Capoeta coadi, a new species of cyprinid fish from the Karun River drainage, Iran based on morphological and molecular evidences (Teleostei, Cyprinidae)

Abstract As presently recognized, the genus Capoeta includes 24 species, nine of which are known to occur in Iran (Capoeta aculeata, Capoeta capoeta, Capoeta buhsei, Capoeta damascina, Capoeta fusca, Capoeta heratensis, Capoeta mandica, Capoeta saadii and Capoeta trutta) and are distributed in almost all Iranian basins except Sistan and Mashkid. Capoeta coadi sp. n. is a new species from the Karun River, southern Iran, draining into the Arvand Rud (Shatt al-Arab) which drains into the Persian Gulf. It is distinguished from all other species of Capoeta by the combination of the following characters: elongate and usually cylindrical body; 8–9 branched dorsal-fin rays; last unbranched dorsal-fin ray weakly to moderately ossified and serrated along 1/3–2/3 of its length; scales small; 70-84 in lateral line (total); 12–17 scales between dorsal-fin origin and lateral line; 9-11 scales between anal-fin origin and lateral line; 26–32 circum-peduncular scales; 10–13 gill rakers on lower limb of first gill arch; 45–47 total vertebrae; one posterior pair of barbels; bright golden-greenish or silvery body coloration in life; length of the longest dorsal-fin ray 15–22% SL; head length 23–26% SL; mouth width 7–10% SL. Capoeta coadi is also distinguished from all other congeners in the Iranian drainages by fixed diagnostic nucleotide substitutions in the mtDNA COI barcode region and cyt b. It is nested in the Capoeta damascina species complex.


Introduction
The Middle East is a transition zone between three major biogeographic units, the Palaearctic, the Afrotropical, and the Oriental realms. It served as an important crossroad of biotic exchange resulting in an outstanding biological diversity of freshwater fishes (Durand et al. 2002, Krupp et al. 2009). Lying between major drainages of the Nile in Africa to the west, the Indus in southern Asia to the east and the Caspian and Black Sea drainages to the north, the Tigris-Euphrates River drainage is the largest river system in the Middle East and has high fish diversity, especially in cyprinid fishes.
Capoeta Valenciennes in Cuvier and Valenciennes 1842 is an example of a cyprinid genus widely spread in the Middle East (Krupp and Schneider 1989). Being found in a wide range of habitats, species of this genus display considerable morphological variability (e.g., scale counts and colour pattern) and the extent of morphological plasticity and genetic variability remain to be determined. As a consequence, there has been considerable disagreement regarding the status of several species. However, Capoeta is considered monophyletic (Krupp 1985, Küçük et al. 2009).
Members of the genus Capoeta are cyprinids characterized by having an elongate, cylindrical body and a short dorsal fin. They have three to five unbranched and 5-9 branched dorsal-fin rays, the last unbranched ray being ossified and serrated. All species have three unbranched and 5 branched anal-fin rays. Scales are usually small. Mouth is inferior and the lower lip is covered with a horny sheath. One pair of barbels (rarely two) is present and the pharyngeal teeth are arranged in three rows. The shape of the mouth as well as the pharyngeal teeth are nearly identical in all species, which indicate their adaptation to the same mode of feeding. This combination of character states distinguishes Capoeta from all other cyprinids (Krupp 1985, Krupp andSchneider 1989).
As presently recognized, the genus Capoeta includes about 24 species (Eschmeyer and Fricke 2016) in different phylogenetic groups widely distributed in many river drainages and basins in southwestern Asia except the Arabian Peninsula (Alwan 2011, Levin et al. 2012. Levin et al. (2012) studied the phylogenetic relationships of the genus Capoeta based on complete mitochondrial gene for cytochrome b sequences obtained from 20 species from the overall range of the genus. Three main groups were detected: the Mesopotamian group (Capoeta trutta group), the Anatolian-Iranian group (Capoeta damascina group) and the Aralo-Caspian group (Capoeta capoeta group).

Material and methods
After anaesthesia, fishes were either fixed in 5% formaldehyde, and stored in 70% ethanol, or directly fixed in 99% ethanol (for molecular studies). Measurements were made with a digital caliper and recorded to 0.01 mm. All measurements were made point to point, and never by projections. Methods for counts and measurements follow Hubbs and Lagler (1958) and Krupp (1983). 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. The last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are counted as "1½".The holotype is included in the calculation of means and SD.
Abbreviations used: SL, standard length; HL, lateral head length. Abbreviations used for museum collections: Zoological Museum of Shiraz University, Collection of Biology Department, Shiraz, Iran (ZM-CBSU), the Senckenberg Research Institute and Natural History Museum (SMF: Frankfurt, Germany), and the private collection of Jörg Freyhof (FSJF: Fischsammlung J. Freyhof).
Purification and sequencing of the PCR products were conducted at Macrogen Korea Laboratories using the aforementioned primer sets.

Molecular data analyses
Data processing and sequence assembly was done in BioEdit 7.2.5 (Hall 1999); MEGA6 (Tamura et al. 2013) was used to create a DNA sequence alignment. No indications of unexpected stop-codons or nuclear copies of mitochondrial fragments occurred in any sequences. All generated DNA barcodes and cyt b were deposited in the NCBI GenBank. The most appropriate sequence evolution model for the given data was determined with Modeltest (Posada and Crandall 1998) as implemented in the MEGA6 software, treating gaps and missing data with the partial deletion option under 95% site coverage cut-off. The model with the lowest BIC (Bayesian Information Criterion) score is considered the best model to describe the substitution pattern for each gene. To explore species phylogenetic relationships, trees were generated using Maximum Likelihood analysis with 10,000 bootstrap replicates in RaxML 7.2.5 (Stamatakis 2006) under the GTR+G model of nucleotide substitution, with fast bootstrap and also Bayesian analysis (BA), using the Markov Chain Monte Carlo method (MCMC), with 6,000,000 generations under the most generalizing model (GTR+G+I) using Mr. Bayes 3.1.1 (Huelsenbeck and Ronquist 2001). Screening for diagnostic nucleotide substitutions was performed manually from the sequence alignment. As an appropriate outgroup to root the constructed phylogenetic hypothesis, we included the distantly related Cyprinus carpio.
Description. General body shape and appearance are shown in Figs 1-3, morphometric data in Table 1 and meristic data are summarized in Tables 2-9. Body elongate and cylindrical; predorsal body profile smoothly convex with no marked discontinuity between head and body except when a nuchal hump is present in few specimens; greatest body depth at level of dorsal-fin origin; snout rounded (in 20 specimens) or pointed (in 14 specimens) and not size dependent; mouth inferior; lips slightly fleshy, especially at the mouth corners; lower lip covered with a sharp-edged horny sheath, its anterior margin straight in adult specimens and rounded to almost crescent-shaped in juveniles, with a considerable degree of individual variation.
Dorsal-fin origin anterior to pelvic-fin origin, its outer margin usually straight to concave with 3-5 unbranched and 8-9 branched rays (3 and 8 in holotype, respectively); last unbranched dorsal-fin ray weakly to moderately ossified, flexible and soft at the tip, serrated in 1/2-2/3 of its length (Fig. 4); pectoral fins not extending to pelvic-fin base; their outer margins usually slightly convex with 16-22 rays in total (19 in holotype) ( Table 2); pelvic fins not extending to anal fin base, their outer margin straight or slightly convex and blunt with 7-11 rays in total (8 in holotype) ( Table 2); pelvic axillary scale present; anal fin with 3 unbranched and 5 branched rays, outer margin straight or slightly convex; caudal fin forked with 16-19 branched rays (17 in holotype) (Table 3), its tip pointed and its upper lobe often longer than lower one.
Scales small, total lateral-line scales 70-84; 12-17 scales between dorsal-fin origin and lateral line (Table 4); 9-11 scales between anal-fin origin and lateral line (Table 4); 26-32 circum-peduncle scales (Table 5); ventral midline and pectoral region covered with deeply embedded scales of reduced size; gill rakers slightly hooked, total gill rakers 14-18 (10-13 gill rakers on lower limb) of first gill arch (Table 8-9); 45-47 total vertebrae; usually one posterior pair of barbels present (very rarely two, 1 out of 51 individual); pharyngeal teeth arranged in 3 rows in the following manner: 2.3.5-5.3.2 and very similar in shape to those of C. damascina; teeth in the main row spatulate or spoon-shaped and crowns flat, narrow and curved.
Preserved specimens. Dorsum, head and sides grey or brownish-grey dorsally and beige or yellow ventrally; dorsal and caudal fins dusky grey; pectoral, pelvic and anal fins white or beige with or without grey tinge; blotches and spots well discernible (Figs 1-2).
Sexual dimorphism. Breeding tubercles present in both sexes, being bigger and more pronounced in males. Tubercles present on the sides of the snout but may also cover the entire body surface, on and above the lateral line with one or two tubercles per scale but not on each scale, below the lateral line especially in the area above the anal fin and on the branched anal-fin rays; tip of anal fin reaching to or beyond the vertical of the caudal-fin base in females and to about 2/3 of the caudal peduncle in males.  Habitat and distribution. Capoeta coadi sp. n. occurs in medium-fast flowing rivers with usually gravel substrates and clear waters (Fig. 5). At the Beshar River sampling site, the river is about 25 m wide, with substrate consisting of coarse gravel and boulders, and fast-flowing and semi-transparent waters. The physicochemical parameters at the spot were: dissolved oxygen, 9.89 mg/L; total dissolved solids, 190.2 mg/L; salinity, 0.19‰; conductivity, 395 µs/cm; pH: 8.5 and water temperature 23.4 °C. It is known only from the Karun River drainage, a system that constitutes the southeastern part of the Tigris-Euphrates River system.
Etymology. The new species is named after Brian W. Coad, a well-known ichthyologist for his valuable contribution to the knowledge of freshwater fishes of Iran.
Comparative remarks. The presence of one pair of barbels in Capoeta coadi sets the species apart from C. antalyensis, C. baliki, C. banarescui, C. tinca, and C. heratensis, all of which have two pairs of barbels based on data from Turan et al. (2006a) and this study. The new species is further distinguished from C. antalyensis by the presence of serrae on the last unbranched dorsal-fin ray (vs. absence) (Fig. 4), and by number of scales between dorsal-fin origin and lateral line (12-17 vs. 10-12 in C. antalyensis) Figure 5. Beshar River at Taleh Gah village, Karun River drainage, type locality of C. coadi. (Table 4), between anal-fin origin and lateral line (9-11 vs. 7), and by total number of the lateral-line scales (70-84 vs. 51-57) ( Table 7). Capoeta coadi is distinguished from C. banarescui by number of scales between anal-fin origin and lateral line (9-11 vs. 8-9) ( Table 4). Data for C. antalyensis and C. banarescui are from Turan et al. (2006a).

Molecular phylogenetic assessments
We generated COI barcode and cyt b sequences for a total of 76 and 61 Capoeta specimens, respectively (Tables 10-11). Two phylogenetic approaches including Maximum Likelihood and Bayesian analyses for species of Capoeta are given in Figs 6-7. Tables 12-13 provide the diagnostic nucleotide substitutions found in the mtDNA COI barcode region and cyt b, respectively.
For inter-specific differences, the greatest pairwise genetic divergence between C. coadi and its congeners was found to be 6.5 by C. erhani and lowest by C. buhsei (0.4) for COI and greatest 9.7 by C. mandica and lowest (1.5) by C. buhsei for cyt b (Tables 14-15).

Discussion
Based on morphological and molecular results, C. saadii and C. coadi are distinct species in the Capoeta damascina species complex group formerly known as C. damascina in Iranian water bodies. Phylogenetic analyses recovered three main groups inside the genus Capoeta: the Mesopotamian group (C. trutta group), the Anatolian-Iranian group (C. damascina group) and the Aralo-Caspian group (C. capoeta group) which is in agreement with Levin et al. (2012). The genus Capoeta is monophyletic (Levin et al. 2012). Based on the previous published data, the Capoeta damascina species complex group diverged from the C. capoeta group about 9.1 MYA (95% CI: 6.4-10.9) in the Tortonian period (Levin et al. 2012). Iranian members of the C. damascina group (buhsei, saadii and coadi) formed a clade sister to other C. damascina species complex group members.