The impact of library preparation protocols on the consistency of allele frequency estimates in Pool‐Seq data

Sequencing pools of individuals (Pool‐Seq) is a cost‐effective method to determine genome‐wide allele frequency estimates. Given the importance of meta‐analyses combining data sets, we determined the influence of different genomic library preparation protocols on the consistency of allele frequency estimates. We found that typically no more than 1% of the variation in allele frequency estimates could be attributed to differences in library preparation. Also read length had only a minor effect on the consistency of allele frequency estimates. By far, the most pronounced influence could be attributed to sequence coverage. Increasing the coverage from 30‐ to 50‐fold improved the consistency of allele frequency estimates by at least 27%. We conclude that Pool‐Seq data can be easily combined across different library preparation methods, but sufficient sequence coverage is key to reliable results.     

A new method for studying population genetics of cyst nematodes based on Pool‐Seq and genomewide allele frequency analysis

Cyst nematodes are important agricultural pests responsible for billions of dollars of losses each year. Plant resistance is the most effective management tool, but it requires a close monitoring of population genetics. Current technologies for pathotyping and genotyping cyst nematodes are time‐consuming, expensive and imprecise. In this study, we capitalized on the reproduction mode of cyst nematodes to develop a simple population genetic analysis pipeline based on genotyping‐by‐sequencing and Pool‐Seq. This method yielded thousands of SNPs and allowed us to study the relationships between populations of different origins or pathotypes. Validation of the method on well‐characterized populations also demonstrated that it was a powerful and accurate tool for population genetics. The genomewide allele frequencies of 23 populations of golden nematode, from nine countries and representing the five known pathotypes, were compared. A clear separation of the pathotypes and fine genetic relationships between and among global populations were obtained using this method. In addition to being powerful, this tool has proven to be very time‐ and cost‐efficient and could be applied to other cyst nematode species.     

Experimental evolution reveals habitat‐specific fitness dynamics among Wolbachia clades in Drosophila melanogaster

The diversity and infection dynamics of the endosymbiont Wolbachia can be influenced by many factors, such as transmission rate, cytoplasmic incompatibility, environment, selection and genetic drift. The interplay of these factors in natural populations can result in heterogeneous infection patterns with substantial differences between populations and strains. The causes of these heterogeneities are not yet understood, partly due to the complexity of natural environments. We present experimental evolution as a new approach to study Wolbachia infection dynamics in replicate populations exposed to a controlled environment. A natural Drosophila melanogaster population infected with strains of Wolbachia belonging to different clades evolved in two laboratory environments (hot and cold) for 1.5 years. In both treatments, the rate of Wolbachia infection increased until fixation. In the hot environment, the relative frequency of different Wolbachia clades remained stable over 37 generations. In the cold environment, however, we observed marked changes in the composition of the Wolbachia population: within 15 generations, one Wolbachia clade increased more than 50% in frequency, whereas the other two clades decreased in frequency, resulting in the loss of one clade. The frequency change was highly reproducible not only among replicates, but also when flies that evolved for 42 generations in the hot environment were transferred to the cold environment. These results document how environmental factors can affect the composition of Wolbachia in D. melanogaster. The high reproducibility of the pattern suggests that experimental evolution studies can efficiently determine the functional basis of habitat‐specific fitness among Wolbachia strains.     

Genome scans on experimentally evolved populations reveal candidate regions for adaptation to plant resistance in the potato cyst nematode Globodera pallida

Improving resistance durability involves to be able to predict the adaptation speed of pathogen populations. Identifying the genetic bases of pathogen adaptation to plant resistances is a useful step to better understand and anticipate this phenomenon. Globodera pallida is a major pest of potato crop for which a resistance QTL, GpaV_vrn, has been identified in Solanum vernei. However, its durability is threatened as G. pallida populations are able to adapt to the resistance in few generations. The aim of this study was to investigate the genomic regions involved in the resistance breakdown by coupling experimental evolution and high‐density genome scan. We performed a whole‐genome resequencing of pools of individuals (Pool‐Seq) belonging to G. pallida lineages derived from two independent populations having experimentally evolved on susceptible and resistant potato cultivars. About 1.6 million SNPs were used to perform the genome scan using a recent model testing for adaptive differentiation and association to population‐specific covariables. We identified 275 outliers and 31 of them, which also showed a significant reduction in diversity in adapted lineages, were investigated for their genic environment. Some candidate genomic regions contained genes putatively encoding effectors and were enriched in SPRYSECs, known in cyst nematodes to be involved in pathogenicity and in (a)virulence. Validated candidate SNPs will provide a useful molecular tool to follow frequencies of virulence alleles in natural G. pallida populations and define efficient strategies of use of potato resistances maximizing their durability.     

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.     

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.     

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.     

Genomics of natural populations: Evolutionary forces that establish and maintain gene arrangements in Drosophila pseudoobscura

The evolution of complex traits in heterogeneous environments may shape the order of genes within chromosomes. Drosophila pseudoobscura has a rich gene arrangement polymorphism that allows one to test evolutionary genetic hypotheses about how chromosomal inversions are established in populations. D. pseudoobscura has >30 gene arrangements on a single chromosome that were generated through a series of overlapping inversion mutations with >10 inversions with appreciable frequencies and wide geographic distributions. This study analyses the genomic sequences of 54 strains of Drosophila pseudoobscura that carry one of six different chromosomal arrangements to test whether (i) genetic drift, (ii) hitchhiking with an adaptive allele, (iii) direct effects of inversions to create gene disruptions caused by breakpoints, or (iv) indirect effects of inversions in limiting the formation of recombinant gametes are responsible for the establishment of new gene arrangements. We found that the inversion events do not disrupt the structure of protein coding genes at the breakpoints. Population genetic analyses of 2,669 protein coding genes identified 277 outlier loci harbouring elevated frequencies of arrangement‐specific derived alleles. Significant linkage disequilibrium occurs among distant loci interspersed between regions with low levels of association indicating that distant allelic combinations are held together despite shared polymorphism among arrangements. Outlier genes showing evidence of genetic differentiation between arrangements are enriched for sensory perception and detoxification genes. The data presented here support the indirect effect of inversion hypothesis where chromosomal inversions are favoured because they maintain linked associations among multilocus allelic combinations among different arrangements.     

The adaptive significance of chromosomal inversion polymorphisms in Drosophila melanogaster

Chromosomal inversions, structural mutations that reverse a segment of a chromosome, cause suppression of recombination in the heterozygous state. Several studies have shown that inversion polymorphisms can form clines or fluctuate predictably in frequency over seasonal time spans. These observations prompted the hypothesis that chromosomal rearrangements might be subject to spatially and/or temporally varying selection. Here, we review what has been learned about the adaptive significance of inversion polymorphisms in the vinegar fly Drosophila melanogaster, the species in which they were first discovered by Sturtevant in 1917. A large body of work provides compelling evidence that several inversions in this system are adaptive; however, the precise selective mechanisms that maintain them polymorphic in natural populations remain poorly understood. Recent advances in population genomics, modelling and functional genetics promise to greatly improve our understanding of this long‐standing and fundamental problem in the near future.     

Inference of sex‐specific expansion patterns in human populations from Y‐chromosome polymorphism

Studying the current distribution of genetic diversity in humans has important implications for our understanding of the history of our species. We analyzed a set of linked STR and SNP loci from the paternally inherited Y chromosome to infer the past demography of 55 African and Eurasian populations, using both the parametric and nonparametric coalescent‐based methods implemented in the BEAST application. We inferred expansion events in most sedentary farmer populations, while we found constant effective population sizes for both nomadic hunter‐gatherers and seminomadic herders. Our results differed, on several aspects, from previous results on mtDNA and autosomal markers. First, we found more recent expansion patterns in Eurasia than in Africa. This discrepancy, substantially stronger than the ones found with the other kind of markers, may result from a lower effective population size for men, which might have made male‐transmitted markers more sensitive to the out‐of‐Africa bottleneck. Second, we found expansion signals only for sedentary farmers but not for nomadic herders in Central Asia, while these signals were found for both kind of populations in this area when using mtDNA or autosomal markers. Expansion signals in this area may result from spatial expansion processes and may have been erased for the Y chromosome among the herders because of restricted male gene flow. Am J Phys Anthropol 157:217–225, 2015. © 2015 Wiley Periodicals, Inc.     

Population genomic analysis uncovers African and European admixture in Drosophila melanogaster populations from the south‐eastern United States and Caribbean Islands

Drosophila melanogaster is postulated to have colonized North America in the past several 100 years in two waves. Flies from Europe colonized the east coast United States while flies from Africa inhabited the Caribbean, which if true, make the south‐east US and Caribbean Islands a secondary contact zone for African and European D. melanogaster. This scenario has been proposed based on phenotypes and limited genetic data. In our study, we have sequenced individual whole genomes of flies from populations in the south‐east US and Caribbean Islands and examined these populations in conjunction with population sequences from the west coast US, Africa, and Europe. We find that west coast US populations are closely related to the European population, likely reflecting a rapid westward expansion upon first settlements into North America. We also find genomic evidence of African and European admixture in south‐east US and Caribbean populations, with a clinal pattern of decreasing proportions of African ancestry with higher latitude. Our genomic analysis of D. melanogaster populations from the south‐east US and Caribbean Islands provides more evidence for the Caribbean Islands as the source of previously reported novel African alleles found in other east coast US populations. We also find the border between the south‐east US and the Caribbean island to be the admixture hot zone where distinctly African‐like Caribbean flies become genomically more similar to European‐like south‐east US flies. Our findings have important implications for previous studies examining the generation of east coast US clines via selection.     

Rapid divergence and convergence of life‐history in experimentally evolved Drosophila melanogaster

Laboratory selection experiments are alluring in their simplicity, power, and ability to inform us about how evolution works. A longstanding challenge facing evolution experiments with metazoans is that significant generational turnover takes a long time. In this work, we present data from a unique system of experimentally evolved laboratory populations of Drosophila melanogaster that have experienced three distinct life‐history selection regimes. The goal of our study was to determine how quickly populations of a certain selection regime diverge phenotypically from their ancestors, and how quickly they converge with independently derived populations that share a selection regime. Our results indicate that phenotypic divergence from an ancestral population occurs rapidly, within dozens of generations, regardless of that population's evolutionary history. Similarly, populations sharing a selection treatment converge on common phenotypes in this same time frame, regardless of selection pressures those populations may have experienced in the past. These patterns of convergence and divergence emerged much faster than expected, suggesting that intermediate evolutionary history has transient effects in this system. The results we draw from this system are applicable to other experimental evolution projects, and suggest that many relevant questions can be sufficiently tested on shorter timescales than previously thought.     

Investigating the genomic basis of discrete phenotypes using a Pool‐Seq‐only approach: New insights into the genetics underlying colour variation in diverse taxa

While large‐scale genomic approaches are increasingly revealing the genetic basis of polymorphic phenotypes such as colour morphs, such approaches are almost exclusively conducted in species with high‐quality genomes and annotations. Here, we use Pool‐Seq data for both genome assembly and SNP frequency estimation, followed by scanning for F_ST outliers to identify divergent genomic regions. Using paired‐end, short‐read sequencing data from two groups of individuals expressing divergent phenotypes, we generate a de novo rough‐draft genome, identify SNPs and calculate genomewide F_ST differences between phenotypic groups. As genomes generated by Pool‐Seq data are highly fragmented, we also present an approach for super‐scaffolding contigs using existing protein‐coding data sets. Using this approach, we reanalysed genomic data from two recent studies of birds and butterflies investigating colour pattern variation and replicated their core findings, demonstrating the accuracy and power of a Pool‐Seq‐only approach. Additionally, we discovered new regions of high divergence and new annotations that together suggest novel parallels between birds and butterflies in the origins of their colour pattern variation.     

Abdominal pigmentation in Drosophila melanogaster females from natural Indian populations

Females of Drosophila melanogaster collected from five geographically distant populations in India were analysed for the intensity of pigmentation in the 5th, 6th and 7th segments of the abdomen. In all three segments, this intensity was found to vary among individuals of any given population, and, furthermore, different populations differ with respect to this phenotypic trait. Statistical analysis revealed significant intra- and interpopulational variation. A clinical pattern was detected: females from populations closer to the equator tended to have lighter cuticle, in which case differences between the three segments could not be detected and all three segments responded both independently and jointly to latitudinal variation, as indicated by a statistically significant positive correlation between latitude and pigmentation score. This is the first report on abdominal pigmentation analysis in natural populations of D. melanogaster that provides evidence that phenotypic flexibility reflects temperature differences, as a result of which abdominal pigmentation shows geographic differentiation.     

Palaeoclimatic variation, adaptation and biogeography of inversion polymorphisms in natural populations of Drosophila robusta

Studies of natural and sexual selection in wild populations of Drosophila have historically provided strong inference for the maintenance of inversion polymorphism. Analysis of geographical variation in the Drosophila robusta chromosomal data collected over more than 50 years from 133 natural populations across eastern North America has confirmed several north–south and east–west clines in the frequencies of some gene arrangements and linked arrangement combinations. Patterns of geographical variation, including several north–south clines, revealed by regression and spatial autocorrelation analyses are concordant with palaeoclimatic shifts, Pleistocene glaciations and historical changes in the composition of North American forest communities. Because D. robusta is a sap-breeder, using the microbe-infested sap exudates of a number of deciduous tree species in which they carry out their life cycle, shifts in climate and palaeovegetation types since the formation of the eastern deciduous forests in the Miocene are hypothesized to be major factors influencing patterns of inversion polymorphisms across the range of this drosophilid species. In areas where sharp deviations in frequencies have been observed, particularly in the mid-western and western portions of the range, these divisions parallel historical geographical disjunctions in the species range that have yet to promote divergence and species formation despite the long history of D. robusta in North America.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 395–411.     

Increase in the degree of inversion polymorphism in Drosophila bipectinata populations transfereed to laboratory conditions

Thirteen laboratory populations (six mass cultures and seven isofemale lines) of Drosophila bipectinata were established from files collected from six different geographic localities (four in the north and two in the south) in India. These mass cultures and isofemale lines were maintained in food bottles in the laboratory for varying number of generations by transferring about 50 files (females and males in equal number) in each generation. After several generations, all the laboratory populations were analysed chromosomally to determine the frequency of different inversions. The results indicate that all the laboratory populations remained chromosomally polymorphic due to the persistence of inversions which were originally present ion the populations. The quantitative data on the frequency of inverted gene orders and the level of inversions heterozygosity in laboratory populations are compared with those in corresponding natural populations. This comparison clearly shows that there is a considerable increase in the frequency of inverted gene orders and the level of inversion heterozygosity in laboratory populations when compared with corresponding natural populations. This is most likely to be due to selective advantage of inversion heterozygotes under stringent competition in laboratory populations. Thus heterotic buffering is associated with chromosome inversion of Drosophila bipectinata.