2 Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, China
Pantropical spotted dolphin,Stenella attenuata(Gray,1846),is a tropical-to-subtropical widely distributed oceanic species occurring between 40°N and 40°S(Jefferson et al., 1993; Perrin and Hohn, 1994; Best,2007; Perrin,2009; Qin et al., 2009),though individuals are occasionally found in colder water(Perrin et al., 1987; Worthy et al., 1993). This species is listed as Least Concern by the International Union for Conservation of the Nature and Natural Resources(IUCN,2014), and is included on Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora(CITES,2013). In Chinese waters,this species was listed as a Grade II National Key Protected Animal since 1988. It occurs mainly in the southern East China Sea,South China Sea and off the Taiwanese coast(Wang,1999; Li et al., 2005), and is recognized as one of the most common cetaceans along the eastern Taiwanese coast(Yeh,2000). It also occurs in Shantou waters of Guangdong Province, and Beibu Gulf of Guangxi Province(Wang,1999; Qin et al., 2009).
Pantropical spotted dolphin is a slender and streamlined species with a dark dorsal cape; the lowersides and bellies of adults are gray; it also has a long and narrow beak,the tip of which is bright white,similar to its lips. A dark gray b and encircles each eye and continues forward to the apex of the melon; it alsohas a dark gape-to-flipper stripe(Jefferson et al., 1993); born spotless,these develop with age,though the degree of spotting varies geographically(Perrin and Hohn, 1994; Edwards et al., 2013): in the PacificOcean,coastal individuals are more spotted thanoffshore forms,while dolphins in the southwesternCaribbean are only lightly spotted or not spotted at all(Mignucci-Giannoni et al., 2003). Despite thesedistinguishing features,this species is at times difficultto differentiate from similar-looking spinner,Atlanticspotted and Indo-Pacific bottlenose dolphins(Stenellalongirostris,Stenella frontalis and Tursiops aduncusrespectively),given variable or otherwise ambiguouscolour morphs.
This species has a life expectancy of over 40 years(Perrin et al., 1987),with sexual maturity attainedbetween 9 and 11 years in females and 12 and 15years in males(Perrin, 2001,2009). Body length alsovaries geographically,ranging from 1.6 to 2.4 minfemales and 1.6 to 2.6 min males(Leatherwood and Reeves, 1983; Reeves and Leatherwood, 1987; Jefferson et al., 1993). Diet in the eastern tropical Pacific and off eastern Taiwan Isl and is dominated bylanternfishes(Myctophidae) and squid(Ommastrephidae and Enoploteuthidae)(Perrin et al., 1973; Robertson and Chivers, 1997;Wang et al., 2003a; Archer and Robertson, 2004),with significantseasonal differences(Wang et al., 2003a).
Although Pantropical spotted dolphins are widelydistributed,most research has been undertaken onanimals from the eastern Pacific Ocean,where theyare frequently caught as bycatch in the yellowfin tuna(Thunnus albacares)fishery,causing one populationto become depleted(Smith,1983; Edwards et al., 2013). Studies on this species from,or proximal toChinese waters include evaluation of heavy metalsfrom Beibu Gulf(Qin et al., 2009),accumulation ofbutyltin compounds and feeding habits from coast ofTaiwan Isl and (Liu et al., 2003; Wang et al., 2003a),cranial variation from the Pacific Ocean(Yao et al., 2008), and age determination and growth from Pacificcoast of Japan(Li et al., 2005).
On October 1,2009,sixteen dolphins were obtainedfrom fishermen by accidentally caught at once in agroup in the Yellow Sea,following which they weresent to the Sh and ong University(Weihai)for speciesidentification and preservation(Fig. 1). Morphologicalparameters were measured and a partial mitochondrialDNA cytochrome b gene amplified by polymerasechain reaction(PCR)to confirm the identification ofthe species.2 MATERIAL AND METHOD 2.1 Samples
Sixteen dolphins were obtained from fishermen byincidental catching when they were engaged in thetrawl fishing in the Yellow Sea and later preserved inSh and ong University at Weihai.2.2 Morphological measurements
Morphological parameters,including sex,toothcount and body measurements were recorded in accordance with recommendations and definitions in Chen et al.(1992),Wang(1999),Zhu et al.(2004),Zhu et al.(2008), and Wang et al.(2013).2.3 DNA samples processing and anlysis
Skin samples were obtained from the flukes of thesixteen dolphins, and were then stored and labeled inseparate containers in low temperature(-20°C)forsubsequent analysis. Total genomic DNAs wereisolated from the skin samples using a phenol/chloroform organic extraction method(Kalmár et al., 2000; Tebbutt et al., 2000; Sambrook and Russell, 2001; Rastogi et al., 2004; Ma et al., 2007).
Amplification reactions were performed in a 25 μLtotal volume containing 10×Buffer(50 mmol/L KCl,10 mmol/L of Tris-HCl,pH 8.4),2.0 mmol/L MgCl2,200 μmol/L each of dATP dTTP dCTP and dGTP,4×10-4 μmol/L each of forward and reverse primers,2.5 units of Taq DNA polymerase, and approximately50–100 ng of extracted genomic DNA templates. Thetemperature profile for the amplifications included thefollowing: an initial hot-start denaturation of 94°C for5 min,followed by 35 amplification cycles of 40 s at94°C,annealing temperature at 56°C(40 s), and extension at 72°C(30 s). An additional 5 min intervalat 72°C was added at the end of the cycle series toensure the complete extension of the PCR products.Primers for the mitochondrial DNA cytochrome bgene used were from Wang et al.(2003b),namely,DP1-L(5′-ACGACGCATTCATTGATCT-3′) and DP2-H(5′-GTGCTCAGGGTAGGACGTA-3′).
All PCR products were sequenced in SangonBiotech Co.,Ltd.(Shanghai,China). The sequenceswere first edited using BioEdit,aligned using thecomputer software Clustal X multiple alignment(Thompson et al., 1997), and corrected by eye. Primer and ambiguous sequences were deleted. Cytochromeb nucleotide sequences were submitted to GenBank.To perform haplotype sequence comparisons,fourhomologous cytochrome b sequences with the besthits of Pantropical spotted dolphins in the blast were added to sequences obtained in the present study.2.4 Species identification
Morphological identification of the dolphins wasbased on the morphometric data previously reported(Perrin et al., 1987; Jefferson et al., 1993; Perrin and Hohn, 1994; Mignucci-Giannoni et al., 2003; Best,2007). Molecular identification was performed using amitochondrial DNA cytochrome b gene sequencesimilarity search under BLAST(Basic Local AlignmentSearch Tool)as implemented in GenBank. Homologoussequences with the highest similarity were downloaded.3 RESULT 3.1 Morphological characteristics
The 16 specimens,8 males and 8 females,wereslender and streamlined with long and narrow beaks,with ambiguous skin coloration. The body lengthranged 174 cm to 228 cm and the jaws had 72–104teeth per dolphin(Table 1). An additional 46 externalmorphological parameters were summarized in Table 2.3.2 Cytochrome b gene sequence
Primers DP1-L/DP2-H generated the PCR productsof all 16 specimens with mitochondrial DNAcytochrome b gene sequences as long as 328-bp. Asmany as six haplotypes(HPSD1-6)were definedamong the 16 specimens(Table 3), and the mount ofthe most common haplotype(HPSD2)was seven,shared by 43.8% of the samples. By comparing thehomologous sequences of the same species availablein the portals of GenBank,we found that the sixhaplotypes had maximal genetic similarity withPantropical spotted dolphins. Two haplotypes hadabsolute genetic similarity with the ones reportedpreviously,including one(HPSD1)which matchedthree reference sequences from two unknownlocations,namely,GenBank accession Nos.EF093030.1 and X56294.1 and eastern tropicalPacific(GenBank accession No. AF084096.1). Theother haplotype(HPSD4)was the same as that of aChinese sample(GenBank accession No.EU557096.1). The blasting results of the others(HPSD2,HPSD3,HPSD5, and HPSD6)in GenBankshowed that the samples had maximal geneticsimilarity(99%)with Pantropical spotted dolphinsfrom other places(Table 4).
An examination of the 328-bp segment and alignment with the four best-hit sequences in the blastdownloaded from GenBank revealed six variablesites(Table 5),accounting for 1.83% of totalnucleotide acids of our Yellow Sea Pantropical spotteddolphin samples. All variable sites were transitionsubstitutions. The average proportions of nucleotidesA,T,C and G were about 27.4%,30.3%,28.0%, and 14.3%,respectively. The proportion of(A+T)(57.7%)was more than that of(G+C)(42.3%).
All cytochrome b sequences were deposited inGenBank under accession numbers JN232942–JN232953 and JN225498–JN225501(Table 5).4 DISCUSSION
The species encountered in the present study wasidentified as Pantropical spotted dolphin based onmorphology and mitochondrial DNA cytochrome bsequences,with the main characteristics matchingthose reported previously(Jefferson et al., 1993; Perrin and Hohn, 1994; Perrin,2009). To the contrary,mitochondrial DNA cytochrome b sequence analysisrevealed as many as six haplotypes amongst ourspecimens,including two haplotypes with absolutegenetic similarity to those reported earlier.
The Yellow Sea cetacean fauna is now recognizedto comprise eight species: finless porpoise(Neophocaena phocarnoides),false killer whale(Pseudorca crassidens),killer whale(Orcinus orca),common dolphin(Delphinus delphis),commonbottlenose dolphin(Tursiops truncatus), and spinner and Pantropical spotted dolphins(Stenellalongirostris, and Stenella attenuata),Minke whale(Balaenoptera acutorostrata)(Wang,1979; Wang et al., 1989; Zhu et al., 2006,2007). In Chinese waters,Pantropical spotted dolphin occurs in the southernEast China Sea and South China Sea,particularly inBeibu Gulf and waters around Taiwan Isl and (Wang,1999). To the best of our knowledge,the presentreport is the first documentation of Pantropical spotteddolphins in the Yellow Sea.
In China,although Pantropical spotted dolphin is aGrade II National Key Protected species(Wang,1999),studies on this species are relatively limited,which cause the lack of basic knowledge on theirstatus and biology. There are no estimates ofpopulation size for this species in Chinese waters,no indication of residency duration or migratory behavior, and very little is known about what impactsfisheries might be having on it,either directly(throughincidental capture),or indirectly(such,but not limitedto competition for prey resources).
This new record of Pantropical spotted dolphinsfrom the Yellow Sea may indicate a range expansionof this species due to environmental change,but moredata are required to more fully evaluate this possibility.Additional research on abundance,populationstructure,ecology and conservation problems causingby the various anthropogenic activities is also neededto better underst and and protect this species inChinese waters.5 ACKNOWLEDGEMENT
We are grateful to ZHAO Qi,ZHOU Can,CUIMengxuan,MA Qinglin, and XING Xiang ofSh and ong University for their generous help and assistance on sample collection and measurements.
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