FishTaxa (2016) 1(2): 94-107 E-ISSN: 2458-942X Journal homepage: www.fishtaxa.com © 2016 FISHTAXA. All rights reserved Article Oxynoemacheilus freyhofi, a new nemacheilid species (Teleostei, Nemacheilidae) from the Tigris basin, Iran Arash JOULADEH-ROUDBAR, Soheil EAGDERI*, Tayyeb HOSSEINPOUR Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, P.O. Box 4111, Iran. *Corresponding author: E-mail: [email protected] Abstract A new species of Nemacheilidae, Oxynoemacheilus freyhofi sp. nov. is described from the Roudbar River, a tributary of the Bakhtiyari River, Karoun River drainage, Iran. The species differs from its congeners in the combination of the following characters: dorsal fin with 10½ branched rays; forked caudal fin with equal size of lobes; slender and compressed caudal peduncle and complete lateral line; very small or absent median incision in upper lip; absence of suborbital groove in male. Oxynoemacheilus freyhofi is also distinguished from its congeners by a K2P distance of 4.5% to O. argyrogramma and 12.9% to O. bergianus. Keywords: Freshwater fish, Taxonomy, Morphology, COI, Loach. Zoobank: urn:lsid:zoobank.org:pub:B9CC32FB-1251-4518-9F89-EA013F203070 urn:lsid:zoobank.org:act:3A899D53-95FE-44C2-A624-249AA8A77946 Introduction Stone loaches, family Nemacheilidae, are small fishes occurring in fresh waters of Asia and its islands, Europe, and northeast Africa (Coad 2015). They inhabit a variety of water bodies from turbulent mountain streams to salty rivers in dry lowlands (Mafakheri et al. 2015a, b, 2016). Nemacheilidae has about 48 genera and more than 661 species (Nelson et al. 2016; Eschmeyer and Fong 2011; Freyhof et al. 2015), with more expected to be described (Coad 2015; Kamangar et al. 2014). This family has a great diversity in Iranian inland waters (Coad 1995, 2006; Jouladh-Roudbar et al. 2015) with about 44 reported species that about 25 of them are endemic to Iran indicating their importance (Esmaeili et al. 2010; Kamangar et al. 2014; Esmaeili et al. 2014; Freyhof et al. 2014, 2015; Jouladh-Roudbar et al. 2015). Stoumboudi et al. (2006) and Prokofiev (2009) placed most Nemacheilid loaches from Eastern Europe and the Middle East in the genus Oxynoemacheilus (Freyhof et al. 2011). The members of this genus is distinguished from the other genus of the family Nemacheilidae by a caudal fin emarginate or truncate if caudal fin forked, the body and caudal peduncle cylindrical, and usually 7-9½ branched dorsal-fin rays (Freyhof et al. 2011). The genus Oxynoemacheilus has 11 reported species from Iranian inland waters, including O. angorae, O. bergianus, O. brandtii, O. frenatus, O. kiabii, O. longipinnis, O. persa and O. tongiorgii, as well as three recently described species from the Choman River drainage of the Tigris basin i.e. O. kurdistanicus, O. chomanicus and O. zagrosensis (Kamangar et al. 2014). In the present paper, we describe an additional species of the genus Oxynoemacheilus collected from the Roudbar River, a tributary of the Bakhtiyari River (Karoun River drainage, Lorestan Province, Iran) in 2015 that demonstrated some morphological and molecular features compared to other members of this genus. Methods Morphological examinations: Measurements follow Kottelat and Freyhof (2007). Standard length (SL) is measured from the tip of the snout to the end of the hypural complex. The length of the caudal peduncle is 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 last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are noted as “1½”. Simple rays of dorsal and anal fins are not counted as they are deeply embedded. Accepted: 3 September 2016; Published: 27 September 2016 DOI: http://dx.doi.org/10.7508/fishtaxa.2016.02.005 Jouladeh-Roudbar et al.-Oxynoemacheilus freyhofi, a new nemacheilid species from Iran 95 Measurements were taken using an electronic caliper and rounded to the nearest 0.1 mm. The data are presented as standard length and head length. Type specimens are deposited in the Ichtyological Museum of Natural Resources Faculty, University of Tehran (IMNRF-UT). DNA extraction and PCR: DNA was extracted from muscle tissue at base of the dorsal fin using a Genomic DNA Purification Kit (#K0512; Thermo Scientific Corporation, Lithuania) following the manufacturer’s protocol. The COI gene was amplified using primers FishF1-(5'-TCAACCAACCACAAAGACATTGGCAC-3') and FishR1- (5'-TAGACTTCTGGGTGGCCAAAGAATCA-3'), designed by (Ward et al. 2005). 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) MgCl2,1 μl of (10 mM) deoxynucleotide triphosphate (dNTP), 1 μl (10 μm) of each primer, 1 μl -1 of Taq polymerase (5 Uμl ), 7 μl of total DNA and 33 μl of H2O. 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 purification Kit (Expin Combo GP – mini; Macrogen incorporation, Korea). The PCR products were sequenced using Sanger method by a robotic ABI- 3130xl sequencer using manufacturer’s protocol. The forward and revers primer was used to single strand sequencing. Molecular data analysis: The sequences were compared to published Oxynoemacheilus sequences using (BLASTn) basic local alignment search tool (Altschul et al. 1990). All sequence data were aligned using MEGA6 software (Tamura et al. 2013). To unify the length of the sequences, the common 650 bp length segments were selected and used for phylogenetic analysis. Modeltest (Posada and Crandall 1998), implemented in the MEGA 6 software (Tamura et al. 2011) was used to determine the most appropriate sequence evolution model for the given data, treating gaps and missing data with the partial deletion option under 95 % site coverage cut-off. The model with the lowest BIC scores (Bayesian Information Criterion) is considered to best describe the substitution pattern (Nei and Kumar 2000; Posada and Crandall 2001). Bayesian analyses of nucleotide sequences were run with the parallel version of MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) on a Linux cluster with one processor assigned to each Markov chain under the most generalizing model (GTR+G+I) because over parametrization apparently does not negatively affect Bayesian analyses (Huelsenbeck and Ranala 2004). Each Bayesian analysis comprised two simultaneous runs of four Metropolis-coupled Markov-chains at the default temperature (0.2). Analyses were terminated after the chains converged significantly, as indicated by the average standard deviation of split frequencies <0.01. Estimates of evolutionary divergence over sequence pairs between species were conducted in Mega6 (Tamura et al. 2013). Analyses were conducted using the Kimura 2-parameter model (Kimura 1980). The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). Codon positions included were 1st+2nd+3rd. All positions containing gaps and missing data were eliminated. As appropriate outgroup to root the constructed phylogenetic hypothesis, Paracobitis molavii (accession number: KJ23516, KJ23517), was included. Abbreviations used: SL, standard length; HL, lateral head length; K2P, Kimura 2-parameter; IMNRFI-UT, Ichtyological Museum of Natural Resources Faculty, University of Tehran; ZM-CBSU, Zoological Museum of Shiraz University, Collection of Biology Department, Shiraz. Results Oxynoemacheilus freyhofi, sp. nov. (Figs. 1-7) Holotype: IMNRF-UT-1036-1, male, 60.3 mm SL; Iran: Lorstan prov.: Roudbar River, tributary of Bakhtiyari River, near Kazem Abad village, 33°08'17.8"N 49°40'43.9"E, S. Eagderi & A. Jouladeh-Roudbar, 19 August 2015. 96 FISHTAXA (2016) 1(2): 94-107 Figure 1. Oxynoemacheilus freyhofi sp. nov., IMNRF-UT- 1036-1, holotype, 55 mm SL; Iran: Lorestan prov.: Roudbar River. Figure 2. Oxynoemacheilus freyhofi, IMNRF-UT- 1036-1, holotype, 60.3 mm SL; Iran: Roudbar River River. Paratypes: IMNRF-UT-1036, 3, 44-45 mm SL; Iran: Lorstan prov.: Roudbar River, tributary of Bakhtiyari River, near Kazem Abad village, 33°08'17.8"N 49°40'43.9"E, S. Eagderi & A. Jouladeh-Roudbar, 19 August 2015. IMNRF-UT-2036, 7, 43-44 mm SL; Iran: Lorstan prov.: Roudbar River, tributary of Bakhtiyari River, near Dehmirza gholi village, 33°07'05.8"N 49°40'03.4"E, S. Eagderi & A. Jouladeh Roudbar, 19 August 2015. Diagnosis: Oxynoemacheilus freyhofi sp. nov. is distinguished from the other species of Oxynoemacheilus in Iran by a combination of characters, none of them unique. Dorsal fin with 10½ branched rays; forked caudal fin Jouladeh-Roudbar et al.-Oxynoemacheilus freyhofi, a new nemacheilid species from Iran 97 Table 1. Morphometric data of Oxynoemacheilus freyhofi (holotype, IMNRF-UT-1036-1; paratypes, IMNRF-1036, 3 specimens, IMNRF-UT- 2036, 7 specimens). Paratypes Holotype Range Mean SD Standard length (mm) 60.3 51.4-55.6 In percent of standard length (SL) Body depth maximal 17.2 17.3-17.9 17.6 0.3 Caudal peduncle depth 9.4 9.6-10 9.8 0.2 Predorsal length 49.9 50.4-51.9 51.1 0.7 Postdorsal length 49.1 51.3-52.4 52.0 0.7 Prepelvic length 54.5 51.7-54.9 52.8 1.7 Preanal length 75.5 74-74.4 74.1 0.3 Caudal peduncle length 15.9 17.2-18 17.5 0.4 Dorsal-fin base length 17.4 16.6-19.7 18.0 1.6 Dorsal-fin depth 15.8 19.5-21.1 20.3 0.8 Anal-fin base length 8.5 6.6-7.6 7.1 0.5 Anal-fin depth 16.2 15.1-16.3 15.7 0.6 Pectoral fin length