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Turkish Journal of Zoology Turk J Zool (2017) 41: 250-258 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1509-52

Phylogenetic status and genetic diversity of corsac ( corsac) in Golestan Province,

1, 2 3 2 Jalil IMANI HARSINI *, Hamid-Reza REZAEI , Saeid NADERI , Hosein VARASTEH MORADI 1 Department of Environment and Energy, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Fisheries and Environmental Science, Faculty of Environmental Science, Gorgan University of Agricultural Sciences & Natural Resources, Gorgan, Iran 3 Department of Environmental Science, Faculty of Natural Resources, University of Guilan, Rasht, Iran

Received: 29.09.2015 Accepted/Published Online: 19.07.2016 Final Version: 04.04.2017

Abstract: The lack of detailed and precise reports about the presence of corsac fox has caused ambiguities about the presence or absence or even status of this in its distribution range in Iran. Therefore, this is one of the complicated issues in Iran’s wildlife fauna and there has been no clear scientific answer to this question to date. In this study, 7 mitochondrial DNA control region haplotypes were identified in 10 corsac fox samples (4 tissue and 6 scat) taken from Golestan Province in Iran, and haplotype diversity was equal to 0.93. The genetic distance between individuals showed large differences between Iranian samples and the samples existing in GenBank, which are from northern . The phylogenetic tree resulting from Bayesian analysis and network analysis showed two Chinese samples and Iranian samples were clustered in two major groups. The analysis of molecular variation between Iranian samples and GenBank samples estimated the genetic structure changes between these two populations at up to 79.36% of total genetic changes.

Consequently, the F-statistic (Fst) index significantly separated the populations of Iran and China in terms of genetic construction. The taxonomic status and also the genetic diversity were clarified using molecular methods for the first time for this threatened species. The obtained data can be used for different aspects of its biological and ecological management.

Key words: Corsac fox, phylogeny, genetic diversity, mitochondrial genome, control region, Iran

1. Introduction (Clark et al., 2009). Iran was not listed as a country within The corsac fox (Vulpes corsac Linnaeus, 1768) is one the distribution range of the corsac fox by Wozencraft of the small adapted to dry climates and has a (2005), but it was cited later by Murdoch (2014). The relatively wide distribution in different ranges, , various distribution maps of the species often include two and arid and semiarid in Central and North separate patches as its distribution range in Iran: first, on (Heptner and Naumov, 1992; Clark et al., 2009). It has a the southeastern coast of the Caspian Sea near the border varied diet throughout all of its distribution range (Allen, with , and second, around the Tejen Basin 1938; Heptner and Naumov, 1992). This varied diet and near the border with , although there is no other compatibility characters have helped this species record for the latter (Heptner et al., 1967; Poyarkov and to adapt to different climates and habitats (Clark et al., Ovsyanikov, 2004; Clark et al., 2009; Murdoch, 2014). 2009; Murdoch et al., 2010). However, illegal hunting (i.e. Khalatbari et al. (2015) succeeded in capturing several poaching) and also habitat destruction have threatened this individuals of this species in the Turkmensahra region species (Reading et al., 1998; Poyarkov and Ovsyanikov, and in Golestan Province in Iran and the first presence of 2004). Based on previous studies, three subspecies have this species was recorded after several years. It should be been introduced for this species (Wozencraft, 2005): V. noted that two carcasses of this species were collected by c. corsac (Linnaeus, 1768), V. c. kalmykorum (Ognev, the Department of Environmental Protection of Golestan 1935), and V. c. turkmenicus (Ognev, 1935). The third Province in 2010–2012, and two other carcasses were also subspecies, which is known as the turkmenicus subspecies, collected and identified in this research during 2014 and was reported by Heptner et al. (1967) in and 2015. semidesert regions of Turkmenistan, , the east Limited studies have been conducted on this species of the Kope-dagh Mountains, and around the Tejen River to date. Some of these studies involved studying the * Correspondence: [email protected] 250 IMANI HARSINI et al. / Turk J Zool seasonal food habits of corsac and red foxes in 2. Materials and methods and the potential for competition (Murdoch et al., 2010) 2.1. Sample collection and surveying the pattern of seed presence in corsac Due to the lack of precise reports about the presence of this and diets in Mongolia (Murdoch et al., 2009). In species, random sampling of scats was done in the probable this regard, some limited genetic studies have also been range of distribution, and 35 scat samples were carried out, including the phylogenetic study of Canidae collected and surveyed in standard conditions. Moreover, based on 6 nuclear loci (Bardeleben et al., 2005), survey four tissue samples were taken from two suspected of the phylogenetic relationships of foxlike canids based samples of corsac fox available from the Department of on the cytochrome b gene (Geffen et al., 1992), and Environment of Golestan Province and two other tissue identification of the complete mitochondrial genome samples were also collected from nature. Figure 1 shows of corsac fox (Zhao et al., 2016). Despite these studies, the geographic position of the study area. many biological and ecological aspects of this species 2.2. DNA extraction, PCR cycle, and sequencing remain unknown (Poyarkov and Ovsyanikov, 2004). This Total genomic DNA from each sample was extracted species has been ranked as ‘Least Concern’ in the IUCN using an AccuPrep Kit and following the Bioneer Red List, but it is listed as ‘Endangered’ and ‘Protected’ by manufacturer’s instructions, and this was used as the Iranian Department of Environment, which is largely the template to amplify a 335-bp fragment of the due to lack of accurate records about its presence and mitochondrial control region using the primers other characteristics like the demographic situation of this L15995 (5′-CTCCACTATCAGCACCCAAAG-3′) species (Ziaie, 2008). (Taberlet et al., 1994) and H16498 Nowadays one of the best ways to protect a species is to (5′-CCTGAAGTAAGAACCAGATG-3′) (Fumagalli et al., preserve its genetic resources and genetic diversity besides 1996). the preservation of the ecosystem, which is conducted by PCR reaction was carried out in a thermal cycler conservation genetics science (Hedrick, 2001). Evaluation (Applied Biosystems 2720 Thermal Cycler) as follows: of population genetic diversity is one of the most important 5 min of initial denaturation at 95 °C; 35 cycles of 30 s goals of population genetic studies and these studies have of denaturation at 95 °C, annealing at 54 °C for 30 s, and formed the basis of any management plan to protect extension at 72 °C for 60 s; and final extension at 72 °C species (Avise and Hamrick, 1996; Frankham et al., 2002; for 7 min. The PCR mix (AccuPower PCR Premix Kit, Hrbek et al., 2005) Bioneer) in a volume of 25 µL included 1 U of Euro Taq There is no evidence of any genetic study on this specific DNA polymerase, 10 µM Tris-HCl, 30 µM KCl, 1.5 µM species, but several studies done based on mitochondrial MgCl2, 250 µM of each dNTP, and 2 pmol of each primer. genomes in closely related species like red fox have shown In order to confirm the amplification of the desired that it is possible to demonstrate the genetic diversity of this region in PCR reactions, products were detected using 2% species in its distribution biogeographic range. Accordingly, agarose gel electrophoresis stained with ethidium bromide. mitochondrial genome sequencing, particularly its control The sequencing products were analyzed using an ABI region, is a powerful tool to study the genetic diversity PRISM3730xl automatic sequencer (Applied Biosystems). and phylogeny of this species (Frati et al., 1998; Inoue et 2.3. Data analysis al., 2007; Perrine et al., 2007; Aubry et al., 2009; Teacher et The Basic Local Alignment Search Tool (Altschul et al., al., 2011; Edwards et al., 2012; Yu et al., 2012). Regarding 1997) in the NCBI database was used to determine the the close relationship between these two species and similarity of sequences. SeqScape software, version 2.6 their similarities, it seems that using the D-loop region (Applied Biosystems), was used to check and edit the would have suitable efficiency in phylogenetic and genetic errors, and then realignment was done in MEGA software structure studies of corsac fox as well. version 5 (Tamura et al., 2011) using Clustal W (Thompson Considering the lack of any information about the et al., 1994). Final adjustment of data was done manually population genetics of corsac foxes in Iran, in the present (Librado and Rozas, 2009). The sequences were deposited research we have tried to confirm the presence of this in GenBank with accession nos. KU378387–KU37396. species in Iran using the analysis of a partial sequence The D-loop region sequence of two corsac foxes and of the control region of mtDNA. We would thus be able also other species of the genus Vulpes retrieved from to determine the genetic structure and genetic diversity GenBank were used to draw the phylogenetic tree (Table of this species. Identification of mtDNA haplotypes and 1). JModelTest Software (Posada, 2008) and AIC/BIC their nucleotide diversity would help us to understand the statistic indexes were used to determine the best model of genetic relationships with its close relatives in more detail the phylogenetic tree. Then, based on the selected model, and allow us to protect this species. we constructed Bayesian phylogenetic analyses in MrBayes

251 IMANI HARSINI et al. / Turk J Zool

Figure 1. Geographic location of collected samples.

Table 1. The retrieved sequences in GenBank.

Accession number Species Accession number Species KJ140137 V. corsac AF036475 V. macrotis NC023958 V. corsac KJ846673 V. macrotis KJ846672 V. zerda AF036477 V. macrotis KJ603240 V. zerda KJ597985-86 V. pallida NC026529 V. lagopus KT033906 V. ferrilata KP342451 V. lagopus KP342452 V. v ulpes KP200876 V. lagopus KF387633 V. v ulpes KJ597987–94 V. rueppellii HF677223 V. v ulpes AF036479 V. velox GQ374180 V. v ulpes AF036472 V. velox AB292751 V. v ulpes AF036476 V. velox HF677208 V. v ulpes KP129091–92 cinereoargenteus KF723525–26 lupus

252 IMANI HARSINI et al. / Turk J Zool version 3.2.2 (Huelsenbeck and Ronquist, 2001) to draw The homology of the sequences was determined using the the Bayesian tree. Analysis was performed with Markov BLAST tool, and the results showed that from a total of 25 chain Monte Carlo sampling for 30 million generations scat samples, 8 samples belonged to golden jackal (Canis sampled every 100 generations. Finally, the drawn trees aureus), 11 samples were related to red fox (V. v ulpes), and were condensed by the majority rule. six samples had the most similarity to corsac fox. Finally, Haplotype frequencies were estimated by Arlequin those six samples and four tissue samples were used in 3.1 Software (Excoffier and Lischer, 2010), and haplotype different analyses (Table 2). and nucleotide diversity was also estimated (±SE) using A total of 335 base pairs from the D-loop region of the the NEI coefficient (1987) in this software. Analysis of mitochondrial genome of 10 samples were sequenced and molecular variance (AMOVA; Excoffier et al., 2005) analyzed. The nucleotide frequencies in this segment were performed using Arlequin (with 10,000 permutations) and A (27.86%), T (28.10%), C (28.89%), and G (15.15%). The the Fst index was used to estimate the differences of the rate of the first substitution (substitution of purine bases hypothetical population pairs. Evolutionary relationships (A-G) and substitution of pyrimidine bases (C-T)) and the among the haplotypes identified were visualized using a second substitution (succession of purine and pyrimidine median-joining network generated using Network 4.6.1.3 bases to each other) was calculated in the studied samples (Bandelt et al., 1999). (Table 3). The results of the Bayesian information criterion 3. Results (BIC) and Akaike information criterion (AIC) showed From 39 collected samples (35 scat and 4 tissue), a total that the HKY+G (gamma = 0.3730) model was the of 29 samples (25 scat and 4 tissue) had the appropriate best-fitted evolutionary nucleotide model for the DNA quality and quantity for PCR and sequencing. The considered sequences. Canis lupus (accession number: differences in the size of the amplified segments made KF723525-26) and Urocyon cinereoargenteus (accession it possible to separate and identify most species (Kitano number: KP129091-92) were used as the out group in et al., 2007). As was mentioned in previous studies, the the phylogenetic tree. The topology of the Bayesian segments amplified by these primers in Vulpes species are phylogenetic tree based on the selected evolutionary very close to each other in size and are even the same size model indicated that corsac fox samples were separated in some species (Zamani et al., 2014). Thus, the size of the from the other species by 1 posterior probability support amplified segment does not tell us which species of fox it (Figure 2). belongs to. In this research, because of the same size of Seven haplotypes were defined by 10 polymorphic the fragments, all amplified segments were sequenced. sites and haplotype diversity was equal to 0.93 in Iranian

Table 2. Details of the samples obtained from corsac fox in Golestan Province, Iran.

Variable site 0 0 0 0 0 0 1 1 2 2 Accession numbers Samples type Haplotype 2 4 4 5 5 5 1 5 0 4 0 0 5 0 1 2 9 3 5 9 KU378387 Tissue H A G A - - - C T C C KU378388 Tissue C . . . - - - . . A . KU378389 Tissue B G . . - - - T . A . KU378390 Tissue G . C G C C A . C A T KU378391 Scat D . . . - - - . C A T KU378392 Scat E . . G C C A . C A T KU378393 Scat F . . . - - - . C A T KU378394 Scat E . . . - - - . . A T KU378395 Scat B . . . - - - . . A . KU378396 Scat C G . . - - - T . A .

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Table 3. Maximum likelihood estimate of substitution matrix. Different rates of transitional substitutions are shown in bold and those of transversional substitutions are shown in italics. The substitution matrix and its rate were estimated by the Tamura and Nei model (Tamura and Nei, 1993).

Nucleotide A T/U C G A - 0.03 0.03 14.59 T/U 0.03 - 29.42 0.02 C 0.03 28.98 - 0.02 G 26.8 0.03 0.03 -

Figure 2. Bayesian phylogenetic tree reconstructed from the genus Vulpes and the position of corsac fox.

254 IMANI HARSINI et al. / Turk J Zool samples. The results of haplotype network analysis 4. Discussion confirmed the separation of 10 Iranian samples of two Carnivorous species are characterized by a high degree retrieved corsac samples in GenBank and revealed that of spatial distribution and low number of individuals. these two groups were separated by 12 mutations. The Management and conservation plans for these species have most frequent haplotypes were B (20%), C (20%), and E always faced many challenges. The situation of the corsac (20%) and 4 of the 7 haplotypes were represented by one fox as a carnivore is ambiguous and more complicated. individual (Figure 3). Although the only authentic habitat of this species in Iran Genetic distance between the samples and also among is in Golestan Province, due to lack of any observations, the haplotypes was calculated with standard deviation as information, or reports on this species in recent years, not 0.023 ± 0.003 (Tables 4 and 5). The results of AMOVA much is known about whether this species still lives there between Iranian samples and the samples in GenBank or has become extinct. However, the final results of this estimated the genetic structure changes between these research sequencing 335 bp of the D-loop region and the two populations (Iran and China) at up to 79.36% of total homology of sequences have shown that populations of genetic changes. Within-population changes would thus the corsac fox are still extant in Iran. be equal to 20.64% (Table 6).

Figure 3. Haplotype network of corsac fox samples. Haplotype A included samples existing in GenBank from northern China (KJ140137 and NC0239580); other haplotypes belong to Iranian samples.

Table 4. Estimates of evolutionary divergence between sequences. The numbers of base substitutions per site between sequences are shown. Standard error estimate(s) are shown above the diagonal. Analyses were conducted using the Tamura and Nei model.

KJ140137 NC023958 KU378387 KU378388 KU378389 KU378390 KU378391 KU378392 KU378393 KU378394 KU378395 KU378396

KJ140137 0.000 0.011 0.010 0.009 0.012 0.010 0.010 0.011 0.010 0.009 0.010

NC023958 0.000 0.011 0.010 0.009 0.012 0.010 0.010 0.011 0.010 0.009 0.010

KU378387 0.047 0.047 0.003 0.005 0.005 0.004 0.004 0.005 0.004 0.005 0.003

KU378388 0.044 0.044 0.003 0.004 0.005 0.003 0.004 0.005 0.004 0.004 0.000

KU378389 0.037 0.037 0.009 0.006 0.007 0.005 0.006 0.006 0.006 0.000 0.004

KU378390 0.057 0.057 0.015 0.012 0.018 0.005 0.004 0.003 0.004 0.007 0.005

KU378391 0.047 0.047 0.006 0.003 0.009 0.009 0.003 0.004 0.003 0.005 0.003

KU378392 0.051 0.051 0.009 0.006 0.012 0.006 0.003 0.003 0.000 0.006 0.004

KU378393 0.054 0.054 0.012 0.009 0.015 0.003 0.006 0.003 0.003 0.006 0.005

KU378394 0.051 0.051 0.009 0.006 0.012 0.006 0.003 0.000 0.003 0.006 0.004

KU378395 0.037 0.037 0.009 0.006 0.000 0.018 0.009 0.012 0.015 0.012 0.004

KU378396 0.044 0.044 0.003 0.000 0.006 0.012 0.003 0.006 0.009 0.006 0.006

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Table 5. Estimates of evolutionary divergence over sequence pairs between haplotypes. The numbers of base substitutions per site from averaging over all sequence pairs between haplotypes are shown. Analyses were conducted using the Tamura and Nei model.

Haplotype A Haplotype B Haplotype C Haplotype D Haplotype E Haplotype F Haplotype G Haplotype H

Haplotype A

Haplotype B 0.0375

Haplotype C 0.0440 0.0061

Haplotype D 0.0473 0.0091 0.0030

Haplotype E 0.0506 0.0122 0.0061 0.0030

Haplotype F 0.0540 0.0153 0.0091 0.0061 0.0030

Haplotype G 0.0572 0.0184 0.0122 0.0091 0.0061 0.0030

Haplotype H 0.0472 0.0091 0.0030 0.0061 0.0091 0.0122 0.0152

Table 6. The results of the analysis of molecular variance (AMOVA) between the Iranian samples and the GenBank samples.

Source of variance d.f. Sum of squares Variance components Percentage of variation Among species 1 23.350 6.49800 79.36 Within species 10 16.900 1.69000 20.64 Total 11 40.250 8.18800

Fixation Index (FST): 0.87915

According to the results of this 335-bp segment and considered a local adaptation index of species with habitat the existence of seven different haplotypes in ten identified (Zink, 2004). This type of adaptation has helped the long- samples, haplotype diversity is equal to 0.93 and reveals term survival of the species in different habitats. Based that, despite the lack of sufficient information about the on previous taxonomic studies, V. c. turkmenicus (Ognev, number and population of this species, there is relatively 1935) is the subspecies of Iran (Wozencraft, 2005) and V. c. high genetic diversity between individuals. This is a sign corsac (Linnaeus, 1768) is the subspecies of northern China that conditions for this species are good and promising. (Smith and Xie, 2008), and this explains the differences Genetic distance between individuals shows large between the samples. Genetic distance between two differences among Iranian samples and the samples in populations of Iran and China is relatively high, so it might GenBank from northern China. This is also true about be better that the taxonomic and phylogenetic statuses genetic differences between haplotypes, and Iranian of these two populations be checked more accurately haplotypes are genetically very distant from Chinese using other mitochondrial and nuclear genomes. haplotypes (Figure 3). In the phylogenetic tree, two The drawn phylogenetic tree of corsac and other Chinese samples, despite being in the Iranian samples’ samples of the genus Vulpes shows that despite the high , were placed in a totally separate branch. The results variability between corsac samples and even the existence of AMOVA between Iranian samples and the samples in of two different subspecies of this species, the samples are GenBank estimated the genetic structure changes between placed in a totally separate clade and are well separated these two populations at up to 79.36% of total genetic from the other fox species, and that an independent genetic changes. Consequently, the F-statistic index significantly structure will be identified between the other members of separated the populations of Iran and China in terms of this genus. genetic construction. The probable reason for these large The obtained data in present study represent the first differences should be surveyed in the different subspecies molecular information on this rare and threatened species of corsac in Iran and northern China. in Iran that can be used for its different aspects of biological The concept of “subspecies” is considered a functional and ecological management by scientific communities taxonomic unit in protection plans and would be and wildlife managers. The clarifying of taxonomic status

256 IMANI HARSINI et al. / Turk J Zool and also the genetic diversity is important not only for which comprises mostly nocturnal and secretive species. documenting current spatial and temporal biological The presence of the corsac fox in Golestan Province is diversity patterns, but also for making fundamental confirmed now. The mt-DNA genome data as basic and ecological decisions in conservation plans. applied markers resolved many issues of this species in the Our results demonstrate that the mitochondrial control studied region, although more detailed studies of other region gene is an efficient marker to distinguish different loci such as nuclear DNA, plus habitat evaluation, are species and also different populations of the genus Vulpes. needed to provide more reliable information for ecological On the other hand, this marker could be confidently management and protection of a valuable part of the used in identifying scat samples of the family Canidae, biodiversity of the country.

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