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Authentication of Tenualosa Species in Perak River, Malaysia

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Authentication of Tenualosa Species in Perak River, Malaysia Authentication of Tenualosa species in Perak River, Malaysia: application of morphological measurement and molecular analysis of partial CO1 and 16S genes to resolve species ambiguity 1Nurul N. Arjunaidi, 2Muhammad F. Zakaria, 1Abdul H. Abdul Aziz, 1,3M. Sheriff Shahreza, 1,3Tun N. Aimi Mat Jaafar, 1,4Y. Giat Seah, 1,3Ariffin Nur Asma 1 School of Fisheries and Aquaculture Sciences, Universiti Malaysia Terengganu, Terengganu, Malaysia; 2 School of Marine Science and Environment, Universiti Malaysia Terengganu, Terengganu, Malaysia; 3 Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, Terengganu, Malaysia; 4 Fish Division South China Sea Repository and Reference Center, Institute of Oceanography and Environment, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia. Corresponding author: A. Nur Asma, [email protected] Abstract. Malaysia possessed two species of Tenualosa sp.; the Tenualosa toli and Tenualosa macrura, which can only be found in Sarawak. Recently, samples that resembled the Tenualosa sp. were found in Perak River of Peninsular Malaysia. The absence of comprehensive study of Tenualosa sp. in Peninsular Malaysia had urged this study to be conducted to authenticate the samples found. Identification were done using morphological analysis with the integration of molecular assessment using partial genes of cytochrome c oxidase subunit 1 (CO1) and 16S rRNA. Morphological analysis of 40 samples showed the closest correlation with Tenualosa ilisha (Hamilton, 1822). However, variations were found in meristic counts and the samples were differentiated into two groups. The meristic counts also overlapped with other species of the same genus. Molecular assessments on 10 samples performed using partial CO1 and 16S rRNA genes had obtained 500-bp and 573-bp sequences respectively, that has high similarity with T. ilisha. Further genetic assessment through phylogenetic analysis showed that all 10 samples formed a monophyletic lineage with T. ilisha. However, the samples were significantly separated with others of the same species from different countries. The findings suggested that there is a possibility the samples might migrate from other countries and had experienced domestic speciation towards the local environment. The findings of this study would be useful in recognizing the potential fisheries resources. This will greatly help in planning and implementing a healthy and sustainable exploitation of this species. Key Words: Tenualosa sp., morphological assessments, cytochrome C oxidase subunit 1 gene, 16S rRNA gene. Introduction. Tenualosa species or known as Terubok is an important and high commercial value clupeid fish. There are five species of Tenualosa spp. can be found worldwide and two species are common in Malaysia. These two species are Tenualosa toli and Tenualosa macrura which can be found only in Sarawak waters (Blaber et al 1996; Phillip 2001). The Sarawak Tenualosa species is one of the most significant estuarine fish that is considered as one of the Malaysian commercial fish species that contributes to the national economy. In Peninsular Malaysia, catching of Tenualosa species is rare and inconsistent in the past few decades (Gambang 1988). It has been presumed that the Tenualosa species has subsequently extinct, however no such records has ever been confirmed. Recently, catch landings of Tenualosa species in Peninsular Malaysia has been proclaimed by several local fishermen in Perak River. The reappearance of Tenualosa species in Perak River has known to be dissappeared for almost 20 years has raised several research questions such as its distribution, taxonomy, and systematics. However, Tenualosa species in Perak River has never received enough attention, hence AACL Bioflux, 2016, Volume 9, Issue 6. 1355 http://www.bioflux.com.ro/aacl no comprehensive studies are done. Consequently, there is lack of information regarding the Tenualosa species in Perak River. To date, this species is still unidentified and there is no current study to officially validate its species even though the Tenualosa species in Perak bare resemblance with the Tenualosa species in Sarawak. Thus, the current status of Tenualosa species in Perak River is still poorly understood. As the identification of this species that are caught in Perak River remains unclear, this might lead to the difficulty of collecting any information regarding the species. Insufficient knowledge and information may leave the Tenualosa species in Perak unattended without any efforts of its preservation and conservation management in Perak River. Pollution and overfishing might occur and could lead to unsustainable exploitation and the declination in numbers of this species that might render its extinction. Such incidents have been observed in the other two species; the T. toli and T. macrura in Sarawak as the numbers have been reported drastically decreasing in the past few years (Phillip 2001; Rahim et al 2014). Abdul Aziz et al (2015) reported that genetic diversity of T. toli was relatively low for two populations in Sarawak and might cause a genetic deprivation towards its population. Therefore, as an initial step, identifying the species in Perak River will acknowledge the local community and authorities regarding the value of the species and efforts to manage the species can be implemented. Moreover, Tenualosa species in Perak River can become an alternative source of Malaysian Tenualosa species supply besides Sarawak. Herein, it is hypothesized that the Tenualosa species in Perak River is of different species from the T. toli and T. macrura from Sarawak. One of the best ways to precisely identify species is through morphometric study accompanied by molecular identification. Kartavtsev et al (2006) reported that molecular approach is more accurate in species identification compared to morphological character identification alone. Several studies have been done through the application of both approaches to precisely validate and identify the aquatic species (Watanabe et al 2008; Kolangi-Miandare et al 2013; Haniffa et al 2014). The development of species-specific morphological and molecular markers has been done to successfully monitor natural population stocks (Tinti et al 2003). It has been demonstrated that both markers can be applied to investigate the species divergence and genetic variation within geographical regions, hence relatively mapping the distribution (Motamedi et al 2014). As a result, efforts to manage natural fisheries population stocks can be implemented and applied to maintain sustainability. Such studies have been done in natural populations of Rastrelliger spp. and Channa striata in Malaysia (Darlina et al 2011; Song et al 2013). However, as any information regarding the Tenualosa species in Perak, identification has been poorly documented, the efforts to manage the natural populations cannot be done. This might cause unsustainable exploitations of fisheries resources and once again causing the dissappearance of valuable Tenualosa species resources in the future. Therefore this study was conducted to identify the Tenualosa species in Perak through morphological and molecular approaches. Material and Method. Samples of Tenualosa species were collected from the Perak River in Bagan Datoh, Perak from January - March 2014. Samples were caught using drift net by the fishermen. A total of 40 samples were collected and used for the experiment. White muscle tissue and fin clips were collected and preserved in 95% ethanol and stored at -20oC for further genetic analysis. The tissue and clips were taken on the right side of the samples to allow the left side to be used for morphological analysis. Morphological analysis. The samples were identified morphologically according to the verification of Narejo et al (2008), Munroe et al (1999) and Hong et al (2013). Twenty five morphometric characters were measured using digimatic digital caliper (Mitutoyo, Japan) with an accurancy of 0.01 mm and seven meristic characters were counted using standard methods. The procedure of fixation, photograph and preservation for the specimen are followed Seah et al (2011). Methods of counting and measuring are generally followed Hubbs & Lagler (1964) and terminology of morphological features and descriptions are modified from Seah et al (2009). Measurements of the morphometric AACL Bioflux, 2016, Volume 9, Issue 6. 1356 http://www.bioflux.com.ro/aacl characters were standardized in order to eliminate any size effect: Standardized measurement = Morphometric Parameter / Standard Length*100. DNA extraction, Polymerase Chain Reaction (PCR) amplification and sequencing. Total genomic DNA of 10 samples were extracted using DNA extraction kits, the Cell/Tissue DNA Extraction Kit (Spin Column) (BioTeke Corporation, China). Two molecular markers were used, the partial cytochrome c oxidase subunit 1 (CO1) and 16S gene marker. The CO1 gene was amplified using two primers based on Jannatul-Farihah et al (2011) which were CO1f (5’-CCTGCAGGAGGAGGAGAYCC-3’) and CO1r (5’- CCAGAGATTAGACCGAAATCAGTG-3’). A PCR mixture containing 2.5 μL of 10× PCR buffer; 0.8 μL of 25 mM MgCl2; 0.6 μL of 100 pmol of both primers; 0.4 μL of 5U Taq DNA polymerase (Vivantis, Malaysia); 2.4 μL of 10,000 uM dNTP; and 3.0 μL of 100 ng DNA sample was prepared in a 25 μL reaction. The cycling profile for CO1 began with the initial step of 3 minutes at 94oC, followed by 30 cycles of denaturation (at 94oC for 30 seconds), annealing (at 52oC for 40 seconds) and extension
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