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.     

RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays

Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.     

Novel molecular markers differentiate Oncorhynchus mykiss (rainbow trout and steelhead) and the O. clarki (cutthroat trout) subspecies

A suite of 26 PCR‐based markers was developed that differentiates rainbow (Oncorhynchus mykiss) and coastal cutthroat trout (O. clarki clarki). The markers also differentiated rainbow from other cutthroat trout subspecies (O. clarki), and several of the markers differentiated between cutthroat trout subspecies. This system has numerous positive attributes, including: nonlethal sampling, high species‐specificity and products that are easily identified and scored using agarose gel electrophoresis. The methodology described for developing the markers can be applied to virtually any system in which numerous markers are desired for identifying or differentiating species or subspecies.     

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.     

Discovery and characterization of novel genetic markers for use in the management of Lahontan cutthroat trout (Oncorhynchus clarkii henshawi)

The Lahontan cutthroat trout (Oncorhynchus clarkii henshawi) is threatened by habitat destruction, over‐harvest and hybridization with nonnative trout. Currently, three Geographic Management Units (GMUs) are recognized within the taxon. Here, we describe a suite of 68 single‐nucleotide polymorphism (SNP) genetic markers for use in the study and management of Lahontan cutthroat trout and a closely related subspecies, the Paiute cutthroat trout (O. c. seleneris). These include markers variable within the two subspecies (n = 35), diagnostic for the two subspecies (n = 23) and diagnostic for Yellowstone cutthroat trout (O. c. bouvieri) and other closely related subspecies (n = 10). Sixty‐three markers were discovered by Sanger sequencing of 171 EST loci in an ascertainment panel including Lahontan cutthroat trout from four populations representing all GMUs. Five markers were identified in a secondary sequencing effort with a single population of Lahontan cutthroat trout. TaqMan assays were validated on six Lahontan cutthroat trout populations and a diverse panel of other trout. Over 90% of the markers variable in Lahontan cutthroat trout were polymorphic in at least two populations, and 66% were variable within all three GMUs. All Lahontan diagnostic markers were also fixed for the Lahontan allele in Paiute cutthroat trout. Most of the Yellowstone diagnostic markers can also be used for this purpose in other cutthroat trout subspecies. This is the first set of SNP markers to be developed for Lahontan cutthroat trout, and will be an important tool for conservation and management.     

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.     

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.     

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.     

Discovery and characterization of a large number of diagnostic markers to discriminate Oncorhynchus mykiss and O. clarkii

Hybridization of cutthroat trout and steelhead/rainbow trout is ubiquitous where they are sympatric, either naturally or owing to introductions. The ability to detect hybridization and introgression between the two species would be greatly improved by the development of more diagnostic markers validated across the two species' many phylogenetic lineages. Here, we describe 81 novel genetic markers and associated assays for discriminating the genomes of these sister species. These diagnostic nucleotide polymorphisms were discovered by sequencing of rainbow trout expressed sequence tags (ESTs) in a diverse panel of both cutthroat trout and steelhead/rainbow trout. The resulting markers were validated in a large number of lineages of both species, including all extant subspecies of cutthroat trout and most of the lineages of rainbow trout that are found in natural sympatry with cutthroat trout or used in stocking practices. Most of these markers (79%) distinguish genomic regions for all lineages of the two species, but a small number do not reliably diagnose coastal, westslope and/or other subspecies of cutthroat trout. Surveys of natural populations and hatchery strains of trout and steelhead found rare occurrences of the alternative allele, which may be due to either previous introgression or shared polymorphism. The availability of a large number of genetic markers for distinguishing genomic regions originating in these sister species will allow the detection of both recent and more distant hybridization events, facilitate the study of the evolutionary dynamics of hybridization and provide a powerful set of tools for the conservation and management of both species.     

Bi‐parentally inherited species‐specific markers identify hybridization between rainbow trout and cutthroat trout subspecies

Eight polymerase chain reaction primer sets amplifying bi‐parentally inherited species‐specific markers were developed that differentiate between rainbow trout (Oncorhynchus mykiss) and various cutthroat trout (O. clarki) subspecies. The primers were tested within known F₁ and first generation hybrid backcrosses and were shown to amplify codominantly within hybrids. Heterozygous individuals also amplified a slower migrating band that was a heteroduplex, caused by the annealing of polymerase chain reaction products from both species. These primer sets have numerous advantages for native cutthroat trout conservation including statistical genetic analyses of known crosses and simple hybrid identification.     

The Dual Challenges of Generality and Specificity When Developing Environmental DNA Markers for Species and Subspecies of Oncorhynchus

Environmental DNA (eDNA) sampling is a powerful tool for detecting invasive and native aquatic species. Often, species of conservation interest co-occur with other, closely related taxa. Here, we developed qPCR (quantitative PCR) markers which distinguish westslope cutthroat trout (Oncorhynchus clarkii lewsi), Yellowstone cutthroat trout (O. clarkii bouvieri), and rainbow trout (O. mykiss), which are of conservation interest both as native species and as invasive species across each other’s native ranges. We found that local polymorphisms within westslope cutthroat trout and rainbow trout posed a challenge to designing assays that are generally applicable across the range of these widely-distributed species. Further, poorly-resolved taxonomies of Yellowstone cutthroat trout and Bonneville cutthroat trout (O. c. utah) prevented design of an assay that distinguishes these recognized taxa. The issues of intraspecific polymorphism and unresolved taxonomy for eDNA assay design addressed in this study are likely to be general problems for closely-related taxa. Prior to field application, we recommend that future studies sample populations and test assays more broadly than has been typical of published eDNA assays to date.     

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.     

Environmental and anthropogenic correlates of hybridization between westslope cutthroat trout (Oncorhynchus clarkii lewisi) and introduced rainbow trout (O. mykiss)

Hybridization with introduced taxa is one of the major threats to the persistence of native biodiversity. The westslope cutthroat trout (Oncorhynchus clarkii lewisi) is found in southeastern British Columbia and southwestern Alberta, Canada, and adjacent areas of Montana, Idaho, and Washington State, USA. Through much of this area, native populations are threatened by hybridization with introduced rainbow trout (O. mykiss). We surveyed 159 samples comprising over 5,000 fish at 10 microsatellite DNA loci to assess the level of admixture between native westslope cutthroat trout (wsct) and introduced rainbow trout in southwestern Alberta. Admixture levels (q_wsct of 0 = pure rainbow trout, q_wsct of 1.0 = pure westslope cutthroat trout) ranged from <0.01 to 0.99 and averaged from 0.72 to 0.99 across seven drainage areas. Regression tree analyses indicated that water temperature, elevation, distance to the nearest stocking site, and distance to the nearest railway line were significant components of a model that explained 34 % of the variation across sites in q_wsct across 58 localities for which habitat variables were available. Partial dependence plots indicated that admixture with rainbow trout increased with increasing water temperature and distance to the nearest railway line, but decreased with increasing elevation and distance from stocking site to sample site. Our results support the hypothesis that westslope cutthroat trout may be less susceptible to hybridization with rainbow trout in colder, higher elevation streams, and illustrate the interaction between abiotic and anthropogenic factors in influencing hybridization between native and introduced taxa.     

Genetic Integrity and Microgeographic Population Structure of Westslope Cutthroat Trout, Oncorhynchus clarki lewisi, in the Pend Oreille Basin in Washington

Five microsatellite DNA loci (Ots-101 ^*,Ots-107 ^*,Oki-10 ^*, Ogo-3 ^*, and FGT-3 ^*) were screened to evaluate the genetic characteristics and population structure for cutthroat trout from eight tributaries of the Pend Oreille River in northeastern Washington and to compare these collections with two hatchery stocks of westslope cutthroat trout, Oncorhynchus clarki lewisi, Yellowstone cutthroat trout, Oncorhynchus clarki bouvieri and a hatchery rainbow trout, Oncorhynchus mykiss, strain that have been stocked in northeastern Washington. Relatively high levels of variation (numbers of alleles and heterozygosity) were observed in all collections and allele frequencies were quite variable among collections. Evidence of limited introgression by rainbow and/or Yellowstone cutthroat was found at several locations. Both F_ST values and tests of genetic differentiation indicated the existence of numerous, reproductively isolated populations. The population in Slate Creek was very similar to the Kings Lake Hatchery strain, and we conclude that this similarity is the result of historical introductions of this hatchery strain into what was presumably a stream without a native cutthroat population. In one stream, differences in introgression and allele frequencies were found above and below a barrier falls. Because of the substantial level of population differentiation observed among the various collections, we recommend that management and conservation actions be focused at the level of individual streams in order to maintain the productivity and genetic character of the existing populations of cutthroat trout.     

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.     

Characterization of 22 novel single nucleotide polymorphism markers in steelhead and rainbow trout

Thirty‐two individuals representing coastal and inland populations of steelhead and rainbow trout (Oncorhynchus mykiss) were sequenced at 18 expressed sequence tags and nine microsatellite loci to identify single nucleotide polymorphisms. A total of 98 polymorphisms were discovered during the screen and 22 were developed into 5′ exonuclease assays (Taqman assays). Genotypes from TaqMan assays were compared to sequence data from individuals in the ascertainment panel to confirm proper allele designations. A larger number of samples (n = 192) from six regions were tested with the validated assays. Per‐locus F_ST values ranged from 0.001 to 0.414.     

Next-generation RAD sequencing identifies thousands of SNPs for assessing hybridization between rainbow and westslope cutthroat trout

The increased numbers of genetic markers produced by genomic techniques have the potential to both identify hybrid individuals and localize chromosomal regions responding to selection and contributing to introgression. We used restriction-site-associated DNA sequencing to identify a dense set of candidate SNP loci with fixed allelic differences between introduced rainbow trout (Oncorhynchus mykiss) and native westslope cutthroat trout (Oncorhynchus clarkii lewisi). We distinguished candidate SNPs from homeologs (paralogs resulting from whole-genome duplication) by detecting excessively high observed heterozygosity and deviations from Hardy-Weinberg proportions. We identified 2923 candidate species-specific SNPs from a single Illumina sequencing lane containing 24 barcode-labelled individuals. Published sequence data and ongoing genome sequencing of rainbow trout will allow physical mapping of SNP loci for genome-wide scans and will also provide flanking sequence for design of qPCR-based TaqMan(?) assays for high-throughput, low-cost hybrid identification using a subset of 50-100 loci. This study demonstrates that it is now feasible to identify thousands of informative SNPs in nonmodel species quickly and at reasonable cost, even if no prior genomic information is available.     

Parallel introgression and selection on introduced alleles in a native species

As humans cause the redistribution of species ranges, hybridization between previously allopatric species is on the rise. Such hybridization can have complex effects on overall fitness of native species as new allelic combinations are tested. Widespread species introductions provide a unique opportunity to study selection on introgressed alleles in independent, replicated populations. We examined selection on alleles that repeatedly introgressed from introduced rainbow trout (Oncorhynchus mykiss) into native westslope cutthroat trout (Oncorhynchus clarkii lewisi) populations in western Canada. We found that the degree of introgression of individual single nucleotide polymorphisms from the invasive species into the native is correlated between independent watersheds. A number of rainbow trout alleles have repeatedly swept to high frequency in native populations, suggesting parallel adaptive advantages. Using simulations, we estimated large selection coefficients up to 0.05 favoring several rainbow trout alleles in the native background. Although previous studies have found reduced hybrid fitness and genome‐wide resistance to introgression in westslope cutthroat trout, our results suggest that some introduced genomic regions are strongly favored by selection. Our study demonstrates the utility of replicated introductions as case studies for understanding parallel adaptation and the interactions between selection and introgression across the genome. We suggest that understanding this variation, including consideration of beneficial alleles, can inform management strategies for hybridizing species.     

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.     

Characterization of twenty‐four microsatellite markers for rainbow trout (Oncorhynchus mykiss)

Twenty‐four new microsatellite markers were developed for genome mapping and population genetics studies in rainbow trout (Oncorhynchus mykiss). The amount of polymorphism, percentage heterozygosity and ability of each marker to amplify genomic DNA from other salmonids were recorded. Seven markers were observed to be duplicated in the rainbow trout genome by containing more than one allele in homozygous (clonal) fish.