Report prepared for the Freshwater Fisheries Society of British Columbia. Contributing authors:

Transcription

1 Initial microsatellite analysis of kokanee (Oncorhynchus nerka) population structure in the Kootenai/y River Basin, Idaho, Montana, and British Columbia Report prepared for the Freshwater Fisheries Society of British Columbia Contributing authors: Paul Anders Cramer Fish Sciences, University of Idaho 121 Sweet Avenue, Moscow, Idaho Joyce Faler, Matt Powell University of Idaho, Hagerman Fish Culture Experiment Station National Fish Hatchery Road, Hagerman, Idaho Harvey Andrusak Redfish Consulting Ltd Hwy 3A Nelson, British Columbia, Canada BC V1L6N6 Charlie Holderman Kootenai Tribe of Idaho, P.O. Box 1269, Mission Road Bonners Ferry, Idaho June 2007 CORPORATE OFFICE OAKDALE OFFICE AUBURN OFFICE IDAHO OFFICE 600 NW Fariss Road 636 Hedburg Way, # High Street, #2 121 W. Sweet Ave., #118 Gresham, Oregon Oakdale, CA Auburn, CA Moscow, ID (503) (209) (530) (208)

2 Executive Summary Seven tetranucleotide microsatellite loci were used to define and assess kokanee (Oncorhynchus nerka) populations and their potential stock structure in the Kootenai River Basin in Idaho and Montana, and in the Kootenay Basin (Koocanusa Reservoir) in Montana and British Columbia. A total of 277 samples were analyzed: 60 from Koocanusa Reservoir tributaries in British Columbia, 30 from Kootenai River tributaries in Idaho, and 187 from Kootenay Lake or Kootenay River tributaries in British Columbia. Samples were primarily collected from kokanee spawning tributaries. DNA was extracted from individual fish fin samples using Qiagen kits and extraction methods. PCR products were genotyped on Applied Biosystems Model 3100 and Model 3730 genetic analyzers. Alleles were scored with Genescan, Genotyper, and Genemapper software from Applied Biosystems. Representative PCR products previously scored on the 3100 were rerun on the 3730 to harmonize data sets between the two genetic analyzers. The number and frequency of alleles per locus and per sample collection, and estimates of heterozygosity were calculated using the Excel Microsatellite Tools program. Allelic richness (average number of alleles per locus corrected for sample size) was determined using the FSTAT program. The Genpop program was used to test for heterozygote deficiency and linkage disequilibrium, to estimate pairwise genetic variance between and among sample collections sites and geographic regions, and to test for departure from to Hardy-Weinberg equilibrium for each of the seven loci. The program STRUCTURE was used to determine the number of distinct clusters (or populations) in the Kootenai River Basin. STRUCTURE analyses of all 277 samples combined indicated two distinct clusters (populations) in the Kootenai River Basin. Using inferred membership assignments of 60% or greater to assign an individual to a particular cluster, Koocanusa Reservoir individuals assigned primarily to Cluster 1 (87%), and Kootenay Lake individuals assigned primarily to Cluster 2 (56%). Nearly half (47%) of the Kootenai River individuals assigned to Cluster 1, whereas about 23% of those individuals were classified as in between the two clusters (inferred membership assignment < 60% to either cluster). Distinct genetic structuring was also revealed among the three stocks in the Kootenay Lake region, consistent with past stock structure assignments based on morphometric and spatial and temporal isolation among these stocks (Vernon 1957). Thus, caution should be used in any thought of stocking North Arm Meadow Creek kokanee into West Arm tributaries. Alternatively, the perceived risk of stocking Meadow Creek fish into South Arm tributaries in Idaho is considerably less because the historic South Arm stock appears to be functionally extinct (thus nullifying introgression concerns), and because previously approved introductions of eyed eggs from Meadow Creek have already occurred during the past decade. Except for Long Canyon Creek, the Idaho sample region lacked adequate sample numbers required for statistical comparisons among tributaries within the region or to individual tributaries in other regions. Regional comparisons revealed that the Kootenai River may be a transitional area between the two lakes (Koocanusa Reservoir and Kootenay Lake) in terms of the percent of samples assigned to each kokanee population. Long Canyon Creek appears to be genetically distinct from the Koocanusa Reservoir i

3 population, and to date from two of the Kootenay Lake collections. However, this conclusion regarding genetic intermediacy is tentative and may change with increased sample collections from Long Canyon Creek and other Idaho tributaries. More samples from Idaho could confirm intermediacy (a hypothetical Cluster 3), or could characterize a population as more similar to Cluster 1 or Cluster 2. Acknowledgements This work was funded by the Freshwater Fisheries Society of British Columbia (FFSBC) and was made possible by the efforts of Bryan Ludwig (FFSBC) and Ken Ashley, Ted Down, and Jeff Burrows (B.C. Ministry of Environment). The authors also acknowledge the Kootenai Tribe of Idaho, the Idaho Department of Fish and Game, Montana Fish Wildlife and Parks, the British Columbia Ministry of Environment, and their various subcontractors for collecting and providing kokanee samples for genetic analysis. All lab work and data analyses were conducted at the University of Idaho s Fish Genetics Lab in Hagerman, Idaho. ii

4 Table of Contents Table of Contents...iii List of Tables... iv I. Introduction... 1 II. Goals and Objectives... 4 III. Methods... 5 IV. Results... 8 V. Discussion VI. Summary VII. Recommendations VIII. References Appendix 1: Origins of Kokanee in Lake Koocanusa, MT Appendix 2: Brief history of Kootenay Lake kokanee stocks (Provided by Harvey Andrusak) iii

5 List of Tables Table 1. Sample size (n) and site description for each collection of kokanee by region.. 5 Table 2. Percent of samples by collection and region assigned to a cluster by estimated membership frequency of 60% or greater. Samples assigned at less than 60% inferred membership to either cluster were recorded to the "in between" category Table 3. Number of samples (n), and number of alleles per locus and across all loci by region and collection site Table 4. Sample size (n), number of alleles (NA), mean number of alleles per locus (MNA), expected heterozygosity (He), observed heterozygosity (Ho), average allelic richness (AR), number of unique alleles, and number of missing alleles by collection Table 5. Identity (bin name) of unique alleles by locus and collection, and number of unique alleles summed across loci and collections Table 6. Identity (bin name) and frequency of alleles specific* to a region, the number and percentage of collections within the region exhibiting the allele, and the total number of specific alleles by locus and region Table 7. Identify (bin name) and total number of missing* alleles by locus and collection Table 8. Individual P-values for Hardy-Weinberg exact tests of equilibrium by locus and collection site, region and Basin, across all loci, and across all collections and regions. 14 Table 9. Individual P-values for Hardy-Weinberg exact tests of heterozygote deficiency by locus and collection site, region and Basin, across all loci, and across all collections and regions Table 10. Individual P-values* for pairwise tests of loci by collection and across all collections for genotypic disequilibrium Table 11. Sample size (n) and pairwise genetic differentiation index (F ST ) of 14 collections of kokanee from the Kootenai/y Basin. Values in bold italics on the diagonal are the average of 13 pairwise FST values for each collection Table 12. Pairwise genetic differentiation index (Fst) of kokanee collected from three regions of the Kootenai River Basin. Values in bold italics on the diagonal are the average of two pairwise Fst values for each location iv

6 List of Figures Figure 1. Map of the Kootenai River Basin in British Columbia, Montana, and Idaho Figure 2. General kokanee sampling areas in British Columbia upstream from Koocanusa Reservoir in Montana, Idaho, and Kootenay Lake (BC tributaries). See Table 1 for more details...6 Figure 3. Percent of individuals in a region assigned to a cluster from a STRUCTURE run for two populations (k) using burn-in of 500,000 repetitions followed by 5,000,000 repetitions... 9 Figure 4. Percent of individuals in a collection (sample size n > 12) assigned to a cluster from a STRUCTURE run for two populations (k) using burn-in of 500,000 repetitions followed by 5,000,000 repetitions v

7 I. Introduction Kokanee (Oncorhynchus nerka) in the Kootenai River Basin in Idaho, Montana, and British Columbia (spelled Kootenay in Canada) are currently represented by an admixture of native and introduced stocks. The Kootenai/y River Basin (Basin) is located between 48 and 51 North latitude and 115 and 118 West longitude, and includes parts of southeastern British Columbia, northern Idaho, and northwestern Montana (Figure 1). The Basin measures approximately 238 miles by 153 miles with an area of 16,180 square miles. Roughly two thirds of the river s 485-mile-long channel and nearly 70% of its watershed area are located within the province of British Columbia. The Montana portion of the Basin comprises about 23% of the watershed and the Idaho portion accounts for about 7% percent (Knudson 1994). Idaho. A native kokanee stock that historically spawned upstream in tributaries of the lower Kootenai River in Idaho after rearing to maturity in the South Arm of Kootenay Lake was reported as functionally extinct by the early 1990s (Ashley and Thompson 1994). Spawning runs (escapement estimates) from this stock numbered into the thousands of fish as recently as the early 1980s (Partridge 1983; Anders 1993; Ashley and Thompson 1993). Since 1996, visual observations and annual redd counts in five Idaho tributaries found no spawners returning to Trout, Smith, and Parker Creeks, while Long Canyon and Boundary Creeks had very few kokanee returns (Andrusak 2007). During 2006, total South Arm annual escapement to all streams in Idaho and British Columbia upstream from Kootenay Lake was < 1,000 spawners (Andrusak 2007). Andrusak et al. (2004) summarized the available historic escapement data available for this population and reported that total annual escapement even prior to hydrodevelopment impacts did not likely exceed 200,000 fish. Montana. Koocanusa Reservoir is the impoundment created by Libby Dam on the Kootenai River near Libby, Montana (lower right in Figure 1). The dam was completed in 1972 and the new reservoir reached full pool during Due to introduction via discharge of nearly 1.5 million presumed moribund fish from British Columbia s Kootenay Hatchery near Wardner between 1969 and 1978 into Norbury Creek (an upper Kootenay River tributary in BC), kokanee entered the Koocanusa Reservoir as early as 1973 (Appendix 1; Appendix Table 1). These fish represented stocks from southeast, south central, and southwestern British Columbia (Okanogan, Chilliwack and Meadow Creek stocks; Appendix Table 1). This non-native kokanee population rapidly expanded in the newly created Koocanusa Reservoir following initial observations of kokanee spawners in Norbury Creek. By 2002 a cumulative peak count of 450,000 spawners was made for eleven index tributary streams in the upper Kootenay River in BC (Westover 2003). Quantitative population abundance and escapement data are lacking for kokanee in Montana waters of the Kootenai Subbasin because Koocanusa Reservoir kokanee reproduce primarily upstream from the Montana-British Columbia border. 1

8 Figure 1. Map of the Kootenai River Basin in British Columbia, Montana, and Idaho. 2

9 Entrainment studies at Libby Dam revealed that approximately 98% of all entrained fish sampled in the dam s draft tubes were kokanee (primarily Age-0 fish), along with a few Age-1 fish (Dunnigan et al. 2003). Review of recapture data indicated that survival of these entrained fish may be as high as 70% (Brian Marotz, MFWP personal communication). After entraining, some fish appear to residualize in the Libby Dam tail waters areas where zooplankton provide suitable forage. Following entrainment, kokanee can remain in Montana waters upstream from Kootenai Falls to the dam, they can pass below the falls and remain in the canyon reach, or they can pass below the falls and migrate further downstream to rear and mature in Kootenay Lake. Entrained kokanee that survive to maturity upstream from Kootenai Falls converge as spawners in the Libby Dam tailrace which blocks further upstream migration (Libby Dam has no upstream fish passage facilities). Entrained kokanee that survive to maturity after rearing downstream from Kootenai Falls (presumably in Kootenay Lake), converge on the falls following upstream spawning migrations because the falls act as an upstream migration barrier for kokanee (Brian Marotz, MFWP, personal communication). During some years, kokanee snag fisheries in Montana produced harvests ranging from thousands to tens of thousands of fish, depending on production, entrainment, and on survival rates during post-entrainment rearing years. It is currently unclear how many entrained Koocanusa Reservoir kokanee migrate downstream to Kootenay Lake and survive to maturity. It is thought that considerable numbers may be accounted for in summer hydroacoustic surveys in the Kootenay Lake s South Arm (Andrusak 2007). However, the frequency and magnitude of Libby Dam s power peaking operations were greatly reduced since the early 1990s to address downstream ecosystem and fisheries needs downstream. This reduction in power peaking is temporally correlated with a considerable drop in Age-0 kokanee entrainment through Libby Dam (Brian Marotz, MFWP personal communication 2006). British Columbia. Three morphologically distinct stocks of kokanee in Kootenay Lake, British Columbia were originally described by Vernon (1957): a North Arm stock that spawned at age 4 (mean size 21.5 cm), a South Arm stock that spawned at age 3 (mean length 18.5 cm), and a West Arm stock that spawned at age 3 (mean length 24.5 cm). All three stocks were fall spawners. The South Arm stock spawned first (early August to mid-september) followed by West Arm (mid-august to mid-september) and North Arm fish (late August to mid-october). The North Arm Kootenay Lake kokanee population has been monitored for over 40 years (Sebastian et al. 2006). Escapement in this population has been estimated to be as high as 4.1 million (Bull 1964) and as low as 200,000 fish (Andrusak 2003). Population abundance of North Arm kokanee increased following nutrient enhancement that began in the North Arm in 1992 (Sebastian et al. 2006). However, escapement during the next few years may decrease due to increased kokanee predation by more abundant piscivorous fish populations in Kootenay Lake. Smaller numbers of kokanee currently spawn in several West Arm tributaries. Based on body size and spawn timing these fish may even be distinct from the upper West Arm population. Escapements to local streams in the lower West Arm have been periodically 3

10 monitored with only a few 100 to 1,000 spawners observed annually (Andrusak et al. 2004a). Following review of the limited historic South Arm stock escapement data, Andrusak et al. (2004b) reported that total annual South Arm kokanee escapement numbers even prior to hydro-development impacts likely did not exceed 200,000. In 2006 the total escapement to all streams was < 1,000 spawners (Andrusak 2007). For a more detailed account of Kootenay Lake kokanee dynamics, see Appendix 2 at the end of this report. Also see Andrusak (2007) for an informative summary of South Arm Kootenay Lake kokanee trends. A main purpose of this study was to provide a baseline for Basin kokanee population identity and stock structure that represent conditions prior to experimental fertilization of Kootenay Lake s South Arm (began during summer 2004) and nutrient restoration in the Kootenai River in Idaho (began during July 2005). Future genetic analysis using samples collected annually in a standardized way could also be used to monitor and assess temporal changes in kokanee population parameters and stock-recruitment dynamics in response to the broader suite of ongoing and future kokanee management activities in Idaho, Montana, and British Columbia. All results presented in this report involved fish collected from or near spawning areas, whereas future work could also involve in-lake sampling during non-reproductive periods to determine the extent that upstream stocks rear and mature in Kootenay Lake. II. Goals and Objectives The goal of this study was to provide a better understanding of kokanee population and stock structure in the Kootenai/y Basin fisheries and ecosystem management and restoration programs. Based on the above introductory information and the need to address a series of critical uncertainties about potential stock structure for Basin kokanee, this study had four objectives, to: 1) Define and delineate kokanee populations, 2) Assess potential stock structure within populations, 3) Determine the magnitude of contribution from kokanee entrainment at Libby Dam to spawner contributions in downstream areas, and 4) Assess future stock-specific response(s) to various kokanee and ecosystem management strategies and practices in Idaho, Montana, and British Columbia. These objectives were designed to help determine which Basin kokanee stocks may benefit from increased productivity following nutrient restoration in the South Arm and the Kootenai River, and whether future stock responses are consistent with state, tribal and provincial restoration plans and management strategies. 4

11 III. Methods Sample collections. Fin clips were taken from kokanee on spawning runs on or near from spawning grounds at 14 collection sites in three regions in the Kootenai River Basin (Figure 12). Three collections occurred in Koocanusa Reservoir tributaries in BC, six from Kootenai River, Idaho tributaries, and five from Kootenay Lake, BC tributaries (Table 1). Table 1. Sample size (n) and site description for each collection of kokanee by region. Region Collection Site n Description Location Kootenay Lake, BC Goat (Akokli) Creek 30 South Arm Tributary 2 Gray Creek 35 Eastern (central) Tributary 3 Kokanee Creek 32 West Arm Tributary 4 Meadow Creek 60 North Arm Tributary 5 Redfish Creek 30 West Arm Tributary Kootenai River, ID 30 6 Cow Creek 6 Braided reach, mainstem Kootenai River RKM Hemlock Bar 1 Canyon reach, mainstem Kootenai River RKM Long Canyon Creek 15 Meander reach, lower Kootenai River RKM N.F. Trout Creek 2 Meander reach, lower Kootenai River RKM S.F. Trout Creek 2 Meander reach, lower Kootenai River RKM Shorty's Island; KR4 4 Meander reach, mainstem Kootenai River RKM 230 Koocanusa Reservoir, BC North Point Elk North and West of Kikoman South of Bridge 13 Total: 277 5

12 L Kootenay Lake Nelson WA BC ID Creston MT BC Lake Koocanusa Bonners Ferry Libby Figure 2. General kokanee sampling areas (ovals) in British Columbia upstream from Koocanusa Reservoir in Montana, and downstream in Idaho and Kootenay Lake (BC tributaries). See Table 1 for more sampling site details. 6

13 Laboratory analysis. DNA was extracted from fin clips using a salt extraction method modified from (Hillis et al. 1996), or a DNeasy Tissue extraction kit from Qiagen. Samples were genotyped at seven tetranucleotide microsatellite loci using primers developed by Olsen et al. (2000): One103, One104, One108, One111, One112, One114, and One115. Amplification reactions consisted of 10ul reactions using 1X PCR buffer II (supplied with Taq polymerase), mm MgCl 2, U/ul Taq polymerase (Applied Biosystems), 0.5 ug/ul BSA, um forward fluorescently labeled primer, um reverse primer, 0.8 mm dntp s (0.2 mm each), all at final concentrations. One103, One111, One112 used 3.0 mm final concentration of MgCl 2 ; One104, One108, and One115 used 3.5 mm; and One114 used 4.0 mm. Amplifications of One104 from Qiagen kit extractions used a 1.6 mm final concentration of MgCl 2. Polymerase chain reactions (PCR) were performed on Perkin-Elmer (GeneAmp PCR System 9700) and MJ Research (PTC-100) thermal cyclers. Cycling conditions included initial denaturation of 2 min at 92 C, followed by 35 cycles of 30 s at 92 C, 30 s at 56 C (One103, One104, One108, One111, One114, and One115), 58 C (One112), or 61 C (One111 and One115), and 30 s at 72 C. A final extension at 72 C for 10 s (One108), 30 min (One103), or 45 min (One104, One111, One112, One114, and One115) was used. PCR products were genotyped on Applied Biosystems Model 3100 and Model 3730 genetic analyzers. Alleles were scored with Genescan, Genotyper, and Genemapper software from Applied Biosystems. Representative PCR products previously scored on the 3100 were rerun on the 3730 to harmonize data sets between the two genetic analyzers. Statistical analysis. Estimates of heterozygosity and the numbers and frequencies of alleles per locus and per sample collection were generated using the Excel Microsatellite Tools program. Unique alleles (those found only in one collection at a frequency of > 2% of the collection) and missing alleles (found in all but one of the collections of sample size n > 12) were determined from allele frequencies. Small sample collections (n<12) were not used to determine missing alleles. The minimum sample size of 12 for analysis allowed inclusion of the smallest sample group from the upstream BC samples (South of Bridge site, n=13; Table 1) and reduced undesirable effects of small sample bias in analysis. Allelic richness estimates (average number of alleles per locus corrected for sample size) were calculated using the FSTAT program (Goudet 2001). Tests for departures of loci and collections from Hardy-Weinberg equilibrium were performed using the GENEPOP program (Raymond and Rousset 1995) that involved a Marko chain Monte Carlo (MCMC) algorithm to estimate exact P-values (Guo and Thompson 1992). Tests for heterozygote deficiency in loci and collections (Rousset and Raymond 1995) and tests for linkage disequilibrium between all pairs of loci were performed using the MCMC method in GENEPOP. Significance levels for multiple tests were adjusted using sequential Bonferroni corrections (Rice 1989). Pairwise genetic variance of collections (F ST ) was calculated as a weighted analysis of variance (Weir and Cockerham 1984) using GENEPOP. To determine the significance of F ST, exact tests of genic and genotypic differentiation of all 14 collections were performed in GENEPOP using the MCMC method. Significance levels for multiple tests were adjusted using sequential Bonferroni corrections (Rice 1989) and the less stringent (Benjamini and Yekutieli 2001) false discovery rate correction method (B-Y) as discussed by Narum (2006). 7

14 The program STRUCTURE (Pritchard et al. 2000) was used to determine the number of distinct populations or clusters (k) in the Kootenai/y River Basin. The number of distinct populations was determined by averaging three iterations of k between 1 and 14 (only 2 iterations of k = 12 and 13, and one iteration of k = 14 due to memory constraints of computer used) at a burn-in of 500,000 iterations, followed by a run of 1,000,000 iterations and selecting the k value with the highest likelihood. Another final run at k = 2 using a burn-in of 500,000 iterations followed by 5,000,000 iterations was used to generate inferred cluster membership for individual samples. All 277 samples were included in STRUCTURE with no a priori population or sample location identity. IV. Results Population definition and stock structure. Two distinct kokanee populations and stock structure were revealed by this study both within and among geographic regions. Results of the STRUCTURE analyses of all 277 samples combined revealed that kokanee in the Kootenai/y Basin exist as two distinct but admixed clusters or populations, with a possible geographically and genetically intermediate admixed stock in Idaho represented by a small sample from a single collection location (Long Canyon Creek). Using inferred membership assignments of 60% or greater to assign an individual to the particular cluster, Koocanusa Reservoir individuals assigned primarily to Cluster 1 (87%), and Kootenay Lake individuals assigned primarily to Cluster 2 (56%) (Figure 33). Cluster 1 in turn was dominated by Meadow Creek stock. Kootenai River individuals assigned primarily to Cluster 1 (47%), with about 30% Cluster 2 fish; the remaining ~23% of individuals were classified as in between (inferred membership assignment < 60% to either Cluster 1 or Cluster 2). Recall that the Kootenai collections were limited in representation, so this group simply reflects Long Canyon Creek collections (n = 15). Greater sample representation is needed to better characterize kokanee population identity and stock structure in Idaho waters of the Kootenai Basin. Results from individual collections within the three geographic regions further reflected this trend illustrated in Figure 3. For example, Cluster 1 fish accounted for 81.8 to 92.0% of samples from the three upstream BC collection sites from Koocanusa Reservoir tributaries (Figure 4, Table 3). Cluster 1 was dominated by Meadow Creek fish, likely resulting from founding effects of Kootenay Hatchery releases or escapees, dominated during the last few years of kokanee operations by Meadow Creek stock (Figure; Table 1; Appendix 1). Cluster 2 dominated 4 of the 5 Kootenay Lake collection sites ( %) with the exception of Meadow Creek, in which over half (51.7%) the fish were assigned to Cluster 1 (i.e. Meadow Creek stock; Figure). Kokanee stocks from the three geographic regions were characterized by varying degrees of genetic admixture (Figure). Admixture was greatest in the Kootenai River (Long Canyon) collections (n=15) whereas Cluster 1 dominated the upstream BC region and Cluster 2 dominated the downstream Kootenay Lake region (Figure 4). 8

15 % of Samples Assigned to Figure 3. Percent of individuals in a region assigned to a cluster from a STRUCTURE run for two populations (k) using burn-in of 500,000 repetitions followed by 5,000,000 repetitions. % of Samples Assigned to Goa t Cluster 1 in between Cluster 2 Gra y Kokane e CLUSTER ASSIGNMENT Structure K=2 Collections 5,500,000 of Sample Size n>12 British Columbia Meado w Redfis h Lon Canyo g n Cree N. Point Elk North & West Kikoma of South of Bridg e k n Figure 4. Percent of individuals in a collection (sample size n > 12) assigned to a cluster from a STRUCTURE run for two populations (k) using burn-in of 500,000 repetitions followed by 5,000,000 repetitions. Idaho MT/B C 9

16 Table 2. Percent of samples by collection and region assigned to a cluster by estimated membership frequency of 60% or greater. Samples assigned at less than 60% inferred membership to either cluster were recorded to the "in between" category. Collection Site Cluster 1 in between Cluster 2 Goat 26.7% 10.0% 63.3% Gray 18.2% 21.2% 60.6% Kokanee 26.9% 3.8% 69.2% Meadow 51.7% 18.3% 30.0% Redfish 6.9% 10.3% 82.8% Cow Creek 50.0% 16.7% 33.3% Hemlock Bar 100.0% 0.0% 0.0% Long Canyon Creek 26.7% 40.0% 33.3% N. Fork Trout 100.0% 0.0% 0.0% S. Fork Trout 100.0% 0.0% 0.0% Shorty's Island 50.0% 0.0% 50.0% N. Point Elk 92.0% 4.0% 4.0% North & West of Kikoman 81.8% 18.2% 0.0% South of Bridge 84.6% 7.7% 7.7% Region Kootenay Lake, B.C. 30.3% 14.0% 55.6% Kootenai River, ID 46.7% 23.3% 30.0% Koocanusa Reservoir, B.C. 86.7% 10.0% 3.3% Basin All collections combined 44.8% 14.2% 41.0% Allelic statistical results. All 277 kokanee samples collectively yielded 177 alleles from the seven loci, averaging 25.3 alleles/locus (Table 3). Allele numbers/locus ranged from 21 (One114) to 42 (One103). Allelic richness among collections of sample size n > 12 (Table 4) was highest in Meadow Creek (8.4) and lowest in Redfish Creek (7.1). Unique alleles (alleles occurring in only one collection) totaled 23, but decreased to 18 when rare alleles (frequency in collection < 2%) were removed (details by population in Table 4, and by locus in Table 5). Three of the unique alleles occurred in collections of sample size n < 3, and may prove to be rare or not unique if more collections are made from the Kootenai River. Among collections with sample size n > 12, expected heterozygosity (He) averaged and observed heterozygosity (Ho) averaged (Table 4). Redfish Creek exhibited the lowest He (0.879), with Meadow Creek and North & West of Kikoman having the highest He (0.928). Gray Creek exhibited the lowest Ho (0.827) and South of Bridge the highest Ho (0.912). 10

17 Table 3. Number of samples (n), and number of alleles per locus and across all loci by region and collection site. Table 4. Sample size (n), number of alleles (NA), mean number of alleles per locus (MNA), expected heterozygosity (He), observed heterozygosity (Ho), average allelic richness (AR), number of unique alleles, and number of missing alleles by collection. Collection n NA MNA He Ho AR 1 Unique alleles Missing alleles 2 Goat Gray Kokanee Meadow Redfish Cow Creek Hemlock Bar * Long Canyon Creek N. Fork Trout * S. Fork Trout * Shorty's Island N. Point Elk North & West of Kikoman South of Bridge Totals and averages ( a ): from all 14 collections a a 18 from collections n> a a a 8.3 a 15 1 Based on minimum sample size of six diploid individuals (comparison of collections having sample size n > 12). 2 Alleles present in all but one collection (only collections of sample size n > 12 compared). *Due to small sample size (n < 3) the frequency is > 2%; allele may prove to be rare or not unique if more collections are taken from the Kootenai River. 11

18 Table 5. Identity (bin name) of unique alleles by locus and collection, and number of unique alleles summed across loci and collections. LOCUS: Collection One103 One104 One108 One111 One115 # Unique alleles across loci Goat Kokanee , ,187 5 Meadow Hemlock Bar 173 1* LCC N. Fork Trout 255 1* S. Fork Trout 208 1* N. Point Elk 212, N. & W. of Kikoman 220, # Unique alleles across collections *Small sample size (n < 3) increases frequency to above 2%. Alleles specific to a geographic region (occurring in only one region at a frequency of 2% or greater) totaled 10: four each in Kootenay Lake and Koocanusa Reservoir, and two in Kootenai River (Table 6). Table 6. Identity (bin name) and frequency of alleles specific* to a region, the number and percentage of collections within the region exhibiting the allele, and the total number of specific alleles by locus and region. *Specific alleles occur at 2% or greater frequency within the region, but do not occur in other regions. Fifteen alleles were missing (occurring in all but one collection of sample size n > 12, at frequency of 2% or greater of the combined Basin collections) from the following collections: Kokanee Creek (5), Redfish Creek (1), Long Canyon Creek (1), North and West of Kikoman (3), and South of Bridge (5). Details by locus and population are presented in Table 7. 12

19 Table 7. Identify (bin name) and total number of missing* alleles by locus and collection. LOCUS: Collection One103 One104 One108 One111 One112 One114 One115 # Missing alleles by collection Kokanee Redfish LCC N. & W. of Kikoman ,219 3 South of Bridge , # Missing alleles by locus *Missing alleles occur in all but one of the collections of sample size n>12. Smaller collections not included. Hardy-Weinberg test results. The Koocanusa Reservoir collections and four of the Kootenay Lake collections were in Hardy-Weinberg equilibrium (Table 8) after sequential Bonferroni corrections (α = = 0.05/13 tests). Goat Creek was not in equilibrium (0.0030). Heterozygote deficiency tests (Table 9) were significant after sequential Bonferroni corrections (α = = 0.05/13 tests) for Goat, Gray and Kokanee creeks, and for all three Kootenay Lake collections. Of the six Kootenai River collections, Long Canyon Creek was not in equilibrium (0.0002); Hemlock Bar had only one sample and so was not tested. The remaining four collections were in equilibrium but power to detect departures was limited due to small sample sizes (n varied from 2 to 6). Long Canyon Creek was not significant for heterozygote deficiency after Bonferroni correction. Individual locus P-values for the 13 collections tested for Hardy-Weinberg ranged from to 1, and none were out of equilibrium after sequential Bonferroni correction (α = = 0.05/91 tests). Heterozygote deficiency tests yielded significant results after sequential Bonferroni correction for locus One103 for Gray Creek (α = = 0.05/91 tests), and for five of the seven loci across all collections: One112, One114, One103, One108, and One115 (α = = 0.05/13 tests). When samples were tested for Hardy-Weinberg equilibrium by region instead of collection, Kootenay Lake (0.0000) and Kootenai River (0.0001) were not in equilibrium after sequential Bonferroni correction (α = = 0.05/3 tests), and locus One114 for both populations was significant (α = = 0.05/21 tests). Koocanusa Reservoir did not prove to be out of Hardy-Weinberg equilibrium. However, all three regions yielded significant heterozygote deficiency tests after sequential Bonferroni correction (α = = 0.05/3 tests). Four of the seven loci for Kootenay Lake were significant for heterozygote deficiency: One112, One114, One103, and One108. Six of the seven loci were significant for heterozygote deficiency when tested across all regions. Tests for genotypic disequilibrium for each pair of loci in each collection (Table 10) were not significant after sequential Bonferroni correction (α = = 0.05/273 tests). Tests were not possible for Hemlock Bar, and there was no information for Cow Creek, N. Fork Trout Creek, S. Fork Trout Creek, Shorty s Island, South of Bridge and much of North and West of Kikoman, Kokanee Creek, and Gray Creek. The no information result in the GENEPOP test for genotypic disequilibrium indicates tables for which all rows or all columns yield marginal sums of one. 13

20 Table 8. Individual P-values for Hardy-Weinberg exact tests of equilibrium by locus and collection site, region and Basin, across all loci, and across all collections and regions. 1 Significant after sequential Bonferroni correction for 13 tests ( ). 2 Significant after sequential Bonferroni correction for 1 test ( ). 3 Significant after sequential Bonferroni correction for 21 tests ( ). 4 Significant after sequential Bonferroni correction for 3 tests ( ). 5 Significant after sequential Bonferroni correction for 7 tests ( ). Bonferroni correction for 90 tests ( ); no P-values for collection by locus were found significant. 14

21 Table 9. Individual P-values for Hardy-Weinberg exact tests of heterozygote deficiency by locus and collection site, region and Basin, across all loci, and across all collections and regions. 15

22 Table 10. Individual P-values* for pairwise tests of loci by collection and across all collections for genotypic disequilibrium. Kootenay Lake Collections Kootenai River Collections N. Long Fork Canyon Trout S. Fork Trout Koocanusa Reservoir Collections North N. & W. Point of S. of Elk Kikoman Bridge Locus#1 Locus#2 Goat Gray Kokanee Meadow Redfish Cow Hemlock Bar Shorty's Island Across all collections One112 One N.I. 1 1 N.I. N.P N.I. N.I. N.I. 1 N.I. N.I One112 One103 1 N.I. N.I. 1 1 N.I. N.P N.I. N.I. N.I N.I One114 One103 1 N.I. N.I. 1 1 N.I. N.P N.I. N.I. N.I N.I. N.I One112 One N.I. N.P N.I. N.I. N.I. 1 1 N.I One114 One N.I. 1 1 N.I. N.P N.I. N.I. N.I. 1 N.I. N.I One103 One N.I. N.I. 1 1 N.I. N.P N.I. N.I. N.I N.I One112 One111 1 N.I N.I. N.P N.I. N.I. N.I. 1 N.I. N.I One114 One111 1 N.I. N.I. 1 1 N.I. N.P N.I. N.I. N.I. 1 N.I. N.I One103 One N.I. N.I. 1 N.I. N.I. N.P N.I. N.I. N.I. 1 N.I. N.I One108 One N.I. N.P N.I. N.I. N.I. 1 N.I. N.I One112 One104 1 N.I N.I. N.P. N.I. N.I. N.I. N.I. 1 1 N.I One114 One104 1 N.I. N.I N.I. N.P. N.I. N.I. N.I. N.I. 1 N.I. N.I One103 One104 1 N.I. N.I. 1 N.I. N.I. N.P. 1 N.I. N.I. N.I. 1 1 N.I. 1 One108 One104 1 N.I. N.I. 1 1 N.I. N.P. 1 N.I. N.I. N.I. 1 1 N.I. 1 One111 One104 1 N.I. N.I. N.I. 1 N.I. N.P. N.I. N.I. N.I. N.I. 1 N.I. N.I. 1 One112 One N.I. N.P N.I. N.I. N.I. 1 1 N.I One114 One N.I. 1 1 N.I. N.P N.I. N.I. N.I. 1 N.I. N.I One103 One115 1 N.I. N.I. 1 N.I. N.I. N.P N.I. N.I. N.I. 1 1 N.I One108 One N.I. N.P N.I. N.I. N.I. 1 1 N.I. 1 One111 One115 1 N.I N.I. N.P N.I. N.I. N.I. 1 N.I. N.I One104 One115 1 N.I. N.I. 1 1 N.I. N.P. N.I. N.I. N.I. N.I. 1 1 N.I. 1 *N.I. = no information; N.P. = not possible. 16

23 F ST test results. Pairwise F ST tests (Table 11) among collections within the Koocanusa Reservoir tributaries in BC and among collections within the Kootenai River were not significant after sequential Bonferroni correction (α = = 0.05/91 tests), and after the less stringent B-Y correction method (α = for 91 tests). Six of ten pairwise comparisons among the Kootenay Lake collections were significant after sequential Bonferroni correction; B-Y did not yield further significant tests. Meadow Creek had significant pairwise tests against all other Kootenay Lake collections. Gray Creek had significant pairwise tests against all Kootenay Lake collections except Goat Creek. The numbers of significant tests by collection were: Meadow Creek (4), Gray Creek (3), Kokanee Creek (2), Redfish Creek (2), and Goat Creek (1). Pairwise F ST tests between collections of sample size n > 12 from differing geographic regions yielded significant results between all three Koocanusa Reservoir collections and all five Kootenay Lake collections after sequential Bonferroni correction (α = = 0.05/91 tests) for 14 comparisons, and after B-Y correction (α = for 91 tests) for one comparison. (If collections of sample size n < 12 are removed from testing, the number of pairwise tests drops from 91 to 36, changing the Bonferroni critical value from to , causing South of Bridge pairwise test against Kokanee Creek to become significant after sequential Bonferroni correction, instead of only after B-Y correction). Long Canyon Creek, the only Kootenai River region collection of sample size n > 12, had significant pairwise tests against all three Koocanusa Reservoir collections (one after B-Y correction), and against Kokanee and Redfish Creeks of the Kootenay Lake region. Pairwise F ST tests between the five Kootenai River collections of sample size n < 12 and collections from other regions yielded significant differences between all five and Redfish Creek (after sequential Bonferroni correction), between Cow Creek, Hemlock Bar, N. Fork Trout and Kokanee Creek (after B-Y correction), and between Cow Creek and Gray Creek (after B-Y correction). There were no significant pairwise tests between Kootenai River sample collections and Koocanusa Reservoir collections. 17

24 Table 11. Sample size (n) and pairwise genetic differentiation index (F ST ) of 14 collections of kokanee from the Kootenai/y Basin. Values in bold italics on the diagonal are the average of 13 pairwise FST values for each collection. 18

25 Collections combined by region yielded significant pairwise F ST tests after sequential Bonferroni correction for all three tests of Kootenay Lake, Kootenai River, and Koocanusa Reservoir (Table 12). Table 12. Pairwise genetic differentiation index (Fst) of kokanee collected from three regions of the Kootenai River Basin. Values in bold italics on the diagonal are the average of two pairwise Fst values for each location. Region Kootenay Lake, B.C Kootenai River, ID Koocanusa Reservoir, B.C Significant after sequential Bonferroni correction for 3 tests ( ). V. Discussion The goal of this study, to provide a better understanding of kokanee population and stock structure in the Kootenai/y Basin fisheries and ecosystem management and restoration program, was successfully met. In short, this study identified two distinct, differentially admixed kokanee populations: one in Koocanusa Reservoir tributaries in BC (Cluster 1), and the other an admixture of three distinct stocks in Kootenay Lake s North, West and South Arms and their tributaries (Cluster 2). A tentatively intermediate third group of fish, both geographically and in terms of genetic admixture was represented by the sole Idaho sample group large enough for statistical analysis, Long Canyon Creek. Additional analyses with more kokanee samples from Kootenai River tributaries in Idaho are needed to better characterize these groups in Idaho. Of the study s four objectives, the first two (1) define and delineate population and stocks, and 2) assess stocks structure, were preliminarily satisfied as summarized in the results section and briefly in the above paragraph. However, the remaining two objectives were not conclusively satisfied. The third objective (to determine the magnitude of contribution from kokanee entrainment at Libby Dam to downstream rearing and/or spawning populations) remains unresolved due to the fact that Koocanusa/BC (Cluster 1) fish, predominantly represented by introduced Meadow Creek stock, with minor alternative stock contributions, cannot be currently distinguished from other Meadow Creek fish that have been planted as eyed eggs into Idaho tributaries during the past 10 years. Satisfaction of the fourth study objective (to assess future responses to various kokanee and ecosystem management strategies in the study area) was also compromised by this inability to decipher Meadow Creek fish produced in Idaho, Montana, or British Columbia. Future analysis enabled by annual kokanee sampling from the same collection sites in Idaho, Montana, and British Columbia could provide valuable information about temporal trends in genetic profiles and population abundance as well as important insight into the effect(s) of various management strategies on stock structure, abundance, and escapement dynamics. 19

26 Koocanusa/BC population. The Upper Kootenay River in BC (upstream from Montana and the Koocanusa Reservoir) did not contain kokanee prior to the construction of Libby Dam (Appendix 1). Presumed dead kokanee that were flushed out from the Kootenay Hatchery intermittently from 1969 through 1978 into Norbury Creek started a successful stock of kokanee downstream, which was first noticed by the B.C. Fisheries Branch in These escaped kokanee were most recently Meadow Creek stock, preceded by non-native kokanee from Chilliwack Lake (Paleface Creek in the Fraser drainage), Okanogan River (Skaha Lake), Meadow Creek, and Moyie Lake (Lamb Creek) (Appendix 1). Pairwise F ST tests show no significant differences among the Koocanusa Reservoir collections, a finding supported by a population recently founded by a common group. Accordingly, STRUCTURE results assigned 87% of these individuals to Cluster 1, and 3% to Cluster 2. Although Meadow Creek is a likely source of the Koocanusa Reservoir population, and assigns primarily to Cluster 1 (52% of individuals), pairwise F ST tests revealed significant differentiation between Meadow Creek and each of the three Koocanusa Reservoir collections (P-values of ). The alternative sources of nonnative kokanee in the Upper Kootenay River BC and in Koocanusa Reservoir may account for this divergence (Appendix 1; Appendix Table 1). Idaho. All Idaho collections except Long Canyon Creek (n=15) contained too few samples to statistically analyze (they ranged from 1-6 samples per collection site). Insufficient sample representation from Idaho streams also prohibits resolving the question of whether any remnant South Arm Kootenay Lake stock exists. Ashley et al. (1994) reported that this South Arm stock was functionally extinct in the early 1990s. Furthermore, extremely low annual escapement to Idaho tributaries to the Kootenai River during the past several decades, including from eyed egg introductions from Meadow Creek stocks, suggest a low probability of existence for a remnant South Arm stock. Long Canyon Creek was the only tributary to Kootenai River, Idaho that could reasonably be tested against tributaries in other regions. Pairwise F ST tests of Long Canyon Creek fish were significant against the West Arm tributaries of Kokanee and Redfish Creeks, and against all three Koocanusa Reservoir collections. STRUCTURE results for Long Canyon Creek were divided: 27% of individuals assigned to Cluster 1, 33% to Cluster 2, and 40% to the in between category (inferred membership <60% to any cluster). Since there have been a number of Meadow Creek origin eyed-egg plants in Kootenai River tributaries during recent years, including Long Canyon Creek (Andrusak 2007), it would be expected that this group of fish may increasingly display Cluster 2 characteristics as more samples from Idaho tributaries are sampled and analyzed. Thus, increased sampling representation from Idaho tributaries to the Kootenai River is required to determine whether Idaho fish constitute a separate admixed population (i.e. a new Cluster 3) or are more closely related to upstream (Cluster 1) or downstream (Cluster 2) populations. British Columbia. Although Kootenay Lake West Arm tributaries (Kokanee and Redfish creeks) were stocked with Meadow Creek stock throughout the early part of the 20 th century through 1952 (BC Ministry of Environment database), pairwise F ST tests revealed significant differentiation between Meadow Creek and fish in these tributaries (P-values of ). Pairwise F ST tests also revealed significant differentiation between Meadow 20

27 Creek and South Arm tributaries of Goat and Gray Creeks (P-values of and , respectively). Meadow Creek also differed from West and South Arm tributaries in STRUCTURE cluster assignments. These results are consistent with the early work of Vernon (1957). Meadow Creek individuals assigned to Cluster 1 at 52% and Cluster 2 at 30%. The other tributaries assigned at half the rate to Cluster 1 (6-27%), and double the rate to Cluster 2 (61-83%). Therefore, based on the pairwise F ST test results and STRUCTURE runs, we do not recommend using North Arm Meadow Creek eggs to stock the West and South Arm tributaries. Stocking Meadow Creek origin fish into several tributaries in Idaho is currently experimental to mitigate for extirpation of native stocks. Vernon (1957) estimated a 2.8% straying rate among Kootenay Lake stocks within the lake, with most straying occurring between West and South Arm stocks. The few fish currently observed in the South Arm escapements are likely Meadow Creek stock because virtually no fish have been counted in upstream historical spawning areas upstream in BC or Idaho (Andrusak 2007). VI. Summary This study revealed the presence of two kokanee populations, with individuals of one population (Cluster 1) in Koocanusa Reservoir, and individuals of the second population (Cluster 2) in Kootenay Lake. Distinct genetic structuring was also revealed among the three stocks in the Kootenay Lake region, consistent with past stock structure assignments based on morphometrics, and spatial and temporal isolation among these stocks (Vernon 1957). Thus, absolute caution should be used in any thought of stocking North Arm Meadow Creek kokanee into West and South Arm tributaries. However, the perceived risk of stocking Meadow Creek stock into South Arm tributaries in Idaho is considerably less because the historic South Arm stock appears to be functionally extinct (thus nullifying introgression concerns), and because previously approved introductions of eyed eggs from Meadow Creek have already occurred during the past decade. Except for Long Canyon Creek, the Idaho sample region lacked adequate sample numbers required for statistical comparisons among tributaries within the region or to individual tributaries in other regions. Regional comparisons suggested that the Kootenai River may be a transitional area between the two lakes, in terms of the percent of samples assigned to each kokanee population. Long Canyon Creek appears to be genetically distinct from the Koocanusa Reservoir population, and to date from two of the Kootenay Lake collections. However, this conclusion regarding genetic intermediacy is tentative and may change with increased sample collections from Long Canyon Creek and other Idaho tributaries. Finally, more samples from Idaho could confirm intermediacy (a hypothetical Cluster 3), or could characterize a population as more similar to Cluster 1 or Cluster 2. 21