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Fishery Bulletin 101(4) 



Some studies of population structure using genetic anal- 

 yses have not revealed the presence of discrete stocks along 

 the Pacific Ocean. Barret and Tsuyuki (1967) used transfer- 

 rin analysis and did not find differences in allele frequen- 

 cies between samples from Hawaii and eastern Pacific 

 samples, although heterogeneity was detected within the 

 eastern Pacific samples (lATTC, 1975). Allozyme variation 

 studies in the esterase locus (Fujino, 1970) did not show 

 enough evidence of genetic differentiation between east- 

 ern Pacific and Hawaii samples. Furthermore, Scoles and 

 Graves (1993) used restriction fragments length polymor- 

 phisms (RFLP) and analysis of mitochondrial (mt) DNA to 

 examine five samples collected across the Pacific Ocean and 

 one from the Atlantic Ocean. Although they detected 34 

 haplotypes and considerable genetic variation, no evidence 

 of genetic differentiation among samples was found. 



However, more recent genetic studies have provided lim- 

 ited evidence of genetic heterogeneity. Ward et al. (1994) 

 analyzed four polymorphic allozyme loci and 18 mtDNA 

 haplotypes in yellowfin tuna from the Pacific Ocean. Al- 

 though no unique haplotypes were found in the analyzed 

 populations through RFLP analysis, the eastern Pacific 

 samples were found to be different from the central and 

 western Pacific samples in frequency differences at a sin- 

 gle locus GPI-F*, suggesting that the signal of population 

 structure exhibited is due to selective factors contributing 

 to the divergence. Eastern Pacific samples (n=41) were 

 collected in the northeast Pacific off California and at an 

 unspecified site off Mexico (n=40). Comparisons of GPI-F* 

 allele frequencies from eastern Pacific also included two 

 samples previously analyzed by Sharp (1978) from Roca 

 Partida (Central America) and Ecuador. Their results 

 showed population homogeneity at the GPI-F* locus for 

 this region. 



To date, the methods and logistics used to study diver- 

 gence in the Pacific yellowfin tuna have been focused on 

 a global rather than a local scale, and sampling has been 

 focused on the wide areas of the west and central Pacific. 

 Local structure in the eastern Pacific yellowfin tuna has 

 not been addressed through a more intense sampling 

 strategy to examine genetic homogeneity in this region. 

 Because tagging studies have shown restricted longitudi- 

 nal movements by yellowfin tuna, population structure and 

 isolation by distance hypotheses can be tested. To evaluate 

 the stock structure of yellowfin tuna in eastern Pacific, we 

 employed analyses of allozymes and of randomly amplified 

 polymorphic DNA (RAPDs). 



RAPDs have proven to be useful genetic markers because 

 of their high levels of polymorphisms (Williams et al., 

 1990; Welsh et. al., 1991). They have been used to estimate 

 population structure in fishes, including the cod (Kenji, 

 1998), red mullet (Mamuris et al., 1998), and striped bass 

 (Bielawski and Pumo, 1997). The use of RAPDs, considered 

 as neutral markers, and the simultaneous use of allozyme 

 analyses with intense sampling in a more local area, might 

 provide evidence about the relationship between gene flow 

 and spatial distribution of the eastern Pacific yellowfin 

 tuna, as well as evidence of the presence of local selective 

 factors responsible for the divergence suggested by Ward 

 etal.(1994). 



Materials and methods 



Sampling 



A total of 327 tissue samples from specimens of ten loca- 

 tions were obtained from commercial tuna boats fishing 

 in the tropical eastern Pacific from 1994 to 1996 (Fig. 1). 

 Muscle tissue samples were dissected from specimens at 

 the time of landing and were transported in liquid nitrogen 

 or on dry ice to the Laboratorio de Genetica de Organismos 

 Acuaticos of the Instituto de Ciencias del Mar y Limnologia 

 in Mexico City. Samples were maintained at -70°C until 

 processing. 



Allozyme analysis 



For allozyme analysis, 1 cm-* (about one gram) of tissue 

 sample was ground with a manual homogenizer in 1.5 

 mL of extraction buffer (O.OIM Tris-O.OOIM EDTA, pH 

 6.8, and 1% NADP) and centrifuged at 2500 g at 4°C. 

 Electrophoretic runs were performed in 12% (w/v) starch 

 gels ( Sigma Chemicals, St. Louis, MO ). Four buffer systems 

 were used to analyze nineteen enzymes that resolved 28 

 loci, eight of which showed polymorphism: Aat-S* (aspar- 

 tate aminotransferase), Glud (glutamate dehydrogenase), 

 Gpi-F* and Gpi-S* (glucose phosphate isomerase). La 

 (leucil-L-alanine), Lgg (L-leucil-glycil-glycine), Pap-F* 

 (L-leucil-L-proline) and 6-Pgd (phosphogluconate dehy- 

 drogenase). Enzymes AK (adenilate kinase), CK (cre- 

 atinine kinase), GAPDH (glyceraldehyde-3-phosphate 

 dehydrogenase), LDH (lactate dehydrogenase), MDH 

 (mMalate dehydrogenase), ME (malic enzyme) and SOD 

 (superoxide dismutase), displayed twenty more loci that 

 were presumably monomorphic. Buffer systems for enzyme 

 analysis were 1) amino-citrate: 0.04M citric acid, 15mL/L 

 of N-3-aminopropyl-morpholine, pH 6.5 (AAT, GPI, and 

 LA); 2) 0.008M Tris, 0.003 M citric acid, pH 6.7 (GLUD 

 and 6-PGD); 3) 0.025 M Tris, 0.192 glycine, pH 8.5 (GPI 

 and LGG); 4) 0.0.076 M Tris, 0.005 M citric acid, pH 8.7 

 (PAP). Enzyme assays were performed following Harris 

 and Hopkinson (1976). Enzymes showing polymorphism 

 were analyzed for all samples and subjected to population 

 genetic analysis. 



RAPD analysis 



For RAPD analyses, genomic DNA was extracted from 

 muscle tissue by using standard phenol-chloroform proto- 

 cols (Sambrooketal., 1989), resuspendedinTE buffer (lOmM 

 Tris-O.lmM EDTA pH 8.0), and quantified with a Hoefer 

 DyNA quant 200 fluorometer. DNA was amplified with 

 primer F-10 (Operon® Alameda, CA; 5'-GGAAGCTTGG-3'). 

 Amplifying reactions were performed in a final volume of 

 22 //L consisting of 0.7 to 1 ng///L of DNA in amplification 

 buffer, 10 mM Tris-HCl, 50 niM KCl, 1.5 mM MgCl.^, 33 

 ng of primer, 10 mM dNTPs, and 1 U of Taq polymerase. 

 Amplification of genomic DNA was performed in a Perkin 

 Elmer®, Foster City, CA (mod. 480), thermal cycler. The pro- 

 gram was set for 1 cycle of 1 min. at 36°C, followed by 44 

 cycles of 1 min. at 36°C; 1 min. at 94°C; 2 min. at 72°C, and 



