Graves et al : Genetic analysis of Cynosaon regalis stock structure 



471 



National Marine Fisheries Service In- 

 shore Trawl Survey. Dates and locations 

 of capture, and the size composition of 

 the collections are presented in Table 1. 

 Freshly-caught weakfish were measured 

 for standard length and then dissected. 

 Ovaries were removed and quickly frozen 

 at -20°C. 



Mitochondrial DNA was obtained by 

 the rapid isolation procedure of Chapman 

 and Powers (1984) for the initial survey 

 of mtDNA genetic heterogeneity. After 

 ethanol precipitation, the mtDNA-en- 

 riched DNA pellet was rehydrated in 75 

 ^L of distilled water, and the yield from 

 ~7g of ovarian tissue was usually suffi- 

 cient for at least 7 restriction digestions 

 visualized with ethidium bromide. 



The 1988 and 1989 samples were sur- 

 veyed with the following six restriction 

 endonucleases: Aval, BamUl, Bgll, 

 Hindlll, Neil, and Pvull. Restriction 

 fragments were separated by horizontal 

 gel electrophoresis on 0.8-1.5% agarose 

 gels run at 2 v/cm for 16 h. Gels of restric- 

 tion digestions of isolations containing 

 high yields of mtDNA were visualized 

 after ethidium bromide staining with 

 ultraviolet light illumination (Maniatis et 

 al. 1982) and photographed with a Pola- 

 roid CU-5 camera using a Wratten #5 

 filter. For those samples in which there 

 was not sufficient mtDNA for direct 

 visualization, restriction digestions were 

 endlabeled before electrophoresis with 

 the appropriate ^^S nucleotide triphos- 

 phate using the Klenow fragment (Maniatis et al. 1982). 

 After electrophoresis, the gels were rinsed with a scin- 

 tillation enhancer, dried, and autoradiographed at 

 -70°C for 5d. 



To compare the genetic relationship of weakfish from 

 the northern and southern ends of their range in great- 

 er detail, mtDNA was purified from ovarian tissues 

 using the CsCl density-gradient centrifugation protocol 

 of Lansman et al. (1981). The mtDNA from these 

 samples was surveyed with the 6 restriction endonu- 

 cleases listed above and the following 7 enzymes: Apal, 

 Avail, Banl, Bell, EeoRV, Hindi, and Hhal. The 

 restriction digestions were endlabeled, separated on 

 agarose gels, and autoradiographed as described above. 



The different fragment patterns produced by each 

 restriction endonuclease were each assigned a letter. 

 A composite mtDNA genotype, consisting of the letters 

 representing the fragment patterns generated by each 

 restriction endonuclease, was then constructed for each 



individual. The nucleon diversity (Nei 1987) was cal- 

 culated for each sample and for the pooled samples. 

 The percent nucleotide sequence divergence between 

 mtDNA genotypes was estimated by the site approach 

 of Nei and Li (1979) and the percent mean nucleotide 

 sequence divergences within and among weakfish 

 samples were calculated following the method of Nei 

 (1987), with the latter value being corrected for within- 

 group heterogeneity. The distribution of genotypic 

 frequencies was evaluated for homogeneity between 

 collections using the G-test (Sokal and Rohlf 1981). 



Results 



Weakfish mtDNA demonstrated very little variation. 

 Of the 370 weakfish surveyed with 6 restriction en- 

 donucleases, 345 shared a common mtDNA genotype 

 (Table 2). Ten variant genotypes were encountered in 



