Iran. J. Ichthyol. (December 2015), 2(4): 235–243 Received: September 21, 2015 © 2015 Iranian Society of Ichthyology Accepted: Novembers 17, 2015 P-ISSN: 2383-1561; E-ISSN: 2383-0964 doi: http://www.ijichthyol.org

First record of Paraschistura alta (Nalbant and Bianco, 1998) from Eastern Iran and providing its COI barcode region sequences (Teleostei: )

Arash JOULADEH-ROUDBAR*1, Soheil EAGDERI2, Saber VATANDOUST3

1Department of Fisheries, Sari University of Agriculture Sciences and Natural Resources, Mazandran, Iran. 2Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran. 3Department of Fisheries, Babol Branch, Islamic Azad University, Babol, Iran. *Email: [email protected]

Abstract: Paraschistura alta, formerly reported from the eastern part of Sistan basin in Afghanistan, is reported for the first time from the Zahak River in Iranian part of this basin and its morphological characteristics, COI barcode and its phylogenetic relationship within other members of the Paraschistura from Iran are provided. All the measured meristic and morphometric characters of seven collected specimens of P. alta (SL= 65.8-112.1mm) from the Zahak River were in the range of its original description. The results of phylogenetic analysis based on the COI barcode region places the Iranian members of the genus Paraschistura into 11 groups, which show between 3.05% and 14.10% K2P sequence divergence. Paraschistura alta from Zahak River corresponds to a distinct clade, sister to P. bampurensis + P. hurmozensis. Keywords: Mitochondrial gene, Phylogenetic analysis, Helmand River, Sistan basin, Iran.

Introduction Pakistan and Afghanistan. Nemacheilid loaches with about 67 genera and more The members of this genus with a dark black than 652 valid species are found across Eurasia with spot or strip at the base of the anterior dorsal fin rays one species in northeast Africa (Eschmeyer & Fong (Coad 2015; Freyhof et al. 2015). They are small 2011). This family has a great diversity in Iranian fishes and characterised by an elongated body with Inland waters (Coad 2015; Freyhof et al. 2015; dorsal and ventral profiles almost parallel. The body Jouladeh-Roudbar et al., 2015a,b; Mafakheri et al., is compressed anteriorly and the head is compressed 2015; Azimi et al., 2015a,b; Mousavi-Sabet et al., or depressed. The caudal peduncle is short and 2015a,b; Ghasemi et al., 2015). Among the member moderately deep. The snout is usually blunt. The of family Nemacheilidae, Paraschistura Prokofiev, dorsal and anal fins usually have 7 and 5 branched 2009 is a newly described genus, and therefore, not rays, respectively. The caudal fin is slightly to deeply all of its species are fully examined and ascribed emarginated. The lateral line is incomplete, may be (Coad 2014) till Freyhof et al. (2015) reviewed the very short and rarely reaches the end of the anal fin genus Paraschistura from Iran and described six new level. Scales arc absent or rarely present but weakly species and provided new information on this taxon. developed on the caudal peduncle (Coad 2015). They also provided an identification key for all 11 Kottelat (2012) listed 14 species in recognized species. However, it seems that still Paraschistura, based mostly on the proposals of members of this genus are poorly known from some Prokofiev (2009) which are described from River drainages in Iran, Turkey, Turkmenistan, Afghanistan (P. lindbergi), Pakistan (P. alepidota,

235

Iranian Journal of Ichthyology (December 2015), 2(4): 235-243

P. kessleri, P. lepidocaulis, P. microlabra, P. naseeri, P. pakistanica, P. prashari, and P. punjabensis), Turkey (P. chrysicristinae), and Turkmenistan (P. turcomana = P. turcomanus), and the three species from Iran, including P. bampurensis, P. nielseni and P. sargadensis. Vatandoust & Eagderi (2015) described P. ilamensis from Tigris River drainage as first species of the genus of Paraschistura from Iranian part of this basin. Freyhof et al. (2015) reviewed the genus Paraschistura from Iran and described of six new species including P. abdolii (from the Sirjan basin and the western tributaries of the Hamun-e Jaz Murian basin), P. aredvii (from the Zohreh drainage), P. hormuzensis (from the Minab drainage), P. naumanni (from the Kol drainage, the Fig.1. Map of Eastern Iran showing sampling station Mond drainage and the Lake Maharlo basin), (∆, Zahak River) in the Sistan basin. P. pasatigris (from the Karun and Karkheh deeply forked caudal fin and emarginated dorsal fin- drainages), and P. susiani (from the Jarahi drainage) free margin. Paraschistura alta has been reported only based on the morphological and molecular (the from Helmand River basin in Afghanistan and now mtDNA COI barcode region) data set. Recently found in the Zahak River of the Sistan basin of Iran Mousavi-Sabet & Eagderi (2015) also described which may be due to recent floods caused entered this P. delvari from Mond River drainage (Persis basin). species to the Sistan basin. Therefore, this study is Based on the described characteristics of aimed to report first record of this species in Iran by Paraschistura pasatigris, this species can be providing its morphological characteristics, suggested as synonym of P. ilamensis. In addition, diagnostic nucleotide substitutions in the mtDNA Freyhof et al. (2015) treated Metaschistura COI barcode region and its phylogentic relationship Prokofiev, 2009 as a synonym of Paraschistura within the members of the genus Paraschistura in Prokofiev, 2009, P. sargadensis as synonym of Iran. P. kessleri and P. turcomanus as synonym of P. turcmenica. Mousavi-Sabat et al. (2015) reviewed Materials and Methods the genus Paraschistura in the Hari River basin, and Seven specimens of P. alta were collected from the revalidated P. turcomana (= P. turcomanus). In Zahak River, Sistan and Baluchestan Province, near addition, P. baluchiorum described from Pakistan is Zabol 30°49'32"N, 61°45'36"E, in September 2014 treated as the synonym of P. bampurensis according during fieldwork on the ichthyofauna of Sistan basin to Nalbant and Bianco (1998). Also, P. alta described by electro-fishing (Fig. 1). After anesthesia with 1% from Afghanistan is retained in the genus Schistora clove solution, three specimens were fixed in 96% by Kottelat (2012), whereas Coad (2014) considered ethanol and the other four specimens were preserved it as P. alta. in 4% buffered formaldehyde and transferred to the The type locality of P. alta is Helmand River laboratory for further investigations. The taxonomic basin in Afghanistan at the Kajaki, north east of key given by Coad (2014) were used to identify the Girisk and it was originally described as Schistura specimens. alta Nalbant & Bianco, 1998. This species is Meristic characteristics of the specimens were characterised by a deep body, a complete lateral line, counted using a stereomicroscope. A total of 26 236 Jouladeh-Roudbar et al.-First record of Paraschistura alta from Iran

of Cobitis avicennae Table 1. Morphaometric data of Paraschistura alta (n=7).

range mean SD Standard length (mm) 65.8-112.1 94.6 7.5 In percent of standard length Head length 14-23.4 18.7 6.6 Body depth at dorsal-fin origin 12.1-20.2 16.1 5.7 Body width at dorsal-fin origin 7.6-12.6 10.1 3.6 Predorsal length 33.1-55.2 44.2 5.6 Postdorsal length 35.6-59.3 47.4 6.8 Prepelvic length 36-60.1 48.0 7.0 Preanal length 54.4-90.7 72.6 7.7 Distance between pectoral and pelvic-fin origins 21.5-35.9 28.7 8.1 Distance between pelvic and anal-fin origins 17.1-28.5 22.8 8.1 Depth of caudal peduncle 8.6-14.3 11.4 4.0 Length of caudal peduncle 8.1-13.5 10.8 3.8 Dorsal-fin depth 14.9-24.9 19.9 7.0 Pectoral-fin length 11.6-16.4 13.5 5.5 Pelvic-fin length 12-20.1 16.1 5.7 Distance between anus and anal fin origin 1.2-2 1.6 0.6 In percent of head length Head depth at nape 47-53 51.2 1.8 Head depth at eye 40-45 42.6 1.2 Snout length 42-48 43.1 2.6 Eye diameter 12-16 13.8 1.8 Postorbital distance 43-47 45.1 1.7 Maximum head width 51-59 53.2 2.1 Interorbital width 22-29 26.1 1.8 Length of inner rostral barbell 20-26 22.1 2.8 Length of outer rostral barbell 27-35 30.1 1.6 Length of maxillary barbell 26-38 32.1 2.8 morphometric features were measured by a caliper to containing 5μl of 10X Taq polymerase buffer, 1μl of the nearest 0.01mm (Table 1). All measurements are (50mM) MgCl2,1μl of (10mM) deoxynucleotide made point to point based on Kottelat & Freyhof triphosphate (dNTP), 1μl (10μm) of each primer, 1μl (2007). The percentage ratios of morphometric of Taq polymerase (5Uμl-1), 7μl of total DNA and characters in relations to SL and HL were calculated. 33μl of H2O. Amplification cycles were as follows: The last two branched rays articulating on a single denaturation for 10 min at 94°C; 30 cycles at 94°C pterygiophore in the dorsal and anal fins are noted as for 1 min, 58.5°C for 1 min, 72°C for 1 min and a “1½”. Unbranched rays of dorsal and anal fins are not final extension for 5min at 72°C. PCR products were counted as they are deeply. purified using purification Kit (Expin Combo GP – DNA extraction and PCR: DNA was extracted from mini; Macrogen incorporation, Korea). The PCR muscle tissue at base of dorsal fin using a Genomic products were sequenced using Sanger method by a DNA Purification Kit (#K0512; Thermo Scientific robotic ABI-3130xl sequencer using manufacturer’s Corporation, Lithuania) following the manufacturer’s protocol. The forward primer FCOI20 was used to protocol. The COI gene was amplified using primers single strand sequencing. FCOI20 (5′-AACCTCTGTCTTCGGGGCTA-3′) Molecular data analysis: The sequences were and RCOI20III (5′-TTGAGCCTCCGTGAAGTGT compared to the published Paraschistura sequences G-3′), designed by (Hashemzadeh-Segherloo et al. using (BLASTn) basic local alignment search tool 2014). Polymerase chain reaction (PCR) conditions (Altschul et al. 1990). All sequence data were aligned were as follows: a 50μl final reaction volume using MEGA6 software (Tamura et al. 2013). To 237 Iranian Journal of Ichthyology (December 2015), 2(4): 235-243

Table 1. Accession numbers of the new materials and materials from NCBI (mainly from Freyhof et al. 2015).

Number Gen Bank No. Species Number Gen Bank No. Species 1 KM603300 P. abdolii 20 KM603297 P. naumanni 2 KM603299 P. abdolii 21 KM603311 P. naumanni 3 KM603298 P. abdolii 22 KM603309 P. naumanni 4 KU314412 P. alta 23 KM603306 P. nielseni 5 KU314413 P. alta 24 KM603280 P. nielseni 6 KU314414 P. alta 25 KM603275 P. nielseni 7 KM603274 P. aredvii 26 KM603295 P. pasatigris 8 KM603289 P. aredvii 27 KM603313 P. pasatigris 9 KM603301 P. aredvii 28 KM603308 P. pasatigris 10 KM603230 P. bampurensis 29 KM603290 P. susiani 11 KM603284 P. bampurensis 30 KM603283 P. susiani 12 KM603231 P. bampurensis 31 KM603261 P. susiani 13 KM603263 P. bampurensis 32 KM603302 P. turcmenica 14 KM603282 P. cristata 33 KM603305 P. turcmenica 15 KM603293 P. cristata 34 KM603286 P. turcmenica 16 KM603312 P. cristata 35 KJ179266 Paracobitis malapterura 17 KJ179267 P. hormuzensis 36 KJ179259 T. hafezi 18 KM603296 P. hormuzensis 37 KJ179254 Turcinoemacheilus hafezi 19 KM603202 P. malapterura unify the length of the sequences, the common 650bp constructed phylogenetic hypothesis, the length segments were selected and used for geographically adjacent loach genera i.e. phylogenetic analysis. Turcinoemacheilus, Paracobitis and Oxynoemache- Maximum likelihood reconstructions were ilus were included. performed using RAxML 7.2.5 (Stamatakis 2006) under the GTR+G+I model of nucleotide Results substitution, with CAT approximation of rate Morphological analysis: Seven specimens of P. alta heterogeneity and bootstrap (10000 bootstrap ranged 65.8-112.1mm of SL (Fig. 2) were collected replicates). Bayesian analysis was performed using from the Zahak River (Sistan and Baluchestan MrBayes 3.1.2. Starting from a random tree, Province, near Zabol) in September 2014. The Metropolis-coupled Markov chain Monte Carlo general body shape of this species is displayed in sampling was performed with four chains run for Figure 2. Morphometric characteristics are provided 1.5×106 generations for COI data set of with a in Table 2. All the measured meristic and sampling frequency of 100. Bayesian posterior morphometric characters of Iranian population of probabilities were obtained from the 50% majority P. alta were in the range of its description provided rule consensus trees. The resulting BI topology was by Coad (2014). The other morphological features applied for both presented trees. (Huelsenbeck et al. and its coloration are provided as following: 2004). All constructed phylograms were displayed Description: Medium sized, slender species with and edited in the programmes FigTree, v1.2.1 large and depressed head. Body deepest at dorsal-fin (Rambaut, 2009). The evolutionary divergence over origin, depth slightly decreasing towards caudal-fin sequence pairs between groups of the studied species base. Greatest body width at middle between were estimated using the Kimura 2-parameter model pectoral- and pelvic fin bases. Section of the head (Kimura 1980). The retrieved sequences of the other roundish, flattened on ventral surface. Caudal members of the genus Paraschistura from GenBank peduncle compressed laterally and short, 0.9-1.2 database (NCBI) following Blast search is shown in (mean 1.1) times longer than deep. Pectoral fin Table 2. As appropriate outgroup to root the reaching approximately 50-55% of distance from 238 Jouladeh-Roudbar et al.-First record of Paraschistura alta from Iran

of Cobitis avicennae

Fig.2. Lateral view of Paraschistura alta collected from the Zahak River (Sistan basin of Iran).

Fig.3. Lateral view of the cleared and stained specimen of Paraschistura alta. pectoral-fin origin to pelvic-fin origin. Pelvic axillary Coloration: Body olive-green or beige in life and lobe ovoid, fully attached to body or tip of axillary yellowish in preserved individuals. Body with 9-10 lobe free. Pelvic fin origin below second or third regularly shaped dark bars (7-10 based on Coad branched dorsal fin ray. Pelvic fin not reaching to (2014)), including 4 predorsal, 1 at dorsal-fin origin, anus. Anal fin origin about one eye diameter behind 1 at middle of dorsal-fin base, 1 at posterior margin anus. Anal-fin origin is at vertical of middle between of dorsal fin-base and 3 (in one specimen 2) bars on dorsal- and caudal fin origins and reaches to caudal caudal peduncle . Bars often dissociate on flank but fin origin. A shallow dorsal adipose keel on caudal usually meeting their homologues on back. Bars on peduncle, without procurrent rays. Margin of dorsal flank in front of the dorsal-fin origin sometimes fin concave. Caudal fin forked with pointed lobe. faded or absent in some individuals. A large black Usually upper lob longer than lower lobe. Dorsal fin spot at base of unbranched and first branched dorsal- with 3 unbranched and 7½ and 8½ branched rays. fin rays. Head olive-green on top and side with cheek Anal fin with 3 unbranched and 5½ branched rays. pale-olive. Dorsal and caudal fins with sporadic Pectoral fin with 9-10 (usually 9) and pelvic fin with black spots on rays and other fins hyaline. 6-7 (usually 7) branched rays (Fig. 3.). Small, Molecular analysis: We generated COI barcodes for rounded, deeply embedded scales on caudal peduncle 3 specimens of P. alta collected from Zahak River. and flanks. Lateral line incomplete, reaching to The estimation of the phylogenetic relationships dorsal-fin origin. Mouth small, strongly arched. Lips based on the COI barcode region places the used fleshy, with many deep furrows. A median sequenced of the members of the genus Paraschistura interruption in lower lip. Upper lip with median into 11 groups (Fig. 4), which show between 3.05% incision. There is no Processus dentiformis. Anterior and 14.1% K2P sequence divergence in their COI nostril opening at tip of a pointed and flap-like tube. barcode region. Paraschistura alta from Zahak River Barbels long. Largest known specimen 112mm SL. corresponds a distinct clade, forming a monophyletic

239 Iranian Journal of Ichthyology (December 2015), 2(4): 235-243

Fig.4. Bayesian consensus tree inferred from COI data. Bayesian posterior probabilities followed by Maximum likelihood bootstrap values are listed above the nodes. group with sister group of P. bampurensis + current study provided the morphometric and P. hurmozensis (Fig. 4). All analysed species of the meristic characteristics, COI barcoding of the Iranian genus Paraschistura corresponds to distinct clades population of this species as well as its phylogenetic (Fig. 4). Table 3 lists the nucleotide substitutions relationship with other members of the genus found in the 650 base pairs long mtDNA COI Paraschistura from Iran. barcode region. Table 4 lists the average estimates of The Sistan basin consists of several lakes of the evolutionary divergence between the varied extent i.e. Hamune puzak, Hamun-e Saberi Paraschistura species. and Hamun-e hirmand and connections straddling the Iran-Afghanistan border. Paraschistura alta has been Discussion described and reported only from Helmand River This study reports the presence of Paraschistura alta basin (the eastern part of the Sistan basin) in for first time from Iranian inland waters, Zahak River Afghanistan at the Kajaki, north east of Girisk. The (Sistan basin), showing its range extension further to Helmand River basin consists about 36800km2 and the western part of the Sistan basin. In addition, the originate from Baba Mountain (east of Farakhulum) 240 Jouladeh-Roudbar et al.-First record of Paraschistura alta from Iran

of Cobitis avicennae Table 3. Nucleotide substitutions found in mtDNA COI barcode region some of Paraschistura species in Iran.

Nucleotide position relative to Oryzias latipes complete mitochondrial genome (AP004421)

5507 5508 5515 5525 5537 5546 5549 5551 5555 5558 5561 5564 5573 5579 5582 5585 5606 5621 5639 5642 5645 5660 5663 5666 5672 5675 5678 5681 5684 5687 5696 5699 5702 5705 5708 5711 5714 5726 5729 5732 5735 5741 5744 5747 5756 P. alta T C T G C T C G T A T A T G A G A T T T A G A T A T A A T A C A G A G T G G A T A G C C C P.abdolii T C A G C C T G A A T A A A A A A T T A A A G C C C G A T G C G A A A C G G G T A G C C T P.aredvii T T T G C T T C T A T G A G A A A T T T G A G C A C G G T A T A A A A T G A A C A A C C C P.bampurensis T C T G C T C G T T C A T G A A A T C T A G A T G C G A T A T A G G A C G G A T A A C C C P.cristata T C T A C T T G T A T A A A G G G T T T A A G C C C A G T A C G A A A C G G A C G G T C C P.hormuzensis T C T G C T C G T T C G T A A A A T T T A A A T G C G A T A T A A A A C A G A T A A C C C P.naumanni T C T A C C T G A A T A A G A A A T T A A A G C C C G A T G C G A A A C A G G T A G C C T P.nielseni C T T G C T T G T A T G A G A A A T T T A A G C A C G G T A T A G A A C G A A T A A C T C P.pasatigris T T T G T T T G T A T A A G A A A C C T A A A C A C G G C A T A A A A C A A A C A A C C C P.susiani T T T A C T T G T A T A A G A A A T T T A A G C A C G A T A T G A A A T G A A C A A C C C P.turcmenica T C T G C T T G T A T A A A A A G T T T A A G C C T A G C A C G G G A C G G A C G G C C C

5759 5763 5765 5771 5777 5780 5786 5787 5789 5795 5798 5807 5810 5813 5816 5819 5825 5831 5834 5837 5840 5844 5846 5847 5849 5852 5856 5858 5861 5864 5867 5870 5879 5891 5897 5900 5903 5906 5909 5912 5913 5915 5921 5924 5925

P. alta A C G A C C T C G A T A A A T C A G T C G C A G G A T A C T A T A T A A T G A T G C A C T P.abdolii A C C C T T C C G A T G G A T A G G T T C T A G A A C G T T A G A T G G T G A T G C A C C P.aredvii A C G T C C T T A A T A A A T A G A T T A T G G A T C A C T A T G T A A C A A T G T A T T P.bampurensis A T G A C C C C G A T A A G T A G A T C G C A G A A T A C T A T A C G A C A A T G C A C T P.cristata A C A C T C T C A A T G G G T A G G T T C T A G A A C A A T G G A T G A C A A T G C G C T P.hormuzensis A C G A C C C C G A T A G G T A A G T C G C A G A A T A C C A T A C G A C A A C G C A C T P.naumanni A C C C T T C C G A T G A G T A G G T T C T A G A A C G C T A A A T G A T G A T G C A C T P.nielseni A C G T C C T C G A G A G G T A A G T T G T G G A T C A C T A T A T G A C A A T G T A T T P.pasatigris A C A A C C T C A G T A T A T A A G T C A T A G A G C A C T A T A T G A C A T T G T A C T P.susiani G C G A C C T C G A T A T G T A A A T C A T G G A A C A C T G T A T G A C A T T G T A C T P.turcmenica A C A C T T T C A G T G G G C A A A C T C T A G A A C A A T A G A T G A C A A T G C G C T

5927 5930 5936 5945 5951 5954 5957 5978 5981 5984 5993 5996 5997 6002 6005 6008 6014 6023 6032 6035 6038 6039 6041 6044 6045 6047 6050 6053 6059 6062 6071 6083 6086 6089 6095 6098 6101 6104 6107 6113 6119 6125 6128 6131

P. alta G G C T A T T T C A C A C C A A T T C A T C G C C A G T C C C A C G G C T A C C C T A C P.abdolii A G T C A C C C T A C G C T G G C C C G C C A C C A G C T C C A T G A C C A C C C T A C P.aredvii A A C T A T T C C A C A C C A A T T T A C C G T C A A T C C C A C G A C T A T C G T A T P.bampurensis A G C T A T T T C A C A C T A A T T C A T C A T C A G T C T T A C A A C T A C C C T A C P.cristata G G T T A C C T C G C A C T G A C C C G C C G C T A A T C C C G T A A C C A C C C T G C P.hormuzensis G G C T A T T C C A C G C T A A T T C A T C G T C A A C C C C A C A A T T A C T C T A C P.naumanni G A T C A C C C C G C G T T G G C C C G C C A C C A G C T C C A T G A C C A C C C T G C P.nielseni A A T T A T T C C A T A C T A A T T T A C C G C C G A T C C C A C G A C C A C C G T A T P.pasatigris A G C C G T T C C A C G C C G A T T T A C T G C C A A T C C C A C G A C C G C T A T A C P.susiani A G C T A T T C C A C A C C G A T T T A T C A C C A G T C C C G C G A C T A C C G T A T P.turcmenica G G T T A C C T C A C A C T G A C C C G C C G C C A A T C C C A T G A C C A C C C C G C

Table 3. Estimates of the average evolutionary divergence between the Iranian Paraschistura species, expressed as number of base substitutions per site. All positions with less than 95 % site coverage were eliminated before analysis, leading to a total of 650 nucleotide positions.

No. Species 1 2 3 4 5 6 7 8 9 10 1 P. alta 2 P. abdolii 12.91 3 P. aredvii 8.84 13.28 4 P. bampurensis 5.92 14.10 9.12 5 P. cristata 11.05 8.31 11.18 12.59 6 P. hormuzensis 6.72 12.23 9.16 4.00 12.01 7 P. naumanni 12.59 3.30 13.28 13.45 8.87 12.23 8 P. nielseni 8.78 13.18 3.05 9.63 11.09 8.53 13.18 9 P. pasatigris 9.41 13.50 6.18 9.70 12.32 8.87 13.18 7.44 10 P. susiani 8.01 11.62 4.20 7.74 9.63 7.50 11.92 4.90 4.75 11 P. turcmenica 11.97 9.16 12.11 12.91 3.54 12.96 10.32 12.01 12.01 10.50

241 Iranian Journal of Ichthyology (December 2015), 2(4): 235-243

and drain south-west for about 1300km (Dupree 410. 1973; Coad 2014) before Sistan (Hamoun) lakes. The Azimi, H.; Mousavi-Sabet, H. & Eagderi, S. 2015a. previous studies have reported little relationship Osteological characteristics of Paraschistura between the western (Iran) and eastern (Afghanistan) nielseni (, Nemacheilidae). Iranian parts of the Sistan basin (Coad 2014). Our results also Journal of Ichthyology 2(3): 155-164. Azimi, H.; Mousavi-Sabet, H.; Eagderi, S. & Vatandoust, confirm the absence of 10 species (Garra wanae, S. 2015b. Osteological characteristics of Schizothorax esocinus, S. labiatus, S. plagiostomus, Turkmenian stone loach, Paraschistura cristata Paracobitis boutanensis, P. gazniensis, Paraschistura (Cypriniformes: Nemacheilidae). International lindbergi, Triplophysa farwelli, and T. griffithii, Journal of Aquatic Biology 3(5):290-300. T. kullmanni) out of 22 reported species from eastern Coad, B.W. 2014. Fishes of Afghanistan. Pensoft part of the Sistan basin (Afghanistan) and vice versa Publishers, Sofia-Moscow. 393 pp. absence of Capoeta fusca in eastern part of this basin Coad, B.W. 2015. Freshwater Fishes of Iran (Available at that presents in Iranian part (Coad 2014; Jouladeh- http://www.briancoad.com) (accessed on 10 Roudbar et al. 2015a). The Iranian part of Sistan October 2015). basin is a network basin and the flooding of the lake Dupree, L. 1984. Afghanistan in 1983: and still no could be led to fish movement toward western part of solution. Asian Survey 229-239. Eschmeyer, W.N. & Fong, J.D. 2011. Pisces. In: Zhang, this basin, where the most of lakes lies in Iran. Z.-Q. (Ed.). biodiversity: An outline of The result of the phylogenetic relationships of higher level classification and survey of taxonomic the Iranian members of the genus Paraschistura richness. Zootaxa 3148:26-38. (except P. delvarii) based on COI barcode region Freyhof, J.; Sayyadzadeh, G.; Esmaeili, H.R. & Geiger, revealed that P. alta is distinct clade sister group to M. 2015. Review of the genus Paraschistura from P. hurmuzensis (from Makran basin, southeast of Iran with description of six new species (Teleostei: Iran) + P. bampurensis (from Jaz-e Murian and Nemacheilidae). Ichthyological Exploration Makran basins, southeast of Iran). The nucleotide Freshwaters 26(1): 1-48. substitution of studied 650bp mtDNA COI barcode Ghasemi, H.; Jouladeh-Roudbar, A.J.; Eagderi, S.; region showed 5.92% and 12.91% differences Abbasi, K.; Vatandoust, S. & Esmaeili, H.R. between P. alta with P. bampurensis and P. abdolii, (2015). Ichthyofauna of Urmia basin: Taxonomic diversity, distribution and conservation. Iranian receptivity. The Sistan, Jaz-e Murian and Makran Journal of Ichthyology 2(3):177-193. basins are connected and therefore their close Hashemzadeh-Segherloo, I.; Abdoli, A.; Purahmad, R.; relationships can be verified based on close Puria, M. & Golzarianpour, K. 2014. Genetic geographical distance. barcoding of Capoeta species in Karoon and Tigris tributaries. New Genetics 9(2): 171-178. (In Farsi) Acknowledgement Huelsenbeck, J.P.; Larget, B. & Alfaro, M.E. 2004. We would like to thank University of Tehran for Bayesian phylogenetic model selection using financial support. We are pleased to thank H. Gohari reversible jump Markov chain Monte Carlo. Moghadam, A. Bahalkeh, and P. Jalili for helping in Molecular Biology and Evolution 21(6): 1123- fish collection and clearing and staining of the 1133. specimens. Jouladeh-Roudbar, A.; Eagderi, S. & Esmaeili, H.R. 2015a. Fishes of the Dasht-e Kavir basin of Iran: an

updated checklist. International Journal of Aquatic References Biology 3(4): 263-273. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W. & Jouladeh-Roudbar, A.; Vatandoust, S.; Eagderi, S.; Jafari- Lipman, D.J. 1990. Basic local alignment search Kenari, S. & Mousavi-Sabet, H. 2015b. Freshwater tool. Journal of Molecular Biology 215(3): 403- fishes of Iran; an updated checklist. AACL Bioflux 242 Jouladeh-Roudbar et al.-First record of Paraschistura alta from Iran

of Cobitis avicennae 8(6): 855-909. likelihood-based phylogenetic analyses with Kimura, M. 1980. Estimation of evolutionary distances thousands of taxa and mixed models. between homologous nucleotide sequences. Bioinformatics 22(21): 2688-2690. Proceedings of the National Academy of Sciences Stamatakis, A. 2006. RAxML-VI-HPC: maximum of the United States of America 78(1): 454-458. likelihood-based phylogenetic analyses with Kottelat, M. & J. Freyhof. 2007. Handbook of European thousands of taxa and mixed models. Freshwater Fishes. Kottelat Cornol and Freyhof, Bioinformatics 22(21): 2688-2690. Berlin, 646 pp. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kottelat, M. 2012. Conspectus cobitidum: an inventory of Kumar, S. 2013. MEGA6: molecular evolutionary the loaches of the world (Teleostei: Cypriniformes: genetics analysis version 6.0. Molecular biology Cobitoidei). The Raffles Bulletin of Zoology 26: 1- and evolution 30(12): 2725-2729. 199. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A. & Mafakheri, P.; Eagderi, S.; Farahmand, H. & Mousavi- Kumar, S. 2013. MEGA6: Molecular Evolutionary Sabet, H. 2015. Osteological structure of Kiabi Genetics Analysis version 6.0. Molecular Biology loach, Oxynoemacheilus kiabii (: and Evolution 30: 2725-2729. Nemacheilidae). Iranian Journal of Ichthyology Vatandoust, S. & Eagderi, S. 2015. Paraschistura 1(3): 197-205. ilamensis, a new species of loach from the Tigris Mousavi-Sabet, H. & Eagderi, S. 2015. Paraschistura River drainage (Teleostei: Nemacheilidae). delvarii spec. nov. – a new species of stone loach International Journal of Aquatic Biology 3(3): 177- from the Persian Gulf basin, southern Iran 182. (Teleostei: Nemacheilidae). Vertebrate Zoology 65 (3): 297-303. Mousavi-Sabet, H.; Vatandoust, H.; Jouladeh-Roudbar, A. & Eagderi, S. 2015a. Taxonomic Status of the Genus Paraschistura (Teleostei: Nemacheilidae) in the Hari River Basin, with Re-validation of P. turcomana. Journal of Applied Biological Sciences (3): 01-05. Nalbant, T.T. & Bianco, P.G. 1998. The loaches of Iran and adjacent regions with description of six new species (Cobitoidea). Italian Journal of Zoology 65: 109-123. (Proceedings of the Ninth Congress of European Ichthyologists (CEI-9) "Fish Biodiversity" organised in Naples at the University Federico II and held in Trieste - Italy, 24-30 August 1997). Prokofiev, A.M. 2009. Problems of the classification and phylogeny of Nemacheiline loaches of the group lacking the preethmoid I (Cypriniformes: Balitoridae: Nemacheilinae). Journal of Ichthyology 49(10): 874-898. Rambaut A. 2009. FigTree, Available from/http://tree.bio. ed.ac.uk/software/figtree. Ronquist, F. & Huelsenbeck, J.P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19(12): 1572-1574. Stamatakis, A. 2006. RAxML-VI-HPC: maximum 243