Contrasting Histories of Three Gene Regions Associated with In(3l)payne of Drosophila Melanogaster

In the present report, we studied nucleotide variation in three gene regions of Drosophila melanogaster, spanning >5 kb and showing different degrees of association with the cosmopolitan inversion In(3-L)Payne. The analysis of sequence variation in the regions surrounding the breakpoints and the heat shock 83 (Hsp83) gene locus, located close to the distal breakpoint, revealed the absence of shared polymorphisms and the presence of a number of fixed differences between arrangements, indicating absence of genetic exchange. In contrast, for the esterase-6 gene region, located in the center of the inversion, we observed the presence of shared polymorphisms between arrangements suggesting genetic exchange. In the regions close to the breakpoints, the common St arrangement is 10 times more polymorphic than inverted chromosomes. We propose that the lack of recombination between arrangements in these regions coupled with genetic hitchhiking is the best explanation for the low heterozygosity observed in inverted lines. Using the data for the breakpoints, we estimate that this inversion polymorphism is around 0.36 million yr old. Although it is widely accepted that inversions are examples of balanced polymorphisms, none of the current neutrality tests including our Monte Carlo simulations showed significant departure from neutral expectations.     

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).     

Mechanisms of genetic exchange within the chromosomal inversions of Drosophila pseudoobscura

We have used the inversion system of Drosophila pseudoobscura to investigate how genetic flux occurs among the gene arrangements. The patterns of nucleotide polymorphism at seven loci were used to infer gene conversion events between pairs of different gene arrangements. We estimate that the average gene conversion tract length is 205 bp and that the average conversion rate is 3.4 x 10(-6), which is 2 orders of magnitude greater than the mutation rate. We did not detect gene conversion events between all combinations of gene arrangements even though there was sufficient nucleotide variation for detection and sufficient opportunity for exchanges to occur. Genetic flux across the inverted chromosome resulted in higher levels of differentiation within 0.1 Mb of inversion breakpoints, but a slightly lower level of differentiation in central inverted regions. No gene conversion events were detected within 17 kb of an inversion breakpoint suggesting that the formation of double-strand breaks is reduced near rearrangement breakpoints in heterozygotes. At least one case where selection rather than proximity to an inversion breakpoint is responsible for reduction in polymorphism was identified.     

Interchromosomal exchange of genetic information between gene arrangements on the third chromosome of Drosophila pseudoobscura

During the last 60 years, the inversion polymorphism on the third chromosome of Drosophila pseudoobscura has become a case study of the evolution of linked blocks of genes, isolated from each other by the suppression of recombination in heterozygotes for different inversions. Due to its location within inverted regions in most gene arrangements, the amylase (Amy) gene region can be used to elucidate the molecular pattern of evolution in these inversions. We studied this region in the Tree Line phylad of gene arrangements, with regard to both restriction site polymorphisms (RSP) and nucleotide sequences. The analysis of restriction maps, encompassing 26 kb, corroborates the cytogenetic phylogeny established on the basis of inversion breakpoints. However, we found that the 2.7 kb of nucleotide sequences of the AmyI gene are identical in both Estes Park and Hidalgo arrangements, despite the fact that these inversions arose independently from Tree Line. These contrasting results suggest that a homogenizing force, most likely gene conversion, is able to bring about localized exchanges between otherwise isolated gene arrangements.      

Evaluation of the genomic extent of effects of fixed inversion differences on intraspecific variation and interspecific gene flow in Drosophila pseudoobscura and D. persimilis

There is increasing evidence that chromosomal inversions may facilitate the formation or persistence of new species by allowing genetic factors conferring species-specific adaptations or reproductive isolation to be inherited together and by reducing or eliminating introgression. However, the genomic domain of influence of the inverted regions on introgression has not been carefully studied. Here, we present a detailed study on the consequences that distance from inversion breakpoints has had on the inferred level of gene flow and divergence between Drosophila pseudoobscura and D. persimilis. We identified the locations of the inversion breakpoints distinguishing D. pseudoobscura and D. persimilis in chromosomes 2, XR, and XL. Population genetic data were collected at specific distances from the inversion breakpoints of the second chromosome and at two loci inside the XR and XL inverted regions. For loci outside the inverted regions, we found that distance from the nearest inversion breakpoint had a significant effect on several measures of divergence and gene flow between D. pseudoobscura and D. persimilis. The data fitted a logarithmic relationship, showing that the suppression of crossovers in inversion heterozygotes also extends to loci located outside the inversion but close to it (within 1-2 Mb). Further, we detected a significant reduction in nucleotide variation inside the inverted second chromosome region of D. persimilis and near one breakpoint, consistent with a scenario in which this inversion arose and was fixed in this species by natural selection.     

Effect of inversion polymorphism on the neutral nucleotide variability of linked chromosomal regions in Drosophila

Recombination is a main factor determining nucleotide variability in different regions of the genome. Chromosomal inversions, which are ubiquitous in the genus Drosophila, are known to reduce and redistribute recombination, and thus their specific effect on nucleotide variation may be of major importance as an explanatory factor for levels of DNA variation. Here, we use the coalescent approach to study this effect. First, we develop analytical expressions to predict nucleotide variability in old inversion polymorphisms that have reached mutation-drift-flux equilibrium. The effects on nucleotide variability of a new arrangement appearing in the population and reaching a stable polymorphism are then studied by computer simulation. We show that inversions modulate nucleotide variability in a complex way. The establishment of an inversion polymorphism involves a partial selective sweep that eliminates part of the variability in the population. This is followed by a slow convergence to the equilibrium values. During this convergence, regions close to the breakpoints exhibit much lower variability than central regions. However, at equilibrium, regions close to the breakpoints have higher levels of variability and differentiation between arrangements than regions in the middle of the inverted segment. The implications of these findings for overall variability levels during the evolution of Drosophila species are discussed.     

The foldback-like transposon Galileo is involved in the generation of two different natural chromosomal inversions of Drosophila buzzatii

Chromosomal inversions are the most common type of genome rearrangement in the genus Drosophila. Although the potential of transposable elements (TEs) for generating inversions has been repeatedly demonstrated in the laboratory, little is known on their role in the generation of natural inversions, which are those effectively contributing to the adaptation and/or evolution of species. We have cloned and sequenced the two breakpoints of the polymorphic inversion 2q7 of D. buzzatii. The sequence analysis of the breakpoint regions revealed the presence in the inverted chromosomes of large insertions, formed by complex assemblies of transposons, that are absent from the chromosomes without the inversion. Among the transposons inserted, the Foldback-like element Galileo, that was previously found responsible of the generation of the widespread inversion 2j of D. buzzatii, is present at both 2q7 breakpoints and is the most likely inducer of the inversion. A detailed study of the nucleotide and structural variation in the breakpoint regions of six chromosomal lines with the 2q7 inversion detected no nucleotide differences between them, which suggests a monophyletic and recent origin. In contrast, a remarkable degree of structural variation was observed in the same six chromosomal lines. It thus appears that the two breakpoints of the inverted chromosomes have become genetically unstable hotspots, as was previously found for the 2j inversion breakpoints. The possibility that this instability is caused by structural properties of Foldback elements is discussed.     

Localization and characterization of X chromosome inversion breakpoints separating Drosophila mojavensis and Drosophila arizonae

Ectopic exchange between transposable elements or other repetitive sequences along a chromosome can produce chromosomal inversions. As a result, genome sequence studies typically find sequence similarity between corresponding inversion breakpoint regions. Here, we identify and investigate the breakpoint regions of the X chromosome inversion distinguishing Drosophila mojavensis and Drosophila arizonae. We localize one inversion breakpoint to 13.7 kb and localize the other to a 1-Mb interval. Using this localization and assuming microsynteny between Drosophila melanogaster and D. arizonae, we pinpoint likely positions of the inversion breakpoints to windows of less than 3000 bp. These breakpoints define the size of the inversion to approximately 11 Mb. However, in contrast to many other studies, we fail to find significant sequence similarity between the 2 breakpoint regions. The localization of these inversion breakpoints will facilitate future genetic and molecular evolutionary studies in this species group, an emerging model system for ecological genetics.     

Coalescent patterns for chromosomal inversions in divergent populations

Chromosomal inversions allow genetic divergence of locally adapted populations by reducing recombination between chromosomes with different arrangements. Divergence between populations (or hybridization between species) is expected to leave signatures in the neutral genetic diversity of the inverted region. Quantitative expectations for these patterns, however, have not been obtained. Here, we develop coalescent models of neutral sites linked to an inversion polymorphism in two locally adapted populations. We consider two scenarios of local adaptation: selection on the inversion breakpoints and selection on alleles inside the inversion. We find that ancient inversion polymorphisms cause genetic diversity to depart dramatically from neutral expectations. Other situations, however, lead to patterns that may be difficult to detect; important determinants are the age of the inversion and the rate of gene flux between arrangements. We also study inversions under genetic drift, finding that they produce patterns similar to locally adapted inversions of intermediate age. Our results are consistent with empirical observations, and provide the foundation for quantitative analyses of the roles that inversions have played in speciation.     

Genetic exchange versus genetic differentiation in a medium-sized inversion of Drosophila: the A2/Ast arrangements of Drosophila subobscura

Chromosomal inversion polymorphism affects nucleotide variation at loci associated with inversions. In Drosophila subobscura, a species with a rich chromosomal inversion polymorphism and the largest recombinational map so far reported in the Drosophila genus, extensive genetic structure of nucleotide variation was detected in the segment affected by the O(3) inversion, a moderately sized inversion at Muller's element E. Indeed, a strong genetic differentiation all over O(3) and no evidence of a higher genetic exchange in the center of the inversion than at breakpoints were detected. In order to ascertain, whether other polymorphic and differently sized inversions of D. subobscura also exhibited a strong genetic structure, nucleotide variation in 5 gene regions (P236, P275, P150, Sxl, and P125) located along the A(2) inversion was analyzed in A(st) and A(2) chromosomes of D. subobscura. A(2) is a medium-sized inversion at Muller's element A and forms a single inversion loop in heterokaryotypes. The lower level of variation in A(2) relative to A(st) and the significant excess of low-frequency variants at polymorphic sites indicate that nucleotide variation at A(2) is not at mutation-drift equilibrium. The closest region to an inversion breakpoint, P236, exhibits the highest level of genetic differentiation (F(ST)) and of linkage disequilibrium (LD) between arrangements and variants at nucleotide polymorphic sites. The remaining 4 regions show a higher level of genetic exchange between A(2) and A(st) chromosomes than P236, as revealed by F(ST) and LD estimates. However, significant genetic differentiation between the A(st) and A(2) arrangements was detected not only at P236 but also in the other 4 regions separated from the nearest breakpoint by 1.2-2.9 Mb. Therefore, the extent of genetic exchange between arrangements has not been high enough to homogenize nucleotide variation in the center of the A(2) inversion. A(2) can be considered a typical successful inversion of D. subobscura according to its relative length. Chromosomal inversion polymorphism of D. subobscura might thus cause the genome of this species to be highly structured and to harbor different gene pools that might contribute to maintain adaptations to particular environments.     

Haplotype-based genomic sequencing of a chromosomal polymorphism in the white-throated sparrow (Zonotrichia albicollis)

Inversion polymorphisms have been linked to a variety of fundamental biological and evolutionary processes. Yet few studies have used large-scale genomic sequencing to directly compare the haplotypes associated with the standard and inverted chromosome arrangements. Here we describe the targeted genomic sequencing and comparison of haplotypes representing alternative arrangements of a common inversion polymorphism linked to a suite of phenotypes in the white-throated sparrow (Zonotrichia albicollis). More than 7.4 Mb of genomic sequence was generated and assembled from both the standard (ZAL2) and inverted (ZAL2(m)) arrangements. Sequencing of a pair of inversion breakpoints led to the identification of a ZAL2-specific segmental duplication, as well as evidence of breakpoint reusage. Comparison of the haplotype-based sequence assemblies revealed low genetic differentiation outside versus inside the inversion indicative of historical patterns of gene flow and suppressed recombination between ZAL2 and ZAL2(m). Finally, despite ZAL2(m) being maintained in a near constant state of heterozygosity, no signatures of genetic degeneration were detected on this chromosome. Overall, these results provide important insights into the genomic attributes of an inversion polymorphism linked to mate choice and variation in social behavior.     

Molecular population genetics of the rp49 gene region in different chromosomal inversions of Drosophila subobscura.

Nucleotide variation at the ribosomal protein 49 (rp49) gene region has been studied in 75 lines of Drosophila subobscura belonging to four chromosomal arrangements (Ost, O3+4, O3+4+8, and O3+4+23). The location of the rp49 gene region within the inversion loop differs among heterokaryotypes: it is very close to one of the breakpoints in heterozygotes involving Ost chromosomes, while it is in a more central position in all other heterokaryotypes. The distribution of nucleotide polymorphism in the different arrangements is consistent with a monophyletic origin of the inversions. The data also provide evidence that gene conversion and possibly double crossover are involved in shuffling nucleotide variation among gene arrangements. The analyses reveal that the level of genetic exchange is higher when the region is located in a more central position of the inverted fragment than when it is close to the breakpoints. The pairwise difference distributions as well as the negative values of Tajima''s and Fu and Li''s statistics further support the hypothesis that nucleotide variation within chromosomal arrangements still reflects expansion after the origin of the inversions. Under the expansion model, we have estimated the time of origin of the studied inversions.     

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.     

Recombination and Gene Flux Caused by Gene Conversion and Crossing over in Inversion Heterokaryotypes

A theoretical analysis of the effects of inversions on recombination and gene flux between arrangements caused by gene conversion and crossing over was carried out. Two different mathematical models of recombination were used: the Poisson model (without interference) and the Counting model (with interference). The main results are as follows. (1) Recombination and gene flux are highly site-dependent both inside and outside the inverted regions. (2) Crossing over overwhelms gene conversion as a cause of gene flux in large inversions, while conversion becomes relatively significant in short inversions and in regions around the breakpoints. (3) Under the Counting model the recombination rate between two markers depends strongly on the position of the markers along the inverted segment. Two equally spaced markers in the central part of the inverted segment have less recombination than if they are in a more extreme position. (4) Inversions affect recombination rates in the uninverted regions of the chromosome. Recombination increases in the distal segment and decreases in the proximal segment. These results provide an explanation for a number of observations reported in the literature. Because inversions are ubiquitous in the evolutionary history of many Drosophila species, the effects of inversions on recombination are expected to influence DNA variation patterns.     

Polytene chromosome map and inversion polymorphism in Drosophila mediopunctata

Drosophila mediopunctata belongs to the tripunctata group, and is one of the commonest Drosophila species collected in some places in Brazil, especially in the winter. A standard map of the polytene chromosomes is presented. The breakpoints of the naturally occurring chromosomal rearrangements are marked on the map. The distribution of breaking points through the chromosomes of D. mediopunctata is apparently non-random. Chromosomes X, II and IV show inversion polymorphisms. Chromosome II is the most polymorphic, with 17 inversions, 8 inversions in the distal region and 9 in the proximal region. Chromosome X has four different gene arrangements, while chromosome IV has only two.     

Recombination and selection in the maintenance of the adaptive value of inversions

A huge amount of data seem to confirm the adaptive value of inversions in Drosophila. The inhibition of recombination in heterokaryotypes mediated by inversions seems fundamental in maintaining their adaptive role. This study shows that recombination is highly suppressed in Drosophila subobscura because of chromosomal inversions, not only inside the inversions but also outside them. It seems that the region outside the inversion where recombination is inhibited is asymmetrical and independent of the inversion length. Despite the difficulty of crossovers taking place near inversion breakpoints, the only two recombination events detected inside inversions were located close to the breakpoint. Thus, selection could be largely responsible for the recombination reduction maintaining sets of adaptive alleles inside the inverted region. Heterokaryotype descendants were always in higher frequency than inbred or outbred homokaryotypes, regardless of the geographical origin of the chromosome, suggesting that chromosomes carrying the same arrangement, although with a different set of alleles for neutral markers, could be submitted to the same selection processes.     

Inferring the demographic history of Drosophila subobscura from nucleotide variation at regions not affected by chromosomal inversions

Drosophila subobscura presents a rich and complex chromosomal inversion polymorphism. It can thus be considered a model system (i) to study the mechanisms originating inversions and how inversions affect the levels and patterns of variation in the inverted regions and (ii) to study adaptation at both the single‐gene and chromosomal inversion levels. It is therefore important to infer its demographic history as previous information indicated that its nucleotide variation is not at mutation–drift equilibrium. For that purpose, we sequenced 16 noncoding regions distributed across those parts of the J chromosome not affected by inversions in the studied population and possibly either by other selective events. The pattern of variation detected in these 16 regions is similar to that previously reported within different chromosomal arrangements, suggesting that the latter results would, thus, mainly reflect recent demographic events rather than the partial selective sweep imposed by the origin and frequency increase of inversions. Among the simple demographic models considered in our Approximate Bayesian Computation analysis of variation at the 16 regions, the model best supported by the data implies a population size expansion soon after the penultimate glacial period. This model constitutes a better null model, and it is therefore an important resource for subsequent studies aiming among others to uncover selective events across the species genome. Our results also highlight the importance of introducing the possibility of multiple hits in the coalescent simulations with an outgroup.     

Evidence of Extensive Genetic Exchange in the Rp49 Region among Polymorphic Chromosome Inversions in Drosophila Subobscura

Restriction map variation in 107 lines extracted from two natural populations of Drosophila subobscura was investigated with seven four-nucleotide-recognizing enzymes in a 1.6-kb region including the rp49 gene, that is located very close to the proximal breakpoint of inversion O3. Fourteen restriction site and 8 length polymorphisms, resulting in 73 haplotypes, have been identified. Estimated heterozygosity per nucleotide, pi = 0.0045, is comparable to the average nucleotide variation observed in Drosophila melanogaster. Because of the location of the rp49 region in D. subobscura, variation in three different gene arrangements-Ost, O3 + 4 and O3 + 4 + 8-has been compared. Out of 14 restriction site polymorphisms, 3 are shared by Ost, O3 + 4 and O3 + 4 + 8, and 3 additional ones are shared by Ost and O3 + 4, evidencing extensive genetic exchange among these polymorphic inversions. In agreement with previous data, the higher level of variation of O3 + 4 (as measured by haplotype diversity and nucleotide variation) suggests that O3 + 4 may be ancestral in relationship to extant gene arrangements.     

Chromosomal inversion polymorphism leads to extensive genetic structure: a multilocus survey in Drosophila subobscura

The adaptive character of inversion polymorphism in Drosophila subobscura is well established. The O(ST) and O(3+4) chromosomal arrangements of this species differ by two overlapping inversions that arose independently on O(3) chromosomes. Nucleotide variation in eight gene regions distributed along inversion O(3) was analyzed in 14 O(ST) and 14 O(3+4) lines. Levels of variation within arrangements were quite similar along the inversion. In addition, we detected (i) extensive genetic differentiation between arrangements in all regions, regardless of their distance to the inversion breakpoints; (ii) strong association between nucleotide variants and chromosomal arrangements; and (iii) high levels of linkage disequilibrium in intralocus and also in interlocus comparisons, extending over distances as great as approximately 4 Mb. These results are not consistent with the higher genetic exchange between chromosomal arrangements expected in the central part of an inversion from double-crossover events. Hence, double crossovers were not produced or, alternatively, recombinant chromosomes were eliminated by natural selection to maintain coadapted gene complexes. If the strong genetic differentiation detected along O(3) extends to other inversions, nucleotide variation would be highly structured not only in D. subobscura, but also in the genome of other species with a rich chromosomal polymorphism.