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The blue sea star Linckia laevigata is strictly marine and ubiquitous on coral reefs throughout the tropical oceans of the world, predominantly in the Indo West Pacific Region and around the Northern part of Australia (Williams 2000) and is commonly found in dense number all through the Philippine archipelago. It is characterized by firm texture, posing slightly tubular, elongated arms, and possessing short yellowish tube feet. Number of arms ranges from 4-7 pieces of sub equal length often due to the regeneration, with fairly stout and blunt ends. Posses' different colour morph of which royal-blue or greenish royal blue is the most commonly observed in live L. laevigata in the Pacific region (Williams, 2000). Mode of reproduction is sexual and with planktonic larval duration of 22-28 days (Yamagutchi 1977). L. laevigata, an inhabitant of shallow reef areas exposed to the sunlight, on rocks, dead coral or rubble at depths of 0 to 60 m. The sedentary adult has limited dispersal potential; it is slow moving macrobenthos creeping in corals and rocky crevices and migrating between reef patches for grazing. Nonetheless, having a long larval stage is the advantageous mechanism of this asteroid for wider range of distribution through passive dispersion with the water current and circulation. Frequently, population of L. laevigata in reef flats are made up of mature individuals, and juvenile are rarely observed. Juvenile-to-adult transformation is estimated to take place in average of about 2 years of age which connotes low turnover rate with a low level of recruitment (Yamagutchi 1977).
The dispersal ability of the species coupled with the physical characteristic of the area defines recruitment success. This is one of the reasons for broad scope dispersal which is thought to give rise to high gene flow and low genetic structuring between populations but high genetic diversity within populations (Williams et al. 1993). Having long planctonic larval stage and high dispersal potential, number of L. laevigata studies revealed high genetic affinity among populations compared to other sedentary species (Williams & Benzie 1993; Juinio-Meñez et al 2003; Crandall et al. 2008). However, other studies reveals differentiation on population meddled with physical factors such as hydrographic conditions, land masses and geological history; the discontinuity of L. laevigata populations in Eastern Indian Ocean Central Indo-Malay Archipelago and Western Pacific (Kochzius et al. 2009) and in Great Barrier Reef , West Australia and Fiji (Williams & Benzie 1997). The same as through with Tridacna crocea in the Coral Triangle (Kochzius & Nuryanto 2008) and eastern seaboard of the Philippines (Ravago-Gotanco et al. 2007). Past and present physical processes clearly influence the structuring of the population of marine species. Fast flowing oceanic currents, intricate current system of seascapelike that of the Indonesian flow through may provide a means or be a barrier for larval transfer and/or isolation that will cause high gene flow or bottleneck of populations.
Eastern Visayas is characterized by local and major oceanographic conditions; it is a frontier to the West Pacific Ocean in the eastern side where North Equatorial Current (NEC) bifurcates to Kuroshio to the north and Mindanao Current to the south (Qu & Lukas 2003). Island of the area is delineated with different bodies of water such as Samar Sea, Camotes Sea, Leyte Gulf and the straits of San Bernardino and San Juanico (Fig. 1). Coupled with dispersal ability of a species, genetic structure of population in this area may have been significantly influenced by these complex hydrographic characteristics. Presence of Mindanao Current, an offshoot of a large-scale oceanic current NEC that surge far down south of the Philippines may facilitate a high transport of plantonic propagules over the stretch of the eastern seaboard which may cause connectivity with northern and eastern Samar populations.
Previous studies on population genetics have shown suitability of cytochrome oxidaze I (COI) gene as a genetic marker to deduce information on connectivity pattern for array of marine invertebrate species: the oyster Crassostrea argulata (Foihil et al. 1998), Tridacna crocea (Kochzius & Nuryanto 2008), Periclimenes soror, Thyca cristallina and L. laevigata (Crandall et al. 2008, Kochzius et al. 2009, and Williams 2000). To allow some degree of comparison with preceding studies, this study utilized partial sequences of mitochondrial COI to investigate L. laevigata's genetic population structure and connectivity pattern in the islands of the Eastern Visayas (Philippines) correlating to the major physical processes in the region.
Figure. 1: Sample collection sites for Linckia laevigata in Eastern Visayas region of Philippines [i.e. Northern Samar; Western Samar; Eastern Samar; Leyte] with the oceanographic features
MATERIALS AND METHODS
Sample collection. One centimetre arm amputation was done to avoid destructive sampling of 157 Linckia laevigata. Among the four described color morph (blue, orange, green and violet) only blue was sampled and analyzed to prevent fallacy result on genetic differentiation from this color variation (Williams 2000). Samples were collected from 5 stations in Eastern Visayas representing the Pacific side and the internal waters of the region: 1 in N. Samar, Eastern Samar 1, W. Samar 2 and 1 in Leyte (Fig. 1 and Table 1). Sampling stations were selected on the basis of land and water body separation by hundreds of kilometres. Collected samples were individually preserved with absolute ethanol and stored at 4Â°c.
DNA extraction, amplification and sequencing. The DNA from tissue samples were extracted using the NucleoSpinÂ® Tissue extraction kit according to the protocol of the manufacturer. Partial sequences of the mitochondrial cytochrome oxidase I gene (COI) was used as genetic marker. Amplification was conducted with the universal DNA primers described by Folmer et al. (1994) through PCR in a volume of 50Î¼l and contained 2Î¼l DNA template, 10mM Tris-HCl (pH 9), 50mM KCl, 1.5mM MgCl2, 0.4Î¼M of each primer, 0.2 mM dNTPs, 2 Î¼l BSA (2 mg/ml) and 1 U Taq polymerase following temperature profile 94Â°C for 5 min, followed by 35 cycles of1 min at 94Â°C, 1.5 min at 45Â°C and 1 min at 72Â°C. Final extension was conducted at 72Â°C for 5 min as described by Kochzius et al.(2009). Purification of the PCR products was done using the QIAqiuck spin column PCR purification kit (Qiagen) following the prescribed protocol of the manufacturer. Purified DNA strands will be sequenced using the DyeDeoxy terminator chemistry (PE Biosystem) and with an automated sequencer (ABI PRISM 310 and 3100, Applied bio systems).
Genetic diversity. Sequence Navigator (version 1.0.1 Applied Biosystems) will be used to edit all sequences and result will be translated to amino acid using the program Bioedit (version 22.214.171.124) to insure that functional mitochondrial DNA was obtained and not nuclear pseudogene. After which multiple alignment will be conducted with Clustal W through Bioedit software. Gene diversity will be analyzed through calculation of haplotype (Nei 1987 as cited by Kochzius et al. 2009) and nucleotide diversity (Nei & Jin 1989) with the program Arlequin (http://cmpg.unibe.ch/software/arliquin3).
Genetic population structure and connectivity. To evaluate presence and significance of population structure data will be analyzed using analysis of molecular variance (AMOVA; Excoffier et al. 1992) and pairwise É¸ST-values with the package Arlequin. Data set will be tested with a hierarchical analysis of molecular variance to elucidate genetic variance among population and within samples considering the geography of the area. Programme TCS (version 1.21; Clement et al. 2000 as cite by Kochzius et al. 2009) will be utilized for calculation of haplotype network.
Table 1: Sample site in Eastern Visayas and number of Linckia laevigata samples collected.
Date of Sampling
No. Of Samples
Marabut, Western Samar
Salcedo, Eastern Samar
San Jose, Northern Samar
Almagro, Western Samar