Philippine Journal of Science 139 (1): 43-50, June 2010 ISSN 0031 - 7683

A Pilot Study on the Genetic Variation of Eastern Little Tuna (Euthynnus affinis) in Southeast Asia

Mudjekeewis D. Santos1,2,*, Grace V. Lopez1, and Noel C. Barut1

1 Marine Fisheries Research and Development Division, National Fisheries Research and Development Institute, 940 Quezon Ave., Quezon City 1103 2 Department of Biology, School of Science and Engineering, Ateneo de Manila University, Katipunan Ave., Quezon City 1108 Philippines

Eastern little tuna or kawakawa (Euthynnus affinis) is one of the commercially important tunas in the Philippines and in the Southeast Asian region. Therefore, its sustainability needs to be ensured by effective management. Unfortunately, knowledge on the stock structure of this migratory species in the region, needed for management, is unclear. Here, we studied its genetic population structure using a 300 bp mitochondrial DNA control region (D-loop) marker. Thirty- five samples collected in five areas around the Philippines (Aparri, Palawan, Cagayan de Oro, Tawi-Tawi, and Davao) and thirteen from Peninsular Malaysia (Pangkor Island, Penang) were sequenced and analyzed. Forty five haplotype sequences were homologous (99%) to each other while three (all from Malaysia) were divergent (80%) sequences indicating misidentification.

Haplotype distribution, low overall FST value and non-concordant phylogeographic grouping of the haplotypes indicated that eastern little tuna is panmixing in Southeast Asia.

Key Words: control region, Philippines, population genetics, stock identification

INTRODUCTION already shown signs of overexploitaton and decline (Barut et al. 2003; Barut et al. 1997). Eastern little tuna or kawakawa, Euthynnus affinis (Cantor 1849), a small epipelagic, migratory, neritic tuna is one Because of its commercial importance and migratory of the major commercial tuna species being caught nature, eastern little tuna should be properly studied worldwide. Its production has steadily increased from and managed to ensure its sustainability. To do this, the 20,000 tons in 1950 to about 280,000 tons in 2006 (FAO structure of its stock in the Philippines and in Southeast 2009). In the Philippines, eastern little tuna remains an Asia needs to be elucidated. It is extremely important important commercial fish species being landed in the to know whether 2 or more local communities or, on country in terms of volume (BAS 2006). Majority of a regional scale, countries, share one stock of eastern eastern little tuna caught by the commercial fisheries of little tuna do that collaborative type of management the country is exported in processed/ canned form because strategy has to be initiated. A stock consists of randomly there is no existing fresh or dried export market. On the interbreeding members whose genetic integrity persists other hand, catches from municipal fisheries goes directly whether they remain spatially and temporally isolated as to the domestic market for local consumption. The eastern a group or whether they alternately segregate breeding little tuna population is relatively healthy in spite of the and otherwise mix freely with members of other unit fact that many of the country’s fishery resources have stocks of the same species. Population or stock structuring could therefore refer to whether or not the total stock or *Corresponding author: [email protected] population exhibit subpopulations or sub-set breeding

43 Philippine Journal of Science Santos et al.: Genetic Variation of Eastern Vol. 139 No. 1, June 2010 Little Tuna, Euthynnus affinis groups where migration and gene flow between the groups de Oro- CO) (Figrue 1). Identity of sampled fish were is restricted to a significant degree. confirmed by the distinct morphologica; characteristic of eastern little tuna i.e. having finlets, keel at the caudal The stock structure of eastern little tuna in the Philippines peduncle, presence of broken oblique stripes in the dorsal and in Southeast Asia is conjecture at best and unclear part of the body and dark spots below the pectoral fin (Devaraj and Vivekanandan 1997). Differential spawning (Collete and Nauen 1983). Fish samples were bought patterns of the species at different geographical location whole and fresh from landing centers. Approximately 300 suggests the presence of distinct subpopulations across g of tissues was dissected out from the left posterior side its geographical range. Two recruitment pulses i.e. during of each individual fish. These were then frozen overnight the first and the second half of the year was noted for the and/or stored in ethanol before processing. On the other Northern Hemisphere e.g. Thailand and Philippines and hand, tissue samples in absolute ethanol from Penang, a single spawning activity towards the middle of the year Malaysia were kindly provided by Dr. Abhu Thalib of the was observed in higher altitudes of the tropical zone e.g. Fisheries Research Institute of Malaysia. Hongkong (Yesaki 1994). Other reports observed varying seasonal spawning peaks according to regions: i.e. March to May in Philippine waters; October-November to April- Mitochondrial DNA extraction and sequencing May (during the NW Monsoon) around the Seychelles; The ethanol preserved tissues were rinsed with de-ionized

January to July (from the middle of the NW monsoon H2O. After which approximately 150 mg were sliced off period to the beginning of the SE monsoon) off East with an uncontaminated disposable razor. The tissues were Africa; and probably from August to October off Indonesia then be minced and placed in a properly marked 1.5 mL (FAO date here). Eppendorf tubes containing Cetyl Trimethyl Ammonium Bromide (CTAB) Extraction buffer (600 uL 2% CTAB Stock structures in tunas have been determined previously pH 8.5, 30 uL of 1% Proteinase K) incubated overnight using different methods including biochemical studies, in a water bath at 55oC with occasional shaking. After tagging experiments, morphometrics, meristics, length incubation, 600 uL of chloroform: isoamyl (3:1) solution frequencies and genetic markers (allozyme, RFLP, RAPD, was added to each of the sample, shaken by hand for about mitochondrial DNA and microsatellites) (Fujino 1976; 3 min. and then centrifuged for 5 min. at 8,000 rpm. The Fujino et al. 1981; Ward 1995; Waples 1990). Among upper aqueous supernatant was then transferred in new, the genetic markers, mitochondrial DNA (MtDNA) has marked 1.5 mL tubes, avoiding inclusion of the organic been employed a number of times because it can be easily phase. Described steps of clean-up will be done twice. extracted, purified and sequenced (if required), it has DNA precipitation was then be carried out by mixing a relatively fast rate of evolution i.e. base substitution 50 uL of 3M Sodium Acetate (NaOAc) and 900 uL 95 compared to genomic DNA (5-10X faster) and it is % Ethanol to the tubes containing the supernate. These maternally inherited. It is therefore not surprising that were then hand shaken for 3 min. and placed overnight the MtDNA control region has been successfully used in in a –20 oC freezer. After precipitation, the tubes are then previous studies on tunas and tuna-like species (Chow et spun in a microcentrifuge at 13,000 rpm for 30 min and al. 1997; Ward et al. 1997; Ward 1995). then the aqueous phase was carefully pipetted out leaving In this study, we present a first report on the genetic the DNA pellet at the bottom of the tube. The pellet was variation of eastern little tuna population in the Philippines then rinsed by adding 500 uL 70% ethanol and spun for and Southeast Asia using mitochondrial DNA control 13,000 rpm for 3 min. before removal of ethanol. Rinsing region (D-loop) analysis. was done twice. Drying was carried out by using a vacuum dryer (Speed-Vac) for 10 minutes. DNA pellets were then rehydrated in 100 uL of 1X TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0). Resulting stock DNA extracts o MATERIALS AND METHODS were stored in cryo vials at -80 C. A 300 bp mitochondrial DNA fragment covering a Sample collection and preservation portion of the variable 5’ end of the control region Eastern little tuna samples were sampled from 1997-1999 (D-loop) was amplified through PCR machine (Perkin- in landing areas or markets around the Philippines that were Elmer Cetus 480). Forward primer CB3R420 (5’ - adjacent to the following / channels- South China CCCCCTAACTCCCAAAgCTAgg – 3’) and reverse (Macarascas, Palawan - M); (Aparri, primer 12Sar430 (5’ – gCCTgCggggCTTTCTAgggCC Cagayan- AC); (Tawi-Tawi, Sulu- TS); Celebes – 3’) designed by the author and manufactured by Sea- (Davao- DG); (Cagayan GENSET, USA were used in the amplification. A 25 uL

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reaction mixture was prepared containing the following RESULTS reagents; 11.4 uL of milliQ H20, 2.5 uL Kocher’s Buffer, 5 uL dNTP’s, 2.5 uL each of 10 uM primers, 0.4 uL taq Designed PCR primers CB3R420 and 12Sar430 amplified and 1 uL of DNA template. Two (2) drops of mineral oil a 300 bp fragment sequence (haplotype) located at the will be added to cover the reaction mixture. After the start of the 5’ variable area of the control region. Eastern reaction, amplicons were electrophoresed through a 1 % little tuna samples, 35 from Philippines and 14 from agarose gel in TBE buffer (90mM Tris-Boric acid, 2mM Malaysia, of varying sizes were subjected to PCR and EDTA). The gel was stained with Ethidium Bromide and sequencing. Clean, readable haplotype sequences were bands were visualized by ultraviolet (UV) illumination. A produced from all the samples except for 1 Malaysian Polaroid camera was utilized in documenting the resultant sample, which failed to amplify. Of the 48 haplotypes, bands immediately after electrophoresis. Size of DNA 45 showed intraspecific identities of 99% while 3 from fragments were estimated by comparing with 100 bp DNA Malaysia (Hap AA, Hap AB and Hap AC) were observed ladder (GIBCO BRL). to be only about 80% compared to the main group of 45 samples (Figure 1). Both sense and antisense part of the 300 bp fragment were sequenced. Cycle sequencing was done by The diversity of haplotypes among the 45 samples was preparing a 12 uL cycle sequence reaction using, 2 uL high, ranging from 0.67 +/- 0.16 to 1.00 +/- 0.08 with of dRhodamine dye, 1 uL of 10uM of the primer, 4 uL of nucleotide diversity from 1.24% to 5.17% (Table 1). Interestingly, 16 (35.6%) exhibited haplotype A (Hap clean DNA and 5 uL of mQ H2O. These were then ran in a PCR machine (Perkin-Elmer GeneAmp PCR System A), 5 (11.1%) showed haplotype B (Hap B) and the rest 9600) at the following profile: 25 cycles, 96oC 0:10, 55oC were unique haplotypes. The common Hap A was found 0:10, 60oC 4:00 and 4oC. Ethanol precipitation was used to be present or shared in all the sampling sites except to clean the cycle sequence reactions. Entire contents of for Davao Gulf, while Hap B could only be found in the extension product was pipetted out and placed into a 4 sites in the Philippines – Aparri, Macarascas, Opol tube containing 1.2 uL of sodium acetate (NaOAc) pH and Tawi-Tawi) (Table 1 and Fig. 1). Hap B was not 5.2 and 26 uL of 95% ethanol (ETOH). The tubes were seen in Davao Gulf, similar to Hap A and in Penang, Malaysia. Global F of the sequences was 0 with a then vortexed and were place on ice for 15 minutes. ST Afterwhich, these were spun for 30 min. at 13,000 rpm mean P-value of 0.166. on an eppendorf benchtop microcentrifuge 5415C or The Minimum Spanning Network (MSN) showed 5417C. Supernatant was then carefully aspirated and no structuring and a dispersed location of individual discarded. Resulting pellet was then rinsed with 250 haplotypes across the network, failing to show any uL of 70% ethanol, vortexed briefly and then spun grouping or clade among different geographic locations again for 2 minutes at 13,000 rpm. The supernatant was (Figure 3). The MSN, however, did show a star-like aspirated and discarded again and the pellet was dried appearance. in a vacuum centrifuge (Speed-vac Savant) for 10 to 15 min. Amplicons were sequenced using ABI PRISM Model 377 ver. 3.3 Automated Sequencer. DISCUSSION Data Analysis Eastern little tuna is widely distributed in the tropical and Raw sense and antisense sequence data were assembled, subtropical waters of the Indo-Pacific region extending analyzed and aligned using Sequencher Ver. 3.0 from about 34oN to 40oS and from Cape St Francis, South (GeneCodes) and GENETYX Ver. 8.0. (Japan). Haplotypic Africa in the West Indian Ocean to the Marquesas Islands in frequency, genetic diversity, and global fixation index the Pacific Ocean. It is found at temperatures ranging from o o (FST) of the 45 confirmed samples were computed using 18 to 28 C and is strictly a coastal fish living its entire life Arlequin ver. 3.11 package (Excoffier et al. 2005). cycle in the neritic regime. Moreover, this species showed Percent identities between haplotypes were computed preference to areas with narrow continental shelves like using Megablast (for highly similar sequences) under the Sri Lanka and the Philippines and is suspected to spawn BLAST 2 sequence (bl2seq). A 99% homology was set to inshore (Yesaki 1994). confirm species identity. Stock structuring was analyzed using the Analysis of Molecular Variance (AMOVA) In this pilot study, we investigated the genetic stock structure (Excoffier et al. 1992), plotted in Minimum Spanning of eastern little tuna, a small, migratory, commercially Network (MSN) in Arlequin, and the tree drawn using the important neritic tuna species in Southeast Asia in support Phylogenetic Analysis Using Parsimony or PAUP ver. 4. of its management. Three haplotypes from Malaysia were

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Figure 1. Map of Southeast Asia showing the sampling sites in the Philippines and Malaysia: Aparri, Cagayan (AC), Macarascas, Palawan (M), Opol, Cagayan (OC), Tawi-Tawi, Sulu (TS), Davao Gulf (DG) and Pangkor Island, Penang, Malaysia (PM). Distribution and frequency of eastern little tuna mitochondrial DNA haplotypes across sampling sites as well as the existing Pacific Ocean currents in the Eastern side of the Philippines; Pacific North Equatorial Current, Kuroshio Current and the Mindanao Current are likewise shown.

Table 1. Eastern little tuna mitochondrial DNA control region haplotypes and its genetic and nucleotide diversity of in each of the 6 sampling sites in Southeast Asia. The 3 divergent haplotypes from Malaysia are not included. Average No. of Average Fork Hap Hap Expected Nucleotide Sampling Site Sampling Date Weight Unique Hap Samples Length (cm) A B Heterozygosity Diversity (%) (grams) Aparri, Cagayan 7 06/20/1998 26 730 4 2 1 (C) 0.67 +/- 0.16 1.24 (AC) Macarascas, 6 10/23/1997 26 290 3 1 2 (D,E) 0.80 +/- 0.18 2.13 Palawan (M) Opol, Cagayan 8 04/02/1997 36 723 3 1 4 (F,G,H,I) 0.89 +/- 0.11 2.61 (OC) Tawi-Tawi, Sulu 7 05/18/1997 27 342 3 1 3 (J,K,L) 0.86 +/- 0.14 2.14 (TS) Davao Gulf (DG) 7 06/30/1997 28 386 - - 7 (M,N,O,P,Q,R,S ) 1.00 +/- 0.08 4.19 Pangkor Is., Penang, Malaysia 10 01/14/1999 32 573 3 - 7(T,U,V,W,X,Y,Z) 0.93 +/- 0.08 5.17 (PM) Hap: haplotypes, (-): absent

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Figure 2. Nucleotide sequences of the identified D-loop region of the mitochondrial DNA haplotypes of eastern little tuna. Number per haplotype are shown in parenthesis. The 45 haplotype sequences with 99% homology among them were deposited in the DNA Data Bank of Japan (DDBJ) with accession numbers from AB519197 to AB519294.

found to be of different species. In the absence of detailed studies. Interestingly, based on genetic and nucleotide information on the collection process for the contributed diversity and geographic location, the haplotypes could be tissue, we speculate that some of the source of the samples divided into four regions: the mid-level diversity Balintang was from juvenile tunas, which are difficult to differentiate Channel, the high-level diversity central Philippines, the because their morphological characteristics are very similar. very high-level diversity Southeastern Philippines and the Such mis-identification is not uncommon for tuna species. very high-level diversity Western Peninsular Malaysia. This For example, yellowfin tuna that have been identified observation could have implications to the population or manually in the field were found to consist of bigeye tuna to the biogeography of the region and is worth studying using mtDNA at frequencies of 0% to 30% (Grewe and in the future. Hampton 1998). Miyabe et al. (2005) likewise observed that eight (1.6%) of the 502 tunas identified in the field Varying recruitment pulses of eastern little tuna in the were misidentified. This discovery actually underscores general Southeast Asian region (Yesaki 1994), indicated the utility of the 300 bp mtDNA control region fragment, the presence of sub-set breeding groups for the species. as well as the primers used, for further taxonomic and However, our present data suggest otherwise, albeit assessment studies. our sample size is relatively small. We observed that 2 dominant mtDNA haplotypes are dispersed all throughout The high genetic diversity of the 300 bp mtDNA marker in the general area of Southeast Asian region. Morever,

this study (about 86%) was similar to the genetic diversity of the haplotypes exhibited a very low FST value and there mtDNA markers used in previous studies on tuna population is no apparent structuring in the Minimum Spanning at 0.60 (Chow and Ushiama 1995) and billfishes at 0.922 Network. All of this suggests that the eastern little tuna in (Chow et al. 1997), suggesting that this region is a useful the Philippines and in Southeast Asia is near “panmixia” genetic marker for this particular species for population or mixing. The star-like appearance of the MSN though

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Thailand and the (Yesaki 1994). The continuous continental shelf connecting the countries in Southeast Asia could also facilitate migration of the said species since they were found to have strong affinities to continental shelves (Yesaki 1994). Graves (1996) suggested that several pelagic fishes including skipjacks, albacore, yellowfin and dolphin fishes have exhibited little spatial partitioning within and between ocean basins because of the occurrence of continuous, circumtropical pelagic environment and a wide range of suitable spawning grounds. Tunas in particular have also been suggested by Ward (1995) to exhibit a low population divergence and that migration of just a few individuals per generation could produce near-genetic homogeneity. Very recently, Magsino and Juinio-Meñez (2008) found no significant genetic stock structuring in the pelagic rabbitfish, Siganus argenteus sampled form Northern to Southern Philippines even though the geographic location of the fish is bifurcated by the Northern Equatorial Current (NEC). While the evidence for panmixia is strong, we could not ignore the absence of haplotypes A and/or B in the Davao Gulf samples. Is it possible that we could be seeing some evidence of a subpopulaton in this area, which is not significantly detectable with the present number of samples in the study? This is similar to the observation of Arnaud et al. (1999) where they detected genetic homogeneity in scad mackerel Decapterus macarellus from Molucca Strait to Banda Sea and for Figure 3. Minimum Spanning Network (MSN) of the mitochondrial DNA haplotypes of eastern little tuna. AC- Aparri, D. macrosoma from South China Sea to Java Sea and Cagayan; M- Macarascas, Palawan; OC- Opol, Cagayan; Makassar Strait but found distinct populations of D. TS- Tawi-Tawi, Sulu; DG- Davao Gulf, PM-Pangkor macrosoma in Sunda Strait, a sharp transition between Islang, Penang, Malaysia. H1 and H2 corresponds the Indian Ocean and Sunda Shelf. Genetic homogeneity the dominant Haplotype A (Hap A) and B (Hap B), does not also mean that there is no population subdivision respectively. The 3 divergent Malaysian samples were as has been observed in the stock identity of horse not included. mackerel (Trachurus trachurus) in Northeast Atlantic and Mediterranean Sea where stocks were identified using morphometrics, parasites and life history but not indicates a recent population expansion of the species (a with genetic markers (Abaunza et al. 2008). It is possible common inference for species in the Indo-West Pacific that the Davao Gulf population could be affected by the (Crandall E. pers. comm.). prevailing current system (Mindanao Current) in the The observed homogeneity in the population of eastern Southeastern Philippines (Figure 1) (Lukas et al. 1991), little tuna in Southeast Asia is similar to other tunas which in some studies was implicated to the limited and billfishes caught from different oceans (Chow et larval dispersal and gene flow in mantis shrimp found al. 1997; Rosel and Block 1996; Ward et al. 1997). in Northern Indonesia and Papua (Barber et al. 2006). “Panmixing” of the species in the region is very possible Arnaud et al. (1999) also implicated the geographic because of the high mobility of the species, the play of isolation, mortality of larvae due to hydrological the prevailing currents in the region (Wyrtki 1961) and variations and homing of adults as probable factors accompanying larval dispersal. This small tuna is widely that brought about distinctiveness of the population of distributed in the tropical and subtropical waters of the D. macrosoma in Sunda Strait, Indonesia. It is thus, Indo-Pacific Region and its larvae are found to be widely interesting to pursue studies on this in the future using dispersed throughout the Philippines (Abuso 1988; Wade a larger sample size and additional sampling sites in the 1950) and was observed to spawn in Hongkong, India, area e.g. Sulawesi and Papua New Guinea.

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Indeed the number of samples analyzed in the study East Asia. Mar Biol 135:699-707 needs to be increased to attain more significant results. BARBER PH, ERDMANN MV, PALUMBI SR. 2006. Furthermore, there are limitations to the use of mtDNA Comparative phylogeography of three codistributed in analyzing populations because it does not account for stomatopods: origins and timing of regional lineage paternal inheritance. Nevertheless, this study provides diversification in the coral triangle. Evolution 60: important initial evidence of the sharing of eastern little 1825-1839. tuna among the countries in Southeast Asia, which would warrant co-management strategies among the member BARUT NC, SANTOS MD, GARCES LR. 1997. countries to ensure the sustainability of this important Overview of Philippine marine fisheries. In: Status fishery resource. It also provides some impetus for and management of tropical coastal fisheries in Asia. looking further into a possible subpopulation in Southern Silvestre G, Pauly D. eds. ICLARM Conference Philippines and adjacent areas that has also implications Proceedings 53: 208. to the management of the stocks separate from the greater BARUT NC, SANTOS MD, MIJARES LL, SUBADE R, Southeast Asian region. ARMADA B, GARCES LR. 2003. Philippine Coastal Fisheries Situation. In: Assessment, Management and Future Directions for Coastal Fisheries in Asian Countries. Silvestre G, Garces L, Stobutzki I, Ahmed ACKNOWLEDGEMENT M, Valmonte-Santos RA, Luna C, Lachica-Alino L, The authors would like to thank the Southwest Fisheries Munro P, Christensen V, Pauly D. eds. Assessment, Science Center, NOAA, La Jolla, California through Management and Future Directions for Coastal Dr. Andrew Dizon and Dr. William Perrin for providing Fisheries in Asian Countries. Worldfish Conference travel funds as well as the Genetics Laboratory facility Proceedings 67. p. 885-914. and office; Mr. Abhu Thalib of the Fisheries Research [BAS] Bureau of Agricultural Statistics. 2006. Fisheries Institute in Penang, Malaysia for collecting and providing Statistics of the Philippines 2005-2007. Quezon Ave., the Malaysian samples; Dr. Karl Marx Quiazon for Quezon City, Philippines: Department of Agriculture- his help in drawing the Southeast Asian map; Dr. Eric Bureau of Agricultural Statistics. 383 p. Crandall for his valuable comments and his assistance in analyzing the mitochondrial haplotypes using Arlequin; CANTOR. 1849. Thynnus affinis. J. Asiatic Soc Bengal and UNESCO Short Term Fellowship on Biotechnology 18:1088-1090. for the fellowship grant. CHOW S, OKAMOTO H, UOZOMI Y, TAKEUCHI Y, TAKEYAMA H. 1997. Genetic stock structure of the swordfish (Xiphias gladius) inferred by PCR-RFLP analysis of the mitochondrial DNA control region. Mar REFERENCES Biol 127: 359 – 367. ABAUNZA P, MURTA AG, CAMPBELL N, CHOW S, USHIAMA H. 1995. Global population CIMMARUTA R, COMESANA AS, DAHLE G, structure of albacore (Thunnus alalunga) inferred by GARCIA SANTAMARIA MT, GORDO LS, IVERSEN RFLP analysis of the mitochondrial ATPase gene. Mar SA, MCKENZIE K et al. 2008. Stock identity of horse Biol 123: 39-45. mackerel (Trachurus trachurus) in the Northeast Atlantic and Mediterranean Sea: Integrating the results COLLETE BB, NAUEN CE. 1983. FAO species from different stock identification approaches. Fish catalogue. Vol.2. Scombrids of the world. An annotated Res 89: 196-209. and illustrated catalogue of tunas, mackerels, bonitos and related species known to date. FAO Fish Synop ABUSO ZV. 1988. Occurrence and distribution of (125)2: 137. larval tunas in , Pass, , and waters north of . Indo-Pacific Tuna DEVARAJ M, VIVEKANANDAN E. 1997. A Development and Management Programme (IPTP) comparative account of the small pelagic fisheries in 88(15): 164-179. the APFIC Region. In: Small Pelagic Resources and their Fisheries in the Asia-Pacific region. Devaraj ARNAUD S, BONHOMME F, BORSA P. 1999. M, Martosubroto P, eds. Proceedings of the APFIC Mitochondrial DNA analysis of the genetic relationships Working Party on Marine Fisheries, First Session; amon.g populations of scad mackerel (Decapterus 1997 May 13-16: Bangkok, Thailand: RAP Publication macarellus, D. macrosoma and D. ruselli) in South- 445 p.

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