MOLECULAR GENETIC INVESTIGATION OF YELLOWSTONE CUTTHROAT TROUT AND FINESPOTTED SNAKE RIVER CUTTHROAT TROUT A REPORT IN PARTIAL FULFILLMENT OF: AGREEMENT # 165/04 STATE OF WYOMING WYOMING GAME AND FISH COMMISSION: GRANT AGREEMENT PREPARED BY: MARK A. NOVAK AND JEFFREY L. KERSHNER USDA FOREST SERVICE AQUATIC, WATERSHED AND EARTH RESOURCES DEPARTMENT UTAH STATE UNIVERSITY AND KAREN E. MOCK FOREST, RANGE AND WILDLIFE RESOURCES DEPARTMENT UTAH STATE UNIVERSITY TABLE OF CONTENTS TABLE OF CONTENTS__________________________________________________ii LIST OF TABLES _____________________________________________________ iv LIST OF FIGURES ____________________________________________________ vi ABSTRACT _________________________________________________________ viii EXECUTIVE SUMMARY ________________________________________________ ix INTRODUCTION _______________________________________________________1 Yellowstone Cutthroat Trout Phylogeography and Systematics _________________2 Cutthroat Trout Distribution in the Snake River Headwaters ____________________6 Study Area Description ________________________________________________6 Scale of Analysis and Geographic Sub-sampling ____________________________8 METHODS____________________________________________________________9 Sample Collection ____________________________________________________9 Stream Sample Intervals ____________________________________________10 Stream Sampling Protocols __________________________________________10 Fish Species Identification ___________________________________________10 Fish Metrics, Photographs, and Tissue Samples __________________________13 Genetic Analysis ____________________________________________________13 Extraction of DNA __________________________________________________13 Methods by Objective _______________________________________________14 Objective 1 – Develop cost-effective, reliable, and repeatable molecular tools that will answer the study questions _____________________________________14 Objective 2a – Determine morphological differences between the two morphotypes of cutthroat trout (YSC & SRC) in the study landscape ________18 Objective 2b – Determine genetic differentiation between the two morphotypes of cutthroat trout (YSC & SRC) in the study landscape _____________________19 Objective 3 – Describe patterns of genetic variation in cutthroat trout within and among major drainages in the study landscape _________________________19 Objective 4 – Assess introgression with rainbow trout using both morphologic and genetic tools_________________________________________________20 Results______________________________________________________________20 Survey Results ______________________________________________________20 Genetic Structuring __________________________________________________22 Results by Study Objective___________________________________________22 Objective 1 – Develop cost-effective, reliable, and repeatable molecular tools that will answer the study questions _____________________________________22 Objective 2b – Determine genetic differentiation between the two morphotypes of cutthroat trout (YSC & SRC) in the study landscape _____________________26 Objective 3 – Describe patterns of genetic variation in cutthroat trout within and among major drainages in the study landscape _________________________36 Objective 4 – Detection of Rainbow Trout Introgression___________________41 Discussion ___________________________________________________________44 Develop cost-effective, reliable, and repeatable molecular tools that will answer the study questions _____________________________________________44 ii Genetic Differentiation among Morphotypes __________________________45 Genetic Differentiation among Major Drainages _______________________46 Detection of Rainbow Trout Introgression____________________________47 Management Recommendations __________________________________48 References Cited ______________________________________________________49 APPENDIX A _________________________________________________________55 APPENDIX B _________________________________________________________64 iii LIST OF TABLES Table 1 Common and scientific names1 of fishes and amphibians in the Snake Headwaters basin of Wyoming, and species abbreviations as identified by the Wyoming Game and Fish Department. _________________________________12 Table 2 Polymerase chain reaction (PCR) primers used to amplify and sequence the ND1-2 region in cutthroat trout. Unpublished primer sources are noted: IDFG = Idaho Fish & Game Eagle Fish Health Lab; USU = Utah State University. ______15 Table 3 Sample subset used to assess landscape-scale sequence variation in the mitochondrial ND1-2 region and to design internal primers to capture this variation. ________________________________________________________________16 Table 4 Polymerase chain reaction (PCR) primers used to amplify and assess polymorphism at nDNA microsatellite loci in cutthroat trout. Unpublished primer sources are noted by place of origin: GIS = Genetic Identification Services._____17 Table 5 Summary by river drainage for numbers of streams and stream reaches, and stream length (km) surveyed for cutthroat trout presence/absence between 1998 and 2003 in the Snake River headwaters of northwest Wyoming. River drainages are listed as they flow into the Snake River proceeding upstream from Palisades Reservoir. ________________________________________________________21 Table 6 Number of streams with cutthroat, brook, and rainbow trout present and the stream length (km) occupied, based on presence/absence surveys between 1998 and 2003 in the Snake River headwaters, Wyoming._______________________21 Table 7 Presence of Yellowstone cutthroat trout (large spotted morphotype) and Snake River cutthroat trout (fine spotted morphotype) in streams surveyed, and stream length (km) occupied in the Snake River headwaters, Wyoming. _____________22 Table 8 Average pairwise genetic distances (and standard errors) between individuals within (along diagonal) and between morphotypic groups of cutthroat trout in the upper Snake River drainage, Wyoming. Samples were pooled across all drainages. Distances within and between groups are expressed as average number of mutational differences (below diagonal, italicized) or average percent of mutational differences (above diagonal). ________________________________26 Table 9 Average pairwise genetic distances (and standard errors) between individuals within (along diagonal) and between morphotypic groups of cutthroat trout in the Jackson Hole segment of the Snake River, Wyoming. Distances within and between groups are expressed as average number of mutational differences (below diagonal, italicized) or average percent of mutational differences (above diagonal). ________________________________________________________26 Table 10 Average pairwise genetic distances (and standard errors) between individuals within (along diagonal) and between morphotypic groups of cutthroat trout in the Gros Ventre River drainage, Wyoming. Distances within and between groups are expressed as average number of mutational differences (below diagonal, italicized) or average percent of mutational differences (above diagonal). ______________27 Table 11 Average pairwise genetic distances (and standard errors) between individuals within (along diagonal) and between morphotypic groups of cutthroat trout in the Hoback River, Wyoming. Distances within and between groups are expressed as iv average number of mutational differences (below diagonal, italicized) or average percent of mutational differences (above diagonal).________________________27 Table 12 Average pairwise genetic distances (and standard errors) between individuals within (along diagonal) and between morphotypic groups of cutthroat trout in the Snake River Canyon segment of the Snake River, Wyoming. Distances within and between groups are expressed as average number of mutational differences (below diagonal, italicized) or average percent of mutational differences (above diagonal). ________________________________________________________27 Table 13 Average pairwise genetic distances (and standard errors) between individuals within (along diagonal) and between morphotypic groups of cutthroat trout in the Greys River, Wyoming. Distances within and between groups are expressed as average number of mutational differences (below diagonal, italicized) or average percent of mutational differences (above diagonal).________________________28 Table 14 Average pairwise genetic distances (and standard errors) between individuals within (along diagonal, shaded) and between geographic groups of cutthroat trout in the Snake River headwaters, Wyoming. Distances within and between groups are expressed as average number of mutational differences (below diagonal, italicized) or average percent of mutational differences (above diagonal). ______36 Table 15 Genetic diversity indices for cutthroat trout in Snake River headwaters drainages. Nucleotide diversity (π), haplotype diversity (Hd), and number of haplotypes are presented for each drainage. _____________________________37 Table 16 Genetic differentiation among cutthroat trout in Snake River headwaters drainages, based on haplotype distributions, characterized using the GST statistic (Nei 1987; Hudson et al. 1992). _______________________________________37 Table 17 Locations and fish metrics for five rainbow trout (RBT) and seven rainbow- cutthroat trout hybrids (RXC) captured in the Snake River