Advance Publication The Journal of Veterinary Medical Science Accepted Date: 12 Apr 2018 J-STAGE Advance Published Date: 5 Sep 2018 Wildlife Science 1 Full paper 2 Title 3 Population structure of the raccoon dog (Nyctereutes procyonoides) using microsatellite 4 loci analysis in South Korea: Implications for disease management 5 6 Running head 7 RACCOON DOGS IN SOUTH KOREA 8 9 Authors and Affiliations 10 YoonJee Hong1, Kyung Seok Kim2, Mi-Sook Min1 and Hang Lee1 11 12 1Conservation Genome Resource Bank for Korean Wildlife (CGRB), Research Institute 13 for Veterinary Science and College of Veterinary Medicine, Seoul National University, 14 Seoul 08826, Korea 15 2Department of Natural Resource Ecology and Management, Iowa State University, Ames, 16 IA 50011, USA 17 18 Corresponding authors: Mi-Sook Min and Hang Lee 19 Mi-Sook Min 20 Conservation Genome Resource Bank for Korean Wildlife (CGRB), Research 21 Institute for Veterinary Science and College of Veterinary Medicine, Seoul National 22 University, Gwanak_1, Gwanak-Ro, Gwanak-Gu, Seoul 08826, Korea 23 Tel: 82.2.880.1240, Fax: 82.2.888.2754, E-mail: [email protected] 24 Hang Lee 1 Wildlife Science 1 Conservation Genome Resource Bank for Korean Wildlife (CGRB), Research 2 Institute for Veterinary Science and College of Veterinary Medicine, Seoul National 3 University, Gwanak_1, Gwanak-Ro, Gwanak-Gu, Seoul 08826, Korea 4 Tel: 82.2.880.1274, Fax: 82.2.888.2754, E-mail: [email protected] 5 2 Wildlife Science 1 Abstract 2 The prevention and control of infectious diseases transmitted by wildlife are gaining 3 importance. To establish effective management strategies, it is essential to understand the 4 population structure of animals. Raccoon dogs (Nyctereutes procyonoides) in South Korea 5 play a key role in the maintenance of food web stability and possess genetic compositions 6 that are unique compared to those in other areas. However, wild raccoon dogs play another 7 role as the main host of various infectious diseases. To establish long-term strategies for 8 disease management, we investigated the genetic structure and possible geographic 9 barriers that influence the raccoon dog population in South Korea by analyzing 16 10 microsatellite loci. The present study showed that mountains were the major factors 11 responsible for genetic structuring, along with distance. We proposed potential 12 management units (MUs) for raccoon dogs based on the genetic structuring and gene-flow 13 barrier data obtained in this study. Four MUs were suggested for the Korean raccoon dog 14 population (northern, central, southwestern, and southeastern). The Korean raccoon dog 15 population structure determined in this study and the proposed MUs will be helpful to 16 establish pragmatic strategies for managing Korean raccoon dog population and for 17 preventing the transmission of infectious diseases. 18 19 Keywords: management unit (MU), microsatellites, Nyctereutes procyonoides, population 20 structure, raccoon dog 21 3 Wildlife Science 1 INTRODUCTION 2 The raccoon dog is a true omnivore [11] that maintains the food web stability in 3 ecosystems [1] and also functions as an effective scavenger [21]. Raccoon dogs play an 4 important role as one of the medium-sized predators or scavengers because of the 5 reduction or extinction of their competitors and other major terrestrial predators, assisting 6 in maintaining the ecological balance in the Korean Peninsula [8]. A phylogeographic 7 study using complete mtDNA cytochrome b sequences suggested that Korean raccoon 8 dogs are a unique population that have adapted to the particular environment of Korean 9 Peninsula in northeast Asia and are different from the other continental and Japanese 10 raccoon dog populations [14]. This study showed that the Korean raccoon dog population 11 should be considered as a valuable biological resource requiring proper management and 12 conservation strategies. 13 Wild raccoon dogs (Nyctereutes procyonoides) play another role as the main host of 14 various infectious diseases, such as rabies, canine distemper, and parasites, which can be 15 transmitted among domestic dogs and wild Canidae species [11, 35, 37]. Its high 16 adaptability to various environments and the reduction of its competitors and predators 17 might assist in increasing its population size in South Korea [9]. The increasing Korean 18 raccoon dog population has raised public concerns over their potential role as disease and 19 parasite vectors, particularly for contagious zoonotic diseases [8, 22]. Some pathogens and 20 parasites are transmitted between dogs and wild canids, and from rodents to foxes, raccoon 21 dogs, dogs, and even humans [11]. Potential issues regarding the prevention and control of 22 wildlife-related infectious diseases are becoming increasingly important. Hence, as part of 23 the effort to establish proper disease risk management strategies, it is essential to 24 understand the population structure and connectivity among the animal populations of 25 epidemiological interest [3, 31]. Lack of information on the movement of individuals of 4 Wildlife Science 1 the vector and/or host population makes it difficult to control and prevent disease 2 outbreaks and transmission. In South Korea, rabies has been mostly reported in wild and 3 domestic animals, and in humans in the northern part of Seoul/Gyeonggi and Gangwon 4 Provinces of South Korea; and preventive measures against rabies are focused only in 5 these areas [35]. However, limited scientific data are available on the dispersal or 6 movement pattern of raccoon dogs, which is the major wild rabies host in South Korea 7 with respect to the geographical features such as rivers and mountain ranges in South 8 Korea. This information might be critical for establishing appropriate strategies to prevent 9 and manage infectious diseases transmitted by raccoon dogs. The genetic structure of wild 10 animal population is formed by limited dispersal owing to artificial or natural barriers. 11 Therefore, investigation on the genetic structure of populations will be of immense help to 12 provide information for the proper management of wild animal populations and their 13 diseases. 14 Several studies have been conducted on wild animal population structure in South Korea 15 by using microsatellites: wild boar (Sus scrofa) [2], water deer (Hydropotes inermis) [16], 16 Siberian roe deer (Capreolus pygargus) [17], and striped field mouse (Apodemus agrarius) 17 [10]. In case of wild boars, the authors did not show a clear population structure but a 18 gradual geographic gradient from north to south and the high mountain range 19 (Baekdudaegan: Taebaek, and Sobaek mountains, Fig. 1) was suggested as the geographic 20 barrier. Generally, raccoon dogs prefer lower altitude (below 300 m) [21] and low 21 temperature (below 0 °C) that limit their migration [12]. Hong et al. [8] also reported the 22 possibility that high altitude mountains (Sobaek mountains, Fig. 1) can act as a 23 geographical barrier for raccoon dogs. In this study, we mainly investigated whether 24 mountain ranges affected raccoon dog population in South Korea. In addition, impacts of 25 the geographical distance on the population differentiation were tested. 5 Wildlife Science 1 2 Microsatellite markers are widely used to identify individuals and to study the 3 phylogeographic relationships, genetic variation, population structuring, and genetic 4 differentiation in various wildlife species [8, 17]. Information obtained by analyzing 5 microsatellite loci is used for determining the genetic structure and recent gene flow 6 among natural populations and serves as the basis for establishing management and 7 conservation strategies for a population, e.g., for defining conservation or management 8 units (MUs) [23]. To help establish long-term strategies for effective population and 9 disease management of raccoon dogs in South Korea, we examined the genetic structure of 10 seven regional populations, by focusing on their genetic diversity and the potential barriers 11 of gene flow among the populations. The findings of the study would provide a valuable 12 resource that might be applied to other mammalian species and related infectious disease 13 for developing better management strategies. 14 15 16 MATERIALS AND METHODS 17 DNA samples and genotyping 18 We analyzed the tissue samples of 194 raccoon dogs collected from seven provinces of 19 South Korea (SG: Seoul/Gyeonggi, WG: Western Gangwon, EG: Eastern Gangwon, CC: 20 Chungcheong, JB: Jeonbuk, JN: Jeonnam, and GS: Gyeongsang; Fig. 1). All the 21 experimental materials were collected legally and provided by the Conservation Genome 22 Resource Bank for Korean Wildlife (CGRB) for this study. All procedures followed when 23 working with animal samples were in accordance with the guidelines of Seoul National 24 University Institutional Animal Care and Use Committee (SNU-IACUC). Details of 25 sampling locations and sample sizes are shown in Table 1. In the case of Gangwon 6 Wildlife Science 1 individuals, the western area was very close to Seoul/Gyeonggi; therefore, these 2 individuals were analyzed separately from the eastern individuals of Gangwon. Sixteen 3 microsatellite loci were used to determine the genotypes. Four microsatellite loci (ZuBeCa 4 15, ZuBeCa 17, ZuBeCa 18, and ZuBeCa 26) were originally developed for the dog, Canis 5 lupus [29, 30]. The other 12 microsatellite loci (Nyct1 to Nyct12) were developed for the 6 Korean raccoon dog, Nyctereutes procyonoides koreensis [8]. The genomic DNA of all 7 dogs was extracted using a DNeasy Tissue kit (QIAGEN, Valencia, USA) and amplified 8 using PCR under the following touchdown conditions: initial denaturation for 3 min at 9 94 °C; followed by 20 cycles at 94 °C for 1 min, 60–50 °C (decreased by 0.5 °C per cycle) 10 for 1 min, 72 °C for 1 min; again followed by 20 cycles at 94 °C for 1 min, 50 °C for 1 11 min, 72 °C for 1 min; and a final extension at 72 °C for 10 min. The PCR reaction mixture 12 comprised 1.5 mM MgCl2, 200 µM of each dNTP, 0.5 U i-Star Taq DNA polymerase 13 (iNtRON Inc, Seongnam, Gyeonggi, Korea), 0.3 µM each of the fluorescent labeled 14 forward primer and non-fluorescent reverse primers, and 10–50 ng template DNA.