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Updates on the Status of Tridacna Crocea in the Philippines with Notes on Molecular Approach Analysis

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Updates on the Status of Tridacna Crocea in the Philippines with Notes on Molecular Approach Analysis Updates on the status of Tridacna crocea in the Philippines with Notes on Molecular Approach Analysis Jane Abigail Santiago1, Raymond Vincent Castillo1, Lota Creencia 2 and Ma. Carmen Lagman1,* 1 Biology Department, De La Salle University 2 College of Fisheries and Aquatic Sciences, Western Philippines University *corresponding author: [email protected] Abstract: The collection sites of Tridacna crocea in the Philippines was listed based on the previous studies from 1986 up to the present date. Eighteen (18) studies showed the wide distribution of T. crocea within the country. New samples were also added to the collection by the present study from Ulugan Bay and Honda Bay in Puerto Princesa Palawan. First record of T. crocea in Batanes Island was also presented by this study using molecular approach identification. The amplified mitochondrial DNA cytochrome c oxidase I gene (COI) sequences of T. crocea and the sequences from Genbank of the Tridacna maxima, Tridacna squamosa, Tridacna noae and outgroup were used to construct Neighbour Joining and Maximum Likelihood tree. The information in this study contributes to the growing data on genetics of the giant clams (Cardiidae: Tridacninae). This can serve as a guide to the possible population structure and adaptation patterns of habitat associated marine invertebrate species. Key Words: Tridacna crocea, Giant clam, Phylogenetics, Philippines, Checklist, COI 1. INTRODUCTION (IUCN) Red List of Threatened Species. Brood-stock imported from Palau and Solomon Islands of Tridacna Giant clams (Cardiidae: Tridacninae) are were made to protect its population (Gomez and locally known as taklobo in the Philippines. Two Alcala, 1988; Gomez et al., 2006) although most of the extant genera are under Tridacninae subfamily which restocking efforts in the Philippine mainly focuses are Hippopus (2 species) and Tridacna (9 extant with Tridacna gigas. Culturing giant clams could be species) (Su et al., 2014). The nine giant clam species the best way to conserve the population by reseeding in the Philippines are T. crocea Lamarck, 1819, T. depleted reefs (Beckvar 1981) and also help coastal derasa (Röding, 1798), T. gigas (Linnaeus, 1758), T. communities for aquaculture industry. A larger maxima (Röding, 1798), T. noae (Röding, 1798), T. amount of capital fund and investment is needed for squamosa Lamarck, 1819, Hippopus hippopus the nursery facilities for T. gigas. It could take 7-year (Linnaeus, 1758) and Hippopus porcellanus growing period for T. gigas to grow and be suitable for (Rosewater, 1982) (Ragaza et al. 2020). The giant meat harvesting which could be a disadvantage for clams are listed in the Convention on International many coastal community villagers (Foyle et al. 1997). Trade in Endangered Species (CITES) appendix II and Smaller species T. crocea, T. maxima, and T. International Union for Conservation of Nature squamosa will be a better option for villagers because it only takes 5-7 months to be ready for export (Foyle et al. 1997). DNA template, 17.1 µL ddH2O, 5 µL 5X Vivantis Tridacna crocea is the smallest among the TaqBuffer, 1.5 µL 25MM MgCl2, 0.5 µL 10 MM giant clam species. It is the most abundant tridacnid dNTPs, 0.4 µL of each primers (10µM). Amplified PCR species in the Philippines reefs (Gotangco et al. 2007) products were sent to Asiagel-Malaysia for which makes it a better candidate for aquaculture- aquarium trade. T. crocea has been popular for sequencing. Additional sequences from GenBank were aquarium enthusiast for its size and colorful bright also utilized for comparison: T. maxima (Nuryanto mantle. T. crocea is recommended giant clam for and Kochzius 2009); T. squamosa (DeBoer et al., community aquaculture industry. In order to do this, 2014); Tridacna sp. (Lizano and Santos, 2014); T. noae identification of the sites where it can be found in the (Neo et al. 2018). Philippines is essential for seeding purposes and 2.3 Genetic analysis. target stocking areas. This study list the sites of existing T.crocea in the Philippines with first record The sequences from the collected Tridacna and additional notes on its phylogeny for future crocea were identified using NCBI-blast (Altschul et. reference of biodiversity hotspot studies. al., 1990). All sequences with additional sequences from GenBank and Laevicardium crassum (Lundin 2. METHODOLOGY 2019) as the outgroup were utilized for molecular phylogenetic analysis. Nucleotide sequences were 2.1 Sample Collection. aligned using MUSCLE using default parameters. Literatures were compiled from 1986 up to Phylogenetic trees were done using Molecular date where Tridacna crocea was collected and Evolution Genetic Analysis (MEGA) version 10.0 in recorded in the Philippines (Table 1). two different phylogenetic modes of Neighbor-Joining Small piece of mantle tissue from T. crocea (NJ) and Maximum-Parsimony (MP). Kimura’s 2- were collected through scuba diving or snorkeling parameter distance model was utilized to construct from Ulugan Bay and Honda Bay in Puerto Princesa, Neighbor- Joining trees (Kimura 1980). Maximum- Palawan and Chanarian, Basco, Batanes in April Parsimony tree was acquired using the Close- 2019. The collected specimens were preserved in 1X Neighbor-Interchange algorithm. NJ and MP trees Phosphate-Buffered Saline (PBS) solution in a 1.5ml are distance-based methods that transform the microcentrifuge tube. Voucher specimens were sequence data into pairwise distances, and then use initially stored in an ice chest and were deposited to - the matrix during tree building. The bootstrap values 20°C freezer in De La Salle University-Practical indicate the robustness of nodes in the result of Genomics Laboratory. phylogeny trees inferred in 1000 replicates. 2.2 DNA extraction, PCR and sequencing. 3. RESULTS AND DISCUSSION DNA extractions were done using 10% chelex (biorad) From 1986, occurrences of T. crocea has been solution (Walsh et al. 1991). A fragment of observed within the Philippine reefs. A total of 18 mitochondrial cytochrome oxidase subunit-I gene studies has been done with T. crocea that includes (COI) was amplified using the tridacnid specific morphological and molecular characterization, primer (forwards: LCO: 5’-GGG TGA TAA TTC GAA distribution and genetic structure. High densities of T CAG AA-3’ and reverse: RCO: 5’-TAG TTA AAG CCC .crocea has been repeatedly observed in Tubbataha CAG CTA AA-3’) (Nuryanto et al., 2007). Polymerase reefs, Palawan (Conales, 2015). The provinces and Chain Reaction (PCR) reactions were performed in a regions of Samar, Tubbataha Reefs, Palawan, total volume of 25 µL containing approximately: 1 µL Sorsogon, Western and Central Visayas, Batangas, Maximum - Neighbor - Likelihood Joining Tree Tree T. crocea T. crocea T. squamosa T. squamosa T. maxima T. maxima T. noae T. noae Figure 1. Phylogenetic tree of giant clams based on 400 bp of the mitochondrial DNA COI gene using genetic distances Kimura 2-parameter on NJ approach; bootstrap analysis with 1000 replicates. Similar topology was constructed with Maximum likelihood tree. Table 1. List of occurrences of Tridacna crocea in the Pamilacan, Bohol; Naguit Morphology Philippines Negros: Tanon Strait, 2009; and Genetic Place of Collection Reference Remarks Carbin; Camiguin Markers Central and Western Alcala 1986 Survey data Island; Southeastern Visayas; Cagayan, Sulu Samar; Spratlys Island Sea; Palawan Regions Ulugan Bay, Palawan DeBoer and Genetic Cagayancillo, Junio et al., Survey data Barber, Marker Palawan;Western 1987 2010 Pangasinan; Polillo; Tubbataha Reef, Dolorosa Survey Zambales Palawan and Jontila, Albay; Sorsogon; 2012 Calatagan; Lubang Apulit Island, Taytay, Gonzales et Survey Island; Apo Reef; Palawan al., 2014 Puerto Galera; North East Negros; El Nido, Ulugan and Honda Bay DeBoer et Population Camiguin Palawan; Romblon; al. 2014 Genetics Dinagat; Carbin, and Negros Occidental; Structure Tubbataha Reef, Estacion et Survey Camarines; Quezon; Palawan al., 1993 Tawi-Tawi Tubbataha Reef, Yamaguchi, Survey Guiuan, Samar Lizano & Morphology Palawan 1996 Santos, 2014 and Genetic Markers Palawan Pangasinan Yu et al., Genetic Tañon Strait, Bohol Naguit, Genetic 2000 Marker Sea, Visayan Sea, 2015 Markers Southeastern Samar Linapacan Strait in Juinio- Genetic Tubbataha Reef, Dolorosa et Survey northern Palawan, the Menez et al., Markers and Palawan al., 2015 Balabac Strait in 2003 Structure Saint Paul Bay, Gonzales, Survey southern Palawan, the Western Palawan 2015 Kalayaan island group Tubbataha Reefs Conales et Morphology in the South China Natural Park, al., 2015 Sea, and the Cagayancillo, Palawan Tubbataha shoals in the Sulu Sea Ulugan Bay This study Genetic Honda Bay Marker Chanarian, Batanes Tubbataha Reef, Dolorosa Survey Mindoro, Quezon Province, Negros Occidental, and Palawan and Davao are the locations where T. crocea has been Schoppe, studied (refer Table 1). 2005 Eastern Philippine Ravago- Population Seaboard: Cagayan, Gotanco et Genetics Most of the survey and genetic studies were Isabela, Aurora,Polillo, al. 2007 conducted in Palawan regions. DeBoer et al. (2014) Catanduanes, Masbate, presented the population genetic structure of T. crocea Balicuatro, Divinubo, in the western side of the Philippines while Ravago- Homonhon, Dinagat, Gotanco et al. (2007) provided for the eastern side. Lianga and Mati Naguit (2015) has showed both morphological and population genetics analysis for T. crocea in Bohol and Negros region. This information demonstrated that clams. This data showed that the four species are
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