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.     

Testing chromosomal phylogenies and inversion breakpoint reuse in Drosophila

A combination of cytogenetic and bioinformatic procedures was used to test the chromosomal phylogeny relating Drosophila buzzatii with D. repleta. Chromosomes X and 2, harboring most of the inversions fixed between these two species, were analyzed. First, chromosomal segments conserved during the divergence of the two species were identified by comparative in situ hybridization to the D. repleta chromosomes of 180 BAC clones from a BAC-based physical map of the D. buzzatii genome. These conserved segments were precisely delimited with the aid of clones containing inversion breakpoints. Then GRIMM software was used to estimate the minimum number of rearrangements necessary to transform one genome into the other and identify all possible rearrangement scenarios. Finally, the most plausible inversion trajectory was tested by hybridizing 12 breakpoint-bearing BAC clones to the chromosomes of seven other species in the repleta group. The results show that chromosomes X and 2 of D. buzzatii and D. repleta differ by 12 paracentric inversions. Nine of them are fixed in chromosome 2 and entail two breakpoint reuses. Our results also show that the cytological relationship between D. repleta and D. mercatorum is closer than that between D. repleta and D. peninsularis, and we propose that the phylogenetic relationships in this lineage of the repleta group be reconsidered. We also estimated the rate of rearrangement between D. repleta and D. buzzatii and conclude that rates within the genus Drosophila vary substantially between lineages, even within a single species group.     

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.     

Cytogenetic Mapping of Enzyme Loci on Chromosomes J and U of DROSOPHILA SUBOBSCURA

Enzyme loci located on chromosome J and U were mapped cytologically by means of a Y translocation technique. A linkage map of the two chromosomes was established in a parallel experiment and the recombination frequency in different regions of the chromosomes determined. A comparison of the cytogenetic localization of the enzyme genes in D. subobscura and D. melanogaster indicates that many paracentric inversions must have taken place in the course of divergent evolution. However, no displacements of genes from one element to another due to pericentric inversions, reciprocal translocations or transposing elements can be observed. In spite of the large number of structural rearrangements that have occurred in the phylogeny of the genus Drosophila, gross similarities of banding pattern in homologous regions of the chromosomes of the two species become apparent.     

Variation of Spontaneous Occurrence Rates of Chromosomal Aberrations in the Second Chromosomes of DROSOPHILA MELANOGASTER

After accumulating mutations by the aid of marked inversions, spontaneous occurrence rates of chromosome aberrations were estimated for 1148 chromosome lines that originated from five stem line second chromosomes of Drosophila melanogaster. In chromosome lines originating from three stem chromosomes (CH, PQ, and RT), mutations were accumulated for 7550, 7252, and 7256 chromosome generations, respectively, but no structural change was detected. For the chromosome lines that originated from the other two stem chromosomes, the situation was different: Twenty aberrations (19 paracentric inversions and 1 translocation between the second and the third chromosomes) during 45990 chromosome generations took place in the 500 chromosome lines derived from stem line chromosome (AW), and 92 aberrations (83 paracentric inversions, 6 pericentric inversions, 2 translocations between the second and the third chromosomes and 1 transposition) arose during 45006 chromosome generations in the 500 chromosome lines derived from stem line chromosome (JH). For the AW group the occurrence rate becomes 0.00043 per chromosome per generation for all aberrations and 0.00041 for inversions. For the JH group the corresponding rates are 0.00204 and 0.00198, respectively.-A non-random distribution of the breakpoint on the salivary gland chromosome was observed and the breakpoints were concentrated in the regions 26, 29, 33, and 34.-The cytoplasms and the chromosomes (other than the second chromosomes) were made approximately uniform throughout the experiments. Thus, this remarkable variability in the occurrence rate is most probably due to the differences in one or more chromosomal elements on the original five stem chromosomes. The mutable chromosomes (AW and JH) appear to carry a kind of mutator factor such as hi (Ives 1950).     

Cytogenetic mapping of marker genes on the chromosome elements C and E of Drosophila pseudoobscura and D. subobscura

Enzyme loci, visible marker genes and -cloned DNA-sequences from a D. miranda library were mapped cytologically on the chromosome elements C and E of D. pseudoobscura and D. subobscura. New data are incorporated into the linkage maps of the two species. Homologous segments can now be localized in the polytene chromosomes with these markers. A comparison of the chromosome elements E of D. melanogaster and D. subobscura shows 12 conserved subsections which have been rearranged by paracentric inversions in the evolution of the two lineages.     

Whole arm inversions of chromosome 4 in Drosophila species

Inversions of genetic segments during the evolution of Drosophila are well documented in the X chromosome and most autosomes, but little attention has been paid to chromosome 4, the smallest autosome or "dot chromosome" present in many Drosophila species. From our previous mapping we have defined probes that mark proximal, intermediate, and distal locations of chromosome 4 in D. melanogaster. In situ hybridizations on salivary gland polytene chromosomes with these probes show that the whole right arm, including genes within cytological region 101EF-102F, is inverted relative to D. simulans. We also used these probes to determine the orientation of the arm of the dot chromosome in nine species of Drosophila, including eight from the melanogaster subfamily. To account for the observed whole arm inversions of chromosome 4 in five of the nine species examined, we propose that three inversion events have occurred during the evolution of these species. These whole arm inversions may explain some of the unusual features of this chromosome.     

Comparative genome analysis of the yellow fever mosquito Aedes aegypti with Drosophila melanogaster and the malaria vector mosquito Anopheles gambiae

An in silico comparative genomics approach was used to identify putative orthologs to genetically mapped genes from the mosquito, Aedes aegypti, in the Drosophila melanogaster and Anopheles gambiae genome databases. Comparative chromosome positions of 73 D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosomal regions, suggesting that some ancestral chromosome elements have been conserved. However, the two genomes also reflect extensive reshuffling within and between chromosomal regions. Comparative chromosome positions of A. gambiae orthologs indicate unequivocally that A. aegypti chromosome regions share extensive homology to the five A. gambiae chromosome arms. Whole-arm or near-whole-arm homology was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. The two genomes contain large conserved chromosome segments that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within. Only very tightly linked genes are likely to retain conserved linear orders within chromosome segments. The D. melanogaster and A. gambiae genome databases therefore offer limited potential for comparative positional gene determinations among even closely related dipterans, indicating the necessity for additional genome sequencing projects with other dipteran species.     

Localization of ecs, dor and swl genes in eight Drosophila species]

The DNA sequences from Drosophila melanogaster early ecdysterone-inducible puff 2B have been located in 8 Drosophila species by in situ hybridization. The location site of the ecs, dor and swi genes in D. funebris, D. virilis, D. hydei, D. repleta, D. mercatorum, D. paranaensis is a puff on the telomeric and of X chromosome; in D. kanekoi it is the puff in distal part of X chromosome; and in D. pseudoobscura it is the puff in proximal portion of X chromosome. So, conservative organization of DNA sequences located in D. melanogaster 2B puff could be suggested. Dispersed distribution of some DNA segments from the region studied in D. hydei chromosomes was revealed.     

A photographic map of Drosophila madeirensis polytene chromosomes.

A photographic map of salivary gland polytene chromosomes of Drosophila madeirensis has been constructed showing homologies and differences with respect to the standard gene arrangement of D. subobscura. Only two paracentric inversions in the X chromosome and some slight minor dissimilarities of one or two bands in the autosomes differentiate the chromosomes of these species.     

Molecular phylogeny and divergence times of drosophilid species

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.      

Molecular evolution of the Sex-Ratio inversion complex in Drosophila pseudoobscura: analysis of the Esterase-5 gene region

The Sex-Ratio chromosome in Drosophila pseudoobscura is subject to meiotic drive. It is associated with a series of three nonoverlapping paracentric inversions on the right arm of the X chromosome. The esterase-5 gene region has been localized to section 23 within the subbasal inversion of the Sex-Ratio inversion complex, making esterase- 5 a convenient locus for molecular evolutionary analyses of the Sex- Ratio inversion complex and the associated drive system. A 504-bp fragment of noncoding, intergenic DNA from the esterase-5 gene region was amplified and sequenced from 14 Sex-Ratio and 14 Standard X chromosomes of D. pseudoobscura, and from 9 X chromosomes of its two sibling species, Drosophila persimilis and Drosophila miranda. There is extensive sequence differentiation between the Sex-Ratio and Standard chromosomal types. The common Standard chromosome is highly polymorphic, while, as expected from either the neutral mutation theory or the selective sweep hypothesis, the rarer Sex-Ratio chromosome has much less within-chromosome nucleotide polymorphism. We estimate that the Standard and Sex-Ratio chromosomes in D. pseudoobscura diverged between 700,000 and 1.3 Mya, or at least 2 million generations ago. The clustering of D. pseudoobscura Sex-Ratio chromosomes in a neighbor- joining phylogeny indicates a fairly old, monophyletic origin in this species. It appears from these data that Sex-Ratio genes were present prior to the divergence of D. pseudoobscura and D. persimilis and that both the Standard and Sex-Ratio chromosomes of D. persimilis were derived from the Standard chromosome of D. pseudoobscura after the inversion events that isolated the D. pseudoobscura Sex-Ratio chromosome.      

Evolutionary rearrangement of the amylase genomic regions between Drosophila melanogaster and Drosophila pseudoobscura

Two Drosophila pseudoobscura genomic clones have sequence similarity to the Drosophila melanogaster amylase region that maps to the 53CD region on the D. melanogaster cytogenetic map. The two clones with similarity to amylase map to sections 73A and 78C of the D. pseudoobscura third chromosome cytogenetic map. The complete sequences of both the 73A and 78C regions were compared to the D. melanogaster genome to determine if the coding region for amylase is present in both regions and to determine the evolutionary mechanism responsible for the observed distribution of the amylase gene or genes. The D. pseudoobscura 73A and 78C linkage groups are conserved with the D. melanogaster 41E and 53CD regions, respectively. The amylase gene, however, has not maintained its conserved linkage between the two species. These data indicate that amylase has moved via a transposition event in the D. melanogaster or D. pseudoobscura lineage. The predicted genes within the 73A and 78C regions show patterns of molecular evolution in synonymous and nonsynonymous sites that are consistent with previous studies of these two species.     

Regulation of the segmentation gene fushi tarazu has been functionally conserved in Drosophila.

An evolutionary approach was applied to identify elements involved in the regulation of the segmentation gene fushi tarazu (ftz) by comparing the Drosophila melanogaster ftz gene with its Drosophila hydei homologue. The overall organization of the ftz gene is very similar in both species. Surprisingly, ftz proved to be inverted in the ANT-C of D. hydei with respect to D. melanogaster. Strong homologies extend over the entire 6 kb of the ftz upstream region with the best match in the 'upstream element'. We identified several highly conserved boxes embedded in unrelated sequences that correspond extremely well to two germ layer specific enhancers in the upstream element. Transformation experiments revealed that D. hydei ftz gene products can restore D. melanogaster ftz function and, furthermore, that trans-acting factors from D. melanogaster recognize and control D. hydei ftz regulatory elements. These findings indicate a conservation of the entire regulatory network among segmentation genes for several millions of years during the evolution of Drosophila.     

Genetic mapping of the Adh locus in the repleta group of Drosophila by in situ hybridization

A biotinylated probe of the Adh (alcohol dehydrogenase) gene of Drosophila melanogaster was used for in situ hybridization on polytene chromosomes of D. mojavensis and D. buzzatii, two species of the repleta group of the genus Drosophila. Hybridization showed that the Adh gene maps at the G1a band of the third chromosome. This is in accordance with a previous result obtained through the use of interspecific hybrid asynapsis as a cytological marker and establishes the limits of the precision of this method.     

Are chromosomal inversions induced by transposable elements? A paradigm from the malaria mosquito Anopheles gambiae

Chromosomal rearrangements abound in nature and can be studied in detail in organisms with polytene chromosomes. In Drosophila and in Anopheline mosquitoes most speciation processes seem to be associated with the establishment of chromosomal rearrangements, particularly of paracentric inversions. It is not known what triggers inversions in natural populations. In the laboratory inversions are commonly generated by X-rays, mutagens or after the activity of certain transposable elements (TEs). The Anopheles gambiae complex is comprised of six sibling species, each one characterized by the presence of fixed paracentric inversions on their chromosomes. Two of these, An. gambiae s.s. and An. arabiensis, are the most important vectors of human malaria and are structured into sub-populations, each carrying a characteristic set of polymorphic chromosomal inversions. We have cloned the breakpoints of the naturally occurring polymorphic inversion In(2R)d' of An. arabiensis. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a transposable element that we called Odysseus. Characteristics of Odysseus' terminal region and its cytological distribution in different strains as well as within the same strain indicate that Odysseus is an actively transposing element. The presence of Odysseus at the junction of the naturally occurring inversion In(2R)d' suggests that the inversion may be the result of the TEs activity. Cytological evidence from Drosophila melanogaster has also implicated the hobo transposable element in the generation of certain Hawaiian endemic inversions. This picture supports the hypothesis of the important role of TEs in generating natural inversions.     

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.     

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.     

Molecular population genetics of X-linked genes in Drosophila pseudoobscura

This article presents a nucleotide sequence analysis of 500 bp determined in each of five X-linked genes, runt, sisterlessA, period, esterase 5, and Heat-shock protein 83, in 40 Drosophila pseudoobscura strains collected from two populations. Estimates of the neutral migration parameter for the five loci show that gene flow among D. pseudoobscura populations is sufficient to homogenize inversion frequencies across the range of the species. Nucleotide diversity at each locus fails to reject a neutral model of molecular evolution. The sample of 40 chromosomes included six Sex-ratio inversions, a series of three nonoverlapping inversions that are associated with a strong meiotic drive phenotype. The selection driven by the Sex-ratio meiotic drive element has not fixed variation across the X chromosome of D. pseudoobscura because, while significant linkage disequilibrium was observed within the sisterlessA, period, and esterase 5 genes, we did not find evidence for nonrandom association among loci. The Sex-ratio chromosome was estimated to be 25,000 years old based on the decomposition of linkage disequilibrium between esterase 5 and Heat-shock protein 83 or 1 million years old based on the net divergence of esterase 5 between Standard and Sex-ratio chromosomes. Genetic diversity was depressed within esterase 5 within Sex-ratio chromosomes, while the four other genes failed to show a reduction in heterozygosity in the Sex-ratio background. The reduced heterogeneity in esterase 5 is due either to its location near one of the Sex-ratio inversion breakpoints or that it is closely linked to a gene or genes responsible for the Sex-ratio meiotic drive system.