We used genetic techniques to evaluate a series of natural waterfalls for their potential to serve as barriers to prevent nonnative salmonids from entering a proposed reintroduction area for federally threatened Greenback cutthroat trout Oncorhynchus clarkii stomias. Genetic samples were collected from nonnative Brook Trout Salvelinus fontinalis at 11 sampling reaches above and below natural waterfalls (height: ~1-3 m under baseflow conditions) along a 33-km segment of Colorado’s upper Cache la Poudre River near the outflow of the proposed reintroduction area. To evaluate whether upstream movement of Brook Trout is restricted by any of these waterfalls, we characterized longitudinal trends in genetic diversity along the river corridor and examined patterns of genetic differentiation and population structure in relation to waterfall locations using a panel of microsatellites. We found no evidence that the waterfalls served as complete movement barriers for nonnative Brook Trout based on genetic clustering analyses, estimates of population differentiation, and longitudinal genetic patterns. Our multi-locus assessment did not identify alleles restricted to downstream reaches, and the river segment was genetically homogenized. This evaluation suggests that the existing waterfalls do not fully prevent upstream movement by nonnative Brook Trout, and thus barrier modification would be needed to establish an isolated Greenback cutthroat trout population in the proposed wilderness area.

Brook Trout tissue samples (fin clips) were collected from 11 sampling sites separated by waterfalls along a 33km segment of Colorado's Cache la Poudre River. 269 individual Brook Trout were genotyped at 12 previously described microsatellite loci: SfoC113, SfoC115, SfoC129, SfoC38, SfoC88, SfoD91, SfoB52, SfoC24, SfoC28, SfoC79, SfoC86, and SfoD75 (King et al. 2012). Genomic DNA was extracted using Qiagen Dneasy Blood and Tissue Kits following the manufacturer’s protocol. Microsatellite markers were amplified using polymerase chain reaction (PCR) in two 10 µl multiplexes, each containing 2 µl genomic DNA, 5 µl Qiagen Multiplex PCR Mastermix, 0.04- 0.1 µl of each forward and reverse PCR primer (Table S4), and 2.18-2.2 µl nuclease free water. The thermal profile for PCR amplification consisted of denaturing at 95°C for 15 minutes, 35 cycles of denaturation at 95°C for 45 seconds, annealing at 56°C for 45 seconds, and extension at 72°C for 2 minutes, followed by a final extension of 60°C for 30 minutes. PCR products were treated with a solution of formamide and GeneScan 600 LIZ size standard and visualized using an Applied Biosystems 3500 genetic analyzer. Alleles were scored using GeneMapper version 6.

King, T. L., B. A. Lubinski, M. K. Burnham-Curtis, W. Stott, and R. P. Morgan. 2012. Tools for the management and conservation of genetic diversity in Brook Trout (Salvelinus fontinalis): tri- and tetranucleotide microsatellite markers for the assessment of genetic diversity, phylogeography, and historical demographics. Conservation Genetics Resources, 4:539–543.