Fungal Diversity (2010) 45:81–98 DOI 10.1007/s13225-010-0062-0 The genus Macrolepiota (Agaricaceae, Basidiomycota) in China Z. W. Ge & Zhu L. Yang & Else C. Vellinga Received: 21 June 2010 /Accepted: 13 September 2010 /Published online: 30 September 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com Abstract Species of the genus Macrolepiota (Agaricaceae) Introduction in China were investigated on the basis of morphology and DNA sequences data. Six species, i.e., M. detersa, M. The genus Macrolepiota (Agaricaceae, Agaricales, Basi- dolichaula, M. mastoidea, M. orientiexcoriata, M. procera, diomycota) was established by Singer (1948). Macroscopi- and M. velosa are recognized, of which M. detersa and M. cally, basidiomata of species in this genus are typically big, orientiexcoriata are new species. All of them are described fleshy, and often with squamules on the pileus; lamellae are and illustrated with line drawings, and a key is provided to white to cream; a prominent annulus is usually present those recognized species. The taxonomic uncertainty of M. which is often movable. Microscopically, clamp connec- crustosa, originally described from China, is also discussed. tions are present on the septa of the hyphae in lamellae; ITS sequences were used to support the new species basidiospores are thick-walled, relatively big, white to delimitations and to test the conspecificity between the cream when accumulated, and the inner spore-wall is Chinese specimens and their relatives from other conti- metachromatic in cresyl blue (Singer 1948). nents. Phylogenetic analyses identify three clades within Originally, Macrolepiota only accommodated non- Macrolepiota: /macrolepiota, /macrosporae, and /volvatae volvate species. Species with a well-formed cup-like volva clade. /macrolepiota clade and /macrosporae clade respec- were placed in a separate genus, namely Volvolepiota tively correspond to section Macrolepiota and section Singer (Singer 1959). A recent study indicated that a volva Macrosporae in Bon’s infrageneric classification. Section at the base of the stipe does not warrant a separate genus, Volvatae is proposed to accommodate species with a volva and thus, Volvolepiota is synonymous with Macrolepiota but without clamp connections within Macrolepiota. (Vellinga and Yang 2003). Accordingly, the genus Macro- lepiota in the current sense also contains species with a Keywords Agaricales . Chlorophyllum . Morphology . cup-like volva. Systematics . Taxonomy Based on morphological and molecular data, Johnson (1999) investigated the traditional classification of the light- spored Lepiota s.l., and found Macrolepiota is not : * Z. W. Ge Z. L. Yang ( ) monophyletic. Later on, Vellinga et al. (2003) evaluated Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, the generic level of Macrolepiota, which was shown to be a Kunming 650204, People’s Republic of China monophyletic genus after transferring species with pileal e-mail: [email protected] squamules made up of a hymenidermal layer, spores with truncated germ pore or without a germ pore, and a smooth Z. W. Ge Harvard University herbaria, stipe to Chlorophyllum Massee. Consequently, representa- Cambridge 02138 MA, USA tives of Macrolepiota in the present sense are characterized by the combination of the following characters: pileal E. C. Vellinga squamules of a trichodermal layer made up of long Department of Plant and Microbial Biology, University of California, subcylindric elements, spores with a germ pore caused by Berkeley, CA 94720-310, USA an interruption of the episporium covered by a hyalinous 82 Fungal Diversity (2010) 45:81–98 cap, and the presence of stipe squamules, often visible as eppendorf tube using a pestle. DNA was isolated with a colored bands in the full-grown specimens (Vellinga 2003 modified Cetyltrimethylammonium bromide (CTAB) Vellinga et al. 2003). procedure of Doyle and Doyle (1987). ITS/5.8S rDNA Currently, there are about 30 species recognized world were amplified using primers ITS1F and ITS4 (White et wide (Kirk et al. 2008). Although the genus contains some al. 1990; Gardes and Bruns 1993). PCR was performed in edible species, which have been an interest to cultivate by a total volume of 25 μl containing 1 U Taq DNA researchers (e.g. Ding and Huang 2003), knowledge of this polymerase, 2.5 μl of 10×Taq polymerase reaction buffer, genus in East Asia is poor and fragmentary. Although a few 1 μl of 25 mM magnesium chloride (QIAGEN Inc., species of Macrolepiota were recorded from China (Shao Valencia, California, USA), 5 nmol of each dNTP, 0.6 μl and Xiang 1981; Zang et al. 1996; Bi et al. 1997; Mao of 10 μM each of the two primers and 1 μl of the DNA 2000; Teng 1996; Vellinga and Yang 2003), literature on extract. PCR reactions were performed with 4 min initial some of these records has very limited information in the denaturationat95°C,followedby34cyclesof50sat descriptions, and information on voucher specimens is 94°C, 40 s at 53°C, 50 s at 72°C, and a final extension of lacking (e.g. Mao 2000, 2009; Teng 1996). Based on 7 min at 72°C followed the last cycle. PCR products were extensive morphological examination and molecular phy- purified using a QIAquick PCR purification kit (QIAGEN logenetic analyses, species diversity of Macrolepiota in Inc., Valencia, California, USA). Sequencing was per- China and the affinities to other species of Macrolepiota are formed using a Bigdye terminator cycle sequencing kit presented. In addition, the infra-generic classification of (Applied Biosystems, Foster City, California, USA) Macrolepiota is also discussed. following the manufacturer’s protocol. Sequencing pri- mers for the ITS regions were ITS1F and ITS4. Sequenc- ing reactions were purified using Pellet Paint (Novagen, Materials and methods Madison, Wisconsin, USA) and were run on an Applied Biosystems 377 XL automated DNA sequencer. Sequence Morphological studies chromatograms were compiled with Sequencher 4.1 software (GeneCodes Corporation, Ann Arbor, Michigan, The examined materials were collected in China, and USA). deposited in KUN (with HKAS numbers), HMAS, GDGM, BPI and HMJAU. Herbarium codes used follow Thiers Phylogenetic analyses (2010). Color notations indicated in the descriptions are from Kornerup and Wanscher (1978), and Color codes are Sequences were aligned using CLUSTAL X 1.81 followed according to the Online Auction Color Chart™,indicated by manual inspection and correction in MacClade by ‘oac’ before a number. The descriptions of species are in (Thompson et al. 1997; Maddison and Maddison 2000). alphabetical order by species epithet. In the description, The resulting ITS data set was evaluated using two tree- macromorphology is based on the field notes and color slides building methodologies: the maximum parsimony (MP) of the material; micromorphology is based on observation of criterion in PAUP* and the Bayesian criterion. Gaps were the material under microscope. Melzer’sreagentwasusedto treated as missing data in all analyses. test the amyloidy of spores. Other structures (e.g. pileal Maximum Parsimony analysis was performed using structure, cheilocystidia and basidia) were observed in 5–10 PAUP* 4.0b10 (Swofford 2004). One thousand heuristic %KOHandwithCongo–red before making line drawings. searches were conducted with random sequence addition The abbreviation [n/m/p] shall mean n basidiospores and tree bisection-reconnection (TBR) branch-swapping measured from m fruit bodies of p collections in 5–10 % algorithms, collapsing zero-length branches and saving all KOH solution. At least 20 basidiospores were measured for minimal-length trees (MulTrees). To measure relative each collection. Dimensions for basidiospores are given as support for the resulting clades, 500 bootstrap replications (a-) b-c (-d). The range b-c contains a minimum of 90% of were performed with the same parameters as for the the measured values. Extreme values (a and d) are given in parsimony analyses (Felsenstein 1985). parentheses. Q is used to mean “length/width ratio” of a To test alternative phylogenetic relationships, the Bayes- spore in side view; avQ means average Q of all basidio- ian analysis were performed using MCMC with Mr. Bayes spores ± sample standard deviation. V3.0b3 (Ronquist and Huelsenbeck 2003). Bayesian analyses were repeated 4.2 million generations and sampled DNA isolation and amplification every 100. The first 25% of generations were discarded as burn-in, and Bayesian posterior probabilities (PP) were then Genomic DNA was extracted from dried material. calculated from the posterior distribution of the retained Small parts of the pileus tissue were ground in an Bayesian trees. Fungal Diversity (2010) 45:81–98 83 Results Macrolepiota velosa, described from southern China, is sister to M. eucharis, a species described from Australia. Morphological observations Clade 2, here referred to as /macrosporae clade, includes M. excoriata, M. mastoidea, M. orientiexcoriata, M. 115 putative Macrolepiota specimens were examined, and phaeodisca, M. konradii, M. psammophila, and M. sub- 87 specimens of Macrolepiota are cited in this paper. These squarrosa. This clade got 100% bootstrap and 1.00 examined specimens represent six Macrolepiota species of bayesian PP support. Within this clade, collections of M. which two are new to science. The six recognized species mastoidea from China clustered with collections from other are Macrolepiota detersa, M. dolichaula, M.