Mycological Progress

, Volume 10, Issue 4, pp 453–479

Species recognition in Pluteus and Volvopluteus (Pluteaceae, Agaricales): morphology, geography and phylogeny

Authors

    • Biology DepartmentClark University
  • Andrew M. Minnis
    • Systematic Mycology & Microbiology LaboratoryUSDA-ARS
  • Stefano Ghignone
    • Instituto per la Protezione delle PianteCNR Sezione di Torino
  • Nelson MenolliJr.
    • Núcleo de Pesquisa em MicologiaInstituto de Botânica
    • Instituto Federal de EducaçãoCiência e Tecnologia de São Paulo
  • Marina Capelari
    • Núcleo de Pesquisa em MicologiaInstituto de Botânica
  • Olivia Rodríguez
    • Departamento de Botánica y ZoologíaUniversidad de Guadalajara
  • Ekaterina Malysheva
    • Komarov Botanical Institute
  • Marco Contu
    • Via Marmilla 12
  • Alfredo Vizzini
    • Dipartimento di Biologia VegetaleUniversità di Torino
Original Article

DOI: 10.1007/s11557-010-0716-z

Cite this article as:
Justo, A., Minnis, A.M., Ghignone, S. et al. Mycol Progress (2011) 10: 453. doi:10.1007/s11557-010-0716-z

Abstract

The phylogeny of several species-complexes of the genera Pluteus and Volvopluteus (Agaricales, Basidiomycota) was investigated using molecular data (ITS) and the consequences for taxonomy, nomenclature and morphological species recognition in these groups were evaluated. Conflicts between morphological and molecular delimitation were detected in sect. Pluteus, especially for taxa in the cervinus-petasatus clade with clamp-connections or white basidiocarps. Some species of sect. Celluloderma are apparently widely distributed in Europe, North America and Asia, either with (P. aurantiorugosus, P. chrysophlebius, P. fenzlii, P. phlebophorus) or without (P. romellii) molecular differentiation in collections from different continents. A lectotype and a supporting epitype are designated for Pluteus cervinus, the type species of the genus. The name Pluteus chrysophlebius is accepted as the correct name for the species in sect. Celluloderma, also known under the names P. admirabilis and P. chrysophaeus. A lectotype is designated for the latter. Pluteus saupei and Pluteus heteromarginatus, from the USA, P. castri, from Russia and Japan, and Volvopluteus asiaticus, from Japan, are described as new. A complete description and a new name, Pluteus losulus, are given for the African P. cervinus var. ealaensis. The American Volvopluteus michiganensis is described in detail. Taxonomic comments and a morphology-based key to all known species of Volvopluteus are provided.

Keywords

BiodiversityITSPhylogenyPluteusSpecies delimitationVolvopluteus

Introduction

The status of the genera traditionally classified in the Pluteaceae Kotl. & Pouzar (Agaricales, Basidiomycota) has been reassessed using molecular data, and two genera, Pluteus Fr. and Volvopluteus Vizzini et al., are now recognized and classified in the family, while Volvariella Speg. has been placed outside the Pluteoid clade (Justo et al. 2010). The traditional subdivision of Pluteus into three sections (Pluteus, Celluloderma Fayod and Hispidoderma Fayod) is essentially supported by molecular data, allowing the inclusion of taxa with partial veil and/or pileipellis as a cutis in sect. Celluloderma. The genus Volvopluteus has been erected to accommodate species formerly placed in Volvariella characterized by a gelatinous pileipellis and average basidiospore size over 11 μm (Justo et al. 2010). During the study of the phylogeny of this group we have gained some insights into several species-complexes of Pluteus and Volvopluteus and discovered new taxa that are described here.

Pluteus cervinus is the type species of the genus, but the limits of the morphological concept for this taxon have been a matter of debate for several decades (Banerjee and Sundberg 1995; Singer 1956; Smith 1968; Vellinga 1990). Around a dozen morphologically similar species or infraspecific taxa have been described. These differ from P. cervinus mostly by general aspect of the basidiocarps, habitat, presence of clamp-connections and shape and size of cheilocystidia. A similar situation involves the closely related species P. petasatus and taxa in this group with white basidiocarps such as P. pellitus and P. nothopellitus. Morphological delimitation of Pluteus species is also controversial for many taxa in sect. Celluloderma and sect. Hispidoderma, and the boundaries between European and North American taxa in these groups are unclear.

The conflicts between morphological, biological and phylogenetic species recognition in Fungi have been addressed in the literature (Taylor et al. 2000). In the case of Pluteus, the practical application of a biological species recognition is even more challenging than for other groups of Agaricales, due to the hardship of growing members of the genus in culture (Banerjee and Sundberg 1993a). Here we focus on the comparison between the classical morphological/biogeographic delimitation of species and the results of the phylogenetic analyses of molecular data. The nuclear ribosomal ITS region (including ITS1, 5.8S and ITS2) has been repeatedly used as a species rank genetic marker for different groups of Agaricales (Frøslev et al. 2006; Hughes et al. 2007; Vellinga 2003, 2007), and despite some limitations (Hughes et al. 2009; Nilsson et al. 2008), it offers a good level of resolution for the species rank.

The present paper addresses the following topics in detail:
  1. (i)

    Species delimitation and conflicts between morphological and phylogenetic species recognition in several lineages of the genus Pluteus: cervinus/petasatus clade, salicinus/albostipitatus clade, atromarginatus/losulus clade, chrysophlebius/phlebophorus clade, fenzlii/mammillatus clade, romellii/aurantiorugosus clade, plautus/longistriatus clade and leoninus clade.

     
  2. (ii)

    Description of three new species of Pluteus, P. saupei, P. heteromarginatus and P. castri, found during the molecular study of Pluteus. A detailed description and a new name, P. losulus, are also given for the African P. cervinus var. ealaensis.

     
  3. (iii)

    Description of the new species Volvopluteus asiaticus and the type collection of Volvopluteus michiganensis. Additional comments on the morphological variability of Volvopluteus gloiocephalus and Volvopluteus earlei and a morphology-based key of the known species of Volvopluteus are also provided.

    For these purposes phylogenetic analyses were performed on separate ITS datasets for each section of Pluteus (Pluteus, Celluloderma and Hispidoderma) and Volvopluteus.

     

Materials and methods

Fungal collections

A total of 185 fresh or dried specimens of Pluteus and Volvopluteus were selected for molecular sampling (Table 1). Additional sequences retrieved from GenBank are listed in Table 2. The geographic origin of the collections includes Africa, Asia, Europe, North and South America. In many cases, morphological study and identification of the collections was done previously to this work (Justo and Castro 2007a, 2010a; Menolli et al. 2010, Minnis and Sundberg 2010), but most specimens were re-examined during the course of this study. Newly collected materials and all the Asian and African herbarium collections were studied using standard procedures for morphological examination of the Pluteaceae (e.g. Justo and Castro 2007b; Minnis and Sundberg 2010). Descriptive terms for morphological features follow Vellinga (1988). The notation [60, 3, 2] indicates that measurements were made on 60 basidiospores from 3 basidiocarps in 2 collections. Color codes are from Munsell Soil-Color Charts (Munsell Color 2009). The following abbreviations are used in the descriptions: avl for average length, avw for average width, Q for quotient of length and width and avQ for average quotient. Herbarium acronyms follow Thiers (2010).
Table 1

New sequences generated for this study with GenBank accession numbers. P = Pluteus, V = Volvopluteus. An asterisk (*) indicates names being published concurrently with the present article. The symbol "!" indicates a type collection (holotypus, epitypus or paratypus)

Taxon

ITS

Collection (Herbarium)

Geographic origin

P. aff. cervinus

HM562151

REG13658

Germany

P. aff. cervinus

HM562168

REG13664

Germany

P. aff. cervinus

HM562128

TNSF897

Japan

P. aff. cervinus

HM562103

TNSF12370

Japan

P. aff. cervinus

HM562102

TNSF12352

Japan

P. aff. cervinus

HM562126

TNSF16091

Japan

P. aff. cervinus

HM562100

TNSF12361

Japan

P. aff. cervinus

HM562101

TNSF12351

Japan

P. aff. cervinus

HM562034

AJ78 (LOU)

Spain

P. aff. cervinus

HM562085

AJ148 (MICH)

USA (FL)

P. aff. cervinus

HM562098

AJ181 (MICH)

USA (MA)

P. aff. cervinus (white basidiocarp)

HM562178

SF7 (BPI)

USA (IL)

P. aff. cinereofuscus

HM562115

TNSF12400

Japan

P. aff. ephebeus

HM562080

Shaffer4673 (MICH)

France

P. aff. ephebeus

HM562198

Pearson s.n. (MICH, as P. plautus)

UK (England)

P. aff. leoninus I

HM562188

SF19 (BPI)

USA (IL)

P. aff. leoninus I

HM562190

SF21(BPI)

USA (MO)

P. aff. leoninus II

HM562139

TNSF11908

Japan

P. aff. nothopellitus

HM562060

AHS42452 (MICH)

USA (MI)

P. aff. nothopellitus

HM562177

SF5 (BPI)

USA (MI)

P. aff. phlebophorus

HM562186

SF16 (SIU)

USA (IL)

P. aff. podospileus

HM562196

Lundell2541 (MICH)

Sweden

P. aff. pouzarianus

HM562167

REG13620

Germany

P. aff. pouzarianus

HM562156

REG13683

Germany

P. aff. semibulbosus

HM562090

TNSF12393

Japan

P. albostipitatus (Dennis) Singer

HM562130

GF5374 (BR)

Democratic Rep. of Congo

P. albostipitatus (Dennis) Singer

HM562106

AJ187 (MICH)

USA (FL)

P. albostipitatus (Dennis) Singer

HM562057

AJ154 (MICH)

USA (FL)

P. atromarginatus (Singer) Kühner

HM562061

AJ75 (LOU)

Spain

P. atromarginatus (Singer) Kühner

HM562040

AJ76 (LOU)

Spain

P. atromarginatus (Singer) Kühner

HM562083

Sundberg3657 (SIU)

USA (OR)

P. atropungens A.H. Sm. & Bartelli

HM562059

A.H.Smith62033!(MICH)

USA (MI)

P. aurantiorugosus (Trog) Sacc.

HM562041

AJ219 (LOU)

Spain

P. aurantiorugosus (Trog) Sacc.

HM562081

ILLS42433

USA (IL)

P. aurantiorugosus (Trog) Sacc.

HM562072

Bigelow19232 (NY)

USA (MA)

P. aurantiorugosus (Trog) Sacc.

HM562074

Hoseny1740 (MICH)

USA (MI)

P. aurantiorugosus (Trog) Sacc.

HM562121

TNSF12391

Japan

P. aureovenatus Menolli & Capelari

HM562160

SP394388

Brazil

P. brunneidiscus Murrill

HM562042

AJ61 (MA)

Spain

P. castri Justo & E.F. Malysheva

HM562092

TNSF17602!

Japan

P. castri Justo & E.F. Malysheva

HM562099

TNSF17081

Japan

P. cervinus (Schaeff.) P. Kumm.

HM562165

REG13652

Germany

P. cervinus (Schaeff.) P. Kumm.

HM562166

REG13662

Germany

P. cervinus (Schaeff.) P. Kumm.

HM562155

REG13616

Germany

P. cervinus (Schaeff.) P. Kumm.

HM562150

REG16651

Germany

P. cervinus (Schaeff.) P. Kumm.

HM562152

REG13641!

Germany

P. cervinus (Schaeff.) P. Kumm.

HM562171

REG13661

Germany

P. cervinus (Schaeff.) P. Kumm.

HM562169

REG13622

Germany

P. cervinus (Schaeff.) P. Kumm.

HM562153

REG13618

Germany

P. cervinus (Schaeff.) P. Kumm.

HM562104

TNSF12347

Japan

P. cervinus (Schaeff.) P. Kumm.

HM562134

AJ192 (LOU)

Spain

P. cervinus (Schaeff.) P. Kumm.

HM562135

AJ79 (LOU)

Spain

P. cervinus (Schaeff.) P. Kumm.

HM562133

AJ191 (LOU)

Spain

P. cervinus (Schaeff.) P. Kumm.

HM562035

AJ82 (LOU)

Spain

P. cervinus (Schaeff.) P. Kumm.

HM562136

AJ77 (LOU)

Spain

P. cervinus (Schaeff.) P. Kumm.

HM562043

AJ106 (MICH)

USA (MA)

P. cervinus (Schaeff.) P. Kumm.

HM562176

SF4 (BPI)

USA (MI)

P. cervinus (Schaeff.) P. Kumm. (white basidiocarp)

HM562200

SF1 (BPI)

USA (IL)

P. cervinus var. scaber J.E. Lange

HM562075

A.H.Smith 39846 (MICH)

USA (WA)

P. cf. eugraptus

HM562116

TNSF12042

Japan

P. cf. nanus

HM562046

AJ216 (LOU)

Spain

P. chrysophlebius (Berk. & Ravenel) Sacc.

HM562125

TNSF12383

Japan

P. chrysophlebius (Berk. & Ravenel) Sacc.

HM562088

TNSF12388

Japan

P. chrysophlebius (Berk. & Ravenel) Sacc.

HM562064

AJ45 (MA)

Spain

P. chrysophlebius (Berk. & Ravenel) Sacc.

HM562180

SF10 (BPI)

USA (IL)

P. chrysophlebius (Berk. & Ravenel) Sacc.

HM562182

SF12 (BPI)

USA (IL)

P. chrysophlebius (Berk. & Ravenel) Sacc.

HM562181

SF11 (SIU)

USA (IL)

P. cinereofuscus J.E. Lange

HM562108

AJ229 (LOU)

Portugal

P. cinereofuscus J.E. Lange

HM562124

AJ34 (MA)

Spain

P. conizatus var. africanus E. Horak

HM562142

Thoen5250

Democratic Rep. of Congo

P. densifibrillosus Menolli & Capelari

HM562159

SP393696!

Brazil

P. dietrichii Bres.

HM562143

JLS1624 (J.L. Siquier)

Spain

P. eliae Singer

HM562076

Isaacs2460 (MICH)

USA (FL)

P. eludens E.F. Malysheva, Minnis & Justo*

HM562118

MA50497!

Portugal (Madeira)

P. eludens E.F. Malysheva, Minnis & Justo*

HM562185

SF15 (BPI)

USA (IL)

P. ephebeus (Fr.) Gillet

HM562044

AJ234 (LOU)

Spain

P. fenzlii (Schulzer) Corriol & P.-A. Moreau

HM562091

TNSF12376

Japan

P. fenzlii (Schulzer) Corriol & P.-A. Moreau

HM562111

Kotlaba F1020647 (F)

Slovakia

P. glaucotinctus E. Horak

HM562131

GF5274! (BR)

Democratic Rep. of Congo

P. glaucotinctus E. Horak

HM562147

SP394380

Brazil

P. glaucotinctus E. Horak

HM562157

SP394384

Brazil

P. glaucotinctus E. Horak

HM562132

Thoen5546 (BR)

Democratic Rep. of Congo

P. granularis Peck

HM562189

SF20 (BPI)

USA (IL)

P. granularis Peck

HM562069

Strack7 (SIU)

USA (IL)

P. granulatus Bres.

HM562048

AJ203 (LOU)

Spain

P. heteromarginatus Justo

HM562058

AJ172! (MICH)

USA (FL)

P. leoninus (Schaeff.) P. Kumm.

HM562077

Josserand s.n (MICH, as P. luteomarginatus)

France

P. leoninus (Schaeff.) P. Kumm.

HM562071

Halling6546 (NY)

USA (NY)

P. leoninus (Schaeff.) P. Kumm.

HM562045

AJ212 (LOU)

Spain

P. leoninus (Schaeff.) P. Kumm.

HM562187

SF17 (BPI)

USA (MI)

P. leoninus(Schaeff.) P. Kumm.

HM562215

DrewH (BPI)

USA(ID)

P. longistriatus (Peck) Peck

HM562149

SP394004

Brazil

P. longistriatus (Peck) Peck

HM562172

SP394386

Brazil

P. longistriatus (Peck) Peck

HM562158

SP393700

Brazil

P. longistriatus (Peck) Peck

HM562082

Minnis309203 (SIU)

USA (MO)

P. losulus Justo(≡ P. cervinus var. ealaensis Beeli)

HM562129

GF5273 (BR)

Democratic Rep. of Congo

P. magnus McClatchie

HM562087

CBM36790

Japan

P. mammillatus (Longyear) Minnis, Sundb. & Methven

HM562120

Singer244A (F)

USA (FL)

P. mammillatus (Longyear) Minnis, Sundb. & Methven

HM562086

Minnis309202 (SIU)

USA (MO)

P. mammillatus (Longyear) Minnis, Sundb. & Methven

HM562119

ASM7916! (EIU)

USA (MO)

P. multiformis Justo, A. Caball. & G. Muñoz*

HM562201

AC4249! (AH)

Spain

P. nothopellitus Justo & M.L. Castro

HM562063

AJ58!(MA)

Spain

P. pallescens P.D. Orton

HM562056

AJ214 (LOU)

Spain

P. pallidus Homola

HM562193

A.H.Smith62487! (MICH)

USA (MI)

P. pantherinus Courtec. & M. Uchida

HM562089

TNSF12882

Japan

P. pellitus (Pers.) P. Kumm.

HM562036

AJ72 (LOU)

Italy

P. pellitus (Pers.) P. Kumm.

HM562047

AJ74 (LOU)

Spain

P. pellitus (Pers.) P. Kumm.

HM562037

AJ202 (LOU)

Spain

P. petasatus (Fr.) Gillet

HM562084

Ammirati3103 (MICH)

USA (MI)

P. petasatus (Fr.) Gillet

HM562065

AJ143 (MICH)

USA (MA)

P. petasatus (Fr.) Gillet

HM562038

AJ201 (LOU)

Spain

P. petasatus (Fr.) Gillet

HM562109

AJ145 (MICH)

USA (FL)

P. petasatus (Fr.) Gillet

HM562175

SF3 (BPI)

USA (IL)

P. petasatus (Fr.) Gillet

HM562070

Mueller-Strack1 (SIU)

USA (IL)

P. petasatus (Fr.) Gillet

HM562073

Harrison10325 (MICH)

USA (MI)

P. petasatus (Fr.) Gillet

HM562179

SF9 (BPI)

USA (MO)

P. phaeoleucus E. Horak

HM562141

GF5102! (BR)

Democratic Rep. of Congo

P. phlebophorus (Ditmar) P. Kumm.

HM562112

Homola1849 (MICH)

USA (MI)

P. phlebophorus (Ditmar) P. Kumm.

HM562039

AJ81 (MA)

Spain

P. phlebophorus (Ditmar) P. Kumm.

HM562117

TNSF12394

Japan

P. phlebophorus (Ditmar) P. Kumm.

HM562138

AJ228 (LOU)

Spain

P. phlebophorus (Ditmar) P. Kumm.

HM562137

AJ194 (LOU)

Spain

P. phlebophorus (Ditmar) P. Kumm.

HM562144

AJ193 (LOU)

Spain

P. phlebophorus (Ditmar) P. Kumm.

HM562184

SF14 (SIU)

USA (MI)

P. plautus (Weinm.) Gillet

HM562055

AJ209 (LOU)

Spain

P. podospileus Sacc. & Cub.

HM562122

TNSF12398

Japan

P. podospileus Sacc. & Cub.

HM562049

AJ204 (LOU)

Spain

P. pouzarianus Singer

HM562170

REG13619

Germany

P. pouzarianus Singer

HM562154

REG13626

Germany

P. pouzarianus Singer

HM562096

TNSF12371

Japan

P. pouzarianus Singer

HM562050

AJ208 (LOU)

Spain

P. puttemansii Menolli & Capelari

HM562164

SP393698!

Brazil

P. riberaltensis var. conquistensis Singer

HM562162

SP393704

Brazil

P. romellii (Britzelm.) Sacc.

HM562123

TNSF12387

Japan

P. romellii (Britzelm.) Sacc.

HM562054

AJ215 (LOU)

Spain

P. romellii (Britzelm.) Sacc.

HM562183

SF13 (SIU)

USA (IL)

P. romellii (Britzelm.) Sacc.

HM562078

Shaffer3715 (MICH)

USA (MI)

P. romellii (Britzelm.) Sacc.

HM562105

Sundberg24198112 (SIU)

USA (MO)

P. romellii (Britzelm.) Sacc.

HM562062

AJ232 (LOU)

Spain

P. rugosidiscus Murrill

HM562079

Homola109 (MICH)

USA (MI)

P. salicinus (Pers.) P. Kumm

HM562051

MA67874

Spain

P. salicinus (Pers.) P. Kumm

HM562174

SF2 (BPI)

USA (MI)

P. sandalioticus Contu & Arras

HM562052

AJ200 (LOU)

Spain

P. sandalioticus Contu & Arras

HM562107

AJ60 (MA)

Spain

P. saupei Justo & Minnis

HM562113

ILLS42441!

USA (IL)

P. seticeps (G.F. Atk.) Singer

HM562199

Shaffer798 (MICH)

USA (IL)

P. seticeps (G.F. Atk.) Singer

HM562192

SF24 (SIU)

USA (WI)

P. seticeps (G.F. Atk.) Singer

HM562191

SF23 (SIU)

USA (MO)

P. sp. I (sect. Hispidoderma)

HM562127

TNSF12372

Japan

P. sp. I (sect. Hispidoderma)

HM562114

TNSF12365

Japan

P. sp. II (sect. Celluloderma)

HM562148

SP394382

Brazil

P. sp. II (sect. Celluloderma)

HM562146

SP394387

Brazil

P. sp. III (sect. Celluloderma)

HM562145

SP394379

Brazil

P. sp. IV (sect. Celluloderma)

HM562173

SP394383

Brazil

P. sp. V (sect. Celluloderma)

HM562161

SP394389

Brazil

P. sp. VI (sect. Celluloderma)

HM562216

DrewU (BPI)

USA (IL)

P. spegazzinianus Singer

HM562194

SingerM3377 (MICH)

Argentina

P. stirps subcervinus

HM562093

TNSF12349

Japan

P. stirps subcervinus

HM562094

TNSF12348

Japan

P. stirps subcervinus

HM562097

TNSF12356

Japan

P. stirps subcervinus

HM562095

TNSF12360

Japan

P. stirps subcervinus

HM562217

DrewP (BPI)

USA (ID)

P. stirps subcervinus (white basidiocarp)

HM562068

Thiers39341 (SIU)

USA (CA)

P. thomsonii (Berk. & Broome) Dennis

HM562053

AJ206 (LOU)

Spain

P. thomsonii (Berk. & Broome) Dennis

HM562066

MA54629

Spain

P. thomsonii (Berk. & Broome) Dennis

HM562067

AMD120 (SIU)

USA (IL)

P. thomsonii (Berk. & Broome) Dennis

HM562197

Homola930 (MICH)

USA (MI)

P. umbrosus (Pers.) P. Kumm.

HM562140

AJ213 (MA)

Spain

P. viscidulus Singer

HM562110

SingerT797! (MICH)

Argentina

P. xylophilus (Speg.) Singer

HM562163

SP393707

Brazil

V. asiaticus Justo & Minnis

HM562206

TNSF15191!

Japan

V. earlei (Murrill) Vizzini, Contu & Justo

HM562205

Mamet 7 (BR)

Democratic Rep. of Congo

V. earlei (Murrill) Vizzini, Contu & Justo

HM562204

MA22816

Spain

V. earlei (Murrill) Vizzini, Contu & Justo

HM246496

TOAV133 (as Volvariella cookei)

Italy

V. earlei (Murrill) Vizzini, Contu & Justo

HM246498

TOHG2001

Italy

V. earlei (Murrill) Vizzini, Contu & Justo

HM246499

TOHG1973 (as Volvariella acystidiata)

Italy

V. earlei (Murrill) Vizzini, Contu & Justo

HM246497

TOAV134

Italy

V. gloiocephalus (DC.) Vizzini, Contu & Justo

HM562207

LOU18619

Portugal

V. gloiocephalus (DC.) Vizzini, Contu & Justo

HM562209

LOU18247

Spain

V. gloiocephalus (DC.) Vizzini, Contu & Justo

HM562208

LOU13710

Spain

V. gloiocephalus (DC.) Vizzini, Contu & Justo

HM562202

AJ239 (LOU)

Spain

V. gloiocephalus (DC.) Vizzini, Contu & Justo

HM562203

PBM2272

USA (CA)

V. gloiocephalus (DC.) Vizzini, Contu & Justo

HM246495

TOAV136

Italy

V. gloiocephalus (DC.) Vizzini, Contu & Justo

HM246490

TOAV135

Italy

V. michiganensis (A.H. Sm.) Justo & Minnis

HM562195

A.H.Smith32-590! (MICH)

USA (MI)

Table 2

Sequences retrieved from GenBank. Original names for misidentified sequences are indicated in parentheses. P = Pluteus. An asterisk (*) indicates names being published concurrently with the present article. The symbol "!" indicates a type collection (holotypus, epitypus or paratypus)

Taxon

ITS

Collection

Geographic origin

P. aff. nothopellitus (as "P pellitus")

FJ774078

LE 217548

Russia

P. aff. romellii (as "P. romellii")

AY854065

ECV3201

USA (CA)

P. albostipitatus

FJ816656

SP393713

Brazil

P. albostipitatus

FJ816661

SP 393714

Brazil

P. atromarginatus

EF530926

UBC F16254

Canada

P. atromarginatus

FJ774075

LE 246081

Russia

P. atromarginatus

DQ494687

HKAS 31573

China

P. aureovenatus

FJ816663

SP 393697

Brazil

P. castri (as "P. aurantiorugosus")

FJ774077

LE 216873

Russia

P. cervinus

EU486448

UBC F16293

Canada

P. cf. nanus

FJ774081

LE 213093

Russia

P. dominicanus var. hyalinus Menolli & Capelari

FJ816665

SP 393695!

Brazil

P. eludens* (as "P. podospileus")

FJ774085

LE 213015

Russia

P. fenzlii

FJ774082

LE 246083

Russia

P. fluminensis Singer

FJ816664

SP 393711

Brazil

P. fluminensis Singer

FJ816655

SP 393710

Brazil

P. fuligineovenosus E. Horak

FJ816662

SP 393705

Brazil

P. granulatus

FJ774086

LE 212990

Russia

P. harrisii Murrill

FJ816666

SP 393709

Brazil

P. harrisii Murrill

FJ816654

SP 393708

Brazil

P. jamaicensis Murrill

FJ816657

SP 393706

Brazil

P. petasatus

AF085495

CBS441.85

Czech. Rep.

P. plautus

FJ774076

LE 213024

Russia

P. romellii

FJ774073

LE 217944

Russia

P. salicinus

FJ774087

LE 215427

Russia

P. semibulbosus (Lasch) Gillet

FJ774080

LE 227534

Russia

P. sp. VII (as "P. exiguus")

FJ774083

LE 226543

Russia

P. sublaevigatus (Singer) Menolli & Capelari

FJ816667

SP 393694

Brazil

P. thomsonii

FJ774084

LE 234787

Russia

P. xylophilus

FJ816659

SP 393701

Brazil

P. xylophilus

FJ816660

NMJ147 (SP)

Brazil

Uncultured Basidiomycete

DQ672275

Environmental sample (soil)

Australia

Uncultured Basidiomycete

AM901879

Environmental sample (house dust)

Finland

Uncultured Basidiomycete

AY969369

Environmental sample (hardwood litter)

USA(NC)

Uncultured fungus

FM999644

Environmental sample (sporocarp)

USA(OH)

Uncultured fungus

FM999650

Environmental sample (sporocarp)

USA(OH)

Uncultured fungus

FM999562

Environmental sample (sporocarp)

USA(OH)

Uncultured fungus

FM999557

Environmental sample (sporocarp)

USA(OH)

Information about the morphology and ecology of the taxa here discussed is based not only on the specimens sampled for molecular analyses, but also on the study of many European and American collections of Pluteus and Volvopluteus (Justo and Castro 2007a, 2010a; Menolli and Capelari 2010; Menolli et al. 2010; Minnis and Sundberg 2010; Rodríguez and Guzmán-Dávalos 2007).

DNA extraction, PCR, sequencing and alignments

Approximately 0.05–0.10 g of tissue (preferably gills) from each collection were ground directly in a 1.5 ml eppendorf tube, using plastic pestles, or in a mortar with liquid nitrogen. DNA was extracted using 3% SDS extraction buffer and was then isolated by the sequential addition of phenol chloroform and chloroform-isoamyl alcohol; finally, isopropyl alcohol and 3 M sodium acetate were added to precipitate the DNA, which was washed with 70% ethanol and resuspended in sterile water.

Primer pairs ITS1F–ITS4 were used to amplify the ITS region (Gardes and Bruns 1993; White et al. 1990). The amplification products were sequenced using ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction reagents with the same primer combination. Sequencing was carried out on an ABI model 3130 Genetic Analyzer. Raw data were processed using Sequencher 4.7 (GeneCodes, Ann Arbor, Michigan).

Sequences were aligned using MAFFT (http://align.bmr.kyushu–u.ac.jp/mafft/online/server/). The alignments were then examined and manually corrected using MacClade 4.05 (Maddison and Maddison 2002). Alignments have been deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S10654).

Sequence divergence was calculated using MatGAT (Campanella et al. 2003).

Phylogenetic analyses

Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Analysis (BA) were performed with the following parameters: (i) MP: Equally weighted parsimony analysis was performed using PAUP *4.0.b10 (Swofford 2002). One thousand heuristic search replicates were performed with starting trees generated by stepwise addition with random addition sequences followed by Tree Bisection Reconnection branch swapping. Up to two trees were kept in each replicate. Parsimony bootstrap analysis was performed with 1000 replicates, each with 10 random taxon addition sequences and branch swapping set to subtree pruning and regrafting; (ii) ML: The analysis was run in the RAxML servers (http://phylobench.vital–it.ch/raxml–bb/index.php; which implements the search protocol of Stamatakis et al. 2008), under a GTR model with one hundred rapid bootstrap replicates; (iii) BA: The analysis was run using MrBayes 3.1 (Ronquist and Huelsenbeck 2003) for ten million generations, under a GTR model, with four chains, and trees sampled every 100 generations; after examining the graphic representation of the likelihood scores, using Tracer (http://tree.bio.ed.ac.uk/software/tracer/), the burn-in period was set to 1.5 million generations for all datasets. The three analyses were performed in all the datasets presented here. The following abbreviations are used: Most Parsimonius Trees (MPT), Bootstrap (BS) and Posterior Probability (PP).

Results

Analyses of section Pluteus (Fig. 1)

The dataset includes 98 Pluteus sect. Pluteus ITS sequences and 2 of sect. Celluloderma (P. cinereofuscus, P. chrysophlebius) that were used as an outgroup. The final dataset consists of 649 characters (gaps included), of which 229 are parsimony informative. In the MP analysis, 1840 MPT were recovered (Length = 699; CI = 0.58; RI = 0.90). One of the 1840 MPT is shown in Fig. 1.
https://static-content.springer.com/image/art%3A10.1007%2Fs11557-010-0716-z/MediaObjects/11557_2010_716_Fig1_HTML.gif
Fig. 1

One of the1840 MPT from the analysis of sect. Pluteus. Branch lengths are shown on the branches

Three major clades are recovered, with moderate to high support, in all the analyses.

cervinus/petasatus clade. Nine major lineages (Fig. 1, clades I to IX) can be recognized: P. cervinus, P. aff. cervinus, P. pouzarianus (including P. pouzarianus, P. brunneidiscus, P. spegazzinianus and P. aff. pouzarianus), P. stirps subcervinus, P. petasatus (including P. petasatus, P. viscidulus, P. magnus), P. xylophilus, P. pellitus, sandalioticus and P. aff. nothopellitus. All these clades are present in the strict consensus tree of MP analysis, the best tree from the ML analysis and the 50 percent majority rule consensus tree from the BA analysis. The topological relations between these groups are usually the same in the three different analyses (Fig. 1), with the following exceptions:

In the ML and BA trees, P. aff pouzarianus is sister to P. spegazzinianus and P. pouzarianus, while in the strict consensus tree of the MP analysis the relations among these three clades are unresolved (Fig. 1).

In the ML and BA analyses P. aff. nothopellitus is placed as the sister clade to P. petasatus, while in the strict consensus tree of the MP analysis it is basal to P. petasatus, P. xylophilus, P. pellitus and P. sandalioticus (Fig. 1).

The internal topology of P. cervinus, P. aff. cervinus and P. petasatus is poorly resolved in all the analyses, though some relationships are consistently recovered:

In P. cervinus, the collection identified as P. cervinus var. scaber and the GenBank sequences of P. cervinus and an environmental sample from housedust are always grouped together and separately from the other sequences in this clade. The Japanese sequence of P. cervinus always clusters separately from all other sequences. The remaining sequences always cluster together, though with many possible alternative topologies in the 1840 MPT.

In P. aff. cervinus, only two pairs of sequences are constantly grouped together: P. nothopellitus AJ58 & P. aff. cervinus AJ78 and P. aff. cervinus TNSF 12352 & TNSF 12370.

In P. petasatus, the sequence of P. magnus is placed as sister to the remaining sequences in this clade in the ML tree and in the 50 percent consensus tree of the MP analysis but this relationship is not recovered in the BA tree or the strict consensus tree of the MP analysis.

salicinus/albostipitatus clade. Five distinct lineages (clades X to XIV in Fig. 1) are recognized: P. albostipitatus (including P. albostipitatus, P. phaeoleucus, P. densifibrillous), P. salicinus, P. harrisii (including P. harrisii, P. puttemansii), P. saupei, P. glaucotinctus. Except for the internal topology of the P. albostipitatus, P. harrisii and P. glaucotinctus clades, all topological relations between and inside these clades are the same in all the analyses (Fig. 1).

In P. albostipitatus, the two collections from Florida (AJ154, AJ187) cluster together in all the analyses, but the general internal topology of this clade changes among the different analyses and the 1840 MPT.

In P. harrisii, the sequence of P. harrisii FJ816654 alternatively clusters with the other sequence of P. harrisii or with the sequence of P. puttemansii.

In P. glaucotinctus, the sequence from the collection Thoen5546 alternatively clusters with the Brazilian collections (SP394380, SP394384) or with the holotype of P. glaucotinctus from Africa (GF 5274). In the 50 percent majority rule consensus tree from the MP analysis the clustering of Thoen5546 and the Brazilian collections is recovered.

atromarginatus/losulus clade. This clade (lineages XV and XVI in Fig. 1) receives 91% BS in the ML analysis, but much lower support in the MP (79% BS) and the BA (0.78 PP) analyses. In all analyses P. losulus (≡ P. cervinus var. ealaensis, see Taxonomy section) is placed as the sister group of the P. atromarginatus clade. The sequence from the holotype of P. atropungens is placed as basal to all sequences of P. atromarginatus (Fig. 1).

Analyses of section Celluloderma (Fig. 2)

The dataset includes 79 Pluteus sect. Celluloderma ITS sequences and 2 of sect. Pluteus (P. glaucotinctus, P. losulus) that were used as an outgroup. The final dataset consists of 675 characters (gaps included) of which, 270 are parsimony informative. In the MP analysis, 259 MPT were recovered (Length = 1294; CI = 0.42; RI = 0.82). One of the 259 MPT is shown in Fig. 2.
https://static-content.springer.com/image/art%3A10.1007%2Fs11557-010-0716-z/MediaObjects/11557_2010_716_Fig2_HTML.gif
Fig. 2

One of the 259 MPT from the analysis of sect. Celluloderma. Branch lengths are shown on the branches

Seven well supported clades are recognized in all the analyses: chrysophlebius/phlebophorus clade, thomsonii clade, cinereofuscus clade, podospileus/seticeps clade, romellii/aurantiorugosus clade, fenzlii/mammillatus clade and ephebeus clade. Three species are placed outside these major lineages: (i) P. dietrichii is placed as the sister taxon to all other species in sect. Celluloderma in all the analyses. (ii) P. eliae is placed as the sister taxon to the podospileus/seticeps clade in the ML and MP analyses but with no statistical support. In the BA analysis it is placed in an unresolved position together with the podospileus/seticeps, ephebeus, fenzlii/mammillatus, romellii/aurantiorugosus and cinereofuscus clades. (iii) Pluteus sp. V (SP394389) is placed as the sister taxon of the romellii/aurantiorugosus clade in the ML and BA analyses, but this relationship does not get statistical support. In all 259 MPT, this taxon is placed as sister to all taxa in sect. Celluloderma except P. dietrichii (Fig. 2).

chrysophlebius/phlebophorus clade (Fig. 2, clade I). Two major clades corresponding to P. chrysophlebius (including P. chrysophaeus sensu Vellinga (1990), see Discussion section) and P. phlebophorus (including P. pallidus and P. luctuosus) are recovered in all the analyses with essentially the same topology depicted in Fig. 2. In all analyses P. chrysophlebius and P. phlebophorus collections are grouped are according to their geographic origin (see Discussion section). P. rugosidiscus is placed as sister to the P. chrysophlebius clade. Four additional taxa are placed in the same subclade as P. phlebophorus: P. cf. eugraptus, P. aff. phlebophorus, Pluteus sp. VI and Pluteus cf. nanus.

thomsonii clade (Fig. 2, clade II). P. dominicanus var. hyalinus is placed as sister to the sequences of P. thomsonii in all analyses. Except for the clustering of P. thomsonii LE234787 from Russia and P. thomsonii AJ206 from Spain, the internal topology of P. thomsonii varies among the different analyses.

cinereofuscus clade (Fig. 2, clade III). It includes P. cinereofuscus, P. eludens, P. fluminensis, P. fuligineovenous, P. jamaicensis, P. multiformis and P. pallescens. Several potentially undescribed taxa are also included here: P. aff. cinereofuscus, Pluteus sp. II (SP394382, SP394387), Pluteus sp. III (SP 394379). One of the two collections included in this study morphologically assignable to P. nanus (AJ216) and two enviromental sequences are also placed in this clade.

podospileus/seticeps clade (Fig. 2, clade IV). P. podospileus, a closely related taxon (Pluteus aff. podospileus Lundell 2541) and P. seticeps are placed here. The internal topology of this clade is the same in all the analyses.

romellii/aurantiorugosus clade (Fig. 2, clade VI). It includes three well-supported clades, corresponding to P. romellii, P. aurantiorugous and a third, more diverse, clade that contains P. sublaevigatus, P. aureovenatus and the Californian sequence previously identified as P. romellii. The Brazilian collection SP394383 is also included in this clade, and it is placed as sister to all other taxa.

fenzlii/mammillatus clade (Fig. 2, clade VII). It contains P. fenzlii and P. mammillatus (three sequences each). In all the analyses the internal topology for each species is unresolved as the sequences are almost 100% identical (Fig. 2).

ephebeus clade (Fig. 2, clade VIII). It contains two distinct clades of P. ephebeus sensu lato and P. riberaltensis var. conquistensis. The internal topology of this clade is the same in all the analyses.

Analyses of section Hispidoderma (Fig. 3)

The dataset includes 29 Pluteus sect. Hispidoderma ITS sequences and 2 of sect. Celluloderma (P. seticeps, P. dietrichii) that were used as an outgroup. The final dataset consists of 682 characters (gaps included) of which, 222 are parsimony informative. In the MP analysis, only 1 equally most parsimonious tree (MPT) was recovered (Length = 666; CI = 0.66; RI = 0.86). The MPT is shown in Fig. 3.
https://static-content.springer.com/image/art%3A10.1007%2Fs11557-010-0716-z/MediaObjects/11557_2010_716_Fig3_HTML.gif
Fig. 3

The MPT from the analysis of sect. Hispidoderma. Branch lengths are shown on the branches

Three major well supported clades are recovered in all the analyses tough the relations between them are not resolved.

leoninus clade (Fig. 3, clade I). It includes the P. leoninus complex (P. leoninus, P. aff. leoninus I, P. aff. leoninus II), P. conizatus var. africanus and P. castri. P. aff. leoninus II appears as basal to P. leoninus and P. aff. leoninus I (MP) or as basal to P. conizatus var. africanus and P. castri (ML and BA).

umbrosus/granularis clade (Fig. 3, clade II). It includes the European isolate of P. umbrosus and the North American P. granularis.

longistriatus/plautus clade (Fig. 3, clade III). Includes P. granulatus, P. plautus, P. semibulbosus, P. longistriatus and a potentially undescribed species from Japan (Pluteus sp. I TNSF12372, TNSF 12365). The species here described as P. heteromarginatus is placed in all the analyses as the sister taxon of P. longistriatus. The internal topology of this clade is essentially the same in all the analyses.

The Japanese P. pantherinus is placed as sister to all other members of sect. Hispidoderma (MP), as sister to the leoninus clade (ML) or in an unresolved position together with the leoninus and the umbrosus/granularis clades (BA), but none of these placements gets statistical support.

Analyses of Volvopluteus (Fig. 4)

The dataset includes 15 Volvopluteus ITS sequences and 2 of Pluteus sect. Hispidoderma (P. longistriatus, P. heteromarginatus) that were used as an outgroup. The final dataset consists of 689 characters (gaps included), of which 125 are parsimony informative. In the MP analysis, 4 MPT were recovered (Length = 219; CI = 0.89; RI = 0.94). One of the MPT is shown in Fig. 4.
https://static-content.springer.com/image/art%3A10.1007%2Fs11557-010-0716-z/MediaObjects/11557_2010_716_Fig4_HTML.gif
Fig. 4

One of the 4 MPT from the analysis of Volvopluteus. Branch lengths are shown on the branches

Three clades are recognized in the analyses:
  • V. gloiocephalus clade. The European and Californian sequences of V. gloiocephalus are placed together in all the analyses. The seven sequences are 99.1-100% identical.

  • V. earlei clade. Sequences of this species from Spain, Italy and the Democratic Republic of Congo are included here. Collections originally identified under the names Volvariella cookei Contu (TOAV133) and Volvariella acystidiata N.C. Pathak (TOHG1973) fall within the molecular variation of V. earlei (see Taxonomic descriptions below). The six sequences are 99.2-100% identical.

  • V. michiganensis/V. asiaticus clade. The North American V. michiganensis and the Japanese V. asiaticus are placed together in all the analyses.

Each one of these clades is well-supported in the analyses but the relationships between them do not receive statistical support. In the all MPT (Fig. 4) V. gloiocephalus. is the the most early diverging clade and V. earlei is sister to V. michiganensis/V.asiaticus. In the ML tree V. earlei is the earliest diverging clade and V. michiganenis/V. asiaticus is sister to V. gloiocephalus. In the BA analysis relationships between the three clades are unresolved.

Discussion

The correspondence between the lineages recovered in the ITS analyses with morphological species and conflicts between morphological/molecular delimitation are discussed below.

Section Pluteus (Fig. 1)

cervinus-petasatus clade (clades I-IX in Fig. 1)

  1. (I)

    Pluteus cervinus

    Collections included here correspond to the most widely accepted morphological concept of P. cervinus (Schaeff.) P. Kumm. (Vellinga 1990): a taxon with brown to dark brown pileus; growing on wood of angiosperms or more rarely terrestrially; metuloids with 2-4(6) hooks at apices, not or only rarely bifid; cheilocystidia forming a well-developed strip, clavate or sphaeropedunculate, less than 70 μm long; clamp-connections absent in all parts of the basidiocarp. The only exceptions to this pattern are the collections SF1 (ILL, USA) with white basidiocarps and Smith 39846 identified as P. cervinus var. scaber J.E. Lange (WA, USA), which is characterized by the very dark (almost black) pileus.

    The highest percentage of sequence divergence (2.3%) occurs between the GenBank sequence of P. cervinus (EU486448, Canada) and some of the European collections. All other sequences have less than 2% sequence divergence and, almost identical sequences exist in distant geographic areas (e.g. AJ191 from Spain and AJ106 from Massachusetts are 99.7% identical).

    All collections sampled in this study grew on angiospermous wood or terrestrially (associated with organic debris), though P. cervinus has also been reported to occur on coniferous wood (Justo and Castro 2007a, Vellinga 1990).

    P. cervinus was originally described from the state of Bavaria (SE Germany) from material growing on decayed wood (Schaeffer 1774). No type collection exists for Pluteus cervinus. Fourteen modern collections from Bavaria were examined and sampled for molecular analyses. Eight collections represent P. cervinus in both morphological and molecular characters while the remaining six collections represent different taxa (P. aff. cervinus, P. pouzarianus, P. aff. pouzarianus). Since these taxa are distinguishable only by using microscopic and/or molecular characters, there is no way to know to which clade the original Agaricus cervinus (Schaeffer 1774) should be assigned.

    To avoid greater and continued confusion about the identity of P. cervinus, the species is lectotypified below by an illustration included in the original work and epitypified by a supporting modern collection, found near the topotype, from which DNA data were obtained.

    Pluteus cervinus (Schaeff.) P. Kumm., Führ. Pilzk.: 99. 1871.

    Agaricus cervinus Schaeff., Fung. Bavar. Palat. 4: 6. 1774 (basionym).

    Lectotypus of Agaricus cervinus Schaeff. (hic designatus): icon. in Schaeffer, Fung. Bavar. Palat. 1: Tab. X. 1762.

    Epitypus of Agaricus cervinus Schaeff. (hic designatus): Germany: Bavaria, near Bodenwöhr, railroad embankment in direction Schwandorf, on wood chips, under Frangula alnus, 7.IX.1995, A. Bresinsky, REG 13641.

    A full morphological description of Pluteus cervinus, in the sense accepted here, can be found in Vellinga (1990).

     
  2. (II)

    Pluteus aff. cervinus

    Collections in this clade are not distinguishable from P. cervinus based on macroscopic characters, ecology or geographic distribution, though they show a greater variation on microscopical characters. The pleurocystidia are commonly provided with bifid or coralloid hooks at the apices in some collections (TNSF 12361, 12351, 12370, 12352, 12347, AJ148), but they have non-divided hooks in the remaining collections. The cheilocystidia are scarce in some collections (TNSF 12351, 12352, REG 13664, 13658) not forming a well-developed strip that completely covers the lamellar edges like in P. cervinus, but in other collections they are frequent. The holotype of P. nothopellitus Justo & M.L. Castro (AJ58), a taxon characterized by white basidiocarps, and an additional collection with the same characteristics (SF7) are included here, but the remaining collections have pigmented basidiocarps.

    The maximum percentage of sequence divergence in this clade is 1.2%. The separation of P. cervinus and P. aff. cervinus is recovered in all the analyses with high statistical support. The sequence divergence between both clades varies between 2 and 4.2%. Representatives of P. cervinus and P. aff. cervinus may occur in the same locality, e.g. P. cervinus AJ106 and P. aff. cervinus AJ181 from Harvard Forest (MA, USA). Though some individual collections of P. aff. cervinus may be separated from P. cervinus based on the characteristics of the cystidia, separation of both clades is not possible when all morphological and geographic variation of each clade is compared. Therefore, P. aff. cervinus is considered to represent a cryptic phylogenetic lineage related to P. cervinus.

     
  3. (III)

    Pluteus pouzarianus and (IV) Pluteus stirps subcervinus

    Collections in both clades differ morphologically from P. cervinus and P. aff cervinus mainly by the presence of clamp-connections, especially at the base of cheilocystidia and in the pileipellis.

    By applying very strict criteria for lineage recognition, three groups can be distinguished in the pouzarianus clade:

    P. pouzarianus/P. brunneidiscus. Includes four collections that fit the morphological/ecological concept of P. pouzarianus Singer (Vellinga 1990) and one that corresponds to P. brunneidiscus Murrill (Justo and Castro 2007b). The main difference between these two taxa is the habitat, on conifers (P. pouzarianus) vs. angiosperms (P. brunneidiscus).

    P. spegazzinianus Singer. This taxon is characterized by the partially pigmented cheilocystidia (Singer 1958).

    P. aff. pouzarianus. These collections have no morphological or ecological differences with the other pouzarianus/brunneidiscus collections, though they are placed separately in all the analyses.

    Sequence divergence in the pouzarianus clade varies between 0-3%.

    Collections in the stirps subcervinus clade fit the morphological/ecological concept of either P. subcervinus (Berk. & Broome) Sacc., P. brunneidiscus and/or P. pouzarianus. The collection TNSF12356 has predominantly hyaline cheilocystidia, intermixed with some cystidia provided with pigment, and would fit the morphological concept of P. spegazzinianus. Sequence divergence in the stirps subcervinus clade varies between 0-1.3%.

    Whether the three lineages in the pouzarianus clade are considered to represent three different taxa or all the sequences are considered to represent one single taxon, separation of the phylogenetic lineages recognized in the analyses is not possible using morphological or ecological characters. Collections growing on coniferous wood may have identical sequences to collections growing on angiospermous wood (AJ61, AJ208). Collections with pigmented cheilocystidia occur in different clades (Singer M3377, TNSF 12356). On the other hand, the presence of clamp-connections is indeed a good morphological character to separate these taxa from P. cervinus and P. aff. cervinus.

    With the current sampling, it is not possible to decide which one of the over ten validly published species names in this group (Justo and Castro 2007b; Singer 1986) should be applied to each of the phylogenetic lineages. Additional collections, especially from regions of the world underrepresented in the present study (Australia, tropical Asia), should be sampled before a final taxonomic decision is made.

     
  4. (V)

    Pluteus petasatus

    Pluteus petasatus (Fr.) Gillet is accepted here in the sense of Vellinga (1990), including collections with a viscid, smooth pileus and collections with a dry, squamulose pileus, which are recognized by others as a separate species, viz. Pluteus patricius (Schulzer) Boud. (Banerjee and Sundberg 1995; Singer 1986). Both characters have a wide range of variation and intergrade to a great extent in the collections studied here. Microscopically, P. petasatus is characterized by the (usually) poorly developed strip of cheilocystidia and the predominantly magnus-type metuloid pleurocystidia (i.e. fusiform and unhooked) near the lamellar edge (Vellinga 1990; Justo and Castro 2007c). The isotype of P. viscidulus Singer, a similar looking Argentinian species, with slightly smaller basidiospores and abundant cheilocystidia (Singer 1958; Justo and Castro 2007c), falls within the molecular variation of P. petasatus.

    The Japanese collection of Pluteus magnus McClatchie (Takehashi and Kasuya 2009a), a species characterized by magnus-type metuloid pleurocystidia and abundant, up to 80 μm long cheilocystidia, is placed either as sister to all P. petasatus sequences (in the ML tree) or falls within the petasatus group (BA, MP strict consensus tree). There is 0.9-1.5% sequence divergence between P. magnus and the P. petasatus sequences. More collections of P. magnus, especially near the topotype in California, should be sampled for molecular data, to make a final decision about its possible synonymy with P. petasatus.

     
  5. (VI)

    Pluteus pellitus,

     
  6. (VII)

    Pluteus sandalioticus and

     
  7. (VIII)

    Pluteus xylophilus.

    Pluteus xylophilus (Speg.) Singer is a South American species, similar to P. petasatus in the presence of magnus-type pleurocystidia near lamella edge and the scarcity of cheilocystidia. It differs from P. petasatus mainly by the brown to dark-brown pileus (Menolli et al. 2010). Pluteus pellitus (Pers.) P. Kumm. is accepted here as a white species with clamp-connections and basidiospores 5-7.5 × 3.5-5 μm (Justo and Castro 2007c). Pluteus sandalioticus Contu & Arras is a South European species mainly associated with Quercus wood characterized by the long (>70 μm) cheilocystidia and presence of clamp-connections (Contu 2001, Justo et al. 2006).

    The degree of sequence divergence between P. pellitus and P. sandalioticus is only 0.9-1.5% and both clades are placed as sister taxa in the MP trees. Therefore, they could be considered as one single, morphologically variable species. On the other hand, the sister taxa relationship of these clades is not recovered in the ML and BA trees, and both clades can be differentiated according to the coloration of the basidiocarps and shape and size of the cheilocystidia. For the time being, both taxa are considered to represent two closely related, but different, species.

     
  8. (IX)

    Pluteus aff. nothopellitus

    This clade includes collections morphologically assignable to P. nothopellitus, a taxon characterized by white basidiocarps and the absence of clamp-connections (Justo and Castro 2007c). However, the holotype of P. nothopellitus (AJ58) is, in fact, a white variant of the normally pigmented taxon here named as P. aff. cervinus. The fact that usually pigmented taxa (P. cervinus, P. aff. cervinus) may exhibit albinistic phenotypes greatly complicates the recognition of P. aff. nothopellitus as a morphological species, though it is indeed a distinct phylogenetic lineage.

     

salicinus-albostipitatus clade (clades X-XIV in Fig. 1)

  1. (X)

    Pluteus albostipitatus (Dennis) Singer

    Molecular data support the treatment of the later described P. phaeoleucus E. Horak and P. densifibrillosus Menolli & Capelari as synonyms of P. albostipitatus. In the present concept, P. albostipitatus is a variable species regarding presence of fibrils on the pileus (from densely fribrillose to completely smooth) and size and shape of basidiospores. Modern descriptions of this species can be found in the works by Horak and Heinemann (1978), Menolli and Capelari (2010) as "P. densifibrillosus", Menolli et al. (2010) and Pegler (1983). Pluteus phaeoleucus supposedly differs from P. albostipitatus by the absence of pleurocystidia in the former (Horak and Heinemann 1978). However examination of the holotype of P. phaeoleucus (Goossens-Fontana 5102 at BR) revealed the presence of frequent pleurocystidia, morphologically similar to those found in P. albostipitatus.

    The internal topology of this clade varies among the different analyses, except for the grouping of the two collections from Florida, USA (AJ154, AJ187). These collections show the highest level of sequence divergence between any pair of sequences in this clade with 2.6% between AJ154 and FJ816661 from Brazil. However, there are no morphological differences between the Florida collections and the rest of the collections studied here. In view of the relatively wide, and non-correlated, morphological and molecular variation, this clade is considered to represent one single morphological species that spans across the paleo and neotropical regions, though it is possible that it harbors different, but morphologically cryptic, phylogenetic species.

     
  2. (XI)

    Pluteus salicinus (Pers.) P. Kumm.

    The combination of blue-green tinges on the basidiocarp, habitat on angiospermous wood, metuloids with distinct hooks at apex, clavate cheilocystidia and presence of clamp-connections define this species (Vellinga 1990). This taxon occurs in both Europe and North America. One of the American collections identified as P. salicinus represent a different species, described below as Pluteus saupei, which is placed in the analyses as the sister taxon to P. harrisii.

     
  3. (XII)

    Pluteus harrisii Murrill

    P. harrisii is usually described as possessing metuloids with poorly developed hooks (Banerjee and Sundberg 1995; Pegler 1983, 1997), and the two Brazilian collections studied here do fit this morphological concept. Examination of the holotypus (Jamaica, Troy and Tyre, on dead wood, 12-14.I.1909, WA Murrill & W Harris 956, NY) and the paratypus (Cuba, El Yunque, III.1903, Underwood & Earle 425, NY) revealed the presence of metuloids with coralloid hooks in the holotypus and metuloids with poorly developed hooks and with a thinner wall in the paratypus (Rodríguez 2010). The type collection of P. puttemansii Menolli & Capelari, which has metuloids with well-developed hooks, shows an ITS sequence almost identical to P. harrisii (0.4-1.1% divergence). Therefore, P. harrisii is considered here as a very variable species regarding the morphology of the pleurocystidia. See Menolli and Capelari (2010) "as P. puttemansii" and Menolli et al. (2010) for a full morphological comparison.

     
  4. (XIII)

    Pluteus saupei (see Taxonomic descriptions)

     
  5. (XIV)

    Pluteus glaucotinctus E. Horak

    This species is characterized by the thin-walled pleurocystidia and the blue-green tinges at the base of the stipe. The molecular variation in the ITS sequences, up to 4.3% sequence divergence between the holotypus (GF5274, Dem. Rep. of Congo) and the Brazilian collections (SP394380, 394384), is not correlated with the morphological variability of the collections. In the molecular analyses, the other African collection (Thoen 5546) alternatively clusters with the holotypus or with the Brazilian collections. As with P. albostipitatus, all collections studied here are considered to represent one morphological species, though cryptic phylogenetic lineages may exist inside this morphological concept.

     

atromarginatus-losulus clade (clades XV and XVI in Fig. 1).

  1. (XV)

    Pluteus atromarginatus / Pluteus atropungens

    P. atromarginatus (Konrad) Kühner is accepted here as a species with dark brown pileus, habitat on coniferous wood, brown pigmented lamellar edges, hooked metuloid pleurocystidia, pigmented cheilocystidia and clamp-connections in all tissues (Vellinga 1990). P. atromarginatus has also been reported as growing on angiospermous wood in North America (Banerjee and Sundberg 1995). P. atropungens A.H. Sm. & Bartellii is characterized by the unhooked pleurocystidia and the habitat on sawdust or angiospermous wood (Smith and Bartelli 1965; Justo and Castro 2007a). In all analyses the sequence from the holotype of P. atropungens is placed in a sister relationship to all the P. atromarginatus sequences, but more sampling is needed to make a taxonomic conclusion about the separation or synonymy of both species.

     
  2. (XVI)

    Pluteus losulus (see Taxonomic descriptions)

     

Section Celluloderma (Fig. 2)

Many taxa in this group (e.g. P. nanus, P. cinereofuscus, P. ephebeus, P. thomsonii) need significantly more collection sampling to address the correspondence between morphological species and molecular lineages, but some observations can be made for several taxa in this group.

Differentiation of Pluteus phlebophorus, P. chrysophlebius and P. nanus

The morphological delimitation, and putative synonymy, of these three species has been discussed at length in the literature for over 50 years (Homola 1972; Kühner and Romagnesi 1956; Minnis and Sundberg 2010; Orton 1986; Singer 1956; Vellinga 1990). Molecular data support both the separation and the morphological species concepts of P. chrysophlebius (Berk. & Ravenel) Sacc. (as "Pluteus chrysophaeus (Schaeff.) Quél.", see Discussion below) and P. phlebophorus (Ditmar) P. Kumm. proposed by Vellinga (1990). The situation of P. nanus (Pers.) P. Kumm. needs further study since collections fitting the broad morphological concept proposed by Vellinga (1990) are placed separately from each other in the phylogenetic analyses (Fig. 2).

The morphological concept of P. phlebophorus proposed by Vellinga (1990) needs to be slightly expanded in view of the molecular data. Pluteus luctuosus Boud. is separated from other taxa in this group by the dark brown lamellar edges with pigmented cheilocystidia. A collection with this characteristic (AJ81) shows an identical ITS sequence with the Spanish collections of P. phlebophorus with colorless cheilocystidia. P. pallidus Homola, characterized by the almost pigmentless basidiocarps, also falls within the molecular variation of P. phlebophorus.

North American collections, that are here considered to represent P. chrysophlebius (SF10, SF11, SF12, Fig. 2), are usually identified in North America under the names P. chrysophlebius or P. admirabilis (Peck) Peck. (Minnis and Sundberg 2010). However, Asian and European collections are typically identified in recent times as P. chrysophaeus sensu Vellinga (1990) by authors including Takehashi and Kasuya (2009b) and Justo and Castro (2007a). Morphological separations of collections from Asia, Europe and North America identified under those three names is not possible. P. chrysophaeus has been variously applied, by European workers, as representing a species with a brown or a yellow pileus, a key morphological character in this group. The absence of a type collection or illustration has caused considerable confusion, summarized by Singer (1956) and Orton (1986). We have reexamined the protologue of Agaricus chrysophaeus (Schaeffer 1774) and the accompanying illustration (Schaeffer 1771). The illustration depicting this species clearly indicates a brown colored pileus with yellowish tints in the stipe, which contrasts with the diagnosis that suggests the possibility of yellow hues in the pileus. This illustration, which is selected below as the lectotype of Agaricus chrysophaeus, does not represent the yellow-capped species known in the literature under the names P. chrysophaeus, P. chryshophlebius or P. admirabilis. It may represent P. phlebophorus or P. romellii, as circumscribed by Vellinga (1990), but in our opinion it is better to definitely abandon the epithet "chrysophaeus", as was done by Singer (1956) and Orton (1986). The name Pluteus chrysophlebius should be used for P. chrysophaeus in the sense of Vellinga (1990). Additional data on P. chrysophlebius, its synonymy and type study can be found in Minnis and Sundberg (2010).

Pluteus chrysophaeus (Schaeff.) Quél., Mém. Soc. Émul. Montbéliard, Sér. 2, 5: 116. 1872.

Agaricus chrysophaeus Schaeff., Fung. Bavar. Palat. 4: 67. 1774 (basionym).

Lectotypus of Agaricus chrysophaeus Schaeff. (hic designatus): icon. in Schaeffer, Fung. Bavar. Palat. III: Tab. CCLIII. 1771, ‘1770’.

P. phlebophorus and P. chrysophlebius collections are usually grouped according to their geographic origin. In both cases intercontinental sequence divergence is less than 2%. No morphological differences were found between collections from different continents. In the absence of biological intercompatibility tests, the decision to consider these geographic clades as different populations of the same species or different species that are distinguishable only by geographic distribution is, ultimately, arbitrary.

Pluteus rugosidiscus Murrill is considered to differ from P. chrysophlebius by the presence of greenish colors on the pileus (Homola 1972). In all analyses the sequence of P. rugosidiscus is placed in a sister relationship to all P. chrysophlebius sequences, with a percentage sequence divergence between 4.8-5.5% . This collection may represent an independent taxon, but more intensive sampling is needed to check the stability of the pileus color as a reliable diagnostic character to separate both taxa.

Pluteus mammillatus vs. Pluteus fenzlii

Both taxa are separated mainly by the color of the lamellar edge, yellow in P. fenzlii (Schulzer) Corriol & P.-A. Moreau, and white to greyish-red in P. mammillatus (Longyear) Minnis, Sundb. & Methven (Corriol and Moreau 2007; Malysheva et al. 2007, Minnis et al. 2006). Separation based on this character is not as straightforward as it seems, since collections of P. fenzlii may have white lamellar edges (Corriol and Moreau 2007; Malysheva et al. 2007). Other characters mentioned by Corriol and Moreau (2007) such as basidiospore size, morphology of pleurocystidia and color of the annulus, show a wide range of overlapping variation between both species.

Eurasian collections of P. fenzlii and North American collections of P. mammillatus cluster separately in all the analyses. The maximum percentage of sequence divergence between both clades is 2.5%. If they are considered as one taxon, the name Pluteus fenzlii has priority over P. mammillatus. If they are considered as separate species, geographic distribution is the only reliable character to tell them apart.

romellii/aurantiorugosus clade

Two clades corresponding to the morphological concepts of P. romellii (Britzelm.) Sacc. and P. aurantiorugosus (Trog) Sacc. (Minnis and Sundberg 2010, Vellinga 1990) are recovered in all the analyses. Collections of P. romellii from Europe, Asia and North America are not grouped according to their geographic origin. Morphological variation between P. romellii and collections identified by Minnis and Sundberg (2010) as P. fulvibadius Murrill is not correlated with species rank differences in the phylogenetic analyses. North American collections of P. aurantiorugosus cluster separately from the European and Asian collections sampled here, though the maximum percentage of sequence divergence between any pair of sequences (Bigelow19232 from Massachusetts and TNSF 19391 from Japan) is 1.4%. Both P. romellii and P. aurantiorugosus are considered to be widely distributed, at least, in the Northern Hemisphere (Minnis and Sundberg 2010; Vellinga 1990).

The Californian collection identified as P. romellii ("AY854065 aff. romellii", Fig. 2) does not represent this species, but a different taxon, related, or perhaps identical, to the South American P. sublaevigatus (Menolli et al. 2010).

Many taxa in this group share the conspicuous yellow, orange or red colors in the basidiocarps, though this characteristic also occurs in other lineages of Pluteus.

Sect. Hispidoderma (Fig. 3)

plautus/longistriatus clade

The wide morphological concept of P. plautus proposed by Vellinga and Schreurs (1985) is not supported by molecular data, as taxa considered synonymous by those authors (P. semibulbosus (Lasch) Gillet and P. granulatus Bres.) represent different species. More sampling in this group is needed to establish how many taxa can be recognized based on both morphological and molecular data.

leoninus clade

P. leoninus (Schaeff.) P. Kumm. is accepted here in the sense of Vellinga (1990), which includes small to large variants, with smooth or rugose pileus surfaces, yellow or yellow-brown pilei, and white to yellow lamellar edges. The North American P. flavofuligineus G.F. Atk. usually has darker brown colors in the pileus, but collections morphologically corresponding to P. flavofuligineus sampled in this study (Halling6546, SF17) do not show molecular differentiation with European specimens of P. leoninus.

Two North American collections (P. aff. leoninus I, SF19, SF21) represent a distinct molecular lineage, but they do not show morphological differentiation from P. leoninus. A collection from Japan (P. aff. leoninus II, TNSF11908) is also molecularly distinct from P. leoninus. This collection differs from P. leoninus in the presence of caulocystidia and probably represents a distinct species, but it is not formally described here due to the incomplete knowledge of its morphological characteristics.

Volvopluteus (Fig. 4)

Four species are recognized in all the analyses: V. asiaticus, V. earlei, V. gloiocephalus and V. michiganensis. The four taxa of Volvopluteus are clearly distinct on molecular grounds. Morphological separation of the species, based on colors of the basidiocarp, basidiospore size and shape, and morphology of the pleuro- and cheilocystidia, is usually possible but there is some degree of overlapping variation, especially between V. gloiocephalus and V. earlei (see Taxonomic descriptions and key below).

Taxonomic descriptions

Pluteus castri Justo & E.F. Malysheva, sp. nov. (Fig. 5)

MycoBank 518595
https://static-content.springer.com/image/art%3A10.1007%2Fs11557-010-0716-z/MediaObjects/11557_2010_716_Fig5_HTML.gif
Fig. 5

Pluteus castri. a. Basidiospores; b. Basidia; c. Pleurocystidia; c2; Pleurocystidia near lamellar edges. d. Cheilocystidia; e. Caulocystidia; f. Pileipellis elements. All from holotype (TNSF 17602). Photographs from LE212090. All scale bars = 10 μm

Latin Diagnosis. Pluteo leonino similis sed differt in cellulae in epicute pilei minoribus, caulocystidiis praesentibus et in spatii interne transcripti sequentia ("ITS").

Etymology. Named after Dr. Marisa Castro in honor of her outstanding contribution to the knowledge of the Pluteaceae and her dedication, spanning over three decades, to training new mycologists.

Pileus 10–50 mm, obtusely conical to campanulate-convex or plano-convex, with a low, broad umbo; surface smooth or covered with small squamules, at center venose and/or densely squamose, bright yellow to yellow-orange (approx. Mu. 10YR 8/8, 2.5Y 8/8) weakly hygrophanous; margin translucently striate. Lamellae crowded, free, ventricose, up to 10 mm broad, white-cream when young, later pink, with concolorous or whitish, flocculose, edges. Stipe 20–70 × 2–5 mm, cylindrical, broadened towards base (up to 7 mm); surface white or white-cream in the upper part, yellowish towards base (approx. Mu. 2.5Y 8/6), smooth or slightly pruinose at apex, longitudinally fibrillose towards base, with distinct squamules and white basal mycelium in the lower part. Context, in stipe and pileus, white to yellowish, yellow-orange under pileipellis. Smell and taste indistinct. Spore print not recorded.

Basidiospores [60, 4, 2] 5.2–7.0 (8.0) × 4.5–5.5 (6.0) μm, avl × avw = 6.1 × 5.0 μm, Q = 1.0–1.40, avQ = 1.21, mostly broadly ellipsoid, more rarely globose or ellipsoid. Basidia 20–35 × 6–10 μm, tetrasterigmate, clavate. Pleurocystidia, 40–85 × 12–22 μm, fusiform, narrowly lageniform or narrowly utriform, commonly provided with 1-3 finger-like excrescences at apices, near lamellar edges mostly obovoid or clavate and without excrescences, hyaline, thin-walled, frequent all over lamella faces. Lamellar edges sterile. Cheilocystidia 20–45 × 6–15 μm, fusiform or lageniform, with elongated apex or apical excrescence, hyaline, thin-walled, with thin, smooth walls, forming a well-developed strip. A second type of cheilocystidia, similar in shape and size to the pleurocystidia occurring near the lamellar edges, can be observed intermixed with the predominant fusiform-lageniform cheilocystidia. Pileipellis an euhymeniderm composed of elements 25–65 × 11–22 μm, (narrowly) clavate, conical, fusiform, spheropedunculate, sometimes with elongated apex or with apical excrescences, filled with yellow or yellow-brown intracellular pigment, with thin, smooth walls. Stipitipellis a cutis; hyphae 5–15 μm wide, cylindrical, colorless or with brown pigment, with thin, smooth walls. Caulocystidia (15)30–68 × 7–17 μm, fusiform or lageniform, commonly with mucronate or rostrate apices, with brown intracellular pigment, thin-walled, mostly fasciculate. Clamp-connections absent in all tissues.

Habit, habitat and distribution: Gregarious, sometimes in fascicles of up to six basidiocarps, on decayed wood of hardwoods (including Populus), also on piled wood chips. Known from Central Russia (Moscow and Samara Regions) and Japan (Kyushu and Honshu). Collected in July-August (Russia), May (Japan) and October (Japan).

Collections examined: JAPAN, Kyushu, Fukuoka prefecture, on piled wood chips, 5.V.2007, M. Shintani & S. Takehashi, TNSF 17602 (holotypus); Honshu, Ibaraki prefecture, Tsukuba, 25.X.2007, K. Osaku, TNSF 17081; RUSSIA: Central Russia, Moscow Region, Prioksko-Terrasny State Reserve, on decaying wood of Populus tremula, 15.VIII.1991, G.E. Levitskaya, LE 216873; Samara Region, near Pribrezhny, on decaying wood of deciduous tree, 1.VII.2007, E.F. Malysheva, LE 212090.

Pluteus castri closely resembles P. leoninus (Schaeff.) P. Kumm. in its external appearance and the pleurocystidia provided with small apical excrescences. Both taxa can be separated by the structure of the pileipellis: an euhymeniderm made up to clavate elements, 25–65 μm long, in P. castri, and a trichohymeniderm made up of narrowly fusiform elements, 60–230 μm long in P. leoninus (Vellinga 1990; pers. obs.). P. castri has caulocystidia, which are absent in P. leoninus. Molecular data clearly support the separation of P. castri from the P. leoninus complex (Fig. 3).

P. conizatus var. africanus E. Horak appears as sister to P. castri in all the analyses. Both taxa are characterized by the short elements in the pileipellis, in comparison to other members of sect. Hispidoderma. P. conizatus var. africanus has even shorter pileipellis elements, 16.5–40 μm long, than P. castri, the pleurocystidia are not provided with apical excrescences, and the cheilocystidia are slightly thick-walled (Horak and Heinemann 1978; pers. obs.). It is not known if P. conizatus var. africanus has caulocystidia as the stipitipellis has not been described for this taxon (Horak and Heinemann 1978). No caulocystidia were observed in the holotype and an additional collection of this taxon studied by us (GF 623, Thoen 5250, both at BR) but newly collected specimens should be checked to verify these observations.

Pluteus heteromarginatus Justo, sp. nov. (Fig. 6)

MycoBank 518596
https://static-content.springer.com/image/art%3A10.1007%2Fs11557-010-0716-z/MediaObjects/11557_2010_716_Fig6_HTML.gif
Fig. 6

Pluteus heteromarginatus. a. Basidiospores; b. Basidia; c. Pleurocystidia; d. Cheilocystidia; e. Caulocystidia; f. Pileipellis elements. All from Holotype (AJ172, MICH). All scale bars = 10 μm

Latin Diagnosis. Pluteo longistriato similis sed differt in pileo brunneo, cheilocystidiis raris, et in spatii interne transcripti sequentia ("ITS").

Etymology. The epithet makes reference to the heterogeneous nature of the lamellar edges, provided with sparse cheilocystidia, basidioles and fertile basidia.

Pileus 20–36 mm, convex to plano-convex, with or without a low, broad umbo; surface radially sulcate almost up to the center, smooth or only slightly rugose-squamulose at center, without conspicuous fibrils, brown (in the range of Mu. 7.5YR 6/6-6/8, 5/6-5/8, 4/6), darker at center and paler towards margin, hygrophanous, pallescent on drying, especially at center. Lamellae crowded, free, ventricose, up to 8 mm broad, white when young, later pink, with concolorous or whitish edges. Stipe 35–50 × 2–4 mm, cylindrical, slightly tapering towards apex; surface white or white-cream (approx. Mu. White Page 2.5Y 9.5/2), glabrous, longitudinally striate, under lens with brown dots all over. Context in stipe and pileus white, slightly cream colored at stipe base. Smell and taste indistinct. Spore print pink (Mu. 5YR 7/4-8/4).

Basidiospores [60, 3, 1] 5.3–7.2 (8.0) × 4.5–6.0 (7.0) μm, avl × avw = 6.2 × 5.3 μm, Q = (1.0) 1.15–1.30 (1.40), avQ = 1.18, broadly ellipsoid, rarely globose or ellipsoid. Basidia 24–34 × 9.5–11 μm, 4-spored, narrowly utriform or clavate. Pleurocystidia 40–65 × 10–17 μm, mostly fusiform or narrowly lageniform, with elongated apex or an apical flexuous excrescence 5–10 μm long, more rarely ovoid or (narrowly) utriform, with evenly disolved pale brown intracellular pigment or colorless, with thin, smooth walls, common all over Lamellar sides. Lamellar edge heterogeneous, with scattered cheilocystidia, basidioles and fertile basidia near the sides. Cheilocystidia 20–45 × 9.5–15 μm, (narrowly) clavate, narrowly utriform, more rarely obovoid with long peduncle or broadly fusiform, without apical appendages, colorless, with thin, smooth walls, scattered along lamellar edge, sometimes in clusters, not forming a well-developed strip. Pileipellis a trichohymeniderm mostly composed of elements 56–100 × 14–20 μm: individual elements very variable in shape, fusiform, narrowly clavate, lageniform, obovoid, utriform, filled with brown intracellular pigment, with thin, smooth walls. At the base of that elements, a second morphological type occurs: 24–28 × 14–16 μm clavate elements, also filled with brown intracellular pigment. Stipitipellis a cutis; hyphae 5–15 μm wide, cylindrical, colorless or with brown pigment; with thin, smooth walls. Caulocystidia 45–75 × 7–17 μm, fusiform or lageniform, sometimes with elongated apex, with brown intracellular pigment, with thin, smooth walls, scattered or in clusters. Clamp-connections absent in all tissues.

Habit, habitat and distribution. Gregarious, on decayed, unidentified (probably coniferous) wood. Collected in Northern Florida (USA) during summer (August).

Collection examined: USA Florida, St. Marks Wildlife Refuge, 26.VIII.2009, D. Floudas, AJ172 (MICH, holotypus).

P. heteromarginatus is morphologically similar to P. longistriatus (Peck) Peck. Both taxa share the hygrophanous, sulcate pileus, colored pleurocystidia and presence of short clavate elements in the pileipellis. P. longistriatus has a grey-brown, fibrillose, pileus, differently shaped pleuro-, cheilo- and caulocystidia, and the lamellar edge is not heterogeneous (Menolli and Capelari 2010). P. longistriatus has been reported from North and South America and molecular data confirm that this taxon occurs in both hemispheres. Morphological and molecular differences between the two support the recognition of P. heteromarginatus as a separate taxon.

All taxa placed by Singer (1986) in stirps longistriatus, mainly because of the sulcate-pectinate margin of pileus, differ from P. heteromarginatus in the hyaline pleurocystidia and crowded cheilocystidia. Other morphological differences are: (i) P. neophlebophorus Singer has strongly venose cap and longer (up to 145 μm) elements of pileipellis; (ii) P. sanctixaverii Singer has a pileipellis composed mostly of short sphaeropedunculate or clavate elements (up to 28.5 μm long), intermixed with dermatocystidia up to 72 μm long; (iii) P. oligocystis Singer has scarce pleurocystidia and a pileipellis mostly of short sphaeropedunculate or clavate elements (up to 41 μm long), intermixed with dermatocystidia up to 150 μm long (Singer 1958).

Other taxa of section Hispidoderma, that may superficially resemble P. heteromarginatus, in the non-fribrillose, striate-sulcate and hygrophanous pileus, with predominantly brown colors (viz. P. atriavellaneus Murrill, P. compressipes Murrill, P. sulcatus Singer) are morphologically distinct from P. heteromarginatus, among other characters, in the crowded cheilocystidia and longer pileipellis elements, up to 150 μm long (Pegler 1983, Singer 1958).

Pluteus losulus Justo, nom. nov. (Fig. 7a)

Pluteus cervinus var. ealaensis Beeli, Bull. Soc. Royal Bot. Belg. 61: 81. 1928 (replaced synonym); non Pluteus ealaensis Beeli Bull. Soc. Royal Bot. Belg. 61: 80. 1928 (= Pluteus congolensis Beeli).
https://static-content.springer.com/image/art%3A10.1007%2Fs11557-010-0716-z/MediaObjects/11557_2010_716_Fig7_HTML.gif
Fig. 7

a. Pluteus losulus (GF5273, BR); b. Pluteus saupei (ILLS42441); c. Volvopluteus asiaticus (TNSF 15191); dVolvopluteus michiganensis (Smith32-590, MICH). a. Basidiospores; b. Basidia; c. Pleurocystidia; c2; Pleurocystidia near lamellar edges. d. Cheilocystidia; e. Caulocystidia; f. Pileipellis elements. All scale bars = 10 μm

MycoBank 518597

Etymology. The epithet makes reference to one of the common names ("losulu") that this species receives in the Democratic Republic of Congo.

Pileus 30–90 mm, obtusely conical to convex or plano-convex, with a low, broad umbo; surface covered with radially arranged fibrils, glabrescent in older specimens, usually with small squamules at center, brown, redish-brown or greyish-brown, slightly pallescent with age; margin entire, not striate. Lamellae crowded, free, ventricose, approx. 10 mm broad, whitish when young, later pink, with whitish, flocculose, edges. Stipe 35–100 × 3–10 mm, cylindrical, broadened towards base; surface whitish, covered with longitudinal fibrils, concolorous with pileus. Context white. Smell acrid, taste very bitter. Spore print pinkish-brown.

Basidiospores [30, 1, 1] 6.0–9.0 (10.0) × 5.0–8.5 (9.0) μm, avl × avw = 7.5 × 6.6 μm, Q = 1.0–1.45, avQ = 1.16, globose to ellipsoid. Basidia 20–30 × 6–8 μm, tetrasterigmate, clavate, some with median constriction. Pleurocystidia metuloid, 50–87 × 15–25 μm, fusiform or narrowly utriform, provided with 2-4 hooks at apices, a few irregularly shaped and with bifid apices, near lamellar edges many metuloids without distinct hooks and with rounded apices, hyaline or with vacuolar brown pigment, with up to 4 μm thick wall, frequent all over lamellar faces. Lamellar edge sterile. Cheilocystidia 40–70 × 10–20 μm, (narrowly) clavate, lageniform, narrowly utriform or subcylindrical, hyaline, thin-walled, crowded, forming a well-developed strip. Pileipellis a cutis, with terminal elements 90–120 × 10–17 μm: individual elements cylindrical, some strongly tapering towards apex, mostly filled with brown intracellular pigment, with thin, smooth walls. Stipitipellis a cutis; hyphae 5–15 μm wide, cylindrical, colorless or with brown pigment, with thin, smooth walls. Caulocystidia 40–73 × 9–15 (–20) μm, fusiform, lageniform, narrowly utriform or clavate, some with elongated apices, with brown intracellular pigment, thin-walled, scattered. Clamp-connections present in all tissues, common at the base of cheilocystidia, caulocystidia and in pileipellis.

Habit, habitat and distribution: Gregarious, on decayed wood, in forests and coffee plantations. Common in the Democratic Rep. of Congo (Horak and Heinemann 1978).

Collections examined: DEMOCRATIC REP. OF CONGO. Panzi-Kivu, May 1953, Goosens-Fontana 5273 (BR).

The macroscopic description is adapted from Horak and Heinemann (1978), and the microscopic data are from our observations on the collection Goosens-Fontana 5273. Three additional collections of this species, including the holotype, are deposited at BR but they are very poorly preserved and no further observations could be made.

P. losulus was originally described as a variety of P. cervinus, but both molecular and morphological data indicate that it represents a distinct species, not closely related to P. cervinus or any of the clamp-bearing species in that group. P. losulus is characterized by the hooked and partially pigmented metuloid pleurocystidia; morphologically variable cheilocystidia and the presence of clamp-connections and caulocystidia. The latter character is rather unusual in sect. Pluteus as it has been described for only three species in the section. P. amphicystis Singer has unhooked metuloid pleurocystidia with crystals at the apices, no clamp-connections and metuloid caulocystidia. It has been reported from Bolivia, Martinique, Mexico and India (Rodríguez and Guzmán-Dávalos 2007; Pegler 1983; Pradeep et al. 2002; Singer 1958). P. martinicensis Singer & Fiard has predominatly unhooked metuloid pleurocystidia and pigmented cheilocystidia. It is known from México, Martinique and India (Rodríguez and Guzmán-Dávalos 2007; Pegler 1983; Pradeep et al. 2002). Pluteus triplocystis Singer has smaller basidiospores (5.5–6 × 5–5.5 μm) and differently shaped cheilocystidia and caulocystidia. It is known only from Mexico (Rodríguez et al. 2004).

Pluteus saupei Justo & Minnis, sp. nov. (Fig. 7b)

MycoBank 518598

Latin Diagnosis. Pluteo salicino similis sed differt in pleurocystidiis sine grandis cornus, cheilocystidiis lageniformibus, et in spatii interne transcripti sequentia ("ITS").

Etymology. Named after Dr. Stephen G. Saupe, the collector of the holotype, in honor of his discovery of the presence of psilocybin in Pluteus.

Pileus 10–35 mm, convex or plano-convex, with shallow depression at center; surface innately radially fibrillose, with blackish, spinulose squamules at center, grey to grey-brown, bruised regions become blue, with olive-green tinges in older specimens; margin translucently striate in older specimens. Lamellae crowded, free, ventricose, approx. 5 mm broad, pink, with slightly paler edges. Stipe 10–50 × 3–5 mm, cylindrical, broadened towards base; surface white, with some longitudinal fibrils near the base, turning blue when bruised, with olive-green tinges in older specimens. Context color not recorded, probably whitish. Smell and taste not recorded. Spore print pinkish-brown.

Basidiospores [30, 1, 1] 6.5–9.0 (10.0) × 5.0–6.5 (7.0) μm, avl × avw = 7.7 × 5.8 μm, Q = 1.15–1.60 (1.70), avQ = 1.33, (broadly) ellipsoid, rarely oblong. Basidia 17.5–26 × 7.5–10 μm, tetrasterigmate, rarely bisterigmate, clavate, oblong or ellipsoid, some with median constriction. Pleurocystidia metuloid, 70–85 × 17–22 μm, fusiform or narrowly utriform, with 2-4 hooks at apices, but hooks usually poorly developed, hyaline, with up to 2 μm thick walls, frequent all over lamellar faces. Lamellar edges sterile. Cheilocystidia 45–85 × 9.5–19.5 μm, mostly lageniform with elongated apices, some fusiform or ovoid, hyaline, thin-walled, crowded, forming a well-developed strip. Pileipellis a cutis, with terminal elements 80–135 × 10–16 μm: individual elements cylindrical, some strongly tapering towards apex, mostly filled with brown intracellular pigment, with thin, smooth walls. Stipitipellis a cutis; hyphae 5–15 μm wide, cylindrical, colorless or with brown pigment, with thin, smooth walls. Caulocystidia absent. Clamp-connections present in all tissues, more abundant at the base of cheilocystidia and on pileipellis.

Habit, habitat and distribution: Solitary, on wood in mixed mesophytic upland forest. Collected in central Illinois during summer (August).

Collections examined. USA Illinois, Urbana, Brownfield Woods, on decaying log, 5.VIII.1980, SG Saupe 230 (ILLS 42441, holotypus); idem, 7.VIII.1980, SG Saupe 264 (ILLS 42442).

The macroscopic description is based on the dried herbarium material and Saupe's notes, especially on staining reactions. These collections were used to demonstrate the presence of psilocybin in P. salicinus (Saupe 1981). However, both molecular data and microscopical examination revealed that they represent a different, undescribed, species in sect. Pluteus (salicinus/albostipitatus clade).

Pluteus saupei differs from P. salicinus in the poorly developed hooks on the pleurocystidia and the morphology of the cheilocystidia. P. salicinus var. achloes Singer lacks blue-green tinges in the basidiocarps, has pleurocystidia with well-developed hooks at the apices and shorter (up to 55 μm), predominantly clavate, cheilocystidia (Banerjee and Sundberg 1995; Singer 1956). P. salicinus var. americanus P. Banerjee & Sundb. has pleurocystidia provided with coralloid hooks at the apices and shorter (up to 45 μm) predominantly clavate cheilocystidia (Banerjee and Sundberg 1993b). P. saupei appears in all analyses as sister to P. harrisii. Both taxa have similar pleurocystidia but P. harrisii lacks clamp-connections and has differently shaped cheilocystidia.

Volvopluteus asiaticus Justo & Minnis, sp. nov. (Fig. 7c)

MycoBank 518599

Latin Diagnosis. Volvopluteo gloiocephalo similis sed differt in pleurocystidiis rostratis, cheilocystidiis lageniformibus et in spatii interne transcripti sequentia ("ITS").

Etymology. The epithet makes reference to the geographic origin of the type collection.

Pileus 72–89 mm, broadly conical in young specimens, later applanate, with umbonate center; surface rough, rugose-striate, with some powdery, minute, whitish scales, glutinous when wet, shiny on drying, greyish brown to brown, dark brown at center; margin entire. Lamellae crowded, free, (broadly) ventricose, up to 8.6 mm broad; dirty pinkish, with ciliate edges. Stipe 85–99 × 9–21 mm, cylindrical, enlarged towards the base, whitish in the upper part, with yellowish tints towards the base, longitudinally fibrillose-striate. Volva white, membranous, 38 mm high, separated from the stipe in the upper part, with abundant rizomorphs. Context white in pileus and stipe, blackish under pileipellis. Smell, taste and spore print not recorded.

Basidiospores [30, 1, 1] 12.0–14.5 × 7.0–8.5 (9.0) μm, avl × avw = 13.2 × 8.0 μm, Q = 1.50–1.80, avQ = 1.66, ellipsoid to oblong. Basidia 29–45 × 10–15 μm, tetrasterigmate, clavate. Pleurocystidia 45–85 × 12–30 μm, fusiform, narrowly utriform, commonly rostrate, provided with an apical excrescence up to 10 μm long, hyaline, thin-walled, frequent all over lamellar faces. Lamellar edges sterile. Cheilocystidia 45–70 × 14–25 μm, mostly lageniform, but also clavate, ovoid or narrowly utriform, some with an apical papilla or excrescence up to 10 μm long, hyaline, thin-walled, crowded, forming a well-developed strip. Pileipellis an ixocutis, composed of hyphae 3–7 μm wide; hyphae cylindrical or irregular in outline, embedded in a 250–450 μm thick gelatinous matrix, hyaline or with pale intracellular brown pigment, with thin, smooth walls. Stipitipellis a cutis; hyphae 4–12 μm wide, cylindrical, colorless or with brown pigment, with thin, smooth walls. Caulocystidia 100–250 × 10–15 μm, cylindrical or flexuose, some with internal septa or bifurcate apices. Volva composed of densely interwoven cylindrical hyphae, 10–15 μm wide. Clamp-connections absent in all tissues.

Habit, habitat and distribution. Gregarious, on the ground, near dead trunks of broad-leaved trees. Collected in northern Japan during Autumn (October)

Collections examined. JAPAN. Hokkaido, Iwamizawa-shi, Midorigaoka, Tonebetsu Nature Park, 5.X.2005, S. Takehashi, TNSF 15191 (holotypus).

The macroscopic description is adapted from Takehashi et al. (2010), as "Volvariella gloiocephala", supplemented with our observations on the type collection. The microscopic characters are from our observations on the type. This collection was originally identified as Volvariella gloiocephala, though molecular data indicate that it represents a distinct species. See below for a morphology-based key of the four species of Volvopluteus.

Volvopluteus michiganensis (A.H. Sm.) Justo & Minnis, Fungal Biology (in press) (Fig. 7d)

Pileus 70–90 mm, broadly conical to plano-convex, with or without a low, broad umbo; surface covered with radially arranged fibrils, viscid; margin entire, rimose; ash grey, similar to the color of Tricholoma terreum (Schaeff.) P. Kumm. Lamellae crowded, free, (broadly) ventricose; up to 15 mm broad; whitish when young, later pink, with whitish, flocculose or slightly eroded, edges. Stipe 80–110 × 10–15 mm, clavate with bulbous base (up to 30 mm broad); whitish, minutely tomentose all over. Volva white, membranous. Context white. Smell earthy, fragrant, taste not recorded. Spore print not recorded.

Basidiospores [30, 1, 1] 10.5–13.5 × 6.5–8 μm, avl × avw = 11.9 × 7.1 μm, Q = 1.4–1.9, avQ = 1.70, ellipsoid to oblong. Basidia 35–55 × 10–15 μm, tetrasterigmate, (narrowly) clavate. Pleurocystidia 70–110 × 25–45 μm, fusiform, narrowly utriform, or lageniform, many provided with an apical excrescence up to 15 μm long; hyaline, thin-walled, frequent all over lamellar faces. Lamellar edges sterile. Cheilocystidia 60–75 × 15–27 μm, fusiform, ovoid with long peduncle, narrowly utriform, many with an apical excrescence up to 15 (–20) μm long; hyaline, thin-walled; crowded, forming a well-developed strip. Pileipellis an ixocutis, composed of hyphae 2–10 μm wide; hyphae cylindrical or irregular in outline, embedded in a 150–300 μm thick gelatinous matrix, hyaline or with pale intracellular brown pigment, with thin, smooth walls. Stipitipellis a cutis; hyphae 3–15 μm wide, cylindrical, colorless or with brown pigment; with thin, smooth walls. Caulocystidia (80–) 100–360 × 10–20 μm, cylindrical or flexuose, some with bifurcate apices. Volva composed of densely interwoven cylindrical hyphae, 5–15 μm wide. Clamp-connections absent in all tissues.

Habit, habitat and distribution. Gregarious, on sawdust. Collected in Michigan (USA), during the Autumn (October).

Collections examined: USA Michigan, Washtenaw Co., Ann Arbor, Jackson Rd., 17.X.1932, A.H. Smith 32-590 (holotypus, MICH).

The macroscopic description is adapted from Smith (1934) and supplemented with our observations of the type collection. This taxon was originally described as a Pluteus, and no mention of a volva was given in the original description, though a conspicuous membranous, white volva is present in the holotype. Since its original description, it was never reported again in the literature, and Smith did not include his own species in the revision of the North American types of Pluteus (Smith and Stuntz 1958). Both molecular and morphological data confirm that this collection represents a species of Volvopluteus. North American collections of V. gloiocephalus should be carefully re-examined as some may represent V. michiganensis.

Morphological variation in Volvopluteus earlei (Murrill) Vizzini, Contu & Justo

V. earlei is usually depicted as a species with small basidiocarps (pileus < 5 cm in diameter), relatively wide basidiospores (avQ < 1.60), absent or scarce pleurocystidia and rostrate cheilocystidia (Contu 2007, Justo and Castro 2010b, Shaffer 1957). It has overall white colors but greyish and ochraceous tinges may be present in the pileus. Variation in color led Contu (1998) to describe Volvariella cookei, characterized by pure white basidiocarps, as a separate species. However, Volvopluteus earlei was originally described as having a "white, discoloring with age" pileus (Murrill 1911: 282) and basidiocarps with pure white or slightly grey-ochre pileus may occur in the same collection (Justo and Castro 2010b, Shaffer 1957). The collection originally identified as V. cookei sampled for molecular data (TOAV133) shows no molecular differentiation from other collections with grey or ochraceous tinges in the basidiocarps. Therefore, Volvariella cookei is considered a synomyn of Volvopluteus earlei.

Volvariella acystidiata N.C. Pathak is macroscopically very similar to V. earlei, differing by the absence of hymenial cystidia (Heinemann 1975). This species has been recently collected in Italy (Vizzini and Contu 2010) and sampled for molecular data. The results from the phylogenetic analyses (Fig. 4, collection TOHG1973) show that it is a morphological variant of V. earlei without cystidia. Therefore this taxon is reduced here to a form of V. earlei:

Volvopluteus earlei f. acystidiatus (N.C. Pathak) Vizzini & Contu, comb. & stat. nov.

Mycobank 518901

Basionym: Volvariella acystidiata N.C. Pathak, Bull. Jar. Bot. Natl. Belg. 45: 195. 1975

An additional collection from Italy (TOHG2001) with spores 11–17 × 7–8 μm and non-rostrate cheilocystidia falls also within the molecular variation of V. earlei (Fig. 4). Morphological differentiation of this collections of V. earlei from white forms of V. gloiocephalus can be challenging, though usually V. gloiocephalus has larger basidiocarps (pileus > 5 cm in diameter).

Morphological variation in Volvopluteus gloiocephalus (DC. : Fr.) Vizzini, Contu & Justo

V. gloiocephalus is accepted here in the sense of Boekhout (1990) including specimens with grey-brown colors and also white variants recognized by others (Shaffer 1957; Singer 1986) as a separate taxon (viz. Volvariella speciosa (Fr.: Fr.) Singer).

Both V. michiganensis and V. asiasticus share with V. gloiocephalus the pileus > 5 cm in diameter with grey-brown colors. V. michiganensis can be separated form V. gloiocephalus by the rimose pileus and average basidiospore length < 12.5 μm. In V. asiaticus the majority of pleurocystidia are rostrate and the cheilocystidia are lageniform (Fig. 7c).

White forms of V. gloiocephalus can be confused with V. earlei. The latter has usually smaller basidiocarps (pileus < 5 cm), basidiospores with avQ < 1.6, absent or very scarce pleurocystidia and rostrate cheilocystidia. However, there is a great variation on the basidiospores and cystidial characteristics of V. earlei (see comments above) that may complicate the morphological separation of both species.

Examination of additional collections of all taxa of Volvopluteus is still needed to establish more precisely the morphological limits of the species. Here we provide a tentative key based on our current knowledge of the genus.

Key to the species of Volvopluteus

  1. 1.

    Hymenial cystidia absent V. earlei f. acystidiatus

     
  2. 1.
    Hymenial cystidia (at least cheilocystidia) present
    1. 2.

      Average basidiospore length < 12.5 μm. Margin of pileus rimose. V. michiganensis

       
    2. 2.
      Average basidiospore length > 12.5 μm. Margin of pileus not rimose
      1. 3.
        Pileus with predominantly white colors
        1. 4.

          Cheilocystidia rostrate, usually each one provided with a flexuous apical appendage up to 40 μm long. Pleurocystidia absent or scarce and then similar to the cheilocystidia V. earlei

           
        2. 4.
          Cheilocystidia not rostrate. Pleurocystidia common, not rostrate
          1. 5.

            Pileus < 5 cm in diameter V. earlei

             
          2. 5.

            Pileus > 5 cm in diameter V. gloiocephalus

             
           
         
      2. 3.
        Pileus with predominantly grey or grey-brown colors (Mu. 10YR 4/1-4/3, 2.5Y 5/1-5/6, 7/1-7/4)
        1. 6.

          The majority of pleurocystidia provided with an apical excrescence up to 15 μm long. Most of the cheilocystidia lageniform. V. asiaticus

           
        2. 6.

          The majority of pleurocystidia without apical excrescences, a few with apical papilla. Most of the cheilocystidia fusiform to clavate. V. gloiocephalus

           
         
       
     

Acknowledgments

The curators of BR, CBM, EIU, F, ILLS, LOU, MA, MICH, NYBG, REG, SIU, SP and TNSF are gratefully acknowledged for managing the loans of their respective collections. The assistance of Nathan Kallen in the lab bench made this work much easier. The technical support of and helpful discussions with Manfred Binder are greatly appreciated. Dimitris Floudas and Brian Seitzman helped with the collecting trips in Massachusetts and Florida. Comments from two reviewers helped to improve the manuscript. Financial support was received from a postdoctoral grant of the Autonomous Government of Galicia (Spain) to A. Justo and from the NSF grant DEB0933081. A. Minnis acknowledges the support from the Alexander H. and Helen V. Smith Research Fund (Mycological Society of America) and also D. Nickrent and S. Sipes (SIU) for providing lab supplies for earlier molecular work. N. Menolli and M. Capelari thank FAPESP (grant 04/04319-2) for the financial support.

Copyright information

© German Mycological Society and Springer 2010