Iranian Journal of Fisheries Sciences 20(3) 628-645 2021 DOI: 10.22092/ijfs.2021.350276.0 Research Article Molecular genetic divergence of five genera of cypriniform fish in Iran assessed by DNA barcoding

Nazari S.1,3*; Paknejad H.3; Jalali A.2,3; Khorshidi Z.3

Received: August 2018 Accepted: November 2020 Abstract The present study represents a comprehensive molecular assessment of some family of freshwater fishes in Iran. We analyzed cytochrome oxidase I (COI) sequences for five genus of cypriniform fishes from Iran. The present investigation provides data on genetic structure of some species of Nemachilidae including Paraschistura bampurensis, kiabii and Turcinemacheilus saadii and Leuciscine cyprinids Alburnoides bipunctatus and Alburnus alburnus from inland waters of Iran. The DNA sequences of the mitochondrial COI gene in all fishes were amplified, and the resulting sequences were compared to entries in GenBank using the Basic Local Alignment Search Tool for nucleotide data. Individuals were assigned to groups using COI gene sequence divergence analysis which was determined using Kimura 2- parameter distances. Results revealed two major clusters which were inconsistent characterized by mediocre genetic divergence (mean 0.8%). The sequences of P. bampurensis showed that specimens collected from four rivers (Beshar, Khersan, Shapour and Fahlian Rivers, southwest of Iran), with 1.89% for Nemacheilian loaches and 0.8% for two Leuciscine cyprinids within-species Kimura two parameters distance, Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 shared haplotype in Neighbor-joining (NJ) tree. Some clusters showed haplotype sharing, or low levels of divergence between species, hindering reliable identification. We discussed the importance of further DNA barcode studies for native and invasive cyprinid species and subsequent submission to GenBank databases for more reliable species match and inference.

Keywords: DNA barcodes, mitochondrial DNA, Leuciscine cyprinids, Nemachilidae, sequence divergence

1-Shahid Motahary Cold-water Fishes Genetic and Breeding Research Center, Iranian Fisheries Sciences Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Yasouj, Iran 2-Center for Integrative Ecology, School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, Victoria 3280, Australia. 3-Faculty of Fisheries and Environmental Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. *Corresponding author's Email: [email protected] 629 Nzari et al., Molecular genetic divergence of five genera of cypriniform fish in Iran assessed...

Introduction consisting mostly of small benthic Fishes are the most species-rich group fishes inhabiting freshwaters of Europe, of vertebrates with about 30,000 species Asia and Ethiopia and inhabit a variety and the family Cyprinidae of Iranian inland waters, e.g. turbulent (), with about 2,010 mountain streams to salty rivers in dry species in 210 genera, is the largest lowlands (Abdoli, 2000, 2011; family among freshwater fishes (Coad, Golzarianpour et al. 2011a). Little is 2006). According to Esmaeili et al. known about the Nemacheilid loaches (2018), the most diverse family is the of western Asia because of their small Cyprinidae with 111 confirmed species size and low value for marketing. (43.19%) followed by Although species of Nemacheilidae are (44 species, 17.12%), Gobiidae (24 not commercially important, the family species, 9.34%), Cyprinodontidae (14 is of high ecological significance and is species, 5.45%), Clupeidae (10 species, a main part of river ecosystems in the 3.89%), Cobitidae (7 species, 2.72%) region. In western Asia, the and Salmonidae (7 species, 2.72%). The Nemacheilid roaches include numerous genera Alburnus and Alburnoides are species that are morphologically Leuciscine cyprinids found in Europe similar. Recent findings regarding and northern parts of southwest Asia Nemacheilidae have led to record of and encompass 22 species, eight of new species in Iran (Golzarianpour et which can find in Iran (Coad, 2006; al., 2011b, 2013; Esmaeili et al., 2013; Pourkazemi et al., 2010). Leuciscine Freyhof et al., 2014), which means cyprinids species complex, are there is still considerable ambiguity in widespread in temperate and widely terms of identifying species of distributed in Iran, commonly in small Nemachilidae. In other words, what is Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 streams and less frequently in the main known about them, is mostly based on areas of large river basins of the limited data, and previous studies have Caspian Sea, Lake Orumiyeh, Tedzhen mainly focused on morphometric River, Kavir, Namak Lake, Zayandeh aspects of the species from this family and Shur Rivers (Esfahan), and Kor (Askari and Shabani, 2013). River (Kiabi et al., 1999; Abdoli, 2000; Considerable research has addressed Nazari et al., 2009). Although the the development of molecular markers taxonomy of Leuciscine cyprinids for identifying fish species (Meyer and species (Alburnoides bipunctatus and Paulay 2005; Roe and Sperling 2007; Alburnus alburnus) were described Zhang and Hanner, 2012) and (Nazari et al., 2011; Seifali et al. 2012), distinguishing cryptic hybridization (Li genetic structure of these species still et al., 2019). Molecular methods and has many gaps. their application for genetic The Nemacheilidae is a species-rich conservation and species differentiation lineage of the order Cypriniforms, studies have been promoted remarkably Iranian Journal of Fisheries Sciences 20(3) 2021 630

over the past 20 years (Xiao et al., many uses in the field of evolution 2009; Mabragana et al., 2014; Burghart because of its higher mutation rate and et al., 2014; Jo et al., 2014; Nazari, et lower effective population size al., 2016; Peoples et al., 2017). Various compared to nuclear DNA (Brown et molecular markers, especially markers al., 1979). The COI is proposed as a derived from mitochondrial DNA, have standard barcode for (Hebert et been evaluated for their potential use as al., 2003a) and could identify a large DNA barcodes (Amiri et al., 2020; variety of species (Steinke et al., 2005, Sadeghi et al., 2020); such markers 2009; Pegg et al., 2006). DNA include restriction digests or direct barcoding is also a helpful approach to sequencing of amplicons from the find new species of threatened NADH dehydrogenase 2 gene (Cashner freshwater fishes (Torres et al., 2013). and Bart, 2010), the cytochrome-c Moreover, mtDNA sequences can be oxidase I gene (COI; Ward et al. 2005; utilized for studying population Kolangi-Miandare et al., 2013), or the genetics and phylogenetics of fish cytochrome-b gene (Zardoya et al. (Peng et al., 2004; Liu and Chen, 2003; 1999; Herbert and Gregory, 2005; Khoshkholgh and Nazari, 2019). Hajibabaei et al., 2007). These Considering difficulties in the molecular markers can provide a simple identification of Nemacheilidae and solution for identification of fish Leuciscine cyprinid species based on species to develop better management their morphological characteristics, the and conservation policies (Lakra et al., aim of this study was to provide 2011). barcoding data for some species of DNA barcoding has known as a Nemachilidae and two Leuciscine reliable molecular tool for species cyprinid fishes in order to rapid and Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 identification. It consists of using a accurate species identification as well small portion of mitochondrial DNA as looking for the internal species from a standard agreed-upon position in marker. In this study, we used DNA the genome which can be searched and barcode methodology to delineate and compared with sequences deposited in identify the five genera of the databases such as NCBI GenBank and cypriniform fish species from BOLD. Nearly, 655-bp-long fragment freshwater environments. DNA barcode from the 5′ region of mitochondrial records generated in this study will be cytochrome c oxidase subunit I (COI) available to researchers for monitoring gene was proposed as the standard and conservation of fish diversity in this region for DNA barcoding of region. species (Hebert et al., 2003a,b; Moritz and Cicero, 2004; Hebert and Gregory, 2005; Wong et al., 2011; Khoshkholgh and Nazari, 2015). The mtDNA has

631 Nzari et al., Molecular genetic divergence of five genera of cypriniform fish in Iran assessed...

Materials and methods discharge into the Persian Gulf. Species sampling Gamasyab River is one of the longest As reference for the extant freshwater permanent rivers in Iran. It originates fish fauna in Iran, we used the current from the northern slopes of Garin list of Iranian freshwater fishes (Coad, Mountain called the Garin headwater. It 2016). The fishes were collected from 5 flows through a few provinces sampling sites located in Shapour, (Hamedan, Kermanshah, Lorestan and Fahlian, Gamasyab, Beshar and Zohre Khuzestan) and ends to the Hoor Al- Rivers in the west and southwest of Azim International Wetland. Three Iran. The Beshar River is the most Nemacheilian roaches were included important source of water in the city, Paraschistura bampurensis from which originates from the southeast of Shapour and Fahlian Rivers, and Boyer Ahmad and the mountains of the Oxynoemacheilus kiabii and Sepidan and Mamasani in Fars province Turcinemacheilus saadii from (Fig. 1). Shapour and Fahlian Rivers are Gamasyab River. The fish were two important permanent rivers in Fars collected by using nets during the May - province as these rivers are the main July, 2012. All specimens were fixed in water sources in the region, especially ethanol (96%) and then transferred to for agricultural activities. After joining the genetic laboratory at Gorgan the other seasonal and permanent University of Agricultural Sciences and streams of routes, the rivers enter to Natural Resources for later analysis. Bouhsher province and finally

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Figure 1: Map of the studied rivers (Gamasyab, Fahlian and Shapour) and sampling locations overlaid on the digital elevation model (DEM)- derived hillshade (80 m resolution) representing the topography of the earth’s surface in the west and southwest of Iran.

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DNA extraction and PCR amplification were labeled using BigDye® Five individuals from three species of Terminator v.3.1 Cycle Sequencing Kit Nemachilidae were sequenced, and data (Applied Biosystems, Inc.) and then were downloaded for further 23 sequenced using an ABI 3730 apillary individuals of Turcinoemacheilae sequencer following manufacturer’s (Teleostei: Nemacheilidae) from NCBI instructions. As shown in Table 2, 652 and EMBL GenBank databases (Table bp of COI consensus barcodes for each 1). No sequence of O. kiabii was species were treated as discrete units to recognized in GenBanks. Total DNA estimate the pairwise level of genetic was extracted from pectoral and pelvic divergence using K2P correction model fin using the traditional proteinase-K (Nei and Kumar, 2000). digestion and standard phenol/chloroform protocol storing at - Data analysis 20°C (Hillis et al., 1996). In order to Nucleotide sequences were manually amplify fragment of mitochondrial COI aligned, edited, and checked for gene, PCR reactions were conducted unexpected stop-codons using the using primer cocktails of FishF2-5′ SeqScape 2.6 (Applied Biosystems), TCGACTAATCATAAAGATATCGG and then submitted to the GenBank CAC3′ and FishR2- Barcode database with the accession 5′ACTTCAGGGTGACCGAAGAATC numbers KP342063-74. The sequences AGAA3′ (Ward et al., 2005). The 25 from GenBank and BOLD databases µL PCR reaction mixes included 18.75 and also from our dataset were aligned µL of ultrapure water, 2.25 µL of 10 × using Mega 5.0 (Tamura et al., 2007).

PCR buffer, 1.25 µL of MgCl2 (50 Afterwards, pairwise genetic distances mM), 0.25 µL of each primer (0.01 were quantified according to Kimura 2- Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 mM), 0.125 µL of each dNTP parameter (K2P) distance model (0.05mM), 0.625 U of Taq polymerase (Kimura, 1980) for sequence and 0.5–2.0 µL of DNA template. comparisons. The COI gene tree was Amplifications were performed using a constructed by Neighbour-Joining (NJ) Mastercycler® Eppendorf gradient method in Mega 5.0 to provide a thermal cycler (Brinkmann Instruments, graphic representation of the patterning Inc.). The thermal regime consisted of of divergence among the species an initial step of 2 min at 95˚C followed (Saitou and Nei, 1987). The robustness by 35 cycles of 0.5 min at 94˚C, 0.5 of the NJ tree was assessed in Mega 5.0 (Tamura et al., 2007) by performing min at 54˚C, and 1 min at 72˚C, bootstrapp analysis with 1000 followed in turn by 10 min at 72˚C and replications. The number of haplotypes, then held at 4˚C. PCR products were haplotype diversity, and nucleotide visualized on 1.2% agarose gels diversity per site for each species were containing ethidium bromide (10 computed by DnaSP v. 6.12.03 mg/mL) and the most intense products software (Rozas et al., 2017). were selected for sequencing. Products 633 Nzari et al., Molecular genetic divergence of five genera of cypriniform fish in Iran assessed...

Table 1: List of specimens included in the analyses, their accession numbers, origin, and source of sequence. Taxon Origin Accession Source number Paraschistura bampurensis. Iran, Shapour River in KP342063 Tissue Fars Province Paraschistura bampurensis. Iran, Shapour River in KP342064 Tissue Fars Province Paraschistura bampurensis. Iran, Shapour River in KP342065 Tissue Fars Province Paraschistura bampurensis. Iran, Shapour River in KP342066 Tissue Fars Province Paraschistura bampurensis. Iran, Shapour River in KP342067 Tissue Fars Province Oxynoemacheilus kiabii Iran, Gamasiab River in KP342068 Tissue Kermanshah Province Oxynoemacheilus kiabii Iran, Gamasiab River in KP342069 Tissue Kermanshah Province Oxynoemacheilus kiabii Iran, Gamasiab River in KP342070 Tissue Kermanshah Province Oxynoemacheilus kiabii Iran, Gamasiab River in KP342071 Tissue Kermanshah Province Oxynoemacheilus kiabii Iran, Gamasiab River in KP342072 Tissue Kermanshah Province Turcinemacheilus saadii Iran, Gamasiab River in KP342073 Tissue Kermanshah Province Turcinemacheilus saadii Iran, Gamasiab River in KP342074 Tissue Kermanshah Province Turcinemacheilus kosswigi Iran Sirvan KJ179245 Esmaeili et al., 2014 Turcinemacheilus kosswigi Iraq Great Zab KJ179255 Esmaeili et al., 2014 Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 Turcinemacheilus kosswigi Iran Sirvan KJ179258 Esmaeili et al., 2014 Turcinemacheilus kosswigi Iraq Little Zab KJ179260 Esmaeili et al., 2014 Turcinemacheilus kosswigi Iraq Little Zab KJ179262 Esmaeili et al., 2014 Turcinemacheilus kosswigi Iraq Little Zab KJ179265 Esmaeili et al., 2014 Turcinemacheilus saadii Iran Karoun KJ179250 Esmaeili et al., 2014 Turcinemacheilus saadii Iran Karkheh KJ179253 Esmaeili et al., 2014 Turcinemacheilus saadii Iran Karoun KJ179257 Esmaeili et al., 2014 Turcinemacheilus saadii Iran Karoun KJ179248 Esmaeili et al., 2014 Turcinemacheilus saadii Iran Karoun KJ179261 Esmaeili et al., 2014 Turcinemacheilus minimus Turkey Euphrates KJ179251 Esmaeili et al., 2014 Turcinemacheilus minimus Turkey Euphrates KJ179263 Esmaeili et al., 2014 Turcinemacheilus minimus Turkey Euphrates KJ179249 Esmaeili et al., 2014 Turcinemacheilus minimus Turkey Euphrates KJ179256 Esmaeili et al., 2014 Turcinemacheilus hafezi Iran Karoun KJ179259 Esmaeili et al., 2014

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Table 1 continued: Taxon Origin Accession Source number Turcinemacheilus hafezi Iran Karoun KJ179252 Esmaeili et al., 2014 Turcinemacheilus hafezi Iran Karoun KJ179254 Esmaeili et al., 2014 Turcinemacheilus hafezi Iran Karoun KJ179264 Esmaeili et al., 2014 Paraschistura bampurensis. Iran Baluchestan KJ179269 Esmaeili et al., 2014 Paraschistura bampurensis. Iran Baluchestan KJ179268 Esmaeili et al., 2014 Paraschistura malapterura. Iran Namak KJ179267 Esmaeili et al., 2014 Paraschistura malapterura. Iran Namak KJ179266 Esmaeili et al., 2014 Turcinemacheilus bahaii Iran Zayandehroud KJ179246 Esmaeili et al., 2014 Turcinemacheilus bahaii Iran Zayandehroud KJ179247 Esmaeili et al., 2014 Alburnus alburnus Iran, Zagros HM392001 Tissue Alburnoides bipunctatus Iran, Zagros KJ552440 Tissue Alburnus mossulensis Iran, Zagros 29994152 Tissue

Table 2: Estimating of Pairwise Genetic Distances among Nemacheilidae species under Kimura 2- Parameter Model (Kimura, 1980). E1_P. bampurensis E3_P. bampurensis 0.000 E2_P. bampurensis 0.032 0.032 E4_P. bampurensis 0.032 0.032 0.000 E5_P. bampurensis 0.032 0.032 0.000 0.000 F1_O. kiabii 0.172 0.172 0.167 0.167 0.167 F2_O. kiabii 0.172 0.172 0.167 0.167 0.167 0.000 F3_O. kiabii 0.172 0.172 0.167 0.167 0.167 0.000 0.000 F4_O. kiabii 0.172 0.172 0.167 0.167 0.167 0.000 0.000 0.000

Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 F5_O. kiabii 0.173 0.173 0.169 0.169 0.169 0.002 0.002 0.002 0.002 G1_T. saadii 0.158 0.158 0.151 0.151 0.151 0.190 0.190 0.190 0.190 0.192 G2_T. saadii 0.158 0.158 0.151 0.151 0.151 0.190 0.190 0.190 0.190 0.192 0.002

Results independently. Bootstrap values of The COI region of samples was species separations were mostly around successfully amplified by using PCR. 100 for O. kiabii and P. bampurensis, The resulting phenogram of 37 and around 70 for T. saadii. NJ tree was sequences were obtained (Fig. 2). The consistent in defining the separation read lengths were 652 bp long for all. among O. kiabii and other species, According to the NJ tree (Fig. 2), the whose clusters were supported by the species in this study were clustered high bootstrap values (Fig. 2).

635 Nzari et al., Molecular genetic divergence of five genera of cypriniform fish in Iran assessed...

Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 Figure 2: Phylogenetic consensus tree of Nemacheilidae species constructed with 652 nucleotide of cytochrome oxidase I (COI) gene using Neighbor-Joining method. Paracobitis malapterura was used as an out group. Bootstrap values greater than 50 are shown.

Genetic variations were found within Alburnoides and Alburnus species the species with a mean K2P distance of ranged from 4.1% for Alburnus 0.006-1.89. Five individuals of O. kiabii alburnus to 5.8% for Alburnoides could not be separated from each other. bipunctatus. The mean genetic The K2P distance between species distances between Alburnus alburnus, ranged from the least value at 15.36 (P. Alburnus mossulensis and Alburnoides bampurensis and T. saadii) to a bipunctatus species are given in Table maximum value at 19.06% (O. kiabii 2. Largest mean intraspecific distance and T. saadii). was observed in Alburnoides The mean genetic distance between bipunctatus (0.58%; Table 3; Fig. 3). Iranian Journal of Fisheries Sciences 20(3) 2021 636

Table 3: Intraspecific genetic variation of Alburnoides and Alburnus Mean intra- Max intra- Genetic Species N h± SD π ± SD sp ± SE sp Distance Alburnus alburnus 6 0.93 ± 0.08 0.0038 ± 0.0051 0.003 ± 0.012 0.006 0.041 ± 0.025

Alburnus mossulensis 7 0.91 ± 0.04 0.0028 ± 0.0036 0.003 ± 0.005 0.009 0.034 ± 0.011

Alburnoides bipunctatus 6 0.89 ± 0.62 0.0046 ± 0.0014 0.003 ± 0.008 0.003 0.058 ± 0.035

N : sample size; h±SD: haplotype diversity ± standard deviation; π±SD: nucleotide diversity ± standard deviation; Mean intra-sp±SE: mean intraspecies K2P distance±standard error; Max intra-sp: maximum intraspecies K2P distance; Genetic distance±standard error (based on 1000 bootstrap replications).

Figure 3: The Neighbour-Joining tree based on COI gene sequences K2P‏ model.

Discussion COI. All samples of the sequenced

Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 In many studies, DNA barcoding species were recognized with no approach served as a powerful and exception and were amplified with reliable tool for the identification of fish DNA barcoding primer. Three species, comprising all major taxa specimens of T. saadii failed to including marine and freshwater species amplify, which might be due to DNA from different geographic regions degradation. The phylogenetic tree (Ward et al., 2005; Hubert et al., 2008; reconstruction suggested that Steinke et al., 2009; April et al., 2011; Turcinemacheilus minimus is most Zhang and Hanner, 2012; McCusker et closely related to T. kosswigi from al., 2013). According to the objectives Tigris drainage and T. minimus is the of the work, COI sequences were sister group of T. kosswigi. While it had investigated primarily to understand the been initially identified as T. kosswigi phylogenetic relationships among the (Breil and Bohlen, 2001); however it is Cyprinid fishes. In this study, introduced as a new species of T. specimens from Cyprinid fishes were minimus from Turkey (Esmaeili et al., sequenced for the barcode region of 2014). Although T. bahaii has been 637 Nzari et al., Molecular genetic divergence of five genera of cypriniform fish in Iran assessed... recorded as a new species in of Leuciscine species, i.e. Alburnus Zayanderoud River from Isfahan, Iran alburnus versus Alburnus mossulensis, and T. hafezi in Bashar River from was noticed to constitute 1.28±0.05%. Yasouj, Iran (Esmaeili et al., 2014), Within the group 1 (Alburnoides these species as sister group are closely bipunctatus) and group 2 (Alburnus related to each other. Turcinemacheilus alburnus), the distances were hafezi is distributed in the Bashar River 1.12±0.03% and 0.094±0.02%, which is connected to Karoun and Dez respectively. Thus, COI alone could not Rivers drainages (Golzarianpour et al., be an appropriate marker to infer 2013), flowing to the delta area of phylogenetic relationships. Similar Arvand River in Khuzestan province, results have been obtained by some Iran. other researchers who investigated the The NJ method permitted ability of COI barcode sequences to unambiguous clustering of obtained draw phylogenies (Montagna et al., sequences within groups with high 2016), while the other authors have statistical support (85–99%). observed it useful for phylogeny Phylogenetic analysis of COI data was reconstruction (Persis et al., 2009). generally congruent with recent studies The Nemeachilus phenogram of Leuciscine cyprinids (Roudbar et al., revealed that O. kiabii and P. 2016; Stierandov et al., 2016) in bampurensis are more similar however, determination of closely related species. O. kiabii is a separate clade. The COI In the present study, we detected some data clearly supported that O. kiabii, shared haplotypes between Leuciscine which has been recorded as a new cyprinids. The difference between the species in Iran (Golzarianpour et al., causes of the shared haplotypes with the 2011a) is different from the other Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 mitochondrial DNA barcoding Nemchilous species with acceptable approach cannot be discriminated. bootstrap value (100%). Indeed, there Thus, it is not known whether shared were great genetic divergence among haplotypes represents the natural, this species and other Nemachilous ancestral state, or whether the shared species. Contrary to P. bampurensis, haplotypes are already due to human- other conspecific samples collected mediated introgression among lineages. from the same area, thus we might have In order to estimate the differences in somewhat underestimated the extent of distance within Leuciscine cyprinids, within species diversity. It is suggested individuals of the genus Alburnoides that sampling should be included were analyzed separately to see if three individuals from different watersheds species really have distinct gene pools. for freshwater fishes as previously So that, they were divided into two mentioned by Ward et al. (2005). groups, according to topology of the Confusion in taxonomic assignments built trees. The maximum p-distance does not probably occur as a result of between the first and the second groups

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inter specific hybridization (Verspoor identification of the five genus of and Hammart, 1991). cypriniform fish species from In the present study, the application freshwater environments. Cytochrome of DNA barcoding has been oxidase I -based DNA barcodes are also demonstrated as the powerful tool for useful to uncover intraspecies identifying marine and freshwater fish divergence and to assign potentially species from different geographic new species in the all genus studied. regions as it has been stated previously (Hubert et al., 2008; McCusker et al., Acknowledgments 2013; Victor et al., 2009; Kim et al., We are grateful to Gorgan University of 2010; Keskin and Atar, 2013). Agricultural Sciences and Natural Likewise, the other studies indicated Resources for providing equipments more than 98% of the analyzed species, and access to the genetic laboratory. especially marine species, could be The authors would like to thank clearly delimitated through DNA Hashem Noferesti for his assistance in barcoding (Zhang and Hanner, 2012; collecting samples of fishes. This Costa et al., 2012). Although our results project was founded by a grant from showed a high rate of efficiency, DNA Gorgan University of Agricultural barcoding has been shown less efficient Sciences and Natural Resources compared to marine species in some awarded to Dr. H. Paknejad. freshwater fish species (April et al., 2011). This is likely due to the References fragmentation of rivers and lakes from Abdoli, A., 2000. The inland water continental freshwater networks. Such fishes of Iran (in Farsi). Iranian fragmentation may consequently lead to Natural and Wildlife Museum Downloaded from jifro.ir at 19:28 +0330 on Friday October 8th 2021 a more pronounced genetic structure Tehran, 378 P. among the populations and deeper Abdoli, A., Golzarianpour, K., Kiabi, divergence among haplotypes than in B.H., Naderi, M. and Patimar, R., the marine realm (Ward et al., 1994). 2011. Status of the endemic loaches Nonetheless, this study is one of the of Iran. Folia Zoologica, 60, 362– first steps of using a DNA barcoding 367. DOI: approach to enhance genetic 10.25225/fozo.v60.i4.a2.2011 understanding of relationships among Amiri, S., Ghavam Mostafavi, P., the species of Nemachilidae in Iran. Nabavi, M. and Shahhosseini M., In this study, we utilized standard 2020. DNA barcoding of Nereididae DNA barcode methodology to delineate polychaetes based on COI gene between five genus of cypriniform barcoding in intertidal shores of fishes from Iran. The results of the Bushehr and Bandar Abbas present study indicated that DNA- provinces, Iran. IJFS. 19(3), 1193- barcoding is a reliable approach for 1213.

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