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 (), 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 rainbowcutthroat 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
v
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 AM. 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,
vi
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
vii
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 nonrandom distribution of haplotypes among drainages (2 = 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 rainbowcutthroat 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.
viii
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 nonnative 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
ix
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
x
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 (2 = 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.
xi
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, overfishing, 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
1
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
2
Approximate historical
range of Snake River
cutthroat trout
Continental Divide
Figure 1 Historical transcontinental range of Yellowstone cutthroat trout, with finespotted Snake River
cutthroat trout historical range indicated.
3
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.
4
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.
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.
Figure 4 A cutthroat trout exhibiting a common intermediate spot pattern, with small to medium spots that
are concentrated toward the caudal peduncle.
5
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
6
Jack
son
Lake
Jackson Hole
Gro
s Ve
ntre
Snake River
Canyon
es
ad
lis
Pa
Ho
b
ac
k
oir
rv
se
Re
Wyoming
Idaho
ys
Gre
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
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);
8
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
<1.0 m. Angling or boat electrofishing surveys were conducted with a single fisher or
netter on larger streams where backpack electrofishers were ineffective.
Our sampling goal was to provide a reasonable probability of detecting YSC
versus SRC within any one stream occupied by cutthroat trout. When sampling across
an environmental gradient (e.g., a stream flowing down an elevation gradient), or the
logistical demands or cost of systematic sampling approaches that of random sampling,
systematic sampling may be pursued (Krebs 1999). Assuming a sampling efficiency of
0.50 for each site, a Poisson sampling distribution, and a minimum of 150 YSC in
10,000 m of stream, the probability of detection should exceed 0.80. Assuming 50 m of
stream sampled at 1,000 m intervals, the expected minimum number of fish detected is:
 = (150/10,000 m) x 500 m sampled x 0.50, and the probability of detection no fish is
given by Equation 1 as;
Equation 1
P0  e   = 0.02 (Zar 1999)
then P(1 or more) = 0.98 (after Rieman and McIntyre 1995).
9
Stream Sample Intervals
Systematic sample intervals were based on total perennial stream length on
1:24,000 topographic maps. A minimum of ten reach intervals were delineated (e.g.,
5.0 km map length divided by 10 = minimum 10 sample reaches at 500-m intervals) by
rounding to the nearest 50 or 100 m. Maximum interval distance regardless of total
stream length is 2000 m. For streams with intervals <1,000 m, all reaches are
measured using a drag tape while walking the stream bank or paced. Streams with
sample intervals >1,000 m were accessed directly by road or trail without actual
measuring or pacing of the stream length. One crew member generally walked the
stream bank to confirm mapped sample reach breaks, while the remaining crew
members drove or hiked to the next reach.
Stream Sampling Protocols
Single-pass electrofishing was conducted by subsampling 50-m to 100-m within
each reach. Subsample length was determined by capture of a minimum of three fish
>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.
10
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 – Young-of-the-year (age 0; 25-65 mm) and age I+ fish
(approximately 80-110 mm) were often encountered and cannot be positively identified
as YSC or SRC. Once confirmed as cutthroat trout (e.g., streams where juvenile or
adult BKT or RBT have been captured), YOY or I+ were noted in comments and
species recorded as CUT. In addition, cutthroat trout 100-150 mm were common and
could not be confirmed as either YSC or SRC due to lack of a distinct spot pattern and
retention of parr marks (a distinct series of dark vertical bars along each side of the fish,
elongate-oval in shape, that appear as a shadow and may exhibit some reddish or
brown coloration). These fish were recorded as CUT with comments on the characters
resulting in no subspecies determination.
Rainbow Trout – Rainbow trout typically are dark green to blue-green dorsally, with
silvery sides, and a distinct bright red or pink lateral stripe. Small irregular black spots
are dense on the head, body and fins. The paired fins have white margins. The
cutthroat “slash” may be present, though typically less conspicuous in rainbow trout or
RXC hybrids.
Introgression with rainbow trout, though rare in the Snake River headwaters, is
possible. Rainbow trout are known to be present in the Gros Ventre River below Lower
Slide Lake, Stump Lake and Fawn Creek in the Greys River drainage, and in Rainbow
Lake and the headwaters of the South Fork of the Buffalo Fork River where they have
been stocked in the past. A good field identification feature of hybridization with
cutthroat trout is white margins along the leading edge of the pectoral, pelvic or anal fins
(Baxter and Stone 1995); white fin margins may be accompanied by profuse spotting on
the head. More difficult to assess in the field are the presence of basibranchial teeth on
the vomer (a characteristic of cutthroat trout). Such hybrids were recorded as RXC and
the characters suggesting hybridization noted.
11
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.
Species
ID
BHS
BKT
BNT
Native
Game
Common Name
Genus
Species
Subspecies
Drainage2
Fish
Catostomus
discobolus
bluehead sucker
N
1,4,7
Salvelinus
fontinalis
brook trout
Y
Salmo
trutta
brown trout
Y
Bonneville
(Bear River)
Oncorhynchus clarkii
utah
BRC
cutthroat trout
Y
9
Colorado River
Oncorhynchus clarkii
pleuriticus
CRC
cutthroat trout
Y
4,7
Oncorhynchus clarkii
CUT
cutthroat trout
Y
Pimephales
promelas
FHM
fathead minnow
N
2,3,5,6
Oncorhynchus mykiss
aguabonita
GDT
golden trout
Y
Thymallus
arcticus
GRL
grayling
Y
2
Poecilia
reticulata
GUP
guppy
N
Oncorhynchus nerka
KOE
kokanee
Y
Salvelinus
namaycush
LAT
lake trout
Y
Rhinichthys
cataractae
LND
longnose dace
N
1,2,3,5,6,8
Snyderichthys
copei
LSC
leatherside chub
N
1,9
Cottus
bairdii
MSC
mottled sculpin
N
1,4,7,9
Catostomus
platyrhynchus
MTS
mountain sucker
N
1,2,4,7,8,9
williamsoni
MWF
mountain whitefish Prosopium
Y
1,2,3,4,7,8,9
NOD
No Data/Unknown
N
OOO
No fish Present
N
Cottus
beldingii
PSC
Paiute sculpin
N
1,9
Oncorhynchus mykiss
RBT
rainbow trout
Y
cutbow
RXC
(RBT x CUT)
Y
Richardsonius balteatus
RSS
redside shiner
N
1,9
Rhinichthys
osculus
SPD
speckled dace
N
1,4,7,9
splake
SPK
(BKT x LKT)
Y
finespotted
Snake River
Oncorhynchus clarkii
subspecies
SRC
cutthroat trout
Y
1
tiger trout
TGT
(BKT x BNT)
Y
TRT
any trout
Y
Gila
atraria
UTC
Utah chub
N
1,9
Catostomus
ardens
UTS
Utah sucker
N
1,9
Yellowstone
Oncorhynchus clarkii
bouveri
YSC
cutthroat trout
Y
1,2,3,8
1 Sources included Nelson et. al. 2004. Baxter and Stone 1995, and Behnke 1992.
2 Drainage Code: 1 - Snake River; 2 - Big Horn River, Shoshone River, Wind River; 3 - Powder River; 4 Green River; 5 - North Platte River; 6 - Little Missouri River, Cheyenne River, Niobrara River, Belle
Fouche River; 7 - Little Snake River; 8 - Yellowstone River; 9 - Bear River.
12
Fish Metrics, Photographs, and Tissue Samples
Up to 10 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 <175
mm. In those cases, not more than one-quarter of the caudal fin was removed.
Photographs and fin clips were identified by a common alpha-numeric code as follows:




SA = Salt subbasin; GH = Greys-Hoback subbasin; GV = Gros Ventre
subbasin; SH = Snake headwaters subbasin;
Year = 98, 99, 00, 01, 02, or 03;
Camera = 01, 02, 03;
1, 2, 3….. k = sequential number of photograph during a sample year with
a given camera.
For example, the 25th fish collected in the Greys-Hoback subbasin using camera
#3 during 2002 was identified as ‘GH020325’. The alpha-numeric was recorded on
the data sheet for the respective fish, and on the fin clip bottle. Each bottle was labeled
with the date, stream name, sample reach distance, and field identification as SRC,
YSC, or CUT.
Genetic Analysis
Tissue samples were collected from throughout the study area as described
above between 1999 and 2003, and selected for inclusion based on the criteria as
stated previously. Additional samples from the Snake River collected in 2004 by WGFD
personnel were included upon request; those samples were screened specifically for
rainbow-cutthroat trout hybrids. The approximate number of samples available for this
and subsequent analyses are presented in Appendix A, Table 18.
Extraction of DNA
Total genomic DNA was extracted from approximately 1,280 samples (Appendix
A Table 19) using a salting out technique adapted from Sunnucks and Hales (1996),
and DNA quality and quantity were assessed using 0.7% agarose gels with appropriate
size (100-base-pair [bp] ladder) and concentration ( Hind III digest) standards.
Samples were diluted to approximately 10 ng/ul in 1X Te buffer (10 mM Tris, 0.1 mM
EDTA; pH 8.0). The DNA extractions are located at Utah State University, archived in a
-80°C freezer, and available for future research.
13
Methods by Objective
Objective 1 – Develop cost-effective, reliable, and repeatable molecular tools that
will answer the study questions
Primer design and optimization for mitochondrial sequence data - An
existing set of polymerase chain reaction (PCR) primers (ND12L and ND12H; Table 2;
Toline et al. 1999) was available for amplification of an ~3,500 bp region of the
mitochondrial NADH gene (including portions of subunits 1 and 2; ND1-2) in trout.
Amplicons produced using these primers have previously been used in restriction
fragment length polymorphism (RFLP) analysis in cutthroat and rainbow trout (Campbell
et al. 2002; Toline et al.1999). We sought to sequence this entire region in a subset of
fish from our study landscape, identify the region containing the most variation, and
design new primers to amplify this (these) smaller region(s). We chose this approach
because sequence data can be more informative than RFLP data in assessing
phylogeographic relationships among populations.
Our sample subset included sixteen samples representing the four major river
drainages in the study area (Greys R, Gros Ventre R, Hoback R, and Snake R) (Table
3). Streams within drainages, and individuals within streams, were selected based on
the presence of L, F, and I morphotypes. A series of internal primers developed by the
Idaho Fish & Game (IDFG) Eagle Genetics Laboratory (Table 2) and our laboratory was
used to obtain sequences from the ND1-2 region. Contiguous sequences for these 16
individuals were constructed and aligned using DNAStar software (Lasargene, Inc.). In
this subset of fish, 15 polymorphic sites were identified in the ~3,500 bp ND1-2 region.
Ten of these sites were concentrated in a 670-bp region of the ND2 gene. We designed
primers and optimized conditions for the amplification of this region using only two PCR
primers (Table 2).
Amplification of the mitochondrial DNA ND2 gene region – An approximately 1,100
base-pair (bp) amplicon containing the NADH dehydrogenase 2 (ND2) gene region of
the mitochondrial genome was amplified using polymerase chain reaction (PCR).
Primers specific for the ND2 gene region – (NDintF4) TAA GCT TTC GGG CCC ATA
CC and (NDvarR) GCT TTG AAG GCT CTT GGT CT – were purchased from Integrated
DNA Technologies, Inc (Coralville, IA). Each 25-L PCR reaction contained 20-50 ng of
extracted DNA template, 1 X PCR buffer, 0.2 mM deoxynucleotide triphosphates
(dNTPs), 2.5 mM MgCl2, 5.0 M of each primer, and 1.25 units (U) of Taq polymerase.
The reaction was denatured at 95o C for 2 min, followed by 30 cycles of 94o C for 1 min,
58o C for 1 min, and 72o C for 1 min 20 sec, with a final 10-min extension at 72o C.
The entire amplified product was sent to the Nevada Genomic Center (NGC) at
the University of Nevada – Reno to be purified, quantitated, and sequenced. Amplicons
were purified using a Qiagen MinElute filter plate on the Qiagen BioRobot 3000, and
quantified with a fluorescent nucleic acid stain (PicoGreen®) and read on a Labsystems
Fluoroskan Ascent fluorescence plate reader. Using the primers described above,
sequencing reactions were performed from both ends of the
14
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.
Primer Name
Sequence
Source
ND12L
5’ GCCTCGCCTGTTTACCAAAAACAT
Toline et al. 1999
ND12H
5’ CCGGCTCAGGCACCAAATAC
Toline et al. 1999
NDintR1
5’ CCTGATCCAACATCGAGGT
IDFG
NDIntF2
5’ ACCTCGATGTTGGATCAGG
IDFG
NDIntR3
5’ GCGTACTCGGCTAGGAAAAA
IDFG
NDIntF4
5’ GGGCAGTGGCACAAACTATT
IDFG
NDIntR5
5’ GGTATGGGCCCGAAAGCTTA
IDFG
NDIntF6
5’ TAAGCTTTCGGGCCCATACC
IDFG
NDIntR7
5’ GGGTCGGGGATTTAGTTCAT
IDFG
NDIntF8
5’ ATGAACTAAATCCCCGACCC
IDFG
Jess16sF
5’ ACCAAAAACATCGCCTCTTG
USU
JesstRNAR
5’ GGGGGAAAGTAGATGGATGC
USU
NDvarF
5’ GAC AAA AAC TCG CAC CCT TC
USU
NDvarR
5’ GCT TTG AAG GCT CTT GGT CT
USU
amplicons with an ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1,
and the reactions are then run on an ABI3730 DNA Analyzer. Two sequencing
reactions, one each forward and reverse, were sufficient to provide complete coverage
of the ND2 gene. For each individual, these two sequences were used to assemble a
contiguous sequence with DNASTAR SeqMan software (Lasergene). These
contiguous sequences were aligned with DNASTAR MegAlign software (Lasergene),
and the aligned sequences trimmed to a total length of 945 bp.
Primer selection and optimization for nuclear microsatellite data – Microsatellite
loci from three different sources were screened for utility in cutthroat trout from the study
landscape (Table 4): 1) LHC loci (10 loci developed for use in Lahontan cutthroat trout
[Peacock et al. 2004]); 2) IDFG loci (6 loci developed for a mix of species and used by
the Idaho Fish and Game Department, Eagle Genetics Laboratory, to assess
Yellowstone cutthroat trout population genetic structure in Idaho; Rexroad et al. 2002;
Wenberg and Bentzen 2001; Nelson and Beacham 1999; Olsen et al. 1998; Sakamoto
et al. 1994; Condrey and Bentzen 1988); 3) RGC loci (14 unpublished loci developed for
use in Rio Grande cutthroat trout for the New Mexico Department of Game and Fish
[K.Jones, Genetic Identification Services]). These loci were initially assessed for their
ability to produce an amplicon of the
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.
Drainage/Stream
Location
(m)
Species
Length
(mm)
Weight
(gm)
Sample ID
Cabin Creek
6,000
SRC
153
44.0
GH-01-02-06
Cabin Creek
6,000
CUT
161
40.0
GH-01-02-08
Flat Creek
48,000
CUT
192
64.0
GH-02-01-60
Flat Creek
48,000
SRC
168
48.0
GH-02-01-61
Pacific Creek
42,000
CUT
230
112.0
SH-03-02-467
Pacific Creek
32,000
SRC
238
110.0
SH-03-02-494
North Fork Fish Creek
8,000
SRC
360
410.0
GV-99-35-18
North Fork Fish Creek
8,000
CUT
172
55.0
GV-99-35-19
Bondurant Creek
2,000
SRC
206
86.0
GH-01-05-29
Bondurant Creek
2,000
YSC
194
71.0
GH-01-05-30
Bull Creek
3,000
YSC
199
105.0
GH-01-15-12
Bull Creek
36,00
SRC
190
90.0
GH-01-15-14
Blind Trail Creek
7,000
SRC
219
108.0
GH-00-24-08
Blind Trail Creek
7,000
YSC
225
104.0
GH-00-24-10
Flat Creek
3,000
SRC
184
73.0
GH-00-17-08
Flat Creek
3,500
YSC
218
81.0
GH-00-14-17
SNAKE RIVER
GROS VENTRE RIVER
HOBACK RIVER
GREYS RIVER
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.
Microsatellite
Group/Locus
Source
PCR
Successfully
Optimized
Polymorphic in
Study Samples
Protocols
Developed
Peacock et al. 2004
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
YES
NO
NO
YES
NO
YES
YES
NO
NO
NO
NO
NO
NO
NO
NO
YES
YES
NO
NO
NO
NO
NO
NO
YES
NO
YES
YES
NO
NO
NO
Sakamoto et al. 1994
Condrey and Bentzen 1988
Olsen et al. 1998
Rexroad et al. 2002
Nelson and Beacham 1999
Wenberg and Bentzen 2001
NO
YES
YES
YES
YES
YES
NO
YES
YES
YES
YES
NO
NO
YES
YES
YES
YES
YES
GIS
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
Ibid.
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
LHC GROUP
OCH5
OCH6
OCH9
OCH10
OCH11
OCH13
OCH14
OCH15
OCH16
OCH17
IDFG GROUP
Fgt3
Ocl1
Ogo4
Omm1036
Ots107
Ssa85
RGC GROUP
G20
H12
H18
H114
H118
H126
H204
H220
J3
J14
J103
J132
K216
17
expected size under standard PCR conditions. Limited protocol optimization was
attempted for loci producing visible amplicons of the appropriate size (based on 1%
agarose gel electrophoresis, with ethidium bromide staining and visualization under UV
light). Fluorescent dye-labeled primers were ordered for LHC and IDFG primers
yielding reliable amplification, and amplicons were assessed for polymorphism in the
same subset of cutthroat trout samples as described above (Table 3). The trial subset
varied from locus to locus. Optimization of RGC loci was suspended after confirmation
of amplicons, and therefore, no polymorphism or protocol information is presented.
Objective 2a – Determine morphological differences between the two
morphotypes of cutthroat trout (YSC & SRC) in the study landscape
We used the only recognized morphological pattern that identifies SRC from YSC, spot
size and distribution, as an initial filter for field determinations during sample collection.
A second level filter was applied that we based on further spot pattern analysis of our
photographic records (scanned 35 mm slide or digital). This
classification of cutthroat trout resulted in fish being binned as one of three distinct spot
pattern morphotypes: Type 1 – large-sparse (L), Type 2 – fine-dense (F), and Type 3 –
intermediate (I) to 1 and 2. Intermediate fish could not be clearly binned as 1 or 2 due
to exhibiting shared characteristics of YSC and SRC as described in the Fish Species
Identification section. The photograph of each specimen was reviewed and assigned a
morphotype, prior to inclusion of its tissue sample for DNA extraction. A 6-cell grid was
visualized on the fish as follows (after Quadri 1959 as adapted by Kruse 1998):
1) Anterior insertion of adipose fin below the lateral line;
2) Same as area 1 except above the lateral line;
3) Behind anterior insertion of the dorsal fin below the lateral line back to boundary
of area 1;
4) Same as three except above the lateral line back to boundary of area 2;
5) Below lateral line forward from boundary of area 3 to opercle; and
6) Same as area 5 except above the lateral line forward from boundary of area 4.
Fish assigned to morphotype 1 had spots predominantly 3-5 mm in diameter, and
concentrated in cells 1-2 and 4. Those assigned to morphotype 2 had spots
predominantly 1-2 mm in diameter, and typically well distributed throughout at least 5 of
the 6 cells; cell 5 typically had comparatively fewer spots. Fish assigned to morphotype
3 generally exhibited an intermediate spot size (2-4 mm diameter) with spots either well
distributed or concentrated toward the caudal peduncle.
Spotting patterns were also used by Kruse (1998) to accurately classify
genetically pure YSC and SRC, but were of little utility in identifying hybrid individuals
(YSCxSRC). However, Kruse found that visual field classifications based on spotting
patterns, and the presence of throat slashes and white fin margins or tips performed
better than discriminant models with either meristic features or spotting patterns in
identifying genetically pure cutthroat trout and RBT, as well as indicating genetic
introgression (i.e., rainbow-cutthroat hybrids [RXC]).
18
Objective 2b – Determine genetic differentiation between the two morphotypes of
cutthroat trout (YSC & SRC) in the study landscape
Individual fish were assigned a morphotypic category (fine-dense, F;
intermediate, I; large-sparse, L; or anamolous) as described in Objective 2a methods
above. Photographs of these fish, and their assigned categories, are provided in
(Appendix B, CD - name files). Mitochondrial ND2 sequences (~1,100 bp) were
obtained for 324 fish representing all major drainages and all morphotypic categories
(Appendix B, CD - name files), following the protocol developed in Objective 1 (see
Results section, below). These sequences were trimmed to 945 bp and aligned using
DNAStar (Lasargene, Inc.) software. Individual sequences were collapsed into 13
distinct haplotypes.
Genetic differentiation between morphotypic categories was assessed by
comparing the distribution of haplotypes among these categories, first by pooling all
samples across the entire study area and second by comparing morphotypic groups
within geographic areas (Jackson Hole, Gros Ventre, Hoback, Snake River Canyon,
and Greys; Figure 5). Comparisons among morphotypic groups were based on the
average sequence divergence among all pairs of individuals within and between groups,
expressed as the number or proportion of pairwise nucleotide differences, using MEGA
v3.0 software (Kumar et al. 2004). Standard errors for these distances were estimated
in MEGA using 500 bootstrap replicates.
Objective 3 – Describe patterns of genetic variation in cutthroat trout within and
among major drainages in the study landscape
Five geographic areas were represented in our sampling scheme (Figure 5):
Jackson Hole (n=139), Gros Ventre (n=62), Hoback (n=40), Snake River Canyon
(n=26), and Greys (n=57). Because genetic differentiation among morphotypes was not
detected (see Results), morphotypes were pooled for the purpose of assessing
differentiation among drainages. Genetic differentiation among drainages was
assessed using three methods: a) average pairwise nucleotide differences within and
between drainages was characterized as described above for morphotypic groupings,
using MEGA software (Kumar et al. 2004); b) A Chi-square test of the distribution of
haplotypes (without regard to haplotype sequence divergence) among drainages was
performed using DnaSP software (Rozas et al. 2003); and c) Genetic differentiation
among pairwise drainages was characterized via the GST statistic (Nei 1987; Hudson et
al. 1992) using DnaSP software (Rozas et al. 2003).
Relationships among haplotypes were assessed by a) constructing a neighborjoining dendrogram based on the number of nucleotide differences with MEGA software
(Kumar et al. 2004), and b) constructing a haplotype network, based on statistical
parsimony, using TCS software v1.18 (Clement et al. 2000).
19
Genetic diversity within drainages was assessed by estimating nucleotide
diversity (π), haplotype diversity (Hd), and enumerating haplotypes, using DnaSP
software (Rozas et al. 2003).
Objective 4 – Assess introgression with rainbow trout using both morphologic
and genetic tools
For field identification we used the morphological features described in the Fish
Species Identification section for rainbow trout, and the following as the primary
indicator of rainbow trout hybridization with cutthroat trout (Kruse 1998; Baxter and
Stone 1995): white margins along the leading edge or tips of the pectoral, pelvic or anal
fins.
Genetic identification of rainbow trout and introgression with cutthroat in the
study landscape was assessed with the mitochondrial ND2 gene region and protocol
developed in Objective 1. Putative rainbow trout and rainbow-cutthroat hybrid
sequences were trimmed, aligned, and included in the haplotype collapse described in
Objective 2b. Relationships to the cutthroat trout haplotypes we identified were
assessed by inclusion of each rainbow trout and rainbow-cutthroat haplotype in the
neighbor-joining dendrogram we constructed; the rainbow trout ND2 sequence was
obtained from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/).
RESULTS
Survey Results
Our distribution surveys conducted in the Snake River headwaters since 1998
indicate present occupancy of cutthroat trout in >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 <14 km of
habitat (Table 6), and do not appear to have displaced native cutthroat trout in any of
the sample streams. Brook trout were found in all areas (Table 6). Brook trout may
have displaced cutthroat trout from 13 of the 81 streams they occupied, totaling 17 km
of habitat.
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, Table 7).
20
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.
Streams1
Reaches
Length (km)
Snake River 2
125
803
790
Greys River
93
678
500
Hoback River
64
393
385
Gros Ventre River
110
611
553
Buffalo Fork River 3
1
16
32
393
2,501
2,260
Drainage
Total
1
Includes main stem and tributaries unless otherwise noted.
Largely tributaries; only 14.0 km surveyed on main stem Snake River between Palisades Reservoir and
Jackson Lake dam.
3 Includes no tributaries. Only 32.0 km of Buffalo Fork River were surveyed between Snake River
confluence and bridge on Forest Road 30050.
2
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.
Cutthroat Trout 1 2
Drainage
Brook Trout 2
Hybrid Trout 2
All Trout
Stream
Length
Stream
Length
Stream
Length
Stream
Length
Snake River
87
410
42
123
-
-
96
489
Greys River
82
373
8
26
1
0.83
83
380
Hoback River 3
45
268
8
42
1
2.50
47
289
Gros Ventre
River3
79
408
22
71
2
10.0
80
437
Buffalo Fork River
1
24
-
-
-
-
1
24
294
1,483
80
261
4
13.33
307
1,619
4
Total
1
Includes YSC, SRC, and unidentified juvenile (<150 mm) or adult (>150 mm) cutthroat trout.
Occupied stream length for species indicated is total whether allopatric or in sympatry with other species
given.
3 Only rainbow-cutthroat trout hybrids were captured.
4 Includes no tributaries. Only 32.0 km of Buffalo Fork River were surveyed between Snake River
confluence and bridge on Forest Road 30050.
2
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.
Yellowstone
cutthroat trout
(Morphotype L)
Drainage
Snake River
cutthroat trout
(Morphotype F)
Yellowstone and
Snake River
Morphotypes
Streams
Length
Streams
Length
Streams
Length
Snake River
33
119
75
304
33
92
Greys River
17
21
77
328
16
16
Hoback River
13
28
37
230
12
24
Gros Ventre River
38
103
68
363
36
87
Buffalo Fork River
1
6
1
24
1
6
102
277
258
1,249
98
225
Total
Both large and fine spotted morphotypes were co-located in 98 streams, representing
225 km. Yellowstone cutthroat trout occur almost exclusively in sympatry with Snake
River cutthroat trout.
Genetic Structuring
Results by Study Objective
Objective 1 – Develop cost-effective, reliable, and repeatable molecular tools that
will answer the study questions
Amplification of the mitochondrial DNA gene region – A pair of PCR primers were
developed for use in upper Snake River cutthroat trout genetic analyses. These primers
reliably amplify a ~1,100 bp region of the ND2 mitochondrial gene, and can be used
both for amplification and sequencing:
Forward Primer: NDintF4 5’ GGGCAGTGGCACAAACTATT (IDFG)
Reverse Primer: NDvarR 5’ GCTTTGAAGGCTCTTGGTCT
PCR Reaction (25 ul volume): 4.0 ul of 5.0 mM dNTPs (final concentration 0.2
mM for each dNTP); 2.5 ul of 10x buffer; 2.5 ul of 25.0 mM MgCl; 0.5 ul of each
primer (10 uM stock); 0.25 ul of Taq polymerase (5.0 U/ul stock); 2.5 ul (20.0ng)
template.
22
PCR Thermal Profile: Hold @ 95o C for 2 min; 30 cycles of – 94o C for 1
min.,58oC for 1 min., 72o C for 1 min. 20 sec.; Final extension @ 72o C for 10
min.
Amplification of the nuclear DNA gene regions – Six polymorphic microsatellite loci
were identified for use in Snake River headwaters cutthroat trout genetic analyses. The
primer pairs reliably amplified these 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 the RGC primers:
Locus: OCH 13
Forward Primer: 5’ GGA GGT GAT TCT ATG GGT AAA T
Reverse Primer: 5’ CAG ATG GGC ACT TAG ATT GTT
Label: HEX green FILTER SET D
Clone Length: 144-262 bp
Locus: OCH 14
Forward Primer: 5’ CGG GCT ATA TGA AGG TGA TCC
Reverse Primer: 5’ GCT ACG CAA ATG AAC AAA CCA
Label: TAMN- yellow FILTER SET D
Clone Length: 263-419 bp
PCR Reaction OCH 13 and 14
Reagent
ddH20
dNTP (ea.)
PCR buffer
MgCl2
primer forward
primer reverse
Taq
template (1:10)
Stock
Final
1.25 mM
10X
25 mM
10 mM
10 mM
5 units/μL
20.0 ng/μL
0.20 mM
1X
1.8 mM
1 mM
1 mM
20.0ng
Total Volume
x1 (μL)
9.33
1.2 (0.3 ea.)
1.5
1.08
0.345
0.345
0.2
1.0
15.0 μL
PCR Thermal Profile OCH 13 and 14: 30 cycles of – 95oC for 30s, 58oC for 1
min 45s; Final extension @ 72o C for 10 min.
Locus: Ocl1
Forward Primer: 5’ ACT ACT AAC CAG CCC ACC ACC C
Reverse Primer: 5’ AGA CAG AGA GGG AGG GAA GC
Label: HEX green FILTER SET D
Clone Length: 100-160 bp
23
PCR Reaction Ocl 1
Reagent
ddH20
dNTP (ea.)
PCR buffer
MgCl2
primer forward
primer reverse
Taq
BSA
template (1:10)
Stock
Final
1.25 mM
10X
25 mM
10 mM
10 mM
5 units/μL
10 mM
20.0 ng/μL
0.20 mM
1X
2.5 mM
0.2 mM
0.2 mM
0.038 mM
0.8 mM
20.0ng
Total Volume
x1 (μL)
11.25
1.2 (0.3 ea.)
2.0
2.0
0.4
0.4
0.15
1.6
1.0
20.0 μL
PCR Thermal Profile Ocl 1: 33 cycles – 95oC for 30s, 63oC for 30s, 72oC for
30s; Final extension @ 72o C for 10 min.
Locus: Ogo 4
Forward Primer: 5’ GTC GTC ACT GGC ATC AGC TA
Reverse Primer: 5’ GAG TGG AGA TGC AGC CAA AG
Label: HEX green FILTER SET D
Clone Length: 120-130 bp
PCR Reaction Ogo 4
Reagent
ddH20
dNTP (ea.)
PCR buffer
MgCl2
primer forward
primer reverse
Taq
BSA
template (1:10)
Stock
Final
1.25 mM
10X
25 mM
10 mM
10 mM
5 units/μL
10 mM
20.0 ng/μL
0.20 mM
1X
2.5 mM
0.5 mM
0.5 mM
0.038 mM
0.8 mM
20.0ng
Total Volume
x1 (μL)
10.05
1.2 (0.3 ea.)
2.0
2.0
1.0
1.0
0.15
1.6
1.0
20.0 μL
PCR Thermal Profile Ogo 4: 33 cycles – 95oC for 30s, 59oC for 30s, 72oC for
30s; Final extension @ 72o C for 10 min.
Locus: Omm 1036
Forward Primer: 5’ TGT AGC AGG TGA GAA TAC CCA
Reverse Primer: 5’ CAC CAT CTC CAT CCT AGG C
Label: HEX green FILTER SET D
Clone Length: TBD
24
PCR Reaction Omm 1036
Reagent
ddH20
dNTP (ea.)
PCR buffer
MgCl2
primer forward
primer reverse
Taq
BSA
template (1:10)
Stock
Final
1.25 mM
10X
25 mM
10 mM
10 mM
5 units/μL
10 mM
20.0 ng/μL
0.20 mM
1X
2.5 mM
0.5 mM
0.5 mM
0.038 mM
0.8 mM
20.0ng
Total Volume
x1 (μL)
10.05
1.2 (0.3 ea.)
2.0
2.0
1.0
1.0
0.15
1.6
1.0
20.0 μL
PCR Thermal Profile Omm 1036: 33 cycles of – 95oC for 30s, 59oC for 30s,
72oC for 30s; Final extension @ 72o C for 10 min.
Locus: Ots 107
Forward Primer: 5’ ACA GAC CAG ACC TCA ACA
Reverse Primer: 5’ ATA GAG ACC TGA ATC GGT A
Label: HEX green FILTER SET D
Clone length: 160-225bp
PCR Reaction Ots 107
Reagent
ddH20
dNTP (ea.)
PCR buffer
MgCl2
primer forward
primer reverse
Taq
BSA
template (1:10)
Stock
Final
1.25 mM
10X
25 mM
10 mM
10 mM
5 units/μL
10 mM
20.0 ng/μL
0.20 mM
1X
2.5 mM
0.2 mM
0.2 mM
0.038 mM
0.8 mM
20.0ng
Total Volume
x1 (μL)
11.25
1.2 (0.3 ea.)
2.0
2.0
0.4
0.4
0.15
1.6
1.0
20.0 μL
PCR Thermal Profile Ots 107: 33 cycles of – 95oC for 30s, 50oC for 30s, 72oC
for 30s; Final extension @ 72o C for 10 min.
25
Objective 2b – Determine genetic differentiation between the two morphotypes of
cutthroat trout (YSC & SRC) in the study landscape
Genetic differentiation among morphotypes was not apparent, either within
drainages or pooling across the entire study area (Tables 8-13). Differences in
haplotypic composition among groups were likely due to sample size differences or
stochastic sampling error (Figures 6 -12). There was no evidence that the morphotypic
groups represent distinct lineages.
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).
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
0.005 (0.001)
5.125 (1.362)
4.740 (1.320)
0.005 (0.001)
0.005 (0.001)
0.004 (0.001)
4.135 (1.276)
0.005 (0.001)
4.964 (1.387)
4.580 (1.265)
0.005 (0.001)
4.926 (1.276)
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).
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
0.003 (0.001)
3.26 (1.034)
2.608 (0.900)
0.003 (0.001)
0.003 (0.001)
0.002 (0.001)
2.067 (0.929)
0.002 (0.001)
2.496 (0.856)
1.993 (0.881)
0.002 (0.001)
1.888 (0.87)
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).
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
0.003 (0.001)
2.821 (0.846)
2.865 (0.802)
0.003 (0.001)
0.004 (0.001)
0.003 (0.001)
2.955 (0.895)
0.003 (0.001)
3.429 (0.970)
3.282 (0.931)
0.004 (0.001)
3.757 (1.090)
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).
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
0.007 (0.002)
6.533 (1.741)
5.540 (1.431)
0.006 (0.002)
0.006 (0.002)
0.006 (0.002)
5.610 (1.488)
0.006 (0.002)
5.462 (1.398)
5.685 (1.455)
0.006 (0.002)
5.846 (1.543)
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).
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
Large Spotted
Morphotype
n/a
n/a
0.001
0.002 (0.001)
Fine Spotted
Morphotype
1.286 (0.422)
0.003 (0.001)
2.571 (0.804)
0.003 (0.001)
Intermediate
Morphotype
1.857 (0.579)
2.673 (0.836)
0.003 (0.001)
3.275 (0.970)
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).
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
Large Spotted
Morphotype
Fine Spotted
Morphotype
Intermediate
Morphotype
0.006 (0.002)
6.095 (1.687)
5.476 (1.494)
0.006 (0.002)
0.006 (0.002)
0.005 (0.001)
4.915 (1.426)
0.005 (0.001)
5.328 (1.440)
4.600 (1.302)
0.005 (0.001)
4.559 (1.298)
28
D
J
YSC
L
K
C
B
M
H
CLADE 1
?
G
?
?
BRC
E–
?
?
?
I
Large Spotted Morphotype
Fine Spotted Morphotype
F
Intermediate Morphotype
?
Anomalous Morphotype
=5
=1
A
CLADE 2
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
MORPHOTYPES
All Drainages
Ho
le
Ja
ck
so
n
Ho
le
n = 275
Fine-Dense
kso
n
ntre
Jac
Large-Sparse
Gros
Ve
Intermediate
er
Riv
ke yon
a
Sn an
C
Ho
b
ac
HAPLOTYPES
All Drainages
k
n=324
ys
Gre
A
B
C
D
E
F
G
H
I
J
K
L
M
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
MORPHOTYPES
Jackson Hole
Large-Sparse
Fine-Dense
n = 93
Intermediate
HAPLOTYPES
Large-Sparse Morphotype
ific
ac
Cr
n = 11
P
Leigh Canyon
Paintbrush
Canyon
e
ak
Sn
R
South Fork Spread Cr
Cascade Cr
Fine-Dense Morphotype
A
n = 57
B
Middle Fork Ditch Cr
C
Death Canyon
D
ke
R
H
Sna
J
M
Flat Cr
Intermediate Morphotype
n = 25
Flow
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
HAPLOTYPES
Large-Sparse Morphotype
n = 13
MORPHOTYPES
Gros Ventre
n = 62
Large-Sparse
Fine-Dense
Moccasin Cr
ork
Fi s
R
Calf Cr
No
rt
hF
e
ak
Sn
hC
r
Intermediate
Gros Ventre R
Papoose Cr
Flow
Park Cr
Tepee Cr
Fine-Dense Morphotype
n = 28
A
B
C
D
n tre
Gros Ve
R
Raspberry Cr
Strawberry Cr
H
K
L
Intermediate Morphotype
n = 21
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
HAPLOTYPES
Large-Sparse Morphotype
n=6
A
Boulder Cr
B
e
ak
Sn
D
R
J
Bull Cr
Flow
Hoback R
MORPHOTYPES
Hoback
Dell Cr
Kerr Cr
Fine-Dense Morphotype
n = 40
n = 21
Large-Sparse
Fine-Dense
Intermediate
r
nt C
ura
d
n
Bo
c
ba
Ho
kR
Intermediate Morphotype
n = 13
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
HAPLOTYPES
Large-Sparse Morphotype
n=5
A
D
I
L
Sn
ak
eR
North F
o
So
rk Fall
uth
F
s
Hor
Cr
ll
Fa
ork
r
eC
Cr
r
nC
bur
o
C
Ho
ba
ck
Cab
in
R
HAPLOTYPES
Fine-Dense Morphotype
Cr
n=7
Snake
Flow
anyon
River C
MORPHOTYPES
Snake River Canyon
HAPLOTYPES
Intermediate Morphotype
n = 26
n = 14
Large-Sparse
Fine-Dense
Intermediate
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
eR
Snak
Little Greys R
Flow
HAPLOTYPES
Large-Sparse Morphotype
Greys R
Ste
er C
r
n=7
Blind
l Cr
Trai
Fine-Dense Morphotype
n = 30
Unnamed Tributary
A
B
D
F
Upper Cabin Cr
H
North Three Forks Cr
MORPHOTYPES
Greys
Intermediate Morphotype
ys
Gre
n = 54
R
Large-Sparse
n = 17
Fine-Dense
Intermediate
Flat Cr
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
Objective 3 – Describe patterns of genetic variation in cutthroat trout within and
among major drainages in the study landscape
Genetic differences among drainages were apparent in all analyses, as
evidenced by a) average pairwise nucleotide differences within and between drainages
(Table 14), b) a non-random distribution of haplotypes among drainages (2 = 232.67; P
< 0.00001), and c) an overall pairwise GST of 0.14. The differences in haplotype
distribution among drainages can be visualized in Figures 13 and 15. These differences
were quantified with pairwise population GST values (Table 16).
Two distinct haplotype clades were present in the dataset (Figures 14 and 15).
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
(Figures 13 and 15). Based on these observations, drainages were grouped into the
two clades; Clade 1 including Jackson Hole and the Gros Ventre, and Clade 2 including
the Hoback, Snake River Canyon, and Greys. When these two groups were compared,
haplotypes were found to be non-randomly distributed between them (2 = 105.64; P <
0.00001), and genetic differentiation between these groups was detectable (G ST =
0.07218).
Genetic diversity differed among drainages (Table 15). The Hoback and Greys
drainages had the highest levels of nucleotide diversity ( ; which takes haplotype
divergence into account), but Jackson Hole and the Greys had the highest haplotypic
diversity (Hd) and the largest number of haplotypes represented.
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).
Jackson Hole
Gros Ventre
Hoback
Snake River
Canyon
Greys
All Drainages
Jackson
Hole
Gros Ventre
Hoback
Snake River
Canyon
Greys
All
Drainages
0.002 (0.001)
1.993 (0.85)
2.935
(0.969)
5.432
(1.480)
8.475
(2.413)
4.422
(1.230)
n/a
0.003
(0.001)
0.003 (0.001)
3.191 (0.91)
5.289
(1.486)
7.584
(2.221)
4.438
(1.257)
n/a
0.006
(0.002)
0.006
(0.002)
0.006 (0.002)
5.651 (1.47)
5.479
(1.551)
5.441
(1.499)
n/a
0.009
(0.003)
0.008
(0.002)
0.006
(0.002)
0.003 (0.001)
2.428 (0.68)
6.331
(1.812)
n/a
0.005
(0.001)
0.005
(0.001)
0.006
(0.002)
0.007
(0.002)
0.005 (0.001)
5.122 (1.34)
n/a
n/a
36
n/a
n/a
n/a
n/a
0.005 (0.001)
4.361 (1.19)
Table 15 Genetic diversity indices for cutthroat trout in Snake River headwaters drainages. Nucleotide
diversity (), haplotype diversity (Hd), and number of haplotypes are presented for each drainage.
 (SD)
Hd (SD)
# Haplotypes
0.0021 (0.00013)
0.00337(0.00058)
0.00598(0.00020)
0.00292(0.00110)
0.00542(0.0010)
0.711(0.021)
0.601(0.063)
0.660(0.039)
0.286(0.112)
0.717(0.040)
8
7
4
4
7
Population
Jackson Hole
Gros Ventre
Hoback
Snake River Canyon
Greys
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).
Jackson Hole
Gros Ventre
Hoback
Snake River
Canyon
Greys
Jackson Hole
n/a
Gros Ventre
0.05500
n/a
Hoback
0.07087
0.05890
n/a
Snake River
Canyon
Greys
0.16493
0.24884
0.10040
n/a
0.05014
0.01183
0.00926
0.14504
n/a
All Drainages
n/a
n/a
n/a
n/a
n/a
37
All
Drainages
0.13737
HAPLOTYPES
Jackson Hole
A
n = 93
B
C
D
HAPLOTYPES
Gros Ventre
n = 62
H
ol
e
H
J
B
Ja
ck
so
n
M
A
C
D
H
HAPLOTYPES
Snake River Canyon
Gros
Vent
K
re
L
n = 26
so
ck
Ja
A
D
n
I
le
Ho
L
Sn
e
ak
on
ny
a
rC
ve
i
R
HAPLOTYPES
Hoback
n = 40
Ho
b
ac
k
A
B
D
J
ys
Gre
HAPLOTYPES
Greys
n = 57
A
B
C
D
E
F
H
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
YSCT1
SRC02
M
L
YSC55
J
D
K
84
H
Clade1
C
YSC53
B
98
G
YSCA1
I
Clade 2
A
43
70
F
E
BRC
70
LHC
74
WSC
CRC
GBC
100
RXC
RBT
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
D
J
YSC
L
K
C
B
M
H
CLADE 1
?
G
?
?
BRC
E–
?
?
?
F
I
Jackson Hole
Gros Ventre
=5
Hoback
=1
Snake River Canyon
?
Greys
A
CLADE 2
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
Objective 4 – Detection of Rainbow Trout Introgression
A total of 12 fish were captured between 1998 and 2003 that were field identified
as either rainbow trout (RBT) or rainbow-cutthroat trout hybrids (RXC; Table 17). Six
fish were captured in the Gros Ventre and field identified as RXC. Five of those were
captured in the Gros Ventre R, and one fish in Crystal Cr. A single fish was captured in
the Hoback River and identified in the field as a RXC. Five fish captured in Fawn Cr,
within the Greys River drainage were identified in the field as RBT. The main identifying
feature for all of the hybrid fish was either white margins or tips on the pelvic and anal
fins. Review of each of the available photographs (one film slide from Fawn Cr was too
poor quality and subsequently discarded) supported the field identifications.
Tissue from 8 of the 12 fish identified as RBT or RXC were included in our
analyses. No photograph or tissue was collected from 3 of the RBT in Fawn Creek due
to their being <150 mm TL. The RXC captured in Crystal Creek was not included in this
analysis. Genetic differences were observed in 6 of the 8 fish that distinguished them
from cutthroat trout. Those 6 fish grouped with RBT as depicted in the neighbor-joining
dendrogram (Fig. 14). While four distinct haplotypes were observed from the 6 fish,
only one haplotype was used in constructing the tree. The two remaining RXC, both Dhaplotype 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.
No other instance of an RXC haplotype was observed in the sample set. The
thirty samples from the Snake River specifically screened due to concerns of potential
hybridization did not exhibit any RXC haplotypes. 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, the main exception being the fish
captured in the Hoback (Figure 16). Also, the RXC captured upstream of Lower Slide
Lake in Crystal Cr and the Gros Ventre R were the first documented hybrid trout being
present above the lake and indicate a greater range in the Gros Ventre than was
previously known (Figure 16).
41
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.
Drainage/Stream
Location
(m)
Species
Length
(mm)
Weight
(gm)
Sample ID
Crystal Cr1
10,000
RXC
374
495
GV-03-02-344
Gros Ventre R
20,000
RXC
327
350
GV-03-03-284
Gros Ventre R
GROS VENTRE RIVER
20,000
RXC
341
445
GV-03-03-287
R1
34,500
RXC
357
490
GV-03-02-273
Gros Ventre R1
48,000
RXC
318
265
GV-03-02-221
Gros Ventre R1
50,000
RXC
378
520
GV-03-01-209
20,000
RXC
373
510
GH-03-01-123
Fawn Cr
750
RBT
220
100
GH-00-07-02
Fawn Cr
1,500
RBT
153
39
GH-00-07-04
Fawn Cr
1,500
RBT
110
16.0
NA2
Fawn Cr
1,500
RBT
105
12.0
NA
Fawn Cr
1,500
RBT
94
8.0
NA
Gros Ventre
HOBACK RIVER
Hoback R
GREYS RIVER
1
Capture locations upstream of Lower Slide Lake were outside of previous known range in the Gros
Ventre River drainage.
2 No photograph or fin clip collected for fish <150 mm TL.
42
Ho
le
n
Ja
ck
so
Gros
Ve
ntre
so
ck
Ja
n
le
Ho
ke
na
ve
Ri
an
rC
n
yo
Ho
b
S
ac
k
ys
Gre
rainbow-cutthroat trout hybrids
rainbow trout
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
DISCUSSION
Develop cost-effective, reliable, and repeatable molecular tools that will answer
the study questions
A set of robust primers were designed and optimized that allowed us to amplify
and sequence the most variable region of the ND1-2 gene. Nearly all (>95%) of the
approximately 530 samples selected from our study landscape, plus 75 out group
samples, amplified using this protocol. The sequences obtained from the 324 cutthroat
trout from our study landscape and 65 individuals from the out groups represent 92% of
the amplicons attempted being successfully sequenced. Within the entire data set, 110
of the 945 base pairs were variable. Eighteen variable sites defined the 13 different
haplotypes within the cutthroat trout from our study landscape (Appendix A Table 21).
Numbers of fish bearing a specific haplotype are listed by stream in Appendix A (Table
21).
The primer set was similarly successful in amplifying and sequencing the
cutthroat trout out groups, rainbow trout, and rainbow-cutthroat trout hybrids. The
number of base pair substitutions between cutthroat trout in the Snake River
headwaters and the Yellowstone cutthroat trout out group (from Yellowstone Lake and
Yellowstone River headwaters upstream of the lake) increased by one to 19, where as
there were 59 variable sites among the Colorado River, greenback, Lahontan, and west
slope cutthroat trout out groups. The number of base pair substitutions increased to 77
between cutthroat trout in the Snake River headwaters and rainbow trout.
Such a high rate of intraspecific variation as was found in the mtDNA ND2 gene
of the cutthroat trout (as noted above, 59 variable sites among the five subspecies), is
one of three key properties that make mtDNA so favorable to phylogeographic studies.
The remaining key properties are maternal transmission, and absence of genetic
recombination in this haploid genome (Avise 2000). That higher intraspecific variation
in mtDNA is more likely to show differences among populations than single-copy
nuclear DNA, arises in part, from the smaller effective population size (one-quarter that
of the bisexually inherited diploid nuclear genome; Billington 2003). While the mtDNA
sequences were useful in confirming hybridization in morphologically suspect fish, a
particular deficiency is the inability to address degree of cutthroat trout introgression
with rainbow trout, also a result of the single-locus haploid genome.
The nuclear marker system, microsatellites, suggested similar successes. A total
of 17 loci from the three primer sets were optimized, and polymorphism was identified at
five of the seven loci evaluated. Specifically, four microsatellite loci were found to be
robust and polymorphic (6-14 alleles/locus) in our optimization sample set (n=16). Two
of the four were OCH loci, and 2 of the loci were obtained from IDFG. One locus
suggested to us by the IDFG lab, while variable, was much less polymorphic (3 alleles),
and one locus showed no variability within our cutthroat trout samples. These four
polymorphic microsatellite loci, and the protocols for their amplification, are available for
future studies on fine-scale (geographically and temporally) genetic structuring in
44
cutthroat trout in the Snake River headwaters. The development and optimization of
primers is commonly the most time consuming portion of generating microsatellite data,
and this work is an important contribution to future research. Microsatellite data could
tell us more about introgression with rainbow trout, and barriers to gene flow discussed
below because they are nuclear, codominant, and have a high mutation rate (Brown
and Epifanio 2003; Gharrett and Zhivotovsky 2003). It is quite likely that very
pronounced structuring would be determined due to identification of unique alleles, and
fixation of alleles (Gharrett and Zhivotovsky 2003; Shaklee and Currens 2003);
identification of diagnostic markers in out groups is also possible.
Genetic Differentiation among Morphotypes
Three recognized sub-species of cutthroat trout have been described within
Wyoming that 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 a fine-spotted
morphotype (finespotted Snake River, SRC) has been identified in the Snake River
headwaters of northwest Wyoming (Baxter and Simon 1970; Behnke 1992), we were
unable to genetically and morphologically differentiate this morphotype from the
Yellowstone cutthroat trout.
Previous morphological and genetic investigations have examined differences in
the fine-spotted and large spotted morphotypes, but have not conclusively differentiated
the two types (Loudenslager 1978; Loudenslager and Kitchin 1979; Loudenslager and
Gall 1980). The presence of a large number of fish with intermediate spotting patterns
within our survey suggests that these fish are clearly not reproductively isolated and that
there is considerable overlap in morphology and meristics. Behnke (1992) suggested
that variation and overlap in meristic counts and observations of intermediate spotting
may indicate continued gene flow and acknowledged that the difference in spotting
pattern, and observed intermediate spotting, may result from simultaneous expression
of two co-dominant alleles at one locus. 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.
Genetic differentiation among morphotypes was not apparent, either within
drainages or when samples were pooled across the entire study area. We could find
no recognizable separation of morphotypes within the mitochondrial haplotypes we
identified. All spotting patterns were represented in the upper Snake River, as well as
each of the major drainages that were sampled. There was no evidence that these
morphotypes represented distinct lineages. Previous 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.
While we were unable to differentiate two distinct morphotypes, the conservation
of unique color and spotting patterns may be important for future management. Unique
phenotypes and life histories in westslope cutthroat trout, and physiological adaptations
45
in related interior cutthroat are examples of such variation that exists and that should be
maintained (Carl & Stelfox 1989; Taylor et al. 2003).
Genetic Differentiation among Major Drainages
While we were unable to discriminate differences between the finespotted and
large spotted morphotype, we were able to detect haplotype frequency differences
among the major drainages that we surveyed. The differences we observed indicate
two major clades that are loosely represented by a haplotype group associated with the
two northern geographic areas (Jackson Hole, Gros Ventre) and a haplotype that
occurs predominantly in the three southern geographic areas (Snake River Canyon,
Hoback, Greys). These clades are likely to have evolved in response to different
hydrogeographic conditions than those that exist today. A large landscape analysis
would be necessary to make statements about ancient barriers that may have produced
these clades. Caution should be exercised when making conclusions regarding the
Snake River Canyon fish because no cutthroat trout from the main Snake River were
included in our sample.
Haplotypes appear to be unsorted among different drainages, suggesting that
there are no differences in lineages among different drainages. There are two possible
explanations; first, that the current patterns of isolation have not been in place long
enough for lineages to arise and second, that barriers among drainages are not
complete, but sufficient to prevent homogenization of haplotype frequencies. Most likely
the current situation is a combination of the two. Taylor et al. (2003) found that there
was significant genetic divergence of westslope cutthroat populations in the upper
Kootenay River and that populations appeared to be demographically independent
despite the ability of fish to move freely among some of the drainages. They
recommended that these populations be treated as distinct biological units for the
purposes of management.
There appear to be an anomalous morphotype associated with haplotype C.
These fish occur almost exclusively above barriers, and in four canyons within the Teton
Mountains, in Jackson Hole. The morphotype exhibits very large (>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
46
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
47
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.
48
REFERENCES CITED
Allendorf, F. W., and R. F. Leary. 1988. Conservation and distribution of genetic
variation in a polytypic species, the cutthroat trout. Conservation Biology 2:170184.
Baxter, G. T., and J. R. Simon. 1970. Wyoming fishes. Wyoming Game and Fish
Commission Bulletin 4.
Baxter, G. T., and M. D. Stone. 1995. Fishes of Wyoming. Wyoming Game and Fish
Department, Cheyenne, WY.
Behnke, R. J. 1992. Native trout of western North America. American Fisheries Society
Monograph 6.
Behnke, R. J. 2002. Trout and salmon of North America. The Free Press. New York.
Carl, L.M. and J.D. Stelfox. (1989). A meristic, morphometric and electrophoretic
analysis of cutthroat trout Salmo clarki from two mountain lakes in Alberta
Canada. Canadian Field Naturalist, 103, 80–84.
Carlsson, J. and J. Nilsson. (1999). Effects of geomorphological structures on genetic
differentiation among brown trout populations in a northern Boreal river drainage.
Transactions of the American Fisheries Society, 130, 36–45.
Campbell, M. R., J. Dillon, and M. S. Powell. 2002. Hybridization and introgression in a
managed, native population of Yellowstone cutthroat trout: Genetic detection and
management implications. Transactions of the American Fisheries Society
131:364-375.
Clement, M., D. Posada, and K. A. Crandall. 2000. TCS: a computer program to
estimate gene genealogies. Molecular Ecology 9:1657-1660.
Condrey, M.J., and P. Bentzen. 1988. Characterization of coastal cutthroat trout
(Oncorhynchus clarki clarki) microsatellites and their conservation in other
salmonids. Molecular Ecology 7:783-792.
Costello AB, Down TE, Pollard SM, Pacas CJ, Taylor EB (2003). The influence of
history and contemporary stream hydrology on the evolution of genetic diversity
within species: an examination of microsatellite DNA variation in bull trout,
Salvelinus confluentus (Pisces: Salmonidae). Evolution, 57, 328–344.
49
Fausch, K. D., C. E. Torgerson, C. V. Baxter, and H. W. Li. 2002. Landscapes to
riverscapes: bridging the gap between research and conservation of stream
fishes. Bioscience 52(6):483-498.
Folt, C. L., K. H. Nislow, and M. E. Power. 1998. Implications of temporal and spatial
scale for Frissell, C. A. 1997. Ecological principles. Pages 96-115 in J.E.
Williams, C.A. Wood, and M.P. Dombeck, editors. Watershed restoration:
principles and practices. American Fisheries Society, Bethesda, Maryland.
Gresswell, R. E. 1988. Status and management of interior stocks of cutthroat trout.
American Fisheries Society Symposium 4.
Gresswell, R. E. 1995. Yellowstone cutthroat trout. Pages 36-54 in M.K. Young,
technical editor. A conservation assessment for inland cutthroat trout. USDA
Forest Service, Rocky Mountain Forest and Range Experiment Station, Fort
Collins, Colorado. General Technical Report RM-GTR-256.
Hagenbuck, W. W. 1970. A study of the age and growth of the cutthroat trout from the
Snaje River, Teton County, Wyoming. Masters Theses, University of Wyoming,
Laramie.
Harper, D. D., and A. M. Farag. 2002. Winter habitat utilization and movements by
cutthroat trout in the Snake River near Jackson, Wyoming 2000-2001. USDI,
U.S. Geological Survey, Columbia Environmental Research Center, Jackson
Field Station. Final Report to U.S. Bureau of Reclamation Project 70L31.
Harper, D. D., and A. M. Farag. 2004. Winter habitat use by cutthroat trout in the Snake
River near Jackson, Wyoming. Transactions of the American Fisheries Society
133:15-25.
Hayden, P. S. 1967. Snake River cutthroat trout study. Part I: the reproductive
behavior of the Snake River cutthroat trout in three tributary streams in Wyoming.
Wyoming Game and Fish Commission and University of Wyoming. Cooperative
Research Project 4.
Henderson, R.C., J.L. Kershner, and C.A. Toline. 2000. Timing and location of
spawning of non-native wild rainbow trout and native cutthroat trout in the South
Fork Snake River, Idaho, with implications for hybridization. North American
Journal of Fisheries Management 20: 584-596.
Hitt, N.P., C.A. Frissell, C.C. Muhlfeld, and F.W. Allendorf. 2003. Spread of
hybridization between native westslope cutthroat trout, Oncorhynchus clarki
lewisi, and nonnative rainbow trout, Oncorhynchus mykiss. Can. J. Fish. Aquat.
Sci. 60: 1440.1451.
50
Hubbs, C. L. 1955. Hybridization between fish species in nature. Systematic Zoology
4:1–20.
Hudson, R. R., D. D. Boos, and N. L. Kaplan 1992. A statistical test for detecting
geographic subdivision. Molecular Biology and Evolution 9:138-151.
Huston, J. E., P. Hamlin, and B. May. 1984. Lake Koocanusa investigations final report.
Montana Department of Fish, Wildlife and Parks, Helena.
Irwin, D. E. 2002. Phylogeographic breaks without geographic barriers to gene flow.
Evolution 56:2383-2394.
Jones, K. C. Unpublished microsatellite primers. Genetic Identification Services.
http://www.genetic-id-services.com/
Kiefling, J. W. 1972. An analysis of stock densities and harvest of the cutthroat trout of
the Snake River, Teton County, Wyoming. Master’s Thesis, University of
Wyoming, Laramie.
Kiefling, J. W. 1978. Studies on the ecology of the Snake River cutthroat trout.
Wyoming Game and Fish Commission, Cheyenne, Wyoming. Fisheries
Technical Bulletin 3.
Krebs, C.J. 1999. Ecological methodology. 2nd Ed. Addison-Wesley Longman
Educational Publishers, Inc. Menlo Park, CA.
Kruse, C. G. 1998. Influence of nonnative trout and geomorphology on distributions of
indigenous trout in the Yellowstone River drainage of Wyoming. PhD
Dissertation, University of Wyoming, Laramie.
Kruse, C. G., Hubert, W.A., and F. J. Rahel. 1996. Sources of variation in counts of
meristic features of Yellowstone cutthroat trout (Oncorhychus clarki bouvieri).
Great Basin Naturalist 56:300-307.
Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: Integrated software for Molecular
Evolutionary Genetics Analysis and sequence alignment. Briefings in
Bioinformatics 5:150-163.
Labbe, T. R., and K. D. Fausch. 2000. Dynamics of intermittent stream habitat regulate
persistence of a threatened fish at multiple scales. Ecological Applications
10:1774-1791.
Leary, R. F., F. W. Allendorf, S. R. Phelps, and K. L. Knudsen. 1987. Genetic
divergence and identification of seven cutthroat trout subspecies and rainbow
trout. Transactions of the American Fisheries Society 116:580-587.
51
Leary, R. F., F. W. Allendorf, and G. K. Sage. 1995. Hybridization and introgression
between introduced and native fish. Pages 91–101 in H. L. Schramm, Jr., and R.
G. Piper, editors. Uses and effects of cultured fishes in aquatic ecosystems.
American Fisheries Society, Symposium 15, Bethesda, Maryland.
Levin, S. A. 1992. The problem of pattern and scale in ecology. Ecology 73:1943-1967.
Likenes, G. A., and P. J. Graham. 1988. Westslope cutthroat trout in Montana: life
history, status, and management. Pages 53–60 in R. E. Gresswell, editor. Status
and management of interior stocks of cutthroat trout. American Fisheries Society,
Symposium 4, Bethesda, Maryland.
Loudenslager, E. J. 1978. Variation in the genetic structure of populations of two
polytypic vertebrates, the cutthroat trout (Salmo clarki) and the deer mouse
(Peromyscus maniculatus). Doctor of Philosophy Thesis. University of Wyoming.
Loudenslager, E. J., and E. M. Kitchin. 1979. Genetic similarity of two forms of cutthroat
trout, Salmo clarki, in Wyoming. Copeia 1979:673-678.
Loudenslager, E. J., and G. A. E. Gall. 1980. Geographic patterns of protein variation
and subspeciation in cutthroat trout, Salmo clarki. Systematic Zoology 29:27-42.
Love, J. D., J. C. Reed, Jr., and K. L. Pierce. 2003. Creation of the Teton Landscape, A
Geologic Chronicle of Jackson Hole and the Teton Range. Grand Teton Natural
History Association, Moose, WY.
May, B. 1996. Yellowstone cutthroat trout Oncorhynchus clarki bouvieri. Pages 11-34
in D.Duff, technical editor. Conservation assessment for inland cutthroat trout:
distribution, status and habitat management implications. USDA Forest Service,
Intermountain Region, Ogden, Utah.
Montgomery, M.R. 1995. Many rivers to cross - of good running water, native trout, and
the remains of wilderness. Simon and Schuster. New York, New York.
Murphy, T. C. 1974. A study of Snake River cutthroat trout. Master's Thesis, Colorado
State University, Fort Collins.
Nei, M. 1987. Molecular evolutionary genetics. Columbia University Press, New York.
Nelson, J. S., E. J. Crossman, H. Espinosa-Perez, L. T. Findley, C. R. Gilbert, R. N.
Lea, and J. D. Williams. 2004. Common and scientific names of fishes from the
United States, Canada, and Mexico. American Fisheries Society, Special
Publication 29, Bethesda, Maryland. USA.
Nelson, R. J., and T. D. Beacham 1999. Isolation and cross species amplification of
microsatellite loci useful for study of Pacific salmon. Animal Genetics 30:228-229.
52
Olsen, J. B., P. Bentzen and J. E. Seeb. 1998. Characterization of seven microsatellite
loci derived from pink salmon. Molecular Ecology 7:1087-1089.
Peacock, M. M., H. M. Neville, and V. S. Kirchoff. 2004. Ten species-specific
microsatellite loci for Lahonton cutthroat trout Oncorhynchus clarki henshawi.
Molecular Ecology Notes 4:557-559.
Quadri, S. U. 1959. Some morphological differences between the subspecies of
cutthroat trout, Salmo clarki clarki, and Salmo clarki lewisi, in British Columbia.
Journal of the Fisheries Research Board of Canada. 16:903-915.
Rexroad, C. E., R. L. Coleman, A. L. Gustafson, W. K. Hershberger, and J. Killefer.
2002. Development of rainbow trout microsatellites markers from repeat enriched
libraries. Marine Biotechnology 4(1):12-16.
Rieman, B. E., and J. D. McIntyre. 1995. Occurrence of bull trout in naturally
fragmented habitat patches of varied size. Transactions of the American
Fisheries Society 124:285-296.
Rozas, J., J. C. Sanchez-DelBarrio, X. Messeguer, and R. Rozas. 2003. DnaSP, DNA
polymorphism analyses by the coalescent and other methods. Bioinformatics
19:2496-2497.
Sakamoto, T., Okamoto, N., Ikeda, Y., Nakamura, Y., and T. Sato. 1994. Dinucleotiderepeat polymorphism in DNA of rainbow trout and its application in fisheries
science. Journal of Fish Biology 4:1093-1096.
Shiozawa, D. K., and R. N. Williams. 1992. Geographic variation, isolation and
evolution of cutthroat trout (Salmo clarki). Final Report to The National
Geographic Society. Grant No. 3653-87.
Skaala, O., and K. E. Jorstad. 1988. Inheretence of the fine-spotted pigmentation
pattern in brown trout. Polish Archives of Hydrobiology 35:295-304.
Spruell, P., M. L. Bartron, N. Kanda, and F. W. Allendorf. 2001. Detection of hybrids
between bull trout (Salvelinus confluentus) and brook trout (Salvelinus fontinalis)
using PCR primers complimentary to interspersed nuclear elements. Copeia
4:1093-1099.
Sunnucks, P., and D. F. Hales. 1996. Numerous transposed sequences of
mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion
(Hemiptera: Aphididae). Molecular Biology and Evolution 13:510-524.
Thurow, R. F., C. E. Corsi, and V. K. Moore. 1988. Status, ecology, and management
of Yellowstone cutthroat trout in the upper Snake River drainage,
53
Idaho. Pages 25–36 in R. E. Gresswell, editor. Status and management of
interior stocks of cutthroat trout. American Fisheries Society, Symposium 4,
Bethesda, Maryland.
Toline, C. A., T. Seamons, and J. M. Hudson. 1999. Mitochondrial DNA analysis of
selected populations of Bonneville, Colorado River, and Yellowstone cutthroat
trout. Department of Fisheries and Wildlife, Utah State University, Logan, UT.
Trojnar, J. R., and R. J. Behnke. 1974. Management implications of ecological
segregation between two introduced populations of cutthroat trout in a small
Colorado lake. Transactions of the American Fisheries Society 103:423-430.
Varley, J. D., and R. E. Gresswell. 1988. Ecology, status, and management of the
Yellowstone cutthroat trout. American Fisheries Society Symposium 4:13-24.
Warren, M. L., and B. M. Burr. 1994. Status of freshwater fishes of the United States:
overview of an imperiled fauna. Fisheries 19:6-18.
Wenburg, J. K., and P. Bentzen. 2001. Genetic and behavioral evidence for restricted
gene flow among coastal cutthroat trout populations. Transactions of the
American Fisheries Society 130:1049-1069.
Wiley, R. W. 1969. An ecological evaluation of the Snake River cutthroat fishery with
emphasis on harvest. Masters Thesis, University of Wyoming, Laramie.
Williams, J. E., J. E. Johnson, D. A. Hendrickson, S. Contreras-Balderas, J. D. Williams,
M. Navarro-Mendoza, D. E. McAllister, and J. E. Deacon. 1989. Fishes of North
America endangered, threatened, or of special concern: 1989. Fisheries 14: 220.
Wyoming Game and Fish Department. 1997. A stocking history: The Snake River
drainage, Wyoming. Wyoming Game and Fish Department, Cheyenne,
Wyoming.
Wyoming Game and Fish Department. 1999. Yellowstone cutthroat trout: Historical
management applications and status and management. Wyoming Game and
Fish Department, Cheyenne, Wyoming.
Young, M. K. 1995. Conservation assessment for inland cutthroat trout. General
Technical Report RM-256. Fort Collins, CO: U.S. Department of Agriculture,
Forest Service, Rocky Mountain Forest and Range Experiment Station.
Zar, J. H. 1999. Biostatistical analysis. 4th Edition. Prentice-Hall, Upper Saddle River,
New Jersey.
54
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.
Buffalo Fork
Snake River
Gros Ventre River
Hoback River
Greys River
Salt River
Total
Fish
Photographs
Tissue
58
3,744
2,254
1,131
1,597
364
9,148
58
1,302
1,051
508
905
138
3,962
58
1,230
709
495
881
124
3,497
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.
Drainage and Stream
Total
Type 1
Type 2
Type 3
Totals
1279
123
907
249
Subtotal
9
11
44
19
18
11
3
12
18
25
25
195
Subtotal
12
38
32
20
94
12
11
26
9
23
19
3
5
304
Historically
Stocked2
JACKSON HOLE
Cascade Cr3
Death Canyon3
Ditch Cr, Middle Fork4
Flat Cr
Leigh Canyon3
Mosquito Cr
Mosquito Cr, North Fork
Pacific Cr4
Paintbrush Canyon3
Snake R
Spread Cr, South Fork4
GROS VENTRE
Calf Cr
Cottonwood Cr
Fish Cr, North Fork
Fish Cr, South Fork
Gros Ventre R
Leeds Cr
Maverick Cr
Moccasin Cr
Papoose Cr
Park Cr
Raspberry Cr
Strawberry Cr
Tepee Cr
21
3
11
1
11
3
5
0
14
38
20
3
54
4
18
5
8
103
4
8
5
5
0
4
0
4
1
3
3
1
2
40
4
21
19
13
81
7
7
15
5
17
10
0
1
200
4
9
8
2
13
1
4
7
3
3
6
2
2
64
(continued)
56
9
11
23
8
17
N
N
N
Y 1990
Y
Y 1970
N
Y 1980
N
Y 1960
Y 1960
N
Y
N
Y 1990
Y 2000
N
N
N
N
N
N
N
Y
Table 19 Continued.
Drainage and Stream
Total
HOBACK
Bare Cr
Bondurant Cr
Boulder Cr
Bull Cr
Cliff Cr
Dell Cr
Fisherman Cr
Fisherman Cr, Middle Fork
Fisherman Cr, North Fork
Hoback R
Jack Cr
Kerr Cr
Little Granite Cr
Mumford Cr
Phosphate Cr
Rim Draw
Shoal Cr
Snag Cr
West Shoal Cr
Willow Cr
Subtotal
2
18
22
23
9
26
4
6
12
85
14
3
17
10
6
2
13
1
6
35
314
Subtotal
7
2
11
19
7
16
12
7
12
5
6
104
SNAKE RIVER CANYON
Bailey Cr
Bailey Cr, West
Cabin Cr
Coburn Cr
Dog Cr
Fall Cr
Fall Cr, North Fork
Fall Cr, South Fork
Horse Cr
Pine Cr
Pritchard Cr
Type 1
3
1
4
1
2
1
2
14
1
1
3
3
1
9
(continued)
57
Type 2
2
13
19
14
9
25
4
6
11
82
12
1
17
9
6
2
13
5
35
285
5
2
6
4
6
15
5
2
10
4
3
62
Type 3
2
2
5
1
1
1
1
1
1
Historically
Stocked2
N
N
N
N
Y
Y 1970
Y 1970
N
Y 1970
Y 1990
Y 1960
Y
Y 1960
N
N
N
Y 1970
N
N
Y
15
2
4
14
1
1
4
2
1
1
3
33
Y
N
Y
Y
Y
Y
Y
Y
Y
N
Y
Table 19 Continued.
Drainage and Stream
GREYS
Blind Trail Cr
Firebox Cr
Flat Cr
Greys R
Little Greys R
Little Greys R, South Fork
Lynx Cr
Murphy Cr
Murphy Cr, North Fork
North Corral Cr
Three Forks Cr, North5
Sheep Cr
Spring Cr
Squaw Cr
Steer Cr
Stewart Cr
Unnamed Tributary to
Lower Cabin Cr5
Upper Cabin Cr5
White Cr
Subtotal
Total
Type 1
Type 2
Type 3
38
2
14
113
24
24
3
4
4
18
29
11
15
4
25
13
3
34
2
9
110
15
15
1
4
4
18
19
11
15
4
23
12
1
8
9
4
362
1
3
2
5
3
306
5
1
1
34
3
1
3
3
4
1
22
1
2
2
6
6
2
6
1
1
Historically
Stocked2
Y
N
N
Y 1990
Y
Y
N
Y
Y
N
Y 1960
Y 1970
Y
Y
Y
N
N
N
N
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 lifestage of cutthroat trout.
58
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
Total
Type 1
Type 2
Type 3
543
91
256
196
9
9
20
11
18
11
17
55
23
0
0
11
0
0
3
0
0
14
0
0
0
5
1
4
0
50
3
9
9
9
6
17
4
17
5
6
173
28
63
82
12
17
49
21
7
9
15
3
5
4
5
0
4
1
3
3
1
2
4
7
38
10
3
6
6
0
1
4
5
11
7
3
0
6
2
2
138
23
75
40
8
6
14
3
40
5
3
4
1
2
1
1
1
2
2
3
7
2
27
3
1
2
2
5
0
12
1
0
79
12
45
22
JACKSON HOLE
Cascade Cr2
Death Canyon2
Ditch Cr, Middle Fork3
Flat Cr
Leigh Canyon2
Pacific Cr3
Paintbrush Canyon2
Snake R
Spread Cr, South Fork3
SUBTOTAL
GROS VENTRE
Calf Cr
Fish Cr, North Fork
Gros Ventre R
Moccasin Cr
Papoose Cr
Park Cr
Raspberry Cr
Strawberry Cr
Tepee Cr
SUBTOTAL
HOBACK
Bondurant Cr
Boulder Cr
Bull Cr
Dell Cr
Hoback R
Jack Cr
Kerr Cr
SUBTOTAL
(continued)
59
Table 20 Continued.
Drainage and Stream
Total
Type 1
Type 2
Type 3
7
13
6
7
3
1
1
2
2
1
3
2
2
2
1
3
10
2
3
1
36
7
10
19
9
8
48
14
4
4
19
3
3
3
3
1
0
4
5
3
31
6
2
3
9
1
2
14
5
1
1
6
5
6
1
3
2
2
2
1
117
21
63
33
SNAKE RIVER CANYON
Cabin Cr
Coburn Cr
Fall Cr, North Fork
Fall Cr, South Fork
Horse Cr
SUBTOTAL
GREYS
Blind Trail Cr
Flat Cr
Greys R
Little Greys R
Steer Cr
Stewart Cr
Three Forks Cr, North4
Unnamed Tributary, Lower
Cabin Cr4
Upper Cabin Cr4
SUBTOTAL
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 largesparse 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 lifestage of cutthroat trout.
60
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
Total
JACKSON HOLE
Cascade Cr2
Death Canyon2
Ditch Cr, Middle Fork
Flat Cr
Leigh Canyon2
Pacific Cr
Paintbrush Canyon2
Snake R
Spread Cr, South Fork
Subtotal
GROS VENTRE
Calf Cr
Fish Cr, North Fork
Gros Ventre R
Moccasin Cr
Papoose Cr
Park Cr
Raspberry Cr
Strawberry Cr
Tepee Cr
Subtotal
A
B
C
D
E
F
G
H
I
J
K
L
M
Total
64
41
50
132
2
1
1
19
1
8
1
3
1
324
6
9
4
2
1
1
2
3
1
15
1
22
3
1
5
9
3
14
2
1
48
1
1
4
8
6
1
1
5
7
6
5
25
3
53
0
7
6
21
4
1
8
7
3
5
62
1
5
0
0
1
6
0
4
3
7
6
5
10
4
2
2
3
5
37
1
9
9
11
9
18
10
14
52
7
139
1
0
0
1
2
1
2
2
5
0
0
(continued)
61
0
7
0
0
Table 21 Continued.
Haplotypes
Drainage and Stream
HOBACK
Bondurant Cr
Boulder Cr
Bull Cr
Dell Cr
Hoback R
Jack Cr
Kerr Cr
A
B
C
Subtotal
2
18
SNAKE RIVER CANYON
Cabin Cr
Coburn Cr
Fall Cr, North Fork
Fall Cr, South Fork
Horse Cr
Subtotal
1
8
5
7
1
22
E
F
G
H
I
J
K
L
M
Total
0
2
2
11
2
16
4
3
40
0
3
9
5
7
2
26
2
1
1
11
4
D
4
3
7
0
2
7
1
1
14
1
0
0
0
0
0
1
0
1
0
1
1
0
0
1
2
0
0
(continued)
62
0
0
1
0
0
1
Table 21 Continued.
Haplotypes
Drainage and Stream
GREYS
Blind Trail Cr
Flat Cr
Greys R
Little Greys R
Steer Cr
Three Forks Cr, North3
Unnamed Tributary to
Lower Cabin Cr3
Upper Cabin Cr3
Subtotal
A
1
5
3
2
1
1
2
15
B
C
D
E
F
G
H
I
J
K
L
M
5
4
1
1
1
6
11
1
3
1
6
5
16
13
4
8
2
1
2
1
2
6
1
26
2
1
1
0
6
Total
0
0
0
0
0
3
2
57
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.
63
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.
64