A preliminary phylogenetic analysis of golden jackals ( Canis aureus ) ( Canidae : Carnivora : Mammalia ) from Turkey based on mitochondrial D-loop sequences

In the present study, partial sequences (439 bp) of mitochondrial DNA including D-loop region were obtained from seven golden jackals, Canis aureus, collected in Turkey. They were compared to the D-loop sequences registered in the GenBank database under the name Canis aureus. We determined four D-loop haplotypes (333 bp) among the seven Turkish sequences. Despite the limited number of sequences, our analysis indicated that Canis aureus consists of two allopatric haplogroups (a major haplogroup representing Austria, Bulgaria, Croatia, Italy, Serbia and Turkey, and a minor haplogroup containing one haplotype from India) within the sampling area. Interestingly, one haplotype from Senegal was clustered close to grey wolves, used as out-group, and this haplotype might not belong to golden jackal as suggested in previous studies. Our work presented the important data obtained from the Turkish samples to reveal the phylogenetic relationships among golden jackals, and it has suggested that there is a relatively high genetic variability in Turkish golden jackals.


Introduction
The golden jackal (Canis aureus L., 1758) is distributed in South-East Europe, the Levant, Arabian Peninsula, the Middle East region, the Indian subcontinent, and South-East Asia and Africa (Wilson & Reeder 1993, 2005, Jhala & Moehlman 2008).However, populations of small canids occurring in Africa may all belong to the cryp tic African wolf, Canis lupus lupaster, as have been con firmedalreadyforEgypt,EritreaandSenegal(Rue ness et al. 2011al. , Gaubert et al. 2012)).
Mitochondrial DNA has a tendency for a higher rate of evolution when compared to nuclear DNA, and is a usefulmarkerfortheelucidationofintraspecificgenetic structure (Sunnucks 2000).The D-loop (control region) is the non-coding region of the mammalian mitochondri-al DNA, which includes substitutions, indels of various lengths and copies of tandem repeats as well as others, and it is highly variable (Sbisà et al. 1997).
Despite the fact that it is widely distributed, the golden jackal is known as the least investigated species using mitochondrial and nuclear markers.The genetic structure of the golden jackal populations was previously investigated using the mitochondrial control region sequences and microsatellite data from Austria and Serbia by Zachos et al. (2009), and from Bulgaria, Croatia, Eastern Italian Alps and Serbia by Fabbri et al. (2014), and recently from Poland by Kowalczyk et al. (2015).To characterize the genetic structure of the golden jackal populations in Israel, Cohen et al. (2013) used micro-satellite loci.Genetic differentiation among the Croatian golden jackals, grey wolves and dogs were investigated by using sequence variation in Y chromosome (Gomerčić et al. 2013).Galov et al. (2014) also tried to develop a Y chromosome marker to determine hybridization between the golden jackals and dogs.Interestingly, Rueness et al. (2011) showed that mitochondrial DNA sequences of C. aureus lupaster from Egypt did not belong to the golden jackal, but to the cryptic African wolf, C. lupus lupaster.Furthermore, relying on a large data set including mitochondrial and nuclear DNA analysis of Koepfli et al. (2015) revealed that conspecific populations in Africa and Eurasia of golden jackal are different species.They also stated that African population of golden jackal probably belongs to a distinct species.However, no genetic analysis has hitherto been performed on samples of the Turkish golden jackal.
Turkey, consisting of both the European and Asian parts, is a zoogeographical land bridge among Africa, Asia and Europe, and currently hosts more than 150 mammalian species (Johnson 2002, Kryštufek &Vohralik 2001, 2009).Of these species, the golden jackal (C.aureus), "the Turkish coyote" (see Johnson 2002), is an inhabitant in the considerable part of Turkey (Kryštufek & Vohra lik 2001, 2009, see Johnson 2002).
As genetic diversity and phylogenetic relationships of the Turkish golden jackal were unknown, in the present study we amplified a partial fragment of mitochondrial DNA including the D-loop region (control region) of seven golden jackals from the Black Sea region in the Asian part of Turkey.Furthermore, the Turkish D-loop sequences were compared to sequences obtained from the GenBank database.

Materials and Methods
Tissues samples (ear, tail, muscle) were collected from seven road-killed individuals belonging to the Turkish golden jackal (Table 1, Fig. 1).All tissues were preserved at -20 °C and in 99% ethanol before total DNA extraction.To extract total DNA, we used a commercial ex traction kit (The DNeasy Blood and Tissue Kit, Qiagen).Using the total DNA, the partial fragment of mitochondrial DNA included D-loop region (control region) was amplified with a specific PCR (The Polymerase Chain Reaction) primer pair (Forward: DLH 5'-CCTG-AAGTAAGAACCAGATG-3' and Reverse: LF15926F 5'-ATATAAAATACTTTGGTC TTGTAAACC-3') (Kir sch ning et al. 2007).PCRamplificationswereperformedinatotalof50µl reaction mixture; 10 × Taq buffer with (NH 4 ) 2 SO 4 :5µl, dNTPmix:1µl,TaqDNApolymerase(5u/µl)(Thermo Scientific):0.3µl,MgCl 2 :3µl,BSA:3µl,5µlofeach primer,DNAextract:1µl,dH 2 O:26.7µl).ThePCRprogram comprised of a pre-denaturation procedure consisting of 5 min.at 95 °C by 1 cycle, a denaturation step of 40 sec.at 95 °C, an annealing step of 1 min.at 54 °C, an extension step of 90 sec.at 72 °C by 30 cycles and an ending step of 10 min.at 72 °C by 1 cycle.To verify the quality of total DNA and PCR products, 1% agarose gel wasrunandstainedwithethidiumbromide.Purification of PCR products was carried out with the Macherey-Nagel Nucleospin Gel and PCR Clean-up kit.The purified products were sequenced in forward and reverse directions with PCR primers by using a sequencer (ABI 3100 Genetic Analyzer).
Relying on mitochondrial D-loop sequences, phylogenetic relationships of C. aureus were revealed using Neighbor-Joining (NJ) and Maximum Likelihood (ML) methods with MEGA6 (Tamura et al. 2013).The bootstrap value for each branch on the ML and NJ trees was calculated with 10000 pseudoreplicates.
Bayesian analysis (BI: Bayesian Inference) using the MCMC (Markov Chain Monte Carlo) technique was carried out with MrBayes v.3.2 (Ronquist et al. 2012), discarding the first 25% of samples as burn-in (The Average Standard Deviation of split Frequencies < 0.01) and calculating the Bayesian posterior probabilities for 0.2 million generations with tree sampled every 100 generations.After discarding burn-in, the remaining samples were held to generate the consensus tree (50% majority rule), and to calculate 95% Bayesian credibility interval and posterior probability.Bayesian tree diagram was drawn using FigTree v1.3.1 (Rambaut 2009).

Results
Weamplifiedthe439bpfragment(tRNA Thr + tRNA Pro + D-loop) of the mitochondrial DNA from the seven Turkish golden jackals, including 333 bp for D-loop (control region) (Table 1, Fig. 1).Genetic analysis revealed four Turkish haplotypes (TrCa1-TrCa4) of the 439 bp fragments, including five variable (polymorphic) sites and two parsimony informative sites.The most common haplotype was TrCa3 (57.1%).Four haplotypes of the Turkish golden jackal (TrCa1 -TrCa4) are deposited in the GenBank database (Accession numbers: KT988006 -KT988009).Based on the 439 bp fragments, the haplotype (Hd) and nucleotide diversities (Pi) of the Turkish samples were 0.7143 and 0.00412, respectively.Sequence divergences of the four Turkish haplotypes ranged from 0.002 to 0.012, with a mean of 0.0066, relying on K2P.
When mitochondrial D-loop region (333 bp) of the seven Turkish golden jackals was analyzed, four D-loop haplotypes were determined as stated above, and the most common haplotype that was shared by four samples obtained from four localities was TrCa3 (Table 1, Fig. 1).Haplotype and nucleotide diversities for the mitochondrial D-loop region of Turkish golden jackal were Hd: 0.7143 and Pi: 0.00543, respectively.
Three phylogenetic trees (BI, ML and NJ) (Figs. 2 -4) were constructed using the mitochondrial D-loop sequences of 269 bp obtained from Turkey and the GenBank database (Table 1), including Senegal sequences, which were probably deposited under the name Canis aureus.The phylogenetic trees showed consistency in their branching topologies that are supported with high posterior probabilities (Fig. 2) and bootstrap values (Figs. 3 -4).In these trees (Figs. 2 -4), the samples of the golden jackal were separated into two haplogroups.From these haplogroups, Haplogroup 1 consisted of haplotypes from Austria, Bulgaria, Caucasia, Croatia, Italy, Poland, Serbia and Turkey, which did not show any geographical clustering.Haplogroup 2 contained a single haplotype, including two sequences of Indian golden jackal, and it composed a separate lineage (Figs. 2 -4).Haplogroup 1 was diverged from Haplogroup 2 with an average distance value of 1.9%.However, Senegalese samples (Senegalese group), which consisted of a single haplotype including two sequences, clustered together with an out-group containing the Canis lupus sequences (Figs. 2 -4).
Besides the three phylogenetic trees (Figs. 2 -4), a median-joining network with the seven mitochondrial D-loop haplotypes (269 bp) of C. aureus from Turkey and the GenBank database was shown in Figure 5.The haplotype network (Fig. 5) also depicted that there was a relatively similar relationship among the Turkish and Austrian, Bulgarian, Caucasian, Croatian, Italian, Polish and Serbian samples, as shown the three phylogenetic trees (Figs. 2 -4).In both trees (Figs. 2 -4) and network (Fig. 5), a single haplotype obtained from the Senegalese sequences registered in the GenBank database under the name C. aureus showed a different grouping from the other golden jackal haplotypes.
In this study, we investigated the genetic diversity and phylogenetic relationships of golden jackal from Turkey using the partial mitochondrial DNA sequences, including D-loop (control region).In search of available datafromtheGenBankdatabase,wecouldnotfindasatisfactory number of sequence to compare with our data (Randi et al. 2000, Aggarwal et al. 2007, Zachos et al. 2009, Rueness et al. 2011, Gaubert et al. 2012, Fabbri et al. 2014, Pilot et al. 2014, Kowalczyk et al. 2015).This study suggests only that the golden jackal (Canis aureus) consists of two allopatric haplogroups (Figs. 2 -4) within the sampling area; besides the sequences from Turkey Haplogroup 1 including sequences from Austria, Bulgaria, Caucasia, Croatia, Italy, Poland and Serbia (Randi et al. 2000, Zachos et al. 2009, Fabbri et al. 2014, Pilot et al. 2014, Kowalczyk et al. 2015, This Study), and Ha plo group 2 comprising one haplotype from India (Ag gar wal et al. 2007, Rueness et al. 2011).However, one haplotype from Senegal (Gaubert et al. 2012) was clustered close to grey wolves, which were used as out-group (Figs. 2 -4).Furthermore, the haplotype network (Fig. 5) also depicted that there was a similar relationship among the Turkish and Austrian, Bulgarian, Caucasian, Croatian, Italian, Polish and Serbian samples (Randi et al. 2000, Zachos et al. 2009, Fabbri et al. 2014, Pilot et al. 2014, Kowalczyk et al. 2015, This Study), as shown in Figures 2-4.In all analyses, which based on the 269 bp D-loop sequences, the Turkish haplotype (TrCa3, the most common haplotype) was identical to other sequences (zachos et al. 2009, Fabbri et al. 2014, Kowalczyk et al. 2015) (Figs. 2 -5).In a study comparing genetic variability of the Caucasian grey wolves to those of Europe and The Middle East, Pilot et al. (2014) reported two haplotypes belonging to golden jackal from Caucasus region.In their report, the two haplotypes from Caucasia, one of which was shared with one of the Turkish haplotypes and where is neighboring to the Northeast part of Turkey, were grouped in Haplogroup 1 (Figs. 2 -5).
Of samples sequenced, the Austrian, Bulgarian, Croatian, Italian and Serbian golden jackals that were report- ed to not display a genetic variation at the mitochondrial control region (D-loop) (Zachos et al. 2009, Fabbri et al. 2014).In contrast to the above-mentioned samples of golden jackal, the Turkish samples were relatively polymorphic, as it contained different D-loop haplotypes, all of which were linked to Haplogroup 1 in phylogenetic trees (Figs. 2 -4).
Inpreviousphylogeneticstudiesfocusedongreyand/ or African wolves from Asia and Africa, a few samples of golden jackals (Aggarwal et al. 2007, Rueness et al. 2011, Gaubert et al. 2012, Pilot et al. 2014) were included.Initially, Aggarwal et al. (2007) used two sequences of golden jackal from Central India to construct a phylogenetic tree of the relationships among the Indian wolves.Haplogroup 2 of the present study consisted of the Indian haplotype found in the two sequences obtained from South Asia (Aggarwal et al. 2007), which was basal to Haplogroup 1 (Figs. 2 -4).In the study of Rueness et al. (2011), which included two Indian sequences, the Canis species occurring in Egypt, formerly named C. aureus lupaster, was found to be related to wolves rather than the golden jackal (C.aureus).The systematic position of this cryptic African wolf (Canis lupus lupaster) was later confirmed by Gaubert et al. (2012).These authors revealed also that the Senegalese samples previ-ouslyidentifiedasgoldenjackalbelongedtotheAfrican wolf lineage.Recently, based on a large date set obtained from mitochondrial and nuclear genomes, Koepfli et al. (2015) revealed that African and Eurasian populations of golden jackal are genetically different lineages, and that African population of golden jackal probably belongs to a different species.Our study showed that the Senegalese sequences registered in the GenBank database under the name C. aureus (Gaubert et al. 2012) grouped together with the wolves used as out-group (Figs. 2 -4).
In conclusion, our work contributed to the understanding of genetic diversity and the phylogenetic relationships of golden jackals based on the variability in their mitochondrial D-loop region.As a result of the mitochondrial DNA analysis, we found that there were four haplotypes in the seven Turkish golden jackals (one different haplo-typeover7/4=1.75samples).Inopposetothestudiesof Zachos et al.(2009)(121/1=121samples)andFabbri et al.(2014)(120/1=120samples),valuefoundinthisstudy (one different haplotype over 7/4=1.75samples) was higher than the values found in the Bulgarian, Croatian, Italian and Serbian golden jackals.In this context, haplotype (Hd) and nucleotide (Pi) diversities were also much higher in the Turkish golden jackal (0.7143 and 0.00412, respectively) than in the Bulgarian, Croatian, Italian and Serbian golden jackals (both were zero).When comparing the genetic diversity of the Turkish golden jackal to the Austrian, Bulgarian, Caucasian, Croatian, Italian, Polish and Serbian golden jackals, it appeared that the Turkish golden jackal had higher genetic diversity.In genetic diversity, the Caucasian population also has resemblance to Turkish population therewithal.
As an indication of higher genetic diversity, the mitochondrial D-loop sequence polymorphism might have resulted from difference of biotic and abiotic factors that affect the golden jackals in Turkey and the other geographical regions (Austria, Bulgaria, Caucasia, Croatia, Italy,PolandandSerbia).Inordertofurtherconfirmthis, more advanced genetic analyses need to be performed by using different mitochondrial and nuclear DNA markers.

Fig. 2 .
Fig. 2. Bayesian tree constructed from haplotypes of the mitochondrial D-loop region (269 bp) of golden jackals, rooted with C. lupus.The bold haplotypes indicate the Turkish golden jackal.

Fig. 3 .
Fig. 3. ML tree constructed from haplotypes of the mitochondrial D-loop region (269 bp) of golden jackals, rooted with C. lupus.The bold haplotypes indicate the Turkish golden jackal.

Fig. 4 .
Fig. 4. NJ tree constructed from haplotypes of the mitochondrial D-loop region (269 bp) of golden jackals, rooted with C. lupus.The bold haplotypes indicate the Turkish golden jackal.

Fig. 5 .
Fig. 5. Median-joining network constructed from haplotypes of the mitochondrial D-loop region (269 bp) of golden jackals.The bold haplo types indicate the Turkish golden jackal.

Table 1 .
The Turkish golden jackal samples and sequences obtained from the GenBank database.
Fig. 1.Localities of the Turkish golden jackal samples (seeTable 1 for map numbers).