GENETIC DIFFERENTIATION OF THE KAMPAR RIVER ' S GIANT FEATHERBACK ( Chitala lopis BLEEKER 1851 ) BASE ON MITOCHONDRIAL DNAANALYSIS

Although the giarrt featherback Chitala /opls is an important fish in Kampar River, the population structure has not been investigated. In this study, genetic diversity and population structure of giant featherback were examined using nucleotide sequence analysis of mitochondrial DNA control region for 54 fish collected from Kampar River. As a comparison, samples from Barito River (South Kalimantan Province), Penyak River (Bangka-Belitung Province), and Indragiri Hilir (Riau Province) were also used in this analysis. The Kuala Tolam (lower reaches of Kampar River) samples had higher nucleotide diversity (6) (0.0033)than Kutopanjang Reservoir (0.0011) and Teso (0.00)(upper reaches of Kampar River), the nucleotide diversity all samples varied from 0.000-0.0033. The genetic differentiation and genetic structure among Kampar River's giant featherback were also supported by pairwise F-statistic value and hierachical analysis of molecular variance, indicating that the Kuala Tolam population is genetically isolated from the population in upper reaches of Kampar River. KEW\IORDS: giant featherback, control region, mitochondrial DNA, genetic variation, Kampar River


INTRODUCTION
Indonesian giant featherback is a member of Notopteridae family (Kottelat et al., 1993; 1997), which has economic value and culture.This fish is very popular because its meat has delicious taste and distinctive primarily because of high fat content (Sunarno, 2002), as well as protein content and high vitamin A (MnO, 2005), placing giant featherback as exclusive and expensive freshwater fish with enough price (more than Rp.50 000/kg).As an illustration, the demand for giant featherback homemade industry ofabout 200 kg/day and used for ornamental fish and the consumption of 40 kg/day.While fishermen just only supply less than 2% (Anonymous, 2003).
Giant featherback fish oroduction decreased d ue to the fishing activities for consumption and ornamental fish uses.Giant featherback annual production has been declined, both at the national level (8,000 tonnes (1991), 5,000 tons (1995), and 3,000 tons (1998) (Directorate General of Fisheries, 2000)), and regionally (Sungai Kampar, Province Riau).The annual production of giant featherback in the Kampar River has been decreased, from 50.2 tons (in 2003) to 7.6 tons in 2007 (Agency of  Marine and Fisheries, 2008).This condition seems to be continued since the demand of this fish is continuously for human need.Consequently, the giant featherback species would be edangerous, while few information is available on the genetic variation and population differences among giant featherback populations.
Actually, almost all genetic studies of giant featherback were based on morphological characteristics (Sunarno et a|.,2007;Wibowo, 2008a)   with the exception of Madang (1999) and Wibowo (2008b), which simply studying giant featherback genetic.There were no researches related to the variation of giant featherback populations in large geographic areas (along river), a more detailed level, sequense DNA.
Knowledge of genetic population is important in planning and implementing appropriate management strategies for the giant fetaherback that is more and more endangered.Furthermore, efforts regarding to reduce pressures on natural populations through domestication and increase production through selective breeding, the basic information related to genetic conditions of this species is neccessary.Here, the genetic differences in geography are analyzed to provide a clear picture of the structure of giantfeatherback populations in Kampar River, Riau Province.The results can provide important biological information as basic information, necessary to plan proper policy and management of giant featherback in the Kampar River.

Fish Samples
Total of 51 rndrviduals were collected from five locations in Kampar River, details of each location and genetic data show in  has been previously stored in absolute ethanol, using genomic extraction kit GENEAID.DNA was purified, and then dried in 100 uL dissolve in the elusion buffer and stored in refrigerator-20"C.Some regions of mtDNA control region fragment were amplified using species specific primers of L-15 940-Thr (S'-MGG TGTMTCCGMGATTG-3') and CR- H (5-TAACGAACTTATGTACGACG-3) (Takag i ef a/., 2006).PCR performed with a thermal cycler BIOER.
30uL PCR reaction mix containing 1.2 units of Tag DNA polymerase (Kappa), 1 x reaction buffer, 5 mM MgClr, 0.4 pM primer, 250 pM dNTPs, and 50 ng genomic DNA.PCR cycles included a pre-condition of 3 min denaturation at 94'C and 28 cycles of 15 sec 94"C, 15 sec 70 sec at 48'C and 68"C, followed by final extension for 5 min at 72"C.PCR product then sent to DNA sequencing services, 1st Base, Singapore for the sequencing.In each sample, sequence done in both directions (forward and reverse), so there are total 108 single-pas DNA sequences fitted for 54 individuals.

Sequence Variation and Haplotype Distribution
Nucleotide sequences were obtained from all 51 fish individuals, sequence length varied from 566-936 bp.The length difference was caused by different numbers of tandem repeat segments.Since there was very few signal for population separation in number of tandem repeat segments (Table 1).The tandem repeat segments were removed and the remaining 560 bp sequences were used for subsequent population analysis.Result analysis from 48 fish specimens in Kampar River, reveal the existence of 8 haplotypes which formed from 10 variable sizes and 3 speciation haplotypes (the very ditferent Kampar River's giant featherback haplotypes formed by more than 118 50 variables site) identified from 3 specimen Kampar River's giant featherback.
Haplotypes composition and the position of bp variation of Kampar River's giant featherback based on control region mtDNA presented in Table 3 and 4.Among the 8 haplotypes, two haplotypes shared in different samples (shared), t haplotype is found in all sample locations; another six are unique of haplotypes.Kutopanjang reservoir sample has 1 site specific haplotype; meanwhile Kuala Tolam has Tolam samples.The haplotype distribution map is 5 site specific haplotypes.Speciation haplotypes shown in Figure 1.
AMOVAAnalysis indicates that the proportion of total genetic variation was found within samples (92.88o/o),only (7.12%) the variations is found among samples (Table 5), and there is a difference between 5 samples location eventhough the magnitude is small.Fixation index (Fst) and slatkin's genetic distances (d) between paired samples shown in Table 6.These data inform that there is a significant genetic difference (base on 10.000 permutation test) between samples from Kuala Tolam with Kutopanjang Reservoir and Teso site (Fst=O.1121and Fst=0.0713;d=0.126 and   d=0.0767).Sample between Kutopanjang and Langgam location have the most genetic similar (Fst=-0.037),while samples between Kuala Tolam with Teso and Kutopanjang reservoir have most different genetically (Fst=O.1121 and Fst=O.0713).
Fixation index (Fst) (bottom) and genetic distance (above) five samples of giant featherback in Kampar River Remarks: * values were significant on '10.000 permutation test -P<0.05)Samples Kuala Tolam seems to be a unique, when the four upper stream samples (Rantau Baru,   Langgam, Teso, and Kutopanjang) were grouped and compared to a group consisting of Kuala Tolam samples.The amount of variance between the two groupswas a large 24.85% (Table 7).On the contrary when all river samples (Rantau Baru, Kuala Tolam, Langgam, and Teso) were grouped and compared to a group reservoir consisting of Kutopanjang samples.
The amount of variance between two groups was a relatively small -6.58 (Table 7).

AA/n\
Remarks: * values *"r" .ignifi""nt on 10.000 prrmation test *P<0.05)These analysis reveal the existence of two reproduction units of giant featherback in Kampar River; one population was in upper reaches of Kampar River which is representative by samples from Kutopanjang, Rantau Baru, Langgam, and Teso and downstream that is representative by samples from Kuala Tolam.This is apparently due to isolation by distance, that gen flow may be restricted among giant featherback population in Kampar River.The same phenomenon (in which more distant location of the sample the greater the genetic differences) was observed by Takagi et al. (2006) in bronze featherback Notophterus nothopterus in the Mekong River, Thailand (Fst -002-0.08),that genetic similarity in Macrobrachium nipponense is associated with the distance of sample location.
In general, the active movement of adult giant featherback and passive movement of eggs and larvae would accelerate the occurrence of gene flow among populations (Slatkin, 1987).However, the low u -6.58 0.09484 0.03527ns -0.065 swimming ability (this could be seen from the shape) of adult giant featherback who live in the standing waters and slow moving of habitat type in the lake, Oxbow marshes, ditches and ponds (Rainboth, 1996) and eggs that are not easily spread out (fish eggs attached to solid submerged vegetation) (Talwar &   Jhingran, 1991), give a consequence, gene flow will be inversely proportionalto the sample location.
In this work, giant featherback samples from Kuala Tolam (downstream) are a distinct population, different from the other samples in the upper reaches the Kampar River which is another population.On the management of water resources, it is important to manage each population separately.The results of this study is clearly reveal that presence of two reproductive units of giantfeatherback in Kampar River water basin are genetically separated.Each giant featherback populations should be managed and evaluated separately for sustainable utilization.Morphology performance of Kampar River's giant featherback lives in acid water (2 and 3) charachteristic by haplotype speciation 1.
Kampar River giant featherback haplotype ind.Attributed with red colour and motif pattern in speciation 2 known by local fishermen as Kumbuhan.caudalfin, the giant featherback with this size is never The size of Kumbuhan can reach more than 30 kg/ reported before (Figure 4).Morphology performance of Kumbuhan, giant featherback that can reaches more than kg/ind.attributed with red colour and motif pattern in caudal fin.
Phylogeny tree inform, the existence of genetic Kampar River's giant featherbacks have different structure along the river (Kampar River) and in haplotype properties than lndragiri River (Riau different river (example Kampar River and Indragiri Province), Penyak River (Bangka Belitung Province) River).In Kampar River, giant featherback lives in and Barito River (South Kalimantan Province).
downstream (Kuala Tolam) has a significant genetic difference with the giant featherback that lives in In order to know ancestral haplotype, haplotype upper reaches of Kampar River (Teso, Rantau Baru, position, and frequency were constructed using Langgam, and Kutopanjang).However, the genetic haplotype network, sees in Figure 5. Base on network structure is not as clearly as another fish, such as construction, Kampar River's giant featherback has Genetic polymorphism, from mtDNA prespective determine population health in natural population.As more variety of its mtDNA then the population has more main prime (mother) and more healthy the population.Base on this statement Kuala Tolam samples are the healtiest population, they have 7 mother (7 haplotype), include haplotype speciation 1.On the contrary giant featherback from Teso samples, they are the lowest population health because they only have t haplotype.CONCLUSIONS 1.There are 8 haplotypes of Kampar River's giant featherback which formed from 10 variable sizes and 3 speciation haplotypes ( very different Kampar River's giant featherback haplotypes formed by more than 118 variables site) identified from 3 specimen of Kampar River's giant featherback. 104bp have not yet repoded (in the position 16,143-16,247).

Table
1.As a comparison, samples from Barito River(South Kalimantan

Table 1 .
Number tandem repeat on giant featherback control region mtDNA

Table 5 .
Analysis of molecular variance for giant featherback samples in Kampar RiverSource of variation d.f.Sum of squares Variance components Percentage of variation