The Australasian species of Lactarius subgenus Gerardii ( Russulales )

The Australasian species of Lactarius subgenusGerardii (Russulales)

Dirk Stubbe & Huyen Than Le & Xiang-Hua Wang &

Jorinde Nuytinck & Kobeke Van de Putte &

Annemieke Verbeken

Received: 4 March 2011 /Accepted: 28 May 2011 /Published online: 2 July 2011# Kevin D. Hyde 2011

Abstract This paper provides an overview and identifica-tion key of the Australasian species of Lactarius subg.Gerardii. A molecular phylogeny based on the nuc DNAmarkers ITS, LSU and rpb2, combined with detailedmorphological observations resulted in the delimitation of12 strongly supported and morphologically recognizablespecies. Five new agaricoid species are described here:Lactarius hora, L. coniculus, L. limbatus, L. leae and L.

leonardii. Other agaricoid species belonging to subg.Gerardii are L. atrovelutinus, L. bicolor, L. ochrogalactusand L. reticulatovenosus. Lactarius venosus is also consid-ered as a member but could not be included in themolecular analysis. Two new pleurotoid species are discov-ered: L. genevievae and L. conchatulus, both close relativesof L. uyedae, but each with distinct characters. Theirposition within subg. Gerardii is supported in the phylog-eny and by their microscopic characters. This study alsoconfirms the existence of multiple cryptic species andspecies complexes for which species recognition or speciesdelimitation remains problematic as is the case for theAustralian species L. wirrabara. In general, detailedmacroscopic and microscopic observations are needed toidentify species of L. subg. Gerardii.

Keywords Basidiomycota . Lactifluus .Plinthogalus .

Taxonomy . Phylogeny . Asia

Introduction

Lactarius, also known as the milkcaps, is a genus ofbasidiomycete macrofungi that, except for Antarctica, isfound on all continents. It is well represented in temperateas well as tropical climates and is one of the prominentsymbionts in many ectomycorrhizal vegetations. Milkcapsare mostly firm and medium-sized mushrooms, with paleand adnate to decurrent lamellae, exuding a latex-likesubstance when damaged. Although essentially an agari-coid genus, some gasteroid and pleurotoid species exist. In2001 Verbeken (2001) estimated that about 400 specieswere known at the time, but this has increased to nearly 500

D. Stubbe (*)Department of Biology, Ghent University,K.L. Ledeganckstraat 35,9000 Ghent, Belgiume-mail: [email protected]

D. StubbeSection Mycology and Aerobiology,Scientific Institute of Public Health,Juliette Wytmanstraat 14,1050 Brussels, Belgium

H. T. LeFaculty of Environment,Hanoi University of Natural Resources and Environment,41A Street K1, Cau Dien, Tu Liem,Hanoi, Vietnam

X.-H. WangKunming Institute of Botany, Chinese Academy of Sciences,Lanhei Road 132#,Kunming 650204, People’s Republic of China

J. Nuytinck :K. Van de Putte :A. VerbekenResearch Group Mycology, Department of Biology,Ghent University,K.L. Ledeganckstraat 35,9000 Ghent, Belgium

Fungal Diversity (2012) 52:141–167DOI 10.1007/s13225-011-0111-3

in less than a decade, especially due to the discovery ofmany new tropical species (Buyck et al. 2007; Das andSharma 2004; Das et al. 2003; Le et al. 2007a, b, c;Montoya and Bandala 2008; Nuytinck et al. 2006; Stubbeet al. 2007, 2008; Van de Putte et al. 2010; Van Rooij et al.2003; Verbeken et al. 2002, 2008; Wang and Verbeken2006). Lactarius has always been considered a sister genusof Russula, but a molecular phylogenetic study of theRussulaceae by Buyck et al. (2008) has revealed that bothgenera are in fact paraphyletic and that the family should besplit up into four closely related genera. Therefore, the newand distinct genus Multifurca was created for a handful offormer Lactarius and Russula species, which resulted in themonophyly of the remaining Russula species. Lactarius iscurrently still paraphyletic and composed of two majorclades: one containing subgenera Russularia, Plinthogalusand Piperites, and the other containing subgenera Lactar-ius, Lactifluus, Lactariopsis, sect. Edules and the recentlyestablished subg. Gerardii (Buyck et al. 2008; Stubbe et al.2010). Buyck et al. (2010) propose to conserve the nameLactarius for the first clade and to use the name Lactifluusfor the latter. Since there is not yet a formal decision on thematter, we will continue to treat Lactarius here as aparaphyletic genus, referring to the first clade as /Lactarius1 and the second clade as /Lactarius 2. When the proposalis approved, the species concerned in this paper will all beplaced in Lactifluus.

Lactarius gerardii Peck is a North American specieswith a velvety brown cap and stipe, and distant whitishlamellae (Peck 1873). The species has been many timesreported from all-over Asia (Das and Sharma 2005; Imazekiet al. 1988; Le et al. 2007b; Lee et al. 2002; Wang et al.2004; Wu and Mueller 1997). However, molecular analysesof Asian specimens demonstrated that they form in fact anaggregate of multiple species, all related to, but nonethelessclearly different from the North American L. gerardii(Stubbe et al. 2010). Lactarius gerardii has long beenconsidered a member of subg. Plinthogalus, but a globalphylogeny demonstrated that L. gerardii and its closerelatives bear no direct kinship to subg. Plinthogalus andare best accommodated in a separate subgenus of their own(Stubbe et al. 2010), which even belongs to a differentgenus, /Lactarius 2. Morphologically L. subg. Gerardii isdistinguished from subg. Plinthogalus by the white sporeprint and well-developed cellular layer in the palisadicpileipellis. Other characters include a brown pileus andstipe, contrasting whitish and usually distant lamellae. Thebasidiospores are always reticulately ornamented and inmost species macrocystidia are absent in the hymenium. Aspecies with a deviating macromorphology is L. uyedae

Singer, originally described from Japan, which is pleurotoidand completely white.

So far, no representatives of the new subgenus Gerardiiare known from Europe, Africa or South America. Northand Central America contain at least six species, which ismost likely an underestimation due to incomplete sampling,while in Asia, together with the Australian region, theexistence of at least 24 species could be demonstrated(Stubbe et al. 2010). Further investigation of the subgenusin America would be welcome, especially because recentand well documented collections are not available forseveral American species (e.g. L. pseudogerardii Hesler &A.H. Sm., L. xanthydrorheus Singer, L. subtomentosusBerk. & Ravenel) preventing a comprehensive study. Thisis not so much the case for Asia and the Australian region,for which recent collections of most described species areavailable, next to many collections resembling L. gerardii.Although vast areas in Australasia remain underexplored,like large parts of China or India, samples from 11different countries, spanning a wide geographical range,could be analysed for this paper (Sri Lanka, India, Nepal,China, Thailand, Malaysia, Indonesia, Japan, South Korea,Australia and New Zealand). Until now, most Asianspecimens have been identified using American names(Das and Sharma 2005; Imazeki et al. 1988; Le et al.2007b; Lee et al. 2002; Wang et al. 2004; Wu and Mueller1997), but this should be revised since intercontinentalconspecificity in this subgenus has been rejected (Stubbeet al. 2010). Seven species of subg. Gerardii have beenoriginally described from Asia and the Australian region:L. bicolor Massee, L. uyedae, L. atrovelutinus J.Z. Ying, L.wirrabara Grgur., L. sepiaceus McNabb, L. reticulatove-nosus Verbeken & E. Horak and L. ochrogalactus Hashiya.The Papua New Guinean species L. venosus Verbeken &E. Horak is also considered a member of subg. Gerardii,but this could not be confirmed molecularly since DNAcould not successfully be extracted from the availablematerial. In this paper, additional data based on recentcollections is provided for L. bicolor and L. atrovelutinus,and observations concerning L. wirrabara and L. sepia-ceus are discussed. Further, the paper includes a phylog-eny with focus on the Australasian species of subg.Gerardii, and describes seven more, molecularly andmorphologically supported species which are here pro-posed as new. An identification key is given that includesall described species of the Australasian region. Some ofthe remaining species could not be described due to a lackof sufficient macromorphological information. Other taxain the phylogeny belong to species complexes or could notbe distinguished morphologically.

142 Fungal Diversity (2012) 52:141–167

Material and methods

Specimens and morphology

The complete list of specimens included in this study isgiven in Table 1. Herbarium abbreviations are adoptedfrom Index Herbariorum (http://sciweb.nybg.org/science2/IndexHerbariorum.asp, accessed 2 May 2011). Specimenscollected by the authors were described and photographedunder daylight conditions. Macroscopic descriptions ofother specimens were based on notes of the collectors.Density of lamellae (L) and lamellulae (l) is expressed as(L+l)/cm and measured at pileus mid-radius. Colourcoding refers to Kornerup and Wanscher (1978) or to theBritish Fungus Flora (BFF) (Henderson et al. 1969).Terminology is mainly adopted from Vellinga (1988) andspecifically for pellis structures from Verbeken (1998a).Microscopic features were studied from exsiccate. Sec-tions of the pileipellis were taken at approximately pileusmid-radius. Drawings were made with a drawing tube at640 × magnification for pellis structures, at 1,600 × forhymenial elements and at 6,000 × for spores. Pellisstructures and hymenial elements were observed andillustrated in a 1 % Congo Red L4 solution (Clémençon1973) after shortly heating the slide with a flame tointensify coloration and improve rehydration. Whennecessary, pre-treatment in a 10% aqueous potassiumhydroxide solution was made. Basidiospores were ob-served and illustrated in Melzer’s reagent and measured inlateral view. Averages (x) and standard deviations (SD)were calculated per collection (20 basidiospores measuredper collection). Measurements exclude ornamentation andare given as an interval: xMIN � 2� SD½ � � xMIN�xMAX � xMAX þ 2� SD½ �. The lowest and highest mea-sured values are mentioned in brackets when they exceedthe interval values. Q stands for the length/width ratio andis also given as an interval.

Molecular phylogenetic analyses

Phylogenetic analysis in this article is for the purpose ofconfirming the systematic position of the discussed specieswithin the classification of Russulaceae. The majority ofsequences were obtained from GenBank, though additionalsequences have been produced for this analysis (Table 1).DNA extraction and amplification are as in Stubbe et al.(2010). Three nuclear DNA markers were used: (1) the ITSregion, which includes ITS1, 5.8S and ITS2, defined byprimers ITS1-F and ITS4 (White et al. 1990); (2) the LSUregion defined by primers LR0R and LR5 (R. Vilgalys lab

‘http://www.biology.duke.edu/fungi/mycolab/primers.htm’);(3) the region between conserved domains 6 and 7 of thesecond largest subunit of the RNA polymerase II (rpb2),using primers bRPB2-6f and fRPB2-7cR (Liu et al. 1999;Matheny 2005). The alignment was constructed as detailedin Stubbe et al. (2010) and is available on the TreeBASEwebsite (www.treebase.org, study ID: S11558). For thephylogenetic analyses, the dataset was subdivided into fivepartitions: ITS1+ITS2, 5.8S, LSU, rpb2 first + secondcodon positions, rpb2 third codon positions. A MaximumLikelihood analysis (ML) was executed with the programRAxML v7.0.3, using the GTRMIX model with parametersoptimized for each partition. The Rapid Bootstrappingalgorithm was implemented for 500 replicates, followedby a ML tree search (Stamatakis 2006a, b; Stamatakis et al.2008).

Results

The phylogenetic analyses (Fig. 1) confirm the mono-phyly of Lactarius subg. Gerardii, as defined by Stubbe etal. (2010), with the L. volemus species complex as itssister clade. The genus Lactarius is split up in twoparaphyletic clades: /Lactarius 1 containing subg. Russu-laria, subg. Piperites and subg. Plinthogalus, and /Lactarius 2 containing subg. Lactifluus, subg. Lactariop-sis, subg. Lactarius and subg. Gerardii. Among theAustralasian representatives of subg. Gerardii, 12 stronglysupported, monophyletic clades and long terminalbranches are retrieved that coincide with morphologicallyidentifiable species (indicated in bold in Fig. 1). Lactariusvenosus and L. sepiaceus are not included in thephylogeny because no useful DNA could be extractedfrom the type material. What is considered as L. wirrabarais grouped into three different clades that cannot bedistinguished morphologically from one another. Of someof the remaining Australasian clades and branches, namely1, 2, 7, 8 and 9 (Fig. 1) (corresponding to clades 23, 19, 8,7 and 11, resp., in Stubbe et al. (2010)), the specimenslacked sufficient macromorphological information toallow a full description despite having distinguishingmicroscopic characters. Detailed information on theseclades is given in Stubbe et al. (2010). For clades 3, 4,5, 6 and 10 (Fig. 1) (corresponding to clades 16, 6, 3, 5and 9, resp., in Stubbe et al. (2010)), species delimitationwas not possible because they exhibited considerablemorphological variability or contained specimens thatwere indistinguishable from specimens belonging to otherclades.

Fungal Diversity (2012) 52:141–167 143

http://sciweb.nybg.org/science2/IndexHerbariorum.asp

http://sciweb.nybg.org/science2/IndexHerbariorum.asp

http://www.biology.duke.edu/fungi/mycolab/primers.htm

http://www.treebase.org

Table 1 Specimens and GenBank accession numbers of DNA sequences used in the molecular analysis. Newly submitted sequences are in bold

Species Voucher Location ITSGenBankaccession no.

LSUGenBankaccession no.

rpb2GenBankaccession no.

Lactarius

L. aff. gerardii E. Nagasawa,TMI15558 (TMI)

Aomori, Japan GU258230 GU265587 GU258324

L. aff. gerardii A. Verbeken/R. Walleyn04-088 (GENT)

Chiang Mai, Thailand GU258239 GU265597 GU258334

L. aff. gerardii D. Stubbe 07-390 (GENT) Chiang Mai, Thailand GU258252 GU265613 GU258350

L. aff. gerardii H.T. Le 317 (GENT) Chiang Mai, Thailand GU258243 GU265604 GU258341





L. aff. gerardii H.T. Le 246 (GENT) Chiang Mai, Thailand EF560686 GU265601 GU258338

L. aff. gerardii D. Stubbe/R. Walleyn07-373 (GENT)




L. aff. gerardii H.T. Le 270 (GENT) Chiang Rai, Thailand EF560685 GU265598 GU258335

L. aff. gerardii KIINA 126 (GENT) Hunan, China GU258227 GU265584 GU258321

L. aff. gerardii R. Watling 24828 (E) Negeri Sembilan, Malaysia GU258275 GU265636 GU258374

L. aff. gerardii R. Watling 24783 (E) Negeri Sembilan, Malaysia – GU265637 GU258375

L. aff. gerardii R. Watling 26708 (E) Negeri Sembilan, Malaysia – GU265590 GU258327

L. aff. gerardii M. Christensen 04-259(GENT)

Nepal GU258234 GU265592 GU258329

L. aff. gerardii S.N. Yahya, FRIM1357(FRIM)

Selangor, Malaysia GU258233 GU265591 GU258328

L. aff. gerardii S.S. Lee, FRIM1098 (FRIM) Selangor, Malaysia GU258232 GU265589 GU258326

L. aff. gerardii X.H. Wang 1768 (HKAS) South Korea GU258225 GU265583 GU258319

L. aff. gerardii E. Nagasawa, TMI15534(TMI)

Tottori, Japan GU258229 GU265586 GU258323

L. aff. gerardii K. Das 4073 (BSD) Uttaranchal, India GU258274 – GU258373

L. aff. gerardii K. Das 4062 (BSD) Uttaranchal, India GU258228 GU265585 GU258322

L. aff. gerardii H.D. Zheng 101 (HKAS) Yunnan, China GU258226 – GU258320

L. aff. volemus H.T. Le 321 (CMU) Thailand GU265662 GU258302 GU258402

L. aff. volemus A. Verbeken 05-394(GENT)

U.S.A. GU265660 GU258300 GU258400

L. atromarginatus H.T. Le 144 (CMU) Thailand EF560674 – –

L. atrovelutinus D. Stubbe 06-003 (GENT) Selangor, Malaysia GU258231 GU265588 GU258325

L. baliophaeus A. Verbeken 05-155(GENT)

Malawi GU258277 GU265576 GU258312

L. bicolor D. Stubbe 06-229(GENT)

Negeri Sembilan, Malaysia GU258221 GU265577 GU258313

L. camphoratus UE 04.09.2004-5 (UPS) Sweden DQ422009 DQ422009 DQ421933

L. cfr. gerardii var.fagicola

J. Nuytinck 07-029(GENT)

Newfoundland, Canada GU258224 GU265582 GU258318

L. cfr. gerardii var.fagicola

D.E. Desjardin 3564 Tennessee, U.S.A. GU258273 GU265635 GU258372

L. chiapanensis V.M. Bandala 4374A(GENT)

Chiapas, Mexico GU258297 GU265580 GU258316

L. clarkeae JET 980 (MEL) Australia EU019924 EU019924 –

L. conchatulus sp. nov. H.T. Le 457 (GENT) Chiang Mai, Thailand GU258296 GU265659 GU258399

L. coniculus sp. nov. D. Stubbe 07-496 (GENT) Sri Lanka GU258236 GU265594 GU258331

L. coniculus sp. nov. D. Stubbe 07-497 (GENT) Sri Lanka GU258237 GU265595 GU258332


Table 1 (continued)




L. deceptivus A.Verbeken 04-181 (GENT) U.S.A. DQ422020 DQ422020 DQ421935

L. edulis A.Verbeken 99-041 Zimbabwe AY606973 DQ421977 DQ421916

L. emergens A. Verbeken 99-005 (GENT) Zimbabwe AY606979 – DQ421919

L. genevievae sp. nov. G. Gates/D. Ratkowsky17-02-05

Tasmania, Australia GU258294 GU265657 GU258397

L. gerardii s.l. P.R. Leacock 5338 (F) Indiana, U.S.A. GU258261 GU265623 GU258360

L. gerardii s.l. A.E. Franco-Molano27-6-89 (NY)

New York, U.S.A. – – GU258377

L. gerardii s.l. A. Verbeken 05-355 (GENT) North Carolina, U.S.A. GU258253 GU265615 GU258352

L. gerardii s.l. A. Verbeken 05-283 (GENT) Tennessee, U.S.A. GU258259 GU265621 GU258358

L. glaucescens H.T. Le 66 (GENT) Thailand GU258298 GU265639 GU258378

L. hora sp. nov. D. Stubbe 07-502 (GENT) Sri Lanka GU258238 GU265596 GU258333

L. leae sp. nov. H.T. Le 295 (GENT) Chiang Mai, Thailand EF560687 GU265600 GU258337

L. leae sp. nov. D. Stubbe/R. Walleyn07-426 (GENT)


L. leae sp. nov. A. Verbeken/R. Walleyn04-090 (GENT)


L. leae sp. nov. H.T. Le 404 (GENT) Chiang Mai, Thailand GU258245 GU265606 GU258343

L. leae sp. nov. H.T. Le 411 (GENT) Chiang Mai, Thailand GU258250 GU265611 GU258348

L. leonardii sp. nov. P. Leonard 34204 New Zealand GU258288 GU265651 GU258391

L. leonardii sp. nov. P. Leonard 35607 Queensland, Australia GU258295 GU265658 GU258398

L. leonardii sp. nov. G. Gates 29-01-02 Tasmania, Australia GU258304 GU265664 GU258404

L. leonardii sp. nov. G. Gates 07-02-04 Tasmania, Australia GU258308 GU265668 GU258408

L. leucophaeus A. Verbeken 97-382(GENT)

Papua New Guinea GU258299 GU265640 GU258379

L. lignyotus UE 06.09.2003-5 (UPS) Sweden DQ421993 DQ421993 DQ421926

L. limbatus sp. nov. D. Stubbe 06-230 (GENT) Negeri Sembilan, Malaysia GU258222 GU265578 GU258314

L. limbatus sp. nov. D. Stubbe 06-247 (GENT) Negeri Sembilan, Malaysia GU258223 GU265579 GU258315

L. longisporus A. Verbeken 99-197 (GENT) / Zimbabwe DQ421971 DQ421971 –

B. Buyck 00.1519 (PC) Madagascar – – DQ421910

L. madagascariensis B. Buyck 99–409 (PC) Madagascar AY606977 DQ21975 DQ421914

L. montoyae K. Das 1065 = typus (BSD) India EF560673 GU265641 GU258380

L. ochrogalactus E. Nagasawa 80-102 = typus(TMI)

Tottori, Japan GU258280 – GU258381

L. panuoides T. Henkel 6843 (DUKE) Guyana – AF218566 –

L. pelliculatus B. Buyck 00-1335 Madagascar AY606978 DQ421974 DQ421913

L. petersenii A.Verbeken 05-267 (GENT) North Carolina, U.S.A. GU258282 GU265643 GU258383

L. petersenii D.P. Lewis 6099 Texas, U.S.A. – GU265647 GU258387

L. phlebophyllus B. Buyck 00-1388 (PC) Madagascar AY606974 DQ421979 DQ421918

L. picinus R.Walleyn 3703 (GENT) Czech Republic GU258279 GU265648 GU258388

L. piperatus UE 09.08.2004-6 (UPS) Sweden DQ422035 DQ422035 DQ421937

L. pubescens UE 15.09.2002-2 (UPS) DQ421996 DQ421996 DQ421929

L. reticulatovenosus E. Horak 6472 = typus(GENT)

Java, Indonesia GU258286 GU265649 GU258389

L. rubroviolascens B. Buyck 97.266 Madagascar – DQ421972 DQ421911

L. subgerardii A.Verbeken 05-269 (GENT) North Carolina, U.S.A. GU258263 GU265625 GU258362

L. subgerardii A.Verbeken 04-200 (GENT) Tennessee, U.S.A. GU258266 GU265628 GU258365

L. uyedae MCA 584 (VPI) Japan – AF218562 –

L. vellereus U. Eberhardt 20.09.2004-22(UPS)

Sweden DQ422034 DQ422034 DQ421936


Taxonomy

Key to the species

1. Basidiocarp agaricoid, with dark or pale brown pileus and stipe.....................................................................................21. Basidiocarp pleurotoid, white, small, not exceeding 30 mm diam.................................................................................152. Lilac, pinkish or brownish discolorations of latex, context or bruised lamellae................................................................92. No discolorations within one hour.....................................................................................................................................33. Basidiospores with ornamentation never exceeding 1 μm in height.................................................................................43. Basidiospores with ornamentation more than 1 μm high..................................................................................................54. Pileus and stipe predominantly greyish brown; terminal elements of pileipellis subcylindrical, up to 70×6μm....1. L. limbatus4. Pileus and stipe dark brown; terminal elements of pileipellis subfusiform to subclavate, up to 45×10μm................2.L. bicolor5. Basidiospores ornamented with interconnected, conical to triangular warts, up to 1.5 μm high; lamellar edge

concolorous with lamellar surface...............................................................................................................3. L. coniculus5. Basidiospore ornamentation not spiny but a partial or complete reticulum composed of ridges; lamellar edge

concolorous with lamellar surface or coloured like pileus...............................................................................................66. Basidiocarp rather firm, pileus of mature basidiocarp 50 mm diam. or more.................................................................76. Basidiocarp with fragile habit, pileus not exceeding 50 mm diam., strongly, radially wrinkled......................................8

Table 1 (continued)




L. velutissimus A. Verbeken 99-185 Zimbabwe AY606982 DQ421973 DQ421912

L. volemus U. Eberhardt 09.08.2004-5(UPS)

Sweden DQ422008 DQ422008 DQ421932

L. wirrabara s.l. 1 S.H. Lewis 908 (MEL) New South Wales,Australia

GU258290 GU265653 GU258393

L. wirrabara s.l. 1 P. Leonard 7308 Queensland, Australia GU258309 GU265669 GU258409

L. wirrabara s.l. 1 P. Leonard 40509 South Island, New Zealand GU258287 GU265650 GU258390

L. wirrabara s.l. 1 S.J. McMullan-Fisher 1913(MEL)


L. wirrabara s.l. 1 H. Weatherhead 39 (MEL) Victoria, Australia GU258289 GU265652 GU258392

L. wirrabara s.l. 1 J.E. Tonkin 1000 (MEL) Victoria, Australia GU258292 GU265655 GU258395

L. wirrabara s.l. 2 P. Leonard 10409 Maroochy B.B.C.,Australia

JF731001 JF731003 JF731005

L. wirrabara s.l. 2 R.E. Halling 6800 Queensland, Australia JF731000 JF731002 JF731004

L. wirrabara s.l. 3 G. Gates/D. Ratkowsky17-01-2002






L. wirrabara s.l. 3 G. Gates 30-01-2007 Tasmania, Australia GU258303 GU265663 GU258403

L. wirrabara s.l. 3 J.E. Tonkin 943 (MEL) Victoria, Australia GU258291 GU265654 GU258394

Multifurca

M. furcata REH 7804 (NY) Costa Rica DQ421994 DQ421994 DQ421927

M. ochricompacta BB 02.107 (PC) U.S.A. DQ421984 DQ421984 DQ421940

Russula

R. camarophylla P.A.M. 01081108 DQ421982 DQ421982 DQ421938

R. nigricans UE 20.09.2004-07 (UPS) Sweden DQ422010 DQ422010 DQ421952

R. persicina UE 21.09.2003 (UPS) DQ422019 DQ422019 DQ421960


1. Lactarius limbatus Stubbe & Verbeken sp. nov.(Figs. 2, 11a, b)Mycobank: MB 561025Etymology: limbatus (Latin), referring to the broad, dark

grey coloured edges of the lamellae.Pileus 30–60 mm diam., planoconvexus ad concavum, in

centro depressus ad papillatum, interdum rugulosus, mar-gine undato vel crenulato, velutinus, brunneogriseus. Stipes25–40×5–10 mm, leviter velutinus, minute rimulosus, pileoconcoloris. Lamellae decurrentes, distantissimae, 4–7 mmlatae, albae, margine atrogriseobrunneo. Contextus albi-dus, gustu mitis. Latex moderate abundans, aquoso-albidus, immutabilis. Basidiosporae subglobosae ad lateellipsoideae, (6.5) 6.8–7.9–8.9×(6) 6.1–6.9–7.8 μm, Q=1.08–1.14–1.24, reticulatae, cristis 0.5–0.8 μm altis ornatae,macula suprahilaris centrale, interdum complete, amyloidea.Macrocystidia absentia. Pleuropseudocystidia 5–10 μmdiam., moderate abundantia, clavata ad subfusiformia.Pileipellis bistrata, 80–120μm crassa, pigmento intracellularis

brunneo; elementa suprapellis 15–70×4–6 μm, gracilia,subcylindrata.

Typus: “Malaysia, state of Negeri Sembilan, PasohNational Forest Reserve, along main trail, between streamand Old Tree Tower, on soil, alt. 117 m, N02°58.88′ E102°18.24′, 17 September 2006, D. Stubbe 06-230 (GENT;isotypus: KEP)”.

Pileus 30–60 mm diam., planoconvex to concave, centredepressed and papillate, smooth to faintly rugulose incentral area, indistinctly grooved towards margin; marginirregularly undulate to faintly crenate; surface dry, finelyvelutinous, concentrically rimulose in patches, brownishgrey in centre (5D4, but darker and browner), paler andgreyer towards margin. Stipe 25–40×5–10 mm, cylindricalto slightly compressed, equal or tapering downwards;surface dry, finely velutinous, concolorous with pileus(between 5D4–5E4), minutely rimulose. Lamellae decurrentto deeply decurrent, very distant, 4–7 mm broad, white, notstaining when bruised; edge smooth, conspicuously dark

7. Pileipellis a distinct palisade; terminal elements mostly 1-celled, not exceeding 35 μm; basidiospore ornamentation acomplete reticulum of firm and straight ridges up to 1 μm high.........................................................................4. L. leae

7. Pileipellis a trichopalisade; terminal elements mostly longer than 35 μm and septate; Australia or NewZealand...................................................................................................................................................................................14

8. Basidiospores 8.1–10.5×7.3–9.0 μm; lamellar edge concolorous with lamellar surface.....................5. L. reticulatovenosus8. Basidiospores 7.1–8.1×6.4–7.6 μm; lamellar edge brown..........................................................................6. L. venosus9. Pileus brown or yellowish brown; injured context staining bright vinaceous pink; latex yellowish brown or becoming

brown; basidiospores ornamented with blunt, interconnected warts...............................................................................109. Pileus dark brown; context unchanging or staining pale lilac or brown; latex unchanging or becoming pinkish;

basidiospores ornamented with a reticulum of ridges......................................................................................................1110. Latex yellowish brown; terminal elements of pilei- and stipitipellis thick-walled (± 1 μm thick).................7.L. ochrogalactus10. Latex watery white or slowly becoming pale brown; terminal elements of pilei- and stipitipellis thin-walled (<0.5 μm

thick)............................................................................................................................................................8. L. leonardii11. Latex white, becoming pink to cinnamon; pileipellis a well-developed palisade with short, terminal elements not

exceeding 35 μm; basidiospores 6.3–8.3×5.6–6.9 μm........................................................................9. L. atrovelutinus11. Not with this combination of characters; pileipellis a hymeniderm or a trichopalisade with terminal elements more than

35 μm long......................................................................................................................................................................1212. Basidiocarp with fragile habit; pileus up to 40 mm diam., strongly wrinkled; context and latex becoming lilac tinged;

pileipellis resembling a hymeniderm with few slender, terminal elements....................................................6. L. venosus12. Basidiocarp firm; context or latex discolouring differently; pileipellis a trichopalisade with subfusiform, terminal

elements...........................................................................................................................................................................1313. Context remaining white but lamellae staining pale brown or pinkish brown when bruised; basidiospore ornamentation

a closed reticulum composed of heavy ridges 1 μm thick; Sri Lanka............................................................10. L. hora13. Context faintly and slowly turning pink when exposed; basidiospore ornamentation either a very dense reticulum or

nearly open reticulum; Australia or New Zealand..........................................................................................................1414. Basidiospore ornamentation an almost complete reticulum composed of more or less acute, triangular ridges, 1–1.5 μm

high.....................................................................................................................................................11. L. wirrabara s.l.14. Basidiospore ornamentation a dense reticulum of low ridges, not higher than 1 μm................................12. L. sepiaceus15. Basidiospores 6.1–7.8×5.1–6.6 μm, ornamentation a reticulum of ridges; macrocystidia with obtuse apex, 40–65 μm

long.......................................................................................................................................................14. L. conchatulus15. Basidiospores larger, ornamentation composed of interconnected warts; macrocystidia more than 100 μm long...................1616. Macrocystidia with obtuse or mucronate to slightly rostrate apex...............................................................13. L. uyedae16. Macrocystidia narrowly cylindrical and gradually tapering towards apex.............................................15. L. genevievae


Russula persicina U.E.21-09-03Russula nigricans U.E.20-09-04-7 SWEDEN

Russula camarophylla P.A.M.01081108Multifurca furcata R.E.H.7804 COSTA RICA

Multifurca ochricompacta B.B.02-107 U.S.A.L. camphoratus U.E.04-09-04.5 SWEDEN

L. pubescens U.E.15-09-02.2 L. baliophaeus A.V.05-155 MALAWI

L. picinus R.W.3703 CZECH REPUBLICL. montoyae K.D.1065 typus INDIAL. atromarginatus H.T.L.144 THAILAND

L. lignyotus U.E.06-09-03.5 SWEDENL. clarkeae J.E.T.908 AUSTRALIA

L. chiapanensis V.M.B.4374A MEXICOL. panuoides T.H.6843 GUYANA

L. longisporus A.V.99-197/B.B.00-1519 ZIMB./MADAG.L. rubroviolascens B.B.97.266 MADAGASCAR

L. deceptivus A.V.04-181 U.S.A.L. vellereus U.E.20-09-04.22 SWEDEN

L. phlebophyllus B.B.00-1388 MADAGASCARL. edulis A.V.99-41 ZIMBABWE

L. madagascariensis B.B.99-409 MADAGASCARL. emergens A.V.99-5 ZIMBABWE

L. pelliculatus B.B.00-1335 MADAGASCARL. velutissimus A.V.99-185 ZIMBABWE

L. piperatus U.E.09-08-04.6 SWEDENL. glaucescens H.T.L.66 THAILAND

L. leucophaeus A.V.97-382 PAPUA NEW GUINEAL. volemus U.E.09-08-04.5 SWEDEN

L. aff. volemus H.T.L.321 THAILANDL. aff. volemus A.V.05-394 U.S.A.

L. petersenii A.V.05-267 U.S.A.L. petersenii D.P.L.6099 U.S.A.

L. ochrogalactus E.N.80-102 typus JAPANL. ochrogalactus E.N.80-102 typus JAPANL. leonardii sp. nov. P.L.34204 NEW ZEALANDL. leonardii sp. nov. P.L.34204 NEW ZEALANDL. leonardii sp. nov. P.L.35607 AUSTRALIAL. leonardii sp. nov. P.L.35607 AUSTRALIAL. leonardii sp. nov. G.G.29-01-02 AUSTRALIAL. leonardii sp. nov. G.G.29-01-02 AUSTRALIAL. leonardii sp. nov. G.G.07-02-04 AUSTRALIAL. leonardii sp. nov. G.G.07-02-04 AUSTRALIA

L. genevievae sp. nov. G.G/D.R.17-02-05 AUSTRALIAL. genevievae sp. nov. G.G/D.R.17-02-05 AUSTRALIAL. conchatulus sp. nov. H.T.L.457 THAILANDL. conchatulus sp. nov. H.T.L.457 THAILAND

L. uyedae MCA 584 JAPANL. uyedae MCA 584 JAPANL. aff. gerardii R.W.24828 MALAYSIA

L. reticulatovenosus E.H.6472 typus INDONESIAL. reticulatovenosus E.H.6472 typus INDONESIAL. subgerardii A.V.05-269 U.S.A.L. subgerardii A.V.04-200 U.S.A.

L. cfr. gerardii var. fagicola J.N.07-029 CANADAL. cfr. gerardii var. fagicola D.E.D.3564 U.S.A.

L. aff. gerardii R.W.24783 MALAYSIAL. wirrabara s.l. 1 P.L.40509 NEW ZEALANDL. wirrabara s.l. 1 S.H.L.908 AUSTRALIAL. wirrabara s.l. 1 S.J.M.-F.1913 AUSTRALIAL. wirrabara s.l. 1 P.L.7308 AUSTRALIAL. wirrabara s.l. 1 J.E.T.1000 AUSTRALIAL. wirrabara s.l. 1 H.W.39 AUSTRALIA

L. atrovelutinus D.S.06-003 MALAYSIAL. atrovelutinus D.S.06-003 MALAYSIAL. aff. gerardii K.D.4062 INDIA

L. aff. gerardii K.D.4073 INDIAL. aff. gerardii M.C.04-259 NEPAL

L. aff. gerardii H.T.L.317 THAILANDL. aff. gerardii H.T.L.400 THAILAND

L. aff. gerardii D.S.07-390 THAILANDL. aff. gerardii A.V./R.W.04-088 THAILAND

L. aff. gerardii TMI15534 JAPANL. aff. gerardii X.H.W.1768 SOUTH KOREAL. aff. gerardii TMI15558 JAPAN

L. aff. gerardii KIINA126 CHINAL. gerardii s.l. A.V.05-283 U.S.A.

L. gerardii s.l. P.R.L.5338 U.S.A.L. gerardii s.l. A.V.05-355 U.S.A.L. gerardii s.l. A.E.F.M.27-6-89 U.S.A.

L. leae sp. nov. H.T.L.404 THAILANDL. leae sp. nov. H.T.L.404 THAILANDL. leae sp. nov. D.S./R.W.07-426 THAILANDL. leae sp. nov. D.S./R.W.07-426 THAILAND

L. leae sp. nov. H.T.L.411 THAILANDL. leae sp. nov. H.T.L.411 THAILANDL. leae sp. nov. A.V./R.W.04-090 THAILANDL. leae sp. nov. A.V./R.W.04-090 THAILANDL. leae sp. nov. H.T.L.295 THAILANDL. leae sp. nov. H.T.L.295 THAILANDL. aff. gerardii H.T.L.394 THAILANDL. aff. gerardii H.T.L.431 THAILAND

L. aff. gerardii FRIM1098 MALAYSIAL. aff. gerardii FRIM1357 MALAYSIAL. aff. gerardii R.W.26708 MALAYSIAL. bicolor D.S.06-229 MALAYSIAL. bicolor D.S.06-229 MALAYSIA

L. hora sp. nov. D.S.07-502 SRI LANKAL. hora sp. nov. D.S.07-502 SRI LANKAL. wirrabara s.l. 2 P.L.10409 AUSTRALIAL. wirrabara s.l. 2 R.H.6800 AUSTRALIA

L. wirrabara s.l. 3 G.G./D.R.24-01-04 AUSTRALIAL. wirrabara s.l. 3 G.G.30-01-07 AUSTRALIAL. wirrabara s.l. 3 J.E.T.943 AUSTRALIAL. wirrabara s.l. 3 G.G./D.R.17-01-02 AUSTRALIA

L. wirrabara s.l. 3 G.G./D.R.12-07-03 AUSTRALIAL. coniculus sp. nov. D.S.07-497 SRI LANKAL. coniculus sp. nov. D.S.07-497 SRI LANKA

L. coniculus sp. nov. D.S.07-496 SRI LANKAL. coniculus sp. nov. D.S.07-496 SRI LANKAL. limbatus sp. nov. D.S.06-247 MALAYSIAL. limbatus sp. nov. D.S.06-247 MALAYSIAL. limbatus sp. nov. D.S.06-230 MALAYSIAL. limbatus sp. nov. D.S.06-230 MALAYSIA

L. aff. gerardii H.D.Z.101 CHINAL. aff. gerardii H.T.L.270 THAILAND

L. aff. gerardii H.T.L.302 THAILANDL. aff. gerardii H.T.L.101 THAILANDL. aff. gerardii H.T.L.246 THAILAND

L. aff. gerardii H.T.L.343 THAILANDL. aff. gerardii D.S./R.W.07-373 THAILAND

0.01

Russula (outgroup)

Multifurca

/Lactarius 1

/Lactarius 2

L. sg. RussulariaL. sg. Piperites

L. sg. Plinthogalus

L. sg. Lactifluus

L. sg. Lactifluus

L. sg. Lactariopsis

L. sg. Lactariopsis

L. sg. Lactarius

L. sg. Lactifluus

L. sg. Gerardii

1

2

3

4

5

6

7

8

9

10


greyish brown over the entire length, concolorous withpileus margin and stipe apex. Context thin in pileus, stuffedin stipe, white; smell unremarkable; taste mild. Latexmoderately abundant, watery white, unchanging on lamellaeor context; colour fading when isolated on glass plate.Basidiospores (6.5) 6.8–7.6–7.9–8.9×6.0–6.8–6.9–7.8 μm,Q=1.06–1.12–1.14–1.24 (n=40), subglobose to broadlyellipsoid; ornamentation a wide-meshed reticulum composedof low and rounded ridges 0.5–0.8 μm high; isolated wartsmostly absent; plage centrally, sometimes completely, amy-loid. Basidia 50–65×11–14 μm, clavate to narrowly clavate,4-spored; sterigmata 5–7×1–2 μm. Macrocystidia absent.Pleuropseudocystidia 5–10 μm diam., moderately abundant,clavate to subfusiform, not branching, occasionally mucro-nate. Lamellar edge sterile, completely composed of cheilo-leptocystidia, 30–70×3–5 μm, narrowly subcylindrical andmostly tapering towards apex. Lamellar trama composed ofsphaerocytes and to a lesser extent of inflated hyphae;lactifers moderately abundant. Pileipellis a palisade, 80–120 μm thick, containing brown, intracellular pigmentation;terminal elements 15–70×4–6 μm, slender and subcylin-drical, 1- to 3-celled, thin-walled or with slightly refringentcell walls, erect, oblique or somewhat repent, often stickingtogether; subpellis 30–50 μm thick, composed of 2–3 layerssubglobose cells, 10–20×10–30 μm, thin-walled or withslightly refringent cell walls. Stipitipellis a trichoderm, 30–70 μm thick, containing intracellular, brown pigmentation;terminal elements 3–6 μm diam., thin-walled, recumbent orrepent.

Habitat: solitary or in small groups, on soil in dipterocarpdominated, lowland rain forest.

Known distribution: Peninsular Malaysia.Material examined: Malaysia—state of Negeri Sembilan,

Pasoh National Forest Reserve, along main trail, betweenstream and Old Tree Tower, on soil, alt. 117 m, N02°58.88′E102°18.24′, 17 September 2006, D. Stubbe 06-230 (typus:GENT; isotypus: KEP); ibidem, 19 September 2006, D.Stubbe 06-247 (GENT; KEP).

Notes: The most striking character of L. limbatus is thebroad, dark coloured edge of the lamellae. Colouredlamellar edges are not uncommon in subg. Gerardii, butare usually thinner, as in L. reticulatovenosus or L. bicolor,both formerly belonging to subg. Plinthogalus. Thischaracter exists also in some genuine members of subg.Plinthogalus, like the North American species L. lignyotus

var. marginatus Hesler & A.H. Sm. and L. fallax var. fallaxA.H. Sm. & Hesler, or the Asian L. atromarginatusVerbeken & E. Horak.

It should be noted that in the European L. lignyotus Fr., darklamella edges are an inconsistent character (Heilmann-Clausen et al. 1996). In the Thai clade 10 (Fig. 1) this isalso the case, but it is clear from the molecular analyses thatwe are dealing with a species complex. Whether or not L.lignyotus should also be considered a species complex, hasnot yet been investigated molecularly. For the Thai L. leaeStubbe & Verbeken, which is morphologically and molecu-larly well delimited (Fig. 1), the dark lamella edges are astable character. In L. limbatus, the coloured lamella edges areexceptionally well developed, so although only two collec-tions (from the same locality) were included in the currentanalysis, we believe it is a good character for this species.

Lactarius limbatus and L. bicolor could be confusedwith one another in the field and both species have similarbasidiospores. However, L. limbatus differs by its greytinge and the longer terminal elements in the pileipellis. Themolecular analysis also clearly separates them.

2. L. bicolor Massee, Bull. Misc. Inf. (Royal Gard.,Kew) Vol. 1914 (2): 73 (1914)

(Figs. 3, 11e)Pileus 20–80 mm diam., planoconvex to applanate and

concave; centre slightly depressed, subumbonate but oftenpapillate, rugulose to wrinkled in central area, often concen-trically rugose near the margin, radially grooved and slightlytranslucently striate; margin smooth to irregularly crenate;surface dry, slightly shiny, minutely velutinous in patches,evenly dark brown or fuliginous umber, paler towards themargin when mature (6F3–6F8, 7F3–7F8). Stipe 10–80×4–14 mm, more or less cylindrical, tapering downwards, ratherslender; surface dry, slightly rugulose to almost smooth,sometimes slightly velutinous, often rimulose; dark brown andconcolorous with cap (6EF3–6EF8, 7EF3–7EF8) or paler andgreyer (5E3–5E8, 6E3–6E8 ); base sometimes with thin,whitish tomentum. Lamellae broadly subdecurrent, distant,rather broad (3–10 mm), white; edge smooth, distinctlymarginate and concolorous with stipe. Context very thin inpileus, hollow in stipe, white, unchanging; smell slightlyfruity; taste mild. Latex abundant, white or watery white,unchanging on context or when isolated; taste mild. Basidio-spores 7.5–8.0–8.4–8.8 (9.6)×6.4–7.1–7.8–8.4 (8.6) μm, Q=1.03–1.08–1.13–1.20 (n=50), globose to subglobose; orna-mentation up to 0.8 μm high, mostly 0.3–0.5 μm high,composed of regular ridges forming a complete reticulum,occasionally with isolated warts and free terminals of ridges;plage very weakly distally amyloid. Basidia 55–75×9–14 μm, clavate, 4-spored. Macrocystidia absent. Pleuro-pseudocystidia 4–6 μm diam., not emergent, narrowlycylindrical. Cheiloleptocystidia 20–35×6.0–10.0 μm, subcla-

Fig. 1 The best ML tree produced in RAxML by applying 500replicates of the Rapid Bootstrapping Algorithm followed by aheuristic ML tree search. Semi-thickened branches are supported byminimum 75% BS support, thickest branches by minimum 95% BSsupport. Names in bold are Australasian species that are morpholog-ically and molecularly well-defined. Lactarius wirrabara s.l. ismarked as a group of cryptic species. Numbered clades arecommented on in the text

�


vate, with basal cells often swollen to subglobose, about 10 μmin diam. Pileipellis a palisade; terminal cells 15–35 (45)×7.0–10.0 μm, often tapering towards the apex; subpellis 70–80 μmthick, composed of 3–4 layers of isodiametric cells which are

15–30 μm wide, with brownish intracellular pigmentation.Stipitipellis a trichopalisade; terminal cells 15–35 (45)×7–10 μm, tapering towards apex; subpellis 20–30 μm thick,composed of subglobose cells of 12–18 μm diam.

Fig. 2 Lactarius limbatus (D. Stubbe 06-230, typus): a basidiocarps; b basidiospores; c basidia; d cheiloleptocystidia; e pleuropseudocystidia; fpileipellis; g stipitipellis. (scale bar basidiocarps: 1 cm; scale bar microscopy: 10 μm)


Study of the holotype: “Pileus depressed, moderatelyfleshy, dark brown, with margin waved, 4–5 cm diam.Lamellae ventricose, distant, attached with deeply decurrenttooth; edge smooth, grey. Stipe terete, glabrous, concolorouswith pileus, 50–60×6–8 mm” (translated from Massee 1914:73). Basidiospores 8.1–9.1–10.1×(6.1) 6.7–8.1–9.5 μm, Q=1.06–1.12–1.37 (n=20), globose to subglobose; ornamenta-tion up to 0.8 μm high, mostly 0.3–0.5 μm high, composedof regular ridges forming a complete reticulum, occasionallywith isolated warts and free terminals of ridges; plage veryweakly distally amyloid. Basidia 70–90×10–14 μm, 4-spored, narrowly clavate. Macrocystidia absent. Pleuro-pseudocystidia 4–6 μm diam., not emergent, narrowlycylindrical. Cheiloleptocystidia 25–60×7–12 μm, fusiform,with dark intracellular pigment and covered with dark brown

incrustations. Pileipellis a palisade; terminal elementsbroadly cylindrical to slightly clavate, rarely narrowlycylindrical, 17–35×5–11 μm, with brownish, intracellularpigmentation. Trama of lamellae, pileus and stipe contain-ing rosettes; many hyphae with striking dark pigmentationand incrustations.

Habitat: on soil in lowland rain forest.Known distribution: Peninsular Malaysia, Singapore.Material examined: Malaysia—state of Negeri Sembilan,

Pasoh National Forest Reserve, along main trail, betweenstream and Old Tree Tower, near Shorea and Dipterocarpus,N02°58.58′ E102°18.19′, 134 m alt., 17 September 2006, D.Stubbe 06-229 (KEP, GENT, proposed here as epitype).Singapore—Jungle part of the Botanical Gardens, on theground, 30 August 1939, E.J.H. Corner. Lactarius no 6,

Fig. 3 Lactarius bicolor (E.M. Burkill 73, typus): a basidiospores; b basidia; c cheiloleptocystidia; d pleuropseudocystidia; e pileipellis. (scalebar: 10 μm)


E00186027 (E); among fallen leaves in the jungle, E.M.Burkill 73 (typus: K).

Notes: The poor condition of the type specimen makes itdifficult to observe all micromorphological characters. Thematerial rehydrated well in a 10 % aqueous potassiumhydroxide solution but dark incrustations were everywherebetween the hyphae and the elements. We presume theseshould be considered an artifact. The specimens of Cornerand Stubbe both provided full macromorphological descrip-tions and are similar in every aspect; therefore we proposeD. Stubbe 06-229 as an epitype of L. bicolor. Cornermentioned the name “L. bicolor” on the colour paintings hemade, annotated with “Lactarius 6”, and we consider hisand our collections conspecific with the type specimen of L.bicolor. The species is characterized by its dark browncolour, the distinctly coloured lamellar edges, basidiosporeswith blunt ridges forming a fairly complete reticulum andthe relatively short terminal elements in the pileipellis.

3. Lactarius coniculus Stubbe & Verbeken sp. nov.(Figs. 4, 11c, d)Mycobank: MB 561026Etymology: coniculus (Latin), referring to the small,

cone-shaped warts on the basidiospores.Pileus 10–60 mm diam., irregulare convexus, umbonatus

ad depressum, rugulosus, velutinus ad laevis, atrobrunneusad brunneum. Stipe 8–40×3–5 mm, velutinus, brunneus,basin versus mycelio griseocremeo. Lamellae adnatae adsubdecurrentes, distantes, crassae, albidae, margine con-colori. Contextus albidus ad pallide griseum, gustu mitis.Latex moderate abundans, aquoso-albidus,immutabilis.Basidiosporae subglobosae ad late ellipsoideae, 7.1–8.5–9.9 (10.2)×6.4–7.4–8.5 μm, Q=1.1–1.14–1.28, verrucisconicis usque ad 1.5 μm altis interconnectibus ornatae,macula suprahilaris non vel centrale amyloidea. Macro-cystidia absentia. Pleuropseudocystidia 5–7 μm diam.,rara, subcylindrata. Pileipellis bistrata, 100–150 μmcrassa, pigmento intracellularis brunneo; elementa supra-pellis 15–40×4–7 μm, subcylindrata ad subfusiformia.

Typus: “Sri Lanka, Kudawa, just outside the entrance ofSinharaja Forest Reserve, 14 December 2007, D. Stubbe07-496 (GENT)”.

Pileus 10–60 mm diam., irregularly convex, somewhatumbonate when young, but centre depressed when mature;margin smooth or irregularly waving, sometimes fissured;surface rugulose with radial wrinkles more pronounced whenolder, completely and strongly velutinous when young, butwhen older or in weathered condition for the most partglabrous and faintly shiny and only velutinous in patches,predominantly blackish brown when young, becoming palerto cigar brown (BFF 16, 5F3) and snuff brown (BFF 17,5E5) when mature. Stipe 8–40×3–5 mm, cylindrical andtapering downwards, rather short and slender compared to

pileus diam.; surface dry, strongly velutinous, cigar (BFF 16,5F3) or snuff brown (BFF 17, 5E5), upwards paler to paledrab (BFF 33, 5E3); base sometimes covered with dense andmatted, greyish cream mycelium. Lamellae adnate tosubdecurrent, moderately distant to distant, 7–9 L+l/cm,rather thick and up to 6 mm broad, sometimes withinconspicuous, anastomosing veins at the base, white ordirty white, contrasting with the dark stipe; edge smooth andconcolorous. Context dirty white to pale grey and thin in cap(1 mm in mature specimen), white and solid in stipe; smellunremarkable or faintly raphanoid; taste mild, neutral;indistinct reaction with ferrous sulphate. Latex moderatelyabundant, whey-like to watery with specks of white,unchanging; no reaction with aqueous 10% potassiumhydroxide solution. Basidiospores 7.1–8.5–8.8–9.9 (10.2)×6.4–7.4–7.7–8.8 μm, Q=1.07–1.14–1.28 (n=40), subglo-bose to broadly ellipsoid; ornamentation composed ofinterconnected conical to triangular, rounded to acute wartswhich are up to 1–1.5 (1.8) μm high; plage inamyloid orcentrally amyloid. Basidia 50–70×9–13 μm, narrowlyclavate, 4-spored, rarely 2-spored; sterigmata 5–10×1.5–2 μm, firm. Macrocystidia absent. Pleuropseudocystidia 5–7 μm diam., rare, subcylindrical with apex rounded orweakly mucronate, not or barely emergent. Lamellar edgeoccasionally with few basidia but predominantly composedof cheiloleptocystidia 20–45×5–7 μm, narrowly subfusi-form, 1- to 3-celled. Lamellar trama predominantly cellular;inflated hyphae also present; lactifers moderately abundant.Pileipellis a trichopalisade in young specimens, a palisadewhen mature, up to 100–150 μm thick, with dark brown,intracellular and diffuse pigmentation in upper cell layers,completely thin-walled; suprapellis composed of subcylin-drical to narrowly subfusiform terminal elements which are15–40×4–7 μm, 1- or 2-celled, sometimes tapering towardsapex but always blunt; subpellis composed of 3–6 layers ofsubglobose to ovoid cells, mostly 10–20×15–30 μm. Stip-itipellis a rather thin trichoderm, 50 μm thick, completelythin-walled, composed of somewhat inflated hyphae 5–8 μmdiam.; terminal elements subcylindrical to narrowly fusiform,mostly erect, sometimes repent, containing dark brown,intracellular and diffuse pigmentation, with rounded apex.

Habitat and ecology: lowland tropical rainforest, in asmall Shorea sp. stand, on humid soil and on a mosscovered tree base.

Known distribution: Sri Lanka.Material examined: Sri Lanka—Kudawa, outside the

entrance of Sinharaja Forest Reserve, 14 December 2007,D. Stubbe 07-496 (typus: GENT); ibidem, 14 December2007, D. Stubbe 07-497 (GENT).

Notes: Pegler’s comprehensive work on the agaric flora ofSri Lanka, does not mention any species of Lactarius (Pegler1986). As far as we know, L. coniculus and L. hora are thefirst Sri Lankan Lactarius species to be published. Apart from


the lack of any colour changes, L. coniculus is characterizedby basidiospores ornamented with interconnected, conical

warts. Similar basidiospores are also found in L. ochrogalac-tus, L. petersenii, L. uyedae and L. genevievae, but these

Fig. 4 Lactarius coniculus (D. Stubbe 07-496, typus): a basidiocarps; b basidiospores; c basidia; d cheiloleptocystidia; e pleuropseudocystidia; fpileipellis; g stipitipellis. (scale bar basidiocarps: 1 cm; scale bar microscopy: 10 μm)


species are not closely related to L. coniculus and can beeasily distinguished by their different habit, colour reactions orthe presence of macrocystidia.

4. Lactarius leae Stubbe & Verbeken sp. nov.(Figs. 5, 11f)Mycobank: MB 561027

Fig. 5 Lactarius leae (D. Stubbe 07-426, typus): a basidiocarp; b basidiospores; c basidia; d cheiloleptocystidia; e pleuropseudocystidia; fpileipellis; g stipitipellis. (scale bar basidiocarps: 1 cm; scale bar microscopy: 10 μm)


Etymology: named after Huyen Than Le, who studiedthe genus Lactarius in northern Thailand.

Pileus 35–120 mm diam., convexus ad concavum, incentro depressus vel papillatus,margine crenulato, primolaevis, tum venosus, velutinus, ex luteo atrobrunneus. Stipes40–95×6–20 mm, minute velutinus, rimulosus, pileo con-coloris. Lamellae late adnatae ad decurrentes, distantes,crassae, 5–12 mm latae, pallide cremeae, margine griseo-brunneo. Contextus albidus, immutabilis. Latex moderateabundans, albidus, immutabilis. Basidiosporae subglobo-sae ad late ellipsoideae, 7.7–8.4–9.2×(6.5) 6.7–7.5–8.3 μm, Q=1.06–1.12–1.26, reticulatae, cristis 0.5–1 μmaltis ornatae, macula suprahilaris interdum distale amyloi-dea. Macrocystidia absentia. Pleuropseudocystidia dis-persa, subcylindrata. Pileipellis bistrata, 70–120 μmcrassa, pigmento intracellularis brunneo; elementa supra-pellis 5–20×5–10, subcylindrata ad subclavata.

Typus: “Thailand, Chiang Mai Province, Doi SuthepNational Park, Sangrasabhasri Lane to Huai Kok Mavillage, N 18° 48.68′ E 98° 54.60′, 1,150 m alt., in littledisturbed montane rainforest dominated by dipterocarpsand Fagaceae, 2 July 2007, D. Stubbe 07-426 (CMU;isotypus: GENT)”.

Pileus 35–120 mm diam., convex with depressed centreor irregularly concave; centre with small papilla; margincoarsely waving and crenate; surface smooth when youngbut later becoming strongly radially wrinkled, grooved andreticulately veined towards margin, dry, velutinous with afaint sheen, faintly translucent, dark yellowish brown(5DE4–5DE7, 5EF8) with fuliginous brown veins (5F5),colours becoming paler in older specimens. Stipe 40–95×6–20 mm, more or less cylindrical and equal or taperingdownwards, often slightly curved; surface dry, faintlylongitudinally wrinkled, minutely velutinous, concolorouswith pileus or less yellowish and a deeper brown, darker inthe apex (5EF7–5EF8, 6F5) and paler downwards (5DE5,6E4–6E5) to brownish white at the base, pellis oftencracked or minutely flaked off, leaving tiny, cream colouredspots. Lamellae decurrent or broadly adnate with a deeplydecurrent tooth, distant, 3–6L+l/ cm, rather thick and broad(5–12 mm), feeling dry or somewhat waxy, pale creamcoloured (3A2, 4A2), not as white as context; edge smoothand greyish brown (5E4), sometimes inconspicuouslycoloured or not over the entire length; decurrent teethcompletely brownish and concolorous with stipe apex.Context thin in pileus, fistulose in stipe, white, unchangingwhen exposed; smell faint; taste unremarkable; pinkish(5A4) reaction with ferrous sulphate, yellowish (2A3–2A4)with 10% aqueous potassium hydroxide solution. Latexmoderately abundant, white or watery white, unchanging.Spore print white. Basidiospores 6.6–8.2–9.1–11.3 (12.1)×5.9–7.5–7.6–8.8 (9.1) μm, Q=1.04–1.11–1.21–1.40 (n=100), subglobose to broadly ellipsoid; ornamentation a

complete reticulum of rather straight ridges, 0.5–1 μm high,often with large meshes on lateral basidiospore sides,isolated warts very few to none; plage often with denseand low reticulations, sometimes distally or diffuselyamyloid. Basidia 40–70×9–12 μm, narrowly clavate, 4-spored with few 1-, 2- and 3-spored basidia present;sterigmata 5–8×1.5–2 μm, firm and relatively long. Macro-cystidia absent. Pleuropseudocystidia rare to moderatelyscarce, subcylindrical and smooth, apex obtuse or some-what tapering, contents with few, often needle-like andrefringent particles. Lamellar edge sterile and completelycomposed of cheiloleptocystidia 10–20 (30)×5–10 μm,broadly clavate, subfusiform or subcylindrical, occasionallyseptate, arising from inflated basal cells (resembling thepellis structure), containing intracellular, diffuse to some-what agglutinated brown pigmentation. Lamellar tramaalmost entirely cellular; lactifers moderately abundant.Pileipellis a palisade, 70–120 μm thick, completely thin-walled; upper layers containing intracellular, diffuse oragglutinated brown pigmentation; suprapellis composed oferect and oblique terminal elements, 5–20×5–10 μm,subcylindrical, sometimes subclavate or broadly conical,sometimes 2-celled; subpellis composed of 2–4 layers ofsubglobose to broadly ellipsoid cells, 15–40 (50)×10–30 μm; underlying layer composed of densely packed,parallel hyphae. Stipitipellis a palisade, 40–70 μm thick,with 1–3 cell layers in subpellis, otherwise completelysimilar to pileipellis.

Habitat: primary or little disturbed montane rain forestswith Dipterocarpus, Castanopsis, Lithocarpus.

Known distribution: northern Thailand.Material examined: Thailand—Chiang Mai province,

Doi Suthep National Park, Sangrasabhasri Lane to HuaiKok Ma village, N18°48.68′ E98°54.60′, 1,150 m alt., 2July 2007, D. Stubbe 07-426 (typus: CMU; isotypus:GENT); ibidem, N19°48.62′ E98°54.60′, 24 June 2004, A.Verbeken/R. Walleyn 04-090 (CMU, GENT); ibidem, N18°48.62′ E98°54.60′, 24 June 2005, H.T. Le 295 (CMU,GENT); ibidem, 7 June 2006, H.T. Le 411; Chiang Maiprovince, Doi Inthanon, near 22 km marker, 5 June 2006,H.T. Le 404 (CMU, GENT).

Notes: The molecular, phylogenetic analyses haverevealed at least four similar species of subg. Gerardii innorthern Thailand. Based on our current knowledge, L. leaeis the only Thai species that can be delimited bothmolecularly and morphologically. The remaining specieseither lack distinctive macromorphological data or exhibitstrong morphological and molecular variability. Lactariusleae is distinguished by the short, terminal elements in thepileipellis (<35 μm long) and the short cheiloleptocystidiawith inflated basal cells. Although a coloured lamellar edgewas not always visible in the field, brown pigmentation waspresent in the cheiloleptocystidia of all collected specimens


when observed with the microscope. The closest relative ofL. leae is the North American L. gerardii s.l.

5. L. reticulatovenosus Verbeken & E. Horak, Sydowia53 (2): 285 (2001)

The species was described from montane rain forests inJava, Indonesia, and so far is only known from the typecollection. It is characterized by its small stature (up to 30 mmdiam.), strongly wrinkled cap and rather large basidiospores(8.1–10.5×7.3–9.0 μm) bearing an irregular reticulum ofridges. A full description is given in Verbeken et al. (2001).

6. L. venosus Verbeken & E. Horak, Austr. Syst. Bot. 13:692 (2000)

The only known collection so far is the type collectionwhich was found in the montane rain forests of Papua NewGuinea and was described in Verbeken and Horak (2000).Since it did not yield any useful DNA, the phylogeneticposition of L. venosus in subg. Gerardii has not yet beenconfirmed molecularly. However, its general habit, basidio-spore characteristics and close resemblance to L. reticulatove-nosus, make it a likely member. Because of the lilacdiscoloration in the context, it has been compared to L.atromarginatus which belongs to subg. Plinthogalus (Fig. 1).

7. L. ochrogalactus Hashiya, Mycoscience 42 (4): 232(2006)

Lactarius ochrogalactus Hashiya was invalidly publishedin 1994, but later validated, and described in detail by Wanget al. (2006). It is a pale brown mushroom with yellowishbrown latex, exhibiting a vinaceous pink colour change in thecontext and lamellae when these are damaged. The basidio-spores are ornamented with high warts, interconnected bylines and low ridges. It is further characterized by thepresence of narrow, sublanceolate macrocystidia up to100 μm long, and terminal elements with thickened cellwalls in the pellis structures and on the lamellar edge.

The species was described from Japan; it has also beenreported from China (Wang et al. 2006) and from Malaysiaon the island of Borneo (Stubbe et al. 2008). The Malaysianspecimen is somewhat distinctive in being dark brown, butotherwise fits the description perfectly.

8. Lactarius leonardii Stubbe & Verbeken sp. nov.misapplied: L. wirrabara ss. Fuhrer 2005(Figs. 6, 11j)Mycobank: MB 561028Etymology: leonardii, named after Patrick Leonard who

collected and described the type specimen.Pileus 60 mm diam., applanatus ad depressum in centro,

siccus, pallide brunneus. Stipes 40×8 mm, cylindratus,siccus, punctatus, pileo concoloris vel atrobrunneus.Lamellae decurrentes, distantes, cremeae, rosascentes.

Contextus albidus, rosascens. Latex aquoso-albidus. Basi-diosporae subglobosae ad late ellipsoideae, 7.7–8.6–10(10.4)×6.2–7.2–8.2 (8.3) μm, Q=1.12–1.23–1.35, incom-plete reticulatae, verrucis usque ad 1 μm altis interconnec-tibus ornatae, macula suprahilaris centrale amyloidea.Macrocystidia 50–90×5–10 μm, dispersa, subfusiformiavel subcylindricata. Pleuropseudocystidia rara, usque ad7 μm diam. Pileipellis palisade, 100–150 μm crassa,pigmento intracellularis brunneo; elementa suprapellis15–55×4–10, subcylindrata vel subfusiformia.

Typus: “Australia, state of Queensland, Lamington NationalPark, 800 m alt., on soil, close to Nothofagus moorei (F.Muell.) Krasser, in beech forest, May 2007, P. Leonard 35607(GENT; isotypus: pers. herb. P. Leonard)”

Pileus 60 mm, applanate to centrally depressed, dry, lightbrown (5D7). Stipe 40×8 mm, cylindrical, dry, punctate,concolorous with pileus but darker. Lamellae decurrent,distant, cream-coloured, staining pink when injured; lamellu-lae of 3 different lengths. Context thin in pileus, white,staining pink. Latex watery white. Basidiospores 7.7–8.6–9.2–11 (11.5)×6.2–7.2–7.4–8.4 (8.7) μm, Q=1.12–1.20–1.26–1.43 (n=60), broadly ellipsoid to subglobose; ornamentationan incomplete reticulum composed of high and blunt warts, ±1 μm high (<1.5 μm), sometimes concatenated but predom-inantly interconnected with faint, sometimes isolated, amyloidlines; plage often centrally amyloid. Basidia 50–65×8–13 μm, mainly 4-spored, also 2- and 3-spored, few 1-spored; sterigmata 5–8×1.5–2 μm. Macrocystidia 50–90×5–10 μm, not to rather abundant, narrowly subfusiform orsubcylindrical with tapering and refringent apex, thin-walled;sometimes arising from within the lamellar trama, notprotruding beyond full-grown basidia. Pleuropseudocystidiaup to 7 μm diam., rare, smooth, not branching, with obtuse tomucronate apex. Lamellar edge sterile, occasionally withbasidia; cheiloleptocystidia 30–55×4–13 μm, clavate, fusi-form or subcylindrical with tapering apex, thin-walled.Lamellar trama predominantly cellular; lactifers few. Pilei-pellis a palisade, 100–150 μm thick, with brown, intracellularand diffuse or somewhat coagulated pigmentation in uppercell layers; terminal elements 15–55×4–10 μm, mostlyslender and subcylindrical with 1–2 septa, sometimes broadlysubfusiform, with cell walls thin or refringent, less than0.5 μm thick; subpellis composed of 3–4 layers of isodia-metric or broadly ellipsoid cells, 15–30×25–50 μm, thin-walled. Stipitipellis a trichoderm, 40–50 μm thick, brown,intracellular pigmentation in upper layers; terminal elements20–50×4–8 μm, mostly subcylindrical but also broadlysubfusiform, 1- or 2-celled with basal cell sometimes slightlyinflated; cell walls thin or refringent.

Habitat: on soil or rotten wood, under Nothofagus orEucalyptus.

Known distribution: Australia (Queensland, Tasmania),New Zealand.


Material examined: Australia—state of Queensland,Lamington National Park, 800 m alt., on soil in beech

forest, near Nothofagus moorei, May 2007, P. Leonard35607 (typus: GENT; isotypus: pers. herb. P. Leonard); state

Fig. 6 Lactarius leonardii (P. Leonard 35607, typus): a basidiospores; b basidia; c macrocystidia; d cheiloleptocystidia; e pileipellis; f stipitipellis.(scale bar: 10 μm)


of Tasmania, Shingle bend, Styx River, 29 January 2002, G.Gates & D. Ratkowsky 29-01-02 (GENT; pers. herb. G.Gates); state of Tasmania, Leap Loop, 7 February 2004, G.Gates & D. Ratkowsky 07-02-04 (GENT; pers. herb. G.Gates). New Zealand—South Island, Caanan, Native Forest,near Nothofagus menziesii, on (rotten) wood, 3 February2004, P. Leonard 34204 (GENT; pers. herb. P. Leonard).

Notes: The molecular phylogeny shows a sister relation-ship between L. ochrogalactus and L. leonardii. This closekinship is reflected in the many morphological similarities.Most remarkable are the vinaceous pink colour reaction ofthe lamellae and context, the basidiospore ornamentationwith high warts and the presence of subcylindrical and thin-walled macrocystidia. Lactarius ochrogalactus differs by itspale brown colour, the yellowish brown latex and the thick-walled cells in the pellis structures. The latex in L. leonardiiis watery white, but a slow, brown discoloration has beenobserved in some collections (Fig. 11). The morphology ofL. leonardii corresponds with the description Fuhrer (2005)gives for L. wirrabara. However, Fuhrer’s concept of L.wirrabara cannot be accepted. The pink colour change,which is actually quite conspicuous, is not mentioned in theoriginal description of L. wirrabara and, more importantly,the basidiospores of all the pink staining specimens areornamented with interconnected, blunt warts, while those ofthe type of L. wirrabara are ornamented with acute ridges.Lactarius leonardii is found both in Australia and NewZealand and can easily be recognized by its colour reactionsand basidiospores.

9. Lactarius atrovelutinus J.Z. Ying, Acta Mycol. Sin.10: 191. 1991.

(Figs. 7, 11k)Pileus 20–40 mm diam., planoconvex with depressed

centre, sometimes with inconspicuous papilla; marginsmooth, enrolled when young; surface dry and stronglyvelutinous, smooth when young but becoming radiallyrugose to somewhat grooved towards the margin, at firstuniformly dark blackish brown (8F3, but darker andbrowner), the central area slightly paler (8F4) when mature,the extreme margin a contrasting, cream-coloured line.Stipe 30–45×8–12 mm, cylindrical, equal or slightlybroadening towards base or apex; surface strongly veluti-nous, smooth to minutely rugulose especially in bottom halfof the stipe, predominantly concolorous with pileus centreor paler (6E5–6E6); apex sometimes with tiny, whitishspots; base greyish white. Lamellae decurrent, very distant,5–10 L+l/cm at mid-radius, thick, 1–4 mm broad, withoutinterconnecting veins, cream coloured when young (5A2),becoming more yellow when mature (4A4), unchanging orslightly pink when bruised; margin smooth, concolorouswith lamellar surface but with a pale greyish tinge,sometimes distinctly greyish brown near the stipe. Context

rather firm, thin to moderately thick in pileus, fistulose tobroadly fistulose in stipe, even in young specimens,whitish, becoming slightly pale cream-coloured (4A2) butwithout striking colour change; smell pleasantly fruity,sweet; taste mild, neutral or with a subtle, acrid aftertaste.Latex abundant, watery white, sticky, unchanging whenisolated on glass plate, droplets on lamellae and stipedrying old rose or brownish pink. Basidiospores 6.3–7.3–8.3×5.6–6.3–6.9 μm, Q=1.09–1.17–1.33 (n=20), subglo-bose to broadly ellipsoid; ornamentation a complete oralmost complete reticulum composed of blunt to sub-acuteridges and warts of irregular height up to 1 (1.3) μm, withfew isolated warts present; plage sometimes centrallyamyloid. Macrocystidia absent. Pleuropseudocystidia 3–4 μm diam., rather abundant, subcylindrical, rarely branch-ing, smooth, apex obtuse. Lamellar edge entirely composedof cheiloleptocystidia, 20–40×3 μm, subcylindrical withobtuse apex, mostly hyaline, sometimes containing brown-ish intracellular pigmentation. Lamellar trama composed ofsphaerocytes and inflated hyphae; lactifers abundant.Pileipellis a palisade, 80–100 μm thick, containing diffuseor agglutinated, brown pigmentation only in suprapellis andupper layers of subpellis; suprapellis composed of erect tooblique terminal elements, 10–25×3–5 μm, subfusiform ornarrowly conical, thin-walled; subpellis 60–70 μm thick,composed of several layers of subglobose to somewhatelongate cells, 3–12×3–8 μm, thin-walled. Stipitipellisvariable, from a well-developed palisade with 2–3 layerssubglobose cells in the subpellis to a trichoderm withslightly inflated subterminal cells, 35–70 μm thick, thin-walled, containing brown, intracellular and diffuse oragglutinated pigmentation; terminal elements 15–30×3–6(8) μm, subfusiform or subcylindrical, erect or oblique, 1-to 3-celled, mostly obtuse apices, thin-walled.

Habitat and ecology: on soil, with Shorea parviflora andS. sumatrana.

Known distribution: China; Peninsular Malaysia.Material examined: Malaysia—Selangor province,

Kepong, FRIM domain, dipterocarp arboretum, 17 August2006, D. Stubbe 06-003 (GENT; KEP).

Notes: The species was originally described fromGuizhou province, China, and a type study by Wang waspublished in 2007 (Wang 2007). The recent Malaysiancollection consists of young basidiocarps, so maturebasidiospores were not abundant and fully developedbasidia were difficult to find. Nonetheless the morpholog-ical characters perfectly match the description and drawingsof L. atrovelutinus. Of course, without a Chinese specimenin the phylogeny, the possibility remains that Malaysian andChinese specimens belong to two separate, cryptic species.The species is characterized by distant lamellae, a pink orbrownish pink colour reaction of the latex, small basidio-spores (7.3×6.3 μm) and short terminal elements. Both the


type and the Malaysian collection have rather smallsphaerocytes in the subpellis. The type specimen hasmarginal cells that are slightly ventricose and have

indistinct content (Wang 2007). Similar marginal cells canbe found in the Malaysian specimen, though they aremostly subcylindrical and hyaline. The cream-coloured

Fig. 7 Lactarius atrovelutinus (D. Stubbe 06-003): a basidiocarps; b basidiospores; c cheiloleptocystidia; d pleuropseudocystidia; e pileipellis; fstipitipellis (young basidiocarp); g stipitipellis (mature basidiocarp). (scale bar basidiocarps: 1 cm; scale bar microscopy: 10 μm)


margin of the pileus of the Malaysian specimen is strikingbut was not mentioned by Ying (1991). However, themargin became hardly visible after drying the specimensand the same could have happened with the type collection,which would explain why it was not mentioned in the typestudy byWang (2007). Ying (1991) mentioned a white sporeprint for L. atrovelutinus, which agrees with its position insubg. Gerardii. In the field, there might be some resem-blance with L. leonardii, though the pink discoloration ofthis species is much more vibrant. A comparison of thebasidiospores will leave little doubt. The North Americanspecies L. gerardii var. subrubescens also exhibits a pinkcolour change, but this is in the context, and not the latexitself. The lamellae of L. gerardii var. subrubescens are atmost cream-coloured and the basidiospores are larger (7–10×6.5–8 μm) (Hesler and Smith 1979).

10. Lactarius hora Stubbe & Verbeken sp. nov.(Figs. 8, 11g, h)Mycobank: MB 561029Etymology: referring to Hora, the Sinhalese name of the

host tree Dipterocarpus zeylanicus.Pileus 55–60 mm diam., planoconvexus, in centro

depressus vel leviter infundibuliformis, rugosus, velutinus,atrobrunneus ad brunneum. Stipes 95–12×12–14 mm,longitudinale rugosus, velutinus, atrobrunneus, localerimosus. Lamellae decurrentes, moderate confertae, ad6 mm latae, pallide cremeae, pallide roseobrunnescentes.Contextus albus, immutabilis. Latex aquoso-albidus, immu-tabilis, gustu astringens. Basidiosporae subglobosae adlate ellipsoideae, (7.8) 8.0–9.3–10.6 (10.7)×(6.8) 7.0–8.2–9.5 μm, Q=1.05–1.13–1.30, reticulatae, cristis usque ad1.3 μm altis, 1 μm crassis ornatae, macula suprahilariscentrale amyloidea. Macrocystidia absentia. Pleuropseudo-cystidia dispersa, 2–6 μm diam. Pileipellis bistrata, 100–120 μm crassa, pigmento intracellularis brunneo; elementasuprapellis 10–45×5–8, gracile conica vel subcylindrata.

Typus: “Sri Lanka, Kudawa, near Sinharaja ForestReserve, in D. zeylanicus stand, 15 December 2007, D.Stubbe 07-502 (GENT)”

Pileus 55–60 mm diam., planoconvex with depressedcentre or slightly infundibuliform; margin more or lessstraight; surface radially rugose with faint concentricwrinkles near the margin, strongly velutinous, dark brownto warm blackish brown, paler and cigar brown (BFF 16) inmarginal zone. Stipe 95×12–14 mm, more or less cylindri-cal and straight, slightly broader at the apex, otherwiseequal, longitudinally rugulose, entirely and strongly velu-tinous, concolorous with pileus but paler near apex andbase, with scattered and tiny, cream coloured spots wherepellis appears chipped; extreme base with whitish tomen-tum. Lamellae decurrent, moderately dense, 6–10 L+l/cm atmid-radius, up to 6 mm broad, occasionally forking, with

inconspicuous, anastomosing veins at lamella base, palecream-coloured (BFF 3), staining pale pinkish brown (‘claypink’, BFF 30) where bruised; edge smooth and entirelycigar or snuff brown (BFF 16–17). Context moderatelythick in pileus, fistulose in stipe, white, unchanging; smellunremarkable, faint; taste neutral; pale pinkish brown (BFF30) with ferrous sulphate, yellow (BFF 50) with 10 %aqueous potassium hydroxide solution. Latex copious,watery white, sticky when drying but not discolouring,taste astringent but not acrid, like chestnuts. Spore printwhite. Basidiospores (7.8) 8.0–9.3–10.6 (10.7)×(6.8) 7.0–8.2–9.5 μm, Q=1.05–1.13–1.30 (n=20), subglobose tobroadly ellipsoid; ornamentation a complete reticulum withfirm ridges up to 1 (1.3) μm high and 1 μm thick, someisolated warts present, basidiospore surface always withminutely granular, amyloid material in the meshes; plagemostly centrally amyloid. Basidia 50–60×10–12 μm, nar-rowly clavate, 4-spored, rarely 3-spored; sterigmata 5–10×1.5–3 μm, firm. Macrocystidia absent. Pleuropseudocysti-dia 2–6 μm diam., scarce, not emergent, subcylindrical,straight or tortuous. Lamellar edge sterile, composed ofcheiloleptocystidia 20–30×3–6 μm, subfusiform to sub-clavate, mostly 2-celled, containing brown, intracellular anddiffuse pigmentation. Lamellar trama predominantly com-posed of inflated hyphae, intermixed with filamentoushyphae and sphaerocytes; lactifers up to 8 μm broad,abundant. Pileipellis a palisade or trichopalisade, 100–120 μm thick, containing intracellular, diffuse or somewhatagglutinated, brown pigmentation; suprapellis composed oferect terminal elements, 10–45×5–8 μm, 1- to 3-celled,narrowly conical or subcylindrical, often with slightlyacuminate apex, thin-walled; subpellis composed of multi-ple layers of subglobose or somewhat elongated cells, 5–25×5–10 μm, cell walls refringent or slightly thickened.Stipitipellis a palisade, 60–80 μm thick, containing diffuseor somewhat agglutinated, brown pigmentation, cell wallsthin or somewhat refringent; suprapellis composed ofnarrowly conical or subfusiform terminal elements, 15–35×3–8 μm, 1- or 2-celled, often with slightly acuminateapex; subpellis composed of 1–2 layers of subglobose orelongate cells, usually not exceeding 15×15 μm.

Habitat and ecology: solitary, on soil, in a Dipterocarpuszeylanicus stand on a slope.

Known distribution: Sri Lanka.Material examined: Sri Lanka—Kudawa, near Sinhar-

aja Forest Reserve, on a forest slope, on soil in a D.zeylanicus stand, 15 December 2007, D. Stubbe 07-502(typus: GENT).

Notes: Lactarius hora is recognized by the dark brownpileus and stipe, the rather dense lamellae that stain pinkishbrown when bruised, and by the basidiospores with firmand rather broad ridges. Although this description is basedon a single basidiocarp, it was mature and in excellent


condition. With its slender appearance and dense lamellae withcoloured edges, the species is reminiscent of L. atromarginatus.

The latter is also an Asian species but has violet staining latexand is a confirmed member of subg. Plinthogalus, as

Fig. 8 Lactarius hora (D. Stubbe 07-502, typus): a basidiocarp; b basidiospores; c basidia; d cheiloleptocystidia; e pleuropseudocystidia; fpileipellis; g stipitipellis. (scale bar basidiocarps: 1 cm; scale bar microscopy: 10 μm)


demonstrated by the molecular analyses (Fig. 1). Thespecimen of L. hora was found growing out of the entranceof a small burrow, which might have resulted in anexceptionally long stipe and subsequent slender appearance.

11. Lactarius wirrabara Grgur., Larger Fungi of SouthAustralia: 58 (1997)

12. Lactarius sepiaceus McNabb, New Zealand J. Bot.9: 50 (1971)

Lactarius sepiaceus was described from New Zealand byMcNabb (1971) who made a detailed description based onseveral of his own collections. Lactarius wirrabara wasdescribed from Australia by Grgurinovic (1997), based onexsiccate which were provided with limited macroscopicnotes. Although L. wirrabara strongly resembles L. sepia-ceus, Grgurinovic did not provide a comparison of the twospecies. Lactarius sepiaceus should be the darker of the two,being sepia coloured to brownish black, while L. wirrabarashould be a paler brownish umber. According to the originaldescription, L. sepiaceus occasionally has dark lamellaredges, while this was not described for L. wirrabara. Nocontext colour changes were mentioned for L. wirrabara,while on the exposed context of L. sepiaceus a very faintsalmon tinge should slowly appear. Verbeken et al. (2010)examined the holotype specimens of these two species andremarked clear differences in the ornamentation of thebasidiospores. One of the paratypes of L. sepiaceus, PDD31236, however, has basidiospores that rather resemble thoseof the holotype of L. wirrabara. For this particular collectionMcNabb mentioned the pink staining context and whencomparing the notes for PDD 31236 with the description ofL. sepiaceus, it seems that McNabb based the macro-morphological description of this species mostly on this

paratype. We therefore suspect that the original description ofL. sepiaceus might be based on more than one species.Unfortunately, we did not succeed to extract useful DNAfrom the type and paratype. When taking in consideration allAustralian and New Zealand specimens in the subg. Gerardiiphylogeny, only L. leonardii and L. genevievae are clearlydistinguishable both molecularly and morphologically. Theremaining specimens fall in two strongly supported clades,one of them consisting of two subclades. Although there issome basidiospore variability among these specimens, theyall have basidiospores that closely resemble those of the typeof L. wirrabara rather than those of the type of L. sepiaceus.Thus, because none of the specimens in the phylogenycomplied to the holotype of L. sepiaceus, we consideredthem all as L. wirrabara s.l. Recent collections with detaileddescriptions will be necessary to achieve a clear-cutdelimitation between L. sepiaceus and L. wirrabara and tounderstand the variability within these species.

13. Lactarius uyedae Singer, Nova Hedwigia 40: 436.1985 (“1984”)

This small, white, pleurotoid Lactarius species is striking-ly different from most species in subg. Gerardii. Threesimilarly looking species in subg. Gerardii have beendiscovered so far, but L. uyedae can be distinguished by itsbasidiospore ornamentation of warts interconnected by lowridges and by its macrocystidia up to 100 (120) μm longwith a rounded or somewhat mucronate apex. So far it isonly known from Japan. Detailed descriptions of the speciesare given by Verbeken (1998b) and by Henkel et al. (2000).

14. Lactarius conchatulus Stubbe & H.T. Le sp. nov.(Fig. 9)

Fig. 9 Lactarius conchatulus (H.T. Le 457, typus): a basidiospores; b basidia; c macrocystidia; d pileipellis. (scale bar: 10 μm)


Mycobank: MB 561031Etymology: conchatulus (Latin) is the diminutive form of

conchatus, meaning shaped like a small shell (of a bivalve).Basidiocarpus pleurotoideus. Pileus 2–7 mm diam.,

primo convexus, tum planoconvexus et leviter depressusad subinfundibuliformis, margine regulari tum crenulato,squamosulus, velutinus, ad marginem hirsutus, albus,pallide flavescens, tum brunneogrisescens. Stipes 3–5×1–1.5 mm, lateralis, pruinosus, albus. Lamellae subde-currentes, subdistantes, 0.5–1.5 mm latae, albae, flavo-brunnescentes. Contextus albus, gustu acris. Latexaquosus, immutabilis. Basidiosporae late ellipsoideae,6.1–7.0–7.8 (8.0)×5.1–5.9–6.6 μm, Q=1.08–1.19–1.35,subcomplete reticulatae, cristis usque 1 μm altis ornatae,macula suprahilaris inamyloidea vel centrale ad com-plete amyloidea. Macrocystidia 40–65×5–6 μm, abun-dantia, cylindricata, emergentia. Pleuropseudocystidianon emergentia. Pileipellis bistrata, 80–100 μm crassa;elementa suprapellis 20–70×3–6 μm, longa, subfusifor-mia, crassitunicata.

Typus: “Thailand, Chiang Mai province, Mae Taengdistrict , Ban Pha Deng village, Pathuranikaram temple, 11July 2007, H.T. Le 457 (legit E.C. Vellinga) (CMU;isotypus: GENT, SFSU)”

Pileus 2–7 mm diam., pleurotoid, convex when young,becoming plano-convex and slightly depressed to almostinfundibuliform, margin regular, somewhat wavy whenolder; surface with faint velutinous scales, hirsute towardsmargin, white, staining pale yellowish (1A2–3, 2A4) whenbruised, after 1 h becoming brownish grey or moreyellowish (4BC5), ultimately dark yellowish grey (4B8)after 2 h. Stipe 3–5×1–1.5 mm, lateral, cylindrical ortapering upwards; surface smooth, pruinose, concolorouswith pileus. Lamellae subdecurrent, subdistant (ca. 20lamellae), 0.5–1.5 mm broad, concolorous with pileus orpaler, brownish yellow when bruised; lamellulae of 2lengths. Context very thin, white in pileus, hollow tostuffed in stipe, white; smell fragrant when young, likegreen grass; taste slightly acrid to acrid; olivaceous yellowreaction (3B5) with 10% aqueous potassium hydroxidesolution. Latex watery, unchanging; yellowish with 10%aqueous potassium hydroxide solution. Basidiospores 6.1–7.0–7.8 (8.0)×5.1–5.9–6.6 μm, Q=1.08–1.19–1.35 (n=20),broadly ellipsoid; ornamentation an almost complete retic-ulum composed of ridges with irregular height up to 0.5–0.9 μm, mostly subacute and with finer, interconnectinglines; plage inamyloid or centrally to completely amyloid.Basidia 35–45×6–10 μm, clavate, 4-spored; sterigmata 5–7×1–1.5 μm. Macrocystidia 40–65×5–6 μm, very abun-dant, cylindrical with obtuse apex, emergent, containingtiny, needle-shaped and refringent granules. Pleuropseudo-cystidia not emergent. Hymenophoral trama composed offilamentous to inflated hyphae. Pileipellis a palisade, 80–

100 μm thick; suprapellis composed of long, subfusiformterminal elements, 20–70×3–6 μm, thick-walled, occa-sionally septate; subpellis composed of several layersisodiametrical to somewhat elongate sphaerocytes, 10–15×4–10 μm, thin-walled.

Habitat: growing on wood, in mixed forests dominatedby Castanopsis and Dipterocarpus.

Known distribution: northern Thailand.Material Examined: Thailand—Chiang Mai province,

Mae Taeng district, Ban Pha Deng village, near Pathummi-karam Temple, 1,076 m alt., forest dominated by Dipterocar-pus spp. and Castanopsis armata, on wood, 11 July 2007, H.T. Le 457 (typus: CMU; isotypus:GENT, SFSU); Chiang MaiProv., Mae Taeng Distr., Tung Joaw village, N19°08.07′E.098°38.09′, 1,300 m alt., rainforest dominated by Casta-nopsis armata, Lithocarpus sp. and Pinus kesiya, solitary onwood, 18 July 2007, H.T. Le 478 (CMU, SFSU)

Notes: The description of the microscopic features isbased on H.T. Le 457. Further remarks are given belowtogether with L. genevievae.

15. Lactarius genevievae Stubbe & Verbeken sp. nov.(Figs. 10, 11i)Mycobank: MB 561032Etymology: named after Genevieve Gates who collected

the species. One of the presumed meanings of the nameGenevieve is ‘white wave’, which is an allusion to theshape and colour of the pilei.

Basidiocarpus pleurotoideus. Pileus 8–18 mm diam.,primo conchiformis, tum flabelliformis, margine regularitum undulato, laevis ad leviter rugulosus, siccus, interdumminute velutinus, albus, aurantiobrunnescens. Stipes usquead 2×2 mm, lateralis, albus. Lamellae furcatae, usque ad1.5 mm latae, albae, aurantiacae ubi contusae. Latex albus.Basidiosporae subglobosae ad late ellipsoideae, 7.1–8.3–9.6 (10.1)×6.5–7.3–8.2 μm, Q=1.07–1.14–1.24, completeet dense reticulatae, verrucis interconnectibus 0.5–0.9 μmaltis ornatae, macula suprahilaris centrale ad completeamyloidea. Macrocystidia 70–110×5–7 μm, abundantia,subcylindricata, emergentia. Pleuropseudocystidia nonemergentia. Pileipellis bistrata, 60–120 μm crassa; ele-menta suprapellis 25–70×2–6 μm, longa, gracile conica,crassitunicata.

Typus: “Australia, state of Tasmania, Growling Swallet,17 February 2005, G. Gates & D. Ratkowsky 17-02-05(GENT)”.

Pileus 8–18 mm broad, conchiform when young,becoming flabelliform; margin entire and straight, becom-ing undulating with age; surface smooth to faintly andconcentrically rugulose, dry, glabrous or minutely veluti-nous, white, bruising orange brown. Stipe about 2×2 mm,reduced, white. Lamellae up to 1.5 mm broad, often forkednear pileus margin, white, bruising orange; lamellulae of 2


lengths. Smell tinny; taste neutral. Latex white. Basidio-spores 7.1–8.3–9.6 (10.1)×6.5–7.3–8.2 μm, Q=1.07–1.14–1.24 (n=20), subglobose to broadly ellipsoid; ornamentationa closed and very dense reticulum composed of broad andblunt warts up to 0.5–0.9 μm high, interconnected by lowerand thinner ridges; plage centrally to completely amyloid.Basidia 40–55×10–13 μm, clavate, 4-spored, few 3-spored;sterigmata 6–10×1.5–2 μm. Macrocystidia 70–110×5–7 μm, very abundant and strongly emergent, long andnarrowly subcylindrical with gradually tapering apex, thin orrefringent cell wall, minutely granular contents. Pleuro-pseudocystidia not emergent. Hymenophoral trama com-posed of inflated hyphae and sphaerocytes; lactifers ratherabundant. Pileipellis a palisade, 60–120 μm thick; supra-pellis composed of long and narrowly conical terminalelements, 25–70×2–6 μm, rarely septate, tapering towardsapex, erect to recumbent or somewhat repent, thick-walled;subpellis composed of 3–4 layers of sphaerocytes 15–20×10–15 μm, thin-walled.

Habitat: on wood.Known distribution: Tasmania.Material examined: Australia—state of Tasmania,

Growling Swallet, 17 February 2005, G. Gates & D.Ratkowsky 17-02-05 (typus: GENT).

Notes: With L. conchatulus and L. genevievae thenumber of pleurotoid Lactarius species has grown to six.

The three Australasian species, L. uyedae, L. conchatulusand L. genevievae are characterized by the white pileus,abundant macrocystidia and absence of a subiculum. Thismorphological similarity agrees with the outcome of themolecular analysis which unites them in a monophyleticgroup. Their placement in subg. Gerardii is remarkablesince this subgenus has an otherwise conserved, agaricoidhabitus with dark and brownish pigmentation. Moreover,macrocystidia and thickened cell walls are rare in thissubgenus though they occur also in L. ochrogalactus. Thethree remaining pleurotoid species, L. panuoides Singer, L.brunellus S.L. Mill., Aime & T.W. Henkel and L. multicepsS.L. Mill., Aime & T.W. Henkel, are Latin Americanspecies, and have been reported from Guyana, Guadeloupe,Trinidad and Brazil (Miller et al. 2002; Singer 1984). Theyare orange to brown coloured, have a subiculum and lackmacrocystidia. Two additional pleurotoid species weredescribed but L. campinensis Singer was later transferredto the genus Russula (Henkel et al. 2000) and L. igapoensis

Fig. 10 Lactarius genevievae (G. Gates & D. Ratkowsky 17-02-05, typus): a basidiospores; b basidia; c macrocystidia; d pileipellis. (scale bar:10 μm)

Fig. 11 a, b Lactarius limbatus (D. Stubbe 06-230, typus); . Lactariusconiculus (D. Stubbe 07-496, typus); d Lactarius coniculus (D. Stubbe07-497); e Lactarius bicolor (D. Stubbe 06-229, epitypus); f Lactariusleae (D. Stubbe 07-426); g, h Lactarius hora (D. Stubbe 07-502,typus); i Lactarius genovievae (G. Gates & D. Ratkowsky 17-02-05,typus) (photo by G. Gates); j Lactarius leonardii (photo by G. Gates);k Lactarius atrovelutinus (D. Stubbe 06-003) (photo by R. Walleyn)

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Singer was ultimately synonymized with L. panuoides(Miller et al. 2002). Phylogenetic analyses of the availableLSU sequence for L. panuoides place the species close to L.clarkeae Cleland, excluding it from subg. Gerardii. It isevident that morphology alone is insufficient to determinethe phylogenetic position of these small, pleurotoid species.It appears that pleurotoid milkcaps have developed at leasttwice, independently from one another on each side of thePacific Ocean.

General discussion

Lactarius subg. Gerardii contains many similar species. Itis therefore essential that detailed observations are madewhen identifying or describing specimens. On freshbasidiocarps, accurate observations of initial latex colourand subsequent discolorations of context and of bruisedlamellae provide valuable information. The colour reactioncaused by ferrous sulphate on the context has not beentested systematically, but might give additional insights.Microscopy is indispensable. Basidiospore characteristicsshould be observed in detail, especially basidiosporeornamentation (density of the reticulum, height and shapeof ridges and warts, etc.). Dimensions of the terminalelements in pellis structures also allow one to distinguishseveral species. Macrocystidia are only present in thepleurotoid clade, where their shape is species specific, andin the clade containing L. leonardii, L. ochrogalactus and L.petersenii.

If the proposal to conserve the genus name Lactarius for/Lactarius 1 and use Lactifluus for /Lactarius 2 is accepted,then all members of subg. Gerardii will be transferred toLactifluus.

With the new species presented in this paper, the totalnumber of described species that with certainty belongsto subg. Gerardii amounts to 16: L. gerardii, L. peterseniiand L. subgerardii from North America, L. ochrogalactus,L. uyedae, L. atrovelutinus, L. leae, L. conchatulus, L.bicolor, L. limbatus, L. hora, L. coniculus and L.reticulatovenosus from Asia and L. wirrabara, L. leonardiiand L. genevievae from Australia. The morphology ofanother eight species implies a taxonomic position insubg. Gerardii, but they still need confirmation bymolecular phylogenetic analyses: L. atro-olivaceus, L.pseudogerardii, L. louisii, L. subtomentosus, L. subisabel-linus var. murrillianus and L. xanthydrorheus from NorthAmerica and L. venosus from Papua New Guinea and L.sepiaceus from New Zealand (Stubbe et al. 2010). Most ofthese species are poorly known and additional samplingand observations would be useful. No doubt, with furtherexploration of poorly studied regions, additional newspecies will be discovered. Lactarius brunellus and L.

multiceps, two small pleurotoid species from SouthAmerica resembling L. uyedae, also need to be testedalthough L. brunellus, with its spiny basidiospore orna-mentation lacking a reticulum, is a less likely candidate forsubg. Gerardii (Stubbe et al. 2010). Apart from well-defined species, the subgenus also harbours many speciescomplexes—which are difficult to delimit, molecularly ormorphologically—and cryptic species—which cannot bedistinguished from each other morphologically.

Acknowledgements We are grateful to following persons for theirhelp during fieldwork: Ruben Walleyn, Samantha Karunarathna,Phongeun Sysouphanthong, Ruilin Zhao, Nopparat Wannathes, NilamWulandari, Thida Win Ko Ko, Dennis Desjardin, Else Vellinga, JeanWeber and Khadijah Rambe. We are indebted to Dr. SaisamornLumyong of Chiang Mai University (Thailand) to provide a MaterialTransfer Agreement and we thank Dr. Kevin Hyde for arranging ourstay at the Mushroom Research Centre. For allowing research inMalaysia we are grateful to the Economic Planning Unit and to Dr.Lee Su See. We would like to thank Ursula Eberhardt for valuableadvice on the molecular work. The Malaysian expedition was fundedby the Foundation of Scientific Research-Flanders (FWO-Vlaanderen).We thank the King Leopold III Foundation for financial support for theexpedition in Sri Lanka. This paper would not have been possible withoutmany herbaria and people providing specimens: Kanad Das (BSD),Dennis Desjardin (SFSU), Lee Su See (KEP), Roy Watling (E), David P.Lewis, Eiji Nagasawa (TMI), Roy Halling (NY), Christen Mortensen,Gregory Mueller (CHIC), Leticia Montoya, Josephine Milne (MEL)and Kew Herbarium (K). We also thank Genevieve Gates andPatrick Leonard for providing specimens, photographs and helpfuldiscussions. Dirk Stubbe was funded by a scholarship of theInstitute for the Promotion of Innovation through Science andTechnology (IWT-Vlaanderen). A PEET-grant of the NationalScience Foundation (USA) (DEB-0118776) to Prof. DennisDesjardin and a scholarship of the Chiang Mai University madeit possible for Huyen Than Le to conduct her research in Thailand.Xiang-Hua Wang was funded by National Natural ScienceFoundation of China (NSFC 30970020, 30300002).

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The Australasian species of Lactarius subgenus Gerardii ( Russulales )

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