Amanita cinis and A . olivovaginata ( Basidiomycota , Amanitaceae ) , two new species , and the first record of A . emodotrygon , from Northwestern Pakistan

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.


Introduction
To date, the genus Amanita Pers. comprises about 650 accepted species and is estimated to contain overall 1000 or more species Cui et al., 2018). The genus is important because it contains sought-after edible species, but also deadly poisonous species. They are ecologically relevant because the majority of them form ectomycorrhizal associations with vascular plants and play important roles in ecosystems and forest development (Yang, 1997). Taxonomic studies of Amanita from Asia have largely focused on China, India, and Japan (Nagasawa and Hongo, 1984;Yang, 1997;Oda et al., 1999;Moncalvo et al., 2000;Tulloss et al., 2001;Chen et al., 2001;Yang, 2001Yang, , 2002Oda et al., 2002;Yang, 2004;Jie et al., 2009;Zhang et al., 2010;Chen, 2014;Deng et al., 2014;Zhang et al., 2015;Yang, 2015;Endo et al., 2017;Cui et al., 2018;Yang et al., 2018). About 60 species of Amanita have been reported from various parts of India, and more than 160 species have been reported from China (Yang, 1997(Yang, , 2000Semwal et al., 2014;Singh and Kaur, 2016;Cui et al., 2018;Yang et al., 2018). Despite these contributions to the knowledge of Amanita in Asia, many regions of the continent remain understudied (Tulloss, 2005).
Pakistan has Himalayan moist temperate forests which are generally suitable for Amanita and their ectomycorrhizal partners. The Shangla District of Pakistan lies on the western edge of the Himalayan range and under the Hindukush mountain range with average elevation 2000-3500 m above sea level (asl) (Ullah et al., 2019a). Almost 90% of the area consists of high mountains, with 10% of the area occurring as plains along the Indus river side (Ullah et al., 2019b). Climatically, District Shangla is a moist temperate area (Figure 1), bordering the Himalayan range (Champion et al., 1965). The highest temperature in summer is 38 o C, while in winter it goes down to −2 to −5 °C. The observed relative humidity of the area is 65.9% annually (Ullah, 2018). Mansehra District is east of Shangla District, with a similar average elevation (2000-3500 m a.s.l.). The climate is also similar (moist temperate), with seasonal periods of rainfall, snow, and drought. Most of the rainfall occurs during the monsoon season (July-August). The total annual rainfall of the district is 1828 mm, and the temperature ranges from 2 to 36 °C (Fiaz, 2013).
According to the standard classification of forest types of Pakistan, the forests fall under the category "Montane temperate forests" (Champion et al., 1965). The mountains of both districts consist of mostly coniferous forests containing Pinus roxburghii Sarg., P. wallichiana A. B. Jackson, Abies pindrow (Royle ex D. Don) Royle, Picea smithiana (Wall.) Boiss., and Taxus wallichiana Zucc.
Broadleaved tree species include Aesculus indica (Wall. ex Camb.) Hook., Acer caesium Wall. ex Brandis, Juglans regia L., Quercus incana Roxb., Q. semicarpifolia Smith, and Ulmus wallichiana Planch. (Ullah, 2018: Ullah et al., 2019a. The moist temperate climate has in average 1778 mm of precipitation annually (Ullah, 2018). While the habitat is ideal for Amanita, few studies of the genus have been done here. Ahmad (1956) reported a species list containing only 2 taxa of Amanita (A. nana Sing. and A. vaginata [Bull.: Fr.] Lamarck). Tulloss et al. (2001) published 9 taxa of Amanita from the Himalayan moist temperate forests of northwestern Pakistan. Niazi et al. (2009) produced a comprehensive report of a rubescent species of section Validae (Fr.) Quél. (possibly A. orsonii Kumar and Lakhanpal) growing ectomycorrhizal on Himalayan spruce (Picea smithiana). Kiran et al. (2017) reported A. pallidorosea and its ectomycorrhizal association with Quercus oblongata. Jabeen et al. (2017)  To date, only 15 species of Amanita have been documented from Pakistan. This study increases the existing taxonomic knowledge of Amanita from Pakistan by describing 2 new species and a new record from the northwestern region of the country using morphological descriptions and molecular phylogenetic data.

Morphological characterization
Specimens were collected from the Shangla and Mansehra districts ( Figure 1) during 2013-2016. Fresh specimens were photographed using a Nikon DS3300 digital camera and tagged. Macromorphological features such as the color of the basidiome, size and shape of the pileus, the stipe, the annulus, volva, lamellar features, and associated vegetation of the area were noted. The specimens were dried and stored in labeled boxes. Color codes were designated using the Munsell Color System (1975). Micromorphological features were described according to Tulloss and Rodríguez-Caycedo (2011). Features were described from sections prepared in 3% KOH, 1% Congo Red, and Melzer's reagent, and observed using a compound light microscope (MX4300H Techno Co., Ltd., Japan) at a magnification of 1000× with oil-immersion. Measurements were recorded using a Carl Zeiss Jena ocular micrometer, and line drawings were made using a camera lucida.
At least 100 basidiospores were measured per species. Values in brackets (= [a/b/c]) represent the number of spores measured (a), per number of basidiomes (b), per number of collections (c). Basidiospores dimensions are shown as (k-) m-n (-p) -The values "m" and "n" represent the 5% and 95% quantiles of spore size distribution (length and width), respectively, with "k" as the smallest and "p" as the largest observed value. We use the biometric variables defined by Tulloss (Tulloss and Rodríguez-Caycedo, 2011). The value L is the range of average spore lengths per specimen examined, and L´ is the overall average spore length, W the range of average spore widths per specimen examined, W´ is the overall average spore width, Q is the ratio of length/width for one spore, Q is the average value of the length/width ratio for spores per specimen examined,; and Q´ is the average length/width ratio for all spores measured. Biometric variables for describing the lamella trama are also those of Tulloss (Tulloss and Rodríguez-Caycedo, 2011): w cs is the width of the central stratum, w st-near is the distance from an outer edge of the central stratum to the nearest base of a basidium on the nearest hymenial surface, and w st-far is the distance from an outer edge of the central stratum to the most distant base of a basidium on the nearest hymenial surface.
The specimens were deposited in the Hazara University Herbarium (HUP), Pakistan, and the LAH Herbarium, Department of Botany University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan.

Molecular analysis
Extractions were obtained from 5-15 mg of dried sporocarp material using a Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA. http://www.qiagen.com/). PCR and cycle sequencing were performed to obtain sequences of nuclear ribosomal internally transcribed spacer regions ITS1, 5.8S, and ITS2 (ITS) using primer pairs ITS1F/ITS4 or ITS2/ITS3 (White et al., 1990;Gardes and Bruns, 1993). Amplification of the nuclear ribosomal large subunit (LSU) genes was done by the primer pair LR0R/LR5 (Vilgalys and Hester, 1990). A 25 µL volume PCR reaction was performed, containing 9 µL ddH 2 O, 12.5 µL PCR master mix, 1.25 µL forward primer, 1.25 µL reverse primer, and 1 µL DNA sample. Amplification protocols and PCR conditions used an initial denaturation step of 2 min at 95 °C, followed by 30 cycles of the following steps: a denature step of 1 min at 95 °C, an annealing step of 30 s at 50 °C, and an extension step of 2 min at 72 °C; the reaction was finalized by a final extension for 5 min at 72 °C. Cycle sequence reaction was performed using the above PCR primers, with separate reactions for forward and reverse primers, and the Big Dye Terminator Kit 3.1 (Applied Biosystems, Foster City, CA, USA) with thermocycler protocols using an annealing temperature of 55 °C. PCR products and cycle sequence reactions were purified using a standard ethanol precipitation prep. DNA extraction, PCR, and cycle sequencing reactions were performed at the Chicago Botanic Garden's Center for Plant Biology and Conservation, Glencoe, Illinois. Sequencing was performed using an ABI-3730-XL DNA Analyzer (Applied Biosystems) in the Pritzker Laboratory at the Field Museum of Natural History, Chicago, Illinois. Sequences produced for this study have been deposited in GenBank.
The sequences were processed, edited, and assembled using Codon Code Aligner 3.5 and Bioedit 7.0. They were then screened for percentage sequence identity using a BLAST search of GenBank and UNITE databases. Nearest matches from both databases were downloaded for phylogenetic analysis. Automated alignment of individual ITS and LSU datasets (Table) was done using MUSCLE 3.8 (Edgar, 2004), followed by additional manual alignment in MESQUITE 2.75 (Maddison and Maddison, 2005). The nucleotide alignments were deposited as Nexus files in TreeBASE (23906).
Phylogenetic inference was conducted using Bayesian and maximum likelihood (ML) methods. For Bayesian inference, we used BEAST 1.8.4 (Drummond and Rambaut, 2007) with a Markov chain Monte Carlo (MCMC) coalescent approach. A Yule tree prior (Gernhard, 2008) was used in all simulations, and the starting tree was randomly generated. Four independent runs were undertaken. Chain length was 10 million generations,  with a sampling frequency of 1000. Tracer 1.6 was used to check the effective sample size (ESS), and burn-in values were adjusted to achieve an overall ESS of ≥200. Maximum clade credibility tree (20% burn-in) was generated using TreeAnnotator 1.6.2 (Drummond and Rambaut, 2007). ML analyses were performed with RAxML-HPC2 on XCED (8.2.10) (Stamatakis, 2006), using 1000 bootstrap replicates and the default parameters as implemented on the CIPRES web portal. Fig Tree 1.4.2 was used for tree visualization, with additional tree annotation done through Adobe Illustrator CS6. Rapid bootstrap analysis/ search for best-scoring ML tree (-f a) was configured. For the bootstrapping phase, the GTRCAT model was selected. One thousand rapid bootstrap replicates were run. Nodes were considered strongly supported when maximum likelihood bootstrap (MLB) were ≥70% and Bayesian posterior probability (BPP) were ≥0.95.

Results
Phylogenetic and morphological analysis of Amanita from the Shangla and Mansehra districts of Pakistan revealed that of the 3 taxa studied, 2 are new species, and 1 is a new  record for the country. All 3 species are phylogenetically characterized and morphologically described below. The final aligned ITS dataset was 920 characters, and the aligned LSU dataset was 965 characters after being trimmed (trimming was done manually in MESQUITE 2.75). The results of phylogenetic analyses of the ITS and LSU datasets are summarized in Figures 2 and 3. The ITS phylogram resolved in 2 clades, representing section Vaginatae (Fr.) Quél. (MLB 100% and BPP nonsignificant) and section Roanokenses Singer ex Singer (MLB 86% and BPP nonsignificant) as delimited by Cui et al. (2018) (Figure 2). Likewise, the LSU phylogram also resolved into sections Vaginatae (MLB 100% and BPP nonsignificant) and Roanokenses (MLB 84% and BPP nonsignificant) (Figure 3).
In the ITS phylogeny (Figure 2), A. cinis is resolved in section Roanokenses and is represented by 3 specimens: LAH-SUA733 (MF489726) = type, LAH-SUA752A Figure 2. Phylogenetic Tree of Amanita species based on ITS sequence data from 57 nucleotide sequences. Sequences generated for this study are indicated in bold. Numbers above or below the branches indicate maximum likelihood bootstrap percentages followed by Bayesian posterior probabilities. In the LSU phylogeny (Figure 3)  Etymology: "cinis" (ashes), refers to the color of universal veil remnants on the pileus surface.
Diagnosis: Pileus yellowish-brown and areolate, often completely covered by olive grayish ashen universal veil in the juvenile stage. At maturity the pileus has a well-formed cuticle, with large patches at the center that become light olive-brown pulverulence away from the center, pruinose to pulverulent at the periphery, and often lost with wind and rain. Stipe with small shredded apical annulus and a grayish, fugacious ring-like structure below. Basidiospores oblong or subcylindrical to rarely ellipsoid, (8.5-) 10-13 (-13.5) × (5.2-) 6-7.5 (-8.5) µm.
Etymology: Named "olivovaginata" because of its "light olive brown to light grayish olive" pileal color and its placement in Amanita sect. Vaginatae.