Two new Oxynoemacheilus species in western Anatolia (Teleostei, Nemacheilidae)

Oxynoemacheilus sakaryaensis sp. nov. , is restricted to the Sakarya River basin, and O. melenicus sp. nov. , is distributed in both the Sakarya River and Büyükmelen 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 is distinguished 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.


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% formaldehyde 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 caudal-fin base.The last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are counted as "1½".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 several Oxynoemacheilus show an exposed lachrymal bone, which is often called a suborbital flap or groove.We distinguish 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.Therefore, we provide keys for western Anatolia only.Differential species diagnoses are given against related species as derived by the COI molecular analysis presented here and against geographically adjacent species.No differential diagnosis is provided against largely unrelated and/or geographically distant species.
Abbreviations used: SL, standard length; K2P, Kimura 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.

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 sequences were submitted to NCBI GenBank with accession numbers OQ332806-OQ332835.Phylogenetic relationships 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 model 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 haplotype network.We used three single-locus species delimitation 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 using 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/abgd/abgdweb.html,accessed on 1 May 2023) and ASAP species delimitation was performed via the ASAP web server (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html,accessed on 1 May 2023) and the following parameters: Pmin = 0.001, Pmax = 0.1, 1000 replicates, and the Kimura evolutionary model, with TS/TV = 2.0.The same parameters 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-ciation:Yule process as the tree prior with 5 million generations, and sampling frequency of 1000.The resulting ultrametric 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).

Results
Key to Oxynoemacheilus bergianus species group in the Anatolia  Diagnosis.Oxynoemacheilus sakaryaensis is distinguished from O. melenicus by having the flank plain yellowish or numerous irregularly shaped pale brownish bars (vs. the flank with 10-13 irregular shaped dark brownish bars or blotches, 0-2 irregularly shaped brownish saddle in front of dorsal-fin origin (vs.3-4) and caudal peduncle depth 2.8-3.2 times in its length (vs.1.9-2.8).Oxynoemacheilus sakaryaensis is distinguished from O. angorae 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 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) Oxynoemacheilus sakaryaensis is distinguished from O. simavicus 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).Oxynoemacheilus sakaryaensis is 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.5-10 black or dark brown bars or blocks on flank (Fig. 6).Oxynoemacheilus sakaryaensis is 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 sakaryaensis is 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 distance 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 marmorate 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,          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 midlateral 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.Diagnosis.Oxynoemacheilus melenicus is distinguished from O. angorae by having a more slender caudal pedincle (caudal pedinuncle depth 1.9-2.8times 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 emarginate), 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 always shorter than outer rostral barbells (vs.equal or longer).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,.Oxynoemacheilus melenicus is distinguished from O. samanticus by having the with 10-13 irregu-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).Oxynoemacheilus melenicus is distinguished from O. fatsaensis by having the flank plain brownish or with 10-13 irregular 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 sp. nov.
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 prominent 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-  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. 1.4-2.0).
Description.See Figs 11, 12 for general appearance and Table 3 for morphometric data.Adult size large (maximum 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 maxillary barbel; outer almost not reaching to vertical through anterior eye margin.Maxillary barbel, not reaching to posterior 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 adipose crest on caudal peduncle.Pelvic fin not reaching vertical 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.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.Upper 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 Büyükmelen Stream and Sakarya River drainage in western Anatolia (Fig. 10).
Etymology.The name of the species is derived from the Stream Büyükmelen.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. sakaryaensis.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, respectively.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 recursive and initial partition predicts ten subsets.ABGD analysis determined that the barcode gap is 0.012.

Figure 10 .
Figure 10.Distribution of Oxynoemacheilus species in the western Anatolia.

(Figure 13 .
Figure 13.Maximum likelihood tree based on mitochondrial cytochrome oxidase subunit I (COI; 645 bp) gene sequences of Oxynoemacheilus spp.Maximum likelihood and Neighboor Joining analyses resulted in congruent trees.Bootstrap and posterior probability values are shown above nodes on tree if 70% or higher.ABGD, GMYC and ASAP clusters were indicated in vertical bars.

Figure 14 .
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.
Phylogenetic positions of Oxynoemacheilus melenicus and Oxynoemacheilus sakaryaensis COI barcode region sequences were analyzed in seven Oxynoemacheilus species in western Anatolia.Oxynoemcaheilus species were divided into three main clades in the all phylogenetic analysis supported by high bootstrap values.The first clade consisted of O. bergianus group species which are O. melenicus, O. sakaryaensis and O. simavicus O. banarescui, O. bergianus, O. samanticus.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).K2P distance is 1.7% between O. melenicus 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 differentiated from all other Oxynoemacheilus species in western Anatolia by two diagnostic and unique nucleotide substitution sites in the COI barcoding region (Table

Table 4 .
Pairwise distance Kimura's two parameters (K2P) values based on cytochrome oxidase sequences of Oxynoemacheilus species. O.