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.     

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.     

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.     

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).     

Six diagnostic single nucleotide polymorphism markers for detecting introgression between cutthroat and rainbow trouts

Ten primer pairs were screened to develop single nucleotide polymorphism (SNP) TaqMan assays that will distinguish California golden trout and some rainbow trouts (Oncorhynchus mykiss sspp., O. m. aguabonita) from the Paiute and Lahontan cutthroat trouts (Oncorhynchus clarkii seleniris, O. c. henshawi). From these 10 primer pairs, one mitochondrial and five nuclear fixed SNP differences were discovered and developed into TaqMan assays. These six assays will be useful for characterizing and monitoring hybridization between these groups. Additional Oncorhynchus clarkii sspp. and Oncorhynchus mykiss sspp. were assayed to determine if these assays are useful in closely related species.     

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.     

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.     

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.     

Estimation of introgression in cutthroat trout populations using microsatellites

Introgressive hybridization, mediated by anthropogenic activity, poses a threat to numerous and diverse taxa. The management of introgressed individuals or populations within species of conservation concern is currently the subject of scientific and political debate. We investigate the utility of 10 non-diagnostic microsatellite loci for investigating admixture from introduced Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (O. mykiss) within 25 putative Rio Grande cutthroat trout (O. c. virginalis) populations. We apply five different approaches (correspondence analysis, maximum-likelihood assignment tests, an admixture estimator based on allele frequencies, an admixture estimator based on coalescent theory and an admixture estimator implementing a Bayesian method) and use two alternative O. c. virginalis reference samples. All approaches were capable of identifying one population that consisted entirely of introduced O. c. bouvieri, and three out of five approaches enabled us to discriminate those populations with relatively high levels of non-native introgression from those populations with little or none. Actual estimates of admixture coefficients within a test population, varied, however, with the approach and reference sample used. These results have important implications for policies dividing populations into different management categories according to the estimated proportion of non-native genetic material that they contain.     

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.     

Intraspecific taxonomy and ecology characterize morphological divergence among cutthroat trout (Oncorhynchus clarkii ssp. Richardson) populations

We compared the proportion of morphological variation accounted for by subspecies categories with the proportion encompassed by ecologically based categories in cutthroat trout ( Oncorhynchus clarkii ssp.), as a means of assessing the relative importance of each approach in identifying intraspecific diversity. We used linear and geometric morphometrics to compare measures of body shape, fin length, and head features between and within subspecies of cutthroat trout. Both categories accounted for a significant proportion of the variation between and within the subspecies; however, the larger proportion was explained by subspecific differences, with the greatest morphological divergence between coastal cutthroat trout ( Oncorhynchus clarkii clarkii ) and interior subspecies. Ecotypic categories within each subspecies also explained significant morphological differences: stream populations had longer fins and deeper, more robust bodies than lake populations. The largest ecotypic differences occurred between stream and lake populations of Yellowstone cutthroat trout ( Oncorhynchus clarkii bouvieri ). Given that many cutthroat trout subspecies are of conservation concern, our study offers a better understanding of intraspecific variation existing within the species, providing precautionary evidence of incipient speciation, and a framework of describing phenotypic diversity that is correlated with ecological conditions.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 266–281.     

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.     

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.     

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.     

Across the great divide: genetic forensics reveals misidentification of endangered cutthroat trout populations

Accurate assessment of species identity is fundamental for conservation biology. Using molecular markers from the mitochondrial and nuclear genomes, we discovered that many putatively native populations of greenback cutthroat trout (Oncorhynchus clarkii stomias) comprised another subspecies of cutthroat trout, Colorado River cutthroat trout (Oncorhynchus clarkii pleuriticus). The error can be explained by the introduction of Colorado River cutthroat trout throughout the native range of greenback cutthroat trout in the late 19th and early 20th centuries by fish stocking activities. Our results suggest greenback cutthroat trout within its native range is at a higher risk of extinction than ever before despite conservation activities spanning more than two decades.     

An analysis of spatial and environmental factors influencing hybridization between native westslope cutthroat trout (Oncorhynchus clarki lewisi) and introduced rainbow trout (O. mykiss) in the upper Kootenay River drainage, British Columbia

Westslope cutthroat trout (Oncorhynchus clarki lewisi, WCT) and introduced rainbow trout (O. mykiss, RBT) readily hybridize and introgression has occurred in many drainages across the historic native range of WCT. In British Columbia (Canada), the upper Kootenay River drainage is the heart of the westslope cutthroat trout (WCT, Oncorhynchus clarki lewisi) distribution in Canada this drainage harbours native WCT gene pools that are thought to be under threat from hybridization with introduced rainbow trout (RBT, O. mykiss). In this study, we assess the extent and distribution of WCT × RBT hybridization in the upper Kootenay River drainage. We used four diagnostic nuclear loci to determine the extent of hybridization in 981 fish collected from 23 sample localities across 12 different streams in the upper Kootenay River drainage. About 14% (142/981) of individuals were identified as hybrids (an individual with both RBT and WCT alleles), 3.4% (33/981) were identified as pure RBT, and the remaining individuals were identified as pure WCT. Although pure RBT were absent from the majority of locales (20/23), we found evidence of hybridization at 78% (18/23) of the localities and the percentage of heterospecific alleles (% I) ranged from 0.7% to 97.1%. Only 22% (5/23) of the localities showed no evidence of hybridization. Spatial analysis showed clustering among hybridized locations and decreasing hybridization with increasing distance from Koocanusa Reservoir, suggesting that the reservoir acts as a RBT source. We found no evidence that stream order, stream magnitude, or stream elevation influenced the extent of hybridization among localities. We compared our results to an analysis conducted in 1986, which indicated that hybridization is relatively recent in the upper Kootenay River drainage and that it is increasing in magnitude and distribution. In the absence of timely management intervention, the genetic integrity of WCT populations in the heart of their Canadian range may be lost. Our results indicate the dynamic nature of hybridization in fluvial systems and that for closely related taxa such as WCT and RBT, hybridization appears to be largely influenced by physical barriers to dispersal and contact between species.     

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.     

Population structure and genetic management of Rio Grande cutthroat trout (Oncorhynchus clarkii virginalis)

The Rio Grande cutthroat trout, Oncorhynchus clarkii virginalis, has declined precipitously over the past century, and currently exhibits a highly fragmented distribution within the Canadian, Pecos and Rio Grande river systems of the western United States. The relationships between populations in the three river drainages, and between O. c. virginalis and the closely related taxa O. c. pleuriticus and O. c. stomias, are not well understood. In order to guide management decisions for the subspecies, we investigated the distribution of variation at 12 microsatellite loci and two regions of the mitochondrial genome. We observed a high level of genetic differentiation between O. c. virginalis populations occupying different headwater streams (global F_st = 0.41). However, we found evidence for previous gene flow within the Rio Grande drainage, indicating that inter-population differentiation may have been exacerbated by the recent effects of population fragmentation. Despite large-scale anthropogenic movement of individuals from the Rio Grande into the Canadian and Pecos, the genetic signature of long-term evolutionary independence between the three drainages has been retained.     

Y chromosome phylogeny for cutthroat trout (Oncorhynchus clarkii) subspecies is generally concordant with those of other markers

Sequence divergence was evaluated in the non-recombining, male-specific OmyY1 region of the Y chromosome among the subspecies of cutthroat trout (Oncorhynchus clarkii) in the western United States. This evaluation identified subspecies-discriminating OmyY1-haplotypes within a ～1200 bp region of the OmyY1 locus and localized the region to the end of the Y chromosome by FISH analysis. OmyY1 sequences were aligned and used to reconstruct a phylogeny of the cutthroat trout subspecies and related species via maximum-parsimony and Bayesian analyses. In the Y-haplotype phylogeny, clade distributions generally corresponded to the geographic distributions of the recognized subspecies. This phylogeny generally corresponded to a mitochondrial tree obtained for these subspecies in a previous study. Both support a clade of trout vs. Pacific salmon, of rainbow trout, and of a Yellowstone cutthroat group within the cutthroat trout. In our OmyY1 tree, however, the cutthroat “clade”, although present topologically, was not statistically significant. Some key differences were found between trees obtained from the paternally-inherited OmyY1 vs. maternally-inherited mitochondrial haplotypes in cutthroat trout compared to rainbow trout. Other findings are: The trout OmyY1 region evolves between 3 and 13 times slower than the trout mitochondrial regions that have been studied. The Lahontan cutthroat trout had a fixed OmyY1 sequence throughout ten separate populations, suggesting this subspecies underwent a severe population bottleneck prior to its current dispersal throughout the Great Basin during the pluvial phase of the last ice age. The Yellowstone group is the most derived among the cutthroat trout and consists of the Yellowstone, Bonneville, Colorado, Rio Grande and greenback subspecies. Identification of subspecies and sex with this Y-chromosome marker may prove useful in conservation efforts.     

Trophic relationships of nonnative brown trout, Salmo trutta, and native Bonneville cutthroat trout, Oncorhynchus clarkii utah, in a northern Utah, USA river

Nonnative trout invasions have caused the widespread decline of cutthroat trout populations in western North America. In contrast to other nonnative salmonids, the role of nonnative brown trout in native cutthroat trout decline is poorly understood. Specifically, the level of ecological similarity that occurs between these species and the importance of other trophic mechanisms (e.g., predation) in their interactions are key uncertainties. We evaluated the trophic relationships of brown trout and cutthroat trout in a northern Utah river using a combination of diet and stable isotope analyses. We compared the dietary habits of these two species using multiple and complementary measures. Based on both stomach contents and δ¹³C signatures, we found that these species consumed a similar and opportunistic diet (i.e., they were nonselective in their foraging patterns). However, at most sizes, brown trout ingested larger prey—including fishes—and occupied a higher relative trophic position (i.e., δ¹⁵N) than cutthroat trout. Overall, these results demonstrate a high degree of dietary similarity and therefore strengthen earlier conclusions regarding interspecific competition between these two species. Our study, when considered alongside the work of others, suggests there is potential for predatory interactions between these species (i.e., brown trout preying on small cutthroat trout). We believe that future research on brown trout–cutthroat trout interactions should consider predatory effects in greater detail.