General Discussion Phylogeography of tropical eel (Anguilla spp) in Indonesia Waters

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 PCR developed in the present study has successfully distinguished seven species that inhabit the waters of Indonesia through one-step PCR Fahmi et al. 2012. With this method, one can drastically reduce the necessary number of sequences, while the results are very reliable for species determination we easily identified 1,112 specimens. There are several advantage of this identify technique 1 more accurate and sensitive, because we used species and subspecies specific primers, 2 quick, because only by one-step PCR was enough to distinguish seven species, 3 lower cost detection by using simple agarose gel electrophoresis no acrylamide migration and 4 also applicable to leptocephali and glass eels missing adult morphological characters. Specimens used in this study were collected all around Indonesian waters, covering the geographic distribution of the Anguilla species that were expected to occur in Indonesia water. With this technique, it was possible to determine around 1115 specimens. After finding out and solve the taxonomic uncertainties, next step in conservation genetics is producing a map of distribution and species composing tropical eels in Indonesia. The 16 species of freshwater eels are widely distribution in the world, there are 5 temperate and 11 tropical anguillid species, all of them have unique catadromous life histories. According to Ege 1939 and Watanabe and Miller 2012, Indonesian waters are inhabited by seven tropical eel species and subspecies including A. celebesensis, A. borneensis, A. marmorata, A. interioris, A. bicolor bicolor, A. b. pacifica, and A. nebulosa nebulosa. That means that two-thirds of tropical eel species inhabit Indonesian waters. Because all unexpected semi-multiplex result led to sequencing, it is possible to affirm that no eel other species was present in the sampled localities. We presented the distribution and species composition of tropical eels in Indonesia in Figure 13. This map is the first detailed distribution of Indonesian eels published. Based on the distribution map, several species showed a wide distribution such as A. marmorata, A. b. bicolor and A. interioris, and some have a limited distribution, known as endemic species such as A. celebesensis and A. borneensis. The distribution pattern is not permanent for each species in geologic time. Moreover, due to their particular life cycle, distribution patterns can change seasonally, linked with the dispersion of their leptocephali driven by the current system in their habitat Miller 2003. So the current system has an 68 important western o nebulosa morpholog there are A. b. pac characters molecular role on an f Sumatra a and A. b gical charac four sympa cifica, the s. In these r determinat nguillid distr and south o b. bicolor, cters. The s atric species first three e two cas tion is now ribution. Th of Java, the the three same case i s: A. marmo species a ses of ove indispensab ere are fou ey are A. ma e first spe is also foun orata, A. in also have erlapping n ble. ur species armorata, A cies havin d in Sulawe terioris, A. overlapping early siblin sympatric i A. interioris, ng most s esi waters w celebesens g morpholo ng species n the A. n. imilar where si and ogical , the Figure 13. Distribution and species s composition n of tropical e eel in Indone esia water. Th determine developm more usef zone whic important no fishing he accurate e dispersal ent and su full to infor ch is not to politically activity. taxonomy and endem ustainable m m the fishi present an y or govern status of ee mic status o manageme ng compan n endanger ment decid el in this stu of species, nt of eel c nies the loc red species de that the r udy have be that useful conservation cation where s. This ma rare or ende een succes to decision n. This m e is their fi ap also is emic specie ssfully ns for ap is shing more es are Th extinction, inbreeding endemic s change. H species. T increased he endemic , because a g depressio species was Here, we pr The necessa their popu eel species as the gene on and less s reduced th roposed the ary precurs lation size. s with typica etic conseq s of populat he ability of e managem sor for gene One efforts ally small po quences of tion size. L f species to ment and co tic manage s could be opulation ar small popu Loss of gen respond th onservation ement of end done is res re more pro ulation are netic divers he environm n of eel end demic spec strict the ca one to more sity in mental demic cies is apture 69 and trading the species through in situ conservation. Based on the mapping of eel distribution we were suggested there are three locations that could be an in situ conservation of eel; Mentawai, Poso and Mahakam. These locations were suspected as the spawning area and grow up of eel both endemic and non- endemic species. While the economic important and widespread of eel, A. marmorata and A. bicolor, can be utilized under controlled or quota fisheries. The most important consideration on the utilization of economically eel is maintenance of genetic diversity and maintenance the population from endangered species. In temperate areas where eel is economically important fish, conservation and management glass eel has been managed. Management on utilization of the glass eel in temperate ares included natural recruitment was low, restriction on fishing, fishing gear control, imposing size limits or requiring fishing licenses and facilitating the passage of glass eel over dams Moriarty and Dekker 1997. Next discussion we would like to emphasize that Indonesian waters is a center of the origin of Anguillia of eels because there are a lot of species. But to propose this hypotheses, we must find the more basal lineage of eels in the phylogeny of the genus and distribution of this basal species. Accordance to Minegishi et al. 2005 was constructed the phylogenetic tree based on complete mtDNA that suggested A. mossambica as basal species, followed by the six species of A. borneensis and the Atlantic two species and Oceanian three species species with vary distribution. And another clade is 11 Indo-Pacific species were the geographic distributions adjacent to each other. So there are two large phylogenetic groups of anguillid eels which have different evolutionary histories. Therefore, the evolutionary process of the genus Anguilla might not be simple, and the present geographic distribution could be attributed to, for example, multiple dispersal events, multidirectional dispersion, or past extinctions. As the result, the question the origin of freshwater anguillid eels, genus Anguilla not really clear and that is always intriguing for scientists The last question only tested in this survey is the research of structure inside the large distribution species. In Indonesia, A. interioris is scarcely observed and no intraspecific structure could be investigated. But A. marmoratus and A. b. bicolor have been sampled in most parts of the country and deserve more detailed investigations. These species have been recently analyzed in their whole distribution from western Indian Ocean to eastern Pacific Ocean Gagnaire 70 71 et al. 2009 2011; Minegishi et al. 2012 but a detailed phylogeography using both mtDNA and nuclear markers in Indonesian, crossroad country between several oceans, is still missing. A preliminary study of A. b. bicolor is proposed in this thesis.

VII. GENERAL CONCLUSION AND SUGGESTION A. Conclusion

1. Nine species subspecies specific primer succesfully amplified fragment 230 bp for A. b. bicolor, 372 bp for A. n. nebulosa, 450 bp for A. borneensis, 620 bp for A. marmorata, 670 bp for A. b. pacifica, 720 bp for A. celebesensis, and 795 bp for A. interioris by using semi- multiplex PCR. 2. Semi-multiplex PCR successfully reduced the number of necessary sequences, so this methods has proven to be most simple, quicker, lower cost no acrilamide migration, specificsensitive, and highly reliable way than previously identification genetically study. 3. We recognized four species and subspecies with wide distribution: Anguilla bicolor bicolor, A. b. pacifica, A. marmorata and A. interioris , two species with limited distribution, close to endemism: A. celebesensis and A. borneensis and one subspecies A. nebulosa nebulosa that is only spread in river flowing into Indian Ocean . 4. There are high genetic diversity of tropical eel in Indonesia water calculated based almost whole of cyt b gene with indicated by high haplotype and nucleotide diversity. 5. There are limited genetic divergence and no genetic structure of A. bicolor in Indonesia water, the divergence between Pacific and Indian Ocean of this species consistent with the classical subspecies designation.

B. Suggestion

1. Based on our results, we suggested using a semi-multiplex PCR assay for identification species and subspecies of tropical eel that inhabit the waters of Indonesia eels. 2. Our study just used one gene marker on phylogenetic analyses and this not enough to understand the evolution and dispersal route, so we need more gene marker to understand the evolution of this genus in Indonesia water 3. There are four species sympatric in the western of Sumatra, south of Java and Sulawesi, three of them having most similar morphological characters, so we must take care on the conservation management because some of them are species endemic of Indonesia water with a narrow spread. 72 REFERENCES Aoyama J, Tsukamoto K. 1997. Evolution of the freshwater eels. Naturwissenschaften; 84:17-21. Aoyama J, Watanabe S, Ishikawa S, Nishida M, Tsukamoto K. 1999. Are morphological characters effective enough to discriminate two species of freshwater eels, Anguilla celebesensis and A. interioris? Ichthyologycal Research. 47: 157–161. Aoyama J, Watanabe S, Nishida M, Tsukamoto K. 2000. Discrimination of catadromous eel species, genus Anguilla, using PCR-RFLP analysis of the mitochondrial 16SrRNA domain. Trans. Amer. Fish. Soc. 129:873-878. Aoyama J, Nishida M, Tsukamoto K. 2001a. Molecular Phylogeny and evolution of freshwater eel, genus Anguilla. Mol. Phylogenet Evol 20: 450-459. Aoyama J, Ishikawa S, Otake T, Mochioka N, Suzuki Y, Watanabe S, Shinoda A, Inoue J, Lokman PM, Inagaki T, Oya M, Hasumoto H, Kubokawa K Lee TW, Fricke H, Tsukamoto K. 2001b. Molecular approach to species identification of eggs with respect to determination of the spawning site of the Japanese eel Anguilla japonica. Fisheries Science 67: 761-763. Aoyama J. 2003. Origin and evolution of the freshwater eels genus Anguilla. In: Aida K,Tsukamoto K, Yamauchi K eds. Eel Biology. Springer, Tokyo. pp.19-29. Aoyama J, Wouthyuzen S, Miller MJ, Inagaki T, Tsukamoto K. 2003. Short- distance spawning migration of tropical freshwater eels. Biol Bull; 204: 104- 108. Aoyama J, Wouthuyzen S, Miller MJ, Minegishi Y, Minagawa G, Kuroki M, Suharti SR, Kawakami T, Sumardihargo KO, Tsukamoto K. 2007. Distribution of the lepthocephali of the freshwater eels, genus Anguilla in the waters off west Sumatera in the Indian Ocean. Environ.Biol.Fish; 80: 445-452. Aoyama J. 2009. Life History and evolution of migratio in catadromous eels Genus Anguilla. Aqua-Bio.Sci.Monogr. 2:1-42. Arai T, D Limbong, T Otake, K Tsukamoto. 1999. Metamorphosis and inshore migration of tropical eels, Anguilla spp in the Indo-Pacific. Marine Ecology Progress Series, 182,283-293. Arai T, D Limbong, T Otake, K Tsukamoto. 2001. Recruitment mechanisms of tropical eels Anguilla spp. and implications for the evolution of oceanic migration in the genus Anguilla. Marin Ecology Progress Series 216: 253- 264. Avice JC, Helfman GS, Sauders NC, Hales LS. 1986. Mitochondrial DNA differentiation in North Altantic eels: Population genetic consequences of an unusual life history pattern. Proc.Natl.Acad.Sci. 83: 4350-4354 . Bastrop R, Strehlow B, Jurss K, Sturmbauer C. 2000. A New Molecular Phylogenetic hypothesis for the Evolution of Freshwater Eels. Mol. Phylogenet. Evol; 14: 250-258. Castle PHJ, Williamson GR. 1974. On the validity of the freshwater eel species Anguilla ancestralis Ege, from Celebes. Copeia. 2: 569-570. 73