Li et al : Comparison of the identification of Sebostes spp by restriction site analysis and by morphological characteristics 



381 



species. The presumed intraspecific variation observed 

 in S. hopkinsi and S. goodei did not interfere with the 

 application of interspecific variation in species identifica- 

 tion, and our other work (Gharrett et al., 2001; Li, 2004) 

 indicates that intraspecific variation generally does not 

 obscure delineation of species. The key we constructed 

 for mtDNA restriction site variation can be especially 

 helpful in learning about variation in the distinguish- 

 ing morphological characters of the early life stages of 

 Sebastes. 



One factor that prevented identification of some ju- 

 venile specimens was poor DNA quality. Specimens 

 must be properly preserved to provide high quality DNA 

 for genetic analysis. Our success in amplifying DNA 

 from specimens preserved in several volumes of 95% 

 ethanol immediately after capture was substantially 

 higher than that of specimens that had been frozen 

 after capture and subsequently thawed for morphologi- 

 cal studies. 



Several small groups of species had similar restric- 

 tion site patterns. Consequently, there was ambiguity 

 in the genetic analysis of those groups. Specifically, 

 we were unable to separate S. carnatus from S. chrys- 

 omelas, and we were unable to distinguish among S. 

 chlorostictus, S. eos, and S. rosenblatti. However, mor- 

 phological characteristics could not reveal a difference 

 between S. carnatus and S. chrysomelas, nor could they 

 distinguish any of the 13 Sebastomus species, including 

 S. chlorostictus, S. eos, and S. rosenblatti. 



Sebastes carnatus and S. chrysomelas are very closely 

 related and, as adults, differ morphologically only in 

 body coloration. Sebastes carnatus has flesh-colored 

 blotches on an olive-brown background, and S. chrys- 

 omelas has yellow blotches on a black background (Love 

 et al., 2002). They are found across similar geographic 

 ranges, from northern California to Baja California, 

 and are often sympatric, inhabiting rocky substrates, 

 although they have different depth distributions (Lar- 

 son, 1980), and their segregation is maintained by the 

 social dominance of S. chrysomelas (Larson, 1980; Hoel- 

 zer, 1987). The two species may also differ slightly in 

 gill-raker counts: S. carnatus typically has 28-30, and 

 S. chrysomelas typically has 27-28 (Hallacher and Rob- 

 erts, 1985). Their taxonomic status as distinct species 

 is generally recognized from their different colorations, 

 even though no fixed biochemical or genetic differences 

 have been reported (Seeb, 1986; Seeb and Kendall, 

 1991; Hunter, 1994; Alesandrini, 1997). A recent study 

 with the use of microsatellites in the nuclear DNA and 

 mtDNA sequence variation has provided evidence that 

 the two species S. carnatus and S. chrysomelas are 

 genetically divergent (Narum, 2000). 



Sebastes chlorostictus, S. eos, and S. rosenblatti are 

 also morphologically similar, are present sympatrically, 

 and are closely related (Chen, 1971; Love, 1996; Rocha- 

 Olivares et al., 1999; Love et al., 2002). As adults, these 

 species segregate by depth to some extent; S. rosenblatti 

 occupies a somewhat narrower range than the other 

 two (Love et al., 1990). Cladistic analyses indicate that 

 S. rosenblatti is the most ancient of the three species 



(Rocha-Olivares et al., 1999). Estimates of divergence 

 times of the subgenus Sebastomus may indicate that 

 the three species are the result of the most recent spe- 

 ciation events within the subgenus, which may have 

 begun less than 140,000 years ago (Rocha-Olivares et 

 al., 1999). 



The assignment of four specimens to multispecies 

 groups in this study indicates the need for further ef- 

 fort to develop markers that will delineate the species 

 in question. Approaches include screening additional 

 regions of the mtDNA and application of additional 

 restriction enzymes. If additional mtDNA regions and 

 restriction enzymes do not provide species-specific in- 

 formation, other molecular techniques, such as the use 

 of microsatellites, should be considered. 



Acknowledgments 



This work represents, in part, the master's thesis work 

 of Z. Li at the University of Alaska Fairbanks. The proj- 

 ect was supported by funding from the U.S. Geological 

 Service (Biological Resources Division) Western Regional 

 Office in Seattle, WA (R.W.O. 32) to AJG. Funding for 

 MMN and MSL was provided by the Biological Resources 

 Division under a cooperative agreement no. 1445-CA09- 

 95-0836 with the Marine Science Institute, University 

 of California, Santa Barbara. 



Literature cited 



Alesandrini, S. 



1997. Genetic differentiation and population structure in 

 the gopher rockfish {Sebastes carnatus) and the black- 

 and-yellow rockfish (S. chrysomelas) along the California 

 coast. M.S. thesis, 33 p. Univ. California. Santa Cruz. 

 Santa Cruz, CA. 



Aoyama, J., S. Watanabe, M. Nishida, and K. Tsukamoto. 



2000. Discrimination of catadromous eels of genus 

 Anguilla using polymerase chain reaction-restriction 

 fragment length polymorphism analysis of the mito- 

 chondrial 168 ribosomal RNA domain. Trans. Am. 

 Fish. Soc. 129:873-878. 



Chen, L.-C. 



1971. Systematics, variation, distribution, and biol- 

 ogy of rockfishes of the subgenus Sebastomus (Pisces, 

 Scorpaenidae, Sebastes). Bull. Scripps. Inst. Oceaogr. 

 18:1-115. 



Chow, S., M. E. Clarke, and P. J. Walsh. 



1993. PCR-RFLP analysis on thirteen western Atlantic 

 snappers (subfamily Lutjaninae): a simple method for 

 species and stock identification. Fish. Bull. 91:619- 

 627. 



Gharrett, A. J., A. K. Gray, and J. Heifetz. 



2001. Identification of rockfish (Sebastes spp.) from 

 restriction site analysis of the mitochondrial ND-3/ 

 ND-4 and 12S/16S rRNA gene regions. Fish. Bull. 

 99:49-62. 



Hallacher, L. E., and D. A. Roberts. 



1985. Differential utilization of space and food by the 

 inshore rockfishes (Scorpaenidae: Sebastes) of Carmel 

 Bay, California. Environ. Biol. Fish. 12:91-110. 



