Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=ujfm20

Download by: [Idaho Dept of Fish & Game] Date: 15 March 2017, At: 10:29

North American Journal of Fisheries Management

ISSN: 0275-5947 (Print) 1548-8675 (Online) Journal homepage: http://www.tandfonline.com/loi/ujfm20

Distinguishing Yellowstone Cutthroat Trout,Rainbow Trout, and Hybrids by Use of Field-BasedPhenotypic Characteristics

Kevin A. Meyer, Patrick Kennedy, Brett High & Matthew R. Campbell

To cite this article: Kevin A. Meyer, Patrick Kennedy, Brett High & Matthew R. Campbell (2017)Distinguishing Yellowstone Cutthroat Trout, Rainbow Trout, and Hybrids by Use of Field-BasedPhenotypic Characteristics, North American Journal of Fisheries Management, 37:2, 456-466

To link to this article: http://dx.doi.org/10.1080/02755947.2017.1280572

Published online: 15 Mar 2017.

Submit your article to this journal

View related articles

View Crossmark data

ARTICLE

Distinguishing Yellowstone Cutthroat Trout, Rainbow Trout,and Hybrids by Use of Field-Based PhenotypicCharacteristics

Kevin A. Meyer* and Patrick KennedyIdaho Department of Fish and Game, 1414 East Locust Lane, Nampa, Idaho 83686, USA

Brett HighIdaho Department of Fish and Game, 4279 Commerce Circle, Idaho Falls, Idaho 83401, USA

Matthew R. CampbellIdaho Department of Fish and Game, 1800 Trout Road, Eagle, Idaho 83616, USA

AbstractNative and nonnative salmonids within the same genus sometimes hybridize, and the hybrids are often

difficult to visually distinguish from parental species. We compared phenotypic delineations (based on severalvisual characteristics) and genotypic screening (using seven nuclear DNA loci) for 323 fish collected from anIdaho stream where Yellowstone Cutthroat Trout O. clarkii bouvieri were introgressed with Rainbow Trout O.mykiss to evaluate our ability to visually distinguish Yellowstone Cutthroat Trout from Rainbow Trout andhybrids. Assuming that the genotypes were 100% accurate, correct phenotypic classification was highest forYellowstone Cutthroat Trout (the genotype confirmed the phenotype 94% of the time), followed by hybrids (79%)and Rainbow Trout (71%). All errors were between pure and hybrid fish. All of the measured phenotypiccharacteristics were useful for differentiating Yellowstone Cutthroat Trout from hybrids, but the most informa-tive characteristics were the lack of a white leading tip on the anal and dorsal fins, the presence of fewer than fivespots on the top of the head, and the presence of a bright red-orange throat slash. Fish size did not influencecorrect phenotypic identification, although fry were excluded from our study. A logistic regression modelpredicted that biologists were more than 50% likely to visually detect Rainbow Trout traits in a hybrid whenits level of hybridization was greater than 22% (95% confidence interval = 4–30%). For an additional 189 fish inthree other Idaho streams, phenotypic differentiation of Yellowstone Cutthroat Trout from Rainbow Trout andhybrids was 91% accurate. These results can be used to screen for introgression in Yellowstone Cutthroat Troutpopulations or to selectively remove Rainbow Trout and hybrids from Yellowstone Cutthroat Trout populations.However, in most cases, genetic confirmation of purity or introgression would still be valuable for identifying lowlevels of hybridization in individuals or in the population at large.

Throughout the world, nonnative salmonids have been propa-gated to expand and improve recreational sportfishing opportu-nities (reviewed by Cambray 2003). Unfortunately, whennonnative salmonids are stocked in waters where native congenersalready exist, hybridization between native and nonnative speciesoften results (Leary et al. 1995). Hybridization with hatchery-stocked Rainbow Trout O. mykiss is a leading cause of decline in

many subspecies of Cutthroat Trout Oncorhynchus clarkii inwestern North America (Young 1995; Mayhood 2000; Behnke2002). Such declines have led to numerous subspecies-specificmanagement plans to help conserve as many unhybridizedCutthroat Trout populations as possible (e.g., Coffin and Cowan1995; Hirsch et al. 2005;Mayhood 2014). Common goals outlinedin such plans are to (1) identify pure Cutthroat Trout populations

*Corresponding author: kevin.meyer@idfg.idaho.govReceived September 19, 2016; accepted December 29, 2016

456

North American Journal of Fisheries Management 37:456–466, 2017© American Fisheries Society 2017ISSN: 0275-5947 print / 1548-8675 onlineDOI: 10.1080/02755947.2017.1280572

and protect them from introgression and (2) eradicate or reduceintrogression where feasible by removing Rainbow Trout andhybrids from the system. Both goals require an ability to accuratelydifferentiate between pure and hybrid fish.

Differentiation between Cutthroat Trout and Rainbow Trout isusually straightforward; however, the identification of CutthroatTrout × Rainbow Trout hybrids (hereafter, “hybrids”) can bedifficult. Historically, this was done using morphometrics andmeristics, such as coloration; spotting patterns; the presence orabsence of basibranchial teeth; and counts of scales, fin rays,caeca, and vertebrae (Behnke 1979). Cutthroat Trout andRainbow Trout have separation in most of these characteristics,whereas hybrids tend to have intermediate characteristics,although they also often overlap in both parental species(Behnke 1979). Recent advances in genetic tools, such as theidentification of diagnostic molecular markers to differentiateparental species (Baker et al. 2002; Ostberg and Rodriguez2002; Baumsteiger et al. 2005), have made the detection ofhybridization in fish populations much easier and more accurate(Scribner et al. 2001). However, the cost of genetically analyzingenough fish to confidently generate conclusions about the purityof Cutthroat Trout populations prohibits sole reliance on thistechnique in many instances (Della Croce et al. 2016).

Consequently, there is still a need for phenotypic differentia-tion of Cutthroat Trout from Rainbow Trout and hybrids in thefield. The ability of biologists to visually differentiate such fishdepends on the degree of overlap in phenotypic characteristicsexhibited by Cutthroat Trout and Rainbow Trout. SomeCutthroat Trout subspecies, such as the Coastal Cutthroat TroutO. clarkii clarkii, are particularly difficult to phenotypicallydifferentiate from Rainbow Trout or steelhead (anadromousRainbow Trout; Pollard et al. 1997; Baumsteiger et al. 2005;Kennedy et al. 2009), probably because they evolved in sympa-try. For other subspecies, such as the Yellowstone CutthroatTrout O. clarkii bouvieri, phenotypic differentiation has beenmore successful. Seiler et al. (2009) recently summarized 29morphological measurements, which they used to distinguishbetween groups of Yellowstone Cutthroat Trout, RainbowTrout, and hybrids with a high degree of accuracy. However,their results were based on digitized photographs taken in thefield but processed in the laboratory, and processing involvednumerous meristic measurements, such as eye diameter and thelength of each fin. This technique can be used to retrospectivelyidentify pure or hybridized populations with high accuracy, but itcannot be used in real time. For example, at a spawning weirinstalled to interrogate upstream-migrating fish, where CutthroatTrout are passed upstream to spawn in natal tributaries butRainbow Trout and hybrids are not (e.g., High 2010), a weiroperator cannot count fin rays and scales and measure tediousexternal characteristics on dozens if not hundreds of fish eachday. External characteristics that are more qualitative in nature,such as body coloration, white-tipped fins, the presence of athroat slash, the number of spots on the top of the head, andother spotting patterns or coloration on various regions of the

fish’s body, have been used to differentiate YellowstoneCutthroat Trout from Rainbow Trout and hybrids (e.g.,Campbell et al. 2002; Meyer et al. 2003, 2006). However, noprevious studies have provided the proportional incidence ofspecific phenotypic traits in genetically identified YellowstoneCutthroat Trout, Rainbow Trout, and hybrids.

Because hybrids can be difficult to identify accurately and canexhibit a range of parental phenotypes (Neff and Smith 1979),combining genetic analyses with specific field-based phenotypictraits that are easy to measure would help to create an accuratemetric for quickly differentiating Yellowstone Cutthroat Troutfrom Rainbow Trout and hybrids. Thus, our primary objectivewas to determine the phenotypic traits that could be used to mostaccurately differentiate Yellowstone Cutthroat Trout fromRainbow Trout and hybrids in the field, with an emphasis oncharacteristics that were easiest and fastest to measure. Wedetermined these characteristics from one stream and validatedthem in three additional streams to verify their diagnostic utilityacross a broader geographic range. As a secondary objective, weevaluated whether the ability of biologists to identify RainbowTrout traits in an individual hybrid was influenced by the degreeof hybridization within the fish.

METHODSFish sampling.—This study was primarily conducted in

Palisades Creek, Idaho, a fourth-order (Strahler 1964; at the1:24,000 hydrologic scale) tributary of the South Fork SnakeRiver near the Idaho–Wyoming border. The stream has a meanwidth of about 10 m and an average gradient of about 2% in thearea of study. Although the stream is 30 km long, a high-gradient,steep cascading section near river kilometer 11 presently appearsto form a natural velocity barrier to fish, as Rainbow Trout andhybrids are absent upstream of this section (Meyer et al. 2006; K.A. Meyer, unpublished data).

Palisades Creek is one of four primary spawning tributaries forYellowstone Cutthroat Trout in the South Fork Snake River. Bythe early 1980s, wild Rainbow Trout had begun to appear inPalisades Creek and the main-stem South Fork Snake River,prompting cessation of all hatchery Rainbow Trout stocking.Since 2000, weirs have been installed and operated on PalisadesCreek in an attempt to halt upstream spawning migrations ofRainbow Trout and hybrids (Yellowstone Cutthroat Trout arepassed over the weirs). However, most of the weirs were nothighly efficient or could not be operated effectively at very highspring flows during the critical period of the spawning migrationrun (High 2010). As a result, a permanent electric weir wasinstalled on Palisades Creek during 2009 at a site 0.7 km upstreamfrom the confluence with the South Fork Snake River.

For the present study, fish were collected via backpack elec-trofishing in September 2012 throughout the lower 11 km ofPalisades Creek (but above the weir). Random samples ofabout 30 Oncorhynchus individuals from every 800 m of streamwere collected. Age-0 fish were excluded from our study for two

DISTINGUISHING TROUT HYBRIDS BASED ON PHENOTYPE 457

reasons. First, for all three taxa (i.e., Yellowstone CutthroatTrout, Rainbow Trout, and hybrids), age-0 fish were small (i.e.,<60 mm TL) at the time of sampling and were extremely rare inthe catch. Second, some of the phenotypic traits that distinguishCutthroat Trout from Rainbow Trout are not fully expressed inage-0 fish (Miller 1950; Martinez 1984; Seiler et al. 2009).

Fish were sedated in a solution of peppermint oil (Danner et al.2011) and water (using a 1:10 stock solution of peppermint oil andethanol; 0.3–0.5mL of the solutionwas used per liter of water) andwere measured for TL. Means and ranges of TLs were similarbetween Yellowstone Cutthroat Trout, Rainbow Trout, andhybrids, although Rainbow Trout were slightly smaller on averagethan the other two groups (Figure 1). A suite of phenotypiccharacteristics was then recorded for each fish (Table 1;Figure 2); selected characteristics were largely based on the resultsof previous studies that differentiated Yellowstone Cutthroat Trout

from Rainbow Trout and hybrids (Campbell et al. 2002; Meyeret al. 2003, 2006). Most of the characteristics are self-explanatory,but spot counts on the top of the head require further description.Biologists specifically counted the spots that were present directlyon the top of the head, above the eyes, starting from just anterior ofthe nares and extending posterior to the point of scale formation(Figure 2). On pure Yellowstone Cutthroat Trout, there is often onespot next to each nare, and if present those spots were not counted.Based on the aforementioned characteristics, one of three experi-enced biologists classified the fish as a Yellowstone CutthroatTrout, a Rainbow Trout, or a hybrid. A small fin clip was collectedfrom each fish for use in genetic analyses.

Genetic analyses.—All genetic samples were screened forRainbow Trout hybridization/introgression by using sevendiagnostic nuclear DNA (nDNA) markers (Occ34, Occ35,Occ36, Occ37, Occ38, Occ42, and OM55; Ostberg andRodriguez 2002). Individual genetic classification was based oncomposite nDNA genotypes following procedures similar tothose outlined by Ostberg and Rodriguez (2006) and Kozfkayet al. (2007). Samples were classified as pure YellowstoneCutthroat Trout if they were homozygous for O. clarkiibouvieri alleles at all loci, pure Rainbow Trout if they werehomozygous for O. mykiss alleles at all loci, and hybrids if theypossessed a mixture of alleles from the two parental species.Hybrids were further classified as first-generation hybrids (F1)if they were heterozygous at all loci, or >F1 hybrids (i.e., eitherRainbow Trout or Yellowstone Cutthroat Trout backcrosses) ifthey possessed a mix of heterozygous and homozygous loci.With seven codominant nDNA loci, our probability ofmistaking a backcross hybrid (>F1) as an F1 hybrid was lessthan 1% (Boecklen and Howard 1997). The use of seven locigave us a 90% probability of detecting a 15% or greater level ofintrogression in an individual fish (Rasmussen et al. 2010).

Statistical analyses.—Logistic regression was used todetermine which phenotypic characteristics (as the independentvariables) best distinguished Yellowstone Cutthroat Trout fromhybrids. We considered this differentiation the most important

TABLE 1. Variations in eight phenotypic characteristics that were used to distinguish Yellowstone Cutthroat Trout from Rainbow Trout and hybrids in PalisadesCreek, Idaho. See Figure 2 for illustrations of these characteristics; letters correspond to the particular traits that are depicted in the figure.

Phenotypic characteristics by taxon

Phenotypic metric Yellowstone Cutthroat Trout Hybrids Rainbow Trout

Belly coloration Orange (A) Variable WhiteSpots on side of body Large; clustered dorsally and posteriorly (B) Variable Small; distributed evenlyThroat slash Bright red-orange (C) Dull or faint (E) AbsentWhite leading edge of pelvic fins Absent Variable Present (F)White leading edge of anal fin Absent Variable Present (G)White leading edge of dorsal fin Absent Variable Present (H)Spots on top of heada Very few or no spots (D) Variable Many spots (I)Coloration on side of body No reddish hue Variable Reddish hue (J)

aOne spot is often present next to each nare, but those spots are not included in the count.

FIGURE 1. Box-and-whisker plots depicting the TLs of fish that were genotypi-cally identified as Rainbow Trout, Yellowstone Cutthroat Trout, or hybrids inPalisades Creek, Idaho (line inside the box = median; boundaries of box = 25thand 75th percentiles; whiskers = 10th and 90th percentiles; black circles = 5th and95th percentiles). Fish smaller than 60 mm TL were excluded from the study.

458 MEYER ET AL.

A C B

G

C E

D

F

H

H

G FJI

FIGURE 2. Representative phenotypes for Yellowstone Cutthroat Trout (upper fish), Rainbow Trout (lower fish), and Yellowstone Cutthroat Trout × RainbowTrout hybrids (middle fish), with various phenotypic characteristics indicated by uppercase letters that correspond to the letters in Table 1 (full descriptions areprovided in the table). The lower three panels depict the head spot counting area and two throat slash variants.

DISTINGUISHING TROUT HYBRIDS BASED ON PHENOTYPE 459

for two reasons. First, pure fish of either species were neverconfused with the opposite parental species, so thedifferentiation of Yellowstone Cutthroat Trout from RainbowTrout was unambiguous. Second, differentiating betweenRainbow Trout and hybrids was considered immaterial sincedetecting either one would confirm the presence of RainbowTrout alleles in any Yellowstone Cutthroat Trout populationbeing evaluated.

We coded our response variable as 0 for hybrids and 1 forYellowstone Cutthroat Trout. Most of the independent variableswere either “yes” or “no” categories (e.g., the leading tip of thedorsal fin either had white coloration or it did not), so we codedthem similarly. For the throat slash, we used only two categories(i.e., “bright red-orange” or “other”). The number of spots on thetop of the fish’s head was categorized as either ≥5 or <5; althoughthe cutoff value was somewhat arbitrary, it was based on years ofexperience in differentiating these three taxa. All possible candi-date models were tested, and Akaike’s information criterion (AIC)was used to identify the best models. We only present the mostplausible models—those for which AIC values were within 2 unitsof the best-performing model (Burnham and Anderson 2004). Wecalculated AIC weights (wi) to judge the relative plausibility ofeach model within the set of most plausible models, and theadjusted R2 for discrete models (~R2; Nagelkerke 1991) was usedto assess the amount of variation explained by each model. TheHosmer–Lemeshow goodness-of-fit statistic (Hosmer et al. 2013)was used to verify that the most plausible logistic regressionmodels adequately fit the data.

A separate logistic regression model was constructed to eval-uate whether fish TL (as the independent variable) was related tothe accuracy of our phenotypic classification; for the responsevariable, a value of 0 was used for inaccurate phenotypic classi-fications and a value of 1 was used for accurate classifications.

Validation of phenotypic characteristics in additionalstreams.—During August and September 2015, we evaluatedwhether the phenotypic characteristics that best distinguishedYellowstone Cutthroat Trout from hybrids in Palisades Creekalso worked effectively at other locations in eastern Idaho. Thisevaluation was conducted in three rivers (Pine Creek, ToponceCreek, and the South Fork Snake River) that were known tocontain Yellowstone Cutthroat Trout, Rainbow Trout, and hybrids.

For each reach sampled in 2015, several-hundred meters ofeach stream were electrofished to gather a sample of fish.Immediately after immobilized fish were netted, an experiencedbiologist examined the fish cursorily to select which individualsto retain for our sample and which fish to return immediately tothe stream. During this initial screening, we retained fish from alltaxa based on overall appearance rather than any specific distin-guishing characteristics. We selected fish in this manner—ratherthan genetically sampling all captured fish until an adequatesample size was achieved—because it allowed us to obtain asample with a wider range of fish sizes composed of all three taxaof interest. From these retained fish, we sampled randomly untilan adequate sample size (n ≥ 30 fish) was obtained for each

stream. Fish were classified as Yellowstone Cutthroat Trout,Rainbow Trout, or hybrids based on the phenotypic characteris-tics included in the top models developed for Palisades Creekfish. Fish were screened genetically via the same methods asdescribed above, and the genotype and phenotype were com-pared to evaluate the accuracy of phenotypically basedclassifications.

Relating visual detection of Rainbow Trout alleles tointrogression levels.—A final logistic regression was used toevaluate whether visual detection of O. mykiss traits inindividual hybrids was related to the degree of hybridizationin the fish. Hybrids from all four streams (n = 144) werecombined for this analysis. We coded the response variableas 0 for inaccurate phenotypic classification and 1 for accuratephenotypic classification of fish that were geneticallyconfirmed to be hybrids, and the independent variable wasthe number of alleles (ranging from 1 to 13 out of the 14alleles screened) that were identified as originating fromRainbow Trout. All statistical analyses were conducted inSAS (SAS 2009), with α set at 0.05.

RESULTSDuring September 2012 in Palisades Creek, a total of 323 fish

were randomly sampled for phenotype–genotype comparisons.Based on genotype, 218 fish were identified as YellowstoneCutthroat Trout, 27 were identified as Rainbow Trout, 34 wereF1 hybrids, and 44 were >F1 hybrids. Assuming 100% genotyp-ing accuracy, our phenotypically based classifications inPalisades Creek were 94% accurate for Yellowstone CutthroatTrout, 79% accurate for hybrids, and 71% accurate for RainbowTrout. All phenotypic errors were between pure parental fish andhybrids (i.e., we never confused a Yellowstone Cutthroat Troutfor a Rainbow Trout or vice versa). Fish that were misidentifiedphenotypically as Rainbow Trout (n = 5) were all Rainbow Troutbackcross hybrids, with each fish having at least 11 RainbowTrout alleles (mean = 12.2 O. mykiss alleles out of the 14 thatwere screened). Fish that were misidentified phenotypically ashybrids (n = 16) were all pure Rainbow Trout except for oneYellowstone Cutthroat Trout. Fish that were misidentified phe-notypically as Yellowstone Cutthroat Trout (n = 14) were allhybrids, and all but one were Yellowstone Cutthroat Trout back-cross hybrids. Of the 14 hybrids that were misclassified asYellowstone Cutthroat Trout, 29% had only one Rainbow Troutallele, and their average number of RainbowTrout alleles was 2.9(out of 14). Phenotypic accuracy in differentiating YellowstoneCutthroat Trout from Rainbow Trout and hybrids was 95%.

All three biologists were accurate in phenotypically distin-guishing between taxa. Two of the biologists were 92% and96% accurate; the third biologist was 83% accurate, with 15 of20 mistakes involving the misidentification of pure RainbowTrout as hybrids.

All of the field-based phenotypic characteristics we analyzedwere useful for differentiating Yellowstone Cutthroat Trout

460 MEYER ET AL.

from Rainbow Trout and hybrids (Table 2). For example, 95%of Yellowstone Cutthroat Trout had fewer than our a prioriarbitrary cutoff of five spots on the top of the head, whereasonly 8% of Rainbow Trout and hybrids had fewer than fivespots on the head (Table 2; Figure 3). In fact, the degree ofhybridization in individual hybrids directly affected the numberof head spots (Figure 4). Least-squares linear regression pre-dicted that at the lowest level of hybridization we could detectwithin an individual hybrid (i.e., 1 of 14 alleles, or 7%), the fishwould have six spots on average.

The absence of white leading tips on the pelvic, anal, anddorsal fins was also particularly useful for correctly classifyingYellowstone Cutthroat Trout (Table 2). For example, noYellowstone Cutthroat Trout had white tips on the dorsal fin,and only 1 of 71 fish with white tips on the pelvic fins and 2 of 81fish with white tips on the anal fin were Yellowstone CutthroatTrout (assuming that those individuals did not represent errors ingenotyping or data recording). However, these characteristicswere less useful for correctly classifying hybrids: 45% of hybridslacked white tips on the pelvic fins, 33% of hybrids lacked whitetips on the anal fin, and 47% of hybrids lacked white tips on thedorsal fin. The throat slash, body spotting pattern, and sidecoloration were also good traits for separation, although somehybrids exhibited Yellowstone Cutthroat Trout characteristics inall of the meristics we examined.

Phenotypic characteristics that were most commonly included(and usually statistically significant) in the best logistic regressionmodels for separating Yellowstone Cutthroat Trout from hybridswere as follows: (1) the absence of white on the leading tip of theanal fin, (2) the occurrence of fewer than five spots on the top ofthe head, (3) the presence of a bright red-orange throat slash, and(4) spots on the side of the body that were clustered dorsally andposteriorly (Table 3). White on the leading tip of the dorsal fin (theabsence of which was a distinguishing trait for YellowstoneCutthroat Trout) was included as a variable in all of the topcandidate models, but this trait was not statistically significant inany of the models. In a separate logistic regression model, fish TLwas not related to the accuracy of our phenotypic classifications(Wald χ2 = 1.21, P = 0.27), indicating that correct classificationsbased on phenotype were not more difficult for smaller fish.

Validation of Phenotypic Characteristics in AdditionalStreams

The phenotypic characteristics that were most diagnostic indifferentiating taxa at Palisades Creek were used to phenotypicallyclassify 189 fish collected from three other streams in easternIdaho during August and September 2015. Assuming 100% gen-otyping accuracy, our phenotypically based classifications in thefield were 89% accurate for Yellowstone Cutthroat Trout (n =110), 85% accurate for Rainbow Trout (n = 13), and 58% accuratefor hybrids (n = 66). Two fish that were misidentified phenotypi-cally as Rainbow Trout were both hybrids with an average of 9Rainbow Trout alleles (out of the 14 screened alleles). Individualsthat were misidentified phenotypically as hybrids (n = 16) were

mostly Rainbow Trout, although some were YellowstoneCutthroat Trout (n = 5). Fish that were misclassified phenotypi-cally as Yellowstone Cutthroat Trout (n = 12) were all YellowstoneCutthroat Trout backcross hybrids; half of these fish had only oneRainbow Trout allele (mean = 2.2 O. mykiss alleles out of 14screened). Phenotypic accuracy for differentiating YellowstoneCutthroat Trout from Rainbow Trout and hybrids in the threestreams was 91%.

Relating Visual Detection of Rainbow Trout Alleles toIntrogression Levels

Including data from all four streams, it was evident that forgenetically confirmed hybrids, the ability of biologists to phe-notypically detect Rainbow Trout traits in an individual wasaffected by the fish’s degree of hybridization at the seven lociwe screened (Figure 5). Using logistic regression (Wald χ2 =30.0, P < 0.001), we predicted that biologists were more than50% likely to visually detect Rainbow Trout traits in a hybridwhen the level of hybridization in the fish was greater than22% (95% confidence interval = 4–30%).

DISCUSSIONResults from this study demonstrate that Rainbow Trout and

hybrids can readily be distinguished from Yellowstone CutthroatTrout based on simple phenotypic characteristics. In PalisadesCreek, only 0.5% of the captured Yellowstone Cutthroat Troutwere mistakenly identified as hybrids, and only 41 of the 897Rainbow Trout alleles detected in the fish we analyzed geneti-cally were missed in individuals that were misclassified asYellowstone Cutthroat Trout. Moreover, the characteristics thatworked best at differentiating between taxa in Palisades Creekwere also effective in other streams, suggesting that our resultscan be used across a broad geographic scale—and perhaps acrossthe range of Yellowstone Cutthroat Trout—for differentiatingRainbow Trout and hybrids from Yellowstone Cutthroat Trout.If the phenotypic characteristics that we identified as most usefulcan be tested in other portions of the Yellowstone CutthroatTrout’s range, this would reveal how broadly the present findingscan be applied. How easily these characteristics can be used bynovice biologists or seasonal field staff to differentiate taxawould also be a useful avenue for future research; consideringthe simplicity of the characteristics we identified as most useful,we expect that inexperienced field staff would have little diffi-culty in applying our findings.

Our phenotypic accuracy was highest for YellowstoneCutthroat Trout. This concurs with the work of Campbell et al.(2002), who found that hatchery personnel at Henrys Lake,Idaho, could visually distinguish Yellowstone Cutthroat Trout(94% accuracy) much better than hybrids (48% accuracy) basedon a similar array of phenotypic characteristics, although wewere more accurate at identifying hybrids (79% in PalisadesCreek; 58% in other streams) than Campbell et al. (2002). Onedifference between the two studies was that Campbell et al.

DISTINGUISHING TROUT HYBRIDS BASED ON PHENOTYPE 461

(2002) did not record the phenotypic characteristics of eachindividual fish. By comparing these characteristics to genotypicresults via logistic regression, we were able to pinpoint whichtraits were most useful for separating taxa. Unfortunately, nocombination of phenotypic characteristics could have differen-tiated Yellowstone Cutthroat Trout from hybrids with 100%

accuracy. In fact, across all four streams, none of the 27 hybridsthat were phenotypically misclassified as Yellowstone CutthroatTrout had white leading tips on any of their fins; all but four hadfewer than five spots on the top of the head; all but four had spotson the side of the body that were clustered dorsally and poster-iorly; and all but five had a strongly discernible throat slash. Most

TABLE 2. Frequency of occurrence (%) of the variations in phenotypic characteristics used to distinguish Yellowstone Cutthroat Trout from Rainbow Trout andhybrids (>F1 hybrids = backcross hybrids) in Palisades Creek and three additional streams in Idaho.

Genotype

Meristic Phenotypic characterYellowstone

Cutthroat Trout >F1 hybrid F1 hybrid Rainbow Trout

Palisades CreekHead spots Five or more 4.6 68.2 91.2 96.3

Fewer than five 95.4 31.8 8.8 3.7Belly Orange hue 26.1 9.1 5.9 0.0

White hue 73.9 90.9 94.1 100.0Pelvic fins White tips present 0.5 52.3 58.8 100.0

White tips absent 99.5 47.7 41.2 0.0Anal fin White tips present 0.9 56.8 79.4 100.0

White tips absent 99.1 43.2 20.6 0.0Dorsal fin White tips present 0.0 38.6 70.6 88.9

White tips absent 100.0 61.4 29.4 11.1Throat slash Bright red-orange and prominent 89.9 34.1 17.6 0.0

Dull but continuous 4.1 11.4 17.6 0.0Faint and barely visible 6.0 43.2 64.7 59.3Absent 0.0 11.4 0.0 40.7

Body spots Smaller; distributed evenly on sides 3.7 70.5 76.5 100.0Larger; clustered dorsally and posteriorly 96.3 29.5 23.5 0.0

Side coloration Presence of reddish hue 5.5 61.4 85.3 96.3Absence of reddish hue 94.5 38.6 14.7 3.7

Other streamsHead spots Five or more 12.6 32.6 85.7 91.7

Fewer than five 87.4 67.4 14.3 8.3Belly Orange hue 41.7 8.7 42.9 5.7

White hue 58.3 91.3 57.1 94.3Pelvic fins White tips present 1.9 52.2 42.9 91.7

White tips absent 98.1 47.8 57.1 8.3Anal fin White tips present 0.0 63.0 57.1 100.0

White tips absent 100.0 37.0 42.9 0.0Dorsal fin White tips present 2.9 52.2 85.7 88.9

White tips absent 97.1 47.8 14.3 11.1Throat slash Bright red-orange and prominent 89.3 39.1 28.6 0.0

Dull but continuous 0.0 2.2 14.3 8.3Faint and barely visible 10.7 47.8 42.9 61.1Absent 0.0 10.9 14.3 30.6

Body spots Smaller; distributed evenly on sides 10.7 50.0 28.6 75.0Larger; clustered dorsally and posteriorly 89.3 50.0 71.4 25.0

Side coloration Presence of reddish hue 95.1 58.7 57.1 91.7Absence of reddish hue 4.9 41.3 42.9 8.3

462 MEYER ET AL.

of these hybrids had only one Rainbow Trout allele or a fewRainbow Trout alleles at the loci we included in our geneticscreening, so it is not surprising that they displayed phenotypiccharacteristics that were mostly (or sometimes entirely) the sameas those of pure Yellowstone Cutthroat Trout.

Surprisingly, our ability to phenotypically differentiateYellowstone Cutthroat Trout from Rainbow Trout and hybridswas not diminished at smaller fish sizes. Although this waspartly due to the fact that we excluded fry from our analyses(since they were generally too small to capture), nevertheless84 of the 511 fish examined across all four streams weresmaller than 150 mm TL, and error rates were essentiallyequivalent between small fish (18%) and large fish (15%).Spotting patterns and other phenotypic characteristics arehighly variable or not fully expressed in juvenile trout(Seiler et al. 2009), and small Cutthroat Trout are notoriouslydifficult to distinguish from Rainbow Trout (Miller 1950;Martinez 1984; Campton and Utter 1985), so we expected amarked difference in error rates between smaller and largerfish. However, previous studies have also demonstrated suc-cessful differentiation between small fish of these two speciesbased on phenotypic traits (e.g., Weigel et al. 2002; Seileret al. 2009). For fry, we suspect that white leading tips onthe anal, dorsal, or pelvic fins may be the most fully expressed

0.00

0.20

0.40

0.60

0.80

1.00Rainbow Trout

0.00

0.20

0.40

0.60

0.80

1.00Hybrids

0.00

0.20

0.40

0.60

0.80

1.00

0 2 4 6 8 10 12 14 16 18 ≥20

Number of spots on the top of the head

Yellowstone Cutthroat Trout

Pe

rce

nt

of f

ish

FIGURE 3. Percentages of Yellowstone Cutthroat Trout, Rainbow Trout, andhybrids sampled in Palisades Creek that exhibited various numbers of spots onthe top of the head.

y = 23.9x + 3.8

R² = 0.16

P < 0.001

0

10

20

30

40

50

60

70

80

0.00 0.20 0.40 0.60 0.80 1.00

Nu

mb

er o

f h

ea

d s

po

ts

Percent O. mykiss alleles

FIGURE 4. Relationship between the level of introgression (percentage ofRainbow Trout O. mykiss alleles among the 14 alleles that were screened) andthe number of spots on the top of the head for Yellowstone Cutthroat Trout ×Rainbow Trout hybrids in Palisades Creek. The line, equation, and statisticswere obtained by fitting a least-squares linear regression to the data.

TABLE 3. Top candidate logistic regression models that best differentiatedYellowstone Cutthroat Trout from Yellowstone Cutthroat Trout × RainbowTrout hybrids in Palisades Creek, Idaho, based on phenotypic characteristics(AIC = Akaike’s information criterion; ΔAIC = difference in AIC valuebetween the specified model and the best-performing model; wi = Akaikeweight; ~R2= adjusted R2 for discrete models). Independent variables (definedin Table 2) that were statistically significant in the models (at α = 0.05) areindicated by bold italic font, and the variables are listed in order of their Waldχ2 value (highest to lowest).

Model AIC ΔAIC wi~R2

Anal fin + Head spots + Throatslash + Body spots + Dorsal fin

128.38 0.00 0.23 0.68

Head spots + Anal fin + Throat slash+ Side coloration + Dorsal fin

128.77 0.39 0.19 0.68

Anal fin + Head spots + Throatslash + Body spots + Sidecoloration + Dorsal fin

129.14 0.76 0.16 0.68

Anal fin + Head spots + Throatslash + Body spots + Bellycoloration + Dorsal fin

129.70 1.32 0.12 0.68

Head spots + Anal fin + Throatslash + Side coloration + Bellycoloration + Dorsal fin

130.02 1.64 0.10 0.68

Head spots + Anal fin + Throatslash + Dorsal fin

130.07 1.69 0.10 0.68

Head spots + Throat slash + Bodyspots + Anal fin + Pelvic fins +Dorsal fin

130.09 1.71 0.10 0.68

DISTINGUISHING TROUT HYBRIDS BASED ON PHENOTYPE 463

characteristics for distinguishing Rainbow Trout and hybridsfrom Yellowstone Cutthroat Trout at that size; future researchto confirm or refute this suspicion would be useful.

Most of the phenotypic characteristics we used to distinguishtaxa were clear-cut, whereas the a priori cutoff for the number ofhead spots required for phenotypic classification of fish asYellowstone Cutthroat Trout was admittedly subjective, despitebeing based on years of experience in phenotypic separation ofthese taxa. The results of this study substantiated our preconceivednotion that a count of about four to six spots on the top of the headis a reasonable cutoff for separating Yellowstone Cutthroat Troutfrom hybrids, although a few Yellowstone Cutthroat Trout hadmore than six head spots. The gap between one YellowstoneCutthroat Trout with eight head spots and three YellowstoneCutthroat Trout with 20 or more head spots (Figure 3), in additionto the three Yellowstone Cutthroat Trout with white leading tips oneither their pelvic fins or anal fin, suggests that a few errors ingenotyping or data recording were made. Because (1) phenotypicdifferentiation of parental taxa was unambiguous, (2) errors inclassification between Rainbow Trout and hybrids were unimpor-tant (either one indicated O. mykiss alleles, which were to beremoved from the stream), and (3) errors on genotypically con-firmed hybrids would be nonsensical (i.e., if alleles from bothparental types were identified, then the fish was unmistakably ahybrid), genotyping errors were only relevant for the 328

Yellowstone Cutthroat Trout (across all four streams) that weregenotyped as pure but that nevertheless might have been hybrids.Among the six Yellowstone Cutthroat Trout with at least 20 headspots or with white tips on pelvic fins or the anal fin, all werephenotyped as hybrids but no Rainbow Trout alleles were ampli-fied; this suggests that at least a few genotyping errors were made,but it is also possible that Yellowstone Cutthroat Trout exhibitwider variation in some of the measured phenotypic traits.Considering that we screened only seven nDNA loci, some geno-typing errors in individual fish should indeed be expected in ourresults. Based on genetic sample size formulas provided byBoecklen and Howard (1997) and Rasmussen et al. (2010), theability to detect a low level of introgression (5%) with a high levelof confidence (95%) for individual fish would require the screen-ing of 29 diagnosticmarkers, and even thenwewould expect somehybrids to be missed. The relationship we found between a fish’slevel of Rainbow Trout introgression and our probability ofvisually detecting Rainbow Trout characteristics in that fish(Figure 4) suggests that any errors in genotyping were likelymade on individuals with very low levels of introgression, thusminimizing the consequences of mistakenly releasing thosehybrids back into the Yellowstone Cutthroat Trout population.Recent identification of single-nucleotide polymorphism lociwith fixed allelic differences between Yellowstone CutthroatTrout and Rainbow Trout (e.g., Hohenlohe et al. 2011) shouldhelp to alleviate genotyping mistakes except for fish with extre-mely low levels of introgression.

When trying to phenotypically detect introgression in aYellowstone Cutthroat Trout population, the misclassificationof a sampling reach as containing no fish with Rainbow Troutalleles would be highly unlikely unless very few fish are sur-veyed or unless introgression is extremely low in that reach.However, because genotypic screening is more likely than phe-notypic delineation to detect low levels of introgression inYellowstone Cutthroat Trout, we encourage any broad-scalestatus surveys of this subspecies (e.g., Kruse et al. 2000; Meyeret al. 2006) to incorporate ample genetic sampling, in as manylocations as possible, to support the classification of reaches ascontaining unhybridized fish. For phenotypic detection of hybri-dization in individual fish, such as during a culling program orwhen operating a weir at which fish with Rainbow Trout traits areblocked from spawning tributaries, it could be argued that acci-dentally culling or blocking access to a few YellowstoneCutthroat Trout would be more acceptable than accidentallyreleasing or allowing passage to fish with Rainbow Trout alleles.Unfortunately, our results indicate that the latter mistakes wouldprobably be more common. Regardless of the design and goal ofthe program, any phenotypically based classification ofYellowstone Cutthroat Trout individuals or populations mustacknowledge that some level of error is unavoidable.

Removal of Rainbow Trout and hybrids from Cutthroat Troutpopulations is a common conservation strategy across westernNorth America to preserve native Cutthroat Trout populationsthat are threatened with hybridization (e.g., Metcalf et al. 2008;

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

Pr (

ph

en

otyp

ica

lly

de

te

ctin

g O

. m

ykis

stra

its)

Percent of screened alleles of O. mykiss origin

FIGURE 5. Logistic regression relationship between the level of introgres-sion (percentage of Rainbow Trout O. mykiss alleles among the 14 alleles thatwere screened) and the probability of visually detecting Rainbow Troutcharacteristics in Yellowstone Cutthroat Trout × Rainbow Trout hybridssampled from four study streams in eastern Idaho (dashed lines = 95%prediction limits). The dotted vertical and horizontal lines depict the level ofhybridization at which biologists were more than 50% likely to visually detectRainbow Trout traits in an individual hybrid.

464 MEYER ET AL.

Muhlfeld et al. 2009; High 2010; Al-Chokhachy et al. 2014). Suchactivities imply that hybridized populations of Cutthroat Trouthave conservation value, although debate on this subject hasbeen fervent (Allendorf et al. 2004, 2005; Campton and Kaeding2005). Many governmental agencies in the western USA collec-tively delineate Cutthroat Trout populations into three categories(UDWR 2000), which include core (<1% introgressed), conserva-tion (1–10% introgressed), and sport fish (>10% introgressed)populations. According to this delineation, all three categoriesare considered valuable for several reasons. First, at the scale ofmost existing data, it is usually difficult to describe the extent andlevel of introgression throughout a particular Cutthroat Troutpopulation. Second, even in Cutthroat Trout populations wherehybridization is extensive, contrary to scientific perception(Allendorf and Leary 1988), empirical evidence suggests thathybrid swarms in Cutthroat Trout populations rarely develop(McKelvey et al. 2016). For populations in which hybrid swarmshave not formed and in which pure Cutthroat Trout still persist, theremoval of RainbowTrout and hybrids should reduce the level andspread of introgression (e.g., Host 2003; Al-Chokhachy et al.2014) and may eventually restore the Cutthroat Trout populationto a genetically pure or near-pure condition (Leary et al. 1995).Genetic testing of each fish and releasing individuals only aftertheir genetic purity has been established will rarely be feasible.Our results suggest that the use of several simple phenotypiccharacteristics to visually identify Rainbow Trout and hybrids inintrogressed populations of Yellowstone Cutthroat Trout willresult in minimal failures to detect Rainbow Trout alleles. Suchphenotypic characteristics should prove useful until more reliablegenetic analyses can be conducted within the population of interestto confirm or refute conclusions that have been drawn fromphenotypic classifications.

ACKNOWLEDGMENTSNumerous people were involved in obtaining fin clips and in

laboratory processing of the genetic samples, most notably C.Sullivan, E. Larson, C. Traughber, S. Elle, K. Nelson, K. Heindel,and A. Boone. We thank K. Heim and D. Schill for providingmanuscript comments and suggestions that greatly improved thepaper. This study was funded in part by anglers and boatersthrough their purchase of Idaho fishing licenses, tags, and permitsand from federal excise taxes on fishing equipment and boat fuelthrough the Federal Aid in Sport Fish Restoration Program.

REFERENCESAl-Chokhachy, R., C. C. Muhlfeld, M. C. Boyer, L. A. Jones, A. Steed, and J.

L. Kershner. 2014. Quantifying the effectiveness of conservation measuresto control the spread of anthropogenic hybridization in stream salmonids:a climate adaptation case study. North American Journal of FisheriesManagement 34:642–652.

Allendorf, F. W., and R. F. Leary. 1988. Conservation and distribution ofgenetic variation in a polytypic species, the Cutthroat Trout. ConservationBiology 2:170–184.

Allendorf, F. W., R. F. Leary, N. P. Hitt, K. L. Knudsen, M. C. Boyer, and P.Spruell. 2005. Cutthroat Trout hybridization and the U.S. EndangeredSpecies Act: one species, two policies. Conservation Biology 19:1326–1328.

Allendorf, F. W., R. F. Leary, N. P. Hitt, K. L. Knudsen, L. L. Lundquist, andP. Spruell. 2004. Intercrosses and the U.S. Endangered Species Act:should hybridized populations be included as Westslope Cutthroat Trout?Conservation Biology 18:1203–1213.

Baker, J., P. Bentzen, and P. Moran. 2002. Molecular markers distinguishCoastal Cutthroat Trout from Coastal Rainbow Trout/steelhead and theirhybrids. Transactions of the American Fisheries Society 131:404–417.

Baumsteiger, J., D. Hankin, and E. J. Loudeslager. 2005. Genetic analyses ofjuvenile steelhead, Coastal Cutthroat Trout, and their hybrids differ sub-stantially from field identifications. Transactions of the American FisheriesSociety 134:829–840.

Behnke, R. J. 1979. The native trouts of the genus Salmo of western NorthAmerica. Report to the U.S. Fish and Wildlife Service, Denver.

Behnke, R. J. 2002. Trout and salmon of North America. Free Press, New York.Boecklen, W. J., and D. J. Howard. 1997. Genetic analysis of hybrid zones:

numbers of markers and power of resolution. Ecology 78:2611–2616.Burnham, K. P., and D. R. Anderson. 2004. Multimodel inference: under-

standing AIC and BIC in model selection. Sociological Methods andResearch 33:261–304.

Cambray, J. A. 2003. The global impact of alien trout species—a review withreference to their impact in South Africa. African Journal of AquaticScience 28:61–67.

Campbell, M. R., J. Dillon, and M. S. Powell. 2002. Hybridization andintrogression in a managed, native population of Yellowstone CutthroatTrout: genetic detection and management implications. Transactions of theAmerican Fisheries Society 131:364–375.

Campton, D. E., and L. R. Kaeding. 2005. Westslope Cutthroat Trout, hybri-dization, and the U.S. Endangered Species Act. Conservation Biology19:1323–1325.

Campton, D. E., and F. M. Utter. 1985. Natural hybridization between steel-head trout (Salmo gairdneri) and Coastal Cutthroat Trout (Salmo clarkiclarki) in two Puget Sound streams. Canadian Journal of Fisheries andAquatic Sciences 42:110–119.

Coffin, P. D., and W. F. Cowan. 1995. Lahontan Cutthroat Trout(Oncorhynchus clarki henshawi) recovery plan. U.S. Fish and WildlifeService, Region 1, Portland, Oregon.

Danner, G. R., K. W. Muto, A. M. Zieba, C. M. Stillman, J. A. Seggio, and S.T. Ahmad. 2011. Spearmint (l-carvone) oil and wintergreen (methyl sali-cylate) oil emulsion is an effective immersion anesthetic of fishes. Journalof Fish and Wildlife Management 2:146–155.

Della Croce, P., G. C. Poole, and G. Luikart. 2016. Detecting and quantifyingintrogression in hybridized populations: simplifying assumptions yield over-confidence and uncertainty. Molecular Ecology Resources 16:1287–1302.

High, B. 2010. Yellowstone Cutthroat Trout conservation efforts on the South ForkSnake River. Pages 275–284 in R. F. Carline and C. LoSapio, editors. WildTrout X: conserving wild trout. Wild Trout Symposium, Bozeman, Montana.

Hirsch, C. L., S. Q. Albeke, and T. P. Nesler. 2005. Range-wide status ofColorado River Cutthroat Trout, Oncorhynchus clarki pleuriticus.Colorado Parks and Wildlife, Fort Collins.

Hohenlohe, P. A., S. J. Amish, J. M. Catchen, F. W. Allendorf, and G. Luikart.2011. Next-generation RAD sequencing identifies thousands of SNPs forassessing hybridization between Rainbow and Westslope Cutthroat trout.Molecular Ecology Resources 11:117–122.

Hosmer, D. W., S. Lemeshow Jr., and R. X. Sturdivant. 2013. Appliedlogistical regression, third edition. Wiley, Hoboken, New Jersey.

Host, S. A. 2003. Reducing introgression with the use of a barrier in tribu-taries of the South Fork Snake River, Idaho. Master’s thesis. University ofIdaho, Moscow.

Kennedy, B. M., J. Baumsteiger, W. L. Gale, W. R. Ardren, and K. G.Ostrand. 2009. Morphological, physiological, and genetic techniques forimproving field identification of steelhead, Coastal Cutthroat Trout, and

DISTINGUISHING TROUT HYBRIDS BASED ON PHENOTYPE 465

hybrid smolts. Marine and Coastal Fisheries: Dynamics, Management, andEcosystem Science [online serial] 1:45–56.

Kozfkay, C. C., M. R. Campbell, S. P. Yundt, M. P. Peterson, and M. S.Powell. 2007. Incidence of hybridization between naturally sympatricWestslope Cutthroat Trout and Rainbow Trout in the Middle ForkSalmon River drainage, Idaho. Transactions of the American FisheriesSociety 136:624–638.

Kruse, C. G., W. A. Hubert, and F. J. Rahel. 2000. Status of YellowstoneCutthroat Trout in Wyoming waters. North American Journal of FisheriesManagement 20:693–705.

Leary, R. F., F. W. Allendorf, and G. K. Sage. 1995. Hybridization andintrogression between introduced and native fish. Pages 91–101 in H. L.Schramm Jr. and R. G. Piper, editors. American Fisheries Society,Symposium 15, Bethesda, Maryland.

Martinez, A. M. 1984. Identification of Brook, Brown, Rainbow, and Cutthroattrout larvae. Transactions of the American Fisheries Society 113:252–259.

Mayhood, D. W. 2000. Provisional evaluation of the status of WestslopeCutthroat Trout in Canada. Pages 579–586 in L. M. Darling, editor.Proceedings of the biological and management of species and habitats atrisk. British Columbia Ministry of Environment, Lands, and Parks, Victoria,and University College of the Cariboo, Kamloops, British Columbia.

Mayhood, D. W. 2014. Conceptual framework and recovery guidelines forrestoring Westslope Cutthroat Trout populations in Alberta. FreshwaterResearch Limited, Technical Report 2014/03-1, Calgary, Alberta.

McKelvey, K. S., M. K. Young, T. M. Wilcox, D. M. Bingham, K. L. Pilgrim,and M. K. Schwartz. 2016. Patterns of hybridization among CutthroatTrout and Rainbow Trout in northern Rocky Mountain streams. Ecologyand Evolution 6:688–706.

Metcalf, J. L., M. R. Siegle, and A. P. Martin. 2008. Hybridization dynamicsbetween Colorado’s native Cutthroat Trout and introduced Rainbow Trout.Journal of Heredity 99:149–156.

Meyer, K. A., D. J. Schill, F. S. Elle, and W. C. Schrader. 2003. A long-termcomparison of Yellowstone Cutthroat Trout abundance and size structurein their historical range in Idaho. North American Journal of FisheriesManagement 23:149–162.

Meyer, K. A., D. J. Schill, J. A. Lamansky Jr., M. R. Campbell, and C. C.Kozfkay. 2006. Status of Yellowstone Cutthroat Trout in Idaho.Transactions of the American Fisheries Society 135:1329–1347.

Miller, R. R. 1950. Notes on the Cutthroat and Rainbow Trouts with adescription of a new species from the Gila River, New Mexico.University of Michigan, Museum of Zoology, Occasional Paper 529,Ann Arbor.

Muhlfeld, C. C., T. E. McMahon, M. C. Boyer, and R. E. Gresswell. 2009.Local-habitat, watershed, and biotic factors in the spread of hybridization

between native Westslope Cutthroat Trout and introduced Rainbow Trout.Transactions of the American Fisheries Society 138:1036–1051.

Nagelkerke, N. J. D. 1991. A note on a general definition of the coefficient ofdetermination. Biometrika 78:691–692.

Neff, N. A., and G. R. Smith. 1979. Multivariate analysis of hybrid fishes.Systematic Biology 28:176–196.

Ostberg, C. O., and R. J. Rodriguez. 2002. Novel molecular markers differ-entiate Oncorhynchus mykiss (Rainbow Trout and steelhead) and the O.clarki (Cutthroat Trout) subspecies. Molecular Ecology Notes 2:197–202.

Ostberg, C. O., and R. J. Rodriguez. 2006. Hybridization and cytonuclearassociations among native Westslope Cutthroat Trout, introduced RainbowTrout and their hybrids within the Stehekin River drainage, NorthCascades National Park. Transactions of the American Fisheries Society135:924–942.

Pollard, W. R., G. F. Hartman, C. Groot, and P. Edgell. 1997. Field identifica-tion of coastal juvenile salmonids. Harbour Publishing, Madeira Park,British Columbia.

Rasmussen, J. B., M. D. Robinson, and D. D. Heath. 2010. Ecologicalconsequences of hybridization between native Westslope Cutthroat(Oncorhynchus clarkii lewisi) and introduced Rainbow (Oncorhynchusmykiss) trout: effects on life history and habitat use. Canadian Journal ofFisheries and Aquatic Sciences 67:357–370.

SAS Institute. 2009. SAS/STAT 9.2 user’s guide, second edition. SASInstitute, Cary, North Carolina.

Scribner, K. T., K. S. Page, and M. L. Burton. 2001. Hybridization in fresh-water fishes: a review of case studies and cytonuclear methods of biolo-gical inference. Reviews in Fish Biology and Fisheries 10:293–323.

Seiler, S. M., K. Gunnell, M. B. Ptacek, and E. R. Keeley. 2009.Morphological patterns of hybridization between Yellowstone CutthroatTrout and introduced Rainbow Trout in the south fork of the Snake Riverwatershed, Idaho and Wyoming. North American Journal of FisheriesManagement 29:1529–1539.

Strahler, A. N. 1964. Quantitative geomorphology of drainage basins andchannel networks. Pages 4–40 in V. T. Chow, editor. Handbook of appliedhydrology, section 4, part II. McGraw-Hill, New York.

UDWR (Utah Division of Wildlife Resources). 2000. Cutthroat Trout man-agement: a position paper, genetic considerations associated withCutthroat Trout management. UDWR, Publication 00-26, Salt Lake City.

Weigel, D. E., J. T. Peterson, and P. Spruell. 2002. A model using phenotypiccharacteristics to detect introgressive hybridization in wild WestslopeCutthroat Trout and Rainbow Trout. Transactions of the AmericanFisheries Society 131:389–403.

Young, M. K., editor. 1995. Conservation assessment for inland CutthroatTrout. U.S. Forest Service General Technical Report RM-256.

466 MEYER ET AL.