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Two Species of Clitopilus (Entolomataceae, Agaricales) from Northern Thailand Kritsana Jatuwong [A,B] , Kevin D

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Two Species of Clitopilus (Entolomataceae, Agaricales) from Northern Thailand Kritsana Jatuwong [A,B] , Kevin D Chiang Mai J. Sci. 2017; 44(1) 115 Chiang Mai J. Sci. 2017; 44(1) : 115-124 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper Two Species of Clitopilus (Entolomataceae, Agaricales) from Northern Thailand Kritsana Jatuwong [a,b] , Kevin D. Hyde [a,b,c,d,e], Samantha C. Karunarathna [a,b,c,d,e], Sunita Chamyoung [a,b] and Pattana Kakumyan*[a,b] [a] Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand. [b] School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand. [c] Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201, China. [d] World Agroforestry Centre, China & East-Asia Office, 132 Lanhei Road, Kunming 650201, China. [e] Mushroom Research Foundation, 128 M.3 Ban Pa Deng T. Pa Pae, A. Mae Taeng, Chiang Mai 50150, Thailand. *Author for correspondence; e-mail: [email protected] Received: 25 May 2015 Accepted: 8 February 2016 ABSTRACT As part of our efforts to study the saprobic mushrooms in Asia, we made three collections of Clitopilus in northern Thailand. Two of the three collections were identified, based on morphological and molecular analyses, as Clitopilus chalybescens described from Khao Yai National Park, Thailand. The third collection is a new species, Clitopilus doimaesalongensis, which we introduce in this paper. The new species C. doimaesalongensis and C. chalybescens are documented with line drawings, photographs and description and compared with similar taxa and molecular data is provided for C. chalybescens for the first time. Keywords: basidiomycetes, molecular analyses, mushrooms, new species, saprobes 1. INTRODUCTION Clitopilus (Fr. ex Rabenh.) P.Kumm. is a Agaricus prunulus Scop., but in 1871, P. Kummer small genus in the family Entolomataceae, transferred it to the new genus Clitopilus and order Agaricales traditionally including designated the type as Clitopilus prunulus (Scop.) saprotrophic or mycoparasitic species, P. Kummer [3]. characterized by basidiomes with omphaloid The genus Clitopilus has a widespread or clitocyboid to pleurotoid habits, subcurrent distribution, especially in northern temperate to decurrent lamellae, pink spore prints, and areas, and was earlier included within Rhodocybe basidiospores distinctively furrowed by 3-12 [4]. Although the 2008 estimate suggested longitudinal ribs [1, 2]. Clitopilus species can that there are about 30 species in the genus easily be recognized by a characteristic [5], a more recent publication in 2009 using farinaceous odor. The type species of Clitopilus molecular phylogenetics suggests that the was described by J.A. Scopoli in 1772 as genus should be redefined to include many 116 Chiang Mai J. Sci. 2017; 44(1) former Rhodocybe species [4]. A good not very common. The basidiomata were dichotomous key to species of the genus photographed in situ (Figure 1). Specimens Clitopilus in Tasmania has recently been were placed in a plastic box after encasing provided [6]. with aluminium foil to prevent damage and In Thailand, there have been very few were taken to the laboratory for further studies on Clitopilus, with only C. apalus studies. Significant information about (Berk. & Br.) Petch, C. prunulus [7] and the specimens and macromorphological C. chalybescens T.J. Baroni & Desjardin [8] characteristics of specimens were annotated known. In this study, we describe a new species in the laboratory and then the specimens of Clitopilus from Thailand and a collection were dried in a standard food dryer (MARTA of C. chalybescens with the support of MT-1941) at 40°C for 24-48 hours. The colour morphological characters and ITS rDNA terminology used for macromorphological sequence data. identification is from Kornerup and Wanscher [9]. The specimens were sealed in 2. MATERIALS AND METHODS Zip lock plastic bags containing dehydrated 2.1 Sample Collection and Macro- silica gel to control humidity. All herbarium morphological Character Examination specimens are deposited in the Mae Fah Luang Three specimens of Clitopilus were made University Herbarium (MFLU), Chiang Rai, during numerous field excursions during Thailand. Faces of Fungi and Index Fungorum 2012 to 2013 in Chiang Rai and Lampang numbers were obtained as in Jayasiri et al. provinces, Thailand, indicating the genus is [10] and Index Fungorum [11]. Figure 1. Basidiomata in the field A, B). Clitopilus chalybescens, C). Clitopilus doimaesalongensis, Scale bars: A, B=25 mm, C=10 mm. 2.2 Micro-morphological Character digital camera. All these evaluations were Examination carried out with a Nikon Eclipse 80i Microscopic work was carried out with microscope at 200x, 400x and 1000x the dried specimens by taking free hand magnification and drawings made. In order sections under a dissecting microscope to chronicle basidiospores (spore quotient), (Motic SMZ-171). Slide preparation was Q is used to mean “length/width ratio” of a implemented with 3-5% KOH and Congo basidiospore in side view. Basidiospore red. A Nikon Eclipse 80i microscope dimensions are based on the measurements was used for microscopic evaluation of of at least 25 basidiospores made in side view basidiospores, basidia, and pileipellis. Image and measured excluding the hilar appendix acquisition was made with a Canon 550D or apiculus at 1000x magnification. Chiang Mai J. Sci. 2017; 44(1) 117 2.3 DNA Extraction, PCR and provider (Beijing Bai Mai Hui Kang Sequencing Biological Engineering Technology Co., P.R. Genomic DNA was extracted from China). dried specimen using the Biospin Fungus Genomic DNA Extraction Kit (Bioer 2.4 Sequence Alignment and Molecular Technology Co., Ltd., Hangzhou, P.R. China). Phylogenetic Analysis The Internal Transcribed Spacer (ITS) Sequenced data were checked and region of the ribosomal DNA was amplified assembled using BioEdit 7.0.9.0 [12], and and sequenced using primers ITS5 and ITS4. blasted with those available in GenBank. PCR reactions were completed in 25 μl The taxa information and GenBank volumes containing 1.0 μl template DNA, accession numbers used in the molecular 9.5 μl double distilled water, 1.0 μl of each work are listed in Table 1. Sequences for each primer and 12.5 μl of 2x power Taq PCR strain were aligned using Clustal X [13]. Master Mix (Genomics BioSci & Tech.) Alignments were manually adjusted to allow [A premix and ready to use solution, including maximum sequence similarity. Gaps were 0.1 Units/μl Taq DNA Polymerase, 500 μm treated as missing data. Phylogenetic analysis dNTP Mixture each (dATP, dCTP, dGTP, were performed using PAUP* 4.0b10 [14]. dTTP), 20 mM Tris-HCL pH8.3, 100 Mm Ambiguously aligned regions were excluded KCl, 3 mM MgCl2, stabilizer and enhancer]. from all analyses. Trees were inferred using The reaction was carried out with 35 cycles the heuristic search option with TBR using the following conditions: denaturation branch swapping and 1000 random sequence (95°C, 30 s), annealing (52°C, 30 s), extension additions. Maxtrees were unlimited, branches (72°C, 1 min) and final extension (72°C, of zero length were collapsed and all multiple 10 min). The primers used for sequencing parsimonious trees were saved. Clade stability were the same as those for amplification. of the trees resulting from the parsimony Amplified products were confirmed with analyses were assessed by bootstrap analysis 1% agarose gel electrophoresis stained with 5000 replicates, each with 100 replicates with ethidium bromide. Sequencing was of random stepwise addition of taxa [15]. performed in a commercial sequencing Trees were figured in Treeview. Table 1. Taxon information used for molecular analyses. Taxon Country of origin GenBank accession Reference numbers (ITS) Clitopilus giovanellae Madrid, Spain EF413030 [16] C. scyphoides Bristol BS8 1UG, UK FJ770390 [17] C. passeckerianus Bristol BS8 1UG, UK FJ770406 [17] C. abortivus Bristol BS8 1UG, UK FJ770404 [17] C. truncate* Bristol BS8 1UG, UK FJ770393 [17] C. crispus Guangdong, China JQ281489 [17] C. scyphoides Bristol BS8 1UG, UK FJ770401 [17] C. hobsonii Bristol BS8 1UG, UK FJ770402 [17] C. kamaka New Zealand KJ461903 [18] C. scyphoides Bristol BS8 1UG, UK KC176282 [19] 118 Chiang Mai J. Sci. 2017; 44(1) Table 1. Continued. Taxon Country of origin GenBank accession Reference numbers (ITS) C. cystidiatus Switzerland HM623130 [20] C. chrischonensis Switzerland HM623128 [20] C. austroprunulus Tasmania, Australia KC139084 [21] C. fallax* Vaud, Switzerland AF357018 [22] C. prunulus Jiangsu, China EU273512 Unpublished C. brunnescens TN, USA HQ222033 Unpublished C. amarus Leide, Netherlands KC885963 Unpublished C. hirneolus Leide, Netherlands KC710132 Unpublished C. chalybescens Lampang, Thailand KP938184 This study C. chalybescens Lampang, Thailand KP938185 This study C. doimaesalongensis Chiang Rai, Thailand KP938183 This study Lyophyllum decastes Vaud, Switzerland AF357060 [22] Note: * in GenBank these taxa are placed under the genus Rhodocybe. 3. RESULTS retention index (RI) of 0.638, and rescaled 3.1 Phylogenetic Analyses consistency index (RC) of 0.271. This most To obtain an understanding on the likely MP tree is presented and it was chosen phylogenetic relationships of our novel species to represent the phylogenetic position of C. doimaesalongensis, with other known Clitopilus C. doimaesalongensis. species, we retrieved ITS sequence data from Sequences were aligned and the alignment GenBank. The Clitopilus dataset comprised 21 was subjected to Maximum parsimony collections (Table. 1), with 18 from GenBank,
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