First genetic record and phylogenetic relationship of Rasbora einthovenii (Bleeker, 1851) (Cyprinidae : Danioninae) from Bangka Island, Indonesia

. Brilliant Rasbora ( Rasbora einthoveni ) called “Seluang” is the one of local fish in Bangka Island. This fish widely distributes from Malaysia Peninsular, Sumatera (North Sumatera-Lampung Province), Kalimantan and the record of morphological character is until Sri Lanka, Laos to Thailand. The genetic status of this fish was unrecorded from Bangka Island. This research aimed to record the genetic status of Rasbora einthovenii compared with the same species in another region using cytochrome oxidase c subunit one (COI) gene. Analysis of maximum likelihood phylogenetic reconstruction of the Kimura 2-parameter model, which is implemented in MEGA 10 with 1000 replicates bootstrapping data sets. The result showed that phylogenetic tree of Rasbora einthovenii from Bangka Island is related closely with the sample from Selangor, Malaysia, and Aquarium Retailers in UK with the genetic distance about 0% means very similar. The samples from Pahang, Malaysia showed the genetic distance is about 3% and samples from Jambi, Indonesia is about 6%. It means that the genetic profile of Rasbora einthovenii from Bangka Belitung Island are the same species as well as from Selangor, Malaysia also the samples from Aquarium Retailers in UK.


Introduction
Rasbora is a genus of pelagic, freshwater fish related to carp, which are relatively small, elongated, and slightly flat, with a geographical distribution covering India, southern China, Indochina, the Sunda Shelf, and the islands of Palawan and Mindanao in the Philippines [1].Rasbora generally lives in almost all types of freshwater habitats and is dominant throughout its distribution area, especially in the Sunda Shelf.One of the genera of Rasbora is Rasbora einthovenii (Bleeker, 1851), this fish is native to the fresh waters of Indonesia and widely distributed in Southeast Asia.This fish's distribution covers Peninsular Malaysia, northern Sumatra, Lampung, and Kalimantan.It is recorded morphologically in Sri Lanka, Laos, and Thailand.Known in some areas as "Seluang", while internationally, it is called Brilliant Rasbora.
Rasbora einthovenii (Bleeker, 1851) is a fish that can live in waters with a pH range of less than 3 to 7 with a temperature range of 22-27 o C and is an extremophile fish category [2].So that this fish found in almost all freshwater areas of Bangka Belitung Islands, including 'kolong' (ex-tin mining).The presence of this fish on Bangka Island is still not widely utilized, but it has the potential as a local ornamental fish, and some people still consume it.Some research on the use of this fish has reached the development of aquaculture [3], trials on cultured conditions with various environmental parameters [4], induction of gonadal maturation through topical gill and oral [5,6].
A population's genetic information is needed to improve seed quality, manage ecosystem biodiversity, and manage genetic diversity in species gene pools [7].This basic information is a reference to determine which species were selected as candidates in the development of domestication and conservation for both aquaculture and aquatic biota conservation efforts [8].Complete species information is needed to ensure that the development of fish species is appropriate and correct.Molecular identification can help identify of this fish species.The cytochrome c oxidase subunit I (COI) gene is the protein-coding gene used to identify the species.COI is a short gene sequence selected from among the many genes used as normal genes for the specific identification of animal species based on DNA barcoding [9].Genetic records of Rasbora einthovenii (Bleeker, 1851) available at the National Centre for Biotechnology Information only come from a few regions in Malaysia (Sarawak, Pahang, and Selangor), Sumatra (Jambi & Riau), Kalimantan (Central & East Kalimantan) and Aquarium retailers in the UK [10].Especially on the Bangka Island, there is no genetic record of this fish in the Gene Bank, so this research could produce the first genetic record.

Materials and methods
The ten samples of fishes were caught using trap fishing gear located in the waters around Balunijuk Village, Merawang District, Bangka Regency.The collected fish samples were then stored in 96% ethanol [11], and sample preparation was carried out for genetic analysis.The fish caught do not require special permits because they are in the Least Concern (LC) status based on the list on the IUCN Redlist [12] and there is no conflict of interest in this research or the samples of fishes.

Molecular identification
Molecular identification begins with the process of tissue destruction and purification of fish DNA.The DNA extraction and purification using a silica column consisting of 4 stages, including lysis, binding, washing, and elution, followed by the DNeasy Blood and Tissue Kit procedure.Part of the fish body was extracted, namely pieces of meat weighing 5-10 mg.The amplification of the COI gene (Cytochrome c Oxidase Subunit I) used a forward primer FishF2_t1 and a reverse primer FishR2_t1 [9].DNA amplification was treated with initial denaturation temperature at 95 o C for 1 minute, denaturation at 95 o C for 15 seconds, annealing at 48-54 o C for 15 seconds, extension at 72 o C for 10 seconds (35 cycles), and final extension at 72 o C for 10 minutes [13].
The mixture of genetic materials used is GoTaq®Green Master Mix (25-50 μl), forward primer (0,5-5 μl), reverse primer (0,5-5 μl), DNA extract (1-5 μl), and Nuclease Free Water (50 μl).These materials are put into Eppendorf and run according to the above temperature treatment.Furthermore, the electrophoresis process moves charged molecules in an electric field to separate macromolecules (such as proteins and nucleic acids) [14] using 1% agarose gel.The DNA sequencing process was carried out to analyse the DNA sequences of the PCR amplicon results with an automated DNA sequencer after purification.Cycle sequencing was carried out using the Big Dye TerminatorR v3.1 kit (Applied Biosystem).The results of the BLAST homology sequencing were performed by entering the genetic code into GenBank.Sequencing results in the form of base sequences were analysed using GenBank database, which is under the NCBI (National Centre for Biotechnology Information).The database is a primary database that holds all the DNA sequences deposited by researchers worldwide.Search technique based on the relationship between data using the search program contained in the database.Two programs that are widely used are Basic Local Alignment Search Tool (BLAST) and FASTA.

Molecular phylogenetic analysis
The Tamura-Nei model-based Maximum Likelihood method was used to infer the evolutionary history [15].The highest log likelihood tree is shown (-2339,6613).The branches show the percentage of trees in which the associated taxa clustered together.A matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach was applied to automatically generate the initial tree(s) for the heuristic search, and the topology with the superior log likelihood value was then selected.With branch lengths measured as the number of substitutions per site, the tree is scaled.There were 16 nucleotide sequences in this analysis.Positions containing gaps and missing data were all eliminated.The final dataset had 617 positions overall.Applications using MEGA 10 conducted evolutionary analyses.[16].

Results and discussions
The phylogenetic tree construction showed that Rasbora einthovenii (Bleeker, 1851) from Bangka Island clustered in the same clade as Rasbora einthovenii (Bleeker, 1851) from Aquarium retailers in the UK and Selangor, Malaysia, with a similarity reaching 100%.It has shown that the fish samples obtained are of the same population and were found on Bangka Island.The genetic gap of Rasbora einthovenii (Bleeker, 1851) specimens from Aquarium retailers in the UK and Sarawak, Malaysia, is 0%.According to [17], a value below 2% indicates a very high genetic similarity between 98-100%.This population is identically very similar in terms of morphology, but needs to be proven by looking at morphometric characters and body shape [18].The samples of Rasbora 1260 (2023) 012002 IOP Publishing doi:10.1088/1755-1315/1260/1/0120024 einthovenii from Pahang, Malaysia showed the genetic distance is about 3,5%, the samples from Jambi, Indonesia is about 5,3%, the samples from Kalimantan Tengah, Indonesia is about 9,8%, the samples from Serawak, Malaysia is about 9,6% and from Kalimantan Timur is about 7,8%.The Chana striata from the Malay Peninsula, Sumatra, and Borneo show on the same branch with low bootstrap values, indicating that the same freshwater fish species may have high genetic distances between individuals separated by distance and geographic barriers.[19].The genetic distance between the Mystus singaringan species from Java and Thailand in the Asia continental region, which are more than 2.000 kilometers apart, varied from 14 to 26%.[20].

Conclusion
The genetic profile of Rasbora einthovenii (Bleeker, 1851) from Bangka Belitung Island is the same species as well as from Selangor, Malaysia also the samples from Aquarium Retailers in UK.The samples of Rasbora einthovenii from Pahang and Serawak, Malaysia, Jambi, Kalimantan Tengah, and from Kalimantan Timur, Indonesia have a genetic distance is about 3,5-9,8% compared with samples from Bangka, Indonesia.Genetic records of Rasbora einthovenii (Bleeker, 1851) indicate that no fish samples have been collected from the island of Bangka and become the first record.

Figure 1 .
Figure 1.A maximum-likelihood phylogenetic tree with 1000 bootstrap replicates.Specimens from Aquarium retailers in UK and Selangor, Malaysia are clustered together with a strong bootstrap value (100%).Rasbora daniconius is the genus of Cyprinidae used as outgroup.

Table 1 .
The distance of Rasbora einthovenii sequences from Bangka with other species in Rasbora with Cytochrome c oxidase subunit I (COI) gene.