Turkish Journal of Botany Turk J Bot (2019) 43: 831-849 http://journals.tubitak.gov.tr/botany/ © TÜBİTAK Research Article doi:10.3906/bot-1903-21 Amanita cinis and A. olivovaginata (Basidiomycota, Amanitaceae), two new species, and the first record of A. emodotrygon, from Northwestern Pakistan 1,3, 2,3 4 Sadiq ULLAH *, Andrew William WILSON , Rodham Elliott TULLOSS , 1 3 5 Muhammad FIAZ , Gregory Michael MUELLER , Abdul Nasir KHALID 1 Department of Botany, Hazara University, Dhodial, Pakistan 2 Denver Botanic Gardens, Denver, CO, USA 3 Chicago Botanic Garden, Glencoe, IL, USA 4 Herbarium Amanitarum Rooseveltensis, Roosevelt, NJ, USA 5 Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan Received: 17.03.2019 Accepted/Published Online: 26.07.2019 Final Version: 21.11.2019 Abstract: Two new species found in Northwestern Pakistan in the mushroom genus Amanita are described and illustrated. Phylogenetic data derived from nuclear ribosomal ITS and LSU regions along with morphological characterizations indicate that these species are novel. Amanita cinis is a member of section Roanokenses, while A. olivovaginata is representative of the section Vaginatae. Amanita emodotrygon, recently described from the state of Uttarakhand, India, is new to Pakistan. Key words: Agaricales, Ectomycorrhizae, Himalaya, Mansehra, Shangla 1. Introduction ectomycorrhizal partners. The Shangla District of Pakistan To date, the genus Amanita Pers. comprises about 650 lies on the western edge of the Himalayan range and under accepted species and is estimated to contain overall 1000 the Hindukush mountain range with average elevation or more species (Yang et al., 2018; Cui et al., 2018). The 2000–3500 m above sea level (asl) (Ullah et al., 2019a). genus is important because it contains sought-after edible Almost 90% of the area consists of high mountains, with species, but also deadly poisonous species. They are 10% of the area occurring as plains along the Indus river ecologically relevant because the majority of them form side (Ullah et al., 2019b). Climatically, District Shangla is a ectomycorrhizal associations with vascular plants and play moist temperate area (Figure 1), bordering the Himalayan important roles in ecosystems and forest development range (Champion et al., 1965). The highest temperature in (Yang, 1997). Taxonomic studies of Amanita from Asia summer is 38o C, while in winter it goes down to −2 to have largely focused on China, India, and Japan (Nagasawa −5 °C. The observed relative humidity of the area is 65.9% and Hongo, 1984; Yang, 1997; Oda et al., 1999; Moncalvo et annually (Ullah, 2018). Mansehra District is east of Shangla al., 2000; Tulloss et al., 2001; Chen et al., 2001; Yang, 2001, District, with a similar average elevation (2000–3500 m 2002; Oda et al., 2002; Yang, 2004; Jie et al., 2009; Zhang et a.s.l.). The climate is also similar (moist temperate), with al., 2010; Chen, 2014; Deng et al., 2014; Zhang et al., 2015; seasonal periods of rainfall, snow, and drought. Most of the Yang, 2015; Endo et al., 2017; Cui et al., 2018; Yang et al., rainfall occurs during the monsoon season (July–August). 2018). About 60 species of Amanita have been reported The total annual rainfall of the district is 1828 mm, and the from various parts of India, and more than 160 species temperature ranges from 2 to 36 °C (Fiaz, 2013). have been reported from China (Yang, 1997, 2000; Semwal According to the standard classification of forest types et al., 2014; Singh and Kaur, 2016; Cui et al., 2018; Yang of Pakistan, the forests fall under the category “Montane et al., 2018). Despite these contributions to the knowledge temperate forests” (Champion et al., 1965). The mountains of Amanita in Asia, many regions of the continent remain of both districts consist of mostly coniferous forests understudied (Tulloss, 2005). containing Pinus roxburghii Sarg., P. wallichiana A. B. Pakistan has Himalayan moist temperate forests Jackson, Abies pindrow (Royle ex D. Don) Royle, Picea which are generally suitable for Amanita and their smithiana (Wall.) Boiss., and Taxus wallichiana Zucc. * Correspondence: [email protected] 831 This work is licensed under a Creative Commons Attribution 4.0 International License. ULLAH et al. / Turk J Bot Figure 1. Map of Pakistan based on the Köppen climate classification. The collection sites are given: Shangla District is shown by a rectangle of red boundaries and Mansehra District is shown by a rectangle of yellow boundaries. Broadleaved tree species include Aesculus indica (Wall. produced a comprehensive report of a rubescent species ex Camb.) Hook., Acer caesium Wall. ex Brandis, Juglans of section Validae (Fr.) Quél. (possibly A. orsonii Kumar regia L., Quercus incana Roxb., Q. semicarpifolia Smith, and Lakhanpal) growing ectomycorrhizal on Himalayan and Ulmus wallichiana Planch. (Ullah, 2018: Ullah et al., spruce (Picea smithiana). Kiran et al. (2017) reported 2019a). The moist temperate climate has in average 1778 A. pallidorosea and its ectomycorrhizal association with mm of precipitation annually (Ullah, 2018). While the Quercus oblongata. Jabeen et al. (2017) reported a new habitat is ideal for Amanita, few studies of the genus have species, A. glarea S. Jabeen et al., from northwestern been done here. Pakistan. More recently, Kiran et al. (2018) reported a new Ahmad (1956) reported a species list containing species, A. griseofusca J. Khan and M. Kiran, from Swat, only 2 taxa of Amanita (A. nana Sing. and A. vaginata Pakistan. [Bull.: Fr.] Lamarck). Tulloss et al. (2001) published 9 To date, only 15 species of Amanita have been taxa of Amanita from the Himalayan moist temperate documented from Pakistan. This study increases the forests of northwestern Pakistan. Niazi et al. (2009) existing taxonomic knowledge of Amanita from Pakistan 832 ULLAH et al. / Turk J Bot by describing 2 new species and a new record from the 2.2. Molecular analysis northwestern region of the country using morphological Extractions were obtained from 5–15 mg of dried descriptions and molecular phylogenetic data. sporocarp material using a Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA. http://www.qiagen.com/). 2. Materials and methods PCR and cycle sequencing were performed to obtain 2.1. Morphological characterization sequences of nuclear ribosomal internally transcribed Specimens were collected from the Shangla and Mansehra spacer regions ITS1, 5.8S, and ITS2 (ITS) using primer districts (Figure 1) during 2013–2016. Fresh specimens pairs ITS1F/ITS4 or ITS2/ITS3 (White et al., 1990; Gardes were photographed using a Nikon DS3300 digital camera and Bruns, 1993). Amplification of the nuclear ribosomal and tagged. Macromorphological features such as the large subunit (LSU) genes was done by the primer pair color of the basidiome, size and shape of the pileus, the LR0R/LR5 (Vilgalys and Hester, 1990). A 25 µL volume PCR reaction was performed, containing 9 µL ddH O, stipe, the annulus, volva, lamellar features, and associated 2 12.5 µL PCR master mix, 1.25 µL forward primer, 1.25 vegetation of the area were noted. The specimens were µL reverse primer, and 1 µL DNA sample. Amplification dried and stored in labeled boxes. Color codes were protocols and PCR conditions used an initial denaturation designated using the Munsell Color System (1975). step of 2 min at 95 °C, followed by 30 cycles of the following Micromorphological features were described according steps: a denature step of 1 min at 95 °C, an annealing to Tulloss and Rodríguez-Caycedo (2011). Features step of 30 s at 50 °C, and an extension step of 2 min at were described from sections prepared in 3% KOH, 1% 72 °C; the reaction was finalized by a final extension for Congo Red, and Melzer’s reagent, and observed using a 5 min at 72 °C. Cycle sequence reaction was performed compound light microscope (MX4300H Techno Co., Ltd., using the above PCR primers, with separate reactions for Japan) at a magnification of 1000× with oil-immersion. forward and reverse primers, and the Big Dye Terminator Measurements were recorded using a Carl Zeiss Jena Kit 3.1 (Applied Biosystems, Foster City, CA, USA) with ocular micrometer, and line drawings were made using a thermocycler protocols using an annealing temperature camera lucida. of 55 °C. PCR products and cycle sequence reactions At least 100 basidiospores were measured per species. were purified using a standard ethanol precipitation prep. Values in brackets (= [a/b/c]) represent the number of DNA extraction, PCR, and cycle sequencing reactions spores measured (a), per number of basidiomes (b), per were performed at the Chicago Botanic Garden’s Center number of collections (c). Basidiospores dimensions are for Plant Biology and Conservation, Glencoe, Illinois. shown as (k–) m–n (–p) – The values m“ ” and “n” represent Sequencing was performed using an ABI-3730-XL DNA the 5% and 95% quantiles of spore size distribution (length Analyzer (Applied Biosystems) in the Pritzker Laboratory and width), respectively, with “k” as the smallest and “p” as at the Field Museum of Natural History, Chicago, Illinois. the largest observed value. We use the biometric variables Sequences produced for this study have been deposited in defined by Tulloss (Tulloss and Rodríguez-Caycedo, GenBank. 2011). The value L is the range of average spore lengths per The sequences were processed, edited, and assembled specimen examined, and L´ is the overall average spore using Codon Code Aligner 3.5 and Bioedit 7.0. They were length, W the range of average spore widths per specimen then screened for percentage sequence identity using a examined, W´ is the overall average spore width, Q is BLAST search of GenBank and UNITE databases. Nearest the ratio of length/width for one spore, Q is the average matches from both databases were downloaded for value of the length/width ratio for spores per specimen phylogenetic analysis.