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.     

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.     

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).     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

Assessing thermal adaptation using family‐based association and FST outlier tests in a threatened trout species

Discovering genetic markers associated with phenotypic or ecological characteristics can improve our understanding of adaptation and guide conservation of key evolutionary traits. The Lahontan cutthroat trout (Oncorhynchus clarkii henshawi) of the northern Great Basin Desert, USA, demonstrated exceptional tolerance to high temperatures in the desert lakes where it resided historically. This trait is central to a conservation hatchery effort to protect the genetic legacy of the nearly extinct lake ecotype. We genotyped full‐sibling families from this conservation broodstock and samples from the only two remaining, thermally distinct, native lake populations at 4,644 new single nucleotide polymorphisms (SNPs). Family‐based genome‐wide association testing of the broodstock identified nine and 26 SNPs associated with thermal tolerance (p < 0.05 and p < 0.1), measured in a previous thermal challenge experiment. Genes near the associated SNPs had complex functions related to immunity, growth, metabolism and ion homeostasis. Principal component analysis using the thermotolerance‐related SNPs showed unexpected divergence between the conservation broodstock and the native lake populations at these loci. F_ST outlier tests on the native lake populations identified 18 loci shared between two or more of the tests, with two SNPs identified by all three tests (p < 0.01); none overlapped with loci identified by association testing in the broodstock. A recent history of isolation and the complex genetic and demographic backgrounds of Lahontan cutthroat trout probably limited our ability to find shared thermal tolerance loci. Our study extends the still relatively rare application of genomic tools testing for markers associated with important phenotypic or environmental characteristics in species of conservation concern.     

MITOCHONDRIAL GENOTYPES HAVE NO DETECTABLE EFFECTS ON MERISTIC TRAITS IN CUTTHROAT TROUT HYBRID SWARMS

Efforts to detect effects of cytoplasmic genes are confounded by the problem of partitioning nuclear and cytoplasmic effects. In this study we test for effects of mtDNA haplotype on development in hybrid populations of cutthroat trout (Oncorhynchus clarki) with randomly associated nuclear and mtDNA genotypes. We have previously described several intraspecific hybrid swarms formed by interbreeding of westslope cutthroat trout (O. c. lewisi) and Yellowstone cutthroat trout (O. c. bouvieri). Genetic distance between these subspecies is high (Nei's D = 0.30; mtDNA P = 0.02), and diagnostic alleles at multiple nuclear loci and two distinct mtDNA haplotypes are present in the hybrids. Historical associations between alleles at nuclear loci and between cytotypes and nuclear alleles have largely decayed. We test for differences in meristic characters between fish with alternate mtDNA haplotypes. Counts and fluctuating bilateral asymmetry for these characters have been previously shown to be sensitive indicators of genetic differences that affect development. No differences were found between mtDNA types in meristic counts or fluctuating asymmetry. Therefore, the alternate mtDNA haplotypes had no detectable effect on development as measured by meristic counts in these hybrid populations. However, diagnostic alleles at one nuclear allozyme locus (CK-CI) were associated with several fin ray counts.     

Development and evaluation of a genome‐wide Coffee 8.5K SNP array and its application for high‐density genetic mapping and for investigating the origin of Coffea arabica L.

Coffee species such as Coffea canephora P. (Robusta) and C. arabica L. (Arabica) are important cash crops in tropical regions around the world. C. arabica is an allotetraploid (2n = 4x = 44) originating from a hybridization event of the two diploid species C. canephora and C. eugenioides (2n = 2x = 22). Interestingly, these progenitor species harbour a greater level of genetic variability and are an important source of genes to broaden the narrow Arabica genetic base. Here, we describe the development, evaluation and use of a single‐nucleotide polymorphism (SNP) array for coffee trees. A total of 8580 unique and informative SNPs were selected from C. canephora and C. arabica sequencing data, with 40% of the SNP located in annotated genes. In particular, this array contains 227 markers associated to 149 genes and traits of agronomic importance. Among these, 7065 SNPs (~82.3%) were scorable and evenly distributed over the genome with a mean distance of 54.4 Kb between markers. With this array, we improved the Robusta high‐density genetic map by adding 1307 SNP markers, whereas 945 SNPs were found segregating in the Arabica mapping progeny. A panel of C. canephora accessions was successfully discriminated and over 70% of the SNP markers were transferable across the three species. Furthermore, the canephora‐derived subgenome of C. arabica was shown to be more closely related to C. canephora accessions from northern Uganda than to other current populations. These validated SNP markers and high‐density genetic maps will be useful to molecular genetics and for innovative approaches in coffee breeding.     

Effects of an Introduced Piscivore on Native Trout: Insights from a Demographic Model

Introductions of exotic species pose a significant threat to the persistence of many native populations, including many inland fishes. In 1994, piscivorous lake trout (Salvelinus namaycush) were discovered in Yellowstone Lake, Yellowstone National Park, Wyoming, USA, one of the last strongholds of the native Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri). Predation by lake trout is expected to lead to a substantial decline in the native cutthroat trout population, which may have significant negative consequences for terrestrial predators that depend on cutthroat trout for prey and for the recreational fishery of the Park. We developed a matrix demographic model for the cutthroat trout population in Yellowstone Lake to identify the life stages that are most critical for understanding population dynamics. Parameter estimates (vital rates) were manipulated to explore the possible consequences of lake trout invasion. Comparisons of our results with current estimates of population trend and age structure suggested that our model reflected current conditions of the system. Elasticity analysis of the model revealed that population growth was most sensitive to annual survival of young trout, the group that is expected to be most vulnerable to lake trout predation. Projection of our deterministic model suggested that, in addition to a decline in abundance of cutthroat trout, the effects of lake trout may be manifest as changes in age and breeding structure of the population. Simulations of a stochastic version of the model indicated that a 60% or greater decline in the cutthroat trout population could be expected within 100 years if the lake trout population were permitted to grow uncontrolled. However, an effective control strategy that prevented the establishment of a large population of lake trout substantially reduced population decline, although the reduction in the availability of adult trout to terrestrial predators and anglers may be still be substantial (20–40%). In addition to current control activities in place in the Park, we recommend a renewed emphasis on understanding and monitoring juvenile life stages of cutthroat trout. Our results demonstrate the value of existing data sets for developing models to estimate the potential impact of biological invasions on the management and conservation of native populations, especially when opportunities and resources for additional empirical studies are limited.     

Patterns of hybridization among cutthroat trout and rainbow trout in northern Rocky Mountain streams

Introgressive hybridization between native and introduced species is a growing conservation concern. For native cutthroat trout and introduced rainbow trout in western North America, this process is thought to lead to the formation of hybrid swarms and the loss of monophyletic evolutionary lineages. Previous studies of this phenomenon, however, indicated that hybrid swarms were rare except when native and introduced forms of cutthroat trout co‐occurred. We used a panel of 86 diagnostic, single nucleotide polymorphisms to evaluate the genetic composition of 3865 fish captured in 188 locations on 129 streams distributed across western Montana and northern Idaho. Although introgression was common and only 37% of the sites were occupied solely by parental westslope cutthroat trout, levels of hybridization were generally low. Of the 188 sites sampled, 73% contained ≤5% rainbow trout alleles and 58% had ≤1% rainbow trout alleles. Overall, 72% of specimens were nonadmixed westslope cutthroat trout, and an additional 3.5% were nonadmixed rainbow trout. Samples from seven sites met our criteria for hybrid swarms, that is, an absence of nonadmixed individuals and a random distribution of alleles within the sample; most (6/7) were associated with introgression by Yellowstone cutthroat trout. In streams with multiple sites, upstream locations exhibited less introgression than downstream locations. We conclude that although the widespread introduction of nonnative trout within the historical range of westslope cutthroat trout has increased the incidence of introgression, sites containing nonadmixed populations of this taxon are common and broadly distributed.     

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.     

The cutthroat trout Y chromosome is conserved with that of rainbow trout

Five genetic markers previously shown to be located on the sex chromosomes of rainbow trout (Oncorhynchus mykiss) were tested for linkage with the sex locus of Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) in a genetic cross created from a rainbow x cutthroat male hybrid. We show that the sex locus of both rainbow and cutthroat trout is on the same homologous linkage group. Fluorescence in situ hybridization (FISH) using a probe for the microsatellite marker Omm1665, which maps close to the sex locus of Yellowstone cutthroat trout, was used to identify the Y chromosome of cutthroat trout in the hybrid. The Y chromosome of cutthroat trout is sub-telocentric and lacks a DAPI band found on the short arm of the Y chromosome of some rainbow trout males.     

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.     

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.     

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.