77 European Journal of Taxonomy 888: 77–96 ISSN 2118-9773 https://doi.org/10.5852/ejt.2023.888.2215 www.europeanjournaloftaxonomy.eu 2023 · Asif M. et al. This work is licensed under a Creative Commons Attribution License (CC BY 4.0). R e s e a r c h a r t i c l e Molecular and morphological studies reveal a new species of Panaeolus (Agaricales, Basidiomycota) from Punjab, Pakistan Muhammad ASIF 1,*, Qudsia FIRDOUS 2, Aiman IZHAR 3, Abdul Rehman NIAZI 4, Samina SARWAR 5 & Abdul Nasir KHALID 6 1,2,3,4,6 Fungal Biology and Systematics Research Laboratory, Institute of Botany, University of the Punjab, Quaid-e-Azam Campus 54590, Lahore, Pakistan. 1 Plant Mycology Research Laboratory, Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan. 5 Department of Botany, Lahore College for Women University, Lahore, 54590, Pakistan. * Corresponding author: asifgondal101@gmail.com 2 Email: qudsiafirdous26@gmail.com 3 Email: aimanizhar25@gmail.com 4 Email: mushroomniazi@gmail.com 5 Email: samina_boletus@yahoo.com 6 Email: drankhalid@gmail.com Abstract. Panaeolus punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi & Khalid sp. nov. was collected from three different localities (Bahawalnagar, Kasur, and Lahore) in Punjab, Pakistan. Morphological observations and phylogenetic analyses based on nuclear encoded internal transcribed spacers (ITS1- 5.8S-ITS2 = ITS) and D1/D2 domain of large subunit (28S) rDNA confirmed the taxonomic distinctness of this species. The new species is potentially hallucinogenic and characterized by a parabolic pileus with a light brown center, broadly fusiform basidiospores, presence of cheilocystidia, pileocystidia, and caulocystidia, and absence of pleurocystidia and clamp connections. The DNA sequences of the species clustered together in a well-supported distinct clade. We present a detailed description, photographs, and line drawings, and elucidate and discuss the phylogenetic position of the new species. Morphological comparisons with phylogenetically and morphologically allied species are discussed. Keywords. Agarics, hallucinogenic, molecular systematics, saprotrophic, taxonomy. Asif M., Firdous Q., Izhar A., Niazi A.R., Sarwar S. & Khalid A.N. 2023. Molecular and morphological studies reveal a new species of Panaeolus (Agaricales, Basidiomycota) from Punjab, Pakistan. European Journal of Taxonomy 888: 77–96. https://doi.org/10.5852/ejt.2023.888.2215 Introduction The genus Panaeolus (Fr.) Quél (1872: 151) has been placed in different families, including Coprinaceae Overeem & Weese (Doveri 2011), Bolbitiaceae Singer (Tóth et al. 2013), and Psathyrellaceae Vilgalys, Moncalvo & Redhead (Amandeep et al. 2014), or has been treated as incertae sedis (He et al. 2019). More recently, Panaeolus along with Copelandia Bres., Panaeolina Pers., and Panaeolopsis Singer have been https://doi.org/10.5852/ejt.2023.888.2215 http://www.europeanjournaloftaxonomy.eu/index.php/ejt/index https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0001-7327-2072 mailto:asifgondal101%40gmail.com?subject= mailto:qudsiafirdous26%40gmail.com?subject= mailto:aimanizhar25%40gmail.com?subject= mailto:mushroomniazi%40gmail.com?subject= mailto:samina_boletus%40yahoo.com?subject= mailto:drankhalid%40gmail.com?subject= https://doi.org/10.5852/ejt.2023.888.2215 https://orcid.org/0000-0002-3096-0198 https://orcid.org/0000-0002-1118-1148 https://orcid.org/0000-0002-6377-4498 https://orcid.org/0000-0002-5635-8031 https://orcid.org/0000-0002-8739-2881 European Journal of Taxonomy 888: 77–96 (2023) 78 placed in a distinct family Galeropsidaceae Singer (Kalichman et al. 2020). These genera share several taxonomically important features such as the pileus covering, spore color, and coprophilous ecology (Singer 1986; Tóth et al. 2013; Kalichman et al. 2020). The members of the family Galeropsidaceae are mostly found growing on open lawns, in steppes and prairies, in mountain deserts and are characterized by a cutis that is rarely and slightly gelatinized, partly enclosed or ovoid pileus, lacunar hymenophore or in some cases regularly developed lamellae often connected by anastomoses, passive spore discharge, ochre-brown spores with germ pore, pileipellis with pileocystidia, the presence in some species of hymenial cystidia, and clamped hyphae (Zeller 1943; Singer & De Leon 1982; Malysheva et al. 2019). Species in the genus are characterized by their typically coprophilous or nitrophilous habitat, slender fruiting body with the hemispherical pileus, cartilaginous and relatively long stipe, bluing context, epithelial pileipellis, and a black spore print that does not fade in concentrated sulphuric acid (Watling & Gregory 1987; Gerhardt 1996; Stamets 1996; Strauss et al. 2022). Panaeolus includes some of the most hallucinogenic species after Psilocybe (Fr.) P.Kumm., for example, P. subbalteatus (Berk. & Broome) Sacc. and P. cambodginiensis Ola’h & R.Heim whose basidiomata contain two hallucinatory compounds, i.e., psilocybin and psilocin (Stamets 1996; Andersson et al. 2009). There are sixteen species in the genus Panaeolus reported in literature (He et al. 2019; Hu et al. 2020) however, in Index Fungorum, 189 records are associated with this genus (accessed on 18 September 2022). Species of Panaeolus are mostly reported from Asia and Europe in tropical to sub-tropical and temperate habitats (Senn-Irlet et al. 1999; Halama et al. 2014; Kaur 2014; Wang & Tzean 2015; Desjardin & Perry 2017; Karunarathna et al. 2017; Akata et al. 2019). Some species of the genus have also been reported from North and South America and Africa in temperate habitats (Adeniyi et al. 2018; Silva-Filho et al. 2019; Teke et al. 2019). To date, 1293 macrofungal species belonging to 411 genera, 115 families, and 24 orders have been reported from Pakistan (Aman et al. 2022). Out of these, 1117 species, 338 genera, 83 families, and 16 orders belong to Basidiomycota, and 176 species, 73 genera, 32 families, and eight orders belong to Ascomycota (Ahmad et al. 1997: Aman et al. 2022). So far, five species of Panaeolus, i.e., P. fimicola (Pers.) Gillet (1878: 621), P. papilionaceus (Bull.) Quél. (1872: 152), P. sphinctrinus (Fr.) Quél. (1872: 151), P. semiovatus (Sowerby) S.Lundell & Nannf. (1938: 537) and P. rickenii Hora (1960: 454) have been reported from Pakistan (Ahmad et al. 1997; Razaq et al. 2012). During the past two decades, both morphological and phylogenetic analyses have been used in mycological research for the identification of new species of the genus Panaeolus (Drehmel et al. 1999; Zhang et al. 2004; Zhao et al. 2011; Razaq et al. 2012; Jayasiri et al. 2015; Wang & Tzean 2015; Li et al. 2016; Zhao et al. 2016). But, only a few species of Panaeolus have been reported based on both morphological and phylogenetic analyses (Ma 2014; Ediriweera et al. 2015; Wang & Tzean 2015; Undan 2016) and most Panaeolus species have been described based only on morphology (Gerhardt 1996; Amandeep et al. 2014; Halama et al. 2014; Silva-Filho et al. 2019). In the present study, some interesting collections of Panaeolus were made from three different locations in Punjab, Pakistan. All the localities lie in the semi-arid region with a maximum average temperature of 45°C and a long rainy season, i.e., July to September (Ahmad et al. 2019). Both morphological characteristics and phylogenetic analyses of ITS and 28S sequence data were used to determine the taxonomic position of the new Panaeolus species which is subsequently described here in detail. Material and methods Type locality The type specimen was collected from Haroonabad, Bahawalnagar District, Punjab, Pakistan (29°60′81″ N, 73°14′68″ E, 163 m a.s.l.) during the monsoon rainy season of August 2019. The ASIF M. et al., New species of Panaeolus from Punjab, Pakistan 79 temperature of the collection site varies from a minimum of 11°C to a maximum of 50°C and the average annual rainfall is 99 mm (Ahmed et al. 2014a, 2014b). The main vegetation of the area includes Dalbergia sissoo Roxb., Vachellia nilotica (L.) P.J.H.Hurter & Mabb., Eucalyptus camaldulensis Dehnh., Azadirachta indica A.Juss. and Albizia lebbeck (L.) Benth. (Ahmed et al. 2014b). The region falls under a hot semi-arid climate (BSh) following the climate map and classification (Peel et al. 2007; Belda et al. 2014). The second collection site is Kasur, Punjab, Pakistan (31°12′79″ N, 74°44′08″ E, 218 m a.s.l.). Climatic conditions are tremendously variable and described as scorching hot summers and cold winters. Monsoons occur towards the end of June with the rainy season lasting 2–3 months. The common woody flora of the district includes Capparis decidua Edgew. (Forssk.), Dalbergia sissoo Roxb., Prosopis cineraria (L.) Druce, Senegalia modesta (Wall.) P.J.H.Hurter, and Vachellia nilotica (L.) P.J.H.Hurter & Mabb. (Nasir et al. 1995; Zabihullah et al. 2006; Lateef et al. 2008; Durrani & Shakoori 2009; Anwar et al. 2012; Waheed et al. 2020). The third collection site is Lahore, the capital of Punjab, Pakistan (32°52′04″ N, 74°35′87″ E, 217 m a.s.l.). It has a hot semi-arid climate (Köppen climate classification BSH) with long, wet, and exceptionally hot summers, dry and warm winters, annual monsoons and dust storms. The monthly mean temperature ranges between 10 and 38°C during the year in Lahore (https://rmcpunjab.pmd.gov.pk/). It has a long rainy season (from the end of June to mid-September), with annual mean rainfall of 838 mm, which increases the humidity of the area (Siddiqui et al. 2020; Tanveer et al. 2020). Morphoanatomical study Basidiomata were photographed in the field, and morphological features such as size, shape, and color of basidiomata were recorded at the time of collection. Munsell’s soil color chart (1975) was used for color notations and for morphological terminologies, Vellinga (2001) was followed. Specimens were air-dried, kept in zipper bags, and deposited in the Herbarium of the Institute of Botany, University of the Punjab, Lahore (LAH). For the microscopic study, slides were prepared (lamellae, pileus, stipe) using free-hand sections of the dried materials rehydrated in 5% aqueous KOH (percentage weight/volume (w/v)) and stained with Congo Red (2%) and Melzer’s reagent following the microscopic procedures of Liang et al. (2011, 2018) and Cai et al. (2018) and observed under the light microscope (CXRII, Labomed Labo America Inc., Fremont, CA, USA) equipped with a camera to examine the following microscopic characteristics under 400× and oil immersion 1000× magnification: size and shape of basidiospores, basidia, cheilocystidia, pileipellis, and stipitipellis. Data for morphoanatomical characteristics was based on at least 25 measurements each of basidia, cheilocystidia, and basidiospores. Length and width ratios of basidiospores is designated as ‘Q’, while the average length and average width ratios of all the basidiospores measured is given as ‘avQ’. The notation ‘n/b/p’ is given, where ‘n’ is the number of basidiospores measured, from ‘b’ basidiomata and from ‘p’ collections (Bas 1969; Yu et al. 2020). DNA extraction, PCR amplification, and sequencing Genomic DNA was extracted using the CTAB method (Porebski et al. 1997). We amplified the ITS and 28S regions of nuclear ribosomal DNA, using the primer combination ITS1F/ITS4 and LR0R/LR5 for ITS and 28S, respectively (White et al. 1990; Vilgalys & Hester 1990). Polymerase chain reaction (PCR) was performed in a 25 μL reaction volume following the protocol given by Warnke (2020). The (PCR) products were sequenced with the same primers in both directions at ©Macrogen Inc. (238, Teheran-ro, Gangnam-gu, Seoul, Republic of Korea). The newly generated sequences of P. punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi & Khalid sp. nov. were submitted to GenBank: KY636363, MZ265142, MZ823627, OP681142 (ITS) and ON116490, ON116491, ON116492 (28S). https://rmcpunjab.pmd.gov.pk/ European Journal of Taxonomy 888: 77–96 (2023) 80 Phylogenetic analyses For phylogenetic analysis, ITS and 28S sequences of nrDNA, generated from the Pakistani collections were compared with sequences in GenBank using the BLAST tool, priority was given to those sequences which showed high bootstrap value in phylogenetic analyses (Altschul et al. 1990). The datasets were created by adding newly generated sequences of P. punjabensis sp. nov. plus the highest-scored BLAST hits were chosen from GenBank and the other sequences of the genus from previous studies (Malysheva et al. 2019; Hu et al. 2020; Voto & Angelina 2021). Four ITS and three 28S sequences were newly generated during this study, and 39 ITS sequences of family Galeropsidaceae including Psathyrella vesterholti Örstadius & E.Larss. (Örstadius et al. 2015: 29) (KC992938) as an outgroup and 30 LSU sequences of family Galeropsidaceae including Psathyrella vesterholti as an outgroup, were used for phylogenetic analyses. The sequence alignment of both datasets was done separately using MUSCLE ver. 3.8 (Edgar 2004), and initial phylogenetic analyses were performed using MEGA-X software (Tamura et al. 2011) using the Maximum Likelihood (ML) method. To calculate the appropriate model of nucleotide evolution, the nrITS dataset was segmented into three partitions, ITS1, 5.8S, and ITS2. The best fit model of nucleotides substitution based on the lowest BIC (Bayesian information criterion) values for each partition and for 28S based dataset was chosen with jModelTest2 on XSEDE via CIPRES science gateway (Darriba et al. 2012). The final phylogenetic analyses of the ITS and 28S datasets were carried out separately using RAxML- HPC2 ver. 8.1.11 under the CIPRES Science Gateway (Miller et al. 2010). In the ML analysis, 1000 bootstrap repetitions were obtained as statistical support with rapid bootstrapping for both datasets. Phylogenetic trees generated by Bayesian Inference (BI) analyses were performed with a Markov chain Monte Carlo (MCMC) coalescent approach implemented in BEAST ver. 1.8.2 (Drummond & Rambaut 2007). Both analyses resulted in a similar topology. Significant support was considered to be ≥ 80%. In the resulting trees, bootstrap values obtained from maximum likelihood analyses and values of Bayesian posterior probabilities > 0.7 were reported. FigTree ver. 1.4.3 (Rambaut 2014) was used for displaying the phylogenetic trees, and both trees were annotated using Adobe Illustrator 2020 ver. 24.1.2.408. Results Phylogenetic analysis of ITS dataset The fragment size of the target region was 758 bp long. From BLAST results, the ITS sequences of Panaeolus punjabensis sp. nov. show 99% similarity with P. papilionaceus. In the ITS phylogenetic tree, the four sequences of the new species formed a separate lineage with strong statistical support. It formed a sister clade with these species of Panaeolus: P. papilionaceus, P. campanulatus (Bull.) Quél., and P. sphinctrinus. In this analysis, P. guttulatus Bres. (KU725993) is also closely related to the new species (Fig. 1; Table 1). Phylogenetic analysis of 28S dataset The 28S alignment contained 892 total characters of which 815 were conserved and 73 were variable. In the 28S phylogenetic tree, the three sequences of the new species also formed a separate lineaged with good bootstrap support. It also formed a sister clade with these species of Panaeolus: P. papilionaceus, P. campanulatus, and P. sphinctrinus. In this analysis, P. semiovatus (MH868191) is also closely related to the new species (Fig. 2; Table 1). ASIF M. et al., New species of Panaeolus from Punjab, Pakistan 81 Fig. 1. Molecular phylogenetic placement of Panaeolus punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi & Khalid sp. nov. based on Maximum Likelihood (ML) method of ITS sequences. Newly generated sequences are in bold. Panaeolus punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi & Khalid sp. nov. (LAH36793, T = Type specimen) is referring to the holotype. Bootstrap values > 80% and Bayesian posterior probabilities > 0.7 are shown above the branches. 0.04 AB158633 Panaeolus cambodginiensis HM035081 Panaeolus sphinctrinus JF908514 Panaeolus fimicola HM035084 Panaeolus cyanescens JF908517 Panaeolus semiovatus MH592651 Panaeolus guttulatus JF908518 Panaeolus acuminatus MF497586 Panaeolus antillarum OP681142 Panaeolus punjabensis LAH37417 OP549249 Panaeolina foenisecii KF830093 Panaeolus papilionaceus KY559329 Panaeolus rickenii MH285992 Panaeolus olivaceus KY636363 Panaeolus punjabensis LAH36792 MH593015 Panaeolus olivaceus MZ265143 Panaeolus punjabensis LAH36793 T MK397578 Panaeolus plantaginiformis JF908515 Panaeolus antillarum ON561653 Panaeolina foenisecii KC992938 Psathyrella vesterholti MF497585 Panaeolus antillarum FJ755227 Panaeolus sphinctrinus MZ856314 Panaeolus mexicanus JF908522 Panaeolus campanulatus MG966283 Panaeolus bisporus JF961376 Panaeolus campanulatus DQ182503 Panaeolus sphinctrinus KM982723 Panaeolus alcis MK397577 Panaeolus plantaginiformis KU725993 Panaeolus guttulatus MN482689 Panaeolus axfordii EU834287 Panaeolus cyanescens JF961377 Copelandia tropicalis MG250381 Copelandia sp. JF961370 Panaeolus subbalteatus JF908521 Panaeolus retirugis MZ823627 Panaeolus punjabensis LAH36794 JF908516 Panaeolus rickenii FJ478119 Panaeolus retirugis 82/0.75 88/1 100/1 100/1 98/1 93/1 98/0.87 100/1 90/1 80/0.75 99/1 87/0.91 98/0.85 97/0.92 98/0.81 91/0.87 100/1 95/1 86/1 82/1 98/1 100/1 Family Galeropsidaceae European Journal of Taxonomy 888: 77–96 (2023) 82 0.01 DQ071696 Panaeolina foenisecii MK278436 Panaeolus sphinctrinus MH867057 Panaeolus subbalteatus ON116492 Panaeolus punjabensis LAH36794 DQ470817 Panaeolus sphinctrinus ON116490 Panaeolus punjabensis LAH36793 T ON116491 Panaeolus punjabensis LAH36792 KF830082 Panaeolus papilionaceus MK397600 Panaeolus plantaginiformis MH867059 Panaeolus subbalteatus MK278435 Panaeolus papilionaceus MH867785 Panaeolus retirugis DQ071694 Panaeolus semiovatus MK278427 Panaeolopsis nirimbii MH867781 Panaeolus acuminatus AY207263 Panaeolus acuminatus MK397599 Panaeolus plantaginiformis MK278429 Panaeolus cyanescens MH867058 Panaeolus subbalteatus MH867786 Panaeolus retirugis MH867787 Panaeolus retirugis U11924 Panaeolina foenisecii MH868191 Panaeolus semiovatus AB104646 Panaeolus sphinctrinus MH867784 Panaeolus retirugis MH867056 Panaeolus subbalteatus KC992938 Psathyrella vesterholti AY207265 Panaeolus papilionaceus MK278431 Panaeolus fimicola MH867557 Panaeolina foenisecii 97/1 83/1 82/1 81/0.99 91/0.87 97/1 93/1 92/1 80/1 81/0.92 95/1 85/1 86/0.87 85/0.79 97/1 84/1 Family Galeropsidaceae Fig. 2. Molecular phylogenetic placement of Panaeolus punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi & Khalid sp. nov. based on Maximum Likelihood (ML) method of 28S sequences. Newly generated sequences are in bold. Panaeolus punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi & Khalid sp. nov. (LAH36793, T = Type specimen) is referring to the holotype. Bootstrap values > 80% and Bayesian posterior probabilities > 0.7 are shown above the branches. ASIF M. et al., New species of Panaeolus from Punjab, Pakistan 83 Copelandia sp. 294130 MG250381 No data USA Unpublished C. tropicalus – JF961377 No data China Unpublished Panaeolina foenisecii FO 46609 No data DQ071696 – Garnica et al. 2007 P. foenisecii CBS 142.40 No data MH867557 – Vu et al. 2018 P. foenisecii – No data U11924 Chapela et al. 1994 P. foenisecii PUL00038201 ON561653 No data USA Unpublished P. foenisecii – OP549249 No data USA Unpublished Panaeolopsis nirimbii PERTH 7680368 No data MK278427 Australia Varga et al. 2019 Panaeolus acuminatus 4084 JF908518 No data Italy Osmundson et al. 2013 P. acuminatus GLM 45986 No data AY207263 Germany Walther et al. 2005 P. acuminatus CBS 268 No data MH867781 Hungary Varga et al. 2019 P. alcis 88085 KM982723 No data Canada Moser 1984 P. antilarum 748 JF908515 No data – – P. antilarum CORT:013830 MF497586 No data Dominican Desjardin & Perry 2017 Republic P. antilarum SFSU:DED 7874 MF497585 No data Thailand Desjardin & Perry 2017 P. axfordii MFLU 19-2367 MN482689 No data China Hu et al. 2020 P. bisporus 188954 MG966283 No data USA Ediriweera et al. 2015 P. cambodginiensis NBRC30222 AB158633 No data – Maruyama et al. 2006 P. campanulatus 10141 JF908522 No data Italy Osmundson et al. 2013 P. campanulatus No data JF961376 No data China Ma 2014 P. cyanescens No data HM035084 No data – Broussal & Dumesny 2015 P. cyanescens 6576 AQUI EU834287 No data Italy Han et al. 2010 P. cyanescens NL-0429 No data MK278429 Hungary Varga et al. 2019 P. fimicola  474 JF908514 No data Italy Wang & Tzean 2015 P. fimicola  NL-0232 No data MK278431 Hungary Varga et al. 2019 P. guttulatus 137 MH592651 No data – Unpublished P. guttulatus AMB n. 18101 KU725993 No data – Unpublished P. mexicanus ANGE1557 MZ856314 No data Dominican Voto & Angelini 2021 Republic P. olivaceus 89608 MH285992 No data USA Unpublished P. olivaceus – MH593015 No data Iran Unpublished P. plantaginiformis LE 2864 MK397578 MK397600 Uzbekistan Malysheva et al. 2019 Table 1. (continued on next page) Taxa information and GenBank accession numbers of nrITS and LSU sequences of Panaeolus (Fr.) Quél. used in the molecular phylogenetic analyses. Sequences generated for this study are shown in bold. Species Voucher No. GenBank Accession No. Origin Reference ITS LSU European Journal of Taxonomy 888: 77–96 (2023) 84 P. plantaginiformis LE 2862 MK397577 MK397599 Russia Malysheva et al. 2019 P. papilionaceus DNA1940 KF830093 KF830082 USA Ediriweera et al. 2015 P. papilionaceus DNA1940 KF830093 KF830082 USA Ediriweera et al. 2015 P. papilionaceus DB 4552 No data MK278435 Austria Varga et al. 2019 P. papilionaceus GLM 45989 No data AY207265 Germany Walther et al. 2005 P. punjabensis LAH36792 KY636363 ON116491 Pakistan This study P. punjabensis LAH36794 MZ823627 ON116492 Pakistan This study P. punjabensis T LAH36793 MZ265143 ON116490 Pakistan This study P. punjabensis LAH37417 OP681142 No data Pakistan This study P. reckenii TENN:054965 KY559329 No data Argentina Unpublished P. retirugis 7070 JF908521 No data Italy Osmundson et al. 2013 P. retirugis xsd08077 FJ478119 No data China Undan 2016 P. retirugis CBS 271 No data MH867784 France Vu et al. 2019 P. retirugis CBS 273 No data MH867786 France Vu et al. 2019 P. retirugis CBS 274 No data MH867787 France Vu et al. 2019 P. retirugis CBS 272 No data MH867785 France Vu et al. 2019 P. rickenii 749 JF908516 No data Italy Osmundson et al. 2013 P. semiovatus 4083 JF908517 No data Italy Osmundson et al. 2013 P. semiovatus GLM 51235 No data DQ071694 – Garnica et al. 2007 P. semiovatus CBS 388 No data MH868191 France Vu et al. 2019 P. sphinctrinus CBS 582 HM035081 No data Pakistan Razaq et al. 2012 P. sphinctrinus PBM 2009 DQ182503 DQ470817 Pakistan Razaq et al. 2012 P. sphinctrinus CZ519-3 FJ755227 No data Pakistan Razaq et al. 2012 P. sphinctrinus KY7130 No data AB104646 Japan Maruyama et al. 2003 P. sphinctrinus NL-3955 No data MK278436 Slovakia Varga et al. 2019 P. subbalteatus No data JF961370 No data China Sette et al. 2010 P. subbalteatus CBS 331 No data MH867059 France Vu et al. 2019 P. subbalteatus CBS 329 No data MH867058 France Vu et al. 2019 P. subbalteatus CBS 327 No data MH867056 France Vu et al. 2019 P. subbalteatus CBS 328 No data MH867057 France Vu et al. 2019 Outgroup Psathyrella vesterholtii JHP10.086 KC992938 KC992938 Denmark Örstadius et al. 2015 Species Voucher No. GenBank Accession No. Origin Reference ITS LSU Table 1. (continued). ASIF M. et al., New species of Panaeolus from Punjab, Pakistan 85 Taxonomy Phylum Basidiomycota R.T.Moore Class Agaricomycetes Doweld Order Agaricales Underw. Family Galeropsidaceae Singer Genus Panaeolus (Fr.) Quél. Panaeolus punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi & Khalid sp. nov. MycoBank MB 840898 Figs 3–4 Diagnosis The new species turns bluish on handling so it is hallucinogenic and can be distinguished by its broadly fusiform basidiospores, claviform cheilocystidia with rounded tips, and clavate caulocystidia. Etymology Specific epithet ‘punjabensis’ refers to the type locality, Punjab Province, Pakistan. Type material Holotype PAKISTAN • Punjab Province, Haroonabad City, District Bahawalnagar; 29°60′81″ N, 73°14′67″ E; alt. 163 m a.s.l.; on nutrient-rich loamy soil; 4 Aug. 2019; Muhammad Asif, BWN-45; GenBank nos MZ265143 (nrITS); ON116490 (28S); LAH[36793]. Additional material examined PAKISTAN • Punjab Province, Lahore, 32°52′04″ N, 74°35′87″ E; alt. 217 m a.s.l.; on loamy soil; 10 Jul. 2015; Qudsia Firdous, BRB S-01; GenBank nos KY636363 (nrITS); ON116491 (LSU); LAH[36792] • same collection data as for preceding; 28 Jul. 2016; Qudsia Firdous, BRB S-22; GenBank nos MZ823627 (nrITS); ON116492 (LSU); LAH[36794] • Punjab Province, Kasur District, 31°12′79″ N, 74°44′08″ E; alt. 218 m a.s.l.; on fallen plant debris; 6 Sep. 2020; Aiman Izhar, KS-0018; GenBank no. OP681142 (nrITS); LAH[37417]. Description Basidiomata 4.4–5.8 cm tall. Pileus 1–1.5 cm diam, conic to parabolic when young, becoming convex with maturity, dry; surface light brown at the center (7.5YR8/4), light grayish green (7.5GY8/1) toward margins, smooth when young becoming rugulose at maturity; margin straight in young stage, striate at maturity (Fig. 3A, D). Lamellae free, olive black (5GY2/1), even margins, distantly placed, two tiers of regularly arranged lamellulae (Fig. 3B–C). Stipe 3.8–5.3 × 0.3–0.6 cm, surface light grayish-green (10GY8/1), central, equal, surface smooth and glabrous, dry, slightly bulbous base, bruising blue on handling (Fig. 3E). Annulus and volva absent. Odor is indistinct. Basidiospores [75/3/3] (13.2–)13.4–16.4(–16.7) × (7.5–)8.2–9.6(–11.4) µm, on average 15 × 9.5 µm, Q = 1.4–1.6, Qav = 1.5, broadly fusiform, smooth, apiculus absent, thick-walled, hyaline in KOH, no colour change in Melzer‘s reagent, non-guttulate, germ pore obvious (Fig. 4B). Basidia (24.8–)24.9–27.9 (–28.1) × (14.3–)14.4–15.6(–15.9) µm, on average 26.2 × 15.1 µm, broadly clavate, mostly bi-spored, rarely tri- or tetra-spored, thick-walled, hyaline in KOH, non-guttulate (Fig. 4A). Cheilocystidia (30.5–) 32.1–41.4(–44.3) × (6–)6.1–9.1(–9.5) µm, on average 37.4 × 7.8 µm, claviform with flexuous neck and rounded apices, thin-walled, hyaline in KOH, non-guttulate (Fig. 4C). Pileocystidia (18–)19.2–32 https://www.mycobank.org/page/Name%20details%20page/840898 European Journal of Taxonomy 888: 77–96 (2023) 86 Fig. 3. Morphology of Panaeolus punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi & Khalid sp. nov. (LAH36793, A–B holotype). Scale bars: A–D = 1 cm, E = 10 cm. Photos by Muhammad Asif. ASIF M. et al., New species of Panaeolus from Punjab, Pakistan 87 Fig. 4. Microscopic characters of Panaeolus punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi & Khalid sp. nov. (LAH36793, holotype). A. Basidia. B. Basidiospores. C. Cheilocystidia. D. Caulocystidia. E. Pileocystidia. Scale bars: A–D = 10 µm, E = 20 µm. Drawings by Aiman Izhar. European Journal of Taxonomy 888: 77–96 (2023) 88 P . p un ja be ns is 10 –1 5 pa ra bo lic to lig ht b ro w n ol iv e bl ac k 38 –5 3 × lig ht 13 .2 –1 6. 7 × br oa dl y 30 .5 –4 4. 3 × cl av if or m w ith T hi s st ud y sp . n ov . co nv ex at th e ce nt er , 3– 6 gr ay is h 7. 5– 11 .4 fu si fo rm 6– 9. 5 fle xu ou s ne ck lig ht g ra yi sh gr ee n an d ro un de d gr ee n to w ar d ap ic es m ar gi ns P. a cu m in at us 17 –2 0 br oa dl y co ni c da rk re dd is h gr ay is h bl ac k 62 –7 5 × 2 re dd is h 11 .4 –1 5 × le m on - 15 .5 –2 5. 5 × fu so id -v en tr ic os e K au r e t a l. to b ro ad ly br ow n to br ow n 7. 8– 11 sh ap ed 5. 5– 8. 5 to ir re gu la rl y 20 14 ; O s- be ll- sh ap ed gr ay is h cy lin dr ic m un ds on et a l. 20 13 br ow n P. a lc is 4– 10 co m pa nu la te pa le g ra y da rk g ra y 20 –9 0 × pe x pa le 18 –2 1 × el lip so id 25 –3 5 × 4– 6 ve rs if or m M os er 1 98 4 to c on ic al 0. 5– 1. 5 oc hr ac eo us 9. 5– 12 gr ay , b as e br ow ni sh P. a xf or di i 16 –2 1 he m i- re dd is h m ot tle d 42 –5 1 × gr ay is h 8. 8– 11 .4 × lim on i- 24 .6 –4 2. 7 × na rr ow ly u tr i- H u et a l. sp he ri ca l t o br ow n to da rk g ra y 1. 5– 2. 5 or an ge to 6. 3– 9 fo rm to 5. 9– 10 .7 fo rm 20 20 ca m pa nu la te gr ay is h lig ht b ro w n el lip so id w hi te P. c am bo dg in ie ns is 12 –2 5 co nv ex to ch oc ol at e pa lli d, th en 55 –9 5 × w hi tis h to 10 .5 –1 2 × le m on - 26 –3 9 × po ly m or ph ic St am et s 19 96 ; br oa dl y co nv ex br ow n be - gr ay is h bl ac k 3. 5– 5 cr ea m , 6. 5– 9 sh ap ed 10 –1 2. 2 W ee ks e t a l. co m in g ye l- to b la ck br ow n ne ar 19 79 lo w is h br ow n th e ba se P. c ya ne sc en s 10 –1 5 he m is ph er ic to pu re w hi te da rk g ra yi sh 50 × 2 –3 un if or m ly 11 –1 8 × lim on i- ab se nt ab se nt W ar tc ho w e t co nv ex br ow n w hi te 9– 13 .7 fo rm to al . 2 01 0 el lip so id P.  fi m ic ol a  10 –2 0 ca m pa nu la te di ng y gr ay m ot tle d gr ay 60 –1 00 × di ng y pa le 11 –1 4 × su b- am yg - 22 .7 –2 7 × cy lin dr ic to St am et s 19 96 ; to c on ve x to b la ck , 1– 2 to w hi tis h 7– 9. 5 da lif or m 5. 7– 10 su bl ag en if or m W an g & sl ig ht re dd is h T ze an 2 01 5 Ta bl e 2. ( co nt in ue d on n ex t p ag e) C om pa ri so n of m ac ro - an d m ic ro -c ha ra ct er is tic s of P an ae ol us p un ja be ns is M . A si f, Q . F ir do us , A . I zh ar , N ia zi & K ha lid s p. n ov ., an d its c lo se ly re la te d ta xa . A bb re vi at io ns : L = L en gt h; W = W id th . Sp ec ie s na m e P ile us P ile us P ile us L am el la e St ip e St ip e Sp or e Sp or e C he ilo - C he ilo - R ef er en ce di am et er sh ap e co lo r co lo r L × W co lo r L × W sh ap e cy st id ia cy st id ia (m m ) (m m ) (µ m ) L × W (µ m ) sh ap e ASIF M. et al., New species of Panaeolus from Punjab, Pakistan 89 P. g ut tu la tu s 10 co nv ex da rk o liv e bl ac k 40 × 2 sn uff 7. 5– 10 × el lip tic al 25 × 5 cy lin dr ic al Se id m oh am - br ow n br ow ni sh 4– 6 m ad i e t a l. 20 19 ; S ul ia - m an 2 01 9 P . p ap ili on ac eu s 10 –5 0 co ni ca l t o br ow ni sh to gr ay to 60 –1 20 × w hi tis h to 12 –1 4 × ci tr if or m 35 –5 3 × ve rm if or m , u rn - A br ah am ca m pu la na te gr ay is h br ow n en tir el y bl ac k 2– 4 sn uff b ro w n 7– 8 7– 12 .5 sh ap ed o r t ub ul ar 20 07 ; K uo 20 07 ; A m an - de ep e t a l. 20 14 ; E di ri - w ee ra e t a l. 20 15 P. r ic ke ni i 18 –2 0 ca m pa nu la te to da rk b ro w n m ot tle d gr ay - 15 –1 40 × bu ff , l at e 9– 12 × el lip so id 19 .5 –3 7 × cl av at e to M a 20 14 co nv ex is h br ow n 1– 2 da rk b ro w n 7– 9. 5 to b ro ad ly 7– 13 la ge ni fo rm w ith w hi tis h to re dd is h el lip so id m ar gi n br ow n P. s em io va tu s 30 –6 0 eg g- sh ap ed to ci nn am on b uff pa le b ro w n, 10 0– 16 0 × w hi tis h to 18 .5 –2 1 × el lip tic al ab se nt ab se nt St am et s 19 96 ; co ni co -c on ve x to p in ki sh b uff la te r m ot tle d 6– 10 pa lli d bu ff 10 –1 1. 5 O sm un ds on an d fa di ng to bl ac ki sh et a l. 20 13 w hi tis h P. s ph in ct ri nu s 10 –2 0 be ll sh ap ed to br ow ni sh w he n gr ay a nd 25 –9 0 × sn uff b ro w n 0. 4– 11 .3 × ci tr if or m 11 .0 –1 6. 5 × po ly m or ph ic E di ri w ee ra um bo na te im m at ur e, m ot tle d bl ac k, 1– 2 6. 6– 7. 5 to le m on - 5. 5– 9. 2 et a l. 20 15 gr ay is h br ow n l at er e nt ir e sh ap ed w he n m at ur e bl ac k Ta bl e 2. (c on tin ue d) . Sp ec ie s na m e P ile us P ile us P ile us L am el la e St ip e St ip e Sp or e Sp or e C he ilo - C he ilo - R ef er en ce di am et er sh ap e co lo r co lo r L × W co lo r L × W sh ap e cy st id ia cy st id ia (m m ) (m m ) (µ m ) L × W (µ m ) sh ap e European Journal of Taxonomy 888: 77–96 (2023) 90 (–34.4) × (10.4–)11.9–13.9(–17.4) µm, on average 25.5 × 13.4 µm, clavate to vesiculose, thin-walled, hyaline (Fig. 4E). Caulocystidia (25.3–)26.7–36.9(–37.5) × (6–)8.1–9.7(–10.5) µm, on average 31.4 × 8.7 µm), clavate, thick-walled (Fig. 4D). Clamp connections are absent in all tissues. Habitat Solitary or in small groups on loamy soil containing herbivore (cattle) dung. Known distribution Known only from three localities, Bahawalnagar, Kasur, and Lahore, Punjab, Pakistan. Discussion The genus Panaeolus is quite similar in appearance to Panaeolina in the field (Kalichman et al. 2020). The two genera can be differentiated on the basis of basidiospores morphology and lamellae color. Lamellae in Panaeolus are grayish-black and basidiospores are smooth, in Panaeolina spores are ornamented and lamellae are dark brown (Kaur et al. 2014). All previously reported species of Panaeolus from Pakistan are not hallucinogenic, but our new species turns bluish on handling which indicates that it is a hallucinogenic species. Several different tests can be performed to test the hallucinogenic various mushrooms including Panaeolus, such as Amplified Fragment Length Polymorphism (AFLP) and High-Resolution Melting essays (HRM) (Lee et al. 2000; Zhang et al. 2022). A detailed comparison of macro- and micro-characteristics of all the closely related species of Panaeolus is given in Table 2, and from the molecular phylogenetic analyses of ITS and 28S and morphoanatomical comparison given in Table 2, we conclude that Panaeolus punjabensis sp. nov. is a new species. In the ITS-based phylogenetic analysis, P. papilionaceus (KF830093), P. retirugis (Fr.) Gillet (JF908521), P. sphinctrinus (HM03581, DQ182503), P. companulatus (L.) Quél. (JF908522), and P. alcis M.M.Moser (KM982723) lie in the same clade and are closly related to the newly described species, while in the 28S-based phylogenetic analysis, P. semiovatus (MH868191, DQ071694), P. acuminatus (P.Kumm.) Quél. (MH867781, AY207263), P. sphinctrinus (MK278436, AB104646), P. papilionaceus (KF830082, MK278435, AY207265), and P. retirugis (MH867784, MH867785, MH867786, MH867787) are close relatives of our new species. In both phylogenetic analyses, different sequences of the same species appear on different positions such as P. retirugis (Gillet 1878: 621) (current name, P. papilionaceus), P. sphinctrinus, and P. campanulatus (Quélet 1872: 151), and this could result to misidentification of species owing to close similarities among the species in the genus Panaeolus (Razaq et al. 2012). Acknowledgements The authors are grateful to Dr. Shah Hussain (Sultan Qaboos University, Muscat, Oman) and Dr. Thatsanee Luangharn (Mae Fah Luang University, Chiang Rai, Thailand) for their critical review, valuable comments, and suggestions on an earlier version of the manuscript which helped us a lot to improve the article. We are thankful to Dr. Francis Q. Brearley (Manchester Metropolitan University, United Kingdom) for the linguistic review of the manuscript. We are also highly obliged to all the anonymous reviewers for their corrections and suggestions to improve this paper. References Abraham W.R. 2007. Bioactive sesquiterpenes produced from fungi: Possibilities and limitations. In: Rai M. (ed.) Mycotechnology: Present Status and Future Prospects: 264–287. I.K. International, Delhi, India. ASIF M. et al., New species of Panaeolus from Punjab, Pakistan 91 Adeniyi M., Odeyemi Y. & Odeyemi O. 2018. Ecology, diversity and seasonal distribution of wild mushrooms in a Nigerian tropical forest reserve. Biodiversitas Journal of Biological Diversity 19 (1): 285–295. https://doi.org/10.13057/biodiv/d190139 Ahmad A., Khan M., Shah S.H.H., Kamran M., Wajid S.A., Amin M., Khan A., Arshad M.N., Cheema M.J.M., Saqid Z.A. & Ullah R. 2019. Agro-ecological zones of Punjab, Pakistan. Food and Agriculture Organization of United Nations, Rome. Ahmad S., Iqbal S.H. & Khalid A.N. 1997. Fungi of Pakistan. Sultan Ahmad Mycological Society of Pakistan, Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore. Ahmed N., Mahmood A., Mahmood A., Tahir S.S., Bano A., Malik R.N., Hassan S. & Ishtiaq M. 2014a. Relative importance of indigenous medicinal plants from Layyah district, Punjab Province, Pakistan. Journal of Ethnopharmacology 155 (1): 509–523. https://doi.org/10.1016/j.jep.2014.05.052 Ahmed N., Mahmood A., Tahir S.S., Bano A., Malik R.N., Hassan S. & Ashraf A. 2014b. Ethnomedicinal knowledge and relative importance of indigenous medicinal plants of Cholistan desert, Punjab Province, Pakistan. Journal of Ethnopharmacology 155 (2): 1263–1275. https://doi.org/10.1016/j.jep.2014.07.007 Abraham W.R. 2007. Bioactive sesquiterpenes produced from fungi: Possibilities and limitations. In: Rai M. (ed.) Mycotechnology: Present Status and Future Prospects: 264–287. I.K. International, Delhi, India. Adeniyi M., Odeyemi Y. & Odeyemi O. 2018. Ecology, diversity and seasonal distribution of wild mushrooms in a Nigerian tropical forest reserve. Biodiversitas Journal of Biological Diversity 19 (1): 285–295. https://doi.org/10.13057/biodiv/d190139 Akata I., Altuntaş D. & Kabaktepe Ş. 2019. Fungi determined in Ankara University Tandoğan Campus area (Ankara-Turkey). Trakya University Journal of Natural Sciences 20 (1): 47–55. https://doi.org/10.23902/trkjnat.521256 Altschul S.F., Gish W., Miller W., Myers E.W. & Lipman D.J. 1990. Basic local alignment search tool. Journal of Molecular Biology 215 (3): 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2 Aman N., Khalid A.N. & Moncalvo J.-M. 2022. A compendium of macrofungi of Pakistan by ecoregions. MycoKeys 89: 171–233. https://doi.org/10.3897/mycokeys.89.81148 Amandeep K., Atri N.S. & Munruchi K. 2014. Two new species of Panaeolus (Psathyrellaceae, Agaricales) from coprophilous habitats of Punjab, India. Mycosphere 3: 125–132. https://doi.org/10.5943/mycosphere/4/3/13 Andersson C., Kristinsson J. & Gry J. 2009. Occurrence and Use of Hallucinogenic Mushrooms Containing Psilocybin Alkaloids. Nordic Council of Ministers. Anwar W., Khan S.N., Tahira J.J. & Suliman R. 2012. Parthenium hysterophorus: an emerging threat for Curcuma longa fields of Kasur District, Punjab, Pakistan. Pakistan Journal of Weed Science Research 18: 91–97. Bas C. 1969. Morphology and subdivision of Amanita and a monograph of its section Lepidella. Persoonia 5: 96–97. https://repository.naturalis.nl/pub/531781 Belda M., Holtanová E., Halenka T. & Kalvová J. 2014. Climate classification revisited: from Köppen to Trewartha. Climate Research 59: 1–13. https://doi.org/10.3354/cr01204 Broussal M. & Dumesny E. 2015. Une récolte française de Stagnicola perplexa. Bulletin de la Société Mycologique de France 131: 237–243. Cai Q., Chen Z.H., He Z.M., Luo H. & Yang Z.L. 2018. Lepiota venenata, a new species related to toxic mushroom in China. Journal of Fungal Research 16: 63–69. https://doi.org/10.13057/biodiv/d190139 https://doi.org/10.1016/j.jep.2014.05.052 https://doi.org/10.1016/j.jep.2014.07.007 https://doi.org/10.13057/biodiv/d190139 https://doi.org/10.23902/trkjnat.521256 https://doi.org/10.1016/S0022-2836(05)80360-2 https://doi.org/10.3897/mycokeys.89.81148 https://doi.org/10.5943/mycosphere/4/3/13 https://repository.naturalis.nl/pub/531781 https://doi.org/10.3354/cr01204 European Journal of Taxonomy 888: 77–96 (2023) 92 Chapela I.H., Rehner S.A., Schultz T.R. & Mueller U.G. 1994. Evolutionary history of the symbiosis between fungus-growing ants and their fungi. Science 266: 1691–1694. Darriba D., Taboada G.L., Doallo R. & Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772. https://doi.org/10.1038/nmeth.2109 Desjardin D.E. & Perry B.A. 2017. Panaeolus antillarum (Basidiomycota, Psathyrellaceae) from wild elephant dung in Thailand. Current Research in Environmental & Applied Mycology 7: 275–281. https://doi.org/10.5943/cream/7/4/4 Doveri F. 2011. Additions to “Fungi Fimicoli Italici”: An update on the occurrence of coprophilous Basidiomycetes and Ascomycetes in Italy with new records and descriptions. Mycosphere 2: 331–427. Drehmel D., Moncalvo J.M. & Vilgalys R. 1999. Molecular phylogeny of Amanita based on large- subunit ribosomal DNA sequences: implications for taxonomy and character evolution. Mycologia 91: 610–618. https://doi.org/10.1080/00275514.1999.12061059 Drummond A.J & Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7: 214. https://doi.org/10.1186/1471-2148-7-214 Durrani A.Z. & Shakoori A.R. 2009. Study on ecological growth conditions of cattle Hyalomma ticks in Punjab, Pakistan. Iranian Journal of Parasitology 4: 24–30. Edgar R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792–1797. https://doi.org/10.1093/nar/gkh340 Ediriweera S., Wijesundera R., Nanayakkara C. & Weerasena J. 2015. First report of Panaeolus sphinctrinus and Panaeolus foenisecii (Psathyrellaceae, Agaricales) on elephant dung from Sri Lanka. Frontiers in Environmental Microbiology 1: 19–23. https://doi.org/10.11648/j.fem.20150102.12 Garnica S., Weiss M., Walther G. & Oberwinkler F. 2007. Reconstructing the evolution of agarics from nuclear gene sequences and basidiospore ultrastructure. Mycological Research 111: 1019–1029. https://doi.org/10.1016/j.mycres.2007.03.019 Gerhardt E. 1996. Taxonomische Revision der Gattungen Panaeolus und Panaeolina (Fungi, Agaricales, Coprinaceae). Bibliotheca Botanica 147: 1–149. Gillet C.C. 1878. Les Hyménomycètes ou description de tous les champignons qui croissent en France. Description et iconographie, propriétés utiles ou vénéneuses: 561–828. JB Baillère & fils, Paris. Halama M., Witkowska D., Jasicka-misiak I. & Poliwoda A. 2014. An adventive Panaeolus antillarum in Poland (Basidiomycota, Agaricales) with notes on its taxonomy, geographical distribution, and ecology. Cryptogamie, Mycologie 35: 3–22. https://doi.org/10.7872/crym.v35.iss1.2014.3 Han K.S., Volk T.J. & Kim H.K. 2010. Identification of Lacrymaria velutina (Pers. ex Fr.) Konrad & Maubl. from Micheon-myeon, Jinju-city, Korea. Mycobiology 38: 249–255. He Z., Su Y., Li S., Long P., Zhang P. & Chen Z. 2019. Development and evaluation of isothermal amplification methods for rapid detection of lethal Amanita species. Frontiers in Microbiology 10: 1523. https://doi.org/10.3389/fmicb.2019.01523 Hora F.B. 1960. New check list of British agarics and boleti: part IV. Validations, new species and critical notes. Transactions of the British Mycological Society 43: 440–459. https://doi.org/10.1016/S0007-1536(60)80067-8 Hu Y., Mortimer P.E., Karunarathna S.C., Raspé O., Promputtha I., Yan K., Xu J. & Hyde K. 2020. A new species of Panaeolus (Agaricales, Basidiomycota) from Yunnan, Southwest China. Phytotaxa 434: 22–34. https://doi.org/10.11646/phytotaxa.434.1.3 https://doi.org/10.1038/nmeth.2109 https://doi.org/10.5943/cream/7/4/4 https://doi.org/10.1080/00275514.1999.12061059 https://doi.org/10.1186/1471-2148-7-214 https://doi.org/10.1093/nar/gkh340 https://doi.org/10.11648/j.fem.20150102.12 https://doi.org/10.1016/j.mycres.2007.03.019 https://doi.org/10.7872/crym.v35.iss1.2014.3 https://doi.org/10.3389/fmicb.2019.01523 https://doi.org/10.1016/S0007-1536(60)80067-8 https://doi.org/10.11646/phytotaxa.434.1.3 ASIF M. et al., New species of Panaeolus from Punjab, Pakistan 93 Jayasiri S.C., Hyde K.D., Ariyawansa H.A., Bhat J., Buyck B., Cai L., Dai Y.-C., Abd-Elsalam K.A., Ertz D., Hidayat I., et al. 2015. The faces of Fungi database: fungal names linked with morphology, phylogeny and human impacts. Fungal diversity 74: 3–18. https://doi.org/10.1007/s13225-015-0351-8 Kalichman J., Kirk P.M. & Matheny P.B. 2020. A compendium of generic names of agarics and Agaricales. Taxon 69: 425–447. https://doi.org/10.1002/tax.12240 Karunarathna S.C., Mortimer P.E., Xu J. & Hyde K.D. 2017. Overview of research of mushrooms in Sri Lanka. Revista Fitotecnia Mexicana 40: 399–403. https://www.redalyc.org/articulo.oa?id=61054247004 Kaur A., Atri N.S. & Kaur M. 2014. Diversity of coprophilous species of Panaeolus (Psathyrellaceae, Agaricales) from Punjab, India. Biodiversitas Journal of Biological Diversity 15: 115–130. https://doi.org/10.13057/biodiv/d150202 Kuo M. 2007. The genus Panaeolus. Retrieved from the MushroomExpert.Com. Website: http://www.mushroomexpert.com/panaeolus.html [accessed 25 Apr. 2022]. Lateef M., Gondal K.Z., Younas M., Sarwar M., Mustafa M.I. & Bashir M.K. 2008. Milk production potential of purebred Holstein Friesian and Jersey cows in subtropical environment of Pakistan. Pakistan Veterinary Journal 28: 9–12. Lee J.CI., Cole M. & Linacre A. 2000. Identification of hallucinogenic fungi from the genera Psilocybe and Panaeolus by amplified fragment length polymorphism. Electrophoresis: An International Journal 21: 1484–1487. Li H., Ma X., Mortimer P.E., Karunarathna S.C., Xu J. & Hyde K.D. 2016. Phallus haitangensis, a new species of stinkhorn from Yunnan Province, China. Phytotaxa 280: 116–128. https://doi.org/10.11646/phytotaxa.280.2.2 Liang J.F., Yang Z.L. & Xu D.P. 2011. A new species of Lepiota from China. Mycologia 103: 820–830. https://doi.org/10.3852/10-216 Liang J.F., Yu F., Lu J.K., Wang S.K. & Song J. 2018. Morphological and molecular evidence for two new species in Lepiota from China. Mycologia 110: 494–501. https://doi.org/10.1080/00275514.2018.1464333 Lundell S. & Nannfeldt J.A. 1938. Fungi Exsiccati Suecici. Fasc. 11–12: 501–600. Uppsala University. Ma T. 2014. Taxonomy of Psilocybe s.l. and Panaeolus in Yunnan, Southwest China, with Notes on Related Genus Protostropharia. Chinese Academy of Forestry, China. Malysheva E., Moreno G., Villarreal M., Malysheva V. & Svetasheva T. 2019. The secotioid genus Galeropsis (Agaricomycetes, Basidiomycota): a real taxonomic unit or ecological phenomenon? Mycological Progress 18: 805–831. https://doi.org/10.1007/s11557-019-01490-6 Maruyama T., Yokoyama K., Makino Y. & Goda Y. 2003. Phylogenetic relationship of psychoactive fungi based on the rRNA gene for a large subunit and their identification using the TaqMan assay. Chemical and Pharmaceutical Bulletin 51: 710–714. Maruyama T., Kawahara N., Yokoyama K., Makino Y., Fukiharu T. & Goda Y. 2006. Phylogenetic relationship of psychoactive fungi based on rRNA gene for a large subunit and their identification using the TaqMan assay (II). Forensic Science International 163: 51–58. https://doi.org/10.1016/j.forsciint.2004.10.028 Miller M.A., Holder M.T., Vos R., Midford P.E., Liebowvitz T., et al. 2010. The CIPRES Portals. Available from https://www.phylo.org/ [accessed 17 Apr. 2022]. Moser M. 1984. Panaeolus alcidis, a new species from Scandinavia and Canada. Mycologia 76: 551– 554. https://doi.org/10.1080/00275514.1984.12023878 https://doi.org/10.1007/s13225-015-0351-8 https://doi.org/10.1002/tax.12240 https://www.redalyc.org/articulo.oa?id=61054247004 https://doi.org/10.13057/biodiv/d150202 http://www.mushroomexpert.com/panaeolus.html https://doi.org/10.11646/phytotaxa.280.2.2 https://doi.org/10.3852/10-216 https://doi.org/10.1080/00275514.2018.1464333 https://doi.org/10.1007/s11557-019-01490-6 https://doi.org/10.1016/j.forsciint.2004.10.028 https://www.phylo.org/ https://doi.org/10.1080/00275514.1984.12023878 European Journal of Taxonomy 888: 77–96 (2023) 94 Munsell. 1975. Munsell soil color charts. Macbeth Division of Kollmorgen Corporation. Baltimore, Maryland. Nasir Y.J., Rafiq R.A. & Roberts T.J. 1995. Wildflowers of Pakistan. Oxford University Press. Örstadius L., Ryberg M. & Larsson E. 2015. Molecular phylogenetics and taxonomy in Psathyrellaceae (Agaricales) with focus on psathyrelloid species: introduction of three new genera and 18 new species. Mycological Progress 14: 1–42. Osmundson T.W., Robert V.A., Schoch C.L., Baker L.J., Smith A., Robich G., Mizzan L. & Garbelotto M.M. 2013. Filling gaps in biodiversity knowledge for macrofungi: contributions and assessment of an herbarium collection DNA barcode sequencing project. PLoS One 8: e62419. https://doi.org/10.1371/journal.pone.0062419 Peel M.C., Finlayson B.L. & McMahon T.A. 2007. Updated world map of the Köppen Geiger climate classification. Hydrology and Earth System Science 11: 1633–1644. https://doi.org/10.5194/hess-11-1633-2007 Porebski S., Bailey L.G. & Baum B.R. 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 15: 8–15. Quélet L. 1872. Les Champignons du Jura et des Vosges. Mémoires de la Société d’Émulation de Montbéliard 2: 43–332. Rambaut A. 2014. FigTree 1.4.2 software. Institute of Evolutionary Biology, University of Edinburgh [accessed 11 Mar. 2022]. Razaq A., Khalid A.N. & Ilyas S. 2012. Molecular identification of Lyophyllum connatum and Paneolus sphinctrinus (Basidiomycota, Agaricales) from Himalyan moist temperate forests of Pakistan. International Journal of Agriculture and Biology 14: 1001–1004. Seidmohammadi E., Abbasi S. & Asef M.R. 2019. The first report of Panaeolus olivaceus and Panaeolus guttulatus from Iran. Taxonomy and Biosystematics 11: 23–30. Senn-Irlet B., Nyffenegger A. & Brenneisen R. 1999. Panaeolus bisporus – an adventitious fungus in central Europe, rich in psilocin. Mycologist 13: 176–179. Sette L.D., Passarini M.R.Z., Rodrigues A., Leal R.R., Simioni K.C.M., Nobre F.S., De Brito B.R., Da Rocha A.J. & Pagnocca F.C. 2010. Fungal diversity associated with Brazilian energy transmission towers. Fungal Diversity 44: 53–63. https://doi.org/10.1007/s13225-010-0048-y Siddiqui R., Siddiqui S., Javid K. & Akram M. 2020. Estimation of rainwater harvesting potential and its utility in the educational institutes of Lahore using GIS techniques. Pakistan Geographical Review 75: 1–9. Silva-Filho A.G.S., Seger C. & Cortez V.G. 2019. Panaeolus (Agaricales) from Western Paraná state, South Brazil, with a description of a new species, Panaeolus sylvaticus. Edinburgh Journal of Botany 76: 297–309. https://doi.org/10.1017/S0960428619000064 Singer R. 1986. The Agaricales in Modern Taxonomy. 4th Ed. Koeltz Scientific Books, Federal Republic of Germany. Singer R. & De Leon P. 1982. Galeropsidaceae west of the Rocky Mountains. Mycotaxon 14: 82–90. Stamets P. 1996. Psilocybin Mushrooms of the World. Ten Speed Press, Berkeley, California, USA. Strauss D., Ghosh S., Murray Z. & Gryzenhout M. 2022. An overview on the taxonomy, phylogenetics and ecology of the psychedelic genera Psilocybe, Panaeolus, Pluteus and Gymnopilus. Frontiers in Forests and Global Change 5: 813998. https://doi.org/10.3389/ffgc.2022.813998 https://doi.org/10.1371/journal.pone.0062419 https://doi.org/10.5194/hess-11-1633-2007 https://doi.org/10.1007/s13225-010-0048-y https://doi.org/10.1017/S0960428619000064 https://doi.org/10.3389/ffgc.2022.813998 ASIF M. et al., New species of Panaeolus from Punjab, Pakistan 95 Suliaman S.Q. 2019. First record of three mycofungal Basidiomycota from Iraq. Plant Archives 19: 313–318. Tamura K., Peterson D., Peterson N., Stecher G., Nei M. & Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739. https://doi.org/10.1093/molbev/msr121 Tanveer M., Ahmed S.R., Aslam R.W., Khalid M.B., Ullah H., Aziz A., Abbas W. & Mirza A.I. 2020. Assessment of irrigated land transformations in Lahore. International Journal of Agriculture & Sustainable Development 2: 114–126. Teke A.N., Kinge T.R., Bechem E.E.T., Ndam L.M. & Mih A.M. 2019. Mushroom species richness, distribution and substrate specificity in the Kilum-Ijim forest reserve of Cameroon. Journal of Applied Biosciences 133: 13592–13617. https://doi.org/10.4314/jab.v133i1.11 Tóth A., Hausknecht A., Krisai-Greilhuber I., Papp T., Vágvölgyi C. & Nagy L.G. 2013. Iteratively refined guide trees help improving alignment and phylogenetic inference in the mushroom family Bolbitiaceae. PLoS One 8: e56143. https://doi.org/10.1371/journal.pone.0056143 Undan R. 2016. Molecular identification and phylogeny of some wild microscopic fungi from selected areas of Jaen, Nueva Ecija, Philippines. Advances in Environmental Biology 10: 153–158. Varga T., Krizsán K., Földi C., Dima B., Sánchez-García M., Sánchez-Ramírez S., Szöllösi G.J., Szarkándi J.G., Papp V., Albert L., et al. 2019. Megaphylogeny resolves global patterns of mushroom evolution. Nature Ecology & Evolution 3: 668–678. https://doi.org/10.1038/s41559-019-0834-1 Vellinga E.C. 2001. Agaricaceae. In: Noordeloos M.E., Kuyper T.W. & Vellinga E.C. (eds) Flora Agaricina Neerlandica 5. Rotterdam, Balkema Publishers. Vilgalys R. & Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4239–4246. Voto P. & Angelina C. 2021. First record of Copelandia mexicana in Dominican Republic and notes on Panaeolus. Mycological Observations 1: 44–58. Vu D., Groenewald M., De Vries M., Gehrmann T., Stielow B., Eberhardt U., Al-Hatmi A., Groenewald J.Z., Cardinali G., Houbraken J., et al. 2019. Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation. Studies in Mycology 91: 135–154. https://doi.org/10.1016/j.simyco.2018.05.001 Waheed M., Arshad F., Iqbal M., Fatima K. & Fatima K. 2020. Ethnobotanical assessment of woody flora of district Kasur (Punjab), Pakistan. Ethnobotany Research and Applications 20: 1–13. https://doi.org/10.32859/era.20.33.1-13 Walther G., Garnica S. & Weiẞ M. 2005. The systematic relevance of conidiogenesis modes in the gilled Agaricales. Mycological Research 109 (5): 525–544. https://doi.org/10.1017/S0953756205002868 Wang Y.W. & Tzean S.S. 2015. Dung-associated, potentially hallucinogenic mushrooms from Taiwan. Taiwania 60 (4): 160–168. https://doi.org/10.6165/tai.2015.60.160 Warnke S.E. 2020. PCR‐based detection of the epibiotic fungus Atkinsonella hypoxylon associated with its host grass Danthonia spicata. Crop Science 60: 1660–1665. https://doi.org/10.1002/csc2.20149 Wartchow F., Carvalho A.S. & Sousa M.C.A. 2010. First record of the psychotropic mushroom Copelandia cyanescens (Agaricales) from Pernambuco State, Northeast Brazil. Brazilian Journal of Bioscience 8: 59–60. https://doi.org/10.1093/molbev/msr121 https://doi.org/10.4314/jab.v133i1.11 https://doi.org/10.1371/journal.pone.0056143 https://doi.org/10.1038/s41559-019-0834-1 https://doi.org/10.1016/j.simyco.2018.05.001 https://doi.org/10.32859/era.20.33.1-13 https://doi.org/10.1017/S0953756205002868 https://doi.org/10.6165/tai.2015.60.160 https://doi.org/10.1002/csc2.20149 European Journal of Taxonomy 888: 77–96 (2023) 96 Watling R. & Gregory N.M. 1987. British Fungus Flora. Agarics and Boleti. 5. Strophariaceae & Coprinaceae p.p. Hypholoma, Melanotus, Psilocybe, Stropharia, Lacrymaria & Panaeolus: 76–93. Weeks R.A., Singer R. & Hearns W.L. 1979. A new species of Copelandia. Lloydia 42: 469–474. White T.J., Bruns T., Lee S. & Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a Guide to Methods and Applications: 315–322. Academic Press, San Diego. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 Yu W.J., Chang C., Qin L.W., Zeng N.K., Wang S.X. & Fan Y.G. 2020. Pseudosperma citrinostipes (Inocybaceae), is a new species associated with Keteleeria from southwestern China. Phytotaxa 450: 8–16. https://doi.org/10.11646/phytotaxa.450.1.2 Zabihullah Q., Rashid A. & Akhtar N. 2006. Ethnobotanical survey in kot Manzaray Baba valley Malakand agency, Pakistan. Pakistan Journal of Plant Sciences 12: 115–121. Zeller S.M. 1943. North American species of Galeropsis, Gyrophragmium, Longia, and Montagnea. Mycologia 35: 409–421. Zhang L.F., Yang J.B., Yang Z.L., Zhang L.F. & Yang J.B.A. 2004. Molecular phylogeny of eastern Asian species of Amanita (Agaricales, Basidiomycota): taxonomic and biogeographic implications. Fungal Diversity 17: 219–238. Zhang X., Yu H., Wang Z., Yang Q., Xia R., Qu Y., Tao R., Shi Y., Xiang P., Zhang S. & Li C. 2022. Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM). Forensic Sciences Research 7: 490–497. https://doi.org/10.1080/20961790.2021.1875580 Zhao R., Karunarathna S., Raspé O., Parra L.A., Guinberteau J., Moinard M., De Kesel A., Barroso G., Courtecuisse R., Hyde K.D., et al. 2011. Major clades in tropical Agaricus. Fungal Diversity 51: 279–296. https://doi.org/10.1007/s13225-011-0136-7 Zhao R.-L., Zhou J.-L., Chen J., Margaritescu S., Sánchez-Ramírez S., Hyde K.D., Callac P., Parra L.A., Lie G.-J. & Moncalvo J.M. 2016. Towards standardizing taxonomic ranks using divergence times – a case study for reconstruction of the Agaricus taxonomic system. Fungal Diversity 78: 239–292. https://doi.org/10.1007/s13225-016-0357-x Manuscript received: 6 June 2022 Manuscript accepted: 2 February 2023 Published on: 10 August 2023 Topic editor: Frederik Leliaert Desk editor: Connie Baak Printed versions of all papers are also deposited in the libraries of the institutes that are members of the EJT consortium: Muséum national d’histoire naturelle, Paris, France; Meise Botanic Garden, Belgium; Royal Museum for Central Africa, Tervuren, Belgium; Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Natural History Museum of Denmark, Copenhagen, Denmark; Naturalis Biodiversity Center, Leiden, the Netherlands; Museo Nacional de Ciencias Naturales-CSIC, Madrid, Spain; Leibniz Institute for the Analysis of Biodiversity Change, Bonn – Hamburg, Germany; National Museum of the Czech Republic, Prague, Czech Republic. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 https://doi.org/10.11646/phytotaxa.450.1.2 https://doi.org/10.1080/20961790.2021.1875580 https://doi.org/10.1007/s13225-011-0136-7 https://doi.org/10.1007/s13225-016-0357-x