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Genetic discrimination of twoCapoeta species in northeasternAnatolia, using mitochondrial16S rRNA geneYusuf Bektaş a , Yılmaz Çiftçi b , Oğuzhan Eroğlu c &

Ali Osman Beldüz da Faculty of Fisheries, Rize University , P. O. Box53100 , Rize , Turkeyb Faculty of Marine Sciences, Ordu University , 52400 ,Fatsa , Ordu , Turkeyc Fisheries Research Institute , P.O. Box 129 , 61001Trabzon , Turkeyd Department of Biology , Faculty of Arts andSciences, Karadeniz Technical University , 61080 ,Trabzon , TurkeyPublished online: 28 Feb 2013.

To cite this article: Yusuf Bektaş , Yılmaz Çiftçi , Oğuzhan Eroğlu & Ali Osman Beldüz(2011) Genetic discrimination of two Capoeta species in northeastern Anatolia,using mitochondrial 16S rRNA gene, Zoology in the Middle East, 53:1, 61-70, DOI:10.1080/09397140.2011.10648862

To link to this article: http://dx.doi.org/10.1080/09397140.2011.10648862

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Genetic discrimination of two Capoeta species in north-eastern Anatolia, using mitochondrial 16S rRNA gene

(Osteichthyes: Cyprinidae)

Yusuf Bekta�, Y�lmaz Çiftçi, O�uzhan Ero�lu, Ali Osman Beldüz

Abstract. The genetic discrimination of two species of the genus Capoeta, C. tinca (Heckel, 1843) and C. banarescui Turan et al., 2006, which is represented by many endemic species in An-atolian Turkey, has been investigated by analysing the partial 16S ribosomal DNA gene (525 bp). A total of 85 fish was sampled from two localities in the Marmara basin and 5 localities in the Black Sea basin. The amount of 16S rDNA sequence divergence separating these two taxa (mean 1.19%) is within the range observed for 16S rDNA variation between other species of freshwater fishes. The derived haplotypes (h: 28) were strictly local and were not shared between species or populations within species. Three types of phylogenetic tree (Bayesian, MP and ML) clearly showed C. banarescui and C. tinca as distinct species separated with significant bootstrap values (BI:94, MP:88, ML:81), confirming previous conclusions based on morphometric and meristic characters. Furthermore, four novel SNPs were identified, allowing discrimination between two species. AMOVA tests revealed that populations of Capoeta in Northeastern Anatolia can be di-vided into two main groups: Coruh River group, including SVS, TRT and ISP populations, and Yesilirmak-Harsit group, consisting of ALC and HRS populations. These results indicate the ef-fectiveness of mitochondrial 16S rDNA gene sequences for both species identification and the phylogenetic analysis of Capoeta species.

Key words. Capoeta, mtDNA, genetic identification, 16S rRNA.

Introduction The members of the genus Capoeta (Valenciennes in Cuvier & Valenciennes, 1842) belong-ing to the family Cyprinidae are distributed widely in freshwater sources, such as lakes and fast flowing streams (GELDIAY & BALIK 1996) in a wide geographic area, including Anatolia (Turkey) (BANARESCU 1991). Capoeta tinca (Heckel, 1843) with two pairs of barbels has been considered as a single species with a broad geographic distribution covering some large rivers in the western, central and northern Anatolia (KURU 1975, BALIK 1979, ERK’AKAN 1981). BANARESCU & HERZIG-STRASCHIL (1999) reported that the distribution of this species encompasses most rivers in Anatolia and western Transcaucasia; yet populations show con-siderable morphological differences. TURAN et al. (2006) compared different populations referred to C. tinca, and described the Coruh population as a new species under the name Capoeta banarescui Turan et al., 2006, based on morphometric and meristic characters.

In the last decade, molecular approaches have helped to support taxonomic conclusions in fishes based on morphological traits (ORTI & MEYER 1997). The mitochondrial 16S rRNA gene sequence was used as an effective genetic marker for the discrimination and genetic identification of fish species (BROWN 1985, KOCHZIUS et al. 2003, SANTOS et al. 2003) and

Zoology in the Middle East 53, 2011: 61–70. ISSN 0939-7140 © Kasparek Verlag, Heidelberg

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62 Zoology in the Middle East 53, 2011

for the determination of interspecific variation in several families of the Cypriniformes (BOROWSKI & MERTZ 2001, SIMONS et al. 2003, TURAN 2008).

Because of the recent taxonomic changes in the genus, it is necessary to assess the taxo-nomic status of Capoeta species with two pairs of barbels in Northeastern Anatolia. The objectives of this study, therefore, were i) to identify Capoeta banarescui through genetic comparisons, ii) to determine the genetic relationship among these two taxa of Capoeta, and iii) to determine the taxonomic status of an unidentified Khramulya population in northeastern Anatolia basin, based on mitochondrial 16S ribosomal RNA gene.

Material and methods Fish Sampling and DNA Extraction A total of 85 Capoeta specimens representing two species was collected from seven different localities in Anatolia, which are shown in Fig. 1. Collection locations and the number of fish from each location are indicated in Table 1. The caudal fin was taken from each individual and either stored at -20°C or kept in absolute ethanol.

Total DNA was extracted from a piece of the caudal fin by using the tissue protocol of Wizard ®Genomic DNA Purification Kit (Promega Corporation, Madison, WI, U.S.A). The concentration of extracted DNA was determined by using Nanodrop 2000C (Thermo Fisher Scientific, Wil-mington, DE, USA) and extractions were stored at -20°C.

PCR amplification and sequencing PCR amplification was carried out to obtain partial sequences of mitochondrial 16S rRNA gene, in a total of 50 �l volumes containing 5 �l of 10X buffer (Promega), 2.5 mM MgCl2, 0.2mM of each dNTPs, 1mM of each primer, 2 mM MgC12, 1 unit of Taq polymerase (Go Taq, Promega) and 2 �l template. The partial mitochondrial 16S rRNA gene was amplified by polymerase chain reaction using the primers Cpt16SF (5'-CCCATGCCCACATGGAAGAG-3') and Cpt16SR (5'-CCCTTATAGCGGCTGCAC-3') that were designed here, based on comparison of Capoeta sequences retrieved from GenBank (Accession numbers EU707345, EU707371, EU707372 and EU707352). PCR cycling conditions included 3 min of initial denaturation at 94°C, followed by 35 amplification cycles (denaturing at 92°C for 1 min, annealing at 53°C for 45s, and extending at 72°C for 1 min) and a final extension step at 72°C for 5 min on a Techne® TC-3000G Gradient Thermal Cycler (Bibby Scientific, Cambridge, U.K., U.S.A.). The size of the polymerase chain reaction (PCR) products was estimated with a 100 bp DNA ladder (Gibco BRL, Madison, WI, USA.) in a 1.2% agarose gel, run in 1X TBE buffer, stained with 0.5 μg μl-1 ethidium bromide and exposed under UV lights. The amplified products of the partial 16S rRNA was 524 bp in length. The partial 16S rRNA gene was sequenced with degenerate Cpt16SF primer designed by the authors. PCR product purification and DNA sequence analysis were performed by Macrogen Inc. (Seoul, Korea) using BigDye (Applied Biosystems Inc., Foster City, CA, U.S.A.) on an Automatic Sequencer (ABI 3730x1; Applied Biosystems) by a contract laboratory. Data Analysis DNA sequences of the 16SrRNA gene were edited using DNASTAR package (Seqman), and the sequences datasets were aligned using BioEdit version 7.0.5 (HALL 1999). No gaps, insertions, or deletions were found in the aligned sequences, and all sequences were translated into amino acid sequences to verify the alignments. The final sequences included partial sequence of the 16SrRNA gene (524bp). The number of haplotypes, haplotype diversity (h), and nucleotide di-versity (�), nucleotide composition, were calculated with DnaSP 4.0 (ROZAS et al. 2003).

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Osteichthyes 63

Fig. 1. Map of the sampling locations. 1. �av�at; 2. Tortum; 3. �spir; 4. Harsir; 5. Alucra; 6. Harmanc�k. 7. Kocaçay.

Phylogenetic trees were rooted with the Capoeta ekmekciae (Turan, 2006) (GenBank FJ648323 and FJ648324) haplotypes and Barbus barbus (Linnaeus) (Danube River drainage) (GenBank AB238965) as outgroup. Sequences of the 16S rRNA genes of the Capoeta species sequenced in this study have been deposited in GenBank under Accession Numbers FJ648295–FJ648322 (Table 2).

An Analysis of Molecular Variance (AMOVA) (EXCOFFIER et al. 2005) was performed using Arlequin version 3.1 software (SCHNEIDER et al. 2000) for measuring variance within and among geographic groups of Capoeta populations from Northeastern Anatolia, applying the estimator of Weir and Cockerham (WEIR & COCKERHAM 1984). Additionally, we calculated the degree of genetic differentiation between two Capoeta populations in this region with pairwise FST (WEIR & COCKERHAM 1984) and their significance by performing 1000 permutations.

Base frequencies, sequence variation and divergence values were determined using MEGA4 (TAMURA et al. 2007). The sequence data were analyzed using maximum parsimony (MP), max-imum likelihood (ML) in PAUP v4.0b10 (SWOFFORD 2002), and Bayesian inference as imple-mented in MrBayes v3.1b (HUELSENBECK & RONQUIST 2001). Maximum parsimony analysis was performed with TBR branch swapping and 100 random taxa addition replicates were obtained by a heuristic search. 1000 bootstrap pseudo-replicates were analyzed. All characters were treated as unordered and used in an unweighted parsimony analysis. To determine the optimal model of DNA substitution for maximum likelihood analyses, an akaike information criterion (AIC; Akaike, 1974) (POSADA & BUCKLEY 2004) was performed using ModelTest v3.06 (POSADA & CRANDALL 1998). Furthermore, GTR+I+G model was identified as best fit model to our data using AIC criteria in Modeltest v3.06 (POSADA & CRANDALL 1998). 10 heuristic searches with random addition of taxa and TBR branch swapping were performed in PAUP v4.0b10 (SWOF-FORD 2002). ML nodal support was restricted to 300 pseudo-replicates. Bayesian analysis was performed with MrBayes 3.0b3 (RONQUIST & HUELSENBECK 2003), with random starting trees and unlinked parameters, and run for 2 × 106 generations, sampling the Markov chains at

intervals of 100 generations. One cold and three heated Markov chains were used in the analysis. The “burn-in” value was conservatively set at 2000; the first 2000 (200,000 generations) trees were eliminated from the approximation of posterior probabilities. HKY model was identified as

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64 Zoology in the Middle East 53, 2011

best fit model to our data using Bayesian Information Criterion (BIC; SCHWARZ 1974, KASS & WASSERMAN 1995) in Modeltest v3.06 (POSADA & CRANDALL 1998). The trees retained from each run were combined and a 50% majority rule consensus tree was produced to determine nodal posterior probabilities. Bootstrap analyses were initiated using 1000 replicates, each with 10 random addition sequences and TBR branch-swapping using PAUP v4.0b10 (SWOFFORD 2002).

Results and discussion The variable nucleotide sequences for all taxa included in this study are given in Table 2. The 16S rRNA alignment consisted of 524 sites, 31 of which were variable (5.9%) and 8 were parsimony informative (1.5%) (Table 2). The sequence analysis of the 16S rRNA for 85 individuals of the two species revealed 28 different haplotypes (Table 2). Twelve differ-ent haplotypes (Genbank Accession Numbers: FJ648311-22) were found for C. tinca and 16 haplotypes (Genbank Accession Numbers: FJ648295-FJ648310) for C. banarescui, and each of the 28 haplotypes was present in only one location.

Haplotypic diversities, based on partial 16S rRNA gene sequence, were h = 0.923 in C. tinca, 0.885 in C. banarescui and 0.821 in Capoeta sp. (Table 1). Nucleotide diversities estimated within and between populations for the three taxa were in C. tinca (average, 0.0035 and 0.0038, respectively), C. banarescui (average, 0.0033 and 0.0036) and Capoeta sp. (average, 0.0032 and 0.0034) (Table 1). A high level of haplotype diversity was also found within populations of C. tinca and C. banarescui (0.923 and 0.885, respectively). The fact that there are no shared mitochondrial haplotypes indicates an absence of gene flow among the three populations and thereby causes a clear geographic tendency among haplo-types of the studied Capoeta samples. This tendency may be attributed to the existence of isolated regional populations where gene flow might be impeded by geographic barriers. Genetic results showed a noteworthy differentiation among the two Capoeta groups: i) C. banarescui (ISP-TRT-SVS); ii) Capoeta sp. (HRS and ALC) in Northeastern Anatolia. This differentiation may also be attributed to the interruption of gene flow during an adequate number of generations and to the physical geographic barriers within the studied areas. Such a pattern of differentiation between populations found in the eastern Black Sea region has previously been reported for Turkish Brown Trout (BARDAKCI et al. 2006). Furthermore, the sequence analysis of the 16S rRNA revealed fixed differences between C. tinca and C. bana-rescui.

Four diagnostic SNPs allowing the separation of C. tinca from C. banarescui were spe-cies-specific positions and generate species-specific patterns for the discrimination of these two Capoeta species. It seems that the existence of species-specific positions makes the 16S rRNA gene a suitable molecular marker for the genetic identification/discrimination of these two species and for the constitution of their phylogenetic relationships. Within the northeast-ern clade, two distinct subclades were noted: i) Coruh River system; ii) Yesilirmak River and Harsit Stream, based on the SNP at position 173 (G) and 342(T), respectively. The principal basins in this region are the Çoruh, eastern Black Sea and Ye�il�rmak. The eastern Black Sea region of Turkey is split into two. north and south, by mountains running parallel to the sea with heights varying between 1500 and 3700 m. As the eastern Black Sea basin, which contains the Harsit stream, is located in the north of the main mountain chain and the Ye�il�rmak stream and Çoruh River are situated in the southern aspect of these mountains, they flow northwards from three different points and reach the sea. Consequently, the

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Osteichthyes 65

Table 1. Collection locations of Capoeta species with abbreviations, sample size, number of haplotypes, haplotype diversity (h) and nucleotide diversity (�) in the partial 16S rRNA sequenc-es (S.D. in parentheses). N = sample size.

Species Location N Nunmber of haplotypes h (�S.D.) � (�S.D.)

C. banarescui Ispir (ISP) 12 3 0.621±0.1176 0.00136±0.0000 Tortum (TRT) 10 3 0.644±0.1012 0.00246±0.0001 Savsat (SVS) 11 3 0.709±0.0827 0.00270±0.0003 Mean 0.821±0.0236 0.00334±0.0002 Capoeta sp. Alucra (ALC) 11 4 0.745±0.0978 0.00284±0.0001 Harsit (HRS) 10 3 0.644±0.1012 0.00246±0.0002 Mean 0.885±0.0110 0.00320±0.0001 C. tinca Harmancik (HRM) 16 6 0.833±0.0560 0.00317±0.0003 Kocacay (KCY) 15 6 0.850±0.0537 0.00324±0.0000

Mean 0.923±0.0200 0.00351±0.0000

Table 3. AMOVA analysis of northeastern Capoeta populations based on 16S rRNA sequences. FST = 0.33587%, P<0.05.

Source of variation d.f. Sum of squares

Variance components

Percentage of variation

Among groups 2 37.799 0.76024 Va 36.55 Among populations within groups 2 15.667 0.65333 Vb 31.41 Within populations 49 32.659 0.66651 Vc 32.04 Total 53 86.125 2.08008

validity of these diagnostic positions was supported by the geographic distributions of the analysed samples.

Several single nucleotide polymorphisms (SNPs) in the 16S rRNA sequences showed fixed differences between taxa. Four differences were found between C. tinca and C. bana-rescui, which can be used to identify individuals of the two species. C. banarescui had A nucleotide at positions 64, 451 and 496, whereas C. tinca had G nucleotide in these posi-tions. At position 38, C. banarescui had T nucleotide, whereas C. tinca had G nucleotide (Table 2).

The pairwise sequence divergences values between 16S rRNA haplotypes within species ranged from 0.19% and 0.38% (mean 0.38%) for C. tinca and 0.19% and 0.96% (mean 0.85%) for northeastern Anatolia samples. On the other hand, within the northeastern group, there was a notable pairwise sequences divergence (0.7%) among C. banarescui haplotypes from the Çoruh River basin and ALC–HRS haplotypes from Yesilirmak and the eastern Black Sea basin. Additionally, the observed 16S rDNA sequence divergence values (0.96–1.35%, mean 1.19%, between C. tinca and C. banarescui haplotypes, and 0.96–1.54%, mean 1.4%, between Capoeta tinca and Capoeta sp. haplotypes) represent some of the

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66 Zoology in the Middle East 53, 2011

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2335670012244773667944556677999

4582473954548394364413130345267

Gen

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N

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HR

M

KC

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ISP

TRT

SVS

ALC

H

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Capoeta banarescui

ISP_I

ACTAACAAAACGGAGTTTACCAATTTGGAAA

FJ64

8295

7

ISP_II

...............A...............

FJ64

8296

3

ISP_III

.........................G.....

FJ64

8297

2

TRT_I

...................G...........

FJ64

8307

5

TRT_II

........G......................

FJ64

8305

4

TRT_III

.........C.....................

FJ64

8306

1

SVS_I

..............A......T.........

FJ64

8310

5

SVS_II

..............A...........A....

FJ64

8309

3

SVS_III

..........A...A................

FJ64

8308

3

12

10

11

Capoeta sp.

ALC_I

....................T.......G..

FJ64

8298

2

ALC_II

......T.............T..........

FJ64

8299

3

ALC_III

..................C.T..........

FJ64

8300

1

ALC_IV

T...................T..........

FJ64

8301

5

HRS_I

.............G......T........C.

FJ64

8302

4

HRS_II

.............G......T...A......

FJ64

8303

5

HRS_III

.............G...A..T..........

FJ64

8304

1

11

10

Capoeta tinca

KCY_I

..G.GT................G......G.

FJ64

8311

5

KCY_II

..G.G.................G......GC

FJ64

8316

3

KCY_III

..G.G......T..........G......G.

FJ64

8312

2

KCY_IV

..G.G.................G....A.G.

FJ64

8313

3

KCY_V

..G.G..T..............G......G.

FJ64

8314

1

KCY_VI

..GCG.................G......G.

FJ64

8315

2

HRM_I

.GG.G.................G......G.

FJ64

8317

3

HRM_II

..G.G.............C...G......G.

FJ64

8318

4

HRM_III

..G.G.......T.........G......G.

FJ64

8319

1

HRM_IV

..G.G.................GA.....G.

FJ64

8320

2

HRM_V

..G.G.................G....C.G.

FJ64

8321

1

HRM_VI

..G.G...........A.....G......G.

FJ64

8322

4

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Osteichthyes 67

Fig. 2. Phylogenetic relationships based on analyses of 16S rRNA dataset of C. tinca, C. banarescui and Capoeta sp. The nodes at branches correspond to statistical supports resulting from MP, Bayesian inference and ML analyses, respectively. Only bootstrap values based on 1000 replications higher than 50% are dis-played. Dash means no significance for the corresponding method. lowest values reported among other congeneric freshwater fish species (1.4–16%) (STEINKE et al. 2005, LI et al. 2005) as well as within the reported ones (0.5 to 9.4%) for species with-in the genus Capoeta from central Anatolia recorded by TURAN (2008). The degree of se-quence divergence between these two species is therefore comparable to that between other well accepted species.

All sequences of mitochondrial 16S rRNA haplotypes in both species were included in the phylogenetic analysis with the sequences of Capoeta ekmekciae as outgroups. The topolo-

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68 Zoology in the Middle East 53, 2011

gies of the bayesian inference, MP and ML trees estimated were almost identical, supporting the relationship between C. tinca and the C. banarescui-Capoeta sp. group, with significant bootstraps values (MP:88, BI:94, ML:81) (Fig. 2). The present phylogenetic analysis re-vealed two major clusters, one exclusively containing the haplotypes of C. banarescui and Capoeta sp. separated into two main groups, and one with the haplotypes of C. tinca. The present results obtained from the partial sequences of the 16S rRNA gene indicate that C. tinca and C. banarescui are genetically distinct species, supporting the �ndings of TURAN et al. (2006). In the northeastern clade, the haplotypes of C. banarescui from the Çoruh River system and the haplotypes of Capoeta sp. from the Ye�il�rmak River and Harsit stream are not shared between groups. Phylogenetic reconstruction carried out with the partial sequence of mtDNA 16S rDNA gene (525 bp) indicates the existence of two distinct haplotype groups: Capoeta sp. (Ye�il�rmak and Harsit populations) identi�ed in the northeastern Black Sea basin and C. banarescui (Fig. 2). The obtained phylogenetic topology (MP:72, BI:92, ML:69) and notable pairwise genetic divergence (D= 0.002) between C. banarescui and HRS-ALC group in this 16S rRNA dataset are above 0.7%, suggesting one new subgroup might be recognised.

In addition to no shared haplotypes, population heterogeneity was also evident by both pairwise FST and AMOVA comparisons between two Black Sea populations. The AMOVA analysis results for 16S rRNA markers showed that a significant part of the variation was found between the two geographic groups (Çoruh river system and Ye�il�rmak River–Harsit stream). Of the total molecular variance, 36.55% was attributable to regional divergence, 31.41% to population differences within regional, and 32.04% to individual differences within populations (Table 3).

The pairwise FST values comparisons indicated that the northeastern Anatolian Capoeta populations were significantly differentiated (FST = 0.33587%, P<0.05; Table 3). F-statistics and AMOVA indicate that C. banarescui and the Yesilirmak–Harsit populations may be genetically diverse taxa. This suggests that these populations are genetically isolated and that there is restricted gene flow between them. Hence, the distribution area of the two pair bar-belled Capoeta in the northeastern Black Sea region of Turkey can be separated into two regions.

In conclusion, the present results show that the 16S rRNA sequences are genetically a suitable marker for the discrimination of C. banarescui from C. tinca in Anatolia and pro-vide insights into their phylogenetic relationships. C. banarescui was confirmed to be genet-ically distinct from C. tinca, and proven to be a valid species. The present genetic data showed correlation with the previous morphological traits. On the other hand, F-statistics and AMOVA results demonstrated significant differences between C. banarescui and the HRS-ALC group. Additionally, further studies must be developed in order to evaluate differ-entiation of the two pair barbelled species of Capoeta and to clarify the taxonomic situation of HRS and ALC populations in the northeastern Anatolia using different genetic markers, such as mtDNA and nuclear markers like microsatellite.

Acknowledgements. We would like to thank D. TURAN for his technical support and valuable suggestions and the Scientific Research Fund of Karadeniz Technical University (Project No: 2006.111.04.1) and Rize University (Project No: 2011.103.01.2) for financial support

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References AKAIKE, H. (1974): A new look at statistical model identification. – IEEE Transactions on Automatic

Control 19: 716-723. ALLARD, M. W., M. M. MIYAMOTO, L. JARECHE, F. KRAUS & M. R. TENNANT (1992): DNA systemat-

ics and evolution of the artiodactyl family Bovidae. – Proceeding of the National Academy of Sci-ences of the United States of America 89: 3972-3976.

BALIK, S. (1979): Taxonomical and ecological investigations upon freshwater �shes of Western Anato-lia. – Ege Üniversitesi Fen Fakültesi Ilmi Raporlar Serisi 236: 1-69.

BANARESCU, P. M. (1991): Zoogeography of fresh waters. Distribution and Dispersal of Freshwater Animals in North America and Eurasia. Vol. 2. – Wiesbaden, p. 512-1091.

BANARESCU, P. M. & B. HERZIG-STRASCHIL (1999): Capoeta tinca (Heckel 1843). p. 413-420. In: P. M. BANARESCU (Ed.), The freshwater fishes of Europe. Vol. 5/1 (Cyprinidae 2), Part I: Rhodeus to Capoeta. – Wiebelsheim.

BARDAKÇI, F., N. DEGERLI, O. ÖZDEMIR & H. H. BA�IBÜYÜK (2006): Phylogeography of the Turkish brown trout Salmo trutta L.: mitochondrial DNA PCR-RFLP variation. – Journal of Fish Biology 68: 36-55.

BOROWSKI, R. & L. MERTZ (2001): Genetic differentiation among populations of the cave fish Schistura oedipus (Cypriniformes: Balitoridae). – Environmental Biology of Fish 62: 225-231.

BROWN, W. M. (1985): The mitochondrial genome of animals: molecular evolutionary genetics. p. 95-130. In: R. J. MACINTYRE (Ed.), Molecular evolutionary genetics. – New York.

ERK’AKAN, F. (1981): Studies on systematics and bioecology of �shes (Pisces) of Sakarya Basin. – PhD Thesis, Hacettepe Üniversitesi, Ankara, 144 pp.

EXCOFFIER, L., G. LAVAL & S. SCHNEIDER (2005): Arlequin (version 3.0): An integrated software package for population genetics data analysis. – Evolutionary Bioinformatics Online 1: 47-50.

GELDIAY, R. & S. BALIK (1996): Türkiye Tatl�su Bal�klar� [The freshwater fishes of Turkey]. – Ege Üniversitesi Su Ürünleri Fakültersi Yay�nlar� No: 46 (II. Bask�), �zmir, 532 pp.

HALL, T. A. (1999): BioEdit: a user-friendly biological sequence alignment editor and analysis pro-gram for Windows 95/98/NT. – Nucleic Acids Symposium Series 41: 95-98.

HUELSENBECK, J. P. & F. R. RONQUIST (2001). MrBayes: Bayesian inference of phylogeny. –Bioinformatics 17: 754-755.

KASS, R. E. & L. WASSERMAN (1995): A reference Bayesian test for nested hypotheses and its relationship to the Schwarz criterion. – Journal of the American Statistical Association 90: 928-934.

KOCHZIUS, M., R. SOLLER, M. A. KHALAF & D. BLOHM (2003): Molecular phylogeny of the lionfish genera Dendrochirus and Pterois (Scorpaenidae, Pteroinae) based on mitochondrial DNA sequences. – Molecular Phylogenetics and Evolution 28: 396-403.

KURU, M. (1975): Systematics and zoogeographical study of �shes (Pisces) living in Tigris-Euphrates, Kura-Arax, Van Lake and Black Sea basins. – Ph.D. Thesis, Atatürk University, Faculty of Scienc-es, Erzurum, 181 pp.

LI, J., X. WANG, S. P. HE & Y. Y. CHEN (2005): Phylogenetic studies of Chinese labeonine fishes (Teleostei: Cyprinidae) based on the mitochondrial 16S rRNA gene. – Progress in Natural Science 15: 213-219.

ORTI, G. & A. MEYER (1997): The radiation of characiform fishes and the limits of resolution of mito-chondrial ribosomal DNA sequences. – Systems Biology 46: 75-100.

POSADA, D & T. R. BUCKLEY (2004): Model selection and model averaging in phylogenetics: advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests. – Systems Biology 53: 793-808.

POSADA, D & K. A. CRANDALL (1998): Modeltest: testing the model of DNA substitution. –Bioinformatics 14 (9): 817-818.

RONQUIST, F. & J. P. HUELSENBECK (2003): MrBayes 3: Bayesian phylogenetic inference under mixed models. – Bioinformatics 19: 1572-1574.

ROZAS, J., J. C. SANCHEZ-DELBARRIO, X. MESSEGUER & R. ROZAS (2003): DnaSP, DNA polymor-phism analyses by the coalescent and other methods. – Bioinformatics 19: 2496-2497.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

olor

ado

at B

ould

er L

ibra

ries

] at

02:

34 2

0 D

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ber

2014

70 Zoology in the Middle East 53, 2011

SANTOS, S., H. SCHNEIDER & I. SAMPAIO (2003): Genetic differentiation of Macrodon ancylodon

(Sciaenidae, Perciformes) populations in Atlantic coastal waters of South America as revealed by mtDNA analysis. – Genetics and Molecular Biology 26: 151-161.

SCHNEIDER, S., D. ROESSLI & L. EXCOFFIER (2000): Arlequin: a software for population genetics data analysis (v. 3.01). – University of Geneva, Department of Anthropology, Geneva.

SCHWARZ, G. (1974): Estimating the dimensions of a model. – Annals of Statistics 6: 461-464. SIMONS, A. M., P. B. BERENDZEN & R. L. MAYDEN (2003): Molecular systematics of North American

phoxinin genera (Actinopterygii: Cyprinidae) inferred from mitochondrial 12S and 16S ribosomal RNA sequences. – Zoological Journal of the Linnean Society 139: 63-80.

STEINKE, D., M. VENCES, W. SALZBURGER & A. MEYER (2005): Taxi: a software tool for dna barcoding using distance methods. – Philosophical Transactions of the Royal Society, B: Biological Sciences 360 (1462): 1975-1980.

SWOFFORD, D. L. (2002): PAUP*. Phylogenetic Analysis using Parsimony (* and Other Methods), version 4. Sinauer Associates, Sunderland, MA.

TAMURA, K., J. DUDLEY, M. NE� & S. KUMAR (2007): MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. – Molecular Biology and Evolution 24: 1596-1599.

TURAN, C. (2008): Molecular systematics of the Capoeta (Cypriniformes: Cyprinidae) species complex inferred from mitochondrial 16S rDNA sequence data. – Acta Zoologica Cracoviensia 51: 1-14.

TURAN, D., M. KOTTELAT, F. G. EKMEKC� & H. O. IMAMO�LU (2006): A review of Capoeta tinca, with descriptions of two new species from Turkey (Teleostei: Cyprinidae). – Revue Suisse de Zoologie 113: 421-436.

WEIR, B. S. & C. C. COCKERHAM (1984): Estimating F-Statistics for the analysis of population struc-ture. – Evolution 38: 1358-1370.

Authors’ address: Yusuf Bektas, Faculty of Fisheries, Rize University, P. O. Box 53100, Rize, Tur-key. – Y�lmaz Ciftci, Faculty of Marine Sciences, Ordu University, 52400, Fatsa, Ordu, Turkey. – Oguzhan Eroglu, Fisheries Research Institute, P.O. Box 129, 61001 Trabzon, Turkey. – Ali Osman Belduz, Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey. – Email: yusuf.bektas.tr@gmail.com.

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