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.

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.

Molecular population genetics of the Polycomb genes in Drosophila subobscura.

Polycomb group (PcG) proteins are important regulatory factors that modulate the chromatin state. They form protein complexes that repress gene expression by the introduction of posttranslational histone modifications. The study of PcG proteins divergence in Drosophila revealed signals of coevolution among them and an acceleration of the nonsynonymous evolutionary rate in the lineage ancestral to the obscura group species, mainly in subunits of the Pcl-PRC2 complex. Herein, we have studied the nucleotide polymorphism of PcG genes in a natural population of D. subobscura to detect whether natural selection has also modulated the evolution of these important regulatory genes in a more recent time scale. Results show that most genes are under the action of purifying selection and present a level and pattern of polymorphism consistent with predictions of the neutral model, the exceptions being Su(z)12 and Pho. MK tests indicate an accumulation of adaptive changes in the SU(Z)12 protein during the divergence of D. subobscura and D. guanche. In contrast, the HKA test shows a deficit of polymorphism at Pho. The most likely explanation for this reduced variation is the location of this gene in the dot-like chromosome and would indicate that this chromosome also has null or very low recombination in D. subobscura, as reported in D. melanogaster.

Sex combs reduced (Scr) regulatory region of Drosophila revisited.

The Hox gene Sex combs reduced (Scr) is responsible for the differentiation of the labial and prothoracic segments in Drosophila. Scr is expressed in several specific tissues throughout embryonic development, following a complex path that must be coordinated by an equally complex regulatory region. Although some cis-regulatory modules (CRMs) have been identified in the Scr regulatory region (~75 kb), there has been no detailed and systematic study of the distinct regulatory elements present within this region. In this study, the Scr regulatory region was revisited with the aim of filling this gap. We focused on the identification of Initiator elements (IEs) that bind segmentation factors, Polycomb response elements (PREs) that are recognized by the Polycomb and Trithorax complexes, as well as insulators and tethering elements. To this end, we summarized all currently available information, mainly obtained from high throughput ChIP data projects. In addition, a bioinformatic analysis based on the evolutionary conservation of regulatory sequences using the software MOTEVO was performed to identify IE and PRE candidates in the Scr region. The results obtained by this combined strategy are largely consistent with the CRMs previously identified in the Scr region and help to: (i) delimit them more accurately, (ii) subdivide two of them into different independent elements, (iii) identify a new CRM, (iv) identify the composition of their binding sites and (v) better define some of their characteristics. These positive results indicate that an approach that integrates functional and bioinformatic data might be useful to characterize other regulatory regions.

Evidence of neofunctionalization after the duplication of the highly conserved Polycomb group gene Caf1-55 in the obscura group of Drosophila.

Drosophila CAF1-55 protein is a subunit of the Polycomb repressive complex PRC2 and other protein complexes. It is a multifunctional and evolutionarily conserved protein that participates in nucleosome assembly and remodelling, as well as in the epigenetic regulation of a large set of target genes. Here, we describe and analyze the duplication of Caf1-55 in the obscura group of Drosophila. Paralogs exhibited a strong asymmetry in evolutionary rates, which suggests that they have evolved according to a neofunctionalization process. During this process, the ancestral copy has been kept under steady purifying selection to retain the ancestral function and the derived copy (Caf1-55dup) that originated via a DNA-mediated duplication event ~18 Mya, has been under clear episodic selection. Different maximum likelihood approaches confirmed the action of positive selection, in contrast to relaxed selection, on Caf1-55dup after the duplication. This adaptive process has also taken place more recently during the divergence of D. subobscura and D. guanche. The possible association of this duplication with a previously detected acceleration in the evolutionary rate of three CAF1-55 partners in PRC2 complexes is discussed. Finally, the timing and functional consequences of the Caf1-55 duplication is compared to other duplications of Polycomb genes.

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.

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.

Structure and population genetics of the breakpoints of a polymorphic inversion in Drosophila subobscura.

Drosophila subobscura is a paleartic species of the obscura group with a rich chromosomal polymorphism. To further our understanding on the origin of inversions and on how they regain variation, we have identified and sequenced the two breakpoints of a polymorphic inversion of D. subobscura--inversion 3 of the O chromosome--in a population sample. The breakpoints could be identified as two rather short fragments (∼300 bp and 60 bp long) with no similarity to any known transposable element family or repetitive sequence. The presence of the ∼300-bp fragment at the two breakpoints of inverted chromosomes implies its duplication, an indication of the inversion origin via staggered double-strand breaks. Present results and previous findings support that the mode of origin of inversions is neither related to the inversion age nor species-group specific. The breakpoint regions do not consistently exhibit the lower level of variation within and stronger genetic differentiation between arrangements than more internal regions that would be expected, even in moderately small inversions, if gene conversion were greatly restricted at inversion breakpoints. Comparison of the proximal breakpoint region in species of the obscura group shows that this breakpoint lies in a small high-turnover fragment within a long collinear region (∼300 kb).

Nonrecombining genes in a recombination environment: the Drosophila "dot" chromosome.

Rate of recombination is a powerful variable affecting several aspects of molecular variation and evolution. A nonrecombining portion of the genome of most Drosophila species, the "dot" chromosome or F element, exhibits very low levels of variation and unusual codon usage. One lineage of Drosophila, the willistoni/saltans groups, has the F element fused to a normally recombining E element. Here, we present polymorphism data for genes on the F element in two Drosophila willistoni and one D. insularis populations, genes previously studied in D. melanogaster. The D. willistoni populations were known to be very low in inversion polymorphism, thus minimizing the recombination suppression effect of inversions. We first confirmed, by in situ hybridization, that D. insularis has the same E + F fusion as D. willistoni, implying this was a monophyletic event. A clear gradient in codon usage exists along the willistoni F element, from the centromere distally to the fusion with E; estimates of recombination rates parallel this gradient and also indicate D. insularis has greater recombination than D. willistoni. In contrast to D. melanogaster, genes on the F element exhibit moderate levels of nucleotide polymorphism not distinguishable from two genes elsewhere in the genome. Although some linkage disequilibrium (LD) was detected between polymorphic sites within genes (generally <500 bp apart), no long-range LD between F element loci exists in the two willistoni group species. In general, the distribution of allele frequencies of F element genes display the typical pattern of expectations of neutral variation at equilibrium. These results are consistent with the hypothesis that recombination allows the accumulation of nucleotide variation as well as allows selection to act on synonymous codon usage. It is estimated that the fusion occurred ∼20 Mya and while the F element in the willistoni lineage has evolved "normal" levels and patterns of nucleotide variation, equilibrium may not have been reached for codon usage.

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps.

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.

The dynamics of the roo transposable element in mutation-accumulation lines and segregating populations of Drosophila melanogaster.

We estimated the number of copies for the long terminal repeat (LTR) retrotransposable element roo in a set of long-standing Drosophila melanogaster mutation-accumulation full-sib lines and in two large laboratory populations maintained with effective population size approximately 500, all of them derived from the same isogenic origin. Estimates were based on real-time quantitative PCR and in situ hybridization. Considering previous estimates of roo copy numbers obtained at earlier stages of the experiment, the results imply a strong acceleration of the insertion rate in the accumulation lines. The detected acceleration is consistent with a model where only one (maybe a few) of the approximately 70 roo copies in the ancestral isogenic genome was active and each active copy caused new insertions with a relatively high rate ( approximately 10(-2)), with new inserts being active copies themselves. In the two laboratory populations, however, a stabilized copy number or no accelerated insertion was found. Our estimate of the average deleterious viability effects per accumulated insert [E(s) < 0.003] is too small to account for the latter finding, and we discuss the mechanisms that could contain copy number.

Chromosomal evolution of elements B and C in the Sophophora subgenus of Drosophila: evolutionary rate and polymorphism.

The locations of 77 markers along the chromosomal elements B (41 markers) and C (36 markers) of Drosophila subobscura, D. pseudoobscura, and D. melanogaster were obtained by in situ hybridization on polytene chromosomes. In comparisons between D. subobscura and D. pseudoobscura, 10 conserved segments (accounting for 32% of the chromosomal length) were detected on element B and eight (17% of the chromosomal length) on element C. The fixation rate of paracentric inversions inferred by a maximum likelihood approach differs significantly between elements. Muller's element C (0.17 breakpoints/Mb/million years) is evolving two times faster than element B (0.08 breakpoints/Mb/million years). This difference in the evolutionary rate is paralleled by differences in the extent of chromosomal polymorphism in the corresponding lineages. Element C is highly polymorphic in D. subobscura, D. pseudoobscura, and in other obscura group species such as D. obscura and D. athabasca. In contrast, the level of polymorphism in element B is much lower in these species. The fixation rates of paracentric inversions estimated in the present study between species of the Sophophora subgenus are the highest estimates so far reported in the genus for the autosomes. At the subgenus level, there is also a parallelism between the high fixation rate and the classical observation that the species of the Sophophora subgenus tend to be more polymorphic than the species of the Drosophila subgenus. Therefore, the detected relationship between level of polymorphism and evolutionary rate might be a general characteristic of chromosomal evolution in the genus Drosophila.

Sequences upstream of the homologous cis-elements of the Adh adult enhancer of Drosophila are required for maximal levels of Adh gene transcription in adults of Scaptodrosophila lebanonensis.

The evolution of cis-regulatory elements is of particular interest for our understanding of the evolution of gene regulation. The Adh gene of Drosophilidae shows interspecific differences in tissue-specific expression and transcript levels during development. In Scaptodrosophila lebanonensis adults, the level of distal transcripts is maximal between the fourth and eighth day after eclosion and is around five times higher than that in D. melanogaster Adh(S). To examine whether these quantitative differences are regulated by sequences lying upstream of the distal promoter, we performed in vitro deletion mutagenesis of the Adh gene of S. lebanonensis, followed by P-element-mediated germ-line transformation. All constructs included, as a cotransgene, a modified Adh gene of D. melanogaster (dAdh) in a fixed position and orientation that acted as a chromosomal position control. Using this approach, we have identified a fragment of 1.5 kb in the 5' region, 830 bp upstream of the distal start site, which is required to achieve maximal levels of distal transcript in S. lebanonensis. The presence of this fragment produces a 3.5-fold higher level of distal mRNA (as determined by real time quantitative PCR) compared with the D. melanogaster dAdh cotransgene. This region contains the degenerated end of a minisatellite sequence expanding farther upstream and does not correspond to the Adh adult enhancer (AAE) of D. melanogaster. Indeed, the cis-regulatory elements of the AAE have been identified by phylogenetic footprinting within the region 830 bp upstream of the distal start site of S. lebanonensis. Furthermore, the deletions Delta-830 and Delta-2358 yield the same pattern of tissue-specific expression, indicating that all tissue-specific elements are contained within the region 830 bp upstream of the distal start site.