A FURTHER DISTRIBUTION RECORD OF THE GENUS COBITIS (ACTINOPTERYGII: CYPRINIFORMES: COBITIDAE) IN IRANIAN INLAND WATERS WITH A NOTE ON ITS ZOOGEOGRAPHIC IMPORTANCE IN THE URMIA LAKE BASIN

This study reports the presence of the genus Cobitis in the Zarrineh River, Urmia Lake basin, Iran. Its taxonomy is clarified and zoogeography of Urmia basin is discussed. This study provides morphological characteristics, mtDNA COI barcode sequences of the collected Cobitis specimens and analyses phylogenetic relation within the members of this genus in Iran. The results revealed that morphometric and molecular (COI) characters of the collected Cobitis specimens are largely overlapping or completely identical with those of Cobitis saniae Eagderi, Jouladeh-Roudbar, Jalili, Sayyadzadeh et Esmaeili, 2017. Sequences of C. saniae from the Urmia Lake Basin clustered with published sequences of C. saniae showing a K2P genetic distance of 0.76% to the population from the Caspian Sea basin. This record shows a range extension of this species toward the western part of Iran. Based on first record of C. saniae from the Zarrineh River, the ichthyofauna of the Urmia Lake basin includes 30 species. The presence of C. saniae in Urmia Lake basins reveals the past connection of this basin with the Caspian Sea basin.


INTRODUCTION
Members of the genus Cobitis Linnaeus, 1758 represent one of the most widely distributed Palearctic fish groups, primary freshwater (Freyhof et al. 2018) with four valid species in Iran (Joulade-Roudbar et al. 2017, Eagderi et al. 2017a, 2017bFreyhof et al. 2018), including Cobitis linea (Heckel, 1849) from the Kor River and upper Kol River drainages, Cobitis avicennae Mousavi-Sabet, Vatandoust, Esmaeili, Geiger et Freyhof, 2015, occurring in the Karkheh and Karun, two sub-tributaries of the Tigris River drainage, Cobitis faridpaki Mousavi-Sabet, Vasil′eva, Vatandoust et Vasil′ev, 2011 from the eastern part of the Iranian Caspian Sea and Namak Lake basins, and Cobitis saniae Eagderi, Jouladeh-Roudbar, Jalili, Sayyadzadeh et Esmaeili, 2017 from the western part of the Caspian Sea basin (Mousavi-Sabet et al. 2015, Eagderi et al. 2017a, 2017b. The species of the genus Cobitis are identified based on diagnostic characters of the color pattern including black spots at the caudal-fin base and four longitudinal pigment zones on the flank (Gambetta's zones 1-4), sexual dimorphism (males have 1-2 Canestrini's scales on the dorsal surface of the anterior pectoral fin rays), suborbital spine morphology, barbel and mental lobe morphology (Menon 1992, Kottelat andFreyhof 2007). In addition to morphological identification, mitochondrial DNA (mtDNA) differentiation, known as DNA barcoding, plays an important role in taxonomic studies and phylogenetic inferences (Yang et al. 2010). The purpose of DNA barcoding, based on partial COI sequence, is to improve the identification of species and to discover new species, and it has been used successfully for delimitation of species in more than 90% of animal species studied (Ward et al. 2005, Hubert et al. 2008.
We collected spined loaches of the genus Cobitis from the Zarrineh River, Urmia Lake basin, Iran in 2016. Therefore, this study was aimed to report for the first-time presence of the genus Cobitis in Urmia Lake basin and clarify its taxonomic status by providing morphological characteristics, mtDNA COI barcode sequences and revealing its phylogenetic relation with the members of the genus Cobitis in Iran with a note on its zoogeographic importance.

MATERIALS AND METHODS
Five specimens of the genus Cobitis were collected from the Zarrineh River, Urmia Lake basin, Kurdistan Province, Iran in May 2016 by electrofishing device. After anesthesia, the fin clips of two specimens were fixed in 96% ethanol for molecular studies and all specimens were preserved in 5% buffered formaldehyde. Morphological analysis. A total of 32 morphometric characteristics were measured by a digital caliper to the nearest 0.01 mm (Table 1), then percentage ratios of the morphometric characters in relations to SL and HL were calculated. All measurements were made point to point based on Kottelat and Freyhof (2007). The 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, at mid-height of the caudal-fin base. The percentage ratios of morphometric characters in relations to SL and HL were calculated. The terminology of the pigmentation pattern follows Kottelat and Freyhof (2007). DNA extraction and PCR. DNA was extracted from the collected pectoral-fin tissues using a modified phenolchloroform method (Sambrook et al. 1989). The COI gene was amplified using primers FCOI20 (5′-AACCTCTG TCTTCGGGGCTA-3′) and RCOI20III (5′-TTGAGCC TCCGTGAAGTGTG-3′) (Hashemzadeh Segherloo et al. 2012). Polymerase chain reaction (PCR) conditions were as follows: a 50 μL final reaction volume containing 5 μL of 10X Taq polymerase buffer, 1 μL of (50 mM) MgCl 2 , 1 μL of (10 mM) deoxynucleotide triphosphate (dNTP), 1 μL (10 μm) of each primer, 1 μL of Taq polymerase (5 U · μL -1 ), 7 μL of total DNA and 34 μL of H 2 O. Amplification cycles were as follows: denaturation for 10 min at 94°C; 30 cycles at 94°C for 1 min, 58.5°C for 1 min, 72°C for 1 min, and a final extension for 5 min at 72°C. PCR products were purified using a purification Kit (Expin Combo GP -mini; Macrogen incorporation, Korea). The PCR products were sequenced using the Sanger method by a robotic ABI-3130xl sequencer using the manufacturer's protocol. The forward and reverse primers were used for single-strand sequencing. Molecular data analysis. We newly generated two DNA barcodes and the sequences were compared to the published Cobitis sequences using a basic local alignment search tool (BLASTn) (Altschul et al. 1990). The retrieved sequences of the other members of the genus Cobitis from the GenBank database (NCBI) following the blast search are shown in Table 2. Sequence data were aligned using BioEdit software. For phylogenetic reconstructions, the datasets were analyzed by Bayesian Inference (BI) using MrBayes 3.1.2 (Ronquist et al. 2012) and maximum likelihood (ML) method in IQTREE 1.6.0 (Hoang 2018). We determined the best-fit model of molecular evolution for the given data using the Bayesian information criterion scores (BIC) in ModelFinder (Kalyaanamoorthy et al. 2017).
MrBayes was run with 6 substitution types (nst = 6) and considered gamma-distributed rate variation across sites with a proportion of invariable sites (GTR + I). Four simultaneous Monte Carlo Markov Chains were run for 10 000 000 generations, sample frequency every 1000 generations, chain temperature 0.2. Log-likelihood stability was attained after 10 000 generations, and we excluded the first 1000 trees as burn-in. The remaining trees were used to compute a 50% majority-rule consensus tree. For ML analyses, we conducted heuristic searches (1000 runs) according to TPM2 + F + G4 model. The genetic distances were investigated based on Kimura two-parameter

RESULTS
The general body shape of the collected Cobitis specimens is displayed in Fig. 1 and the morphological characteristics in Table 1. The morphometric characters of the Cobitis specimens from the Zarrineh River overlapped to those of C. saniae from the Sefid River, the Caspian Sea basin (Table 1) except postdorsal length (34.3-40.8 vs. 45.6%-49.9% SL) and outer rostral barbel length (18.2-21.4 vs. 9.7%-16.2% HL). In addition, Cobitis specimens of the Zarrineh Rivers bear a single lamina circularis on the pectoral fin in males with its base widely connected to pectoral-fin ray as C. saniae key character. The color patterns i.e., Gambetta's zones of the pigmentations of the Urmia lake basin specimens were similar to those of C. saniae with a single black spot on the upper caudal-fin base and type II pigmentations pattern of Z4 i.e., merged blotches not forming a dark stripe. Two phylogenetic approaches Bayesian Inference (BI) and Maximum Likelihood (ML), gave the same tree topologies and thus one is presented (Fig. 2). Our analysis of the COI sequence data clusters the newly found Cobitis population from the Zarrineh River with specimens of C. saniae from the Caspian Sea basin (Fig. 2). In addition, in our tree, Iranian members of the genus Cobitis including C. saniae, C. faridpaki, and C. avicennae into the Cobitis taenia Linnaeus, 1758 species group (Freyhof et al. 2018). Table 3 lists the mean genetic divergences found in the mtDNA COI sequences of the studied species. The generated DNA barcode sequences of this Cobitis population showed more than 99% similarity with the available sequences of C. saniae. In addition, it is distinguished by having two diagnostic nucleotide substitutions in the mtDNA COI barcode region from other members of the genus Cobitis in Iran (Table 4) and a minimum K2P genetic distance of 0.7% with C. saniae from the Caspian Sea basin.

DISCUSSION
Endorheic Urmia Lake basin, particularly its center wetland i.e., Urmia Lake is an important ecosystem in north-western Iran (Ghasemi et al. 2015). Drastic changes in the lake environment are suggested to be the consequences of aggressive regional water resources development plans, intensive agricultural activities, and upstream competition for water (AghaKouchak et al. 2015). Inflows of the lake are also under severe pressure due to the above-mentioned factors and lack of perspective management. Their ecological conditions are strongly affected, and the biodiversity of running waters is threatened (Ghasemi et al. 2015). Ghasemi et al. (2015) reported 29 species belonging to 25 genera and 7 families from the Urmia Lake basin. However, there are some changes in the taxonomic status of some species that is worth to be mentioned here. From the superfamily Cobitoidea, three species of the family Nemacheilidae, including Oxynoemacheilus bergianus (Derjavin, 1934), Oxynoemacheilus brandtii (Kessler, 1877), and Paracobitis malapterura (Valenciennes, 1846) were reported (Ghasemi et al. 2015). Based on the recent works (Eagderi et al. 2018a), those of the reported O. brandtii from the Urmia lake basin were described as Oxynoemacheilus elsae Eagderi, Jalili et Çiçek, 2018. In addition, those of Barbus cf. cyri (see Jalili et al. 2015, Ghasemi et al. 2015, Nikmehr et al. 2016, Khaefi et al. 2017, Çiçek et al. 2018   , and Capoeta sevangi De Filippi, 1865 (see Zareian et al. 2018, respectively. Therefore, based on the above-mentioned changes with the first record of C. saniae from the Zarrineh River, the ichthyofaunal of the Urmia lake basin is included 30 species. Members of the genus Cobitis show interspecific variations in terms of morphological characters and color patterns (Eagderi et al. 2017a, 2017b, Freyhof et al. 2018). The presently reported study revealed that morphometric characters (Table 1) and color pattern of the Cobitis specimens from the Zarrineh River were largely overlapping with those of C. saniae from the Caspian Sea basin except post-dorsal and outer rostral barbel lengths. These morphological differences could be a result of phenotypic plasticity caused by different environmental conditions of their habitats , Radkhah et al. 2017. In a species with a large distribution area as C. saniae, we expect some intraspecific morphological differences in different environmental conditions (Marcil et al. 2006).
The Middle East, species of the genus Cobitis had been reviewed recently by Freyhof et al. (2018), who recognized 30 species in four species groups. Cobitis saniae occurs from the Sefid River in Iran north-west to the Kura-Aras River system in Azerbaijan, Armenia, and Turkey (Eagderi et al. 2017a, 2017b, Freyhof et al. 2018. Furthermore, Freyhof et al. (2018) reported C. saniae from the Rioni River, the Georgian Black Sea basin. In the presently reported study, C. saniae sequences from the Urmia Lake Basin clustered with published sequences of C. saniae from the Caspian Sea basin with a K2P genetic divergence of 0.7%. The presently provided data constitute the evidence that the distribution range of this species reaches to the western part of Iran. Low genetic distance found between Urmia Lake and the Caspian Sea basin fall in an intraspecific range of distance among Cobitis species (Freyhof et al. 2018) Despite many efforts to collect more specimens of the genus Cobitis in the Zarrineh River, we could able to collect only five specimens, however, these few records show its zoogeographic importance. The presence of C. saniae in the Caspian Sea basin (the Aras River) and Urmia Lake basins suggests their connection in the past due to geological events. Possible corridors between these basins   T T C   5904  5907  5916  5929  5940  5952  5955  5957  5958  5961  5967  5970  5976  6001  6002  6024  6033  6042  6043  6048  6049  6051  6054  6057  6063  6069  6072 6078 6081

C. linea G C A T T C A T T T A A A T T G T A T A T A T T T A T A A C. avicennae
A C G C C C A C A C G G C C T A C G C T C G C C C T C G C C. faridpak A C G C C C T C A T G G C C T A T T C T T G C T C T T G C C. saniae (Caspian) A T G C C C A C A T G G C C T A T C C T T G C T C T T G C C. saniae (Urmia) A T G C C C A C A T G G C C G A T C C T A G C T C T T G C could be inter-drainage connections in the northern part of the Urmia Lake basin in Khoy (Ghara-Tappeh) via the Aras River, which drains into the Caspian Sea, and stream capture events occurring in the southeast of this basin, as the headwaters of some rivers draining to the Caspian Sea (e.g., Qezel-Ozan River) are located in the proximity to the south-eastern Urmia Lake basin (Zarrineh River) (Jalili et al. 2017, Khaefi et al. 2017). This conclusion is supported by geological reports indicating the origin of the modern