Microsatellite Evolution at Two Hypervariable Loci Revealed by Extensive Avian Pedigrees

Nadeena R. Beck, Michael C. Double and Andrew Cockburn

Evolutionary Ecology Group, School of Botany and Zoology, Australian National University, Canberra, Australia


    Abstract
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Genealogies generated through a long-term study of superb fairy-wrens (Malurus cyaneus) were used to investigate mutation within two hypervariable microsatellite loci. Of 3,230 meioses examined at the tetranucleotide locus (Mcyµ8), 45 mutations were identified, giving a mutation rate of 1.4%. At the dinucleotide locus (Mcyµ4) 30 mutations were recorded from 2,750 meioses giving a mutation rate of 1.1%. Mutations at both loci primarily (80%; 60/75) involved the loss or gain of a single repeat unit. Unlike previous studies, there was no significant bias toward additions over deletions. The mutation rate at Mcyµ8 increased with allele size, and very long alleles (>70 repeats) mutated at a rate of almost 20%. The length of the mutating allele and allele span, however, were strongly correlated so it was not possible to isolate the causative factor. Allele size did not appear to affect mutation rate at Mcyµ4, but the repeat number was considerably lower at this locus. The gender of the mutating parent was significant only at Mcyµ8, where mutations occurred more frequently in maternal alleles. However, at both loci we found that alleles inherited from the mother were on average larger than those from the father, and this in part drove the higher mutation rate among maternally inherited alleles at Mcyµ8.

Key Words: microsatellites • Malurus cyaneus • mutation


    Introduction
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The most widely accepted mechanism of microsatellite mutation is the slipped-strand mispairing model, in which the misaligned reassociation of replicating DNA strands after DNA polymerase slippage results in the insertion or deletion of one or more repeat units (Levinson and Gutman 1987; reviewed by Eisen 1999). Microsatellite mutations are estimated to occur between 10-3 and 10-5 times per locus per generation (Crozier et al. 1999; Hancock 2000), a rate several orders of magnitude higher than at other loci. However, previous studies on microsatellite evolution have revealed a large variation in mutation rates, not only between loci, but also between alleles at the same locus (Primmer et al. 1996; Wierdl, Dominska, and Petes 1997; Chakraborty et al. 1997; Primmer and Ellegren 1998; Primmer et al. 1998; Neff and Gross 2001).

Mutation rates have been found to be influenced primarily by the sex of the individual, the nature of the repeat motif, and the length of the allele. Mutation rates have been reported to be up to five times as high in paternally transmitted alleles, probably because of the greater number of cell divisions involved in spermatogenesis relative to oogenesis (Ellegren and Fridolfsson 1997; Primmer et al. 1998; Ellegren 2000). The effect of repeat motif on mutation rate is unclear. Weber and Wong (1993) found that the mutation rate at tetranucleotide loci in humans was four times higher than at dinucleotide loci, a conclusion that has some support (Hastbacka et al. 1992; Zahn and Kwiatkowski 1995). Subsequent studies, however, suggest that mutation rate is inversely related to motif size (Chakraborty et al. 1997; Anderson et al. 2000). Although larger repeat units might be less prone to slippage, there is some evidence that mismatch repair mechanisms are less able to recognize misalignments involving four or more base pairs (Eisen 1999). Finally, many studies in different organisms have found a mutational bias in favor of long alleles over short alleles within the same locus (Primmer et al. 1996; Wierdl, Dominska, and Petes 1997; Primmer et al. 1998; Anderson et al. 2000). In an analysis of 592 AC microsatellite loci from five vertebrate classes, Neff and Gross (2001) also found that mutation rate, inferred from microsatellite variability, increased with average allele length. Amos et al. (1996) suggested that heterozygotes with a large size difference between alleles (large allele span) have an elevated rate of mutation rate. However, neither Primmer et al. (1998) nor Ellegren (2000) found any evidence to support this conclusion, suggesting instead that a large allele span is likely to involve a long allele which is more prone to replication errors due to slippage.

Previous studies have found that most mutations involve single repeat changes consistent with a step-wise model, and that there is significant bias toward additions over deletions (Amos et al. 1996; Primmer et al. 1996, 1998; Neff and Gross 2001). Longer alleles are, however, more likely to display deletions than shorter alleles, and these deletions are likely to involve repeat numbers larger than two; perhaps providing a "length ceiling" preventing infinite growth (Weirdl et al. 1997; Primmer et al. 1998; Ellegren 2000). Primmer et al. (1998) also identified mutational differences between male and female barn swallows (Hirundo rustica), finding that additions are more common in males, but the magnitude of size alterations are greater in females.

Clearly, the mechanisms of microsatellite mutations are still not fully understood and need to be further investigated to devise more accurate models of mutation for use in population and paternity analysis (Primmer et al. 1998). This study takes advantage of extensive pedigrees resulting from a long-term study of superb fairy-wrens (Malurus cyaneus) to investigate the mutation characteristics of two hypervariable microsatellite loci. Hypervariable loci such as these represent only a very small proportion of the total microsatellite loci present in the genome and are therefore likely to be subject to different mutational processes, resulting in a higher mutation rate (Webster, Smith, and Ellegren 2002). However, it is important to have an understanding of the processes affecting the evolution of these unusual loci, as highly polymorphic microsatellites are preferentially selected for use in population studies and genome mapping.


    Methods
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Study Species and Population
This study was based on the population of superb fairy-wrens (Malurus cyaneus) resident in the Australian National Botanic Gardens (ANBG) and immediate surrounds. Superb fairy-wrens are a cooperatively breeding species in which males display extreme natal philopatry (Mulder 1995). In contrast, females are obligate dispersers. Whereas 43% of females disperse within 14 weeks of fledging, the remainder are forced off their natal territory at the onset of the following breeding season when the dominant female no longer tolerates them (Mulder 1995). Females disperse an average of 11.8 territory widths and are not known to travel or settle with close relatives. Dispersing females that obtain a breeding vacancy breed in the first year, producing up to two clutches of three to four eggs. Although socially monogamous, this species has the highest known rate of extra-pair fertilization of any bird, with 76% of offspring sired by males outside the social group (Mulder et al. 1994).

The ANBG population of superb fairy-wrens has been the subject of an extensive behavioral and genetic study for over 12 years, resulting in a demographic data set including social and reproductive information for over 4,000 individuals (Langmore and Mulder 1992; Mulder et al. 1994; Mulder 1995; Dunn and Cockburn 1999; Double and Cockburn 2000). The study area encompasses approximately 75 territories but is embedded in a large, panmictic population that extends from the study area in all directions. All individuals within the study area are color banded, and year-round censuses have determined group histories and social relationships. To assign paternity to offspring within this population, more than 2,500 individuals have been genotyped at five or more microsatellite loci (Double et al. 1997b). The extreme natal philopatry observed in males and the breeding philopatry of females of this species have allowed the construction of large pedigrees for use in the verification of genotypes and the identification of microsatellite mutation events.

Microsatellite Genotyping
Throughout the 12-year study, blood was collected from all nestlings on territories within the study area and from adults dispersing into the ANBG. DNA was isolated from blood samples by ammonium acetate extraction (Richardson et al. 2001) after digestion with proteinase K (Progen). Microsatellite loci were amplified in polymerase chain reaction (PCR) mixes consisting of approximately 50 µg of template DNA, 1.2 µl of Opti-prime 10x PCR buffer, 1.2 µl of 2 mM dNTPs, 1.2 µl of 2 µM forward and fluorescently labeled reverse primers, 1.0 µl of 25 mM MgCl, 0.1 µl AmpliTaq DNA polymerase (PerkinElmer) and ddH2O to a total reaction volume of 12 µl. PCR was performed on an FTS-960 Thermal Sequencer (Corbett Research) using the following profile: initial template DNA denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, 55°C for 30 s, polymerization at 72°C for 30 s or 45 s, and a final extension at 72°C for 3 min.

PCR products were visualized on a 5.3% polyacrylamide gel using an ABI Prism 377 automated sequencer (PerkinElmer). All samples were run with TAMRA 500 internal size standard (PerkinElmer), and allele sizes were determined using GeneScan 3.1 and Genotyper 2.0 software (PerkinElmer/ABI).

Genetic fathers were identified by exclusion (Double and Cockburn 2000). Every potential father within the study area has been sampled and genotyped. Up to seven microsatellite loci were used for paternity assignment in the study population; six dinucleotide repeat loci and one tetranucleotide repeat locus (GenBank accession numbers U82385U82382; Double et al. 1997b). With allele frequencies from the five most commonly used loci, the probability of false assignment to a male unrelated to the true sire was 9.8 x 10-5. Using a simulation-based approach (Double et al. 1997a), we estimated the probability of false assignment to a single first-order relative of the true sire was 0.08. This probability fell to 0.04 if all seven loci were used. Because all males within the study area are genotyped, a false assignment could occur only if the true sire was outside our study area and had never been sampled or if the true sire produced a mutant allele and another male matched all the offspring's paternal alleles.

Mutation Detection
The present study sought mutations at two of the microsatellite loci used to assign paternity; the dinucleotide Mcyµ4 and the tetranucleotide Mcyµ8 (table 1). Sequences for these two microsatellite loci are available from GenBank (accession numbers U82388 and U82392). The two loci were chosen because of the clarity of their GeneScan profiles. Mutations were detected by comparing the genotype of an offspring with that of both its parents. Only individuals for whom both maternal and paternal genotypes were available were included in the analysis. PCR conditions for Mcyµ4 were changed during the study and improved the clarity of the genotyping profiles. Previous ambiguous profiles were discarded, and therefore the number of meioses examined at Mcyµ8 was greater than at Mcyµ4 (3230 versus 2750).


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Table 1 Characterization of the Two Microsatellite Loci Used in This Analysis of Mutation Events

 
The reliable identification of mutations depends on being confident of correctly matching offspring to parents for genotype comparison. Observations of hundreds of laying sequences over the last 12 breeding seasons have revealed no evidence for intraspecific brood parasitism (Mulder et al. 1994). Therefore the maternal alleles of the offspring could always be confidently assigned.

Only those individuals whose paternity had been assigned based on matching genotypes at five or more loci were included in the analysis of mutation events. If a putative mutation was identified with five loci, then the remaining two loci were examined to further lower the probability of incorrect paternity assignment. Individuals with ambiguous genotypes or in whom the putative father was mismatched at more than one locus were excluded from the analysis. Where possible, individual genotypes and mutations were confirmed by tracking the inheritance of each allele through genealogies. For example, if an offspring inherited a mutated allele from its mother, we first checked the genotypes of other offspring produced by the mother and, if possible, the mother's parents to confirm the size of the progenitor allele. In an attempt to verify the existence of a mutant allele, we also checked if the offspring had successfully reproduced and if so whether the mutated allele had been inherited.

Initially we used three different protocols to identify the progenitor allele: (1) assume that the smallest mutational change in allele size was most likely (Primmer et al. 1998; Crozier et al. 1999; Ellegren 2000); (2) assume that the largest mutational change allele size was most likely; and (3) random assignment of the progenitor allele. The distribution of mutational changes produced by these three protocols was then compared to the distributions expected under single-step and multi-phase models of microsatellite evolution.

Statistical analyses were performed with the software package JMP (SAS Institute). We used logistic regression to analyze a discrete response (e.g., mutation yes/no) to a continuously distributed explanatory variable (e.g., allele length). Terms were assessed by the change in deviance which approximates a {chi}2 distribution. We used two-tailed tests and rejected the null hypothesis when P < 0.05.


    Results
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Mutation Rate
At the dinucleotide Mcyµ4 locus, 1,375 individuals were examined, representing 2,750 meioses. Of these, 30 mutations were recorded, yielding a mutation rate of 1.1%. These meioses were derived from 236 females and 258 males. The maximum number of offspring produced by any one individual was 51, with the average being 5.3 ± 5.9 SD for males and 5.8 ± 5.0 SD for females. The mutation rate at the tetranucleotide Mcyµ8 locus was found to be 1.4% (45 mutations from 1,615 individuals: 3,230 meioses). At this locus, the meioses were derived from 254 females and 288 males. Again, the maximum number of offspring produced by any one individual was 51, with the average being 5.6 ± 6.1 SD for males and 6.4 ± 5.4 SD for females. In 65 of the 75 mutation events, the size of the suspected progenitor allele could be confirmed by examination of the genotypes from the progenitor's own parents and/or non-mutant offspring. In eight cases the size of the mutant allele could be verified because the mutant allele was passed on to the following generation. In all other cases the individual with the mutant allele failed to breed successfully within our study area or exclusively transmitted the nonmutant allele.

Magnitude and Directionality of Mutations
Initially, we identified progenitor using three different assumptions (fig. 1). Under the first assumption, that the smallest change in allele size was most likely, the average size of mutations was 1.7 ± 1.4 SD repeat units at Mcyµ4, and 1.5 ± 2.6 SD repeat units at Mcyµ8. The majority of mutant alleles (Mcyµ4: 20/30; Mcyµ8: 40/45) differed from the parental allele by an increase or decrease of just one repeat unit. Assuming the largest change in allele size was most likely, the average mutation size was 5.2 ± 2.7 SD repeat units at Mcyµ4 and 20.4 ± 13.1 SD repeat units at Mcyµ8. Finally, random assignment of the progenitor allele resulted in an average mutation size of 3.9 ± 3.0 SD at Mcyµ4 and 7.6 ± 9.6 SD at Mcyµ8. For both loci, only the assumption of the smallest change in allele size produced a distribution of mutational changes that is easily reconciled with the expectation of a predominance of single-step mutations as predicted by single-step and multi-phase models of microsatellite evolution (e.g., Di Rienzo et al. 1994). We followed this assumption in all subsequent analyses.



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FIG. 1. Mutational changes in repeat number presented as a percentage of the total number of mutation events detected (Mcyµ4: N = 30; Mcyµ8: N = 45). We identified the change in size between progenitor and mutant alleles by assuming (1) that the largest size change was most likely, (2) that the smallest size change was most likely, or (3) by random assignment of the progenitor allele

 
The difference between the number of additions and deletions at Mcyµ4 (15 versus 13, respectively; {chi}2 = 0.14, df = 1, P = 0.71) and Mcyµ8 (23 versus 21, respectively; {chi}2 = 0.09, df = 1, P = 0.76) was not significant. For two mutations at Mcyµ4 and one at Mcyµ8, the mutant allele lay equidistant between two potential progenitor alleles and so it was uncertain if an addition or deletion had occurred. For each locus only two mutations involved more than two repeat units. At Mcyµ4, mutations deleted four and six repeats, whereas at Mcyµ8, a mutation added four repeats and another removed 18 repeats.

Allele Length
At Mcyµ4, allele length did not influence mutation rate (logistic regression: {chi}2 = 0.25, df = 1, P = 0.6; fig. 2a). In contrast, the mutation rate at Mcyµ8 was greatly influenced by allele length, with the longest alleles mutating at a rate of almost 20% of meioses (logistic regression: {chi}2 = 31.4, df = 1, P < 0.001; fig. 2b). At both loci, allele length did not influence the frequency of deletions (logistic regression: Mcyµ4: {chi}2 = 1.4, df = 1, P = 0.2; Mcyµ8: {chi}2 = 0.04, df = 1, P = 0.8).



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FIG. 2. Allele frequency distribution (shaded) and mutation frequency distribution (black) at Mcyµ4 and Mcyµ8. At Mcyµ8 alleles were pooled into groups of two repeat units. The allele frequency represents the proportion of meioses observed in a particular allele class. The mutation rate was calculated from the number of mutations in an allele size class, divided by the total number of meioses within that class

 
Interpretation of the effects of allele length may be biased if the more common alleles often occur in heterozygotes with adjacent alleles. Mutations are masked if an allele mutates to the other allele carried by the individual. As longer alleles are generally rare, heterozygotes are less likely to have adjacent alleles. Therefore, mutations might be recognized more easily in rare, longer alleles. At Mcyµ8 we found that longer alleles were less likely to occur in a heterozygote with an allele of adjacent length (logistic regression: {chi}2 = 8.9, df = 1, P > 0.005). However, even the most common alleles had only a 20% chance of occurring with an adjacent allele. Given a mutation rate of 1.4%, fewer than 1 in 1,000 mutations is likely to be masked in this way. Therefore, we conclude that the effect of allele size on mutation rate at this locus is real.

Allele Span
Allele span refers to the difference in length between the alleles of a heterozygote (Amos et al. 1996). At Mcyµ4, parents with large allele spans were no more likely to pass on mutant alleles than those with small allele spans (logistic regression: {chi}2 = 0.3, df = 1, P = 0.6). Seventeen mutations originated from the parent with the largest difference in allele size, whereas 11 occurred in the parent with the smallest allele span (contingency table analysis: {chi}2 = 1.3, df = 1, P = 0.26). One mutation occurred in an individual whose parents had identical allele spans, and the parental origin of another could not be determined because the parents had identical alleles.

At Mcyµ8, parents with a large allele span were more likely to pass a mutant allele on to their offspring (logistic regression: {chi}2 = 11.1, df = 1, P < 0.001), and 68% (28/41) of mutations occurred in the parent with the largest allele span ({chi}2 = 5.49, df = 1, P = 0.02). However, allele span and the length of the progenitor allele were highly correlated (r2 = 0.19, F = 8.99, df = 1, P = 0.005), so it was not possible to determine whether allele span or allele length had the greater influence on mutation rate. Similarly, the effect of allele span and length could not be dissected by analyzing the frequency data; 17 of the 41 mutations did not involve the largest parental allele, of which 10 (58%) were inherited from the parent with the largest allele span ({chi}2 = 1.8, df = 1, P = 0.17). At Mcyµ8 the parental origin of three mutations could not be identified, and one mutation occurred in an individual whose parents had identical allele spans.

Influence of Gender on Mutation
At Mcyµ4 more mutations originated from a paternal allele (66%; 19/29), although the difference was not significant ({chi}2 = 2.8, df = 1, P = 0.09). The parental origin of one mutation could not be identified at this locus. By contrast, mutant alleles at Mcyµ8 were more likely to be maternally derived (74%; 31/42; {chi}2 = 9.5, df = 1, P = 0.002). This result could be driven by a tendency for females to pass on larger alleles (see below). However, in a logistic regression model which initially included, and therefore controlled for, allele length, we still found that the mutation rate was significantly influenced by gender of the parent donating the allele ({chi}2 = 7.9, df = 1, P = 0.02). The parental origin of three mutations at Mcyµ8 could not be identified because the parents had identical alleles at this locus.

Gender bias in mutation rates could be explained by a difference in the average allele length. We found no difference in allele length at either Mcyµ4 or Mcyµ8 either between mothers and fathers or between male and female offspring (table 2). At both loci, however, alleles inherited from the mother were on average longer that those from the father (table 2). Although the effect is slight (about half of one repeat at each locus), it is highly significant. The longest allele was maternally derived in 53.5% (691/1291) of offspring at Mcyµ4 and 52.9% (824/1557) of offspring at Mcyµ8. This result is most likely due to differential reproductive success of males and females within the study area. When only a single offspring was randomly selected for each mother and father, there was no significant difference between alleles inherited from males or females for either locus (Mcyµ4: t = 0.8, df = 498, P = 0.4; Mcyµ8: t = 0.8, df = 541, P = 0.4). There was no evidence to suggest that either sex was preferentially passing on their larger or smaller alleles at either locus (all P > 0.1).


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Table 2 Effect of Gender on Allele Sizes at Mcyµ4 and Mcyµ8

 
At both loci the gender of the parent with the progenitor allele did not influence whether the mutation was an addition or a deletion (table 3; Mcyµ4: contingency table analysis: {chi}2 = 0.02, df = 1, P = 0.9; Mcyµ8: {chi}2 = 1.1, df = 1, P = 0.29). There was also no evidence at either locus that mutations of more than a single repeat unit were more commonly inherited from fathers or mothers (Mcyµ4: Fisher Exact test, P = 0.2; Mcyµ8: Fisher Exact test, P = 0.1).


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Table 3 Summary of Mutations at Mcyµ4 and Mcyµ8

 

    Discussion
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Correct Paternity Assignment
Identifying mutations based on comparisons with parental genotypes poses some potential problems, particularly in a species that exhibits high levels of extra-pair paternity and strong male philopatry. In these circumstances the possibility of incorrectly assigning a close relative as the sire becomes more likely (Double et al. 1997a). For example, it is possible for the true sire to pass on a mutated allele, causing paternity to be assigned to his brother. This phenomenon may occur infrequently, but it could result in either an underestimate of mutation rate, where a mutation results in an allele of a high frequency in the population, or an overestimate of mutation rate where the correct sire has not been sampled. Although errors are possible, the nature of the mutations themselves argues for their reliable identification. First, we found that almost all mutated alleles differed by a single repeat from the progenitor allele, an unlikely result if false paternity assignment were the cause of a mismatch. Second, the size distribution of mutating alleles at Mcyµ8 is very different from the allele frequency distribution, and it is consistent with the pattern reported in other studies (e.g., Primmer et al. 1996, 1998). Third, the majority of mutations detected in this study occurred in the maternal line. As there is no evidence of intraspecific brood parasitism in superb fairy-wrens (Mulder 1994), mutations occurring in the maternal line can be identified with some confidence. Finally, offspring with mutant alleles were evenly distributed throughout our study area. If incorrect paternity assignments had led to incorrect attribution of mutation, we would expect the majority of mutations to be clustered on the periphery of the study area, where potential fathers are less likely to have been sampled.

Other potential sources of bias in the identification of mutations are genotyping errors and null alleles. We are able to exclude genotyping errors as a potential source of bias, because all offspring genotypes are cross-checked with the maternal genotype, ruling out loading errors. In addition, where paternity is ambiguous, individuals are regenotyped for clarification.

The presence of null alleles may artificially inflate mutation estimates, because an individual possessing a null allele will be scored as a homozygote. However, the proportion of mutant individuals that were homozygous was not significantly different from the proportion of homozygotes in the general population ({chi}2 = 1.26, df = 1, P > 0.05). Also there was no heterozygote deficiency (table 1), which would be expected if null alleles were frequent.

The Influence of Motif on Mutation Rates
Mutation rates were similar at both Mcyµ4 and Mcyµ8 (1.1% and 1.4%, respectively). Weber and Wong (1993) found that tetranucleotide loci had higher mutation rates than dinucleotide loci, whereas more recent studies have found that mutation rate is inversely proportional to motif size (Chakraborty et al. 1997; Anderson et al. 2000). Although it makes intuitive sense that a longer motif would be more stable, many microsatellite systems (including that used in this study) include highly variable tetranucleotide loci (Li, Huang, and Brown 1997; Primmer et al. 1998), and such hypervariability itself implies a higher mutation rate. When selecting microsatellites for population analyses, however, there is always a bias toward more variable, and hence more informative, loci.

The Influence of Sex on Mutation Rates
The effect of the sex of the mutating parent was only significant at Mcyµ8, where almost three times as many mutations occurred in the maternal line as in the paternal line. This is in contrast to previous studies where microsatellite mutation rates have been found to be up to five times as high in males as in females, a difference that is generally attributed to the higher number of mitoses involved in spermatogenesis (Ellegren 2000). There is some evidence that sex affects different loci differently, with other studies finding more uniform mutation rates with respect to sex (e.g., Jeffreys et al. 1988; Talbot et al. 1995). Although the conservative identification of paternal mismatches as mutations might contribute to an excess of maternal mutations, the significant difference in average length of alleles inherited from each sex contributes to an elevated mutation rate in females, particularly at this locus, where allele length has such a significant effect on mutation rate.

Directionality and Magnitude of Mutations
Our study found no significant difference between the number of additions and deletions at either locus. This result is contrary to the findings of Amos et al. (1996) and Primmer et al. (1996, 1998), who found marked directionality in microsatellite mutations, favoring additions. This result led Primmer et al. (1998) to hypothesize that there must be a microsatellite length ceiling preventing uncontrolled growth. It has been suggested that a mechanism counteracting gradual expansion would involve very large deletions of 50 to 100 repeat units (Weber and Wong 1993). Primmer et al. (1998) found no evidence for deletions of such magnitude and suggested that such events are probably rare, and so would not be detected in typical samples sizes. However, the practice of assigning mutations to the allele requiring the smallest number of repeat changes could, in some cases, mask large changes in repeat number.

However, our findings are consistent with observations by Crozier et al. (1999), who found no evidence of directionality in an ant microsatellite. They suggested that this locus was approaching an equilibrium frequency distribution of allele sizes. If microsatellites eventually reach an equilibrium allele frequency distribution where there is no pronounced directionality in mutations, then a mechanism preventing infinite microsatellite growth through large deletions becomes unnecessary. If this is the case, however, it might be expected that deletions would be concentrated among the largest alleles at a locus. This pattern has been found in other studies (e.g., Wierdl, Dominska, and Petes 1997; Ellegren 2000) but appears not to be the case in fairy-wrens, where deletions are not more common in longer alleles.

Most of the mutations in this study involved length changes of only one repeat unit, consistent with the two-phase model of microsatellite mutation (Di Rienzo et al. 1994). Our findings are concordant with previous studies and support the applicability of the two-phase model to population and genetic analyses.

Allele Length
Investigation of the distribution of mutations at the two loci reveals significant differences. At Mcyµ8, the size distribution of mutating alleles is extremely skewed toward higher repeat numbers, with the longest alleles mutating at rates of almost 20%. However, allele size does not appear to affect mutation rate at Mcyµ8 until repeat numbers extend beyond 70. Below this threshold, mutations rates are similar to those at Mcyµ4, where mutating alleles are more evenly distributed with respect to allele size but allele length does not exceed 40 repeat units. A similar pattern was found by Primmer et al. (1996), who found that mutation rates were significantly higher in alleles greater than 80 repeat units.

The significant difference in allele lengths inherited from mothers and fathers was unexpected but may reflect the biology of this species. There is extreme skew in the reproductive success of superb fairy-wrens. Within our study area 5% of males sire 50% of offspring, and a single male is related to over 500 of the 2,500 individuals genotyped in this study (unpublished data). This skew appears to have generated the significant difference in allele lengths inherited from mothers and fathers for the two loci examined here.

The similarity between the mutation rates at these two loci may reflect the influence of a combination of length and motif. We suggest that while the tetranucleotide motif may be more stable, the mutation rate at Mcyµ8 is driven primarily by allele length. In contrast, at Mcyµ4, the stability of comparatively short alleles counters a less stable dinucleotide motif. Thus similar mutation rates at different loci may obscure the operation of different mutational mechanisms.

Allele Span
Amos et al. (1996) suggested that microsatellite mutations were more likely to occur in heterozygous individuals with a larger difference between the sizes of their two alleles. This effect of allele span differed between Mcyµ4 and Mcyµ8. Significantly more mutations at Mcyµ8 occurred in the parent with the largest span; however, this effect was not seen at Mcyµ4. Primmer et al. (1998) suggested that large allele spans are more likely to involve large alleles whose length would be the predominant factor affecting mutation rate. In our study allele span and allele length were strongly correlated, so we could not decipher which factor had the greater influence on mutation rate.

Conclusions
The results of this study provide further support for the influence of allele length (or allele span) on the mutation rates of microsatellite loci and that stepwise mutations predominate. However, we did not find a predominance of additions resulting in an increase in allele size—a phenomenon that has been consistently reported in other studies. We also found, in contrast to previous studies, that a disproportionate number of mutations at Mcyµ8 originated from maternal meiotic events. This seems to be due in part to the surprising finding that in our study population maternally derived alleles tended to be longer than those inherited from the father. Although the mutation rate at these two microsatellite loci appears similar, the mutational processes involved appear to differ. At the tetranucleotide locus allele length or span seems to have the greatest influence whereas for Mcyµ4 perhaps the dinucleotide repeat motif raises the mutation rate across the entire allele range. It seems that different underlying mechanisms influence different loci, and further studies are needed to more fully understand the complexities of microsatellite evolution.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
This study was funded by the Australian Research Council and the Australian National University. We thank Rod Peakall and two anonymous referees for their insightful comments and guidance.


    Footnotes
 
E-mail: mike.double{at}anu.edu.au. Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 

    Amos, W., S. J. Sawcer, R. W. Feakes, and D. C. Rubinsztein. 1996. Microsatellites show mutational bias and heterozygote instability. Nat. Genet 13:390-391.[ISI][Medline]

    Anderson, T. J. C., X. Z. Su, A. Roddam, and K. P. Day. 2000. Complex mutations in a high proportion of microsatellite loci from the protozoan parasite Plasmodium falciparum. Mol. Ecol 9:1599-1608.[CrossRef][ISI][Medline]

    Chakraborty, R., M. Kimmel, D. N. Stivers, L. J. Davison, and R. Deka. 1997. Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci. Proc. Natl. Acad. Sci. USA 94:1041-1046.[Abstract/Free Full Text]

    Crozier, R. H., M. E. Kaufmann, M. E. Carew, and Y. C. Crozier. 1999. Mutability of microsatellites developed for the ant Camponotus consobrinus. Mol. Ecol 8:271-276.[CrossRef][ISI][Medline]

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Accepted for publication September 6, 2002.