A landmark environmental law looks ahead.

In late December 1973, the United States enacted what some would come to call "the pitbull of environmental laws." In the 50 years since, the formidable regulatory teeth of the Endangered Species Act (ESA) have been credited with considerable successes, obliging agencies to draw upon the best available science to protect species and habitats. Yet human pressures continue to push the planet toward extinctions on a massive scale. With that prospect looming, and with scientific understanding ever changing, Science invited experts to discuss how the ESA has evolved and what its future might hold. -Brad Wible.

Inbreeding depression explains killer whale population dynamics.

Understanding the factors that cause endangered populations to either grow or decline is crucial for preserving biodiversity. Conservation efforts often address extrinsic threats, such as environmental degradation and overexploitation, that can limit the recovery of endangered populations. Genetic factors such as inbreeding depression can also affect population dynamics but these effects are rarely measured in the wild and thus often neglected in conservation efforts. Here we show that inbreeding depression strongly influences the population dynamics of an endangered killer whale population, despite genomic signatures of purging of deleterious alleles via natural selection. We find that the 'Southern Residents', which are currently endangered despite nearly 50 years of conservation efforts, exhibit strong inbreeding depression for survival. Our population models suggest that this inbreeding depression limits population growth and predict further decline if the population remains genetically isolated and typical environmental conditions continue. The Southern Residents also had more inferred homozygous deleterious alleles than three other, growing, populations, further suggesting that inbreeding depression affects population fitness. These results demonstrate that inbreeding depression can substantially limit the recovery of endangered populations. Conservation actions focused only on extrinsic threats may therefore fail to account for key intrinsic genetic factors that also limit population growth.

Influence of RNA-Seq library construction, sampling methods, and tissue harvesting time on gene expression estimation.

RNA sequencing (RNA-Seq) is popular for measuring gene expression in non-model organisms, including wild populations. While RNA-Seq can detect gene expression variation among wild-caught individuals and yield important insights into biological function, sampling methods can also affect gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq library types: 3' RNA-Seq (QuantSeq) and whole mRNA-Seq (NEB). We evaluated effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 min after euthanasia on estimated gene expression in blood, gill, and muscle. We found no significant differences in gene expression between sampling methods or tissue collection times with either library type. When library types were compared using the same blood samples, 58% of genes detected by both NEB and QuantSeq showed significantly different expression between library types, and NEB detected 31% more genes than QuantSeq. Although the two library types recovered different numbers of genes and expression levels, results with NEB and QuantSeq were consistent in that neither library type showed differences in gene expression between sampling methods and tissue harvesting times. Our study suggests that researchers can safely rely on different fish sampling strategies in the field. In addition, while QuantSeq is more cost effective, NEB detects more expressed genes. Therefore, when it is crucial to detect as many genes as possible (especially low expressed genes), when alternative splicing is of interest, or when working with an organism lacking good genomic resources, whole mRNA-Seq is more powerful.

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.

Whole-genome resequencing confirms reproductive isolation between sympatric demes of brown trout (Salmo trutta) detected with allozymes.

The sympatric existence of genetically distinguishable populations of the same species remains a puzzle in ecology. Coexisting salmonid fish populations are known from over 100 freshwater lakes. Most studies of sympatric populations have used limited numbers of genetic markers making it unclear if genetic divergence involves certain parts of the genome. We returned to the first reported case of salmonid sympatry, initially detected through contrasting homozygosity at a single allozyme locus (coding for lactate dehydrogenase A) in brown trout in the small Lakes Bunnersjöarna, Sweden. First, we verified the existence of the two coexisting demes using a 96-SNP fluidigm array. We then applied whole-genome resequencing of pooled DNA to explore genome-wide diversity within and between these demes; nucleotide diversity was higher in deme I than in deme II. Strong genetic divergence is observed with genome-wide FST  ≈ 0.2. Compared with data from populations of similar small lakes, this divergence is of similar magnitude as that between reproductively isolated populations. Individual whole-genome resequencing of two individuals per deme suggests higher inbreeding in deme II versus deme I, indicating different degree of isolation. We located two gene-copies for LDH-A and found divergence between demes in a regulatory section of one of these genes. However, we did not find a perfect fit between the sequence data and previous allozyme results, and this will require further research. Our data demonstrates genome-wide divergence governed mostly by genetic drift but also by diversifying selection in coexisting populations. This type of hidden biodiversity needs consideration in conservation management.

Genomic evidence for inbreeding depression and purging of deleterious genetic variation in Indian tigers.

Increasing habitat fragmentation leads to wild populations becoming small, isolated, and threatened by inbreeding depression. However, small populations may be able to purge recessive deleterious alleles as they become expressed in homozygotes, thus reducing inbreeding depression and increasing population viability. We used whole-genome sequences from 57 tigers to estimate individual inbreeding and mutation load in a small-isolated and two large-connected populations in India. As expected, the small-isolated population had substantially higher average genomic inbreeding (FROH = 0.57) than the large-connected (FROH = 0.35 and FROH = 0.46) populations. The small-isolated population had the lowest loss-of-function mutation load, likely due to purging of highly deleterious recessive mutations. The large populations had lower missense mutation loads than the small-isolated population, but were not identical, possibly due to different demographic histories. While the number of the loss-of-function alleles in the small-isolated population was lower, these alleles were at higher frequencies and homozygosity than in the large populations. Together, our data and analyses provide evidence of 1) high mutation load, 2) purging, and 3) the highest predicted inbreeding depression, despite purging, in the small-isolated population. Frequency distributions of damaging and neutral alleles uncover genomic evidence that purifying selection has removed part of the mutation load across Indian tiger populations. These results provide genomic evidence for purifying selection in both small and large populations, but also suggest that the remaining deleterious alleles may have inbreeding-associated fitness costs. We suggest that genetic rescue from sources selected based on genome-wide differentiation could offset any possible impacts of inbreeding depression.

The crucial role of genome-wide genetic variation in conservation.

The unprecedented rate of extinction calls for efficient use of genetics to help conserve biodiversity. Several recent genomic and simulation-based studies have argued that the field of conservation biology has placed too much focus on conserving genome-wide genetic variation, and that the field should instead focus on managing the subset of functional genetic variation that is thought to affect fitness. Here, we critically evaluate the feasibility and likely benefits of this approach in conservation. We find that population genetics theory and empirical results show that conserving genome-wide genetic variation is generally the best approach to prevent inbreeding depression and loss of adaptive potential from driving populations toward extinction. Focusing conservation efforts on presumably functional genetic variation will only be feasible occasionally, often misleading, and counterproductive when prioritized over genome-wide genetic variation. Given the increasing rate of habitat loss and other environmental changes, failure to recognize the detrimental effects of lost genome-wide genetic variation on long-term population viability will only worsen the biodiversity crisis.

Conservation genetics.

Natural populations currently face a wide variety of threats including climate change, habitat loss, over-harvesting, invasive species and disease. The most recent report by the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) found that ecosystems have declined by approximately 50% relative to historical pristine conditions, and that approximately 25% of species are now threatened by extinction. This human-caused biodiversity crisis calls for using all available scientific tools to understand and reverse the increasing rate of extinction. While extinction is inherently a demographic process, being driven by changes in the population growth rate, the field of genetics plays an important role in the conservation of biodiversity. 'Conservation genetics' is a diverse field that applies genetic principles and methods to characterize and advance the preservation of biodiversity. Here, I first provide a short history of the development of the field and then list examples of the most important ways that genetics contributes to conservation.

The Genetic Architecture of Fitness Drives Population Viability during Rapid Environmental Change.

AbstractThe rapid global loss of biodiversity calls for improved predictions of how populations will evolve and respond demographically to ongoing environmental change. The heritability (h2) of selected traits has long been known to affect evolutionary and demographic responses to environmental change. However, effects of the genetic architecture underlying the h2 of a selected trait on population responses to selection are less well understood. We use deterministic models and stochastic simulations to show that the genetic architecture underlying h2 can dramatically affect population viability during environmental change. Polygenic trait architectures (many loci, each with a small phenotypic effect) conferred higher population viability than genetic architectures with the same initial h2 and large-effect loci under a wide range of scenarios. Population viability also depended strongly on the initial frequency of large-effect beneficial alleles, with moderately low initial allele frequencies conferring higher viability than rare or already-frequent large-effect alleles. Greater population viability associated with polygenic architectures appears to be due to higher short-term evolutionary potential compared with architectures with large-effect loci. These results suggest that integrating information on the trait genetic architecture into quantitative genetic and population viability analysis will substantially improve our understanding and prediction of evolutionary and demographic responses following environmental change.

Evaluating the outcomes of genetic rescue attempts.

Augmenting gene flow is a powerful tool for the conservation of small, isolated populations. However, genetic rescue attempts have largely been limited to populations at the brink of extinction, in part due to concerns over negative outcomes (e.g., outbreeding depression). Increasing habitat fragmentation may necessitate more proactive genetic management. Broader application of augmented gene flow will, in turn, require rigorous evaluation to increase confidence and identify pitfalls in this approach. To date, there has been no assessment of best monitoring practices for genetic rescue attempts. We used genomically explicit, individual-based simulations to examine the effectiveness of common approaches (i.e., tests for increases in fitness, migrant ancestry, heterozygosity, and abundance) for determining whether genetic rescue or outbreeding depression occurred. Statistical power to detect the effects of gene flow on fitness was high (≥0.8) when effect sizes were large, a finding consistent with those from previous studies on severely inbred populations. However, smaller effects of gene flow on fitness can appreciably affect persistence probability but current evaluation approaches fail to provide results from which reliable inferences can be drawn. The power of the metrics we examined to evaluate genetic rescue attempts depended on the time since gene flow and whether gene flow was beneficial or deleterious. Encouragingly, the use of multiple metrics provided nonredundant information and improved inference reliability, highlighting the importance of intensive monitoring efforts. Further development of best practices for evaluating genetic rescue attempts will be crucial for a responsible transition to increased use of translocations to decrease extinction risk.

Evaluación de los Resultados de los Intentos de Rescate Genético Resumen El aumento del flujo génico es una herramienta poderosa para la conservación de poblaciones pequeñas y aisladas. Sin embargo, los intentos de rescate genético en su mayoría se han limitado a las poblaciones que se encuentran al borde de la extinción, en parte debido a la preocupación que existe por los resultados negativos (es decir, la depresión exogámica). La creciente fragmentación del hábitat puede requerir un manejo genético más proactivo. La aplicación más extensa del flujo génico aumentado requerirá a su vez una evaluación rigurosa para incrementar la confianza e identificar las dificultades de esta estrategia. A la fecha, no ha habido una evaluación de las mejores prácticas de monitoreo para los intentos de rescate genético. Usamos simulaciones explícitas basadas en individuos para examinar la efectividad de las estrategias comunes (es decir, análisis del incremento en adaptabilidad, ascendencia migratoria, heterocigosidad y abundancia) para determinar si ocurrió el rescate genético o la depresión exogámica. El poder estadístico para detectar los efectos del flujo génico sobre la adaptabilidad fue elevado (≥0.8) cuando el tamaño de los efectos fue grande, un hallazgo consistente con aquellos realizados en estudios previos sobre poblaciones con una endogamia severa. Sin embargo, los efectos menores del flujo génico sobre la adaptabilidad pueden afectar de manera apreciable la probabilidad de persistencia, pero las estrategias actuales de evaluación no proporcionan resultados de los cuales se puedan hacer inferencias confiables. El poder de las medidas que examinamos para evaluar los intentos de rescate genético dependió del tiempo desde que inició el flujo génico y de si el flujo génico fue benéfico o perjudicial. De manera alentadora, el uso de múltiples medidas proporcionó información no redundante y mejoró la confiabilidad de la inferencia, resaltando así la importancia de los esfuerzos intensivos de monitoreo. El futuro desarrollo de mejores prácticas para la evaluación de los intentos de rescate genético será de suma importancia para la transición responsable hacia el mayor uso de reubicaciones para reducir el riesgo de extinción.

The Peril of Gene-Targeted Conservation.

The genomics revolution has sparked interest in using our increased understanding of the loci involved in phenotypic variation and adaptation to advance the conservation of biodiversity. Despite much interest and discussion, it remains unclear whether, when, and how such analyses should be used to guide conservation action. Such 'gene-targeted' conservation strategies, while promising, are complicated by several factors including the complex genomic architecture of phenotypic variation and the strong potential for undesirable outcomes such as the loss of genome-wide genetic variation and evolutionary potential. We caution against relying on gene-targeted approaches as a conservation silver bullet and propose rigorous criteria to identify situations where gene-targeted approaches are likely to benefit conservation.

Genomic consequences of intensive inbreeding in an isolated wolf population.

Inbreeding (mating between relatives) is a major concern for conservation as it decreases individual fitness and can increase the risk of population extinction. We used whole-genome resequencing of 97 grey wolves (Canis lupus) from the highly inbred Scandinavian wolf population to identify 'identical-by-descent' (IBD) chromosome segments as runs of homozygosity (ROH). This gave the high resolution required to precisely measure realized inbreeding as the IBD fraction of the genome in ROH (F ROH). We found a striking pattern of complete or near-complete homozygosity of entire chromosomes in many individuals. The majority of individual inbreeding was due to long IBD segments (>5 cM) originating from ancestors ≤10 generations ago, with 10 genomic regions showing very few ROH and forming candidate regions for containing loci contributing strongly to inbreeding depression. Inbreeding estimated with an extensive pedigree (F P) was strongly correlated with realized inbreeding measured with the entire genome (r 2 = 0.86). However, inbreeding measured with the whole genome was more strongly correlated with multi-locus heterozygosity estimated with as few as 500 single nucleotide polymorphisms, and with F ROH estimated with as few as 10,000 single nucleotide polymorphisms, than with F P. These results document in fine detail the genomic consequences of intensive inbreeding in a population of conservation concern.

Inferring Individual Inbreeding and Demographic History from Segments of Identity by Descent in Ficedula Flycatcher Genome Sequences.

Individual inbreeding and historical demography can be estimated by analyzing runs of homozygosity (ROH), which are indicative of chromosomal segments of identity by descent (IBD). Such analyses have so far been rare in natural populations due to limited genomic resources. We analyzed ROH in whole genome sequences from 287 Ficedula flycatchers representing four species, with the objectives of evaluating the causes of genome-wide variation in the abundance of ROH and inferring historical demography. ROH were clearly more abundant in genomic regions with low recombination rate. However, this pattern was substantially weaker when ROH were mapped using genetic rather than physical single nucleotide polymorphism (SNP) coordinates in the genome. Empirical results and simulations suggest that high ROH abundance in regions of low recombination was partly caused by increased power to detect the very long IBD segments typical of regions with a low recombination rate. Simulations also showed that hard selective sweeps (but not soft sweeps or background selection) likely contributed to variation in the abundance of ROH across the genome. Comparisons of the abundance of ROH among several study populations indicated that the Spanish pied flycatcher population had the smallest historical effective population size (Ne) for this species, and that a putatively recently founded island (Baltic) population had the smallest historical Ne among the collared flycatchers. Analysis of pairwise IBD in Baltic collared flycatchers indicated that this population was founded <60 generations ago. This study provides a rare genomic glimpse into demographic history and the mechanisms underlying the genome-wide distribution of ROH.

Genomic Variation of Inbreeding and Ancestry in the Remaining Two Isle Royale Wolves.

Inbreeding, relatedness, and ancestry have traditionally been estimated with pedigree information, however, molecular genomic data can provide more detailed examination of these properties. For example, pedigree information provides estimation of the expected value of these measures but molecular genomic data can estimate the realized values of these measures in individuals. Here, we generate the theoretical distribution of inbreeding, relatedness, and ancestry for the individuals in the pedigree of the Isle Royale wolves, the first examination of such variation in a wild population with a known pedigree. We use the 38 autosomes of the dog genome and their estimated map lengths in our genomic analysis. Although it is known that the remaining wolves are highly inbred, closely related, and descend from only 3 ancestors, our analyses suggest that there is significant variation in the realized inbreeding and relatedness around pedigree expectations. For example, the expected inbreeding in a hypothetical offspring from the 2 remaining wolves is 0.438 but the realized 95% genomic confidence interval is from 0.311 to 0.565. For individual chromosomes, a substantial proportion of the whole chromosomes are completely identical by descent. This examination provides a background to use when analyzing molecular genomic data for individual levels of inbreeding, relatedness, and ancestry. The level of variation in these measures is a function of the time to the common ancestor(s), the number of chromosomes, and the rate of recombination. In the Isle Royale wolf population, the few generations to a common ancestor results in the high variance in genomic inbreeding.

Genomics advances the study of inbreeding depression in the wild.

Inbreeding depression (reduced fitness of individuals with related parents) has long been a major focus of ecology, evolution, and conservation biology. Despite decades of research, we still have a limited understanding of the strength, underlying genetic mechanisms, and demographic consequences of inbreeding depression in the wild. Studying inbreeding depression in natural populations has been hampered by the inability to precisely measure individual inbreeding. Fortunately, the rapidly increasing availability of high-throughput sequencing data means it is now feasible to measure the inbreeding of any individual with high precision. Here, we review how genomic data are advancing our understanding of inbreeding depression in the wild. Recent results show that individual inbreeding and inbreeding depression can be measured more precisely with genomic data than via traditional pedigree analysis. Additionally, the availability of genomic data has made it possible to pinpoint loci with large effects contributing to inbreeding depression in wild populations, although this will continue to be a challenging task in many study systems due to low statistical power. Now that reliably measuring individual inbreeding is no longer a limitation, a major focus of future studies should be to more accurately quantify effects of inbreeding depression on population growth and viability.

SNP discovery in candidate adaptive genes using exon capture in a free-ranging alpine ungulate.

Identification of genes underlying genomic signatures of natural selection is key to understanding adaptation to local conditions. We used targeted resequencing to identify SNP markers in 5321 candidate adaptive genes associated with known immunological, metabolic and growth functions in ovids and other ungulates. We selectively targeted 8161 exons in protein-coding and nearby 5' and 3' untranslated regions of chosen candidate genes. Targeted sequences were taken from bighorn sheep (Ovis canadensis) exon capture data and directly from the domestic sheep genome (Ovis aries v. 3; oviAri3). The bighorn sheep sequences used in the Dall's sheep (Ovis dalli dalli) exon capture aligned to 2350 genes on the oviAri3 genome with an average of 2 exons each. We developed a microfluidic qPCR-based SNP chip to genotype 476 Dall's sheep from locations across their range and test for patterns of selection. Using multiple corroborating approaches (lositan and bayescan), we detected 28 SNP loci potentially under selection. We additionally identified candidate loci significantly associated with latitude, longitude, precipitation and temperature, suggesting local environmental adaptation. The three methods demonstrated consistent support for natural selection on nine genes with immune and disease-regulating functions (e.g. Ovar-DRA, APC, BATF2, MAGEB18), cell regulation signalling pathways (e.g. KRIT1, PI3K, ORRC3), and respiratory health (CYSLTR1). Characterizing adaptive allele distributions from novel genetic techniques will facilitate investigation of the influence of environmental variation on local adaptation of a northern alpine ungulate throughout its range. This research demonstrated the utility of exon capture for gene-targeted SNP discovery and subsequent SNP chip genotyping using low-quality samples in a nonmodel species.

Cross-Species Application of SNP Chips is Not Suitable for Identifying Runs of Homozygosity.

Cross-species application of single-nucleotide polymorphism (SNP) chips is a valid, relatively cost-effective alternative to the high-throughput sequencing methods generally required to obtain a genome-wide sampling of polymorphisms. Kharzinova et al. (2015) examined the applicability of SNP chips developed in domestic bovids (cattle and sheep) to a semi-wild cervid (reindeer). The ancestors of bovids and cervids diverged between 20 and 30 million years ago (Hassanin and Douzery 2003; Bibi et al. 2013). Empirical work has shown that for a SNP chip developed in a bovid and applied to a cervid species, approximately 50% genotype success with 1% of the loci being polymorphic is expected (Miller et al. 2012). The genotyping of Kharzinova et al. (2015) follows this pattern; however, these data are not appropriate for identifying runs of homozygosity (ROH) and can be problematic for estimating linkage disequilibrium (LD) and we caution readers in this regard.