Molecular phylogeny of the genus Capoeta (Teleostei: Cyprinidae) in Anatolia, Turkey

Biochemical Systematics and Ecology 70 (2017) 80e94

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Molecular phylogeny of the genus Capoeta (Teleostei: Cyprinidae) in Anatolia, Turkey Yusuf Bektas a, *, Davut Turan a, Ismail Aksu a, Yılmaz Ciftci b, Oguzhan Eroglu c, Gokhan Kalayci a, Ali Osman Belduz d a

Department of Basic Sciences, Faculty of Fisheries, Recep Tayyip Erdogan University, 53100 Rize, Turkey Faculty of Fisheries, Faculty of Marine Sciences, Ordu University, 52400, Ordu, Turkey c Central Fisheries Research Institute, 61001 Trabzon, Turkey d Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 August 2016 Received in revised form 1 November 2016 Accepted 5 November 2016

Phylogeny of Capoeta genus distributed in Anatolia were carried out by analysing mitochondrial cytochrome b gene (1140 bp) sequences from 332 samples representing 59 populations of 15 species across their geographical distribution. Haplotype network and phylogenetic analysis (neighbor-joining, maximum-likelihood, maximum parsimony, and bayesian inference) of the 103 cytochrome b haplotypes detected in Capoeta species resulted in similar tree topologies including four distinct clades, in congruent with taxonomic classification of Capoeta based on morphological characteristics such as scale size, mouth shape, and body spotting. Based on cyt b nucleotide sequences, the present study suggests that four undescribed Capoeta species may exist in Anatolia freshwater; one species in the Kizilirmak River, the second species in the Dirgine River, the third species B. Menderes River, and the fourth species in the some Yesilirmak tributaries that run into the Black Sea Basin. Capoeta taxa distributed in the rivers of Anatolian freshwater basins are isolated from each other during middle Miocene (Serravallian)-late Pleistocene (Ionian) (about 13.75e0.41 million years). This suggests that distribution and presence of Capoeta species were shaped under paleogeographic conditions such as Pleistocene climate changes in Quarternary period as well as tectonic uplift and faulting, which probably has not changed up to now. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Capoeta Mitochondrial DNA Cytochrome b Genetic identification Phlogeny Anatolia

1. Introduction The Cyprinidae is the largest freshwater fish family in the world, with a remarkable number of over 2000 species and 210 genera (Nelson, 1994). The world distribution of the cyprinid genus Capoeta (Valenciennes in Cuvier and Valenciennes, 1842)
na
rescu and Coad, 1991; ranges from East Europe to West Asia, including Anatolia, covering a wide geographic area (Ba
na
rescu, 1999). Capoeta is usually rheophile and some species can be found in lacustrine environment (Geldiay and Ba
rescu, 1999; Kottelat and Freyhof, 2007). Balık, 2007; B
ana

* Corresponding author. E-mail address: [email protected] (Y. Bektas). http://dx.doi.org/10.1016/j.bse.2016.11.005 0305-1978/© 2016 Elsevier Ltd. All rights reserved.

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Currently, 22 Capoeta species have been described worldwide although there are taxonomic inconsistencies resulting from the fact that some species have been reported as synonym and some local populations have been identified as subspecies. The genus is represented by 19 species (C. angorae, C. antalyensis, C. baliki, C. banarescui, C. barroisi, C. bergamae, C. caelestis, C. capoeta, C. damascina, C. ekmekciae, C. erhani, C. kosswigi, C. mauricii, C. pestai, C. sieboldi, C. tinca, C. trutta, C. turani, and C. umbla), 7 of which being endemic in Turkish freshwaters (Turan et al., 2006a, 2006b, 2008; Geldiay and Balık, 2007; Turan et al., 2008; Levin et al., 2012). On the other hand, some species present in Turkish freshwaters (C. tinca, C. baliki, C. banarescui ve C. antalyensis) are separated based on having two pairs of barbels compared to other species in the Capoeta genus (Turan et al., 2008; Levin et al., 2012). Although geological history and evolution of the region, which may have determined the distribution of cyprinids in Anatolia, is well known, there is very little information about the fossil history and record of the Anatolian bioprovince. Only a handful studies such as Hrbek and Meyer (2003), Durand et al. (2002), Zardoya and Doadrio (1999) proposed a palaeobiogeographical story of Anatolia based on molecular studies on Aphanius and Barbus species. Nevertheless, there were a few genetic studies on Capoeta species mentioned among the limited number of studies (Durand et al., 2002; Tsigenopoulos et al., 2003) focused on the phylogeny and biogeography of cyprinids from Turkish freshwaters until 2006. After new Capoeta species (C. balıki, C. banarecsui, C. caelestis, C. ekmekciae, C. mauricii ve C. erhani) were recently reported in Anatolia, taxonomical status within genus has been investigated using different genetic markers by some scientists (Turan, 2008; Bektas et al., 2011; Levin et al., 2012). However, these consecutive genetic studies were not enough to unearth the phylogenetic relationships and specification of Capoeta distributed in Anatolia. Moreover, genetic structures of populations belonging to endemic Capoeta species having two pair barbels in Turkey have not been analysed in detail. Therefore, there is a need to clarify the taxanomic status of Capoeta species that have been reported in the literature and previously undefined populations. The main goals of this study; (1) to clarify the phylogenetic relationships in Capoeta species (especially in those having two barbels) distributed in Turkey, (2) to find out the genetic identification of potential new populations and (3) to delineate phlyogeographical history of Capoeta genus in Anatolia. To achieve these goals, mitochondrial cytochrome (cyt) b gene polymorphism is preferred because of its proven use in former studies (Zardoya and Doadri, 1999; Durand et al., 2002; Tsigenopoulos et al., 2003; Levin et al., 2012) in order to reveal phylogenetic relationships among various groups in Euro Asiatic cyprinids.

2. Material and methods 2.1. Sample collection and DNA extraction Overall, 332 individuals belonging to nineteen Capoeta taxa were collected from different geographical regions of Anatolia (Fig. 1; Table 1). Collection locations and the number of fish samples are shown in Table 1. Caudal fin tissue was removed from each specimen, preserved in 96% ethanol and then transferred to the Laboratory of Fish Genetics, Faculty of Fisheries, Recep Tayyip Erdogan University, and stored at
20  C. Total DNA was extracted from 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 (ThermoFisher Scientific, Wilmington, DE, USA). Extractions were quantified in a 1% agarose-TAE (Tris-acetate-EDTA) gel containing 0.5 mg/L of ethidium bromide and examined under UV light, and stored at
20  C.

2.2. Polymerase chain reaction (PCR) amplification and DNA sequencing PCR amplification of the mitochondrial cytochrome b gene (1140 bp) was carried out using primers GluDG.L (50 TGACTTGAARAACCAYCGTTG-3'; Palumbi, 1996) and H16460 [50 -CGAYCTTCGGATTAACAAGAC CG-3' (Perdices et al., 2004);]. The PCR reactions were performed with 10 ml of 10x Taq polymerase reaction buffer, 5 ml of 2.5 mM magnesium chloride (MgCl2), 10 ml of dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP) (Promega, USA), 1 ml of Taq DNA polymerase (1U), 2 ml each of forward and reverse primers (10pM), and 5 ml of template DNA (25e50 ng), for 50 ml reaction mixture. PCR amplification reactions were conducted on BioRad T100 Gradient Thermal Cycler (Biorad, Hercules, CA, USA). The thermocycling program was consisted of an initial denaturation of 94  C for 3 min, followed by 35 cycles of 94  C for 45 s, 58  C for 45 s, 72  C for 45 s; and then a final extension of 72  C for 7 min and a final hold at 4  C. The amplified PCR products were separated on a 1,2% agarose gel stained with ethidium bromide and visualized on a UV transilluminator in Quantum-Capt ST4 system (Vilber Lourmat, France). Amplification products were purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and were bi-directionally sequenced using two amplification primers (Cpt_SF: 50 -ATGGCAAGCCTACGAAAAACACCC-30 and Cpt_SR: 50 -TTTCTARCCATCCTGCTAGTGG-30 ), designed for this study, with an ABI PRISM 3730  1 Genetic Analyser (Applied Biosystem, Foster City, CA, USA) using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystem) at the Macrogen Inc. (Amsterdam, Netherlands).

82 Y. Bektas et al. / Biochemical Systematics and Ecology 70 (2017) 80e94

Fig. 1. The map of the sampling locations of Capoeta species. Numbers represent sampling localities are given in Table 1.

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Table 1 Origin and number of individuals from 24 Capoeta species sequenced for mtDNA cytochrome b gene. Population index (P.I.), sample numbers (N), Haplotype numbers (H), Altitudes (m), GPS coordinates, and GenBank accession numbers. Clade, Subclade, Species, River, Locality, Drainage

P. I.

N

Clade I Subclade I C. banarescui R. Bulanık at Savsat, Coruh, Black Sea Basin

1

R. Tortum at Erzurum, Coruh, Black Sea Basin

4

_ R. Coruh at Ispir, Coruh, Black Sea Basin

5

4 2 2 1 1 2 3 1 1 1 1 3 1 2 1 1 Hd ¼ 2 6 1 5 1 1 1 3 1 1 1 1 1 1 1 5 4 5 Hd ¼

Capoeta sp.1 R. Kastel at Trabzon, Black Sea Basin

6

R. Harsit at Dogankent, Giresun, Black Sea Basin

7

R. Alucra at Giresun, Yes¸ilırmak, Black Sea Basin

8

R. Kelkit at Gümüs¸hane, Yes¸ilırmak, Black Sea Basin

9

R. Yesilirmak at Refahiye, Erzincan, Black Sea River Basin

10

Clade, Subclade, Species, River, Locality, Drainage

P. I.

N

Subclade II Capoeta sp.2 R. Narlidere at Vezirkopru, Samsun, Black Sea Basin

13

R. Devrez at Ilgaz, Cankiri, Black Sea Basin

14

R. Delice at Yerkoy, Yozgat, Black Sea Basin R. Kilicozu at Ozbag, Kirsehir, Black Sea Basin

15 16

R. Kesikkopru at Kirsehir, Black Sea Basin

17

R. Pecenek at Sereflikochisar, Ankara, Black Sea Basin _ R. Kizilirmak at Imranlı, Kizilirmak, Black Sea Basin

18 20

Capoeta sp.3 R. Dirgine at Devrek, Zonguldak, Black Sea Basin

C. baliki R. Balikli at Hendek, Sakarya, Black Sea Basin

21

22

H

GPS coordinates

GenBank No.

1 383 2 3 4 5 6 1182 7 8 9 10 1163 11 12 13 14 15 16 0,954 (±0,021)

41 15.5300 N/42 19.3720 E

GQ423977 GQ423978 GQ423979 GQ423980 GQ423981 GQ423982 GQ423983 GQ423984 GQ423985 GQ423986 GQ423987 GQ423988 GQ423989 GQ423990 GQ423991 GQ423992

17 207 18 19 162 20 21 22 23 24 1430 25 26 27 28 29 30 31 32 1366 33 34 1562 0,933 (±0,017)

40 54.2180 N/40 10.7450 E

H

Altitude (m)

40 31.6560 N/41 02.2730 E

p (10¡2) ¼ 0,374 (±0,028)



0



0

40 49.928 N/38 54.440 E

40 20.3830 N/38 44.8630 E

40 07.3690 N/39 21.1890 E 39 55.1920 N/38 45.5930 E p (10¡2) ¼ 0,468 (±0,035)

KY065239 KY065240 GQ423972 GQ423973 GQ423974 GQ423975 GQ423976 GQ423993 GQ423994 GQ423995 GQ423996 GQ423997 GQ423998 GQ423999 GQ424000 KY065241 KY065242 KY065243

GPS coordinates

GenBank No.

4 35 474 1 36 15 35 844 1 37 10 35 780 6 35 1048 3 38 1 39 5 35 866 1 36 10 40 1063 5 41 1719 3 42 Hd ¼ 0,595 (±0,064)

41 7.1230 N/35 13.6420 E

KY065244 KY065245 KY065244 KY065246 KY065244 KY065244 KY065247 KY065248 KY065244 KY065245 KY065249 KY065250 KY065251

3 43 224 1 44 Hd ¼ 0,500 (±0,265)

41 2.0440 N/31 52.6360 E

5 3 2 1 1

40 46.0600 N/30 46.2600 E

45 46 47 48 49

Altitude (m)

40 31.4640 N/41 33.4690 E

82

40 54.2770 N/33 38.2500 E 39 37.3280 N/34 29.3990 E 39 14.4990 N/34 07.6760 E

38 57.6570 N/34 11.9580 E 38 51.9860 N/33 42.4150 E 39 50.4600 N/38 17.1110 E

p (10¡2) ¼ 0,076 (±0,011) KY065252 KY065253

¡2

p (10 ) ¼ 0,044 (±0,014) GQ424011 GQ424012 GQ424013 GQ424014 GQ424015

(continued on next page)

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Table 1 (continued ) Clade, Subclade, Species, River, Locality, Drainage

P. I.

N

GPS coordinates

GenBank No.

R. Burcin at Kızılcahamam, Sakarya, Black Sea Basin

23

4 50 957 1 51 4 52 1 53 Hd ¼ 0,887 (±0$036)

40 28.9380 N/32 39.1530 E

GQ424016 GQ424017 GQ424018 GQ424019

3 54 41 4 55 6 56 1 57 1 58 2 59 761 3 60 1 57 1 61 967 1 62 Hd ¼ 0,500 (±0,265)

40 01.7480 N/27 50.9010 E

C. tinca R. Koca at Manyas, Balıkesir, Susurluk, Marmara Basin

24

R. Emet at Harmancık, Bursa, Susurluk, Marmara Basin

25

Eber Lake at Bolvadin, Afyon, Eber Lake Basin

26

H

Altitude (m)

Clade, Subclade, Species, River, Locality, Drainage

P. I.

N

H

C. antalyensis R. Aksu at Gokdere, Mediterranean Sea Basin

30

11 1 1 1 1 Hd ¼

63 14 64 65 66 67 0,476 (±0,155)

Subclade III C. mauricii Beys¸ehir Lake at Isparta, Turkish Lake District

31

Altitude (m)

p (10¡2) ¼ 0,163 (±0,016)

39 40.4020 N/29 07.8550 E

38 41.0310 N/31 07.7110 E

GQ424001 GQ424002 GQ424003 GQ424004 GQ424005 GQ424006 GQ424007 GQ424008 GQ424009 GQ424010

p (10¡2) ¼ 0,044 (±0,014) GPS coordinates

GenBank No.

37 01.7120 N/30 54.7590 E

GQ424020 GQ424021 GQ424022 GQ424023 GQ424024

p (10¡2) ¼ 0,047 (±0,017)

3 85 1123 2 86 Hd ¼ 0,600 (±0,0175)

37 55.2700 N/31 20.6530 E

KY065271 KY065272

p (10¡2) ¼ 0,053 (±0,015)

Subclade IV C. bergamae R. Karabol at Kayagil, Usak, Gediz, Aegean Sea Basin

27

5 87 750 Hd ¼ 0,000 (±0,000)

38 37.8890 N/29 17.7740 E p (10¡2) ¼ 0,000 (±0,000)

KY065273

Capoeta sp.4 R. Banaz at Sivaslı, Usak, B. Menderes, Aegean Sea Basin R. Kayirli, Yatagan, Mugla, B. Menderes, Aegean Sea Basin

28 29

3 88 785 4 89 267 Hd ¼ 0,571 (±0.119)

38 32.9940 N/29 37.2240 E 37 25.5100 N/28 08.3090 E p (10¡2) ¼ 0,036 (±0,010)

KY065274 KY065275

SubcladeV C.damascina saadi Rodan River, Makran Basin, Iran Kor River, Makran Basin, Iran C. buhsei Taghra Rud stream, Namak lake Basin, Iran C. caelestis R. Goksu at Mersin, Mediterranean Sea Basin

C. damascina R. Zamanti at Tomarza, Seyhan, Mediterr. Sea Basin R. Horu at Bahçe, Ceyhan, Mediterranean Sea Basin R. Firniz at K.Maras¸, Ceyhan, Mediterranean Sea Basin R. Asi at Serinyol, Hatay, Orontes, Mediterranean Sea Basin € prü Dam (Asi) at Hatay, Orontes, Mediterranean Sea Basin Tahtako R. Kueik at Kilis, Kueik, Mediterranean Sea Basin

JF798327 JF798326 JF798283 32

33 35 37 38 39 41

5 74 133 2 75 Hd ¼ 0,476 (±0,171)

36 39.2820 N/33 21.9040 D

2 2 4 4 3 3 Hd ¼

1362 512 565 334 380 560 (±0,000)

38 25.8100 N/35 37 10.6610 N/36 37 45.4280 N/36 36 21.9310 N/36 36 51.1050 N/36 36 41.0830 N/37 p (10¡2) ¼ 0,000

76 76 76 76 76 76 0,000

KY065260 KY065261

p (10¡2) ¼ 0,042 (±0,010)

Clade, Subclade, Species, River, Locality, Drainage

P. I.

N

H

Altitude (m)

GPS coordinates

C. umbla R. Tohma at Gürün, Sivas, Euphrates, Persian Gulf Basin R. Toprakkale at Aziziye, Erzurum, Euphrates, Persian Gulf Basin R. Taslicay at Tas¸lıcay, Agri, Euphrates, Persian Gulf Basin R. Salat at Bismil, Diyarbakır, Tigris, Persian Gulf Basin R. Horoz at Hizan, Bitlis, Tigris basin, Persian Gulf Basin R. Zilan at Ercis¸, Van Lake Basin R. Merzimen at Yavuzeli, G.antep, Euphrates, Persian Gulf Basin R. Karasu at Araban, Gaziantep, Euphrates, Persian Gulf Basin R. Kangal at Çetinkaya, Sivas, Euphrates, Persian Gulf Basin €l, Tigris, Persian Gulf Basin R. Çaysuyu at Genç, Bingo R. Erziki at Hakkari, Tigris, Persian Gulf Basin

44 48 49 51 53 55 42 43 45 52 54

6 2 2 2 2 3 2 2 4 1 2

77 78 79 80 80 81 82 83 82 82 84

1331 2186 1750 536 1347 1684 529 529 1402 1008 1409

38 40 39 37 38 39 37 37 39 38 37

43.2570 N/37 14.4600 N/40 38.7870 N/43 51.7790 N/40 14.4240 N/42 03.6960 N/43 17.5370 N/37 24.8350 N/37 15.1870 N/37 34.2300 N/40 40.3930 N/43

58.3310 E 29.5750 E 41.8520 E 12.8280 E 41.1790 E 10.9380 E (±0,000)

KY065262 KY065262 KY065262 KY065262 KY065262 KY065262 GenBank No.

16.2580 E 59.3650 E 21.9910 E 52.3510 E 27.3690 E 18.6590 E 34.3720 E 37.5750 E 37.6160 E 18.6020 E 51.8850 E

KY065263 KY065264 KY065265 KY065266 KY065266 KY065267 KY065268 KY065269 KY065268 KY065268 KY065270

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Table 1 (continued ) Clade, Subclade, Species, River, Locality, Drainage Clade II C. sieboldii Borçka Dam at Artvin, Coruh, Black Sea Basin R. R. R. R.

Tersakan at Havza, Yesilirmak, Black Sea Basin Tozanlı at Erbaa,Yesilirmak, Black Sea Basin Devrez at Ilgaz, Cankiri, Kizilirmak, Black Sea Basin Kizilirmak at Zara, Kizilirmak, Black Sea Basin

Clade III Subclade A C. capoeta heratensis R. Keltechinar, Turkmenistan R. Murghab, Turkmenistan C. aculeate R. Sevah, Iran Beshar stream, Karun basin, Iran Subclade B C. ekmekciae R. Coruh at Borçka, Artvin, Coruh, Black Sea Basin

P. I.

2 11 12 14 19

N

H

Altitude (m)

1 3 2 2 4 3 Hd ¼

41 12.1910 N/42 01.0210 E

68 69 70 71 72 73 0,867

379 647 589 844 1344 (±0,048)



0



40 59.352 N/35 40 20.4880 N/36 40 54.2770 N/33 39 54.0920 N/37 p (10¡2) ¼ 0,241

0

43.029 E 30.1700 E 38.2500 E 45.7840 E (±0,092)

KY065254 KY065255 KY065256 KY065257 KY065258 KY065259

JF798318 JF798319 JF798265 JF798266

3

4 3 1 Hd ¼

90 119 91 92 0,679 (±0,122)

P. I.

N

C. capoeta R. Digor at Kars, Aras, Caspian Sea Basin R. Selim at Kars, Aras, Caspian Sea Basin R. Kars at Kars, Aras, Caspian Sea Basin R. Hanak at Ardahan, Kura, Caspian Sea Basin

56 57 58 59

3 93 1 93 2 93 4 94 1 95 Hd ¼ 0,618

R. Aksu at K.Maras¸, Ceyhan, Mediterranean Sea Basin

GenBank No.

p (10¡2) ¼ 0,146 (±0,039)

Lineage, Species, River, Locality, Drainage

C. capoeta sevangi R. Arpa, Aras, Caspian Sea, Armenia Sevan lake, Caspian Sea, Armenia R. Mezamor, Aras, Caspian Sea, Armenia Clade IV C. erhani R. Ucurgene at Bahçe, Osmaniye, Ceyhan, Mediterranean Sea Bas.

GPS coordinates

Hd ¼ 0,870 (±0,033)

H

41 21.9310 N/41 40.4580 E

GQ424025 GQ424026 GQ424027

p (10¡2) ¼ 0,097 (±0,023)

Altitude (m)

GPS coordinates

GenBank No.

1485 1862 1754 1806

40 40 40 41

(±0,104)

p (10¡2) ¼ 0,140 (±0,024)

15.8070 N/43 28.2880 N/42 35.7050 N/43 13.8380 N/42

32.3460 E 48.0310 E 03.9610 E 50.5050 E

KY065276 KY065276 KY065276 KY065277 KY065278

JF798301 JF798290 JF798295

34 36

1 96 211 3 97 2 98 458 1 99 Hd ¼ 0,810 (±0,130)

37 10.4490 N/36 30.3870 E 

0



0

37 29.376 N/36 53.697 E

KY065279 KY065280 KY065281 KY065282

p (10¡2) ¼ 0,142 (±0,107)

C. barroisi €prü3 Dam (Asi) at Hatay, Orontes, Mediterranean Sea Basin Tahtako R. Afrin at Kilis, Orontes, Mediterranean Sea Basin

39 40

5 100 380 5 101 406 Hd ¼ 0,555 (±0,075)

36 51.1050 N/36 41.1790 E 36 48.3680 N/36 58.9310 E p (10¡2) ¼ 0,049 (±0,007)

KY065283 KY065284

C. trutta R. Merzimen at Yavuzeli, G.antep, Euphrates, Persian Gulf Basin R. Murat at Mus, Euphrates, Persian Gulf Basin R. Ambar at Bismil, Diyarbakır, Tigris, Persian Gulf Basin R. Karasu at Ilic, Erzincan, Euphrates, Persian Gulf Basin

42 47 50 46

3 102 2 102 4 102 5 103 Hd ¼ 0,560

37 17.5370 N/37 38 51.9350 N/41 37 53.0160 N/40 39 28.7890 N/38 p (10¡2) ¼ 0,139

KY065285 KY065285 KY065285 KY065286

529 1252 588 918 (±0,125)

34.3720 E 30.0540 E 29.1430 E 35.5650 E (±0,045)

Outgroups Luciobarbus esocinus, R. Tigris, Turkey, Persian Gulf Basin Luciobarbus pectoralis, R. Goksu, Turkey, Medit. Sea Basin Barbus tauricus eschericii, R. Karasu, Turkey, Black Sea Basin Barbus tauricus eschericii, R. Kelkit, Turkey, Black Sea Basin Barbus tauricus eschericii, R. Corukh, Turkey, Black Sea Basin Cyrprinus carpio

KP712264 AF145933 AY331029 AY331030 AY331033 DQ868871

*Locality numbers correspond to numbers in Fig. 1. Haplotypes numbers found in each locality correspond to numbers in Fig. 2.

2.3. Data analyses Sequences were manually checked for correction with the Bioedit 7.2.5 software (Hall, 1999) and the alignment of nucleotide sequences coding the mitochondrial cytochrome b gene was performed with the Clustal W method implemented in MegAlign, version 7.2 (LaserGene; DNASTAR, Madison, WI, USA). DnaSP v5 (Librado and Rozas, 2009) software packages

86 Y. Bektas et al. / Biochemical Systematics and Ecology 70 (2017) 80e94 Fig. 2. Haplotype network for cytochrome b (cyt b) sequences of the Anatolian Capoeta species. Each square represents haplotype and its size is proportional to the haplotype frequency. Within the network, numbers on the lines connecting haplotypes indicate the number of mutational step. Single step connections are not labeled. The location of the all haplotypes is color-coded to indicate different basins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Y. Bektas et al. / Biochemical Systematics and Ecology 70 (2017) 80e94

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were used to calculate summary statistics including number of haplotypes (H), haplotype diversity (Hd), and nucleotide diversity (p) for each species of Capoeta. For sequence comparisons, pairwise genetic distances between species were calculated using the Kimura 2-parameter (K2P) distance method (Kimura, 1980) as implemented in MEGA 6.06 (Tamura et al., 2013). The sequences of cyt b haplotype generated are deposited in GenBank (GenBank accession numbers: KY065239-KY065286) (Table 1). Divergence times (for K2P genetic distance) among the major Turkish clades for cyt b data and the dating of the principal events leading to speciation in genus Capoeta were calculated using calibration point time separation between genus Luciobarbus and Capoeta species complex [the early to middle Miocene; 14.6e20.7, (mean 17.0) Mya] since Capoeta is suggested to arise through hybridization of Luciobarbus and Hemigrammocapoeta by Levin et al. (2012). The genealogical relationships among haplotypes was illustrated by the median-joining (MJ) network algorithm (Bandelt et al., 1999) with Network (version 4.6.1.0; www.fluxus-engineering.com) that allows finding the number of mutations, number of individuals that share a particular haplotype and the geographical distribution of identified clades. Phylogenetic trees were generated by applying algorithms based on maximum parsimony (MP) and maximum likelihood (ML) methods as implemented in PAUP* version 4.0b10 for Macintosh (Swofford, 2003), as well as Bayesian Markov Chain Monte Carlo (MCMC) inference (BI) methods using MrBayes v.3 (Ronquist and Huelsenbeck, 2003). The most appropriate evolution models for the given data were selected using jModelTest v.0.1.1 (Posada, 2008) under the Akaike information criterion (AIC) (Akaike, 1973) and Bayesian Information Criterion (BIC) (Swartz, 1978). MP trees were estimated utilizing with tree-bisectionereconnection (TBR) branch swapping and 100 random addition replicates under the heuristic search option of the parsimony program of PAUP*4.0b10 for Macintosh. ML analysis was performed using the optimal evolutionary model (GTR þ I þ G) selected by jModelTest v.0.1.1 (Posada, 2008) under the AIC criterion. The 1000 bootstrap replicates (Felsenstein, 1985) were used to evaluate tree branch reliability in MP and ML. The Bayesian inference (BI) analysis was performed using the GTR þ I þ G model selected by jModelTest v.0.1.1 (Posada, 2008) under the BIC criterion. Four MCMC Markov chains were run simultaneously for 2 million generations. The trees and likelihood scores were sampled each 1000 generations. Burn-in was based on inspection of the number of generations required to reach stationarity in log-likelihood scores. Based on the plotting, first 5000 trees were discarded from further analyses and the remaining (post burn-in) trees were used to build a 50% majority consensus tree. The consensus tree created through this process was visualized using MEGA 6.06 (Tamura et al., 2013). All phylogenetic trees were rooted with Luciobarbus esocinus (KP712264), Luciobarbus pectoralis (AF145933), Barbus tauricus eschericii (AY331029, AY331030, and AY331033), and Cyprinus carpio (DQ868871) representing outgroup taxa (Fig. 3; Table 1).

3. Results and discussion 3.1. Genetic diversity and haplotype analysis The whole mtDNA cytochrome b gene (1140 bp) was determined for 332 individuals, which represents 59 localities placed in different geographical regions (see Fig. 1; the sample localities are also listed in Table 1). A total of 103 cyt b haplotypes was found among the nineteen species (C. antalyensis, C. balıki, C. banarescui, C. barroisi, C. bergamae, C. caelestis, C. capoeta, C. damascina, C. ekmekciae, C. erhani, C. mauricii, C. sieboldii, C. tinca, C. trutta, C. umbla, Capoeta sp.1, Capoeta sp.2, Capoeta sp.3 and Capoeta sp.4) of Capoeta, with haplotype CB-HRS1 [see Table 1 for sample size (N), number of haplotypes (Hd)]. Up to now, the nineteen Capoeta species have been identified in Anatolia, seven of which are endemic to the region. Four of the nineteen species - C. kosswigi, C. turani, C. pestai, and C. angorae - are taxonomically problematic and are probably to be synonyms of other Capoeta species; C. umbla, C. erhani, C. mauricii, and C. damascina, respectively. Therefore, the necessary analysis and evaluation were carried out with nineteen Capeota taxa with the addition of four new groups (Capoeta sp.1, Capoeta sp.2, Capoeta sp.3, and Capoeta sp.4) identified in this study. The genetic variation within each population and species was described as haplotype diversity (h) and nucleotide diversity (p) indices (Table 1). The total haplotype diversity was found to be medium/high ranging from 0.47619 ± 0.00015 (C. antalyensis) to 0.95442 ± 0.021 (C. banarescui) except for C. bergamae and C. damascina (h ¼ 0.000 and p ¼ 0.000) having monomorphic populations. The nucleotide diversity was low, ranged from 0.00000 ± 0.00000 (C. damascina) to 0.374 ± 0.028 (C. banarescui) except for Capoeta sp.1 (0.556 ± 0.035) 10
2 that showed a higher value of nucleotide diversity (Table 1). The combination of medium/high haplotype diversity (Hd ¼ 0.476e0.954) and low nucleotide diversity (p ¼ 0.042e0.241), determined in most Capoeta species distribution in Anatolia as observed in other cyprinids species (Hrbek et al., 2002; Dastan et al., 2012; Dogac et al., 2015), except C. bergamae ve C. damascina whose populations were monomorphic, may be a sign of a rapid population growth from small effective populations caused by genetic bottleneck events or founder effect (Frankham, 1996; Grant, 1998; Avise, 2000). Genetic uniformity among C. damascina populations in Kueik, Seyhan, Ceyhan and Orontes Rivers in the southeastern Anatolia that drains into the Mediterranean Sea (h ¼ 0.000 and p ¼ 0.000) (Table 1), and the fact that there is no significant decline in their population owing to threats in the area by human activity such as dam construction and climate change such as droughts (Freyhof, 2014) suggest that this area may have been colonized by monomorphic populations. Capoeta bergameae was also monomorphic (Hd ¼ 0.000 and p ¼ 0.000) (Table 1) due to the lack of the number of samples and sampling stations for this species.

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Fig. 3. Phylogenetic tree rendered by maximum likelihood analysis of the mitochondrial cyt b data set, using as outgroup Cyprinus carpio. Only bootstrap values based on 1000 replications higher than 50% are displayed. The bayesian analysis and maximum-parsimony method resulted in similar and congruent trees. Numbers above branches means posterior probabilities of BI/Bootstrap values of ML/Bootstrap values of MP. Clade IV is composed of spotted body Capoeta species; C. trutta, C. barroisi and C. erhani incorporated into the Mesopotamian Capoeta group (Fig. 3).

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3.2. Network mapping and phylogenetic analysis Haplotype network analysis revealed that the species of Anatolian Capoeta form three haplogroups, separated by at least sixty-four mutational steps (Fig. 2). Within the clade I containing Capoeta with small scale, there are five subclades separated by at least thirty-seven mutational steps (Fig. 2): The first subclade includes totally thirty-four haplotypes from the estaern Black Sea basin (Capoeta sp.1 and C. banarescui). The second subclade comprise of thirty-three haplotypes from the south Marmara Sea basin and Eber lake basin (C. tinca), from the Black Sea Basin (C. baliki, Capoeta sp.2 and Capoeta sp.3), and from the Mediterranean Sea Basin (C. antalyensis) (Fig. 2). The third subclade includes two haplotypes from Turkish Lake District (C. mauricii). The fourth subclade encloses three haplotypes from the Aegean River Basin (C. bergamae and Capoeta sp.4). The fifth subclade consists of fourteen haplotypes from the Mediterranean Sea Basin (C. caelestis and C. damascina), the Persian Gulf Basin (C. umbla), Makran Basin (C. damascina saadi), and Namak lake Basin (C. buhsei), which is separated by at least five mutational steps. The clade II contains six haplotypes of C. sieboldii which is the only Capoeta species with one pair of barbels distributed in the Black Sea Basin, and which is characterized by pleated lips that distinguish it from all other Capoeta species (Fig. 2). The clade III (capoeta with large scale) is composed of sixteen haplotypes of from the Black Sea basin (C. ekmekciae), the Caspian Sea Basin (C. capoeta), Caspian Sea Basin (C. heratensis), Karun Basin (C. aculeata), and Aras river and Sevan Lake Basins. (C. capoeta sevangi), which is separated by eight mutational steps and formed two subclades: C. heratensis and C. aculeata (Subclade A) and C. ekmekciae, C. capoeta, and C. capoeta sevangi (Subclade B) (Fig. 2). The clade IV (spotted Capoeta) comprised totally eight haplotypes from the Persian Gulf Basin (C. trutta) and the Mediteranean Sea basin (C. barroisi and C. erhani). This clade formed three distinct groups that were separated by two, nine and eight mutational steps from one another (Fig. 2). The phylogenetic trees derived from the maximum parsimony, maximum likelihood and bayesian analyses of cyt b haplotypes showed that four major clades (small scale, pleated lips, large scale, and spotted body) corresponding to the twenty-five species were identified within Anatolian Capoeta, supported by high posterior probability and bootstrap values (>67 bootstrap and >0.8 posterior probability) (Fig. 2) similar to the clades identified in the haplotype network. Within Clade I, Capoeta species which have two pairs of barbels are supported by high bootstrap values (MP/ML/BI: 94/83/0.9) and divided into two main groups representing east and west Anatolia (Fig. 3). While the east group (Capoeta sp.1 and C. banarescui; subclade I) contains Harsit, Kastel, Coruh, Yesilirmak rivers and tributaries draining into Black Sea, the west group (Capoeta sp.2, Capoeta sp.3, C. tinca, C. balıki, and C. antalyensis; subclade II) covers Eber lake and rivers that runs into Mediterranean (Aksu River), Black Sea (Dirgine, Kızılırmak, Sakarya, and tributaries), Marmara (Koca River) Sea. However, in a study by Levin et al. (2012) aiming to determine the phylogeny of Capoeta, C. antalyensis was shown to be in a different phylogenetic position, clustered with species belonging to the Mediterranean basin (Fig. 3) even though this species has two pairs of barbels. This condradicts with our results as species with two pairs of barbels are found only in subclade I and II in this study (Fig. 3). Since the number of barbels may even vary in populations of C. steindachneri (Nikol’skii, 1938; Levin et al., 2005), it is suggested that the number of barbel can not be related to sophistication and could be evolutionarily a reversible character in Capoeta species complex (Levin et al., 2012). Despite these suggestions and the difficulty of explaining the presence of the taxa in Mediterranean, South Marmara and Black Sea basins with different biogeographic stories, the number of barbels in Capoeta can be a sign of a group. By considering the phylogenetic tree topologies of Capoeta species distributed in Anatolia, it can be seen that main Capoeta clades are also consistent with grouping based on morphological properties such as scale size, mouth shape and body spotting (Fig. 3). Moreover, the haplotypes of two Capoeta species collected from the different rivers or regions as C. baliki (Sakarya river drainage and Lake Eber) and C. tinca (Susurluk drainage) were closely related to one another (>69% bootstrap and 0.6 posterior probabilities) (subclade II; Fig. 3). While all northwestern Anatolia including Marmara Sea basin was a highland draining to Black Sea during late Pliocene (Yıldırım and Emre, 2004), neotectonic events in the region (current Susurluk river basin) under the influence of the southern branch of North Anatolian Fault may have caused isolation in the direction of DB and KG (Barka and Reilinger, 1997; Selim et al., 2013). In our study, the genetic divergence dated at least in early Pleistocene (about 1.34 Mya) may indicate that gene flow may have interrupted in Capoeta populations of Eber Lake, Sakarya and Susurluk river basins by geographical isolations due to regional neotectonic events. Subclade III contains two haplotypes of C. mauricii obtained from the Lake Beys¸ehir drainage in Turkish Lake District. While subclade IV comprises two species; C. bergamae and Capoeta sp.4 from Aegean Sea basin and Iran river basin, subclade V includes six species; C. umbla, C. damascina, C. caelestis, C. buhsei, and C. damascina saadi from Iran-Anatolian group. Clade II includes only one species; C. sieboldii, with pleated lips, and only Capoeta species with two pair of barbels in Black Sea basin (Fig. 3). Clade III is represented by C. heratensis, C. aculeata, C. c. sevangi, and C. capoeta species (with large scale). The results of our study do not coincide with many points in that of Turan (2008) whose results were based on the partial sequences of 16S rRNA, examining the biogeography of Anatolian Capoeta. The first disagreement concerns the taxonomic situations of C. koswigi and C. umbla. Namely, while Turan (2008) suggests that C. koswigi and C. umbla are two closely related subspecies of C. trutta, according to present results, C. koswigi is synonym of C. umbla that is settled a different clade from that of C. trutta (Fig. 3) The second: Having formed genetically identical in Turan's tree, C. barroisi and C. damascina are embedded into two well-separated clades (I and IV) in the present study. Third: Turan (2008) suggests that C. angorae is a valid species. On the contrary, C. angorae is determined to be synonym of C. damascina in our study. Turan suggests that C. c. capoeta and C. c. sieboldii are closely related subspecies, however, C. capoeta ve C. sieboldii are not congeneric (well separated) species which are located in two different clades by our study. The mitochondrial cyt b region is a protein-coding gene with a faster

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evolutionary rate compared to the 16S rRNA (RNA gene) (Meyer, 1993; Simon et al., 1994), so it is a very useful molecular marker for understanding the phylogenetic relationships at the species-level and can improve species identification (Briolay et al., 1998). Despite the mitochondrial cyt b and 16S genes have different evolutionary rates, the differences in genetic relationships among species in genus Capoeta are quite interesting. However, the genetic distance among species within the genus Capoeta found here is consistent, and it coincides with the genetic distance between species in previous studies (Bektas et al., 2011; Levin et al., 2012). Due to high hybridization potential as a probable source of phylogenetic error, the possibility of hybridization of closely related taxa (genus Barbus and Luciobarbus) should be taken into account when determining the phylogeny of Anatolian Capoeta. To eliminate phylogeny errors that may result from hybridization, Anatolian haplotypes of L. esocinus (KP712264), L. pectoralis (AF145933), and B. tauricus eschericii (AY331029, AY331030, and AY331033) (Figs. 2 and 3; Table 1) which were retrieved from GenBank, representing the genus Barbus and Luciobarbus, which are close relatives of Capoeta were added to the phylogenetic tree and network analysis (Fig. 3). The resulting phylogenetic tree topology and haplotype network shows that none of these species is an intergeneric hybrid of Barbus and/or Luciobarbus species that are distributed in the same area (Figs. 2 and 3). Moreover, Capoeta species have been traditionally identified based on their anatomical features. Nevertheless, the presence of morphological characteristics (evidence) that could suggest a possible hybridization with closely related taxa is not observed in Anatolian Capoeta species. 3.3. Divergence times Because the Balkanian-Anatolian-Iranian archipelago took place between the Tetyhs and Paratethys Seas, Anatolia land has established palaeobiogeographic connections with the Paratethys region of the Central Europe (24-18 Mya), central Asia (18e13.5 Mya), Central Europe and Asia (13.5e11.1 Mya), the South-Asian and African (11.1e9 Mya), Greece and Mediterranean Sea (9e5.3 Mya), and Mesopotamia (5.3e2 Mya) during Early Oligocene-early Pleistocene (24-2 Mya), respectively €hme et al., 2003). These palaeobiogeographic connection timeline suggests that Anatolian fish fauna were probably (Bo formed because of interaction with the fish fauna in adjacent areas. Therefore, Anatolia has become a center of endemism and biodiversity for freshwater fish over time (Hrbek et al., 2004; Perea et al., 2010; Küçük et al., 2009) because it is located at the crossroads of continents with different biogeographic and hydrological factors. The high species diversity that stems from different eco-regions formed in Anatolia freshwater shows itself in Capoeta genus too. Overall, Anatolia freshwater fish fauna shows faunal proximity to the adjacent basin while Capoeta genus distributed in all East Asia except South Arabian Peninsula, but not found in adjacent Europe (Levin et al., 2012). Morover, the absence of Capoeta in the European parts of Turkey (Thrace) as shown in this study could be a sign that khramulya may have colonized Anatolia from Asia, and the western border of the colonization seems to be the Aegean and Marmara Seas located between the present-day Greece and Western Anatolia since € gl, 1999; Cagatay et al., 2006). Middle Miocene (Ro The pairwise sequence differences among and within clades in related to Capoeta complex were 4.56e8.96% and 0.2e2.0% (Table 2), respectively. In this study, calibration point proposedby Levin et al. (2012) suggests that the timing of spliting-events of Anatolian Capeota clades took place about 13.75e7.81 million years ago from middle Miocene (Serravallian stage) to late Miocene (Ionian stage) (Fig. 4; Table 2) when important geographical events that shaped the Anatolian Plateau of the present€ gl, 1984). Pairwise genetic distances among the Anatolian Capoeta haploday happened (Quennell, 1984; Steininger and Ro types revealed a structure, pointing Capoeta groups having small scale (Capoeta sp.1, C. banarescui, C. tinca, C. baliki, Capoeta sp.2 and Capoeta sp.3, C. antalyensis, C. caelestis, C. damascina, C. umbla, C. bergamae, Capoeta sp.4, C. mauricii, C. damascina saadi, and C. buhsei; Clade I), pleated lips (C. sieboldii; Clade II), large scale (C. ekmekciae, C. capoeta, C. heratensis, C. capoeta sevangi, and C. aculeata; Clade III), and black spotted body (C. trutta, C. barroisi and C. erhani; Clade IV) (Table 2). As shown in Fig. 4, spotted Capoeta groups including C. trutta, C. barroisi and C. erhani are initially separated from Anatolian Capoeta complex about 13.75 Mya (Serravallian). Spotted Capoeta (Clade IV) distributed in Tigris, Euphrates, Orontes and Ceyhan River drainages are also known as Mesopotamia group (Levin et al., 2012; Zareian et al., 2016). As a result of the uplift of Eastern Anatolia in the intersection point of the present-day Coruh, Aras and Upper Euphrates basins (Vasilyan et al., 2014), some of the streams in uplifted Eastern Anatolia discharged into the plaeo-Euphrates and Tigris Rivers, which were colonized by freshwater fish in the late Pliocene, 3.6 to 2.58 Mya (Krupp, 1983; Hrbek et al., 2004). Both this discharge to the upper Tigris € hme et al., 2003) and also early and Euphrates basin and the fossil records of the genus Capoeta with Pliocene-aged (Bo

Table 2 Genetic distances between and within the identified mtDNA clades of Capoeta.

Clade Clade Clade Clade

I II III IV

Clade I

Clade II

Clade III

Clade IV

0.2095 ± 0.313 4.566 6.311 8.710

0.00276 ± 0.128 6.091 8.034

0.1727.037 ± 0.179 8.966

0.00953

Note: The mean pairwise differences in percents of nucleotide divergence ±SD are given below diagonal; values on diagonal (underlined with grey) indicate within-group divergences.

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Fig. 4. Estimated time of separation among the clades/species within Capoeta species complex.

Pleistocene-aged (Vasilyan et al., 2014) show that Plio-Pleistocene-aged fish fauna of Anatolia is quite similar to the present€hme et al., 2003). The fact that Orontes fish fauna diverged from that of the day Anatolian and Mesopotamia fish fauna (Bo Euphrates about 2 Mya (Heller, 2007) and from that of the Ceyhan in the late Pliocene (Tarı et al., 2014) explains the divergence times (2.63e1.85 Mya) (Fig. 4) among C. barroisi (Orontes River basin), C. erhani (Ceyhan River basin) and C. trutta (Euphrates River basin) in the spotted Capoeta group. Large scaled Capoeta (Clade III), also known as Aralo-Caspain group due to bearing species distributed in Aral and Caspian Sea basins (Levin et al., 2012; Zareian et al., 2016), was estimated to be separated from the rest of Capoeta clades (I and II) about 10.49 million years ago (Fig. 4). In addition to findings in previous studies, large scaled C. ekmekciae haplotypes endemic to the Coruh river system are nested together with C. capoeta haplotypes from Aras and Kura Rivers in terms of both haplotype network and phylogenetic tree (Fig. 2; 3). This close genetic relationship suggests that there could be a connection between upper Aralo-Caspian basin and Black Sea drainage system before Pliocene mountain formation (Por and Dimentman, 1989; Levin et al., 2012), but this connection could be abrupted in recent times. C. heratensis, C. aculeata ve C. capoeta sevangi haplotypes (Kura-Aras and Caspian Sea basin) obtained from the Genbank was included in large scaled group. Clade II contains only C. sieboldii, which is easily distinguishable from all other Capoeta because it has pleated lips. Although most Capoeta species distubuted in Anatolian rivers that drain to Black Sea have two pairs of barbels, C. sieboldi with a single pair of barbels is distubuted in Black Sea river system including Borcka Dam of Coruh river basin in the east and Yesilirmak (Tozanlı and Tersakan rivers) and Kizilirmak (its main body and Devrez river) tributaries in the middle Black Sea. This clade (II) was estimated to be separated from small scale Capoeta (Clade I) about 7.81 million years ago (Fig. 4). Small-scaled Capoeta, most of which are endemic to Anatolian, inhabit widely in major river systems throughout Anatolia. €
and Freyhof, 2011; Ilhan In the Central Anatolia basin that contains many local endemic species (Hrbek et al., 2004; Ozulu g et al., 2014), C. mauricii is endemic to Lake Beys¸ehir, a graben lake with the Miocene and Pliocene in age (Kazancı et al., 2011; Hakyemez et al., 2013). In consistent with this dating, C. mauricii separated from sister taxa: C. bergamae (Gediz River Basin) and Capoeta sp.4 (B. Menderes River Basin) approximately 6.23 Mya (Fig. 4). Although most species in Mesopotamia show large distribution throughout the Tigris/Euphrates river system from Turkey (Smith et al., 2014), C. caelestis is endemic to Goksu river that drains Mut basin and was settled in mid-Taurus after Pliocene-Pleistocene epochs when there was marine environment (Yıldız et al., 2003). Colonization of the region by monomorphic populations may have resulted in C. damascina

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[a typical species of Mesopotamia (Krupp, 1985)] being represented with only one haplotype in the rivers of Orontes, Seyhan and Ceyhan (separated from each other 3.22e0.95 Mya) that drain to Mediterranean Sea. C. umbla having the high haplotype number and haplotype/nucleotide diversity levels (Table 1) was nested together with its sister group C. damascina, which indicates that they are evolutionarily a very close group. C. damascia saadi and C. buhsei haplotypes (Kura-Aras and Caspian Sea basin) obtained from the Genbank were included in small-scaled group (Fig. 3). With the formation of current Pontids in late Miocene (about 6.00 Mya) (Robinson et al., 1995), large rivers of Anatolia such as Sakarya, Kızılırmak, Yesilırmak, and Coruh that formed in probably late Miocene and late Pliocene created current river basins (Dogan, 2010, 2011; Yıldırım et al., 2011). These rivers flows into the Black Sea by the drain of Anatolian plateau in the € r et al., 2005) that force the rivers change their course south side of Pliocene mountain belt by crossing NAF and Pontids (S¸engo on the way. On the other hand, Black Sea became a freshwater lake during Pleistocene period (2.588e0.117 Mya) including repeated glaciation events in recent times and these rivers probably behaved as glacial refuge for freshwater fauna like other rivers of Black Sea basin (Kotlik et al., 2004; Perea et al., 2010; Akhan et al., 2014). During Pliocene-Early Pleistocene prevailing freshwater environmental conditions in paleo-central Anatolian plateau (Beker et al., 2008), Capoeta, which has two pairs of barbels, generally found in the current rivers flowing into Black Sea might have colonized Aksu basin. After colonization, due to northward movement of Arabian plate and Alanya massif, MenderesTaurus block was isolated from other Anatolian basin (Quennell, 1984; Waldron, 1984), which may explain why C. antalyensis with two pairs of barbels is localized only in middle Mediterranean region located in the south of this block. Acknowledgments This work was funded in part by the scientific research funds of Recep Tayyip Erdogan University (RTEÜ BAP, Project No: 2011.103.01.2). We are grateful to Cüneyt Kaya and Esra Bayçelebi for help with sample collection. References Akaike, H., 1973. Information theory and an extension of the maximum likelihood principle. In: Petrov, B.N., Csaki, F. (Eds.), Proceedings of the 2nd International Symposium on Information Theory. Akademiai Kiado, Budapest, pp. 267e281. Akhan, S., Bektas, Y., Berber, S., Kalayci, G., 2014. Population structure and genetic analysis of narrow-clawed crayfish (Astacus leptodactylus) populations in Turkey. Genetica 142, 381e395. http://dx.doi.org/10.1007/s10709-014-9782-5. Avise, J.C., 2000. Phylogeography: the History and Formation of Species. Harvard University Press, USA. €hl, A., 1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16, 37e48. http://dx.doi.org/10.1093/ Bandelt, H.J., Forster, P., Ro oxfordjournals.molbev.a026036. Barka, A.A., Reilinger, R., 1997. Active tectonics of the Mediterranean region: deduced from GPS, neotectonic and seismicity data. Ann. Geophis XI, 587e610.
lu, C., Ertekin, I.K., 2008. Pliocene- lower Pleistocene Ostracoda fauna from insuyu limestone (Karapınar-Konya/Central Turkey) and its Beker, K., Tunog paleoenvironmental implications. Geol. Bull. Turk. 51 (1), 1e31. Bektas, Y., Ciftci, Y., Eroglu, O., Belduz, A.O., 2011. Genetic discrimination of two Capoeta species in north-eastern Anatolia, using mitochondrial 16S rRNA gene (Osteichthyes: Cyprinidae). Zool. Middle East 53, 61e70. http://dx.doi.org/10.1080/09397140.2011.10648862. € hme, M., Reichenbacher, B., Schulz-Mirbach, T., 2003. Neogene süsswasserfischfauna anatoliens - ein schlüssel zum verst€ Bo andnis der (Palaeo-) bio€ischer süsswasserfische. In: International Symposium of Fisheries and Zoology (In Memory of Ord. Prof. Dr. Curt Kosswig in His 100th geographie europa Birth Anniversary), pp. 23e26. October 2003, Istanbul. Briolay, J., Galtier, N., Brito, R.M., Bouvet, Y., 1998. Molecular phylogeny of cyprinidae inferred from cytochrome b DNA sequences. Mol. Phy. Evol. 9, 100e108. http://dx.doi.org/10.1006/mpev.1997.0441.
rescu, P.M., 1999. The Freshwater Fishes of Europe. 5. Cyprinidae 2. Part I. Rhodeus to Capoeta. Aula, p. 426. Wiesbaden. B
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