New indicators for monitoring genetic diversity applied to alpine brown trout populations using whole genome sequence data.

International policy recently adopted commitments to maintain genetic diversity in wild populations to secure their adaptive potential, including metrics to monitor temporal trends in genetic diversity - so-called indicators. A national programme for assessing trends in genetic diversity was recently initiated in Sweden. Relating to this effort, we systematically assess contemporary genome-wide temporal trends (40 years) in wild populations using the newly adopted indicators and whole genome sequencing (WGS). We use pooled and individual WGS data from brown trout (Salmo trutta) in eight alpine lakes in protected areas. Observed temporal trends in diversity metrics (nucleotide diversity, Watterson's Ï´ and heterozygosity) lie within proposed acceptable threshold values for six of the lakes, but with consistently low values in lakes above the tree line and declines observed in these northern-most lakes. Local effective population size is low in all lakes, highlighting the importance of continued protection of interconnected systems to allow genetic connectivity for long-term viability of these populations. Inbreeding (FROH ) spans 10%-30% and is mostly represented by ancient (<1 Mb) runs of homozygosity, with observations of little change in mutational load. We also investigate adaptive dynamics over evolutionarily short time frames (a few generations); identifying putative parallel selection across all lakes within a gene pertaining to skin pigmentation as well as candidates of selection unique to specific lakes and lake systems involved in reproduction and immunity. We demonstrate the utility of WGS for systematic monitoring of natural populations, a priority concern if genetic diversity is to be protected.

Assessment of the Global Variance Effective Size of Subdivided Populations, and Its Relation to Other Effective Sizes.

The variance effective population size ([Formula: see text]) is frequently used to quantify the expected rate at which a population's allele frequencies change over time. The purpose of this paper is to find expressions for the global [Formula: see text] of a spatially structured population that are of interest for conservation of species. Since [Formula: see text] depends on allele frequency change, we start by dividing the cause of allele frequency change into genetic drift within subpopulations (I) and a second component mainly due to migration between subpopulations (II). We investigate in detail how these two components depend on the way in which subpopulations are weighted as well as their dependence on parameters of the model such a migration rates, and local effective and census sizes. It is shown that under certain conditions the impact of II is eliminated, and [Formula: see text] of the metapopulation is maximized, when subpopulations are weighted proportionally to their long term reproductive contributions. This maximal [Formula: see text] is the sought for global effective size, since it approximates the gene diversity effective size [Formula: see text], a quantifier of the rate of loss of genetic diversity that is relevant for conservation of species and populations. We also propose two novel versions of [Formula: see text], one of which (the backward version of [Formula: see text]) is most stable, exists for most populations, and is closer to [Formula: see text] than the classical notion of [Formula: see text]. Expressions for the optimal length of the time interval for measuring genetic change are developed, that make it possible to estimate any version of [Formula: see text] with maximal accuracy.

Monitoring genetic diversity with new indicators applied to an alpine freshwater top predator.

Genetic diversity is the basis for population adaptation and long-term survival, yet rarely considered in biodiversity monitoring. One key issue is the need for useful and straightforward indicators of genetic diversity. We monitored genetic diversity over 40 years (1970-2010) in metapopulations of brown trout (Salmo trutta) inhabiting 27 small mountain lakes representing 10 lake systems in central Sweden using >1200 fish per time point. We tested six newly proposed indicators; three were designed for broad, international use in the UN Convention on Biological Diversity (CBD) and are currently applied in several countries. The other three were recently elaborated for national use by a Swedish science-management effort and applied for the first time here. The Swedish indicators use molecular genetic data to monitor genetic diversity within and between populations (indicators ΔH and ΔFST , respectively) and assess the effective population size (Ne -indicator). We identified 29 genetically distinct populations, all retained over time. Twelve of the 27 lakes harboured more than one population indicating that brown trout biodiversity hidden as cryptic, sympatric populations are more common than recognized. The Ne indicator showed values below the threshold (Ne ≤ 500) in 20 populations with five showing Ne < 100. Statistically significant genetic diversity reductions occurred in several populations. Metapopulation structure appears to buffer against diversity loss; applying the indicators to metapopulations suggest mostly acceptable rates of change in all but one system. The CBD indicators agreed with the Swedish ones but provided less detail. All these indicators are appropriate for managers to initiate monitoring of genetic biodiversity.

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.

Moose genomes reveal past glacial demography and the origin of modern lineages.

BACKGROUND: Numerous megafauna species from northern latitudes went extinct during the Pleistocene/Holocene transition as a result of climate-induced habitat changes. However, several ungulate species managed to successfully track their habitats during this period to eventually flourish and recolonise the holarctic regions. So far, the genomic impacts of these climate fluctuations on ungulates from high latitudes have been little explored. Here, we assemble a de-novo genome for the European moose (Alces alces) and analyse it together with re-sequenced nuclear genomes and ancient and modern mitogenomes from across the moose range in Eurasia and North America. RESULTS: We found that moose demographic history was greatly influenced by glacial cycles, with demographic responses to the Pleistocene/Holocene transition similar to other temperate ungulates. Our results further support that modern moose lineages trace their origin back to populations that inhabited distinct glacial refugia during the Last Glacial Maximum (LGM). Finally, we found that present day moose in Europe and North America show low to moderate inbreeding levels resulting from post-glacial bottlenecks and founder effects, but no evidence for recent inbreeding resulting from human-induced population declines. CONCLUSIONS: Taken together, our results highlight the dynamic recent evolutionary history of the moose and provide an important resource for further genomic studies.

Parallel adaptive evolution of geographically distant herring populations on both sides of the North Atlantic Ocean.

Atlantic herring is an excellent species for studying the genetic basis of adaptation in geographically distant populations because of its characteristically large population sizes and low genetic drift. In this study we compared whole-genome resequencing data of Atlantic herring populations from both sides of the Atlantic Ocean. An important finding was the very low degree of genetic differentiation among geographically distant populations (fixation index = 0.026), suggesting lack of reproductive isolation across the ocean. This feature of the Atlantic herring facilitates the detection of genetic factors affecting adaptation because of the sharp contrast between loci showing genetic differentiation resulting from natural selection and the low background noise resulting from genetic drift. We show that genetic factors associated with timing of reproduction are shared between genetically distinct and geographically distant populations. The genes for thyroid-stimulating hormone receptor (TSHR), the SOX11 transcription factor (SOX11), calmodulin (CALM), and estrogen receptor 2 (ESR2A), all with a significant role in reproductive biology, were among the loci that showed the most consistent association with spawning time throughout the species range. In fact, the same two SNPs located at the 5' end of TSHR showed the most significant association with spawning time in both the east and west Atlantic. We also identified unexpected haplotype sharing between spring-spawning oceanic herring and autumn-spawning populations across the Atlantic Ocean and the Baltic Sea. The genomic regions showing this pattern are unlikely to control spawning time but may be involved in adaptation to ecological factor(s) shared among these populations.

Do estimates of contemporary effective population size tell us what we want to know?

Estimation of effective population size (Ne ) from genetic marker data is a major focus for biodiversity conservation because it is essential to know at what rates inbreeding is increasing and additive genetic variation is lost. But are these the rates assessed when applying commonly used Ne estimation techniques? Here we use recently developed analytical tools and demonstrate that in the case of substructured populations the answer is no. This is because the following: Genetic change can be quantified in several ways reflecting different types of Ne such as inbreeding (NeI ), variance (NeV ), additive genetic variance (NeAV ), linkage disequilibrium equilibrium (NeLD ), eigenvalue (NeE ) and coalescence (NeCo ) effective size. They are all the same for an isolated population of constant size, but the realized values of these effective sizes can differ dramatically in populations under migration. Commonly applied Ne -estimators target NeV or NeLD of individual subpopulations. While such estimates are safe proxies for the rates of inbreeding and loss of additive genetic variation under isolation, we show that they are poor indicators of these rates in populations affected by migration. In fact, both the local and global inbreeding (NeI ) and additive genetic variance (NeAV ) effective sizes are consistently underestimated in a subdivided population. This is serious because these are the effective sizes that are relevant to the widely accepted 50/500 rule for short and long term genetic conservation.  The bias can be infinitely large and is due to inappropriate parameters being estimated when applying theory for isolated populations to subdivided ones.

Are we underestimating the occurrence of sympatric populations?

Sympatric populations are conspecific populations that coexist spatially. They are of interest in evolutionary biology by representing the potential first steps of sympatric speciation and are important to identify and monitor in conservation management. Reviewing the literature pertaining to sympatric populations, we find that most cases of sympatry appear coupled to phenotypic divergence, implying ease of detection. In comparison, phenotypically cryptic, sympatric populations seem rarely documented. We explore the statistical power for detecting population mixtures from genetic marker data, using commonly applied tests for heterozygote deficiency (i.e., Wahlund effect) and the structure software, through computer simulations. We find that both tests are efficient at detecting population mixture only when genetic differentiation is high, sample size and number of genetic markers are reasonable and the sympatric populations happen to occur in similar proportions in the sample. We present an approximate expression based on these experimental factors for the lower limit of FST , beyond which power for structure collapses and only the heterozygote-deficiency tests retain some, although low, power. The findings suggest that cases of cryptic sympatry may have passed unnoticed in population genetic screenings using number of loci typical of the pre-genomics era. Hence, cryptic sympatric populations may be more common than hitherto thought, and we urge more attention being diverted to their detection and characterization.

Effective sizes and time to migration-drift equilibrium in geographically subdivided populations.

Many versions of the effective population size (Ne) exist, and they are important in population genetics in order to quantify rates of change of various characteristics, such as inbreeding, heterozygosity, or allele frequencies. Traditionally, Ne was defined for single, isolated populations, but we have recently presented a mathematical framework for subdivided populations. In this paper we focus on diploid populations with geographic subdivision, and present new theoretical results. We compare the haploid and diploid versions of the inbreeding effective size (NeI) with novel expression for the variance effective size (NeV), and conclude that for local populations NeV is often much smaller than both versions of NeI, whenever they exist. Global NeV of the metapopulation, on the other hand, is close to the haploid NeI and much larger than the diploid NeI. We introduce a new effective size, the additive genetic variance effective size NeAV, which is of particular interest for long term protection of species. It quantifies the rate at which additive genetic variance is lost and we show that this effective size is closely related to the haploid version of NeI. Finally, we introduce a new measure of a population's deviation from migration-drift equilibrium, and apply it to quantify the time it takes to reach this equilibrium. Our findings are of importance for understanding the concept of effective population size in substructured populations and many of the results have applications in conservation biology.

Metapopulation inbreeding dynamics, effective size and subpopulation differentiation--A general analytical approach for diploid organisms.

Motivated by problems in conservation biology we study genetic dynamics in structured populations of diploid organisms (monoecious or dioecious). Our analysis provides an analytical framework that unifies substantial parts of previous work in terms of exact identity by descent (IBD) and identity by state (IBS) recursions. We provide exact conditions under which two structured haploid and diploid populations are equivalent, and some sufficient conditions under which a dioecious diploid population can be treated as a monoecious diploid one. The IBD recursions are used for computing local and metapopulation inbreeding and coancestry effective population sizes and for predictions of several types of fixation indices over different time horizons.

Population-scale sequencing reveals genetic differentiation due to local adaptation in Atlantic herring.

The Atlantic herring (Clupea harengus), one of the most abundant marine fishes in the world, has historically been a critical food source in Northern Europe. It is one of the few marine species that can reproduce throughout the brackish salinity gradient of the Baltic Sea. Previous studies based on few genetic markers have revealed a conspicuous lack of genetic differentiation between geographic regions, consistent with huge population sizes and minute genetic drift. Here, we present a cost-effective genome-wide study in a species that lacks a genome sequence. We first assembled a muscle transcriptome and then aligned genomic reads to the transcripts, creating an "exome assembly," capturing both exons and flanking sequences. We then resequenced pools of fish from a wide geographic range, including the Northeast Atlantic, as well as different regions in the Baltic Sea, aligned the reads to the exome assembly, and identified 440,817 SNPs. The great majority of SNPs showed no appreciable differences in allele frequency among populations; however, several thousand SNPs showed striking differences, some approaching fixation for different alleles. The contrast between low genetic differentiation at most loci and striking differences at others implies that the latter category primarily reflects natural selection. A simulation study confirmed that the distribution of the fixation index F(ST) deviated significantly from expectation for selectively neutral loci. This study provides insights concerning the population structure of an important marine fish and establishes the Atlantic herring as a model for population genetic studies of adaptation and natural selection.

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.

Quasi equilibrium, variance effective size and fixation index for populations with substructure.

In this paper, we develop a method for computing the variance effective size N eV, the fixation index F ST and the coefficient of gene differentiation G ST of a structured population under equilibrium conditions. The subpopulation sizes are constant in time, with migration and reproduction schemes that can be chosen with great flexibility. Our quasi equilibrium approach is conditional on non-fixation of alleles. This is of relevance when migration rates are of a larger order of magnitude than the mutation rates, so that new mutations can be ignored before equilibrium balance between genetic drift and migration is obtained. The vector valued time series of subpopulation allele frequencies is divided into two parts; one corresponding to genetic drift of the whole population and one corresponding to differences in allele frequencies among subpopulations. We give conditions under which the first two moments of the latter, after a simple standardization, are well approximated by quantities that can be explicitly calculated. This enables us to compute approximations of the quasi equilibrium values of N eV, F ST and G ST. Our findings are illustrated for several reproduction and migration scenarios, including the island model, stepping stone models and a model where one subpopulation acts as a demographic reservoir. We also make detailed comparisons with a backward approach based on coalescence probabilities.

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.

Quasi equilibrium approximations of the fixation index under neutrality: the finite and infinite island models.

The fixation index F(ST) and the coefficient of gene differentiation G(ST) are analyzed for the finite island model under short time spans, ignoring mutations. Dividing the reproduction cycle into the three steps-gamete formation, fertilization, and migration-we develop a new approach for computing quasi equilibrium formulas for F(ST) (and G(ST)). Our formulas generalize earlier ones and reveal that the equilibrium value of F(ST) is influenced not only by the migration rate and local effective population size, N(e), but also by the local census size N, particularly so when the migration rate is high. The order of migration and fertilization is found to have a smaller effect on F(ST). A major advantage compared to previous approaches is that stochastic allele frequency of migrants is easily accommodated, thereby avoiding underestimation of F(ST) for large migration rates.

Characteristics of the variance effective population size over time using an age structured model with variable size.

The variance effective population size (NeV) is a key concept in population biology, because it quantifies the microevolutionary process of random genetic drift, and understanding the characteristics of NeV is thus of central importance. Current formulas for NeV for populations with overlapping generations weight age classes according to their reproductive values (i.e. reflecting the contribution of genes from separate age classes to the population growth) to obtain a correct measure of genetic drift when computing the variance of the allele frequency change over time. In this paper, we examine the effect of applying different weights to the age classes using a novel analytical approach for exploring NeV. We consider a haploid organism with overlapping generations and populations of increasing, declining, or constant expected size and stochastic variation with respect to the number of individuals in the separate age classes. We define NeV, as a function of how the age classes are weighted, and of the span between the two points in time, when measuring allele frequency change. With this model, time profiles for NeV can be calculated for populations with various life histories and with fluctuations in life history composition, using different weighting schemes. We examine analytically and by simulations when NeV, using a weighting scheme with respect to reproductive contribution of separate age classes, accurately reflect the variance of the allele frequency change due to genetic drift over time. We show that the discrepancy of NeV, calculated with reproductive values as weights, compared to when individuals are weighted equally, tends to a constant when the time span between the two measurements increases. This constant is zero only for a population with a constant expected population size. Our results confirm that the effect of ignoring overlapping generations, when empirically assessing NeV from allele frequency shifts, gets smaller as the time interval between samples increases. Our model has empirical applications including assessment of (i) time intervals necessary to permit ignoring the effect of overlapping generations for NeV estimation by means of the temporal method, and (ii) effects of life table manipulation on NeV over varying time periods.

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.

Variance effective population size is affected by census size in sub-structured populations.

Measurement of allele frequency shifts between temporally spaced samples has long been used for assessment of effective population size (Ne ), and this 'temporal method' provides estimates of Ne referred to as variance effective size (NeV ). We show that NeV of a local population that belongs to a sub-structured population (a metapopulation) is determined not only by genetic drift and migration rate (m), but also by the census size (Nc ). The realized NeV of a local population can either increase or decrease with increasing m, depending on the relationship between Ne and Nc in isolation. This is shown by explicit mathematical expressions for the factors affecting NeV derived for an island model of migration. We verify analytical results using high-resolution computer simulations, and show that the phenomenon is not restricted to the island model migration pattern. The effect of Nc on the realized NeV of a local subpopulation is most pronounced at high migration rates. We show that Nc only affects local NeV , whereas NeV for the metapopulation as a whole, inbreeding (NeI ), and linkage disequilibrium (NeLD ) effective size are all independent of Nc . Our results provide a possible explanation to the large variation of Ne /Nc ratios reported in the literature, where Ne is frequently estimated by NeV . They are also important for the interpretation of empirical Ne estimates in genetic management where local NeV is often used as a substitute for inbreeding effective size, and we suggest an increased focus on metapopulation NeV as a proxy for NeI .

Range-wide and temporal genomic analyses reveal the consequences of near-extinction in Swedish moose.

Ungulate species have experienced severe declines over the past centuries through overharvesting and habitat loss. Even if many game species have recovered thanks to strict hunting regulation, the genome-wide impacts of overharvesting are still unclear. Here, we examine the temporal and geographical differences in genome-wide diversity in moose (Alces alces) over its whole range in Sweden by sequencing 87 modern and historical genomes. We found limited impact of the 1900s near-extinction event but local variation in inbreeding and load in modern populations, as well as suggestion of a risk of future reduction in genetic diversity and gene flow. Furthermore, we found candidate genes for local adaptation, and rapid temporal allele frequency shifts involving coding genes since the 1980s, possibly due to selective harvesting. Our results highlight that genomic changes potentially impacting fitness can occur over short time scales and underline the need to track both deleterious and selectively advantageous genomic variation.

Genomic dynamics of brown trout populations released to a novel environment.

Population translocations occur for a variety of reasons, from displacement due to climate change to human-induced transfers. Such actions have adverse effects on genetic variation and understanding their microevolutionary consequences requires monitoring. Here, we return to an experimental release of brown trout (Salmo trutta) in order to monitor the genomic effects of population translocations. In 1979, fish from each of two genetically (F ST = 0.16) and ecologically separate populations were simultaneously released, at one point in time, to a lake system previously void of brown trout. Here, whole-genome sequencing of pooled DNA (Pool-seq) is used to characterize diversity within and divergence between the introduced populations and fish inhabiting two lakes downstream of the release sites, sampled 30 years later (c. 5 generations). Present results suggest that while extensive hybridization has occurred, the two introduced populations are unequally represented in the lakes downstream of the release sites. One population, which is ecologically resident in its original habitat, mainly contributes to the lake closest to the release site. The other population, migratory in its natal habitat, is genetically more represented in the lake further downstream. Genomic regions putatively under directional selection in the new habitat are identified, where allele frequencies in both established populations are more similar to the introduced population stemming from a resident population than the migratory one. Results suggest that the microevolutionary consequences of population translocations, for example, hybridization and adaptation, can be rapid and that Pool-seq can be used as an initial tool to monitor genome-wide effects.