Graves et al  Biochemical genetics of P^^lsbrsx 



63 



Est-3, Icdh-S, Pgdh, Pro-2, Sod); and P. iiehitlifer and 

 P. maculatofasciatus were distinguished at six loci 

 (Adh-2, Est-3, Gapdh-2, G6pdh, Pro-2, Pro-4). A 

 number of loci were also identified that are capable of 

 separating one species from each of the other two. For 

 example, P. clatfrratus is fixed for alleles not found in 

 P. nebulifer or P. maculatofasciatus at four gene loci 

 (Ck-A, Icdh-S, Pgdh, Sod). Thus all three species can 

 be distinguished using only two loci: one of the four 

 for which P. clathratus has unique alleles, and one of 

 the six that separates P. nebulifer and P. ■maculato- 

 fasciatus. A similar procedure can be used with the two 

 loci (Adh-2, G(ipdh) that distinguish P. nebuUfer from 

 the other two species or the two loci (Est-3, Pro-2) for 

 which P. maculatofasciatus has unique alleles. No 

 single locus was identified that by itself completely 

 distinguishes all three Paralabrax species. The closest 

 to a completely diagnostic locus is Ada; at this locus, 

 a different allele predominates in each of the species 

 at a frequency of 0.80 or more. 



Considerable mtDNA sequence differentiation was 

 demonstrated between the three Paralabrax species 

 (Table 4). Six of the restriction endonucleases (Bglll. 

 Clal, Kpnl, Sail, Smal, and A'6al) failed to cleave the 

 circular mtDNA molecule more than once in each of 

 three species and were therefore uninformative. Of the 

 13 restriction endonucleases which produced two oi' 

 more fragments in the Paralabrax species, eight {Am I, 

 Avail, BstEll. Hindi, Hindlll, Hinjl. Hpall, and 

 Xhol) were able to distinguish all three species, while 

 three enzymes (Bam HI, EcoRl, and Sstl) were able to 

 distinguish one of the species from the other two, and 

 two enzymes (Pstl and P(mll) produced similai- 

 mtDNA fragments in all three species. Thus 8 of the 

 13 informative restriction endonucleases were diag- 

 nostic for all three species. 



The mean nucleotide sequence divergences between 

 the three Paralabrax species, in pairwise comparisons, 

 are presented in Table 3. Because digestions with 

 Hinfl, Hpall, and Avail (restriction endonucleases 

 which recognize four or five nucleotide base pairs) pro- 

 duced so many fragments, it was not possible to assure 

 homology of similarly migrating fragments, and the 

 results from these enzymes were not used in the cal- 

 culation of interspecific divergences. 



Little intraspecific mtDNA nucleotide sequence vari- 

 ation was detected in this study. Of the 18 fish analyzed 

 with the 13 informative restriction endonucleases, only 

 two variants were encountered, each in a single in- 

 dividual (Table 4). 



The species-specific fragment patterns produced 

 from the digestion of Paralabrax mtDNA with the 

 restriction endonuclease Hivdlll (Fig. 1) were con- 

 sidered the most practical to use for specific identifica- 

 tion because there were diagnostic differences in the 



larger (most easily visualized) bands. Fresh or frozen 

 tissue samples as small as 0.01 g could be identified 

 from a Hindlll digestion of the mtDNA enriched in 

 a miniprep procedure and run on a 1.0% agarose 

 minigel stained with ethidium bromide. 



It was not possible to identify tissue samples smaller 

 than 0.01 g on ethidium bromide-stained minigels 



