한수지 50(6), , 2017 Note Korean J Fish Aquat Sci 50(6), ,2017 제주도사계연안어란의분자동정과격월별출현양상 한송헌 김맹진 김준상 1 송춘복 2 * 국립수산과학원서해수산연구소, 1 한국수산자원관리공단제주지사

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1 한수지 50(6), , 2017 Note Korean J Fish Aquat Sci 50(6), ,2017 제주도사계연안어란의분자동정과격월별출현양상 한송헌 김맹진 김준상 1 송춘복 2 * 국립수산과학원서해수산연구소, 1 한국수산자원관리공단제주지사, 2 제주대학교해양과학대학 Molecular Identification and Bimonthly Abundance of Fish Eggs Collected in the Coastal Waters of Sagye, Jejudo Island Song-Hun Han, Maeng Jin Kim, Joon Sang Kim 1 and Choon Bok Song 2 * Fisheries Resources and Environment Division, National Fisheries Research and Development Institute, Incheon 22383, Korea 1 Korea Fisheries Resources Agency, Jeju Branch, Jeju 63005, Korea 2 College of Ocean Sciences, Jeju National University, Jeju 63243, Korea This study investigated the species composition and abundance of floating fish eggs to determine the timing and location of spawning of fish inhabiting the coastal waters of Sagye, Jejudo Island. Eggs were collected with a Bongo net bimonthly from May 2009 to February Identifications were based on nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Eggs were determined to belong to 43 distinct taxa, 35 of which were identified to the species level. The assemblage spanned eight orders, 23 families, and 32 genera. The number of taxa collected varied from month to month, with 14 taxa (12 species) found in June 2009, 11 taxa (10 species) in October 2009, 10 taxa (nine species) each in August 2009 and February 2010, eight taxa (six species) in April 2009, and five taxa (four species) in December Five abundant species (Branchiostegus japonicus, Engraulis japonicus, Pseudolabrus sieboldi, Goniistius zonatus, and Halichoeres tenuispinis) together represented 52.8% of the total number of eggs collected during the study. Key words: Fish eggs, Identification, Molecular biology, Jejudo Island 서론,,,. DNA barcode, (Hebert et al., 2003)., mitochondrial DNA cytochrome c oxidase subunit I (COI) (species specific), (Ratnasingham and Hebert, 2007). COI (Lee and Kim, 2014a; Lee and Kim, 2014b; Lee and Kim, 2016)., PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism)., (Cocolin et al., 2000; Dooly et al., 2005) (Bivalvia) (Gastropoda) (Wang et al., 2006)., PCR-RFLP (pre-screening). COI PCR RFLP,,,, Korean J Fish Aquat Sci 50(6) , December 2017 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 19 October 2017; Revised 27 November 2017; Accepted 5 December 2017 *Corresponding author: Tel: Fax: address: cbsong@jejunu.ac.kr Copyright 2017 The Korean Society of Fisheries and Aquatic Science 829 pissn: , eissn:

2 830 한송헌ㆍ김맹진ㆍ김준상ㆍ송춘복. 재료및방법 시료채집 (33 13 N, E) (Fig. 1). ( 0.6 m, 2 m, 333 m) 2-3 knots 10, (HYDRO-BIOS flowmeter) 1,000 m 3 (ind./1,000 m 3 ). 70% ethanol. (YSI, USA). Total DNA 추출 DNA 70% ethanol 1.5 ml eppendorf tube micropipet tip. tissue lysis buffer (Bioneer Inc., Korea) 50 μl 0.2 μg/μl proteinase K (Bioneer Inc., Korea) 4 μl binding buffer (Bioneer Inc., Korea) 50 L 60 10, 100% isopropanol (Sigma, USA) 25 μl, DNA binding column (Bioneer Inc., Korea). Accuprep Genomic DNA Extraction Kit (Bioneer Inc., Korea). DNA Unicam UV/VIS Spectrometer (He ios, Unicam Ltd., UK). PCR 증폭 PCR COI trna forward primer (Asn_F, 5'-YGATCCYRCAAAHWCTTAGT- TAACAGCTAA-3') reverse primer (Ser_R, 5'-GGGGTTC- DAYTCCYYCCTTTCT-3'). PCR volume 40 μl, 10 mm Tris-HCl (ph 8.3) 50 mm KCl, 2 mm MgCl 2, 0.2 mm dntp mix, 0.5 mm of each primer, 50 ng template DNA solution, 1 U Ex Taq DNA polymerase (Takara, Japan) TaKaRa PCR Thermal Cycler Dice (TP600, Takara Bio Inc.). PCR denaturing step , 94 30, primer annealing 54 1, primer extension , 72 7 extension. PCR 1X TAE buffer 0.5 mg/ L ethidium bromide 0.8% agarose (LE agarose, Cambrex co.) gel. PCR AccuPrep Gel Pruification Kit (Bioneer Inc., Korea). 제한효소처리 Korea East Sea Fig. 1. A map showing a sampling site located at the coastal waters of Sagye, Jejudo Island. COI HinfI (Bioneer Inc., Korea) Hpy188I (NEB Inc., UK). HinfI 50 mm Tris-HCl (ph 7.6) 10 mm MgCl 2, 100 mm NaCl, 1 mm DTT, 10 ng PCR, 1 U HinfI volume 15 μl. Hpy188I volume 15 μl, 20 mm Tris-acetate (ph 7.9) 50 mm potassium acetate, 10 mm Mg acetate, 1 mm DTT, 10 ng PCR, 1 U Hpy188I X TAE buffer 0.5 μg/μl ethidium bromide 2% agarose (LE agarose, Cambrex co.) gel band. band, PCR. 염기서열결정및종동정 Sampling location Jejudo Island PCR-RFLP PCR Big Dye Terminator v3.1 Cycle Sequencing Kits ABI 3730xl DNA Analyzer (Applied Biosystems, USA). COI ( 1,551 bp) DNAssist program 2.2 (Patterton and Graves, 2000). (species identification)

3 제주도사계연안어란의분자동정과격월별출현양상 831 GenBank database (species), (genus), (family) (order). Kimura-2-parameter distance model (Kimura, 1980), MEGA6 (Tamura et al., 2013) Neighbor- Joining (Saitou and Nei, 1987). 군집구조분석및환경요인과의상관관계 (Shannon and Weaver, 1963), (Pielou, 1966), (McNaughton, 1967), SPSS (Ver 14.0 SPSS Inc.) (Pearson correlation coefficient Pearson s r),. 결과 수온과염분 Fig , , psu ( ) 34.3 psu ( ), 33.0 psu. (r=-0.916, P<0.01). 종동정과출현현황, COI PCR (91.4%). PCR RFLP,., Table 1. 43, 35 ( ) (81.8%). 8 2 (family), 1 (order), 5. (Order) (Perciformes) ( 1 ), (Pleuronectiformes) 2 3 ( 1 ), (Aulopiformes) 2 Temperature ( ) Apr. Jun. Aug. Oct. Dec. Feb. Fig. 2. Bimonthly variation of surface water temperature ( ) and salinity (psu) at the sampling location. 3. (Cupeiformes), (Ophidiiformes), (Gadiformes), (Scorpaeniformes), (Tetraodontiformes). (Family), (Labridae) 7, (Sparidae) 3. (Synodontidae), (Moridae), (Malacanthidae), (Cheilodactylidae), (Callionymidae) 2, 1. 출현종및출현량 Month Temp. Sal. (ind./1,000 m 3 ) Fig. 3 Table 1. 5,590, ,344, ,693, , , , (12 ), (10 ), (9 ), (6 ) 5 (4 ). (Branchiostegus japonicus) , 16.3%. (Engraulis japonicus) , (11.9%), (Pseudolabrus eoethinus) (11.3%), (Goniistius zonatus) (6.9%), (Halichoeres tenuispinis) (6.4%), % Salinity (psu)

4 832 한송헌ㆍ김맹진ㆍ김준상ㆍ송춘복 Table 1. Species occurrence and number of individuals of fish eggs (ind./1,000 m 3 ) collected bimonthly in the coastal waters of Sagye, Jejudo Island from April 2009 to February 2010 Order Family Species Total no. Apr Jun Aug Oct Dec Feb of eggs (%) Cupeiformes Engraulidae Engraulis japonicus (11.9) Aulopiformes Aulopodidae Aulopus japonicus (0.6) Synodontidae Saurida wanieso (0.1) Trachinocephalus myops (3.8) Ophidiiformes Ophidiidae Hoplobrotula armata (1.2) Gadiformes Moridae Lotella phycis (0.7) Moridae sp (1.5) Scorpaeniformes Platycephalidae Platycephalus indicus (0.1) Perciformes Moronidae Lateolabrax japonicus (1.5) Serranidae Caprodon schlegelii (4.1) Callanthiidae Callanthias japonicus (4.9) Malacanthidae Branchiostegus albus (0.5) Branchiostegus japonicus (16.3) Sparidae Acanthopagrus schlegeli (1.0) Evynnis japonica (0.6) Sparus sarba (0.1) Mullidae Upeneus japonicas (0.3) Kyphosidae Girella punctate (0.8) Oplegnathidae Oplegnathus fasciatus (1.4) Cheilodactylidae Goniistius quadricornis (1.9) Goniistius zonatus (6.9) Labridae Halichoeres tenuispinis (6.4) Pseudolabrus eoethinus (11.3) Pseudolabrus sieboldi (5.7) Semicossyphus reticulatus (0.3) Stethojulis terina (3.4) Suezichthys gracilis (0.2) Xyrichtys dea (1.8) Pinguipedidae Pinguipedidae sp (0.1) Callionymidae Neosynchiropus ijimae (0.1) Repomucenus virgis (0.5) Trichiuridae Trichiurus japonicus (1.2) Scombridae Auxis rochei (1.1) Pleuronectiformes Paralichthyidae Paralichthys olivaceus (2.8) Pseudorhombus pentophthalmus (0.1) Pleuronectidae Eopsetta grigorjewi (1.0) Pleuronectiformes sp (0.1) Tetraodontiformes Diodontidae Diodon holocanthus (1.7) Unidentified sp. A (0.1) Unidentified sp. B (0.1) Unidentified sp. C (1.1) Unidentified sp. D (0.6) Unidentified sp. E (0.1) Total no. of eggs (%) 219 (3.9) 406 (7.3) 2,343 1,693 (41.9) (30.3) 254 (4.5) 254 (4.5) 5,590 (100.0) Number of taxa examined Number of species identified , non-appearance.

5 제주도사계연안어란의분자동정과격월별출현양상 833 Apr, 2009 Jun, 2009 Aug, , Oct, 2009 Dec, 2009 Feb, , ,000 1,000-2,000 >2,000 (ind./1,000 m) Fig. 3. Bimonthly abundance of fish eggs collected at the sampling location of the coastal waters of Sagye, Jejudo Island. Values in parentheses indicate the number of species (Callanthias japonicus) 4 98 (44.7%), 6 (Oplegnathus fasciatus) 76, % (38.9%) , 494 (29.2%) 137 (53.9%) (Caprodon schlegelii) 164 (24.3%). 생태지수의변동,, Fig , , , , , , , , , , , , 환경요인과의상관관계 Diversity Evenness Dominant Apr Jun Aug Oct Dec Feb Month Fig. 4. Diversity, evenness and dominance indices of fish eggs collected during this study.

6 834 한송헌ㆍ김맹진ㆍ김준상ㆍ송춘복 Table 2. Comparison between present and previous studies on the mean abundance and number of identified species of fish eggs Sampling area Sampling period (Sampling times, no. of location) Mean abundance of fish eggs No. of species (No. of taxa) Reference Yeongil Bay Feb-Dec, 2001 (4, 8) (9) Han et al., 2003 Saemangeum Jan-Aug, 2003 (5, 9-12) (8) Cha et al., 2004 Uljin Jan-Oct, 2002 (4, 5) (7) Han and Kim, 2007 Jejudo Island Aug, 2006-Jul, 2007 (12, 4) (43) Han et al., 2015 Sagyeoi Apr, 2009-Feb, 2010 (6, 1) (43) Present study Pearson s r (P<0.05), (P<0.05)., (P>0.05). 주요어종의출현시기및수온범위 (Table 1) (Fig. 2), , 6, 12, , , , 8, , ( ), 8. (Paralichthys olivaceus) , 6, , (Family) (29.1%) (7 ), , (28.2 ) 1, (14.9 ). (Lateolabrax japonicus), (Acanthopagrus schlegeli), (Girella punctata),, (Trichiurus japonicus), (Eopsetta grigorjewi)., ,, , , 고찰 PCR. COI 94.7% cytochrome b (cyt b) PCR 68.3% (Han et al., 2015). cyt b trna (glutamic acid trna 5' threonine proline trna 3' ) trna (Inoue et al., 2001) trna Thr. Ivanova et al. (2007) COI PCR 96.8%. COI Ivanova et al. (2007) 650 bp, Lee and Kim (2014a) 510 bp, Lee and Kim (2016) 515 bp. PCR-RFLP band prescreening 2-3 1,550 bp COI.. (Table 2)., 932, 574 (Han et al., 2003), 610 (Cha et al., 2004), 299 (Han and Kim, 2007), 506 (Han et al., 2015). (Kim et al., 2014) (kg/1,000 m 2 ) CPUE (Catch Per Unit Effort),,,

7 제주도사계연안어란의분자동정과격월별출현양상 , , (H =2.31), (53.9%) (H =1.18) , (Fig. 4). Han et al. (2015) 34 (43 ), Lee et al. (2006) 59, Go et al. (1991) 64.,,. 사사 (R ). References Cha SS, Chun SJ, Yang JS and Choi KW Species composition and abundance of ichthyoplankton in the waters of Saemangeum, Korea Korean J Ichthyol 16, Cocolin L, D Agaro E, Manzano M, Lanari D and Comi G Rapid PCR-RFLP method for the identification of marine fish fillets (seabass, seabream, umbrine, and dentex). J Food Sci 65, org/ /j tb10604.x. Dooley JJ, Sage HD, Clarke MA, Brown HM and Garrett SD Fish species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: application to detect white fish species in food products and an interlaboratory study. J Agric Food Chem 53, org/ /jf047917s. Go YB, Go GM and Kim JM Occurrence of fish larvae at Hamduck costal area, northern part of Cheju Island. Korean J Ichthyol 3, Han KH and Kim DG Quantitative variation and species composition of ichthyoplankton in coastal waters of Uljin, Korea. Korean J Ichthyol 19, Han KH, Hong JS, Kim YS, Jeon KA, Kim YS, Hong BK and Hwang DS Species composition and seasonal variations of ichthyoplankton in coastal waters of Yeongil Bay, Korea. Korean J Ichthyol 15, Han SH, Kim MJ and Song CB Molecular identification and distribution pattern of fish eggs collected around Jejudo Island. Korean J Ichthyol 27, Hebert PDN, Cywinska A, Ball SL and dewaard JR Biological identifications through DNA barcodes. Proc R Soc Lond B 270, rspb Inoue JG, Miya M, Tsukamoto K and Nishida M Complete mitochondrial DNA sequence of Conger myriaster (Teleostei: Anguilliformes): novel gene order for vertebrate mitochondrial genomes and the phylogenetic implications for anguilliform families. J Mol Evol 52, dx.doi.org/ /s Ivanova NV, Zemlak TS, Hanner RH and Hebert PDN Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes 7, Kim MJ, Han SH, Kim JS, Kim BY and Song CB Species composition and bimonthly changes of fish community in the coastal waters of Sagyeoi, Jeju Island. Korean J Ichthyol 26, Kimura M A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequence. J Mol Evol 16, org/ /bf Lee SJ, Go YB and Kim BJ Seasonal variation of species composition and distribution of fish eggs and larvae in the western part of Jeju Island, Korea. Korean J Ichthyol 18, Lee SJ and Kim JG. 2014a. Identification of Trichiurus (Pisces: Trichiuridae) eggs and larvae from Korea, with a taxonomic note. Fish Aquat Sci 17, FAS Lee SJ and Kim JG. 2014b. Molecular identification and morphological comparison of juveniles of two Collichthys species (Pisces: Sciaenidae) from the Yellow Sea. Kor J Fish Aquat Sci 47, KFAS Lee SJ and Kim JG Morphological characteristics and distribution of Pleuronectidae (Pisces) eggs in the western margin of the East Sea. Ocean Sci J 51, org/ /s McNaughton SJ Relationship among functional prop-

8 836 한송헌ㆍ김맹진ㆍ김준상ㆍ송춘복 erties of California grassland. Nature 216, dx.doi.org/ /216168b0. Patterton HG and Graves S DNAssist: the integrated editing and analysis of molecular biology sequences in windows. Bioinform Appl Note 16, org/ /bioinformatics/ Pielou EC The measurement of diversity in different types of biological collection. J Theor Biol 13, Ratnasingham S and Hebert PDN BOLD: The Barcode of Life Data System ( Mol Ecol Notes 7, Saitou N and Nei M The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4, Shannon CE and Weaver W The mathematical theory of communication. Univ Illinois Press, Urbana, U.S.A. 125pp. Tamura K, Stecher G, Peterson D, Filipski A and Kumar S MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30, org/ /molbev/mst197. Wang S, Bao Z, Zhang L, Li N, Zhan A, Guo W, Wang X and Hu J A new strategy for species identification of planktonic larvae: PCR-RFLP analysis of the internal transcribed spacer region of ribosomal DNA detected by agarose gel electrophoresis or DHPLC. J Plankton Res 28,

제주연안해역어란의분자동정과분포양상 285 Saitoh et al., 2009; Lelièvre et al., 2010). 이연구에서종동정에사용된 mitochondrial DNA 내 cytochrome b 유전자 (cyt b) 는해당단백질의구조와기능이잘알려져있으며어류의계

제주연안해역어란의분자동정과분포양상 285 Saitoh et al., 2009; Lelièvre et al., 2010). 이연구에서종동정에사용된 mitochondrial DNA 내 cytochrome b 유전자 (cyt b) 는해당단백질의구조와기능이잘알려져있으며어류의계 284 Korean 한송헌 Journal 김맹진 of 송춘복 Ichthyology, Vol. 27, No. 4, 284-292, December 2015 ISSN: 1225-8598 (Print), 2288-3371 (Online) Received: October 1, 2015 Revised: December 20, 2015 Accepted: December