Research Article
Print
Research Article
Chondrostoma smyrnae, a new nase from the Tahtalı reservoir drainage in the Aegean Sea basin (Teleostei, Leuciscidae)
expand article infoFahrettin Küçük, Yılmaz Çiftçi§, Salim Serkan Güçlü, Davut Turan|
‡ Isparta University of Applied Sciences, Isparta, Turkey
§ Ordu University, Ordu, Turkey
| Recep Tayyip Erdoğan University, Rize, Turkey
Open Access

Abstract

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.

Key Words

Cytochrome b, Cytochrome oxidase I, freshwater fish, taxonomy, Western Anatolia

Introduction

Nases of the genus Chondrostoma Agassiz, 1832 are medium-to large-sized leuciscid fishes 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 (Kottelat and 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, 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, Güçlü, Mutlu & Çiftci, 2017 from the Göksu River, and C. ceyhanensis Küçük, Turan, Güçlü, Mutlu & Çiftci, 2017 from the Seyhan and Ceyhan rivers (Küçük et al. 2017). Here, we describe an additional species from the Tahtalı River drainage in Turkey.

Materials and methods

The care of experimental animals was consistent with the Republic of Turkey animal welfare laws, guidelines and policies approved by 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).

Abbreviations used

SL standard length;

BI Bayesian Inference;

ML Maximum Likelihood;

mt mitochondrial.

Collection codes:

IFC-ESUF Inland Fishes Collection, Eğirdir Fisheries Faculty of Isparta University of Applied Sciences;

FFR Recep Tayyip Erdogan University Zoology Museum of the Faculty of Fisheries, Rize;

FSJF Fischsammlung J. Freyhof, Berlin.

DNA extraction, PCR amplification and sequencing

Total DNA was isolated from ethanol-preserved tissue samples using the Invitrogen PureLink Genomic DNA Mini Kit according to the manufacturer’s instructions and stored at −20 °C prior to use. The mitochondrial cytochrome b (cyt b) gene (1141 bp) was amplified using Forward (5’– AAT GAC TTG AAG AAC CAC CGT-3’) and Reverse (5’– CAA CGA TCT CCG GTT TAC AAG AC-3’) (Robalo et al. 2007) primers. Cytochrome c oxidase subunit 1 (coI) barcode region (652 bp) was amplified using the FishF1 (5’-TCAACCAACCACAAAG ACATTGGCAC-3’) and FishR1 (5’-TAGACTTCTGGGTGGCCAAAGAATCA-3’) (Ward et al. 2005) primer pairs. PCR reactions contained 5 µl of template DNA (25–50 ng/µl), 2 µl each of forward and reverse primers (10 pM of each primer), 25 µl of PCR Master Mix (2X) (Promega) and ddH2O for 50 µl reaction mixture. PCR amplifications were performed using a Techne (TC-Plus) thermal cycler with the conditions as follows: after a preliminary denaturation at 94 °C for 4 min, each of the 35 cycles consisted of denaturation at 94 °C for 1 min, annealing (for cyt b, 30 s at 60 °C and for coI, 30 s at 54 °C), and primer extension at 72 °C for 1.5 min (1 min for coI) and a final extension at 72 °C for 10 min, followed by cooling to 4 °C. The PCR products were purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, and bidirectional sequencing of purified PCR products was performed using the same PCR primers on an Applied Biosystems 3730 XL Genetic Analyser (Applied Biosystem, Foster City, CA, USA) using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystem) at the Macrogen Inc. (Amsterdam, Netherlands) (https://dna.macrogen-europe.com/eng/).

Phylogenetic reconstruction

For the phylogenetic analyses, two data sets were used, an 1140 bp fragment of cyt b and 652 bp fragment of 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: MT387055MT387058 for the cyt b and MW719591MW719611 and MW722822MW722824 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).

Table 1.

List of Chondrostoma species analysed, sampling locality, sources and GenBank accession numbers.

Species River/Drainage Country Sources Accession numbers
Cyt b CoI
C. holmwoodii Gölmarmara/ Akpınar Turkey Çiftci et al. (2020) MT387078
Gediz river/ Derbent Turkey Çiftci et al. (2020) MT387099
Bakırçay/ Bergama Turkey Durand et al. (2003) AF533765
Gediz river/ Uşak Turkey Durand et al. (2003) AF533762
Bakır stream Turkey Geiger et al. (2014) KJ553066.1
Gediz river /Uşak Turkey This study MW719600
Gediz river /Uşak Turkey This study MW719598
Gediz river /Uşak Turkey This study MW719599
Bakır stream Turkey Geiger et al. (2014) KJ552962.1
C. smyrnae Şaşal stream/ Tahtalı Turkey This study MT387055 MW722822
Şaşal stream/ Tahtalı Turkey This study MT387056 MW722823
Şaşal stream/ Tahtalı Turkey This study MT387057 MW722824
Şaşal stream/ Tahtalı Turkey This study MT387058
C. turnai Çine stream Turkey Çiftci et al.(2020) MT387091
Çine stream Turkey Çiftci et al. (2020) MT387093
Çine stream Turkey Çiftci et al. (2020) MT387092
Çine stream Turkey Çiftci et al. (2020) MT387095
Çine stream Turkey Çiftci et al. (2020) MT387094
Büyük Menderes Turkey Geiger et al. (2014) KJ553058.1
Yayla lake/Buldan Turkey This study MW719601
Yayla lake/Buldan Turkey This study MW719602
Yayla lake/Buldan Turkey This study MW719603
Büyük Menderes Turkey Geiger et al. (2014) KJ553026.1
Büyük Menderes Turkey Geiger et al. (2014) KJ552989.1
C. angorense Akin stream/ Sakarya Turkey Çiftci et al. (2020) MT387066
Porsuk stream/ Sakarya Turkey Çiftci et al. (2020) MT387114
Porsuk stream/ Sakarya Turkey Çiftci et al. (2020) MT387112
Kızılırmak river Kırşehir Turkey Perea et al. (2010) HM560078
Porsuk stream/ Sakarya Turkey Çiftci et al. (2020) MT387113
Sakarya river/ Kızılcahamam Turkey This study MW719604
Sakarya river/ Kızılcahamam Turkey This study MW719605
Kızılırmak river/ Zara Turkey This study MW719606
Kızılırmak river/Zara Turkey This study MW719607
Delice stream/Yerköy Turkey This study MW719608
Delice stream /Yerköy Turkey This study MW719609
Kızılırmak river/ Boyabat Turkey This study MW719610
Kızılırmak river/ Boyabat Turkey This study MW719611
C. meandrense Işıklı spring/ Çivril Turkey Çiftci et al. (2020) MT387085
Işıklı spring/ Çivril Turkey Çiftci et al. (2020) MT387087
Işıklı spring/ Çivril Turkey Çiftci et al. (2020) MT387088
Işıklı spring/ Çivril Turkey Çiftci et al. (2020) MT387086
Büyük Menderes Turkey Geiger et al. (2014) KJ553109
Büyük Menderes /Çivril Turkey This study MW719591
Büyük Menderes/ Çivril Turkey This study MW719592
Büyük Menderes/ Dinar Turkey This study MW719593
Büyük Menderes/ Dinar Turkey This study MW719594
Büyük Menderes/ Dinar Turkey This study MW719595
Büyük Menderes/ Akçay Turkey This study MW719596
Büyük Menderes Turkey Geiger et al. (2014) KJ553083
C. nasus Diina River Serbia Schönhuth et al. (2014) MG806833
Soca drainage Slovenia Geiger et al. (2014) KJ553248
Simav drainage Turkey Geiger et al. (2014) KJ552881
Danube River Romania Org (2018) MF135845
Susurluk, Kocacay Turkey Durand et al. (2003) AF533760
Danube River Austria Durand et al. (2002) AY026402
Simav Stream, Yanıkburnu Turkey Çiftci et al. (2020) MT387104
Simav Stream, Yanıkburnu Turkey Çiftci et al. (2020) MT387105
Simav Stream, Yanıkburnu Turkey Çiftci et al. (2020) MT387106
Simav Stream, Bigadiç Turkey Çiftci et al. (2020) MT387109
Simav Stream, Bigadiç Turkey Çiftci et al. (2020) MT387110
C. beysehirense Beyşehir lake Turkey Çiftci et al. (2020) MT387079
Beyşehir lake Turkey Çiftci et al. (2020) MT387080
Beyşehir drainage Turkey Geiger et al. (2014) KJ552898
Beyşehir lake Turkey This study MW719597
C. fahirae Tefenni Burdur Turkey Çiftci et al. (2020) MT387128
Pseudochondrostoma willkommii KF529128 KJ554279
Achondrostoma arcasii KF529113 KJ552518

Results

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).

Table 2.

Pairwise distance of cyt b and coI data set within and among Chondrostoma species based on 1.000 bootstrap replications using the Kimura 2-parameter distance method; values in lower left cells = percent difference among taxa for cyt b, values in upper right cells = percent difference among taxa for coI and diagonal = percent difference within taxa for cyt b/coI.

C. holmwoodii C. smyrnae C. turnai C. angorense C. meandrense C. nasus C. beysehirense
C. holmwoodii 0.53 /0.12 1.19 1.47 0.77 0.95 0.93 0.85
C. smyrnae 2.95 0.13 /0.21 1.32 1.03 1.21 1.19 1.11
C. turnai 2.91 2.20 0.33 /0.09 1.01 1.18 1.16 1.08
C. angorense 2.47 2.61 2.48 0.35 /0.00 0.48 0.46 0.38
C. meandrense 2.87 2.77 2.67 1.26 0.26 /0.04 0.64 0.56
C. nasus 2.96 2.64 2.45 1.47 1.53 0.50 /0.00 0.54
C. beysehirense 3.29 2.86 3.00 2.30 2.28 2.18 0.26 /0.15
C. fahirae 9.67 9.88 9.95 9.18 8.75 9.36 9.08

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).

Figure 1. 

Maximum Likelihood (ML) estimation of the phylogenetic relationships of Chondrostoma species based on the mitochondrial cyt b sequence data. The tree was created with the TIM3+G substitution model. Branches are labelled with maximum likelihood bootstrap ≥50%.

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.

Figure 2. 

Maximum Likelihood (ML) estimation of the phylogenetic relationships of Chondrostoma species based on the mitochondrial coI barcode region. The tree was created with the TPM1uf+G substitution model. Branches are labelled with maximum likelihood bootstrap ≥50%.

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.

Key to species of Chondrostoma in the Anatolian Aegean Sea basin

1 12–14 gill rakers on first gill arch; no keratinised cutting edge on lower jaw C. fahirae
19–28 gill rakers on first gill arch; a keratinised cutting edge on lower jaw 2
2 Lateral line with 56–68+2–3 scales 3
Lateral line with 43–52+1–2 scales 5
3 Lower jaw markedly arched and keratin not well developed C. meandrense
Lower jaw slightly arched and keratin is well developed 4
4 5–7 scale rows between lateral line and pelvic-fin origin. In life, a longitudinal violet stripe (band) in middle of flank from caudal-fin base to dorsal-fin base C. holmwoodii
5 scale rows between lateral line and pelvic-fin origin. Not a longitudinal violet stripe in life (band) C. angorense
5 Body width at dorsal-fin origin 13–15% SL; body width at mid-point of caudal peduncle 4–6% SL C. turnai
Body width at dorsal-fin origin 16–19% SL; body width at mid-point of caudal peduncle 6–8% SL C. smyrnae

Chondrostoma smyrnae sp. nov.

Fig. 3a–c

Holotype

IFC-ESUF 03-1566, 191 mm SL; Turkey: İzmir prov.: Tahtalı reservoir about 2 km north of Değirmendere, 38°08'19"N, 27°07'10"E.

Paratypes

IFC-ESUF 03-1567, 22, 152–205 mm SL; IFC-ESUF 03-1568, 22, 181–272 mm SL; same data as holotype. – IFC-ESUF 03-1550, 2, 92.68–109.02 mm SL; Turkey: İzmir prov.: stream Şaşal about 1 km south of Küner, 38°11'57"N, 27°08'09"E. – FFR 2079, 1, 92 mm SL; Turkey: İzmir prov.: stream Balaban at Küner, 38.213950 27.101505.

Material used in molecular genetic analysis

IFC-ESUF DNA-03-1550, 7, Turkey: İzmir prov.: stream Şaşal about 1 km south of Küner, 38°11'57"N, 27°08'09"E (GenBank accession number: MT387055MT387058; MW719591MW719611; MW722822MW722824).

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).

Figure 3. 

a. Chondrostoma smyrnae, IFC-ESUF 03–1567, just after fixation form, 167 mm SL, Turkey: Tahtalı reservoir; b. IFC-ESUF 03–1566, holotype, 191 mm SL, Turkey: Tahtalı reservoir; c. IFC-ESUF 03–1567, paratype 205 mm SL, Turkey: Tahtalı reservoir; d. Chondrostoma turnai, IFC-ESUF 03–1557, 197 mm SL, Turkey: Çine stream.

Figure 4. 

Jaws bones of C. smyrnae (a.) and C. turnai (b.) (Dn: dentary, Pmx: premaxilla, Mx: maxilla, Crp: Coronoid process).

Figure 5. 

Hyomandibular, quadrate and pharyngeal teeth of C. smyrnae (a.) and C. turnai (b.) (Hym: hyomandibulare, qu: quadrate, Pht: Pharyngeal teeth).

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).

Figure 6. 

Ventral view of head: a. C. smyrnae, IFC-ESUF 03–1567, paratype, 189 mm SL, Turkey: Tahtalı reservoir; b. C. turnai, IFC-ESUF 03–1524, 174 mm SL, Turkey: Çine Stream; c. C. meandrense, IFC-ESUF 03–1519, 183 mm SL, Turkey: Işıklı Spring; d. C. holmwoodii, IFC-ESUF 03–1513, 156 mm SL, Turkey: Gediz River; e. C. angorense, IFC-ESUF 03–1502, 152 mm SL, Turkey: Sakarya River; f. C. fahirae, IFC-ESUF 03–1512, 109 mm SL, Turkey: Dalaman River; g. C. beysehirense, IFC-ESUF 03–1505, 288 mm SL, Turkey: Beyşehir Lake.

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.

Table 3.

Morphometric data of Chondrostoma smyrnae (holotype, IFC-ESUF 03-1566 and paratypes, IFC-ESUF 03-1567; n = 22).

Holotype Holotype & paratypes
mean Min Max SD
Standard length (mm) 190.5 174.7 152.3 205.0
In percent of standard length
Head length 21.9 21.1 19.9 22.7 0.7
Body depth 26.6 25.9 24.2 28.4 1.4
Body width at dorsal-fin 17.5 17.4 16.0 19.4 1.2
Body width at caudal peduncle 6.8 6.8 5.7 8.0 0.9
Predorsal distance 51.2 50.8 49.0 52.2 1.2
Prepelvic distance 52.6 53.1 51.1 54.6 1.8
Preanal distance 71.4 73.2 70.4 75.7 2.6
Pectoral-fin origin to anal-fin 49.6 52.8 49.7 58.7 1.7
Pectoral-fin origin to pelvic-fin 29.8 32.0 29.8 36.2 1.5
Pelvic-fin origin to anal-fin 19.6 22.1 19.8 24.6 0.9
Dorsal-fin depth 18.4 18.4 17.1 20.6 0.8
Anal-fin depth 16.2 15.4 14.4 16.9 0.9
Pectoral-fin length 16.9 17.3 16.4 19.9 1.0
Pelvic-fin length 15.9 14.9 11.3 17.3 0.9
Caudal peduncle length 17.9 18.5 16.5 20.4 1.4
Caudal peduncle depth 11.8 11.0 10.2 11.8 0.9
In percent of head length
Snout length 31.6 33.1 31.1 35.3 1.2
Eye diameter 18.5 20.7 17.5 22.8 1.4
Interorbital distance 41.8 41.7 38.7 46.4 2.1
Head width1 56.9 59.7 54.9 65.3 2.3
Head depth1 54.7 54.6 50.9 57.8 2.4
Head depth2 81.1 79.9 75.0 85.2 2.6
Mouth width 27.2 27.2 24.1 27.9 1.0
Table 4.

Morphometry of C. turnai (IFC-ESUF 03-1558. n = 43), C. meandrense (IFC-ESUF 03-1519. n = 24), C. holmwoodii (IFC-ESUF 03-1513. n = 19), and C. fahirae (IFCESUF 03-1512. n = 36).

C. turnai C. meandrense C. holmwoodii C. fahirae
SL (mm) 132.0–191.8 120.6–200.9 88.8–161.3 82.8–96.8
Mean min max SD mean min max SD mean Min max SD mean min max SD
In percent of standard length
Head length 21.9 20.7 23.4 0.8 22.6 21.9 23.6 0.5 23.1 21.5 24.0 0.4 24.1 23.3 25.1 0.6
Body depth 24.6 22.9 27.0 1.0 23.9 21.8 25.5 1.0 23.6 22.0 24.8 0.7 24.0 21.9 25.3 1.1
Body width at dorsal-fin 14.3 12.8 15.1 0.8 14.4 12.5 16.7 0.8 13.0 12.3 14.6 0.5 12.6 10.7 14.6 0.9
Body width at caudal peduncle 5.3 4.0 5.8 1.4 5.00 3.5 5.9 0.9 5.6 4.7 6.6 0.7 5.6 4.1 6.9 1.2
Predorsal distance 51.5 50.0 54.5 1.2 51.3 49.6 52.6 1.5 52.2 50.7 54.0 1.1 53.5 51.8 55.0 1.6
Prepelvic distance 52.7 50.1 53.8 0.9 52.4 50.7 54.0 1.6 52.7 50.6 54.2 1.4 52.5 49.8 53.8 1.2
Preanal distance 73.4 71.8 75.1 1.4 72.1 69.4 74.8 1.3 72.2 70.4 73.4 1.5 70.3 67.3 72.9 1.4
Pectoral-fin origin to anal-fin 52.9 48.7 54.3 1.4 49.9 46.1 52.4 0.9 49.9 48.2 51.1 0.6 47.1 45.7 49.9 0.6
Pectoral-fin origin to pelvic-fin 31.4 29.0 34.3 1.0 29.2 26.5 31.5 1.2 28.9 28.2 29.4 0.7 28.0 25.4 30.3 0.6
Pelvic-fin origin to anal-fin 22.0 20.5 23.0 1.2 20.9 19.5 23.5 0.5 20.9 19.8 21.6 0.4 28.0 25.4 30.3 0.6
Dorsal-fin depth 19.2 17.5 21.4 0.8 18.3 16.2 20.2 1.0 19.5 17.2 20.9 0.7 21.0 20.0 22.4 1.1
Anal-fin depth 15.3 13.5 16.4 0.9 14.9 13.4 16.8 0.8 16.2 14.8 17.1 0.6 16.6 15.0 18.8 0.9
Pectoral-fin length 18.0 16.0 20.1 0.8 17.5 15.4 19.1 0.9 18.4 18.0 19.4 0.9 20.5 18.7 21.9 0.9
Pelvic-fin length 15.6 14.1 17.5 1.0 15.4 13.8 17.5 0.9 15.4 14.6 16.2 0.8 17.3 14.1 19.0 1.2
Caudal peduncle length 17.4 14.5 18.5 0.5 18.2 16.7 20.3 0.9 17.8 16.2 19.4 0.6 19.4 18.2 20.9 0.9
Caudal peduncle depth 10.5 9.70 11.7 0.8 10.5 9.9 11.2 0.4 10.2 9.4 10.9 0.2 10.8 9.9 11.9 0.5
In percent of head length
Snout length 30.6 27.2 33.8 1.6 32.5 29.1 34.8 1.5 31.4 26.9 33.3 1.0 31.2 28.0 32.6 1.2
Eye diameter 22.2 20.1 24.7 1.3 21.9 19.7 26.3 1.7 20.6 17.6 22.4 0.8 25.7 23.2 29.1 1.7
Interorbital distance 38.2 33.1 41.9 2.1 36.4 33.7 38.7 1.4 36.1 32.7 39.0 1.1 37.8 35.1 39.6 1.6
Head width1 54.4 49.8 57.0 2.4 43.4 37.6 48.2 2.2 44.0 40.5 47.7 1.7 42.6 38.9 48.1 1.6
Head depth1 50.6 47.1 56.1 1.9 50.1 47.6 54.8 1.7 50.1 47.3 52.5 0.8 56.8 51.1 62.4 3.0
Head depth2 74.3 70.6 78.9 1.9 73.6 69.8 79.4 2.2 72.7 71.2 75.4 0.8 75.0 72.2 76.8 1.7
Mouth width 26.1 23.1 27.7 1.3 25.8 23.8 28.3 1.1 27.4 25.3 29.3 1.4 25.4 24.4 27.8 1.1
Figure 7. 

Top view of Chondrostoma smyrnae (a.) IFC-ESUF 03-1567, 194.2 mm SL; (b.) 190.0 mm SL, Tahtalı Reservoir; and C. turnai (c.) IFC-ESUF 03-1524,195.1 mm SL; (d.) 181.3 mm SL, Stream Çine.

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 56.

Table 5.

Frequency of lateral line scales, scale rows between the lateral line and dorsal-fin origin, and the lateral line and pelvic-fin origin in Eastern Aegean Chondrostoma species.

lateral line scales
N 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Mean
C. smyrnae 29 1 6 4 7 6 5 50.9
C. turnai 17 1 4 1 3 3 2 2 1 47.4
C. meandrense 14 2 2 2 6 2 58.2
C. holmwoodii 14 1 1 4 1 2 2 3 63.4
C. fahirae 24 1 1 5 6 5 3 1 1 1 47.6
scale rows between lateral line and dorsal-fin origin scale rows between lateral line and pelvic-fin origin
N 8 81/2 9 91/2 10 11 Mean 31/2 4 41/2 5 51/2 6 7 Mean
C. smyrnae 28 19 9 8.3 28 4.0
C. turnai 15 8 2 4 1 8.4 1 10 1 3 4.2
C. meandrense 14 1 11 2 9.0 14 5.0
C. holmwoodii 20 2 13 5 10.2 5 13 2 5.8
C. fahirae 10 5 5 8.5 7 3 4.3
Table 6.

Frequency of gill rakers, branched dorsal-fin rays, and branched anal-fin rays in Eastern Aegean Chondrostoma species.

Gill Rakers
N 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Mean
C. smyrnae 16 3 5 3 3 2 20.8
C. turnai 11 1 2 4 3 1 24.2
C. meandrense 20 2 2 3 6 6 1 25.8
C. holmwoodii 14 2 8 2 2 22.3
C. fahirae 7 1 3 3 13.3
Branched dorsal-fin rays Branched anal-fin rays
N 7 8 9 10 Mean 8 9 10 11 12 Mean
C. smyrnae 36 2 34 7.9 2 5 29 9.8
C. turnai 18 18 8.0 11 7 9.4
C. meandrense 14 12 2 8.1 8 6 9.4
C. holmwoodii 16 16 8.0 14 2 9.1
C. fahirae 10 10 7.0 3 7 8.7

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 attempts to find it there have thus far proven unsuccessful and it may have been extirpated.

Figure 8. 

Distribution of Chondrostoma species in western Anatolia.

Etymology

The species is named for Smyrna, the historic name of the city known today as Izmir. A noun in genitive, indeclinable.

Discussion

The identification of Chondrostoma symrnae is a significant contribution to the genus Chondrostoma. There are six species of Chondrostoma (C. angorense, C. beysehirense, C. fahirae, C. holmwoodii, C. meandrense and C. turnai) in western Anatolia. The differences between Chondrostoma smyrnae, C. fahirae, C. holmwoodii, C. meandrense and C. turnai, which occur in the Aegean Sea basin of Turkey, are given in detail in the diagnosis section.

Chondrostoma smyrnae is distinguished from C. angorense and C. beysehirense, which occur adjacent to the Aegean Sea basin of Turkey, by having 48–53 lateral line scales (vs. 59–68 in C. angorense, 60–67 in C. beysehirense), 8–9 scale rows between the lateral line and the dorsal-fin origin (vs. 9–10 in C. angorense, vs. 10–11 in C. beysehirense), four scale rows between lateral line anal-fin origin (vs. 5 in C. angorense, 5–6 in C. beysehirense) and 19–23 gill rakers on first gill arch (vs. 33–39 in C. beysehirense).

In other words, their high genetic distance and position in the phylogenetic trees with high bootstrap values easily differentiated C. smyrnae from other species. The results on comparative morphological and genetic studies based on cyt b and coI genes showed that the new species differ from formerly described Chondrostoma species.

Comparative Material

Material used for morphometric and meristic comparison

Chondrostoma angorense : IFC-ESUF 03-1501, 32, 80–174 mm SL; Turkey: Eskişehir prov.: Porsuk River about 2 km west of Yörükkırka, 39°36'00"N, 30°25'09"E. – IFC-ESUF 03-1502, 11, 35–162 mm SL; Turkey: Eskişehir prov.: stream Akin 0.5 km south of Akin, 39°20'02"N, 30°30'59"E. – IFC-ESUF 03-1503, 2, 245–300 mm SL; Turkey: Kütahya prov.: stream Emet about 10 km north of Eğriöz, 39°28'10"N, 29°15'17"E. – IFC-ESUF 03-1537, 23, 151–216 mm SL; Turkey: Kütahya prov.: stream Yanıkburnu about 25 km east of Dursunbey, 39°33'04"N, 28°56'55"E. – IFC-ESUF 03-1538, 3, 137–185 mm SL; Turkey: Balıkesir prov.: stream Bigadiç west of Bigadiç, 39°23'48"N, 28°04'50"E. – IFC-ESUF 03-1549, 1, 264 mm SL; Turkey: Afyonkarahisar prov.: stream Kali about 15 km west of Çay, 38°32'28"N, 30°50'41"E.

Chondrostoma beysehirense : IFC-ESUF 03-1505, 16, 156–251 mm SL; Turkey: Konya prov.: Beyşehir Lake about 20 km south of Şarkikaraağaç, 37°52'42"N, 31°20'46"E.

Chondrostoma fahirae : IFC-ESUF 03-1512, 36, 60–127 mm SL, Turkey: Burdur prov.: Başpınar 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 Yusufça, 37°13'37"N, 29°32'57"E.

Chondrostoma holmwoodii : IFC-ESUF 03-1513, 19, 68–160 mm SL; Turkey: Manisa prov.: Gediz River at Derbent, 38°46'37"N, 29°12'41"E. – IFC-ESUF 03-1514, 7, 58–118 mm SL; Turkey: Manisa prov.: Gediz River about 16 km east of Kula, 38°35'46"N, 28°48'30"E. – IFC-ESUF 03-1515, 1, 112 mm SL; Turkey: Manisa prov.: Gediz River about 15 km north of Kula, 38°40'08"N, 28°36'14"E. – IFC-ESUF 03-1516, 1, 145 mm SL; Turkey: Manisa prov.: Gediz River about 5 km east of Gölmarmara, 38°42'08"N, 27°58'10"E. – IFC-ESUF 03-1517, 3, 85–102 mm SL; Turkey: İzmir prov.: Gediz River about 8 km east of Menemen, 38°37'42"N, 27°10'41"E.

Chondrostoma meandrense : IFC-ESUF 03-1519, 45, 120–209 mm SL, Turkey: Denizli prov.: Işıklı Spring, 38°19'19"N, 29°51'10"E. – IFC-ESUF 03-1522, 19, 96–151 mm SL, Turkey: Denizli prov.: streem Küfi about 4 km north of Işıklı, 38°21'48"N, 29°50'56"E. – IFC-ESUF 03-1523, 20, 110–219 mm SL, Turkey: Afyonkarahisar prov.: Stream Karasandıklı 0.5 km east of Karasandıklı, 38°31'40"N, 30°10'39"E. – IFC-ESUF 03-1525, 4, 65–138 mm SL, Turkey: Afyonkarahisar prov.: Suçıkan 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.: Büyük Menderes River about 2 km west of Çıtak, 38°09'23"N, 29°38'24"E. – IFC-ESUF 03-1562, 3, 125–154 mm SL, Turkey: Denizli prov.: Büyük Menderes River about 1 km north of Hançalar, 38°07'54"N, 29°23'19"E.

Chondrostoma turnai : IFC-ESUF 03-1524, 44, 75–210 mm SL; Turkey: Aydın prov.: stream Çine about 8 km south of Aydın, 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 Güney, 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: Aydın prov.: Akçay Stream about 3 km east of Sırma, 37°36'18"N, 28°29'34"E. – IFC-ESUF 03-1569, 1, 239 mm SL; Turkey: Denizli prov.: Vali Recep Yazıcıoğlu reservoir about 3 km east of Denizli, 37°46'14"N, 29°07'39"E.

Material used in molecular genetic analysis

Chondrostoma angorense : IFC-ESUF DNA-03-1502, 1; Turkey: Eskişehir prov.: stream Akin 0.5 km south of Akin, 39°20'02"N, 30°30'59"E (GenBank accession number: MT387066). – IFC-ESUF DNA-03-1501, 3; Turkey: Eskişehir prov.: Porsuk River about 2 km west of Yörükkırka, 39°36'00"N, 30°25'09"E (GenBank accession number: MT387112MT387114). FFR DNA CH5-6, 2; Turkey: Kızılcahamam, Sakarya River drainage, (GenBank accession number: MW719604, MW719605). IFC-ESUF DNA-03-1500, 2; Turkey: Sivas prov.: Kızılırmak River about 3 km east of Zara, 39°54'27"N, 37°48'25"E (GenBank accession number: MW719606, MW719607). FFR DNA CH18-19, 2; Turkey: Yerköy, Delice stream, (GenBank accession number: MW719608, MW719609).

Chondrostoma beysehirense : IFC-ESUF DNA-03-1505, 2; Turkey: Konya prov.: Beyşehir Lake about 20 km south of Şarkikaraağaç, 37°52'42"N, 31°20'46"E (GenBank accession number: MT387079MT387080). IFC-ESUF DNA-03-1505, 1; Turkey: Konya Prov.: Beyşehir, Beyşehir Lake, 37°55'43"N, 31°21'24"E (GenBank accession number: MW719597).

Chondrostoma fahirae : IFC-ESUF DNA-03-1512, 1; Turkey: Burdur prov.: Başpınar Spring about 13 km south of Tefenni, 37°11'08"N, 29°45'16"E (GenBank accession number: MT387128).

Chondrostoma holmwoodii : IFC-ESUF DNA-03-1513, 1; Turkey: Manisa prov.: Gediz River at Derbent, 38°46'37"N, 29°12'41"E (GenBank accession number: MT387099). – IFC-ESUF DNA-03-1516, 1; Turkey: Manisa prov.: Gediz River about 5 km east of Gölmarmara, 38°42'08"N, 27°58'10"E (GenBank accession number: MT387078). IFC-ESUF DNA-03-1513, 3; Turkey: Derbent Bridge, Manisa Prov.: Gediz River drainage, 38°46'40"N, 29°12'45"E (GenBank accession number: MW719598MW719600).

Chondrostoma meandrense : IFC-ESUF DNA-03-1519, 4; Turkey: Denizli prov.: Işıklı Spring, 38°19'19"N, 29°51'10"E (GenBank accession number: MT387085MT387088). IFC-ESUF DNA-03-1519, 2; Turkey: Işıklı Village, Denizli Prov.: Işıklı Spring, Büyük Menderes River drainage, 38°19'21"N, 29°51'07”E (GenBank accession number: MW719591MW719592). IFC-ESUF DNA-03-1525, 3, Turkey: Afyonkarahisar prov.: Suçıkan Spring, Büyük Menderes River east of Dinar, 38°04'14"N, 30°10'38"E (GenBank accession number: MW719593MW719595). FFR DNA CH13, 1; Turkey: Akçay, Büyük Menderes River drainage (GenBank accession number: MW719596).

Chondrostoma turnai : IFC-ESUF 03-1557, 5; Turkey: Aydın prov.: Stream Çine about 8 km south of Aydın, 37°45'43"N, 27°50'12"E. IFC-ESUF DNA-03-1524, 3; Turkey: Çiftlikburnu Village, Aydın Prov.: Çine Stream, Büyük Menderes River, 37°42'52"N, 27°50'04"E (GenBank accession number: MW719601MW719602).

Acknowledgements

This study was supported by a grant from the Scientific and Technological Research Council of Turkey (TÜBİTAK) (Project No: KBAG-111T900). We are pleased to thank Müfit Özuluğ (İstanbul) for allowing Jörg Freyhof to participate in his many years of fieldwork. We thank Utku Avcı (Rize) for proofreading the manuscript and Esra Bayçelebi (Rize) for producing the distribution map (Fig. 8).

References

  • Bogutskaya N (1996) Contribution to the knowledge of leuciscine fishes of Asia Minor. Part 1. Morphology and taxonomic relationships of Leuciscus borysthenicus (Kessler, 1859), Leuciscus smyrnaeus Boulenger, 1896 and Ladigesocypris ghigii (Gianferrari, 1927) (Cyprinidae, Pisces). Publicaciones Especiales. Instituto Espanol de Oseanografia 21: 25–44.
  • Çiftci Y, Mutlu AG, Küçük F, Güçlü SS, Turan D (2020) Molecular phylogeny and phylogeography of genus Chondrostoma Agassiz, 1835 (Teleostei: Leuciscidae) determined by mitochondrial DNA sequences in Anatolia. Zoology in the Middle East 66(3): 206–221. https://doi.org/10.1080/09397140.2020.1788255
  • Durand JD, Bianco PG, Laroche J, Gilles A (2003) Insight into the origin of endemic Mediterranean ichthyofauna: phylogeography of Chondrostoma genus (Teleostei, Cyprinidae). Journal of Heredity 94(4): 315–328. https://doi.org/10.1093/jhered/esg074
  • Durand JD, Tsigenopoulos CS, Ünlü E, Berrebi P (2002) Phylogeny and biogeography of the family Cyprinidae in the Middle East inferred from cytochrome b DNA – evolutionary significance of this region. Molecular Phylogenetics and Evolution 22(1): 91–100. https://doi.org/10.1006/mpev.2001.1040
  • Elvira B (1997) Taxonomy of the genus Chondrostoma (Osteichthyes, Cyprinidae): An Updated review. Folia Zoologica 46: 1–14.
  • Farris JS, Kallersjo M, Kluge AG, Bult C (1995) Constructing a significance test for incongruence. Systematic Biology 44(4): 570–572. https://doi.org/10.2307/2413663
  • 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, Kärst H, Kottelat M, Kovačić M, Laporte M, Lorenzoni M, Marčić Z, Özuluğ M, Perdices A, Perea S, Persat H, Porcelotti S, Puzzi C, Robalo J, Šanda R, Schneider M, Šlechtová V, Stoumboudi M, Walter S, Freyhof J (2014) Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes. Molecular Ecology Resources 14: 1210–1221. https://doi.org/10.1111/1755-0998.12257
  • Güçlü SS, Küçük F, Turan D, Çiftci Y, Mutlu AG (2018) A new Chondrostoma species from the Büyük Menderes River basin, Turkey (Teleostei: Cyprinidae). Zoology in the Middle East 64(3): 315–321. https://doi.org/10.1080/09397140.2018.1511293
  • 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]
  • Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 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.
  • 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
  • Küçük F, Turan D, Güçlü SS, Mutlu AG, Çiftçi Y (2017) Two new species of Chondrostoma Agassiz, 1832 (Teleostei: Cyprinidae) from the Ceyhan, Seyhan and Göksu Rivers in the East Mediterranean Region of Turkey. Turkish Journal of Fisheries and Aquatic Sciences 17: 795–803. https://doi.org/10.4194/1303-2712-v17_4_15
  • Org CG (2018) Freshwater Diversity Identification for Europe (FREDIE)-funded by the German Leibniz association (SAW-2011-ZFMK-3).
  • Freyhof J, Özuluğ M (2009) Pseudophoxinus evliyae, a new species of spring minnow from Western Anatolia with remarks on the distribution of P. ninae and the systematic position of P. fahirae (Teleostei: Cyprinidae). Ichthyological Exploration of Freshwaters 20(4): 309–318.
  • Robalo JI, Almada VC, Levy A, Doadrio I (2007) Re-examination and phylogeny of the genus Chondrostoma based on mitochondrial and nuclear data and the definition of 5 new genera. Molecular Phylogenetics and Evolution 42(2): 362–372. https://doi.org/10.1016/j.ympev.2006.07.003
  • Schönhuth S, Vukić J, Šanda R, Yang L, Mayden RL (2018) Phylogenetic relationships and classification of the Holarctic family Leuciscidae (Cypriniformes: Cyprinoidei). Molecular Phylogenetics and Evolution 127: 781–799. https://doi.org/10.1016/j.ympev.2018.06.026
  • Swofford DL, Olsen GJ, Waddell PJ (1996) Phylogenetic inference. in “molecular systematics”. [Hillis DM, Moritz C, Mable BK (Eds)]Sinauer, Sunderland 14: 407–514.
  • 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 matrix choice. Nucleic Acids Research 22(22): 4673–4680. https://doi.org/10.1093/nar/22.22.4673
  • Ward RD, Zemlak TS, Innes BH, Last PR, Heber PDN (2005) DNA barcoding Australia′s fish species. Philosophical Transactions of the Royal Society B 360: 1847–1857. https://doi.org/10.1098/rstb.2005.1716