DNA barcode of Acropora hyacinthus of Karimunjawa Archipelago

Karimunjawa is one of the earliest marine parks in Indonesia. Karimunjawa National Park (KNP) was designated as a marine conservation area to conserve marine resources from destructive fishing activities. Scleractinian corals in the genus Acropora are among the most dominant distributed in the KNPs, including the species of Acropora hyacinthus. Here, we present a comprehensive analysis of intra- and interspecific COI variabilities in A. hyacinthus to analyze genetic diversity and to describe the kinship relationship of the coral between 5 localities of the reefs. Genetic marker Cytochrome Oxidase I of the mitochondrial genome DNA (mtDNA) was used to analyze genetic diversity. Reconstruction of phylogenetic tree and genetic diversity were made by using software MEGA 5.05 (Molecular Evolutionary Genetics Analysis). The results indicate corals A. hyacinthus from five localities of Karimunjawa Archipelago are in the high category of genetic diversity. However, the five populations showed 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. The results may aid managers of the park in the selection of appropriate propagules sources which can help to restore important data for conservation and sustain coral reef resources.


Introduction
Karimunjawa waters are one of the earliest marine parks in Indonesia. The park lies in the Java Sea, approximately ± 45 nautical miles from Jepara, on the north coast of Central Java, Indonesia. The park was designated as a Strict Natural Reserve on 9 April 1986 by Minister of Forestry under decree (PHKA No. 123/Kpts-II/1986) as an effort to conserve marine resources from destructive fishing activities. In 2001, all marine waters of Karimunjawa National Park (KNP) were designated as a marine conservation area by the Ministry of Forestry Decree No. 74/Kpts-II/2001 [1]. The park consists of 27 big and small islands, seven of which are inhabited. Coral communities are found vigorously to a depth about 15 m with Pocillopora and Acroporidae being the dominant family. According to survey conducted by Wildlife Conservation Society (WCS), there are 63 genera of 15 families of scleractinian corals. Recent trends in the average of coral cover were moderate (around 40-60 %) [2] with similarity index between 0.43 to 0.91 [3].
KNP represents well the population-related reef management issues that are common in developing countries [4], particularly in the Indo-Pacific where efforts to mitigate local impacts that reduce reef 2 1234567890 ''"" resilience are currently scarce [5]. With most populated island in Indonesia is nearby (Java Island, > 60 % of total nation population), KNP pose a significant challenge for reef managers to address issues of balancing demands for reef-generated resources with conservation efforts [6]. Though it was reported that co-management frameworks as adaptive measures to gain trust, collaboration and a sense of ownership by reef users, improved some ecological condition of the reefs [1], however, extensive human activity related to tourism [7], coupled with emergence of coral disease [8,9,10], coral bleaching [11,12] and increasing local demands of coral reefs resources by a growing local population [13] can contribute to reefs"s decline condition. For coral reefs, potential adaptation ability to re-establish after disturbances is important. There are various factors that may facilitate local adaptation including genetic factors. Understanding coral diversity is essential in managing coral reefs ecosystem especially when many of the reefs are in degraded condition [14]. When MPA network is designated, it is important to understand genetic connectivity among reefs and which areas are particularly rich in species to ensure that the designated protected areas are self-sustaining and possibly export propagules to neighboring reefs [15]. Most corals release gametes in a great event namely mass corals spawning [16] which were then dispersed by ocean currents into various distances from the natal reefs. Knowledge of genetic diversity is a need to understand the coral distribution and the source of coral larvae which is important to replenish coral reefs ecosystem. However, genetic diversity has rarely been corporate in designing a Marine Protected Area [14].
DNA barcoding (DBC) is an alternative to traditional taxonomic methods that could become a useful tool for coral reef conservation [17]. Cytochrome c oxidase subunit 1 (COI) has been proposed as the main barcoding gene for metazoans [18]. DNA barcoding has been extensively used to discriminate between closely related species, to identify new, cryptic or invasive species, and to assess biodiversity across many animal phyla [19]. The effectiveness of this approach clearly depends on the availability of extensive databases of COI-5 barcode sequence standards [17]. Based on the results of DNA barcode of 90 species of corals from 44 genera belonging to 14 families, suggested that the criterion was not being appropriate for most scleractinian corals (or anthozoans in general) because this region evolves very slowly in these organisms and consequently both inter-and intra-specific variation are extremely low [20]. However, taxonomy of Stylophora pistillata was successfully demonstrated comprises of four deeply divergent clades based on COI divergence and phylogeny of the coral [21]. Genus Acropora, are by far the most diverse living reef-building coral genus, with 114 species recognized worldwide [22] and others possibly unnamed. The genus distributes on most tropical reefs throughout the world, except the eastern Pacific Reefs [23]. Acropora is distinguished from all other extant coral genera by a unique form of polyp structural. Corallites are small, divide into two types axial and radial. Corallites have septa in two cycles or less and columellae are seldom developed [24]. Acropora plays a dominant role in the species composition and abundance of many Indonesian reefs [25]. Acroporid is a good example of studying reefs connectivity since they have the most genetic information available for any coral genus worldwide. Acroporids also knew to have largest geographical ranges compare to other corals family [26].
Here, we present a comprehensive analysis of intra-and interspecific COI variabilities in Acropora hyacinthus to determine connectivity patterns and genetic diversity of the coral on Karimunjawa Archipelago reefs. The results may aid managers of the park in the selection of appropriate propagules sources which can help to restore important data for future coral reef conservation and sustain coral reef resources.  [27]. Each coral colony as a sample was documented for identification. The samples from branches were cut for 1-2 cm of random colony using cutting pliers. At least 1 m distanced from one colony to another colony to avoid collected clone colony. As branches colony, A. hyacinthus can reproduce through fragmentation [28]. Small pieces were then put into a 2 mL labeled cryovial. To avoid resampled, each colony that was already cut was tagged using the small plastic number and cable ties. Table 1 has specific site information along with the number of corals collected at each site. Samples were preserved in 95 % ethanol until used. In total, 104 coral branches were obtained from KNP.

PCR Amplification
Amplifications were performed in 25 μl volume master mix PCR solution. Universal primer for COI gene, consisted of forward primer LCOI1490: 5'-CAAATCATAAAGATATTGG-3' and reverse primer HCOI2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' [29] were used for amplification. Master mix PCR solution containing 1 μl of each primer, 1 μl of DNA Template (< 250 ng·μl -1 ), 12.5 μl of GoTaq ® Green Master Mix (Promega, USA) and 9.5 μl of Nuclease-Free Water (Promega, USA). Thermal cycle condition was following protocol by [20]. PCR products were analyzed using 1 % agarose gel. PCR products that have DNA band were directly sequenced at 1 st Base Laboratory using both of primers. Sequencing results were analyzed using MEGA 6.0 for alignment process and Phylogenetic tree was constructed using Phylogenetic Analysis Using Parsimony (PAUP) software [30].

Results and Discussion
In total, there were 32 sequences resulted from Menjangan Kecil Is., Cilik Is., Genting Is., Seruni Is., and Sambangan Is., Karimunjawa Archipelago, Java Sea. The resulting Oxidase I (CO I) of the mitochondrial genome DNA (mtDNA) sequences were corresponding to the genotype that was analyzed for homologies of sequences in the database using BLAST searches. BLAST analysis showed that all 32 sequences were closely related to Acropora hyacinthus under 99 % similarity. All nucleotide sequences have been deposited in the GeneBank database under accession number LC189268-LC189316.

Phylogenetic Tree
Reconstruction of the phylogenetic tree was done using PAUP* 4.0 (Phylogenetic Analysis Using Parsimony) [30] with parsimony method and 1,000 times boots trap. Kinship relationship was observed using TreeView program.
Phylogenetic construction based on COI sequences found 6 distinct clusters of A. hyacinthus. Outgroup using DNA data base of Montipora sp. Phylogenetic tree of A. hyacinthus was shown in figure 1. When comparison was made with A. hyacinthus population from Raja Ampat, a similar result was obtained. A. hyacinthus of Raja Ampat has grouped into 6 different clades [31]. 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 5 1234567890 ''""

Analysis of Genetic Diversity
Genetic diversity of A. hyacinthus was observed using DNAsp [32]. Based on statistic analysis of genetic diversity of 32 samples showed 15 haplotypes (table 2). There were 11 haplotypes (73.3 % of total samples) are unique haplotypes that consisted only one sample each. While four haplotypes (26.7 %) are more common haplotypes with haplotype 1 as the highest member of samples (13 samples). Estimation of haplotype diversity value of A. hyacinthus was 0.8286 which categorized has a high genetic diversity [33]. It is likely that population of A. hyacinthus at Karimunjawa Archipelago showed high genetic diversity.  Distributions of species of the coral genus Acropora in the Indonesian archipelago show a duality reminiscent of the Wallace"s Line patterns seen in terrestrial animals and plants, rather than the concentric pattern predicted by the "center of origin" model [34]. This duality is due to an overlap of Indian Ocean species distributions diminishing eastwards and Pacific Ocean species distributions diminishing westwards within the archipelago [34]. Additionally, a large number of species with broad Indo-Pacific distribution, as well as some regional endemics, occur within the archipelago. Thus although Indonesia has a high species number overall, this is due to the presence of a composite fauna with strong regional differences.
Using multi-locus nucleotide sequence data, [26] recently described two, previously unsuspected, cryptic species complexes within the Acropora syngameon (i.e., groups of species connected through a genetic exchange). Acropora is categorized as the most diverse and abundant genera of reef-building corals [22,24]. The genus is notorious for high levels of shared polymorphism between species, which is due, in part at least, to widespread genetic exchange through introgression [35,36,37]. Despite the absence of any fixed sequence differences, utilized allele frequency variation at 10 loci to demonstrate that two common coral species in Australia, A. cytherea and A. hyacinthus, actually represent species complexes minimum two and four cryptic species. These cryptic species were discovered in complete sympatry and form part of an extended syngameon with a complex network of gene flow among species. Cryptic species were identified by the "genotypic cluster" species definition, which defines species as distinguishable genotypic groups.
The results of this research indicate that corals A. hyacinthus from Karimunjawa Island is categorized as high of genetic diversity, though they showed a relatively close genetic relationship of kinship. We need run a second gene such as microsatellite or intron sequencing to confirm possibility that population of A. hyacinthus at Karimunjawa Archipelago may have a cryptic variation as suggested by [26]. However, the possibility that population of A. hyacinthus at Karimunjawa Archipelago has high diversity could not be excluded. Therefore, managers of the KNP need more appropriate approach to resources include the genetic diversity.