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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
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
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 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 headwaters, Wyoming. 42
Table 18 Summary of the number of records, by river drainage, of individual fish, and the approximate number of those fish that were photographed and/or a caudal fin clip collected. River drainages are listed as they generally occur from north to south. 55
Table 19 Total genomic DNA extractions for several streams1 in each of five geographic areas. The number of extractions per stream varied due to stream length, numbers of fish captured over the minimum size (>150 mm), and number of samples available for each putative cutthroat trout morphotype1. The geographic areas are arranged as they generally occur from north to south. A history of cutthroat trout stocking in each stream is provided. 56
Table 20 Lists the streams and samples, by geographic area1, selected for sequencing the mtDNA ND2 gene region. Contiguous sequences (~1,100 bp) of n=324 samples were completed. 59
Table 21 Number of cutthroat trout1 of the haplotypes A-M, per stream, within five geographic areas in the Snake River study area. The Snake River is split into two geographic areas, Jackson Hole and Snake River Canyon. The geographic areas are arranged as they generally occur from north to south. 61
LIST OF FIGURES
FIGURE 1 HISTORICAL TRANSCONTINENTAL RANGE OF YELLOWSTONE CUTTHROAT TROUT, WITH FINESPOTTED SNAKE RIVER CUTTHROAT TROUT HISTORICAL RANGE INDICATED. 3
Figure 2 Yellowstone cutthroat trout Oncorhynchus clarki bouvieri (YSC). This fish exhibits the YSC spotting pattern, with larger spots that are concentrated towards the caudal peduncle. 5
Figure 3 The finespotted Snake River cutthroat trout Oncorhynchus clarki subspecies (SRC) remains taxonomically undescribed. This fish shows the classic SRC pattern with small well distributed spots. 5
Figure 4 A cutthroat trout exhibiting a common intermediate spot pattern, with small to medium spots that are concentrated toward the caudal peduncle. 5
Figure 5 Snake River headwaters study area, in northwest Wyoming (approximately 9,440 km2). The five geographic areas between Palisades Reservoir and Jackson Lake are: Jackson Hole, Gros Ventre, Hoback, Snake River Canyon, and Greys. 7
Figure 6 Occurrence of cutthroat trout morphotypes within mitochondrial haplotypes A-M. YSC is a single occurrence haplotype from Yellowstone Lake; BRC is Bonneville cutthroat trout. Haplotype network was produced using statistical parsimony. 29
Figure 7 Frequencies of three morphotypes and thirteen haplotypes in the Snake River headwaters study landscape. Occurrence of morphotypes was similar throughout each of the five geographic areas, whereas four haplotypes were dominant. 30
Figure 8 Displays locations of streams in Jackson Hole from which samples were selected. Frequencies of the three morphotypes are on the upper left. Haplotype frequencies by specific morphotype are on the right. 31
Figure 9 Displays locations of streams in the Gros Ventre River drainage from which samples were selected. Frequencies of the three morphotypes are on the upper right. Haplotype frequencies by specific morphotype are on the left. 32
Figure 10 Displays locations of streams in the Hoback River drainage from which samples were selected. Frequencies of the three morphotypes are on the lower left. Haplotype frequencies by specific morphotype are on the right. 33
Figure 11 Displays locations of streams in the Snake River Canyon from which samples were selected. Frequencies of the three morphotypes are on the left. Haplotype frequencies by specific morphotype are on the right. 34
Figure 12 Displays locations of streams in the Greys River drainage from which samples were selected. Frequencies of the three morphotypes are on the lower left. Haplotype frequencies by specific morphotype are on the right. 35
Figure 13 Frequencies of haplotypes A-M varied among the five geographic areas within the Snake River headwaters, Wyoming. Four haplotypes were dominant, with two (B and D) occurring throughout the study landscape. Haplotype A was present mainly in the Hoback, Snake River Canyon and Greys. Haplotype C occurs mainly in tributaries in the Teton Mountains within Jackson Hole. 38
Figure 14 Dendrogram of haplotypes A-M identified in the Snake River headwaters, Wyoming. Cutthroat trout out groups include: BRC – Bonneville, CRC – Colorado River, GBC – greenback, LHC – Lahontan, SRC02 – finespotted Snake River, WSC – west slope, YSCA1, YSCT1, YSC53 and YSC55 – Yellowstone. Rainbow trout (RBT) and rainbow-cutthroat hybrid (RXC) haplotypes are included in this unrooted neighbor-joining tree. Values at branches are the relative strengths of nodes (percent) assessed by bootstrapping 1,000 times; scale is 5 base pair difference. 39
Figure 15 Network of cutthroat trout haplotypes A-M, with frequency of occurrence for each of five geographic areas indicated by symbols. YSC is a single occurrence haplotype from Yellowstone Lake; BRC is Bonneville cutthroat trout. Haplotype network was produced using statistical parsimony. 40
Figure 16 Presence of rainbow trout or rainbow-cutthroat trout hybrids in Gros Ventre and Greys River drainages were previously known or suspected. The capture of rainbow-cutthroat trout hybrids in the Hoback R, and upstream of Lower Slide Lake in the Gros Ventre were the first documented. 43
ABSTRACT
WE USED A LANDSCAPE SCALE APPROACH TO FACILITATE THE SYNTHESIS OF GEOMORPHIC, ECOLOGICAL, AND GENETIC INFORMATION REGARDING THE DISTRIBUTION AND ORGANIZATION OF YELLOWSTONE CUTTHROAT TROUT, ONCORHYNCHUS CLARKI BOUVIERI, AND FINESPOTTED SNAKE RIVER CUTTHROAT TROUT, ONCORHYNCHUS CLARKI SUBSPECIES, IN THE SNAKE RIVER HEADWATERS OF NORTHWEST WYOMING. SELECTION CRITERIA ALLOWED US TO HIERARCHICALLY ANALYZE FOR MORPHOLOGICAL OR GEOGRAPHIC STRUCTURING FROM THE BASIN SCALE, TO THE STREAM REACH SCALE. WHILE WE WERE UNABLE TO DIFFERENTIATE TWO DISTINCT MORPHOTYPES, THE CONSERVATION OF UNIQUE COLOR, SPOTTING PATTERNS, AND LIFE HISTORIES MAY BE IMPORTANT FOR FUTURE MANAGEMENT. GENETIC DIFFERENCES AMONG DRAINAGES WERE APPARENT, AS EVIDENCED BY A) AVERAGE PAIRWISE NUCLEOTIDE DIFFERENCES WITHIN AND BETWEEN DRAINAGES, B) A NON-RANDOM DISTRIBUTION OF HAPLOTYPES AMONG DRAINAGES (χ2 = 232.67; P < 0.00001), AND C) AN OVERALL PAIRWISE GST OF 0.14. TWO DISTINCT HAPLOTYPE CLADES WERE PRESENT IN THE DATASET. CLADE 1 HAPLOTYPES TENDED TO BE MORE COMMON IN JACKSON HOLE AND THE GROS VENTRE, AND CLADE 2 HAPLOTYPES WERE MORE COMMON IN THE HOBACK, SNAKE RIVER CANYON, AND GREYS. MORPHOLOGICAL AND GENETIC DIFFERENCES WERE OBSERVED IN RAINBOW-CUTTHROAT HYBRIDS THAT DISTINGUISHED THEM FROM CUTTHROAT TROUT. HYBRIDIZATION WAS LIMITED TO THOSE LOCALES PREVIOUSLY SUSPECTED OF HARBORING RBT OR RXC. FURTHER WORK IS RECOMMENDED USING THE EXISTING SAMPLES AND MARKERS DEVELOPED FROM THIS EFFORT, COMBINED WITH ADDITIONAL COLLECTIONS FROM THE SNAKE RIVER.
EXECUTIVE SUMMARY
TO DATE THERE HAVE BEEN NO CONCERTED EFFORTS TO DETERMINE WHETHER YELLOWSTONE CUTTHROAT TROUT, ONCORHYNCHUS CLARKI BOUVIERI (YSC), AND FINESPOTTED SNAKE RIVER CUTTHROAT TROUT, ONCORHYNCHUS CLARKI SUBSPECIES (SRC), IN THE SNAKE RIVER HEADWATERS DIFFER WITH RESPECT TO ECOLOGY, MORPHOLOGY, AND/OR GENETIC CHARACTERISTICS. THE MAJORITY OF THE RESEARCH WITHIN THE SNAKE RIVER HEADWATERS HAS DESCRIBED THE BIOLOGY AND ECOLOGY OF THE SRC FISHERY IN THE MAINSTEM SNAKE RIVER IN JACKSON HOLE (HAYDEN 1967; WILEY 1969; HAGENBUCK 1970; KIEFLING 1972, 1978; HARPER AND FARAG 2002; HARPER AND FARAG 2004). PREVIOUS GENETIC INVESTIGATIONS OF FISH FROM BOTH THE MAINSTEM AND HEADWATER TRIBUTARIES WERE USED TO DESCRIBE THE PHYLOGEOGRAPHY OF THE INTERIOR CUTTHROAT TROUT (MURPHY 1974), EXAMINE GENETIC DIFFERENCES BETWEEN YSC AND SRC (LOUDENSLAGER 1978), AND USED TO COMPILE A BIOLOGICAL CLASSIFICATION OF NATIVE TROUT OF WESTERN NORTH AMERICA (BEHNKE 1992). CONCERN OVER THE STATUS OF THESE FISH PROMPTED A STATUS REVIEW OF YSC FOLLOWING A 1998 PETITION TO LIST THEM AS A THREATENED SPECIES UNDER THE ENDANGERED SPECIES ACT (WGFD 1999). THE YSC WAS EVENTUALLY FOUND TO BE “NOT WARRANTED” FOR LISTING, BUT THE US FISH AND WILDLIFE SERVICE IS UNDER COURT ORDER TO RE-EXAMINE THE DATA USED FOR THE INITIAL FINDING AND PROCEED WITH A 1-YEAR STATUS REVIEW.
The Snake River above Palisades dam has been identified as a large, relatively intact basin that represents one of the last strongholds of YSC and the potentially unique SRC morphotype. However, the status of cutthroat trout populations and the threats to these populations have never been formally investigated. This work examines three major questions that need to be answered in order to determine the status of cutthroat trout in the Snake River above Palisades dam. First, are there morphometric and genetic differences between YSC and SRC that would indicate that these are unique subspecies and that these fish should be managed separately? Second, if there are no apparent subspecies differences, are there differences in the genetic structure among geographic units that would indicate separate management units within the basin? Third, given the stocking history of rainbow trout and other non-native cutthroat trout, where have populations within the basin been compromised by hybridization and where are the potential threats? We used these questions to develop the following study objectives:
1) Develop cost-effective, reliable, and repeatable molecular tools that will answer the study questions.
2) Determine morphometric and genetic differentiation between the two morphotypes of cutthroat trout (YSC & SRC) in the study landscape.
3) Describe patterns of genetic variation in cutthroat trout within and among major drainages in the study landscape.
4) Assess introgression with rainbow trout using both morphologic and genetic tools.
No definitive description of the historical range of cutthroat trout in the Snake River headwaters exists. Behnke (1992) hypothesizes that only YSC were historically present in tributaries of the Snake River upstream of the Gros Ventre River confluence and in headwater streams of the Gros Ventre River drainage. He speculates that the finespotted morphotype historically occupied the Snake River downstream of Jackson Lake and all tributaries downstream of the Gros Ventre River. However, our distribution surveys suggest a pattern of headwater occupancy by YSC persists in each of the river drainages, as well as in many of the smaller tributaries to the Snake River. Present occupancy of cutthroat trout is >95% of streams, and >90% of stream length inhabited by trout. Cutthroat trout were found in 294 streams in 1,483 km of habitat. Nine rainbow-cutthroat hybrids were found in the Greys (2), Hoback (1), and Gros Ventre (6) areas during the survey. Brook trout were found in all areas. Yellowstone cutthroat trout (large spotted morphotype) were found in considerably fewer streams (102 streams, 277km) and in fewer locations than Snake River cutthroat trout (fine spotted morphotype; 258 streams, 1,249 km). The large and fine spotted forms were sympatric in 98 streams, representing 225 km.
The SRC and YSC morphotypes are closely related (Loudenslager and Kitchin 1979; Loudenslager and Gall 1980), and F1 progeny exhibiting intermediate spotting patterns have been observed when hatchery stocks were combined with wild populations (Behnke 1992). Montgomery (1995) proposed to name the SRC subspecies Oncorhynchus clarki behnkei. However, the name was invalidated due to omission of a type specimen (D. Shiozawa, personal communication). The actual recognition of the SRC as a subspecies distinct from the YSC remains unresolved (Behnke 2002). Prior to our work, no information on genetic status of these fishes (e.g., introgression by rainbow trout) was available for our study streams. While non-native trout introductions have been widespread throughout the basin, displacement of cutthroat trout (e.g., by brook trout), and presumably introgression (e.g., with rainbow trout) are assumed to have occurred on a limited basis.
Our landscape scale approach facilitated the synthesis of geomorphic, ecological, and genetic information regarding the distribution and organization of cutthroat trout within the river drainage, watershed, stream, or stream reach. Samples were selected for inclusion in this analysis based on trout external morphology (spot patterns). Streams were selected to be representative of five geographic areas comprised of four river drainages in the riverscape. Selection criteria allowed us to hierarchically analyze for morphological or geographic structuring from the basin scale, to the stream reach scale.
Polymerase chain reaction (PCR) primers were developed for use with mitochondrial DNA in upper Snake River cutthroat trout. These primers reliably amplify a ~1,100 bp region of the ND2 mitochondrial gene, and can be used both for amplification and sequencing. Six polymorphic microsatellite loci were identified for use in Snake River headwaters cutthroat trout genetic analyses. The primer pairs reliably amplified these nuclear loci, and allele sizes ranged from approximately 100 to 400 bp. Multiplexing (simultaneous amplification) of several loci was not attempted pending determination of polymorphism for several primer sets.
Genetic differentiation among morphotypes was not apparent, either within drainages or pooling across the entire study area. Differences in haplotypic composition among groups were likely due to sample size differences or stochastic sampling error. While we were unable to differentiate two distinct morphotypes, the conservation of unique color, spotting patterns, or life histories may be important for future management. Unique phenotypes and life histories in westslope cutthroat trout, and physiological adaptations in related interior cutthroat trout are examples of such variation that exists and that should be maintained (Carl & Stelfox 1989; Taylor et al. 2003).
Genetic differences among drainages were apparent in all analyses, as evidenced by a) average pairwise nucleotide differences within and between drainages, b) a non-random distribution of haplotypes among drainages (χ2 = 232.67; P < 0.00001), and c) an overall pairwise GST of 0.14.
Two distinct haplotype clades were present in the dataset. Clade 1 was distributed throughout the study area, but there was a tendency for this group of haplotypes to be more common in Jackson Hole and the Gros Ventre, while clade 2 haplotypes were more common in the Hoback, Snake River Canyon, and Greys. These clades are likely to have evolved in response to different hydrogeographic conditions than those that exist today.
The main identifying feature for all of the rainbow-cutthroat hybrid fish was white margins or tips on the pelvic and anal fins. Genetic differences were observed in 6 of the 8 hybrid fish that distinguished them from cutthroat trout. The two remaining RXC, both D-haplotype fish, clearly exhibited white on the pelvic and/or anal fins. Lack of a RXC haplotype in these two fish emphasizes that mtDNA only expresses maternal inheritance. Sequencing of the mtDNA ND2 gene essentially functioned as a fine-filter screening of all 324 samples from throughout the study landscape. This suggests that hybridization is largely limited to those locales previously suspected of harboring RBT or RXC.
Recommendations based on these results include: 1) Initiate a landscape level analysis using this sample set and nuclear markers (microsatellites) to understand historic geologic and hydrologic conditions that may explain the patterns of genetic variability observed in this study; 2) While not conclusive, frequency differences among drainages suggest that there should be caution in translocating cutthroat trout among drainages; and 3) Initiate mainstem Snake River investigations to better determine the presence, location, and extent of hybridization in the river between Palisades Reservoir and Jackson Lake.
INTRODUCTION
THE CUTTHROAT TROUT ONCORHYNCHUS CLARKI IS THE ONLY TROUT NATIVE TO WYOMING (BAXTER AND STONE 1995). THREE RECOGNIZED SUB-SPECIES OF CUTTHROAT TROUT HAVE BEEN DESCRIBED WITHIN THE STATE AND INHABIT TRIBUTARIES OF THE BEAR RIVER (BONNEVILLE CUTTHROAT, O. C. UTAH), COLORADO/GREEN RIVER (COLORADO CUTTHROAT, O.C. PLEURITICUS), AND THE YELLOWSTONE RIVER AND SNAKE RIVER (YELLOWSTONE CUTTHROAT, O. C. BOUVIERI, YSC). WHILE THE DIFFERENTIATION AMONG SUB-SPECIES HAS BEEN WELL DOCUMENTED, A MORPHOMETRICALLY DISTINCT FISH HAS BEEN IDENTIFIED WITHIN THE SNAKE RIVER SYSTEM THAT MAY ALSO BE UNIQUE. A FINESPOTTED MORPHOTYPE (FINESPOTTED SNAKE RIVER, SRC) HAS BEEN IDENTIFIED IN THE SNAKE RIVER HEADWATERS OF NORTHWEST WYOMING THAT IS A VISUALLY DISTINCT, YET TAXONOMICALLY PUZZLING NATIVE FISH (BEHNKE 1992) THAT HAS BEEN PROPOSED FOR SUBSPECIES STATUS (BAXTER AND SIMON 1970; BEHNKE 1992). HOWEVER, THERE HAVE BEEN NO ATTEMPTS TO DETERMINE WHETHER YSC AND SRC IN THE SNAKE RIVER HEADWATERS DIFFER WITH RESPECT TO ECOLOGY, MORPHOLOGY, AND/OR GENETIC CHARACTERISTICS.
Though Yellowstone cutthroat are one of the more well-studied subspecies of interior cutthroat trout (see Behnke 1992; Gresswell 1988; and Gresswell 1995), the majority of work to date has been conducted in segments of large rivers or lakes of Idaho, Montana, and Wyoming outside of the Snake River basin. The majority of the research within the Snake River has described the biology and ecology of the SRC fishery in the mainstem Snake River in Jackson Hole (Hayden 1967; Wiley 1969; Hagenbuck 1970; Kiefling 1972, 1978; Harper and Farag 2002; Harper and Farag 2004). Previous genetic investigations of fish from both the mainstem and headwater tributaries were used to describe the phylogeography of the interior cutthroat trout (Murphy 1974), examine genetic differences between YSC and SRC (Loudenslager 1978), and used to compile a biological classification of native trout of western North America (Behnke 1992). Little research has occurred in the remaining tributaries (Greys River, Hoback River, Gros Ventre River, Buffalo Fork) flowing into the Snake River between Palisades Reservoir and Jackson Lake dam.
The identification of the current distribution and status of populations of YSC has become a management priority for state and federal agencies due to their continued decline in distribution and abundance throughout most of the historical range. Causes for these declines include the loss of habitat due to poor land use practices, over-fishing, and the introduction of non-native species that have successfully invaded and occupied YSC habitat (Behnke 1992). Concern over the status of these fish prompted a status review of YSC following a 1998 petition to list them as a threatened species under the Endangered Species Act (WGFD 1999). The YSC was eventually found to be “not warranted” for listing, but the US Fish and Wildlife Service is under court order to re-examine the data used for the initial finding and proceed with a 1-year status review.
The Snake River above Palisades dam has been identified as a large, relatively intact basin that represents one of the last strongholds of YSC and the potentially unique SRC morphotype. However, the status of cutthroat trout populations and the threats to these populations have never been formally examined. This work examines three major questions that need to be answered in order to determine the status of cutthroat trout in the Snake River above Palisades dam. First, are there morphometric and genetic differences between YSC and SRC that would indicate that these are unique subspecies and that these fish should be managed separately? Second, if there are no apparent subspecies differences, are there differences in the genetic structure among geographic units that would indicate separate management units within the basin? Third, given the stocking history of rainbow trout and other non-native cutthroat trout, where have populations within the basin been compromised by hybridization and where are the potential threats? We used these questions to develop the following study objectives:
1) Develop cost-effective, reliable, and repeatable molecular tools that will answer the study questions.
2) Determine morphometric and genetic differentiation between the two morphotypes of cutthroat trout (YSC & SRC) in the study landscape.
3) Describe patterns of genetic variation in cutthroat trout within and among major drainages in the study landscape.
4) Assess introgression with rainbow trout using both morphologic and genetic tools.
Yellowstone Cutthroat Trout Phylogeography and Systematics
Yellowstone cutthroat trout are native to the Yellowstone River and Snake River headwaters in Idaho, Montana, and Wyoming (Figure 1). Their trans-continental divide range in Montana and Wyoming likely resulted from headwater connection 20,000 to 50,000 years ago, similar to the present connectivity of Atlantic Creek and Pacific Creek at Two Ocean Pass (Behnke 1992). This connection between the Columbia and Missouri River basins remains, and fish are not restricted from inter-basin movement, even today. Yellowstone cutthroat trout became isolated in the headwaters of the Snake River following creation of Shoshone Falls (between 30,000 and 60,000 years ago). The finespotted morphotype is hypothesized to have originated from the YSC in present-day Jackson Hole during the Pinedale glacial period 15,000 to 25,000 years ago while isolated by glacially dammed lakes (Loudenslager and Kitchin 1979, Love et al. 2003).
Behnke (1992) postulates that, “after several thousand years of isolation, the ancestral Yellowstone cutthroat trout and the new form, both slightly differentiated after isolation, came together again, but instead of freely hybridizing, they partitioned
the Snake River headwaters environment and maintained their distinctions through reproductive isolation. Once in contact again, evolutionary mechanisms governed by natural selection probably resulted in their spotting differences.” Alternatively, continued partitioning of the riverscape after breakdown of the hypothesized barrier may depend more upon low average dispersal distance of individuals from populations (Irwin 2002). The finespotted morphotype was considered a taxonomically unidentified native fish (Behnke 1992) with a historical range limited to the Snake River headwaters of northwestern Wyoming between Palisades Reservoir
[pic]
Figure 1 Historical transcontinental range of Yellowstone cutthroat trout, with finespotted Snake River cutthroat trout historical range indicated.
and Jackson Lake, and possibly eastern Idaho. Montgomery (1995) proposed to name it a subspecies; Oncorhynchus clarki behnkei. However, the name was invalidated due to omission of a type specimen (D. Shiozawa, personal communication). The actual recognition of the SRC as a subspecies distinct from the YSC remains unresolved (Behnke 2002).
The SRC and YSC morphotypes are closely related (Loudenslager and Kitchin 1979; Loudenslager and Gall 1980), and F1 progeny exhibiting intermediate spotting patterns have been observed when hatchery stocks were combined with wild populations (Behnke 1992). Behnke (1992) suggests that reproductive isolation is not complete between SRC and YSC when sympatric. His analyses have shown variation and overlap in meristic counts, and observations of intermediate spotting suggest continued gene flow. Behnke (1992) also acknowledged that the difference in spotting pattern, and observed intermediate spotting, may result from simultaneous expression of two co-dominant alleles at one locus, as shown by Skaala and Jorstad (1988) in brown trout Salmo trutta.
Genetic comparisons of YSC and SRC (Leary et al. 1987, Allendorf and Leary 1988) with allozyme electrophoresis did not discern diagnostic markers at the many loci analyzed. More recent analyses by Kruse et al. (1996) of YSC in the Greybull River drainage (Missouri River drainage) of northwest Wyoming showed no consistent difference in counts of seven meristic features of fish sampled from 18 streams. Seven of the 18 sample locations were selected due to close proximity to known locations of past SRC introductions by state fishery personnel. They also compared fish among streams thought to contain “pure” YSC based on the absence of an allele (AK-1*333; common among SRC in the Snake River drainage; Wild Trout and Salmon Genetics Lab, University of Montana, Missoula), and streams where protein electrophoresis confirmed presence of the allele; its presence was assumed to be indicative of integration with stocked SRC.
Relatively little recent work has occurred on the phylogenetic classification of these fishes (Shiozawa and Williams 1992), and differences in spot size and numbers remain the only means to distinguish between YSC and SRC. Yellowstone cutthroat trout have medium to large sized spots (3-5 mm diameter) that are concentrated toward the caudal peduncle (Figure 2). Snake River cutthroat trout have a profusion of smaller spots (1-2 mm diameter) that are well distributed across the side of the fish (Figure 3). Variations in these spotting patterns (i.e., fewer medium size spots more evenly distributed) are common, and suggest mixing, or possibly environmental influences, such that distinguishing between these fishes is not possible in all cases (Figure 4). Spotting patterns have been shown to correctly classify YSC and SRC (>95%) in blind tests where there was no hybridization with rainbow trout, but were not useful in identifying fish with intermediate spotting patterns (Kruse 1998).
Application of recent advances in molecular genetic techniques have identified mtDNA haplotypes that distinguish populations of YSC isolated by distance (Campbell et al. 2002), and nDNA markers distinguishing YSC from the other inland cutthroat trout subspecies (Spruell et al. 2001) and rainbow trout (RBT), though the techniques have not been applied to the closely related YSC and SRC morphotypes.
[pic]
Figure 2 Yellowstone cutthroat trout Oncorhynchus clarki bouvieri (YSC). This fish exhibits the YSC spotting pattern, with larger spots that are concentrated towards the caudal peduncle.
[pic]
Figure 3 The finespotted Snake River cutthroat trout Oncorhynchus clarki subspecies (SRC) remains taxonomically undescribed. This fish shows the classic SRC pattern with small well distributed spots.
[pic]
Figure 4 A cutthroat trout exhibiting a common intermediate spot pattern, with small to medium spots that are concentrated toward the caudal peduncle.
Cutthroat Trout Distribution in the Snake River Headwaters
No definitive description of the historical range of cutthroat trout in the Snake River headwaters exists. Behnke (1992) hypothesizes that only YSC were historically present in tributaries of the Snake River upstream of the Gros Ventre River confluence and in headwater streams of the Gros Ventre River drainage. He speculates that the finespotted morphotype historically occupied the Snake River downstream of Jackson Lake and all tributaries downstream of the Gros Ventre River. However, our early distribution surveys suggest a pattern of headwater occupancy by YSC persists in each of the river drainages, as well as in many of the smaller tributaries to the Snake River.
A 1996 habitat conservation assessment by May (1996) suggested YSC and the finespotted morphotype may be present in 100% of their historically occupied streams and lakes in the Snake River headwaters of Wyoming, and occupy additional lakes as a result of past and current stocking practices. Due to the coarse scale of analysis and use of “best available information”, caution must be exercised in interpreting May’s findings. Allendorf and Leary (1988), and Varley and Gresswell (1988), and others (Young 1995) have identified the introduction of non-native fishes as posing the greatest danger to native cutthroat trout conservation, due mainly to interbreeding, and the primary cause for decline of YSC in other portions of their range. Prior to our work, no information on genetic status of these fishes (e.g., introgression by rainbow trout) was available for our study streams. While non-native trout introductions have been widespread throughout the basin, displacement of cutthroat trout (e.g., by brook trout), and presumably introgression (e.g., with rainbow trout) are assumed to have occurred on a limited basis.
Study Area Description
At the broad scale this work assessed the landscape distribution and organization of cutthroat trout populations between Palisades Reservoir and Jackson Lake, Wyoming (Figure 5). The distribution surveys were conducted in all named streams, including the Greys River, Hoback River, and Gros Ventre River drainages. All named tributaries to the Snake River were surveyed upstream to Jackson Lake Dam.
Mapping of mtDNA haplotypes by geographic areas or major river drainages was completed after initial analysis of samples from across the basin. Five geographic areas were identified a priori for mapping and analyses. These areas, as they generally occur from north to south, include Jackson Hole, Gros Ventre, Hoback, Snake River Canyon, and Greys. The Snake River and its tributaries were split due to the stark geomorphological break between the broad mountain valley of Jackson Hole, and the Snake River Canyon. The remaining three areas are comprised of the
[pic]
Figure 5 Snake River headwaters study area, in northwest Wyoming (approximately 9,440 km2). The five geographic areas between Palisades Reservoir and Jackson Lake are: Jackson Hole, Gros Ventre, Hoback, Snake River Canyon, and Greys.
major tributary river drainages; the Salt River drainage was excluded due to being highly fragmented by water developments and not meeting the stream selection criteria (see following section). Surveys were conducted throughout the length of streams occupied by fish, including above and below natural barriers. Analyses were largely constrained to connected stream networks, except for several isolated stream segments above natural barriers in the Teton Mountains.
Scale of Analysis and Geographic Sub-sampling
The landscape scale approach facilitated the synthesis of geomorphic, ecological, and genetic information regarding the distribution and organization of cutthroat trout within the river drainage, watershed, stream, or stream reach. Samples were selected for inclusion in this analysis based on trout external morphology (spot patterns). Streams were selected to be representative of the five geographic areas comprised of four river drainages in the riverscape. Selection criteria allowed us to hierarchically analyze for morphological or geographic structuring from the basin scale, to the stream reach scale. Specifically, samples were selected by stream based on the following criteria:
1) Ensure variation in spotting patterns within each stream was documented by surveys throughout the occupied length of a stream;
2) There was no history of stocking, or at least recent stocking, in each stream to the extent possible;
3) Connectivity existed among all streams selected, both within and between the geographic areas;
4) Minimize spatial clustering of samples within a stream, to the extent possible, by selecting samples from throughout the occupied length of each stream;
5) Minimize spatial clustering of streams, to extent possible, by selecting streams from throughout each geographic area;
6) Ensure that streams were stratified across the five geographic areas;
7) Ensure that streams were stratified within each of the five geographic areas;
8) Include samples from each of the available age classes or size groups within each stream;
9) Include a minimum of n=30 fish from each geographic area or river drainage, where possible, that exhibit the large-sparse spotting pattern (i.e., YSC);
10) Segregate fish into three distinct spot pattern morphotypes: Type 1 – large-sparse (L), Type 2 – fine-dense (F), and Type 3 – intermediate (I) to 1 and 2;
11) Morphotypes present must be confirmed based on photographic records from stream surveys; and
12) Samples to be included in the analysis should be only from those streams where the large-sparse morphotype was observed.
Exceptions to these criteria were allowed only in the case where number of streams with the large-sparse spotting pattern were limited, necessitating the inclusion of streams or samples within streams that were above man-made barriers to upstream movement or stocking was documented in the last 50 years. Furthermore, availability of tissue samples from cutthroat trout with photographic documentation in large rivers was inconsistent, specifically in the case of the Snake River. Samples were clustered in two areas between the Gros Ventre River confluence and Jackson Lake, and no samples were available in the southern portion of Jackson Hole or the Snake River Canyon (Figure 5).
METHODS
SAMPLE COLLECTION
Systematic electrofishing surveys were completed to verify fish presence and distribution in all named streams on National Forest, National Park, and National Refuge system lands between Palisades Reservoir and Jackson Lake dam (Figure 5). Sampling was conducted with a model 12B Smith-Root battery powered backpack electrofisher. Crews consisted of one person operating the electrofisher and two netters. A single netter was employed only where the wetted width of the stream was 150 mm (for subspecies identification, minimum total length for YSC and SRC to develop their spotting pattern, and loss of parr marks). Sampling ceased after no fish were captured in three consecutive reaches, a barrier to upstream occupancy was encountered, headwaters were reached, or when water flow appeared insufficient to support resident fish. In each case the reason for terminating the survey was documented. Stream distance was recorded for notable physical features encountered (i.e., Forest System road or trail crossings, named and unnamed tributary confluences, water falls and high gradient riffles or cascades).
Sampling began at the reach interval break, except where electrofishing conditions dictated beginning elsewhere within the reach. For example, when a reach break fell within a large beaver pond or dam complex that cannot be sampled effectively with a backpack electrofisher due to water volume, depth, or difficult access (i.e., silt bottom too difficult to safely wade), sampling began at the next accessible point upstream. A minimum 50-m sample was measured. It was often necessary to sample 75 m or 100 m in an attempt to capture a minimum of 3 cutthroat trout >150 mm. When variations in spotting pattern were observed (i.e., both YSC and SRC are identified), sampling continued up to 100 m in an attempt to capture >3 each of YSC and SRC to measure, photograph, and collect fin clips. Sampling ceased when 10 cutthroat trout >150 mm were captured.
Fish Species Identification
Prior to sampling a stream, the Wyoming Game and Fish Department (WGFD) stream and lake catalogue were reviewed and all previously documented fish species noted. Also, the WGFD stocking history (WGFD 1997) for the stream was reviewed, and introduced fish species noted regardless of the time since stocking. These notes provided a list of non-game species to anticipate and identify (e.g., speckled dace vs. long-nose dace vs. mountain sucker), as well as an alert to the possibility of encountering brook trout Salvelinus fontinalis (BKT), rainbow trout Oncorhynchus mykiss (RBT), rainbow trout-cutthroat trout hybrids (RXC), or cutthroat trout with confusing spot patterns (e.g., Bonneville cutthroat trout). Species were recorded in the field, as well as database entries, using the 3-letter abbreviations in Table 1.
Cutthroat Trout >150 mm – The cutthroat trout name arises from the red or orange slash present under each side of the lower jaw. Differences in spot size and distribution are the only recognized means to distinguish between YSC and SRC. Yellowstone cutthroat trout have medium to large sized spots (3-5 mm) that are conspicuous and rounded, and concentrated toward the caudal peduncle; color is typically yellowish brown, silvery or brassy (Figure 2). Bright golden-yellow, orange or red colors are absent (Behnke 1992; Baxter and Stone 1995). Snake River cutthroat trout have a profusion of small (1-2 mm) irregular spots that are well distributed across the side of the fish and somewhat concentrated in the caudal peduncle (Figure 3). Color is typically more yellowish brown, with orange or red on the lower fins (Behnke 1992; Baxter and Stone 1995). Cutthroat trout were identified as YSC, SRC, or CUT (delineates undetermined cutthroat trout), and comments recorded on unusual characteristics or decision criteria.
Cutthroat Trout 150 mm were measured and photographed in each sample reach; a caudal fin clip is collected from all photographed cutthroat trout and RBT (and all RBT or CUT exhibiting characters of hybridization). Fish were measured and photographed lying on their right side, dorsal fin up, and facing to the left, using a standardized measuring board. Total length was recorded to the nearest millimeter, and weight to the nearest 5 gm. After photographing the fish, a caudal clip was collected. Fin clips were >5 mm in diameter, although this proved difficult for fish 95% of streams and >90% of stream length inhabited by trout. This includes 393 streams between Palisades Reservoir and Jackson Lake dam, with approximately 2,500 sample locations in more than 2,260 km of habitat surveyed (Table 5). Cutthroat trout were found in 294 streams in 1,483 km of habitat (Table 6). Nine rainbow-cutthroat hybrids were found in the Greys (2), Hoback (1), and Gros Ventre (6) areas during the survey. They were present in 5 mm), and disperse spots, not observed anywhere else in the study landscape. While we cannot determine the origin of these differences, it is likely a result of isolation of fish with the more ancient haplotype C (Figure 15). Review of the stocking records for the four streams in question (Cascade Cr, Death Canyon, Leigh Canyon, and Paintbrush Canyon) indicates that only Leigh Canyon had a documented introduction, likely of finespotted Snake River cutthroat trout. Although no record exists, it is assumed cutthroat trout have been stocked in Cascade Canyon due to the presence of brook trout above the barrier falls, and the likelihood of stocking Lake Solitude (the headwater source) due to its popularity as a back packing destination and trail access. Thus, it is quite likely that presence of haplotype D in Cascade Cr and haplotypes B and D in Leigh Canyon resulted from introductions of cutthroat trout via stocking. Regardless, the fact that these anomalous fish are extant in Cascade Cr and Leigh Canyon, and are the only morphotype and haplotype present in Death Canyon and Paintbrush Canyon, is indicative of the historical genetic variation present in cutthroat trout within the study landscape. Significant portions of within species diversity may be partitioned between populations above and below barriers (Carlsson and Nilsson 1999; Costello et al. 2003). Their demographic independence may indicate that conservation and recovery plans should take into account the importance of these isolated populations (Taylor et al. 2003).
Detection of Rainbow Trout Introgression
There is little evidence for the widespread hybridization of cutthroat trout with rainbow trout in the Snake River headwaters of Wyoming. Our data suggests that hybridization is largely limited to those areas that were previously suspected of harboring RBT or hybrids due to rainbow trout stocking. Two notable exceptions are the single hybrid captured in the Hoback River, and several hybrid captured upstream of Lower Slide Lake in the Gros Ventre River drainage. Some caution must be exercised relative to the genetic techniques used in this study. The use of mitochondrial DNA may underestimate the true hybridization extent because of maternal inheritence. Other techniques (e.g., specifically nuclear markers such as allozymes, microsatellites, PINES) may be more appropriate to further examine the full extent of hybridization (Henderson et al. 2000, Hitt et al. 2003). Two fish that were identified in the field as hybrids, but were genetically represented as pure using mtDNA should be screened using a different technique to confirm the initial call.
Hybrid identification using the appearance diagnostics was corroborated by genetic analysis in 6 of 8 fish, indicating those diagnostics are useful to quickly screen potential hybrids for genetic analysis. Henderson et al. (2000) found that field techniques using spotting pattern, body color, mandible length, and presence or absence of coloration below the gill covers were effective at screening YSCxRBT hybrids with a misclassification rate of 2%. The addition of these characteristics in future sampling of YSC and SRC to examine the invasion of hybrids could increase reliability in identification of hybrids in the field.
There are a number of potential reasons why hybridization may not be widespread in the upper Snake River. Rainbow trout stocking occurrences were limited to discrete areas within the drainage and were not as widespread or repeated as stocking in the river below Palisades Reservoir (Henderson 1998). Reproductive isolation may be important in preventing hybridization between related fish species (Hubbs 1955; Leary et al. 1995). Spatial or temporal separation (Thurow 1988; Huston et al. 1984; Likenes and Graham 1988) during spawning may prevent hybridization in the few streams where introduced rainbow trout and native cutthroat trout coexist. For example, fewer YSCxRBT hybrids were found in tributaries to the Snake River below Palisades Reservoir when compared to the number of hybrids in the mainstem (Henderson et al. 2000). Hybrids that were found in tributaries were typically in the lower portions of tributaries, while pure YSC were found higher in the drainage. Given the limited numbers of hybrids that we found, it is difficult to determine the direction of hybridization within the headwaters tributaries. Follow up studies should be conducted in the areas where hybrids were currently identified to determine whether the proportion of hybrids is changing.
One caution that should be noted is that very few of our fish came from the mainstem Snake River. Given our sampling we have little idea of the extent of hybridization that exists in the main river from Jackson Lake to Palisades Reservoir. If patterns of invasion below Palisades Reservoir are any indication, the initial invasion front may be located in the main river and not further up the tributaries. There appears to be no environmental gradient that would retard or stop RBT or YSCxRBT hybrids from occupying portions of the main river or tributaries. This is a concern given that a wild, reproducing RBT population (including RXC) is present and connected (via Palisades Reservoir) in the Salt River drainage (Figure 1). Observations on the spread of hybridization in the Flathead River system indicate that the presence of neighboring populations of hybrids is more important than environmental characteristics, particularly when environmental conditions are favorable for both species (Hitt et al. 2003). Further investigation of the mainstem should be attempted to determine the presence and extent of hybrids to inform future management.
Management Recommendations
Initiate a landscape level analysis using this sample set and nuclear markers (microsatellites) to understand historic geologic and hydrologic conditions that may explain the patterns of genetic variability observed in this study.
While not conclusive, frequency differences among drainages suggest that there should be caution in translocating cutthroat trout among drainages.
Initiate mainstem Snake River investigation to better determine the presence, location, and extent of hybridization in the river between Palisades Reservoir and Jackson Lake.
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APPENDIX A
Table 18 Summary of the number of records, by river drainage, of individual fish, and the approximate number of those fish that were photographed and/or a caudal fin clip collected. River drainages are listed as they generally occur from north to south.
| |Fish |Photographs |Tissue |
| | | | |
|Buffalo Fork |58 |58 |58 |
|Snake River |3,744 |1,302 |1,230 |
|Gros Ventre River |2,254 |1,051 |709 |
|Hoback River |1,131 |508 |495 |
|Greys River |1,597 |905 |881 |
|Salt River |364 |138 |124 |
|Total |9,148 |3,962 |3,497 |
Table 19 Total genomic DNA extractions for several streams1 in each of five geographic areas. The number of extractions per stream varied due to stream length, numbers of fish captured over the minimum size (>150 mm), and number of samples available for each putative cutthroat trout morphotype1. The geographic areas are arranged as they generally occur from north to south. A history of cutthroat trout stocking in each stream is provided.
|Drainage and Stream |Total |Type 1 |Type 2 |Type 3 |Historically |
| | | | | |Stocked2 |
| | | | | | |
|Totals |1279 |123 |907 |249 | |
| | | | | | |
|JACKSON HOLE | | | | | |
|Cascade Cr3 |9 | | |9 |N |
|Death Canyon3 |11 | | |11 |N |
|Ditch Cr, Middle Fork4 |44 |21 | |23 |N |
|Flat Cr |19 | |11 |8 |Y 1990 |
|Leigh Canyon3 |18 | |1 |17 |Y |
|Mosquito Cr |11 | |11 | |Y 1970 |
|Mosquito Cr, North Fork |3 | |3 | |N |
|Pacific Cr4 |12 |3 |5 |4 |Y 1980 |
|Paintbrush Canyon3 |18 | | |18 |N |
|Snake R |25 |0 |20 |5 |Y 1960 |
|Spread Cr, South Fork4 |25 |14 |3 |8 |Y 1960 |
|Subtotal |195 |38 |54 |103 | |
| | | | | | |
|GROS VENTRE | | | | | |
|Calf Cr |12 |4 |4 |4 |N |
|Cottonwood Cr |38 |8 |21 |9 |Y |
|Fish Cr, North Fork |32 |5 |19 |8 |N |
|Fish Cr, South Fork |20 |5 |13 |2 |Y 1990 |
|Gros Ventre R |94 |0 |81 |13 |Y 2000 |
|Leeds Cr |12 |4 |7 |1 |N |
|Maverick Cr |11 |0 |7 |4 |N |
|Moccasin Cr |26 |4 |15 |7 |N |
|Papoose Cr |9 |1 |5 |3 |N |
|Park Cr |23 |3 |17 |3 |N |
|Raspberry Cr |19 |3 |10 |6 |N |
|Strawberry Cr |3 |1 |0 |2 |N |
|Tepee Cr |5 |2 |1 |2 |Y |
|Subtotal |304 |40 |200 |64 | |
(continued)
Table 19 Continued.
|Drainage and Stream |Total |Type 1 |Type 2 |Type 3 |Historically |
| | | | | |Stocked2 |
| | | | | | |
|HOBACK | | | | | |
|Bare Cr |2 | |2 | |N |
|Bondurant Cr |18 |3 |13 |2 |N |
|Boulder Cr |22 |1 |19 |2 |N |
|Bull Cr |23 |4 |14 |5 |N |
|Cliff Cr |9 | |9 | |Y |
|Dell Cr |26 |1 |25 | |Y 1970 |
|Fisherman Cr |4 | |4 | |Y 1970 |
|Fisherman Cr, Middle Fork |6 | |6 | |N |
|Fisherman Cr, North Fork |12 | |11 |1 |Y 1970 |
|Hoback R |85 |2 |82 |1 |Y 1990 |
|Jack Cr |14 |1 |12 |1 |Y 1960 |
|Kerr Cr |3 |2 |1 | |Y |
|Little Granite Cr |17 | |17 | |Y 1960 |
|Mumford Cr |10 | |9 |1 |N |
|Phosphate Cr |6 | |6 | |N |
|Rim Draw |2 | |2 | |N |
|Shoal Cr |13 | |13 | |Y 1970 |
|Snag Cr |1 | | |1 |N |
|West Shoal Cr |6 | |5 |1 |N |
|Willow Cr |35 | |35 | |Y |
|Subtotal |314 |14 |285 |15 | |
| | | | | | |
|SNAKE RIVER CANYON | | | | | |
|Bailey Cr |7 | |5 |2 |Y |
|Bailey Cr, West |2 | |2 | |N |
|Cabin Cr |11 |1 |6 |4 |Y |
|Coburn Cr |19 |1 |4 |14 |Y |
|Dog Cr |7 | |6 |1 |Y |
|Fall Cr |16 | |15 |1 |Y |
|Fall Cr, North Fork |12 |3 |5 |4 |Y |
|Fall Cr, South Fork |7 |3 |2 |2 |Y |
|Horse Cr |12 |1 |10 |1 |Y |
|Pine Cr |5 | |4 |1 |N |
|Pritchard Cr |6 | |3 |3 |Y |
|Subtotal |104 |9 |62 |33 | |
(continued)
Table 19 Continued.
|Drainage and Stream |Total |Type 1 |Type 2 |Type 3 |Historically |
| | | | | |Stocked2 |
| | | | | | |
|GREYS | | | | | |
|Blind Trail Cr |38 |3 |34 |1 |Y |
|Firebox Cr |2 | |2 | |N |
|Flat Cr |14 |3 |9 |2 |N |
|Greys R |113 |1 |110 |2 |Y 1990 |
|Little Greys R |24 |3 |15 |6 |Y |
|Little Greys R, South Fork |24 |3 |15 |6 |Y |
|Lynx Cr |3 | |1 |2 |N |
|Murphy Cr |4 | |4 | |Y |
|Murphy Cr, North Fork |4 | |4 | |Y |
|North Corral Cr |18 | |18 | |N |
|Three Forks Cr, North5 |29 |4 |19 |6 |Y 1960 |
|Sheep Cr |11 | |11 | |Y 1970 |
|Spring Cr |15 | |15 | |Y |
|Squaw Cr |4 | |4 | |Y |
|Steer Cr |25 |1 |23 |1 |Y |
|Stewart Cr |13 | |12 |1 |N |
|Unnamed Tributary to |8 |1 |2 |5 |N |
|Lower Cabin Cr5 | | | | | |
|Upper Cabin Cr5 |9 |3 |5 |1 |N |
|White Cr |4 | |3 |1 |N |
|Subtotal |362 |22 |306 |34 | |
| | | | | | |
1 Streams were selected based on the following criteria:
1) Assume no genetic structure associated with spotting pattern;
2) No history of stocking, to extent possible;
3) Connectivity both within and among river drainages;
4) Streams stratified across the 5 geographic areas;
5) Streams spatially representative within each major river drainage;
6) Maximize age-class or size-groups within each stream;
7) Samples selected from throughout occupied length of stream;
8) Select up to 30 samples per stream for analysis; and
9) In longest streams, segregate sample populations according to stream segments.
10) Spotting pattern morphotypes include: Type 1=large-sparse spots; Type 2=fine-dense spots; and Type 3=intermediate to types 1 and 2.
2 Streams with record of stocking any form of cutthroat trout (WGFD 1997). Last stocking after 1960 is indicated by decade.
3 CUT samples from above confirmed natural barriers to upstream fish movement.
4 Additional tributaries to stream are available with fish exhibiting large-sparse spotting morphotype.
5 Samples from above road culvert identified as barrier to upstream movement of at least one life-stage of cutthroat trout.
Table 20 Lists the streams and samples, by geographic area1, selected for sequencing the mtDNA ND2 gene region. Contiguous sequences (~1,100 bp) of n=324 samples were completed.
|Drainage and Stream |Total |Type 1 |Type 2 |Type 3 |
| | | | | |
|TOTAL |543 |91 |256 |196 |
| | | | | |
|JACKSON HOLE | | | | |
|Cascade Cr2 |9 |0 |0 |9 |
|Death Canyon2 |9 |0 |0 |9 |
|Ditch Cr, Middle Fork3 |20 |11 |0 |9 |
|Flat Cr |11 |0 |5 |6 |
|Leigh Canyon2 |18 |0 |1 |17 |
|Pacific Cr3 |11 |3 |4 |4 |
|Paintbrush Canyon2 |17 |0 |0 |17 |
|Snake R |55 |0 |50 |5 |
|Spread Cr, South Fork3 |23 |14 |3 |6 |
|SUBTOTAL |173 |28 |63 |82 |
| | | | | |
|GROS VENTRE | | | | |
|Calf Cr |12 |4 |4 |4 |
|Fish Cr, North Fork |17 |5 |7 |5 |
|Gros Ventre R |49 |0 |38 |11 |
|Moccasin Cr |21 |4 |10 |7 |
|Papoose Cr |7 |1 |3 |3 |
|Park Cr |9 |3 |6 |0 |
|Raspberry Cr |15 |3 |6 |6 |
|Strawberry Cr |3 |1 |0 |2 |
|Tepee Cr |5 |2 |1 |2 |
|SUBTOTAL |138 |23 |75 |40 |
| | | | | |
|HOBACK | | | | |
|Bondurant Cr |8 |4 |2 |2 |
|Boulder Cr |6 |1 |3 |2 |
|Bull Cr |14 |2 |7 |5 |
|Dell Cr |3 |1 |2 |0 |
|Hoback R |40 |1 |27 |12 |
|Jack Cr |5 |1 |3 |1 |
|Kerr Cr |3 |2 |1 |0 |
|SUBTOTAL |79 |12 |45 |22 |
(continued)
Table 20 Continued.
|Drainage and Stream |Total |Type 1 |Type 2 |Type 3 |
| | | | | |
|SNAKE RIVER CANYON | | | | |
|Cabin Cr |7 |1 |3 |3 |
|Coburn Cr |13 |1 |2 |10 |
|Fall Cr, North Fork |6 |2 |2 |2 |
|Fall Cr, South Fork |7 |2 |2 |3 |
|Horse Cr |3 |1 |1 |1 |
|SUBTOTAL |36 |7 |10 |19 |
| | | | | |
|GREYS | | | | |
|Blind Trail Cr |9 |3 |5 |1 |
|Flat Cr |8 |3 |3 |2 |
|Greys R |48 |3 |31 |14 |
|Little Greys R |14 |3 |6 |5 |
|Steer Cr |4 |1 |2 |1 |
|Stewart Cr |4 |0 |3 |1 |
|Three Forks Cr, North4 |19 |4 |9 |6 |
|Unnamed Tributary, Lower Cabin Cr4 |5 |1 |2 |2 |
|Upper Cabin Cr4 |6 |3 |2 |1 |
|SUBTOTAL |117 |21 |63 |33 |
| | | | | |
1 Streams were selected based on the following criteria:
1) Ensure variation in spotting patterns within each stream was documented by surveys throughout the occupied length of a stream;
2) There was no history of stocking, or at least recent stocking, in each stream to the extent possible;
3) Connectivity existed among all streams selected, both within and between the geographic areas;
4) Minimize spatial clustering of samples within a stream, to the extent possible, by selecting samples from throughout the occupied length of each stream;
5) Minimize spatial clustering of streams, to extent possible, by selecting streams from throughout each geographic area;
6) Ensure that streams were stratified across the five geographic areas;
7) Ensure that streams were stratified within each of the five geographic areas;
8) Include samples from each of the available age classes or size groups within each stream;
9) Include a minimum of n=30 fish from each geographic area or river drainage, where possible, that exhibit the large-sparse spotting pattern (i.e., YSC);
10) Segregate fish into three distinct spot pattern morphotypes: Type 1 – large-sparse, Type 2 – fine-dense, and Type 3 – intermediate to 1 and 2;
11) Morphotypes present must be confirmed based on photographic records from surveys; and
12) Samples to be included in the analysis should be only from those streams where the large-sparse morphotype was observed.
2 CUT samples from above natural barriers to upstream fish movement.
3 Additional tributaries available with fish exhibiting large-sparse spotting morphotype.
4 Samples from above road culvert identified as barrier to upstream movement of at least one life-stage of cutthroat trout.
Table 21 Number of cutthroat trout1 of the haplotypes A-M, per stream, within five geographic areas in the Snake River study area. The Snake River is split into two geographic areas, Jackson Hole and Snake River Canyon. The geographic areas are arranged as they generally occur from north to south.
| | | | | |Haplotypes | | | | | | | |Drainage and Stream |A |B |C |D |E |F |G |H |I |J |K |L |M |Total | | | | | | | | | | | | | | | | | |Total |64 |41 |50 |132 |2 |1 |1 |19 |1 |8 |1 |3 |1 |324 | | | | | | | | | | | | | | | | | |JACKSON HOLE | | | | | | | | | | | | | | | |Cascade Cr2 | | |6 |2 | | |1 | | | | | | |9 | |Death Canyon2 | | |9 | | | | | | | | | | |9 | |Ditch Cr, Middle Fork |1 |1 |4 |5 | | | | | | | | | |11 | |Flat Cr | |2 | |7 | | | | | | | | | |9 | |Leigh Canyon2 | |3 |9 |6 | | | | | | | | | |18 | |Pacific Cr | |1 |3 |5 | | | |1 | | | | | |10 | |Paintbrush Canyon2 | | |14 | | | | | | | | | | |14 | |Snake R | |15 |2 |25 | | | |5 | |4 | | |1 |52 | |Spread Cr, South Fork | | |1 |3 | | | | | |3 | | | |7 | |Subtotal |1 |22 |48 |53 |0 |0 |1 |6 |0 |7 |0 |0 |1 |139 | | | | | | | | | | | | | | | | | |GROS VENTRE | | | | | | | | | | | | | | | |Calf Cr | | |1 |6 | | | | | | | | | |7 | |Fish Cr, North Fork | |1 | |5 | | | | | | | | | |6 | |Gros Ventre R |3 |5 | |10 | | | | | | |1 |2 | |21 | |Moccasin Cr | | | |4 | | | | | | | | | |4 | |Papoose Cr |1 | | | | | | | | | | | | |1 | |Park Cr |4 | | |2 | | | |2 | | | | | |8 | |Raspberry Cr | | | |2 | | | |5 | | | | | |7 | |Strawberry Cr | | | |3 | | | | | | | | | |3 | |Tepee Cr | | | |5 | | | | | | | | | |5 | |Subtotal |8 |6 |1 |37 |0 |0 |0 |7 |0 |0 |1 |2 |0 |62 | |
(continued)
Table 21 Continued.
| | | | | |Haplotypes | | | | | | | |Drainage and Stream |A |B |C |D |E |F |G |H |I |J |K |L |M |Total | | | | | | | | | | | | | | | | | |HOBACK | | | | | | | | | | | | | | | |Bondurant Cr | | | |2 | | | | | | | | | |2 | |Boulder Cr |1 | | |1 | | | | | | | | | |2 | |Bull Cr |11 | | | | | | | | | | | | |11 | |Dell Cr | | | |2 | | | | | | | | | |2 | |Hoback R |4 |4 | |7 | | | | | |1 | | | |16 | |Jack Cr | |3 | |1 | | | | | | | | | |4 | |Kerr Cr |2 | | |1 | | | | | | | | | |3 | |Subtotal |18 |7 |0 |14 |0 |0 |0 |0 |0 |1 |0 |0 |0 |40 | | | | | | | | | | | | | | | | | |SNAKE RIVER CANYON | | | | | | | | | | | | | | | |Cabin Cr |1 | | |1 | | | | | | | |1 | |3 | |Coburn Cr |8 | | | | | | | |1 | | | | |9 | |Fall Cr, North Fork |5 | | | | | | | | | | | | |5 | |Fall Cr, South Fork |7 | | | | | | | | | | | | |7 | |Horse Cr |1 | | |1 | | | | | | | | | |2 | |Subtotal |22 |0 |0 |2 |0 |0 |0 |0 |1 |0 |0 |1 |0 |26 | |
(continued)
Table 21 Continued.
| | | | | |Haplotypes | | | | | | | |Drainage and Stream |A |B |C |D |E |F |G |H |I |J |K |L |M |Total | | | | | | | | | | | | | | | | | |GREYS | | | | | | | | | | | | | | | |Blind Trail Cr |1 | | |5 | | | | | | | | | |6 | |Flat Cr |5 | | | | | | | | | | | | |5 | |Greys R |3 |4 | |6 | |1 | |2 | | | | | |16 | |Little Greys R | |1 | |11 | | | |1 | | | | | |13 | |Steer Cr |2 |1 | |1 | | | | | | | | | |4 | |Three Forks Cr, North3 |1 | |1 |3 |2 | | |1 | | | | | |8 | |Unnamed Tributary to
Lower Cabin Cr3 |1 | | | | | | |2 | | | | | |3 | |Upper Cabin Cr3 |2 | | | | | | | | | | | | |2 | |Subtotal |15 |6 |1 |26 |2 |1 |0 |6 |0 |0 |0 |0 |0 |57 | | | | | | | | | | | | | | | | | |1 Samples were selected by stream based on the following criteria:
1) Ensure variation in spotting patterns within each stream was documented by surveys throughout the occupied length of a stream;
2) No history of stocking, or at least recent stocking, in each stream to the extent possible;
3) Connectivity existed among all streams selected, both within and between the geographic areas;
4) Minimize spatial clustering of samples within a stream, to the extent possible, by selecting samples from throughout the occupied length of each stream;
5) Minimize spatial clustering of streams, to extent possible, by selecting streams from throughout each geographic area;
6) Ensure that streams were stratified across the five geographic areas;
7) Ensure that streams were stratified within each of the five geographic areas;
8) Include samples from each of the available age classes or size groups within each stream;
9) Include a minimum of n=30 fish from each geographic area or river drainage, where possible, that exhibit the large-sparse spotting pattern (i.e., YSC);
10) Segregate fish into three distinct spot pattern morphotypes: (1) large-sparse, (2) fine-dense, and (3) intermediate to 1 and 2;
11) Morphotypes present must be confirmed based on photographic records from stream surveys; and
12) Samples to be included in the analysis should be only from those streams where the large-sparse morphotype was observed.
2 Samples from above confirmed natural barriers to upstream fish movement.
3 Samples from above road culvert identified as barrier to upstream movement of at least one life-stage of cutthroat trout.
APPENDIX B
FISH PHOTOGRAPH LIBRARY: JPEG FILES ARE THE COMPLETE SET OF PHOTOGRAPHS ASSOCIATED WITH TISSUE SAMPLES. EACH INDIVIDUAL FISH PHOTOGRAPH HAS A UNIQUE IDENTIFIER, PIC_NUM, ON THE FISH SHEETS IN THE DATABASE LIBRARY (SEE BELOW) IS USED AS THE FILE NAME.
Database Library: separate EXCEL files for each river drainage with complete data set from presence-absence surveys. One additional file is metadata for survey data.
Cutthroat trout sample location and extraction information: the file ext_samp_data_060105.xls contains individual sheets by river drainage that includes each sample identified for extraction, as well as a summary table. Also contains individual sheets for each river drainage that include each sample identified for amplification and use in mtDNA sequencing, as well as a summary table.
Out group sample sources and extraction information: the file gen_samp_outgroup_053105.xls contains individual sheets for each out group. Each sheet provides source and individual sample data when available.
Mitotype data generated from mitochondrial DNA sequences: the file mitotype _data_053105.xls is the raw data generated from the unique mtDNA sequences.
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