Brief Description of iVfethods: 



Polymerase chain reaction (PCR) amplification of paired interspersed nuclear DNA elements (PlNEs) was 

 used to determine each fish's genetic characteristics at multiple regions of the nuclear DNA. This method 

 produces DNA fragments that can be used to distinguish between various cutthroat trout subspecies 

 (Oncorhynchus clarki spp.), rainbow trout {O. mykiss) and their hybrids, and between bull trout (Salvelinus 

 conjluentiis). brook trout {S. fontinalis), and their hybrids. The presence of a PfNE marker is dommant to 

 absence. First-generation (Fi) hybrids will have all the diagnostic markers characteristic of the two 

 hybridizing species. Backcrossed (F2+) individuals will possess some, but not all, markers characteristic of 

 both parental species. The appearance of a marker indicates the individual is either heterozygous or 

 homozygous for that marker, which precludes us from directly calculating allele frequencies. However, in 

 order to provide comparative values, we have assumed the samples conform to Hardy- Weinberg 

 expectations in order to estimate the average genetic contribution from each species. 



It is critical to note that in all hybrid swarms, regardless of the percent contribution fi-om the non-native 

 species, all individuals are of hybrid origin, even those that appear "pure" at our diagnostic loci. It is not 

 possible to "rescue" pure individuals from these populations as they likely do not exist. Due to the random 

 reshuffling of alleles during sexual reproduction, many individuals will appear pure for one or the other 

 parental species due to the limited number of marker loci used. It has been shown that 6 markers are 

 adequate to provide coarse classification of hybridization, but upwards of 70 markers are required to 

 discriminate be^veen pure individuals, if they exist, and backcrossed individuals in hybrid swarms (Boecklen 

 and Howard 1997). 



Literature Cited: 



Boecklen \VJ, and Howard DJ (1997) Genetic analysis of hybrid zones: numbers of markers and power of 



resolution. Ecology 78 (8) pp. 261 1-2616 



Sample Details: 



Chimney Creek: A total of nine of eleven samples amplified fi"om this sample. All nine individuals displayed only 

 PINE fragments diagnostic of westslope cutthroat trout. With a sample size of nine individuals, we have a 66% chance 

 of detecting as little as 1% hybridization between westslope cutthroat and rainbow trout. A larger sample size is 

 necessary for a more accurate analysis. 



Twelveniile Creek: A total of seventeen of thirty samples amplified at all three primer pairs. .\11 individuals displayed 

 PINE fragments diagnostic of westslope cutthroat trout. Five individuals also displayed PINE fragments diagnostic of 

 rainbow trout. The majority of individuals were re-extracted without success. With a sample size of seventeen 

 individuals, we have an 87% chance of detecting as little as 1% hybridization between westslope and rainbow trout. 

 The successfully amplified individuals that displayed PINE fragments diagnostic of rainbow trout and their 

 corresponding site nunibcr follows: Samplef^(Site#) 2(4), 3(4), 4(4), 9(5), 23(7) 



Skalkaho Creek: All twenty-five successfully amplified individuals in this sample displayed PINE fragments 

 diagnostic of westslope cutthroat trout. Two individuals also displayed PINE fragments diagnostic of rainbow trout. 

 With a .sample size of twenty-five individuals, we have a 95% chance of detecting as little as 1% hybridization 

 between westslope cutthroat and rainbow trout. 



