Chiang Mai J. Sci. 2017; 44(4) 1201

Chiang Mai J. Sci. 2017; 44(4) : 1201-1209 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

DNA Barcoding of the Economically Important Leucocalocybe mongolica to Prevent Mislabeling Peng Zhao* [a], Wen-Ying Zhuang [b], Tolgor Bau* [a, c] and Xiao-Dan Yu [d] [a] Key Laboratory of Shandong Province for Edible Mushroom Technology, Institute of Mycological Science and Technology, Ludong University, Yantai 264025, Shandong, China. [b] State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. [c] Institute of Mycology, Jilin Agricultural University, Changchun 130118, Jilin, China. [d] College of Biological Sciences and Technology, Shenyang Agricultural University, Shenyang 110866, Liaoning, China. * Author for correspondence; e-mail: [email protected]; [email protected]

Received: 4 August 2016 Accepted: 7 March 2017

ABSTRACT Morphological identification of the L. mongolica species, which is traded in edible wild mushroom markets, can be challenging for citizens and mycologists who are not experts on this fungal group. For the exploration of a rapid and reliable approach of L. mongolica species discrimination, nuclear ribosomal internal transcribed spacer (ITS), which is the proposed universal DNA barcode for fungi, was tested in this study to investigate the feasibility of discriminating L. mongolica from 11 low-priced that are frequently mislabeled and sold as the former in markets in North China (Inner Mongolia and Hebei Province). The appropriate intra- and inter-specific sequence divergences are considered to be important criteria for judging the suitability of the candidate DNA marker. ITS sequence showed high species identification levels within and among the 12 species. The largest intra-specific ITS variation among the investigated taxa was 2.2%, and the smallest inter-specific variation was 5.9%, which was much higher than the intra-specific variation. No overlap was observed. ITS successfully discriminated L. mongolica from the confusing mushrooms. Thus, we present ITS as the DNA barcode for the famous, expensive mushroom and its frauds or easily mislabeled fungi.

Keywords: DNA barcoding, intra-specific variation, inter-specific variation, barcoding gap, DNA mini-barcode

1. INTRODUCTION The edible wild mushroom Leucocalocybe Hebei Province, etc.) and is a well-known, mongolica (S. Imai) X.D. Yu & Y.J. Yao is delicious mushroom with nutritional and distributed in North China (Inner Mongolia, medicinal purposes, particularly in folk 1202 Chiang Mai J. Sci. 2017; 44(4)

medicine; it was formerly considered to be mitochondrial gene cytochrome c oxidase I a member of the genus Tricholoma (Fr.) (COI), the nuclear ribosomal RNA internal Staude (, ) [1]. Recently, transcribed spacer (ITS), LSU, etc. [10-13]. Tricholoma mongolicum S. Imai was transferred ITS, the most widely used molecular from Tricholoma to a newly established genus, marker in fungal identification and Leucocalocybe X.D. Yu & Y.J. Yao, based on phylogeny studies, was confirmed as the its morphological features and nuclear official and universal barcode for fungi [14]. ribosomal large subunit (LSU) sequence [2]. For macrofungi, i.e., mushrooms, DNA Leucocalocybe mongolica is extraordinarily barcoding also has been incorporated to rare in the wild. Thus far, it has not been facilitate the species recognition of some artificially cultivated. In addition, it is taxonomic groups in recent years [15-18]. delicious and medicinal. Therefore, it is In this study, ITS was tested to assess highly valuable in the wild edible mushroom its feasibility to discriminate L. mongolica market. Currently, quite a few low-priced from 11 low-priced mushrooms which mushrooms, which are similar to L. mongolica are mislabeled and traded as L. mongolica in appearance, are sold by some market traders in the commercial mushroom markets of as L. mongolica. Consequently, the L. mongolica North China. purchased by consumers is commonly a fraud. The identification of L. mongolica merely 2. MATERIALS AND METHODS based on morphological characteristics is 2.1 Materials challenging not only for citizens but also Species tested in this study belong to for mycologists who are not experts on (9 species), Agaricaceae this fungal group. Even for taxonomists, (2 species), and Lyophyllaceae (1 species) of species identification of L. mongolica is usually Agaricales, Basidiomycota. The closely related time-consuming, especially for individual species of L. mongolica [2] --- Lepista irina collections that lack sufficient diagnostic and member(s) of some genera , features. Thus, the identification technique , Tricholoma, Calocybe and Leucopaxillus with a rapid, efficient and correct species were examined here. Considering the aim discrimination of L. mongolica is essential. of the present study was to differentiate Due to the decreasing team of professional L. mongolica from its frauds rather than to taxonomic mycologists [3] and the limits of infer the phylogenetic relationships, all the morphological identification approaches, closely related species of L. mongolica were a promising and standard molecular tool, not analyzed. DNA barcoding, is being increasingly applied A total of 32 sequences from 12 species, to distinguish biological samples through including Leucocalocybe mongolica and 11 suitable intra- and inter-specific sequence low-priced species (Agaricus arvensis, Agaricus variation, which provides rapid and precise bernardii, Calocybe gambosa, Clitocybe nebularis, species identification for the purposes of Lepista irina, Lepista personata, Leucopaxillus quarantine control of exotic species, ecology, giganteus, Leucocybe connata, Tricholoma album, and biodiversity, among others [4-9]. Tricholoma japonicum and Tricholoma populinum) Fungal DNA barcoding has drawn that are frequently sold as L. mongolica in great attention. Several barcode genes were markets in North China (Inner Mongolia demonstrated in these studies, such as the and Hebei Province), were listed in Table 1. Chiang Mai J. Sci. 2017; 44(4) 1203

Table 1. Materials used in this study. Species Collection number Geographical GenBank accession or sourcea origin number of ITS Agaricus arvensis ARV1 Hungary AY484691 KCCM11246P Korea HM004552 CBS 166.33 Germany AJ133391 CA640 France JF797194 A. bernardii ARP173 USA AF432880 WC772 USA AY484678 Calocybe gambosa HC78/64 Switzerland AF357027 8064 Italy JF907775 Clitocybe nebularis CBS 362.65 Italy AF357063 PBM 2259(WTU) USA DQ486691 ZBH(3) China HQ436121 Lepista irina AFTOL-ID 815 USA DQ221109 dd08025 China FJ810142 OTA61646 New Zealand HM237136 L. personata IFO 7717 - AF241522 Leucocalocybe mongolica - - AB121014 M10 China HQ446483 Z3 China HQ446485 HMJAU24938 China, Xilingol KC413941* League (Inner Mongolia) Leucocybe connata DUKE-JM90c - EF421104 5182 Italy JF908332 SR-32 Pakistan HE819396 Lcxin - HM119488 Leucopaxillus giganteus GC 98046 Italy JQ639151 GC 94133 Italy JQ639150 Tricholoma album CBS 360.47 France AF241516 T. japonicum MR27 Japan AB036900 Tj 1 Japan AF204809 Tj 3 Japan AF204810 T. populinum AT2004265 Sweden JN019589 O-F72983 Norway JN019591 O-F85576 Norway JN019593 O-F70087 Norway JN019599 a CBS= Centraal-bureau voor Schimmelcultures, Utrecht, The Netherlands; HMJAU= Herbarium of Mycology of Jilin Agricultural University, China; KCCM = the Korean Culture Center of Microorganisms, Korea; IFO = Institute for Fermentation Culture Collection, Japan; PBM = P.B. Matheny *The GenBank accession number in boldface indicates the newly submitted sequence; the others were retrieved from GenBank. 1204 Chiang Mai J. Sci. 2017; 44(4)

2.2 DNA Extraction, PCR Amplification from ITS, ITS1 and ITS2 sequences were and Sequencing constructed using the K2P model with Genomic DNA was extracted from each MEGA 5.2 [24] to show species divergence specimen [19]. ITS was amplified and among the 12 tested species. sequenced with the primer pairs ITS1 and ITS4 [20]. PCR was performed with a 2720 3. RESULTS AND DISCUSSION Thermal Cycler (Applied Biosystems, Foster The fragments analyzed are 514-582 City, California, USA) using a 25 μL reaction base pairs (bp) for ITS, 184-247 bp for ITS1, system comprising 16 μL of double-distilled 164-184 bp for ITS2. The intra- and inter- water, 2.5 μL of 10× PCR buffer, 2 μL of specific sequence variations for the individual MgCl2 (25 mmol/L), 1.25 μL of each primer species were shown in Figure 1. The intra- (10 μmol/L), 0.5 μL of dNTP (10 mmol/L specific variations were obviously lower than each), 1.25 μL of DNA template, 0.25 μL of the inter-specific variations. The largest Taq DNA polymerase (5 U/μL). The PCR intra-specific ITS variation among the conditions were an initial step of 5 min at investigated taxa was 2.2%, and the smallest 94 °C, 30 cycles of 30 s at 94 °C, 30 s at inter-specific variation was 5.9%, which was 53 °C, and 30 s at 72 °C, followed by 10 min much higher than the intra-specific variation. at 72 °C. PCR reaction system, all other The average, maximum and minimum intra- chemical products were purchased from and inter-specific ITS sequence divergences is Sangon Biotech (Shanghai) Co., Ltd. The 0.6±0.7% & 14.9±10.0%, 2.2% & 37.2%, obtained amplicons were sequenced in both 0 & 5.9%, respectively, calculated from directions with ITS1 and ITS4 using an ABI Table 2 which was generated by TaxonGap 3730 XL DNA Sequencer (SinoGenoMax software. ITS sequence divergence of all Co., Ltd.). individuals tested in this study was shown as supplementary data. 2.3 ITS Barcode Estimation for All of the tested species could be Leucocalocybe mongolica and its separated from one another in the NJ tree Confusing Species generated by the ITS, ITS1 and ITS2 Newly obtained sequences and those sequences (Figure 2, Supplemental Figures 1, retrieved from GenBank were aligned using 2). Sequences of the same species showed high ClustalX 1.81 [21] and manually edited to adjust identity, and the other 11 mushroom species the aligned sequences using BioEdit 7.0 [22]. in markets were distinctly discriminated from The aligned sequences were input into L. mongolica. DNAStar 7.1.0 (Lasergene, WI, USA) to To determine a DNA barcode for any calculate the similarity matrix and to visually fungal group, appropriate intra- and inter- illustrate the intra- and inter-specific variations specific sequence variations are required and of the potential ITS barcode marker for are treated as significant criteria [25]. In the L. mongolica and the 11 confusing species using present study, comparisons of nucleotide TaxonGap 2.4.1 software [23]. The data was variation within and among species divided into two subsets for evaluation of demonstrated the presence of a universal feasibility of DNA mini-barcode. Therefore, barcode gap [26], i.e., a space between intra- ITS1 and ITS2 regions were also estimated, and inter-specific sequence divergences (Figure respectively. 1). In other words, no overlapping was Neighbor-joining (NJ) trees inferred observed. Consequently, the ITS sequences Chiang Mai J. Sci. 2017; 44(4) 1205

could be used to successfully discriminate ITS marker as a feasible DNA barcode to L. mongolica from other mushrooms distinguish L. mongolica from mislabeled (Figure 2). Therefore, our study suggested fungi.

Table 2. The intra- and inter-specific variations among ITS from Leucocalocybe mongolica and its confusing species generated by TaxonGap software. T. 0.2 16.8 populinum 0 T. 18.6 japonicum - 18.1 album Tricholoma 0.3 12.0 connata Leucocybe 2.2 37.2 giganteus Leucopaxillus 0.3 5.9 mongolica Leucocalocybe - L. 5.9 personata 0.6 7.5 irina Lepista 0.8 6.9 nebularis Clitocybe 0.5 30.6 gambosa Calocybe A. 1.1 9.8 bernardii 0 9.8 arvensis Agaricus Species intra-specific inter-specific ITS sequence ITS sequence variations (%) variations (%) 1206 Chiang Mai J. Sci. 2017; 44(4)

Figure 1. Comparisons of intra- and inter-specific variations among ITS, ITS1 and ITS2 from Leucocalocybe mongolica and its confusing species generated by TaxonGap software. The grey and black bars represent the intra- and inter-specific variations, respectively. The thin black lines indicate the smallest inter-specific variation. The names next to the dark bars indicate the closest species. The numbers on the scale show the percentage intra- and inter-specific variations.

Figure 2. Neighbor-joining tree based on ITS sequences from Leucocalocybe mongolica and its confusing species. Chiang Mai J. Sci. 2017; 44(4) 1207

The threshold of DNA barcode is not investigated species comprising L. mongolica exactly the same for all the organisms and its frauds. For other expensive [4, 13, 27, 28]. This is due to the distinctive mushrooms which are faked in markets, variation patterns of different barcode similar to L. mongolica in this study, DNA markers. For ITS barcode, the proposed barcoding may provide a valid approach DNA barcode of Kingdom Fungi, the largest to distinguish them. intra-specific variation obtained in this study A shorter barcode, namely DNA (2.2%) agrees approximately with that in mini-barcode [29, 30], can be applied via genus Lachnum (2.13%) [27]. Whereas, it is high-throughput sequencing platforms in not consistent with family Nectriaceae (1.30%) DNA barcode identification device in future. [28]. Actually, a mobile DNA barcoding vehicle Ideally, there is a significant barcode (BioBus) engineered by University of gap among species when DNA barcoding Guelph is in service. We explored the feasibility method are employed [26]. In this work, of ITS1 and ITS2 regions as we found that barcode gap exists within the DNA mini-barcodes of the tested species. investigated taxa. Nevertheless, overlapping As shown in Figure 1, Supplemental Figure 1, frequently occurred between the intra- and 2, both ITS1 and ITS2 can be useful to inter-specific distances [26, 28]. L. mongolica discriminate L. mongolica from its frauds or and its frauds are not all closely related easily mislabeled fungi as well as ITS species. Some of these frauds have a little barcode and consequently may be utilized far genetic relationship from L. mongolica. as DNA mini-barcode for L. mongolica and its Therefore, a clear barcde gap appeared here. mislabeled mushrooms. Of the 23 samples of ‘L. mongolica’ DNA barcoding is a powerful tool bought from the markets in Inner Mongolia, in the evaluation of food authenticity. five samples (21.7%) were identified as true Fraud fish have been recognized by DNA L. mongolica on the basis of morphological barcoding [31, 32]. The current work features by taxonomic specialists. The other suggests that DNA barcoding methodology 18 samples of the so-called ‘L. mongolica’ has potential for discriminating wild edible turn out to be 11 different low-priced mushrooms from frauds that are traded in species, resembling L. mongolica, which were mushroom commercial markets. This tool can mislabeled and sold as or mixed with offer third-party organizations or L. mongolica. Their fruit bodies may be cut governments a new opportunity for assessing into slices to avoid correct recognition. edible mushroom authenticity and controlling For most common consumers, it is difficult commercial frauds. to discriminate true L. mongolica from the We propose founding of the frauds regardless of whether the mushroom Mushroom-BOL (Mushroom Barcoding of is fresh or dried because the frauds are Life initiative) as a member of CBOL similar in color and shape, especially the (the Consortium for the Barcode of Life), sliced mushroom products. In this study, a branch of the Fungal Working Group, attempts were made to evaluate the possibility similar to the currently existing FISH-BOL, of using ITS, the universal DNA barcode to integrate the mushroom research groups of fungi, to identify L. mongolica presently and projects concerned with DNA barcoding found in the mushroom market. Fortunately, worldwide for the accelerated development ITS barcode successfully discriminated the of macrofungal species identification. 1208 Chiang Mai J. Sci. 2017; 44(4)

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