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Diversity and Phylogeny of Suillus (Suillaceae; Boletales; Basidiomycota) from Coniferous Forests of Pakistan.

Byline: Samina Sarwar and Abdul Nasir Khalid

Abstract

Suillus (Boletales; Basidiomycota) is an ectomycorrhizal genus generally associated with Pinaceae. Coniferous forests ofPakistan are rich in mycodiversity and Suillus species are found as early appearing fungi in the vicinity of conifers. This studyreports the diversity of Suillus collected during a period of three (3) years (2008-2011). From 32 basidiomata of Suilluscollected 12 species of this genus were identified. These basidiomata were characterized morphologically andphylogenetically by amplifying and sequencing the ITS region of rDNA. Copyright 2014 Friends Science Publishers

Keywords: Moist temperate forests; PCR; rDNA; Ectomycorrhizae.

Introduction

Suillus (Suillaceae Basidiomycota Boletales) formsectomycorrhizal associations mostly with members of thePinaceae and is characterized by having slimy capsglandular dots on the stipe large pore openings that areoften arranged radially and a partial veil that leaves a ring ortissue hanging from the cap margin (Kuo 2004). This genusis mostly distributed in northern temperate locationsalthough some species have been reported in the southernhemisphere as well (Kirk et al. 2008). Wu et al. (2000)discussed the bio-geographic pattern and phylogeneticrelationship of Suillus species from Eastern Asian (Chinaand Nepal) and North American territories. Knowledge ofSuillus species diversity is important because of their majorroles in natural and managed ecosystems as ectomycorrhizalfungi. This fungal group becomes an important factor forreforestation program worldwide. Furthermore they areimportant as a food source for human being and animals(Brundrett et al. 1996). They are also used as a bio-indicator of environmental quality. Studies on the diversityand taxonomy (base on morphological characters andmolecular analyses) of Suillus is lacking and needs moreinvestigation.The purpose of this research was to study thediversity and phylogeny of Suillus collected fromconiferous forests of Pakistan which are located at 1373to 3050 m. altitude. The most important component ofthese forests are coniferous trees i.e. Pinus wallichianaA.B. Jackson P. roxburghii Sargent Abies pindrow Roxb.(Royle) Cedrus deodara (Roxb.) Loud. Picea smithiana(Wall.) Boiss. Taxus wallichiana Zucc mixed withdeciduous trees (Hussain 1995). Heavy rainfall and adequate temperature make the environment suitable forthe growth of mushrooms in these forests.This paper described the diversity of Suillus (BoletesFungi) with the help of the anatomical morphological andgenetic analyses as little knowledge is available from forestsin Pakistan.

Materials and Methods

Sporocarp Collection

Table 1: Distribution of Suillus taxa collected from different sites of coniferous forests of Pakistan

Scientific names###Host tree/Substrate###Locality###Date###of Collector name###Identification###Comments

###collection###method

Suillus bovinus###Coniferous trees###Mushkin###forests 2007###Razaq###Morphological###Previously reported

###District Astore

Suillus grevillei###Coniferous trees###Khanspur###1996###Morphological

###Iqbal and Khalid###Previously reported

Suillus###cf###Abies pindrow###Pinus Khanspur Helipad###2008###Sarwar Morphological###New record and Molecular

granulatus###wallichiana###and Molecular###analysis first time from Pakistan

Suillus granulatus###coniferous trees###Murree Malakundi 1969 1992 Ahmad Shibata Morphological###Previously reported

###Pirchinasi###1993###and Murakami

###2010###andSarwar

Suillus luteus###On soil along sides of Dashkin###District 2007###Razaq###Morphological###Previously reported

###canals###Astore

Suillus placidus###Juglans regia Pinus Dhirkot###(AJK) 1993###and Murakami Iqbal Morphological###Previously reported

###wallichiana and Abies Sharan###Nathiagali 1996###and Khalid

###pindrow###Dungagali

Suillus sibiricus Abies pindrow Pinus KPK Ayubia Khera 1962 1993 Ahmad###Morphological###Molecular analysis first time from

###wallichiana Populus spand gali###Kuzagali 2008###and Murakami###and Molecular###Pakistan

###Salix alba###Banjoosa###(AJK) 2010###Niazi Sarwar

###Batakundi Pirchinasi

Suillus tomentosus Coniferous trees under Nathiagali Malakundi 1992 1996 Shibata Iqbal and Morphological###Previously reported

###Abies###pindrow###and Dungagali###and 2008###Khalid Niazi

###herbaceous vegetation

Suillus viscidus###Various trees especially Mushkin###forests 2007###Razaq###Morphological###Previously reported

###pine trees###District Astore

Suillus collinitus Pinus wallichiana###Helipad and Khanspur 2008 2010 Sarwar###Morphological###Molecular analysis first time from

###(KPK)###and Molecular###Pakistan

Suillus brevipes###Quercus incana###KPK Khanspur###2008###Sarwar###Morphological###New record

Suillus flavidus###Pinus wallichiana###KPK###Khanspur 2008 2010 Sarwar###Morphological###New record

###Ayubia

Specimens were collected from the selected areas (Fig. 1;Table 1) beginning early summer (2008-2011) whensporocarp production was first observed in July untilproduction ceased at the end of September. Sporocarps weretaken with the help of sharp digger. A special designation(collection number) was given to each sample. Field noteswere made of fresh fruiting bodies including colormeasurements shape and bruising reactions. Photographs offresh sporocarps were also taken to view various parts suchas pileus surface stipe and pore surface. Afterphotographing the sporocarps were dried by keeping themnear a fan heater. After drying each specimen was placed ina separate paper bag and labeled.

Morphoanatomic Characterization of Sporocarps

For morphological characterization the followingcharacteristics of fresh sporocarps were taken: Color shapemeasurements (width length thickness) of pileus and stipe;context of pileus and stipe and color changing of contextupon bruising; ornamentation of stipe and pileus surface;attachment of stipe; shape of pileus margin color; presenceof ring on stipe; color of pore surface pore and tube sizeand bruising reaction of pore surface.For anatomical characterization of sporocarps acompound microscope was used and the following characterswere noted by preparing slides in KOH Meltzer's TrypanBlue and Lactic Acid: Shape length width cytoplasmiccontents of basidia cystidia basidiospores pileipellis andterminal cells of pileipellis and color reaction.

Molecular Characterization

DNA Extraction and Amplification

DNA was extracted from dried sporocarps andectomycorrhizae (ECM) using the enzymatic digestion andglassfibre filtration (EDGF) protocol in Dentinger et al.(2010). The nuclear ribosomal internal transcribed spacer(ITS) region was amplified following PCR conditions inDentinger et al. (2010) using the fungal specific anduniversal primers (White et al. 1990; Dentinger et al.2010). PCR products were purified using ExoSAPIT(R)(Affymetrix High Wycombe UK) and dyeterminatedunidirectional sequencing was performed using a BigDye(R)Terminator V3.1 Cycle Sequencing Kit (LifeTechnologies/ABI California USA) in 10 L reactionswith respective primers following the protocol in Dentingeret al. (2010). Sequencing reactions were cleaned usingethanol precipitation following the manufacturer'sinstructions re-suspended in 30 L of distilled water andrun on an ABI 3730 DNA sequencer in the JodrellLaboratory Royal Botanic Gardens Kew.

Editing of Sequences and BLAST Analysis of ITSSequences

ITS sequences were compared using Basic Local AlignmentSearch Tool (BLAST) network service using NationalCenter for Biotechnology Information (NCBI) to compareor confirm identifications. These sequences were edited and cleaned at BioEdit where required and were aligned withother sequences present in GenBank using the musclealignment tool (www.ebi.ac.uk/Tools/msa/muscle). Inaligned sequences all characters were equally weighed andgap positions were treated as missing data. Percent Identityand divergence of species was calculated using thecomputer program MegAlign (DNASTAR Inc.) and percentgenetic characters of different species were calculated withJalview software. The preferable cutoff value for speciesdelimitation was 97% below which the sequences wereconsidered to represent different species.

Phylogenetic Analysis

Phylogenetic trees were made separately for each speciessequence(s) because either their ITS1 ITS2 parts orcomplete ITS region was amplified successfully so combinephylogenetic tree was not reliable. Maximum Likelihood(ML) analysis was done using Molecular EvolutionaryGenetic Analysis (MEGA 5.0) with default settings ofprogram i.e. JukesCantor Model and for ML HeuristicNearestNeighborInterchange (NNI) method was used(Tamura et al. 2011). 1000 bootstrapping replicates wereperformed for analysis. Phylogenetic position of somespecies was confirmed by making Maximum ParsimonyTree with bootstrapping using PAUP Version 4.0b10.

Results

Tweleve Suillus species are identified morpho-anatomicallyfor this study; species which had previously been describedare listed at the end of the results. Attempts were made toidentify all species by sequencing but only 4 species werecharacterized successfully by molecular analysis. Resultsare given alphabetically.Suillus brevipes (Peck) Kuntze Revis. Gen. Pl.(Leipzig) 3(2): 535 (1898) Fig. 2.Pileus 2.56 cm convex to hemispheric to planechocolate brown shiny smooth glabrous sticky flesh thickand offwhite margins slightly incurved entire smooth ofsame color like pileus surface.Context whitish to light yellowish no color changeupon bruising.Stipe 36 cm long 12 cm thick centric clavatesmooth whitish with brown small patches at some pointssemihollow ring and volva absent context whitish nocolor change upon bruising.Pore surface adnate and ascending whitish to cream tolight yellowish pores rounded to irregular about 2 per mmtubes 49 mm deep yellowish no color change uponbruising.Basidispores ellipsoid to fusiform to subfusiformsmooth thick walled 610 A- 36 m (8.2 1.27 A- 4.8 0.93; Q m = 2.1 0.76).Basidia clavate 24 sterigmate thin walled 1826 A-68 m.Cystidia cylindrical to clavate to subfusoid toampullaceous thick walled dark brown contents 3549 A-59 m.Pileipellis cylindrical with rounded ends thinwalled4055 A- 58 m most terminal elements subclavate tocylindrical 4958 A- 79 m.Smell and Taste not distinctive.Edibility edible.Chemical reactions pileipellis stains olive in FeSO 4dark brown in KOH Meltzer reagent and Lactic acid sporesbrown in Meltzer reagent.MATERIAL EXAMINED: Pakistan: KHYBER PAKHTUNKHWAKhanspur 2250 m.a.s.l. under Quercus incana Roxb.solitary on ground 19thJune 2008 Sarwar S.B. # 12(LAH0608).Suillus c.f. granulatus (L.) Roussel Fl. Calvados Edn2: 34 (1806) Fig. 3.Pileus 613 cm wide plane to convex surface viscidsticky smooth yellowish brown to camel brown marginssmooth straight or flaring.Context yellowish no color change upon bruising.Stipe: 47 cm long 12 cm thick equal centric solidyellowish to yellowish brown brownish glandular dots onupper half ring absent.Pore surface yellowish adnate and horizontal colorchange to brownish when bruised pores angular to irregularand frequent about 12 per mm tubes 311 mm deep.Basidispores subfusiform smooth 811 A- 46 m(9.4 0.97 A- 5.1 0.7; Q m = 1.9 0.5).Basidia clavate 34 sharp sterigmate thick walled1618 A- 67.5 m. Cystidia cylindrical to fusoid toampullaceous granular contents visible thick walled darkbrown 5055 A- 7.59 m. Pileipellis clavate to irregular5369A-1113 m terminal elements of Pileipellis clavate toirregular hyphae septate at end 4962 A- 1012 m.Smell and Taste not distinctive.Edibility edible.Chemical reactions pileipellis stains bluish gray inFeSO 4 olive gray in KOH; spores brown in Meltzer reagent.Material examined: Pakistan: Khyber PakhtunkhwaKhanspur 2350 m a.s.l. under P. wallichiana solitary onground 17thJune 2008 Sarwar S.B. # 72(LAH0608)(Holotype); Helipad 2350 m a.s.l. under A. pindrowsolitary on ground 18thJune 2008 Sarwar S.B. #72A(LAH0608).Suillus collinitus (Fr.) Kuntze Revis Gen. Pl. (Leipzig)3(2): 536 (1898) Fig. 4.Pileus 2 cm wide hemispheric to convex viscid whenwet brown to dark brown margins smooth deflexed tostraight. Context yellowish white no color change uponbruising.Stipe 5.67.4 cm long about 1 cm thick nearly equalcylindrical yellowish to brownish yellow whitish pink nearbase with pinkish mycelia at base ring absent dry centricocassionally slightly curved with brown glandular dots.Pore surface bright yellow to yellowish brownish upon bruising adnate to decurrent pores rounded toangular 12 per mm tubes shorter near margins of pileus.Basidispores ellipsoid to fusiform thick walledsmooth light honey brown (7) 913 A- 57 m (11.4 1.25 A- 6.12 0.75; Q m = 1.9 0.4).Basidia clavate 24 sterigmate 1315 A- 79 m.Cystidia clavate to subglobose dark brown thick walledwith brownish contents (26) 3541 A- 710 m. Pileipelliscylindrical to slightly clavate thick walled 7079 A- 1317m most terminal elements of pileipellis cylindrical toclavate some are globose from above dark brown contentsvisible thick walled 6067 A- 912 m.Smell and Taste: not distinctive.Edibility edible.Chemical reactions pileipellis stains olive in FeSO 4dark brown in KOH Meltzer reagent and Lactic acid sporesbrown to brown in Meltzer reagent.Material examined: Pakistan: Khyber PakhtunkhwaKhanspur Helipad 2250 m a.s.l. under P. wallichianasolitary on ground 26thJuly 2008 Sarwar S.B. #03(LAH0708) (Holotype); 18thJune 2010 Sarwar S.B. #03A(LAH0610).Suillus flavidus (Fr.) J. Presl Wsobecny Rostl. (Praha)2: 1917 (1846) Fig. 5.Pileus 39 cm wide convex to hemispherical to nearlyplane occasionally slightly umbonate at maturityoccasionally margins straight and flaring to slightly deflexedwith whitish remnants of veil surface viscid to glutinouswhen wet glabrous yellow to yellowish brown. Contextlight yellow changes brown when bruising not bluing.Stipe 310 cm long 1.52 cm thick nearly equalcylindrical centric and curved solid slightly dry reddishwhen young yellow to white with reddish tinge whenmature whitish glandular dots in some case whitish thickband like ring present above centre of stipe color above ringyellow.Pore surface yellow becomes slightly brown uponbruising adnate and horizontal pores angular to irregularinfrequent about 2 per mm tubes 39 mm deep.Basidispores ellipsoid to fusoid smooth 913 A- 46m (11.3 1.2 A- 5.2 0.6; Q m = 2.26 0.17).Basidia cylindric to long clavate thick walledyellowish brown contents visible in Meltzer reagent 14sterigmate 2226 A- 810 m. Cystidia cylindrical tofusoidventricose brown contents visible thick walleddark brown 3234 A- 910 m. Pileipellis long cylindricalto slightly clavate thick walled brown 7784 A- 1820 mmost terminal elements of pileipellis cylindrical toclavate in clusters and separate also some are globosefrom above dark brown thick walled 7177 A- 810 (14) m.Smell and Taste not distinctive.Edibility edible.Chemical reactions pileipellis stains reddish in KOH spores brownish in Meltzer reagent light yellow to honeyyellow in Lactic acid.Material examined: Pakistan: Khyber PakhtunkhwaAyubia 2350 m a.s.l. under P. wallichiana gregarious onground 19thJune 2008 Sarwar S.B. # 06(LAH0608)(Holotype); Khanspur 2250 m a.s.l. solitary on ground17thJuly 2010 Sarwar S.B. # 06A(LAH0710).Suillus sibiricus (Singer) Singer Farlowia 2: 260(1945) Fig. 6

Genbank: JN11974854

Pileus 48 cm pulvinate to obtuse sticky slimy shinyglabrous yellowish brown smooth sometimes withbrownish scales on yellowish to dull yellowish colormargins entire slightly darker color than pileus surfacedeflexed. Context pale yellowish not bluing on exposing.Stipe about 10 cm long 1.21.8 cm thick centralequal ring present yellowish to offwhite from apex toring reddish brown from ring towards base whitish nearbase whitish to brown glandular dots rough solid curved.Pore surface yellowish adnate and ascending colorchange to brownish when bruised pores angular andfrequent about 2 per mm tubes 717 mm deep.Basidispores ellipsoidfusiform thin walled smooth1012 A- 3.55 m (10.6 2.5 A- 4.1 0.53; Q m = 2.94 0.34).Basidia clavate 24 sterigmate thick walledbrownish contents visible in Meltzer reagent 2539 A- 79m. Cystidia cylindrical to subfusiform thick walledyellowish brown in Meltzer reagent yellowish browncontents 3345 (62) A- 79 m. Pileipellis a tangled layerof repent hyphae thin walled granular contents septate6074 A- 810 m most terminal elements subclavateclavatecylindrical with pointed ends thick walled 6899 A-1320 m.Smell and Taste not distinctive.Edibility edible.Chemical reactions pileipellis stains yellowish brownin FeSO 4 dark brown to black in KOH hyaline to lighthoney in Meltzer reagent; spores light yellowish brown inMeltzer reagent light yellow to honey yellow in Lactic acid.Material examined: Pakistan: Khyber PakhtunkhwaKhaira Gali 2347 m a.s.l. under P. wallichiana solitary onground 18thJune 2010 Sarwar S.B. # 53(LAH0610)(Holotype); Nathiagali 2520 m a.s.l. 19thJuly 2010 SarwarS.B. # 53A(LAH0710); Khaira gali 2347 m a.s.l. underSalix alba L. scattered or in groups on ground 7thAugust2010 Sarwar S.B. # 53B(LAH0810).Suillus tomentosus (Kauffman) Singer Mycologia51(4): 570 (1960) [1959] Fig. 7.Pileus 46 cm wide convex becoming nearly planewith age yellowish surface viscid irregular circular patchesof graybrown to dark brown tomentum or squamules allover the pileus surface margins incurved when young gradually becoming deflexed to straight to uplifted with agesmooth. Context light yellowish bluing whenexposed. Odor and taste not distinctive.Stipe 47 cm long 12 cm thick nearly equal drysolid centric cylindrical yellowish with brown patchessometimes yellowish glandular dots near apex basalmycelium salmonbuff volva and annulus absent contextyellowish bluing upon exposure.Pore surface adnate and horizontal to arcuate brightyellowish pores infrequent pores angular to irregular withwide openings 1 per mm tubes 611 mm deep changingslightly blue then brown upon bruising.Basidispores oblongellipsoidinequilatteral slightlyapiculate thin walled smooth 1415 A- 57 m (14.5 0.40 A- 5.9 0.73; Q m = 2.4 0.39).Basidia clavate to irregular 23 sterigmate hyalinethin walled contents visible 3336 A- 1415 m. Cystidiaelongated cylindrical to sub clavate 3442 A- 910 m.Pileipellis cylindrical elongated thin walled septate 72103 A- 1621 m terminal elements of pileipellis cylindricalto subclavate to irregular shaped septate in some cases 5995 A- 1521 m. FeSO 4 creamish to pinkish in KOH.Material examined: Pakistan: Khyber PakhtunkhwaAyubia 2350 m a.s.l. under P. wallichiana in groups onground 15thAugust 2006 A.R. Niazi # 38(LAH0806).

Phylogenetic Analysis Figs. 811

Four Suillus species S. c.f. granulatus S. collinitus S.flavidus and S. sibiricus were characterized molecularly andphylogenetically. During molecular analysis of S. c.f.granulatus 379 bp long sequence belonging to 5.8S andITS2 showed maximum 99% similarity and 99% querycoverage with sequence of S. c.f. granulatus (L54121)during BLAST. For phylogenetic analysis 31 sequencescontaining 412 genetic characters were used after aligningand trimming at both ends. These contains 275 conserved113 variable and 67 parsimony informative sites. Allcharacters were equally weighted and unordered.Phylogenetic tree was made by maximum likelihoodcriteria. S. c.f. granulatus from Pakistan form clade with S.c.f. granulatus (L54121) (Fig. 8) supported by 92%bootstrap value and shared maximum 100% genetic characters and 0.0% genetic divergence with the same.These values are well supportive to confirm our sequence asS. c.f. granulatus.Molecular analysis of S. collinitus was carried out with3 different fruiting bodies by using 5.8S and ITS2 part ofnrDNA region. All these showed more than 97% similaritywith S. collinitus (HM347658) and (JQ685733) duringBLAST. During phylogenetic analysis 27 sequencescontaining 412 genetic characters were used in the finalaligned datasheet. These sequences contained 270conserved sites 118 variable sites and 64 parsimonyinformative sites. The aligned data was analyzed bymaximum lkelihood using MEGA 5.0. All characters wereequally weighted and unordered. S. collinitus sequencesfrom Pakistan form a clade with S. collinitus (HM347658)and (JQ685733) retrieved from GenBank (Fig. 9). S.collinitus sequences from Pakistan shared above 99%genetic characters with other with 0.00.3% geneticdivergence with each other and with S. collinitus(HQ406820) and S. collinitus (HM347658) these sharedabove 99% genetic characters with genetic divergence 0.00.3%. Phylogenetically these sequences have beenconfirmed S. collinitus.When ITSrDNA sequence of Suillus flavidus fromPakistan was submitted for similarity in GenBank it wasidentified as uillus flavidus with 98% maximum identity and100% query coverage with S. flavidus (FJ845439) fromCanada. The phylogenetic analysis included 25 sequencesbelonging to 13 species. For phylogenetic analysis a total of426 genetic characters were used in an aligned datasheet.These sequences contained 330 conserved sites 91 variablesites 66 parsimony informative sites. The phylogram basedon maximum likelihood criterion represented by 2 majorclades. Clade I is formed by 11 sequences and theirclustering is not highly resolved (42% bootstrapping).Topologically S. flavidus occupied the top position in thephylogram. This species is represented by 9 sequences 6from sporocarps and 3 from P. wallichiana ectomycorrhizalroots from Pakistan and 2 sequences retrieved fromGenBank. Sequences from Pakistan shared 100% of theirgenetic characters (rDNAITS sequences) with each otherand shared about 98% with S. flavidus (FJ845439). It shared92.1% of its genetic characters with S. lakei (Murrill) A.H.Sm. and Thiers (DQ367912) and 91.6% with S.caerulescens A.H. Sm. and Thiers (EU486453). Geneticdivergence was also measured for S. flavidus with all thesequences included in the analysis. No genetic divergencewas found among the rDNAITS of S. flavidus fromPakistan (Fig. 10). There was little genetic divergence (0.52.5) compared with S. flavidus (FJ845439).The sequences of S. sibiricus from Pakistan showed99% similarity with isolates of S. sibiricus from China andAmerica confirming the morphological identification. Thephylogenetic analysis for S. sibiricus was carried out usingparsimony as optimality criterion. The sequences included in this analysis had around 659 genetic characters fromwhich 494 characters were used for further analysis afteralignment and trimming from both 3' and 5' sites of rDNAITS. After that none of characters were excluded from finalanalysis.All characters were of type 'unord'. There were 53parsimonyinformative sites 415 constant sites and 26variable sites. All the gaps were treated as "missing" data.Multistate taxa were interpreted as uncertainty. The startingtree(s) was obtained via stepwise addition with randomaddition of sequence and 1000 number of replicates. Therewere 49145891 starting seeds for the tree generated. Only01 tree held at each step during stepwise addition of thesequences. Treebisectionreconnection (TBR) was used asbranchswapping algorithm. A total of 6457051rearrangements were tried for the best tree. Only 27 treeswere retained for analysis. The genetic distance matrix wasderived from Maximum Parsimony (MP) analysis generateda consensus tree from the best 144 trees showing thefollowing scores: Tree length (TL) = 146 consistency index(CI) = 0.6438 homoplasy index (HI) = 0.3562 CIexcluding uninformative characters = 0.5517 HI excludinguninformative characters = 0.4483 retention index (RI) =0.7977 rescaled consistency index (RC) = 0.5136.Phylogenetic analysis showed the various species of Suillus.Maximum Parsimony consensus tree indicating three majorclades and one independent clade.A Maximum Parsimony consensus tree wasconstructed exclusively for Suillus species fromgeographically different localities specially from EasternAsia (China and Nepal) Eastern North America and fromPakistan to resolve exact identification. The cladogramrepresents (Fig. 11) a major polytomous clade formed by S.americanus (Peck) Snell and S. sibiricus species. All of thespecies of this clade shared 9899% of characters studied sofar for this analysis and thus identified as S. sibiricus. Both S.sibiricus and S. americanus occupied topologically differentpositions in the same polytomous clade.The Maximum Parsimony analysis resulted in a majorpolytomous clade comprising sixteen isolates of S.americanus and S. sibiricus. All these species aremonophyletic along with S. flavidus (FJ845439) S.megaporinus Snell and E.A. Dick (GQ249400) and S.umbonatus Dick and Snell (L541115). S. sibiricus has beenpublished by the author in ICMBMP7.

Discussion

Suillus is an important ectomycorrhizal bolete characterizedby a slimy pileus stipe with glandular dots and ring widepore openings smooth spores and usually associated withconifers (Bessette et al. 2000; Kuo 2004). Many scientistshave done molecular and Phylogenetic analyses of Suillusspecies. Kretzer et al. (1996) analyzed 38 sequences ofSuillus species for phylogenetic and taxonomic studies. Wuet al. (2000) discussed the bio-geographic pattern andphylogenetic relationship of Suillus species. Manian et al.(2001) investigated the genetic diversity and relationshipsbetween Suillus species based on ribosomal DNAsequences. S. quiescens T.D. Bruns and Vellinga was firsttime reported and described morpho-anatomically andmolecularly by Bruns et al. (2010).From Pakistan nine (9) species of Suillus such asSuillus bovinus S. grevillei S. granulatus S. luteus S.placidus S. sibiricus S. tomentosus and S. viscidus havealready been reported (Ahmad 1962; Shibata 1992;Murakami 1993; Iqbal and Khalid 1996; Razaq 2007;Niazi 2008; Sultana et al. 2011). S. sibiricus (Singer)Singer was also analyzed phylogenetically from Pakistan(Sarwar et al. 2011).The present investigation explores the status of Suillusfrom the high mountains of Pakistan. S. brevipes and S.flavidus were compared with closely related species. S.brevipes from Pakistan has maximum similarity with S.brevipes reported from other countries due to convex tohemispheric pileus chocolate brown smooth shiny pileussurface whitish to light yellowish context with no colourchange upon bruising clavate stipe without ring andglandular dots whitish to light yellowish pore surface andsmooth spores. S. brevipes is similar with S. albidipes(Peck) Singer S. c.f. granulatus and S. pallidiceps A.H. Sm.and Thiers due to convex pileus no ring on stipe andsmooth spores. The major differences between these speciesis that S. c.f. granulatus has a shorter stipe and distinctlyraised granules on the stipe while S. brevipes has a smoothwhite stipe. Similarly S. brevipes is differentiated from S.albidipes by having stipe without glandular dots and largerspores in former. Major difference of S. brevipes from S.pallidiceps is white to pale yellow pileus of latter while S.brevipes has chocolate brown (Thiers 1975; Bessette et al.2000; Santana et al. 2007; Bruns et al. 2010).S. flavidus is characterized by a convex to hemisphericpileus with some reddish brown spots on the margin andsmall hanging veil remnants. Cap color is yellowish withprominent ring on stipe and smooth spores which range incolor from light to dark brown. S. flavidus resembles S. lakeibut the pileus surface of the latter is covered with dullreddish brown small scales without glandular dots on stipe.S. flavidus also resembles to S. caerulescens A.H. Sm andThiers but S. caerulescens does not have glandular dots onstipe. S. flavidus is similar to S. grevillei (Klotzsch) Singerbut S. grevillei has a reticulated stipe and glandular dotscharacteristic of S. flavidus (Thiers 1975; Bessette et al.2000) are absent.S. collinitus S. c.f. granulatus S. sibiricus and S.tomentosus were characterized morpho-anatomically andthese shares many characters but can be differentiated fromeach other due to some major differences. S. sibiricus can bedifferentiated due to its ring on the stipe which is absent inother three. S. sibiricus is often confused with the NorthAmerican species S. americanus. The latter has largersporocarps as compared with S. sibiricus. Despite this minor difference other macro and micro features resemble eachother. The only other feature that separates these two speciesis their geographical distribution (Wu et al. 2000). S.collinitus can be identified due to its pinkish basal mycelia.S. c.f. granulatus has glandular dots on upper half of thestipe. The context and pore surface in S. tomentosus stainsblue upon exposing which is distinguishing character of thisspecies (Thiers 1975; Bessette et al. 2000; Santana et al.2007).In conclusion according to the results of this studymany trees like Abies Cedrus Pinus Populus Quercus andother coniferous trees are dependent upon mycorrhizal fungifor their survival. Knowledge of the mycorrhizal symbiontslike Suillus species give us a better understanding of theecology of these important timber trees. Also understandingthe host preference of Suillus species has aided local peoplein locating a new source of edible species of this mushroomwhich in turn benefits the local economy. Speciesdocumented from this study compared with those fromother areas of the world give us a better understanding ofbiogeography patterns and address questions concerningspecies dispersal. Phylogenetic studies of this genus arecurrently available only for a limited number of speciesfrom other parts of the world. Phylogenetic studies of AsianSuillus species give us a better understanding of theevolution of this genus on a worldwide scale and aid in thebiogeographic analysis. The present work is the firstmolecular analysis of this genus in Pakistan.

Acknowledgments

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

References

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