New tetranucleotide microsatellites for fine‐scale discrimination among endangered chinook salmon (Oncorhynchus tshawytscha)

The unambiguous identification of Central Valley spring‐run chinook salmon has become imperative since their proposed listing in 1998. The accuracy of methods used to assign individuals to their stock of origin is critical for understanding juvenile migration patterns and determining the success of protection measures. Existing microsatellites discriminate between the endangered winter‐run and other chinook but are insufficient to characterize phylogenetically less distinct runs. Here, we isolated and developed highly variable tetranucleotide microsatellites for the specific goal of increasing discriminatory power among closely related populations, providing a new power towards the reliable differentiation of nonwinter runs     

The impact of supplementation in winter-run chinook salmon on effective population size

Supplementation of young raised at a protected site, such as a hatchery, may influence the effective population size of an endangered species. A supplementation program for the endangered winter-run chinook salmon from the Sacramento River, California, has been releasing fish since 1991. A breeding protocol, instituted in 1992, seeks to maximize the effective population size from the captive spawners by equaling their contributions to the released progeny. As a result, the releases in 1994 and 1995 appear not to have decreased the overall effective population size and may have increased it somewhat. However, mistaken use of non-winter-run chinook spawners resulted in artificial crosses between runs with a potential reduction in effective population size, and imprinting of the released fish on Battle Creek, the site of the hatchery, resulted in limiting the contribution of the released fish to the target mainstem population. Rapid genetic analysis of captured spawners and a new rearing facility on the Sacramento River should alleviate these problems and their negative effect on the effective population size in future years.     

Evolution of a perfect simple sequence repeat locus in the context of its flanking sequence

Microsatellites, which have rapidly become the preferred markers in population genetics, reliably assign individual chinook salmon to the winter, fall, late-fall, or spring chinook runs in the Sacramento River in California's Central Valley (Banks et al. 2000. Can. J. Fish. Aquat. Sci. 57:915-927). A substantial proportion of this discriminatory power comes from Ots-2, a simple CA repeat, which is expected to evolve rapidly under the stepwise mutation model. We have sequenced a 300-bp region around this locus and typed 668 microsatellite-flanking sequence haplotypes to explore further the basis of this microsatellite divergence. Three sites of nucleotide polymorphism in the Ots-2 flanking sequence define five haplotypes that are shared by the Californian and Canadian populations. The Ots-2 microsatellite alleles are nonrandomly distributed among these five haplotypes in a pattern of gametic disequilibrium that is also shared among populations. Divergence between the winter run and other Central Valley stocks appears to be caused by a combination of surprisingly static evolution at Ots-2 within a context of more rapidly changing haplotype frequencies.     

Genetic Variation within and Between Domesticated Chinook Salmon, Oncorhynchus tshawytscha, Strains and their Progenitor Populations

Domesticated chinook salmon strains in British Columbia (BC), Canada are believed to have originated primarily from populations of the Big Qualicum (BQ) River and Robertson Creek (RC) on Vancouver Island in the early 1980s. The number of parental fish that gave rise to the domesticated strains and their subsequent breeding history during approximately five ensuing generations of domestication were not documented. Genetic variation at 13 microsatellite loci was examined in samples from two domesticated strains and the two progenitor populations to determine the genetic relationships among them. The domesticated strains had lower allelic diversity and tended to have lower levels of expected heterozygosity than did the BQ and RC progenitor populations. Only three alleles over all 13 loci were detected in the domesticated strains that were not present in the BQ and RC samples, whereas the progenitor strains possessed over 25 (BQ) and 43 (RC) private alleles. Genetic distance and F_ST values also indicated a closer relationship of the domesticated strains with the BQ than the RC population. One domesticated strain had a significant excess of heterozygosity compared with that expected under conditions of mutation-drift equilibrium, indicative of a recent genetic bottleneck. Genetic differentiation between the domesticated strains was as great as that distinguishing them from the progenitor populations, indicating that the genetic base of domesticated chinook salmon could be increased by hybridization. The existence of genetically distinct domesticated strains of chinook salmon in coastal BC generates the need for an evaluation of potential genetic interactions between domesticated escapees and natural spawning populations.     

Effects of natural selection on patterns of DNA sequence variation at the transferrin, somatolactin, and p53 genes within and among chinook salmon (Oncorhynchus tshawytscha) populations

This paper describes DNA sequence variation within and among four populations of chinook salmon (Oncorhynchus tshawytscha) at the transferrin, somatolactin and p53 genes. Patterns of variation among salmon species at the transferrin gene have been hypothesized to be shaped by positive natural selection for new alleles because the rate of nonsynonymous substitution is significantly greater than the rate of synonymous substitution. The twin goals of this study were to determine if the history of selection among salmon species at the transferrin gene is also reflected in patterns of intraspecific variation in chinook salmon, and to look for evidence of local adaptation at the transferrin gene by comparing patterns of nonsynonymous and synonymous variation among chinook salmon populations. The analyses presented here show that unlike patterns of variation between species, there is no evidence of greater differentiation among chinook salmon populations at nonsynonymous compared to synonymous sites. There is also no evidence of a reduction of within-species variation due to the hitchhiking effect at the transferrin gene, although in some populations nonsynonymous and synonymous derived mutations are both at higher frequencies than expected under a simple neutral model. Population size weighted selection coefficients (4Ns) that are consistent with both the inter and intraspecific data range from approximately 10 to approximately 235, and imply that between 1 and 40% of new nonsynonymous mutations at the transferrin gene have been beneficial.     

Bimodal run distribution in a northern population of sockeye salmon (Oncorhynchus nerka): life history and genetic analysis on a temporal scale

Life history variation and genetic differentiation were analysed in sockeye salmon in Klukshu River, Yukon Canada over 7 years (1994-2000). Sockeye salmon return to the Klukshu River in two distinct runs, with a small 'early run' in June-August, and a larger 'late run' in August-September. A maximum likelihood test for clusters indicated that the return frequency distribution was bimodal in all the years analysed. Life history differences (fork length, sex ratio, age at maturity, fresh- and saltwater residency times) were found between the early and late runs; however, inconsistent patterns suggest that environmental effects outweigh, or strongly interact with, genetic effects for the life history characters evaluated. Analysis of variation at eight microsatellite loci showed that the early and late runs are genetically differentiated in all years examined (exact test). FST estimates between runs within years were significantly greater than zero (range: 0.018-0.041) for all years except one (0.004). The genetic variance explained by early vs. late runs (2.27%) was twice the variance among years (1.16%) based on analysis of molecular variance. Our neighbour-joining tree showed early and late runs generally clustering separately, indicating higher gene flow among the early or late run fish across years relative to between-run gene flow. Two years did not fit the general clustering pattern; although the early and late runs in 1995 and 2000 were genetically differentiated, they clustered separately from the rest of the groups. We cannot offer a definitive explanation for these anomalies; however, an analysis of possible cryptic population structure in early and late runs indicated that at least a few fish strayed between the runs in each year, and the highest rate of mixing was in 1995 and 2000. Our data indicate that the runs are at least partially reproductively isolated as a result of temporal and/or spatial isolating mechanisms. Such reproductive isolation has important implications for conservation and management of the Klukshu sockeye salmon, and make them an evolutionarily interesting group because of parallels with incipient speciation.     

Effects of a temperature control device on nutrients, POM and plankton in the tailwaters below Shasta Lake, California

A temperature control device (TCD) was installed by the U.S. Bureau of Reclamation on Shasta Dam, California, in March 1997 for controlling downstream river temperatures. Temperature modification was required to aid recovery of the endangered winter run chinook salmon (Oncorhynchus tshawytscha) in the Sacramento River, and to minimize loss of generating capacity as a result of releasing deeper, colder water through low level outlet works to meet downstream temperature criteria. This study began two years prior to operation of the TCD, to compare pre- and post-operational changes on downstream tailwaters, including nutrients, particulate organic matter (POM) and plankton. During epilimnetic withdrawals from January to mid-June, and mid-level withdrawals through August, operation of the TCD was associated with decreases in dissolved nitrate–nitrate concentrations, localized increases in small particulate organic matter (SPOM) at Shasta tailwaters, increases of bacillariophyta (<25 m size fraction), and increases in copepod biomass. These changes can potentially influence the food base of the river and therefore fish production in the Upper Sacramento River, including the chinook salmon.     

Assessment of genetic variation between reproductive ecotypes of Klamath River steelhead reveals differentiation associated with different run-timings

Seven microsatellite DNA loci were optimized to assess genetic differentiation in coastal steelhead (Oncorhynchus mykiss irideus) sampling groups from the lower Klamath River (California, USA). Genetic relationships among three winter‐run and two summer‐run groups were investigated. The different groups displayed high levels of allelic variation. Pairwise F_ST comparisons and Nei's genetic distance supported low, yet significant, genetic differentiation between summer and winter run‐timings similar to other studies of temporal variation in salmonids. Analysis of molecular variance showed that most of the genetic variation was at the individual level (97.9%), although significant genetic variation existed between timing of runs (2.59%). Additionally, at least one locus in each group was out of Hardy–Weinberg equilibrium due to a deficiency in heterozygotes, and significant F_IS values were observed in three temporal collections. Together, these results suggest stock admixture, caused by multiple populations of origin in each sampling group, better known as the Wahlund Effect. These observations are preliminary evidence for isolation by time between Klamath River steelhead runs during distinct periods.     

Denaturing gradient gel electrophoresis (DGGE): a rapid and sensitive technique to screen nucleotide sequence variation in populations

We describe a rapid and sensitive method for the detection of nucleotide sequence variation that can be used for large-scale screening of population markers. Denaturing gradient gel electrophoresis (DGGE) detects sequence variants of amplified fragments by the differences in their melting behavior. DGGE detects most single-base substitutions when carried out on products amplified with a primer to which a GC clamp has been added. Although DGGE has been primarily used for the detection of limited numbers of single-base mutations in disease studies, it offers great potential for use in population analysis of genetic markers with greater levels of sequence variation. The methodology described was developed to identify the number and distribution of MHC class I alpha 1 alleles among chinook salmon (Oncorhynchus tshawytscha) populations. DGGE detects 28 of 31 identified alpha 1 sequences, which differ by between 1 and 16 nucleotides and a two-codon indel. By creating a network of control alleles, 22-23 of the MHC alleles can be resolved rapidly and accurately by a single gel run condition, and 27 alleles can be resolved by two gel run conditions. This techniques has been used in surveys scoring alleles from two MHC markers (class I alpha 1 and alpha 2) in 20,000 individuals of chinook and coho (O. kisutch) salmon. A single person in our laboratory now analyzes 160 salmon from one MHC locus per day with DGGE.     

A single-locus minisatellite discriminates chinook salmon (Oncorhynchus tshawytscha) populations

A knowledge of genetic structure in natural populations is often necessary for conservation and management purposes, especially in declining Pacific salmon populations. To test for genetic differentiation between nine populations of chinook salmon, Oncorhynchus tshawytscha, from south-western British Columbia, Canada, DNA was extracted from 603 fish and hybridized with a single-locus minisatellite probe. Multivariate statistical analyses of the resulting allele size data permitted successful overall population identification of 52% (individual population range: 24–78%; P < 0.005), indicating a high level of genetic differentiation among the nine populations. Two of the nine populations were further analysed using data from a second minisatellite locus. The discrimination success rate improved from 81.1% (one-locus analyses) to 90.0% (two-locus analyses), indicating the potential for greatly increased resolution gained by the addition of more loci. These results indicate that variation at minisatellite loci can be used for assessing population-level genetic structure, even with artificial gene flow.     

Ontogenetic variations in the type and size of prey consumed by juvenile coho,Oncorhynchus kisutch,and chinook,O. tshawytscha,salmon

Synopsis Stomach contents of juvenile coho,Oncorhynchus kisutch, and chinook,O. tshawytscha, salmon collected in purse seines off the coast of Washington and Oregon were examined for variations related to predator size. There was a general trend toward increasing consumption of fish with increasing body size, due mainly to the increase in northern anchovy biomass consumed by the larger salmon. Most of the major prey taxa showed significant differences among the size classes examined for both salmon species. There was a direct relationship between predator and prey size for both coho and chinook, but considerable variation was found in prey length consumed within each size class. Prey width did not provide as good a fit as prey length for either species. In general, coho consumed larger fish prey in relation to their body length than chinook but there were substantial differences by month or year of collection.     

Genetic diversity of north Okhotsk Sea chum salmon populations with natural and artificial reproduction

The variability of 32 enzyme loci was studied in chum salmon populations with different types of reproduction—natural, mixed, and artificial—in some Magadan Region rivers. Among the populations studied, the values of mean heterozygosity and allele number per locus did not differ significantly. We found evidence of definite temporal stability of the populations, and also found that their genetic variability was expressed only slightly but still remained in spite of periodic egg transplantations between rivers. Statistically significant spatial genetic differentiation of the populations accounted for 0.55 to 0.76% of the total variation and the mean inter-year differentiation accounted for 0.30% of the total. Significant temporal (seasonal) genetic subdivision was revealed in chum salmon of the Tauy River. The populations of the Okhotsk Sea coast are very similar genetically to the east Sakhalin populations. The industrial chum salmon population founded and reproduced artificially in the Kulkuty River preserves the genetic similarity of the donor Yama River chum salmon. In the industrial population, we observed a tendency toward reduction of genetic variation over time. The contribution of the Yama population to the gene pool of the Ola chum salmon, (both by natural reproduction and by farming) is small in spite of many large-scale transplantations. However, the consequences of those transplantations are revealed by means of linkage disequilibrium analysis.     

Xenoma formation during microsporidial gill disease of salmonids caused by Loma salmonae is affected by host species (Oncorhynchus tshawytscha,O. kisutch,O. mykiss) but not by salinity

Host species and salinity often affect the development of disease in aquatic species. Eighty chinook salmon Oncorhynchus tshawytscha, 80 coho salmon O. kisutch and 80 rainbow trout O. mykiss were infected with Loma salmonae. Forty of each species were reared in seawater and 40 in freshwater. The mean number of xenomas per gill filament was 8 to 33 times greater in chinook salmon than in rainbow trout (RBT). Coho salmon had a mean xenoma intensity intermediate to that of chinook salmon and RBT. In contrast to the differences between species, salinity had no significant effect on xenoma intensity in any of these host species. The onset of xenoma formation occurred at Week 5 postexposure (PE) for chinook salmon and RBT, and at Week 6 PE for coho salmon. RBT had cleared all visible branchial xenomas by Week 9 PE, whereas xenomas persisted in coho and chinook salmon at Week 9 PE. Histologically, xenomas were visible in the filament arteries of the branchial arch in chinook and coho salmon gills but were absent from RBT gills. Fewer xenomas were seen in the central venous sinusoids of RBT than in chinook and coho salmon. The lower xenoma intensity, shorter duration of infection and pathological characteristics, common to microsporidial gill disease in RBT, suggest a degree of resistance to clinical disease that is not seen in coho and chinook salmon.     

Inorganic pyrophosphatase: a new polymorphic allozyme locus in Pacific salmon

Genetic polymorphism of inorganic pyrophosphatase was investigated in 2799 individuals in four species of Pacific salmon: chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch), kokanee (O. nerka), and steelhead (O. mykiss), using horizontal starch gel electrophoresis. This enzyme system appears to be an isolocus system with electrophoretically indistinguishable allozymes encoded by two loci (PP-1,2*) expressed in retinal tissue. Mendelian inheritance was observed with a breeding study in three family crosses. Population variability in four species was characterized in 44 populations from the U.S. Pacific coast. Three alleles were found in chinook salmon; two alleles each were found in coho salmon, kokanee, and steelhead. Chinook salmon and kokanee populations differed enough with respect to PP-1,2* frequencies that this isolocus is useful for genetic stock identification in these species.     

Molecular evolution at Mhc genes in two populations of chinook salmon Oncorhynchus tshawytscha

The DNA sequences of four exons of the MHC (major histocompatibilty complex) were examined in chinook salmon ( Oncorhynchus tshawytscha ) from an interior (Nechako River) and a coastal (Harrison River) population in the Fraser River drainage of British Columbia. Mhc class I A1, A2 and A3 sequences and a class II B1 sequence were obtained by PCR from each of 16–20 salmon from each population. The class I A1 and a pair of linked A2–A3 exons were derived from two different classical salmonid class I genes, Sasa-A and Onmy-UA , respectively. Allelic variation for B1, A1 and A2 was characterized by the high levels of nonsynonymous substitution indicative of the effects of natural selection on Mhc domains that contain peptide binding regions. The number of alleles detected at each of the four exons ranged from three ( B1 ) to 22 ( A1 ), but levels of nucleotide sequence divergence at all four exons were low relative to classical mammalian Mhc genes. The nucleotide similarity among alleles ranged between 89 and 99% over all exons, and all four domains possessed only two major sequence motifs. Allelic distributions at B1, A1 and A3 confirmed the genetic distinctiveness of the Harrison and Nechako chinook salmon populations revealed in previous studies. The two major allelic motifs of B1 and A1 segregated strongly between the populations. In spite of evidence that allelic diversity at these chinook salmon Mhc exons has been generated by selection, the level and distribution of diversity in the two salmon populations strongly reflected the demographic history of the species, which has been characterized by repeated bottlenecks and isolation-by-distance in glacial refugia.     

Fecundity and egg size variation in North American Pacific salmon (Oncorhynchus)

We examined regional and latitudinal variation in fecundity and egg weight for five species of Pacific salmon ( Oncorhynchus ) along the Pacific coast of North America. Data were examined for 24 chum salmon, 15 pink salmon, 34 sockeye salmon, 44 chinook salmon, and 40 coho salmon populations from published sources, unpublished Canadian hatchery records, our own laboratory investigations, and other unpublished sources. Substantial regional variation in fecundity and egg weight was observed, with salmon on the Queen Charlotte Islands and Vancouver Island in British Columbia generally having lower fecundity and larger egg size than nearby mainland populations. The relative distance of freshwater migration to the spawning grounds generally had a marked effect on both fecundity and egg size, with populations spawning in the upper portions in the drainages of large rivers like the Fraser River in British Columbia having reduced fecundity and egg size compared with coastal spawning populations. Fecundity was generally higher and egg size generally lower in more northern populations of sockeye, chinook, and coho salmon compared with southern ones. We suggest that egg size tends to be lower in northern populations of some species as a result of increased fecundity due to their older ages at maturity and a limited amount of energy that can be expended on egg production.     

Evidence for a carrier state of infectious hematopoietic necrosis virus in chinook salmon Oncorhynchus tshawytscha.

In British Columbia, Canada, infectious hematopoietic necrosis virus (IHNV) is prevalent in wild sockeye salmon Oncorhynchus nerka and has caused disease in seawater net-pen reared Atlantic salmon Salmo salar. In this study, chinook salmon Oncorhynchus tshawytscha experimentally exposed to an isolate of IHNV found in British Columbia became carriers of the virus. When Atlantic salmon were cohabited with these virus-exposed chinook salmon, IHNV was isolated from the Atlantic salmon. Identification of chinook salmon populations that have been exposed to IHNV may be difficult, as virus isolation was successful only in fish that were concurrently infected with either Renibacterium salmoninarum or Piscirickettisia salmonis. Also, IHNV-specific antibodies were detected in only 2 of the 70 fish experimentally exposed to the virus. Two samples collected from chinook salmon exposed to IHNV while at a salt water net-pen site had a seroprevalence of 19 and 22%; however, the inconsistencies between our laboratory and field data suggest that further research is required before we can rely on serological analysis for identifying potential carrier populations. Because of the difficulty in determining the exposure status of populations of chinook salmon, especially if there is no concurrent disease, it may be prudent not to cohabit Atlantic salmon with chinook salmon on a farm if there is any possibility that the latter have been exposed to the virus.     

Indirect genetic effects underlie oxygen-limited thermal tolerance within a coastal population of chinook salmon

With global temperatures projected to surpass the limits of thermal tolerance for many species, evaluating the heritable variation underlying thermal tolerance is critical for understanding the potential for adaptation to climate change. We examined the evolutionary potential of thermal tolerance within a population of chinook salmon (Oncorhynchus tshawytscha) by conducting a full-factorial breeding design and measuring the thermal performance of cardiac function and the critical thermal maximum (CT_max) of offspring from each family. Additive genetic variation in offspring phenotype was mostly negligible, although these direct genetic effects explained 53% of the variation in resting heart rate (f_H). Conversely, maternal effects had a significant influence on resting f_H, scope for f_H, cardiac arrhythmia temperature and CT_max. These maternal effects were associated with egg size, as indicated by strong relationships between the mean egg diameter of mothers and offspring thermal tolerance. Because egg size can be highly heritable in chinook salmon, our finding indicates that the maternal effects of egg size constitute an indirect genetic effect contributing to thermal tolerance. Such indirect genetic effects could accelerate evolutionary responses to the selection imposed by rising temperatures and could contribute to the population-specific thermal tolerance that has recently been uncovered among Pacific salmon populations.     

Characterization of microsatellite loci in chinook salmon (Oncorhynchus tshawytscha) and cross‐species amplification in other salmonids

Previous studies of population genetic structure of fall‐run chinook salmon (Oncorhynchus tshawytscha) in California’s Central Valley have either not focused on or have been unable to resolve intertributary differences within the San Joaquin River basin. The authors describe the isolation, the polymerase chain reaction conditions, and characterize the cross‐species amplification of 17 microsatellite loci in six species of salmonids. Fourteen of these loci are polymorphic in fall‐run chinook from the San Joaquin River drainage. These results indicate the potential utility of microsatellite markers developed for one species, for both congenerics and species within a closely related genus.     

Diel movements of out-migrating Chinook salmon (Oncorhynchus tshawytscha) and steelhead trout (Oncorhynchus mykiss) smolts in the Sacramento/San Joaquin watershed

We used ultrasonic telemetry to describe the movement patterns of late-fall run Chinook salmon (Oncorhynchus tshawytscha) and steelhead trout (O. mykiss) smolts during their entire emigration down California’s Sacramento River, through the San Francisco Bay Estuary and into the Pacific Ocean. Yearling hatchery smolts were tagged via intracoelomic surgical implantation with coded ultrasonic tags. They were then released at four upriver locations in the Sacramento River during the winters of 2007 through 2010. Late-fall run Chinook salmon smolts exhibited a nocturnal pattern of migration after release in the upper river. This is likely because individuals remain within a confined area during the day, while they become active at night and migrate downstream. The ratio between night and day detections of Chinook salmon smolts decreased with distance traveled downriver. There was a significant preference for nocturnal migration in every reach of the river except the Estuary. In contrast, steelhead smolts, which reside upriver longer following release, exhibited a less pronounced diel pattern during their entire migration. In the middle river, Delta, and Estuary, steelhead exhibited a significant preference for daytime travel. In the ocean Chinook salmon preferred to travel at night, yet steelhead were detected on the monitors equally during the night and day. These data show that closely related Oncorhynchus species, with the same ontogenetic pattern of out-migrating as yearlings, vary in migration tactic.