celebesensis marmorata b. pacifica

46 Table 9. Matrix genetic distance between all of species-subspecies on genus Anguilla. Grey highlights one is species-subspecies during this study. Standar errors SE was showed above diagonal. Analysis conducted under Kimura 2-parameter 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1. A. b. bicolor 0,004 0,011 0,008 0,008 0,008 0,008 0,010 0,009 0,011 0,010 0,010 0,011 0,011 0,008 0,010 0,009 0,010 2. A. b. pacifica 0,023 0,011 0,008 0,008 0,009 0,008 0,010 0,009 0,011 0,010 0,010 0,011 0,011 0,008 0,010 0,010 0,010 3. A. borneensis 0,103 0,100 0,010 0,009 0,010 0,010 0,011 0,010 0,011 0,011 0,010 0,010 0,010 0,010 0,010 0,010 0,010 4. A. celebesensis 0,077 0,074 0,089 0,008 0,008 0,008 0,008 0,009 0,011 0,010 0,008 0,008 0,010 0,009 0,009 0,008 0,009 5. A. interioris 0,067 0,063 0,081 0,062 0,007 0,007 0,009 0,006 0,011 0,010 0,009 0,010 0,011 0,007 0,010 0,009 0,010 6. A. marmorata 0,072 0,070 0,087 0,065 0,048 0,008 0,010 0,007 0,011 0,011 0,008 0,009 0,011 0,008 0,011 0,010 0,010 7. A. n. nebulosa 0,063 0,063 0,092 0,068 0,051 0,057 0,009 0,008 0,011 0,010 0,009 0,009 0,010 0,008 0,010 0,009 0,010 8. A. megastoma 0,089 0,087 0,096 0,061 0,075 0,072 0,080 0,011 0,010 0,011 0,010 0,010 0,012 0,010 0,010 0,009 0,010 9. A. luzonensis 0,075 0,070 0,088 0,075 0,043 0,053 0,056 0,090 0,011 0,010 0,009 0,010 0,010 0,008 0,011 0,010 0,011 10. A. mossambica 0,101 0,101 0,097 0,096 0,098 0,096 0,099 0,093 0,102 0,010 0,010 0,011 0,011 0,011 0,010 0,010 0,010 11. A. rostrata 0,097 0,094 0,098 0,094 0,096 0,090 0,092 0,104 0,093 0,095 0,010 0,011 0,007 0,010 0,010 0,010 0,010 12. A. reinthardtii 0,083 0,083 0,087 0,070 0,076 0,063 0,068 0,079 0,075 0,088 0,091 0,009 0,011 0,009 0,010 0,009 0,010 13. A. japonica 0,102 0,096 0,094 0,073 0,082 0,074 0,076 0,079 0,085 0,093 0,094 0,062 0,011 0,010 0,011 0,009 0,011 14. A. anguilla 0,108 0,101 0,095 0,101 0,095 0,095 0,097 0,104 0,092 0,102 0,046 0,096 0,099 0,011 0,011 0,010 0,011 15. A. obscura 0,068 0,062 0,088 0,074 0,056 0,060 0,063 0,083 0,058 0,104 0,092 0,075 0,084 0,096 0,011 0,010 0,011 16. A. a. australis 0,095 0,089 0,079 0,076 0,085 0,086 0,091 0,085 0,096 0,084 0,084 0,080 0,087 0,086 0,097 0,008 0,001 17. A. diefenbachii 0,079 0,075 0,077 0,061 0,073 0,079 0,069 0,074 0,076 0,081 0,078 0,072 0,074 0,079 0,080 0,061 0,008 18. A. a. scimidthi 0,094 0,088 0,080 0,075 0,084 0,085 0,090 0,083 0,095 0,084 0,082 0,079 0,088 0,085 0,095 0,001 0,060 Table 10 Genetic distance within species and sub species. Species Genetic distance within species SE

A. marmorata

0,0055 0,0013 A. interioris 0,0058 0,0015

A. n. nebulosa

0,0035 0,0011

A. b. pacifica

0,0040 0,0010 A. b. bicolor 0,0080 0,0018 A. celebesensis 0,0063 0,0015 A. borneensis 0,0013 0,0010 A. marmorata LOM.1 A. marmorata PAP.12 SANG.5 A. marmorata BAL.42 A. marmorata AMB.21 LAS.89 PAL.5 PAP.2.8 BAL.29 POIG.1.17 INO.47 A. marmorata BAL.5 LOM.3.4 LAS.161.32 POS.18 A. marmorata PAP.6 A. marmorata BAL.30 A. marmorata PAL.3 A. marmorata OBI.11 A. marmorata BAL.1 AMU.32 A. marmorata LAS.149.3 A. marmorata BAL.51.7 LAS.120.77 LOM.6.7 PAP.1 SANG.10 PEL.501.126 POS.24 A. marmorata PAL.1 A. marmorata BAL.8 A. marmorata POS.20 A. marmorata LOM.5 BAL.12.21A MEN.2 POS 57B OBI.4 A. marmorata POS.1 A. marmorata SANG.8 A. marmorata PAL.4 A. marmorata LAS.1 POS.7 A. marmorata PAP.7 A. marmorata AMU.46 A. marmorata LAS.124 A. marmorata POIG.8 A. marmorata PEL.12 Cla de 1 A. marmorata BAL.50 LAS.104 SANG.1 INO3 A. marmorata INO.1 A. marmorata A. marmorata POS.72 A. marmorata MEN.5 A. marmorata PAP.16 A. marmorata BEN.6 PEL.183 A. marmorata PAP.5 A. marmorata AMB.63 A. marmorata PAP.9 A. marmorata BEN.23 A. marmorata AMB.7 A. marmorata BEN.20.24.30.2 MEN.23.41.17 PEL.257.17.192 ACE.35 A. marmorata BEN.31 A. marmorata ACE.36 A. marmorata MEN.27 A. marmorata PAP.4 A. marmorata MEN.32A OBI.1 A. marmorata POS.2 A. marmorata POS.3 Cla de 2 C Figure 7. Neighbor Joining NJ phylogentic tree of all sequence in this study based on 950 bp of mitochondrial DNA cyt b gene fragmen under Kimura 2-parameter model up 75. la de 1 A.interioris MEN.4 A.interioris MEN.44 A.interioris PAP.15 A.interioris MEN.2A POS.57A A.interioris PAP.11 Cla de 2 A.interioris MEN.13 A.interioris MEN.26 A.interioris MEN.1 A.interioris A.interioris MEN.12 A.interioris PAP.13 A.interioris PAP.14 POS.9 Cla de 3 A. n. nebulosa PEL.96 A. n. nebulosa PEL.161 A. n. nebulosa PEL.113 A. n. nebulosa BEN.15 A. n. nebulosa PEL.52 A. n. nebulosa A. n. nebulosa ACE.24 LOM.2 PEL.132 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 A. celebesensis BEN.33 A. celebesensis INO.20 A. celebesensis POS.16A Cla de 1 A.celebesensis POS.25 AMU.49 A. celebesensis POS.9 A.celebesensis Cla de 2 A. celebesensis POS.17 A. celebesensis POS.10 A. celebesensis AMU.14.1 POS.11 INO.14 A. celebesensis POS.19 Cla de 3 A.mossambica A. borneensis MAH.1 A. borneensis MAH.2 A.borneensis A. borneensis MAH.3 Serrivomer sector 99 99 90 99 99 90 99 99 90 99 99 86 95 0.08 0.06 0.04 0.02 0.00 47 Relationship among and within species was also supported by Kimura 2- parameter genetic distances we present genetic distance within and between species on Table 9 and Table 10 respectively. A. b. bicolor and A. b. pacifica has closed relationship genetic distance 0,023 than other pairs. Whereas farthest genetic relationship we found between A. borneensis with other species, there were 0.081, 0.087, 0.089, 0.092, 0.100 and 0.103 with A. interioris, A. marmorata, A. celebesensis, A. n. nebulosa, A. b. pacifica and A. b. bicolor respectively. Allopatric Divergence of A. bicolor The construction a NJ phylogenetic tree of all speciemen on this study confirming a significant genetic differentiation between A. b. bicolor and A. b. pacifica that supported by strong bootstrap value were 100 replications and low genetic distance between two subspecies. A. b. pacifica is composed the population from eastern part of Indonesia water Bengalon, Donggala, Poigar and Obi, see location in Appendix 1. The nucleotide divergence within this subspecies is ranked between 0-3. Accordance table frequence haplotype, A. b. pacifica have one common haplotype Hap. 70 that share on all population. While A. b. bicolor is composed the population from western part of Indonesia water Aceh, Padang, Bengkulu, Pelabuhan Ratu, Cilacap, Pangandaran and Bali, see location in Appendix 1. The nucleotide differentiation within this subspecies is ranked between 0,2-3 Table.11 Accordance table frequence haplotype, A. b. bicolor also have one common haplotype Hap. 120 that share on all population. The NJ phylogenetic tree of cyt b also reveal important genetic divergence for two clade of A. b. bicolor with strong bootstraps; 82 bootstrap replications for clade B1 and 86 for clade B2 Figure 7. Both of clade was supported by nonsynonymous mutation on codon 161 with a change Glycine G on clade 1 into Tryptophan W on clade 2. Genetic distance of two clade is 0,0180 SE=0,003 under K2P analysis. Even A. b. bicolor separated two distinct monophyletic clade however not supported by unique distribution, probably they plays the sympatric speciations. Allopatric speciation has led mutations on cyt b sequences of A. b. bicolor were 16 nucleotide of 1042 sequence. Table 12 showed the mutation position on cyt b sequence on this study. Nonsynonymous mutation n=3 also found on cyt b 48 sequence of A. b. bicolor and A. b. pacifica that alter amino acid differences Table 12. Table 11 Intraspecific genetic differentiation measured within A. bicolor species No Sub species Sample site n specimen analysed Nhp π 1 A. b. bicolor Aceh 6 6 0,871 2 A. b. bicolor Padang 16 10 0,572 3 A. b. bicolor Bengkulu 2 2 1,344 4 A. b. bicolor Pelabuhan Ratu 27 22 2,632 5 A. b. bicolor Pangandaran 5 4 0,557 6 A. b. bicolor Cilacap 5 5 0,729 7 A. b. bicolor Bali 6 5 0,723 8 A. b. pacifica Bengalon 4 4 0,464 9 A. b. pacifica Donggala 8 6 0,247 10 A. b. pacifica Obi 2 1 0,000 11 A. b. pacifica Poigar 4 4 3,439 Total 16 85 69 11,578 Average 6,27 1,053 nucleotide divergence π of A. b. bicolor n=67 = 1,061 and A. b. pacifica n=18 = 1,037 Table 12. Mutation on nucleotide sequence above and amino acid below between A. b. bicolor and A. b. pacifica 198 306 447 464 582 600 675 693 709 723 750 780 828 840 882 900 A. b. bicolor T G C C G G G A A T T T C T C G

A. b. pacifica

C A T T A A A G T C C C T C T A A. b. bicolor

A. b. pacifica

Methionin M Isoleucine I Leucine L Nucleotide mutations nt‐ Amino acid mutations codon‐ Serine S Leucine L Isoleucine I 153 231 237 A Wide Distribution of A. marmorata A. marmorata was found nearly in all sampling locations except Padang, Cilacap, Pangandaran and Bengalon due to the limitations of this study. Alignment of 1042 bp of cyt b from 92 individuals of A. marmorata which collected from 16 locations has 99 polymorphic sites 10,42. The quantities of nucleotide diversity from each population presented in Table 13 with rank 0,08- 6,9. Nucleotide diversity average were 0,973±0,004 . A branch of A. marmorata on NJ phylogenetic tree Figure 4 was supported by 100 replications bootstrap. This branch separated into two clades but by weak support bootstrap 72 and 56 for clade 1 and clade 2 respectively. Clade 2 run into nonsynonymous mutation from clade 1 where Isoleucine I on codon 120 change being Methionine M. 49 A. marmorata has three common haplotypes Hap5, 6, 17 and 27 were share on wide populations. Nevertheless this species also have a specific haplotype that found only in specific region, such as Hap48 only found in the western part of Indonesian waters and absent in eastern part of Indonesia, whereas Hap13 found only in eastern part of Indonesian waters and absent in western part of Indonesia. Table 13 Intraspecific genetic differentiation measured within A. marmorata species No Code Sample site n speciment analized Nhp 1 Ace Aceh 2 2 6,910 ± 0,03455 2 Men Mentawai 8 5 0,295 ± 0,00073 3 Ben Bengkulu 7 4 0,084 ± 0,00028 4 Pel Pelabuhan Ratu 7 4 2,221 ± 0,01246 5 Bal Bali 12 10 1,401 ± 0,00902 6 Lom Lombok 6 5 0,313 ± 0,00103 7 Las Lasusua 9 6 0,288 ± 0,00060 8 Pal Palu 4 4 0,400 ± 0,00086 9 Pos Poso 9 8 0,538 ± 0,00107 10 Sang Sangata 5 5 0,365 ± 0,00079 11 Poig Poigar 3 2 0,192 ± 0,00090 12 Amu Amurang 2 2 0,384 ± 0,00192 13 Ino Inobonto 3 3 0,448 ± 0,00083 14 Obi Obi 3 3 0,576 ± 0,00202 15 Amb Ambon 3 3 0,576 ± 0,00193 16 Pap Papua 9 8 0,581 ± 0,00073 16 92 74 15,572 ± 0,0697 4,63 0,973 ± 0,0044 π Total Average Discussion This is the first study of phylogenetic relationship and genetic diversity based on mitochondria DNA cytochrome b gene of Anguilla that covering all of the geograpical distribution in Indonesian waters. According to Avice et al. 1986, providing an adequate description of the genetic structure of populations that responsible for a given genetic architecture and evolutionary factors is an important objective of population genetics. In this study we present genetic diversity and phylogenetic relationship among and within species in genus Anguilla to understanding the evolutionary process of Anguillid that inhabit in Indonesia waters. 50 Genetic diversity Seven species of Indonesian tropical eel shows a higher haplotype diversity at the cyt b locus range 0,93 to 1,00 compare to same locus in another species, Anguilla anguilla in eropa range 0,62 to 0,82, Daemen et al. 2001; 0,78 by Avise et al. 1998 and almost as same as with A. japonica 0,97 by Sang et al. 1994 and 0,96 by Ishikawa et al. 2001. The haplotype variation we observed at the cyt b locus was 63,4 considerable 135 haplotype, in 213 eel specimens, this different haplotype is lower than other species as follow Japanese flounder 98, N=82, Fuji and Nishida 1997, Japanese eel 98, N=55, Ishikawa et al. 2001. The molecular marker used in this study cyt b showed high level of polymorphic site 20,48 compared with D-loop locus on other Anguilla, A. japonica 11 , Sang et al. 1994 and 17, Ishikawa et al. 2001 and A. anguilla 0,4 by Daemen et al. 2001. Sequence divergence in this study 0,1-1,06 lower with A. japonica 1,1-1,6, Ishikawa et al. 2001 and higher than A. anguilla 0,2-0,5, Daemen et al. 2001. The comparison indicated that Indonesian tropical eel has various of haplotype and frequency of variable site relatively higher than temperate eel A. anguilla and as same as Japan eel A. japonica, nevertheless the degree of sequence difference and haplotype different among Indonesia ees almost lower than other species. If there was any genetic heterogenity on Indonesia eel species, the level of nucleotide diversity should be also high, this variability pattern probably reflect the history of the Anguilla that they were migrated from the sea to the freshwater on their life cycle, so the specimen collection should be represent the seasion or using more specimen is needed. It is could be seen on genetic diversity of A. boorneensis and A. n. nebulosa that have a few amount of specimen, so the genetic diversity of both species is still questionable. In addition, the causes of low nucleotide diversity of A. borneensis may be due to single panmictic population or single spawning population, A. borneensis is an endemic species from Borneo Aoyama et al. 2001 and Minegishi et al. 2005 with narrow dispersal. According to Ryman and Utter 1986, mating between related individuals is inbreeding, this caused by narrow dispersal of species, so high probability to mates in the next generation. Inbreeding will be decreasing genetic diversity Frankham 2002. The cyt b sequence of Indonesian tropical eel reveal high and significant differences of genetic variation among seven species that observed on this study, 51 this is expected for marine fish which often using mtDNA as genetic markers because their ability to detect the population structure Ryman and Utter 1986. Number transitions and transversions suggested that few multiple substitutions have occurred at most nucleotide position. This suggests that substitutions at the variable position are not saturated, indicating that interspecific genetic distance observed in this study could be used to phylogenetic construction and that genetic divergence among the taxa could be used for molecullar clokcevolutionary time calibrations Lydeard and Roe 1997. Phylogenetic and Evolution According to Minegishi et al 2005 and Aoyama 2003 one the one hand which causing the discrepancies determine of the basal species on the previous phylogenetic study be caused the outgroup setting. In this study we used three species suggested by Inoue et al. 2004 that conger eel Conger myriaster, cutthroat eel Synaphobrancus kaupii and sawtooth eel Serrivomer sector as outgroup, if the construction of phylogenetic trees using the three species separately as outgroup, will be obtained three phylogenetic with different of topologies and basal species candidates. However, if three species used simultaneously as outgroup will be obtained phylogenetic tree as same as topologies of Synaphobranchus kaupi species as out group, so it concluded using Synaphobranchus kauphi as outgroup most reliable. A. b. bicolor A. b. pacifica Figure 8. Dendogram of genetic distance of Indonesian tropical eel based on cyt b sequence, red font : cyt b sequence from this study and black one: based on sequence from GenBank. AP007247 A. obscura A. interioris A B 4 6 9 4 3 7 A. luzonensis A. n. nebulosa A. marmorata A. celebesensis A P 0 0 7 2 4 3 A. megastoma A B 0 3 8 5 5 6 A. japonica A P 0 0 7 2 4 8 A. reinhardthii Indo-Pacific Group A P 0 0 7 2 4 0 A. diefenbachii A P 0 0 7 2 3 4 A. a. australis A P 0 0 7 2 3 5 A. a. scimidthi Oceanic Group A. borneensis A P 0 0 7 2 3 3 A. anguilla A P 0 0 7 2 4 9 A. rostrata Atlantic-Group AP007244 A. mossambica A P 0 0 7 2 5 0 Serrivomer s A B 0 3 8 3 8 8 1 Conger myaster 0.12 0.10 0.08 0.06 0.04 0.02 0.00 52 53 The topology of tree suggested that A. mossambica as basal species. This topology almost same with the phylogenetic tree that constructed by Minegishi et al. 2005 based whole mt-DNA. Beside that the same topology of tree, this study also found three clade of geographical distributions; Atlantic group A. anguilla and A. rostrata, Oceanic group A. dieffenbachii, autralis A. australis and A. a. schmidtii and Indo-Pacific group A. celebesensis, A. megastoma, A. nebulosa nebulosa, A. bicolor bicolor and A. b. pacifica Figure 8. Indicating that marker cyt b which used in this study is more reliable for phylogenetic relation. Estimation of the divergence time of Anguilla had been calculated by Minegishi et al. 2005. The beginning of speciestion of extant angillid species was around 20 million years ago Mya and the estimate divergence between genus Anguilla and Serivormer sector was 52 Mya. Whereas A. b. bicolor and A. b pacifica indicated as two last species which separated around 1,6 Mya. Interestingly, the branch topology of A. b. bicolor on Figure 7 was separated into two mayor clades that supported by the high bootstrap 93, each clade have two nucletide diagnostic Table 8 even they dont have subspecies nucleotide diagnostic. This indicated A. b. bicolor had two population structures. According to Li 1942 a basic of evolution process in the evolutions of DNA sequence is the change in the nucleotide with time. The change of nucleotide sequence will separate the new sequence from the old one. After the separation of two nucleotide sequences, each sequence will start accumulating nucleotide subtitutions and two sequences will diverge from each other. Initially, every subtitution will increase the dissimilarity between two sequences.

V. Population Genetic Structure of Tropical eel Anguilla bicolor in Indonesia Water

Abstract The objective of this study was to reveal the population structure of shortfinned eel, Anguilla bicolor, that inhabit in Indonesia water. The genotypes of seven microsatellite DNA were analysed for 180 specimens collected from 10 representative location where two subspecies have been found. Analysis with seven microsetallite loci showed Expected He and observed Ho heterozigosities of each locus range range from 0,594 to 0,921 and from 0,250 to 1,000 respectively. Significant deviation from H-Wequilibrium were detected in 26 out of 70 cases which AjTR 04 and Aro 63 locus were more informative demonstrated with the higher average number of alleles. Whereas the genetic divergence and genetic distance between A. bicolor was from 0,20 to 5,52 and 0,241 to 0,869 respectively. Key word; Microsatellite, A. bicolor bicolor, A. bicolor pacifica, population structure and tropical eel. Introduction The shortfinned eel, Anguilla bicolor, has a relatively wide geographic distribution compared to the 19 species and subspecies of genus Anguilla, it is distributed longitudinally from the eastern coasts of Africa through the seas around Indonesia to New Guinea in Pacific Ocean Ege 1993. The wide distribution of A. bicolor is one of the characteristics of marine fishes including large population sizes and pelagic eggs and larvae Palumbi 1994; Ward 1994. These characteristics, combined with the lack of physical barrier in the oceans, lead to the expectation of weak or no population structure Waples 1998. Freshwater eels of the genus Anguilla are catadromous fish with long spawning migrations at various scales, expanding from a few hundred for tropical eel to thousands of kilometers for temperate eel Aoyama 2009. Besides the distance migration, differences between tropical and temperate eel species occur in the pelagic larval duration, distribution range and spawning season Arai et al. 1999, 2000. Geographic distribution of each tropical Anguillid species partly or completely overlaps with at least one other congener in spawning area, growth 54 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