Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis

Homoplasy has recently attracted the attention of population geneticists, as a consequence of the popularity of highly variable stepwise mutating markers such as microsatellites. Microsatellite alleles generally refer to DNA fragments of different size (electromorphs). Electromorphs are identical in state (i.e. have identical size), but are not necessarily identical by descent due to convergent mutation(s). Homoplasy occurring at microsatellites is thus referred to as size homoplasy. Using new analytical developments and computer simulations, we first evaluate the effect of the mutation rate, the mutation model, the effective population size and the time of divergence between populations on size homoplasy at the within and between population levels. We then review the few experimental studies that used various molecular techniques to detect size homoplasious events at some microsatellite loci. The relationship between this molecularly accessible size homoplasy size and the actual amount of size homoplasy is not trivial, the former being considerably influenced by the molecular structure of microsatellite core sequences. In a third section, we show that homoplasy at microsatellite electromorphs does not represent a significant problem for many types of population genetics analyses realized by molecular ecologists, the large amount of variability at microsatellite loci often compensating for their homoplasious evolution. The situations where size homoplasy may be more problematic involve high mutation rates and large population sizes together with strong allele size constraints.     

Variation of Microsatellite Size Homoplasy Across Electromorphs, Loci, and Populations in Three Invertebrate Species

Size homoplasy was analyzed at microsatellite loci by sequencing electromorphs, that is, variants of the same size (base pairs). This study was conducted using five interrupted and/or compound loci in three invertebrate species, the honey bee Apis mellifera, the bumble bee Bombus terrestris, and the freshwater snail Bulinus truncatus. The 15 electromorphs sequenced turned out to hide 31 alleles (i.e., variants identical in sequence). Variation in the amount of size homoplasy was detected among electromorphs and loci. From one to seven alleles were detected per electromorph, and one locus did not show any size homoplasy in both bee species. The amount of size homoplasy was related to the sequencing effort, since the number of alleles was correlated with the number of copies of electromorphs sequenced, but also with the molecular structure of the core sequence at each locus. Size homoplasy within populations was detected only three times, meaning that size homoplasy was detected mostly among populations. We analyzed population structure, estimating F _st and a genetic distance, based on either electromorphs or alleles. Whereas little difference was found in A. mellifera, uncovering size homoplasy led to a more marked population structure in B. terrestris and B. truncatus. We also showed in A. mellifera that the detection of size homoplasy may alter phylogenetic reconstructions. Received: 21 July 1997 / Accepted: 29 January 1998     

微卫星跨物种交叉PCR扩增的一个新问题:扩增非同源产物

微卫星已被广泛应用于群体遗传学、生态学和进化生物学研究。然而,一些物种微卫星尚未克隆。为了节省时间和经费,研究人员往往使用一个物种已发表的微卫星引物扩增其近缘物种的微卫星。该研究对属于3个不同科(Clariidae、Heteropneustidae 和Pimelodidae)的7个鲶鱼物种的微卫星跨物种PCR扩增产物进行了序列分析,研究发现扩增非同源（non-orthologous）产物是微卫星跨物种PCR扩增的一个新问题。该研究共采用4对胡子鲶微卫星座位引物对7个鲶鱼物种进行了跨物种PCR扩增。对获得的204个PCR产物的序列分析结果表明,两对微卫星座位引物扩增了所有7个物种的同源特异产物。而其他两个座位的引物扩增了特异但非同源的多态产物,对近缘物种的扩增也获得类似结果。另外,除胡子鲶等位基因大小异源同型(size homoplasy)的特征不明显外,其他物种在3个微卫星座位都具有这一非常明显的特征。这些数据表明,微卫星跨物种间交叉扩增能产生非同源产物;等位基因大小异源同型与微卫星座位本身有关,而与物种间的亲缘关系无明显的相关性。微卫星跨物种扩增产生的非同源产物和等位基因大小异源同型将使系统发育、群体遗传学和进化研究明显复杂化。因此,在应用微卫星跨物种交叉扩增数据以前,最好对跨物种交叉扩增产物进行测序验证。     

What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles

Sixty-five microsatellite alleles amplified from ancestral citrus accessions classified in three separate genera were evaluated for sequence polymorphism to establish the basis of inter- and intra-allelic genetic variation, evaluate the extent of size homoplasy, and determine an appropriate model (stepwise or infinite allele) for analysis of citrus microsatellite alleles. Sequences for each locus were aligned and subsequently used to determine relationships between alleles of different taxa via parsimony. Interallelic size variation at each SSR locus examined was due to changes in repeat copy number with one exception. Sequencing these alleles uncovered new distinct point mutations in the microsatellite region and the region flanking the microsatellite. Several of the point mutations were found to be genus, species, or allele specific, and some mutations were informative about the inferred evolutionary relationships among alleles. Overall, homoplasy was observed in alleles from all three loci, where the core microsatellite repeat was changed causing alleles of the same size class to be identical in state but not identical by descent. Because nearly all changes in allele size (with one exception) were due to expansion or contraction of the repeat motif, this suggests that a stepwise mutation model, which assumes homoplasy may occur, would be the most appropriate for analyzing Citrus SSR data. The collected data indicate that microsatellites can be a useful tool for evaluating Citrus species and two related genera since repeat motifs were reasonably well retained. However, this work also demonstrated that the number of microsatellite alleles is clearly an underestimate of the number of sequence variants present.     

Complex mutational patterns and size homoplasy at maize microsatellite loci

Microsatellite markers have become one of the most popular tools for germplasm characterization, population genetics and evolutionary studies. To investigate the mutational mechanisms of maize microsatellites, nucleotide sequence information was obtained for ten loci. In addition, Single-Strand Conformation Polymorphism (SSCP) analysis was conducted to assess the occurrence of size homoplasy. Sequence analysis of 54 alleles revealed a complex pattern of mutation at 8/10 loci, with only 2 loci showing allele variation strictly consistent with stepwise mutations. The overall allelic diversity resulted from changes in the number of repeat units, base substitutions, and indels within repetitive and non-repetitive segments. Thirty-one electromorphs sampled from six maize landraces were considered for SSCP analysis. The number of conformers per electromorph ranged from 1 to 7, with 74.2% of the electromorphs showing more than one conformer. Size homoplasy was apparent within landraces and populations. Variation in the amount of size homoplasy was observed within and between loci, although no differences were detected among populations. The results of the present study provide useful information on the interpretation of genetic data derived from microsatellite markers. Further efforts are still needed to determine the impact of these findings on the estimation of population parameters and on the inference of phylogenetic relationships in maize investigations. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The impact of microsatellite electromorph size homoplasy on multilocus population structure estimates in a tropical tree (Corythophora alta) and an anadromous fish (Morone saxatilis)

Microsatellite allelic states are determined by electrophoretic sizing of polymerase chain reaction fragments to define electromorphs. Numerous studies have documented that identical microsatellite electromorphs are potentially heterogeneous at the DNA sequence level, a phenomenon called electromorph size homoplasy. Few studies have examined the impact of electromorph size homoplasy on estimates of population genetic parameters. We investigated the frequency of microsatellite electromorph size homoplasy for 12 loci in the tropical tree Corythophora alta and 11 loci in the anadromous fish Morone saxatilis by sequencing 14-23 homozygotes per locus sampled from multiple populations for a total of 453 sequences. Sequencing revealed no homoplasy for M. saxatilis loci. Seven C. alta loci exhibited homoplasy, including single and compound repeat motifs both with and without interruptions. Between 12.5 and 42.9% of electromorphs sampled per locus showed size homoplasy. Two methods of correction for homoplasy in C. alta generally produced little or no change in single-locus estimates of RST, except for two loci in which some additional differentiation among populations was revealed. Twelve-locus estimates of RST (including the seven loci corrected for homoplasy) were slightly greater than estimates from uncorrected data, although the 95% confidence intervals overlapped. The frequency of methodological errors such as clerical mistakes or sample mislabelling per genotype scored was estimated at 5.4 and 7.3% for C. alta and M. saxatilis, respectively. Simulations showed that the increase in RST produced by homoplasy correction was only slightly larger than variation in RST estimates expected to be caused by methodological errors.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in Lake Malawi cichlids

Recent studies have suggested that size homoplasy is a prevalent feature of microsatellites and is expected to increase with time of divergence among populations and taxa. In this study, we performed sequence analysis of alleles from a complex microsatellite locus (Pzeb4, initially isolated from Pseudotropheus (Maylandia) zebra) from 1 midwater-feeding and 10 rock-dwelling cichlid fish species from Lake Malawi, East Africa, to investigate how widespread size homoplasy is among closely related taxa at this locus. All cichlid fishes endemic to this lake are believed to have originated within the last 700,000 years, and some species may be less than 200 years old. The number of eletromorphs found per species varied from 3 to 13. Sequence analysis of 95 cloned Pzeb4 PCR products (representing 18 electromorphs) revealed 13 new alleles. Ten of the 13 electromorphs (77%) were found to show size homoplasy due to either single nucleotide substitutions/indels or large indels. To investigate how well this locus fits the single-step mutation model (SMM), the minimum number of mutations required to explain the length differences between pairs of alleles was plotted against their size differences. Of the 300 comparisons, 166 (55.3%) corresponded to SMM expectations and 86 (28.7%) required a smaller number of mutations, and for 48 (16.0%) pairwise comparisons, a larger number of mutations were required to explain the length differences as compared with SMM expectations. Finally, a large deletion in the microsatellite sequence observed in the three rock-dwelling species Pseudotropheus lucerna, Pseudotropheus (Tropheops) 'band,' and Pseudotropheus (Tropheops) 'rust' and the midwater-feeding species Copadichromis sp. is believed to represent a shared ancestral polymorphism.     

Microsatellite size homoplasy, SSCP, and population structure: a case study in the freshwater snail Bulinus truncatus

The extent of microsatellite size homoplasy, as well as its effect on several population genetics statistics, was investigated in natural populations using the single-strand conformation polymorphism (SSCP) method. The analysis was conducted using 240 individuals from 13 populations of the freshwater snail Bulinus truncatus at a GT(n)CT(m) compound microsatellite locus. We showed that SSCP can be used to uncover, at least partly, size homoplasy in the core sequence of this category of loci. Eight conformers (SSCP variants) were detected among the three size variants (electromorphs). Sequencing revealed that each conformer corresponded to a different combination of repeats in the GT(n) and CT(m) arrays. Part of this additional variability was detected within populations, resulting in a substantial increase in gene diversity in four populations. Additional variability also changed the values of parameters used to analyze population differentiation among populations: pairwise tests of differentiation were significant much more often with conformers than with electromorphs. On the other hand, pairwise estimates of F(st) were either smaller or larger with conformers than with electromorphs, depending on whether or not electromorphs were shared among populations. However, estimates of F(st) (or analogs) over all populations were very similar, ranging between 0.66 and 0.75. Our results were consistent with the theoretical prediction that homoplasy should not always lead to stronger population structure. Finally, conformer sequences and electromorph size distribution suggested that single-point and/or stepwise mutations occurring simultaneously in the different repeated arrays of compound microsatellites produce sequence variation without size variation and hence generate more size homoplasy than expected under a simple stepwise mutation model.     

Mutation and evolution of microsatellite loci in Neurospora

The patterns of mutation and evolution at 13 microsatellite loci were studied in the filamentous fungal genus Neurospora. First, a detailed investigation was performed on five microsatellite loci by sequencing each microsatellite, together with its nonrepetitive flanking regions, from a set of 147 individuals from eight species of Neurospora. To elucidate the genealogical relationships among microsatellite alleles, repeat number was mapped onto trees constructed from flanking-sequence data. This approach allowed the potentially convergent microsatellite mutations to be placed in the evolutionary context of the less rapidly evolving flanking regions, revealing the complexities of the mutational processes that have generated the allelic diversity conventionally assessed in population genetic studies. In addition to changes in repeat number, frequent substitution mutations within the microsatellites were detected, as were substitutions and insertion/deletions within the flanking regions. By comparing microsatellite and flanking-sequence divergence, clear evidence of interspecific allele length homoplasy and microsatellite mutational saturation was observed, suggesting that these loci are not appropriate for inferring phylogenetic relationships among species. In contrast, little evidence of intraspecific mutational saturation was observed, confirming the utility of these loci for population-level analyses. Frequency distributions of alleles within species were generally consistent with the stepwise mutational model. By comparing variation within species at the microsatellites and the flanking-sequence, estimated microsatellite mutation rates were approximately 2500 times greater than mutation rates of flanking DNA and were consistent with estimates from yeast and fruit flies. A positive relationship between repeat number and variance in repeat number was significant across three genealogical depths, suggesting that longer microsatellite alleles are more mutable than shorter alleles. To test if the observed patterns of microsatellite variation and mutation could be generalized, an additional eight microsatellite loci were characterized and sequenced from a subset of the same Neurospora individuals.     

Evolutionary history of microsatellites in the obscura group of Drosophila

The evolutionary origins of microsatellites are not well understood. Some investigators have suggested that point mutations that expand repeat arrays beyond a threshold size trigger microsatellites to become variable. However, little empirical data has been brought forth on this and related issues. In this study, we examine the evolutionary history of microsatellites in six species within the obscura group of Drosophila, tracing changes in microsatellite alleles using both PCR product size and sequence data. We found little evidence supporting a general role of point mutations triggering initial microsatellite expansion, and no consistent threshold size for expansion was observed. Flanking region length variation was extensive when alleles were sequenced in distantly related species, and some species possessed altogether different repeat arrays between the same primer binding sites. Our results suggest extreme caution in using microsatellite allele sizes for phylogenetic analyses or to infer divergences between populations.     

Nine microsatellite DNA primers for <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> ssp. <Emphasis Type="Italic">sinensis</Emphasis> (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the arid regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.     

Microsatellite Allelic Homoplasy Due to Variable Flanking Sequences

Microsatellite DNA sequences have become the dominant source of nuclear genetic markers for most applications. It is important to investigate the basis of variation between alleles and to know if current assumptions about the mechanisms of microsatellite mutation (that is to say, variations involving simple changes in the number of repeat) are correct. We have characterized, by DNA sequencing, the human alleles of a new highly informative (CA)n repeat localized approximately 20 kb centromeric to the HLA-B gene. Although 12 alleles were identified based on conventional length criteria, sequencing of the alleles demonstrated that differences between alleles were found to be more complex than previously assumed: A high degree of microsatellite variability is due to variation in the region immediately flanking the repeat. These data indicate that the mutational process which generates polymorphism in this region has involved not only simple changes in the number of dinucleotide CA repeats but also perturbations in the nonrepeated 5′ and 3′ flanking sequences. Three families of alleles (not visible from the overall length of the alleles), with presumably separate evolutionary histories, exist and can yield to homoplasy of size. Effectively, we can observe alleles of the same size with different internal structures which are separated by a significant amount of variation. Although allelic homoplasy for noninterrupted microsatellite loci has been suggested between different species, it has not been unequivocally demonstrated within species. A strong association is noted between alleles defined at the sequence level and HLA-B alleles. The observation of several families of alleles at the population level provides information about the evolutionary history and mutation processes of microsatellites and may have implications for the use of these markers in phylogenetic, linkage disequilibrium studies, and gene mapping. Received: 14 May 1996 / Accepted: 9 September 1996     

Evolutionary factors affecting the cross‐species utility of newly developed microsatellite markers in seabirds

Microsatellite loci are ideal for testing hypotheses relating to genetic segregation at fine spatio‐temporal scales. They are also conserved among closely related species, making them potentially useful for clarifying interspecific relationships between recently diverged taxa. However, mutations at primer binding sites may lead to increased nonamplification, or disruptions that may result in decreased polymorphism in nontarget species. Furthermore, high mutation rates and constraints on allele size may also with evolutionary time, promote an increase in convergently evolved allele size classes, biasing measures of interspecific genetic differentiation. Here, we used next‐generation sequencing to develop microsatellite markers from a shotgun genome sequence of the sub‐Antarctic seabird, the thin‐billed prion (Pachyptila belcheri), that we tested for cross‐species amplification in other Pachyptila and related sub‐Antarctic species. We found that heterozygosity decreased and the proportion of nonamplifying loci increased with phylogenetic distance from the target species. Surprisingly, we found that species trees estimated from interspecific F_ST provided better approximations of mtDNA relationships among the studied species than those estimated using D_C, even though F_ST was more affected by null alleles. We observed a significantly nonlinear second order polynomial relationship between microsatellite and mtDNA distances. We propose that the loss of linearity with increasing mtDNA distance stems from an increasing proportion of homoplastic allele size classes that are identical in state, but not identical by descent. Therefore, despite high cross‐species amplification success and high polymorphism among the closely related Pachyptila species, we caution against the use of microsatellites in phylogenetic inference among distantly related taxa.     

Digital fragment analysis of short tandem repeats by high‐throughput amplicon sequencing

High‐throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high‐throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high‐throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost‐effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence‐aware microsatellite data.     

Nine porcine microsatellite loci tested for size homoplasy in genetically diverse breeds

Kind and probability of homoplasy across allelic microsatellite fragments can be investigated using DNA of genetically diverse pig breeds. In this study, nine microsatellite loci (SW1897, SW2427, SW489, SW957, TNFB, IFNG, SW2410, SW2019 and S0215) were analysed using DNA samples of pigs from Vietnam (Indigenous breeds Co, Meo, Muong Khuong, Tap Na) and Germany (European Wild Boar, Pietrain). In a total of 39 sequences, 20 differences within isomorphic alleles were observed in comparison with the respective reference sequences. They affected five of the nine tested microsatellite loci. The majority (18) of SNPs occurred in the 5'-flanking regions of the microsatellite repeats, 10 were found in the 3'-flanking regions and only one SNP occurred within the repeat of the Wild Boar sequence of SW2427. The compound microsatellites IFNG and S0215 were unaffected by size homoplasy (SH) within our material. We conclude that the fragment length analysis of microsatellites is a reliable tool for intraspecific phylogenetic studies because SH rates within a species were low.     

New polymorphic microsatellite loci for the Greek smooth newt, Lissotriton vulgaris graecus, and their utility in the nominotypical subspecies

Eight polymorphic microsatellites were isolated from the Greek smooth newt (Lissotriton vulgaris graecus) using a microsatellite enrichment protocol and selective hybridization with a biotinylated (AC)(11) probe. The loci showed different variation patterns in a single breeding population (32 individuals) with mean number of alleles at 5.0 and mean observed heterozygosity at 0.520. The amplification success also in the nominotypical subspecies favours the use of these microsatellite loci in population genetic analyses as well as in the study of contact zones between smooth newt subspecies.     

Population structure within and between subspecies of the Mediterranean triplefin fish Tripterygion delaisi revealed by highly polymorphic microsatellite loci

Although F(ST) values are widely used to elucidate population relationships, in some cases, when employing highly polymorphic loci, they should be regarded with caution, particularly when subspecies are under consideration. Tripterygion delaisi presents two subspecies that were investigated here, using 10 microsatellite loci. A Bayesian approach allowed us to clearly identify both subspecies as two different evolutionary significant units. However, low F(ST) values were found between subspecies as a consequence of the large number of alleles per locus, while homoplasy could be disregarded as indicated by the standardized genetic distance G'(ST). Heterozygosity saturation was observed in highly polymorphic loci containing more than 15 alleles, and this threshold was used to define two loci pools. The less variable loci pool revealed higher genetic variance between subspecies, while the more variable pool showed higher genetic variance between populations. Furthermore, higher differentiation was also observed between populations using G'(ST) with the more variable loci. Nonetheless, a more reliable population structure within subspecies was obtained when all loci were included in the analyses. In T. d. xanthosoma, isolation by distance was detected between the eight analysed populations, and six genetically homogeneous clusters were inferred by Bayesian analyses that are in accordance with F(ST) values. The neighbourhood-size method also indicated rather small dispersal capabilities. In conclusion, in fish with limited adult and larval dispersal capabilities, continuous rocky habitat seems to allow contact between populations and prevent genetic differentiation, while large discontinuities of sand or deep-water channels seems to reduce gene flow.     

A test for concordance between the multilocus genealogies of genes and microsatellites in the pathogenic fungus Coccidioides immitis

Uncovering the correct phylogeny of closely related species requires analysis of multiple gene genealogies or, alternatively, genealogies inferred from the multiple alleles found at highly polymorphic loci, such as microsatellites. However, a concern in using microsatellites is that constraints on allele sizes may occur, resulting in homoplasious distributions of alleles, leading to incorrect phylogenies. Seven microsatellites from the pathogenic fungus Coccidioides immitis were sequenced for 20 clinical isolates chosen to represent the known genetic diversity of the pathogen. An organismal phylogeny for C. immitis was inferred from microsatellite-flanking sequence polymorphisms and other restriction fragment length polymorphism-containing loci. Two microsatellite genetic distances were then used to determine phylogenies for C. immitis, and the trees found by these three methods were compared. Congruence between the organismal and microsatellite phylogenies occurred when microsatellite distances were based on simple allele frequency data. However, complex mutation events at some loci made distances based on stepwise mutation models unreliable. Estimates of times of divergence for the two species of C. immitis based on microsatellites were significantly lower than those calculated from flanking sequence, most likely due to constraints on microsatellite allele sizes. Flanking-sequence insertions/deletions significantly decreased the accuracy of genealogical information inferred from microsatellite loci and caused interspecific length homoplasies at one of the seven loci. Our analysis shows that microsatellites are useful phylogenetic markers, although care should be taken to choose loci with appropriate flanking sequences when they are intended for use in evolutionary studies.     

Investigating the allelic evolution of an imperfect microsatellite locus in the Hawaiian mushroom Rhodocollybia laulaha

The evolutionary mechanisms that give rise to microsatellite alleles remain poorly understood in general and are especially understudied for fungal microsatellite loci. The unusual G28 microsatellite locus was developed from the Hawaiian mushroom Rhodocollybia laulaha. Here, we employ a novel approach to test for allele size homoplasy and examine competing mechanistic models of microsatellite evolution in the context of biogeographic expectations for this locus based on Hawaiian geologic history. Seven G28 alleles have been identified from a sampling of 153 individuals. The G28 locus is composed of a trinucleotide imperfect motif, which permits examination of the relationships between alleles and allows for detection of potential size homoplasy within the repetitive element. Alignment of G28 allele sequence data across multiple unrelated individuals suggests that alleles of like size are homologous within Hawaii. A variety of gap coding methods are explored in the inference of allele evolution. Length differences between alleles appear to be the result of polymerase slippage at multiple positions in the repetitive element, suggesting an intricate process of allelic evolution, which is not necessarily stepwise. Complex migration scenarios must be invoked to explain the current geographic distribution of alleles if their evolution was in fact sequential (from longest to shortest or from shortest to longest) as predicted by the "progression rule."