Morphological and Molecular Evidence Supports the Occurrence
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Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54 DOI: 10.1007/s13131-014-0457-y http://www.hyxb.org.cn E-mail: [email protected] Morphological and molecular evidence supports the occurrence of a single species of Zebrias zebrinus along the coastal waters of China WANG Zhongming1,2,4, KONG Xiaoyu1*, HUANG Liangmin1, WANG Shuying1,4, SHI Wei1, KANG Bin3 1 Key Laboratory of Marine Bio-resources Sustainable Utilization, Marine Biodiversity Collection of South China Sea, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China 2 Key Laboratory of Sustainable Utilization of Technology Research for Fishery Resource of Zhejiang Province, Marine Fisheries Research Institute of Zhejiang, Zhoushan 316100, China 3 Asian International Rivers Center, Yunnan University, Kunming 650091, China 4 University of Chinese Academy of Sciences, Beijing 100049, China Received 11 November 2012; accepted 28 May 2013 ©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2014 Abstract The so-called zebra sole includes a group of small flatfishes characterized by transverse band pairs on the ocular side and distributed throughout shallow waters along the coast of the Indo-West Pacific Ocean. Sev- eral species of the zebra sole have been recorded from the coastal waters of China. Morphological analysis of 1 107 specimens of the zebra sole from 15 successive localities along the China’s coast demonstrated that no significant variations among these localities were found on the basis of meristic counts and morphometric characters. Phylogenetic analysis based on COI gene sequences of 14 individuals and D-loop of 22 indi- viduals from eight localities showed that they were indistinguishable among these localities. Therefore, both morphological and molecular evidence supported the occurrence of a single species of the zebra sole along the China’s coast. The available name for this species is Zebrias zebrinus (Temminck and Schlegel, 1846) in- stead of Z. zebra (Bloch, 1787). Zebrias fasciatus (Basilewsky, 1855) and Solea ommatura (Richardson, 1846) are considered here as two synonyms of Z. zebrinus. Key words: Zebrias, zebra sole, China’s coast, morphological and molecular evidence, species validity, synonyms Citation: Wang Zhongming, Kong Xiaoyu, Huang Liangmin, Wang Shuying, Shi Wei, Kang Bin. 2014. Morphological and molecular evidence supports the occurrence of a single species of Zebrias zebrinus along the coastal waters of China. Acta Oceanologica Si- nica, 33(8): 44–54, doi: 10.1007/s13131-014-0457-y 1 Introduction pairs of transverse dark bands on the ocular side and yellow The genus Zebrias belongs to the family Soleidae of the Pleu- marks on the caudal fin; interorbital space narrow and scaly; ronectiformes, and includes a group of species characterized by dorsal and anal fins branched and completely confluent with a combination of the following characters: eyes and color on the caudal fin (the last dorsal and anal rays as long as adjacent cau- right side, with black transverse bands more or less arranged in dal rays); and pectoral fin on the ocular side longer than the one pairs; pectoral fin of the blind side rudimentary or wanting, dor- on the blind side (Li and Wang, 1995). However, the taxonomy of sal and anal fins confluent with caudal fin; teeth minute, only the zebra sole is poorly understood. present on blind side or absent (Jordan and Starks, 1906; Cheng Bloch (1787) initially described specimens of the zebra sole and Chang, 1965). A total of 22 nominal species have been de- from Tranquebar, east coast of India, as Pleuronectes zebra [here scribed and included in Zebrias (Bloch, 1787; Richardson, 1846; Zebrias zebra]. Later, three other species were described on the Temminck and Schlegel, 1846; Basilewsky, 1855; Kaup, 1858; basis of similar specimens from seas of the Northwest Pacific: Bleeker, 1860; Macleay, 1882; Alcock, 1890; Regan, 1903; Gil- Solea zebrina (Temminck and Schlegel, 1846) [here Z. zebrinus, christ, 1906; Jenkins, 1910; McCulloch, 1916; Cheng and Chang, type locality: Nagasaki, Japan]; Solea ommatura (Richardson, 1965; Whitley, 1966; Rama-Rao, 1967; Talwar and Chakrapany, 1846) [type locality: Canton (presently Guangdong Province, 1967; Joglekar, 1976; Oommen, 1977; Seigel and Adamson, 1985; China)], and Solea fasciata (Basilewsky, 1855) [here Z. fasciatus, Gomon, 1987; Randall, 1995), and they are widely distributed type locality: Shantung (i.e., Shandong Province, China)]. Spe- in the Indian Ocean and western central Pacific Ocean. Zebrias cies delineation of the zebra sole has long been a controversial zebra, commonly called the zebra sole, is a kind of small flatfish issue. Some authors argued that two distinct species existed: living in shallow waters throughout the coast of the Indo-West the northern species named as Z. fasciatus, and the southern Pacific. It is characterized by having the following features: 11 one as Z. zebrinus (Jordan and Metz, 1913; Ochiai, 1955; Naka- Foundation item: The Key Innovation Project of Chinese Academy of Sciences under contract No. KSCX2-YW-Z-0929; the National Natural Science Foundation of China under contract Nos 30870283 and 31071890. *Corresponding author, E-mail: [email protected] WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54 45 bo, 2002) or Z. zebra (Ochiai, 1963, 1984; Yamada and Okamura, d1-8, d4-6, d4-8, d6-7, d6-8, d6-9, d6-10, d8-9, d8-10, and d9- 1986). A majority of researchers held that the zebra sole repre- 10 (Fig. 1). All measurements were expressed as proportions of sented a single species, but two specific names were used for the standard length, except for d6-7 and d6-8, and those related it. This species was identified either as Z. zebra (Günther, 1862 with the head, such as ED, SE, d1-2, and d1-3, which were given as Synaptura zebra; Jordan and Evermann, 1902; Hubbs, 1915; as proportions of the head length. Subsequently, SPSS v.16.0 Chu, 1931; Wu, 1932; Wang, 1933; Cheng, 1955, 1962; Tchang was employed for the basic statistical analysis of morphological and Wang, 1963; Cheng and Chang, 1965; Li, 1987; Shen, 1993; data. Principal component analysis (PCA) was also performed Li and Wang, 1995; Shen and Wu, 2011) or as Z. zebrinus (Jordan on the covariance-variance matrix of the log10-transformed and Starks, 1906; Jordan and Hubbs, 1925; Evermann and Shaw, measurements (Xie et al., 2003). 1927; Fowler, 1929; Wu, 1929). Apparently, there are controver- The examined specimens from the coastal waters of China sies over how many zebra sole species occur in China’s seas and were housed in the Marine Biodiversity Collections of South which name(s) are available. The present investigation tries to China Sea, Chinese Academy of Sciences, Guangzhou, Guang- provide morphological and molecular evidence to confirm the dong Province, China. Type specimens of Z. zebra and Z. zebri- presence of a single zebra sole species in China’s seas, and com- nus are located in the Museum für Naturkunde, Germany (ZMB ment on the available name for this species. 2423) and Netherlands Centre for Biodiversity Naturalis (RMNH D.1308, RMNH D.1306 and 1307), respectively. 2 Materials and methods 2.3 Molecular analysis 2.1 Sample collection The sequences of mitochondrial COI gene and control re- Specimens of the zebra sole were collected from local fish- gion (D-loop) were used for molecular analysis. Sample infor- ing harbors by benthic trawling. A total of 1 107 specimens were mation is shown in Table 2. Total genomic DNA was extracted obtained from 15 sites along the China’s coast (Table 1 and using marine animal tissue DNA kits (TIANGEN Biotech, Fig. S1). Fresh specimens were stored in ice after collection and Beijing, China) following the manufacturer’s protocol. Prim- then kept in 95% ethanol or frozen at −20°C in the laboratory. ers for amplifications of mitochondrial DNA sequences were as follows: R-COI-6754 (CTAAGCCATCCTACCTGTG) and F- 2.2 Morphological analysis COI-8350 (TCAACTCCTCCCTTTCTCG) for COI; and L-17114 Measurements and counts were made on the ocular side of (RCGCCCAAAGCTAGDATTC), F-95 (GACAGTAAAGTCAGGAC- individuals. Each measurement was taken from point to point CAAGCCTTTGTGC), and F-2753 (TAGATAGAAACTGACCTG- with digital calipers and the data were recorded with precision of GATTACTCCGGT) for D-loop. The PCR was performed in a 25 0.1 mm. Meristic counts mainly followed those of Ochiai (1955) μL reaction volume containing 0.2 mmol/L dNTP, 0.5 μmol/L and Cheng and Chang (1965), including dorsal-fin rays (D), of each primer, 1 U/25 μL LA Taq (Takara, Dalian, China), 2.5 μL anal-fin rays (A), lateral-line scales (LLS, from above the pos- 10 × LA Taq Buffer II (Mg+ Plus), and approximately 50 ng DNA terior margin of operculum to caudal-fin base) and vertebrae template. PCR cycling conditions included an initial denatur- number. Additional counts were pectoral-fin rays (P). Twenty- ation at 95°C for 3 min, followed by 35 cycles of a denaturation one measurements were made, including six morphometric at 95°C for 30 s, an annealing at 48°C for 40 s, and elongation at measurements: standard length (SL), head length (HL), eye 68°C for 1−2 min, with a final extension at 72°C for 10 min. The diameter (ED, the horizontal diameter of the lower eye), space PCR products were detected in 1.0% agarose gels and purified between eyes (SE, minimal distance between the two eyes), using a Takara Agarose Gel DNA Purification Kit (Takara, China), pectoral-fin length (PL), space between band pairs (SB, the first and sequenced in both directions using the ABI 3730 Genetic space between band pairs just behind the pectoral-fin base). Analyzer (Invitrogen Corporation). Fragments that could not be There were 15 truss network measurements made between 10 directly sequenced were inserted into the PMD18-T vector (Ta- landmarks set on the ocular side: d1-2, d1-3, d1-4, d1-5, d1-6, kara, China), and transformed into E.