DNA Authentication of Indonesian Leaffish Pristolepis grooti from Kelekar River and Ogan River in South Sumatra Based on Cytochrome C Oxidase Subunit I (COI) Gene

Indonesian leaffish Pristolepis grooti , an endemic species, are distributed in the region of Sumatra, Riau, Bangka Belitung and Kalimantan. However, there has been a decline in the population recently. This research purposed to investigate the mitochondrial DNA cytochrome c oxidase subunit I (COI) gene, the ge-netic distance, the genetic tree of the leaffish and characterize the chemical physics of water of its habitat in the Kelekar River Muara Enim Regency and the Ogan River, Ogan Ilir Regency. The method used in species authentication was DNA isolation, amplification using PCR (Polymerase Chain Reaction) and sequencing of COI gene. The size of the COI mtDNA gene fragment was 704 bp (PM 1, PM 4, PP 2 and PP 4) and 723 bp (PM 2, PM 3, PP 1 and PP 3). A cryptic diversity of the species P. grooti is found based on the genetic distance value of 4.5-6%, both in the Kelakar and Ogan Rivers. The phylogenetic tree of the leaffish of this study formed 2 separate sub-clusters with a bootstrap value of 50%. The properties of water qualities in the two rivers included temperatures 28.3-31.8°C, pH 5.6-8.3, dissolved oxygen 4.82-10.89 mg L -1 , alkalinity 10-28 mg L -1 CaCO 3 , water transparency 16-45 cm, ammonia 0.47-0.70 mg L -1 , water current 0.17-0.30 m s -1 and TDS 7-44 mg L -1 . Copyright: © 2023, J. Tropical Biodiversity Biotechnology (CC BY-SA 4.0)

0.014) in Kelekar floodplain, Ogan Ilir Regency (Muslim & Syaifudin 2022). Identification and characterization of leaffish are essential, as they are related to the adaptability, cultivation techniques and conservation of the leaffish in its natural habitat, which is still very limited, so thus it is important to apply a relatively easy and more accurate method to identify the species using the molecular technique, relatively. One of which is through DNA barcoding by using mitochondrial DNA that matches the target for analysis on various species-level target genes (Masters et al. 2007). The samples of leaffish for DNA barcoding were taken from the Kelekar River, Muara Enim Regency and the Ogan River in Pemulutan, Ogan Ilir Regency.
Cytochrome C oxidase subunit 1 (COI) is one of the genes in the mitochondrial genome (mtDNA), which is precisely used as a barcode. This molecular technique can be used for determining genetic analysis systematically and at the taxonomic level of species, populations and also plays a role in the selection process of fish genetic diversity. The application of DNA barcoding has been conducted on several aquatic organisms i.e Hemibagrus nemurus (Syaifudin et al. 2017), flatfish from Vietnam (Truong et al. 2020), snake-skin gourami, blue gourami (Syaifudin et al. 2019), Pristolepis fasciata from Malaysia (Noikotr 2019) striped snakehead, catfish Mystus singaringan (Pramono et al. 2019), and ocellated snakehead (Syaifudin et al. 2020). In this study, the application of DNA barcodes has an important role in determining COI gene sequences as a taxonomic tool to reveal the genetics authentication of species among Indonesian leaffish from the Kelekar and the Ogan River in South Sumatra.

MATERIALS AND METHODS Materials
Indonesian leaffish ( Figure 1) and water samples were taken from two locations ( Figure 2) i.e, Kelekar River in Segayam Village, Muara Enim Regency (given the PM code) and the leaffish from the Ogan River in Pemulutan District, Ogan Ilir Regency at sampling coordinates 3°03¢17¢ ¢S 104°46¢32¢¢E (given the PP code). Four individuals were taken from each river. All specimens were collected in the wild and determined morphologically in situ by visual observation based on manual identification (Saanin 1984). The length of leaffish from the Kelekar River ranged from 6.6-10.9 cm and weighed of 4.37-34.12 g while the leaffish from the Ogan River measures 11.5-13.5 cm in length and 28.66-50.53 grams of weight. Fin samples were put into a 1.5 ml tube with a 96% ethanol solution, then stored in a freezer at -20ºC until DNA isolation was carried out.

DNA Isolation
The total genome of the leaffish DNA was isolated using the extraction kit of Genomic DNA (GeneAid) as described in the protocol. The DNA was extracted in six stages, i.e. the preparation of specimens, the lysis of cell, RNAse addition, DNA precipitation, cleaning and DNA dissolution. The extracted DNA of fin samples were stored in a freezer (-20 o C), until the electrophoresis process was carried out to check DNA integrity and PCR (Polymerase Chain Reaction).
DNA Amplification DNA genome was amplified using Polymerase Chain Reaction (PCR) to target a 655 bp of COI gene with primer pairs of FishF2 (forward)-5' TCGACTAATCATAAAGATATCGGCAC 3' dan FishR2 (reverse)-5' ACTTCAGGGTGACCGAAGAATCAGAA 3' (Ward et al. 2005). PCR was performed in a final volume of 50 µL. Each reaction contained 17 µL ddH 2 O, 25 µL My Taq Red Mix, 10 µL Fish R2 primer (10 pmol/µL), 10 µL Fish F2 primer (10 pmol/µL) and 6 µL DNA template. DNA amplification was carried out in stages: initiation cycle at 94 o C for 1 minute in 1 cycle, denaturation at 94 o C for 30 seconds, annealing at 52 o C for 30 seconds, extension or elongation at 72 o C for 15 seconds in 35 cycles and post extension 72 o C for 4 minutes in 1 cycle. Furthermore, the PCR product was visualized by electrophoresis of 1% agarose gel in 75 voltage for 35 minutes. The size of the DNA from the PCR was measured using a 1 kb DNA marker. The PCR products were sequenced bi-direction with both primers through the services of PT Genetics Science (Jakarta).

Data Analysis
The COI gene sequences in fasta format are then aligned using MEGA X software, then continue to BLAST (Basic Local Alignment Search Tool) for determining the homology of a DNA sequence with the data in NCBI (National Center for Biotechnology Information). Furthermore, all sequences were aligned to analyze the genetic distance and phylogenetic tree, including a sequence of Oreochromis niloticus (GenBank: KM438528) from Stirling collection as an outgroup. The phylogenetic tree was constructed using the Neighbor-Joining (NJ) method and the Maximum Composite Likelihood model on MEGA software X Version (Kumar et al. 2018;Stecher et al. 2020) and the genetic distance was analyzed using the Pairwise Distance method (Kimura 1980).

RESULTS AND DISCUSSION DNA Authentication
The amplified DNA of the COI gene of leaffish was electrophoresed using 1% agarose gel, showing a size of 704 bp for PM 1, PM 4, PP 2 and PP 4 samples code and 723 bp for PM 2, PM 3, PP 1 and PP 3. All sequences of PCR product have been submitted in Barcode of Life Database Identification (BOLD-ID) under the accession number BOLD:ADO0531 and BOLD:ADN7493. The COI gene sequence of the leaffish is analyzed through nucleotide BLAST on the website of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) for comparison with other species in the GenBank database. The percentages of nucleotide similarity of the leaffish were presented in Table 1.
Nine individuals of P. grooti were barcoded successfully using COI gene with universal primer followed (Ward et al. 2005) at annealing temperature optimization of 52ºC for 30 seconds in 35 cycles of PCR. Annealing temperature in the PCR used to usually calculated from Tm-5ºC to Tm+5ºC (Muladno 2010 There was none of the leaffish from the Kelekar River and the Ogan River 100% similar to other fish samples or the same species in Gen-Bank (Table 1). Pristolepis grooti is very similar to P. fasciata, with slight differences in the presence of 3½ rows (fasciata: 4½ rows) of scales separating dorsal fin mid spines from lateral line scales; ventral fin that does not reach the anal canal, coloration, and profile of the top of the head towards the nape which is more convex (fasciata: tends to be straight) (Weber & Beaufort. 1936).

Genetic Distance and Phylogenetics
The genetic distance of the leaffish from the Kelekar and the Ogan River with other species in the GenBank data was presented in Table 3.
The genetic distance of the leaffish P. grooti from both rivers showed a range of 1.6-8.4% with P. fasciata from Genbank database. Sam- ples PP 2, PP 3, PP 4, PM 1 have a genetic distance of 0.016 (1.6%) against PM3 and 0.045 (4.5%) to PM 4. The genetic distance of samples PM 1, PM 3, PP 2, PP 3 and PP 4 was 0.047 (4.7%) to P. fasciata from Malaysia (KT001055.1) and 0.049 (4, 9%) from Vietnam (MH721176.1). The genetic distance within population from Kelekar or Ogan River is around 4.5-6%. This finding showed cryptic diversity of the fish species. Cryptic species are taxa which are distinguished by unique genetic differences, distinctive ecological preferences and the complete or practically complete absence of morphological discrepancies (Bączkiewicz et al. 2017). Therefore, they can be distinguished with the use of molecular methods, as it has been done for sea cucumbers (Muliani et al. 2020), triplophysa (Wang et al. 2020), and Asian bronze featherback (Lavoué et al. 2020).
The genetic construction of the leaffish from the Kelekar River and the Ogan River is presented in Figure 3. The genetic tree of the leaffish consisted of four clusters, namely first cluster of research samples from the Kelekar, Ogan River and Pristolepis fasciata from Vietnam (accession code: MH721176.1) and Malaysia (KT001055.1, KT001054.1) with bootstrap value of 91%. There were three sub clusters of the first cluster. Samples PP1, PP2, PP3, PP4, PM1 and PM3 were in separate sub cluster from PM2 and PM4 (bv= 91). The second cluster was a group of P. fasciata from Thailand (MK448080.1, MK049487.1, MK628422, and MK 049486.1) (bv=98), the third cluster was P. rubripinnis from India, while the fourth cluster was an outgroup of O. niloticus (KM438528). The phylogenetic of the leaffish of this study formed 2 separate sub-clusters consisting of the first sub-cluster, namely PP 2, PP 3, PP 4, PP 1, PM 1 and  PM 3 with P. fasciata from Vietnam (MH721176) and Malaysia (KT001055) and the second sub-cluster, namely PM 2 and PM 4 with P. fasciata from Malaysia (KT001054) with a bootstrap value of 50%.
The genetic distance indicated that P. grootii differed genetically more than 3% than P.fasciata, genetically. The smaller the value of genetic distance denotes that the species is more related to other species and has a close relationship, while the greater the value of genetic distance indicates that the species is increasingly divergent. If the genetic distance is more than 3%, it indicates that in the group there are species that are different from other members, on the contrary, if the genetic distance value is equal to or less than 3%, it indicates that the group or cluster still comes from one species or the same species (Wong & Hanner 2008).
The construction of the phylogenetic relationship between species or related taxa was supported by the availability of a sequence database in many species (Hedrick 2005). However, when only one COI gene is used, a gene tree is produced, and it will not describe the broad evolutionary history of a group of species (Rubin et al. 2012). The genetic tree of all specimens of P. grooti in this study was separated from P. fasciata Thailand, but showed an indefinite cluster with the specimen from Malaysia and Vietnam. P. fasciata from Malaysia and Vietnam were located between two clusters of samples PP1, PP2, PP3, PP4, PM1, PM3 and PM2 and PM4 with low bootstrap value (bv=50). Bootstrapping is used to make inferences and evaluate the robustness of the tree (Holmes 2003). The data is relatively stable when the bootstrap value is greater than 70% (Lemey et al. 2009) relatively. Furthermore, P. grooti of this study also indicated different sub-cluster between Kelekar and Ogan River, where PM2 and PM4 from Kelekar River were separated from PM1 and PM3 (bv=91), where they belong to the same sub-cluster to PP1, PP2, PP3 and PP4 (originally from Ogan River). The proximity of the leaffish of the Kelekar and the Ogan River is interconnected because the Kelekar River flows into the Ogan River and empties into the Musi River (Wijaya 2001). The high similarity and genetic relationship of the leaffish are in line with the high level of diversity of these fish species that inhabit the Sunda shelf which consists of interconnected islands of Sumatra, Kalimantan, Java, Bangka Belitung, which are dominated by fish that inhabit peatlands and freshwater, especially those that are endemic (Hubert et al. 2008), so that mating occurs in the distribution of the leaffish, geographically.

Water Quality
The water quality of the habitat of the leaffish in Kelekar and Ogan River was presented in Table 4. the pH value of Kelekar and Ogan River ranged 5.6-8.3, temperature values of 28.3-31.8ºC, dissolved oxygen was between 4.82-10.89 mg L -1 , total dissolved solid (TDS) was about 7-44 mg L -1 . The water transparency value was approximately 16-40 cm, while the total alkalinity was about 10-28 mg L -1 . Mutagenic substances in the Reservoir 1 in the Canela National Forest have been altering the genetic integrity of the aquatic organisms that may be a threat for that aquatic ecosystem (Bühler et al. 2014). Furthermore, pollutants present in Madin Reservoir water were genotoxic and cytotoxic to C. carpio (Pérez-Coyotl et al. 2017).
Water pH media, dissolved oxygen and TDS in Kelekar and Ogan River were in accordance with the water quality standard class II for fishing activities, mainly for fish to grow and reproduce (Government Implementation of Environmental Protection and Management. 2021). The value of ammonia (NH 3 ) was 0.47-0.70 mg L -1 . This range was higher than required for fish production in freshwater based on the quality standard for grades 2 (< 0.2 mg/L (Central Government 2021). In this study, water current ranges from 0.17 to 0.30 m s -1 , indicated that the Kelekar River was classified as a slow current, while the Ogan River (0.19-0.21 m s -1 ) was categorized as a slow current and water current of 0.25-0.5 m s -1 was at medium current (Supartiwi 2000). The water transparency value was approximately 16-40 cm, while the optimal value for water transparency required for the growth of freshwater fish is around 30-60 cm (Cholik et al. 1991). The total alkalinity was about 10-28 mg L -1 and still supports the survival of the leaffish. The total value of good alkalinity in waters is between 5-500 mg L -1 CaCO 3 (Effendi 2003).

CONCLUSIONS
This investigation utilizes the COI barcode for the molecular authentication of endemic fish P. grooti from Southern Sumatra. These results indicated the leaffish had the highest identity to P. fasciata from Malaysia (KT001055) with different percentages of each sample ranging from 95.29 to 96.13%. The genetic tree supported that P. grooti in this study was separated from P. fasciata in Thailand, but showed an indefinite cluster with the specimen from Malaysia and Vietnam.

AUTHOR CONTRIBUTION
The study was designed by MSF. ETG and MW were conducted the laboratory works. The data was analysed by MSF and ETG. All the authors wrote original draft, edited and approved the manuscript.