156 Korean 고명훈 Journal 방인철 of Ichthyology, Vol. 28, No. 3, 156-163, September 2016 ISSN: 1225-8598 (Print), 2288-3371 (Online) Received: September 8, 2016 Revised: September 24, 2016 Accepted: September 26, 2016 한국고유종남방종개 Iksookimia hugowolfeldi ( 미꾸리과 ) 의난발생및초기생활사 고명훈 방인철 1, * 이화여자대학교에코과학부, 1 순천향대학교생명시스템학과 Egg Development and Early Life History of the Korean Southern Spine Loach, Iksookimia hugowolfeldi (Pisces: Cobitidae) by Myeong-Hun Ko and In-Chul Bang 1, * (Division of EcoScience, Ewha Womans University, Seoul 03760, Republic of Korea; 1 Department of Life Sciences and Biotechnology, Soonchunhyang University, Asan 31538, Republic of Korea) ABSTRACT Egg development and early life history of Korean southern spine loach, Iksookimia hugowolfeldi, were observed in the present study. Eggs were obtained after injecting females with 0.5 ml/kg of Ovarprim. Eggs were artificially fertilized using the dry method in the laboratory. Number of spawned eggs were 1,933±530 per individual. Mature eggs were slightly adhesive with light yellowish coloring, and they measured 1.35±0.03 mm in diameter. Hatching of the embryo occurred 56 h (50%) after fertilization at water temperature 25 C, and newly hatched larvae were averaged of 5.6±0.18 mm in total length. At 5 days after hatching, larvae averaged 7.8±0.31 mm in total length and their yolk sacs had been completely absorbed. Beginning at 15 days after hatching, the fish entered the juvenile stage and reached 13.2±0.87 mm in total length. At 100 days after hatching, the band patterns and external form of juvenile were similar to those of adults, and they averaged 49.2±4.29 mm in total length. Key words: Iksookimia hugowolfeldi, spine loach, egg development, early life history 서 미꾸리과 (Cobitidae) 어류는잉어목 (Cypriniformes) 에속하 며유라시아와아프리카북부에널리분포하는담수어류로 21 속 171 종이보고되었고 (Kottelat, 2012), 우리나라에는 5 속 16 종이서식한다 (Kim, 2009). 이중참종개속 (Iksookimia) 은우 리나라고유속으로참종개 I. koreensis 와부안종개 I. pumila, 왕종개 I. longicorpa, 남방종개 I. hugowolfeldi, 동방종개 I. yongdogensis, 북방종개 I. pacifica 6 종이지리적으로나뉘어 서서식하고있다 (Kim, 2009). 남방종개는 1993 년 Nalbant 에의해신종보고된우리나라 고유종으로영산강과탐진강, 서남부의남해로흐르는하천에 론 * Corresponding author: In-Chul Bang Tel: 82-41-530-1286, Fax: 82-41-530-1638, E-mail: incbang@sch.ac.kr 서식하는것으로알려져있으며, 외형적으로왕종개와유사하나첫번째반문이진하지않고체측중앙반문이배쪽까지가늘고길게이어져있어차이를보인다 (Nalbant, 1993; Kim, 1997; Kim and Park, 2007). 남방종개에관한연구는형태분석및핵형분석 (Choi, 2003), 생태 (Choi, 2003; Park, 2016), 난막구조 (Park, 1996; Park and Kim, 2001), 분자계통 (Kim et al., 2000; Šlechtová et al., 2008; Kwan, 2015; Perdices et al., 2016) 등이있으나아직까지난발생및초기생활사에대한연구는이루어지지않았다. 어류의난발생및초기생활사연구는종의산란기특징과난발달및자치어발달등의특유의생식전략등을분석하여종의특징을규명하고유사종과의분류학적유연관계를밝히는데적용되어왔으며 (Blaxter, 1974; Balon, 1985; Shimizu et al., 1998; Ko et al., 2015), 자원증강및멸종위기종의복원을위한인공증식기술확립의중요한기초연구로폭넓게연구되 156 http://www.fishkorea.or.kr
한국고유종남방종개의난발생및초기생활사 157 고있다 (ME, 2009; MLTM, 2010, 2011). 따라서본연구에서는아직까지조사되지않은우리나라고유종남방종개의난발생및초기생활사를조사하여생물학적특징을밝히고미꾸리과어류들과유연관계를논의하고자한다. 재료및방법 남방종개 Iksookimia hugowolfeldi는 2011년 6월 27일부터 7월 2일까지전라북도장성군북하면용두리의영상강상류북하천에서족대 ( 망목 4 4 mm) 를이용하여채집하였으며, 이후실험실로운반하여수온 23 로사육 관리하였다. 채집된개체중성숙한개체들을골라암 수모두 1 kg당 Ovaprim (Syndel, Canada) 0.5 ml를주사한후, 12시간경과후에암컷의복부를압박하여채란하고여기에수컷으로부터얻은정액을 Ringer solution에 100배희석시켜건식법으로수정시켰다. 산란수는개체별로모두계수하였으며, 난경은인공배란된난을무작위로 30개를선정하여 0.01 mm까지측정하였다. 수정란은직경 15 cm 패트리디쉬에분산수용하여난발생과 전기자어기를관찰하였으며, 이후수조 (20, 50, 100 L) 에순차적으로옮겨사육하면서후기자어기와치어기를관찰하였다. 난황흡수가끝난후기자어기부터부화후 30일까지는알테미아 (Artemia sp.) 유생을공급하였고, 31일이후부터는배합사료와실지렁이를순차적으로공급하여사육하였다. 사육용수는초기매일 1/2씩환수하다 30일이후부터순환여과식으로전환하였으며, 사육수온은 25 로관리하였다. 난발생과정과자치어의발달단계는 Kim (1997) 과 Kim et al. (2005) 을따라전기자어기, 후기자어기, 치어기로구분하였고, 발달과정은디지털카메라 (Olympus DP72, Japan) 가부착된해부현미경 (Olympus SZX9, Japan) 하에서관찰하고촬영하였다. 자 치어는부화직후부터부화후 100일까지각단계별무작위로 10개체를선별한후마취제 MS-222 (Sindel, Canada) 로마취하여관찰하고전장을측정하였다. 결과 1. 성숙개체크기및성숙란의특징채집된남방종개 Iksookimia hugowolfeldi의수컷 (n=3) 은 Table 1. Egg development of Iksookimia hugowolfeldi at water temperature 25 Stage Elaspsed Characters Fig. 1 Zygote period Insemination 00 h 00 min Sperm and egg are inseminated Swelling 00 h 20 min Swelling A Blastodisc 01 h 00 min Blastodisc B Cell cleave period 2 cells 01 h 30 min 2 blastodisc is cleavage C 4 cells 01 h 50 min 2-2 array of blastomeres D 8 cells 02 h 10 min 2-4 array of blastomeres E 16 cells 02 h 30 min 4-4 array of blastomeres F 32 cells 02 h 45 min 4-8 array of blastomeres G 64 cells 03 h 05 min 8-8 array of blastomeres H 128 cells 03 h 20 min 8-16 array of blastomeres Morula (256 cells) 03 h 40 min 16 regular tiers of blastomeres I Blastula 04 h 40 min Flattening produces an elliptical shape J Gastrula period Early gastrulation 08 h 20 min Early gastrulation (50% epiboly) K Middle gastrulation 11 h 00 min Middle gastrulation (75% epiboly) - Late gastrulation 12 h 30 min Late gastrulation (90% epiboly) L Embryoric period Formation of the embryo 14 h 00 min Formation of the embryo M 3-4 myotomes 15 h 00 min 3-4 myotomes, formation of optic vesicles N 9-10 myotomes 17 h 10 min 9-10 myotomes O 16-17 myotomes 19 h 30 min 17-18 myotomes, formation of auditory vesicles and Kupffer s vesicles P 28-30 myotomes 22 h 00 min 28-30 myotomes, disappear Kupffer s vesicles, formation of eye lens and specialization of brain Q Formation of heart 30 h 00 min Formation of heart R 48 h 00 min Hatching start Hatching period 56 h 00 min Hatching (50%) S 60 h 00 min Hatching complete
158 고명훈 방인철 A B C D E F G H I J K L M N O P Q R S Fig. 1. Egg development and hatching of Iksookimia hugowolfeldi at water temperature 25. Time required for each developmental stage is shown in Table 1. The bar indicate 1 mm. 전장 94~105 mm로 모두 정액을 가지고 있었고, 암컷 (n = 5) 은 전장 105~133 mm로 모두 산란을 하지 않았다. Ovaprim을 주사한 후 11~12시간에 모두 배란되었으며 난질은 모두 양호 1.35±0.03 (1.25~1.39) mm였다. 2. 난발생 과정 하였다. 산란된 성숙란은 밝은 노란색의 분리침성난으로, 산란 수 (n = 5)는 1,933±530 (1,350~2,680)개였고, 난경 (n = 30)은 남방종개의 수정란은 수정 20분 후에 물을 흡수하여 직
한국고유종 남방종개의 난발생 및 초기생활사 L1 L7 L2 L8 L3 L9 L4-1 L10 L4-2 L11 L5 L12 L6-1 L13 L6-2 L14 159 Fig. 2. Larval and juvenile development of Iksookimia hugowolfeldi. The bar indicates 1 mm. L1: 0 day, pre-larva, total length 5.6±0.18 mm (mean±sd); L2: 1 day, 6.5±0.21 mm; L3: 2 days, 7.0±0.33 mm; L4: 3 days, 7.2±0.24 mm; L5: 4 days, 7.4±0.24 mm; L6: 5 days, post-larva, 7.8± 0.30 mm; L7: 7 days, 8.3±0.12 mm; L8: 12 days, 11.4±0.78 mm; L9: 15 days, juvenile, 13.0±0.87 mm; L10: 20 days, 18.6±0.0.85 mm; L11: 30 days, 23.6±1.44 mm; L12: 50 days, 30.5±3.09 mm; L13: 70 days, 39.5±2.83 mm; L14: 100 days, 49.2±4.3 mm. 4세포기는 1시간 50분 후에 경할을 통하여 형성되었고 (Fig. 경 1.9±0.03 mm (n = 30)으로 팽창하였다 (Fig. 1A). 수정 후 1시간 후에 세포질이 동물극 (animal pole)쪽으로 끌려 배반 1D), 8세포기는 2시간 10분 후에 난할을 통하여 (Fig. 1E), 16 시간 30분 후에 배반에 난할이 일어나 형성되었다 (Fig. 1C). 1F). 32세포기는 2시간 45분 후에 (Fig. 1G), 64세포기는 3시 (blastodisc, 1세포기)이 형성되었으며 (Fig. 1B), 2세포기는 1 세포기는 2시간 30분 후에 경할을 통하여 형성되었다 (Fig.
160 고명훈 방인철 Table 2. Comparisons of early life historical characteristics of the genus Iksookimia species Stages Species I. hugowolfeldi I. longicorpa I. koreensis I. pumila I. pacifica Spawning (maturity) period July June June~July June~July July Total length of matured female (mm) 105~133 96~150 102~127 67~110 116±22.0 (n=5) (n =30) (n=5) (n=16) (n=10) Matured egg Number of spawned eggs 1,933±530 1,992±925 2,020±518 1,017±334 (1,350~2,680) (717~3,862) (1,339~2,635) (352~1,440) 2,968±502 Egg size (mm) 1.35±0.03 1.53±0.07 1.40±0.02 1.27±0.02 (1.25~.39) (1.4~1.7) (1.27~1.54) (1.21~1.36) 1.09±0.04 Color of eggs light yellowish light yellowish light yellowish light yellowish faint white Egg development Water temperature ( ) 25 23~25 23 23 21~24 Time until blastodisc 1 h 40 min 50 min 45 min 45 min Time until morula (256 cell) 3 h 40 min 3 h 3 h 3 h 20 min 3 h 30 min Time until blastula 4 h 40 min 4 h 4 h 5 min 4 h 10 min 4 h 30 min Time until early gastrulation (50% epiboly) 8 h 20 min 7 h 10 min 7h 5 min 7 h 40 min 8 h 30 min Time until formation of the embryo 14 h 15 h 13 h 40 min 14 h 40 min 15 h 10 min Time until formation of auditory vesicles 19 h 30 min 20 h 20 h 21 h 30 min 17 h 50 min Time of beginning of heart beating 30 h 30 h 31 h 33 h 30 h Time of hatching (50%) 48~60 (56) h 50~72 h 45~60 (50) h 47~55 (52) h 48 h Larva and juvenile Hatching size (mm) 5.6±0.18 (5.3~.9) 5.1±0.23 (4.8~5.2) 4.7±0.14 (4.6~5.0 ) 4.7±0.21 (4.5~4.9) 2.9±0.05 (2.8~2.9) Apparence period (day) of outer gill filament 0~5 2~5 1~5 1~5 2~7 Days until postlarva 5 5 5 5 7 Days until juvenile 15 25 17 17 26 References Present study Ko et al., 2009 Kim, 1978; Ko et al., 2012 Ko et al., 2013 Lee et al., 2011 간 5분후에형성되었으며 (Fig. 1H), 128세포기는 3시간 20 분, 상실기 (morula) 인 256세포기는 3시간 40분후에형성되었고 (Fig. 1I), 이후난할을계속하여 4시간 40분후에는포배기 (blastula) 가형성되었다 (Fig. 1J). 8시간 20분후에낭배기 (gastrula) 가형성되어식물극 (vegetal pole) 쪽으로덮기시작하였으며 (Fig. 1K), 이후 11시간후에낭배중기에 (75%), 12시간 30분후에는 90% 이상을덮어낭배말기에도달하였다 (Fig. 1L). 14시간후에는원구 (blastopore) 가폐쇄되어배 (embryo) 가형성되었으며 (Fig. 1M), 15시간후에는근절 (myotomes) 이 3~4개가생기고안포 (optic vesicle) 가형성되었다 (Fig. 1N). 17시간후에는근절이 9~10개가형성되었으며 (Fig. 1O), 19시간 30분후에는근절이 16~17개이고이포 (auditory vesicle) 와 Kupper s vesicle이형성되었다 (Fig. 1P). 이후꼬리가난황에서분리되면서움직이기시작하였으며, 22 시간후에는근절이 28~30개가생기고눈의렌즈가형성되며뇌가분화하였고 Kupper s vesicle이사라졌다 (Fig. 1Q). 30시간후에는심장이형성되어뛰기시작하였다 (Fig. 1R). 48시간 후부터꼬리를이용하여난막 (chorion) 을뚫고부화하였고, 56 시간후에 50% 가, 60시간이내에모두부화하였다 (Fig. 1S). 3. 자어와치어의발달 1) 전기자어기부화직후의자어는전장 5.6±0.18 (5.3~5.9) mm (n=10) 로몸은무색으로입과항문은열리지않았고외새 (external gill) 1쌍이나타났다. 가슴지느러미가형성되기시작하였으며앞쪽에구형의난황이있고몸의중앙과뒤쪽에막지느러미 (embryo fin) 가형성되었다 (Fig. 2L 1). 1일후에는전장 6.5± 0.21 mm (n =10) 로눈은검게착색되었고앞쪽의난황이흡수되어거의 1자로바뀌었다. 외새는 5쌍이나타났고몸에흑색포가침적되기시작하였으며수염 1쌍이나타나기시작하였다 (Fig. 2L 2). 2일후에는전장 7.0±0.33 mm (n =10) 로난황이 1/2 이상흡수되었고온몸에흑세포가침적되었으며뒤쪽의막지느러미가넓어졌다 (Fig. 2L 3). 3일후에는전장 7.2±0.24
한국고유종남방종개의난발생및초기생활사 161 mm (n =10) 로외새가짧아졌고수염이안경만큼길어졌으며가슴지느러미는두장보다길어졌다 (Fig. 2L 4). 4일후에는전장 7.4±0.24 mm (n =10) 로난황이대부분흡수되었고수염은 3쌍이관찰되었으며꼬리지느러미원기가나타나기시작하였다. 또한입과항문이열리면서처음으로먹이 ( 알테미아부화유생 ) 를섭식하였다 (Fig. 2L 5). 2) 후기자어기부화후 5일째에는전장 7.8±0.30 mm (n =10) 로난황이모두흡수되었고외새는짧아져아가미뚜껑속으로모두들어갔으며등지느러미와뒷지느러미원기가나타났다 (Fig. 2L 6). 7일후에는전장 8.3±0.12 mm (n=10) 로꼬리지느러미기조는 7~10개가관찰되었으며수염이길게신장되었다 (Fig. 2L 7). 12일후에는전장 11.4±0.78 mm (n =10) 로등지느러미기조가 2~4개, 꼬리지느러미기조는 12~15개가관찰되었다 (Fig. 2L 8). 3) 치어기부화후 15일째에는전장 13.0±0.87 mm (n =10) 로등지느러미기조 7~8개, 뒷지느러미기조 5~7개가되면서치어기로이행하였고두부에는입과눈으로연결되는검은줄이나타났으며체측증앙부에흑색포가모이기시작하였다 (Fig. 2L 9). 20일후전장 18.6±0.85 mm (n =10) 로체측상부와중앙부에흑소포들이모여 7~10개의반문을형성하였고미병부에뚜렷한검은점이나타났으며꼬리지느러미에검은줄 1개가형성되었다 (Fig. 2L 10). 30일후전장 23.6±1.44 mm (n =10) 로막지느러미가축소되면서미병부부근에만있었고등지느러미에 1줄, 꼬리지느러미에 2줄의무늬가나타났다 (Fig. 2L 11). 50일후전장 30.5±3.09 mm로등쪽과체측중앙의반문은커지고뚜렷해졌으며등지느러미 2줄, 꼬리지느러미에 3줄의무늬가있었다 (Fig. 2L 12). 70일후전장 39.5±2.83 mm (n =10) 로체측중앙부에긴타원형의반문이 8~12개가나타나고꼬리지느러미에 3~4줄의무늬가있었다 (Fig. 2L 13). 100후에는전장 49.2±4.3 mm (n =10) 로체측상부에 10~12개의반문이있고체측중앙부에배까지이어지는가늘고긴반문 8~12개가나타났으며배쪽은은백색을띄어전체적인외형이비교적성어와유사하였다 (Fig. 2L 14). 고찰 미꾸리과 (Cobitidae) 의참종개속 Iksookimia는 1993년 Nalbant에의해신속으로기재된속으로가슴지느러미가새의부리모양으로뾰족하며체측중앙의반문은횡으로길게신장되어있어기름종개속 Cobitis, 미꾸리속 Misgurnus, 새코미꾸리속 Koreocobitis, 좀수수치속 Kichulchoia속과구별되며 6종 이보고되어있다 (Nalbant, 1993; Kim, 1997; Kim, 2009). 이중난발생및초기생활사에관한연구는왕종개 Iksookimia longicorpa (Ko et al., 2009) 와북방종개 I. pacifica (Lee et al., 2011), 참종개 I. koreensis (Ko et al., 2012), 부안종개 I. pumila (Ko et al., 2013) 4종에대해보고되어있으며, 본종과비교하면 Table 2와같다. 본조사에서남방종개는 6월말에서 7월초에채집된개체가성숙되어있었으나모두산란하지않아산란기는 7월로추정되어 Choi (2003) 와 Park (2016) 의보고와비교적유사하였으며, 같은속의왕종개와참종개, 부안종개, 북방종개등의산란시기 (6월 ~7월 ) 와도대체로비슷하였다. 암컷의전장범위는 105~133 mm로참종개와비교적유사하였으며, 성숙란은밝은노란색으로왕종개와참종개, 부안종개와동일하였으나회색의북방종개와는차이를보였다. 배란된난경은 1.35± 0.03 mm로참종개 (1.40±0.02 mm) 와비교적유사하나왕종개 (1.53±0.07 mm) 보다는작고부안종개 (1.27±0.02 mm), 북방종개 (1.09±0.04 mm) 보다는큰편이었다. 포란수는 1,933± 530개로왕종개 (1,992±925개) 및참종개 (2,020±518개) 와비슷하나부안종개 (1,017±334개) 보다는많고북방종개 (2,968±502개) 보다는적은편이었다. 난발달의각단계별이행시간을근연종과비교하여보면, 남방종개는배반 (blastodisc) 부터낭배기 (gastrulation) 까지는근연종보다 40~60분늦었지만이후배 (embryo) 발달단계부터는비교적유사한경향을보였고, 부화시간은 56 (48~60) 시간으로왕종개 (50~72시간) 와참종개 (50시간), 부안종개 (52 시간 ) 와비슷하였으나북방종개 (48시간) 보다는느린편이었다. 부화직후자어크기는전장 5.6±0.18 mm로왕종개 (5.1± 0.23 mm) 와비교적유사하였지만참종개 (4.7±0.14 mm) 와부안종개 (4.7±0.21 mm), 북방종개 (2.9±0.05 mm) 보다는큰편이었다. 비슷한수온 (23~25 ) 에서유사한먹이 ( 알테미아, 배합사료등 ) 로사육된참종개속의부화후발달단계를비교하면, 난황이흡수된후기자어기로의이행은남방종개가부화후 5일로왕종개와참종개, 부안종개와동일하였으나, 북방종개 7일보다는빠른편이었고, 치어기로의이행시기는 15일로나타나참종개 (17일) 와부안종개 (17일) 와비교적유사하였으나왕종개 (25일) 와북방종개 (26일) 보다는빠른편이었다. 미꾸리과어류중미꾸리속 (Uchida, 1939; Kim et al., 1987) 과기름종개속 (Okada, 1959-1960; Saitoh and Hosoya, 1988; Shimizu et al., 1998; Song et al., 2008; Ko and Park, 2012; Ko and Won, 2015), 참종개속 (Ko et al., 2009, 2012, 2013; Lee et al., 2011), 새코미꾸리속 (Song et al., 2009), 좀수수치속 (Kim and Lee, 1995; Ko et al., 2014) 등은발달단계중전기자어기 (pre-larva stage) 에외새 (external gill) 가나타나지만 Niwaella delicata (Suzuki, 1966) 는외새가나타나지않아다른속어류
162 고명훈 방인철 와차이를보였다. 외새의출현기간은남방종개가부화 0 일부 터 4 일까지로 5 쌍이관찰되어참종개속의왕종개와참종개, 부안종개 (Ko et al., 2009, 2012, 2013) 와거의동일하였으나 2 일부터 7 일까지나타나는북방종개와는차이를보였으며 (Lee et al., 2011), 그외미꾸리과어류에서는기름종개속의기름종 개 C. hankugensis 와줄종개 C. tetralineata, 미호종개 C. choii 와비교적유사하였다 (Song et al., 2008; Ko and Park, 2012; Ko and Won, 2015). 따라서남방종개의난발생및초기생활사특징은왕종개와 가장가깝고그다음으로참종개와부안종개와근연관계로나타났으나북방종개와는난경과부화직후자어크기, 외새출현시기, 후기자어이행시기등에서큰차이를보였다. 최근분자계통학적연구가진행되었는데, Šlechtová et al. (2008) 과 Perdices et al. (2016) 은참종개속어류가다계통으로보고하였으나 Kwan (2015) 은참종개속이단계통으로묶이고, 남방종개는왕종개와동방종개와가장근연관계이고그다음은참종개와부안종개이며북방종개가가장먼근연관계로보고하여본결과는비교적 Kwan (2015) 의계통학적유연관계와유사하였다. 요 한국고유종남방종개 Iksookimia hugowolfeldi의난발생및초기생활사를밝히기위하여조사를실시하였다. 성숙한개체복강에 Ovaprim (0.5 ml/kg) 을주사하여 12시간경과후복부압박법으로채란하여건식법으로인공수정시켰다. 산란된난은평균 1,933±530개로, 약한점착성을띤밝은노란색의분리침성난이었으며, 난경은 1.35±0.03 mm였다. 수온 25 에서수정후 56시간 (50%) 에부화하였으며, 크기는전장 5.6±0.18 mm였다. 부화후 5일에는전장 7.8±0.31 mm로난황을모두흡수하여후기자어기로넘어갔으며, 부화후 15일에는전장 13.2±0.87 mm로지느러미기조가모두정수가되어치어기로이행하였다. 부화후 100일에는전장 49.2±4.29 mm로성장하고외부형태및체측반문이성어와비교적유사하였다. 약 REFERENCES Balon, E.K. 1985. Early Life Histories of Fishes: New developmental, ecological and evolutionary perspectives. Dr. W. Junk Publisher, Dordrecht, 280pp. Blexter, J.H.S. 1974. The Early Life History of Fish. Spring-Verlag, Berlin, 765pp. Choi, E.K. 2003. Biology of the southern spined loach, Iksookimia hugowolfeldi (Pisces, Cobitidae). Master Thesis, Chonbuk National University, 42pp. (in Korean) Kim, I.S. 1997. Illustarated Encyclopedia of Fauna & Flora of Korea. Vol. 37 Freshwater Fishes. Ministry of Education. Youngi 629pp. (in Korean) Kim, I.S. 2009. A review of the spined loaches, family Cobitidae (Cypriniformes) in Korea. Korean J. Ichthyol., 21(Suppl.): 7-28. Kim, I.S. and E.H. Lee. 1995. Studies on early embryonic development of Niwaella multifasciata (Pisces: Cobitidae). Korean J. Limnol., 28: 455-462. (in Korean) Kim, I.S. and J.Y. Park. 2007. Freshwater Fishes of Korea. Kyohak Publishing, Seoul 465pp. (in Korean) Kim, I.S., Y. Choi, C.L. Lee, Y.J. Lee, B.J. Kim and J.H. Kim. 2005. Illustrated Book of Korean Fishes. Kyohak Publishing, Seoul, 615pp. (in Korean) Kim, S.Y., I.S. Kim, K.Y. Jahng and M.H. Chang. 2000. Molecular phylogeny of Korean loaches inferred from mitochondrial DNA Cytochrome b Sequences. Korean J. Ichthyol., 12: 223-229. Kim, Y.U., Y.S. Park and D.S. Kim. 1987. Development of eggs, larvae and juveniles of loach, Misgurnus mizolepis Günther. Bull. Korean Fish. Soc., 20: 16-23. (in Korean) Ko, M.H. and I.C. Bang. 2014. Spawning character and early life history of the endangered Korean dwarf loach, Kichulchoia brevifasciata (Teleostei: Cobitidae). Korean J. Ichthyol., 26: 89-98. (in Korean) Ko, M.H., J.Y. Park and I.S. Kim. 2009. Development of eggs and early life history of Iksookimia longicorpa (Pisces: Cobitidae) from Nakdong River of Korea. Korean J. Ichthyol., 21: 15-22. (in Korean) Ko, M.H., S.Y. Park and I.C. Bang. 2012. Egg development and early life history of Korean spined loach, Iksookimia koreensis (Pisces: Cobitidae). Korean J. Limnol., 45: 93-101. (in Korean) Ko, M.H., S.Y. Park and I.C. Bang. 2013. Egg development and early life history of the endangered Korean spine loach, Iksookimia pumila (Pisces: Cobitidae). Korean J. Ichthyol., 25: 65-73. (in Korean) Ko, M.H. and Y.J. Won. 2015. Egg development and early life history of the Korean spine loach, Cobitis tetralineata (Pisces: Cobitidae). Korean J. Ichthyol., 27: 95-103. (in Korean) Kottelat, M. 2012. Conspectus Cobitidum: an inventory of the loaches of the world (Teleostei: Cypriniformes: Cobitoidea). Raffles Bull. Zool., 26(Suppl.): 1-199. Kwan, Y.S. 2015. Molecular phylogenetic and population genetic studies of the freshwater fish family Cobitidae (Teleostei: Cypriniformes) in Korea. Doctoral Thesis, Ewha Womans University, 143pp. Lee, W.O., K.H. Kim, J.M. Baek, Y.J. Kang, H.Z. Jeon and C.H. Kim. 2011. Embryonic development and early life history of the northern loach, Cobitis pacifica (Pisces: Cobitidae). Korean J. Limnol., 44: 1-8. (in Korean) ME (Ministry of Environment). 2009. Development of Genetic
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