Excess Polymorphism at the Adh Locus in DROSOPHILA MELANOGASTER

The evolutionary history of a region of DNA encompassing the Adh locus is studied by comparing patterns of variation in Drosophila melanogaster and its sibling species, D. simulans. An unexpectedly high level of silent polymorphism in the Adh coding region relative to the 5' and 3' flanking regions in D. melanogaster is revealed by a populational survey of restriction polymorphism using a four-cutter filter hybridization technique as well as by direct sequence comparisons. In both of these studies, a region of the Adh gene encompassing the three coding exons exhibits a frequency of polymorphism equal to that of a 4-kb 5' flanking region. In contrast, an interspecific sequence comparison shows a two-fold higher level of divergence in the 5' flanking sequence compared to the structural locus. Analysis of the patterns of variation suggest an excess of polymorphism within the D. melanogaster Adh locus, rather than lack of polymorphism in the 5' flanking region. An approach is outlined for testing neutral theory predictions about patterns of variation within and between species. This approach indicates that the observed patterns of variation are incompatible with an infinite site neutral model.     

Nucleotide sequence divergence in the A+T-rich region of mitochondrial DNA in Drosophila simulans and Drosophila mauritiana

We have determined the nucleotide sequences of two regions within the A+T-rich region of mitochondrial DNA (mtDNA) in the siIII type of Drosophila simulans and the maI type of D. mauritiana. The sequences of the two regions in siIII and maI are almost identical. The sequences include elements corresponding to the type I and type II repeats elements and the T-stretches as reported in D. melanogaster; an approximately 340-bp region (A region) adjacent to the tRNA(Ile) gene includes a part of the type II repeat element, and an approximately 440- bp region (B region) includes a central portion of the A+T-rich region between the type I and type II repeat arrays. Each sequence of the two species was compared with those of D. melanogaster and D. yakuba. The sequences of the A region are relatively well conserved among the four species. The alignment of the two sequences of the B region with those of D. melanogaster and D. yakuba requires numerous insertions/deletions. For both regions, nucleotide differences between D. simulans or D. mauritiana and D. melanogaster are similar to those between the two and D. yakuba. The tendency is obvious in a subregion within the type II repeat element in the A region. These findings suggest that the rate of nucleotide substitution in the subregion is accelerated in the lineage leading to D. melanogaster. Loss of functional constraint in the stem-loop-forming sequence is proposed for this acceleration.      

The molecular evolution of the alcohol dehydrogenase and alcohol dehydrogenase-related genes in the Drosophila melanogaster species subgroup

The DNA sequences of the Adh genes of three members of the Drosophila melanogaster species subgroup have been determined. This completes the Adh sequences of the eight species of this subgroup. Two species, D. yakuba and D. teissieri, possess processed Adh pseudogenes. In all of the species of the subgroup, a gene of unknown function, Adhr, is located about 300 bp 3' to Adh. Although this gene is experiencing a higher rate of synonymous substitution than Adh, it is more constrained at the amino acid level. Phylogenetic relationships between all eight members of the melanogaster subgroup have been analyzed using a variety of methods. All analyses suggested that the D. yakuba and D. teissieri pseudogenes have a single common ancestor, rather than evolving independently in each species, and that D. melanogaster is the sister species to D. simulans, D. sechellia, and D. mauritiana. The evolutionary relationships of the latter three species remain equivocal.      

Molecular Population Genetics of the Distal Portion of the X Chromosome in Drosophila: Evidence for Genetic Hitchhiking of the Yellow-Achaete Region

We have estimated DNA sequence variation and differentiation within and between Drosophila melanogaster and its sibling species, Drosophila simulans, using six-cutter restriction site variation at yellow-achaete (y-ac), phosphogluconate dehydrogenase (Pgd), and period (per). These three gene regions are of varying distance from the telomere of the X chromosome and range from very low to moderate rates of recombination in D. melanogaster. According to Tajima's test of neutrality, the Pgd region has been influenced by balancing selection in D. melanogaster. This is consistent with previous data suggesting the allozyme polymorphism at this locus is visible to selection. The Hudson, Kreitman, Aguadé test of neutrality reveals a significant departure from neutrality for the y-ac region compared to the per or rosy regions in D. simulans. There is also a significant departure for the y-ac region compared to the Adh 5' flanking region in D. melanogaster. In both species the departure appears to be due to reduced variation at y-ac compared to that expected from divergence between D. simulans and D. melanogaster. We conclude that recent hitchhiking associated with the selective fixation of one or more advantageous mutants in the y-ac region is the best explanation for reduced variation at y-ac.     

Patterns of Microsatellite Variability in the Drosophila Melanogaster Complex

Forty-seven microsatellite loci were amplified in Drosophila melanogaster, Drosophila simulans, Drosophila mauritiana and Drosophila sechellia. The two cosmopolitan species D. melanogaster and D. simulans were found to be the most variable ones, followed by D. mauritiana and D. sechellia. A model based clustering algorithm was applied to the population samples of D. melanogaster, D. simulans and D. sechellia. No evidence for population substructure was detected within species--most likely due to insufficient power. A Markov chain Monte Carlo method developed for demographic inference based on microsatellites provided unambiguous evidence for population contraction in D. melanogaster, D. simulans and D. sechellia, despite that the D. melanogaster and D. simulans population samples were of non-African origin and represented recently expanded populations.     

Hsp70 duplication in the Drosophila melanogaster species group: how and when did two become five?

To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10--15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.     

Analysis of nucleotide substitutions of mitochondrial DNAs in Drosophila melanogaster and its sibling species

To study the rate and pattern of nucleotide substitution in mitochondrial DNA (mtDNA), we cloned and sequenced a 975-bp segment of mtDNA from Drosophila melanogaster, D. simulans, and D. mauritiana containing the genes for three transfer RNAs and parts of two protein- coding genes, ND2 and COI. Statistical analysis of synonymous substitutions revealed a predominance of transitions over transversions among the three species, a finding differing from previous results obtained from a comparison of D. melanogaster and D. yakuba. The number of transitions observed was nearly the same for each species comparison, including D. yakuba, despite the differences in divergence times. However, transversions seemed to increase steadily with increasing divergence time. By contrast, nonsynonymous substitutions in the ND2 gene showed a predominance of transversions over transitions. Most transversions were between A and T and seemed to be due to some kind of mutational bias to which the A + T-rich mtDNA of Drosophila species may be subject. The overall rate of nucleotide substitution in Drosophila mtDNA appears to be slightly faster (approximately 1.4 times) than that of the Adh gene. This contrasts with the result obtained for mammals, in which the mtDNA evolves approximately 10 times faster than single-copy nuclear DNA. We have also shown that the start codon of the COI gene is GTGA in D. simulans and GTAA in D. mauritiana. These codons are different from that of D. melanogaster (ATAA).      

Evolution of the A+T-rich region of mitochondrial DNA in the melanogaster species subgroup of Drosophila

We determined the nucleotide sequences of two regions in the A+T-rich region of mitochondrial DNA (mtDNA) in the siI and siII types of D. simulans, the maII type of D. mauritiana, and D. sechellia. The sequences were aligned with those of the corresponding regions of siIII of D. simulans and maI of D. mauritiana, D. melanogaster, and D. yakuba. The type I and type II elements and the T-stretches were detected in all eight of the mtDNA types compared, indicating that the three elements are essential in the A+T-rich region of this species subgroup. The alignment revealed several short repetitive sequences and relatively large deletions in the central portions of the region. In the highly conserved sequence elements in the type II elements, the substitution rates were not uniform among lineages and acceleration in the substitution rate might have been due to loss of functional constraint in the stem–loop-forming sequences predicted in the type II elements. Patterns of nucleotide substitutions observed in the A+T-rich region were further compared with those in the coding regions and in the intergenic regions of mtDNA. Substitutions between A and T were particularly repressed in the highly conserved sequence elements and in the intergenic regions compared with those in the A+T-rich region excluding the highly conserved sequence elements and in the fourfold degenerate sites in the coding regions. The functional and structural characteristics of the A+T-rich region that might be involved in this substitutional bias are discussed.     

Distribution and conservation of sequences homologous to the 1731 retrotransposon in Drosophila

The distribution of 1731 retrotransposon-hybridizing sequences in the family Drosophilidae has been studied using a 1731 probe from Drosophila melanogaster. Squash blot and Southern blot analyses of 42 species reveal that the 1731 sequences are widespread within both the Sophophora and Drosophila subgenera and are also present in the genera Scaptomyza and Zaprionus. Hence the 1731 retrotransposon family appears to have a long evolutionary history in the Drosophilidae genome. Differences of hybridization signal intensity suggested that the 1731 sequence is well conserved only in the three species most closely related to D. melanogaster (D. simulans, D. mauritiana, and D. sechellia). A survey of insertion sites in numerous different populations of the previous four species by in situ hybridization to polytene chromosomes has shown in all cases both chromocentric hybridizations and a low number of sites (0-5) on the chromosomal arms. This number of sites is among the lowest observed in D. melanogaster and D. simulans when 1731 is compared with other retrotransposon families. In addition, we have observed species-specific patterns of the chromocentric hybridization signal, suggesting rapid modifications of the beta-heterochromatin components since the radiation of the melanogaster subgroup.      

Differences in Crossover Frequency and Distribution among Three Sibling Species of Drosophila

Comparisons of the genetic and cytogenetic maps of three sibling species of Drosophila reveal marked differences in the frequency and cumulative distribution of crossovers during meiosis. The maps for two of these species, Drosophila melanogaster and D. simulans, have previously been described, while this report presents new map data for D. mauritiana, obtained using a set of P element markers. A genetic map covering nearly the entire genome was constructed by estimating the recombination fraction for each pair of adjacent inserts. The P-based genetic map of mauritiana is ~1.8 times longer than the standard melanogaster map. It appears that mauritiana has higher recombination along the entire length of each chromosome, but the difference is greatest in centromere-proximal regions of the autosomes. The mauritiana autosomes show little or no centromeric recombinational suppression, a characteristic that is prominent in melanogaster. D. simulans appears to be intermediate both in terms of total map length and intensity of the autosomal centromeric effect. These interspecific differences in recombination have important evolutionary implications for DNA sequence organization and variability. In particular, mauritiana is expected to differ from melanogaster in patterns and amounts of sequence variation and transposon insertions.     

Molecular evolution of duplicated amylase gene regions in Drosophila melanogaster: evidence of positive selection in the coding regions and selective constraints in the cis-regulatory regions

In this study, we randomly sampled Drosophila melanogaster from Japanese and Kenyan natural populations. We sequenced duplicated (proximal and distal) Amy gene regions to test whether the patterns of polymorphism were consistent with neutral molecular evolution. F(st) between the two geographically distant populations, estimated from Amy gene regions, was 0.084, smaller than reported values for other loci, comparing African and Asian populations. Furthermore, little genetic differentiation was found at a microsatellite locus (DROYANETSB) in these samples (G'st = -0.018). The results of several tests (Tajima's, Fu and Li's, and Wall's tests) were not significantly different from neutrality. However, a significantly higher level of fixed replacement substitutions was detected by a modified McDonald and Kreitman test for both populations. This indicates that positive selection occurred during or immediately after the speciation of D. melanogaster. Sliding-window analysis showed that the proximal region 1, a part of the proximal 5' flanking region, was conserved between D. melanogaster and its sibling species, D. simulans. An HKA test was significant when the proximal region 1 was compared with the 5' flanking region of Alcohol dehydrogenase (Adh), indicating a severe selective constraint on the Amy proximal region 1. These results suggest that natural selection has played an important role in the molecular evolution of Amy gene regions in D. melanogaster.     

Analysis of copia sequence variation within and between Drosophila species

The sequences of the 5' long-terminal repeat (LTR) and adjacent leader regions of 27 full-length copia elements isolated from natural populations of Drosophila melanogaster, D. simulans, and D. mauritiana are presented. Phylogenetic analyses indicate that although D. melanogaster copia elements are distinct from those of D. simulans and D. mauritiana, the elements of these latter two species are not distinguishable from one another. LTRs and adjacent 5' leader regions of elements isolated from D. simulans and D. mauritiana are structurally similar to one another and carry substantial deletional variation mapping to regions previously identified as being of potential importance for copia expression.      

Paleo-demography of the Drosophila melanogaster subgroup: application of the maximum likelihood method

The species divergence times and demographic histories of Drosophila melanogaster and its three sibling species, D. mauritiana, D. simulans, and D. yakuba, were investigated using a maximum likelihood (ML) method. Thirty-nine orthologous loci for these four species were retrieved from DDBJ/EMBL/GenBank database. Both autosomal and X-linked loci were used in this study. A significant degree of rate heterogeneity across loci was observed for each pair of species. Most loci have the GC content greater than 50% at the third codon position. The codon usage bias in Drosophila loci is considered to result in the high GC content and the heterogenous rates across loci. The chi-square, G, and Fisher's exact tests indicated that data sets with 11, 23, and 9 pairs of DNA sequences for the comparison of D. melanogaster with D. mauritiana, D. simulans, and D. yakuba, respectively, retain homogeneous rates across loci. We applied the ML method to these data sets to estimate the DNA sequence divergences before and after speciation of each species pair along with their standard deviations. Using 1.6 x 10(-8) as the rate of nucleotide substitutions per silent site per year, our results indicate that the D. melanogaster lineage split from D. yakuba approximately 5.1 +/- 0.8 million years ago (mya), D. mauritiana 2.7 +/- 0.4 mya, and D. simulans 2.3 +/- 0.3 mya. It implies that D. melanogaster became distinct from D. mauritiana and D. simulans at approximately the same time and from D. yakuba no earlier than 10 mya. The effective ancestral population size of D. melanogaster appears to be stable over evolutionary time. Assuming 10 generations per year for Drosophila, the effective population size in the ancestral lineage immediately prior to the time of species divergence is approximately 3 x 10(6), which is close to that estimated for the extant D. melanogaster population. The D. melanogaster did not encounter any obvious bottleneck during the past 10 million years.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

DNA Sequence Variation at the Period Locus within and among Species of the Drosophila Melanogaster Complex

A 1.9-kilobase region of the period locus was sequenced in six individuals of Drosophila melanogaster and from six individuals of each of three sibling species: Drosophila simulans, Drosophila sechellia and Drosophila mauritiana. Extensive genealogical analysis of 174 polymorphic sites reveals a complex history. It appears that D. simulans, as a large population still segregating very old lineages, gave rise to the island species D. mauritiana and D. sechellia. Rather than considering these speciation events as having produced ``sister' taxa, it seems more appropriate to consider D. simulans a parent species to D. sechellia and D. mauritiana. The order, in time, of these two phylogenetic events remains unclear. D. mauritiana supports a large number of polymorphisms, many of which are shared with D. simulans, and so appears to have begun and persisted as a large population. In contrast, D. sechellia has very little variation and seems to have experienced a severe population bottleneck. Alternatively, the low variation in D. sechellia could be due to recent directional selection and genetic hitchhiking at or near the per locus.     

Evolution of the Transposable Element Mariner in Drosophila Species

The distribution of the transposable element mariner was examined in the genus Drosophila. Among the eight species comprising the melanogaster species subgroup, the element is present in D. mauritiana, D. simulans, D. sechellia, D. yakuba and D. teissieri, but it is absent in D. melanogaster, D. erecta and D. orena. Multiple copies of mariner were sequenced from each species in which the element occurs. The inferred phylogeny of the elements and the pattern of divergence were examined in order to evaluate whether horizontal transfer among species or stochastic loss could better account for the discontinuous distribution of the element among the species. The data suggest that the element was present in the ancestral species before the melanogaster subgroup diverged and was lost in the lineage leading to D. melanogaster and the lineage leading to D. erecta and D. orena. This inference is consistent with the finding that mariner also occurs in members of several other species subgroups within the overall melanogaster species group. Within the melanogaster species subgroup, the average divergence of mariner copies between species was lower than the coding region of the alcohol dehydrogenase (Adh) gene. However, the divergence of mariner elements within species was as great as that observed for Adh. We conclude that the relative sequence homogeneity of mariner elements within species is more likely a result of rapid amplification of a few ancestral elements than of concerted evolution. The mariner element may also have had unequal mutation rates in different lineages.     

Horizontal transfer of hobo transposable elements within the Drosophila melanogaster species complex: evidence from DNA sequencing.

The hobo family of transposable elements, one of three transposable-element families that cause hybrid dysgenesis in Drosophila melanogaster, appears to be present in all members of the D. melanogaster species complex: D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. Some hobo-hybridizing sequences are also found in the other members of the melanogaster subgroup and in many members of the related montium subgroup. Surveys of older isofemale lines of D. melanogaster suggest that complete hobo elements were absent prior to 50 years ago and that hobo has recently been introduced into the species by horizontal transfer. To test the horizontal transfer hypothesis, the 2.6-kb XhoI fragments of hobo elements from D. melanogaster, D. simulans, and D. mauritiana were cloned and sequenced. The DNA sequences reveal an extremely low level of divergence and support the conclusion that the active hobo element has been horizontally transferred into or among these species in the recent past.