Li et a\ Comparison of the identification of Sebastes spp, by restriction site analysis and by morpfiological cfiaracteristics 



377 



because of its high cost, is usually limited to 

 a much smaller span of DNA. Closely related 

 species of several genera have been identi- 

 fied with this method; for example, snap- 

 pers (Chow et al., 1993), eels (Aoyama et 

 al., 2000; Lin et al., 2002), billfishes (Innes 

 et al., 1998), and rockfishes (Gharrett et al., 

 2001). For identification of larvae and ju- 

 veniles, restriction site patterns of adults 

 can be used as references to ensure the re- 

 liability of the results. A limited database 

 was used and a small number of samples 

 were tested for a previous report on larval 

 and juvenile identification of Sebastes, also 

 based on restriction patterns of the mtDNA 

 (Taylor, 1998). 



The objective of this study was to compare 

 the results of identifying pelagic juvenile 

 rockfishes by the application of a key for de- 

 lineating species based on mtDNA variation 

 of adult rockfishes (Li et al., 2006) and by 

 using only morphological characteristics. The 

 questions we addressed were the following: 

 Are the results for both genetic identifica- 

 tions and morphological identifications con- 

 cordant? If not, how did these two methods 

 differ? The targets of the comparison were 

 samples of pelagic juvenile rockfishes col- 

 lected from southern California. Both ge- 

 netic and morphological analyses were con- 

 ducted without knowledge of the results of 

 the other. 



Materials and methods 



Collection of juveniles 



Juvenile rockfishes were collected from the coast of 

 Santa Barbara, California in June of 1998 and 2000 

 (Table 1). Fish were captured at night with a modified 

 Cobb mid-water trawl with a nominal 12.2 mxl2.2 m 

 opening and 9-mm mesh net. Average trawling depth 

 ranged from 18 to 29 m below the surface and above 

 bottom depths ranged from 500 to 800 m, except for one 

 specimen that was caught over a bottom depth of 88 m. 

 Generally, specimens were sorted and then frozen on 

 board the vessel. However, specimens from two hauls 

 were sorted and immediately put in 95% ethanol aboard 

 the vessel. Similar collection methods were used for both 

 years. Details of the 1998 survey are provided in Nishi- 

 moto and Washburn (2002). The standard lengths (mm) 

 of the specimens ranged from 16 to 42 mm. 



Genetic identification 



Following collection, each fish was identified in the labo- 

 ratory by using morphological characters and meristics 

 and pigmentation patterns. Specimens were chosen for 

 genetic examination based on morphological differences. 



Fish representing different developmental stages (e.g., 

 transition from larval to pelagic juvenile stage) of a spe- 

 cies were included in the sample. We examined forty-nine 

 specimens that included species that were easily identi- 

 fied based on pigmentation and morphological characters 

 and individuals for which identification was less certain. 

 All specimens were provided for genetic analysis without 

 information on the morphological assignment. 



For the genetic analysis, muscle tissue and skin from 

 one side of the body was removed from each speci- 

 men in September 2000. Total genomic DNA was iso- 

 lated by using a Purgene DNA^-^' isolation kit (Gentra 

 Systems, Inc., Minneapolis, MN). Total DNA of each 

 specimen was PCR-amplified and digested with specific 

 restriction enzymes as directed in the key (Li et al., 

 2006). In some cases, digests with additional restric- 

 tion enzymes were conducted to increase certainty of 

 the identifications. Restriction fragments were sepa- 

 rated electrophoretically on 1.5% agarose gels (one part 

 agarose [Sigma-Aldrich, St. Louis, MO] and two parts 

 SynergeF*"' [Diversified Biotech Inc., Boston, MA]) in 

 0.5xTBE buffer (TBE is 90 mM Tris-boric acid, and 



