Genetic-environment associations explain genetic differentiation and variation between western and eastern North Pacific rhinoceros auklet (Cerorhinca monocerata) breeding colonies.

Animals are strongly connected to the environments they live in and may become adapted to local environments. Examining genetic-environment associations of key indicator species, like seabirds, provides greater insights into the forces that drive evolution in marine systems. Here we examined a RADseq dataset of 19,213 SNPs for 99 rhinoceros auklets (Cerorhinca monocerata) from five western Pacific and 10 eastern Pacific breeding colonies. We used partial redundancy analyses to identify candidate adaptive loci and to quantify the effects of environmental variation on population genetic structure. We identified 262 candidate adaptive loci, which accounted for 3.0% of the observed genetic variation among western Pacific and eastern Pacific breeding colonies. Genetic variation was more strongly associated with pH and maximum current velocity, than maximum sea surface temperature. Genetic-environment associations explain genetic differences between western and eastern Pacific populations; however, genetic variation within the western and eastern Pacific Ocean populations appears to follow a pattern of isolation-by-distance. This study represents a first to quantify the relationship between environmental and genetic variation for this widely distributed marine species and provides greater insights into the evolutionary forces that act on marine species.

Effects of stocking at the parr stage on the reproductive fitness and genetic diversity of a wild population of Atlantic salmon (Salmo salar L.).

Captive-breeding programs are among the most adopted conservation practices to mitigate the loss of biodiversity, including genetic diversity. However, both genetic and nongenetic changes occurring in captivity can reduce the fitness of supplemented individuals, which complicate rehabilitation efforts. In the case of Atlantic salmon, the intensity of changes that occur in captivity and their impact on fitness will vary with the stocking practice adopted. In this study, we test whether salmon stocked at the parr stage have reduced reproductive success compared with their wild conspecifics and whether they contribute to increase genetic diversity in the targeted population. To do so, we use high-throughput microsatellite sequencing of 38 loci to accurately assign 2381 offspring to a comprehensive set of possible parents from a supplemented Atlantic salmon population in Québec, Canada. Captive-bred salmon stocked at the parr stage had fewer mates than their wild conspecifics, as well as a reduced relative reproductive success (RSS) compared with their wild counterparts. Nonetheless, in comparison with previous studies, stocking at the parr stage significantly improved RSS compared with salmon stocked as smolts and they displayed a reduction in reproductive success similar to salmon stocked as fry, which spend less time in captivity than parr. Moreover, supplementation of captive-bred salmon significantly contributed to increasing genetic diversity. These results should contribute to informing resource managers in determining the best stocking practice to enhance Atlantic salmon populations.

Thermal regime during parental sexual maturation, but not during offspring rearing, modulates DNA methylation in brook charr (Salvelinus fontinalis).

Epigenetic inheritance can result in plastic responses to changing environments being faithfully transmitted to offspring. However, it remains unclear how epigenetic mechanisms such as DNA methylation can contribute to multigenerational acclimation and adaptation to environmental stressors. Brook charr (Salvelinus fontinalis), an economically important salmonid, is highly sensitive to thermal stress and is of conservation concern in the context of climate change. We studied the effects of temperature during parental sexual maturation and offspring rearing on whole-genome DNA methylation in brook charr juveniles (fry). Parents were split between warm and cold temperatures during sexual maturation, mated in controlled breeding designs, then offspring from each family were split between warm (8°C) and cold (5°C) rearing environments. Using whole-genome bisulfite sequencing, we found 188 differentially methylated regions (DMRs) due to parental maturation temperature after controlling for family structure. By contrast, offspring rearing temperature had a negligible effect on offspring methylation. Stable intergenerational inheritance of DNA methylation and minimal plasticity in progeny could result in the transmission of acclimatory epigenetic states to offspring, priming them for a warming environment. Our findings have implications pertaining to the role of intergenerational epigenetic inheritance in response to ongoing climate change.

Strong Parallel Differential Gene Expression Induced by Hatchery Rearing Weakly Associated with Methylation Signals in Adult Coho Salmon (O. kisutch).

Human activities and resource exploitation led to a massive decline of wild salmonid populations, consequently, numerous conservation programs have been developed to supplement wild populations. However, many studies documented reduced fitness of hatchery-born relative to wild fish. Here, by using both RNA sequencing and Whole Genome Bisulfite Sequencing of hatchery and wild-born adult Coho salmon (Oncorhynchus kisutch) originating from two previously studied river systems, we show that early-life hatchery-rearing environment-induced significant and parallel gene expression differentiation is maintained until Coho come back to their natal river for reproduction. A total of 3,643 genes differentially expressed and 859 coexpressed genes were downregulated in parallel in hatchery-born fish from both rivers relative to their wild congeners. Among those genes, 26 displayed a significant relationship between gene expression and the median gene body methylation and 669 single CpGs displayed a significant correlation between methylation level and the associated gene expression. The link between methylation and gene expression was weak suggesting that DNA methylation is not the only player in mediating hatchery-related expression differences. Yet, significant gene expression differentiation was observed despite 18 months spent in a common environment (i.e., the sea). Finally, the differentiation is observed in parallel in two different river systems, highlighting the fact that early-life environment may account for at least some of the reduced fitness of the hatchery salmon in the wild. These results illustrate the relevance and importance of considering both epigenome and transcriptome to evaluate the costs and benefits of large-scale supplementation programs.

Environment-driven reprogramming of gamete DNA methylation occurs during maturation and is transmitted intergenerationally in Atlantic Salmon.

An epigenetic basis for transgenerational plasticity in animals is widely theorized, but convincing empirical support is limited by taxa-specific differences in the presence and role of epigenetic mechanisms. In teleost fishes, DNA methylation generally does not undergo extensive reprogramming and has been linked with environmentally induced intergenerational effects, but solely in the context of early life environmental differences. Using whole-genome bisulfite sequencing, we demonstrate that differential methylation of sperm occurs in response to captivity during the maturation of Atlantic Salmon (Salmo salar), a species of major economic and conservation significance. We show that adult captive exposure further induces differential methylation in an F1 generation that is associated with fitness-related phenotypic differences. Some genes targeted with differential methylation were consistent with genes differential methylated in other salmonid fishes experiencing early-life hatchery rearing, as well as genes under selection in domesticated species. Our results support a mechanism of transgenerational plasticity mediated by intergenerational inheritance of DNA methylation acquired late in life for salmon. To our knowledge, this is the first-time environmental variation experienced later in life has been directly demonstrated to influence gamete DNA methylation in fish.

Artificial Rearing of Atlantic Salmon Juveniles for Supportive Breeding Programs Induces Long-Term Effects on Gut Microbiota after Stocking.

In supportive breeding programs for wild salmon populations, stocked parr experience higher mortality rates than wild ones. Among other aspects of phenotype, the gut microbiota of artificially raised parr differs from that of wild parr before stocking. Early steps of microbiota ontogeny are tightly dependent upon environmental conditions, both of which exert long-term effects on host physiology. Therefore, our objective was to assess to what extent the resilience capacity of the microbiota of stocked salmon may prevent taxonomic convergence with that of their wild congeners after two months in the same natural environment. Using the 16S SSU rRNA marker gene, we tested the general hypothesis that environmental conditions during the very first steps of microbiota ontogeny imprint a permanent effect on later stages of microbiota recruitment. Our results first showed that gut microbiota composition of stocked and wild parr from the same genetic population, and sharing the same environment, was dependent on the early rearing environment. In contrast, skin microbiota in stocked individuals converged to that of wild individuals. Taxonomic composition and co-occurrence network analyses suggest an impairment of wild bacteria recruitment and a higher instability for the gut microbiota of stocked parr. This study is the first to demonstrate the long-term effect of early microbiota ontogeny in artificial rearing for natural population conservation programs, raising the need to implement microbial ecology.

Rearing environment affects the genetic architecture and plasticity of DNA methylation in Chinook salmon.

Genetic architecture and phenotypic plasticity are important considerations when studying trait variation within and among populations. Since environmental change can induce shifts in the genetic architecture and plasticity of traits, it is important to consider both genetic and environmental sources of phenotypic variation. While there is overwhelming evidence for environmental effects on phenotype, the underlying mechanisms are less clear. Variation in DNA methylation is a potential mechanism mediating environmental effects on phenotype due to its sensitivity to environmental stimuli, transgenerational inheritance, and influences on transcription. To characterize the effect of environment on methylation, we created two 6 × 6 (North Carolina II) Chinook salmon breeding crosses and reared the offspring in two environments: uniform hatchery tanks and seminatural stream channels. We sampled the fish twice during development, at the alevin (larval) and fry (juvenile) stages. We measured DNA methylation at 13 genes using a PCR-based bisulfite sequencing protocol. The genetic architecture of DNA methylation differed between rearing environments, with greater additive and nonadditive genetic variance in hatchery fish and greater maternal effects in seminatural channel fish, though gene-specific variation was evident. We observed plasticity in methylation across all assayed genes, as well as gene-specific effects at two genes in alevin and six genes in fry, indicating developmental stage-specific effects of rearing environment on methylation. Characterizing genetic and environmental influences on methylation is critical for future studies on DNA methylation as a potential mechanism for acclimation and adaptation.

Copy number variants outperform SNPs to reveal genotype-temperature association in a marine species.

Copy number variants (CNVs) are a major component of genotypic and phenotypic variation in genomes. To date, our knowledge of genotypic variation and evolution has largely been acquired by means of single nucleotide polymorphism (SNPs) analyses. Until recently, the adaptive role of structural variants (SVs) and particularly that of CNVs has been overlooked in wild populations, partly due to their challenging identification. Here, we document the usefulness of Rapture, a derived reduced-representation shotgun sequencing approach, to detect and investigate copy number variants (CNVs) alongside SNPs in American lobster (Homarus americanus) populations. We conducted a comparative study to examine the potential role of SNPs and CNVs in local adaptation by sequencing 1,141 lobsters from 21 sampling sites within the southern Gulf of St. Lawrence, which experiences the highest yearly thermal variance of the Canadian marine coastal waters. Our results demonstrated that CNVs account for higher genetic differentiation than SNP markers. Contrary to SNPs, for which no significant genetic-environment association was found, 48 CNV candidates were significantly associated with the annual variance of sea surface temperature, leading to the genetic clustering of sampling locations despite their geographic separation. Altogether, we provide a strong empirical case that CNVs putatively contribute to local adaptation in marine species and unveil stronger spatial signal of population structure than SNPs. Our study provides the means to study CNVs in nonmodel species and highlights the importance of considering structural variants alongside SNPs to enhance our understanding of ecological and evolutionary processes shaping adaptive population structure.

Transcriptional Basis of Copper-Induced Olfactory Impairment in the Sea Lamprey, a Primitive Invasive Fish.

Olfaction mediates behaviors necessary for survival and reproduction in fishes. Anthropogenic inputs of contaminants into aquatic environments, specifically copper, are known to disrupt a broad range of olfactory-mediated behaviors and can cause long-lasting damage even at low concentrations that have profound impacts on the biology of aquatic organisms. The sea lamprey (Petromyzon marinus) is a primitive fish species invasive to the North American Great Lakes that relies on olfaction to navigate during natal homing and in mate choice during reproduction. To investigate effects of copper on sea lamprey olfaction and the potential for maintenance of olfactory function during copper exposure, we exposed juvenile sea lamprey to environmentally ecologically relevant copper concentrations (0, 5, 10 and 30 µg/L) for 24 hr and characterized gene transcription response in olfactory tissue (i.e., peripheral olfactory organ and olfactory bulb) and forebrain using whole transcriptome sequencing. Copper exposure induced a pattern of positive dose-dependent transcriptional response. Expression changes primarily reflected up-regulation of genes involved in apoptosis and wound healing. Unlike higher vertebrates, genes specifically related to the olfactory senses of the sea lamprey, e.g., olfactory receptors, exhibited little transcriptional response to copper exposure, suggesting the mechanism of copper-induced olfactory impairment is through necrosis of the olfactory bulb and not copper-selective inhibition of olfactory receptors. Fully two-thirds of the differentially expressed genes at higher doses of copper have no known function and thus represent important candidates for further study of the responses to copper-induced olfactory injury. Our results shed light on the evolution of vertebrate olfactory repair mechanisms and have important implications for the conservation and management of both invasive and native populations of lamprey.

Variation in juvenile Chinook salmon (Oncorhynchus tshawytscha) transcription profiles among and within eight population crosses from British Columbia, Canada.

Phenotypic differences among populations within a species have been reported for a variety of traits, ranging from life history to physiology to gene transcription. Population-level phenotypic variation has been attributed to genetic differences resulting from genetic drift and/or local adaptation as well as environmental differences resulting from plasticity. We studied population- and family-level variation in gene transcription for 22 fitness-related genes, comprising immune, growth, metabolic, and stress processes in Chinook salmon (Oncorhynchus tshawytscha). We created hybrid Chinook salmon families from eight populations and treated them with an immune stimulus, a handling stress challenge, and held some as a no-treatment control group. Population effects, sire effects, and narrow-sense heritability (h2 ) were calculated for each candidate gene within each treatment group. We expected population to have a significant effect on gene transcription for many of our genes; however, we found a population effect for transcription at only one immune gene at rest. The limited number of significant population effects on gene transcription, combined with significant additive genetic variance within each population does not support the expectation of past strong selection pressures acting on heritable transcription profiles among populations. Instead, our results indicate that Chinook salmon likely adapt to their local environment through transcriptional plasticity rather than fixed differences. The expectation for fixed population-level differences in gene transcription at fitness-related genes, reflecting accepted models of local adaptation is high; however, comparisons among multiple populations using half-sibling breeding designs are rare. Our work fills an important gap in our growing understanding of the process of among and within-population divergence.

Chromosomal fusion and life history-associated genomic variation contribute to within-river local adaptation of Atlantic salmon.

Chromosomal inversions have been implicated in facilitating adaptation in the face of high levels of gene flow, but whether chromosomal fusions also have similar potential remains poorly understood. Atlantic salmon are usually characterized by population structure at multiple spatial scales; however, this is not the case for tributaries of the Miramichi River in North America. To resolve genetic relationships between populations in this system and the potential for known chromosomal fusions to contribute to adaptation, we genotyped 728 juvenile salmon using a 50 K SNP array. Consistent with previous work, we report extremely weak overall population structuring (Global FST  = 0.004) and failed to support hierarchical structure between the river's two main branches. We provide the first genomic characterization of a previously described polymorphic fusion between chromosomes 8 and 29. Fusion genomic characteristics included high LD, reduced heterozygosity in the fused homokaryotes, and strong divergence between the fused and the unfused rearrangement. Population structure based on fusion karyotype was five times stronger than neutral variation (FST  = 0.019), and the frequency of the fusion was associated with summer precipitation supporting a hypothesis that this rearrangement may contribute local adaptation despite weak neutral differentiation. Additionally, both outlier variation among populations and a polygenic framework for characterizing adaptive variation in relation to climate identified a 250-Kb region of chromosome 9, including the gene six6 that has previously been linked to age-at-maturity and run-timing for this species. Overall, our results indicate that adaptive processes, independent of major river branching, are more important than neutral processes for structuring these populations.

Standing genetic diversity and selection at functional gene loci are associated with differential invasion success in two non-native fish species.

Invasive species are expected to experience a unique combination of high genetic drift due to demographic factors while also experiencing strong selective pressures. The paradigm that reduced genetic diversity should limit the evolutionary potential of invasive species, and thus, their potential for range expansion has received little empirical support, possibly due to the choice of genetic markers. Our goal was to test for effects of genetic drift and selection at functional genetic markers as they relate to the invasion success of two paired invasive goby species, one widespread (successful) and one with limited range expansion (less successful). We genotyped fish using two marker types: single nucleotide polymorphisms (SNPs) in known-function, protein-coding genes and microsatellites to contrast the effects of neutral genetic processes. We identified reduced allelic variation in the invaded range for the less successful tubenose goby. SNPs putatively under selection were responsible for the observed differences in population structure between marker types for round goby (successful) but not tubenose goby (less successful). A higher proportion of functional loci experienced divergent selection for round goby, suggesting increased evolutionary potential in invaded ranges may be associated with round goby's greater invasion success. Genes involved in thermal tolerance were divergent for round goby populations but not tubenose goby, consistent with the hypothesis that invasion success for fish in temperate regions is influenced by capacity for thermal tolerance. Our results highlight the need to incorporate functional genetic markers in studies to better assess evolutionary potential for the improved conservation and management of species.

Human-mediated and natural dispersal of an invasive fish in the eastern Great Lakes.

The globally invasive Round Goby (Neogobius melanostomus) was introduced to the Great Lakes around 1990, spreading widely and becoming the dominant benthic fish in many areas. The speed and scope of this invasion is remarkable and calls into question conventional secondary spread models and scenarios. We utilized nine microsatellites to identify large-scale genetic structure in Round Goby populations in the eastern Great Lakes, and assessed the role of colonization vs. secondary transport and dispersal in developing this structure. We identified three clusters, corresponding with Lake Huron, eastern Lake Erie, and western Lake Erie plus eastern Lake Ontario, along with three highly divergent populations. Bottleneck analysis identified founder effects in two divergent populations. Regression analyses of isolation by distance and allelic richness vs. distance from the initial invasion site were consistent with limited migration. However, some populations in eastern Lake Erie and Lake Ontario showed anomalously low genetic distance from the original site of colonization, consistent with secondary transport of large numbers of individuals via ballast water. We conclude that genetic structure of Round Goby in the Great Lakes principally resulted from long-distance secondary transport via ballast water with additional movement of individual via bait buckets and natural dispersal. The success of Round Gobies represents an interesting model for colonization characterization; however, those same attributes present significant challenges for conservation and fisheries management. Current management likely prevents many new species from arriving in the Great Lakes, but fails to address the transport of species within the lakes after they arrive; this is an issue of clear and pressing importance.

Environmental and genetic determinants of transcriptional plasticity in Chinook salmon.

Variation in gene transcription is widely believed to be the mechanistic basis of phenotypically plastic traits; however, comparatively little is known about the inheritance patterns of transcriptional variation that would allow us to predict its response to selection. In addition, acclimation to different environmental conditions influences acute transcriptional responses to stress and it is unclear if these effects are heritable. To address these gaps in knowledge, we assayed levels of messenger RNA for 14 candidate genes at rest and in response to a 24-h confinement stress for 72 half-sib families of Chinook salmon reared in two different environments (hatchery and semi-natural stream channel). We observed extensive plasticity for mRNA levels of metabolic and stress response genes and demonstrated that mRNA level plasticity due to rearing environment affects mRNA level plasticity in response to stress. These effects have important implications for natural populations experiencing multiple stressors. We identified genotype-by-environment interactions for mRNA levels that were dominated by maternal effects; however, mRNA level response to challenge also exhibited a non-additive genetic basis. Our results indicate that while plasticity for mRNA levels can evolve, predicting the outcome of selection will be difficult. The inconsistency in genetic architecture among treatment groups suggests there is considerable cryptic genetic variation for gene expression.

Plasticity in gene transcription explains the differential performance of two invasive fish species.

Phenotypic plasticity buffers organisms from environmental change and is hypothesized to aid the initial establishment of nonindigenous species in novel environments and postestablishment range expansion. The genetic mechanisms that underpin phenotypically plastic traits are generally poorly characterized; however, there is strong evidence that modulation of gene transcription is an important component of these responses. Here, we use RNA sequencing to examine the transcriptional basis of temperature tolerance for round and tubenose goby, two nonindigenous fish species that differ dramatically in the extent of their Great Lakes invasions despite similar invasion dates. We used generalized linear models of read count data to compare gene transcription responses of organisms exposed to increased and decreased water temperature from those at ambient conditions. We identify greater response in the magnitude of transcriptional changes for the more successful round goby compared with the less successful tubenose goby. Round goby transcriptional responses reflect alteration of biological function consistent with adaptive responses to maintain or regain homeostatic function in other species. In contrast, tubenose goby transcription patterns indicate a response to stressful conditions, but the pattern of change in biological functions does not match those expected for a return to homeostatic status. Transcriptional plasticity plays an important role in the acute thermal tolerance for these species; however, the impaired response to stress we demonstrate in the tubenose goby may contribute to their limited invasion success relative to the round goby. Transcriptional profiling allows the simultaneous assessment of the magnitude of transcriptional response as well as the biological functions involved in the response to environmental stress and is thus a valuable approach for evaluating invasion potential.

Novel molecular approach demonstrates that turbid river plumes reduce predation mortality on larval fish.

Turbidity associated with river plumes is known to affect the search ability of visual predators and thus can drive 'top-down' impacts on prey populations in complex ecosystems; however, traditional quantification of predator-prey relationships (i.e. stomach content analysis) often fails with larval fish due to rapid digestion rates. Herein, we use novel molecular genetic methods to quantify larval yellow perch (YP) in predator stomachs in western Lake Erie to test the hypothesis that turbidity drives variation in larval predation. We characterize predator stomach content DNA to first identify YP DNA (single nucleotide polymorphism) and then quantify larval YP predation (microsatellite allele counting) in two river plumes differing in turbidity. Our results showed elevated larval YP predation in the less turbid river plume, consistent with a top-down impact of turbidity on larval survival. Our analyses highlight novel ecological hypothesis testing using the power of innovative molecular genetic approaches.

Environmental associations with gene transcription in Babine Lake rainbow trout: evidence for local adaptation.

The molecular genetic mechanisms facilitating local adaptation in salmonids continue to be poorly characterized. Gene transcription is a highly regulated step in the expression of a phenotype and it has been shown to respond to selection and thus may be one mechanism that facilitates the development of local adaptation. Advances in molecular genetic tools and an increased understanding of the functional roles of specific genes allow us to test hypotheses concerning the role of variable environments in shaping transcription at known-function candidate loci. To address these hypotheses, wild rainbow trout were collected in their first summer and subjected to metabolic and immune challenges. We assayed gene transcription at candidate loci that play a role in the molecular genetic response to these stresses, and correlated transcription with temperature data from the streams and the abundance and diversity of bacteria as characterized by massively parallel pyrosequencing. Patterns of transcriptional regulation from resting to induced levels varied among populations for both treatments. Co-inertia analysis demonstrated significant associations between resting levels of metabolic gene transcription and thermal regime (R (2) = 0.19, P = 0.013) as well as in response to challenge (R (2) = 0.39, P = 0.001) and resting state and challenged levels of cytokine gene transcription with relative abundances of bacteria (resting: R (2) = 0.25, P = 0.009, challenged: R (2) = 0.65, P = 0.001). These results show that variable environments, even within a small geographic range (<250 km), can drive divergent selection among populations for transcription of genes related to surviving stress.