152 
Fishery Bulletin 120(2) 
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Figure 1 
Map of the study area of northwest Washington showing the locations where samples of 
California sea lion (Zalophus californianus) and Steller sea lion (Humetopias jubatus) 
scat were collected for the study of the diets of these species by Scordino et al. (2022) 
during 2010—2013. The dashed lines indicate the 200-m isobath. The solid lines indicate 
the perimeters of marine fishing management areas 3, 4, 4B, and 5. 
Genetic analysis 
Salmon bones were pulverized in a BeadBug! homoge- 
nizer (SKU D1030, Benchmark Scientific, Sayreville, NJ) 
by using 2-mL prefilled sterile tubes containing 3-mm zir- 
conium beads (SKU D1032-30, Benchmark Scientific). 
Pulverized bone samples were then digested for 24 h at 
56°C in 500 pL of extraction buffer (0.5 M EDTA pH 8, 1 M 
urea, and 20 mg/L proteinase K) with constant rotation. 
After 24 h, digested samples were centrifuged at 1000 g for 
5 min to pellet bone material. After centrifugation, the 
' Mention of trade names or commercial companies is for identi- 
fication purposes only and does not imply endorsement by the 
National Marine Fisheries Service, NOAA. 
supernatant lysate was carefully pipetted into sterile 
15-mL falcon tubes, and DNA was purified from the super- 
natant lysate by using a QIAquick PCR purification kit 
(catalog no. 28104, Qiagen, Hilden, Germany). To increase 
DNA yield, the elution buffer was heated to 60°C prior to 
it being adding to the column during the elution step. 
We used species-specific primers and Applied Biosys- 
tems TaqMan MGB probes (minor groove binder probes, 
Thermo Fisher Scientific, Waltham, MA) developed by 
Rasmussen Hellberg et al. (2010) for identifying salmon 
bones as chum salmon (O. keta), Chinook salmon, pink 
salmon, steelhead (O. mykiss), sockeye salmon (O. nerka), 
or coho salmon (O. kisutch) on the basis of 915 cytochrome 
c oxidase subunit I DNA barcode sequences (Table 2). 
No TaqMan MGB probes were used for cutthroat trout 
