CONFIRMING THE PRESENCE OF BENNETT ’ S STINGRAY , HEMITRYGON BENNETTII ( ELASMOBRANCHII : MYLIOBATIFORMES : DASYATIDAE ) , FROM THE WESTERN BAY OF BENGAL

The occurrence of Bennett’s stingray, Hemitrygon bennettii (Müller et Henle, 1841), is confirmed from Indian waters, based on a single male specimen (370 mm DW) collected from Visakhapatnam, western Bay of Bengal on 28 January 2017. Existing, updated reports on the distribution of the species, places it in the IndoPacific region with the western limit being the Strait of Malacca. With the presently confirmed occurrence from the western Bay of Bengal, the known distribution of the species extends further westwards to the Indian east coast, although it is likely to be a rare species in Indian waters.

Bennett's stingray, Hemitrygon bennettii, is a poorly known stingray distributed in the Indo-Pacific and is considered to be most abundant in the northwestern Pacific Ocean region.However, its westernmost distribution limit in the Indian Ocean is unknown, with possible distribution range suggested from Japan to India (Valenti 2016).The species can grow to a maximum size of 610 mm disc width (DW) and it occurs mostly at depths of 25-36 m, in coastal soft bottom habitats (Last et al. 2016a, Weigmann 2016) Several Indian studies have reported/listed H. bennettii from Indian waters (Day 1878, Day 1889, Misra 1947, 1952, 1969, Talwar and Kacker 1984).However, recent reports are mostly lacking or misidentified.In a recent checklist of chondrichthyans from India, Akhilesh et al. ( 2014) listed the occurrence of H. bennettii in India as "Needs Confirmation" category, requiring additional specimens along with genetic data, due to the absence of "correctly" identified material in collections in India matching that of H. bennettii and possible misidentification in recent studies/ reports from the region.Furthermore, several misidentified photos have been deposited from India or nearby regions as "H.bennettii" in popular websites * and even FishBase (Froese and Pauly 2018), which are possibly Maculabatis sp. or Himantura sp.Hemitrygon bennettii has been reported from the Persian Gulf and Sea of Oman by Assadi and Dehghani (1997) and Vossoughi and Vosoughi (1999).Reports of the species from Oman Sea, Persian Gulf and Sri Lanka (Anonymous 2018) could not be verified due to lack of easily accessible photographic and morphometric evidence, however, authors positively assume Sri Lankan record could be reliable.
In the monumental work, 'Rays of the world', Last et al. (2016a) too, restricted the western distribution limit of H. bennettii to the Coral Triangle region (Strait of Malacca) only.In this study, we confirm the presence of Hemitrygon bennettii along the east coast of India using morphometrics, photographs and COI data, based on a single male specimen of 370 mm DW collected from Visakhapatnam Fisheries Harbour, Andhra Pradesh, India on 28 January 2017 and landed as trawl bycatch from the western Bay of Bengal.

MATERIALS AND METHODS
A single male specimen of Hemitrygon bennettii was collected from Visakhapatnam Fishing Harbour (Fig. 1) on 28 January 2017, landed by fishery trawl boat which had fished in areas within 100 m depth off Visakhapatnam within the Indian EEZ.The specimen was brought to the laboratory in fresh condition, photographs were taken and tissue samples were collected for genetic analyses.The specimen was identified following Last et al. (2016a).The disc width (DW) in mm and total weight (g) of the specimen was recorded.Tissue samples were collected and preserved in 95% ethanol for further DNA studies.Following this, the specimen was preserved in 10% formalin.All further morphometric measurements were taken of the preserved specimen.Morphometric measurements follow Jacobsen and Bennett (2009), Smith et al. (2009), and Muktha et al. (2018).Molecular analysis.DNA extraction was carried out using a standard phenol/chloroform extraction protocol.A 650 bp region of the Cytochrome C oxidase 1 was amplified using a universal primer (Folmer et al. 1994).The composition of PCR reaction mixture was; 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 200 µM of each dNTP, 0.2 µM of each oligonucleotide, 1 unit of Taq DNA polymerase and 50 ng of template DNA.The fragments were amplified using Biorad T100 thermocycler (Biorad USA) and the PCR conditions were; initial denaturation at 94°C for 4 minutes followed by 33 cycles of; denaturation at 94°C for 30 s, annealing at 42°C for 30 s, extension at 72°C for 40 s and a final extension at 72°C for 7 min.Purification of the PCR products was carried out using Qiagen PCR purification kit and sequencing was carried out with BigDye Terminator Sequencing Ready Reaction v 3.0 kit (Applied Biosystems) using the primers.A 620 bp region of the Cytochrome C Oxidase gene was amplified and the sequence submitted to NCBI, GenBank with accession number MH329420.
The sequence of cytochrome c oxidase 1 (COI) (620 bases) was aligned with available sequences retrieved from GenBank of other species of the genus, Hemitrygon bennettii, H. fluviorum, H. akajei, H. izuensis, H. parvonigra, H. laevigata, and H. longicauda using Clustal W in MEGA 6.A neighbor joining tree was constructed using UPGMA method with 1000 bootstraps.Tree topology was also tested using maximum likelihood, maximum parsimony and neighbour-joining methods.The COI tree was then rooted with the sequence of Scoliodon laticaudus Müller et Henle, 1838 retrieved from GenBank.Genetic divergence between all the species was analysed using Kimura 2 p distance values in MEGA 6.The current specimen is deposited in the fish collections of the Visakhapatnam Research Centre of ICAR-Central Marine Fisheries Research Institute (CMFRI), India with accession number CMFRI V-D.1.

Family Dasyatidae Hemitrygon Müller et Henle, 1838
Hemitrygon bennettii (Müller et Henle, 1841) Figs.2-6 Diagnosis.Medium-sized stingray with brownish to greenish-brown dorsal colouration and dorsal disc with median row of small denticles extending to tail, pair of lateral denticles patches on the scapular region, spines on tail larger; tail length >2 times DW, tail without any bands, tail with thin ventral fold (ribbon like), ventral tail fold origin behind the sting origin.Small denticles seen in the interspiracular region, mid-dorsal region, and shoulder regions of disc.Description.(Figs. 2-6) Disc weakly rhombic, disc width nearly equal to disc length, disc width 1.03 times in disc length; dorsal surface with denticles and row of spines; ventral surface smooth.Body depressed, flat and slightly elevated trunk; snout short, broadly triangular or pointed, with anterior margin nearly straight to slightly concave, outer margins rounded; pre-oral snout length 22.1% in disc width, 0.5 in head length, 2.6 times mouth width.Pre-orbital snout length 0.24 in disc width, 1.8 in inter-orbital; pre-orbital snout width 0.6 in disc width; acute at apex with indistinct, triangular apical lobe; snout   (Wang et al. 2012, Last and White 2013, Lim et al. 2015) showing 100% similarity (n = 11) (Fig. 7).The neighbour-joining tree constructed using 27 sequences of COI of other Hemitrygon spp.showed distinct clustering among species (Fig. 7) with significant bootstrap values.In the COI tree, H. bennettii sequence of the specimen from Visakhapatnam (MH329420) clustered together with H. bennettii from Indonesia, China, and Malaysia.The tree topology was same when analyses were conducted using maximum likelihood, maximum parsimony, and neighbor-joining methods.

REMARKS AND DISCUSSION
Hemitrygon bennettii is a nearshore species having a depth range of 25-36 m (Weigmann 2016).Trawlers operating off Visakhapatnam fish along the coast of India in depths down to150 m and the majority of the major fishing grounds are found within the narrow continental shelf area in the region (Muktha et al. 2018).Since the specimen was landed by a trawler operating in Indian waters, the species is confirmed to have been caught within the Indian EEZ.Hemitrygon bennettii resembles H. akajei, however, H. akajei has a DW 1.1-1.2(vs.0.9-1.0 in H. bennettii).The presently reported H. bennettii material from the Indian coast had a slightly shorter tail (post-cloacal tail) (2.1 times DW), than mentioned as character for species by Last et al. (2016a) as 2.3-2.6 times DW and that by Nishida and Nakaya (1990) and Garman (1913) as >3 times DW.However, it may be noted that Müller and Henle (1841) in the original description had mentioned about shorter tails and Jordan and Richardson (1909) reported similar variable tail length for H. bennettii from Formosa and Zhang et al. (2010) from China reported tail lengths being 1.6-2.6 times DW.So, identification purely based on tail length may be unreliable for Hemitrygon due to its high degree of variability across regions.Genetic analysis and detailed morphometry have unambiguously confirmed the presence of H. bennettii in Indian waters.Since only a single specimen has been identified and recorded from Indian waters, this species may be considered to be rare in the region.
Several authors have reported the presence of Hemitrygon bennettii from Indian waters earlier; however, none of the authors has provided reference materials/collection details.This remains a large problem in elasmobranch taxonomy research in India due to which taxonomy of Indian elasmobranchs still remains largely unresolved.Part of this is due to the fact that no single Indian museum or repository either individually or all collections together hold specimens of all batoids reported from India, which hinders detailed and accurate elasmobranch taxonomic research in India.Assessment of the validity and distinctiveness of taxa requires sampling across the species range and examining materials reported in the same name, including from the type localities (Crandall et al. 2009, Rocha et al. 2014).The

Fig. 1 .
Fig. 1.Map showing Visakhapatnam (presently reported study-rectangle) along western Bay of Bengal, other reports from Indian Ocean (circle-unconfirmed report, star-record from Sri Lanka) and known western most distribution range from Last et al. (2016a) (triangle)

Fig. 7 .
Fig. 7. Neighbour-joining tree of nucleotide sequence divergence of the COI gene following the Kimura two-parameter model (K2P) generated by NCBI database for available Hemitrygon species and Hemitrygon bennettii from India (MH32920) angle 110°.Greatest disc width slightly behind spiracles, towards anterior part of trunk.Head short, pointed at apex; pectoral fin broad, expanded to form trapezoidal disc, apex angular, posterior margin convex.Five gill slits; fifth gill slit smallest; first gill slit length 5.9% of head length; fifth gill slit length 4.2% of head length; distance between first pair of gill slits 2 times inter-narial width; * grouped closest to H. bennettii from China, South China Sea, Malaysia, and Indonesia