354 



Fishery Bulletin 98(2) 



have been proposed to overcome this obstacle (e.g. 

 Seeb and Kendall, 1991; Rocha-Olivares, 1998b). In 

 our paper we describe new developmental stages of 

 two species of Sebastes identified using mitochon- 

 drial DNA (mtDNA) sequence data. 



Materials and methods 



Sample collection 



Specimens were collected in the vicinity of Tanner 

 (32°69'N119°12'W) and Cortes (32°61'N119°33'W) 

 Banks in August 1995 during a rockfish sampling 

 cruise aboard the Scripps Institution of Oceanogra- 

 phy RV Robert Gordon Sproul. Four specimens were 

 sampled in oblique tows with a 5-m^ Isaacs-Kidd 

 midwater trawl. The fifth specimen was retrieved, 

 intact, from the digestive tract of an adult greenspot- 

 ted rockfish, Sebastes chlorostictus, caught with hook 

 and line. Fish were preserved in dBVc ethanol. Except 

 for one specimen that completely dried out upon 

 evaporation of the preserving fluid, the shrinkage 

 effect of ethanol preservation on the length of the 

 specimens has been assumed to be negligible owing 

 to their relatively large size (15.0-30.4 mm SL, 

 Radtke, 1989). The dehydrated specimen was rehy- 

 drated in water before description. 



Molecular analyses 



Total genomic DNA was extracted and purified from 

 liver or muscle tissue with a GlasPac/GS (U.S. 

 National Scientific Supply CO., San Rafael, CA) 

 DNA purification kit as described in Rocha-Olivares 

 ( 1998b). Universal primers, and versions customized 

 for Sebastes, were used for the polymerase chain 

 reaction (PCR) and automated cycle sequencing (see 

 Rocha-Olivares, 1998a for a complete list of primers). 

 A region spanning 781 base pairs (bp) of the mito- 

 chondrial cytochrome b (c_yt-6) was amplified by PCR 

 as described in Rocha-Olivares et al. ( 1999a). Briefly, 

 50-mL reactions were performed following Kocher 

 et al. (1989), with 100 ng of genomic DNA and 2 

 units of Taq DNA polymerase (Perkin Elmer Cetus, 

 Foster City, CA, or Gibco BRL, Rockville, MD). Ther- 

 mal cycling was performed as follows: hot start at 

 90°C for 2 min., followed by 36 cycles of 50 s at 94°C; 

 2 min at 51°C; 1.5 min at 72°C, and a final exten- 

 sion of 3 min at 72°C to ensure complete ampli- 

 fication of products. PCR products were purified 

 with microconcentrators (Microcon(H) 100, Millipore, 

 Bedford, MA) or purification columns (QIAquick® 

 250, Qiagen, Valencia, CA) following manufactur- 

 ers' protocols. Automated DNA sequencing was per- 



formed with ABI PRISM (Perkin Elmer Cetus) 

 DyeDeoxy® dRhodamine chemistry on an ABI 377 

 DNA sequencer in 12 \iL reactions ( 30-100 ng double 

 stranded PCR product, 3 pmol primer, 1.6-2.0 pL 

 terminator ready reaction mix); cycle sequencing 

 annealing was 10 seconds at 55°C; we followed the 

 manufacturer's protocol in respect to all other exper- 

 imental conditions. Sequence data were obtained by 

 sequencing both DNA strands of the PCR products. 



Molecular identification 



The mtDNA data from the unknown pelagic young 

 was aligned with a database of orthologous sequences 

 obtained from adult specimens of congeneric spe- 

 cies generated at the genetics and physiology labo- 

 ratory of the Southwest Fisheries Science Center in 

 La Jolla (Rocha-Olivares, 1998a)." Because the mor- 

 phological and pigmentation patterns of the speci- 

 mens revealed that they belonged to the subgenus 

 Sebastomus (Chen, 1971; 1975), the DNA sequence 

 data were compared to 89 sequences obtained from 

 all 15 species of this subgenus (Rocha-Olivares et al., 

 1999a; 1999c). Species identification was determined 

 on the basis of the most similar haplotype among 

 the species compared. Sequence comparisons were 

 carried out by using pair-wise measures of sequence 

 divergence calculated as the total number of differ- 

 ent nucleotides. 



Results 



Molecular identification 



The mtDNA sequence data confirmed morphologi- 

 cal observations that the five pelagic young were 

 members of the subgenus Sebastomus. The number 

 of conspecific DNA sequences used in the reference 

 database ranged from 1 to 13 (S. notius, S. lentigino- 

 sus, « = 1; S. spinorbis, n-3; S. capensis, n=4; S. ocu- 

 latus, S. exsul, S. helvomaculatus. n=5; S. rosaceus, 

 n=6; S. chlorostictus, S. ensifer n=l; S. umbrosus, S. 

 constellatus, S. simulator, S. eos, n=8; S. rosenblafti, 

 n = 13). Two juveniles (CAS and CA4) had identical 

 mtDNA cyt-6 sequences. On the basis of the number 

 of nucleotide differences, one specimen (CAl, Fig. 

 1) was identified as starry rockfish, S. constellatus, 

 and the other four as swordspine rockfish, S. ensifer 

 (CA2-CA5, Fig. 1). The mitochondrial haplotypes of 

 three specimens (CAl, CA3, and CA4) were identical 

 to adult reference sequences, providing unequivocal 



- Rocha-Olivare.s. A., and R. D. Vetter. 1998. Unpubl. data. 



