66 (3): 427 – 435 © Senckenberg Gesellschaft für Naturforschung, 2016. 21.12.2016 Genetic analysis of the Turkish gray wolf (Canis lupus) based on partial mitochondrial DNA sequences Osman İbiş 1, 2, Eren Aksöyek 3, Servet Özcan 2, 4, Akif Keten 5, Tarkan Yorulmaz 6 & Coşkun Tez 4, * 1 Department of Agricultural Biotechnology, Faculty of Agriculture, Erciyes University, Kayseri, Turkey — 2 Genome and Stem Cell Center, GENKOK, Erciyes University, Kayseri, Turkey — 3 Graduate School of Natural and Applied Sciences, Erciyes University, Kayseri, Turkey — 4 De- partment of Biology, Faculty of Sciences, Erciyes University, Kayseri, Turkey — 5 Department of Wildlife Ecology and Management, Faculty of Forestry, Düzce University, Düzce, Turkey — 6 Department of Biology, Faculty of Sciences, Çankırı Karatekin University, Çankırı, Turkey — * Corresponding author: [email protected] Accepted 19.vii.2016. Published online at www.senckenberg.de / vertebrate-zoology on 13.xii.2016. Abstract In this study, we focused on determining the genetic variability of Turkish gray wolves and also to reveal phylogenetic relationships of the Eurasian wolf populations using mitochondrial DNA sequences. Partial mitochondrial DNA sequences (440 bp) were obtained from 12 Turkish gray wolves, including D-loop region (332 bp). We found seven D-loop haplotypes (332 bp) among the 12 Turkish gray wolves. The D-loop sequences of the Turkish gray wolf were compared to sequences registered in GenBank from Eurasia under the name Canis lupus. Five Turkish haplotypes were shared with conspecific sequences from other regions of Eurasia in the species’ range. Two haplotypes were unique for the Turkish wolves. The existence of shared haplotypes suggests that the gray wolves in Turkey and other regions might be originated from the same gene pool. The present study reports significant data for determining the genetic variability and revealing the phylogenetic relationships of Turkish gray wolves; it is suggested that the genetic variability of Turkish gray wolves is relatively high. Key words Canis lupus, Mitochondrial DNA, D-loop, Turkey. Introduction The gray wolf (Canis lupus L., 1758) is a member of the In order to manage and to conserve wild species, na- family Canidae and one of the most important terres- ture conservationists and researchers make an effort to trial predators in Northern Hemisphere (SILLERO-ZUBIRI draw a comprehensive picture of the genetic variability et al. 2004). It is widely distributed across much of the of populations in different regions. There are numer- Holoarctic region (WILSON & REEDER 1993, 2005, MECH ous studies on extant gray wolves on populations and & BOITANI 2010). Gray wolves also occur in Turkey and on phylogenetic relationships throughout the species’ its surrounding areas (SALVATORI & LINNELL 2005, WILSON range based on mitochondrial DNA (WAYNE et al. 1992, & REEDER 1993, 2005, KRyštufek & VOHRALIK 2009). ELLEGREN et al. 1996, VILÁ et al. 1999, RANDI et al. 2000, The gray wolf has been considered the first mam- FLAGSTAD et al. 2003, Jędrzejewski et al. 2005, PILOT et mal species domesticated by humankind (CLUTTON- al. 2006, 2010, 2014, PANG et al. 2009, FAIN et al. 2010, BROCK 1995), with multiple origins (PANG et al. 2009, Gomerčič et al. 2010, RUTLEDGE et al. 2010, Weckworth VON HOLDT et al. 2010). It is also known to be the most et al. 2010, 2011, ASADI-AGHBOLAGHI et al. 2014, BRAY important wildlife species (SILLERO-ZUBIRI et al. 2004). et al. 2014, Djan et al. 2014, FABRI et al. 2014, JANSSON ISSN 1864-5755 427 İbiş, O. et al.: Genetic analysis of Canis lupus based on partial mitochondrial DNA sequences Fig. 1. Localities of the Turkish gray wolf samples (see Table 1 for numbers). et al. 2014, MATSUMURA et al. 2014, ISHIGURO et al. 2016, al. 2014, JANSSON et al. 2014, MATSUMURA et al. 2014, etc.). ISHIGURO et al. 2016, etc.), no wide-ranging and detailed The present study is focused on the Turkish gray mitochondrial DNA analysis of Turkish gray wolves has wolf (Canis lupus). This large-sized carnivore is widely been performed up to now. Only a few samples of gray distributed throughout Turkey, where forms a zooge- wolves from Turkey have been used in genetic analyses ographical bridge between Africa, Asia, Europe and the (VILÁ et al. 1997, 1999, RANDI et al. 2000, PILOT et al. Arabian Plate (JOHNSON 2002, KRyštufek & VOHRALIK 2006, 2010, 2014). 2001, 2009). The gray wolf is one of well-known large By presenting preliminary results of an analysis of carnivores like red fox (Vulpes vulpes), golden jackal partial mitochondrial sequences, we aim to contribute to (Canis aureus) and brown bear (Ursus arctos) in Turkey the knowledge of the genetic variability of Turkish gray (JOHNSON 2002, CAN 2004, CAN & TOGAN 2004, ALBAYRAK wolves and the relationships with other conspecific popu- 2011, Aşan-BAYDEMİR et al. 2011, İLEMİN 2014, AKSÖYEK lations throughout Eurasia. et al. 2016, AMBARLI et al. 2016). The Turkish wolves inhabit all biomes, which include prey. The population size of the Turkish gray wolf has been estimated at around 5000 – 7000 individuals (CAN Materials and Methods 2004, SALVATORI & LINNELL 2005). Due to the decrease of prey animals such as sheep, gazelle, red deer, roe deer, wild boar and brown hare, habitat fragmentation and il- Sampling legal hunting etc., the Turkish gray wolves have shown a decline in the last century; they occur in small subpopu- The tissue samples (ear, skin, tail, etc.) were collected lations (CAN & TOGAN 2004, SALVATORI & LINNELL 2005, from the 12 road-killed gray wolves in the Asian part of ŞEKERCİoğlu et al. 2011). Therefore, measures for con- Turkey (Table 1, Fig. 1). servation management have been taken by the Ministry of Forestry and Water Affairs in Turkey (see www. ormansu.gov.tr). DNA extraction and PCR conditions Mitochondrial DNA, indicative of maternal inher- itance, is one of the important tools, which have been Tissue samples stored in 99% ethyl alcohol were used to used in genetic analyses of Turkish carnivores (İBİş & obtain genomic DNA, which were extracted by using a Tez 2014, İBİş et al. 2014, 2015a,b, AKSÖYEK et al. 2016, commercial extraction kit (The DNeasy Blood and Tissue DEMİrbaş et al. 2016). While mitochondrial DNA of the Kit, Qiagen) according to the manufacturer’s instructions. gray wolf was analyzed for various populations through- A short fragment of mitochondrial DNA including the out species’ range (ELLEGREN et al. 1996, VILÁ et al. 1999, D-loop (control region) was amplified by using DLH RANDI et al. 2000; FLAGSTAD et al. 2003; Jędrzejewski et (Forward: 5’-CCTGAAGTAAGAACCAGATG-3’) and al. 2005, PILOT et al. 2006, 2010, 2014, PANG et al. 2009, LF15926F (Reverse: 5’-ATATAAAATACTTTGGTCTT FAIN et al. 2010, Gomerčič et al. 2010, RUTLEDGE et al. GTAAACC-3’) primers (KIRSCHNING et al. 2007). 2010, Weckworth et al. 2010, 2011, ASADI-AGHBOLAGHI A total of 50 µl reaction mixture were used for PCR et al. 2014, BRAY et al. 2014, Djan et al. 2014, FABRI et (Polymerase Chain Reactions) amplifications; 10 × Taq 428 VERTEBRATE ZOOLOGY — 66 (3) 2016 Table 1. A list of grey wolf samples (sequences/haplotypes) used in this study. Map no. (Fig. 1) Nr. of Turkish samples Code of Turkish haplotypes Accession number Locality Reference Conspecific sequences Reference 1 1437 Tr.Cl.D1 KY039989 Sarıkaya, Yozgat, Turkey This Study AY570178-AY570181 Ardalan et al. 2004 2 1516 Tr.Cl.D1 KY039989 Kars, Turkey This Study AF008136-AF008142 Vila et al. 1997 3 1518 Tr.Cl.D1 KY039989 Nebioğlu, Köyü, Ardahan, Turkey This Study KJ195895-KJ195898, KJ490942-KJ490944 Pilot et al. 2014 4 1543 Tr.Cl.D1 KY039989 Suveren Köyü, Iğdır, Turkey This Study AF115687-AF115703, AF115707, AF115714 Randi et al. 2000 5 1446 Tr.Cl.D2 KY039990 Sarıkamış, Kars, Turkey This Study AY916804-AY916822 Luo et al. 2005 6 1336 Tr.Cl.D3 KY039991 Sorgun, Yozgat, Turkey This Study AF338803-AF338811, AF487754 Valiere et al. 2003 7 1542 Tr.Cl.D3 KY039991 Tüney Köyü, Çankırı, Turkey This Study KF723519-KF723526 Jansson et al. 2014 8 1448 Tr.Cl.D4 KY039992 Kıbrıscık, Bolu, Turkey This Study FJ978005-FJ978035 Pilot et al. 2010 9 1334 Tr.Cl.D5 KY039993 Tekman, Erzurum, Turkey This Study DQ480503-DQ480507 Bjornerfeldt et al. 2006 10 1559 Tr.Cl.D5 KY039993 Sarıkamış, Kars, Turkey This Study JN182019-JN182092 Hindrikson et al. 2011 11 1460 Tr.Cl.D6 KY039994 Abant Gölü, Bolu, Turkey This Study AB007372-AB007373 Tsuda et al. 1997 12 1441 Tr.Cl.D7 KY039995 Karakurt, Kars, Turkey This Study AY172676-AY172677 Yang et al. 2002 KF857179 Deng and He 2016 KF661038-KF661055, KF661078-KF661082 Thalmann et al. 2013 KF661087, KF661091, KF661095 Thalmann et al. 2013 AB499818-AB499825 Matsumura et al. 2014 AM711902 Arnason et al. 2007 KC540917- KC540929 Asadi-Aghbolaghi et al. 2014 Haplotype codes in Figures 2 – 3; lu.1: AF338807, AF115689; lu.2: KF723519, FJ978017; lu.3: FJ978027, AF115688, AF115687, FJ978028; lu.4: AF115687, FJ978028; lu.5: Tr.Cl.D3; lu.6: Tr.Cl.D4; lu.7: Tr.Cl.D6, AF115696, KJ490942, KC540923, KC540924; lu.8: AF338806, KF723523, AF115694, AF115695, FJ978020, FJ978019; lu.9: FJ978034; lu.10: AY916808, AY916807, FJ978016, KJ195895, FJ978015, AF008142; lu.11: AF115697; lu.12: AY916810; lu.13: Tr.Cl.D7, KJ195897; lu.14: AY916805, AY916814, lu.15: AY916819; lu.16: AY916815; lu.17: FJ978006, AF487754, KF723521, KF723520, AF115693, AF115692, FJ978005, DQ480504, JN182058, JN182032, JN182074, JN182060, JN182036, JN182033, JN182030, JN182075, JN182085, JN182084, JN182049, JN182050, JN182020, JN182026, JN182023, JN182022, JN182080, JN182086,