Mycobiology Research Article A Checklist of the Basidiomycetous Macrofungi and a Record of Five New Species from Mt. Oseo in Korea Won Dong Lee, Hyun Lee, Jonathan J. Fong, Seung-Yoon Oh, Myung Soo Park, Ying Quan, Paul E. Jung and Young Woon Lim* School of Biological Sciences, Seoul National University, Seoul 151-747, Korea Abstract Basidiomycetous macrofungi play important roles in maintaining forest ecosystems via carbon cycling and the mobilization of nitrogen and phosphorus. To understand the impact of human activity on macrofungi, an ongoing project at the Korea National Arboretum is focused on surveying the macrofungi in unexploited areas. Mt. Oseo was targeted in this survey because the number of visitors to this destination has been steadily increasing, and management and conservation plans for this destination are urgently required. Through 5 field surveys of Mt. Oseo from April to October 2012, 116 specimens of basidiomycetous macrofungi were collected and classified. The specimens were identified to the species level by analyzing their morphological characteristics and their DNA sequence data. A total of 80 species belonging to 57 genera and 25 families were identified. To the best of our knowledge, this is the first study to identify five of these species—Artomyces microsporus, Hymenopellis raphanipes, Pholiota abietis, Phylloporus brunneiceps, and Sirobasidium magnum—in Korea. Keywords Basidiomycetous macrofungi, DNA barcoding, Fungal flora, Internal transcribed spacer, Mt. Oseo, Unrecorded species Macrofungi are fungal species that produce fruiting bodies their fruiting body morphologies and microscopic features. visible to the naked eye [1]. Most macrofungi are members Some macrofungi do not have unique characters, and of Basidiomycota, but some belong to Ascomycota. therefore, it is difficult to distinguish them to the species Basidiomycetous macrofungi, such as bracket fungi, level using morphology alone. Many of these problems can mushrooms, and puffballs, play essential roles in maintaining be overcome by using DNA barcoding [11]. For the forest ecosystems. Saprobic macrofungi contribute to carbon kingdom Fungi, the internal transcribed spacer (ITS) region cycling by decomposing woody debris on the forest floor has been formally proposed as the primary fungal barcoding [2], while mycorrhizal macrofungi aid the survival of other gene, because its high sequence variation resolves closely forest species by mobilizing nitrogen and phosphorus [3]. related species across the fungal tree of life [12]. Coupling Additionally, fruiting bodies of basidiomycetous macrofungi morphological data with ITS sequences increases the can be important sources of food [4, 5] and medicine [6- accuracy of species identification and provides more 10] for humans. detailed information on fungal flora. For example, several Macrofungi have traditionally been identified based on Amanita species were confirmed as new records in Korea based on their morphological characteristics and ITS and large subunit rDNA sequence data [13, 14]. In addition, 2 Mycobiology 2014 June, 42(2): 132-139 http://dx.doi.org/10.5941/MYCO.2014.42.2.132 similar Korean species of the genus Russula were delimited pISSN 1229-8093 • eISSN 2092-9323 based on their morphology and ITS sequences [15]. © The Korean Society of Mycology In 2012, the Korea National Arboretum initiated a *Corresponding author survey of macrofungi in the unexploited areas of Korea. E-mail: [email protected] One of the areas targeted in the survey was Mt. Oseo, Received April 4, 2014 located in Chungcheongnam-do, South Korea. In recent Revised April 28, 2014 years, the number of visitors to Mt. Oseo has steadily Accepted May 8, 2014 increased, and the fear that human activity is damaging the ecosystem is increasing. Management and conservation This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// measures, which rely on baseline data from biodiversity creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted surveys, are needed. Although many flora and fauna non-commercial use, distribution, and reproduction in any medium, surveys have recently been conducted at Mt. Oseo [16-21], provided the original work is properly cited. studies on fungal flora have been rare; the only fungal data 132 Fungal Flora of Mt. Oseo 133 available are from a single report based on a survey in a period during which the macrofungi produce fruiting August 2010 [22]. bodies. In the field, color photographs were obtained and The goal of our study was to investigate and catalog the information about the collecting site, habitat, host, substrate, basidiomycetous macrofungi of Mt. Oseo. In addition to and fruiting body was recorded for each specimen. Samples building a checklist of the basidiomycetous macrofungi of were placed in specially prepared paper boxes and brought Mt. Oseo, we discovered 5 species previously not recorded to the laboratory for identification. After being dried in in Korea. air-vented ovens at 55oC for 3~4 days, the specimens were deposited in the Seoul National University Fungus Collection MATERIALS AND METHODS (SFC). The specimens were initially identified on the basis of Study area. Mt. Oseo (36o27' N, 126o40' W), the highest their macro- and microscopic features according to peak of the Geumbuk Mountains, is located in Cheongna- published descriptions [24-30]. Taxonomic classification of myeon, Boryeong-si, Chungcheongnam-do, South Korea. species was as per the Fungal Tree of Life (http://aftol.org), The mountain range runs north to south for 12 km and and the nomenclature was based on Index Fungorum east to west for 18 km and has an average altitude of 790.7 (http://www.indexfungorum.org/Names/Names.asp). m [23]. The surveys were conducted from April to For the new records in Korea, microscopic structures of October 2012 in a region of Mt. Oseo that includes the the specimens suspended in 3% (w/v) KOH, 1% (w/v) Natural Recreation Forest (shaded region in Fig. 1). In phloxine, and Melzer’s reagent (IKI) [31] and mounted on 2012, the prevailing climate of the area was moderate, with slides were measured using a Nikon SMZ1500 dissecting an annual mean temperature of 12.2oC and temperatures microscope and a Nikon Eclipse 80i optical microscope ranging from −1.4oC (mean minimum temperature in (Nikon, Tokyo, Japan). Quotient (Q) was the ratio of the December) to 26.8oC (mean maximum temperature in difference in the mean basidiospore length to the mean August). Precipitation during the spring, summer, and fall spore width of the specimens studied. The ‘Methuen of 2012 was 160.3 mm, 681.3 mm, and 272.7 mm, respectively. Handbook of Colour’ [32] was used as the color standard. All the meteorological data were collected by the Korea Meteorological Administration at the Boryeong meteorological DNA sequence-based identification. Genomic DNA observatory: http://www.kma.go.kr/weather/climate/data_ was extracted using a modified CTAB extraction protocol monthly.jsp (accessed March 2014). [33]. The ITS region was amplified using the primer set ITS1F/ITS4B [34]. PCR was performed in a C1000 thermal Fungal survey and processing. Five surveys were cycler (Bio-Rad, Hercules, CA, USA) using the AccuPower conducted throughout 2012 (April, June, July, August, and PCR PreMix (Bioneer, Daejeon, Korea) in a final volume October). The surveys spanned spring, summer, and fall— of 20 µL containing 10 pmol of each primer and 1 µL of Fig. 1. Geographical location and sampling regions of Mt. Oseo (Chungcheongnam-do, South Korea). The area surveyed in this study is denoted by the shaded region, which includes the Mt. Oseo Natural Recreation Forest. 134 Lee et al. DNA (10 ng/µL) by the method of Park et al. [15]. The RESULTS AND DISCUSSION PCR products were electrophoresed on 1% agarose gel with LoadingSTAR (Dyne Bio, Seoul, Korea) and purified using A total of 116 specimens of basidiomycetous macrofungi the Expin PCR SV purification kit (GeneAll Biotechnology, were collected during the surveys. Based on the Seoul, Korea), according to the manufacturer’s instructions. macromorphological features alone, we were able to Sequencing was performed in the reverse direction using confidently identify 60 species. For the remaining specimens, the ITS4B primer. DNA sequencing was performed at the DNA sequence analysis was performed because the DNA Synthesis and Sequencing Facility, Macrogen (Seoul, morphological identification was inconclusive. For these Korea), by using an ABI3700 automated DNA sequencer. unidentified specimens, a preliminary identification was All the sequences have been deposited in GenBank performed using the ITS sequence analysis, and the (accession Nos. KJ609155~KJ609174). identification was verified by comparing the observed For molecular identification, each sequence was compared morphological details with the published data. An additional with reference sequences in GenBank by using BLAST 20 species were identified using the molecular data (Fig. [35]. Sequences were edited and aligned using MEGA5 2). The morphological features and ITS sequences from 2 [36]. A neighbor-joining phylogenetic analysis was also specimens did not match with the taxonomic information performed in MEGA5 using the Kimura 2-parameter or sequences available in GenBank, and therefore, model of evolution for tree inference and 500 bootstrap identification was limited to the genus level: Xerocomus sp. replicates. (SFC20120725-43) and Marasmiellus sp. (SFC20120821-