Zoosyst. Evol. 99 (2) 2023, 439-455 | DO! 10.3897/zse.99.102575 gee BERLIN Two new Oxynoemacheilus species in western Anatolia (Teleostei, Nemacheilidae) Davut Turan!, Sadi Aksu*, Gdkhan Kalayci? 1 Recep Tayyip Erdogan University, Faculty of Fisheries, 53100 Rize, Tiirkiye 2 Eskisehir Osmangazi University, Vocational School of Health Services, 26700 Eskisehir, Tiirkive https://zoobank. org/8D151C12-8994-4338-BFFA-48F 4DE5DA4A0 Corresponding author: Gékhan Kalayci (gokhan.kalayci@erdogan.edu.tr) Academic editor: Nicolas Hubert # Received 23 February 2023 # Accepted 9 June 2023 @ Published 4 October 2023 Abstract Oxynoemacheilus sakaryaensis sp. nov., is restricted to the Sakarya River basin, and O. melenicus sp. nov., is distributed in both the Sakarya River and Buytikmelen Stream. Oxynoemacheilus sakaryaensis is distinguished by having a flank plain or with numerous irregularly shaped pale brownish bars and a caudal-peduncle depth 2.8—3.2 times in its length. Oxynoemacheilus melenicus 1s distin- guished by having a flank with 10—13 irregular shaped brownish bars or blotches and the caudal peduncle depth 1.9—2.8 times in its length. Oxynoemacheilus banarescui, O. samanticus, O. simavicus, O. fatsaensis, O. sakaryaensis, and O. melenicus are valid, which belong to the O. bergianus species group. O. melenicus and O. sakaryaensis were differentiated from all other Oxynoemacheilus Species in western Anatolia by two diagnostic and unique nucleotide substitution sites in the COI barcoding region. Also, species delineation tests (ABGD, GMYC, ASAP) and phylogenetic analyses support the validity of O. melenicus and O. sakaryaensis as distinct species. Key Words Cytochrome oxidase I, freshwater fish, molecular identification, Northwestern Anatolia, species delineation, taxonomy Introduction Nemacheilid loaches of the genus Oxynoemacheilus are widespread fishes all over the Eastern Mediterranean, the southern Caucasus, Anatolia, Mesopotamia, and Central Iran (Freyhof et al. 2011, 2022; Kottelat 2012). Frey- hof et al. (2011) list 41 species as valid in that genus, and Kottelat (2012) also included O. oxianus from the Central Asian Amu-Darya drainage. Since, Cicek et al. (2018); Erk’akan (2012); Freyhof (2016); Freyhof et al. (2017, 2019, 2021a, 2021b, 2022); Freyhof and Abdullah (2017); Freyhof and Geiger (2021); Freyhof and Ozu- lus (2017); Kamangar et al. (2014); Kaya et al. (2020, 2021); Saygun et al. (2021); Sayyadzadeh et al. (2016); Sungur et al. (2017); Turan et al. (2019); YoSurtcuoglu et al., (2021a, 2021b); YoSurtcuoslu et al. (2022) described and re-validated additional species and we recognize 62 species of Oxynoemacheilus as valid. Oxynoemacheilus is one of the largest genera of freshwater fishes in the Western Palearctic. It has been reviewed comprehensive- ly in the global distribution zone due to the many species. Indeed, most Oxynoemacheilus species are distributed in small ranges except O. bergianus, which lives in both the Caspian Sea basin and the Persian Gulf basin (Freyhof 2016; Freyhof and Abdullah 2017; Freyhof and Ozulug 2017; Freyhof et al. 2017; Freyhof et al. 2022). In recent years, new species (O. veyseli, O. elsae, O. ciceki) have been discovered from stretching Anatolia and nearby ba- sins (Ci¢ek et al. 2018; Eagderi et al. 2018; Sungur et al. 2017; Freyhof et al. 2022). Freyhof et al. (2022) treated four species within O. bergianus species group as valid, corresponding to O. banarescui, O. bergianus, O. fatsaensis, and O. simavi- cus. Bektas et. al (2022) reported O. bergianus, O. saman- ticus, O. fatsaensis, O. banarescui and O. simavicus as valid species. Freyhof et al. (2022) reported O. simavicus Copyright Turan, D. 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. 440 from drainages in the southern shores of the Marmara Sea, as well as the Sakarya and Buyukmelen Stream. In this study, these populations of the species in Sakarya and Buytikmelen rivers are described as new species as O. sakaryaensis and O. melenicus. These two species are included in the O. bergianus species group, which has a slender caudal peduncle and lack the two distinct black or dark-brown spots on the caudal fin base and the presence of suborbital flap or groove. Material and methods The care of experimental animals was consistent with the Republic of Turkey’s animal welfare laws, guidelines, and policies. After anesthesia, fishes were fixed in 5% formalde- hyde stored in 70% ethanol or directly fixed in 99% ethanol. Measurements were made with a dial caliper and recorded to 0.1 mm. All measurements were made point-to-point, never by projections. Methods for counts and measurements followed Kottelat and Freyhof (2007) and the terminology of head canals followed Kottelat (1990). Standard length (SL) was measured from the tip of the snout to the end of the hypural complex. The length of the caudal peduncle was measured from behind the base of the last anal-fin ray to the end of the hypural complex, at mid-height of the cau- dal-fin base. The last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are count- ed as “1/2”. Simple rays of dorsal and anal fins were not counted as they were deeply embedded. The holotype was included in the calculation of means and SD. Males of sev- eral Oxynoemacheilus show an exposed lachrymal bone, which is often called a suborbital flap or groove. We distin- guish between a suborbital flap and a suborbital groove. In the present study, all Oxynoemacheilus having an exposed lachrymal bone possess a suborbital groove. We see no application in developing identification keys for several species in large geographic areas. There- fore, we provide keys for western Anatolia only. Differ- ential species diagnoses are given against related species as derived by the COI molecular analysis presented here and against geographically adjacent species. No differen- tial diagnosis is provided against largely unrelated and/or geographically distant species. Abbreviations used: SL, standard length; K2P, Kimu- ra 2-parameter. Collection codes: FFR, Recep Tayyip Erdogan University Zoology Museum of the Faculty of Fisheries, Rize. Materials examined are listed at the end of the study. DNA extraction, PCR and sequencing Total DNA was extracted from fin clips via Qiacube au- tomated DNA/RNA purification system using Qiagen DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany). DNA quality and quantity were checked on a NanoDrop 2000/c spectrophotometer (Thermo Scientific, Rockford, zse.pensoft.net Turan, D. et al.: Two new Oxynoemacheilus species in western Anatolia IL, USA) and 0.8% agarose gel electrophoresis. The stan- dard vertebrate DNA barcode region of COI gene (645 bp) was amplified using a universal CO/ barcoding primer pair, the FishFl (5’-TCAACCAACCACAAAGACATTG- GCAC-3’) and FishR1 (5’-TAGACTTCTGGGTGGC- CAAAGAATCA-3’) (Ward et al. 2005). PCR reactions were performed in a 50 uL reaction volume containing 5 uL 10x PCR buffer, 100 ng template DNA, 0.5 mM dNTPs mix, 3 mM MgCl, 0.5 mM of each primer, and 1 wL Taq DNA polymerase (New England Biolabs). The polymerization was carried out under the following con- ditions: initial denaturation at 95 °C for 30 s, denaturation at 95 °C for 30 s, annealing at 58 °C for 45 s, extension at 68 °C for 1 min through 35 cycles, and a final extension at 68 °C for 5 min using Biorad T100 (Bio-Rad, Hercules, CA, USA) thermal cycler. The PCR products were also run and visualized under UV Quantum—Capt ST4 system (Vilber Lourmat, France), purified, and sequenced at Mac- rogen Europa Inc. (Amsterdam, Netherlands). Molecular data analysis We have used the newly generated 30 DNA barcodes from the present study and included additional 42 specimens from earlier studies deposited to NCBI GenBank (Geiger et al. 2014, Geiger 2019, Turan et al. 2019, Bektas et al. 2022, Freyhof et al. 2022) (Table 1). The dataset also consisted of DNA barcodes from individuals Seminemacheilus lendlii and Oxynoemacheilus cemali as outgroup taxon. Clustal W algorithm (Thompson et al. 1994) in Bioedit v7.2.5 (Hall 1999) was used to align COI barcode sequences, and the se- quences were submitted to NCBI GenBank with accession numbers OQ332806—0Q332835. Phylogenetic relation- ships among species were carried out using both maximum likelihood (ML) and Neighboor Joining (NJ) analysis using MEGA 11 (Tamura et al. 2021). TrN+G model (Kimura 1980) was chosen as the best nucleotide substitution mod- el according to the Bayesian information criterion (BIC) in jModeltest v. 0.0.1 (Posada 2008). The K2P distance model (Kimura 1980) in MEGA 11 was used to estimate pairwise genetic distances among species. POPART (1.7) (Leigh and Bryant 2015) was used to generate and display the haplo- type network. We used three single-locus species delimi- tation methods relying on different operational criteria for species delimitation being implemented: ABGD, Automatic Barcode Gap Discovery (Puillandre et al. 2012), Assemble Species by Automatic Partitioning (ASAP) (Puillandre et al. 2021) and GMYC, the General Mixed Yule Coalescent method, single-threshold version (Fujisawa and Barraclough 2013). All species delimitation methods were performed us- ing the COI sequences (not haplotypes). The ABGD species delimitation was performed via the ABGD web server with default settings (https://bioinfo.mnhn.fr/abi/public/abed/ abgdweb.html, accessed on 1 May 2023) and ASAP spe- cies delimitation was performed via the ASAP web serv- er (https://bioinfo.mnhn. fr/abi/public/asap/asapweb.html, accessed on 1 May 2023) and the following parameters: Zoosyst. Evol. 99 (2) 2023, 439-455 441 Table 1. List of COI sequences downloaded from NCBI Gen- Species Accession N. Drainage Reference Bank with information on drainage and country of origin. O. fatsaensis OL855790 Tersakan stream, —_| Bektas et. al. 2022 Yesilirmak Species | Accession N. Drainage Reference O. fatsaensis 0L855791 Tersakan stream, —_| Bektas et. al. 2022 O. melenicus 0K316642 Asar stream, Freyhof et. al. 2022 Yesilirmak Buyukmelen River O. banarescui 0K316694 Yenice River Freyhof et. al. 2022 O. melenicus MHO18854 | Buyukmelen drainage | Turan et. al. 2019 O. banarescui MH469261 Devrekani stream Turan et. al. 2019 O. melenicus MHO18856 | Buyukmelen drainage | Turan et. al. 2019 O. banarescui MH469262 Devrekani stream Turan et. al. 2019 O. melenicus 0K316651 Asar stream, Freyhof et. al. 2022 O. samanticus | MH018861 Terme stream, Turan et. al. 2019 Buyukmelen Kizilirmak River O. melenicus OK316688 Dogancay, Sakarya Freyhof et. al. 2022 O. samanticus 0K316652 Kizilirmak River Freyhof et. al. 2022 River O. samanticus | MHO18863 Vezirkoprii stream, Turan et. al. 2019 O. melenicus | MHO18855 | Biiyiikmelen drainage | Turan et. al. 2019 Kizilirmak River O. melenicus 0K316739 Asar stream, Freyhof et. al. 2022 O. bergianus MH469265 Murat River Turan et. al. 2019 Buyukmelen River O. bergianus MH469266 Murat River Turan et. al. 2019 O. melenicus 0K316620 |Allikova stream, Sakarya | Freyhof et. al. 2022 O. bergianus OL855763 Murat River Bektas et. al. 2022 River O. bergianus OL855764 Merziman stream, Bektas et. al. 2022 O. melenicus 0K316798 |Allikova stream, Sakarya| Freyhof et. al. 2022 Euphrates River O. bergianus OL855765 | Goksu river, Euphrates | Bektas et. al. 2022 O. sakaryaensis | OK316616 Bayindir stream, Freyhof et. al. 2022 O. bergianus OL855766 Sogutlucay stream, | Bektas et. al. 2022 Sakarya River Euphrates O. sakaryaensis | OK316622 Bayindir stream, Freyhof et. al. 2022 O. bergianus O0L855767 Sogutlucay stream, | Bektas et. al. 2022 Sakarya River Euphrates O. sakaryaensis | 0K316635 Bayindir stream, Freyhof et. al. 2022 O. bergianus OK316672 | Yalekhlou, Caspian sea |Freyhof et. al. 2022 Sakarya River basin O. sakaryaensis | 0K316735 Bayindir stream, | Freyhof et. al. 2022 O. bergianus 0K316691 Murat River Freyhof et. al. 2022 Sakarya River O. bergianus MK546446 Caspian sea basin Geiger 2019 O. sakaryaensis | OK316743 Bayindir stream, Freyhof et. al. 2022 O. bergianus MK546447 Euphrates Geiger 2019 Sakarya River O. bergianus MK546448 Euphrates Geiger 2019 O. sakaryaensis | OK316796 Bayindir stream, Freyhof et. al. 2022 O. bergianus MK546449 Serhan stream, Geiger 2019 Sakarya River Euphrates O. simavicus KJ553724 Simav stream Geiger et. al. 2014 O. angorae OL855744 Karasu stream, Iznik | Bektas et. al. 2022 O. simavicus KJ553970 Simav stream Geiger et. al. 2014 lake O. fatsaensis OL855789 Tersakan stream, _| Bektas et. al. 2022 O. angorae OL855745 | Purtek stream, Sakarya | Bektas et. al. 2022 Yesilirmak O. angorae OL855747 | Zamanti stream, Seyhan | Bektas et. al. 2022 Pmin = 0.001, Pmax = 0.1, 1000 replicates, and the Kimura evolutionary model, with TS/TV = 2.0. The same parame- ters were applied under ABGD analysis. GMYC analysis was applied by the single-threshold version of the method, which usually outperforms the multiple-threshold version (Fujisawa and Barraclough 2013). The input ultrametric phylogenetic tree was made in BEAST v.1.8.4 (Drummond et al. 2012) with the following parameters: strict clock, Spe- Results ciation: Yule process as the tree prior with 5 million genera- tions, and sampling frequency of 1000. The resulting ultra- metric tree was imported into R 3.1.3 (R Core Team 2013), and the single threshold ST-GMYC analysis was carried out using the R packages (Splits; Ezard et al. (2009) and Ape libraries; Paradis et al. (2004). In the concordant outcome of these methods, the resulting delimitation appears more logical (Dellicour and Flot 2018). Key to Oxynoemacheilus bergianus species group in the Anatolia if 2 Snout length equal or greater than postorbital length Snout length equal or smaller than postorbital length Flank with marmalade pattern or with numerous irregularly shaped dark brown blotches as two or three horizontal ROWS PPP ants d ean Maen trays Ma SNC WN, Puen eR OES: eo INY ee SP eerie RES Ne OS, CON Ee cB PPOCD AES ES. SRP AN Tre Os Gene oe EVE ere AMI a O. fatsaensis The flank plain or with two to numerous irregularly shaped dark brown blotches The flank with plain yellowish or with numerous irregularly shaped pale brown bars in most individuals The flank dark brownish with 2-13 irregular shaped dark brownish bars or blotches Maxillary barbells approximately equal or greater outer rostral barbells Maxillary barbells always shorter than outer rostral barbells The flank dark brownish with 10-13 irregular shaped dark brownish bars or blotches The flank with 2-9 irregularly shaped brown bars or blotches andl eS Raltnttrataoagsiees Ps iasuenadssninty Pie hisiiethdemadAunts Mesanth ecamaetii petite Ne thence O. simavicus eS er aiaiaii eae aa nee aoa Tee Dae em ane Snr nee oaen pee Seer reat Mad Deng oe 2 eyed OU Den eae O. bergianus Interorbital width 15-24% HL Interorbital width 26-34% HL zse.pensoft.net 442 Oxynoemacheilus sakaryaensis sp. nov. https://zoobank.org/6006326D-484D-435C-BD03-AFAE9A 84DEE3 Figs 1, 2 Type material. Holotype. FFR15629, 1,58 mm SL; Tur- key: Ankara prov.: stream Kirmir 3 km north of Gudil, a tributary of Sakarya River, 40.236°N, 32.606°E. Examined materials. Paratypes. FFR15514, 20, 51— 62 mm SL; same data holotype. — FFRO1527, 17, 50-62 mm SL; Turkey: Ankara prov.: stream Kirmir about 3 km north of Gtidiil, 40.236°N, 32.261°E. — FFRO1387, 9, 39-58 mm SL; Turkey: Ankara prov.: stream [Ilhan at Ilhan Village, 40.093°N, 32.245°E. — FFR15621, 27, 37— 65 mm SL; Turkey: Ankara prov.: stream Ilhan at [han Village, 40.097°N, 32.250°E. — FFR15623, 21, 49-60 mm SL; Turkey: Ankara prov.: stream Kirmir about 6 km north of Gidiil 40.259°N, 32.268°E.— FFR15624, 14, 47-60 mm SL; Turkey: Ankara prov.: stream Bayindir at Gimele Village, 40.314°N, 32.466°E. —FFR 1364, 16, 25-64 mm SL; Turkey: Ankara prov: stream Kirmir at Kizilcahamam, 40.483°N, 32.653°E.-FFR 1386, 13, 49— 62 mm SL; FFR 15515, 1, 62 mm SL; Turkey: Ankara prov: stream Oz at Kizilcahamam, 40.463°N, 32.653°E.— FFR 1387, 9, 39-58 mm SL; FFR 1527, 17, 50-62 mm SL; Turkey: Ankara prov: stream Ilhan 5 km west of Gokcebag, 40.093°N, 32.245°E. — FFR 15514, 1, 59 mm SL; Turkey: Ankara prov: stream Kirmir 3 km north of Gidtil, 40.236°N, 32.261°E. Material used in molecular genetic analysis. FFR DNA 15623, 4, Turkey: Ankara prov.: stream Kirmir about 6 km north of Gtidtil, 40.259°N, 32.268°E. (Gen- Bank accession numbers OQ332822—O0Q332825) — FFR DNA 15629, 3, Turkey: Ankara prov: stream Kirmir about 3 km north of Guditl 40.236°N, 32.606°E. (Gen- Bank accession numbers 0Q332826—O0OQ332828) — FFR DNA 15621, 3, Turkey: Ankara prov: stream Ilhan at II- han Village, 40.097°N, 32.250°E. (GenBank accession numbers 0Q332829—0Q332831) — FFR DNA 1527, 2, Turkey: Ankara prov: stream Kirmir about 3 km north of Gudul, 40.236°N, 32.261°E. (GenBank accession num- bers O0Q332820—0Q332821) Diagnosis. Oxynoemacheilus sakaryaensis is distin- guished from O. melenicus by having the flank plain yel- lowish or numerous irregularly shaped pale brownish bars (vs. the flank with 10-13 irregular shaped dark brownish bars or blotches, O—2 irregularly shaped brownish saddle in front of dorsal-fin origin (vs. 3—4) and caudal pedun- cle depth 2.8—-3.2 times in its length (vs. 1.9-2.8). Oxy- noemacheilus sakaryaensis is distinguished from O. an- gorae by having a caudal-peduncle depth 2.8—3.2 times in its length (vs.1.4—1.8), the flank with plain yellowish or with numerous irregularly shaped brown bars in most individuals (vs. showing a dark-brown mid lateral stripe or a series of fused, dark-brown blotches interrupted by a whitish or pale-brown lateral line (Fig. 3), a groove in upper lip in males (vs. absent), an axillary lobe at the base of pelvic fin (vs. absent) and the caudal-fin moderately forked (vs. emarginated) Oxynoemacheilus sakaryaensis zse.pensoft.net Turan, D. et al.: Two new Oxynoemacheilus species in western Anatolia is distinguished from O. banarescui by the flank with plain yellowish or with numerous irregularly shaped brown bars in most individuals (vs. 7-9 brownish blotches on flank (Fig. 4), the caudal peduncle depth 2.8-3.2 times in its length (vs. 1.9—2.8) and maxillary barbells always shorter than outer rostral barbells (vs. equal or shorter) Oxynoe- macheilus sakaryaensis is distinguished from O. simavi- cus by the flank with plain yellowish or with numerous irregularly shaped pale brown bars in most individuals (vs. flank with 2-8 dark brownish blotches (Fig. 5) and 7-9 small and very pale brownish saddles on back (vs. 4—5 large dark brownish saddles on back). Oxynoemachei- lus sakaryaensis 1s distinguished from O. samanticus by having a snout length smaller than postorbital length (vs. the snout length longer than the postorbital length), more slender caudal peduncle (caudal peduncle depth 2.8—3.2 times in its length, vs. 2.2—2.7) and the flank with plain yellowish or with numerous irregularly shaped brown bars in most individuals (vs. S—10 black or dark brown bars or blocks on flank (Fig. 6). Oxynoemacheilus sakaryaensis 1s distinguished from O. fatsaensis by the flank with plain yellowish or with numerous irregularly shaped brown bars in most individuals (vs. the flank with marmalade pattern or with numerous irregularly shaped dark brown blotches as two or three horizontal rows (Fig. 7) and more slender caudal peduncle (caudal peduncle length 2.8—3.2 times greater than its length (vs. 2.0—-2.6). Oxynoemacheilus sa- karyaensis 1s distinguished from O. bergianus by the flank with plain yellowish or with numerous irregularly shaped brown bars in most individuals (vs. 4-9 black or dark brown bars or blocks on flank (Fig. 8) and a greater dis- tance between anus and anal-fin origin (3-5% SL, vs. 2-3). Oxynoemacheilus sakaryaensis is distinguished from O. seyhanensis by having the caudal-peduncle length 2.8—3.2 times greater than its depth (vs.1.2—1.4), the flank with plain yellowish or with numerous irregularly shaped brown bars in most individuals (vs. the body with mar- morate pattern or numerous small irregularly shaped and spaced dark-brown bars on flank (Fig. 9), a suborbital groove in males (vs. absent), an axillary lobe at base of pelvic fin (vs. absent), a forked caudal fin (vs. slightly emarginate), lacking dorsal and ventral adipose crest on caudal peduncle (vs. present) and a more slender caudal peduncle (depth 2.7—3.5 times in its length, vs. 2.0—2.6, vs. 1.2—1.4). Oxynoemacheilus sakaryaensis is distinguished from O. cemali by having the flank with plain yellowish or with numerous irregularly shaped brown bars in most individuals (vs. 9-15 irregularly shaped dark-gray bars on the flank), a forked caudal-fin (vs. slightly forked), and having a slender caudal peduncle (the caudal-peduncle length 2.8—3.2 times greater than its depth (vs.1.4—2.0). Description. See Figs 1, 2 for general appearance and Table 2 for morphometric data. Adult size large (maximum 65 mm SL). Body slender, compressed at caudal peduncle, greatest depth about midline between nape and dorsal-fin origin, slightly decreasing towards caudal-fin base. No hump at nape. Greatest body width at pectoral-fin base. Head pointed, upper head profile slightly convex on snout, Zoosyst. Evol. 99 (2) 2023, 439-455 443 Figure 1. Oxynoemacheilus sakaryaensis, FFR15629 holotype, 58 mm SL; Turkey: stream Kirmir. Figure 2. Oxynoemacheilus sakaryaensis, FFR 15623 paratypes, top to bottom 56 mm SL, 61 mm SL; Turkey: stream Kirmir. zse.pensoft.net AAA Turan, D. et al.: Two new Oxynoemacheilus species in western Anatolia « - - ae i FS = Figure 3. Oxynoemacheilus angorae, FFRO1513, 55 mm SL, Turkey: stream Ber¢in, FFRO1526, 64 mm SL; Turkey: stream Kirmir. Table 2. Morphometric data of Oxynoemacheilus sakaryaensis (holotype, FFR15629, paratypes, FFR 15514; n= 20). Standard length (mm) In percent of standard length Head length 23.8 | 23.1 | 20.4) 24.8] 1.1 Body depth at dorsal-fin origin 17.7| 0.8 Predorsal length 52.7 47.7 | 53.0} 1.7 Postdorsal length 35.9 32.0 | 39.7} 2.0 Preanal length 74.2 68.4 | 74.2] 1.5 Prepelvic length 52.4| 1.4 Distance between pectoral and pelvic-fin origins Distance between pelvic and analfin origins Distance between vent and analfin origin Depth of caudal peduncle Length of caudal peduncle Dorsal-fin depth 27.0 | 32.0} 1.1 18,8; 2755:| 129 0.5 7.2 | 64 | 7.9 | 04 6.3 : : za7/T3 17.6 18.8 | 16.6 | 20.7} 1.2 Anal-fin depth 0.9 Pectoralfin length 26.3] 1.7 Pelvic-fin length 17.8 | 16.8 |14.6}17.9] 0.9 In percent of head length 49.3 | 45.4 | 58.1 | 3.2 Head depth at eye 35.6} 37.9| 3.1 Snout length 17.3 | 24.5} 1.9 Eye diameter 4?7-5-|'55:3:| 3:2 Postorbital distance 62.0 | 54.6 | 69.5 | 3.3 Maximum head width 25.3 28.0 | 23.1 | 35.8] 3.2 23.9 25.9 | 20.5 | 32.7] 2.8 Interorbital width 30.6 | 24.8 | 37.6 | 3.4 Length of outer rostral barbel Length of maxillary barbel Length of inner rostral barbel 26.2 | 26.6 | 22.4) 34.1 | 27 3.0 2:9? 2.89 |! BH | CO.) Caudal peduncle length/depth zse.pensoft.net flattened on ventral surface. Snout slightly pointed at tip. Mouth narrow and arched, lips well developed. A narrower median interruption in lower lip. A narrower median inci- sion in upper lip a very shallow groove. A suborbital groove in males. Barbels short, inner rostral barbel not reaching to base of maxillary barbel; outer almost not reaching to vertical through anterior eye margin. Maxillary barbell not reaching to posterior eye-margin in most individuals. Cau- dal peduncle slender, compressed laterally, length 2.8—3.2 times longer than deep. Axillary lobe presents at pelvic-fin base, fully attached to body. Pelvic-fin origin below first or second branched dorsal-fin ray. Anal-fin origin at vertical- ly equal to dorsal-fin tip. The pectoral fin almost reaching vertical through tip of dorsal-fin origin in males. No dorsal or ventral adipose crest on caudal peduncle. Lateral line complete, reaching caudal-fin base. Body covered by embedded scales on flank, back, and bel- ly. Dorsal fin with 8’ branched rays, its outer margin straight or slightly concave. Anal fin with 5’2 branched rays, its outer margin straight or slightly concave. Pec- toral fin with 10-12 rays, outer margin straight. Pelvic fin with 7-8 rays, outer margin slightly convex. Caudal fin with 8+8, 8+9, and 9+9 branched rays, outer margin moderately forked and lopes slightly pointed. Coloration. Body yellowish or brownish in life and preserved individuals. Head and cheek with small, plain brown mottling on top and cheeks, without color pat- tern ventrally. No pigmentation below a line from pec- toral-fin base to anus. A large, irregularly shaped, dark- brown blotch at dorsal fin-origin. Flank plain yellowish with numerous irregularly shaped brown bars in most Zoosyst. Evol. 99 (2) 2023, 439-455 445 Figure 5. Oxynoemacheilus simavicus, FFR 01544, mm SL, 52 mm SL; Turkey: stream Nilifer. Figure 6. Oxynoemacheilus samanticus, FFRO15518, 64 mm SL, 62 mm SL; Turkey: stream Soruk. zse.pensoft.net 446 Turan, D. et al.: Two new Oxynoemacheilus species in western Anatolia Figure 8. Oxynoemacheilus bergianus, FFRO15506, 59 mm SL, 57 mm SL; Turkey, Murat River at Ballibostan. Figure 9. Oxynoemacheilus seyhanensis, FFRO1577, 53 mm SL, 54 mm SL; Turkey: stream Soruk. zse.pensoft.net Zoosyst. Evol. 99 (2) 2023, 439-455 Bulgaria 30°0'O"E = 30°24'0"E 30°48 D"E 31°12'0"E 31°36'0"E y 36'0"N 40°0'0"N 40°24'0"N 40°48'0"N 5 , aa eh rie aa , a ee : re Ss Fugees ‘ 4 ve ee my * e, es ‘ Pi tte / ; : 1 ‘” ‘ 3 _,-, ‘3 L ¥ See gee he s) Wy ~*~ ~ ‘ ? Ni ; =p Ute eae TMi 30°0'0"E 30°24'0"E 30°48'0"E 31°12'0"E 31°36'0"E 39°12'0"N 39°36'0"N 40°0'0"N 40°24'0"N 40°48'0"N 39°12'0"N 39° 447 Russia Georgia eS AEN Oxynoemacheilus angorae Oxynoemacheilus banarescui A Oxynoemacheilus melenicus @ 9 Oxynoemacheilus simavicus Kilometers 480 * te eeu Oxynoemacheilus sakaryaensis 32°0'0"E a 32°12'0"E 32°24'0"E 32°36'0"E oN 40°24'0"N 40°24'0"N 40°12'0"N 40°12'0"N 40°0'0"N 40°0'0"N 32°12'0"E 32°24'0" 32°0'0"E 32°36'0"E Figure 10. Distribution of Oxynoemacheilus species in the western Anatolia. individuals. Back with zero to 2 pale blotches anterior to dorsal-fin origin. The dorsal part of caudal peduncle with 3-4 irregularly shaped pale saddle, not fused with mid- lateral blotches. One or two irregular shaped small black spots on caudal-fin base. Dorsal-fin with 1—2 and caudal fin with 2-3 fine, irregularly shapes black bands on rays. Anal, pectoral and pelvic fins greyish to yellowish, with numerous small black spots on rays. Distribution. Oxynoemacheilus sakaryaensis was found in the Sakarya drainage in western Anatolia (Fig. 10). Etymology. The name of the species is derived from the Sakarya River. Oxynoemacheilus melenicus sp. nov. https://zoobank.org/F530199C-A29C-4A 1 B-A 3F 1-493CDF0390CF Figs 11, 12 Type material. Holotype. FFR15627, 1, 65 mm SL; Tur- key: Eskisehir prov.: stream Yarilgan at Gemic¢ Village, 39.343°N, 30.463°E. Examined materials. Paratypes. FFR15626, 39, 50-69 mm SL; same data holotype. FFRO1378, 2, 45—57 mm SL; Turkey: Ktitahya prov.: stream Porsuk at Kuta- hya 39.380°N, 30.067°E. — FFRO1525, 1, 55 mm SL; Turkey: Ktitahya prov.: stream Porsuk about 9 km south of Ktttahya, 39.349°N, 30.038°E. — FFR15625, 4, 32-49 mm SL; Turkey: Kutahya prov: stream Porsuk at Porsuk Village, 39.348°N, 30.036°E. -FFR15630, 5, 61-63 mm SL; Turkey: Eskisehir prov.: stream Allikova about 3km south of Kumbet, 39.681°N, 30.157°E. -FFR 01564, 10, 45-61 mm SL; Turkey: Duizce prov: stream Asar about 2 km west of Kaynasli a tributary of Buyiikmelen drainage, 40.781°N, 31.277°E. Material used in the molecular genetic analysis. FFR DNA 15630, 2, Turkey: Eskisehir prov.: stream Allikova about 3km south of Ktiimbet, 39.681°N, 30.157°E (Gen- Bank accession numbers 0Q332806, 0Q332808). — FFR DNA 15625, 3, Turkey: Ktitahya prov.: stream Porsuk at Porsuk Village, 39.348°N, 30.036°E. (GenBank accession numbers 0Q332809-0Q332811). — FFR DNA 15627, 4, Turkey: Eskisehir prov.: stream Yarilgan at Gemic¢ Vil- lage 39.343°N, 30.463°E. (GenBank accession numbers 0Q332812—0Q332815). -FFR DNA 15631, 4, Turkey: Eskisehir prov.: a tributary of Sakarya River about 9 km northeast of Nasreddi Hoca Village, 39.553°N, 31.757°E. (GenBank accession numbers OQ332816—O0Q332819). — FFR DNA 01524, 1, Turkey: Kitahya prov.: stream Porsuk at Porsuk Village 39.350°N, 30.038°E. (GenBank accession number OQ332807). zse.pensoft.net 448 Turan, D. et al.: Two new Oxynoemacheilus species in western Anatolia Figure 11. Oxynoemacheilus melenicus, FFR15627 holotype, 65 mm SL; Turkey: stream Yarilgan. Diagnosis. Oxynoemacheilus melenicus 1s distin- guished from O. angorae by having a more slender cau- dal pedincle (caudal pedinuncle depth 1.9—2.8 times in its length, vs.1.4—1.8), the flank with 6-13 irregular shaped brownish bars or blotches (a dark-brown midlateral stripe or a series of fused, dark-brown blotches interrupted by a whitish or pale-brown lateral line (Fig. 3), a suborbital groove in males (vs. absent), an axillary lobe at pelvic fin base (vs. absent), a forked caudal fin (vs. slightly emargi- nate), a more slender caudal peduncle (depth .5 times in its length, vs. 2.4—1.8).). Oxynoemacheilus melenicus is distinguished from O. banarescui by having the flank with 10-13 irregular shaped brownish bars or blotches (vs. the flank with 7-10 large, irregularly shaped dark-brown, vertically elongated blotches or bars along lateral midline behind dorsal-fin origin (Fig. 4) and maxillary barbells al- ways shorter than outer rostral barbells (vs. equal or lon- ger). Oxynoemacheilus melenicus is distinguished from O. simavicus by having the flank with 10-13 irregularly shaped brown bars or blotches (vs. 2—8 blotches (Fig. 5) and 6-8 irregularly shaped dark brown saddle on back (vs. 4-6) and a longer head (head length 22—26% SL, vs. 19-22). Oxynoemacheilus melenicus is distinguished from O. samanticus by having the with 10-13 irregu- zse.pensoft.net lar shaped brownish bars or blotches (vs. the flank with 5—10 irregular shaped brownish blotches along lateral line (Fig. 6) and the snout length smaller than postorbital length (vs. the snout length longer than postorbital length) and the pelvic-fin origin below first or second unbranched dorsal-fin ray (vs. equal with dorsal fin origin). Oxynoe- macheilus melenicus is distinguished from O. fatsaensis by having the flank plain brownish or with 10-13 irregu- lar shaped brownish bars or blotches (vs. flank marmalade pattern or with numerous irregularly shaped dark brown blotches as one or two horizontal rows (Fig. 7) and the pelvic-fin origin below the last unbranched dorsal-fin ray (vs. first or second branched dorsal-fin ray). Oxynoemacheilus melenicus is distinguished from O. bergianus the flank with 10—13 dark brownish bars on flank (vs. 4-9 bars or blotches (Fig. 8) and the presence promi- nent bars or blotches in front of dorsal fin (vs. mostly absent or slightly prominent) and a greater distance between anus and anal-fin origin (4-6% SL, vs. 2-3). Oxynoemacheilus melenicus is distinguished from O. seyhanensis by having the with 10—13 irregular shaped brownish bars or blotches (vs. the body with marmorate pattern or numerous small irregularly shaped and spaced dark-brown bars on flank (Fig. 9), a suborbital groove in males (vs. absent), an axil- Zoosyst. Evol. 99 (2) 2023, 439-455 449 Figure 12. Oxynoemacheilus melenicus, FFR15626 paratypes, top to bottom 60 mm SL, 63 mm SL; Turkey: stream Yarilgan. lary lobe at pelvic fin base (vs. absent), a forked caudal fin (vs. slightly truncate) and a more slender caudal peduncle (caudal peduncle length 1.9—2.7 times its depth, vs. 1.1- 1.4). Oxynoemacheilus melenicus is distinguished from O. cemali by having a forked caudal-fin (vs. slightly forked) and a more slender caudal peduncle (depth 1.9—2.6 times in its length, vs. 1.42.0). Description. See Figs 11, 12 for general appearance and Table 3 for morphometric data. Adult size large (max- imum 69 mm Ls). Body slender, compressed at caudal peduncle, greatest depth about midline between nape and dorsal-fin origin, slightly decreasing towards caudal-fin base. No hump at nape. Greatest body width at pectoral-fin base. Head pointed, upper head profile slightly convex on snout, flattened on ventral surface. Snout slightly pointed at tip. Mouth narrow and arched, lips well developed. A narrower median interruption in lower lip. A suborbital groove in males. No median incision in upper lip. Barbels short, inner rostral barbel not reaching to base of maxil- lary barbel; outer almost not reaching to vertical through anterior eye margin. Maxillary barbel, not reaching to pos- terior eye-margin in most individuals. Caudal peduncle slender, compressed laterally, length 1.9—2.7 times longer than deep. Axillary lobe present at pelvic-fin base, fully attached to body. Pelvic-fin origin below first or second branched dorsal-fin ray. Anal-fin origin at vertically equal to dorsal-fin tip. Pectoral fin not reaching vertical through tip of dorsal-fin origin in males. No dorsal or ventral adi- pose crest on caudal peduncle. Pelvic fin not reaching ver- tical of dorsal-fin tip, almost reaching to anus. Anal fin not reaching caudal-fin base. Caudal fin moderately forked. Lateral line complete, reaching caudal-fin base. Body covered by embedded scales on flank, back, and belly. Table 3. Morphometric data of Oxynoemacheilus melenicus (holotype, FFR15627; paratypes, FFR 15626; n = 20). paratypes Standard length (mm) In percent of standard length Head length 25.4] 0.9 Body depth at dorsal-fin origin 15.5 | 15.7 |14.3}17.0] 0.8 Predorsal length 52.1 50.9 | 47.4} 53.2] 1.7 Postdorsal length 36.4 | 36.8 | 34.6] 39.8] 1.1 Preanal length Prepelvic length Distance between pectoral and pelvic-fin 748/13 537] 17 origins Distance between pelvic and anal-fin 12 origins ae between vent and analfin AA 46 0.7 Depth of caudal peduncle Length of caudal peduncle a3 [04 205/05 Dorsal-fin depth 18.1 18.3 | 16.8 | 20.3 | 0.9 Anal-fin depth 15.8 | 15.9 |14.2}18.4] 1.0 Pectoral-fin length 20.2 | 21.6 | 19.0} 23.9} 1.4 Pelvic-fin length In percent of head length Head depth at eye Snout length a7 oe 2.8 se? 3.8 2.6 798 |27 30 33 Eye diameter Postorbital distance Maximum head width Interorbital width Length of inner rostral barbel Length of outer rostral barbel Length of maxillary barbel 26.5 | 27.2 |21.3|35.4| 3.6 2:3 2.6 | 2:3, 2:8 |-0:2 zse.pensoft.net Caudal peduncle length/depth 450 Turan, D. et al.: Two new Oxynoemacheilus species in western Anatolia Table 4. Pairwise distance Kimura’s two parameters (K2P) values based on cytochrome oxidase sequences of Oxynoemacheilus species. 0. bergianus O. melenicus O. sakaryaensis O. simavicus O. fatsaensis O. banarescui O. samanticus O. bergianus Bianeatae 0,080 Table 5. List of the variable nucleotide substitutions in the 645 base pairs long mt DNA COI barcode region. Species Variable nucleotide positions 111122 2222222233 3334444444 556 2489078900 0133478915 6891267899 292 1063670214 7314365451 0161387308 247 QO. melenicus CCTYATGGGY RCAGGCGTGR GTTRGYGAYC RCC O. sakaryaensis Tire WARS Of RR la tle eH WI reo" va ee O. simavicus TY We WiGee en” ELACRUVTACE © LCS eAC =BAs (Y= C/T, R= A/G, W= A/T, S= C/G) Dorsal fin with 7-8’ branched rays, its outer margin straight or slightly concave. Anal fin with 54 branched rays, its outer margin straight. Pectoral fin with 10-11 rays, outer margin straight. Pelvic fin with 7-8 rays, outer margin slightly convex. Caudal fin with 8+8, 8+9, and 9+9 branched rays, outer margin forked, and lopes slightly pointed. Coloration. Body yellowish in life and light brown in preserved individuals. Head and cheek plain or with small, plain brown mottling on top and cheeks, without color pattern ventrally. Numerous pigmentations below a line from pectoral-fin base to anus. A dark brown blotch at dorsal fin-origin. Flank plain brownish or with 10-13 irregular shaped brownish bars or blotches. Back in front of dorsal-fin with zero or four dark brownish blotch. Up- per part of caudal peduncle with 3—4 irregularly shaped dark brownish blotches, not fused with midlateral bars or blotches in most individuals. One irregular shaped small black spots on caudal-fin base. Dorsal fin with 1—2 and caudal fin with 2-3 fine, irregularly shapes black bands on rays. Anal, pectoral and pelvic fins plain yellowish, and pectoral fin with few small black spots on rays. Distribution. Oxynoemacheilus melenicus was found in the Biytikmelen Stream and Sakarya River drainage in western Anatolia (Fig. 10). Etymology. The name of the species is derived from the Stream Buytikmelen. Phylogenetic positions of Oxynoemacheilus melenicus and Oxynoemacheilus sakaryaensis COI barcode region sequences were analyzed in seven Oxynoemacheilus species in western Anatolia. Oxy- noemcaheilus species were divided into three main clades zse.pensoft.net in the all phylogenetic analysis supported by high boot- strap values. The first clade consisted of O. bergianus group species which are O. melenicus, O. sakaryaensis and O. simavicus O. banarescui, O. bergianus, O. saman- ticus. The second and third clades included O. fatsaensis and O. angorae, respectively. O. melenicus, constituted a highly supported clade sister to O. sakaryaensis (Fig. 13). Intrageneric K2P distances between species ranged from 1.7% (O. melenicus, O. sakaryaensis) to 8.0% (O. angorae and O. bergianus). K2P distance is 1.7% between O. me- lenicus and its closest relative, O. sakaryaensis and, 2.4% between O. melenicus and O. simavicus (Table 4). Also, the K2P distance is 2.7% between O. sakaryaensis and O. simavicus. O. melenicus differs from its most closely related congeners, O. sakaryaensis, and O. simavicus, by 9 and 12 nucleotide substitution sites and O. sakaryaensis differs from O. simavicus, by 13 nucleotide substitution sites. O. melenicus and O. sakaryaensis were differenti- ated from all other Oxynoemacheilus species in western Anatolia by two diagnostic and unique nucleotide sub- stitution sites in the COI barcoding region (Table 5). In the haplotype network analysis, the 32 distinct haplotypes were determined. The most common haplotype was H9, shared by many populations belonging to O. sakary- aensis. All species have unique haplotypes, as distinctly illustrated in the haplotype network (Fig. 14). We found eight OTUs according to ABGD and GMYC analysis. The ASAP determined nine clusters for studied species of Oxynoemacheilus. The likelihoods of GMYC and null models were 600.9632 and 595.6004, respec- tively. The GMYC analysis was represented by eight ML entities (CI: 8-11). ASAP’s best partition (score = 1.50) results from a p-distance threshold of 0.009 and both re- cursive and initial partition predicts ten subsets. ABGD analysis determined that the barcode gap is 0.012. Zoosyst. Evol. 99 (2) 2023, 439-455 0.02 90/92 98/99 71/ 88/87 [78 96/99 O. melenicus OQ332806 O. melenicus OQ332811 O. melenicus OK316620 O. melenicus 0Q332807 O. melenicus 0Q332810 O. melenicus 0Q332812 O. melenicus O0Q332813 O. melenicus 0Q332814 O. melenicus 0Q332816 O. melenicus 0Q332817 O. melenicus 0Q332818 O. melenicus 0Q332819 O. melenicus 0OQ332815 O. melenicus OK31688 O. melenicus MH018855 O. melenicus OK316739 O. melenicus O1K316642 O. melenicus MH018856 O. melenicus MH018854 O. melenicus 01316651 O. melenicus 0Q332809 O. melenicus 0Q332808 O. melenicus OK316798 O. sakaryaensis 0OQ332826 O. sakaryaensis 0Q332821 O. sakaryaensis OQ332824 O. sakaryaensis OK316616 O. sakaryaensis OK316622 O. sakaryaensis OK316635 O. sakaryaensis O1K316735 O. sakaryaensis OK316743 O. sakaryaensis OK316796 O. sakaryaensis 0OQ332822 O. sakaryaensis 0OQ332823 O. sakaryaensis 0Q332825 O. sakaryaensis 0Q332827 O. sakaryaensis OQ332828 O. sakaryaensis OQ332820 O. sakaryaensis OQ332829 O. sakaryaensis 0Q332830 O. sakaryaensis 0OQ332831 O. simavicus KJ553724 88/96] 0. simavicus KJ553970 98/97 98/98 85/78 97/99 O. simavicus 0Q332835 O. simavicus 0Q332832 O. simavicus 0Q332833 99/99) Q. simavicus 0Q332834 O. banarescui OK316694 O. banarescui MH469262 93/94L O. banarescui MH469261 91/96 O. samanticus MH018863 O. samanticus OK316652 9192L O. samanticus MH018861 O. bergianus MKK546446 O. bergianus MH469265 O. bergianus MH469266 O. bergianus OL855763 O. bergianus OK316672 98/99) OQ. bergianus OK316691 99/100 84/80 O. bergianus MK546449 O.7. bergianus OL855766 O. bergianus MK546448 O. bergianus OL855767 O. bergianus MK546447 O. bergianus OL855765 O. bergianus OL855764 O. fatsaensis OL855789 O. fatsaensis OL855790 O. fatsaensis OL855791 O. angorae OL855744 O. angorae OL855745 O. angorae OL855747 O. cemali MH018866 Seminemacheilus lendii KP 163989 ABGD GMYC ASAP 451 Figure 13. Maximum likelihood tree based on mitochondrial cytochrome oxidase subunit I (COI; 645 bp) gene sequences of Oxy- noemacheilus spp. Maximum likelihood and Neighboor Joining analyses resulted in congruent trees. Bootstrap and posterior prob- ability values are shown above nodes on tree if 70% or higher. ABGD, GMYC and ASAP clusters were indicated in vertical bars. zse.pensoft.net 452 H32 H31 H22 Turan, D. et al.: Two new Oxynoemacheilus species in western Anatolia H13 H11 H12 H10 H14 H15 H17 1 fsa} ples HIS (eae @ O. melenicus @ 0. sakaryaensis @ O. simavicus oO O. fatsaensis e@ O. banarescui Oo O. samanticus Oo bergianus Bo angorae Figure 14. Median-joining network of the COI haplotypes. Circle size corresponds to sample size; one bar indicates an additional mutational step. Small black circles represent median vectors. Each small lines represent one nucleotide difference. Discussion Oxynoemacheilus melenicus and O. sakaryaensis were ge- netically identified in O. bergianus species group. Similar to this study Freyhof et al. 2022 revealed two unnamed molecular clades in the Western Black Sea (Buytkmelen and Sakarya River drainage), which could represent can- didate species. However, they could not describe these species because morphological differences could not be found. In this study, besides morphological differences, the results from the species delineation tests (ABGD, GMYC, ASAP) and phylogenetic analyses support the validity of O. melenicus and O. sakaryaensis as distinct species. Freyhof et al. 2022 detected 15 entities in the PTP analy- sis, while mPTP detected four entities representing putative species, and ASAP predicted 15 entities, which are mainly congruent to the PTP result. According to their results, O. banarescui, O. fatsaensis, O. samanticus, O. simavicus, and specimens from Sakarya and Buytik Melen were deter- mined as different clusters, and O. bergianus was divided into two clusters. In our ABGD and GMYC analysis, eight entities were defined, which are O. melenicus, O. sakary- aensis, O. simavicus, O. banarescui, O. bergianus, O. sa- manticus. O. fatsaensis and O. angorae. Unlike ABGD and GMYC, O. simavicus was divided into two clusters in the ASAP analysis. In this regard, this study reflected that the species delineation tests agree with the previous research. Freyhof et.al. (2022) reported that Oxynoemacheilus ber- zse.pensoft.net gianus group comprises ten molecular clades following congruently well-supported NJ, MP, and ML based enti- ties. Species described as O. bergianus, O. banarescui, O. fatsaensis, and O. simavicus from Turkey, O. lenkoranensis from Azerbaijan, and O. /ongipinnis and O. parvinae from Iran belong to this species group. In addition to these, it also includes four unnamed molecular clades. We did not have the opportunity to examine O. lenkoranensis, O. longipin- nis, O. parvinae, and four unnamed groups due to the lack of specimens from their localities. However, we examined many specimens from their distribution area from Oxynoe- macheilus bergianus, O. banarescui, O. fatsaensis, O. sa- manticus. We concluded that O. bergianus, O. banarescui, O. fatsaensis, O. samanticus, and O. simavicus, together with O. sakaryaensis and O. melenicus are valid species. Freyhof et al. (2022) did not make a detailed compari- son, considering that the Sakarya and Buytikmelen popu- lations are O. simavicus. They gave only brief information about the color and pattern characteristics of the O. sima- vicus. Freyhof et al. 2022 stated that O. simavicus with mottled and blotched individuals are common, including those from the Simav, Biytikmelen, and Sakarya River drainages. According to our results, Oxynoemacheilus sa- karyaensis is characterized by having a flank with plain or with numerous irregularly shaped pale brownish bars and O. melenicus with a flank with 10-13 irregularly shaped brownish bars or blotches. However, O. simavicus is char- acterized by a flank with 2—8 dark brownish blotches. Zoosyst. Evol. 99 (2) 2023, 439-455 Comparative material Oxynoemacheilus angorae: FFROO1389, 2, 49-63 mm SL; Turkey: Ankara prov.: stream Ber¢in at Kizilcaham- am, 40.471N 32.655E. — FFRO1506, 5, 37-63 mm SL; Turkey: Bolu prov.: stream Uludere at Hendek, 40.809°N, 30.758°E. — FFRO1513, 14, 41-60 mm SL; Turkey: An- kara prov.: stream Bercin at Kizilcahamam, 40.482°N, 32.653°E. — FFRO1521, 6, 60-77 mm SL; Turkey: Bilecik prov.: stream Goéynuk 4 km west of Goyntik, 40.386°N, 30.746°E. — FFRO1524, 4, 51-63 mm SL; Turkey: Kiuta- hya prov.: stream Porsuk on road from Kutahya to Usak, 39.350°N, 30.038°E. — FFRO1526, 6, 45-63 mm SL; Tur- key: Ankara prov.: stream Kirmir 3 km north of Gidul, 40.236°N, 32.261°E. — FFRO1547, 9, 46-54 mm SL; Tur- key: Iznik prov.: stream Karasu 6 km southeast of Iznik, 40.468°N, 29.681°E. — FFRO1549, 50, 20-59 mm SL; Turkey: Ankara prov.: stream Pegenek 7 km east of Seref- likoghisar, 40.471°N, 32.655°E. — FFRO1550, 2, 34-51 mm SL; Turkey: Yozgat prov.: stream Delice at 2 km southeast of Yerkoy, 39.622°N, 34.490°E. — FFRO1554,116, 32-62 mm SL; Turkey: Kastamonu prov.: stream Devrez 6 km south- east of Tosya, 40.984°N, 34.099°E. — FFRO1557, 14, 29- 43 mm SL; Turkey: Kursehir prov.: Kizilirmak River at Kes- ikkoprti, 38.961°N, 34.199°E. — FFRO1558, 43, 20-67 mm SL; Turkey: Kirsehir prov.: stream Kilic6zti Kizilcahamam 2 km north of Ozba8, 39.241°N, 34.128°E. — FFRO1559, 4, 51-61 mm SL; Turkey: Cankiri prov.: stream Devrez 2 km south of Ilgaz, 40.904°N, 33.638°E. — FFRO1571, 5, 52- 67 mm SL; Turkey: Eskisehir prov.: stream Purtek 11 km southeast of Sivrihisar, 39.518°N, 31.618°E. Oxynoemacheilus banarescui: FFR15511, 4, 43- 77 mm SL; Turkey: Kastamonu prov.: stream Devrekan1 8 km northeast of Devrekan1, 41.627°N, 33.922°E. — IUSHM 2018-1403, 10, 50 -62 mm SL; Turkey: Karabtik prov.: stream Davulga at 15 km north of Mengen, 41.087°N, 32.023°E. Oxynoemacheilus cemali: FFRO1359, 4, 53-73 mm SL; Turkey: Giresun prov: stream Dereli at Dereli, 40.740°N, 38.450°E. — FFRO1440, 7, 31-64 mm SL; Turkey: Erzin- can prov: stream Koroglu 2 km south of Kurelik, 39.932°N, 38.749°E. — FFRO1441, 12, 52-95 mm SL; Turkey: Gutmiushane prov: stream Degirmen at Yukar16zliice village, 39. 986°N, 39.540°E. — FFRO1470, 16, 47—72 mm SL; Tur- key: Erzincan prov: stream Koroglu 2 km north of Refahiye, 39.920°N, 38.760°E. — FFRO1515, 12, 30-83 mm SL; Tur- key: Samsun prov: stream Tersakan 5 km northeast of Hav- za, 40.989°N, 35.717°E. — FFRO1582, 26, 49-70 mm SL; Turkey: Erzurum prov: Coruh River 500 m northwest of Ad- abas1, 40.380°N, 40.320°E— FFR 01583, 19, 68-88 mm SL; Turkey: Erzurum prov: stream Yagli at 2 km southeast of In- cesu, 40.308°N, 41.007°E. — FFRO1584, 22, 36-88 mm SL; Turkey: Erzurum prov: stream Tortum 4 km west of Demur- ciler, 40.379°N, 40.320°E. — FFRO1585, 3, 70-81 mm SL; Turkey: Erzurum prov: stream Tortum 600 m west of En- guzek Kapi Castle, 40.510°N, 41.523°E. — FFRO1586, 16, 36—64 mm SL; Turkey: Erzurum prov: stream Tortum 2 km west of Altincanak, 40.568°N, 41.596°E. — FFRO1587, 27, 453 51-75 mm SL; Turkey: Erzurum prov: stream Oltu 2 km west of Ayvali, 40.753°N, 41.854°E. — FFRO1588, 8, 60— 86 mm SL; Turkey: Artvin prov: stream Oltu 3 km south- western of Isham, 40.774°N, 41.713°E. — FFRO1589, 18, 48-72 mm SL; Turkey: Artvin prov: Coruh River at 1 km north of Bademli, 40.445°N, 40.901°E. — FFRO1590, 33, 67—78 mm SL; Turkey: Artvin prov: stream Eksinar 1 km north of Eksinar, 41.116°N, 42.056°E. — FFRO1591, 4, 66-68 mm SL; Turkey: Artvin prov: stream Kopriler 3 km northwest of Ardanuc, 41.137°N, 42.038°E. — FFRO1592, 14, 41-84 mm SL Turkey: Artvin prov: stream Okcular 2 km south of Eskikale, 41.263°N, 42.191°E. — FFRO1593, 27, 49-83 mm SL; Turkey: Artvin prov: stream Goknar 1 km north of Cayag&z1, 41.284°N, 42.232°E. — FFRO1594, 54, 46-74 mm SL; Turkey: Gumiishane prov: stream Siran 15 km northwest of Siran, 40.310°N, 39.031°E. — FFRO1596, 17, 60-70 mm SL; Turkey: Giresun prov: stream Bagirsak Alucra, 40.310°N, 38.773°E. Oxynoemacheilus fatsaensis: FFR15513, 1,55 mm SL; Turkey: Samsun prov: stream Tersakan 5 km east of Hav- za 40.990°N, 35.717°E. — FFRO1361, 5, 46-58 mm SL; Turkey: Samsun prov: stream IIhanli at Ayvacik 40.990°N, 36.634°E. — FFRO1362, 10, 37-64 mm SL; Turkey: Gire- sun prov: stream Aksu at Dereli 40.731°N, 38.460°E. — FFRO1362, 10, 37-64 mm SL; Turkey: Tokat prov: stream Kelkit 9 km north of Erba 40.759°N, 36.515°E. — FFRO1516, 31, 38-75 mm SL; same data as holotype. Oxynoemacheilus seyhanensis: FFRO1433, 9, 38— 78 mm SL; Turkey: Sivas prov.: Kizilirmak River at Zara, 39.902°N, 37.763°E. — FFRO1474, 32, 32 -79 mm SL; Turkey: Sivas prov.: Kizilirmak River 7 km north of Zara, 39.961°N, 37.739°E. — FFRO1545, 20, 51-78 mm SL; Turkey: Cankiri prov.: stream Ulusu at Cerkes, 40.820°N, 32.800°E. — FFRO1551, 1, 69 mm SL; Turkey: Yozgat prov.: stream Delice 11 km northwest of Yerk6y, 39.698°N, 34.362°E. — FFRO1552, 7, 38-77 mm SL; Turkey: Yozgat prov.: stream Delice 2 km southeast of Yerkoy, 39.622°N, 34.490°E. — FFRO1578, 8, 50-67 mm SL; Turkey: Cankir1 prov.: stream Ulusu at Cerkes, 40.800°N, 32.883°E. Oxynoemacheilus bergianus: FFRO1457, 11, 64— 72 mm SL; Turkey: Malatya prov.: stream Sultansuyu 8 km east of Akcadag, 38.339°N, 38.064°E. — FFRO1467, 28, 54-64 mm SL; Turkey: Erzurum prov.: stream Bas 10 km east of Askale, 39.948°N, 40.804°E.—FFR 1457, 11, 64 -72 mm SL; Turkey: Malatya prov.: stream Sul- tansuyu 8 km east of Akcada&, 38.3388°N, 38.0620°E. —FFR 15506, 25, 33-59 mm SL; Turkey: Agri prov.: Mu- rat River 17 km west of Taslicay, 39.6785°N, 43.1887°E. Oxynoemacheilus samanticus: FFRO1553, 2, 55-62 mm SL; Turkey: Yozgat prov.: stream Delice, 2 km southeast of Yerkoy, 39.622°N, 34.490°E. — FFRO1556, 3, 37-55 mm SL; Turkey: Sinop prov.: stream Gokirmak at Carsak 11 km east of Boyabat, 41.453°N, 34.889°E. —FFRO15518, 19, 54-62 mm SL; Turkey: Samsun prov.: stream Soruk 20 km east of Vezirkoprii, 41.119°N, 35.227°E. Oxynoemacheilus simavicus: FFRO1380, 12, 38— 50mm SL; Turkey: Balikesir prov: stream Koca at Manyas. —FFRO1505, 28, 32-56 mm SL; Turkey: Balikesir prov: zse.pensoft.net 454 stream Sakar at Manyas 40.050°N, 27.962°E. — FFRO1518, 8, 44-74 mm SL; Turkey: Balikesir prov: stream Koca 4 km north of Balya 39.782°N, 27.596°E. — FFRO1520, 8, 47— 55 mm SL; Turkey: Balikesir prov: stream Degirmenbogaz1 13 km west of Manyas 40.000°N, 27.828°E. — FFRO1522, 4, 47-51 mm SL; Turkey: Balikesir prov: stream Koca at Tlica 39.878°N, 27.780°E. — FFRO1542, 4, 48-49 mm SL; Turkey: Bursa prov: stream Niltifer at Narlidere 40.244°N, 29.298°E. — FFRO1544, 4, 48-49 mm SL; Turkey: Bursa prov: stream Niltifer at Misi 40.180N 28.974E. Material used in the molecular genetic analysis Oxynoemacheilus simavicus FFR DNA 01505, 1, Turkey: Balikesir prov: stream Sa- kar at Manyas 40.050°N, 27.962°E. (GenBank acces- sion number O0Q332835) — FFR DNA 01512, 3, Turkey: Balikesir prov: stream Dursunbey 4 km south of Adaoren 39.609°N, 28.751°E. (GenBank accession numbers 0Q332832—0Q332834) Acknowledgments We thank Jorg Freyhof for his contribution to the manu- script. We thank Esra Baycelebi for taking photos of some specimens, and fieldwork. Also, we are pleased to thank Ciineyt Kaya, Ismail Aksu, Zileyha Akpinar, and Yusuf Bektas for their great help during fieldwork in the studied area. Many thanks also to Baran YogurtcuoSlu (Ankara), Mufit Ozulu® (Istanbul), and Mahmut Elp (Kastamonu) for providing material and editor and reviewers, who took the burden to read the manuscript for valuable comments. This study was supported by the Scientific Research Proj- ect Coordination Unit of Recep Tayyip Erdogan Univer- sity (Project No: FBA-2022-1419). 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