Li et a\. A key to selected Sebastes spp based on mitochondrial DNA restriction fragment analysis 



183 



forts. An increased understanding of life history varia- 

 tion can improve the systematic descriptions of Sebastes 

 species, which has mostly been based on morphology. 



Several methods have been developed to obtain spe- 

 cies-specific information that supplements the use 

 of morphological and meristic characters for species 

 identification. Otolith microstructure and other hard 

 structures have been used to distinguish the late- 

 stage larvae and young-of-the-year pelagic juveniles of 

 some Sebastes species (Laidig and Adams'; Laidig and 

 Ralston, 1994; Laidig et al., 1996). Biochemical genetic 

 methods have been used to identify adults (Barrett et 

 al.. 1966; Seeb, 1986), and may also be used to identify 

 juveniles of some species (Seeb and Kendall, 1991). 

 LeClair and Buckley (2001) used allozyme variation at 

 37 loci to positively identify 155 individuals of juvenile 

 Sebastes diploproa and Sebastes melatwps; but they 

 were unable to identify another 29 individuals, partly 

 because of a limited database. Despite some success 

 with their use, allozymes often have low resolution, 

 and there are complexes of species that cannot be dis- 

 tinguished by using allozymes alone (Seeb and Kendall, 

 1991). 



DNA sequence-based methods have some advantages 

 over the use of allozymes for the identification of juve- 

 nile fish species. They often have greater resolution, due 

 in part to the fact that expression of some metabolic 

 enzymes changes during development, whereas DNA 

 sequences generally do not. DNA is less susceptible to 

 degradation than the enzymes used in allozyme stud- 

 ies. Also, DNA sequence-based methods require a small 

 amount of tissue sample, which is especially suited for 

 work with samples from early life stages (e.g.. Gray 

 et al., 2006). Because it has a relatively high rate of 

 substitution (Moritz et al., 1987), mitochondrial DNA 

 (mtDNA) can be useful for distinguishing closely re- 

 lated species. Mitochondrial DNA is usually inherited 

 maternally in vertebrates and does not recombine with 

 paternal mtDNA if it is leaked into the zygote (Gyllen- 

 sten et al., 1991; but see Rokas et al., 2003). Thus, the 

 mtDNA sequence represents a matrilineal phylogeny 

 and is often useful in delineating phylogenetic rela- 

 tionships of closely related species. Sequences of the 

 mitochondrial cytochrome b gene were used to identify 

 the pelagic young of Sebastes constellatus and Sebastes 

 ensifer (Rocha-Olivares et al., 2000). Multiplex PCR 

 of haplotype-specific regions of mtDNA has also been 

 used to identify early stages of Sebastes species (Rocha- 

 Olivares, 1998). 



The objective of this study was to devise a key for 

 species identification using mtDNA restriction fragment 

 data from both the ND3/ND4 and 12S/16S regions. 

 In a previous study, data for species-specific mtDNA 

 restriction site variation in the ND3/ND4 region were 



' Laidig, T. E., and P. B. Adams (eds. ). 199L Methods used 

 to identify pelagic juvenile rockfish (genus Sebastes) occurring 

 along the coast of central California. NOAA-TM-NMFS- 

 SWFSC-166. 180 p. NMFS Southwest Fisheries Science 

 Center, 110 Shaffer Rd., Santa Cruz, CA 95060. 



presented for 15 Sebastes species (Gharrett et al., 2000). 

 We have included data for 56 additional species using 

 the ND3/ND4 and 12S/16S regions of the mtDNA as the 

 target region of PCR-amplification. We have included 

 specimens of Helicolenus hilgendorfi and Sebastolobus 

 alascanus for contrast. 



Materials and methods 



Adult specimens of 71 species of rockfishes were collected 

 from the Gulf of Alaska, the coastal waters of Califor- 

 nia and Baja California, and from the coast of Japan. 

 Samples of H. hilgendorfi and Sebastolobus alascanus, 

 species from two sister genera, were collected from Japa- 

 nese coastal waters and the Gulf of Alaska, respectively, 

 to provide outgroup comparisons. A sample of heart 

 tissue from each specimen was preserved in either 95% 

 ethanol or a solution that was 80% 0.25M ethylenediami- 

 netetraacetic (EDTA) acid at pH 8 and saturated with 

 NaCl (Seutin et al., 1991) and 20% dimethyl sulfoxide 

 (DMSO). At least five individuals of each species were 

 analyzed, except for a few species for which less than 

 five specimens were available. We did not distinguish 

 between S. ciliatus and S. variabilis, which had not yet 

 been described (Orr and Blackburn, 2004), when we 

 collected samples. 



Total genomic DNA was isolated by using a Purgene 

 DNA^M isolation kit (Gentra Systems, Inc., Minneapolis, 

 MN). Two target regions of mtDNA were amplified by 

 using the polymerase chain reaction. The ND3/ND4 

 region begins in the glycyl tRNA gene and spans the 

 NADH-dehydrogenase subunit-3, arginyl tRNA, NADH- 

 dehydrogenase subunit-4L, and NADH-dehydrogenase 

 subunit-4 genes, ending in the histidyl tRNA gene. The 

 12S/16S region starts near the phenylalanyl tRNA end 

 of the 12S rRNA gene, and runs through the valyl tRNA 

 gene to near the leucyl tRNA end of the 16S rRNA gene. 

 Primers for target regions have been used to amplify 

 these regions in northern Pacific rockfish (Gharrett et 

 al., 2001). The lengths for the amplified ND3/ND4 and 

 12S/16S regions are about 2385 and 2430 base pairs, 

 respectively, based on the aggregate restriction maps. 



Subsamples of the PCR products of each individual 

 were digested with one or more of the restriction endo- 

 nucleases BstN I, BstU I, Cfo I, Dde I, Hind II, Hinf I, 

 Mbo I, Msp I, Rsa I, and Sty I. Fragments were sepa- 

 rated electrophoretically in 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 90mM Tris-boric acid, and 

 2 mm EDTA, pH 7.5). A 100 base-pair ladder provided 

 molecular weight markers to estimate restriction frag- 

 ment sizes. Gels were stained with ethidium bromide 

 and photographed on an ultraviolet light transillumi- 

 nator. Restriction fragments that could not be accu- 

 rately measured from agarose gels were separated on 

 8% polyacrylamide gels and stained with SYBR Green 

 I Nucleic Acid Stain'^^' (Molecular Probes, Eugene, OR) 

 using a 25-bp ladder for a molecular weight standard. 



