Abstract. -Restriction endonu- 

 clease analysis of mitochondrial cy- 

 tochrome b and 12S ribosomal RNA 

 (rRNA) gene fragments amplified by 

 polymerase chain reaction (PCR) 

 was applied to thirteen western At- 

 lantic snapper species (the genera 

 Lutjanus, Ocyurus and Rhom- 

 boplites), in order to assess reliabil- 

 ity of this method for genetic spe- 

 cies and stock identification studies. 

 Eight species (L. apodus, L. 

 buccanella, L. campechanus, L. 

 cyanopterus, L. griseus, L. synagris, 

 L vivanus and R. aurorubens) could 

 be identified by haplotype analysis 

 on either single or both fragments, 

 while the others (L. analis, L. jocu, 

 L. mahogoni, L. purpureus and O. 

 chrysurus) were not separated from 

 one another because of overlapping 

 or identical haplotypes observed be- 

 tween species. High nucleon diver- 

 sity estimates (/; = 40-80 f7 r ) observed 

 within four species (L. campechanus, 

 L. cyanopterus, L. griseus and L. 

 jocu) indicated that sufficient in- 

 traspecific polymorphism could be 

 detected by the PCR-[RFLP] restric- 

 tion fragment length polymorphism 

 analysis making it a useful method 

 for investigating genetic stock 

 structure. 



PCR-RFLP analysis on thirteen 

 western Atlantic snappers (subfamily 

 Lutjaninae): a simple method for 

 species and stock identification 



Seinen Chow 



National Research Institute of Far Seas Fisheries 

 Ondo 5-7-1. Shimizu, Shizuoka 424, Japan 



M. Elizabeth Clarke 

 Patrick J. Walsh 



Division of Marine Biology and Fisheries 



Rosenstiel School of Marine and Atmospheric Science, University of Miami. 



4600 Rickenbacker Causeway. Miami. FL 33149-1098 



Manuscript accepted 14 May 1993. 

 Fishery Bulletin 91:619-627 ( 1993). 



In order to manage fisheries re- 

 sources, it is essential to know the 

 recruitment mechanisms of a given 

 species. Lutjanidae is one of the larg- 

 est teleostean families, comprising 4 

 subfamilies, 17 genera, and 103 spe- 

 cies (Allen, 1985). More than a half 

 of the lutjanid species belong to the 

 subfamily Lutjaninae in which 14 

 species under three genera {Lutjanus, 

 Ocyurus and Rhomboplites) have 

 been described in the western Atlan- 

 tic Ocean. All are fine food fishes and 

 very important for commercial and 

 recreational fisheries, and consider- 

 able concern has been focused on the 

 need to understand recruitment in 

 order to manage these reef species 

 (Huntsman, 1981; Doherty, 1983; 

 Roberts and Polunin, 1991). However, 

 the close morphological similarity of 

 larvae among lutjanid species, espe- 

 cially within the subfamily Lutjan- 

 inae, has made specific identification 

 of larvae a difficult task (Richards, 

 1985; Leis, 1987; Richards and Lin- 

 deman, 1987). Allozyme electro- 

 phoresis has proven useful for 

 identifying some fish species even at 

 their embryonic or larval stages 

 (Morgan, 1975; Smith and Crossland, 

 1977; Mork et al„ 1983; Graves et 

 al., 1989). Since DNA is virtually the 



same in any cell type of an individual 

 and can be extracted from ethanol- 

 preserved specimens, restriction frag- 

 ment analyses of DNA is becoming a 

 preferred method to investigate 

 variation and relationships between 

 fish species (Billington and Hebert, 

 1991). Distinct restriction fragment 

 patterns between three bass species 

 of the genus Paralabra.x could be de- 

 tected in ethanol-preserved indi- 

 vidual eggs and larvae via Southern 

 blot analysis (Graves et al., 1990). 

 Although conventional mitochondrial 

 DNA (mtDNA) methods are power- 

 ful for detecting variation in restric- 

 tion fragment length, intensive DNA 

 analysis would be difficult especially 

 on very small samples, such as fish 

 embryos or larvae, which offer a very 

 small amount of DNA. To overcome 

 this problem, the use of the poly- 

 merase chain reaction (PCR) method, 

 which can amplify DNA sequences 

 more than 10-million fold (Saiki 

 et al., 1988), is neccessary In this 

 paper, we report amplification of 

 two mitochondrial genes (cytochrome 

 b and 12S rRNA) using fresh or 

 frozen samples, ethanol-preserved 

 embryos and larvae, and alcohol- 

 preserved museum samples of thir- 

 teen western Atlantic snapper spe- 

 cies, and results of restriction frag- 

 ment length polymorphism (RFLP) 



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