A latitudinal cline in the Chinook salmon (Oncorhynchus tshawytscha) Clock gene: evidence for selection on PolyQ length variants

A critical seasonal event for anadromous Chinook salmon (Oncorhynchus tshawytscha) is the time at which adults migrate from the ocean to breed in freshwater. We investigated whether allelic variation at the circadian rhythm genes, OtsClock1a and OtsClock1b, underlies genetic control of migration timing among 42 populations in North America. We identified eight length variants of the functionally important polyglutamine repeat motif (PolyQ) of OtsClock1b while OtsClock1a PolyQ was highly conserved. We found evidence of a latitudinal cline in average allele length and frequency of the two most common OtsClock1b alleles. The shorter 335 bp allele increases in frequency with decreasing latitude while the longer 359 bp allele increases in frequency at higher latitudes. Comparison to 13 microsatellite loci showed that 335 and 359 bp deviate significantly from neutral expectations. Furthermore, a hierarchical gene diversity analysis based on OtsClock1b PolyQ variation revealed that run timing explains 40.9 per cent of the overall genetic variance among populations. By contrast, an analysis based on 13 microsatellite loci showed that run timing explains only 13.2 per cent of the overall genetic variance. Our findings suggest that length polymorphisms in OtsClock1b PolyQ may be maintained by selection and reflect an adaptation to ecological factors correlated with latitude, such as the seasonally changing day length.     

Sequencing improves our ability to study threatened migratory species: Genetic population assignment in California's Central Valley Chinook salmon

Effective conservation and management of migratory species requires accurate identification of unique populations, even as they mix along their migratory corridors. While telemetry has historically been used to study migratory animal movement and habitat use patterns, genomic tools are emerging as a superior alternative in many ways, allowing large‐scale application at reduced costs. Here, we demonstrate the usefulness of genomic resources for identifying single‐nucleotide polymorphisms (SNPs) that allow fast and accurate identification of the imperiled Chinook salmon in the Great Central Valley of California. We show that 80 well‐chosen loci, drawn from a pool of over 11,500 SNPs developed from restriction site‐associated DNA sequencing, can accurately identify Chinook salmon runs and select populations within run. No other SNP panel for Central Valley Chinook salmon has been able to achieve the high accuracy of assignment we show here. This panel will greatly improve our ability to study and manage this ecologically, economically, and socially important species and demonstrates the great utility of using genomics to study migratory species.     

Identification of multiple genetically distinct populations of Chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) in a small coastal watershed

Management and restoration planning for Pacific salmon is often characterized by efforts at broad multi-basin scales. However, finer-scale genetic and phenotypic variability may be present within individual basins and can be overlooked in such efforts, even though it may be a critical component for long-term viability. Here, we investigate Chinook salmon (Oncorhynchus tshawytscha) within the Siletz River, a small coastal watershed in Oregon, USA. Adult Chinook salmon were genotyped using neutral microsatellite markers, single nucleotide polymorphisms and “adaptive” loci, associated with temporal variation in migratory behavior in many salmon populations, to investigate genetic diversity based upon both spatial and temporal variation in migratory and reproductive behavior. Results from all three marker types identified two genetically distinct populations in the basin, corresponding to early returning fish that spawn above a waterfall, a spring-run population, and later returning fish spawning below the waterfall, a fall-run population. This finding is an important consideration for management of the species, as spring-run populations generally only have been recognized in large watersheds, and highlights the need to evaluate population structure of salmon within smaller watersheds, and thereby increase the probability of successful conservation of salmon species.     

Characterization of rainbow trout cell lines using microsatellite DNA profiling

Ten microsatellite loci (Omy27DU,Omy325(A3)UoG, OmyFGT5TUF,OmyFGT14TUF, OmyFGT15TUF,OmyFGT23TUF, Omy77DU,Ssa20.19NUIG, Ots1BML, andOne18ASC) were amplified using the polymerase chain reaction to create genetic profiles for nine cell lines (RTG-2, RTH-149,RTL-W1,RTgill-W1, RTS-11, RTS-34st, RTP-2, RTP-91E and RTP-91F) from rainbow trout(Oncorhynchus mykiss) and one cell line (CHSE-214) from Chinook salmon (O. tschawytscha). A cell line (PHL) from anon-salmonid, the Pacific herring (Clupea harengus pallasi), was included as a control. The ten loci clearly revealed the uniqueness of each cell line, except for two cell lines (RTP-91E andRTP-91F) from the same fish. RTP-91E and RTP-91F were identical at all loci except Ssa20.19NUIG. The most useful locus for demonstrating uniqueness was Ots1BML. The information was used to demonstrate that an uncharacterized rainbow trout cell line (Clone 1A)was in fact CHSE-214, illustrating the usefulness of multiplexed microsatellites for the creation of genetic profiles for salmonid cell lines and for the testing of cell line cross-contamination. This revised version was published online in August 2006 with corrections to the Cover Date.     

Discriminating coho salmon (Oncorhynchus kisutch) populations within the Fraser River, British Columbia, using microsatellite DNA markers

Three microsatellite loci were used to examine genetic variation among 16 coho salmon ( Oncorhynchus kisutch ) populations within the Fraser River drainage system, in British Columbia, Canada. Each locus was highly polymorphic with 30 alleles at the Ots 101 locus, 15 alleles at the Ots 3 locus and 38 alleles at the Ots 103 locus. Average observed heterozygosities were 86.1%, 70%, and 56.1%, respectively. With the exception of the Dunn and Lemieux River populations, Chi-square tests and F _ST values indicated that all populations had significantly different allele frequencies. Two distinct population groups within the Fraser River drainage were observed. Lower Fraser River populations were strongly differentiated from populations spawning in the upper Fraser River, which includes the Thompson River (a tributary flowing into the upper Fraser) and the portion of the Fraser River beyond the precipitous Fraser River canyon. This regional population structure may have resulted from colonization of the upper and lower Fraser River regions by different founder populations following Pleistocene glaciation, and be maintained by adaptive differences between the two groups of coho salmon. Coho salmon populations in the upper Fraser and Thompson River drainages form an evolutionarily significant unit (ESU) of importance for conservation of biodiversity in coho salmon. Microsatellite DNA loci show promise as technically simple and highly informative genetic markers for coho salmon population management.     

Characterization of a chromosomal rearrangement responsible for producing "apparent" XY-female fall-run Chinook salmon in California

Fluorescence in situ hybridization (FISH) was used to identify the X and Y chromosomes of offspring produced by normal and "apparent" XY-female fall-run Chinook salmon (Oncorhynchus tshawytscha) from California. FISH experiments were performed using probes to 2 sex-linked loci, growth hormone pseudogene (GH-Psi), and OtY1, as well as a probe to a sex-linked microsatellite (Omy7INRA). Comparison of FISH staining patterns between the offspring produced by normal and apparent XY-females revealed that the apparent XY-female examined transmitted a "Y-like" chromosome with an attenuated OtY1 and GH-Psi signal to half of its offspring. Segregation analysis of microsatellites derived from rainbow trout (Oncorhynchus mykiss) with respect to phenotypic sex was carried out for 2 normal and 2 apparent XY-female Chinook salmon families. Inheritance patterns of Omy7INRA were consistent with this locus being closely linked to GH-Psi in males and in apparent XY-females carrying the Y-like chromosome. Another microsatellite locus (Omm1077) was closely linked to the primary sex-determining locus (SEX) in males but not to GH-Psi/OtY1 in apparent XY-females. The FISH analyses suggest that apparent XY-female fall-run Chinook salmon in California are not the product of a Y chromosome to autosome translocation. Despite the combined FISH and inheritance analyses, we were unable to differentiate between 2 alternative explanations for apparent XY-females, namely, recombination of markers between the sex chromosomes, or a Y chromosome with a dysfunctional or missing sex-determining region.     

Evidence for fine-scale genetic structure and estuarine colonisation in a potential high gene flow marine goby (Pomatoschistus minutus)

Marine fish seem to experience evolutionary processes that are expected to produce genetically homogeneous populations. We have assessed genetic diversity and differentiation in 15 samples of the sand goby Pomatoschistus minutus (Pallas, 1770) (Gobiidae, Teleostei) from four major habitats within the Southern Bight of the North Sea, using seven microsatellite and 13 allozyme loci. Despite its high dispersal potential, microsatellite loci revealed a moderate level of differentiation (overall F(ST)=0.026; overall R(ST)=0.058). Both hierarchical analysis of molecular variance and multivariate analysis revealed significant differentiation (P<0.01) between estuarine, coastal and marine samples with microsatellites, but not with allozymes. Comparison among the different estimators of differentiation (F(ST) and R(ST)) pointed to possible historical events and contemporary habitat fragmentation. Samples were assigned to two breeding units in the estuary and coastal region. Despite this classification, there were indications of a complex and dynamic spatiotemporal structure, which is, most likely, determined by historical events and local oceanic currents.     

Neither philopatric nor panmictic: microsatellite and mtDNA evidence suggests lack of natal homing but limits to dispersal in Pacific lamprey

Most species with lengthy migrations display some degree of natal homing; some (e.g. migratory birds and anadromous salmonids) show spectacular feats of homing. However, studies of the sea lamprey (Petromyzon marinus) indicate that this anadromous species locates spawning habitat based on pheromonal cues from larvae rather than through philopatry. Previous genetic studies in the anadromous Pacific lamprey (Entosphenus tridentatus) have both supported and rejected the hypothesis of natal homing. To resolve this, we used nine microsatellite loci to examine the population structure in 965 Pacific lamprey from 20 locations from central British Columbia to southern California and supplemented this analysis with mitochondrial DNA restriction fragment length polymorphism analysis on a subset of 530 lamprey. Microsatellite analysis revealed (i) relatively low but often statistically significant genetic differentiation among locations (97% pairwise F(ST) values were <0.04 but 73.7% were significant); and (ii) weak but significant isolation by distance (r(2) = 0.0565, P = 0.0450) but no geographic clustering of samples. The few moderate F(ST) values involved comparisons with sites that were geographically distant or far upstream. The mtDNA analysis--although providing less resolution among sites (only 4.7%F(ST) values were significant)--was broadly consistent with the microsatellite results: (i) the southernmost site and some sites tributary to the Salish Sea were genetically distinct; and (ii) southern sites showed higher haplotype and private haplotype richness. These results are inconsistent with philopatry, suggesting that anadromous lampreys are unusual among species with long migrations, but suggest that limited dispersal at sea precludes panmixia in this species.     

Natal philopatry does not lead to population genetic differentiation in Buller's albatross (Thalassarche bulleri bulleri)

Genetic variability in the only two existing populations of Buller's albatross (Thalassarche bulleri bulleri) was assessed using six polymorphic microsatellite loci. Large biological samples were obtained from both the Snares (n = 99) and the Solander Islands (n = 27). Several measures of genetic differentiation including F(ST) and R(ST) and a principal coordinates analysis (PCO) suggest a complete absence of genetic structure between three breeding colonies on the Snares Islands, and between them and one breeding colony on the Solander Islands. Mark/recapture studies of recently banded albatross chicks on the Snares found high natal philopatry in T. b. bulleri, but the microsatellite DNA data suggest that sufficient gene flow still occurs between all four breeding colonies to maintain a genetically homogeneous population overall.     

Reproductive isolation following reintroduction of Chinook salmon with alternative life histories

We evaluated reproductive isolation of Chinook salmon (Oncorhynchus tshawytscha) life history types that have been reintroduced to northern Idaho, USA. Analysis of 1003 samples at six microsatellite loci revealed strong reproductive isolation between ocean- and stream-type Chinook salmon (fall and summer spawn timing, respectively) within the Clearwater River sub-basin (F _ST = 0.148, P < 0.00001). Very little evidence for gene flow among the two life history types was observed as assignment tests correctly assigned 99.6% of individuals in reference collections to either ocean- and steam-type Chinook salmon. Assignment of naturally reared juveniles indicated that both life history types were present with 24.1% stream-type and 75.9% ocean-type. Previous studies suggest high levels of divergence among the two life history types in natural populations, and our study verifies the persistence of reproductive isolation among types following colonization of habitat. Successful colonization of new habitat by (re)introduced species is likely influenced by diversity in life history types and this strategy has lead to naturally spawning populations in a variety of available habitats in the Clearwater River. As many populations of O. tshawytscha are listed as threatened or endangered under the U.S. Endangered Species Act, hope for recovery lies not only in effective management and habitat improvement, but adaptability of this species.     

Evidence for contrasting modes of selection at interacting globin genes in the European rabbit (Oryctolagus cuniculus)

In hybrid zones between genetically differentiated populations, variation in locus-specific rates of introgression may reflect adaptation to different environments or adaptation to different genetic backgrounds. The European rabbit, Oryctolagus cuniculus, is well-suited to studies of such hybrid zone dynamics because it is composed of two genetically divergent subspecies that hybridize in a zone of secondary contact in central Iberia. A species-wide survey of allozyme variation revealed a broad range of locus-specific divergence levels (F(ST) ranged from 0 to 0.54, mean F(ST)=0.16). Interestingly, the two loci that fell at opposite ends of the distribution of F(ST) values, haemoglobin alpha-chain (HBA) and haemoglobin beta-chain (HBB), encode interacting subunits of the haemoglobin protein. The contrasting patterns of spatial variation at these two loci could not be reconciled under a neutral model of population structure. The HBA gene exhibited higher-than-expected levels of population differentiation, consistent with a history of spatially varying selection. The HBB gene exhibited lower-than-expected levels of population differentiation, consistent with some form of spatially uniform selection. Patterns of linkage disequilibrium and allele frequency variation do not appear to fit any simple model of two-locus epistatic selection.     

Time scale matters: genetic analysis does not support adaptation‐by‐time as the mechanism for adaptive seasonal declines in kokanee reproductive life span

Seasonal declines of fitness‐related traits are often attributed to environmental effects or individual‐level decisions about reproductive timing and effort, but genetic variation may also play a role. In populations of Pacific salmon (Oncorhynchus spp.), seasonal declines in reproductive life span have been attributed to adaptation‐by‐time, in which divergent selection for different traits occurs among reproductively isolated temporal components of a population. We evaluated this hypothesis in kokanee (freshwater obligate Oncorhynchus nerka) by testing for temporal genetic structure in neutral and circadian‐linked loci. We detected no genetic differences in presumably neutral loci among kokanee with different arrival and maturation dates within a spawning season. Similarly, we detected no temporal genetic structure in OtsClock1b, Omy1009uw, or OmyFbxw11, candidate loci associated with circadian function. The genetic evidence from this study and others indicates a lack of support for adaptation‐by‐time as an important evolutionary mechanism underlying seasonal declines in reproductive life span and a need for greater consideration of other mechanisms such as time‐dependent, adaptive adjustment of reproductive effort.     

Forensic identification of endangered Chinook Salmon (Oncorhynchus tshawytscha) using a multilocus SNP assay

Upper Columbia River spring-run Chinook salmon are listed as endangered under the Endangered Species Act (ESA). Forensic genetic analyses are needed to assist the National Oceanic and Atmospheric Administration Fisheries Office of Law Enforcement (NOAA Fisheries OLE) during criminal investigations of ESA take violations. Previous genetic studies using allozyme markers have demonstrated that the spring-run and summer-run of Chinook salmon in the Upper Columbia River are genetically differentiated. Because many of the carcasses collected as forensic evidence are of compromised quality, we have developed a PCR based assay to distinguish between the spring-run and the summer-run Chinook salmon in the upper Columbia River Basin. A total of 347 Chinook salmon samples from the upper Columbia River in Washington State were surveyed for single nucleotide polymorphisms (SNPs) using PCR-RFLP at two nuclear genetic loci (Somatolactin and Cytochrome p450A), and a mitochondrial locus (Cytochrome Oxidase III/ND3). We found near fixed differences in our SNP baseline between the summer-run and the spring-run Chinook salmon in the upper Columbia River at these loci enabling us to assign individuals to the most likely population of origin with a high degree of accuracy.     

Microsatellite DNA analyses support an east-west phylogeographic split of American alligator populations

We examined the population genetic structure of American alligators (Alligator mississippiensis) sampled from 12 localities across the southeastern United States. The primary goal of this study was to determine the extent of population differentiation among alligators from four Florida lakes using eight microsatellite loci and compare the results to additional sites located at varying distances from them. Analyses of population structure revealed little differentiation (F(ST)=0.039; Rho=0.012) among the four Florida lakes, Apopka, Griffin, Orange and Woodruff, which are all located in the St. John&'s River watershed in north-central Florida. Further, there was little differentiation among these samples and samples collected from the Everglades National Park (F(ST)=0.044; Rho=0.009) and south Georgia (F(ST)=0.045; Rho=0.032). Therefore, these six samples were pooled together as a "FL/sGA group." Similarly, samples collected in the western extent of the range, Anahuac National Wildlife Refuge in Texas and Salvador Wildlife Management Area, Marsh Island Wildlife Refuge and Rockefeller Wildlife Refuge in Louisiana, also lacked population structure (F(ST)=0.024; R(ST)=0.040). These four populations were pooled into the "TX/LA group." Comparisons of these two groups with samples taken from the Santee Coastal Reserve in South Carolina and Mobile, Alabama yielded three to four times more differentiation among groups (F(ST)=0.131; Rho=0.187). These and other analyses support the hypothesis of an east-west phylogeographic split in American alligator populations and are consistent with studies of many freshwater fish and aquatic and terrestrial turtles distributed throughout this same geographic region.     

Discovery and characterization of single nucleotide polymorphisms in Chinook salmon, Oncorhynchus tshawytscha

Molecular population genetics of non-model organisms has been dominated by the use of microsatellite loci over the last two decades. The availability of extensive genomic resources for many species is contributing to a transition to the use of single nucleotide polymorphisms (SNPs) for the study of many natural populations. Here we describe the discovery of a large number of SNPs in Chinook salmon, one of the world's most important fishery species, through large-scale Sanger sequencing of expressed sequence tag (EST) regions. More than 3 Mb of sequence was collected in a survey of variation in almost 132 kb of unique genic regions, from 225 separate ESTs, in a diverse ascertainment panel of 24 salmon. This survey yielded 117 TaqMan (5' nuclease) assays, almost all from separate ESTs, which were validated in population samples from five major stocks of salmon from the three largest basins on the Pacific coast of the contiguous United States: the Sacramento, Klamath and Columbia Rivers. The proportion of these loci that was variable in each of these stocks ranged from 86.3% to 90.6% and the mean minor allele frequency ranged from 0.194 to 0.236. There was substantial differentiation between populations with these markers, with a mean F(ST) estimate of 0.107, and values for individual loci ranging from 0 to 0.592. This substantial polymorphism and population-specific differentiation indicates that these markers will be broadly useful, including for both pedigree reconstruction and genetic stock identification applications.     

Genetic identity of YOY bluefin tuna from the eastern and western Atlantic spawning areas

We used 320 young-of-the-year (YOY) specimens of the highly migratory and overfished Atlantic bluefin tuna, Thunnus thynnus, Linnaeus 1758, to evaluate the hypothesis that Atlantic bluefin tuna comprises 2 stocks with spawning grounds in the Gulf of Mexico and in the Mediterranean Sea. Significant genetic differentiation at 8 nuclear microsatellite loci (F(ST) = 0.0059, P = 0.0005) and at the mitochondrial control region (Phi(ST) = 0.0129, P = 0.0139) was detected among YOY Atlantic bluefin tuna captured on spawning grounds in the Gulf of Mexico (n = 40) versus the western (n = 255) and eastern (n = 25) basins of the Mediterranean Sea. The genetic divergence among spawning populations, combined with the extensive trans-Atlantic movements reported for juvenile and adult Atlantic bluefin tuna, indicates a high degree of spawning site fidelity. Recognition of genetically distinct populations necessitates independent management of Atlantic bluefin tuna on spawning grounds and warrants evaluation of the level of mixing of populations on feeding grounds. The genetic pattern might not have been detected unless juvenile specimens or actively spawning adults had been sampled.     

Genetic Structure of Chum Salmon (Oncorhynchus keta) Populations in the Lower Columbia River: Are Chum Salmon in Cascade Tributaries Remnant Populations?

The lower Columbia River drainage once supported a run of over a million chum salmon. By the late 1950s, the run had decreased to often a few hundred fish. With the exception of Grays River near the coast and an aggregation of chum salmon spawning in creeks and the main stem near Bonneville Dam in the Columbia Gorge, most populations were thought to be extinct. However, chum salmon consistently return in low numbers to tributaries originating in the Cascade Range: the Cowlitz, Lewis, and Washougal rivers. To assess whether Cascade spawners were strays or remnants of former populations, chum salmon from the Coastal, Cascade and Gorge ecoregional zones were characterized at 17 microsatellite loci. Significant heterogeneity in genotype distributions was detected between zones and collections formed regional groups in a neighbor-joining tree. Cascade collections had higher allelic richness and private alleles, and the Cowlitz River supported genetically divergent fall and summer runs, the only summer chum salmon run extant in the Columbia River drainage. We propose that chum salmon in the Cascade zone are remnants of original populations. We attribute the divergence between zonal groups to diverse ecological conditions in each zone, which promoted regional genetic adaptation, and to genetic drift experienced in small populations.     

Chinook and Coho salmon hybrids linked to habitat and climatic changes on Vancouver Island,British Columbia

Between 2013 and 2019, 63 presumed Chinook salmon Oncorhynchus tshawytscha sampled primarily in the Strait of Georgia (0.63% of total sample) were identified as potential Chinook–Coho (Oncorhynchus kisutch) hybrids by the presence of anomalous microsatellite genotypes. Their hybrid origin was confirmed by single nucleotide polymorphism amplification of two species‐specific amplicons. Mitochondrial DNA indicated that most of these fish resulted from the hybridization of Coho salmon females and Chinook salmon males. Although no diagnostic external features were identified, several individuals displayed an abnormal scale arrangement on the caudal peduncle. One hybrid juvenile examined for meristics exhibited a pyloric caeca count intermediate between published values for Chinook and Coho salmon. Most hybrids originated in the Cowichan River during the 2014 brood year. Their prevalence in the watershed is a naturally occurring event, likely exacerbated by prolonged low water levels which limit habitat and delay Chinook salmon spawning, in addition to the differential abundance of the parental species. This research is the first to document ongoing natural hybridization (Chinook–Coho salmon crosses) and link it to habitat and climatic changes, and includes the identification of eight F1 adults and two juvenile backcross or F2 hybrids. The potential negative impacts of hybridization, particularly in Coho salmon through potential introgression, warrant hybrid identification as an ecosystem monitoring tool within a survey program.     

Self-sustaining populations, population sinks or aggregates of strays: chum (Oncorhynchus keta) and Chinook salmon (Oncorhynchus tshawytscha) in the Wood River system, Alaska

Small populations can provide insights into ecological and evolutionary aspects of species distributions over space and time. In the Wood River system in Alaska, USA, small aggregates of Chinook (Oncorhynchus tshawytscha) and chum salmon (O. keta) spawn in an area dominated by sockeye salmon (O. nerka). Our objective was to determine whether these Chinook and chum salmon are reproductively isolated, self-sustaining populations, population sinks that produce returning adults but receive immigration, or strays from other systems that do not produce returning adults. DNA samples collected from adult chum salmon from 16 streams and Chinook salmon from four streams in the Wood River system over 3 years were compared to samples from large populations in the nearby Nushagak River system, a likely source of strays. For both species, microsatellite markers indicated no significant genetic differentiation between the two systems. Simulations of microsatellite data in a large source and a smaller sink population suggested that considerable immigration would be required to counteract the diverging effects of genetic drift and produce genetic distances as small as those observed, considering the small census sizes of the two species in the Wood River system. Thus, the Wood River system likely receives substantial immigration from neighbouring watersheds, such as the Nushagak River system, which supports highly productive runs. Although no data on population productivity in the Wood River system exist, our results suggest source-sink dynamics for the two species, a finding relevant to other systems where salmonid population sizes are limited by habitat factors.