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.

Genetics and the conservation of natural populations: allozymes to genomes.

I consider how the study of genetic variation has influenced efforts to conserve natural populations over the last 50 years. Studies with allozymes in the 1970s provided the first estimates of the amount of genetic variation within and between natural populations at multiple loci. These early studies played an important role in developing plans to conserve species. The description of genetic variation in mitochondrial DNA in the early 1980s laid the foundation for the field of phylogeography, which provided a deeper look in time of the relationships and connectivity among populations. The development of microsatellites in the 1990s provided much more powerful means to describe genetic variation at nuclear loci, including the ability to detect past bottlenecks and estimate current effective population size with a single temporal sample. In the 2000s, single nucleotide polymorphisms presented a cornucopia of loci that has greatly improved power to estimate genetic and population demographic parameters important for conservation. Today, population genomics presents the ability to detect regions of the genome that are affected by natural selection (e.g. local adaptation or inbreeding depression). In addition, the ability to genotype historical samples has provided power to understand how climate change and other anthropogenic phenomena have affected populations. Modern molecular techniques provide unprecedented power to understand genetic variation in natural populations. Nevertheless, application of this information requires sound understanding of population genetics theory. I believe that current training in conservation genetics focuses too much on the latest techniques and too little on understanding the conceptual basis which is needed to interpret these data and ask good questions.

Cryptic inbreeding depression in a growing population of a long-lived species.

Genetic effects are often overlooked in endangered species monitoring, and populations showing positive growth are often assumed to be secure. However, the continued reproductive success of a few individuals may mask issues such as inbreeding depression, especially in long-lived species. Here, we test for inbreeding depression in little spotted kiwi (Apteryx owenii) by comparing a population founded with two birds to one founded with 40 birds, both from the same source population and both showing positive population growth. We used a combination of microsatellite genotypes, nest observations and modelling to examine the consequences of assessing population viability exclusively via population growth. We demonstrate (i) significantly lower hatching success despite significantly higher reproductive effort in the population with two founders; (ii) positive growth in the population with two founders is mainly driven by ongoing chick production of the founding pair; and (iii) a substantial genetic load in the population founded with two birds (10-15 diploid lethal equivalents). Our results illustrate that substantial, cryptic inbreeding depression may still be present when a population is growing, especially in long-lived species with overlapping generations.

Legacy introductions and climatic variation explain spatiotemporal patterns of invasive hybridization in a native trout.

Hybridization between invasive and native species, a significant threat to worldwide biodiversity, is predicted to increase due to climate-induced expansions of invasive species. Long-term research and monitoring are crucial for understanding the ecological and evolutionary processes that modulate the effects of invasive species. Using a large, multidecade genetics dataset (N = 582 sites, 12,878 individuals) with high-resolution climate predictions and extensive stocking records, we evaluate the spatiotemporal dynamics of hybridization between native cutthroat trout and invasive rainbow trout, the world's most widely introduced invasive fish, across the Northern Rocky Mountains of the United States. Historical effects of stocking and contemporary patterns of climatic variation were strongly related to the spread of hybridization across space and time. The probability of occurrence, extent of, and temporal changes in hybridization increased at sites in close proximity to historical stocking locations with greater rainbow trout propagule pressure, warmer water temperatures, and lower spring precipitation. Although locations with warmer water temperatures were more prone to hybridization, cold sites were not protected from invasion; 58% of hybridized sites had cold mean summer water temperatures (<11°C). Despite cessation of stocking over 40 years ago, hybridization increased over time at half (50%) of the locations with long-term data, the vast majority of which (74%) were initially nonhybridized, emphasizing the chronic, negative impacts of human-mediated hybridization. These results show that effects of climate change on biodiversity must be analyzed in the context of historical human impacts that set ecological and evolutionary trajectories.

Vive la résistance: genome-wide selection against introduced alleles in invasive hybrid zones.

Evolutionary and ecological consequences of hybridization between native and invasive species are notoriously complicated because patterns of selection acting on non-native alleles can vary throughout the genome and across environments. Rapid advances in genomics now make it feasible to assess locus-specific and genome-wide patterns of natural selection acting on invasive introgression within and among natural populations occupying diverse environments. We quantified genome-wide patterns of admixture across multiple independent hybrid zones of native westslope cutthroat trout and invasive rainbow trout, the world's most widely introduced fish, by genotyping 339 individuals from 21 populations using 9380 species-diagnostic loci. A significantly greater proportion of the genome appeared to be under selection favouring native cutthroat trout (rather than rainbow trout), and this pattern was pervasive across the genome (detected on most chromosomes). Furthermore, selection against invasive alleles was consistent across populations and environments, even in those where rainbow trout were predicted to have a selective advantage (warm environments). These data corroborate field studies showing that hybrids between these species have lower fitness than the native taxa, and show that these fitness differences are due to selection favouring many native genes distributed widely throughout the genome.

Genomics and the future of conservation genetics.

We will soon have complete genome sequences from thousands of species, as well as from many individuals within species. This coming explosion of information will transform our understanding of the amount, distribution and functional significance of genetic variation in natural populations. Now is a crucial time to explore the potential implications of this information revolution for conservation genetics and to recognize limitations in applying genomic tools to conservation issues. We identify and discuss those problems for which genomics will be most valuable for curbing the accelerating worldwide loss of biodiversity. We also provide guidance on which genomics tools and approaches will be most appropriate to use for different aspects of conservation.

Dispersal and selection mediate hybridization between a native and invasive species.

Hybridization between native and non-native species has serious biological consequences, but our understanding of how dispersal and selection interact to influence invasive hybridization is limited. Here, we document the spread of genetic introgression between a native (Oncorhynchus clarkii) and invasive (Oncorhynchus mykiss) trout, and identify the mechanisms influencing genetic admixture. In two populations inhabiting contrasting environments, non-native admixture increased rapidly from 1984 to 2007 and was driven by surprisingly consistent processes. Individual admixture was related to two phenotypic traits associated with fitness: size at spawning and age of juvenile emigration. Fish with higher non-native admixture were larger and tended to emigrate at a younger age--relationships that are expected to confer fitness advantages to hybrid individuals. However, strong selection against non-native admixture was evident across streams and cohorts (mean selection coefficient against genotypes with non-native alleles (s) = 0.60; s.e. = 0.10). Nevertheless, hybridization was promoted in both streams by the continuous immigration of individuals with high levels of non-native admixture from other hybrid source populations. Thus, antagonistic relationships between dispersal and selection are mediating invasive hybridization between these fish, emphasizing that data on dispersal and natural selection are needed to fully understand the dynamics of introgression between native and non-native species.

Whole-genome resequencing uncovers molecular signatures of natural and sexual selection in wild bighorn sheep.

The identification of genes influencing fitness is central to our understanding of the genetic basis of adaptation and how it shapes phenotypic variation in wild populations. Here, we used whole-genome resequencing of wild Rocky Mountain bighorn sheep (Ovis canadensis) to >50-fold coverage to identify 2.8 million single nucleotide polymorphisms (SNPs) and genomic regions bearing signatures of directional selection (i.e. selective sweeps). A comparison of SNP diversity between the X chromosome and the autosomes indicated that bighorn males had a dramatically reduced long-term effective population size compared to females. This probably reflects a long history of intense sexual selection mediated by male-male competition for mates. Selective sweep scans based on heterozygosity and nucleotide diversity revealed evidence for a selective sweep shared across multiple populations at RXFP2, a gene that strongly affects horn size in domestic ungulates. The massive horns carried by bighorn rams appear to have evolved in part via strong positive selection at RXFP2. We identified evidence for selection within individual populations at genes affecting early body growth and cellular response to hypoxia; however, these must be interpreted more cautiously as genetic drift is strong within local populations and may have caused false positives. These results represent a rare example of strong genomic signatures of selection identified at genes with known function in wild populations of a nonmodel species. Our results also showcase the value of reference genome assemblies from agricultural or model species for studies of the genomic basis of adaptation in closely related wild taxa.

Effects of crossovers between homeologs on inheritance and population genomics in polyploid-derived salmonid fishes.

A whole genome duplication occurred in the ancestor of all salmonid fishes some 50-100 million years ago. Early inheritance studies with allozymes indicated that loci in the salmonid genome are inherited disomically in females. However, some pairs of duplicated loci showed patterns of inheritance in males indicating pairing and recombination between homeologous chromosomes. Nearly 20% of loci in the salmonid genome are duplicated and share the same alleles (isoloci), apparently due to homeologous recombination. Half-tetrad analysis revealed that isoloci tend to be telomeric. These results suggested that residual tetrasomic inheritance of isoloci results from homeologous recombination near chromosome ends and that continued disomic inheritance resulted from homologous pairing of centromeric regions. Many current genetic maps of salmonids are based on single nucleotide polymorphisms and microsatellites that are no longer duplicated. Therefore, long sections of chromosomes on these maps are poorly represented, especially telomeric regions. In addition, preferential multivalent pairing of homeologs from the same species in F1 hybrids results in an excess of nonparental gametes (so-called pseudolinkage). We consider how not including duplicated loci has affected our understanding of population and evolutionary genetics of salmonids, and we discuss how incorporating these loci will benefit our understanding of population genomics.

How much gene flow is needed to avoid inbreeding depression in wild tiger populations?

The number and size of tiger populations continue to decline owing to habitat loss, habitat fragmentation and poaching of tigers and their prey. As a result, tiger populations have become small and highly structured. Current populations have been isolated since the early 1970s or for approximately seven generations. The objective of this study is to explore how inbreeding may be affecting the persistence of remaining tiger populations and how dispersal, either natural or artificial, may reduce the potentially detrimental effect of inbreeding depression. We developed a tiger simulation model and used published levels of genetic load in mammals to simulate inbreeding depression. Following a 50 year period of population isolation, we introduced one to four dispersing male tigers per generation to explore how gene flow from nearby populations may reduce the negative impact of inbreeding depression. For the smallest populations, even four dispersing male tigers per generation did not increase population viability, and the likelihood of extinction is more than 90% within 30 years. Unless habitat connectivity is restored or animals are artificially introduced in the next 70 years, medium size wild populations are also likely to go extinct, with only four to five of the largest wild tiger populations likely to remain extant in this same period without intervention. To reduce the risk of local extinction, habitat connectivity must be pursued concurrently with efforts to increase population size (e.g. enhance habitat quality, increase habitat availability). It is critical that infrastructure development, dam construction and other similar projects are planned appropriately so that they do not erode the extent or quality of habitat for these populations so that they can truly serve as future source populations.

So long to genetic diversity, and thanks for all the fish.

The world faces a global fishing crisis. Wild marine fisheries comprise nearly 15% of all animal protein in the human diet, but, according to the U.N. Food and Agriculture Organization, nearly 60% of all commercially important marine fish stocks are overexploited, recovering, or depleted (FAO 2012; Fig. 1). Some authors have suggested that the large population sizes of harvested marine fish make even collapsed populations resistant to the loss of genetic variation by genetic drift (e.g. Beverton 1990). In contrast, others have argued that the loss of alleles because of overfishing may actually be more dramatic in large populations than in small ones (Ryman et al. 1995). In this issue, Pinsky & Palumbi (2014) report that overfished populations have approximately 2% lower heterozygosity and 12% lower allelic richness than populations that are not overfished. They also performed simulations which suggest that their estimates likely underestimate the actual loss of rare alleles by a factor of three or four. This important paper shows that the harvesting of marine fish can have genetic effects that threaten the long-term sustainability of this valuable resource.

What does effective population size tell us about loss of allelic variation?

There are two primary measures of the amount of genetic variation in a population at a locus: heterozygosity and the number of alleles. Effective population size (N e) provides both an expectation of the amount of heterozygosity in a population at drift-mutation equilibrium and the rate of loss of heterozygosity because of genetic drift. In contrast, the number of alleles in a population at drift-mutation equilibrium is a function of both N e and census size (N C). In addition, populations with the same N e can lose allelic variation at very different rates. Allelic variation is generally much more sensitive to bottlenecks than heterozygosity. Expressions used to adjust for the effects of violations of the ideal population on N e do not provide good predictions of the loss of allelic variation. These effects are much greater for loci with many alleles, which are often important for adaptation. We show that there is a linear relationship between the reduction of N C and the corresponding reduction of the expected number of alleles at drift-mutation equilibrium. This makes it possible to predict the expected effect of a bottleneck on allelic variation. Heterozygosity provides good estimates of the rate of adaptive change in the short-term, but allelic variation provides important information about long-term adaptive change. The guideline of long-term N e being greater than 500 is often used as a primary genetic metric for evaluating conservation status. We recommend that this guideline be expanded to take into account allelic variation as well as heterozygosity.

Genetic consequences of a century of protection: serial founder events and survival of the little spotted kiwi (Apteryx owenii).

We present the outcome of a century of post-bottleneck isolation of a long-lived species, the little spotted kiwi (Apteryx owenii, LSK) and demonstrate that profound genetic consequences can result from protecting few individuals in isolation. LSK were saved from extinction by translocation of five birds from South Island, New Zealand to Kapiti Island 100 years ago. The Kapiti population now numbers some 1200 birds and provides founders for new populations. We used 15 microsatellite loci to compare genetic variation among Kapiti LSK and the populations of Red Mercury, Tiritiri Matangi and Long Islands that were founded with birds from Kapiti. Two LSK native to D'Urville Island were also placed on Long Island. We found extremely low genetic variation and signatures of acute and recent genetic bottleneck effects in all four populations, indicating that LSK have survived multiple genetic bottlenecks. The Long Island population appears to have arisen from a single mating pair from Kapiti, suggesting there is no genetic contribution from D'Urville birds among extant LSK. The Ne/NC ratio of Kapiti Island LSK (0.03) is exceptionally low for terrestrial vertebrates and suggests that genetic diversity might still be eroding in this population, despite its large census size.

Genomic patterns of introgression in rainbow and westslope cutthroat trout illuminated by overlapping paired-end RAD sequencing.

Rapid and inexpensive methods for genomewide single nucleotide polymorphism (SNP) discovery and genotyping are urgently needed for population management and conservation. In hybridized populations, genomic techniques that can identify and genotype thousands of species-diagnostic markers would allow precise estimates of population- and individual-level admixture as well as identification of 'super invasive' alleles, which show elevated rates of introgression above the genomewide background (likely due to natural selection). Techniques like restriction-site-associated DNA (RAD) sequencing can discover and genotype large numbers of SNPs, but they have been limited by the length of continuous sequence data they produce with Illumina short-read sequencing. We present a novel approach, overlapping paired-end RAD sequencing, to generate RAD contigs of >300-400 bp. These contigs provide sufficient flanking sequence for design of high-throughput SNP genotyping arrays and strict filtering to identify duplicate paralogous loci. We applied this approach in five populations of native westslope cutthroat trout that previously showed varying (low) levels of admixture from introduced rainbow trout (RBT). We produced 77 141 RAD contigs and used these data to filter and genotype 3180 previously identified species-diagnostic SNP loci. Our population-level and individual-level estimates of admixture were generally consistent with previous microsatellite-based estimates from the same individuals. However, we observed slightly lower admixture estimates from genomewide markers, which might result from natural selection against certain genome regions, different genomic locations for microsatellites vs. RAD-derived SNPs and/or sampling error from the small number of microsatellite loci (n = 7). We also identified candidate adaptive super invasive alleles from RBT that had excessively high admixture proportions in hybridized cutthroat trout populations.

Hunting effects on favourable conservation status of highly inbred Swedish wolves.

The wolf (Canis lupus) is classified as endangered in Sweden by the Swedish Species Information Centre, which is the official authority for threat classification. The present population, which was founded in the early 1980s, descends from 5 individuals. It is isolated and highly inbred, and on average individuals are more related than siblings. Hunts have been used by Swedish authorities during 2010 and 2011 to reduce the population size to its upper tolerable level of 210 wolves. European Union (EU) biodiversity legislation requires all member states to promote a concept called "favourable conservation status" (FCS) for a series of species including the wolf. Swedish national policy stipulates maintenance of viable populations with sufficient levels of genetic variation of all naturally occurring species. Hunting to reduce wolf numbers in Sweden is currently not in line with national and EU policy agreements and will make genetically based FCS criteria less achievable for this species. We suggest that to reach FCS for the wolf in Sweden the following criteria need to be met: (1) a well-connected, large, subdivided wolf population over Scandinavia, Finland, and the Russian Karelia-Kola region should be reestablished, (2) genetically effective size (Ne ) of this population is in the minimum range of Ne = 500-1000, (3) Sweden harbors a part of this total population that substantially contributes to the total Ne and that is large enough to not be classified as threatened genetically or according to IUCN criteria, and (4) average inbreeding levels in the Swedish population are <0.1.

Estimation of male gene flow from measures of nuclear and female genetic differentiation.

An approach is provided to estimate male gene flow and the ratio of male to female gene flow, given that there are estimates of diploid, nuclear gene flow and haploid, female gene flow. This approach can be applied to estimates of differentiation (F ST ) from biparentally and maternally inherited markers, assuming the equilibrium island model and equal effective numbers of males and females. Corrections to formulas used previously for California sea lions (González-Suárez M, Flatz R, Aurioles-Gamboa D, Hedrick PW, Gerber LR. 2009. Isolation by distance among California sea lion populations in Mexico: redefining management stocks. Mol Ecol. 18:1088-1099.) and American bison (Halbert ND, Gogan PJP, Hedrick PW, Wahl L, Derr JN. 2012. Genetic population substructure in bison in Yellowstone National Park. J Hered. 103:360-370.) are given and revised values for those species are calculated. The effect of unequal male and female effective population sizes, nonequilibrium conditions, and approximations of differentiation formulas are briefly discussed.

Securing the demographic and genetic future of tuatara through assisted colonization.

Climate change poses a particular threat to species with fragmented distributions and little or no capacity to migrate. Assisted colonization, moving species into regions where they have not previously occurred, aims to establish populations where they are expected to survive as climatic envelopes shift. However, adaptation to the source environment may affect whether species successfully establish in new regions. Assisted colonization has spurred debate among conservation biologists and ecologists over whether the potential benefits to the threatened species outweigh the potential disruption to recipient communities. In our opinion, the debate has been distracted by controversial examples, rather than cases where assisted colonization may be a viable strategy. We present a strategic plan for the assisted migration of tuatara (Sphenodon punctatus), an endemic New Zealand reptile. The plan includes use of extant populations as reference points for comparisons with assisted-colonization populations with respect to demography, phenotypic plasticity, and phenology; optimization of genetic variation; research to fill knowledge gaps; consideration of host and recipient communities; and inclusion of stakeholders in the planning stage. When strategically planned and monitored, assisted colonization could meet conservation and research goals and ultimately result in the establishment of long-term sustainable populations capable of persisting during rapid changes in climate.

Human-induced evolution caused by unnatural selection through harvest of wild animals.

Human harvest of phenotypically desirable animals from wild populations imposes selection that can reduce the frequencies of those desirable phenotypes. Hunting and fishing contrast with agricultural and aquacultural practices in which the most desirable animals are typically bred with the specific goal of increasing the frequency of desirable phenotypes. We consider the potential effects of harvest on the genetics and sustainability of wild populations. We also consider how harvesting could affect the mating system and thereby modify sexual selection in a way that might affect recruitment. Determining whether phenotypic changes in harvested populations are due to evolution, rather than phenotypic plasticity or environmental variation, has been problematic. Nevertheless, it is likely that some undesirable changes observed over time in exploited populations (e.g., reduced body size, earlier sexual maturity, reduced antler size, etc.) are due to selection against desirable phenotypes-a process we call "unnatural" selection. Evolution brought about by human harvest might greatly increase the time required for over-harvested populations to recover once harvest is curtailed because harvesting often creates strong selection differentials, whereas curtailing harvest will often result in less intense selection in the opposing direction. We strongly encourage those responsible for managing harvested wild populations to take into account possible selective effects of harvest management and to implement monitoring programs to detect exploitation-induced selection before it seriously impacts viability.