mycoscience 57 (2016) 150e155 Available online at www.sciencedirect.com journal homepage: www.elsevier.com/locate/myc Note An unexpected discovery of clavarioid fungi: First record of Lepidostroma asianum in China * Gang He, Pei-Ru Wang, Shuang-Lin Chen, Shu-Zhen Yan College of Life Sciences, Nanjing Normal University, Nanjing 210023, PR China article info abstract Article history: A new record species of Lepidostroma collected from Fujian Province in southeastern China, Received 1 May 2015 was identified as L. asianum based on morphological characteristics and phylogenetic an- Received in revised form alyses. Macro- and micro-morphological features of the species are reported and 18 December 2015 illustrated. Accepted 23 December 2015 © 2015 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Available online 25 January 2016 Keywords: Lepidostromatales Phylogeny Taxonomy The genus Lepidostroma Magd.€ & S. Winkl. (Lepidostromatales, usually associate with green algae and have soil-inhabiting Basidiomycota), a genus of clavarioid fungi, is characterized (Oberwinkler 1984; Ertz et al. 2008). However, Lepidostroma by its simple (rarely branched), clavarioid to club-shaped and Multiclavula also have some morphological differences basidiomata with pale yellow to orange or pink salmon between the genera. The basidia of Multiclavula are usually color, thin-walled, clavate to subclavate or cylindrical basidia suburniform and with 4e6(8) sterigmata, but Lepidostroma al- with 2e4 sterigmata, and hyaline, smooth, ellipsoid-ovoid to ways have subclavate to clavate-cylindrical basidia and with lacryform or slightly reniform basidiospores (Ertz et al. 2008; 2e4 sterigmata (Magdefrau€ and Winkler 1967; Oberwinkler Sulzbacher et al. 2012; Hodkinson et al. 2012, 2014; Yanaga 1984; Fischer et al. 2007; Ertz et al. 2008; Sulzbacher et al. et al. 2015). This genus grows on soil or laterite soil with 2012; Hodkinson et al. 2012, 2014; Yanaga et al. 2015). green algae around the base of the basidiomata (Ertz et al. Although these morphological differences can distinguish the 2008). According to latest contributions, seven Lepidostroma two genera, to a certain extent, but it is difficult to correctly species have so far been recorded in the world (Magdefrau€ and distinguish or identify species (especially if Multiclavula Winkler 1967; Oberwinkler 1984; Fischer et al. 2007; Ertz et al. closely resembles Lepidostroma in the yellow-orange fruiting 2008; Sulzbacher et al. 2012; Hodkinson et al. 2012, 2014; body with four sterigmata) (Hodkinson et al. 2014). Molecular Yanaga et al. 2015). Lepidostroma is very similar to the genus data is, then, indispensable for identification. In the past, Multiclavula on morphological and ecological features, both Lepidostroma was placed in the Multiclavula for a long time genera have clavarioid or coral-like fruiting body of small size (Petersen 1967; Oberwinkler 1984, 2001, 2012). Based on * Corresponding author. Tel.: þ86 25 85891571; fax: þ86 25 85891183. E-mail address: [email protected] (S.-Z. Yan). http://dx.doi.org/10.1016/j.myc.2015.12.006 1340-3540/© 2015 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. mycoscience 57 (2016) 150e155 151 molecular data, Lepidostroma belongs to a new family Lep- Phylogenetic analyses were done using maximum likeli- idostromataceae (Lepidostromatales), which formed a distinct hood (ML) inference. Maximum likelihood analyses were lineage and was found close to Atheliales in subclass Agar- performed through PhyML 3.0 (Guindon et al. 2010) with the icomycetidae, but unrelated to Multiclavula (Cantharellales) GTRþIþG model to perform a tree inference and search for a (Moncalvo et al. 2006; Ertz et al. 2008; Sulzbacher et al. 2012; good topology. ML bootstrapping was performed with 1000 Hodkinson et al. 2012, 2014). replicates (Felsenstein 1985). During a clavarioid fungi investigation in southeastern China, surprisingly, L. asianum Yanaga & N. Maek. was Lepidostroma asianum Yanaga & N. Maek., Mycoscience 56: discovered from the Wuyi Mountain in Fujian Province. This 1e9, 2015. Figs. 1, 2. species of Lepidostroma has hitherto not been reported in China. The aim of this work is to make a contribution to the Fruiting bodies cylindrical to slightly fusiform, tapering mycobiota of China by adding a new record. The description and subacute at the apex, usually unbranched or occasionally and molecular phylogenetic analyses are provided. with 1e2 branches at the apex, 12e46 Â 1e1.8 mm, light buff, Collection was dried in the field with silica gel or dried by warm buff and antimony yellow at the base, apricot orange, drying oven at 30e40 C, and then was brought to the labo- ochraceous orange, salmon orange, mikada orange, zinc or- ratory. The specimen examined in the present study was ange or orange chrome above when fresh, and salmon orange, deposited in the Microbial Cultures Center of Nanjing Normal orange chrome, capucine orange and zinc orange, occasion- University (MCCNNU), Nanjing, China. Macroscopic charac- ally ochraceous salmon and ochraceous buff when dry. ters were described from fresh and dried materials. Color was Context solid both flesh and dry, slightly brittle. Taste and described subjectively and noted according to Ridgway (1912). odor none distinctive, or ill-defined. Macrochemical reactions Micromorphological features were observed on dried mate- not recorded. Hyphae of the fresh monomitic, 2e5 mm in diam, rials under a Zeiss microscope (Axio Image A1, Gottingen,€ hyaline, thin-walled, with clamp-connection, forming a dense Germany). Hand-sections were routinely and revived in 5% texture in the subhymenium and trama, tightly packed. potassium hydroxide (KOH). Typical mountant was 5% KOH Basidia 26e47 Â 5e10 mm, clavate, or subcylindrical, clamped, solution, with Melzer's reagent and 1% Congo red solution straight, 2e4 sterigmate; sterigmata 4.5e5 mm long; usually occasionally used as stains. Measurements of basidiospores, with granular-guttulate. Basidiospores subcylindrical to basidia, sterigmata and hyphal features were made in 5% KOH narrowly ellipsoid, or subreniform, flattened to slightly solution. For each element, at least 30 individual structures concave adaxially, 7e10(e15.5) Â 3.5e5(e6.5) mm, hilar ap- were measured. pendix small, 1e1.5 mm in length, smooth, thin-walled, hya- Genomic DNA was extracted from dried basidiomata, ac- line, usually with one or two guttulate contents, non-amyloid. cording to the DNA extraction procedure from Gardes and Cystidia none. Bruns (1993). Universal primers ITS1F/ITS4 were used for the Habit and Habitat: gregarious on red clay and rocks in ITS region (internal transcribed spacer region of the nuclear Fujian Province, southeastern China. ribosomal RNA gene) amplification (White et al. 1990; Gardes Distribution: Japan (Yanaga et al. 2015) and China (In this and Bruns 1993), primers LROR/LR5 (Vilgalys and Hester study, new to China). 1990; Rehner and Samuels 1994) for the LSU rRNA gene Specimen examined: CHINA, Fujian Province, Wuyi (large subunit of the nuclear ribosomal RNA gene) amplifica- Mountain, alt. 205.7 m, N 2637020.200, E 1175902.200, 27 Jun 2014, tion, and primers NSSU97A/NS24 (Gargas and Taylor 1992; collected by Gang He, MCCNNU 140134 (GenBank accession Kauff and Lutzoni 2002) for the SSU rRNA gene (small sub- number: KR186216[ITS], KR186217[LSU], KR186218[SSU]). unit of the nuclear ribosomal RNA gene). The PCR products The diagnostic features of L. asianum and allied species in were examined by electrophoresis in 1% (w/v) agarose gels, Lepidostroma are briefly compared and summarized in Table 2. and sent to the company (Sangon Biotech Co., Ltd., Shanghai, For ITS data, comparison with sequences available in the China) for sequencing. GenBank database revealed that the ITS sequence (Accession The sequences obtained in this study were checked and No. KR186216) show 97e98% homology with that of Lepidos- assembled using MEGA 5.0 (Tamura et al. 2011). The LSU and troma asianum (Accession Nos. AB819622, AB819621, AB819620 SSU sequences related to Lepidostroma (Table 1) were down- and AB819619) in Japan (Yanaga et al. 2015). loaded from GenBank database according to the recent The LSU data matrix comprises a total of 23 sequences phylogenetic analyses on Lepidostroma and allied taxa (Ertz (including 22 from GenBank). This dataset is 853 base pairs et al. 2008; Sulzbacher et al. 2012; Hodkinson et al. 2012, long and contain 257 (30.1%) variable sites. The combined LSU 2014; Yanaga et al. 2015). DNA sequences were aligned using and SSU dataset matrix comprises a total of 20 sequences default conditions for gap openings and gap extension pen- (including 19 from GenBank). This dataset is 2492 base pairs alties with MUSCLE (Edgar 2004; Uehling et al. 2012). Two long and contains 620 (24.9%) variable sites. alignments, the one was LSU dataset, the other was combined The topologies of the ML trees are shown with the boot- LSU and SSU dataset, were then edited with manual adjust- strap support values for clades of analyses (Figs. 3, 4). The ment by MEGA 5.0 (Tamura et al. 2011) for subsequent phylogenetic results indicate that the phylogenetic position of phylogenetic analyses. The alignments were deposited in our sample is in the genus Lepidostroma, which is unrelated to TreeBASE (submission 17544). Due to limited data about Lep- Multiclavula. In the LSU analysis (Fig. 3), our Lepidostroma idostroma sequences in GenBank, the ITS sequence of our collection falls