High density of long dinucleotide microsatellites in Drosophila subobscura

We isolated 96 dinucleotide repeats with five or more tandemly repeated units from a subgenomic Drosophila subobscura library. The mean repeat unit length of microsatellite clones in D. subobscura is 15, higher than that observed in other Drosophila species. Population variation was assayed in 32-40 chromosomes from Barcelona, Spain, using 18 randomly chosen microsatellite loci. Positive correlation between measures of variation and perfect repeat length measures (mean size, most common, and longest allele) is consistent with a higher mutation rate in loci with longer repeat units. Levels of microsatellite variation measured as variance in repeat number and heterozygosity in D. subobscura were similar to those of Drosophila pseudoobscura and higher than those of Drosophila melanogaster and Drosophila simulans. Our data suggest that higher levels of microsatellite variation, and possibly density, in D. subobscura compared with D. melanogaster are due to both a higher average effective population and a higher intrinsic slippage rate in the former species.     

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.      

Isolation and characterization of dinucleotide repeat microsatellites in Drosophila ananassae

Drosophila ananassae is a cosmopolitan species with a geographic range throughout most of the tropical and subtropical regions of the world. Previous studies of DNA sequence polymorphism in three genes has shown evidence of selection affecting broad expanses of the genome in regions with low rates of recombination in geographically local populations in and around India. The studies suggest that extensive physical and genetic maps based on molecular markers, and detailed studies of population structure may provide insight into the degree to which natural selection affects DNA sequence polymorphism across broad regions of chromosomes. We have isolated 85 dinucleotide repeat microsatellite sequences and developed assay conditions for genotyping using PCR. The dinucleotide repeats we isolated are shorter, on average, than those isolated in many other Drosophila species. Levels of genetic variation are high, comparable to Drosophila melanogaster. The levels of variation indicate the effective population size of an Indonesian population of D. ananassae is 58,692 (infinite allele model) and 217,284 (stepwise mutation model), similar to estimates of effective population size for D. melanogaster calculated using dinucleotide repeat microsatellites. The data also show that the Indonesian population is in a rapid expansion phase. Cross-species amplification of the microsatellites in 11 species from the Ananassae, Elegans, Eugracilis and Ficusphila subgroups indicates that the loci may be useful for studies of the sister species, D. pallidosa, but will have limited use for more distantly related species.     

High frequency of microsatellites in Drosophila pseudoobscura

Using 30,000 bp of anonymous sequence data, we note that dinucleotide repeat arrays appear to be much more common in Drosophila pseudoobscura than in D. melanogaster or D. simulans. Repeat arrays bearing five or more units are situated on average once every 3000 bp in D. pseudoobscura, and repeat arrays bearing ten or more units are situated on average once every 7500 bp. We did not detect an association between microsatellite presence and GC-content of flanking regions.     

Interspecific comparison of the period gene of Drosophila reveals large blocks of non-conserved coding DNA.

We have cloned and sequenced the coding region of the period (per) gene from Drosophila pseudoobscura and D. virilis. A comparison with that of D. melanogaster reveals that the conceptual translation products consist of interspersed blocks of conserved and non-conserved amino acid sequence. The non-conserved portion, comprising approximately 33% of the protein sequence, includes the perfect Thr-Gly repeat of D. melanogaster, which is absent from the D. pseudoobscura and D. virilis proteins. Based on these observations and cross-species transformation experiments, we suggest that the interspecific variability in the per primary amino acid sequence contributes to the control of species-specific behaviors.     

Variable Rates of Evolution among Drosophila Opsin Genes

DNA sequences and chromosomal locations of four Drosophila pseudoobscura opsin genes were compared with those from Drosophila melanogaster, to determine factors that influence the evolution of multigene families. Although the opsin proteins perform the same primary functions, the comparisons reveal a wide range of evolutionary rates. Amino acid identities for the opsins range from 90% for Rh2 to more than 95% for Rh1 and Rh4. Variation in the rate of synonymous site substitution is especially striking: the major opsin, encoded by the Rh1 locus, differs at only 26.1% of synonymous sites between D. pseudoobscura and D. melanogaster, while the other opsin loci differ by as much as 39.2% at synonymous sites. Rh3 and Rh4 have similar levels of synonymous nucleotide substitution but significantly different amounts of amino acid replacement. This decoupling of nucleotide substitution and amino acid replacement suggests that different selective pressures are acting on these similar genes. There is significant heterogeneity in base composition and codon usage bias among the opsin genes in both species, but there are no consistent relationships between these factors and the rate of evolution of the opsins. In addition to exhibiting variation in evolutionary rates, the opsin loci in these species reveal rearrangements of chromosome elements.     

DNA sequence variation and the recombinational landscape in Drosophila pseudoobscura: a study of the second chromosome.

The relationship between rates of recombination and DNA sequence polymorphism was analyzed for the second chromosome of Drosophila pseudoobscura. We constructed integrated genetic and physical maps of this chromosome using molecular markers at 10 loci spanning most of its physical length. The total length of the map was 128.2 cM, almost twice that of the homologous chromosome arm (3R) in D. melanogaster. There appears to be very little centromeric suppression of recombination, and rates of recombination are quite uniform across most of the chromosome. Levels of sequence variation (theta(W), based on the number of segregating sites) at seven loci (tropomyosin 1, Rhodopsin 3, Rhodopsin 1, bicoid, Xanthine dehydrogenase, Myosin light chain 1, and ribosomal protein 49) varied from 0.0036 to 0.0167. Generally consistent with earlier studies, the average estimate of theta(W) at total sites is 1.5-fold higher than that in D. melanogaster, while average theta(W) at silent sites is almost 3-fold higher. These estimates of variation were analyzed in the context of a background selection model under the same parameters of mutation rate and selection as have been proposed for D. melanogaster. It is likely that a significant fraction of the higher level of sequence variation in D. pseudoobscura can be explained by differences in regional rates of recombination rather than a larger species-level effective population size. However, the distribution of variation among synonymous, nonsynonymous, and noncoding sites appears to be quite different between the species, making direct comparisons of neutral variation, and hence inferences about effective population size, difficult. Tajima's D statistics for 6 out of the 7 loci surveyed are negative, suggesting that D. pseudoobscura may have experienced a rapid population expansion in the recent past or, alternatively, that slightly deleterious mutations constitute an important component of standing variation in this species.     

Molecular population genetics of sequence length diversity in the Adh region of Drosophila pseudoobscura

Positive and negative selection on indel variation may explain the correlation between intron length and recombination levels in natural populations of Drosophila. A nucleotide sequence analysis of the 3.5 kilobase sequence of the alcohol dehydrogenase (Adh) region from 139 Drosophila pseudoobscura strains and one D. miranda strain was used to determine whether positive or negative selection acts on indel variation in a gene that experiences high levels of recombination. A total of 30 deletion and 36 insertion polymorphisms were segregating within D. pseudoobscura populations and no indels were fixed between D. pseudoobscura and its two sibling species D. miranda and D. persimilis. The ratio of Tajima's D to its theoretical minimum value (D(min)) was proposed as a metric to assess the heterogeneity in D among D. pseudoobscura loci when the number of segregating sites differs among loci. The magnitude of the D/D(min) ratio was found to increase as the rate of population expansion increases, allowing one to assess which loci have an excess of rare variants due to population expansion versus purifying selection. D. pseudoobscura populations appear to have had modest increases in size accounting for some of the observed excess of rare variants. The D/D(min) ratio rejected a neutral model for deletion polymorphisms. Linkage disequilibrium among pairs of indels was greater than between pairs of segregating nucleotides. These results suggest that purifying selection removes deletion variation from intron sequences, but not insertion polymorphisms. Genome rearrangement and size-dependent intron evolution are proposed as mechanisms that limit runaway intron expansion.     

African Drosophila melanogaster and D. simulans populations have similar levels of sequence variability, suggesting comparable effective population sizes

Drosophila melanogaster and D. simulans are two closely related species with a similar distribution range. Many studies suggested that D. melanogaster has a smaller effective population size than D. simulans. As most evidence was derived from non-African populations, we readdressed this question by sequencing 10 X-linked loci in five African D. simulans and six African D. melanogaster populations. Contrary to previous results, we found no evidence for higher variability, and thus larger effective population size, in D. simulans. Our observation of similar levels of variability of both species will have important implications for the interpretation of patterns of molecular evolution.     

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.     

A Reanalysis of Protein Polymorphism in Drosophila Melanogaster, D. Simulans, D. Sechellia and D. Mauritiana: Effects of Population Size and Selection

Comparison of synonymous and nonsynonymous variation/substitution within and between species at individual genes has become a widely used general approach to detect the effect of selection versus drift. The sibling species group comprised of two cosmopolitan (Drosophila melanogaster and Drosophila simulans) and two island (Drosophila mauritiana and Drosophila sechellia) species has become a model system for such studies. In the present study we reanalyzed the pattern of protein variation in these species, and the results were compared against the patterns of nucleotide variation obtained from the literature, mostly available for melanogaster and simulans. We have mainly focused on the contrasting patterns of variation between the cosmopolitan pair. The results can be summarized as follows: (1) As expected the island species D. mauritiana and D. sechellia showed much less variation than the cosmopolitan species D. melanogaster and D. simulans. (2) The chromosome 2 showed significantly less variation than chromosome 3 and X in all four species which may indicate effects of past selective sweeps. (3) In contrast to its overall low variation, D. mauritiana showed highest variation for X-linked loci which may indicate introgression from its sibling, D. simulans. (4) An average population of D. simulans was as heterozygous as that of D. melanogaster (14.4% v.s. 13.9%) but the difference was large and significant when considering only polymorphic loci (37.2% v.s. 26.1%). (5) The species-wise pooled populations of these two species showed similar results (all loci = 18.3% v.s. 20.0%, polymorphic loci = 47.2% v.s. 37.6%). (6) An average population of D. simulans had more low-frequency alleles than D. melanogaster, and the D. simulans alleles were found widely distributed in all populations whereas the D. melanogaster alleles were limited to local populations. As a results of this, pooled populations of D. melanogaster showed more polymorphic loci than those of D. simulans (48.0% v.s. 32.0%) but the difference was reduced when the comparison was made on the basis of an average population (29.1% v.s. 21.4%). (7) While the allele frequency distributions within populations were nonsignificant in both D. melanogaster and D. simulans, melanogaster had fewer than simulans, but more than expected from the neutral theory, low frequency alleles. (8) Diallelic loci with the second allele with a frequency less than 20% had similar frequencies in all four species but those with the second allele with a frequency higher than 20% were limited to only melanogaster the latter group of loci have clinal (latitudinal) patterns of variation indicative of balancing selection. (9) The comparison of D. simulans/D. melanogaster protein variation gave a ratio of 1.04 for all loci and 1.42 for polymorphic loci, against a ratio of approximately 2-fold difference for silent nucleotide sites. This suggests that the species ratios of protein and silent nucleotide polymorphism are too close to call for selective difference between silent and allozyme variation in D. simulans. In conclusion, the contrasting levels of allozyme polymorphism, distribution of rare alleles, number of diallelic loci and the patterns of geographic differentiation between the two species suggest the role of natural selection in D. melanogaster, and of possibly ancient population structure and recent worldwide migration in D. simulans. Population size differences alone are insufficient as an explanation for the patterns of variation between these two species.     

Evolution of the glucose dehydrogenase gene in Drosophila

The glucose dehydrogenase genes (Gld) of Drosophila melanogaster, of D. pseudoobscura, and of D. virilis have been isolated and compared with each other in order to identify conserved and divergent aspects of their structure and expression. The exon/intron structure of Gld is conserved. The Gld mRNAs are similar, with a range of 2.6-2.8 kb among the three species. All three species exhibit peaks of Gld expression during every major developmental stage, although considerable variation in the precise timing of these peaks exists between species. Interspecific gene transfer experiments demonstrate that the regulation and function of the D. pseudoobscura Gld is similar enough to the homologous gene in D. melanogaster to substitute for its essential role in the eclosion process. Comparison of the putative promoter sequences has identified both shared and divergent sequence elements which are likely responsible, respectively, for the conserved and divergent patterns of expression observed. The entire coding sequences of the pseudoobscura and melanogaster Gld genes are presented and shown to encode a 612-amino-acid pre-protein. The inferred amino acid sequences are 92% conserved between the two species. In general the intronic regions of Gld are unusually well conserved.     

A survey of chromosomal and nucleotide sequence variation in Drosophila miranda

There have recently been several studies of the evolution of Y chromosome degeneration and dosage compensation using the neo-sex chromosomes of Drosophila miranda as a model system. To understand these evolutionary processes more fully, it is necessary to document the general pattern of genetic variation in this species. Here we report a survey of chromosomal variation, as well as polymorphism and divergence data, for 12 nuclear genes of D. miranda. These genes exhibit varying levels of DNA sequence polymorphism. Compared to its well-studied sibling species D. pseudoobscura, D. miranda has much less nucleotide sequence variation, and the effective population size of this species is inferred to be several-fold lower. Nevertheless, it harbors a few inversion polymorphisms, one of which involves the neo-X chromosome. There is no convincing evidence for a recent population expansion in D. miranda, in contrast to D. pseudoobscura. The pattern of population subdivision previously observed for the X-linked gene period is not seen for the other loci, suggesting that there is no general population subdivision in D. miranda. However, data on an additional region of period confirm population subdivision for this gene, suggesting that local selection is operating at or near period to promote differentiation between populations.     

The period gene of Drosophila carries species-specific behavioral instructions.

We have analyzed and compared the circadian locomotor activity rhythms of Drosophila melanogaster and D.pseudoobscura. The rhythms of D.pseudoobscura are stronger and the periods shorter than those of D.melanogaster. We have also transformed D.melanogaster flies with a hybrid gene containing the coding region of the D.pseudoobscura period (per) gene. Behavioral assays of flies containing this hybrid gene show that the per protein encoded by the D.pseudoobscura per gene is able to rescue the rhythmic deficiencies of arrhythmic, pero1 D.melanogaster. More important, the rhythms of some of these strains are stronger and the periods shorter than those of D.melanogaster (and those of transformants which carry the equivalent D.melanogaster per gene construct) and hence resemble those of D.pseudoobscura. The results suggest that the primary amino acid sequence of the per gene encodes species-specific behavioral instructions that are detectable when only the per gene is transferred to a different species.     

The Speciation History of Drosophila Pseudoobscura and Close Relatives: Inferences from DNA Sequence Variation at the Period Locus

Thirty-five period locus sequences from Drosophila pseudoobscura and its siblings species, D. p. bogotana, D. persimilis, and D. miranda, were studied. A large amount of variation was found within D. pseudoobscura and D. persimilis, consistent with histories of large effective population sizes. D. p. bogotana, however, has a severe reduction in diversity. Combined analysis of per with two other loci, in both D. p. bogotana and D. pseudoobscura, strongly suggest this reduction is due to recent directional selection at or near per within D. p. bogotana. Since D. p. bogotana is highly variable and shares variation with D. pseudoobscura at other loci, the low level of variation at per within D. p. bogotana can not be explained by a small effective population size or by speciation via founder event. Both D. pseudoobscura and D. persimilis have considerable intraspecific gene flow. A large portion of one D. persimilis sequence appears to have arisen via introgression from D. pseudoobscura. The time of this event appears to be well after the initial separation of these two species. The estimated times since speciation are one mya for D. pseudoobscura and D. persimilis and 2 mya since the formation of D. miranda.     

High mutation rate of a long microsatellite allele in Drosophila melanogaster provides evidence for allele-specific mutation rates

Within recent years, microsatellite have become one of the most powerful genetic markers in biology. For several mammalian species, microsatellite mutation rates have been estimated on the order of 10(- 3)-10(-5). A recent study, however, demonstrated mutation rates in Drosophila melanogaster of at least one order of magnitude lower than those in mammals. To further test this result, we examined mutation rates of different microsatellite loci using a larger sample size. We screened 24 microsatellite loci in 119 D. melanogaster lines maintained for approximately 250 generations and detected 9 microsatellite mutations. The average mutation rate of 6.3 x 10(-6) is identical to the mutation rate from a previous study. Most interestingly, all nine mutations occurred at the same allele of one locus (DROYANETSB). This hypermutable allele has 28 dinucleotide repeats and is among the longest microsatellite reported in D. melanogaster. The allele-specific mutation rate of 3.0 x 10(-4) per generation is within the range of mammalian mutation rates. Future microsatellite analyses will have to account for the dramatic differences in allele-specific mutation rates.      

Desiccation resistance in two species of Drosophila

Three populations of Drosophila pseudoobscura, each one represented by 12 isofemale lines, and one laboratory strain of D. melanogaster were tested for desiccation resistance at two time periods. Except in the case of one population of D. pseudoobscura, the ability to withstand drying was significantly greater in females than in the corresponding males. The males of the three populations of D. pseudoobscura differed significantly among themselves in their resistance to desiccation, as did the females. The females of D. melanogaster exhibited a consistently higher survival rate than those of D. pseudoobscura, but not the males. These results are discussed with reference to the third chromosome inversion polymorphism of D. pseudoobscura and the cosmopolitan distribution of D. melanogaster.     

On the detection of nonrandom associations between DNA polymorphisms in natural populations of Drosophila

The capacity to detect nonrandom associations between restriction-map variants was examined in eight gene regions of Drosophila melanogaster (yellow-achaetescute, white, Zw, Adh, Est6, and rosy) and D. pseudoobscura (Adh and Xdh), on the basis of published population data. The statistical power from individual pairwise tests was both heterogeneous and generally low across gene regions. Sample sizes larger than those currently being used are needed to ensure any power to detect disequilibrium by individual tests. It is found that the heterogeneity in power is mostly explained by large differences in the intensity of sample disequilibrium among regions. The yellow-achaete- scute, Zw, and Adh loci of D. melanogaster displayed both the highest mean power (approximately 0.4) and a very great disequilibrium (mean absolute values of D' were 0.8-1). By contrast, all the other gene regions exhibited lower mean power (approximately 0.2) and moderate levels of disequilibrium (0.4-0.6). Although the proportion of significant pairwise associations, especially for white, Est6, and rosy in D. melanogaster and for Adh and Xdh in D. pseudoobscura, is more or less close to the type I error, simultaneous-inference significance tests show that gametic disequilibrium is occurring at the eight DNA regions examined.      

Length Variation of Cag/Caa Trinucleotide Repeats in Natural Populations of Drosophila Melanogaster and Its Relation to the Recombination Rate

Eleven genes distributed along the Drosophila melanogaster chromosome 2 and showing exonic tandem repeats of glutamine codons (CAG or CAA) were surveyed for length variation in a sample of four European and African populations. Only one gene was monomorphic. Eight genes were polymorphic in all populations, with a total number of alleles varying between five and 12 for 120 chromosomes. The average heterozygozity per locus and population was 0.41. Selective neutrality in length variation could not be rejected under the assumptions of the infinite allele model. Significant population subdivision was found though no geographical pattern emerged, all populations being equally different. Significant linkage disequilibrium was found in four out of seven cases where the genetic distance between loci was <1 cM and was negligible when the distance was larger. There is evidence that these associations were established after the populations separated. An unexpected result was that variation at each locus was independent of the coefficient of exchange, although the latter ranged from zero to the relatively high value of 6.7%. This would indicate that background selection and selective hitchhiking, which are thought to affect levels of nucleotide substitution polymorphism, have no effect on trinucleotide repeat variation.