Mycologia, 106(5), 2014, pp. 1015–1026. DOI: 10.3852/13-338 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

The taxonomic foundation, species circumscription and continental endemisms of Singerocybe: evidence from morphological and molecular data Jiao Qin

Seven species are recognized in the genus, including one new species and four new combinations. Clitocybe trogioides and Clitocybe trogioides var. odorifera are synonyms of Singerocybe humilis and Singerocybe alboinfundibuliformis respectively. Most of these species are geographically restricted in their distributions. Furthermore our study expands the distribution range of Singerocybe from the North Temperate Zone to Australia (Tasmania) and tropical southern Asia. Key words: Allopatric speciation, monophyly, new combinations, Singerocybe umbilicata, synapomorphy

Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China, and University of Chinese Academy of Sciences, Beijing 100049, China

Bang Feng Zhu L. Yang1 Yan-Chun Li Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

David Ratkowsky Genevieve Gates

INTRODUCTION

Tasmanian Institute of Agriculture, University of Tasmania, Hobart, Tasmania 7001, Australia, and School of Plant Science, University of Tasmania, Hobart, Tasmania 7001, Australia

Clitocybe sect. Bulluliferae Singer, characterized by swollen elements in the epicutis of the pileus, originally was placed in Clitocybe subgen. ‘‘EuClitocybe’’ by Singer (1943) with the type species C. kuehneri Singer. This section then was transferred to subgen. Pseudolyophyllum Singer or subgen. Cystoclitus Singer of Clitocybe (Fr.) Staude (Singer 1962, 1975). Because C. kuehneri is an invalid name (due to the lack of a Latin description or a reference), Harmaja (1969) rearranged this group of fungi in section Bulluliferae (Singer) Harmaja of Clitocybe by designation of Clitocybe hydrogramma (Bull.) P. Kumm. as the type species of the section. Harmaja (1974) proposed Singerella as an independent genus to accommodate C. hydrogramma, the single valid species of C. sect. Bulluliferae. Then Harmaja (1976) recognized another species in this genus, Singerella clitocyboides (Cooke & Massee) Harmaja (5Agaricus clitocyboides Cooke & Massee). Unfortunately Singerella Harmaja (1974) is a later homonym of Singeriella Petrak (1959). Consequently Harmaja (1988) renamed his Singerella Singerocybe Harmaja and listed three species, Singerocybe phaeophthalma (Pers.) Harmaja, S. hydrogramma (Bull.) Harmaja (5 C. hydrogramma), and S. viscida Harmaja, in the genus, with the last taxon selected as the generic type, but neglected S. clitocyboides. The taxonomic foundation of Singerocybe or its precursor Singerella, however, has not been widely accepted by mycologists (Singer 1975, Bigelow 1982, Cle´menc¸on 1984, Singer 1986, Corner 1994, Kuyper 1995, Takahashi 2000, Horak 2005, Seok et al. 2009). Using the internal transcribed spacer (ITS) and the nuclear large subunit ribosomal DNA (nrLSU) as

Haruki Takahashi 284-1 Ouhama, Ishigaki, Okinawa 907-0001, Japan

Karl-Heinz Rexer Gerhard W. Kost Systematic Botany & Mycology, FB17, PhilippsUniversity Marburg, Karl-von-Frisch-Straße, 35043, Marburg, Germany

Samantha C. Karunarathna School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand Mushroom Research Foundation, 128 M.3 Ban Pa Deng T. Pa Pae, A. Mae Taeng, Chiang Mai 50150, Thailand

Abstract: The genus Singerocybe (Tricholomataceae, Agaricales, Basidiomycota) has been the subject of controversy since its proposal in 1988. Its taxonomic foundation, species circumscription and geographical distribution have not yet been examined with molecular sequence data. In this study phylogenetic analyses on this group of fungi were conducted based on collections from Europe, eastern Asia, southern Asia, North America and Australia, with four nuclear markers, ITS, nrLSU, tef1-a and rpb2. Molecular phylogenetic analyses, together with morphological observations, strongly support Singerocybe as a monophyletic group and identify the vesicles in the pileal and stipe cuticle as a synapomorphy of this genus. Submitted 10 Nov 2013; accepted for publication 4 Apr 2014. 1 Corresponding author. E-mail: [email protected]

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gene markers, Vizzini et al. (2010) found that C. phaeophthalma (Pers.) Kuyper is not closely related to other taxa of Clitocybe sensu lato (s.l.) and suggested resurrecting the genus Singerocybe for C. phaeophthalma and allied species. Only European species were included in Singerocybe when it was proposed. The species from North America, Clitocybe adirondackensis (Peck) Sacc., C. jalapensis (Murrill) Singer, which were placed in C. sect. Bulluliferae by Singer (1979, 1986), as well as C. caespitosa Peck, C. kuehneri Singer, C. fritilliformis (Lasch) Gillet, and C. anastomosica Velen., were recognized as synonyms of C. phaeophthalma or C. hydrogramma (Bigelow 1955, 1982; Kuyper 1995). The species from Australia and New Zealand, C. clitocyboides (Cooke & Massee) Pegler and Leucopaxillus otagoensis Stevenson, have been reported to resemble closely C. phaeophthalma or C. hydrogramma (Pegler 1965, Horak 1971, Ratkowsky and Gates 2002). Some species from Asia also were found to have vesicles in their cutis. These species include Cantharellus humilis Berk. & Broome from tropical Asia (Berkeley and Broome 1875, Pegler 1986), Clitocybe trogioides Corner from Sri Lanka (Corner 1994), C. trogioides var. odorifera Har. Takah. from Japan (Takahashi 2000), C. alboinfundibuliforme Seok et al. from South Korea (Seok et al. 2009), as well as some unnamed mushrooms in China. Because only limited phylogenetic study on these taxa has been conducted so far, the relationships among them remain largely unclear. Based on collections from eastern Asia, southern Asia, Europe, North America and Australia, we conducted morphological observations and multiloci phylogenetic analyses of C. phaeophthalma and other clitocyboid species mentioned above with vesicles in the cutis. These loci included ITS, nrLSU, partial RNA polymerase II second largest subunit gene (rpb2) and translation elongation factor 1 alpha gene (tef1-a). Our goals in this study are: (i) to detect whether the C. phaeophthalma complex is a monophyletic group and thereby validate the genus Singerocybe, (ii) delimitate the species within Singerocybe if its validity can be proved, (iii) illustrate the distribution pattern and primarily discuss the possible evolutionary history of Singerocybe. MATERIALS AND METHODS Sample collection.—Basidiomata of C. phaeophthalma and its allies were collected during the past 20 y, mainly from southwestern and northeastern China, Australia (Tasmania), Japan and Sri Lanka. Voucher specimens were deposited in the Cryptogamic Herbarium of Kunming Institute of Botany, the Chinese Academy of Sciences

(KUN-HKAS), the Herbaria of Kanagawa Prefectural Museum of Natural History (KPM) and the Natural History Museum and Institute (CBM) of Japan. Samples from Germany, Italy and the American states of Tennessee and Arizona also were included for comparison. In total 50 specimens were obtained and studied. We detailed information about the specimens used in the molecular study (TABLE I). Morphological observation.—Morphological descriptions of the basidiomata were based on color images, field notes and observations in the laboratory. Color codes of the form 7D5, indicating the plate, row, and color block, are from Kornerup and Wanscher (1981). A plate containing six species of Singerocybe used in this study is illustrated (SUPPLEMENTARY FIG. 1). Micromorphological data were obtained from the dried specimens after sectioning and mounting in 5% KOH solution. In the descriptions of basidiospores, the abbreviation [n/m/p] shall mean n basidiospores measured from m basidiomata of p collections; Q is used to mean ‘‘length/width ratio’’ of a spore in side view; Q means average Q of all basidiospores 6 sample standard deviation; the expressions of the form (a)b–c(d) to stand for the dimensions of basidiospores, where the range b–c contains a minimum of 90% of the measured values, while a and d in brackets stand for the extreme values. DNA extraction, PCR and sequencing.—Genomic DNA was isolated from newly collected materials dried with silica gel or herbarium specimens with the CTAB method (Doyle and Doyle 1987). Universal primers ITS1/ITS4 (or ITS5/ITS4) were adopted for amplification of the ITS region (White et al. 1990). For the amplification of the tef1-a and nrLSU region, the primers 983F/1567R (Rehner and Buckley 2005) or 595F/1167R (Kauserud and Schumacher 2001) and the primers LROR/LR5 or LROR/LR7 (Vilgalys and Hester 1990) were employed respectively. For rpb2, primers were designed corresponding to positions 47–66 (rpb2_SF: TGGCT CTCAT GGCAT GTATC) and 755–736 (rpb2_SR: TACGA GGAGA CTGCA ACCAA) according to the sequence of Clitocybe adirondackensis (accession number HQ728532) using online software Primer3 (Rozen and Skaletsky 2000). Amplification reactions were performed in an ABI 2720 thermal-cycler (Applied Biosystems, Foster City, California) or an Eppendorf master-cycler (Eppendorf, Netheler-Hinz, Hamburg, Germany). The PCR program was as follows: predenaturation at 95 C for 3 min, followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 50 C (ITS, nrLSU and tef1-a) or 53 C (for rpb2) for 50 s, and elongation at 72 C for 90 s; afterward a final elongation at 72 C for 8 min was included. PCR products were purified with the Gel Extraction & PCR Purification Combo Kit (Spin-column, Bioteke, Beijing, China), sequenced on an ABI-3730-XL sequence analyzer (Applied Biosystems, Foster City, California) using the same primers as those used in amplifications. Sequence alignments and phylogenetic analyses.—Sequences of the ITS, nrLSU, tef1-a and the rpb2 datasets were aligned with Opal 0.3.7 (Wheeler and Kececioglu 2007) separately

Tasmania, Australia Tasmania, Australia Tasmania, Australia Mt Hanthana, Sri Lanka The Alps, Germany Canton Zurich, Switzerland Lazio, Italy Yunnan Province, China Yunnan Province, China Yunnan Province, China Yunnan Province, China

HKAS 75451 HKAS 75452 HKAS HKAS HKAS ZT HKAS HKAS HKAS HKAS HKAS

S. S. S. S. S. S. S. S. S.

clitocyboides humilis phaeophthalma phaeophthalma phaeophthalma umbilicata umbilicata umbilicata umbilicata

S. clitocyboides

S. clitocyboides

79015 56417 78038 77290 79103

75453 74717 53463

Tasmania, Australia

HKAS 75450

S. clitocyboides

Connecticut, USA Arizona, USA Arizona, USA Arizona, USA Tennessee, USA Tennessee, USA Tennessee, USA Tennessee, USA Tennessee, USA Ooita Prefecture, Japan Yunnan Province, China Yunnan Province, China Yunnan Province, China Beijing, China Yunnan Province, China Yunnan Province, China Yunnan Province, China Yunnan Province, China Jilin Province, China Tasmania, Australia

NYBG NYBG NYBG NYBG TENN064118 TENN064652 TENN061227 TENN060310 TENN064660 HKAS 75448 HKAS 74715 HKAS 73150 HKAS 73262 HKAS 74716 HKAS 78039 HKAS 78046 HKAS 68702 HKAS 70550 HKAS 56000 HKAS 75449

R.E. Halling3529 H.E. Bigelow18244 H.E. Bigelow18199 H.E. Bigelow18252 SAT09-211-07 E.C. Vellinga4065V R.H. Petersen13141 E.B. Lickey12602 P.B. Matheny3320 Atsuko Hadano J. Qin129 J. Qin164 J. Qin276 J. Qin431 J. Qin637 J. Qin644 B. Feng921 Z.W. Ge3046 Y.C. Li1146 G.M. Gates & D.A. Ratkowsky0637 G.M. Gates & D.A. Ratkowsky0638 G.M. Gates & D.A. Ratkowsky0639 G.M. Gates & D.A. Ratkowsky0640 G.M. Gates1592 S.C. Karunarathna K.-H. Rexer8366 J. Schneller M. Gelardi Y. C. Li1584 J. Qin636 Zhu L. Yang5631 B. Feng1225

Singerocybe adirondackensis S. adirondackensis S. adirondackensis S. adirondackensis S. adirondackensis S. adirondackensis S. adirondackensis S. adirondackensis S. adirondackensis S. alboinfundibuliformis S. alboinfundibuliformis S. alboinfundibuliformis S. alboinfundibuliformis S. alboinfundibuliformis S. alboinfundibuliformis S. alboinfundibuliformis S. alboinfundibuliformis S. alboinfundibuliformis S. alboinfundibuliformis S. clitocyboides

Locality

Herbarium

Collection no. or collector

Specimens used in molecular studies and GenBank accession numbers

Name code

TABLE I.

JX514118 JX514131 JX514125 KF208446 KF208447 JX514120 KF208448 KF208450 KF208449

KF208445

JX514117

JX514116

HQ902910 HQ902911 HQ902912 JX514121 — — JX514122 JX514123 HQ728529 JX514132 JX514130 JX514127 JX514126 JX514124 KF208451 KF208452 JX514128 JX514129 JX514119 —

ITS

JX514113 JX514114 JX514107 KF208454 KF208453 JX514101 KF208455 KF208457 KF208456

JX514097

JX514099

JX514096

— HQ902913 HQ902914 HQ902915 JX514102 JX514103 JX514104 JX514105 HQ728530 JX514115 JX514112 JX514109 JX514108 JX514106 KF208458 KF208459 JX514110 JX514111 JX514100 JX514098

LSU

KF208444 — KF208442 — KF208443 KF208437 KF208439 KF208438 —

—

—

—

— — — — — — KF208441 KF208440 — KF208434 KF208432 KF208430 KF208431 KF208433 KF208435 KF208436 KF208428 KF208429 KF208427 —

tef1-a

GenBank accession Nos.

JX514149 JX514150 JX514139 — KF208465 JX514134 KF208461 KF208460 KF208462

—

—

—

— JX514135 JX514136 JX514137 — JX514140 JX514141 JX514142 HQ728532 JX514148 JX514147 JX514144 JX514143 JX514138 KF208463 KF208464 JX514145 JX514146 JX514133 —

rpb2

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and manually checked on Bioedit 7.0.9 (Hall 1999). The ambiguously aligned regions (, 100 bp in total) of the ITS dataset were detected with Gblocks (Castresana 2000) and then excluded. Two introns of the tef1-a matrix were detected by Blast X (http://blast.ncbi.nlm.nih.gov/Blast. cgi?CMD5Web&PAGE_TYPE5BlastHome) and Translator (http://www.fr33.net/translator.php), in which two ambiguously aligned regions (, 55 bp) were excluded, while a finely aligned region (, 32bp) of the second intron was involved in analyses. The sequences of different loci were concatenated with Phyutility 2.2 (Smith and Dunn 2008) to carry out combined analyses. For comparing the C. phaeophthalma complex and other clitocyboid fungi in the Tricholomatoid clade, a matrix of combined ITS and nrLSU sequences of Clitocybe s.l. retrieved from GenBank (SUPPLEMENTARY TABLE I) and generated in this study was made with reference to former phylogenetic work (Vizzini and Ercole 2012). Then, sequences of all four loci were combined to produce a matrix for species recognition within Singerocybe. These sequence alignments were deposited at TreeBASE (http://purl.org/phylo/treebase; submission ID14566). For phylogenetic analyses based on the combined fourloci matrix and two-loci matrix, both maximum likelihood (ML) and Bayesian inference (BI) algorithms were employed. Separate analyses based on sequences of four loci also were conducted (SUPPLEMENTARY FIG. 2) for examining conflict among topologies with ML. The substitution model suite for each dataset was chosen with the Akaike information criterion (AIC) implemented in MrModeltest 2.3 (Nylander 2004). The GTR+I+G model was chosen as the best model for ITS, nrLSU, tef1-a and rpb2 by MrModeltest (Nylander 2004). RAxML 7.2.6 (Stamatakis 2006) and MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) were used in the ML and BI analyses respectively. For the combined analysis, a partitioned mixed model was used by defining the ITS, nrLSU, tef1-a and the rpb2 as four independent partitions. All parameters in the ML analysis were kept at their default levels, and statistical support was obtained using rapid nonparametric bootstrapping with 1000 replicates. The BI analysis using selected models and four chains was conducted by setting generations to 10 000 000 and using the STOPRULE command by setting STOPVAL to 0.01. Chain convergence was determined with Tracer 1.5 (http://tree.bio.ed.ac.uk/software/tracer/) to ensure sufficiently large ESS values (. 200). Burn-in was determined by checking the log likelihood trace plots in Tracer. Posterior probabilities were calculated using the SUMT command implemented in MrBayes.

RESULTS Molecular phylogeny.—Sequence lengths of the ITS, nrLSU, tef1-a and the rpb2 datasets are 565, 811, 530 and 582 bp respectively. Phylogenetic analyses based on the ITS-nrLSU matrix (FIG. 1) and the four-loci matrix (FIG. 2) strongly support C. phaeophthalma and its allies as a monophyletic group significantly divergent from other clitocyboid genera (e.g. Clitocybe sensu stricto [s.s.], Ampulloclitocybe Redhead, Infundibulicybe

Harmaja, Cleistocybe Ammirati et al., Trichocybe Vizzini, Musumecia Vizzini & Contu, Paralepistopsis Vizzini). In total six species (viz. Singerocybe adirondackensis, S. alboinfundibuliformis, S. clitocyboides, S. humilis, S. phaeophthalma, S. umbilicata sp. nov.) are revealed to be members of Singerocybe. Asian species S. alboinfundibuliformis and S. humilis are labeled herein Clade I, while three species distributed in the north temperate zone, viz. S. phaeophthalma from Europe, S. adirondackensis from North America and S. umbilicata from eastern Asia are designated Clade II. Different topologies of four loci indicate the uncertain position of Singerocybe clitocyboides. The phylogeny generated from the nrLSU, the rpb2 datasets (SUPPLEMENTARY FIG. 2) and the two-loci matrix (FIG. 1) suggested that S. clitocyboides is the basal group of Singerocybe. However, the phylogenetic trees inferred from the ITS (SUPPLEMENTARY FIG. 2) and the four-loci matrix (FIG. 2) showed that S. clitocyboides was clustered with S. phaeophthalma, S. adirondackensis and S. umbilicata collected from the north temperate zone. Morphological observation.— Samples clustered in Singerocybe share a common feature, vesicles in the pileal and stipe (although less) cutis (FIGS. 1, 2). For species delimitation, two macromorphological characters, whether the pileus is deeply infundibuliform with a hollow stipe and whether the gills are distant and apparently intervenose, can be employed more effectively than micromorphological characters. A series of morphological differences among all species within Singerocybe are detailed in schematic drawings (FIG. 2) and KEY (see below). Four specimens labeled S. umbilicata were found to be closely related to Singerocybe phaeophthalma but characterized by oneor two-spored basidia, thinner context and more depressed pileus (FIGS. 2, 3, SUPPLEMENTARY FIG. 1). Types of Cantharellus humilis and Clitocybe trogioides were carefully compared and found to be identical, indicating that the latter should thus be a synonym of the former (FIG. 4). The spore size of C. trogioides measured by us is 4.5–6(7) 3 3–4(4.5) mm, Q 5 (1.22)1.41–1.78(1.83), Q 5 1.58 6 0.15, which is bigger than that in Corner’s (1994) original description (4–4.3 3 2.8–3 mm). The spores of Cantharellus humilis and Clitocybe trogioides show little difference from that of its allied species within Singerocybe, and the vesicles in the pileipellis are found to be usually disposed terminally (FIG. 4). Clitocybe trogioides var. odorifera, which was described from Japan (Takahashi 2000), shares identical macro- and micromorphological characters with C. alboinfundibuliforme described from South Korea (Seok et al. 2009) and some specimens collected from China.

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FIG. 1. ML tree of the concatenated ITS and nrLSU alignment. A synapomorphy of Singerocybe, the vesicles in the pileal and stipe cutis, is shown. Bootstrap values (. 50), together with Bayesian posterior probabilities (. 0.90) are indicated along nodes.

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FIG. 2. ML tree inferred from the four-loci (ITS, nrLSU, tef1-a and rpb2) dataset and schematic drawings of Singerocybe, inferred from molecular and morphological data. The synapomorphy of Singerocybe—the vesicles in the pileal and stipe cutis, is shown. Morphological characters of basidiomata for each species are schematically displayed. Bootstrap values (. 50) and Bayesian posterior probabilities (. 0.90) are indicated along nodes.

TAXONOMY This work discovered one new species, Singerocybe umbilicata, from China and indicated that four additional species previously placed in Clitocybe or Cantharellus should be transferred to Singerocybe (see below). They are Cantharellus humilis, Clitocybe adirondackensis, C. alboinfundibuliforme and C. clitocyboides, while C. trogioides and C. trogioides var. odorifera should be treated as synonyms of Cantharellus humilis (5 S. humilis) and Clitocybe alboinfundibuliformis (5 S. alboinfundibuliformis) respectively. Singerocybe umbilicata Zhu L. Yang & J. Qin, sp. nov. FIGS. 3, 4A, B MycoBank MB804928 Holotypus: CHINA. KUNMING, YUNNAN PROVINCE: Yeyahu resort, 2000 m, 24 Sep 2012, Zhu L. Yang 5631 (HKAS 77290).

Etymology: the epithet refers to the umbilicate pileus.

Pileus 20–40 mm diam, plane at first, then umbilicate to slightly infundibuliform, strongly depressed but not hollow when matured; surface white (4A1) to cream (3B2, 4A2) to pale brownish (biscuitcolored, 4B3–4C3); margin paler, often wavy to undulate when mature; context thin (up to 2 mm thick), white to cream. Lamellae decurrent, moderately crowded, cream (4A2) to dirty white (4A1), up to 1.5 mm tall, barely intervenose. Stipe 30–50 3 3– 6 mm, cream (4A2) to biscuit-colored (4B3) to brownish (4C4), cylindrical, slightly thickened at the base, fistulose. Odor strong, unpleasant. Basidiospores [92/4/4] (4.5)5–8(12) 3 3– 4.5(5.5) mm, Q 5 (1.22) 1.33–2.12(2.5), Q 5 1.66 6 0.28, elliptical in face view, lacrymoid in side view, smooth, colorless, hyaline, thin-walled, non-amyloid. Basidia 15–25 3 4–6 mm, clavate, usually one- or twospored, occasionally three- or four-spored, with basal

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FIG. 3. Color images (online only) of basidiomata of Singerocybe umbilicata. A, B. HKAS 78038. C. HKAS 77290 (holotype). D. HKAS 79103.

clamp connections and long sterigmata (to 6 mm). Pileipellis composed of more or less radially arranged filamentous hyphae 3–6 mm wide, but 1.5–3 mm wide near pileal surface, interspersed with vesicles (21–48.5 3 10–25 mm), which also are found in apex of stipe; clamp connections common. Habit and habitat: Summer to autumn, grouped or gregarious, rarely scattered, on rich humus soils or on piles of fallen leaves in Fagaceae or in mixed woods. Additional specimens examined: CHINA. KUNMING, YUNNAN PROVINCE: Xishan, 15 Sep 2012, J. Qin 636 (HKAS 78038); Yunnan Academy of Forestry,

1 Sep 2012, B. Feng 1225 (HKAS 79103); Qiongzhu Temple, 28 Sep 2008, Y. C. Li 1584 (HKAS 56417). Specimens of Singerocybe phaeophthalma examined: GERMANY: the Alps, G. Kost & K.-H. Rexer 8366 (HKAS 53463). ITALY: Lazio, 16 Nov 2012, M. Gelardi s.n. (HKAS 79015). SWITZERLAND: Rappentobel, Meilen, canton Zurich, 740 m, 27 Jul 2010, J. Schneller (ZT). Commentary: Singerocybe umbilicata is similar to S. phaeophthalma and S. adirondackensis on account of its umbilicate to more or less infundibuliform pileus that is not hollow to the base of the stipe. However, it

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FIG. 4. Micromorphological features of Singerocybe umbilicata and S. humilis. A. Basidia and basidiospores of S. umbilicata (holotype). B. Radial-vertical section of pileipellis of S. umbilicata (holotype). C. Spores of S. humilis (K [m] 141908, identified as Cantharellus humilis by T. Petch). D. Spores of S. humilis (isotype of Cantharellus humilis). E. Spores and vesicles in pileipellis of S. humilis (holotype of Clitocybe trogioides). F. Spores and pileipellis of S. humilis (HKAS 42848).

differs from the latter two taxa by its more depressed pileus, thinner context of the pileus and one- or twospored basidia. Singerocybe adirondackensis (Peck) Zhu L. Yang & J. Qin, comb. nov. MycoBank MB801147 Basionym: Agaricus adirondackensis Peck, Ann. Rep. Reg. N.Y. St. Mus. 23:77 (1872) [1870]; Clitocybe adirondackensis (Peck) Sacc., Syll. fung. (Abellini) 5:180 (1887) Specimens examined: USA. TENNESSEE: Blount, 17 Jul 2006, R.H. Petersen 13141 (TENN061227); Sevier, 6 Aug 2009, E.C. Vellinga 4065V (TENN064652); 6 Sep 2004, E.B. Lickey 12602 (TENN060310); 30 Jul 2009, SAT09-211-07 (TENN064118); Cocke, 5 Aug 2009, P.B. Matheny 3320 (TENN064660). USA. CONNECTICUT: 25 Sep 1982, R.E. Halling 3529 (NYBG). USA. ARIZONA: 9 Aug 1980, H.E. Bigelow 18199 (NYBG); 11 Aug 1980, H.E. Bigelow 18244 (NYBG); 11 Aug, 1980, H.E. Bigelow 18252 (NYBG).

Singerocybe alboinfundibuliformis (S.J. Seok et al.) Zhu L. Yang, J. Qin & Har. Takah., comb. nov. MycoBank MB801146 Basionym: Clitocybe alboinfundibuliformis S.J. Seok et al. Mycobiology 37(4):295 (2009, as ‘‘alboinfundibulliforme’’) Taxonomic synonym: Clitocybe trogioides var. odorifera Har. Takah., Mycoscience 41(1):15 (2000), syn. nov. Specimens examined: JAPAN: Ooita-shi, Ooita Prefecture, 22 Sep 2011, A. Hadano (HKAS 75448); Machida, Tokyo metropolis, 19 Jul 1992, H. Takahashi (NC0005003); Machida, Tokyo metropolis, 23 Sep 1992, H. Takahashi (NC0005004); Yamato-shi, Kanagawa Prefecture, 22 Sep 1997, H. Takahashi (PARATYPE of C. trogioides var. odorifera, CBM-FB-24118); Yamato-shi, Kanagawa Prefecture, 15 Jul 1998, H. Takahashi (HOLOTYPE of C. trogioides var. odorifera, CBM-FB-24119); Sakura-shi, Chiba Prefecture, 4 Oct 1997, H. Takahashi (PARATYPE of C. trogioides var. odorifera, CBM-FB-16056). CHINA. YUNNAN PROVINCE: Kunming Botanic Garden, 31 Aug 2007, Zhu L. Yang 4932 (HKAS 52249); Kunming Botanic Garden, 15 Aug 2008,

QIN ET AL.: SYSTEMATICS OF SINGEROCYBE Zhu L. Yang 5143 (HKAS 54440); Kunming Botanic Garden, 28 Aug 2008, Zhu L. Yang 5174 (HKAS 54471); Gaoligongshan, Longyang County, 11 Aug 2010, Q. Cai 364 (HKAS 67928); Heilongtan Park, Kunming, 4 Sep 2011, Y.J. Hao 583 (HKAS 71692); Heilongtan Park, Kunming, 12 Sep 2011, J. Qin 129 (HKAS 74715); Gaoligongshan, Tengchong County, 9 Aug 2011, J. Qin 276 (HKAS 73262); Maji Town, Fugong County, 2 Aug 2011, J. Qin 164 (HKAS 73150); Tengchong County, 10 Aug 2011, J. Qin 302 (HKAS 73288); Xishan, Kunming, 5 Sep 2012, J. Qin 637 (HKAS 78039); Yeyahu, Kunming, 22 Sep 2012, J. Qin 644 (HKAS 78046); Bailongtan, Gucheng district, 19 Aug 2010, B. Feng 921 (HKAS 68702); Lufeng County, 11 Sep 2011, Z.W. Ge 3046 (HKAS 70550). CHINA. JILIN PROVINCE: Moon Lake, Changchun, 30 Aug 2008, Y. C. Li 1146 (HKAS 56000). CHINA. BEIJING: Baiwangshan Forest Park, 22 Sep 2010, J. Qin431 (HKAS 74716). CHINA. HUBEI PROVINCE: Muyu Town, Shennongjia National Nature Reserve, 14 Jul 2012, Q. Cai 776 (HKAS 75454). CHINA. SHANDONG PROVINCE: Yangtianshan National Forest Park, Qingzhou, 22 Aug 2011, X. H. Wang 3010 (HKAS 75446); Beijiushui, Laoshan, Qingdao, 18 Aug 2011, X. H. Wang 2995 (HKAS 75445).

Singerocybe clitocyboides (Cooke & Massee) Zhu L. Yang, J. Qin & G.M. Gates, comb. nov. MycoBank MB801145 Basionym: Agaricus clitocyboides Cooke & Massee, Grevillea 15(no. 76):98 (1887); Pleurotus clitocyboides (Cooke & Massee) Sacc., Syll. fung. (Abellini) 9:46 (1891); Dendrosarcus clitocyboides (Cooke & Massee) Kuntze Revis. gen. pl. (Leipzig) 3(2):463 (1898, as ‘‘clitocybodes’’); Clitocybe clitocyboides (Cooke & Massee) Pegler, Aust. J. Bot. 13:328 (1965); Singerella clitocyboides (Cooke & Massee) Harmaja, Karstenia 15:17 (1976). Specimens examined: AUSTRALIA. TASMANIA: Mount Field National Park, 28 May 2002, G.M. Gates & D.A. Ratkowsky 0637 (HKAS 75449); Wielangta rainforest, 6 Jun 2000, G.M. Gates & D.A. Ratkowsky 0638 (HKAS 75450); Kermandie Falls, 29 May 2001, G.M. Gates & D.A. Ratkowsky 0639 (HKAS 75451); Mount Mangana, Bruny Island, 26 May 2001, G.M. Gates & D.A. Ratkowsky 0640 (HKAS 75452); North West Bay River, 16 Sep 2011, G.M. Gates 1592 (HKAS 75453).

Singerocybe humilis (Berk. & Broome) Zhu L. Yang & J. Qin, comb. nov. FIG. 4C–F MycoBank MB801144 Basionym: Cantharellus humilis Berk. & Broome, J. Linn. Soc., Bot. 14(no. 73):33 (1875) Taxonomic synonym: Clitocybe trogioides Corner, Nova Hedwigia, Beih. 109:126 (1994), syn. nov. Specimens examined: SRI LANKA. KANDY: Mount Hanthana, SK-82, Samantha C. Karunarathna (HKAS 74717); 31 Dec 1968, E.J.H. Corner 236725 (HOLOTYPE of C. trogioides, E 00218606); Peradeniya, Jul 1868, G.H.K. Thwaites 91 [ISOTYPE of Cantharellus humilis, K (M)

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59492)]; Peradeniya, 24 Jul 1912, T. Petch 3520 [K (M) 141908]. CHINA.YUNNAN PROVINCE: Huanglianhe, Yingjiang County, 17 Jul 2003, Zhu L. Yang 3716 (HKAS 42848).

KEY TO THE CURRENTLY KNOWN SPECIES OF SINGEROCYBE 1a. Basidiomata nearly white; pileus deeply infundibuliform to the base of tubular stipe, pileal margin translucent striate when wet; flesh thinner; gills narrow and intervenose . . . . . . . . . . . . . . . . . . . . 2 1b. Basidiomata dirty white, pale isabella to cream buff; pileus not depressed to the base of stipe, viscid or not; flesh thicker; gills broad, intervenose or not . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2a. Gills very narrow, dichotomous, interstices becoming transversely reticulate to subporoid; in tropical Asia . . . . . . . . . . . . . . . . S. humilis 2b. Gills occasionally branching dictomously and shallowly intervenose; in subtropical to temperate East Asia . . . . . S. alboinfundibuliformis 3a. Pileus viscid; gills densely intervenose with low anastomoses; in Europe (Finland and Belgium) . . . . . . . . . . . . . . . . . . . . . . . . . . . S. viscida 3b. Pileus non-viscid; gills rarely intervenose . . . . . . . 4 4a. Mature basidiomata relatively large (pileus up to 15 cm diam) with hollow stipes; in Southern Hemisphere . . . . . . . . . . . . . . . S. clitocyboides 4b. Mature basidiomata relatively small (pileus usually 4–6[11] cm diam) with slightly fistulose to nearly stuffed stipes; in Northern Hemisphere . . . . . . . . . . . . . . . . . . . . . . . . 5 5a. Pileus umbilicate to infundibuliform; pileal margin usually not striate; flesh thin (up to 2 mm thick); gills usually subdistant; basidia usually one- or twospored; in eastern Asia . . . . . . . . . . . S. umbilicata 5b. Pileus applanate to depressed; pileal margin striate or not; flesh thick (more than 2 mm thick); gills subdistant or crowded; basidia usually four-spored; in Europe, North Africa and North America . . . . 6 6a. Pileal margin usually short-striate; gills usually subdistant; in Europe and North Africa . . . . . . . . . . . . . . . . . . . . . . . S. phaeophthalma 6b. Pileal margin usually not striate; gills close or crowded; in North America . . . S. adirondackensis

DISCUSSION Taxonomic foundation of Singerocybe.—Our phylogenetic analyses based on the four-loci DNA sequences strongly support the Clitocybe phaeophthalma complex (viz. C. sect. Bulluliferae) which has vesicles in the pileal and stipe cuticle, as a monophyletic group. Thus, Singerocybe should be accepted as a valid genus both morphologically and molecular phylogenetically with the vesicles as a synapomorphy. Apart from the vesicles, the chicken, strong fishy, spermatic or rancid farinaceous odor, which was reported

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(Bigelow 1955; Kuyper 1981, 1995; Takahashi 2000) as common in most species within Singerocybe, also can be useful for identification. Exceptionally the unpleasant odor has not been mentioned in S. humilis (Berkeley and Broome 1875, Corner 1994), which might be related to the rainy habitat and/or to the collector’s neglect in examining this character. The presence of vesicles was defined as the key character for the identification of Singerocybe when this genus was proposed (Harmaja 1974, 1988). Another group of fungi, Clitocybe sect. Cystoclitus Singer, represented by C. podocarpi (Singer) Singer and C. praeandina Singer, also have been reported with vesicles (or swollen elements), but they differ from C. sect. Bulluliferae by their subhymeniform epicutis (Singer 1986). No material of C. sect. Cystoclitus was available for our study. In recent years new taxa successively found from Clitocybe s.l. of the tricholomatoid clade repeatedly have verified the heterogeneity of this group. They are Ampulloclitocybe, Infundibulicybe, Cleistocybe, Trichocybe, Musumecia, Paralepistopsis (Redhead et al. 2002; Harmaja 2003; Ammirati et al. 2007; Vizzini et al. 2010a, b; Vizzini et al. 2011; Vizzini and Ercole 2012; also in FIG. 1). However, the relationships among these new genera and Clitocybe s.s. (typified by C. nebularis) are far from resolved. Missing data and complicated patterns of evolution might partly contribute to the indefinite backbones in the phylogeny of Tricholomataceae. Further work using a multidisciplinary approach and extensive sampling should solve these problems. Species delimitation.—Our observation on morphological characters of samples from different continents, together with molecular phylogenetic analyses, indicate that the shape of the pileus, the stipe and the lamellae can be used in species recognition of Singerocybe. However, micromorphological differences among species are subtle. For instance, the pileus of S. clitocyboides, S. adirondackensis and S. phaeophthalma is shallowly depressed with a nearly stuffed stipe. However, two species of Clade I, S. alboinfundibuliformis and S. humilis, have a deeply infundibuliform pileus with an open tubular stipe and obviously intervenose gills (FIG. 2). For closely allied species, the geographical distribution can be used as a useful marker for species identification. For example, S. adirondackensis, which occurs in North America, was thought to be identical to S. phaeophthalma (Bigelow 1982, Gminder and Krieglsteiner 2001, Horak 2005), which is known only from Europe. Molecular phylogenetic analyses showed that they indeed are closely related but should be treated as separate species (FIGS. 1, 2). The crowded gills of

Clitocybe adirondackensis (Bigelow 1955; SUPPLEMEN1) is another distinguishing feature. Singerocybe clitocyboides, usually with larger basidiomes than S. adirondackensis and S. phaeophthalma, is known only from Australia and New Zealand. Similarly, S. humilis with shallowly intervenose gills, is distributed in tropical Asia, while its closely allied species, S. alboinfundibuliformis, with shallower, strongly reticulate and sometimes even subporoid gills, is distributed in subtropical and temperate eastern Asia. Both S. phaeophthalma and S. hydrogramma originally were described from Europe. Although Harmaja (1988) retained both species, it has been widely accepted that S. hydrogramma is a synonym of S. phaeophthalma (Kuyper 1981, Bigelow 1982, Cle´menc¸on 1984, Kuyper 1995, Gminder and Krieglsteiner 2001, Horak 2005, Seok et al. 2009, Vizzini et al. 2010). We thus follow Kuyper (1981) and adopt S. phaeophthalma to represent samples from Europe. Singerocybe viscida is known only from Belgium and Finland and has never been collected after being first described (Dr H. Harmaja pers comm). No materials of this taxon were available for our comparison. However, we accept it as a separate species in Singerocybe for the time being in that its viscid cap is significantly different from other species of this genus. Several additional taxa (viz. Clitocybe hydrogramma var. werneri Malenc¸on [; C. phaeophtalma var. werneri (Malenc¸on) Quadr.], Pseudolyophyllum gracilentum Raithelh., P. hydrogramma var. inodora Raithelh., P. hydrogramma var. subumbilicata Raithelh., C. hydrogramma var. gibboides Raithelh. [; Clitocybe gibboides (Raithelh.) Raithelh. or Pseudolyophyllum phaeophthalma var. gibboides (Raithelh.) Bon]), were described from southern and central Europe and reported to be morphologically different from or close to C. phaeophthalma (Malenc¸on 1942; Raithelhuber 1969, 1971; Quadraccia and Rossi 1985; Perezde-Gregorio 2009; Raithelhuber 1995, 1997; Bon 1997). Considering the above-mentioned studies, more taxa of Singerocybe might be delimited in Europe if both morphological and molecular phylogenetic analyses could be employed in the near future.

TARY FIG.

Biogeographic history.—Species within Singerocybe are found to be endemic to certain continents in this study. Despite the uncertain position of S. clitocyboides in the phylogenetic trees obtained from different gene markers, two major clades could be identified in this genus. Clade I is formed by the Asian species S. alboinfundibuliformis and S. humilis, while Clade II contains three species distributed in the north temperate zone (viz. S. phaeophthalma from Europe,

QIN ET AL.: SYSTEMATICS OF SINGEROCYBE S. adirondackensis from North America and S. umbilicata from eastern Asia). The two species of Clade I are more divergent than the three species disjunctively distributed in the Northern Hemisphere. This has two possible explanations. One is that the two Asian species have an earlier speciation process than those three within Clade II. Another is that the ecological adaptation of S. humilis to a tropical environment accelerated its divergence from its closely related species S. alboinfundibuliformis. For Clade II, limited divergence can be detected among three species from different continents, indicating some relatively recent biota interchange among eastern Asia, Europe and North America. On the other hand, this also emphasizes again the importance of allopatric speciation on the evolution of fungi, which was indicated in both ectomycorrhizal and saprotrophic species (Kohn 2005, Giraud et al. 2008, Ryberg and Matheny 2012). For S. clitocyboides, the only species reported from the Southern Hemisphere, its origin is still an open question due to its uncertain position in phylogenetic analyses using different data. However, its strong divergence from other species indicate a similar evolutionary history to that of S. humilis, that is early speciation or/and strong ecological adaptation. No sample of Singerocybe has been reported or collected from southeastern Asia so far. Considering possible biota interchange between the Northern Hemisphere and Australia through southeastern Asia, attention might be paid to this region to address the question about the origin and the dispersal of Singerocybe. ACKNOWLEDGMENTS The authors are grateful for the help of Dr Barbara Thiers from the Herbarium of New York Botanic Garden (NYBG, USA); Dr Ronald H. Petersen from University of Tennessee Herbarium (TENN, USA); Dr Teruo Katsuyama from the Herbarium of Kanagawa Prefectural Museum of Natural History (KPM, Japan); Dr Yuki Mikanagi from the Herbarium of Natural History Museum and Institute (CBM, Japan); Prof Hua Peng and Dr En-De Liu from the Herbarium of Kunming Institute of Botany (KUN) for their kind help on loaning specimens. Prof Egon Horak (Austria), Dr P. Brandon Matheny (University of Tennessee, USA), Dr Matteo Gelardi (Italy), Ms. Beth Heap (Australia), Ms Atsuko Hadano, Mr Yuichi Taneyama (Japan), Dr ZaiWei Ge, Dr Xiang-Hua Wang, Mr Qi Zhao, Ms Qing Cai and Ms Yan-Jia Hao (KUN) are thanked for providing valuable specimens and/or images. Thanks to Dr Harri Harmaja (Finland), Dr Matteo Gelardi and Prof Bellu` Francesco (Italy), for their support and precious documentary material. Thanks also to Dr Jianping Xu (McMaster University, Canada), Dr Brian Perry (University of Hawaii, USA) and the two anonymous reviewers for their constructive comments and suggestions. This study was supported by

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the National Basic Research Program of China (No. 2014CB138305), the Joint Funds of the National Natural Science Foundation of China and Yunnan provincial government (No. U1302263) and the Hundred Talents Program of the Chinese Academy of Sciences.

LITERATURE CITED Ammirati JF, Parker AD, Matheny PB. 2007. Cleistocybe, a new genus of Agaricales. Mycoscience 48:282–289, doi:10.1007/S10267-007-0365-5 Berkeley MJ, Broome CE. 1875. Enumeration of the fungi of Ceylon. 2. Bot J Linn Soc 14:29–140, doi:10.1111/ j.1095-8339.1873.tb00301.x Bigelow HE. 1955. A note on Clitocybe adirondackensis. Mycologia 47:902–908, doi:10.2307/3755513 ———. 1982. North American species of Clitocybe I. Nova Hedwigia Beih 72:1–280. Bon M. 1997. Flore mycologique d’Europe 4. Tricholomataceae (2)-Clitocyboideae (Clitocybes, le´pistes, omphales et ressemblants). Documents mycologiques. Me´moire hors Se´rie 4. CRDP de l’Academie d’Amiens. 181 p. Castresana J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Bio Evol 17:540–552, doi:10.1093/oxford journals.molbev.a026334 Cle´ menc¸ on H. 1984. Kompendium der Blaetterpilze: Clitocybe. Beih Z Mykol 5:1–68. Corner EJH. 1994. Agarics in Malaysia: I Tricholomatoid, II Mycenoid. Nova Hedwigia Beih 109:126–127. Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15. Giraud T, Refre´gier G, le Gac M, de Vienne DM, Hood ME. 2008. Speciation in fungi. Fungal Genet Biol 45:791– 802, doi:10.1016/j.fgb.2008.02.001 Gminder A, Krieglsteiner GJ. 2001. Die Großpilze BadenWu¨rttembergs, Band 3. Stuttgart, Germany: Verlag Eugen Ulmer. 638 p. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acid S 41:95–98. Harmaja H. 1969. The genus Clitocybe (Agaricales) in Fennoscandia. Karstenia 10:5–113. ———. 1974. Singerella n. gen., a separate genus for Clitocybe hydrogramma. Karstenia 14:113–115. ———. 1976. Type studies in Clitocybe 2. Karstenia 15:16–18. ———. 1987 [1988]. Studies on the agaric genera Singerocybe n. gen. and Squamanita. Karstenia 27:71–75. ———. 2003. Notes on Clitocybe s. lato (Agaricales). Ann Bot Fenn 40:213–218. Horak E. 1971. A contribution toward the revision of the Agaricales (Fungi) from New Zealand. NZ J Bot 9:402– 462, doi:10.1080/0028825X.1971.10430193 ———. 2005. Ro¨hrlinge und Bla¨tterpilze in Europa (tubed and gilled fungi in Europe). Mu¨nchen, Germany: Elservier. 555 p. Kauserud H, Schumacher T. 2001. Outcrossing or inbreeding: DNA markers provide evidence for type of

1026

MYCOLOGIA

reproductive mode in Phellinus nigrolimitatus (Basidiomycota). Mycol Res 105:676–683, doi:10.1017/ S0953756201004191 Kohn LM. 2005. Mechanisms of fungal speciation. Ann Rev Phytopathol 43:279–308, doi:10.1146/annurev.phyto. 43.040204.135958 Kornerup A, Wanscher JH. 1981. Taschenlexikon der Farben 3. Aufl. Go¨ttingen: Verlag Muster-Schmidt. 242 p. Kuyper TW. 1981. Notes on Clitocybe I. Persoonia 11:385– 386. ———. 1995. Genus Clitocybe. In: Noordeloos ME, Kuyper TW, eds. Flora Agaricina Neerlandica. Netherlands: CRC Press. p 54–55. Malenc¸on G. 1942. Notes critiques sur quelques Hyme´nomyce`tes d’Europe et d’Afrique du Nord. Bull Soc Mycol Fr 58:36–38. Nylander J. 2004. MrModeltest 2.3. Program distributed by the author. Uppsala, Sweden: Evolutionary Biology Centre, Uppsala University. Pegler DN. 1965. Studies on Australasian Agaricales. Aust J Bot 13:323–356. ———. 1986. Agaric flora of Sri Lanka. Kew Bull Addit Ser 12:1–519. Perez-de-Gregorio MA. 2009. Fungi Non Delineati 44:4–9. Petrak F. 1959 [1958]. Singeriella n. gen., eine neue, mit den Entopeltaceen verwandte Gattung aus den Hochgebirgen Perus. Sydowia 12:252–257. Quadraccia L, Rossi W. 1985 [1984]. Novitates—combinaisons et taxons nouveaux. Docums Mycol 14:27–32. Ratkowsky DA, Gates GM. 2002. A preliminary census of the macrofungi of Mount Wellington, Tasmania—the Agaricales. Pap Proc R Soc Tasman 136:89–100. ¨ ber die Variationsbreite von Raithelhuber J. 1969 [1968]. U Clitocybe hydrogramma. Z Pilzk 34:175–181. ———. 1971. Trichterlinge in Su¨ddeutschland usw 5. Folge. Metrodiana 2:67–78. ———. 1995. Species et combin. nov. Metrodiana 23:3–10. ———. 1997. Trichterlinge Mitteleuropas (Fortsetzung und Schluss). Metrodiana 24:3–123. Redhead SA, Lutzoni F, Moncalvo JM, Vilgalys R. 2002. Phylogeny of agarics: partial systematics solutions for core omphalinoid genera in the Agaricales (euagarics). Mycotaxon 83:19–57. Rehner SA, Buckley E. 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97:84–98, doi:10.3852/mycologia. 97.1.84 Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574, doi:10.1093/bioinformatics/ btg180 Rozen S, Skaletsky HJ. 2000. Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S, eds. Bioinformatics methods

and protocols: methods in molecular biology. Totowa, New Jersey: Humana Press. p 365–386. Ryberg M, Matheny PB. 2012. Asynchronous origins of ectomycorrhizal clades of Agaricales. Proc R Soc B 279: 2003–2011, doi:10.1098/rspb.2011.2428 Seok SJ, Kim YS, Park KM, Kim WG, Yoo KH, Park IC. 2009. New species of Agaricales. Mycobiology 37:295–299, doi:10.4489/MYCO.2009.37.4.295 Singer R. 1943. Das System der Agaricales III. Ann Mycol 41: 1–189. ———. 1962. The Agaricales in modern taxonomy. 2nd ed. Weinheim, Germany: Cramer. 915 p. ———. 1975. The Agaricales in modern taxonomy. 3rd ed. Vaduz, Liechtenstein: Cramer. 912 p. ———. 1979 [1978]. Keys for the identification of the species of Agaricales II. Sydowia 31:229–230. ———. 1986. The Agaricales in modern taxonomy. 4th ed. Koenigstein, Germany: Koeltz Scientific Books. p 236– 247. Smith SA, Dunn CW. 2008. Phyutility: a phyloinformatics tool for trees, alignments and molecular data. Bioinformatics 24:715–716, doi:10.1093/bioinformatics/ btm619 Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688– 2690, doi:10.1093/bioinformatics/btl446 Takahashi H. 2000. Two new species and one new variety of Agaricales from central Honshu, Japan. Mycoscience 41:15–23, doi:10.1007/BF02464381 Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238– 4246. Vizzini A, Contu M, Ercole E. 2011. Musumecia gen. nov. in the Tricholomatoid clade (Basidiomycota, Agaricales) related to Pseudoclitocybe. Nord J Bot 29:734–740, doi:10.1111/j.1756-1051.2011.01169.x ———, ———, Musumeci E, Ercole E. 2010b. A new taxon in the Infundibulicybe gibba complex (Basidiomycota, Agaricales, Tricholomataceae) from Sardinia (Italy). Mycologia 103:203–208, doi:10.3852/10-137 ———, Ercole E. 2012. Paralepistopsis gen. nov. and Paralepista (Basidiomycota, Agaricales). Mycotaxon 120:253–267, doi:10.5248/120.253 ———, Musumeci E, Murat C. 2010. Trichocybe, a new genus for Clitocybe puberula (Agaricomycetes, Agaricales). Fungal Divers 42:97–105, doi:10.1007/s13225-010-0030-8 Wheeler TJ, Kececioglu JD. 2007. Opal 0.3.7: software for aligning multiple biological sequences. Available at http://opal.cs.arizona.edu White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications.. New York: Academic Press. p 315–322.