Lactarius volemus sensu lato (Russulales) from northern Thailand: morphological and phylogenetic...

Lactarius volemus sensu lato (Russulales) from northernThailand: morphological and phylogenetic speciesconcepts explored

Kobeke Van de Putte & Jorinde Nuytinck & Dirk Stubbe &

Huyen Thanh Le & Annemieke Verbeken

Received: 1 June 2010 /Accepted: 18 October 2010 /Published online: 20 November 2010# Kevin D. Hyde 2010

Abstract Lactarius volemus (Fr.: Fr.) Fr. is a well knownand morphologically easily recognizable milkcap of theNorthern hemisphere, forming ectomycorrhiza with bothdeciduous and coniferous trees. It was originally describedfrom Europe, but is also reported in other continents.Although it is characterized by several unique macro- andmicromorphological features, substantial variation in col-our, lamellae spacing and changing and staining of the latexhas been recorded and it is therefore considered as aputatively unresolved species complex. This study exploresthe concordance between morphological and phylogeneticspecies concepts within L. volemus sensu lato of northernThailand, combining a critical morphological scrutiny witha multiple gene genealogy based on LSU, ITS and rpb2nuclear sequences. Twelve strongly supported monophyleticclades and six terminal branches are discernable in allphylogenetic trees and represent 18 phylogenetic species. Sixof the monophyletic clades can be morphologically distin-guished and are described as new species: L. acicularis, L.crocatus, L. distantifolius, L. longipilus, L. pinguis and L.vitellinus. Five other clades also show some morphologicaldifferences, but these are too subtle and do not allow for aclear-cut species delimitation without the corroboration ofmolecular data. Lactarius volemus sensu lato of northern

Thailand is therefore still considered as a partially crypticspecies complex. Pleurolamprocystidia, pileipellis hairs andto a lesser degree also pileus colour are important diagnosticcharacteristics. Spore morphology, latex discoloration andpileus surface texture are less useful as diagnostic features.Whether this rich diversity is the result of in situ Pleistocenesurvival or post-glacial expansion and subsequent radiation,has yet to be revealed.

Keywords Russulaceae . Cryptic species . Speciescomplex .Morphology .Multiple gene phylogeny

Introduction

Fungal species have been mainly diagnosed by theirmorphological characteristics up to the middle of the 20thcentury. Starting from the 1960s and 1970s, the use ofpopulation tools and DNA-based methods ushered in a newera of species delimitation. Numerous reproductively andgenetically isolated clusters within many of the traditionalmorphological species were now revealed (Brasier 1997).This kind of previously overlooked cryptic speciationseems to be a general rather than an exceptional case infungi and as surveys of DNA sequence variation are on therise, so are the records of cryptic species (e.g. Dettman etal. 2006; Hallenberg et al. 2007; Hedh et al. 2008;Kauserud et al. 2006, 2007; Le Gac et al. 2007; Matute etal. 2006; Steenkamp et al. 2002; Stubbe et al. 2010).

Lactarius volemus (Fr.: Fr.) Fr. is such a traditionalmorphological species. It was first described from Sweden(Fries 1838), but since then, the name has been applied formorphological look-a-likes in Guatemala, Mexico, easternNorth America and Asia. It can be found from June toNovember, forming ectomycorrhiza with various host trees

K. Van de Putte (*) : J. Nuytinck :D. Stubbe :A. VerbekenResearch group Mycology, Department of Biology,Ghent University,K.L. Ledeganckstraat 35,9000 Ghent, Belgiume-mail: [email protected]

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

Fungal Diversity (2010) 45:99–130DOI 10.1007/s13225-010-0070-0

including Carpinus, Castanea, Castanopsis, Dipterocarpus,Fagus, Lithocarpus, Quercus, Picea and Pinus (Adhikari etal. 2005; Arora 1986; Heilmann-Clausen et al. 1998; Heslerand Smith 1979; Montoya et al. 1996; Verbeken et al. 1996;Wang 2007).

Being renowned for its delectable taste, L. volemus alsoranks among the best edible mushrooms of the Northernhemisphere. It is sold commercially mainly in Asianmarkets, but also in Europe and North America (Boa2004; Christensen et al. 2008; Colak et al. 2009; Dell et al.2005; Garibay-Orijel et al. 2006, 2007; Wang et al. 2004).

Lactarius volemus can easily be recognized by its dry,often velvety, clay-buff to orange-brown, reddish-brownpileus, greenish reaction with FeSO4, fishy smell andextremely abundant white latex. The latex is unchangingwhen isolated, but when it stays in contact with the flesh, itstains context and lamellae brown while usually slowlybecoming brown itself.

Furthermore, L. volemus exhibits several remarkablemicroscopical features. The key diagnostics are the abun-dant lamprocystidia (cystidia with a distinctly thickenedwall), the pilei- and stipitipellis with a lampropalisadestructure (a layer of anticline, thick-walled and rather longterminal elements on top of a sublayer of rounded cells) andthe presence of numerous rosettes of sphaerocytes in thetrama of the lamellae. The subglobose to globose spores areornamented with amyloid ridges which form a complete oralmost complete reticulate pattern.

Although L. volemus is morphologically well-characterized,a substantial amount of variation in colour, lamellae spacingand habit has been recorded and it is therefore considered as aputatively unresolved worldwide species complex (Le 2007;Shimono et al. 2007; Wang 2007). This variation has led tothe description of ten different varieties to which, in general,no taxonomical importance seems to be attributed (Bouchet1959; Dörfelt et al. 2004; Fries 1838; Heim 1962; Hesler andSmith 1979; Maire 1937; Neuhoff 1956; Peck 1885).Moreover, Wang (2007) endorses the occurrence of manymorphologically intermediary specimens, making morpho-logical species boundaries hard to assess.

Up to now, one variety of L. volemus has been originallydiagnosed from Asia: L. volemus var. asiaticus Dörfelt, Kiet& Berg, characterized by small, matt brown and distinctlyvelvety basidiocarps and small spores (Dörfelt et al. 2004).

Lactarius volemus seems to be not only morphologically,but also genetically very divergent. Recently, Shimono etal. (2007) examined the L. volemus species complex inJapan, where three macromorphological variants of thecommon brown coloured L. volemus have been observed,i.e., a variant with red pileus, one with a yellow pileus andlong stipe, and one with a conspicuously velvety surfacetexture. Using nuclear LSU sequence data, they distin-guished four genetically isolated clades. Since cap colour

supported the delimitation of these clades, they suggested thatthis can be a reliable diagnostic character. Additionally, theymentioned that microscopical characters, taste and fatty acidcomposition of the four groups differed from one another.

In recent years, several mycological studies have beenconducted in northern Thailand, and more specifically inthe surrounding areas of the Mushroom Research Centre(Le et al. 2007a; Karunarathna et al. 2010; Kerekes andDesjardin 2009; Sanmee et al. 2008; Wannathes et al.2009). In this region, the current knowledge of the genusLactarius is mainly based on the work of Le (2007) and Leet al. (2007a, b, c). The L. volemus group is abundantlypresent in the mixed forests (dominated by Dipterocarpa-ceae, Fagaceae and Pinaceae) of northern Thailand. Basedon ITS sequences, Le (2007) distinguished at least fourclades, which also differed in pileus colour, length ofterminal pilei- and stipitipellis hairs and length of lamp-rocystidia. However, due to very limited molecular data set,no taxonomical changes were undertaken. An exhaustivemorphological and multi-locus phylogenetic study of thisgroup in northern Thailand is therefore imperative.

This paper aims to answer the following questions: (1)how many phylogenetic species can be distinguished innorthern Thailand, (2) which DNA regions are the mostsuitable for species delimitation (3) are these phylogeneticspecies cryptic, and if not, (4) which morphologicalfeatures are diagnostic?

Materials and methods

Sampling

This study is based on 79 collections of L. volemus sensu lato,mainly from Chiang Mai Province. Some collections fromChiang Rai and Mae Hong Son Province were also included.Collection sites are listed with details in Table 1. The studiedmaterial is deposited in the Herbarium of Chiang MaiUniversity (CMU), the Herbarium Universitatis Gandavensis(GENT), the Herbarium of Mae Fah Luang University(MFLU) and the Herbarium of San Francisco State University(SFSU). Six collections of L. volemus from North America,nine collections from Europe and one collection from Chinawere also included in the analyses (Table 1).

DNA extraction

Two methods were used for DNA extraction:

(1) DNA was extracted from fresh material using aCTAB-based method as previously described byNuytinck and Verbeken (2003). Pellets were dissolvedin 100 μl ultrapure water.

100 Fungal Diversity (2010) 45:99–130

Tab

le1

collections

used

formolecular

analysis

Species

Originalidentification

Vou

cher

collection

Origin

GenBank

accession

no.ITS

GenBank

accession

no.LSU

GenBank

accession

no.rpb2

Multifurca

M.furcata

R.Hallin

g78

04COSTA

RICA

DQ42

1994

DQ42

1927

M.ochricom

pacta

B.Buyck

2107

USA

DQ42

1984

DQ42

1940

M.zona

ria

D.E.Desjardin

7442

THAILAND

DQ42

1990

DQ42

1942

Lactarius

L.atrovelutin

usD.Stub

be06

-003

(GENT)

MALAYSIA

GU26

5588

GU25

8231

GU25

8325

L.aff.gerardii

E.Nag

asaw

a,TMI15

558(TMI)

JAPA

NGU26

5587

GU25

8230

GU25

8324

L.aff.hygrop

horoides

A.Verbeken/R.Walleyn

04-091

(=H.T.Le13

7)(CMU,GENT,

MFLU,SFSU)

THAILAND

HQ31

8209

HQ32

8937

L.clarkeae

M.Noo

rdeloo

s20

0400

2TA

SMANIA

HQ31

8205

HQ31

8282

HQ32

8933

L.clarkeae

M.Noo

rdeloo

s20

0401

8TA

SMANIA

HQ31

8206

HQ31

8283

HQ32

8934

L.clarkeae

M.Noo

rdeloo

s20

0412

2TA

SMANIA

HQ31

8207

HQ31

8284

HQ32

8935

L.gerardiis.l.

A.Verbeken

05-375

(GENT)

USA

GU26

5616

GU25

8254

GU25

8353

L.glau

cescens

A.Verbeken

04-202

(GENT)

USA

HQ31

8203

HQ31

8280

HQ32

8932

L.hygrop

horoides

A.Verbeken

05-251

(GENT)

USA

HQ31

8208

HQ31

8285

HQ32

8936

L.piperatus

H.T.Le51

(CMU,GENT,

MFLU,SFSU)

THAILAND

HQ31

8204

HQ31

8281

L.pseudo

luteop

usA.Verbeken/R.Walleyn

04-129

(=H.T.Le15

5)(CMU,GENT,

MFLU,SFSU)

THAILAND

HQ31

8210

HQ31

8286

HQ32

8938

Lactarius

volemus

sensulato

L.acicularissp.no

v.L.aff.volemus

H.T.Le19

9(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8170

HQ31

8257

HQ32

8906

L.acicularissp.no

v.L.aff.volemus

H.T.Le26

5(CMU,GENT,

MFLU,SFSU)

TH-K

CHQ31

8196

HQ31

8277

HQ32

8926

L.acicularissp.no

v.L.volemus

H.T.Le27

7(CMU,GENT,

MFLU,SFSU)

TH-D

SHQ31

8183

HQ32

8919

L.acicularissp.no

v.L.volemus

H.T.Le37

0(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8187

HQ31

8270

HQ32

8922

L.acicularissp.no

v.L.aff.volemus

D.Stub

be07

-456

(CMU,GENT)

TH-H

W1

HQ31

8125

HQ31

8224

HQ32

8867

L.acicularissp.no

v.L.volemus

K.Vande

Putte

08-002

(GENT,

MFLU)

TH-H

W1

HQ31

8132

HQ31

8226

HQ32

8869

L.acicularissp.no

v.L.volemus

K.Vande

Putte

08-007

(GENT,

MFLU)

TH-PT

HQ31

8137

HQ31

8230

HQ32

8874

L.acicularissp.no

v.L.volemus

K.Vande

Putte

08-029

(GENT,

MFLU)

TH-PT

HQ31

8147

HQ31

8239

HQ32

8884

L.acicularissp.no

v.L.volemus

K.Vande

Putte

08-033

(GENT,

MFLU)

TH-D

IHQ31

8150

HQ31

8242

HQ32

8887

L.crocatus

sp.no

v.L.volemus

H.T.Le17

(CMU,GENT,

MFLU,SFSU)

TH-M

RC

HQ31

8186

HQ32

8921

L.crocatus

sp.no

v.L.aff.volemus

H.T.Le20

2(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8157

HQ31

8248

L.crocatus

sp.no

v.L.aff.volemus

var.fla

vus

H.T.Le22

2(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8141

HQ32

8878

L.crocatus

sp.no

v.L.aff.volemus

H.T.Le22

3(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8160

HQ32

8896

L.crocatus

sp.no

v.L.aff.volemus

H.T.Le22

4(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8200

HQ32

8930

L.crocatus

sp.no

v.L.aff.volemus

H.T.Le22

5(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8172

HQ32

8908

L.crocatus

sp.no

v.L.aff.volemus

H.T.Le23

4(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8161

Fungal Diversity (2010) 45:99–130 101

Tab

le1

(con

tinued)

Species


Vou

cher

collection

Origin

GenBank

accession

no.ITS

GenBank

accession

no.LSU

GenBank

accession

no.rpb2

L.crocatus

sp.no

v.L.volemus

H.T.Le24

5(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8142

HQ31

8234

HQ32

8879

L.crocatus

sp.no

v.L.volemus

H.T.Le26

6(CMU,GENT,

MFLU,SFSU)

TH-K

CHQ31

8180

HQ31

826

HQ32

8916

L.crocatus

sp.no

v.L.volemus

H.T.Le26

8(CMU,GENT,

MFLU,SFSU)

TH-K

CHQ31

8181

HQ31

8266

HQ32

8917

L.crocatus

sp.no

v.L.volemus

K.Vande

Putte

08-034

(GENT,

MFLU)

TH-D

IHQ31

8151

HQ31

8243

HQ32

8888

L.crocatus

sp.no

v.L.volemus

K.Vande

Putte

08-035

(GENT,

MFLU)

TH-D

IHQ31

8152

HQ32

8889

L.crocatus

sp.no

v.L.volemus

K.Vande

Putte

08-036

(GENT,

MFLU)

TH-D

IHQ31

8153

HQ31

8244

HQ32

8890

L.crocatus

sp.no

v.L.volemus

K.Vande

Putte

08-042

(GENT,

MFLU)

TH-PT

HQ31

8155

HQ31

8246

HQ32

8892

L.distan

tifoliussp.no

v.L.aff.volemus

H.T.Le28

8(CMU,GENT,

MFLU,SFSU)

TH-H

W2

HQ31

8193

HQ31

8274

L.distan

tifoliussp.no

v.L.aff.volemus

H.T.Le28

9(CMU,GENT,

MFLU,SFSU)

TH-H

W2

HQ31

8192

L.distan

tifoliussp.no

v.L.volemus

D.Stub

be07

-461

(CMU,GENT)

TH-H

RT

HQ31

8124

HQ31

8223

HQ32

8866

L.long

ipilu

ssp.no

v.L.volemus

H.T.Le18

4(CMU,GENT,

MFLU,SFSU)

TH-D

IHQ31

8169

HQ31

8256

HQ32

8905

L.long

ipilu

ssp.no

v.L.aff.volemus

H.T.Le20

6(CMU,GENT,

MFLU,SFSU)

TH-D

IHQ31

8171

HQ31

8258

HQ32

8907

L.long

ipilu

ssp.no

v.L.corrug

isH.T.Le27

3(CMU,GENT,

MFLU,SFSU)

TH-TJ

HQ31

8195

HQ31

8276

HQ32

8925

L.long

ipilu

ssp.no

v.L.aff.volemus


04-160

(=H.T.Le16

8)(CMU,GENT,

MFLU,SFSU)

TH-TJ

HQ31

8143

HQ31

8235

HQ32

8880

L.ping

uissp.no

v.L.volemus

var.volemus

H.T.Le25

5(CMU,GENT,

MFLU,SFSU)

TH-M

RC

HQ31

8178

HQ31

8263

HQ32

8914

L.ping

uissp.no

v.L.aff.volemus

var.volemus

H.T.Le27

8(CMU,GENT,

MFLU,SFSU)

TH-D

SHQ31

8184

HQ31

8268

L.ping

uissp.no

v.L.volemus


04-023

(=H.T.Le117)

(CMU,GENT,

MFLU,SFSU)

TH-TJ

HG31

8111

HQ31

8211

HQ32

8858

L.ping

uissp.no

v.L.volemus


04-162

(=H.T.Le16

9)(CMU,GENT,

MFLU,SFSU)

TH-TJ

HQ31

8121

HQ31

8221

HQ32

8863

L.vitellinu

ssp.no

v.L.volemus

var.volemus

H.T.Le26

9(CMU,GENT,

MFLU,SFSU)

TH-K

CHQ31

8182

HQ31

8267

HQ32

8918

L.vitellinu

ssp.no

v.L.aff.volemus

H.T.Le34

8(CMU,GENT,

MFLU,SFSU)

TH-TJ

HQ31

8164

HQ31

8251

HQ32

8900

L.vitellinu

ssp.no

v.L.aff.volemus

var.fla

vus

K.Vande

Putte

08-024

(GENT,

MFLU)

TH-TJ

HQ31

8144

HQ31

8236

HQ32

8881

L.vitellinu

ssp.no

v.L.aff.volemus

var.fla

vus

K.Vande

Putte

08-025

(GENT,

MFLU)

TH-TJ

HQ31

8145

HQ31

8237

HQ32

8882

L.volemus

sensulato

L.volemus

D.E.Desjardin

7577

(CMU,GENT,

SFSU)

TH-D

IHQ31

8188

L.volemus

sensulato

L.volemus

var.volemus

H.T.Le24

7(CMU,GENT,

MFLU,SFSU)

TH-H

KB

HQ31

8175

HQ31

8261

HQ32

8911

L.volemus

sensulato

L.volemus

var.volemus

H.T.Le24

9(CMU,GENT,

MFLU,SFSU)

TH-H

KB

HQ31

8176

HQ32

8912

L.volemus

sensulato

L.aff.volemus

H.T.Le25

0(CMU,GENT,

MFLU,SFSU)

TH-H

KB

HQ31

8162

HQ32

8898

L.volemus

sensulato

L.volemus

H.T.Le22

8(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8198

HQ32

8928

L.volemus

sensulato

L.volemus

var.volemus

H.T.Le23

0(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8174

HQ31

8260

HQ32

8910

L.volemus

sensulato

L.volemus

K.Vande

Putte

08-008

(GENT,

MFLU)

TH-PT

HQ31

8138

HQ31

8231

HQ32

8875

species1

L.volemus

H.T.Le35

9(CMU,GENT,

MFLU,SFSU)

TH-M

HS

HQ31

8168

HQ31

8255

HQ32

8904

species2

L.volemus

M.Härkönen,

KIINA15

8(G

ENT)

CHIN

AHQ31

8126

HQ31

8225

species2

L.aff.volemus

D.Stub

be07

-468

(CMU,GENT)

TH-H

RT

HQ31

8123

HQ32

8865


Tab

le1

(con

tinued)

Species


Vou

cher

collection

Origin

GenBank

accession

no.ITS

GenBank

accession

no.LSU

GenBank

accession

no.rpb2

species3

L.volemus

H.T.Le22

9(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8173

HQ31

8259

HQ32

8909

species3

L.aff.volemus

H.T.Le23

2(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ32

8901

species3

L.aff.volemus

var.volemus

H.T.Le28

4(CMU,GENT,

MFLU,SFSU)

TH-PT

HQ31

8166

HQ31

8253

HQ32

8903

species3

L.volemus

K.Va

nde

Putte

08-026

(GENT,

MFLU)

TH-PT

HQ31

8146

HQ31

8238

HQ32

8883

species3

L.volemus

K.Va

nde

Putte

08-031

(GENT,

MFLU)

TH-PT

HQ31

8148

HQ31

8240

HQ32

8885

species3

L.volemus

K.Va

nde

Putte

08-039

(GENT,

MFLU)

TH-PT

HQ31

8154

HQ31

8245

HQ32

8891

species3

L.volemus

K.Va

nde

Putte

08-043

(GENT,

MFLU)

TH-PT

HQ31

8156

HQ31

8247

HQ32

8893

species4

L.aff.volemus

var.volemus

H.T.Le21

9(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8167

HQ31

8254

species4

L.volemus

K.Va

nde

Putte

08-011

(GENT,

MFLU)

TH-PT

HQ31

8139

HQ31

8232

HQ32

8876

species4

L.volemus

K.Va

nde

Putte

08-032

(GENT,

MFLU)

TH-PT

HQ31

8149

HQ31

8241

HQ32

8886

species4

L.volemus

var.volemus

H.T.Le23

1(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8197

HQ31

8278

HQ32

8927

species4

L.volemus

var.volemus

H.T.Le26

4(CMU,GENT,

MFLU,SFSU)

TH-K

CHQ31

8179

HQ31

8264

HQ32

8915

species4

L.volemus

H.T.Le28

5(CMU,GENT,

MFLU,SFSU)

TH-PT

HQ31

8185

HQ31

8269

HQ32

8920

species4

L.volemus

K.Va

nde

Putte

08-004

(GENT,

MFLU)

TH-PT

HQ31

8134

HQ31

8228

HQ32

8871

species4

L.volemus

K.Va

nde

Putte

08-005

(GENT,

MFLU)

TH-PT

HQ31

8135

HQ32

8872

species5

L.aff.volemus

H.T.Le31

3(CMU,GENT,

MFLU,SFSU)

TH-H

W1

HQ31

8190

HQ31

8272

species6

L.aff.volemus

H.T.Le29

4(CMU,GENT,

MFLU,SFSU)

TH-SR

HQ31

8191

HQ31

8273

HQ32

8923

species8

L.aff.volemus

var.volemus

H.T.Le17

0(CMU,GENT,

MFLU,SFSU)

TH-D

IHQ31

8165

HQ31

8252

HQ32

8902

species8

L.volemus

K.Va

nde

Putte

08-021

(GENT,

MFLU)

TH-PT

HQ31

8140

HQ31

8233

HQ32

8877

species8

L.aff.volemus


04-060

(=H.T.Le12

3)(CMU,GENT,

MFLU,SFSU)

TH-PT

HQ31

8122

HQ31

8222

HQ32

8864

species11

L.aff.volemus

H.T.Le32

1(CMU,GENT,

MFLU,SFSU)

TH-D

IGU26

5662

GU25

8302

GU25

8402

species11

L.aff.volemus

H.T.Le33

7(CMU,GENT,

MFLU,SFSU)

TH-D

SU

HQ31

8189

HQ31

8271

species11

L.aff.volemus

D.Stub

be07

-402

(CMU,GENT)

TH-D

IHQ32

8897

species11

L.aff.volemus


04-087

(=H.T.Le13

3)(CMU,GENT,

MFLU,SFSU)

TH-D

SU

HQ31

8112

HQ31

8212

HQ32

8859

species12

L.volemus

var.volemus

H.T.Le25

1(CMU,GENT,

MFLU,SFSU)

TH-H

KB

HQ31

8177

HQ31

8262

HQ32

8913

species14

L.aff.volemus

K.Va

nde

Putte

08-006

(GENT,

MFLU)

TH-PT

HQ31

8136

HQ31

8229

HQ32

8873

species16

L.aff.volemus

H.T.Le27

5(CMU,GENT,

MFLU,SFSU)

TH-K

CHQ31

8194

HQ31

8275

HQ32

8924

species17

L.aff.volemus

H.T.Le19

(CMU,GENT,

MFLU,SFSU)

TH-TJ

HQ31

8199

HQ31

8279

HQ32

8929

species17

L.volemus

H.T.Le21

4(CMU,GENT,

MFLU,SFSU)

TH-TJ

HQ31

8158

HQ31

8249

HQ32

8894

species17

L.aff.volemus

var.fla

vus

H.T.Le33

3(CMU,GENT,

MFLU,SFSU)

TH-H

ND

HQ31

8163

HQ31

8250

HQ32

8899

species17

L.aff.volemus

var.fla

vus

K.Va

nde

Putte

08-023

(GENT,

MFLU)

TH-TJ

HQ31

8133

HQ31

8227

HQ32

8870

species17

L.aff.volemus


04-089

(=H.T.Le13

9)(CMU,GENT,

MFLU,SFSU)

TH-D

SU

HQ31

8159

HQ32

8895

Lactarius

volemus

sensulato

from

Europ

e

L.volemus

sensulato

L.volemus

J.Vauras

5100

F(G

ENT)

FIN

LAND

HQ31

8115

HQ31

8215

HQ32

8860

L.volemus

sensulato

L.volemus

A.Verbeken

97-510

(GENT)

FRANCE

HQ31

8117

HQ31

8217

L.volemus

sensulato

L.volemus

A.Verbeken

97-512

(GENT)

FRANCE

HQ31

8113

HQ31

8213

L.volemus

sensulato

L.volemus

R.Walleyn

1228

(GENT)

FRANCE

HQ31

8116

AY60

6959

AY60

6966


Tab

le1

(con

tinued)

Species


Vou

cher

collection

Origin

GenBank

accession

no.ITS

GenBank

accession

no.LSU

GenBank

accession

no.rpb2

L.volemus

sensulato

L.volemus

B.Buyck

2620

(GENT)

GERMANY

HQ31

8114

HQ31

8214

L.volemus

sensulato

L.volemus

B.Buyck

2699

(GENT)

GERMANY

HQ31

8119

HQ31

8219

L.volemus

sensulato

L.volemus

SJS/PC/AL(G

ENT)

SCOTLAND

HQ31

8120

HQ31

8220

HQ32

8862

L.volemus

sensulato

L.volemus

A.Verbeken

07-82(G

ENT)

SLOVENIA

HQ31

8118

HQ31

8218

HQ32

8861

Lactarius

volemus

sensulato

from

North

America

L.volemus

sensulato

L.volemus

A.Verbeken

04-165

(GENT)

USA

HQ31

8131

L.volemus

sensulato

L.volemus

A.Verbeken

04-173

(GENT)

USA

HQ31

8128

L.volemus

sensulato

L.corrug

isA.Verbeken

04-185

(GENT)

USA

HQ31

8202

L.volemus

sensulato

L.volemus

A.Verbeken

04-194

(GENT)

USA

HQ31

813

HQ32

8868

L.volemus

sensulato

L.aff.corrug

isA.Verbeken

05-252

(GENT)

USA

HQ31

8201

HQ32

8931

L.volemus

sensulato

L.volemus

A.Verbeken

05-330

(GENT)

USA

HQ31

8129

L.volemus

sensulato

L.volemus

A.Verbeken

05-384

(GENT)

USA

HQ31

8127

L.volemus

sensulato

L.volemus

var.volemus

A.Verbeken

05-394

(GENT)

USA

GU26

5660

GU25

8300

GU25

8400

TH-D

IThailand

,ChiangMai

Province,

Doi

Inthanon

NationalPark,

junctio

nof

Highw

ay10

09androad

toMae

Chem,N19

°31.58

′E98

°29.64

′,alt.17

00m,hu

mid

mon

tane

rainforest

with

Castano

psissp.,Lith

ocarpu

sechino

ps,Quercus

sp.

TH-D

SThailand

,ChiangMai

Province,

Doi

SaKet

district,N18

°54′31

.5″E99

°12′57

.0″,alt.57

4m,rainforestdo

minated

byCastano

psisspp.

TH-D

SU

Thailand

,ChiangMai

Province,

Mae

Taeng

district,Doi

Suthep-Pui

NationalPerk,

SangasahasriLaneto

HuaiKok

Mavillage,N18

°48.62

′E98

°54.60

′,alt.1150

m,rainforest

with

Castano

psisspp.,Lith

ocarpu

spo

lystachyus

TH-H

KBThailand

,ChiangMai

Province,

Mae

Taeng

district,HuaiKom

Bang,

alt.70

0m,foresttrail,rain

forest

TH-H

ND

Thailand

,ChiangMai

Province,

HuaiNam

DangNationalPark,

nature

trail,N19

°18.29

′E98

°35.88

′,alt.15

30m,forestwith

Pinus

kesiya,Dipterocarpus

sp.,Bam

booandscattered

Castano

psisspp.

TH-H

RTThailand

,ChiangMaiProvince,Mae

MaLai,H

uaiR

aiTem

ple,N19

°06.17

0′E98

°53.36

3′,alt.

515m,inteak

plantatio

nwith

Dipterocarpus

obtusifoliu

s,Dipterocarpus

sp.,Sh

orea

spp.,

Pterocarpus

macrocarpus

TH-H

W1Thailand

,ChiangMai

Province,

Mae

Taeng

district,Ban

Mae

Sae

Highw

ay10

95near

50km

marker,N19

°14.59

9′E98

°39.45

6′,alt.10

02m,rainforest

with

Castano

psis

armata,

Castano

psissp.,Lith

ocarpu

ssp.,Pinus

kesiya

TH-H

W2Thailand

,ChiangMai

Province,

Mae

Taeng

district,Ban

Mae

Sae,Highw

ay10

95at

22km

marker,N19

°07.57

′E98

°45.65′,alt.75

0m,xericbroad-leaf

forest

(Dipterocarpus

spp.,

Tecton

agran

dis)

with

Pinus

kesiya

TH-K

CThailand

,ChiangRai

Province,

Khu

nChaeNationalPark,

N19

°04′40

.5″E99

°23′54

.3″,alt.96

3m,rainforestdo

minated

byCastano

psisarmata,

Castano

psissp.,Pinus

kesiya

TH-M

HSThailand

,Mae

Hon

gSon

Province,

onHighw

ay10

95near

198km

marker,op

posite

SuanIm

Chay10

0m,alt.28

8m,forestdo

minated

byDipterocarpus

sp.

TH-M

RCThailand

,ChiangMai

Province,Mae

Taeng

district,Ban

Pha

Deng,

Mushroo

mResearchCentre,N19

°17.12

3′E98

°44.00

9′,alt.90

0m,rainforestwith

Castano

psisarmata,

Erythrina

sp.,Dipterocarpus

sp.,Pinus

kesiya

TH-PTThailand

,ChiangMaiProvince,Mae

Taeng

district,B

anPha

Deng,

Pathu

mmikaram

Tem

ple,N19

°06′28

.8″E98

°44′47

.3″,alt.10

50m,rainforestwith

Castano

psisarmata,

Dipterocarpus

sp.,Lith

ocarpu

ssp.

TH-SRThailand

,ChiangMai

Province,

Mae

Taeng

district,Srilann

aNationalPark,

HuayMae

WaNatureTrail,

wet

forestdo

minated

byDipterocarpus

spp.

andBam

boo

TH-TJThailand

,ChiangMai

Province,

Mae

Taeng

district,Tun

gJoaw

,N19

°08′.07′

E98

°38.09

′,alt.14

23m,rainforestwith

Castano

psisspp.,Lith

ocarpu

ssp.,Pinus

kesiya


(2) DNA was extracted from dried material using theprotocol described by Nuytinck and Verbeken(2003),with the following modifications:

– the incubation with extraction buffer and 10% SDSwas prolonged to 1 h at 65°C. Subsequently, 2 μlof 1 M proteinase K (enhances degradation ofproteins and promotes cell lysis, resulting inextracts with a higher DNA concentration) wasadded to each sample and the samples were againincubated overnight at 55°C.

– pellets were dissolved in 100 μl ultrapure water.– pellets were clean enough after this first extraction.

Therefore, no additional extraction with 2x CTABbuffer was performed because this usually resultsin extracts with too low DNA concentrations.

PCR amplification, sequencing and alignments

Protocols for PCR amplification and for sequencing followLe et al. (2007b). Three nuclear loci were amplified andsequenced: (1) the internal transcribed spacer region ofthe ribosomal DNA (ITS), which includes spacer regionsITS1 and ITS2 and the ribosomal gene 5.8S, (2) a part ofthe ribosomal large subunit 28S region (LSU) and (3) theregion between domains 6 and 7 of the second largestsubunit of RNA polymerase II (rpb2). The ITS regionwas amplified using the ITS-1F and ITS-4 primers (Whiteet al. 1990). When amplification failed, ITS was amplifiedin two parts using primer ITS-1F with ITS-2 (White et al.1990) and primer ITS-3 (White et al. 1990) with ITS-4.The LSU region was amplified using primers LR0R andLR5 (http://www.biology.duke.edu/fungi/mycolab/primers.htm), whereas the brpb2-6F and frpb2-7CR (Liu et al.1999; Matheny 2005) primers were used to amplify the rpb2region.

The software Sequencher™ v4.8 (GeneCodes Corpo-ration, Ann Arbor, Michigan, U.S.A.) was used toassemble forward and reverse sequences into contigs,inspect ABI chromatograms and edit nucleotides whereneeded. All sequences were aligned using the on-lineversion of the multiple sequence alignment programMAFFTv6 (Katoh and Toh 2008) with settings FFT-NS-2, FFT-NS-i or L-INS-i. The alignment was furthermanually refined with BioEdit Sequence Alignment Editorv7.0.9.0 (Hall 1999).

To eliminate ambiguously aligned positions in thealignment as objectively as possible, the on-line programGblocks v0.91b (Castresana 2000) was used. Theprogram was run with settings allowing for smallerblocks, gaps within these blocks and less strict flankingpositions. Alignments are available from the first authorby request.

Outgroup choice

A recent molecular phylogeny of the agaricoid Russulaceaeidentified four distinct clades, representing four genera. Anew genus Multifurca Buyck & V. Hofstetter was created,comprising species of former Russula subsect. Ochricom-pactae and Multifurca furcata (Coker) Buyck & V.Hofstetter, a species which was formerly placed inLactarius. The remainder of Russula was shown to bemonophyletic, while the rest of Lactarius was split up intotwo genera: ‘Lactarius 1’ and ‘Lactarius 3’, with theformer comprising L. volemus and allied species (Buyck etal. 2008). A proposal is submitted to conserve the nameLactarius for ‘Lactarius 1’ and to use the name Lactifluusfor ‘Lactarius 3’. We keep referring to all milkcaps as‘Lactarius’ until the proposal is accepted.

We used an outgroup containing three species of Multi-furca. We also included several genetically closely relatedspecies to L. volemus in our analyses: L. hygrophoroidesBerk. & M.A. Curtis, L. pseudoluteopus X.H. Wang &Verbeken, L. clarkeae Cleland, L. piperatus (L.: Fr.) Pers.,L. glaucescens Crossl. and L. gerardii Peck.

Data analysis

LSU, ITS and rpb2 sequence data were analysed bothseparately and in combination. The LSU gene was analysedas a whole. ITS was partitioned into the spacer regionsITS1 and ITS2 and the ribosomal gene 5.8S. The rpb2sequence was divided into four partitions: the rpb2 intronand the first, second and third codon positions of the exon.

The program RAxML v7.0.3 (Stamatakis 2006) wasused to implement the Rapid Bootstrapping algorithm for500 replicates combined with a ML search (Stamatakis etal. 2008). This analysis was performed on all four data sets(LSU, ITS, rpb2 and combined) and will be further referredto as the ML analysis. An additional ML analysis wasperformed on the LSU data set augmented with the 2Chinese and 19 Japanese L. volemus LSU sequences fromShimono et al. (2007) (GenBank accession no. AB238645–AB238665).

Two Bayesian phylogenetic (BI) analyses were carriedout on the combined data set (LSU-ITS-rpb2) in MrBayesv3.1.2 (Ronquist and Huelsenbeck 2003). For the firstanalysis, the model that best fits the data was determinedfor each partition with MrModeltest v2.3 (Nylander 2004)using Akaike’s Information Criterion. This method isreferred to as the BI-MrModeltest method. For the secondanalysis, a single general time-reversible model with ratevariation across sites and a proportion of invariable siteswas used. Rates and all model parameters were unlinkedbetween all partitions. This method is referred to as the BI-GTR+I+Γ method. Five independent, parallel runs of 1 cold


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

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

and 4 heated chains were run for 20 million generations each.Trees and parameter estimates were saved every 1000thgeneration. The temperature was set to 0.3 allowing the chainsto swap more easily from cold to heated, promotingconvergence of the five runs Convergence, associatedlikelihood and effective sample size values of the differentruns were checked visually with Tracer v1.4.1 (Drummondand Rambaut 2007). An appropriate burn-in value wasdetermined with the method proposed by Beiko et al.(2006), in which successive log-likelihood values along theMarkov chain are compared. The first sample of 100 treeswith a mean log-likelihood that is greater than the mean log-likelihood of the last 100 samples of the entire run is markedas the end of the burn-in phase (Beiko et al. 2006).

Phylogenetic species boundaries were identified by ex-ploring the congruence of the three different gene genealogies(Taylor et al. 2000). Clades are considered to be independentevolutionary lineages, and regarded as phylogenetic specieswhen they are well-supported and occur in all singlegenealogies (Pringle et al. 2005). New species weredescribed when morphology and/or ecology substantiatedthis phylogenetic species concept. Phylogenetic specieslacking morphological characteristics were diagnosed ascryptic species belonging to Lactarius volemus sensu lato.

The evolutionary distance within and among the putativephylogenetic species was measured by estimating thenumber of nucleotide substitutions per site. Therefore, theprogram Mega v4.0 (Tamura et al. 2007) was used tocalculate uncorrected p-distances with uniform gamma ratesamong sites and ML estimates of the corrected distancesusing a Kimura-2-parameter (K2P) model and gamma ratesdistribution with the shape parameter estimated by jMo-delTest v0.1.1 (Posada 2008). All positions containing gapsand missing data were eliminated from the data set(Complete deletion option). Standard error estimates (S.E.)were obtained by a bootstrap procedure with 1000replicates. These evolutionary distance values are providedfor comparative purposes with our future studies on the L.volemus species complex.

Morphological approach

Macromorphological characteristics are based on freshmaterials and document all aspects of size, shape, texture,colour, colour changes, latex features, odour and taste. Thecolours were described in daylight conditions using thecolour codes of the Methuen book of colours (Kornerupand Wanscher 1978) and the Munsell Soil Colour Charts(2000). Latex colour and changes were recorded while itexuded from the mushroom, from a drop placed on a glassslide, from a drop on white paper and tissue. Furthermore,the reaction of the context and the latex with chemicalsFeSO4 and KOH (10%) was noted.

Micromorphological characters were documented fromthe exsiccata of mature specimens. For terminology werefer to Vellinga (1988) and specifically for pileipellisstructures to Verbeken (1998). Spores were observed inMelzer’s reagent and all other elements of pileipellis,hymenium and trama were observed in Congo-Red in L4,in Congo-Red in ammonia or in a 10% KOH solution. Foreach collection, a minimum of 20 spores in side view weremeasured excluding the ornamentation. Spore measure-ments are given as described in Nuytinck and Verbeken(2005). Spore measurements and drawings were made witha Zeiss Axioscop 2 microscope and drawing tube at 6000×magnification.

All elements from the hymenium and pileipellis weremeasured halfway the pileus radius. Basidia were measuredexcluding the sterigmata. Pileipellis hairs were measured onscalps. Drawings of the pileipellis, however, were madefrom sections. Hymenial elements and pileipellis structureswere illustrated at 1600× magnification with the aid of adrawing tube attached to an Olympus CX31 microscope.All microscopical observations and drawings were made bythe first author.

Results

Nucleotide alignments

After alignment and application of Gblocks, the ITSalignment included 85 sequences of 556 bases, the LSUalignment 109 sequences of 789 bases and the rpb2alignment 91 sequences of 781 bases. The concatenateddata set included 111 taxa and the program Gblocksretained 84% or 2211 characters of the original alignment.Most excluded regions came from the ITS region and therpb2 intron.

Maximum likelihood and Bayesian phylogeny

All single-locus ML analyses revealed nearly identicaltopologies and bootstrap values. Only minor conflictsconcerning specimens Le 228, Le 247, Le 249, Desjardin7577, Le 230 and Van de Putte 08-008 were detected. Thefirst four specimens (Le 228, Le 247, Le 249, Desjardin7577) are placed within clade 3 in all analyses but not in therpb2 analysis, where they occupy isolated positions on longbranches. The latter two specimens (Le 230, Van de Putte 08-008) belong to clade 4 in the ITS phylogeny, but are placedseparately in the rpb2 and LSU phylogenies. As expected,the clades supported by single-locus analyses, received evengreater support in the multi-locus ML-analysis.

The two methods applied for the BI analysis did notdiffer in tree topologies and probability values, nor did


these topologies differ from the ML phylograms. Evalu-ation of the BI analysis output showed that run 1 of theBI-MrModeltest analysis converged on a higher loglikelihood value (−12609) than runs 2, 3, 4 and 5. Thelatter four runs all converged on similar log likelihood(−12667 to −12683) and parameter values. Therefore,burn-in values and summary statistics were calculated forrun 1 separately and for runs 2, 3, 4 and 5 together. Theburn-in value for run 1 was determined at 3052000generations, while the burn-in value for runs 2, 3, 4 and5 together at 7.1 million generations. Despite thedifference in log likelihood values between the differentruns, tree topologies and posterior probability values werenearly identical. All five runs of the BI-GTR+I+Γ analysisconverged on similar log likelihood (−12582 to −12599)and parameter values. However, convergence in run 3 wasonly reached after 17.3 million generations, while runs 1,2, 4 and 5 all converged after 4.8 to 7.5 milliongenerations. Therefore, run 3 was discarded and summarystatistics were only calculated for runs 1, 2, 4 and 5together, with a burn-in value of 7.5 million generations.The Effective Sample Size (ESS) for the likelihood valueof the combined runs was slightly higher for the BI-MrModeltest analysis. The latter consisted of 5020.611uncorrelated samples, while the BI-GTR+I+Γ methodcomprised 4856.08 uncorrelated samples.

Phylogenetic species

Figure 1 shows the multi-locus ML tree with the associatedbootstrap values and posterior probability values of the50% majority consensus BI-MrModeltest tree. Only boot-strap values above 75% and posterior probabilities above85% are shown. Lactarius volemus sensu lato forms amonophyletic clade with a confidence value of 100%.Twelve strongly supported monophyletic clades and at leastsix terminal branches represent 18 phylogenetic species innorthern Thailand. The actual number of phylogeneticspecies is likely to be higher than 18 since the phylogeneticposition of specimens Le 228, Le 247, Le 249, Desjardin7577 and specimens Le 230, Van de Putte 08-008 is notresolved. Either these specimens belong to clades 3 and 4respectively or they form distinctive evolutionary lineageson their own.

Conspecificity with Japanese and Chinese L. volemus sensulato

To investigate conspecificity between the Thai, Japaneseand Chinese L. volemus, we constructed a ML phylogrambased on our LSU data set, augmented with the LSUsequences produced by Shimono et al. (2007) (results notshown). The Japanese ‘velvet type’ and ‘common form’ of

L. volemus are so far not recorded from northern Thailand.There are two possible cases of conspecificity: (1) TheJapanese ‘reddish type’ intermingles with species 4,however, with a very low support, (2) The ‘Chinese type’groups together with species 3 (BS 96), and not, as couldbe expected, with species 2 which also comprises aChinese collection. The remaining 15 Thai species are sofar endemic to Thailand. However, these results arepreliminary as (1) more Japanese and Chinese specimensand (2) more genetic markers are prerequisites to confirmconspecificity.

Evolutionary distance

Table 2 shows between-clade and within-clade divergen-ces, calculated as p-distances and ML estimates of K2Pdistances. Only clades comprising more than onespecimen were considered. Both distance measures aredistinctly higher for ITS than for rpb2 and LSU. Thisreflects the rather conserved nature of the rpb2 and LSUgenes in comparison to the higher variability of the ITSgene. Between-clade p-distances range from 0.8% to 3.5%for rpb2, from 0.3% to 2.6% for LSU and from 0.6% to7.6% for ITS. Between- clade K2P distances are higher,ranging from 0.8% to 3.9% for rpb2, from 0.3% to 2.8%for LSU and from 0.6% to 12% for ITS. The geneticdivergence between sister species is very low: p-distances(rpb2/LSU/ITS) between L. longipilus and species 8 areestimated at 1.4/0.4/1.2%, between L. crocatus andspecies 11 at 1.5/0.3/3.8% and between L. pinguis andspecies 17 at 0.8/0.9/2.1%.

Within clade p-distances and K2P distances are identicalor nearly so and very low, ranging from 0% to 0.7% forrpb2, 0–0.5% for LSU and 0–1.6% for ITS.

Concordance between phylogeny and morphology

The Appendix shows pileus colour, spore measurements,pleurolamprocystidia measurements and length of pilei-pellis hairs of the collections which were subjected to acritical microscopical examination. Six out of 18 phylo-genetic species (clades 7, 9, 10, 13, 15 and 18) can bedistinguished based on morphology and are described asthe following new species respectively: L. longipilus sp.nov., L. distantifolius sp. nov., L. crocatus sp. nov., L.vitellinus sp. nov., L. acicularus sp. nov. and L. pinguis sp.nov. The morphological characters diagnosing these newspecies are outlined in detail in the taxonomical chapterbelow.

While species 2 cannot be distinguished morphologically,morphological trends are observed in phylogenetic species 3,4, 8, 11 and 17 (see Appendix). However, the morphologicalvariability is too high to distinguish them from one another


unambiguously. These species remain cryptic, belonging toLactarius volemus sensu lato.

The remaining phylogenetic species (1, 5, 6, 12, 14and 16), representing single specimens (Le 359, Le 313,

Le 294, Le 251, Van de Putte 08-006 and Le 275respectively), are not described as new species. Moresampling is needed to confirm their species status morpho-logically and/or ecologically.

Taxonomy

Lactarius acicularis Van de Putte & Verbeken sp. nov.(Figs. 2, 3, 14o–p–q, and 15a–b)

MycoBank: MB518942Etymology: acicularis, meaning narrow, needle-like,

referring to the very long and slender terminal pileipellis hairs.A speciebus affinibus differt combinatione characterum:

cellulae terminales pileipellis longissimae usque ad130 μm, pileus a brunneo ad aurantiobrunneum etornamentum sporalis usque ad 1.4 μm altum.

Typus: Thailand, Chiang Mai Province, Mae Taengdistrict, Ban Pha Deng, Pathummikaram Temple, N19°06′28.8″ E98°44′47.3″, 1050 m, rainforest with Castanopsisarmata, Dipterocarpus sp., Lithocarpus sp., 15 July 2008,Van de Putte 08-029 (holotype MFLU, isotype GENT)

Basidiocarps medium-sized. Pileus 35–85 mm diameter,convex when immature, becoming applanate with slightlydepressed centre to infundibuliform when mature, marginregular or waving, surface dry and smooth to slightlyrugulose when young, rugose when mature, velutinous ornot, pruinose or not, yellowish orange brown (5C7–5C8),brown (6D5), in the centre darker brown (6F6) or reddishbrown in the centre, getting paler and with or without moreorange (6C8) or yellowish orange tinges towards themargin, young specimens darker. Lamellae subdecurrent

to decurrent, cream (4A3–4A4), discolouring brown (6D5–6E5) to greyish brown (5C3–5C4) when damaged,crowded (11–17L+l/cm), narrow to rather broad (1.5–6 mm), with numerous lamellulae of different length, edgesmooth and concolourous. Stipe 45–85×5–15 mm, cylin-drical to slightly tapering downwards, centrally attached,surface dry, velutinous or not, pruinose or not, smooth orlongitudinally rugulose, yellowish orange (4A5–5A5),brownish orange (6C8–6D8) to greyish brownish orange(6C5–5C6–6B5–6B6–6C6) or brown (6D4–6D5). Latexcopious, sticky, white, slightly watery or not, unchangingwhen isolated, unchanging on lamellae or within fewminutes to half an hour turning brown, no reaction orbecoming cream (4A3–4A4–4B3) or light brown (6C4–6C5) with KOH 10%, taste mild. Context moderately thickin pileus, solid to stuffed in stipe, whitish to cream (2A2–

Fig. 1 ML phylogram of Lactarius volemus sensu lato from northernThailand based on LSU, ITS and rpb2 nuclear sequences. Posteriorprobability values of the BI-MrModeltest analysis (runs 2–5) areadded. Bootstrap values ≥75% and posterior probabilities ≥85% areshown. Underlined taxa occupy uncertain positions. Branches in boldlead to well supported clades. The following abbreviations are used: AV:A. Verbeken, AVRW: A. Verbeken & R. Walleyn, BB: B. Buyck, DED:D.E. Desjardin, DS: D. Stubbe, JV: J. Vauras, KIINA: M. Härkönen,KVP: K. Van de Putte, LTH: H.T. Le, N: M. Noordeloos, RH: R.Halling, RW: R. Walleyn

b

Lactarius volemus sensu lato of northern Thailand: key to the species

1. Gills very distant; pleurolamprocystidia very slender, 7 μm broad maximum; pileus rusty orange ................................................................................................................................................................................................... Lactarius distantifolius sp. nov.1. Gills close; pleurolamprocystidia broader ......................................................................................................................... (2)2. Pleurolamprocystidia very broad, up to 17 μm ................................................................................................................................................................................................................................................................................Lactarius pinguis sp. nov.2. Pleurolamprocystidia more slender ................................................................................................................................... (3)3. Numerous pileipellis hairs up to 100 μm or more ........................................................................................................... (4)3. Pileipellis hairs almost never up to 100 μm ..................................................................................................................... (5)4. Spore ornamentation up to 2.3 μm; pileus reddish-brown or brown ......................................................................................................................................................................................................................................... Lactarius longipilus sp. nov.4. Spore ornamentation up to 1.4 μm; pileus brown or orange-brown ..............................................................................................................................................................................................................................................Lactarius acicularis sp. nov.5. Pleurolamprocystidia often longer than 100 μm, up to 130 μm; pileus with orange tinges ............................................................................................................................................................................................................Lactarius crocatus sp. nov.5. Pleurolamprocystidia rarely longer than 100 μm, up to 103 μm; pileus dominantly yellow with orange tinges ......................................................................................................................................................................... Lactarius vitellinus sp. nov.


Tab

le2

The

numberof

base

substitutions

persite

from

averagingov

erallsequ

ence

pairs

between(A

–B)andwith

in(C–D

)each

cladewhich

comprises

morethan

onespecim

en.Both

uncorrected

p-distancesand

ML

estim

ates

ofcorrected

Kim

ura2-parameter

distancesare

show

nin

percent.Uncorrected

p-distanceswerecalculated

with

uniform

ratesam

ongsites.

K2P

distanceswerecalculated

assuminggammadistribu

tedratesam

ongsites,with

thegamma

parameter

estim

ated

injM

odelTestforeach

dataset.Allpo

sitio

nscontaining

gaps

andmissing

datawereelim

inated

from

thedataset(Com

pletedeletio

nop

tion).S

tand

arderrorestim

ates

(S.

E.)wereob

tained

byabo

otstrapprocedurewith

1000

replicates.Betweencladedistance

S.E.

values

areshow

nabov

ethediagon

al,with

incladeS.E.estim

ates

areshow

nafterdistance

values.T

hepresence

ofn/cin

theresults

deno

tescasesin

which

itwas

notpo

ssibleto

estim

ate

evolutionary

distancesdu

eto

missing

sequ

ence

data

A.Betweencladep-distance

(rpb

2/LSU/ITS)

C.With

incladep-distance

Clade

23

47

89

1011

1315

1718

rpb2

LSU

ITS

20.8/0.5/0.4

0.5/0.3/0.8

0.7/0.6/1.0

0.6/0.6/1.0

0.6/0.5/1.4

0.8/0.5/1.0

0.6/0.5/1.2

0.7/0.6/1.1

0.8/0.6/1.1

0.8/0.5/1.2

0.8/0.6/1.3

n/c

0.0/0.0

n/c

32.0/1.6/0.6

0.4/0.3/0.6

0.6/0.5/0.9

0.5/0.5/0.9

0.5/0.5/1.3

0.7/0.4/1.1

0.5/0.5/1.1

0.7/0.6/1.1

0.7/0.5/1.0

0.7/0.5/1.1

0.7/0.5/1.3

0.1/0.1

0.0/0.0

0.0/0.0

41.6/0.9/2.2

0.9/0.7/1.6

0.6/0.4/0.8

0.4/0.4/0.8

0.5/0.4/1.3

0.7/0.4/1.1

0.5/0.4/1.0

0.7/0.5/1.0

0.7/0.5/1.0

0.6/0.4/1.1

0.7/0.5/1.2

0.3/0.1

0.0/0.0

0.3/0.2

72.5/2.2/3.9

2.3/1.5/3.3

1.9/1.3/2.3

0.5/0.2/0.4

0.6/0.5/1.2

0.8/0.4/1.0

0.6/0.4/1.1

0.8/0.5/1.0

0.8/0.4/1.0

0.8/0.5/1.1

0.8/0.5/1.2

0.1/0.1

0.0/0.0

0.1/0.1

81.9/2.6/4.5

1.7/1.8/3.9

1.3/1.7/3.0

1.4/0.4/1.2

0.5/0.5/1.2

0.7/0.4/1.0

0.5/0.4/1.1

0.7/0.5/1.0

0.7/0.5/1.0

0.7/0.5/1.0

0.7/0.5/1.2

0.7/0.3

0.5/0.2

1.6/0.5

91.8/2.2/7.6

1.6/1.6/7.1

1.2/0.9/6.9

2.0/1.6/6.3

1.5/0.4/6.9

0.7/0.5/1.2

0.5/0.5/1.2

0.7/0.5/1.3

0.7/0.5/1.2

0.6/0.5/1.3

0.7/0.5/1.4

n/c

0.0/0.0

1.2/0.6

103.2/2.7/5.3

2.7/1.5/4.7

2.6/1.0/4.3

3.4/1.2/3.7

2.9/1.6/4.5

2.8/1.6/6.3

0.5/0.2/1.0

0.8/0.4/1.3

0.8/0.4/1.2

0.8/0.4/1.2

0.8/0.5/1.4

0.2/0.1

0.0/0.0

0.0/0.0

111.8/1.9/5.3

1.6/1.5/4.7

1.2/1.0/4.3

1.9/1.5/4.5

1.5/1.8/5.3

1.3/1.6/5.9

1.5/0.3/3.8

0.7/0.4/1.1

0.7/0.4/1.1

0.6/0.4/1.2

0.6/0.5/1.3

0.0/0.0

0.0/0.0

0.0/0.0

132.6/2.7/4.8

2.9/2.4/4.2

2.5/1.9/4.4

3.1/2.2/4.3

2.8/2.6/4.7

2.6/2.2/6.7

3.2/1.5/5.7

2.2/1.5/5.1

0.8/0.5/0.7

0.7/0.6/0.8

0.8/0.6/0.9

0.0/0.0

0.0/0.0

0.1/0.1

153.5/2.4/4.7

2.9/2 .0/4.2

2.9/1.8/4.3

3.8/1.7/4.2

3.2/1.9/4.6

2.9/2.4/6.2

3.3/1.3/5.6

2.7/1.3/5.0

3.5/2.2/1.9

0.7/0.4/0.6

0.7/0.4/0.9

0.2/0.1

0.1/0.0

0.1/0.1

173.0/2.1/5.0

2.9/1.6/4.4

2.4/1.2/4.6

3.2/1.9/4.5

2.7/2.2/4.9

2.2/1.8/6.5

3.2/1.3/5.6

2.2/1.3/5.3

2 .8/2.2/2.1

2.7/1.2/1.5

0.4/0.3/0.8

0.4/0.2

0.1/0.1

0.0/0.0

183.1/2.3/6.5

2.9/1.9/6.0

2.5/1.4/5.5

3.4/1.9/5.6

2.8/2.3/6.3

2.2/2.0/8.3

3.2/1.9/6.7

2.2/1.9/6.3

2.9/1.5/3.1

2.8/1.4/3.1

0.8/0.9/2.1

0.0/0.0

0.1/0.1

0.4/0.2

B.BetweencladeK2P

-distance(rpb

2/LSU/ITS)

D.With

incladeK2P

distance

Clade

23

47

89

1011

1315

1718

rpb2

LSU

ITS

20.6/0.5/0.4

0.6/0 .4/0.9

0.7/0.6/1.5

0.6/0.7/1.6

0.6/0.5/2.8

0.9/0.6/1.8

0.6/0.6/1.8

0.7/0.7/1.7

0.9/0.6/1.6

0.8/0.6/1.8

0.8/0.6/2.4

n/c

0.0/0.0

n/c

32.1/1.7/0.6

0.4/0.3/0.7

0.7/0.5/1.3

0.5/0.5/1.4

0.5/0.5/2.5

0.7/0.5/1.6

0.5/0.5/1.6

0.7/0.6/1.5

0.8/0.6/1.4

0.8/0.5/1.5

0.8/0.5/2.1

0.1/0.1

0.0/0.0

0.0/0.0

41.6/0.9/2.5

1.0/0.8/1.8

0.6/0.4/0.9

0.5/0.5/1.1

0.5/0.4/2.5

0.7/0.4/1.5

0.5/0.4/1.5

0.7/0.6/1.5

0.8/0.5/1.4

0.7/0.4/1.6

0.7/0.5/1.9

0.3/0.1

0.0/0.0

0.4/0.2

72,5/2.3/4.6

2.4/1.5/3.8

1.9/1.4/2.5

0.5/0.2/0.5

0.7/0.5/2.1

0.9/0.4/1.4

0.6/0.4/1.6

0.8/0.6/1.5

0.9/0.5/1.4

0.8/0.6/1.5

0.9/0.6/1.9

0.1/0.1

0.0/0.0

0.1/0.1

81,9/2.7/5.5

1.8/1.9/4.6

1.3/1.7/3.4

1.5/0.4/1.2

0.5/0.5/2.3

0.8/0.4/1.5

0.5/0.5/1.8

0.7/0.6/1.5

0.8/0.5/1.4

0.7/0.6/1.6

0.8/0.6/2.1

0.7/0.3

0.5/0.2

1.7/0.6

91,8/1.5/10.9

1.6/1.7/9.7

1.2/0.9/9.5

2.1/1.7/8.1

1.5/2.1/9.2

0.8/0.5/2.0

0.5/0.5/1.9

0.8/0.6/2.4

0.8/0.6/2.1

0.7/0.5/2.3

0.7/0.6/3.1

n/c

0.0/0.0

1.2/0.6

103,4/2.0/6.5

2.8/1.5/5.6

2.7/1.1/5.1

3.5/1.2/4.3

3.0/1.7/5.4

2.9/1.7/8.1

0.6/0.2/1.5

0.8/0.5/2.0

0.8/0.5/1.9

0.8/0.5/1.9

0.8/0.6/2.3

0.2/0.1

0.0/0.0

0.0/0.0

111,8/2.0/6.6

1.6/1.5/5.7

1.2/1.1/5.1

2.0/1.5/5.4

1.5/1.9/6.6

1.3/1.7/7.4

1.5/0.3/4.5

0.7/0.5/1.8

0.7/0.5/1.7

0.7/0.4/1.8

0.7/0.6/2.1

0.0/0.0

0.0/0.0

0.0/0.0

132,7/2.8/5.9

3.0/2.5/5.0

2.5/2.0/5.3

3.2/2.3/5.1

2.9/2.7/5.7

2.7/2.3/9.0

3.3/1.5/7.1

2.2/1.5/6.2

0.9/0.6/0.8

0.8/0.6/0.9

0.8/0.7/1.2

0.0/0.0

0.0/0.0

0.0/0.0

153,7/2.5/5.7

3.0/2.1/4.9

3.0/1 .9/5.1

3.9/1.7/4.9

3.3/2.0/5.4

3.0/2.5/8.0

3.4/1.4/7.0

2.7/1.4/6.1

3.7/2.3/2.0

0.8/0.4/0.7

0.8/0.4/1.2

0.2/0.1

0.1/0.0

0.1/0.2

173,1/2.2/6.2

3.0/1.7/5.3

2.5/1.2/5.6

3.3/2.0/5.4

2.8/2.3/6.0

2.2/1.9/8.5

3.3/1.4/7.0

2.2/1.3/6.5

2.8/2.3/2.4

2 .8/1.2/1.6

0.4/0.3/0.9

0.4/0.2

0.1/0.1

0.0/0.0

183,2/2.5/8.7

3.0/2.0/7.7

2.5/1.5/7.0

3.4/2.1/7.1

1.3/2.4/8.1

2.2/2.1/12

3.3/2.0/8.8

2.2/2.0/8.2

3.2/2.6/3.5

3.2/1.5/3.5

0.8/0.9/2.4

0.0/0.0

0.1/0.1

0.5/0.2


Fig. 2 Lactarius acicularis: a. basidiocarps (Le 199), b. basidiospores(holotype), c. section through hymenium (Van de Putte 08-033), d.pseudopleurocystidia, e. basidia (Stubbe 07-456), f. pleurolamprocys-

tidia (holotype, Van de Putte 08-033, 08-007, 08-002) g. cheilocystidia(Le 199, Stubbe 07-456, Van de Putte 08-002) (scale bar = 10 μm,scale bar basidiocarps = 1 cm)


2A3), quickly turning brown to greyish brown (5B3–5C3–5C4–6C2–6D4) after cut, smell faint to fishy, taste mild.Macrochemical reactions context almost immediately turn-ing yellowish with KOH 10%, after a few secondsbecoming more yellowish orange, greenish grey to bluishgrey (25E2–25F3–26D2–26E3) or olive brown (4D3–4D4) reaction with FeSO4.

Basidiospores 7.0–7.9–8.5–9.1(–9.3)×6.5–7.2–7.8–8.5 μm, subglobose (Q=1.01–1.08–1.10–1.21); ornamen-tation amyloid; ridges up to 1.4 μm high, with rather sharpedges, forming a rather regular to slightly irregularcomplete or incomplete reticulum, sometimes isolated wartspresent; strong amyloidity between the ridges; plagedistally to almost completely amyloid. Basidia 40–60×9–12 μm, clavate, subclavate to subfusiform, 4-spored.pleurolamprocystidia 35–100×6–9(–11.5) μm; wall 1–4(–5) μm; rather short, subfusiform, subclavate, subcylin-drical, acuminate or with obtuse apex, occasionally furcateor lobed at the top, emergent or not, arising from thesubhymenium, moderately to very abundant. Pleuropseu-docystidia 2–6 μm diam., subcylindrical, often capitate,apex obtuse, occasionally lobed, emergent, scarce toabundant. Lamellae-edge fertile, composed of basidia and

cheilocystidia. Cheilocystidia 15–85×3–8 μm; thin-walledand hyaline to thick-walled [0.5–2.5(–3) μm]; fusiform,subfusiform, subclavate or somewhat irregularly shaped,acuminate or with obtuse apex, outline sometimes wavy.Hymenophoral trama cellular, with abundant lactifers androsettes of sphaerocytes. Pileipellis a lampropalisade;subpellis 50–120 μm thick, composed of rounded to elongatedor irregularly shaped, thick-walled cells (5–55×5–45 μm);terminal elements 15–130×3–8 μm; wall 0.5–1.5 μm; sub-cylindrical to subfusiform with tapering apices, acuminateor obtuse apex, outline often wavy, often septate.

Studied material: THAILAND—Chiang Mai Province,Mae Taeng district, Ban Pha Deng, Pathummikaram Temple,N19°06′28.8″ E98°44′47.3″, alt. 1050 m, rainforest withCastanopsis armata, Dipterocarpus sp., Lithocarpus sp.,8 July 2008, Van de Putte 08-007 (GENT, MFLU)—ibid.,15 July 2008, Van de Putte 08-029 (holotype MFLU, isotypeGENT)—Chiang Mai Province, Mae Taeng district, Ban Mae

Sae Highway 1095 near 50 km marker, N19°14.599′E98°39.456′, alt. 1002 m, rainforest with Castanopsisarmata, Castanopsis sp., Lithocarpus sp., Pinus kesiya, 28July 2004, Le 199 (CMU, GENT, MFLU, SFSU)—ibid., 4July 2007, Stubbe 07-456 (CMU, GENT)—ibid., 5 July2008, Van de Putte 08-002 (GENT, MFLU)—Chiang MaiProvince, Doi Inthanon National Park, junction of Highway1009 and road to Mae Chem, N19°31.58′ E98°29.64′, alt.1700 m, humid montane rainforest with Castanopsis sp.,Lithocarpus echinops, Quercus sp., 16 July 2008, Van dePutte 08-033 (GENT, MFLU)—Chiang Mai. Province, DoiSa Ket district, N18°54′31.5″ E99°12′57.0″, alt. 574,rainforest dominated by Castanopsis spp., 12 June 2006,Le 277 (CMU, GENT, MFLU, SFSU)

Note: L. acicularis can be recognized by the combinationof the very long pileipellis hairs, the brown to reddish-brownpileus colour and the spores with an ornamentation of1.4 μm maximum. It can be confused with L. longipilus,which also has distinctly long pileipellis hairs and a reddish-brown pileus. They differ from one another by the height ofthe spore ornamentation, which can be up to 2.1–2.3 μm inL. longipilus, but only up to 1.1–1.4 μm in L. acicularis.

Lactarius crocatus Van de Putte & Verbeken sp. nov.(Figs. 4, 5, 14j–k, and 15c–d)

Mycobank: MB518943Etymology: crocatus means orange, referring to the

orange tinged pileus.A speciebus affinibus differt combinatione characterum:

cellulae terminales pileipellis moderate brevae usque ad75 μm, pleurolamprocystidia moderate lata sed longa (40–130×6–13 μm) et pileus crocatus, aurantiacus.

Typus: Thailand, Chiang Mai Province, DoiInthanon National Park, junction of Highway 1009 and

road to Mae Chem, N19°31.58′ E98°29.64′, alt. 1700 m,

Fig. 3 Lactarius acicularis: section through pileipellis (Stubbe 07-456) (scale bar = 10 μm)


humid montane rainforest with Castanopsis sp., Lithocar-pus echinops, Quercus sp., 16 July 2008, Van de Putte 08-035 (holotype MFLU, isotype GENT)

Basidiocarps medium-sized to large. Pileus 45–100 mmdiameter, convex when immature, becoming applanate,subumbilicate or infundibuliform when mature, marginregular or slightly waving-undate, surface dry, slightly tostrongly velutinous, pruinose or not, rugose at margin, withcentre rugose or not, light orange (5A6), bright orange withochraceous tinge (5A8–5B8–6B7) to cinnamon coloured(6C8), getting paler towards the margin or not. Lamellaesubdecurrent to decurrent with tooth, whitish cream (3A4–4A4), discolouring brown (5C4–5D4–5D5) to dark brown(6E4–5F7) when bruised, crowded (14–20L+l/cm), narrowto broad (1.5–5 mm), with numerous lamellulae of differentlength, edge smooth and concolourous. Stipe 45–90×8–20 mm, cylindrical to slightly tapering downwards, cen-trally attached, surface dry, slightly velutinous or not,concolourous with pileus or paler, often getting palertowards the top. Latex copious, sticky, white, unchangingwhen isolated, unchanging in contact with lamellae orwithin few minutes to half an hour turning light brown(6D4–7D4), ultimately becoming dark coffee-and-milkcoloured, pale yellowish brown (4A4–4A5) to pale orange

brown (5A4) reaction with KOH 10%, occasionallybecoming dark brown (5F6–5E6) after half an hour, tastemild. Context moderately thick in pileus, solid to stuffed instipe, cream to pale yellow, but quickly turning brownish(5B2–5C3) after cut, smell fishy, taste mild. Macrochem-ical reactions context almost immediately turning yellowish(3A4) with KOH 10%, greenish grey to bluish grey (24D2–25E3) reaction with FeSO4 after a few seconds, then slowlydarkening to dark greenish grey (1F3–1F4–1E3–29E3).

Basidiospores 6.7–7.5–8.3–9.0(–9.1)×6.2–6.8–7.4–8.2(–8.5) μm, subglobose (Q=1.03–1.09–1.12–1.19); orna-mentation amyloid; ridges up to 1.3–1.6 μm high, withrather sharp edges, forming a complete or incomplete, oftenirregular reticulum with rather small meshes, isolated wartsoccasionally present; strong amyloidity between the ridges;plage distally to almost complete amyloid. Basidia 40–70×8–12 μm, clavate to subclavate, 4-spored, occasionally 2-spored. Pleurolamprocystidia 40–130×6–13(–14) μm;wall (1–) 2–6(–7) μm; rather long and broad, subcylin-drical, fusiform, subfusiform to subclavate, acuminate orwith obtuse apex, distinctly or slightly emergent, arisingfrom the subhymenium, moderately to very abundant.Pleuropseudocystidia 2–7 μm diam., cylindrical to lobedor tortuose, apex obtuse and often capitate to moniliform,slightly to distinctly emergent, scarce to very abundant.Lamellae-edge fertile, composed of basidia and cheilocys-tidia. Cheilocystidia 20–105×3–11 μm, thin-walled andhyaline to thick-walled (0.3–4 μm), fusiform, subfusiform,subclavate or somewhat irregularly shaped, acuminate orwith obtuse apex, sometimes with wavy outline. Hymeno-phoral trama cellular, with abundant lactifers and rosettesof sphaerocytes. Pileipellis a lampropalisade; subpellis 35–90 μm thick, composed of rounded to elongated orirregularly shaped, thick-walled cells (5–35×5–30 μm);terminal elements 10–70(–75)×4–11 μm; wall 0.5–2 μm;subcylindrical to subfusiform with tapering apices, acumi-nate or obtuse apex, outline often wavy, often septate.

Studied material: THAILAND—Chiang Mai Province,Mae Taeng district, Ban Mae Sae, Highway 1095 near50 km marker, N19°14.599′ E98°39.456′, alt. 1002 m,rainforest with Castanopsis armata, Castanopsis sp.,Lithocarpus sp., Pinus kesiya, 28 July 2004, Le 202(CMU, GENT, MFLU, SFSU)—ibid., 5 September 2004,Le 222 (CMU, GENT, MFLU, SFSU)—ibid., 5 September2004, Le 224 (CMU, GENT, MFLU, SFSU)—ibid., 5September 2004, Le 225 (CMU, GENT, MFLU, SFSU)—ibid., 5 September 2004, Le 234 (CMU, GENT, MFLU,SFSU)—Chiang Mai Province, Mae Taeng district, BanPha Deng, Mushroom Research Centre, N19°17.123′ E98°44.009′, alt. 900 m, rainforest with Castanopsis armata,Erythrina sp., Dipterocarpus sp., Pinus kesiya, 13 July2003, Le 17 (CMU, GENT, MFLU, SFSU)—Chiang MaiProvince, Mae Taeng district, Ban Pha Deng, Pathummi-

Fig. 4 Lactarius crocatus: section through pileipellis (holotype)(scale bar = 10 μm)


Fig. 5 Lactarius crocatus: a. basidiocarps (holotype, Van de Putte 08-042), b. basidiospores (Le 222), c. basidia (Le 225), d. section throughhymenium (Le 268), e. pleurolamprocystidia (Le 17, holotype, Le 224)

f. cheilocystidia (Le 17, Van de Putte 08-034, Le 268, 224, 234), g.pseudopleurocystidia (Le 17, 224, 202, 225, Van de Putte 08-034)(scale bar = 10 μm, scale bar basidiocarps = 1 cm)


karam Temple, N19°06′28.8″ E98°44′47.3″, 1050 m, rain-forest with Castanopsis armata, Dipterocarpus sp., Litho-carpus sp., 27 July 2008, Van de Putte 08-042 (GENT,MFLU)—Chiang Mai Province, Doi Inthanon NationalPark, junction of Highway, 1009 and road to Mae Chem,N19°31.58′ E98°29.64′, alt. 1700 m, humid montane rain-forest with Castanopsis sp., Lithocarpus echinops, Quercussp., 16 July 2008, Van de Putte 08-034 (GENT, MFLU)—ibid., 16 July 2008, Van de Putte 08-035 (holotype MFLU,isotype GENT)—ibid., 16 July 2008, Van de Putte 08-036(GENT, MFLU)—Chiang Rai Province, Khun Chae Nation-al Park, N19°04′40.5″ E99°23′54.3″, alt. 936 m, rainforestwith Castanopsis armata, Castanopsis sp., Pinus kesiya, 10June 2005, Le 268 (CMU, GENT, MFLU, SFSU).

Note: L. crocatus is characterized by the combination of thedistinctly long and rather broad pleurolamprocystidia, thepileipellis hairs of moderate length and the orange tinged pileus.

Lactarius distantifolius Van de Putte, Stubbe & Verbekensp. nov. (Figs. 6, 7, 14h–i, and 15e–f)

Mycobank: MB518944Etymology: contraction of distans and folius, referring to

the distant lamellae.A speciebus affinibus differt combinatione characterum:

pileus intense rufo-aurantius, lamellae distantissimae etpleurolamprocystidia longa, gracilia, 45–155×5–7 μm,pariete usque ad 3 μm crasso.

Typus: Chiang Mai Province, Mae Ma Lai, Huai RaiTemple, N19°06.170′ E98°53.363′, alt. 515 m, in teakplantation with Dipterocarpus obtusifolius, Dipterocarpusspp., Pterocarpus macrocarpus, Shorea spp., 5 July 2007,Stubbe 07-461 (holotype CMU, isotype GENT).

Basidiocarps medium-sized. Pileus 20–115 mm diameter,centre depressed, with or without an inconspicuous papilla,margin involute when immature, becoming inflexed, deflexedor straight, margin edge smooth, surface dry, entirelyvelutinous, more strong so in the centre, smooth to faintly ormoderately rugulose in the centre, distinctly and concentricallyrugulose towards the margin, intense rusty orange (6C8–7C8),darker in the centre, there with a more brownish tinge,gradually getting paler and only slightly more yellowishtowards the margin (5A8–5B8), discolouring dirty brown(6E4–6E6–7E4–7E6)when bruised. Lamellae decurrent, cream(3A2) with a warm yellowish tinge (4A3–4A4), discolouringpale to dark brown (7D4–7D6–7E4–7E6) when bruised,subdistant (4–5L+l/cm), rather thick and broad (up to6 mm), with three series of lamellulae, furcations occurringnear the pileus margin, edge smooth and concolourous. Stipe15–50×15–20 mm, subcylindrical and somewhat taperingdownwards, robust, centrally to eccentrically attached, surfacedry and matt, weakly to moderately velutinous, completelyand minutely but distinctly longitudinally rugulose, conco-lourous with pileus margin and strongly contrasting with thepale lamellae. Latex copious, sticky, white, unchanging whenisolated, within few minutes to half an hour turning pinkishbrown or dirty brown (6D3–6D5) on the lamellae and stipe,no reaction with KOH 10%, taste mild. Context thick inpileus, solid in stipe, firm, white but quickly turning palebrown or pinkish brown (5C4) after cut, smell likeseafood, taste mild. Macrochemical reactions context withgaiac slowly becoming bluish, almost immediately turningpale yellow (3A4) with KOH 10%, faint reaction orbecoming greyish olive (1E2) with FeSO4.

Basidiospores 7.8–8.8–9.1–10.3(–10.4)×6.9–7.8–8.2–8.9 μm, subglobose (Q=1.01–1.11–1.13–1.23); ornamen-tation amyloid; ridges up to 2–2.1 μm high, with rathersharp edges, forming an irregular complete or incomplete

Fig. 6 Lactarius distantifolius: section through pileipellis (holotype)(scale bar = 10 μm)


Fig. 7 Lactarius distantifolius: a. basidiospores (holotype, Le 289), b.basidiocarps (holotype), c. pseudopleurocystidia (Le 289), d. sectionthrough hymenium (Le 288), e. pleurolamprocystidia (holotype), f.

cheilocystidia (holotype), g. basidia (holotype, Le 289) (scale bar =10 μm, scale bar basidiocarp = 1 cm)


reticulum, occasionally isolated warts present; strongamyloidity between the ridges; plage almost completeamyloid. Basidia 55–70×9.5–12 μm, clavate to subclavate,4-spored. Pleurolamprocystidia 45–155×5–7 μm; wall 1–3 μm; long and very slender, subcylindrical to subfusi-form, acuminate or with obtuse apex, outline often wavy,slightly to distinctly emergent, arising from the subhyme-nium, very abundant. Pleuropseudocystidia 4-6 μm diam.,subcylindrical to lobed or slightly tortuose, often capitate,moderately to very slightly emergent, scarce. Lamellae-edge fertile, composed of basidia and cheilocystidia.Cheilocystidia 25–90×4–5.5 μm; thin-walled and hyalineto thick-walled (0.5–2 μm); subcylindrical to subfusiform,acuminate or with obtuse apex, outline often wavy, rarelyseptate. Hymenophoral trama cellular, with abundantlactifers and rosettes of sphaerocytes. Pileipellis a lamp-ropalisade; subpellis 77–100 μm thick, composed of roundedto elongated or irregularly shaped, thick-walled cells(5–55×5–40 μm); terminal elements 10–60×4–9.5 μm; wall0.5–2 μm; erect, subfusiform with tapering apices, sometimessubcylindrical or subclavate with obtuse apex, occasionallyirregularly shaped and lobed, outline often wavy, not septate.

Studied material: THAILAND—Chiang Mai Province,Mae Taeng district, Mae Ma Lai, Huan Rai Temple, N19°06.170′ E98°53.363′, alt. 515 m, in teak plantation withDipterocarpus obtusifolius, Dipterocarpus spp., Pterocarpusmacrocarpus, Shorea spp., 5 July 2007, Stubbe 07-461(holotype CMU, isotype GENT)—Chiang Mai Province,Mae Taeng district, Ban Mae Sae, Highway 1095 at 22 kmmarker, N19°07.57′ E98°45.65′, alt. 750 m, xeric broad-leafforest (Dipterocarpus spp., Tectona grandis) withPinus kesiya,21 June 2005, Le 288 (CMU, GENT, MFLU, SFSU)—ibid.,21 June 2005, Le 289 (CMU, GENT, MFLU, SFSU).

Note: L. distantifolius can be recognized at a singleglance by the long and strikingly slender, relatively thinwalled pleurolamprocystidia, its intense, deep rusty orangepileus colour and very distant gills. Macroscopically, itresembles the Papua New Guinean species Lactariusaustrovolemus Hongo. Lactarius austrovolemus indeedhas the same distant lamellae, but differs from L. distant-ifolius microscopically by the broader and distinctly shorterpleurolamprocystidia (Hongo 1973). Microscopically, L.distantifolius resembles the Papua new Guinean speciesLactarius lamprocystidiatus Verbeken & Horak by the veryslender pleurolamprocystidia. These are, however, conspic-uously shorter in L. lamprocystidiatus. In the field, however,the latter can be easily distinguished from L. distantifolius bythe crowded lamellae and the darker and deeper reddish tobrownish pileus (Verbeken and Horak 2000).

Lactarius longipilus Van de Putte, Le & Verbeken sp.nov. (Figs. 8, 9, 14e–f, and 15i–j)

Mycobank: MB518946

Etymology: contraction of longi and pilus, referring tothe long terminal pileipellis hairs.

A speciebus affinibus differt combinatione characterum:cellulae terminales pileipellis longissimae usque ad 130 μm,pileus a brunneo ad rubrobrunneum et ornamentum sporalisusque ad 2.3 μm altum.

Typus: Thailand, Chiang Mai Province, Tung Joaw,N19°06′28.8″ E98°38.09, alt. 1300 m, forest with Casta-nopsis spp., Lithocarpus sp., Pinus kesiya, 30 June 2004,Verbeken and Walleyn 04-160 (= Le 168) (holotype CMU,isotype GENT, MFLU, SFSU)

Basidiocarps medium-sized to rather large. Pileus 55–115 mm diameter, depressed to deeply infundibuliform whenmature, margin regular to somewhat irregularly waving-undate, surface dry, velvety, smooth or slightly rugose in thecentre, radially rugose near the margin, dark brown toreddish brown (7E8–7E6 to 8D7–8E7) in the centre,gradually getting paler and with more orange tinge towardsthe margin (6C8–6D7 to 5A–B6) or margin concolourouswith the centre, with or without small papilla. Lamellaesubdecurrent to decurrent with small tooth, pale cream (4A–B4), discolouring pale brown when bruised (6D5–6),crowded (10–16L+l/cm), rather broad (3–4 mm), with 3–4series of lamellulae, edge smooth and concolourous. Stipe75–90×7–15 mm, cylindrical to slightly tapering upwards,centrally attached, surface dry, velvety, longitudinally rugose,brownish orange (6C8–6E7–6E8–7E7–7E8). Latex copious,sticky, white, unchanging when isolated, ultimately becom-ing coffee-and-milk coloured (5A3–5B3) on lamellae, noreaction or becoming pale cream (4A3) with KOH 10%.Context moderately thick in pileus, solid in stipe, cream,turning brownish after cut, smell slightly fishy, taste mild.Macrochemical reactions context turning yellowish withKOH 10%, greyish green reaction with FeSO4.

Basidiospores 7.6–8.4–8.8–9.8(–10.5)×6.9–7.7–8.1–9.0(–9.1) μm, subglobose (Q=1.00–1.10–1.21); ornamenta-tion amyloid; ridges up to 2.1–2.3 μm high, with rather sharpedges and forming a regular to slightly irregular complete oralmost complete reticulum, warts between the ridges rare;strong amyloidity between the ridges; plage distally to almostcompletely amyloid. Basidia 45–65×10–14 μm, clavate tosubclavate, mostly 4-spored, occasionally 2-spored. Pleuro-lamprocystidia 35–115×6–9(–10) μm; wall 1–3.5 μm;fusiform to subfusiform, acuminate, emergent, arising fromthe subhymenium, abundant. Pleuropseudocystidia 4–6.5 μm diam., cylindrical, lobed or strongly tortuose, apexobtuse, emergent, scarce to abundant. Lamellae-edge fertile,composed of basidia and cheilocystidia. Cheilocystidia 15–75×3–9 μm; thin walled and hyaline to thick-walled (0.5–3 μm); fusiform to subfusiform or irregularly shaped,acuminate or with slightly obtuse apex, outline sometimeswavy. Hymenophoral trama cellular, with abundant lactifersand rosettes of sphaerocytes. Pileipellis a lampropalisade;


Fig. 8 Lactarius longipilus: a. basidiocarps (holotype), b. basidio-spores (holotype), c. section through hymenium (holotype), d. basidia(holotype, Le 206), e. pleurolamprocystidia (Le 273, 184), f.

cheilocystidia (Le 184, 206, 273), g. pseudopleurocystidia (Le 273)(scale bar = 10 μm, scale bar basidiocarps = 1 cm)


subpellis 50–100 μm thick, composed of rounded toelongated or irregularly shaped, thick-walled cells (8–65×5–45 μm); terminal elements very long and slender; 10–130×3–6.5 μm; wall 0.5–2 μm; subcylindrical to subfusi-form with tapering apices, slightly acuminate or obtuse apex,outline straight to wavy, often septate.

Studied material: THAILAND—Chiang Mai Province,Doi Inthanon National Park, junction of Highway 1009 androad to Mae Chem, N19°31.58′ E98°29.64′, alt. 1700 m,humid montane rainforest with Castanopsis sp., Lithocar-pus echinops, Quercus sp. etc., 16 July 2004, Le 184(CMU, GENT, MFLU, SFSU)—ibid., July 2004, Le 206(CMU, GENT, MFLU, SFSU)—Chiang Mai Province,

Tung Joaw, N19°06′28.8″ E98°38.09, alt. 1300 m, forestwith Castanopsis spp., Lithocarpus sp., Pinus kesiya, 30August 2003, Le 273 (CMU, GENT, MFLU, SFSU)—ibid.,30 June 2003, Verbeken and Walleyn 04-160 (= Le 168)(holotype CMU, isotype GENT, MFLU, SFSU).

Note: L. longipilus can be confused with L. acicularis.For discussion, see under that species.

Lactarius pinguis Van de Putte & Verbeken sp. nov.(Figs. 10, 11, 14s, and 15g–h)

Mycobank: MB518945Etymology: derived from pingue, meaning ‘thick’ or

‘fat’, referring to the very broad and thick-walled pleuro-lamprocystidia.

A speciebus affinibus differt combinatione characterum:cellulae terminales pileipellis longissimae usque ad 130 μmet pleurolamprocystidia lata (7–17 μm), pariete usque ad7 μm crasso.

Typus: Tung Joaw, N19°08.07′ E98°38.09′, alt. 1400 m,rainforest with Castanopsis armata, Lithocarpus sp., Pinuskesiya, 20 June 2004, Verbeken and Walleyn 04-023(= Le 117) (holotype CMU, isotypes GENT, MFLU, SFSU)

Basidiocarps medium-sized, slender. Pileus 40–80 mmdiameter, convex when immature, becoming applanate todeeply depressed, margin regular, somewhat waving-undatewhen older, surface dry and smooth to slightly rugulosewhen young, rugose when old, slightly velutinous, verypale yellowish white or straw-coloured, locally slightlybrowning, very young specimens more apricot, light orange(5A5) in centre and light yellow (4A5) on margin,discolouring to light brown (6D6) when bruised. Lamellaedecurrent with small tooth, whitish to cream, discolouringpale brown when damaged, crowded (15L+l/cm), ratherbroad (2-4 mm), with numerous lamellulae of differentlength, edge smooth and concolourous. Stipe 40–95×10–15mm, cylindrical to slightly tapering downwards, centrally toeccentrically attached, surface dry, concolourous with pileus.Latex copious, sticky, white, unchanging when isolated,within few minutes to half an hour turning light brown(6D4–7D4) on lamellae, ultimately becoming dark coffee-and-milk coloured, no or pale brownish (4B4–4B5) reactionwith KOH 10%, taste mild. Context moderately thick inpileus, solid to stuffed in stipe, cream to pale yellow, butquickly turning brownish after cut, smell faint to distinctlylike Crustaceae, taste mild, becoming slightly bitter. Macro-chemical reactions context almost immediately turningyellowish with KOH 10%, green blue reaction with FeSO4.

Basidiospores 8.0–9.0–9.1–10.2(–10.5)×7.4–8.3–8.4–9.4(–9.6) μm, subglobose (Q=1.00–1.07–1.10–1.16); or-namentation amyloid; ridges up to 2.0 μm high, forming arather regular complete or incomplete reticulum, isolatedwarts rare; wall strongly amyloid between the ridges; plagedistally to almost complete amyloid. Basidia 40–65×11–

Fig. 9 Lactarius longipilus: section through pileipellis (holotype)(scale bar = 10 μm)


Fig. 10 Lactarius pinguis: a. pleurolamprocystidia (holotype, Le278), b. basidiocarps, c. section through hymenium (holotype), d.basidia (holotype), e. basidiospores (holotype), f. cheilocystidia

(holotype), g. pseudopleurocystidia (Le 255) (scale bar = 10 μm,scale bar basidiocarp = 1 cm)


14 μm, moderately broad, clavate to subclavate, 4-spored.Pleurolamprocystidia 50–120×7–17 μm; wall 3–7 μm;conspicuously broad and thick-walled, subfusiform tosubclavate, acuminate or with obtuse apex, often furcateor lobed at the top, distinctly, slightly or not emergent,arising from the subhymenium, moderately to very abun-dant. Pleuropseudocystidia 3–7 μm diam., cylindrical toconspicuously tortuose, apex obtuse, not or slightlyemergent, scarce to abundant. Lamellae-edge fertile, com-posed of basidia and cheilocystidia. Cheilocystidia 15–96×3.5–12 μm; thin walled and hyaline to thick-walled (0.5–7 μm); subfusiform to subclavate, acuminate or with obtuseapex, outline sometimes wavy. Hymenophoral tramacellular, with abundant lactifers and rosettes of sphaero-cytes. Pileipellis a lampropalisade; subpellis 50–140 μm

thick, composed of rounded to elongated or irregularlyshaped, thick-walled cells [5–65 (–80)×5–45 μm]; termi-nal elements 30–120(–130)×2–8 μm; wall 0.5–1.5 μm;subcylindrical to subfusiform with tapering apices, slightlyacuminate or obtuse apex, often wavy outline, often septate.

Studied material: THAILAND—Chiang Mai Province,Doi Saket district, N18°54′31.5″ E99°12′57.0″, alt. 574 m,rainforest dominated by Castanopsis spp., 12 July 2006, Le278 (CMU, GENT, MFLU, SFSU)—Chiang Mai Province,Mae Taeng district, Ban Pha Deng, Mushroom ResearchCentre, N19°17.123′ E98°44.009′, alt. 900 m, rainforestwith Castanopsis armata, Dipterocarpus sp., Erythrina sp.,Pinus kesiya, 13 July 2003, Le 255 (CMU, GENT, MFLU,SFSU)—Chiang Mai Province, Tung Joaw, N19°08′.07′E98°38.09′, alt. 1400 m, rainforest with Castanopsis spp.,Lithocarpus sp., Pinus kesiya, 20 June 2004, Verbeken andWalleyn 04-023 (= Le 117) (holotype CMU, isotypes GENT,MFLU, SFSU)—ibid., 30 June 2004, Verbeken and Walleyn04-162 (= Le 169) (CMU, GENT, MFLU, SFSU).

Note: Collections Le 117 and Le 169 are fully grown andhave a distinctly pale, yellowish white pileus. Collections Le255 and Le 278, on the other hand, have a brownish pileuscolour. These collections, however, both comprise youngspecimens. As observed in other pale yellowish variants, thecolour of the pileus often evolves from rather dark brown inyoung specimens to conspicuously pale in fully grownspecimens. Therefore, it is possible that the brown pileuscolour in the two young collections would have evolved topale whitish yellow at maturity. Not pileus colour, but thestrikingly broad pleurolamprocystidia and long pileipellishairs, should be used as diagnostic features for this species.

The pale yellowish pileus is also typical for the sisterspecies of L. pinguis (species 17). Microscopically, species17 differs from L. pinguis by the less broad pleuro-lamprocystidia and shorter pileipellis hairs (Appendix).

Both species resemble the North-American pale yellowishL. volemus var. flavus (Hesler and Smith 1979). Lactariuspinguis remains unmistakable due to its very broad pleuro-lamprocystidia and long pileipellis hairs. Species 17 differsfrom L. volemus var. flavus by the longer pleurolamprocys-tidia and pileipellis hairs. Lactarius volemus var. flavus isfurthermore characterized by distinctly smaller and lowerornamented basidiospores (Hesler and Smith 1979).

Lactarius vitellinus Van de Putte & Verbeken sp. nov.(Figs. 12, 13, 14m–n, and 15k–l)

Mycobank: MB518947Etymology: vitellinus, meaning yellowish orange, refer-

ring to the yellowish orange pileus colourA speciebus affinibus differt combinatione characterum:

cellulae terminales pileipellis moderate brevae usque ad85 μm, pleurolamprocystidia moderate lata et brevia (55–105×6–13 μm) et pileus vitellinus, ex aurantiaco luteus.

Fig. 11 Lactarius pinguis: section through pileipellis (holotype)(scale bar = 10 μm)


Fig. 12 Lactarius vitellinus: a. basidiospores (Le 348, holotype, Vande Putte 08-025), b. basidiocarps (Van de Putte 08-025, Le 269), c.section through hymenium (Le 269), d. pseudopleurocystidia (Le

348), e. pleurolamprocystidia (holotype), f. basidia (holotype), g.cheilocystidia (holotype) (scale bar = 10 μm, scale bar basidiocarp =1 cm)


Typus: Thailand, Chiang Mai Province, Tung Joaw,N19°08.07′ E98°38.09, alt. 1400 m, rainforest with Casta-nopsis spp., Lithocarpus sp., Pinus kesiya, 14 July 2008,Van de Putte 08-024 (holotype MFLU, isotype GENT)

Basidiocarps medium-sized. Pileus 35–60 mm diameter,plano-convex with slightly depressed centre, margin regular,surface dry, smooth to rugulose in the centre, rugulose at themargin, not velutinous nor pruinose, pale yellowish orange(4A5–4A4) or orange (5A8–6A8), in the centre slightlybrowning, young specimens darker and more orange,discolouring brownish when bruised. Lamellae decurrent toadnate with decurrent tooth, pale cream (3A2–3A3),discolouring brownish (5D4–5D5–5D7–6E4) when dam-aged, crowded (14–20L+l/cm), narrow to broad (1.5–3.5 mm), with numerous lamellulae of different length, edgesmooth and concolourous. Stipe 40–95×9–11 mm, cylindri-cal to slightly tapering upwards, centrally attached, surfacedry, smooth to longitudinally rugulose, not velutinous norpruinose, orange to yellowish orange (4A4–4A5–4A7).Latex copious, sticky, white, unchanging when isolated,slowly discolouring brownish on lamellae, no reaction orslowly becoming pale yellowish brown with KOH 10%,taste mild. Context moderately thick in pileus, solid in stipe,whitish cream (3A2–3A3), quickly turning brownish orgreyish brown after cut (5C5–5D5–6B2–6C3–6D5), smellfragrant to fishy, taste mild. Macrochemical reactionscontext almost immediately turning yellowish to yellowish

orange with KOH 10%, slowly becoming dark greyish blue(23F3–23F4) or greyish green (1F2–1F3) with FeSO4.

Basidiospores (6.6–)7.1–7.9–8.0–8.9(–9.2)×(6.2–)6.6–7.3–7.4–8.3 μm, subglobose [Q=1.00–1.07–1.09–1.14–(1.19)]; ornamentation amyloid; ridges up to 2.0 μm high,with rounded to rather sharp edges, forming a regularcomplete or incomplete reticulum; strong amyloiditybetween the ridges; plage distally to almost completelyamyloid. Basidia (40–)45–65×9–13 μm, subclavate toclavate, 4-spored. Pleurolamprocystidia 55–105×6–13 μm; wall 2–5 μm; rather short and broad, fusiform tosubfusiform, subcylindrical, subclavate, acuminate or withobtuse apex, emergent, arising from the subhymenium, veryabundant. Pleuropseudocystidia, 2.5–7 μm diam., subcy-lindrical, subclavate, occasionally lobed, apex obtuse, oftencapitate to moniliform, emergent or not, abundant to veryabundant. Lamellae-edge fertile, composed of basidia andcheilocystidia. Cheilocystidia 25–80×4–9 μm; thin walledand hyaline to thick-walled (0.5–3.5 μm); fusiform,subfusiform to subclavate, acuminate or with obtuse apex,outline sometimes wavy. Hymenophoral trama cellular,with abundant lactifers and rosettes of sphaerocytes.Pileipellis a lampropalisade; subpellis 70–110 μm thick,composed of rounded to elongated or irregularly shaped,thick-walled cells (5–40×5–30 μm); terminal elements 10–70(–85)×5–15 μm; wall 0.5–1.5 μm; subcylindrical,fusiform, subfusiform or obclavate, with tapering apices,acuminate or obtuse apex, outline often wavy, often septate.

Studied material: THAILAND—Chiang Mai Province,Tung Joaw, N19°08.07′ E98°38.09, alt. 1400 m, rainforestwith Castanopsis spp., Lithocarpus sp., Pinus kesiya, 16July 2005, Le 348 (CMU, GENT, MFLU, SFSU) - ibid., 14July 2008, Van de Putte 08-024 (holotype MFLU, isotypeGENT)—ibid., 14 July 2008, Van de Putte 08-025 (GENT,MFLU)—Chiang Rai Province, Khun Chae National Park,N19°04′40.5″ E99°23′54.3″, alt. 936 m, rainforest withCastanopsis armata, Castanopsis sp., Pinus kesiya, 10 June2005, Le 269 (CMU, GENT, MFLU, SFSU).

Note: L. vitellinus can be recognized by the combinationof the rather broad but short pleurolamprocystidia, pileipellishairs of moderate length and the pileus with yellowish tinge.

Discussion

Cryptic diversity

This study changed the current circumscription of L.volemus in northern Thailand, which now includes no lessthan 18 phylogenetic species. Both ITS, LSU and rpb2single loci are suitable for species delimitation. However,the combined LSU and rpb2 loci, being rather easy toamplify and far more easy to align than ITS, are the most

Fig. 13 Lactarius vitellinus: section through pileipellis (Van de Putte08-025) (scale bar = 10 μm)


Fig. 14 Species 3: a (Van de Putte 08-031), b (Van de Putte 08-043),species 4: c (Van de Putte 08-004), d (Le 230), L. longipilus: e (Le184), f (Le 206), species 8: g (Le 170), L. distantifolius: h, i(holotype), L. crocatus: j (Van de Putte 08-034), k (Van de Putte 08-

042), species 11: l (Verbeken & Walleyn 04-87), L. vitellinus: m(holotype), n (Van de Putte 08-025), L. acicularis: o (Stubbe 07-456),p (holotype), q (Van de Putte 08-007), species 17: r (Van de Putte 08-023), L. pinguis: s (holotype)


appropriate for species delimitation. Phylogenetic resolution atdeeper levels is still lacking. More genes are needed toelucidate the relatedness between most of the different species.

Six phylogenetic species can be morphologically recog-nized and are fully described and illustrated in this paper.The 6 remaining clades (thus not considering the 6 singlecollections which also represent 6 phylogenetic species),however, are cryptic: although there is a concordancebetween morphology and molecular data, nearly identicalforms are distributed in these clades (see Appendix). Forexample, collections with short pileipellis hairs of up to50 μm and orange tinged pilei are scattered throughout clades3, 4 and 11. Only the breadth of their pleurolamprocystidiaallows for a discrimination between these collections. Theseare up to 11–14 μm broad in clade 11, up to 10–12 μm inclade 4 and up to 9 μm in clade 3. Consequently, a collectionwith an orange pileus, pileipellis hairs of 50 μm andpleurolamprocystidia which are 10 μm broad will be

impossible to place unambiguously. Morphology allowsperhaps for a tentative assignment of specimens to one ofthese clades, but corroboration of molecular data is imperativeto pinpoint species boundaries. Additional studies are neededto investigate possible physiological, chemical, ectomycor-rhizal or ecological differences between these clades.

It is not uncommon that previously presumed crypticdiversity appears to be partially cryptic or even not cryptic atall (e.g. Amato and Montresor 2008; Goodman et al. 2009;Hagino et al. 2009; Murillo et al. 2009; Ragionieri et al. 2009;Verbruggen et al. 2007). One natural explanation is that manycryptic species are morphologically rather simple (Bickford etal. 2007; Stuart et al. 2006), as is the case for most fungi. Fewmorphological diagnostics are therefore available and mor-phological differences among closely related species are oftenrather subtle. These are consequently either regarded asintraspecific variation or easily overlooked when no critical,in-depth morphological examination is conducted.

Fig. 15 SEMphotographs of basidiospores. L. acicularis: a, b (Van de Putte 08-033), L. crocatus: c, d (Le 17), L. distantifolius: e, f (holotype), L. pinguis:g, h (holotype), L. longipilus: i, j (holotype), L. vitellinus: k, l (holotype) Scale bar = 2 μm


Diagnostic morphological characters

Relatively few morphological characters can be used todiscriminate species within the L. volemus group. Pleuro-lamprocystidia and pileipellis hairs are the most importantdiagnostic features. A critical examination of pileipellis hairlength and breadth and length of the pleurolamprocystidia areprerequisites to identify different species. Importantly, theseelements should be measured halfway the cap radius.Accurate lengths of pileipellis hairs should be measured fromscalps taken from the pileus surface, rather than from sectionsin which long hairs are often broken.

Spore morphology is usually very important to diagnosespecies within the genus Lactarius (Nuytinck and Verbeken2005; Stubbe et al. 2008, 2010). This is, surprisingly, not thecase for the L. volemus group due to the phenotypic plasticityof the height and acuteness of the ridges, the regularity of themeshes and the size of the spores. Only in the case of L.acicularis and L. longipilus, the height of the ridges doesbecome an important distinctive character.

In agreement with Shimono et al. (2007), we observed thatpileus colour supports the delimitation of at least somephylogenetic species. However, infraspecific variability can behigh, or the same pileus colour can be observed in severalspecies. Lactarius pinguis and its sister species 17, for example,are both characterized by the same pale yellowish white pileus.An orange tinged pileus is characteristic for both L. crocatusand species 8, but also occurs in species 3 and species 4.Intense, deep reddish colours are observed in L. longipilus andthroughout species 3, 4 and 8 (Figure 14). Pileus colour(observed in fully grown specimens only) can indeed allow fora tentative assignment of the specimens to one or more clades,but it should not be used as a diagnostic character without thecorroboration of micromorphological characters.

The pileus surface texture, the lamellar density and thechanging and staining of the latex have been reported to bequite variable (Hesler and Smith 1979; Le 2007; Shimono etal. 2007; Wang 2007). Based on LSU sequences, Shimono etal. (2007) found that the velvet type of L. volemus in Japancorresponds to a separate phylogenetic taxon. We indeedobserved specimens that were conspicuously more velvetythan others, but these are scattered throughout the differentclades. This is not surprising considering the ephemeral natureof this character. Basidiocarps with a more exposed positionoften have a less velvety pileus in comparison withbasidiocarps standing more sheltered. The presence, absenceor intensity of the velvety aspect also depends on thedevelopmental stage of the basidiocarps. Young basidiocarpsare often distinctly velvety, whereas fully grown basidiocarpsof the same collection can be not velvety at all.

The lamellar density seems to be rather constant. Whileall clades are characterized by close lamellae, L. distant-ifolius has distinctly distant lamellae.

Very little variation, not consistent within clades, has beenobserved concerning colour and colour changes of the latex.

In situ Pleistocene survival or post-glacial expansionand radiation

Although tropical regions have the potential to harborenormous species diversity, revealing 18 phylogenetic specieswithin L. volemus sensu lato in such a relatively small area ofnorthern Thailand, was beyond our expectations. With thisstudy included, no less than 60 species of Lactarius are nowrepresented in northern Thailand (Le 2007; Le et al. 2007a,b, c). This high degree of richness does not only account forLactarius, but also for other groups of macrofungi (e.g.Sanmee et al. 2008; Wannathes et al. 2009).

How did this diversity come about? Let us shed some lighton the evolutionary history of northern Thailand. Going backto the Oligocene and Early Miocene, rainforest in SoutheastAsia was very scarce due to a dry and seasonal climate. Thischanged during the Mid-Miocene as a wet climate becamedominant. The region was now colonized by rainforest, mostlikely accompanied by ectomycorrhizal fungi. This forestmost likely persisted as a continuum until the late Miocene-early Pliocene, when the uplift of the Tibetan plateau and theHimalayas induced its reduction (Morley 2000). Whereasmost records indicate subsequent cooling and drying trendsduring the Quaternary glacial maxima (Anshari et al. 2001;Cannon and Manos 2003; Dam et al. 2001; Gathorne-Hardyet al. 2002; Morley 2000; Penny 2001; van der Kaars et al.2001; White et al. 2004), there exists some contradictionconcerning the extent of the Pleistocene forests in northernThailand. In this regard, two possible scenarios could haveled to the rich diversity in this region.

(1) in situ Pleistocene survival resulted in the current highdiversity in northern Thailand. This hypothesis issupported by a palynological study of a peat-swamp,which revealed unexpected stability in the glacial-agedvegetation of northeast Thailand. Pollen analyses indicatethat the region supported a fagaceous-coniferous forestduring the late Pleistocene (Penny 2001). With Quercusand Pinus as dominating trees (Penny 2001), ectomycor-rhizal fungi could have persisted and diversified in thesein situ refugia.

(2) The current diversity in northern Thailand is the resultof a post-glacial colonization and subsequent radia-tion. According to Gathorne-Hardy et al. (2002), themost of Thailand was severely affected by Pleistocenedrought, with savannah dominating the area. Forestrefugia, were present in northern and eastern Borneo,northern and western Sumatra and the Mentawaiislands (Gathorne-Hardy et al. 2002). Most likely, theectomycorrhizal fungi in northern Thailand either went


extinct or followed the retracting forest southwards.However, after the glacial maxima, as forest was againpresent in northern Thailand, the few survivors couldnow re-colonize the region and diversify rapidly whileoccupying many open niches.

Based on our current data, strong arguments in favor ofone of these two hypotheses are difficult to provide. Futuremolecular clock analyses, statistical phylogeographic ana-lyses, ectomycorrhizal studies and a wide sampling in thewhole of Southeast Asia, are crucial to understand theprocesses which have led to the present day diversity.

Acknowledgements The first author is supported by the “BijzonderOnderzoeksfonds Ghent University” (BOF). The NFS grant (DEB-

0118776) of Prof. Dennis Desjardin made it possible for H.T. Le toconduct her research in Thailand. We would like to express ourgratitude to all who provided collections for this research and helpedduring the fieldwork: Ruben Walleyn, Else Vellinga, Dennis Desjardin,Samantha Karunarathna, Phongeun Sysouphanthong, Jian Kui-Liu,Rui-Lin Zhao and Michael Pilkington. We wish to acknowledge Prof.Kevin Hyde and Mae Fah Luang University, and Dr. SaisamornLumyong of Chiang Mai University for providing a Material TransferAgreement that allowed removal of fungal specimens from Thailand.We would like to thank Heroen Verbruggen for providing the scriptfor calculating MrBayes burn-in values and convergence times and thecomments on the MrBayes analyses. Andy Vierstraete is acknowl-edged for the comments on the DNA-extraction protocol. TheNational Botanical Garden of Belgium is thanked for the use of thescanning electron microscope and in particular Myriam de Haan forassisting in taking the photographs. Finally, we would like to thankthe reviewers for their comments.

Appendix

Appendix Pileus colour, spore measurements, pleuromacrocystidia measurements and length of pileipellis hairs of the morphologically examinedcollections. All measurements are shown in micrometer (μm)

Vouchercollection

Pileus colour Spore measurements Pleurolamprocystidia Pileipellis hairs(l×b)

Size (l×b)a Ornamentationheightb

Size (l×b) Wallthickness

Species 3

Van de Putte08-026

deep reddish brown 7.1–8.0–9.0×6.6–7.5–8.4 2.0 50–100(–106)×6–9 2–4 8–55×3–6

Van de Putte08-043

deep reddish brown 6.9–7.7–8.5×6.5–7.1–7.8(–7.9) 2.1 41–104×6–8(–9) 1.5–3(–4) 11–52×3–9

Le 229 dark brick 7.4–8.3–9.2(–9.3)×(6.7–)6.8–7.6–8.4 1.8 12–55(–67)×3.5–5

Le 232 brick 7.6–8.7–9.8×7.2–8.0–8.9 1.7 46–99×6–9 2–4.5 12–50×3–7

Le 284 deep reddish brown 55–88×5–9 2–4

Species 4

Van de Putte08-004

bright orange 7.1–7.8–8.5×6.5–7.3–8.0 2.0 47–115(–130)×(6)–7–10(–11) 1.5–4 17–38×4–8

Van de Putte08-005

bright orange 58–133×6–10 2–5 11–46×4–8.5

Van de Putte08-011

brown 7.3–8.3–9.3(–9.8)×6.7–7.6–8.4(–8.8) 2.0 39–108×6–10.5 2–4.5 15–40(–48)×3–8

Van de Putte08-032

bright orange 35–125×7–11 1.5–5 10–39(–43)×3.5–7

Le 285 cinnamon 7.5–8.4–9.2(–9.3)×7.2–7.7–8.2(–8.4) 2.2 39–86×5–11 1.5–5 15–36(–43)×3.5–7

L. longipilus

Le 168 brick 7.7–8.8–9.8(–10.5)×(7.1–)7.2–8.1–9.0(–9.1) 2.1 50–106×7–9(–10) 1–3 15–130×4–6.5

Le 184 dark brick 47–105(–116)×6–9 1–3.5 14–110×4–5.5

Le 206 deep reddish brown 7.6–8.4–9.3(–9.6)×6.9–7.7–8.5(–8.6) 2.3 47–106×6–9 1–3 11–130×4–6

Le 273 37–114×6.5–8 1–3 12–116×3–6

Species 8

Le 123 reddish brown 6.8–7.9–9.1×6.5–7.3–8.1 1.7 30–95(–104)×5.5–9 (1–)2–4 10–75×3.5–5

Van de Putte08-021

deep reddish brown 47–85×6–9 1.5–3

Le 170 deep reddish brown 53–122×5–9 1.5–4 9–70×4.5–6

L. distantifolius

Stubbe 07-461 rusty orange 7.8–8.8–9.7×6.9–7.8–8.6(–8.7) 2.1 59–145×5–7 1–3 12–60×4–9.5

Le 288 rusty orange 45–155×5–6 1–2.5 11–59×5–7

Le 289 rusty orange 8.0–9.1–10.3(–10.4)×7.5–8.2–8.9 2 56–128×5–6 1.5–2.5 13–45×5–7


Appendix (continued)

Vouchercollection

Pileus colour Spore measurements Pleurolamprocystidia Pileipellis hairs(l×b)

Size (l×b)a Ornamentationheightb

Size (l×b) Wallthickness

L. crocatus

Van de Putte08-034

deep ochraceousorange

39–132×6–11 2–5 14–69(–75)×5–11

Van de Putte08-036

deep ochraceousorange

53–130×6–12(–14) 2–5(–6) 12–65×4–6

Le 17 7.5–8.3–9.0(–9.1)×6.6–7.4–8.2(–8.5) 1.4 48–130×7–13 2–6

Le 202 yellowish orange 46–130×7–12 3–5(–6) 14–67×4–7

Le 222 yellowish orange 6.7–7.5–8.2(–8.4)×6.2–6.8–7.5 1.6 52–130×6–13 2–5(–6) 10–60(–65)

Le 224 47–128×8–13(–14) 2–5(–6)

Le 225 7.5–8.1–8.8×6.6–7.3–8.0 1.3 54–128×7–12(–13) 3–5(–6) 18–60(–70)×4.5–6

Le 234 yellowish orange toorange

48–128×8–11(–12) 2–5

Le 268 yellowish orange 7.1–7.9–8.6×6.6–7.3–7.9 1.6 45–115×7–12(–14) (1)–3–6(–7) 13–60(–67)×4–6.5

Species 11

Le 133 ochraceous orange 6.8–7.6–8.4(–8.5)×6.1–6.8–7.4 1.8 61–120×6–9 2–4 20–54×4–12.5

Stubbe 07-402 pale brownish orange 7.1–8.2–9.3(–9.6)×6.4–7.4–8.4(–8.8) 1.8 45–115×(5–)6–8(–9) (1–)1.5–3 17–52×4–9

Le 321 ochraceous orange (7.3–)7.5–8.7–9.8×(6.5–)6.7–7.8–9.0 1.6 52–125×(5–)6–8(–9) 2–4 10–60(–73)×4–5.5

Le 337 cinnamon 6.9–8.0–9.2(–9.3)×(6.4–)6.5–7.2–8.0 1.7 / 13–60(–66)×4–7.5

L. vitellinus

Van de Putte08-024

pale orangish yellow 7.3–8.0–8.7×6.6–7.3–8.0(–8.2) 1.8 56–99×7–13 2–5 13–60(–65)×4–12

Van de Putte08-025

pale orangish yellow 7.0–8.0–8.9(–9.2)×6.7–7.4–8.1 1.8 57–95×6–12 2–5 18–60(–72)×4–9

Le 269 58–103×6–11 2–5

Le 348 pale orangish yellow (6.6–)7.1–7.9–8.8×(6.2–)6.5–7.4–8.3 2 54–95×6–12 3–5 12–70(–86)×4–8

L. acicularis

Stubbe 07-456 (7.2–)7.3–8.3–9.2×(6.9–)7.0–7.6–8.2 1.4 46–86×7–11.5 1.5–4(–4.5) 20–131×3.5–8

Van de Putte08-002

brown 33–90(–100)×6–9(–10) 1–2(–3.5) 17–110×3.5–6

Van de Putte08-007

brown 7.3–8.3–9.2(–9.3)×6.9–7.8–8.5 1.3 53–95×6–9(–10) 1–3(–4) 20–105×3–8

Van de Putte08-029

cinnamon 7.3–8.1–8.9×6.6–7.4–8.2 1.3 50–93×6–9 (1.5–)2–3 16–115×3–6.5

Van de Putte08-033

brownish cinnamon 7.0–7.9–8.8×6.5–7.2–7.8 1.3 56–90×(6.5)7–9.5 2–3.5 14–110×3.5–7

Le 199 brick 50–100×(5.5–)6–10.5 2–4.5(–5) 16–109×3–6

Le 277 cinnamon 7.9–8.5–9.1×7.1–7.8–8.4 1.1 20–123×3.5–6.5

Species 17

Le 139 pale yellowish white 41–126×(5–)6–10(–11) 1–4(–5) 19–66×4–6

Van de Putte08-023

pale yellowish white (7.2–)7.8–8.8–9.7(–9.8)×(6.8–)7.1–8.0–8.8 1.8 47–138(–155)×6–10(–11) 2–3(–4) 18–90×4–9

Le 19 pale yellowish white 7.6–8.5–9.4(–9.6)×7.0–7.7–8.5(–8.9) 1.8 55–135×7–10(–11) 2–4 24–80(–105)×3–6

Le 214 pale yellowish white 7.5–8.2–8.9×(6.8–)7.0–7.5–8.2(–8.3) 1.6 44–107×(5)7–10(–12) 1–3 10–85×3.2–7

Le 333 pale yellowish white 48–110(–123)×(5–)7–10(–11) 1.5–3(–4) 10–60(–77)

L. pinguis

Le 117 pale yellowish white 8.1–9.1–10.2×7.4–8.3–9.2(–9.6) 2.0 (48–)75–101×(7)–9–17 3–7 40–120(–130)×2–5

Le 169 pale yellowish white 8.0–9.0–10.0(–10.5)×7.4–8.4–9.4 2.0 57–108×9–16 4–7 32–110×3–8

Le 255 60–114×9–16 2–7 40–110×3.2–4

Le 278 60–118×7–16 3–7 53–115×3–4.5

a Average spore sizes are shown in italicsbMaximal height of the ornamentation, which is composed of ridges


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Lactarius volemus sensu lato (Russulales) from northern Thailand: morphological and phylogenetic...

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