Accepted Manuscript Fungal Communities in a Korean Red Pine Stand, Gwangneung Forest, Korea Chang Sun Kim, Sang-Kuk Han, Jong Woo Nam, Jong Won Jo, Young-Nam Kwag, Jae-Gu Han, Gi-Ho Sung, Young Woon Lim, Seunghwan Oh PII: S2287-884X(17)30093-6 DOI: 10.1016/j.japb.2017.08.002 Reference: JAPB 246 To appear in: Journal of Asia-Pacific Biodiversity Received Date: 2 June 2017 Revised Date: 4 August 2017 Accepted Date: 16 August 2017 Please cite this article as: Kim CS, Han S-K, Nam JW, Jo JW, Kwag Y-N, Han J-G, Sung G-H, Lim YW, Oh S, Fungal Communities in a Korean Red Pine Stand, Gwangneung Forest, Korea, Journal of Asia- Pacific Biodiversity (2017), doi: 10.1016/j.japb.2017.08.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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ACCEPTED MANUSCRIPT 1 Fungal Communities in a Korean Red Pine Stand, Gwangneung Forest, Korea 2 3 Short Title: Fungal Communities in a Korean Red Pine Stand 4 5 Chang Sun Kim 1, Sang-Kuk Han 1, Jong Woo Nam 1, Jong Won Jo 1, Young-Nam Kwag 1, Jae-Gu Han 2, Gi-Ho 6 Sung 3, Young Woon Lim 4 and Seunghwan Oh 1* 7 8 1Forest Biodiversity Division, Korea National Arboretum, Pocheon 487-820, Republic of Korea 9 2Mushroom Research Division, National Institute of Horticultural and Herbal Science, Rural Development 10 Administration, Eumseong 369-873, Republic of Korea 11 3Institute for Bio-medical Convergence, International St. Mary’s Hospital and College of Medicine, Catholic 12 Kwandong University, Incheon 404-834, Republic of Korea 13 4School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea 14 15 *Corresponding author 16 E-mail: [email protected] MANUSCRIPT 17 18 Abstract 19 For the seasonal changes of fungi (belong to Ascomycota and Basidiomycota) diversity, we performed a 20 biweekly survey of macrofungi on defined plot in a Korean red pine ( Pinus densiflora ) stand (Gwangneung 21 Forest, Pochen-si, Korea) from April to December 2014. The plot was surveyed 18 times. In addition, we also 22 investigated the diversity of the soil fungi community during four seasons using by pyrosequencing method. The 23 collected macrofungi (25 specimens) were classified into one phylum, one class, four orders, 10 families, 13 24 genera, and 17 species; the soil fungal communities were classified into two phyla, 15 classes, 43 orders, 91 25 families, and 124 generaACCEPTED (49,937 sequence reads), designated as 124 genus-level operational taxonomic units 26 (GOTUs). Using macrofungal collection data, environmental factor data (n = 10), and pyrosequencing data, we 27 evaluated changes in fungal diversity with seasons and soil layers. Non-metric multidimensional scaling 28 (NMDS) ordination revealed distinct clusters of GOTUs assemblage with season. Two environmental factors 29 (exchangeable K and C/N ratio) were found to be significantly associated with soil fungi communities in the 1 ACCEPTED MANUSCRIPT 1 Korean red pine stand. This study will lead to a better understanding of relationships between Korean red pine 2 stand stands and soil fungal communities. 3 4 Keywords: diversity, environmental factors, macrofungi, pyrosequencing, soil layers 5 6 Introduction 7 Gwangneung Forest (Pocheon-si, Gyeonggi-do) is one of the best natural forests in Korea, and also a 8 precious global repository of bio-diversity (Cho et al 2007; Kim and Han 2008). The Gwangneung Forest has 9 been thoroughly protected and managed since its designation as a mausoleum forest of King Sejo in 1468 (Cho 10 et al 2007). Various plants (over 1,000 species), macrofungi (nearly 700 species), and animals (nearly 3,000 11 species) have been recorded there; therefore, the United Nations Educational, Scientific, and Cultural 12 Organization (UNESCO) designated this place as the fourth Korean biosphere reserve, on June 2, 2010 13 (http://www.kna.go.kr) (Cho et al 2007; Kim and Han 2008). 14 Investigation of macrofungal diversity based solely on macrofungal sampling is limited by the number of 15 collected specimens; further, the specimens might be difficult to discern. Fruit bodies of some macrofungi are 16 small and difficult to see with a naked eye (e.g., AscomycotaMANUSCRIPT species, such as Chlorociboria , Scutellinia , Peziza , 17 etc.). In addition, some small-sized or fragile species (e.g. Clitopilus , Crepidotus , Mycena , etc) generally quickly 18 disappear (Andrew et al 2013). Accordingly, next-generation sequencing is an alternative approach of surveying 19 the fungal diversity and discovering new biological groups (Jumpponen et al 2010; Lim et al 2010; Kim et al 20 2016). Lim et al. (2010) and Kim et al. (2016) attempted to detect soil fungal communities in Korea using one 21 next-generation sequencing technique, pyrosequencing, generating ca . 400 bp for each OTU (operational 22 taxonomic units). They suggested that pyrosequencing is a reliable tool for surveying the fungal diversity in the 23 soil, facilitating the understanding of ecological types (saprophytes, symbionts, and parasites) of fungi. Although 24 fungal DNA barcoding is still unaccomplished and interpretation of the relationship between fungal 25 communities and environmentalACCEPTED factors is plagued, pyrosequencing is a useful tool for investigating soil fungal 26 diversity (Jumpponen et al 2010; Kim et al 2016). 27 We previously investigated the seasonal dynamics of soil fungal communities in a Mongolian oak- 28 dominated forest in Gwangneung Forest (Kim et al 2016). Here, we subsequently investigated a different forest 29 stand, Korean red pine stand. The investigation was based on the sampling of macrofungi, analysis of soil 2 ACCEPTED MANUSCRIPT 1 chemistries, and pyrosequencing, to obtain ecological data. Further, the relationship between the soil fungal 2 community and chemical properties of the soil was examined by multivariate statistical analyses. 3 4 Materials and methods 5 Study site 6 The study site was a Korean red pine stand (37°46 ʹ27.26 ʺ N, 127°11 ʹ14.34 ʺ E, elevation 379 m); ca . 85% 7 of the tree layer coveraged by Korean red pine ( Pinus densiflora Siebold & Zucc.), mixed with few 8 Rhododendron mucronulatum Turcz. and Disporum smilacinum A. Gray, in the Gwangneung Forest, Pocheon-si, 9 Gyeonggi Province, Korea (Figure 1). 10 11 Macrofungi collection and identification 12 Defined plots were surveyed 18 times on biweekly basis, for macrofungi collection, from April to 13 December 2014. The collected specimens were identified based on the macroscopic characteristics (basidiomata; 14 cap size, shape, color, surface texture, and surface moisture; gill color, attachment, spacing, lamellules; stem size, 15 the presence or absence of partial and universal veils, etc.) and microscopic characteristics (the size and shape of 16 basidiospores, basidia, and cystidia, etc.). Microscopic observationsMANUSCRIPT were made using an Olympus BX53 17 microscope and Jenoptik ProgRes C14 Plus Camera (Jenoptik Corporation, Jena, Germany). Microscopic 18 parameters were measured using ProgRes Capture Pro v.2.8.8. software (Jenoptik Corporation). All collected 19 specimens (n = 25) are listed in Table 1; dried specimens were deposited in the herbarium of Korea National 20 Arboretum (KH). 21 22 Soil sampling and chemical analyses of the soil 23 The soil was sampled in the spring (April 24th), summer (August 11th), autumn (October 16th) and winter 24 (December 1st) from a defined plot (20 × 10 m). Three soil frames (ca . 25 × 25 cm) were collected at about 1-m 25 distance from differentACCEPTED dominant plants ( Pinus densiflora ); these frames were divided into an L-layer (litter 26 layer; 0–1 cm), H-layer (humic layer; 1–3 cm), and Ah-layer (a layer between the humic and mineral matter 27 layers; 3–5 cm) (Figure 1). The soil samples were sieved using a 2-mm sieve. The resulting material was 28 combined to yield a composite sample from each horizon (Figure 1). Subsamples for chemical analyses (soil) 29 and DNA extraction were frozen and stored at −80°C. The chemical analyses were conducted in the National 3 ACCEPTED MANUSCRIPT 1 Instrumentation Center for Environmental Management (NICEM, Seoul National University, Seoul, Korea); the + - 2 following environment factors were measured: pH, exchangeable K, total phosphorus (T-P), NH 4 , NO 3 , 3 organic matter (OM), water retention (WR), total organic carbon (TOC), total nitrogen (T-N), and C/N ratio 4 (based on TOC/T-N) (Table 2). 5 6 DNA extraction, 454-pyrosequencing, and taxonomic assignment 7 DNA of the soil microorganisms was extracted using a PowerSoil DNA isolation kit (MoBio Laboratories 8 Inc., Carlsbad, CA). DNA library was prepared using PCR products, according to the GS FLX plus library prep 9 guide (Roche, Basel, Switzerland). The DNA libraries were quantified using Picogreen assay (Victor 3). Clonal 10 amplification of a purified library, emPCR, was carried out using the GS-FLX plus emPCR kit (Roche, Basel, 11 Switzerland). A 20-ng aliquot of each DNA sample was used for a 50-µl PCR reaction. Primers ITS1F/ITS4 12 (White et al 1990) were used to amplify the ITS region of fungal rRNA gene. FastStart High Fidelity PCR 13 system (Roche, Basel, Switzerland) was used for PCR amplification, with the following conditions: 94°C for 3 14 min; followed by 35 cycles of: 94°C for 15 s, 55°C for 45 s, and 72°C for 1 min; and a final elongation step at 15 72°C for 8 min. The PCR products were purified using AMPure beads (Beckman Coulter, Inc., Brea, CA).