Received: 13 April 2019 | Revised: 15 July 2019 | Accepted: 31 July 2019 DOI: 10.1111/jai.13964 ORIGINAL ARTICLE A new record of Iranian subterranean fishes reveals the potential presence of a large freshwater aquifer in the Zagros Mountains Saber Vatandoust1 | Hamed Mousavi‐Sabet2,3 | Matthias F. Geiger4 | Jörg Freyhof5 1Department of Fisheries, Babol Branch, Islamic Azad University, Babol, Iran Abstract 2Department of Fisheries, Faculty of Natural A new locality is reported for the Iranian subterranean fishes Garra typhlops and Resources, University of Guilan, Sowmeh Garra lorestanensis (and probably Eidinemacheilus smithi), near the village Tuveh in the Sara, Iran 3The Caspian Sea Basin Research Dez River drainage. The site is 31 km straight‐line distance away from the only other Center, University of Guilan, Rasht, Iran known locality where these species have been observed previously. The finding sug‐ 4 Zoological Research Museum Alexander gests the presence of a sizeable subterranean aquifer system in the Tigris drainage Koenig, Leibniz Institute for Animal Biodiversity, Bonn, Germany extending for between 31 and 162 km. 5Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, KEYWORDS Berlin, Germany cyprinidae, cytochrome oxidase i, distribution, freshwater fish Correspondence Hamed Mousavi‐Sabet, Department of Fisheries, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, P.O. Box: 1144, Guilan, Iran. Email: [email protected] 1 | INTRODUCTION Loven (Figure 2) and it is the aim of this study to identify these speci‐ mens and discuss them within a geographical context. The Iranian subterranean fishes Garra typhlops (Bruun & Kaiser, 1944), Garra lorestanensis Mousavi‐Sabet & Eagderi, 2016, and Eidinemacheilus 2 | MATERIALS AND METHODS smithi (Greenwood, 1976) were long known only from their type local‐ ity, a well‐like pool called Loven, which is the natural outlet of a sub‐ 2.1 | Sampling and morphological examination terranean limestone aquifer in the Zagros Mountains located in the Ab‐e Sirum (or Ab‐e Serum) valley near Tang‐e Haft, Lorestan (Bruun Sampling was carried out using a hand‐net with mesh size of 2 mm. & Kaiser, 1944; Mousavi‐Sabet & Eagderi, 2016; Smith, 1953; Figure After anaesthesia, fishes were fixed in 5% formaldehyde and stored 1). These species were for around 60 years believed to be endemic to in 70% ethanol or directly fixed in 99% ethanol. Measurements were this site, but fish identified as G. typhlops were later collected about made with a dial caliper and recorded to 0.1 mm. Standard length (SL) 131 km away from the type locality in the Simarreh River drainage, and was measured from the tip of the snout to the end of the hypural com‐ the species was thus assumed to be more widely‐distributed in karst plex. Morphological identification follows Mousavi‐Sabet and Eagderi areas of the Zagros Mountains (Mahjoorazad & Coad, 2009). However, (2016) and Mousavi‐Sabet, Vatandoust, Fatemi, and Eagderi (2016). as all subterranean Garra species in the Middle East are superficially very similar in morphology and general appearance, it cannot be fully 2.2 | Abbreviations used excluded that the Garra from the Simarreh might belong to another species, although the site is no longer accessible after being flooded by SL, standard length; K2P, Kimura 2‐parameter. Collection codes: the Simarreh reservoir (Mahjoorazad & Coad, 2009). During fieldwork VMFC, Vatandoust & Mousavi‐Sabet Fish Collection, Tehran; FSJF, in 2018, we found subterranean fishes at a new site, 31 km away from Fischsammlung J. Freyhof, Berlin. J Appl Ichthyol. 2019;00:1–7. wileyonlinelibrary.com/journal/jai © 2019 Blackwell Verlag GmbH | 1 2 | VATANDOUST ET AL. 2.3 | Material examined Garra lorestanensis: VMFC GL‐H, holotype, 55 mm SL; VMFC GL‐P1 to VMFC GL‐P3, paratypes, 3, 27.2–58.0 mm SL: Iran: Lorestan prov.: Loven, 33°04′39′′N 48°35′33′′. — VMFC GL‐T, 2, 43–56 mm SL: Iran: Khuzestan prov.: spring Tuveh, near Tuveh vil‐ lage, 32°48′48.79′′N 48°43′6.94′′E. — FSJF 4,065, 1. 53 mm SL; Iran: Khuzestan prov.: spring Tuveh, north‐west of Tuveh village, 32°48′48.79′′N 48°43′6.94′′E. Garra typhlops: VMFC GT01, 1, 41 mm SL; VMFC GT02 to VMFC GT07, 6, 33.2–64.0 mm SL: Iran: Lorestan prov.: Loven, 33°04′39′′N 48°35′33′′E. — VMFC GT‐T, 3, 30–52 mm SL: Iran: Khuzestan prov.: spring Tuveh north‐west of Tuveh vil‐ lage, 32°48′48.79′′N 48°43′6.94′′E. — FSJF 4,066, 3, 29–54 mm SL; Iran: Khuzestan prov.: spring Tuveh north‐west of Tuveh village, 32°48′48.79′′N 48°43′6.94′′E. 2.4 | New materials used in the molecular genetic analysis Garra lorestanensis, FSJF 3,266; Iran: spring Tuveh north‐west of Tuveh village, 32°48′48.79′′N 48°43′6.94′′E (GenBank accession numbers: MN166778, MN166779). Garra typhlops, FSJF 3,267; Iran: spring FIGURE 1 Loven, type locality of Eidinemacheilus smithi, Garra Tuveh north‐west of Tuveh village, 32°48′48.79′′N 48°43′6.94′′E lorestanensis and Garra typhlops (GenBank accession numbers: MN166780, MN166781). FIGURE 2 Map of the Iranian portion of the Tigris River drainage, showing sites where subterranean fish have been found: Simarreh Dam, Loven, and Tuveh spring VATANDOUST ET AL. | 3 at 72°C. Second cycle set (25 repeats): 35 s denaturation at 94°C, 2.5 | DNA extraction 90 s annealing at 40°C and 90 s extension at 72°C; final elonga‐ Genomic DNA was extracted using the BioSprint96 magnetic bead tion 10 min at 72°C. The following primers were used: LCO1490‐JJ: extractor and respective kits by Qiagen. Polymerase chain reaction 5′‐CHACWAAYCATAAAGATATYGG‐ 3′ and HCO2198‐JJ: 5′‐ (PCR) was carried out in a total reaction volume of 20 μl, including AWACTTCVGGRTGVCCAAARAATCA‐ 3′ (Astrin & Stüben, 2008). 2 μl of undiluted DNA template, 0.8 μl of each primer (10 pmol/μl), Outhouse bidirectional sequencing followed enzymatic clean‐up 2 μl of “Q‐Solution” and 10 μl of “Multiplex PCR Master Mix”, con‐ using ExoSAP‐IT (USB Corporation) with Macrogen Inc. taining hot start Taq DNA polymerase and buffers (Qiagen, Hilden, Germany). Thermal cycling was performed on GeneAmp PCR 2.6 | Molecular data analysis System 2,700 machines (Life Technologies) as follows: hot start Taq activation: 15 min at 95°C; first cycle set (15 repeats): 35 s denatura‐ We generated four new DNA barcodes and included previ‐ tion at 94°C, 90 s annealing at 55°C (−1°C/cycle) and 90 s extension ously published data from NCBI GenBank for an additional 34 TABLE 1 List of COI‐sequences Species Drainage Country GenBank Reference downloaded from NCBI GenBank with information on drainage and country of Garra amirhosseini Tigris Iran KM214773 Behrens‐Chapuis et al., 2015 origin Garra amirhosseini Tigris Iran KM214780 Behrens‐Chapuis et al., 2015 Garra barreimiae Shawkah UAE KM214779 Hamidan, Geiger, & Freyhof, 2014 Garra barreimiae Wurayah UAE KM214783 Hamidan et al., 2014 Garra ghorensis Ghor Jordan KJ553520 Geiger et al., 2014 Garra ghorensis Alhass Jordan KM214788 Hamidan et al., 2014 Garra gymnothorax Tigris Iran KM214803 Behrens‐Chapuis et al., 2015 Garra gymnothorax Tigris Iran KM214735 Behrens‐Chapuis et al., 2015 Garra jordanica Mujib Jordan KM214753 Hamidan et al., 2014 Garra jordanica Jallain Syria KM214750 Hamidan et al., 2014 Garra longipinnis Jabal‐al‐ Oman KM214752 Hamidan et al., 2014 Akhdar Garra longipinnis Jabal‐al‐ Oman KM214756 Hamidan et al., 2014 Akhdar Garra lorestanensis Loven Iran KM214776 Hamidan et al., 2014 Garra lorestanensis Loven Iran JF416298 Hashemzadeh Segherloo et al., 2012 Garra mondica Mond Iran KM214741 Behrens‐Chapuis et al., 2015 Garra mondica Mond Iran KM214762 Behrens‐Chapuis et al., 2015 Garra persica Rudan Iran KM214746 Sayyadzadeh, Esmaeili, & Freyhof, 2015 Garra persica Shur Iran KM214706 Sayyadzadeh et al., 2015 Garra rossica Kavir Iran KM214768 Hashemzadeh Segherloo et al., 2016 Garra rossica Jazmurian Iran KM214690 Hashemzadeh Segherloo et al., 2016 Garra rufa Firat Turkey KM214800 Hamidan et al., 2014 Garra rufa Tigris Iraq KM214715 Hamidan et al., 2014 Garra tashanensis Tigris Iran KY365750 Mousavi‐Sabet et al., 2016 Garra tashanensis Tigris Iran KY365751 Mousavi‐Sabet et al., 2016 Garra typhlops Loven Iran KM214717 Hamidan et al., 2014 Garra typhlops Loven Iran KM214731 Hamidan et al., 2014 Garra variabilis Orontes Syria KJ553391 Geiger et al., 2014 Garra variabilis Orontes Syria KJ553422 Geiger et al., 2014 Garra widdowsoni Euphrates Iraq KM214769 Hamidan et al., 2014 Garra widdowsoni Euphrates Iraq KM214795 Hamidan et al., 2014 4 | VATANDOUST ET AL. FIGURE 3 Maximum likelihood estimation of phylogenetic relationships based on the mitochondrial COI barcode region (Hasegawa‐Kishino‐Yano model, discrete Gamma distribution for rate differences with five categories + G parameter = 1.5441. Nucleotide positions with less than 98% site coverage were eliminated, resulting in 634 analysed positions. Numbers of major nodes indicate bootstrap values from 1,000 pseudoreplicates from the ML, NJ and MP method above 75% and “+” denotes support above 95% from all three methods. Except for the outgroup, the tree is drawn to scale with branch lengths depicting number of substitutions per site specimens (Table 1). We included as outgroup taxa one indi‐ PAUP4b) and maximum likelihood (ML) phylogenetic trees with vidual each of Eidinemacheilus proudlovei, E. smithi, Tariqilabeo 1,000 bootstrap replicates to explore phylogenetic affinities adiscus and T. diplochilus. Data processing and sequence as‐ of the mitochondrial lineages from the new locality. All codon sembly was done with the software Geneious Pro (Biomatters, positions were included and positions with less than 98% site 2013) and the Muscle algorithm (Edgar, 2004) was used to coverage were eliminated resulting in 634 analysed nucleo‐ align the DNA barcodes after manually screening for unex‐ tide positions. The Species Delimitation Plugin (Masters, Fan, pected indels or stop codons.