 6.3‒7.2 (‒7.3) × 4.2‒4.9 (‒5.1) µm, Q = (1.3‒) 1.33‒1.5 (‒1.7), Me

= 6.7 × 4.6 µm, Qe = 1.5, ellipsoid to broadly ellipsoid, mostly with two oil granules rarely with one, smooth, thick walled (3‒4 µm). Basidia 20‒25 × 8‒10 µm, clavate.

Cheilocystidia 39‒70 × 4‒6 µm (in the top), 9 – 12.2 µm in the broadest part, narrowly lageniform. Pleurocystidia 30‒50 × 5 – 8.5 µm (in the narrowest part) 15 – 20 µm, narrowly lageniform to utriform. Pileipellis composed of cylindrical hyphae with rounded to mucronate apices, individual hyphae 5‒10 µm in diameter.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2300 m a.s.l., in decomposing litter under Abies pindrow

241

(Royle ex D.Don) Royle, Junaid Khan, 14th August, 2014, MM-52 (SWAT

000149).

Comments: There are no previous records of the species from Pakistan and is an addition to the fungal flora of Pakistan.

Figure 99. A‒D. Basidiomata of Volvariella hypopithys (MM-52). A: full basidiome. B: View of the Lamellae. C: Pileus surface showing tomentose nature. D: sliced basidome showing internal features. Bars: 10 mm For A‒D.

242

Figure 100. A – E. Microscopic structures of Volvariella hypopithys. A: Basidiospores. B: Pleurocystidia. C: Basidia. D: Cheilocystidia. E: Pileipellis. Bars: 4.8 µm for A, 4.37 µm for B, 3.5 µm for C, 8.44 for D, 10 V for E.

243

Volvariella bombycina (Schaeff.) Singer, Lilloa 22: 401 (1951) [1949] Figs. 101 &

102

Pileus 50‒120 mm across, starting as egg like enclosed in universal veil, then opening conical and maturing conico‒convex to convex at maturity, white to cream colored, surface smooth, wooly tomentose especially when young, then maturing velvety and drying smooth, cap margin exceeding the lamellae and tomentose, interior fleshy, cream colored, lowly turn pinkish (7.5YR 9/4) upon cutting, thicker at the disc (3‒4mm). Lamellae free, broad (5‒7mm) at the center, close, light pink (7.5YR 8/4) at first, then aging dark reddish pink (10R 5/6), lamellar edge even to serrulate, lamellulae present, in 1‒3 tiers, of diverse lengths. Stipe 40‒60 × 5‒12 mm, central, terete, thickening downward with a slightly bulbous base, smooth, glabrous, interior fleshy, solid, wet, concolorous to the exterior, unchanging, base covered with long membranous volva, 30‒40 mm high, free in the upper part, attached at the base, white at first, brownish to grayish later on.

Basidiospores (6.5‒) 6.8‒8.2 (‒8.3) × (4.2‒) 4.4‒5.2 (‒5.4) µm, Q = (1.4‒) 1.5‒1.7 (‒1.8),

Me = 7.4 × 4.8 µm, Qe = 1.6, broadly ellipsoid to ellipsoid, rarely oval, smooth, thick walled (0.3‒0.4 µm), with one central guttule. Basidia 15‒20 × 5‒7 µm, cylindrical to clavate. Cheilocystidia 35‒45 × 6‒16 µm, fusiform to ventricose to more or less lageniform, thin walled. Pleurocystidia 60‒71 × 10‒18 µm, more or less utriform, rare.

Pileipellis a cutis, hyphae 3‒5 µm in diameter, pileocystidia 40‒50 µm long, cylindrical.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley 2250 m. a.s.l., on decomposing cavities of standing

tree of Abies pindrow, Junaid Khan, 01st August, 2014, MJ-54, (SWAT000150).

244

PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Ingaro Dherai village,

1000 m. a.s.l., on decomposing cavities of Populus nigra L., Junaid Khan, 10th

September, 2014, ING-45, (LAH31414), repeatedly from Same location in the

same year and consecutive years. PAKISTAN, KHYBER PAKHTUNKHWA,

Swat District, Miandam valley, 2200 m a.s.l., on decomposing cavities of standing

tree of Abies pindrow, Junaid Khan, 21st July, 2015, MM-1508, (LAH31415).

Comments: This species was recently reported by Hussain (2016) from Malakand district,

Khyber Pakhtunkhwa, Pakistan and this is the second report of the species from Pakistan.

245

Figure 101. A‒D. Basidiomata of Volvariella bombycina. A: Natural habitat (MM-1508). B: view of the pileus surface and lamellae (ING-45). C: Sliced fruiting body showing context. D: view of the under-surface (MJ-54). Bars: 10 mm for A‒D.

246

Figure 102. A – E. Microscopic structures of Volvariella bombycina. A: Basidiospores. B: Cheilocystidia. C: Basisia. D: Pleurocystidia. E: Pileipellis. Bars: 4.4 µm for A, 5.83 µm for B, 2.8 µm for C, 9.1 µm for D, 8.75 µm for E.

247

VOLVOPLUTEUS Vizzini, Contu & Justo

Volvopluteus earlei (Murrill) Vizzini, Contu & Justo, in Justo, Vizzini, Minnis, Menolli,

Capelari, Rodríguez, Malysheva, Contu, Ghignone & Hibbett, Fungal Biology 115(1): 15

(2011) Figs. 103 & 104

Pileus 20‒40 mm across, parabolic to parabolic-conical when young, plane by maturity with more or less uplifted margins, light yellowish pink (5YR 9/4), moderate yellowish pink (2.5YR 7/4) with a reddish pink margins (10R 6/8), surface moist and viscid in young specimens, dry and dull in mature specimens, surface glabrous to somewhat velvety, smooth in young specimens, bumpy to rugulose in mature specimens with striated margin, striation short almost 1/4th of the total pileus diameter, hygrophanous and two toned then

(with a dark pinkish brown margin and whitish central part), shape of margin crenate, interior soft, whitish, unchanging upon cutting, thin, 1‒2 mm thick at the disc, less than

1mm elsewhere. Lamellae free, broad (4‒6mm at the center), close, cream colored in young specimens, pinkish (7.5YR 8/8) in mature specimens, lamellar edge serrulate to serrate, lamellulae present, in 1‒3 tiers, of diverse lengths, some very short others very long. Stipe 70‒120 × 4‒7 mm, central, terete, thickening downward with somewhat flattened base, smooth, velvety, cream colored, turning slightly pinkish (7.5 YR 9/4) upon maturity and handling, fragile when young, context same colored as exterior, unchanging, centrally hollow, volva present, small, 12‒15 mm high, membranous, 2‒3 lobed, disappearing especially in the upper part.

Basidiospores (8.7‒) 10.7‒12.9 (‒13.8) × (6.6‒) 7.7‒8.9 (‒9.6) µm, Q = (1.2‒) 1.3‒1.6 (‒

1.7), Me = 11.8 × 8.2 µm, Qe = 1.4, broadly ellipsoid to limoniform, apiculus prominent,

248 smooth, thick walled (≤0.4 µm), mostly with one large and many small guttules, hyaline in

KOH solution, non-amyloid. Basidia 30‒40 × 10‒12 µm, cylindrical to somewhat clavate with very little difference in the upper part and lower part, 4-sterigmated, sterigmate 3‒4

µm high, hyaline in KOH solution, somewhat granular in Congo red. Cheilocystidia 20‒

40 × 6‒12 µm, calvate to cylindrical, rare. Pleurocystidia none observed. Pileipellis ixocutis, hyphae 5‒15 µm in diameter, with cylindrical terminal ends and scattered obovoid to broadly cylindrical elements, rarely branched, pileal elements 30‒35 × 14‒18 µm.

Pileocystidia 30‒50 × 20‒25 µm, ovoid to obovoid. Stipitipellis hyphae 7‒10 µm in diameter, more or less parallel, simple septate.

Materials examined: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District,

Ingaro Dherai village, 1000 m. a.s.l., among grasses in humus rich wet soil in the

paddy fields, Junaid Khan, 27th June, 2016, ING-1501, (SWAT000151). Same

location, Junaid Khan, 06th July, 2016, ING-1609, (LAH31416).

Molecular characterization Fig. 105

A 684bp fragment generated by sequencing the ITS region of ING‒1501, matched 99% with V. earlei, V. cookie Contu. and V. acystidiata N.C. Pathak (HM562205; HM246496;

HM246499) at Genbank. The closely matching sequences in the GenBank and those mentioned in Justo et al., (2011) were used in the phylogenetic analysis.

The Pakistani collection clustered with V. earlei and V. cookie with s strong bootstrap value thus confirming the identity of Pakistani collection as V. earlei. The V. cookie once considered as independent species is now regarded as a synonym of V. earlei (Justo et al.,

2011).

249

Comments: there are no records of any Volvopluteus species from Pakistan and the present study is introducing this genus to the fungi of Pakistan by reporting Volvariella earlei.

Volvariella earlei is a rare find in the study area and was collected twice from a single location.

Figure 103. A‒C. Basidiomata of Volvolpluteus earlei. A: ING-1501 in natural habitat. B: ING-1609. C: View of the two toned pileus surface. Bars: 10 mm for A‒C.

250

Figure 104. A – F. Microscopic structures of Volvopluteus earlei. A: Basidiospores. B: Basidia. C: Cheilocystidia. D: Pileocystidia. E: Pileipellis. F: Stipitipellis. Bars: 5.43 µm for A, 9.3 µm for B, 8.1 µm for C, 8.7 µm for D, 32 µm for E, 15 µm for F.

251

FJ774077.1 Pluteus aurantiorugosus voucher LE 216873 KP004941.1 Pluteus leoninus voucher ASIS24550 NR 119871.1 Pluteus castroae TNS F-17602 HM562099.1 Pluteus castroae voucher TNSF17081 99 KP192911.1 Pluteus variabilicolor isolate TL20130521-01 KP192914.1 Pluteus variabilicolor isolate VM20130504-01 KP004940.1 Pluteus leoninus voucher ASIS23019 57 Pluteus (ING-32) 89 Pluteus (ING32) JN603206.1 Pluteus chrysaegis voucher K13587 Pluteus 93 98 HM562142.1 Pluteus conizatus var. africanus JF908628.1 Pluteus granulatus voucher 16148 KF306032.1 Pluteus plautus isolate MSSF234

88 JF908606.1 Pluteus leoninus voucher 601 57 KF306003.1 Pluteus roseipes isolate P27 KC147681.1 Pluteus roseipes voucher UC 1861251 HM562140.1 Pluteus umbrosus voucher AJ213

98 JF908622.1 Pluteus umbrosus voucher 12187 98 HM562189.1 Pluteus granularis voucher SF20 99 HM562069.1 Pluteus granularis voucher Strack7 NR 119876.1 Volvopluteus michiganensis MICH 11761

98 JN182873.1 Volvariella gloiocephala isolate 55 99 HM562209.1 Volvopluteus gloiocephalus voucher LOU18247 NR 119878.1 Volvopluteus asiaticus TNS F-15191 HM562204.1 Volvopluteus earlei voucher MA22816 Volvopluteus HM562205.1 Volvopluteus earlei voucher Mamet7 HM246496.1 Volvariella cookei voucher TO AV133 99 HM246497.1 Volvariella earlei voucher TO AV134 Volvopluteus earlei (ING-1501) Volvopluteus earlei (ING-1609)

99 JN086667.1 Volvariella volvacea strain OE-12 KC142118.1 Volvariella volvacea strain OE-273 97 HM562212.1 Volvariella bombycina voucher AJ244 KC142120.1 Volvariella bombycina 99 EF566874.1 Volvariella bombycina Volvariella Volvariella bombycina (ING-21)

56 JF415136.1 Volvariella dunensis voucher TOAV140 HM562210.1 Volvariella taylorii voucher AJ54 72 HM246491.1 Volvariella taylorii voucher TO AV142

0.02

Figure 105. Molecular Phylogenetic analysis of the taxa of Pluteacea of the present study and associated taxa by Maximum Likelihood method. The analysis involved 39 nucleotide sequences. All positions containing gaps and missing data were eliminated.

252

Family POLYPORACEAE

b) Cerioporus

1. C. squamosus

a) Panus

1. P. rudis

253

CERIOPORUS

Cerioporus squamosus (Huds.) Quél., Enchir. fung. (Paris): 167 (1886) Fig. 106

Fruiting body 50‒80 mm across, semicircular in shape when attached from the side, almost circular when growing on the top of the substrate, center shallowly depressed, surface cream colored to pinkish brown (10R 5/10), covered by concentric brown scales

(2.5YR 8/8), margin inrolled especially in young stages. Pore surface running down the stem, whitish to cream colored, 7‒10 mm deep, pores irregularly arranged, angular, small,

1‒3 per mm. Stipe 10‒20 mm thick, mostly off centric, lateral to central, concolorous with cap or lighter, porous on the lower side.

Basidiospores (14.3‒) 15‒17.4 (‒17.5) × (7.3‒) 7.6‒9.2 (‒9.5) µm, Q = (1.6‒) 1.7‒2.2 (‒

2.3), Me = 16.3 × 8.3 µm, Qe = 2, punctate, sub-cylindric to oblong ellipsoid. Basidia 30‒

50 × 5‒7 µm, cylindrical to irregular. Hyphal system dimitic, generative hyphae with clamp connections, branched, 4‒5.5 µm in diameter, thin walled, binding hyphae simple septate, 4‒5 µm in diameter, rarely branched, with cylindrical terminals.

MATERIALS STUDIED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2250 m a.s.l., on decomposing conifer stump, 25th July,

2015, MM-24 (SWAT000152).

Comments: this appears the first report of this species from Pakistan.

254

Figure 106. A‒H. Morphology of Cerioporus squamosus (MM-24). A – C: Different views of the basidiome. D: Basidiospores. F: Basidia. G: Generative hyphae. D: Bonding hyphae. Bars: 10 mm for A‒C, 5.4 µm for D, 11.25 for F & H, 15 µm for G.

255

PANUS

Panus rudis Fr., Epicr. syst. mycol. (Upsaliae): 398 (1838) [1836‒1838] Figs. 107 & 108

Fruiting body 50‒80 mm across, petaloid to semicircular when growing from the side of the substrate otherwise round in outline, infundibuliform to vase shaped in the latter case, margin incurved to inrolled, purplish at first then passing through different shades of purplish (10R 9/2) and reddish (2.5YR 9/2) turning brownish (5Y 9/4) and finally drying light brown, hispid to villose composed of 1‒2 mm long hairs, context whitish, thin, 1 ‒2 mm in thickness, unchanging upon cutting. Lamellae decurrent, narrow, close, purplish to pinkish (7.5R 9/2) at first then drying light brown, lamellulae absent, unchanging upon cutting or bruising. Stipe 10‒20 × 8‒10 mm in dimensions, central to lateral depending upon the attachment to the substrate, concolorous to the lamellae or slightly whitish, floccose with loosely arranges hairs of 1mm length, base with white mycelium, texture tough, interior solid, fibrous, whitish, unchanging.

Basidiospores (5.5‒) 5.7‒6.2 (‒6.5) × (3.2‒) 3.23‒3.37 (‒3.4) µm, Q = 1.8‒1.89 (‒1.9),

Me = 6 × 3.3 µm, Qe = 1.8, ellipsoid, smooth, hyaline in KOH solution. Basidia 15‒20 ×

4‒5 µm, clavate, hyaline in KOH solution. Cystidia 15‒20 × 6‒8 µm, narrowly clavate to narrowly cylindrical. Tramal hyphae 2‒3 µm in thickness, clamped, thick walled, hyaline in KOH solution. Contextual hyphae 4‒5 µm in thickness, clamped, thick walled, hyaline in KOH solution. Binding hyphae without clamp connections, 2‒4 µm in diameter.

Material examined: PAKISTAN, KHYBER PAKHTUNKHWA, SWAT

DISTRICT, Ingaro Dherai Village, 1000 m a.s.l., on decomposing Populus nigra

256

L., stumps, Junaid Khan, 23rd February, 2014, ING-06 (SWATXXX). Same

locality, Junaid Khan, 13th May, 2015, ING‒16 (Lectotype) (LAHXXX).

Comments: A common species found in the woodland ecosystems of the study area.

Already reported from Kalam and Shogran (Ahmad, 1980).

Figure 107. A –D. Basisiomata of Panus rudis. A: Basidiome in natural habitat. B: View of the pileus surface. C: View of the under surface. E: View of dried specimens. Bars: 10 mm for A‒D.

257

Figure 108. A – E. Microscopic structures of Panus rudis. A: Basidiospores. B: Basidia. C: Cystidia. D: Contextual hyphae. E: Binding hyphae. Bars: 3.93 µm for A, 5.7 µm for B, 4.4 µm for C, 7.9 for D, 6.33 µm for E.

258

Family PTERULACEAE

a) Pterula

1. P. bisporitica nom.prov.

259

PTERULA Fr.

Pterula bisporitica nom.prov. Fig. 109

Etymology: the epithet “bisporitica” refers to the 2-spored basidia characterizing the species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2250 m a.s.l., under Abies pindrow, 10th August, 2014, MJ-75 (Holotype), (SWAT000154),

Diagnosis: fruiting body small to medium sized, frequently branching, resembling antlers, light to moderate pink in color, surface smooth, lightly pruinose, stipe short, attached to the substrate through blackish rhizomorph, basidiospores cylindrical, basidia 2-spored, cystidia present. Fruiting body 25‒35 mm high, 20‒50 mm across, terrestrial, erect, antlers like, frequently branching below, dichotomous above, with acute apices, branches terete, light pink (2.5YR

9/4) with moderate pink (2.5YR 7/4) apices, surface smooth, lightly pruinose, fragile. Stipe short, 4‒7 × 1‒2 mm, central, terete, equal, concolorous with the branches, attached to the substrate through blackish rhizomorph.

Basidiospores (6.5‒) 6.7‒10 (‒10.5) × (3.7‒) 3.72‒4.57 (‒4.6) µm, Q = (1.6‒) 1.63‒2.25

(‒2.3), Me = 7.8 × 4.3 µm, Qe = 1.8, cylindrical, with apiculus, hyaline in KOH solution, smooth walled, with a single guttule. Basidia 30‒45 × 6‒11 µm, clavate, 2-sterigmated, hyaline in KOH solution. Cystidia 20‒25 × 5‒8 µm, narrowly utriform, clavate, cylindrical with capitate heads to somewhat mucronate, thin walled, hyaline in KOH solution. Hyphal system dimitic, generative hyphae 3‒5.5 µm in diameter, with clamp connections, hyaline in KOH, skeletal hyphae 5 – 7 µm in diameter, septa without clamps.

260

Molecular characterization Fig. 110

The target region upon amplification generated a 710 and 743bp fragments which upon

BLAST search matched 96% with Pterula echo J. McLaughlin & E.G. McLaughlin

(DQ494693) and Tephrocybella griseonigrescens Picillo, Vizzini & Contu (NR137975) from USA. Based on close matches in the BLAST search, taxa of other genera were also included in subsequent phylogenetic analysis.

A total of 17 closely matching sequences were downloaded and used in the final analysis.

There were 710 sites in the aligned dataset out of which 233 were conserved, 468 variable containing 376 parsimony informative.

The maximum likelihood analysis resolved species from different lineages into different clades showing a strong relationship. The Pakistani sequences of MJ-75 clustered within the Pterula clade forming a sister clade with Pterula sp. MB67and KP133248.1 Pterula sp. 984, with a robust value of 89 % supporting its independent position. All the Pterula sequences used in the present analysis except for that of Pterula subulata Fr.(KR673516), clustered in a well-supported clade. Similarly the Amylocorticiellum sp. UC2022882

(KP814442) clustered within the Pterula clade and needs confirmation.

Comments: there are no previous records of any Pterula species (Ahmad et al., 1997) and both the genus and species are additions to the mycobiota of Pakistan.

261

Figure 109. A – F. Macro and Micro-morphological characters of Pterula bisporitica. A: Cluster of fruiting bodies in natural habitat. B: Harvested fruiting body showing complete characters. C: Basidiospores. D: Cystidia. E: Basidia. F. generative hyphae. Bars: 3 mm for A & B, 3.1 µm for C, 3.7 µm for D, 4.3 for E, 7.22 µm for F.

262

Pterula bisporitica 100 Pterula bisporitica

89 Pterula bisporitica DQ911591.1 Pterula sp. MB67 Pterula 98 KP133248.1 Pterula sp. 984 KP814442.1 Amylocorticiellum sp. UC2022882 88 91 DQ494693.1 Pterula echo isolate AFTOL-ID 711 KP192551.1 Tephrocybe confusa isolate FR2013224

95 Tephrocybella 96 NR 137975.1 Tephrocybella griseonigrescens TO HG 21112014 99 KR105775.1 Tephrocybella griseonigrescens voucher TO:HG 21112014 KX236475.1 Ceriporia occidentalis Ceriporia 100 KX236473.1 Ceriporia occidentalis

100 KP814386.1 Leptosporomyces galzinii voucher UC2023185 Leptosporomyces KP814385.1 Leptosporomyces galzinii voucher UC2023180 KR673516.1 Pterula subulata voucher KA12-1276

Hyphodontia 52 KJ668514.1 Hyphodontia subalutacea 100 KP814444.1 Hyphodontia subalutacea

0.05 Figure 110. Molecular Phylogenetic analysis of Pterula bisporitica and associated taxa by Maximum Likelihood method inferred from ITS data. The tree with the highest log likelihood (‒1718.4255) is shown. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated.

263

Family RUSSULACEAE

a) Lactarius

1. L. furcatilamellatus nom.prov.

2. L. maireioides nom.prov.

3. L. mediterraneensis

4. L. pterosporus

5. L. sanguifluus

b) Lactifluus

1. L. versiformis

c) Russula

1. R. amythestina

2. R. kangchenjungae

3. R. olivacea

4. R. postiana

5. R. similis

264

LACTARIUS Pers.

Lactarius furcatilamellatus nom.prov. Fig. 113

Etymology: the epithet “fructilamellatus” refers to the forking lamellae characterizing the present species

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2250 m a.s.l., under Abies pindrow (Royle ex D.Don) Royle, 10th August, 2014, Junaid

Khan, MJ-56 (Holotype), (SWAT000156).

Diagnosis: fruiting body medium sized, pileus convex to plano-convex with a

depressed center and incurved margin, color vivid orange, lamellae decurrent,

close, vivid orange yellow, developing greenish spots with age, secreting orange

latex upon cutting, basidoposres sub-globose to broadly ellipsoidal.

Pileus 40‒60 mm across, convex at first then plano-convex with a depressed center and incurved margin, vivid orange (2.5YR 6/18) to vivid orange yellow (7.5YR 7/16) with clear cut whitish concentric zones, cap margin inrolled and scaly in young stages, eroded and wavy in mature stages, develop greenish spots with age or injury especially over the margins, context concolorous with the cap or slightly paler, thin, 2‒3 mm at the center and up to 1 mm at the edge, wet, paler than pileus, color unchanging, secrete orange colored latex upon cutting. Lamellae decurrent, close with large number of short gills, forking, rarely inter-venose, narrow (not more than 2 mm at the center), vivid orange yellow

(7.5YR 7/18) in young specimens, maturing pale orange yellow (7.5YR 8/4), developing grayish greenish yellow (10Y 7/4) to light olive (10Y 5/4) spots with age, fuzzy in appearance especially in young stages, exudes orange (2.5YR 7/12) latex upon cutting, lamellar edge even. Stipe 40‒70 × 10‒16 mm in dimensions, central, terete, cylindrical or tapering towards base, generally thicker at the center, strong orange colored (7.5YR 7/12),

265 smooth without any scrobiculi, covered with a whitish fuzz especially at the center, attached to the soil through whitish rhizomorph, hollow from inside, context thin, flesh concolorous with the stipe with a more brighter (5YR 8/12) basal portion, inner side composed of white cottony white tissues.

Basidiospores (7.4‒) 7.6‒8.4 (‒8.8) × (6.5‒) 6.51‒7.3 (‒7.4) µm, Q = (1.1‒) 1.12‒1.2 (‒

1.3), Me = 8 × 6.9 µm, Qe = 1.2, sub-globose in face view, broadly ellipsoidal in side view, warted, warts up-to 2.5 µm long, ridges forming complete reticulum. Basidia 30‒50 × 10‒

12 µm, clavate, gulltulate, mostly 4-spored, rarely 2-spored, thin walled, hyaline in KOH solution. Cheilocystidia absent. Pleurocystidia 35‒50 × 10‒15 µm, narrowly utriform, clavate to more or less lanceolate, thin walled, hyaline in KOH, guttulate in Congo red.

Pileipellis hyphae 3‒4 µm in size, with scattered lactifers.

MATERIALS STUDIED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2250 m a.s.l., under Abies pindrow, 28th August,

2016, Junaid Khan, MJ-16124 (LAH31418).

Molecular characterization Fig. 114

The target region of MJ-56 upon sequencing yielded a 748bp fragment Upon BLAST analysis it showed 98% similarity with Lactarius abieticola X.H. Wang (KY174941;

KY174945; NR_152988) followed by L. salmonicolor R. Heim & Leclair (AF249287;

JF908283) and L. vividus X.H. Wang, Nuytinck & Verbeken (KT163431; KT163432) with

97% similarity.

Molecular phylogenetic analysis was carried out using MEGA 6 software (Tamura et al.,

2013). A total of 45 sequences were included in the final dataset with 44 in‒group taxa and one outgroup taxon (Lactarius aestivus; KJ769669; KJ769670). Pakistani collection

266 occupied an intermediate position between L. abieticola and L. vividus, supporting the independent position of the present collection.

Comments: based on morphological uniqueness and molecular support, this taxon is being proposed as new to science.

267

Figure 113: A‒D: Morphology of Lactarius furcatilamellatus. A, C & D: MJ-56. B: MJ- 16124. E‒H: microscopic structures. E: Basidiospores. F: Pleurocystidia. G: Pileipellis. H: Basidia. Bars: 12 mm for A‒D, 4.1 µm for E, 9.5 µm for F & G, 11.87 µm for H.

268

74 AF140267 Lactarius deterrimus AF140266 Lactarius deterrimus KX095064 Lactarius fennoscandicus 98 KX095055 Lactarius fennoscandicus KC840610 Lactarius aurantiosordidus EF685094 Lactarius aurantiosordidus KP115211 Lactarius sanguifluus 99 HG797001 Lactarius sanguifluus KY174934 Lactarius hengduanensis KY174933 Lactarius hengduanensis KF133269 Lactarius akahatsu 99 EF141544 Lactarius akahatsu

99 EF685051 Lactarius deliciosus EF685050 Lactarius deliciosus 59 EF685083 Lactarius rubrilacteus 96 EF685085 Lactarius rubrilacteus

99 AY332556 Lactarius semisanguifluus AY332555 Lactarius semisanguifluus

100 EF685077 Lactarius laeticolor EF685091 Lactarius laeticolor KJ705195 Lactarius thyinos 54 100 KJ705196 Lactarius thyinos AF249287 Lactarius salmonicolor JF908283 Lactarius salmonicolor KY174945 Lactarius abieticola 64 99 KY174941 Lactarius abieticola NR 152988 Lactarius abieticola

96 Lactarius furcatilamellatus Lactarius furcatilamellatus KT163432 Lactarius vividus 100 KT163431 Lactarius vividus KY174917 Lactarius pseudohatsudake 99 KY174916 Lactarius pseudohatsudake KJ508896 Lactarius hatsudake 100 JN387078 Lactarius hatsudake KJ769670 Lactarius aestivus 100 KJ769669 Lactarius aestivus

0.005 Figure 114. Molecular phylogenetic analysis of Lactarius furcatilamellatus (MJ-56). The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes‒Cantor model. The tree with the highest log likelihood (‒1773.3081) is shown. There were a total of 545 positions in the final dataset.

269

Lactarius maireioides nom.prov. Fig. 115

Etymology: the epithet “maireioides” refers to the close resemblance of the present taxon with “L. mairei”

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2250 m a.s.l., under Quercus dilatata Royle, in Mixed Quercus‒coniferous forest, Junaid khan, 25th July, 2015, MJ-1590a (Holotype), (SWAT000155).

Diagnosis: fruiting body small to medium sized, pileus hemispherical to plane with inrolled margin, pubescent-tomentose with a smooth disc, often forming concentric rings, margin heavily tomentose, pale yellow to orange yellow, stipe pruinose, basidiospores broadly ellipsoid, pleurocystidia 40‒55 ×10‒13 µm lanceolate. Pileus 20‒40 m across, hemispherical with an inrolled margin when young then turning plane with depressed center, may turn infundibuliform in more mature specimens, color pale yellow (2.5Y 9/4) with a moderate orange yellow (7.5YR 7/8) disc, dry, dull, surface smooth at the disc otherwise pubescent to tomentose, scales radiating from center to the margins, in concentric rings in some specimens, pileus margins tomentose in young specimens, without scales by maturity, interior solid, context concolorous with the exterior or slightly paler, thin, 2‒3 mm at the center, 1 mm at the margins, secreting whitish latex upon cutting, unchanging upon cutting. Lamellae decurrent, slightly running down the stipe, forking, close to crowded, broader at the center (3‒4 mm), light orange (5YR 8/8) to moderate orange (2.5YR 7/8), exudes white to colorless latex upon cutting, lamellar edge even, lamellulae present. Stipe 20‒35 × 7‒10 mm in dimension, central, terete, mostly tapering towards top and bottom, thicker at the center, in other specimens equal or tapering towards base, light orange (2.5YR 8/8) to moderate orange (2.5YR 7/8), fuzzy with whitish

270 powdery mass especially in young stages, exuding white latex upon cutting or breaking, bruising dark brown, interior chalky, context whitish, unchanging.

Basidiospores (6.3‒) 6.5‒7.4 (‒7.7) × 5.4‒6.5 (‒6.7) µm, Me = 6.9 × 5.7 µm, Q = (1.1‒)

1.11‒1.29 (‒1.3), Qe = 1.2, broadly ellipsoid, ornamentation up-to 1 µm. Basidia 25‒40 ×

10‒12 µm, 4-spored, cylindrical to clavate, sterigmata 4‒5 µm. Pleurocystidia 40‒55 ×10‒

13 µm in dimension, lancelotae in shape, scattered. Lamellar trama with abundant ochre color lactifers forming pseudocystidia in some places. Pileipellis a cutis with abundant lactifers, individual hyphae up to 2 µm in diameter.

ADDITIONAL MATERIALS STUDIED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2250 m a.s.l., under

Quercus dilatata, in Mixed Quercus-coniferous forest, Junaid Khan, 31st July,

2016, MJ-1689 (LAH31417).

Molecular characterization Fig. 116

The target region (ITS1, ITS2 and 5.8S of nrDNA) of MJ-1590a yielded a fragment of

759bp. Upon BLAST search at GenBank, the query sequence matched 97% with Lactarius mairie (JF909298; AY336952; AY336950), followed by L. torminosus (Schaeff.) Gray

(KR025613; DQ367908) with 95% similarity.

For phylogenetic reconstruction, closely matching sequences from NCBI together with those mentioned in Nuytinck et al., (2004) were downloaded. The final dataset comprised of 22 in-group taxa with Russula brevipes (AF349714) as outgroup taxon. Maximum likelihood analysis from the highly similar sequences yielded a consensus tree clustering the Lactarius species into different clades. Pakistani collection clustered within the L.

271 mairie clade forming a sister clade with a strong bootstrap value, supporting its unique position.

Comments: this taxon is being proposed as new to science.

272

Figure 115: A‒C: Morphological characters of Lactarius mairieoides. A & B: Holotype collection (MJ-1590a). C: MJ-1689. D: Basidiospores. E: Basidia and Pleurocytidium. F: Cross section of lamellae. G: Pileipellis. H: Sub‒Pellis. Bars: 7mm for A‒C. 4.7 µm for D, 12.4 µm for E, 28.7 µm for F, 9.6 µm for G & H.

273

KR025613 Lactarius torminosus

GU373496 Lactarius torminosus 94 JF908288 Lactarius torminosus

KR082868 Lactarius torminosus

61 DQ367908 Lactarius torminosus

AY606953 Lactarius pubescens

KR082883 Lactarius pubescens 97 12 AF096986 Lactarius tesquorum

65 JF908309 Lactarius tesquorum

Lactarius maireioides (MJ-1590a)

AY336951 Lactarius mairei var il 100 21 84 JF908298 Lactarius mairei

96 AY336952 Lactarius mairei

99 AY336950 Lactarius mairei 41 HQ650733 Lactarius olympianus

98 EF685079 Lactarius olympianus

39 KF432968 Lactarius scrobiculatus

96 EU597079 Lactarius scrobiculatus

JF908268 Lactarius pallidus

97 AY606951 Lactarius pallidus

AY336960 Lactarius spinosulus

99 AY336961 Lactarius spinosulus

AF349714 Russula brevipes

0.02 Figure 116. Molecular Phylogenetic analysis by Maximum Likelihood method of Lactarius maireioides and associated taxa using nrITS data. The tree with the highest log likelihood (‒1552.8177) is shown. The Pakistani collection is represented with mark

274

Lactarius mediterraneensis Llistos. & Bellù, Mycotaxon 57: 176 (1996) Fig. 117

Pileus 75mm across, umbilicate in shape with an incurved margin, may be due to some mechanical injury within the young stages, pale yellow with a whitish tone (5Y 9/6), turning grayish yellow (5Y 7/4) upon handling, surface dry, dull, pubescent, with pale whitish hairs on a pale yellow background, cap margin even, interior chalky, whitish to whitish yellow, unchanging, thicker at the center (3‒4mm), 1‒1.5 mm at the margin.

Lamellae decurrent with some sinuate lamellae, narrow to moderately broad (2‒3 mm), getting thinner on both sides, sub‒distant, pale yellow (2.5Y 9/4), developing brown spots in mature specimens, lamellar edge eroded to serrulate, obtuse, lamellulae present, rare, short. Stipe 25 × 14 mm in dimensions, short, central, terete, tapering towards the base with an attenuated base, whitish, bruising pale yellowish (2.5Y 9/4), texture rigid, interior solid, pale yellow (2.5Y 9/4), without any veil, surface smooth to slightly pubescent.

Basidiopsores (9‒) 9.6‒11.2 (‒12.7) × (8.5‒) 9‒9.6 (‒10.5) µm, Q = (1‒) 1.1‒1.2, Me =

10.4 × 9.4 µm, Qe = 1.1, globose to sub-globose, broadly ellipsoid in side view, warted, amyloid, ridges forming incomplete reticulum, with prominent apiculus. Basidia 60‒70 ×

14‒17 µm, 4-sterigmated, sterigmata 5‒7 µm long. Cheilocystidia 25‒40 × 12‒16 µm, narrowly clavate to narrowly utriform, rarely mammiform. Pleurocystidia 60‒70 × 13‒17

µm, narrowly clavate to narrowly utriform. Pileipellis composed of somewhat branched clampless hyphae with somewhat rounded to sub‒acute tips, individual hyphae 3‒5 µm in diameter. Lactifers unbranched, abundant in lamellar trama often forming pseudocystia, pseudocystidia inter-spread among the basidia, with acute apices.

275

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Marghazar valley, 1100 m a.s.l., under Quercus incana, Junaid Khan, 12th

August, 2016, MZ-1607 (SWAT000158).

Molecular characterization Fig. 118

The target region (ITS) of MZ-1607 yielded a 747bp long segment which on BLAST analysis showed 99% similarity with Lactarius mediterraneensis Llistos. & Bellù

(JF908267; JF908307) from USA followed by L. acerrimus Britzelm. (EF493285;

AJ889958) from Denmark and Sweden respectively.

Phylogenetic analysis included 31 taxa with Russula brevipes Peck. (AF349714) selected as outgroup taxon. The aligned dataset was composed of 706 sites, out of which 502 sites were conserved, 184 variable with 93 variable cites.

Upon maximum likelihood analysis, the Pakistani collection of Lactarius (MZ-1607) clustered within the piperati clade of Lactarius with L. mediterraneensis, confirming its identity as L. mediterraneensis.

Comments: There are no previous records for this species from Pakistan and this appears the first record of the species and a new addition to the Lactarius flora of Pakistan.

276

Figure 117. A – G. Morphology of Lactarius mediterraneensis (MZ-1607). A: view of the pileus showing texture and reaction with KOH. B: View of the lamellae and stipe. C: Basidiospores. D: Basidia. E: Cheilocystidia. F: Pleurocystidia. G: Pileipellis. Bars: 10 mm for A & B, 5.75 µm for C, 10.1 µm for D, 5.4 µm for E, 11.9 µm for F, 16.25 µm for G.

277

99 AY336951 Lactarius mairei var il

98 JF908298 Lactarius mairei AY336952 Lactarius mairei 83 99 AY336950 Lactarius mairei Lactarius aff.mairei HQ650733 Lactarius olympianus 99 EF685079 Lactarius olympianus Lactarius mediterraneensis (MZ-1607)

97 JF908267 Lactarius mediterraneensis 86 JF908307 Lactarius mediterraneensis

67 AF096986 Lactarius tesquorum JF908309 Lactarius tesquorum 63 AY606953 Lactarius pubescens 94 KR082883 Lactarius pubescens JF899564 Lactarius torminosus DQ367908 Lactarius torminosus 71 KR082868 Lactarius torminosus JF908288 Lactarius torminosus 80 KR025613 Lactarius torminosus GU373496 Lactarius torminosus KT583640 Lactarius haugiae EF141543 Lactarius purpureus DQ099898 Lactarius alnicola EU597079 Lactarius scrobiculatus 65 87 KF432968 Lactarius scrobiculatus 99 AF140262 Lactarius scrobiculatus JF908268 Lactarius pallidus 98 AY606951 Lactarius pallidus AY336960 Lactarius spinosulus 98 AY336961 Lactarius spinosulus AF349714 Russula brevipes

0.02 Figure 118. Molecular Phylogenetic analysis of Lactarius mediterraneensis inferred from nITS sequence data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes-Cantor model.

278

Lactarius pterosporus Romagn., Revue Mycol., Paris 14: 108 (1949) Fig. 111

Pileus 40‒70 mm across, convex at first then plano-convex with a depressed center, often distorted with age with an upturned margin, color ranges from grayish brown (7.5YR 4/2) to blackish brown (7.5YR 1/2) in different collection with a moderate brownish (10YR

7/8) margin, cap margin entire, surface dry, dull, smooth when young then wrinkled to bumpy by maturity at least at the disc, some specimens distinctly bumpy, interior solid to chalky, continuous with the stipe, context white, turning pink upon cutting, thicker at the center (2‒4 mm) than margin (up to 1mm). Lamellae decurrent, slightly running down, close to crowded, pale yellow (2.5Y 9/4) at first, pale orange yellow (7.5YR 8/4) at maturity, short gills present, exudes white latex upon cutting or breaking, latex turning pinkish later on. Stipe 20‒90 mm long, 10‒15 mm wide, central, mostly tapering towards base, sometimes equal, smooth in some collections otherwise wrinkled in others especially in mature specimens, concolorous with the pileus or slightly lighter with a whitish base, exudes milky white latex upon cutting or breaking that turn pinkish later on, interior white then turning pinkish upon cutting or breaking, firm at first then chalky at maturity.

Basidiospores (8.9‒) 9.1‒10.7 (‒11) × (8.8‒) 8.9‒9.9 (‒10.1) µm, Q = 1‒1.1, Me = 9.9 ×

9.5 µm, Qe = 1, globose, ornamentation up-to 2 µm high forming a complete reticulum, amyloid. Basidia 40‒60 × 12‒15 µm, clavate, 4-spored, sterigmata up to 5 µm.

Cheilocystidia 50‒70 × 15‒18 µm, fusiform. Pileipellis an ixocutis composed of somewhat interwoven hyphae (3‒5 µm) with blunt apices, lactifers inter-spread and abundant.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 21st

279

July, 2015, MM-1506 (SWAT000157). PAKISTAN KHYBER

PAKHTUNKHWA, Swat District, Miandam valley, 2225 m a.s.l., under Abies

pindrow Junaid Khan, 07th August 2016, MM-1615, (LAH31419).

Molecular characterization Fig. 112

The target region of MM-1536 and MJ-67 yielded a 722bp fragment. After trimming the motifs, a 622bp sequence was left which on BLAST search matched 99% with Lactarius pterosporus Romagn (JQ446151; JQ446152; JQ446153; KF432963) followed and L. pterosporus (Bolton) Gray (KF432962; JQ446084; JQ446985).

For phylogenetic reconstruction, 49 in-group sequences and single outgroup sequence were included. The Pakistani collections (MJ-67 and MM-1536), clustered with L. pterosporus forming a sister clade. However, based on the close morphological resemblance and molecular data (99% similarity), the present collections are treated as L. pterosporus herein.

Comments: with no previous record, this taxon is being reported for the first time from

Pakistan.

280

Figure 111: A – G. Morphological characters of Lactarius pterosporus. A–C: Basidioma D: Basidiospores. E: Basidia. F: Cheilocystidia. G: Pileipellis. Bars: 10 mm for A–C, 3 µm for D, E & F. 8 µm for G.

281

JQ446083 Lactarius acris

85 JQ446085 Lactarius acris JQ446081 Lactarius acris JQ446084 Lactarius acris JQ446141 Lactarius ruginosus JQ446137 Lactarius ruginosus 97 JQ446145 Lactarius ruginosus 51 KF432963 Lactarius pterosporus KR025628 Lactarius pterosporus JQ446151 Lactarius pterosporus JQ446153 Lactarius pterosporus

82 JQ446152 Lactarius pterosporus Lactarius pterosporus MM1506 Lactarius pterosporus MM1615 JQ446098 Lactarius azonites JQ446092 Lactarius azonites 96 JQ446094 Lactarius azonites 23 JQ446095 Lactarius azonites

93 JQ446086 Lactarius atromarginatu 53 JQ446124 Lactarius atromarginatu 61 EF560674 Lactarius lavandulus EF560675 Lactarius lavandulus

99 JQ446101 Lactarius fallax var fa 100 JQ446104 Lactarius fallax var co

95 JQ446103 Lactarius fallax 43 97 JQ446102 Lactarius fallax JQ446114 Lactarius lignyotus 95 JQ446117 Lactarius lignyotus 95 JQ446112 Lactarius lignyotus JF908301 Lactarius lignyotus

99 JQ446143 Lactarius romagnesii KF432964 Lactarius romagnesii JQ446110 Lactarius fuliginosus

93 EF560657 Lactarius fuliginosus 52 JQ446132 Lactarius picinus Q446128 Lactarius fuliginosus

85 55 JQ446129 Lactarius picinus JQ446127 Lactarius picinus JQ446111 Lactarius fuliginosus 78 JQ446131 Lactarius fuliginosus 54 JQ446130 Lactarius picinus

99 JN797631 Lactarius fumosibrunneu 40 JN797632 Lactarius fumosibrunneu EU819483 Lactarius fumosus EF560665 Lactarius friabilis 43 99 KF432961 Lactarius friabilis

99 EF560680 Lactarius oomsisiensis

92 EF560678 Lactarius oomsisiensis 65 EF560679 Lactarius oomsisiensis

0.005 Figure 112. Molecular Phylogenetic analysis of Lactarius pterosporus and associated taxa by Maximum likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes-Cantor model. All positions containing gaps and missing data were eliminated.

282

Lactarius sanguifluus (Paulet) Fr., Epicr. syst. mycol. (Upsaliae): 341 (1838) [1836‒

1838] Fig. 119

Pileus 50‒100 mm across, young specimens plano-convex with a depressed center, umbilicate to infudibuliform by maturity, light reddish brown (10R 6/4) to light grayish reddish brown (2.5YR 6/2) with a light yellowish pink margin (7.5R 9/4), later developing yellow green (7.5GY 9/8) to deep green yellow (7.5GY 4/10) color and finally drying light orange color (7.5YR 9/8), surface viscid to glutinous in young specimens, viscid to dry in mature specimens, shiny, texture smooth, pubescent except at the disc, hairs mostly in concentric rings, context moist, pale yellowish pink (7.5R 9/2) at the center, concolorous with the pileus at the margin, turning grayish red (5R 6/4) upon cutting, thicker at the center

(4‒6 mm) than margin (1 mm), texture spongy, releasing grayish red (5R 5/6) upon cutting.

Lamellae decurrent, narrow to moderately broad at the center (2‒3 mm), close, grayish reddish orange (10R 5/6), greening upon aging and bruising (7.5GY 5/6), lamellar edge entire, moderately acute, arid, lamellulae very frequent, forking, anastomosing at the base, releasing grayish red (5R 5/6) latex upon cutting. Stipe 40‒60 × 10‒13 mm in dimensions, central, terete, equal to slightly thickening towards base, concolorous to the pileus with a whitish tone especially in the young specimens, smooth in the upper part, hairy at the base, hairs whitish, interior spongy to hollow in some specimens, moderate red (5R 5/10) below the surface, pale yellow orange (7.5YR 8/4).

Basidiospores (7.3‒) 7.5‒8.6 (‒9.4) × (5.7‒) 6.5‒7.6 (‒7.9) µm, Q = (1‒) 1.1‒1.2 (‒1.3),

Me = 8.1 × 7 µm, Qe = 1.2, sub-globose to broadly ellipsoid, ornamented, ornamentation low forming incomplete reticulum, amyloid. Basidia 30‒50 × 7‒11 µm, narrowly clavate, guttulate, bases simple septate. Cheilocystidia none observed. Pleurocystidia 35‒65 × 7‒

283

11 µm, narrowly clavate with acute to obtuse apices, narrowly utriform, guttulate in Congo red. Pileipellis composed of narrow hyphae of size 3‒5 µm in diameter with scattered lactifers, lactifers golden brown in KOH, often piercing the cuticle forming pseudocystidia.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2050 m a.s.l., under Pinus wallichiana, Junaid Khan,

27th July, 2014, MJ-11 (SWAT000119). PAKISTAN KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2225 m a.s.l., under Pinus

wallichiana, Junaid Khan, 14th July 2015, MJ-1527a, (LAH31420).

Molecular characterization Fig. 120

A 741bp segment was generated by amplifying the nITS region of MJ-11 and MJ-1527a.

The query sequence on BLAST search matches 99% with Lactarius sanguifluus

(KP115209; KP115210; KP115211 and other sequences) and 98% with L. semisanguifluus

R. Heim & Leclair (HF559377; AF140268; DQ658872).

A total of 47 nucleotide sequences were generated from GenBank to construct the phylogeny of L. sanguifluus with Russula brevipes used as outgroup taxon. The Pakistani collections (MJ-11 and MJ1527a) clustered with other sequences of L. sanguifluus thus confirming the identity.

Comments: this taxon is previously reported from Khanspur by Razzaq et al., (2012). This is the first report of this species from the present study area.

284

Figure 119: A‒G: Morphology of Lactarius sanguifluus. A‒C: Basidiomata. A & B: MJ-11. C: MJ‒1527a. D‒G: Microscopic structures of Lactarius sanguifluus. D: Basidiospores. E: Pleurocystidia. F: Basidia with a pseudocystidium. G: Pileipellis. 4 µm for D, 7.64 µm for E, 9.09 µm for F, 12.5 µm for D.

285

98 EF685056 Lactarius deliciosus KJ769674 Lactarius deliciosus EF685051 Lactarius deliciosus 66 87 EF685050 Lactarius deliciosus EF685058 Lactarius deliciosus KJ769677 Lactarius rubrilacteus 58 EF685083 Lactarius rubrilacteus 95 EF685085 Lactarius rubrilacteus KC840610 Lactarius aurantiosordidus EF685094 Lactarius aurantiosordidus 87 EU711576 Lactarius sp GAL14544 EF141544 Lactarius akahatsu KF133269 Lactarius akahatsu 99 KJ769671 Lactarius akahatsu

62 AY332549 Lactarius vinosus 78 AY332551 Lactarius vinosus 57 AY332552 Lactarius vinosus Lactarius sanguifluus (MJ-11) 58 87 Lactarius sanguifluus (MJ-1527a) 76 KP115211 Lactarius sanguifluus 86 HG797001 Lactarius sanguifluus

99 AY332556 Lactarius semisanguifluus AY332555 Lactarius semisanguifluus JN613159 Lactarius indigo 90 KJ705193 Lactarius indigo KT163437 Lactarius vividus KT163432 Lactarius vividus KT163431 Lactarius vividus 100 KT163429 Lactarius vividus KT163435 Lactarius vividus KT163433 Lactarius vividus 74 KT163428 Lactarius vividus Lactarius furcatilamellatus DQ679801 Lactarius salmonicolor JF908283 Lactarius salmonicolor AF140265 Lactarius salmonicolor 97 AF140264 Lactarius salmonicolor AF140259 Lactarius salmonicolor AF140258 Lactarius salmonicolor AF249287 Lactarius salmonicolor 50 KJ705196 Lactarius thyinos EF685102 Lactarius thyinos KJ705195 Lactarius thyinos 99 KF133271 Lactarius thyinos KJ769680 Lactarius thyinos EF685126.1 Lactarius laeticolor AF349714 Russula brevipes

0.02 Figure 120. Molecular Phylogenetic analysis of Lactarius sanguifluus and associated taxa inferred from ITS data using the Maximum Likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood (‒1747.6205) is shown.

286

Lactifluus (Pers.) Roussel

Lactifluus versiformis Van de Putte, K. Das & Verbeken, in Van de Putte, Nuytinck, Das

& Verbeken, Fungal Diversity 55: 186 (2012) Fig. 121

Pileus 60‒90 mm across, convex when young, palno-convex with a depressed center to infundibuliform in mature specimens, margin incurved at first then uplifted later on, dark reddish orange (10R 10/6) to grayish reddish orange (10R 6/6) at the margin, deep reddish brown at the disc (10R 2/10), surface dry dull, texture wrinkled at the margin otherwise smooth, cap margin even to slightly crenate in mature specimens, context cream colored to light yellowish pink (5YR 9/4), dry, thicker at the center (3‒4mm) than edges (less than

1mm), texture corky, secreting white colored latex upon cutting in young specimens.

Lamellae adnate to slightly decurrent, narrow, broader at the center (2‒3mm), close to crowded, pale orange yellow (7.5YR 8/4 to 10YR 9/4), turning grayish yellowish brown at maturity (10YR 5/2), appears pruinose, lamellar edge serrulate, lamellulae present, of diverse lengths. Stipe 40‒50 × 10‒13 mm in dimensions, central terete, tapering towards base, same colored as lamellae in the upper part, reddish brown (10R 3/12) downward with a whitish pruinose base, texture firm, interior solid to somewhat corky later on, concolorous with the context of the pileus with somewhat darker margins.

Basidiospores (8.4‒) 8.7‒10 (‒10.1) × (8‒) 8.2‒9.2 (‒9.4) µm, Q = 1‒1.08 (‒1.1), Me =

9.3 × 8.8 µm, Qe = 1.1, globose, with amyloid ornamentation, ornamentation forming complete reticulum, apiculus present. Basidia 35‒45 × 10‒13 µm, clavate to subfusiform,

4-spored, guttulate. Cheilocystidia 20 – 100 × 4 – 8 μm, fusiform, sub-fusiform to sub- clavate with obtuse t acuminate tips, mostly thick walled, walls up to 2 µm thick.

Pleurocystidia 50‒100 (mostly 78) × 8‒10 µm, fusiform to sub‒fusiform with acute

287 apices, causally septate at the bases, thick walled, walls up-to 2.5 µm thick.

Pleuropseudocystidia 45‒50 × 5‒8 μm, slightly emergent, somewhat cylindrical with obtuse apices, sometimes lobed. Pileipellis composed of rounded to elongated cells, 5‒30

× 3‒7 μm, with scattered pileocystidia. Pileocystidia 50‒120 × 3‒5 μm, cylindrical with obtuse apices, margin mostly undulating.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 14th

August, 2014, MM-42 (SWAT000160).

Molecular characterization Fig. 122

The ITS region of MM-42 upon sequencing yielded a 692bp long sequence. After trimming the motifs, a 582bp sequence was left which on BLAST search matched 99% with L. versiformis (JN388961; JN388963; JN388964; JN388967) followed by 95% similarity with L. volemus (Fr.) Kuntze (JQ753937; JQ753905; JQ753906).

For phylogenetic reconstruction, 37 closely matching sequences were downloaded from

GenBank and those mentioned in de Putte et al., (2012). Russula brevipes (AF349714) was used as outgroup taxon. Upon maximum likelihood analysis the Pakistani collection clustered with L. versiformis species confirming the identity.

Comments: this taxon is a new record for Pakistan and appears second from around the globe.

288

Figure 121. A‒G: Morphology of Lactifluus versiformis. A & B: Basidiomata in natural habitat. C‒G: Microscopic structures. C: Basidiospores. D: Basidia. E: Cheilocystidia. F: Pleurocystidia. G: Pileipellis. Bars: 10 μm for C, 6.6 μm for D, 11.1 μm for E, 14.28 μm for F, 16.6 μm for G.

289

JQ753907 Lactarius volemus 68 JQ753909 Lactarius volemus

72 JQ753896 Lactarius volemus JQ753900 Lactarius volemus JQ753905 Lactarius volemus JQ753942 Lactifluus sp. KP-2012a JQ753943 Lactifluus sp KP-2012a 99 JQ753933 Lactifluus sp KP-2012a JQ753932 Lactifluus sp KP-2012a JN388969 Lactifluus leptomerus JN388973 Lactifluus leptomerus 100 JN388968 Lactifluus leptomerus HQ318221 Lactarius pinguis 100 HQ318263 Lactarius pinguis JN388964 Lactifluus versiformis JN388963 Lactifluus versiformis JN388961 Lactifluus versiformis

99 Lactifluus versiformis (MM-42) JN388967 Lactifluus versiformis NR119980 Lactifluus versiformis 65 JN388966 Lactifluus versiformis JN388962 Lactifluus versiformis HQ318226 Lactarius acicularis HQ318270 Lactarius acicularis 100 HQ318277 Lactarius acicularis HQ318236 Lactarius vitellinus HQ318251 Lactarius vitellinus 100 HQ318267 Lactarius vitellinus JN388977 Lactarius corrugis 100 EU598154 Lactarius corrugis HQ318276 Lactarius longipilus 100 HQ318235 Lactarius longipilus KF432958 Lactifluus longipilus 63 HQ318234 Lactarius crocatus HQ318243 Lactarius crocatus 100 HQ318246 Lactarius crocatus AF349714 Russula brevipes

0.02 Figure 122. Molecular Phylogenetic analysis of Lactifluus versifromis and associated taxa from nITS data by using the Maximum Likelihood method based on the Jukes- Cantor model. All positions containing gaps and missing data were eliminated. There were a total of 473 positions in the final dataset.

290

RUSSULA Pers.

Russula amethystina Quél., Compt. Rend. Assoc. Franç. Avancem. Sci. 26 (2): 450 + tab.

4, fig. 13 (1898) [1897] Fig. 123

Pileus 40‒60 mm across, convex, maturing plane, purple with a lighter margin (7.5P 6/12) and darker disc (7.5P 4/12), unchanging upon bruising, surface viscid, shiny, surface smooth to slightly bumpy at the margin, glabrous, interior white, unchanging, breaking with a snap, thicker at the center (3‒4mm) than margin (1‒1.5mm). Lamellae adnexed, some lamellae forking near the stipe, broader at the center (3‒4mm), close, pale yellowish white (7.5Y 9/4), lamellar edge even, lamellulae absent. Stipe 40‒50 × 7‒10 mm in dimension, central, terete, equal to slightly thickening downward, white overall, unchanging upon bruising, somewhat striated, texture breaking with a snap, interior corky, concolorous to the exterior, unchanging.

Basidiospores (7.7‒) 8‒9 (‒9.2) × (6.5‒) 6.7‒7.7 (‒7.9) µm, Q = 1.1‒1.2, Me = 8.6 × 7.5

µm, Qe = 1.1, sub-globose to broadly ellipsoidal, ornamentation amyloid, 1‒1.5 µm, ridges forming incomplete reticulum. Basidia 40‒50 × 10‒15 µm in dimensions, clavate, 4- spored, guttulate, bases simple septate. Cheilocystidia 30‒40 × 7‒9 µm, clavate, narrowly cylindrical, flexuous to mucronate, thin walled, hyaline in KOH solution. Pleurocystidia

50‒70 × 8‒10 µm, narrowly lageniform, lanceolate-clavate with capitate heads, thin walled. Pileipellis terminal hyphae 20‒45 × 4‒6 µm, cylindrical to sub-cylindrical with slightly narrowed terminals.

291

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District,

Gabin Jabba valley, 2400 m a.s.l., on soil under Picea smithiana, Junaid Khan, 30th

August, 2015, GJ-1505 (SWAT000161).

Molecular characterization Fig. 124

The target region comprising ITS1 and ITS2 and 5.8 S of rDNA, gave a 586bp fragment after trimming which was subjected to blast search. The query sequence showed 99% similarity with Russula amethystina (KT953612; KT953613; KT953614; KT953616;

KT953617) and Russula postiana (AF230898; KF850410). Other closely matching (98% similar) taxa included R. lutea (HQ604848) and R. risigallina (KX095001).

The final dataset used for phylogenetic analysis contained 31 sequences with 30 in-group taxa and one outgroup taxon (Lactarius versiformis NR119980). Maximum likelihood was conducted in which the Pakistani collection clustered with Russula amethystine, confirming its identity.

Comments: Russula amethystina was recently reported by Sana (2016) with no other records from Pakistan. This appears to be the second report of this species from Pakistan.

292

Figure 123. A‒C. Morphology of Russula amethystina (GJ-1505). A‒C: Different views of Basidiomata. D‒H: Microscopic structures. D: Basidiospores. E: Basidia. F: Cheilocystidia. G: Pleurocystidia. H: Pileipellis. Bars: 5mm for A ‒ C, 9.5 µm for A, B & D, 5 µm for C, 14.25 µm for E.

293

KT953615.1 Russula amethystina KT953613.1 Russula amethystina KT953617.1 Russula amethystina KT953616.1 Russula amethystina 93 KT953614.1 Russula amethystina KT953612.1 Russula amethystina AF230898.1 Russula postiana Russula amethystina (GJ-1505)

79 JF908682.1 Russula amethystina HQ604848.1 Russula lutea 69 KX537643.1 Russula helios KX095001.1 Russula risigallina 73 KX095012.1 Russula risigallina 62 AY061713.1 Russula risigallina JQ711959.1 Russula decolorans 81 KJ705228.1 Russula decolorans 81 GU234063.1 Russula chamiteae AY245542.2 Russula californiensis 50 JF899571.1 Russula xerampelina KX267626.1 Russula rosacea 90 KP149066.1 Russula paludosa 99 KP149065.1 Russula paludosa 67 KP149064.1 Russula paludosa

100 KF850413.1 Russula roseipes 84 AY061716.1 Russula roseipes KM069449.1 Russula turci

97 HQ604850.1 Russula murrillii EF530935.1 Russula turci 52 KP783462.1 Russula turci 100 KF002747.1 Russula turci NR 119980.1 Lactifluus versiformis

0.02 Figure 124. Molecular Phylogenetic analysis of Russula amethystina and associated taxa inferred from nITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes‒Cantor model. All positions containing gaps and missing data were eliminated. There were a total of 446 positions in the final dataset.

294

Russula kangchenjungae Van de Putte, K. Das & Buyck [as 'khanchanjungae'], Cryptog.

Mycol. 31(4): 381 (2010) Figs. 125 & 126

Pileus 60‒100 mm across, plano-convex with a depressed center and inrolled margin when young, uplifted, somewhat infundibuliform to irregular in mature specimens, grayish reddish brown (2.5 YR 4/2) when young, light brown (7.5YR 5/4) to dark brown (5YR

2/4) in one collection, light yellowish brown (10YR 7/4) in other collection, slowly turning moderate reddish (5R 4/8) upon cutting and decomposition, surface dry, dull, velutinous when young, areolate in mature specimens exposing the light yellowish brown (2.5Y 9/6) background, cap margin undulating and eroded in young specimens, context cream colored, unchanging, thicker at the center (3‒4mm) than margins (less than 1mm), breaking easily, moist to dry. Lamellae decurrent short, narrow, 1‒2mm at the center, narrowing outward, close, cream colored in young stages, pale yellowish with a pinkish tone (2.5Y

9/4) in mature specimens, turning moderate yellow (5Y 7/6) upon reaction with KOH and decomposing strong reddish orange (7.5R 4/8), lamellar edge even, crisped near stipe in some mature specimens, lamellulae present, very frequent, of diverse lengths, dry. Stipe

50‒80 × 10‒17 mm in dimensions, central, terete in young specimens, irregular in mature with more or less lacunose base, clavate (especially in young specimens) to thickened at the base, cream colored when young, light brown (7.5YR 6/4) to light yellowish brown

(10YR 7/4) in mature stages with a cream colored background, bruising moderate yellowish brown (10YR 4/4), decomposing strong reddish orange (7.5R 4/8), turning moderate yellow upon reaction with KOH (5Y 7/6), surface smooth to slightly fibrillose in young specimens, later on cracking giving the it areolae appearance, interior solid, context cream colored, unchanging, breaking with a snap.

295

Basidiospores (6.1‒) 6.6‒7.5 (‒8.6) × (5.8‒) 5.9‒6.8 (‒7.4) µm, Q = (1‒) 1.1‒1.15 (‒1.2),

Me = 7.1 × 6.4 µm, Qe = 1.1, globose to sub-globose, non-amyloid, ornamented, ridges forming incomplete reticulum, apiculus present. Basidia 30‒40 × 7‒9 µm, clavate, 4- sterigmated, sterigmata, sterigmata 2‒3 µm in length. Cheilocystidia 40‒75 × 6‒9 µm, abundant, sub-fusiform to sub-cylindrical with acute, obtuse, mucronate to capitate apices, contents oily in Congo red and Melzer’s reagent. Pileipellis hyphae 5‒9 µm in diameter, with obtuse to somewhat pointed end, hyaline in KOH, staining in KOH. Pileocystidia

100‒130 × 5‒7 µm, elongated, content similar to cheilocystidia. Stipitipellis hyphae 3‒6

µm in diameter, caulocystidia scattered, 70‒100‒150 × 10‒12 µm in dimension, content resemble the contents of cheilocystidia.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2250 m a.s.l., on soil under Abies pindrow, Junaid

Khan, 09th August, 2015, MM-1634 (SWAT000162). Same locality, 2050 m a.s.l.

Junaid Khan, 08th July, 2016, MM-1607 (LAH31421).

Molecular characterization Fig. 127

The ITS region of MM-1634 and MM-1607 yielded a 707bp sequence, which showed 99% similarity with voucher specimens of Russula kangchenjungae (KR364129) from India, followed by R. subnigricans (AB291738) with 92% similarity. Other matching taxa included R. eccentric Peck., (KC699768; KC699769; KC699778) with 90% similarity, R. nigricans (JQ711972; JQ711892) and R. albonigra (Krombh.) Fr. (DQ422029; KF306042) with 88% similarity.

296

Molecular phylogenetic analysis involved 31 nucleotide sequences with Lactarius pterosporus Romagn (AY331013) used as outgroup. Pakistani collections (MM-1634 and

MM-1607) clustered within the section compacta with Russula kangchenjungae with a strong bootstrap value of 100%, supporting conspecificity of Pakistani collection with

Indian collection.

Comments: There are no previous records of Russula kangchenjungae from Pakistan and is being reported for the first time from northern areas of Khyber Pakhtunkhwa Pakistan.

297

Figure 125. A‒D. Basidiomata of Russula kangchenjungae. A: (MM-1607) Young fruiting body showing velvety pileus and clavate basal part. B: collection MM‒1607 in natural habitat. C: view of the context and areolate pileus (MM-1534). D: view of fruiting body showing turning dark reddish upon decomposition of the stipe. Bars: 6 mm for A ‒ D.

298

Figure 126. A‒E: Microscopic structures of Russula kangchenjungae (MM-1644). A: Basidiospores. B: Pileocystidia. C: Cheilocystidia. D: Basidia. E: Caulocystidia. Bars: 4 µm for A, C & D, 10 µm for B & E.

299

KC699769 Russula eccentrica KC699773 Russula eccentrica

99 KC699768 Russula eccentrica KC699780 Russula eccentrica 99 KC699779 Russula eccentrica KC699771 Russula eccentrica 84 KP033478 Russula eccentrica KR364129 Russula khanchanjungae 87 100 Russula kangchenjungae (MM-1634) 77 Russula kangchenjungae (MM-1607) 52 AB291738 Russula subnigricans KP033481 Russula polyphylla 87 100 DQ422027 Russula polyphylla KF306037 Russula cantharellicola 100 KF306036 Russula cantharellicola

100 KT933979 Russula dissimulans EU598198 Russula cf nigricans 99 KC699756 Russula nigricans 70 KC952682 Fagus sylvatica EF126735 Russula subnigricans

100 EF534351 Russula subnigricans 98 AB291732 Russula subnigricans JQ888194 Russula adusta 86 50 JF908669 Russula adusta 100 FJ845430 Russula densifolia AY061652 Russula adusta 85 DQ422029 Russula albonigra JF834364 Russula aff albonigra 100 65 KF306040 Russula albonigra 88 JF834355 Russula albonigra AY331013 Lactarius pterosporus

0.02 Figure 127. Molecular Phylogenetic analysis of Russula kangchenjungae and associated taxa using nr ITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes‒Cantor model. All positions containing gaps and missing data were eliminated.

300

Russula olivacea (Schaeff.) Fr., Epicr. syst. mycol. (Upsaliae): 356 (1838) [1836‒1838]

Figs. 128 & 129

Pileus 50‒70 mm across, convex to pulvinate in young specimens, plano-convex to umbilicate in mature specimens with a depressed center, moderate greenish yellow (10Y

7/6) at margin, strong greenish yellow (7.5Y 7/10) at the disc, surface dry, dull to shiny, texture smooth with an even to slightly crenate cap margin, context white, dry, thicker at the disc (3‒4mm) than margin (not more than 1mm), unchanging upon bruising, turning light pink colored upon reaction with KOH. Lamellae adnexed, narrow to moderately broad (2‒3 mm at the center), close, cream colored to pale yellow (5Y 9/4), lamellar edge even, lamellulae absent. Stipe 60‒100 × 15‒30 mm in dimensions, central, terete to irregular compressed in some specimens, tapering towards base, white, turning moderate reddish orange (10R 5/8) upon bruising, unchanging or slightly getting light olivaceous with KOH, texture chalky, interior concolorous to the exterior, unchanging.

Basidiospores (7.4‒) 8.1‒9.7 (‒11) × (6.8‒) 7‒8.6 (‒9.2) µm, Q = (1‒) 1.1‒1.2 (‒1.3), Me

= 9 × 7.8 µm, Qe = 1.2, sub-globose to broadly ellipsoid, ornamented with spines, ridges forming incomplete reticulum, spines up‒to 1 µm long, amyloid, with prominent apiculus.

Basidia 40‒50 × 12‒14 µm, clavate, 4-spored, guttuate in KOH solution. Cheilocystidia

40‒55 × 6‒10 µm, cylindrical with round, acute to sub-capitate apices, some centrally septate, thin walled to thick walled, hyaline in KOH. Pleurocystidia 50‒80 × 7‒13 µm, narrowly fusiform to cylindrical with acute apices, thin walled to thick walled (up-to 1µm), hyaline to light golden in KOH.

301

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Marghazar, 1100 m a.s.l., under Quercus spp, Junaid Khan, 12th August,

2016, MZ-1602 (SWAT000163).

Comments: this is the first record of this taxon from Pakistan.

Molecular characterization Fig. 130

The ITS region of the nDNA of MZ-1602 yielded a 920bp long sequence which upon

BLAST search matched 97% with Russula olivacea (EU284014; AF418634; AY061699).

Maximum likelihood analysis was carried out MEGA6 and selecting Jukes and Cantor model as the best model for analysis. The analysis involved 49 sequences with 48 in-group taxa and Lactarius salmonicolor as an outgroup taxon. The analysis clustered the Pakistani collection of R. olivacea with other species of R. olivacea (EU284012; EU284014;

AF418634; AY061699; JX425373) confirming the identity as R. olivacea.

Comments: based on morphological and molecular data, this olivaceous collection is being reported here as Russual olivacea, representing the first record for Pakistani mycobiota..

302

Figure 128. A –D. Basidiomata of Russula olivacea (MZ-1602). A: collection MZ-1602. B: reaction of pileus with KOH. C: reaction of KOH on stipe. Bars: 10 mm for A & D. 5mm for B & C.

303

Figure 123. A‒E. Microscopic features of Russula olivacea. A: Basidiospores the one showing incomplete reticulum. B: Pileipellis. C: Range of cheilocystidia. D: Range of pleurocystidia. E: Basidia. Bars: 5 µm for A, 14.2 µm for B, 8.8 µm for C, 12.3 µm for D, 11.9 µm for E.

304

KJ748435 Russula xerampelina EF530944 Russula favrei KJ748432 Russula xerampelina KP454007 Russula xerampelina FJ627037 Russula favrei KC581298 Russula favrei EU597074 Russula vesca KP966376 Russula katarinae KP966377 Russula katarinae KF810135 Russula subsulphurea KT933955 Russula rugulosa KU205269 Russula nitida KX095023 Russula sphagnophila KX095016 Russula sphagnophila 73 KU205349 Russula nitida KF850409 Russula sphagnophila KU205285 Russula faginea JX436949 Russula pascua JX436948 Russula pascua JX436950 Russula pascua JX436944 Russula pascua KT934001 Russula nitida KU205277 Russula nuoljae LN714599 Russula pascua JX436958 Russula pascua JX436951 Russula pascua JX436946 Russula pascua KU205289 Russula faginea AY245542 Russula californiensis AY194601 Russula decolorans 99 KT933992 Russula decolorans KP149062 Russula decolorans AY061670 Russula decolorans JF908700 Russula quercilicis GU234063 Russula chamiteae 64 DQ367913 Russula decolorans FJ845428 Russula vinosa KF306039 Russula eccentrica KU356732 Russula olivacea JX425373 Russula olivacea Russula olivacea 94 Russula olivacea Russula olivacea AY061699 Russula olivacea 90 EU284012 Russula olivacea EU284014 Russula olivacea AF418634 Russula olivacea KT875105 Russula emetica AF140265 Lactarius salmonicolor

0.02 Figure 130. Molecular Phylogenetic analysis of Russula olivacea inferred from nITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes-Cantor model.

305

Russula postiana Romell, Ark. Bot. 11(no. 3): 5 (1911) Figs. 131 & 132

Pileus 30‒70 mm across, convex to hemispherical, turning plane with age, pale greenish yellow (10Y 9/4) to dark greenish yellow (10Y 6/6) at the margins, light yellowish brown

(7.5YR 7/4) at the disc, turning deep yellowish brown (10YR 3/8) later on, dry or slightly viscid especially in the young stages, shiny, smooth or slightly bumpy in some specimens, cap margin even or slightly sulcate, cap cuticle peeling up to ¾ of the total diameter, context white, unchanging upon cutting or bruising, thicker at the center (5‒7 mm) than margin (1‒2 mm), chalky, cap margin short striate. Lamellae adnexed, broader at the center (3‒4 mm), thick (0.8 mm), close to sub‒distant, lamellar edge even, blunt, lamellulae absent, cream colored to light yellow green (5GY 9/4), unchanging. Stipe 90‒120 × 15‒

25 mm in dimensions, central, terete, clavate to sub-clavate, smooth to slightly striate, white, context concolorous with the exterior, texture chalky, unchanging.

Basidiospores (7.4‒) 7.6‒9 (‒9.2) × (6.7‒) 6.8‒7.7 (‒7.8) µm, Q = 1.1‒1.2, Me = 8.5 × 7.4

µm, Qe = 1.1, sub-globose to broadly ellipsoidal, warty, warts 1‒1.5 µm, amyloid, with ridges forming incomplete reticulum. Basidia 45‒55 × 10‒15 µm in dimensions, clavate, guttulate, 4-spored, 5‒7 µm long. Cheilocystidia 30‒45 × 5‒9 µm, clavate, narrowly cylindrical, flexuous to mucronate, thin walled, hyaline in KOH. Pleurocystidia 40‒70 ×

8‒10 µm, narrowly lageniform, lanceolate to clavate with capitate heads, thin walled.

Pileipellis terminal hyphae 15‒45 × 4‒6 µm, cylindrical to sub-cylindrical with slightly narrowed terminals. Stipitipellis composed of parallel simple septate hyphae, 4‒6 µm in diameter, caulocystidia scattered, cylindrical with sub-capitate heads, 30‒40 µm in length.

306

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Gabin Jabba valley, 2250 m a.s.l., under Picea smithiana, Junaid Khan,

30th August, 2015, GJ-1512 (SWAT000164). Same locality, 2200 m a.s.l. Junaid

Khan, 31st August, 2015, GJ-1510 (LAH31422).

Molecular characterization Fig. 133

The target region of GJ-1512 gave approximately 591bp fragment after trimming and editing. Upon BLAST search at NCBI and UNITE database, the Pakistani sequence of present Russula collection matches 98% with R. postiana (AF230898; UDB011312;

UDB015977) R. lutea (KF679818; HQ604848).

The analysis involved 26 nucleotide sequences. Molecular phylogenetic analysis places

Pakistani collection of Russula species (GJ-1512) with Russula postiana. The present collection also conforms morphologically to R. postiana and is treated as R. postiana herein.

Comments: There are no previous records of this species from Pakistan and is being reported as new record for Pakistani mycobiota.

307

Figure 131. A‒D: Basidiomata of Russula postiana. A: View of GJ‒1512 showing full basidiome and sticky pileus. B: View of the lamellae and somewhat striate stipe (GJ‒1512). C: GJ‒1510 in natural habitat. D: View of the peeled off cuticle, context and striate margin. Bars: 10 mm for A‒D.

308

Figure 132. A‒F. Microscopic features of Russula postiana. A: Basidiospores. B: Basidia. C: Cheilocystidia. D: Pleurocystidia. E: Caulocystidia. F: Pilocystidia. Bars: 6.53 µm for A, 8.7 µm for B & D, 5.75 µm for C, 10.8 µm for E, 11.1 µm for F.

309

UDB011316 Russula Postiana UDB016074 Russula postiana

98 UDB011312 Russula postiana KF679818 Russula lutea AF230898 Russula postiana

93 Russula postiana (GJ-1512) AY061713 Russula risigallina 100 DQ422022 Russula risigallina 63 HQ604848 Russula lutea 64 KX537643 Russula helios EF530935 Russula turci 99 KP783462 Russula turci

58 75 GU373485 Russula paludosa 99 GU373486 Russula paludosa

99 KP901249 Russula paludosa DQ367916 Russula xerampelina 99 JF899571 Russula xerampelina DQ367913 Russula decolorans 81 94 FJ845432 Russula decolorans HM189839 Russula integra HM189950 Russula velenovskyi 87 HM189949 Russula velenovskyi 100 HM189951 Russula velenovskyi KT968586.1 EU598157 Russula compacta NR119980 Lactifluus versiformis

0.02 Figure 133. Molecular Phylogenetic analysis of Russula postiana and associated taxa. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes‒Cantor model. The tree with the highest log likelihood (‒2541.3060) is shown. There were a total of 557 positions in the final dataset.

310

Russula similis nom.prov. Fig. 134

Etymology: the epithet “similis” refers to similar, based on the close resemblance of this species with Russula violeipes

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2050 m a.s.l., on soil in Mixed Quercus-coniferous forest, Junaid Khan, 01st July, 2016,

MJ-1605 (SWAT000165).

Diagnosis: pileus medium sized, parabolic to pulviate, deep purplish red to dark

purplish red, moderate red to cream colored clavate stipe, globose to sub-globose

basidiospores with warts forming incomplete reticulum.

Pileus 40‒60 mm across, parabolic to pulvinate when young, umbilicate with an uplifted margin when mature, color very deep purplish red (10RP 1/8) in young specimens, dark purplish red (7.5RP 3/4) in mature specimens, surface dry, dull to shiny, texture smooth, glabrous to slightly granular, context firm in young specimens, corky in mature specimens, purplish (7.5P 4/6) beneath the cuticle in young specimens otherwise creamy‒whitish overall, unchanging. Lamellae adnexed, broad at the center (3‒4mm) than at the margin

(1‒2mm) at the margin, close to crowded, white to creamy white in young stages, light orange yellow (10YR 9/8) when mature, lamellar edge even, obtuse, lamellulae absent.

Stipe 40‒50 × 15‒25 mm in dimensions, central, terete, clavate, color moderate red (5R

4/8) above, cream colored at the extreme base, drying white, smooth in most of the specimens, striate in age specimens, interior corky, concolorous with the exterior or somewhat whitish, unchanging.

311

Basidiospores (7.9‒) 8‒10.3 (‒10.6) × (7.1‒) 7.6‒9.6 (‒9.9) µm, Q = 1 - 1.1, Me = 9.1 ×

8.4 µm, Qe = 1.1, globose to sub-globose, ornamentation amyloid, isolated warts forming incomplete reticulum. Basidia 15‒20 × 4‒6 µm, clavate, 4-spored, guttulatae.

Cheilocystidia 50‒60 × 6‒20µm, cylindrical with round to acute apices, rarely clavate or mucronate. Pleurocystidia 30‒50 × 5‒8 µm, narrowly lanceolate-clavate, thin walled.

Pileipellis with numerous pileocystidia, 40‒60 × 3-5 µm, elongated, with cylindrical- narrowly clavate terminals

ADDITIONAL MATERIAL EXAMINED: PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2250 m a.s.l., under Picea

smithiana, Junaid Khan, 30th August, 2016, MJ-16155 (LAH31440).

Molecular characterization Fig. 135

The three generated sequence from MJ-1605 were 709bp long and matching 94% with

Russula violeipes (KF361797; KF361783; KF361784) followed by R. nitida (EU598164) and R. mariae (EU819426; EU598199).

The maximum likelihood analysis involved 28 nucleotide sequences containing 27 in- group sequences and one outgroup sequence (Lactarius pterosporus AY331013). The

Pakistani collection formed a separate clade with a strong bootstrap value of 96% close to

R. violeipes, supporting unique nature.

312

Figure 134: A‒H: Macroscopic and Microscopic structures of Russula similis (MJ- 1606). A‒C: Basidiomata in natural habitat. D: Basidiospores. E: Pileocystidia. F: Basidia. G: Cheilocystidia. H: Pleurocystidia. Bars: 10mm for A‒C, 10 µm for D, 2.5 µm for E, 4 µm for F, 8 µm for G & H.

313

EU819426 Russula mariae 99 EU598199 Russula mariae EU598164 Russula nitida AB459514 Russula cf rosacea AY061655 Russula amoenicolor KT824241 Russula intervenosa EU019934 Russula variispora EU019938 Russula rostraticystidia KF361784 Russula violeipes

96 KF361797 Russula violeipes 99 KF361783 Russula violeipes Pakistani collection Pakistani collection 100 Pakistani collection

100 DQ974758 Russula cyanoxantha 57 KF937362 Russula cyanoxantha KT933965 Russula redolens

100 DQ422012 Russula ochrospora JF908701 Russula ochrospora 72 JF908698 Russula anatina 99 DQ422007 Russula parazurea KM373243 Russula crustosa 91 KM373269 Russula crustosa EU598194 Russula crustosa EU819437 Russula virescens

99 EU598193 Russula crustosa 64 KM085411 Russula virescens AY331013 Lactarius pterosporus outgroup

0.01 Figure135. Molecular Phylogenetic analysis of Russula similis inferred form nrITS data using the Maximum Likelihood method based on the Jukes‒Cantor model. The tree with the highest log likelihood (‒1888.0496) is shown. All positions containing gaps and missing data were eliminated. There were a total of 418 positions in the final dataset.

314

Family STROPHARIACEAE

a) Agrocybe

1. A. elatella

b) Pholiota

1. P. microcarpa nom.prov.

315

AGROCYBE Fayod

Agrocybe elatella (P. Karst.) Vesterh., Nordic Jl Bot. 9(3): 317 (1989) Fig. 136

Pileus 30‒40 mm across, convex at first turning plano-convex with age, shape of margin straight, brown with a slightly darker center (5YR 5/8), smooth, hygrophanous, dry dull, context thin, 1mm at the edge, 2‒2.5 mm at the center, brown, not changing on cutting or bruising. Lamellae adnate, sub-distant to close, concolorous with the cap or slightly getting darker with age, Lamellulae frequent, extending up to the middle. Stipe 60‒80 × 2‒4 mm, central terete, equal or slightly thickening downward, brownish (5YR 6/8), fibrillose, annulus present, superior, pendent, evanescent.

Basidiospores (8.6‒) 8.6‒10 (‒11.1) × 5.7‒6.7 (‒6.8) µm, Q = 1.4‒1.6 (‒1.7), Me = 9.6 ×

6.3 µm, Qe = 1.5, ellipsoidal, smooth, with apiculus, germ pore present. Basidia club shaped, 4-spored, 20‒25 × 8‒10 µm, basidioles club shaped, numerous. Cheilocystidia clavate to broadly clavate, often in clusters, 15‒20 × 10‒18 µm. Pleurocystidia lageinform to narrowly utriforme, 40‒55 × 18‒25 µm. Pileipellis irregular epithelium.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, swat

DISTRICT, Ingaro Dherai Village, 1000m a.s.l., in grassy land, Junaid Khan, 12th

April, 2015, ING-17 (SWAT000166).

Comments: Agrocybe elatella is an addition to the fungi of Pakistan.

316

Figure 136: A‒H: Morphology of Agrocybe elatella (ING-17). A‒C: different views of Basidiomata. D‒G: Anatomical features of Agrocybe elatella. D: Basidiospores. E: Basidia. F: Cheilocystidia. G: Pleurocystidia. H: Pileipellis. Bars: 10 mm for A‒C, 10 µm for D, 15 µm for E‒H.

317

PHOLIOTA (Fr.) P. Kumm.

Pholiota microcarpa nom.prov. Figs. 137 & 138

Etymology: the epithet “microcarpa” refers to the small fruiting body, characterizing this species.

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Takhta Band village,

950 m a.s.l., in humus rich sandy soil with nearby Populus nigra and Salix babylonica tress, Junaid Khan, 04th November, 2014, TB-017 (Holotype) (SWAT000157).

Diagnosis: fruiting body small (≤30), pileus conical to broadly convex with a slight

umbo, brown, squamose, lamellae uncinated, yellowish cream colored, stipe scaly-

fibrillose, basidiospores ellipsoidal, cheilocystidia none, pleurocystidia present.

Pileus 20–25 mm across, conical to broadly convex when young, plano–convex to saucer shaped with a small umbo by maturity, light brown (7.5Y 7/4) with a dark brown (5YR

2/4) disc, surface dry, squamose, scales appressed, radiating from the disc, shape of margin appendiculate, interior solid, 2–2.5 in the central region, narrowing towards margins with less than 1mm thickness, flesh whitish with a pinkish tone, unchanging upon cutting.

Lamellae uncinate with a small decurrent tooth, narrow, not more than 2.5 mm at the broadest part, close, yellowish cream colored (7.5Y 9/4), lamellar edge lightly serrulate, lamellulae frequent, of diverse lengths. Stipe 30–35 × 3–5 mm, central, terete, equal, straight to lightly flexuous in some specimens, scaly–fibrillose except at the extreme top where longitudinally striated, scales same colored as pileus on a whitish to cream colored background, base covered with white rhizomorph, interior solid except in the lower half

318 where centrally hollow, brownish white, turning deep yellowish brown (10YR 3/8) at the extreme base upon cutting, unchanging elsewhere. Annulus absent in mature specimens.

Basidiospores (6.2–) 6.5–7.2 (–7.3) × (3.9–) 4–4.5 (–4.7) µm, Q = (1.4–) 1.5–1.7 (–1.9),

Me = 6.9 × 4.3 µm, Qe = 1.6, ellipsoidal with somewhat rounded ends, mostly with guttules, apiculus present, less prominent, thick walled (0.3–0.4 µm), light golden brown in KOH solution. Basidia 15‒20 × 5‒7 µm, clavate, 4-sterigmated, sterigmata ≤4.5 µm.

Cheilocystidia none observed. Pleurocystidia 20‒30 × 5‒6 µm, narrowly lageniform to narrowly utriform with ventricose necks, some with capitae heads, mostly in clusters.

Pileipellis a cutis, hyphae clamped, measuring 5‒7 µm, some smooth walled others roughened, mostly hyaline in KOH with scattered golden brown hyphae. Pileocystidia 30‒

65 × 13‒25 µm, clavate, ovoid to elongated, mostly in chains, some deeply granular inside, others not. Stipitipellis hyphae clamped, frequently branching, elongated measuring 4.5–

7 µm in diameter, hyaline to light golden brown in KOH solution, smooth walled.

Molecular characterization Fig. 139

The ITS region of TB-017 upon amplification with fungal specific primers, ITS1F and

ITS4 resulted in a 620bp sequence. Upon BLAST search the query sequence matched 94% with Pholiota gummosa (Lasch) Singer (JF908580) and P. caespitosa A.H. Sm. & Hesler

(NR119908). It also showed 93% similarity with P. adiposa (Batsch) P. Kumm.

(AB470888) and P. burkei A.H. Sm. & Hesler (HQ222016). All the closely matching taxa belongs to the sub-genus Pholiota of the genus Pholiota and accordingly sequences from the same sub-genus were downloaded for further analysis.

319

For phylogenetic reconstruction, 23 nucleotide sequences were used in the maximum likelihood analysis with 22 in-group taxa and one outgroup taxon (Stropharia aeruginosa

(Curtis) Quél. AB985291). The sequences of TB-017 formed a sister clade with P. adiposa

(AB470888) and P. gummosa (JF908580) with a strong bootstrap value of 96%, confirming its unique identity. Sequences of P. gummosa and P. adiposa occupied two different positions and the sequences of P. adiposa (AB470888), P. gummosa (JF908580),

P. burkei (HQ222016) and P. caespitosa (NR119908) needs confirmation.

Comments: Pholiota microcarpa is proposed as new to science supported by molecular and morphological evidences.

320

Figure 137. A‒D. Different views of Basidiomata of Pholiota microcarpa (Holotype). For size refer to the scale.

321

Figure 138. A–F. Microscopic structures of Pholiota microcarpa (TB-017). A: Basidioapores. B: Pleurocystidia. C: Basidia. D: Pileocystidia. E: Pileipellis. F: Stipitipellis. Bars: 4.98 µm for A, 5.4 µm for B & C, 10.65 µm for D, 15.75 for E, 21 µm for F.

322

AB470888.1 Pholiota adiposa

41 JF908580.1 Pholiota gummosa

Pholiota microcarpa 93 96 Pholiota microcarpa

NR 119908.1 Pholiota caespitosa 35 83 HQ222016.1 Pholiota burkei

99 KC122896.1 Pholiota terrestris

49 KU363737.1 Pholiota terrestris

JF908581.1 Pholiota gummosa 98 JF908590.1 Pholiota jahnii voucher 16840

KC581317.1 Pholiota limonella subgenus Pholiota 83 JF908584.1 Pholiota cerifera 89 FJ596875.1 Pholiota aurivella

65 FJ596881.1 Pholiota aurivella 99 94 KM496470.1 Pholiota limonella

KJ609166.1 Pholiota abietis 56

35 JQ283956.1 Pholiota adiposa 54 JQ283955.1 Pholiota adiposa

FJ810174.1 Pholiota nameko

100 AY251304.1 Pholiota nameko

FJ596876.1 Pholiota squarrosoides

100 JF908591.1 Pholiota squarrosoides

AB985291.1 Stropharia aeruginosa Outgroup

0.02 Figure 139. Molecular Phylogenetic analysis of Pholiota microcarpa and associated taxa using nITS data by Maximum Likelihood method. There were a total of 525 positions in the final dataset.

323

TRICHOLOMATACEAE

a) Lepista

1. L. nuda

2. L. panaeolus

b) Leucopaxillus

1. L. giganteus

c) Melanoleuca

1. M. cinereifolia

d) Tricholoma

1. T. conicosulphureum nom.prov.

e) Tricholomopsis

1. T. robustus nom.prov

2. T. umbonatus nom.prov

324

LEPISTA (Fr.) W.G. Sm.

Lepista nuda (Bull.) Cooke, Handb. Brit. Fungi 1: 192 (1871) Fig. 140

Pileus 40‒60 mm across, plano-convex to plane with an inrolled margin and slight central umbo, purple to light purple (10PB 7/4) with a purplish brown (7.5RP 7/4) disc, surface dry, shiny, cap margin even, surface smooth to slightly pubescent, interior concolorous to the exterior, moist, thin, 1‒3 mm at the center, less than 1mm at the margins, unchanging upon cutting. Lamellae sinuate to slightly decurrent in some specimens, broad, 3‒4 mm near the stipe, close, darker than the pileus, purple (10PB 4/6), lamellar edge even to slightly serrulate, acute, lamellulae present, of diverse lengths. Stipe 50‒70 × 5‒10 mm in dimension, central, terete, equal to slightly thickening downward, same colored as lamellae, surface fibrillose to finely floccose, attached to the substrate through lilac to whitish mycelium, texture firm, interior solid, context concolorous to the exterior, unchanging.

Basidiospores (5.9‒) 6.4‒8.7 (‒8.8) × (4.3‒) 4.4‒5.4 (‒6.2) µm, Q = (1.3‒) 1.35‒1.68 (‒

1.7), Me = 7.4 × 4.9 µm, Qe = 1.5, ellipsoidal, lightly roughened, with apiculus, hyaline in

KOH solution. Basidia 35‒40 × 7‒8 µm, narrowly clavate, 4-sterigmated, guttlate, clamped at the bases. Cystidia none observed. Pileipellis a cutis, hyphae 3‒6 µm in diameter, clamped at septa.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2250 m a.s.l., under Pinus wallichiana, 21st July, 2015,

MM-1507 (SWAT000168). PAKISTAN, KHYBER PAKHTUNKHWA, Swat

325

District, Gabin Jabba valley, 2300 m a.s.l., under Pinus wallichiana, 01st August,

2016, GJ-1650. (LAH31423).

Comments: Lepsita nuda is previously reported from Kaghan valley (Sultana et al., 2011) and Mansehra (Fiaz, 2013) with no records from the present study area.

326

Figure 140. A–E. Macro and Micro-morphological characters of Lepista nuda. A: View of the lamellae. B: View of the pileus surface. C: Basidiospores. D: Basidia. E: Pileipelis. Bars: 10 mm for A & B, 4.7 µm for C, 12.57 µm for D, 22 µm for E.

327

Lepista panaeolus (Fr.) P. Karst., Bidr. Känn. Finl. Nat. Folk 32: 481 (1879) Figs. 141 &

142

Pileus 50‒80 mm across, pulvinate in shape, cream colored, bruising grayish, surface dry, dull, smooth to lightly rugulose, glabrous, rarely with scattered hairs, cap margin even, interior solid, context whitish to cream colored, unchanging, thicker at the disc (3‒5mm), narrow at the margins (≤ 1mm). Lamellae short decurrent, narrow (1mm at the thickest part), crowded, cream colored at first, turning pinkish (7.5YR 7/4) later on and finally maturing brownish (5YR 5/8), forking near the stipe, lamellar edge even, lamellulae frequent, of diverse lengths. Stipe 40‒75 × 10‒20 mm, central, round to bumpy, thickening downward, color pinkish (7.5YR 7/4), to cream colored, surface pruinose-fibrillose, interior stuffed in the upper part, hollow below, context concolorous with the exterior.

Basidiospores (5.3‒) 5.5‒5.9 (‒6.4) × 3.4‒3.6 (‒3.8) µm, Q = 1.6‒1.75 (‒1.8), Me = 5.8 ×

3.5 µm, Qe = 1.7, ellipsoidal, hyaline in KOH solution, with apiculus, smooth to lightly roughened. Basidia 20‒30 × 6‒8 µm, clavate, mostly 4-sterigmated, rarely with two, with basal clamps, contents granular. Cystidia 20‒30 × 4‒7 µm, cylindrical, narrowly utriform or mucronate. Pileipellis a cutis, hyphae 7‒8 µm, clamped, terminal elements cylindrical.

MATERIALS STUDIED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 21st

August, 2014, MJ-88 (SWAT000169). Same location, Junaid Khan, 28th August,

2016, MJ-16115 (LAH31424).

328

Molecular Characterization Fig. 143

The 696bp long fragment generated by amplification of the ITS region of MJ-88 and MJ-

1615 matched 99% Lepista panaeolus (as L. panaeola) (KP195725; KJ681007.1), L. paxilloides (Esteve-Rav. & M. Villarreal) Consiglio & Contu, (KJ681012; KJ681013), L. irina (Fr.) H.E. Bigelow (HM237136; FJ810142), L. subconnexa (Murrill) Harmaja

(KJ681021; KJ681023) and L. caespitosa (Bres.) Singer (KJ680998; KJ680997).

Based on the BLAST result, 42 closely matching nucleotide sequences were downloaded and used in the Maximum Likelihood analysis using MEGA6 software. The aligned dataset was composed of 691 sites out of which 527 were conserved, 151 variable with 106 parsimony informative and 43 singleton. After removal of ambiguous position there were total of 465 position which were used in the final analysis.

The analysis clustered the included sequences into two clades viz. Lepista and Collybia and allies, which are in conformity with the findings of Alvarado et al., (2015). The sequences of MJ-88 and MJ-1615 and the closely matching sequences of L. subconnexa,

L. paxilloides, L. caespitosa and L. irina clustered together. The name L. paneolous is adopted for MJ-88 and MJ-1615 and L. subconnexa, L. paxilloides and L. caespitosa are treated here as synonyms of L. following Alvarado et al., (2015). These taxa were once treated as different based on habitat and lamellar attachment (Bon, 1997) which are now considered as of little taxonomic value. The sequences of L. irina (HM237136; FJ810142) are also doubtful as mentioned by Alvarado et al., (2015) and should be considered as L. panaeolus.

329

Comments: Lepista panaeolus is an addition to the fungi of Pakistan with no previous records. This species should be considered as a rare find in the study area with only two collections from a common site in Malam Jabba in two different years.

Figure 141. A – F. Basidiomes of different collections of Lepista panaeolus A: Natural habitat (MJ-88) showing lightly rugulose nature of the pileus and shape. B: Basidiome showing habit and complete fruiting body. C: View of the context. D: View of the pileus context and narrow lamellae. E & F: View of the different coloration of the lamellae.

330

Figure 142. A – D. Microscopic structures of Lepista panaeolus. A: Basidiospores. B: Cystidia. C: Basidia. D: Pileipellis. Bars: 4.1 µm for A, 5.62 µm for B, 5.23 µm for C, 13.1 µm for D.

331

Figure 143. Molecular Phylogenetic analysis of Lepista panaeolus and associated taxa by Maximum Likelihood method using nITS data. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes-Cantor model. The tree with the highest log likelihood (-1661.8815) is shown.

332

LEUCOPAXILLUS Boursier

Leucopaxillus giganteus (Sowerby) Singer, Schweiz. Z. Pilzk. 17: 14 (1939) Fig. 144

Pileus 70‒150 mm across, saucer shaped with an inrolled margin in young stages, the margin may be upturned in mature specimens and the edge then even, light brown gray

(10Y 8/2) to greyish brown (5GY 9/2), background creamy, turn darker upon bruising, surface moist, shiny, texture smooth, cap margin wavy, interior solid, context white, moist, thicker at the center (5‒7 mm) than margin (not more than 1mm), unchanging upon cutting.

Lamellae slightly decurrent, narrow, 1‒2 mm at the center, close to crowded, cream colored in young stages then with a pinkish tone later on, lamellulae present, mostly in one tier. Stipe 80‒100 × 20‒35 mm in dimensions, central, terete, equal to slightly tapering at the base, punctate in the upper portion, fibrillose below, fibrils greyish, background cream colored, white at the base with white mycelium, texture spongy, interior solid, white, unchanging.

Basidiospores (5.7‒) 5.71‒7.7 × 4‒4.1 µm, Q = (1.4‒) 1.43‒1.86 (‒1.9), Me = 6.5 × 4 µm,

Qe = 1.6, ellipsoid, smooth, thin walled, hyaline in KOH solution, lightly amyloid. Basidia

20‒25 × 6‒8 µm, clavate, mostly 4-spored, rarely 2-spored, sterigmata 3‒4 µm long, guttulate in Congo red, clamped at bases. Cystidia cylindrical with somewhat irregular walls and apices. Pileipellis a trichoderm of clamped hyphae, clamps very prominent, terminal elemsts clavte-cylindrical, 3‒5 µm in diameter. Stipitipellis hyphae 5‒7 µm wide, ending in cylindrical-spoon shaped terminals, septate, clamped, clamps less prominent compared to the pileipellis hyphae.

333

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Gabin Jabba valley, 2250 m a.s.l., under Pinus wallichiana, Junaid Khan,

23rd August, 2014, MM-76 (SWAT000171). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Gabin Jabba valley, 2200 m a.s.l., under Pinus

wallichiana, Junaid Khan, 08th July, 2016, GJ-1605, (LAH31325). PAKISTAN,

KHYBER PAKHTUNKHWA, Swat District, Kalam valley, 2300 m. a.s.l., under

Cedrus deodara, Junaid Khan, 08th September, 2016, KM-06, (LAH31326).

Molecular Characterization Fig. 145

A 670bp fragment was generated by amplifying the ITS region of MM-76 and GJ-1605 using fungal specific primers of ITS1F and ITS4. The query sequences showed 99% similarity with Leucopaxillus giganteus (KP453698; JQ639151; KP453711).

A total of 28 nucleotide sequences were used in the maximum likelihood phylogenetic analysis. The Pakistani sequences clustered with voucher sequences of L. giganteus, confirming the identity.

Comments: There are no previous records of this taxon from Pakistan and is an addition to the fungi of Pakistan.

334

Figure 144. A‒G. Morphology of Leucopaxillus giganteus (MM-76). A & B. Basidiomes growing in natural habitat. C–G. Microscopic structures. C: Basidiospores. D: Cystidia. E: Basidia. F: Pileipellis. G: Stipitipellis. Bars: 8.7 µm for C & D, 7.56 µm for E, 19.1 µm for F, 11.5 µm for F.

335

KJ417280.1 Leucopaxillus amarus

99 KJ417278.1 Leucopaxillus amarus KJ417293.1 Leucopaxillus laterarius KJ417294.1 Leucopaxillus laterarius KJ417286.1 Leucopaxillus eucalyptorum 99 KJ417285.1 Leucopaxillus eucalyptorum KJ417289.1 Leucopaxillus gracillimus 100 KJ417288.1 Leucopaxillus gracillimus KJ417295.1 Leucopaxillus lilacinus KJ417296.1 Leucopaxillus paradoxus 56 JQ639147.1 Leucopaxillus alboalutaceus 100 KJ417275.1 Leucopaxillus alboalutaceus KJ417323.1 Leucopaxillus tricolor JQ639156.1 Leucopaxillus monticola 93 JQ639148.1 Leucopaxillus cerealis 78 KJ417274.1 Leucopaxillus albissimus 69 KJ417273.1 Leucopaxillus albissimus KP453711 Leucopaxillus giganteus JQ639150.1 Leucopaxillus giganteus 100 JQ639151.1 Leucopaxillus giganteus

64 KP453698 Leucopaxillus giganteus Leucopaxillus giganteus (MM-76) 67 Leucopaxillus giganteus (GJ-1605) EU819413.1 Leucopaxillus tricolor JQ639153.1 Leucopaxillus mirabilis KP453712.1 Leucopaxillus pulcherrimus JX679360.1 Paxillus involutus 100 EU819416.1 Paxillus involutus

0.02 Figure 145. Molecular Phylogenetic analysis of Leucopaxillus giganteus and associated taxa using nITS data by Maximum Likelihood method. The analysis involved 28 nucleotide sequences. All positions containing gaps and missing data were eliminated.

336

MELANOLEUCA Pat.

Melanoleuca cinereifolia (Bon) Bon, Docum. Mycol. 9(33): 71 (1978) Figs. 146 & 147

Pileus 45‒60 mm across, plano-convex to plane with slightly uplifted margins in mature specimens, umbonate, shape of margin straight, blackish brown to chocolate brown with a darker disc in some specimens light pinkish brown in other collection, dry, dull smooth or somewhat bumpy near umbo, glabrous, cap margin regular, context greyish white or slightly brownish below the pellis, whitish below, moist to dry, thicker at the disc (4‒6mm) thinner (1‒1.5mm) at the edge, brittle. Lamellae adnate, emarginated to slightly sinuate, broad (3‒4 mm at the center), thickness average (0.5‒0.8 mm), sub-distant to close, whitish to off white with a grayish hue, gill edge even or slightly scalloped, dry, unequal with frequent short lamellulae, lamellulae in four to six tiers. Stipe 50‒70 × 5‒8 mm, central, terete, equal or slightly thickening towards base, base covered with white rhizomorph, fibrillose to longitudinally striated, texture fragile, interior solid, context concolorous with the pileal context, moist or dry, fibrillose, unchanging upon cutting.

Basidiospores (7.3‒) 7.5‒8.7 (‒9.1) × (4.3‒) 4.5‒5.5 (‒6), Q = (1.5‒) 1.58‒1.66 (‒1.7),

Me = 8.1 × 5, Qe = 1.6, ellipsoid, apiculus present, appears smooth and hyaline in KOH, lightly ornamented in Melzer’s reagent. Basidia 20‒25 × 7‒10 µm, clavate, mostly 4- spored with scattered 2-spored, thin walled. Cheilocystidia 50‒70 × 10‒15 µm, narrowly conical to setiform, crystalliferous, thick walled (≤ 3 µm). Pleurocystida 60‒70 × 10‒15

µm, narrowly lageniform to conical with a short pedicel, thick walled, crystalliferous or not. Pileipellis a cutis with cylindrical hyphae of size 8‒10 µm wide, thin walled, hyaline in KOH solution, staining in Congo red.

337

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2250 m a.s.l., in decomposing needle litter under

Abies pindrow, Junaid Khan, 10th August, 2014, MJ-59 (SWAT000171).

Molecular characterization Fig. 148

The ITS region of MJ-59 yielded a 730bp fragment which showed 99% similarity with sequences of Melanoleuca cinereifolia (JN052137; JN052138) from USA and KJ182965 from Pakistan upon BLAST search.

A total of 34 ITS nucleotide sequences were used in the phylogenetic analysis, including

33 in-group taxa and one outgroup taxon (Limnoperdon incarnatum G.A. Escobar,

DQ097363). The aligned dataset had 743 sites of which 448 sites were conserved, 285 variable with 197 parsimony informative sites.

Jukes and Cantor model was selected as the best model for maximum likelihood analysis which clustered all the sequences into two well supported clades. The Pakistani sequences of MJ-59 clustered with the included sequences of Melanoleuca cineriefolia in clade A confirming the identity.

Comments: Melanoleuca cinereifolia is reported from Shangla district by Saba & Khalid

(2014) with no other records from Pakistan. This is the first report of this species from district Swat and second report from Pakistan and Asia.

338

Figure 146. A – E. Different views of the basidiomes of Melanoleuca cinereifolia (MJ- 59). Bars: 10 mm for A‒E.

339

Figure 147. A–E. Microscopic structures of Melanoleuca cinereifolia (MJ-59). A: Basidiospores. B: Cheilocystidia. C: Basidia. D: Pileipellis terminal elements. E: Pleurocystidia. Bars: 4.6 µm for A, 9.54 µm for B, 5.2 µm for C, 12.5 µm for D, 8.3 µm for E.

340

KY318763 Melanoleuca cinereifolia JX429108 Melanoleuca aff cinereifolia JX429210 Melanoleuca aff cinereifolia KJ182965 Melanoleuca cinereifolia JN052137 Melanoleuca cinereifolia 66 JN052138 Melanoleuca cinereifolia

68 JN616472 Melanoleuca subpulverulenta JN616473 Melanoleuca subpulverulenta JN616450 Melanoleuca nivea clade A 84 JN616444 Melanoleuca heterocystidiosa JN616445 Melanoleuca heterocystidiosa 98 JN392452 Melanoleuca nivea JX429226 Melanoleuca communis JX429204 Melanoleuca communis 64 JX429207 Melanoleuca communis JN616465 Melanoleuca strictipes 98 JN616466 Melanoleuca strictipes

72 JN616433 Melanoleuca exscissa 100 JN616436 Melanoleuca exscissa JN616474 Melanoleuca substrictipes

94 JN616439 Melanoleuca grammopodia 90 JN616440 Melanoleuca grammopodia JF908352 Melanoleuca brevipes

100 JN616458 Melanoleuca pseudoluscina JN616459 Melanoleuca pseudoluscina clade B 98 JN616452 Melanoleuca paedida

80 JN616453 Melanoleuca paedida

100 JN616425 Melanoleuca cognata JF908360 Melanoleuca cognata 64 JN616426 Melanoleuca decembris JN616428 Melanoleuca decembris JN616420 Melanoleuca angelesiana 98 JN616421 Melanoleuca angelesiana DQ097363 Limnoperdon incarnatum outgroup

0.02 Figure 148. Molecular Phylogenetic analysis of Melanoleuca cinereifolia by Maximum Likelihood method inferred from nITS data using MEGA6. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes‒Cantor model. The tree with the highest log likelihood (‒1485.3407) is shown.

341

TRICHOLOMA (Fr.) Staude

Tricholoma conicosulphureum nom. prov. Figs. 149 & 150

Etymology: the epithet “conicosulphureum” refers to the conical shape of the pileus and close resemblance with Tricholoma sulphureum

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Miandam valley, 2250 m a.s.l., in decomposing needle litter under Abies pindrow, Junaid Khan, 09th August,

2015, MM-1534 (SWAT000172).

Diagnosis: pileus conical with a revolute margin, color light orange red to light

yellowish, surface appressed scaly, scaly-fibrillose stipe with a whitish basal

rhizomorph, ellipsoidal to broadly ellipsoidal basidiospores.

Pileus 50‒60 mm across, conical with revolute margins, color light orange red (2.5YR 8/8) with a light yellowish tan margin (5Y 9/6), surface dry, appressed scaly, scales small and less concentrated on the margins, margin crenated. Lamellae uncinated, attached with a tooth, broad (3‒5mm) at the middle, close, pale greenish yellow (7.5Y 9/4), lamellae edge serrulate, lamellulae present, of diverse lengths. Stipe 70‒90 × 10‒12 mm, central, terete, equal or very slightly thickening downward, concolorous with the pileus, surface scaly- fibrillose, base with white rhizomorph.

Basidiospores (9.2‒) 9.9‒11.6 (‒11.9) × (5.7‒) 6‒6.9 (‒7) µm, Q = (1.5‒) 1.55‒1.77 (‒

1.8), Me = 10.5 × 6.4 µm, Qe = 1.6, ellipsoidal to broadly ellipsoidal, with apiculus, smooth, hyaline in KOH. Basidia 35‒45 × 8‒10 µm, club shaped, mostly 4-spored, sterigmata ≤ 0.7 µm, guttulate, basal clamps present. Cheilocystidia 35‒45 × 3‒7 µm,

342 cylindrical with sub-capitate heads, flexuous to threads like, hyaline in KOH.

Pleurocystidia 30‒35 × 3‒5 µm, cylindrical. Pileipellis hyphae 2‒3 µm in diameter, simple septate, golden brown to reddish brown in KOH. Elements of the scales 20‒45 ×

4‒12 µm, cylindrical to more or less obovoid, in chains, hyaline to lightly grayish in KOH solution. Stipitipellis hyphae 4‒6 µm in diameter, simple septate, terminal elements narrowly clavate to cylindrical, with scattered beaded/knotted hyphae, light golden to reddish brown in KOH.

Molecular characterization Fig. 151

The ITS region of MM-1534 upon amplification with fungal specific primers ITS1F and

ITS4 yielded a 650 and 713bp fragments that showed 93% similarity with Tricholoma sulphureum (Bull.) P. Kumm., (LT000090; AY462036) from Denmark and Germany, respectively and T. bufonium (Pers.) Gillet, (AY462029) from Germany. These species belong to the section Sericella in the genus Tricholoma and sequences of other species of this section were downloaded for phylogenetic reconstruction.

There were a total of 17 nucleotide sequences in the final dataset with 16 in-group taxa and one outgroup taxon (Hypsizygus marmoreus (Peck) H.E. Bigelow, HM561970). In the aligned dataset there were 668 positions out of which 384 characters were conserved, 280 variable and 221 parsimony informative.

Upon maximum likelihood analysis, the Pakistani sequences of MM-1534 formed a sister clade with the sequences of T. sulphureum and T. bufonium with a strong bootstrap value of 99% thus supporting the independent position of the Pakistani collection. The species

343 of T. sulphureum clustered within two different clades viz. one with T. bufonium and others with T. hemisulphureum (Kühner) A. Riva.

Comments: this taxon is being proposed as a new to science.

Figure 149. Basidiome of Tricholoma conicosulphureum (MM‒1534). A: Natural habitat. B: View of the lamellae. C: View of the Stipe surface and basal mycelium. D: full basidiome.

344

Figure 150. A–G. Microscopic structures of Tricholoma conicosulphureum (MM- 1535). A: Basidiospores. B: Cheilocystidia. C: Pleurocystidia. D: Basidia. E: Stipitipellis. F: Pilocystidia. G: Pileipellis. Bars: 9.1 µm for A , 11.5 µm for B & E, 10.4 µm for C, 13.3 µm for D, 8.5 µm for F, 7.1 for G.

345

AY462036 Tricholoma sulphureum 64

AY462035 Tricholoma sulphureum

100 AY462029 Tricholoma bufonium

99 LT000090 Tricholoma sulphureum

Tricholoma conicosulphureum

100 47 Tricholoma conicosulphureum

JN021105 Tricholoma inamoenum

LT000174 Tricholoma inamoenum 100 100

LT000173 Tricholoma inamoenum

LT222026 Tricholoma sp KR-2015b

80 AF377245 Tricholoma sulphureum

96 AY462037 Tricholoma sulphureum

100 LT000065.1 Tricholoma hemisulphureum

LT222027 Tricholoma hemisulphureum

65 AF377205 Tricholoma lascivum

HM561970.1 Hypsizygus marmoreus

0.02 Figure 151. Molecular Phylogenetic analysis of Tricholoma conicosulphureum and associated taxa based of nrITS data by Maximum Likelihood method. The tree with the highest log likelihood (‒2234.7654) is shown.

346

TRICHOLOMOPSIS Singer

Tricholomopsis robustus nom.prov. Fig. 153

Etymology: “robustus” refers to large size of the fruiting body, compared to the associated taxa

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2250 m a.s.l., among decomposing needle litter under Pinus wallichiana, Junaid Khan, 25th

July, 2015, MJ-1586 (Holotype), (SWAT000174).

Diagnosis: fruiting body large compared to other species of the genus, convex to

saucer shaped, stipe smaller compared to the pileus diameter, ellipsoidal to oval

basidiospores, narrowly utriform pleurocystidia.

Pileus 100‒120 mm across, convex when young plane to saucer shaped with a central umbo, color light yellow (10YR 9/8) with a reddish (5R 8/10) central part, surface dry, lightly velutinous, scales small, appressed, more concentrated in the central part, cap margin even at first, cracking with age. Lamellae adnate to adnexed, close, color yellowish brown (2.5Y 9/4), lamellar edge serrulate, lamellulae present, of diverse lengths, mostly short. Stipe 60‒70 × 10‒12 mm, central, terete, thickening downward, straight or bent, concolorous to the lamellae, with or without reddish pink coloration, surface more or less pruinose in young specimens, smooth to lightly fibrillose in mature specimens, base with a whitish mycelium.

Basidiospores (4.7) 5.1‒6.3 (6.9) × (3.4) 3.6‒4.3 (4.7) µm, Q = (1.2) 1.3‒1.6 (1.7), Me =

5.7 × 4 µm, Qe = 1.4, variable in shape, broadly ellipsoidal, ellipsoidal to oval, smooth,

347 content granular in KOH, with apiculus. Basidia 20‒30 × 4‒7 µm, clavate, guttulate, 2‒4 sterigmated. Cheilocystidia none observed. Pleurocystidia same sized as basidia, narrowly utriform. Pileipellis a cutis, hyphae 4‒8 µm in diameter, clamped at the bases, with clavate-cylindrical terminal elements. Pileipellis hyphae 4‒8 µm in diameter, rarely clamped, with cylindrical terminals.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Malam Jabba valley, 2150 m a.s.l., among decomposing needles under

Pinus wallichiana, Junaid Khan, 25 August, 2015, MM-15195 (LAH31426).

Molecular characterization Fig. 154

The ITS region of MJ-1586 and MM-15195 generated a 510 and 560bp fragments, respectively. The generated sequences were subjected to BLAST analysis which matched

95% with unknown species of Tricholomopsis viz. Tricholomopsis sp.IB-2015b voucher

UPS-F-646220 and voucher TUB11582 (KP058984; KP058981) from Sweden. It also showed 94% similarity with Tricholomopsis rutilans (KF692082; KF668308) from Korea.

For phylogenetic analysis a total of 17 nucleotide sequences were retrieved from Genbank including 16 in‒group taxa. Pluteus romellii (AY854065.1) was used as outgroup taxon following Holec & Kolařík (2012). There were total of 669 sites in the aligned dataset, of which 396 sites were conserved, 246 variable with 122 parsimony informative. The

Pakistani sequences of MJ-1586 and MM-15195 formed a separate clade along-with other collection (MJ-16116) which clearly support the unique identity of both the collections.

Comments: this taxon is being proposed as new to science as Tricholomopsis robustus.

348

Figure 154. A–F. Morphology of Tricholomopsis robustus. A: view of the pileus (MJ- 1586). B. view of the lamellae (MJ-15195). C ‒ F: Microscopic structures (MJ-1595). C: Basidiospores. D: Basidia. E: Pleurocystidia. F: Pileipellis. Bars: A & B: refer to scale, 6.5 µm for C, 6.78 µm for D, 7.3 µm for E, 15.2 µm for F.

349

Tricholomopsis umbonatus nom.prov. Fig. 152

Etymology: the epithet “umbonatus” refers to the umbonate pileus characterizing the species.

Type: PAKISTAN, KHYBER PAKHTUNKHWA, Swat District, Malam Jabba valley,

2250 m a.s.l., in decomposing needle litter under pinus wallichiana, Junaid Khan, 08th

August, 2015, MJ‒15116 (Holotype), (SWAT000173).

Diagnosis: fruiting body medium sized, pileus broadly convex with a central umbo,

pale yellowish pink with reddish pink appressed scales, pruinose-fibrillose reddish

yellow stipe, broadly ellipsoid basidiospores, narrowly utriform to narrowly clavate

cheilocystidia.

Pileus 35‒50 mm across, broadly convex with a central umbo, color pale yellowish pink

(2.5YR 9/2) covered with reddish pink scales (7.5R 6/6), scales appressed, more concentrated on the center, pileus margin crenate. Lamellae adnexed, broader at the middle

(2‒4 mm), narrowing outward and less than 1mm at the extreme end, close, color pale yellowish cream (7.5Y 9/4), lamellar edge serrulate, lamellulae present, in 3‒6 tiers, of diverse lengths. Stipe 60‒80 × 10‒12 mm, central, terete, thickening downward or lightly clavate, color pale reddish yellow (10YR 9/8), pruinose-fibrillose.

Basidiospores (4.8) 5.1‒6.4 (6.6) × (3.6) 4‒4.6 µm, Q = (1.2) 1.3‒1.46 (1.5), Me = 5.7 ×

4.3 µm, Qe = 1.3, broadly ellipsoid, smooth walled, hyaline in KOH, with a single central guttule, apiculus present. Basidia 15‒25 × 4‒6 µm, clavate, 4-sterigmated, guttulate in

KOH solution, basal clamps present. Cheilocystidia none observed. Pleurocystidia 20‒

30 × 4‒6 µm, narrowly utriform to narrowly clavate with acute apices, contents granular

350 in KOH solution, clamped at the bases. Pileipellis hyphae 6‒9 µm in diameter, clamped at the septa, with cylindrical terminals. Stipitipellis elongated, 7‒11 µm in diameter, clamped at the septa, there are scattered green colored areas in the stipitipellis with scattered elaters.

Molecular characterization Fig. 154

The ITS region of MJ-15116 resulted in a 714bp sequence which was subjected to BLAST search. The query sequence matched 95% with Tricholomopsis rutilans (KY435938;

KF692082) from Czech Republic and Korea respectively. It also matched 92% with

Tricholomopsis sp. IB2015b voucher UPS‒F‒646220 and voucher TUB11582

(KP058984; KP058981) from Sweden.

For phylogenetic analysis a total of 17 nucleotide sequences were retrieved from Genbank including 16 in-group taxa. Pluteus romellii (AY854065.1) was used as outgroup taxon following Holec & Kolařík (2012). There were a total of 669 sites in the aligned dataset, of which 396 sites were conserved, 246 variable with 122 parsimony informative.

The Pakistani sequences of MJ-15116 formed a separate clade along-with other Pakistani collection (MJ-1586), confirming the unique identity.

Comments: this taxon is being proposed as new to science.

351

Figure 152. A – F. Morphology of Tricholomopsis umbonatus (15116). A & B: Basidiomata. A: Natural habitat. B: View of the lamellae. C ‒ F: Microscopic structures. C: Basidiospores. D: Basidia. E: Pleurocystidia.F: Pileipellis. Bars: 10 mm for A & B, 7.31 µm for C, 8.33 µm for D, 6.42 µm for E, 16.2 µm for F.

352

KP058984.1 Tricholomopsis sp. IB-2015b 99

KP058981.1 Tricholomopsis sp. IB-2015b 65

KF692082.1 Tricholomopsis rutilans

98 KF668308.1 Tricholomopsis rutilans 88

Tricholomopsis robustus 98

Tricholomopsis robustus

61 100 Tricholomopsis umbonatus

100 Tricholomopsis umbonatus

32 HE649945.1 Tricholomopsis osiliensis

100 HE649943.1 Tricholomopsis osiliensis

KY010824.1 Tricholomopsis ornaticeps voucher PDD:102769 60

100 KY010822.1 Tricholomopsis ornaticeps voucher PDD:102517

FN554893.1 Tricholomopsis flammula

99 HE649941.1 Tricholomopsis flammula genomic DNA containing

KJ146733.1 Tricholomopsis decora voucher UBC F23918

92 KJ146732.1 Tricholomopsis decora voucher UBC F23917

AY854065.1 Pluteus romellii isolate AFTOL-ID 625

0.02 Figure 154. Molecular Phylogenetic analysis of Tricholomopsis taxa reported during present investigation and associated taxa by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jukes‒Cantor model. The tree with the highest log likelihood (‒1460.1783) is shown.

353

ASCOMYCOTA

Family CALOSCYPHACEAE

a) Genus CALOSCYPHA

1. C. fulgens

354

CALOSCYPHA Boud.

Caloscypha fulgens (Pers.) Boud., Bull. Soc. mycol. Fr. 1: 103 (1885) Fig. 155

Fruiting body 10‒30 mm across, cup shaped, mostly distorted with age. Inner surface yellowish orange to yellow (10YR 7/12), smooth. Outer surface ochre brown to light orange (2.5 YR 6/18) when young, turning bluish green (10BG 7/12) on bruising and aging, smooth to slightly rugulose. Stipe mostly absent, rudimentary if present, attached to the substrate through a network of white mycelia.

Ascospores (7.9‒) 8‒9 (‒9) × (7.9‒) 8‒9 (‒9) µm, Q = 1, Me = 8.5 × 8.5 µm, Qe = 1, globose, smooth. Asci 130‒150 × 8‒10 µm, 8-spored, cylindrical. Paraphysis 130‒150 ×

1‒2 µm, tapering toward top with wavy ends; never projecting beyond asci; filamentous, bearing orange colored granules. Excipular cells 5‒8 µm in diameter, irregular, with golden yellowish oily contents.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 26th

April, 2015, MM-02 (SWAT000175). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Miandam valley, 2150 m a.s.l., under Abies

pindrow, Junaid Khan, 01st May, 2015, MM-1502 (LAH31428).

Comments: both the genus and species are reported for the first time from Pakistan.

355

Figure 155. A – F. Morphology of Caloscypha fulgens (MM-02). A: Ascomata in natural habitat. B: View of the inner and outer layers. C: View of the bluing reaction upon bruising and handling. D: Ascomata of Caloscypha fulgens. E: Close‒up of Asci showing round Ascopsores and paraphysis with wavy terminals. F: Asci. Bars: 8mm for A-C, 32 µm for D&F, 16 µm for E.

356

Family HELVALLACEAE

a) Helvella

1. H. crispa

2. H. elastica

3. H. leucopus

4. H. macropus

5. H. maculata

6. H. solitaria

357

HELVELLA L.

Helvella crispa (Scop.) Fr., Syst. mycol. (Lundae) 2(1): 14 (1822) Fig. 156

Fruiting body terrestrial, saddle shaped. Pileus 30‒50 mm, irregularly lobed, inrolled.

Upper surface cream colored (10Y 9/4), surface smooth to bumpy, glabrous, unchanging upon bruising, interior cream colored, thin (1mm). Under surface cream colored in young stages, turning light brown to brown later on, finely hairy. Stipe 60‒100 × 15‒20 mm, attached centrally or off‒centrically, white to cream colored when young, with yellowish brown coloration in mature specimens, surface glabrous, furrowed, ribs with blunt edges, interior hollow, flesh thin, pliable.

Ascospores (17‒) 17.5‒20 × 11‒13 µm, elliptical, with a single and large oil droplet in the center, some may have few other small oil droplets. Asci 250‒300 × 14‒17 µm in width.

Paraphyses same length as asci, up to 3‒6 µm in width, cylindric with clavate apices, tapering towards base.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 09th

August, 2015, MM-1537 (SWAT000176). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Malam Jabba valley, 2150 m a.s.l., under Abies

pindrow, Junaid Khan, 01st July, 2016, MJ-15112, (LAH31429).

Comments: this taxon is widely distributed in the forests of Pakistan and is reported locations including Murree, Shogran, Sharan, Kalam, Patriata (Ahmad, 1955a; 1956b;

1969a). This is second report of this taxon from the present study area.

358

Figure 156. A‒D. Morphology of Helvella crispa. A: Ascomata in natural habitat (MM- 1537). B: Ascocpores, asci and paraphyses. C: Excipular cells. D: Ascomata (MJ-15112). Bars: 15mm for A & D, 15 µm for B, 30 µm for C.

359

Helvella elastica Bull., Herb. Fr. 6: tab.242 (1785) Fig. 157

Fruiting body terrestrial, mostly single, saddle shaped. Pileus 20‒25 mm across, mostly bi-lobed resembling liver, rarely irregularly lobed, inrolled. Upper surface glabrous, smooth to lightly bumpy, light brown (2.5Y 9/4) to yellowish brown (5Y 9/4), interior cream colored, thin (≤1mm), unchanging upon breaking, rubbery. Undersurface white, fuzzy. Stipe 50‒80 × 4‒6 mm, mostly central, terete to lightly compressed, thickening downward, white overall or with a yellowish brown base, surface smooth, glabrous, tapering towards top, glabrous, interior hollow, flesh thin (less than 1mm), rubbery.

Ascospores (13.4‒) 14.5‒17.9 (‒18.9) × (8.7‒) 9.2‒12.5 (‒12.9) µm, Q = (1.3‒) 1.4‒1.67

(‒1.7), Me = 16.4 × 11.3 µm, Qe = 1.5, broadly ellipsoid, smooth walled, with a single central oil droplet. Asci 180‒250 × 20‒25 μm, 8-spored, cylindrical with a tapering base.

Paraphyses of same length as asci, 8–10 μm in the upper part, 3.5 – 4.5 μm in the basal part, narrowly clavate. Excipular cells 20‒35 × 15‒25 µm, variously shaped, globular to broadly cylindrical, in chains.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 14th

August, 2015, MM-38 (SWAT000177). PAKISTAN, KHYBER

PAKHTUNKHWA, Swat District, Miandam valley, 2150 m a.s.l., under Abies

pindrow, Junaid Khan, 01st May, 2015, MM-1560 (LAH31430).

Comments: this species is already reported from Pakistan (Ahmad et al., 1997) and represent first record for the present study area.

360

Figure 157. A – F. Morphology of Helvella elastica (MM-38). A: Ascomata in natural habitat. B: View of the underside. C: View of the asci and paraphysis with clavate apices. D: View of the hollow stipe with yellowish brown base. E: Asci with ascspores. F: Excipular cells. Bars: 15mm for A, B & D, 40 µm for C & E, 26 µm for F. Helvella leucopus Pers. Mycol.eur. (Erlanga) 1: 213 (1822) Fig. 158

361

Fruiting body saddle shaped, terrestrial, single or in clusters. Pileus 25‒40 mm across, mostly three lobed, lobes irregularly folded and joined at their ends, inrolled towards the stipe, upper surface chocolate brown (5YR 3/4) to blackish brown (5YR 1/2) with a whitish brown margin, smooth, glabrous, interior cream colored, thin (≤ 1mm), under surface light brown to grayish brown, glabrous, attached to the stipe through several points. Stipe 25–

50 × 8‒15 mm, central, cream colored, some with a brownish to pinkish tone (10YR 9/4), thickening downward with a swollen base, smooth above, furrowed at the base, interior hollow, flesh whitish to creamy, unchanging.

Ascospores (13.3‒) 15.2‒18.5 (‒20.4) × (9.8‒) 10.1‒13.1 (‒14.5) µm, Q = 1.4‒1.48 (1.5),

Me = 17.4 × 12.2 µm, Qe = 1.4, broadly ellipsoidal, smooth walled, with a single oil granule. Asci 250‒350 × 18‒23 µm, cylindrical with a slightly tapering base, 8-spored, thin walled, content granular in Congo red. Paraphyses of same length as asci, cylindrical with swollen tips (6‒8 µm), 3–4.5 µm in the lower part, septate and there sometimes lightly inflated, smooth and thin walled. Excipular cells 20‒25 × 14–16 µm, cylindrical with somewhat rounded tips, in chains.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Takhta Band village, 1000 m a.s.l., on burnt soil, Junaid Khan, 01st April,

2014, TW-08 (SWAT000178). PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Ingaro Dherai village, 1000 m a.s.l., on burnt soil, Junaid Khan, 14th April,

2015, ING-07 (LAH31430).

Comments: this taxon is a new addition to the fungi of Pakistan.

362

Figure 158. A–F. Macroscopic and microscopic features of Helvella leucopus. A: Ascomata in clusters TW-08). B: Natural habitat (ING-07). C ‒ F: Microscopic structures (ING-07). C: Asci with Ascospores. D: Clavate and septate paraphysis. E & F. Excipular cells. Bars: 10mm for A & B, 21.4 µm for C, 15 µm for D, 22.8 µm for E & F.

363

Helvella macropus (Pers.: Fr. ) P. Karst. Fig. 159

Fruiting body terrestrial, cup shaped, in scattered groups. Pileus 20‒35 mm across, cup shaped when young, almost flat by maturity, margin slightly inrolled and wavy in appearance, upper surface smooth to finely fuzzy, light brown in color, margins dark brown

(10YR 4/4) to blackish brown (10YR 5/4), undersurface darker in color, dark brownish

(10YR 3/4), hairy, hairs small and more prominent on the margins. Sitpe 30‒40 × 2‒3 mm, central, terete, slightly thickened downward, straight to flexuous in the lower part, color light brownish in the upper half, cream colored in the lower half, surface finely hairy especially in the upper part, glabrous downward.

Ascopsores (17.9‒) 19‒22 (‒22.5) × (10‒) 11.4‒12.5 (‒13) µm, Q = 1.6 – 1.7 (1.76), Me

= 20 × 12.1 µm, Qe = 1.66, fusoid to sub-fusoid, lightly verrucose, mostly with single guttules, rarely with few (one large and other mall). Asci 250‒350 × 15‒18 μm, cylindrical, tapering downward, thin walled. Paraphysis same length as asci, 5‒8 μm wide, with clavte to sub-clavate apices, contents granular.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 09th

August, 2015, MM-1532 (SWAT000179).

Comments: this taxon is a new record for ascomycetes of Pakistan.

364

Figure 159. A‒D: Macroscopic and microscopic features of Helvella macropus (MM- 1532). A & B. Acomata in natural habitat. C & D: Microscopic structures. C: Excipular cells. D: Asci with ascospores and Paraphyses. Bars: 10mm for A & B, 20 µm for C & D.

365

Helvella maculata N.S. Weber, Beih. Nova Hedwigia 51: 27 (1975) Fig. 160

Fruiting body more or less saddle shaped, terrestrial. Pileus 15‒20 mm across, irregularly lobed, inrolled towards the stipe and touching that mostly, color of the upper surface light brownish (10YR 6/4) with dark brownish (10YR 3/4) spots giving a dotted appearance, surface bumpy especially at the center otherwise smooth, glabrous, under surface cream colored to light yellowish brown (10YR 5/6), lightly hairy, costate at the center, interior, whitish, thin (≤ 1mm). Stipe 25–45 × 5‒12 mm, mostly central and equal, pure white in color, deeply ribbed, often inter-venose, ribs with blunt edges, interior hollow, flesh white and brittle.

Acospores (18–) 18.5–21.6 (–22) × (11–) 11.6 – 13.7 (–14.1) µm, Q = 1.55 – 1.6 (–1.62),

Me = 20 × 12.1 µm, Qe = 1.6, broadly ellipsoidal, smooth walled, with a single and large oil granule in the center. Asci 200‒300 × 15‒20 µm, cylindrical, tapering downward, thin walled. Paraphyses same length as asci, 5‒8 µm wide, with clavate to sub-clavate apices.

Excipular elements 20‒30 × 10‒15 µm, cylindrical, terminal elements clavate to sub- globose.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 09th

August, 2015, MM-38 (SWAT000180).

Comments: This taxon is a new record for Pakistani mycobiota.

366

Figure 160. A ‒ D. Macroscopic and microscopic features of Helvella maculata (MM- 38). A & B: Ascomata. A: Natural habitat. B: View of the underside of cap. C & D: Microscopic structures. C: Asci with spores and paraphyses. D: excipular cells. Bars: 5mm for A & B, 30 µm for C & D.

367

Helvella solitaria P. Karst., Bidr, Kann, Finl. Nat. Folk 19: 37 (1871) Fig. 161

Fruiting body terrestrial, cup shaped, mostly in scattered group. Pileus 20‒35 mm across, hemispherical with an inrolled margin at first, expanding later on, often cracking irregularly, inner surface chocolate brown (10YR 5/4) to dark brown (10YR 3/4), margin concolorous or yellowish in some specimens, smooth to lightly bumpy and glabrous, interior rubbery, flesh cream colored, unchanging, darker than inner surface, outer surface rough, blackish brown to black. Stipe 30‒40 × 5‒7 mm, thickening downward, light brown

(10YR 6/4) to chocolate brown (10YR 2/4) with a darker upper part, furrowed, furrows straight, rarely inter-venose, interior hollow.

Ascospores (16.9‒) 17‒19.1 (‒19.7) × (11.3‒) 11.4‒12.5 (‒13.1) µm, Q = 1.5 (1.6), Me =

18.2 × 12.1 µm, Qe = 1.5, broadly ellipsoidal, smooth walled, with a large single oil granule. Asci 250‒300 × 15‒17 µm, cylindrical, 8-spored, contents granular in Congo red, thin walled. Paraphysis same length as asci, 4‒6 µm wide, cylindrical with sub-capitate to lightly flexuous apices, septate at the bases. Excipular cells 20‒30 × 15‒20 µm, cylindrical to broadly clavate, rarely globose to sub-globose, in chains.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Takhta Band village, 1000 m a.s.l., on soil with nearby tree of Populus

nigra L., Junaid Khan, 01st April, 2014, TB-03 (SWAT000181). PAKISTAN,

KHYBER PAKHTUNKHWA, Swat District, Ingaro Dherai village, 1000 m a.s.l.,

on soil with nearby tree of Populus nigra L, Junaid Khan, 19th April, 2015, ING-

05 (LAH31431).

Comments: this taxon is being reported for the first time from Pakistan.

368

Figure 161. A–F. Macroscopic and microscopic features of Helvella solitaria. A: Ascomata in natural habitat (ING-05). B: View of the stipe and under surface ING-05). C: Asci with ascospores. D: View from the top (TB-03). E & F. Excipular cells (ING-05). Bars: 6 mm for A, B & D, 22 µm for C, 45 µm for E & F.

369

Family MORCHELLACEAE

a) Verpa

1. V. conica

370

VERPA Sw.

Verpa conica (O.F. Müll.) Sw., K. svenska Vetensk‒Akad. Handl. 36: 129 (1815) Fig. 162

Pileus 15‒20 mm high, 10‒15 mm across, conical to more or less obtuse-conical, attached to the stipe only at the apex, color dark brown (10YR 5/6) to light chocolate brown (10YR

4/4) with a lighter margin, margin incurved towards the stipe at first, then irregular in some specimens with age, surface glabrous, more or less smooth at the disc, otherwise bumpy, shiny when wet, under surface cream to whitish brown colored (2.5Y 5/4), wet in appearance, lightly hairy otherwise appear smooth, context thin, fragile, lighter than exterior. Stipe 40‒90 × 5‒8 mm, central, terete, lightly thickening downward, cream colored with a light pinkish tone in some specimens, fistulous, glabrous, context thin, fragile, cream colored.

Ascospores (24.9‒) 25.9‒28.6 (‒28.8) × (13.9‒) 16‒17.1 (‒17.4) µm, Q = (1.5‒) 1.6‒1.7

(‒1.8), Me = 27.2 × 16.4 µm, Qe = 1.7, elliptical, smooth walled. Asci 150‒220 × 20‒25

µm, cylindrical, smooth walled. Paraphyses same length as asci, 10‒12 µm in diameter, septate, with clavate terminals.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 26th

April, 2015, MM-01 (SWAT000182).

Comments: this taxon is an addition to the fungi of Pakistan.

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Figure 162. A–D. Macroscopic and microscopic features of Verpa conica (MM-01). A: Ascomata in natural habitat. B: Ascus with ascospores. C: Asci and paraphysis. D: View of the under surface of pileus. Bars: 12 mm for A & D, 25 µm for B, 17 µm for C.

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Family PYRONEMATACEAE

a) Otidea

1. O. leporina

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Otidea leporina (Batsch) Fuckel Jb. nassau. Ver. Naturk. 23‒24: 329 (1870) [1869‒70]

Fig. 163

Fruiting body 50‒70 × 30‒40 mm across, growing in clusters, mostly spoon shaped with a cleft on one side, rarely cup shaped. Inner surface yellowish brown (10YR 6/4) to dark brown (10YR 5/6), smooth, wet, slippery especially in wet conditions, sterile, margin incurved in most of the specimens, outer surface concolorous to the inner surface or slightly paler, smooth or very finely hairy, interior concolorous to the exterior, thin, not more than

1.5 mm, unchanging. Stipe 3‒7 × 3‒5 mm in dimensions, compressed, bumpy, paler than the aerial part, whitish at the extreme base, flesh concolorous to the exterior or slightly paler, unchanging upon cutting.

Ascospores (13.7‒) 14‒16 (‒16.2) × (6.5‒) 6.5‒7.7 (‒7.8) µm, Q = (1.8‒) 1.9‒2.2 (‒2.3),

Me = 14.9 × 7.1 µm, Qe = 2.1, oblong or elliptical, smooth, mostly with two oil guttules rarely with one, thick walled. Asci 150‒180 × 10‒12 µm, 8-spored, cylindrical, smooth.

Paraphyses 150‒190 × 2‒3 µm, septate, with curved tips. Excipular hyphae, branched, septate and there constricted, 3‒8 µm in diameter, excipular cells swollen, in chains, 12‒

17 × 10‒15 µm, septate and there constricted.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2200 m a.s.l., under Abies pindrow, Junaid Khan, 01st

August, 2014, MM-04 (SWAT000183).

Comments: this taxon is previously reported from Murree and Sharan (Ahmad, 1956b;

Khalid, 1992). This is the first report of this species from the present study area,

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Figure 163. A–F. Macroscopic and microscopic features of Otidea leporina (MM-04). A: Ascoma in natural habitat. B: View of the outer surface. C: Asci with paraphysis. D: Ascospores. E: Excipular cells. E: Excipular hyphae. Bars: 5mm for A & B, 30 µm for C, 35 µm for D, 34 µm for E, 53 µm for F.

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Family XYLARIACEAE

a) Xylaria

1. X. hypoxylon

2. X. polymorpha

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XYLARIA Hill ex Schrank

Xylaria hypoxylon (L.) Grev., Fl. Edin.: 355 (1824) Figs. 164 (B&C) & 165 (B&D)

Fruiting body 30‒60 × 2‒4 mm above the ground, 15‒30 mm underground, cylindrical to narrowly fusiform, mostly round, flattened in some specimens, tips always flattened and tapering with acute apices, mostly branched at the base with 3‒5 fruiting bodies arising from a common base, unbranched to forked above, fuzzy, color whitish silvery to blackish silvery with completely black base/ underground part and yellow to yellowish brown

(10YR 9/8) apices, in some places stripped like zebra, smooth in the upper half, longitudinally furrowed downward to somewhat roughened with ostiolar papillae, interior solid, cream colored, turning grayish upon cutting. Base 15‒30 × 2‒5 mm in dimensions, mostly irregular, rarely cylindrical, round to flattened, tapering downward with pointed end, black, smooth above hairy tomentose downward.

Ascospores (9–) 9.7–13(–13.5) × (4–) 4.7–5.8(–6.2) µm, Q = (1.9–)2.0–2.5 (–2.9), Me =

11 × 4.9 µm, Qe = 2.25, mostly ellipsoid with somewhat rounded ends, smooth, guttulate, two in number, medium brown to somewhat blackish in KOH. Asci 100‒150 × 6‒8 µm, cylindrical with long basal peduncle, peduncle 50‒100 µm long, 8‒spored. Paraphyses

130‒150 × 1‒2 µm, septate, hyaline in KOH.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Miandam valley, 2250 m a.s.l., in decomposing needle litter under Abies

pindrow, Junaid Khan, 21st July, 2015, MM‒1510 (SWAT000184). PAKISTAN,

KHYBER PAKHTUNKHWA, Swat District, Ingaro Dherai village, 1000 m a.s.l.,

on soil from decomposing plant debris, Junaid Khan, 04th July, 2015, ING‒1504,

(LAH31433).

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Comments: this taxon is reported from several locations including Changa manga, Daphar plantation and Gujranwala (Ahmad, 1969a, 1978; Mirza & Qureshi, 1978). This is the first report of this taxon from Khyber Pakhtunkhwa.

Xylaria polymorpha (Pers.) Grev., Fl. Edin.: 355 (1824) Figs. 164 (A&D) & 165 (A&C)

Fruiting body 30‒60 × 3‒6 mm, cylindrical to clavate with round apices and unbranched, color dark gray to blackish gray, surface rough, interior solid, compact, white to cream colored. Stipe 20‒30 mm stipe, tapering downward, black in color, less rough compared to the upper part.

Ascospores (18–) 20‒28 (–30) × (5–) 6‒7.5 (–9), Q = (3–) 3.3 –3.6 (–3.7), Me = 24 × 6.75

µm, Qe = 3.3, ellipsoid to navicular, ends rounded to acute, germpore present, straight to oblique, surface smooth, color dark brown in KOH. Asci 100‒150 × 8‒12 µm, cylindrical with a long pedicel, pedicel 70‒100 × 1.5‒3 µm, 8-spored.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat

District, Gabin Jabba valley, 2350 m a.s.l., on the base of a standing tree of Picea

smithiana, Junaid Khan, 30th August, 2015, GJ-1517 (SWAT000185).

Molecular characterization Fig. 166

A fragment of 581bp generated by amplifying the ITS region of GJ-1517, matched 99% with Xylaria polymorpha (FN666412; KF897015; FM164944) from Italy, China and

Germany. It also showed 97 % similarity with X. hypoxylon (L.) Grev. (AM993133;

AM993134).

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A total of 23 nucleotide sequences were used for the reconstruction of phylogeny of

Pakistani collection of Xylaria polymorpha. The maximum likelihood analysis clustered the included sequences into three clades. The Pakistani sequences of GJ-1517 clustered with sequences of X. polymorpha from Italy, China and Germany together with X. anisopleura (Mont.) Fr. and X. scruposa (Fr.) Fr. in Clade II.

Comments: new record for the present study area.

Figure 164. A‒ D. Ascomata of Xylaria polymorpha (A & D) and X. hypoxylon (B & C).

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Figure 165. A – D. Microscopic structures of Xylaria polymorpha and X. hypoxylon. A: Asci of Xylaria polymorpha. B. Ascospores of X. polymorpha. C: Asci of X. hypoxylon. D: Ascospores of Xylaria hypoxyon. Bars: 10 µm for A & C, 15 µm for B, 7 µm for D.

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JN225908.1 Xylaria castorea

GU322456.1 Xylaria digitata

29 JQ936295.1 Xylaria berteri

98 KP133344.1 Xylaria berteri

57 JX160062.1 Xylaria primorskensis

46 FJ707473.1 Xylaria primorskensis Clade I

96 JX256816.1 Xylaria allantoidea

KP133349.1 Xylaria cuneata

JQ341087.1 Xylaria grammica 70

100 AM993134.1 Xylaria hypoxylon

99 AM993133.1 Xylaria hypoxylon

KF897015 Xylaria polymorpha 88

FM164944.1 Xylaria polymorpha

100 FN666412 Xylaria polymorpha

Xylaria polymorpha

Xylaria polymorpha Clade II 74 KP133319.1 Xylaria anisopleura 100

KP133318.1 Xylaria anisopleura

98 KP133497.1 Xylaria scruposa

KP133498.1 Xylaria scruposa 73

KP133499.1 Xylaria scruposa

KP133429.1 Xylaria globosa Clade III 100 KP133428.1 Xylaria globosa

0.02 Figure 166. ITS based Molecular Phylogenetic analysis of Xylaria polymorpha and associated taxa by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2‒parameter model. The tree with the highest log likelihood (‒1740.7589) is shown. The Pakistani collection of X. polymorpha is represented by

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Chapter 4

DISCUSSION

Present investigation aimed at the characterization of selected taxa of macrofungi in the woodland and forest ecosystem of Northern parts of Khyber Pakhtunkhwa, Pakistan. A total of 88 taxa belonging to 30 families and 57 genera of Ascomycota and Basidiomycota are reported. Six species viz., Albatrellus roseus, Clavariadelphus elongatus, Descolea quercina, Gymnopilus swaticus, and Phaeocollybia pakistanica have been published as new species alongwith 25 other species proposed as new to science. Abortiporus biennis and Lactifluus volemus (presenting third and first record for Pakistani mycobiota, respectively) have been published with the introduction of these generic names to the fungi of Pakistan. Amanita pallidorosea is also published as a new record for Pakistani mycobiota.

Agaricaceae is a large and diverse family of Agaricales containing a large number of different saprotrophic groups of fungi (Qasim, et al., 2015). During present study, this family is represented by six genera (Chlorophyllum, Echinoderma, Lepiota,

Leucoagaricus, Lycoperdon and Macrolepiota) and seven species (Chlorophyllum hortense, Echinoderma asperum, Lepiota cristata, Leucoagaricus campestris nom.prov.,

Lycoperdon microsporon nom.prov., L. perlatum and Macrolepiota excoriate).

Chlorophyllum is a poorly reported genus from Pakistan with only two species

(Chlorophyllum hortense (Murrill) Vellinga and Cholorophyllum molybdites (G. Mey.)

Massee) reported so far (Nawaz, 2012; Qasim, 2013). The present study report

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Chlorophyllum hortense for the third time from Pakistan and first time from the present study area.

Echinoderma (Locq. ex Bon) Bon, is a newly erected genus (Bon, 1991) in the Agaricacea which was first circumscribed as a subgenus of Cystolepiota Singer (Bon, 1981).

Echinoderma asperum (Pers.) Bon, representing the second record of this species from

Pakistan and first from the present study area is previously reported from Sharan

(Murakami, 1993) as Lepiota aspera (Pers.) Quél. The genus name Echinoderma is introduced for the first time to the fungi of Pakistan.

Lepiota is a large genus of Agaricacea represented by almost 400 species of worldwide distribution (Kirk et al., 2008; Malysheva et al., 2013). The genus is represented by 33 species from Pakistan (Ahmad et al., 1997; Ahmad, 1980; Shibata, 1992; Murakami, 1993;

Sultana et al., 2011, Razaq et al., 2012a, Nawaz et al., 2013; Saba, 2016) and different species are frequently encountered in a wide geographic ranges across Pakistan. Lepiota cristata (Bolton) P. Kumm., presented during present study, is a commonly encountered species in the woodlands and forest ecosystems of Pakistan and has been previously reported by Iqbal & Khalid (1996) and Razaq, (2012).

Leucoagaricus is a polyphyletic genus (Vellinga, 2004), characterized by free lamellae, non-striate pileus margins, free lamellae, absence of clamp connections and presence of pseudoparaphyses (Singer 1986, Vellinga 2001). The genus is worldwide in distribution with more than 100 taxa concentrated mostly in temperate regions (Malysheva et al. 2013).

From Pakistan, nine species including L. asiatica T. Qasim, R. Nawaz and A.N. Khalid, L. badius, L. lahorensiformis, L. lahorensis Qasim, Amir & Nawaz, L. leucothites (Vittad.)

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Wasser, L. pakistaniensis, L. serenus (Fr.) Bon & Boiffard), L. sultanii and L. umbonatus are reported so far (Ahmad et al., 1997; Qasim et al., 2015; Ge et al., 2015; Hussain et al.,

2018). During present study, L. campestris nom. prov., is proposed as new to science based on some discrete morphological characteristic and supporting molecular data. This species is characterized by medium to large sized fruiting body, grayish brown to yellowish brown campanulate to plane pileus having an inrolled margin, appressed scaly surface with appendiculate margin, clavte grayish brown stipe, ellepsoid to amygdaliform basidiospores and large number of different kinds of cheilocystidia. Although, very close to L. leucothites morphologically, presence of multiple types of cheilocystidia and molecular data from nITS regions confirms its unique position. Upon maximum likelihood analysis, L. campestris formed a sister clade with L. leucothites with a longer branch length and a significant bootstrap value (Figure 9), supporting its independent position.

Lycoperdon is a well-known genus characterized by pear shaped to sub-globose fruiting bodies with sterile bases and apical pore (Akata & Halici, 2010). The genus once classified in Lycoperdaceae, is now shifted to Agaricaceae (Matheny et al., 2006). It is represented by 32 taxa from Pakistan (Ahmad et al., 1997; Yousaf, 2015; Saba, 2015). During present investigation two species of this genus viz. Lycoperdon microsporon nom.prov., and L. perlatum are being reported. Lycoperdon perlatum is one of the common puffball found in a wide range of forests across the study area. Morphologically, this species is closely matching with L. excipuliforme (Scop.) Pers. which can however be differentiated by a thicker stipe equivalent to almost half of the head diameter.

Lycoperdon microsporon nom.prov. is characterized by a pyriform fruiting body that ranges in color from pure white to cream with a pinkish tone, flaking exoperidium, and

384 smaller basidiospores (3.2‒3.6 × 2.9‒3.29 µm) with a short pedicel (7‒10 µm) compared to the L. mammiforme the closely matching taxon. Macroscopically and phylogenetically,

L. mammiforme appears most resembling species to L. microsporon in having whitish flacking outer peridium and granular inner peridium. However, both the species can be microscopically differentiated by difference in their basidiospores size and length of pedicel. Lycoperdon microsporon has smaller basidiospores (3.2‒3.6 × 2.9‒3.3 µm) and pedicel (7‒10 µm) compared to L. mammiforme having larger basidiospores measuring

4.4‒5 (‒6.5) µm and a much longer (≤23 µm) pedicel (Pegler et al., 1995). Another closely falling species is L. molle Pers. which is morphologically distinct by its turbinate to stipitate pyriforme fruiting body, spiny and grey-brown exoperidium, cream to yellow-brown endoperidium and larger basidiospores (3.8‒) 4‒5.1 (‒6.2) µm (Pegler et al., 1995).

Macrolepiota is characterized by large fleshy basidiomata, mostly but not always with squamules on the pileus, white to cream colored spore print and a prominent annulus

(Singer 1948). The genus is represented by almost 30 recognized species of worldwide distribution (Kirk et al., 2008). Knowledge of the genus is fragmentary in Asia (Ge et al.,

2010) with only two species (M. excoriata (Schaeff.) Wasser; M. procera (Scop.) Singer) reported from Pakistan (Ahmad et al., 1997; Fiaz, 2013). Macrolepiota excoriata is the only Macrolepiota species collected and presented during present investigation. Previously reported from District Mansehra, Khyber Pakhtunkhwa (Fiaz, 2013), this species is being reported for the second time from Pakistan and first from the present study area. Based on the available reports and distribution data, this species may be considered as a rare find in

Pakistan.

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Albatrellaceae is a polyporoid family within the Russulales clade (Audet, 2010) represented by 9 genera and more than 25 species (Smith et al., 2013). With no previous records, the family may be considered quite rare for Pakistan. During our present study we were able to collect different collections of Albatrellopsis, Albatrellus and Jahnoporus form different locations in the study area.

Species now treated in Albatrellopsis were part of Albatrellus until recently when Audet

(2010) re-erected it as a new independent genus. Albatrellopsis flettiodes nom.prov. representing the first report of this genus from Pakistan was encountered from Miandam valley, Pakistan during present investigation. This taxon is characterized by a terrestrial, more or less circular shiny, grey to rusty greyish brown pileus, pileus edge exceeding tube layer, whitish grey decurrent pore surface and inamyloid, sub-globose to ellipsoid basidiospores (4.2‒5.4 × 3.4‒4.4 µm). Morphologically, A. flettii (Morse ex Pouzar) Audet, appears the closest counterpart having a grayish pileus and similar hyphal anatomy.

However, A. flettii is characterized by apricot to cream colored pore surface which are in clear contrast to the whitish grey pore surface and stipe of A. flettiodes (Gilbertson &

Ryvarden, 1986). Moreover, A. flettiodes is distinctive in having the exceeding cap margin and larger basidiospores compared to A. flettii (3.5 – 4 × 2.5-3 µm) (Morse, 1941).

Molecular data also evidently supports the independent position of A. flettiodes (Figure.

17). Upon maximum likelihood it separates from A. flettii forming a separate branch with a strong bootstrap value of 93%.

Albatrellus is an ecologically important genus in Albatrellaceae is also reported for the first time from Pakistan. Macroscopically, the genus is characterized by medium to large sized basidiomes, glabrous to scaly pileal surfaces and a poroid hymenophore having small

386

3–5 pores per millimeter (Stalpers, 1992; Ginns, 1997; Zheng & Liu, 2008).

Microscopically, it is characterized by monomitic hyphal system and smooth, broadly ellipsoid to sub-globose basidiospores which can be amyloid and inamyloid (Miller &

Miller, 1980; Wang & Liu, 2002). Presence or absence of clamp connections, amyloidy of the spores, structure of contextual hyphae and reaction of flesh to different chemicals are also regarded as taxonomically important characters (Pouzar, 1975). Albatrellus roseus

J.Khan, Sher & Khalid is the only Albatrellus species reported and described from Pakistan herein. This species is characterized by a yellowish brown to pinkish red squamulose pielus, whitish pore surface that turn yellow upon bruising and pinkish red upon aging and reaction with KOH solution, ellipsoid to ovoid amyloid basidiospores and contorted contextual hyphae. Diagnostically, A. avellaneus, a North American species, is a close counterpart to A. roseus in its squamulose to areolate pileal surface, white pore surface that turn yellow upon handling, smooth, small, inamyloid basidiospores of almost same size and shape and clampless generative hyphae (Ginns, 1997). However, A. avellaneus differ from the latter by its pale purple brown color that finally fades to pale orange tan with age, tubes that turn brown upon drying and grows in different season of the year (October to

January). Both species also behave differently when reacted with KOH, A. avellaneus turn bright yellow (Ginns, 1997) in contrast to A. roseus which turns pinkish red. Albatrellus ovinus (Schaeff.) Kotl. & Pouzar can be differentiated from A. roseus by a creamy white pileal color (Gilbertson & Ryvarden 1986; Volk et al., 1995; Ryman et al., 2003) and turning of olive brown upon reaction with KOH (Gannaz, 2012). Also, A. roseus has larger basidiospores (4.9‒6 × 4‒4.58 µm) compared to the basidiospores of A. ovinus (3.8‒4.6 ×

3.3‒3.5 µm). Albatrellus piceiphilus can be distinguished by fused fruiting bodies, pale

387 yellow to yellowish pore surface, pale brownish vinaceous stipe, yellowish context and amyloidy of the basidiospores (Cui et al., 2008). Molecular phylogenetic analysis also supports independent nature of this taxon. Upon maximum likelihood analysis, it clusters along-with A. avellaneus forming a separate branch supported by a robust bootstrap value of 96%.

Jahnoprus, the third representative genus of Albatrellaceae in Pakistan, is a new record for Pakistan. Jahnoporus oreinus Spirin, Vlasák & Miettinen is the only species of this genus collected and described during present investigation. This species was recently described in 2015 by Spirin et al., (2015) from Russia with no records from any ther part of the world. This is apparently the first report of this species from outside the type locality.

The Pakistani collection is morphologically similar to the original description by Spirin et al., (2015) with minor differences. Most importantly, the stipe of the Pakistani collection is hirsute and covered by more dense and longer hairs compared to the pileus, which is different from smooth to rarely hairy stipe of the Russian collections. Molecular data of nITS region was retrieved to confirm the identity of this taxon, which evidently supported it as J. oreinus by placing it with other sequences of this species from Russia (Figure. 19).

Based on morphology and molecular data, J. hirtus seems the most resembling species, however this species can be distinguished by larger size (up-to 150mm) and hispid to warted pileus surface.

Amanitaceae is a species rich family comprised of two genera namely Amanita Pers. and

Limacella Earle. Amanita is the larger and best known genus among the two with expected

1000 species of worldwide distribution

(http://www.amanitaceae.org/?About+Amanitaceae). The genus is represented in Pakistan

388 by 17 accepted taxa (Ahmad et al., 1997; Razzaq, 2013; Saba, 2016; Sana, 2016). During our macrofungal investigation of Northern parts of Khyber Pakhtunkhwa, we collected many collections of Amanita species, seven of which are presented here. Out of these

Amanita griseofusca J.Khan & M,Kiran, is published as new to science while A. ahmadii nom.prov., and A. cinerea nom.prov., are proposed new to science. Amanita pallidorosea

P. Zhang & Zhu L. Yang is reported for the first time from Pakistan and Amanita cinnamomescens, Amanita pakistanica., Amanita pantherina are already reported species.

Amanita ahmadii nom.prov. is proposed as a new species based on morphological and molecular data. It is characterized by overall grayish coloration, warted pileus which breaks with age and amyloid, sub-globose basidiospores. Molecular data clearly supports the independent position of this taxon and places it in validate section of the sub-genus

Lepidella. Morphologically and molecularly, A. frittilaria Sacc. appears a close counterpart which can be separated by its brownish gray pileus and ellipsoidal basidiospores. Other species within the same clade (A. franchetii and A. aspera recently named as A. augusta

Bojanchtev & R.M. Davis) are also morphologically separable by their yellowish brown pileus and ellipsoidal basidiospores.

Amanita cinerea nom.prov., another proposed new to science species is characterized by a slate gray ashy-felty pileus surface which is with or without warts, slightly striated margin, bulbous stipe with volval remnants arranged in rings, broadly ellipsoid basidiospores and sphaeropedunculate cystidia. Morphologically, it is very similar to

Amanita spissacea, differing in very few anatomical characters, importantly in basidiospore size. The Pakistani species has smaller basidiospores compared to the basidiospores of A. spissacea (7‒10 × 5‒9 μm). Cystidia of both also differ from each other,

389 sphaeopedunculate in A. cinerea and clavate in A. spissacea. Amanita australis G. stev., another similar species can be differentiated by honey buff coloration and larger basidiospores (8.0‒) 9.0 ‒ 12.0 (‒14.5) × (7.0‒) 8.0 ‒ 10.5 µm. Molecular data of nITS region also support its independent position by placing it in a separate clade along-with A. spissacea and A. australis.

Amanita griseofusca is characterized by light brown color with a darker disc, one third striation on the cap margin and loose and breaking volva at the base. Maximum likelihood analysis inferred from nITS data shows that, this species is closely related to Amanita beckeri, A. crocea and A. oblongospora, from which it can however be differentiated by some macroscopic and microscopic features. The light brown pileus of Pakistani collection is different from saffron orange pileus of A. crocea, golden walnut of A. beckeri and light pearl grey of A. oblongospora. Striation on the cap margin of present taxon is almost one third which is different from one fourth of A. oblongospora. The lamellae are free, crowded and creamy to beige colored which are different from close to sub-crowded lamellae of A. crocea, and sub-distant and white to orange tan lamellae of A. oblongispora.

Amanita pallidorosea Zhang & Zhu L. Yang, an Asian taxon is published as a new record for Pakistani mycobiota. This species is characterized by umbonate, rosy pileus and smaller globose to sub-globose basidiospores. Amanita subpallidorosea Hai J. Li with its somewhat similar rosy pileus can be misguided with A. pallidorosea in the field. However, the former can be easily distinguished by lacking central umbo (Zhang et al., 2010) and microscopically through larger Basidiospores (8.5–11 × 8–10 μm). Upon maximum likelihood analysis, the Pakistani collection of A. pallidorosea clustered with specimens from USA, Russia and Republic of Korea respectively confirming its identity (Figure. 32).

390

Amanita cinnamomescens Tulloss, Iqbal, Khalid & Bhandary nom.prov. was identified by referring to the literature on www.amanitaceae.org. It is a common find in the moist temperate coniferous forests of the study area and was collected from several locations repeatedly. This species can be easily recognized in the field by larger size, orange yellow coloration, striated pileus margin and yellowish annulate and volvate stipe.

Amanita pantherina (DC.) Krombh., an already reported species from Pakistan (Ahmad, et al., 1997) was collected from various localities in District Swat, is rather rare but widespread species of Amanita in Pakistan. This species is fairly recognizable in the field by it its brown pileus bearing pure white volval remnants which are regularly arranged in most of the cases, striated margin, volvate and annulate white stipe and white unchanging context. Microscopically it is noteworthy in having non‒amyloid spores.

Amanita pakistanica Tulloss, Iqbal & Khalid was identified by referencing to the literature of Tulloss et al., (2001). Morphologically, Amanita longistriata Imai (1938) appears a close counterpart. However that species can be easily differentiated by having sub-distant lamellae and broader spores with lower spore l/w ratio (1.9‒1.22). The species was first reported by Tullos et al., (2001) and this appears to be its second report from Pakistan.

Bolbitiaceae is a represented by a single genus (Descolea) with a single species (D. quercina) during present investigation. Descolea Singer, is a cortinarioid genus of sequestrate fungi that form agaricoid basidiomes (Horak, 1971). The genus once thought concentrated in southern hemisphere is now represented by species of wide geographic distribution (South America, New Guinae, New Zealand, Siberia, Australia, India, Japan and Korea, Pakistan) (Bougher & Malajzczuk, 1985). Descolea quercina J.Khan &

391

A.Naseer is characterized by medium to large sized, light yellowish brown to deep yellowish brown basidiomes, hygrophanous and squamose-granulose pileus, stipe with a strongly striated annulus and limoniform, coarsely verrucose basidiospores with a clear papilla and partly concrescent verrucae. Morphologically, D. quercina and D. pretiosa E.

Horak, are connected in particular because of their same sized fruiting bodies, matching color, scaly pileus and limoniform basidiospores. However, the latter is differentiable by its strongly rugulose pileus, somewhat larger basidiospores (12‒14.5 × 7‒8 µm) with isolated warts lacking plage and habitat under conifers (Horak, 1971). Descolea flavoannulata (Lj.N. Vassiljeva) E. Horak, another same sized taxon already reported from

Pakistan (Niazi et al., 2007), resemble D. quercina by its somewhat similar color and limoniform basidopores, however it has a radially wrinkled pileus and larger basidiospores

(12‒16 × 8‒9 µm) lacking plage. Phylogenetically, the present species cluster close to D. maculata Bougher with which it is connected by reason of the appressed squamulose pileus, color of the basidiomes and basidiospores of almost same size (10-13 × 6-7.5 µm).

Both can however be separated by many morphological characters. Descolea maculata has a rippled or wrinkled pileus surface and amygdaliform to sublimoniform basidiospores which are minutely verrucose (Bougher & Malajczuk 1985).

Boletaceae is a large family of basidiomycota characterized by a porous hymenium rather than lamellae in its members. During the present investigation 6 species belonging to 6 genera were investigated out of which three species are proposed as new to science.

Butyriboletus is a newly erected genus in Bolectaceae and is being reported for the first time from Pakistan represented by Butyriboletus pakistanicus nom.prov. Upon phylogentic analysis, Butyriboletus pakistanicus nom. prov., clustered close to B. sanicibus Arora &

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Frank and B. fechtneri (Velen.) Arora & Frank forming a separate branch supported by a strong bootstrap value of 96% (Figure. 40). Morphologically it is connected with B. sanicibus particularly in having similar coloration of the pileus, pore surface and reticulated stipe, and poses a difficulty to distinguish among the two in the field. However, B. pakistanicus with its characteristic longer stipe (almost double to the pileus diameter) is in clear contrast to B. sanicibus having a stipe longer as the diameter of the pileus (Arora &

Frank, 2014). Microscopically, B. sanicibus is differentiable by its larger basidiospores

(11−15 × 4−5μm) with a spore Q of 2.8 compared to the 2.5 Q of the Pakistani collection

(Arora & Frank, 2014). Butyriboletus yicibus D. Arora & J.L. Frank is another matching taxon having brown pileus, yellowish brown to pale yellow hymenophore that usually stains blue-gray when bruised and yellow reticulate stipe (Wu et al., 2016). However, it is differentiable from B. pakistanicus in particular of its association with Abies/Picea, dark red to purplish red slender and shorter stipe, and larger basidiospores measuring 13–15 ×

4–5 µm (Wu et al., 2016).

Caloboletus alpinus nom.prov. is characterized by yellow pore surface, red colored reticulated scaly-areolate stipe and association with Picea smithiana in alpine altitudes. In the molecular phylogentic tree (Figure. 43) it formed a sister clade to Caloboletus panniformis. Both these share same habitat and association with conifers including Abies spp., and Picea spp., in high altitudes. However, both are easily differentiable from each other due to some morphological differences. Caloboletus panniformis has a felty tomentose pileus having velvety squamules, smooth stipe and basidiospores with suprahilar depression (Zhao et al., 2014; Wu et al., 2016) in contrast to C. alpinus having a scaly areolate pileus, reticulated stipe, and basidiospores lacking suprahilar depression.

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Caloboletus calopus (Pers.) Vizzini is similar in particular beacuase of reddish reticulate stipe and yellow to pallid yellow pore surface (Wu et al., 2016), however, it is a European species and has larger basidospores (9–19 × 5–6 μm) compared to smaller basidiospores of C. alpinus (10.4‒14.4 × 5.4‒5.9). Another taxon C. inedulis (Murrill) Vizzini is also morphologically similar to C. alpinus in having reddish reicula on the stipe and brownish pileus surface. However, it is differentiable from C. alpinus by its relatively conspicuous slender stipe and conspicuous depression in basidiospores (Zhao et al., 2014). Both the morphological differences and molecular evidence support independent position of

Caloboletus alpinus as a new to science species. The genus Caloboletus is also an addition to the fungi of Pakistan.

Lanmaoa is another newly erected genus in the Boletaceae (Wu et al., 2015). This genus is characterized by its thin hymenophore (thickness of hymenophore 1/3–1/5 times that of pileal context at the position halfway to the pileus center) which stains blue when bruised, a light yellow context which stains pale blue slowly when cut and an interwoven trichodermium to a subcutis pileipellis (Wu et al., 2015). This genus is reported for the first time from Pakistan with the description of Lanmaoa angustispora for the first time from

Pakistan. This species is characterized by medium to large sized basidiomata, pulvinate to plano-convex pileus, yellowish red to yellowish orange pore surface, stipe bruising bluish in the upper parts, reddish in the basal, cylindrical basidiospores and fusiform cystidia.

Morphologically, Lanmaoa angustispora is similar to L. flavorubra with which it also form a sister clade (Figure. 46), however it is differentiable by its broader basidiospores measuring 9.1–11.9×4.2–4.9 μm and a darker dtipe with fine reticula at the stipe apex

(Halling & Matta, 2004; Wu et al., 2005).

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Veloporphyrellus Gómez & Singer, is introduced for the first time for Pakistani mycobiota.

A single species (Veloporphyrellus purpureus nom.prov.) of this genus is being proposed as new to science during present investigation. It is characterized by a convex to plano- convex, deep red to reddish pink pileus, finely fibrillose and concolorous stipe, cylindrical basidiospores measuring 12.6 × 6 µm and clavate cystidia with acute apices.

Morphologically, V. alpinus Li & Yang and V. pseudovelatus Li & Yang seems the most comparable. However, V. pseudovelatus has a coaco brown to chestnut brown tomentose pileus and concolorous membranous veil remnants on the pilea margin (Li et al., 2014), which are in clear contrast to red to reddish pink pileus which is roughened to granulose and lack any remnants of membranous veil. Veloporphyrellus alpinus has a yellowish orange to grayish orange or brown to reddish brown stipe surface, and is easily differentiable from V. purpureus. Molecular data also evidently supports the independent position of present taxon. Upon maximum likelihood analysis, it formed a branch of its own along-with some unnamed species of Veloporphyrellus supported by robust bootstrap value of 100%.

Tylopilus porphyrosporus, the only species in the genus from Pakistan is previously reported from Shogran (Murakami, 1993), represent a new record for the present study area. It is one of the commonly encountered bolete in the forests of study area and may be regarded as common and widespread.

Clavariadelphus Donk. is a well-known genus within the Clavariadelphaceae and order

Gomphales. It is fairly easy to recognize in the field due to its unique characteristics including tall, erect, and narrowly clavate basdiomes, solitary growth, and yellow-brown to pallid brown coloration. Microscopically it is characterized by smooth and clamped

395 tramal hyphae having medallion or ampulliform inflations at the septa, presence of gloeoplerous hyphae and ellipsoid to amygdaliform basidiospores (Methven 1990). It is represented by 19 species of widespread geographic distribution in temperate regions of the world (Kirk et al. 2008; Methven, 1990). Currently only three species of

Clavaraiadelphus are reported from Pakistan including C. pistillaris (L.) Donk, C. truncatus Donk, and C. pakistanicus Hanif & Kahlid (Ahmad et al., 1997; Hanif et al.,

2014). During present study a very interesting species “Clavariadelphus elongatus J.Khan,

Sher & Khalid” is described and published as new to science. It is characterized by sub- cylindrical large fruiting body, ovoid to ellipsoid basidiospores and large basidia measuring

55‒85 × 7‒10 µm. Clavariadelphus occcidentalis Methven is clustering along-with the present taxon and is morphologically connected by having a large fruiting body (≤ 250mm tall) and rugose to rugulose surface. However both the species can be differentiated by some macro and micro-characters. Fruiting body of C. elongatus is sub-cylindrical, tapering upwards and has a yellowish pink to grayish reddish orange coloration which is in clear contrast to the more or less clavtae fruiting body of C. occidentalis having yellow- white, orange-white to yellow coloration Microscopically both can be differentiated by spore size and shape, broadly ellipsoid, broadly ovate or amygdaliform and larger basidiospores (10.5‒14 × 6‒7.5 µm) in C. occidentalis compared to the ovate to broadly ellipsoid and smaller basidiospores of (8.4‒9.9 × 6.2‒7.3 µm) of C. elongatus (Methven,

1990). It also seems reasonable to compare the newly described taxon with those already reported from Pakistan. Clavraidelphus truncatus can be differentiated by a clavate fruiting body with a turbinate apex and elliptical to oblong larger basidiospores measuring 9–13 m× 5–7 µm. Clavariadelphus pakistanicus can be distinguished by its smaller, somewhat

396 clavate fruiting body (≤ 120 mm tall) with broader obtuse tips, broadly ellipsoid basidiospores compared to the ovoid to broadly ellipsoid basidiospores of C. elongatus.

Clavariadelphus pistillaris can also be differentiated by its larger basidopores measuring

10.5–14 × 6–7.5 µm.

Cortinariaceae is an ectomycorrhizal family of Agaricomycota represented by more than

2000 species of worldwide distribution (Kirk et al., 2008). Members of this family are characterized by the presence of a Cortinate veil that is present especially in young stages covering the lamellae. Cortinarius is the largest genus of Cortinariaceae, which has remained poorly reported from Pakistan (Ahamd et al., 1997). During our present venture of macofungal surveys of northern areas of Pakistan, we report three previously undescribed species supported by morphological and molecular data.

Cortinarius clavatus nom.prov. is characterized by convex to plano-convex light brown to strong brown umbonate pileus, clavate to lightly thickened fibrillose stipe and broadly ellipsoid to sub-amygdaliform, ornamented basidiospores with suraphilar depression.

Maximum likelihood analysis inferred from nITS data clusters it within the Hinnulaie clade with C. hinnuleus, C. garciae and C. hinnuleoarmillatus supported by a strong bootstrap value. No data can be found for C. garciae for comparison among the species, however, a detailed comparison is given for C. clavatus C. hinnuleoarmillatus Reumaux and C. hinnuleus Fr. Morphologically all the three species share hyhrophanous nature of the pileus and stipe. Cortinarius hinnuleus is differentiable by strongly verrucose and smaller basidiospores measuring 7.0–9.5 × 5.4–6.5 μm (Breitenbach & Kranzlin, 2000) compared to the somewhat larger basidiospores measuring 9.9‒11.6 × 6.7‒7.7 µm. Cortinarius hinnuleoarmillatus has a similar spore length but less broader compared to the C. clavatus

397 and can also be differentiated by the presence of cheilocystidia which are absent in

Pakistani collection (8.8–10.5 × 5.4–6.3 μm). This species can also be differentiated by a persistent annulus which is in clear contrast to the ephemeral annulus of C. clavatus leaving a ring zone.

The other Pakistani collection (C. striatus nom.prov.) lies close to C. annae-maritae

Bendiksen & Brandrud, both morphologically and phylogenetically. Both the species are connected in particular because of small sized basidiomes (≤ 30 mm), hygrophanous nature and reddish brown coloration. However C. annae-maritae has smaller basidiospores measuring 7–7.5 × 5–5.5 µm compared to the basidiospores of C. striatus (8.3‒9.7 × 4.7‒

5.3 µm) and are of different shapes (ellipsoid to sub-amygdaliform in C. striatus and ellipsoid-subglobose in C. annae-maritae).

The third Pakistani collection, C. longistipes nom.prov clustered with C. torvus with which it share some morphology as well. Both have the lilac/purplish coloration in young stages, decurved margin, smooth to velvety pileus surface and club shaped stipe (Fries, 1838).

Both the species also share the same range of basidiospores size especially in length, 8‒

11.5 × 4.5‒6 µm in C. torvus and 9‒11.1 × 5.4‒7.2 µm. However C. torvus has ellipsoidal basidiospores which has a narrow apicular end and are heavily ornamented. These characters contrasting C. longistipes having broadly ellipsoid to amygdaliform basidiospores which are heavily ornamented.

Entolomataceae Kotl. & Pouzar is a cosmopolitan pink-spored and species-rich family in the euagarics (Co-David et al., 2009). During present investigation, a single species of

Rhodocybe genus was collected and proposed as new to science. Molecular analysis places

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Rhodocybe luteus nom.prov in the well-known species complex of Rhodocybe popinalis within the Rhodocybe-Clitopilus clade. The newly described species is closely similar to other species of Rhodcybe popinalis complex and cannot be separated by morphological traits, except for the unique coloration (pale orange yellow color in young stages). The ITS based maximum likelihood analysis confirm the unique identity of R. luteus, forming a separate branch with a significant bootstrap value.

The genus Hericium Pers. is one of the important genus in the family Hericiaceae (Larsson

& Larsson, 2003). It is worldwide in distribution and is considered as rare throughout the globe. Macroscopically it is characterized by annual, fleshy fruiting bodies with spiny hymenial surface. Important microscopic characters include amyloid, globose to ellipsoid basidiospores and monomitic hyphal system with scattered gloeocystidia. In Pakistan, the genus is represented by five species including H. caput-ursi (Fr.) Corner, H. clathriodes

(Pall.) Pers, H. coralloides (Scop.) Pers, H. erinaceus (Bull.) Pers., and H. ramosum (Fr.)

Pers (Ahmad et al., 1997). During our macrofungal survey, H. cirrhatum (Pers.) Nikol representing the first record for fungi of Pakistan was collected from various localities in the Swat district. Hericium cirrhatum is a very rare find in Britain (Reilly, 2011) and

Europe (Parfitt et al., 2005) and has a very few reports from Asia. This has been reported from Russia (Volobuev, 2013) and India (Das & Sharma, 2009). With no previous records from Pakistan (Ahmad et al., 1997) this species may be considered as rare find in Pakistan as well. Morphoogically, H. erinaceus is the most resembling species, which can however be differentiated by its longer and compact spines and fairly circular and fleshier fruiting body. Microscopically both can be differentiated by the spores walls (slightly roughened in H. erinaceus and smooth in H. cirrhatum). Hericium caput-ursi is another comparable

399 species, however it has a branched fruiting body with spines hanging in tufts from tips of branches. Phylogenetic analysis nested Pakistani collection with H. cirrhatum (EU784261) from Britain and Sweden (AF506385).

Hymenochaetaceae, is considered as one of the most important families in

Hymenochaetales of Basidiomycota with 27 genera and 487 species of worldwide distribution (Kirk, et al., 2008). Coltricia abieticola, is the only species being reported during present investigation and present the first report of the genus from Pakistan. The

Pakistani collections morphologically fits well with the description of Dai (2010), with slight contrast microscopically. The color of the hyphae in KOH solution is pale yellowish to golden yellow as reported by Dai (2010) and lilaceous to blackish cinnamon-lilaceous during present study. Identity of the present taxon was also confirmed through molecular data of the ITS region. The maximum likelihood analysis based on nITS data cluster the

Coltricia species into 5 well-supported clades. The Pakistani collection of C. abieticola nested within clade A along-with C. pyrophila (Wakef.) Ryvarden and C. weii Dai. The analysis evidently supports the monophyletic nature of Coltricia genus.

Hymenogastraceae is a large family of Agaricomycetes containing both lamellated and false truffle like genera (Matheny et al., 2006). During present investigation this family is represented by four genera (Galerina; Gymnopilus; Hebeloma; Phaeocollybia) and six species. Galerina marginata (Batsch) Kühner is being reported for the first time from the present study area and is previously reported only once from Pakistan (Iqbal & Khalid,

1996).

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Gymnopilus is a saprophytic genus in Hymenogastraceae characterized by: yellow to ferruginous fruiting bodies, saprotrophic nature, bitter taste, presence of cortinoid to membranous veil and rusty brown spore print (Kaur et al., 2015). The genus is represented by more than 200 different species of worldwide distribution (Kirk et al, 2008). This genus is little explored in Pakistan and only six species including Gymnopilus aeruginosus (Peck)

Singer, G. chrysites (Berk.) Singer, G. chrysimyces (Berk.) Manjula, G. hybridus (Gillet)

Maire, G. junonius (Fr.) Orton and G. sapineus (Fr.) Murrill are previously reported

(Ahmad et al., 1997). In this study we present two more species of Gymnopilus using morphological and molecular tools. Gymnopilus penetrans (Fr.) Murrill, presenting the first record of this species from Pakistan is strictly lignicolous, has a paler pileus without a tomentose surface and narrower hyphae of the pileus cuticle. It is reported from different geographical regions of the world including Europe, North Africa, and many parts of North

America (Davis et al., 2012).

Gymnopilus swaticus J.Khan, Sher & Khalid is another newly described taxon clustered within fulgens/decipiens clade (G. decipiens (W.G. Smith) Orton, G. fulgens (J. Favre et

Maire) Singer, G. odini (Fr.) Bon & Roux., and G. turficola Moser & Ladurner).

Morphologically, it resembles with G. decipiens in its somewhat similar pileus and stipe surface which is tomentose fibrillose pileal surface, and finely floccose to fibrillose scaly stipe, basidiospores of almost same size, and presence of cheilocystidia and pleurocystidia

(Holec, 2005; Høiland 1990; Orton, 1993). However, it differs from G. swaticus in its habitat (on soil especially sandy and burnt soil in Picea Mill. to conifer dominated forests), has a smaller fruiting body (≤ 30 mm), cheilocystidia of variable shape (narrowly lageniform-fusiform, brodly lageniform, utriform, fusiform-cylindrical), and rare, and

401 narrowly utriform pleurocystida (Høiland 1990; Orton 1993). Gymnopilus turficola also resembles G. swaticus in its involute margin, tomentose fibrillose pileal surface, color of young and mature specimens, fibrillose stipe without annulus, presence of cheilocystidia and pleurocystidia, and basidiospores of almost same size and shape. Yet, this species can be differentiated by its habitat in palsa mires growing on peat in subarctic areas, greenish lamellae in young state, the development of an iodoform odor when kept closed for some hours, bottle shaped cheilocystidia and pleurocystidia, and presence of caulocystidia

(Moser et al., 2001).

Hebeloma present the third genus of Hymenogastraceae during present study. Up-till now, only four species of this genus are reported from Pakistan (Ahmad et al., 1997; Sana, 2016).

Hebeloma rostratum Beker, Vesterh. & U. Eberh., in Beker, Eberhardt, Vesterholt &

Schütz, presented here is the first record of this species from outside Europe (Eberhardt et al., 2015). It is characterized by spores characters (spiny and strongly dextrinoid) and cheilocystidia (≤ 7.5 µm). In all macro and micro-characters except for the presence of pleurocystidia, the present collection conform well to the original description of Eberhardt

(et al., 2015).

Phaeocollybia R. Heim, is a genus of agaricoid fungi in the family Hymenogastraceae

(Matheny et al., 2006) is being reported for the first time from Pakistan. The genus is easy to recognize in the field due to the presence of the deeply rooted cartilaginous stipe, moist to viscid umbonate caps, and brown spores. Microscopically the genus is characterized by gelatinous tissues, roughened brown spores with an apical callus, the presence of cheilocystidia, and tibiiform diverticula (Norvell & Exeter 2008). It is widely distributed especially in temperate regions with about 95 worldwide known species

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(http://www.indexfungorum.org/). Nowhere common, the genus is quite rare for Pakistan

(Khan et al., 2016). During our present investigation, two novel taxa of Phaeocollybia were collected and characterized, out of which Phaeocollybia pakistanica Khan, Sher & Khalid has been published (Khan et al., 2016) and P. varicolor nom.prov is proposed as new to science. Phaeocollybia varicolor is characterized by different coloration of the fruiting bodies in different stages of life cycle and amygdaliform to broadly ellipsoidal, densely ornamented basidiospores with a plage and germpore. Molecular phylogenetic analysis inferred from nITS data places this taxon in a clade along-with P. olivacea A.H. Sm., P. gregaria A.H. Sm. & Trappe, and P. fallax A.H. Sm., forming a separate branch. P. olivacea a North American taxon is easily distinguishable by its olivaceous coloration

(Norvell, 1998), completely absent in P. varicolor. Phenotypically, this species is also similar to P. ammiratii Norvell in having frequent clamp connections especially in the pileipellis and acute umbo. However both the species can be differentiated by their different coloration (reddish orange in P. varicolor and tawny ochraceous in P. ammiratii) and shape of the pileus (conical to conico-campanulate in P. variacolora and convex to campanulate in P. ammiratii) (Norvell, 2000). It is also reasonable to compare this taxon with P. pakistanica, from which it is clearly differentiable by lacking the purplish lamellae characterizing the latter (Khan et al., 2016).

Inocybaceae is an ectomycorrhizal family with more than 500 worldwide species (Kirk et al., 2008). During our investigation, three interesting species of Incoybe genus are being reported, two of which (I. pallidorimosa nom.prov and I. salicetum nom.prov) are proposed as new to science while I. putilla is a new record for Pakistan.

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Inocybe pallidorimosa, a proposed new to science species belong to the section Rimosae.

The section Rimosae is placed in sub-genus Inosperma of genus Inocybe (Kühner, 1980;

Stangl, 1989). This section is named after the rimose pileus surface of its members. Other characters of the members of this section include, ellipsoid to bean shaped basidiospores and absence of metuloid pleurocystidia. Its members mostly have a bulbous base, the flesh turn reddish and develop yellow to olivaceous tinges on the lamellae (Larsson et al., 2009).

Upon maximum likelihood analysis using the nITS data, I. pallidorimosa nested within sub-clade A, along-with I. rimosa (Bull.) P. Kumm, I. melliolens Kühner, I. sororia

Kauffman, I. bulbosissima (Kühner) Bon, I. arenicola (R. Heim) Bon and I. guttulifer

Kühner. Morphologically I. rimosa seems the most comparable, which can however be separated strongly rimose pileus and mostly ellipsoidal basidiospores in contrast to less rimose pileus and mostly phaseoliform basidiospores. Inocybe arenicola another morphologically comparable species can be differentiated by its habitat in dunes, whitish pileus and larger basidiospores (12–16 × 6–8.5 µm) compared to I. pallidorimosa. Inocybe bulbosissima an arctic to alpine species has a yellow to ochraceous pileus and larger basidiospores in comparison to the present Pakistani collection.

Inocybe salicetum nom.prov falls within the sub-genus Mallocybe of Inocybe. Mallocybe species are generally characterized by a fibrous-scaly pileus, ochre-brown to red-brown coloration, adnate lamellae (most) and absence of spermatic odor. Microscopically these are characterized by smooth basidiospores, absence of metuloid cheilocystidia and pleurocystidia and presence of necropigment on basidia especially in fresh specimens

(Stangl, 1989; Jacobsson, 2008). Inocybe salicetum is characterized by a small to medium sized fruiting body growing in sandy soils under Salix babylonica L., hemispherical to

404 convex pileus with or without a short umbo, squamulose pileal surface, fibrillose to fibrillose-pruinose stipe and broadly ellipsoidal to phaseoliform basidiospores. The maximum likelihood analysis clustered I. salicetum with I. malenconii R. Heim, forming a sister clade, with which it is also morphologically comparable. Both the species are morphologically connected especially in having somewhat similar coloration, scaly pileus and almost same sized cheilocystidia. However, I. malenconii is differentiable by somewhat smaller fruiting body (≤20mm) and larger basidiospores (approx. 13 μm) compared to the larger fruiting body (≤35 mm) and smaller basidiospores (8.8‒10.2 × 5.3‒

6.1 µm) of I. salicetum. Inocybe leucoloma has a distinctive habitat, growing in alpine and arctic habitats above the treeline and is differentiable from all other species of the clade.

Inocybe agardhii (N. Lund) P.D. Orton also grows with Salix, share similar pileus surface and shape of cheilocystidia with I. salicetum, which however can be differentiated by its larger fruiting body (≤60 mm).

Inocybe putilla, a European taxon, is reported for the first time from Pakistan.

Morphologically the Pakistani collections (MJ-23 & M-24) conform well to the European collections (Kokkonen & Vauras, 2012). However the European collection are reported to be associated with Quercus, while the Pakistani collection were associated with Pinus wallichiana thus new host range is reported for this species.

Omphalotaceae is presented by a single genus and species (Gymnopus dysodes (Halling)

Halling) during this work. Previously, 7 species of Gymnopus are reported from Pakistan

(Ahmad, 1980; Iqbal & Khalid, 1996; Shibata, 1992; Sultana et al., 2011; Saba & Khalid,

2014a). Gymnopus dysodes is being reported for the first time from Pakistan growing in lowland sites not more than 1100 m a.s.l. Molecular phylogenetic analysis cluster this

405 species within the section Impudicae which is characterized by unpleasant smell resembling rotten cabbage, onion, garlic etc. This taxon is generally a North American species, is characterized mainly by its growth on decomposing wood, dark reddish brown to cinnamon brownish basidiomata, cinnamon brown lamellae, dark reddish to light reddish brown stipe, unpleasant smell and presence of cylindrical to flexuous cheilocystidia

(Halling, 1997). The present collection was collected from underground buried roots of

Alnus nitida (Spach) Endel and morphologically conform to the original description.

Molecularly, Gymnopus impudicus (Fr.) Antonín, Halling & Noordel, is a resembling species which can be separated by a darker brown pileus, black stipe base, smaller basidiospores (5.5–7.5 × 3.5–4) μm and presence of coralloid to moniliform cheilocystidia measuring 20–40×3–9 μm (Noordeloos, 1995).

Oudemansiella Speg., is the only genus representing Physalacriaceae during this study. It is presented by O. sparslamellata nom.prov. which is characterized by a light brown to whitish pileus, punctate to lacerate radicating stipe, distant lamellae, mostly 4-spored basidia, globose to sub-globose basidiospores (13.8‒15.3 × 13‒14.3 μm), oblong, fusiform to cylindrical cheilocystidia, widely cylindric, oblong, narrowly ellipsoid to flexuous pleurocystidia, ixohymenidermal pieipellis, conical to setiform caulosetae and capitate to sub-capitate caulocystidia. In the phylogenetic tree inferred from nITS data, O. sparslamellata nested between O. raphanipes (Berk.) Pegler & Young, O. chiangmaiae

Zhu L. Yang, G.M. Muell., G. Kost & Rexer and O. radicata (Relhan) Singer.

Oudemansiella chiangmaiae and O. raphanipes can be distinguished by their dark coloration, sub-globose to ellipsoid basidiospores and a cuticular pileipellis structure

(Yang & Zang, 1993; Petersen & Hughes, 2010). Oudemansiella radicata is differentiable

406 by its smooth to furfuraceous stipe and ellipsoid to limoniform larger basidiospores measuring 13‒24 × 9‒14 μm (Petersen & Nagasawa, 2005). It also seems reasonable to compare O. sparslamellata with some Asian taxa for which no sequence data are available in the GenBank. Oudemansiella ahmadii (Dörfelt) R.H. Petersen, a taxon from Pakistan, is characterized by comparatively larger basidiospores (14–20 × 12–19 μm), lack of sphaeropedunculate elements in the pileipellis (Petersen & Nagasawa, 2005) and lack of pleurocystidia (Saba, 2016; Pegler & Young, 1987). These characters are in contrast to O. sparslamellata having smaller basidiospores (13.4–16.8 × 13.1–16.5 μm), presence of sphaeropedunculate elements in pileipellis and presence of pleurocystidia. Oudemansiella japonica (Dörfelt) Pegler & Young, another Asian taxon is morphologically similar to O. sparslamellata in having somewhat similar stipe and sub-globose spores. However, the former can be differentiated by a dark brown pileus with reticulate black veins radiating from disc to margin, free to adnate lamellae without decurrent tooth, rotund pleurocystidia with refringent capitulum and stout subutriform coherent cheilocystidia (Petersen &

Nagasawa, 2005).

Pluteaceae is presented by 3 genera viz. Pluteus, Volvariella and Volvopluteus out of

Volvopluteus is an addition to the fungi of Pakistan. Pluteus populicola nom.prov. is proposed as new to science and is characterized by: light orange yellow to dark yellow, glabrous to lightly velvety, pulvinate to plano-convex pileus with a central umbo, longitudinally striate to pruinose stipe, mostly sub-globose, rarely oval to broadly ellipsoidal basidiospores and utriform cheilocystidia. The maximum likelihood analysis clustered P. populicola within the leoninus clade, forming a sister branch with P. variabilicolor Babos, which also is a close phenotypic counterpart. Both these taxa can be

407 differentiated by some macro and micro-morphological characteristics, especially the smooth pileus surface of P. populicola is different from the centrally rugose-veined pileus of P. variabilicolor. Basidiospores shape and Q value also appears promising: sub-globose to broadly ellipsoidal with a spore Q of 1.1 in P. populicola and broadly ellipsoidal to sub- globose with a Q of 1.22 in P. variabilicolor. Also the pileipellis structure in both the species is differentiable, ixocutis with branched hyphae suspended in gelatinous matrix, lacking intracellular pigment in Pakistani collection and a hymeniderm consisting of clavate, rounded terminal elements with yellow intracellular pigment in P. variabilicolor

(Lezzi et al., 2014).

Volvariella is presented by 2 species including V. bombycina and V. hypopythis.

Volvariella bombycina is one of the common edible species found in the study area. It is widely distributed in the study area and was collected from different locations growing on host ranging from deciduous to coniferous decomposing trees. Volariella hypopithys is a small Volvariella collected once form moist temperate forest of Miandam valley during present investigation. This is the first report of this taxon from Pakistan and may be considered very rare in the study area. Volvariella pusilla (Pers.) Singer is the most resembling species to V. hypopithys, which can be differentiated by smooth stipe compared to velvety-canescent pileus of V. hypopithys.

Volvopluteus, the third genus representing Pluteacea during present investigation, is reported for the first time from Pakistan by introducing Volvopluteus earlei to the fungi of

Pakistan. It is characterized by medium sized basidiomes (≤50mm), broadly ellipsoid basidiospores and absence of pleurocystidia in most of the cases (Justo et al., 2011).

Morphologically, V. gloiocephala (DC.) Boekhout & Enderle appears comparable however

408 it has a larger basidiome (pileus 50–150 mm), larger basidiospores (generally 13.0–16.5 ×

8.0–9.3 μm, Qe = 1.6–1.85 and clavate to fusiform pleurocystidia, making it different from

V. earlei.

Polyporacea is a wood rotting poroid family belonging to basidiomycota. Cerioporus and

Panus are the two genera reported during present venture. Cerioporus squamosus (Huds.)

Quél., Enchir. is an edible species previously reported from Pakistan (Ahmad, et al., 1997) as Polyporus squamosus. Panus rudis is a common encounter in the study area growing on decomposing Populus stumps.

Pterulaceae Corner, is an important family of basidiomycota typified by Pterula Fr.,

(Corner, 1970). Members of this family are characterized by having resupinate to effused or coralliform fruiting bodies, monomitic to dimitic hyphal structure, and globose, ellipsoid or fusiform, smooth and inamyloid basidiospores (Donk, 1964; Corner, 1970; Oberwinkler

1977, Bernicchia & Gorjon, 2010). Species of Pterulaceae are of great ecological importance, some are key players in the carbon cycle (Floudas et al., 2012), and some have potential applications in biomedical engineering and biodegradation (Lang et al. 2006,

Floudas et al. 2015). During present study an interesting species of Pterula viz. P. bisporitica nom.prov. was collected which is being proposed as new to science herein.

Morphologically, Pterula bisporitica resemble P. verticillata in having similar coloration and branching pattern. However the latter can be differentiated by having smaller basidiospores (6–7 × 3–3.5 µm), 2‒4 sterigmated basidia and hyphoid cystidia with long rostrate apex (Senthilarasu, 2013). Identity of the present taxon was also confirmed using molecular data generated from nITS region. There are very few sequences of Pterula species in the GenBank and the query sequences showed significant similarity with taxa of

409 other genera (Amylocorticiellum, Hyphodontia, Leotosporomyces, Tephrocybe and

Tephrocybella). Subsequently, based in high similarity sequences of these genera were also used in the phylogenetic analysis. The maximum likelihood analysis showed a strong relationship among taxa these genera and resolved them into different clades. The Pakistani sequences of P. bisporitica clustered within the Pterula clade forming a sister clade with unnamed Pterula sp. MB67and KP133248.1 Pterula sp. 984, with a robust value of 89 % supporting its independent position. All the Pterula sequences used in the present analysis except for that of Pterula subulata Fr.(KR673516), clustered in a well-supported clade.

Similarly the Amylocorticiellum sp. UC2022882 (KP814442) clustered within the Pterula clade and needs confirmation.

Russulaceae in one of the species rich family of ectomycorrhizal basidiomycetes, playing important roles in forest ecosystems. Until recently Lctarius and Russula were the only two agaricoid genera identified in the family. However recent investigations reveals four main agaricoid genera viz. Lactarius, Lactifluus, Multifurca, and Russula (Verbeken &

Nuytinck, 2013) along-with some corticioid species (Buyck et al., 2008). During present investigation this family proved to be the most abundant represented by 11 species encompassing 3 genera.

The genus Lactarius, with a significant character of exuding milk was once thought as monophyletic (Miller et al., 2006) is paraphyletic and has been splitted into Lactarius and

Lactifluus (Verbeken & Nuytinck, 2013). Of these Lactifluus seems the most variable with species mostly concentrated in temperate regions of the world and has a large cryptic diversity within the genus (Putte et al., 2009; Stubbe et al., 2010). Morphologically,

410 members of Lactifluus can be differentiated from those of Lactarius by the absence of zonate and glutinose pilei and include all annulate species (Buyck et al., 2008).

Among the species of Lactarius, L. furcatilamellarus is proposed as new to science, L. pterosporus, L. mediterraneensis are new records for Pakistan and L. sangufluus is an already reported taxon (Ahmad et al., 1997).

Lactifluus maireiodes nom.prov. is characterized by small to medium sized fruiting body, pubescent-tomentose pileus with a smooth disc and inrolled and heavily tomentose margin, pruinose stipe and lanceolate pleurocystidia measuring 40‒55 ×10‒13 µm. Maximum likelihood analysis places this taxon within the section Piperati forming a separate branch along-with L. mairei. A significant bootstrap value of 84% clearly support independent position of L. maireiodes. Phenotypically Lactarius mairei is strikingly similar to L. maireiodes and poses a great difficulty in telling apart both the species in the field.

However, L. maireiodes has a smaller size (≤40mm), pale brown to tawny cap, small yellowish brown stipe of size not more than 35 mm. Microscopically it can be differentiated by broadly ellipsoid and smaller basidiospores measuring 6.5–7.5 × 5–6 µm, and pleurocystidia of size not more than 60 µm compared to the larger basidiospores (7.5‒9 ×

6‒7µm) and pleurocystidia (≥60 mm).

Lactarius furcatilamellatus nom.prov. is proposed as new to science based on morpho- anatomical and molecular evidences. Morphologically the current taxon is characterized by forking lamellae, greening of the lamellae and pileus upon aging and bruising and association with Abies pindrow. Molecular phylogenetic analysis placed this taxon at an intermediate position between L. abieticola X.H. Wang and L. vividus Wang, Nuytinck &

Verbeken and supported its independent position. Morphologically L. furcatilamellatus is

411 a close relative of Lactarius abieticola sharing same coloration of the fruiting body, latex and association with Abies (Wang, 2016). The forking lamellae and the greening tendency of the former are however enough to distinguish among the two in the field. In addition L. abieticola has a strongly hygrophanous pileus which is greasy to sticky, different from non- hygrophanous and non-greasy pileus of L. furcatilamellatus. Lactarius abieticola is also microscopically distinct from L. furcatilamellatus by its larger basidiospores 7.5-10.0 ×

6.0-8.0 µm (Wang, 2016). Lactarius deterrimus Groger is somewhat comparable in having forked lamellae, however the purplish red turning of the flesh upon cutting make it different from L. furcatilamellatus. Lactarius vividus is extremely variable morphologically, however it is readily differentiable by its vivid color, subdistant to distant lamellae and association with Pinus subgenus Pinus (Wang et al., 2015).

Lactifluus versifromis, is described from India with no other records from any part of the globe (Putte et al., 2012). This is the first report of this taxon from Pakistan and appears second from outside type locality. The Pakistani collection matches closely with Indian type collection and is also evidently supported by molecular phylogenetic analysis.

In the genus Russula, 5 species are being reported comprising one new taxon (R. similis nom.prov.), 3 new records for Pakistan (R. kangchenjungae, R. olivacea & R. postiana) and R. amythestina, second report from Pakistan.

Russula similis nom.prov. is characterized by parabolic to pulviate, deep purplish red to dark purplish red pileus, moderate red to cream colored clavate stipe, globose to sub- globose basidiospores with warts forming incomplete reticulum, cylindrical cheilocystidia with round apices and narrowly lanceolate-clavate pleurocystidia. Molecular data of nITS data places this taxon near to R. violeipes Quél with which it also share most of the

412 morphological characters. Both of the taxa share similar habitat (under Oak), are similar in color and some micro-characters especially shape of cystidia and pileipellis. Both the taxa are quite inseparable in the field, except for the yellow coloration of the young specimens of Russula violeipes which is absent in Russula similis. Microscopically, R. similis has larger basidiospores (8‒10.6 × 7.6‒9.6 µm) compared to R. violeipes (5.6‒6.3 × 7.2‒7.9

µm).

Russula kangchenjungae, a new record for Pakistan was reported in 2010 from India by

Das et al., (2010). The Pakistani collection resemble the Indian collection morphologically.

Molecular analysis identify the present collection as Russula kangchenjungae and the same name is followed in the present work.

Strophariaceae is represented by two genera (Agrocybe and Pholiota) and two species (A. elatella (P. Karst.) Vesterh and P. microcarpa nom.prov.) during present work. Agrocybe is a genus of brown-spored mushrooms of about 100 species of worldwide distribution

(Kirk et al., 2008). Agrocybe elatella is being reported as a new record for Pakistani mycobiota. This species is microscopically characterized by having lageniform to narrowly utriform cheilocystidia.

Pholiota (Fr.) P. Kumm. is characterized by (pale) yellow to brown basidiomes, a viscid to glutinous and often scaly pileus, an annulus, rusty to dark brown smooth to slightly roughened spores with a germ pore, cheilocystidia and pleurocystidia (in a number of species as chrysocystidia), and typically lignicolous habit (Jacobsson, 2008b). A single species, P. microcarpa nom.prov. is being reported during present work. It is characterized by small basidiomes not more than 25 mm with light brown squamose pileus.

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Phylogenetically P. microcarpa lies close to P. adiposa (Batsch) P. Kumm and P. gummosa (Lasch) Singer, which can be differentiated in the field by larger fruiting bodies

(≥ 25mm). Moreover, P. gummosa has a squamulose pileus with very appressed scales which may not be apparent in mature stages (Smith & Hesler, 1968) and is different from the squamose pileus of P. microcarpa with scales more prominent and present in all stages of basidiomata. Similarly P. adiposa has gelatinous stipe scales compared to the non- gelatinous pileal scales of P. microbasidiomata (Smith & Hesler, 1968).

Tricholomataceae is a large and diverse family of Agaricales with 78 genera and 1020 accepted species (Kirk et al., 2008). This family is encompassed by 5 genera (Lepista,

Leucopaxillus, Melanoleuca, Tricholoma and Tricholomopsis) and 7 species during present investigation. Lepista nuda (Bull.) Cooke, present the first record from the study area and third from Pakistan, is previously reported from Kaghan and Mansehra district by

Sultana et al., (2011) and Fiaz (2013), respectively. Lepista panaeolous (Fr.) P. Karst is a new addition to the fungi of Pakistan.

Leucopaxillus giganteus, the sole species of Leucopaxillus reported during present study is an addition to the fungi of Pakistan. It is characterized by large sized basidomes that can attain a size of 45 cm and a brownish to grayish brown pileus in mature specimens.

Clitocybe candida Bres., a comparable species can also attain such a big size however this species lack the brown to grayish brown coloration in mature stages and remain white throughout the life cycle.

Melanoleuca Pat. is a cosmopolitan genus characterized by collybioid to tricholomatoid basidomata, convex to slightly depressed pilei, adnate to emarginate and shortly decurrent

414 lamellae, absence of annulus, hyaline basidiospores with amyloid ornamentations, and absence of clamp connections (Singer, 1986; Vizzini et al., 2012). It is presented by three species in Pakistan (Ahmad et al., 1997; Saba & Khalid, 2014b). Melanoleuca cinereifolia, presented here is previously reported from Shangle district by Saba & Khalid (2014b). This species is characterized by a short stipe and lageniform cystidia and habitat in sand dunes

(Vizzini et al., 2012). The present collection although fits well morphologically with the description of (Vizzini et al., 2012), however it was collected from humus rich forest floor in moist temperate forest of Malam Jabba. The present habitat of M. cinereifolia in coniferous forest was also reported by Saba & Khalid (2014) from Pakistan and a new habitat is being reported for this species.

Tricholoma conicosulphureum nom.prov. is characterized by a light orange red to light yellowish and conical pileus with a revolute margin, appressed scaly surface, scaly- fibrillose stipe with a whitish basal rhizomorph and ellipsoidal to broadly ellipsoidal basidiospores. Molecular phylogenetic analysis of the nITS data places it section Sericella.

It is a well-known species complex within the genus Tricholoma Comandini et al. (2004).

Tricholoma bufonium clustering within the first clade with T. sulphureum has been regarded as T. sulphureus (Comandini et al., (2004), however T. sulpureum species clustering in different clades represent cryptic species and need further phylogenetic studies. Pileus color is considered poorly suited for differentiation among the species

(Christensen & Heilmann-Clausen, 2013), rather color of the lamellae and basal mycelium appears promising (Heilmann-Clausen et al., 2017). Tricholoma conicosulphureum has pale greenish yellow lamellae, a white mycelium at the base and is boreal in nature making it different from T. sulphureum and other species of the complex.

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Within the Tricholomopsis, two unique species are being reported from Pakistan supported by morphological and molecular evidence. Tricholomopsis umbonatus nom.prov. is characterized by an umbonate pileus whose diameter is smaller than pileus length. The other species viz. T. robustus is characterized by the large pileus diameter (≤ 120 mm) almost double sized as stipe length. The ratio of pileus diameter to the stipe length is of significance in separating both the species from each other: almost double in T. robustus, half to that of stipe’s length in T. umbonatus. Similarly the narrowly clavate pleurocystidia with acute apices characterizing T. umbonatus are absent in T. robustus further separating both species. Basidiospores shape in both the species is somewhat different (broadly ellipsoid in T. umbonatus and broadly ellipsoid to oval in T. robustus) and can be used for separating both the species. Other characters including basidiospores size and pileipellis structures appears similar in both the species.

It also seems reasonable to compare the present Pakistani collection with T. rutilans falling close in molecular analysis. It is characterized by the presence of red-violet fibrils on the stipe (Holec & Kolařík, 2012) which are absent in both of the Pakistani species.

Ascomycota is presented by 11 species distributed among 5 genera. Caloscypha is a monotypic genus typified by C. fulgens. It is easily recognizable in the filed by having a characteristic bluing reaction on bruising. Microscopically it can be confirmed by the presence of globose ascospores and paraphyses with wavy ends. Aleuria aurantia (Pers.)

Fuckel resembling C. fulgens in its appearance is a close counterpart. The former can however be very easily distinguished by lacking characteristic bluing reaction characterizing C. fulgens. Microscopically both of these can be distinguished by the shape of the ascospores (globose in C. fulgens and elliptical and reticulate in A. aurantia)

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Helvella proved to be the largest genus of Ascomycota with six species being reported during present investigation. Among these Helvella leucopus, H. macropus, H. solitaria and H. maculata are new record for Pakistani mycobiota while H. crispa and H. elastica new records for the present study area.

The genus Verpa is represented by a single species viz V. bohemica (Krombh.) J. Schröt.,

(Ahmad et al., 1997). Recently another species, V. asiatica nom.prov. was proposed as new to science from Pakistan (Sana, 2016). Verpa conica presented here is the first record of the species and second in the genus from Pakistan. Verpa conica is characterized by more or less conical, smooth to bumpy pileus. This species can be easily differentiated from other two taxa from Pakistan (V. bohemica and V. asiatica nom. prov.) by the absence of furrowed pileus characterizing the both.

Otidea leporina is previously reported from Murree and Kaghan valley by Ahmad (1955a;

1956b; 1978) and Khalid (1992) and represent the single species of Otidea from Pakistan.

This is the fifth record of this species from Pakistan and first from present study area.

Morphologically, O. onotica (Pers.) Fuckel tends to be morphologically similar species and can be misidentified with O. leporina. However, O. onotica has a larger fruiting body, is more yellowish with a pinkish cast on inner surface when young, and has narrower basidiospores.

Xylaria hypoxylon is an already reported taxon from Pakistan which was reported from various locations in Punjab province by Ahmad (1969a; 1978) and Mirza & Qureshi

(1978). There are no records of this species from Khyber Pakhtunkhwa and this study reports and describe this species from District Swat for the first time. This species should be regarded as one of the widespread species in Pakistan inhabiting variety of habitats.

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Xylaria polymorpha is a common species reported frequently form North America (Rogers

& Callan, 1986). Morphologically it can be confused with X. longipes, however, X. polymorpha can de differentiated by roughened surface and larger ascospores with short, straight to somewhat oblique germpore. This taxon is already reported from Patriata and

Murre hills, parts of the Punjab province by Ahmad (1956b; 1978). The present report of this taxon is the fourth for Pakistan and first for Khyber Pakhtunkhwa province and present study area. There is no previous description and molecular data of this taxon from Pakistan and this study is providing these details for the first time.

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CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS

The present PhD project was based on the hypothesis that “Northern parts of Khyber

Pakhtunkhwa are less explored in terms of macrofungal surveys and there are many species awaiting documentation”. This was the first serious effort to explore the macrofungal diversity in the current study area. A total of 88 taxa were dealt, of which 35% are proposed as new to science. Beside the large number of previously undescribed taxa, 32% are new records for Pakistani mycobiota and 25% are new records for current study area. The research output include the publication of Albatrellus roseus, Clavariadelphus elongatus,

Descolea quercina, Gymnopilus swaticus and Phaecollybia pakistanica as new species and

Amanita pallidorosea and Lactifluus volemus as new records for Pakistan. Abortiporus biennis is published with the introduction of the genus name “Abortiporus” to the fungi of

Pakistan.

The use of molecular tools for identification of fungi is a newly adopted practice in Pakistan and the present study has been able to establish a fungal molecular systematic lab at the

Center for Plant Sciences and Biodiversity, University of Swat, Pakistan. The study also has been able to deposit voucher specimens to the Herbarium, University of Swat, Pakistan

(SWAT). Reference sequences of macrofungi dealt during present investigation are deposited to the GenBank to represent Pakistan.

The project results are clearly in accordance with our hypothesis and indicate that, present study area has a rich biodiversity of macrofungi but has remained neglected so far. Based on the large number of undescribed taxa encountered during present investigation, I am

419 convinced that there may be many more taxa awaiting documentation. Due to certain limitation all the potential sites were not surveyed and it is assumed that these sites may harbor some more interesting species. In the light of these conclusions, I propose the following recommendations for future studies

• More detailed and in-depth research should be carried out to explore the macrofungal wealth in the Northern areas of Khyber Pakhtunkhwa, Pakistan.

• The government should support projects regarding the exploration of this neglected but important group of fungi.

• Macrofungi are especially important for their food value and health of forest ecosystems, agriculture and forest departments are therefore require to collaborate with universities for potential benefits.

 An atlus of the reported macrofungi (fruiting bodies) is highly desirable, indicating, (1) poisonous fungi, (2) non-edible fungi, and, (3) edible fungi for the benefits of people.

420

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ANNEXURE-A (LIST OF PUBLICATIONS)

1. Hassan Sher, Junaid Khan, & Abdul Nasir Khalid. (2018).Clavariadelphus elongatus sp. nov. (Basidiomycota; Gomphales; Clavariadelphaceae) - Addition to the Club Fungi of Pakistan. Phytotaxa 365 (2): 182–188. 2. Munazza Kiran, Junaid Khan, Hassan Sher, Donald H. Pfister, & Abdul Nasir Khalid. (2018). Amanita griseofusca: A new species of Amanita in section Vaginatae from Malam Jabba, Swat, Pakistan. Phytotaxa 364 (2): 181–192. 3. Junaid Khan, Hassan Sher, & Abdul Nasir Khalid. (2018). Albatrellus roseus sp. nov. (Albatrellaceae; Basidiomycota), the first representative of the genus from Pakistan. Mycoscience 59, 12-17. 4. Junaid Khan, Hassan Sher, Arooj Naseer, & Abdul Nasir Khalid. (2018). Descolea quercina (Bolbitiaceae), a new species from moist temperate forests in Pakistan. MycoKeys 27: 65–76. 5. Junaid Khan, Munazza Kiran, Sana Jabeen, Hassan Sher, & Abdul Nasir Khalid. (2017). Gymnopilus penetrans and G. swaticus sp. nov. (Agaricomycota: Hymenogastraceae); a new record and a new species from northwest Pakistan. Phytotaxa 312 (1): 060–070. 6. Munazza Kiran, Junaid Khan, Arooj Naseer, Hassan Sher, & Abdul Nasir Khalid. (2017). Amanita pallidorosea in Pakistan and its ectomycorrhizal association with Quercus oblongata. Mycotaxon, 132, 799–811. 7. Junaid Khan, Hassan Sher, & Abdul Nasir Khalid. (2016). Phaeocollybia Pakistanica sp. nov. The first representative of the genus from Pakistan. Mycotaxon, 131 (4):899-896. 8. Junaid Khan, Hassan Sher, & Abdul Nasir Khalid. (2016). Abortiporus biennis: the third record of this fungus and a new genus name for the Pakistan funga. Österr. Z. Pilzk. 25 (2016) – Austrian J. Mycol. 25. 9. Junaid Khan & Hassan Sher. (2016). Lactifluus volemus (Fr.) Kuntze; An addition to the fungi of Pakistan. International Journal of Agriculture and Biology, International Journal of Agriculture and Biology, 18: 1095‒1097.

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