Zoosyst. Evol. 99 (1) 2023, 1-13 | DO! 10.3897/zse.99.91275 yee BERLIN Taxonomic review of the Chondrostoma (Ieleostei, Leuciscidae) species from inland waters of Turkey: an integrative approach Fahrettin Kiictik!, Yilmaz Ciftci*, Salim Serkan Giiclii!, Ayse Giil Mutlu°, Davut Turan* Isparta University of Applied Sciences, Egirdir Fisheries Faculty, 32200 Isparta, Turkey Ordu University, Fatsa Marine Science Faculty, 52400 Ordu, Turkey Burdur Mehmet Akif Ersoy University, Faculty of Science and Literature, Department of Molecular Biology and Genetics, 15100 Burdur, Turkey BB W MY fF Recep Tayyip Erdogan University, Fisheries Faculty, 53000 Rize, Turkey https://zoobank. org/BB27D943-CA 3B-45 78-98 55-S59A 39BE2E397 Corresponding author: Fahrettin Kiictik (fahrettinkucuk@isparta.edu.tr) Academic editor: Nicolas Hubert # Received 4 August 2022 Accepted 8 September 2022 @ Published 6 January 2023 Abstract The genus Chondrostoma (Leuciscidae: Leuciscinae), composed of small to medium-sized fish with a scraper feeding characteristic, is distributed in the West and Middle East, Caucasus, Europe and Northern Mediterranean drainages. This genus spreads across Anatolia and Thrace, with the exception of Goksu and Esen rivers in Turkey’s Mediterranean basin. It is also difficult to understand the systematics of Chondrostoma, which is complicated morphologically. Therefore, in this study, an identification key was made by evaluating external morphology, osteology (some jaw bones and 5" ceratobrachial) and molecular features together. A total of 13 valid species have been so far recorded from Turkish inland waters, among which are C. beysehirense, C. ceyhanensis, C. colchicum, C. cyri, C. holmwoodii, C. kinzelbachi, C. meandrense, C. nasus, C. regium, C. smyrnae, C. toros, C. turnai and C.vardarense. Our molecular data showed that C. angorense (Kizilirmak and Sakarya rivers) is a synonym of C. colchicum (Coruh and Yesilirmak rivers). In addition, C. angorense was morphologically similar to C. colchicum. Therefore, we explored the systematic position of C. vardarense (from Merig River) and C. nasus (from Simav River) in this study. Key Words Chondrostoma, freshwater fish, phylogeny, taxonomy Introduction The family Leuciscidae is one of the most diversified monophyletic families in the Holarctic Cypriniformes order and has a wide distribution area. Despite abundant research, there have still been different hypotheses con- cerning their speciation and intrafamilial diversification. They were classified into 7 subfamilies based on mor- phological characters, 6 subfamilies on osteology, and 6 subfamilies on molecular characters. These subfamilies are Pogonichthyinae, Leuciscinae, Plagopterinae, Lavini- inae, Phoxininae and Pseudaspininae. The family consists of 358 taxa, according to molecular phylogenetic classifi- cation (Schonhuth et al. 2018; Van der Laan et al. 2020). Many studies reported that Chondrostoma (Leuciscidae: Leuciscinae) shows a wide geographical distribution across Europe and Asia. It is evident throughout Europe: from the continent’s eastern border to the Atlantic coast, from the southern Mediterranean to the Baltic Sea, the Danube River basin and the Thrace region, and through- out Asia: all of Anatolia, the Caucasus and the Middle East, the Tigris-Euphrates basins, the Caspian Sea basin of Iran, Esfahan and the Kor rivers basins (Esmaeili et al. 2014; Coad 2017; Eagderi et al. 2017; Kiicuk et al. 2017; Guclt et al. 2018). A recent molecular phylogenetic study showed that the genus consisted of two lineage groups, the first of which was the Mediterranean species and the other was the Danube and Mesopotamian species (Durand et al. 2003). Robalo et al. (2007) determined the phylogeny of Copyright Kucuk, F. 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. 2 Chondrostoma using molecular data (mitochondrial gene and nuclear actin gene). It has been reported, however, that rasping during feeding may change mouth structure, resulting in morphology-based misidentification. Also, some bones (jaw, ethmoid, basioccipital and pharynge- al etc.) were reported to be sinomorphic (Robalo et al. (2007). Kuctik et al. (2017) which suggested that oste- ology and molecular data could be more suitable in the clarification of Chondrostoma systematics than classical morphological data. Ciftci et al. (2020) investigated the phylogeny of pre- viously described Chondrostoma species in Turkish in- land waters. Their study found that Regium and Nasus groups consisted of inland waters of Turkey. The first group represents the species in the Caucasus, Black Sea region and Marmara region and, in the Aegean and Cen- tral Anatolian basin, while the second group represents species dispersed in the eastern Mediterranean rivers and Tigris-Euphrates rivers basin. In addition, C. nasus was recorded from the Simav River (Susurluk basin). On the other hand, Chondrostoma fahirae was not included in all molecular studies (Cift¢i et al. 2020). In the latest study (Turan et al. 2021), C. fahirae was re-identified from the Dalaman River basin in south western Turkey as the spe- cies Turcichondrostoma fahirae. As mentioned above, Chondrostoma species, which are distributed in the inland waters of Anatolia and Thrace, have not been fully addressed until recently due to their complex morphology and lack of distinctive taxonomical characters. In this study, we gathered the comprehensive external morphology, osteology (some jaw bones and 5" ceratobrachial) and molecular data on the genus. In ad- dition to traditional morphology, we classified the genus Chondrostoma based on the osteology (premaxilla, max- illa, dentary and fifth brachial gill arc) and molecular data (16S rRNA, Cytb and COI). Furthermore, we addressed the external morphology and osteology of C. nasus, which is widely distributed throughout Europe and recorded from Simav River (Geiger et al. 2014) and of C. vardarense, which was already known from Meri¢ (Evros) River. Material and methods The care of experimental animals was consistent with the Republic of Turkey animal welfare laws, guidelines and policies approved by Sileyman Demirel University Local Ethics Committee for Animal Experiments (permit refer- ence number 2011/6/5). Fish samples were caught with electroshock and fishing nets from all regions of Turkey (Fig. 1). After anaesthesia (anesthesia), fishes were fixed in 4% formaldehyde and stored in 70% ethanol or direct- ly fixed in 99% ethanol. 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 Frey- hof (2007). Standard length (SL) was measured from the tip of the snout to the posterior extremity of the hypural zse.pensoft.net Kucuk, F. et al.: Review of Chondrostoma in Turkey complex. 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. The last two branched rays articulating on a single ptery- giophore in the dorsal and anal fins were counted as “1/2”. The simple dorsal- and anal-fin rays were not counted since the anterior-most rays are deeply embedded. For osteological preparations, one specimen of a Regi- um group (C. regium, 182.5 mm from Tigris River), two specimens Nasus group (C. nasus, 181.2 mm SL from Si- mav River and C. meandrense, 183.8 mm SL from Isikli Lake spring) were cleared and stained with alizarin red S, according to the protocol of Taylor and van Dyke (1985). Osteology of C. meandrense from Turan et al. (2021), C. smyrnae and C. turnai from Kuck et al. (2021). The specimens were studied using a stereomicroscope (Nikon SMZ1500), and photos taken with a digital machine with a glycerol bath. The nomenclature of the skeletal ele- ments was followed Bogutskaya (1996). Vertebral counts were obtained from radiographs and counted as total, pre-dorsal, abdominal and caudal vertebrae following Naseka (1996). Abbreviations used SL, standard length; HL, head length; BI, Bayesian In- ference; ML, Maximum Likelihood; mt, mitochondrial. Collection codes: IFC-ESUF, Inland Fishes Collection, E&irdir Fisheries Faculty of Isparta University of Applied Sciences; FSJF, Fischsammlung J. Freyhof, Berlin. Molecular analyses Total DNA extraction was carried out from ethanol-pre- served tissue samples of each specimen using Invitrogen PureLink ® Genomic DNA Mini Kit following the man- ufacturer’s protocols, and stored at —20 °C prior to use. The mitochondrial cytochrome b (Cytb) 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) prim- ers and sequenced with Forward (5’°— AGG CGG CTT CTC AGT AGA CA -3’) and Reverse (5°— AGA AAT TTT GTC GGC GTC TG -3’) internal primers (Cift¢i et al. 2020). Partial sequence of mitochondrial 16S ribo- somal RNA gene (577 bp) was amplified using the For- ward (5° — AAG CCT CGC CTG TTT ACC AA - 3’) and Reverse (5° —- CTG AAC TCA GAT CAC GTA GG - 3’) (Robalo et al. 2007) primer pairs. The PCR reaction was carried out in a final volume of 50 ul using 25 ul of PCR Master Mix (2X) (Promega Corporation, Madison, WI, USA), 5 ul of template DNA (25-50 ng/uL), 2 ul each of forward and reverse primers (10 pM of each primer), and Zoosyst. Evol. 99 (1) 2023, 1-13 ddH20 to the total volume. PCR amplifications were per- formed using the following protocol: initial denaturation at 94 °C for 1 min, followed by denaturation at 94 °C (1 min), annealing at 60 °C (55 °C for 16S rRNA) (30 s) and primer elongation at 72 °C (1.5 min for Cytb and 1.0 min for 16S rRNA), repeated for 35 cycles, a final elongation cycle at 72 °C for 5 min, and cooling to 4 °C. Successfully amplified PCR products were purified prior to sequencing using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to protocol sup- plied by the manufacturer. The purified DNA fragments were sent to Macrogen (Macrogen Inc., Seoul, Korea; www.macrogen.com) for sequencing of both strands. Se- quences were assembled and edited in BIOEDIT (Hall 1999) and aligned with CLUSTAL W (Thompson, Hig- gins and Gibson 1994) as implemented in BIOEDIT. All sequences of Chondrostoma species were deposited in GenBank under accession numbers given in Suppl. ma- terial 1: Table S1. For the phylogenetic analyses, three data sets were used, 577 bp fragment of 16S rRNA gene, 1140 bp of Cytb and 652 bp fragment of COI sequences. 16S rRNA, Cytb and COI sequences were aligned with the previous sequences from GenBank (Suppl. material 1: Table S1) with the Clustal W algorithm (Thompson et al. 1994 avail- able 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 inspected and corrected visually. To determine whether the DNA sequence datasets (16S rRNA, Cytb and COI) were congruent, an incongruence length difference (ILD) test (Farris et al. 1995) imple- mented as partition homogeneity test in PAUP 4.0b10 (Swofford 1998) was performed using 1000 partition rep- licates, each comprising 100 random sequence addition replicates, and TBR branch swapping. Invariant charac- ters were removed from the data sets prior to performing the ILD test (Cunningham 1997). The partition homoge- neity test revealed no significant difference (P value = 1 - (599/1000) = 0.401) between three data partitions (16S rRNA, Cytb and COI), indicating that the three data sets could be combined for analysis. 16S rRNA, Cytb and COI sequences of Turcichondrostoma fahirae and Telestes souffia were also included in the phylogenetic analysis as outgroup (Suppl. material 1: Table S1). To assess the best fitting nucleotide substitution mod- el for combined data set of Chondrostoma species, we used the jModelTest v.0.1 (Guindon and Gascuel 2003; Posada 2008), based on hierarchical series of likelihood ratio tests. GIR+I+G substitution model (gamma shape = 0.7890; p-inv = 0.6520) was selected as the best fit to the genus Chondrostoma data set by the lowest AIC score in jModeltest 0.1.1 (Posada 2008) and therefore, we used this model for the subsequent phylogenetic analysis. Phylogenetic trees were generated using Bayesian infer- ence (BI), and maximum likelihood (ML) to determine the evolutionary relationships among haplotypes. PhyML 3.0 (Guindon and Gascuel 2003) software was used for ML analyses. Bootstrap tests (Felsenstein 1985) were per- formed with 1000 pseudo replicates for ML analyses with the same software used for phylogenies. Bayesian infer- ences of Chondrostoma phylogeny were performed as im- plemented in MrBayes 3.2 (Ronquist et al. 2012) software. Four independent Markov Monte-Carlo coupled chains were run with 10° generations and sampled every 100 Figure 1. Distribution of Chondrostoma species in inland waters of Turkey (1. C. colchicum — Akin Stream, Sakarya River; 2. C. colchicum — Kaizilirmak River; 3. C. colchicum — SuatUgurlu reservoir, Yesilirmak River; 4. C. colchicum — Coruh River; 5. C. beysehirense — Beysehir Lake; 6. C. ceyhanensis — Sir reservoir, Ceyhan River; 7. C. ceyhanensis — Seyhan reservior, Seyhan River; 8. C. cyri — Kura River; 9. C. cyri — Aras River; 10. C. holmwoodii — Gediz River; 11. C. kinzelbachi — Gélbasi Lake, Asi River; 12. C. meandrense — Isikli Spring, Biiyik Menderes River; 13. C. nasus — Simav Stream; 14. C. regium — Karakaya reservoir, Euphrates River; 15. C. regium — Ongozlii Bridge, Tigris River; 16. C. smyrnae — Tahtali reservoir; 17. C. toros — Goksu River; 18. C. turnai — Cine Stream, Birytik Menderes River; 19. C. vardarense — Merig River). zse.pensoft.net generations to yield tree topologies. The first 25% genera- tions were discarded as burn-in. FigTree v.1.4.4 (Rambaut 2018) was used to build constrained trees. A calculation of pairwise genetic distance among different species with Kimura 2-parameter (K2P; Kimura 1980) model was per- formed using MEGA X (Kumar et al. 2018). Results Morphology of Chondrostoma The body is typically fusiform, deep and somewhat cylin- drical. The mouth position inferior, mouth usually straight or slightly arched, but markedly arched in C. meandrense and C. cyri (Fig. 2a—d), the lower lip is covered with a Kucuk, F. et al.: Review of Chondrostoma in Turkey horny layer. The pre-dorsal distance is between 47.1— 55.0/SL, pelvic-fin origin, usually in front of the dorsal fin origin; pre-pelvic distance /SL ratio is 48.4—-55.0. Body depth markedly high, tts depth 19.3—28.4/SL, the caudal peduncle length/its depth ratio is between 1.73—1.92. The caudal-fin deeply forked and the lobes are pointed (some species have a black band on the outer margin of the cau- dal fin); eye diameter relatively large, the eye diameter/HL ratio is 17.5—25.2 (Table 1). A complete lateral line with 44-74 total scales, 8-11 scale rows between the lateral line and the dorsal-fin origin, 4-7 scale rows between the lateral line and the pelvic fin-origin (Table 2). The scales are partially angular and arranged properly on the body, and rays are positioned anteriorly and posteriorly. The focus closer to the anterior edge of the scale. The angle between the focus and anterior margin varies by species. Figure 2. Mouth shape of Chondrostoma species (a. C. nasus; b. C. meandrense; ¢. C. regium; d. C.cyri). Table 1. Some Morphometric data of Chondrostoma species of Turkey. Species (%) min max X min. max C. colchicum (Sakarya) 2259) 24,598 23:0. A926 -550 C. colchicum (Kizilirmak) 21:8 2372 2207 - ANA 53:8 C. colchicum (Yesilirmak) ail e241 «219! 48-5 2:5 C. colchicum (Coruh) 22°06, “24:92 238 496 508 C. beysehirense (Beysehir) Di ste 252 BA» “AS67509 C. ceyhanensis (Ceyhan) 20:9. 24:52. ©2372," 43:5. 52:9 C. ceyhanensis (Seyhan) 20:1, ~245 "22 RS AB ly 28372 C. cyri (Aras) LO 36 21.8 —20;9% 47.17 6505 C. cyri (Kura) 19.0 21.2 20.0 46.8 50.0 C. holmwoodii (Gediz) 22.0 248 23.6 50.7 54.0 C. kinzelbachi (Asi) 19,7- #22:2. 207 “ASF © 53:3 C. meandrense (B.Menderes) 23.0 25.5 24.1 49.6 52.1 C. nasus (Simav) 234° 25.9 2A 550225 52.7 C. nasus (Danube) 254s 225 Ge e241? S502 e527 C. nasus (Rhine) 235) 2620 24:3 “S052 7525 C. regium (Tigris) 19:8" 2275 —2h.3 “4c 507 C. regium (Euphrates) 196 219 21.6 47.0 50.1 C. smyrnae (Tahtall) 242° 284 25.90 49;0" 5222 C. toros (Goksu) 23.3. 264° 24.5 AGO 53: C. turnai (Cine) 22:9; 20 24-6: 50:07 54.5 C.vardarense (Meric) 227 (25-0 244) ‘473829 zse.pensoft.net Body depth / SL Pre-dorsal / SL (%) Pre-pelvic / SL (%) pel. “Biks 49.4 50.1 BLT “50,7 D2. » 0 50/8) . Dla 9125. O01 50.1 49.9 Eye diameter / HL Caudal peduncle (%) length / depth (%) max X min max x 2:2 1eO9LL VIZ, “179 220 VFA G2 ds 194 23.0 170s 22182) 1e9T 1.3 1:67 1.88" 175 20.0" UGS ML Ale 1-95 1k 86 SOs =. 16.9 °.23.8°- 22:3 sl.61, «194° 1:80 50.2 53.4 19.2 23.4 1.57 1.90 1.84 52.3 20:0 p23 22a do 2020 194. OL.6 O6- = 73:0) 1.68 2.03 1.94 54.2 17.6 22.4 1.62 1.94 1.78 51.2 18.4 22.2 L80 2703" 1,92 OaAS BA.3% 62021, 23.6 L700 1292" 180 O353 LBs 28 1.64 1.81 1.73 03:3 18.2 21.0 1.64 1.81 1.73 he Bs 13,06" 2st Vise l.G2 "1020 49.0 52.1 19.0 23.8 LZ POG; = 1085 49.2 52.1 die Brews os 00 1.20; »2:05- 1:81 54,6 S3tl 27:6" 22:8 1.65 2.04 1.85 59.0 19.5 24.9 1.64 1.88 1.74 53.8 52.7 20.1 24.7 1.45 1.85 1.74 he Me Lie «2e.9 159 1.94 .VA7 min max X min HAS JSS eee 55.2 18.7 53:2" 525) 19'5 49.6 51.4 15.8 48.9 51.0 18:3 O19 50.9 50.7 50.6 48.4 50.9 50.9 523 Zoosyst. Evol. 99 (1) 2023, 1-13 Osteology of Chondrostoma In the classification of Chondrostoma species, some jaw bones (premaxilla, maxilla and dentary), hyomandibular bone, the 5" ceratobrachial arc and pharyngeal teeth, and the numbers of vertebrae were examined. The anterior part of the dentary medially curved at an angle of about 90 degrees or slightly contorted. There is a cavity on its outer surface where the keratin layer is found. This cavity is larger and deeper in species, a well-developed keratinized layer. The coronoid process is usually anteriorly inclined or slightly vertical unlike in C. meandrense. Premaxilla deep and without ascendant process, slightly concave for scraping, bottom edge sharp, pos- terior edge 1s thin and long; maxilla is deep and ascend- ing process is vertical (Fig. 3a—c). Hyomandibular bone is long-narrow or short-wide, it varies across species. Upper edge of 5" ceratobranchial enlarged and axe- shaped. Pharyngeal teeth knife-like in one row; 5-5, 5-6, 6-6, 6-7, 7-7; some species slightly serrated and slightly hooked at tip (Fig. 4a—c); in C. colchicum, C. cyri and C. meandrense usually 5-5 or 5-6 (Table 2). The operculum bone is square-shaped; the preopercu- lum is “L” shaped and interoperculum is short, where- as the suboperculum is narrow and long. There are 20-39 gill rakers on first gill arch, vertebrae formula: 42—49:25—29+17-21, the lowest number of vertebrae are in C. meandrense (42) and the highest in C. regium (49) (Table 2). Table 2. Meristic characters of Chondrostoma species in Turkey. a * g a Species = F A ® ‘bo A ® Sie eee) ee S83 Ra oo C. colchicum (Sakarya) 59-68 9-10 5 C. colchicum (Kizilirmak) 59-66 9-10 5 C. colchicum (Yesilirmak) 59-62 9 4-5 C. colchicum (Coruh) 57-63 9-10 4-5 C. beysehirense (Beysehir) 60-71 9-11 5-6 C. cyri (Aras) 54-59 8 4 C. cyri (Kura) 56-67 8-9 4-5 C. holmwoodii (Gediz) 60-66 9-11 5-7 C. ceyhanensis (Seyhan) 59-68 9-1 4-6 C. ceyhanensis(Ceyhan) 59-66 10-11 4-6 C. kinzelbachi (Golbas)) 59-73 10-11 5-6 C. meandrense (B.menderes) 55-60 8-10 4-5 C. regium (Tigris) 63-69 9-11 4-6 C. regium (Euphrates) 62-73 10-11 4-6 C. nasus (Simav) 64-72 9-10 5 C. nasus (Danube) 53-60 8-9 3-4 C. nasus (Rhine) 57-63 8-9 5-6 C. smyrnae (Tahtali) 48-53 8-9 4 C. toros (GOksu) 56-64 10-11 4-6 C. turnai (Cine) 44-51 8-9/3-4 3-4 C.vardarense (Meric) 57-64 8-9/4-5 4-5 fin origin External morphology and osteology of Chondrostoma did not support the divergence of two lineage groups, Na- sus and Regium, based on molecular (16S rRNA, Cytb and COI) data. However, with a few exceptions in all Regium group species (C. regium, C. ceyhanensis, C. kinzelbachi, C. toros and C. vardarense); the mouth straight or slight- ly arched (Fig. 2c), the keratinised layer in the lower jaw and the cavity in the anterior edge of the dentary well-de- veloped, coronoid process of dentary slightly anteriorly in- clined (Fig. 3c). In the Nasus lineage group, the mouth is slightly arched but straight in C. nasus (Fig. 2a, b, d) the ke- ratinised layer on the lower jaw slightly developed and den- tary coronoid process slightly vertical in C. beysehirense, C. cyri, C. meandrense, C. smyrnae and C. turnai, albeit ante- riorly inclined in C. colchicum and C. holmwoodii (Fig. 3b). C.fahirae, which was included inthe genus Chondrostoma by Freyhof and Ozulug (2015), was identified as Turcichon- drostoma fahirae (Turan et al. 2021). This new genus is clearly distinguished from other Chondrostoma species by their external morphology and osteology. 7’ fahirae also has reduced lateral line scales, gill rakers on first gill arch and pharyngeal teeth, 45-52, 9-14 and 5—5, respectively. Mouth arched, no keratinized layer on lower jaw; dentary short and its anterior edge not contorted, the cavity in the ante- rior edge of the dentary is slightly developed and coronoid prosses markedly vertical, with ascending process of pre- maxilla well developed and vertical, pharyngeal teeth ex- cept one row 5—5, thin-long and hooked (Turan et al. 2021), as are the genus A/burnus, Pseudophoxinus and Squalius, upper edge of 5" ceratobranchial not enlarged. 2 8 0 $ (he eS — eT) eo a < ro) <3 eg 8-9 9-11 22-25 6-5/6-6 46-47 9 9-11 20-25 6-6 47-48 8-9 9-11 22-26 6-5/6-6 44-46 8-9 9-10 24-26 5-5/6-6 46 8 8-10 33-39 6-7/7-7 45 8 9-10 20-24 6-5 45-46 8-9 9-10 21-24 6-5/6-6 45-46 8 9-10 21-24 6-6 45-46 8-9 9-10 25-29 5-6/6-6 46-48 8-9 9-11 24-28 6-6 47-48 8-9 10-11 32-34 6-6/7-7 46-47 8-9 9-10 23-28 6-5/6-6 42 8-10 10-12 25-32 6-6/7-6 46-47 9 10 27-30 6-6/7-6 48-49 8 9 24-29 6-5/7-6 46-48 8-10 9-11 27-31 6-6 48 8-9 9-10 30-31 6-6 48 7-8 8-10 19-23 5-6/5-5 43 8-9 9-11 26-29 6-6 47 8 9-10 22-2] 5-5/6-6 43 8 9-10 28-31 6-6 47 zse.pensoft.net Kucuk, F. et al.: Review of Chondrostoma in Turkey Figure 4. Hiyomandibular and pharyngeal teeth of some Chondrostoma species (a. C. nasus; b. C. meandrense; ¢. C. regium). Results of molecular analyses A total of 146 specimens from Anatolian Chondrosto- ma species were sequenced and deposited in GenBank with accession numbers ON796577—ON796688 for 16S rRNA and OL870982—OL871061 for Cytb gen regions. In total, 77 published cyt b sequences and 63 published COI sequences from GenBank were downloaded (Suppl. material 1: Table S1). The 16S rRNA, Cytb and COI se- quences of each species were concatenated to generate a combined data set. The final data set had a total of 2369 (for 16S rRNA: 577 bp, Cytb: 1140 bp and COI: 652 bp) nucleotide positions without insertion, deletion, gap and stop codon. Phylogenetic relationships between sequenc- es were reconstructed for the combined data set using the BI and ML method. The phylogenetic trees (BI and ML) showed congruent topologies with high posterior proba- bility (PP) and bootstrap (BP) values ranging from 0.51 tol.0 and 60.5% to 100%, respectively and were consis- zse.pensoft.net tently divided into two major lineages (Regium and Nasus lineages) (Fig. 5). Regium lineage at the base of the tree represents sixteen localities (locality codes: A, Ma, Q, U, Z, FIR, E, P, GO, D, H, J, M, Sr, BE and ME) from the Tigris and Euphrates rivers to Ceyhan, Seyhan, Berdan and Goksu rivers, the Orontes River and its main branch- es. Nasus lineages consists of seventeen localities (locality codes: Ak, V, Y, CO, KI, YE, I, S, T, EK O, R, L, K, C, B and G) from rivers draining into the Sea of Marmara, the southwest of the Anatolian Peninsula, the Black Sea Basin and Caucasian basin from north-eastern Turkey. Within Regium lineage, there were three well-supported clades. The first clade (CI) contains species (C. vardarense) from the Meric¢ River (Evros), the longest river flowing entire- ly within the borders of the Balkan region, eventually emptying into the Aegean Sea near Enez in Turkey. The second clade (CII) includes the monotypic species (C. re- gium) from the Tigris and Euphrates River basins in the eastern part of Turkey. The third clade (CHI) comprises Zoosyst. Evol. 99 (1) 2023, 1-13 the species of the eastern Mediterranean represented by three monophyletic, easily distinguished subclades, name- ly C. ceyhanensis, C. kinzelbachi and C. toros. Within the Nasus lineage there were four clades. The first clade (CIV) includes C. beysehirense from Lake Beysehir in the prov- inces of Isparta and Konya in south-western Turkey. The second clade (CV) includes C. cyri from Lake Cildir and the tributaries of the Kura River in Ardahan Province. The third clade (CVI) consisted of three subclades of Aegean species; the first subclade included only C. holmwoodii from Lake Marmara and Gediz River, the second subclade consisted of C. smyrnae from the Sagal Stream, which was recently described (Kuictik et al. 2021), and the third sub- clade was formed only by C.turnai from the Cine Stream. The last clade (CVII) included three species, C. colchicum, C. meandrense and C. nasus, from rivers draining into the Sea of Marmara, the southwest of the Anatolian Peninsula and the Black Sea basin. For the combined mitochondrial genes (16S rRNA, Cytb and COI), interspecific and intraspecies genetic dis- tance values (K2P) are given in Table 3. The molecular analysis in this study shows that the interspecific genetic distances between the analysed Chondrostoma species ranged from 0.866% (between C. colchicum and C. me- andrense) to 3.911% (between C. toros and C. smyrnae). On the other hand, intraspecific genetic distances within Chondrostoma species ranged from 0.015% for C. holm- woodii and 0.664% for C. kinzelbachi (Table 3). Anatolia, for example, genus Pseudophoxinus (Hrbek et al. 2002, 2004). The Chondrostoma species showing allopatric spe- ciation in inland waters of Turkey have diversified in all river basins (except between the Goksu and Esen rivers in the Mediterranean region) (Fig. 1). Of these species, C. regium is the most widely distributed species, includ- ing the Tigris, Euphrates basins and Sinnep Stream (up- per basin of the Quveik River, Northern Syria). There are three Chondrostoma species in the Eastern Mediter- ranean rivers’ basin; these are C. kinzelbachi (Asi River and Balikli Lake), C. ceyhanensis (Ceyhan, Seyhan and Berdan rivers) and C. toros (Géksu River) (Fig. 6). C. angorense was described by Elvira (1997) from Sa- karya River. The same researcher also reported this species from Kizilirmak and Yesilirmak rivers. This species is dis- tributed between Sakarya and Kizilirmak rivers drainage in Black Sea basin. Therefore, it is possible to see morpholog- ical variations among the populations of C. angorense; for example, the number of the lateral line scales (57—68), the number of gill rakers on first gill arch (20—26) (Table 2), and the development of the keratinised layer in the lower jaw. We compared C. angorense specimens from Sakarya (Porsuk River and Akin Stream) and Kuzilirmak rivers with C. colchicum. No morphological or genetic differ- ences were found between these populations (Cift¢i et al. 2020). The number of scales on the lateral line (59-68 in Sakarya, 59-66 in Kizilirmak, 59-62 in Yesilirmak and Table 3. Average intergeneric and intrageneric distance of Cyt b, 16S rRNA and COI combined data set for Chondrostoma species based on 1,000 bootstrap replications using K2P distance method; values in lower left cells = percent difference among taxa and diagonal = percent difference within taxa. N 1 2 3 4 5 1 C. colchicum 16 es 2 C.meandrense 9 0.866 0.162 3 C. nasus 10 1.051 1.041 eee 4 C. turnai 7 1.868 1.942 1.836 0.228 5 C. smyrnae 4 1.922 2.004 1.975 1.574 ee 6 C. holmwoodii 8 1.740 1.950 2.042 2.002 2.057 7 C.cyri 8. An7639 806850 2.3154 2.432. 8 C.beysehirense 2 1.731 1.685 1.644 2.160 2.124 9 C.ceyhanensis 16 2.852 2.394 2.783 3.588 3.489 10 C. toros Mew Si LUe 32872 3.30" 13967 «2 329.14. 11 C. kinzelbachi 3 3023, 82.751" eS. 131884: 23.665 12 C. regium 22 2.642 2.643 2.673 3.232 3.224 13 C. vardarense 3 3.060 3.196 3.282 3.849 3.678 6 7 8 9 10 11 12 13 0.015 2.285 See 2.282 1.867 0.4091 3.509 2.903 2.6604 0.464 3.635 3.272 3.174 1.792 Boe 3.523 3.041 3.001 1.685 1.948 0.664 2.981 2.562 2.467 1.777 2.276 1.932 0.438 3.527 3.366 3.439 3.015 3.187 3.158 2.845 0.078 Zoogeography of Chondrostoma species in the inland water of Turkey Anatolia is well isolated from its surroundings. There- fore, it has complex lentic, lotic systems and endorheic basins for the diversification of fish taxa. The Anatolian fish species are Tethys Sea origins, Glacier relicts, Cen- tral and Western Europe origins, Central and Western Asia origins, Mesopotamia origins, Sarmatian Sea origins and have entered Inland Waters from the Seas (Kuru et al. 2014), but the diversification centre of many species is 57-63 in Coruh), scales rows between lateral line and dorsal-fin origin (9-10 in Sakarya, Kizilirmak and Coruh, 9 in Yesilirmak) and scales rows between lateral line and pelvic-fin origin (5 in Sakarya and Kizilirmak, 4—5 in Yesilirmak and Coruh), gill rakers on first gill arch (20- 25 in Kizilirmak and Sakarya, 22—26 in Yesilirmak and Coruh) were similar. However, the number of vertebrae was slightly different (46-48 in Kizilirmak and Sakarya, and 4446 in Yesilirmak and Coruh) (Table 2). Further- more, it has been reported that any differentiation did not exist between these populations at molecular level. There- zse.pensoft.net 0.97/97.2 0.72/76.1 1.0/99.8] 0.85/89.4 110/97.7 0.95/82.3 1.0/94.8 1]0/94.1 0.51/60.5 Nasus Lineage 1.0/100 1.0/99.4 1.0/99.5 1.0/89 0.90/68.2 Regium Lineage 0.005 1.0/94.8 .0/95.8 Kucuk, F. et al.: Review of Chondrostoma in Turkey N796580/ MT. ute MWw719606 C. colchicum ON7965 81/ HM560078/ MW719609 ON796582/ OL871000/ MW719607 ON796583/ OL871001/ MW719610 ON796584/ OL871002/ MW719611 ON796585/ MT387126/ MWS911280 ON796592/ MT387113 ON796579/ Mise71ii thiw719608 ON796588/' M1387 N7a6508/4 /7387087/ W719592 Ob NT aeeea) O87 1004) MWT19s44 ON796597/ MT387088 ae MW911281 ON 796583) TAEESEOT ON796596/ OL871007, ON796594/ Miao) ON 7965094 TOO! 1/ OL871012, Kiso3088 ONT26603/ ee KJS52788 CVIl Cc. meandrense ON796608/ OL871017 QN726604/ MT387106/ KJ552997 ON796609/ MT387106/ - ON rear tad M37 10/ KJS553248 a rw M73: eCH 12 ae r ON796602/ MT387104, N796605/ MT38 1097 ass3171 ON796607/ OL871016/ - ON796612/ MT387091/ MW719601 ON796614/ MT387093/ MW719603 ON796618/ OL871033/ KJ553026 ONSeeta Mirae 002 MW719602 38709 ai 88 C. nasus 1.0/100 Cc. turnai 1.0/100 ON79662 O/T 38705 [Mire 2M MT387057/ MW729824 c. smyrnae ON Bea? 796628/ OL871046/ KIS52962 29 ee K1552922 CVi 1.0/100 C. holmwoodii Np56623 1387078) MW719598 ON7966337 aE /KU729242 ON796637, ON796638/ MT387072 96631/ mira 7069/ MW564534 96636/ atts 09/ - TaB970 MW564465 6635/ 01871100/ - ON796639/ Mr387079/ Kl ON796640/ MT387080/ MW71aso7 Oe C. cyri eysehirense mcliVv ON796647/ ae «De 285 Bj ONaeeaar att Mar /- 87118/ MW911286 ON7 Faecal f Mr3a7074) KJ553283 ON796646/ MT38 O72 Ks 2768 6682 ae tied 090/ - oh ON796644/ MT387089/ KJSS3017 on7aee437 Att iMngarGaark K553158 ON79665 reo POL8710e1 - 6643/ MT387083/ KISS3177 ON786655/ O18 3657) MT387100/ MW911283 1.0/100 F on796658/ mr387101/ MW911284 QN796699/ M1387081/ MW31 1283 ON796661, Mirs87079/ tise3133 ON796663/ MI387077/'K)583200 1.0/100 C. ceyhanensis Cill Ol On? Cc toros 1.0/100 C. kinzelbachi Cc. regium ON796673/ 387 096/ - cil ON796664/ MT387059/ - jis Ree T3871 ne) 669/ MT387063/ - -/ 3871 19/ - 6684/ MT387120/ - aia hE hee (a 10/100 ON796685/ M7387122/ 14553218 TPAasert34/ OSs K1553294 Ic. vardarense ONT 86687) 1387124, 252 aeeeR) MT387128/ AS09861) KI5S4711 Cl MWS112919 Qutgroup Figure 5. Bayesian inference tree of Chondrostoma species based on the combined data set. Bayesian and ML methods yielded identical topologies and so only the Bayesian tree is shown. The numbers above nodes are Bayesian posterior probabilities and maximum likelihood (ML) bootstrap values, respectively (those above 50% are shown). fore, Ciftci et al. (2020) proposed that C. colchicum spec- imens from Yesilirmak and Coruh rivers are similar to C. angorense (Sakarya and Kizilirmak). We recommend that this species is re-examined at morphological and genetic levels by collecting samples of all populations(Tuapse, Coruh, Yesilirmak, Kizilirmak and Sakarya rivers). Kura River is the type location of C. cyri, which is only distributed in the Aras and Kura rivers basin. In our study, two localities were sampled; these were the upper tributary of Kura River (Cakir Dere near Gole) and Lake Cildir (Aras basin). Kaya et al. (2020) report- ed from Lake Cildir, Kars Stream, B-20 Canal at Aralik (Aras basin) and Stream Cakir (Kura basin). However, the fossil record of C. cyri was found in the Erzurum region during the Pleistocene (1.55—1.0 Mya) period (Bohme and Ilg 2003). C. beysehirense is distributed across Beysehir Lake basin (Beysehir and Su&la lakes, zse.pensoft.net and Apa reservoir). This species is consumed in limit- ed quantities by the local people in the Beysehir region (Konya province) (Fig. 7). Four Chondrostoma species are distributed in the Ae- gean Region rivers (Bakircay, Gediz, Biytk Menderes and Tahtali reservoir, near Kuctk Menderes basin) of Turkey. C. holmwoodii is distributed in the Gediz and Bakircay rivers basin. This species differs from other Anatolian Chondrostoma species by the pale-pink thin band between the operculum and the caudal fin base. The lower basin of the Gediz River is highly polluted so that C. holmwoodii populations exist only in the upper basin of this river (upper region of Demirk6prti reservoir). On the other hand, Ilhan et al. (2020) reported that this spe- cles 1S widespread in all basins of Bakircay. Two species are diversified in Btiiywk Menderes, the largest river in the Aegean region. Of them, C. meandrense is found in the Zoosyst. Evol. 99 (1) 2023, 1-13 middle and upper river basin, and C. turnai in the Cine Stream (lower river basin). In previous studies, it was re- ported that only C. meandrense was distributed in this river basin (Elvira 1997; Fricke et al. 2007). However, Geiger et al. (2014) and our project results (TUBITAK, KBAG-111T900) led to a new unnamed species different from C.meandrense being recorded in the Buytk Men- deres River. The new species C. turnai, which was de- scribed by Giic¢lii et al. (2018), is known from the lower and middle branches of the Btiytk Menderes basin (Cine Stream, Akcay Stream near Nazilli, Yenicekent near Sarayk6oy) (Fig. 8). Figure 6. Eastern Mediterranean and South-eastern Anatolia Chondrostoma species (all Turkey); a. C. foros, IFC-ESUF 03- 1555, 142.76 mm SL; Mersin prov.: Hamamkéy Village, Mut, Goksu River; b. C. ceyvhanensis, IFC-ESUF 03-1556, 208.49 mm SL; Kahramanmaras prov.: Sir reservoir, Ceyhan River; ec. C. kinzelbachi 1FC-ESUF 03-1518, 214.6 mm SL; Hatay prov.: Lake Golbasi, Asi River; d. C. regium, IFC-ESUF 03-1533, 214.7 mm SL; Sivas prov.: Kangal, Euphrates River. Distribution area of C. nasus and C. vardarense in Turkey and its taxonomic status C. nasus has a wide distribution area that spans from the Black Sea basin to the southern Baltic Sea and southern North Sea. It has also been recorded as an invasive spe- cies or introduced in some streams in France, Italy and Slovenia, although it has not been recorded from Meri¢ River (Turkey and Greece) which originates from Eu- rope and flows into the Aegean Sea (Kottelat and Frey- hof 2007). However, C. nasus is distributed in Simav and Kocagay rivers (Susurluk River basin) in the Northern Aegean and Marmara regions (Geiger et al. 2014; Cift¢1 Figure 7. Central Anatolia, Marmara and Western Black Sea regions Chondrostoma species; a. C. beysehirense, IFC-ESUF 03-1505, 265.3 mm SL, Turkey: Konya prov.: Lake Beysehir; b. C. cyrii, IFC-ESUF 03-1511, 144.2 mm SL; Turkey: Kars prov.: Lake Cildir, Aras River; ec. C. colchicum, IFC-ESUF 03- 1501, 169.4 mm SL; Turkey: Eskisehir prov.: Porsuk Stream; d. C. nasus, IFC-ESUF 03-1536, 256.8 mm SL; Turkey: Kiitahya prov.: Yanikburnu Stream, Simav River; e. C. nasus FSJF-781, ZFMK-ICH 84668-94679, 221 mm SL; Germany: Rhine River. et al. 2020), and has an interesting zoogeographical dis- tribution pattern. We compared the morphological characters of C. na- sus specimens from Rhine (Germany) and Danube (Ro- mania) rivers with those of Simav River specimens. The Simav River specimens are distinguished from Danube River specimens by having more lateral line scales (64— 72, vs. 53-60), more scale rows between lateral line and pelvic-fin origin (4-5, mode 5, vs. 3-4, mode 4), a more slender body (body depth at dorsal-fin origin 22—26% SL, mean 24.0, vs. 25—28, mean 27), a more slender caudal peduncle (9—-11% SL, vs. 11-12), a longer snout (33-38% HL, vs. 28-32) and a smaller eye diameter (17—24% HL, vs. 25-27). In addition to the above-mentioned differenc- es, the Simav River specimens are distinguished from Danube River specimens by the shape of the head (the upper profile of the head is convex in interorbital space, vs. straight or slightly convex) and markedly concave at level of nostrils (vs. straight or slightly concave). The Si- mav River specimens are distinguished from Rhine Riv- er specimens by having more lateral line scales (64—72, zse.pensoft.net 10 Figure 8. Aegean and Thrace region Chondrostoma species (all Turkey); a. Chondrostoma smyrnae, IFC-ESUF 03-1567, 167 mm SL, Izmir prov.: Tahtali reservoir; b. C. turnai, IFC-ESUF 03- 1557, 197 mm SL, Aydin prov.: Cine Stream in the lower Biiyiik Menderes River; ¢. C. meandrense, IFC-ESUF 03-1519, 180.21 mm SL; Denizli prov.: Isikli Spring in the Upper Biiyitik Menderes watershed; d. C. holmwoodii, IFC-ESUF 03-1513, 149.5 mm SL; Manisa prov.: Gediz River; e. Chondrostoma vardarense, IFC-ES- UF 03-1534, 195 mm SL; Edirne prov.: Merig River. vs. 57-63), a longer head (22—26, mean 24.4% SL, vs. 21—23, mean 21.5), a more slender body (body depth at dorsal-fin origin 22—26% SL, mean 24.6, vs. 25—29, mean 27.3), a more slender caudal peduncle (caudal peduncle depth 9-11% SL, vs. 11-12), a narrow interorbital dis- tance (34-40% HL, mean 38.0, vs. 39-45, mean 41.6) and a smaller mouth gape (26-30, mean 29.4% HL, vs. 29-34, mean 30.2) (Tables 1, 2). C. nasus (Simav, Danube and Rhine rivers) is distin- guished from C. colchicum from Sakarya and Kizilirmak rivers by having more gill rakers on the outer side of the first gill arch (24-31, mode 27.8, vs. (20) 22—25, mode 23.5) and a wider mouth gape (24-34% HL, vs. 24—26). Moreover, the lower jaw in C. nasus is characterized by a well-developed keratinized edge (vs. slightly devel- oped) and slightly arched (vs. straight). In addition to the above-mentioned differences, it is distinguished from C. colchicum by having a deeper body (body depth at dor- sal fin origin 22-29% SL, mean 26.2, vs. 21-25, mean 22.9) and a slightly wider head (interorbital distance 34— 45% HL, mean 39.2 vs. 33-39, mean 35.3) (Tables 1, 2). zse.pensoft.net Kucuk, F. et al.: Review of Chondrostoma in Turkey Moreover, the molecular structure of the Meri¢ River samples has been compared with that of the Aoos River in north-western Greece, the Erzen River in central Albania, the Lepenac River in northern Macedonia, and the Angitis River in northern Greece, where the distribution of C. vardarense was established by the same researchers (Cift¢i et al. 2020). They found that the samples from the Meri¢ River were closer to the samples from the Angitis and Lep- enac rivers (Aegean region) than the samples from the Aoos and Erzen rivers (Adriatic region). Moreover, intraspecific and interspecific genetic distance estimates do not support the current classification of C. vardarense. Meri¢ River population with a very high intraspecies heterogeneity was recorded as Chondrostoma sp. (Cift¢i et al. 2020). How- ever, we assumed that this species could be C. vardarense since a new taxonomic revision is currently unavailable. Vardar (Axios) River (Aegean Sea region) is the type locality of C. vardarense. However, this species has been documented from the rivers Pinios, Aliakmon, Strymon, Nestos and Meri¢ (Crivelli 2006). In addition, some stud- ies reported that this species inhabits the Aoos River, which is a part of the Adriatic Sea basin. However, we have no adequate information as to whether this species is present in other rivers. Therefore, determining its precise distribution regions merits further investigation. Conclusion We here reviewed Chondrostoma species in Turkish in- land waters using external morphology, osteology and molecular data. As stated by Elvira (1997), taxonomical status of Chondrostoma species is quite complex. Some morphological characters are functional in taxonomy, among which are the gill rakers’ first gill arch and lat- eral line scales. Although these characters do not apply to many species, they are useful in identifying species together with osteology (dentary and premaxilla) and molecular data. In conclusion, our study has confirmed the existence of 13 valid species, namely C. colchicum, C. beysehirense, C. ceyhanensis, C. cyri, C. holmwoodii, C. kinzelbachi, C. meandrense, C. nasus, C. regium, C. smyrnae, C. toros, C. turnai and C. vardarense in Turkish inland waters. On the other hand, the taxonom- ic status of C. angorense remains uncertain. We consider that re-examination of all Chondrostoma populations be- tween Sakarya (Turkey) and Tuapse (Russia), where the species 1s dispersed, could resolve this uncertainty. Comparison material Chondrostoma angorense: IFC-ESUF 03-1501, 32, 80-174 mm SL; Turkey: Eskisehir prov.: Porsuk River about 2 km west of Yortikkirka, 39°36'00"N, 30°25'09"E.—IFC-ESUF 03-1502, 11, 35-162 mm SL; Turkey: Eskisehir prov.: stream Akin 0.5 km south of Akin, 39°20'02"N, 30°30'59"E —IFC-ES- UF 03-1503, 2, 245-300 mm SL; Turkey: Kutahya Zoosyst. Evol. 99 (1) 2023, 1-13 prov.: stream Emet about 10 km north of Egri6z, 39°28'10"N, 29°15'17"E—IFC-ESUF 03-1538, 3, 137-185 mm SL; Turkey: Balikesir prov.: Bigadi¢ Stream west of Bigadi¢, 39°23'48"N, 28°04'S0"E.— IFC-ESUF 03-1549, 1, 264 mm SL; Turkey: Afyon- 11 1516, 1, 145 mm SL; Turkey: Manisa prov.: Gediz River about 5 km east of Gélmarmara, 38°42'08"N, 27°58'10"E.—IFC-ESUF 03-1517, 3, 85-102 mm SL; Turkey: Izmir prov.: Gediz River about 8 km east of Menemen, 38°37'42"N, 27°10'41"E. karahisar prov.: Kali Stream about 15 km west of | Chondrostoma_ kinzelbachi: YFC-ESUF 03-1518,19, Cay, 38°32'28"N, 30°50'41"E. 173.6-220.58 mm SL, Turkey: Hatay prov.: Golbasi Chondrostoma beysehirense: IFC-ESUF 03-1505, 16, Lake, 36°30'13.0"N, 36°29'45.3"E. 156-251 mm SL; Turkey: Konya prov.: Beyse- Chondrostoma meandrense. IFC-ESUF 03-1519, 45, hir Lake about 20 km south of Sarkikaraagac, 37°52'42"N, 31°20'46"E. Chondrostoma ceyhanensis: IFC-ESUF 03-1556, 208.49 mm SL; Turkey: Kahramanmaras prov.: Sir Dam Lake, Ceyhan River, 37°34'30.09"N, 36°45'43.60"E—IFC-ESUF 03-1545, 22, 191.76— 264.77 mm SL; Turkey: Kahramanmaras prov.: Sir Dam Lake, Ceyhan River, 37°34'30.09"N, 36°45'43.60"E—IFC-ESUF 03-1546, 26, 77.76— 143.15 mm SL; Turkey: Osmaniye prov.: Tecir- li Bridge, Ceyhan River drainage, 37°11'41.4"N, 36°04'59.3"E—IFC-ESUF 03-1539, 14, 140.85— 205.36 mm SL; Turkey: Adana prov.: Seyhan Dam Lake, 37°03'58.9"N, 35°17'46.7"E.—IFC-ESUF 03- 1540, 3, 59.54—-97.73 mm SL; Turkey: Adana prov.: Cakit Stream, Seyhan River drainage, 37°06'10.4"N, 35°06'34.7"E—IFC-ESUF 03-1541, 16, 71.42- 81.88 mm SL; Turkey: Adana prov.: Eglence Stream, Seyhan River drainage, 37°17'30.8"N, 35°13'28.7"E. Chondrostoma_ colchicum. IFC-ESUF 03-1506, 9, 228.74-277.73 mm SL; Turkey: Erzurum prov.: Ispir, Coruh River, 40°31'50.0"N, 41°02'19.2"E—IFC-ES- UF 03-1507, 7, 191.71-242.77 mm SL; Turkey: Artvin prov.: Borcka, Coruh River, 41°21'53.4"N, 41°40'38.1"E. Chondrostoma cyri. IFC-ESUF 03-1508, 7, 129.05— 159.85 mm SL; Turkey: Ardahan prov.: Cakir Stream, Kura River, 40°58'01.4"N, 42°35'12.7"E— IFC-ESUF 03-1509, 53, 118.70-155.51 mm SL; Turkey: Ardahan prov.: Gole, Kura River, 40°54'32.6"N, 42°39'08.9"E.—IFC-ESUF 03-1510, 4, 95.38—-128.96 mm SL; Turkey: Kars prov.: Ak¢alar Creek, Arpacay1 Stream, Aras River, 40°46'20.8"N, 43°17'40.6"E—IFC-ESUF 03-1511, 3, 130.20— 153.47 mm SL; Turkey: Kars prov.: Cildir Lake, Aras River, 41°02'32.3"N, 43°13'15.5"E. Turcichondrostoma 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°32'57"E. Chondrostoma holmwoodii: JFC-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- 120-209 mm SL, Turkey: Denizli prov.: Isikli Spring, 38°19'19"N, 29°51'10"E—IFC-ESUF 03- 1522, 19, 96-151 mm SL, Turkey: Denizli prov.: Kufi Stream about 4 km north of Isikli, 38°21'48"N, 29°50'56"E.—IFC-ESUF 03-1523, 20, 110-219 mm SL, Turkey: Afyonkarahisar prov.: Karasandikli Stream 0.5 km east of Karasandikli, 38°31'40"N, 30°10'39"E.—IFC-ESUF 03-1525, 4, 65-138 mm SL, Turkey: Afyonkarahisar prov.: Su¢gikan 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: Denizli prov.: Buytk Menderes River about 2 km west of Citak, 38°09'23"N, 29°38'24"E—IFC-ES- UF 03-1562, 3, 125-154 mm SL, Turkey: Denizli prov.: Buiytk Menderes River about 1 km north of Hangalar, 38°07'54"N, 29°23'19"E. Chondrostoma nasus. IFC-ESUF 03-1536, 17, 138.62- 170.62 mm SL, Turkey: Kutahya prov.: Yanikburnu Stream about 25 km east of Dursunbey, 39°33'04"N, 28°56'55"E.—IFC-ESUF 03-1537, 23, 151-216 mm SL; Turkey: Kttahya prov.: Yanikburnu Stream about 25 km east of Dursunbey, 39°33'04"N, 28°56'55"E. Chondrostoma regium: IFC-ESUF 03-1527, 21,177.96— 263.07 mm SL; Turkey: Sut prov.: Botan Stream, Tigris River drainage, 37°51'09"N, 41°53'14"E— IFC-ESUF 03-1528, 20, 151.97-196.37 > mm SL;Turkey: Batman prov.: Botan Stream, Tigris River drainage, 37°51'09"N, 41°53'14"E—IFC-ES- UF03-1529, 15, 151.43-228.79 mm SL; Turkey: Batman prov.: Botan Stream, Tigris River drain- age, 37°51'09"N, 41°53'14"E—IFC-ESUF 03- 1530, 9, 181.67—204.74 mm SL; Turkey: Dityar- bakir prov.: Ongozlt Bridge, Tigris River drainage, 37°53'13.6"N, 40°13'42.4"E—IFC-ESUF 03-1531, 9, 157.68—202.35 mmSL; Turkey: Ilisu Village, Kaplicalar, Tigris River drainage, 37°31'06.3"N, 41°50'17.5"E.—IFC-ESUF03-1533, 20, 137.06— 235.95 mm SL; Turkey: Sivas prov.: Kangal, Delihaci Village, Euphrates River drainage, 39°17'45.5"N, 37°28'47 4"E.—IFC-ESUF 03-1552, 1, 70.82 mm SL; Turkey: Kilis prov.: Sinnep (Kuveik) Stream, 36°44'50.2"N, 37°14'40.4"E. Chondrostoma smyrnae: IFC-ESUF 03-1566, 190 mm SL; Turkey: Izmir prov.: Tahtali reservoir about 2 km north of Degirmendere, 38°08'19"N, 27°07'10"E—IFC-ES- UF 03-1567, 22, 152-205 mm SL; Turkey: Izmir prov.: Tahtali reservoir about 2 km north of DeSirmen- dere, 38°08'19"N, 27°07'10"E—IFC-ESUF 03-1568, 22, 181-272 mm SL; Turkey: Izmir prov.: Tahtali res- ervoir about 2 km north of Degirmendere, 38°08'19"N, zse.pensoft.net 12 27°07'10"E—IFC-ESUF 03-1550, 2, 92.68—-109.02 mm SL; Turkey: Izmir prov.: Sasal Stream about 1 km south of Kiiner, 38°11'57"N, 27°08'09"E. Chondrostoma toros: IFC-ESUF 03-1555, 142.76 mm SL; Turkey: Mersin (Icel) prov.: Hamamkéy Village, Mut, Goksu River, 36°37'51.74"N, 33°22'03.18"E.— IFC-ESUF 03-1547, 34, 54.95-163.77 mm SL; Tur- key: Mersin (icel) prov.: Hamamkoy Village, Mut, Goksu River, 36°37'51.74"N, 33°22'03.18"E— IFC-ESUF 03-1554, 10, 142.37-189.92 mm SL; Turkey: Mersin prov.: Eustarin Zone, Silifke, Gdksu River drainage, 36°20'51.0"N, 34°01'09.9"E. Chondrostoma turnai: IFC-ESUF 03-1524, 44, 75- 210 mm SL; Turkey: Aydin prov.: Cine Stream about 8 km south of Aydin, 37°45'43"N, 27°50'12"E— IFC-ESUF 03-1563, 1, 145 mm SL; Turkey: Denizli prov.: Cindere reservoir about 8 km south of Guney, 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-ESUF 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; Turkey: Denizli prov.: Vali Recep Yazicioglu reservoir about 3 km east of Deni- zli, 37°46'14"N, 29°07'39"E. Chondrostoma vardarense. IFC-ESUF 03-1534, 26, 195-210 mm SL; Turkey: Edirne prov.: Meri¢ River, 41°39'39.1"N, 26°33'41.1"E. Acknowledgements This study was supported by a grant from Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: KBAG-111T900). We thank Dr Mustafa OZTOP (Burdur) for proofreading the manuscript. References Berg LS (1949) Freshwater fishes of the U.S.S.R. and adjacent coun- tries. No. 29: 467-925. Bogutskaya N (1996) Contribution to the knowledge of leuciscine fish- es of Asia Minor. Part 1.Morphology and taxonomic relationships of Leuciscus borysthenicus (Kessler, 1859), Leuciscus smyrnae- us Boulenger, 1896 and Ladigesocypris ghigii (Gianferrari, 1927) (Cyprinidae, Pisces). Publicaciones Especiales. Instituto Espanol de Oseanografia 21: 25-44. Bohme M, Ilg A (2003) Database of Vertebrates: fossil Fishes, Amphib- ians, Reptiles and Birds (fosFAR-base) localities and taxa from the Triassic to the Neogene. www.wahre-staerke.com [accessed on 24 December 2021] Ciftci Y, Mutlu AG, Gigli SS, Turan D, Kuctik F (2020) Phylogeogra- phy of the genus Chondrostoma Agassiz, 1835 (Teleostei: Leucis- cidae) in Anatolia, as inferred from mitochondrial DNA analysis. Zoology in the Middle East 66(3): 206—221. https://doi.org/10.1080 /09397140.2020.1788255 zse.pensoft.net Kucuk, F. et al.: Review of Chondrostoma in Turkey Coad BW (2017) Freshwater Fishes of Iran. http://www.briancoad.com [accessed on 24 December 2021] Crivelli AJ (2006) Chondrostoma_ beysehirense. In: TUCN 2008. 2008 IUCN Red List of Threatened Species. www.iucnredlist.org [Downloaded on 04 March 2009] Cunningham CW (1997) Can three incongruence tests predict when data should be combined? Molecular Biology and Evolution 14(7): 733-740. https://doi.org/10.1093/oxfordjournals.molbev.a0258 13 Durand JD, Bianco PG, Laroche J, Gilles A (2003) Insight into the origin of endemic Mediterranean ichthyofauna: Phylogeography of Chondrostoma genus (Teleostei, Cyprinidae). The Journal of Heredity 94(4): 315-328. https://doi.org/10.1093/jhered/esg074 Eagderi S, Jouladeh-Roudbar A, Sungur Birecikligil S, Cigek E, Coad BW (2017) Chondrostoma esmaeilii, a new cyprinid species from the Tigris river drainage in Iran (Teleostei: Cyprinidae). Vertebrate Zoology 67(2): 125-132. Elvira B (1997) Taxonomy of the genus Chondrostoma (Osteichthyes, Cyprinidae): An updated review. Folia Zoologica 46(Supplementum 1): 1-14. Esmaeili HR, Coad BW, Mehraban H, Masoudi M, Khaefi R, Abbasi K, Mostavavi H, Vatandoust S (2014) An updated checklist of fishes of the Caspian Sea basin of Iran with a note on their zoogeography. Iranian Journal of Ichthyology 1(3): 152-184. Farris JS, Kallersjo M, Kluge AG, Bult C (1995) Constructing a signif- icance test for incongruence. Systematic Biology 44(4): 570-572. https://doi.org/10.2307/2413663 Felsenstein J (1985) Confidence-Limits on phylogenies — an approach using the bootstrap. Evolution 39(4): 783-791. https://doi. org/10.1111/).1558-5646.1985.tb00420.x Fricke R, Bilecenoglu M, Sar1 HM (2007) Annotated checklist of fis- hand lamprey species (Gnathostomata and Petromyzontomorphi) ofTurkey, including a Red List of threatened and declining species. Stuttgarter Beitrage zur Naturkunde Seria A 706: 1-172. Geiger MF, Herder F, Monaghan MT, Almada V, Barbieri R, Bariche M, Berrebi P, Bohlen J, Casal-Lopez M, Delmastro GB, Denys GPJ, Dettai A, Doadrio I, Kalogianni E, Karst H, Kottelat M, Kovacic M, Laporte M, Lorenzoni M, Maréié Z, Ozulug M, Perdices A, Perea S, Persat H, Porcelotti S, Puzzi C, Robalo J, Sanda R, Schneider M, Slechtova V, Stoumboudi M, Walter S, Freyhof J (2014) Spatial het- erogeneity in the Mediterranean Biodiversity Hotspot affects barcod- ing accuracy of its freshwater fishes. Molecular Ecology Resources 14(6): 1210-1221. https://doi.org/10.1111/1755-0998.12257 Gigli SS, Kucgtk F, Turan D, Ciftci Y, Mutlu AG (2018) A new Chondrostoma species from the Buytik Menderes River basin, Turkey (Teleostei: Cyprinidae). Zoology in the Middle East 64(3): 315-321. https://doi.org/10.1080/09397140.2018.1511293 Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Bi- ology 52(5): 696-704. https://doi.org/10.1080/10635 150390235520 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In. Nucleic Acids Symposium Series 41(41): 95-98. [London: Information Retrieval Ltd., c1979-—c2000] Hall BG (2008) Phylogenetic trees made easy: a how-to manual. Sinau- er Associates, Sunderland, Massachusetts, [xiv +] 230 pp. https:// doi.org/10.1080/10635 150802303458 Hrbek T, Kiiciik F, Frickey T, Stdlting KN, Wildekamp RH, Meyer A (2002) Molecular phylogeny and historical biogeography of the Zoosyst. Evol. 99 (1) 2023, 1-13 Aphanius (Pisces, Cyprinodontiformes) species complex of central Anatolia, Turkey. Molecular Phylogenetics and Evolution 25(1): 125-137. https://doi.org/10.1016/S1055-7903(02)00203-8 Hrbek T, Stdlting KN, Bardakgi F, Kuciik F, Wildekamp RH, Meyer A (2004) Plate tectonics and biogeographical patterns of the Pseudophoxinus (Pisces: Cypriniformes) species complex of central Anatolia, Turkey. Molecular Phylogenetics and Evolution 32(1): 297-308. https://doi.org/10.1016/j.ympev.2003.12.017 Ilhan A, Sarit HM, Kurtul I (2020) Fish fauna of Bakircay Stream (North Eagean, Turkey). Ege Journal of Fisheries and Aquatic Sciences 37(3): 309-312. https://doi.org/10.12714/egejfas.37.3.14 Kaya C, Turan D, Baycelebi E (2020) Taxonomic assessment and dis- tribution of fishes in upper Kura and Aras river drainages. Zoosys- tematics and Evolution 96(2): 325-344. https://dot.org/10.3897/ zse.96.52241 Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide se- quences. Journal of Molecular Evolution 16(2): 111-120. https:// doi.org/10.1007/BF01731581 Kottelat M, Freyhof J (2007) Handbook of European freshwater fishes. Kottelat, Cornol and Freyhof, Berlin, [xiv +] 646 pp. Kuciik F, Turan D, Gigli SS, Mutlu AG, Ciftci Y (2017) Two new spe- cies of Chondrostoma Agassiz, 1832 (Teleostei: Cyprinidae) from the Ceyhan, Seyhan and Goksu rivers in the East Mediterranean re- gion of Turkey. Turkish Journal of Fisheries and Aquatic Sciences 17(4): 795-803. https://doi.org/10.4194/1303-2712-v17_4_15 Kuciik F, Ciftci Y, Giglti SS, Turan D (2021) Chondrostoma smyrnae, a new nase from the Tahtali reservoir drainage in the Aegean Sea basin (Teleostei, Leuciscidae). Zoosystematics and Evolution 97(1): 235-248. https://doi.org/10.3897/zse.97.6369 1 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547-1549. https://doi.org/10.1093/molbev/msy096 Kuru M (1981) Revision of Chondrostoma species of Turkey. Hacettepe bulletin of natural sciences and engineering 10: 111-121. Kuru M, Yerli SV, Mangit F, Unlii E, Alp A (2014) Fish biodiversity in inland waters of Turkey. Joumal of Academic Documents for Fish- eries and Aquaculture 3: 93-120. Naseka AM (1996) Comparative study on vertebral column in the Gobi- oninae (Cyprinidae, Pisces) with special reference to its systematics. Instituto Espanol de Oceanografia 21: 149-167. Posada D (2008) jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution 25(7): 1253-1256. https://dot.org/10.1093/ molbev/msn083 Rambaut A (2018) FigTree vl. 4.2, a graphical viewer of phylogenet- ic trees. http://tree.bio.ed.ac.uk/software/figtree [Downloaded on 26.11.2018] Robalo JI, Almada VC, Levy A, Doadrio I (2007) Re-examination and phylogeny of the genus Chondrostoma based on mitochondrial and 13 nuclear data and the definition of 5 new genera. Molecular Phylo- genetics and Evolution 42(2): 362-372. https://doi.org/10.1016/. ympev.2006.07.003 Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Hohna S, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phy- logenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sys- bio/sys029 Schénhuth S, Vuki¢ J, Sanda R, Yang L, Mayden RL (2018) Phylo- genetic relationships and classification of the Holarctic family Leuciscidae (Cypriniformes: Cyprinoidei). Molecular Phyloge- netics and Evolution 127: 781-799. https://doi.org/10.1016/. ympev.2018.06.026 Swofford DL (1998) PAUP* Phylogenetic Analysis Using Parsimony (*and other methods). Version 4 beta 10. Sinauer Associates, Sun- derland, MA. Taylor WR, van Dyke GC (1985) Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. Cybium 9: 107-119. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight ma- trix choice. Nucleic Acids Research 22(22): 4673-4680. https://dot. org/10.1093/nar/22.22.4673 Turan D, Kuciik F, Giiclii SS, Aksu I (2021) Zurcichondrostoma, a new genus for the Leuciscidae (Teleostei: Cypriniformes) from South- western Anatolia. Journal of Fish Biology 99(6): 1968-1977. https:// doi.org/10.1111/jfb.14903 Van der Laan R, Fricke R, Eschmeyer WN [Eds] (2020) Eschmeyer’s catalog of fishes: classification. http://www.calacademy.org/scien- tists/catalog-of-fishes-classification [accessed on 24.12.2021] Supplementary material | Table SI Authors: Fahrettin Ktctk, Yilmaz Cift¢i, Salim Serkan Guclt, Ayse Gul Mutlu, Davut Turan Data type: MS Word file Explanation note: Table S1. List of 16S rRNA, Cytb and COI sequences used in molecular data analyses. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.99.91275.suppl1 zse.pensoft.net