Removal of Microsatellite Interruptions by DNA Replication Slippage: Phylogenetic Evidence from Drosophila

Bettina Harr, Barbara Zangerl and Christian Schlötterer1,

Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Austria


    Abstract
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Microsatellites are tandem repetitions of short (1–6 bp) motifs. It is widely assumed that microsatellites degenerate through the accumulation of base substitutions in the repeat array. Using a phylogenetic framework, we studied the evolutionary dynamics of interruptions in three Drosophila microsatellite loci. For all three loci, we show that the interruptions in a microsatellite can be lost, resulting in a longer uninterrupted microsatellite stretch. These results indicate that mutations in the microsatellite array do not necessarily lead to decay but may represent only a transition state during the evolution of a microsatellite. Most likely, this purification of interrupted microsatellites is caused by DNA replication slippage.


    Introduction
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Recently, the mutational dynamics of microsatellite DNA has gained substantial attention. The widespread use of microsatellites in behavioral ecology (Schlötterer and Pemberton 1998Citation ), population genetics (Jarne and Lagoda 1996Citation ), and phylogenetic reconstruction (Harr et al. 1998Citation ) requires a better understanding of microsatellite mutational processes. While single-copy DNA evolves primarily through the accumulation of base substitutions, the predominant mutation mechanism of microsatellites is DNA replication slippage (Tautz and Schlötterer 1994Citation ). This mutation process is caused by the special structure of microsatellites, which consists of a tandem repetition of short repeat units (1–6 bp). During DNA synthesis, the two DNA strands can slip against each other and realign out of register, which results in unpaired repeat loops. Most of these loops are corrected by the mismatch repair system, and only the small fraction which was not repaired results in gains or losses of one or more repeat units (Eisen 1999Citation ). Experimentally determined microsatellite mutation rates range from 10-6 to 10-2 (Levinson and Gutman 1987Citation ; Dallas 1992Citation ; Weber and Wong 1993Citation ; Schug, Mackay, and Aquadro 1997Citation ; Wierdl, Dominska, and Petes 1997Citation ; Schlötterer et al. 1998Citation ; Schug et al. 1998aCitation ), which is considerably higher than the nucleotide substitution rate.

A very useful approach to the study of microsatellite evolution is to map sequence changes in the microsatellite onto a phylogenetic tree. Because uninterrupted microsatellites are on average more polymorphic than interrupted ones (Weber 1990Citation ; Goldstein and Clark 1995Citation ), there is a preference to select loci with an uninterrupted repeat in the focal species (Ellegren, Primmer, and Sheldon 1995Citation ). Caused by this ascertainment bias, the generally emerging picture from cross-species comparisons is that microsatellite loci seem to decay with increasing phylogenetic distance through the accumulation of point mutations in the repeat array. Here, we demonstrate that interruptions of the repeat motif can also be removed, thus providing a mechanism that counteracts the decay of microsatellites.


    Material and Methods
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Fly Stocks
Drosophila melanogaster and D. simulans flies were taken from our laboratory collection. The remaining lines were obtained from the Bloomington Drosophila Species Stock Center and the UMEA Drosophila Stock Center.

Molecular Biology
PCR primers were designed from D. melanogaster sequences. Identical PCR conditions were used for all species. Primer sequences and annealing temperatures are given in table 1 .


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Table 1 Microsatellite Loci and Amplification Conditions

 
Genomic DNA was extracted from single flies with a high-salt method (Miller, Dykes, and Polesky 1988Citation ). To identify homozygous individuals from D. melanogaster and D. simulans, we typed 30 individuals following standard protocols (Schlötterer 1998Citation ). A typical cycling profile consisted of 30 cycles with 50 s at 94°C, 50 s at 55–57°C (depending on the primer pair), and 90 s at 72°C.

For DNA sequencing, 50 µl PCR reactions with 100 ng of genomic DNA, 1.5 mM MgCl2, 200 µM dNTPs, 1 µM of each primer, and 2.5 U Taq polymerase were gel-purified and directly sequenced in both directions using the BigDye sequencing chemistry on an ABI 377 automated sequencer. The sequences have been deposited in GenBank (accession numbers AJ246181AJ246213).

Data Analysis
To test for orthology, we amplified the microsatellite with an alternative set of primers and sequenced the PCR product. In all cases, the sequences obtained with both primer sets were identical for each species.

Sequences were aligned using CUSTAL W (Thompson, Higgins, and Gibson 1994Citation ). A parsimony criterion was used for the alignment of the repetitive microsatellite structure. Considering that base substitutions in the microsatellite repeat are less frequent than slippage mutations, the alignment of the microsatellite region was constructed by reducing the number of base substitutions in the repeat while allowing for indels to account for variation in repeat number (which is generated by DNA slippage). For one locus (DMTOR), sufficient flanking sequence was available to reconstruct the phylogeny of this locus. This alignment is available from the authors' webpage (http://i122server.vu-wien.ac.at). The microsatellite itself (i.e., positions 51–91 of the D. melanogaster sequence AJ246186) and additional repetitive sequences in the flanking region that showed length variability across species (i.e., positions 247–298, 360–369, 408–416, and 468–478 of the D. melanogaster sequence AJ246186) were excluded from the analysis. The phylogenetic tree was obtained by applying the maximum-likelihood criterion using the Puzzle, version 4.0.1 (Strimmer and von Haeseler 1996Citation ), software and the Tamura-Nei model for sequence evolution (Tamura and Nei 1993Citation ). The obtained phylogenetic grouping was largely consistent with another data set based on rDNA sequences (Pélandakis and Solignac 1993Citation ). As the locus DMTOR did not amplify in the species Drosophila ananassae and Drosophila kikkawai, we included these species in the tree according to the rDNA phylogeny (fig. 1 ).



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Fig. 1.—Maximum-likelihood tree based on the flanking sequence of microsatellite locus DMTOR. The dashed line indicates the phylogenetic grouping of Drosophila ananassae and Drosophila kikkawai according to the rDNA phylogeny published by Pélandakis and Solignac (1993)Citation .

 

    Results
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
PCR primer-binding sites of the loci studied were conserved to a variable extent. While locus DS01001 amplified in the melanogaster species complex only, the primer-binding sites of the remaining loci were conserved over a larger phylogenetic range. Locus DS06335b amplified the most distantly related species, including species from the montium and ananassae subgroups.

Locus DMTOR
To determine whether an interruption of a microsatellite sequence represents an ancestral or derived state, a reliable phylogeny is required. We used the flanking sequence at DMTOR to reconstruct the phylogenetic grouping of the species in which DMTOR could be amplified (fig. 1 ). The obtained species phylogeny of locus DMTOR is largely consistent with a previously published phylogeny (Pélandakis and Solignac 1993Citation ).

The (CA)n repeat motif of locus DMTOR is conserved in the melanogaster subgroup and some more distantly related species (fig. 2 ). At character 27, the (CA)n microsatellite is interrupted by a T (fig. 2 ). Figure 3A shows the character evolution of this substitution. Within the melanogaster subgroup, all species except D. melanogaster harbor a T at position 27, indicating that T represents the ancestral character state. Thus, D. melanogaster has lost the T substitution and gained an uninterrupted stretch of microsatellite repeats. Sequencing of four D. melanogaster alleles with different repeat numbers always revealed an uninterrupted microsatellite. Sequence analysis of Drosophila mauritiana (N = 4) showed that the A->G substitution at position 37 was not fixed; two individuals did not carry this interruption. Most likely, this is not the result of a purification event in the microsatellite, but a recent substitution which is specific to D. mauritiana. The microsatellite region of more distantly related species cannot be aligned unambiguously (homologous positions could not be identified with certainty) in the microsatellite region; therefore, no statement about gain or loss of interruptions in the microsatellite can be made. It should be noted, however, that the most distantly related species, Drosophila eugracilis and Drosophila elegans, have a relatively long stretch of seven or nine uninterrupted CA repeats, respectively, possibly indicating that some mechanisms are counteracting base substitutions in the microsatellite.



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Fig. 2.—Microsatellite region at locus DMTOR. Lowercase letters indicate the flanking region, and capital letters indicate the microsatellite region. Bold letters indicate the positions of microsatellite interruptions subjected to purification events.

 


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Fig. 3.—Character state evolution of interruptions in the microsatellite at loci DMTOR, DS01001, andDS06335b. A, Locus DMTOR, character 27 in figure 2 . B, Locus DS01001, character 30 in figure 5 . C, Locus DS06335b, character 22 in figure 6 . D, Locus DS06335b, character 45 and 46 in figure 6 .

 
Within the takahashii subgroup, the most frequent interruption in the microsatellite is a G. Drosophila prostipennis had three A->G substitutions (positions 33, 35, and 39). While multiple independent transitions cannot be excluded, an alternative explanation would be the duplication of the G substitution by DNA slippage (see fig. 4 ).



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Fig. 4.—Mechanistic model for the duplication of interruptions in the microsatellite DMTOR. Imperfections in the repeat array are indicated in bold letters. A, Leading-strand slippage of one repeat unit results in two adjacent microsatellite interruptions. B, Leading-strand slippage of two repeat units results in microsatellite interruptions that are separated by one repeat unit

 
Locus DS01001
The microsatellite region at locus DS01001 was sequenced in the melanogaster species complex. For D. melanogaster, 110 individuals from several natural populations were analyzed. Drosophila simulans, D. mauritiana, and Drosophila sechellia were each represented by a single individual. In all species, the microsatellite repeat was interrupted by the insertion of a single nucleotide at position 30 (fig. 5 ). In D. melanogaster, two individuals from Kenya were identified which did not carry this interruption. Considering that the T interruption occurs at high frequency in D. melanogaster, as well as in all members of the simulans clade, it can be assumed that it represents the ancestral state (fig. 3B ). Hence, both individuals from Kenya have lost the interruption and restored a pure microsatellite. Uncertainty about the ancestral character state for two additional interruptions of the microsatellite in D. simulans, D. sechellia, and D. mauritiana (positions 41 and 48; fig. 5 ) precluded their further analysis.



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Fig. 5.—Microsatellite region at locus DS01001. Lowercase letters indicate the flanking region, and capital letters indicate the microsatellite region. Bold letters indicate the positions of microsatellite interruptions subjected to purification events.

 
Locus DS06335b
A T interruption in the microsatellite at position 22 represents the ancestral state within the melanogaster subgroup (figs. 3C and 6 ). This interruption was lost in all members of the simulans clade through a purification event in their common ancestor. In some members of the takahashii/suzuki subgroup, a T interruption at a similar position could be observed. Assuming that this interruption is homologous to the interruption at position 22, it can be regarded as ancestral. Thus, the lack of the T interruption in Drosophila lutescens indicates a purification event in this species (fig. 3C ). In D. prostipennis, the T interruption may be homologous to either position 22 or position 48; therefore, the mutational history of the microsatellite in D. prostipennis cannot be unambiguously resolved.

A second interruption in the microsatellite (an insertion of AG at positions 45 and 46) is conserved across the melanogaster and takahashii subgroup. While this interruption is fixed in the melanogster subgroup, Drosophila mimetica and D. prostipennis have lost it. Mapping this character on the phylogenetic tree (fig. 3D ) suggests that this interruption has been lost twice (in D. mimetica and in D. prostipennis). Most important, phylogenetic analysis of the flanking sequence of microsatellite locus DS06335b resulted in the same topology for the suzuki/takahashii group (not shown). Thus, it can be excluded that the repeated loss of an interruption in the microsatellite is an artifact of an inaccurate phylogenetic assumption (i.e., that the gene tree of DMTOR differs from the gene tree of DS06335b).

For comparison, we also included the sequences of two more distantly related Drosophila species, D. ananassae and D. kikkawai. Drosophila kikkawai, the most distantly related species, has a long microsatellite with eight uninterrupted CA repeats at this locus. Interestingly, only the focal species, D. melanogaster, carries a longer uninterrupted stretch of repeats.


    Discussion
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Phylogenetic Reconstruction Based on Flanking Sequence of Microsatellites
Here, we used flanking sequence of the microsatellite locus DMTOR to reconstruct the phylogeny of the microsatellite locus. The resulting relationships were very similar to those previously published (Pélandakis and Solignac 1993Citation ). For comparison, we also sequenced the flanking sequence of microsatellite locus DS06335b for D. lutescens, D. prostipennis, D. takahashii, and D. eugracilis and obtained the identical branching pattern, as shown in figure 1 . Despite this overall consistency and the high bootstrap support values, the phylogenetic tree also shows some discrepancy with a previously published consensus tree of the melanogaster complex (Harr et al. 1998Citation ). While D. mauritiana and D. simulans were more closely related in the microsatellite-based phylogeny (Harr et al. 1998Citation ), the DNA sequence–based phylogeny shown in figure 1 grouped D. sechellia and D. simulans together. The reason for this discrepancy is likely to be the well-described problem that gene trees may differ from species trees, a phenomenon that is particularly pronounced for closely related species (Pamilo and Nei 1988Citation ).

While the obtained high bootstrap support values may suggest that noncoding sequences, such as flanking regions of microsatellites, are potentially useful for phylogenetic reconstruction, determination of the correct alignment of these regions can be quite challenging.

How General Is the Phenomenon of Microsatellite Purification?
In this study, we report evidence for microsatellite purification at three different loci. These data were extracted from a survey of 53 microsatellites in the melanogaster group (unpublished data). In total, approximately 300 locus/species combinations were amplified and sequenced. It is difficult to infer the frequency of purification events from this data set without certainty about the ancestral state of the imperfection in the microsatellite. Very often, this proof was not possible, because either the relevant species did not amplify or the homologous positions could not be unambiguously identified, particularly for more diverged taxa. Thus, we did not include them in this report.

Purification of imperfections in a microsatellite repeat was also described in other studies. The analysis of a dinucleotide (DQCAR) in a phylogenetic framework identified the loss of an interruption at the allele DQB1 *0202 (Jin et al. 1996Citation ; Taylor, Durkin, and Breden 1999Citation ). Laken et al. (1997)Citation described the loss of an interrupting base substitution in a mononucleotide run in the APC (adenomatous polyposis coli) gene, which was associated with colorectal cancer. Similarly, two reports which studied the evolution of an interrupted microsatellite in a plasmid in yeast also identified mutational events which restored a pure microsatellite stretch (Petes, Greenwell, and Dominska 1997Citation ; Maurer, O'Callaghan, and Livingston 1998Citation ). Hence, purification of an imperfect microsatellite is apparently a general phenomenon of microsatellite DNA and needs to be considered for the long-term evolution of microsatellite DNA.

Mechanisms of Microsatellite Purification
Average rates of DNA slippage mutation in D. melanogaster are on the order of 10-6 (Schug, Mackay, and Aquadro 1997Citation ; Schlötterer et al. 1998Citation ), which is about 100 times as frequent as base substitutions (Powell 1997) and 1,000 times as frequent as deletions (Petrov and Hartl 1998Citation ). Hence, the high frequency of DNA slippage makes this mutation process a good candidate for the removal of interruptions in a microsatellite array. Purification may occur through the following mechanism: the template strand slips back before the interruption is synthesized on the new DNA strand, and the interruption is placed upstream of the 3' end of the nascent DNA strand (fig. 7 ). When DNA synthesis continues on the new DNA strand, this strand loses the interruption in the microsatellite. Experimental evidence from microsatellites with imperfections strongly suggests that microsatellite interruptions are lost by an intramolecular process, such as DNA slippage (Petes, Greenwell, and Dominska 1997Citation ; Maurer, O'Callaghan, and Livingston 1998Citation ). Nevertheless, the important difference between the plasmid-based studies and our observations in D. melanogaster is that the plasmids carried a large number of repeats (>25 repeats). While large deletions are common in plasmid systems with long microsatellites (mean deletion size of 12.8 repeats [Petes, Greenwell, and Dominska 1997Citation ] or 51.5 repeat units [Maurer, O'Callaghan, and Livingston 1998Citation ]), they are not expected to be a major evolutionary factor in D. melanogaster, because the microsatellites in this species are, on average, shorter than the deletions observed in the plasmid studies (Schug et al. 1998bCitation ; Bachtrog et al. 1999Citation ).



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Fig. 7.—Model for the purification of an interrupted microsatellite. Interruptions in the repeat array are indicated in bold.

 
One prediction of DNA slippage as the driving force of microsatellite purification is that duplications of interruptions in the microsatellite structure should also be detectable. In a larger data set (unpublished data), we identified eight loci with putative duplications of an interruption. One of these duplications occurred at locus DMTOR (fig. 2 ), where the G interruption is duplicated twice in D. prostipennis (see fig. 4 for a mechanistic model). Other published data sets also indicate that interruptions in a microsatellite can be duplicated. One particularly good example is given by the microsatellite locus A113 in Apis melifera, where a TT insertion is repeated up to three times (Estoup et al. 1995Citation ).

In principle, the slippage process leading to the removal of an interruption in the microsatellite repeat does not differ from a slippage event, which merely alters the repeat number. Comparisons of microsatellite mutation rates for interrupted and pure microsatellites indicate that interrupted microsatellites have a lower mutation rate (Weber 1990Citation ; Jin et al. 1996Citation ; Petes, Greenwell, and Dominska 1997Citation ). One possible explanation for this phenomenon is that microsatellite mutation rates are length- dependent (Jin et al. 1996Citation ; Wierdl, Dominska, and Petes 1997Citation ; Brinkmann et al. 1998Citation ; Schlötterer et al. 1998Citation ) and that the mutation rate increases more than would be expected by a linear function of microsatellite length (Wierdl, Dominska, and Petes 1997Citation ; Brinkmann et al. 1998Citation ). Because the interruption of a microsatellite is often asymmetric, the mutation generates two arrays of different lengths. Studies of interrupted microsatellites showed that most of the slippage events occur in the longer stretch of the subdivided microsatellite (Loridon et al. 1998Citation ; Schlötterer and Zangerl 1999Citation ). Thus, the lower mutation rate of interrupted microsatellites and the predominance of slippage mutations in its longer stretch strongly suggest that an interruption in the microsatellite poses a significant barrier to DNA slippage. Hence, despite the general similarity to ‘normal’ slippage events, it can be assumed that purification events occur more rarely than per-repeat-unit slippage events in an uninterrupted microsatellite.

Implications for the Long-Term Evolution of Microsatellites
The simplest model of microsatellite evolution assumes that a random mutation process generates short "proto" microsatellites. With an increasing repeat number, the probability of a DNA slippage event increases. Under the conservative assumption that the gain of repeat units is as likely as their loss, there is a certain probability that DNA slippage can generate a long stretch of microsatellite DNA. Similarly, DNA slippage may also reduce the length to a few repeat units. Theoretical studies using general models of microsatellite evolution indicated that the persistence time of microsatellites can be quite large (Tachida and Iizuka 1992Citation ; Stephan and Kim 1998Citation ).

With increasing persistence times, base substitutions in the microsatellite array become an important evolutionary factor. Provided that interruptions in a microsatellite repeat have a dramatic impact on its mutational behavior, base substitutions in a microsatellite repeat have been regarded as an essential component determining their long-term evolution. While several studies have assumed that selection maintains microsatellites below a certain size, it has recently been suggested that base substitution in the microsatellite array would also prevent infinite growth of microsatellites (Bell and Jurka 1997Citation ; Kruglyak et al. 1998Citation ). Hence, the length distribution of microsatellites is assumed to be an equilibrium state affected by the combination of two mutation processes occurring at different rates: DNA slippage and base substitutions. Even if a biased mutation model is accepted, under which the gain of repeat units is more likely than the loss of repeat units, computer simulations have shown that interruptions in the microsatellite are an effective mechanism for prevention of infinite growth of microsatellites (Palsbøll, Bérubé, and Jørgensen 1999Citation ).

Under the models used by Bell and Jurka (1997)Citation , Kruglyak et al. (1998)Citation , and Palsbøll, Bérubé, and Jørgensen (1999)Citation , interruptions in the microsatellite can be lost by back mutations only. Provided that the rate of base substitutions or non-DNA slippage-based indels is much lower than DNA slippage rates, interruptions of a microsatellite are effectively unidirectional if slippage-based removal of microsatellite interruptions is not considered. A recent study put an even stronger emphasis on the consequences of an interruption in a microsatellite (Taylor, Durkin, and Breden 1999Citation ). Based on known phylogenies, Taylor, Durkin, and Breden (1999)Citation used seven loci to map changes in the microsatellite stretch on these phylogenies and concluded that once an interruption of a microsatellite occurred, the locus degenerated quickly. This model of interruption-induced microsatellite decay provides an interesting hypothesis, even though it remains unknown whether the persistence time would have been longer without the initial interruption.

Consistent with previous observations, we also detected several interruptions in the microsatellite. Our data, however, indicate that an interruption is often a transitory state in the evolution of a microsatellite locus and does not always result in its degeneration. Interestingly, even though the orthologous microsatellite sequence from distantly related species (such as D. elegans [DMTOR] or D. kikkawai [DS06335b]) was not completely pure, parts of uninterrupted microsatellite DNA of lengths similar to that found in the focal species D. melanogaster could still be observed.

The evolutionary importance of purging interruptions in a microsatellite array is essentially dependent on their frequency. Based on our data set, we were able to demonstrate that the phenomenon exists, but whether this is a rare or frequent event remains open to further investigations.



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Fig. 6.—Microsatellite region at locus DS06335b. Lowercase letters indicate the flanking region, and capital letters indicate the microsatellite region. Bold letters indicate the positions of microsatellite interruptions subjected to purification events

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Many thanks to Steven Weiss and the members of C.S.'s lab for helpful comments and discussion. We are grateful to the UMEA stock center and the Drosophila Species Stock Center for providing flies. Special thanks to C. Aquadro for his attentive editorship. This work was supported by a grant from the Fonds zur Förderung der wissenschaftlichen Forschung (FWF) to C.S.


    Footnotes
 
Charles Aquadro, Reviewing Editor

1 Keywords: Drosophila microsatellite interruption slippage Back

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


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 Material and Methods
 Results
 Discussion
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Accepted for publication March 2, 2000.