Amanitaahmadii, a new species of Amanita subgenus Amanitina section Validae from Pakistan

Abstract A new species from coniferous forests in Pakistan, Amanitaahmadii, is described on the basis of morpho-anatomy and molecular data set analyses. This species is characterized by its medium-sized to large basidiomata, grayish brown to brown pileal surface and rimose pileus margin with gray to dark brown verrucose veil remnants, a cream stipe with bulbous base having grayish brown or brown longitudinal striations above the annulus, a scaly surface towards the base, globose to broadly ellipsoid and amyloid basidiospores, and the absence of clamped septa in all tissues. Molecular phylogenetic analyses based on ITS and LSU sequences confirmed its identity as a new taxon nested within subgen. Amanitinasect.Validae.


Introduction
Amanitaceae E. J. Gilbert is a large family of agaricoid fungi that has been classified by many mycologists and split into various genera subgenera and sections (Corner and Bas 1962;Bas 1969). During recent years, it has been split into two genera, Amanita Pers., a genus of putatively ectomycorrhizal fungi, and Saproamanita Redhead, Vizzini, Drehmel & Contu, a genus of putatively saprotrophic fungi (Redhead et al. 2016). This generic split has been rejected by Tulloss et al. (2016) based in part on the guidelines of Vellinga et al. (2015) for introducing new genera. Concise amended characterizations have been provided for the monophyletic family Amanitaceae and its two monophyletic genera, Amanita and Limacella Earle. This declaration is based on the current use of next-generation sequencing in studies of fungal ecology opposing the splitting of the genus. Recently Cui et al. (2018) and Yang et al. (2018) inferred the phylogeny of Amanitaceae based on multi-locus sequencing data. The results indicated that Amanitaceae is monophyletic and consists of five genera. The genus Amanita consists of 95% of the species which are characterized by agaricoid basidiomata, colorless and hyaline, ballistosporic and smooth basidiospores, free lamellae, presence of volval remnants (Persoon 1797). A total of 540 known species of Amanita are distributed worldwide (Yang 2000, Kirk et al. 2008, Menolli et al. 2009, Tulloss 2009, Wartchow et al. 2009, Justo et al. 2010, Wartchow and Gamboa-Trujillo 2012, Cho et al. 2015, Hosen et al. 2015, Tang et al. 2015, Wartchow and Cortez 2016, Jabeen et al. 2017, Kiran et al. 2018a. From Pakistan, 19 species of Amanita are known to date (Ahmad et al. 1997, Jabeen et al. 2017, Kiran et al. 2018a. Tulloss et al. (2001) described one new species, A. pakistanica Tulloss, S.H. Iqbal & Khalid, but refrained from describing two more due to lack of materials. The work on these species is in progress by several workers, and it is estimated that the total number of Amanita from Pakistan could be above 50. Many taxa of the genus have been reported as edibles (Tulloss and Bhandary 1992, Buyck 1994, Montoya-Esquivel 1997, though some others are deadly poisonous (Yang 2015, Cai et al. 2016). Most of the species are ecologically important forming mycorrhizal symbiosis (Yang 1997, 2000, Kiran et al. 2018a.
Members of Amanita subgen. Amanitina (E. J. Gilbert) E. J. Gilbert have nonstriated pileus margins, attenuate lamellulae and amyloid basidiospores ). Six sections in this subgenus are recognized , based on the morphology of the remnants of the universal veil and the pileal margin. The sect. Validae is characterized by pilei that are usually distinctly colored, margins that are non-appendiculate and do not exceed the gill margin, non-fragile and membranous annuli and basal bulbs that are usually small (Tulloss and Yang 2018, Yang 1997. During our ongoing studies of ectomycorrhizal fungi in Khyber Pakhtunkhwa province, we collected specimens of an unknown Amanita species belonging to Amanita subgen. Amanitina sect. Validae. The aim of the present study was to characterize and identify the taxon based on molecular phylogeny using the sequence data of the internal transcribed spacer (ITS) and partial large subunit (LSU) of ribosomal RNA. Here, we describe this taxon as a new species.

Sampling sites
Specimens were collected from three different areas in two districts of Khyber Pakhtunkhwa province of Pakistan. One of these, the Swat district, has a very rich biodiversity. The mountains are covered with snow throughout the winter and in summer temperature ranges between 16-33 °C. The average annual precipitation in Swat district ranges from 1000 mm to 1200 mm. The first area, Gabin Jabba, is a lush green valley in Swat district, which is characterized by a moist temperate vegetation with Picea smithiana (Wall.) Boiss. and Abies pindrow Royle as the dominant tree species. Mashkun, the second area in Swat district, is in the western part of the Himalayas. This collection site is a dry temperate forest with A. pindrow, P. smithiana and Cedrus deodara (Roxb. ex D. Don) G. Don as the dominant tree species along with Pinus wallichiana A. B. Jacks.
The third area is Kumrat valley, which lies at the extreme North of the Dir Upper district. It is located in the foothills of the Hindu Kush mountains with an elevation of about 950-2440 m (Siddiqui et al. 2013). Snowfall occurs frequently in winter, rainfall during monsoon season ranges from 100 mm to 255 mm. Forests are dominated by a mixture of C. deodara, A. pindrow, Picea smithiana, and Pinus wallichiana, and Populus nigra L. is the main broad-leaved tree.

Macroscopic and microscopic characterization
Specimens were collected during routine macrofungal surveys and photographed in their natural habitats using a Nikon D3200 camera. Morphological features of fresh specimens were recorded and colors were designated using Munsell Soil Color Charts (Munsell 1975) and then forced-air dried for long term preservation. For detailed anatomical descriptions, tissues from different parts of the basidiomata were mounted on glass slides in 5% Potassium Hydroxide solution (KOH; w/v). Phloxine (1% w/v aqueous solution) was used for a better contrast. Melzer's reagent was used to check the amyloidity of basidiospores. Anatomical features were noted under a compound microscope (MX4300H, Meiji Techno Co., Ltd, Japan). Measurements were recorded using a Carl Zeiss (Jena) ocular micrometer and line drawings were made using Leitz Wetzlar camera lucida. Size and shape of basidiospores are presented in a form following the description of ranges for biometric variables according to Tulloss (2016). Voucher specimens are deposited in the Herbarium at the University of the Punjab (LAH), Quaid-e-Azam Campus, Lahore, Pakistan and at the Swat University Herbarium (SWAT), Swat, Pakistan.

DNA extraction, PCR and sequencing
For genomic DNA extraction, a standard CTAB method (Bruns 1995) was followed. Internal transcribed spacer regions along with central 5.8S region of nuclear ribosomal DNA (nrDNA) were amplified (Gardes and Bruns 1993) using forward primer ITS1F and reverse primer ITS4 (White et al. 1990). For LSU amplification, LR0R as forward and LR5 as reverse primers were used (Ge et al. 2014). The PCR products were sent to Macrogen Inc. (Korea) for sequencing.

Sequence alignment and phylogenetic analyses
Consensus sequences were generated from the sequences obtained by both primers (forward and reverse) in BioEdit software v. 7.2.5 (Hall 1999). Sequences of Amanita subgen. Amanitina sect. Validae at NCBI (http://www.ncbi.nlm.nih.gov/) and from published literature (Kim et al. 2013, Cai et al. 2014) were added to the datasets. Taxa from the sect. Phalloideae were chosen as outgroup . Shorter ITS and LSU sequences were omitted from the final matrices. Species and specimens used for the molecular phylogenetic analyses are given in Table 1. Multiple sequences were aligned using online webPRANK by EMBL-EBI, Wellcome Trust Genome Campus, UK (https://www.ebi.ac.uk/goldman-srv/webprank/). The phylogeny was inferred by maximum likelihood (ML) analysis using model selection for best DNA analysis for each dataset in MEGA6 software (Tamura et al. 2013). Models with the lowest BIC scores (Bayesian Information Criterion) were considered to describe the substitution pattern the best. Non-uniformity of evolutionary rates among sites may be modeled by using a discrete gamma distribution (+G) with 5 rate categories and by assuming that a certain fraction of sites are evolutionarily invariable (+I). The phylogenetic analyses were performed at 1000 bootstrap replicates. Percentage identity and divergence in nrDNA-ITS of the taxa were analyzed using MegAlign (DNAStar, Inc.). Sequences generated in this study were submitted to GenBank under accession numbers KY996724, KY996755, MF116158 and MF070490 for ITS and KY996725 and MK166021 for LSU.

Phylogeny
Consensus sequences of the ITS region were BLAST searched at NCBI. These sequences showed 98% identity to A. aff. fritillaria (KJ466372 and KJ466373) sequences from China (Cai et al. 2014) with 94-100% query cover. It also showed 95% identity with an A. franchetii (JX515561) sequence from Bulgaria with 100% query cover and 0.0 E value. The LSU consensus sequence BLAST at NCBI showed 99% identity to A. aff. flavoconia (HQ539663) and Amanita sp. (KT072738) sequences from the eastern USA and A. fritillaria (KF245897) sequences from South Korea with 99% query cover.  Taxa from subgen. Amanitina sect. Phalloideae (Fr.) Quél. were chosen as the outgroup (Kim et al. 2013). The sequences generated during this study clustered with the similar taxa in sect. Validae (Figs 1-3). Our species clustered with A. aff. fritillaria, A. citrinoindusiata, A. franchetii f. franchetii , A. franchetii f. lactella (as A. franchetii in GenBank), A. franchetii f. queletii (as A. aspera in GenBank) and A. spissa in phylogenetic analysis. However, A. ahmadii separated from A. aff. fritillaria with a strong bootstrap value of 95%, 49% and 100% in ITS, LSU and ITS+LSU sequence dataset analyses, respectively (Figs 1-3).  (Tamura 1992). The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.4454)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 88 nucleotide sequences. There were a total of 1018 positions in the final dataset. Sequences generated during the present investigation are marked with bullets. Red represents the holotype.  (Kimura 1980). A discrete gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.2164)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 81 nucleotide sequences. There were a total of 871 positions in the final dataset. Sequences generated during the present investigation are marked with bullets. Red represents the holotype.  (Tamura and Nei 1993). A discrete gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.2250)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 43.3848% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 52 nucleotide sequences. There were a total of 1760 positions in the final dataset. Sequences generated during the present investigation are marked with bullets. Red represents the holotype. Diagnosis. Small to medium-sized basidiomata, grayish brown to brown pileal surface having rimose and non-appendiculate pileal margins, verrucose, gray to dark bluish or brown veil remnants, dry and split stipe surface at the base forming scales, globose to subglobose, smooth, amyloid basidiospores.
Etymology. The species epithet ahmadii refers to Sultan Ahmad, the pioneer Pakistani mycologist.
Habitat and distribution. In coniferous forests of Pakistan with a moist temperate to dry temperate climate.

Discussion
Amanita ahmadii is characterized by its grayish brown to brown pileus surface with abundant gray to dark brown verrucose veil remnants and by its rimose margins. Anatomically it is characterized by its globose to broadly ellipsoid basidiospores. The species is morphologically similar to A. fritillaria Sacc. by its grayish to brownish gray pileus surface, and verrucose volval remnants. Amanita fritillaria differs by bearing ellipsoid basidiospores (Corner and Bas 1962, Yang 1997, 2005, 2015. In phylogenetic trees based on ITS, LSU and combined sequence datasets of both regions, A. fritillaria was inferred as a distinct lineage from A. ahmadii.
Amanita aff. fritillaria (HKAS56832 and HKAS57649, Cai et al. 2014) forms a sister clade to A. ahmadii (Figs 1-3), but it is morphologically distinct. The former taxon possesses a brownish and purplish pileus surface (Zhu L. Yang pers. comm.) while the latter has a grayish brown or brown pileus surface with highly rimose margins (Cai et al. 2014). Amanita fritillaria f. malayensis Corner & Bas was described from Singapore (Corner and Bas 1962), but more recently was also found in subtropical, evergreen, broad-leaved forests in China; it differs from A. ahmadii in having a dark umber to rather pale grayish umber pileus (Yang 2005(Yang , 2015. The European sequences labeled as "A. franchetii" and "A. aspera" in GenBank are close relatives of A. ahmadii in the ITS phylogenetic analysis. Amanita franchetii (Boud.) Fayod is somewhat variable in appearance and there are three morphological infraspecific taxa, including A. franchetii f. franchetii (Boud.) Fayod (JX515562 and JX515563), A. franchetii f. lactella Neville & Poumarat (JX515561) and A. franchetii f. queletii (Bon & Dennis) Neville & Poumarat (AF085485) (Neville and Poumarat 2004). The last taxon most closely resembles A. ahmadii but differs in having more yellow hues on the stipe and pronounced reddening on the bulb with age. Amanita augusta Bojantchev & R. M. Davis, as "A. franchetii" in GenBank (GQ250398), another species from western North America looks similar to A. ahmadii but its yellowish brown pileus with yellow universal veil remnants and ellipsoid spores (Bojantchev and Davis 2013) distinguishes it from A. ahmadii. During phylogenetic analyses, all these taxa were inferred as distinct species.
The novel species also showed differences from A. castanea Thongbai, Tulloss, Raspé & K. D. Hyde from Thailand. Amanita castanea bears a viscid, shiny and sericeous pileal surface, which is dark brown at center and light brown to brownish orange towards margin, with universal veil mostly towards the margin, rarely over disc, as scattered gray to brownish gray, reddish brown to grayish brown warts or small floccose patches and globose basidiospores (Thongbai et al. 2016). All these characters distinguish A. castanea from A. ahmadii. In molecular phylogenetic analyses, A. castanea is clustered with the species in a distant clade within sect. Validae (Figs 1-3). Amanita ahmadii also showed morphological distinctions from A. citrinoindusiata Zhu L. Yang, Y. Y. Cui & Q. Cai, a newly reported species in the same section from China. This species is characterized by its robust, brownish gray, gray to dark gray pileus and a stipe bearing a citrine to yellowish annulus. This suggests it is a separate species from A. ahmadii . Molecular data also supports the separation of these two taxa in phylogenetic trees (Figs 1-3).
The European A. excelsa Gonn. & Rabenh is also morphologically close to A. ahmadii in having a gray-brown pileus. However, A. excelsa differs from A. ahmadii in having mealy, gray irregular and non-persistent patches of volval remnants on the pileus. The volva in A. excelsa has 2-5 pale ochre brown zones of friable material above the bulb, and lastly, the broadly ellipsoid to ellipsoid, occasionally elongate basidiospores also distinguish A. excelsa from A. ahmadii (Neville & Poumarat, 2004). The phylogenetic position of these taxa also indicates that they are separate. Based on morphological characters and molecular phylogenic analysis, our new species belongs to Amanita subgen. Amanitina sect. Validae.