A single generation in the wild increases fitness for descendants of hatchery-origin Chinook salmon (Oncorhynchus tshawytscha).

Reintroduction is an important tool for the recovery of imperiled species. For threatened Pacific salmonids (Oncorhynchus spp.) species, hatchery-origin (HOR) individuals from a nearby source are often used to reestablish populations in vacant, historically occupied habitat. However, this approach is challenged by the relatively low reproductive success that HOR Pacific salmonids experience when they spawn in the wild, relative to their natural-origin (NOR) counterparts. In this study, we used genetic parentage analysis to compare the reproductive success of three groups of adult Chinook salmon (Oncorhynchus tshawytscha) reintroduced above Cougar Dam on the South Fork McKenzie River, Oregon: HOR Chinook salmon from an integrated stock; first-generation, wild-born descendants (hereafter F 1s) of Chinook salmon produced at the same hatchery; and NOR Chinook salmon that are presumed to have been produced below the dam, on the mainstem McKenzie River, or elsewhere and volitionally entered a trap below Cougar Dam. We found that F 1s produced nearly as many adult offspring as NORs, and 1.8-fold more adult offspring than HORs. This result suggests that, for the South Fork McKenzie reintroduction program, a single generation in the wild increases fitness for the descendants of HOR Chinook salmon. Although these results are encouraging, care must be taken before extrapolating our results to other systems.

Implications of Large-Effect Loci for Conservation: A Review and Case Study with Pacific Salmon.

The increasing feasibility of assembling large genomic datasets for non-model species presents both opportunities and challenges for applied conservation and management. A popular theme in recent studies is the search for large-effect loci that explain substantial portions of phenotypic variance for a key trait(s). If such loci can be linked to adaptations, 2 important questions arise: 1) Should information from these loci be used to reconfigure conservation units (CUs), even if this conflicts with overall patterns of genetic differentiation? 2) How should this information be used in viability assessments of populations and larger CUs? In this review, we address these questions in the context of recent studies of Chinook salmon and steelhead (anadromous form of rainbow trout) that show strong associations between adult migration timing and specific alleles in one small genomic region. Based on the polygenic paradigm (most traits are controlled by many genes of small effect) and genetic data available at the time showing that early-migrating populations are most closely related to nearby late-migrating populations, adult migration differences in Pacific salmon and steelhead were considered to reflect diversity within CUs rather than separate CUs. Recent data, however, suggest that specific alleles are required for early migration, and that these alleles are lost in populations where conditions do not support early-migrating phenotypes. Contrasting determinations under the US Endangered Species Act and the State of California's equivalent legislation illustrate the complexities of incorporating genomics data into CU configuration decisions. Regardless how CUs are defined, viability assessments should consider that 1) early-migrating phenotypes experience disproportionate risks across large geographic areas, so it becomes important to identify early-migrating populations that can serve as reliable sources for these valuable genetic resources; and 2) genetic architecture, especially the existence of large-effect loci, can affect evolutionary potential and adaptability.

A duplicated amh is the master sex-determining gene for Sebastes rockfish in the Northwest Pacific.

Teleost fish are the most diverse group of vertebrates and provide opportunities to study the evolution of sex determination (SD) systems. Using genomic and functional analyses, we identified a male-specific duplication of anti-Müllerian hormone (amh) gene as the male master sex-determining (MSD) gene in Sebastes schlegelii. By resequencing 10 males and 10 females, we characterized a 5 kb-long fragment in HiC_Scaffold_12 as a male-specific region, which contained an amh gene (named amhy). We then demonstrated that amhy is a duplication of autosomal amh that was later translocated to the ancestral Y chromosome. amha and amhy shared high-nucleotide identity with the most significant difference being two insertions in intron 4 of amhy. Furthermore, amhy overexpression triggered female-to-male sex reversal in S. schlegelii, displaying its fundamental role in driving testis differentiation. We developed a PCR assay which successfully identified sexes in two species of northwest Pacific rockfish related to S. schlegelii. However, the PCR assay failed to distinguish the sexes in a separate clade of northeast Pacific rockfish. Our study provides new examples of amh as the MSD in fish and sheds light on the convergent evolution of amh duplication as the driving force of sex determination in different fish taxa.

Adaptive markers distinguish North and South Pacific Albacore amid low population differentiation.

Albacore (Thunnus alalunga) support an economically valuable global fishery, but surprisingly little is known about the population structure of this highly migratory species. Physical tagging data suggest that Albacore from the North and South Pacific Ocean are separate stocks, but results from previous genetic studies did not support this two stock hypothesis. In addition, observed biological differences among juveniles suggest that there may be population substructure in the North Pacific. We used double-digest restriction site-associated DNA sequencing to assess population structure among 308 Albacore caught in 12 sample areas across the Pacific Ocean (10 North, 2 South). Since Albacore are highly migratory and spawning areas are unknown, sample groups were not assumed to be equivalent to populations and the genetic data were analyzed iteratively. We tested for putatively adaptive differences among groups and for genetic variation associated with sex. Results indicated that Albacore in the North and South Pacific can be distinguished using 84 putatively adaptive loci, but not using the remaining 12,788 presumed neutral sites. However, two individuals likely represent F1 hybrids between the North and South Pacific populations, and 43 Albacore potentially exhibit lower degrees of mixed ancestry. In addition, four or five cross-hemisphere migrants were potentially identified. No genetic evidence was found for population substructure within the North Pacific, and no loci appeared to distinguish males from females. Potential functions for the putatively adaptive loci were identified, but an annotated Albacore genome is required for further exploration. Future research should try to locate spawning areas so that life history, demography, and genetic population structure can be linked and spatiotemporal patterns can be investigated.

Sex matters: Otolith shape and genomic variation in deacon rockfish (Sebastes diaconus).

Little is known about intraspecific variation within the deacon rockfish (Sebastes diaconus), a recently described species found in the northeast Pacific Ocean. We investigated population structure among fish sampled from two nearshore reefs (Siletz Reef and Seal Rock) and one offshore site (Stonewall Bank) within a <50-km2 area off the Oregon coast. Fish from the three sample sites exhibited small but statistically significant differences based on genetic variation at >15,000 neutral loci, whether analyzed independently or classified into nearshore and offshore groups. Male and females were readily distinguished using genetic data and 92 outlier loci were associated with sex, potentially indicating differential selection between males and females. Morphometric results indicated that there was significant secondary sexual dimorphism in otolith shape, but further sampling is required to disentangle potential confounding influence of age. This study is the first step toward understanding intraspecific variation within the deacon rockfish and the potential management implications. Since differentiation among the three sample sites was small, we consider the results to be suggestive of a single stock. However, future studies should evaluate how the stock is affected by differences in sex, age, and gene flow between the nearshore and offshore environments.

Characterizing neutral and adaptive genomic differentiation in a changing climate: The most northerly freshwater fish as a model.

Arctic freshwater ecosystems have been profoundly affected by climate change. Given that the Arctic charr (Salvelinus alpinus) is often the only fish species inhabiting these ecosystems, it represents a valuable model for studying the impacts of climate change on species life-history diversity and adaptability. Using a genotyping-by-sequencing approach, we identified 5,976 neutral single nucleotide polymorphisms and found evidence for reduced gene flow between allopatric morphs from two high Arctic lakes, Linne'vatn (Anadromous, Normal, and Dwarf) and Ellasjøen (Littoral and Pelagic). Within each lake, the degree of genetic differentiation ranged from low (Pelagic vs. Littoral) to moderate (Anadromous and Normal vs. Dwarf). We identified 17 highly diagnostic, putatively adaptive SNPs that differentiated the allopatric morphs. Although we found no evidence for adaptive differences between morphs within Ellasjøen, we found evidence for moderate (Anadromous vs. Normal) to high genetic differentiation (Anadromous and Normal vs. Dwarf) among morphs within Linne'vatn based on two adaptive loci. As these freshwater ecosystems become more productive, the frequency of sympatric morphs in Ellasjøen will likely shift based on foraging opportunities, whereas the propensity to migrate may decrease in Linne'vatn, increasing the frequency of the Normal morph. The Dwarf charr was the most genetically distinct group. Identifying the biological basis for small body size should elucidate the potential for increased growth and subsequent interbreeding with sympatric morphs. Overall, neutral and adaptive genomic differentiation between allopatric and some sympatric morphs suggests that the response of Arctic charr to climate change will be variable across freshwater ecosystems.

Evidence for interannual variation in genetic structure of Dungeness crab (Cancer magister) along the California Current System.

Using a combination of population- and individual-based analytical approaches, we provide a comprehensive examination of genetic connectivity of Dungeness crab (Cancer magister) along ~1,200 km of the California Current System (CCS). We sampled individuals at 33 sites in 2012 to establish a baseline of genetic diversity and hierarchal population genetic structure and then assessed interannual variability in our estimates by sampling again in 2014. Genetic diversity showed little variation among sites or across years. In 2012, we observed weak genetic differentiation among sites (FST range = -0.005-0.014) following a pattern of isolation by distance (IBD) and significantly high relatedness among individuals within nine sampling sites. In 2014, pairwise FST estimates were lower (FST range = -0.014-0.007), there was no spatial autocorrelation, and fewer sites had significant evidence of relatedness. Based on these findings, we propose that interannual variation in the physical oceanographic conditions of the CCS influences larval recruitment and thus gene flow, contributing to interannual variation in population genetic structure. Estimates of effective population size (Ne ) were large in both 2012 and 2014. Together, our results suggest that Dungeness crab in the CCS may constitute a single evolutionary population, although geographically limited dispersal results in an ephemeral signal of IBD. Furthermore, our findings demonstrate that populations of marine organisms may be susceptible to temporal changes in population genetic structure over short time periods; thus, interannual variability in population genetic measures should be considered.

Testing advances in molecular discrimination among Chinook salmon life histories: evidence from a blind test.

The application of DNA-based markers toward the task of discriminating among alternate salmon runs has evolved in accordance with ongoing genomic developments and increasingly has enabled resolution of which genetic markers associate with important life-history differences. Accurate and efficient identification of the most likely origin for salmon encountered during ocean fisheries, or at salvage from fresh water diversion and monitoring facilities, has far-reaching consequences for improving measures for management, restoration and conservation. Near-real-time provision of high-resolution identity information enables prompt response to changes in encounter rates. We thus continue to develop new tools to provide the greatest statistical power for run identification. As a proof of concept for genetic identification improvements, we conducted simulation and blind tests for 623 known-origin Chinook salmon (Oncorhynchus tshawytscha) to compare and contrast the accuracy of different population sampling baselines and microsatellite loci panels. This test included 35 microsatellite loci (1266 alleles), some known to be associated with specific coding regions of functional significance, such as the circadian rhythm cryptochrome genes, and others not known to be associated with any functional importance. The identification of fall run with unprecedented accuracy was demonstrated. Overall, the top performing panel and baseline (HMSC21) were predicted to have a success rate of 98%, but the blind-test success rate was 84%. Findings for bias or non-bias are discussed to target primary areas for further research and resolution.

Adaptive genetic markers discriminate migratory runs of Chinook salmon (Oncorhynchus tshawytscha) amid continued gene flow.

Neutral genetic markers are routinely used to define distinct units within species that warrant discrete management. Human-induced changes to gene flow however may reduce the power of such an approach. We tested the efficiency of adaptive versus neutral genetic markers in differentiating temporally divergent migratory runs of Chinook salmon (Oncorhynchus tshawytscha) amid high gene flow owing to artificial propagation and habitat alteration. We compared seven putative migration timing genes to ten microsatellite loci in delineating three migratory groups of Chinook in the Feather River, CA: offspring of fall-run hatchery broodstock that returned as adults to freshwater in fall (fall run), spring-run offspring that returned in spring (spring run), and fall-run offspring that returned in spring (FRS). We found evidence for significant differentiation between the fall and federally listed threatened spring groups based on divergence at three circadian clock genes (OtsClock1b, OmyFbxw11, and Omy1009UW), but not neutral markers. We thus demonstrate the importance of genetic marker choice in resolving complex life history types. These findings directly impact conservation management strategies and add to previous evidence from Pacific and Atlantic salmon indicating that circadian clock genes influence migration timing.

Clock polymorphism in Pacific salmon: evidence for variable selection along a latitudinal gradient.

Seasonal timing of life-history events is often under strong natural selection. The Clock gene is a central component of an endogenous circadian clock that senses changes in photoperiod (day length) and mediates seasonal behaviours. Among Pacific salmonids (Oncorhynchus spp.), seasonal timing of migration and breeding is influenced by photoperiod. To expand a study of 42 North American Chinook salmon (Oncorhynchus tshawytscha) populations, we tested whether duplicated Clock genes contribute to population differences in reproductive timing. Specifically, we examined geographical variation along a similar latitudinal gradient in the polyglutamine domain (PolyQ) of OtsClock1a and OtsClock1b among 53 populations of three species: chum (Oncorhynchus keta), coho (Oncorhynchus kisutch) and pink salmon (Oncorhynchus gorbuscha). We found evidence for variable selection on OtsClock1b that corresponds to latitudinal variation in reproductive timing among these species. We evaluated the contribution of day length and a freshwater migration index to OtsClock1b PolyQ domain variation using regression trees and found that day length at spawning explains much of the variation in OtsClock1b allele frequency among chum and Chinook, but not coho and pink salmon populations. Our findings suggest that OtsClock1b mediates seasonal adaptation and influences geographical variation in reproductive timing in some of these highly migratory species.

Clock genes localize to quantitative trait loci for stage-specific growth in juvenile coho salmon, Oncorhynchus kisutch.

In most organisms, an internal circadian clock coordinates the expression of biological rhythms and enables individuals to anticipate and respond to the seasonally changing environment. There is remarkable conservation of function in the molecular machinery underlying this circadian clock across taxa with 4 canonical proteins interacting to form an autoregulatory feedback loop: CLOCK, CRYPTOCHROME, PERIOD, and BMAL. We mapped duplicated copies of Clock and Cryptochrome in coho salmon (Oncorhynchus kisutch) to determine if these genes localize to quantitative trait loci (QTL) for hatch timing, weight, length, and growth rate measured throughout the juvenile life-history stage. We found that Cryptochrome2b mapped to a QTL region for growth (measured at 304 days post-hatching) on linkage group OKI06. The percentage of variation (PEV) explained by this QTL was 15.2%. Cryptochrome2b was also associated with a marginally nonsignificant QTL for length (measured at 395 days post-hatching). OtsClock1b mapped to a QTL region for growth rate (PEV 10.1%) and length (PEV 10.5%) on linkage group OKI24 (measured at 479 days posthatching). Neither gene localized to QTL for hatch timing or weight. Our findings indicate that the growth rate and length QTL associated with OtsClock1b and Cryptochrome2b are development stage-specific and may result from temporally differentiated gene expression patterns.

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.

Candidate loci reveal genetic differentiation between temporally divergent migratory runs of Chinook salmon (Oncorhynchus tshawytscha).

Local adaptation is a dynamic process driven by selection that can vary both in space and time. One important temporal adaptation for migratory animals is the time at which individuals return to breeding sites. Chinook salmon (Oncorhynchus tshawytscha) are excellent subjects for studying the genetic basis of temporal adaptation because their high seasonal homing fidelity promotes reproductive isolation leading to the formation of local populations across diverse environments. We tested for adaptive genetic differentiation between seasonal runs of Chinook salmon using two candidate loci; the circadian rhythm gene, OtsClock1b, and Ots515NWFSC, a microsatellite locus showing sequence identity to three salmonid genes central to reproductive development. We found significant evidence for two genetically distinct migratory runs in the Feather River, California (OtsClock1b: F(ST)=0.042, P=0.02; Ots515NWFSC: F(ST)=0.058, P=0.003). In contrast, the fall and threatened spring runs are genetically homogenous based on neutral microsatellite data (F(ST)=-0.0002). Similarly, two temporally divergent migratory runs of Chinook salmon from New Zealand are genetically differentiated based on polymorphisms in the candidate loci (OtsClock1b: F(ST)=0.083, P-value=0.001; Ots515NWFSC: F(ST)=0.095, P-value=0.000). We used an individual-based assignment method to confirm that these recently diverged populations originated from a single source in California. Tests for selective neutrality indicate that OtsClock1b and Ots515NWFSC exhibit substantial departures from neutral expectations in both systems. The large F(ST )estimates could therefore be the result of directional selection. Evidence presented here suggests that OtsClock1b and Ots515NWFSC may influence migration and spawning timing of Chinook salmon in these river systems.