An Unexpected Discovery of Clavarioid Fungi: First Record of Lepidostroma Asianum in China

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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 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 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) 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 into a clade consisting of two sequences of L. sample (MCCNNU140134, GenBank no. KR186216) has not asianum obtained from GenBank, which forms a distinct and been used for phylogenetic analyses. well-supported clade within Lepidostroma (with 91% bootstrap 152 mycoscience 57 (2016) 150e155

Table 1 e Taxon and GenBank accession numbers for the LSU, SSU and ITS 3-gene sequences used in this study. Taxon GenBank accession no. Strain or herbarium no.a Location LSU SSU ITS

Lepidostroma asianum KR186217 KR186218 KR186216 MCCNNU 140134 China Lepidostroma asianum (1) AB767245 AB767250 AB819619 TUMH 50299 Japan Lepidostroma asianum (2) AB767248 AB767249 AB819622 TUFC 14267 Japan Lepidostroma akagerae (1) FJ171733 FJ171729 e Ertz et al. 8556 (BR) Rwanda Lepidostroma akagerae (2) FJ171734 FJ171730 e Ertz et al. 7673 (BR) Rwanda Lepidostroma caatingae (1) KC170318 eeUFRN Fungos 1479 Brazil Lepidostroma caatingae (2) KC170319 eeUFRN Fungos 1478 Brazil Lepidostroma calocerum FJ171737 FJ171727 e R05 (F) Costa Rica Lepidostroma rugaramae (1) FJ171735 FJ171731 e Ertz et al. 8544 (basidiomata; BR) Rwanda Lepidostroma rugaramae (2) FJ171736 FJ171732 e Ertz et al. 8544 (squamules; BR) Rwanda Lepidostroma vilgalysii JN698908 eeDUKE RV-MX16 Mexico Lepidostroma winklerianum FJ171738 FJ171728 e Egan 18705 (OMA) Mexico Serpula himantioides AJ440943 AJ440948 e P99 USA Serpula lacrymans GU187596 GU187649 e REG 383 e Calocera cornea AY701526 AY771610 e AFTOL-ID 438 e Dacrymyces sp. AY691892 AY705954 e AFTOL-ID 528 e Multiclavula mucida (1) AY885163 DQ521416 e AFTOL-ID 1130 e Multiclavula mucida (2) AF287875 AF026613 e DSH 96-056 e Hydnum albidum AY293186 AY293135 e MB11-6024/2 e Athelia arachnoidea GU187557 GU187616 e CBS 418.72 The Netherlands Leptosporomyces raunkiaeri GU187588 GU187640 e CFMR HHB-7628 USA Tylospora asterophora JN938828 JN939064 e TU TU110326 Estonia Fibulorhizoctonia sp. AY635779 AY654887 e AFTOL-ID 576 e

a Herbarium for source of collections: BR ¼ Herbarium of the National Botanical Garden of Belgium, Meise, Belgium; CBS ¼ Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; CFMR ¼ Center for Forest Mycology Research, Forest Products Laboratory, USDA Forest Service, Madison, Wisconsin, USA; DSH ¼ Personal collection of Dr David S. Hibbett, Biology Department, Clark University, Worcester, USA; DUKE ¼ Duke University Herbarium, Durham, USA; F ¼ Field Museum of Natural History, Chicago, USA; MCCNNU ¼ Microbial Cultures Center of Nanjing Normal University, Nanjing, China; OMA ¼ University of Nebraska Omaha, Omaha, Nebraska, USA; REG ¼ Herbarium, Regens- burgische Botanische Gesellschaft, Universitat€ Regensburg, Regensburg, Germany; TU ¼ University of Tartu, Tartu, Estonia; TUMH ¼ Tottori University Mycological Herbarium, Tottori, Japan; TUFC ¼ Tottori University Fungal Culture Collection, Tottori, Japan; UFRN ¼ Universidade Federa do Rio Grande do Norte, Rio Grande do Norte, Brazil.

Fig. 1 e Lepidostroma asianum (MCCNNU140134). A: Habitat in the ﬁeld, arrows indicating the place where the fungus was found. B: Basidiomata in natural habitat on rock; C, D: Basidiomata in natural habitat on soil. Bars:BeD10mm. mycoscience 57 (2016) 150e155 153 € agdefrau and Fischer et al. (2007) Winkler (1967) Petersen (1967) Yanaga et al. (2015) Sulzbacher et al. (2012) Fischer et al. (2007) Hodkinson et al. (2012) Hodkinson et al. (2014) M Habitats References lateritic soil and rocks bank soil rocks 5.5 On soil e 5 On red clay 6.5 On clay 3.8 On sandy soil e 5 On open 6 On soil 5 On roadside e e 3.7 e e 4 e m) 4 3.5 4 3 2.5 m ( Size of 10 14 e 10 10 9 11 12 e e e e e e basidiospores 7 6 7.2 Shape of suballantoid or slightly reniform to elongate pip shaped basidiospores sterigmata Number of 10 Four Ovoid 9 e 8 10 Four Cylindrical to 76 Four Ellipsoid Four Suballantoid 1010 Two Four Elongate ovoid 11 Ellipsoid 7 e e e e e

e Lepidostroma asianum m) 105 Four Ovoid 8 4 5 Fig. 2 Microscopic features of 7 6 44) m ( e (MCCNNU140134). A: Basidiospores. B: Basidia. C: Hyphae. 40 47 40( 45 34 45 45 e e e e e Bars:AeC10mm. e e Size of basidia 25

support). In the combined LSU and SSU dataset analysis

(Fig. 4), our sample and two samples of L. asianum from Japan basidia Shape of also cluster together closely in the same clade with 100% Subclavate 35 Subclavate or clavate 35 bootstrap support, which therefore conﬁrmed the identiﬁca- somewhat truncate tion. Comparison with the bootstrap support values in the 1) Subclavate or clavate 30 species in the world. phylogenetic analyses, the combined gene (LSU and SSU) e 1.5 Subclavate to clavate 23 0.4 0.4 0.6( 0.7 Subclavate to clavate 25 e 2 Clavate 26 dataset was higher than that of single gene LSU dataset. 2.5 Subclavate, e e e e e e 1 0.5 1 0.3 0.2 Hence, the combined LSU and SSU dataset provided better 0.3 0.4 (mm) resolution and support than the single gene LSU dataset. 50 36 21 15 13 12 15 basidiomata e e e e e According to description by Yanaga et al. (2015), L. asianum e e Lepidostroma (when dry) 5 10 (when dry) 3 is distinguished by its pale orange to reddish orange, occasionally with 2e3 branches at apex, cylindrical to narrowly fusiform fruiting body with subulate apex, usually presence of clamp connections at hyphal septa, and basidiospores 7e10 3.5e5 mm, cylindrical to suballantoid. For example, the phylogenetic relationship between L. caatingae Sulzbacher & Lu¨ cking and L. asianum based on LSU sequences is very close Filiform, simple, very rarely branched Cylindrical narrowly fusiform Filiform, cylindrical to clavate, simple ClavarioidClavate to cylindrical 12 18 (Fig. 3), and L. asianum is also similar to L. caatingae in mac- Club shaped, simpleClub 5 shaped, simple 8 romorphological characters (Yanaga et al. 2015). However, L.

asianum is distinct from L. caatingae by the hyphal septa usu- The main characteristics of ally has clamp connection, and the size of basidiospores in L. e asianum is wider than that of L. caatingae (Yanaga et al. 2015). Lepidostroma vilgalysii B.P. Hodk., distributed in Mexico, has Species Shape of basidiomata Size of Table 2 L. akagerae L. asianum L. rugaramae L. caatingae L. calocerum elongate-ovoid and larger basidiospores (11e14 4e6.5 mm) L. vilgalysii L. winklerianum 154 mycoscience 57 (2016) 150e155

(15e24 3e6 mm), ellipsoid and slightly small sized basidiospores (6.5e8 3.5e4.5 mm), and inconspicuous clamp connections (Petersen 1967). Multiclavula sinensis, originally described from China, is distinguished from L. asianum by having club-shaped basidiomata with round apex, cylindrical to slightly reniform and smaller basidiospores (6e7 3e3.5 mm), and with mild of carrots taste (Petersen and Zang 1986). Multiclavula vernalis is separated from L. asianum by its clavate basidiomata with round apex, very short basidia (7e20 4e7 mm), and ellipsoid to elongate-ovoid and narrowly sized basidiospores (8e12 2.5e3.5 mm) (Petersen 1967). Morphological research of our collection indicates that this specimen most closely resembles L. asianum from Japan. Un- fortunately, the needle-like crystalloid materials were not observed in our collection, and occasionally the number of e Fig. 3 Maximum likelihood phylogeny depicting sterigmata was two. However, the molecular results present Lepidostroma phylogenetic relationships of species and here strongly supported previous morphological identifica- other allied genera based on LSU sequence. Bootstrap tion. The molecular phylogeny shows that our collection values above 50% are given above branches. The scale closely clustered together with two sequences of L. asianum, represents the number of nucleotide substitutions per site. and this clade separated from the other species of Lepidos- troma. The placement in a distinct clade with this species is congruent with morphological analyses. Based on pairwise distance analyses of LSU sequences from all known Lepidos- than L. asianum (Hodkinson et al. 2012). Lepidostroma calocerum troma species, the genetic distances between our specimen (G.W. Martin) Oberw. obviously differs from L. asianum in its and sequences from L. asianum suggest that it is conspecific. squamous thalli associated with the basidiomata (Petersen So, molecular data provides a very useful supplement for 1967). Moreover, Lepidostroma asianum is clearly distin- identification. guished from both L. rugaramae (Eb. Fisch., Ertz, Killmann & In conclusion, we unexpectedly collected a Lepidostroma- Serus.) Ertz, Eb. Fisch., Killmann, Serus. & Lawrey and L. win- like specimen from Fujian Province in southeastern China. klerianum B.P. Hodk. & Lu¨ cking by having cylindrical to Based on morphological and molecular evidences, we narrowly fusiform basidiomata with subulate apex and by confirmed that our collection is L. asianum. To our knowledge, producing basidiomata associated with film-like (crustose) this is the first report of L. asianum in China, which has been thalli (Fischer et al. 2007; Hodkinson et al. 2014). Under Multi- only discovered in Japan before (Yanaga et al. 2015). With the clavula, there are some species macromorphologically closely addition of China the number of Asian Lepidostroma distribu- related to L. asianum with yellow to pale orange basidiomata, tions increased to two. such as M. clara (Berk. & M.A. Curtis) R.H. Petersen, M. sinensis R.H. Petersen & M. Zang and M. vernalis (Schwein.) R.H. Petersen (Petersen 1967; Petersen and Zang 1986). Multiclavula Disclosure clara, differs from L. asianum in having short sized basidia

The authors declare no conﬂict of interest. All the experi- ments undertaken in this study comply with the current laws of the People's Republic of China.

Acknowledgments

We are sincerely grateful to the anonymous referees whose comments made our paper more accessible to the readers. This study was supported by the fund of the National Basic Priorities Program of China (2013FY110400) & A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Fig. 4 e Maximum likelihood phylogeny depicting references phylogenetic relationships of Lepidostroma species and other allied genera based on combined analysis of LSU and SSU sequences. Bootstrap values above 50% are given Edgar RC, 2004. MUSCLE: a multiple sequence alignment method above branches. The scale represents the number of with reduced time and space complexity. BMC Bioinformatics 5: nucleotide substitutions per site. 113. http://dx.doi.org/10.1186/1471-2105-5-113. mycoscience 57 (2016) 150e155 155

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