60 



Fishery Bulletin 88(1). 1990 



been used to demonstrate intra- and interspecific 

 genetic relationships among and within various marine 

 and freshwater fishes (Ferris and Berg 1987). This 

 technique provides an additional tool for the investiga- 

 tion of egg and larval identities. Although original pro- 

 tocols were costly, time-consuming, and generally not 

 applicable to the small amount of tissue available with 

 early-life-history forms, a modification of existing 

 methodologies has provided a relatively simple method 

 for distinguisliing the eggs and larvae of closely related 

 (congeneric) species. In this paper, we report the 

 results of an investigation of water-soluble protein and 

 mtDNA restriction fragment differentiation among the 

 three southern California species ofParalabrax to find 

 a reliable genetic marker to discriminate the early-life- 

 history stages of the three species. A cost-effective 

 means of analyzing mtDNA restriction fragments of 

 both fresh and ethanol-preserved individual eggs is 

 presented. 



Materials and methods 



Adult specimens of the three Paralabrax species were 

 collected by hook and line, pole spear, and beach seine 

 off San Diego and La Jolla, California. After collection, 

 fish were transported to the laboratory alive or on ice. 

 Samples of liver, eye, and muscle tissue were removed 

 and frozen at -25°C until electrophoretic analysis was 

 undertaken. Gonads and livers were removed and used 

 fresh or stored at -70°C for mtDNA analysis. 



Eggs and larvae of the three species were obtained 

 from a captive brood stock maintained at the Southern 

 California Edison research facility in Redondo Beach, 

 California. Eggs were collected within 12 hours of 

 spawning and either transported to the laboratory in 

 San Diego in seawater or preserved in 95% ethanol. 



Protein electrophoresis 



Four specimens each of Paralabrax niacidatofasciatus 

 and P. nebulifer were collected for the electrophoretic 

 investigation. The sample of P. clathrntu:^ (45) was 

 larger because individuals were collected as part of a 

 more extensive investigation of gene flow within south- 

 ern California coastal fishes (Waples and Rosenblatt 

 1987). Samples of muscle, liver, and eye tissue were 

 individually macerated in an approximately equal vol- 

 ume of 0.1 M potassium phosphate buffer, pH 7.0, 

 before centrifugation for 10 minutes at 16,000 g, 5°C. 

 Supernatants were loaded on horizontal starch gels 

 using procedures similar to those described by Selander 

 et al. (1971). Staining recipes for the 24 enzyme and 

 protein systems studied (Table 1) were modified from 

 Shaw and Prasad (1970) and Harris and Hopkinson 



(1976). A more detailed description of the electrophor- 

 etic procedures, including all staining recipes, is pre- 

 sented in Waples (1986). 



Proteins encoded by multiple genes (isozymes) were 

 numbered according to decreasing anodal mobility. For 

 each gene locus the mobility of the most common allele 

 in Paralabrax clathratus was arbitrarily designated 

 100, and alternate alleles were numbered in accordance 

 with their mobility relative to this standard. Genetic 

 similarity and genetic distance were computed from the 

 allele frequency data using Nei's (1978) method, which 

 corrects for small sample size. 



Mitochondrial DMA analysis 



Mitochondrial DNA for restriction analysis was jiuri- 

 fied from fresh or frozen gonad or liver samples from 

 six individuals of each species following the protocols 

 for equilibrium density gradient ultracentrifugation of 

 Lansman et al. (1981), with minor modifications. Typi- 

 cal yields were 1 microgram of mtDNA per gram of 

 fresh tissue. 



A mini-prep procedure was developed for isolating 

 mtDNA from small tissue samples based on the tech- 

 niques described by Chapman and Powers (1984). 

 Tissue samples as small as an individual egg (0.6 mg) 

 were homogenized in 0.15 mL 10 mmol/L TRIS, 10 

 mmol/L EDTA, pH 7.4 (TE buffer) in a 0.2-mL ground 

 glass homogenizer. The homogenate was transfered to 

 a 1.5 mL microfuge tube and an additional 0.5 ml of 

 TE buffer added. The tube was spun at 800 g, 4°C, for 

 3 minutes to remove nuclei and cellular debris. The 

 supernatant was transferred to a second microfuge 

 tube and centrifuged at 12,000 g, 4°C, for 20 minutes 

 to pellet mitochondria. The mitochondrial pellet was 

 resuspended in 0.4 mL TE buffer and lysed with 0.04 

 mL 10% SDS. The mitochondrial lysate was extracted 

 with an equal volume of a 25:24:1 phenol/chloroform/ 

 isoamyl alcohol solution and then an equal volume of 

 24:1 chloroform/isoamyl alcohol. The DNA from the 

 aqueous layer, after the addition of ammonium acetate, 

 was precipitated in two volumes of 95% ethanol at 

 -70°C for at least 2 hours. 



The following restriction endonucleases employed in 

 this study were purchased from Bethesda Research 

 Laboratories (BRL) and used according to the sup- 

 plier's instructions: Aral. ArnW, BamWl, B(fl\\, 

 BslEll, Clal, Ecom, HuicU, HitidlU, Hinjl, HpaU, 

 Kpnl, Pstl, PvhU. Sail. Smal, SkU. Xbal. andXhoL 

 Endlabelling was performed accoi-ding to the protocols 

 of Brown (1980), with the exception that an ethanol 

 precipitation was not necessary for digestions with 

 restriction endonucleases that recognized six liase 

 pairs. Electrophoresis of restriction fragments was per- 

 formed on both large and mini-submarine horizontal 



