Molecular Identification and Genetic Diversity of Acropora hyacinthus from Boo and Deer Island, Raja Ampat, West Papua

Indonesia lies at the centre of biodiversity for corals. However, the reefs suffered from extensive human exploitation. Marine Protected Areas is thought to be best solution to protect coral reefs ecosystem. Understanding genetic diversity is crucial for effective management of the MPAs, however genetic diversity is rarely been corporate in designing an MPA. Moreover, many MPAs are uneffectively manage due to poor designated and demarcated.Raja Ampat which is located in western tip of West Papua, was designated as a park to mitigatethreatsand protect the valuable marine resources.Scleractinian corals in the genus Acropora are among the most dominant distributed in Raja Ampat waters, including the species of Acroporahyacinthus. The research aimed to analyze genetic diversity and to describe the kinship relationship of Acroporahyacinthus between 2 populations: Boo Island and Deer Island, Raja Ampat. Genetic marker Cytochrome Oxidase I (CO I) of the mitochondrial genome DNA (mtDNA) was used to analyze genetic diversity. Reconstruction of phylogenetic tree and genetic diversity were made by usingsoftware MEGA 5.05 (Moleculer Evolutionary Genetics Analysis). The results of this research indicatecorals A. hyacinthus from Boo Island and Deer Island Raja Ampat are in the low category of genetic diversity and overall had a close genetic relationship of kinship. This is likely due to the small size of the population and few numbers of samples that may not represent the population.


Introduction
Indonesia lies at the centre of biodiversity for corals [1]. Though the reefs support life of more than 67.500 villages, however it suffered from extensive human exploitation [2]. The occurrence of bleaching events [3] coupled with emergence of coral diseases [4] even in remote area [5] made reef's condition rapidly declining. Therefore, there is an urgent need for reefs protection.
Marine Protected Area (MPA) is thought to be one of the best strategies for protecting biological diversity, increase fisheries yields, and protect particularly vulnerable life stages of marine species [6]. For ensure protection, it is important to understand the genetic connectivity among reefs and identify designated protected areas that are self-sustaining and possibly export young propagules to the adjacent reefs [7]. Raja Ampat is located in the heart of the 'Coral Triangle', an area encompassing Malaysia, Philippines, Solomon, Timor Leste, Papua New Guinea and Indonesia, which has the highest coral diversity in the world. The archipelago is comprised of several large mountainous islands including Waigeo, Batanta, Salawati and Misool, and hundreds of small islands such as Kofiau, Gam, Fam and Ayau. The land and surrounding sea occupy approximately 43.000 km2. While many of these are uninhabited, most of the population (~43,435) lives on 34 islands in the archipelago [8]. Almost all of the populations depend on their marine resources to support their livelihood [9]. Raja Ampat was designated as Marine Proctected Areas (MPA) in order to mitigate threats and protect valuable marine resources. The MPA was established under Goverment Decree No. 27/2008 which protect more than million hectares of coastal and marine in Raja Ampat areas.
Reefs were generally in very good condition compared to most areas of Indonesia with high live coral diversity and minimal stress due to natural phenomenon such as cyclones, predation (i.e. crownof-thorns starfish), and freshwater runoff [8]. However, 17% of reefs were considered to be in poor condition, mostly because of high levels of silting and the increase explosive use for fishing. Marine resources in Raja Ampat are also under threat from increasing frequency of disturbances associated with climate change (i.e. coral bleaching).
Documenting the diversity of marine life is challenging because many species are cryptic, small, and rare, and belong to poorly known groups10]. A genetic identification system would be particularly useful for such organisms, especially whose identification may be uncertain due to phenotypic plasticity and lack of identification tools at early stages of life [11]. DNA barcoding has become an attractive method to identify species over a wide taxonomic range [12]. Cytochrome c oxidase subunit 1 (COI) has been proposed as the main barcoding gene for metazoans [11]. Though was reported as rapid, inexpensive and need little expertise, however, the method was thought controversial since it simplified various taxonomic characters into one single character of identification only [13].Yet the method was reported can readily discriminate among closely related species across most animal phyla. The method was successfully documented enormous diversity of various cryptic animals that only 10.9% of the sequences were matched to reference barcodes in public databases [10].However, it was suggested that DNA barcode was not approriate for most scleractinian corals because this region evolves very slowly in these organisms and consequently both inter-and intra-specific variation are extremely low [14].In contrast, DNA barcode was successfully revealed the taxonomy of Stylophora pistillata by examining COI divergence and phylogeny of the coral. By analyzing Cytochrome Oxidase I sequences, from 241 samples across 4 different geographic ranges, showed this taxon in fact comprises four deeply divergent clades [15].
Acropora, differ from all other extant coral genera has unique form of polyp which formed by one or more central or axial corallites. The axial corallites bud the radial corallites as the coral grow [16].Speciesof Acropora are also highly polymorphic andhave always carried problems of identification [17]. The genera also plays a dominant role in the species composition and abundance of many Indonesian reefs [18]. Scleractinian corals in the genus Acropora are among the most dominant distributedgenera in Raja Ampat waters.This publication aims to determine the connectivity patterns and genetic diversity of Acropora hyacinthus of Raja Ampat in order to understand the genetic diversity of the coral to help conservation program of the MPA. Acropora hyacinthus was collected from core zone area of Kofiau Marine Park Area. Since sampling was conducted at MPAs area, prior the sampling, a letter of sampling permission was obtained from the Raja Ampat Bureau of National Marine Park (Simaksi, BTN). Samples were taken from the core zone of Kofiau District. Two islands were chosen as sampling location namely Deer Island (-1.155234, 129.846921) and Boo Island (-1.175641, 129.337620). A. hyacinthus at the sampling location showed various colony shape, though most of the colony has tabulate form (Fig.1).Twenty fragments were obtained from each island from each sampling location at the two islands.

Sample collections
Fragments are cut from the middle branches of the coral colony using cutting pliers. The length of fragments approximately 1-3 cm. Fragment were then put inside a labelled ziplock plastic. After collection, fragments were removed and put into 15 ml falcon tube and preserved in 95% ethanol. Fragments are then transported to Integrated Lab (CORESDU) Diponegoro University. To avoid to sample clone, distance of one colony to another colony at least 1 m. Both species are capable to reproduce through fragmentation since the colony shape is branching. Samples are preserved until used.

DNA Extraction
A quick and simple DNA extraction method with the resin, Chelex 100 (BioRad) is done. A 0.2mm section of tissues was scrapped using sterile spatula and place into 1.5 ml tubes filled with 300uL 10% Chelex. Vortexed samples in Chelex slurry for 10-15 seconds. Spinned samples briefly (10-15 sec) at high speed in a microcentrifuge. Incubated samples for 20 minutes at 95°C. Vortexed samples again for 10-15 seconds. Spinned tubes again at high speed in microcentrifuge to ensure that all contents are in the bottom of the microcentrifuge tube. Supernatant was directly used for PCR amplification. DNA concentration was then quantified using Nanodrop with spectral length on 260/280 µm and 260/230 µm.

PCR Amplification
Universal primer of DNA barcode were used for this research, there were LCOI1490: 5′-CAAATCATAAAGATATTGG-3′ as forward primer and reverse primer : HCOI2198: 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ [19]. PCR was performed using the following thermal The PCR products will be electrophoresed in a 1% agarose (FMC Bioproduct, Rockland, ME, USA) gel in 1x TAE buffer to assess the yield. The PCR products were then genotype using ABI PRISM 3730xl Genetic Analyzer used standard protocol developed by the manufacturer.

Sequencing and Alignment
Sequencing was conducted at Genetika Science Indonesia. Prominent bands of PCR samples were sent to the company. According to the company protocol, sequencing were conducted using Big Dye Terminator v.3.1 Cycle Sequencing Kit follows the PCR cycle sequen. The formula of sequen procedure were as follows : 8 μl big dye, DNA template, 3.2 pmol primer, add millipore water into the final volume 20 μl. While sequencing cycles are as follows: initial denaturation at 95 °C for 5 minutes, then following by 50 cycles of (denaturation at 95 °C for 30 second; annealing at 48 °C for10 second; and extention at 60 °C for 40 minutes). The results were then kept in 4 °C until purification.
Purification was done by purify the amplified DNA using 5 µl EDTA 100 mMinto PCR products. And then was added60 µl of 100% ethanol. Mixed samples were then be homogenized and were incubated at room temperature for 15 minutes. Samples were then centrifuged at 2000 -3000 rpmfor 30 minutes. Then added60 µl of 70% ethanol into the samples tube. Samples were then be centrifuged again at 4 °C with 1650 rpm speedfor 15 minutes. Purified DNA then were sequenced using ABI PRISM 3730xl Genetic Analyzer following the protocol available from the Applied Biosystems, USA. The most prominent band of purified PCR products are used as sequencing samples.
The sequenced results were then alignmented and were analyzed using BLAST(Basic LocalAlignmentSearchTool) based on genetic data base of NCBI (NationalInstituteforHealth, USA (www.ncbi.nlm.nih.gov). Phylogenetic tree was constructed using MEGA5. Neighbor Joining Tree was used to construct a tree to analyse genetic distance. An outgroup was used when constructed the three. Genetic diversity was observed using DNAsp software [20] and Arlequin: http://cmpg.unibe.ch/software/arlequin35/Arlequin35.html.

Results and Discussion
In total, there were 25 sequenced resulted from Deer and Boo Island samples. The resulting Oxidase I (CO I) of the mitochondrial genome DNA (mtDNA) sequences corresponding to the genotype were analyzed for homologies with sequences in the data base using BLAST searching. BLAST analysis showed that all 25 sequences were closely related to Acropora hyacinthus under 99% similarity ( Table  1). The accession numbers of COI mtDNA of the sources that used as comparison was presented as well in Table 1. All nucleotide sequences of the COI were than deposited to Genebank. All nucleotide sequences have been deposited in the GeneBank database under accession number LC187992 -LC188016 (Table 1). \

Phylogenetic Tree
Reconstruction of phylogenetic tree was done using PAUP* 4.0 (Phylogenetic Analysis Using Parsimony) [21]with parsimonymethod and 1000 times bootstrap. Kinship relationship was observed using TreeView program.
Phylogenetic construction based on COI sequences found 6 distinct cluster ofA. hyacinthus. Outgroup using DNA data base of Montipora sp. Phygenetic tree of A. hyacinthus was shown at  (Table 2).
When comparison was made with A. hyacinthus population from Karimunjawa Archipelago, similar result was obtained. A. hyacinthus of Karimunjawa Archipelago was grouped into 5 differents clades [22]. Samples of A. hyacinthus from Kofiau District was collected from Deer Island and Boo Island that was separated by more than 100 km, while samples from Karimunjawa Archipelago was collected from two different cluster islands namely Seruni, Sambangan and Genting cluster and Cilik and Menjangan Kecil cluster islands that separate by most far 60 km only. However, the Menjangan Kecil Island and Cilik Island was set behind the main Karimunjawa Island that face of open sea while Seruni, Sambangan and Genting was set inside lagoonal area of Karimunjawa main island.

Analysis of Genetic Diversity
Genetic diversity of A. hyacinthus was observed using DNAsp [20]. Based on statistic analysis of genetic diversity of 25 samples showed 6 haplotypes. Distribution of haplotypes was shown at Table 3. Estimation of haplotype diversity value of A. hyacinthus was 0,3667. When haplotype diversity was observed from each island it was showed much greater value. Haplotype diversity of A. hyacinthus collected from Boo Island was 0, 9238 (n=15) while haplotype diversity of A. hyacinthus collected from Deer Island was 0,9333 (n=10).