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Addition to the Boletes: Boletus pakistanicus sp. nov. from the Coniferous Forests of Pakistan.

Byline: Samina Sarwar and Abdul Nasir Khalid

Abstract

Boletus pakistanicus (Fungi Basidiomycota Boletaceae) characterized by uniformly pale yellowish flesh; is described as a new species from the coniferous forests of Pakistan based on morphological and molecular analysis. A description illustrations phylogenetic analysis and comparison with related taxa are presented. Copyright 2014 Friends Science Publishers.

Keywords: Boletes; Basidiospore; Ectomycorrhizae; Pileipellis.

Introduction

The Boletaceae Chevall. is a monophyletic family of mushrooms (Binder and Besl 2000) having pores on the underside of the pileus. There are 35 genera and approximately 787 species in this family (Kirk et al. 2008). The genus Boletus sensu auct has almost 300 species (Kirk et al. 2008). Boletes like many other macrofungi are an important part of forest ecosystems because they form ectomycorrhizae with a variety of green plant symbionts. In this role they are an integral part of all forest systems since they are involved with basic processes such as nutrient cycling nutrient uptake and decomposition of organic matter (Halling et al. 2007).

From Pakistan until present only twenty-one Boletus species i.e. B. aestivalis (Paulet) Fr. B. appendiculatus Schaeff. B. barrowsii Thiers and A.H. Sm. B. calopus Pers. B. chrysenteron Bull. B. edulis Bull. B. elegans Bull. B. erythropus sensu Pers. B. fraternus Peck B. luridus Sowerby B. parasiticus Bull. B. placidus Bonord. B. piperatus Bull. B. pulverulentus Opat. B. queletii Schulzer B. reticulatus Boud. B. rubellus Krombh. B. spadiceus Schaeff. B. subtomentosus sensu Bolton B. subvelutipes Peck and B. ustalis Berk. have been reported (Ahmad et al. 1997; Razaq 2007; Niazi 2008).

Recently an additional Boletus species resembling B. dryophilus Thiers was collected from the moist coniferous forests of Pakistan associated with Pinus wallichiana A.B. Jacks. Based on macro- and micromorphological features in combination with sequence data of rDNA-ITS this bolete represents a new species. A description illustrations phylogenetic analyses and comparison with related taxa are presented herein.

Materials and Methods

Basidiocarps were collected during the rainy season (July- August 2010) and macromorphological characteristics were recorded in the field. Specimens were studied macroscopically and microscopically in the laboratory. In the description of spore dimensions the first values corresponds to the range of the lengths and widths the values in the parentheses indicate the mean spore length and width their standard deviation followed by Qm value which represents the mean Q value the standard deviation. The mean Q was taken as the mean of all E values where E= length/width of each spore measured. Similarly measurements of other structures were taken in range with exceptional values in parentheses.

After drying with a fan heater specimens were deposited in the Punjab University herbarium Botany department (LAH).

Molecular Analysis

DNA was extracted from dried basidiocarps using Extract N. Amp.TM Plant kit (SIGMA) and the nrDNA i.e. ITS1-5.8S-ITS2 was amplified using fungal specific primers pairs (ITS1F/ITS4). Amplification parameters were - denaturation at 94C for 4 min followed by 35 cycles of 45 s at 94C 45 s at 54C and 1 min 30 s at 72C and a final extension at 72C for 2 min. After purification of PCR products bidirectional sequencing was done by Macrogen (South Korea) and the sequence subjected to BLAST and searched in GenBank for comparing with available sequences. For calculating percent identity similarity and divergence the selected sequences were aligned using Clustal W and corrected manually. All ambiguous insertions and deletions were removed prior to further analyses. Percent Identities (PID) and DNA divergence were calculated by DNAStar. Maximum Likelihood and Maximum Parsimony criteria were used to describe the phylogenetic placement with other species included in the present study. Phylograms were made using Mega5.

Results

B. pakistanicus Sarwar and Khalid nov. sp. (Plate 1) GenBank Number: JQ178324.Number of Mykobank: MB 564279.Pileus usque 7 cm siccus tomentosus saepe rimosus rufus vel rufo-brunneus. Tubuli flavi tactu coerulei; pori05-1 mm lati. Stipes siccus subtiliter flocculosus-striatus sursum ochraceousbasim versus rubris floccis ornatus basiflavidus. Contextus pallide citrinus in sectione cyanescens. Spores 14-18 x 6-7 m L/l ellipsidoideae leaves. Pileipellis trichodermica articulis terminalis sub-diametricis lageniformibus laevibus.Habitat sub Pinis in locis montanis mensibusaestivalis. Holotypus in the University of Punjab Herbarium sub LAH S. B. # 49 conservatus.

Etymology

Based on the country where the new species was found.

Macrocharacers

PILEUS 6-7 cm wide depressed to infundibuliform to irregularly shaped at maturity surface dry rough tomentose patches present on pileus surface dull red to brownish red margins uplifted to slightly recurved of same color as pileus not entire but cracked. CONTEXT creamish to light yellowish bluing when exposed. STIPE 4-7 cm long 1- 2 cm thick central equal dry solid curved brownish yellow near hymenium dull red below becoming yellowish near base smooth lacking reticulations sometimes slightly longitudinally striated context light yellowish throughoutthe stipe slowly bluing when exposed like pileus context. PORE SURFACE yellow to olive yellow free and approximate to adnexed readily bluing when bruised pores circular to elliptical to irregularly shaped 1-2 per mm tubes 5-9 mm deep. SMELL AND TASTE not distinctive. EDIBILITY not known.

Microcharacers

BASIDIOSPORES fusiform to sub-fusoid smooth olive brown 14 - 18 x 6 - 8 m (16.06 1.4 A- 7.15 0.5; Qm=2.25 0.14). BASIDIA broadly clavate long 2 - 4 sterigmate sterigmata long and sharp (34-) 38 - 43 A- (7-)11 - 12 (-15) m yellowish brown contents visible inMeltzer's. CYSTIDIA cylindrical to subclavate (26-) 33 - 40A- 7 - 8 m. PILEIPELLIS trichoderm consisting of slender cylindrical to subclavate septate more or less interwoven hyphae 95 - 110 (-130) A- 10 - 11 m terminal elements clavate lageniform 57 - 80 A- 22 - 24 m. CLAMP CONNECTIONS not observed. CHEMICAL REACTIONSPlate. 1: Boletus pakistanicus. A and B Sporocarps; C Basidia; D Cystidia; E Terminal elements of Pileipellis hyphae; F Pileipellis hyphae; G Basidiospores. Scale Bars: for A and B = 1 cm; C = 13 m; D = 11 m; E = 34 m; F = 36 m; G = 7 m.

pileipellis stains dark blue with the application of FeSO4 hyaline to light yellowish in KOH Meltzer's and Lactic acid spores olive brown to brown in Meltzer's light yellow to honey yellow in Lactic acid.

Material Examined Pakistan Khyber Pakhtunkhwa Khera Gali

2347 m.a.s.l. under Pinus wallichiana A.B. Jacks. solitary on ground 17th June 2010 LAH S. B.# 49 (holotypus) (GenBank accession. JQ178324): Helipad 2438 m.a.s.l. under Pinus wallichiana A.B. Jacks solitary on ground 6th July 2010 (S. B.# 62); Nathia gali 2493 m.a.s.l. under Pinus wallichiana A.B. Jacks solitary on ground 7th July 2010 (S. B.# 76).

Molecular and Phylogenetic Analyses

When B. pakistanicus was submitted for closest matches

Table 1: rDNA sequences downloaded from GenBank for molecular characterization and phylogenetic analysis

FUNGAL SPECIES###COLLECTION NO.###ORIGIN###ACCESSION NO.

Boletus cf. chrysenteron###JMP0007###USA###EU819456.1

B. chrysenteron###TENN60896###USA###FJ596906.1

B. dryophilus###3035###USA###AY185181.1

B. dryophilus###3049###USA###AY185183.1

B. erythropus###SU46###Italy###DQ131633.1

B. erythropus###SU47###Italy###DQ131634.1

B. subtomentosus###C 1064254###USA###EU526005.1

B. zelleri###src444###USA###DQ974704.1

B. pakistanicus###S.B.49###PAK###JQ178324

Octaviania columellifera###TNS-F-11695###Japan###EF183532.1

O. columellifera###TNS-F-11697###Japan###EF183542.1

X. armeniacus###MA-Fungi 46678###Spain###AJ419221.1

X. chrysenteron###KGP62###USA###DQ822793.1

X. chrysenteron###TDB635###USA###DQ533981.1

X. cisalpinus###BB28_409_Oh_161006 (DNA660)###Germany###HM190085.1

X. cisalpinus###BB28_409_Oh_161006 (DNA479)###Germany###HM190084.1

X. porosporus###BB28_403_Ah_161006 (DNA451)###Germany###HM190086.1

X. pruinatus###BB28_305_Of_Fa_070507 (DNA799)###Germany###HM190106.1

X. pruinatus###BB06_105_Of_Pi_230407 (DNA750)###Germany###HM190107.1

Xerocomus sp. MHM129###MHM129###USA###EU569235.1

X. zelleri###KGP68###USA###DQ822794.1

the top BLAST results were B. subtomentosus (Accession No. EU526005.1) Xerocomus zelleri (Accession No. DQ822794.1) and Xerocomus chrysenteron (Accession No. DQ822793.1) with 89% similarity and B. dryophilus (Accession No. AY185183.1) with 88% similarity. These matches are well below the 97% cuttoff value for species identification. Phylogenetic placement of B. pakistanicus was confirmed by making phylogenetic trees using maximum parsimony and maximum likelihood criteria.

The sequences included in this analysis had approximately 925 characters from which 729 characters were used for further analysis after alignment and trimming from both 3' and 5' sites of rDNA-ITS. After that none of characters were excluded from final analysis.

All characters were of type 'unord'. There were 209 parsimony-informative sites 438 constant sites 282 variable characters were parsimony-uninformative. All the gaps were treated as "missing" data. Multistate taxa were interpreted as uncertain.

Phylogenetic trees were constructed for B. pakistanicus along with species from geographically different localities (Table 1). The phylograms represent a major polytomous Clade I formed by B. subtomentosus (EU526005.1) Xerocomus zelleri (DQ822794.1) B. zelleri (DQ974704.1) Xerocomus chrysenteron (DQ533981.1 DQ822793.1) B. chrysenteron (FJ596906.1) Boletus cf. chrysenteron (U819456.1) Xerocomus sp. MHM129 (EU569235.1) Xerocomus porosporus (HM190086.1) Xerocomus cisalpinus (HM190084.1. HM190085.1) Xerocomus pruinatus (HM190106.1 HM190107.1) and B. dryophilus (AY185181.1 AY185183.1).B. pakistanicus shares maximum of 88.87 of genetic characters and has 78.6% identity compared with B. dryophilus (AY185181.1). B. pakistanicus shares 88.84% genetic characters and has 77.8% identity compared with B. subtomentosus (EU526005.1). B. pakistanicus shares minimum genetic characters of 82.44% and has 71.6% identity compared with B. erythropus (DQ131633.1). There is significant genetic divergence of rDNA-ITS among B. pakistanicus and other rDNA sequences included in the present study (Fig. 3). The nearest rDNA sequence is of B. dryophilus (AY185183.1). Both phylogenetic analyses placed B. pakistanicus with B. dryophilus (AY185181.1 AY185183.1) with comparatively short branches and supportive bootstrapping (Fig. 1 and 2) as sub-clade II of major clade I. This polytomous clade formed by 16 species was included in both analyses. The rDNA-ITS of B. pakistanicus was found distinct when the rDNA sequence divergence was compared with other species included in the analyses. Minimum divergence (9.5) was found among the rDNA of B. pakistanicus and X. zelleri (DQ822794.1) and maximum (19.0) with B. erythropus (DQ131634). There was great variability in the ITS1-5.8S-ITS2 region among all the species included in the present investigation.

Comments

Based on available shared genetic characters analyzed in the present study B. pakistanicus was closest to B. subtomentosus B. dryophilus and X. chrysenteron. None of these species were found to be identical to B. pakistanicus. In the phylogenetic analyses B. pakistanicus appeared as a sister clade with B. armeniacus B. erythropus and Octaviana columellifera (Fig. 2). From the calculated PIDs for the present study the cutoff value (97%) for species delimitation did not match with B. pakistanicus (Fig. 3) and thus designated as a new species.Morphologically B. pakistanicus is characterized by dull red to brownish red depressed to irregular shaped pileus yellow to olive-yellow pores which turns blue upon bruising with central equal solid curved stipe which is smooth with light longitudinal striations. Within the boletes it appears morphologically closest to B. subtomentosus Xerocomus zelleri X. chrysenteron and B. dryophilus with which it shares many common characters. But this species is clearly distinct from other similar taxa of Boletus for its uniformly pale yellow flesh before the blue color and terminal elements of pileipellis lageniform.Morpho-anatomic characterization as well as molecular analysis strongly suggested that this species is distinct from closely related species and is a previously undescribed taxon.

Acknowledgments

We sincerely thank Higher Education Commission Pakistan (HEC) for funding this project and Jodrell Laboratories Royal Botanic Gardens Kew for providing facilities for molecular work.

References

Ahmad S. S.H. Iqbal and A.N. Khalid 1997. Fungi of Pakistan pp:120121. Sultan Ahmad Mycological Society of Pakistan Department of Botany University of Punjab Quaid-e-Azam Campus Lahore PakistanBinder M. and H. Besl 2000. 28S rDNA sequence data andchemotaxonomical analyses on the generic concept of Leccinum (Boletales). In: Micologia Duemila pp: 7182. Papetti C. and G. Consiglio (eds. ). Associazione Micologica Bresadola Trento ItalyHalling R.E. T.W. Osmundson and M.A. Neves 2007. Pacific boletes:implications for biogeographic relationships. Mycological Res. 112:437447Kirk P.M. P.F. Cannon D.W. Minter and J.A. Stalpers 2008. Dictionary of the Fungi 10th edition. CABI Wallingford UKNiazi A.R. 2008. Biodiversity of Ectomycorrhizas in Conifers fromHimalayan Moist Temperate Forests of Pakistan. Ph.D. Thesis Department of Botany University of Punjab Lahore PakistanRazaq A. 2007. Taxonomic Studies on Basidiomycota from NorthernAreas of Pakistan. Ph.D. thesis University of Karachi Karachi Pakistan
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Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9PAKI
Date:Jun 30, 2014
Words:2071
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