TAXONOMY, DIVERSITY AND PHYLOGENY OF SOME FROM PAKISTAN

A dissertation submitted to the University of the Punjab in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

IN BOTANY BY

NOUSHEEN YOUSAF DEPARTMENT OF BOTANY UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN AUGUST, 2014

Dedicated to My loving Mother & Father Without whom none of my success would be possible

TABLE OF CONTENTS Chapter Title Page No. no. Summary i Acknowledgements iv List of Plates vi List of Tables Ix List of Figures x 1.0 INTRODUCTION 1 2.0 AIMS AND OBJECTIVES 6 3.0 MATERIALS AND METHODS 7 3.1 Description of sampling sites 7 3.2 Collection of samples 7 3.3 Assigning labels to samples 7 3.4 Drying and preservation of sporocarps 8 3.5 Morpho–anatomical characterization 8 3.5.1 Observations of , capillitium and paracapillitium 8 3.5.2 Observations of peridium 8 3.6 Molecular analyses 9 3.6.1 Genomic DNA extraction 9 3.6.2 PCR, Agarose Gel Electrophoresis and Sequencing 9 3.7 Analyses of molecular data 10 3.7.1 Editing of sequences and BLAST analysis 10 3.7.2 Phylogenetic analyses 10 3.8 Analyses of Diversity 10 4.0 RESULTS 13 4.1 14 4.1.1 Taxa belonging to 14 4.1.2 Taxa belonging to 218 4.1.3 Taxa belonging to Phelloriniaceae 225 4.1.4 Taxa belonging to 228 4.1.5 Taxa belonging to 233 41.6 Taxa belonging to 241 4.1.7 Taxa belonging to Geastraceae 265 4.1.8 Taxa belonging to 299 4.1.9 Taxa belonging to 309 4.2 Taxonomy of Herbarium Gasteroid Taxa 311 4.3 Phylogeny of Gasteroid Fungi 333 4.4 Diversity of Gasteroid Fungi 369 4.4.1 Diversity of gasteroid fungi in different forests 369 4.4.2 Richness (SR) of sampling sites 371 4.4.3 Taxonomic diversity of gasteroid fungi 371 4.4.4 Species Frequency (SFl) of gasteroid fung 372 5.0 DISCUSSION 388 6.0 REFERENCES 430

i

SUMMARY

This investigation deals with taxonomy, phylogeny and diversity of gasteroid fungi from different areas of Pakistan. Sampling was carried out from the regions with varying climates, forests types and altitudes. During this study, 228 specimens were collected and identified using morphological and molecular techniques, comprising 101 different species of gasteroid fungi belonging to 20 genera, 9 families and 5 orders. Among these, fifty taxa (50) seem previously undescribed and twenty four (24) are new records for Pakistan.

Total gasteroid fungal taxa of studied in this investigation are seventy (70) represented by three families, i.e. Agaricaceae, Nidulariaceae and Phellorineaceae. Out of these, thirty eight (38) taxa seem new to science (17 of , 15 of , 3 of , 1 of , , and each) and sixteen (16) are first time reported from Pakistan.

Agaricaceae includes one (1) species of Arachnion (A. olivoflavus), one (1) species of (B. phalloides), twenty four (25) of Bovista (B. aestivalis, B. ahmadii nom. prov., B. areolata nom. prov., B. ayubiensis nom. prov., B. brunnescenta nom. prov., B. bovistoides, B. concinna, B. flavescens nom. prov., B. flavum nom. prov., B. fusca, B. himalaica, B. kreiselli nom. prov., B. lahorensis nom. prov., B. levisspora nom. prov., B. longispora, B. minima nom. prov., B. nigrescens var. rectapedicellata nom. prov., B. oblongispora, B. pakistanica nom. prov., B. pesudonigrescens nom. prov., B. plumbea, B. promontori, B. trachyspora and B. viridispora nom. prov., Bovista sp. (BPK92), four (4) of Calvatia (C. cyathiformis var. fragilis nom. prov., C. desosaiensis nom. prov., C. lilacina, C. pseudocyathiformis nom. prov.), 1 of Disciseda (D. hyalothrix), twenty eight (28) of Lycoperdon (L. albiceratum nom. prov., L. altimontanum, L. aurea-bruneum nom. prov., L. curtisii var. ovalisporum nom. prov., L. dermoxanthum var. album nom. prov., L. dermoxanthum var. flavum., L. excipuliforme, L. gilgitii nom. prov., L. japonicum, L. lahorense nom. prov., L. lahorense var. parvasporum nom. prov., L. lignum-bruneum nom. prov., L. lineum nom. prov., L. mammiforme, L. mammiforme var. magnasporum, L. molle, L. nigrescens, L. niveum, L. olivoflavum nom. prov., L. parvasporum nom. prov., L. perlatum, L. pratense var. flavum nom. prov., L. pseudoniveum nom. prov., L. pyriforme, L. sharanense nom. prov., L. sclerocystis nom. prov., L. utriforme, and Lycoperdon sp. (LPK74), one (1) of (M. arenarea), 1 of (P. pistillaris), five (5) of ii

Tulostoma (T. fimbriatum, T. morenoii nom. prov., T. squamosum, Tulostoma sp. 1 (TPK6), Tulostoma sp. 2 (TPK4). Nidulariaceae includes two (2) of Cyathus (C. olla, C. pakistanicus nom. prov.), and Phellorineaceae includes one (1) species of Phellorinia (P. herculeana).

Gasteroid fungal taxa of order are thirteen (13) belonging to three families, i.e. Diplocystaceae, Rhizopogonaceae, and Sclerodermataceae. Among these, three (3) taxa seem new to science (2 of and 1 of ) and five (5) are new records from Pakistan.

Diplocystaceae includes one (1) species of (A. hygrometricus). Rhizopogonaceae includes three (3) species of Rhizopogon (R. flavescens nom. prov., R. himalayensis nom. prov., R. roseolus). Sclerodermataceae includes six (6) species of (S. areolatum, S. bovista, S. chevelerii, S. dictyosporum, S. fuscum, Scleroderma sp. SPK21), 3 species of Pisolithus (P. flavescens, P. tinctorious, Pisolithus sp. PPK16).

Among twelve (12) gasteroid taxa of belong to one family, geastraceae which includes ten (10) species of (G. fimbriatum, G. himlayensis nom. prov., G. lycogalopsus nom. prov., G. pakistanicus nom. prov., G. parvulus nom. prov., G. patulus nom. prov., G. pseudolimbatum, G. pseudosaccatum nom. prov., P. saccatum, G. triplex), one (1) of (M. coliforme), one (1) of Radiigera (R. ayubiensis nom. prov.).

In this family, seven (7) species are presented here as new and one (1) taxon is first time being reported from Pakistan.

Phallales is represented by phallaceae in this study. It includes five (5) species of (P. ahmadii nom. prov., P. galericulatus, P. hadriani, P. roseus, and P. rubicundus). Among these, one (1) is proposed as a new species, and two (2) are new records for Pakistan.

One sequestrate i.e. R. pakistanica nom. prov., belonging to Russulaceae of is also included in this study and seems previously undescribed.

Some gasteroid specimens were taken as a loan from LAH herbarium, Pakistan, morphological descriptions of which are given at the end. iii

Taxonomy is correlated with phylogenetic analysis of each based on ITS-nr- DNA region. Diversity of gasteroid species studied in different forests is also given. During this investigation, maximum diversity of gasteroid species was found in Himalayan moist temperate forests.

Published work during this investigation has been appended as Annexure.

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ACKNOWLEDGEMENTS I would like to pay my heartiest gratitude to my respected teacher and PhD supervisor, Dr. Abdul Nasir Khalid for his continuous guidance, support, kindness and advices throughout the duration of my research work.

I am thankful to Prof. Dr. Muhammad Saleem, Chairman, Department of Botany, and Prof. Dr. Khan Rass Masood, Ex-Chairman, for providing me conducive environment and facilities for carrying out this research project.

Thanks are also due to Prof. Donald H. Pfister (Harvard University, Cambridge, MA, USA) for providing me excellent opportunity to work in his lab under his supervision, allowing me to use library and herbarium facilities for six months.

I am indebted to Higher Education Commission of Pakistan for funding this research project and awarding me the scholarships under indigenous 5000 fellowships (Batch IV) and IRSIP (International Research Support Initiative Program).

I am thankful to my foreign collaborators, Prof. Hanns Kreisel (Germany), Prof. F. D. Calonge (Spain), Prof. G. Moreno (University of Alcalá, Madrid, Spain), Dr. Scott. T. Bates (USA), Ms. Larissa Trierveiler-Pereira (), Dr. Takamichi Orihara (Curator, Kanagawa Prefectural Museum of Natural History, Japan) for their help, valuable comments and suggestions for improvement of my work and publications.

I must thank Genevieve E. Tocci (Curatorial Assistant, Harvard University Herbaria) for helping me getting access to gasteroid collections.

Thanks are also due to Dr. Abdul Rehman Niazi (Deptt. of Botany, PU, Lahore) and Dr. Najam-ul-Sehar Afshan (Center for Undergraduate Studies, PU, Lahore) for their moral support and assistance in field tours.

I extend my thanks to Dr. Katherine Lobuglio for her guidance and supervision in molecular work at Pfister lab, Harvard University, USA.

I sincerely thank Prof. Meredith Blackwell (Radcliffe Institute for Advanced Study, Harvard University, USA), Dr. Rosane Healy (Postdoctoral Fellow, Pfister Lab. USA), Dr. Teresa Iturriaga (Venezuela), Mr. Danny Haelewaters, and Mr. Jason for their affection and simulating discussions during my work at Harvard University. v

I highly acknowledge my friends especially Ms. Syeda Bint-e-Zahira, Ms. Aamna Ishaq, and Ms. Sana Jabeen for always being there for me. I also acknowledge all my lab fellows especially Dr. Muhammad Fiaz, Dr. Abdul Razaq, Dr. Sobia Ilyas, Dr. Samina Sarwar, Dr. M. Hanif, Ms. Malka Saba, and Ms. Tasleem Javaid for their cheerful company.

This project would have impossible without the prayers and support of my parents. I am thankful to my sisters, brothers, brother in law, and all my family for their endless support and unconditional love.

Nousheen Yousaf vi

LIST OF PLATES Plate no. Title Page no. 1–3 Morphology of Arachnion olivoflavus 15–17 4–5 Morphology of 19–20 6–7 Morphology of Bovista areolata 22–23 8–10 Morphology of B. bovistoides 25–27 11–13 Morphology of B. fusca 29–31 14–16 Morphology of B. kreiselli 33–35 17–18 Morphology of B. nigrescens var. rectapedicellata 37–38 19–20 Morphology of B. plumbea 41–42 21–22 Morphology of B. pseudonigrescens 44–45 23–25 Morphology of B. aestivalis 47–49 26–27 Morphology of B. ahmadii 51–52 28 Morphology of B. ayubiensis 54 29–30 Morphology of B. brunnescenta 56–57 31–33 Morphology of B. concinna 59–61 34–35 Morphology of B. flavescens 63–64 36 Morphology of B. flavus 66 37–39 Morphology of B. himalaica 68–70 40–41 Morphology of B. lahorensis 72–73 42–43 Morphology of B. levispora 75–76 44–45 Morphology of B. longispora 78–79 46–47 Morphology of B. minima 82–83 48–49 Morphology of B. oblongispora 84–85 50–51 Morphology of B. pakistanica 87–88 52 Morphology of B. promontorii 90 53–54 Morphology of B. trachyspora 92–93 55–56 Morphology of B. viridispora 95–96 57–58 Morphology of Bovista sp. (BPK92) 98–99 59–60 Morphology of var. fragilis 101–102 61–63 Morphology of C. deosaensis 104–106 64–66 Morphology of C. lilacina 109–111 67–68 Morphology of C. pseudocyathiformis 113–114 69–70 Morphology of Disciseda hyalothrix 116–117 71–72 Morphology of Lycoperdon pyriforme 120–121 73–74 Morphology of L. japonicum 123–124 75 Morphology of L. utriforme 126 76 Morphology of L. parvasporum 128 77–78 Morphology of L. perlatum 131–132 79–80 Morphology of L. albiceratum 134–135 81–82 Morphology of L. altimontanum 137–138 83–84 Morphology of L. aurea–bruneum 140–141 85–86 Morphology of L. dermoxanthum var. album 143–144 87–88 Morphology of L. dermoxanthum var. flavum 146–147 89 Morphology of L. excipuliforme 149 90–91 Morphology of L. lignum–brunneum 151–152 92–93 Morphology of L. lineum 154–155 94 Morphology of L. mammiforme 157 95 Morphology of L. mammiforme var. magnasporum 159 96 Morphology of L. molle 161 97–99 Morphology of L. nigrescens 163–165 100–101 Morphology of L. niveum 167–168 vii

Plate no. Title Page no. 102–103 Morphology of L. pseudoniveum 170–171 104–105 Morphology of L. sclerocystis 173–174 106–107 Morphology of L. sharanense 176–177 108 Morphology of Lycoperdon sp. (LPK74) 179 109–111 Morphology of L. curtisii var. ovalisporum 181–183 112 Morphology of L. gilgitii 185 113–115 Morphology of L. lahorense 187–189 116 Morphology of L. lahorense var. parvasporum 191 117–118 Morphology of L. olivoflavum 193–194 119–120 Morphology of L. pratense var. flavum 196–197 121–122 Morphology of 199–200 123–125 Morphology of 203–205 126 Morphology of Tulostoma fimbriatum 207 127 Morphology of T. morenoii 209 128–129 Morphology of T. squamosum 211–212 130–131 Morphology of Tulostoma sp. 1 (TPK6) 214–215 132 Morphology of Tulostoma sp. 2 (TPK4) 217 133–135 Morphology of Cyathus olla 219–221 136–137 Morphology of C. pakistanicus 223–224 138–139 Morphology of Phellorinia herculeana 226–227 140–142 Morphology of 230–232 143 Morphology of Rhizopogon flavescens 234 144–145 Morphology of R. himalayensis 236–237 146–147 Morphology of R. roseolus 239–240 148–149 Morphology of Pisolithus flavus 242–243 150–151 Morphology of P. tinctorious 245–246 152 Morphology of Pisolithus sp. (PPK16) 248 153–154 Morphology of Scleroderma areolatum 250–251 155–156 Morphology of S. bovista 253–254 157–158 Morphology of S. chevalieri 256–257 159 Morphology of S. dictyosporum 259 160–161 Morphology of S. fuscum 261–262 162 Morphology of Scleroderma sp. (SPK21) 264 163 Morphology of 266 164–165 Morphology of G. himalayensis 268–269 166–168 Morphology of G. lycogalopsus 271–273 169 Morphology of G. pakistanicus 275 170–171 Morphology of G. parvulus 277–278 172–173 Morphology of G. patulus 280–281 174 Morphology of G. pseudolimbatum 283 175 Morphology of G. pseudosaccatum 285 176–177 Morphology of G. saccatum 287–288 178–179 Morphology of G. triplex 290–291 180–181 Morphology of Myriostoma coliforme 294–295 182–183 Morphology of Radiigera ayubiensis 297–298 184 Morphology of Phallus ahmadii 300 185 Morphology of P. galericulatus 302 186 Morphology of P. hadriani 304 187 Morphology of P. roseus 306 188 Morphology of P. rubicundus 308 189 Morphology of Russula pakistanica 310 190–191 Morphology of 312–313 viii

Plate no. Title Page no. 192–193 Morphology of C. cyathiformis 315–316 194–196 Morphology of Disciseda cervina 318–320 197 Morphology of Lycoperdon pusillum 322 198 Morphology of Schizostoma mundkurii 324 199 Morphology of Scleroderma flavidum 326 200 Morphology of Tulostoma evanescens 328 201 Morphology of T. volvulatum 330 202 Morphology of T. vulgare 332

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LIST OF TABLES

Table Title Page no. no. 1. Sequences of primers used for PCR/sequencing reactions in this study. 12 2. Gasteroid fungal species collected from Himalayan moist temperate 373 forest. 3. Gasteroid fungal species collected from Subtropical forests. 375 4. Gasteroid fungal species collected from Tropical forests. 376 5. Gasteroid fungal species collected from Artificial/ Irrigated forests. 377 6. Gasteroid fungal species collected from Alpine pasture 377 7. Species Richness (SR) of sampling sites. 377 8. Species frequency (SFl) of gasteroid fungi 379

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LIST OF FIGURES Fig. no. Title Page no. 1. Molecular phylogenetic analysis of Arachnion and Disciseda 335 2. Molecular phylogenetic analysis of Battarrea spp. 337 3. Molecular phylogenetic analysis of subgenus Bovista of Bovista 339 4. Molecular phylogenetic analysis of subgenus Globaria of Bovista 340 5. Molecular phylogenetic analysis of Calvatia spp. 342 6. Molecular phylogenetic analysis of subgenera , 344 , Lycoperdon and of genus Lycoperdon 7. Molecular phylogenetic analysis subgenus Utraria of Lycoperdon 346 8. Molecular phylogenetic analysis of Podaxis and related taxa 348 9. Molecular phylogenetic analysis of Tulostoma spp. 350 10. Molecular phylogenetic analysis of Cyathus spp. 352 11. Molecular phylogenetic analysis of Astraeus spp. 354 12. Molecular phylogenetic analysis of Rhizopogon spp. 356 13. Molecular phylogenetic analysis of Pisolithus spp. 358 14. Molecular phylogenetic analysis of Scleroderma spp. 360 15. Molecular phylogenetic analysis of Geastrum spp. 362 16. Molecular phylogenetic analysis of Geastrum lycogalopsus 364 17. Molecular phylogenetic analysis of Phallus spp. 366 18. Molecular phylogenetic analysis of Russula spp. 368 19. Graphical presentation of diversity of gasteroid fungi in different 382 forests of Pakistan. 20. Graphical presentation showing Species Richness (SR) of visited 382 forests of Pakistan. 21. Graphical presentation of gasteroid fungal taxa studied. 383 22. Graphical presentation of percentage of gasteroid fungal taxa studied. 383 23. Graphical presentation of gasteroid fungal taxa studied in families of 384 Agaricales 24. Graphical presentation of percentage of fungal taxa studied in 384 families of Agaricales 25. Graphical presentation of families studied in Boletales 385 26. Graphical presentation of percentage of taxa studied in families of 385 Boletales 27. Graphical presentation of gasteroid fungal taxa studied in Geastrales 386 28. Graphical presentation of gasteroid fungal taxa studied in 386 29. Graphical presentation of gasteroid fungal taxa studied in Russulales 386

30. Graphical presentation of distribution of no. of gasteroid fungal taxa 387 studied in nine families. 31. Graphical presentation of distribution of no. of gasteroid fungal taxa 387 in each genus.

1

1.0. INTRODUCTION

Gasteroid fungi formerly known as “Gasteromycetes” is a heterogeneous group of fungi belonging to phylum . This group of fungi produces spores inside their fruit bodies rather than on their outer surface (Kirk et al., 2008). Species of this group form strikingly visible fruiting bodies but in all cases spores mature internally and do not discharge forcibly like other members of Basidiomycota, rather passively by variety of methods (Pegler et al., 1995; Reijnders, 2000).

This is an artificial assemblage because the fungi included in this group are polyphyletic in origin but still mycologists find it convenient to study these fungi under one group because of morpho-anatomical resemblance between the members of this group. Major sub groups that are included in gasteroid fungi are puff balls, earth balls, earth stars, stink horns, birds nest fungi and false etc. (Ellis & Ellis, 1990).

Gasteroid fungi are diverse and cosmopolitan in distribution (Dring, 1980) occurring in temperate to tropical regions but commonly found in warmer areas. These fungi are also reported from plains with lower altitudes to hilly and mountainous regions with high altitude, however, there are some species which are restricted to the deserts and sand dunes of temperate areas (Miller & Miller, 1988; Muhsin et al., 2012; Yousaf et al., 2012a; Yousaf et al., 2013c). They have been recorded from all over the world including Asia, , Europe, North America and (Cunningham, 1944; Smith, 1951; Kreisel, 1967; DeVilliers et al., 1989; Ellis & Ellis, 1990; Yoshimi & Hagiwara, 1992; Ahmad et al., 1997; Anderson et al., 1998; Priest & Lenze, 1999; Binder & Bresinsky, 2002; Calonge et al., 2004; Khalid & Iqbal, 2004; Iqbal et al., 2006; Canon & Kirk, 2007; Kasuya, 2007; Kasuya & Katumoto, 2008; Coetzee & van Wyk, 2009; Cortez et al., 2009; Moreno et al., 2009; Phosri et al., 2009; Sermenli & Isiloglu, 2009; Piña et al., 2010; Jeppson et al., 2012; Alves & Cortez, 2013; da Silva, 2013; Phosri et al., 2013, Yousaf et al., 2014).

Members of this group are saprotrophic growing on dead decaying organic matter or rotten wood and logs (Richter et al., 2003). Their fruiting bodies usually appear during rainy season from June to October. The major importance of this group lies in its role to form ectomycorrhizal association with coniferous and broad-leaved trees e.g., Astraeus, Pisolithus, Rhizopogon and Scleroderma (Bandou et al., 2006; Chen et al., 2006; Wilson 2

et al., 2012). However, some of the members of this group are non ectomycorrhizal (Sims et al., 1997; Turjaman et al., 2005; Webster & Weber, 2007). Several members of this group have wide spread use amongst the various cultures worldwide especially as sources of food when young and in traditional medicines (Deng et al., 2007; Zhao et al., 2007; Coetze & van Wyk, 2009). They have also been used as a source for the compounds with and activity (Roland et al., 1960; Lam et al., 2001; Jonathan & Fasidi, 2003; Stanikunaite et al., 2008; Tsay et al., 2009; Chung et al., 2010; Ramesh & Pattar, 2010; Redzic et al., 2010; Rogers, 2011).

Studies on gasteroid fungi may date back to 1753 when Linnaeus named some members belonging to that group. First scientific and critical treatment to gasteriod fungi was given by CH Persoon when he wrote “Synopsis methodica fungorum” of 1801. In 1821, the name Gasteromycetes was given to this group of fungi by in “Systema Mycologicum of 1821”. Later on, conspicuous shape and appearance of the basidiomata in gasteromycetes have long attracted the attention of mycologists who studied this group morphologically, wrote many books and developed keys for identification of taxa in Gasteromycetes. Several books, keys and descriptions on gasteroid fungi are available (Johnson, 1928; Cunningham, 1932; Zeller, 1949; Ahmad, 1952; Kreisel, 1967; Miller & Miller, 1988, 2006; Ellis & Ellis, 1990; Pegler et al., 1995).

Although by early twentieth century scientists began to produce alternative taxonomies based on evolutionary theories but till the middle of twentieth century, same convenient division “Gasteromycetes” was retained and followed and same traditional morphological methods were being used to study this group. In late twentieth century, the introduction of molecular techniques in the field of fungi revolutionized the concept of Gasteromycetes which was previously based on traditional and morphological taxonomy. Now days using molecular analysis has established the fact that members of this group have polyphyletic origin (Hibbett et al., 1997). It is confirmed that “Gasteromycetes” represents the heterogeneous assemblage of the species derived from multiple ancestors (Krüger et al., 2001; Miller et al., 2001; Binder & Hibbett, 2006; Hosaka et al., 2006). So they can collectively referred to as a single group, i.e., “Gasteroid fungi” but cannot be classified as same group, i.e., “Gasteromycetes” (Hibbett et al., 1997; Krüger et al., 2001; Binder & Bresinsky, 2002). 3

Although the artificial nature of this group has been known or suspected for many decades, it still comes as a shock to the most mycologists to realize just how strongly convergent the evolution of this fungi have been (Webster & Weber, 2007).

All gasteroid fungi can be grouped under four orders based on molecular phylogenetic analysis i.e. Agaricales, Boletales, Geastrales and Phallales. Some of the gasteroid forms are also included in Russulales. Members of Lycoperdaceae are forming a separate gasteroid lineage within Agaricaceae (Hibbet et al., 1997; Krüger et al., 2001; Binder & Bresinsky, 2002). Members of sclerodermataceae now confirmed to be phylogenetically linked to boletales (Binder & Bresinsky, 2002). Epigeous and hypogeous gasteromycetes occur in four of the 8 of homobasidiomycetes recognized by Hibbett & Thorn in 2001 (Kirk et al., 2008).

Molecular analysis of fungi was previously based upon the ITS, LSU, SSU of rDNA as genetic markers for identification and delimitation between the genera and within the species and phylogenetic placements (Kruger et al., 2001; Binder & Bresinsky, 2002; Louzan et al., 2007; Larsson & Jeppson, 2008; Fangfuk et al., 2010a; Jeppson et al., 2012). Use of many other genes of nuclear and mitochondrial DNA is now becoming a popular approach. Multigene phylogenies of gasteroid fungi are being derived to produce authentic taxonomies of this group in recent years (Binder & Hibbett, 2006; Hosaka et al., 2006, Giachini et al., 2010).

In Pakistan, exploration efforts for this group of fungi never remained uniform; it only faced its climax from 1939 to 1956 as afterwards number of published reports of gasteromycetes became scant (Ahmad, 1952; Ahmad et al., 1997; Sultan et al., 2001; Khalid & Iqbal, 2004; Razzaq & Shahzad, 2004, 2007; Iqbal et al., 2006; Sultana et al., 2007; Moreno et al., 2009, 2010b; Yousaf et al., 2012a, b; Yousaf et al., 2013a, b, c; Yousaf et al., 2014).

Pakistan is one of the floristically richest country, located in between latitudes 23° and 37° N (a small area is north of 37°), and longitudes 61° and 78° E (a small area is west of 61°) with a wide range of climate: tropical to temperate with arid conditions existing in the coastal south, characterized by a monsoon season with adequate rainfall and a dry season with lesser rainfall (FAO, 1987; Pakistan online encyclopedia, 2004). Heavy rainfall supports the growth of gasteroid fungi both in temperate and tropical regions. Variations in 4

the climate support a wide variety of ecosystem and species diversity. It also allows a wide variety of fungi to flourish in the region. In addition to climatic variations, also a wide range of habitat exists in Pakistan, from coastal regions along the Arabian Sea, to arid scrubland in the Punjab region, to high altitude forests, and tundra of the Himalayas. Climatic conditions of Pakistan are variable and range from extremely arid to temperate which are ideal and offer a rich heritage of biological diversity in rainy seasons. Considering these factors, it is not surprising that a biologically diverse and species rich assortment of gasteroid fungi is present in this country.

A big contribution in this field was made by renowned Pakistani mycologist, Late Dr. Sultan Ahmad, who remained engaged in the study of gasteromycetes for several years in last century. He started working on this group fifteen years before independence and continued till 1980. At the time of partition, he migrated from to Pakistan leaving behind a rich collection of gasteroid fungi. Later on, work was resumed and he wrote a hand book, “Gasteromycetes of West Pakistan” in 1952 (Ahmad, 1952). This book is still being used as a source of taxonomic description and also for comparing the unidentified gasteroid fungi in Pakistan and neighboring countries.

Ahmad contributed a significant number of species to this group and remained the sole contributor till 1964 (Ahmad et al., 1997). He published about 91 species. He reported 16 species from different regions of subcontinent before independence in 1939 and later in 1950, among which 7 species belong to genus Tulostoma Pers., and 1 species to each genus i.e. Colus Cav., Cyathus Haller, Dictyophora Desvaux, Disciseda Czem., Lanopila Fr., Fr., Phellorinia Berk., and Sphearobolus Tode (Ahmad 1939a, 1939b, 1950a, 1950b, 1950c).

Most of the Ahmad’s contribution to this field was accomplished in 1952 when he contributed 48 species to the gasteromycetous flora of Pakistan. In these reports, 13 and 10 spp. belong to Tulostoma and Lycoperdon Pers., respectively, 4 to Geastrum Pers, 3 to Phallus Junius ex Linnaeus and Bovista Pers. each, 2 to Melanogaster Corda and Schizostoma Ces. Et de Not., and 1 belongs to each genus Battarrea Pers, Calvatia Fr., Mycenastrum Desv., and Scleroderma Pers. Later on, twenty one (21) new records to this group were added by Ahmad (1952, 1956, 1962, 1964, 1969a, 1969b). After that till 1978, there was no published report of gasteroid fungi from Pakistan. Mirza and Qureshi (1978) contributed 1 species of genus Lycoperdon to myco-flora of Pakistan. Ahmad (1980) 5

contributed 2 new records to gasteromycetes flora of Pakistan (Ahmad et al., 1997). During the last and first decade of twentieth and twenty first century respectively, eleven (11) gasteroid fungi were added as new species to the world mycoflora from Pakistan (Yoshimi & Hagiwara, 1992; Khalid & Iqbal, 1996; 2004; Iqbal et al., 2006; Moreno et al., 2009, 2010a, b; Yousaf et al., 2013a).

In short, gasteroid fungi have been documented from the nation in several publications (Long & Ahmad, 1947; Ahmad, 1952; Yoshimi & Hagiwara, 1992; Khalid & Iqbal, 1996, 2004; Ahmad et al., 1997; Sultan et al., 2001; Razzaq & Shahzad, 2004, 2007; Iqbal et al., 2006; Sultana et al., 2007; Moreno et al., 2009, 2010b; Yousaf et al., 2012a, b, Yousaf et al., 2013a, b, c, Yousaf et al., 2014). In total, about 120 species belonging to approximately 30 genera have been reported from Pakistan so far.

Paksitani gasteroid flora has never been analyzed phylogenetically by mycologists of the country. As far as attempts regarding molecular and phylogenetic work of gasteroid fungi in Pakistan are concerned, there are only two reports about the use of molecular tools for the identification of gasteroid fungi (Moreno at al., 2009, 2010b) before this study. During this investigation, one new gasteroid taxon have been published using molecular tools (Yousaf et al., 2013a). This investigation is first serious attempt to see the phylogenetic placement and relationships of gasteroid fungi of the country with world gasteroid fungal flora on molecular basis. During present study, 101 gasteroid taxa belonging to 20 genera have been documented which were collected from different areas of Pakistan. A total of 129 DNA sequences (127 of ITS-nrDNA, 2 of LSU-nrDNA) were generated from the fruiting bodies as a supporting tool with morphology and for producing phylogenetic trees. Diversity of gasteroid fungi reported in this work has been calculated in terms of Species Richness (SR) in different forest types of Pakistan.

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2.0. AIMS AND OBJECTIVES Aims and the objectives of this project were;  To collect and identify the gasteroid fungi from different regions of Pakistan.  To obtain data on the occurrence, habitat and diversity of gasteroid fungi in Pakistan.  Up gradation of the existing descriptions of gasteroid fungi of Pakistan.  Introduction of molecular techniques for study of gasteroid fungi in Pakistan.

 To combine the use of conventional and modern techniques for the accurate identification of gasteroid fungi.  Assessing phylogenetic relationships of gasteroid fungi known from different regions of Pakistan.  To find out phylogenetic position of Pakistani gasteroid fungal flora among world fungi.

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3.0. MATERIALS AND METHODS

3.1. Description of sampling sites

For sampling of gasteroid fungi, periodic and frequent field tours were conducted during rainy seasons (June to October) from 2008–2013. Sampling sites, which were hypothesized to support rich diversity of fungi were selected. Sampling was carried out from plains, deserts and hilly areas. Vegetation of sampling sites varied from irrigated plantations to tropical, subtropical forests, moist temperate forests, to high tundra of Himalayas reaching up to alpine pastures.

Major sampling sites includes some regions of Khyber Pakhtunkhwa province: Abbottabad district (Ayubia National Park, Khanspur, Simla hill, Thandiani); Galyat of Pakistan (Nathia Gali); Swat district (Kalam, Malam Jabba, Mankial, Miandam, Ushu); Kaghan Valley (Battakundi, Nadibunglaw, Sharan); Mansehra district (Batrasi, Chattar plains, Jabori, Musaka Musala, Ucharhi, Ughi forest); Punjab province: Bahawalpur district (Lal Sohanra Park, Cholistan desert); Chakwal district; Dera Ghazi Khan district (Fort Munro); Kasur district (Changa Manga forest); Khushab district (Sakesar); Lahore district; Mianwali district; Sialkot district (Daska); Azad Kashmir (Neelum Valley, Sharda); Gilgit- Baltistan province (Fairy Meadows, Deosai National Park); Sindh province (Nawabshah).

3.2. Collection of samples

Basidiomata of gasteroid fungi were collected with the help of a sharp digger (knife or small trowel) avoiding the loss of rhizomorphs. Field notes related to morphology, habitat, gregariousness, and host tree were prepared at the time of collection. Both mature and young fruiting bodies were collected for the same specimen whenever available. Basidiomata have dry, fleshy to very fleshy textures so those were kept in separate boxes.

3.3. Assigning labels to samples

Collected specimen were photographed and labelled by giving them special codes for identification. Each label included the locality, date of collection and collector’s name, tentative name, habitat, host tree/ of vegetation present around them. A log of all the collected samples with their details was prepared for each year.

8

3.4. Drying and preservation of sporocarps

Samples were dried using fan heater/dryer for the purpose of preservation. Collections were kept with their field labels while drying to avoid mixing. The specimens were preserved in paper boxes (containing blotting papers), and in zip-lock bags. Samples are then brought back to laboratory for further analysis.

3.5. Morpho-anatomical characterization

Macroscopic analysis included recording of macro-morphological data by the use of hand lens or stereo microscope. Color and shape of , texture of peridium (exoperidium, endoperidium), surface ornamentation, color and texture of , presence or absence of sub-gleba/ sterile base, and morphology of rhizomorphs were observed.

3.5.1. Observations of spores, capillitium and paracapillitium

Small amount of gleba was mounted on a slide using 5%KOH, H2O, Lacto-phenol as mounting media. Trypan blue was used to observe the hyaline paracapillitial hyphae and ornamentation.

3.5.2. Observations of peridium

A small portion of exoperidium was scratched from the basidiomata and observed using 5% KOH and Melzer’s reagent. Hard tissue of the basidioma was softened by dipping in 95% aqueous ethanol. On the other hand, arrangement of mesoperidium and endoperidium was observed by cutting the free hand sections of peridium and by mounting in 5% KOH.

Measurements of all above mentioned characters were made using Carl-Ziess Digital Ocular Micrometer. At least thirty mature spores were randomly measured for each specimen.

Images of the were drawn at 100× and rest of the characters were drawn at 40×. LM (Light Microscope) phototographs and Scanning Electron Micrographs (SEM) were also taken for some taxa. Standard taxonomic and descriptive methodologies for gasteroid fungi were followed (Ahmad, 1952; Bates, 2004). Ainsworth and Bisby’s Dictionary of the Fungi (Kirk et al., 2008) and CABI (http;//www.indexfungorum.org/Names/Names.asp) and literature were followed for 9

accurate taxonomy of taxa. All the specimens were deposited in LAH Herbarium, Department of Botany, University of the Punjab, Lahore, Pakistan.

3.6. Molecular analysis

3.6.1 Genomic DNA extraction

For DNA analysis, small portion of glebal material containing spores and capillitium was removed from interior part of dried basidioma. Genomic DNA was extracted using three methods, modified CTAB method (Gardes & Bruns, 1996), Extract- N-Amp™ Plant PCR Kit by Sigma (Product no. XNAP2), DNeasy Plant Mini Kit by Qiagen (Cat. no. 69104). Standard manufacturer’s protocols were followed for these kits.

3.6.2. PCR, Agarose Gel Electrophoresis and Sequencing

For PCR, serial dilutions were done for each DNA extraction/template (1:10, 1:100, and 1:1000). PCR was performed to amplify the Internal Transcribed Spacers (ITS-1 and ITS-2) in addition to the 5.8S regions of the nr-DNA gene using different primer pairs (ITS1-F & ITS4/ ITS1 & ITS4). PCR conditions for ITS-rDNA included: Initiation denaturation at 95.0° for 2:00 min, then 95.0° for 1:00 min, 52° for 1:00 min, 72° for 00: 45 s, and 72° for 10:00 min followed by a 4° soak.

PCR was also performed to amplify the LSU region of nr-DNA using LROR & LR5 primer pairs for some specimens. PCR conditions for LSU-rDNA included: 35-40 cycles at 94° for 00:30 s, 60° for 00:30 s, and 72° for 1:30 min, followed by a 4° soak. Sequences of the primers are given in Table 1. LSU and ITS regions were amplified using ECONO Taq Polymerase by Lucigen (Catalogue # 30031).

PCR products were then loaded on 1% Agarose gel stained with SYBR Safe DNA Gel Stain/ ethidium bromide using 1kb DNA ladder. Bands were then visualized under UV in Gel documentation system with default settings.

After necessary amplification, PCR products were purified using QIAquick PCR Purification Kit–Qiagen (Catalogue # 28104) following manufacturer’s protocol. For sequencing reactions, Cycle sequencing, using BigDye terminator (Applied Biosystems, Foster city, CA), was done in a Peltier Thermal Cycler PTC-200 (Pfister lab, Harvard University, MA), using the following program: 96° for 3 min, then 25 cycles of 96° for 10 s, ramping 1.0/s to 60°, 60° for 4 min, followed by a 4° soak. Cycle sequencing 10

reaction volumes were 8 µl. Sequencing reactions were purified using CENTRI-SEP spin columns-Princeton separations (Product no. CS-901) following manufacturer’s protocol. Products were sequenced with ITSl/LROR and ITS4/LR5 with the ABI BigDye Terminator Sequencing Kit (v3.1) for ITS and LSU sequences, respectively. Sequences were read using an AB11373Oxl capillary sequencer (Applied Biosystems, Foster City, CA, USA) at the Harvard University, Cambridge, MA, USA. Some of the PCR products were sent to Macrogen, South Korea for purification and sequencing. For some specimens, more than one sequence were obtained from different collections.

3.7. Analyses of molecular data

3.7.1. Editing of sequences and BLAST analysis

After getting the results of sequencing, sequences were edited and contigs were assembled using Sequencher 3.0 (GeneCodes Inc., Ann Arbor, Michigan, USA). BioEdit software was also used for editing the sequences manually (www.mbio.ncsu.edu/bioedit/bioedit.htm). The consensus sequences were then BLAST (Basic Local Alignment Search Tool) searched in NCBI database (Genbank) for the closely related sequences.

3.7.2. Phylogenetic analyses

Phylogenetic trees were constructed for each genus during this study. Sampling of taxa for phylogenetic analyses was based upon retrieving closely related sequences from Genbank after BLAST search. Sequences from already published data were tried to include in each alignment to bring the accuracy. At least one out group sequence was selected for each tree. Multiple alignments were performed using MUSCLE alignment software (Edgar, 2004). Phylogenetic tree were constructed with the Maximum Likelihood algorithm and Jukes & Cantor (1969) model of sequences evolution using Model testing feature of MEGA5 software (Tamura et al., 2011). Bootstrap consensus tree inferred from 1000 replicates is presented here to describe the phylogeny of taxa and corresponding bootstrap values >50 % are cited in the tree.

3.8. Analysis of Diversity

Diversity in the terms of Species Richness (SR) of sampling sites and Species

Frequency (SFl) was calculated using formulas (Chen, 2006). 11

Species Richness (SR)

SR = No. of taxa in one locality/ Total no. of taxa

Species Frequency (SFl)

SFl = (Sl /Stl ) × 100 Where,

Sl = No. of sites from which species is collected

Stl = Total no. of sampling sites 12

Table 1. Sequences of primers used for PCR/sequencing reactions in this study.

Serial No. Primer Sequence Nature

01 ITS1 TCCGTAGGTGAACCTGCGG Forward 02 ITS1-F CTTGGTCATTTAGAGGAAGTAA Forward

03 ITS4 TCCTCCGCTTATTGATATGC Reverse

04 LROR ACCCGCTGAACTTAAGC Forward 05 LR5 TCCTGAGGGAAACTTCG Reverse

13

4.0. RESULTS

During this investigation, 101 taxa of gasteroid fungi have been described belonging to 9 families and 5 orders. Taxonomy, phylogeny and diversity of gasteroid taxa studied are given in this chapter.

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4.1. Taxonomy (Morphological characterization)

Taxonomy of gasteroid taxa based on morphology is presented here family wise.

4.1.1. Taxa belonging to Agaricaceae

Arachnion olivoflavus nom. prov. (Plates 1–3) Etymology: On the basis of olivaceous yellow color of basidioma and spores

Basidiomata globose, yellow to brown, up to 15 mm in diam., solitary, attached to the substratum by short, mycelial base. Peridium double. Exoperidium adherent to endoperidium, persistant. Endoperidium fragile, broken away easily. Ostiole not observed. Gleba yellow, pulverulent.

Basidiospores globose to sub-globose, smooth in 3% KOH, asperulate in Trypan blue, olivaceous, 3.3–4.9 × 3.3–4.4 µm in diam., with a short stump of pedicel (up to 0.6 µm). Eucapillitium olivaceous, branched, pores numerous, big, slit like sometimes (up to 0.6 µm), tips attenuate, sub-undulate, aseptate, up to 4.4 µm in diam., with wall thickness up to 0.6 µm. Exoperidium composed of globose to sub-globose sphaerocysts, 16–35 × 8.0–32 µm in diam. Endoperidium composed of hyaline to olivaceous, septate, thin walled tightly packed hyphae, up to 5 µm.

Material examined: PAKISTAN: Punjab, Narowal, Naula Basantar, 10th July, 2013, solitary, on ground, at 261m (857 ft) a.s.l, N. Yousaf, BPK59 (Holotype) (LAH1000027). 15

A B

C D

E F

Plate 1. Figs. A–F. Arachnion olivoflavus (BPK59). A & B. Mature basidioma. C. Basidiospores. D. Porous eucapillitial . E. Exoperdial hyphal elements. F. Endoperidial hyphae. Bar: = 0.25 cm for A & B, = 7 µm for C, = 16 µm for D, = 10 µm for E, = 13 µm for F. 16

A

B C Plate 2. Figs. A–C. LM photographs of Arachnion olivoflavus (BPK59). A. Basidiospores. B & C. Olivaceous yellow, porous eucapillitial hyphae. Bar: = 7.5 µm for A, = 9 µm for B & C. 17

A

B

Plate 3. Figs. A–B. LM photographs of Arachnion olivoflavus (BPK59). A. Exoperdial hyphal elements. B. Septate endoperidial hyphae. Bar: = 10 µm for A, = 18 µm for B. 18

Battarrea phalloides (Dicks.) Pers., Syn. meth. fung. (Göttingen) 1: xiv, 129 (1801)

(Plates 4–5)

Basidiomata consists of spore case and a stalk, up to 170 mm long. Spore-case discoid, concave edges curving inward. Peridium double. Exoperidium cream to light brown, thin, semi-decidous, rough due to adherent sand particles. Endoperidium thick. Stalk tough, woody, scaly, firmly attached to underside of spore case, 155mm long, pale yellow, encrusted with sand particles. Volva lost during collection. Gleba reddish brown, pulverulent, exposed by rupturing of peridium.

Basidiospores globose to sub globose, ornamented, finely verrucose, dark brown, up to 7 µm in diam.

Material examined: PAKISTAN: Punjab, Bahawalpur district, Cholistan desert, 29th Sep.

2005, solitary, at 461 m (1512 ft) a.s.l., A. R. Niazi, ARN108 (BPK69) (LAH LAH290805); Sindh, Nawabshah, at 107 ft a.s.l., K. Sultana, BPK93 (LAH1000028).

Comments: B. phalloides is commonly called as scaly stalked puff ball (Arora, 1986), usually found growing solitary to gregarious in arid, semi-arid to desert environments. It has been reported previously as B. stevenii by Ahmad (1952) from Peshawar. It is a new record to Cholistan desert of Bahawalpur district and Nawabshah (Sindh).

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B

A C A

Plate 4. Figs. A–C. Battaraea phalloides (BPK69). A. A mature basidioma. B. Line drawing of basidioma. C. Smooth basidiospores under 40 ×. Bar: = 0.9 cm for A, = 1.5 cm for B, = 5.5 µm for C.

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A

B

Plate 5. Figs. A–B. LM photographs of Battarrea phalloides (BPK69). A. Spores and Elators. B. Magnified view of basidiospores. Bar: = 30 µm for A, = 2.5 µm for B.

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Bovista areolata nom. prov. (Plates 6–7)

Etymology: on the basis of areolate structure of peridium

Basidiomata globose to subglobose, off-white to light yellowish when young, gregarious, up to 1.76 –3 × 1–2.7 cm high, attached to the substratum by a small mycelial base, light yellow to brown above & off-white below; dehiscence not recorded in the specimen, possibly by an apical pore. Peridium < 1 mm, double. Exoperidium white to pale yellow when young, yellowish to pale brown with age, persistent, composed of polygonal verrucae, more & bigger above, smooth below. Endoperidium not exposed from upper side, light yellow brown. Gleba yellowish olive to olivaceous. Sub gleba absent.

Basidiospores subglobose to ovoid, pedicellate, olivaceous, brown inside, 5.2–7.0 × 5.0–6.5 µm; Pedicel olivaceous, undulating, tapering, 3.8–13.3 µm. Eucapillitium Bovista type, consists of isolated units, each unit with thick main axis with branches arising from it, up to 16.6 µm in diam. wall thickness up to 5.9 µm, interwoven, branches tapering, knob like projections present, 2 µm thin (wall thickness up to 0.8 µm). Exoperidium composed of hyaline to light olivaceous spahaerocysts. Endoperidium composed of hyaline to olivaceous, aseptate, branched hyphae, up to 5 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 1st Aug. 2010, gregarious, on ground, at 1192 m (3912 ft) a.s.l., M. Fiaz, MPK4 (Holotype), (LAH10000029).

22

A

B Plate 6. Figs. A–B. Bovista areolata (MPK4). A. Mature basidiomata. B. L.S of mature basidiomata. Bar: = 0.4 cm for A, = 0.3 cm for B. 23

A

C

B

D E

Plate 7. Figs. A–E. Bovista areolata (MPK4). A. Pedicellate basidiospores. B. Exoperidial elements. C. Thick main axis of eucapillitium. D. Bovista type capillitial threads. E. Endoperidial hyphae. Bar: = 4.7 μm for A, = 7.3 μm for B, = 13 μm for C, = 35 μm for D, = 10 μm for E.

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Bovista bovistoides (Cooke & Massee) S. Ahmad, Publ. Dept. Bot. Panjab Univ. 11: 15 (1952) (Plates 8–10)

Basidiomata globose to subglobose, off-white when young, grey to brown when mature, 20-30 mm broad × 20-35 mm high, gregarious; attached to the substratum by a small mycelial base having rhizomorphs; rhizomorphs off white, branched, encrusted with soil particles and other debris material. Dehiscence by an apical pore, pore develops while sloughing off the exoperidium, up to 3 mm in diam. Peridium double. Exoperidium off- white, smooth, later peels off irregularly from all over the gasterocarp exposing the endoperidium, sometimes remains in the form of patches all over the gasterocarp after peeled off. Endoperidium grey dull brown, exposed in mature specimens with some remnants of exoperidium, papery. Gleba brown, compact, cottony. Sub gleba absent.

Basidiospores globose to sub globose, brown, pedicellate, central oil droplet present up to 4.3–6.0 µm; pedicel hyaline, curved, up to 15.6 µm long. Eucapillitium consists of thick main axis, with branches arising from it, chestnut brown, aseptate, 19 µm in diam., with wall thickness up to 6.4 µm, branches attenuate, thick main axis up to, tips up to µm 3.5 thin. Endoperidium composed of hyaline to light brown, aseptate to rarely septate, branched, tightly packed hyphae, up to 6 µm in diam.

Material examined: PAKISTAN: Khyber Pakhunkhwa, Nathia gali, 24th Aug. 2010, on ground, at 2500 m (8205 ft) a.s.l., N. Yousaf, BPK94 (LAH10000030).

Additional material examined: PAKISTAN: Punjab, Murree, 20th Aug. 1948, on ground, Sultan Ahmad, BH1 (LAH2520).

Comments: B. bovistoides was previously recorded by Ahmad in 1952 from Murree of Punjab province. It is also known from India and Germany (Sharma & Thind, 1990; Bresinsky et al., 2000).

25

A

C B

Plate 8. Figs. A–C. Bovista bovistoides (BPK94). A. Mature basidiomata. B. Eucapillitium, thick walled main axis. C. Endoperidial hyphae. Bar: = 1 cm for A, = 7 µm for B, = 1.3 µm for C. 26

A

B Plate 9. Figs. A–B. Bovista bovistoides. A. Basidiospores. B. Eucapillitium, an overview. Bar: = 3.3 µm for A, = 25 µm for B. 27

A B

C

Plate 10. Figs. A–C. LM photographs of Bovista bovistoides (BPK94). A. Basidiospores. B & C. Eucapillitial hyphae. Bar: = 16 µm for A, = 100 µm for B, = 25 µm for C.

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Bovista fusca Lév., Annls Sci. Nat., Bot., sér. 3 5: 303 (1846) (Plates 11–13) Basidiomata globose to slightly subglobose, off-white with ochraceous brown coloration when young, turning pale yellow to brown when mature, up to 40 mm broad × 35 mm high, solitary; attached to the substratum by small base, encrusted with soil particles and other debris material. Dehiscence by an apical pore; pore develops while sloughing off the exoperidium, up to 2 mm in diam. Peridium double. Exoperidium off-white to cream, brown with age, smooth to granular, persistant. Endoperidium pale yellowish brown, not exposed in mature specimens, papery. Gleba brown, compact, cottony. Sub gleba absent.

Basidiospores oblong, smooth, greenish brown, 6.0–7.0 × 6.0–6.6 µm, pedicellate, pedicel hyaline, up to 15 µm long, central oil droplet present, brown in water mounts. Eucapillitium dark brown, Bovista type, with thick main axis, up to 13 µm in diam., with wall thickness up to 5.5 µm. frequently branched, dichotomous when occurring, aseptate, straight to sub-undulate, pores absent, branches with tapering ends, sub-septal ramifications present. Exoperidium composed of hyaline to olivaceous, subglobose hyphal elements, 31 × 22 µm in diam. Endoperidium composed of olivaceous brown, septate, sub undulate, rarely branched, tightly packed hyphae, up to 6 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Mashkoon, 9th Aug. 2012, in groups, on ground, at 3540 m (11615 ft) a.s.l., N. Yousaf, BPK85 (LAH10000031), BPK86 (LAH10000032).

Comments: B. fusca is characterized by brown basidioma with no subgleba, ovoid basidiospores having long pedicels (up to 16 µm). Morphologically it is close to B. nigrescens, but it has globose to subglobose basidiospores in contrast to B. fusca. It has been known from Asia, Europe and North America (Kreisel, 1967; Demoulin & Dring, 1975; Sharma & Thind, 1990; Moreno et al., 2010a). It is first record of B. fusca from Pakistan.

29

A

B

Plate 11. Figs. A–B. Bovista fusca. A. Basidiosmata (BPK85). B. Basidioma (BPK86). Bar: = 1.4 cm for A, = 1 cm for B. 30

A

B

Plate 12. Figs. A–B. Bovista fusca (BPK86). A. Basidiospores. B. Thick-walled eucapillitial hyphae. Bar: = 3.2 µm for A, = 8.8 µm for B. 31

A

B

Plate 13. Figs. A–B. Bovista fusca. A. Endoperidial hyphae. B. Exoperidial elements. Bar: = 6.6 µm for A, = 5.5 µm for B.

32

Bovista kreiselii nom. prov. (Plates 14–16) Etymology: after the name of European mycologist “Hanns Kreisel” Basidiomata sub-globose, light brown, 20–25 mm in diam. × 25–35 mm in height, gregarious, often in a group of 3–4; attached to the substrate by hard, tough, small mycelial cord, adherent with the soil particles and debris. Osteole medium, up to 4 mm in diam. Peridium double. Exoperidium dark brown in mature specimen (only visible under high magnification), peeled off, in the form of warts; warts minute, dark brown, distant from one another. Endoperidium papery, dull to light brown, smooth. Gleba brown, cottony. Sterile base present.

Basidiospores ovoid, pedicellate, brown, 6.0–8.0 × 5.9–6.0 µm; pedicel hyaline, tapering, up to 13 µm long. Eucapillitium Bovista type, not composed of isolated units, composed of thick main axis, up to 13.7 µm in diam., with wall thickness up to 4.3 µm, branches arising from the main axis, branches up to 6.6 µm with attenuate tips, (tips 4 µm in diam.), aseptate, pores absent. Exoperidium composed of hyaline, globose to sub-globose and elongated hyphae elements mixed with common hyphae, up to 36.5 µm. Endoperidium composed of hyaline, aseptate, thin walled, rarely branched, tightly packed, straight hyphae, up to 8.5 µm.

Material examined: Pakistan, Khyber Pakhtunkhwa, Thandiani, 16th Sep. 2012, in groups, among grass, at 2672 m (8766 ft) a.s.l., N. Yousaf, BPK10 (Holotype), (LAH10000033). 33

A

B

Plate 14. Figs. A–B. Bovista kreiselii (BPK10). A. Basidiomata. B. Eucapillitial hypha. Bar: = 0.55 cm for A, = 7.0 cm for B. 34

A

B C Plate 15. Figs. A–C. Bovista kreiselii. A. Pedicellate basidiospores. B. Eucapillitium type. C. Endoperidial hypahe. Bar: = 3.0 µm for A, = 26 µm for B, = 17 µm for C. 35

Plate 16. Fig. Bovista kreiselii, exoperidial elements. Bar: = 6 µm.

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Bovista nigrescens var. rectapedicellata nom. prov. (Plates 17–18)

Etymology: on the basis of straight pedicels of basidiospores Basidiomata sub-globose, dark voilet brown, 25–30 mm in diam. × 25–35 mm in height, solitary; attached to the substrate by small mycelial base, adherent with the soil particles and debris. Osteole medium, up to 3 mm in diam. Peridium double. Exoperidium not observed. Endoperidium papery, violet brown. Gleba brown, cottony. Sterile base absent.

Basidiospores globose to sub globose, thick-walled, pedicellate, dark brown, 4–6 µm in diam.; pedicel 11.3 – 17.0 µm long. Eucapillitium Bovista type, dark, encrusted, branched, aseptate, attenuate at tips, up to 20 µm in diam, wall thickness up to 4.5 µm. Paracapillitium present, hyaline, fragile, septate, branched. Exoperidium not observed. Endoperidium composed of olivaceous to brown, septate, branched and tightly packed hyphae, up to 5 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Mashkoon, 9th Aug. 2012, in groups, on ground, at 3540 m (11615 ft) a.s.l., N. Yousaf, BPK27 (LAH10000034).

37

A

B

Plate 17. Figs. A–B. Bovista nigrescens var. rectapedicellata (BPK27). A. Mature basidioma, BPK27. B. LM photograph of pedicellate basidiospores. Bar: = 0.9 cm for A, = 6.6 µm for B. 38

A

B C Plate 18. Figs. A–C. LM photographs of Bovista nigrescens var. rectapedicellata (BPK27). A. Bovista type capillitial threads. B. Thick main axis; Bovista type capillitium. C. Endoperidial hyphae. Bar: = 110 µm for A, = 35 µm for B, = 55 µm for C.

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Bovista plumbea Pers., Ann. Bot. (Usteri) 15: 4 (1795) (Plates 19–20)

Basidiomata globose to subglobose, off-white when young, brown when mature, up to 30 mm broad × 25 mm high, solitary, sometimes gregarious; attached to the substratum via thick tuft of ; rhizomorphs black, sometimes white, heavily encrusted with soil particles, dehiscence by an apical pore; pore small to medium, up to 4 mm in diam., develops as exoperidium sloughs off. Exoperidium white when young, grayish off white when mature, thick, persistent when young, peeling off upon maturity, in the form of sheets, firstly from apical portion then below, sometimes patches can be found all over the gasterocarp, can be seen attached to the basal part in mature specimens in the form of weathered, thin membranous layer. Endoperidium mouse gray when exposed, smooth to rough, papery, sometimes covered with dust, encrusted with sand particles, Gleba grayish black, cottony to powdery, pulverulent. Sterile base absent.

Basidiospores sub-globose to oval, dark brown, 3.5–6 × 4.5–7.5 µm, with central oil droplet, pedicellate, pedicel up to 12 µm long, brown to hyaline, tapering, straight to slightly curved. Capillitium brown, prominent, well developed, in the form of isolated units, not much interwoven, thick main axis, with branches originating from the axis, branches tapers at the ends, aseptate, pores absent, main axis up to 17 µm thick, branches up to 1.8– 4.7 µm thick, wall thickness from 1.8–5.7 µm.

Material Examined: PAKISTAN: Kyber Pakhtunkhwa, Khanspur, 8th Aug. 2010, at 2250 m (7381 ft) a.s.l., solitary, N. Yousaf, BPK38 (LAH10000035); Khanspur, 19th July. 2011, solitary, N. Yousaf, BPK14 (LAH10000036), BPK46 (LAH10000037); Khanspur, Halipad, 8th Aug. 2011, in groups, among grass, N. Yousaf, BPK80 (LAH10000038); Kaghan Valley, Nadibunglaw, 13th Aug. 2011, at 2134 m (7001 ft), in groups, among grass, on ground, N. Yousaf, BPK81 (LAH10000039); Gilgit-Baltistan, Fairy meadows, 22nd July. 2010, solitary, at 13751 m (4191 ft) a.s.l., A.N. Khalid, BPK84 (LAH10000040); Gilgit-Baltistan, Deosai National Park, 11th Sep. 2011, at 4114 m (13497 ft) a.s.l., in groups, on ground, A. N. Khalid, BPK22 (LAH10000041), BPK23 (LAH10000042), BPK24 (LAH10000043), BPK25 (LAH10000044), BPK83 (LAH10000045); Swat district, Miandam, 7th Aug. 2012, at 1832 m (6012 ft) a.s.l., solitary, N. Yousaf, BPK17 (LAH10000046); Mashkoon, 9th Aug. 2012, at 3540 m (11615 ft) a.s.l., in groups, N. Yousaf, BPK82 (LAH10000047); Malam Jabba, 10 Aug. 2012, at 2485 m (8152 ft) a.s.l., solitary, N. Yousaf, BPK28 (LAH10000048), Ushu, 4th Sep. 2013, solitary, under Cedrus 40

deodara, at 2418m (7933 ft) a.s.l., S. Jabeen, U1 (LAH10000049); Mansehra district, Musaka-Musala, at 4062 m a.s.l., solitary, among grass, Aug. 2011, M. Fiaz, M3, (HUP Herbarium No. MFG–323.) (LAH100102).

Additional material examined: PAKISTAN: Khyber Pakhtunkhwa, Kaghan Valley, 28th Aug. 1949, on ground, S. Ahmad (LAH 250806a); Changla gali, 24th Sep. 1949, on ground, S. Ahmad (LAH 250806b); Changla gali, 25th Sep. 1950, on ground, S. Ahmad (LAH 250806c); Abbottabad district, Ayubia, Mukshpuri, 28th Sep. 1949, on ground, S. Ahmad (LAH250807); Mirajani above Nathia gali, 16th Aug. 1954, on the ground, S. Ahmad (LAH15723, duplicate in KEW Herbarium); Swat district, Kalam, 9th Sep. 1963, S. Ahmad (LAH15724); Gilgit-baltistan, Skardu, 8th Sep. 1966, on ground, S. Ahmad (LAH19372).

Comments: B. plumbea is the type species of genus Bovista. It is characterized by globose fruiting bodies with white exoperidium and lead grey endoperidium, oval, long pedicellate basidiospores. It is usually found growing on grounds and among grasses. It is commonly occurring gasteroid taxon, reported from different localities of Pakistan. It has previously been reported from Changla gali, Bata Kundi (Kaghan Valley), and Kalam (Swat district) of KP by Ahmad (1952, 1956). It occurs solitary, sometimes gregarious on ground, among grasses from dry to moist climates, from Himalayan moist temperate forests of Khyber Pakhtunkhwa and in alpine vegetation of Gilgit-Batistan. B. plumbea has worldwide distribution, commonly occurring gasteroid taxon being reported from different localities of Pakistan. It has been collected and reported from Changla gali, Kaghan valley, Skardu, Mukshpuri etc. Here it is being reported from Deosai plains, Khanspur and Sharan. It occurs solitary, sometimes gregarious on ground, among grasses in Himlayan Moist Temperate Forests of Punjab, Khyber Pakhtunkhwa and in alpine vegetation of Gilgit-Batistan. 41

A B

C

D E Plate 19. Figs. A–E. (A & B) Mature basidiomata after exoperidium has peeled off (BPK22, BPK84). C. Young basidiomata with white exoperidium (BPK14). D. BPK80. E. BPK81. Bar: = 0.8 cm for A, B, D & E, = 0.6 cm for C. 42

B A

C

D

Plate 20. Figs. A–D. Bovista plumbea. A. Pedicellate basidiospores. B. Bovista type capillitial threads. C. Thick main axis of Bovista type capillitium. D. Endoperidial hyphae. Bar: = 4.5 µm for A, = 16.5 µm for B, = 33 µm for C, = 11 µm for D.

43

Bovista pseudonigrescens nom. prov. (Plates 21–22)

Etymology: on the basis of its affinity with B. nigrescens Basidiomata solitary, globose to subglobose, brown, up to 30 × 30 mm in diam., attached to the substratum by a mycelial cord with little rhizomorphs; Rhizomorphs white, encrusted with the particles of soil and other debris material. Dehiscence by an apical pore; Pore medium sized, up to 2 mm in diam. Peridium double. Exoperidium off-white at early stages, completely lost at maturity. Endoperidium papery, rigid, tough, persistent, brown to grey. Gleba grayish black compact. Sub gleba absent.

Basidiospores globose to ovoid, brown, 5.6–6.7 × 5.0–6.0 µm, smooth to slightly echinulate, pedicellate; pedicel hyaline, not tapering, up to 14 µm long. Eucapillitium Bovista type, with some hyphae resembling the thick main axis of typical Bovista type capillitium, pores absent, aseptate to sub-septal ramifications present, slightly encrusted with some amorphous material, up to 11 µm in diam., with wall thickness up to 5.5 µm, branches with tapering ends, somewhat undulate, branches with false septa. Paracapillitium present, rare, hyaline, septate, branched. Exoperidium composed of sub globose to irregular sphaerocysts. Endoperidium composed of olivaceous to light brown, frequently septate, unbranched, tightly packed hyphae, up to 10 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Kaghan Valley, Nadi Bunglow, 14th Aug. 2011, at 1314 m (4313 ft) a.s.l., N. Yousaf, GPK28 (Holotype), (LAH10000050).

44

A B

C

D

Plate 21. Figs. A–D. Bovista pseudonigrescens (GPK28). A & B. Two views of basidiomata. C. basidiospores. D. Endoperidial hyphae. Bar: = 0.5 cm for A & B, = 4.0 μm for C, = 18 μm for D. 45

A

B

Plate 22. Figs. A–B. Bovista pseudonigrescens. A. Intermediate type of eucapillitium. B. Eucapillitial hyphae. Bar: = 40 μm for A, = 7 μm for B. 46

Bovista aestivalis (Bonord.) Demoulin, Beih. Sydowia 8: 143 (1979) (Plates 23–25) Basidiomata globose, olivaceous yellow to brown when mature, brown above, yellow below, 12–30 × 15–30 mm diam., solitary, in groups of two sometimes; attached to the substratum by a well-developed rhizomorphs, heavily encrusted with soil particles. Dehiscence by an apical pore, pore up to 5 mm in diam. Peridium double. Exoperidium olivaceous yellow to brown in the form of small granules all over the basidioma, persistent, later on peeling off first from the top to the base leaving a crack like pattern on the basidioma. Endoperidium light brown to yellow, papery. Gleba olivaceous brown, compact, pulverulent. Sub gleba absent, rudimentary sterile base present, up to 4 mm in height.

Basidiospores globose, smooth to asperulate, yellowish brown, 4.0–4.7 µm in diam., a short remnant of pedicel attached sometimes, thick walled. Eucapillitium brown, intermediate type, pores present, branched, hyphae resembling main axis, up to 9.4 µm in diam., wall thickness up to 3.5 µm, aseptate, sub-undulate at tips, (up to 3 µm) bulging at some points, sub-septal ramifications absent, branches with tapering ends, tips undulate. Paracapillitium absent. Exoperidium composed of light greenish, sub-globose to slightly elongated hyphal elements, 14 × 9.7 µm. Endoperidium composed of hyaline to light greenish, straight, septate, rarely branched, thick-walled, tightly packed hyphae, up to 3.7 µm in diam., wall thickness up to 1.2 µm bulging at some points.

Material examined: PAKISTAN: Khyber Paktun Khwa, Swat district, Mankial Top, 5th Sep. 2013, at 3822 m (12542 ft) a.s.l., in groups, under Cedrus deodara, S. Jabeen, BPK33 (LAH10000051); Kalam, 3rd Sep. 2013, at 2003 m (6573 ft) a.s.l., solitary, under Cedrus deodara, S. Jabeen, BPK70 (LAH10000052).

Comments: B. aestivalis is characterized by yellowish brown basidiomaa with granulose peridium, intermediate type capillitium with pits, smooth to asperulate, globose basidiospores. It has been reported from many countries (Kreisel, 1967; Bates et al., 2009; Larsson et al., 2009; Moreno et al., 2010a). It is a new record for Pakistan. 47

A B

C

Plate 23. Figs. A–C. . A & C. Mature basidioma (BPK33). B. Basidioma (BPK70). Bar: = 0.9 cm for A & B, = 1.3 cm for C. 48

A

B C

D E Plate 24. Fig. A–E. LM photographs of Bovista aestivalis (BPK33). A & B. Asperulate basidiospores with a stump of pedicel. C. Basidiospores in Trypan blue. D & E. Olivaceous, non porous eucapillitial hyphae. Bar: = 12 µm for A, = 4.0 µm for B, = 5.0 µm for C, = 66 µm for D, = µm 7.6 for E. 49

Plate 25. Fig. Bovista aestivalis. A. Exoperidial elements. B. Endoepridial hyphae. Bar: = 4.0 µm

50

Bovista ahmadii nom. prov. (Plates 26–27)

Etymology: on the name of Asian mycologist “S. Ahmad”

Basidiomata sub-globose to slightly ob-ovoid, brown with olivaceous tint, 28–35 mm broad × 30–40 mm high, gregarious; attached to the substratum by well-developed mycelial cord. Rhizomorphs white, well developed, heavily encrusted with the soil particles and other debris material. Dehiscence by an apical osteole, pore up to 4 mm in diam. Peridium double. Exoperidium persistent, peeling off at maturity, remains in the form of small granular warts, Endoperidium brown, papery, persistent, covered with warts of exoperidium. Gleba olivaceous brown, pulverulent. Sub gleba absent.

Basidiospores globose, asperulate, olivaceous to brown, 4.0–6.2 × 4.0–5.7 µm, central oil droplet present, brownish in water mounts, pedicelate; pedicel hyaline to olivaceous, up to 1.5 µm long. Eucapillitium olivaceous browm, intermediate type, frequently branched, dichotomous when occurring, aseptate, up to 3.5 µm wide, thick walled (wall thickness up to 1.33 µm), sub-undulate, pores present, bulging at some points, tapering at the ends. Exoperidium composed of globose to sub-globose elements, up to 30 µm. Endoperidium composed of hyaline, branched, septate, tightly packed hyphae, up to 5 µm.

Material examined: PAKISTAN: Punjab, Sakesar, 15th Aug. 2006, at 1522m (4993 ft) a.s.l., gregarious, on ground, A. N. Khalid, BPK52 (Holotype), (LAH10000053); Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 1st Aug. 2010, at 1088m (3571 ft) a.s.l., solitary, on ground, N. Yousaf, BPK35 (LAH10000054).

51

A

B C

Plate 26. Figs. A–C. Bovista ahmadii. A. Basidiomata (BPK35). B. Basidioma (BPK52). C. Asperulate basidiospores. Bar: = 0.55 cm for A & B, = 4.5 µm for C. 52

A

B

C

Plate 27. Figs. A–C. Bovista ahmadii. A. Exoperidial elements. B. Eucapillitial hyphae. C. Endoperidial hyphae. Bar: = 6.6 µm for A, = 5.5 µm for B, = 5.0 µm for C.

53

Bovista ayubiensis nom. prov. (Plate 28)

Etymology: on the name of type locality “Ayubia”

Basidioma sub globose, grayish brown, 35 mm in diam. × 20 mm in height, solitary; attached to the substrate by small, white mycelial cord. Dehiscence by an apical pore, apical torn aperture in this case. Sterile base absent. Peridium double. Exoperidium peeled off, not persistent, entirely rubbed off from the endoperidium in mature specimens, small granular form left at the basal portion; granules blackish. Endoperidium exposed, papery, brownish-grey, shiny, smooth. Gleba grayish brown, cottony. Sub gleba absent.

Basidiospores globose, olivaceous, 3.23–4.0 μm in diam., with a stump of pedicel, pedicel hyaline, up to 0.95 μm, sterigmal remnants absent from mounts. Capillitium intermediate type, olivaceous brown, aseptate, without pores, frequently branched, branches seems originating from the thick axis, up to 3.7–7.0 μm in diam., thick-walled, 0.9–1.14 μm, attenuate at tips, tips up to 1.5 μm. Paracapillitium absent. Exoperidium not observed. Endoperidium composed of thin walled, septate, branched and hyaline hyphae, up to 5 μm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Ayubia, Khanspur, Halipad, 1st Sep. 2009, solitary, among grass, at 2250 m (7381 ft) a.s.l., N. Yousaf, BPK3 (Holotype), (LAH10000055). 54

A

B

C D

A Plate 28. Figs. A–D. Bovista ayubiensis (BPK3). A. Mature basidioma. B. Apedicellate B basiodiopsores. C. Eucapillitial hyphae. D. Endoperidial hyphae. Bar: = 0.5 cm for A, = 2.2 µm for B, = 11 µm for C, = 11 µm for D. 55

Bovista brunnecenta nom. prov. (Plates 29–30) Etymology: on the basis of brown color of basidioma

Basidioma globose, brown, up to 25 mm high, solitary; attached to the substratum by a small mycelial base, encrusted with debris and soil particles. Dehiscence by an apical pore. Peridium ≤ 1 mm, thin, double. Exoperidium not recorded in young specimens, peeled off with age, in the form of remnants in mature specimens, granules more below, less above. Endoperidium exposed in mature specimens, brown, papery. Gleba brown, cottony. Sub gleba absent.

Basidiospores ovoid to ellipsoid, apedicellate, brown, 3.5–5.3 × 4.5–6.1 µm. Eucapillitium intermediate type, brown, frequently branched, up to 6.0 µm in diam. wall thickness up to 2 µm, interwoven, branches tapering, knob like projections present, 2 µm thin (wall thickness up to 0.8 µm). Exoperidium composed of hyaline to light olivaceous sphaerocysts, up to 20 µm. Endoperidium composed of hyaline to olivaceous, aseptate, branched hyphae, up to 5 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Dadar, 15th Sep. 2012, solitary, among grass, at 636 m (2089 ft) a.s.l., N. Yousaf, BPK86 (Holotype), (LAH10000056). 56

A

B C

Plate 29. Figs. A–C. Bovista brunnecenta (BPK86). A. A mature basidioma. B. Basidiospores. C. Exoperidial elements. Bar: = 0.7 cm for A, = 2.2 µm for B, = 7 µm for C. 57

A

B C Plate 30. Figs. A–C. Bovista brunnecenta. A. Intermediate type eucapillitium. B. Endoperidial hyphae. C. Eucapillitial hypahe. Bar: = 33 µm for A, = 11 µm for B, = 6.6 µm for C.

58

Bovista concinna S. Ahmad, Sydowia 3(1-6): 335 (1949) (Plates 31–33) Basidiomata globose to slightly sub globose, grayish brown above, orange brown below when mature up to 35 mm broad × 30 mm high, gregarious; attached to the substratum by a well-developed mycelial cord, consisting of rhizomorphs; rhizomorphs off white, branched, encrusted with soil particles and other debris material. Dehiscence by an apical pore, pore develops while sloughing off the exoperidium, up to 4 mm in diam. Peridium double. Exoperidium off-white to cream, smooth to granular, later peeling off irregularly exposing the endoperidium, in the form of granules. Endoperidium dull grayish brown, exposed in mature specimens, papery. Gleba brown, compact, cottony. Sub gleba present sometimes.

Basidiospores globose, verrucose, brown, numerous sterigmal remnants present in the mounts, 5.7–7.0 µm in diam., verrucae up to 0.8 µm. Eucapillitium Lycoperdon type, branched, sub-undulate, encrusted with some amorphous material, septate, up to 5.0 µm in diam., wall thickness up to 0.8 µm. Paracapillitium present, hyaline, branched, septate, up to 6.3 µm in diam. Exoperidium composed of thick walled, dark brown, elongated hyphal elements, up to 63 × 28 µm, wall thickness up to 8 µm. Endoperidium composed of hyaline, thin walled, septate, tightly packed hyphae, up to 5 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Abbottabad district, Ayubia, Khanspur, 22nd Aug. 2012, solitary, among grass, at 2250 m (7381 ft) a.s.l., N. Yousaf, BPK2 (LAH10000057).

Additional material examined: Bovista concinna, Punjab, Murree, 31st Aug. 1949, in vegetable debris, Sultan Ahmad, BH33 (LAH19373).

59

A B

C

Plate 31. Figs. A–C. Bovista concinna. A & B. Basidioma (BPK2). C. Basidiomata (BH3). Bar: = 0.7 cm for A & B, = 1.3 cm for C.

60

A

B Plate 32. Figs. A–B. LM photographs of Bovista concinna, A. Verrucose basidiospores. B. Lycoperdon type eucapillitium. Bar: = 7.0 µm for A, = 25 µm for B. 61

A

B C Plate 33. Figs. A–C. LM photographs of Bovista concinna. Encrusted and septate eucapillitial threads. Bar: = 18 µm for A, = 4 µm for B & C.

62

Bovista flavescens nom. prov. (Plates 34–35)

Etymology: on the basis of yellowish color of basidioma

Basidiomata sub globose to slightly obovoid, yellow with brown lines when young, dull brown with age, up to 37 mm broad × 40 mm high, solitary, apical portion with cracking pattern with reticulate appearance, basal portion smooth, without cracking or reticulate appearance; attached to the substratum by well-developed mycelial cord. Rhizomorphs not recorded or lost during collection. Dehiscence by developing osteole at apical portion. Peridium double. Exoperidium pale yellowish, persistent, covered with shiny sand like paricles, granular at upper portion of basidiomata. Endoperidium brown, papery, persistent, entirely covered with exoperidium. Gleba yellowish brown, compact, cottony. Sub gleba present.

Basidiospores globose to ovoid, slightly warted, olivaceous to brown, 4.3–6.2 × 4.3–5.7 µm, central oil droplet present, brownish in water mounts, yellowish in KOH pedicellate; pedicel hyaline to olivaceous, up to 1.7 µm long. Eucapillitium olivaceous brown, intermediate type, frequently branched, dichotomous when occurring, aseptate, up to 3.5 µm wide, thick walled (wall thickness up to 1.33 µm), sub-undulate, pores absent, bulging at some points, tapering at the ends. Exoperidium composed of hyaline, subglobose hyphal elements, up to 19 × 15.8 µm. Endoperidium composed of light brown, septate, branched, tightly packed hyphae, up to 6 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 1st Aug. 2010, at 1088 m (3571 ft) a.s.l., solitary, on ground, M. Fiaz, BPK83 (Holotype), (LAH10000058).

63

A

A B

D

C

D

Plate 34. Figs. A–D. Bovista flavescens (BPK83). A. Mature basidiomata. B. Line drawing of basidioma. C. Smooth basidiospores. D. Eucapillitial hyphae. Bar: = 0.7 cm for A & B, = 3.4 µm for C, = 5 µm for D. 64

A

B

Plate 35. Figs. A–B. Bovista flavescens. A. Exoperidial elements. B. Endoperidial hyphae. Bar: = 6 µm for A, = 6.6 µm for B.

65

Bovista flavus nom. prov. (Plate 36) Etymology: on the basis of yellow color of basidioma Basidiomata globose, yellow when young, yellowish brown when mature, 15–18 mm broad × 15–18 mm high, gregarious; attached to the substratum by a mycelial base, up to 5 mm high, encrusted with soil particles and other debris material, consists of off white, branched rhizomorphs. Dehiscence by an apical pore, up to 3 mm in diam. Peridium doubles. Exoperidium yellow, smooth to granular, in form of granules all over the gasterocarp, persistent. Endoperidium brown, papery. Gleba brown, compact, cottony. Sub gleba absent.

Basidiospores globose, smooth, olivaceous brown, 5.5–6.0 µm in diam., with a stump of pedicel, central oil droplet present, brown in water mounts. Eucapillitium yellowish brown, Lycoperdon type, frequently branched, dichotomous when occurring, aseptate, straight to sub-undulate, pores absent, branches with tapering ends, tips undulate, , sub-septal ramifications present, up to 5 µm in diam. in diam. wall thickness up to 2.3 µm. Paracapillitium absent. Exoperidium composed of hyaline, subglobose hyphal elements, up to 21 × 14 µm in diam. Endoperidium composed of hyaline, frequently septate, branched, thin walled, tightly packed hyphae, up to 6.6 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Dadar, 15th Sep. 2012, gregarious, on ground, under Pinus wallichiana, at 636 m (2089 ft) a.s.l., N. Yousaf, BPK91 (Holotype), (LAH10000059). 66

A B

C D

E Plate 36. Figs. A–E. Bovista flavus (BPK91). A. Basidiomata, B. Basidiospores. C. Endoperidial hyphae. D. Eucapillitial threads. E. Exoperidial elements. Bar: = 0.66 cm for A, = 3 µm for B, = 14 µm for C, = 5.5 µm for D, = 8.8 µm for E.

67

Bovista himalaica Yousaf, Kreisel & Khalid, in [2], Mycol. Progr. (2013) (Plates 37– 39) Etymology: on the basis of type locality “Himalayan moist temperate forest” Basidiomata globose to pyriforme, pale brown, up to 30 mm high × 23 mm broad, no rhizomorphs or very little mycelial base; sterile base absent. Gleba olivaceous, compact, with olivaceous brown spore mass; dehiscence by an apical pore. Sub gleba rudimentary, indistinct. Peridium double. Exoperidium persistent, homogenous acute-granular configuration, brown, composed of globose to subglobose hyphae, soon dehiscing irregularly in minute granules or patches, covered with black debris. Endoperidium shiny, papery, golden brown to pale brown, composed of branched, hyaline, thin walled and septate hyphae, opens by a circular mouth about 3 mm in diam.

Basidiospores subglobose to oval, slightly ellipsoid, smooth, olivaceous, 3.8–6.0 × 3.15–4.5 µm, pedicel 5–8 × 1.2 µm, sterigmal remnants absent from mounts. Capillitium intermediate type, prominent, well developed, sub elastic, with no pits, with dichotomous and frequent but sub septal ramifications, breaking at these septa and sometimes irregularly, branched, olivaceous, tapering at the ends, up to 7 µm wide with wall thickness 0.7–2.75 µm, tapering branches up to 1.15 µm wide, with wall thickness up to 0.475 µm. Paracapillitium absent.

Material examined: PAKISTAN: Khyber PakhtunKhwa, Khanspur, 1st Aug. 2009, solitary to gregarious, among grass, at 2250 m (7381 ft) a.s.l., N. Yousaf, NYGL5 (LAH 010809); Khanspur, 8th Aug. 2010, N. Yousaf, NYG3 (LAH080810); Khanspur, Halipad, 22nd Aug. 2010, among grass, N. Yousaf, NYG5 (LAH220810); Khanspur, 10th Aug. 2011, N. Yousaf, NYG2. (LAH100811); Khanspur, 16th Sep. 2012, A. R. Niazi, BPK47 (LAH10000060); Swat, Miandam, 8th Aug. 2012, gregarious, among grass, on slope, at 2042 m (6225 ft) a.s.l., N. Yousaf, BPK51 (LAH10000061).

68

z Plate 37. Figs. a–d. Fruiting bodies of Bovista himalaica. a. A collection no. NYGL5. b. Collection no. NYG3. c. Collection no. NYG5. d. Collection no. NYG2. Bar: = 0.75 for a–d.

69

Plate 38. Figs. a–e. Bovista himalaica. a. Exoperidial elements. b. Minutely ornamented basidiospores. c. Intermediate type capillitial threads. d. Endoperidial hyphae. e. Eucapillitial hyphae. Bar: = 12 µm for a, 3.5 µm for b, 70 µm for c, 13 µm for d, 16 μm for e. 70

Plate 39. Figs. a–b. Scanning Electron Micrographs of basidiospores of Bovista himalaica.

71

Bovista lahorensis nom. prov. (Plates 40–41) Etymology: on the basis of type locality “Lahore”

Basidioma globose, off-white when young, yellowish brown when mature, up to 10 mm broad × 10 mm high, solitary; attached to the substratum by a very short, delicate mycelial base, encrusted with soil particles and other debris material, rhizomorphs white, up to 8 mm high. Dehiscence by an apical pore, pore develops while sloughing off the exoperidium, up to 1 mm in diam. Peridium double. Exoperidium not persistent, in the form of brown warts all above gasterocarp. Endoperidium yellowish brown, papery. Gleba olivaceous brown, compact, cottony. Sub gleba absent.

Basidiospores globose, ornamented, asperulate, yellowish to olivaceous brown, 3.5–4.4 µm in diam., central oil droplet present, yellowish brown in water mounts. Eucapillitium olivaceous, Lycoperdon type, frequently branched, aseptate, straight to sub- undulate, pores numerous, branches with attenuate tips, aseptate, thin walled, up to 5.0 µm in diam. (wall thickness 0.7 µm). Paracapillitium present, scarce, hyaline, branched, septate, up to 3.3 µm in diam. Exoperidium light brown, globose, subglobose and elongated hyphal elements attached with septate hyphae, 25 × 24 µm, with hyphae up to 4.7 µm in diam. Endoperidium composed of yellowish brown, thin walled, aseptate, branched, straight, branched, tightly packed hyphae, up to 5 µm in diam.

Material examined: PAKISTAN: Punjab, Lahore, University of the Punjab, Department of Botany lawn, 19th July. 2013, solitary, on ground, at 217 m (713 ft) a.s.l., N. Yousaf, BPK85 (Holotype), (LAH10000062).

72

A B

C

D

Plate 40. Figs. A–D. Bovista lahorensis (BPK85). A & B. Basidioma. C. Asperulate basidiospores. D. Endoperidial hyphae. Bar: = 0.7 cm for A & B, = 2.7 µm for C, = 5.5 µm for D. 73

A

B

Plate 41. Figs. A–B. Bovista lahorensis. A. Exoperidial elements. B. Porous eucapillitial hyphae. Bar: = 8.5 µm for A, = 7.8 µm for B.

74

Bovista levispora nom. prov. (Plates 42–43)

Etymology: on the basis of smooth basidiospores

Basidiomata sub-globose to slightly pyriform, solitary, up to 28 mm broad × 30 mm high, off-white when young, yellowish brown when mature; attached to the substratum by a short mycelial base, encrusted with soil particles and other debris material, rhizomorphs white. Dehiscence by an apical pore, pore develops while sloughing off the exoperidium, up to 5 mm in diam. Peridium double. Exoperidium off-white when young, yellowish brown with age, persistent in mature specimens, in the form of granules. Endoperidium dull yellow to brown, not exposed in mature specimens, covered with exoperidium more above than below papery. Gleba brown, compact, cottony. Sub gleba present.

Basidiospores globose, yellowish to olivaceous brown, smooth, 6.0–7.5 µm in diam., with a stump of pedicel, up to 2.3 µm high, central oil droplet present, brown in water mounts. Eucapillitium yellowish brown, Lycoperdon type, frequently branched, dichotomous when occurring, aseptate, up to 7 µm high, wall thickness up to 2.3 µm, straight to sub-undulate, pores present, numerous, branches with tapering ends, tips undulate, bulging at some points, sub-septal ramifications present. Paracapillitium absent. Exoperidium composed of hyaline, sub-globose to elongated hyphal elements, 19–9 µm. Endoperidium composed of hyaline, frequently septate, rarely branched, tightly packed hyphae, up to 4.3 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Malam Jabba, 10th Sep. 2012, solitary, on ground, at 2485 m (8155 ft) a.s.l., N. Yousaf, BPK87 (Holotype), (LAH10000063).

75

A

B

Plate 42. Figs. A–B. Bovista levispora (BPK87). A. Basidiomata. B. Exoperidial elements. Bar: = 1.2 cm for A, = 6 µm for B.

76

A B

C

Plate 43. Figs. A–C. Bovista levispora (BPK87). A. Basidiospores. B. Eucapillitial threads. C. Endoperidium. Bar: = 3.7 µm for A, = 7.7 µm for B, = 4.7 µm for C.

77

Bovista longispora Kreisel, Nova Hedwigia, Beih. 25: 74 (1967) (Plates 44–45) Basidiomata globose to slightly subglobose, off-white to cream when young, dull grayish brown when mature, 15-43 mm broad × 15-40 mm high, gregarious; attached to the substratum by a mycelial cord, encrusted with soil particles and other debris material. Dehiscence by an apical pore, pore develops while sloughing off the exoperidium, up to 4 mm in diam. Peridium double. Exoperidium off-white to cream, smooth to granular, later peeling off irregularly from all over the gasterocarp exposing the endoperidium, in the form of granules all over the gasterocarp Endoperidium dull grayish brown, exposed in mature specimens, papery. Gleba olivaceous, compact, cottony, consisting of 3/4th of the gasterocarp. Sub gleba present, pale yellow to off-white, compact, covering almost 1/4th of gasterocarp.

Basidiospores oblong, smooth, brownish green to olivaceous, up to 3.0 × 5.0 µm in diam., with a stump of pedicel, up to 1 µm, central oil droplet present, brown in water mounts. Eucapillitium olivaceous brown, Lycoperdon type, frequently branched, dichotomous when occurring, aseptate, straight to sub-undulate, pores present, branches with tapering ends, tips undulate, septa present at the tips sometimes, sub-septal ramifications present, up to 5 µm. Exoperidium composed of hyaline to brown, sub-globose hyphal elements. Endoperidium composed of hyaline, frequently septate, rarely branched, tightly packed hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 2nd Aug. 2010, at 1088 m (3571 ft) a.s.l., in groups, on ground, M. Fiaz, BPK84 (LAH10000064). 78

A

B

Plate 44. Figs. A–B. Bovista longispora (BPK84). A. Young and mature basidiomata. B. Exoperidial arrangement of sphaerocysts. Bar: = 0.8 cm for A, = 5.5 µm for B. 79

A

B C

Plate 45. Figs. A–C. Bovista longispora. A. Basidiospores. B. Eucapillitial hyphae. C. Endoperidial hyphae. Bar: = 2.0 µm for A, = 9 µm for B, = 11 µm for C. 80

Bovista minima nom. prov. (Plates 46–47) Etymology: on the basis of small size of basidiomata

Basidiomata globose to subglobose, brown in mature specimens, 7–15 in diam. × 10–18 mm height, gregarious; attached to the substratum by a small mycelial base, light yellow to brown above and off-white below; dehiscence not recorded in the specimen, possibly by developing an apical pore as exoperidium sheds off. Peridium <1 mm, double. Exoperidium peeled off in mature specimens, remnants in the form of black spots, pale brown with age. Endoperidium not exposed from upper side, light yellow brown. Gleba brown, cottony. Sub gleba absent.

Basidiospores subglobose, short pedicellate, brown, 3.6–4.3 µm; pedicel up to 1 µm. Eucapillitium intermediate type, brown, sometimes with thick main axis with branches arising from it, aseptate to rarely septate, septa usually present in the branches with attenuate tips, main axis up to 7.6 µm in diam. wall thickness up to 3 µm, branches up to 3.3 µm thin. Exoperidium composed of hyaline subglobose to irregular, to somewhat elongated, up to 11 µm wide, up to 20 µm in length. Endoperidium composed of tightly packed, hyaline to olivaceous, septate and branched hyphae, up to 4.2 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Jabori, 14th Sep. 2013, gregarious, among grass, at 1366 m (4482 ft) a.s.l., N. Yousaf, BPK90 (Holotype), (LAH10000065).

81

A

B C

Plate 46. Figs. A–C. Bovista minima (BPK90). A. Basidioma. B. Basidiospores. C. Intermediate type eucapillitial hyphae. Bar: = 1 cm for A, = 27 µm for B, = 2.6 µm for C.

82

A B

C

Plate 47. Figs. A–C. Bovista minima. A. Endoperidial hyphae. B. Eucapillitial hyphae. C. Exoperidial hyphae. Bar: = 6 µm for A, = 6 µm for B, = 7.8 µm for C. 83

Bovista oblongispora (Lloyd) Bottomley, Bothalia 4(3): 580 (1948) (Plates 48–49) Basidiomata globose to subglobose, olivaceous brown, up to 20 mm high × 25 mm broad; attached to the substratum by well developed rhizomorphs. Dehiscence by an apical pore. Peridium double. Exoperidium persistent, offwhite when young, brown in mature specimens, in the form of granules in mature specimens. Endoperidium shiny, papery, olivaceous brown. Gleba brown, compact. Sub gleba present, rudimetary.

Basidiospores ellipsoid, smooth, olivaceous brown, 4.7–6.0 × 4.0–4.7 µm, with a stump of pedicel, sterigmal remnants absent from mounts. Capillitium Lycoperdon type, olivaceous, prominent, well developed, pits present, numerous, branched, tapering at the ends, subundulate, up to 7.3 µm wide with wall thickness 0.1 µm, tapering branches up to 2.3 µm wide. Paracapillitium absent. Exoperidium composed of olivaceous to hyaline, subglobose with elongated hyphal elements, up to 19 ×14 µm. Endoperidium composed of olivaceous, branched, thin walled, and septate hyphae, up to 5.6 µm, with wall thickness up to 0.7 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Miandam, 7th Aug. 2012, in groups, on ground, at 1832 m (6012 ft) a.s.l., N. Yousaf, BPK74 (LAH10000066); Malam Jabba, 10th Aug. 2012, solitary, on ground, at 2584 m (8152 ft) a.s.l., N. Yousaf, BPK4 (LAH10000067), BPK13 (LAH10000068).

Commnets: B. oblongispora is a morphologically more similar species to B. himalaica. It is characterized by ellipsoid, smooth basidiospores and intermediate/Lycoperdon type capillitium. Phylogenetically, it lies close and segregate from B. himalaica, maintaining a separate position. It is an addition to gasteroid fungal flora of Pakistan.

84

A B

C

D

Plate 48. Figs. A–D. Bovista oblongispora. A. Basidioma (BPK4). B. Basidioma (BPK13). C. Basidiomata (BPK74). D. Basidiospores. Bar: = 0.7 cm for A–C, = 3 µm for D. 85

A B

C

Plate 49. Figs. A–C. Bovista oblongispora. A. Exoperidial elements. B. Endoperidial elemsnts. C. Eucapillitial hyphae. Bar: = 8 µm for A, = 7.5 µm for B, = 7.7 µm for C. 86

Bovista pakistanica nom. prov. (Plates 50–51) Etymology: on the basis of country name “Pakistan”

Basidiomata globose to slightly subglobose, off-white when young, turning greyish brown with age, up to 25 mm broad × 20 mm high, solitary to gregarious,; attached to the substratum by a small mycelial base consists of rhizomorphs; rhizomorphs off white, encrusted with soil particles. Dehiscence by an apical pore, pore develops while sloughing off the exoperidium, up to 2 mm in diam. Peridium double. Exoperidium off-white with greyish tint, smooth to slightly rough, later peeling off exposing the endoperidium. Endoperidium dull grayish brown, exposed in mature specimens, papery. Gleba greyish brown, compact, cottony. Sub gleba absent.

Basidiospores globose, olivaceous, with brown outer wall, oil droplet present, with a stump of pedicel, 4.0–5.7 µm in diam. Eucapillitium intermediate type, septate, mostly braches with septa, olivaceous, pores present, sub-undulate, bulging at some points, branched, attenuate at tips, slightly encrusted, 1.5–7.3 µm in diam., wall thickness up to 1.0 µm. Paracapillitium present. Exoperidium composed of globose to sub-globose sphaerocysts. Endoperidium composed of hyaline, tightly packed hyphae.

Material examined: Khyber Pakhtunkhwa, Swat district, Kalam, 4th Sep. 2013, solitary to gregarious, among decaying needles of forest floor, at 2123 m (6965 ft) a.s.l., S. Jabeen, BPK71 (Holotype), (LAH10000069), BPK72 (LAH10000070), BPK73 (LAH10000072), BPK75 (LAH10000073), BPK76 (LAH10000074).

87

A

B C

Plate 50. Figs. A–C. Bovista pakistanica A. Young Basidiomata (BPK71). B. Basidioma (BPK72). C. Mature basidioma with greyish endoperidium (BPK73). Bar: = 1.2 cm for A, = 1.1 cm for B & C.

88

A B

C D

E F

Plate 51. Figs. A–F. LM photographs of Bovista pakistanica. A & E. Basidiopsores. B–D. Intermediate type capillitial threads. E–F. Asperulate basidiospores. Bar: = 22 µm for A, = 28 µm for B & C, = 15.5 µm for D, = 4.5 µm for E, 6.5 µm for F 89

Bovista promontorii Kreisel, Nova Hedwigia, Beih. 25: 225 (1967) (Plate 52) Basidioma subglobose, orange brown, 25 mm in diam.; attached to the substratum by small rhizomorphs. Dehiscence by an apical pore, up to 2 mm in diam. Peridium double. Exoperidium persistent, in the form of granules on the endoperidium in mature specimen. Endoperidium light brown, papery. Gleba cottoy, olivaceous brown.

Basidiospores ellipsoid to oblong, olivaceous, with a stump of pedicel, central oil droplet present, 4.7–6.0 × 4.0–5.0 µm in diam., pedicel up to µm. Eucapillitium olivaceous brown, intermediate, pores present, non elastic, thin-walled with attenuate tips, frequently branched, septate mostly at tips, up to 5.0 µm, with wall thickness up to 1.4 µm, tips up to 1.8 µm, sub-septal ramifications absent. Exoperidium composed of brown to hyaline, subglobose sphaerocysts, up to 48 × 40 µm. Endoperidium composed of olivaceous, branched, septate, thin-walled tightly packed hyphae, up to 5.6 µm.

Material examined: PAKISTAN, Khyber Pakhtunkhwa, Khanspur, 1st Aug. 2006, among grass, at 2250 m (7500 ft) a.s.l., A. R. Niazi, BPK58 (LAH10000075).

Comments: B. promontorii is another related but a rare species, which has characteristic features of intermediate capillitium type and smooth to asperulate oblong basidiospores. It bears morphological resemblance with B. aestivalis (Larsson & Jeppson, 2008). B. promontorii is a new record from Pakistan.

90

A B

C

D E

Plate 52. Figs. A–E. Bovista promontorii (BPK58). A. Basidioma. B. Endoperidial hyphae Basidiospores. C. Exoperidial elements. D. Eucapillitial hyphae. E. Basidiospores. Bar: = 0.45 cm for A, = 11 µm for B, = 12 µm for C, = 14.5 µm for D, = 4.0 µm for E. 91

Bovista trachyspora (Lloyd) Kreisel, Feddes Repert. 69: 202 (1964) (Plates 53–54)

Basidiomata globose to slightly subglobose, grayish brown when mature gregarious, 10–12 mm broad × 10 mm high; attached to the substratum by a mycelial cord, up to 5 mm high, consists of off white, branched rhizomorphs, encrusted with soil particles and other debris material. Dehiscence by an apical pore, pore develops while sloughing off the exoperidium, up to 2 mm in diam. Peridium double. Exoperidium peeled off in mature specimens, small granules left over the endoperidium. Endoperidium off white to grayish above near the pore, brown below, exposed in mature specimens, papery. Gleba olivaceous, compact, cottony. Sub gleba absent.

Basidiospores globose, asperulate, light brown, 4.4–6.4 µm, with only a stump of pedicel, pedicel up to 1.9 µm, hyaline. Eucapillitium Lycoperdon type, light brown, frequently branched, branching tips attenuate, straight to sub-undulate, aseptate to rarely septate, pores absent, up to 6 µm in diam., wall thickness up to 1.7 µm, not encrusted with amorphous material. Paracapillitium present; composed of hyaline, branched, septate, up to 2.5 µm in diam. Exoperidium not recorded. Endoperidium composed of thin walled, light brown, frequently septate, rarely branched, tightly packed hyphae, up to 3.3 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Kaghan valley, Nadi Bunglow, 14th Aug. 2011, at 1314 m (4313 ft) a.s.l., solitary, on grass, N. Yousaf, BPK6 (LAH10000076). 92

A B

C

Plate 53. Figs. A–C. Bovista trachyspora (BPK6). A & B. Mature basidioma. C. Lycoperdon Type eucapillitial hyphae. Bar: = 0.2 cm for A & B, = 3.7 µm for C. 93

A B

C

Plate 54. Figs. A–C. Bovista trachyspora. A. Asperulate basidiospores. B. Endoperidial hyphae. C. Paracapillitial hyphae. Bar: = 3.3 µm for A, = 7.2 µm for B, = 3.5 µm for C. 94

Bovista viridispora nom. prov. (Plates 55–56) Etymology: on the basis of greenish color of basidiospores Basidiomata globose to slightly sub globose, ochraceous brown when young, dull brown when mature, up to 15–20 mm broad × 15–20 mm high, gregarious; attached to the substratum by a well-developed mycelial base, rhizomorphs branched, white, encrusted with soil particles and other debris material. Dehiscence by an apical pore, up to 3 mm in diam. Peridium double. Exoperidium ochraceous brown when young, dull brown with age, granular, in the form of granules all over the gasterocarp, light brown above, blackish below, persistent. Endoperidium brown, not exposed in mature specimens, papery. Gleba yellowish brown, compact, cottony. Sub gleba absent

Basidiospores globose, smooth to asperulate, light brown to greenish brown, 5.0– 5.6 µm in diam., short pedicelate, with a stump of pedicel, pedicel up to 2.4 µm, central oil droplet present, brown in water mounts. Eucapillitium brown, intermediate type, frequently branched, dichotomous when occurring, up to 6 µm in diam., wall thickness up to 3.0 µm, aseptate, straight to sub-undulate, bulging at some points, pores absent, sub-septal ramifications absent, branches with tapering ends, tips undulate, up to 2.5 µm thin. Paracapillitium absent. Exoperidium composed of hyaline to brown, sub-globose to elongated hyphal elements, 26 × 9 µm. Endoperidium composed of hyaline, straight, frequently septate, rarely branched, tightly packed hyphae, up to 6 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Shimla hill, 14th Sep. 2012, solitary, on ground, at 1972 m (6473 ft) a.s.l., N. Yousaf, BPK88 (LAH10000077); Dadar, 15th Sep. 2012, gregarious, on ground, at 636 m (2089 ft) a.s.l., N. Yousaf, BPK89 (Holotype) (LAH10000078). 95

A

B

Plate 55. Figs. A–B. Bovista viridispora. A. Basidioma (BPK89). B. Exoperidial elements. Bar: = 1 cm for A, = 7.3 µm for B. 96

A

B

C

Plate 56. Figs. A–C. Bovista viridispora. A. Basidiospores. B. Paracapillitial hyphae. C. Eucapillitial hyphae. Bar: = 3.55 µm for A, = 8.0 µm for B & C.

97

Bovista sp. (BPK92) (Plates 57–58) Basidioma sub globose, brown when mature, up to 10 mm broad × 12 mm high, solitary; attached to the substratum by a short mycelial base, encrusted with soil particles and other debris material, rhizomorphs white. Dehiscence by an apical pore, pore develops while sloughing off the exoperidium, up to 1 mm in diam. Peridium double. Exoperidium peeled off completely in mature specimens. Endoperidium brown, papery. Gleba olivaceous brown, compact, cottony. Sub gleba absent

Basidiospores globose, smooth, yellowish to olivaceous brown, 5.0–6.4 µm in diam., with a stump of pedicel, central oil droplet present, brown in water mounts. Eucapillitium brown, Lycoperdon type, frequently branched, aseptate, straight to sub- undulate, pores absent, branches with tapering ends, tips undulate, attenuate, aseptate, up to 5.2 µm in diam. (wall thickness 2.6 µm). Paracapillitium present, scarce, hyaline, branched, septate, up to 3.8 µm in diam. Exoperidium not observed. Endoperidium composed of brown, frequently septate, rarely branched, not straight, branched, tightly packed hyphae, up to 15.3 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Dadar, 15th Sep. 2012, solitary, among grass, at 636 m (2089 ft) a.s.l., N. Yousaf, BPK92 (LAH10000079).

98

A

B

Plate 57. Figs. A–B. Bovista sp. A. Basidioma (BPK92). B. Eucapillitial hyphae. Bar: = 0.4 cm for A, = 4.2 µm for B.

99

A B

C

Plate 58. Figs. A–C. Bovista sp. A. Basidiospores. B. Paracapillitium. C. Endoperidial hyphae. Bar: = 3.6 µm for A, = 8.3 µm for B, = 7.5 µm for C.

100

Calvatia cyathiformis var. fragilis nom. prov. (Plates 59–60) Etymology: Due to its similarity with “C. fragilis” Basidioma sub-globose to ob-pyriforme (turbinate), slightly tapering at the base, off-white to cream when young with brown spotty appearance at some places, basal portion brown, up to 72 mm broad × 57 mm high, solitary, encrusted with the soil particles; attached to the substratum by small mycelial cord (as recorded in the specimen). Dehiscence by irregular rupturing of the apical part exposing the glebal portion. Peridium double. Exoperidium off-white, slightly tough, encrusted with soil particles outside, lost in mature specimen, non-persistent. Endoperidium brown. Gleba dark purplish brown (2.5YR1/2), compact, becoming powdery with age. Sub gleba absent.

Basidiospores rounded, ornamented, echinulate, dark brown, 5.0–7.0 µm in diam. including ornamentation, 4.2–5.2 µm in diam excluding ornamentation, echines up to 1.7 µm, sterigmal remnants absent from mounts, brownish in water mounts. Eucapillitium brown, broken in to segments, septate, broken where septa present, up to 5 µm in diam., branched, straight to sub-undulate, pores present, numerous, medium sized. Paracapillitium present, hyaline, septate, branched, not abundant, bulging at some points. Exoperidium composed of thick walled, brown, irregular shaped hyphal elements. Endoperidium composed of brown, aseptate, branched, tightly packed hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 10th Aug. 2010, at 3325 m (10908 ft) a.s.l., gregarious, on ground, M. Fiaz, CPK12 (LAH10000080).

101

A

B Plate 59. Figs. A–B. Calvatia cyathiformis var. fragilis (CPK12). A. A mature basidioma. B. Verrucose basidiospores. Bar: = 0.6 cm for A, = 3.0 µm for B.

102

A

B C

Plate 60. Figs. A–C. Calvatia cyathiformis var. fragilis. A. Exoperidial elements. B. Capillitial threads with pores. C. Endoperidial hyphae. Bar: = 6.4 µm for A, = 9.0 µm for B & C.

103

Calvatia deosaiensis nom. prov. (Plates 61–63)

Etymology: on the basis of type locality “Deosai”

Basidiomata sub-globose, off-white when young, up to 35 mm broad × 30 mm high, solitary; attached to the substratum by a rooting base with rhizomorphs; rhizomorphs white, branched, well developed, encrusted with soil particles. Dehiscence by an irregular rupturing of apical part. Exoperidium white when young, yellowish brown with age, thick (< 1 mm), composed of conspicuous polygonal verrucae or warts, bigger above, gradually becoming smaller below, attached to the endoperidium in young specimen. Endoperidium not exposed persistent. Gleba yellowish brown, compact, cottony, non-pulverulent. Sub- gleba composed of 1/3rd of basidioma, up to 10 mm in high, grayish brown, consists of small chambers, soft, compact. Basidiospores sub-globose to slightly ovoid, smooth under LM, finely verrucose under SEM, yellowish brown in water mounts, olivaceous in KOH medium, 4.8–7.6 × 4.5– 7.6 µm, pedicellate, pedicel hyaline, small, up to 1.5 µm long, sterigmal remnants absent from mounts. Eucapillitium olivaceous brown, broken into segments, branched, antler like, branching frequent, numerous pores present, undulate, bulging at some places, heavily encrusted with some amorphous material, up to 8 µm in diam., wall thickness up to 2.5 µm. Sub-gleba composed of yellowish brown, aseptate, long, thick walled, branched (branching frequent), undulate tightly packed hyphae with tapering ends. Exoperidium composed of globose, sub-globose to elongated hyphal elements. Endoperidium composed of dark orange to brown, thick walled, aseptate, undulate branched hyphae.

Material Examined: PAKISTAN: Gilgit-Baltistan, Deosai Plains, Deosai National Park, 9th Sep. 2011, solitary, among grass, at 4114 m (13497 ft) a.s.l., A. N. Khalid, CPK11 (Holotype), (LAH10000081). 104

A B

C

D

Plate 61. Figs. A–D. Calvatia deosaiensis (CPK11). A & B. Two views of basidiomata. C. Basidiospores. D. Exoperidial elments. Bar: = 0.7 cm for A, = 0.66 cm for B, = 5.9 µm for C, = 18 µm for D. 105

A

C B Plate 62. Figs. A–C. Calvatia deosaiensis. A. Eucapillitial threads. B. Endoperidial hyphae. C. Sub-glebal hyphae. Bar: = 17.6 µm for A, = 11 µm for B & C.

106

A

B

Plate 63. Figs. A–B. Calvatia deosaiensis. Scanning Electron Micrograph showing basidiospores with rough surface and a short pedicel.

107

Calvatia lilacina (Mont. & Berk.) Henn., Hedwigia 43: 205. 1904. (Plates 64–66)

Basidiomata consists of upper spore case and lower well developed sterile base, spore case not recorded in this collection, weathered (glebal material belonging to upper part is available); attached to the substrate by thick, white, branching rhizomorphs, encrusted with the particles of soil. Sterile base with diaphragm, 60–90 mm in diam. × 45– 60 mm in height, obovoid, sub-globose to broadly ob-pyriform, slightly tapering at the base which ends in a rounded pseudo- below, separated from the gleba by a prominent diaphragm. Peridium weathered from upper side. Gleba remnants are present, brown, powdery, pulverulent.

Basidiospores globose, verrucose, brown in water mounts, 7.2–9.0 µm in diam., including verrucose ornamentation, 4.5–5.4 µm in diam., excluding ornamentation; pedicel absent or rudimentary, sterigmal remnants absent from mounts. Eucapillitium Calvatia- type, brown, up to 6.4 µm in diam., thick walled (walls up to 1 µm), smooth to slightly encrusted, tapering at the ends sometimes (1.2 µm in diam.), straight to sub undulate, numerous small pores present, small to medium sized pores, often bulging at some points, not fragile, aseptate (rarely septate, broken where septa present), rare septa can be seen, frequently branched (dichotomous when occurring), brown in water mounts. Paracapillitium branched, hyaline, septate, and scarce. Exoperidium composed of spherical, oval to irregular sphaerocysts, elongated elements also present, deep brown. Endoperidiumcomposed of light brown, thin walled, branched and aseptate (sometimes joint like septa present) hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Khabbal, Ughi Forest, 1st Sep. 2008, solitary under Pinus roxburghii Sarg., on ground, at 975 m (3198 ft) a.s.l, M. Fiaz, NYG207 (LAH10000082); Gilgit-Baltistan, Deosai plains, Deosai National Park, 10th Sep. 2011, among grass, at 4114 m (13497 ft) a.s.l., A. R. Niazi, CPK1 (LAH10000083).

Comments: C. lilacina (CPK1) described here as a new record from Pakistan, is characterized by the presence of sterile base and conspicuous diaphragm at the base (Cunningham, 1925). Fragile peridium enclosing the upper glebal part and strongly verrucose spores are also important distinguishing features of this species. It is usually found growing in rainy season from July to September, also recorded causing fairy rings 108

on green pastures. It is cosmopolitan, mainly a European taxon however its distribution from Asia is not much known and found to be reported from , Srilanka, North America, South Africa, Southern Europe, Australia and New Zealand (Cunningham, 1925).

109

A H

G

Plate 64. Figs. a–c. Calvatia lilacina. a. Basidiomata with conspicuous diaphragm and remnants of gleba (CPK1). b. Side view of basidioma (NYG207). c. Longitudinal section of sterile base and diaphragm. Bar: = 1.4 cm for a–c.

110

A B

C

D Plate 65. Figs. A–D. Calvatia lilacina. A. Exoperidial elements. B. Verrucose basidiospores. C. Eucapillitial threads having pores. D. Endoperidial hyphae. Bar: = 12 μm for A, = 4 μm for B, = 6.6 μm for C, = 11 μm for D. 111

A

B C

D Plate 66. A–D. LM photographs of Calvatia lilacina (CPK1). A & B. Short capillitial threads. C. Porous capillitial hypha. D. Verrucose basidiopsores. Bar: = 16 µm for A–C, = 3.0 µm for D. 112

Calvatia pseudocyathiformis nom. prov. (Plates 67–68) Etymology: Due to its resemblance with C. cyathiformis Basidioma ob-pyriforme (turbinate), tapering at the base, brown with off-white granules over the surface, up to 100 mm broad × 90 mm high, solitary; attached to the substratum by small rhizomorphs. Dehiscence by irregular rupturing of the apical part exposing the glebal portion. Peridium double. Exoperidium brown, persistent. Endoperidium off-white to light brown. Gleba dark purplish brown, compact, becoming powdery with age. Sterile base present, up to 20 mm high.

Basidiospores globose, ornamented, echinate, violaceous, 5.5–6.0 µm in diam. including ornamentation, 4.2–4.4 µm in diam., excluding ornamentation, echines up to 0.7 µm, thick-walled, sterigmal remnants absent from mounts. Eucapillitium brown, broken in to segments, septate, broken where septa present, 2.2–4.0 µm in diam., wall thickness up to 0.5 µm, branched, straight to sub-undulate, pores present, encrusted with some amorphous material.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 10th Aug. 2010, at 3325 m (10908 ft) a.s.l., gregarious, on ground, M. Fiaz, CPK4 (Holotype), (LAH10000084).

113

A

B

C

Plate 67. Figs. A–C. Calvatia pseudocyathiformis (CPK4). A. A mature basidioma. B. Basidiospores. C. Eucapillitial hyphae. Bar: = 0.6 cm for A, = 3 μm for B, = 4.4 μm for C. 114

A

B

Plate 68. A–B. LM photographs of Calvatia pseudocyathiformis (CPK4). A. Basidiospores. B. Septate and porous capillitial hyphae with spores. Bar: = 6.6 µm for A, = 4 µm for B.

115

Disciseda hyalothrix (Cooke & Massee) Hollós, Növ. Közl. 1: 107 (1902) (Plates 69–70) Basidioma globose, blackish brown when mature, up to 20 mm broad × 20 mm high, encrusted with soil and other debris material, solitary; rhizomorphs not observed, lost during collection. Dehiscence by an apical pore, up to 5 mm in diam. Peridium double. Exoperidium not observed, lost. Endoperidium blackish brown, papery, rough. Gleba blackish brown, compact, cottony. Sub gleba absent

Basidiospores globose, ornamented, verrucose, brown, 8.0–11.3 µm in diam. including verrucae, 6.9–7.8 µm in diam. (ornamentation excluded), verrucae up to 2.8 µm long; pedicel light brown to hyaline, curved at tips, tapering, up to 19.5 µm long, 2.4 µm thick, dark brown in water mounts. Eucapillitium brown, broken in to segments, short threads, branched, aseptate, straight to sub-undulate, pores absent, thin walled, up to 5.0 µm in diam. (wall thickness 1.6 µm). Paracapillitium absent. Exoperidium not recorded. Endoperidium composed of brown, thin walled, aseptate to rarely septate, branched, tightly packed hyphae, up to 4 µm in diam.

Material examined: PAKISTAN: Punjab, Kasur district, Changa Manga forest, 24th Aug. 2013, at 201 m (660 ft) a.s.l., solitary, on ground, N. Yousaf, DPK1 (LAH10000085).

Comments: It is characterized by globose to sub-globose basidioma with ornamented and pedicellate . It is wide spread taxon and known from many countries (Laferrière & Gilbertson 1992; Esqueda et al., 1995; Ochoa & Moreno, 2006; Moreno et al., 2007, 2010a).

116

A B

C D

Plate 69. Figs. A–D. Disciseda hyalothrix (DPK1). A. A mature Basidioma. B. Pedicellate Basidiospores. C. Eucapillitial hyphae. D. Peridial hyphae. Bar: = 0.55 µm for A, = 6.4 µm for B, = 3.4 µm for C, = 14.5 µm for D. 117

A

B

C

Plate 70. Figs. A–C. LM photographs of Disciseda hyalothrix (DPK1). A. Spores and short eucapillitial hyphae. B & C. Verrucose, pedicellate basidiospores. Bar: = 70 µm for A, = 12.5 µm for B, = 5.5 µm for C.

118

Lycoperdon pyriforme Schaeff., Fung. Bavar. Palat. 4: 128 (1774) (Plates 71–72)

Basidiomata pear shaped, off white to cream when young, turning brown with age, up to 30 mm broad × 40 mm in height, gregarious; attached to the substratum by well- developed rhizomorphs; rhizomophs thick, white, heavily encrusted with soil particles and other debris material. Dehiscence by an apical pore. Peridium double. Exoperidium off white to cream, brown when matures, slightly warty to granulose when young. Endoperidium papery, pale brown. Gleba off-white in young specimens, olivaceous brown in mature, compact, cottony. Pseudocolumella present well developed. Sterile base present.

Basidiospores globose, thick walled, smooth, olivaceous brown, 3.9–4.6 µm in diam., detached sterigmal remnants absent from mounts. Eucapillitium well developed, olive brown, aseptate, branched, dichotomous branching, elastic, straight, up to 3.7 µm in diam., (wall thickness up to 1.6 µm), pores absent. Paracapillitium abundant, hyaline, septate, branched. Exoperidium composed of irregular shaped hyphal elements. Endoperidium composed of hyaline, branched, aseptate tightly packed hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Ayubia, Khanspur, 10th Aug. 2010 & 5th July, 2011, gregarious, on wood of Pinus wallichiana, at 2250 m (7500 ft) a.s.l., N. Yousaf, LPK71 (LAH10000086), LPK72 (LAH10000087); 16th Aug. 2011, gregarious, N. Yousaf, LPK8 (LAH10000088), LPK15 (LAH10000089), LPK57 (LAH10000090); Halipad, 20th July, 2011, solitary, A. R. Niazi, LPK10 (LAH10000091), LPK12 (LAH10000092), LPK45 (LAH10000093), KPK210 (LAH10000094); 21st August 2011, LPK48 (LAH10000095); Mukhspuri, 3rd Aug., 2011, under Abies pindrow, at 2755 m (9040 ft) N. Yousaf, LPK62 (LAH10000096); Kaghan valley, Nadibunglaw, 15th August, 2011, on ground, semi hypogeous, in groups, under Pinus wallichaina, at 1314 m (4313 ft) a.s.l., N. Yousaf, LPK14A, LPK17, LPK43, (LAH 108210); 13th August, 2011, N. Yousaf, LPK44 (LAH); Swat district, Malam Jabba, 7th August. 2012, solitary, at 2485 m (8152 ft) a.s.l., N. Yousaf, LPK49, LPK50, (LAH 108210); 4th Sep. 2013, gregarious, under Pinus wallichiana, N. Yousaf, LPK60 (LAH 108210); Abbottabad district, Thandiani, 8th August 2012, gregarious, on wood of Pinus wallichiana, 2750 m (8858 ft), N. Yousaf, LPK51 (LAH10000097); Mansehra district, Dadar, 15th Sep. 2012, gregarious, under Pinus wallichiana, at 636 m (2089 ft) a.s.l., N. Yousaf, LPK38 (LAH10000098), LPK54 (LAH10000099). 119

Commnets: L. pyriforme is an edible species of the genus commonly known as pear- shaped , found growing with preference for wood (Kuo, 2004; Lincoff, 1981, Larsson & Jeppson, 2008). This has previously been reported by Ahmad from Murree in 1952. It is a new record from Abbottabad district, Mansehra district, Kaghan valley and Swat district of Khyber Pakhtunkhwa, Pakistan.

120

B A

C D

Plate 71. Figs. A–D. Lycoperdon pyriforme. A. Mature basidiomata (LPK17). B. Basidiomata (LPK43). C. LPK72. D. LPK60. Bar: = 2 cm for A–D. 121

A

C

B

D E

Plate 72. Figs. A–E. Lycoperdon pyriforme. A. Basidiospores. B. Thick walled exoperidial elements. C. Eucapillitial hyphae. D. Paracapillitial hyphae. E. Endoperidial hyphae. Bar: = 3.0 µm for A, = 11 µm for B, = 8 µm for C, = 6.2 µm for D, = 11 µm for E. 122

Bovistella japonica Lloyd, Mycol. Writ. 2: 281 (1906) (Plates 73–74)

≡ Lycoperdon japonicum

Basidiomata globose to sub-globose, 55–85 mm diam.  85 mm high, white when young, turning reddish brown with age. Sterile Base well developed, up to 30 mm high, spongy, chambered, basally attached to the substratum by well developed, thick mycelial strand, having white, branched rhizomorphs heavily encrusted with soil particles; ostiole lacking; dehiscence irregular, fruiting body breaking apart apically. Peridium <1mm thick. Exoperidium white when young, becoming dull brown with age, thin, formed of granular matter which covers the endoperidium, persistent. Endoperidium entirely covered by exoperidium even in older specimen, light brown, thin, papery. Gleba brown, cottony, compact.

Basidiospores subglobose to ovoid, hyaline to greenish in KOH, 4.0–5.5 × 3.8–4.7 µm, pedicellate, pedicels short to very long, up to 19 µm long. Capillitium Bovista type, well developed, separate units having a thick main axis (up to 20 µm diam.), thick walled (walls up to 2.85 µm), branches originating from the main axis, 1.2–6.0 µm diam. (walls up to 1.0 µm), with attenuate tips. Exoperidium composed of sub-globose to irregular sphaerocysts. Endoperidium composed of hyaline, unbranched, and aseptate hyphae. Sterile base composed of pigmented, thick walled, branched, and aseptate hyphae.

Material examined: Pakistan: Khyber Pakhtunkhwa, Nathia gali, 13th Sep. 2006, at 2500 m (8205 ft) a.s.l., solitary, on ground, under Pinus wallichiana A. B. Jacks., in Himalayan moist temperate forest, A. R. Niazi, BPK78 (LAH130906).

123

Plate 73. Figs. A–H. Lycoperdon japonicum. A. Young basidioma. B. Mature basidioma. C. Pedicellate basidiospores. D. Typical Bovista type capillitial units. E. Exoperidial elements (Sphaerocysts). F. Thick, main axis of capillitial threads. G. Aseptate endoperidial hyphae. H. Hyphae of sterile base. Bar: = 1.5 cm for A & B, = 2.5 μm for C, = 60 μm for D, = 3 μm for E, = 20 μm for F, = 10 μm for G & H. 124

A B

C

D E Plate 74. Figs. A–E. LM photographs of Lycoperdon japonicum (BPK78). A & B. Eucapillitial threads. C & D. Pedicellate Basidiospores. E. Thick main axis of eucapillitial hypha. Bar: = 72 µm for A, = 60 µm for B, = 50 µm for C, = 2.75 µm for D, = 12 µm for E. 125

Lycoperdon utriforme Bull., Hist. Champ. Fr. (Paris): 153 (1791) (Plate 75)

Basidioma pyriforme, off-white to cream when young, up to 100 mm broad × 70 mm high, solitary; attached to the substratum by rhizomorphs. Dehiscence by irregular rupturing of the apical part exposing the glebal portion. Peridium double. Exoperidium off- white, forming cracks at the apical portion, hexagonally chambered, lost in mature specimen exposing the brown endoperidium. Endoperidium brown, papery. Gleba brown, compact, becoming powdery with age. Sterile base present.

Basidiospores rounded, smooth, brown, 3.8–5.5 µm in diam. Eucapillitium brown, broken in to segments, septate, broken where septa present, up to 6.5 µm in diam., wall thickness up to 0.7 µm, branched, straight to sub-undulate, pores present, numerous, medium sized. Paracapillitium absent. Exoperidium composed of thick walled, brown, globose to sub-globose, hyphal elements, up to 31 × 21 µm in diam. Endoperidium composed of brown, aseptate, branched, tightly packed hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Kalam, 9th Sep. 2013, at 5287 m (17347 ft) a.s.l., solitary, on ground, S. Jabeen, CPK5 (LAH100000100).

Comments: L. utriforme is characterized by big fruiting bodies with distinct diaphragm separating gleba from subgleba. Gleba has characteristic type of fragile capillitium with numerous pits and smooth basidispores (Larsson & Jeppson, 2008; Jalink, 2010).

126

B A C

F D E

Plate 75. A–F. Lycoperdon utriforme (CPK5). A. A young basidioma. B. LM photograph of basidiospores. C. LM photograph of eucapillitial hypha. D. Exoperidial elements. E. Illustration of basidiospores F. Illustration of capillitial thread. Bar: = 1.2 cm for A, = 6.5 µm for B, = 8 µm for C, = 20 µm for D, = 4 µm for E, = 4.5 μm for F.

127

Lycoperdon parvasporum nom. prov. (Plate 76) Etymology: on the basis of small size of the spores Basidiomata pyriforme, off white to cream when young, grayish brown with age, consists of upper sporecase and pseudo-stipe below, up to 35 mm broad × 60 mm in height; spore-case sub-globose, up to 35 mm broad × 25 mm in height; pseudo-stipe up to 15 mm broad × 40 mm in height, gregarious; attached to the substratum by well-developed rhizomorphs; rhizomophs thin, white, branched, heavily encrusted with soil particles and other debris material. Dehiscence by an apical pore, up to 5 mm in diam. Peridium double. Exoperidium consists of off-white to grayish black convergent spines, later peeling off from the surface leaving reticulate pattern of scars, more spines on upper spore case, lesser below. Endoperidium papery, greyish brown, exposed in mature specimens. Gleba white when young, brown with age, compact, cottony. Pseudocolumella present, well developed. Sterile base present, well developed, compact.

Basidiospores globose, finely verrucose, thick walled, olivaceous green, up to 3.5 µm in diam. including verrucae, detached sterigmal remnants absent in the mounts. Eucapillitium well developed, olivaceous brown, aseptate, false septa present, branched, ocassional dichotomous branching, straight to sub-undulate, branches with tapering ends, encrusted with amorphous material, pores rare, up to 4 µm in diam., thick-walled, (wall thickness up to 1.4µm). Paracapillitium present, hyaline, septate, branched. Exoperidium composed of hyaline, globose to sub-globose sphaerocysts. Endoperidium composed of hyaline, branched, rarely septate tightly packed hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Miandam, 7th Aug. 2012, gregarious, at 1889m (6200 ft) a.s.l., N. Yousaf, LPK46 (Holotype), (LAH100000101). 128

A

B

C Plate 76. Figs. A–C. Lycoperdon parvasporum (LPK46). A. Basidiomata. B. LM photograph of basidiospores. C. LM photograph of eucapillitial hyphae. Bar: = 1.3 cm for A, = 7.7 µm for B, = 14.6 µm for C.

129

Lycoperdon perlatum Pers., Observ. mycol. (Lipsiae) 1: 145 (1796) (Plates 77–78) Basidiomata pyriforme, off white to cream when young, brown with age, consists of upper sporecase and lower pseudo-stipe below, up to 35 mm broad × 60 mm in height; spore-case subglobose, up to 35 mm broad × 25 mm in height; pseudostipe up to 15 mm broad × 40 mm in height, gregarious, solitary sometimes; attached to the substratum by well-developed rhizomorphs. Rhizomophs thin, white, branched, heavily encrusted with soil particles and other debris material. Dehiscence by an apical pore; pore medium up to 10 mm in diam. Peridium double. Exoperidium consists of off-white convergent spines, later peeling off from the surface leaving reticulate pattern of scars, more spines on upper spore case, lesser below. Endoperidium papery, greyish brown, exposed in mature specimens. Gleba white when young, brown with age, compact, cottony, becoming pulverulent with age. Pseudocolumella present, well developed. Sterile base present, well developed, compact, alveolate, grayish brown.

Basidiospores globose, ornamented, verrucose, thick walled, brown, 3.9–5.0 µm in diam. including verrucae, 3.8–4.4 µm in diam. excluding verrucae, verrucae up to 1.3 µm long, detached sterigmal remnants present in the mounts, spore deposit olivaceous in water mounts. Eucapillitium well developed, yellowish brown, aseptate, branched, dichotomous branching, straight to sub-undulate, branches with tapering ends, encrusted with some amorphous material, pores rare, up to 5.3 µm in diam., (wall thickness up to 2.2 µm). Paracapillitium present, hyaline, septate, branched. Exoperidium composed of hyaline, globose to sub-globose sphaerocysts. Endoperidium composed of hyaline, branched, rarely septate tightly packed hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Abbottabad district, Ayubia, Khanspur, 11th Aug. 2010 & 14th Aug. 2010, gregarious, on ground, at 2250 m (7500 ft) a.s.l., N. Yousaf, LPK40 (LAH100000102), LPK41 (LAH100000103); 18th July, 2011, solitary, N. Yousaf, LPK42 (LAH100000104); 10th & 11th Aug. 2012, gregarious, A. R. Niazi, LPK24 (LAH100000105), LPK25 (LAH100000106), LPK68 (LAH100000107), LPK69 (LAH100000108), LPK65 (LAH100000109), A. R. Niazi; Nathia gali, 10th July, 2010, gregarious, on ground, among grass, at 2423 m (7951 ft) a.s.l., N. Yousaf, LPK11 (LAH100000110); Swat district, Ushu, 4th Sep. 2013, solitary, under Cedrus deodara, at 2418 m (7933 ft) a.s.l., S. Jabeen, LPK75 (LAH100000111); Kalam, 7th Sep. 2013, at 2123m (6965 ft) a.s.l., under Cedrus deodara, S. Jabeen, LPK82 (LAH100000112). 130

Mansehra district, Chattar plains, 15th September 2012, gregarious, under Pinus wallichiana A. B. Jacks.,, at 1661 m (5451 ft) a.s.l., LPK36 (LAH100000113), LPK39 (LAH100000114); Mansehra district, Khabbal Paien, Ucharhi, 1st Aug. 2010, in groups, at 1825 m (5987 ft) a.s.l., M. Fiaz, F-04, (LAH100104).

Comments: is called warted puffball and usually found growing gregariously in fields. It is characterized by off white, pear shaped fruiting bodies with stipe like base. Exoperidium is composed of whitish warts which rubbed off easily as specimen ages leaving a reticulate pattern over the sporocarp. It is edible when internal flesh is white and young (Dickinson & Lucas, 1982, Kuo, 2007). It is most commonly occurring puffball in different regions of world and also in Himalayan moist temperate forests of Pakistan. Lycoperdon excipuliforme is a similar species in the field and can be confused with L. perlatum. Both species form reticulate pattern after sloughing off warts but fruiting bodies of L. excipuliforme are considerably bigger compared to L. perlatum (Yousaf et al., 2012a). It is the most commonly occurring Lycoperdon sp. in moist temperate region of Pakistan. It has been reported from Patriata (Murree), Changla gali, Bahrain (Swat), Shogran, Sharan (Kaghan valley) by Ahmad in 1952 and 1956. It is first report of this taxon from Mansehra district and Khanspur.

131

B A

C D E

G F

Plate 77. Figs. A–G. Lycoperdon perlatum. A. Basidiomata, collection no. LPK68 B. LPK69. C. LPK70. D. LPK73. E. LPK82. F. LM photograph of basidiospores. G. LM photograph eucapillitial hyphae. Bar: = 2 cm for A–E, = 7.5 µm for F, = 14 µm for G. 132

A B

C

D Plate 78. Figs. A–D. Lycoperdon perlatum. A. Basidiospores. B. Eucapillitial hyphae. C. Exoperidial elements. D. Endoperidial hyphae. Bar: = 3.6 µm for A, = 18.3 µm for B, = 17 µm for C, = 7 µm for D. 133

Lycoperdon albiceratum nom. prov. (Plates 79–80)

Etymology: on the basis of off white to yellow color of exoperidium.

Basidiomata sub-globose to pyriforme, sometimes turbinate, sometimes forming a pseudo-stipe, off-white to light yellow when young, turning light brown when mature, up to 40 mm broad × 60 mm high, gregarious; attached to the substratum by a well-developed mycelial base, rhizomorphs branched, white, encrusted with soil particles and other debris material. Dehiscence by an apical pore, pore up to 5 mm in diam., Peridium double. Exoperidium off-white when young, yellow to brown with age, granular. Endoperidium brown, papery. Gleba olivaceous brown, compact, cottony. Sterile base present, up to 30 mm high.

Basidiospores globose, verrucose, olivaceous brown, 3.3–4.8 µm in diam., sterigmal remnants attached sometimes, numerous sterigmal remnants present in mounts, verrucae >1 µm, central oil droplet present, olivaceous in water mounts. Eucapillitium brown, long threads, pores present, branched, up to 2.5–11.8 µm in diam., wall thickness up to 0.7 µm, aseptate, straight to sub-undulate, bulging at some points, sub-septal ramifications absent, branches with tapering ends, tips undulate. Paracapillitium absent. Exoperidium composed of hyaline, globose to sub-globose hyphal element. Endoperidium composed of hyaline, straight, frequently septate, rarely branched, thin-walled, tightly packed hyphae, up to 4 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Thandiani, 16th Sep. 2012, on ground, among grass, at 2672 m (8766 ft) a.s.l., A. N. Khalid, LPK37 (Holotype) (LAH100000115); Mansehra district, Ughi forest, 5th July, 2011, in groups, among grass, at 1174 m (3852 ft) a.s.l., M. Fiaz, MPK2 (LAH100000116).

Additional material examined: Lycoperdon sp. Mansehra district, Ughi forest, 1st July, 2011, M. Fiaz, MPK5 (LAH100000117); Thandiani, 17th Aug. 2013, on ground, among grass, N. Yousaf, LPK47 (LAH100000118). 134

A B

C D

E

Plate 79 Figs. A–E. Lycoperdon albiceratum (LPK47). A. Basidiomata. B. LM photograph of basidiospores. C–E. LM photographs of eucapillitium. Bar: = 1.3 cm for A, = 8 µm for B, = 33 µm for C, = 13 µm for D, = 10 µm for E. 135

B A

C D

Plate 80. Figs. A–D. Lycoperdon albiceratum. A. Basidiospores. B. Exoperidial elements. C. Capillitial threads. D. Septate endoperidial hyphae. Bar: = 3.8 µm for A, = 15 µm for B, = 10 µm for C, = 6 µm for D.

136

Lycoperdon altimontanum Kreisel, Feddes Repert. Spec. Nov., Beih. 87(1-2): 97 (1976) (Plates 81–82) Basidiomata turbinate, pale yellowish to light brown, up to 60 mm broad × 60 mm high, solitary; attached to the substratum by a small mycelial cord with some rhizomorphs, encrusted with the particles of soil and other debris material, up to 15 mm high. Dehiscence by an apical pore, osteole up to 15 mm in diam., Peridium double. Exoperidium persistent, entirely covering the endoperidium even in mature specimen, adherent to the endoperidium¸ pale yellow o brown, granular, granules less above, more below, Endoperidium brown. Gleba compact, cottony, brown with olivaceous tint. Sterile base present, well-developed, olivaceous brown, up to 25 mm high, chambered.

Basidiospores globose, verrucose, olivaceous, 6.0–9.5 × 5.7–9.5 µm including ornamentation, 3.6–7.4 × 3.6–7.6 µm excluding ornamentation, echines up to 1.7 µm, sterigmal remnants attached, up to 5.7 µm, hyaline, numerous sterigmal remnants present in mounts. Eucapillitium brown, well-developed, long separate threads, aseptate, false septa present, frequently branched, pores present, medium sized, up to 5 µm in diam., sub- septal ramifications present, wall thickness up to 2 µm, branches with tapering ends, straight to sub undulate. Paracapillitium absent. Exoperidium composed of hyaline, globose to subglobose to elongated hyphal elements, up to 13 µm high × 8.6 µm broad. Endoperidium composed of thin walled, septate, unbranched, tightly packed hyphal elements, up to 2 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Mankial, 4th Sep. 2013, under Cedrus deodara (Roxb.) G.Don, at 3822 m (12542 ft) a.s.l., S. Jabeen, LPK63 (LAH100000119).

Comments: L. altimontanum is characterized by turbinate basidiomata with well- developed sterile base. Eucapillitium in this Lycoperdon species is very distinct, well developed, frequently branched, having false septa, subseptal ramification. Morphologically, this taxon is close to Lycoperdon molle but spores are more echinulated in L. altimontanum as compared to the former. It is an addition to gasteroid fungi of Pakistan. 137

A

B

Plate 81. Figs. A–B. Lycoperdon altimontanum (LPK63). A. Mature basidioma. B. Basidiospores. Bar: = 0.9 cm for A, = 3 µm for B.

138

B A

C

Plate 82. Figs. A–C. Lycoperdon altimontanum. A. Exoperidial elements. B. Eucapillitial hyphae. C. Endoperidial hyphae. Bar: = 10 µm for A, = 8 µm for B, = 3.5 µm for C.

139

Lycoperdon aurea-brunneum nom. prov. (Plates 83–84)

Etymology: on the basis of golden brown color of endoperidium of basidiomata

Basidiomata sub globose to turbinate, white when young, turning brown with age, 40 mm in diam. × 45 mm in height; attached to the substratum by well-developed thick white rhizomorphs, adherent with soil and other debris material. Dehiscence by an apical pore; pore not observed. Peridium double. Exoperidium white, in the form of pointed warts, which separates from endoperidium in sheets, yellowish brown at maturity, sloughing off, first peeling from the upper side of spore case, and then from the lower side; warts slightly bigger above than below. Endoperidium relatively smooth when exoperidium peels off, brown, papery. Gleba brown, cottony. Sterile base present, well developed, chambered; chambers < 1mm wide.

Basidiospores globose, minutely ornamented, echinulated, dark olivaceous brown, brown in water mounts, 4.75–6.0 µm in diam. Eucapillitium well developed, long separate threads, dark brown, branched, branching not frequent, rare, pores present, 1.9–5.0 µm in diam., thick walled (walls up to 1.0 µm), with attenuate tips, undulated, rarely septate, bulging at some points. Exoperidium composed of hyaline, globose to subglobose sphaerocysts. Endoperidium composed of hyaline, unbranched and septate hyphae.

Material examined: PAKISTAN: Gilgit-Baltistan, Fairy meadows, 22nd July. 2010, in groups, at 4191 m (13751 ft) a.s.l., A.N. Khalid, LPK1 (Holotype), (LAH100000120). 140

A

B

Plate 83. Figs. A–B. Lycoperdon aurea-brunneum (LPK1). A & B. Young and mature basidioma. Bar: = 0.7 cm for A & B. 141

B A

C D

Plate 84. Figs. A–D. Lycoperdon aurea-brunneum. A. Basidiospores. B. Exoperidial elements. C. Endoperidial hyphae. D. Encrusted eucapillitial hyphae. Bar: = 2 µm for A, = 12 µm for B, = 7 µm for C, = 4 µm for D.

142

Lycoperdon dermoxanthum var. album nom. prov. (Plates 85–86)

Etymology: on the basis of white color of basidioma

Basidioma globose, off-white when young, brown when mature, up to 17 mm diam., solitary; attached to the substratum by a short mycelial base, encrusted with soil particles and other debris material. Dehiscence by an apical pore. Peridium double. Exoperidium off-white when young, brown with age, later on in the form of granules all over the gasterocarp. Endoperidium brown, papery. Gleba dull brown, compact, cottony. Sub gleba absent

Basidiospores globose, smooth to asperulate, light brown, 3.5–4.0 µm in diam., with a stump of pedicel, central oil droplet present, light brown in KOH mounts. Eucapillitium brown, intermediate type, pores numerous, branched, up to 3 µm in diam., wall thickness up to 0.9 µm, aseptate, thin walled, straight to sub-undulate, bulging at some points, sub-septal ramifications absent, branches with tapering ends, tips undulate. Paracapillitium branched, hyaline, septate. Exoperidium composed of light yellowish brown, subglobose to elongated hyphal elements intermixed with hyphae, 60 × 9 µm. Endoperidium composed of light yellowish brown, straight, frequently septate, branched, thin-walled, tightly packed hyphae, up to 4.7 µm in diam., bulging at some points.

Material examined: PAKISTAN: Punjab, Dera Ghazi Khan district, Fort Munro, 17th Aug. 2013, on sandy soil, distributed throughout the sampling sites at various maturity stages, under Eucalyptus sp. at 123.75 m (406 ft) a.s.l., N. Yousaf, BPK36 (LAH100000121). 143

A B

C

Plate 85. Figs. A–C. Lycoeprdon dermoxanthum var. album (BPK36). A & B. Different views of basidioma. C. Exoperidial elements. Bar: = 0.6 cm for A & B, = 8 µm for C. 144

A

B C

Plate 86. Figs. A–C. Lycoperdon dermoxanthum var. album. A. Basidiospores B. Eucapillitial hypha. C. Endoperidial elements. Bar: = 2 µm for A, = 3.5 µm for B, = 8 µm for C.

145

Lycoperdon dermoxanthum var. flavum nom. prov. (Plates 87–88)

Etymology: on the basis of yellow color of basidioma

Basidiomata globose to slightly sub-globose, yellowish when young, turning brown when mature, up to 13–17 mm broad × 13–17 mm high, gregarious; attached to the substratum by a well-developed mycelial base, rhizomoprhs branched, white, encrusted with soil particles and other debris material. Dehiscence by an apical pore, up to 1 mm in diam. Peridium double. Exoperidium off-white when young, yellow to brown with age, later on in the form of small granules all over the basidioma, persistent. Endoperidium yellowish brown, not exposed in mature specimens, papery. Gleba olivaceous brown, compact, cottony. Sub gleba present

Basidiospores globose, asperulate, olivaceous brown, brown in water mounts, 4.3– 4.8 µm in diam., with a stump of pedicel, central oil droplet present. Eucapillitium brown, long threads, pores present, branched, up to 4 µm in diam., wall thickness up to 0.6 µm, aseptate, straight to sub-undulate, bulging at some points, sub-septal ramifications absent, branches with tapering ends, tips undulate. Paracapillitium branched, hyaline, septate, up to 5 µm in diam. Exoperidium composed of hyaline, sub globose to elongated hyphal elements intermixed with hyphae 40 × 19 µm. Endoperidium composed of olivaceous yellow, straight, frequently septate, branched, thin-walled, tightly packed hyphae, up to 3 µm in diam., bulging at some points.

Material examined: PAKISTAN: Punjab, Dera Ghazi Khan District, Fort Munro, 17th Aug. 2013, on sandy soil, distributed throughout the sampling sites at various maturity stages, under Eucalyptus sp. at 123.75m (406 ft) a.s.l., N. Yousaf, BPK31 (LAH100000122). 146

A

B

Plate 87. Figs. A–B. Lycoperdon dermoxanthum var. flavum (BPK31). A. Different views of basidiomata. B. Exoperidial elements. Bar: = 0.6 cm for A, = 8 µm for B.

A

147

Plate 88. Figs. A–C. Lycoperdon dermoxanthum var. flavu. A. Basidiospores B. Endoperidial elements. C. Eucapillitial hyphae. Bar: = 2.6 µm for A, = 8 µm for B, = 2.5 µm for C. 148

Lycoperdon excipuliforme (Scop.) Pers., Syn. meth. fung.: 143 (1801) (Plate 89) Basidiomata elliptical to turbinate, 30–50 mm diam. × 50 mm high; spore case broad, globose to subglobose; ostiole present, irregular. Sterile base present, and forming a well-developed pseudostipe, attached to the substrata by well developed rhizomorphs, these white, branched, thick, and heavily encrusted with soil particles, and woody debris. Peridium layered, thickness up to 1 mm. Exoperodium dull brown, fragile, in the form of granules which later slough off. Endoperidium dull, golden brown, papery. Gleba dark brown, pulverulent.

Basidiospores globose to subglobose, dark brown, 5.3–6.7 µm diam. (4.4–5.5 µm excluding ornamentation), verrucose (verrucae up to 1 µm), numerous sterigmal remnants present in mounts, these up to 35.6 µm long. Eucapillitium capillitial threads 1.14–6.8 µm diam., branched, (dichotomous when occurring), thick-walled (walls up to 2 µm), sub- undulated to undulate, aseptate, having slit-like pores, with attenuate tips. Paracapillitium absent. Exoperidium composed of subglobose sphaerocysts. Endoperidium composed of unbranched, septate hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Nathia gali, 26th Aug. 2006, solitary, on ground, under bamboo tree, at 2500 m (8205 ft) a.s.l., N. Yousaf, LPK71 (LAH100000123), NYG209 (LAH268006); Swat district, Miandam, 7th Aug. 2012, gregarious, at 1889 m (6200 ft) a.s.l., N. Yousaf, LPK72 (LAH100000124); Mansehra district, Ughi forest, 5th July. 2011, in groups, among grass, at 1091 m (3582 ft) a.s.l., M. Fiaz, LPK56 (LAH100000125); Nadibunglaw, on ground, among grass, N. Yousaf, LPK58 (LAH100000126); Swat district, Mankial, 4th Sep. 2013, on forest floor with decaying needles of Cedrus deodara, at 3822 m (12542 ft) a.s.l., S. Jabeen, LPK80 (LAH100000127).

Comments: Lycoperdon excipuliforme, commonly called the “pestle puffball”, usually fruits in groups from summer to autumn in coniferous, and deciduous woodlands. It is characterized by large basidiomes consisting of upper spore case, long pseudostipe, and by the presence of slit-like pores in capillitial threads. Lycoperdon perlatum Pers. resembles L. excipuliforme in that they develop the same reticulate pattern over the surface of endoperidium as the larger verrucae slough off. However, L. perlatum has smaller stature, and lacks a well-developed, elongated pseudo stipe. This species is a new addition to myco flora of Pakistan. 149

F

A Plate 89. Figs. A–F. Lycoperdon excipuliforme. A & B. Basidiomata (LPK71). C. Verrucose F basidiospores. D. Capillitial threads. E. Exoperidial elements (sphaerocysts). F. Endoperidial E hyphae. Bar: = 0.8 cm for A & B, = 2.5 µm for C, = 10 µm for D & F, = 4 µm for E. 150

Lycoperdon lignum-brunneum nom. prov. (Plates 90–91)

Etymology: on the basis of wood-brown color of basidiomata

Basidiomata pyriforme, wood-brown above, brownish at base, consists of upper spore case and lower short pseudostipe below, up to 30–40 mm broad × 35–45 mm in height; pseudostipe up to 15 mm broad × 40 mm in height, gregarious, solitary some times; attached to the substratum by well-developed mycelial cord, consists of rhizomorphs; rhizomophs off-white, branched, up to 20 mm high, heavily encrusted with soil particles and other debris material. Dehiscence by an irregular rupturing of an apical part. Peridium double. Exoperidium olivaceous, persistent, entirely covering the endoperidium even in mature specimens. Endoperidium papery, light brown. Gleba yellowish to light brown with age, compact, cottony. Pseudocolumella absent. Sterile base present, well developed, compact, spongy, light yellowish brown, up to 10 mm in diam.

Basidiospores globose, thick walled, verrucose, brown, 5.6–7.5 µm in diam. including verrucae, verrucae up to 1.5 µm long, spore yellowish brown in water mounts. Eucapillitium well developed, yellowish brown, aseptate, branched, dichotomous branching, straight to subundulate, branches with tapering ends, pores absent, up to 6 µm in diam., (wall thickness up to 3.5 µm), tips up to 3.5 µm thin. Paracapillitium absent. Exoperidium composed of hyaline to light brown, sub-globose to elongated sphaerocysts, up to 23.5 µm in diam. Endoperidium composed of hyaline to light brown, thin walled, branched, rarely septate tightly packed hyphae, up to 4 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Abbottabad district, Ayubia, Khanspur, 22th Aug. 2010, solitary, among grass, at 2250 m (7500 ft) a.s.l., N. Yousaf, CPK3 (Holotype), (LAH100000128); 13th Sep. 2012, among grass, solitary, N. Yousaf, LPK27 (LAH100000129), LPK28 (LAH100000130), LPK33 (LAH100000131). 151

A

B

C

Plate 90. A–C. Lycoperdon lignum-brunneum (CPK3). A. Mature basidiomata. B. LM photograph of basidiospores. C. LM photograph of thick-walled capillitial hyphae. Bar: = 0.9 cm for A, = 10 µm for B, = 8 µm for C. 152

A B

C D

Plate 91. A–D. Lycoperdon lignum-brunneum (CPK3). A. Thick-walled eucapillitial hyphae. B. Endoperidial hyphae. C. Basidiospores. D. Exoperidial elements. Bar: = 4 µm for A, = 14 µm for B, = 2 µm for C, = 17 µm for D.

153

Lycoperdon lineum nom. prov. (Plates 92–93)

Etymology: on the basis of linen color of basidioma

Basidiomata subglobose to ob-pyriforme, consists of upper spore case and basal pseudostipe sometimes, linen color to grayish brown above, off-white below, 15–47 × 25– 55 mm in diam.; attached to the substratum by white mycelial strands, heavily encrusted with debris material and soil particles. Exoperidium in the form of minute warts, persistent, much dense towards the base, warts off-white to brownish, smaller, short to longer, up to 1 mm long, narrower towards tips, peeling off at some places exposing the endoperidium. Endoperidium off-white to brown, papery. Gleba light brown, cottony. Sterile base well developed, prominent, 10–23 mm wide × 10–28 mm high.

Basidiospores globose, ornamented, echinulate, brown, 5.2–6.5 μm in diam. including ornamentation, 4.0–5.0 μm in diam. (ornamentation excluded), echines 0.76– 1.33 µm high, pedicellate, pedicel up to 0.95 µm, numerous sterigmal remnants present in mounts. Eucapillitium well-developed, prominent, dark brown, non elastic, branched, branching occasional, aseptate, but a few septa present at some points, branches hyaline, attenuate at tips, pores present, up to 1.33–6.6 µm in diam., with wall thickness 0.6–1.4 µm. Paracapillitium scarce, septate, branched, hyaline, up to 5.9 µm in diam. Exoperidium consists of subglobose to elongated, hyaline elements. Endoperidium consists of reddish brown, septate hyphae, up to 4.72 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Khanspur, 18th July 2011, gregarious, among grass at 2250 m (7500 ft) a.s.l., N. Yousaf, LPK19 (Holotype), (LAH100000132); Nathia gali, 10th Aug. 2009, in groups, among grass, at 2423 m (7951 ft) a.s.l., N. Yousaf, BPK68 (LAH100000133); Mansehta, 1st July, 2011, solitary, among grass, on ground, at 1661m (5451 ft) a.s.l., N. Yousaf, BPK45 (LAH100000134). 154

A

B C D

Plate 92. Figs. A–D. Lycoperdon lineum (LPK19). A. Mature basidiomata. B. Exoperidial elements. C. LM photographs of spores and eucapillitial hyphae. D. LM photograph of branched hyphae. Bar: = 1 cm for A, = 10 µm for B, = 11 µm for C & D. 155

A

B C

D Plate 93. Figs. A–D. Lycoperdon lineum. A. Basidiospores. B. Paracapillitial hyphae. C. Eucapillitial hyphae D. Endoperidial hyphae. Bar: = 3 µm for A, = 3.3 µm for B, = 3.5 µm for C, = 8 µm for D. 156

Lycoperdon mammiforme Pers. [as 'mammaeforme'], Syn. meth. fung. (Göttingen) 1: 146 (1801) (Plate 94) Basidiomata pyriforme, consisting of upper spore case and lower stipe like portion below; spore case sub-globose, 35 mm broad × 45 mm high; stipe 25 mm high × 10 mm broad, ochraceous brown in mature specimens, solitary; attached to the substratum by a small mycelial base, encrusted with the particles of soil. Dehiscence by an apical pore. Exoperidium orange brown with small patches peeling off upon maturity, some remnants of patches evident on stipe. Endoperidium orange brown. Gleba brown, compact, cottony. Sterile base present, developed.

Basidiospores globose, echinulated, olivaceous brown, 4.8–6.2 µm in diam. including ornamentation, 3.5–4.0 µm excluding ornamentation, echines up to 0.7 µm, sometimes with short sterigmal remnants attached like small pedicel, brown in water mounts. Eucapillitium Lycoperdon type, long separate threads, dark brown, branched, branching not frequent, pores absent, up to 6.8 µm in diam., thick walled (walls up to 1.5 µm), with attenuate tips, septate, bulging at some points, short to long threads. Exoperidium composed of hyaline, globose to sub-globose sphaerocysts, up to 16 × 13 µm in diam. Endoperidium composed of hyaline to light brown, rarely branched and septate hyphae, up to 4 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Khansur, Halipad, 10th Aug. 2012, solitary, among grass, at 2250 m (7500 ft) a.s.l., N.Yousaf, LPK24A (LAH100000135).

Comments: L. mammiforme Pers is also known as flaky puffball because exoperidium peels off in forms of sheets. It is characterized by ochraceous brown basidiomata, with aculeate basidiospores and capillitium lacking false septa (Kreisel, 1962). It is a new record for Pakistan.

157

A B

C D

E

G F

Plate 94. Figs. A–G. (LPK24). A & B. Mature basidiomata. C. Illustration of basidiospores. D. Illustration of eucapillitial hyphae. E. LM photograph of basidiospores. F & G. LM photograph of eucapillitial hyphae. Bar: = 0.7 cm for A & B, = 4 µm for C, = 6 µm for D, = 2.3 µm for E, = 13 µm for F & G.

158

Lycoperdon mammiforme var. magnasporum nom. prov. (Plate 95)

Etymology: on the basis of larger basidiospores than L. mammiforme

Basidiomata cream to pinkish with off-white to ochraceous patches on basidiomata, turning yellowish with age, fruiting bodies of variable sizes, turbinate to pyriforme, consisting of upper spore case and lower stipe like portion below; spore case sub-globose, 18–38 mm broad × 13–30 mm high; stipe 10–25 mm high × 8–15 mm high, gregarious; attached to the substratum by well-developed mycelial cord with rhizomorphs, encrusted with the particles of soil; rhizomorphs white. Dehiscence by an apical pore; osteole up to 5 mm in diam. Exoperidium off-white to cream with pinkish tint when young, turning pale yellowish with brown small patches upon maturity, peeling off in the form of sheets, first from the spore case then from the stipe, peeling exoperidium forming a ring under the spore case. Endoperidium orange to light brown with age. Gleba brown with light olivaceous tint, compact, cottony. Sterile base present, occupying 1/3rd of the gasterocarp, light brown, soft, chambered, chambers small, well developed.

Basidiospores globose, echinulated, olivaceous brown, 5.8–7.3 µm in diam. including ornamentation, 5.3–5.8 µm excluding ornamentation, echines up to 1.5 µm, sometimes with short sterigmal remnants attached like small pedicel, brown in water mounts. Eucapillitium long separate threads, dark brown, branched, branching not frequent, pores absent, 1.9–9.7 µm in diam., thick walled (walls up to 2 µm), with attenuate tips, false septate, sub-septal ramifications present, bulging at some points, short to long threads. Exoperidium composed of hyaline, globose to subglobose sphaerocysts, up to 21 µm in diam. Endoperidium composed of hyaline to light brown, rarely branched and septate hyphae, up to 5.7 µm. Sterile base composed of olivaceous, thick-walled septate hyphae, up to 5.0 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 5th July. 2011, in groups, among grass, at 1091 m (3582ft) a.s.l., M. Fiaz, LPK55 (LAH100000136) 159

A

C B

D E

F

Plate 95. Figs. A–F. Lycoperdon mammiforme var. magnasporum (LPK55). A. Mature basidioma. B. Basidiospores. C. Eucapillitial hyphae. D & E. LM photograph of eucapillitial hyphae. F. LM photograph of basidiospores. Bar: = 0.5 cm for A, = 4.5 µm for B, = 20 µm for C– E, = 3 µm for F. 160

Lycoperdon molle Pers., Syn. meth. fung. (Göttingen) 1: 150 (1801) (Plate 96) Basidioma obpyriform, pseudo stipitate, puncticulate (minutely dotted) at the base, brown when mature, 20–40 mm in diam. × 65 mm in height; attached to the substrate by thin branching rhizomorphs or mycelial mats, covered or adherent with soil particles and debris, osteole medium, irregular torn aperture, upto 33 mm in diam. Peridium double, exoperidium white when young, dark brown in mature specimen, persistant, in the form of echines, echines long at some places, covering the whole gasterocarp, more dense on the upper portion, and less on the pseudo stipe, darker towards the apical side and lighter towards the base, endoperidium light yellow, shiny, papery, wholly covered by echinulate exoperidium, sterile base well developed, occupying 2/3 of the gasterocarp, seems to form a pseudo-stipe. Gleba cottony, brown.

Basidiospores globose, verrucose, brown, (3.9-) 4.56–6.27 µm in diam., including ornaments and 3.7–5.225 µm in diam., excluding ornaments, verrucae up to 0.7–1.3 µm, sterigmal remnants present in the mounts, branching frequent, dichotomously branched, straight to sub undulate, tips attenuate, aseptate, pores present, olivaceous. Exoperidiµm composed of orbicular or somewhat ovoid or elongated sphaerocysts. Endoperidium composed of tightly interwoven hyphal elements.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Khanspur, 1st Aug. 2007, gregarious, on soil, among grass, on ground, at 2250 m (7500 ft), A. R. Niazi, LPK52 (LAH100000137).

Comments: L. molle was previously reported by Razzaq & Shahzad in 2005 from Kaghan valley. This is a second report of this taxon from Pakistan. 161

A B

C D

E

Plate 96. Figs. A–E. Lycoperdon molle (LPK52). A. A Basidioma. B. Basidiospores. C. Exoperidial elements. D. Endoperidial hyphae. E. Eucapillitial hyphae. Bar: = 1 cm for A, = 3 µm for B, = 15 µm for C, = 9.5 µm for D, = 9 µm for E. 162

Lycoperdon nigrescens Pers., Neues Mag. Bot. 1: 87 (1794) (Plates 97–99) Basidiomata sub globose to slightly pyriforme, dark brown, up to 27–40 × 38–45 mm, spore case brown, globose with sterile base below, solitary to gregarious, usually in the group of two. Sterile base well developed, off-white, up to 15 mm high, 10–15 mm wide; attached to the substratum by well-developed rhizomorphs. Rhizomorphs white, branched, encrusted with the particles of soil and debris. Peridium double. Exoperidium persistent, in the form of warts; warts brown to blackish grey, up to 1 mm long, more dense towards upper side, less dense towards the base; many small rounded depressions over the surface of spore case. Endoperidium off-whitish to brown, papery, heavily encrusted with warts. Gleba light brown to olivaceous, pulverulent. Sterile base present. Dehiscence by an apical pore (not recorded in this case).

Basidiospores globose, verrucose, hyaline to olivaceous, 4.3–5.3 μm in diam., including ornamentation, 3.8–4.5 μm in diam., (ornamentation excluded), verrucae less dense, up to 1 μm long, sterigmal remnants present in mounts. Eucapillitium aseptate, prominent, branched, branching less frequent, elastic, well developed, attenuate at tips, straight to sub undulate, sub-septal ramifications present, thick walled, up to 7.5 (–13.7) μm in diam., with wall thickness 2.5–3.5 μm. Paracapillitium abundant, hyaline, branched, septate. Exoperidium consists of thin walled, hyaline, subglobose elements. Endoperidium consists of aseptate, hyaline hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Khanspur, 18th July. 2011, gregarious, among grass at 2250 m (7500 ft) a.s.l., N. Yousaf, LPK73 (LAH100000138).

Comments: L. nigrescens is commonly known as blackish puffball and characterized by having warts that are more than 1 μm long, leaving a reticulate pattern over the peridium. It can be confiused with L. umbrinum in the field. It is known from many countries (Calonge & Syvokon, 2008). It is an addition to myco flora of Pakistan.

163

A

B

Plate 97. Figs. A–B. Lycoperdon nigrescens (LPK73). A. Basidiomata. B. Thick-walled eucapillitial hypha. Bar: = 0.7 cm for A, = 6.5 µm for B. 164

A

B

Plate 98. Figs. A–B. Lycoperdon nigrescens. A. Basidiospores. B. Exoperidial elements. Bar: = 2.2 cm for A, = 7 µm for B. 165

A

B C Plate 99. Figs. A–C. Lycoperdon nigrescens. A. Eucapillitial hyphae. B. Paracapillitial hyphae. C. Endoperidial hyphae. Bar: = 8 µm for A, = 7 µm for B, = 9 µm for C.

166

Lycoperdon niveum Kreisel, Khumbu Himal 6(1): 30 (1969) (Plates 100–101) Basidiomata turbinate, off-white to yellow when young, turning brown with age, 30 mm in diam. × 35 mm in height; attached to the substratum by thick rhizomorphs, adherent with soil and other debris material. Dehiscence by an apical pore; pore not observed. Peridium double. Exoperidium off-white to yellow, turning light brown with age, in the form of granules. Endoperidium relatively smooth when exoperidium peels off, brown, papery. Gleba brown, cottony. Sterile base present, up to 10 mm high.

Basidiospores globose, finely verrucose, light brown, 4.3–5.6 µm in diam., brown in water mounts. Eucapillitium well-developed, brown, branched, slit like pores present, 1.7–5.2 µm in diam., thick walled (walls up to 1.5 µm), long threads, with attenuate tips, encrusted with some amorphous material. Exoperidium composed of hyaline, globose to sub-globose sphaerocysts up to 44 µm × 17 µm. Endoperidium composed of hyaline to light brown, branched and septate hyphae, up to 4.5 µm. Sub-gleba composed of olivaceous, thick-walled septate hyphae, up to 4.5 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Malam Jabba, 10th Aug. 2012, solitary, at 2484 m (8152 ft) a.s.l., N. Yousaf, LPK61 (LAH100000139).

Comments: L. niveum is characterized by off-white exoperidium which do not change color at maturity. It is close to L. umbrinum Pers., but in this case spines turns dark brown at maturity. It has been reported from many countries (Kreisel, 1969; Demoulin, 1971). It is a new record for Pakistan.

167

B A

C D

Plate 100. Figs. A–D. Lycoperdon niveum. A. Mature basidioma (LPK61). B. LM photograph of basidiospores. C & D. LM photographs of eucapillitial hyphae. Bar: = 0.8 cm for A, = 15 µm for B, = 9 µm for C, = 3.5 µm for D. 168

A B

C

Plate 101. Figs. A–C. Lycoperdon niveum. A. Eucapillitial hyphae and spores. B. Exoperidial elements. C. Endoperidial hyphae. Bar: = 9 µm for A, = 22 µm for B, = 10 µm for C.

169

Lycoperdon pseudoniveum nom. prov. (Plates 102–103)

Etymology: on the basis of resemblance with L. niveum

Basidiomata globose to subglobose, pale yellowish brown, up to 30 cm high × 25 cm broad; attached to the substratum by rhizomorphs; rhizomorphs white; dehiscence by an apical pore; pore 5 mm in diam. Exoperidium brown, not persistent, in the form of minute granules over the endoperidium, sloughing off, first from the upper side of the spore case, still present on lower portion. Endoperidium papery, brown. Gleba olive brown, cottony, becoming pulverulent with age. Sterile base rudimentary.

Basidiospores globose to sub-globose, olive brown, 3.0–6.0 µm in diam., pedicellate, pedicel up to 4 µm long, finely echinulate, olive brown in water mounts. Eucapillitium well-developed, long separate threads, aseptate to rarely septate, olivaceous brown, cytoplasmic contents get constricted at some points, bulging occasionally, with attenuate tips, rarely branched, pores present, 1.7–8.4 µm wide, thick-walled (walls thickness 0.6–0.9 µm), undulated to sub undulate. Paracapillitium absent. Exoperidium composed of sub-globose to slightly elongated sphaerocysts. Endoperidium composed of hyaline, aseptate, and unbranched hyphae.

Material examined: PAKISTAN: Gilgit-Baltistan, Fairy meadows, 22nd July. 2010, in groups, at 13751m (4191 ft) a.s.l., A.N. Khalid, LPK2 (Holotype), (LAH100000140).

170

A

B

Plate 102. Figs. A–B. Lycoperdon pseudoniveum (LPK2). A. Basidioma. B. Basidiospores. Bar: = 0.5 cm for A, = 2 µm for B. 171

A B

C

Plate 103. Figs. A–C. Lycoperdon pseudoniveum. A. Exoperidial elements. B. Eucapillitial hyphae. C. Endoperidial hyphae. Bar: = 7 µm for A, = 5 µm for B, = 9 µm for C.

172

Lycoperdon sclerocystis nom. prov. (Plates 104–105) Etymology: on the basis of thick-walled mycosclereids on the exoperidium

Basidiomata pear shaped, dark brown above, light brown below, 35 × 40 mm high and 35 mm broad; attached to the substratum by a very small mycelial base; dehiscence by an apical pore, up to 5mm in diam. Peridium double. Exoperidium persistent, not peeling off in the form of warts, forming a continuous layer on the entire endoperidium, warts more thick and crowded, pale yellow at basal sterile portion. Endoperidium brittle, papery, entirely covered with exoperidium, becoming smooth more towards the base. Gleba furfuraceous, powdery. Sterile base present, up to 20 mm high.

Basidiospores rounded, ornamented, verrucose, brown, 5.2–7.2 µm in diam. including ornamentation and 4.0–5.0 µm excluding ornamentation, verrucae up to 1.2 µm long, pedicellate, pedicel up to 30.5 µm, numerous pedicels in the mounts. Eucapillitium prominent, branched (branching occasional), aseptate, thick-walled, 1.7–7.0 µm wide, wall thickness 0.8–1.5 µm, attenuate at tips, undulating at some points mostly at ends, often bulging. Paracapillitium absent. Exoperidium composed of olivaceous to hyaline, thick walled, irregular shaped sclerieds. Endoperidium composed of aseptate, unbranched and hyaline hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Ayubia, Khanspur, 22th Aug. 2010, solitary, among grass, at 2250 m (7500 ft) a.s.l., N. Yousaf, LPK35 (Holotype), (LAH100000141); 13th Sep. 2012, among grass, solitary, N. Yousaf, LPK31 (LAH100000142); 8th Aug. 2013, among grass, solitary, N. Yousaf, LPK23 (LAH100000143). 173

A B

C D

Plate 104. Figs. A–D. Lycoperdon sclerocystis. A & B. Mature basidioma (LPK35). C. LM photograph of basidiospores. D. LM photograph of eucapillitium. Bar: = 1 cm for A & B, = 6.5 µm for C, = 25 µm for D. 174

A B

C D

Plate 105. Figs. A–D. Lycoperdon sclerocystis. A. Basidiospores. B. Thick-walled exoperidial elements. C. Eucapillitial hyphae. D. Endoperidial hyphae. Bar: = 3.8 µm for A, = 7 µm for B, = 13 µm for C, = 11 µm for D. 175

Lycoperdon sharanense nom. prov. (Plates 106–107) Etymology: on the basis of type locality “Sharan”

Basidiomata pyriforme, brown when mature, 20–60 mm broad × 30–80 mm high, in groups; attached to the substratum by a well-developed rhizomorphs, rhizomoprhs branched, white, encrusted with soil particles and other debris material. Dehiscence by an apical pore, up to 3 mm in diam. Peridium double. Exoperidium in the form of granules all over the basidioma, persistent. Endoperidium light brown, not entirely exposed in mature specimens, papery. Gleba brown, compact, cottony. Sub gleba present, up to 20 mm high.

Basidiospores globose, echinulate, olivaceous brown, 6.5–7.3 µm in diam., including ornamentation, up to 5 µm in diam. (ornamentation excluded), with a stump of pedicel, up to 2 µm long, central oil droplet present, olivaceous in water mounts. Eucapillitium brown, long threads, pores present, (up to 0.6 µm wide), branched, up to 2– 6.4 µm in diam., wall thickness up to 2.3 µm, aseptate, straight to sub-undulate, bulging at some points, sub-septal ramifications absent, branches with tapering ends, tips undulate. Sub-gleba composed of brown, branched, thick walled, septate, hyphae, up to 4.0 µm in diam. Exoperidium composed of hyaline, globose to sub-globose hyphal elements, 23 × 14 µm. Endoperidium composed of hyaline to light brown, straight, frequently septate, branched, thin-walled, tightly packed hyphae, up to 5.7 µm in diam., bulging at some points.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Sharan, 14th Aug. 2011, on ground, in groups, at 1314m (4313 ft) a.s.l., N. Yousaf, LPK59 (Holotype), (LAH100000144). 176

A

B C Plate 106. Figs. A–C. Lycoperdon sharanense (LPK59). A. Basidiomata. B. Exoperidial elements. C. Endoperidial hyphae. Bar: = 11 cm or A, = 12 µm for B, = 9 µm for C. 177

A

B Plate 107. Figs. A–B. LM photographs of Lycoperdon sharanense (LPK59). A. Basidiospores B. Porous eucapillitial hyphae. Bar: = 4.5 µm for A, = 10 µm for B.

178

Lycoperdon sp. (LPK74) (Plate 108)

Basidioma sub globose, slightly tapering towards the base, light brown, 20–25 mm in diam. 25–35 mm in height, gregarious, often in a group of 3–4, attached to the substrate by hard, tough, small mycelial cord, adherent with the soil particles and debris. Osteole medium, up to 4 mm in diam. Peridium double. Exoperidium peeled off, in the form of warts; warts minute, dark brown, distant from one another. Endoperidium papery, dull light brown, smooth. Gleba brown, cottony. Sub gleba present.

Basidiospores rounded, smooth, olivaceous brown, 2.9–3.6 μm in diam., apedicellate, sterigmal remnants absent from mounts. Eucapillitium brown, olivaceous, prominent, well developed, straight to sub undulate, aseptate, 2.5–5.7 μm in diam., wall thickness 0.76–1.4 μm, attenuate at tips; tips not narrower than 1.4 μm, frequently branched, bulging at some points. Paracapillitium absent. Exoperidium composed of globular, hyaline, thin walled sphaerocysts. Endoperidium composed of thin walled, hyaline, septate and branched hyphae.

Material examined: PAKISTAN, Khyber Pakhtunkhwa, Abbottabad district, Ayubia, Khanspur, 22nd Aug. 2010, at 2250 m (7381 ft) a.s.l., N. Yousaf, LPK74 (LAH100000145). 179

A

B C D Plate 108. Figs. A–D. Lycoperdon sp. (LPK74). A. Mature basidiomata. B. Endoperidial hyphae. C. Basidiospores. D. Eucapillitial threads. Bar: = 0.6 µm for A, = 7 µm for B, = 2.5 µm for C, = 12 µm for D.

180

Lycoperdon curtisii var. ovalisporum nom. prov. (Plates 109–111)

Etymology: on the basis of ovoid shape of spores than L. curtisii Basidiomata sub-globose, pale yellow, up to 10–15 mm in diam. × 15mm in height; attached to the substratum by small mycelial pad, heavily encrusted with the particles of soil; osteole may develop with sloughing of exoperidium, not recorded in the specimen. Exoperidium in the form of small conical warts, off-white at first, becoming brown with age, more above, lesser below, often peeling off in form of patches or plates, persistent from above. Endoperidium pale yellowish brown, breaking away sometimes to expose the inner part. Gleba olivaceous, cottony first, soon becoming pulverulent. Sub gleba present yellowish white, up to 7 mm in height from the base. Basidiospores sub-globose to ovoid, asperulate, short pedicellate, olivaceous, 5.2– 6.6 × 5.0–5.7 µm in diam., pedicel up to 3.3 µm in height. Eucapillitium present, scarce, thin walled, brown, branched, aseptate to rarely septate, sub-undulate, pores absent, up to 4.7 µm in diam., with wall thickness up to 2.35 µm. Paracapillitial hyphae abundant, hyaline, with granular cytoplasmic contents, thin walled, branched, septate, 6.6 µm in diam. Exoperidium composed of sub-globose to elongated, hyaline to light brown hyphal sphaerocysts, up to 30 µm × 22 µm in diam. Endoperidium composed of hyaline to light brown, thick-walled, aseptae, up to 15 µm in diam., with wall thickness up to 5.6 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Abbottabad district, Dadar, 15th Sep. 2012, in groups, among grass, at 1972 m (6473 ft) a.s.l., N. Yousaf, VPK1 (LAH100000146).

181

A B

C

Plate 109. Figs. A–C. var. ovalisporum (VPK1). A. Mature basidiomata. B. Asperulate sub-globose basidiospores. C. Paracapillitial hyphae. Bar: = 0.5 cm for A, = 3.7 μm for B, = 8 μm for C. 182

A B

C Plate 110. Figs. A–C. Lycoperdon curtisii var. ovalisporum. A. Eucapillitial hyphae. B. Exoperidial sphaerocysts. C. Endoperidial hyphae. Bar: = 8.8 μm for A, = 13 μm for B, = 2.2 μm for C. 183

A

B

Plate 111. Figs. A–B. LM photographs of basidiospores of Lycoperdon curtisii var. ovalisporum. Bar: = 14 µm for A, = 2.5 µm for B.

184

Lycoperdon gilgitii nom. prov. (Plate 112) Etymology: on the name of type locality “Gilgit”

Basidiomata pyriforme to globose, off-white, 18 mm broad × 20 mm high, epigeous, small; attached by a narrow small, off white mycelial base. Diaphragm well delineated. Peridium double. Exoperidium peeling off, in the form of warts, more on basal portion, deciduous at maturity, forms a reticulate network on endoperidium. Endoperidium thin, white, bronze at the base, lighter in color above. Ostiole present, up to 2 mm in diam. Gleba light greyish brown, compact. Sub-gleba present, up to 10 mm high, brownish grey, chambered, chambers small to medium.

Basidiospores globose, minutely ornamented, echinulated, olivaceous, 3.5–4.0 µm in diam., basidia not observed. Eucapillitium Lycoperdon type, branched, dichotomous branching, aseptate, thick walled, up to 5 µm. Paracapillitium present, abundant, septate. Exoperidium consists of globose to sub globose sphaerocysts. Exoperidium consists of tightly packed branched, aseptate hyphae, up to 4 µm.

Material examined: PAKISTAN: Gilgit-Blatistan, Fairy meadows, 1st Aug. 2007, gregarious, on soil, among grass, on ground, at 4031 m (13225 ft) a.s.l., N. Yousaf, VPK9 (Holotype), (LAH100000147).

185

A B

C D

F E

Plate 112. Figs. A–F. Lycoperdon gilgitii (VPK9). A. Basidioma. B. Exoperidial elements. C. Basidiospores. D. Eucapillitial hyphae. E. Endoperidial hyphae. F. Paracapillitial hyphae. Bar: = 0.8 cm for A, = 10 µm for B, = 2.5 µm for C, = 3.5 µm for D, = 8.8 µm for E, = 4 µm for F.

186

Lycoperdon lahorense nom. prov. (Plates 113–115)

Etymology named for the type locality “Lahore”

Basidiomata globose to somewhat pyriforme, off white to pale yellow above when young, pale brown to grayish brown with age, off-white to light yellow below, small to medium, 8–20 mm in diam. × 9–25 mm in height; attached to the substratum by white, branched rhizomorphs, these encrusted with the particles of soil; ostiole present, up to 4 mm in diam. Peridium double. Exoperidium pale yellowish to grayish brown with age, not persistent, fragile, peeling off in the form of patches, patches encrusted with pale brown to dark brown warts, sub-flocculate, sometimes entire exoperidium is in the form of minute, prominent warts, these more above, and lesser below, encrusted with debris material. Endoperidium papery, grayish brown to grey towards apical portion when mature, off- white to pale yellow downwards, entirely exposed in mature specimens. Gleba olivaceous to brown, cottony. Sub gleba pale yellow, up to 10 mm high, comprising only 1/3rd of gasterocarp.

Basidiospores globose, minutely verrucose, olivaceous, 3.5–4.3 μm in diam., with a stump of pedicel. Paracapillitium hyaline to olivaceous, septate, bulging at some points, cytoplasmic contents abundant in hyphae, encrusted with some amorphous material, 2.3– 7.6 μm in diam., branched (dichotomous branching absent), septa present, thick walled, (walls up to 1 μm), with attenuate tips (up to 1 μm). Eucapillitium absent. Exoperidium composed of hyaline to olivaceous, globose to subglobose sphaerocysts. Endoperidium composed of tightly interwoven, olivaceous to hyaline, aseptate, branched, thick walled hyphae.

Material examined: PAKISTAN, Punjab, Lahore, University of the Punjab, New Campus, Botanical Garden, 5th July. 2011, in groups, among grass, on ground, at 217 m (712 ft) a.s.l., N. Yousaf, VPK4 (Holotype), (LAH100000148); 2nd Sep. 2012, in groups, among grass, N. Yousaf, VPK6 (LAH100000149). 187

A B

C

Plate 113. Figs. A–C. Fruiting bodies of Lycoperdon lahorense A & B. VPK4. A. young fruiting bodies with exoperidium intact. B. Mature fruiting bodies after exoperidium peeled off. C. VPK6, Young fruiting bodies (2012 collection). Bar: = 0.55 cm for A & B, = 0.8 cm for C. 188

A B

C D

E

Plate 114. Figs. A–E. Lycoperdon lahorense. A. Asperulate basidiospores. B. Globose to subglobose exoperidial elements. C. Parcapillitial threads. D. Aseptate hyphae of sub gleba. E. Endoperidial hyphae. Bar: = 2.5 µm for A, = 10 µm for B–E.

189

A B

C

D Plate 115. Figs. A–D. LM photographs of Lycoperdon lahorense. A–C. Paracapillitial hyphae. D. Globose asperulate basidiospores. Bar: = 14 µm for A, = 10 µm for B, = 7 µm for C, = 14 µm for D.

190

Lycoperdon lahorense var. parvasporum nom. prov. (Plates 116–117)

Etymology: on the basis of small spores than L. lahorense

Basidiomata sub-globose, off-white when young turning grey with age, up to 10– 25 mm in diam. × 25 mm in height, sometimes forming a pseudostipe below; attached to the substratum by small mycelial pad, heavily encrusted with the particles of soil; dehiscence by an apical pore or slit, osteole develop with sloughing of exoperidium, up to 5 mm in diam. Exoperidium off-white, granular. Endoperidium grayish brown, papery. Gleba olivaceous, cottony. Sub gleba present. Basidiospores sub globose, asperulate, olivaceous, 2.8–3.8 µm in diam. Eucapillitium absent. Paracapillitial hyphae abundant, hyaline, thin walled, branched, septate, 3.0–7.5 µm in diam. Exoperidium composed of sub-globose to elongated, hyaline hyphal sphaerocysts. Endoperidium composed of hyaline to light brown, thick-walled, aseptae, up to 5 µm in diam. Material examined: PAKISTAN, Punjab, Lahore, University of the Punjab, New Campus, 25th July, 2013, on ground, among grass, in groups, among grass, at 217m (712 ft). a.s.l., N. Yousaf, VPK3 (LAH100000150).

191

A

B

C

Plate 116. Figs. A–C. Lycoperdon lahorense var. parvasporum (VPK3). A. Basidiomata. B. LM photograph of basidiospores. C. LM photograph of paracapillitial hyphae. Bar: = 0.7 cm for A, = 4.7 µm for B, = 8.5 µm for C.

192

Lycoperdon olivoflavum nom. prov. (Plates 117–118)

Etymology named for the characteristic olive-yellow coloration of the basidiomata

Basidiomata sub globose to depressed globose, occasionally plicate at the base, bright olive-yellow to pale olive yellow, up to 20 mm in diam. × 17 mm in height; attached to the substratum by small mycelial pad, heavily encrusted with soil particles; ostiole developed by sloughing of the exoperidium, later torn away irregularly with age. Exoperidium whitish at first, becoming yellowish white to pale yellow, turning olive yellow at maturity, ornamented, flocculose, fragile, persistant. Endoperidium pale yellowish white, breaking away sometimes to expose the inner part. Gleba olivaceous, cottony first, soon becoming pulverulent. Sub gleba yellowish white, comprising the basal 2/3 rd of gasterocarp, composed of cellular chambers.

Basidiospores globose, asperulate to verrucose, pedicellate, dark olivaceous, (2.85– ) 3.3–4.3 (–5.32) μm in diam., pedicel up to 1.33 μm long, olivaceous in water mounts. Eucapillitium scarce, mostly present towards the inner side of endoperidium, lining the periphery, Lycoperdon type, brown, 2.0–4.4 μm in diam., tapering towards tips, thick walled, walls up to 1 μm, aseptate to rarely septate, branched, straight to sub-undulate. Paracapillitium hyaline, abundant, septate, encrusted with some amorphous material. Exoperidium composed of globose to subglobose to irregular shaped, hyaline, thin walled exoperidial elements. Endoperidium composed of hyaline, branched, septate and bulging hyphae.

Material examined: PAKISTAN, Khyber Pakhtunkhwa, Khanspur, Helipad, 8th Aug. 2011, in groups, under Pinus wallichiana, at 2250 m (7500 ft) a.s.l., N. Yousaf, VPK2 (Holotype), (LAH100000151). 193

A

B

D C Plate 117. Figs. A–D. Lycoperdon olivoflavum. A. Mature basidiomata (VPK2). B. LM photograph of basidiospores. C. LM photograph of eucapillitial hypha. D. LM photograph of paracapillitial hyphae. Bar: = 0.4 cm for A, = 6 µm for B, = 10 µm for C, = 15 µm for D.

194

A B

C D

E

Plate 118. Figs. A–E. Lycoperdon olivoflavum (VPK2). A. Asperulate basidiospores. B. Exoperidial elements. C. Parcapillitial threads. D. Eucapillitial hyphae. E. Endoperidial hyphae. Bar: = 1.7 µm for A, = 10 µm for B & C, = 5 µm for D, = 10 μm for E. 195

Lycoperdon pratense var. flavum nom. prov. (Plates 119–120) Etymology: due to yellow color of basidioma

Basidiomata sub-globose, pale yellow, 10–25 mm in diam. × 15 mm in height; attached to the substratum by a small mycelial pad, heavily encrusted with the particles of soil; osteole present. Exoperidium in the form of small conical warts, off-white at first, becoming brown with age. Endoperidium pale yellowish brown, papery. Diaphragm poorly developed. Gleba olivaceous, cottony. Sub gleba present olivaceous.

Basidiospores globose, smooth to asperulate, olivaceous brown, 4.0–5.0 µm. Eucapillitium absent. Paracapillitium present, hyaline, branched, septate, encrusted with some amorphous material, up to 5.0 µm. Exoperidium composed of hyaline, globose to sub-globose sphaerocysts, up to 25 ×16 µm Endoperidium composed of hyaline, septate, thick walled, non elastic, tightly packed hyphae, up to 5 µm, wall thickness up to 1.88 µm.

Material examined: PAKISTAN, Khyber Pakhtunkhwa, Swat district, Kalam, 7th Sep. 2013, at 2123m (6965 ft) a.s.l., under decaying needles of Cedrus sp., VPK8 (LAH100000152). 196

A

B

Plate 119. Figs. A–B. Lycoperdon pratense var. flavum (VPK8). A. Basidioma. B. Paracapillitial hyphae. Bar: = 0.5 cm for A, = 5 µm for B. 197

A B

C

Plate 120. Figs. A–C. Lycoperdon pratense var. flavum. A. Basidiospores. B. Exoperidial elements. C. Endoperidial hyphae. Bar: = 3 µm for A, = 6 µm for B, = 8 µm for C.

198

Montagnea arenaria (DC.) Zeller [as 'arenarius'], Mycologia 35(4): 418 (1943) (Plates 121–122) Basidiomata hypogeous, stipitate at maturity, up to 14 cm tall. 18 mm high × 28 mm broad, in the form of expanded disc, with lamellae extending from it; Disc grayish black, 13 mm in diam., dissected into 4-5 irregular rays, each ray consisting of numerous radial lamellae, lamellae black with grayish tint; Stipe 13 cm high, whitish, hollow, hard, scaly, scales with brownish tint, ridges and furrows present, more prominent towards the pileus, tapers upwards. Vovla subglobose, up to 1 cm high, 1.2 cm broad, encrusted with soil particles.

Basidiospores blackish brown, with prominent germ pore, ovoid, up to 11.6–16.5 × (3.6-) 7.4–8.55 μm.

Material examined: PAKISTAN: Punjab, Lahore, University of the Punjab, New Campus, 25th June, 2011, solitary, among grass, on ground, at 217 m (712 ft) a.s.l., N. Yousaf, MPK1 (LAH100000153); Chakwal district, Dhuman, 6th July, 2012, on ground, at 498 m (1634 ft) a.s.l., R. Fatima, MPK3 (LAH100000154)

Additional material examined: Montagnites montagnei Fr. In sandy soil, Ladhar, Sheikhupura, 18th Aug. 1952, S. Ahmad (LAH19374).

Commnets: Montagnea arenarea is a type species of the genus It is worldwide in distribution and found growing solitary or in groups with an adaptation to arid and sandy places (Zeller, 1943; Rauschert, 1964; Kreisel, 1971; Reid & Eicker, 1991; Chen, 1999; Minter et al., 2001; Baseia & Milanez, 2002a; Madrid & Muñoz, 2006; Nieves-Rivera & Aime, 2006; Dörfelt & Gube, 2007). It is previously reported from Ladhar, Lahore, Muzaffargarh, Palla, Panjnad, Sheikhupura, Pakistan (Ahmad, 1952). It is a first report of this fungus from district Chakwal but reported second time from district Lahore.

199

A B

C

Plate 121. Figs. A–C. Montagnea arenaria (MPK1). A. A mature basidioma. B. Line drawing of basidioma. C. Basidiospores. Bar: = 1.2 cm for A & B, = 2.5 μm for C. 200

A

B

Plate 122. Figs. A–B. LM photographs of Montagnea arenaria. A. Basidiospores. B. Crushed basidia in the . Bar: = 4.0 µm for A & B.

201

Podaxis pistillaris (L.) Fr. [as 'Podaxon'], Syst. mycol. (Lundae) 3(1): 63 (1829)

(Plates 123–125)

Basidiomata stipitate, 85–135 mm tall, scattered to gregarious. Pileus 30–50 mm high, 14–18 mm broad, off-white, ovoid, scaly, margins leaving the stipe with age, curved upwards in mature specimens; outer layer not persistant, fragile, soft, composed of furfuraceous scales, peeled off with age; Inner layer smooth, light to dark brown, papery to membranous. Gleba pulverulent, dark brown to black with age. Stipe 8-10 mm high, 5mm in diam., whitish, rough, scaly, hollow, attached to the substrate by a bulbous base, heavily encrusted with soil particles.

Basidiospores subglobose to ovoid, thick walled, double walled, dark brown, 8.2– 12.7 µm × 7.3–11.6 µm, apical thickness (up to 3.5 µm), germ pore well marked, sterigmal remnants absent from mounts. Capillitium hyaline to pale brown hyphae, branched, septate, fragile, thin walled. Exoperidium composed of elongated hyphal elements, 8.5–21 µm in diam. Endoperidium hyaline, branched, septate, tightly packed hyphae, up to 6 µm in diam.

Material examined: PAKISTAN: Punjab, Lahore, University of the Punjab, New Campus, 5th July, 2011, 13th & 16th July, 2013; 23rd August, 2013, solitary or in groups, among grass, on ground, along road sides, at 217 m (712 ft) a.s.l., N. Yousaf, PPK6 (LAH100000155), PPK18 (LAH100000156), PPK19 (LAH100000157), PPK20 (LAH100000158); Lahore district, Bakharmandi, 20th June, 2013, in groups, at 217 m (712 ft) a.s.l., M. Anees, PPK21 (LAH100000159); Bahawalpur, Lal Sohanra Park, 18th August, 2013, on ground, 461 m (1512 ft) a.s.l., N. Yousaf, PPK22 (LAH100000160); Chakwal, Dhuman, 15th July, 2012, at 498 m (1,634 ft) a.s.l., R. Fatima, PPK24 (LAH100000161); Neela Dulha, 13th Aug. 2013, along the road side, at 498m (1,634 ft) a.s.l., N. Yousaf, PPK25 (LAH100000162); Khyber Pakhtunkhwa, Nathia gali, 20th July, 2011, along the track, among grass, N. Yousaf, PPK23 (LAH100000163).

Comments: Podaxis pitillaris is the type species of the genus and is commonly called as desert shaggy mane because its young fruiting bodies have close similarity with shaggy ink cap ( comatus). It exhibit a secotoid habit and is found growing solitary or in groups in arid and semi-arid regions (Massee, 1890; Morse, 1933; Dring, 1973; Keirle et al., 2004; Muhsin, 2012). 202

Previously it was reported by Ghaffar & Kafi in 1968 from Karachi. It is first report of P. pistillaris from Punjab and Khyber Pakhtunkhwa province. 203

B A

Plate 123. Figs. A–B. Podaxis pistillaris (PPK23). A. Basidiomata. B. Line drawing of basidioma. Bar: = 1 cm for A & B. 204

B A

C

C

D E

Plate 124. Figs. A–E. Podaxis pistillaris. A. Oval thick walled basidiopsores. B. Club shaped basidia. C. Eucapillitial threads. D. Endoperidial hyphae. E. Exoperidial hyphae. Bar: = 4.5 μm for A, = 20 μm for B, = 5 μm for C & E, = 10 μm for D. 205

A

B C Plate 125. Figs. A–C. LM photographs of Podaxis pistillaris. A. Basidiospores. Collection no. PPK6. B. Basidiospores, collection no. PPK7. C. Hyaline glebal hyphae. Bar: = 6 µm for A, = 15 µm for B & C.

206

Tulostoma fimbriatum Fr., Syst. mycol. (Lundae) 3(1): 43 (1829) (Plate 126)

Sporophore having stipe and sporocarp, up to 20–45 mm high. Sporocarp ovate to subglobose, up to 10–17 mm high × 10–15 mm wide. Exoperidium granular, persistent to deciduous, Endoperidium tough, often covered with exoperidium. Peristome fibrillose, Stipe scaly, rough with adherent patches of sand, visible portion 10-30 mm high, attached to underside of sporocarp. Volva absent, base slightly bulbous. Gleba wood brown, pulverulent.

Basidia not found. Basidiospores globose to sub-globose, verrucose, brown, 6.0– 7.5 × 5.7–7.0 µm in diam., verrucae up to 2.0 µm long. Eucapillitium olivaceous to hyaline, elastic, thick walled, branched, long threads, not broken in to segments, septate, joints like septa present, up to 11.3 µm in diam., wall thickness up to 6 µm. Exoperidium composed of globose to subglobose, hyaline, hyphal elements, up to 16 × 10 µm in diam. Endoperidium composed of hyaline, branched, septate, tightly packed hyphae, up to 8.7 µm in diam. Stipe composed of brown, thin walled, septate, unbranched, straight, tightly packed hyphae.

Material examined: PAKISTAN: Punjab, Lahore, University of the Punjab, Quaid-e- Azam campus, 10th Aug. 2006, in groups, at (217 m) 712 ft a.s.l., A. N. Khalid, TPK8 LAH100000164).

Comments: T. fimbriatum is known from many countries and also included in the red list of Bulgaria (Gyosheva et al., 2006). It is a second report for Pakistan.

207

A B

C D

E F

Plate 126. Figs. A–F. Tulostoma fimbriatum (TPK8). A. Basidiomata. B. Basidiospores. C. Eucapillitial hyphae. D. Endoperidial hyphae. E. Exoperidial elements. F. Stipe hyphae. Bar: = 1.3 µm for A, = 5 µm for B, = 11 µm for C, = 15 µm for D & E, = 11 µm for F.

208

Tulostoma morenoii nom. prov. (Plate 127)

Etymology: on the name of European mycologist “G. Moreno”

Basidiomata up to 35 mm long, consists of spore case and a stipe. Spore case off- white to light brown, up to 10 mm in diam, mouth circular. Stipe up to 25 mm high, hollow, encrusted with soil particles. Mouth circular, small. Peridium doble. Exoperidium peeled off in mature specimens. Endoperidium off-white, papery. Gleba light brown to cinnamon brown, powdery.

Basidiospores subglobose, verrucose, olivaceous brown, 7.3–10.5 µm including ornamentation, 6.0–7.0 µm excluding ornamentation, verrucae up to 1.6 µm. Eucapillitium olivaceous, branched, thick-walled, joint like septa present, up to 11.6 µm in diam., wall thickness up to 3.5 µm. Endoperidium composed of hyaline to light green, thick-walled, aseptate, non-elastic, up to 5.6 µm, wall thickness up to 2.0 µm. Stipe composed of parallel, septate hyphae.

Material examined: PAKISTAN, Punjab, Bahawalpur, 18th Aug. 2013, gregarious, on sandy soil, at 126m (413 ft) a.s.l., A. N. Khalid, TPK1 (Holotype), (LAH100000165). 209

A

B C

D

Plate 127. Figs. A–D. Tulostoma morenoii (TPK1). A. Basidiomata. B. Eucapillitial hyphae. C. Endoepridial hyphae. D. Basidiospores. Bar: = 0.5 cm for A, = 24 µm for B, = 9 µm for C, = 4 µm for D.

210

Tulostoma squamosum Pers., Syn. meth. fung. (Göttingen) 1: 139 (1801)

(Plates 128–129)

Sporophore having sporocarp, stipe and radicating base, up to 50 mm high. Sporocarp subglobase to globose, 10 mm high, 13 mm broad, attached to substratum by small mycelial base. Dehiscence is by an apical mouth; Mouth raised, definite, 1 mm in diam., tubular. Exoperidium rough, persistent, hyphal, dull brown, warty, slowly deciduous. Endoperidium light brown, slightly tough, long covered by exoperidium. Stipe central, scaly, brown, long, hollow, 0.3 cm wide. Base fibrillary. Gleba brown.

Basidiospores globose to sub globose, verrucose, brown, 7.3–8.7 × 6.8–7.8 µm in diam., verrucae up to 1.4 µm long. Eucapilitium hyaline to olivaceous, branched, septate, joint like septa present, tips rounded, up to 12.7 µm in diam., wall thickness up to 3.8 µm. Endoperidial hyphae composed of hyaline, septate, branched, thick walled hyphae, up to 5.2 µm in diam. Stipe composed of light brown, thin walled, frequently septate, tightly packed hyphae, up to 10 µm in diam.

Material examined: PAKISTAN: Punjab, Khushab district, Salt range, Sakesar hills, solitary, on ground under Pinus rouxburghii, at a.s.l., 10 Aug. 2006, A.N. Khalid, TPK2 (LAH100000167).

211

A

A B

Plate 128. Figs. A–B. Tulostoma squamosum (TPK2). A. Mature basidiomata. B. Line drawings of basidioma. Bar: = 0.6 cm for A & B. 212

B A

C D

Plate 129. Figs. A–D. Tulostoma squamosum. A. Basidiospores. B. Eucapillitial hyphae. C. Endoperidial hyphae. D. Stipe hyphae. Bar: = 3 µm for A, = 9 µm for B–D.

213

Tulostoma sp. 1 (TPK6) (Plates 130–131) Spore-case globose to sub globose, up to 10 mm diam., off-white. Exoperidium peeled off in mature specimens, in the form of light brown granules over the surface, encrusted with sand particles or plant debris. Endoperidium off white, papery, tough. Mouth short, tubular, circular. Gleba ochraceous brown. Stipe up to 35 × 5 mm, hollow, sub-undulate to straight, off-white to light brown, rough, exoperidium peeling off in the form of scales exposing the off white endoperidium.

Basidiospores sub globose, smooth, brown to greenish brown, 6.1–7.5 × 5.4–7.0 µm. Eucapillitium hyaline to greenish brown, thick walled, branched, septate, clamp connections present, up to 9.4 µm in diam., wall thickness up to 4.7 µm. Peridium composed of hyaline to light green, undulate, septate, highly branched hyphae, with clamp connections present, up to 16.5 µm in diam. Stipe composed of hyaline, straight, septate, rarely branched, tightly packed hyphae, up to 6.8 µm.

Material examined: PAKISTAN, Punjab, Sialkot district, Daska, 15th Sep. 2012, on bare ground, sandy soil, at 236 m (775 ft) a.s.l., A. Zulfiqar, TPK6 (LAH100000168).

214

A

B

Plate 130. Figs. A–B. Tulostoma sp. 1 (TPK1). Basidiomata. Bar: = 0.5 cm for A & B.

215

B A

C D

E

Plate 131. Figs. A–E. Tulostoma sp. 1. A. Line drawing of basidioma. B. Stipe hyphae. C. Basidiospores. D. Peridial hyphae. E. Eucapillitial hyphae. Bar: = 0.8 cm for A, = 14 µm for B, = 4 µm for C, = 25 µm for D, = 7 µm for E.

216

Tulostoma sp. 2 (TPK4) (Plate 132) Sporophore having stipe and spore case, up to 40 mm high. Spore case globose to sub globose, up to 10 mm in diam., attached to the substratum by well-developed mycelial base, heavily encusted with soil particles and debris material. Dehiscence by an apical mouth; circular, definite, raised. Exoperidium deciduous, Endoperidium tough, papery. Stipe scaly, rough with adherent patches of sand, visible portion 10-30 cm high, attached to underside of sporocarp. Volva absent, base slightly bulbous. Gleba wood brown, pulverulent.

Basidia not found. Basidiospores sub globose, verrucose, brown, 5.6–8.0 µm in diam. Eucapillitium hyaline to light greenish, elastic, thick walled, branched, septate, joints like septa present, up to 7.0 µm in diam., wall thickness up to 3.0 µm. Exoperidium not observed. Endoperidium composed of hyaline to light greenish, branched, septate, tightly packed hyphae, up to 5.5 µm in diam., wall thickness up to 2.5 µm. Stipe composed of brown, thin walled, septate, unbranched, straight, tightly packed hyphae.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Balakot tehsil, Batta Kundi, 15th Aug. 2009, in groups, at 2707 m (8882 ft) a.s.l., N. Yousaf, TPK4 (LAH100000169). 217

A

B C

D Plate 132. Figs. A–D. Tulostoma sp. 2 (TPK4). A. Basidiomata. B. Basidiospores. C. Eucapillitial hyphae. D. Endoperidial hyphae. Bar: = 0.6 cm for A, = 6 µm for B, = 11 µm for C, = 6 µm for D.

218

4.1.2. Taxa belonging to Nidulariaceae

Cyathus olla (Batsch) Pers., Syn. meth. fung. (Göttingen) 1: 237 (1801) (Plates 133–135)

Basidioma vase shaped, gregarious, 5–10 mm wide by 10–13 mm tall, grayish brown, exterior rough, encrusted with soil particles on the outer side, attached to the substratum by a small mycelial base, on logs. Ectoperidium grey, encrusted with soil, rough. Endoperidium smooth, striated, dark grey at the base, light grey near the apex. Peridioles black, 3 mm wide, sub globose.

Basidiopores ellipsoid, thick-walled, hyaline to light green, 9.0–13 × 6.6–10.6 µm. Peridiole hyphae hyaline to light brown, brown when aggregated, aseptate, straight, tightly packed, up to 4.5 µm in diam. Ectoperidium composed of hyaline, septate, thin walled hyphae, mixed with some debris material, up to 10 µm in diam. Mesoperidium composed of hyaline, irregular, branched, septate, clamped hyphae, up to 11.8 µm in diam. Endoperidium composed of hyaline to light green, septate, clamps present, branched hyphae, up to 13 µm in diam.

Material examined: Pakistan, Khyber Pakhtunkhwa, Khyber Pakhtunkhwa, Nadi Bunglow, 15th August, 2011, on wood, in groups, at 1314m (4313 ft) a.s.l., N. Yousaf, CPK7 (LAH100000170).

Comments: C. olla (CPK36) is characterized by silver to grey, smooth inner surface and wide opening usually found growing on woody debris. C. striatus (Huds.) Willd., is a close taxon but it has ribs on inner surface of fruiting body (Ellis & Ellis, 1990). Its distribution is known from Asia, Australia, Europe, North America and South Africa (Liu & Li 1989; Yang et al., 2001; Zhou et al., 2004; Dorjey et al., 2013).

219

A

B

Plate 133. Figs. A & B. Cyathus olla (CPK7). Basidiomata. Bar: = 0.6 cm for A, = 0.2 cm for B.

A

B

220

A

B

C D

Plate 134. Figs. A–D. Cyathus olla. A. Basidiospores. B. Ectoperidial hyphae. C. Mesoperidial hyphae. D. Endoperidial hypahe. Bar: = 4.7 µm for A, = 11 µm for B, = 7.5 µm for C, = 4.5 µm for D.

221

A

B

Plate 135. Figs. A–B. LM photographs of Cyathus olla (CPK7). A. Basidiospores. B. Close view of basidiospores. Bar: = 8 µm for A, = 2.7 µm for B.

222

Cyathus pakistanicus nom. prov. (Plates 136–137)

Etymology: on the name of country “Pakistan” it is being reported.

Fruiting body funnel shaped, gregarious, 5–10 mm in diam., 10–15 mm in height. Ectoperidium consists of hyphal hairs in young specimens, off-white to brown. Endoperidium smooth, dark grey, light grey. Peridioles black, 2 mm in diam., sub globose, numerous.

Basidiospores ellipsoid to oblong, hyaline, 10 × 15 µm. Basidia clavate, up to 14 µm wide. Ectoperidium composed of thin-walled, hyaline to olivaceous, branched, frequently septate, not uniform, clamp connections present, with pointed tips, up to 8.5 µm in diam. Endoperidium composed of light brown to hyaline, branched, aseptate, undulate hyphae, up to 3.5 µm. Peridiole covering composed of thick-walled, brown, branched, sub- undulate, non elastic hyphae. Peridiole hyphae hyaline, thin-walled, branched.

Material examined: PAKISTAN: Punjab, Chakwal, 4th Aug. 2013, at 522 m (1714 ft) a.s.l., gregarious, on cow dung, R. Fatima, CPK10 (Holotype), (LAH100000171). 223

A B

C

D

Plate 136. Figs. A–D. Cyathus pakistanicus (CPK10). A & B. Basidiomata. C. LM photograph of ectoperidial hyphae D. LM photograph of hyphae of peridiole wall. Bar: = 1 cm for A & B, = 15 µm for C & D. 224

A B

C D

Plate 137. Figs. A–D. Cyathus pakistanicus (CPK10). A. Basidiospores. B. with two spores. C. Ectoperidial hyphae. D. Endoperidial hyphae. Bar: = 7.5 µm for A, = 6.5 µm for B, = 11 µm for C, = 9.5 µm for D.

225

4.1.3. Taxa belonging to Phelloriniaceae

Phellorinia herculeana (Pers.) Kreisel, Česká Mykol., 15: 196 (1961) (Plates 138–139)

≡ Phellorinia inquinans Berk, London J. Bot., 2: 521 (1843).

Basidiomata consisting of spore case, stipe, and bulbous base, ≤ 85 mm tall; spore case ≤ 50 mm diam. × 35 mm high, basal portion encrusted with soil particles; dehiscing as apical portions wear away to expose the powdery spore mass. Stipe woody, continuous with spore case, not uniform, having a wide, bulbous base, and tapering toward the apex, with parallel, broad ridges and furrows running from apex to base, and permanent scales scatter intermittently; rhizomorphs not observed. Exoperidium off-white when immature, pale yellowish to brown with age, thick, entirely covered with hard scales of variable sizes. Endoperidium continuous with stipe, pale yellow. Gleba pulverulent, brown.

Basidiospores globose to sub-globose, apedicellate, hyaline, 4.5–8.0 μm diam., verrucose (verrucae <1 μm long). Capillitium hyaline, rarely present in mounts. Exoperidium composed of branched, hyaline hyphae, with inflated, globose elements at their tips. Endoperidium composed of thin, hyaline, branching, septate hyphae.

Material examined: PAKISTAN, Punjab, Bahawalpur, Rahim Yaar Khan, Cholistan Desert, 29th Sep. 2006, solitary, on ground, at 115 m (380 ft) a.s.l., N. Yousaf, NYG210, (LAH 290900).

226

A B

C

D

Plate 138. Figs. A–D. Phellorinia herculeana (NYG210). A. Mature basidiomata. B. Illustration of a basidioma. C & D. LM photographs of basidiospores. Bar: = 1cm for A & B, = 6.5 µm for C, = 3.5 µm for D. 227

A

B

C

Plate 139. Figs. A–C. Phellorinia herculeana. A. Veruucose basidiospores. B. Exoperidial hyphae with globular elements. C. Endoperidial hyphae. Bar: = 2 µm for A, = 9 µm for B, = 11 µm for C.

228

4.1.4. Taxa belonging to Diplocystaceae

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

(Plates 140–142)

Immature basidiomata globose to slightly sub-globose, off-white to cream to pink, 16–38 mm broad × 15–22 mm high, semi hypogeous, in groups, numerous thread like structures at the basal portion of basidiomes, cracked at upper portion, heavily encrusted with debris material; dehiscence pattern not recorded in the specimen. No basal portion or attachment point found. Peridium very hard, < 1 mm thick, three layered. Exoperidium clay colored, membranous, encrusted with soil, peeling off in the form of patches. Mesoperidium thicker than exoperidium, pale brown, entirely covered with exoperidium, not exposed. Endoperidium lining inside the mesoperidium facing fertile portion, off-white to grey, thinner than mesoperidium. Gleba powdery, grayish black.

Mature basidiomata globose to depressed globose, sessile, 25–50 mm in diam. when mature, splitting in a stellate pattern, encrusted with soil particles and other debris material, odour not recorded. Peridium double. Exoperidium up to 1.5 mm thick, splitting in to 5–12 acute rays; rays expanded when fresh, recurved inward upon drying; composed of three layers; mycelial layer, thin, brown, not persistent; middle layer off-white to grey, thicker than mycelial layer; inner layer off-white to grey to brown with age, as thick as middle layer, extensively cracked in mature specimens. Endoperidium sessile, off-white to grey, opens by a pore; pore small to medium, up to 3 mm in diam. Gleba light brown to brown, cottony.

Basidiospores globose to sub globose, densely verrucose, dark brown, 7–11 μm in diam. including ornaments (6–11 μm in diam. ornamentation excluded), verrucae up to 1– 1.5 µm long. Capillitium hyaline, thick walled, with narrow lumen, branched, rarely septate, up to 5.0–12.8 μm in diam., (walls up to 1.9–4.5 μm), clamp connections present.

Material examined: Pakistan: Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 1st Sep. 2006, in groups, among grass, at 975.36 m (3200 ft) a.s.l., A. N. Khalid, APK4 (LAH100000172); 10th Aug. 2010, gregarious, on ground, M. Fiaz, APK13 (LAH100000173); Abbottabad district, Shimla hill, 14th Sep. 2012, in groups, on the sloppy ground, under Pinus wallichiana, at 1972 m (6473 ft) a.s.l., N. Yousaf, APK5 229

(LAH100000174), APK6 (LAH100000175), APK7 (LAH100000176); Mansehra district, Jabori, 15th Sep. 2012, in groups, on ground, among leaf litter and grass, at 1367 m (4487 ft) a.s.l., N. Yousaf, APK8 (LAH100000177); Dadar, 15th Sep. 2012, in groups, on sloppy ground, under Pinus wallichiana, at 636 m (2089 ft) a.s.l., N. Yousaf, APK2 (LAH100000178), APK9 (LAH100000189), APK10 (LAH100000180), APK11 (LAH100000181), APK12 (LAH100000182); Batrasi, 25th April, 2012, among grass, at 1241 m (4073 ft) a.s.l., N. Yousaf, APK14 (LAH100000183); Punjab, Dera Ghazi Khan, Fort Munro, 17th Aug. 2013, under Pinus sp. on ground, sandy soil, at 123.75 m (406 ft) a.s.l., N. Yousaf, APK3 (LAH100000184); Swat district, Ushu, 4th Sep. 2013, solitary, under Cedrus deodara, at 2418 m (7933 ft) a.s.l., S. Jabeen, APK15 (LAH100000185).

Additional Material Examined: Geastrum hygrometricum, Pakistan: Punjab, Murree, 25th March1964, on ground, S. Ahmad (LAH16974).

Comments: Astraeus hygrometricus was reported first time by Ahmad in 1956 as Geastrum hygrometricum, from different regions of Pakistan viz., Patriata (Murree) of Punjab, Kalam (Swat district), Kaghan valley of KP. It is reported first time from Khabbal Paien, and Dadar of district Mansehra, and district Abbottabad. Fort Munro (Dera Ghazi Khan) is also new location of A. hygrometricus.

A

B C

230

A

B C

D E

Plate 140. Figs. A–E. Astraeus hygrometricus. A–C. Immature basidiomata (APK8, APK5, APK8). D & E. Mature basidiomata (APK4 & APK3). Bar: = 0.8 cm for A–C, = 1 cm for D–E. 231

A

B C

Plate 141. Figs. A–C. Astraeus hygrometricus. A. Verrucose basidiospores. B. Eucapillitial threads. C. Joint like septa in capillitial threads. Bar: = 2.7 µm for A, = 5 µm for B, = 11 µm for C.

232

A B

C

Plate 142. Figs. A–B. LM photographs of Astraeus hygrometricus (APK2). A. Thick walled capillitial hyphae and spores. B. Basidiospores. C. Capillitial hyphae showing thick walls, and joint like septa. Bar: = 100 µm for A, = 6.7 µm for B, = 18 µm for C. 233

4.1.5. Taxa belonging to Rhizopogonaceae

Rhizopogon flavescens nom. prov. (Plate 143)

Etymology: on the basis of yellowish color of basidioma

Basidiomata globose to sub-globose, 7–20 mm in diam., yellowish, semi hypogeous, in groups, turning brown with age, no change in color when bruised or cut, attached to the substratum by small rhizomorphs. Peridium Peridium elastic when fresh, wrinkled when dry. Gleba songy, brown when mature.

Basidiospores cylindrical to ellipsoid, light green, 6.7–11.5 × 3.2–4.6 µm, one to two oil droplets present, amyloid. Peridium composed of yellow, thin walled, branched, septate tightly packed hyphae.

Material examined: Pakistan, Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 3rd Sep. 2013, hypogeous, at 1091 m (3582 ft) a.s.l., N. Yousaf, RPK14 (Holotype), (LAH100000186).

234

A

B

C Plate 143. Figs. A–C. Rhizopogon flavescens (RPK14). A. Basidiomata, B & C. Basidiospores. Bar: = 0.7 cm for A, = 13 cm for B, = 4.5 cm for C.

235

Rhizopogon himalayensis nom. prov. (Plates 144–145) Etymology: on the name of type locality “Himalayan moist temperate forest”

Basidiomata subglobose, yellow to brown, 25–40 mm high and 20–40 mm wide, semi-hypogeous when collected, in groups, yellow when fresh, turning brown with age, covered with thin membrane of hyphae, and adherent with soil particles and debris, no change in color when bruised or cut, anchored in the soil by well-developed rhizomophs present at the base. Rhizomorphs well developed, white, branched, rhizomorph system up to 20 mm in length, encrusted with debris and soil particles, dehiscence not observed. Peridium elastic when fresh. Exoperidium pale yellowish, persistent. Gleba off-white to yellow, firmly spongy.

Basidiospores elliptical to oblong, smooth, light greenish with brown wall, 4.6–8.0 × 3.0–4.0 µm, non amyloid. Peridium is composed of hyaline to brown, thin walled, branched, and septate hyphae.

Material examined: Pakistan, Khyber Pakhtunkhwa, Swat district, Kalam, 3rd Sep. 2013, hypogeous, in groups, under Cedrus deodara, at 3596 m (11800 ft) a.s.l., S. Jabeen, RPK8 (Holotype), (LAH100000187).

236

A

B C Plate 144. Figs. A–C. Rhizopogon himalayensis (RPK8). A. Basidiomata. B. Basidiospores. C. Peridial hyphae. Bar: = 0.9 cm for A, = 4 µm for B, = 6.8 µm for C. 237

A

B Plate 145. Figs. A–B. LM photographs of Rhizopogon himalayensis (RPK8). A. Hymenium and basidiospores. B. Basidiospores. Bar: = 11 µm for A, = 6.5 µm for B.

238

Rhizopogon roseolus (Corda) Th. Fr., Svensk bot. Tidskr. 3: 282 (1909) (Plates 146–147)

Basidiomata sub globose to somewhat elongated, growing on ground, in groups, semi hypogeous, whitish yellow to brown to red when fresh, changing color with age, covered with soil particles, gasterocarp cracking at the base somewhat chambered gleba a little, changing color when bruised or cut, 25-40 mm high and 35- 40 mm wide, surface rough, patchy some times when mature, anchored in the soil by well-developed rhizomorphs present at the base. Rhizomorphs white, branched, rhizomorph system up to 30 mm in length, delicate, may lost when mishandled, encrusted with debris and soil particles, dehiscence irregular first from the apical portion showing somewhat stellate pattern. Peridium elastic when fresh. Exoperidium thin, light brown, in form of membrane, constricted at some points, somewhat granular covered with particles of soil, peeling off exposing underlying endoperidium, persistent somewhat; Endoperidium thick, whitish yellow, persistent; Gleba off-white to yellow, firmly spongy, chambered, chambers becoming small towards the base.

Basidiospores elliptic, cylindrical, smooth, light greenish in 5% KOH, 4.6–8.5 ×3.0–4.3 µm, two oil droplets present, and bluish grey in Melzer’s reagent.

Material examined: Pakistan, Punjab, Lahore, University of the Punjab, Botanical Garden, 14th March, 2011, 20th March, 2012, on ground, semi hypogeous, in groups, under Pinus rouxburghii, at 215 m (705 ft) a.s.l., RPK11 (LAH100000187), RPK12 (LAH100000188); Khyber Pakhtunkhwa, Nadi Bunglow, 15th August, 2011, on ground, semi hypogeous, in groups, under Pinus wallichaina, at 1314 m (4313 ft) a.s.l., RPK13 (LAH100000189).

Additional material examined: Rhizopogon flavus, Punjab, Murree, 31st Aug. 1960, S. Ahmad, RH3 (LAH15211). B

239

A

A

B C

D

Plate 146. Figs. A–D. (RPK11). A–C. Different views of mature basidioma. B D. L.S of mature basidioma. Bar: = 0.5 cm for A, = 0.8 cm for B & C, = 0.7 cm for D. 240

A

C B Plate 147. Figs. A–C. Rhizopogon roseolus. A. Basidiomata (RPK13). B. Peridial hyphae. C. Basidiospores. Bar: = 0.9 cm for A, = 9 µm for B, = 2.2 µm for C.

241

4.1.6. Taxa belonging to Sclerodermataceae

Pisolithus flavus nom. prov. (Plates 148–149)

Etymology: on the basis of yellow color of basidioma

Basidiomata subglobose to elongated, off white to bright yellow, up to 80 mm in diam. × 120 mm in height; attached by a solid pseudostipe that ends in bright yellow rhizomorphs, up to 60 mm in height; dehiscence by irregular rupturing of the apical part exposing the glebal portion. Exoperidium off-white to yellow, smooth, membranous to elastic. Endoperidium grayish lights brown, becoming hard with maturity, eventually cracking apart apically to allow dehiscence. Gleba dull olivaceous brown at maturity, interior lined with thick tramal plates that create chambers, these containing dark brown, granular pseudoperidioles; pseudoperidioles composed of hyphal aggregations containing spores, distributed throughout the gleba, more apparent at the apex of the basidiomata and becoming pulverulent with age.

Basidiospores globose, olivaceous brown in water mounts, 9.0–11.5 µm in diam. (ornamentation included or 6.8–8.8 µm excluding ornamentation), verrucose (verrucae up to 1.7 µm). Eucapillitium lacking. Pseudo-perdiole hyphae up to 5.0 µm in diam., thin walled, hyaline, septate and branched.

Material examined: PAKISTAN: Punjab, Dera Ghazi Khan district, Fort Munro, 17th Aug. 2013, on sandy soil, distributed throughout the sampling sites at various maturity stages, under Eucalyptus sp. at 123.75 m (406 ft) a.s.l., N. Yousaf, PPK11 (Holotype), (LAH100000189), District Bahawalpur, Lal Sohanra Park, 18th Aug. 2013, on humus rich soil, PPK12 (LAH100000190), PPK14 (LAH100000191).

242

A B

C D

Plate 148. Figs. A–D. Pisolithus flavus. A & B. Young and mature basidiomata (PPK11). C. Basidiomata (PPK14). D. Basidiospores. Bar: = 3 cm for A, = 2 cm for B, = 2.5 cm for C, = 5.5 µm for D. 243

A

B

Plate 149. Figs. A–B. LM photograph of basidiospores. A. Pisolithus flavus (PPK11). B. Pisolithus tinctorious (PPK10). Bar: = 4 μm for A, = 8 μm for B.

244

Pisolithus tinctorius (Mont.) E. Fisch., (1900) (Plates 150–151) Basidiomata subglobose to globose, brown, 50–60 mm in diam. × 60–70 mm in height; pseudo-stipe up to 30 mm long, basally attached to the substratum by thick; attached by a solid pseudostipe that is typically buried below ground, yellowish mycelial strands encrusted with soil particles; ostiole lacking. Exoperidium off-white to brownish, smooth, eventually peeling away in patches to expose the endoperidium. Endoperidium dark grayish brown, firm, rugulose, eventually cracking apart apically to allow dehiscence. Gleba pale to reddish brown, interior lined with thick tramal plates that create chambers, these containing dark brown, granular pseudoperidioles; pseudoperidioles composed of hyphal aggregations containing spores, distributed throughout the gleba, more apparent at the base of the gasterocarp and becoming pulverulent toward the apex.

Basidiospores globose to subglobose, apedicellate, yellowish brown in water mounts, 7.0–10.5 µm in diam. (ornamentation included or 6.5–8.8 µm excluding ornamentation), strongly verrucose (verrucae up to 2 µm), sterigmal remnants present in mounts. Eucapillitium lacking. Pseudoperdiole hyphae up to 1.8–4.8 µm in diam., thin walled, hyaline, septate and branched. Exoperidium composed of branching, septate hyphae, clamp connections present. Endoperidium composed of unbranched, septate, unclamped hyphae

Material examined: PAKISTAN: Lahore, Quaide Azam Campus, University of Punjab, 26th Aug. 2006, gregarious, on ground, near Bamboo tree, at 215 m (705 ft) a.s.l., N. Yousaf, PPK17 (LAH100000192); Mansehra district, Ughi forest, 10th Aug. 2011, on sandy soil, at 1192 m (3912 ft) a.s.l., M. Fiaz, PPK10 (LAH100000193).

245

A

B C

Plate 150. Figs. A–C. Pisolithus tinctorious. A. Basidiomata (PPK10). B & C. Different views of basidioma (PPK17). Bar: = 1 cm for A–C.

246

B A

AB AAC

C

D

Plate 151. Figs. A–D. Pisolithus tinctorious. A. Verrucose basidiospores. B. Glebal hyphae. C. Exoperidial hyphae. D. Endoperidial hyphae. Bar: = 2.7 µm for A, = 10 µm for B–D.

247

Pisolithus sp. (PPK16) (Plate 152) Basidiomata sub-globose to pyriforme, 23–35 mm in diam. × 20–35 mm in height; attached by a solid pseudo-stipe that is typically buried below ground; pseudo-stipe up to 10 mm long, basally attached to the substratum by rhizomophs, rhizomorphs off white, remnants attached at the base, encrusted with soil particles; ostiole lacking. Exoperidium ochraceous brown when young, dark grey with age, rough, peeling away in patches to expose the endoperidium. Endoperidium dark grayish brown, firm, eventually cracking apart apically to allow dehiscence. Interior part consists of two layers interior lined with thick tramal plates that create chambers, these containing dark brown, granular pseudoperidioles; pseudoperidioles composed of hyphal aggregations containing spores, distributed throughout the gleba, more apparent at the apex of the basidioma, base composed of tightly packed brown hyphae.

Basidiospores sub-globose, brown in water mounts, 11.5–13.0 µm in diam. (ornamentation included or 8.5–10.5 µm excluding ornamentation), strongly verrucose (verrucae up to 3 µm), sterigmal remnants absent in mounts. Eucapillitium lacking. Pseudo- peridiole hyphae up to 9.9 µm in diam., thin walled, hyaline, septate and branched. Sterile base composed of brown (orange brown when aggregated), branched, aseptate hyphae, up to 8 µm in diam. Peridium orange brown hyphae when aggregated, tightly packed, irregular, aseptate, branched, 4.7 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Jabori, 14th Sep. 2012, in groups, on ground, at 1010 m (3312 ft) a.s.l., N. Yousaf, PPK16 (LAH100000194). 248 A

A B

C D

E F

Plate 152. Figs. A–F. Pisolithus sp. (PPK16). A & B. Basidioma. C. Basidiospores. D. Peridial hyphae. E. Pseudo-peridioles hyphae. F. Hyphae of sterile base. Bar: = 0.85 cm for A, = 0.9 cm for B, = 6.5 µm for C, = 10 µm for D & E, = 6.5 µm for F.

249

Scleroderma areolatum Ehrenb., Sylv. Mycol. Berol. (Berlin) 15: 27 (1818) C (Plate 153–154) Basidiomata epigeous, up to 30 mm broad, 25 mm high, subglobose, attached to the substratum by a thick mycelial base, forming a pseudostipe; pseudostipe tough, often two basidiomata arise from the same tuft of mycelium. Rhizomorphs white, thick, growing individually or in small groups. Peridium thin, <1mm thick. pale yellow to brown; basal portion smooth, without scales, off white to pale yellow, upper part with brown spots like scales, dehiscence by tearing away the apical portion. Gleba powdery, dark purplish. Basidiospores globose, echinulate, never reticulate, brown, 12–18 µm in diam., echines up to 2.5 µm long. Tramal hyphae rarely septate, branched, hyaline, with few amorphous material encrusted, up to 5.5 µm wide, wall thickness up to 1 µm. Exoperidium composed of aseptate, branched hyphae, up to 7 µm wide, wall thickness up to 2.5 µm. Mesoperidium composed of aseptate, branched hyphae, wall encrusted, up to 7 µm wide, wall thickness 2.6 µm. Endoperidium composed of septate, branched hyphae, up to 6.6 µm wide, wall thickness up to 2.8 µm. Material examined: PAKISTAN: Khyber Pakhtunkhwa, Abbottabad district, Ayubia, Khanspur, 12th Aug. 2007, solitary, on ground, in Himalayan moist temperate forest, at 2575 m (8448 ft) a.s.l., N. Yousaf, NYG04 (SPK10) (LAH 120807).

Comments: Scleroderma areolatum is ectomycorrhizal, and a poisonous species (Yamada & Katsuya, 1995; Mason et al., 2000; Chen et al., 2006a, 2006b). It is characterized by having non reticulate, densely echinulate basidiospores, a distinctive feature which separates the species from other Scleroderma spp. examined, which have reticulate and sub reticulate basidiospores. The species is widely distributed in Australia, Europe, Japan, , North America, Southern Brazil, South China and South America (Lu et al., 1999; Giachini et al., 2000; Kasuya et al., 2002; Barroetavena et al., 2005; Chen et al., 2006b). It is a new record from Pakistan.

250

B A

C D

E F

Plate 153. Figs. A–F. Scleroderma areolatum. A. Illustration of a basidioma, SPK10. B. Basidiospore. C. Exoperidial hyphae. D. Mesoperidial hyphae. E. Endoperidial hyphae. F. Tramal hyphae. Bar: = 0.5 cm for A, = 3.5 µm for B; = 28 µm for C; = 14 µm for D & F, = 18 µm for E.

251

A

B Plate 154. Figs. A–B. LM photographs of Scleroderma areolatum (SPK10). A. Echinulate to verrucose basidiospores. B. Tramal hyphae. Bar: = 9.5 µm for A, = 10 µm for B.

252

Scleroderma bovista Fr., Syst. mycol. (Lundae) 3(1): 48 (1829) (Plates 155–156) Basidiomata epigeous, up to 30 mm broad × 30 mm high, globose to slightly sub- globose, yellowish to dull brown, with well-developed mycelial base. Peridium <1 mm thick, tough, relatively rough, elastic, scales present; scales black, thin, more dense on top, spot-like; dehiscence by an irregular rupturing of an apical part. Gleba firm, compact, powdery with age, grayish black. Basidiospores partially to completely reticulate, ornamented, verrucose, dark brown, 13–19 µm (av. 16 µm) in diam. including ornamentation, 11–14.5 µm in diam. excluding ornamentation, verrucae up to 4.7 µm high. Tramal hyphae hyaline, thin walled, septate, branched, clamp connections present, slightly encrusted with some amorphous material, branched, up to 10.5 µm in diam. Exoperidium composed of hyaline, septate, branched hyphae, up to 6 µm in diam. Mesoperidium composed of hyaline, branched, septate hyphae with clamp connections, up to 9.5 µm in diam. Endoperidium composed of hyaline, thin walled, branched, septate hyphae with clamp connection, up to 6 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Kalam, 5th Sep. 2013, in groups, under Cedrus deodara, at 2638 m (8655 ft) a.s.l., S. Jabeen, SPK1, SPK19, (LAH100000195); Mansehra district, Ughi forest, 5th July. 2011, in groups, among grass, at 1091.8 m (3582 ft) a.s.l., M. Fiaz. 32 (SPK18) (LAH100000196); Khanspur, 22nd August, 2010, in groups, among grass, at 2250 m (7500 ft) a.s.l., N. Yousaf, SPK6, (LAH100000197); Khanspur, PU Suites, 22nd Aug. 2010, in groups, gregarious, N. Yousaf, SPK5 (LAH100000198); Helipad, 15th Aug. 2009, on ground, gregarious, N. Yousaf, SPK9 (LAH100000199); Nadi Bunglow, 15th Aug. 2011, on ground, in groups, at 1314 m (4313 ft) a.s.l., SPK20 (LAH100000200).

Comments: S. bovista has been reported from Patriata (Murree hills) in 1956 by Ahmad. It is a new record for Abottabad, Mansehra, and Swat districts. 253

A B

C D Plate 155. Figs. A–D. Scleroderma bovista. A. Basidiomata (SPK1). B. Basidiomata (SPK5). C. Basidioma, (SPK20). D. LM photograph of basidiospores with complete reticulum alongwith hyaline tramal hyphae. Bar: = 1 cm for A–C, = 20 μm for D.

254

A

B C

D Plate 156. Figs. A–D. Scleroderma bovista. A. Basidiospores. B. Exoperidial hyphae. C. Tramal hyphae. D. Endoperidial hyphae. Bar: = 4.5 μm for A, = 11 μm for B & C, = 10 μm for D. 255

Scleroderma chevalieri Guzmán, Ciencia Mex. 25: 202(1967) (Plates 157–158) Basidioma epigeous, 45 mm broad, 40 mm high, sub-globose, attached to the substratum via a tuft of mycelium, latter some time aggregated to form pseudostipe. Rhizomorphs white, thick. Peridium <1 mm thick, hard, rough, covered with patches of scales, mature specimen cracked at the upper portion, dehiscence by the irregular splitting of the peridium. Gleba pulverulent, pale yellow, brown to olivaceous. Basidiospores globose, echinulate, partially reticulate or sub reticulate, brown, 11– 17 µm in diam., echines up to 3 µm long. Tramal hyphae aseptate, branched, hyaline, with clamp connections, up to 5.3 µm wide, wall thickness up to 1.3 µm. Exoperidium composed of septate, unbranched hyphae; up to 8.8 µm wide, wall thickness up to 2.0 µm. Mesoperidium composed of aseptate, unbranched hyphae, clamp connections rarely present; up to 9.5 µm wide, wall thickness up to 2.4 µm. Endoperidium composed of aseptate, unbranched hyphae, up to 9.5 µm wide, wall thickness 2.5 µm. Material examined: PAKISTAN: Khyber Pakhtunkhwa, Nathia gali, 25th Aug. 2006, in groups, on ground, under Abies pindrow in Himalayan moist temperate forest, at 2501 m (762 ft) a.s.l., N. Yousaf, NYG03 (SPK7) (LAH 250806). Comments: Like other members of Scleroderma, S. chevalieri also has hard, tough peridium and is characterized by a subglobose basidioma with short pseudostipe. It has larger ornamented basidiospores (up to 17 µm in diam.) with sub reticulum and peridium less than 1 mm thick. These features of peridial thickness and partially reticulate spores are shared by other two taxa, e.g. S. sinnamariense Mont. and S. stellatum Berk., however colored peridium with lemon yellow to black scales and smaller spores in S. sinnamarense are not found in S. chevalieri. S. stellatum has a star-shaped opening and smaller spores (Sims et al., 1995). It is a new record for Pakistan.

256

AA AB

AD AC

AE AF

Plate 157. Figs. A–F. Scleroderma chevalieri. A. Illustration of a basidioma (weathered), SPK7. B. Basidiospores. C. Exoperidial hyphae. D. Mesoperidial hyphae. E. Tramal hyphae. F. Endoperidial hyphae. Bar: = 0.8 cm for A, = 3.5 µm for B, = 10 µm for C, = 11 µm for D, = 10 µm for E, = 20 µm for F.

257

A

B Plate 158. Figs. A–B. LM photographs of Scleroderma chevalieri (SPK7). A. Basidiospores. B. hyaine tramal hyphae. Bar: = 5 µm for A, = 11 µm for B.

258

Scleroderma dictyosporum Pat., Bull. Soc. Mycol. Fr. 12(3): 135 (1896) (Plate 159) Basidiomata epigeous, up to 30 mm broad and 30 mm high, globose to sub-globose, dull brown, with well-developed mycelial base often produced into a pseudo-stipe; several fruit bodies arising from a single stalk. Peridium <1 mm thick, tough, relatively smooth, elastic, brittle in dried specimen cuticle breaking into scales; scales black, thin, more on top, spot like; dehiscence by an irregular rupturing of an apical part. Gleba firm, compact, powdery with age, grayish to sepia, lighter towards the center. Basidiospores globose, echinulate-reticulate, brown, 12–15 µm in diam., echines up to 4 µm long, young spores without reticulum, nurse cells prominent in the mount. Tramal hyphae hyaline, thin walled, with clamp connections. Exoperidium composed of fascicles of erect hyphae, dark orange to brown, up to 6.5 µm, thick walled, encrusted. Mesoperidium with globose elements, hyaline (yellowish in mass), up to 6 µm in diam., thin walled, mixed with long hyaline hyphae. Endoperidium composed of septate, branched, hyaline hyphae, surrounding the gleba, hyphae up to 6 µm wide, thin walled. Material examined: PAKISTAN: Khyber Pakhtunkhwa, Naran, 23rd Sep. 2009, in groups, on sandy soil, at 3350 m (10990 ft) a.s.l., N. Yousaf, NYG01 (SPK17) (LAH 230909).

Comments: Scleroderma dictyosporum is an ectomycorrhizal species (Mukerji et al., 2000; Rai et al., 2009) and is distinguished by its globose basidioma with shorter pseudo- stipe, thin peridium (<1mm), echinulate-reticulate basidiospores (12.5–15.0 µm) with echines up to 4 µm. This species is close to S. fuscum (Corda) E. Fisch., but the latter has smooth peridium, absence of stipe and larger basidiospores (up to 17 µm). It is a new record for Pakistan.

259

B A

C D

Plate 159. Figs. A–D. Scleroderma dictyosporum (SPK17). A. Illustration of a basidioma. B. Basidiospores. C. Exoperidial and mesoperidial hyphae. D. Endoperidial hyphae. Bar: = 0.6 cm for A, = 3.3 µm for B, = 17 µm for C, = 11 µm for D.

260

Scleroderma fuscum (Corda) E. Fisch., in Engler & Prantl, Nat. Pflanzenfam., Teil. I (Leipzig) 1**: 336 (1900) (Plates 160–161)

Basidioma sub-globose to elongated, up to 50 mm long, shortly stipitate. Peridium 1 mm thick, without scales, smooth wrinkled when dry. Base pale yellow. Dehiscence not observed, attached to the substratum by small rhizomorphs, Gleba powdery, grayish brown.

Basidiospores globose, ornamented, reticulate, dark brown, up to 16.3 µm in diam., echines up to 3 µm long, young spores without reticulum. Eucapillitium absent. Tramal hyphae present, hyaline, septate, branched, up to 5 µm, nurse cells evident in the mount. Endoperidium composed of light brown, hyaline, septate, branced and tightly packed, up to 5 µm.

Material examined: PAKISTAN: Azad Kashmir, Neelum Valley, Sharda, 17th October 2009, gregarious, on ground, at 1981 m (6488 ft) a.s.l., N. Yousaf, SPK8 (LAH100000201).

Comments: S. fuscum belongs to section Scleroderma and very close to S. bovista because of similar spore morphology however peridium is different in both species. S. bovista has peridium with brown scales whereas latter has smooth peridium without scales. It is a new record for Pakistan.

261

C A B

D E

Plate 160. Figs. A–E. Scleroderma fuscum (SPK8). A. A basidioma. B. Tramal hyphae. C. AD C AF Mesoperidium. D. Basidiospores. E. Endoperidial hyphae surrounding gleba. Bar: = 0.7 cm for A, = 9.5 µm for B, = 13 µm for C, = 4.5 µm for D, = 9 µm for E. 262

A

B

Plate 161. Figs. A–B. LM photographs of basidiospores of Scleroderma fuscum (SPK8). Bar: = 8.8 µm for A, = 6.5 µm for B.

263

Scleroderma sp. (SPK21) (Plate 162)

Basidiomata epigeous, up to 35 mm broad, 25 mm high, subglobose, light brown above to grayish brown below, covered with brownish black scales all over the peridium, smooth below, attached to the substratum by a small mycelial base, lacking a pseudostipe. Rhizomorphs not observed in the collection. Peridium thin, <1mm thick, dehiscence not observed. Gleba powdery, dark grayish green

Basidiospores globose, echinulate, completely reticulate, brown, 12–17 µm in diam. including ornamentation, 9–14, echines up to 5 µm long. Tramal hyphae septate, branched, thin walled, hyaline, with few amorphous material encrusted, up to 5.5 µm wide. Exoperidium composed of aseptate, thin walled, branched hyphae, up to 10.3 µm wide. Endoperidium composed of hyaline to light brown, septate, irregular, branched hyphae, clamps present, up to 9.7 µm wide.

Specimen examined: PAKISTAN: Khyber Pakhtunkhwa, Abbottabad district, Thandiani, 12th Sep. 2012, at 2672 m (8766 ft) a.s.l., solitary, on ground, N. Yousaf, SPK21 (LAH100000202). 264

A B

C D E

Plate 162. Figs. A–E. Scleroderma sp. (SPK21). A. A basidioma. B. Tramal hyphae. C. AD C AF Mesoperidium. D. Basidiospores. E. Endoperidial hyphae surrounding gleba. Bar: = 0.6 cm for A, = 8.5 µm for B, = 5 µm for C, = 16 µm for D, = 13 µm for E.

265

4.1.7. Taxa belonging to Geastraceae

Geastrum fimbriatum Fr., Syst. mycol. (Lundae) 3(1): 16 (1829) (Plate 163) ≡ Geastrum sessile (Sowerby) Pouzar Dried basidiomata 37–45 mm in width × 15–35 mm in height, exoperidium splitting to the middle forming 7 rays, rays very narrow at tips, tips curved. Peridium double. Exoperidium composed of three layers; Mycelial layer very thin, membranous, peeling off at some points, often revealing fibrous layers which is lighter in color; Fibrous layer brown, glabrous; Pseudoparenchymatous layer light brown. Endoperidial body 25 mm in diam., × 25 mm in height, stalk absent; Sporecase globose, light brown to grey, glabrous with occasional hyphal protrusions (visible under magnification); Peristome fibrillose, not delimited, slightly raised in the central portion to a short narrow point to conical. Gleba grayish black.

Basidiospores globose, 4.0–4.5 µm in diam., including ornamentation, 3.0–3.8 µm without ornamentation, verrucose, with dense verrucae, up to 1.0 µm long, yellowish brown. Eucapillitium Geastrum type, 2.5–9.0 µm in diam., thick walled, smooth to slightly encrusted, attenuate at tips, straight to sub-undulate, aseptate, often knob like projections present, unbranched.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Abbottabad district, Ayubia National Park, 24th August, 2010, in groups, along the track, among grass, at 2438 m (8000 ft) a.s.l., N. Yousaf, GPK6 (LAH100000203).

Comments: G. fimbriatum has previously been reported from Gilgit-Baltistan by Razzaq & Shahzad as G. saccatum in 2007. It is first report from Abbottabad district, and Swat and second report for Pakistan.

266

A

B C

D Plate 163. Figs. A–D. Geastrum fimbriatum (GPK6). A. Dried basidiomata. B. Illustration of basidiospores. C. LM photograph of eucapillitium. D. LM photograph of basidiospores. Bar: = 0.8 cm for A, = 3.3 µm for B, 22 µm for C, 2 µm for D. 267

Geastrum himalayensis nom. prov. (Plates 164–165)

Etymology: on the basis of type locality of Himalayan moist temperate forest Basidiomata up to 30 mm in width × 25 mm in height, exoperidium splitting to the middle or less forming 7-8 rays, not splitting again at tips, rays non hygroscopic, slightly curving on the underside. Peridium double. Exoperidium composed of three layers; Mycelial layer persistent, thin, slightly encrusted with leafy debris or soil particles; Pseudoparenchymatous layer pallid at first, brown, thin, smooth; Fibrous layer off white, thin, smooth. Endoperidial body, up to 15 mm in diameter and 15 mm in height, (including peristome), globose, endoperidium grayish brown to light brown; Peristome plicate, distinctly delimited, depression present around the peristome, conical, up to 1 mm in height. Gleba grayish brown, cottony.

Basidiospores olivaceous brown, globose, echinulate, up to 3.9–4.6 µm in diameter including verrucae and 2.88–3.0 µm excluding verrucae, verrucae up to 1.2 um long. Eucapillitium prominent, well developed, aseptate, rarely branched, encrusted, long straight threads, tips attenuate, brown, 2.9–6.0 µm wide, wall thickness 1.2–2.6 µm. Paracapillitium present.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Mansehra district, Dadar, 15th Sep. 2012, in groups, among grass, at 1127 m (3700 ft) a.s.l., N. Yousaf, GPK16 (Holotype), (LAH100000204), GPK21 (LAH100000205).

268

A

B

Plate 164. Figs. A–B. Geastrum himalayensis. A. Mature basidiomata (GPK16). B. Mature basidiomata (GPK21). Bar: = 0.7 cm for A, = 1 cm for B. 269

A B

C D

E F

Plate 165. Figs. A–F. LM photographs of Geastrum himalayensis. A–D. Different views of eucapillitial hyphae. E & F. Echinulate basidiospores. Bar: = 60 µm for A, = 30 µm for B, = 20 µm for C, = 15 µm for D, = 1.5 µm for E, = 2 µm for F.

270

Geatrum lacogalopsus nom. prov. (Plates 166–168)

Etymology: on the basis of resemblance with “Lycogalopsis”

Basidiomata globose to subglobose, gregarious, up to 15 mm broad × 15 mm high, grey to off white; attached to the substratum by a well developed mycelial base, forming a whitish mat over the debris and soil particles, rhizomorphs white, up to 5 mm high. Dehiscence by an apical pore, pore develops while sloughing off the exoperidium. Peridium three layered. Exoperidium persistent, in the form of brown pachtes. Mesoperidium offwhite to yellowish brown. Endoperidium brown, papery. Gleba brown, compact. Sub gleba absent

Basidiospores globose, smooth, olivaceous brown, 2.8–3.3 µm in diam., central oil droplet present. Eucapillitium olivaceous to brown, Geastrum type, branched rarely, aseptate, straight, heavily encrusted with some amorphous material, pores absent, attenuate at tips, tips undulate sometimes, aseptate, thick walled, up to 6.0 µm in diam. Paracapillitium present, scarce, hyaline, branched, septate. Exoperidium hyaline, branched curled aseptate hyphae, hyphae with rounded ends, up to 4.3 µm in diam. Mesoperidium composed of hyaline, aseptate, thick walled, occassionly branched straight hyphae, up to 6 µm in diam Endoperidium composed of light brown to olivaceous, thick walled, aseptate, branched, tightly packed hyphae, up to 4 µm in diam.

Material examined: PAKISTAN: Punjab, Kasur district, Changa Manga forest, 24th Aug. 2013, at 201 m (660 ft) a.s.l., solitary, on ground, N. Yousaf, DPK3 (Holotype), (LAH100000206). 271

B A

C

Plate 166. Figs. A–C. Geatrum lacogalopsus (DPK3). A. A basidioma B. Basidiospores. C. AD C AF Eucapillitial hyphae. Bar: = 1 cm for A, = 2.5 µm for B, = 7 µm for C.

272

A B

C Plate 167. Figs. A–C. Geatrum lacogalopsus. A. Exoperidial hypahe. B. Exoperidial hyphae. C. AD Mesoperidial hypahe. Bar: = 11 µm for A & C, = 13 µm for B. C AF 273

AD C AF

A

B

C D E Plate 168. Figs. A–E. LM photographs of Geatrum lacogalopsus. A–D. Different views of AD eucapillitial hyphae. E. Basidiospores. Bar: = 13 µm for A & D, = 6.6 µm for B, = 60 µm for C, = C AF 6 µm for E.

274

Geastrum pakistanicus nom. prov. (Plate 169) Etymology: on the name of the country from where it is first time described. Basidiomata up to 30 mm in width × 15–30 mm in height, exoperidium splitting to the middle or less forming 6rays, rays very narrow at tips, tips not curved inside, making a cup like case for endoperidial body. Peridium double. Exoperidium composed of three layers; Mycelial layer brown, very thin, striated, not encrusted with soil and debris material; Fibrous layer thin, off white to light brown, glabrous; Pseudoparenchymatous layer gray to brown. Endoperidial body 20 mm in diam, stalk absent; Sporecase globose, light brown to grey; Peristome plicate, distinctly delimited, slightly raised in the central portion, light coloration around sometimes. Gleba grayish black. Basidiospores globose, echinulate, olivaceous brown, 3.3–4.5 µm in diam., including ornamentation, 2.6–3.4 µm without ornamentation, echines up to 1 µm long. Eucapillitium Geastrum type, brown, up to 5.2 µm in diam., thick walled (up to 2.2 µm), encrusted with amorphous material, attenuate at tips, straight, aseptate, rarely branched.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Nadi Bunglow, 13th Aug. 2011, in groups, on the ground, among grass, at 2752 m (9028 ft) a.s.l., N. Yousaf, GPK32 (Holotype), (LAH100000207), GPK33 (LAH100000208), GPK34 (LAH100000209), GPK35 (LAH100000210).

275

A

B

C Plate 169. Figs. A–C. Geastrum pakistanicus (GPK32). A. Basidiomata. B. LM photograph of echinulated basidiospores. C. LM photograph of encrusted eucapillitial hyphae. Bar: = 0.8 cm for A, = 1.8 µm for B, = 13 µm for C. 276

Geastrum parvulus nom. prov. (Plates 170–171) Etymology: on the basis of small size of fruiting body Basidiomata 25–35 mm in width × 15 mm in height, exoperidium splitting to the middle forming 10 rays, rays very narrow at tips, tips curved. Peridium double. Exoperidium composed of three layers; Mycelial layer very thin, membranous, not encrusted with debris material and soil; Fibrous layer off white; Pseudoparenchymatous layer off white to light brown. Endoperidial body 12–15 mm in diam., × 15 mm in height, stalk absent; Sporecase globose, grey; Peristome fibrillose, delimited, slightly raised in the central portion to a short narrow point to conical. Gleba grayish black. Basidiospores globose, minutely warted, olivaceous to dark brown, 4.0–5.5 µm in diam., including ornamentation. Eucapillitium Geastrum type, up to 6 µm in diam., thick walled (wall thickness up to 3 µm), with narrow lumen, smooth to slightly encrusted, attenuate at tips, straight to sub-undulate, aseptate, mostly unbranched.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Abbottabad district, Shimla hill, 14th September, 2012, in groups, along the slopes, among grass, at 1972 m (6473 ft) a.s.l., N. Yousaf, GPK13 (Holotype), (LAH100000211).

277

A

B

Plate 170. Figs. A–B Geastrum parvulus (GPK13). Mature basidiomata. Bar: = 1.5 cm for A, = 0.7 cm for B. 278

B A

C

D

E

Plate 171. Figs. A–E. LM photographs of Geastrum parvulus. A & B. Eucapillitial hyphae. C. Thick walls of eucapillitial hypahe. D & E. Minutely warted basidiospores. Bar: = 22 µm for A, =

16 µm for B, = 11 µm for C, = 12 µm for D, = 2 µm for E.

279

Geastrum patulus nom. prov. (Plates 172–173) Etymology: on the basis of spreading and fragile characteristics of rays Basidiomata 20–30 mm in width × 15–20 mm in height, exoperidium splitting to the middle forming 10 rays, rays very narrow at tips, tips not curved inside, spreads straight parallel to spore case. Peridium double. Exoperidium composed of three layers; Mycelial layer very thin, membranous, not much encrusted with soil and debris material; Fibrous layer off white to light brown, glabrous; Pseudoparenchymatous layer gray to brown. Endoperidial body 20 mm in diam, stalk absent; Spore case globose, light brown to grey,; Peristome fibrillose, not delimited, slightly raised in the central portion to a short narrow point to conical, light coloration around. Gleba grayish black. Basidiospores globose, olivaceous to light brown, 4.0–5.0 µm in diam., including ornamentation, 3.0–4.0 µm without ornamentation, echinulate, up to 1.0 µm long. Eucapillitium Geastrum type, light brown, up to 5.5 µm in diam., thick walled (up to 2.5 µm), smooth to slightly encrusted, attenuate at tips, straight to sub-undulate, aseptate, often knob like projections present, mostly unbranched.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Abbottabad district, Shimla hill, 14th September, 2012, in groups, along the slopes, among grass, at 1972 m (6473 ft) a.s.l., N. Yousaf, G15 (Holotype), (LAH100000212), G18 (LAH100000213).

280

B A

C

Plate 172. Figs. A–C. Geastrum patulus. A. Mature basidiomata (GPK14). B. Mature basidiomata (GPK15). C. Collection no. GPK18. Bar: = 0.8 cm for A–C.

281

A

B

C

Plate173. Figs. A–C. LM photographs of Geastrum patulus. A & B. Verrucose basidiospores. C. Encrusted eucapillitial hyphae. Bar: = 7.8 µm for A, = 1 µm for B, = 11 µm for C.

282

Geastrum pseudolimbatum Hollós [as 'Geaster'], Mathem. Természettud. Közlem. 19: 507 (1901) (Plate 174) Basidiomata up to 30 mm in width × 25 mm in height, exoperidium splitting to the middle or less forming 8 rays, curving again upward near the base of endoperidial body. Peridium double. Exoperidium composed of three layers; Mycelial layer persistent, off- white to gray, slightly encrusted with leafy debris or soil particles; Pseudoparenchymatous layer brown, smooth; Fibrous layer off white, thin. Endoperidial body, up to 20 mm in diameter and 15 mm in height, globose, endoperidium grayish brown; Peristome not well marked in the specimen due to mishandling. Gleba grayish brown, cottony.

Basidiospores globose, verrucose, violet brown, 4.8–5.6 µm in diameter including verrucae and up to 4.6 µm excluding verrucae, verrucae up to 1 um long. Eucapillitium prominent, well developed, hyaline to light brown, aseptate, rarely branched, slightly encrusted with amorphous material, mostly smooth, long straight threads, tips attenuate, 2.7–6.7 µm wide, wall thickness up to 2.7 µm.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Ushu, 4th September, 2013, solitary, among grass, at 3200 m (10498 ft) a.s.l., M. Saba, GPK51 (LAH100000214)

283

A B

C

D E Plate 174. Figs. A–E. Geastrum pseudolimbatum (GPK51). A & B. Basidioma. C. LM photographs of thick walled encrusted eucapillitial hyphae. D & E. LM photographs of verrucose basidiospores. Bar: = 0.5 cm for A & B, = 12 µm for C, = 2.4 µm for D, = 1.5 µm for E.

284

Geastrum pseudosaccatum nom. prov. (Plate 175) Etymology: on the basis of similarities with G. saccatum Basidiomata up to 60 mm in diam. when fresh, exoperidium splitting to the middle forming 8 rays, not splitting again at tips, rays inarching; rays non-hygroscopic. Peridium double layered. Exoperidium composed of three layers; Mycelial layer thin, persistant; Pseudoparenchymatous layer brown, pallid at first, glabrous near the endoperidial body, rough at the tips or the end of the rays; Fibrous layer white, thin. Collar absent. Endoperidial body up to 25 mm in height and 18 mm in width; stalk absent; grayish brown, open by a peristome; Peristome fibrillose, delimited, paler area present around the peristome, up to 2 mm in height. Gleba greyish brown, cottony. Basidiospores globose, ornamented, truncate, brown, 4.5–5.5 µm in diam., including ornamentation, 3.7–4.7 µm in diameter (ornamentation excluded), brown; verrucae up to 1.5 µm long. Eucapillitium Geastrum type, up to 8 µm in diameter, wall thickness up to 3.5 µm, glabrous to slightly encrusted, unbranched, mostly straight, aseptate, with long narrow attenuate tips, lacking pores.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Miandam, 6th Sep. 2013, in groups, among grass, at 1936 m (6351 ft) a.s.l., M. Saba, GPK2 (Holotype), (LAH100000215).

285

B A

C

D E Plate 175. Figs. A–E. Geastrum pseudosaccatum (GPK2). A. Basidiomata. B. LM photographs of eucapillitial hyphae. C–E. LM photographs of basidiospores. Bar: = 0.5 cm for A, = 45 µm for B, = 6 µm for C, = 1.6 µm for D, = 2.5 µm for E.

286

Geastrum saccatum Fr., Syst. mycol. (Lundae) 3(1): 16 (1829) (Plates 176–177) Dried basidiomata up to 40 mm in width × 25 mm in height, exoperidium splitting to the middle or less forming 9 rays, rarely splitting again at tips, rays non hygroscopic, rolling under the endoperidial body. Peridium double. Exoperidium composed of three layers; Mycelial layer persistent, thin, heavily encrusted with leafy debris or soil particles; Pseudoparenchymatous layer, pallid at first, becoming brown to dark brown, thin, less than 1 mm when dried, rough, becoming rimose with age; Fibrous layer off white, thin, smooth. Endoperidial body, up to 20 mm in diameter and 15 mm in height, (including peristome), globose to depressed globose, endoperidium grayish brown to light brown; Peristome plicate, distinctly delimited, depression present around the peristome, conical, up to 1 mm in height. Gleba grayish brown, cottony.

Basidiospores globose, verrucose, brown, 4.5–5.5 µm in diameter including verrucae and 3.5–4.5 µm excluding verrucae, verrucae up to 1.33 um long. Eucapillitium prominent, well developed, aseptate, unbranched, encrusted, long straight threads, tips attenuate, brown, 3.0–7.0 µm wide.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Nathia gali, 22nd Aug. 2010, in groups, among grass, on ground, at 2501 m (8205 ft) a.s.l., N. Yousaf, GPK26 (LAH100000216), GPK27 (LAH100000217); Abbottabad district, Ayubia National Park, 23rd Aug. 2010, in groups, along the track, among grass, at 2438 m (8000 ft) a.s.l., N. Yousaf, GPK29 (LAH100000218).

Comments: G. saccatum has previously been reported from Kaghan valley by Ahmad in 1952 and currently from district Mansehra by Yousaf et al., 2014. It is first report for Nathia gali.

287

A

B C Plate 176. Figs. A–C. . Different views of mature basidiomata. A. Collection no. GPK26. B. Collection no. GPK27. C. Collection no. GPK29. Bar: = 0.8 cm for A, = 0.7 cm for B & C. 288

A

B

C Plate 177. Figs. A–C. LM photographs of Geastrum saccatum. A. Encrusted eucapillitial hyphae. B & C. Verrucose basidiospores. Bar: = 11 µm for A, = 6 µm for B, = 2.5 µm for C.

289

Geastrum triplex Jungh. [as 'Geaster'], Tijdschr. Nat. Gesch. Physiol. 7: 287 (1840) (Plates 178–179)

Basidiomata up to 50 mm in diam. when fresh, up to 40 mm in width × 25 mm in height when dried, exoperidium splitting to the middle forming 5-7 rays, not splitting again at tips, rays inarching, and revolute, involute, extending towards the middle basal portion, often tips of the rays touching the mycelial layer; rays non-hygroscopic. Peridium double layered. Exoperidium composed of three layers; Mycelial layer thin, persistant; Pseudoparenchymatous layer brown, pallid at first, glabrous near the endoperidial body, rough at the tips or the end of the rays; Fibrous layer white, thin. Collar present, forming a cup like structure around the endoperidial body. Endoperidial body up to 25 mm in height and 18 mm in width; stalk absent; grayish brown, open by a peristome; Peristome fibrillose, distinctly delimited, paler area present around the peristome, up to 2 mm in height. Gleba greyish brown, cottony. Basidiospores globose, ornamented, verrucose, brown, 4.0–5.5 µm in diam., including ornamentation and up to 3.2–4.5 µm in diameter (ornamentation excluded), yellowish brown in water mounts; verrucae up to 0.95 µm long. Eucapillitium Geastrum type, 2–6 µm in diameter, wall thickness up to 1.2 µm, glabrous to slightly encrusted, unbranched, mostly straight, aseptate, with long narrow attenuate tips, lacking pores.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Nathia gali, 8th Aug. 2008, in groups, among grass, on ground, at 2501 m (8205 ft). a.s.l., A. R. Niazi GPK22 (LAH100000219); Ayubia National Park, 15th August, 2009, 22nd August, 2010, solitary to in groups, along the track among grass, at 2438 m (8000 ft) a.s.l., A.R. Niazi, GPK23 (LAH100000220), GP28 (LAH100000221); Khanspur, 10th August 2009, in groups, among grass, at 2250 m (7500 ft) a.s.l., N. Yousaf, GPK25 (LAH100000222); 23rd August, 2010, solitary, among grass, at 2250 m (7500 ft) a.s.l., N. Yousaf, GPK24 (LAH100000223).

Comments: G. triplex is characterized by large basidiomata, presence of a collar, and cracked exoperidum. It has previously been reported from Kalam (Swat), Shogran (Kaghan valley), Nathia gali, Patriata (Murree hills) by Ahmad in 1952 and 1956, also by Khan in 1968 (Ahmad et al., 1997). It has been recently reported from district Mansehra (Yousaf et al., 2014). It is the first report from Khanspur and Ayubia National Park and second report for Nathia gali. 290

A B

C D

E F Plate 178. Figs. A–F. Geastrum triplex. Different views of mature basidioma. A. Collection no. GPK22. B. Collection no. GPK23. C. Collection no. GPK24. D. Collection no. GPK28. E. Collection no. GPK25. F. Basidiospores. Bar: = 1.3 cm for A, 1.5 cm for B, 0.8 cm for C & D, 1.2 cm for E, = 5 µm for E. 291

A

B C Plate 179. Figs. A–C. LM photographs of Geastrum triplex. A. Verrucose basidiospores. B & C. Eucapillitial threads. Bar: = 9 µm for A, = 8.5 µm for B & C.

292

Myriostoma coliforme (Dicks.) Corda, Anleit. Stud. Mykol., Prag: 131 (1842)

(Plates 180–181)

Dried basidioma up to 55 mm in width × 60 mm in height, solitary, consists of upper stalked spore case and radiating rays below. Peridium bilayered; Exoperidium splitting from the middle by forming 10 or more rays, rays revolute, united from the base near the endoperidial body, free at the tips, up to 30 mm long, tips curving towards the central basal portion, often touching the mycelial layer, rays rarely splitting at tips; Exoperidium three layered; Mycelial layer blackish brown, thin, patchy throughout, persistent, encrusted heavily with the debris material; Fibrous layer brown; Pseudo- parenchymatous layer yellowish brown with some dark brown, dotty powdery mass attached, thick; Endoperidium body (including multiple stalks and spore case), spore case greyish brown, up to 15 mm in height and 30 mm in diameter; endoperidium grayish brown, stalks, many attached, up to 5 mm in height; spore case opens by many pores through which spore escapes, up to 2 mm in diam. Gleba brown, cottony, later becoming pulverulent.

Basidispores globose, heavily ornamented, verrucose, brown, up to 7 µm in diam., including verrucae, up to 5 µm in diam. (ornamentation excluded), verrucae up to 2 µm long. Eucapillitial threads brown, aseptate, straight, thin walled, up to 5 µm in diam. in diam. Exoperdium composed of hyaline, aseptate to rarely septate, unbranched, thick walled, tightly packed hyphae, up to 6 µm in diam., wall thickness up to 2.5 µm. Endoperidium composed of brown, unbranched, aseptate, tightly packed hyphae, up to 5 µm

Material Examined: PAKISTAN, Khyber Pakhtunkhwa, Khanspur, Halipad, 8th Aug. 2011, in groups, under Pinus wallichiana, 2250 m (7500 ft) a.s.l., N. Yousaf, NYG6 (LAH100812).

Comments: M. coliforme is commonly called as pepper pot earth star or salt shaker because of the shape of its fruiting body which splits open from the middle forming many rays revealing endoperidium body (Long, 1942, Pegler & Spooner, 1992). Corda (1842) replaced the name M. anglicum, given by Desvaux with Myriostoma coliforme which is currently accepted name of this species. Many species have been considered as a 293

synonymy, which include Lycoperdon coliforme Dicks., Geastrum coliformis (Dicks.) Pers., Polystoma coliforme (Dicks.) Gray, and Myriostoma coliforme var. capillisporum V.J. Staněk. European Council for Conservation of Fungi enlisted M. coliforme as a threatened species that is facing the danger of habitat extinction. Authors have been surveying different parts of the country since last 10 years for exploring diversity of the fungi. Explored areas range from plains and deserts of Punjab to higher altitude of Himalayas, pastures of fairy meadows, Gilgit, Baltistan to Deosai plains, second highest plateau of the world. Among all the fungi collected, M. coliforme is thought to be very rare as its distribution in this region is concerned, when compared with other gasteroid fungi. 294

D C

E F

Plate 180. Figs. A–F. Myriostoma coliforme. A & B. Basidioma. C. Basidiospores. D Endoperidial hyphae. E. Eucapillitial hyphae. F. Exoperidial hyphae. Bar: = 0.8 cm for A & B, = 3.5 µm for C, = 14 µm D & F, = 15 µm for E. 295

A

B

C

Plate 181. Figs. A–C. LM photographs of basidiospores of Myriostoma coliforme. A & B. Globose verrucose basidiospores. C. Eucapillitial thick-walled hyphae. Bar: = 12 µm for A, = 2.3 µm for B, = 6 µm for C.

296

Radiigera ayubiensis nom. prov. (Plates 182–183)

Etymology: on the name of type locality

Basidioma hypogeous at first, sub-globose, up to 4 cm in diam., ochraceous brown when fresh, dark brown with age; encrusted with debris material; dehiscence by irregular rupturing of the apical part. Perdium 2 layered. Exoperidium ochraceous, hard, peeled off in form of patches exposing endoperidium. Endoperidium fibrous, off-white, thin, membranous. Gleba dark brown to black at maturity, powdery.

Basidiospores rounded, verrucose, dark brown, 6.9–8.0 µm in diam. including verrucae, 6.0–7.08 µm in diam. excluding verrucae, verrucae up to 2.4 µm long. Peridium composed of hyaline to olivaceous, thick walled, aseptate, branched, mixed with dirt, up to 8.3 µm in diam., wall thickness up to 4.5 µm. Eucapillitial hyphae hyaline to brown, fragile, septate, branched hyphae, up to 4.5 µm in diam.

Material examined: Pakistan, Khyber Pakhtunkhwa, Abbottabad district, Mukhspuri, 10th Sep. 2006, on ground, at 2813 m (9232 ft) a.s.l., A. R. Niazi, RPK7 (Holotype), (LAH100000224).

297

A

A B

C D

E

Plate 182. Figs. A–E. Radiigera ayubiensis. A & B. Different views of mature basidioma. C. Basidiospores. D. Tramal hyphae. E. Peridial hyphae. Bar: = 0.7 cm for A & B, = 3.5 µm for C, = 4.5 µm for D, = 6.5 µm for E.

298

A

B C

D

Plate 183. Figs. A–D. LM photographs of basidiospores of Radiigera ayubiensis. A–C. Verrucose basidiospores. D. Eucapillitial hyphae. Bar: = 15 µm for A, = 3 µm for B, = 5.5 µm for C, = 25 µm for D.

299

4.1.8. Taxa belonging to Phallaceae

Phallus ahmadii nom. prov. (Plate 184)

Etymology: on the name of Asian mycologist “S. Ahmad”

Egg off white, sub-globose, outer surface relatively smooth, slightly encrusted with soil particles. Mature fruiting body in the group of two, up to 70 mm tall and 20 mm wide at the top and 25 mm at the base; attached to the ground by less numerous branched, white, thin rhizomorphs, up to 20 mm high. Receptaculum bearing pileus and pseudostipe; Receptaculum surface merulioid. Pileus campanulate with pointed apex, up to 20 mm high × 8 mm in diam., hollow, having glebal mass. Gleba olivaceous, less stinky when collected, wet, sticky. Pseudostipe off-white, hollow, spongy, porous, soft, uniform, up to 7 mm in diam. Volva ovoid, saccate, 20 mm in height, 25 mm in diam., prominent, off-white, texture soft, outer surface rough, encrusted with soil particles, membranous, gelatinous from inside.

Basidiospores ellipsoid, greenish, 4.5–5.0 × 2.5–3.0 µm, amyloid.

Material examined: PAKISTAN: Punjab, Lahore, University of the Punjab, New Campus, Botanical Garden, 15th July, 2013, in groups, on ground, among grass, at 217 m (712 ft) a.s.l., N. Yousaf, PPK4 (Holotype), (LAH100000225). 300

A B C

D E Plate 184. Figs. A–E. Phallus ahmadii (PPK4). A. Off-white eggs of basidioma. B. Line drawing of basidiospores. C. LM photograph of basidiospores. D & E. Different views of basidiomata. Bar: = 1 cm for A, D & E, = 4 µm for B, = 11 µm for C. 301

Phallus galericulatus (Möller) Kreisel, Czech Mycol. 48(4): 275 (1996) (Plate 185)

= Itajahya galericulata Möller

Egg off white, sub-globose, outer surface relatively smooth, slightly encrusted with soil particles. Mature fruiting body solitary, up to 90 mm tall and 15 mm wide at the top and 25 mm at the base; attached to the ground by conspicuous base consists of branched, white, thin rhizomorphs, up to 15 mm high. Receptaculum bearing pileus and stipe; Receptaculum surface rugulose; Pileus sub-globose with round apex, up to 20 mm high × 8 mm in diam., having glebal mass; Calyptra present, disk like structure present at top. Gleba dark green, wet, sticky, off-white gelatinous material covering glebal portion from the top, strongly foetid. Pseudostipe off-white, hollow, spongy, porous, soft, uniform, up to 15 mm in diam. Volva ovoid, saccate, 30 mm in height, 25 mm in diam., prominent, off- white, medium thickness, texture soft, outer surface rough, encrusted with soil particles, membranous, gelatinous from inside.

Basidiospores ellipsoid, greenish, up to 4.0 × 2.5 µm, amyloid.

Material examined: PAKISTAN: Punjab, Lahore, University of the Punjab, New Campus, Botanical Garden, 18th July, 2013, solitary, on ground, among grass, at 217 m (712 ft) a.s.l., N. Yousaf, PPK5 (LAH100000226). 302

A B

C

Plate 185. Figs. A–C. Phallus galericulatus. A & B. Different views of basidioma (PPK5). C. LM photograph of basidiospores. Bar: = 0.8 cm for A & B, = 5 µm for C. 303

Phallus hadriani Vent., Mém. Inst. nat. Sci. Arts 1: 517 (1798) (Plate 186)

Egg stage up to 50 mm high and 30 mm in diam., subglobose to ovoid, sheathed by a tough, brown membrane. Mature fruiting body grayish to violet, up to 125 mm high, attached to the substratum by well developed thick branched rhizomorphs at the base. Receptaculum with off white reticulate pileus head and pseudostipe. Pileus slightly campanulate, having glebal mass, up to 30 mm in height, 25 mm in diam., cylindrical, narrowing at the top, hollow. Gleba olivaceous green wet, sticky, deliquescent, not much smelly. Pseudostipe off white, spongy, hollow, porous, up to 80 mm in height, 17–20 mm in diam., cylindrical, almost equal, firm, hard. Volva thick, texture soft, cup shaped, covering 1/3rd of the stipe, globose, grayish to violet with off white tones, outer surface relatively smooth, not encrusted with soil particles, lined with grayish to violet gelatinous and sticky material inside, up to 37 mm in height and 35 mm in diam.

Basidiospores cylindrical, smooth, hyaline, up to 4.0 × 2.0 µm.

Material examined: Pakistan, Khyber Pakhtunkhwa, Mansehra district, Ughi forest, 10th September 2011, on ground, at 3325 m a.s.l., PPK1 (LAH100000227); Chattar plains, 14th August, 2012, on slopes, gregarious, among grass, N. Yousaf, PPK15 (LAH100000228).

304

B

A E

C D F Plate 186. Figs. A–F. Phallus hadriani. A. a reticulate receptaculum surface. B. Eggs of basidiomata (PPK15). C. A mature basidioma (PPK15). D. Off white pseudostipe and purplish volva. E. Purplish egg of basidiomata (PPK1). F. A mature basidioma (PPK1). Bar = 0.5 cm for A, = 1.5 cm for B & C, 0.8 cm for D, E & F.

305

Phallus roseus Delile, Hist. Nat. 2:300, 1823. (Plate 187)

≡ Itajahya rosea (Delile) E. Fisch.

Egg off white to light pink, sub-globose, rough, membranous, up to 80 mm in diam. and 60 mm high. Basidiomata gregarious, up to 80 mm tall and 20 mm wide at the top and up to 45 mm at the base; attached to the substratum by white, thick, branched rhizomorphs. Receptaculum consists of pileus and pseudostipe. Pileus sub-globose, up to 20 mm high and 35 mm in dia., hollow, with sticky glebal mass. Gleba olivaceous turning grey with age, smell foetid when collected, sticky and wet. Pseudostipe off white to light pink, spongy, rough, hard, up to 30 mm in diam., tapering towards the pileus. Calyptra extended at the top of the pileus, irregular shaped, pinkish, up to 20 mm high. Volva up to 45 × 45 mm in diam., off-white, rough, membranous, gelatinous inside.

Basidiospores ellipsoid, greenish to olivaceous.

Material Examined: PAKISTAN: Punjab, Lahore, University of the Punjab, Quaid-e- Azam campus, 15th Aug. 2010, 10th July, 2011, gregarious, on ground, at 217 m (713 ft) a.s.l., 15th July 2012, 21st July 2013. N. Yousaf, PPK3 (LAH100000229), PPK26 (LAH100000230), PPK27 (LAH100000231), PPK28 (LAH100000232), PPK29 (LAH108210); Mianwali, 12th Aug. 2013, in groups, on bare ground, under Acacia sp. at 210 m (689 ft) a.s.l., A. R. Niazi, PPK17 (LAH290808); Bahawalpur, Lal Sohanra Park, gregarious, at 126 m (413 ft) a.s.l., N. Yousaf, PPK30 (LAH100000233).

Comments: Phallus roseus has previously been reported by Ahmad in 1952 as Itahjaya rosea from Pakistan. This species is adapted to arid to semi-arid regions in Pakistan. It has been reported previously from Bahawalpur state: Palla, Gunjranwala, Ladhar, Lahore, and Sheikhupura (Ahmad, 1952). It grows abundantly in groups among the grass. It is first record of this taxon from Mianwali.

306

E

Plate 187. Figs. A–E. Phallus roseus .A. A group of mature basidiomata and eggs. B. A mature basidioma with prominent calyptra at the top of pileus. C. Line drawing of a longitudinal section of an egg. D. Illustration of basidiospores. E. LM photograph of basidiospores. Bar: = 1.5 cm for A, = 1 cm for B, = 0.8 cm for C, = 4 µm for D, = 8 µm for E. 307

Phallus rubicundus (Bosc) Fr., Syst. mycol. (Lundae) 2(2): 284 (1823) (Plate 188) Egg off white, sub-globose, outer surface relatively smooth, turning greyish at maturity, encrusted with soil particles, 50 mm in diam. and 35 mm high. Mature fruiting body solitary or gregarious, up to 70 mm tall and 5–8 mm wide at the top and 6–12 mm at the base; attached to the ground by less numerous branched, white, thick rhizomorphs. Receptaculum bearing pileus and stipe; Receptaculum surface rugulose; Pileus campanulate, 16 mm high and 5–8 mm in diam., hollow, having glebal mass, operculum like structure present above pileus head at the top of receptaculum; operculum concave & globose from up ward & flattened from downward facing the pileus, 6–8 mm in diam.; Gleba purple turning purplish black with age, smell foetid when collected, soft, wet, sticky. Stipe yellowish to orange, hollow, spongy, porous, soft, not entirely enclosed by volva, broader and wider at the base (8.5– 10 mm in diam.) tapering upwards (6.5 mm in diam.). Volva ovoid, saccate, 20 mm in height, 12–15 mm in diam., prominent, well developed, off-white, medium thickness, texture soft, outer surface rough, slightly having smooth appearance, encrusted with soil particles, membranous, gelatinous from inside, remnants of volva absent on the stipe.

Material examined: PAKISTAN: Punjab, Lahore, University of the Punjab, New Campus, near Healy College of Commerce, 25th June, 2011, in groups, among grass, on ground, at 217 m (712 ft) a.s.l., N. Yousaf, PPK2 (LAH100000234).

Comments: P. rubicundus was reported by Ahmad in 1952 from Balakot. This is a second report of this species from Pakistan and first report from Lahore.

308

A B

C

Plate 188. Figs. A–C. Phallus rubicundus (PPK2). A & B. Different views of mature basidiomata. C. Line drawing of basidioma. Bar: = 1 cm for A–C.

309

4.1.9. Taxa belonging to Russulaceae

Russula pakistanica nom. prov. (Plate 189)

Etymology: on the name of the country it is being reported

Basidiomata hypogeous, 15 × 20 mm in diam., thickness up to 10 mm in diam., subglobose to irregular, lobed. Peridium attached to the glebal part, inseparable, ochraceous with off-white to pale yellow patches, staining reaction not observed. Odor not distinctive. Taste not recorded. Gleba off-white in fresh specimens, brown when mature, chambered.

Hymenium up to 130 µm broad, tramal hyphae up to 3.5 µm, Sub-hymenium composed of 2–3 tiers of isodiametric cells 3.0 (–11.6) µm in diam., up to 30 µm in diam. Basidia clavate, light greenish, up to 33 × 11 µm, with 2–4 sterigmate, up to 2.5 µm long. Cystidia clavate, 25 × 11 µm, Basidiospores light green, rounded, ornamented, verrucose, up to 8.5–9.5 µm in diam. including verrucae, up to 7.7–9.0 µm in diam. excluding verrucae, verrucae up to 1.25 µm long, amyloid.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Mankial Top, 2nd Sep. 2013, in groups, hypogeous, under Cedrus deodara, at 3822 m (12542 ft) a.s.l., Sana Jabeen, RPK10 (Holotype), (LAH100000235).

310

A B

C D E

F G H

Plate 189. Figs. A–H. Russula pakistanica (RPK10). A. Basidiomata. B. An overview of hymenium. C. Basidia and cystidia. D–H. Verrucose basidiospores. Bar: = 0.5 µm for A, = 35 µm for B, = 20 µm for C, = 13 µm for D, = 7.5 µm for E, = 2.5 µm for F, = 2.4 µm for G & H.

311

4.2. Taxonomy of Herbarium Gasteroid Taxa During this investigation, some gasteroid specimens were taken as a loan from LAH herbarium, Lahore, Pakistan for comparisons with recently collections. Descriptions and illustrations of some of the specimens are given here.

Calvatia craniiformis (Schwein.) Fr., Summa veg. Scand., Section Post. (Stockholm): 442 (1849) (Plates 190-191) Basidioma sub globose, tapering at the base, solitary, up to 75 mm broad × 70 mm high, brown, encrusted with the soil particles, apical portion lost; attached to the substratum by a well-developed mycelial cord, up to 20 mm high, dehiscence by irregular rupturing of the apical part exposing the glebal portion. Peridium double. Exoperidium brown, encrusted with soil particles outside, persistant. Endoperidium dull brown. Gleba brown, compact, cottony. Sub gleba present, greyish brown, chambered, up to 40 mm high.

Basidiospores globose to sub globose, smooth, thick walled, oil droplet present, dark brown, 5.4–6.5 µm in diam., sterigmal remnants absent from mounts, brownish in water mounts. Eucapillitium hyaline to light greenish brown, broken in to segments, septate, broken where septa present, up to 11.5 µm in diam., with wall thickness up to 3.3 µm, branched, straight to sub-undulate, pores present, numerous, medium sized. Paracapillitium absent. Exoperidium composed of hyaline, subglobose to elongated, thin walled hyphal elements, 40 × 30 µm. Endoperidium composed of hyaline, septate, rarely branched, tightly packed hyphae, up to 6 µm in diam.

Material examined: PAKISTAN: Khyber Pakhtunkhwa, Swat district, Kalam, 23rd Aug. 1952, S. Ahmad (LAH12546).

312

A B

C D

E F Plate 190. Figs. A–F. Calvatia craniiformis. A & B. Different views of basidioma. C, E–F. LM photographs of basidiospores. D. LM photograph of eucapillitial hyphae. Bar: = 1.2 cm for A & B, = 9.5 µm for C, = 11 µm for D, = 4 µm for E & F. 313

A B

C D

Plate 191. Fig. A–D. Calvatia craniiformis. A. Basidiospores. B. Exoperidial elements. C. Eucapillitial threads. D. Endoperidial hyphae. Bar: = 4.5 µm for A, = 12 µm for B & D, = 9 μm for C.

314

Calvatia cyathiformis (Bosc) Morgan, J. Cincinnati Soc. Nat. Hist. 12(4): 168 (1890) (Plates 192–193) Basidioma sub-globose to ob-pyriforme (turbinate), slightly tapering at the base forming pseudostipe, brown with violet tint, up to 100 mm broad × 150 mm high, solitary; attached to the substratum by small mycelial cord. Diaphragm absent. Dehiscence by irregular rupturing of the apical part exposing the glebal portion. Peridium double. Exoperidium intact at maturity, persistent, brown with age, form ridges and furrows at the apical portion. Endoperidium brown. Gleba brown, cottony and becoming powdery with age. Sterile base present.

Basidiospores globose to subglobose, thick walled, verrucose, brown, 5.0–8.0 µm in diam. including ornamentation, up to 4.6–6.6 µm in diam excluding ornamentation, verrucae up to 1.3 µm, sterigmal remnants absent from mounts, brownish in water mounts. Eucapillitium light brown, broken in to segments, septate, broken where septa present, up to 5 µm in diam., thin walled, branched, straight to sub-undulate, pores present, wall thickness up to 1 µm. Paracapillitium not observed. Exoperidium composed of thick walled, brown, irregular shaped hyphal elements. Endoperidium composed of brown, aseptate, branched, tightly packed hyphae.

Material examined: PAKISTAN: Punjab, Zafarwal, 8th Sep. 1964, in groups, on ground, S. Ahmad, CH13, (LAH19983).

Comments: This taxon is re described and illustrated to compare it with the collection of C. cyathiformis var. fragilis (CPK12), C. lilacina (CPK1), and C. pseudocyathiformis (CPK4) which are also described in this study.

315

A

B C

D E Plate 192. A–E. Calvatia cyathiformis. A–C. A young basidioma. D. Basidiospores. E. Eucapillitial hypha. Bar: = 2 cm forA–C, = 5.5 μm for D, = 11 μm for E. 316

A

B

C

Plate 193. A–C. LM photographs of Calvatia cyathiformis. A. Verrucose Basidiospores. B. Eucapillitial hypha. C. Close view of verrucose basidiopsores. Bar: = 11 µm for A & B, = 5.5 µm for C.

317

Disciseda cervina (Berk.) G.H. Cunningham, Proc. Linn. Soc. N.S.W. 52(3): 238 (1927) (Plates 194–196)

Basidiomata globose to sub-globose, brown, up to 20 mm in diam., hypogeous, later becoming exposed after maturity. Dehiscence by an apical pore, pore up to mm in diam. Peridium double. Exoperidium in the form of sandy covering around endoperidium, later peeling away exposing the upper part of spore case, leaving a cup like structure at the base. Endoperidium brown, papery, smooth. Gleba brown, pulverulent.

Basidiospores globose, verrucose, brown, 5.5–6.0 µm in diam. Eucapillitium brown, branched, aseptate to rarely septate, pores absent, up to 5 µm. Endoperidium composed of hyaline to brown, aseptate, branched, tightly packed hyphae, up to 5 µm.

Material examined: Pakistan: Punjab, Ladhar, Sheikhupura, in sandy soil, 25-08-1948, S. Ahmad (LAH).

Comments: It is characterized by brownish spore case embedded within an exoperidial cup. Basidiospores are apedicellate, round and verrucose. It was formerly described by S. Ahmad from Sheikhupura (1952). It is again being described here based on the same collection. D. candida has also been studied for comparison.

318

A

B

Plate 194. Figs. A & B. Disciseda cervina. Different views of basidiomata. Bar: = 0.7 cm for A & B.

319

A B

C

Plate 195. Figs. A–C. Disciseda cervina. A. Basidiospores. B. Peridial hyphae. C. Eucapillitial hyphae. Bar: = 1.8 µm for A, = 11 µm for B, = 7 µm for C.

320

A B

C D

Plate 196. Figs. A–D. LM photographs. A. Verrucose basidiospores of Disciseda cervina. B. Eucapillitial hyphae of D. cervina. C. Pedicellate basidiospores of D. candida. D. Eucapillitial hyphae of D. candida. Bar: = 9 µm for A, = 25 µm for B, = 8 µm for C, = 22 µm for D.

321

Lycoperdon pusillum Fr., Sylloge Fungorum VII: 487 (1888) (Plate 197) Basidiomata globose to subglobose, gregarious, off white to cream when young, brown with age, 15–30 mm broad × 10–30 mm in height; attached to the substratum by a mycelial cord, up to 10 mm high. Rhizomophs not recorded in the collection, heavily encrusted with soil particles and other debris material. Dehiscence by an apical pore; pore small up to 2 mm in diam. Peridium double. Exoperidium consists of orange to brown granules or warts in mature specimens, later peeling off from the surface in the form of sheet, mostly persistent. Endoperidium papery, light brown. Gleba light brown, cottony. Pseudocolumella absent. Sterile base absent.

Basidiospores globose, ornamented, verrucose, thick walled, brown, 4–5 µm in diam. including verrucae, spores brown in water mounts. Eucapillitium well developed, brown, aseptate, branched, dichotomous branching, subundulate, bulging at some points, branches with tapering ends, encrusted with some amorphous material, pores present, up to 6.7 µm in diam., (wall thickness up to 1.5 µm). Paracapillitium absent. Exoperidium not observed. Endoperidium composed of hyaline to light brown, branched, frequently septate, tightly packed hyphae, up to 7 µm in diam.

Material examined: PAKISTAN: Punjab, Ladhar, Sheikhupura, 10th Aug. 1952, on ground, S. Ahmad, LH10 (LAH181218).

Comments: S. Ahmad reported the taxon in 1939 from Lahore and Rawalpindi districts of Punjab. 322

A

B C

D Plate 197. Figs. A–D. Lycoperdon pusillum. A. Basidiomata. B. Basidiospores. C. Eucapillitial hyphae. D. Endoperidial hyphae. Bar: = 0.9 cm for A, = 2.8 µm for B, = 9 µm for C, = 15 µm for D.

323

Schizostoma mundkurii (S. Ahmad) Long & Stouffer [as 'mundkuri'], Mycologia 35(1): 27 (1943) (Plate 198)

Spore-case globose to subglobose, 18–35 × 14–30 mm. in diam., dehiscence by an irregular rupturing of the apical part; exoperidium off white with some brownish tone, persistent, peeling off in the form of scales at the stipe; endoperidium papyraceous maroon to dark brown. Stem up to 15 cm. long, 10 mm. thick, uniform, slightly tapering towards the base, longitudinally striated, scales present; scales off white to light brown, inserted on the under-side of the sporo case, attached to the substratum by a well developed base. Gleba umber brown, with purple tone, cottony to powdery with age;

Basidiospores sub globose, thick walled, dark brown, (4.2–) 6.0–9.5 µm in diam. Eucapillitium consists of isolated threads, orange brown, septate, thick walled, branched, rounded at the tips, up to 13 µm wide, wall thickness up to 3.8 µm. Peridium composed of hyaline, septate, branched, up to 11.8 µm wide. Stipe hyphae tightly packed, straight, septate, light brown, up to 9 µm wide.

Material examined: PAKISTAN: Punjab, Jhang, 7th Aug. 1952, on sandy soil, S. Ahmad, (LAH12509). 324

A B

C D E

F G Plate 198. Figs. A–G. Schizostoma mudkurii. A. Basidiomata. B. Basidiospores. C. Thick-walled eucapillitial hyphae. D. Perdial hyphae. E & G. LM photographs of eucapillitial hyphae. F. LM photograph of basidiospores. Bar: = 2 cm for A, = 4.5 µm for B, = 11 µm for C, = 22 µm for D & G, = 18 µm forE, = 12 µm for F.

325

Scleroderma flavidum Ellis & Everh., J. Mycol. 1(7): 88 (1885) (Plate 199) Basidiomata epigeous, up to 55 mm broad, 50 mm high, sub-globose, attached to the substratum by a thick mycelial cord, forming a pseudo-stipe; pseudo-stipe tough, 20 mm high × 20 mm broad. Rhizomorphs not observed. Peridium thin, <1mm thick. Off white to ochraceous; rough, hard, without scales. Dehiscence by tearing away the apical portion. Gleba compact, becoming pulverulent with age, greyish black.

Basidiospores globose, echinulate, completely reticulate, brown, 13.0–18.5 µm in diam. including ornaments, 10.6–14.4 µm in diam. excluding ornaments, echines up to 4 µm long. Tramal hyphae rarely septate, branched, hyaline, with few amorphous material encrusted, up to 6.6 µm wide. Exoperidium composed of hyaline, septate, branched hyphae, clamp connections present, up to 7 µm wide. Mesoperidium composed of aseptate, branched hyphae, with rounded tips, up to 7 µm wide. Endoperidium composed of aseptate, branched hyphae, up to 6.5 µm wide. Material examined: Pakistan, Khyber Pakhtunkhwa, Shogran, 25th July 1957, S. Ahmad (LAH11238).

326

A B

C

D E Plate 199. Figs. A–E. Scleroderma flavidum. A & B. Basidioma. C. Reticulate basidiospores. D. Exoperidial hyphae. E. Mesoperidial and Endoperidial hyphae. Bar: = 1 cm for A & B, = 5.5 µm for C, = 11 µm for D & E. 327

Tulostoma evanescens Long & S. Ahmad, Farlowia 3: 235 (1947) (Plate 200)

Spore case globose to subglobose, up to 05–15 × 04–12 mm diam., off-white to brown. Exoperidium in the form of off-white to brown granules over the surface, heavily encrusted with sand particles or plant debris. Endoperidium off white, smooth, with some remnants of exoperidial granules; papyraceous, tough. Mouth small, slightly tubular, up to 1 mm long. Socket slightly conspicuous. Gleba brown. Stipe 2–5 × 5–30 mm, off white to light brown, hollow, straight, tapering towards the base, rough, striated, exoperidium peeling off in the form of scales exposing the off white endoperidium, 1/5th of the portion buried in the soil, encrusted with sand particles. Volva absent.

Basidiospores globose to subglobose, smooth, hyaline, chestnut brown in water mounts, 3.8–5.0 µm in diam. Eucapillitium hyaline, thick walled, swollen at septa, branched, up to 6.4 µm in diam. Exoperidium not observed. Endoperidium composed of hyaline, thick walled, branched, septate, (joint like septa present), tightly packed hyphae up to 6.4 µm in diam. with wall thickness up to 3.5 µm. Stipe hyphae straight, septate, branched and tightly packed hyphae, encrusted with soil particles, up to 6 µm.

Material examined: Pakistan, Punjab, Ladhar, Sheikhupura, August, 1948, on sandy soil, S. Ahmad, TH2 (LAH23651).

328

A B

C

D E F

Plate 200. Figs. A–F. Tulostoma evanescens. A & B. Basidiomata. C. Basidiospores. D. Peridial hyphae. E. Eucapillitial hyphae. F. Stipe hyphae. Bar: = 1.9 cm for A & B, = .5 µm for C, = 15 µm for D, = 12 µm for E, = 20 µm for F.

329

Tulostoma volvulatum I.G. Borshch., 7: 189 (1865) (Plate 201)

Spore case globose to sub-globose, up to 18–25 × 10–20 mm diam., off-white. Exoperidium not much distinct, peeled off in the form of off-white granules over the surface, encrusted with sand particles or plant debris. Endoperidium off white, smooth, with some remnants of exoperidial granules; papyraceous, tough. Mouth short, slightly projected. Socket inconspicuous, appressed to the stem. Gleba cinamomum brown. Stipe 3–10 × 130 mm, off white to light brown, hollow, straight, tapering towards the base, rough, striated, exoperidium peeling off in the form of scales exposing the off white endoperidium, ½ of the portion buried in the soil, encrusted with sand particles. Volva present, up to 25 mm high.

Basidiospores subglobose to ellipsoid, thick walled, smooth, brown, 4.2–8.7 µm in diam. Eucapillitium brown, thick walled, aseptate to rarely septate, branched, up to 14 µm in diam., wall thickness up to 4.3 µm, tips rounded. Exoperidium not observed. Endoperidium composed of hyaline, branched aseptate, tightly packed hyphae, up to 6.6 µm. Stipe hyphae branched, straight, septate and tightly packed, up to 6 µm.

Material examined: Pakistan, Punjab, Chiniot, 11th Feb. 1965, on sandy soil, S. Ahmad, TH6, (LAH23956). 330

A B C

D E F Plate 201. Figs. A–F. Tulostoma volvuatum. A & B. Basidiomata. B. Endoperidial hyphae. C. Basidiospores. D. Eucapillitial hyphae. E. Stipe hyphae. Bar: = 2 cm for A & B, = 13 µm for C & F, = 6 µm for D, = 20 µm for E.

331

Tulostoma vulgare Long & S. Ahmad, Farlowia 3: 248 (1947) (Plate 202) Spore case globose to subglobose, up to 13–25 × 5–10 mm diam., dull brown to ochraceous brown, attached with the stipe. Exoperidium brown, darker towards the base, persistent, heavily encrusted with sand particles. Endoperidium light brown, rough, not exposed in mature specimens; papyraceous, tough. Mouth short, not raised, concolorous. Socket conspicuous. Gleba cinamomum brown. Stipe 35–55 × 3–6 mm, solid, sub-undulate to straight, striated, light brown, rough, exoperidium peeling off in the form of scales exposing the brown endoperidium. Base bulbous with well developed radiating root, up to 10 mm high.

Basidiospores globose to subglobose, slightly ellipsoid, thick walled, smooth, brown, 6.0–8.0 µm in diam. Eucapillitium brown, thick walled, aseptate to rarely septate, joint like septa present, branched, up to 7 µm in diam., wall thickness up to 2.8 µm. Exoperidium not observed. Endoperidium composed of hyaline, branched, aseptate, joint like septa when present, tightly packed hyphae, up to 6.6 µm. Stipe hyphae hyaline to light brown, straight, septate and tightly packed, up to 6 µm.

Material examined: Pakistan, Punjab, Ladhar, Sheikhupura, 25th Aug. 1948, on sandy soil, S. Ahmad, TH1 (LAH21360). 332

A B

C

D E F

Plate 202. Figs. A–F. Tulostoma vulgare. A & B. Basidiomata. C. Basidiospores. D. Eucapillitial hyphae. E. Endoperidial hyphae. F. Stipe hyphae. Bar: = 2 cm for A & B, = 6 µm for C, = 8.5 µm for D, = 16 µm for E & F.

333

4.3. Phylogeny of Gasteroid Fungi Phylogenetic analyses of taxa studied during this investigation is presented here genus wise.

334

Phylogenetic analysis of Arachnion and Disciseda (Fig. 1)

In present investigation, three (3) ITS-nrDNA sequence of Arachnion sp. (BPK59) and 2 sequences of Disciseda sp. (DPK1) were successfully amplified.

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank and literature (Larsson & Jeppson, 2008). The analysis involved 50 nucleotide sequences, 49 in group and 1 out group sequence. Out of 49 in group sequences, 31 belong to genus Lycoperdon, 7 of Arachnion, 5 of Disciseda, 4 of Calvatia, and 2 of Bovista. One (1) sequence of Mycenastrum corium (DQ112628) was taken as an out group.

Final alignment matrix was 697 nucleotide long, which included 462 conserved, 229 variable and 150 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All ambiguous positions were removed for each sequence pair.

Phylogram represents the gasteroid taxa of Agaricaceae, with distinct status of genus Arachnion within the family. 335

92 Lycoperdon turneri DQ112594 54 Lycoperdon umbrinum DQ112591 Lycoperdon lambinonii DQ112576 Lycoperdon cretaceum DQ112598 Lycoperdon altimontanum DQ112588 67 Lycoperdon excipuliforme DQ11259 Lycoperdon niveum DQ112569 EU833663 Lycoperdon frigidum DQ112559 Lycoperdon decipiens DQ112583 54 Lycoperdon rimulatum EU833664 DQ112578 Lycoperdon lividum DQ112600 Lycoperdon nigrescens DQ112577

58 Lycoperdon rupicola DQ112581 51 Lycoperdon dermoxanthum DQ112579 Lyco p e rd o n Lycoperdon subumbrinum JN572906 Lycoperdon muscorum DQ112604 Lycoperdon ericaeum DQ112606 Lycoperdon radicatum DQ112608 Bovistella japonica BPK78 52 97 Bovistella_radicataAJ237624 69 Lycoperdon utriforme DQ112607 Lycoperdon subcretaceum JN572908

100 VascellumpratenseDQ112554 Lycoperdon pratense DQ112556 Lycoperdon norvegicum DQ112631 DQ112632 94 Lycoperdon perlatum AJ237627 Lycoperdon caudatum DQ112633 Lycoperdon pyriforme DQ112558

98 Bovista paludosa AJ237630 Bo vista Bovista graveolens DQ112618

51 Arachnion olivoflavus BPK59 100 Arachnion olivoflavus BPK59 Arachnion olivoflavus BPK59

87 Arachnion album HQ235049 Ara chnio n Arachnion album EU833649 100 Arachnion album HQ235046 Arachnion album HQ235044 55 Calvatia craniiformis KF551248 Calvatia fenzlii FJ772413 Ca lva tia 99 AJ617492 52 Calvatia pachydermica EU833653 DQ11262

72 Disciseda candida DQ112626 Discise d a Disciseda candida EU833654 Disciseda hyalothrix DPK1 100 Disciseda hyalothrix DPK1

Mycenastrum corium DQ112628 Out g ro up

0.01 Fig. 1. Molecular phylogenetic analysis of Arachnion and Disciseda by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –4493.94). ■ indicate species collected from Pakistan. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree.

336

Phylogenetic analysis of Battarrea spp. (Fig. 2) In present investigation, two ITS-nrDNA sequences from one specimen of Battarrea phalloides (BPK69) collected during this study were produced.

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank. The analysis involved 23 nucleotide sequences, 22 in group and 1 out group sequence. Out of 22 in group sequences, 8 belong to genus Battarrea, 7 of , 3 of , and 2 of Bovista, Leucoagaricus and Tulostoma each and one (1) sequence of Mycenastrum corium. One (1) sequence of Geastrum triplex (KC581978) was taken as an out group.

Final alignment matrix was 780 nucleotide long, which included 344 conserved, 401 variable and 259 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions with less than 0% site coverage were eliminated. That is, fewer than 100% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 539 positions in the final dataset.

In this analysis, two clades were resolved, i.e. I and Clade II. Clade I consists of taxa belonging to genera Bovista, Lepiota, Leucoagaricus, Mycenastrum and Tulostoma. Clade II consists of sequences of B. phalloides (= B. stevenii) from different countries.

337

53 EU081959 Lepiota cristata EU081948 100 Lepiota cristata EU081945 Lepiota cristata JN944090 Lepiota boudieri FJ998388 Lepiota lilacea FJ998394

86 100 Lepiota lilacea GQ203822 CLADE I 78 Leucoagaricus griseodiscus GQ329059 Leucoagaricus bresadolae GQ329047

75 80 Mycenastrum corium DQ112628

66 Bovista limosa DQ112614 100 Bovista pusilla EU915073 Tulostoma kotlabae DQ112629 57 Tulostoma squamosum DQ415732 Battarrea phalloides BPK69 Battarrea stevenii DQ184689 Battarrea phalloides BPK69

99 Battarrea stevenii AF215658 CLADE II Battarrea stevenii AF215648 63 Battarrea phalloides AF215657 Battarrea phalloides AF215656 Battarrea stevenii AF215650

Geastrum triplex KC581978 Out g ro up

0.05 Fig. 2. Molecular phylogenetic analysis of Battarrea spp. and related taxa by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –3190.61). ■ indicate species collected from Pakistan. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree. 338

Phylogenetic analysis of Bovista spp. (subgenera Bovista and Globaria)

(Figs. 3 & 4) In present investigation, ITS-nrDNA regions of 13 species were successfully amplified and twenty (29) sequences were generated. This included 7 sequences of B. himalaica, 5 of B. pakistanica, 3 each of B. ahmadii, B. plumbea, 2 each of B. aestivalis, B. oblongispora and 1 each of B. areolata, B. flavescens, B. kreiselii, B. nigrescens var. rectapedicellata, B. promontorii, B. pseudonigrescens and Bovista sp.

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank and literature (Larsson & Jeppson, 2008, Yousaf et al., 2013a). The analysis involved 50 nucleotide sequences, 49 in group and 1 out group sequence. Among 49 in group sequences 22 sequences belong to subgenus Bovista, 21 belong to subgenus Globaria, while 6 sequences did not follow any subgenus based clustering. Mycenastrum corium (DQ112628) was taken as an out group.

Final alignment matrix was 686 nucleotide long, which included 483 conserved, 189 variable and 92 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 531 positions in the final dataset.

In this analysis, two clades were resolved. Sub clade I and II of Clade I represent two subgenera Bovista and Globaria of Bovista, respectively. Subgenus Clade II only consists of 5 sequences of B. pakistanica.

339

Bovista plumbea BPK54 68 Bovista plumbea Pak2 94 Bovista plumbea Pak1 Bovista plumbea DQ112613 Bovista sp. GPK1 Bovista areolata MPK4 Bovista graveolens DQ112618 Bovista nigrescens DQ112612 Bovista kreiselii BPK10

58 Bovista pseudonigrescens GPK28 54 Bovista nigrescens var. rectapedicellata BPK27 Sub Clade I 91 Bovista cretacea DQ112610 Bovista cretacea DQ112611 CLADE I

58 Bovista limosa EU915075 Bovista limosa EU915074 99 Bovista limosa DQ112615 Bovista paludosa DQ112609 65 Bovista pusilla DQ112614

98 Bovista pusilla EU915072 76 Bovista pusilla EU915073 Bovista tomentosa DQ112616 75 Bovista minor DQ112617

Sub Clade II 94

CLADE II 95 Bovista furfuracea DQ112622 Mycenastrum corium DQ112628

0.01 Fig. 3. Molecular phylogenetic analysis of subgenus Bovista by the Maximum Likelihood (ML) method (ML) based on ITS-nrDNA sequences. (Ln likelihood = –2341.67). ■ indicate species studied and sequenced. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree.

340

65 Sub Clade I

Bovista himalaica BPK47 Bovista himalaica Pak1 Bovista himalaica Pak3 57 Bovista himalaica Pak2 Bovista himalaica Pak4 Bovista himalaica BPK51 Bovista himalaica Pak5 Bovista oblongispora BPK13 53 Bovista oblongispora BPK74 94 Bovista promontorii BPK58 CLADE I

Bovista promontorii DQ112621 Sub Clade II Bovista ahmadii BPK35 99 Bovista ahmadii BPK35 Bovista ahmadii BPK52 Bovista aestivalis EU833650 Bovista flavescens BPK83 Bovista aestivalis FJ438477 Bovista aestivalis BPK33 55 Bovista aestivalis BPK70 Bovista polymorpha AJ237613 Bovista aestivalis DQ112620 Bovista pakistanica BPK75 Bovista pakistanica BPK76

Bovista pakistanica BPK71 CLADE II 95 Bovista pakistanica BPK73 Bovista pakistanica BPK72 Bovista furfuracea DQ112622 Mycenastrum corium DQ112628

0.01

Fig. 4. Molecular phylogenetic analysis of subgenus Globaria by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –2341.67). ■ indicate species studied and sequenced. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree.

341

Phylogenetic analysis of Calvatia spp. (Fig. 5) In present investigation, ITS-nrDNA regions of 3 species were successfully amplified and four sequences were generated. This included 2 sequences of Calvatia lilacina (CPK1) and 1 each of C. pseudocyathiformis (CPK4) and C. cyathiformis var. fragilis (CPK12).

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank. The analysis involved 34 nucleotide sequences. This included 33 in group and 1 out group sequence. All of 33 in group sequences belong to genus Calvatia. One (1) sequence of Mycenastrum corium (DQ112628) was taken as an out group.

Final alignment matrix was 698 nucleotide long, which included 495 conserved, 181 variable and 103 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 34 nucleotide sequences. All positions with less than 0% site coverage were eliminated. That is, fewer than 100% alignment gaps, missing data, and ambiguous bases were allowed at any position.

In the phylogram, distinct status of fifteen (15) Calvatia spp. was tried to resolve. 342

AJ486864

85 AJ486868 AJ486867

88 AJ486866 Calvatia cyathiformis var. cyathiformis AJ486870 55 AJ486872 84 82 AJ486865 CPK12 Calvatia cyathiformis var. fragilis CPK4 Calvatia pseudocyathiformis 97 53 AJ486959 81 AJ486958 AJ486960 Calvatia fragilis 60 82 AJ486957

66 AJ486870 63 AJ486871 CPK1_1 Calvatia lilacina 100 CPK1_2

EU833652 Calvatia cf. leiospora

100 AJ486966 Calvatia chilensis AJ486965

EU833651 Calvatia bicolor 54 FJ772413 Calvatia fenzlii EU833653 Calvatia pachydermica

79 EF190316 97 AJ617492

95 EF190318 Calvatia gigantea EF190317 85 EF190313 68 EF190314

DQ112625 Calvatia craniiformis DQ112624 Calvatia candida JQ734547 Calvatia holothurioides 84 AF485064 Calvatia rubroflava DQ112628 Mycenastrum corium (out group)

0.01 Fig. 5. Molecular phylogenetic analysis of Calvatia spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –2439.95). ■ indicate species collected from Pakistan. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree.

343

Phylogenetic analysis of Lycoperdon spp. (subgenera Aioperdon, Bovistella, Lycoeprdon, Vascellum) (Fig. 6)

In present investigation, ITS-nrDNA regions of 12 species were successfully amplified and twenty (20) sequences were generated. This included 6 (six) sequences of L. pyriforme, 3 of L. lahorense, 2 of L. perlatum and 1 each of L. curtisii var. ovalisporum, L. gilgitii, L. japonicum, L. lahorense var. parvasporum, L. pratense var. flavum, L. olivoflavum, Lycoperdon sp. BK1, Lycoperdon parvasporum, and L. utriforme.

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank and literature (Larsson & Jeppson, 2008). The analysis involved 43 nucleotide sequences. Final alignment matrix was 684 nucleotide long, which included 518 conserved, 156 variable and 91 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding.

In this analysis, four clades were resolved. Each clade represents a subgenus of Lycoperdon, i.e. Apioperdon, Bovistella, Lycoperdon, and Vascellum. Clade of subgenus Vascellum is further divided in two sub clades. Each clade contains sequenes of taxa studied during this investigation.

344

75 Lycoperdon pratense DQ112555 Lycoperdon pratense AJ237625 52 Lycoperdon pratense DQ112556 Lycoeprdon pratense DQ112554

Lycoperdon pratense var. flavum VPK8 Sub Clade I

84 Lycoperdon gilgitii VPK9 Lycoperdon olivoflavus VPK2 Lycoperdon sp. BK1 Vascellum intermedium EU833667 Subgenus Vascellum 98 Lycoperdon lahorense VPK4 58 Lycoperdon lahorense VPK6 Lycoperdon lahorense VPK6 Vascellum curtisii HQ235045 58 Vascellum pratense FJ481033 Sub Clade II Lycoperdon curtisii var. ovalisporum VPK1 65 Vascellum curtisii HQ235043 Lycoperdon lahorense var. parvasporum VPK3 52 Lycoperdon wrightii HM237177 Lycoperdon marginatum DQ112632 Lycoperdon norvegicum DQ112631

94 Lycoperdon parvasporum LPK46 90 Lycoperdon perlatum DQ112630 75 Subgenus Lycoperdon Lycoperdon perlatum KF551249 99 Lycoperdon perlatum AJ237627 98 Lycoperdon perlatum LPK73 79 Lycoperdon perlatum LPK82 Lycoperdon radicatum DQ112608 Lycoperdon japonicum BPK78 98 AJ617490 84 Subgenus Bovistella Lycoperdon utriforme DQ112607 63 83 Lycoperdon utriforme CPK5 Handkea utriformis EU833659 Lycoperdon pyriforme LPK10 Lycoperdon pyriforme LPK14A Lycoperdon pyriforme LPK62 Lycoperdon pyriforme AJ237619 100 Lycoperdon pyriforme LPK48 Subgenus Apioperdon Lycoperdon pyriforme AY854075 Lycoperdon pyriforme AJ237620 Lycoperdon pyriforme AJ237618 Lycoperdon pyriforme LPK17 Lycoperdon pyriforme DH2 Mycenastrum corium DQ112628

0.01 Fig. 6. Molecular phylogenetic analysis of subgenus Apioperdon, Bovistella, Lycoperdon and Vascellum of genus Lycoperdon by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –2341.67). ■ indicate species studied and sequenced. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree.

345

Phylogenetic analysis of Lycoperdon spp. (subgenus Utraria) (Fig. 7) In present investigation, ITS-nrDNA regions of fourteen (14) species were successfully amplified and twenty seven (27) sequences were generated. This included seven (7) sequences of L. lineum, 3 of L. excipuliforme, and 2 each of L. albiceratum, L. dermoxanthum var. album, L. dermoxanthum var. flavum, L. sclerocystis and 1 each of L. altimontanum, L. aurea-brunneum, L. mammiforme, L. mammiforme var. magnasporum, L. niveum, L. pseudoniveum, L. sharanense, Lycoperdon sp. (MPK5), Lycoperdon sp. (LPK47).

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank and literature (Larsson & Jeppson, 2008). The analysis involved 66 nucleotide sequences. Among these, 65 are in group sequences belonging to Lycoperdon subgenus Utraria. One (1) sequence of Mycenastrum corium (DQ112628) was taken as an out group.

Final alignment matrix was 749 nucleotide long, which included 572 conserved, 189 variable and 85 parsimony informative sites.

Phylogenetic tree was constructed using Maximum Likelihood method based on Jukes and Cantor model. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. In this analysis, all the species belonging to subgenus Utraria were included. 346

Lycoperdon excipuliforme LPK80 Lycoperdon excipuliforme LPK58 Lycoperdon sp.1 MPK5 Lycoperdon sp.2 LPK47 Lycoperdon albiceratum MPK2 Lycoperdon albiceratum LPK37 Lycoeprdon excipuliforme LPK56 Lycoperdon excipuliforme DQ112590 Lycoperdon altimontanum DQ112588 60 65 Lycoperdon altimontanum LPK3 Lycoperdon lineum BPK45 Lycoperdon lineum BPK45 87 Lycoperdon lineum LPK19 Lycoperdon lineum LPK14 Lycoperdon lineum BPK68 Lycoperdon lineum BPK68 Lycoperdon lineum LPK19 Lycoperdon pseudoniveum LPK2 Lycoperdon sharanense LPK59

76 Lycoperdon cretaceum DQ112598 88 Lycoperdon cretaceum DQ112597

99 Lycoperdon sclerocystis LPK23 Lycoperdon sclerocystis LPK35

84 Lycoperdon mammiforme DQ112567 65 Lycoperdon mammiforme AJ237621 87 Lycoperdon mammiforme LPK24 Lycoperdon mammiforme var. magnasporum LPK55 Lycoperdon niveum DQ112569

87 Lycoperdon niveum LPK61 61 Lycoperdon niveum DQ112571 Lycoperdon pulcherrimum EU833663 Lycoperdon decipiens DQ112583 Lycoperdon rimulatum EU833664

75 Lycoperdon frigidum DQ112563 Lycoperdon frigidum DQ112559 Lycoperdon atropupureum DQ112586 56 Lycoperdon molle DQ112565

60 Lycoperdon molle EU833662 Lycoperdon molle DQ112566 Lycoperdon lambinonii DQ112575 Lycoperdon lambinonii DQ112576

57 Lycoperdon umbrinum DQ112592 Lycoperdon umbrinum DQ112591 87 Lycoperdon turneri DQ112596 90 Lycoperdon turneri DQ112594 Lycoperdon lividum DQ112599 99 Lycoperdon lividum DQ112600 Lycoperdon echinatum DQ112578 Lycoperdon nigrescens DQ112577

97 Lycoperdon subumbrinum DQ112601 75 Lycoperdon subumbrinum JN572906 Lycoperdon aurea-brunea LPK1

98 Lycoperdon ericaeum DQ112605 51 Lycoperdon ericaeum DQ112606 Lycoperdon muscorum DQ112604 98 Lycoperdon muscorum JN572904

100 Lycoperdon rupicola DQ112580 Lycoperdon rupicola DQ112581 50 Lycoperdon dermoxanthum DQ112579 55 69 Lycoperdon dermoxanthum var. album BPK36 91 Lycoperdon dermoxanthum var. album BPK36 98 Lycoperdon dermoxanthum var. flavum BPK31 53 Lycoperdon dermoxanthum var. flavum BPK31 Lycoperdon caudatum DQ112633 Lycoperdon pusillum AB067724 Mycenastrum corium DQ112628 Fig. 7. Molecular phylogenetic analysis of Lycoperdon spp. of subgenus Utraria by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –3127.98). ■ indicate species from Pakistan.

347

Phylogenetic analysis of Podaxis spp. and related taxa (Fig. 8) In present investigation, ITS-nrDNA region from two collection of Podaxis pistillaris (PPK6, GPK33) was successfully amplified and 1 sequence from each specimen was generated.

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank. The analysis involved 34 nucleotide sequences. Among 33 in group sequences, 8 sequences belong to Lepiota and Podaxis each, 4 each of Lycoperdon and Macrolepiota, 3 of Calvatia, 2 of Mycenastrum and Bovista and 1 each of Disciseda and Holocotylon. One (1) sequence of Geastrum triplex (KC581978) was taken as an out group.

Final alignment matrix was 818 nucleotide long, which included 402 conserved, 369 variable and 253 parsimony informative sites.

Phylogenetic tree was constructed using Maximum Likelihood method based on Jukes and Cantor model. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

348

100 Lepiota cristata JN944090 95 Lepiota cristata EU081948 Lepiota cristata AF391027 Lepiota castaneidisca AF391063 99 Le p io ta 100 Lepiota castaneidisca AF391058 90 Lepiota hymenoderma AF391028 Lepiota coloratipes KC900377 100 Lepiota coloratipes KC819622 Podaxis pistillaris PPU85336 Podaxis pistillaris GPK33 98 Podaxis pistillaris PPK6 83 Podaxis pistillaris DQ311082 Po d a xis Podaxis pistillaris DQ311086 Podaxis pistillaris AM234060 Podaxis pistillaris AF059285 91 Podaxis pistillaris GQ249882

100 Mycenastrum corium EU833666 Mycenastrum Mycenastrum corium DQ112628

100 Macrolepiota clelandii AY083201 Macrolepiota clelandii AY083195 Macrolepiota 78 Macrolepiota neomastoidea AF482845 100 Macrolepiota neomastoidea KF551251

62 Lycoperdon rimulatum EU833664

98 Lycoperdon pulcherrimum EU833663 Lyco p e rd o n Lycoperdon niveum DQ112563 71 78 Lycoperdon frigidum DQ112559

Holocotylon brandegeeanum EU8336 Holocotylon

96 Bovista paludosa AJ237630 Bo vista 88 Bovista plumbea JX183695 Disciseda candida EU833654 Discise d a Calvatia pachydermica EU833653

94 Calvatia gigantea EF190314 Ca lva tia 90 Calvatia gigantea EF190316

Geastrum triplex KC581978 Out g ro up

0.05 Fig. 8. Molecular phylogenetic analysis of Podaxis and related taxa by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –5271.87). ■ indicate species from Pakistan. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree. 349

Phylogenetic analysis of Tulostoma spp. (Fig. 9) In present investigation, ITS-nrDNA regions from three Tulostoma speices (TPK1, TPK2, TPK8) were successfully amplified and 1 sequence was generated from each specimen.

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank and literature (Caffot et al., 2011). The analysis involved 17 nucleotide sequences, 16 in group and one out group sequence. All in group sequences belong to genus One (1) Tulostoma. Lycoperdon perlatum (AY264919) was taken as an out group.

Final alignment matrix was 722 nucleotide long, which included 387 conserved, 289 variable and 160 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. 350

100 Tulostoma domingueziae HQ667594 Tulostoma domingueziae HQ667593 Tulostoma squamosum TPK2 59 99 Tulostoma squamosum DQ415732 Tulostoma melanocyclum EU784435 99 100 Tulostoma melanocyclum EU784436 Tulostoma sp HQ667596 100 Tulostoma sp HQ667595 53 Tulostoma morenoii TPK1 Tulostoma fimbriatum TPK8 Tulostoma kotlabae DQ112629 Tulostoma brumale EU784433

100 Tulostoma beccarianum AF097752 97 Tulostoma brumale EU784434 Tulostoma xerophilum HQ667592 50 Tulostoma niveum EU784437 Lycoperdon perlatum AY264919

0.05

Fig. 9. Molecular phylogenetic analysis of Tulostoma spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –3838.22). ■ indicate species from Pakistan. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree

351

Phylogenetic analysis of Cyathus spp. (Fig. 10) In present investigation, ITS-nrDNA regions two Cyathus spp. collected were successfully amplified and two sequences were generated. This included 1 sequence each of Cyathus olla (CPK36) and C. pakistanicus (CPK10).

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank and literature (Zhao et al., 2007). The analysis involved 32 nucleotide sequences. This included 29 in group sequences and 3 out group sequences. All of 29 in group sequences belong to Cyathus. Among three out group sequences two belong to (DQ463357, CA1) and 1 of Nidula (DQ463358).

Crucibulum sp. (CA1) was collected from Harvard Arboratum, Forest hills, MA, USA and its ITS-nrDNA sequence was generated and included in the study as out group.

Final alignment matrix was 745 nucleotide long, which included 402 conserved, 334 variable and 243 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All ambiguous positions were removed for each sequence pair. In this analysis, two clades were produced, i.e. Clade I and Clade II. Cyathus olla (CPK36) falls in Clade I and C. pakistanicus falls in Clade II. 352

65 Cyathus olla KC989088 Cyathus olla DQ463345 Cyathus olla CPK36 63 Cyathus olla DQ463343 Cyathus hookeri DQ463346 Cyathus colensoi DQ463344 Cyathus hookeri KC005989

99 Cyathus jiayuguanensis DQ463341 CLADE I 68 Cyathus africanus var. latisporus DQ463342 Cyathus africanus DQ463340 69 Cyathus africanus DQ463347 Cyathus berkeleyanus DQ463355 Cyathus pallidus DQ463356 67 Cyathus gansuensis DQ463348 100 Cyathus gansuensis KC869661 100 59 Cyathus crassimurus DQ463350 51 Cyathus crassimurus NR119587 98 Cyathus triplex DQ463353 Cyathus setosus DQ463349

99 Cyathus annulatus DQ463351 Cyathus annulatus NR119588 63 99 Cyathus renweii DQ463352 CLADE II 99 Cyathus renweii NR119589

87 Cyathus striatus EU784195 Cyathus striatus DQ486697 Cyathus pakistanicus CPK10 Cyathus stercoreus DQ463354 100 Cyathus stercoreus FJ478125 99 Cyathus stercoreus EU784193 Nidula niveotomentosa DQ463358 Crucibulum sp. CA1 100 Crucibulum laeve DQ463357

0.02

Fig. 10. Molecular phylogenetic analysis of Cyathus spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –4045.76). ■ indicate species sequenced for this study. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree.

353

Phylogenetic analysis of genus Astraeus (Fig. 11) In present investigation, two ITS-nrDNA sequences of Astraeus hygrometricus (APK2 and APK3) were generated.

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank and from published literature (Phosri et al., 2007, 2013; Fangfuk et al., 2010a). The analysis involved 34 nucleotide sequences, 33 in group and 1 out group sequence. All of 33 in group sequences belong to genus Astraeus. One (1) sequence of was taken as an out group.

Final alignment matrix was 884 nucleotide long, which included 467 conserved, 368 variable and 227 parsimony informative sites.

In phylogram, branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 382 positions in the final dataset.

In this analysis, nine clades were resolved. Each clade represents one Astraeus sp. Pakistani collections of A. hygrometricus form a clade with two other sequences of same species from France. 354

75 AB535105_Japan

99 AB507398_Japan AB535106_Japan 87 A. koreanus 89 AB507407_Japan AB507399_Japan 66 99 AB535107_Japan AJ629408_Spain A. telleriae 96 80 AJ629404_Greece AJ629398_USA AJ629402_USA A. smithii 64 97 AJ629403_USA AJ629399_USA

60 APK2_Pakistan APK3_Pakistan 70 A. hygrometricus 73 AJ629394_France 88 AJ6294064_France AB535110_Japan AB507402_Japan 99 AB535112_Japan Astraeus sp. 84 AB507396_Japan 90 AB535114_Japan

100 AJ629880_Thailand A. odoratus AJ629875_Thailand

74 EU718088_no data 96 AJ629393_Canary Islands A. pteridis AJ629407_USA 63 AJ629392_Mexico

95 AJ629409_USA A. morganii 94 AJ629410_USA

63 AJ629381_Thailand 9AB507404_Thailand A. asiaticus 100 AJ629386_Thailand AJ629382_Thailand

AJ629886 Scleroderma verrucosum (Out Group)

0.02 Fig. 11. Molecular phylogenetic analysis of Astraeus spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –1634.59). ■ indicate species collected from Pakistan. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree.

355

Phylogenetic analysis of Rhizopogon spp. (Fig. 12) In present investigation, out of 5 Rhizopogon collections, ITS-nrDNA regions of 3 species were successfully amplified and three sequences were generated. This included one sequence each of R. flavescens, R. himalayensis, and R. roseolus. Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank. The analysis involved 33 nucleotide sequences comprising 32 in group and 1 out group sequence. All of 32 in group sequences belong to Rhizopogon. One (1) sequence of clavipes (JF899572) was taken as an out group.

Final alignment matrix was 679 nucleotide long, which included 484 conserved, 183 variable and 108 parsimony informative sites.

In phylogenetic tree, branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding.

In this analysis, three clades were resolved. These clades represent different sections of Rhizopogon based on morphology, i.e. Clade I (Section Rhizopogon) and Clade II (Section Vilosulli) and Clade II (Section Amylopogon). All of the sequences of taxa studied falls in section Rhizopogon.

356

Rhizopogon roseolus AJ810051 68 Rhizopogon roseolus AM084707 99 Rhizopogon roseolus AJ810052 86 Rhizopogon roseolus RPK13 KF482486 Rhizopogon mohelnensis AJ810039

90 75 Rhizopogon rubescens JX907816 99 Rhizopogon rubescens DQ068965 CALDE I (Rhizopogon) Rhizopogon flavescens RPK14 58 94 Rhizopogon himalayensis RPK8 Rhizopogon luteorubescens GQ267482 98 51 Rhizopogon pseudoroseolus KC152203 93 Rhizopogon pseudoroseolus KC152205 Rhizopogon vulgaris JN858081

50 95 Rhizopogon abietis KC346843

99 Rhizopogon hawkerae KC306745 Rhizopogon pseudovillosus KC306764

96 HQ385852 82 95 Rhizopogon ochraceisporus AF071439 Rhizopogon vesiculosus HQ385854 CALDE II (Villosuli) Rhizopogon zelleri KC306760 53 63 Rhizopogon villescens KC306767

75 Rhizopogon parksii KC306761

81 Rhizopogon subareolatus KC306765 67 Rhizopogon colossus KC306752

99 Rhizopogon ellenae AF224276 Rhizopogon ellenae AH011350

88 Rhizopogon subpurpurasce AF062929 99 Rhizopogon milleri NR_119445 CALDE III (Amylopogon) Rhizopogon salebrosus NR_119438

86 Rhizopogon brunsii AY971841 100 Rhizopogon brunsii AY971839 Suillus cavipes JF899572

0.01 Fig. 12. Molecular phylogenetic analysis of Rhizopogon spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –2941.04). indicate species collected from Pakistan. GenBank accession number/Collection number are given at the ends. Bootstrap values = or >50 % are cited in the tree.

357

Phylogenetic analysis of Pisolithus spp. (Fig. 13) In present investigation out of three Pisolithus spp. described, ITS-nrDNA region of one species (P. flavus/ PPK11) was successfully amplified and one sequence was generated. All the other collections did not produce any results even after repeated extractions and amplification. Some of them produced noisy sequences, therefore excluded from the analysis.

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank. The analysis involved 33 nucleotide sequences, 32 in group and 1 out group sequence. Out of 32 in groups sequences, 6 sequences belong to P. albus complex, 2 each of P. abditus, P. arhizus, P. aurantioscabrosus, P. calongei, P. capsulifer, P. marmoratus, P. microcarpus, P. orientalis, P. tinctorious, Pisolithus sp. 3, Pisolithus sp. 8, Pislothus sp. 10 and one (1) sequence belongs to each P. indicus, and P. flavus (PK11). One (1) sequence of (GU373495) was taken as an out group.

Final alignment matrix was 771 nucleotide long, which included 282 conserved, 438 variable and 320 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 344 positions in the final dataset.

In this analysis, three clades were resolved, i.e. Clade I, Clade II and Clade III. Topology of the phylogenetic tree is consistent with Phosri et al., 2012 and Martin et al., 2013. 358

FR748122 52 FR748123 FR748121 72 P. albus complex AF228654 81 AF228656 64 AF228655 61 PPK11_Pak P. flavus * AF374661 Pisolithus sp. 8 (Phosri et al., 2012) AF374708 99 70 AF004735 P. microcarpus 100 AF228657 AF374624 CLADE I Pisolithus sp. 10 (Phosri et al., 2012) 73 AF374646 FR748119 P. abditus 100 FR748120 FR748135 P. capsulifer 100 FR748137 FR748128 98 P. arhizus FR748132 FR748143 98 P. orientalis FR748144 99 AF143233 P. tinctorious 100 AF143234 AY756113 P. indicus AF415227 CLADE II P. aurantioscabrosus 100 AF415226 AF004733 99 P. marmoratus AF374719 HE578141 97 P. calongei CLADE III HE578141 97 AF228641 Pisolithus sp. 3 (Phosri et al., 2012) 99 AF228642 GU373495 Suillus luteus (outgroup)

0.05

Fig. 13. Molecular phylogenetic analysis of Pisolithus spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –2393.85). * indicate species collected from Pakistan. Bootstrap values = or >50 % are cited in the tree.

359

Phylogenetic analysis of Scleroderma spp. (Fig. 14) Out of five different Scleroderma spp. which were collected and described during this study, only one collection of one Scleroderma sp. viz., S. bovista (SPK1) gave successful amplification and produced reliable sequence. Other collections were either not successful at DNA extraction or produced noisy sequences after PCR, therefore excluded from the analysis.

However, three other collections were obtained from three different sites of USA. One ITS-nrDNA sequence from each of these specimens was produced for their identification. These are S. areolatum (SDH2 from USA), S. citrinum (SHA1 from Arnold Arboretum, Harvard University, USA), and S. septentrionale (SHJ1, USA).

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank and following literature (Phosri et al., 2009; Nouhra et al., 2012). The analysis involved 39 nucleotide sequences. This included 38 in group and 1 out group sequence. Out of 39 in group sequences, 5 belong to each S. bovista, S. septentrionale, 4 to S. citrinum, 3 each of S. areolatum, S. bermudense, S. sinnamariense, S. verrucosum, 2 of S. cepa, S. dictyosporum, S. meridionale, S. michiganense, S. patagonicum, S. polyrhizum each. One (1) sequence of Pisolithus arhizus (FM213365) was taken as an out group.

Final alignment matrix was 758 nucleotide long, which included 400 conserved, 352 variable and 279 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 411 positions in the final dataset.

In this analysis, topology of phylogenetic tree is consistent with Phosri et al., 2009 and Nouhra et al., 2012. Most of the Scleroderma spp. grouped according to their similar basidiospores morphology. 360

99 JX030276 57 EU819517 SPK1 S. bovista 81 FM213340 HF933242 70 HQ688788 100 S. patagonicum HQ688789 70 FM213336 61 S. septentrionale ? 100 JX434684 EU819441 100 S. michiganense CLADE I FM213346

70 SHA1 90 EU784414 100 S. citrinum FJ824090 63 HM189957 HF933239 100 S. meridionale HF933238

61 95 SHJ1

100 FM213337 S. septentrionale ? 65 FM213338 EU718115 99 FM213351 S. areolatum SDH2 61 FM213355 99 99 S. cepa CLADE II DQ453694

81 AJ629886

67 HF933237 S. verrucosum 66 71 HF933241 FJ840448 S. dictyosporum 100 FJ840447 EU718123 80 S. polyrhizum FM213350

55 KJ209680 KJ209679 S. bermudense 100 KJ209678 FM213364 FM213361 S. sinnamariense 100 HQ687222

FM213365 Pisolithus arhizus

0.02 Fig. 14. Molecular phylogenetic analysis of Scleroderma spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –2253.16). ■ indicate species collected and sequenced during this study. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree. 361

Phylogenetic analysis of Geastrum and Myrisostoma (Fig. 15) In the present phylogenetic analysis of Geastraceae, fourteen (14) sequences of ITS- nrDNA regions were produced. Out of these, 13 sequences belong to genus Geastrum and 1 to Myriostoma.

Out of 13 sequences of Geastrum spp., 4 sequences belong to G. pakistanicus sp. nov., 3 to G. patulus sp. nov. 1 each to G. fimbriatum, G. himalayensis sp. nov., G. parvulus, G. pseudolimbatum, G. pseudosaccatum sp. nov., and G. triplex

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank and from literature (Jeppson et al., 2013). This analysis involved 47 nucleotide sequences, 45 are in group belonging to Geastrum and 2 sequences of Myriostoma are taken as out groups.

Final alignment matrix was 766 nucleotide long, which included 360 conserved, 393 variable and 285 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The analysis involved 47 nucleotide sequences. All positions with less than 0% site coverage were eliminated. That is, fewer than 100% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 397 positions in the final dataset.

In this analysis, three clades were resolved, i.e. Clade I, Clade II and Clade III. Clade I and II consist of sequences belonging to genus Geastrum. Clade III is a small clade consisting of two sequences of Myriostoma.

Clade I is further sub-divided in two sub clades, i.e. Sub clade I and Sub Clade II. G. fimbriatum, G. himalayensis sp. nov., G. pseudolimbatum and G. triplex fall in Clade I. G. pakistanicus sp. nov. G. parvulus, G. patulus sp. nov., and Geastrum sp. 1 clustered in Clade II. G. pseudosaccatum segregated from the species of Clade 1 and Clade II.

362

87 Geastrum triplex KC581979 Geastrum triplex KC581978 59 Geastrum triplex_Pak 97 Geastrum indicum JN845097 Geastrum smardae KC581976 77 100 Geastrum smardae KC581977 KC582012 Geastrum sessile JN845123 Geastrum fimbriatum_Pak 99 59 Geastrum fimbriatum KC582015 73 Geastrum fimbriatum KC582016 Geastrum himalayensis_Pak

100 Geastrum coronatum KC581965 Sub Clade I Geastrum coronatum JN845092

96 Geastrum kotlabae KC582004 73 Geastrum pouzarii KC582002 CLADE I Geastrum campestre KC581998 75 Geastrum pseudostriatus KC581996

92 Geastrum berkeleyi KC581988 98 Geastrum berkeleyi KC581987

99 Geastrum elegans KC582014 Geastrum elegans KC582013 Geastrum floriforme KC581983 Geastrum parvistriatum JN943160 61 Geastrum striatum EU784249 Geastrum morganii KC581971 Geastrum corollinum KC581972

100 KC582011 Geastrum rufescens KC582010 Sub Clade II Geastrum pseudolimbatum KC581974 Geastrum pseudolimbatum_Pak 100 Geastrum pseudolimbatum KC581973 KC581962

100 Geastrum lageniforme EU784235 Geastrum lageniforme KC581966 Geastrum saccatum KC581968 68 Geastrum patulus_GPK18 54 Geastrum patulus_GPK15 52 99 CLADE II Geastrum patulus_GPK14 Geastrum parvulus_Pak Geastrum pakistanicus_Pak4 Geastrum pakistanicus_Pak1 98 Geastrum pakistanicus_Pak2 71 Geastrum pakistanicus_Pak3 Geastrum pseudosaccatum_Pak Myriostoma coliforme_USA CLADE III 100 Myriostoma coliforme JN845203

0.02 Fig. 15. Molecular phylogenetic analysis of Geastrum spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –-3592.03). ■ indicate species collected and sequenced during this study. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree

363

Phylogenetic analysis of Geastrum lycogaolpsus (Fig. 16) In the present phylogenetic analysis, 2 sequences of LSU-nrDNA regions were produced from a collection of Geastrum lycogaolpsus.

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank. This analysis involved 44 nucleotide sequences. Out of these, in group sequences are 41, 39 belonging to Geastrum and 2 of Radiigera. Three sequences were selected as out groups, these are Sclerogaster xerophilus (FJ435978), Lycogalopsis solmsii (KF017599), and Trappea darkeri (DQ218651).

Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 44 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair.

Final alignment matrix was 964 nucleotide long, which included 688 conserved, 214 variable and 136 parsimony informative sites. 364

99 Geastrum pectinatum JN845232 55 Geastrum pectinatum JN845228 Geastrum pectinatum KC581962 Geastrum striatum JN939558 Geastrum striatum_KC581960 99 Geastrum striatum JN939557 Geastrum coronatum KC581965 KC581958 JN845223 Geastrum corollinum KC581972 Radiigera flexuosa KC581970 Radiigera taylorii JN845329 Geastrum fornicatum KC582012 57 Geastrum velutinum JN845297 Geastrum morganii KC581971 Geastrum indicum JN845215

100 Geastrum campestre JN939555 Geastrum campestre JN939556 58 Geastrum kotlabae KC582005 99 Geastrum kotlabae KC582004

96 Geastrum floriforme KC581984 Geastrum floriforme KC581983

99 Geastrum lycogalopsus DPK3 Geastrum lycogalopsus DPK3

100 Geastrum rufescens KC582010 Geastrum rufescens KC582009 Geastrum schmidelii KC582007 100 Geastrum schmidelii JN845239 Geastrum xerophilum KC581975 Geastrum mirabile JN845225 100 Geastrum mirabile JN845227

99 Geastrum saccatum KC581969 Geastrum saccatum KC581968 100 Geastrum triplex JN845272 Geastrum triplex JN845248 94 81 Geastrum smardae KC581977 93 Geastrum smardae KC581976 Geastrum fimbriatum KC582016 Geastrum sessile JN845241 98 Geastrum fimbriatum JN84521 90 Geastrum sessile FJ644508 Sclerogaster xerophilus FJ435978 Lycogalopsis solmsii KF017599 100 Trappea darkeri DQ218651

0.01 Fig. 16. Molecular phylogenetic analysis of Geastrum lycogalopsus by the Maximum Likelihood (ML) method based on LSU-nrDNA sequences. (Ln likelihood = –-4250.51). ■ indicate species collected and sequenced during this study. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree

365

Phylogenetic analysis of Phallus spp. and related taxa (Fig. 17) In present investigation out of five Phallus spp. collected, ITS-nrDNA regions of 4 species were successfully amplified and 7 sequences were generated. This included 3 sequences of Phallus ahmadii, 1 of P. galericulatus (= Itajahya galericulata), 2 of P. hadriani (1 from each year collection), and 1 of P. roseus (= I. rosea).

Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank. The analysis involved 26 nucleotide sequences, 25 in group and 1 out group sequence. Out of 26 in groups sequences, 11 belong to genus Phallus, 5 of Dictyophora (= Phallus), 3 of Mutinus, and 2 of , Lysurus, and Protubera each. One (1) sequence of Scleroderma verrucosum (HF933241) was taken as an out group.

Final alignment matrix was 858 nucleotide long, which included 269 conserved, 522 variable and 375 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 312 positions in the final dataset.

In this analysis, two clades were resolved, i.e. Clade I and Clade II. Clade I consists of further two sub clades. It consists of sequences belonging to genera Phallus, Dictyophora and Mutinus. Dictypohora is now considered as synonymous to Phallus. Clade II consists of sequences belonging to genera Clathrus, Lysurus, and Protubera. All of the sequences of taxa collected during this study falls in clade I. 366

73 Dictyophora indusiata f. lutea HQ414538 100 Dictyophora indusiata JN182874

82 Dictyophora indusiata f. lutea AF324173 Dictyophora echinovolvata AF324166 58 100 Dictyophora echinovolvata AF324164 AF324171 Phallus hadriani DQ404385 50 Phallus hadriani PPK1 65 99 92 Phallus hadriani PPK15

82 Phallus roseus PPK3 CLADE I Phallus galericulatus PPK5

99 Phallus rugulosus AF324170 70 Phallus rugulosus AF324169

57 Phallus ahmadii PPK4 Phallus ahmadii PPK4 100 Phallus ahmadii PPK4 Mutinus caninus GQ981513

95 Mutinus sp KC128651 100 Mutinus sp KC128650

100 Lysurus cruciatus AJ878736 Lysurus cruciatus AJ878735

97 GQ981501 54 CLADE II Clathrus ruber GQ981500 99 Protubera canescens GQ981521 100 Protubera canescens GQ981520 Scleroderma verrucosum HF933241

0.05 Fig. 17. Molecular phylogenetic analysis of Phallus spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –2346.36). ■ indicate species collected from Pakistan. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree

367

Phylogenetic analysis of Gymnomyces (= Russula) spp. (Fig. 18) In present investigation one ITS-nrDNA regions of 1 species was successfully amplified and generated. Sampling of taxa for phylogenetic analysis was based on retrieving closely related sequences from Genbank. The analysis involved 20 nucleotide sequences. This included 19 in group and 1 out group sequence. Out of 19 in group sequences, 13 belong to genus Russula, and 6 to Gymnomyces. One (1) sequence of Tylopilus pseudoscaber (KC797154) was taken as an out group.

Final alignment matrix was 798 nucleotide long, which included 498 conserved, 224 variable and 70 parsimony informative sites.

Initial tree(s) for the heuristic search were obtained by applying the Neighbor- Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 20 nucleotide sequences. All ambiguous positions were removed for each sequence pair.

In this analysis, sequences of Gymnomyces and Russula did not show any particular pattern of clustering with respect to growth habit of fruiting bodies, viz. gasteroid habit and mushroom like habit. 368

59 Gymnomyces fallax AY239349 65 Gymnomyces fallax KC152107 Gymnomyces brunnescens AY239327 Russula foetens AY061677 76 100 Russula cf. foetens DQ422023 Gymnomyces fragrans AY239331 58 77 Gymnomyces parksii AY239335 85 Gymnomyces subfulvus AY239321 Russula subfoetens KF002757 Russula vinaceocuticulata GU222258 60 Russula pakistanica RPK10 Russula granulata JQ272365 68 Russula cf. ventricosipes EU598187 Russula pulverulenta AY061736

89 Russula livescens JN129398 86 Russula livescens GU371295 Russula cerolens HQ604832 Russula cerolens HQ604830 100 Russula cerolens HQ604829 Tylopilus pseudoscaber KC797154

0.05 Fig. 18. Molecular phylogenetic analysis of Russula spp. by the Maximum Likelihood (ML) method based on ITS-nrDNA sequences. (Ln likelihood = –2543.82). ■ indicate species collected from Pakistan. GenBank accession number/Collection number are given at the end of species names. Bootstrap values = or >50 % are cited in the tree.

369

4.4. Diversity of Gasteroid Fungi

To work out the diversity of gasteroid fungi, a number of field trips to different regions of different forest types were conducted. Sampling was done randomly in rainy season during 2008-2013. Data on diversity of gasteroid fungi was compiled for different forest types of country. A total of 225 samples were collected and identified up to species level. They belonged to 101 species, 20 genera, 9 families and 5 orders (Figs. 21–29).

4.4.1. Diversity of gasteroid fungi in different forests

Forests of Pakistan are heterogeneous and reflect great physiographic, climatic and edaphic contrasts. There are twelve types of forests based on climate, altitude and composition of forest species (Champion et al., 1965).

Gasteroid fungi were collected from different regions falling under five major forest types, i.e. temperate, tropical, subtropical, alpine and irrigated. Temperate forest is represented by Himalayan moist temperate forest. Tropical forests are Tropical dry deciduous forests, Tropical thorn forest, Tropical scrub forest. Subtropical and Alpine forest studied are Subtropical pine forest, and Alpine pastures, respectively. Changa Manga and Lal Sohanra forests are two artificially irrigated forests (Champion et al., 1965).

Diversity of gasteroid fungi in Himalayan moist temperate forest

Sampling sites in this forest are Ayubia National Park, Khanspur, Muskhpuri, Nathia gali; Swat district (Kalam, Malam Jabba, Mankial, Mashkoom, Miandam, Ushu), Kaghan valley (Nadibunglaw, Batta kundi), Sharan, Naran; Abottabad district (Thandiani, Shimla hill); Gilgit-Baltistan (Fairy Meadows); Azad Kashmir (Neelum valley, Sharda).

During this study, highest diversity of gasteroid fungi was found in this forest with 59 species belonging to 12 genera (Fig. 19). Among these, members of agaricaceae are more abundant and rich in diversity in this forest as compared to other groups of gasteroid fungi. So, Lycoperdon and Bovista were two dominant genera with 21 and 16 number of species, respectively in this forest. Out of a total of 20 genera reported in present investigation, 12 genera are known to occur from this forest (Table 2).

370

Diversity of gasteroid fungi in subtropical pine forest

Areas which were explored for the diversity of gasteroid fungi in subtropical pine forest are Batrasi, Chattar plains, Dadar, Jabori, Musaka- musala, Ucharhi, and Ughi forest of district Mansehra.

Second highest number of taxa was found in this forest with 27 species belonging to 9 genera (Table 3). Here, it was noted that however total no. of different species are less as compared to Himalayan moist temperate forests, however this forest is more diverse with respect to the presence of different genera. Since it is a pine dominating forest, it was found that Astraeus hygrometricus was the most abundant and frequent species. A number of collections of this taxon was made at different maturity stages, found growing under Pine trees of district Mansehra (Fig. 19)

Diversity of gasteroid fungi in Tropical forests

Tropical forests include, Tropical thorn forest, Tropical dry deciduous forest, Tropical scrub forest and dry tropical forest. Some sites of Punjab province (Daska, Fort Munro, Lahore, Mianwali, Narowal) and Sindh provinve (Nawab Shah) are included in tropical thorn forest whereas Chakwal district (Dumhan, Neela dulha), Sakesar are the areas under vegetation type of tropical dry deciduous/ scrub forest, respectively. Cholistan desert is under dry tropical forest. All of these sites were studied to find out gasteroid fungal diversity in regions with tropical climate and vegetation. Total no. of gasteroid fungi collected are 24 belonging to 13 genera (Table 4). This forest is more diverse because of having highest no. of genera when compared to other forests, however number of different species is less than aforementioned forests. Deserticolous gasteroid species, i.e. Battarrea, Montagnea, Pisolithus, Podaxis and Tulostoma are found in this forests. Out of 5 Phallus spp. reported in this investigation, 4 belonged to tropical forests. Genera Bovista and Lycoperdon are also found but species richness and abundance is very less here when compared their presence in other forests (Fig. 19).

Diversity of gasteroid fungi in Artificial/irrigated plantations

Two irrigated plantations were visited under this category. viz., Changa Manga forest of Kasur district of Punjab and Lal Sohanra National park of Bahawalpur district. Only 6 species belonging to 6 genera were found (Table 5, Fig. 19). 371

Diversity of gasteroid fungi in Alpine pasture

Deosai national park is the example of alpine pastures. Deosai is known as land of giants and known as second highest plateau of the world. This area was studied and visited for the diversity of gasteroid fungi. Three species of were collected belonging to Bovista and Calvatia (Table 6).

4.4.2. Species Richness (SR) of sampling sites

When species richness of studied forests were calculated, Himalayan moist temperate forests showed maximum SR value of 0.565, followed by 0.21 and 0.171 of Subtropical pine forest and tropical forests, respectively (Fig. 20).

In Himalayan moist temperate forest, Khanspur is the most species rich sites for gasteroid fungi as compared to other sites in this forest. In Subtropical forests, Ughi forest and Lahore in the tropical forests are the comparatively species rich sites (Table 7).

4.4.3. Taxonomic diversity of gasteroid fungi

Gasteroid fungi presented in this study belonged to five orders; Agaricales, Boletales, Geastrales, Phallales, and Russulales. When taxonomic diversity of gasteroid fungi was calculated, it was found that maximum no. of collected samples belong to Agaricales. Out of 225 collected samples were, highest number of specimens belonged to Agaricales followed by boletales, geastrales, phallales and russulales (Figs. 21 & 22). Almost 69% of taxa studied were in order agaricales. Among Agaricales, three families were studied with maximum no. of specimens (98%) in Agaricaceae (Figs. 23 &24).

Gasteroid fungi belonging to boletales are grouped in three families with highest number of taxa studied in Sclerodermataceae (69%) (Figs. 25 & 26).

Geastrales, Phallales and Russulales are represented by one family each (Figs. 27– 29). Among all 9 families of 5 orders of gasteroid fungi studied, Agaricaceae has highest no. of taxa i.e. 70 (Fig. 30).

As regard the diversity of species within the genus is concerned, Lycoperdon and Bovista are the genera with highest no. of species, i.e. 28 and 25 species, respectively (Fig. 31).

372

4.4.4. Species Frequency (SFl) of gasteroid fungi

SFl was calculated for all 101 taxa. Among these, highest SFl of 21.62% was shown by Bovista plumbea. It is followed by Astraeus hygrometricus, Lycoperdon pyriforme, L. perlatum, L. excipuliforme, Podaxis pistillaris, S. bovista, Geastrum triplex, L. lineum, and

Phallus roseus, with SFl values of 18.9%, 16.2%, 16.2%, 13.5%, 10.8%, 10.8%, 8.1%, 8.1%, 8.1%, respectively (Table 8). 373

Table 2. Gasteroid fungal species collected from Himalayan moist temperate forest.

Himalayan moist temperate forest

Sr. no. Species 1* 2* 3* 4* 5* 6* 7* 8* 9* 10* 11* 12* 13* 14* 15* 16* 17* 18* 1. Bovista bovistoides − − − + − − − − − − − − − − − − − − 2. B. fusca − − − − − − + − − − − − − − − − − − 3. B. kreiselii − − − − − − − − − − − − − + − − − − 4. B. nigrescens var. rectapedicellata − − − − − − + − − − − − − − − − − − 5. B. plumbea − + − − − − + + + + − + − − − − + − 6. B. pseudonigrescens − − − − − − − − − − − + − − − − − − 7. B. aestivalis − − − − + + − − − − − − − − − − − − 8. B. ayubiensis − + − − − − − − − − − − − − − − − − 9. B. concinna − + − − − − − − − − − − − − − − − − 10. B. himalaica − + − − − − − + − − − − − − − − − 11. B. levisspora − − − − − − − + − − − − − − − − − 12. B. oblongispora − − − − − − − + + − − − − − − − − − 13. B. pakistanica − − − − + − − − − − − − − − − − − − 14. B. promontorii − − − − − − − − − − − − − − − − − − 15. B. trachyspora − − − − − − − − − − − + − − − − − − 16. B. viridispora − − − − − − − − − − − − − − + − − − 17. Lycoperdon pyriforme − + + − − − − + − − − + − + − − − − 18. L. japonicum − − − + − − − − − − − − − − − − − − 19. L. utriforme − − − − + − − − − − − − − − − − − − 20. L. parvasporum − − − − − − − − + − − − − − − − − − 21. L. perlatum − + − + + − − − − + − − − − − − − − 22. L. albiceratum − − − − − − − − − − − − − + − − − − 23. L. altimontanum − − − − − + − − − − − − − − − − − − 24. L. aurea−bruneum − − − − − − − − − − − − − − − − + − 25. L. excipuliforne − − − + − + − − + − − + − − − − − − 26. L. khanspurense − + − − − − − − − − − − − − − − − − 27. L. lignum−bruneum − + − − − − − − − − − − − − − − − − 28. L. lineum − + − + − − − − − − − − − − − − − − 374

29. L. mammiforme − + − − − − − − − − − − − − − − − − 30. L. molle − + − − − − − − − − − − − − − − − − 31. L. nigrescens − + − − − − − − − − − − − − − − − − 32. L. niveum − − − − − − − + − − − − − − − − − − 33. L. pseudoniveum − − − − − − − − − − − − − − − − + − 34. L. sharanense − − − − − − − − − − − − + − − − − − 35. L. gilgitii − − − − − − − − − − − − − − − − + − 36. L. olivoflavum − + − − − − − − − − − − − − − − − − 37. L. pratense var. flavum − − − − + − − − − − − − − − − − − − 38. Podaxis pistillaris − − − + − − − − − − − − − − − − − − 39. Tulostoma sp. − − − − − − − − − − + − − − − − − − 40. Cyathus olla − − − − − − − − − − − + − − − − − − 41. Astraeus hygrometricus − − − − − − − − − − − − − − + − − − 42. Rhizopogon himalayensis − − − − + − − − − + − − − − − − − − 43. R. roseolus − − − − − − − − − − − + − − − − − − 44. Scleroderma areolatum − + − − − − − − − − − − − − − − − − 45. S. bovista − + − − + − − − − − − + − + − − − − 46. S. chevalieri − − − + − − − − − − − − − − − − − − 47. S. dictyosporum − − − − − − − − − − − − − − − + − − 48. S. fuscum − − − − − − − − − − − − − − − − − + 49. Geastrum fimbriatum + − − − − − − − − − − − − − − − − − 50. G. pakistanicus − − − − − − − − − − − + − − − − − − 51. G. parvulus − − − − − − − − − − − − − − + − − − 52. G. patulus − − − − − − − − − − − − − − + − − − 53. G. pseudolimbatum − − − − − − − − − + − − − − − − − − 54. G. pseudosaccatum − − − − − − − − + − − − − − − − − − 55. G. saccatum + − − + − − − − − − − − − − − − − − 56. G. triplex + + − + − − − − − − − − − − − − − − 57. Myriostoma coliforme − + − − − − − − − − − − − − − − − − 58. Radiigera ayubiensis − + + − − − − − − − − − − − − − − − 59. Gymnomyces pakistanicus − − − − − + − − − − − − − − − − − − 1*. Ayubia National Park, 2*. Khanspur, 3*. Mushkpuri, 4*. Nathia gali, 5*. Kalam, 6*. Mankial, 7*. Mashkoom, 8*. Malam Jabba, 9*. Miandam, 10*. Ushu, 11*. Batta kundi, 12*. Nadi bunglow, 13*. Sharan, 14*. Thandiani, 15*. Shimla hill, 16*. Naran, 17*. Fairy meadows, 18*. Azad Kashmir 375

Table 3. Gasteroid fungal species collected from Subtropical pine forests.

Subtropical pine forest Sr. no. Species 1* 2* 3* 4* 5* 6* 7* 1. Bovista areolata − − − − − − + 2. B. plumbea − − − − + − − 3. B. ahmadii − − − − − − + 4. B. brunnescenta − − + − − − − 5. B. flavus − − + − − − − 6. B. flavescens − − − − − − + 7. B. longispora − − − − − − + 8. B. minima − − − + − − − 9. B. viridpora − − + − − − − 10. Bovista sp. (BPK92) − − + − − − − 11. Calvatia cyathiformis var. fragilis − − − − − − + 12. C. lilacina − − − − − − + 13. C. pseudocyathiformis − − − − − − + 14. Lycoperdon pyriforme − − + − − − − 15. L. perlatum − + − − − + + 16. L. albiceratum − − − − − − + 17. L. excipuliforme − − − − − − + 18. L. lineum − − − − − − − 19. L. mammiforme var. magnasporum − − − − − − + 20. Lycoperdon curtisii var. ovalisporum − − + − − − − 21. Astraeus hygrometricus + − + + − − + 22. Rhizopogon flavescens − − − − − − + 23. Pisolithus tinctorious − − − − − − + 24. Pisolithus sp. PPK16 − − − + − − − 25. Scleroderma bovista − − − − − − + 26. Geastrum himalayensis − − + − − − − 27. Phallus hadriani − + − − − − + 1*. Batrasi, 2*. Chattar plains, 3*. Dadar, 4*. Jabori, 5*. Musaka−Musala, 6*. Ucharhi, 7*. Ughi forest 376

Table 4. Gasteroid fungal species collected from Tropical forests.

Tropical forests Sr. Species Tropical dry deciduous forest Tropical scrub forest Dry tropical forest Tropical thorn forests no. 1* 2* 3* 4* 5* 6* 7* 8* 9* 1. Arachnion olivoflavus − − − − − − + − − 2. Battarrea phalloides − − + − − − − − + 3. Bovista lahorensis − − − − − + − − − 4. B. ahmadii − + − − − − − − 5. B. dermoxanthum var. album − − − − + − − − − 6. B. dermoxanthum var. flavum − − − − + − − − − 7. Lycoperdon curtisii var. − − − − − − − − − ovalisporum 8. L. lahorense − − − − − + − − − 9. L. lahorense var. parvasporum − − − − − + − − − 10. Montagnea arenaria + − − − − + − − − 11. Podaxis pistillaris + − − − − + − − − 12. Tulostoma fimbriatum − − − − − + − − − 13. Tulostoma sp. (TPK6) − − − + − − − − − 14. T. squamosum − + − − − − − − − 15. Cyathus pakistanicus + − − − − − − − − 16. Phellorinia herculeana − − + − − − − − − 17. Astraeus hygrometricus − − − − + − − − − 18. Rhizopogon roseolus − − − − − + − − − 19. Pisolithus flavus − − − − + − − − 20. P. tinctorious − − − − − + − − − 21. Phallus ahmadii − − − − − + − − − 22. P. galericulatus − − − − − + − − − 23. P. roseus − − − − − + − + − 24. P. rubicundus − − − − − + − − − 1*. Chakwal, 2* Sakesar, 3*. Cholistan, 4*. Daska, 5*. Fort Munro, 6*. Lahore, 7*. Narowal, 8*. Mianwali, 9*. Nawab Shah 377

Table 5. Gasteroid fungal species collected from Artificial/ Irrigated forests. Artificial/Irrigated Forests Sr. no. Species 1* 2* 1. Disciseda hyalothrix + − 2. Podaxis pistillaris − + 3. Tulostoma morenoii − + 4. Geastrum lycogalopsus + − 5. Pisolithus flavus − + 6. Phallus roseus − + 1*. Changa Manga, 2*. Lal Sohanra Park Table 6. Gasteroid fungal species collected from Alpine pasture. Alpine pasture (Deosai National park) Sr. no. Species 1. Bovista plumbea 2. Calvatia lilacina 3. C. deosaiensis

Table 7. Species Richness (SR) of sampling sites. Sr. No. Forest types Locality SR 1. Ayubia National Park 0.0197 2. Khanspur 0.1184

3. Mushkpuri 0.0131 4. Nathia gali 0.0592 5. Kalam 0.046 6. Mankial 0.0263 7. Mashkoom 0.0197 8. Malam Jabba 0.0328

9. moist Himalayan forest temperate Miandam 0.0395 378

10. Ushu 0.0263 0.565 11. Batta kundi 0.0065 12. Nadi bunglaw 0.0592 13. Sharan 0.0065 14. Thandiani 0.0263 15. Shimla hill 0.0263 16. Naran 0.0044 17. Fairy meadows 0.0263 18. Azad Kashmir 0.0044 19. Batrasi 0.0044 20. Chattar plains 0.0131 21. Dadar 0.0526 22. Jabori 0.0197 0.21

23. Ucharhi 0.0044 24. Musaka-Musala 0.0044

25. Pine Subtropical forest Ughi forest 0.105 26. Dry tropical forests Chakwal 0.0197 27. Sakesar 0.0131 28. Cholistan 0.0044 29. Daska 0.0044 30. Fort Munro 0.0263 0.171 31. Lahore 0.0789 32. Narowal 0.0044 33. Mianwali 0.0044 34. Nawabshah 0.0488 35. Artificial irrigated forests Changa Manga 0.0131 0.0328 36. Lal Sohanra park 0.0197 37. Alpine pasture Deosai National park 0.0197 0.0197 SR = No. of taxa collected from one locality over total no. of taxa. 379

Table 8. Species frequency (SFl) of gasteroid fungi

Sr. No. Orders Families Genus Subgenus Species name Collections No. of SFl sites % 1. Agaricales Agaricaceae Arachnion Arachnion olivoflavus 1 1 2.702 2. Battarrea Battarrea phalloides 2 2 5.555 3. Bovista Bovista Bovista areolata 1 1 2.703 4. B. bovistoides 1 1 2.703 5. B. fusca 2 1 2.703 6. B. kreiselli 1 1 2.703 7. B. nigrescens var. rectapedicellata 1 1 2.703 8. B. plumbea 16 8 21.62 9. B. pseudonigrescens 1 1 2.703 10. Globaria B. aestivalis 2 2 5.405 11. B. ahmadii 2 2 5.405 12. B. ayubiensis 1 1 2.703 13. B. brunnescenta 1 1 2.703 14. B. concinna 1` 1 2.703 15. B. flavescens 1 1 2.703 16. B. flavus 1 1 2.703 17. B. himalaica 6 2 5.405 18. B. lahorensis 1 1 2.703 19. B. levisspora 1 1 2.703 20. B. longispora 1 1 2.703 21. B. minima 1 1 2.703 22. B. oblongispora 2 2 5.405 23. B. pakistanica 5 1 2.703 24. B. promontorii 1 1 2.703 25. B. trachyspora 1 1 2.703 26. B. viridispora 2 2 5.405 27. Bovista sp. (BPK92) 1 1 2.703 28. Calvatia Calvatia cyathiformis var. fragilis 1 1 2.703 29. C. deosaiensis 1 1 2.703 30. C. lilacina 2 2 5.405 31. C. pseudocyathiformis 1 1 2.703 32. Disciseda Disciseda hyalothrix 1 1 2.703 380

33. Lycoperdon Apioperdon Lycoperdon pyriforme 21 6 16.21 34. Bovistella L. japonicum 1 1 2.703 35. L. utriforme 1 1 2.703 36. Lycoperdon L. parvasporum 1 1 2.703 37. L. perlatum 14 6 16.21 38. Utraria L. albiceratum 2 2 5.405 39. L. altimontanum 1 1 2.703 40. L. aurea-brunneum 1 1 2.703 41. L. dermoxanthum var. album 1 1 2.703 42. L. dermoxanthum var. flavum 1 1 2.703 43. L. excipuliforme 5 5 13.51 44. L. lignum-brunneum 4 1 2.703 45. L. lineum 3 3 8.108 46. L. mammiforme 1 1 2.703 47. L. mammiforme var. magnasporum 1 1 2.703 48. L. molle 1 1 2.703 49. L. nigrescens 1 1 2.703 50. L. niveum 1 1 2.703 51. L. pseudoniveum 1 1 2.703 52. L. sclerocystis 2 1 2.703 53. L. sharanense 1 1 2.703 54. Lycoperdon sp. (LPK74) 1 1 2.703 55. Vascellum L. curtisii var. ovalisporum 1 1 2.703 56. L. gilgitii 1 1 2.703 57. L. lahorense 2 1 2.703 58. L. lahorense var. parvasporum 1 1 2.703 59. L. olivoflavum 1 1 2.703 60. L. pratense var. flavum 1 1 2.703 61. Montagnea Montagnea arenaria 2 2 5.405 62. Podaxis Podaxis pistillaris 9 4 10.81 63. Tulostoma Tulostoma fimbriatum 1 1 2.703 64. T. morenoii 1 1 2.703 65. T. squamosum 1 1 2.703 66. Tulostoma sp. 1 1 1 2.703 67. Tulostoma sp. 2 1 1 2.703 68. Nidulariaceae Cyathus Cyathus olla 1 1 2.703 381

69. C. pakistanicus 1 1 2.703 70. Phelloriniaceae Phellorinia Phellorinia herculeana 1 1 2.703 71. Boletales Diplocystaceae Astraeus Astraeus hygrometricus 14 7 18.91 72. Rhizopogonaceae Rhizopogon Rhizopogon flavescens 1 1 2.703 73. R. himalayensis 1 1 2.703 74. R. roseolus 3 2 5.405 75. Sclerodermataceae Pisolithus Pisolithus flavus 3 2 5.405 76. P. tinctorious 2 2 5.405 77. Pisolithus sp. (PPK16) 1 1 2.703 78. Scleroderma Scleroderma areolatum 1 1 2.703 79. S. bovista 7 4 10.81 80. S. chevalieri 1 1 2.703 81. S. dictyosporum 1 1 2.703 82. S. fuscum 1 1 2.703 83. Scleroderma sp. (SPK21) 1 1 2.703 84. Geastrales Geastraceae Geastrum Geastrum fimbriatum 1 1 2.703 85. G. himalayensis 2 1 2.703 86. G. lycogalopsus 1 1 2.703 87. G. pakistanicus 4 1 2.703 88. G. parvulus 1 1 2.703 89. G. patulus 2 1 2.703 90. G. pseudolimbatum 1 1 2.703 91. G. pseudosaccatum 1 1 2.703 92. G. saccatum 3 2 5.405 93. G. triplex 5 3 8.108 94. Myriostoma Myriostoma coliforme 1 1 2.703 95. Radiigera Radiigera ayubiensis 1 1 2.703 96. Phallales Phallaceae Phallus Phallus ahmadii 1 1 2.703 97. P. galericulatus 1 1 2.703 98. P. hadriani 2 2 5.405 99. P. roseus 7 3 8.108 100. P. rubicundus 1 1 2.703 101. Russulales Russulaceae Russula Russula pakistanica 1 1 2.703

382

70 59 60

50

40

27

30 24 No. of species No.of species 20 No. of genera 12 13 9 10 6 6 3 2 0 Himalayan Subtropical Tropical Artificial Alpine forest moist Pine forest forests irrigated temperate forests forest

Fig. 19. Graphical presentation of diversity of gasteroid fungi in different forests of Pakistan.

0.6 0.565

0.5 (SR) 0.4 0.3 ichness 0.21 R 0.171 0.2 Himalayan moist temperate forest

0.1 0.0328 0.0197 Subtropical Pine forest Species 0 Tropical forests Artificial irrigated forests Alpine forest

Forest type studied

Fig. 20. Graphical presentation showing Species Richness (SR) of visited forests of Pakistan. 383

180

160 155

140

120

100

80 70 No.of species 60

37 40 23 20 11 13 12 12 5 4 3 1 1 1 1 0 Agaricales Boletales Geastrales Phallales Russulales Orders of gasteroid fungal taxa studied

Total No. of taxa collected Total no. of different species Total no. of genera

Fig. 21. Graphical presentation of gasteroid fungal taxa studied.

80.00% 69.30% 70.00% 67.98%

60.00%

50.00%

40.00%

30.00% No.of species

20.00% 16% 12.87% 11.88% 10.00% 10.08% 5% 4.95% 0.44% 0.99% 0.00% Agaricales Boletales Geastrales Phallales Russulales Percentage of orders of gasteroid fungal taxa studied

Percentage of taxa collected Total no. of different species

Fig. 22. Graphical presentation of percentage of gasteroid fungal taxa studied. 384

160 152

140

120

100

80 67

No.of species 60

40

20 9 2 2 1 1 1 1 0 Agaricaceae Nidulariaceae Phellorineaceae Families in Agaricales

Total no. of taxa collected No. of Different species No. of genera

Fig. 23. Graphical presentation of gasteroid fungal taxa studied in families of Agaricales

120.00%

98.06% 100.00% 95.70%

80.00%

60.00% Percentage 40.00%

20.00%

1.29% 2.86% 0.65% 1.42% 0.00% Agaricaceae Nidulariaceae Phellorineaceae

Families of Agaricales Percentage of taxa collected Percentage of different species

Fig. 24. Graphical presentation of percentage of fungal taxa studied in families of Agaricales 385

20 18 18 16 14 14 12 10 9 8 5 No.of species 6 3 4 2 2 1 1 1 0 Diplocystaceae Rhizopogonaceae Sclerodermataceae Families in Bolatales

Total no. of taxa collected No. of Different species No. of genera

Fig. 25. Graphical presentation of families studied in Boletales

80.00% 69.00% 70.00% 60.00% 49% 50.00% 37.80% 40.00%

30.00% 23.00% Percentage 20.00% 13.50% 7.70% 10.00% 0.00% Diplocystaceae Rhizopogonaceae Sclerodermataceae Families in Bolatales

Percentage of taxa collected Percentage of different species

Fig. 26. Graphical presentation of percentage of taxa studied in families of Boletales

386

25 21 20 15 12 10 5 3 0

No.of species Geastraceae Families in Geastrales

Total no. of taxa collected No. of different species No. of genera

Fig. 27. Graphical presentation of gasteroid fungal taxa studied in Geastrales

12 11

10

8

6 5

4 No.of species 2 1

0 Phallaceae Family in Phallales Total no. of taxa collected No. of different species No. of genera

Fig. 28. Graphical presentation of gasteroid fungal taxa studied in Phallales

1.2 1 1 1 1 0.8 0.6 0.4

No.of species 0.2 0 Russulaceae Family in Russulales

Total no. of taxa collected No. of different species No. of genera

Fig. 29. Graphical presentation of gasteroid fungal taxa studied in Russulales

387

80 67 70 60 50 40 30 20 9 12

No.of species 5 10 2 1 1 3 1 0

Families of gasteroid fungi studied

Agaricaceae Nidulariacae Phellorineaceae Diplocystaceae Rhizopogonacace Sclerodermataceae Geastraceae Phallaceae Russulaceae

Fig. 30. Graphical presentation of distribution of no. of gasteroid fungal taxa studied in nine families.

30 28 25 25

Agaricales 20 Boletales 15 Geastrales 10

No.of species 10 Phallales 6 5 5 4 Russullales 5 3 3 2 1 1 1 1 1 1 1 1 1 1 0

Genera of gasteroid fungal taxa studied Fig. 31. Graphical presentation of distribution of no. of gasteroid fungal taxa in each genus.

388

5.0. DISCUSSION During this investigation, two hundred and twenty eight (228) gasteroid taxa belonging to five orders Agaricales, Boletales, Geastrales, Phallales and Russulales were collected from different regions of Pakistan and studied using morphological and molecular techniques. Sampling was done from the areas with variable climate, altitude and vegetation type.

Taxonomy and phylogeny of almost each genus is discussed here order wise.

Agaricales is commoly known as gilled , however it also contain some non- gilled members like gasteroid fungi (Matheny et al., 2006). Gasteroid fungi belonging to order Agaricales are confined to three families of Agaricoid clade, viz. Agaricaceae, Nidulariaceae, and Phelloriniaceae.

Gasteroid members of Agaricales so far known from Pakistan are the species belonging to genera, Battarrea Persoon, Bovista Pers., Calvatia Fries, Crucibulum Tul. & C. Tul., Cyathus Haller, Disciseda Czern., Lanopila Fr., Lycoperdon Pers., Montagnea Fr., Podaxis Desv., Tulostoma Pers., and Phellorinia Berk. (Ahmad 1952, 1956, Ahmad et al., 1997, Khalid & Iqbal, 2004).

In this study, seventy taxa were studied belonging to three families of Agaricales.

In Agaricaceae, studied genera include Arachnion Schwein., Battarrea Persoon, Bovista, Calvatia, Disciseda, Lycoperdon, Montagnea and Podaxis.

Arachnion Schwein.

Genus Arachnion was first proposed by Schwein., in 1822 by describing A. album as type species. It is characterized by the small fruiting body, fragile peridium, peridiolar gleba and rare to absent capillitum (Lloyd, 1906a; Lander, 1934; Long, 1941; Zeller, 1949). This genus is widespread and found in subtropical regions. It is represented by six species worldwide (Cunningham, 1944; Bottomley, 1948; Demoulin, 1972; Pérez-Silva et al., 1994; Quadraccia, 1996; Calonge et al., 2004; Kasuya et al., 2006; Kirk et al., 2008; Trierveiler-Pereira & Baseia, 2009; Cortez et al., 2010). 389

During this investigation, one species of this genus is collected from semi-arid region of Punjab. Arachnion olivoflavus nom. prov. (BPK59) does not bear any characteristic resemblance with any of the reported Arachnion spp.

Arachnion olivoflavus nom. prov., (BPK59) characterized by small olivaceous yellow basidioma, smooth to asperulate basidiopsores and highly porous olivaceous yellow eucapillitial hyphae. Presence of well-developed capillitium suggests its distinct status within the genus. It differs by A. drummondii Berk., A. iulii Quadraccia, and A. tener (Berk.) Long, beacause the latter three lack capillitium and endoperidium (Kasuya et al., 2006).

In phylogenetic analysis (Fig. 1), this taxon maintained its distinct status from other taxa by good 87% bootstrap support. Three sequences of A. olivoflavus fall in the clade with four sequences of A. album (only available in genbank). It shares a maximum of 97.5% genetic characters and least divergence of 2.4% with one sequence of A. album (EN833649).

Morphologically, it shares little characters with A. album such as fragile peridium upon maturity. Gleba in A. olivoflavus has a slight rough feel but peridioles are absent which is a characteristic feature of A. album. Eucapillitium which is well-developed having numerous pores is present in A. olivoflavus (BPK59), is absent in A. album. Spores size is also variable in both cases, A. olivoflavus has smaller basidiospores than those of A. album (3.3–4.9 × 3.3–4.4 µm vs 4.2–6.0 × 3.5–5.0 µm) (Kasuya et al., 2006).

It is therefore described here as a new species. It is aslo a first documentation of this genus from Pakistan. In addition, phylogenetic placement of Arachnion within agaricaceae was not confirmed till now. Phylogenetic analysis (Fig. 1) confirms position of Arachnion in Agaricaceae.

Battarrea Persoon

Battarrea, a cosmopolitan genus, was first established by Persoon in 1801 by describing B. phalloides, its type species (Esqueda et al., 2002). It is widespread and represented by three (3) species world wide (Kirk et al., 2008). This genus was originally reported from Btitish Isles, but it is also found in Africa, Asia, Australia, North America, and South America (Guzmán, 1958; Herrera, 1959; Pegler et al., 1995; Watling et al., 1995a; Calonge, 1998; Jacobson et al., 1999; Martin & Johanesson, 2000; Liu & Fan, 2006; 390

Cortez et al., 2009a). Members of the genus are usually found in hot and tropical regions but also found in cool, humid regions as well (Fortey, 1998; Esqueda et al., 2002). This genus is characterized by sub-globose spore case and a stipe, sometimes with volva at the base. Many uses of Battarrea are known in different cultures, e.g. use as medicine in treatment of sores and swellings (Burk, 1983; Felger & Moser, 1991).

In present investigation, one specimen of Battarrea phalloides was collected from Cholistan desert of Pakistan and identified using morphological and molecular methods.

During BLAST, both sequences of B. phalloides (BPK62) showed a maximum similarity of 100% with B. stevenii (DQ184689/ Kenya) with 92% query coverage. However, 99% similarity was also shown with five more sequences of B. phalloides (= B. stevenii) (AF215648, AF215650, AF215656, AF215657, AF215658) from Spain, with query coverage ranges of 86–90 %.

In phylogenetic tree, B. phalloides falls in Clade II along with above mentioned sequences of Battarrea. When sequence distances were computed, it was found that among all of these sequences Battarrea used in the analysis, B. phalloides showed 100% genetic similarity and 0.0% divergence with B. stevenii (AF215657) from Kenya (Fig. 2).

This is also a first report of molecular characterization of Battarrea sp. from Pakistan.

Bovista Pers.

Genus Bovista Pers. is characterized by having epigeous gasteroid basidiomata without conspicuous sterile base, and globose to oval basidiopsores (Miller & Miller, 1988; Ellis & Ellis, 1990). Members of the genus are widely distributed, being found primarily in temperate regions. The genus contains approximately 55 species (Kirk et al., 2008), which have been reported previously from Eastern Africa (Demoulin & Dring, 1975), Finland (Haeggstrom, 1997), Iceland (Hallgrimsson, 1988), India. (Sharma et al., 2007), Japan (Yoshie et al., 2004; Miwa & Kasuya, 2009), Mexico (Calonge et al., 2004), North America (Coker & Couch, 1928; Bates et al., 2009), northern Europe (Larsson et al., 2009), Pakistan (Ahmad 1952; Ahmad et al., 1997), Panama (Gube & Piepenbring, 2009), South Africa (Bottomley, 1948; DeVilliers et al., 1989), and South America (Suarez & Wright, 1994; Baseia, 2005; Trierveiler-Pereira et al., 2010). 391

Kreisel (1967) categorized Bovista spp. in two subgenera, subgenus Bovista and subgenus Globaria. Bovista spp. with characteristics Bovista type capillitium fall in subgenus Bovista whereas species having intermediate and Lycoperdon type capillitium belong to subgenus Globaria.

Four species, B. bovistoides (Cooke & Massee) S. Ahmad, B. concinna S. Ahmad, B. lycoperdoides (Cooke) S. Ahmad, and B. plumbea Pers., have been reported from Pakistan, so far (Ahmad, 1952; Ahmadet al., 1997).

In present investigation, twenty four (25) Bovista species were studied and described here. Among these, seven (7) taxa belong to subgenus Bovista, and seventeen (18) taxa to subgenus Globaria.

Phylogenetic analyses of some of the Bovista spp. has also been given and morphology is supported by molecular phylogeny.

Subgenus Bovista B. areolata nom. prov., B. bovistoides, B. fusca Lév., B. kreiselii nom. prov., B. nigrescens var. rectapedicellata nom. prov., B. plumbea, B. pesudonigrescens nom. prov., have been placed in subgenus Globaria. Among these, three (3) taxa seem new to science, one (1) taxon is described here as a new variety. B. fusca is a new record for Pakistan. B. bovistoides, and B. plumbea are being reported from different areas of the country during this study.

Three taxa which seems previously undescribed have been discussed here.

Bovista areolata nom. prov. (MPK4) belongs to section Nanobovista of subgenus Bovista. It is characterized by areolate endoperidium, absence of sub-gleba and aseptate, non-pitted capillitium. Due to these features it falls in series Fuscae. In this series, these characters are only shared by B. fulva Massee, B. flaccida (Lloyd) Kreisel, B. bovistoides, and B. fusca. Among these, B. fulva is more close to Bovista sp. (MPK4) because both have areolate endoperdium. There are some other features of B. fulva shared with taxon under study, which includes olive brown gleba, dimensions of spores and capillitial hyphae. But presence of distinct main stem, absence of false septa in capillitium are character in current Bovista species which make it different from B. fulva. Phylogenetically, this taxon appears as sister species of B. plumbea because they share same basic characters and follow 392

Kreisel’s taxonomy based on morphology. However, both taxa are quite different from each other in some morphological features. B. plumbea is known as grayish puffball on the basis of grayish endoperidium and gleba, whereas B. areolata has olivaceous peridium and gleba. These morphological differences and distinct phylogenetic status suggest this taxon as new species.

Bovista kreiselii nom. prov. (BPK10) have basic characters in common with the Bovista spp. under subgenus Bovista. This taxon is more close to B. pseudonigrescens (GPK28), B. nigrescens var. rectapedicellata (BPK27) and B. nigrescens Pers. However, B. kreiselii different from first two because of having spores with pointed pedicels. This character of tapering pedicel is shared by B. nigrescens Pers. Characters of capillitial threads with no subseptal ramifications and false septa in B. nigrescens and B. nigrescens var. rectapedicellata (BPK27), is also found in B. kreiselii (BPK10). Despite of having close resemblance with B. nigrescens, and B. nigrescens var. rectapedicellata, there are some other morphological differences between them, i.e. presence of sterile base which is very conspicuous in B. kreiselii but never found in B. nigrescens and B. nigrescens var. rectapedicellata. Molecular and phylogenetic results also segregated these species.

Among Section Bovista of subgenus Bovista, B. pseudonigrescens nom. prov., (GPK28) seems to be a close relative of Bovista nigrescens Pers. but the major difference in both species is the type of capillitium present in both. i. e. isolated units of typical Bovista type capillitium in B. nigrescens and capillitium (without isolated units) with main stems in Bovista pseudonigrescens (GPK28). Dehiscence of basidioma take place by a definite apical pore in current species whereas irregular dehiscence pattern is the characteristic of B. nigrescens. Other differences are the presence of paracapillitium, sub-septal ramification and fasle septa in current species. However these features are not found in B. nigrescens. Pedicel of the spores are pointed, and mostly 4–9 µm in B. nigrescens, however pedicels do not taper and straight in this case. These features suggest an addition of a new Bovista species in Section Bovista of genus Bovista.

B. nigrescens var. rectapedicellata nom. prov., (BPK27) shares most of the characters with B. nigrescens. But the major difference is that, pedicel in B. nigrescens is always tapering and pointed whereas pedicel in current species is straight. Therefore it is proposed here as a variety of B. nigrescens. 393

During molecular analysis, 5 out of 7 species were successfully amplified and therefore used for phylogenetic analysis. All of the Bovista spp.in subgenus Bovista fall in a subclade 1 of Clade 1 (Fig. 3).

B. areolata (MPK4) is phylogenetically close to B. plumbea and appear as sister species to it. Although it enjoys its distinct status within this clade but without a good bootstrap support.

In phylogenetic analysis, B. kreiselii (BPK10), B. pseudonigrescens (GPK28) and B. nigrescens var. rectapedicellata (BPK27) clustered together forming a minor clade (B. nigrescens species complex) within subclade1 of Clade I having B. greveolens, B. nigrescens, and B. plumea as closely related taxa. Morphological comparisons with the closely related taxa are given above in detail.

In the phylogram, three (3) sequences of B. plumbea fit best with a reported sequence of B. plumbea (DQ112613) with a good bootsrap value to 90%. Morphological identification was therefore confirmed by molecular data.

Subgenus Globaria

Taxa included in subgenus Globaria are B. aestivalis (Bonord.) Demoulin, B. ahmadii nom. prov., B. ayubiensis nom. prov., B. brunnescenta nom. prov., B. concinna, B. flavescens nom. prov., B. flavus nom. prov., B. himalaica Yousaf, Kreisel and Khalid, B. lahorensis nom. prov., B. levispora nom. prov., B. longispora Kreisel, B. minima nom. prov., B. oblongispora (Lloyd) Bottomley, B. pakistanica nom. prov., B. promontorii Kreisel, B. trachyspora (Lloyd) Kreisel, B. viridispora nom. prov., and Bovista sp. (BPK92).

Among these, 10 taxa seems previously undescribed, one new species (B. himalaica) has been published during this study by the author, four (4) are new records for Pakistan (B. aestivalis, B. longispora, B. promontorii, B. trachyspora). B. concinna is second time reported from Pakistan. Bovista sp. (BPK92) remained unidentified and require more detailed analysis to determine its fate.

In phylogenetic analysis, 21 sequences of 10 different species falls in Subclade II of Clade I, and a smaller Clade II. Sub clade I is labelled as subgenus Globaria (Fig. 4) 394

B. ahmadii nom. prov., (BPK35, BPK52) is characterized by the frequent pits in eucapillitial hyphae, indistinct sub gleba and asperulate basidiospores. It belongs to series Polymorhae of section Globaria. In phylogram (Fig. 3b), three sequences of B. ahmadii form a small clade with B. aestivalis (EU833650, FJ438477, AJ237613, DQ112620, BPK33, BPK70) and B. flavescens (BPK83) by maintaining its distinct status. Among all of these, one sequence, EU833650 is a close taxon, both share 97.2% genetic characters and show divergence of 2.3%, when sequence distances were computed. It is also morphologically close to B. aestivalis (Bonord.) Demoulin. Spores are round and asperulate in both taxa but size is different. Spores in B. aestivalis are smaller than those of B. ahmadii (4.0–4.7 µm vs 4.0–6.2).

B. ayubiensis nom. prov., (BPK3) bears no key resemblance with any of the reported Bovista spp. This species is characterized by brown fruiting bodies with brown gleba, having no sub gleba, smooth sub-pedicellate basidiospores, non-pitted intermediate type capillitium. Characters of this Bovista sp. suggest its placement in series Albosquamosae of section Globaria of Subgenus Globaria. Above combinations of characters are only found in B. oblongispora (Lloyd) Bottomley, and B. californica Kreisel. However, the former is separated from current taxon mainly due to having oblong basidiospores. Latter has the spores which are ornamented in contrast to smooth spores in currently studied taxon. Eucapillitiam threads are also septate and paracapillitium is also present in both taxa unlike current Bovista sp. Combination of these characters suggest Bovista sp. a novel taxon and as a new addition to series Albosquamosae.

Morphologically, it can also be confused with B. pila in the field but both the taxa are different anatomically. B. pila has typical Bovista type capillitium in contrast to the presence of intermediate type capillitium.

Bovista brunnescenta nom. prov., (BPK86) is characterized by brown endoperidium, absence of sub gleba, thick-walled, non-pitted intermediate type capillitium and ovoid basidiospores.This taxon does not bear any characteristic resemblance with any of the reported Bovista spp. in subgenus globaria On the other side, when compared with the species of subgenus Bovista, this taxon can be confused with B. plumbea Pers. and B. nigrescens in the field due to similar color of endoperidium. But anatomically, later two are distantly related with currently studied Bovista spp. because of presence of typical Bovista type capillitium in both species. This taxon is also close to B. longispora due to 395

similar shape of spores but latter has pitted capillitium. Characters of this taxon suggest its placement in series Trachysporae as a new taxon.

B. flavescens nom. prov., (BPK83) is characterized by globose to ovoid, finely warted basidiospores. Macroscopically it has characteristic yellowish brown cracking exoperidium and a compact sub gleba. It belongs to section Globaria, of subgenus Globaria. Due to absence of pits in the capillitium, it falls in series Albosquamosae. Eight Bovista species are included in this series by Kreisel viz., B. oblongispora (C. G. Lloyd) Bottomley, B. albosquamosa Kreisel, B. heterocapilla Kreisel, B. californica Kreisel, B. citrina (Berk. & Broome) Bottomley, B. abyssinica Mont. B. colorata (Peck) Kreisel, and B. coprophila (Cooke & Mass.) G. H. Cunn. Among these, B. oblongispora, B. californica, B. coprophila, B. abyssinica, B. colorata share the character of apedicellate to sub- pedicellate spores. First two species are distinguished from current species by the absence of sub gleba, but the presence of subpedicellate spores is a shared characer. Sub gleba is present in rest of the four Bovista spp., but spores are pedicellate in this case which is present in our specimen. Presence of sub gleba and subpedicellate basidiospores is a new combination in this series which is represented by currently studied taxon and represents a novel taxon. In phylogenetic tree, this taxon appears distinct and a sister speices to B. aestivalis (as mentioned above). When analyzed genetically, all of sequences of B. aestivalis and B. flavscens share up to 87.5% genetic characters, and show divergence of 8.5% suggesting big differences in both taxa.

B. himalaica has been published in Yousafet al., 2013a.

B. levispora nom. prov. (BPK87) is characterized by the small fruiting bodies with distinct sub gleba, smooth basidiospores, and frequently pitted, Lycoperdon type capillitial hyphae. It belongs to the series Asperae of section Lagoperdon of subgenus Globaria. These features are also shared by B. aspera Lév., and B. africana Kreisel. But a major difference suggesting segregation of these species from Bovista sp. in this study is size of basidiospores. Spores are bigger in presently studied Bovista sp. as compared to spores of both above mentioned species (up to 4.1 µm & 5.2 µm vs 7.5 µm). This seems to be a novel taxon and an addition to the series Asperae of section Lagoperdon. 396

B. lahorensis nom. prov. (BPK85), B. viridispora nom. prov. (BPK89), B. minima nom. prov. (BPK90), B. flavus nom. prov. (BPK91) and Bovista sp. (BPK92) are Bovista species with fruiting bodies less than 20 mm in diameter. These are segregated from all other reported Bovista sp. by their fruiting bodies’ small size. All of these can be grouped easily in two sections of subgenus Globaria. i.e. Lagoperdon, and Xyloperdon

B. lahorensis (BPK85), B. flavus (BPK91), and Bovista sp. (BPK92) have Lycoperdon type eucapillitium, therefore belong to section Lagoperdon, while B. viridispora (BPK89), and B. minima (BPK91) have intermediate type of eucapillitium, so members of section Xyloperdon. B. minor Morgan is a species known for its small size of basidiomata but have Bovista type capillitium different from currently studied taxa.

B. lahorensis nom. prov. (BPK85) is characterized by a small basidioma with no subgleba, paracapillitium present, asperulate basidiospores and Lycoperdon type capillitium with pores. It can be compared with B. flavus (BPK91), Bovista sp. (BPK92). However, it can be differentiated from B. flavus because latter have eucapillitium without pores, no paracapillitium, smooth and larger basidiospores (3.5–4.4 vs 5.5–6.0 µm). Enoperidium is aseptate in B. lahorensis whereas frequently septate in B. flavus.

It also seems close to Bovista sp. (BPK92) beacause both share basidiomata with no subgleba and presence of paracapillitium. But there are also some characters which are different in both. Spores are smooth and larger (3.5–4.4 vs 5.0–6.4 µm), pores are absent in eucapillitial hyphae and endoperidium is frequently septate in Bovista sp. (BPK92) compared to aseptate endoperidium in B. lahorensis.

B. flavus nom. prov. (BPK91) is characterized by small basidiomata without subgleba, smooth basidiospores and Lycoperdon type capillitium with no pores. It is close to Bovista sp. (BPK92) because both have small fruiting bodies with no sub gleba, smooth basidiospores, Lycoperdon type eucapillitium without pores and septate endoperidial hyphae. However, B. flavus is differentiated from it by color of basidioma which is characteristic yellow versus brown in Bovista sp. (BPK92) and absence of paracapillitium which is present in Bovista sp. (BPK92) suggesting to treat both taxa separately.

B. viridispora nom. prov. (BPK89) is characterized by small, yellowish brown fruiting bodies with no subgleba. Spores are greenish brown, globose and asperulate, with intermediate type, thick-walled eucapillitium. Apedicellate basidiospores and absence of 397

pits and similar diameter of eucapillitium brings this taxon close to B. abyssinica Mont. However, sub gleba is present in B. abyssinica and absent in B. viridispora. It is also comparable to B. minima (BPK90). Both have no subgleba and intermediate type of capillitium without pores. However, spores are asperulate in B. viridispora and smooth in B. minima.

B. minima nom. prov. (BPK90) is characterized by small basidioma with no sub gleba, sub globose smooth spores, intermediate type of eucapillitium without pores. Its comparison with B. viridispora is given in above paragraph.

In phylogram, B. promontorii (BPK58) and one sequence of same species from genbank (DQ112621) clustered with each other (Calonge & Syvokon, 2008).

B. pakistanica nom. prov., has intermediate type of capillitium, and asperulate, globose basidiospores with fruiting bodies less than 25 mm in diam., having no sub gleba. Among small Bovista spp. described in this study, it appears close to B. lahorensis, and B. viridispora because all of these have asperulate basidiospores. It can be differentiated from B. lahorensis because latter have Lycoperdon type capillitium. B. viridispora has capillitium with wall thickness up to 3 µm, however, wall of capillitium is very thin, up to 1 µm in B. pakistanicus. In phylogenetic analysis, four (4) sequences of B. pakistanica form a separate clade showing its distinct status from the rest of Bovista species of Clade I.

Calvatia Fr. Genus Calvatia has been established by Fries 1849 by describing C. cyathiformis as a type species. It is characterized by medium to large sized basidioma with a reduced or defined sterile base. Dehiscence occurs by irregular rupturing of peridium, not through a definite pore. (Calonge & Martín, 1990; Li, 2011; Cortezet al., 2012). In the beginning, many species being treated under the genus Lycoperdon were transferred to Calvatia by Morgan (1980). Zeller and Smith (1964) had written a book “Genus Calvatia in North America” and provided keys, classification status of 32 Calvatia species of the region belonging to 7 stripes with some taxonomic details. This genus has been extensively studied by many mycologists (Kreisel, 1992, 1994; Calonge & Martı´n, 1990; Lange, 1990, 1993, 1994; Calonge, 1998; Gube, 2007; Kasuya & Kotumoto, 2008; Cortez & Alves, 2012). 398

Species of this genus are distributed world-wide, reported from Asia (Ahmad, 1952; Eckblad, 1957, 1970; Ito, 1959; Liu, 1984; Imazekiet al., 1988; Fan et al., 1994; Ahmadet al., 1997; Moreno et al., 1998; Khalid & Iqbal, 2004; Kasuya & Retnowati, 2006; Shrestha & Kropp, 2009; Kumar & Sharma, 2011; Kasuya, 2010, Kasuya & Kotumoto, 2008), Africa (Bottomley, 1948; Dring, 1964), Australia (Grgurinovic, 1998), Europe (Kreisel, 1973; Calonge, 1990; Morenoet al., 1996; Cortezet al., 2012), North America (Morgan, 1890; Coker & Couch, 1928; Zeller, 1947; Garner, 1956; Zeller & Smith, 1964; Ponce de Leon, 1976; Burk, 1983; Urista et al., 1985; Calonge et al., 2003; Miller & Miller, 2006; Bates, 2004; Richter & Morse, 2008; Bates et al., 2009; Kasuya, 2010; Gabel & Gabel, 2011); South America (Rick, 1961; Silveira, 1943; Baseia, 2003; Wartchow & Silva, 2007; Baseia & Calonge, 2008; Suárez et al., 2009; Trierveiler-Pereira & Baseia, 2009; Cortez et al., 2012; Alves & Cortez, 2013). Total number of the Calvatia species reported to be forty (40) so far (Hawksworth et al., 1996; Kirk et al., 2003, 2008; Coetzee & van Wyk, 2007; Alves & Cortez, 2013).

Many species of this genus are of wide importance with reference to its edibility and its biotechnological potential (Gasco et al., 1974; Umezawa et al., 1975; Burk, 1983; Liu, 1984; Kawahara et al., 1995; Pegler et al., 1995; Lam et al., 2001; Coetzee & van Wyk, 2009; Li, 2011; Leffingwell & Alford, 2011). Genus Calvatia in Pakistan is represented by five species, i.e., C. ahmadiana, C. ahmadii. C. craniiformis, C. cyathiformis, and C. excipuliformis var. excipuliformis (Ahmad, 1952, 1956; Khalid and Iqbal, 1996, 2004). In this investigation, four Calvatia species were collected from different regions of the country and described here on morphological and molecular basis. These are C. cyathiformis var. fragilis nom. prov., C. deosaiensis nom. prov., C. lilacina (Mont. & Berk.) Henn., and C. pseudocyathiformis nom. prov. Among these, two taxa are presented here as new species. One taxon is proposed as a new variety of C. cyathiformis. One taxon, C. lilacina is a new record for Pakistan. C. cyathiformis var. fragilis nom. prov. (CPK12) is characterized by off-white, turbinate basidioma with reduced sterile base. It has verrucose basidiospores and pitted capillitium. Morphologically, it is also very close to C. cyathiformis but spores are comparatively smaller in current spcies (up to 7.0 vs 8.0 µm). Paracapillitium which is present in C. cyathiformis var. fragilis is absent in C. cyathiformis. It is also very close to 399

C. fragilis beacause both have similar basidioma and basidiospore’s morphology. Size of basidiopsores and eucapillitial hyphae is also same in both cases. However, phylogenetically, it is segregated from C. fragilis, therefore presented here as a variety of C. cyathiformis.

C. pseudocyathiformis nom. prov. (CPK4) is characterized by sub-globose to turbinate basidioma with short sterile base, and olivaceous echinate basidiospores. Morphologically C. cyathiformis and C. fragilis seems to be the closest taxa. Calvatia sp. (CPK4) shares the characters of fragile peridium with both the taxa but latter both have larger basidiospores (up to 8.0 µm) as compared to those of C. pseudocyathiformis (up to 6.0 µm). In addition, echines are smaller, capillitial threads are less in diam., and less thick walled in current taxon when compared with both C. cyathiformis and C. fragilis. It is proposed here as a new species.

During phylogenetic analysis of Calvatia, C. pseudocyathiformis (CPK4) and C. cyathiformis var. fragilis (CPK12) exhibit a distinct position with moderate to weak bootstrap support, 82% and 55 BS, respectively (Fig. 5). Both taxa form a clade with 7 sequences of C. cyathiformis var. cyathiformis, and 6 sequences of C. fragilis. They share 93.4% of genetic similarity and 1.9% divergence from each other.

C. pseudocyathiformis shares a maximum of 98.6% genetic similarity and a minimum of 0.8% genetic divergence whereas C. cyathiformis var. fragilis shows a maximum of 90.3% similarity and a minimum of 1.9% divergence with with all of seven sequences of C. cyathiformis var. cyathiformis.

CPK12 is phylogenetically more close to C. cyathiformis var. cyathiformis and morphologically close to C. fragilis so described as a variety of C. cyahiformis var. fragilis.

In phylogenetic analysis, 2 sequences of C. lilacina (CPK1) bear a distinct status but with weak bootstrap support, (60%). These 2 sequences form a small sister clade with C. cyathiformis var. cyathiformis, C. cyathiformis var. fragilis, C. fragilis and C. pseudocyathiformis (Fig. 5).

C. deosaiensis nom. prov. (CPK14) is characterized by thick, white exoperidium forming conspicuous polygonal warts or pyramids, attached to the endoperidium when young. It has smooth to finely warted, pedicellate basidiospores and aseptate, frequently pitted eucapillitium. The feature of having thick verrucose or warty peridium is also shared 400

by C. booniana which is a bigger specimen, up to 60 cm high compared to smaller specimen of C. deosaiensis (up to 3.5 cm high). Feature of having smooth spores (under LM) is also shared by C. craniiformis (Schwein.) Fr. But spores are apedicellate and small in C. craniiformis as compared to those of C. deosaiensis (4.8–7.6 vs 5.4–6.5 µm). It is proposed here as a new species.

Disciseda Czern. Genus Disciseda was first established by Czern. in 1845, by describing D. collabescens Czern. as type species. It has worldwide distribution and contains 15 species, known to occur in Asia, Africa, Australia, Europe and North America (Coker and Couch, 1928; Rick, 1961; Kers, 1975; Morales and Kimbrough, 1978; Jeppson, 1983; Aparicio- Navarro et al.,. 1994; Esqueda et al., 1995; Pérez-Silva et al., 2000; Gilbertson et al., 2001; Dörfelt and Nowak, 2002; Sarasini, 2005; Bates, 2006; Ochoa and Moreno, 2006; Esqueda et al., 2006; Wright & Albertó, 2006; Kirk et al., 2008; Lizárraga et al., 2010; Hernández- Navarro et al., 2013).

Members of this genus are characterized by hypogeous to semi hypogeous basidioma with circumscissile rupture of the exoperidium leaving half of it forming a cup at the basal portion and open by basal opening. They are adapted to deserticolous and sandy habitats (Mitchel et al., 1975; Morales & Kimbrough, 1978; Miller & Miller, 1988; Jeppson, 1996; Calonge, 1998; Moreno et al., 2003, 2010a; Jordal et al., 2007).

Previously, two (2) Disciseda species have been reported form arid regions of Punjab. These are D. candida (Schwein.) Lloyd and, D. cervina (Berk.) G. H. Cunningham (Ahmad, 1939, 1950; 1952; Ahmad et al., 1997).

In present investigation, one Disciseda sp. (DPK1) was collected from the floor of manmade forest “Changa Manga” and identified as D. hyalothrix. Its phylogenetic position is also given. D. cervina was taken as a loan from LAH herbarium and re-described and given at the end.

DPK1 is morphologically identified as D. hyalothrix. In phylogenetic analysis, two sequences of Disciseda sp. (DPK1) appears on a separate line distinguished from rest of the gasteroid genera of agaricaceae. It shows a maximum genetic similarity of 91.7% and divergence of 6.2% with D. candida (EU833654). With the other two sequences of D. bovista and D. candida (DQ11262, DQ112626), it shares less than 90% of genetic 401

characters and a least of 7% divergence. Due to this confusion, same morphological based identification is retained until more species of Disciseda will be sequenced and phylogeny of the genus will be carried out to make the status clear (Fig. 1).

It is an addition to gasteroid fungal flora of Pakistan.

Lycoperdon Pers.

Genus Lycoperdon was first established by Persoon in 1796 by describing L. perlatum as a type species. It is represented by 52 species worldwide (Kirk et al., 2008; Jeppson et al., 2012). It is characterized by sub globose to pyriforme basidomata with conspicuous sterile base and dehiscence by an apical pore. It has been studied extensively by many mycologist (Hollòs, 1904; Perdeck, 1950; Amhad, 1952; Smarda, 1958; Kreisel, 1962; Demoulin, 1971, 1972, 1979; Akata & Halici, 2010). Recent molecular phylogenetic studies have widened the concept of the genus by including the species of genus Bovistella, Handkea, , and Vascellum (Bates, 2004; Bates et al., 2009; Gube, 2009; Larsson & Jeppson, 2008; Jeppson et al., 2012).

Larsson & Jeppson (2008) established new limits of Lycoperdon by proposing subgenera within Lycoperdon, viz., Apioperdon, Bovistella, Lycoperdon, Morganella, Utraria and Vascellum.

In Pakistan, twenty one (21) Lycoperdon spp. are known reported from different regions (Ahmad, 1939a, 1939b, 1952, 1956, 1980a, 1980b; Mirza & Qureshi, 1978; Ahmad et al., 1997; Gardezi, 2005).

In this investigation, 28 Lycoperdon spp. have been identified and described. Among these, 17 Lycoperdon species belong to subgenus Utraria, six of subgenus Vascellum, 2 of subgenera Bovistella and Lycoperdon, and 1 of subgenus Apioperdon (Fig. 6).

L. pyriforme belongs to subgenus Apioperdon. Five sequences were generated from six different collections (LPK10, LPK14A, LPK17, LPK62, LPK48). One specimen of L. pyriforme (DH2) was collected from Boston Harbour Island, USA and its ITS-rDNA sequence was generated and included in the study. During phylogenetic analysis, all of the sequences fall in the clade of subgenus Apioperdon along with other sequences of L. pyriforme (AJ237618, AJ237619, AJ237620, AY854075) from Genbank. 402

Two species viz., L. japonicum (= Bovistella japonica), L. utriforme (= L. utriformis) belong to subgenus Bovistella during this study. According to Larsson and Jeppson (2008) it was proposed to accommodate the taxa of medium sized fruiting bodies with well-developed diaphragm.

L. japonicum (previously known as B. japonica) appears to cluster with other Lycoperdon species in subgenus Bovistella, in phylogenetic analysis based on ITS-nrDNA region and maintained its status as distinct species with moderate bootstrap support (84%). It is therefore shifted to Lycoperdon and renamed as L. japonicum. It forms clade with L. radicatum (DQ112608) and L. utriforme (AJ617490, DQ112607, EU833659).

L. japonicum (≡ B. japonica) is characterized by the presence of short, separate capillitial units, pedicellate spores, and sterile base with gleba having capillitial units, typical of Bovista, These characters are also shared with B. ohiensis (Ellis & Morgan) Morgan, B. radicata (Durieu & Mont.) Pat., B. lycoperdoides (Schwein.) Lloyd, B. paludosa (Lév.) Pat., and B. ammophila (Lév.) Lloyd. Among these taxa, B. japonica is more closely allied with B. ohiensis and B. paludosa, which all share the same general spore dimensions (~4–5 µm). Bovistella japonica, however, can be distinguished from these taxa as it has a more compact sterile base. This species is a new addition to the mycoflora of Pakistan.

L. utriforme (CPK5) falls in the same clade and fits well with two sequences of L. utriforme (EU833659, DQ112607) from Genbank, with a bootstrap support of 83%, therefore named the same (Fig. 6).

Areolate structure of exoperidium after falling off spines, absence of diaphragm, abundant eucapillitium are the key features of Lycoperdon species belonging to subgenus Lycoperdon (Larsson & Jeppson, 2008).

In this investigation, two Lycoperdon species belong to subgenus Lycoperdon, these are L. parvasporum nom. prov., and L. perlatum (Fig. 6).

L. parvasporum nom. prov., (LPK46) is characterized by pyriforme basidiomata having prominent exoperidial warts leaving a reticulate pattern on the endoperidium after sloughing off. Morphologically it can be confused with L. perlatum because of having similar morphology but both taxa have anatomical differences. Major difference is the ornamentation of basidiopsores in both Lycoperdon species. Basidiospores are olivaceous 403

and finely verrucose and smaller in present taxon whereas spores are brown and distinctly verrucose and larger in L. perlatum (3.9–5.0 vs 3.5 µm).

One sequence of L. parvasporum (LPK46) falls in the clade of subgenus Lycoperdon along with L. marginatum (DQ112630), L. norvegicum (DQ112631), L. perlatum (KF551249, DQ112630, AJ237627, LPK73, LPK82). Among these, it appear as as a distinct species and as sister species to L. perlatum with moderate bootstrap support of 75%. Morphologically, it is also distinct from L. perlatum. It shares maximum genetic similarity of 94.6% and divergence of 2.2% with L. perlatum (AJ237627). It is described here as a new species in subgenus Lycoperdon.

Two collections of L. perlatum (LPK73, LPK82) fits well in a clade of subgenus Lycoperdon with other sequences of L. perlatum (KF551249, DQ112630, AJ237627). Among all of these, LPK82 and LPK73 shows maximum genetic similarity (99.6% and 99.3%) with L. perlatum (DQ112630), respectively.

Total number of Lycoperdon species belonging to subgenus Utraria of Lycoperdon described in this study is seventeen (17). In addition, three (2) Lycoperdon species (MPK5, LPK47) are included only in phylogenetic analysis (Fig. 7).

Among seventeen species, seven species are described as new to science, three are presented as new varieties, five species are new records for the country, and one species is described from new locality of Pakistan. One taxon (L. pusillum) is re described based on Sultan Ahmad’s collection for comparison with other taxa.

New Lycoperdon species are L. albiceratum nom. prov. (LPK37/MPK2), L. aurea- brunneum nom. prov. (LPK1), L. lignum-brunneum nom. prov. (CPK3), L. lineum nom. prov., (LPK19/ LPK14/ BPK45/ BPK68), L. pseudoniveum nom. prov., (LPK2), L. sclerocystis nom. prov. (LPK23/ LPK35), and L. sharanense nom. prov. (LPK59).

L. albirceratum nom. prov. (LPK37/ MPK2) is characterized by sub-globose to pear shaped basidiomata, which often form a pseudo-stipe. It has the characteristic off-white to light yellow exoperidium which resembles with peridium of L. pratense, but L. pratense has a diaphragm and smaller sized fruiting bodies. In phylogenetic analysis, it is close to L. excipuliforme (DQ112590). Anatomically, this species also matches with L. excipuliforme because both have verrucose basidiospores and have detached sterigmal remnants in mounts but size of basidiospores differ in both cases. Spores in L. excipuliforme are larger 404

(4.4–5.5 µm vs 3.3–4.8 µm) than spores in L. albiceratum. It is presented here as a new species.

L. aurea-brunneum nom. prov. (LPK1) is characterized by pear shaped medium sized basidiomata having exoperidium in the form of warts that converge with each other. Exoperidium peels off in sheets with age exposing the golden brown smooth endoperidium. This particular appearance of exoperidium bring this taxon close to L. marginatum Vittad., in which exoperidium also peels off in the form of sheets (also known as peeling puff ball). However both of these taxa differ in anatomical details. Spores are up to 5.0 in L. marginatum as compared to larger spores (6.0 µm) of Lycoperdon sp. (LPK1). It is presented here as a new species. Phylogenetically, it has distinct status with 75% bootstrap support. It appears close and as a sister species of L. subumbrinum. It shares 91.5% and 91.6% genetic similarity and 1.3% and 1.4% divergence with L. subumbrinum (DQ112601, JN572906), respectively. Anatomically, LPK1 has larger spores as compared to those of L. subumbrinum (4.75–6.0 vs 4.0–4.5 µm).

L. lignum-brunneum nom. prov. (CPK3) characterized by medium sized, sub- globose to pear-shaped basidiomata, verrucose basidiospores and thick-walled eucapillitial hyphae. It is very close to L. rimulatum beacause of similar spore morphology and size but eucapillitial hyphae and more thick walled in current Lycoperdon sp. as compared to those of L. rimulatum (1.0 vs 3.5 µm). It is proposed here as new species.

L. lineum nom. prov. (LPK19/ LPK14/ BPK45/ BPK68) is characterized by sub- globose to turbinate basidiomata with small to well-developed sterile base. Basidiomata is grayish brown to linen with small brownish black warts and granules on exoperidium. Dehiscence is typical by an apical pore. Morphologically it is very close to L. lambinonii because of same shape of basidiomata. However spores are larger in L. lineum than L. lambinonii (6.5 µm vs 4.5 µm). Phylogenetically, it is close to L. altimontanum (DQ112588) and L. excipuliforme (DQ11259) but without a satisfactory bootstrap support. It is presented here as new species.

L. pseudoniveum nom. prov. (LPK2) is characterized by small, olivaceous brown, basidioma, with short basal rhizomorphs and finely echinulate basidiopsores. Morphologically it is close to L. niveum because of same size and morphology of basidioma. Both taxa have finely ornamented basidiospores and pitted eucapillitium. 405

However it is differentiated from L. niveum by poorly developed sterile base. Another difference is the presence of thin walls of eucapillitial hyphae in LPK2 as compared to thick walled capillitium of L. niveum. Clustering of LPK2 with L. cretaceum is no supported with bootstrap value and is therefore, questionable. This taxon is presented here as a new species

L. sclerocystis nom. prov. (LPK23/ LPK35) is characterized by medium sized, sub- globose to pear-shaped basidiomata. Distinguishing features of this taxon includes thick- walled, polymorphic, mycosclerieds as exoperidium and presence of amorphous material attached to the verrucae of the spores. These two features does not bear any characteristic resemblance with reported Lycoperdon sp. However, B. sclerocystis has this feature of having exoperidium composed of mycosclereids (Calonge et al., 2004). It is presented here as a novel taxon.

L. sharanense nom. prov. (LPK59) is characterized by pear-shaped, grayish brown basidiomata with granular exoperidium and olivaceous brown pedicellate spores. Lycoperdon cretaceum is a closely related species but peridium in this case is composed of polygonal warts with clusters of spines in upper part and cracking into irregular areas. However peridium is almost smooth without forming polygonal warts or patches in current species. Basidiospores are smaller in L. cretaceum (up to 5 µm) as compared to spores of Lycoperdon sp. (LPK59) which are up to 7.3 µm in diameter. It is described here as a new species. Its segregation from L. cretaceum as a distinct species is supported by moderate bootrap value of 76%.

Three new varieties are proposed, viz., L. dermoxanthum var. album (BPK36), L. dermoxanthum var. flavum (BPK31), L. mammiforme var. magnasporum (LPK55

L. dermoxanthum var. flavum (BPK31) is characterized by small, yellowish basidioma with a little rhizomorphs at the base. Spores are asperulate with pitted capillitium. These characters bring this taxon close to L. dermoxanthum but color of peridium brings difference in both taxa. Peridium is buff to brown in L. dermoxanthum whereas it is yellow in present taxon. It is therefore treated as the variety of L. dermoxanthum. Exoperidium peels off in the form of furfuraceous scales in L. dermoxanthum but exoperidium is smooth to granular in current taxon (BPK31).

L. dermoxanthum var. flavum (BPK31) is characterized by off-white basidioma with dehiscence by an apical pore. It has small sub gleba and light brown spores and 406

capillitium. Both the taxa (BPK31 and BPK36) share close resemblance with each other and also with L. dermoxanthum. In phylogenetic tree, these species are segregated from L. dermoxanthum by 91% bootstrap value. However, they also differ from each other in a tree with good bootstrap support of 98%. This is also supported by morphological data. In addition to difference in color of basidioma which is yellow in BPK31 and off-white in BPK36, they also have some anatomical differences. BPK31 have larger spores (4.3–4.8 µm) that are olivaceous yellow, than smaller basidiospores (3.5–4.0 µm) of BPK36, where spores and capillitium are light brown.

Both of these taxa have been proposed here as variety of L. dermoxanthum, i.e. L. dermoxanthum var. album (BPK36), L. dermoxanthum var. flavum (BPK31).

L. mammiforme var. magnasporum (LPK55) is characterized by off-white to ochraceous basidiomata, with exoperidium peeling off in forms of sheets or patches first from the sporecase then from the pseudo-stipe. These characters bring it close to L. mammiforme Pers., also known as flaky puffball. But L. mammiforme has smaller basidiospores as compared to the former (4.0–5.0 vs 5.8–7.3 µm). During phylogenetic analysis, it is segregated from three sequences of L. mammiforme with bootstrap support of 87%. Current Lycoperdon sp. (LPK55) is therefore proposed to be the variety of L. mammiforme.

L. altimontanum Kreisel, L. excipuliforme (Scop.) Pers., L. mammiforme Pers., L. nigrescens Pers., and L. niveum Kreisel are new records for the country. L. molle Pers., is being described from new localities of Pakistan.

Lycoperdon sp. (LPK74) remained unidentified and requires further analysis.

Presence of a papyraceous diaphragm between the glebal and subglebal tissues, reduced or absent eucapillitium, abundant paracapillitium and asperulate basidiospores are important and taxonomic features in subgenus Vascellum and play a crucial role in the identification of taxa.

Subgenus Vascellum corresponds to genus Vascellum Smarda. It has been extensively studied by many mycologists (Kreisel, 1962; Ponce de Leon, 1970, Kreisel & Dring, 1967; Smith et al., 1973; Smith, 1974; Homrich & Wright, 1988; Kreisel, 1993; Kreisel & Hausknecht, 2001; Krüger et al., 2001; Bates et al., 2009; Coetzee & van Wyk 2010). Habitat of the species ranges from pastures, meadows and savanas of lower altitude 407

to prairies and grasslands of higher elevations which are distributed worldwide. Some of the species are involved in the formation of fairy rings in turf grass (Miller JR, 2010).

In this investigation, six (6) Lycoperdon species belong to subgenus Vascellum. Among these three (3) species are new to science, and three (3) new varieties are proposed (Fig. 6). Lycoperdon spp. new to science are L. gilgitti nom. prov. (VPK9), L. lahorense nom. prov. (VPK4, VPK6), and L. olivoflavum nom. prov. (VPK2). New varieties are L. curtisii var. ovalisporum nom. prov. (VPK1), L. lahorense var. parvasporum nom. prov. (VPK3), and L. pratense var. flavum nom. prov. (VPK8).

All of the Lycoperdon spp. with more or less definite diaphragm collected in this study clustered together in two sub clades of clade of subgenus Vascellum. Sub clade I consists of four sequences of L. pratense (DQ112554, DQ112555, DQ112556, AJ237625), and one sequence of V. intermedium (EU833667), L. pratense var. flavum (VPK8), and L. olivoflavum (VPK2).

L. giltitii nom. prov., (VPK9) is characterized by a small basidioma with off-white exoperidium and light grayish gleba and sub-gleba. It has well defined diaphragm and gleba has true capillitium in addition to paracapillitium. In phylogenetic analysis, L. gilgitti (VPK9) shows its distinct position but without good bootstrap support. It is phylogenetically and anatomically close to L. pratense and L. olivoflavum due to the presence of eucapillitial hyphae in the gleba. But color of endoperidium and gleba of L. gilgitii (VPK9) is different from the others. It is off-white to grayish in VPK9 whereas it ranges from brown to olivaceous yellow in the latter, respectively.

L. lahorense nom. prov. (VPK4, VPK6) is characterized by sub-flocculate to slightly warted exoperidium, bi-colored exoperidium with wel-defined apical pore, asperulate basidiospores and absence of eucapillitial threads in the gleba. Three sequences of L. lahorense form a sub clade II (Clade I) with L. curtisii (HQ235043, HQ235045), L. wrightii (= L. curtisii), (HM237177) having 97% bootstrap support. Both of these taxa share the characters of dehiscence of basidiomata through apical pore and presence of asperulate globose basidiospores. However these species also bear morphological differences. Exoperidium in both species is fragile and deciduous but different in visual appearance. It is composed of prominent, convergent fibrils in L. curtisii when young in contrast to powdery appearance in present species. Characteristics bi-colored endoperidium 408

is also a unique feature in L. lahorense. In L. curtisii gleba contains true eucapillitial threads along with abundant paracapillitium, but true capillitium is absent in LPK6. Diaphragm is another feature different in both species. It is very distinct in L. lahorense and lacking in L. curtisii.

L. olivoflavum nom. prov. (VPK2) is characterized by subglobose, off-white to pale yellow to olive-yellow basidiomata with irregular to slit-like dehiscence. Fruiting bodies often remained attached with each other even at maturity. In phylogenetic tree, its distinct position in a sub clade I is supported by 72% bootstrap value. Morphologically L. olivoflavum and L. pratense share the characters of presence of eucapillitium, asperulate, globose basidiospores and irregular dehiscence pattern. However these species are different by size and color of basidiomata and texture of exoperidium. Diaphragm which is quite distinct in L. pratense, is not much developed in L. olivoflavum.

L. pratense var. flavum nom. prov. (VPK8) maintained its unique status in phylogenetic analysis but without satisfactory bootstrap value. It is close to L. pratense morphologically and moleculary. Both taxa though have a slight difference in the color of basidioma. Yellowish basidioma in VPK8 suggested a variance from L. pratense in which basidioma is light brown upon maturity. L. pratense have true capillitium which is absent in VPK8. Therefore it is described here as a variety of L. pratense.

L. lahorense var. parvasporum nom. prov. (VPK3) is close to V. lahorense both morphologically and phylogenetically. In both taxa, only difference is the smaller size of the basidiospores in VPK3 than those of L. lahorense (2.8–3.8 µm vs 3.5–4.3). It is therefore described as a variety of L. lahorense.

L. curtisii var. ovalisporum nom. prov. (VPK1) is phylogenetically close to L. curtisii (HQ235045, HQ235043) but differs anatomically. L. curtisii has globose spores while VPK1 has subglobose to ovoid spores. It is treated here as a variety of L. curtisii.

Montagnea Fr. This genus was first established by Fries in 1836 (Baseia & Milanez, 2002a) and usually found growing in hot and arid regions. It has worldwide distribution and represented by 5 species (Kirk et al., 2008). It is characterized by a pileus with radial lamellae, hard stipe and bulbous base. Basidiospores are dark and have prominent germ 409

pore. Morphologically it is considered closely related to genus Coprinus (Redhead et al., 2001; Simpson & Grgurinovic, 2001).

This genus in Pakistan is represented by only one species, M. arenaria (DC.) Zeller from different regions of Punjab.

In present investigation, two specimens of M. arenaria were collected again from different localities of Punjab with arid to semi-arid climate and identified using morphological methods.

Podaxis Desv.

This genus belongs to family agaricaceae, first established by Desv. in 1809 by describing P. senegalensis Desvaux as a type species. It has worldwide distribution and is characterized by larger basidiomata with a pileus, stalk and bulbous base (Massee, 1890; Morse, 1933; Brasfield, 1937; Bessey, 1950; Martínez, 1971; Dring, 1973; Moustafa, 1975; McKnight & Stransky, 1980; Hilton & Kenneally, 1981; Ewaze & Muhsin, 1988; Miller & Miller, 1988; DeVilliers et al., 1989; Priest & Lenz, 1999; Baseia & Galvao, 2002; Young et al., 2002; Al-Thani, 2010). This genus is well adapted to arid regions. Previously, it was thought that this genus resembles with and this was also confirmed using molecular techniques and found close to Agaricus/Lepiota clade (Smith, 1973; Hopple & Vilgalys, 1994, 1999; Moncalvo et al., 2002; Keirle et al., 2004).

Several uses of the genus are also known as medicines, food, and face paints (Cleland & Johnston, 1933; Bottomley, 1984; Gupta & Singh, 1991; Hashem & Al- Rahmah, 1993; Mao, 2000; Jiskani, 2001; Diallo et al., 2002; Al-Fatimi et al., 2006; Khalaf, 2008).

In Pakistan, this genus was only represented by one species, i.e. Podaxis pistillaris. In this investigation, many fruiting bodies of P. pistillaris were collected from different regions of Punjab and Khyber Pakhtunkhwa and identified using morphological and molecular methods.

In phylogenetic analysis, two sequences of P. pistillaris (PPK6, GPK33) form sister clade with Lepiota Clade and cluster with other sequences of P. pistillaris (AF059285, PPU85336, DQ311082, DQ311086, AM234060, GQ249882) reported from different countries (Fig. 8). 410

Tulostoma Fr. Tulostoma was first proposed by Persoon in 1801, characterized by sporophore consisting of spore case and a stalk. Members of this genus are called stalked puffballs usually found in arid and semi-arid regions (Miller & Miller 1988, Calonge, 1998; Valle et al., 2000). This genus comprises 81 species having worldwide distribution (White, 1901; Cunningham, 1944; Long & Ahmad, 1947; Demoulin, 1968; Wright et al., 1972; Calonge & Demoulin, 1975; Brochmann et al., 1981; Kreisel, 1984; Binyamini & Wright, 1986; Wright, 1987a, 1987b; Altés & Moreno, 1991; Bilgrami et al., 1991; Calonge & Martín, 1992; Moreno et al., 1992; Altés & Moreno, 1993; Moreno et al., 1994, 1995, 1997; Coetzee et al., 1997; Esqueda et al., 1998; Altes & Moreno, 1999; Sesli et al., 2000; Baseia & Galvão, 2002; Baseia & Milanez, 2002b; Calonge & Carranza, 2003; Asai, 2004; Kirk et al., 2008; Cortez et al., 2010; Esqueda et al., 2010; Piña et al., 2010).

Tulostoma in Pakistan comprises twenty five species (Ahmad, 1939, 1952, 1956; Ahmad et al., 1997). Most of these species are reported from sub-tropical, arid regions of the country, Punjab (Bahawalpur, Sargodha, Sheikhupura, Zafarwal), Sindh (Kirana hills) and a few species have been reported from Murree (Punjab), Swat (northern areas), and Gilgit-Baltistan.

In this investigation, four Tulostoma spp. were collected from different regions of Pakistan and described here using morphological and molecular methods. These are T. fimbriatum, T. morenoi nom. prov., T. squamosum, Tulostoma sp. 1 (TPK6), and Tulostoma sp. 2 (TPK4).

T. morenoi seems previously undescribed, T. squamosum is a new record for the country and T. fimbriatum has been reported this time from different locality of the country.

T. morenoi nom. prov. (TPK1) is characterized by off-white, smooth endoperidium, circular peristome and verrucose basidiospores. In phylogenetic tree, it forms a small clade along with one other sequence of T. fimbriatum (TPK8) and appear as a sister species of it. When pairwise sequences distances of both of these were calculated, it was found that they both share 82.8% of genetic characters and a difference in sequence was 9.2%, therefore suggesting both sequences are very different from each other.

In phylogram, T. squamosum clustered with a sequence of T. squamosum (DQ415732) already available from genbank with 99 % bootstrap support. It is an addition 411

to fungal flora of Pakistan. T. fimbriatum also maintained its distict status with the genus in phylogenetic analysis. Tulostoma sp. 1 (TPK6), and Tulostoma sp. 2 (TPK4) needs further analysis and remained unidentified during this study.

Cyathus Haller

Cyathus is a common genus of birds nest fungi and can be distinguished from Nidula by the presence of a funiculus cord at point of attachment of peridiole with the peridium. It is widely distributed and found growing on wooden logs, animal dung and on soil rich in organic matter (Brodie, 1966, 1967, 1971, 1977; Ren & Zhou, 1992; Chen et al., 2002; Zhou et al., 2004; Dorjey et al., 2013). This genus comprises 45 species worldwide (Kirk et al., 2008). Several morphological groups within the genus were proposed by many mycologists based on the structure of peridium and the color of perdioles (Tulasne & Tulasne, 1844; White, 1902; Lloyd, 1906b). Phylogenetic studies on genus Cyathus suggested three groups of Cyathus spp. according to clustering of taxa in the clades. These are ollum, striatum, and pallidum groups (Zhao et al., 2007).

Species of the genus have also been studied extensively for their antimicrobial, antioxidant and decomposition potential, and ability to decolorize dyes (Allbutt et al., 1971; Johri & Brodie, 1971; Johri et al. 1971; Ayer & Taube, 1973; Ayer & Paice, 1976; Ayer & Taylor, 1976; Anke et al., 1976, 1977; Anke & Oberwinkler, 1977; Ayer & Yoshida, 1978; Ayer & Lee, 1979; Ayer & Singer, 1980; Abbott & Wicklow, 1984; Saxena et al., 1994; Vasdev & Kuhad, 1994a, 1994b; Sethuraman et al., 1998; Shinners-Carnelley et al., 2002; Liu & Zhang, 2004; Blenis & Chow, 2005; Vasdev et al., 2005; Mishra et al., 2006; Petrova et al., 2007; Chhabra et al., 2008; Kang et al., 2007, 2008).

Cyathus stercoreus (Schwein.) De Toni is reported to be a threatened species and included in the red list of many countries (Gyosheva et al., 2006).

This genus in Pakistan comprises only two species C. limbatus Tul. & C. Tul., and C. stercoreus. These species have been reported from arid regions of Punjab province by Ahmad (1939, 1980).

In this investigation, two Cyathus spp. were collected from two different climatic regions and identified on morphological and molecular basis. Cyathus olla is a new record for Pakistan and C. pakistanicus is presented here as a new species (Fig. 10). 412

In phylogenetic tree, one sequence of C. olla (CPK36) clustered with three other sequences of C. olla (DQ463345, KC989088, DQ463343) from Genbank (Fig. 10).

Cyathus pakistanicus nom. prov. (CPK10) is characterized by fruiting bodies growing on dung having numerous black peridioles. C. stercoreus (Schwein.) De Toni is a closely resembling taxon because of similar habit of growing on dung. However both taxa are different anatomically. Basidiospores are ellipsoid to oblong and small in CPK10 while spores in C. stercoreus are roughly spherical and large (35 × 25 µm vs 15 × 10 µm). Phylogenetically it is also a distinct species with a good bootstrap support of 100%, and appears as a sister species of C. stercoreus. C. pakistanicus shares 94.1% genetic characters with three sequences of C. stercoreus (DQ463354, FJ478125, EU784193) with divergence up to 3.6%.

Phellorinia Berk. Phellorinia was erected by Berkeley in 1843 with P. inquinans Berk., as the type species. It is a monotypic genus and widely distributed across arid to semi-arid regions of all continents, including countries such as Australia (Fuhrer, 2005; Williams & Woinarski, 1997), Pakistan (Ahmad, 1952; Ahmad et al., 1997), Spain, and Yemen (Kreisel & Fatimi, 2004). This genus is Pakistan is represented by P. inquinans described from many arid to semi-arid regions.

In present investigation two fruiting bodies of P. herculeana was collected from cholistan desert, Bahawalpur and identified on morphological basis. It has previously been reported from different regions of Pakistan viz., Sargodha, Multan, Jhang by Ahmad (1939, 1952). It is a first record of this taxon from Cholistan desert of Bahawalpur district.

Boletales was first established to accommodate genus Boletus. Recent molecular analysis revealed that this order also contain some non- as well. These are some gilled mushrooms and gasteroid fungi. Among the gasteroid fungi, members belonging to family Diplocystaceae, Rhizopogonaceae, and Sclerodermataceae are included (Binder & Bresinsky, 2002; Binder & Hibbett, 2006).

This order contains a big number of taxa that are edible and ectomycorrhizal. Nearly all of the gasteroid fungi included in this order are known to form ectomycorrhizal associations with wide range of trees, these are Astraeus spp., Pisolithus spp., Rhizopogon spp., and Scleroderma spp. 413

Gasteroid fungi of boletales group in Pakistan is also represented by species of aforementioned genera. This includes six (6) species of Scleroderma, 1 each of genus Astraeus, Rhizopogon and Pisolithus (Ahmad, 1952; Ahmad et al., 1997; Razzaq & Shahzad, 2004).

In present study, 13 taxa belonging to boletales have been described and identified. Among these, 6 species belong to genus Scleroderma, 3 of Pisolithus, 3 of Rhiopogon and 1 of genus Astraeus.

Astraeus Morgan

Genus Astraeus was first proposed by Morgan by describing A. hygrometricus as a type species in 1989. This genus is characterized by star shaped splitting of exoperidium in the form of rays and rays are hygroscopic in nature (Miller & Miller, 1988). Formerly this was thought belonging to Geastrum by several mycologists (Coker & Couch, 1928). However, it differs from Geastrum due to many reasons. Morphologically, Astraeus does not have a definite peristome as in Geastrum, anatomically absence of columella, larger basidiospores and thick-walled, branched, septate capillitium in Astraeus supports its segregation from Geastrum.

Phylogenetically, this genus falls in sub order of order boletales along with Boletinellus Murrill, Desv., Gyroporus Quél., Phlebopus (R.Heim) Singer, Pisolithus Alb. & Schwein, Scleroderma Pers., and Veligaster Guzmán (Binder & Bresinsky, 2002).

This genus is common and usually found in tropical and temperate regions (Lloyd, 1902). This genus is well known for forming ectomycorrhizal associations with a wide range of host trees (Trappe, 1967; Molina, 1979; Malajczuk et al., 1982; Cannon and Kirk, 2007). This genus is widespread and reported from Asia, Australia, Europe, and North America (Nouhra & De Toledo, 1998; Baseia & Galvão, 2002; Fangfuk et al., 2010a, 2010b; Giri et al., 2012; Kennedy et al., 2012; Phosri et al., 2013). This genus has also been studies for its antimicrobial activities and medicinal value (Stanikunaite et al., 2008; Biswas et al., 2011; Giri et al., 2012; Lai et al., 2012). Its edibility is also known in different cultures (Petcharat, 2003; Fangfuk et al., 2010b).

Morphologically, it was thought that there are only two species of this genus, A. hygrometricus (Pers.) Morgan and A. pteridis (Shear) Zeller. (Bisby et al., 2001). Later on 414

molecular study based on ITS rDNA region’s sequences obtained from many collections from different countries revealed many taxonomic complexities within the genus. Phosri et al. (2013) and Fangfuk et al., (2010a) concluded that there are more than two species within the genus and A. hygrometricus also show complex behavior when many collections were compared molecularly. This genus is now represented by eight species, viz., A. asiaticus Phosri, M.P. Martín & Watling, A. hygrometricus (Pers.) Morgan, A. koreanus (V.J.Staněk) Kreisel, A. morganii Phosri, Watling & M.P.Martín, A. odoratus Phosri, Watling, M.P.Martín & Whalley, A. pteridis (Shear) Zeller, A. smithii Watling, M.P.Martín & Phosri, A. telleriae M.P.Martín, Phosri & Watling.

In Pakistan this genus is represented by only one species, i.e. A. hygrometricus (Ahmad. 1956; Ahmad et al., 1997).

In this study, many specimens of A. hygrometricus were collected from both temperate and tropical climates from number of sites. They are all identified on morphological basis and few of those were analyzed molecularly.

Astraeus hygrometricus was reported first time by Ahmad in 1956 as Geastrum hygrometricum, from different regions of Pakistan viz., Patriata (Murree) of Punjab, Kalam (Swat), Kaghan Valley of KPK. It is reported first time from Khabbal Paien, and Dadar of district Mansehra, and district Abbottabad. Fort Munro (Dera Ghazi Khan) is also new location of A. hygrometricus.

In phylogenetic analysis, nine (9) small clades were resolved and each clade is represents one Astraeus sp. Origin of the species was written with the accession numbers in the tree. The clade labeled as Astraeus sp. was based on the collections that Fangfuk et al., (2010a) named as Japanese group II (Fig. 11).

Initially genus Astraeus hygrometricus was considered as the only species of the genus which was widespread. Many collections from the different countries were named the same.

In recent literature it was found, that phylogenetic analysis based species delimitation revealed the complexity within this genus and it was concluded that are more no. of species than expected morphologically. Many collections which were previously named as A. hygrometricus were different when analyzed molecularly so their new names 415

have been proposed recently. The phylogenetic tree produced in this study is consistent with the phylogenetic analysis of Phosri et al., 2013; Fangfuk et al., 2010a.

Pakistani collections of Astraeus spp. (APK2 and APK3) fall in a clade along with A. hygrometricus French collections with 73 bootstrap value. APK3 (which is more reliable sequence than APK2) shows 98% similarity and 1 % divergence with A. hygrometricus (AJ629406). However it shares 93.8 % genetic characters and 1% divergence with the other A. hygrometricus of the same clade (AJ629394).

All of the eight species under this genus has been characterized molecularly so a good picture of the genus is available. Still there is a need to study more collections known from different parts of the world on molecular basis to find their accurate identification and placement. There is a need to get more ITS rDNA sequences of Pakistani Astraeus spp. to reveal the species diversity of this genus in the country.

Rhizopogon Fr.

Rhizpogon is a hypogeous genus of belonging to family Rhizopogonaceae, represented by 150 species worldwide (Kirk et al., 2008). It is characterized by globose to subglobose basidiomata with peridium enclosing the gleba that is in the form of locules lined with hymenium, and lacks columella. It has ellipsoid, cylindrical to ovoid spores (Grubisha, 1998).

The taxonomy of Rhizopogon is extensively studied by many mycologists (Fries, 1817; Zeller & Dodge, 1918; Coker & Couch, 1928; Zeller, 1939, 1941, 1948; Smith, 1966; Trappe & Guzmán, 1971; Hosford & Trappe, 1980; Trappe, 1975; Miller, 1986; Cazares et al., 1992; Molina & Trappe, 1994; Martin, 1996; Allen et al., 1999; Trappe et al., 2007). Systematic classification of the genus in a particular family remained subject of debate for many years (Dodge, 1931; Fischer, 1933; Zeller, 1939, 1941, 1948; Lange, 1956; Smith, 1971, 1973; Hawker, 1975; Miller, 1983; Miller & Miller, 1988; Castellano et al., 1989; Bruns & Szaro, 1992; Molina et al., 1992; Hawksworth et al., 1996; Martin, 1996; Allen et al., 1998).

There were many hypothesis regarding origin of Rhizopogon from boletoid ancestor and its relationships with genus Suillus. Both concepts exists its origin from mushroom shape boletoid fruiting body and vice versa (Thiers, 1971, 1975, 1984; Bruns et al., 1989, 1990, 1992, 1998; Baura et al., 1992; Binder & Hibbett, 2006) 416

Most of the species of Rhizopogon are ectomycorrhizal fungal symbionts of forest trees especially with and Douglas fir, and sometimes found host specific (Kessel, 1927; Castellano &Trappe, 1985; Molina & Trappe, 1982a, 1982b, 1994; Molina et al., 1997, 1998; Taylor & Bruns, 1999; El Karkouri et al., 2003). Some of the species also known as edible (Yun & Hall, 2004). It also plays an important role in forest ecosystem by providing itself as food for rodents (Maser & Maser, 1988; Colgan & Claridge, 2002; Izzo et al., 2005; Ashkannejhad & Horton, 2006)

Four sections of the genus have been developed for species identification. These are Amylopogon, Fulviglebae, Rhizopogon, Roseoli and Villosuli (Smith, 1964; Grubisha et al., 2002).

This genus is poorly known in Pakistan due to lack of focus on collection of this organism. Two Rhizopogon spp. are known from the country, these are R. flavus Petch, and R. roseolus (Corda) Th. Fr., from temperate regions (Ahmad, 1952, 1956).

In this investigation, three Rhizopogon spp. were collected and described on morphological and molecular basis. These are R. flavescens nom. prov. R. himalayensis nom. prov. and R. roseolus (Fig. 12).

Rhizopogon himalayensis nom. prov. (RPK8) is characterized by yellowish basidiomata surrounded by hyphal mat, ellipsoid to oblong, non amyloid basidiospores and with hypogeous habit under Cedrus deodara. It belongs to section Rhizopogon. Morphologically it is close to R. truncatus Linder beacause of yellow color of fruiting body and surrounding hyphal mat, but it can be differentiated by the spore size which is bigger in R. truncatus (9.0–13 × 3.5–5 µm vs 4.6–8.0 × 3.0–4.0 µm). Molecular data also support its distinct position in the tree. It is represented here as new species.

R. flavescens nom. prov. (RPK13) is characterized by small and yellowish basidioma with amyloid basidiospores.

In phylogenetic analysis, R. flavescens (RPK14), R. himalayensis (RPK8), and R. roseolus (RPK13) falls in section Rhizopogon along with R. abietis (KC346843), R. luteorubscens (GQ267482), R. mohelensis (AJ810039), R. pseudoroseolus (KC152203, KC152205), R. rubescens (JX907816, DQ068965), R. roseolus (AM084707, AJ810051, AJ810052), R. vulgaris (JN858081). 417

RPK8 and RPK14 are close and appear as sister species with 94% bootstrap support. Both share 98.5% genetic similarity and show 1.4% divergence from each other. These taxa are separated from all of the taxa in section Rhizopogon with good bootstrap support of 90%.

Among all of these, RPK8 and RPK14 show maximum genetic similarity of 97.5%, 96.5% with R. rubescens (JX907816, DQ068965) and genetic divergence of 2.2%, 3.0%, respectively. On the basis of current analysis, this taxon seems previously undescribed.

Rhizopogon roseolus (RPK13) also falls in section Rhizopogon, shows 99.7% similarity and 0.3% divergence with three sequences of R. roseolus (AM084707, AJ810051, AJ810052) supporting all sequences the same.

R. himalayensis (RPK8), R. flavescens (RPK14) seem new to science. R. roseolus is reported by Ahmad from Murree; Changla gali; Shogran, Kagan Valley (Ahmad et al., 1997). This is first time reported from arid regions of Lahore, Punjab. No. of reports of Rhizopogon spp. from Pakistan after this study increased from 2 to 4.

Pisolithus Alb. & Schwein

Pisolithus is first established by Alb. & Schwein in 1805 by describing P. arhizus as a type species. Species of this genus are distributed worldwide both in temperate and tropical areas (Cullings & Makhija, 2001; Thomas et al., 2003; Moyersoen & Beever, 2004; Reddy et al., 2005; Kasuya et al., 2010; Moreno et al., 2010; Phosri et al., 2012; Martín et al., 2013). Several polymorphisms and variations exist in Pisolithus in terms of basidiomata and spores morphology, culture characteristics, enzymes and mycorrhizal activities (Ho, 1987; Kope & Fortin, 1990; Lamhamedi et al., 1990; Burgess et al., 1994; Watling et al., 1995b; Anderson et al., 1998; Singla et al., 2004; Kasuya et al., 2007, 2010)

P. tinctorious is also studied for its reducing activities as ectomycorrhizal symbionts in forest soils (Cairney & Ashford, 1989). It is known to form ectomycorrhizal associations with many gymnospermic and angiospermic trees (Marx et al., 1970, 1977; Momoh & Gbadegesign, 1980; Massicotte et al., 1987; Garbaye et al., 1988; Kammerbauer et al., 1989; Weiss, 1992; Cairney & Chambers, 1997; Duponnois & Ba, 1999; Díez et al., 2001; Moyersoen & Beever, 2004). 418

Molecular techniques used for delimitation of Pisolithus spp. is now becoming common practice among mycologists and supported the hypothesis of greater species diversity of the genus (Kope & Fortin, 1990; Farmer & Sylvia, 1998; Junghans et al., 1998; Gomes et al., 1999, 2000; Anderson et al., 2001; Martin et al., 2002; Kanchanaprayudh et al., 2003b; Moyersoen et al., 2003). Previously only one species, P. tinctorious has been reported by Razzaq and Shahzad in 2007 from Karachi. In this study, three Pisolithus species are presented. These are P. flavus nom. prov., P. tinctorious and Pisolithus sp. Pak1. Former is presented here as a new species whereas P. tinctorious is reported first time from Punjab and KP provinces of the country. Previously it was thought that genus Pisolithus is monotypic and represented by only one species, i.e. Pisolithus tinctorious. But recent phylogenetic analysis based on many collections of Pisolithus spp. from different parts of the world rejected this concept and proved that this genus comprises several biological species. In phylogenetic analysis on the basis of ITS-rDNA regions, Pisolithus spp. clustered together based on geography and host with which they form ectomycorrhizal association. Phylogenetic tree produced in this study is consistent with published literature (Phosri et al., 2012, Martin et al., 2013).

Pisolithus flavus (PPK11) is characterized by medium to large, off-white to bright yellow basidioma, well developed rhizomorphs, often forming pseudo-stipe, and olivaceous yellow basidiopsores and growing under Eucalyptus sp. It does not bear any characteristic resemblance with any Pisolithus species except with P. albus. However, it enjoys its distinct position within the genus when analyzed molecularly.

In phylogenetic tree, it falls in Clade I. Its distinct position in Clade I is supported by 81 bootstrap support. However it appears as a sister species to taxa in P. albus complex. P. albus only share the character of off-white basidioma, but bright yellow basidioma in the latter is not characteristic of P. albus. PPK11 shares a maximum of 94.2% genetic characters with P. albus sp. complex whereas it shows a least of 5.3% divergence from these. Both genetic and morphological characters confirms the distinct status of P. flavus PPK11 and it is represented here as a new taxon (Fig. 13).

Scleroderma Pers. 419

Scleroderma was first established by Persoon in 1801and it is represented by S. verrucosum Pers. as type species. Generic description is now amended after including Veligaster as a synonym by Guzmán et al., 2013. It is an extensively studied genus (Guzmán, 1970; Sims et al., 1997; Baseia & Galvão, 2002; Kasuya et al., 2002). This genus comprises 32 species worldwide (Šebek, 1953; Guzmán, 1970; Demoulin, 1974; Miller & Miller, 1988; Baseia & Milanez, 2000; Guzmán & Ovrebo, 2000; Guzmán et al., 2004, 2013; Gurgel et al., 2008; Kirk et al., 2008; Moreno et al., 2010; Alfredo et al., 2012; Nouhra et al., 2012; Rusevska et al., 2014). Scleroderma species are thick-skinned earth balls that are separated primarily based on basidiospore characters and peridium structure and dehiscence (Sims et al., 1995; Alfredo et al., 2012). These gasteroid basidiomycetes form ectomycorrhizal associations with various trees (Marshall, 1904; Rifai, 1987; Marx et al., 1991; Jeffries & McLain, 2004; Chen, 2006a, 2006b; Watling, 2006; Chen et al., 2007; Sanon et al., 2009).

Biochemical studies on a few taxa are also being done which found healing potential of polysaccharides isolated from Scleroderma species (Nascimento et al., 2012)

On the basis of molecular phylogeny, Scleroderma found belonging to boletales, so Sclerodermatales is no longer a taxonomic term (Binder & Bresinsky, 2002; Hughey et al., 2000; Louzan et al., 2007; Wilson et al., 2011). Guzmán et al. (2013) have described three sections of Scleroderma based on spore morphology and presence or absence of clamp connections. Recent studies in molecular analysis and phylogeny of this genus, it was found a strong correlation between spore morphology and clustering of taxa in the clades of phylogenetic tree (Phosri et al., 2009; Nouhra et al., 2012; Rusevska et al., 2014).

Five Scleroderma species have been recorded previously from Pakistan (Ahmad et al.,. 1997). These are S. aurantium (= Pers.), S. bovista Fr., S. cepa Pers., S. flavidum Ellis & Everh., S. sinnamariense Mont., S. verrucosum (Bull.) Pers.

In this study, we collected and identified five more Scleroderma species viz., S. areolatum Ehrenb., S. bovista, S. chevalieri Guzmán, S. dictyosporum Pat., S. fuscum (Corda) E. Fisch. One Scleroderma sp. (SPK21) remained unidentified and require further analysis.

In the phylogram, taxa are clustered on the basis of their basidiospore’s ornamentation. Sims et al., (1995) described a key of 25 Scleroderma spp., in which he 420

grouped the taxa in to three sections. Three sections were Aculeatispora, Sclerangium and Scleroderma. Scleroderma spp. with non reticulate and echinulate or verrucose spores and hyphae without clamp connection belong to section aculeatispora, whereas those with subreticulate to reticulate basidiospores and hyphae with clamp connections belong to other two sections. He included S. areolatum, S. albidum, S. cepa, S. columnare, S. leave, S. leptopodium, S. texens, S. uruguayense, and S. verrucosum in section Aculeatispora, S. chevalieri, S. citrinum, S. echinatum, S. floridanum, S. polyrhizum, S. reae, S. sinnamariense, S. stellatum in Sclerangium (with sub-reticulate spores). However S. bovista, S. dictyosporum, S. citrinum, S. fuscum, S. hypogeum, S. meridionale and S. michiganense in section Scleroderma (with reticulate spores). Recently Guzmán et al., (2013) amended the genus description by including Veligaster’s characters, proposing Veligaster a synonym of Scleroderma. They grouped taxa in to 3 sections on the basis of spore morphology and presence or absence of clamp connections. To adjust the S. verrucosum, which is a type species, section Aculeatispora was renamed as section Scleroderma. Previous section Scleroderma was given a new name of Reticulatae, and section Sclerangium remained same.

In this phylogenetic analysis, Scleroderma spp. with completely reticulate basidiopores are clustered within Clade I, whereas those with non reticulum and echinulate to verrucose basidiospores were clustered together in Clade II. Rest of the taxa included in the study have sub-reticulate basidiopsores but these Scleroderma spp. did not follow any pattern of clustering. More detailed study on these taxa with respect to morphology and molecular phylogeny is required (Fig. 14).

All of the sequences generated during this study were clustered in Clade I and Clade II, within their respective group of sequences of a particular species.

It is first report of study of molecular phylogeny of Scleroderma spp. from Pakistan.

Geastraceae is a family of earth stars in order Geastrales. Geastrum is the only big genus in this family comprising more than 50 species worldwide. Other genera are Myriostoma (Pepper pot earth star) which is monotypic, Radiigera and Sphaerobolus (Kirk et al., 2008) 421

In traditional classification family of earthstars was classified under but recently Hosaka et al., (2006) proposed new phylogenetic classification of Gomphoid- Phalloid group and proposed the placement of this family in order Geastrales.

Before this study, Geastraceae in Pakistan included nineteen species, 18 belonging to Geastrum Pers., and 1 of Sphaerobolus (Ahmad, 1939, 1952, 1969; Ahmad et al., 1997; Gardezi 2005; Razzaq & Shahzad, 2007)

In this investigation, 12 species of Geastraceae belonging to genera Geastrum (ten species), Myriostoma (1 species) and Radiigera (1 species) have been collected from different regions of Pakistan, described and identified morphologically and molecularly.

Geastrum Pers.

Genus Geastrum was first described by Persoon in 1801 by describing G. pectinatum as a type species (Castiglia et al., 2013). It is represented by 50 species worldwide (de Toledo , 1996; Soto & Wright, 2000; Baseia & Milanez, 2002c; Esqueda et al., 2003; Bates, 2004; Calonge & Matta, 2004; Douanla-Meli et al., 2005; Baseia & Calonge, 2006; Rocabado et al., 2007; Zamora & Calonge, 2007; Kirk et al., 2008; Cortez et al., 2009b; Zhang & Papinutti, 2009; Friedrich, 2011; Trierveiler-Pereira et al., 2011; Srivastava et al., 2014; Zamora et al., 2014). This genus is characterized by the behavior of outer layer of peridium which splits off in the form of rays like a star exposing the inner peridium layer. Spores are born in the spore case formed by endoperidium and released through mouth in the form of a pore. Mouth can be raised, depressed, fibrillose or plicate, an important morphological tool for identifying species. Phylogenetically this genus is placed in Gomphoid-Phalloid group by Hosaka et al., 2006.

In this study, nine (10) Geastrum spp. were collected from different areas of country and identified on morphological and molecular basis. These are G. fimbriatum Fr., G. himalayensis nom. prov., G. lycogalopsus nom. prov., G. pakistanicus nom. prov., G. parvulus nom. prov., G. patulus nom. prov., G. pseudolimbatum Hollos, G. pseudosaccatum nom. prov., G. saccatum Fr., G. triplex Jungh.

Out of these, five (6) taxa seems previously undescribed, 1 taxon (G. pseudolimbatum) is new record for the country, and 3 taxa (G. fimbriatum, G. saccatum, G. triplex) are already reported from Pakistan. 422

All of these 10 Geastrum species are aslo molecularly characterized except for G. saccatum which was identified only on morphological basis (Fig. 15).

G. himalayensis nom. prov., is characterized by small to medium sized fruiting body, plicate peristome and distinctly ornamented basidiospores. Morphologically, it resembles with G. fimbriatum, G. saccatum and G. triplex. In the phylogenetic analysis, it came as sister species with G. fimbriatum (= G. sessile), G. smardae, G. fornicatum, G. indicum, and G. triplex in sub clade I of Clade I. Out of these, it is relatively closer to G. fimbriatum. When analyzed, it shares 76.9% and 79% genetic characters with G. fimbriatum (KC582015, KC582016) respectively. However, it shows 17.4% and 17.2 % divergence with above two sequences of G. fimbriatum, respectively. This divergence is also supported by morphological characters. Spores of G. himalayensis (GPK16) are distinctly verrucose as compared to shortly warted basidiospores of G. fimbriatum. On the basis of morphological and molecular differences with the other Geastrum spp., this taxon is presented here as a new taxon.

G. pakistanicus nom. prov., is characterized by thin exoperidium, olivaceous, and verrucose basidiospores. In phylogenetic analysis, four sequences of G. pakistanicus clustered together in a minor clade in Sub clade II of Clade I with 98 bootstrap support. G. parvulus and G. patulus are sister species to G. pakistanicus. Sequenes of G. pakistanicus showed up to 92% shared genetic characters and up to 6.4% divergence with the sequences of G. patulus nom. prov., G. parvulus also exhibit up to 87% similarity and up to 12% divergence with G. pakistanicus, so the latter is described here as a new species.

G. parvulus (GPK13) and G. patulus (GPK14, GPK15, GPK18) which are described here as new species are morphologically close to each other and also close to G. saccatum. In phylogenetic analysis, one sequence of G. parvulus and 3 sequences of G. patulus are segregated from each other as sister species in sub clade II of Clade I and this is also supported by 99 bootstrap value. G. parvulus shared up to 90% genetic characters and up to 5.0% divergence with G. patulus sequences. Both are presented here as new species.

G. parvulus nom. prov. is characterized by small basidiomata, and minutely warted basidiospores. Morphologically it can be confused with G. saccatum because both have 423

basidiospores with minute warts, but the basidiomata in G. saccatum are quite bigger than this taxon.

G. patulus nom. prov. is characterized by small basidioma with rays spreading straight around the spore case, and densely ornamented basidiospores. This is close to recently studied taxon GPK13, because both have small basidiomata but the former has densely ornamented basidiospores as compared to the latter which has minutely ornamented.

G. pseudosaccatum nom. prov. (GPK2) is characterized by large basidiomata and densely ornamented and large basidiospores. Morphologically it is similar to G. saccatum, both have densely ornamented basidiospores but former has large spores and verrucae as compared to the latter. G. saccatum has relatively smaller basidiomata than G. pesudosaccatum. It does not bear any resemblance with any of the Geastrum spp. in the phylogenetic analysis. It showed different behavior in the tree by segregating itself from all the species of Clade I and Clade II. It is characterized by large basidiomata and distinct spore’s ornamentation. This taxon seems previously undescribed.

Morphological identification of G. fimbriatum and G. pseudolimbatum and G. triplex are also confirmed by molecular phylogenetic analysis with a good bootstrap support.

G. pseudolimbatum is a first time reported from Pakistan, and remaining three taxa, viz., G. fimbriatum, G. saccatum and G. triplex are being reported from new localities of the country.

G. lycogalopsus nom .prov. (DPK3) is characterized by small fruiting bodies with thick peridium, smooth basidiospores, growing gregariously forming white mycelial mat on forest floor. This character of forming white mat over forest floor brings this taxon close to genus Lycogalopsis which is represented by L. solmosii. ITS-nrDNA sequences of DPK3 did not give any significant similarity with any sequence after BLAST. However, LSU- nrDNA sequences produced matches with Geatrum spp. Mophologically, this taxon only shared thick peridium with Geastrum (which remained unopened in this case). Anatomically, Geastrum spp. always have ornamented spores, however spores are smooth in present taxon (DPK3). Therefore, on phylogenetically basis, this taxon is named as 424

Geastrum lycogalopsus until more results from other genes will come and make taxonomic status of this taxon clear.

Myriostoma Desv.

Myriostoma Desv. is a monotypic genus that belongs to order geastrales. It was first established by Desv. (1809) when he published Myriostoma anglicum Desv. [=Myriostoma coliforme (Dicks.) Corda] as type species.

Myriostoma coliforme has cosmopolitan and worldwide distribution It is found growing either solitary or in groups on humus rich soil. Most of the studies carried out so far on M. coliforme were related to taxonomy and an attempt for its placement in an appropriate taxonomic family. Initial studies on M. coliforme was done by John Ray in 1696, although it was first scientifically described as new species in 1776 by James Dickson. Phylogeny of genus Myriostoma faced many shifts between Geastraceae and Astraeaceae by many mycologists however it is currently being treated under the former (Spegazzini, 1908; Sunhede, 1989; Minter et al., 1990; Yousaf et al., 2013b). Molecular and phylogenetic studies using rDNA and other genes as genetic markers have also confirmed its position within Geastraceae. It is a sister genus to Geastrum (Kruger et al., 2001; Hosaka et al., 2006)

During the exploration of macro fungi from different areas of Pakistan, find of a rare fungal specimen has been remained a successful venture. Because this species has been included in list of 33 fungal species facing the threats of extinction (Dahlberg & Croneborg, 2003). It also poses a need to explore more areas for looking for more flora in the view of conversation of macro fungal biodiversity. M. coliforme is first time reported as a new record from Pakistan.

In this investigation, one specimen of M. coliforme was collected from Khanspur, KP, Pakistan. It is described and identified on morphological basis.

M. coliforme (MPK2), collected and described in this study was not successfully amplified for ITS-nrDNA region. Freshly collected specimens of M. coliforme (MC1) were examined from Texas, USA. It’s one (1) ITS sequence was generated and incorporated in the phylogenetic analysis (Fig. 15). M. coliforme is an addition to gasteroid fungal flora of Pakistan. 425

Radiigera Zeller Genus Radiigera was first reported by Zeller in 1944 with R. fuscogleba as a type species. It belongs to Geastraceae and only known from Europe and North America. It is widespread and represented by 4 species. These are R. bushnellii L.S. Domínguez & Castellano, R. flexuosa L.S. Domínguez & Castellano, R. fuscogleba Zeller, R. taylorii (Lloyd) Zeller (Kirk et al., 2008). However, Index fungorum treats all these four species under Geastrum after Jeppson et al., 2013 who proposed to include these Radiigera sp. under Geastrum on the basis on molecular phylogeny.

According to Index fungorum, currently recognized species under Radiigera are only three (3). These are R. asperata D.A. Reid, R. cinnamomea Zeller, R. tropica Orihara & T. Kasuya.

In this investigation, one Radiigera sp. was collected from Ayubia, Pakistan and described on morphological basis. This taxon seems previously undescribed and represents new species. Radiigera ayubiensis nom. prov. is characterized by ochraceous brown basidioma, with dark brown, verrucose basidiospores. This taxon does not bear any resemblance with the know Radiigera spp. R. cinnamomea, R. tropica have smaller basidiospores as compared to recently described Radiigera sp.

Phallales includes spectacular members of macro fungi commonly called stinkhorns. It contains great diversity of genera, distributed worldwide and usually found in tropical regions of the world (Coker & Couch, 1928; Cunningham, 1944; Rea, 1955; Dring, 1964; Burk, 1979; Blanton & Berk, 1980; Dring, 1980; Arora & Berk, 1982; Zang & Peterson, 1989; Guzmán et al., 1990; Calonge & Kreisel, 2002; Baesia et al., 2003; Calonge et al., 2005; Vargas-Rodriguez & Vázquez-García, 2005; Baseia et al., 2006; Hemmes & Desjardin, 2009).

In traditional classification stinkhorns were classified under class gasteromycetes. Now this group can be placed in a morphological group “Gasteroid fungi”. Recent studies based on multigene phylogenies suggested that stinkhorns are now included in Gomphoid- Phalloid group along with some non gasteroid members (Hosaka et al., 2006).

Phallales in Pakistan includes ten species, 6 belonging to Phallus Junius ex Linnaeus, 3 of Lysurus Fr. (= Simblum Klotzsch ex Hook), and 2 of Protubera Möller (Ahmad, 1952, 1969; Moreno et al., 2009). 426

Phallus Junius ex L.

Genus Phallus is characterized by foul smelling mushroom and represented by P. impudicus L. as a type species. Species of this genus are distributed worldwide, mostly in tropical regions (Long, 1907; Lee, 1957; Oso, 1976; Kreisel, 1996; Baseia et al., 2003, 2006; Liu et al., 2005; Calonge et al., 2008; Kasuya, 2008; Desjardin & Perry, 2009; Moreno et al., 2009; Ottoni et al., 2010; Cortez et al., 2011). A comprehensive study on this genus was done by Hanns Kreisel in 1996. A key for the identification of Phallus spp. is also developed by Calonge (2005). According to Dictionary of Fungi, this genus is represented by 18 species (Kirk et al., 2008).

In this study, five (5) Phallus spp. were collected from different climatic regions of Pakistan and identified on morphological and molecular basis. These are P. ahmadii nom. prov., P. galericulatus (Möller) Kreisel, P. hadriani Vent., P. roseus Delile, and P. rubicundus (Bosc) Fr. (Fig. 17).

Phallus ahmadii nom. prov., (PPK4) is characterized by off-white to olivaceous pseudostipe, merulioid receptacle surface, conical pileus, with less stinky, olivaceous gleba and off-white volva. In sub clade I of Clade I, it forms a small sister clade with P. rugulosus (= P. rubicundus). Both species have conical pileus, and rugulose or merulinoid receptacle surface. However, P. rugulosus has orange while P. ahmadii has off-white pseudostipe.

P. caliendricus Dring seems to be very close to P. ahmadii morphologically. Both the species have merulinoid receptacle surface, olivaceous gleba, dirty off-whitte volva. But former is strongly foetid but latter is less stinky. P. ahmadii has greenish, ellipsoid, amyloid spores whereas P. caliendricus has hyaline spores (Dring & Rayner, 1967). These differences suggest a distict status and novelty of P. ahmadii.

P. galericulatus (PPK5) and P. roseus (PPK3) appears as sister species, sub clade I of Clade I, have most of the features in common. They both have characteristic features of having rugulose receptacle surface with calyptra at the top of wig-like pileus. These characteristerics are distinguishing features of genus Itajahya. Itajahya is now treated as synonymous to Phallus. Index Fugorum still treats these two species under Itahjaya. Differences between P. galericulatus and P. roseus are that the former has off white and latter have pinkish pseudostipe. 427

P. hadriani (PPK1 & PPK15) is characterized by reticulate receptacle surface and pinkish to purplish volva. Its position in clade I with already reported P. hadriani sequence (DQ404385) is strongly supported with 99 bootstrap value.

P. ahmadii seems previously undescribed. P. galericulatus is new record from Paksitan. P. hadriani represents second report of its occurrence in Pakistan. P. roseus and P. rubicundus are being described from different localities of the country this time. No. of reports of Phallus spp. after this study increased from 5 to 7.

Out of total of 25 Phallus spp. reported worldwide, only 9 taxa have been characterized molecularly for ITS-nrDNA region so far. Further work is needed in this regard to build up a complete phylogenetic tree of genus Phallus.

Russulaes contains some gasteroid members along with agaricoid taxa, one of which is Gymnomyces Massee & Rodway (≡ Russula Pers.). It is characterized by gasteroid basidiomata having non-reticulate to reticulate and ornamented basidiospores belonging to family Russulaceae (Smith et al., 2006). Like other members of Russulaceae, a few sequestrate species of this genus are also found to form ectomycorrhizal relationships with the forest trees e.g. Pinus and Quercus (Trappe & Castellano, 1986; Stendel et al., 1999; Horton & Bruns, 2001; Avis et al., 2003; Smith et al., 2006).

In this study, basidiomata were found hypogeous and collected from Mankial located in north of Swat valley. Mankial is famous for its several summits and peaks. This taxon was collected from the Mankial top’s forest floor which was dominated with tress of Cedrus Trew and Pinus L.

Morphologically, it is close to G. pallidus Massee and Rodway due to presence of globose basidiospores with verrucose ornamentation, however both can be differentiated o the basis of color and size of basidiospores. Spores are hyaline in G. pallidus whereas greenish in RPK10, in addition, spores in present taxon are slightly smaller than G. pallidus (7.7–9.0 µm vs 9–10 µm). Peridium is also fragile in the latter which is very hard and persistent in RPK10.

In the phylogenetic analysis (Fig. 18), it forms a minor clade with both agaricoid and sequestrate forms of Russula. It lie close to Russula vinaceocuticulata (GU222258) with a bootstrap support of 60%. However, when sequence distances were calculated, both sequence show 2.5% genetic divergence from each other and share 93.6% genetic 428

characters, therefore suggesting a segregation from R. vinaceocuticulata. It shares a maximum of up to 97% genetic characters and divergence of l.9–2.0% with sequences of sequestrate forms (AY239321, AY239335, AY239331, AY239349, KC152107). It is presented here as a new species.

For diversity analysis, field trips to thirty seven (37) sampling sites were made and 228 specimens were collected which belonged to 5 orders, 9 families, 20 genera and 101 species. Highest Species Richness (SR) was found in Himalayan moist temperate forest. All the sites in this forest are characterized by adequate rain fall throughout the year and soil rich in organic matter and humus. These conditions favors the growth of macro fungi in these sites. This forest is expected to have enormous diversity of puffballs as observed in this study.

It was also noted that abundance of gasteriod taxa is more than the species richness in these sites. It was a trend that many taxa had sufficient no. of fruiting bodies growing at a place, also one particular taxon was found many times in one sampling site. However this trend was not found in tropical and subtropical forests. Here, number of fruiting bodies and frequency of occurrence of a species in one particular sampling site is limited, like no. of taxa belonging to different genera are more rather than many taxa belonging to few genera.

Species that were found more common are B. plumbea, L. pyriforme, L. perlatum, and Astraeus hygrometricus. B. plumbea was found from most of the localities of Himalayan moist temperate forest, with altitude ranging from 2100 m to 4200 m. Fruiting bodies were also abundant. Lycoperdon pyriforme and L. perlatum are also commonly occurring gasteroid fungal species from same forest but also found in subtropical pine forest. Both of these were not found above 2700 m a.s.l.

A. hygrometricus, another taxon with many collections, is a common species of subtropical pine forest and rarely found in Himlayan forest. During the analysis of data, it was also observed that some taxa showed dedication for particular climatic conditions. Phallus spp. seem to be the taxa of lower altitude, found mostly in tropical to subtropical regions with few collections from medium altitude (2200 m) were also found. All Geastrum sp. except 1 were found from Himalayan forest. 429

Battarrea, Tulostoma spp., Phellorinia are species of dry and arid areas and so are found in this study. However one Tulostoma sp. was collected from Batta kundi having moist temperate climate.

Pisolithus showed its dedication to tropical and subtropical sites and never to moist temperate climate. Podaxis pistillaris is considered as desert adapted species, reported from amny sites of tropical forests in this study but was also collected from Nathia gali. These taxa which were found growing in outside their particular climatic zones shows that they have high plasticity and adaptability and broad ecological amplitude.

It is concluded that there is an enormous potential of finding a big number of gasteroid fungi from unexplored sites in rainy seasons which can lead to raise the number of world gasteroid flora since many new species are expected to come in future explorations.

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