Exploring a Pool‐seq‐only approach for gaining population genomic insights in nonmodel species

Developing genomic insights is challenging in nonmodel species for which resources are often scarce and prohibitively costly. Here, we explore the potential of a recently established approach using Pool‐seq data to generate a de novo genome assembly for mining exons, upon which Pool‐seq data are used to estimate population divergence and diversity. We do this for two pairs of sympatric populations of brown trout (Salmo trutta): one naturally sympatric set of populations and another pair of populations introduced to a common environment. We validate our approach by comparing the results to those from markers previously used to describe the populations (allozymes and individual‐based single nucleotide polymorphisms [SNPs]) and from mapping the Pool‐seq data to a reference genome of the closely related Atlantic salmon (Salmo salar). We find that genomic differentiation (F_ST) between the two introduced populations exceeds that of the naturally sympatric populations (F_ST = 0.13 and 0.03 between the introduced and the naturally sympatric populations, respectively), in concordance with estimates from the previously used SNPs. The same level of population divergence is found for the two genome assemblies, but estimates of average nucleotide diversity differ ( ≈ 0.002 and  ≈ 0.001 when mapping to S. trutta and S. salar, respectively), although the relationships between population values are largely consistent. This discrepancy might be attributed to biases when mapping to a haploid condensed assembly made of highly fragmented read data compared to using a high‐quality reference assembly from a divergent species. We conclude that the Pool‐seq‐only approach can be suitable for detecting and quantifying genome‐wide population differentiation, and for comparing genomic diversity in populations of nonmodel species where reference genomes are lacking.     

Population genomic analyses from low‐coverage RAD‐Seq data: a case study on the non‐model cucurbit bottle gourd

Restriction site‐associated DNA sequencing (RAD‐Seq), a next‐generation sequencing‐based genome ‘complexity reduction’ protocol, has been useful in population genomics in species with a reference genome. However, the application of this protocol to natural populations of genomically underinvestigated species, particularly under low‐to‐medium sequencing depth, has not been well justified. In this study, a Bayesian method was developed for calling genotypes from an F₂ population of bottle gourd [Lagenaria siceraria (Mol.) Standl.] to construct a high‐density genetic map. Low‐depth genome shotgun sequencing allowed the assembly of scaffolds/contigs comprising approximately 50% of the estimated genome, of which 922 were anchored for identifying syntenic regions between species. RAD‐Seq genotyping of a natural population comprising 80 accessions identified 3226 single nuclear polymorphisms (SNPs), based on which two sub‐gene pools were suggested for association with fruit shape. The two sub‐gene pools were moderately differentiated, as reflected by the Hudson's F_ST value of 0.14, and they represent regions on LG7 with strikingly elevated F_ST values. Seven‐fold reduction in heterozygosity and two times increase in LD (r²) were observed in the same region for the round‐fruited sub‐gene pool. Outlier test suggested the locus LX3405 on LG7 to be a candidate site under selection. Comparative genomic analysis revealed that the cucumber genome region syntenic to the high F_ST island on LG7 harbors an ortholog of the tomato fruit shape gene OVATE. Our results point to a bright future of applying RAD‐Seq to population genomic studies for non‐model species even under low‐to‐medium sequencing efforts. The genomic resources provide valuable information for cucurbit genome research.     

Utility of pooled sequencing for association mapping in nonmodel organisms

High‐density genome‐wide sequencing increases the likelihood of discovering genes of major effect and genomic structural variation in organisms. While there is an increasing availability of reference genomes across broad taxa, the greatest limitation to whole‐genome sequencing of multiple individuals continues to be the costs associated with sequencing. To alleviate excessive costs, pooling multiple individuals with similar phenotypes and sequencing the homogenized DNA (Pool‐Seq) can achieve high genome coverage, but at the loss of individual genotypes. Although Pool‐Seq has been an effective method for association mapping in model organisms, it has not been frequently utilized in natural populations. To extend bioinformatic tools for rapid implementation of Pool‐Seq data in nonmodel organisms, we developed a pipeline called PoolParty and illustrate its effectiveness in genetic association mapping. Alignment expectations based on five pooled Chinook salmon (Oncorhynchus tshawytscha) libraries showed that approximately 48% genome coverage per library could be achieved with reasonable sequencing effort. We additionally examined male and female O. tshawytscha libraries to illustrate how Pool‐Seq techniques can successfully map known genes associated with functional differences among sexes such as growth hormone 2. Finally, we compared pools of individuals of different spawning ages for each sex to discover novel genes involved with age at maturity in O. tshawytscha such as opsin4 and transmembrane protein19. While not appropriate for every system, Pool‐Seq data processed by the PoolParty pipeline is a practical method for identifying genes of major effect in nonmodel organisms when high genome coverage is necessary and cost is a limiting factor.     

Genome‐wide selection components analysis in a fish with male pregnancy

A major goal of evolutionary biology is to identify the genome‐level targets of natural and sexual selection. With the advent of next‐generation sequencing, whole‐genome selection components analysis provides a promising avenue in the search for loci affected by selection in nature. Here, we implement a genome‐wide selection components analysis in the sex role reversed Gulf pipefish, Syngnathus scovelli. Our approach involves a double‐digest restriction‐site associated DNA sequencing (ddRAD‐seq) technique, applied to adult females, nonpregnant males, pregnant males, and their offspring. An F_ST comparison of allele frequencies among these groups reveals 47 genomic regions putatively experiencing sexual selection, as well as 468 regions showing a signature of differential viability selection between males and females. A complementary likelihood ratio test identifies similar patterns in the data as the F_ST analysis. Sexual selection and viability selection both tend to favor the rare alleles in the population. Ultimately, we conclude that genome‐wide selection components analysis can be a useful tool to complement other approaches in the effort to pinpoint genome‐level targets of selection in the wild.     

Genome‐wide single nucleotide polymorphisms reveal population history and adaptive divergence in wild guppies

Adaptation of guppies (Poecilia reticulata) to contrasting upland and lowland habitats has been extensively studied with respect to behaviour, morphology and life history traits. Yet population history has not been studied at the whole‐genome level. Although single nucleotide polymorphisms (SNPs) are the most abundant form of variation in many genomes and consequently very informative for a genome‐wide picture of standing natural variation in populations, genome‐wide SNP data are rarely available for wild vertebrates. Here we use genetically mapped SNP markers to comprehensively survey genetic variation within and among naturally occurring guppy populations from a wide geographic range in Trinidad and Venezuela. Results from three different clustering methods, Neighbor‐net, principal component analysis (PCA) and Bayesian analysis show that the population substructure agrees with geographic separation and largely with previously hypothesized patterns of historical colonization. Within major drainages (Caroni, Oropouche and Northern), populations are genetically similar, but those in different geographic regions are highly divergent from one another, with some indications of ancient shared polymorphisms. Clear genomic signatures of a previous introduction experiment were seen, and we detected additional potential admixture events. Headwater populations were significantly less heterozygous than downstream populations. Pairwise F_ST values revealed marked differences in allele frequencies among populations from different regions, and also among populations within the same region. F_ST outlier methods indicated some regions of the genome as being under directional selection. Overall, this study demonstrates the power of a genome‐wide SNP data set to inform for studies on natural variation, adaptation and evolution of wild populations     

Inference of chromosomal inversion dynamics from Pool‐Seq data in natural and laboratory populations of Drosophila melanogaster

Sequencing of pools of individuals (Pool‐Seq) represents a reliable and cost‐effective approach for estimating genome‐wide SNP and transposable element insertion frequencies. However, Pool‐Seq does not provide direct information on haplotypes so that, for example, obtaining inversion frequencies has not been possible until now. Here, we have developed a new set of diagnostic marker SNPs for seven cosmopolitan inversions in Drosophila melanogaster that can be used to infer inversion frequencies from Pool‐Seq data. We applied our novel marker set to Pool‐Seq data from an experimental evolution study and from North American and Australian latitudinal clines. In the experimental evolution data, we find evidence that positive selection has driven the frequencies of In(3R)C and In(3R)Mo to increase over time. In the clinal data, we confirm the existence of frequency clines for In(2L)t, In(3L)P and In(3R)Payne in both North America and Australia and detect a previously unknown latitudinal cline for In(3R)Mo in North America. The inversion markers developed here provide a versatile and robust tool for characterizing inversion frequencies and their dynamics in Pool‐Seq data from diverse D. melanogaster populations.     

Comparing Pool‐seq,Rapture, and GBS genotyping for inferring weak population structure: The American lobster (Homarus americanus) as a case study

Unraveling genetic population structure is challenging in species potentially characterized by large population size and high dispersal rates, often resulting in weak genetic differentiation. Genotyping a large number of samples can improve the detection of subtle genetic structure, but this may substantially increase sequencing cost and downstream bioinformatics computational time. To overcome this challenge, alternative, cost‐effective sequencing approaches, namely Pool‐seq and Rapture, have been developed. We empirically measured the power of resolution and congruence of these two methods in documenting weak population structure in nonmodel species with high gene flow comparatively to a conventional genotyping‐by‐sequencing (GBS) approach. For this, we used the American lobster (Homarus americanus) as a case study. First, we found that GBS, Rapture, and Pool‐seq approaches gave similar allele frequency estimates (i.e., correlation coefficient over 0.90) and all three revealed the same weak pattern of population structure. Yet, Pool‐seq data showed F_ST estimates three to five times higher than GBS and Rapture, while the latter two methods returned similar F_ST estimates, indicating that individual‐based approaches provided more congruent results than Pool‐seq. We conclude that despite higher costs, GBS and Rapture are more convenient approaches to use in the case of species exhibiting very weak differentiation. While both GBS and Rapture approaches provided similar results with regard to estimates of population genetic parameters, GBS remains more cost‐effective in project involving a relatively small numbers of genotyped individuals (e.g., <1,000). Overall, this study illustrates the complexity of estimating genetic differentiation and other summary statistics in complex biological systems characterized by large population size and migration rates.     

The genomic footprint of climate adaptation in Chironomus riparius

The gradual heterogeneity of climatic factors poses varying selection pressures across geographic distances that leave signatures of clinal variation in the genome. Separating signatures of clinal adaptation from signatures of other evolutionary forces, such as demographic processes, genetic drift and adaptation, to nonclinal conditions of the immediate local environment is a major challenge. Here, we examine climate adaptation in five natural populations of the harlequin fly Chironomus riparius sampled along a climatic gradient across Europe. Our study integrates experimental data, individual genome resequencing, Pool‐Seq data and population genetic modelling. Common‐garden experiments revealed significantly different population growth rates at test temperatures corresponding to the population origin along the climate gradient, suggesting thermal adaptation on the phenotypic level. Based on a population genomic analysis, we derived empirical estimates of historical demography and migration. We used an F_ST outlier approach to infer positive selection across the climate gradient, in combination with an environmental association analysis. In total, we identified 162 candidate genes as genomic basis of climate adaptation. Enriched functions among these candidate genes involved the apoptotic process and molecular response to heat, as well as functions identified in studies of climate adaptation in other insects. Our results show that local climate conditions impose strong selection pressures and lead to genomic adaptation despite strong gene flow. Moreover, these results imply that selection to different climatic conditions seems to converge on a functional level, at least between different insect species.     

Localizing FST outliers on a QTL map reveals evidence for large genomic regions of reduced gene exchange during speciation‐with‐gene‐flow

Populations that maintain phenotypic divergence in sympatry typically show a mosaic pattern of genomic divergence, requiring a corresponding mosaic of genomic isolation (reduced gene flow). However, mechanisms that could produce the genomic isolation required for divergence‐with‐gene‐flow have barely been explored, apart from the traditional localized effects of selection and reduced recombination near centromeres or inversions. By localizing F_ST outliers from a genome scan of wild pea aphid host races on a Quantitative Trait Locus (QTL) map of key traits, we test the hypothesis that between‐population recombination and gene exchange are reduced over large ‘divergence hitchhiking’ (DH) regions. As expected under divergence hitchhiking, our map confirms that QTL and divergent markers cluster together in multiple large genomic regions. Under divergence hitchhiking, the nonoutlier markers within these regions should show signs of reduced gene exchange relative to nonoutlier markers in genomic regions where ongoing gene flow is expected. We use this predicted difference among nonoutliers to perform a critical test of divergence hitchhiking. Results show that nonoutlier markers within clusters of F_ST outliers and QTL resolve the genetic population structure of the two host races nearly as well as the outliers themselves, while nonoutliers outside DH regions reveal no population structure, as expected if they experience more gene flow. These results provide clear evidence for divergence hitchhiking, a mechanism that may dramatically facilitate the process of speciation‐with‐gene‐flow. They also show the power of integrating genome scans with genetic analyses of the phenotypic traits involved in local adaptation and population divergence.     

Identifying outlier loci in admixed and in continuous populations using ancestral population differentiation statistics

Finding genetic signatures of local adaptation is of great interest for many population genetic studies. Common approaches to sorting selective loci from their genomic background focus on the extreme values of the fixation index, F_ST, across loci. However, the computation of the fixation index becomes challenging when the population is genetically continuous, when predefining subpopulations is a difficult task, and in the presence of admixed individuals in the sample. In this study, we present a new method to identify loci under selection based on an extension of the F_ST statistic to samples with admixed individuals. In our approach, F_ST values are computed from the ancestry coefficients obtained with ancestry estimation programs. More specifically, we used factor models to estimate F_ST, and we compared our neutrality tests with those derived from a principal component analysis approach. The performances of the tests were illustrated using simulated data and by re‐analysing genomic data from European lines of the plant species Arabidopsis thaliana and human genomic data from the population reference sample, POPRES.     

Assembly and RNA‐free annotation of highly heterozygous genomes: The case of the thick‐billed murre (Uria lomvia)

Thanks to a dramatic reduction in sequencing costs followed by a rapid development of bioinformatics tools, genome assembly and annotation have become accessible to many researchers in recent years. Among tetrapods, birds have genomes that display many features that facilitate their assembly and annotation, such as small genome size, low number of repeats and highly conserved genomic structure. However, we found that high genomic heterozygosity could have a great impact on the quality of the genome assembly of the thick‐billed murre (Uria lomvia), an arctic colonial seabird. In this study, we tested the performance of three genome assemblers, ray /sscape , soapdenovo 2 and platanus , in assembling the highly heterozygous genome of the thick‐billed murre. Our results show that platanus , an assembler specifically designed for heterozygous genomes, outperforms the other two approaches and produces a highly contiguous (N50 = 15.8 Mb) and complete genome assembly (93% presence of genes from the Benchmarking Universal Single Copy Ortholog [BUSCO] gene set). Additionally, we annotated the thick‐billed murre genome using a homology‐based approach that takes advantage of the genomic resources available for birds and other taxa. Our study will be useful for those researchers who are approaching assembly and annotation of highly heterozygous genomes, or genomes of species of conservation concern, and/or who have limited financial resources.     

Population genomic footprints of selection and associations with climate in natural populations of Arabidopsis halleri from the Alps

Natural genetic variation is essential for the adaptation of organisms to their local environment and to changing environmental conditions. Here, we examine genomewide patterns of nucleotide variation in natural populations of the outcrossing herb Arabidopsis halleri and associations with climatic variation among populations in the Alps. Using a pooled population sequencing (Pool‐Seq) approach, we discovered more than two million SNPs in five natural populations and identified highly differentiated genomic regions and SNPs using F_ST‐based analyses. We tested only the most strongly differentiated SNPs for associations with a nonredundant set of environmental factors using partial Mantel tests to identify topo‐climatic factors that may underlie the observed footprints of selection. Possible functions of genes showing signatures of selection were identified by Gene Ontology analysis. We found 175 genes to be highly associated with one or more of the five tested topo‐climatic factors. Of these, 23.4% had unknown functions. Genetic variation in four candidate genes was strongly associated with site water balance and solar radiation, and functional annotations were congruent with these environmental factors. Our results provide a genomewide perspective on the distribution of adaptive genetic variation in natural plant populations from a highly diverse and heterogeneous alpine environment.     

Pool‐hmm: a Python program for estimating the allele frequency spectrum and detecting selective sweeps from next generation sequencing of pooled samples

Due to its cost effectiveness, next generation sequencing of pools of individuals (Pool‐Seq) is becoming a popular strategy for genome‐wide estimation of allele frequencies in population samples. As the allele frequency spectrum provides information about past episodes of selection, Pool‐seq is also a promising design for genomic scans for selection. However, no software tool has yet been developed for selection scans based on Pool‐Seq data. We introduce Pool‐hmm, a Python program for the estimation of allele frequencies and the detection of selective sweeps in a Pool‐Seq sample. Pool‐hmm includes several options that allow a flexible analysis of Pool‐Seq data, and can be run in parallel on several processors. Source code and documentation for Pool‐hmm is freely available at https://qgsp.jouy.inra.fr/ .     

Sequence‐tagged high‐density genetic maps of Zoysia japonica provide insights into genome evolution in Chloridoideae

Zoysiagrass (Zoysia spp.), belonging to the genus Zoysia in the subfamily Chloridoideae, is widely used in domestic lawns, sports fields and as forage. We constructed high‐density genetic maps of Zoysia japonica using a restriction site‐associated DNA sequencing (RAD‐Seq) approach and an F₁ mapping population derived from a cross between ‘Carrizo’ and ‘El Toro’. Two linkage maps were constructed, one for each of the parents. A map consisting of 2408 RAD markers distributed on 21 linkage groups was constructed for ‘Carrizo’. Another map with 1230 RAD markers mapped on 20 linkage groups was constructed for ‘El Toro’. The average distance between adjacent markers of the two maps was at 0.56 and 1.4 cM, respectively. Comparative genomics analysis was carried out among zoysiagrass, rice and sorghum genomes and a highly conserved collinearity in the gene order was observed among the three genomes. Chromosome collinearity was disrupted at centromeric regions for each chromosome pair between zoysiagrass and sorghum genomes. However, no obvious synteny gaps were observed across the centromeric regions between zoysiagrass and rice genomes. Two homologous chromosomes for each of the 10 sorghum chromosomes were found in the zoysiagrass genome, indicating an allotetraploid origin for zoysiagrass. The reduction of the basic chromosome number from 12 to 10 in chloridoids and panicoids took place via independent single‐step nested chromosome fusion events after the two subfamilies diverged from a common ancestor. The genetic maps will assist in genome sequence assembly, targeted gene isolation and comparative genomic analyses among grasses.     

High degree of genetic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus)

Populations of widespread marine organisms are typically characterized by a low degree of genetic differentiation in neutral genetic markers, but much less is known about differentiation in genes whose functional roles are associated with specific selection regimes. To uncover possible adaptive population divergence and heterogeneous genomic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus), we used a candidate gene‐based genome‐scan approach to analyse variability in 138 microsatellite loci located within/close to (<6 kb) functionally important genes in samples collected from ten geographic locations. The degree of genetic differentiation in markers classified as neutral or under balancing selection—as determined with several outlier detection methods—was low (F_ST = 0.033 or 0.011, respectively), whereas average F_ST for directionally selected markers was significantly higher (F_ST = 0.097). Clustering analyses provided support for genomic and geographic heterogeneity in selection: six genetic clusters were identified based on allele frequency differences in the directionally selected loci, whereas four were identified with the neutral loci. Allelic variation in several loci exhibited significant associations with environmental variables, supporting the conjecture that temperature and salinity, but not optic conditions, are important drivers of adaptive divergence among populations. In general, these results suggest that in spite of the high degree of physical connectivity and gene flow as inferred from neutral marker genes, marine stickleback populations are strongly genetically structured in loci associated with functionally relevant genes.     

QST–FST comparisons with unbalanced half‐sib designs

Q_ST, a measure of quantitative genetic differentiation among populations, is an index that can suggest local adaptation if Q_ST for a trait is sufficiently larger than the mean F_ST of neutral genetic markers. A previous method by Whitlock and Guillaume derived a simulation resampling approach to statistically test for a difference between Q_ST and F_ST, but that method is limited to balanced data sets with offspring related as half‐sibs through shared fathers. We extend this approach (i) to allow for a model more suitable for some plant populations or breeding designs in which offspring are related through mothers (assuming independent fathers for each offspring; half‐sibs by dam); and (ii) by explicitly allowing for unbalanced data sets. The resulting approach is made available through the R package QstFstComp.     

Chironomus riparius (Diptera) genome sequencing reveals the impact of minisatellite transposable elements on population divergence

Active transposable elements (TEs) may result in divergent genomic insertion and abundance patterns among conspecific populations. Upon secondary contact, such divergent genetic backgrounds can theoretically give rise to classical Dobzhansky–Muller incompatibilities (DMI), thus contributing to the evolution of endogenous genetic barriers and eventually causing population divergence. We investigated differential TE abundance among conspecific populations of the nonbiting midge Chironomus riparius and evaluated their potential role in causing endogenous genetic incompatibilities between these populations. We focussed on a Chironomus‐specific TE, the minisatellite‐like Cla‐element, whose activity is associated with speciation in the genus. Using a newly generated and annotated draft genome for a genomic study with five natural C. riparius populations, we found highly population‐specific TE insertion patterns with many private insertions. A significant correlation of the pairwise F_ST estimated from genomewide single‐nucleotide polymorphisms (SNPs) and the F_ST estimated from TEs is consistent with drift as the major force driving TE population differentiation. However, the significantly higher Cla‐element F_ST level due to a high proportion of differentially fixed Cla‐element insertions also indicates selection against segregating (i.e. heterozygous) insertions. With reciprocal crossing experiments and fluorescent in situ hybridization of Cla‐elements to polytene chromosomes, we documented phenotypic effects on female fertility and chromosomal mispairings. We propose that the inferred negative selection on heterozygous Cla‐element insertions may cause endogenous genetic barriers and therefore acts as DMI among C. riparius populations. The intrinsic genomic turnover exerted by TEs may thus have a direct impact on population divergence that is operationally different from drift and local adaptation.     

Divergent selection and drift shape the genomes of two avian sister species spanning a saline–freshwater ecotone

The role of species divergence due to ecologically based divergent selection—or ecological speciation—in generating and maintaining biodiversity is a central question in evolutionary biology. Comparison of the genomes of phylogenetically related taxa spanning a selective habitat gradient enables discovery of divergent signatures of selection and thereby provides valuable insight into the role of divergent ecological selection in speciation. Tidal marsh ecosystems provide tractable opportunities for studying organisms' adaptations to selective pressures that underlie ecological divergence. Sharp environmental gradients across the saline–freshwater ecotone within tidal marshes present extreme adaptive challenges to terrestrial vertebrates. Here, we sequence 20 whole genomes of two avian sister species endemic to tidal marshes—the saltmarsh sparrow (Ammospiza caudacutus) and Nelson's sparrow (A. nelsoni)—to evaluate the influence of selective and demographic processes in shaping genome‐wide patterns of divergence. Genome‐wide divergence between these two recently diverged sister species was notably high (genome‐wide F_ST = 0.32). Against a background of high genome‐wide divergence, regions of elevated divergence were widespread throughout the genome, as opposed to focused within islands of differentiation. These patterns may be the result of genetic drift resulting from past tidal march colonization events in conjunction with divergent selection to different environments. We identified several candidate genes that exhibited elevated divergence between saltmarsh and Nelson's sparrows, including genes linked to osmotic regulation, circadian rhythm, and plumage melanism—all putative candidates linked to adaptation to tidal marsh environments. These findings provide new insights into the roles of divergent selection and genetic drift in generating and maintaining biodiversity.