Population Genetic Structure of Tropical eel Anguilla bicolor in Indonesia Water

habitat or both, during their life cycle Aoyama 2009, questionning the origin of species distinction. Most of freshwater eels are considered to form randomly mating panmictic populations. The mechanism producing panmixia is that individual from different area spawn in common area in the ocean Avise et al. 1986. Two or more species have been regarded as completely panmictic, each comprising a single and randomly mating population A. anguilla and A. rostrata as temperate species; A. borbneensis at least, as tropical species. However, recent evidence from studies of microsatellite variation of European eel has challenged this model Wirth and Bernatchez, 2001; Daemen et al. 2001; Maes Volckaert 2002. Wirth and Bernatchez 2001 found that populations of European eel show slight but significant, genetic differentiation and genetic distance between population, this was interpreted as geographical heterogeneity and geographic distance. This implies restricted gene flow and lack of random mating between eels from different population. But this hypothesis has then been contreadicted by considering that the observed disequilibrium should be due to temporal differences between samples Dannewitz et al. 2005 ; Maes et al. 2006; Pujolar et al. 2006. This question is still discussed. Ege 1939 has described A. bicolor as composed of two subspecies, A. bicolor bicolor in the Indian Ocean and A. bicolor pacifica in the western Pacific Ocean and the sea around the northern part of Indonesia. Allopatric distribution has led to differences in the morphology of both subspecies, Ege 1939 noticed slight differences in their means and ranges of total number of vertebrae. Indonesia is an important biogeographic crossroad for ichthyofauna, the western part of Indonesia is connected with Indian Ocean and the eastern one was connected with the Pacific Ocean. Concerning the widespread distribution of A. bicolor, this exceptional geographic distribution and structure made this species an important model for eel biology understanding. While investigated at its whole distribution scale Minegishi et al. 2011, nothing is known in Indonesian waters, such as the exact distribution and lineages differentiation among Indonesian rivers. Beside, this species was also an important commercial fish because of its distribution and abundance. In recent years, scientists have begun using microsatellites as a way to study within species evolution. Microsatellites are widely used as markers for studying genetic mapping, population structures and evolutionary genetics Whirt 55 and Bernatchez 2001. This aims at revealing the genetic connectivity and population structure of shortfinned A. bicolor in Indonesian waters using microsatellites. Material and Methods Specimens A total of 180 specimens of A. b. bicolor were collected from ten sampling stations along Indonesian waters that represent the sea connecting Pacific Ocean and Indian Ocean. As follow, were considered here: Ace 24 individuals, Padang 24, Bali 24, Pelabuhan Ratu 24, Obi 7, Poigar 4, Donggala 18, Pangandaran 21, Bengalon 11 and Bengkulu 23 Figure 9 and Table 14. All specimens were collected between 2008 and 2011. Total genomic DNA was extracted by using gSYNC Mini Kit Tissue Geneaid. The protocol, according to the manufacturer’s recommendations, is as follow: approximately 25 mg of eel tissue were incubated with lysis buffer and Proteinase-K at 60 o C for 1 hour or until lyses was completed, and then incubated with a second lysis buffer at 70 o C. The next steps were binding DNA with ethanol and separate DNA from undesirable material by using GD column. The last step was washing the DNA twice by different wash buffers. The genomic DNA was eluted by Nuclease Free-Water. The quantity of DNA was checked by electrophoresis. Figure 9 Sampling location of “inter-ocean” Indian Ocean with black circles and Pacific Ocean red circle 56 Table 14 List of specimen used for microsatellite analyses in the present study Sampling Location Code Sampling date No specimen Indian Ocean Estuary of Tadu, Aceh Ace January 2010 24 Estuary of Bungus River, Padang Pad August 2008 24 Estuary of Ketaun River, Bengkulu, Sumatera Ben December 2009 23 Estuary of Cimadiri, Pelabuhan Ratu, Java Pel August 2009 24 Estuary of Pangandaran, Java Pang June 2009 21 Estuary of Mengereng River, Bali Bal June 2009 24 Pacific Ocean Estuary of Bengalon River, Borneo Beng October 2010 11 Estuary of Donggala River, Celebes Dong October 2010 18 Obi Island, Maluku Obi November 2011 7 Estuary of Poigar, North Celebes Poig January 2012 4 Total specimens 180 Microsatellite genotyping Eight microsatellite loci, which have been published for other anguillid species, were genotyped as follow Table 15. Polymerase chain reactions PCR were performed on a Mastercycler Eppendorf, Hamburg, Germany in a 10 µl reaction volume containing 1,5 mM MgCl 2 , 0,2 mM of each dNTP, 1µ l 10x PCR buffer Promega, Fitchurg WI, USA, 0,5 µM of forward and reverse fluorescent-labelled primers, 0,2 units of Taq polymerase, and 2,0 µl of template DNA. Amplification parameters were 35 cycles of denaturation at 95 o C for 30 s, annealing for 30 s see Table 2 for annealing temperatures and extension at 72 o C for 30 s after heating at 95 o C for 30 s. Labelled PCR product were electrophoresed with a DNA ladder Promega on an 8,0 acrylamide gel, which was later scanned on a FMBIO II Multi-View Hitachi, Tokyo, Japan scanner. Fragment size were determined using the software FMBIO Analysis 8,0 Hitachi. Each molecue size produced by the software is then assimilated to an allele after checking the gel by eyes Statistical Analyses Most statistical analyses were performed using the program GENETIX 4.05 Belkhir et al. 1998. The software GENETIX 4.05 was used for two types of classical parameters calculations: i calculation of the number of alleles, allele 57 58 frequencies, observed heterozygosity Ho, expected heterozygosity He for each locus in each locality for all samples, ii estimation of Fis Wright 1965 and its statistical significance following Weir and Cockerham 1984 and iii the linkage disequilibrium among the loci analyses for all samples in this study significance estimated with 5000 permutation test. Then calculation of the exact test deviations from Hardy-Weinberg equilibrium HWE conducted under POPGENE 32. The genetic differentiations between localities and Oceans were characterized using hierarchical F-statistic theta Fst of Wright and genetic distance characterized under Nei 1972 as implemented in POPGENE 32. The presence of intraspecific genetic structure was tested using the model-based clustering method of Pritchard et al. 2000 implemented in STRUCTURE VERSION 2.1 for each value of K, which is the number of genetically distinct populations. The Markov chain Monte Carlo scheme was run with a burn-in period of 5000 steps and a chain length of 50000 replicates following the admixture model. Result Eight pairs of primer were tested and seven of them gave easily interpretable and highly polymorphic patterns. The PCRs were carried out using the annealing temperature proposed in the original paper for each pair of primers. Alleles frequencies at each locus are presented in Annex 4. The linkage disequilibrium LD allow to detect non-random associations of alleles at two or more loci. LD was calculated only on samples of more than 15 specimens, three populations Obi, Poigar and Bengalon which less than 15 specimens were not included in this analyse. There is no linkage disequilibrium between genotypes of the seven microsatellite loci among seven populations, indicating that all loci are independent. Linkage disequilibria were observed at p0,05 for seven pairs of loci. A total 172 alleles were observed across seven microsatellite loci with a number by locus ranging from 3 in AjTR 12 to 22 in Aro63 for all populations. The population parameters at the 7 microsatellite loci is presented in Table 16. Expected He and observed Ho heterozygosities of each locus range from 0,594 to 0,921 and from 0,250 to 1,000 respectively. Significant deviation from Table 15. List locus that using during this study Locus Name Repeat motifs Primer Name Primer sequence Range bp Primer labeling Anneling temperature oC Reference FAro054 CTCAACTCCAGCACACTGGA RAro054 ACAAAATAGCTCCGTAACAC FAro95 GGCTGTTATTCTGGACGTCG RAro95 CCCTAAGTATCCTACATACAG FAro063 CCAGATACCTTGACAACGGC RAro063 TCAAGAGCTTCCTGACCCTC FAjTR04 CACCCTTGCCCTATTTTGATA RAjTR04 GCGTAGTCATGATCACCTGT FAjTR12 AACGTTAGTCCCTAGGTTCC RAjTR12 TAAGGGTGTTATATGTTCAG FAjTR45 GCGCATGGAGAACTCTAAT RAjTR45 CAATAGAGTGAGGACAGTAGA FAjTR37 AGACCTTATGTCACCTTATGCT RAjTR37 AAGATGTTAAATTCAATTGTGC FAJMS10 TGTCTAACACTAAGAAAAGGAGAGG RAJMS10 GGCTGCCAGTATCTTCTCAAAG Tseng et al 2001 Wirth Bernatchez 2001 Wirth Bernatchez 2001 Ishikawa et al 2001 Ishikawa et al 2001 Ishikawa et al 2001 Wirth Bernatchez 2001 Ishikawa et al 2001 AjTR-04 GT 15 167-217 5-CY3 55---57 Aro054 136-200 5-fluorescein 55---58 AjTR-45 CT 3 T TC 2 CCT 6 141-165 5-CY5 55---60 AjTR-12 AG 14 154-214 5-fluorescein 55 AjTR-37 TG 14 188-202 5-CY3 55 Aro95 86-136 5-CY5 55---57 Aro063 160-230 5-CY5 58 AJMS-10 GA 17 137-175 5-fluorescein 58 59 Hardy-Weinberg equilibrium HWE were detected in 26 out of 70 cases 7 loci x 10 populations. AjTR 04 and Aro 63 were more polymorphic than the five other loci, with higher average numbers of alleles, 13.7 and 13.6 respectively and average of heterozygote were also higher than other loci, there are 0.885 and 0.886 respectively. In addition the meaningful of locus of AjTR04 and Aro63 also was demonstrated with the significant of deviation HWE, there were two of ten populations non-significance of deviation HWE with both loci above. The ten samples are presented here in two groups: the Indian Ocean IO and the Pacific Ocean PO groups. The Indian Ocean group is represented by Aceh, Padang, Bengkulu, Pelabuhan Ratu, Pangandaran and Bali and the Pacific Ocean group by Donggala, Poigar, Bengalon and Obi. These two clusters correspond respectively to A. b. bicolor Indian Ocean part and A. b. pacifica inter-oceans zone. The average number of alleles, He and Ho of Indian Ocean groups were from 13 to 15, from 0,860 to 0,891 and from 0,702 to 0,777 respectively, while group of Pacific Ocean were from 5 to13, from 0,767 to 0,873 and from 0,594 to 0,671 respectively. That means that the populations from Indian Ocean group are globally more polymorphic than those belonging to the Pacific Ocean group. F ST values, giving the genetic divergences between populations that revealed geographical differentiation and structuration. F-statistic F IS , F IT and F ST analysis of all locus among ten populations we present in Table 17. Inbreeding rate between F IS and within F IT populations are 0.194 19.4 and 0.209 20.9 respectively, whereas genetic differentiation F ST between population is 0.018 1.8. Accordance to Wright 1978 has suggested the guidelines for the interpretation of Fst that the range value of F ST between 0 to 0,05 indicated little genetic differentiation. The same result was also showed by the structuration under STRUCTURE programme analyses, there is no structure among 10 population of A. bicolor in Indonesia water Figure 10. 60 Table 16. Genetic variability of population parameters at 7 microsatellite loci on tropical eel in Indonesia water Microsatellite locus no allele Parameters observed Ace n=24 Pad n=24 Ben n=22 PelR n=24 Pang n=21 Bal n=23 Obi n=7 Poig n=4 Dong n=18 Beng n=11 Average per locus AjTR12 No. of alleles 8 15 15 13 13 15 7 3 8 9 11 23 Allele range 150-170 146- 186 140- 180 146- 172 142- 178 142- 174 146- 174 156- 170 146- 164 146- 168 140-186 He, 0,835 0,885 0,862 0,880 0,855 0,862 0,796 0,594 0,753 0,855 0,818 Ho, 0,636 0,750 0,864 0,667 0,810 0,783 0,857 0,250 0,389 0,455 0,646 H-W test n.s n.s n.s n.s n.s AjTR45 No. of alleles 8 10 10 8 10 8 6 6 9 7 8,2 11 Allele range 135-149 135- 153 135- 155 137- 151 135- 155 135- 149 137- 151 137- 151 135- 151 137- 151 135-155 He, 0,833 0,846 0,838 0,848 0,816 0,824 0,694 0,750 0,825 0,744 0,802 Ho, 0,792 0,958 0,818 1,000 0,850 0,864 0,714 0,750 0,765 0,636 0,815 H-W test n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s Aro63 No. of alleles 16 17 15 22 17 15 7 6 12 9 13,6 34 Allele range 168-22 172- 214 186- 230 160- 230 166- 230 170- 222 182- 208 188- 204 182- 224 182- 208 160-230 He, 0,898 0,921 0,906 0,933 0,910 0,914 0,806 0,813 0,909 0,845 0,885 Ho, 0,750 0,583 0,619 0,636 0,500 0,304 0,500 0,500 0,067 0,700 0,516 H-W test 0,0 0,0 n.s 0,0 AjTR37 No. of alleles 14 14 14 13 15 16 7 6 18 11 12,8 23 Allele range 188-218 188- 222 186- 220 188- 214 188- 218 188- 218 192- 222 188- 210 188- 234 192- 230 186-234 He, 0,879 0,888 0,878 0,885 0,900 0,912 0,816 0,813 0,923 0,888 0,878 Ho, 0,826 0,708 1,000 0,917 0,905 0,958 0,857 1,000 0,833 0,909 0,891 H-W test n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s Aro54 No. of alleles 16 16 12 12 13 9 5 5 12 12 11,2 26 Allele range 148-194 136- 184 148- 184 148- 178 146- 194 150- 166 156- 172 156- 166 150- 200 152- 180 136-200 He, 0,902 0,893 0,830 0,687 0,859 0,815 0,781 0,778 0,897 0,884 0,833 Ho, 1,000 0,625 0,714 0,792 0,750 0,650 0,750 0,667 0,875 0,909 0,773 H-W test n.s n.s n.s n.s n.s n.s n.s n.s n.s Aro95 No. of alleles 16 17 15 14 13 12 4 6 11 8 11,6 24 Allele range 94-130 86-122 100- 136 86-120 96-124 98-120 104- 114 92-118 96-120 96-130 86-136 He, 0,885 0,888 0,896 0,869 0,893 0,865 0,694 0,813 0,879 0,826 0,851 Ho, 0,958 0,875 0,682 0,696 0,619 0,833 0,286 1,000 0,563 0,455 0,697 H-W test n.s n.s 0,0 n.s n.s n.s n.s n.s AjTR04 No. of alleles 13 16 15 17 18 14 8 6 18 12 13,7 31 Allele range 187-225 177- 227 183- 221 181- 225 177- 229 183- 225 187- 233 187- 229 183- 241 179- 247 177-247 He, 0,891 0,918 0,899 0,921 0,904 0,899 0,806 0,813 0,926 0,888 0,886 Ho, 0,478 0,417 0,455 0,708 0,762 0,625 0,429 0,500 0,667 0,636 0,568 H-W test 0,0 0,0 0,0 n.s n.s 0,0 Average He 0,875 0,891 0,873 0,860 0,877 0,870 0,770 0,767 0,873 0,847 Ho 0,777 0,702 0,736 0,774 0,742 0,717 0,628 0,667 0,594 0,671 No.of alleles 13 15 14 14 14 13 6 5 13 10 Note; Alelle range, n = number specimen on population, number under the loci name are the total number allele found, He = heterozygosities expected, Ho = heterozygosities observed. 61 Table 17. F IS , F IT and F ST for 7 loci among 10 population of A. bicolor in Indonesia water Locus F IS F IT F ST AjTR12 0,210 ± 0,051 0,229 ± 0,055 0,0230 ± 0,010 AjTR45 -0,002 ± 0,036 0,020 ± 0,041 0,0219 ± 0,009 Aro63 0,440 ± 0,075 0,446 ± 0,074 0,0102 ± 0,005 AjTR37 0,037 ± 0,038 0,037 ± 0,039 0,0002 ± 0,005 Aro54 0,105 ± 0,061 0,156 ± 0,047 0,0576 ± 0,031 Aro95 0,188 ± 0,062 0,195 ± 0,062 0,0080 ± 0,008 AjTR04 0,382 ± 0,049 0,384 ± 0,050 0,0022 ± 0,004 Average 0,194 ± 0,053 0,209 ± 0,052 0,018 ± 0,010 The genetic distances between population were calculated according to Nei 1972, and are given in Table 18. The stemming dendrogram UPGM construction with PHYLIP Ver.3.5 is showed on Figure 10. Genetic distance between population ranged from 0,238 between Pelabuhan Ratu and Bali to 0,869 between Obi and Poigar. Genetic distance between population within Indian Ocean group range from 0,238 to 0,458, within Pacific Ocean group 0,343 to 0,869 and betwen Pacific and Indian group 0,392 to 0,839. According to the dendogram on Figure 10, Anguilla bicolor is divided into three group: first group consist of 5 populations Aceh, Padang, Pelabuhan Ratu, Pangandaran and Bali, second group consist of three populations Donggala, Bengalon and Bengkulu and third group consist of two population Obi and Poigar. The geographic position of the samples are in agreement with the dendrogramme except the Bengkulu sample Indian Ocean clustered with the Danggala one inter-oceans sample. Poigar and then Obi samples are positioned at the basis of the tree Figure 11. Figure 10. Clustering analyses K=2 for the A. bicolor data set 180 individu, 10 population and 7 loci performed using STRUCTURE programme Pritchard et al. 2000 62 Table 18. Genetic distance among population during present study calculated under Nei 1972 Ace Pad Bal PelR Obi Poig Dong Pang Beng Ben Ace Pad 0.267 Bal 0.268 0.260 PelR 0.398 0.395 0.238 Obi 0.695 0.665 0.839 0.590 Poig 0.778 0.703 0.781 0.706 0.869 Dong 0.429 0.392 0.630 0.472 0.444 0.633 Pang 0.288 0.284 0.361 0.473 0.837 0.638 0.462 Beng 0.494 0.500 0.655 0.557 0.607 0.705 0.343 0.588 Ben 0.346 0.254 0.396 0.458 0.851 0.603 0.389 0.241 0.576 pop1 pop2 pop8 Aceh Padang pop3 pop4 Pangandaran Bali PelabuhanRatu pop7 pop10 pop9 Donggala pop6 Bengkulu pop5 Bengalon Poigar Obi 5 Figure 11. Dendogram based on genetic distances Nei 1972 between A. bicolor samples, using UPGMA method. Discussion Microsatellite as marker Microsatellite loci have been used in recent studies of population structure of anguillidae because of their higher degree of sensitivity to detect slight genetic differentiation. According to previous studies, A. bicolor is distributed from the Indian Ocean to the Pacific Ocean Jespersen 1942, Ege 1939 and Watanabe et al. 2005 and is composed of two subspecies : Anguilla bicolor bicolor and Anguilla bicolor pacifica. However, according to Watanabe et al. 2005 this taxonomy is not well supported by clear differences in genetics and morphology. It was so expected that, using microsatellites as markers in the population 63 structure studies of A. bicolor, a more reliable picture of the populations structure will be obtained Wirth and Bernatchez 2001, Mank and Avise 2003. Microsatellite have been successful to solve the long controversial issue of panmixia and population genetic structure of European eels Anguilla anguilla and American eels Anguilla rostrata Dannewitz et al. 2005; Tomas et al. 2011; Mank and Avise 2003 and Wirth and Bernatchez 2001 because both of species have sligh differences genetic and morphological characters. In the tropical area, using of microsatellite also have managed to solve scientific mystery of population structure of wide geograpical ditribution of tropical eel A. marmorata Ishikawa et al. 2004; Minegishi et al. 2008 and Gagnaire et al. 2009. The preliminary study of population structure of A. bicolor using microsatellite marker has been done by Minegishi et al. 2012. This study showed that A. bicolor has diverged between the Indian Ocean and Pacific Ocean, which consisten with the classical subspecies designation, but apparently genetically homogeneous in Indian Ocean. Population structure The assignation analysis of population structure STRUCTURE software was unable to detect geographic organization among the ten samples analysed. That was indication a lack of genetic differentiation within A. bicolor species in Indonesian waters. Low genetic differentiation Fst 0,05 for A. bicolor in both inter-ocean Indian Ocean dan Pacific Ocean was observed in this study that cause there are no private locus or diagnostic locus of each subspecies. Minegishi et al. 2012 stated based on bayesian demographic history reconstruction and neutrality tests that are indicated population growth in each ocean after the Indo-Pacific divergence. However, the dendrogramme construction, based on Nei 1972 distance clearly divided the samples into a Indian Ocean cluster 5 locations and a inter- oceans cluster of 3 locations, one of them Bengkulu being aberrant. In the Indian ocean, this species appears to be divided into two clusters: the first, composed of Aceh, Padang and Pangandaran, positioned rather at the north of the Indian Ocean cost. The second group, composed of Bali and Pelabuhan Ratu, represents the southern part of the Indian Ocean sampled zone. Despite the observed geographic and genetic organization of these samples, they represent probably the same panmictic population of the west of Indonesian 64 waters. It has been questioned whether there were georaphically divergent populations in western Indonesia because the spawning area of this species recently only know around of Mentawai sea, west Sumatera. These results seems to confirm this hypothesis. Interestingly, the population of Bengkulu, based on genetic divergence and genetic distance, seems closely related to Donggala and Bengalon populations. The phylogenetic study on chapter IV showed that the population Bengkulu has no close relationship with A. b pacifica see Figure 12. However Minegishi et al. 2012 suggested that no absolute physical barriers to gene flow exists in this zone, limiting the population divergence in eel. Perhaps, the aberrant classification of the Bengkulu sample corresponds to this possible gene flow. The total inefficiency of STRUCTURE assignations demonstrates that the genetic differences between the samples and between the sub-species is very limited. A. b. pacifica DONG.11 A. b. pacifica POIG.15 A. b. pacifica BEG.3 A. b. pacifica BEG.4 A. b. pacifica DONG.1.14.16 POIG.5 OBI.3.9 A.b.pacifica A. b. pacifica BEG.1 A. b. pacifica DONG.2 A. b. pacifica DONG.4 A. b. pacifica OBI.7 A. b. pacifica BEG.8 A. b. bicolor PEL.425 A. b. bicolor BAL.16 A. b. bicolor ACE.32 PANG.11 A. b. bicolor PAD.73 PEL.238 A. b. bicolor BEN.63 A. b. bicolor ACE.5 A. b. bicolor PEL.85.96 A. b. bicolor PAD.9 A. b. bicolor CIL.13 A. b. bicolor PANG.3 A. b. bicolor PAD.87 PEL.35 A. b. bicolor POS.16 A. b. bicolor PEL.8 A. b. bicolor ACE.9 A. b. bicolor PEL.298 A. b. bicolor BAL.21 A. b. bicolor BAL.3 A. b. bicolor PEL.105 A. b. bicolor PEL.14 Cla de 1 A. b. bicolor PAD.48 A. b. bicolor ACE.17 A. b. bicolor CIL.5 A. b. bicolor PEL.360 A. b. bicolor MEN.32 A. b. bicolor PAD.17 A. b. bicolor ACE.11 A. b. bicolor PAD.96 A. b. bicolor PAD.14 A. b. bicolor AN.927 PEL.404 A. b. bicolor PEL.63 A. b. bicolor PEL.92 A. b. bicolor PAD.3 PEL.71 A. b. bicolor ACE.29 A. b. bicolor BAL.49 A. b. bicolor CIL.2 A. b. bicolor PEL.4 A. b. bicolor BEN.39 A. b. bicolor PANG.18 A. b. bicolor PAD.60 A.b.bicolor A. b. bicolor PANG.2 A. b. bicolor PEL.339 A. b. bicolor AN923.930 CIL.10 PAD.16.31.7.56 PANG.8 PEL.500.175.390 BAL 35 A. b. bicolor BAL 55 A. b. bicolor PAD.3.75 A. b. bicolor PEL.8.308 Cla de 2 99 86 95 0.000 0.002 0.004 0.006 0.008 0.010 Figure 12. Phylogenetic relationship of A. bicolor based on cytochrome b gene Fahmi 2013 chapter IV 65

VI. General Discussion

Several topics on phylogeography of tropical eel in Indonesia has been investigated in the present study, including the establishment of accurate taxonomy of tropical eels by using molecular approach, distribution and dispersal pattern, phylogenetic relationship among species that inhabit in Indonesian water and population genetic structure of species that have widespread distribution, all information obtained in this study are needed for conservation management of anguillid in Indonesian water. Conservation of eel nowadays was become a big issue in the world. Historically it seems that little attention was given by fisheries managers to eel and their fisheries in most part of the world, and only recently, as recruitment has drastically declined in some regions, awareness raised about eel conservation issue. After these species continued to show drastic recruitment decline in some areas and was listed in the Appendix II of the Convention on International Trade on Endangered species of Wild Fauna and Flora CITES Red List, there has been increasing concern among researchers Watanabe and Miller 2012. Accordance to the decrease of eel population in the Northern Hemisphere temperate, so researchers suggested the management and conservation of tropical eel should be more cautious. The classical conservation theory focused on population abundance and diversity of species, but since molecular genetics develop recently the classical conservation also switching towards to conservation genetic. Conservation genetics is conservation and management of species by using molecular genetics analyses to understand aspects of species biology. Conservation genetic was involved interdisciplinary science such as; ecology, molecular biology, population genetics, mathematical modeling and evolutionary systematic. The first aim of conservation genetics is to reduce extinction risk by minimize inbreeding depression, loss genetic diversity and ability to evolve in response to environmental change, resolving taxonomic uncertainties and defining management units within species Frankham 2003. A critical first step in species conservation is to gain a clear understanding of its taxonomy. The taxonomy status must be accurately established for several reasons: such as legal protection of endangered species and populations, identification of species dispersal, determination of species that have high 66 economic value, stock assessment, determination of endemic or invasive status of species, help to decisions for development and sustainable management of eel farming and to determination of the fitness level of population from variation of species or composition species Frankham et al 2007. A comprehensive taxonomy study of the genus Anguilla has been conducted by Ege 1939 based on morphological characters. The systematic classification proposed by Ege remains widely accepted by many biologists for long period. However, Watanabe et al 2004 showed that the morphological characters used by Ege were insufficient for discriminating all the Anguilla species without considering their geographic distribution. It can be a problem when sampling location is unknown, especially in the tropical area, where there are some case of sympatric distributions between two species with all morphological characters overlapping. In these cases, two species cannot be strictly identified. In addition, potential problems of the geographic distribution of species will be increased dramatically considering passive transport of leptocephali and international trading of young eel. Passive transport of leptocephali for long periods can easily change the range of glass eels recruitment, thus the geographic distribution of the species can change year after year. Furthermore the international trade of glass eels and young eels for aquaculture purpose lead to introduction of non-native species eels in several areas of the world Watanabe et al 2004, Aoyama 2009. After establishment that morphological characters were not sufficient to identify all species, molecular genetics have been recommended Aoyama et al 2005. Since then various molecular identification methods of eels began to be developed; some of them using non specific primer such as RAPD Kim et al 2009; Lehmann et al. 2000 and RFLP Lin et al 2001, 2002; Aoyama et al 2001, whereas the specific primers were used to distinguish two different species such as A. japonica and A. anguilla Sezaki et al 2005, A. interioris and A. celebesensis Aoyama et al. 2000 or A. anguilla and A. rostrata Trautner 2006. Most of them use simple PCR followed by sequencing, while sequencing is not convenient for large samples because of its cost in time and money. In addition most of these researches concerned temperate area only. Considering Indonesian waters that are inhabited by several sympatric species of tropical eels, almost all morphological character in this genus are overlapping. So a rapid and efficient identify technique is needed. Semi multiplex 67