Mar. Biotechnol. 2, 587–600, 2000 DOI: 10.1007/s101260000045 © 2000 Springer-Verlag New York Inc. Microsatellite DNA Population Structure and Stock Identification of Steelhead Trout (Oncorhynchus mykiss) in the Nass and Skeena Rivers in Northern British Columbia Terry D. Beacham,1,* Susan Pollard,2 and Khai D. Le1 1Department of Fisheries and Oceans, Science Branch, Pacific Biological Station, Nanaimo, BC., V9R 5K6, Canada 2Ministry of Fisheries, 780 Blanshard Street, Victoria, BC., V8V 1X4, Canada Abstract: Population structure and the application to genetic stock identification for steelhead (Oncorhynchus mykiss) in the Nass and Skeena Rivers in northern British Columbia was examined using microsatellite markers. Variation at 8 microsatellite loci (Oki200, Omy77, Ots1, Ots3, Ssa85, Ots100, Ots103, and Ots108) was surveyed for approximately 930 steelhead from 7 populations in the Skeena River drainage and 850 steelhead from 10 populations in the Nass River drainage, as well as 1550 steelhead from test fisheries near the mouth of each river. Differentiation among populations within rivers accounted for about 1.9 times the variation observed among years within populations, with differences between drainages less than variation among populations within drainages. In the Nass River, winter-run populations formed a distinct group from the summer-run populations. Winter-run populations were not assessed in the Skeena River watershed. Simulated mixed-stock samples suggested that variation at the 8 microsatellite loci surveyed should provide relatively accurate and precise estimates of stock composition for fishery management applications within drainages. In the Skeena River drainage in 1998, Babine River (27%) and Bulkley drainage populations (31%) comprised the main components of the returns. For the Nass River in 1998 steelhead returning to Bell-Irving River were estimated to have comprised 39% of the fish sampled in the test fishery, with another 27% of the returns estimated to be derived from Cranberry River. The survey of microsatellite variation did not reveal enough differentiation between Nass River and Skeena River populations to be applied confidently in estimation of stock composition in marine fisheries at this time. Key words: British Columbia, microsatellite loci, population structure, steelhead, stock composition. INTRODUCTION in British Columbia. There are no directed commercial fish- eries for steelhead in British Columbia, and significant ef- Steelhead (Oncorhynchus mykiss), the anadromous form of forts are made to reduce the bycatch of steelhead in com- rainbow trout, are found in all major coastal river systems mercial salmon fisheries because this species is considerably less abundant naturally than all other Pacific salmon spe- Received January 14, 2000; accepted July 13, 2000. *Corresponding author: telephone 250-756-7149; fax 250-756-7053; e-mail cies. This low level of abundance can lead to special and [email protected] often contentious measures for steelhead conservation to 588 Terry D. Beacham et al. reduce exploitation in fisheries. One area where there MATERIALS AND METHODS has been substantial concern for steelhead is the Skeena River drainage in northern British Columbia. Measures Collection of DNA Samples and Polymerase have been taken to reduce steelhead exploitation dur- Chain Reaction ing fisheries for the more abundant sockeye salmon (O. nerka), which have run timing very similar to that For the characterization of the baseline populations, DNA of summer-run steelhead in the lower river. Run timing was extracted from adipose fin clips of adult steelhead pre- for specific steelhead populations is uncertain, and there served in 90% ethanol. The main method of sampling is currently no method to identify the presence of spe- adults was by angling, although enumeration fences were cific populations during their passage through the lower used at some locations (Sustut, Babine, and Toboggan in river. Skeena River drainage) in some years. The Sustut River Determination of population structure of exploited samples were derived mainly from the upper Sustut drain- species is an essential component in successful management age, but 13 fish from the lower Sustut drainage were also of fisheries. Specifically, this information can be used for included in the 1997 samples. Juveniles were included in the applications ranging from the determination of appropriate analysis for the 1997 Bell-Irving River sample (61 of 96 fish conservation units to estimation of stock composition in analyzed). Approximately 930 steelhead were sampled from mixed-stock fisheries. In British Columbia, surveys of ge- 7 Skeena River populations and 850 steelhead from 10 Nass netic variation have been used to describe population struc- River populations, with most of the populations sampled in ture in steelhead (Parkinson, 1984a; Taylor, 1995; Beacham at least 2 years (Table 1 and Figure 1). All populations in the et al., 1999), and can also be used to estimate stock com- Skeena River were summer run. In the Nass River, 5 sum- position in mixed-fishery samples (Parkinson, 1984b; mer-run and 5 winter-run populations were surveyed Beacham et al., 1999). (Table 1). Laboratory methods detailing DNA extraction Microsatellites are rapidly developing into a tool procedures, details of the 8 loci amplified (Omy77, Oki200, of choice to survey genetic variation among salmonid Ots1, Ots3, Ots100, Ots103, Ots108 and Ssa85), and details populations. Nonlethal sampling and the abundance of polymerase chain reaction (PCR) were outlined in of loci make this technology very effective for describing Beacham et al. (1999). population structure in Pacific salmon (Beacham et Fishery samples were collected in both the Nass and al., 1998; Seeb et al., 1998; Small et al., 1998) and steel- Skeena Rivers. In the lower Nass River, fish wheels were head (Nielsen et al., 1994, 1997; Wenburg et al., 1996; used to conduct the 1998 test fishery, with general details of Ostberg and Thorgaard, 1999). Microsatellite DNA loci the test fishery outlined by Link and Gurak (1997). All can also be very useful in estimating stock compo- samples analyzed were derived from 2 fish wheels. In the sition in mixed-stock salmon fisheries (Beacham and lower Skeena River, samples were collected from steelhead Wood, 1999) and have been previously applied to steelhead caught in the Tyee gillnet test fishery in 1996 and 1998 near caught incidentally in commercial salmon fisheries the mouth of the river, with details of the test fishery out- (Beacham et al., 1999). We evaluated microsatellite varia- lined by Jantz et al. (1990). All samples collected in 1996 tion among steelhead populations in the Nass River and were analyzed, whereas in 1998 samples were generally ana- Skeena River drainages to address stock identification lyzed in proportion to run abundance, with approximately questions. 50% of the fish captured in the test fishery surveyed for The objectives of the present study were to determine microsatellite variation. population structure of steelhead in the Nass and Skeena Rivers using microsatellite variation and evaluate potential Gel Electrophoresis and Band Analysis applications for stock identification within each watershed. We also evaluated whether microsatellite variation can pro- PCR products were size fractionated on 16-cm by 17-cm vide accurate and precise estimates of stock composition for nondenaturing polyacrylamide gels visualized by staining steelhead from the two watersheds when they occur to- with 0.5 mg/ml ethidium bromide in water and illuminat- gether in marine mixed-stock fisheries. We then estimated ing with ultraviolet light. Nelson et al. (1998) and Beacham stock composition of steelhead caught in test fisheries in the et al. (1999) provided a more complete description of gel lower portions of the two rivers. electrophoretic conditions and estimation of allele size, as Steelhead Population Structure and Stock ID 589 Table 1. Steelhead Samples Collected and Analyzed from 7 Skeena River and 10 Nass River Populations in Northern British Columbia, and From a Test Fishery Near the Mouth of Each River* Population Years sampled N* Total N Skeena River summer run 1. Sustut River 1994, 1996, 1997, 1998 27, 50, 84, 50 211 2. Babine River 1991, 1992, 1995, 1996, 1997, 1998 19, 18, 39, 31, 29, 128 264 3. Bulkley River 1995, 1996, 1997 7, 36, 20 63 4. Morice River 1991, 1992, 1995, 1998 20, 30, 15, 46 111 5. Toboggan Creek 1998 128 128 6. Kispiox River 1992, 1995, 1998 20, 30, 35 85 7. Zymoetz River 1993, 1995, 1997 16, 18, 38 72 8. Test fishery 1996, 1998 190, 926 1116 Nass River summer run 9. Bell-Irving River 1997, 1998 96, 92 188 10. Damdochax Creek 1994, 1995, 1996, 1997, 1998 27, 18, 36, 20, 47 148 11. Meziadin River 1996, 1997, 1998 9, 5, 40 55 12. Kwinageese River 1995, 1997, 1998 11, 8, 92 111 13. Cranberry River 1995, 1996, 1997 19, 29, 147 195 14. Test fishery 1998 436 436 Nass River winter run 15. Chambers Creek 1997, 1998 24, 24 48 16. Ishkheenickh River 1997, 1998 14, 19 33 17. Kwinamass River 1997, 1998 17, 4 21 18. Tseax River 1998 16 16 19. Kincolith River 1996, 1997, 1998 16, 15, 10 41 *Sample sizes are for years sampled. well as a figure of a gel. Precision of estimated allele size, GENEPOP. The dememorization number was set at 1000, evaluated with the standard fish analyzed for each locus, and 50 batches were run for each test with 1000 iterations was similar to that outlined by Beacham et al. (1999). per batch. Critical significance levels for simultaneous tests were evaluated with the sequential Bonferroni adjustment Data Analysis (Rice, 1989). A neighbor-joining analysis illustrating genetic relationships among populations was conducted with Each population at each locus was tested for departure from PHYLIP (Felsenstein, 1993). The allele frequency matrix Hardy-Weinberg equilibrium (HWE) using GENEPOP was resampled 1000 times, and Cavalli-Sforza and Edwards Version 3.1 (Raymond and Rousset, 1995), as was temporal (1967) chord distance was used to estimate distance among stability of allele frequencies.