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.     

Early Marine Migration Patterns of Wild Coastal Cutthroat Trout (Oncorhynchus clarki clarki), Steelhead Trout (Oncorhynchus mykiss), and Their Hybrids

Background

Hybridization between coastal cutthroat trout (Oncorhynchus clarki clarki) and steelhead or rainbow trout (Oncorhynchus mykiss) has been documented in several streams along the North American west coast. The two species occupy similar freshwater habitats but the anadromous forms differ greatly in the duration of marine residence and migration patterns at sea. Intermediate morphological, physiological, and performance traits have been reported for hybrids but little information has been published comparing the behavior of hybrids to the pure species.

Methodology/Principal Findings

This study used acoustic telemetry to record the movements of 52 cutthroat, 42 steelhead x cutthroat hybrids, and 89 steelhead smolts, all wild, that migrated from Big Beef Creek into Hood Canal (Puget Sound, Washington). Various spatial and temporal metrics were used to compare the behavior of the pure species to their hybrids. Median hybrid residence time, estuary time, and tortuosity values were intermediate compared to the pure species. The median total track distance was greater for hybrids than for either cutthroat or steelhead. At the end of each track, most steelhead (80%) were located near or north of the Hood Canal, as expected for this seaward migrating species, whereas most cutthroat (89%) were within 8 kilometers of the estuary. Most hybrids (70%) were detected leaving Hood Canal, though a substantial percentage (20%) remained near the Big Beef Creek estuary. More hybrids (7.5%) than pure cutthroat (4.5%) or steelhead (0.0%) were last detected in the southern reaches of Hood Canal.

Conclusions/Significance

Given the similarity in freshwater ecology between the species, differences in marine ecology may play an important role in maintaining species integrity in areas of sympatry.     

Erosion of interspecific reproductive barriers resulting from hatchery supplementation of rainbow trout sympatric with cutthroat trout

The frequency of hybridization between cutthroat (Onchorhynchus clarki clarki) and rainbow (O. mykiss irideus) trout from coastal habitats in British Columbia, Canada, was examined in seven populations where the two species are sympatric with no history of rainbow trout stocking and compared with areas where native rainbow trout populations have been supplemented with hatchery fish (three populations). Four nuclear markers were used to identify each species and interspecific hybrids and one mitochondrial marker showed the direction of gene exchange between species. The frequency of hybrids was significantly higher (Fisher exact test, P < 0.001) in river systems where hatchery rainbow trout have been introduced (50.6% hybrids) than in populations where the two species naturally co-occur without supplementation (9.9% hybrids).     

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

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

Hybrid zone structure and the potential role of selection in hybridizing populations of native westslope cutthroat trout (Oncorhynchus clarki lewisi) and introduced rainbow trout (O. mykiss)

Introgressive hybridization is a common feature of many zones of contact between divergent lineages of fishes. This is particularly common when taxa that are normally allopatric come into artificial (human-induced) secondary contact. We examined 18 native populations of westslope cutthroat trout (Oncorhynchus clarki lewisi, WCT) to determine the extent of introgressive hybridization with introduced rainbow trout (O. mykiss, RBT) and the genetic structure of hybridizing populations in the upper Kootenay River, southeastern British Columbia, Canada. Using four diagnostic nuclear loci we calculated a hybrid index, inbreeding coefficient, FIS, and the linkage disquilibrium correlation coefficient, Rij, for each locality to determine the distribution of genotypes in each population. We also categorized the 142 hybrid individuals found across localities into four hybrid classes based on their genotypes. The majority of localities (11/18) showed a unimodal distribution of genotypes skewed towards genotypes of WCT. Two localities, however (lower Gold Creek and Lodgepole Creek) showed a flat to bimodal distribution and one site (lower Bull River) showed a unimodal distribution skewed towards RBT genotypes. The majority of hybrid individuals were classified genotypically as WCT backcrosses (59%) and post-F1 individuals (24%). We found a skewed ratio of pure WCT to pure RBT (17:1) and only four F1 hybrids (3%), suggesting that the spread of RBT alleles may be facilitated by hybrids straying to neighbouring populations. We also tested for the action of selection in one population using cohort analyses, but found little evidence of differential selection between pure WCT and hybrid individuals. Pooled across age classes there were significant differences in genotypic frequencies among loci suggesting differential introgression. There was no asymmetry to the hybridization between rainbow trout and westslope cutthroat trout because both species' mitochondrial DNA haplotypes were observed at similar frequencies in the hybrids. Our analyses suggest that hybrid swarms are likely to form in the upper Kootenay River drainage and that certain native WCT populations in British Columbia are at risk of local genomic extinction.     

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.     

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.     

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.     

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.     

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.     

Diversity of movements by individual anadromous coastal cutthroat trout Oncorhynchus clarkii clarkii

Wild, downstream‐migrating cutthroat trout, Oncorhynchus clarkii clarkii, smolts and adults were captured at a weir in Big Beef Creek, Hood Canal, Washington, surgically implanted with acoustic tags and tracked to identify spring and summer movements using stationary receivers in order to test the assumption that the species moves little while in marine waters. Overall, 93–96% migrated from the stream into the east side of the long narrow fjord, where they dispersed north and south along the shoreline. Most O. c. clarkii were detected nearshore within 10 km of the release site, with declining detection rates to 77 km. Over one‐third (36%) crossed c. 2–4 km of deep water to the other side but only one O. c. clarkii left the Hood Canal basin. Movements and behaviour patterns did not differ between smolts and adults but cluster analysis revealed two modes of distribution, here categorized as residents and migrants. Within these categories of overall distribution, a range of finer‐scale behaviour patterns was observed, including sedentary individuals, daily moving between proximate sites and more continuous long‐distance travel. Diel movement patterns varied markedly among individuals but overall activity increased near dawn, peaked around mid‐day and declined but continued at night. These patterns contrast with sympatric and closely related steelhead trout, Oncorhynchus mykiss, providing new insights into the diversity of salmonid behaviour.     

Habitat diversity and species diversity: testing the competition hypothesis with juvenile salmonids

I experimentally tested two predictions of the hypothesis that the positive relationship between habitat diversity and species diversity arises from a reduction in the negative effects of interspecific competition. By allowing species to partition habitat and avoid competition, habitat diversity should 1) facilitate the addition of an ecologically intermediate species into an existing community, and 2) reduce the negative effects of that species on existing members of the community. I tested these predictions with juveniles of three sympatric salmonid species: coho salmon ( Oncorhynchus kisutch ), steelhead trout ( O. mykiss ), and cutthroat trout ( O. clarki ), which in natural streams occupy deep low-velocity pools, shallow high-velocity riffles, and intermediate habitats, respectively. I introduced two (coho and steelhead) and three species communities into each of three artificial stream habitats: pools, riffles, and diverse. The results provide partial support for the predictions. Cutthroat trout grew fastest in the diverse stream habitat. Though habitat diversity did not eliminate the negative effects of competition, in the three species community coho and steelhead grew as fast in the diverse habitat as in either homogeneous habitat. The results are consistent with data on species number and evenness from natural communities, where variation along other niche axes confounds the relationship between habitat diversity, interspecific competition, and species diversity.     

Changes in use of lake habitat by experimentally segregated populations of cutthroat trout and Dolly Varden char

The role of competition in a lacustrine community of two salmonid species, cutthroat trout Oncorhynchus clarki and Dolly Varden char Salvelinus malma , was studied in three coastal British Columbia lakes Habitat use by the species alone (allopatric) and in coexistence with each other (sympatric) was investigated by gill netting at 0-40 m depth contours (surface to bottom) so that several habitats (littoral, epipelagic. pelagic, epibenthic) were sampled From June to October, trout used mainly littoral and epipelagic habitats in sympatry and allopatry Char used all habitats in allopatry. and deep habitats (pelagic, epibenthic) not frequented by trout in sympatry The two species were thus spatially segregated with depth in sympatry Diel (day, night) and seasonal changes (spring, summer, autumn) in habitat use were not pronounced The shift in habitat use by experimentally allopatric char but not trout suggests that the effects of competition between sympatric trout and char for habitat resources are greater on the char     

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.     

Variable hybridization outcomes in trout are predicted by historical fish stocking and environmental context

Hybridization can profoundly affect the genomic composition and phenotypes of closely related species, and provides an opportunity to identify mechanisms that maintain reproductive isolation between species. Recent evidence suggests that hybridization outcomes within a species pair can vary across locations. However, we still do not know how variable outcomes of hybridization are across geographic replicates, and what mechanisms drive that variation. In this study, we described hybridization outcomes across 27 locations in the North Fork Shoshone River basin (Wyoming, USA) where native Yellowstone cutthroat trout and introduced rainbow trout co‐occur. We used genomic data and hierarchical Bayesian models to precisely identify ancestry of hybrid individuals. Hybridization outcomes varied across locations. In some locations, only rainbow trout and advanced backcrossed hybrids towards rainbow trout were present, while trout in other locations had a broader range of ancestry, including both parental species and first‐generation hybrids. Later‐generation intermediate hybrids were rare relative to backcrossed hybrids and rainbow trout individuals. Using an individual‐based simulation, we found that outcomes of hybridization in the North Fork Shoshone River basin deviate substantially from what we would expect under null expectations of random mating and no selection against hybrids. Since this deviation implies that some mechanisms of reproductive isolation function to maintain parental taxa and a diversity of hybrid types, we then modelled hybridization outcomes as a function of environmental variables and stocking history that are likely to affect prezygotic barriers to hybridization. Variables associated with history of fish stocking were the strongest predictors of hybridization outcomes, followed by environmental variables that might affect overlap in spawning time and location.     

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

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

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.     

Resource utilization by bull char and cutthroat trout in a mountain stream in Montana,U.S.A.

Resource utilization of sympatric populations of bull char,Salvelinus confluentus, and west-slope cutthroat trout,Oncorhynchus clarki lewisi, were studied by underwater observations of foraging behaviour and microhabitat use, and dietary analysis in a mountain stream of the Flathead River Basin, northwest Montana, U.S.A. Nearly 70% of bull char were categorized as benthic foragers, which moved constantly and captured prey primarily from the streambed, while all cutthroat trout were drift foragers, which held relatively fixed focal points in the midwater layers of pools during foraging. The composition of stomach contents was markedly different between the two species. Bull char fed primarily on baetid mayflies captured from the benthos or drift, whereas cutthroat trout ate primarily terrestrial invertebrates. The species also used different microhabitats. Bull char held positions close to the streambed and rarely strayed far from overhead cover, whereas cutthroat trout held focal points farther above the bed and far from overhead cover. Dietary segregation between these two salmonids appeared to result not only from differences in foraging tactics but also in the foraging microhabitats. Resource partitioning is considered to be one of important mechanisms allowing coexistence of these two stream salmonids.     

Hybridization between the escaped Rosa rugosa (Rosaceae) and native R. blanda in eastern North America

Rosa rugosa, a vigorous ornamental shrub introduced from Asia in the 19th century, is now naturalized in coastal northeastern North America, where it occasionally grows in sympatry with the native R. blanda. To document hybridization between these species, evaluate its extent across the area of sympatry, and examine the use of morphology as a field monitoring tool, we sampled 179 individuals of parental species and putative hybrids in 13 pure and 11 mixed populations. We developed allele-specific primers to assay single nucleotide polymorphisms (SNPs) markers from one chloroplast region and four low-copy nuclear introns. Our results revealed frequent bidirectional hybridization and infrequent introgression in sympatric populations of these species. The recurrent presence of F(1) hybrids in mixed populations indicated the weakness of early-acting reproductive barriers. Morphological data were concordant with molecular data and provided additional evidence for the presence of a few backcrosses. Morphological analyses yielded diagnostic characters for identifying hybrids and monitoring the hybrid zone. Such hybridization could ultimately lead to the genetic assimilation of R. blanda in mixed populations and to the formation of invasive hybrid genotypes, a phenomenon that is of economic and ecological concern because of the increasing number of exotic species worldwide.