Barcoding of Fin Clip Samples Revealed High Exploitation of Plectropomus leopardus in Spermonde Archipelago

Spermonde Archipelago in South Sulawesi is among groupers sources in Indonesia for International Marine Live Trade. It was not much known which species are mostly exploited in those areas during the study because only fin clip samples could be obtained. Here we performed DNA forensic analysis to groupers fin clips collected from Spermonde Archipelago using barcode marker to know what species were exploited and which ones was the most exploited species. A total of 110 fin clip samples were obtained during the field trips. Sequencing of the cytochrome c oxidase 1 gene was successful for only 36 samples. Species determination was based on sequences similarity to conspecific sequence available in barcoding of life database (BOLD). BOLD similarity test placed the 36 fin clip samples into four different species, namely Cromileptes altivelis, Epinephelus ongus, Plectropomus leopardus, and Plectropomus maculatus. The K-2P taxonomic tree showed clear separation among species which was supported by high K2P genetic distances among species. Most of fin clips were identified as Plectropomus leopardus indicates that this species was the most exploited in Spermonde Archipelago. This proved that molecular identification not only can be used to biodiversity study in certain area but also in forensic analysis of a threatened wildlife. This study has important contribution for conservation effort of those groupers species, especially P. leopradus and provides new approach for taxonomist and conservation managers to identify samples, especially when only incomplete specimens are available.

cially when only incomplete specimens are available.

METHODS Study area, sampling technique, and sample handling
Grouper samples were collected randomly by the fisherman from all around Spermonde Archipelago (Figure 1).Small piece of fin clip tissue samples were cut off randomly from life grouper individuals owned by middlemen.This sampling technique was applied to avoid the individual getting die, otherwise the price become lowered.Tissue samples were collected during the field trips in 2012.The samples were then preserved directly in 96 % ethanol and afterwards they were stored on temperature of 4 °C until the DNA analysis.The DNA processing was performed in 2015.

DNA extraction and barcode marker amplification
Genomic DNA was isolated using slightly modified Chekex®100 method by Kochzius and Nuryanto (2008).Approximately of 2 mm 2 tissue samples were extracted in 5% of 100 mL chelex, mixed with 0.5 mM dithiotreithol (DTT) and 2 µL proteianse-K.The mixtures were incubated in thermomixer at 56ºC for approximately of 6 hours and homogenized manually after each hour incubation.The success of DNA isolations were checked by migrating the extracted DNA in agarose 1%, stained using 1% ethidium bromide and visualized under the UV-light.
The cytochrome c oxidase I (COI) gene fragments of the samples were amplified using

INTRODUCTION
Grouper is the most popular fish group in international marine live trade.It has high economic values as well as high export demands (Harikrishnan et al., 2010).In Southeast Asia, grouper has very high prices (Pierre et al., 2008).In Spermonde Achipelago, the prices of life individuals reach 250,000 IDR per kilogram, while dead individuals only priced for 50,000 IRD per kg (personal observation).Therefore, grouper has been widely farmed in Southeast Asian countries, including Indonesia.However, grouper farming was unable to meet market demand.This condition makes natural capture is still representing the largest portion of grouper exports in the world (Pierre et al., 2008) with Indonesia that supports a largest portion to global grouper catch (Ottolenghi et al., 2004).Grouper stock in Indonesia become overfished (Scales et al., 2007) and put Plectropomus leopardus, Epinephelus coioides listed as near threatened, while Chromileptes altivelis as vulnerable species (http://www.iucnredlist.org/details/44684/0).In this case, conservation efforts are urgently needed.Fundamental research such as proper identification of target species into species level become a vital step.
In the case of incomplete individual samples were found during the field trips, forensic analysis through DNA barcoding is the only approach suitable to reveal taxonomic status of the samples.The cytochrome c oxidase 1 (CO1) is widely used as barcode marker in such studies (Mabragaña et al., 2011;Radulovici et al., 2010) and it is believed as highly precise marker for species identification (deWaard et al., 2011).It has been reported that CO1 barcode could properly identify fish (von der Heyden et al., 2014;Hubert et al., 2012;Muricy, 2011;), even from fillet samples (Wong and Hanner, 2008); and fish larvae with limited morphological features (Pegg et al., 2006;Nuryanto et al., 2017).
Due to the explanation above, this study was conducted to figure out what species were exploited and which one was the most exploited in Spermonde Archipelago, South Sulawesi.The partial sequences of the COI gene were used as a forensic barcode to reveal taxonomic status of fin clip samples of groupers that region.This research provides valuable information about the application of animal systematics, especially in forensic analysis of wildlife specimen.In addition, information from this study is also important for conservation effort of those groupers species and provides new approach for taxonomist and conservation managers to identify samples, espe-forward primer 5'atctttggtgcatgagcaggaatagt3', instead of using FishF2 and reverse primer FishR2-5'tagacttctgggtggccaaagaatca3' (Ward et al., 2005).The PCR reactions was carried out in a total volume of 50 µl contained 29.8 µl ultrapure water; 1X PCR buffer, MgCl 2 5 mM, 0.01 mM of each primer, 0.05 mM of each dNTP, 1 U Taq polymerase, and 4 µl of template DNA.The thermal cycles were as follow: one cycle at 95 °C for 5 minutes and followed by 35 cycles of 1 minute at 94 °C, 1minute annealing at 54 °C, and 1.5 minutes at 72 °C.The final extension was carried out at 72 °C for 10 minutes.Amplification products were visualized on 1 % Agarose gel over the UVligh and photographed.

Data analysis
Taxonomic status of the fin clip samples was defined by comparing the CO1 sequences of the samples to previously published sequences listed in barcode of life database (BOLD) (Ratnasingham & Hebert, 2007) using BIN value of 3% according to species level database.Genetic divergences within and among species were estimated based on Kimura 2 parameters (K2P) substitution model as implemented in Arlequin software version 3.5.(Excoffier & Lischer, 2010).The K2P neighbour-joining (NJ) tree was reconstructed in MEGA software 6.0 versions (Kumar et al., 2008).Topology of the tree was supported by 1000 non-parametric bootstrap replicates.

PCR amplification
A total of 110 fin clip samples were obtained during the field trips.However, amplification and sequencing proccesses was only successful for 36 samples.This minor success of amplification was due to the use of old preserved fin clip samples.The fin clips samples were stored for at least three years until DNA processing since they were collected in 2012, while DNA analysis was performed in 2015.Although theoretically, DNA can still be amplified from ancient material, in fact it is more difficult to amplify DNA from either ancient samples or even from old preserved samples compared to the fresh collected ones.This could be due to that DNA in long preserved tissue will be fragmented.Fragmented DNA sometimes become very difficult to be amplified.According to Hird et al. (2006) amplification of highly degraded DNA was very hard, especially if target fragment quite long and the success of amplification rate reduced with the increased of target fragments.
In one hand, our result was different to the result of a study by Sutrisno (2012) and Zimmermann et al. (2008) that reported a successful amplification of less than six years preserved moths.In other hand, our result is also similar to Sutrisno (2012) and Zimmermann et al. (2008) for older samples.These difference and similarity of our success to Sutrisno (2012) and Zimmermann et al. (2008) on PCR amplification of old preserved samples could be due to that we used different animal and tissues types.Here we used fish and fin clips tissue samples while Sutrisno (2012) and Zimmermann et al. (2008) used body tissues of moths.This argument is supported by Srinivasan et al. (2002) which proved that fixative affect the success of DNA amplification from preserved tissue and leads to the amplification of artifacts amplicons.Moreover, DNA quality of preserved tissues linearly decreased with preservation times and become more fragmented.Fragmented DNA would be very difficult to be amplified.This means the success of DNA amplification from old preserved samples are varied among organismal groups and tissue types.Therefore, it is not surprising if there will be some differences and similarities of the success of PCR amplification from one study to the others.

Homology test and genetic species concept
Homology test of the samples to the sequences available di bold system databases showed that the samples had sequences similarity ranges from 97.24 % to 100% to the sequences of conspecific.These mean that our samples had sequences divergences of less than 3% as the standard divergence value in bold systems.These similarity values mean that all of our samples could be identified accurately into species level.sub sub-clade E. The remaining samples identified as Cromileptes altivelis were also combined together with KJ594967 to form another monophyletic group called as sub sub-clade F.
If we go backward in the branching pattern of the tree (Figure 2), we came to genus levels.It is clearly found that samples belong to single genus are placed together in a single monophyletic group (sub-clade A) with rather short branch length.The placement of E. ongus and C. altivelis into a single monophyletic group was due to that they have closer relationship compared to P. leopardus and P. maculatus with genetic divergence 0.146 (Table 2).Since the both E. ongus and C. altivelis belong to the different genera, they are combined because no congenera species are available but by rather long branch length (sub-clade B).This means that both species are combined together in a taxonomic category above genus.This separation is congruence with the result of molecular identification that showed that genus Plectropomus consist of P. leopardus and P. maculatus.The separation of both subclade and sub-subclade were supported by high bootstraps values.These mean that the separation of those groups was statistically reliable and improve the quality of identification result.
A clear discrimination among species and above species level as indicated in the taxonomic tree (Figure2) provides additional scientific data about the reliability of the COI gene as barcode marker in species delimitation, especially in fish.The power of this gene as a species barcode marker because it is changeable enough (Nuryanto et al., 2007;Nuryanto and Kochzius, 2009) due to its high mutation rate which is higher compared to other mitochondrial genome (Hebert et al., 2003).Clear separation of different species and above species category was also reported in fish larvae (Nuryanto et al., 2017), or even among strain within species (Nuryanto et al., 2018).
The K-2P genetic divergences among fin clip samples within species range from 0.000 in P. maculatus up to 0.016 in C. Altivelis (Table 2).These mean that low genetic divergences were found among individuals from single species.Similar low genetic divergences were also reported Among 36 sequences of fin clips, a total of 18 fin clip samples were genetically identified as Plectropomus leopardus.Nine samples were identified as Cromileptes altivelis, seven fin clips were identified as Epinephelus ongus, and the remaining two fin clips were identified as P. maculatus (Table 1).
There were no doubt to assign the fin clip samples into those four species due to all samples show sequences similarity of more than 97% either to sequences in bold systems or to genbank databases.This means that sequences divergence between our samples with their conspecific sequences available in boldsystems and genbank databases less than 3%.According to Pegg et al. (2006) different fish species can be delineated based on sequence divergence of 1% to 3%.Even if we refer to Pereira et al. (2013), maximum genetic divergence within species might reach 8.5% depend on fish group.In other words, genetic similarity among individuals within species can be less than 97%.Therefore, our assignment of the fin clips samples into four species based on 97.24% sequences similarity was very convincing.
The Kimua 2 Parameters (K-2P) neighbour-joining tree topology (Figure 2) supported the result of DNA barcoding.Similar support was also obtained when the trees were constructed using maximum parsimony analysis based on Kimua 2 Parameters.In higher category, the ingroup samples formed a monophyletic group or monophyletic clade compared to the out-group samples (Figure 2).If we look into detail to our samples in Figure 2, the samples were divided into two sub-monophyletic groups (sub-clades) and we call as sub-clade A and B. It can also be found on the tree that each individual sample formed monophyletic groups with their conspecific relatives taken from database.For example, all fin clips identified as P. leopardus was put together in a same monophyletic group with P. leopardus JN021314 from genbank and referred to as sub sub-clade C. All individual samples which were identified as P. maculatus formed a monophyletic clade with P. maculatus KR863514 (sub subclade D).The fin clip samples identified as Epinephelus ongus were placed together with E. ongus KP194568 in one monophyletic group, called as  Inter-species genetic divergences were range from 0.05 between P. leopardus and P. ongus up to 0.226 between P. maculatus and E. ongus (Table 2).These observed high values of inter-species genetic divergences are common phenomena.Several studies also reported high genetic divergences among species in various animal groups, such as in fish (Peg et al., 2006;Pereira et al., 2013;Nuryanto et al., 2017;Nuryanto et al., 2018); and Crustacea (Barber et al., 2002).
Molecular identification had also been performed on Epinephelus from India (Sachithanandam et al., 2012) and from Israel (Dor, 2012).In general, molecular barcoding was carried out on individuals with clear or definite taxonomic status.Whereas, molecular barcoding for forensic analysis on unidentified tissue samples of Indonesia's groupers was rather rare, especially in regard to the most exploited species.Therefore, the result of this study proved that molecular identification is not only valuable for taxonomic diversity study in certain area, but it can be also addressed as DNA forensic analysis to trace illegal fishing of near threatened and vulnerable species such as Plectropomus leopardus and Cromileptes altivelis, respectively.This research provides valuable information about the application of animal systematics, especially in wildlife forensic analysis.In addition, information from this study was also important for conservation effort of those groupers species.This study also provides new approach for taxonomist and conservation managers to identify samples, especially when only incomplete specimens are available.

CONCLUSION
Forensic analysis using COI barcode successfully identified fin clips samples into species level.A total of four groupers species was exploited during the sampling and most of them were genetically identified as P. leopardus, means that P. leopardus was the most exploited grouper in Spermonde Archipelago.

Figure 1 .
Figure 1.Sulawesi Island with sampling site in Spermonde Archipelago.

Figure 2 .
Figure 2. Phylogenetic tree based on 633 bp of the mitochondrial DNA COI gene using genetic distances Kimura 2-parameter; bootstrap analysis with 1000 replicates

Table 1 .
Species assigned based on BOLD database, similarity value, and number of individual (N) Hubert et al. (2012)7) (2007)on Tridacna spp with the values range from 0.011 up to 0.028.Higher intra-specific genetic divergence was reported byHubert et al. (2012)on other fish group with the value reach up to 0.080.Therefore, the placement of fin clips samples into four different species was