Reader: Please note the following erratum for this article. Page 56, line 5 Read “S. melanops” instead of “S. maliger” so that the sen- tence begins “Similarly, the haplotypes of S. melanops and S. fl avidus (subgenus Sebastosomus) were tightly clustered . .”. 49 Abstract–Species-specifi c restriction Identifi cation of rockfi sh (Sebastes spp.) site variation in the 12S/16S rRNA and ND-3/ND-4 mtDNA regions was by restriction site analysis of the mitochondrial used to distinguish among 15 rockfi sh species of the genus Sebastes common ND-3/ND-4 and 12S/16S rRNA gene regions to the waters of Alaska. Intraspecifi c variation exhibited by eight of the spe- Anthony J. Gharrett cies (based on fi ve individuals of each species) did not obscure the interspe- Andrew K. Gray cifi c variation, except possibly between Fisheries Division, School of Fisheries and Ocean Sciences S. zacentrus and S. variegatus. Intra- University of Alaska Fairbanks specifi c nucleotide diversity averaged 11120 Glacier Highway 0.0024 substitutions per nucleotide, Juneau, Alaska 99801 whereas interspecifi c nucleotide diver- E-mail address (for A. J. Gharrett): [email protected] gence averaged 0.0249. In contrast, the average nucleotide divergences between Sebastes and two other scorpaenid spe- Jonathan Heifetz cies, Helicolenus hilgendorfi and Sebas- Auke Bay Laboratory tolobus alascanus, were 0.0805 and Alaska Fisheries Science Center 0.1073, respectively. Cladistic and phe- National Marine Fisheries Service, NOAA netic analyses supported some, but not 11305 Glacier Highway all, of the subgenera assignments of Juneau, Alaska 99801-8626 Sebastes. A scheme for distinguishing among the species studied was pre- sented. Restriction sites of 10 restric- tion endonucleases were mapped in the two PCR-amplifi ed mtDNA regions by using double digests. In all, we The species-rich genus of Sebastes rock- species have also contributed to a cha- detected 153 sites corresponding to 640 fi sh has challenged both fi sheries scien- otic history of their systematics and (13.5%) of the 4815 nucleotides in the two regions combined. The ND-3/ND-4 tists and ichthyologists since they were many aspects of the phylogeny have 2 region exhibited substantially more fi rst described from Alaskan waters by not been resolved (Kendall and re- intraspecifi c, interspecifi c, and interge- Tilesius (S. ciliatus; 1813, cited in Esch- viewed in Cramer [1895] and Phillips neric variation than the 12S/16S rRNA meyer, 1998) and Richardson (S. cauri- [1957]. Cuvier (1829, cited in Eschmey- region. nus; 1845). Both the large number of er,1998) fi rst described the genus Se- species, about 100 worldwide (Ishida, bastes for northern Atlantic specimens. 1984; Kendall, 1991), and the metamor- The number of genera recognized for phoses that occur in larval and juve- the species presently placed in Sebastes nile fi sh produce a confusing number has expanded and contracted repeated- of forms. The diversity of species and ly, reaching a maximum of 15 (Jordan forms combine to limit our knowledge et al., 1930) and now these genera are of the biology, including life histories, of generally considered subgenera. When rockfi shes. To date, identifi cation to spe- combined with fi ve northwestern Pa- cies is not possible for many larvae and cifi c Ocean (Matsubara, 1943) and one juveniles (e.g. Kendall, 1991; Moser, northern Atlantic Ocean subgenus, Se- 1996), and distinguishing between some bastes comprises about 22 subgenera adult species may be diffi cult. For exam- (Kendall, 1991). ple, adult S. variegatus is similar to Identifi cation and systematics of fi sh S. zacentrus and adult S. mystinus, S. depend largely on morphological char- melanops, and S. ciliatus are often mis- acters; morphology alone, however, does identifi ed (Love1). The inability to iden- not always provide suffi cient criteria, tify species constrains surveys of larval especially for identifi cation of larval abundance and, consequently, ecologi- and juvenile forms. Genetic informa- cal studies that are important for con- tion, obtained by using biochemical or servation and management of rockfi sh molecular methods, has been used to and other species. In addition, the ques- address systematic problems. In some tions facing biologists and fi shery man- agers require tools that can resolve 1 intraspecifi c population (stock) struc- Love, M. 2000. Personal commun. Marine Sciences Institute, University of California, ture, as well as methods for identifying Santa Barbara, CA. 93106. species. 2 Kendall, A.W. 2000. Personal commun. Manuscript accepted 22 August 2000. The size of the genus and the pau- NMFS Alaska Fisheries Center, 7600 Sand- Fish. Bull. 99:49–62 (2001). city of information about some of the point Way NE, Seattle, WA 98115. 50 Fishery Bulletin 99(1) instances, genetic differences can be used to differentiate restriction site analyses on fi ve individuals from each of 15 between species that have overlapping morphologies. For different Sebastes species common in Alaskan waters and example, cryptic species of southern Atlantic Ocean Se- mapped the sites using double digests to determine indi- bastes species were recognized from mtDNA analysis (Ro- vidual-based haplotypes. From these data, we examined cha-Olivares et al., 1999a). Genera and many species of intra- and inter-specifi c divergences and used both phe- rockfi sh can be distinguished from protein electrophoresis netic and cladistic procedures to examine relationships differences (e.g. Tsuyuki et al., 1968; Johnson et al., 1972). among the haplotypes. We also mapped the sites for short- More recently, allozyme data (Seeb, 1986) and mtDNA spine thornyhead (Sebastolobus alascanus) and Helicole- variation (Johns and Avise, 1998; Rocha-Olivares, 1998a; nus hilfendorfi to facilitate analysis. Finally, we developed Seeb, 1998; Rocha-Olivares et al. 1999b, 1999c) have been a mtDNA restriction fragment-based strategy for identify- used to address questions about the evolution and sys- ing Sebastes species. tematics of Sebastes. Genetic differences may provide the means for identifying rockfi sh larvae and juveniles that cannot be identifi ed from their morphology (Seeb and Ken- Materials and methods dall, 1991). Recently, Rocha-Olivares (1998b) devised a PCR-based approach for identifi cation of Sebastes species. Adult specimens of 15 different species of Sebastes rockfi sh The advantage of his approach is that it is fast. The disad- and Sebastolobus alascanus were collected from the east- vantage is that failed PCR reactions are part of the iden- ern Gulf of Alaska (Table 1). These species are the most tifi cation scheme. However, failed reactions can also result abundant of the approximately 25 species reported in the from poor quality DNA or intraspecifi c variation and lead region. In the fi eld, species identifi cation was confi rmed by to misidentifi cation of the specimens. Intraspecifi c genetic using the pictoral guide of Kramer and O’Connell (1988) variation can also provide information about population and the key and descriptions in Hart (1973). H. Ida (Kita- structure (e.g. Wishard et al., 1980; Seeb et al., 1988; Ro- sato University, Sanriku, Japan) provided samples of Heli- cha-Olivares and Vetter, 1999). colenus hilgendorfi from Japanese coastal waters. Samples Vertebrate mitochondrial DNA (mtDNA) is compact of heart tissue from each specimen were preserved in 95% (about 16,500 base pairs) and has been completely se- ethanol or a solution of 20% dimethyl sulfoxide (DMSO), quenced in a variety of organisms including carp (Cyprinus 0.25M ethylenediaminetetraacetic acid (EDTA) at pH 8 carpio; Chang et al., 1994) and rainbow trout (Oncorhyn- and saturated with NaCl (Seutin et al., 1991). chus mykiss; Zardoya et al., 1995). Because mitochondria Total cellular DNA was isolated by phenol-chloroform are transmitted primarily through maternal genes (Gyl- extraction (Wallace, 1987) or with Puregene DNATM iso- lensten et al., 1991), mtDNA is haploid and clonally in- lation kits (Gentra Systems, Inc., Minneapolis, MN). Two herited (Meyer, 1993). Restriction fragment analyses of target regions were PCR-amplifi ed from total cellular PCR-amplifi ed regions of mtDNA provide a rapid and DNA with primers that we developed for coho salmon (On- practical method for detecting nucleotide sequence varia- corhynchus kisutch) mtDNA studies. The ND3/ND4 region tion in mtDNA between individuals or species. Sequence begins in the glycyl tRNA gene and spans the NADH- variation detected by restriction enzymes produces bina- dehydrogenase subunit-3, arginyl tRNA, NADH-dehydro- ry character-state data that can be used in phylogenetic genase subunit-4L, and NADH-dehydrogenase subunit-4 analyses (e.g. Dowling et al., 1992). An advantage of re- genes, ending in the histidyl tRNA gene. The 12S/16S re- striction site surveys over sequencing is that they are gion extends from near the phenylalanyl tRNA end of the practical for detecting variation in large sequence spans. 12S rRNA gene through the valyl tRNA gene to near the The number of nucleotides screened in restriction site sur- leucyl tRNA end of the 16S rRNA gene (Table 2). From veys depends on the number of restriction enzymes used restriction digests, we estimated that the ND3/ND4 and and their match with the DNA sequence. 12S/16S regions comprised 2385 and 2430 base pairs (bp), We have developed primers that can be used to PCR respectively, as compared with 2331 and 2402, respective- amplify regions of Sebastes mtDNA. The amplifi ed regions ly, for O. mykiss. Target sequences were amplifi ed by heat- provide material for addressing species identifi cation and ing to 94°C for 5 min, followed by 30 cycles for 1 min stock identifi cation questions about rockfi sh. In addition, at 94°C, 1 min at 55°C, and 3 min at 72°C using Taq the haplotypes observed provide information for address- polymerase from Perkin Elmer (Norwalk, CT) according ing systematic relationships among Sebastes.