Drosophila virilis Has Long and Highly Polymorphic Microsatellites

Christian Schlötterer and Bettina Harr

Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Vienna, Austria


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Comparative genomics is a powerful approach to inference of the dynamics of genome evolution. Most information about the evolution of microsatellites in the genus Drosophila has been obtained from Drosophila melanogaster. For comparison, we collected microsatellite data for the distantly related species Drosophila virilis. Screening about 0.5 Mb of nonredundant genomic sequence from GenBank, we identified 239 dinucleotide microsatellites. On average, D. virilis dinucleotides were significantly longer than D. melanogaster microsatellites (7.69 repeats vs. 6.75 repeats). Similarly, direct cloning of microsatellites resulted in a higher mean repeat number in D. virilis than in D. melanogaster (12.7 repeats vs. 12.2 repeats). Characterization of 11 microsatellite loci mapping to division 40–49 on the fourth chromosome of D. virilis indicated that D. virilis microsatellites are more variable than those of D. melanogaster.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Microsatellites are short, tandemly repeated sequence motifs of 1–6 bp which are distributed over the euchromatic part of the genome. Both the frequency and the length (number of repeats) of microsatellites significantly exceed the expectations based on the nucleotide composition of the genome. It is widely accepted that short protomicrosatellites, which arise by chance in the genome, are expanded by replication slippage, a mutation process which is specific to microsatellite DNA. Slippage mutations occur during DNA replication by displacement of the nascent strand, which subsequently realigns out of register. If DNA synthesis continues on this misplaced DNA molecule, the repeat number of the microsatellite is altered (Tautz and Schlötterer 1994Citation ). Based on this mechanism, the distribution of microsatellites is expected to be fairly constant across genomes of different taxa. Nevertheless, significant differences were described between species. The abundance of different dinucleotide repeat types varies among species, with (AT)n repeats predominating in Arabidopsis thaliana, (GT/CA)n in mammals and Drosophila melanogaster, and (CT/GA)n in Caenorhabditis elegans (Schlötterer 2000Citation ). Furthermore, the genomic length distribution of microsatellites was also found to differ between species. A particularly illustrative example is that of the comparison of human and D. melanogaster microsatellites. Direct cloning experiments and GenBank surveys indicate that D. melanogaster has significantly shorter microsatellites than do humans (Kruglyak et al. 1998Citation ; Schug et al. 1998bCitation ; Bachtrog et al. 1999Citation ). Recently, it has been suggested that the genomic length distribution of microsatellites is determined by the genome size; species with larger genomes tend to have longer microsatellites (Hancock 1996Citation ). This trend would be consistent with the observation of shorter microsatellites in D. melanogaster. Nevertheless, yeast and C. elegans have a smaller genome than D. melanogaster but, on average, longer microsatellites (Harr and Schlötterer 2000Citation ). Furthermore, (GT/CA)n microsatellites are longer in Fugu rubripes than in humans despite a smaller genome (Edwards et al. 1998Citation ). To further investigate the relationship between genome size and microsatellite length distribution, we studied Drosophila virilis, which has a larger genome than D. melanogaster (Powell 1997Citation ).


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Flystocks
Flies were obtained from the National Drosophila Species Resource Center, the Umeå Drosophila Stock Center, J. Vieira, and J. Aspi. The following lines were analyzed (if known, geographic origin is given in parentheses): D. virilis—15010-1051.9 (Sendai, Japan), 15010-1051.8 (Truckee, Nev.), 15010-1015.38 (Japan), 15010-1051.47 (Hangchow, China), 15010-1051.48 (Texmelucan, Mexico), 15010-1051.49 (Chaco, Argentina), 15010-1051.51 (Santiago, Chile), 15010-1051.52 (Russia), S170, S172, S173, S171, strain 2 (Kutaisi, Georgia), strain 9 (Batumi, Georgia), W157 (Mexico), W158 (Japan), W159 (the Netherlands); Drosophila lummei—15010-1011.1 (Moscow, Russia), 15010-1011.2 (Overhalix, Sweden), 15010-1011.4 (Kukkola, Finland), 15010-1011.5 (Karesjoki, Finland), 15010-1011.7 (Oulu, Finland), S070 (Kuopio, Finland), S071 (Vaajasalo, Finland), S072 (Moscow, Russia), luJapFu (Japan), 1101, 1100.

GenBank Search
Drosophila virilis sequences were retrieved from GenBank in June 1998. Redundant sequences were removed, as were sequences that were obtained from a search for long simple sequence stretches (Tautz and Renz 1984Citation ). Mitochondrial sequences were also excluded from the analysis. Only microsatellites with five or more repeats were counted. To test if the mean repeat number in D. virilis was significantly higher than that in D. melanogaster, we resampled (200 times) the number of microsatellite loci that we detected in D. virilis (239) from the D. melanogaster data set described in Bachtrog et al. (1999)Citation . We then calculated the mean repeat number of the resampled data sets. P values indicated the fraction of pseudoreplicates, which had at least as many repeats as observed in the D. virilis sample.

Cloning of Microsatellites
Genomic DNA was isolated from D. virilis and D. melanogaster by a high salt extraction method (Miller, Dykes, and Polesky 1988Citation ). Genomic DNA was digested separately with AluI, HaeIII, and RsaI and subsequently pooled. Size-fractionated fragments 500–1,100 bp were cloned into M13mp18. Clones carrying a microsatellite were identified by hybridization at 37°C with (GT)7G and (AG)7A oligonucleotides, which were end-labeled with {gamma} 32P. Filters were washed 3 times in 5 x SSC, 0.1% SDS, at 37°C. Positive clones were identified by autoradiography. Filters from both species were processed together. Hence, the screening procedures were identical for both species. Positive clones were sequenced on an ABI 377 automated sequencer. (AT/TA)n repeats were not screened because of the self-complementarity and low Tm of an (AT/TA)n probe. Nevertheless, no dramatic bias in relative microsatellite density is expected, as the frequencies of (AT/TA)n microsatellites determined in GenBank surveys are very similar for D. melanogaster and D. virilis (28.9% vs. 33.9%).

Typing of Microsatellites
To determine microsatellite variability in D. virilis and D. lummei, 11 microsatellites mapping to the fourth complement were typed radioactively following standard protocols (Schlötterer 1998bCitation ). In brief, end-labeled ({gamma} 32P) PCR primers were used in a 10-µl reaction volume (1.5 mM MgCl2, 200 µM dNTPs, 1 µM of each primer, 50–100 ng template DNA, and 0.5 U Taq polymerase). Initial denaturation for 4 min at 94°C was followed by 30 cycles of 1 min at 94°C, 1 min at 50–56°C (depending on the primer combination), and 1 min at 72°C. We used a final extension of 72°C for 45 min to assure a quantitative terminal transferase activity of the Taq polymerase. PCR products were separated on a 7% denaturing polyacrylamide gel (32% formamide, 5.6 M urea). PCR products were sized by running a sizing ladder next to the amplified microsatellites (Schlötterer and Zangerl 1999Citation ). Summary results of the microsatellite analysis are available on the authors' web page (http://i122server.vu-wien.ac.at/).

Statistical Analyses
Heterozygosity and variance in repeat number was determined with the software package Microsat (Minch et al. 1995Citation ). Statistical tests were carried out with the SPSS software.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Analysis of Cloned Microsatellites
Since slight differences in the cloning procedure may influence the result, we cloned microsatellites from D. melanogaster and D. virilis in parallel to compare their genomic distribution. All experimental steps were carried out in parallel, and screening for positive clones was done with the same hybridization probe and washing solutions.

Microsatellite Density
We screened 4.2 x 103 D. melanogaster and 2.2 x 103 D. virilis clones carrying an insert. Eighteen positive clones were identified in D. melanogaster, and 65 were identified in D. virilis. Assuming an average insert size of 800 bp, this translates into 0.54 microsatellites per 100 kb in D. melanogaster and 3.64 microsatellites per 100 kb in D. virilis. While the microsatellite density obtained for D. melanogaster was lower than that in previous reports (England, Briscoe, and Frankham 1996Citation ; Schug et al. 1998bCitation ), the microsatellite density in D. virilis greatly exceeded all reported values for D. melanogaster. Consistent with our observation of an approximately sevenfold higher microsatellite density in D. virilis are experiments in which genomic DNA hybridized with microsatellite probes also detected a stronger hybridization signal in D. virilis (Pardue et al. 1987Citation ).

Microsatellite Length in Cloned Microsatellites
In total, 10 D. melanogaster and 26 D. virilis microsatellite clones were sequenced. We used two different measurements to compare the lengths of the cloned microsatellites. First, the longest uninterrupted dinucleotide stretch was counted in each clone. Second, we allowed for one imperfection (base substitution, insertion, change in repeat type) in the microsatellite stretch and counted the total number of repeats. On average, D. virilis clones carried 12.7 uninterrupted repeats, while D. melanogaster clones had 12.2 repeats (table 1 ). This difference, however, was not statistically significant (P = 0.37, Mann-Whitney U-test). Allowing for one imperfection in the microsatellite stretch, the average microsatellite lengths changed to 15.2 in D. virilis and 12.8 in D. melanogaster. The difference between both species was still not statistically significant (P = 0.15, Mann-Whitney U-test).


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Table 1 Microsatellite Repeat Numbers Obtained by Direct Cloning from Genomic DNA

 
Interestingly, 8 out of 26 D. virilis clones contained at least two microsatellites longer than four repeats which were not adjacent to each other. In D. melanogaster, 1 out of 10 clones contained more than a single microsatellite repeat. These results are consistent with the observation of Bachtrog et al. (1999)Citation , who showed that microsatellites are not completely randomly distributed, but have some tendency to cluster. While our data suggested that D. virilis shows a more pronounced clustering of microsatellites, this was not significant (P = 0.22, Fisher's exact test), particularly if the higher density of D. virilis microsatellites is accounted for. It should be noted, however, that clustering of microsatellites could strongly affect our estimated microsatellite densities. The more pronounced the clustering of microsatellites is, the higher the mean microsatellite density would be.

Data Bank Survey
We conducted a GenBank search for dinucleotide microsatellites in D. virilis. Like in D. melanogaster (Schug et al. 1998bCitation ; Bachtrog et al. 1999Citation ), (GT/CA)n was the most frequent repeat type (45.6%) in D. virilis, followed by (AT)n (33.9%) and (GA/CT)n (19.7%). Only two (GC)n microsatellites with five repeats each were detected.

Microsatellite Density
Two hundred thirty-nine D. virilis dinucleotide microsatellites with five or more repeats were identified in approximately 0.5 Mb of nonredundant genomic sequence. Hence, the microsatellite density would be about 48 microsatellites per 100 kb, which is about twice as high as that in D. melanogaster (22 microsatellites per 100 kb; Bachtrog et al. 1999Citation ). The microsatellite density in D. melanogaster was determined from large contiguous sequences. In contrast, for D. virilis, we concatenated short GenBank entries. Therefore, coding sequences were overrepresented in the D. virilis data set, which may explain why the GenBank survey detected only a twofold higher microsatellite density as compared to the sevenfold higher density obtained from the direct cloning experiments.

Microsatellite Length
The mean length of D. virilis microsatellites was 7.69 repeats. Compared with D. melanogaster, which has a mean length of 6.75 repeats, D. virilis microsatellites are longer. This difference is statistically significant (P < 0.005). In contrast to the estimated microsatellite density, this result is not expected to be biased by a higher representation of coding sequence in the data set. From the length distribution of microsatellites in the D. melanogaster and D. virilis genome (fig. 1 ), it is apparent that short microsatellites are less abundant in D. virilis, while longer ones are slightly more frequent.



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Fig. 1.—Frequency distribution of the different size classes of microsatellites in Drosophila melanogaster and Drosophila virilis. Drosophila melanogaster data are based on about 3,000 microsatellite loci taken from Bachtrog et al. (1999) Citation

 
Analysis of Natural Variation
To determine the variability of D. virilis microsatellites, we typed 17 D. virilis individuals for 11 loci mapping to division 40–49 on the fourth chromosome (unpublished data). It should be noted that those loci were isolated independently of those discussed above. Thus, they represent an independent data set. The average variability of the analyzed microsatellite loci was high. Heterozygosities ranged from 0.53 to 0.84, averaging 0.70 (table 2 ). The mean variance in repeat number was 14.2. Based on the allele distribution of the microsatellite loci typed, we calculated an average length of 11.1 repeats. This measurement assumes that all length variation can be attributed to changes in repeat number only. For comparison, we also analyzed 14 individuals of D. lummei, a close relative of D. virilis. The average variance in repeat number was 21.2, and the average heterozygosity was 0.63 (table 2 ), suggesting similar levels of variability in D. lummei.


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Table 2 Microsatellite Variability in Drosophila virilis and Drosophila lummei

 
In comparison, average heterozygosities in the D. melanogaster species group are lower than 0.5 (Bachtrog et al. 2000Citation ), suggesting that microsatellite variability is higher in the D. virilis species group. Similarly, the mean variance (V) in repeat number of microsatellites is significantly smaller in D. melanogaster (1.98; Bachtrog et al. 2000Citation ) than in D. virilis (14.2; P = 0.001, Mann-Whitney U-test). The mean repeat number of the 11 D. virilis microsatellites was 11.1 repeats, which is longer than the 8.85 repeats observed in a population survey of D. melanogaster (Bachtrog et al. 2000Citation ) (table 3 ).


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Table 3 Average Repeat Numbers and Microsatellite Density

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The genomic distribution of microsatellites is one of the key parameters in understanding their evolution. Accordingly, many studies have provided estimates for microsatellite density and mean length. Nevertheless, those numbers have often been obtained by different approaches. In this report, we used three different ways to characterize microsatellites in D. virilis: direct cloning of microsatellites, a GenBank survey, and analysis of natural variation. Our data, which were all independently obtained, show a large variance (table 3 ). In the following discussion, we will argue that this is a reflection of the methods used, as well as the underlying definition of a microsatellite.

It is widely accepted that estimates of microsatellite length distributions from population data suffer from the problem of ascertainment bias (Ellegren, Primmer, and Sheldon 1995Citation ), with the problem being that PCR primers are preferentially designed for those loci with more repeats, as they are normally more polymorphic. Direct cloning is therefore often regarded as a more objective way to characterize the length distribution of microsatellites. Hence, it is surprising that we obtained a large difference between the mean repeat number obtained from cloning experiments compared with those from GenBank surveys (table 3 ). Such a difference could be caused by a cloning bias in favor of longer microsatellites. Since longer microsatellites are less abundant, the estimated density of microsatellites will be lower. In addition, heterochromatic regions of the genome, which are depauperate of microsatellite DNA (Pardue et al. 1987Citation ), could further reduce the density of microsatellites as estimated by direct cloning.

It should be noted, nevertheless, that the mean length of a microsatellite is strongly affected by the definition of a microsatellite. We set the minimum length of a microsatellite to five repeats, which resulted in a mean length of 7.69 repeats for D. virilis microsatellites. Increasing the lower boundary to six repeats changes the mean repeat number to 8.77. Furthermore, the length distribution of microsatellites will also be affected when imperfections in the microsatellite structure are permitted. Very often, reports about the cloning of microsatellites also include interruptions in the repeat structure. GenBank surveys, however, only report the number of uninterrupted repeats. Therefore, our data suggest that microsatellite lengths should not be regarded as absolute values which can easily be compared.

Regardless of these differences, our comparison of the two species D. melanogaster and D. virilis consistently showed the same trend, strongly supporting the existence of a real biological phenomenon.

Microsatellites Are Longer in D. virilis than in D. melanogaster
In GenBank surveys, we observed that microsatellites in D. virilis are longer than those in D. melanogaster. Also, direct cloning experiments (table 3 ) found a higher mean microsatellite length in D. virilis. While the latter result was not statistically significant, it has to be noted that the difference in mean repeat number between D. melanogaster and D. virilis, as inferred from the GenBank survey, is about one repeat unit. Given the large variance in repeat number (table 1 ), a large sample size is required to obtain statistical significance for such a small difference in mean repeat number. Thus, we obtained a statistically significant difference only for our GenBank survey.

While a difference in mean microsatellite repeat number of less than one repeat unit may appear small given the large variance, the comparison with human data indicates that such a difference has important implications. Previously, we used 33 Mb of nonredundant genomic sequence in humans and calculated the mean repeat number of all dinucleotide microsatellites with five or more repeat units (Harr and Schlötterer 2000Citation ). Interestingly, the obtained mean repeat number of human microsatellites is 7.64, which is almost identical to the mean repeat number observed in D. virilis (7.69). Based on the genomic length distribution of microsatellites, Kruglyak et al. (1998) Citation recently calculated that the slippage rate (per repeat unit) of human microsatellites is about 21-fold higher than that in D. melanogaster if the same base substitution rate is assumed. Hence, it may be expected that D. virilis microsatellites should have a higher mutation rate than D. melanogaster loci.

In agreement with the prediction of the model of Kruglyak et al. (1998)Citation , we found microsatellites in D. virilis to be more variable than those in D. melanogaster. The interpretation of this result, however, is complicated by the fact that the effective population size and the mutation rate determine microsatellite variability. Presuming a constant base substitution rate in D. melanogaster and D. virilis, the population sizes of both species are very similar (Hilton and Hey 1996, 1997Citation ; Vieira and Charlesworth 1999Citation ). Hence, it could be concluded that the higher variability of D. virilis microsatellites is the result of a higher mutation rate. Nevertheless, it has previously been shown that microsatellite mutation rate is positively correlated with repeat number (Goldstein and Clark 1995Citation ; Schlötterer et al. 1998Citation ; Schug et al. 1998aCitation ; Harr and Schlötterer 2000Citation ). Hence, longer microsatellites are more variable than short ones. In our data set, the microsatellites of D. virilis were longer on average than those of D. melanogaster (table 3 ). Accordingly, we can not determine whether the slippage rate per repeat unit of D. virilis microsatellites is higher than that in D. melanogaster.

Our observation of longer D. virilis microsatellites fits the general trend for longer repetitive sequences in this species. The neurogenic gene mastermind has high levels of cryptic simplicity in its coding region. As described for cryptic simple regions in general (Tautz, Trick, and Dover 1986Citation ), mastermind also has a high frequency of indels between species (Newfeld, Smoller, and Yedvobnick 1991Citation ). The comparison between D. melanogaster and D. virilis indicates a longer mastermind protein in D. virilis (1,596 vs. 1,655 amino acids). Further support for a general trend of longer repetitive DNA in D. virilis comes from the segmentation gene hunchback. Similar to mastermind, hunchback contains regions of high cryptic simplicity, and the hunchback protein in D. virilis is also longer (816 vs. 758 amino acids in D. melanogaster) (Treier, Pfeifle, and Tautz 1989Citation ).

Consistent with the observation that the general level of repetition seems to be related to genome size (Hancock 1996Citation ), D. virilis has a larger genome size than D. melanogaster (0.34–0.38 pg per haploid genome compared with 0.18–0.21 pg in D. melanogaster) (Powell 1997Citation ). Even if only the euchromatic genome sizes are compared, the D. virilis genome is still approximately 36% larger than the D. melanogaster genome. The length difference between D. virilis and D. melanogaster microsatellites, however, is too small to account for the difference in genome size. More likely, size differences in long introns are an important factor in determining the genome sizes of both species (Moriyama, Petrov, and Hartl 1998Citation ).

Why Does the Distribution of Microsatellites Differ Between Species?
Recent results indicate that the intraspecific size distribution of microsatellites is not constrained by selection, but through a size-dependent mutation mechanism: long microsatellite alleles have a downward mutation bias, while shorter alleles do not show such a trend (Schlötterer 1998aCitation ; Harr and Schlötterer 2000Citation ). Interestingly, this mutation pattern has been described for a wide range of species, including yeast (Wierdl, Dominska, and Petes 1997Citation ), D. melanogaster (Schlötterer et al. 1998aCitation ; Harr and Schlötterer 2000Citation ), and humans (Ellegren 2000Citation ; Xu, Peng, and Fang 2000Citation ). Although D. melanogaster and humans share the allele-specific mutation spectrum described above, the two species differ in their mean microsatellite lengths. Harr and Schlötterer (2000)Citation recently suggested that each species has a characteristic (species-specific) microsatellite length at which the mutation spectrum changes. Rather than no mutation bias or a slightly upward mutation bias, as is typical for short alleles, those alleles, which are longer than the critical length, have a downward mutation bias. Assuming that different species have characteristic lengths at which the mutation behavior changes, the differences in microsatellite length distribution could be explained. This model is fully applicable to the observed difference between D. virilis and D. melanogaster. Given that long microsatellite alleles are described for both D. melanogaster and D. virilis, their mutation spectra could be compared to verify the model of Harr and Schlötterer (2000)Citation .


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Many thanks to J. Vieira and G. Muir for helpful comments on earlier versions of the manuscript. We are grateful to Renate Ritter for technical assistance. Two anonymous referees made helpful suggestions. The National Drosophila Species Resource Center, the Umeå Drosophila Stock Center, J. Vieira, and J. Aspi provided flies. This work was supported by grants of the Oesterreichische National Bank and Fonds zur Förderung der Wissenschuftlichen Forschung to C.S.


    Footnotes
 
Pierre Capy, Reviewing Editor

1 Keywords: microsatellites Drosophila virilis, Drosophila melanogaster, genome evolution Back

2 Address for correspondence and reprints: Christian Schlötterer, Institut für Tierzucht und Genetik, Josef-Baumann Gasse 1, 1210 Vienna, Austria. E-mail: christian.schloetterer{at}vu-wien.ac.at Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 

    Bachtrog, D., M. Agis, M. Imhof, and C. Schlötterer. 2000. Microsatellite variability differs between dinucleotide repeat motifs—evidence from Drosophila melanogaster. Mol. Biol. Evol. 17:1277–1285.[Abstract/Free Full Text]

    Bachtrog, D., S. Weiss, B. Zangerl, G. Brem, and C. Schlötterer. 1999. Distribution of dinucleotide microsatellites in the Drosophila melanogaster genome. Mol. Biol. Evol. 16:602–610.[Abstract]

    Edwards, Y. J., G. Elgar, M. S. Clark, and M. J. Bishop. 1998. The identification and characterization of microsatellites in the compact genome of the Japanese pufferfish, Fugu rubripes: perspectives in functional and comparative genomic analyses. J. Mol. Biol. 278:843–854.[ISI][Medline]

    Ellegren, H. 2000. Heterogeneous mutation processes in human microsatellite DNA sequences. Nat. Genet. 24:400–402.[ISI][Medline]

    Ellegren, H., C. R. Primmer, and B. C. Sheldon. 1995. Microsatellite ‘evolution’: directionality or bias? Nat. Genet. 11:360–362.[Medline]

    England, P. R., D. A. Briscoe, and R. Frankham. 1996. Microsatellite polymorphisms in a wild population of Drosophila melanogaster. Genet. Res. 67:285–290.

    Goldstein, D. B., and A. G. Clark. 1995. Microsatellite variation in North American populations of Drosophila melanogaster. Nucleic Acids Res. 23:3882–3886.

    Hancock, J. M. 1996. Simple sequences and the expanding genome. Bioessays 18:421–425.

    Harr, B., and C. Schlötterer. 2000. Long microsatellite alleles in D. melanogaster have a downward mutation bias and short persistence times, which cause their genome wide under-representation. Genetics 155:1213–1220.

    Hilton, H., and J. Hey. 1996. DNA sequence variation at the period locus reveals the history of species and speciation events in the Drosophila virilis group. Genetics 144:1015–1025.

    ———. 1997. A multilocus view of speciation in the Drosophila virilis species group reveals complex histories and taxonomic conflicts. Genet. Res. 70:185–194.[ISI]

    Kruglyak, S., R. T. Durret, M. Schug, and C. F. Aquadro. 1998. Equilibrium distributions of microsatellite repeat length resulting from a balance between slippage events and point mutations. Proc. Natl. Acad. Sci. USA 95:10774–10778.

    Miller, S. A., D. D. Dykes, and H. F. Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16:1215.

    Minch, E., A. Ruiz-Linares, D. Goldstein, M. Feldman, and L. L. Cavalli-Sforza. 1995. Microsat (version 1.4d): a computer program for calculating various statistics on microsatellite allele data.

    Moriyama, E. N., D. A. Petrov, and D. L. Hartl. 1998. Genome size and intron size in Drosophila. Mol. Biol. Evol. 15:770–773.[Free Full Text]

    Newfeld, S. J., D. A. Smoller, and B. Yedvobnick. 1991. Interspecific comparison of the unusually repetitive Drosophila locus mastermind. J. Mol. Evol. 32:415–420.[ISI][Medline]

    Pardue, M. L., K. Lowenhaupt, A. Rich, and A. Nordheim. 1987. (dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function. EMBO J. 6:1781–1789.[Abstract]

    Powell, J. R. 1997. Progress and prospects in evolutionary biology: the Drosophila model. Oxford University Press, Oxford, England.

    Schlötterer, C. 1998a. Are microsatellites really simple sequences? Curr. Biol. 8:R132–R134.

    ———. 1998b. Microsatellites. Pp. 237–261 in A. R. Hoelzel, ed. Molecular genetic analysis of populations: a practical approach 2/e. Oxford University Press, Oxford, England.

    ———. 2000. Evolutionary dynamics of microsatellite DNA. Chromosoma (in press).

    Schlötterer, C., R. Ritter, B. Harr, and G. Brem. 1998. High mutation rates of a long microsatellite allele in Drosophila melanogaster provides evidence for allele-specific mutation rates. Mol. Biol. Evol. 15:1269–1274.[Abstract/Free Full Text]

    Schlötterer, C., and B. Zangerl. 1999. The use of imperfect microsatellites for DNA fingerprinting and population genetics. Pp. 153–165 in J. T. Epplen and T. Lubjuhn, eds. DNA profiling and DNA fingerprinting. Birkhäuser, Basel, Switzerland.

    Schug, M. D., C. M. Hutter, K. A. Wetterstrand, M. S. Gaudette, T. F. Mackay, and C. F. Aquadro. 1998a. The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster. Mol. Biol. Evol. 15:1751–1760.

    Schug, M. D., K. A. Wetterstrand, M. S. Gaudette, R. H. Lim, C. M. Hutter, and C. F. Aquadro. 1998b. The distribution and frequency of microsatellite loci in Drosophila melanogaster. Mol. Ecol. 7:57–69.

    Tautz, D., and M. Renz. 1984. Simple DNA sequences of Drosophila virilis isolated by screening with RNA. J. Mol. Biol. 172:229–235.[ISI][Medline]

    Tautz, D., and C. Schlötterer. 1994. Simple sequences. Curr. Opin. Genet. Dev. 4:832–837.[Medline]

    Tautz, D., M. Trick, and G. A. Dover. 1986. Cryptic simplicity in DNA is a major source of genetic variation. Nature 322:652–656.

    Treier, M., C. Pfeifle, and D. Tautz. 1989. Comparison of the gap segmentation gene hunchback between Drosophila melanogaster and Drosophila virilis reveals novel modes of evolutionary change. EMBO J. 8:1517–1525.[Abstract]

    Vieira, J., and B. Charlesworth. 1999. X chromosome DNA variation in Drosophila virilis. Proc. R. Soc. Lond. B Biol. Sci. 266:1905–1912.

    Wierdl, M., M. Dominska, and T. D. Petes. 1997. Microsatellite instability in yeast: dependence on the length of the microsatellite. Genetics 146:769–779.

    Xu, X., M. Peng, and Z. Fang. 2000. The direction of microsatellite mutations is dependent upon allele length. Nat. Genet. 24:396–399.[ISI][Medline]

Accepted for publication June 30, 2000.