First record and DNA Barcoding of Oman cownose ray, Rhinoptera jayakari Boulenger, 1895 from Andaman Sea, India

zoosystema art. 40 (4) — Published on 27 February 2018 www.zoosystema.com 2018  40  4 First record and DNA Barcoding of Oman Cownose Ray, Rhinoptera jayakari Boulenger, 1895 from Andaman Sea, India Hosahalli Divakar PRADEEP, Shivdas Shirke SWAPNIL, Musaliyarakam NASHAD, Sasidharan VENU, Kumar RAVI RANJAN, Gopalakrishnan SUMITHA, Sukham MONALISHA DEVI & Mahesh Kumar FAREJIYA Directeur De la publication : Bruno David Président du Muséum national d’Histoire naturelle réDactrice en chef / Editor-in-chiEf : Laure Desutter-Grandcolas assistants De réDaction / AssistAnt Editors : Anne Mabille ([email protected]), Emmanuel Côtez Mise en page / PAgE lAyout : Anne Mabille coMité scientifique / sciEntific boArd : James Carpenter (AMNH, New York, États-Unis) Maria Marta Cigliano (Museo de La Plata, La Plata, Argentine) Henrik Enghoff (NHMD, Copenhague, Danemark) Rafael Marquez (CSIC, Madrid, Espagne) Peter Ng (University of Singapore) Gustav Peters (ZFMK, Bonn, Allemagne) Norman I. Platnick (AMNH, New York, États-Unis) Jean-Yves Rasplus (INRA, Montferrier-sur-Lez, France) Jean-François Silvain (IRD, Gif-sur-Yvette, France) Wanda M. Weiner (Polish Academy of Sciences, Cracovie, Pologne) John Wenzel (The Ohio State University, Columbus, États-Unis) couverture / covEr : Underside of the head Rhinoptera jayakari Boulenger, 1895. Zoosystema est une revue en flux continu publiée par les Publications scientifiques du Muséum, Paris / Zoosystema is a fast track journal published by the Museum Science Press, Paris Les Publications scientifiques du Muséum publient aussi / The Museum Science Press also publish: Adansonia, Anthropozoologica, European Journal of Taxonomy, Geodiversitas, Naturae. 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Le CFC est membre de l’International Federation of Reproduction Rights Organisations (IFRRO). Aux États-Unis d’Amérique, contacter le Copyright Clearance Center, 27 Congress Street, Salem, Massachusetts 01970. PHOTOCOPIES: The Publications scientifiques du Muséum adhere to the Centre Français d’Exploitation du Droit de Copie (CFC), 20 rue des Grands Augustins, 75006 Paris. The CFC is a member of International Federation of Reproduction Rights Organisations (IFRRO). In USA, contact the Copyright Clearance Center, 27 Congress Street, Salem, Massachusetts 01970. 67 ZOOSYSTEMA • 2018 • 40 (4) © Publications scientifiques du Muséum national d’Histoire naturelle, Paris. www.zoosystema.com urn:lsid:zoobank.org:pub:30D5CE6B-8578-4822-90C1-B51E9937845A Pradeep H. D., Swapnil S. S., Nashad M., Venu S., Ravi Ranjan K., Sumitha G., Monalisha Devi S. & Farejiya M. K. 2018. — First record and DNA Barcoding of Oman cownose ray, Rhinoptera jayakari Boulenger, 1895 from Andaman Sea, India. Zoosystema 40 (4): 67-74. https://doi.org/10.5252/zoosystema2018v40a4. http://zoosystema.com/40/4 ABSTRACT Strategically located to the southeast of the Bay of Bengal and northwest of the Malay Peninsula, the Andaman Sea houses rich and varied biological diversity both in its flora and fauna which is under explored. The present study reports the occurrence of Oman cownose ray, Rhinoptera jayakari Boulenger, 1895 as a new record from this region. A male specimen with a disc width of 494 mm was landed by a motorized longline boat in Junglighat fishing harbour. Port Blair, Andaman and Nicobar Islands fills the wide gap in the known distribution range of the species. A detailed morphological description of freshly collected specimen in comparison with those from the previous published reports elsewhere was carried out. Molecular marker based taxonomical annotation using Mitochondrial COI gene sequencing confirmed the identity of the specimen. Hosahalli Divakar PRADEEP Shivdas Shirke SWAPNIL Musaliyarakam NASHAD Fishery Survey of India, Port Blair Zonal Base, Port Blair-744101, Andaman and Nicobar Islands (India) [email protected] (corresponding author) Sasidharan VENU Kumar RAVI RANJAN Department of Oceanography & Marine Biology, Brookshabad Campus, Pondicherry University, PB No. 1, Chakkargaon P. O, Port Blair-744112, Andaman and Nicobar Islands (India) Gopalakrishnan SUMITHA Department of Aquaculture and Fishery Microbiology, Research Centre Calicut University, MES Ponnani College, Ponnani, Malappuram, Kerala (India) Sukham MONALISHA DEVI Fisheries Resource Management Division, Central Islands Agriculture Research Institute, ICAR, Port Blair-744105, Andaman and Nicobar Islands (India) Mahesh Kumar FAREJIYA Fishery Survey of India, Plot N° 2A, Unit No.12, New Fishing Harbour, Sassoon Dock, Colaba, Mumbai-400005 (India) Submitted on 29 March 2017 | Accepted on 26 October 2017 | Published on 27 February 2018 First record and DNA Barcoding of Oman cownose ray, Rhinoptera jayakari Boulenger, 1895 from Andaman Sea, India KEY WORDS Rays, myliobatiformes, Andaman, Nicobar waters, first report. 68 ZOOSYSTEMA • 2018 • 40 (4) Pradeep H. D. et al. RÉSUMÉ Première signalisation et premier barcode pour la raie mourine à queue courte Rhinoptera jayakari Boulenger, 1895 en mer d’Andaman, Inde. Stratégiquement située au sud-est de la baie du Bengale et au nord-ouest de la péninsule malaise, la mer d’Andaman abrite une diversité biologique riche et variée tant pour sa flore que pour sa faune qui est sous explorée. La présente étude mentionne la présence de la raie mourine à queue courte, Rhinoptera jayakari Boulenger, 1895 comme une nouvelle signalisation dans cette région. Un spécimen mâle avec une largeur de disque de 494 mm a été débarqué par un palangrier motorisé dans le port de pêche de Junglighat. Port Blair, Andaman et Nicobar Islands comblent le large fossé dans la distribution connue de l’espèce. Une description morphologique détaillée des spécimens fraîchement collectés a été réalisée en comparaison avec les publications précédentes. L’identité de l’échantillon a été confirmée par annotation taxonomique moléculaire en séquençant le gène mitochondrial COI. INTRODUCTION Rays of the family Rhinopteridae Bonaparte, 1835, are distributed in warm temperate and tropical seas except around oriental Pacific Island (McEachran & Fechhelm 1998; Last & Stevens 2009; Domingues etal. 2009; Froese& Pauly 2017). They are semi pelagic and gregarious, often forming large schools (Bogorodsky etal. 2014). Weigmann (2016) listed eight species under genus Rhinoptera Cuvier, 1829 and mentioned that R.jayakari Boulenger, 1895 is represented by three unresolved subgroups in the western Indian Ocean, based on the NADH2gene (Naylor etal. 2012). Rhinoptera sewelli Misra, 1946from the Arabian Sea, questionably valid following Compagno & Last (1999), was considered to be possibly conspecific with R. jayakari (Last etal. 2010). These species are strongly allopatric with widespread geographic ranges believed to reflect their high mobility (Schwartz 1990). Species of cownose rays are very similar to one another and most of the classification work is based on number of tooth plate rows or series. However, dentition is highly variable in some species and the family is in urgent need of revision (Bigelow & Schroeder 1953; James 1970; Sandoval & Olivares 2011). Presently eight species under the genus Rhinoptera are considered valid (Bailly 2014; Last etal. 2016). Previous records of the Oman Cownose Ray (Rhinoptera jayakari Boulenger, 1895) in the north-western Indian Ocean include those from the Gulf of Oman (type locality), southeastern Arabian Sea coast of Oman (Henderson& Reeve 2011), Gulf of Aden (Bonfil & Abdallah 2004) and Red Sea (Bogorodsky etal. 2014). From Indian waters, it has been listed by Akhilesh et al. (2014) and Joshi etal. (2016), but without morphological details. DNA barcoding has previously been performed for the specimens collected from Kochi, Southwest coast of India (Bineesh etal. 2016) and deposited in the Genbank, NCBI. The aim of the current study is to utilize the mitochondrial COI molecular marker to confirm the taxonomic identity of a specimen identified as R.jayakari collected from the Andaman Sea and report the species occurrence in that area for the first time, thus filling the gap in the known distribution range of this species. MATERIAL AND METHODS A single specimen of Rhinoptera jayakari was collected from Junglighat fishing landing centre, Port Blair, South Andaman (Fig. 1). It was caught by a motorised fishing boat with longline gear at a depth of 30-50m in the waters of the Andaman and Nicobar islands of the Andaman Sea. Morphometric measurements were recorded and expressed as proportion of disc width (DW), according to Last & Stevens (2009) and White etal. (2010). These measurements were compared with the published data by Bogorodsky etal. 2014 (Table 1). The specimen was preserved in the museum of the zonal base of Fishery Survey of India, Port Blair. The tissue sample of R.jayakari was collected and preserved in 99.5% ethanol at ambient temperature and DNA was extracted by standard protocols (Miller etal. 1988). The extracted DNA was checked by agarose (0.8%) gel electrophoresis. The partial sequence of mitochondrial COI gene was amplified using specific universal primers Fish F1 (5’- TCA ACC AAC CAC AAA GAC ATT GGC AC-3’) and Fish R1 (5’-TAG ACT TCT GGG TGG CCA AAG AAT CA-3’) (Ward etal. 2005) in 25μl reaction volume containing 1× assay buffer (100mM Tris, 500mM KCl, 0.1% gelatin, pH9.0) with 1.5mM MgCl2, 0.5μl of each primer, 200μM of each dNTPs (Genei, Bangalore, India), 1.5UTaq DNA polymerase, and 20ng of template DNA. PCR reactions were carried out in Veriti TM 96-Well Thermal Cycler (Applied Biosystems, USA) following initial preheating at 95°C for 5min, denaturation 94°C for 30s, annealing 53°C for 45s, extension 72°C for 1min, repeated for 30cycles, followed by a final extension for 10min at 72°C. The PCR products were electrophoresed on 1.5% agarose gel, stained with ethidium bromide (10μg/ ml) to ensure that a fragment of the correct size had been amplified. The gel was visualized on a Gel documentation system (Bio-Rad, USA). The purified PCR Products were labeled using Big Dye Terminator V.3.1cycle sequencing kit (Applied Biosystems Inc.) and sequenced bidirectionally using ABI3730capillary sequencer using the primers Fish F1and Fish R1. MOTS CLÉS Raies, myliobatiformes, mers d’Andaman et de Nicobar, signalisation nouvelle. 69 ZOOSYSTEMA • 2018 • 40 (4) First record of Oman cownose ray from Andaman Sea Sequences were analyzed and the Basic Local Alignment Search Tool (BLAST) algorithm (Altschul etal. 1990) was used to search the GenBank database for homologous sequences (http://www.ncbi.nlm.nih.gov/). The sequences were multiple aligned and were used to construct a phylogenetic tree of K2P distance were created to provide graphic representation of divergence by the neighbor-joining (NJ) method (Saitou & Nei 1987), using the MEGA V. 6.0package (Tamura etal. 2013). Bootstrap analysis was based on 1000replicates. SYSTEMATICS Family RhinopteRidae Bonaparte, 1835 Genus RhinopteraCuvier, 1829 Rhinoptera jayakariBoulenger, 1895 Rhinoptera jayakari Boulenger, 1895: 1520-1523. M ateRial exaMined . — MUS/FSI/PB/EB/09/2016, 1 ♂ Rhinoptera jayakari of Disc Width (DW) 494mm and weight2.9kg (Fig2A), collected from Junglighat fishing landing centre, Port Blair, South Andaman. The specimen was caught by a motorized fishing boat with longline gear at a depth of 30-50m in the Andaman and Nicobar waters of the Andaman Sea. descRiption of speciMen fRoM the andaMan sea Disc length 1.5in disc width; near straight anterior margin, posterior margin concave, apices acutely pointed (Fig.2); disc mostly naked, except for small and stellate-based prickles dorsally on snout and inter orbital space (Fig.3B); trunk thick and broad; head relatively broad and its width between posterior margin of spiracles 5.3in disc width; upper part of the snout bilobed, separated almost at level of mouth from a lower, forward-projecting and also the bilobed part by a continuous deep groove around front of head; eyes moderately large, lateral on head and just anteromedial to spiracles (Fig.2B), eyes 1.8in spiracle length; mouth transverse, straight, and narrow (Fig.3A), preoral distance8.4in disc width; anterior nasal flaps long medially expanded and fused into a broad, elongated fringed nasal curtain overlapping mouth; nostrils slightly anterior to mouth, connected by broad nasoral grooves with mouth (Fig.3A); tail short, 1.4in disc length; base of tail with relatively high, rounded dorsal fin originating over pelvic-fin insertions; posterior margin of the dorsalfin almost upright; a serrate spine on tail behind dorsal fin (Fig.3D), its base beneath dorsal-fin inner margin; teeth flat and pavement-like, in9series in upper jaw (Fig.3C), 3medial rows of broad teeth and3-5rows of narrower diamond-shaped teeth on each side. Colour Dorsal surface of disc dark olivaceous, ventral surface of disc white, tail dark olivaceous gradually becoming black distally. DNA barcoding The obtained partial sequence of COI gene of was submitted to the NCBI database with an accession number KY574494. fig. 1. — Junglighat Fishing Harbour (Sampling station), South Andaman. INDIA ANDAMAN & NICOBAR ISLANDS Junglighat Fishing Harbour SOUTH ANDAMAN 150 km 90°E 92°15’E 92°30’ 92°45’ 12° 12°00’11°45’ 11°30’ 11°15’N 10°8°N 92° 94° 5 km Andaman sea Andaman sea Bay of Bengal Bay of Bengal N N 70 ZOOSYSTEMA • 2018 • 40 (4) Pradeep H. D. et al. SMF 34988 Yemen SMF 34989 Jizan KAUMM 36 Jizan Present specimen %DW %DW %DW %DW mm Disc width 727 684 771 494 Total length, from medial point of snout 100.8 108.2 91.4 111.13 549 Tail length 44 45.3 36.5 47.77 236 Disc length (direct) (snout to rear edge of pectoral fin along axis) 61.5 66.7 62.1 64.57 319 Disc thickness 11.7 11.7 11.9 11.34 56 Disc, anterior margin of spiracle to pectoral insertion 49.1 53 51 55.87 276 Disc, snout to maximum width 36.9 37.4 36.4 38.87 192 Head length 26.7 28.2 26.1 26.52 131 Snout-preobital 11.2 10.9 11.5 10.73 53 Snout preoral 11 11.5 10.8 11.94 59 Snout-prenasal 11.9 11.4 12 11.34 56 Lower-flap length 12.8 12.4 12.8 12.35 61 Eye Diameter 2.3 2.3 2.2 2.23 11 Inter-eye width 17.5 17.7 17 17.61 87 Orbit and spiracle length 8.5 9.4 8.5 9.31 46 Spiracle length 4.2 4.2 44.05 20 Interspiracular width, anteriorly 15.5 16.2 15.6 15.99 79 Interspiracular width, posteriorly 18.2 19.1 18 19.03 94 Mouth width, between edges of teeth 8.4 8.8 8.5 8.70 43 Mouth width, between folders 13.1 13.1 13.2 13.16 65 Nostril length 1.5 1.3 1.4 1.21 6 Nasal curtain width 14.7 15.3 14.6 15.38 76 Nasal curtain length 4.7 4.85 4.4 4.86 24 SMF 34988 Yemen SMF 34989 Jizan KAUMM 36 Jizan Present specimen Internasal width, anteriorly (between bases of nasal curtain) 9.4 9.9 9.3 9.72 48 Internasal width, posteriorly (between posterior out ends of nostrils) 11.3 11.7 11.2 11.74 58 Width 1st gill slit 2.8 2.8 2.6 2.83 14 Width 5st gill slit 1.9 1.9 1.7 1.82 9 Distance between 1st gill slits 18.2 18.5 18.8 18.62 92 Distance between 5st gill slits 12.3 13 12.6 12.75 63 Pelvic fin, length 13.8 13.5 11.5 13.66 67.5 Pelvic fin. Base width 6.3 6.7 5.8 6.56 32.4 Tail width, axil of pelvic fin 3.5 3.5 3.2 3.36 16.6 Tail width, base of sting 21.9 1.8 1.82 9 Tail height, base of sting 2.3 2.1 2.2 2.15 10.3 Spine length broken 7.3 7.1 0.00 broken Snout to origin of cloaca 52.4 58.8 53.6 58.50 289 Cloaca origin to sting 11.5 13.1 11.4 12.45 61.5 Cloaca origin to tail tip 48.8 49.1 40 48.79 241 Cloaca length 3.6 5.1 2.7 4.86 24 Clasper, post cloacal length (from posterior edge of cloaca to tip of clasper) 14.6 13.5 13.4 13.36 66 Clasper, length from pelvic axil (from insertion of pelvic fin to tip of clasper) 12.9 13.1 13 12.75 63 Dorsal-fin base length 5.8 76.6 6.48 32 Dorsal-fin height 2.3 5.3 5.3 5.16 25.5 Teeth rows on upper jaw 79 9 9 AB fig. 2. — A, Dorsal view of Rhinoptera jayakari Boulenger, 1895; B, antero-lateral view of the head with upper and lower lobes. Size of specimen: 494 mm DW. table 1. — Morphometric details of Rhinoptera jayakari Boulenger, 1895 (in mm and % Disc Width [DW]) in comparison with previous records. 71 ZOOSYSTEMA • 2018 • 40 (4) First record of Oman cownose ray from Andaman Sea The partial sequence of mitochondria COI gene produced a mean value of 623nucleotide base pairs. BLAST result shows a 99 % match with the existing COI sequences of R.jayakari from India (GenBank: KF899679.1, KF899683.1, and KM0703015.1) in the NCBI database. It also forms a distinctive clade in Neighbour Joining (NJ) phylogenetic tree inferred from mitochondrial COI gene sequences (Fig.4). The pair wise genetic distances values (K2P) based on COI sequences are given in Table2. The interspecies distance ranged from0.002to0.008and the genetic distance within the genus Rhinoptera ranged from0.002to0.093. DISCUSSION In present scenario, the importance of biological collections with clear and descriptive morphological details in conjunction with the state of art molecular marker based studies in taxonomy has become the need of the hour. The limited basic taxonomical studies in the batoid members have paved hurdles in front of researchers round the globe. The Andaman Sea had remained prestine in this context and had come to limelight with upcoming development of basic science research. Hence, the morphological and molecular identification of organisms, AB C D fig. 3. — Rhinoptera jayakari Boulenger, 1895: A, underside of the head; B, location of prickles on dorsal surface of the head; C, nine series of teeth, upper jaw; D, dorsal fin along with spine. Size of specimen: 494 mm DW. 72 ZOOSYSTEMA • 2018 • 40 (4) Pradeep H. D. et al. especially batoids, had been difficult due to the limited available descriptions in literature globally. Rhinoptera jayakari had been previously reported from gulf of Oman to Red Sea along northwestern Indian Ocean (Bonfil& Abdallah2004; Henderson& Reeve2011; Bogorodsky etal. 2014). Ahmad etal. (2013) has mentioned a number of identification characters of R. Jayakari in a field guide on look-alike Sharks and Rays species of the Southeast Asian Region but they are not enough to confirm the identity of the specimen. From, Indian waters R.jayakari has been listed by Akhilesh etal. (2014), reported from Gulf of Mannar (Joshi etal. 2016) and DNA barcoding of R. Jayakari based on partial COI gene sequences has been performed from Kerala coast (Bineesh etal. 2016). Only a single species of Rhinoptera (R.javanica Müller & Henle, 1841) has been reported from Andaman and Nicobar waters of the Andaman Sea (Rajan etal. 2013). Differentiation of closely related members of Rhinoptera clade is quite difficult solely based on morphological characteristics. Compagno & Last (1999) separated R.adspersa from R.javanica in having fewer rows of teeth in upper jaw (7vs 9). However, the number of rows appears to be a variable character, so R.adspersa has been placed in the synonymy of R.javanica. The Australian species R.neglecta can be distinguished from both R.javanica and R.jayakari in having a smooth disc without prickles, tail as long as the disc length and origin of the dorsal fin in front of the pelvicfin insertions. Recent works by Bogorodsky etal. in 2014on the morphological studies of three specimens from Red Sea mentioned the difference in the number of teeth rows in the upper jaw: one specimen has ninerows; two specimens have 12rows of teeth (with the outermost rows variable present). Thus, posing another hurdle on morphological identification keys. Hence, the molecular marker based taxonomic annotation becomes the perfect tool for species level confirmation. Rhinoptera jayakari has been confused with R.javanica but it differs in having a broader head (17.6% disc width vs 14.516% disc width in R.javanica), shorter tail (1.4in disc length vs0.5-0.8in disc length in R.javanica), and dorsal-fin origin over pelvic-fin insertions (dorsal-fin origin behind pelvic-fin insertions in R.javanica). Morphometrical characters of the present specimen like, disc length (1.5), head (5.3), preoral distance (8.4), tail (1.5) in disc width and eyes 1.8in spiracle length, fall well within the range given by Bogorodsky etal. (2014) for R.jayakari collected from Red Sea. This is the first study of mitochondrial COI gene sequence of R.jayakari from the Andaman Sea. The DNA barcode sequences based on partial sequence information of COI gene has been widely Rhinoptera jayakari Boulenger, 1895 Andaman isoloate KF899679.1 Rhinoptera jayakari KF899683.1 Rhinoptera jayakari KM073015.1 Rhinoptera jayakari KM364907.1 Rhinoptera steindachneri (Evermann & Jenkins, 1891) DQ108133.1 Rhinoptera javanica (Müller & Henle, 1841) KX060788.1 Mobula japanica (Müller & Henle, 1841) KT307247.1 Mobula mobular (Bonnaterre, 1788) EU398918.1 Mobula thurstoni (Lloyd, 1908) JF493899.1 Mobula kuhlii (Müller & Henle, 1841) EU398913.1 Mobula tarapacana (Philippi, 1892) JQ765531.1 Manta alfredi (Krefft, 1868) EU398902.1 Manta birostris (Walbaum, 1792) EF607354.1 Dasyatis zugei (Müller & Henle, 1841) JQ305802.1 Dasyatis hypostigma (Santos & Carvalho, 2004) EU398987.1 Pristis clavata (Garman, 1906) EU398507.1 Aetobatus ocellatus (Kuhl, 1823) FJ812196.1 Aetobatus narinari (Euphrasén, 1790) 100 81 98 99 38 89 98 98 98 98 100 100 100 100 100 KM364888.1 Mobula eregoodootenkee (Bleeker, 1859) 0.02 fig. 4. — Neighbour Joining (NJ) phylogenetic tree of Rhinoptera jayakari Boulenger, 1895 and related species inferred from mitochondrial COI gene sequences. 73 ZOOSYSTEMA • 2018 • 40 (4) First record of Oman cownose ray from Andaman Sea used in identification and validation of species (Ward etal. 2005). The phylogenetic tree derived from mitochondrial COI gene strongly confirms the present specimen was R.jayakari which helps to avoid the ambiguity in identification based on external characteristics. Hence, the present study not only adds a new species to fish diversity of Andaman and Nicobar waters but also fills the wide gap in the known distribution range of this species. Acknowledgements The authors are thankful to the fishermen operating from the Junglighat Fish Landing center, Port Blair, Andaman and Nicobar Islands, India for handing over the specimen. The help rendered by DrRonald Fricke, Senior Curator of Ichthyology, State Museum of Natural History Stuttgart, Baden-Württemberg, Germany in identifying the specimen and the valuable suggestions for improving the molecular biology part by Mr Solly Solomon, FSI, Mormugao Zonal Base, Goa, India are thankfully acknowledged. 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