Eva Puerma

Multilocus analysis of variation using a large empirical data set: phenylpropanoid pathway genes in Arabidopsis thaliana

Detecting the signature of adaptation on nucleotide variation is often difficult in species that like Arabidopsis thaliana might have a complex demographic history. Recent re‐sequencing surveys in this species provided genome‐wide information that would mainly reflect its demographic history. We have used a large empirical data set (LED) as well as multilocus coalescent simulations to analyse sequence variation at loci involved in the phenylpropanoid pathway of this species. We surveyed and examined DNA sequence variation at nine of these loci (about 19.7 kb) in 23 accessions of A. thaliana and one accession of its closely related species Arabidopsis lyrata. Nucleotide variation was lower at nonsynonymous sites than at silent sites in all loci, indicating generalized functional constraint at the protein level. No association between variation and position in the metabolic pathway was detected. When the data were contrasted against the standard neutral model, significant deviations for silent variation were detected with Tajimas D, Fus FS and Fay and Wus H multilocus test statistics. These deviations were in the same direction than in previous large‐scale multilocus analyses, suggesting a genome‐wide effect. When the nine‐locus data set was contrasted against the large empirical data set, the level (Wattersons θ) and pattern of variation (Tajimas D) detected in these loci did not deviate either at the single‐locus or multilocus level from the corresponding empirical distributions. These results would support an important role of the demographic history of A. thaliana in shaping nucleotide variation at the nine studied phenylpropanoid loci. The potential and limitations of the empirical distribution approach are discussed.

Characterization of the breakpoints of a polymorphic inversion complex detects strict and broad breakpoint reuse at the molecular level

Inversions are an integral part of structural variation within species, and they play a leading role in genome reorganization across species. Work at both the cytological and genome sequence levels has revealed heterogeneity in the distribution of inversion breakpoints, with some regions being recurrently used. Breakpoint reuse at the molecular level has mostly been assessed for fixed inversions through genome sequence comparison, and therefore rather broadly. Here, we have identified and sequenced the breakpoints of two polymorphic inversions-E1 and E2 that share a breakpoint-in the extant Est and E1 + 2 chromosomal arrangements of Drosophila subobscura. The breakpoints are two medium-sized repeated motifs that mediated the inversions by two different mechanisms: E1 via staggered breaks and subsequent repair and E2 via repeat-mediated ectopic recombination. The fine delimitation of the shared breakpoint revealed its strict reuse at the molecular level regardless of which was the intermediate arrangement. The occurrence of other rearrangements in the most proximal and distal extended breakpoint regions reveals the broad reuse of these regions. This differential degree of fragility might be related to their sharing the presence outside the inverted region of snoRNA-encoding genes.

The origin of chromosomal inversions as a source of segmental duplications in the Sophophora subgenus of Drosophila

Chromosomal inversions can contribute to the adaptation of organisms to their environment by capturing particular advantageous allelic combinations of a set of genes included in the inverted fragment and also by advantageous functional changes due to the inversion process itself that might affect not only the expression of flanking genes but also their dose and structure. Of the two mechanisms originating inversions —ectopic recombination, and staggered double-strand breaks and subsequent repair— only the latter confers the inversion the potential to have dosage effects and/or to generate advantageous chimeric genes. In Drosophila subobscura, there is ample evidence for the adaptive character of its chromosomal polymorphism, with an important contribution of some warm-climate arrangements such as E1+2+9+12. Here, we have characterized the breakpoints of inversion E12 and established that it originated through the staggered-break mechanism like four of the five inversions of D. subobscura previously studied. This mechanism that also predominates in the D. melanogaster lineage might be prevalent in the Sophophora subgenus and contribute to the adaptive character of the polymorphic and fixed inversions of its species. Finally, we have shown that the D. subobscura inversion breakpoint regions have generally been disrupted by additional structural changes occurred at different time scales.

Multiple and diverse structural changes affect the breakpoint regions of polymorphic inversions across the Drosophila genus

Chromosomal polymorphism is widespread in the Drosophila genus, with extensive evidence supporting its adaptive character in diverse species. Moreover, inversions are the major contributors to the genus chromosomal evolution. The molecular characterization of a reduced number of polymorphic inversion breakpoints in Drosophila melanogaster and Drosophila subobscura supports that their inversions would have mostly originated through a mechanism that generates duplications —staggered double-strand breaks— and has thus the potential to contribute to their adaptive character. There is also evidence for inversion breakpoint reuse at different time scales. Here, we have characterized the breakpoints of two inversions of D. subobscura —O4 and O8— involved in complex arrangements that are frequent in the warm parts of the species distribution area. The duplications detected at their breakpoints are consistent with their origin through the staggered-break mechanism, which further supports it as the prevalent mechanism in D. subobscura. The comparative analysis of inversions breakpoint regions across the Drosophila genus has revealed several genes affected by multiple disruptions due not only to inversions but also to single-gene transpositions and duplications.

Monitoring chromosomal polymorphism in Drosophila subobscura over 40 years

The inversion chromosomal polymorphism of Drosophila subobscura is considered to be adaptive as a result of its responses at different time scales to temperature changes. This work reports the longest‐term study of chromosomal polymorphism for a single population of D. subobscura with climatic data from the collecting site itself. The chromosomal analysis of D. subobscura samples collected six times over a 40‐year period at the same location and in the same seasonal interval has revealed the continuous presence of 16 common and six moderately rare chromosomal arrangements through the period. This analysis also corroborates the previously detected negative relationship between the frequencies of the standard (cold‐climate) arrangement on each of its five chromosomes and temperature, as well as between a comprehensive measure of cold adaptation (the total autosomal proportion of standard arrangement) and temperature. These and previous results would support that species harboring cold‐ and warm‐adapted polymorphic chromosomal arrangements, like D. subobscura, can rapidly respond to environmental changes.

Genomic analysis of European Drosophila melanogaster populations on a dense spatial scale reveals longitudinal population structure and continent-wide selection

Abstract Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatio-temporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterise variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.Genetic variation is the fuel of evolution. However, analyzing evolutionary dynamics in natural populations is challenging, sequencing of entire populations remains costly and comprehensive sampling logistically difficult. To tackle this issue and to define relevant spatial and temporal scales of variation, we have founded the European Drosophila Population Genomics Consortium (DrosEU). Here we present the first analysis of 48 D. melanogaster population samples collected across Europe in 2014. Our analysis uncovers novel patterns of variation at multiple levels: genome-wide neutral SNPs, mtDNA haplotypes, inversions, and TEs showing previously cryptic longitudinal population structure; signatures of selective sweeps shared among populations; presumably adaptive clines in inversions; and geographic variation in TEs. Additionally, we document highly variable microbiota and identify several new Drosophila viruses. Our study reveals novel aspects of the population biology of D. melanogaster and illustrates the power of extensive sampling and pooled sequencing of populations on a continent-wide scale.Genetic variation is the fuel of evolution. However, analyzing dynamics of evolutionary change in natural populations is challenging, genome sequencing of entire populations remains costly and comprehensive sample collection logistically challenging. To tackle this issue and to define relevant spatial and temporal scales of variation for a population genetic model system, the fruit fly Drosophila melanogaster, we have founded the European Drosophila Population Genomics Consortium (DrosEU). Our principal objective is to employ the strengths of this collaborative consortium to extensively sample and sequence natural populations on a continent-wide scale and across distinct timescales. Here we present the first analysis of the first DrosEU pool-sequencing dataset, consisting of 48 population samples collected across the European continent in 2014. The analysis of this comprehensive dataset uncovers novel patterns of variation at multiple levels: genome-wide neutral SNPs, mtDNA haplotypes, inversions and TEs that exhibit previously cryptic longitudinal population structure across the European continent; signatures of selective sweeps shared among the majority of European populations; presumably adaptive clines in inversions; and geographic variation in TEs. Additionally, we document highly variable microbiota among European fruit fly populations and identify several new Drosophila viruses. Our study reveals novel aspects of the population biology of D. melanogaster and illustrates the power of extensive sampling and pooled sequencing of natural populations on a continent-wide scale.

Dense gene physical maps of the non-model species Drosophila subobscura

The comparative analysis of genetic and physical maps as well as of whole genome sequences had revealed that in the Drosophila genus, most structural rearrangements occurred within chromosomal elements as a result of paracentric inversions. Genome sequence comparison would seem the best method to estimate rates of chromosomal evolution, but the high-quality reference genomes required for this endeavor are still scanty. Here, we have obtained dense physical maps for Muller elements A, C, and E of Drosophila subobscura, a species with an extensively studied rich and adaptive chromosomal polymorphism. These maps are based on 462 markers: 115, 236, and 111 markers for elements A, C, and E, respectively. The availability of these dense maps will facilitate genome assembly and will thus greatly contribute to obtaining a good reference genome, which is a required step for D. subobscura to attain the model species status. The comparative analysis of these physical maps and those obtained from the D. pseudoobscura and D. melanogaster genomes allowed us to infer the number of fixed inversions and chromosomal evolutionary rates for each pairwise comparison. For all three elements, rates inferred from the more closely related species were higher than those inferred from the more distantly related species, which together with results of relative-rate tests point to an acceleration in the D. subobscura lineage at least for elements A and E.

Polymorphism at genes involved in salt tolerance in Arabidopsis thaliana (Brassicaceae).

PREMISE OF THE STUDY Genes involved in relevant functions for environmental adaptation can be considered primary candidates for their variation having been shaped by natural selection. Detecting recent selective events through their footprint on nucleotide variation constitutes a challenging task in species with a complex demographic history such as Arabidopsis thaliana. We have surveyed nucleotide variation in this species at nine genes involved in salt tolerance. The available genomewide information for this species has allowed us to contrast the levels and patterns of variation detected at the candidate genes with empirical distributions obtained from noncandidate regions. METHODS We sequenced nine genes involved in salt tolerance (~32 kb) in 20 ecotypes of A. thaliana and analyzed polymorphism and divergence at the individual gene and multilocus levels. KEY RESULTS Variation at the nine genes studied was characterized by a generalized skew toward polymorphisms with low-frequency variants. Except for genes RCD1 and NHX8, this pattern was similar to that generally detected in the A. thaliana genome and could thus be primarily explained by the species demographic history. The more extreme deviation at the NHX8 gene and its excess of polymorphism relative to divergence points to the recent action of selection on this gene. CONCLUSIONS The analysis of nucleotide polymorphism and divergence at nine genes involved in salt tolerance provided little evidence for the recent action of positive selection. Only the signals detected at NHX8 from both polymorphism and divergence were suggestive of the putative contribution of this gene to local adaptation.

Evidence for a Gene Involved in Multiple and Diverse Rearrangements in the Drosophila Genus

In Drosophila, chromosomes have been extensively reorganized during evolution, with most rearrangements affecting the gene order in chromosomal elements but not their gene content. The level of reorganization and the evidence for breakpoint reuse vary both between and within elements. The subito gene stands out as a gene involved in multiple rearrangements both because of its active single-gene transposition and because it is the nearest gene to diverse rearrangements breakpoints. Indeed, subito has undergone three single-gene transpositions and it is the nearest gene to the breakpoints of other single-gene transpositions and of two chromosomal inversions. Given that subito is involved in meiosis and therefore active in the female germ line, the high number of nearby fixed breakages might be related among others to the presumed high accessibility of the subito region to the machinery associated with double-strand breaks repair. A second important contributor would be the reduced and simple regulatory region of subito, which would imply that a fraction of the rearrangements originating from subito nearby breakages would have not affected either its pattern or timing of expression and would have, thus, not resulted in reduced fitness.

The High-Quality Genome Sequence of the Oceanic Island Endemic Species Drosophila guanche Reveals Signals of Adaptive Evolution in Genes Related to Flight and Genome Stability

Abstract Drosophila guanche is a member of the obscura group that originated in the Canary Islands archipelago upon its colonization by D. subobscura. It evolved into a new species in the laurisilva, a laurel forest present in wet regions that in the islands have only minor long-term weather fluctuations. Oceanic island endemic species such as D. guanche can become model species to investigate not only the relative role of drift and adaptation in speciation processes but also how population size affects nucleotide variation. Moreover, the previous identification of two satellite DNAs in D. guanche makes this species attractive for studying how centromeric DNA evolves. As a prerequisite for its establishment as a model species suitable to address all these questions, we generated a high-quality D. guanche genome sequence composed of 42 cytologically mapped scaffolds, which are assembled into six super-scaffolds (one per chromosome). The comparative analysis of the D. guanche proteome with that of twelve other Drosophila species identified 151 genes that were subject to adaptive evolution in the D. guanche lineage, with a subset of them being involved in flight and genome stability. For example, the Centromere Identifier (CID) protein, directly interacting with centromeric satellite DNA, shows signals of adaptation in this species. Both genomic analyses and FISH of the two satellites would support an ongoing replacement of centromeric satellite DNA in D. guanche.