Identification of Cynoglossus joyneri eggs and larvae by DNA barcoding and morphological method

doi:10.17520/biods.2017094

Abstract

Abstract:

This study was designed to accurately identify Cynoglossus eggs and larvae (Cynoglossus sp.), and to accumulate base data for further study on the taxonomy and early identification of Cynoglossus fishes. Cynoglossus sp. samples were collected in Qingshan Bay, Qingdao, between May and October 2015. Three eggs, 3 larvae and a total of 5 individuals from 2 species (C. joyneri and C. lighti) in the Jiaozhou Bay area were barcoded using COI, sequenced, and compared with 6 Cynoglossus species found in the Shandong Peninsula and recorded in GenBank. Under a light microscope, the egg had a circular shape and floated within a wider perivitelline space; eggs ranged in diameter from 0.68 to 0.87 mm with a mean diameter of 0.73±0.03 mm; eggs had a single layer membrane and were smooth and transparent; the oil globules were located opposite the yolk sac, ranged from 6 to 15 in number, and diameter ranged from 0.04 to 0.10 mm (mean diameter: 0.07±0.01 mm); there were obvious characteristics including the 2 dorsal fin rays and pigmentation and body length changes in the period of metamorphosis for larvae identification. Results of genetic analysis showed that the average K2P distances pairwise-species in Cynoglossus sp., C. joyneri and C.lighti ranged from 0.006 to 0.009, and was 0.002 for C. abbreviatus and C. purpureomaculatus. The two groups were placed in two different clades with the support of a high bootstrap value and had a close genetic relationship, separately. The neighbor-joining (NJ) phylogenetic trees were constructed for COI gene sequences of 8 species using MEGA 6.0, and Zebrias zebra was included as an outgroup. The above analysis and results showed that the Cynoglossus sp. is C. joyneri.

Key words: Cynoglossus, synonym, ichthyoplankton, COI, identification

Xiaomeng Zhou, Shuxin Guo, Na Song, Xiumei Zhang. Identification of Cynoglossus joyneri eggs and larvae by DNA barcoding and morphological method[J]. Biodiv Sci, 2017, 25(8): 847-855.

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URL: https://www.biodiversity-science.net/EN/10.17520/biods.2017094

https://www.biodiversity-science.net/EN/Y2017/V25/I8/847

Figures/Tables 7

References 33

[1]	Ahlstrom EH, Moser HG (1976) Eggs and larvae of fishes and their role in systematic investigations and in fisheries. Revue des Travaux de l’Institut des Pêches Maritimes, 40, 379-398.
[2]	Ball SL, Hebert PDN, Burian SK, Webb JM (2005) Biological identifications of mayflies (Ephemeroptera) using DNA barcodes. Journal of the North American Benthological Society, 24, 508-524.
[3]	Bian XD, Zhang XM, Xiao YS, Gao TX, Wan RJ (2007) Identification of fish eggs of Japanese halfbeak (Hyporhamphus sajori) using mtDNA sequencing. Periodical of Ocean University of China, 37(S1), 111-116. (in Chinese with English abstract)
	[卞晓东, 张秀梅, 肖永双, 高天翔, 万瑞景 (2007) 线粒体DNA序列在沙氏下鱵鱼鱼卵鉴别上的应用. 中国海洋大学学报(自然科学版), 37(S1), 111-116.]
[4]	Carpenter KE, Niem VH (2001) FAO Species Identification Guide for Fishery Purposes: The Living Marine Resources of the Western Central Pacific, pp. 3890-3891. FAO, Rome.
[5]	Chen YX, Wu RX, Liang N, Liu J (2015) Phylogenetic relationship in family Sparidae of China in mitochondrial COI gene sequence. Oceanologia et Limnologia Sinica, 46, 611-619. (in Chinese with English abstract)
	[陈咏霞, 吴仁协, 梁娜, 刘静 (2015) 基于线粒体COI基因序列的中国鲷科鱼类系统进化关系. 海洋与湖沼, 46, 611-619.]
[6]	Gao DK (2015) Applying Basic Research on the Morphological and Molecular Approaches to Identifying the Ichthyoplankton. PhD dissertation, Ocean University of China, Qingdao. (in Chinese with English abstract)
	[高东奎 (2015) 鱼类浮游生物形态及分子鉴定方法的应用基础研究. 博士学位论文, 中国海洋大学, 青岛.]
[7]	General Administration of Quality Supervision, Inspection and Quarantine of China, Standardization Administration of China(2007) Specifications for Oceanographic Survey-- Part 6: Marine Biological Survey. China Standards Press, Beijing. (in Chinese)
	[中华人民共和国国家质量监督检验检疫总局, 中国国家标准化管理委员会(2007) 海洋调查规范第6部分: 海洋生物调查. 中国标准出版社, 北京.]
[8]	Gu SO, Hwang HJ, Kim JK (2009) Morphological description of Cynoglossus joyneri larvae collected from the Yellow Sea. Korean Journal of Fisheries and Aquatic Sciences, 42, 725-728. (in Korean with English abstract)
[9]	Hebert PDN, Cywinska A, Ball SL (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London B: Biological Sciences, 270, 313-321.
[10]	Li SZ, Wang HM (1995) Fauna Sinica: Osteichthyes. Pleuronectiformes. Science Press, Beijing. (in Chinese)
	[李思忠, 王惠民 (1995) 中国动物志硬骨鱼纲鲽形目. 科学出版社, 北京.]
[11]	Li XM, Ye ZJ, Li ZG, Hu HS, Zou Q (2016) Spatial and temporal changes of the egg size changes of Japanese anchovy (Engraulis japonicus) inhabiting the near shore spawning ground of central Yellow Sea. Periodical of Ocean University of China, 46(2), 54-60. (in Chinese with English abstract)
	[李秀梅, 叶振江, 李增光, 胡海生, 邹全 (2016) 黄海中部近岸产卵场日本鳀卵子大小的时空变化. 中国海洋大学学报(自然科学版), 46(2), 54-60.]
[12]	Liu SF, Liu JX, Zhuang ZM, Gao TX, Han ZQ, Chen DG (2010a) Monophyletic origin and synonymic phenomena in the sub-family Cynoglossinae inferred from mitochondrial DNA sequences. Biodiversity Science, 18, 275-282. (in Chinese with English abstract)
	[柳淑芳, 刘进贤, 庄志猛, 高天翔, 韩志强, 陈大刚 (2010a) 舌鳎亚科鱼类单系起源和同种异名的线粒体DNA证据. 生物多样性, 18, 275-282.]
[13]	Liu SF, Chen LL, Dai FQ, Zhuang ZM (2010b) Applicaction of DNA barcoding gene CO1 for classifying family Sciaenidae. Oceanologia et Limnologia Sinica, 41, 223-232. (in Chinese with English abstract)
	[柳淑芳, 陈亮亮, 戴芳群, 庄志猛 (2010b) 基于线粒体CO1基因的DNA条形码在石首鱼科(Sciaenidae)鱼类系统分类中的应用. 海洋与湖沼, 41, 223-232.]
[14]	Menon AGK (1977) A Systematic Monograph of the Tongue Soles of the Genus Cynoglossus Hamilton-Buchanan (Pisces, Cynoglossidae). Smithsonian Institution Press, Washington, DC.
[15]	Miao XG, Jiang JX, Shi W, Wang ZM, Wang SY (2013) Phylogenetic relationship of Cynoglossinae based on COI barcoding marker. Journal of Tropical Oceanography, 32(5), 85-92. (in Chinese with English abstract)
	[苗宪广, 江金霞, 时伟, 王忠明, 王淑英 (2013) 舌鳎亚科Cynoglossinae鱼类基于COI条形码的系统发育关系. 热带海洋学报, 32(5), 85-92.]
[16]	Minami T (1983) The early life history of a tongue fish Cynoglossus joyneri. Bulletin of the Japanese Society of Scientific Fisheries, 49, 719-724. (in Japanese with English abstract)
[17]	Nakabo T (2000) Fishes of Japan with Pictorial Keys to the Species, 2nd ed. Tokai University Press, Tokyo. (in Japanese)
[18]	Ni HE, Zheng ZM (2002) Comparison of shapes and growth features of three species in Cynoglossus. Marine Sciences, 26(12), 61-64. (in Chinese with English abstract)
	[倪海儿, 郑忠明 (2002) 几种舌鳎的形态和生长特性的比较. 海洋科学, 26(12), 61-64.]
[19]	Pasco-Viel E, Charles C, Chevret P, Semon M, Tafforeau P, Viriot L, Laudet V (2010) Evolutionary trends of the pharyngeal dentition in Cypriniformes (Actinopterygii: Ostariophysi). PLoS ONE, 5, e11293.
[20]	Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning. Cold Spring Harbor Laboratory Press, New York.
[21]	Sharina SN, Kartavtsev YP (2010) Phylogenetic and taxonomic analysis of flatfish species (Teleostei, Pleuronectiformes) inferred from the primary nucleotide sequence of cytochrome oxidase 1 gene (Co-1). Russian Journal of Genetics, 46, 356-361.
[22]	Song C, Yu YN, Zhang T, Yang G, Zhang LZ (2014) Phylogenetic analysis of Cynoglossidae in the Yangtze Estuary based on partial sequence of mitochondrial COI. Chinese Journal of Zoology, 49, 716-726. (in Chinese with English abstract)
	[宋超, 于亚男, 张涛, 杨刚, 章龙珍 (2014) 基于线粒体COI基因部分序列的长江口舌鳎科鱼类系统分类研究. 动物学杂志, 49, 716-726.]
[23]	Su JX (1995) Fish and Marine Fish Culture, 2nd ed. China Agriculture Press, Beijing. (in Chinese)
	[苏锦祥 (1995) 鱼类学与海水鱼类养殖, 第二版. 中国农业出版社, 北京.]
[24]	Walsh PS, Metzger DA, Higuchi R (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques, 10, 506-513.
[25]	Wan RJ, Jiang YW (2000) The species and biological characteristics of the eggs and larvae of osteichthyes in the Bohai and Yellow Sea. Journal of Shanghai Fisheries University, 9, 290-297. (in Chinese with English abstract)
	[万瑞景, 姜言伟 (2000) 渤、黄海硬骨鱼类鱼卵与仔稚鱼种类组成及其生物学特征. 上海水产大学学报, 9, 290-297.]
[26]	Wan RJ, Jiang YW, Zhuang ZM (2004) Morphological and developmental characters at the early stages of the tonguefish Cynoglossus semilaevis. Acta Zoologica Sinica, 50, 91-102. (in Chinese with English abstract)
	[万瑞景, 姜言伟, 庄志猛 (2004) 半滑舌鳎早期形态及发育特征. 动物学报, 50, 91-102.]
[27]	Wang ZD, Guo YS, Chen RL, He XY, Liu CW, Liu Y (2009) COI barcoding sequences of teleosts in the South China Sea. Oceanologia et Limnologia Sinica, 40, 608-614. (in Chinese with English abstract)
	[王中铎, 郭昱嵩, 陈荣玲, 何晓莹, 刘楚吾, 刘筠 (2009) 南海常见硬骨鱼类COI条码序列. 海洋与湖沼, 40, 608-614.]
[28]	Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PD (2005) DNA barcoding Australia’s fish species. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 360, 1847-1857.
[29]	Xu H, Li J, Kong XY, Xiao ZZ, Li YL, Zhang YC, Shi W, Wei ZP, Yu ZN (2008) Phylogenetic relationship and length variation in the first ribosomal internal transcribed spacer of Cynoglossinae species. Oceanologia et Limnologia Sinica, 39, 35-41. (in Chinese with English abstract)
	[徐晖, 李军, 孔晓瑜, 肖志忠, 李玉龙, 张艳春, 时伟, 位正鹏, 喻子牛 (2008) 6种舌鳎亚科鱼类ITS1序列长度多态性及系统分析. 海洋与湖沼, 39, 35-41.]
[30]	Yagi Y, Kinoshita I, Fujita S, Ueda H, Aoyoma D (2009) Comparison of the early life histories of two Cynoglossus species in the inner estuary of Ariake Bay, Japan. Ichthyological Research, 56, 363-371.
[31]	Yang DL, Wu GZ, Pang HY (1983) The morphology of the early stage of longuefishes, Cynoglossus semilaevis Günther and C. joyneri Günther, in the Bohai Bay. Marine Sciences, 7(2), 29-32. (in Chinese with English abstract)
	[杨东莱, 吴光宗, 庞红艳 (1983) 渤海半滑舌鳎及焦氏舌鳎的鱼卵和仔稚鱼的形态. 海洋科学, 7(2), 29-32.]
[32]	Zhou CW, Cheng QT (1997) Ichthyography of Shandong. Shandong Science and Technology Press, Ji’nan. (in Chinese)
	[周才武, 成庆泰 (1997) 山东鱼类志. 山东科学技术出版社, 济南.]
[33]	Zhou MY, Chen X, Yang SY (2015) Identification of several fish eggs and larvae by DNA barcoding in Xiamen Water. Marine Environmental Science, 34(1), 120-125, 135. (in Chinese with English abstract)
	[周美玉, 陈骁, 杨圣云 (2015) 采用DNA条形码技术对厦门海域鱼卵、仔稚鱼种类的鉴定. 海洋环境科学, 34(1), 120-125, 135.]

形态特征 Morphological characters	短吻红舌鳎 C. joyneri			长吻红舌鳎 C. lighti
形态特征 Morphological characters	Li & Wang, 1995	Zhou & Cheng, 1997	Nakabo, 2000	Li & Wang, 1995	Zhou & Cheng, 1997	Nakabo, 2000
上、中侧线鳞间鳞数 Scales between upper and middle lateral line	12-13	11-12	/	10-11	10-11	/
体长/体高 Body length/body depth	3.6-4.4	3.8-4.0	3.4-4.1	3.8-4.5	4.2-5.0	3.8-4.5
头长/头高 Head length/head depth	≤1	/	/	>1	/	/

形态特征 Morphological characters	短吻红舌鳎 C. joyneri			长吻红舌鳎 C. lighti
形态特征 Morphological characters	Li & Wang, 1995	Zhou & Cheng, 1997	Nakabo, 2000	Li & Wang, 1995	Zhou & Cheng, 1997	Nakabo, 2000
上、中侧线鳞间鳞数 Scales between upper and middle lateral line	12-13	11-12	/	10-11	10-11	/
体长/体高 Body length/body depth	3.6-4.4	3.8-4.0	3.4-4.1	3.8-4.5	4.2-5.0	3.8-4.5
头长/头高 Head length/head depth	≤1	/	/	>1	/	/

编号Number	种类 Species	序列编号Sequence numbers	序列来源 Sources of sequences
1	舌鳎属样品 Cynoglossus sp.	egg.1-egg.3; lar.1-lar.3	本研究 This study
2	短吻红舌鳎 C. joyneri	joy.1-joy.5	本研究(1-3); JQ738430.1; KF979127.1
3	长吻红舌鳎 C. lighti	lig.1-lig4	本研究(1-2); DQ116752.1; HQ711865.1
4	宽体舌鳎 C. robustus	rob.1-rob.3	KT718591.1; HM180552.1; HM180553.1
5	半滑舌鳎 C. semilaevis	sem.1-sem.3	JN242742.1; JN242743.1; JN242744.1
6	紫斑舌鳎 C. purpureomaculatus	pur.1-pur.3	DQ116749.1; JQ738570.1; JQ738571.1
7	短吻三线舌鳎 C. abbreviatus	abb.1	DQ116750.1
8	带纹条鳎 Zebrias zebra	zeb.1-zeb.3	JQ738566.1; JQ738567.1; JQ738568.1

编号Number	种类 Species	序列编号Sequence numbers	序列来源 Sources of sequences
1	舌鳎属样品 Cynoglossus sp.	egg.1-egg.3; lar.1-lar.3	本研究 This study
2	短吻红舌鳎 C. joyneri	joy.1-joy.5	本研究(1-3); JQ738430.1; KF979127.1
3	长吻红舌鳎 C. lighti	lig.1-lig4	本研究(1-2); DQ116752.1; HQ711865.1
4	宽体舌鳎 C. robustus	rob.1-rob.3	KT718591.1; HM180552.1; HM180553.1
5	半滑舌鳎 C. semilaevis	sem.1-sem.3	JN242742.1; JN242743.1; JN242744.1
6	紫斑舌鳎 C. purpureomaculatus	pur.1-pur.3	DQ116749.1; JQ738570.1; JQ738571.1
7	短吻三线舌鳎 C. abbreviatus	abb.1	DQ116750.1
8	带纹条鳎 Zebrias zebra	zeb.1-zeb.3	JQ738566.1; JQ738567.1; JQ738568.1

碱基 Base	总频率 Total	密码子第1位点 The first position of codon	密码子第2位点 The second position of codon	密码子第3位点 The third position of codon
T	31.3	18.0	43.8	32.1
C	26.5	24.5	28.1	27.1
A	25.6	27.4	14.5	34.8
G	16.6	30.1	13.6	6.0
A+T	56.9	45.4	58.3	66.9
G+C	43.1	54.6	41.7	33.1