Journal of Asia-Pacific Biodiversity 8 (2015) 305e308

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Journal of Asia-Pacific Biodiversity

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Original article Intraspecific diversity of saltuarius (Gebler) based on DNA barcode analysis

Jun Hyoung Jeon a,b, Bong-Kyu Byun b, Young-Bok Cho c, Suh Yun Woo d, Cheol-Hak Kim e, Jongok Lim f, Doo-Sang Park a,* a Microbiological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea b Department of Biological Science and Biotechnology, Hannam University, Daejeon, South Korea c Natural History Museum, Hannam University, Daejeon, South Korea d Division of Forestry Research, Chung cheong buk-do Institute of Forest Protection and Management Research, Cheongju, South Korea e Natural Enemy Business Division, Osang K-Insect, Guri, South Korea f Division of Forest Biodiversity, Korea National Arboretum, Pocheon, South Korea article info abstract

Article history: Monochamus saltuarius has a morphological polymorphism, but there is no standard phenotype to Received 22 October 2015 distinguish the differences in M. saltuarius species. To investigate molecular diversity of M. saltuarius, Received in revised form mitochondrial cytochrome c oxidase I 5’ sequence were analyzed against specimens collected from 28 October 2015 Chungbuk, Gyeonggi, and Gangwon province. The DNA barcode results showed that the specimens make Accepted 30 October 2015 two groups with a 1.68%e3.1% K2P distance, but cannot find a specific phenotype difference among the Available online 10 November 2015 specimens. Copyright Ó 2015, National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA). Keywords: COI Production and hosting by Elsevier. This is an open access article under the CC BY-NC-ND license (http:// DNA barcode creativecommons.org/licenses/by-nc-nd/4.0/). K2P Monochamus Monochamus saltuarius

Introduction Morewood et al 2002). M. saltuarius occurred in Pinus koraiensis in the central area of Korea, Chungnam and Gangwon province (Lee The Cerambycidae are an abundant species in the family of and Lee 2000; Kwon et al 2006). Meanwhile, Monochamus alter- Chrysomeloidea (Insecta: Coleoptera), and all members are nor- natus had been collected at various sites covering the whole seaside mally known as longhorn . The family consists of w 25,000 area of Gyeongnam and Jeonnam province in the survey from 1989 described species in nine subfamilies all over the world (Sama et al to 1994 (Moon et al 1995). 2010; Bouchard et al 2011) and 357 species are known from the In Korea, M. alternatus is known to be a vector of the pinewood Korean fauna (Lee 1987; Danilevsky 1992; Paek et al 2010; nematode, Bursaphelenchus xylophilus, but M. saltuarius has the Danilevsky 2013; Lim et al 2012, 2013a, 2013b; Oh 2013; Oh and ability to be a vector of this nematode. According to a recent report, Lee 2013; Jang et al 2015). Eight species in the genus Mono- it is accepted that M. saltuarius transmits the pinewood nematode chamus were recorded from Korea (Paek et al 2010). Among them, to Korean white pine trees at Gwangju, which is located in Monochamus saltuarius is an economic important pest of conifer in Gyeonggi province (Korea Forest Research Institute 2007; Han et al Korea, Japan, China, Taiwan, Western Europe, and Northern 2007). America regions (Dominik 1982; Shao et al 1988; Vallentgoed 1991; M. saltuarius, as well as M. alternatus, are important pests because they transmit the pinewood nematode and cause serious damage to conifer species which are very abundant in Korean * Corresponding author. Tel.: þ82 42 860 4657; fax: þ82 42 860 4625. peninsula as well as Japan and North-Eastern China. Therefore, E-mail address: [email protected] (D.-S. Park). precise ecological study and taxonomic records are especially Peer review under responsibility of National Science Museum of Korea (NSMK) and important to control the pests. Korea National Arboretum (KNA). http://dx.doi.org/10.1016/j.japb.2015.10.016 pISSN2287-884X eISSN2287-9544/Copyright Ó 2015, National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA). Production and hosting by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 306 JH Jeon et al. / Journal of Asia-Pacific Biodiversity 8 (2015) 305e308

Table 1. PCR primers used in this study. Table 3. Pairwise distance of COI between populations.

Primer name Primer sequence (50e30) Primer source Region Intragroup Intergroup

LCO1490 GGTCAACAAATCATAAAGATATTGG Folmer et al (1994) Min Max Min Max HCO2198 TAAACTTCAGGGTGACCAAAAAATCA Folmer et al (1994) GWA 0.00 0.30 1.68 3.10 MHemF1 GCATTYCCACGAATAAATAAYATAAG Park et al (2011) GWB 0.00 0.00 1.80 3.10 MHemR1 GGTGGATAAACTGTTCAWCC Park et al (2011) CBB 0.00 0.00 1.68 3.10 PCR ¼ Polymerase chain reaction GGB 0.00 0.00 1.80 3.10 YPA 0.00 0.40 1.68 3.10 YPB 0.00 0.00 1.90 3.10 GPA 0.00 0.30 1.68 3.10 GPB 0.00 0.00 1.90 3.10 M. saltuarius have morphological polymorphism, but there are Average 0.00 0.15 1.76 3.10 no data to distinguish the diversity of the pest. Recently, molecular COI ¼ cytochrome c oxidase I; CBB ¼ Chungbuk B group; GGB ¼ Gyeonggi B group; identification, so called DNA barcoding (Hebert et al 2003), is get- GPA ¼ Gapyeong A group; GPB ¼ Gapyeong B group; GWA ¼ Gangwon A group; ¼ ¼ ¼ ¼ ting popular to discriminate species. In this study, we collected over GWB Gangwon B group; Min minimum; Max maximum; YPA Yangpyeong A group; YPB ¼ Yangpyeong B group. a hundred specimens in the central region of Korean Peninsula and analyzed the molecular diversity of M. saltuarius.

Materials and methods same software. A neighbor-joining (NJ) analysis (Saitou and Nei, 1987) was performed with MEGA 6.0 (Tamura et al 2013). The specimens examined in this study were collected from five locations from 2011 to 2015 in Korea. Most of the specimens from the collection sites were collected mainly by sweeping nets in the Results and discussion field located in Gangwon, Gyunggi, and Chungbuk province (Table 2). All specimens were stored under air-dried conditions. The A total of 69 DNA barcodes were secured among a total of 104 materials were preserved at the following institutions: Systematic individual specimens. Phylogenetic analysis of the secured DNA Entomology Laboratory, Hannam University, Daejeon, Korea (SEL/ barcode showed the specimens make two phylogenetic groups HNU). with an average K2P distance 1.68e3.1%. A group, included three DNA was extracted from either dried or ethanol-fixed leg sam- sites including the Gangwon A group (GWA), Yangpyeong A group ples using a standard Glass Fiber extraction protocol (Ivanova et al (YAP), and Gapyeong A group (GPA). B group was composed of the 2006). Furthermore, genomic DNA extraction was performed using Gangwon B group (GWB), Chungbuk B group (GBB), Gyeonggi B a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Mitochondrial group (GGB), Yangpyeong B group (YPB), and Gapyeong B group cytochrome c oxidase I (COI) a 658-bp fragment was amplified (GPB). In the COI sequences, the average nucleotide composition using the primer set, LCO149/HCO2198 (Folmer et al 1994), was as follows: A, 30.70%; T 38.45%; G, 15.05%; and C, 15.05% within LCO1490/MhemR1 or MhemF1/HCO2198 (Park et al 2011; Table 1). the A Group taxa but A, 30.55%; T 38.75%; G, 15.81%; and C, 14.89% Polymerase chain reaction (PCR) amplifications were done in a 20- when the B Group taxa were included. mL volume including 5 mL of 20% trehalose, 2.8 mL of distilled water, Diversity of intergroup specimens averaged 1.3% K2P distance, 2 mLof10 PCR buffer [20mM Tris-HCL (pH 8.0), 40mM NaCL, 2mM ranged from 1.68% to 3.1%, whereas it ranged from 0% to 0.4% in sodium phosphate, 0.1mM EDTA, 1mM DDT, stabilizers, 50% (v/v) the intragroup, with an average of 0.15%. The two groups glycerol], 2 mL of MgCl₂ (50mM), 2 mL of each primer (10mM), 2 mLof collected in Gangwon province, GWA and GWB showed 2.4% 25mM dNTP, 0.2 mL of Taq polymerase (PlatinumÒ Taq, Invitrogen, difference and it was 2.3% and 2.5% between GWA and CBB, GWA Carlsbad City, CA, USA) and 2 mL of extracted DNA. PCR thermocy- and GGB, respectively. Also, GBA and YPB showed 2.4% difference cling was done under the following conditions: 5 minutes at 94 C; of K2P distance and it was 2.8% between GWA and GPB (Tables 3 five cycles of 30 seconds at 94 C, 30 seconds at 45 C, 60 seconds at and 4). 72 C; 40 cycles of 30 seconds at 94 C, 30 seconds at 51 C, 60 sec- Average body lengths of Group A are 18.12 mm and 17.59 mm, onds at 72 C; 7 minutes at 72 C; held at 4 C. Contigs were respectively, for males and females, and it was 18.40 mm and assembled using CodonCode aligner version 2.0.6 (CodonCode Co., 18.15 mm for males and females, respectively, in Group B specimen. Centerville City, MA, USA) and were subsequently aligned by the However, there is no significant difference in morphological char- acter by microscopic examination, we confirm the fact that A group sample’s parameter length is a little shorter and wider. A and B groups are collected from each area and the regional difference is Table 2. Sample list of the Monochamus saltuarius used in the third study. not considered to be shown.

Group Region No. Host plant Date Stage The habitat distribution data showed that A group specimens were collected only in the Yangpyeong and Gapyeong region in A Gangwon A 6 Jun 2011 Adults Gangwon province whereas B group specimens were distributed Gapyeong A 24 Nut pine Jun 2014eJun 2015 Adults Yangpyeong A 15 Nut pine Jun 2014 Adults most in the collection region (Figure 1). B Gangwon B 1 Jun 2011 Adults In conclusion, it is clear that there are two genetically different Chungbuk B 14 Rigida, nut pine, Jun 2011 Adults groups of M. saltuarius with a different regional distribution. pine However, we cannot conclude where the two group of the pest Gyeonggi B 4 Breed Aug 2011 Adults fi Gapyeong B 4 Nut pine Jun 2014eJun 2015 Adults distributed to a speci c region because of limited specimen Yangpyeong B 1 Nut pine Jun 2014 Adults collection areas. Expanding the collection to the southern part of the Korean peninsula, Jeju Island, Japan, and East-Northern Asia is JH Jeon et al. / Journal of Asia-Pacific Biodiversity 8 (2015) 305e308 307

Figure 1. Collection site of Monochamus Saltuarius: CB, Chungbuk; GG, Gyeonggi; GP, Gapyeong; GW, Gangwon; YP, Yangpyeong.

Table 4. Pairwise distance average of COI between populations. necessary to confirm the distribution and genetic diversity of the pest (Figure 2). Between populations

GWA GWB CBB GGB YPA YPB GPA GPB

GWA d 0.024 0.023 0.025 d 0.024 d 0.028 Acknowledgments GWB 0.024 ddd0.023 d 0.024 d CBB 0.023 ddd0.022 d 0.023 d This research was supported by a fund (project Code No. Z- ddd d d GGB 0.025 0.023 0.025 1541745-2013-15-01) from the Research of and Plant YPA d 0.023 0.022 0.023 d 0.023 d 0.025 YPB 0.024 ddd0.023 d 0.025 d Quarantine Agency, Anyang, South Korea. GPA d 0.024 0.023 0.025 d 0.025 d 0.028 GPB 0.028 ddd0.025 d 0.028 d

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