Bull 611 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hadi et al. Bull. Iraq nat. Hist. Mus. (2023) 17(4): 611-628. https://doi.org/10.26842/binhm.7.2023.17.4.0611 ORIGINAL ARTICLE MORPHOLOGICAL AND MOLECULAR STUDIES OF KAIS KINGFISH CYPRINION KAIS HECKEL, 1843 (PISCIES, CYPRINIFORMES, CYPRINIDAE) FROM THE MIDDLE OF IRAQ Hind Dyia Hadi*♦, Abed Hassan Baraaj** and Atheer Hussain Ali*** *Iraq Natural History Research Centre and Museum, University of Baghdad, Baghdad, Iraq. ** Department of Biology, College of Science, Baghdad University, Iraq. ***Department of Fisheries and Marine Resources, College of Agriculture, University of Basrah, Basrah, Iraq. ♦Correspondence: e-mail: hinddhiaa86@gmail.com Recived Date: 07 May 2023, Accepted Date 12 July 2023, Published Date:20 December 2023 This work is licensed under a Creative Commons Attribution 4.0 International License ABSTRACT Cyprinidae species are the most abundant and widely distributed fish species in the inland waters of Iraq. Cyprinids are complex species, and it is difficult to identify them on the basis of morphology. Thus, the morphological characteristics must be achieved and confirmed by molecular analysis. Twenty specimens of Cyprinion kais Heckel, 1843 (Piscies, Cypriniformes, Cyprinidae) were collected from two localities at Tigris River in the middle of Iraq: five specimens from Al-Tharthar Lake, Saladin Province, and 15 specimens from Al- Zubaydiyah sub-district, Wasit Province. The DNA sequences of C. kais were done using the mitochondrial DNA cytochrome b (cytb) gene. After analysis, the sequences were compared with sequences of other fish genera and species available in GenBank. The barcoding result (DNA sequencing) showed that it fit with C. kais. We conclude the results of the current study confirm that the validity of the morphological diagnosis of C. kais, using DNA sequencing analysis. Keywords: Cyprinidae, Cytochrome, Fishes, Morphology, Tigris River. INTRODUCTION Kais kingfish Cyprinion kais Heckel, 1843, is a freshwater fish belonging to the largest family of freshwater fish Cyprinidae, and this species is distributed in the inland waters of Iraq, Turkey, Syria, and Iran. In Iraq, it is recorded from marshes such as Al-Chabaish, large rivers such as the Tigris, Shatt Al- Arab River, and Lesser Zab, smaller rivers such as the Khalis near Baghdad, in ponds such as those on Za’faraniyah fish farm south of Baghdad, and in reservoirs such as the Dukan Dam (Coad, 2010; Afrasiab et al., 2013; Froese and Pauly, 2023). BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Iraq Natural History Research Center & Museum, University of Baghdad https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home Copyright © Bulletin of the Iraq Natural History Museum Online ISSN: 2311-9799-Print ISSN: 1017-8678 https://doi.org/10.26842/binhm.7.2023.17.4.0611 https://orcid.org/0000-0002-0779-0194 https://orcid.org/0000-0001-8842-8519 https://orcid.org/0000-0002-2541-968x mailto:hinddhiaa86@gmail.com https://creativecommons.org/licenses/by/4.0/ https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home 612 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Morphological and molecular studies of kais This fish is edible and a valuable species for sport fishing, and it could be used as an aquarium fish (Nasri et al., 2010). Despite its wide distribution and importance, few studies have been conducted on this species, due to its lower abundance, compared to C. macrostomum. Some researchers considered C. kais and C. macrostomum to be synonymous (Berg, 1949; Karaman, 1971), but Bianco and Banarescu (1982) indicated that they are two different species; as well as Coad (2010) used the most important characteristics to differentiate C. kais from C. macrostomum, the shape of the mouth is one such character, being narrower and more arched and the lateral lobes present in C. kais, while the mouth is broader and lacking lateral lobes in C. macrostomum. Freshwater groups are morphologically diverse, indicating their ability to adapt to local selection pressures, but marine groups are globally uniform, reflecting the stability of marine environments. In the case of the sympatric species C. macrostomum and C. kais, the strongest selective factor appears to be food competition, as all other environmental factors are the same (Nasri et al., 2018; Al- Thahaibawi et al., 2019). The nature of the feed related to one species of fish determines the size and shape of the mouth of fish; the mouth shape, is a trophic character in C. kais (Gazwan et al., 2021; Coad, 2023); it can be considered as developmental plasticity as well as a few related morphological adaptations to feed on benthic invertebrates (the deeper body, the least head height, dorsal fin base length, dorsal-fin height, and longer pectoral-fin); but osteological, new genetic method, and morphological information rejected their synonymy (Daştan et al., 2012; Nasri et al., 2013; Coad, 2023). To recognize fish species, there are numerous taxonomic methods, the morphological study being the conventional one, which is helpful, speedy, and includes many characters, morphometric and meristic characters, for species identification (Al-Janabi, 2014; Stein et al., 2014). The morphological similarities, particularly between species belonging to the same genus, have been one of the most common causes of confusion in recognizing the Iraqi fish fauna (Faddagh et al., 2012a). C. kais is morphologically similar to C. macrostomum, so molecular techniques are necessary to elucidate species identification in this case. With the improvement of new genetic advances that utilize information obtained from the molecular components of the cell, the mitochondria cytochrome b (mt-cyb) gene is one of 11 components of a group of proteins called complex III. Cytb is generally utilized as a locale of mitochondrial DNA to identify evolutionary connections between different groups, and because of its sequence diversity it is valuable inside families (Caine et al., 2006). There are a few uncertainties about the taxonomic and developmental status of Cyprinion species, and many authors considered the taxonomic status of Cyprininae species and genera with their genetic links to remain doubtful (Howes, 1982). The aim of the current study is to confirm the identification of C. kais by combining the morphological and molecular characters. 613 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hadi et al. MATERIALS AND METHODS Study area Two localities at the Tigris River were chosen to collect studied fish species as follows: A. Al-Tharthar Lake The biggest natural lake in Iraq; which lies within the middle of Iraq, around 120 km northwest of Baghdad, between the Tigris and the Euphrates rivers, within coordinates 33°58′N 43°11′E. The Lake; with an area ranging between 1875 - 2710 km2 and a maximum depth of 68.4 m, is fed by the Tigris River; the water is discharged from the lake to the Euphrates River (Szczerbowski et al., 2001; Zdanowski et al., 2001). B. Al- Zubaydiyah Sub-district It is one of the sub-districts of the Al-Suwaira District in Wasit Province. Al- Zubaydiyah is located 50 km to the south of the city of Essaouira and 85 km to the north of the city of Kut, the capital of Wasit Province, southeastern Baghdad, within coordinates 32°45'46.6"N 45°10'48.7"E. The sampling localities are illustrated on the Iraqi map (Map.1). Map (1): Sampling areas. 614 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Morphological and molecular studies of kais Specimens' collection A total of 20 specimens of fishes, 17 specimens for morphology study, and three specimens for molecular study) were collected using gill nets by fishermen; during the period from June; 2022 until December 2022. Fishes were transported in a cool box with crush of ice to the laboratory of the Iraq Natural History Research Centre and Museum, University of Baghdad. Morphological study Each specimen is measured using 1-meter measuring board graduated in millimetres (mm) and a digital calliper, and the weight of each fish individual was done immediately by digital balance. The morphometric features were measured from the left side of each fish. The morphometric and meristic characters are measured according to those in Hubbs and Lagler (1964) as shown in Table (1). The specimens were photographed using a Samsung camera of Korean origin. Table (1): Morphometric and meristic characters and their abbreviations. No. Morphometric characters Abbreviated name 1 Total Length TL 2 Fork Length FL 3 Standard Length SL 4 Head Length HL 5 Head Height HH 6 Snout Length SnL 7 Eye Diameter ED 8 Interorbital Distance IOD 9 Postorbital Length POL 10 Maximum Body Height MAXH 11 Minimum Body Height MINH 12 Caudal Peduncle Length CPL 13 Dorsal Fin Length DFL 14 Dorsal Fin Height DFH 15 Pectoral Fin Length PFL 16 Pelvic Fin Length PFL 17 Pectoral-Pelvic Distance PPD 18 Pelvic-Anal Distance PAD 19 Anal Fin Length AFL 20 Anal Fin Height AFH 21 Pre Dorsal Length PDL 22 Post Back Distance PBD 23 Upper Caudal Fin Length UCFL 24 Lower Caudal Fin Length LCFL 25 Centre of Caudal Fin Length CCFL 26 Caudal Peduncle Height CPH 27 Mouth Width MW 28 Prepectoral Length PPL 29 Prepelvic Length PVL 30 Preanal Length PAL 615 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hadi et al. 31 Preanus Length PASL Meristic characteristics 32 Scales of Lateral Line SLL 33 Up Scales of Lateral Line USLL 34 Down Scales of Lateral Line DSLL 35 Dorsal Fin Spines DFS 36 Dorsal Fin Rays DFR 37 Anal Fin Spines AFS 38 Anal Fin Rays AFR 39 Pectoral Fin Rays PFR 40 Pelvic Fin Rays PVFR 41 Caudal Fin Rays CFR 42 Barbels NB 43 Scales around the least circumference of the caudal peduncle SCP 44 Gill Rakers GR Plate (1): The morphometric measurements carried out of C. kais. [From Kashefi et al. (2012) with own authors modification]. 616 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Morphological and molecular studies of kais Plate (2): The meristic characters carried out on Cyprinion kais [From Coad (2010) with own authors modification] DNA Extraction Total DNA was extracted from the muscle dorsal side of each fish species, and specimens were preserved in absolute ethanol 100%, using the Genomic DNA Extraction Kit (addbio/Korea, Cat.no. 10023). Genomic DNA were extracted following Sambrook et al. (1989) and tested using electrophoresis on 1% agarose gel stained with ethidium bromide dye. Amplification and polymerase chain reaction PCR Approximately 508 bp were amplified from 5’ region of the cytb gene using universal fish primers (design in this study). The forward primer FishF1 5’ (CAAGCCTACGAAAAACMCAC) 3’, and the reverse primer 5’ FishR1 (TCTACTGAGAAKCCRCCTCA) 3’. Polymerase Chain Reaction PCR reactions are given as 50 μl total volumes containing 25 μl of 2xTaq DNA Polymerase Master Mix, 2 μl of each primer (FishF1 and FishR1), 17 μl free water, and 4μl of DNA template by Optimus 96G thermal cycler. The PCR thermal cycling conditions were: 94ºC for five min; 35 cycles of 94ºC for 30s, 52ºC for 30 s, 72ºC for 30 s; final extension of 72ºC for 5 min; and hold at 4ºC. The PCR products were then electrophoresed on 2% agarose gel stained with ethidium bromide dye. A ladder of 100 bp (promega) was utilized with this test. The profiles were tested on UV light Transilluminator and documented by photographing with Canon Camera with a gel documentation tool. Sequencing of PCR product sent to (Macrogen / Korea) with forward primer. The results were in the form of special files that were evaluated with Bioedit software. Version 7; 2013; https://bioedit.software.informer.com/7.2/. The alignment and comparison of sequences generated from sequencing findings were also compared with data from the same organism genes that were found in the gene bank at the National Center for Biotechnology Information https://bioedit.software.informer.com/7.2/ 617 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hadi et al. (NCBI), which had previously been investigated in various nations across the world by using the Basic Local Alignment Search Tool (BLAST). The results of sequencing showed 99 - 100 % agreement with reference sequences. The phylogenetic tree of the species was drawn by using the sequence data of the samples under study and compared with the previously studied species using the MEGA X: Molecular Evolutionary Genetics Analysis (Kumar et al., 2018). The nucleotide location value of variance is calculated using the maximum likelihood (ML), and a molecular phylogenetic tree is generated. The bootstrap method with 500 repeats was used to assess the accuracy of the estimated phylogenies. RESULTS Morphology The morphometric and meristic characters of C. kais are indicated in Tables (2, 3), to show the general morphology of Kais kingfish. Table (2): The results of morphological characters for Cyprinion kais. Characters min-max (Mean ±SD) in mm Proportional measurements as expressed as a percentage of Standard Length or of Head length* Weight 320- 584 (404.8± 74.5) - Total Length TL 121- 184 (143±1.71) - Fork Length FL 108-166 (128±1.62) - Standard Length SL 100-144 (116±1.42) - Maximum Body Height MAXH 33-47 (39.6 ± 4.4) 31.25-37.38 (34.32 ± 1.7) Minimum Body Height MINH 9-15 (11.6 ± 1.5) 8.88-11.0 (10.2 ± 0.69) Caudal Peduncle Length CPL 11-33 (15.4 ± 5.4) 10.9-24.4 (13.21 ± 3.59) Dorsal Fin Length DFL 21-36 (28.7 ± 4.0) 21- 27.69 (24.80 ±1.71) Dorsal Fin Height DFH 13-28 (22.1 ± 4.2) 12.59-22.1 (19.07± 2.30) Pectoral Fin Length PFL 16-24 (19.9 ± 2.2) 14.58-20.58 (17.28 ± 1.33) Pelvic Fin Length VFL 15-26 (18.7 ± 2.7) 14.52-19.60 (16.19 ± 1.34) Pectoral-pelvic Distance PV 27-45 (34.5 ± 4.9) 26.73-33.0 (29.79 ± 1.64) Predorsal distance 50-73 (60.9 ± 7.7) 47.61- 56.86 (52.58±2.65) Prepelvic Distance PAD 52-75 (61.7 ± 7.2) 49.52-59.81 (53.38±2.45) Prepectoral Distance PAD 22-34 (26.8 ± 3.6) 18.80-26.73 (23.26±2.10) Preanal Distance PAD 76-113 (90.4 ± 10.8) 73.07-82.24 (78.11±2.47) Pelvic-Anal Distance VA 23-38 (31.2 ± 4.1) 22.30-34.56 (27.45±2.82) 618 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Morphological and molecular studies of kais Anal Fin Length AFL 8-16 (11.9±2.2) 8.33-13.76 (11.04±2.30) Anal Fin Height AFH 13-23 (18.3±3.3) 11.11-22.54 (15.91 ±3.18) Preanus Distance 73-108 (85.3±9.8) 67.69-77.57 (73.73±2.70) Post Back Distance POB 45- 78 (61.5±8.6) 42.88- 58.93 (50.90±4.99) Upper Caudal Fin Length UCFL 23- 38 (29.1±4.4) 21.36-29.41 (25.16±2.25) lower Caudal Fin Length DCFL 23-35 (27.6±3.9) 19.65-27.45 (23.92±2.01) Center Caudal Fin Length CCFL 9-15 (12.6±3.7) 8.25-12.87 (10.97±1.29) Caudal Peduncle Height 11-16 (14.2±1.4) 10.89-14.01 (12.32±1.02) Head Length 22-32(25.4±3.1) (HL/SL)*100 Head Height 15-26(18.4±2.7) 65.38-81.25 (72.36*±4.23) Snout Length 6-11(7.8±1.3) 26.92-36.66 (30.84*±3.26) Orbit diameter 4-6 (5.3±0.6) 17.24-25.00 (21.10*±2.46) Eye Diameter 3-3.9 (3.5±8.8) 9.09-16.95 (12.35*±1.95) Post Orbit Distance 9-15 (11.6±1.5) 39.28-52.17 (45.99*±3.29) Between Eye Distance 7-11.2 (9.1±1.2) 31.03- 41.30 (35.96*±3.29) Mouth width 5-9.4 (7.0±1.6) 20.83-33.91 (27.41*±4.07) Table (3): The results of meristic characters for C. kais. Meristic characters Min-max (Mean ±SD) Lateral Line Scales 36-39 (37.5 ±1.12) Up Scales of Lateral Line 7-8 (7.5 ± 0.49) Down Scales of Lateral Line 3-4 (3.5± 0.52) Dorsal Fin Spines 1-1(1±0) Dorsal Fin Rays 13-14 (13.5± 0.35) Pectoral Fin Rays 12-13 (12.5± 0.41) Anal Fin Spines 1-1(1±0) Anal Fin Rays 7-8 (7.5± 0.26) Caudal Fin Rays 16-18 (17.5±0.6) Pelvic Fine Spines 1-1 Pelvic Fin Rays 8-8 (8±0) Scales around the least circumference of the caudal peduncle 8-9 (8.5±0.5) Gill rakers 11-12 (11.5± 0.52) 619 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hadi et al. Plate (3): Cyprinion kais (Whole body) from Al-Tharthar Lake, Saladin Province. Plate (4): Mouth of Cyprinion kais (ventral view). Molecular study PCR primers of the cytochrome b gene, and specific primers are successfully amplified, and gel electrophoresis is performed to show PCR amplification of the cyto b, which yielded 508 bp product. These amplifications are used for species diagnosis and give comparative data for developmental taxonomy studies and family development research at the species level. Sequencing of these genes is performed in order to determine the genotype of C. kais is collected from the Al-Zubaydia sub-district. The sequence of one gene examined forward and reverse primer is performed; this is part of the sequence process requirements and the use of PCR technology in the context of the method of genetic analysis. The results of nucleotide alignment with the sequences in the gene bank showed that the identities ranged from 99- 100%. Partial cds, and cytochrome b gene mitochondria are compatible with the same sequence fragment marker, which is available at the Gene Bank in the National Center for Biotechnology Information (NCBI). Plate (5) showed a partial sequence and pair-wise analysis of the fish specimens. The current results of a total 508 bp at the 5 ends for the cytb mtDNA region for three specimens were sequenced, and the best likelihood tree resulting from partitioned maximum likelihood analysis indicated that the tree of genetic relations between the studied specimens showed in Plate (6) that there are two sub-branches. The bootstrap value (BP) between these two branches is equal to 59%. One of the branches showed that the C. kais (Local sample T1) 1 0 m m 620 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Morphological and molecular studies of kais formed a sister group with C. macrostomum from AF 180836.1 with a bootstrap value (BP) is equal to 54% and There was a similarity between local samples (T1), and C. macrostomum with standard sequences of C. kais of the same gene in France (AF180860.1) with a bootstrap value (BP) is equal to 33% and the another branched showed that the C. kais (Local sample - T2) formed a sister group with C. kais (Local sample –T3) with a bootstrap value (BP) is equal to 50% and there was a similarity between local samples (T1 and T2) with standard sequences of Cyprinion sp. of the same gene from Saudi Arabia (MT 157380.1) with a bootstrap value (BP) is equal to 33%. Plate (5): Pair wise alignment of partial cds, cytochrome b (cyt b) gene of C. kais Query is the study or sample sequence and subject is the GenBank sequence. 621 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hadi et al. Luciocyprinus langsoni NC 066657.1 China Barbonymus altus JX066773.1 Cambodia Cyprinion semiplotum MN603795.1 India Acrossocheilus longipinnis NC 047455.1 China Acrossocheilus stenotaeniatus KJ994699.1 China Barbus apoensis AF180859.1 France Cyprinion acinaces MT157379.1 Saudi Arabia Cyprinion kais (Local sample-T1) Cyprinion macrostomus AF180826.1 France Cyprinion kais AF180860.1 France Cyprinion sp. MT157380.1 Saudi Arabia Cyprinion kais (Local sample-T2) Cyprinion kais (Local sample-T3) 100 54 50 43 37 98 62 33 33 59 Plate (6): Phylogenetic tree analysis of Cyprinion kais local samples resemble for others in different countries; draw by MEGA X using Maximum like hood (ML) method with bootstrap value 500 repeats. DISCUSSION Morphology Morphometric and meristic traits are practical tools of fish morphology for new species definition (Strauss and Bond, 1990) and species identification (Nelson et al., 2016). In addition, fish morphology can influence their swimming performance, reproductive, camouflage, and feeding activities, etc. (Sfakiotakis et al., 1999). The fish species in the current study belong to the family Cyprinidae. The description of the current specimens of C. kais similar to those explained by Coad (2010). The dorsal fin rays in this study are similar to those in Banarescu and Herzig-Straschil (1995), but the number of lateral line scales in the current study is lesser (36-39 vs 41-44), and the width of the mouth is expressed as percentage to the head length in this study is larger (22.7-29.3% vs 13.5-22.0%). As a comparison of some traits of C. kais in the current study, such as number of the anal-fin ray, the number of the dorsal-fin ray, the number of the pelvic- fin ray, lateral line scales, and gill rakers which are 7, 13-14, 8, 36-39, and 11-12 respectively which are in agreement with the results of Coad (2010), which are 7, 12-16, 8, 36-43, 8-12 respectively, the number of the pectoral-fin rays in this study is lower in the number (12-14 rays) with 14-18 rays in Coad (2010). As a comparison of some features of C. kais in the current study, such as total length, standard length, body depth, head length, number of the dorsal-fin ray, number of the pelvic- fin ray, and number of the anal-fin ray are 14.3, 11.58, 3.9, 2.54, (13-14), 8, 7 respectively are in agreement with the results of Agha (2017), which are 15.7, 12.76, 3.84, 2.86, (13-14), 8,7 but the number of the scales of lateral line, and gill rakers in this study lesser (36-39 vs 39-40), and 11-12 vs 13-14 respectively. The form of a mouth, dorsal fin ray, and pectoral fin ray in the current study are similar to the results of Nasri et al. (2013), but the standard length of C. kais in the current study is larger than the results of Nasri et al. (2013). The dorsal fin ray, pelvic fin ray, pectoral fin ray, 622 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Morphological and molecular studies of kais and gill rakers in this study are in agreement with the result of Kaya et al. (2016) but the lateral scale line of Iraqi specimens has lesser than that of Turkish specimens (36-39 vs 40- 43). The numbers of the dorsal-fin ray, the anal-fin ray, the pelvic-fin ray, pectoral fin ray, lateral line scale, scale above lateral line, and scale below lateral line are 13-14, 7, 8, 12-13, 36-39, 7-8, and 3-4 respectively, are in agreement with the results of Nasri et al. (2018), which are 13, 7, 7-8, 12-13, 39, 7, and 4 respectively. As a comparison of some features of C. kais in the current study, such as a small mouth with large lateral lobes, lateral line scales, the number of dorsal fin branched rays, number of the pelvic-fin rays, number of the pectoral fin rays, are: 36-39, 13-14, 8, 12-13 respectively which in agreement with the results of Coad (2023), which are 36-43, 12-16, 7-8, 12-18 respectively. Molecular study In the present study, we classified, and identified conventionally on the basis of external morphological traits. However, the various developmental stages of fish are difficult to identify by morphological characteristics. Therefore, the traditional identification and classification results were confirmed by mitochondrial DNA sequencing, and fortunately, the results matched and there was no confusion in the scientific nomenclature. DNA sequencing is one of the most important methods that contributed to the rapid diagnosis of species (Pons et al., 2006). The cyto b gene as a code for animal species identification (Hebert et al., 2004); especially the fish species, which have been attracting attention lately. This study demonstrated the effectiveness of the cyto b gene in identifying Cyprinidae species. Banarescu and Herzig-Straschil (1995) reported that there are some specimens that are to a certain degree intermediate between 'typical' C. macrostomus and C. kais, being closer to C. macrostomus based on some morphological traits; these specimens have mouth width as expressed as a percentage of head length and number of dorsal fin rays. This is similar to the current study, but DNA sequencing revealed and confirmed that the fish species is C. kais. The present phylogenetic analysis showed that the cyt b genome sequencing results of C. kais showed a strong association with C. macrostomum and a sister group formed with a BP of 54%. These results agreed with Faddagh et al. (2012a), who determined that BP has 89 % common ancestry. Faddagh et al. (2012b) also determined that taxonomical status of some cyprinid species in Iraqi waters using the mitochondrial 16S rRNA gene: Barbus kersin (=L. kersin), B. grypus (=L. grypus), B. barbulus (=L. barbulus), B. xanthopterus (= L. xanthopterus), Cyprinus carpio, B. sharpeyi (=M. sharpeyi), and C. luteus, the results confirmed that the six fish species genetically belong to the Cyprinidae. Aziz (2015) used the same gene cyto b to identify nine species of Cyprinidae. The DNA sequencing result showed that all species belong to Cyprinidae, and the phylogenetic relationship degree with this family for C. macrostomum, L. esocinus, C. trutta and L. xanthopterusa was a BP of 90%, for C. luteus was a BP of 87%, for grypus (A. grypus) was a BP of 76%, and C. regium, C. carpio and C. carassius was a BP of 75%; this finding is also in agreement with the previous study (Zardoya et al., 1999), who found that the origin of Barbus sp. belongs to Cyprinidae. 623 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Hadi et al. Durand et al. (2002) used the cyt b gene to conduct phylogenetic and zoogeographical studies on C. macrostomum and C. kais in the Middle East Region and indicated that the level of difference between them is less than 0.4% among the studied cyprinids. In this study, the results are in agreement with Daştan et al. (2012), who used mtDNA PCR-RFLP based on pairwise comparison to study the phylogeographic and phylogenetic relationships among C. kais, C. macrostomus, and Carasobarbus chantrei and indicated that the divergence rates among haplotypes of fish populations from each river basin ranged from 0.023 to 7.35%. Yang et al. (2012) used cyto b gene as a molecular marker to identify Barbus sp. and Carasobarbus sp. DNA sequencing results indicate that there is a genetically close relationship among them, and both species belong to the Cyprinidae family, which in turn belongs to the order Cypriniformes. CONCLUSIONS In view of the results of the current study, the identification of Cyprinion kais is confirmed morphologically. The DNA sequencing results showed and confirmed the validity of the fish species and indicated that the correctly sequenced species were identified using cyto b gene. Locally, the number of freshwater native fishes that have been genetically studied reaches 29 out of 53 (54.7%) known species. As well, seven species out of 14 (50%) known species of Cyprinidae are genetically established. ACKNOWLEDGEMENTS We extend our sincere thanks to Prof. Dr Hula Younis Fadhil Al-Sadi, College of Science, University of Baghdad, for her great contribution and advice about molecular work. CONFLICT OF INTEREST STATEMENT These results are a part of Ph. D. Dissertation, in the Department of Biology, College of Science, University of Baghdad for the first author. Further, the authors of this manuscript confirm no existence other relationship with any official institution. LITEREATURE CITED Afrasiab, S. R., Mohammad, K. M., Ali, H. H., Al- Moussawi, A. A. and Abd Al-Rasul, M. S. 2013. Fauna and flora of Hawraman mountain (Part One) Hawraman lowest zone, Kurdistan Province North East of Iraq. Bulletin of the Iraq Natural History Museum, 12(4): 7-34. [Click here] Agha, G. 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Cyprinion Kais (Heckel, 1843) دراسة مظهرية وجزيئية ألسماك بنيني صغير الفم (Piscies, Cypriniformes, Cyprinidae) في وسط العراق ***و اثير حسين علي **, عبد حسن براج*هند ضياء هادي .العراق ,قسم االسماك, مركز بحوث و متحف التاريخ الطبيعي, جامعة بغداد, بغداد * العراق. ,**قسم علوم الحياة, كلية العلوم, جامعة بغداد, بغداد العراق., البصرة***قسم االسماك و الثروة البحرية, كلية الزراعة, جامعة البصرة, 20/12/2023، تأريخ النشر: 12/7/2023القبول: ، تأريخ 7/5/2023تأريخ االستالم: الخالصة , وهي بانها تضم أكثر أنواع األسماك وفرة Cyprinidaeتمتاز عائلة الشبوطيات في العديد من املياه الداخلية في العراق. تعتبر هذه االسماك من األنواع املعقدة موزعة . وبالتالي, ً ومن الصعب تشخيصها مظهريا ً يجب التاكد من صحة تشخيص تصنيفيا من البنيني صغير عينة 20الفحص الجزيئي. في هذه الدراسة جمع االنواع باستخدام من بحيرة Cyprinion kais Heckel, 1843 (Piscies, Cypriniformes, Cyprinidae) الفم التابعه الى عينة(15عينة( التابعه الى محافظة صالح الدين ومنطقة الزبيدية ) 5الثرثار ) وسط العراق.محافظة واسط, . DNA cytochrome b (cytb)ين عبارة عن ج C. kaisكان تسلسل الحمض النووي لـ بعد التحليل , تمت مقارنة التسلسالت مع متواليات األجناس واألنواع السمكية األخرى املخزنة في البنك العام. أظهرت نتائج تسلسل الحمض النووي أن األنواع املدروسة نتائج الدراسة الحالية صحة التشخيص املظهري لسمكة تؤكد . هذا النوعتنتمي إلى Cyprinion kais .وذلك باستخدام تحليل تسلسل الحمض النووي