Microsatellite DNA analysis of rainbow trout (Oncorhynchus mykiss) from western Alberta, Canada: native status and evolutionary distinctiveness of “Athabasca” rainbow trout

Molecular genetic assays can contribute to conservation of aquatic taxa by assessing evolutionary and taxonomic distinctiveness, levels of genetic variation within and between populations, and the degree of introgression with introduced taxa. The Athabasca River drainage of␣western Alberta, Canada is one of only three (and the largest) drainages flowing east of the continental divide that contain native populations of rainbow trout (Salmonidae: Oncorhynchus mykiss). The “Athabasca” rainbow trout has been considered a preglacial relict worthy of special conservation measures. In addition, the native range of Athabasca rainbow trout has seen many instances of introductions of non-native populations since the beginning of the 20th century. We assayed rainbow trout from the Athabasca River drainage, from hatchery populations, and from representative populations in adjacent regions (N = 49 localities) for variation at 10 microsatelite loci to assess the level of evolutionary distinctiveness of Athabasca rainbow trout, and to assess the levels of introgression with non-native hatchery fish. We found that native Athabasca rainbow trout did not form a distinctive genetic assemblage and that the greatest amount of allele frequency variation was attributable to contemporary drainage systems (29.3%) rather than by a Athabasca/non-Athabasca distinction (12.6%). We found that 78% of all fish were confidently assigned to a “wild” rather than a “hatchery” genetic grouping and that most of the inferred introgression with hatchery fish was restricted to a few localities (N = 6). Our results suggest that: (i)␣Athabasca River rainbow trout are likely postglacial immigrants from adjacent populations of the Fraser River, and (ii) that there is no evidence of widespread introgression of hatchery alleles into native Athabasca River drainage rainbow trout.     

Genetic variability and components of fitness in hatchery strains of rainbow trout

Two fitness components, development rate and egg size, were examined in six hatchery strains of rainbow trout, Oncorhynchus mykiss (syn. Salmo gairdneri Richardson), with different amounts of enzyme heterozygosity. The average expected heterozygosities per strain ranged from 4 to 8%, based upon an electrophoretic analysis of the protein products of 42 loci. Strains with higher heterozygosities had faster development rates, as measured by hatching time, than strains with lower heterozygosity. Concordance between hatching time and another measure of development rate, degree of yolk-sac resorption, suggests that hatching time is a valid measure of embryonic development rate in salmonid fishes. Earlier-hatching embryos were longer and heavier at the time of yolk-sac resorption than later-hatching fish. Females from more-heterozygous strains also had a tendency to have larger eggs. These data suggest that genetic variation is an important biological resource to be conserved in hatchery stocks.     

Effect of stress on blood coagulation and haematology in rainbow trout (Salmo gairdneri)

The effect of short-term stress on blood clotting times, cell counts, haematocrits, and blood glucose levels was studied in a hatchery strain and wild type rainbow trout, Salmo gairdneri. Blood clotting times declined and thrombocyte counts, haematocrits, and blood glucose levels increased after stress in both strains of trout. The wild type of rainbow trout, however, required less time to recover from the stress than the hatchery strain of rainbow trout. The implications of a stress activated blood coagulation system in fish are discussed.     

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.     

Sixty years of anthropogenic pressure: a spatio-temporal genetic analysis of brown trout populations subject to stocking and population declines

Analyses of historical samples can provide invaluable information on changes to the genetic composition of natural populations resulting from human activities. Here, we analyse 21 microsatellite loci in historical (archived scales from 1927 to 1956) and contemporary samples of brown trout ( Salmo trutta ) from six neighbouring rivers in Denmark, to compare the genetic structure of wild populations before and after population declines and stocking with nonlocal strains of hatchery trout. We show that all populations have been strongly affected by stocking, with admixture proportions ranging from 14 to 64%. Historical population genetic structure was characterized by isolation by distance and by positive correlations between historical effective population sizes and habitat area within river systems. Contemporary population genetic structure still showed isolation by distance, but also reflected differences among populations in hatchery trout admixture proportions. Despite significant changes to the genetic composition within populations over time, dispersal rates among populations were roughly similar before and after stocking. We also assessed whether population declines or introgression by hatchery strain trout should be the most significant conservation concern in this system. Based on theoretical considerations, we argue that population declines have had limited negative effects for the persistence of adaptive variation, but admixture with hatchery trout may have resulted in reduced local adaptation. Collectively, our study demonstrates the usefulness of analysing historical samples for identifying the most important consequences of human activities on the genetic structure of wild populations.     

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

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

Identifying footprints of selection in stocked brown trout populations: a spatio‐temporal approach

Studies of interactions between farmed and wild salmonid fishes have suggested reduced fitness of farmed strains in the wild, but evidence for selection at the genic level is lacking. We studied three brown trout populations in Denmark which have been significantly admixed with stocked hatchery trout (19–64%), along with two hatchery strains used for stocking. The wild populations were represented by contemporary samples (2000–2006) and two of them by historical samples (1943–1956). We analysed 61 microsatellite loci, nine of which showed putative functional relationships [expressed sequence tag (EST)‐linked or quantitative trait loci]. F_ST‐based outlier tests provided support for diversifying selection at chromosome regions marked by three loci, two anonymous and one EST‐linked. Patterns of differentiation suggested that the loci were candidates for being under diversifying hitch‐hiking selection in hatchery vs. wild environments. Analysis of hatchery strain admixture proportions showed that in one wild population, two of the loci showed significantly lower admixture proportions than the putatively neutral loci, implying contemporary selection against alleles introduced by hatchery strain trout. In the most strongly admixed population, however, there was no evidence for selection, possibly because of immigration by stocked trout overcoming selection against hatchery‐derived alleles or supportive breeding practices allowing hatchery strain trout to escape natural selection. To our knowledge, this is the first study demonstrating footprints of selection in wild salmonid populations subject to spawning intrusion by farmed fish.     

Analysis of microsatellite variation in the rainbow trout (Parasalmo (Oncorhynchus) mykiss) from Kamchatka

Genetic variation of Kamchatka rainbow trout Parasalmo (O.) mykiss was examined using 10 microsatellite DNA loci, and phylogeographic comparison with other representatives of the species across the distribution range was performed. It was demonstrated that Kamchatka populations differed from other geographic groups of rainbow trout in a number of microsatellite loci. These populations also displayed distinct clustering and were characterized by lower genetic diversity. Analysis of a set of 26 different microsatellite loci (personal and literature data) demonstrated that most of the populations within the Kamchatka region were separated from one another, characterized by marked geographic differentiation, and affiliation to certain river basins. In Kamchatka rainbow trout, with high degree of probability, three geographic clusters (northwestern, southwestern, and eastern) were identified. In general, analysis of microsatellite DNA supported the data on low genetic diversity of the Kamchatka group Parasalmo (O.) mykiss, based on the variation estimates for a number of genes of nuclear and mitochondrial DNA, and allozyme loci.     

Analysis of microsatellite variation in the rainbow trout <Emphasis Type="Italic">Parasalmo (Oncorhynchus) mykiss</Emphasis> from Kamchatka

Genetic variation of Kamchatka rainbow trout Parasalmo (O.) mykiss was examined using 10 microsatellite DNA loci, and phylogeographic comparison with other representatives of the species across the distribution range was performed. It was demonstrated that Kamchatka populations differed from other geographic groups of rainbow trout in a number of microsatellite loci. These populations also displayed distinct clustering and were characterized by lower genetic diversity. Analysis of a set of 26 different microsatellite loci (personal and literature data) demonstrated that most of the populations within the Kamchatka region were separated from one another, characterized by marked geographic differentiation, and affiliation to certain river basins. In Kamchatka rainbow trout, with high degree of probability, three geographic clusters (northwestern, southwestern, and eastern) were identified. In general, analysis of microsatellite DNA supported the data on low genetic diversity of the Kamchatka group Parasalmo (O.) mykiss, based on the variation estimates for a number of genes of nuclear and mitochondrial DNA, and allozyme loci.     

Microsatellite and mitochondrial DNA polymorphism reveals life-history dependent interbreeding between hatchery and wild brown trout (Salmo trutta L.)

The effects of stocking hatchery trout into wild populations were studied in a Danish river, using microsatellite and mitochondrial DNA (mtDNA) markers. Baseline samples were taken from hatchery trout and wild trout assumed to be unaffected by previous stocking. Also, samples were taken from resident and sea trout from a stocked section of the river. Genetic differentiation between the hatchery strain and the local wild population was modest (microsatellite FST = 0.06). Using assignment tests, more than 90% of individuals from the baseline samples were classified correctly. Assignment tests involving samples from the stocked river section suggested that the contribution by hatchery trout was low among sea trout (< 7%), but high (46%) among resident trout. Hybrid index analysis and a high percentage of mtDNA haplotypes specific to indigenous trout observed among resident trout that were assigned to the hatchery strain suggested that interbreeding took place between hatchery and wild trout. The latter result also indicated that male hatchery trout contributed more to interbreeding than females. We suggest that stronger selection acts against stocked hatchery trout that become anadromous compared to hatchery trout that become resident. As most resident trout are males this could also explain why gene flow from hatchery to wild trout appeared to be male biased. The results show that even despite modest differentiation at neutral loci domesticated trout may still perform worse than local populations and it is important to be aware of differential survival and reproductive success both between life-history types and between sexes.     

Biochemical genetic variation in populations of golden trout, Salmo aguabonita: evidence of the threatened Little Kern River golden trout, S.a. whitei.

Eight wild populations of the High Sierra golden trout, Salmo aguabonita, and one domestic stock of rainbow trout, Salmo gairdneri, were examined for biochemical-genetic variation in eight protein systems. Variation within the eight systems was determined by at least 10 loci in both golden and rainbow trout and all the alleles identified in rainbow trout were observed as electro-phoretically identical phenotypes in golden trout. Variation was observed at an average of 51 percent of the loci in the golden trout samples and for five of the 10 loci in the rainbow trout. Average heterozygosity ranged from 12.6 to 13.9 percent for seven of the golden trout populations with one showing a low value of 5.4 percent. A comparable estimate of 12.1 percent was found for the rainbow stock. On the basis of genetic variation and allele frequencies at three loci, the eight golden trout populations were divided into two distinct groups. Three populations sampled from the Little Kern River basin tended to be genetically distinct from two additional Little Kern River basin populations and from three geographically distinct populations sampled from the eastern Kern River area. The former three populations were hypothesized to be of a recent rainbow-golden hybrid origin. Trout in the other two Little Kern River basin populations, sampled in head-waters of a stream tributary to the Little Kern River, were considered to be the threatened Little Kern golden trout, S. a. whitei Evermann, due to their high degree of genetic similarity to the geographically distinct subspecies S. a. aguabonita sampled from the eastern Kern River area. The finding of substantial genetic variation in the wild golden trout populations indicates that this threatened species is not at present genetically impoverished and thus does not appear to be in immediate danger of extinction through lack of adaptive capability.     

Diagnostic single nucleotide polymorphisms for identifying westslope cutthroat trout (Oncorhynchus clarki lewisi), Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (Oncorhynchus mykiss)

We describe 12 diagnostic single nucleotide polymorphism (SNP) assays for use in species identification among rainbow and cutthroat trout: five of these loci have alleles unique to rainbow trout (Oncorhynchus mykiss), three unique to westslope cutthroat trout (O. clarkii lewisi) and four unique to Yellowstone cutthroat trout (O. clarkii bouvieri). These diagnostic assays were identified using a total of 489 individuals from 26 populations and five fish hatchery strains.     

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

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

Variability in swimming performance and underlying physiology in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta)

We investigated intra- and interspecific variation in swimming performance and related physiological parameters in two members of the salmonid family. For our comparisons, we sourced juvenile brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) from one hatchery and a second strain of rainbow trout from another. The hatcheries maintain genetic stocks obtained several decades ago from very different environments. We tested competing hypotheses: that there would be greater interspecific (across species) variation or that there would be greater intraspecific (within species) variation, owing to regional adaptations. To test these hypotheses, individual and small schools of five fish were taken to fatigue using the critical swimming speed test (U(crit)), and three post-exercise physiological metrics, packed red cell volume (hematocrit), citrate synthase and lactate dehydrogenase activity, were assessed. The majority of the results in swimming performance and hematocrit support that intraspecific variation was greater than interspecific variation, i.e. the location had a stronger effect than did genus. Variation in lactate dehydrogenase activity supported neither intra- nor interspecific variation as determining factors. In sum, our findings suggest that the performance of different species of salmonids from the same locale can be more similar than those of the same species from different areas.     

Population genetic structure and ancestry of Oncorhynchus mykiss populations above and below dams in south-central California

Genetic analyses of coastal Oncorhynchus mykiss, commonly known as steelhead/rainbow trout, at the southern extreme of their geographic range in California are used to evaluate ancestry and genetic relationships of populations both above and below large dams. Juvenile fish from 20 locations and strains of rainbow trout commonly planted in reservoirs in the five study basins were evaluated at 24 microsatellite loci. Phylogeographic trees and analysis of molecular variance demonstrated that populations within a basin, both above and below dams, were generally each other’s closest relatives. Absence of hatchery fish or their progeny in the tributaries above dams indicates that they are not commonly spawning and that above-barrier fish are descended from coastal steelhead trapped at dam construction. Finally, no genetic basis was found for the division of populations from this region into two distinct biological groups, contrary to current classification under the US and California Endangered Species Acts.     

Wild and aquaculture populations of the eastern oyster compared using microsatellites

Five new microsatellite markers were developed for the eastern oyster (Crassostrea virginica), and allelic variability was compared between a wild Chesapeake Bay population (James River) and a hatchery strain (DEBY). All loci amplified readily and demonstrated allelic variability with the number of alleles ranging from 16 to 36 in the wild population and from 11 to 19 in the DEBY strain. Average observed and expected heterozygosities were estimated at 0.66 and 0.80 in the hatchery sample. The corresponding estimates were 0.91 and 0.75 in the wild sample. Results indicated lower genetic variability in the DEBY strain and significant genetic differentiation between the wild population and hatchery strain. These microsatellite loci will prove valuable for future population genetic studies and in tracking of hatchery strains used in restoration.     

Molecular Evidence for Introgression and Loss of Genetic Variability in Salmo (trutta) macrostigma as a Result of Massive Restocking of Apennine Populations (Northern and Central Italy)

We assessed structural gene variation (allozymes and mtDNA) of brown trout to evaluate the genetic variability of Apennine stream populations (Northern and Central Italy) and the possibility of introgression by alien genomes after massive restocking with hatchery strains (Atlantic stocks). Genetic variability within and between Apennine populations was extremely low in our samples. Only two allozyme loci were polymorphic and mean hetero-zygosity was also reduced compared to other brown trout populations. Allelic frequencies determined for both loci were similar to the ones detected in the corresponding hatchery spawners. The reduction or total absence of the Mediterranean nuclear (LDH-5) and mitochondrial (16S rDNA) diagnostic markers suggests the domestic origin of most populations, and the introgression effects carried out by non-native genomes. From a taxonomic point of view, a clear differentiation emerges among basins placed on opposite sides of the Apennine chain (Tyrrhenian and Adriatic regions). In particular, the presence of Mediterranean genotypes and haplotypes characterizing Salmo (trutta) macrostigma is sporadic along the eastern Apennine side, adding additional doubts on the original presence and wide distribution of this salmonid along the Adriatic side of the mountain chain. In spite of conservation programs devoted to preservation of local genetic characteristics of S. t. macrostigma, massive restocking practices with hatchery strains obtained by a few spawners is the major cause of significant `founder effect' and `inbreeding depression' even in Apennine regions.     

HETEROZYGOSITY‐FITNESS CORRELATIONS IN RAINBOW TROUT: EFFECTS OF ALLOZYME LOCI OR ASSOCIATIVE OVERDOMINANCE?

Previous studies with rainbow trout (Oncorhynchus mykiss) have shown that increased heterozygosity at allozyme loci is correlated with several phenotypic traits associated with fitness. We expected to find a similar effect of heterozygosity at other nuclear loci if these associations are due to loci in linkage disequilibrium with the allozyme loci (i.e., associative overdominance), rather than the allozymes themselves. We examined the association between multiple locus heterozygosity and condition factor at 10 allozyme and 10 microsatellite loci. Individuals that were more heterozygous at allozyme loci had significantly greater condition factor in two hatchery cohorts of rainbow trout (1996 P = 0.006; 1997 P < 0.001). In contrast, there was no evidence at microsatellite loci that increased heterozygosity was associated with greater condition factor. Our results suggest that the observed relationship between heterozygosity at allozyme loci and condition factor in rainbow trout appears to be due to the allozyme loci themselves, rather than associative overdominance. We cannot, however, rule out that differences in the mutation process between allozymes and microsatellites may be responsible for these observations. Regardless of the underlying mechanism, these results support the view that allozymes and microsatellites are differentially affected by natural selection.     

Lower fitness of hatchery and hybrid rainbow trout compared to naturalized populations in Lake Superior tributaries

We have documented an early life survival advantage by naturalized populations of anadromous rainbow trout Oncorhynchus mykiss over a more recently introduced hatchery population and outbreeding depression resulting from interbreeding between the two strains. We tested the hypothesis that offspring of naturalized and hatchery trout, and reciprocal hybrid crosses, survive equally from fry to age 1+ in isolated reaches of Lake Superior tributary streams in Minnesota. Over the first summer, offspring of naturalized females had significantly greater survival than offspring of hatchery females in three of four comparisons (two streams and 2 years of stocking). Having an entire naturalized genome, not just a naturalized mother, was important for survival over the first winter. Naturalized offspring outperformed all others in survival to age 1+ and hybrids had reduced, but intermediate, survival relative to the two pure crosses. Averaging over years and streams, survival relative to naturalized offspring was 0.59 for hybrids with naturalized females, 0.37 for the reciprocal hybrids, and 0.21 for hatchery offspring. Our results indicate that naturalized rainbow trout are better adapted to the conditions of Minnesota's tributaries to Lake Superior so that they outperform the hatchery-propagated strain in the same manner that many native populations of salmonids outperform hatchery or transplanted fish. Continued stocking of the hatchery fish may conflict with a management goal of sustaining the naturalized populations.     

Deviation from morphological intermediacy in interstrain hybrids of rainbow trout,Salmo gairdneri

Synopsis Deviations from morphological intermediacy in six first generation hybrids between three hatchery strains of rainbow trout, raised in a common environment, are reported. Hybrids have higher mean counts of four meristic characters than their maternal parental strain in a significantly greater number of cases (18 out of 24). Furthermore, eight of eleven hybrid indices are not intermediate. These results are discussed in reference to several mechanisms and models proposed to account for observed responses of meristic characters to environmental and genetic influences.