Freshwater ecosystems are negatively impacted by a variety of anthropogenic stressors, with concomitant elevated rates of population decline for freshwater aquatic vertebrates. Because reductions in population size and extent can negatively impact genetic diversity and gene flow, which are vital for sustained local adaptation, it is important to measure these characteristics in threatened species that may yet be rescued from extinction. Across its native range, Bull Trout (Salvelinus confluentus) extent and abundance are in decline due to historic overharvest, invasive nonnative species, and habitat loss. In Alberta’s Eastern Slope region, populations at the range margin have progressively been lost, motivating us to better understand the amount and distribution of genetic variation in headwater habitats and some downstream sites where they continue to persist. Across this region, we sampled 431 Bull Trout from 20 sites in the Athabasca and Saskatchewan River basins and assayed 10 microsatellite loci to characterize within- and among-population genetic variation. The Saskatchewan and Athabasca River basins contained similar levels of heterozygosity but were differentiated from one another. Within the Athabasca River basin, five genetically differentiated clusters were found. Despite the evidence for genetic differentiation, we did not observe significant isolation-by-distance patterns among these sites. Our findings of ample genetic diversity and no evidence for hybridization with non-native Brook Trout in headwater habitats provide motivation to ameliorate downstream habitats and remove anthropogenic barriers to connectivity towards the goal of long-term persistence of this species.

Sample collection: Adipose fin tissue samples were non-lethally obtained by clipping and immediately transferred into 95% EtOH at 4°C. Samples obtained from W. Hughson, S. Herman, M. Sullivan, and W. Warnock were collected using similar methods, although the latter three collectors preserved the sample by drying the adipose fin tissue and storing them in separate envelopes in a cool, dry location. All samples were collected within eight years; a time span comparable to a single Bull Trout generation within the region.

Processing: Nuclear DNA was extracted from EtOH-preserved adipose fin tissue using a standard proteinase-K phenol-chloroform protocol and stored in ddH2O at 4°C. DNA quantity was determined using a Nanodrop 1000 Spectrophotometer V3.7and sample concentrations were subsequently adjusted to 50 ng/μl. During PCR, DNA regions, excised by forward fluorescently labeled oligonucleotide primers for 10 microsatellite loci (Omm1128, Sco102, Sco105, Sco106, Sco109, Sco215, Sco216, Sco220, Sfo18, and Smm22), were multiplexed in four optimized groups. These loci were chosen based on the degree of polymorphism, ability to detect Bull Trout × Brook Trout hybrids, and resolution to detect population structure of the samples. Desired fragments were amplified using a C1000 Touch Thermal Cycler. PCR products were run on a 1.5% w/v agarose gel using a standard gel electrophoresis protocol and visualized using DigiDOC IT electrophoresis gel imager. Samples showing clearly defined bands representing the DNA fragment PCR products of the specified microsatellite loci were then analyzed. Fragment lengths were determined using a standard protocol for microsatellite fragment analysis with an ABI 3500XL Capillary Electrophoresis Genetic Analyzer and scaled against GeneScan-500LIZ size standard. For each sample, electropherograms were produced, which were scored using GENEMAPPER v.4.1. Raw genotype scores were assessed by MICROCHECKER v.2.2.3 to uncover genotyping errors and presence of null alleles that did not amplify during PCR. Because null alleles were present in no more than four of the 10 loci, all samples were used for further analyses.

Spreadsheet: The first sheet of the spreadsheet contains notes on (1) the formatting of the entries and (2) how we coded missing values.

Table 1: Microsatellites loci targeted to determine levels of genetic differentiation and population structure of Bull Trout in the Athabasca River basin.

Table 2: Expected heterozygosity, allelic richness and total private alleles at each sampling site.