242 Abstract—Population structure of Population structure of pink salmon pink salmon (Oncorhynchus gorbus- cha) from British Columbia and Wash- (Oncorhynchus gorbuscha) in British Columbia ington was examined with a survey of microsatellite variation to describe and Washington, determined with microsatellites the distribution of genetic variation. Variation at 16 microsatellite loci was Terry D. Beacham (contact author)1 surveyed for approximately 46,500 1 pink salmon sampled from 146 loca- Brenda McIntosh tions in the odd-year broodline and Cathy MacConnachie1 from 116 locations in the even-year 2 broodline. An index of genetic differ- Brian Spilsted 3 entiation, FST, over all populations Bruce A. White and loci in the odd-year broodline was E-mail address for contact author: [email protected] 0.005, with individual locus values 1 ranging from 0.002 to 0.025. Popula- Fisheries and Oceans Canada tion differentiation was less in the Pacific Biological Station 3190 Hammond Bay Road even-year broodline, with a FST value of 0.002 over all loci, and with individ- Nanaimo, B. C., Canada V9T 6N7 ual locus values ranging from 0.001 2 Fisheries and Oceans Canada to 0.005. Greater genetic diversity 417-2nd Avenue West was observed in the odd-year brood- Prince Rupert, B. C. Canada V8J 1G8 line. Differentiation in pink salmon 3 Pacific Salmon Commission allele frequencies between broodlines 600-1155 Robson Street was approximately 5.5 times greater Vancouver, B. C., Canada V6E 1B5 than regional differentiation within broodlines. A regional structuring of populations was the general pat- tern observed, and a greater regional structure in the odd-year broodline than in the even-year broodline. The geographic distribution of microsatel- Pink salmon (Oncorhynchus gor- Estimation of genetic population lite variation in populations of pink buscha) spawn in more than 700 structure has been a key area of salmon likely reflects a distribution rivers in British Columbia (Aro and research in salmon assessment and of broodlines from separate refuges Shepard, 1967), and the distribution management. Identification of a ge- after the last glaciation period. of spawning populations varies in odd- netically distinct group of populations numbered years and even-numbered in the distribution of a species is a years. Spawning occurs primarily key step in conserving and maintain- only in odd years in the Fraser River ing genetic diversity. Genetically dis- drainage, mainly in even years on tinct populations or regional groups of the Queen Charlotte Islands, and in populations (stocks) were determined both years in central and northern through surveys of genetic variation coastal areas of British Columbia to evaluate the population structure (Neave, 1952). Because virtually all of a species (Shaklee and Bentzen, pink salmon mature at two years of 1998). Identification of genetically age (Bilton and Ricker, 1965), two dis- distinct groups in the distribution of tinct broodlines in the species have pink salmon in British Columbia and developed (odd-year and even-year), Puget Sound may lead to the conser- with virtually no gene flow between vation of genetic diversity by fisheries the broodlines. This reproductive iso- and resource management. An evalu- lation of the broodlines has resulted ation of genetic variation is effective in reported differences in body size in describing the population struc- (Godfrey, 1959), morphological char- ture of salmonids, is a key part in the acteristics (Ricker, 1972; Beacham, elucidation of management units or 1985), timing of spawning (Aro and conservation units for a species and Shepard, 1967; Dyagilev and Markev- can be applied to manage fisheries Manuscript submitted 3 October 2011. Manuscript accepted 5 January 2012. ich, 1979), and genetic differentiation exploiting specific stocks of salmon. Fish. Bull. 110:242–256 (2012) (Aspinwall, 1974; Beacham et al., Determination of genetic population 1988). Evaluation of the genetic pop- structure is an important part in de- The views and opinions expressed ulation structure both between and veloping a genetically based method or implied in this article are those of the author (or authors) and do not necessarily within broodlines in British Columbia for estimation of stock composition. reflect the position of the National Marine and Washington form the basis of the Allozymes were initially the key set Fisheries Service, NOAA. current study. of genetic markers used in evaluat- Beacham et al.: Population structure of Oncorhynchus gorbuscha in British Columbia and Washington, determined with microsatellites 243 ing population structure in pink salmon in British Co- lumbia (Beacham et al., 1988; Shaklee et al., 1991) Table 1 and elsewhere in the Pacific Rim distribution of the Microsatellite loci surveyed in pink salmon (Oncorhyn- species (Aspinwall, 1974; Varnavskaya and Beacham, chus gorbuscha) and their associated annealing and 1992; Shaklee and Varnavskaya, 1994; Noll et al., 2001; extension temperatures and times (seconds), as well as Hawkins et al., 2002). Analysis of allozyme variation the number of cycles used in polymerase chain reaction provided the general pattern of marked differentiation amplifications. between the broodlines, but identification of fine-scale regional structure was limited, although estimation of Locus Annealing Extension Cycles stock composition of samples from mixed-stock fisheries was conducted (Beacham et al., 1985; Shaklee et al., Oki10 53°C/30s 70°C/30s 39 1991). Development of DNA markers has led to new Oki101 53°C/45s 68°C/30s 40 avenues of research for using genetic variation in de- One101 50°C/30s 70°C/30s 39 fining population structure. Mitochondrial DNA varia- One102 50°C/30s 70°C/30s 39 tion again showed strong differentiation between the One104 50°C/30s 70°C/30s 36 broodlines, but differentiation among regional groups One109 55°C/30s 70°C/30s 34 of pink salmon was limited (Brykov et al., 1996, 1999; One111 55°C/30s 70°C/30s 34 Churikov and Gharrett, 2002). However, Golovanov et One114 50°C/30s 70°C/45s 38 al. (2009) noted that differentiation among even-year Ots213 52°C/45s 72°C/60s 38 populations was higher than among odd-year popula- Ots7e 51°C/30s 72°C/30s 35 tions in the northern Sea of Okhotsk region. OtsG253b 60°C/45s 72°C/45s 35 Microsatellites are reported to be useful for evaluat- OtsG311 50°C/45s 68°C/45s 34 ing fine-scale population structure in salmonids (Banks OtsG68 50°C/30s 70°C/30s 36 et al., 2000) and have been used to evaluate large- Ssa407 60°C/30s 70°C/30s 39 scale and regional variation in chum salmon (O. keta) Ssa408 60°C/45s 70°C/45s 40 (Beacham et al., 2009). Initial applications of micro- Ssa419 50°C/30s 70°C/30s 40 satellite variation to evaluate individual identification and population structure were reported by Olsen et al. (1998, 2000a). However, surveys of population variation by Small et al. (1998), a Qiagen 96-well DNeasy®1 pro- were quite limited in these studies, and no comprehen- cedure (Qiagen, Mississauga, Ontario), or a Promega sive evaluation of variation at microsatellites has been Wizard SV96 Genomic DNA Purification system (Pro- conducted for pink salmon. A survey of microsatellite mega, Madison, WI). Once extracted DNA was avail- variation over a broader geographic range of pink salm- able, surveys of variation at 16 microsatellite loci were on distribution would likely be valuable for evaluating conducted: Oki10 (Smith et al., 1998), Oki101 (Beacham population structure. et al., 2011), OtsG68, OtsG253b, OtsG311 (Williamson In the current study, we outline the microsatellite- et al., 2002), Ots213 (Greig et al., 2003), Ots7e (Wright based population structure of pink salmon in British et al., 2008), One101, One102, One104, One109, One111, Columbia and Washington as an initial step in evalu- One114 (Olsen et al., 2000b), Ssa407, Ssa408, Ssa419 ating whether higher resolution in estimation of stock (Cairney et al., 2000) composition may be possible when compared with esti- In general, polymerase chain reaction (PCR) DNA mates previously derived with allozymes. This objec- amplifications were conducted by using a DNA En- tive was accomplished by analyzing variation at 16 gine Cycler Tetrad2 (BioRad, Hercules, CA) in 6-µL microsatellite loci to evaluate relationships in popula- volumes consisting of 0.15 units of Taq polymerase, tion structure of pink salmon, as well as by analyzing 1-µL of extracted DNA, 1×PCR buffer (Qiagen), 60 µM regional differences in allelic variation. The distribu- each nucleotide, 0.40 µM of each primer, and deionized tion of genetic diversity among broodlines, regions, and water. The thermal cycling profile involved one cycle populations was estimated in the study, as well as the of Taq activation for 15 minutes at 95°C, followed by a stability of population structure. denaturation cycle of 30 seconds at 94°C, with anneal- ing and extension conditions for each locus as outlined in Table 1. PCR fragments were initially size fraction- Materials and methods ated in denaturing polyacrylamide gels with an ABI 377 automated DNA sequencer, and genotypes were scored Collection of DNA samples and laboratory analysis by Genotyper, vers. 2.5 software (Applied Biosystems, Foster City, CA) by using an internal lane sizing stan- Tissue samples were collected from mature pink salmon. dard. Later in the study, microsatellites were size frac- Samples were preserved in 95% ethanol, and sent to the tionated in an ABI 3730 capillary DNA sequencer, and Molecular Genetics Laboratory