BIODIVERSITAS ISSN: 1412-033X Volume 22, Number 4, April 2021 E-ISSN: 2085-4722 Pages: 1644-1651 DOI: 10.13057/biodiv/d220408 Genetic variation of longtail tuna Thunnus tonggol landed in four fish markets in Indonesia based on mitochondrial DNA IDA AYU ASTARINI1,2, ENEX YUNIARTI NINGSIH3,4, DEVY SIMANUNGKALIT4, SHELLA AYU ARDIANA4, M. DANIE AL MALIK3, NI LUH ASTRIA YUSMALINDA3, ANDRIANUS SEMBIRING3, NI PUTU DIAN PERTIWI3,5, NI KADEK DITA CAHYANI3, ALLEN COLLINS6 1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Udayana. Jl. Raya Kampus Unud, Jimbaran, Badung 80361, Bali, Indonesia. Tel./fax.: +62-361-701954, email: iaastarini@unud.ac.id 2Graduate Program in Environmental Sciences, Universitas Udayana. Jl. P.B. Sudirman, Denpasar 80114, Bali, Indonesia 3Biodiversitas Indonesia (BIONESIA). Jl. Tukad Balian No. 121, Denpasar 80226, Bali, Indonesia 4Department of Marine Sciences, Faculty of Marine and Fisheries, Udayana University. Jl. Raya Kampus Unud, Jimbaran, Badung 80361, Bali, Indonesia 5Biology and Marine Fisheries Department, Faculty of Mathematics and Natural Sciences, Universitas Pendidikan Ganesha. Jl. Udayana, Singaraja, Buleleng 81116, Bali, Indonesia 6National Systematics Laboratory, NOAA’s National Marine Fisheries Service, Smithsonian National Museum of Natural History. Washington DC. 20560, United States of America Manuscript received: 1 December 2020. Revision accepted: 7 April 2021. Abstract. Astarini IA, Ningsih EY, Simanungkalit D, Ardiana SA, Al Malik MD, Yusmalinda NLA, Sembiring A, Pertiwi NPD, Cahyani NKD, Collins A. 2021. Genetic variation of longtail tuna Thunnus tonggol landed in four fish markets in Indonesia based on mitochondrial DNA. Biodiversitas 22: 1644-1651. Longtail tuna (Thunnus tonggol, Family: Scombridae) is an economically valuable neritic species found in tropical and subtropical waters in the Indo-Pacific region. High catch numbers, which have been decreasing, could negatively impact this tuna’s population level. Little research has been conducted on the longtail tuna population in Indonesia. This study aims to determine the genetic diversity and potential population structure of longtail tuna landed in four fish markets in Indonesia (representing three sampling locations because two markets are relatively close to each other) based on sequences of a region of mitochondrial control region (d-loop). A total of 101 samples, out of 110, were identified and confirmed as T. tonggol species by amplifying and sequencing a fragment of d-loop (amplicons ranging from 482 - 523 bp). Neighbor-joining analysis resulted in a topology with all samples grouped into one clade with an average genetic distance of 0.020. Meanwhile, haplotype diversity (Hd) and nucleotide diversity (π) values of the longtail tuna samples were 0.9939 and 0.0192, respectively. The fixation index (Fst) value was - 0.00507, with p> 0.05, which indicates that there is no significant population structure among the longtail tuna collected from four fish markets representing three sampling locations. The results of this analysis can be used as basic data in planning sustainable fisheries management efforts. Keywords: Control region, genetic conservation, genetic distance, haplotype INTRODUCTION and Graves 2019). The fish possesses an upper body that is bluish-black, a long tail base, silvery-white belly with oval Tuna is one of the three largest fisheries commodities in spots arranged horizontally, a finlet that is yellow with gray Indonesia, after shrimp and demersal fish (Habibi et al. edges, 2 dorsal fins, and a yellow anal fin (White et al. 2011). Tuna makes up a significant part of the global 2013; Collette and Graves 2019). seafood market, worth more than $42 billion USD value The longtail tuna has a coastal distribution in the Indo- per year, and are vulnerable to overfishing (Tidd et al. Pacific region (Kumar and Kocour 2015) and is being 2018). Tuna fishing globally has reached 7.7 million exploited by commercial fisheries in several countries tons/year, with total Indonesian catch reaching 16% of total throughout the Indo-Pacific. Throughout the Indian Ocean global take (Alfajri 2017). Indonesia is the largest tuna region, the highest contributions to longtail tuna catches exporting country in Southeast Asia with tuna export were from Iran (34%) and Indonesia (31%), followed by volume of 209,410 tons and production value reaching Taiwan, Thailand, Oman, Pakistan, Malaysia, India, and 768.4 million USD in 2013 (Alfajri 2017). This relatively Australia (Abdussamad et al. 2012). high catch number is feared to cause a decrease in tuna Decreasing numbers of the longtail tuna catch raise populations, especially the longtail tuna (Thunnus tonggol). concerns that genetic variation of longtail tuna is being Longtail tuna (T. tonggol) is a neritic species found in decreased as a result of exploitation (Riccioni et al. 2010; tropical and subtropical waters (Restiangsih and Hidayat Siriraksophon 2017). Also, catch statistics may be 2018; Collette and Graves 2019). General characteristics of inaccurate due to misidentification (Mohri et al. 2013). longtail tuna are a maximum body length of 142 cm and Misidentification of catch estimates of certain tuna species maximum weight of 35.9 kg (Griffiths et al. 2010; Collette may be caused by the similarity of morphological ASTARINI et al. – Genetic variation of longtail tuna (Thunnus tonggol) 1645 characters of several tuna species, i.e., yellowfin tuna (T. This study aims to complement understanding of the albacares) and longtail tuna (T. tonggol) (Mohri et al. genetic diversity of T. tonggol by determining the genetic 2013). Genetic conservation strategies that incorporate diversity and potential population structure of landed molecular identification methods to determine species longtail tuna obtained from four fish markets in Indonesia, identity will more accurately reveal the status and health of including Kedonganan-Bali, Muncar-Banyuwangi, Pabean- fish populations. It is essential to understand the genetic Surabaya, and Sagulung-Batam. The d-loop mitochondria information of longtail tuna as a basis for guiding DNA marker is used to investigate the population structure sustainable fisheries resource management policies. of longtail tuna across the study areas. Understanding DNA barcoding techniques provide fast and accurate longtail tuna populations will help identify genetic stocks, means for identifying cryptic organisms (Rahayu and which would be an important source of information to help Nugroho 2015). One locus that is often used in species policymakers in managing sustainable fisheries of this delimitation and population analysis is the non-coding gene species in Indonesia, especially in these areas of study. known as d-loop or control region. This mtDNA locus is a region involved in the control of mtDNA replication and transcription processes and has relatively high mutation MATERIALS AND METHODS and polymorphism rates, thus rendering nucleotide sequences that vary greatly between individuals (Rahayu Sample collections and Nugroho 2015). D-loop contains the most varied DNA This research is based on collections conducted from sequences of all animal mtDNA genomes (Savira 2012). December 2018 to June 2019. Fin clips of longtail tuna Therefore, it can be informative when studying variation, were collected from four fish markets in Indonesia diversity, and genetic structure of animal populations including Bali (n = 41), Banyuwangi (n=20), Surabaya (n = (Astuti and Kurniati 2010). In the case of tunas, genetic 28) and Batam (21), for a total of 110 samples (Figure 1). analysis of d-loop has been widely employed, for example Samples were preserved in 96% ethanol before being in Thunnus spp. (Joka 2013), Thunnus albacares (Akbar et transported to the laboratory for DNA extraction. al. 2014), Thunnus tonggol (Willette et al. 2016), and Interviews with local fishermen were conducted to confirm Thunnus obesus (Pertiwi et al. 2017). the catch location of the longtail tuna samples, which were within 50 miles offshore of each port. Figure 1. Sampling locations at four fish markets in Indonesia 1646 BIODIVERSITAS 22 (4): 1644-1651, April 2021 Molecular analysis showed that all the longtail tuna samples from the four fish Molecular analysis was carried out at the Yayasan markets grouped into one clade. The resulting topology Biodiversitas Indonesia (Bionesia) Laboratory, Bali. DNA also revealed that the Genbank sequence of T. tonggol extracts were isolated using 10% chelex (Walsh et al. (KC313300.1) nested with the collected samples rather 1991). Extracted DNA was then used to amplify, using than the outgroup species (T. obesus, T. albacares, T. Polymerase Chain Reaction (PCR) methods, a fragment of orientalis, E. affinis), confirming that the samples collected the mitochondrial DNA (mtDNA) control region (d-loop) were longtail tuna (T. tonggol). Genetic distance within the locus. PCR methods were carried out following the T. tonggol specimens is 0.02, whereas the genetic distance methods in Allen et al. (2017) using the forward primer between T. tonggol and its outgroup is 0.199. (CRK: 5 '- AGC TCA GCG CCA GAG CGC CGG TCT For the population genetic analysis, samples from TGT AAA - 3') and reverse primer (CRE: 5– 'CCT GAA Banyuwangi and Bali were treated as one sampling GTA GGA ACC AGA TG - 3') (Lee et al. 1995). location (Bali* population) because of their close Each PCR reaction was 22 μl in volume, consisting of proximity. Genetic diversity analysis shows that among the reagent solution containing 12.5 μl ddH2O, 2.5 μl 10x PCR 101 samples collected, 82 different haplotypes were found, buffer (PE-II), 2.5 μl dNTP, 2.0 μl MgCl2 and 1.25 μl with a haplotype diversity value of 0.9939, and a primary CRK - CRE and 0.125 μl of PE Amplitaq, and 3 ul nucleotide diversity value of 0.0192 (Table 1). The of DNA template. Each microtube was vortexed for 30 haplotype distribution (Figure 3) showed nine (9) seconds to homogenize the samples. PCR reaction and haplotypes to be shared in different sampling locations; thermocycling profile were modified from the methods in eight (8) found to be shared by two sampling locations, and Allen et al. (2017), with an initial denaturation of 94°C for only one (1) haplotype shared among all the sampling 10 s, 38 cycles of 94°C for 15 s, 50°C for 30 s, 72°C for 45 locations. 73 non-singleton haplotypes were found, with s, with final extension of 72°C for 5 min. PCR products 34, 17, and 22 unique haplotypes acquired within the were visualized using 1% gel agarose stained by Biotium® sampling locations of Bali*, Batam, and Surabaya, gel red stain. Successfully amplified products were then respectively. sent to a DNA sequencing facility and sequenced using Big Population genetic analysis using Analysis of Dye Chain Termination. Molecular Variance (AMOVA) showed that the structures of the longtail tuna were not significantly different within Data analysis Bali* (Bali and Banyuwangi), Surabaya, and Batam (FST- Sequences were edited and aligned using the value = -0.00507; p-value >0.05) (Table 3). This result CLUSTAL W algorithm in MEGA X (Kumar et al. 2018). indicates a mixing population of longtail tuna from the four A distance-based topology was obtained using the fish markets. Neighbor-Joining method (Saitou and Nei 1987) with 1000 bootstrap replications. Sequences of Thunnus obesus Discussion (JN572738.1), Thunnus albacares (JN572794.1), Thunnus Longtail tuna (Thunnus tonggol) is a neritic species orientalis (JN631250.1), and Euthynnus affinis found in Indo-Pacific shallow, coastal, tropical and (JN655119.1) from Genbank (http://ncbi.nlm.nih.gov) were subtropical waters (Restiangsih and Hidayat 2018). This used as an outgroup. In addition to the outgroup, one tuna species has a wide distribution across Indonesia, sequence of T. tonggol (KC313300.1) was added to the including Aceh (Rahmah et al. 2019), Java Sea (Widodo et analysis to confirm the samples as T. tonggol. For al. 2011; Fitriani et al. 2020; Hidayat et al. 2020), Bali population genetic analysis, Banyuwangi and Bali samples (Mahmud et al. 2019), Lombok (Setyadji and Nugraha were treated as one population site, with Surabaya and 2015), East Kalimantan (Alfian et al. 2020), Sulu and Batam as different population sites. Genetic diversity Sulawesi Sea (Wanchana et al. 2015). Indonesia is listed as analysis to measure haplotype diversity (Hd) (Nei 1987) one of countries with the highest contributions to longtail and nucleotide diversity (π) was done in DNAsp 5.10 tuna catches (31%) in Asia (Kumar and Kocour 2015; (Rozas et al. 2017). The Analysis of Molecular of Variance Abdussamad et al. 2012). However, this species’ similar (AMOVA) in Arlequin Ver.3.5 (Excoffier and Lischer morphological characters with those of other tuna 2010) was used to assess population genetic structure. compromise accurate identification of the fish, leading to mislabeling and uncertainty in published catch estimates (Pauly and Froese 2012). Thus, molecular identification RESULTS AND DISCUSSION methods can help improve the determination of species identity and the accurate assessment of the status of Result populations. This study aims to determine the genetic From the total of 110 samples from four fish markets, diversity and potential population structure among longtail nine samples were not identified as T. tonggol. Among tuna obtained from four fish markets in Indonesia, those nine samples, four were identified as T. albacares including Kedonganan-Bali, Muncar-Banyuwangi, Pabean- and five as Euthynnus affinis. This left 101 samples Surabaya, and Sagulung-Batam, using the d-loop confirmed as T. tonggol, represented by amplicons 411 mitochondrial DNA marker. base pair (bp) long (Figure 2). All sequences have been Unlike all other Thunnus species, oceanodromous, deposited in Genbank with accession numbers longtail tuna occupy shallow waters and do not undergo (MW658015-MW658124). The neighbor-joining topology diel vertical migration, limiting their niche and migration ASTARINI et al. – Genetic variation of longtail tuna (Thunnus tonggol) 1647 (Griffiths 2020). Longtail tuna has been reported to occupy Table 1. Genetic diversity of longtail tuna (Thunnus tonggol) the coastal areas close to landmasses and is rarely found populations from all study locations beyond continental shelf waters (Yesaki 1994). This information is in-line with our finding where the fishermen Sample locations N Hd π Hn from these four markets also reported catching the longtail Bali and Banyuwangi 52 0.9902 0.0195 43 tuna within 50 miles offshore of each port and accord with Batam 21 1.0000 0.0181 21 our understanding based on surveying other fish markets Surabaya 28 1.0000 0.0197 28 around Indonesia. Longtail tuna is more commonly sold at All 101 0.9939 0.0192 82 fish markets adjacent to ocean beds with extensive shallow Note: N: Number of samples, Hd: Haplotype diversity, π: topography. Nucleotide diversity, Hn: Number of Haplotype Figure 2. Neighbor-Joining (NJ) topology generated from 411 bp of mtDNA control region sequence data from Thunnus tonggol species, with node support assessed using 1000 bootstrap replications 1648 BIODIVERSITAS 22 (4): 1644-1651, April 2021 Figure 3. The distribution of shared haplotypes of longtail tuna (Thunnus tonggol) between study locations Neighbor-joining analysis showed that 101 samples (2016) and Malaysian waters by Kasim et al. (2020). were confirmed as longtail tuna (T. tonggol). The Genbank Wijana and Mahardika (2010) reported a similar result for T. tonggol were nested onto a similar clade with the yellowfin tuna, which exhibited no genetic differences in samples collected (Figure 2). Comparison results with samples from the Philippines and Spain. In our analysis, Genbank data using BLAST (Basic Local Alignment longtail tuna samples nested tightly in one clade, which Search Tool) show the similarity of the samples collected could be a result of the relatively narrow geographic scope with T. tonggol data is 97% - 100%. Thus, this study found in this study. The four sampling locations are connected that 101 of 110 samples collected from four fish markets and are along the migration path of longtail tuna. were accurately identified as Thunnus tonggol, an error rate of about 8%. Genetic distance analysis showed a close Table 2. Distribution of nine shared haplotypes of longtail tuna relationship between samples found in Bali, Banyuwangi, (Thunnus tonggol) between study locations Surabaya, and Batam. Differences in nucleotide sequences between samples could occur due to environmental Sample locations Total sample Haplotype 1 2 3 locations conditions that affect an individual’s genetic material 1 1 1 2 (Verawati 2015). Population genetic analysis indicates a 2 1 1 2 mixing population of longtail tuna from the four fish 3 1 1 2 markets. 4 1 1 1 3 Mitochondrial DNA has commonly been used for tuna 5 1 1 2 population studies, e.g., in yellowfins (Akbar et al. 2014), 6 1 1 2 bigeye (Nugraha 2009; Pertiwi et al. 2017), and longtail 7 1 1 2 (Willette et al. 2016; Kasim et al. 2020). Studies of longtail 8 1 1 2 tuna suggest the existence of panmictic populations among 9 1 1 2 those studied, i.e., in the South China Sea by Willette et al. Table 3. Analysis of Molecular Variance (AMOVA) of longtail tuna across study locations Source of variation d.f Sum of squares Variance component Percentage of variation Among sample location 2 6.680 -0.01995 Va -0.51 Within sample location 98 388.033 3.95952 Vb 100.51 Total 100 394.713 3.93957 FST -0.00507 P-value 0.71652 ± 0.01300 ASTARINI et al. – Genetic variation of longtail tuna (Thunnus tonggol) 1649 For future studies, expanding sampling locations and would be widespread adaptive response by longtail tuna to employing more sensitive genetic markers might better environmental conditions, such as temperature, turbidity, or resolve this species' population patterns. Although little to chlorophyll-a presence. Many factors, including no genetic structure has been revealed within tuna temperature pattern and chlorophyll-a, are influenced by populations of the areas mentioned above, differentiation in upwelling. Upwelling can affect the abundance and mtDNA has been detected between wider regions, such as distribution of phytoplankton in water (Barata et al. 2014), between longtail tuna populations of the Indian Ocean and which determines local productivity. Phytoplankton, in the South China Sea (Willette et al. 2016). Next-Generation turn, will attract small fish, which is the food source for Sequencing (NGS) methods to investigate Single tuna (Padmaningrat 2017). Nucleotide Polymorphisms (SNPs) have also uncovered High genetic diversity in longtail tuna populations genetic differentiation among Atlantic, Indian and Pacific indicates that nucleotide changes are still occurring in their Ocean populations (Pecoraro et al. 2018). This method also mtDNA control region locus and, that all else being equal, found genetic differentiation within the Atlantic Ocean increases the survival chance of the population. High (Eastern Atlantic Ocean and Western Atlantic Ocean) and genetic diversity should be maintained to preserve its the Pacific Ocean (Eastern Pacific Ocean and Western- sustainability in the face of having high commercial value. Central Pacific Ocean) populations of yellowfin tuna But note that Hughes et al. (2008) have argued that genetic (Pecoraro et al. 2018). diversity may or may not directly impact individual Our sampling of longtail tuna in Indonesia revealed a species, populations, communities, and ecosystems. high number of haplotypes, differing in each location Overfishing a tuna species may decrease the genetic (Table 1); among 52 samples collected from Bali and diversity of those species, affecting its population structure. Banyuwangi, 43 haplotypes were found; among 28 Therefore, a strategy to preserve biodiversity through Surabaya samples there were 28 haplotypes; and among the genetic conservation is needed. Several efforts that can be 21 Batam samples, 21 haplotypes were detected. Only a undertaken to maintain sustainable tuna fisheries are (1) handful of haplotypes were sampled between regions, with controlling the minimum size limit on every catch, and (2) the haplotype diversity (Hd) value of 0.9939. Meanwhile, limiting the catch time, where fishing tuna is only allowed the nucleotide diversity value of 0.0192 shows that almost at the peak of the tuna season. every sample has a different haplotype with a small A recent review (Griffiths et al. 2020) posited four difference in nucleotide sequences between samples. The putative stocks across the entire range of longtail tuna: high genetic diversity uncovered in longtail tuna obtained Western Indian, Northern Indian, Oceania and Southeast from four Indonesian fish markets is similar to what was Asia. The hypothesis of a single southeastern Asia stock is reported by Willette et al. (2016) in the South China Sea. based on prior studies by Willette et al. (2016) and Malik et Other research on pelagic tuna fish such as yellowfin tuna al. (2020) that detected no genetic structure in longtail tuna (Akbar et al. 2014) and bigeye tuna (Nugraha 2009; Pertiwi populations in the South China Sea and the Java Sea. In our et al. 2017) around Indonesia and the Indian Ocean have Indonesia-focused study, we also derived a fixation index also shown high genetic diversities. (Fst) from AMOVA analysis indicating that there is no The high genetic diversity exhibited by longtail tuna population genetic structure in the longtail tuna population may be due to several factors, i.e., large population size, between the three populations we sampled. Thus, our work migration, and high adaptability of the species. Large corroborates the idea that longtail tuna populations within population size allows an individual to freely breed the South China Sea and Indonesia constitute a single stock (interbreeding) with other individuals from that population. of population. Similarly, Kunal et al. (2014) found that the This can help increase the frequency of alleles (gene longtail tuna population inhabiting northwest Indian waters couples located at the corresponding locus on the constitutes a single stock population. Further genetic homologous chromosomes that make up gene arrangement) studies of longtail tuna from Oceania and the Western of an offspring. A large-sized population can prevent a Indian Ocean are needed to assess their respective population from declining. Detecting high genetic diversity population structures more fully. of longtail tuna suggests that localized catches do not Although longtail tuna are known to be neritic and not selectively remove sub-populations or genetic diversity of to undergo high levels of migration, this species has a wide the overall species. Similar results were reported by distribution with little known population differentiation Nugraha (2009), who reported that 190 samples of bigeye other than that between populations in the South China Sea tuna from two populations of the Indian Ocean and the and Indian waters (Kunal et al. 2014). Islands and the Pacific Ocean were broken into 5 broadly distributed sub- general complexity of Indonesia's marine ecosystem may groups. Grewe and Hamptom (1998), who conducted a help explain differentiation flow through current between study of 800 samples of the bigeye tuna using mtDNA and Southeast Asian waters and northern Australia (Lee et al. microsatellite DNA analysis, also found sub-populations 2002), which were made separate stock populations within the bigeye tuna populations in the Pacific Ocean. between those locations (Griffiths et al. 2020). Moreover, A second factor that could cause high genetic diversity morphometric, meristic, and electrophoretic observations in longtail tuna is migration, which influences the flow of of longtail tuna (Abdulhaleem 1989) have also indicated genes in a population. This is supported by Nishida et al. that there are two distinct populations between Indian and (1998), who discovered deep oceanic migration of several Pacific. As a result, the two locations should be managed as tuna species by using tagging methods. A third factor different population stocks (Kumar and Kocour 2015). 1650 BIODIVERSITAS 22 (4): 1644-1651, April 2021 Like others focusing on the South China Sea (Willette Mitochondrial DNA and DNA Microsatellite Analysis. Marine et al. 2016) and Java Sea (Malik et al. 2020), this study Research. Commonwealth Scientific and Industrial Research Organisation, Australia. covers only a narrow, small-scale region of the overall Griffiths SP, Fry GC, Manson FJ, Lou DC. 2010. Age and growth of geographic distribution of Thunnus tonggol. However, this longtail Tuna (Thunnus tonggol) in tropical and temperate waters of data could help the government evaluate the policy for the central Indo-Pacific. ICES J Mar Sci 67 (1): 125-134. DOI: sustainable fisheries of this species. In sum, the genetic 10.1093/icesjms/fsp223. 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