Research Article |
Corresponding author: Sana Jabeen ( ectomycorrhizae@gmail.com ) Academic editor: Maria-Alice Neves
© 2019 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.
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
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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
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 (
Members of Amanita subgen. Amanitina (E. J. Gilbert) E. J. Gilbert have non-striated pileus margins, attenuate lamellulae and amyloid basidiospores (
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.
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.
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 (
For genomic DNA extraction, a standard CTAB method (
Consensus sequences were generated from the sequences obtained by both primers (forward and reverse) in BioEdit software v. 7.2.5 (
Species and specimens of Amanita used for the molecular phylogenetic analyses.
Species | Voucher | Country | GenBank accession number | Reference | |
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ITS | LSU | ||||
A. aff. brunnescens | BW_HF 10C | USA | – | HQ539661 | – |
A. aff. citrina | BW_PNC | USA | – | HQ539662 | – |
HKAS 34170 | China | AY436449 | AY436489 |
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A. aff. flavorubens | PSMCC 121 | USA | – | HQ539663 | – |
BW_HF-FR | USA | – | HQ539664 | – | |
A. aff. fritillaria | HKAS56832 | China | KJ466372 | KJ466479 |
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HKAS57649 | China | KJ466373 | KJ466480 |
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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 |
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A. brunneolocularis | ANDES_F313 NVE57 | Colombia | FJ890033 | FJ890044 | Vargas et al. 2011 |
A. brunnescens | RET 637-7 | USA | KT006762 | KT006766 |
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BW_HP12 | USA | – | HQ539674 | – | |
RET 529-10 | USA | KP284273 | KP284284 | – | |
RET 554-1 | USA | KP284275 | KP284285 | – | |
RET 549-9 | USA | – | KP284283 | – | |
JS94/2 | – | – | AF097379 |
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A. castanea | MFLU 15-1424 | Thailand | KU904823 | KU877539 |
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A. cf. flavorubescens | JMP0098 | USA | EU819454 | – |
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A. cf. spissacea | BZ2015-40 | Thailand | KY747464 | – |
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OR1214 | Thailand | KY747469 | KY747478 |
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A. citrina | LEM 960298 | Japan | AB015679 | – |
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JM96/61 | – | – | AF097378 | – | |
TM02_102 | Canada | – | EU522722 |
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KA12-1226 | South Korea | KF245908 | KF245892 |
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JSH s.n. | – | – | AF041547 | – | |
JS94/1 | – | – | AF097377 |
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ANDES_F405 IP25 | Colombia | – | FJ890046 | Vargas et al. 2011 | |
BW JLR 102106-1 | USA | – | HQ539679 | – | |
KA12-1612 | South Korea | KF245909 | KF245893 |
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A. citrinoindusiata | HKAS100522 | China | MH508320 | MH486468 |
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HKAS58884 | China | MH508323 | MH486471 |
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HKAS58886 | China | MH508324 | MH486472 |
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HKAS58796 | China | MH508321 | MH486469 |
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HKAS58888 | China | MH508325 | MH486473 |
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HKAS58874 | China | MH508322 | MH486470 |
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A. excelsa | HKAS 31510 | Germany | AY436453 | AY436491 |
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Ge 816 | China | – | HQ539691 | – | |
A. flavipes | KA12-0685 | South Korea | KF245911 | KF245895 |
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HKAS 36582 | China | AY436455 |
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KA12-1517 | South Korea | KF245912 | KF245896 |
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A. flavoconia | TENN61564 | USA | JF313655 | – | – |
BW_PH22 | – | – | HQ539693 | – | |
ANDESF408CV3 | Colombia | FJ890029 | FJ890041 |
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TM03_435 25S | Canada | – | EU522816 |
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NVE 351 | Colombia | KF937301 | – |
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NVE 242 | Colombia | KF937300 | – |
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A. flavoconia | HKAS 34047 | USA | AY436456 |
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RV5Aug96 | – | – | AF042609 |
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A. flavorubens | RET 295-9 | USA | – | HQ539694 | – |
A. flavorubescens | TENN61660 | USA | JF313650 | – | – |
F:PRL6062 | USA | GQ166902 | – |
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RV96/102 | – | – | AF097380 |
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A. franchetii | JM96/27 | – | – | AF097381 |
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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 | – |
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A. fritillaria | – | China | JF273505 | – |
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HKAS 38331 | China | AY436457 | – |
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KA12-1231 | South Korea | KF245913 | KF245897 |
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A. lavendula | RET 639-7 | USA | KP866163 | KR865979 |
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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 |
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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 |
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RET 093-10 | USA | – | HQ539716 | – | |
NY 00066710 | USA | KJ535437 | KJ535441 | – | |
A. phalloides | GDGM:40312 | Italy | KC755034 | – | |
A. porphyria | LEM960303 | Japan | AB015677 | – |
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DAVFP:26784 | USA | JF899548 | – | ||
RET 079-1 | Switzerland | KP866181 | KP866192 |
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HKAS 31531 | China | AY436471 | AY436500 |
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RET 309-8 | Norway | KP866176 | KP866189 | – | |
RET 404-2 | Czech Republic | KP866171 | KP866184 | – | |
RET 404-9 | Czech Republic | – | KP866185 | – | |
A. rubescens | JMP0003 | USA | EU819464 | – |
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TRTC156957 | Canada | JN020972 | – |
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LE241998 | Russia | JF313652 | – | – | |
RK01-01 | Denmark | AJ889923 | – | – | |
EMF4 | China | JF273507 | – | – | |
LEM950063 | Japan | AB015682 | – |
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ASIS23255 | South Korea | KM052530 | – | – | |
ASIS23444 | South Korea | KM052535 | – | – | |
KA 12-1221 | Korea | KF245919 | KF245903 |
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RET 122-8 | Turkey | – | HQ539735 | – | |
ANDES_F416 NVE160 | Colombia | FJ890031 | FJ890043 | Vargas et al. 2011 | |
RV5Aug96 | – | – | AF042607 |
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RV97/23 | – | – | AF097383 |
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JM96/53 | – | – | AF097382 |
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KA12-0936 | South Korea | KF245918 | KF245902 |
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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 |
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A. spissa | UP541 | – | EF493270 | – |
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KF02-47 | – | AJ889924 | – | – | |
UP542 | – | EF493271 | – |
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KA12-0884 | South Korea | KF245910 | KF245894 |
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NYBG 47779 | Germany | – | HQ539743 | – | |
A. spissacea | LEM960187 | Japan | AB015683 | – |
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ASIS24872 | South Korea | KM052552 | KU139485 | – | |
ASIS26240 | – | KT894841 | KU139454 | – | |
ASIS24978 | – | KM052550 | KU139487 | – | |
ASIS24775 | – | KM052543 | KU139484 | – | |
ASIS24949 | – | KM052546 | KU139486 | – | |
A. virosa | HKAS 56694 | China | JX998030 | JX998058 |
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HMJAU23304 | China | KJ466431 | KJ466498 |
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JM 97/42 | – | – | AF159086 |
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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 (
Taxa from subgen. Amanitina sect. Phalloideae (Fr.) Quél. were chosen as the outgroup (
Molecular phylogenetic analysis of ITS sequences using the maximum likelihood method based on the Tamura 3-parameter model (
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.
Molecular phylogenetic analysis of ITS+LSU sequences by using the maximum likelihood method based on the Tamura-Nei model (
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.
The species epithet ahmadii refers to Sultan Ahmad, the pioneer Pakistani mycologist.
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.
In coniferous forests of Pakistan with a moist temperate to dry temperate climate.
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).
Amanita ahmadii basidiomata. A, B LAH35010 (holotype) C SWAT0001351. Photos by Abdul Nasir Khalid and Junaid Khan. Scale bars: 1 cm (A); 1.2 cm (B); 0.5 cm (C).
Amanita ahmadii LAH35010 (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).
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 (
Amanita aff. fritillaria (HKAS56832 and HKAS57649,
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) (
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 (
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.
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.