Chondrostoma smyrnae, a new nase from the Tahtalı reservoir drainage in the Aegean Sea basin (Teleostei, Leuciscidae)

Chondrostoma smyrnae, a new species, from the Tahtalı reservoir drainage is distinguished by having a slightly arched lower jaw with a well-developed keratinised edge, a deep and cylindric body, a complete lateral line with 47–52+1 total scales, 8–9 scale rows between the lateral line and the dorsal-fin origin, 4 scale rows between the lateral line and the pelvic fin-origin, and 19–23 gill rakers on the first gill arch. Moreover, molecular analyses using full cyt b (1141 bp) and partial coI (652 bp) sequences of the mitochondrial genome from specimens of the new species, C. smyrnae and specimens belonging to other Chondrostoma species from central and western Anatolia demonstrated that the C. smyrnae is easily differentiated by their high pairwise genetic distances of cyt b and coI data set (>2.20 and 1.03%, respectively) and by their position in the phylogenetic trees obtained through Maximum Likelihood (ML) methodology.


Introduction
Nases of the genus Chondrostoma Agassiz, 1832 are medium-to large-sized leuciscid fishes geographically widespread from France to the Volga River and Central Iran (Elvira 1997, Kottelat and Freyhof 2007, Küçük et al. 2017, Güçlü et al. 2018. Some species of Chondrostoma have a wide distribution (as C.nasus) and exhibit ecological and morphological diversity Freyhof 2007, Durand et al. 2003), with some ambiguities relating to the taxonomy of some morphologically defined as C. fahirae (Elvira 1987, Durand et al. 2003, Freyhof and Özuluğ 2009. The major divergence times within Chondrostoma in Turkey coincided with the tectonic and climatic evolution of Anatolia in the Late Pliocene and Early Pleistocene which were seen as the uplift of the Anatolian plateau and an ice age that involved both the migration and isolation of species, respectively (Çiftçi et al. 2020).
Currently, 14 species of the genus lives in Turkey, of which eight are endemic to Anatolia (Güçlü et al. 2018(Güçlü et al. , Çiftçi et al. 2020). In the Eastern Aegean Sea basin, 4 species are known: C. fahirae (Ladiges, 1960) from the Dalaman River drainage, C. holmwoodii (Boulenger, 1896) from the Gediz and Bakır River drainages, C. meandrense Elvira, 1987 from the Lake Işıklı basin, and C. turnai Güçlü, Küçük, Turan, Çiftçi & Mutlu, 2018 from the lower and middle Büyük Menderes River drainage. Additional species distributed adjacent to the Eastern Aegean basin are C. angorense Elvira, 1987, which is widespread in the Southern Marmara and Black Sea basins, and C. beysehirense Bogutskaya, 1997 that is endemic to tributaries of the Lake Beyşehir in Central Anatolia.
During a revision project of the genus Chondrostoma in Turkey, we already described three new Chondrostoma species suggested as evolutionary lineages by Geiger et al. (2014), including C. turnai from the Büyük Menderes River (Güçlü et al. 2018), C. toros Küçük, Turan,

Materials and methods
The care of experimental animals was consistent with the Republic of Turkey animal welfare laws, guidelines and policies approved by Süleyman Demirel University Local Ethics Committee for Animal Experiments (permit reference number 2011/6/5). Samples were collected by electroscoker. After anaesthesia, samples of caudal fin tissue taken from each specimen for the molecular analysis were fixed and stored in 98% ethanol and fish were fixed in 4% formaldehyde. Measurements were made with a dial caliper and recorded to 1 mm. All measurements were made point-to-point, never by projections. Methods for counts and measurements follow Kottelat and Freyhof (2007). Standard length (SL) was measured from the tip of the snout to the posterior extremity of the hypural 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 counted from the first scale touching the shoulder girdle to the posterior-most scale at the end of the hypural complex. Scales on the caudal-fin were indicated by "+". The last two branched rays articulating on a single pterygiophore in the dorsal and anal fins were counted as "1½". The simple dorsal-and anal-fin rays were not counted since the anteriormost rays are deeply embedded.
For osteological preparations, one specimen of a new species (168.8 mm SL) and one specimen of C. turnai (139.4 mm SL) were cleared and stained with alizarin red S, according to the protocol of Taylor and van Dyke (1985). The specimens were examined using a stereomicroscope (Nikon SMZ1500), photos taken with a digital machine with a glycerol bath. The nomenclature of the skeletal elements followed Bogutskaya (1996)

Phylogenetic reconstruction
For the phylogenetic analyses, two data sets were used, an 1140 bp fragment of cyt b and 652 bp fragment of coI sequences. Cyt b and coI sequences were aligned with the previous sequences from GenBank (Table 1) with the Clustal W algorithm (Thompson et al. 1994 available in Bioedit 7.2.5 (Hall 1999)) with default parameters (gap opening: 10.00 and gap extension: 0.10; Hall 2008) and all alignments were inspected and corrected visually. Sequences obtained in this study were deposited in GenBank (accession numbers: MT387055-MT387058 for the cyt b and MW719591-MW719611 and MW722822-MW722824 for the coI).
After translating the nucleotide sequence of protein coding genes to proteins using MEGA X (Kumar et al. 2018), we found no stop codons or indels present in this alignment. Haplotypes were detected using DnaSP v5 software (Librado and Rozas 2009). Duplicate sequences were not used for phylogenetic tree construction. Subsequently, phylogenetic analyses have been performed over aligned nucleotides containing polymorphic sites for two different data sets (cyt b and coI). Computation of phylogenetic tree reconstructions of haplotypes was carried out using Maximum Likelihood (ML) analyses. ML analyses for these two datasets were performed using PhyML version 3.0 (Guindon and Gascuel 2003), with 1000 bootstraps under the best-fit models (TIM3+G for cyt b, TPM1uf+G for coI) which were calculated by the Akaike and Bayesian Information Criteria (AIC and BIC) approaches in the programme jModelTest 0.1.1 (Posada 2008). All of the trees deduced from the Cyt b and CoI sequences were rooted with Achondrostoma arcasii (Steindachner, 1866) and Pseudochondrostoma willkommii as an outgroup taxa. Constrained trees were generated in TreeWiev (Page 1996). A calculation of pairwise genetic distance among different species with Kimura 2-parameter (K2P) distances model (Kimura 1980) was performed using MEGA X (Kumar et al. 2018).

Molecular analysis
The nucleotide sequences of both strands resulted in the full length of mitochondrial cyt b (1141 bp) for the samples. Four haplotype sequences belonging to Chondrostoma smyrnae were compared with other Chondrostoma sequences from Western Anatolia available from GenBank (Table 1). A total of 1140 homologous sites for each of the 32 individual sequenced were aligned, with 215 (18.9%) variable and 119 (10.4%) variable characters parsimony-informative under the maximum parsimony optimality criterion. For cyt b gene, Intrageneric K2P distances among analysed Chondrostoma species from western Anatolia ranged from 1.26% between C. angorense and C. meandrense to 9.95% between C. fahirae and C. turnai ( Table 2). The mean intraspecific divergence ranged between 0.13% for C. smyrnae and 0.53% for C. holmwoodii (Table 2). In addition, the pairwise genetic distances between C. smyrnae and the other species ranged from 2.20% to 9.88% ( Table 2). The dataset for cytochrome c oxidase subunit 1 (coI) included 24 individuals sequenced by this study and eight individuals from GenBank belonging to seven Chondrostoma species (Chondrostoma smyrnae, C. turnai, C. meandrense, C. holmwoodii, C. beysehirense, C. angorense and C. nasus) in western Anatolia (Table 1). A total of 652 characters for each of the 36 individuals sequenced were aligned, 597 (91.6%) characters were constant, 27 (4.14%) variable characters were parsimony-uninformative and 28 (4.29%) characters were parsimony-informative under the maximum parsimony optimality criterion. For coI gene, Intrageneric K2P distances among analysed Chondrostoma species from western Anatolia ranged from 0.38% between C. angorense and C. beysehirense to 1.47% between C. holmwoodii and C. turnai ( Table 2). The mean intraspecific divergence ranged between 0.00% for C. angorense and C. nasus and 0.21% for C. smyrnae (Table 2). In addition, the pairwise genetic distances between C. smyrnae and the other species ranged from 1.03% to 1.32% (Table 2).
Phylogenetic relationships among the sequences were reconstructed independently for cyt b and coI genes using the ML method. In the phylogenetic reconstructions for cyt b, Chondrostoma haplotypes formed three distinct monophyletic clades, hereafter referred to as clades I, II, and III as in Figure 1, which were consistently supported by high bootstrap values (100). The first clade consisted of C. holmwoodii, C. smyrnae and C. turnai while the other clade contains C. angorense, C. meandrense and C. nasus. C. smyrnae formed a well-supported clade sister to C. turnai and both taxa were clustered in a monophyletic group distinguished from C. holmwoodii. In addition, C. fahirae were positioned basal to all species of Chondrostoma (Figure 1).
For coI, the phylogenetic tree demonstrated that Chondrostoma species are divided into four clades ( Figure 2). Clade I is formed by C. meandrense and C. nasus species while clade II is made up of the C. turnai, C. smyrnae and C. holmwoodii. Compared to cyt b results, the position of C. angorense was clearly separated from the clade including C. meandrense and C. nasus species, and formed a distinct and well-supported phylogenetic clade (93). C. smyrnae formed a well-supported lineage (85) and clustered together with C. turnai.
All phylogenetic analysis showed that these species are separated from one another. Also, C. smyrnae individuals from the Tahtalı reservoir drainage in the Aegean Sea basin are concluded to be distinct members of the Chondrostoma species studied in all phylogenetic trees.  Diagnosis. Chondrostoma smyrnae is distinguished from other species occur to adjacent basin by a cylindrical body (body width at dorsal-fin origin 16.8-19.3% SL, vs. 13.3-15.4 in C. turnai (Fig. 3d), 14.1-16.6 in C. meandrense, 12.0-16.3 in C. holmwoodii, 12.4-15.7 in C. fahirae, except C. beysehirense), a wider head (head width at anterior margin of eye 55-65% HL, vs. 42-54), by having less lateral line scales (48-53 vs. 60-67 in C. beysehirense, 60-66 in C. holmwoodii and 56-60 in meandrense, except C. turnai and C. fahirae). Chondrostoma smyrnae is further distinguished from C. turnai by the absence keel between pelvic fin-origin and anus (vs. present in specimens larger than 160 mm SL), a straight or slightly arched lower jaw (vs. arched), more total lateral line scales (48-53, vs. 44-51), and fewer gill rakers on first gill arch (19-23, vs. 22-27). Also, C. smyrnae further differs from C. turnai by the shape of jaws, hyomandibular, quadrate and the fifth brachial gill arc. In C. smyrnae, the dentary thick and coronoid process inclined forward (vs. thin and coronoid process nearly vertical); premaxilla very deep and posterior edge short (vs. slender and posterior edge long); hyomandibular long and narrow (vs. short and wide), the fifth brachial gill arc wide angle (vs. narrow angle) and pharyngeal teeth wide (vs. thin); outer margin of quadrate slightly pointed (vs. rounded) (Figs 4, 5).
The new species is also distinguished from C. meandrense by the body colour silvery in life (silvery, vs. brownish). It is further distinguished from C. holmwoodii by having 8-9 scale rows between the lateral line and dorsal-fin origin (vs. 9-11), four scale rows between the lateral line and pelvic-fin origin (vs. 6-7). Chondrostoma smyrnae is further distinguished from C. fahirae by having of 19-23 gill rakers on the first gill arch (vs. 12-14), well keratinised cutting edge present on the lower jaw (vs. slightly) (Fig. 6).
Description. See Figures (3) for general appearance, Table 3, 4 for morphometric and Table 5, 6 for meristic data. A small-sized individual of Chondrostoma symrnae with a cylindrical body shape is shown on Figure 7. Dorsal and ventral body profiles markedly convex. Interorbital area slightly convex. Mouth inferior, arched, with a keratinized cutting edge on lower jaw. The lower jaw slightly arched. Snout long, length 31-35% HL, with slightly rounded tip. Rostral cap almost covers upper jaw. Lower jaw relatively long, lower jaw-quadrate junction on vertical through eye pupil margin. Eye diameter about equal to interorbital distance. Caudal peduncle depth fits 10-12 times in its length. Dorsal-fin outer margin markedly concave. Anal-fin outer margin concave. Caudal-fin deeply forked, lobes with pointed tips. Outer margins of pectoral and pelvic-fins slightly convex. Pharyngeal teeth in one row, 5-6, 5-5, sharp, serrated, hooked at tip (Fig. 5). Dentary thick and coronoid process inclined forward. Premaxilla very deep and its posterior edge short. Hyomandibular long and narrow. Fifth branchial gill arc wide angle and pharyngeal teeth wide. Outer margin of quadrate slightly pointed.  The number of lateral line scales, scale rows between the lateral line and dorsal-fin origin, scale rows between the lateral line and pelvic-fin origin, branched dorsal-fin rays, branched anal-fin rays and rakers on the outer side of the first gill arch are shown in Tables 5-6.
Colouration. In life: fins pinkish with hyaline margins; back brown; flank silvery with pinkish hue. After fixation: back and upper portion of flank dark greyish; mid-lateral portion of flank and belly yellowish. Dorsal and caudal fins dark grey, pelvic and anal fins yellowish. Peritoneal membrane black.
Distribution. Chondrostoma smyrnae is known from the Tahtalı reservoir basin (Fig. 8). It is also expected to be native to the Küçük Menderes River drainage but at-tempts to find it there have thus far proven unsuccessful and it may have been extirpated.
Etymology. The species is named for Smyrna, the historic name of the city known today as Izmir. A noun in genitive, indeclinable.
In other words, their high genetic distance and position in the phylogenetic trees with high bootstrap values easily differentiated C. smyrnae from other species. The results on comparative morphological and genetic studies based on cyt b and coI genes showed that the new species differ from formerly described Chondrostoma species.

Comparative Material
Material used for morphometric and meristic comparison