11 urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3 MycoKeys MC 1314-4057 1314-4049 Pensoft Publishers 10.3897/mycokeys.56.31819 31819 Research Article Basidiomycota DNA barcoding Molecular systematics Nomenclature Phylogeny Taxonomy Asia Amanita ahmadii, a new species of Amanita subgenus Amanitina section Validae from Pakistan Jabeen Sana ectomycorrhizae@gmail.com https://orcid.org/0000-0001-8839-7716 1 2 Kiran Munazza https://orcid.org/0000-0002-3671-9039 2 Khan Junaid https://orcid.org/0000-0001-6929-1182 3 Ahmad Ishtiaq 1 4 5 Ahmad Habib 5 Sher Hassan 3 Khalid Abdul Nasir https://orcid.org/0000-0002-5635-8031 2 Department of Botany, Division of Science and Technology, University of Education, Lahore, Punjab, Pakistan University of Education Lahore Pakistan Department of Botany, University of the Punjab, Quaid-e-Azam Campus-54590, Lahore, Punjab, Pakistan University of the Punjab Lahore Pakistan Center for Plant Sciences and Biodiversity, University of Swat, Swat, Khyber Pakhtunkhwa, Pakistan University of Swat Swat Pakistan Centre of Plant Biodiversity and Conservation, University of Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan Islamia College University Peshawar Pakistan Islamia College University, Peshawar, Khyber Pakhtunkhwa, Pakistan University of Peshawar Peshawar Pakistan

Corresponding author: Sana Jabeen (sanajabeenue@gmail.com;sanajabeen@ue.edu.pk)

Academic editor: M.-A. Neves

 2019 23 07 2019 56 81 99 C166FFE5-FF8C-FFC6-CD3B-FFF4FF8BFFF5 3355845 21 11 2018 26 12 2018 Sana Jabeen, Munazza Kiran, Junaid Khan, Ishtiaq Ahmad, Habib Ahmad, Hassan Sher, Abdul Nasir Khalid This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

A new species from coniferous forests in Pakistan, Amanita ahmadii, 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. Amanitina sect. Validae.

 Amanitaceae nrDNA Swat Higher Education Commission, Pakistan 501100004681 http://doi.org/10.13039/501100004681 Citation

Jabeen S, Kiran M, Khan J, Ahmad I, Ahmad H, Sher H, Khalid AN (2019) Amanita ahmadii, a new species of Amanita subgenus Amanitina section Validae from Pakistan. MycoKeys 56: 81–99. https://doi.org/10.3897/mycokeys.56.31819

 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, Cui et al. 2018, Kiran et al. 2018a, b). From Pakistan, 19 species of Amanita are known to date (Ahmad et al. 1997, Jabeen et al. 2017, Kiran et al. 2018a, b). 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 non-striated pileus margins, attenuate lamellulae and amyloid basidiospores (Cui et al. 2018)). Six sections in this subgenus are recognized (Cui et al. 2018), 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, Cui et al. 2018).

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.

 Materials and methods 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, Cui et al. 2018) were added to the datasets. Taxa from the sect. Phalloideae were chosen as outgroup (Cui et al. 2018). 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.

Species and specimens of Amanita used for the molecular phylogenetic analyses.

Species Voucher Country GenBank accession number Reference
ITS LSU
A. aff. brunnescens BW_HF 10C USA – HQ539661 –
A. aff. citrina BW_PNC USA – HQ539662 –
HKAS 34170 China AY436449 AY436489 Zhang et al. 2004, Thongbai et al. 2016
A. aff. flavorubens PSMCC 121 USA – HQ539663 –
BW_HF-FR USA – HQ539664 –
A. aff. fritillaria HKAS56832 China KJ466372 KJ466479 Cai et al. 2014, Thongbai et al. 2016
HKAS57649 China KJ466373 KJ466480 Cai et al. 2014
A. aff. spissacea 2C5 Japan AB973749 – –
A. ahmadii LAH35010 Pakistan KY996724 KY996725 –
SWAT0001351 Pakistan MF070490 – –
LAH35241 Pakistan KY996755 MK166021 –
LAH35242 Pakistan MF116158 – –
A. augusta DBB49390 USA JQ937287 –
DBB21873 USA JX515564 – –
A. augusta as “A. franchetii” 07040 USA GQ250398 – –
A. bisporigera RET 377-9 USA KJ466374 KJ466434 Thongbai et al. 2016
A. brunneolocularis ANDES_F313 NVE57 Colombia FJ890033 FJ890044 Vargas et al. 2011
A. brunnescens RET 637-7 USA KT006762 KT006766 Thongbai et al. 2016
BW_HP12 USA – HQ539674 –
RET 529-10 USA KP284273 KP284284 –
RET 554-1 USA KP284275 KP284285 –
RET 549-9 USA – KP284283 –
JS94/2 – – AF097379 Drehmel et al. 1999
A. castanea MFLU 15-1424 Thailand KU904823 KU877539 Thongbai et al. 2016
A. cf. flavorubescens JMP0098 USA EU819454 – Palmer et al. 2008
A. cf. spissacea BZ2015-40 Thailand KY747464 – Cai et al. 2012
OR1214 Thailand KY747469 KY747478 Cai et al. 2012
A. citrina LEM 960298 Japan AB015679 – Oda et al. 1999, Thongbai et al. 2016
JM96/61 – – AF097378 –
TM02_102 Canada – EU522722 Porter et al. 2008
KA12-1226 South Korea KF245908 KF245892 Kim et al. 2013
JSH s.n. – – AF041547 –
JS94/1 – – AF097377 Drehmel et al. 1999
ANDES_F405 IP25 Colombia – FJ890046 Vargas et al. 2011
BW JLR 102106-1 USA – HQ539679 –
KA12-1612 South Korea KF245909 KF245893 Kim et al. 2013
A. citrinoindusiata HKAS100522 China MH508320 MH486468 Cui et al. 2018
HKAS58884 China MH508323 MH486471 Cui et al. 2018
HKAS58886 China MH508324 MH486472 Cui et al. 2018
HKAS58796 China MH508321 MH486469 Cui et al. 2018
HKAS58888 China MH508325 MH486473 Cui et al. 2018
HKAS58874 China MH508322 MH486470 Cui et al. 2018
A. excelsa HKAS 31510 Germany AY436453 AY436491 Thongbai et al. 2016
Ge 816 China – HQ539691 –
A. flavipes KA12-0685 South Korea KF245911 KF245895 Kim et al. 2013
HKAS 36582 China AY436455 Zhang et al. 2004
KA12-1517 South Korea KF245912 KF245896 Kim et al. 2013
A. flavoconia TENN61564 USA JF313655 – –
BW_PH22 – – HQ539693 –
ANDESF408CV3 Colombia FJ890029 FJ890041 Thongbai et al. 2016
TM03_435 25S Canada – EU522816 Porter et al. 2008
NVE 351 Colombia KF937301 – Vasco-Palacios et al. 2014
NVE 242 Colombia KF937300 – Vasco-Palacios et al. 2014
A. flavoconia HKAS 34047 USA AY436456 Zhang et al. 2004
RV5Aug96 – – AF042609 Moncalvo et al. 2000
A. flavorubens RET 295-9 USA – HQ539694 –
A. flavorubescens TENN61660 USA JF313650 – –
F:PRL6062 USA GQ166902 – Thongbai et al. 2016
RV96/102 – – AF097380 Drehmel et al. 1999
A. franchetii JM96/27 – – AF097381 Drehmel et al. 1999
A. franchetii f. lactella as “A. franchetii” DBBJUS01 Spain JX515563 – –
DBB52095 Bulgaria JX515562 – –
DBB51482 Bulgaria JX515561 – –
A. franchetii f. queletii as “A. aspera” IFO-8262 – AF085485 – Lim and Jung 1998
A. fritillaria – China JF273505 – Legendre et al. 2009
HKAS 38331 China AY436457 – Zhang et al. 2004
KA12-1231 South Korea KF245913 KF245897 Kim et al. 2013
A. lavendula RET 639-7 USA KP866163 KR865979 Thongbai et al. 2016
A. luteofusca PSC 1093b Australia – HQ539705 –
A. luteolovelata PSC 2187 Australia – HQ539706 –
A. morrisii RET 672-6 USA KR919762 KR919770 –
RET 271-7 USA KT213441 KT213442 Thongbai et al. 2016
RET 445-10 USA KR919760 KR919768 –
A. novinupta GO-2009-234 Mexico KC152066 – –
GO-2009-315 Mexico KC152065 – –
GO-2009-301 Mexico KC152067 – –
RET 060-2 USA KF561974 KF561978 Thongbai et al. 2016
RET 093-10 USA – HQ539716 –
NY 00066710 USA KJ535437 KJ535441 –
A. phalloides GDGM:40312 Italy KC755034 –
A. porphyria LEM960303 Japan AB015677 – Oda et al. 1999
DAVFP:26784 USA JF899548 –
RET 079-1 Switzerland KP866181 KP866192 Thongbai et al. 2016
HKAS 31531 China AY436471 AY436500 Thongbai et al. 2016
RET 309-8 Norway KP866176 KP866189 –
RET 404-2 Czech Republic KP866171 KP866184 –
RET 404-9 Czech Republic – KP866185 –
A. rubescens JMP0003 USA EU819464 – Palmer et al. 2008
TRTC156957 Canada JN020972 – Dentinger et al. 2011
LE241998 Russia JF313652 – –
RK01-01 Denmark AJ889923 – –
EMF4 China JF273507 – –
LEM950063 Japan AB015682 – Oda et al. 1999
ASIS23255 South Korea KM052530 – –
ASIS23444 South Korea KM052535 – –
KA 12-1221 Korea KF245919 KF245903 Thongbai et al. 2016
RET 122-8 Turkey – HQ539735 –
ANDES_F416 NVE160 Colombia FJ890031 FJ890043 Vargas et al. 2011
RV5Aug96 – – AF042607 Moncalvo et al. 2000
RV97/23 – – AF097383 Drehmel et al. 1999
JM96/53 – – AF097382 Drehmel et al. 1999
KA12-0936 South Korea KF245918 KF245902 Kim et al. 2013
A. sp. ANDES_F241 IP24 Colombia FJ890032 FJ890047 Vargas et al. 2011
RET 516-10 USA KP711830 KP711838 –
RET 516-5 USA KP711836 KP711837 –
RET 530-1 USA KT072736 KT072737 –
RET 539-8 USA KT072735 KT072738 –
HKAS 38419 China AY436474 AY436502 Thongbai et al. 2016
A. spissa UP541 – EF493270 – Nygren et al. 2008
KF02-47 – AJ889924 – –
UP542 – EF493271 – Nygren et al. 2008
KA12-0884 South Korea KF245910 KF245894 Kim et al. 2013
NYBG 47779 Germany – HQ539743 –
A. spissacea LEM960187 Japan AB015683 – Oda et al. 1999
ASIS24872 South Korea KM052552 KU139485 –
ASIS26240 – KT894841 KU139454 –
ASIS24978 – KM052550 KU139487 –
ASIS24775 – KM052543 KU139484 –
ASIS24949 – KM052546 KU139486 –
A. virosa HKAS 56694 China JX998030 JX998058 Cai et al. 2012
HMJAU23304 China KJ466431 KJ466498 Cai et al. 2012
JM 97/42 – – AF159086 Moncalvo et al. 2000
Results 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. citrinoinduciata, 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).

10.3897/mycokeys.56.31819.figure1 3488214 86435D7E-DC34-5CFF-9073-67BCE22F80F9

Molecular phylogenetic analysis of ITS sequences using the maximum likelihood method based on the Tamura 3-parameter model (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.

https://binary.pensoft.net/fig/320217 10.3897/mycokeys.56.31819.figure2 3488216 CF3182C6-4E7A-5AEA-A189-46003A2A1828

Molecular phylogenetic analysis of LSU sequences by using the maximum likelihood method based on the Kimura 2-parameter model (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.

https://binary.pensoft.net/fig/320218 10.3897/mycokeys.56.31819.figure3 3488218 CA5E1EE9-0E72-5CFB-BAFC-2E79FFBEFBCD

Molecular phylogenetic analysis of ITS+LSU sequences by using the maximum likelihood method based on the Tamura-Nei model (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.

https://binary.pensoft.net/fig/320219 Taxonomy Fungi Agaricales Amanitaceae 57168515-F4A7-59C7-8C61-5C6E5D5A4094 Amanita ahmadii MB821204 Jabeen, I. Ahmad, Kiran, J. Khan & Khalid sp. nov. Figs 4 , 5 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.

 Holotype.

Pakistan, Khyber Pakhtunkhwa province, Malakand division, Swat district, Mashkun, 2500 m a.s.l., on soil under Cedrus deodara, 5 Sept. 2013, Sana Jabeen SJ35 (LAH35010; GenBank ITS: KY996724; LSU: KY996725).

 Etymology.

The species epithet ahmadii refers to Sultan Ahmad, the pioneer Pakistani mycologist.

 Description.

Pileus 4–7 cm in diameter, convex to flat at maturity; cuticle gray (2.5BG4/2) to grayish brown (10YR3/2) or brown (2.5Y4/4) with time; surface dry; universal veil remnants on pileus verrucose, aligned in one direction, scattered, gray (2.5Y4/2) to dark brown (2.5Y2/2); margins non-appendiculate, incurved when young, highly rimose by maturity. Lamellae off-white (2.5BG4/2) to cream (5Y9/4) becoming brownish when dry, adnexed, subdistant to close; edges entire. Lamellulae small (1/3 of the lamellae), attenuate, truncate. Stipe 6.7–9 × 0.6–1.5 cm, apex slightly wider and white, with up to 1.5 cm wide bulbous base, central, cylindrical; surface with grayish brown (5GY5/2) striations above the annulus, splitting towards the base forming scales on white (2.5BG4/2) to cream (5Y9/4) context. Annulus superior, membranous, skirt-like, with longitudinal striations on the upper surface, gray (2.5Y4/2) with a darker lower part. Universal veil absent. Ordorless and not changing color upon bruising.

Basidiospores [60/3/3] (6.5) 7–8.5 (9.5) × (6) 6.5–7.5 (8) µm, Q = (1) 1.03–1.22 (1.33), avg Q = 1.10, globose to broadly ellipsoid, amyloid in Melzer’s reagent. Basidia (32) 34.5–59 (67) × 7–8 µm, clavate, frequently 4 sterigmate, 2 sterigmata also observed, thin-walled, hyaline in 5% KOH. Subhymenium pseudoparenchymatous, cells isodiameteric, intermixed and densely packed. Veil remnants made up of hyphae with terminal subglobose to elongated cells (42.5) 49.5–54 (57) × (13) 13–16 (19) µm on a branched filament 3–4 µm wide; septa frequent; clamp connections absent. Pileipellis filamentous, 4–5 µm in diameter, branched, septate; clamp connections absent, light brown with some hyaline tissue in 5% KOH. Universal veil remnants of globose to subglobose cells (6.8) 8–12.2 (12.7) × (4.4) 7.5–10.5 (11) µm with filaments (0.7) 0.9–2.6 (3.5) µm in diameter. Hyphae from stipe 3–24 µm wide, filamentous, branched, hyaline in 5% KOH, septate; clamp connections absent in all tissues.

 Habitat and distribution.

In coniferous forests of Pakistan with a moist temperate to dry temperate climate.

 Additional specimens examined.

Pakistan, Khyber Pakhtunkhwa province, Malakand division, Dir Upper district, Kumrat, 2232 m a.s.l., on soil under conifers, 2 Sept. 2015, Abdul Nasir Khalid FS82 (LAH35241; GenBank ITS: KY996755; LSU: MK166021); Swat district, Mashkun, 2500 m a.s.l., on soil under Cedrus deodara, 4 Aug. 2013, Ishtiaq Ahmad IS213P65 (LAH35242; GenBank ITS: MF116158); Gabin Jabba valley, 2450 m a.s.l., on soil under Picea smithiana, 30 Aug. 2015, Junaid Khan GJ-1508 (SWAT0001351; GenBank ITS: MF070490).

10.3897/mycokeys.56.31819.figure4 3488220 2F6AC63A-F52B-52EF-A0DE-A4DFA4AC4B1C

Amanita ahmadii basidiomata. A, BLAH35010 (holotype) C SWAT0001351. Photos by Abdul Nasir Khalid and Junaid Khan. Scale bars: 1 cm (A); 1.2 cm (B); 0.5 cm (C).

https://binary.pensoft.net/fig/320220 10.3897/mycokeys.56.31819.figure5 3488222 4FEE57B4-032A-5AFB-B500-2DB3E1A3EAD4

Amanita ahmadiiLAH35010 (holotype). A Basidiospores B Basidia, basidioles and subhymenium C Pileipellis D Universal veil remnants on pileus surface E Hyphae from stipe F Partial veil. Drawings by Sana Jabeen. Scale bars: 5.5 µm (A); 8 µm (B–D); 22 µm (E, F).

https://binary.pensoft.net/fig/320221 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, 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 (Cui et al. 2018). 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.

 Acknowledgements

This work was financially supported by the Higher Education Commission (HEC)-Pakistan to Dr Sana Jabeen under Indigenous PhD Fellowships for 5000 Scholars (Phase-II), Dr Sana Jabeen, Dr Ishtiaq Ahmad and Munazza Kiran under International Research Support Initiative Program (IRSIP) and Dr Hassan Sher under Pak-US science and technology promotion program. We sincerely thank Prof. Donald H. Pfister for providing the opportunity to Dr Sana Jabeen, Dr Ishtiaq Ahmad and Munazza Kiran to work in his laboratory at Department of Organismic and Evolutionary Biology, Harvard University, MA, USA. Thanks are also due to Prof. Dr Zhu-Liang Yang (Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China) for providing pictures to compare the morphology of the specimens and review of the manuscript. Special thanks are due to Dr Else C. Vellinga, (Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA) for editing the text. Her useful comments and suggestions greatly improved this article. Authors are grateful to Dr Rosanne Healy (Assistant Scientist, Department of Plant Pathology, University of Florida. Gainesville, FL 32611) for linguistic suggestions and helpful comments resulting in the removal of technical errors. We are also thankful to Dr Abdul Rehman Khan Niazi (Department of Botany, University of the Punjab, Lahore, Pakistan) and all laboratory fellows for accompanying the tours to different areas of Pakistan.

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