213 



Molecular methods for the genetic identification 

 of salmonid prey from Pacific harbor seal 

 (Phoca vitulina richardsi) scat 



Maureen Purcell 



Greg Mackey 



Eric LaHood 



Conservation Biology Molecular Genetics Laboratory 

 Northwest Fisheries Science Center 

 National Marine Fisheries Service. NOAA 

 2725 Montlake Blvd. E. 

 Seattle, Washington 98112-2097 



Harriet Huber 



National Marine Mammal Laboratory 

 Alaska Fisheries Science Center 

 National Marine Fisheries Service, NOAA 

 7600 Sand Point Way NE 

 Seattle, Washington 98115 



Linda Park 



Conservation Biology Molecular Genetics Laboratory 



Northwest Fisheries Science Center 



National Marine Fisheries Service, NOAA 



2725 Montlake Blvd. E. 



Seattle, Washington 98112-2097 



E-mail address (for L. Park, contact author): linda parkig'noaa gov 



Twenty-six stocks of Pacific salmon 

 and trout [Oncorhynchus spp.), rep- 

 resenting evolutionary significant 

 units (ESU), are listed as threatened 

 or endangered under the Endangered 

 Species Act (ESA) and six more stocks 

 are currently being evaluated for 

 listing. 1 The ecological and economic 

 consequences of these listings are 

 large; therefore considerable effort has 

 been made to understand and respond 

 to these declining populations. Until 

 recently. Pacific harbor seals (Phoca 

 vitulina richardsi) on the west coast 

 increased an average of 5% to 1% per 

 year as a result of the Marine Mammal 

 Protection Act of 1972 (Brown and 

 Kohlman 2 ). Pacific salmon are season- 

 ally important prey for harbor seals 

 (Roffe and Mate, 1984; Olesiuk, 1993); 

 therefore quantifying and understand- 

 ing the interaction between these two 

 protected species is important for 

 biologically sound management strat- 

 egies. Because some Pacific salmonid 

 species in a given area may be threat- 



ened or endangered, while others are 

 relatively abundant, it is important 

 to distinguish the species of salmonid 

 upon which the harbor seals are prey- 

 ing. This study takes the first step in 

 understanding these interactions by 

 using molecular genetic tools for spe- 

 cies-level identification of salmonid 

 skeletal remains recovered from Pacific 

 harbor seal scats. 



Most studies of harbor seal food hab- 

 its rely on morphological identification 

 of indigestible parts (e.g. otoliths and 

 bones) from scat. Otoliths can be used 

 to identify fish species (Ochoa-Acuna 

 and Francis, 1995) but are not always 

 present in scats, which can result in an 

 underestimate of the number of species 

 and the number offish consumed (Har- 

 vey, 1989). Skeletal remains in scat are 

 much more common and generally 

 bones can be identified to the species 

 level (Cottrell et al., 1996). Morpho- 

 logical identification is possible to the 

 family level only with Pacific salmonid 

 bones; however, genetic markers have 



the ability to discriminate between 

 species, and the feasibility of extracting 

 DNA from bones has been clearly dem- 

 onstrated (Hochmeister et al., 1991). 



Mitochondrial DNA (mtDNA) has 

 been widely employed in systematic 

 studies (reviewed by Avise, 1994) mak- 

 ing it ideal for animal species identifi- 

 cation. In this study, we explored three 

 regions of the mitochondrial genome 

 that have been previously character- 

 ized in Pacific salmonids (Shedlock 

 et al., 1992; Domanico and Phillips, 

 1995: Parker and Kornfield, 1996). 

 DNA sequencing of these regions 

 provided an unambiguous way to de- 

 termine species identity. Because high 

 throughput sequencing can be prohibi- 

 tively expensive for laboratories with 

 limited facilities, restriction fragment 

 length polymorphism (RFLP) analysis 

 was also explored as an alternative for 

 species identification. A previous study 

 had established a species-specific poly- 

 merase chain reaction (PCR) test for 

 Pacific Northwest salmon and coastal 

 trout species (McKay et al., 1997). The 

 PCR test is based on the initial ampli- 

 fication of an approximately 1000-bp 

 fragment of the nuclear growth hor- 

 mone 2 gene. The degraded state of the 

 DNA isolated from bones recovered 

 from scat has generally limited suc- 

 cessful PCR to amplicons of 300 bp or 

 less (data not shown). Furthermore, 

 the amount of DNA isolated from bone 

 fragments can be quite small; mtDNA 

 is present in higher copy number per 

 cell than is nuclear DNA. Thus, we 

 considered mtDNA it to be a more 



1 http://www.nwr.noaa.gov/lsalmon/salmesay 

 specprof.htm. [Accessed June 17, 2003.] 



- Brown, R. F. and S. G. Kohlman. 1998. 

 Trends in abundance and current status 

 of the Pacific harbor seal tPhoca vitulina 

 richardsi) in Oregon: 1977-1998. ODFW 

 (Oregon Department of Fish and Wildlife i 

 Wildlife Diversity Program Technical 

 Report, 98-6-01. 16 p. [Available from 

 ODFW, 7118 NE Vandenberg Ave. Corval- 

 lis, OR 97333.] 



Manuscript approved for publication 

 9 October 2003 by Scientific Editor. 



Manuscript received 20 October 2003 

 at NMFS Scientific Publications Office. 



Fish. Bull. 102:213-220 (2004). 



