Genetic assessments serve as powerful tools to evaluate the effects of anthropogenic habitat fragmentation on natural populations. In the Klamath River Basin, construction of four dams from 1912 to 1962 had a profound impact on the distribution of anadromous fishes, blocking access to over 751 river kilometers of critical habitat. To characterize the genetic diversity and connectivity among Oncorhynchus mykiss in the basin prior to the historic removal of the four dams in 2024, we genotyped 2466 samples at 193 presumably neutral and 105 putatively adaptive markers. Using complementary population genetic analyses, we found a clear division between coastal and inland O. mykiss lineages located primarily downstream and upstream of the outlet of Upper Klamath Lake, and little genetic structure associated with dams. Further, we detected distinct inland and coastal genetic lineages upstream of the lake outlet supporting the hypothesis that ancestral inland O. mykiss were secondarily invaded by a coastal lineage. We found that neutral genetic diversity was significantly greater in collections consisting primarily of anadromous O. mykiss compared to collections of adfluvial, fluvial, and resident fish while genetic diversity was significantly lower in adfluvial collections. Based on the chromosome Omy5 markers associated with anadromous/resident phenotypes, we found that anadromous and heterozygous genotypes were more prevalent downstream of the lake outlet while resident genotypes were prevalent upstream of the outlet. Based on the chromosome Omy28 markers associated with adult migration timing, we found that late-migration timing and heterozygous genotypes were numerous downstream of the lake outlet while early-migration timing genotypes were prevalent upstream of the outlet. The results of our population genetic analyses highlight the genetic diversity and structure of O. mykiss in the Klamath River Basin and will serve as a critical baseline for future assessments post dam removal.

Steelhead/rainbow trout (Oncorhynchus mykiss) is an imperilled salmonid with two main life history strategies: migrate to the ocean or remain in freshwater. Domesticated hatchery forms of this species have been stocked into almost all California waterways, possibly resulting in introgression into natural populations and altered population structure. We compared whole-genome sequence data from contemporary populations against a set of museum population samples of steelhead from the same locations that were collected prior to most hatchery stocking. We observed minimal introgression and few steelhead-hatchery trout hybrids despite a century of extensive stocking. Our historical data show signals of introgression with a sister species and indications of an early hatchery facility. Finally, we found that migration-associated haplotypes have become less frequent over time, a likely adaptation to decreased opportunities for migration. Since contemporary migration-associated haplotype frequencies have been used to guide species management, we consider this to be a rare example of shifting baseline syndrome that has been validated with historical data. We suggest cautious optimism that a century of hatchery stocking has had minimal impact on California steelhead population genetic structure, but we note that continued shifts in life history may lead to further declines in the ocean-going form of the species.

The increasing feasibility of whole-genome sequencing has been highly anticipated, promising to transform our understanding of the biology of nonmodel species. Notably, dramatic cost reductions beginning around 2007 with the advent of high-throughput sequencing inspired publications heralding the 'genomics revolution', with predictions about its future impacts. Although such predictions served as useful guideposts, value is added when statements are evaluated with the benefit of hindsight. Here, we review 10 key predictions made early in the genomics revolution, highlighting those realised while identifying challenges limiting others. We focus on predictions concerning applied aspects of genomics and examples involving salmonid species which, due to their socioeconomic and ecological significance, have been frontrunners in applications of genomics in nonmodel species. Predicted outcomes included enhanced analytical power, deeper insights into the genetic basis of phenotype and fitness variation, disease management and breeding program advancements. Although many predictions have materialised, several expectations remain unmet due to technological, analytical and knowledge barriers. Additionally, largely unforeseen advancements, including the identification and management applicability of large-effect loci, close-kin mark-recapture, environmental DNA and gene editing have added under-anticipated value. Finally, emerging innovations in artificial intelligence and bioinformatics offer promising new directions. This retrospective evaluation of the impacts of the genomic revolution offers insights into the future of genomics for nonmodel species.

Genetic methods have become an essential component of ecological investigation and conservation planning for fish and wildlife. Among these methods is the use of genetic marker data to identify individuals to populations, or stocks, of origin. More recently, methods that involve genetic pedigree reconstruction to identify relationships between individuals within populations have also become common. We present here a novel set of multiallelic microhaplotype genetic markers for Chinook salmon, which provide excellent resolution for population discrimination and relationship identification from a rapidly and economically assayed panel of markers. We show how this set of genetic markers assayed by sequencing 204 amplicons, in tandem with a reference dataset of 1636 individual samples from 17 populations, provides definitive power to identify all known lineages of Chinook salmon in California. The inclusion of genetic loci that have known associations with phenotype and that were identified as outliers in examination of whole-genome sequence data allows resolution of stocks that are not highly genetically differentiated but are phenotypically distinct and managed as such. This same set of multiallelic genetic markers has ample variation to accurately identify parent-offspring and full-sibling pairs in all California populations, including the genetically depauperate winter-run lineage. Validation of this marker panel in coastal salmon populations not previously studied with modern genetic methods also reveals novel biological insights, including the presence of a single copy of a haplotype for a phenotype that has not been documented in that part of the species range, and a clear signal of mixed ancestry for a salmon population that is on the geographic margins of the primary evolutionary lineages present in California.

Populations composed of individuals descended from multiple distinct genetic lineages often feature significant differences in phenotypic frequencies. We considered hatchery production of steelhead, the migratory anadromous form of the salmonid species Oncorhynchus mykiss, and investigated how differences among genetic lineages and environmental variation impacted life history traits. We genotyped 23,670 steelhead returning to the four California Central Valley hatcheries over 9 years from 2011 to 2019, confidently assigning parentage to 13,576 individuals to determine age and date of spawning and rates of iteroparity and repeat spawning within each year. We found steelhead from different genetic lineages showed significant differences in adult life history traits despite inhabiting similar environments. Differences between coastal and Central Valley steelhead lineages contributed to significant differences in age at return, timing of spawning, and rates of iteroparity among programs. In addition, adaptive genomic variation associated with life history development in this species varied among hatchery programs and was associated with the age of steelhead spawners only in the coastal lineage population. Environmental variation likely contributed to variations in phenotypic patterns observed over time, as our study period spanned both a marine heatwave and a serious drought in California. Our results highlight evidence of a strong genetic component underlying known phenotypic differences in life history traits between two steelhead lineages.

Advances in high-throughput sequencing and bioinformatic data processing have prompted a transition in wildlife and fisheries genetics from the use of allozymes, mtDNA, or microsatellites towards markers that are more amenable to genotyping by sequencing, increasing the amount of data obtained for a lower cost with less time-consuming techniques. Microhaplotypes are novel multi-allelic genetic markers that utilize a high-throughput genomic amplicon sequencing approach to genotype large numbers of individuals for parentage and kinship analysis and population genetic studies, including applications in monitoring and fisheries management. We describe the development of a panel of microhaplotypes for Oncorhynchus mykiss, a species of high cultural and economic importance throughout its native range and globally through introductions for aquaculture and due to its reputation as a prized sport fish among recreational fishers. The panel includes 124 loci presumed to be neutral, a marker for the sex determination locus (SdY), and 10 loci targeting previously identified adaptive genomic variants associated with important life-history traits in this species. We demonstrate that this panel provides high resolution for phylogeographic and other genetic analysis and on initial standardized reference population genetic baseline of California O. mykiss.

Steelhead/rainbow trout ( Oncorhynchus mykiss ) is an imperiled salmonid with two main life history strategies: migrate to the ocean or remain in freshwater. Domesticated hatchery forms of this species have been stocked into almost all California waterbodies, possibly resulting in introgression into natural populations and altered population structure.We compared whole-genome sequence data from contemporary populations against a set of museum population samples of steelhead from the same locations that were collected prior to most hatchery stocking.We observed minimal introgression and few steelhead-hatchery trout hybrids despite a century of extensive stocking. Our historical data show signals of introgression with a sister species and indications of an early hatchery facility. Finally, we found that migration-associated haplotypes have become less frequent over time, a likely adaptation to decreased opportunities for migration. Since contemporary migration-associated haplotype frequencies have been used to guide species management, we consider this to be a rare example of shifting baseline syndrome that has been validated with historical data.We suggest cautious optimism that a century of hatchery stocking has had minimal impact on California steelhead population genetic structure, but we note that continued shifts in life history may lead to further declines in the ocean-going form of the species.### Competing Interest StatementThe authors have declared no competing interest.

Since the first introduction from North America more than a century ago, rainbow trout (Oncorhynchus mykiss) have rapidly established self-sustaining populations in major river basins of Patagonia. Many generations later, only the freshwater resident life history is expressed in the Chubut and Negro rivers of northern Argentinian Patagonia, whereas both the resident and anadromous life histories are found in the Santa Cruz River of southern Argentina. Despite previous studies that have tried to identify the sources of these introduced populations, uncertainty still exists. Here we combined data from many single-nucleotide polymorphisms and microsatellite loci in O. mykiss populations from Argentina and North America to evaluate putative source populations, gene flow between Argentinian river basins, and genetic diversity differences between Argentinian and North American populations. We found that populations from northern and southern Patagonia are highly differentiated and have limited gene flow between them. Phylogeographic analysis also confirmed that they have separate origins, with the northern populations most closely related to the domesticated rainbow trout strains that are raised worldwide and the Santa Cruz River populations most closely related to North American populations from California and Oregon that have an anadromous component. In addition, fish with different life histories in the Santa Cruz River were found to constitute a single interbreeding population. No evidence was found of reduced genetic variation in introduced rainbow trout, suggesting multiple contributing sources. In spite of these advances in understanding, significant questions remain regarding the origins and evolution of the introduced O. mykiss in Patagonia.

Multiple studies in a range of taxa have found links between structural variants and the development of ecologically important traits. Such variants are becoming easier to find due, in large part, to the increase in the amount of genome-wide sequence data in nonmodel organisms. The salmonids (salmon, trout, and charr) are a taxonomic group with abundant genome-wide datasets due to their importance in aquaculture, fisheries, and variation in multiple ecologically important life-history traits. Previous research on rainbow trout (Oncorhynchus mykiss) has documented a large pericentric (∼55 Mb) chromosomal inversion (CI) on chromosome 5 (Omy05) and a second smaller (∼14 Mb) chromosome inversion on Omy20. While the Omy05 inversion appears to be associated with multiple adaptive traits, the inversion on Omy20 has received far less attention. In this study, we re-analyze RAD-seq and amplicon data from several populations of rainbow trout (O. mykiss) to better document the structure and geographic distribution of variation in the Omy20 CI. Moreover, we utilize phylogenomic techniques to characterize both the age- and the protein-coding gene content of the Omy20 CI. We find that the age of the Omy20 inversion dates to the early stages of O. mykiss speciation and predates the Omy05 inversion by ∼450,000 years. The 2 CIs differ further in terms of the frequency of the homokaryotypes. While both forms of the Omy05 CI are found across the eastern Pacific, the ancestral version of the Omy20 CI is restricted to the southern portion of the species range in California. Furthermore, the Omy20 inverted haplotype is comparable in genetic diversity to the ancestral form, whereas derived CIs typically show substantially reduced genetic diversity. These data contribute to our understanding of the age and distribution of a large CI in rainbow trout and provide a framework for researchers looking to document CIs in other nonmodel species.

Life‐history variation is the raw material of adaptation, and understanding its genetic and environmental underpinnings is key to designing effective conservation strategies. We used large‐scale genetic pedigree reconstruction of anadromous steelhead trout (Oncorhynchus mykiss) from the Russian River, CA, USA, to elucidate sex‐specific patterns of life‐history traits and their heritability. SNP data from adults returning from sea over a 14‐year period were used to identify 13,474 parent–offspring trios. These pedigrees were used to determine age structure, size distributions and family sizes for these fish, as well as to estimate the heritability of two key life‐history traits, spawn date and age at maturity (first reproduction). Spawn date was highly heritable (h2 = 0.73) and had a cross‐sex genetic correlation near unity. We provide the first estimate of heritability for age at maturity in ocean‐going fish from this species and found it to be highly heritable (h2 from 0.29 to 0.62, depending on sex and method), with a much lower genetic correlation across sexes. We also evaluated genotypes at a migration‐associated inversion polymorphism and found sex‐specific correlations with age at maturity. The significant heritability of these two key reproductive traits in these imperiled fish, and their patterns of inheritance in the two sexes, is consistent with predictions of both natural and sexually antagonistic selection (sexes experience opposing selection pressures). This emphasizes the importance of anthropogenic factors, including hatchery practices and ecosystem modifications, in shaping the fitness of this species, thus providing important guidance for management and conservation efforts.

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.

The expression of anadromy in partially migratory salmonid populations is influenced by sex-specific interactions among an individual’s genotype, condition, and environment, but genotype–phenotype relationships prior to the expression of migratory type are poorly understood. We examined whether juvenile growth and condition differed with respect to sex and a migration-associated genomic region (Omy05) in a coastal California population of steelhead and rainbow trout (Oncorhynchus mykiss). Sex and Omy05 genotype had additive effects on annual growth from age 0+ to age 1+ (∼6–18 months old), with higher growth in males and individuals with copies of the rearranged–resident haplotype. Condition at age 1+ increased with the number of copies of the rearranged–resident haplotype, with similar support for additive or interactive effects with sex; additive effects suggested that genotype differences occurred in both sexes and also that condition was slightly higher in males, while interactive effects suggested that the genotype differences occurred only in males. Our results indicate that phenotypic differences related to sex and Omy05 genotype occur well in advance of anadromous migration or freshwater maturation in this southern O. mykiss population.

Genetic changes underlying adaptation vary greatly in terms of complexity and, within the same species, genetic responses to similar selective pressures may or may not be the same. We examine both complex (supergene) and simple (SNP) genetic variants occurring in populations of rainbow trout (Oncorhynchus mykiss) independently isolated from ocean access and compared them to each other and to an anadromous below-barrier population representing their ancestral source to search for signatures of both parallel and nonparallel adaptation. All landlocked populations displayed an increased frequency of a large inversion on chromosome Omy05, while 3 of the 4 populations exhibited elevated frequencies of another inversion located on chromosome Omy20. In addition, we identified numerous regions outside these 2 inversions that also show significant shifts in allele frequencies consistent with adaptive evolution. However, there was little concordance among above-barrier populations in these specific genomic regions under selection. In part, the lack of concordance appears to arise from ancestral autopolyploidy in rainbow trout that provides duplicate genomic regions of similar functional composition for selection to act upon. Thus, while selection acting on landlocked populations universally favors the resident ecotype, outside of the major chromosomal inversions, the resulting genetic changes are largely distinct among populations. Our results indicate that selection on standing genetic variation is likely the primary mode of rapid adaptation, and that both supergene complexes and individual loci contribute to adaptive evolution, further highlighting the diversity of adaptive genomic variation involved in complex phenotypic evolution.

Straying has been difficult to study directly in natural steelhead Oncorhynchus mykiss populations. We analyzed an opportunistic sample of seven adult steelhead from a small basin on the Big Sur coast of California to determine their life history traits, including whether they were strays. Otolith natural tags (Sr-87/Sr-86) and genetic stock identification indicated that all seven adults were strays from at least six different sources. Three adults strayed from nearby streams (<72 km) on the Big Sur coast, while three strayed from distant sources, including the Klamath River (680 km to the north); the source of one stray could not be identified. Six strays were progeny of steelhead mothers, but one was the offspring of a nonanadromous mother. Six were female and one was male, and all that could be genotyped were homozygous (n = 4) or heterozygous (n = 2) for the anadromy-associated haplotype in a migration-associated genomic region. While the opportunistic nature and small size of the sample prevents us from inferring the rate of straying into the basin, our study nonetheless demonstrates that steelhead may stray across greater distances than generally has been appreciated and that nonanadromous females can produce anadromous offspring that stray, thus providing connectivity among basins.

Differentiation between ecotypes is usually presumed to be complex and polygenic. Seasonal patterns of life history in salmon are used to categorize them into ecotypes, which are often considered "distinct" animals. Using whole-genome sequencing and tribal fishery sampling of Chinook salmon, we show that a single, small genomic region is nearly perfectly associated with spawning migration timing but not with adiposity or sexual maturity, traits long perceived as central to salmon ecotypes. Distinct migration timing does not prevent interbreeding between ecotypes, which are the result of a simple, ancient polymorphism segregating within a diverse population. Our finding that a complex migratory phenotype results from a single gene region will facilitate conservation and restoration of this iconic fish.

Whole-genome duplications (WGDs) have occurred repeatedly and broadly throughout the evolutionary history of eukaryotes. However, the effects of WGD on genome function and evolution remain unclear. The salmonid WGD that occurred approximately 88 million years ago presents an excellent opportunity for studying the effects of WGD as ∼10-15% of each salmonid genome still exhibits tetrasomic inheritance. Herein, we utilized the rainbow trout (Oncorhynchus mykiss) genome assembly and brain transcriptome data to examine the fate of gene pairs (ohnologs) following the salmonid whole-genome duplication. We find higher sequence identity between ohnologs located within known tetrasomic regions than between ohnologs found in disomic regions, and that tetrasomically inherited ohnologs showed greater similarity in patterns of gene expression and per ohnolog were lower expressed, than disomically inherited ohnologs. Enrichment testing for Gene Ontology terms identified 49 over-represented terms in tetrasomically inherited ohnologs compared to disomic ohnologs. However, why these ohnologs are retained as tetrasomic is difficult to answer. It could be that we have identified salmonid specific "dangerous duplicates", that is, genes that cannot take on new roles following WGD. Alternatively, there may be adaptive advantages for retaining genes as functional duplicates in tetrasomic regions, as presumably, movement of these genes into disomic regions would affect both their sequence identity and their gene expression patterns.

Males and females often differ in their fitness optima for shared traits that have a shared genetic basis, leading to sexual conflict. Morphologically differentiated sex chromosomes can resolve this conflict and protect sexually antagonistic variation, but they accumulate deleterious mutations. However, how sexual conflict is resolved in species that lack differentiated sex chromosomes is largely unknown. Here we present a chromosome-anchored genome assembly for rainbow trout (Oncorhynchus mykiss) and characterize a 55-Mb double-inversion supergene that mediates sex-specific migratory tendency through sex-dependent dominance reversal, an alternative mechanism for resolving sexual conflict. The double inversion contains key photosensory, circadian rhythm, adiposity and sex-related genes and displays a latitudinal frequency cline, indicating environmentally dependent selection. Our results show sex-dependent dominance reversal across a large autosomal supergene, a mechanism for sexual conflict resolution capable of protecting sexually antagonistic variation while avoiding the homozygous lethality and deleterious mutations associated with typical heteromorphic sex chromosomes.

The construction of dams and water diversions has severely limited access to spawning habitat for anadromous fishes. To mitigate for these impacts, hatchery programs rear and release millions of juvenile salmonids, including steelhead (anadromous Oncorhynchus mykiss). These programs sometimes use non-indigenous broodstock sources that may have negative effects on wild populations. In California, however, only one anadromous fish hatchery program currently uses non-native broodstock: the steelhead program at Nimbus Fish Hatchery on the American River, a tributary of the Sacramento River in the California Central Valley. The goal of this study was to determine if potentially appropriate sources of broodstock for the Nimbus Hatchery steelhead program exist in the Upper American River, above Nimbus and Folsom dams. We show that all Upper American River O. mykiss sampled are closely related to other populations in the Central Valley steelhead Distinct Population Segment, with limited introgression from out-of-...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.