Distribution of Tropical Eel Genus Anguilla in Indonesian Waters Based on Semi-multiplex PCR

Tropical eels living in Indonesian waters are known to be composed of several species, but their real listing together with their distribution ranges need to be established. The main difficulties are the very high number of islands with perennial rivers where these species are living during the growth phase of their life cycle. It is difficult, sometimes impossible, to determine the species using morphological characters, moreover on glass eels. In order to establish the geographic distribution of tropical eels of the genus Anguilla in Indonesian waters, a total 1115 specimens were collected between 2008 and 2012. Sample collection was done in the growth habitats that are rivers and estuaries by commercial nets of different categories according to the fish size. All samples were identified genetically using the recently developed semi-multiplex PCR method. Four species and subspecies was recognized 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.


Introduction
The catadromous freshwater eels genus Anguilla is distributed nearly world-wide except the South Atlantic and the Eastern Pacific oceans (Ege 1939).
Freshwater eels spawn in the offshore ocean.After hatching, their larvae migrate to coastal areas as pelagic, floating and transparent (Mochioka 2003).Eel larvae, called leptocephali, are transported passively by warm currents flowing at low latitudes.When they approach the continental shelf, leptocephali metamorphose into glass eels before settling in the continental waters (rivers and lakes) to grow for years until changing into yellow eels or "elver" and then silver eels.The dispersal of leptocephali not only drives the distribution of freshwater eel species on continental areas, but also the phylogeography of the genus and its evolution (Aoyama and Tsukamoto 1997).
After Johannes Schmidt succeeded collecting anguillid leptocephali in the Sargasso Sea in 1922 (Schmidt 1922), he and his colleagues, through Carlsberg Foundation's Oceanographic Expedition, continued their efforts by searching for the spawning areas of freshwater eels in the Indo-Pacific region where most of the species of this genus are found.They successfully collected leptocephali in the Indo-Pacific region during their expedition from 1928to 1930(Jespersen 1942)).However, most of these leptocephali have overlapping morphological characters, hampering exact identifications.Since then, the spawning areas of the Indo-Pacific anguillid species have remained a mystery.As a result, the studies of Indo-Pacific eels are still poorly understood as well as the exact locations of the spawning areas, and their larval migrations and the recruitment mechanisms.
To solve the problems in identifying anguillid leptocephali, genetic approaches, mtDNA sequences or RFLP, has been successfully used (Aoyama et al. 1999(Aoyama et al. , 2001a(Aoyama et al. , 2001b;;Aoyama 2003;Watanabe et al. 2005).Since species identification of anguillid leptocephali has been developed, projects aimed at learning more about the spawning areas, larvae distribution and larval ecology of anguillid in the Indo-Pacific region have been organized.The long scientific cruise of the Baruna Jaya, in central Indonesia sea, around Sulawesi Island, from 2001-2002, successfully collected leptocephali of A. marmorata, A. bicolor pacifica and A. interioris.This survey also collected leptocephali of A. celebesensis and A. borneensis allowing to deduce the spawning areas of these species (Aoyama et al. 2003, Wouthuyzen et al. 2009).In 2003 this cruise also collected young leptocephali A. bicolor bicolor in west Sumatera, positioning a spawning area of . b. bicolor in this zone (Aoyama et al. 2007).
The three Indonesian endemic species spawning areas are also to be discovered: leptocephali of A. interioris have been caught in western Sumatera waters (Aoyama et al. 2007) and Sulawesi waters (Aoyama et al. 2003, Wouthuyzen et al. 2009); leptocephali of A. celebesensis were recognized in Tomini Bay Sulawesi Island and that of A. borneensis were found in Makasar strait (Aoyama et al. 2003;Wouthuyzen et al. 2009).
According to the geographic range of each species, Aoyama et al. (2001) and Lin et al. (2001) who studied genus Anguilla molecular phylogenetics based on partial mtDNA, showed four major subgroups in the world, that are Oceanic, Atlantic, Tropical-Pacific and Indo-Pacific lineages.However, the complete mtDNA sequence of all species of genus Anguilla was determined recently by Minegishi et al. (2005) which suggested the geographic structure of eel as follow: two Atlantic species (A.rostrata and A. anguilla), two Oceanic species (A.differenbachii and A. australis), and nine Indo-Pacific species (A. japonica, A. reinhardtii, A. marmorata, A. nebulosa, A. bicolor, A. interioris, A. celebesensis, A. megastoma and A. obscura).Seven species/subspecies of the Indo-Pacific Anguilla occur in the western Pacific and eastern Indian Ocean around Indonesian waters (Ege 1939;Castle & Williamson 1974).Because of this high diversity, several scientists considered that "Indonesia is homeland of anguilid".
Besides, Indonesia is also known as "the origin of anguillid" because phylogenetic analyses indicated that the endemic tropical species A. borneensis from eastern Borneo (Kalimantan) is one of the most basal species of the genus (Aoyama et al. 2001;Minegishi et al. 2005).
Among the 19 existing eel species and sucspecies, 7 occupy the Indonesian rivers.Half of them are endemic, limited to Indonesia (A. borneensis, A. cebesensis, A. interioris), the others show larger range distribution (A. obscura, A. nebulosa), sometimes established at the Indo-Pacific level (A.bicolor and A. marmorata).However, the species range distribution of the 7 Indonesian species is only an extrapolation of very limited records.
The widespread species A. bicolor and A. marmorata are of interest because their distribution and abundance place them in central economic position, and because their exceptional geographic distribution (over 18,000 km east-west) and structure made them an important model for eel biology understanding, encompassing probably several spawning areas.Short fin eels A.
bicolor are considered to be structured into two subspecies A. b. bicolor in Indian Ocean, especially at the west of Indonesia and A. b. pacifica in Pacific Ocean, at the east of Indonesia (Minegishi et al. 2012).The giant mottled eel A. marmorata is not taxonomically divided into subspecies because of its morphological stability, however molecular studies have demonstrated its structure into four differentiated populations: North Pacific, South Pacific, Indian Ocean, and Mariana (Minegishi et al. 2008;Gagnaire et al. 2009Gagnaire et al. , 2011)).
One of the most exigent parts of this study is sampling strategy.Indonesia is an archipelago country with counts 17000 islands, among them several thousand islands possibly hosting eels so a complete sampling is impossible.The selection of representative sampling location is necessary.A new molecular identification method has been developed recently to distinguish seven tropical eel inhabiting Indonesian waters, called semi-multiplex PCR assay.By using multiple species-specific primers in one PCR reaction, the identification of all Indonesian species is now simple, quick, low cost, sensitive, and highly reliable (Fahmi et al. 2012).

Indonesian sea have a complex topography and connectivity between the
In this study the semi-multiplex PCR methodology was used in order to establish a first map distribution of species and subspecies of tropical eel (Anguilla spp) that inhabit in Indonesian water.

Material and Methods
The 1115 specimens were collected in 28 locations around the Indonesian waters, covering the whole geographic distribution of genus Anguilla as known or expected in Indonesian waters (Appendix 1, Table 4).Specimens collection was conducted in river estuaries along the coast of Indian Ocean, Pacific Ocean and around Arafuru and Celebes Seas.The specimens were collected from 2008 to 2011 by using traps, nets and fishing.Oceanographic data of Indonesian seas (Gardon 2005) used for looking the movement of water and the possible spread of eels.
All of specimens were identified using semi-multiplex PCR protocol according to Fahmi et al (2012 (in press)).Nine species-specific primers were added in one PCR reaction.The PCR was carried out in a total volume of 10 µl containing 2 µl 5x Green GoTaq @ reaction buffer, 0.5 µl MgCl 2 (25 mM), 1.25 µl dNTP (2 mM), 0.5 µl each primer (10 mM) with 1 µl (10 mM), 0.05 µl GoTaq @ DNA polymerase (5 u/µl), 0.2 µl ddH 2 O and 1 µl template DNA (around 20 ng). A. marmorata was found in almost all sampling locations except Station 4, 8,13 and 15 (Fig. 5b).However, by enhancing the sampling frequency, it is likely that this species would be seen everywhere.The highest abundances of A. marmorata are in Sulawesi and Ambon waters, whereas around Sumatra Island this species is commonly found in Bengkulu and Mentawai waters.The last widespread species found in Indonesian waters was A. interioris.
This species is not found in many Indonesian sampling stations as the other two widespread species.A. interioris was found in the waters of Mentawai, Lombok, Poso and the western part of Papua.The abundance of this species was relatively high in the waters of Mentawai and the harvest of adult eels is done intensively for its high economic benefice (Fig. 5c).

Discussion
Producing new data on Indonesian eel species is an important challenge for several reasons.Both of biodiversity knowledge for conservation and economical exploitation need species determination and distribution ranges.
For most of eel species, the timing of the vital cycle of Indonesian populations is not known.The efficient sampling periods are not well known, even by Indonesian people, because eels are not commonly consumed.Moreover, these fish are often considered as sacred or mystic symbols by many people, because of some mystery in their life cycle.
Currently, freshwater eels have been harvested intensively in some regions of Indonesia.In order to, to overcome this excessive exploitation, the Indonesian government established a regulation for exportation, banning juvenile of eels. A. marmorata and A. bicolor are the two most important commercial species since these fish are widespread and abundant in Indonesian waters so catching of both species has been done intensively.However, the number of five other species are not know their distribution and dispersal

Specimen collection design
The most important phase in this study is the samples collection.The reason is Indonesia as an archipelago with more than 17000 islands and the choice of sampling locations was determinant for the survey design.In this study, sampling strategy was influenced by the surface current pattern in Indonesian Sea, which was known as 'migration loops', differentiation and speciation (Tsukamoto et al. 2002).
The larval migration of the Atlantic eels A. anguilla and A. rostrata and Japanese eels A. japonica have been fairly well studied and these species were found to have spawning sites associated with oceanographic frontal feature such as temperature (McCleave 1993) or salinity (Tsukamoto 1992;Kimura et al. 1994).On the contrary, tropical species, and especially Indonesian ones, are waiting for basic researches on the spawning areas and on larvae migration.
The western part of Indonesian Sea is influenced by current SECC that developed during the whole year and lies just crossing the equator (Wyrtki 1973).
Anguillid leptocephali could have been transported into the sampling area from further offshore -around Mentawai-by the flow of the SECC to south and north Sumatra (Aoyama et al. 2007).This current flow generally towards south-east along the coast of Sumatra continued along the coast of Java (SJV).In this study, the eels entering to the rivers of this coast were collected, forming populations which can be found all around the year.Indonesia is strongly influenced by the ITF current.The Indonesian through flow (ITF) is a current entering Indonesia waters from the Pacific, flowing to the Indian distribution patterns of eel has been widely adopted for temperate Anguillid case, but it is not always applicable to tropical eel.
The distribution range of freshwater eel is linked with the distribution of their leptocephali and with the current system in their habitat (Miller 2003).
Temperate eels appear to make longer spawning migrations to spawn in low- Pacific and Indian Oceans, so surface heat fluxes, thermo cline and variability in thermocline waters and patterns of Indonesia current was influenced by Pacific and Indian Ocean fluctuations.The eastern part of Indonesia waters influenced by the Indonesian throughflow (ITF) current.ITF are water mass flow passing through Indonesian waters from Pacific to Indian Ocean.This water mass flow occurs as a result of the pressure difference between the two oceans.The water mass drives upper thermocline water from the North Pacific through the western route of the Makassar Strait directly exit through the Lombok Strait or flow eastward into the Banda Sea and further joined with the south equatorial current (SEC) (Wirtky 1973; Gardon 2005).The western Indonesian waters affected by South Equatorial Counter Current (SECC), and this current going down the Sumatran coast entry to southern coast of Java by South Java Current (SJC).
photographed by a Canon camera digital.
Another species has a wide distribution.The shortfin A. bicolor, according to its geographic distribution, is divided into two subspecies, A. b. bicolor in western Indonesia, in waters connected with Indian Ocean and A. b. pacifica spread in the east of Indonesia receiving Pacific Ocean waters (Fig.5a).A. b. pacifica has been recorded in the north of Sulawesi, eastern of Borneo and Maluku waters (Fig.5a).This paper was constituted the first report of the presence of A. b. pacifica in Borneo and Maluku.The abundance of A. b. pacifica in eastern Borneo is so high, the harvest of this species has been done intensively by fishermen for economic purposes.

Fig 5 .
Fig 5. Distribution of the seven species and subspecies of freshwater eels which were found around Indonesia during this study: a) A. b. bicolor and A. b. pacifica, b) A. marmorata, c) A. interioris, d) A. celebesensis, e) A. borneensis, and f) A. n. nebulosa.The current flow pattern in Indonesia waters, probably influencing the spread of eel larvae, is given through simplified arrows, adapted from Wirtky(1973,  2005).ITF (Indonesian Troughflow), SEC (South Equatorial Current), SECC (South Equatorial Countercurrent), SJC (South Java Current) While in the eastern part of Indonesian Sea influenced majority by current ITF.The first flow of ITF was through the Makassar Strait with one branch entering the Indian Ocean through the Lombok Strait, while the bulk of the Makassar transport turns eastward in the Flores Sea, into the Banda Sea, eventually passing into the Indian Ocean on either side of Timor.The second flow of ITF passages east of Sulawesi and connections of the Banda Sea to the Indian Ocean also on either side of Timor(Gardon 2005).Thus the present study was conducted sampling of specimen along the flow through by ITF.Anguilla bicolor distributionThe semi-multiplex PCR assay was proposed byFahmi et al. (2012)  has been successfully used to distinguish between A. b. bicolor and A. b. pacifica.Previously publications only distinguished both species through their geographic distribution, even though when they used molecular approaches (Aoyama 2007).Using semi-multiplex method, in this study clearly showed that A. b. bicolor was only found in waters connected to the Indian Ocean and A. b. pacifica to the Pacific ocean.The results showed that there was no overlapping area between both subspecies (Fig 5a).This sub-species distributions contrast with that found by Sugeha et al 2008 who recorded A. b. pacifica in the western of Sumatra and south of Java waters by using RFLP-PCR for taxa determination.In accordance with the discovery of leptochepali of A. b. bicolor by Aoyama et al. 2007, in the waters at the west of Sumatra constitute spawning area of this subspecies.The discovery of young leptocephali (TL 44-55 mm) of A. b. bicolor by Aoyama et al. (2007) during their expedition in the west of Sumatera Island, indicated that the spawning area of this subspecies was somewhere in front of the island.The leptochepali of A. b. bicolor are then passively transported by the flow of SECC and SJV into the southern region of Java (Herunadi 2003; Aoyama et al. 2007).The age of glass eel of A. b. bicolor entering Cimandiri river estuary, Pelabuhan Ratu has been determined (at least 118-262 days) by using otolith microstructure (Setiawan et al. 2001).Calculating back the passive migration of leptocephali, taking into account the larvae phase duration and the speed of the flow of SJC, it could be estimated that spawning area of A. b. bicolor was around Mentawai Island (Haerunadi 2003).This author found glass eel of A. b. bicolor in the south west coast of Sumatra, in Bengkulu and Lampung rivers, in the south western coast of Java, in Malimping and Cimandiri rivers, and abundant A. b. bicolor glass eels on the south eastern coast of Java, in Cilacap river and in the northern part of the Sumatra, in Aceh river.It is most probable, based on the whole data obtained on A. b. bicolor, that this species is locally constituted by a single population using a single spawning area.This is supported by the researches conducted by Minegishi et al. (2012) who described the genetic population structure of A. bicolor, showing that A. b. bicolor is genetically homogeneous in the whole Indian Ocean.In the eastern part of Indonesia, A. b. pacifica is widely distributed but less abundant.Allopatric isolation between two Oceans have probably split A. bicolor into two subspecies.According to Wouthuyzen et al. (2009) spawning area of A. b. pacifica occurs at the north of Sulawesi Sea.Arai et al (1999) indicated that their leptocephali can only be sampled in January, March, April, October and December.Similarly with A. b. bicolor, the population structure analysis of A. b. pacifica showed no significant genetic divergence within its south Pacific Ocean distribution (Minegishi et al. 2012).The wide distribution of A. b. pacifica at the east of Indonesian Sea may be due to dispersive transportation of leptocephali in the whole eastern Indonesia.The dynamics of water currents in eastern latitude westward-flowing current such as the North and South Equatorial Currents, and their leptocephali have been collected far from their freswater habitat.Whereas the tropical eel have the unique pattern distribution and migration such as: they have short migration distance, short metamorphosis duration, they spawning whole the years and some of them widely distribution and the other narrow.So, in several things the tropical eel different from temperate eel, the other hand, until now the theory of migration, distribution patterns and evolution of eel has been widely adopted from the temperate Anguillid.Future study on the of distribution pattern, migration theory and evolution of tropical eel is needed and it is most important to complete information of biological eel.

Table 4 .
List of sampling locations of tropical eels in Indonesia