Mitochondrial DNA Part A DNA Mapping, Sequencing, and Analysis
ISSN: 2470-1394 (Print) 2470-1408 (Online) Journal homepage: http://www.tandfonline.com/loi/imdn21
DNA barcoding of three species (Canis aureus, Canis lupus and Vulpes vulpes) of Canidae Eren Aksöyek, Osman İbiş, Servet Özcan, Mohammad Moradi & Coşkun Tez To cite this article: Eren Aksöyek, Osman İbiş, Servet Özcan, Mohammad Moradi & Coşkun Tez (2016): DNA barcoding of three species (Canis aureus, Canis lupus and Vulpes vulpes) of Canidae, Mitochondrial DNA Part A To link to this article: http://dx.doi.org/10.1080/24701394.2016.1180512
Published online: 14 May 2016.
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Date: 14 May 2016, At: 06:17
MITOCHONDRIAL DNA PART A, 2016 http://dx.doi.org/10.1080/24701394.2016.1180512
RESEARCH ARTICLE
DNA barcoding of three species (Canis aureus, Canis lupus and Vulpes vulpes) of Canidae c,d € €yeka, Osman _Ibis¸b,c, Servet Ozcan Eren Akso , Mohammad Moradie and Cos¸kun Tezd
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a Graduate School of Natural and Applied Sciences, Erciyes University, Kayseri, Turkey; bDepartment of Agricultural Biotechnology, Faculty of Agriculture, Erciyes University, Kayseri, Turkey; cGenome and Stem Cell Center, GENKOK, Erciyes University, Kayseri, Turkey; dDepartment of Biology, Faculty of Sciences, Erciyes University, Kayseri, Turkey; eDepartment of Biology, Faculty of Science, University of Zanjan, Zanjan, Iran
ABSTRACT
ARTICLE HISTORY
Sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene have been used for DNA barcoding and determining the genetic diversity of mammal species. In the current study, our intention was to test the validity of COI barcodes for detecting genetic divergence and to reveal whether or not there is a genetic variation at this marker within canids. Three species (Canis aureus, Canis lupus and Vulpes vulpes) from the family Canidae were selected for DNA barcoding using samples collected from Iran and Turkey. All three species had unique barcoding sequences and none of the sequences were shared among these species. The mean sequence divergences within and among the species were 0.61% and 12.32%, respectively, which fell into the mean divergence ranges found in some mammal groups. The genetic diversity of these three canid species was relatively higher than that found in previously reported studies.
Received 25 February 2016 Revised 12 April 2016 Accepted 16 April 2016 Published online 13 May 2016
Introduction Canidae is one of the most interesting families in the class Mammalia from the point of view of evolutionary biology, behavioural ecology, predation and domestication (SilleroZubiri et al. 2004). This family consists of at least 35 species (Sillero-Zubiri et al. 2004; Wilson & Reeder 1993, 2005), three of which are the golden jackal Canis aureus, the grey wolf Canis lupus and the red fox Vulpes vulpes. The golden jackal is distributed throughout North and Northeast Africa, the Arabian Peninsula, Southeastern Europe, Turkey, the Middle East region, the Indian subcontinent and Southeast Asia (Wilson & Reeder 2005; Jhala & Moehlman 2008; but see Koepfli et al. 2015). The grey wolf is more widespread throughout the Northern Hemisphere, occurring in North America, Europe, the Middle East and Asia (Wilson & Reeder 2005; Mech & Boitani 2010). Contrary to the golden jackal and grey wolf, the red fox is the most widespread canid species and it is widely distributed throughout Europe, Asia, North Africa, and North America (Wilson & Reeder 2005; Macdonald & Reynolds 2008). Mitochondrial DNA has a higher rate of evolution compared to the nuclear DNA, and it is a useful marker for the elucidation of intraspecific structure and also for the identification of species (Sunnucks 2000). The mitochondrial cytochrome oxidase c subunit I (COI) gene has been used as the standard DNA barcoding region for representatives of miscellaneous groups belonging to the animal kingdom for more than a decade (Hebert et al. 2003a,b, 2004; Ward et al. 2005; Hajibabaei et al. 2006a,b; Yoo et al. 2006; Clare et al. 2007; Kerr et al. 2007; Robins et al. 2007; Borisenko et al. 2008). The CONTACT Cos¸kun Tez
[email protected]
COI; DNA barcoding; Canidae; Iran; Turkey
first DNA barcoding study on animal samples was performed by Hebert et al. (2003a), who proposed a 648 bp region (from 58 bp to 705 bp) from the 50 end of the mitochondrial COI gene as a marker to differentiate and identify species. The mouse mitochondrial genome was used for the reference in COI barcoding (see Fr ezal & Leblois 2008). DNA barcoding studies including mammal groups such as bovids, primates, bats and other small mammals have demonstrated an increase in COI sequences in recent years (Lorenz et al. 2005; Hajibabaei et al. 2006b; Clare et al. 2007; Robins et al. 2007; ~eda et al. Borisenko et al. 2008; Cai et al. 2011; Alvarez-Casta n 2012). Despite the fact that there have been many genetic analyses based on the mitochondrial sequences of the three canid species mentioned above (Wayne et al. 1992; Vila et al. 1999; Randi et al. 2000; Aggarwal et al. 2003, 2007; Flagstad et al. 2003; Valie`re et al. 2003; Sharma et al. 2004; Kirschning et al. 2007; Ishiguro et al. 2009; Zachos et al. 2009; Fain et al. 2010; Gomercˇic´ et al. 2010; Rutledge et al. 2010; Weckworth et al. 2010; Rueness et al. 2011; Weckworth et al. 2011; Gaubert et al. 2012; Aghbolaghi et al. 2014; Bray et al. 2014; Djan et al. 2014; Fabbri et al. 2014a,b; Galov et al. 2014; Pilot et al. 2014; Koepfli et al. 2015; Yumnam et al. 2015), DNA barcoding studies addressing these three canid species have not been varied out thus far. Specifically, the genetic diversity and species identification of the three canid species found in Turkey (C. aureus, C. lupus and V. vulpes) and the one canid species found in Iran (V. vulpes) have not been previously studied using the mitochondrial COI barcode. Some Turkish populations of Canidae are threatened due to poaching, poisoning, habitat loss, etc. (Johnson 2002; Can 2004; Salvatori & Linnell 2005; Albayrak 2011). Since the numbers of these
Department of Biology, Faculty of Sciences, Erciyes University, Kayseri 38039, Turkey
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
KEYWORDS
2
€ E. AKSOYEK ET AL.
animals are diminishing, there is a need for quick identification of these species. Therefore these medium-sized carnivores are a model group for testing the DNA barcoding method. The present study aims to investigate two topics: (i) To test the validity of the COI gene for DNA barcoding among individuals of three species (C. aureus, C. lupus and V. vulpes) of the family Canidae and (ii) To determine genetic diversity of the three canid species based on the COI gene sequences.
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Materials and methods Tissue samples (ear, tail, skeletal muscle, tongue, etc.) were collected from 72 road-killed individuals throughout Turkey and parts of Iran (Table 1). Tissues were preserved in 99% ethanol and stored at 20 C. Genomic DNA was extracted from tissue samples by using the DNeasy Blood and Tissue Kit (QIAGEN) following the manufacturer’s instructions. To amplify different fragments of the mitochondrial COI gene, we used the following primer pairs: HCO2198–LCO1490 (Folmer et al. 1994), UCOI–DCOI (Li et al. 2011) and H7227–L6569 (Wayne et al. 1997) for C. aureus and C. lupus; and COXVULF–COXVULR (Prusak & Grzybowski 2008) for V. vulpes. PCRs (Polymerase Chain Reactions) were performed in a volume of 50 ll for each primer pair. The PCR reagent cocktails for the four primer pairs were as follows: 1X Taq buffer, 1 lM of each primer, 200 lM of dNTP mix, 2 units of Taq DNA polymerase, 2.5 mM of MgCl2, 2 ll of template DNA (1/5 diluted), 4 ll of BSA and 31.3 ll of sterile water (dH2O) for primer pair HCO2198–LCO1490 (Folmer et al. 1994); 1X Taq buffer, 0.1 lM of each primer, 0.05 lM of dNTP mix, 3 units of Taq DNA polymerase, 2.5 mM of MgCl2, 1 ll of template DNA (1/5 diluted), 4 ll of BSA and 34.45 ll of sterile water (dH2O) for primer pair UCOI–DCOI (Li et al. 2011); 1X Taq buffer, 0.2 lM of each primer, 0.05 lM of dNTP mix, 2 units of Taq DNA polymerase, 1.25 mM of MgCl2, 2 ll of template DNA (1/5 diluted), 4 ll of BSA and 34.05 ll of sterile water (dH2O) for primer pair H7227–L6569 (Wayne et al. 1997); 1X Taq buffer, 0.2 lM of each primer, 0.05 lM of dNTP mix, 2 units of Taq DNA polymerase, 1.25 mM of MgCl2, 2 ll of template DNA (1/5 diluted), 4 ll of BSA and 34.05 ll of sterile water (dH2O) for primer pair COXVULF–COXVULR (Prusak & Grzybowski 2008). Thermocycling parameters were set at pre-denaturation of 1 cycle at 94 C for 1 min, first denaturation of 5 cycles at 94 C for 30 s, first annealing of 5 cycles at 46 C for 90 s, first extension of 5 cycle at 72 C for 1 min, second denaturation of 30 cycles at 94 C for 30 s, second annealing of 30 cycles at 51 C for 90 s, second extension of 30 cycle at 72 C for 60 s and an ending step of 1 cycle at 72 C for 5 min for primer pair HCO2198–LCO1490 (Folmer et al. 1994); pre-denaturation of 1 cycle at 95 C for 3 min, denaturation of 30 cycles at 94 C for 45 s, annealing of 30 cycles at 54–56 C for 90 s, extension of 30 cycles at 72 C for 90 s and an ending step of 1 cycle at 72 C for 10 min for primer pair UCOI–DCOI (Li et al. 2011); pre-denaturation of 1 cycle at 94 C for 3 min, denaturation of 35 cycles at 94 C for 45 s, annealing of 35 cycles at 51–53 C for 30 s, extension of 35 cycles at 72 C for 45 s and an ending step of 1 cycle at 72 C for 10 min for primer pair
H7227–L6569 (Wayne et al. 1997); and pre-denaturation of 1 cycle at 95 C for 10 min, denaturation of 35 cycles at 94 C for 30 s, annealing of 35 cycles at 55 C for 45 s, extension of 35 cycles at 72 C for 60 s and an ending step of 1 cycle at 72 C for 10 min for primer pair COXVULF–COXVULR (Prusak & Grzybowski 2008). In order to verify the quality of genomic DNA extractions and PCR products, 1% and 1.5% agarose gels were run and stained with ethidium bromide. All PCR products were purified using the Macherey-Nagel Nucleospin Gel and PCR Cleanup kit, and were sequenced in both the forward and reverse directions with the same PCR primers by using the ABI 3100 Genetic Analyzer. COI sequences were aligned in Geneious v.R6.1.6 (http:// www.geneious.com), in which MAFFT v7.017 was used for the multiple sequence alignment with default parameters (Katoh et al. 2002), and we used DnaSP v.5.10 (Librado & Rozas 2009) to determine haplotypes and to estimate haplotype and nucleotide diversities within each species. We used MEGA 6.0 (Tamura et al. 2013) to calculate the genetic distances among sequences of the three canid species, based on the Kimura 2parameter (K2P) model of DNA substitution (Kimura 1980) and then to construct an unrooted phylogenetic tree we used the Neighbour-joining (NJ) method (Saitou & Nei 1987).
Results Mitochondrial COI gene and DNA barcoding Mitochondrial COI gene of different fragment lengths was amplified from 68 out of 72 samples belonging to three Turkish species (C. aureus, C. lupus and V. vulpes) and one Iranian species (V. vulpes) of the family Canidae. The newly generated COI sequences have been deposited in the GenBank database (Accession numbers: KX156584–KX156608) (Table 1). The COI sequences of conspecific samples in this study were determined to be either identical or very similar to each other. They were compared to sequences obtained from the GenBank database (Table 1). The three species were observed to have unique barcoding sequences (648 bp) and no barcoding sequences were shared among the three species.
Golden jackal A total of seven Turkish golden jackals were used for the DNA barcoding analysis. We successfully amplified the COI gene in all seven samples, which resulted in a fragment 1506 bp in length (from 40 bp to 1545 bp) (Table 1). We found three COI haplotypes among the seven sequences of the Turkish golden jackal. Haplotype and nucleotide diversities were Hd: 0.6667 (66.67%) and Pi: 0.00051, respectively. When we examined the specific barcoding region of the mitochondrial COI gene (from 58 bp to 705 bp, 648 bp) (Hebert et al. 2003a,b; Hebert et al. 2004; Li et al. 2011), we found only one variable (polymorphic) site and 647 invariable (monomorphic) sites in the seven Turkish golden jackals. Two haplotypes were found in the seven COI barcoding sequences. Haplotype and nucleotide diversities were Hd: 0.4762 (47.62%)
MITOCHONDRIAL DNA PART A
3
Table 1. A list of canid samples (sequences/haplotypes) used in this study (anot used in analysis). Sample Nr
Haplotype code
Sequence length
Accession number
Canis aureus
638 1584 856 1252 1257 1271 1339
Tr.Ca.COI.1 Tr.Ca.COI.1 Tr.Ca.COI.2 Tr.Ca.COI.2 Tr.Ca.COI.2 Tr.Ca.COI.2 Tr.Ca.COI.3
Canis lupus
1336 1448 1542 1441 1460 1437 1446 1518 1543 1559 1516
Tr.Cl.COI.1 Tr.Cl.COI.1 Tr.Cl.COI.1 Tr.Cl.COI.2 Tr.Cl.COI.2 Tr.Cl.COI.3 Tr.Cl.COI.3 Tr.Cl.COI.3 Tr.Cl.COI.3 Tr.Cl.COI.4 *Tr.Cl.COI.5
1506 bp 1506 bp 1506 bp 1506 bp 1506 bp 1506 bp 1506 bp 588 bp 580 bp 1506 bp 1506 bp 1506 bp 1506 bp 1506 bp 1506 bp 1506 bp 1506 bp 1506 bp 1506 bp 1388 bp 16,729 bp 16,691 bp 16,731 bp 16,562 bp 16,731 bp 16,730 bp 16,729 bp 16,730 bp 16,757 bp 16,730 bp 16,710 bp 16,729 bp 16,729 bp 16,730 bp 16,730 bp 16,196 bp 16,196 bp 16,759 bp 657 bp 657 bp 657 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp
KX156584 KX156584 KX156585 KX156585 KX156585 KX156585 KX156586 a AF028186 a KJ887398 KX156587 KX156587 KX156587 KX156588 KX156588 KX156589 KX156589 KX156589 KX156589 KX156590 KX156591 AB499818 AB499819 AB499820 AB499821 AB499822 AB499823 AB499824 AB499825 AM711902 DQ480503 DQ480504 DQ480505 DQ480506 DQ480507 DQ480508 EU789787 EU789788 JF342908 JF443203 JF443204 JF443208 KF661038 KF661039 KF661040 KF661041 KF661042 KF661043 KF661044 KF661045 KF661046 KF661047 KF661048 KF661049 KF661050 KF661051 KF661052 KF661053 KF661054 KF661055 KF661056 KF661057 KF661058 KF661059 KF661060 KF661061 KF661062 KF661063 KF661064 KF661065 KF661066 KF661067 KF661068 KF661069
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Taxon
Haplotype code in Figure 1 (648 bp)
Locality
Reference
C. C. C. C. C. C. C.
aureus_1 aureus_1 aureus_2 aureus_2 aureus_2 aureus_2 aureus_2
Devrek, Zonguldak, Turkey Bafra, Samsun, Turkey Arhavi, Artvin, Turkey Karakoca, Ulubey, Ordu, Turkey Fındıklı, Rize, Turkey Ovacık K€oy€u, Artvin, Turkey Efirli, Ordu, Turkey
C. C. C. C. C. C. C. C. C. C.
lupus_10 lupus_10 lupus_10 lupus_10 lupus_10 lupus_4 lupus_4 lupus_4 lupus_4 lupus_12
Sorgun, Yozgat, Turkey Kıbrıscık, Bolu, Turkey T€uney K€oy€u, C¸ankırı, Turkey Karakurt, Kars, Turkey Abant G€ol€u, Bolu, Turkey Sarıkaya, Yozgat, Turkey Sarıkamıs¸, Kars, Turkey Nebio glu, K€oy€u, Kars, Turkey Suveren K€oy€u, Igdır, Turkey Sarıkamıs¸, Kars, Turkey Kars, Turkey
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
lupus_2 lupus_1 lupus_1 lupus_2 lupus_3 lupus_2 lupus_2 lupus_2 lupus_10 lupus_10 lupus_10 lupus_10 lupus_11 lupus_4 lupus_10 lupus_10 lupus_10 lupus_10 lupus_1 lupus_10 lupus_1 lupus_10 lupus_10 lupus_10 lupus_7 lupus_10 lupus_10 lupus_10 lupus_13 lupus_10 lupus_10 lupus_13 lupus_10 lupus_4 lupus_4 lupus_10 lupus_7 lupus_10 lupus_10 lupus_10 lupus_1 lupus_10 lupus_1 lupus_6 lupus_10 lupus_10 lupus_10 lupus_1 lupus_6 lupus_1 lupus_10 lupus_1 lupus_1
This study This study This study This study This study This study This study Wayne et al. (1997) Sathishkumar (2014) This study This study This study This study This study This study This study This study This study This study This study Matsumura et al. (2014) Matsumura et al. (2014) Matsumura et al. (2014) Matsumura et al. (2014) Matsumura et al. (2014) Matsumura et al. (2014) Matsumura et al. (2014) Matsumura et al. (2014) Arnason et al. (2007) Bjornerfeldt et al. (2006) Bjornerfeldt et al. (2006) Bjornerfeldt et al. (2006) Bjornerfeldt et al. (2006) Bjornerfeldt et al. (2006) Bjornerfeldt et al. (2006) Pang et al. (2009) Pang et al. (2009) Imes and Sacks (2011) Eger et al. (2011) Eger et al. (2011) Eger et al. (2011) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) (continued)
4
€ E. AKSOYEK ET AL.
Table 1. Continued Taxon
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Vulpes vulpes
Haplotype code
Sequence length
Accession number
1406 1332 1377 1408 925 1381 954
Tr.Vv.COI.1 Tr.Vv.COI.2 Tr.Vv.COI.2 Tr.Vv.COI.2 Tr.Vv.COI.3 Tr.Vv.COI.3 Tr.Vv.COI.4
16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,727 bp 16,683 bp 16,682 bp 16,726 bp 16,708 bp 16,691 bp 16,726 bp 16,727 bp 16,664 bp 16,711 bp 16,729 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp
KF661070 KF661071 KF661072 KF661073 KF661074 KF661075 KF661076 KF661077 KF661078 KF661080 KF661081 KF661085 KF661087 KF661088 KF661090 KF661091 KF661095 KF857179 KX156592 KX156593 KX156593 KX156593 KX156594 KX156594 KX156595
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. V. V. V. V. V. V. V.
lupus_10 lupus_1 lupus_1 lupus_1 lupus_1 lupus_10 lupus_10 lupus_10 lupus_8 lupus_9 lupus_10 lupus_10 lupus_13 lupus_5 lupus_10 lupus_10 lupus_14 lupus_10 vulpes_13 vulpes_14 vulpes_14 vulpes_14 vulpes_15 vulpes_15 vulpes_11
1045 1070 1419 1470 1549 539 627 777
Tr.Vv.COI.4 Tr.Vv.COI.4 Tr.Vv.COI.4 Tr.Vv.COI.4 Tr.Vv.COI.4 Tr.Vv.COI.5 Tr.Vv.COI.5 Tr.Vv.COI.5
740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp
KX156595 KX156595 KX156595 KX156595 KX156595 KX156596 KX156596 KX156596
V. V. V. V. V. V. V. V.
vulpes_11 vulpes_11 vulpes_11 vulpes_11 vulpes_11 vulpes_1 vulpes_1 vulpes_1
924 1137
Tr.Vv.COI.5 Tr.Vv.COI.5
740 bp 740 bp
KX156596 KX156596
V. vulpes_1 V. vulpes_1
1345
Tr.Vv.COI.5
740 bp
KX156596
V. vulpes_1
1380 1410 1413 1438 1530 1533 706
Tr.Vv.COI.5 Tr.Vv.COI.5 Tr.Vv.COI.5 Tr.Vv.COI.5 Tr.Vv.COI.5 Tr.Vv.COI.5 Tr.Vv.COI.6
740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp
KX156596 KX156596 KX156596 KX156596 KX156596 KX156596 KX156597
V. V. V. V. V. V. V.
vulpes_1 vulpes_1 vulpes_1 vulpes_1 vulpes_1 vulpes_1 vulpes_3
803 923 952 1326 1356 1389
Tr.Vv.COI.6 Tr.Vv.COI.6 Tr.Vv.COI.6 Tr.Vv.COI.6 Tr.Vv.COI.6 Tr.Vv.COI.6
740 bp 740 bp 740 bp 740 bp 740 bp 740 bp
KX156597 KX156597 KX156597 KX156597 KX156597 KX156597
V. V. V. V. V. V.
vulpes_3 vulpes_3 vulpes_3 vulpes_3 vulpes_3 vulpes_3
1536 610 1361 1575 760 1555 1597 1598 1600 1590 1596 1591 1592 1595 1601 1593 1589 1602 1599
Tr.Vv.COI.6 Tr.Vv.COI.7 Tr.Vv.COI.7 Tr.Vv.COI.8 Tr.Vv.COI.9 Tr.Vv.COI.10 Ir.Vv.COI.1 Ir.Vv.COI.1 Ir.Vv.COI.1 Ir.Vv.COI.2 Ir.Vv.COI.2 Ir.Vv.COI.3 Ir.Vv.COI.3 Ir.Vv.COI.4 Ir.Vv.COI.4 Ir.Vv.COI.5 Ir.Vv.COI.6 Ir.Vv.COI.6 Ir.Vv.COI.7
740 bp 740 bp 740 bp 696 bp 696 bp 696 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp 740 bp
KX156597 KX156598 KX156598 KX156599 KX156600 KX156601 KX156602 KX156602 KX156602 KX156603 KX156603 KX156604 KX156604 KX156605 KX156605 KX156606 KX156607 KX156607 KX156608
V. V. V. V. V. V. V. V. V. V. V. V. V. V. V. V. V. V. V.
vulpes_3 vulpes_5 vulpes_5 vulpes_11 vulpes_1 vulpes_5 vulpes_3 vulpes_3 vulpes_3 vulpes_10 vulpes_10 vulpes_12 vulpes_12 vulpes_12 vulpes_12 vulpes_11 vulpes_14 vulpes_14 vulpes_16
Sample Nr
Haplotype code in Figure 1 (648 bp)
Locality
S€oke C¸ıkıs¸ı, Kus¸adası, Aydın, Turkey Kurucab€uk, Datc¸a, Mu gla, Turkey Sarık€oy, Beys¸ehir, Konya, Turkey Selc¸uk, _Izmir, Turkey Tavas, Denizli, Turkey Serinhisar, Denizli, Turkey Karacakoca K€oy€u, Ulubey, Ordu, Turkey Ilıca, Erzurum, Turkey Kelkit, G€um€us¸hane, Turkey Kars, Turkey Ka gızman Yol Ayırımı, Kars, Turkey Bozat, Kars, Turkey Kızılırmak, C¸ankırı, Turkey Kemerhisar, Bor, Ni gde, Turkey Amasya-Samsun Yolu, Amasya, Turkey E girdir, Isparta, Turkey C¸allıgedik Gec¸idi, C¸alıs¸, Nevs¸ehir, Turkey G€oc¸ebe K€oy€u, Kavak, Samsun, Turkey Bozan, Bozkırı, Konya, Turkey Uzundere, Gaziemir, _Izmir, Turkey Eskis¸ehir, Turkey Topc¸u K€oy€u, Yozgat, Turkey 10 Km. Tosya, Kastamonu, Turkey Gemic¸, Orhangazi, Bursa, Turkey G€oltas¸ C¸imento Yakını, Isparta, Turkey Arguvan, Malatya, Turkey Tas¸k€opr€u, Babaeski, Kırklareli, Turkey Erciyes Da gı, Kayseri, Turkey Afs¸ar K€oy€u, Beys¸ehir, Konya, Turkey Ergani, Diyarbakır, Turkey C¸akırlar K€oy€u, K€opr€ubas¸ı, Manisa, Turkey Korkuteli, Antalya, Turkey Kandıra-Kefken Yolu, Kocaeli, Turkey Kumluca Ba gları, Edirne, Turkey Erzincan, Turkey Karkın-C¸amlıbel, Tokat, Turkey Kuzukaya, C¸ıldır, Ardahan, Turkey Hamadan, Iran Hamadan, Iran Tehran, Iran Kordestan, Iran Lorestan, Iran Kermanshah, Iran Kermanshah, Iran Lorestan, Iran Qazvin, Iran Ilam, Iran Kordestan, Iran Zanjan, Iran Mazandaran, Iran
Reference Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Thalmann et al. (2013) Deng and He (2014) This study This study This study This study This study This study This study This This This This This This This This
study study study study study study study study
This study This study This study This This This This This This This
study study study study study study study
This This This This This This
study study study study study study
This This This This This This This This This This This This This This This This This This This
study study study study study study study study study study study study study study study study study study study (continued)
MITOCHONDRIAL DNA PART A
5
Table 1. Continued
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Taxon
Sample Nr
Haplotype code
Sequence length
Accession number
16,813 bp 725 bp 725 bp 725 bp 657 bp 16,633 bp 16,723 bp 16,688 bp
AM181037 FJ392293 FJ402874 FJ402883 JF499383 JN711443 GQ37418 KF387633
Haplotype code in Figure 1 (648 bp) V. V. V. V. V. V. V. V.
Locality
vulpes_7 vulpes_4 vulpes_4 vulpes_4 vulpes_6 vulpes_8 vulpes_9 vulpes_9
and Pi: 0.00073, respectively. The genetic distance between the two haplotypes of C. aureus from Turkey was very low, 0.0015 (0.15%), based on the K2P substitution model. We compared our sequences with two COI sequences of golden jackals from GenBank (AF028186: 588 bp, from 703 bp to 1290 bp; KJ887398: 580 bp, from 703 bp to 1282 bp) (Table 1). Based on a multiple sequence alignment 580 bp in length (from 703 bp to 1282 bp), the Turkish golden jackal sequences were distinct compared to the sequences (AF028186 and KJ887398) obtained from GenBank (Table 1). We note that the region compared in this alignment lies beyond the typical COI barcoding region (from 58 bp to 705 bp, 648 bp) and therefore, we could not compare the intraspecific differences of the DNA barcoding region between the sequences of this study and those from GenBank.
Grey wolf Fourteen Turkish grey wolves were used in the DNA barcoding analysis. The amplification of the COI gene was successfully performed in 11 out of 14 samples (Table 1). Sequencing of the amplified COI gene resulted in 10 sequences 1506 bp in length (from 40 bp to 1545 bp) and one sequence 1388 bp in length (from 158 bp to 1545 bp). This shorter sequence was not analysed. Four haplotypes were found among the 10 sequences 1506 bp in length. Haplotype and nucleotide diversities were Hd: 0.7778 (77.78%) and Pi: 0.00093, respectively. Based on the barcoding region of the COI gene (from 58 bp to 705 bp, 648 bp), we found three haplotypes in 10 Turkish grey wolves. Haplotype and nucleotide diversities were Hd: 0.8333 (83.33%) and Pi: 0.00154, respectively. There were two variable (polymorphic) sites and 646 invariable (monomorphic) sites. In the COI barcoding region (648 bp), genetic distances among the three haplotypes of C. lupus from Turkey ranged from 0.0015 (0.15%) to 0.0031 (0.31%), with an average of 0.002 (0.2%), based on the K2P substitution model. Seventy-three (73) COI sequences of different lengths were downloaded from GenBank (Table 1). For the COI barcoding region (648 bp), nine of the sequences of the Turkish grey wolf were found to be identical with some of the sequences obtained from the GenBank (Table 1). However, one sequence of the Turkish grey wolf (Tr.Cl.COI.4) was different when compared to these GenBank sequences (Table 1 and Figure 1). Relying on the COI barcoding region (648 bp), genetic distances ranged from 0.0016 (0.16%) to 0.0097 (0.97%) in all the wolves, with an average of 0.0051 (0.51%), based on the K2P substitution model.
Reference Arnason et al. (2006) Prusak and Grzybowski (2008) Prusak and Grzybowski (2008) Prusak and Grzybowski (2008) Lissovsky et al. (2011) Yu et al. (2012) Zhong et al. (2010) Zhang et al. (2013)
Red fox The DNA barcoding analysis of the red fox was performed by using 13 samples from Iran and 37 samples from Turkey (Table 1).
Turkish samples Sequencing of the amplified COI gene resulted in two sequence lengths. One was 696 bp in length (from 40 bp to 696 bp) observed in three samples and the other was 740 bp in length (from 1 bp to 740 bp) observed in 34 samples (Table 1). Seven COI haplotypes were found in the 34 sequences that were 740 bp in length, while three COI haplotypes were found in the three sequences of length 696 bp. In the sequences for the 696 bp and 740 bp lengths, haplotype and nucleotide diversities were Hd: 1 (100%) and 0.7968 (79.68%), and Pi: 0.00670 and 0.00860, respectively. Based on the COI sequences of different lengths (696 bp and 740 bp), a total of eight haplotypes were determined in the 37 COI barcoding sequences (from 58 bp to 705 bp, 648 bp: Hebert et al. 2003a,b, 2004; Li et al. 2011) of the Turkish red foxes. Haplotype and nucleotide diversities were Hd: 0.8018 (47.62%) and Pi: 0.00916, respectively. There were 24 variable (polymorphic) sites and 624 invariable (monomorphic) sites. In the COI barcoding region (648 bp), the mean genetic distance among the eight haplotypes of V. vulpes was 0.0128 (1.28%), ranging from 0.0015 to 0.0302, relying on K2P.
Iranian samples The amplification of mitochondrial COI gene was successfully performed in 13 out of the 14 Iranian samples (Table 1). Sequencing of the amplified COI gene generated 13 sequences with a fragment length of 740 bp (from 1 bp to 740 bp) and seven haplotypes. Haplotype and nucleotide diversities were Hd: 0.9103 (91.03%) and Pi: 0.01102, respectively. Based on the COI barcoding sequences (from 58 bp to 705 bp, 648 bp) (Hebert et al. 2003a; Li et al. 2011), six haplotypes were found in the 13 sequences. Haplotype and nucleotide diversities were Hd: 0.8590 (85.9%) and Pi: 0.01155, respectively. There were 20 variable (polymorphic) sites and 628 invariable (monomorphic) sites. The mean genetic distance between the six haplotypes of V. vulpes was 0.0143 (1.43%), ranging from 0.0015 to 0.0269, relying on K2P.
Comparison of COI barcoding in Iranian and Turkish red foxes In the COI barcoding region (648 bp), three sequences of the Turkish red fox were identical to three Iranian sequences
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Figure 1. Neighbour-Joining tree (K2P, 10,000 bootstrap replicates) constructed for three canid species, based on COI barcodes (648 bp). Numbers above branches are the bootstrap values.
(Table 1). Indeed, when comparing COI sequences of different length obtained from GenBank (Table 1), we found that a total of eight haplotypes from the Turkish and Iranian red foxes were unique. Genetic distances of the COI barcoding region ranged from 0.0016 (0.16%) to 0.0297 (2.97%) with an average of 0.0111 (1.11%), based on K2P.
Phylogenetic analysis of three species based on DNA barcodes To test the competence of barcodes based on the mitochondrial COI gene in determining the boundaries of these three canid species, we analysed 148 sequences of the three species obtained from this study and from GenBank (Table 1). Relying on K2P distances, the mean sequence divergence within the three canid species was 0.0061 (0.61%), while the mean distance among three species was 0.1232 (12.32%). Mean divergence between the two genera, Canis and Vulpes, was 0.1663 (16.63%).
Based on the COI barcodes, an unrooted NJ tree showed that the canid sequences from this study and those of the GenBank database represented three distinct evolutionary clusters (Figure 1). In the NJ tree (Figure 1), V. vulpes showed a deep divergence from two Canis species (C. aureus and C. lupus), whereas divergence between C. aureus and C. lupus was not relatively deep. V. vulpes diverged into two distinct haplogroups; the first group consisted of the Iranian/Turkish samples and those of the GenBank sequences, while the second group consisted of only the Iranian and Turkish samples. In comparison with V. vulpes, C. lupus showed shallow intraspecific divergences (Figure 1).
Discussion Iran and Turkey, located in the southwestern part of the Palearctic region, have both been historically and zoogeographically a centre for mammal diversity. These countries
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host more than 150 mammal species (Karami et al. 2008; Krystufek & Vohralik 2001, 2009). In Turkey (Randi et al. 2000; Pilot et al. 2010, 2014; Statham et al. 2014; _Ibis¸ et al. 2014, 2015) and Iran (Vila et al. 1999; Aghbolaghi et al. 2014; Pilot et al. 2014), there are few studies on the mitochondrial DNA of canids, such as the golden jackal, grey wolf and red fox. Red foxes have endurance to disperse great distances and overcome geographic barriers. Turkish red foxes (V. vulpes) have an interesting phylogeographic pattern, as they were found clustered into two clear lineages (_Ibis¸ et al. 2014), which are allopatrically distributed across Turkey, as the boundaries of the two lineages are currently uncertain. One of the two lineages has only haplotypes of Japanese (from Hokkaido) and Turkish (from Southwest part) red foxes, based on partial sequences of the mitochondrial cytochrome b gene (_Ibis¸ et al. 2014). The sampling of the Turkish grey wolves, golden jackals and Iranian wolves (Randi et al. 2000; Pilot et al. 2010, 2014; Statham et al. 2014; _Ibis¸ et al. 2015) has been relatively low so far compared to red foxes (_Ibis¸ et al. 2014). Also, there is no current mitochondrial DNA barcode data from any of the canids from Iran and Turkey. The COI sequences and barcoding were used to investigate the inter- and intraspecific differences among the sequences and phylogenetic relationships of the three species (C. aureus, C. lupus and V. vulpes). The target of this study was to evaluate the genetic variability and relationship within and among the three canid species with respect to the main components of wildlife stock in Turkey. The sequences obtained in the current study generated a large amount of new data that use in applications of DNA barcoding. Our results showed that the DNA barcodes based on COI sequences could be used to classify individuals into two genera (Canis and Vulpes) or three species (C. aureus, C. lupus and V. vulpes). The findings of this study also indicated that the intraspecific differences were relatively lower than the interspecific differences. Furthermore, the Turkish red foxes were divided into two haplogroups (Figure 1), in agreement with the previous report of _Ibis¸ et al. (2014). During the last decade, to determine cryptic species and to identify species from several geographical regions, DNA barcodes based on the COI sequences have been successfully used as a molecular marker to support the evidence for taxonomic disagreement. There are a considerable amount of DNA barcoding studies on the animal kingdom, such as bovids (Cai et al. 2011), primates (Lorenz et al. 2005; Hajibabaei et al. 2006a), bats (Clare et al. 2007), other mammals (Borisenko ~eda et al. 2012; et al. 2008; Li et al. 2011; Alvarez-Casta n Bondoc 2013; Echi et al. 2013; Bondoc et al. 2014), lepidopterans (Hajibabaei et al. 2006b; Mutanen et al. 2015), marine polychaetes (Maturana et al. 2011), fishes (Ward et al. 2005; Lara et al. 2010) and birds (Kerr et al. 2007). Based on COI barcode sequences, mammals show a higher mean intraspecific divergence values compared to that of other animal groups. These values ranged from 0.60% to 1.1% in mammals (Lorenz et al. 2005; Hajibabaei et al. 2006a; Clare et al. 2007; Borisenko et al. 2008; Cai et al. 2011) but from 0.17% to 0.46% in other animal groups (Ward et al. 2005; Hajibabaei et al. 2006b; Kerr et al. 2007; Maturana et al. 2011). According to the results of Hebert et al. (2003b), the divergence of COI barcode
7
sequences among the 13,320 congeneric species pairs within 11 animal phyla ranged from 0.0% to 53.7%. The mean interspecific divergences were found to be between 5.8% and >100% in mammals (Borisenko et al. 2008; Cai et al. 2011; Li ~eda et al. 2012; Bondoc 2013; Echi et al. 2011; Alvarez-Casta n et al. 2013), although the mean interspecific divergences ranged from 4.41% to 47% in other animals such as lepidopterans (Hajibabaei et al. 2006b), marine polychaetes, (Maturana et al. 2011) and freshwater fish (Ward et al. 2005; Lara et al. 2010). In this study, the mean intraspecific divergence (0.61%) fell within the mean divergence ranges previously found in other mammal groups (Lorenz et al. 2005; Clare et al. 2007; Borisenko et al. 2008; Cai et al. 2011).
Conclusion To reveal genetic divergence and phylogenetic relationships as well as to determine the species boundaries among members of the family Canidae, the mitochondrial COI gene sequences obtained in the present study could be considered a valuable tool for efficient use of DNA barcodes to identify and discriminate species, especially on the basis of non-invasively collected samples (e.g. faecal remains). Furthermore, based on the genetic divergence inferred from the sequences obtained from canid samples in Iran and Turkey, the mitochondrial COI barcodes might be used to determine the precise distribution and dispersal rotes of the three canids within the wildlife stock of Iran and Turkey.
Acknowledgements We would like to thank Ahmet Yesari Selc¸uk, Vedat C¸adır and Tas¸kın Tez € for their assistance in collecting the samples, and Donna Sue Ozcan and Dr. Klaus-Peter Koepfli for editing and improving English.
Disclosure statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Funding information This study was supported by the research fund of Erciyes University (Project nr: FYL-2014-5454) and partly by the research fund (grant) of the University of Zanjan.
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