Genetic variation and phylogenetic analysis of Indonesian indigenous catfish (baung fish) based on mitochondrial 12S rRNA gene

Background and Aim: Baung fish is an essential commodity in Indonesia; however, few studies have explored the genetic diversity of Indonesian catfish. Thus, this study aimed to analyze the genetic variation and phylogenetic relationships among Indonesian catfish based on the mitochondrial 12S ribosomal RNA (rRNA) gene. Materials and Methods: In total, 28 catfish were collected from nine rivers in seven provinces and from the Indian Ocean. Catfish genomes were obtained from epaxial and hepaxial muscle samples. The mitochondrial 12S rRNA gene was amplified by polymerase chain reaction using a pair of primers (Baung12SF and Baung12SR). The 12S rRNA sequences were analyzed using MEGA X to determine genetic variation and phylogenetic relationships. Results: In total, 178 variation sites in the 12S rRNA gene were substituted among Indonesian catfish. The genetic distance between all Indonesian catfish samples was 0.1-16.0%. The closest genetic distance was between MP and PM catfish, whereas the farthest genetic distances were between BF and EM and PD and EM. For the entire population, based on mean diversity calculations, the number of base substitutions per site was 0.08. Conclusion: Indonesian catfish were divided into four clades based on the 12S rRNA gene. The catfish MP, KR, PM, MS, BB, and KS were grouped with Hemibagrus nemurus, the catfish EM was grouped with Mystus vittatus, the catfish BSBJ was grouped with Pangasius pangasius, and the catfish PD and BF were grouped with Netuma thalassina.


Introduction
Indonesia is a country with enormous biodiversity (i.e., "mega biodiversity"). For example, around 16% of the world's fish species are found in Indonesia, and 2000 of these 7000 fish species are freshwater fish. Thus, the freshwater fish population of Indonesia is second only to that of Brazil [1]. Baung fish (Hemibagrus nemurus) is an essential commodity in Indonesia because it is widely consumed and contains essential nutrients. For example, baung fish are a valuable source of protein, lipids (with large amounts of omega-3, omega-6, monounsaturated fatty acids, docosahexaenoic acid, and eicosapentaenoic acid), minerals, albumin, and antioxidants [2][3][4].
The distribution of baung fish is relatively wide in the islands of Java, Sumatra, and Kalimantan [5].
The genetic markers of baung fish have been studied to identify species and preserve genetic resources; however, given the size of the aquaculture industry in Indonesia, relatively few studies have been conducted [7]. The diversity of the nucleotides of each species can be used as genetic markers, which can be used to construct phylogenetic trees and complement current molecular data that are currently lacking [8,9]. Mitochondria are membrane-bound cell organelles that generate most of the chemical energy needed to power biochemical reactions in the cell. Mitochondrial DNA contains 37 genes, all of which are essential for normal mitochondrial function. Transfer RNA (tRNA) and ribosomal RNA (rRNA) are types of RNA that help assemble amino acids into functioning proteins. The 12S and 16S rRNA mitochondrial genes are relatively conserved; they have evolved more slowly than the mitochondrial genome as a whole and can be used as genetic markers for the identification of species and in forensic investigations [10][11][12].
To date, few studies have explored the genetic diversity of Indonesian catfish. Thus, the present study aimed to characterize Indonesian catfish from different provinces (those known as baung fish by local people) and to determine the diversity among them using 12S rRNA gene sequences and comparisons with the available GenBank sequence. Moreover, the genetic variability of the 12S rRNA gene in catfish was measured to determine the variation and relationships among Indonesian catfish from different regions.

Ethical approval
This study was approved by the Animal Ethics Committee for using Animal and Scientific Procedures in Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia.

Study period and location
The collection of catfish samples was carried out from 2017-2020, but for the study of Genetic variation and phylogenetic analysis of Indonesian indigenous catfish (baung fish) based on mitochondrial 12S rRNA gene, it was conducted from January to September 2020 in the Laboratory of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Gadjah Mada University.

Catfish collections
Baung fish DNA was obtained from 28 samples of the epaxial and hepaxial muscles of fish from various rivers in Indonesia and from the Indian Ocean. Table-1 shows the origin, number, and code of the baung fish. All individuals were identified based on morphological characteristics and sample tissues were preserved in RNAlater buffer (Qiagen). The catfish samples in this study were considered to be unrelated genetically because they were taken individually from the rivers and ocean. Catfish were collected from the ocean to determine the relationship and genetic diversity between river catfish and sea catfish.

DNA extraction and 12S rRNA gene amplification
The total DNA of catfish was extracted using a gSYNCTM DNA Mini Extraction Kit (Geneaid Biotech Ltd., Taiwan) following the manufacturer's instructions and then stored at −20°C until use. The 12S rRNA fragments of the target region were amplified by polymerase chain reaction (PCR) using a pair of primers: Baung12SF: 5′-TAA CAC TGA AGA TGT TAA GA-3′ and Baung12SR: 5′-TAG CTA AAT CAT GAT GCA AA-3′. The PCR reaction was conducted in a total volume of 50 µL, comprising 25 µL of master mix (Kapa2G ReadyMix, 1 st Base), 2 µL of DNA template, 1 µL (10 pmol) of each primer, and 21 µL of distilled water. Reaction cycles in an Infinigen Thermocycler comprised an initial denaturing step at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 41°C for 45 s, and 72°C for 90 s, with a final extension at 72°C for 5 min. DNA amplifications were confirmed by 1% agarose gel electrophoresis with a 100 bp DNA ladder (Genaid) used for genotyping.

Sequences and phylogenetic analysis
All purified PCR products were sequenced directly by 1 st Base Sequencing INT using forward and reverse primers. The fragments of forward and reverse 12S rRNA gene sequences were aligned using ClustalW and edited, and then, multiple alignments were performed with data linked to H. nemurus and other catfish from the NCBI database. Fragments of the 12S rRNA gene were analyzed for 956 nucleotides. Genetic distance was determined using the Kimura two-parameter method and phylogenetic relationships were assessed through the neighbor-joining (NJ) method using MEGA X version 10.1 (https:// www.megasoftware.net) [13]. The bootstrap method for genetic distance analysis included 1000 replicates. A phylogenetic tree was constructed based on 12S rRNA sequences, and catfish sequences from other countries were used to reveal relationships and clusters among catfish. To construct the phylogenetic tree and determine relationships among catfish, the sequences of comparison species were obtained from the NCBI database: H. nemurus (KJ573466.1), Mystus cavasius (KU870465.1), Pangasius pangasius (KC572135.1), Pangasianodon gigas (AY762971.1), Arius arius (KX211965.1), and Netuma thalassina (MG587041.1).   Figures-1-3).

Genetic distance analysis based on 12S rRNA gene sequences
Baung fish from Kalimantan (MS, KS, and BB), Sumatra (MP and KR), and Java (PM) were identified as Hemibagrus spp. with a genetic distance of 0.1-6.0% (Table-2). Catfish EM and BSBJ were identified as Mystus spp. and Pangasius spp., respectively.
Fifteen sites were found in the nucleotide variation of the 12S rRNA gene that could be used as genetic markers among Indonesian catfish. Genetic markers distinguished baung fish from the Bomberay River, West Papua (BF1, BF2, BF3, and BF4), which were identified as Netuma spp. with a genetic distance of 5.2%. Fifty sites were found in the nucleotide variation that could be used as genetic markers for fish samples from the Bomberay River. The genetic distance between all samples of Indonesian catfish was 0.1-16.0%.
After averaging across all sequence pairs between groups, the number of base substitutions per site was calculated for the entire population as 0.08 (Table-2). Genetic distances were obtained by bootstrapping (1000 replicates) and analyses were conducted using the Kimura two-parameter model [14]. All ambiguous positions were removed for each sequence pair (i.e., the pairwise deletion option). Although the genetic distances between the Indonesian catfish samples in this study were from 0.001 to 0.161, the closest genetic distance was between catfish MP to PM, whereas the farthest genetic distances were between catfish BF and EM and PD to EM.

Phylogenetic relationships of Indonesian catfish
A phylogenetic tree of Indonesian catfish and other catfish from the NCBI database was constructed using the NJ method [15]. The optimal phylogenetic tree (sum of branch lengths=0.38391001) is shown in Figure-4. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches [16]. The tree is drawn to scale, with its branch lengths in the same units as those of the evolutionary distances used to infer it. The evolutionary distances are shown as the number of base substitutions per site. In total, 34 nucleotide sequences were analyzed. After all ambiguous positions were removed for each sequence pair, 970 positions existed in the final dataset. Evolutionary analyses were conducted in MEGA X [13].
The 12S rRNA nucleotide sequence was used to examine the phylogenetic relationship among all Indonesian catfish samples and some catfish from other countries (Figure-4). First, alignment was performed in ClustalW, and then forward and reverse sequences were edited in MEGA X. A phylogenetic tree containing 28 Indonesian catfish was constructed. In this tree, the Indonesian catfish were divided into four clades with the catfish from other countries. The  Available at www.veterinaryworld.org/Vol.14/March-2021/27.pdf catfish MP, KR, PM, MS, BB, and KS were grouped with H. nemurus, the catfish EM was grouped with Mystus vittatus, the catfish BSBJ was grouped with P. pangasius, and the catfish PD and BF were grouped with N. thalassina. These four groups were supported by bootstrapping at approximately 75-100% NJ.

Determining the species of Indonesian catfish based on the 12S rRNA gene
Throughout their distribution area in Indonesia, Hemibagrus spp. are the fish most widely consumed as food. Thus, species identification is important for the sustainable use of this species complex [17]. Much of the biodiversity in the Indonesian archipelago has yet to be identified and/or characterized including catfish.
Indeed, local Indonesian people typically use the same name for all types of catfish. Morphologically determination of species of catfish is difficult because they are highly similar in this respect. Thus, the genetic analysis provides more accurate information regarding the diversification and evolutionary relationships among species [18,19]. Such analysis is vital as catfish are found throughout the fresh and brackish waters of Asia and Africa, with more than 200 species known to exist in 17 genera, making catfish one of the largest fish families [20].
Mitochondrial DNA is popular as a target for species identification and the study of genetic diversity because it includes more mitochondrial DNA than nuclear DNA, has high variation, and lacks recombination [21]. Based on previous research by Megarani et al. [22] [23] identified freshwater fish in South Sumatra, such as baung (H. nemurus), beringit (M. singaringan), gabus (Channa striata), serandang (Channa pleurophthalma), and sepat (Trichogaster spp.), using the Cytochrome C oxidase subunit I (COI) mtDNA sequence; the COI mtDNA gene can be used to differentiate fish at the species level and shows effective and accurate species relatedness. Thus, both the 12S rRNA and COI mtDNA genes are recommended for the identification and analysis of genetic diversity between species [11,24].
The present study used a similar sample to that researched by Megarani et al. [22], but one group differed, namely, the PD sample. The type of mitochondrial gene studied also differed between the two studies. Here, all sequences of the 12S rRNA gene were blasted in the NCBI database; the results indicated that Indonesian catfish comprise four groups: H. nemurus, M. vittatus, P. pangasius, and N. thalassina/A. arius.

Phylogenetics and phylogeographics of Indonesian catfish
Based on research by Dodson et al. [25], the biogeographical history of Southeast Asia contributed to extensive admixture during the Pleistocene low sea-levels of genetic groups of an obligate the river catfish that isolated during periods of high sea levels. In this study, one type of catfish was taken directly from the Indian Ocean, that is, catfish PD. The 12S rRNA sequence analysis showed that this catfish had a close genetic relationship with catfish BF that originated from Papua Island, and these two catfish had a close genetic relationship with N. thalassina.
H. nemurus, allegedly from Southeast Asia, has previously been reported to have broad genetic subdivisions based on molecular phylogenetic analysis and phylogeography [17,23,25]. The results of the present