J. Ocean Univ. China (Oceanic and Coastal Sea Research) DOI 10.1007/s11802-014-2609-x ISSN 1672-5182, 2015 14 (1) http://www.ouc.edu.cn/xbywb/ E-mail:[email protected] The Taxonomic Status of Japanese Threadfin Bream Nemipterus japonicus (Bloch, 1791) (Perciformes: Nemipteridae) with a Redescription of this Species From the South China Sea Based on Morphology and DNA Barcodes NING Ping1), *, SHA Zhongli1), Paul D. N. HEBERT2), and Barry RUSSELL3) 1) Laboratory of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P. R. China 2) Biodiversity Institute of Ontario, University of Guelph, Guelph ON N1G 2WI, Canada 3) Museum and Art Gallery of the Northern Territory, Darwin NT 0801, Australia (Received May 9, 2013; revised December 20, 2013; accepted January 4, 2014) © Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2014 Abstract Because of its importance as a food source, Nemipterus japonicus (Bloch, 1791) (Nemipteridae) or Japanese threadfin bream is the best studied of these taxa, and numerous investigations have examined its fisheries, its biology and biochemistry. De- spite such intensive work, the taxonomic status of N. japonicus has never been seriously questioned and it is regarded as a common species, widely distributed throughout the Indo-Western Pacific Ocean. In fact, Bloch’s description of the type specimen of N. ja- ponicus has ambiguous collection data and lacks a designation for the type locality, though it is probably Java. In this paper, DNA barcode results based on COI gene support the existence of two geographically separated lineages of the Japanese threadfin bream, both being an Indian Ocean and western Pacific lineage, with 2.7% sequence divergence, and the results indicate a possible existing of some cryptic species. The two lineages also possess a diagnostic difference in their belly color, with specimens in the South China Sea having a silver belly, while those from the Indian Ocean isolate specimen have a yellow coloration. Based upon new collections from the South China Sea, this species from the western Pacific is morphologically redescribed and its details of DNA barcode diver- sity are shown for the future investigations. Key words South China Sea; Indian Sea; Indo-Pacific Barrier; COI gene details DNA barcode diversity in N. japonicus on the ba- sis of specimens from the South China Sea. 1 Introduction Nemipterus Swainson, 1839, the largest genus in the family Nemipteridae, includes about 26 species (Russell, 2 Materials and Methods 1990) which are distributed in the Indo-Western Pacific 2.1 Sample Collection and Examination Ocean. Because of its importance as a food source, Ne- There are 15 examined specimens collected from the mipterus japonicus (Bloch, 1791) is the best studied of southern coastal waters of China from 1950 to 2011 and these taxa and numerous investigations have examined its deposited at the Marine Biological Museum (MBM), In- fisheries, its biology and biochemistry (Krishnamoorthi, stitute of Oceanology, Chinese Academy of Sciences (IO- 1971; Russell, 1990; Chawla et al., 1996; Joshi, 2010; CAS). The other 22 specimens examined are in: Academy Naqasha and Nazeera, 2010). Despite this intensive work, of Natural Sciences (ANSP) Philadelphia, USA; Aca- the taxonomic status of N. japonicus has never been seri- demica Sinica (ASIZ), Taipei, Taiwan, China; the Natural ously questioned and it is regarded as a common species, History Museum (BMNH), London, UK; California Aca- widely distributed throughout the Indo-Western Pacific demy of Sciences (CAS), San Francisco, USA; Museum Ocean (Froese and Pauly, 2013). and Art Gallery of the Northern Territory (NTM), Darwin, While carrying out morphological and DNA barcode Australia; Senckenberg Museum (SMF), Frankfurt, Ger- studies, we discovered evidence for genetic divergence many; Smithsonian Institution (USNM), Washington DC, within N. japonicus that is linked to collection locality. USA; Zoological Museum (ZMB), Berlin, Germany. This paper provides a morphological redescription and 2.2 Morphological Description * Corresponding author. E-mail: [email protected] Color and pigmentation patterns were examined in 2 NING et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2015 14 digital images of freshly collected specimens, and de- “HKY+G+I” for ML was chosen based on results from tailed measurements were made on specimens preserved the option of “Find Best DNA Models”: heuristic method in 95% alcohol or 10% formalin. The technical terms and is Nearest-Neighbor-Interchange (NNI), bootstrap values measurements followed Russell (1990): SL means stan- are computed with 1000 replications. NJ bootstrap values dard length. HL means head length. Distributional data were estimated using 1000 replicates with Kimura’s derived from Russell (1990), FishBase (Froese and Pauly, two-parameter model of substitution (K2P distance) evo- 2013) and the Taiwan Fish Database (Shao, 2013). lution model. The genetic distances were estimated based on Kimura’s two-parameter model using MEGA 5.0 2.3 DNA Extraction, PCR, and Sequencing software. Genomic DNA was extracted from three individuals (IOCAS 135FB00144-1, IOCAS 135FB00144-2, IOCAS 3 Results 135FB00144-3) using Tiangen® Marine Animal Tissue DNA Extraction Kit and following the manufacturer’s 3.1 Synonym instructions. Polymerase chain amplification was con- Nemipterus japonicus (Bloch, 1791) ducted using standard fish primers (Ward et al., 2005) for the barcode region of the COI gene: COI F1 5′- TCAACCAACCACAAAGACATTGGCAC-3′ COI R1 5′- TAGACTTCTGGGTGGCCAAAGAATCA-3′ Each reaction had a total volume of 25μL containing 1μL of template, 2.5μL of 10× PCR buffer, 1.5μL of Mg2+, 1μL of dNTP mix, 1μL of each primer, 0.3μL of Taq DNA polymerase, and 17.7 μL of dH2O. The thermocycler re- gime was 3 min at 95°C, followed by 35 cycles, each at Fig.1 Nemipterus japonicus (IOCAS 135FB00144-1), 94 °C for 30 s, at 55 °C for 30 s, at 72 °C for 1 min, and 151.5mm SL, Sanya City, Hainan Island, China. Scale final extension at 72 °C for 10 min. The reaction products bar=1.0 cm. were separated by electrophoresis on 1.0% agarose gel, Sparus japonicas – Bloch, 1791: 110 (original type local- stained with ethidium bromide, and visualized under ul- ity: lacks a designation for the type locality; the type traviolet light. Sequencing reactions were performed at specimen has ambiguous collection data, but probably the Key Laboratory of Experimental Marine Biology Java) (KLEMB) of IOCAS. Three COI sequences had been ?Coryphaena lutea – Schneider in Bloch and Schneider, deposited into the NCBI database with the access num- 1801: 297, fig. 58 (Tranquebar, India) bers from KF134003 to KF134005, corresponding to Cantharus filamentosus – Rüppell, 1828–30 (1829): 50, voucher ID from 135FB00144-1 to 135FB00144-3. pl. 12, fig. 3 (Massaua, Red Sea) Dentex tambulus – Valenciennes in Cuvier and 2.4 COI Sequences Downloaded from GenBank and Valenciennes, 1830: 249, 558 ('Rade de Pondicherry', Checked by BOLD India) Dentex luteus – Valenciennes in Cuvier and Valenciennes, COI sequences for additional specimens were obtained 1830: 250 ( Pondicherry, India) from GenBank: N. japonicus from the Indian Ocean ?Dentex striatus – Valenciennes in Cuvier and (GenBank accession numbers: EF609553-EF609555) Valenciennes, 1830: 252 (Tranquebar, India) (Lakra et al., 2011); N. mesoprion (GenBank accession Spondyliosoma guliminda – (non Valenciennes) Cantor, numbers: EF609557-EF609559); N. virgatus (GenBank 1849: 1032 (Penang) accession numbers: FJ237833-FJ237835); N. hexodon Dentex Blochii – Bleeker, 1851: 176 (Batavia =Jakarta) (GenBank accession number: EF609414); N. peronii Synagris japonicus – Günther, 1859: 378 (Batavia (GenBank accession number: EF609415); N. furcosus =Jakarta) (GenBank accession number: EF609413). A sequence Synagris filamentosus – Günther , 1859: 378 (Red Sea; from Parascolopsis eriomma (GenBank accession num- coast of Pondicherry, India) ber: HQ945943) was used as an outgroup. The COI se- Dentex japonicus – Bleeker, 1865: 173 (Siam) quences from GenBank and those obtained in this study Synagris grammicus – Day, 1865: 14 (Cochin, Malabar were validated on BOLD (Ratnasingham and Hebert, coast of India) 2007) to ensure that specimens were properly identified. Heterognathodon fiaviventris – Steindachner, 1866: 778, p1. 13, fig. 6 (Zanzibar) 2.5 Sequence Alignment and Phylogenetic Analysis Dentex (Heterognathodon) filamentosus – Steindachner, All sequences were aligned using the CLUSTAL W, 1868: 976 (Mauritius) and then truncated to a uniform length by MEGA 5.0. The Nemipterus japonicus – Jordan and Seale, 1907: 21 aligned sequences were subjected to both maximum- (Cavite, Philippines); Akazaki, 1962: 91 (Tonkin Bay, likelihood (ML), and neighbour-joining (NJ) analyses Vietnam; Iran); Cheng, 1962: 505–510 (South China using MEGA 5.0 (Tamura et al., 2011). The model of Sea); Wu, 1985: 187–193 (East China Sea); Lee, 1986: NING et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2015 14 3 168, pl. 3, fig. 13 (Kaohsiung, Taiwan); Russell, 1986: Synagris flavolinea) (ANSP 53454), Saukiwan, Hong 19–35 (species catalog, Indian Sea); Li, 1987: 336–337 Kong, China; 1 ex. (110.0 mm SL, paratype of S. fla- (China sea); Russell, 1990: 40, pl. II, b, fig. 72 (Indian volinea) (ANSP 53455), Saukiwan, Hong Kong, China; 1 Ocean and West Pacific); Lee, 1993: 369–371 (Tai- ex. (156.0 mm SL) (ASIZ P055432), Kao- hsiung, Tai- wan); Russell, 1993: 295–310