Molecular phylogenetic analysis of commercially important Asian monsoon scallop, Amusium pleuronectes (Linnaeus 1758) from Indonesia

The Asian monsoon scallop, Amusium pleuronectes, is a key member of shellfish communities that are the most commercially harvested in Asia. Materials were obtained from four sites in Indonesia: Semarang, Bali Island, Maros and Buru Island. Nucleotide sequence analysis was performed on PCR-amplified mitochondrial DNA 16S rRNA. In total, 556-558 base pair nucleotide sequences were obtained from 8 individuals. Molecular analyses revealed that the samples belong to the A. pleuronectes species. Phylogenetic analyses were carried out by comparing the gene sequences of A. pleuronectes haplotypes in Indonesia with gene sequences of the same base pair length and with the of A. japonicum which was used as an outgroup. The phylogenetic trees were consistent and indicated in the two sub-clades. The presence of A. pleuronectes and the phylogenetic status of the A. pleuronectes Indonesian haplotype were reported. Clarifying the phylogenetic status of ecologically important species provides basic information for biosecurity studies for possible future conservation and control programs.


INTRODUCTION
Asian monsoon scallop, Amusium pleuronectes, is a bivalve species which belongs to the Pectinidae.The species has very wide distribution from central Ryukyu Island, South China Sea to the South-East Asia, and Australia (Habe, 1964;Morton, 1980), at a depth of 18 to 40 m (Minchin, 2003) and temperatures ranging 28 to 29°C (Del Norte 1986;Cabacaba et al., 2020).A. pleuronectes lies recessed in the substrate, the white colored valve positioned lying on the seabed while brown colored valve is on top and the umbonal region is submerged in the substrate (Morton 1980).
Asian monsoon scallops are often a major component of the catch of fishing vessels.In Indonesia, the species is of significant commercial value, with about 500 to 1100 metric tons/year harvested during 1994-1999 (Suprijanto and Widiowati, 2006).However, the recent catch statistics showed that production declined to 100 to 300 metric tons/year between 2000and 2003(FAO, 2006)).Uncontrolled fishing activities may kill large numbers of stocks, leading to a serious threat to this species as a whole.Assessment of its populations in Indonesian revealed a high exploitation ratio due to the high demand and overfishing.Continuous fishing pressure may ultimately lead these marine species to a high risk of genetic and population bottlenecks if preventive action is not taken (Hardianto et al., 2022a;Hardianto et al., 2022b).Thus, there is a need to establish baseline data to understand the current stock status from this species.
DNA barcoding combined with a molecular phylogenetic tree is a system designed for precise and accurate identification of a species using a short and standardized gene region (Hebert et al., 2003).Research on DNA Barcode and phylogenetic tree has been carried out on marine organism like fish (Khansa et al., 2023;Nursalim et al., 2022;Syaifudin et al., 2021), shellfish (Beden and Karahan, 2020;Praipue et al., 2021), crustacean (Irwani et al., 2020;Vella and Vella, 2022; ) and coral reef (Wijayanti et al., 2017a;Wijayanti et al., 2018).The most promising benefit of DNA barcoding and phylogenetic tree for species authentication lies in the ability to identify early stages that cannot be done by using morphological descriptions and inter species connections.It has been proved to be effective for identifying species in juvenile, larvae and adult organism.This research is conducted to amplify nucleotide sequences of the mitochondrial 16S ribosomal RNA, to identify species, determine genetic distances and analyze phylogenetic relationships of Asian monsoon scallop from Indonesia.Thus, the identification of the species using the 16S rRNA is an effort to develop genetic information that can be used as the basis for developing better conservation strategies for the target species in the future.

METHODS AND MATERIALS
Samples were collected in 2022 from the ocean at the deep 10-30 m from a large proportion of the natural distribution of this species in Indonesia from four different Islands (Java Island; Semarang, Bali Island; South Denpasar, Sulawesi Island; Maros, and Buru Island).Individual A. pleuronectes samples were fixed in 95% ethanol on site and then transported to the laboratory until used for genetic analysis.Muscle tissue (10 mg) from the samples was excised from near the shell using scissors and forceps.DNA was extracted using the Realpure Genomic DNA Extraction Kit (Durviz S.L) following the manufacturer's manual procedure.
The partial mitochondrial 16S ribosomal RNA gene was amplified via polymerase chain reaction (PCR) using universal primers 16sar-L (5'-CGCCTGTTTATCAAAAACAT-3') and 16sbr-H (5'-CCGGTTTGAACTCAGATCACGT-3') (Palumbi et al. 1991).All reactions were carried out in a 25 μl total volume and the following reagents were added to each PCR microtube: 1 μl of template DNA, 25 pmol of each primer, 12.5 μl of Bioline master mix Taq DNA polymerase.Each sample was adjusted to 25 μl with distilled H2O.PCR conditions consisted of a hot start (94°C, 180 s) followed by 30 cycles of denaturation at 94°C (30 s), annealing at 48-50°C for 30 s, extension at 72°C (90 s), and final extension at 72°C (420 s) using a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA).Verification of PCR was performed via electrophoresis in a 1% agarose S gel.After electrophoresis, gels were stained with ethidium bromide and products checked on a transilluminator (Advanced Scientific Products Pty Ltd., Queensland, Australia).PCR products were purified using a PCR product pre-sequencing kit (USB Co., USA).Amplified DNA was sequenced on an ABI 3730xl DNA analyzer using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, USA).
Sequence data were aligned using muscle alignment software, implemented in MEGA X (Kumar et al., 2018) using default alignment parameters, and the sequences were adjusted manually to avoid mismatches.The sequences were analyzed their identity using BLAST (Basic Local Alignment Search Tool) in NCBI (National Center for Biotechnology Information).We also conducted sequence data to calculate Kimura's two-parameter (K2P) distance with 1,000 bootstrap replications, to analysis the pairwise genetic difference, nucleotide composition and to constructed phylogenetic tree by the Neighbor Joining (NJ) methods using MEGA.Haplotype network analysis between the species also constructed by the analysis of minimum spanning network (MSN) using PopART (Leight and Bryant, 2015).

RESULTS AND DISCUSSION
In the sequencing results, we obtained a 556-558 bp mtDNA 16S rRNA sequence.The sequence was obtained after the alignment of eight specimens of A. pleuronectes from four localities across Indonesia (Figure 2).
Nucleotide sequences of all haplotypes were deposited in the DNA Databank of Japan under the accession numbers LC761945-LC761942 (Table 1).BLAST analysis of the sequence results (LC761945-LC761942) has a match range of 97% to 99% with A. pleuronectes haplotypes from other DNA data banks (Table 2).The mean genetic distance in pairwise analysis was calculated to range from 0.01 to 0.04 (Table 3).The maximum genetic distance was observed between SEM-2 and HM630501.1 Australia at 0.04 (Table 3).Result of the haplotype network analysis showed that not many gaps between localities investigated (Figure 2).The most appropriate model was determined as K2+G+I as a result of base substitution model analysis.Bases ratios were calculated: Adenine ratio: 24.7%, Thymine ratio: 30%, Guanine ratio: 27.8%, and Cytosine ratio: 17.5% (Table 2).Phylogenetic analyses indicated that our specimen was evolutionary closer among locality observed despite a two sub clade difference.Neighbor Joining and Maximum Likelihood trees showed the same topology and similar results, and they proved to be consistent (Figure 3).Although the presence of A. pleuronectes has been recorded in different countries of Asia and in Australia, studies on its phylogenetic status are limited in the literature especially the phylogenetics status in Indonesia.In the previous study reported by Mahidol et al. (2007b), only Thailand A. pleuronectes were phylogenetically studied.Salvi et al. (2010), compared the phylogenetic status of Pectinidae, but also only include the A. pleuronectes from Thailand.Mynhardt et al. (2014), studied shell shape convergence and systematically status of family Pectinidae including A. pleuronectes from China, Thailand, the Philippines dan Australia without Indonesia.Determining the phylogenetic status of an organism provides an important pool of information for biosecurity studies.A. pleuronectes is a species that provides important information on environment quality and plays a role in the food chain of marine ecosystems (Mahidol et al., 2007;Cabacaba et al., 2020).If the phylogeny of A. pleuronectes is fully clarified, basic information will be supplied to the studies to be carried out in order to protect the balance of marine ecosystems.
The result of BLASTn analyses in Table 2 denoted that nucleotide sequences of the from four localities observed in Indonesia were identical, showing ranged 97% to 98.69% matched to A. pleuronectes from Thailand (DQ640841.1 and DQ640830.1),the Philippines (KC879122.1,KC879126.1 and KC879114.1),China (AF362387.1),Vietnam (AJ571616.1)and Australia (HM630501.1,JF339128.1 and HM630497.1).Sequence similarity higher than 97% was the criterion for authentication at the species level (Wong & Hanner, 2008) and a similarity lower than that was used for recognition at the genus level.This result denoted that distinguishing species using DNA barcoding is very accurate.The 16S rRNA is effectively used as species authentication method because intraspecific variation is low, but has high interspecific variation values especially in adjacent taxa (Ward et al., 2005;Feng et al., 2011;Lumogdang et al., 2022).DNA barcoding validates to recognize species for international trading, either certifying fisheries products consumption or ornamental fish trade (Dahruddin et al., 2016;Feng et al., 2011;Lumogdang et al., 2022).This method is very useful for sustainable fishery resource management.The genetic distance in this research was also used to determine the genetic relationship between A. pleuronectes observed from Indonesia with other samples from DNA data bank.Genetic distance is varied in the ranged of 0.01 to 0.04.Hubert et al. (2014) stated that a genetic distance difference of less or equal to 3% indicates molecularly identical species.The smaller the genetic distance between individuals in a population, the more uniform the population is.Conversely, the greater the genetic distance of an individual in a population, the more diverse the population will be.The largest genetic distance (0.16 to 0.17) was found between A. pleuronectes from Indonesia and species outgroup A. japonicum from Japan (HM622707.1).Similar to the genetic distance analysis, the haplotype network analysis showed not to big different gap within species analysis (Figure 2).It is clearly denoted that within A. pleuronectes barcode variation was low in compare to the sequence variation between species in genus Amusium.Genetic distance indicates the ratio of a genetic distinction between species or populations (Dogan & Dogan, 2016).Therefore, a smaller genetic distance value creates a more indistinguishable appearance partial sequence of 16S rRNA gene compared between the two species (Mahidol et al., 2007b).

CONCLUSION
In terms of species identification, molecular identification by phylogenetic analysis and DNA barcoding is more reliable and accurate than morphological identification.In the present work, the aim was to produce a definite species identification of the specimens obtained in Indonesia by phylogenetic analysis and DNA barcoding to clarify their phylogenetic position in order to give fundamental data for future biosecurity investigations.The analyses confirmed that the samples originate from the A. pleuronectes species and are most closely related to the GenBank-registered haplotypes KP900978.1,AJ571616.1,DQ640841.1,KC879126.1,and HM630501.1.In the light of these results, it was decided that the programs to be established in the places where phylogenetically linked haplotypes are identified can be included into the research in Indonesia.

Figure 3 .
Figure 3. Haplotype network (implemented in PopART) of Amusium pleuronectes based on 16S rRNA sequences using minimum spanning network analysis.

Figure 4 .
Figure 4. Phylogenetic relationships of Pectinidae inferred by Neighbor Joining analysis (NJ) of 16S rRNA gene regions.

Table 1 .
Details of four sampling sites, number of samples collected (n) and accession number for

Table 2
BLAST Identity percentage of nucleotide of Amusium Pleuronectes based on 16S rRNA

Table 3
Base composition (%) of 16S rRNA gene amplified for each location