Zoosyst. Evol. 97 (1) 2021, 235-248 | DOI 10.3897/zse.97.63691 Arte BERLIN Chondrostoma smyrnae, a new nase from the Tahtali reservoir drainage in the Aegean Sea basin (Teleostei, Leuciscidae) Fahrettin Kiictik!, Yilmaz Ciftci*, Salim Serkan Giiclii!, Davut Turan? 1 Isparta University of Applied Sciences, Egirdir Fisheries Faculty, 32200 Isparta, Turkey 2 Ordu University, Fatsa Marine Science Faculty, 52400 Ordu, Turkey. 3 Recep Tayyip Erdogan University, Fisheries Faculty, 53000 Rize, Turkey http://zoobank. org/5303FE40-D99D-426F-915 7-525 F24F 38187 Corresponding author: Fahrettin Kictk (fahrettinkucuk@isparta.edu.tr) Academic editor: Nicolas Hubert # Received 27 January 2021 # Accepted 24 March 2021 Published 7 April 2021 Abstract Chondrostoma smyrnae, a new species, from the Tahtali reservoir drainage is distinguished by having a slightly arched lower jaw with a well-developed keratinised edge, a deep and cylindric body, a complete lateral line with 47-52+1 total scales, 8—9 scale rows between the lateral line and the dorsal-fin origin, 4 scale rows between the lateral line and the pelvic fin-origin, and 19—23 gill rakers on the first gill arch. Moreover, molecular analyses using full cyt b (1141 bp) and partial co/ (652 bp) sequences of the mitochondrial genome from specimens of the new species, C. smyrnae and specimens belonging to other Chondrostoma species from central and western Anatolia demonstrated that the C. smyrnae is easily differentiated by their high pairwise genetic distances of cyt b and col data set (>2.20 and 1.03%, respectively) and by their position in the phylogenetic trees obtained through Maximum Likelihood (ML) methodology. Key Words Cytochrome b, Cytochrome oxidase I, freshwater fish, taxonomy, Western Anatolia Introduction Nases of the genus Chondrostoma Agassiz, 1832 are medium-to large-sized leuciscid fishes geographical- ly widespread from France to the Volga River and Central Iran (Elvira 1997, Kottelat and Freyhof 2007, Kucuk et al. 2017, Guc¢lii et al. 2018). Some species of Chondrostoma have a wide distribution (as C.nasus) and exhibit ecological and morphological diversity (Kottelat and Freyhof 2007, Durand et al. 2003), with some ambi- guities relating to the taxonomy of some morphological- ly defined as C. fahirae (Elvira 1987, Durand et al. 2003, Freyhof and Ozulug 2009). The major divergence times within Chondrostoma in Turkey coincided with the tec- tonic and climatic evolution of Anatolia in the Late Plio- cene and Early Pleistocene which were seen as the uplift of the Anatolian plateau and an ice age that involved both the migration and isolation of species, respectively (Ciftci et al. 2020). Currently, 14 species of the genus lives in Turkey, of which eight are endemic to Anatolia (Gilt et al. 2018, Cift¢i et al. 2020). In the Eastern Aegean Sea basin, 4 species are known: C. fahirae (Ladiges, 1960) from the Dalaman River drainage, C. holmwoodii (Boulenger, 1896) from the Gediz and Bakir River drainages, C. meandrense Elvira, 1987 from the Lake Isikli basin, and C. turnai Gicli, Kuictk, Turan, Ciftci & Mutlu, 2018 from the lower and middle Buyuk Menderes River drain- age. Additional species distributed adjacent to the Eastern Aegean basin are C. angorense Elvira, 1987, which is widespread in the Southern Marmara and Black Sea ba- sins, and C. beysehirense Bogutskaya, 1997 that is endem- ic to tributaries of the Lake Beysehir in Central Anatolia. During a revision project of the genus Chondrostoma in Turkey, we already described three new Chondrosto- ma species suggested as evolutionary lineages by Geiger et al. (2014), including C. turnai from the Biytk Men- deres River (Guc¢lti et al. 2018), C. toros Kuictk, Turan, Copyright Fahrettin Kuictik et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 236 Guclt, Mutlu & Ciftci, 2017 from the Goksu River, and C. ceyhanensis Kiicgtik, Turan, Gucli, Mutlu & Ciftci, 2017 from the Seyhan and Ceyhan rivers (Kicuk et al. 2017). Here, we describe an additional species from the Tahtali River drainage in Turkey. Materials and methods The care of experimental animals was consistent with the Republic of Turkey animal welfare laws, guidelines and policies approved by Stleyman Demirel University Local Ethics Committee for Animal Experiments (permit reference number 2011/6/5). Samples were collected by electroscoker. After anaesthesia, samples of caudal fin tis- sue taken from each specimen for the molecular analysis were fixed and stored in 98% ethanol and fish were fixed in 4% formaldehyde. Measurements were made with a dial caliper and recorded to 1 mm. All measurements were made point-to-point, never by projections. Methods for counts and measurements follow Kottelat and Freyhof (2007). Standard length (SL) was measured from the tip of the snout to the posterior extremity of the hypural com- plex. The length of the caudal peduncle was measured from behind the base of the posterior anal-fin ray to the posterior extremity of the hypural complex, at mid-height of the caudal-fin base. The lateral line scales were count- ed from the first scale touching the shoulder girdle to the posterior-most scale at the end of the hypural complex. Scales on the caudal-fin were indicated by “+”. The last two branched rays articulating on a single pterygiophore in the dorsal and anal fins were counted as “12”. The simple dorsal-and anal-fin rays were not counted since the anteriormost rays are deeply embedded. For osteological preparations, one specimen of a new species (168.8 mm SL) and one specimen of C. turnai (139.4 mm SL) were cleared and stained with alizarin red S, according to the protocol of Taylor and van Dyke (1985). The specimens were examined using a stereomi- croscope (Nikon SMZ1500), photos taken with a digital machine with a glycerol bath. The nomenclature of the skeletal elements followed Bogutskaya (1996). Abbreviations used SL standard length; BI __ Bayesian Inference; ML Maximum Likelihood; mt mitochondrial. Collection codes: IFC-ESUF Inland Fishes Collection, Egirdir Fisheries Faculty of Isparta University of Applied Sciences; FFR Recep Tayyip Erdogan University Zoology Museum of the Faculty of Fisheries, Rize; FSJF Fischsammlung J. Freyhof, Berlin. zse.pensoft.net KUCUK, F. et al.: A new Chondrostoma species from Turkey DNA extraction, PCR amplification and Sequencing Total DNA was isolated from ethanol-preserved tissue samples using the Invitrogen PureLink Genomic DNA Mini Kit according to the manufacturer’s instructions and stored at —20 °C prior to use. The mitochondrial cy- tochrome b (cyt b) gene (1141 bp) was amplified using Forward (5’— AAT GAC TTG AAG AAC CAC CGT-3’) and Reverse (5’— CAA CGA TCT CCG GTT TAC AAG AC-3’) (Robalo et al. 2007) primers. Cytochrome c ox- idase subunit 1 (co/) barcode region (652 bp) was am- plified using the FishF1 (5’-TCAACCAACCACAAAG ACATTGGCAC-3’) and FishR1 (5’-TAGACTTCTGG- GTGGCCAAAGAATCA-3’) (Ward et al. 2005) primer pairs. PCR reactions contained 5 ul of template DNA (25-50 ng/ul), 2 ul each of forward and reverse prim- ers (10 pM of each primer), 25 ul of PCR Master Mix (2X) (Promega) and ddH2O for 50 ul reaction mixture. PCR amplifications were performed using a Techne (TC- Plus) thermal cycler with the conditions as follows: af- ter a preliminary denaturation at 94 °C for 4 min, each of the 35 cycles consisted of denaturation at 94 °C for 1 min, annealing (for cyt b, 30 s at 60 °C and for col, 30 s at 54 °C), and primer extension at 72 °C for 1.5 min (1 min for co/) and a final extension at 72 °C for 10 min, followed by cooling to 4 °C. The PCR products were purified with the QIAquick PCR purification kit (Qia- gen, Hilden, Germany) according to the manufacturer’s protocol, and bidirectional sequencing of purified PCR products was performed using the same PCR primers on an Applied Biosystems 3730 XL Genetic Analyser (Ap- plied Biosystem, Foster City, CA, USA) using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystem) at the Macrogen Inc. (Amsterdam, Netherlands) (https://dna.macrogen-europe.com/eng/). Phylogenetic reconstruction For the phylogenetic analyses, two data sets were used, an 1140 bp fragment of cyt b and 652 bp fragment of col sequences. Cyt b and col sequences were aligned with the previous sequences from GenBank (Table 1) with the Clustal W algorithm (Thompson et al. 1994 available in Bioedit 7.2.5 (Hall 1999)) with default parameters (gap opening: 10.00 and gap extension: 0.10; Hall 2008) and all alignments were inspect- ed and corrected visually. Sequences obtained in this study were deposited in GenBank (accession numbers: MT387055—MT387058 for the cyt b and MW719591- MW719611 and MW722822-MW722824 for the co/). After translating the nucleotide sequence of protein coding genes to proteins using MEGA X (Kumar et al. 2018), we found no stop codons or indels present in this alignment. Haplotypes were detected using DnaSP v5 software (Librado and Rozas 2009). Duplicate se- quences were not used for phylogenetic tree construc- tion. Subsequently, phylogenetic analyses have been Zoosyst. Evol. 97 (1) 2021, 235-248 Zod Table 1. List of Chondrostoma species analysed, sampling locality, sources and GenBank accession numbers. Species River/Drainage Country Sources Accession numbers Cyt b Col C. holmwoodii Golmarmara/ Akpinar Turkey Ciftci et al. (2020) MT387078 Gediz river/ Derbent Turkey Ciftci et al. (2020) MT387099 Bakircay/ Bergama Turkey Durand et al. (2003) AF533765 Gediz river/ Usak Turkey Durand et al. (2003) AF533762 Bakir stream Turkey Geiger et al. (2014) KJ553066.1 Gediz river /Usak Turkey This study MW719600 Gediz river /Usak Turkey This study MW719598 Gediz river /Usak Turkey This study MW719599 Bakir stream Turkey Geiger et al. (2014) KJ552962 71 C. smyrnae Sasal stream/ Tahtall Turkey This study MT387055 MW722822 Sasal stream/ Tahtall Turkey This study MT387056 MW722823 Sasal stream/ Tahtall Turkey This study MT387057 MW722824 Sasal stream/ Tahtall Turkey This study MT387058 C. turnai Cine stream Turkey Ciftci et al.(2020) MT387091 Cine stream Turkey Ciftci et al. (2020) MT387093 Cine stream Turkey Ciftci et al. (2020) MT387092 Cine stream Turkey Ciftci et al. (2020) MT387095 Cine stream Turkey Ciftci et al. (2020) MT387094 Buyuk Menderes Turkey Geiger et al. (2014) KJ553058.1 Yayla lake/Buldan Turkey This study MW719601 Yayla lake/Buldan Turkey This study MW719602 Yayla lake/Buldan Turkey This study MW719603 Buyuk Menderes Turkey Geiger et al. (2014) KJ553026.1 Buyuk Menderes Turkey Geiger et al. (2014) KIS52989. 11 C. angorense Akin stream/ Sakarya Turkey Ciftci et al. (2020) MT387066 Porsuk stream/ Sakarya Turkey Ciftci et al. (2020) MT387114 Porsuk stream/ Sakarya Turkey Ciftci et al. (2020) MT387112 Kizilirmak river Kirsehir Turkey Perea et al. (2010) HM560078 Porsuk stream/ Sakarya Turkey Ciftci et al. (2020) MT387113 Sakarya river/ Kizilcahamam Turkey This study MW719604 Sakarya river/ Kizilcahamam Turkey This study MW719605 Kizilirmak river/ Zara Turkey This study MW719606 Kizilirmak river/Zara Turkey This study MW719607 Delice stream/Yerkoy Turkey This study MW7 19608 Delice stream /Yerkoy Turkey This study MW7 19609 Kizilirmak river/ Boyabat Turkey This study MW719610 Kizilirmak river/ Boyabat Turkey This study MW719611 C. meandrense Isikli spring/ Civril Turkey Ciftci et al. (2020) MT387085 Isikli spring/ Civril Turkey Ciftci et al. (2020) MT387087 Isikli spring/ Civril Turkey Ciftci et al. (2020) MT387088 Isikli spring/ Civril Turkey Ciftci et al. (2020) MT387086 Buyuk Menderes Turkey Geiger et al. (2014) KJ553109 Buyuk Menderes /Civril Turkey This study MW719591 Buyuk Menderes/ Civril Turkey This study MW719592 BuyUk Menderes/ Dinar Turkey This study MW719593 Buyuk Menderes/ Dinar Turkey This study MW719594 Buyuk Menderes/ Dinar Turkey This study MW719595 Buyuk Menderes/ Akcay Turkey This study MW719596 Buyuk Menderes Turkey Geiger et al. (2014) KJ553083 C. nasus Diina River Serbia Schonhuth et al. (2014) MG806833 Soca drainage Slovenia Geiger et al. (2014) KJ553248 Simav drainage Turkey Geiger et al. (2014) KJ552881 Danube River Romania Org (2018) MF 135845 Susurluk, Kocacay Turkey Durand et al. (2003) AF533760 Danube River Austria Durand et al. (2002) AY026402 Simav Stream, Yanikburnu Turkey Ciftci et al. (2020) MT387104 Simav Stream, Yanikburnu Turkey Ciftci et al. (2020) MT387105 Simav Stream, Yanikburnu Turkey Ciftci et al. (2020) MT387106 Simav Stream, Bigadi¢ Turkey Ciftci et al. (2020) MT387109 Simav Stream, Bigadi¢ Turkey Ciftci et al. (2020) MT387110 C. beysehirense Beysehir lake Turkey Ciftci et al. (2020) MT387079 Beysehir lake Turkey Ciftci et al. (2020) MT387080 Beysehir drainage Turkey Geiger et al. (2014) KJ552898 Beysehir lake Turkey This study MW719597 C. fahirae Tefenni Burdur Turkey Ciftci et al. (2020) MT387128 Pseudochondrostoma willkommii geo babon bee: KJ554279 Achondrostoma arcasii KFS2Z9143 KJ552518 zse.pensoft.net 238 performed over aligned nucleotides containing poly- morphic sites for two different data sets (cyt b and col). Computation of phylogenetic tree reconstructions of haplotypes was carried out using Maximum Likell- hood (ML) analyses. ML analyses for these two data- sets were performed using PhyML version 3.0 (Guin- don and Gascuel 2003), with 1000 bootstraps under the best-fit models (TIM3+G for cyt b, TPM1uf+G for col) which were calculated by the Akaike and Bayes- ian Information Criteria (AIC and BIC) approaches in the programme jModelTest 0.1.1 (Posada 2008). All of the trees deduced from the Cyt b and Col sequenc- es were rooted with Achondrostoma arcasii (Stein- dachner, 1866) and Pseudochondrostoma willkommii as an outgroup taxa. Constrained trees were generat- ed in TreeWiev (Page 1996). A calculation of pairwise genetic distance among different species with Kimura 2-parameter (K2P) distances model (Kimura 1980) was performed using MEGA X (Kumar et al. 2018). Results Molecular analysis The nucleotide sequences of both strands resulted in the full length of mitochondrial cyt b (1141 bp) for the sam- ples. Four haplotype sequences belonging to Chondrosto- ma smyrnae were compared with other Chondrostoma sequences from Western Anatolia available from GenBank (Table 1). A total of 1140 homologous sites for each of the 32 individual sequenced were aligned, with 215 (18.9%) variable and 119 (10.4%) variable characters parsimony-in- formative under the maximum parsimony optimality crite- rion. For cyt b gene, Intrageneric K2P distances among ana- lysed Chondrostoma species from western Anatolia ranged from 1.26% between C. angorense and C. meandrense to 9.95% between C. fahirae and C. turnai (Table 2). The mean intraspecific divergence ranged between 0.13% for C. smyrnae and 0.53% for C. holmwoodii (Table 2). In addi- tion, the pairwise genetic distances between C. smyrnae and the other species ranged from 2.20% to 9.88% (Table 2). The dataset for cytochrome c oxidase subunit 1 (co/) included 24 individuals sequenced by this study and eight individuals from GenBank belonging to seven KUCUK, F. et al.: A new Chondrostoma species from Turkey Chondrostoma species (Chondrostoma smyrnae, C. tur- nai, C. meandrense, C. holmwoodii, C. beysehirense, C. angorense and C. nasus) in western Anatolia (Table 1). A total of 652 characters for each of the 36 individuals sequenced were aligned, 597 (91.6%) characters were constant, 27 (4.14%) variable characters were parsimo- ny-uninformative and 28 (4.29%) characters were parsi- mony-informative under the maximum parsimony opti- mality criterion. For col gene, Intrageneric K2P distances among analysed Chondrostoma species from western Anatolia ranged from 0.38% between C. angorense and C. beysehirense to 1.47% between C. holmwoodii and C. turnai (Table 2). The mean intraspecific divergence ranged between 0.00% for C. angorense and C. nasus and 0.21% for C. smyrnae (Table 2). In addition, the pairwise genetic distances between C. smyrnae and the other spe- cies ranged from 1.03% to 1.32% (Table 2). Phylogenetic relationships among the sequences were reconstructed independently for cyt b and col genes using the ML method. In the phylogenetic reconstructions for cyt b, Chondrostoma haplotypes formed three distinct mono- phyletic clades, hereafter referred to as clades I, II, and III as in Figure 1, which were consistently supported by high bootstrap values (100). The first clade consisted of C. holm- woodii, C. smyrnae and C. turnai while the other clade con- tains C. angorense, C. meandrense and C. nasus. C. smyr- nae formed a well-supported clade sister to C. turnai and both taxa were clustered in a monophyletic group distin- guished from C. holmwoodii. In addition, C. fahirae were positioned basal to all species of Chondrostoma (Figure 1). For col, the phylogenetic tree demonstrated that Chondrostoma species are divided into four clades (Figure 2). Clade I is formed by C. meandrense and C. nasus species while clade II is made up of the C. turnai, C. smyrnae and C. holmwoodii. Compared to cyt b results, the position of C. angorense was clearly separated from the clade including C. meandrense and C. nasus species, and formed a distinct and well-supported phylogenetic clade (93). C. smyrnae formed a well-supported lineage (85) and clustered together with C. turnai. All phylogenetic analysis showed that these species are separated from one another. Also, C. smyrnae indi- viduals from the Tahtali reservoir drainage in the Aegean Sea basin are concluded to be distinct members of the Chondrostoma species studied in all phylogenetic trees. Table 2. Pairwise distance of cyt b and col data set within and among Chondrostoma species based on 1.000 bootstrap replications using the Kimura 2-parameter distance method; values in lower left cells = percent difference among taxa for cyt b, values in upper right cells = percent difference among taxa for co/ and diagonal = percent difference within taxa for cyt b/col. C.holmwoodii C.smyrnae C.turnai C.holmwoodii 0:53-/012 ak S 1.47 C.smyrnae 2195 0.13 /0.21 L3Z C.turnai 2,91 2.20 0.33 /0.09 C.angorense 2.47 2261 2.48 C.meandrense 2.87 27 2.67 C.nasus 2.96 2.64 2.45 C.beysehirense eas) 2.86 3.00 C.fahirae S67 9.88 G95 zse.pensoft.net C.angorense C.meandrense C.nasus C. beysehirense 0.77 0.95 0.93 0.85 Abe ipa 119 Titel 1.01 TLS, 1.16 1.08 0.35 /0.00 0.48 0.46 0.38 1.26 0.26 /0.04 0.64 0.56 1.47 ines 0.50 /0.00 0.54 2.30 2.28 2.18 0.26 /0.15 Oks 5 9736 9.08 Zoosyst. Evol. 97 (1) 2021, 235-248 239 C. holmwoodii AF533765 C. holmwoodii MT387099 100 C. holmwoodii MT387078 C. holmwoodii AF533762 C. smyrnae MT387055 89 | C.smyrnae MT387056 | 100 C.smyrnae MT387057 C. smyrnae MT387058 94 C.turnai MT387093 C.turnai MT387091 C.turnai MT387092 100 C.turnai MT387095 C. turnai MT387094 C. angorense HM560078 C.angorense MT387114 ——— C.angorense MT387066 C. angorense MT387112 C. angorense MT387113 C. meandrense MT387087 ____—C. meandrense MT387085 99 C. meandrense MT387088 100 C. meandrense MT387086 59 C. nasus MT387104 C. nasus MT387109 C. nasus MT387106 C. nasus AF533760 93 86 C. nasus MT387105 C. nasus MT387110 C. nasus AY 026402 74 C. beysehirense MT387079 | = C. beysehirense MT387080 © C. fahirae MT387128 O Pseudochond. willkommii KF529128 | uterou Achond. arcasii KF529113 8 Pp Clade I 78 97 76 Clade II 500 length units Figure 1. Maximum Likelihood (ML) estimation of the phylogenetic relationships of Chondrostoma species based on the mito- chondrial cyt b sequence data. The tree was created with the TIM3+G substitution model. Branches are labelled with maximum likelihood bootstrap >50%. Key to species of Chondrostoma in the Anatolian Aegean Sea basin il 12-14 gill rakers on first gill arch; no keratinised cutting edge on lOWe?r JAW ............ cece eeee cece ee eee eee ee eeseeeeeeeeeeeees C. fahirae - 19-26 sill rakersson first onl archa keratinisedieutting edee-on OWS jaw. ...8escss5.....ctanzeSasose dd ebgas se) piel then ead eedyd-sscartad 2 2 Kateral line Witt iG OS $e =o. SCALES ike eas demaaasivssvuneeeenadinsciaten vutredsen ene dccintprqe raced tee ag fstecipin re PEK CC spbeinnloge a ene Sea PES 3 - batStallwiine With Aig— 52 lee SOAS Fees sects eens nee a nee bh is fees oe he oe enna ae Bean meen cee hl Seen ae ac¥ oko ce enniaie eee 5: 3 Lower jaw markedly arcinectand keratin. Hot well CevElOOe Ss 224 syasues see bert eid. oaett Londen eenersatnneadeegwds a imerteete C. meandrense - Lower jaw slightly arched and keratin iS Well CEVElIOPEC .... eee cecc ccc eecceeeeeeeeeeeeeeeece eee eeeseeeeeaeeseeeaeeseeeeeeeseeeeeseeeeeeeeseees 4 4 5-7 scale rows between lateral line and pelvic-fin origin. In life, a longitudinal violet stripe (band) in middle of flank from Ca vielal HMA Se - CORSA TOSS os c.nin bn Myypin pontine wtauthnit ecinnastanpleedssshuddnantndes tyaenp aber riaaia ite vddehogsaphiedsedasnapecteat at C. holmwoodii - 5 scale rows between lateral line and pelvic-fin origin. Not a longitudinal violet stripe in life (band) ........... C. angorense 5 Body width at dorsal-fin origin 13-15% SL; body width at mid-point of caudal peduncle 4-6% SL.................6. C. turnal - Body width at dorsal-fin origin 16-19% SL; body width at mid-point of caudal peduncle 6-8% SL................ C. smyrnae zse.pensoft.net 240 KUCUK, F. et al.: A new Chondrostoma species from Turkey C. meandrense KJ553083 C. meandrense MW719595 C. meandrense KJ553109 C. meandrense MW719591 C. meandrense MW719594 C. meandrense MW719592 C. meandrense MW719593 C. meandrense MW719596 C. nasus MF 135845 C. nasus KJ553248 88 C. nasus MG806833 C. nasus KJ552881 .turnai MW719603 C.turnai MW719602 C.turnai MW719601 : C. turnai KJ553026 98 C. turnai KJ552989 50 C. turnai KJ553058 C. smyrnae MW722823 85 C. smyrnae MW722822 56 | C. smyrnae MW722824 C. holmwoodii KJ552962 93 C.holmwoodii MW719599 ae C.holmwoodii MW719600 C.holmwoodii MW719598 ——__ C. holmwoodii KJ553066 C. beysehirense MW719597 C. beysehirense KJ552898 C. angorense MW719608 C. angorense MW719610 —_— C. angorense MW719606 C. angorense MW719611 _C. angorense MW719604 C. angorense MW719609 C. angorense MW719605 C. angorense MW719607 Pseudochond. willkommii KJ554279 87 93 61 a Clade I Clade II Clade IV CIll Achond. arcasti KJ552518 500 length units | Outgroup Figure 2. Maximum Likelihood (ML) estimation of the phylogenetic relationships of Chondrostoma species based on the mito- chondrial co/ barcode region. The tree was created with the TPM1uf+G substitution model. Branches are labelled with maximum likelihood bootstrap >50%. Chondrostoma smyrnae, sp. nov. http://zoobank.org/44365C7F-0D20-4983-9E3B-7205046ED1FA Fig. 3a—c Holotype. IFC-ESUF 03-1566, 191 mm SL; Turkey: Izmir prov.: Tahtali reservoir about 2 km north of De@ir- mendere, 38°08'19"N, 27°07'10"E. Paratypes. IFC-ESUF 03-1567, 22, 152—205 mm SL; IFC-ESUF 03-1568, 22, 181-272 mm SL; same data as holotype. — IFC-ESUF 03-1550, 2, 92.68—109.02 mm SL; Turkey: Izmir prov.: stream Sagal about 1 km south of Kuner, 38°11'57"N, 27°08'09"E. — FFR 2079, 1, 92 mm SL; Turkey: Izmir prov.: stream Balaban at Kiuner, 38.213950 27.101505. Material used in molecular genetic analysis. IFC-ESUF DNA-03-1550, 7, Turkey: Izmir prov.: stream Sasal about 1 km south of Ktiner, 38°11'57"N, 27°08'09"E (GenBank accession number: MT387055—M1T387058; MW719591—-MW719611; MW722822-MW722824). zse.pensoft.net Diagnosis. Chondrostomasmyrnae is distinguished from other species occur to adjacent basin by a cylindrical body (body width at dorsal-fin origin 16.8-19.3% SL, vs. 13.3- 15.4 in C. turnai (Fig. 3d), 14.1-16.6 in C. meandrense, 12.0-16.3 in C. holmwoodii, 12.4—15.7 in C. fahirae, except C. beysehirense), a wider head (head width at anterior margin of eye 55-65% HL, vs. 42-54), by having less lateral line scales (48-53 vs. 60-67 in C. beysehirense, 60-66 in C. holmwoodii and 56-60 in meandrense, except C. turnai and C. fahirae). Chondrostoma smyrnae is further distinguished from C. turnai by the absence keel between pelvic fin-origin and anus (vs. present in specimens larger than 160 mm SL), a straight or slightly arched lower jaw (vs. arched), more total lateral line scales (48-53, vs. 44-51), and fewer gill rakers on first gill arch (19-23, vs. 22-27). Also, C. smyrnae further differs from C. turnai by the shape of jaws, hyomandibular, quadrate and the fifth brachial gill arc. In C. smyrnae, the dentary thick and coronoid process inclined forward (vs. thin and Zoosyst. Evol. 97 (1) 2021, 235-248 241 4 PT AA, a hy AA ’ 4 ~~ i MO eNO LO TAO Mee ccd Tein c4t 107 yt eee eee ee ¥ y f- et he : : 3 AA OS amen Mae darane eo or oe re 7 2% ta % e247, gays J ‘ he > Figure 3. a. Chondrostoma smyrnae, IFC-ESUF 03-1567, just after fixation form, 167 mm SL, Turkey: Tahtali reservoir; b. IFC-ESUF 03-1566, holotype, 191 mm SL, Turkey: Tahtali reservoir; c. IFC-ESUF 03-1567, paratype 205 mm SL, Turkey: Tahtali reservoir; d. Chondrostoma turnai, [FC-ESUF 03-1557, 197 mm SL, Turkey: Cine stream. coronoid process nearly vertical); premaxilla very deep and The new species is also distinguished from C. me- posterior edge short (vs. slender and posterior edge long); | andrense by the body colour silvery in life (silvery, vs. hyomandibular long and narrow (vs. short and wide), the brownish). It is further distinguished from C. holmwoodii fifth brachial gill arc wide angle (vs. narrow angle) and _ by having 8—9 scale rows between the lateral line and dor- pharyngeal teeth wide (vs. thin); outer margin of quadrate __ sal-fin origin (vs. 9-11), four scale rows between the lateral slightly pointed (vs. rounded) (Figs 4, 5). line and pelvic-fin origin (vs. 6-7). Chondrostoma smyrnae zse.pensoft.net 242 KUCUK, F. et al.: A new Chondrostoma species from Turkey Dn Figure 4. Jaws bones of C. smyrnae (a.) and C. turnai (b.) (Dn: dentary, Pmx: premaxilla, Mx: maxilla, Crp: Coronoid process). is further distinguished from C. fahirae by having of 19-23 gill rakers on the first gill arch (vs. 12-14), well keratinised cutting edge present on the lower jaw (vs. slightly) (Fig. 6). Description. See Figures (3) for general appearance, Table 3, 4 for morphometric and Table 5, 6 for meristic data. A small-sized individual of Chondrostoma symrnae with a cylindrical body shape is shown on Figure 7. Dorsal and ventral body profiles markedly convex. In- terorbital area slightly convex. Mouth inferior, arched, with a keratinized cutting edge on lower jaw. The low- er jaw slightly arched. Snout long, length 31-35% HL, with slightly rounded tip. Rostral cap almost covers up- per jaw. Lower jaw relatively long, lower jaw-quadrate junction on vertical through eye pupil margin. Eye di- ameter about equal to interorbital distance. Caudal pe- duncle depth fits 10-12 times in its length. Dorsal-fin outer margin markedly concave. Anal-fin outer margin concave. Caudal-fin deeply forked, lobes with pointed tips. Outer margins of pectoral and pelvic-fins slightly convex. Pharyngeal teeth in one row, 5-6, 5-5, sharp, serrated, hooked at tip (Fig. 5). Dentary thick and coro- noid process inclined forward. Premaxilla very deep and its posterior edge short. Hyomandibular long and _nar- row. Fifth branchial gill arc wide angle and pharyngeal teeth wide. Outer margin of quadrate slightly pointed. zse.pensoft.net Table 3. Morphometric data of Chondrostoma smyrnae (holotype, IFC-ESUF 03-1566 and paratypes, IFC-ESUF 03-1567; n = 22). Holotype Holotype & paratypes mean Min Max SD Standard length (mm) L9G PA « NS 2320570 In percent of standard length Head length 21.9 748 eed Mi Ngo ie See A bk OLY Body depth 26.6 25.9 242 28.4 1.4 Body width at dorsal-fin 17.5 17.4 16.0 19.4 1.2 Body width at caudal peduncle 6.8 6.35 «Dif “S80 e909 Predorsal distance BilsZ 5OIS: 249:0- 5272" ~ 152 Prepelvic distance 52.6 5S, 1) bly *54.6" 168 Preanal distance 714. G32" 70d Powe 2:6 Pectoral-fin origin to anal-fin 49.6 5218 G97” -5E. ay Pectoral-fin origin to pelvic-fin 29S TSP 10 7 "2938. “3632 9175 Pelvic-fin origin to anal-fin 19.6 22.1 19.8 246 0.9 Dorsal-fin depth 18.4 18.4 171 620:6 OS Anal-fin depth 16.2 15.4 144 16.9 0.9 Pectoral-fin length 16.9 TF i8e 616. 4- 1 95Q 1.0 Pelvic-fin length Lo La Gre Me ee 09 Caudal peduncle length 17.9 18.5 16.5 20.4 1.4 Caudal peduncle depth 11.8 FIO gO TS" O59 In percent of head length Snout length 31.6 33:14 31.19 935,32 Be? Eye diameter 18.5 20:7) LTS 2238 ths Interorbital distance 41.8 41.7 38.7 46.4 2.1 Head width, 56,9 3 Jeel HATO eG oI Zs Head depth, 54.7 54.6 509 57.8 2.4 Head depth, Sls TIO 1O0. B52 2.6 Mouth width Zia a 2 2 AML! OF Gy OdO Zoosyst. Evol. 97 (1) 2021, 235-248 243 Pht Figure 5. Hyomandibular, quadrate and pharyngeal teeth of C. smyrnae (a.) and C. turnai (b.) (Hym: hyomandibulare, qu: quadrate, Pht: Pharyngeal teeth). The number of lateral line scales, scale rows between the lateral line and dorsal-fin origin, scale rows between the lateral line and pelvic-fin origin, branched dorsal-fin rays, branched anal-fin rays and rakers on the outer side of the first gill arch are shown in Tables 5-6. Colouration. In life: fins pinkish with hyaline mar- gins; back brown; flank silvery with pinkish hue. After fixation: back and upper portion of flank dark greyish; mid-lateral portion of flank and belly yellowish. Dorsal and caudal fins dark grey, pelvic and anal fins yellowish. Peritoneal membrane black. Distribution. Chondrostoma smyrnae is known from the Tahtali reservoir basin (Fig. 8). It is also expected to be native to the Kticik Menderes River drainage but at- tempts to find it there have thus far proven unsuccessful and it may have been extirpated. Etymology. The species is named for Smyrna, the his- toric name of the city known today as Izmir. A noun in genitive, indeclinable. Discussion The identification of Chondrostoma symrnae is a significant contribution to the genus Chondrostoma. There are six species of Chondrostoma (C. angorense, C. beysehirense, C. fahi- rae, C. holmwoodii, C. meandrense and C. turnai) in western Anatolia. The differences between Chondrostoma smyrnae, zse.pensoft.net 244 KUCUK, F. et al.: A new Chondrostoma species from Turkey Table 4. Morphometry of C. turnai (IFC-ESUF 03-1558. n = 43), C. meandrense (IFC-ESUF 03-1519. n = 24), C. holmwoodii (IFC-ESUF 03-1513. n= 19), and C. fahirae (IFCESUF 03-1512. n= 36). C. turnai C. meandrense C. holmwoodii C. fahirae SL (mm) 132.0-191.8 120.6-200.9 88.8-161.3 82.8-96.8 Mean min max SD mean min max SD mean Min max SD mean min max _ SD In percent of standard length Head length 219 2007 = (23.4 ANS W2256 2159 123.6 025 = 23el= 221) I2450 @ OE & 2A le 2323182541) -' OG Body depth 24:6 229. 2EO- 1.0 23:9" 2158 e258 10. “23:6. 22.0 PAS OF ZAR Peo 2x5 -Lyl Body width at dorsal-fin 4.3: A238, Te]. Gs. .144. 12 5 16-2 0%. Leo 123 146 O85. 126 1O7> 1b Os Body width at caudal peduncle 5.3 40 58 14 500 35 59 09 56 47 66 07 56 41 69 1.2 Predorsal distance Bie -5O0e 5A5: ee 536 4ASS 662.6 45 62:2: 60,7 “64.0 » Tl 5375) see: S510 Trp Prepelvic distance 52.7 boas S38 1019 6524: ebO Poa 1465 “52/7 SOG "542 — “WA. ob 2b AG 8 B38 rhe Preanal distance BOAT TS ahold ee el. “OO 74S ss LP eh PO OTS, el 0S Ot hoe lA. Pectoral-fin origin to anal-fin 52.9 48.7 54.3 14 49.9 46.1 524 09 49.9 48.2 51.1 O06 47.1 45.7 499 0.6 Féctoral-fin-origin to-pelvic-fin. S1li4-°29.0- 34.5 10° 29:2 26:55 31.5 1.2 289 28:2. 29:4 0% 280 -25.4 303 ~0.6 Pelvic-fin origin to anal-fin 22.0205 23,0 1.2. 20:9 19:5 323.5 0:5 ~209- 19:8 21.6. 04 28,0° 25.4 30,2 0.6 Dorsal-fin depth To? IS SA OS. Te. 2A R202" Ws Gib. Lae AOS «6.7 P10. 20 Da 2A «Hel Anal-fin depth ISS WSS Tha OS TAO Wa. 16.8: 0.6. “iG. 2: P48. Ifa 0:6-" 166. 150 cee 0:9 Pectoral-fin length LEO. Beso Or, eB. 1725-154 Tek 0:9) Aa, oS) GA PW OL9- “2ZOt5 Tee PALO” G29 Pelvic-fin length r6- 14.1 175) 1.0 154° 1388) 1S) 0.9 Wed. Wao 162 0.8 1733 146 190 T2 Caudal peduncle length 17.4 J4.5 (8:5 @5 I82 16.7 203 0.9 178 162% 19:4 06 194 182 209 0.9 Caudal peduncle depth LO! “OiZO5 I> OSE” TOs Sro> able OA. Oe Sa TOS Oe = HOR B29 “19 Oss In percent of head length Snout length 30:6 .272. 33:8. shi6 82:5 29-1 (34.8) «LS 31:4 "269 33.3- 1.0) 312-260" 32.6 “1.2 Eye diameter PPR BOM Baten ISP e218. Te 26s. te Se OGS iveor 224s whe Pere OSPR) OO kL ap Interorbital distance 38.2 33.1 41.9 2.1 364 33.7 38.7 14 36.1 32.7 39.0 1.1 37.8 35.1 396 1.6 Head width, 5A 4 CASS) SOWA ABA 37.6. ABS 22 ~ 440-405 APY “IW “Fo 389 Asa Pies Head depth, 56-6: “A777. BOM. 192 -5Oi). © 4 2iow SARS. al FeO le AS) 52.5 VOR bans, bln) FoAa™ 3 Head depth, 743 FOG 7BO elo WHOL 696 7OA O28 727) Te Foe OB. TSO) 722-768. Ll Mouth width 2671, 23s) "27 SS 2o8e23.8 26:3 1 274 25.0 29.3 14. 25.4 244° 27:8 Pal Table 5. Frequency of lateral line scales, scale rows between the lateral line and dorsal-fin origin, and the lateral line and pelvic-fin origin in Eastern Aegean Chondrostoma species. lateral line scales N 44 45 46 47 48 49 50 51 52 53 54 55 56 57 458 #£«+59 60 61 62 63 64 65 66 Mean ‘C.smymae 299i (sts—<‘i—s~s—stsCH 80.9 C. turnai Lb A OS Se 2 2 af 47.4 C.meandrense 14 a2 2 6° 2 58.2 C. holmwoodii 14 1 1. A DT 2 2 93 634 C. fahirae 24.71) JS 6° =) 38 1 1 1 47.6 scale rows between lateral line and scale rows between lateral line and dorsal-fin origin pelvic-fin origin N 8 8, 9 9,, 10 11 Mean 3,, 4 4,, 5 5,, 6 7 Mean C. smyrnae 28 19 9 8.3 28 4.0 C, turnai $5.76 52 4" 1 8.4 le TOF 1 93 4.2 C.meandrense 14 1 ahy 2 9.0 14 5.0 C. holmwoodii 20 2 13.6 | 10:2 5 1352 5.8 C. fahirae LOGeS 5 8.5 if 3 4.3 Table 6. Frequency of gill rakers, branched dorsal-fin rays, and branched anal-fin rays in Eastern Aegean Chondrostoma species. Gill Rakers N 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 =Mean C. smyrnae 16 5 3 3 2 20.8 C. turnai 11 1 2 4 3 1 24.2 C. meandrense 20 2 a 3 6 6 1 25.8 C. holmwoodii 14 2 8 2 2 22.3 C. fahirae 7 1 3 3 13.3 Branched dorsal-fin rays Branched anal-fin rays N 7 8 9 10 Mean 8 9 10 11 12 #£4=Mean C. smyrnae 36 2 34 7.9 2 Sy) 29 9.8 C. turnai 18 18 8.0 an 9.4 C. meandrense 14 12 2 8.1 8 6 9.4 C. holmwoodii 16 16 8.0 14. 2 9.1 C. fahirae 10 10 7.0 3 7 8.7 zse.pensoft.net Zoosyst. Evol. 97 (1) 2021, 235-248 245 : : Figure 6. Ventral view of head: a. C. smyrnae, IFC-ESUF 03-1567, paratype, 189 mm SL, Turkey: Tahtali reservoir; b. C. turnai, IFC-ESUF 03-1524, 174 mm SL, Turkey: Cine Stream; ¢. C. meandrense, IFC-ESUF 03-1519, 183 mm SL, Turkey: Isikli Spring; d. C. holmwoodii, IFC-ESUF 03-1513, 156 mm SL, Turkey: Gediz River; e. C. angorense, IFC-ESUF 03-1502, 152 mm SL, Turkey: Sakarya River; f. C. fahirae, IFC-ESUF 03-1512, 109 mm SL, Turkey: Dalaman River; g. C. beysehirense, IFC-ESUF 03-1505, 288 mm SL, Turkey: Beysehir Lake. Figure 7. Top view of Chondrostoma smyrnae (a.) IFC-ESUF 03-1567, 194.2 mm SL; (b.) 190.0 mm SL, Tahtali Reservoir; and C. turnai (¢.) IFC-ESUF 03-1524,195.1 mm SL; (d.) 181.3 mm SL, Stream Cine. zse.pensoft.net 246 gen es tS 36.000 27.000 KUCUK, F. et al.: A new Chondrostoma species from Turkey . angorense ’. beysehirense '. fahirae . holmwoodii ". meandrense ". smyrnae * furnai @ O © ) | @ e | @ }. LIEK ue 30.000 Figure 8. Distribution of Chondrostoma species in western Anatolia. C. fahirae, C. holmwoodii, C. meandrense and C. turnai, which occur tn the Aegean Sea basin of Turkey, are given in detail in the diagnosis section. Chondrostoma smyrnae is distinguished from C. angorense and C. beysehirense, which occur adja- cent to the Aegean Sea basin of Turkey, by having 48-53 lateral line scales (vs. 59-68 in C. angorense, 60-67 in C. beysehirense), 8—9 scale rows between the lateral line and the dorsal-fin origin (vs. 9-10 in C. angorense, vs. 10-11 in C. beysehirense), four scale rows between lat- eral line anal-fin origin (vs. 5 in C. angorense, 5—6 in C. beysehirense) and 19-23 gill rakers on first gill arch (vs. 33-39 in C. beysehirense). In other words, their high genetic distance and position in the phylogenetic trees with high bootstrap values easily differentiated C. smyrnae from other species. The results on comparative morphological and genetic studies based on cyt b and col genes showed that the new species differ from formerly described Chondrostoma species. Comparative Material Material used for morphometric and meristic comparison Chondrostoma angorense: IFC-ESUF 03-1501, 32, 80— 174 mm SL; Turkey: Eskisehir prov.: Porsuk River about 2 km west of Yortkkirka, 39°36'00"N, 30°25'09"E. — IFC-ESUF 03-1502, 11, 35-162 mm SL; Turkey: Eskise- hir prov.: stream Akin 0.5 km south of Akin, 39°20'02"N, zse.pensoft.net 30°30'59"E. — IFC-ESUF 03-1503, 2, 245-300 mm SL; Turkey: Kutahya prov.: stream Emet about 10 km north of E&ri6z, 39°28'10"N, 29°15'17"E. — IFC-ESUF 03-1537, 23, 151-216 mm SL; Turkey: Kitahya prov.: stream Yanikburnu about 25 km east of Dursunbey, 39°33'04"N, 28°56'55"E. — IFC-ESUF 03-1538, 3, 137-185 mm SL; Turkey: Balikesir prov.: stream Bigadi¢ west of Biga- dig, 39°23'48"N, 28°04'50"E. — IFC-ESUF 03-1549, 1, 264 mm SL; Turkey: Afyonkarahisar prov.: stream Kali about 15 km west of Cay, 38°32'28"N, 30°50'41"E. Chondrostoma_ beysehirense: IFC-ESUF 03-1505, 16, 156-251 mm SL; Turkey: Konya prov.: Beysehir Lake about 20 km south of Sarkikaraagac, 37°52'42"N, 31°20'46"E. Chondrostoma fahirae: IFC-ESUF 03-1512, 36, 60-127 mm SL, Turkey: Burdur prov.: Baspinar Spring about 13 km south of Tefenni, 37°11'08"N, 29°45'16"E. — IFC-ESUF 03-1551, 1,92 mm SL, Turkey: Burdur prov.: Dalaman River about 4 km north of Yusuf¢a, 37°13'37"N, 29° S75 Es Chondrostoma holmwoodii: 1FC-ESUF 03-1513, 19, 68-160 mm SL; Turkey: Manisa prov.: Gediz River at Derbent, 38°46'37"N, 29°12'41"E. — IFC-ESUF 03-1514, 7, 58-118 mm SL; Turkey: Manisa prov.: Gediz River about 16 km east of Kula, 38°35'46"N, 28°48'30"E. — IFC-ESUF 03-1515, 1, 112 mm SL; Turkey: Manisa prov.: Gediz River about 15 km north of Kula, 38°40'08"N, 28°36'14"E. —IFC-ESUF 03-1516, 1, 145 mm SL; Turkey: Manisa prov.: Gediz River about 5 km east of Gélmar- mara, 38°42'08"N, 27°58'10"E. — IFC-ESUF 03-1517, 3, Zoosyst. Evol. 97 (1) 2021, 235-248 85-102 mm SL; Turkey: Izmir prov.: Gediz River about 8 km east of Menemen, 38°37'42"N, 27°10'41"E. Chondrostoma meandrense: IFC-ESUF 03-1519, 45, 120-209 mm SL, Turkey: Denizli prov.: Isikli Spring, 38°19'19"N, 29°S1'10"E. — IFC-ESUF 03-1522, 19, 96-151 mm SL, Turkey: Denizli prov.: streem Kufi about 4 km north of Isikli, 38°21'48"N, 29°50'56"E. — IFC-ES- UF 03-1523, 20, 110-219 mm SL, Turkey: Afyonkara- hisar prov.: Stream Karasandikli 0.5 km east of Karas- andikli, 38°31'40"N, 30°10'39"E. — IFC-ESUF 03-1525, 4, 65-138 mm SL, Turkey: Afyonkarahisar prov.: Sucikan Spring 0.5 km east of Dinar, 38°04'14"N, 30°10'38"E. — IFC-ESUF 03-1561, 21, 50-158 mm SL, Turkey: Den- izli prov.: Buyuik Menderes River about 2 km west of Citak, 38°09'23"N, 29°38'24"E. — IFC-ESUF 03-1562, 3, 125-154 mm SL, Turkey: Denizli prov.: Biiytik Men- deres River about | km north of Hancalar, 38°07'54"N, 29223'19"E. Chondrostoma turnai: IFC-ESUF 03-1524, 44, 75-— 210 mm SL; Turkey: Aydin prov.: stream Cine about 8 km south of Aydin, 37°45'43"N, 27°50'12"E. — IFC-ES- UF 03-1563, 1, 145 mm SL; Turkey: Denizli prov.: Cin- dere reservoir about 8 km south of Giiney, 38°05'40"N, 29°01'32"E. — IFC-ESUF 03-1564, 3, 92-99 mm SL; Turkey: Denizli prov.: Yenicekent DSI Pomp about 3 km east of Yenicekent, 38°02'16"N, 28°57'47"E. — IFC-ES- UF 03-1565, 15, 113-175 mm SL; Turkey: Aydin prov.: Akcay Stream about 3 km east of Sirma, 37°36'18"N, 28°29'34"E. — IFC-ESUF 03-1569, 1, 239 mm SL; Tur- key: Denizli prov.: Vali Recep Yazicioglu reservoir about 3 km east of Denizli, 37°46'14"N, 29°07'39"E. Material used in molecular genetic analysis Chondrostoma angorense: IF C-ESUF DNA-03-1502, 1; Turkey: Eskisehir prov.: stream Akin 0.5 km south of Akin, 39°20'02"N, 30°30'59"E (GenBank accession number: MT387066). — IFC-ESUF DNA-03-1501, 3; Turkey: Eskisehir prov.: Porsuk River about 2 km west of Yortikkirka, 39°36'00"N, 30°25'09"E (Gen- Bank accession number: MT387112—MT387114). FFR DNA CHS5-6, 2; Turkey: Kizilcahamam, Sakarya River drainage, (GenBank accession number: MW719604, MW719605). IFC-ESUF DNA-03-1500, 2; Turkey: Si- vas prov.: Kizilirmak River about 3 km east of Zara, 39°S4'27"N, 37°48'25"E (GenBank accession number: MW719606, MW719607). FFR DNA CH18-19, 2; Turkey: Yerkoy, Delice stream, (GenBank accession number: MW719608, MW719609). Chondrostoma_ beysehirense: IFC-ESUF DNA-03- 1505, 2; Turkey: Konya prov.: Beysehir Lake about 20 km south of Sarkikaraagac, 37°52'42"N, 31°20'46"E (GenBank accession number: MT387079—-MT387080). IFC-ESUF DNA-03-1505, 1; Turkey: Konya Prov.: Beysehir, Beysehir Lake, 37°55'43"N, 31°21'24"E (Gen- Bank accession number: MW719597). Chondrostoma fahirae: IFC-ESUF DNA-03-1512, 1; Turkey: Burdur prov.: Baspinar Spring about 13 km south 247 of Tefenni, 37°11'08"N, 29°45'16"E (GenBank accession number: MT387128). Chondrostoma holmwoodii: IFC-ESUF DNA-03- 1513, 1; Turkey: Manisa prov.: Gediz River at Derbent, 38°46'37"N, 29°12'41"E (GenBank accession number: MT387099). — IFC-ESUF DNA-03-1516, 1; Turkey: Manisa prov.: Gediz River about 5 km east of G6lmarma- ra, 38°42'08"N, 27°58'10"E (GenBank accession num- ber: MT387078). IFC-ESUF DNA-03-1513, 3; Turkey: Derbent Bridge, Manisa Prov.: Gediz River drainage, 38°46'40"N, 29°12'45"E (GenBank accession number: MW719598—MW719600). Chondrostoma meandrense: |FC-ESUF DNA-03- 1519, 4; Turkey: Denizli prov.: Isikli Spring, 38°19'19"N, 29°51'10"E (GenBank accession number: MT387085-— MT387088). IFC-ESUF DNA-03-1519, 2; Turkey: Isikh Village, Denizli Prov.: Isikli Spring, Buytk Menderes River drainage, 38°19'21"N, 29°51'07”E (GenBank ac- cession number: MW719591—MW719592). IFC-ESUF DNA-03-1525, 3, Turkey: Afyonkarahisar prov.: Sugikan Spring, Buyuk Menderes River east of Dinar, 38°04'14"N, 30°10'38"E (GenBank accession number: MW719593- MW719595). FFR DNA CH13, 1; Turkey: Akc¢ay, Biytik Menderes River drainage (GenBank accession number: MW719596). Chondrostoma turnai: IFC-ESUF 03-1557, 5; Tur- key: Aydin prov.: Stream Cine about 8 km south of Aydin, 37°45'43"N, 27°50'12"E. IFC-ESUF DNA-03-1524, 3; Turkey: Ciftlikburnu Village, Aydin Prov.: Cine Stream, Buytk Menderes River, 37°42'52"N, 27°50'04"E (Gen- Bank accession number: MW719601—MW719602). Acknowledgements This study was supported by a grant from the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: KBAG-111T900). 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