In Aceh, Indonesia

Fisheries Research 234 (2021) 105796 Contents lists available at ScienceDirect Fisheries Research journal homepage: www.elsevier.com/locate/®shres DNA barcoding of commercially important groupers (Epinephelidae) in Aceh, Indonesia Nur Fadli a,*, Zainal A. Muchlisin a, Mohd N. Siti-Azizah b,c a Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh, Indonesia b School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia c Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Terengganu, Malaysia ARTICLE INFO ABSTRACT Handled by J. Vinas~ Here, we report for the first time a comprehensive survey of the DNA barcoding of commercially important groupers in Aceh waters, Indonesia using the cytochrome oxidase subunit I (COI) gene. The fish samples were Keywords: collected from major fish landing sites and fish markets in nine districts in Aceh, that covered the western and Kerapu eastern coastal ecosystems of Aceh. In total, the 144 COI sequences revealed 26 grouper species with most COI represented by multiple specimens. As expected, genetic distances increased from lower to higher taxonomic Banda Aceh levels: 0 %–1.27 % within species, 3.79 %–19.96 % within genus and 13.84 %–24.15 % within family. Out of the Molecular taxonomy Diversity total species, 23 (88.5 %), were categorised as Least Concern (LC) and three species (11.5 %) were Data Deficient Fisheries (DD) under the IUCN Red List. Furthermore, four species were categorized under ‘decreasing population trend’ (Epinephelus bleekeri, E. coioides, Plectropomus leopardus, and Variola albimarginata). This study is a significant contribution to the DNA barcode library of groupers in this region and expansion of the Indonesian and the global barcode entries in general. 1. Introduction standards. As a result, similar to other countries, the groupers are heavily exploited to supply the local and regional markets. The trend of The groupers is one of the most commercially valuable fish group grouper catches in Aceh reveal a yearly rise. The Department of Marine globally including in many coastal Asian regions such as in Aceh, and Fishery of Aceh reported that the annual grouper catch rose to 4, Indonesia. The biodiverse Indonesian waters support 100 species and 603.4 tons for the year 2014 from 4,056.2 tons in the year 2011. new reports of species discoveries are not uncommon (Tucker et al., However, no species-specific records are available. Groupers are the 2016). In India, out of 70 species, 36 species are commercially important second highest fisheries commodity (32.51 %) exported by Aceh espe (Basheer et al., 2016; Froese and Pauly, 2019). In the Philippines, out of cially to Thailand, Malaysia and Hongkong (http://suhana.web.id/, 112 species, 27 species are highly priced (Alcantara and Yambot, 2014; accessed 18 June 2018). Froese and Pauly, 2019). They are popular due to their delicate, desir An accurate species list is one of the essential information for stra able taste and flavor (Alcantara and Yambot, 2014; Chiu et al., 2008; tegizing an optimal fisheries management plan (Bakar et al., 2018; Sadovy de Mitcheson et al., 2013). There are no reliable records for Basheer et al., 2016; Pavan-Kumar et al., 2016; Ward, 2000). Unfortu grouper diversity or how many of these are commercially important in nately, in most Aceh fish landing sites, members of this group are Aceh. As they are commercially valuable, the groupers are regarded as collectively recorded as "kerapu" instead of the precise scientificspecies one of the firstfish groups to be overexploited in the fisheriesindustry in names. An accurate inventory of this high-valued fishgroup has not been most countries (Sadovy de Mitcheson, 1994). Sadovy de Mitcheson et al. previously carried out in Aceh. Groupers are among the more chal (2013) reported that out of 163 grouper species documented across the lenging fishesto identify. Many groupers have the same morphological globe, it is estimated that 12 % of grouper species (20 species) are at a and phenotypic characteristics (Alcantara and Yambot, 2014; Craig risk of extinction and another 13 % (22 species) are considered to be et al., 2001, 2011). Furthermore, colour and morphometric characters nearly threatened based on the IUCN Red List criteria. In Aceh, 1 kg may vary during ontogenic development and/or environmental condi grouper could fetch 7–10 USD, a highly lucrative value by local tions. Moreover, as the colours of dead and live animals can differ * Corresponding author at: Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh, 23111, Indonesia. E-mail address: [email protected] (N. Fadli). https://doi.org/10.1016/j.fishres.2020.105796 Received 16 April 2020; Received in revised form 8 September 2020; Accepted 21 October 2020 Available online 13 November 2020 0165-7836/© 2020 Elsevier B.V. All rights reserved. N. Fadli et al. Fisheries Research 234 (2021) 105796 considerably, taxonomic identification is even more complicated 2.2. DNA extraction, primer, and PCR assay (Alcantara and Yambot, 2014; Craig et al., 2011; Zhuang et al., 2009). Thus, molecular tools such as DNA barcoding are becoming a necessary The modified CTAB protocol was used to isolate genomic DNA complementary approach to address the ambiguities. (Grewe et al., 1993). Subsequently, the final concentration of the DNA barcoding grouper research based on the cyrochrome oxidase extracted DNA was quantified using the NanoDrop 2000c Spectropho subunit I (COI) gene have been well documented (Alcantara and Yam tometer (Thermo Scientific, Waltham, MA). PCR amplification of the bot, 2014; Aziz et al., 2016; Basheer et al., 2016; Jefri et al., 2015) in COI locus was performed based on Ward et al. (2005). PCR was set up in several regions but not in the Aceh waters. This pioneering study is a 25 μL mixture reaction containing 2.0 μL DNA template, 0.5 μL of each aimed to generate the first comprehensive reference COI sequence li primer, 2.5 μL of 10x i-Taq™ plus PCR Buffer, 2.0 μL of 25 mM MgCl2, brary for grouper species that are landed in Aceh, providing a baseline 1.0 μL of dNTP, 0.25 μL of i-Taq™ plus DNA Polymerase and 16.25 μL of data that will be valuable not only for Aceh fisheriesmanagement plan Milli-Q water and was amplified in a BIO-RAD T100 Thermal Cycler but also for Indonesia. (BioRad Laboratories Inc., USA). The thermal conditions consisted of ◦ initial denaturation at 95 C for 2 min followed by 30 cycles of dena ◦ ◦ 2. Materials and methods turation at 94 C for 45 s; annealing at 47.9–60 C for 45 s; elongation at ◦ ◦ 72 C for 1 min and a final extension of 72 C for 10 min before ◦ 2.1. Sample collection termination of the reaction at 4 C. Successful PCR products as visual ized by clear bands in a 1.7 % agarose gel stained with 2–2.5 μL of Sampling was conducted from May 2015 to December 2016 from RedSafe™ Nucleic Acid Staining Solution (IntRON Biotechnology, major fish landing sites and fish markets in nine districts in Aceh, Gyeonggi-do, Korea) were subsequently sent to the First BASE Labora namely: Weh Island, Banda Aceh, Krueng Raya, Sigli, Lhokseumawe, Idi, tories, Malaysia, for bidirectional sequencing using BigDye Terminator Calang, Meulaboh and Tapak Tuan (Fig. 1, Table 1). The fish samples v3.1 Cycle Sequencing Kit and ABI PRISM 3730xl DNA Analyzer were morphologically identified to species level (Craig et al., 2011; (Applied Biosystems, Foster City, CA). Froese and Pauly, 2019; Heemstra and Randall, 1993). Fin clipping of representative samples of each species were obtained and fixedin 96 % 2.3. Data analysis ethanol placed in 2 mL microcentrifuge tube. Sample sizes ranged from one to 40 individuals with an average of six specimens per species All COI sequences were trimmed and aligned using MEGA 6.06 (twenty-two species were represented by more than one specimen and software (Tamura et al., 2013). The aligned sequences were translated four species represented by a single specimen). Tissue and whole body into protein to ensure accurate alignment and detection of stop codons, vouchering and documentation followed the Fish-BOL collaborator’s if present. This software was also used to investigate base composition protocol (Steinke and Hanner, 2011) and are stored at the Faculty of and to compute the number of variable sites. Pairwise genetic distances Marine and Fisheries Syiah Kuala University at Banda Aceh, Indonesia. (conspecific, congeneric and confamilial) based on the All sequences have been deposited in BOLD systems under the project Kimura-2-parameter (K2P) model were estimated using MEGA 6.06 “DNA barcoding of commercially important groupers in Aceh” (http:// (Kimura, 1980; Tamura et al., 2013) and Sequence Analysis Engine of www.boldsystems.org). Other supplementary data of the collected fish BOLD (www.boldsystems.org). Haplotype distributions were summar specimens, including their BOLD Accession numbers, are presented in ised in DnaSP 5.10 (Librado and Rozas, 2009; Rozas et al., 2003). Table 1. Initial species identifications were conducted by comparing COI Fig. 1. Sampling sites of groupers along the coast of Aceh. 2 N. Fadli et al. Fisheries Research 234 (2021) 105796 Table 1 List of the grouper species from Aceh, Indonesia, and their newly generated BOLD accession numbers. Sample Locality Total No Genus Species Composition BOLD Accession No. WI BA KR CL MB TT SG LM ID Sample 1 Cephalopholis Cephalopholis argus 1 1 31 % FADLI022-17 2 Cephalopholis aurantia 5 5 FADLI023-17 - FADLI027-17 3 Cephalopholis boenak

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