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  • Samples for genetic analysis were small (approximately 2–3 mm) fin clips taken during hook and line or longline fishing operations. Samples were collected during 2005 by the Washington Department of Fish and Wildlife in Willapa Bay, Washington (WA, n = 44), from 2003–2007 by the Monterey Bay Aquarium (SF-1, n = 36), and from 2007–2012 by the Aquarium of the Bay within San Francisco Bay, California (SF-2, n = 53) for a total of 133 tissue samples. The size range for sharks sampled in San Francisco were a mixture of adults and subadults: males averaged 148 cm with 66% mature (mature size range for males is between 150 and 180 cm) and females averaged 133 cm with only 15% mature (mature size range for females is between 192 and 208 cm). The sharks sampled in Washington were also a mixture of adults and subadults and in general were larger than those sampled in San Francisco: males averaged 206 cm with 93% mature and females averaged 195.25 cm with 38% mature. Tissue samples from sharks were preserved in 70–100% ethanol and/or frozen at -20°C or -55°C until analysis. DNA was extracted from tissue using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, California). Conditions for polymerase chain reaction (PCR) were optimized for amplification of the seven microsatellite loci (SG13, SG24, SG25, SG27, SG28, SG30 and SG31) [11]. Each microsatellite locus was amplified separately using a GeneAmp PCR 9600 thermal-cycler (Perkin Elmer, Wellesley, Massachusetts) in a total volume of 10μl containing 1μl of 100–250 ng/μl purified DNA template, 0.5 μM/μl forward and reverse primer, 4μl PCR Mastermix 2X (Taq polymerase with manufacturer’s supplied buffer, dNTPs and MgCl2, Promega, Madison, Wisconsin), and 4μl DNA/RNA free dH2O. The amplification profile of each primer follows that described by Larson et al., 2011 [11]. PCR products were stored at 4°C or -20°C until analysis using an Applied BioSystems (ABI, Foster City, California) 3100 sixteen-capillary system in Genescan mode. Each run contained at least one reference sample as well as several repeated samples to ensure accuracy. There was no multiplexing of loci. Allele scoring was performed using Genescan Software version 3.0 (Applied Biosystems) or Peakscanner Software 1.0 (Applied Biosystems). Tests for departures from Hardy-Weinberg equilibrium were performed using the Hardy-Weinberg probability function with default Markov chain parameters in Option One of GENEPOP 3.1 software [12]. Sequential Bonferroni adjustments over all loci were used to determine significance levels for all simultaneous tests, resulting in a final significant p value of ≤ 0.007 [13]. MICROCHECKER software [14] was used to determine genotyping errors such as allelic dropout, stuttering and null alleles. Genetic diversity estimates, observed and expected heterozygosity (HO and HE), allele diversity, and population structure analyses such as Principal Coordinates Analysis (PCoA) and population assignment cluster analysis were measured using GenAlEx 6.5 [15]. Relatedness within populations was examined using three programs: MLRELATE [16], COLONY [17] and COANCESTRY [18]. MLRELATE calculates the maximum likelihood estimates of relatedness and relationships from microsatellites. The program COLONY uses a maximum likelihood method to assign siblings and parentage using individual genotypes at all markers. It estimates a full- and half-sibling relationship, assigns parentage, and evaluates reproductive skew, or the estimated percentage of each potential parent’s contribution to offspring genotypes. For this analysis both male and female sevengill sharks were assumed to use a polygamous mating system. The program COANCESTRY employs seven relatedness estimators. We choose to use the triadic likelihood estimator (denoted as TrioML), as this likelihood method uses the genotypes of a triad of individuals in estimating pairwise relatedness reducing the chance of genes identical in state (IIS) being mistakenly inferred as identical by descent (IBD). The method allows for inbreeding and accounts for genotype errors in data such as null alleles. Finally BOTTLENECK software [19] was used to determine if the populations had experienced a recent significant population bottleneck that may now be affecting diversity. Specifically the program tests for heterozygosity excess under Hardy-Weinberg equilibrium since allelic diversity is expected to decrease faster than observed heterozygosity. We chose to use the two-phase model (TPM) with 70% and 90% single-step mutations combined with a variance of 30 and the one-tailed Wilcoxon sign rank test to determine statistical significance.
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