Phylogeography pattern of Lutjanus bengalensis (bloch, 1790) in indonesia waters and south china sea

. Lutjanus or snappers usually known as economical fish in Indonesia and worldwide. This research aims to observe the different phylogeography correlation of Lutjanus bengalensis in Indonesia Waters and South China Sea used COI gene marker. The primary data collected from fish port Pulo Aceh, Indonesia (5 sequences), while the population of others region such as Bali (3 sequences) and Maluku (3 sequences) was retrieved from NCBI GenBank. South China Sea represented by sampled from Taiwan (3 sequences) and Hongkong (3 sequences) that also retrieved from NCBI Genbank. In total 17 sequences were analysed the diversity of haplotypes 0-1 and the diversity of nucleotide 0-0.030. Based on this researched data, it will become basic data for spesies management for Lutjanus bengalensis in Indonesia.


Introduction
Snapper is a Lutjanidae family that inhabit tropical and subtropical waters [1].Based on the data cited in [2], there are over 113 species of Lutjanidae family over the world with 58 of them found in Indonesia waters to the South China Sea.One of the most frequently encountered snappers is the Lutjanus bengalensis [3,4].This species dominates the waters of the Indo-Pacific region [5].The term Indo-Pacific refers to a geographic area encompassing a significant portion of the waters around the Asian continent, including regions around Indonesia, Malaysia, the Philippines, Australia, Japan, and China [6,7].
According to Leliaert, Payo, Gurgel, Schils, Draisma, Saunders, Kamiya, Sherwood, Lin and Huisman [8] the Indo-Pacific is a highly productive marine region, harboring diverse fish species and unique marine ecosystems.The marine biodiversity in the Indo-Pacific waters is crucial for global ecological balance and supports the livelihoods and resources for millions of people living in the vicinity.The marine waters of Indonesia and the South China Sea are part of the Central Indo-Pacific region.L. bengalensis is often found in the waters of the Malacca Strait, the Java Sea, and the Banda Sea [9][10][11].The South China Sea is directly connected to Indonesian waters and borders several Asian countries, including Hong Kong and Taiwan [12,13].The South China Sea is considered snappers fishing ground, with Lutjanus bengalensis being one of the widely distributed species in these waters [14].
Due to the similarities and distinctiveness observed in these two marine regions, investigations into the genetic relationships among species exhibiting regional variations have become a compelling subject for exploration.Numerous genetic studies on population diversity have been carried out, encompassing from several species and population such as Katsuwonus pelamis and Thunnus albacares from North Maluku Sea [15], snapper species L. fulvus and L. kasmira from Indo-Pacific waters [16], and L. kasmira from China waters [17].Nevertheless, there is an absence of reported studies regarding the correlation between geographical factors and genetic diversity within the L.bengalensis population in Indonesian waters and the South China Sea.Phylogeography analysis has been applied to provided valuable insights into the historical biogeography of species and how they have responded to environmental changes over time [18].This field holds particular significance in comprehending how climate change and the destruction of habitats impact the distribution and biodiversity of life on Earth.Previous studies have delved into and recorded comparisons of phylogeographic patterns, such as those found in reef fish from the western Indian Ocean and Eden Bay, Arab [19,20], distributed populations of snapper L. jocu and L. analis from Atlantic Ocean [21], mackerel and tuna from Indonesia archipelago [22], reef fish Cephalopolis argus from Indo-Pacific sea [23], and L. kasmira from the Bengal Strait and Arabian Sea [24].The phylogeography investigation holds significance in uncovering the connection biogeography and the life histories of species, providing a foundation for the development of more effective management strategies.Species with close genetic ties can be incorporated into a unified management and conservation plan, while those with greater genetic divergence should be managed with distinct approaches.Consequently, the primary goal of this research was to assess the phylogeographic patterns of L. bengalensis between Indonesia waters and South China Sea populations, serving as fundamental data for devising an improved species management plan in Indonesia.

Collecting samples of tissues
This research employed primary and secondary data.Specifically, the research utilized primary data from Indonesian populations (Aceh) and secondary data sourced by NCBI Genbank for L. bengalensis samples in Indonesian population (Maluku and Bali).In addition, samples from South China Sea were sourced from populations in Hongkong and Taiwan (as depicted in Figure 1), and this data was also obtained by the NCBI Genbank.
A total of five L. bengalensis samples from Aceh population were collected at fish landing (Pulo Aceh) in Aceh Province, Indonesia in January 2023.These fish were documented and about 1 cm of their fin (pectoral) was aseptically excised before preserving the samples in a 96% ethanol solution following the Fish-BOL collaborator's protocol [25].The specimens were conveyed to the laboratory for additional examination.Comparative Phylogeography analysis utilized L. bengalensis sequences sourced Hong Kong and Taiwan, obtained from The GenBank as the secondary data.Figure 1 and Table 1 illustrate the origins of the sample sources.

The extraction of DNA
The process of DNA esxtraction followed established protocols using Cetyltrimethyl Ammonium Bromide (CTAB).Initially, finely chopped pectoral fin was placed in a 1.5 mL sterile tube also added 3 µl of Proteinase K and 700 µl of CTAB.After vortex in 15 seconds, it was cultivated at 60°C for 180 minutes.Following cultivating, mixed 700 µl of Chloroform Isoamyl Alcohol (CIA).Subsequently, the solution underwent centrifugation at 11,000 rpm for 900 seconds.The resulting supernatant was cautiously moved to a new 1.5 mL tube.Then, 100% ethanol was applied, followed by vortexing for 30 seconds and centrifugation at 12,000 rpm for 15 minutes.The supernatant was discarded, and the tube underwent rinsing with 70% ethanol.After inverting the tube and tapping it on a dry tissue to remove excess ethanol, it was left to air-dry at 20-22°C for 600 seconds.Following this, 60 µl of deionized water was added, and the DNA sample was ultimately inventory at -20°C for future utilization.

The Amplification using PCR method
The PCR protocol was executed using a mixture consisting of 25 µl of DNA template, 8.5 µl of ddH2O, 1 µl of F1 and R1 (forward and reverse) and 12.5 µl of Red Master Mix.The PCR process occurred in a PCR machine (Senso Quest Lab cycler) and comprised 30 cycles.Each cycle initiated with an initial pre-denaturation step at 95°C for 2 minutes, followed by denaturation at 94°C for 45 seconds, annealing at 52°C for 45 seconds, extension at 72°C for 60 seconds, and a final extension at 72°C for 10 minutes, concluding with cooling to 4°C [26].The PCR product was subjected to electrophoresis in a 2% agarose gel in the following manner: Two microliters of the PCR product were loaded into wells created in the agarose gel, and electrophoresis was carried out at 100 volts for a duration of 30 minutes.Subsequently, DNA visualization was achieved with UVITEC FIRERIDER V10 system.The DNA bands that appeared clean and well-defined were chosen for sequencing to First Base Laboratories, Malaysia.

Genetic analysis
The sequence data underwent editing and analysis through the utilization of MEGA 6.06.
BlAST genebank for validated the seqeuences.Kimura-2-parameter (K2P) model within the MEGA X software [27] for analysis genetic distance.The Maximum Likelihood (ML) approach with replicating 1000 bootstraps and the Hasegawa-Kishino-Yano model for the phylogenetic tree.Additionally, for analys the haplotype distribution used DNASP 5.10.

Result and Discussion
There was 17 mitochondrial COI gene sequences of L. bengalensis were obtained from five locations, namely Aceh (5 sequences), Maluku (3 sequences), Bali (3 sequences), Hongkong (3 sequences), and Taiwan (3 sequences).Haplotype diversity within these five populations ranged from 0 to 1, while nucleotide diversity ranged from 0 to 0.030.The genetic diversity of the grouper populations in Bali, Hongkong, and Taiwan exhibited high haplotype diversity (Hd) values, reaching 1, whereas the Aceh population had a moderate Hd value of 0.6 (Table 2).The genetic distance that show highest within-population was observed in the Taiwan with 0.032, while the Maluku population shows the lowest with a value of 0 (Table 3).L. bengalensis from all five populations fall within the same clade, indicating a common ancestry (Figure 2).Bali and Maluku populations showed connectivity.Ten haplotypes were derived from the 17 sequences, with haplotype 6 distributed in the Hongkong and Taiwan populations, while haplotype 3 was found in the Bali and Maluku populations (Figure 3).The research demonstrated that the greatest haplotype variety was identified in the Bali, Taiwan and Hongkong population with a value of 1 with 3,2,2 haplotypes, whereas the minimum haplotype variety was identified in the Maluku population with only 1 haplotype, while Aceh with 0.6.Based on [29] Haplotype variety within the range of 0.1 to 0.4 is considered low, while values between 0.5 and 0.7 are classify as moderate, and haplotype diversity ranging from 0.8 to 1.00 was classified to high class.Hence, the diversity of haplotype within the snapper inhabitants in Aceh falls into the moderate classify, whereas Bali, Hong Kong, and Taiwan exhibit high variety.The elevated genetic diversity observed in Hong Kong and Taiwan populations is likely attributed to the currents originating from the South China Sea, flowing directly toward Taiwan and Hong Kong.This facilitates the mixing of L. bengalensis larvae from these regions, resulting in increased a close genetic distance and genetic variety.Juvenile of the fish exhibit limited movement capabilities, making them susceptible to being transported by ocean currents [30].The elevated genetic diversity is attributed to factors like geographic proximity and the influence of ocean currents, whereas diminished genetic diversity within a population results from a combination of factors, including habitat conditions and excessive exploitation [31,32].The 17 sequences from five populations of Aceh, Maluku, Bali, Hongkong and Taiwan it produces one main clade.This suggests the strong familial relationship among these populations that can be traced back to a common ancestor.The genetic distance between clades provided support for this phylogenetic tree [33].The Taiwan population exhibited the highest genetic variation among populations (interspecific) at a value of 0.032, while the lowest divergence was observed in Maluku populations with a value of 0. Populations with lower genetic distance values signify a more intimate familial connection, while higher genetic distance values indicate a more distant kinship relationship [34].
The analysis of population connectivity revealed that populations in the South China Sea exhibit interlinked gene flow, as evidenced by the presence the haplotypes were shared among them.For example, haplotype No. 6 was identified in both the Taiwan and Hongkong inhabitants.According to Saleky and Dailami [35], the connecting of the populations results from shared geographic locations and consistent current patterns.Nevertheless, there is no common haplotype shared between the populations in Indonesian waters and the South China Sea.

Conclusion
The genetic variation within the snapper fish population in Indonesian waters surpasses that in the South China Sea, with the Bali population exhibiting the highest genetic diversity.Nevertheless, the genetic diversity within the Indonesian snapper fish population remains at a moderate level.Although there is gene flow connectivity within the snapper fish population in the South China Sea, it is not linked to the population between Indonesian waters and the South China Sea.

Acknowledgment
This research was supported by the PMDSU research scheme year 2022 with the contract number 062/E5/PG.02.00.PL/2023.Therefore the authors thank the Minister of Education, Culture, Research, and Technology of the Republic of Indonesia for funding this research.

Table 2 .
Comparing the genetic diversity of snapper L. bengalensis among five populations in Indonesian waters and the South China Sea.Evaluating total haplotypes (Hn), nucleotide diversity (π)and haplotype diversity (Hd)

Table 3 .
The genetic distance within population (in bold) and between population of L. bengalensis from populations using COI gene