Complex mutation at a microsatellite locus in sturgeons: Acipenser sinensis, A. schrenckii, A. gueldenstaedtii and A. baerii

Cross‐species amplifications of microsatellite locus Spl‐106, which was originally screened from the genome of shovelnose sturgeon (Scaphirhynchus platorynchus) with a perfect TAGA repeat motif, were carried out in four other species of the genera Acipenser. A total of 34 polymerase chain reaction (PCR) products representing 16 different alleles of this locus was sequenced. Sequence analysis results showed that besides the number changes of repeat units, many mutational events, such as single‐base substitutions and various insertion/deletion (indels) occurred not only at species level but also at individual level, even among the different alleles within the same individual. The repeat motifs varied from perfect (TAGA)n array to perfect compound (TAAA)m (GAAA)n and perfect or imperfect compound (TAAA)m (TAGA)n (TAAA)x arrays in different species and different individuals. The evolution dynamics of this locus in sturgeons was inferred in that it may evolve from a single perfect to different perfect or imperfect compounds.     

Evidence for complex mutations at microsatellite loci in Drosophila.

Fifteen lines each of Drosophila melanogaster, D. simulans, and D. sechellia were scored for 19 microsatellite loci. One to four alleles of each locus in each species were sequenced, and microsatellite variability was compared with sequence structure. Only 7 loci had their size variation among species consistent with the occurrence of strictly stepwise mutations in the repeat array, the others showing extensive variability in the flanking region compared to that within the microsatellite itself. Polymorphisms apparently resulting from complex nonstepwise mutations involving the microsatellite were also observed, both within and between species. Maximum number of perfect repeats and variance of repeat count were found to be strongly correlated in microsatellites showing an apparently stepwise mutation pattern. These data indicate that many microsatellite mutation events are more complex than represented even by generalized stepwise mutation models. Care should therefore be taken in inferring population or phylogenetic relationships from microsatellite size data alone. The analysis also indicates, however, that evaluation of sequence structure may allow selection of microsatellites that more closely match the assumptions of stepwise models.     

Rapid divergence of microsatellite abundance among species of Drosophila

Among major taxonomic groups, microsatellites exhibit considerable variation in composition and allele length, but they also show considerable conservation within many major groups. This variation may be explained by slow microsatellite evolution so that all species within a group have similar patterns of variation, or by taxon-specific mutational or selective constraints. Unfortunately, comparing microsatellites across species and studies can be problematic because of biases that may exist among different isolation and analysis protocols. We present microsatellite data from five Drosophila species in the Drosophila subgenus: D. arizonae, D. mojavensis, and D. pachea (three cactophilic species), and D. neotestacea and D. recens (two mycophagous species), all isolated at the same time using identical protocols. For each species, we compared the relative abundance of motifs, the distribution of repeat size, and the average number of repeats. Dimers were the most abundant microsatellites for each species. However, we found considerable variation in the relative abundance of motif size classes among species, even between sister taxa. Frequency differences among motifs within size classes for the three cactophilic species, but not the two mycophagous species, are consistent with other studied Drosophila. Frequency distributions of repeat number, as well as mean size, show significant differences among motif size classes but not across species. Sizes of microsatellites in these five species are consistent with D. virilis, another species in the subgenus Drosophila, but they have consistently higher means than in D. melanogaster, in the subgenus Sophophora. These results confirm that many aspects of microsatellite variation evolve quickly but also are subject to taxon-specific constraints. In addition, the nature of microsatellite evolution is dependent on temporal and taxonomic scales, and some variation is conserved across broad taxonomic levels despite relatively high rates of mutation for these loci.     

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.     

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.     

Size homoplasy and mutational processes of interrupted microsatellites in two bee species, Apis mellifera and Bombus terrestris (Apidae)

Similar microsatellite electromorphs (PCR products of the same size) can arise from independent mutational events. Such alleles are not identical by descent. This phenomenon, termed size homoplasy, was studied by sequencing electromorphs of two microsatellite loci in which the stretch of basic repeats is interrupted by different short (1-2 bp) DNA motifs. The number and position of these interruptions were established for electromorphs from closely and distantly related populations of honeybees and bumblebees. No sequence difference was found when electromorphs came from the same subspecies or from closely related subspecies, suggesting that they were probably identical by descent. In contrast, sequence differences were often detected in distantly related subspecies, showing that size homoplasy frequently occurs at this level of population differentiation. Size homoplasy is increased by limits to free length variation of alleles, a phenomenon that seems to act on interrupted microsatellites when comparing distantly related taxa, that is, honeybee subspecies from different evolutionary lineages. Electromorph sequences suggest that, within the scope of these limits, large mutation events have occurred frequently at both interrupted loci studied. In good agreement with the molecular data, computations based on the observed heterozygosity and number of electromorphs and simulation studies showed that neither locus fits the one-step stepwise mutant model (SMM). We speculate that interrupted microsatellites in general could be characterized by a higher variance in repeat number and consequently a lower homoplasy rate than pure ones. Hence, interrupted microsatellites should be most appropriate for investigating population differentiation and evolutionary relationship between relatively distant populations.      

Inverse relationship between F and microsatellite polymorphism in the marine fish, walleye pollock (Theragra chalcogramma): implications for resolving weak population structure

Microsatellites have proved to be useful for the detection of weak population structure in marine fishes and other species characterized by large populations and high gene flow. None the less, uncertainty remains about the net effects of the particular mutational properties of these markers, and the wide range of locus polymorphism they exhibit, on estimates of differentiation. We examined the effect of varying microsatellite polymorphism on the magnitude of observed differentiation in a population survey of walleye pollock, Theragra chalcogramma. Genetic differentiation at 14 microsatellite loci among six putative populations from across the North Pacific Ocean and Bering Sea was weak but significant on large geographical scales and conformed to an isolation-by-distance pattern. A negative relationship was found between locus variability and the magnitude of estimated population subdivision. Estimates of F(ST) declined with locus polymorphism, resulting in diminished power to discriminate among samples, and we attribute this loss to the effects of size homoplasy. This empirical result suggests that mutation rates of some microsatellite loci are sufficiently high to limit resolution of weak genetic structure typical of many marine fishes.     

Human Microsatellites: Mutation and Evolution

Microsatellites (MSs) are short tandem DNA repeats with the repetitive motif of two to six nucleotides, forming tracts up to hundreds of nucleotides long. Notwithstanding the active use of MSs in genetic studies of various biological problems, the reasons for their wide occurrence in the genome, their possible functions, and mutational behavior are still unclear. The mutation rate in MS repeats is on average several orders of magnitude higher than in the remaining DNA, which allows for direct estimation of evolutionary transformation rate in nucleotide sequences of the genome. Mutation process in MSs is highly heterogeneous, with distinct differences between species; furthermore, within a species it differs among loci with different repeat size, among alleles of one locus, and among individuals of different sex and age. Most MS mutations are caused by DNA slippage during replication but the probability of this event depends on the locus. In this review, a number of models of MS evolution are discussed, which account for the relationship between mutation rate and allele size, different mutation direction in alleles of different size, and the appearance of point mutations within repeat tracts restricting allele size. The MS evolution is considered mainly in the context of selective neutrality, although there is evidence showing functional significance of some variants of tandem repeats and thus their possible selective value.     

Characterization of polymorphic microsatellite markers in the adder,Vipera berus

Microsatellite DNA markers can yield sufficient resolution for individual identification as well as provide genetic information on a larger, interpopulational scale. Here we present details on six microsatellite primer pairs developed for the adder, Vipera berus. The number of alleles found varied between 2 and 38 per locus. The objectives behind developing these markers included assessment both of paternity and population histories from different parts of the species’ range. Cross‐species amplification indicated that these markers may also be useful for studies of other species within the Viperidae family.     

Mutational bias in penguin microsatellite DNA

Analysis of nucleotide sequence variation at a microsatellite DNA locus revealed extensive size homoplasy of alleles in Adélie penguins (Pygoscelis adeliae). Variation in the flanking regions at this locus allowed discrimination between mechanisms proposed for length changes in microsatellite DNA alleles. We further examined the structure of alleles for the same microsatellite DNA locus across 11 additional species of penguin (Spheniscidae) by mapping allele sequences onto an independent penguin phylogeny. Our analysis indicated that the repeat motifs appear to have evolved independently on several occasions. We observed sequence instability in the region bordering the repeat tract with a transversional bias predominating. We propose that this bias results from inaccurate DNA replication owing to the sequence context of this repeat tract. Because we show that regions flanking repeat sequences exhibit this mutational bias, this cautions against the use of such regions for phylogeny reconstruction.     

Isolation and characterization of six polymorphic microsatellite loci in the western flower thrips Frankliniella occidentalis (Insecta,Thysanoptera)

Highly polymorphic microsatellite markers can provide important demographic information on founder events and range expansion following initial introduction of invasive insect species. Six microsatellite loci were isolated from an enriched DNA library in order to study the invasion patterns of the western flower thrips, Frankliniella occidentalis. All loci tested were found to be polymorphic and successfully amplified in all individuals. The number of alleles per locus ranged from five to nine and heterozygosity ranged from 45 to 73%. Some of the loci were also successfully amplified in other thrips species.     

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     

Human microsatellites: mutation and evolution

Microsatellites (MSs) are short tandem DNA repeats with the repetitive motif of two to six nucleotides, forming tracts up to hundreds of nucleotides long. Notwithstanding the active use of MSs in genetic studies of various biological problems, the reasons for their wide occurrence in the genome, their possible functions, and mutational behavior are still unclear. The mutation rate in MS repeats is on average several orders of magnitude higher than in the remaining DNA, which allows for direct estimation of evolutionary transformation rate in nucleotide sequences of the genome. Mutation process in MSs is species-specific; furthermore, within a species it differs among loci with different repeat size, among alleles of one locus, and among individuals of different sex and age. Most MS mutations are caused by DNA slippage during replication but the probability of this event depends on the locus. In this review, a number of models of MS evolution are discussed, which account for the relationship between mutation rate and allele size, different mutation direction in alleles of different size, and the appearance of point mutations within repeat tracts restricting allele size. The MS evolution is considered mainly in the context of selective neutrality, although there is evidence showing functional significance of some variants of tandem repeats and thus their possible selective value.     

Thermodynamic characteristization of Di-, Tri-, and tetranucleotide loci in parthenogenetic lizards <Emphasis Type="Italic">Darevskia unisexualis</Emphasis>

Allelic polymorphism of three microsatellite loci from the genome of parthenogenetic lizard Darevskia unisexualis was characterized using analysis of free energy (Gibbs energy) of the DNA/DNA duplex formation within the stepwise mutational model. It was demonstrated that the number of microsatellite cluster monomericic units would change to decrease the mean free energy of the locus. In addition, based on the analysis of nucleotide composition, the GC content of each locus was evaluated, and belonging of the loci examined to certain isochore families was suggested.     

Microsatellite DNA analysis of northern pike (Esox lucius L.) populations: insights into the genetic structure and demographic history of a genetically depauperate species

The northern pike Esox lucius L. is a freshwater fish exhibiting pronounced population subdivision and low genetic variability. However, there is limited knowledge on phylogeographical patterns within the species, and it is not known whether the low genetic variability reflects primarily current low effective population sizes or historical bottlenecks. We analysed six microsatellite loci in ten populations from Europe and North America. Genetic variation was low, with the average number of alleles within populations ranging from 2.3 to 4.0 per locus. Genetic differentiation among populations was high (overall θ_ST = 0.51; overall ρ_ST = 0.50). Multidimensional scaling analysis of genetic distances between populations and spatial analysis of molecular variance suggested a single phylogeographical race within the sampled populations from northern Europe, whereas North American and southern European populations were highly distinct. A population from Ireland was monomorphic at all loci, presumably reflecting founder events associated with introduction of the species to the island in the sixteenth century. Bayesian analysis of demographic parameters showed differences in θ (a product of effective population size and mutation rate) among populations from large and small water bodies, but the relative differences in θ were smaller than expected, which could reflect population subdivision within the larger water bodies. Finally, the analyses showed drastic population declines on a time scale of several thousand years within European populations, which we ascribe to either glacial bottlenecks or postglacial founder events.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 91–101.     

Development and characterization of microsatellite DNA markers for the Alpine plant species Campanula thyrsoides

We isolated and characterized eight polymorphic microsatellite markers for the Alpine plant species Campanula thyrsoides (Campanulaceae). Number of alleles per locus ranged from six to 12 and the observed heterozygosity was between 0.529 and 0.900. Observed vs. expected heterozygote deficits were significantly different in one out of eight loci following Bonferroni's correction for multiple tests. We did not find evidence for linkage disequilibrium between locus pairs. The microsatellite markers are being used for the study of genetic variation and gene flow within and among populations of C. thyrsoides in the Swiss Alps.     

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 EVOLUTION IN VERTEBRATES: INFERENCE FROM AC DINUCLEOTIDE REPEATS

Abstract We analyze published data from 592 AC microsatellite loci from 98 species in five vertebrate classes including fish, reptiles, amphibians, birds, and mammals. We use these data to address nine major questions about microsatellite evolution. First, we find that larger genomes do not have more microsatellite loci and therefore reject the hypothesis that microsatellites function primarily to package DNA into chromosomes. Second, we confirm that microsatellite loci are relatively rare in avian genomes, but reject the hypothesis that this is due to physical constraints imposed by flight. Third, we find that microsatellite variation differs among species within classes, possibly relating to population dynamics. Fourth, we reject the hypothesis that microsatellite structure (length, number of alleles, allele dispersion, range in allele sizes) differs between poikilotherms and homeotherms. The difference is found only in fish, which have longer microsatellites and more alleles than the other classes. Fifth, we find that the range in microsatellite allele size at a locus is largely due to the number of alleles and secondarily to allele dispersion. Sixth, length is a major factor influencing mutation rate. Seventh, there is a directional mutation toward an increase in microsatellite length. Eighth, at the species level, microsatellite and allozyme heterozygosity covary and therefore inferences based on large-scale studies of allozyme variation may also reflect microsatellite genetic diversity. Finally, published microsatellite loci (isolated using conventional hybridization methods) provide a biased estimate of the actual mean repeat length of microsatellites in the genome.     

Genetic Structure of Earthworm Populations at a Regional Scale: Inferences from Mitochondrial and Microsatellite Molecular Markers in Aporrectodea icterica (Savigny 1826)

Despite the fundamental role that soil invertebrates (e.g. earthworms) play in soil ecosystems, the magnitude of their spatial genetic variation is still largely unknown and only a few studies have investigated the population genetic structure of these organisms. Here, we investigated the genetic structure of seven populations of a common endogeic earthworm (Aporrectodea icterica) sampled in northern France to explore how historical species range changes, microevolutionary processes and human activities interact in shaping genetic variation at a regional scale. Because combining markers with distinct modes of inheritance can provide extra, complementary information on gene flow, we compared the patterns of genetic structure revealed using nuclear (7 microsatellite loci) and mitochondrial markers (COI). Both types of markers indicated low genetic polymorphism compared to other earthworm species, a result that can be attributed to ancient bottlenecks, for instance due to species isolation in southern refugia during the ice ages with subsequent expansion toward northern Europe. Historical events can also be responsible for the existence of two divergent, but randomly interbreeding mitochondrial lineages within all study populations. In addition, the comparison of observed heterozygosity among microsatellite loci and heterozygosity expected under mutation-drift equilibrium suggested a recent decrease in effective size in some populations that could be due to contemporary events such as habitat fragmentation. The absence of relationship between geographic and genetic distances estimated from microsatellite allele frequency data also suggested that dispersal is haphazard and that human activities favour passive dispersal among geographically distant populations.     

Microsatellite loci from the ectomycorrhizal basidiomycete Suillus pictus associated with the genus Pinus subgenus Strobus

Seven microsatellite loci were isolated and characterized from ectomycorrhizal basidiomycete Suillus pictus associated with soft pine species (Pinus subgenus Strobus) using a dual‐suppression‐PCR technique. Microsatellite variation was assessed using 38 isolates of S. pictus sampled from three different local populations in Japan. The number of alleles per locus ranged from three to 12 within populations. These microsatellite loci can be used for studying the allelic variation and genetic structure in/among local populations of S. pictus.