Chromosomal Homology and Molecular Organization of Muller''s Elements D and E in the Drosophila Repleta Species Group

Thirty-three DNA clones containing protein-coding genes have been used for in situ hybridization to the polytene chromosomes of two Drosophila repleta group species, D. repleta and D. buzzatii. Twenty-six clones gave positive results allowing the precise localization of 26 genes and the tentative identification of another nine. The results were fully consistent with the currently accepted chromosomal homologies and in no case was evidence for reciprocal translocations or pericentric inversions found. Most of the genes mapped to chromosomes 2 and 4 that are homologous, respectively, to chromosome arms 3R and 3L of D. melanogaster (Muller's elements E and D). The comparison of the molecular organization of these two elements between D. melanogaster and D. repleta (two species that belong to different subgenera and diverged some 62 million years ago) showed an extensive reorganization via paracentric inversions. Using a maximum likelihood procedure, we estimated that 130 paracentric inversions have become fixed in element E after the divergence of the two lineages. Therefore, the evolution rate for element E is approximately one inversion per million years. This value is comparable to previous estimates of the rate of evolution of chromosome X and yields an estimate of 4.5 inversions per million years for the whole Drosophila genome.     

Heterochromatic distribution of HeT-A- and TART-like sequences in several Drosophila species

Drosophila melanogaster telomeres contain arrays of two non-LTR retrotransposons called HeT-A and TART. Previous studies have shown that HeT-A- and TART-like sequences are also located at non-telomeric sites in the Y chromosome heterochromatin. By in situ hybridization experiments, we mapped TART sequences in the h16 region of the long arm close to the centromere of the Y chromosome of D. melanogaster. HeT-A sequences were localized in two different regions on the Y chromosome, one very close to the centromere in the short arm (h18-h19) and the other in the long arm (h13-h14). To assess a possible heterochromatic location of TART and HeT-A elements in other Drosophila species, we performed in situ hybridization experiments, using both TART and HeT-A probes, on mitotic and polytene chromosomes of D. simulans, D. sechellia, D. mauritiana, D. yakuba and D. teissieri. We found that TART and HeT-A probes hybridize at specific heterochromatic regions of the Y chromosome in all Drosophila species that we analyzed.     

Chromosomal elements evolve at different rates in the Drosophila genome

Recent results indicate that the rate of chromosomal rearrangement in the genus Drosophila is the highest found so far in any eukaryote. This conclusion is based chiefly on the comparative mapping analysis of a single chromosomal element (Muller's element E) in two species, D. melanogaster and D. repleta, representing the two farthest lineages within the genus (the Sophophora and Drosophila subgenera, respectively). We have extended the analysis to two other chromosomal elements (Muller's elements A and D) and tested for differences in rate of evolution among chromosomes. With this purpose, detailed physical maps of chromosomes X and 4 of D. repleta were constructed by in situ hybridization of 145 DNA probes (gene clones, cosmids, and P1 phages) and their gene arrangements compared with those of the homologous chromosomes X and 3L of D. melanogaster. Both chromosomal elements have been extensively reshuffled over their entire length. The number of paracentric inversions fixed has been estimated as 118 +/- 17 for element A and 56 +/- 8 for element D. Comparison with previous data for elements E and B shows that there are fourfold differences in evolution rate among chromosomal elements, with chromosome X exhibiting the highest rate of rearrangement. Combining all results, we estimated that 393 paracentric inversions have been fixed in the whole genome since the divergence between D. repleta and D. melanogaster. This amounts to an average rate of 0.053 disruptions/Mb/myr, corroborating the high rate of rearrangement in the genus Drosophila.     

Zaprionus tuberculatus: chromosome map and gene mapping by DNA in situ hybridization.

The genus Drosophila has long been used as a model of karyotype evolution, demonstrating change by paracentric inversion and occasional centric fusion of an ancestral karyotype of five rod-shaped and one "dot" chromosome. This study shows, by mapping D. melanogaster probes hybridized to polytene chromosomes of Zaprionus tuberculatus, that this ancestral pattern extends beyond the genus Drosophila. A formal polytene chromosome map of Z. tuberculatus is presented.     

Using comparative genomic data to test for fast-X evolution

Genes may acquire nonsynonymous substitutions more rapidly when X-linked than when autosomal, but evidence for "fast-X evolution" has been elusive. Fast-X evolution could explain the disproportionate contribution of X-linked genes to hybrid sterility and other traits. Here, we use a comparative genomic approach, with sequences of 30-110 genes in four Drosophila species, to test for fast-X evolution. Specifically, the 3L autosome arm in D. melanogaster and D. simulans is homologous to the right arm of the X chromosome in D. pseudoobscura and D. miranda. We executed two paired comparisons to determine how often genes on this chromosome arm exhibit higher rates of nonsynonymous substitution in the D. pseudoobscura species group, as predicted by fast-X evolution. We found a statistically significant pattern consistent with fast-X evolution in one comparison and a similar trend in the other comparison. Variation in functional constraints across genes may have masked the signature of fast-X evolution in some previous studies, and we conclude paired comparisons are more powerful for examining rates of evolution of genes when X-linked over autosomal.     

Sequence-based detection and breakpoint assembly of polymorphic inversions

Inversion polymorphisms have occupied a privileged place in Drosophila genetic research since their discovery in the 1920s. Indeed, inversions seem to be nearly ubiquitous, and the majority of species that have been thoroughly surveyed have been found to be polymorphic for one or more chromosomal inversions. Despite enduring interest, however, inversions remain difficult to study because their effects are often cryptic, and few efficient assays have been developed. Even in Drosophila melanogaster, in which inversions can be reliably detected and have received considerable attention, the breakpoints of only three inversions have been characterized molecularly. Hence, inversion detection and assay design remain important unsolved problems. Here, we present a method for identification and local de novo assembly of inversion breakpoints using next-generation paired-end reads derived from D. melanogaster isofemale lines. PCR and cytological confirmations demonstrate that our method can reliably assemble inversion breakpoints, providing tools for future research on D. melanogaster inversions as well as a framework for detection and assay design of inversions and other chromosome aberrations in diverse taxa.     

Discordant Rates of Chromosome Evolution in the Drosophila Virilis Species Group

In Drosophila, the availability of polytene chromosome maps and of sets of probes covering most regions of the chromosomes allows a direct comparison of the organization of the genome in different species. In this work, we report the localization, in Drosophila virilis, D. montana, and D. novamexicana, of >100 bacteriophage P1 clones containing ~65 kilobase inserts of genomic DNA from D. virilis. Each clone hybridizes with a single euchromatic site in either chromosome 1 or chromosome 3 in D. virilis. From these data, it is possible to estimate the minimum number of inversions required to transform the map positions of the probes in one species into the map positions of the same probes in a related species. The data indicate that, in the D. virilis species group, the X chromosome has up to four times the number of inversions as are observed in chromosome 3. The first photographic polytene chromosome maps for D. montana and D. novamexicana are also presented.     

Inversion polymorphism in Drosophila obscura

The inversion polymorphism of Drosophila obscura Fallen, a European species of the obscura group of the subgenus Sophophora, is described. A total of 21 inversions have been recorded; they are located in the five large chromosomes of the species (a dot chromosome is also present) and form 25 gene arrangements present in the species' natural populations. Strains from five different countries were studied. Two of these inversions were found to be pericentric, and the remaining were paracentric. The presence of "hot" points (multibreaks) was noticed. The distribution of the relative lengths of inversions conformed to the Van Valen-Levins distribution, contrary to what happens in D. subobscura. Observations also showed that there is no crossover inhibition between nonoverlapping inversions. The phylogeny of chromosome C was reconstructed; the existence of several arrangements not found was postulated; and the primitive gene arrangement, linking D. obscura to its closely related species was identified, as well as the primitive gene arrangements of the other chromosomes. Photographic maps of the chromosomes of D. obscura are provided here.     

Inversion polymorphism in Indian natural populations of Drosophila melanogaster

Six natural populations (three urban and three rural) of Drosophila melanogaster from India were analysed for chromosome inversions, revealing the presence of 19 different paracentric autosomal inversions. One new inversion has also been detected in a laboratory stock established from flies collected from Kerala. In total 20 different paracentric inversions in Indian D. melanogaster have been detected during the present study, and of these, 4 are common cosmopolitans; 2 are rare cosmopolitans; 7 are recurrent endemics; and 7 are unique endemics. The quantitative data clearly show that the urban populations are different from the rural ones with respect to inversion polymorphism.     

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.     

Genetic analysis of female mating recognition between Drosophila ananassae and Drosophila pallidosa: application of interspecific mosaic genome lines

Drosophila ananassae and Drosophila pallidosa are closely related species that can produce viable and fertile hybrids of both sexes, although strong sexual isolation exists between the two species. Females are thought to discriminate conspecific from heterospecific males based on their courtship songs. The genetic basis of female discrimination behavior was analyzed using isogenic females from interspecific mosaic genome lines that carry homozygous recombinant chromosomes. Multiple regression analysis indicated a highly significant effect of the left arm of chromosome 2 (2L) on the willingness of females to mate with D. ananassae males. Not only 2L but also the left arm of chromosome X (XL) and the right arm of chromosome 3 (3R) had significant effects on the females' willingness to mate with D. pallidosa males. All regions with strong effects on mate choice have chromosome arrangements characterized by species-specific inversions. Heterospecific combinations of 2L and 3R have previously been suggested to cause postzygotic reproductive isolation. Thus, genes involved in premating as well as postmating isolation are located in or near chromosomal inversions. This conclusion is consistent with the recently proposed hypothesis that "speciation genes" accumulate at a higher rate in non-recombining genome regions when species divergence occurs in the presence of gene flow.     

Molecular Organization of the X Chromosome in Different Species of the Obscura Group of Drosophila

Nine single copy regions located on the X chromosome have been mapped by in situ hybridization in six species of the obscura group of Drosophila. Three Palearctic species, D. subobscura, D. madeirensis and D. guanche, and three Nearctic species, D. pseudoobscura, D. persimilis and D. miranda, have been studied. Eight of the regions include known genes from D. melanogaster (Pgd, zeste, white, cut, vermilion, RNA polymerase II 215, forked and suppressor of forked) and the ninth region (lambda DsubF6) has not yet been characterized. In all six species, as in D. melanogaster, all probes hybridize to a single site. Established chromosomal arm homologies of Muller's element A are only partly supported by present results since two of the probes (Pgd and zeste) hybridize at the proximal end of the XR chromosomal arm in the three Nearctic species. In addition to the centric fusion of Muller's A (= XL) and D (= XR) elements, the metacentric X chromosome of the Nearctic species requires a pericentric inversion to account for this result. Previously proposed homologies of particular chromosomal regions of the A (= X) chromosome in the three species of the D. subobscura cluster and of the XL chromosomal arm in the three species of the D. pseudoobscura cluster are discussed in light of the present results. Location of the studied markers has changed drastically not only since the divergence between the melanogaster and obscura groups but also since the Palearctic and Nearctic species of the obscura group diverged.     

Patterns of inversion polymorphism in three species of the Drosophila melanogaster species group

In Drosophila, chromosomal polymorphism due to paracentric inversions is very common and constitutes an adaptive character. The degree of chromosomal variability varies in different species and also in different populations of the same species. Chromosomal polymorphism in Indian natural populations of three species, D. melaonogaster, D. ananassae and D. bipectinata which belong to the melanogaster species group has been studied and the quantitative data on frequency of inversions have been reported. Behaviour of chromosome inversions has also been studied in laboratory conditions. The present review summarises the work done on inversion polymorphism in Indian populations of three species which clearly demonstrates that these three species vary in their patterns of inversion polymorphism and have evolved different mechanisms for adjustment to their environments although they belong to the same species group.     

Intron length evolution in Drosophila

I present data on the evolution of intron lengths among 3 closely related Drosophila species, D. melanogaster, Drosophila simulans, and Drosophila yakuba. Using D. yakuba as an outgroup, I mapped insertion and deletion mutations in 148 introns (spanning approximately 30 kb) to the D. melanogaster and D. simulans lineages. Intron length evolution in the 2 sister species has been different: in D. melanogaster, X-linked introns have increased slightly in size, whereas autosomal ones have decreased slightly in size; in D. simulans, both X-linked and autosomal introns have decreased in size. To understand the possible evolutionary causes of these lineage- and chromosome-specific patterns of intron evolution, I studied insertion-deletion (indel) polymorphism and divergence in D. melanogaster. Small insertion mutations segregate at elevated frequencies and enjoy elevated probabilities of fixation, particularly on the X chromosome. In contrast, there is no detectable X chromosome effect on fixations in D. simulans. These findings suggest X chromosome-specific selection or biased gene conversion-gap repair favoring insertions in D. melanogaster but not in D. simulans. These chromosome- and lineage-specific patterns of indel substitution are not easily explained by existing general population genetic models of intron length evolution. Genomic data from D. melanogaster further suggest that the forces described here affect introns and intergenic regions similarly.     

Rapid sequence turnover at an intergenic locus in Drosophila

Closely related species of Drosophila tend to have similar genome sizes. The strong imbalance in favor of small deletions relative to insertions implies that the unconstrained DNA in Drosophila is unlikely to be passively inherited from even closely related ancestors, and yet most DNA in Drosophila genomes is intergenic and potentially unconstrained. In an attempt to investigate the maintenance of this intergenic DNA, we studied the evolution of an intergenic locus on the fourth chromosome of the Drosophila melanogaster genome. This 1.2-kb locus is marked by two distinct, large insertion events: a nuclear transposition of a mitochondrial sequence and a transposition of a nonautonomous DNA transposon DNAREP1_DM. Because we could trace the evolutionary histories of these sequences, we were able to reconstruct the length evolution of this region in some detail. We sequenced this locus in all four species of the D. melanogaster species complex: D. melanogaster, D. simulans, D. sechellia, and D. mauritiana. Although this locus is similar in size in these four species, less than 10% of the sequence from the most recent common ancestor remains in D. melanogaster and all of its sister species. This region appears to have increased in size through several distinct insertions in the ancestor of the D. melanogaster species complex and has been shrinking since the split of these lineages. In addition, we found no evidence suggesting that the size of this locus has been maintained over evolutionary time; these results are consistent with the model of a dynamic equilibrium between persistent DNA loss through small deletions and more sporadic DNA gain through less frequent but longer insertions. The apparent stability of genome size in Drosophila may belie very rapid sequence turnover at intergenic loci.     

Rates and patterns of chromosomal evolution in Drosophila pseudoobscura and D. miranda

Comparisons of gene orders between species permit estimation of the rate of chromosomal evolution since their divergence from a common ancestor. We have compared gene orders on three chromosomes of Drosophila pseudoobscura with its close relative, D. miranda, and the distant outgroup species, D. melanogaster, by using the public genome sequences of D. pseudoobscura and D. melanogaster and approximately 50 in situ hybridizations of gene probes in D. miranda. We find no evidence for extensive transfer of genes among chromosomes in D. miranda. The rates of chromosomal rearrangements between D. miranda and D. pseudoobscura are far higher than those found before in Drosophila and approach those for nematodes, the fastest rates among higher eukaryotes. In addition, we find that the D. pseudoobscura chromosome with the highest level of inversion polymorphism (Muller's element C) does not show an unusually fast rate of evolution with respect to chromosome structure, suggesting that this classic case of inversion polymorphism reflects selection rather than mutational processes. On the basis of our results, we propose possible ancestral arrangements for the D. pseudoobscura C chromosome, which are different from those in the current literature. We also describe a new method for correcting for rearrangements that are not detected with a limited set of markers.     

(dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function.

In situ hybridization of (dC-dA)n.(dG-dT)n to the polytene chromosomes of Drosophila melanogaster reveals a clearly non-random distribution of chromosomal sites for this sequence. Sites are distributed over most euchromatic regions but the density of sites along the X chromosome is significantly higher than the density over the autosomes. All autosomes show approximately equal levels of hybridization except chromosome 4 which has no detectable stretches of (dC-dA)n.(dG-dT)n. Another striking feature is the lack of hybridization of the beta-heterochromatin of the chromocenter. The specific sites are conserved between different strains of D. melanogaster. The same overall chromosomal pattern of hybridization is seen for the other Drosophila species studied, including D. simulans, a sibling species with a much lower content of middle repetitive DNA, and D. virilis, a distantly related species. The evolutionary conservation of the distribution of (dC-dA)n.(dG-dT)n suggests that these sequences are of functional importance. The distribution patterns seen for D. pseudoobscura and D. miranda raise interesting speculations about function. In these species a chromosome equivalent to an autosomal arm of D. melanogaster has been translocated onto the X chromosome and acquired dosage compensation. In each species the new arm of the X also has a higher density of (dC-dA)n.(dG-dT)n similar to that seen on other X chromosomes. In addition to correlations with dosage compensation, the depletion of (dC-dA)n.(dG-dT)n in beta-heterochromatin and chromosome 4 may also be related to the fact that these regions do not normally undergo meiotic recombination.     

The effect of developmental temperature on the genetic architecture underlying size and thermal clines in Drosophila melanogaster and D. simulans from the east coast of Australia

Body size and thermal tolerance clines in Drosophila melanogaster occur along the east coast of Australia. However the extent to which temperature affects the genetic architecture underlying the observed clinal divergence remains unknown. Clinal variation in these traits is associated with cosmopolitan chromosome inversions that cline in D. melanogaster. Whether this association influences the genetic architecture for these traits in D. melanogaster is unclear. Drosophila simulans shows linear clines in body size, but nonlinear clines in cold resistance. Clinally varying inversions are absent in D. simulans. Line-cross and clinal analyses were performed between tropical and temperate populations of D. melanogaster and D. simulans from the east coast of Australia to investigate whether clinal patterns and genetic effects contributing to clinal divergence in wing centroid size, thorax length, wing-to-thorax ratio, cold and heat resistance differed under different developmental temperatures (18 °C, 25 °C, and 29 °C). Developmental temperature influenced the genetic architecture in both species. Similarities between D. melanogaster and D. simulans suggest clinally varying inversion polymorphisms have little influence on the genetic architecture underlying clinal divergence in size in D. melanogaster. Differing genetic architectures across different temperatures highlight the need to consider different environments in future evolutionary and molecular studies of phenotypic divergence.