Redefining Dispersal Boundaries of Siganus fuscescens In The Coral Triangle Area

The increasing demand of fish in the Coral Triangle Area has led to overexploitation of some species of fishes. One of the commercial fishes, which is also known to be the source of food and income for local communities, is the Mottled Spinefoot (Siganus fuscescens). Population studies on this species are important in order to manage sustainable stock populations. Genetic variation of the mitochondrial DNA was analyzed to examine the population structure of Siganus fuscescens in Indonesia, as part of the Coral Triangle Area. In total, 789 basepairs of control region mtDNA sequences were determined from 133 specimens collected from six localities, including Seribu Islands (n=27), Karimunjawa (n=19), Komodo (n=39), Selayar (n=20), Lembeh (n=19) and Luwuk (n=9). From the data, 27 variable sites and 24 haplotypes were detected, with most of the haplotypes unique to each location. Haplotype data show that one haplotype was shared among all populations, three haplotypes were shared between two populations (Komodo & Selayar; Lembeh & Seribu; Komodo & Karimunjawa), and 20 were unique to a single population. Haplotype diversity (h=0.444) and nucleotide diversity (π=0.00165) were low. The diversity result, i.e. the ΦST value (0.0658, P < 0.0001) revealed genetic structure in S. fuscescens populations in Indonesia. A non-dispersal strategy led to restricted gene flow and genetic structuring in S. fuscescens. However, both the neutrality test and the mismatch distribution indicated that S. fuscescens might have been in populations at demographic equilibrium, with restriction to the population expansion. Although indicating unexpected minor population structure pattern, the overall result still suggest the management of this species population as a single unit across Indonesia.


Introduction
Indonesia is located at the center of a Coral Triangle, an area with the highest marine biodiversity. Recognized as a biodiversity hotspot, it is home to a remarkable diversity of marine species (Carpenter et al., 2011;Hoeksema, 2007;Veron et al., 2009). Widely known as an archipelagic country, Indonesia has extensive coastal areas that hold high economical value supporting its community. Another wealths of Indonesia's resources is its abundance and diversity of marine species (Hughes et al., 2003;Hoeksema, 2004).
Indonesia is also distinguished by the threats that plague its marine biodiversity (Carpenter et al., 2011). Nowadays, the escalating human population is increasing the demand for fish, which is causing overfishing of some species of marine fishes (Sala & Knowlton, 2006). Directorate General of Capture Fisheries recorded that the volume of fisheries production in Indonesia reached five million tonnes in 2010, while the lowest trend reached three million tonnes in 2000 (KKP, 2011). In order to maintain the supply of fish stock so it can comply with the market demand, marine aquaculture was established in several areas throughout Indonesia. Indonesia is also on the list of the top ten countries with the highest marine aquaculture production in 2010 with 2,304,828 tonnes of fish (FAO, 2012). Siganus is one of the economically important species because of its value as food commodity and aquarium fish (Lam, 1974;Ayson et al., 2002;Kuriiwa et al., 2007). Several Siganus species have been reported as being overfished in the Philippines, including Siganus canaliculatus, Siganus spinus, Siganus argenteus (Soliman et al., 2009) and Siganus fuscescens (Ravago-Gotanco & Juinio-Mendez, 2010). However, information based on a genetic study is still lacking on Siganus sp. population stock in Indonesia. This research aims to study the population structure of Siganus fuscescens in order to manage sustainable stock populations in Indonesia.
Samples were collected from muscle tissue or pectoral fin clip and preserved in 95% ethanol. Each sample were morphologically identified as described by Woodland (1990). DNA was extracted using 10% chelex solution (Walsh et al., 1991). A fragment of control region mitochondrial DNA (mtDNA) was amplified via Polymerase Chain Reaction (PCR) using S.

Population genetics analysis
Sequences were edited and aligned using the CLUSTALW algorithm in MEGA5.05 (Tamura et al., 2011). Phylogenetic analysis of S. fuscescens, S. canaliculatus, and S. argenteus (accession numbers: GU929566-GU929669, GU929670-GU929673, and GU929674-GU929676, respectively) sequences was performed to further confirm that no sampling errors were made. Mitochondrial control region data of S. fuscescens were generated from Genbank (GU929566-GU929669) and used to confirm clades with the previous S. fuscescens populations study (Ravago-Gotanco & Juinio-Mendez, 2010). Neighbor-joining (NJ) analysis using MEGA5.05 was initially performed to confirm clades, followed by Bayesian analysis using BEAST v1.7.5 (Drummond et al., 2013). Boostrapping was performed to test the robustness of the resulting tree in NJ tree (Falsenstain, 1985), while Bayesian Information Criterion (BIC) using JmodelTest 0.1.1 (Guindon & Gascuel, 2003;Posada, 2008) was used to determine model parameters. Bayesian analysis was run for 10 7 generations. The posterior probability estimates from 10,000 tree with a burn-in of 1,000 trees.

Results and Discussion
A total of 133 individuals identified as Siganus fuscescens were collected and analyses using mitochondrial control region DNA, yielding 24 haplotypes (Genebank accession numbers KU365295-KU365318). The length of each sequence were 812 base pairs (bp) consists of 786 homologous sites, two insertions/deletions (indels), and 27 variable sites.
Several reports based on nuclear sequences indicated hybridization between S. fuscescens and S. canaliculatus, two species of Siganus species that has similar morphology (Lam, 1974;Kuriiwa et al,, 2007). To avoid miss-identification of these two species, GenBank sequences were used as a comparison. Genetic identification using GenBank data confirms that samples on this study were on the same clade as clade A (Figure 2) in comparison with the results of the previous research conducted by Ravago-Gotanco & Juinio-Mendez (2010).
Clade A was reported to be widely distributed across the Coral Triangle, while clade B is restricted to the northwest Pacific, and clade C within the southwest Pacific (Borsa et al., 2007). The phylogenetic tree confirms that all of the samples collected from the Indonesia regions belong to S. fuscescens clade A.
The S. fuscescens mtDNA control region data set consists of 24 haplotypes that are shared or unique to each of the locations, including a single haplotype that is shared among all populations, three haplotypes that are shared between two populations (Komodo & Selayar; Lembeh & Seribu; Komodo & Karimunjawa), and 20 haplotypes that are unique to a single population (Figure 3). Haplotype distribution within each of geographical sites is indicated in Figure 3a. The minimumspanning tree concordant with the phylogenetic tree indicated that all haplotypes are nested into one clade with one dominant haplotype, followed by unique haplotypes or shared haplotypes between two populations. The circles size within Figure 3b is proportionally to the frequency of occurrence of each of the haplotypes, with each haplotypes are separated by one mutational step.
Geographical distance limits genetic differences between populations, acting as oceanic barriers that separate habitats. Numerous publications have reported that genetic structure is found in some species due to large geographical distances, including a blennioid fish, Axoclinus nigricaudus (Riginos & Nachman, 2001), temperate seagrass, Zostera marina (Hammerli & Reusch, 2003) and a giant clam, Tridacna crocea (DeBoer et al., 2008). However, it appears that geographical distance does not limit the genetic structure in S. fuscescens populations within this study, which is indicated by the high level of gene flow in S. fuscescens populations in the central part of Indonesia (Figure 3). High indication of gene flow is shown by a large percentage of shared haplotypes between populations. A single panmictic population is probably common in this area, which was documented by Lourie et al. (2009) using three different species of seahorse (Hippocampus barbouri, H. kuda, and H. spinosissimus), coral reef fish, Caesio cunning (Ackiss et al., 2013), blue starfish, Linckia laevigata and gastropod Despite the finding of highly shared haplotypes, the result also shows unique haplotypes in each locality (Figure 3a), which may be caused by the isolation of these haplotypes within a single population due to limited dispersal ability and the effect of ocean currents. The adults of S. fuscescens probably travel over a short distance as indicated by a short home range and territory that is usually less than three kilometers (Green et al., 2015). Despite the limited dispersal potential of individual adults, long distance dispersal potentially occurred at the larval stages (Shanks, 2009). Although pelagic larval duration (PLD) of this species is not known, the sibling species, S. Canaliculatus, is exposed to three weeks planktonic larval periods (Hasse & Madraisau, 1977). The long pelagic larval period potentially facilitated a long dispersal distance of a species (Shanks et al., 2003). Dispersal can also be affected by ocean currents (Nakajima et al., 2014;Raynal et al., 2014;Lo et al., 2014). The immense passage of water conveyed by the Indonesian Throughflow (ITF), which moves water from the Pacific to Indian Oceans, can facilitate connectivity and enhance lineage mixing between sites, reflected by no obvious differentiation in central Indonesia (Lind et al., 2012;Timm et al., 2012).
It is also notable that a higher gene flow among geographical population was associated with major ocean currents (Imron et al., 2007). Connectivity shown by the population of S. fuscescens in Sulawesi (Luwuk, Lembeh, Selayar) and Komodo seemingly follows the ITF pattern. Similar patterns were also found in other fish species such as coral reef fish, Caesio cunning (Ackiss et al., 2013), the false clown anemonefish, Amphiprion ocellaris (Timm et al., 2012), and the humbug damselfish Dascyllus aruanus (Raynal et al., 2014). Meanwhile, connectivity between populations in Java (Seribu and Karimunjawa) and populations in the east (Sulawesi and Komodo) are probably maintained by seasonality reversing current (SRC). Although previous research could not find any relations between a genetic pattern and seasonal current in Sunda shelf (Nelson et al., 2000), this result indicated that the SRC may play an important role in the dispersal of S. fuscescens.
In addition, haplotype diversity (h) and nucleotide diversity (π) for all populations were indicated as low diversity with the value of h = 0.4659 and π = 0.00165, respectively. Low levels of nucleotide diversity might be a signal of recent population expansion (Rohfritsch & Borsa, 2005). The number of haplotypes, haplotype diversity, and an estimate of nucleotide diversity for each site are presented in Table 1.
Meanwhile, the Φ-statistic value of AMOVA analyses (0.0658, p<0.0001) revealed the genetic structure in S. fuscescens population in Indonesia, which is signaled by significant genetic differences in populations on Seribu Islands compared to other populations, including Komodo and Selayar. Significant pairwise ΦST values were indicated in the Table 2. This result also shows the significant genetic differentiation of two out of 15 population comparisons.
The genetic difference result was not concordant with the hypothesis of the Sunda Shelf Barrier, which is believed to be a geographical genetic break that separates the western and eastern parts of Indonesia. Other significant genetic pairwise differences between distinguishable populations also indicate the same result. Meanwhile, several unique haplotypes revealed in this study may be caused by local isolation due to the effect of sea level rise during Pleistocene glacial events (Carpenter et al., 2011). This phenomenon led to localized extinction and recolonization of Siganus sp. populations associated with algal and seagrass meadows in shallow and sheltered reef flats (Woodland, 1990).
The historical demography was analyzed using Tajima's D test and mismatch distribution. The Tajima's D value was significantly negative (p<0.05) for three out of six populations, suggesting an excess of low-frequency haplotypes relative to that expected under mutation-drift equilibrium. Most likely, this indicates a model of rapid population expansion in which several rare alleles have recently derived from one or a few alleles of high frequency. However, the test shows an insignificant negative result for the mean value of all regional populations (p>0.05) (Table 3), indicating the population did not significantly depart from the neutral evolution model. The mismatch distribution for S. Fuscescens appeared to be multimodal. This was supported by   (Benjamini & Hochberg, 1995). the non-significant low values for SSD (0.06778, p-value=0.24167) and Harpending's raggedness index (0. 17631, p-value=0.58333) under the sudden expansion model. This result indicated that the S. fuscescens population in this study is stable.The minimum-spanning tree shape also supported that the population of S. fuscescens is at demographic equilibrium.
This suggests that the population range expansion may have been restricted to a nondispersal strategy that may have restrained S. fuscescens from demographic expansion. Based on these findings, the population of S. fuscescens in Indonesia should be managed as a single natural management unit throughout Indonesia

Conclusion
There is a minor structure in Siganus fuscescens populations in Indonesia. However, this population structure is not limited by the geographical distance shown by a high indication of gene flow and haplotypes shared among populations. The population also shows low haplotype and nucleotide diversity and a stable demographic population. The population structure of Siganus fuscescens in this study is not concordant with the Sunda Shelf Barrier hypothesis, which acts as a geographical genetic break that separates the western and eastern parts of Indonesia. Weir, B.S. Cockerham CC. 1984