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.     

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).     

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.     

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.     

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.     

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.     

Hybridization rapidly reduces fitness of a native trout in the wild

Human-mediated hybridization is a leading cause of biodiversity loss worldwide. How hybridization affects fitness and what level of hybridization is permissible pose difficult conservation questions with little empirical information to guide policy and management decisions. This is particularly true for salmonids, where widespread introgression among non-native and native taxa has often created hybrid swarms over extensive geographical areas resulting in genomic extinction. Here, we used parentage analysis with multilocus microsatellite markers to measure how varying levels of genetic introgression with non-native rainbow trout (Oncorhynchus mykiss) affect reproductive success (number of offspring per adult) of native westslope cutthroat trout (Oncorhynchus clarkii lewisi) in the wild. Small amounts of hybridization markedly reduced fitness of male and female trout, with reproductive success sharply declining by approximately 50 per cent, with only 20 per cent admixture. Despite apparent fitness costs, our data suggest that hybridization may spread due to relatively high reproductive success of first-generation hybrids and high reproductive success of a few males with high levels of admixture. This outbreeding depression suggests that even low levels of admixture may have negative effects on fitness in the wild and that policies protecting hybridized populations may need reconsideration.     

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.     

Adaptation genomics: next generation sequencing reveals a shared haplotype for rapid early development in geographically and genetically distant populations of rainbow trout

Local adaptation occurs when a population evolves a phenotype that confers a selective advantage in its local environment, but which may not be advantageous in other habitats. Restricted gene flow and strong selection pressures are prerequisites for local adaptation. Fishes in the family Salmonidae are predicted to provide numerous examples of local adaptation because of the high fidelity of returning to spawn in their natal streams, which results in highly structured populations, and the wide diversity of environments that salmonids have colonized. These conditions are ideally suited for producing a set of specialist phenotypes, whose fitness is maximized for one specific habitat, rather than a generalist phenotype similarly viable in several environments. Understanding patterns and processes leading to local adaptations has long been a goal of evolutionary biology, but it is only recently that identifying the molecular basis for local adaptation has become feasible because of advances in genomic technologies. The study of shared adaptive phenotypes in populations that are both geographically distant and genetically distinct should reveal some of the fundamental molecular mechanisms associated with local adaptation. In this issue of Molecular Ecology, Miller et al. (2012) make a significant contribution to the development of adaptation genomics. This study suggests that salmonids use standing genetic variation to select beneficial alleles for local adaptations rather than de novo mutations in the same gene or alternative physiological pathways. Identifying the genetic basis for local adaptation has major implications for the management, conservation and potential restoration of salmonid populations.     

Early effects of the strategies of creating a genetic refuge and direct translocation for conserving and restoring populations of native brown trout

1. The conservation of salmonid inter‐ and intra‐specific diversity is a well‐known challenge, and general management guidelines and conservation processes are available. However, research demonstrating the outcomes of practical conservation actions is largely lacking. 2. We monitored the spatiotemporal genetic and demographic evolution of a native Mediterranean brown trout population in a river in the French Alps to assess the efficacy and early effects of genetic refuge (i.e. cessation of stocking) and wild trout translocation strategies. We also studied the use of angling as a tool to limit the introgression of the wild standing population. 3. We found that the rate of non‐native alleles in wild populations was age dependent, underpinning the importance of using age profiles in the design of genetic conservation studies. 4. Genetic refuge and direct translocation of wild trout resulted in a rapid and significant decrease in the percentages of non‐native alleles. Moreover, the genetic refuge strategy resulted in a significant reduction in the number of pure non‐native individuals, without changing trout densities, whilst direct translocations resulted in the establishment of dense, self‐sustaining native trout populations. Direct translocations changed the distribution of genotype categories and increased densities up to 55‐fold in 3 years. Our results also showed that angling resulted in a selective pressure on non‐native trout introduced at fry stage, whereas non‐native trout issued from natural recruitment were not affected. 5. Our study provides insights for improving the efficacy of practical conservation policies and can be used in other native freshwater fish conservation plans. Proactive measures such as direct translocation need to be implemented together with passive approaches such as genetic refuge policies. Before implementing such actions, accurate genetic and demographic studies at small geographical scales are essential to ensure that no self‐sustaining population of non‐native fish is present. To obtain rapid colonisation, we recommend introducing fish along whole river sections rather than concentrating on a few river stretches. Angling pressure can be used as an additional tool to improve restoration.     

Plastid DNA sequencing and nuclear SNP genotyping help resolve the puzzle of central American Platanus

Background and Aims

Recent research on the history of Platanus reveals that hybridization phenomena occurred in the central American species. This study has two goals: to help resolve the evolutive puzzle of central American Platanus, and to test the potential of real-time polymerase chain reaction (PCR) for detecting ancient hybridization.

Methods

Sequencing of a uniparental plastid DNA marker [psbA-trnH^(GUG) intergenic spacer] and qualitative and quantitative single nucleotide polymorphism (SNP) genotyping of biparental nuclear ribosomal DNA (nrDNA) markers [LEAFY intron 2 (LFY-i2) and internal transcribed spacer 2 (ITS2)] were used.

Key Results

Based on the SNP genotyping results, several Platanus accessions show the presence of hybridization/introgression, including some accessions of P. rzedowskii and of P. mexicana var. interior and one of P. mexicana var. mexicana from Oaxaca (= P. oaxacana). Based on haplotype analyses of the psbA-trnH spacer, five haplotypes were detected. The most common of these is present in taxa belonging to P. orientalis, P. racemosa sensu lato, some accessions of P. occidentalis sensu stricto (s.s.) from Texas, P. occidentalis var. palmeri, P. mexicana s.s. and P. rzedowskii. This is highly relevant to genetic relationships with the haplotypes present in P. occidentalis s.s. and P. mexicana var. interior.

Conclusions

Hybridization and introgression events between lineages ancestral to modern central and eastern North American Platanus species occurred. Plastid haplotypes and qualitative and quantitative SNP genotyping provide information critical for understanding the complex history of Mexican Platanus. Compared with the usual molecular techniques of sub-cloning, sequencing and genotyping, real-time PCR assay is a quick and sensitive technique for analysing complex evolutionary patterns.