Microplastic exposure is associated with epigenomic effects in the model organism Pimephales promelas (fathead minnow).

Microplastics have evolutionary and ecological impacts across species, affecting organisms' development, reproduction, and behavior along with contributing to genotoxicity and stress. As plastic pollution is increasing and ubiquitous, gaining a better understanding of organismal responses to microplastics is necessary. Epigenetic processes such as DNA methylation are heritable forms of molecular regulation influenced by environmental conditions. Therefore, determining such epigenetic responses to microplastics will reveal potential chronic consequences of this environmental pollutant. We performed an experiment across two generations of fathead minnows (Pimephales promelas) to elucidate transgenerational epigenetic effects of microplastic exposure. We exposed the first generation of fish to four different treatments of microplastics: two concentrations of each of pre-consumer polyethylene (PE) and PE collected from Lake Ontario. We then raised the first filial generation with no microplastic exposure. We used enzymatic methylation sequencing on adult liver tissue and homogenized larvae to evaluate DNA methylation differences among treatments, sexes, and generations. Our findings show the origin of the plastic had a larger effect in female minnows whereas the effect of concentration was stronger in the males. We also observed transgenerational effects, highlighting a mechanism in which parents can pass on the effects of microplastic exposure to their offspring. Many of the genes found within differentially methylated regions in our analyses are known to interact with estrogenic chemicals associated with plastic and are related to metabolism. This study highlights the persistent and potentially serious impacts of microplastic pollution on gene regulation in freshwater systems.

Integrating otolith and genetic tools to reveal intraspecific biodiversity in a highly impacted salmon population.

Intraspecific biodiversity is vital for species persistence in an increasingly volatile world. By embracing methods that integrate information at different spatiotemporal scales, we can directly monitor and reconstruct changes in intraspecific biodiversity. Here we combined genetics and otolith biochronologies to describe the genotypic and phenotypic diversity of Chinook salmon (Oncorhynchus tshawytscha) in the Yuba River, California, comparing cohorts that experienced a range of hydroclimatic conditions. Yuba River salmon have been heavily impacted by habitat loss and degradation, and large influxes of unmarked hatchery fish each year have led to concern about introgression and uncertainty around the viability of its wild populations, particularly the rarer spring-run salmon. Otolith strontium isotopes showed that Yuba River origin fish represented, on average, 42% (range 7%-73%) of spawners across six return years (2009-2011, 2018-2020), with large interannual variability. The remainder of adult Chinook salmon in the river were primarily strays from the nearby Feather River hatchery, and since 2018 from the Mokelumne River hatchery. Among the Yuba-origin spawners, on average, 30% (range 14%-50%) exhibited the spring-run genotype. The Yuba-origin fish also displayed a variety of outmigration phenotypes that differed in the timing and size at which they left the Yuba river. Early-migrating fry dominated the returns (mean 59%, range 33%-89%), and their contribution rates were negatively correlated with freshwater flows. It is unlikely that fry survival rates are elevated during droughts, suggesting that this trend reflects disproportionately low survival of larger later migrating parr, smolts, and yearlings along the migratory corridor in drier years. Otolith daily increments indicated generally faster growth rates in non-natal habitats, emphasizing the importance of continuing upstream restoration efforts to improve in-river growing conditions. Together, these findings show that, despite a long history of habitat degradation and hatchery introgression, the Yuba River maintains intraspecific biodiversity that should be taken into account in future management, restoration, and reintroduction plans. The finding that genotypic spring-run are reproducing, surviving, and returning to the Yuba River every year suggests that re-establishment of an independent population is possible, although hatchery-wild interactions would need to be carefully considered. Integrating methods is critical to monitor changes in key genetic, physiological, and behavioral traits to assess population viability and resilience.

Genetic diversity goals and targets have improved, but remain insufficient for clear implementation of the post-2020 global biodiversity framework.

Genetic diversity among and within populations of all species is necessary for people and nature to survive and thrive in a changing world. Over the past three years, commitments for conserving genetic diversity have become more ambitious and specific under the Convention on Biological Diversity's (CBD) draft post-2020 global biodiversity framework (GBF). This Perspective article comments on how goals and targets of the GBF have evolved, the improvements that are still needed, lessons learned from this process, and connections between goals and targets and the actions and reporting that will be needed to maintain, protect, manage and monitor genetic diversity. It is possible and necessary that the GBF strives to maintain genetic diversity within and among populations of all species, to restore genetic connectivity, and to develop national genetic conservation strategies, and to report on these using proposed, feasible indicators.

Linking gene expression patterns with survival studies elucidates adaptive potential in changing environments.

A major goal in ecology, evolution and conservation biology is understanding how species adapt to changing conditions and using that information to improve conservation actions. Primary to advancing our understanding of adaptation and adaptive potential is determining the causes and consequences of the genetic and phenotypic intraspecific variation that allows for adaptation. However, few studies have been able to link the variation present in molecular process under differing conditions to intraspecific variation in survival. In a From the Cover article in this issue of Molecular Ecology, Harder et al explore the relationship between molecular process and survival to understand the adaptive variation underlying tolerance to low thiamine (vitamin B1, an essential micronutrient) conditions in Atlantic salmon (Salmo salar). Thiamine is required for metabolic functioning, including energy production and nervous and cardiovascular system functioning. By combining controlled breeding and phenotyping with a survival study and transcriptomics, the authors are able to quantify among-family differences in survival under low thiamine conditions. They find wide variation in survival among families, and this survival is linked to differences in gene expression patterns. Their study elucidates the importance of combining different data types to characterize within-population phenotypic variation and understand how that variation may lead to genetic adaptation under stressful conditions.

Migration-related phenotypic divergence is associated with epigenetic modifications in rainbow trout.

Migration is essential for the reproduction and survival of many animals, yet little is understood about its underlying molecular mechanisms. We used the salmonid Oncorhynchus mykiss to gain mechanistic insight into smoltification, which is a morphological, physiological and behavioural transition undertaken by juveniles in preparation for seaward migration. O. mykiss is experimentally tractable and displays intra- and interpopulation variation in migration propensity. Migratory individuals can produce nonmigratory progeny and vice versa, indicating a high degree of phenotypic plasticity. One potential way that phenotypic plasticity might be linked to variation in migration-related life history tactics is through epigenetic regulation of gene expression. To explore this, we quantitatively measured genome-scale DNA methylation in fin tissue using reduced representation bisulphite sequencing of F2 siblings produced from a cross between steelhead (migratory) and rainbow trout (nonmigratory) lines. We identified 57 differentially methylated regions (DMRs) between smolt and resident O. mykiss juveniles. DMRs were high in magnitude, with up to 62% differential methylation between life history types, and over half of the gene-associated DMRs were in transcriptional regulatory regions. Many of the DMRs encode proteins with activity relevant to migration-related transitions (e.g. circadian rhythm pathway, nervous system development, protein kinase activity). This study provides the first evidence of a relationship between epigenetic variation and life history divergence associated with migration-related traits in any species.

Genomics and 20 years of sampling reveal phenotypic differences between subpopulations of outmigrating Central Valley Chinook salmon.

Intraspecific diversity plays a critical role in the resilience of Chinook salmon populations. California's Central Valley (CV) historically hosted one of the most diverse population complexes of Chinook salmon in the world. However, anthropogenic factors have dramatically decreased this diversity, with severe consequences for population resilience. Here we use next generation sequencing and an archive of thousands of tissue samples collected across two decades during the juvenile outmigration to evaluate phenotypic diversity between and within populations of CV Chinook salmon. To account for highly heterogeneous sample qualities in the archive dataset, we develop and test an approach for population and subpopulation assignments of CV Chinook salmon that allows inclusion of relatively low-quality samples while controlling error rates. We find significantly distinct outmigration timing and body size distributions for each population and subpopulation. Within the archive dataset, spring run individuals that assigned to the Mill and Deer Creeks subpopulation exhibited an earlier and broader outmigration distribution as well as larger body sizes than individuals that assigned to the Butte Creek subpopulation. Within the fall run population, individuals that assigned to the late-fall run subpopulation also exhibited an earlier and broader outmigration distribution and larger body sizes than other fall run fish in our dataset. These results highlight the importance of distinct subpopulations for maintaining remaining diversity in CV Chinook salmon, and demonstrates the power of genomics-based population assignments to aid the study and management of intraspecific diversity.

Remnant salmon life history diversity rediscovered in a highly compressed habitat.

Chinook salmon (Oncorhynchus tshawytscha) display remarkable life history diversity, underpinning their ability to adapt to environmental change. Maintaining life history diversity is vital to the resilience and stability of Chinook salmon metapopulations, particularly under changing climates. However, the conditions that promote life history diversity are rapidly disappearing, as anthropogenic forces promote homogenization of habitats and genetic lineages. In this study, we use the highly modified Yuba River in California to understand if distinct genetic lineages and life histories still exist, despite reductions in spawning habitat and hatchery practices that have promoted introgression. There is currently a concerted effort to protect federally listed Central Valley spring-run Chinook salmon populations, given that few wild populations still exist. Despite this, we lack a comprehensive understanding of the genetic and life history diversity of Chinook salmon present in the Yuba River. To understand this diversity, we collected migration timing data and GREB1L genotypes from hook-and-line, acoustic tagging, and carcass surveys of Chinook salmon in the Yuba River between 2009 and 2011. Variation in the GREB1L region of the genome is tightly linked with run timing in Chinook salmon throughout their range, but the relationship between this variation and entry on spawning grounds is little explored in California's Central Valley. We found that the date Chinook salmon crossed the lowest barrier to Yuba River spawning habitat (Daguerre Point Dam) was tightly correlated with their GREB1L genotype. Importantly, our study confirms that ESA-listed spring-run Chinook salmon are spawning in the Yuba River, promoting a portfolio of life history and genetic diversity, despite the highly compressed habitat. This work highlights the need to identify and protect this life history diversity, especially in heavily impacted systems, to maintain healthy Chinook salmon metapopulations. Without protection, we run the risk of losing the last vestiges of important genetic variation.

Graduate student's guide to necessary skills for nonacademic conservation careers.

Graduate education programs in conservation science generally focus on disciplinary training and discipline-specific research skills. However, nonacademic conservation professionals often require an additional suite of skills. This discrepancy between academic training and professional needs can make it difficult for graduate students to identify the skills and experiences that will best prepare them for the conservation job market. We analyzed job advertisements for conservation-science positions and interviewed conservation professionals with experience hiring early-career conservation scientists to determine what skills employers of conservation professionals seek; whether the relative importance of skills varies by job sector (government, nonprofit, and private); and how graduate students interested in careers in conservation science might signal competency in key skills to potential employers. In job advertisements, disciplinary, interpersonal, and project-management skills were in the top 5 skills mentioned across all job sectors. Employers' needs for additional skills, like program leadership, conflict resolution and negotiation, and technical and information technology skills, varied across sectors. Our interview results demonstrated that some skills are best signaled to employers via experiences obtained outside thesis or dissertation work. Our findings suggest that graduate students who wish to be competitive in the conservation job market can benefit by gaining skills identified as important to the job sector in which they hope to work and should not necessarily expect to be competent in these skills simply by completing their chosen degree path.

A new genomic resource to enable standardized surveys of SNPs across the native range of brook trout (Salvelinus fontinalis).

Understanding how genetic diversity is distributed across spatiotemporal scales in species of conservation or management concern is critical for identifying large-scale mechanisms affecting local conservation status and implementing large-scale biodiversity monitoring programmes. However, cross-scale surveys of genetic diversity are often impractical within single studies, and combining datasets to increase spatiotemporal coverage is frequently impeded by using different sets of molecular markers. Recently developed molecular tools make surveys based on standardized single-nucleotide polymorphism (SNP) panels more feasible than ever, but require existing genomic information. Here, we conduct the first survey of genome-wide SNPs across the native range of brook trout (Salvelinus fontinalis), a cold-adapted species that has been the focus of considerable conservation and management effort across eastern North America. Our dataset can be leveraged to easily design SNP panels that allow datasets to be combined for large-scale analyses. We performed restriction site-associated DNA sequencing for wild brook trout from 82 locations spanning much of the native range and domestic brook trout from 24 hatchery strains used in stocking efforts. We identified over 24,000 SNPs distributed throughout the brook trout genome. We explored the ability of these SNPs to resolve relationships across spatial scales, including population structure and hatchery admixture. Our dataset captures a wide spectrum of genetic diversity in native brook trout, offering a valuable resource for developing SNP panels. We highlight potential applications of this resource with the goal of increasing the integration of genomic information into decision-making for brook trout and other species of conservation or management concern.

Rapid CRISPR-Cas13a genetic identification enables new opportunities for listed Chinook salmon management.

Accurate taxonomic identification is foundational for effective species monitoring and management. When visual identifications are infeasible or inaccurate, genetic approaches provide a reliable alternative. However, these approaches are sometimes less viable (e.g., need for near real-time results, remote locations, funding concerns, molecular inexperience). In these situations, CRISPR-based genetic tools can fill an unoccupied niche between real-time, inexpensive, but error-prone visual identification and more expensive or time-consuming, but accurate genetic identification for taxonomic units that are difficult or impossible to visually identify. Herein, we use genomic data to develop CRISPR-based SHERLOCK assays capable of rapidly (<1 h), accurately (94%-98% concordance between phenotypic and genotypic assignments), and sensitively (detects 1-10 DNA copies/reaction) distinguishing ESA-listed Chinook salmon runs (winter- and spring-run) from each other and from unlisted runs (fall- and late fall-run) in California's Central Valley. The assays can be field deployable with minimally invasive mucus swabbing negating the need for DNA extraction (decreasing costs and labour), minimal and inexpensive equipment needs, and minimal training to conduct following assay development. This study provides a powerful genetic approach for a species of conservation concern that benefits from near real-time management decision-making but also serves as a precedent for transforming how conservation scientists and managers view genetic identification going forward. Once developed, CRISPR-based tools can provide accurate, sensitive, and rapid results, potentially without the prohibitive need for expensive specialty equipment or extensive molecular training. Further adoption of this technology will have widespread value for the monitoring and protection of our natural resources.

Attack of the PCR clones: Rates of clonality have little effect on RAD-seq genotype calls.

Interpretation of high-throughput sequence data requires an understanding of how decisions made during bioinformatic data processing can influence results. One source of bias that is often cited is PCR clones (or PCR duplicates). PCR clones are common in restriction site-associated sequencing (RAD-seq) data sets, which are increasingly being used for molecular ecology. To determine the influence PCR clones and the bioinformatic handling of clones have on genotyping, we evaluate four RAD-seq data sets. Data sets were compared before and after clones were removed to estimate the number of clones present in RAD-seq data, quantify how often the presence of clones in a data set causes genotype calls to change compared to when clones were removed, investigate the mechanisms that lead to genotype call changes and test whether clones bias heterozygosity estimates. Our RAD-seq data sets contained 30%-60% PCR clones, but 95% of RAD-tags had five or fewer clones. Relatively few genotypes changed once clones were removed (5%-10%), and the vast majority of these changes (98%) were associated with genotypes switching from a called to no-call state or vice versa. PCR clones had a larger influence on genotype calls in individuals with low read depth but appeared to influence genotype calls at all loci similarly. Removal of PCR clones reduced the number of called genotypes by 2% but had almost no influence on estimates of heterozygosity. As such, while steps should be taken to limit PCR clones during library preparation, PCR clones are likely not a substantial source of bias for most RAD-seq studies.

Evaluating Bioinformatic Pipeline Performance for Forensic Microbiome Analysis*,†,‡.

Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories. These differences can potentially affect microbial community composition and downstream analyses. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, varying minimum library size or minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that three open-source bioinformatic pipelines, MG-RAST, mothur, and QIIME2, had significant differences in relative abundance, alpha-diversity, and beta-diversity, despite the same input data. Increasing minimum library size and sample size increased the number of low-abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice affect results in important ways. Given the growing potential application of forensic microbiology to the criminal justice system, continued research on standardizing computational methodology will be important for downstream applications.

Dysbiosis in the Dead: Human Postmortem Microbiome Beta-Dispersion as an Indicator of Manner and Cause of Death.

The postmortem microbiome plays an important functional role in host decomposition after death. Postmortem microbiome community successional patterns are specific to body site, with a significant shift in composition 48 h after death. While the postmortem microbiome has important forensic applications for postmortem interval estimation, it also has the potential to aid in manner of death (MOD) and cause of death (COD) determination as a reflection of antemortem health status. To further explore this association, we tested beta-dispersion, or the variability of microbiomes within the context of the "Anna Karenina Principle" (AKP). The foundational principle of AKP is that stressors affect microbiomes in unpredictable ways, which increases community beta-dispersion. We hypothesized that cases with identified M/CODs would have differential community beta-dispersion that reflected antemortem conditions, specifically that cardiovascular disease and/or natural deaths would have higher beta-dispersion compared to other deaths (e.g., accidents, drug-related deaths). Using a published microbiome data set of 188 postmortem cases (five body sites per case) collected during routine autopsy in Wayne County (Detroit), MI, we modeled beta-dispersion to test for M/COD associations a priori. Logistic regression models of beta-dispersion and case demographic data were used to classify M/COD. We demonstrated that beta-dispersion, along with case demographic data, could distinguish among M/COD - especially cardiovascular disease and drug related deaths, which were correctly classified in 79% of cases. Binary logistic regression models had higher correct classifications than multinomial logistic regression models, but changing the defined microbial community (e.g., full vs. non-core communities) used to calculate beta-dispersion overall did not improve model classification or M/COD. Furthermore, we tested our analytic approach on a case study that predicted suicides from other deaths, as well as distinguishing MOD (e.g., homicides vs. suicides) within COD (e.g., gunshot wound). We propose an analytical workflow that combines postmortem microbiome indicator taxa, beta-dispersion, and case demographic data for predicting MOD and COD classifications. Overall, we provide further evidence the postmortem microbiome is linked to the host's antemortem health condition(s), while also demonstrating the potential utility of including beta-dispersion (a non-taxon dependent approach) coupled with case demographic data for death determination.

The future is now: Amplicon sequencing and sequence capture usher in the conservation genomics era.

The genomics revolution has initiated a new era of population genetics where genome-wide data are frequently used to understand complex patterns of population structure and selection. However, the application of genomic tools to inform management and conservation has been somewhat rare outside a few well studied species. Fortunately, two recently developed approaches, amplicon sequencing and sequence capture, have the potential to significantly advance the field of conservation genomics. Here, amplicon sequencing refers to highly multiplexed PCR followed by high-throughput sequencing (e.g., GTseq), and sequence capture refers to using capture probes to isolate loci from reduced-representation libraries (e.g., Rapture). Both approaches allow sequencing of thousands of individuals at relatively low costs, do not require any specialized equipment for library preparation, and generate data that can be analyzed without sophisticated computational infrastructure. Here, we discuss the advantages and disadvantages of each method and provide a decision framework for geneticists who are looking to integrate these methods into their research programme. While it will always be important to consider the specifics of the biological question and system, we believe that amplicon sequencing is best suited for projects aiming to genotype <500 loci on many individuals (>1,500) or for species where continued monitoring is anticipated (e.g., long-term pedigrees). Sequence capture, on the other hand, is best applied to projects including fewer individuals or where >500 loci are required. Both of these techniques should smooth the transition from traditional genetic techniques to genomics, helping to usher in the conservation genomics era.

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.

RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a much improved restriction site-associated DNA (RAD) sequencing protocol and a new method called Rapture ( R: AD c APTURE: ). The new RAD protocol improves versatility by separating RAD tag isolation and sequencing library preparation into two distinct steps. This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses an in-solution capture of chosen RAD tags to target sequencing reads to desired loci. Rapture combines the benefits of both RAD and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. Our results demonstrate that Rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. The methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science.

Transcriptional Response to Acute Thermal Exposure in Juvenile Chinook Salmon Determined by RNAseq.

Thermal exposure is a serious and growing challenge facing fish species worldwide. Chinook salmon (Oncorhynchus tshawytscha) living in the southern portion of their native range are particularly likely to encounter warmer water due to a confluence of factors. River alterations have increased the likelihood that juveniles will be exposed to warm water temperatures during their freshwater life stage, which can negatively impact survival, growth, and development and pose a threat to dwindling salmon populations. To better understand how acute thermal exposure affects the biology of salmon, we performed a transcriptional analysis of gill tissue from Chinook salmon juveniles reared at 12° and exposed acutely to water temperatures ranging from ideal to potentially lethal (12° to 25°). Reverse-transcribed RNA libraries were sequenced on the Illumina HiSeq2000 platform and a de novo reference transcriptome was created. Differentially expressed transcripts were annotated using Blast2GO and relevant gene clusters were identified. In addition to a high degree of downregulation of a wide range of genes, we found upregulation of genes involved in protein folding/rescue, protein degradation, cell death, oxidative stress, metabolism, inflammation/immunity, transcription/translation, ion transport, cell cycle/growth, cell signaling, cellular trafficking, and structure/cytoskeleton. These results demonstrate the complex multi-modal cellular response to thermal stress in juvenile salmon.

Climate change likely to facilitate the invasion of the non-native hydroid, Cordylophora caspia, in the San Francisco Estuary.

Climate change and invasive species can both have negative impacts on native species diversity. Additionally, climate change has the potential to favor invasive species over natives, dealing a double blow to native biodiversity. It is, therefore, vital to determine how changing climate conditions are directly linked to demographic rates and population growth of non-native species so we can quantitatively evaluate how invasive populations may be affected by changing conditions and, in turn, impact native species. Cordylophora caspia, a hydrozoan from the Ponto-Caspian region, has become established in the brackish water habitats of the San Francisco Estuary (SFE). We conducted laboratory experiments to study how temperature and salinity affect C. caspia population growth rates, in order to predict possible responses to climate change. C. Caspia population growth increased nonlinearly with temperature and leveled off at a maximum growth rate near the annual maximum temperature predicted under a conservative climate change scenario. Increasing salinity, however, did not influence growth rates. Our results indicate that C. caspia populations in the SFE will benefit from predicted regional warming trends and be little affected by changes in salinity. The population of C. caspia in the SFE has the potential to thrive under future climate conditions and may subsequently increase its negative impact on the food web.