Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki)

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.     

Microsatellite analysis of genetic population structure in an endangered salmonid: the coastal cutthroat trout (Oncorhynchus clarki clarki)

The genetic population structure of coastal cutthroat trout ( Oncorhynchus clarki clarki ) in Washington state was investigated by analysis of variation in allele frequencies at six highly polymorphic microsatellite loci for 13 anadromous populations, along with one outgroup population from the Yellowstone subspecies ( O. clarki bouvieri) (mean heterozygosity = 67%; average number of alleles per locus = 24). Tests for genetic differentiation revealed highly significant differences in genotypic frequencies for pairwise comparisons between all populations within geographical regions and overall population subdivision was substantial ( F _ST = 0.121, R _ST = 0.093), with 44.6% and 55.4% of the among-population diversity being attributable to differences between streams ( F _SR = 0.054) and between regions ( F _RT = 0.067), respectively. Analysis of genetic distances and geographical distances did not support a simple model of isolation by distance for these populations. With one exception, neighbour-joining dendrograms from the Cavalli-Sforza and Edwards' chord distances and maximum likelihood algorithms clustered populations by physiogeographic region, although overall bootstrap support was relatively low (53%). Our results suggest that coastal cutthroat trout populations are ultimately structured genetically at the level of individual streams. It appears that the dynamic balance between gene flow and genetic drift in the subspecies favours a high degree of genetic differentiation and population subdivision with the simultaneous maintenance of high heterozygosity levels within local populations. Results are discussed in terms of coastal cutthroat trout ecology along with implications for the designation of evolutionarily significant units pursuant to the US Endangered Species Act of 1973 and analogous conservation units.     

A genetic analysis of the London strain of rainbow trout

The London strain of rainbow trout (Oncorhynchus mykiss) was created by interbreeding three other strains of rainbow trout and therefore was expected to have higher levels of genetic variation than other strains of rainbow trout. We examined 129 London strain rainbow trout from Indiana by allozyme electrophoresis to assess levels of genetic variation and to examine the relationship between the London strain and other hatchery strains. When using the same loci to compare with other hatchery strains the London strain showed levels of genetic variation within the range of other hatchery strains: mean heterozygosity of 0.053 (0.031-0.099), 1.27 (1.20-1.60) alleles per locus and 20.0% (20.0-40.0%) of the loci were polymorphic. The London strain is somewhat distinct from other hatchery strains (D=0.009-0.072), in part because of the high frequency of the sIDHP*40 allele.     

A suite of twelve single nucleotide polymorphism markers for detecting introgression between cutthroat and rainbow trout

A suite of 12 subspecies and species-specific single nucleotide polymorphism (species-specific SNP) markers was developed to distinguish rainbow trout (RT) Oncorhynchus mykiss from the four major subspecies of cutthroat trout: westslope cutthroat trout (WCT) Oncorhynchus clarki lewisi, Yellowstone cutthroat trout (YCT) Oncorhynchus clarki bouvieri, coastal cutthroat trout (CCT) Oncorhynchus clarki clarki, Lahontan cutthroat trout (LCT) Oncorhynchus clarki henshawi, and their hybrids. Several of the markers were linked to help strengthen hybrid determinations, and sex-specific species-specific SNP assays were also developed.     

Characterization of 13 microsatellites for Lahontan cutthroat trout (Oncorhynchus clarki henshawi) and cross-amplification in six other salmonids

Thirteen newly developed tri- and tetranucleotide repeat microsatellite markers were developed for Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a threatened subspecies endemic to the Lahontan hydrographic basin in the western USA. These loci are highly polymorphic with five to 30 alleles per locus and observed heterozygosities ranging from 0.4 to 0.7. Cross-species amplification of these markers was most successful in the closely related rainbow trout, Oncorhynchus mykiss, with only three loci amplifying in brown trout, Salmo trutta. Nonoverlapping allelic distributions for many of these loci among the six salmonid species screened suggest these markers may be useful for hybrid determination.     

Acoustic measurement of trout distributions in Spada Lake, Washington, using stationary transducers

Diel patterns in the spatial distribution of rainbow and cutthroat trout were studied with stationary acoustic transducers, gillnets, and setlines in Spada Lake during summer thermal stratification. Four diel periods (dawn, 03.00–07.00 hours; day, 07.00–19.00 hours; dusk, 19.00–23.00 hours; night, 23.00–03.00 hours) and two horizontal strata (nearshore, bottom depth < 8 m; offshore, bottom depth > 8 m) were identified from fish distribution patterns. During the day, trout were almost exclusively offshore and their densities were highest at intermediate depths of the water column (4–16 m). From dusk to dawn, trout were in intermediate and shallow depths (0–4 m) of the offshore and nearshore strata. The mean depth of capture was significantly deeper for cutthroat trout (9 m) than for rainbow trout (3m). Both species primarily inhabited the epilimnion and metalimnion where temperature ranged from 22.5 to 11.0°C and were nearly absent from deeper, cooler waters. Dissolved oxygen levels ranging from 3.9 to 8.6 mg/l did not seem to influence trout distributions.     

A molecular analysis of hybridization between native westslope cutthroat trout and introduced rainbow trout in southeastern British Columbia, Canada

Restriction site variation in the Ikaros gene intron was used to assess the incidence of westslope cutthroat trout ( Oncorhynchus clarki lewisi ), rainbow trout ( O. mykiss ) and interspecific hybrids at 11 localities among eight streams tributary to the upper Kootenay River system in south-eastern British Columbia, Canada. Out of 356 fish assayed by this technique, hybrids ( n =16) were found at seven of the 11 sites across five different streams. Rainbow trout ( n =6) were found at two of the 11 sites. Analysis of hybrids with a second genetic marker (heat shock 71 intron) indicated that most represented either backcrosses to both westslope cutthroat and rainbow trout, or post F₁ hybrids. Mitochondrial DNA analysis indicated that hybrid matings occur between male rainbow trout and female westslope cutthroat trout and vice versa. Comparison of present hybridization in five tributaries relative to an allozyme-based analysis in the mid-1980s, that documented hybrids in only a single tributary of seven that were common to the two studies, suggests that hybridization and introgression has increased in upper Kootenay River tributaries. The present analysis is a conservative estimate of genetic interaction between the species because introgression was not tested in the majority of samples. Identification of genetically pure westslope cutthroat trout populations, and why they might be resistant to introgression from rainbow trout, are crucial conservation priorities for this unique subspecies of cutthroat trout.     

Absence of developmental incompatibility in hybrids between rainbow trout and two subspecies of cutthroat trout

We examined the developmental rate of hybrids between rainbow trout (Salmo gairdneri) and two subspecies of cutthroat trout: westslope cutthroat trout (Salmo clarki lewisi) and Yellowstone cutthroat trout (Salmo clarki bouvieri). These taxa show considerable genetic divergence at 42 structural loci encoding enzymes; the mean Nei's d between the rainbow trout and the two species of cutthroat trout is 0.22. We used four measures of developmental rate: time of hatching and yolk resorption, rate of increase in activity of four enzymes, and time of initial detection of seven isozyme loci. The two cutthroat trout subspecies reached hatching and yolk resorption earlier than rainbow trout. Cutthroat trout had higher relative enzyme activities than rainbow trout from deposition of eye pigment to hatching. There was no difference in the rate of increase in enzyme activity or time of initial expression of these loci between these species. Hybrids showed developmental rates intermediate or similar to that of the parental species using all measures. Our results indicate an absence of regulatory and developmental incompatibility between these taxa.This research was supported by NSF Grants ISP-8011449 and BSR-8300039. M.M.F. was supported by a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada.     

Lower fitness of hatchery and hybrid rainbow trout compared to naturalized populations in Lake Superior tributaries

We have documented an early life survival advantage by naturalized populations of anadromous rainbow trout Oncorhynchus mykiss over a more recently introduced hatchery population and outbreeding depression resulting from interbreeding between the two strains. We tested the hypothesis that offspring of naturalized and hatchery trout, and reciprocal hybrid crosses, survive equally from fry to age 1+ in isolated reaches of Lake Superior tributary streams in Minnesota. Over the first summer, offspring of naturalized females had significantly greater survival than offspring of hatchery females in three of four comparisons (two streams and 2 years of stocking). Having an entire naturalized genome, not just a naturalized mother, was important for survival over the first winter. Naturalized offspring outperformed all others in survival to age 1+ and hybrids had reduced, but intermediate, survival relative to the two pure crosses. Averaging over years and streams, survival relative to naturalized offspring was 0.59 for hybrids with naturalized females, 0.37 for the reciprocal hybrids, and 0.21 for hatchery offspring. Our results indicate that naturalized rainbow trout are better adapted to the conditions of Minnesota's tributaries to Lake Superior so that they outperform the hatchery-propagated strain in the same manner that many native populations of salmonids outperform hatchery or transplanted fish. Continued stocking of the hatchery fish may conflict with a management goal of sustaining the naturalized populations.     

Metabolic traits of westslope cutthroat trout,introduced rainbow trout and their hybrids in an ecotonal hybrid zone along an elevation gradient

In the Upper Oldman River, Alberta, introduced non‐native hatchery rainbow trout (Oncorhynchus mykiss) hybridize with native westslope cutthroat trout (O. clarkii), resulting in a hybrid swarm. Rainbow trout dominate at low elevations (< 1250 m) in the river mainstem, cutthroat in high‐elevation tributaries (> 1400 m), and hybrids are numerically dominant in the mid‐elevation range. We hypothesized that metabolism of rainbow trout would exceed that of cutthroat trout, and that the elevation gradient in genetic makeup would be mirrored by a gradient in metabolic traits, with intermediate traits in the hybrid‐dominated ecotone. Metabolic traits were measured and regressed against the genetic makeup of individuals and elevation. Rainbow trout had higher oxygen consumption rates (OCRs), higher white muscle lactate dehydrogenase (LDH), and citrate synthase (CS) activity, and higher plasma acetylcholinesterase (AchE) than cutthroat trout. Hybrids had intermediate OCRs and AchE, but LDH activity as high as rainbow trout. While hybrid zones are usually modelled as a balance between cross species mating and selection against hybrids, ecotonal hybrid zones, where hybrids proliferate in intermediate habitats and have traits that appear well suited to ecotonal conditions, have been proposed for some plants and animals, and may have important implications for resource management and conservation. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 56–72.     

Hybridization dynamics between Colorado's native cutthroat trout and introduced rainbow trout

Newly formed hybrid populations provide an opportunity to examine the initial consequences of secondary contact between species and identify genetic patterns that may be important early in the evolution of hybrid inviability. Widespread introductions of rainbow trout (Oncorhynchus mykiss) into watersheds with native cutthroat trout (Oncorhynchus clarkii) have resulted in hybridization. These introductions have contributed to the decline of native cutthroat trout populations. Here, we examine the pattern of hybridization between introduced rainbow trout and 2 populations of cutthroat trout native to Colorado. For this study, we utilized 7 diagnostic, codominant nuclear markers and a diagnostic mitochondrial marker to investigate hybridization in a population of greenback cutthroat trout (Oncorhynchus clarkii stomias) and a population of Colorado River cutthroat trout (Oncorhynchus clarkii pleuriticus). We infer that cutthroat-rainbow trout hybrid swarms have formed in both populations. Although a mixture of hybrid genotypes was present, not all genotype combinations were detected at expected frequencies. We found evidence that mitochondrial DNA introgression in hybrids is asymmetric and more likely from rainbow trout than from cutthroat trout. A difference in spawning time of the 2 species or differences in the fitness between the reciprocal crosses may explain the asymmetry. Additionally, the presence of intraspecific cytonuclear associations found in both populations is concordant with current hypotheses regarding coevolution of mitochondrial and nuclear genomes.     

Development and evaluation of 200 novel SNP assays for population genetic studies of westslope cutthroat trout and genetic identification of related taxa

DNA sequence data were collected and screened for single nucleotide polymorphisms (SNPs) in westslope cutthroat trout (Oncorhynchus clarki lewisi) and also for substitutions that could be used to genetically discriminate rainbow trout (O. mykiss) and cutthroat trout, as well as several cutthroat trout subspecies. In total, 260 expressed sequence tag‐derived loci were sequenced and allelic discrimination genotyping assays developed from 217 of the variable sites. Another 50 putative SNPs in westslope cutthroat trout were identified by restriction‐site‐associated DNA sequencing, and seven of these were developed into assays. Twelve O. mykiss SNP assays that were variable within westslope cutthroat trout and 12 previously published SNP assays were also included in downstream testing. A total of 241 assays were tested on six westslope cutthroat trout populations (N = 32 per population), as well as collections of four other cutthroat trout subspecies and a population of rainbow trout. All assays were evaluated for reliability and deviation from Hardy–Weinberg and linkage equilibria. Poorly performing and duplicate assays were removed from the data set, and the remaining 200 assays were used in tests of population differentiation. The remaining markers easily distinguished the various subspecies tested, as evidenced by mean G_ST of 0.74. A smaller subset of the markers (N = 86; average G_ST = 0.40) was useful for distinguishing the six populations of westslope cutthroat trout. This study increases by an order of magnitude the number of genetic markers available for the study of westslope cutthroat trout and closely related taxa and includes many markers in genes (developed from ESTs).     

Subspecies-informative SNP assays for evaluating introgression between native golden trout and introduced rainbow trout

We characterize 20 single nucleotide polymorphism assays for evaluating hybridization between native golden trout subspecies (Oncorhynchus mykiss aguabonita and O. m. whitei) and introduced rainbow trout strains. These assays utilize the 5′‐nuclease reaction, facilitating high‐throughput genotyping of many individuals and making them useful in quantifying and monitoring introgression and potentially applicable to studies of other O. mykiss groups. Minor allele frequency differentials (δq) among native and introduced rainbow groups ranged from 0 to 1, with an average differential of 0.75 for both subspecies aguabonita and whitei relative to the hatchery rainbow trout strain.     

Characterization of tetranucleotide microsatellites for Rio Grande cutthroat trout and rainbow trout,and their cross‐amplification in other cutthroat trout subspecies

We describe the isolation and characterization of 12 tetranucleotide microsatellites for Rio Grande cutthroat trout (Oncorhynchus clarkii virginalis) and rainbow trout (Oncorhynchus mykiss), and subsequently investigate their performance in Colorado River cutthroat trout (Oncorhynchus clarkii pleuriticus), greenback cutthroat trout (Oncorhynchus clarkii stomias) and Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri). All 12 loci are polymorphic in all subspecies of O. clarkii examined.     

Role of genetic background in the introgressive hybridization of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) with Westslope cutthroat trout (<Emphasis Type="Italic">O. clarkii lewisi</Emphasis>)

Conservation and management of endemic species may increasingly involve efforts to prevent hybridization with other species. Native westslope cutthroat trout (Oncorhynchus clarkii lewisi) management in western North America is based largely on admixture estimates with introduced rainbow trout (O. mykiss), with the highest conservation priority given to cutthroat populations that do not exhibit admixture. This study examined the hypothesis that such ancestry quotients are dependent upon the genetic background of reference rainbow trout populations. We used 10 microsatellite loci to estimate admixture within westslope cutthroat trout collected from 39 sites from Alberta, Canada, using three genetically distinct (pairwise F_ST = 0.100–0.281) rainbow trout genetic backgrounds: a wild (introduced) population from Alberta, two wild (native) populations from British Columbia, and a present-day hatchery broodstock line. Ancestry quotients were significantly impacted by genetic background, whereby the extent of admixture was highest with locally introduced (wild, naturalized) rainbow trout lines and lowest with the hatchery lines. Our results suggest that future studies ought to explore the possibility that local adaptation or drift in introduced rainbow trout populations may contribute to decreased reproductive isolation with geographically proximal cutthroat trout populations.     

Ten species specific microsatellite loci for Lahontan cutthroat trout,Oncorhynchus clarki henshawi

Ten polymorphic microsatellite loci (containing tri and tetra‐nucleotide repeats) were developed for the Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a subspecies of cutthroat trout listed as threatened under the United States Endangered Species Act. Polymorphism was assessed for 445 individuals from 12 populations representing eight watersheds spread throughout the three Distinct Population Segments defined for this subspecies. All loci were polymorphic (X? = 19, range 7–26 alleles). All loci were in Hardy–Weinberg equilibrium (HWE) except for one locus (OCH 9) in a single population (P < 0.00014 after Bonferroni correction for multiple tests).     

ASSOCIATIONS BETWEEN MITOCHONDRIAL AND NUCLEAR GENOTYPES IN CUTTHROAT TROUT HYBRID SWARMS

We examined mtDNA and nuclear allozyme genotypes in hybrid populations formed from interbreeding of westslope cutthroat trout (Oncorhynchus clarki lewisi) and Yellowstone cutthroat trout (O. c. bouvieri). These subspecies show substantial genetic divergence (Nei's D = 0.30; mtDNA P = 0.02). Diagnostic alleles at multiple nuclear loci and two distinct mtDNA haplotypes segregate in the hybrids. Nuclear and mtDNA genotypes are largely randomly associated, although there is slight disequilibrium in both nuclear and cytonuclear measures in some samples. Consistent positive gametic disequilibria for three pairs of nuclear loci confirm one previously reported linkage, and indicate two more. Allele frequencies provide no evidence for selection on individual chromosome segments. However, westslope mtDNA haplotype frequencies exceed westslope nuclear allele frequencies in all samples. This may be explained by differences in the frequency of occurrence of reciprocal F₁ matings, by viability, fertility, or sex ratio differences in the progeny of reciprocal matings, or by weak selection on mtDNA haplotypes.     

Gastric evacuation rates and maximum daily rations of rainbow trout fed chironomid larvae at 7.8, 10.0 and 12.8°C

Gastric evacuation rate (GER) and maximum daily ration (CMAX) data for rainbow trout, Oncorhynchus mykiss, are scant, particularly those that apply to field populations of this species. We determined GERs and CMAXs of rainbow trout (approx. 160-300 mm TL, 40-300 g live weight), fed chironomid larvae at 7.8, 10.0 and 12.8°C. Primary GER and CMAX results are provided so they can be readily compared and combined with data from related studies. Disclosure of primary data should facilitate development of predictive equations for GER and CMAX that are applicable to the broad array of conditions under which rainbow trout exist. Our GERs at 10°C were intermediate to but different from most reported in other such studies of rainbow trout at this temperature where different food types were used. Our CMAX values for rainbow trout were substantially higher than those reported in a previous study, and we argue that our values are probably more appropriate for stream-dwelling populations. We also provide evidence that Elliott's (1972, 1975) equations for predicting GER and CMAX in brown trout, Salmo trutta, should not be applied to rainbow trout as has been done in some studies. This revised version was published online in August 2006 with corrections to the Cover Date.     

Diagnostic single nucleotide polymorphisms for identifying westslope cutthroat trout (Oncorhynchus clarki lewisi), Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (Oncorhynchus mykiss)

We describe 12 diagnostic single nucleotide polymorphism (SNP) assays for use in species identification among rainbow and cutthroat trout: five of these loci have alleles unique to rainbow trout (Oncorhynchus mykiss), three unique to westslope cutthroat trout (O. clarkii lewisi) and four unique to Yellowstone cutthroat trout (O. clarkii bouvieri). These diagnostic assays were identified using a total of 489 individuals from 26 populations and five fish hatchery strains.     

DEVELOPMENTAL INSTABILITY AND HIGH MERISTIC COUNTS IN INTERSPECIFIC HYBRIDS OF SALMONID FISHES

Interspecific hybrids have been proposed to have reduced developmental stability in comparison to their parental species because the parental genomes have not undergone selection for the maintenance of developmental stability when they occur together. We present data from four interspecific hybrids of salmonid fishes that support this view. Natural hybrids of bull trout (Salvelinus confluentus) with brook trout (Salvelinus fontinalis) and laboratory hybrids of rainbow trout (Salmo gairdneri) with Yellowstone (Salmo clarki bouvieri), westslope (S. c. lewisi), and coastal (S. c. clarki) cutthroat trout all have higher levels of fluctuating asymmetry than either of their parental species raised in the same environment. Thus, the hybrids have reduced developmental stability. The hybrids do not have meristic counts intermediate to the counts of the parental species. The hybrids usually have counts as high as the species with the higher count for those characters that differ between the parental species and often have higher counts for those characters that do not differ between the parental species. We suggest that the tendency for interspecific hybrids to have high meristic counts may be related to differences between the species in the length and timing of the developmental periods during which the counts of the characters are determined.