Microsatellite Allele Size Research Articles

Intra-cultivar variability at microsatellite loci in date palm cultivars across the GCC countries

In this study, we characterized 30 date palm trees of Khalas cultivar from the GCC countries, along with seven male trees from Qatar, using 14 microsatellite loci. The results showed that the microsatellites [(GA)n] in the date palm cultivar Khalas varied considerably in allele size (range = 120–322 bp, mean = 189.78), which revealed a high degree of gene diversity (range = 0.66–0.85, mean = 0.75) and distinguished all the individual Khalas trees within and among the GCC countries. The extent of polymorphism microsatellite loci was dependent on individual loci itself, which was positively correlated with the number of repeats at the corresponding microsatellite loci. The estimates of the skewness and kurtosis of the allelic distributions showed that none of the distribution of 14 microsatellite loci was considered normal (skewness = 0, kurtosis = 3), which suggested that the shape of the allelic distribution of these loci varied by chance. In the majority of the cases, the microsatellite allele size of the most frequent allele (mode) was very close to the median ( ± 2 bp or ± (GA)1), which indicated that the most frequent allele was the ancestral allele. Nearly half of the allelic distributions of the 14 microsatellite loci were positively skewed and the other half was negatively skewed, which indicated that the alleles evolved respectively by gaining and losing of (GA)n repeats from the ancestral allele, resulting in intra-cultivar variability in Khalas cultivar over the generations. Based on this analysis, we conclude that the Khalas variety did not evolve independently at multiple origins, but rather it evolved from a single origin and, subsequently, the clones were distributed across the GCC countries. The accumulated mutations of these clones over time resulted in drastic changes among them at the microsatellite loci.

Screening of elite eggplant (Solanum melongena) genotypes for bacterial wilt (Ralstonia solanacearum) in field conditions and their genetic association by using SSR markers

Bacterial wilt (Ralstonia solanacearum) of eggplant (Solanum melongena L.) has been a major production constraint in the tropics and sub tropics of the world. Lack of understanding about host, pathogen and environment perhaps one of the reasons for limited success in controlling this disease. In present investigation, six elite genotypes of eggplant were screened against bacterial wilt in field conditions and later SSR screening was done to identify markers linked for resistant and susceptible lines, and their genetic clustering. At field level, the genotypes like CARI-1, IIHR-7 and IIHR-500A had shown resistant to bacterial wilt and recorded good yield. Further, 245 SSRs markers used for screening had shown good amplification, however only 37 primers were polymorphic, and microsatellite allele sizes were determined at their 74 loci. The average polymorphic information content was 0.315 and it ranged from 0.239 to 0.375. The SSR emh21J12 shown 170 bp band for the resistant genotypes and 160 bp band for the susceptible ones. Another SSR emf01K16 gave the unique banding pattern in resistant genotypes at 250 bp and susceptible at 260 bp. Dendrogram analysis classified these six genotypes into three main clusters. Cluster I consists of IIHR-575, IIHR-108 and IIHR-500A, where IIHR-500S was solitary. The cluster II consists Rampur local and IIHR-7 whereas, cluster III was solitary comprising CARI-1. It was revealed that the co-dominant markers such as SSR proved to be high effective tool in discriminating between resistant and susceptible genotypes, and classifying these genotypes based on genetic diversity. Hence, the field and molecular markers screening reveals that eggplant genotypes IIHR-7, IIHR-500A and CARI-1 are resistant to bacterial wilt and these resistant genotypes can be used for further breeding programme, and the identified SSR markers can be useful tool for marker assisted selection for bacterial wilt in eggplant.

Examination of Clock and Adcyap1 gene variation in a neotropical migratory passerine.

Complex behavioral traits, such as those making up a migratory phenotype, are regulated by multiple environmental factors and multiple genes. We investigated possible relationships between microsatellite variation at two candidate genes implicated in the control of migratory behavior, Clock and Adcyap1, and several aspects of migratory life-history and evolutionary divergence in the Painted Bunting (Passerina ciris), a species that shows wide variation in migratory and molting strategies across a disjunct distribution. We focused on Clock and Adcyap1 microsatellite variation across three Painted Bunting populations in Oklahoma, Louisiana, and North Carolina, and for the Oklahoma breeding population we used published migration tracking data on adult males to explore phenotypic variation in individual migratory behavior. We found no correlation between microsatellite allele size within either Clock and Adcyap1 relative to the initiation or duration of fall migration in adult males breeding in Oklahoma. We also show the lack of significant correlations with aspects of the migratory phenotype for the Louisiana population. Our research highlights the limitations of studying microsatellite allelic mutations that are of undetermined functional influence relative to complex behavioral phenotypes.

OGDD (Olive Genetic Diversity Database): a microsatellite markers' genotypes database of worldwide olive trees for cultivar identification and virgin olive oil traceability.

Olive (Olea europaea), whose importance is mainly due to nutritional and health features, is one of the most economically significant oil-producing trees in the Mediterranean region. Unfortunately, the increasing market demand towards virgin olive oil could often result in its adulteration with less expensive oils, which is a serious problem for the public and quality control evaluators of virgin olive oil. Therefore, to avoid frauds, olive cultivar identification and virgin olive oil authentication have become a major issue for the producers and consumers of quality control in the olive chain. Presently, genetic traceability using SSR is the cost effective and powerful marker technique that can be employed to resolve such problems. However, to identify an unknown monovarietal virgin olive oil cultivar, a reference system has become necessary. Thus, an Olive Genetic Diversity Database (OGDD) (http://www.bioinfo-cbs.org/ogdd/) is presented in this work. It is a genetic, morphologic and chemical database of worldwide olive tree and oil having a double function. In fact, besides being a reference system generated for the identification of unkown olive or virgin olive oil cultivars based on their microsatellite allele size(s), it provides users additional morphological and chemical information for each identified cultivar. Currently, OGDD is designed to enable users to easily retrieve and visualize biologically important information (SSR markers, and olive tree and oil characteristics of about 200 cultivars worldwide) using a set of efficient query interfaces and analysis tools. It can be accessed through a web service from any modern programming language using a simple hypertext transfer protocol call. The web site is implemented in java, JavaScript, PHP, HTML and Apache with all major browsers supported.Database URL: http://www.bioinfo-cbs.org/ogdd/

Estimation of taurindicine hybridization of American Zebu cattle in Brazil

Our objective was to estimate Bos primigenius taurus introgression in American Zebu cattle. One hundred and four American Zebu (Nellore) cattle were submitted to mtDNA, microsatellite and satellite analysis. Twenty-three alleles were detected in microsatellite analysis, averaging 4.6 ± 1.82/locus. Variance component comparisons of microsatellite allele sizes allowed the construction of two clusters separating taurus and indicus. No significant variation was observed when indicus and taurus mtDNA were compared. Three possible genotypes of 1711b satellite DNA were identified. All European animals showed the same restriction pattern, suggesting a Zebu-specific restriction pattern. The frequencies of B. primigenius indicus-specific microsatellite alleles and 1711b satellite DNA restriction patterns lead to an estimate of 14% taurine contribution in purebred Nellore.

Adult survival and microsatellite diversity in possums: effects of major histocompatibility complex-linked microsatellite diversity but not multilocus inbreeding estimators

Adult survival is perhaps the fitness parameter most important to population growth in long-lived species. Intrinsic and extrinsic covariates of survival are therefore likely to be important drivers of population dynamics. We used long-term mark-recapture data to identify genetic, individual and environmental covariates of local survival in a natural population of mountain brushtail possums (Trichosurus cunninghami). Rainfall and intra-individual diversity at microsatellite DNA markers were associated with increased local survival of adults and juveniles. We contrasted the performance of several microsatellite heterozygosity measures, including internal relatedness (IR), homozygosity by loci (HL) and the mean multilocus estimate of the squared difference in microsatellite allele sizes within an individual (mean d (2)). However, the strongest effect on survival was not associated with multilocus microsatellite diversity (which would indicate a genome-wide inbreeding effect), but a subset of two loci. This included a major histocompatibility complex (MHC)-linked marker and a putatively neutral microsatellite locus. For both loci, diversity measures incorporating allele size information had stronger associations with survival than measures based on heterozygosity, whether or not allele frequency information was included (such as IR). Increased survival was apparent among heterozygotes at the MHC-linked locus, but the benefits of heterozygosity to survival were reduced in heterozygotes with larger differences in allele size. The effect of heterozygosity on fitness-related traits was supported by data on endoparasites in a subset of the individuals studied in this population. There was no apparent density dependence in survival, nor an effect of sex, age or immigrant status. Our findings suggest that in the apparent absence of inbreeding, variation at specific loci can generate strong associations between fitness and diversity at linked markers.

Genetic structure and differentiation of Oryza sativa L. in China revealed by microsatellites

China is one of the largest centers of genetic diversity of Oryza sativa L. in the world. Using a genetically representative primary core collection of 3,024 rice landraces in China, we analyzed the genetic structure and intraspecific differentiation of O. sativa, and the directional evolution of SSR. The genetic structure was investigated by model-based structure analysis and construction of neighbor-joining phylogenetic tree. Comparison between genetic structure and predefined populations according to Ting's taxonomic system revealed a hierarchical genetic structure: two distinct subspecies, each with three ecotypes and different numbers of geo-ecogroups within each ecotype. Two subspecies evidently resulted from adaptation to different environments. The different cropping systems imposed on the subspecies led to further differentiation, but the variation within each subspecies resulted from different causes. Indica, under tropical-like or lowland-like environments, exhibited clear differentiation among seasonal ecotypes, but not among soil-watery ecotypes; and japonica showed clear differences between soil water regime ecotypes, but not among seasonal ecotypes. Chinese cultivated rice took on evident directional evolution in microsatellite allele size at several aspects, such as subspecies and geographical populations. Japonica has smaller allele sizes than indica, and this may partly be the result of their different domestication times. Allele size was also negatively correlated with latitude and altitude, and this may be interpreted by different mutation rates, selection pressures, and population size effects under different environments and cropping systems.

MsatAllele_1.0: An R Package to Visualize the Binning of Microsatellite Alleles

MsatAllele is a computer package built on R to visualize and bin the raw microsatellite allele size distributions. The method is based on the creation of an R database with exported files from the open-source electropherogram peak-reading program STRAND. Contrary to other binning programs, in this program, the bin limits are not fixed and are automatically defined by the data stored in the database. Data manipulation and graphical functions allow to 1) visualize raw allele size variation, allowing the detection of potential scoring errors, strange bin distributions, and unexpected spacing between the bins; 2) bin raw fragment sizes and write bin summary statistics for each locus; and 3) export genotype files with the resulting binned data.

Effects of a Topically Applied Bioadhesive Berry Gel on Loss of Heterozygosity Indices in Premalignant Oral Lesions

The aim of this study was to assess the effects of topical application of a 10% (w/w) freeze-dried black raspberry (FBR) gel on oral intraepithelial neoplasia (IEN) variables that included histologic diagnoses and loss of heterozygosity (LOH) indices. Microsatellite instability and/or LOH at tumor suppressor gene-associated chromosomal loci have been associated with a higher risk for oral IEN progression to oral squamous cell carcinoma. Previously, our laboratories have shown that FBRs are well tolerated and possess potent antioxidant, apoptotic, and differentiation-inducing properties. Each participant with IEN served as their own internal control. Before treatment, all lesions were photographed, and lesional tissue was hemisected to obtain a pretreatment diagnosis and baseline biochemical and molecular variables. Gel dosing (0.5 g applied four times daily for 6 weeks) was initiated 1 week after the initial biopsy. Genomic DNA was isolated from laser-captured basilar and suprabasilar surface epithelial cells followed by PCR amplification using primer sets that targeted known and presumed tumor suppressor gene loci associated with INK4a/ARF, p53, and FHIT. Allelic imbalance was determined by sequence analysis using normal participant tissues to establish microsatellite marker peak patterns and allele sizes. Confirming earlier phase I data, none of the 27 participants developed FBR gel-associated toxicities. Furthermore, our results show histologic regression in a subset of patients as well as statistically significant reduction in LOH at tumor suppressor gene-associated loci. These preliminary data suggest that further evaluation of berry gels for oral IEN chemoprevention is warranted.

Genetic structuring of the temperate gorgonian coral (Corallium rubrum) across the western Mediterranean Sea revealed by microsatellites and nuclear sequences

In the past decades, anthropogenic disturbance has increased in marine costal habitats, leading to dramatic shifts in population size structure of various marine species. In the temperate region, the gorgonian coral (Corallium rubrum) is one of the major disturbed species, mostly owing to the exploitation of its red skeleton for jewellery purposes. Red coral is a gonocoric species inhabiting subtidal rocky habitats in the Mediterranean and neighbouring Atlantic coasts. In order to investigate the spatial genetic structuring of C. rubrum, five microsatellite markers and the ribosomal internal transcribed spacer 1 (ITS-1) sequences were analysed in 11 samples from the northwestern Mediterranean Sea. Phylogenetic reconstructions obtained from ITS-1 sequences analysis showed that samples from Minorca and Sardinia islands were the most divergent while the three samples from the Tuscan archipelago (Argentario, Giannutri and Elba) appeared genetically homogeneous. Both markers revealed a strong structuring over large spatial scales (though to a different extent) with no isolation by distance pattern. Microsatellite-based F(ST) estimates appeared much smaller than ITS-based estimates and not significantly correlated, likely due to microsatellite allele size homoplasy typical of these highly polymorphic loci. Our study shows that the absence of clear patterns of genetic structuring over large spatial scales together with strong genetic structuring should be interpreted with caution because such patterns may hide underlying small-scale genetic structuring. Our results further confirm that effective larval dispersal in red coral is highly restricted in the northwestern Mediterranean Sea, suggesting that an increase of anthropogenic disturbance could aggravate the disappearance of red coral, not only along the Mediterranean coasts but also, and with more intensity, in the main Mediterranean islands.

Application of microsatellite fingerprints for pedigree analysis of Hungaricum grapevine varieties

the Carpathian Basin were involved into our examination, which aimed at genotyping their accessions. DNA fingerprints of 101 varieties were determined with 6 microsatellite markers till 2005, resulting in successful discrimination of the accessions. Based on these results for pedigree determination, even more cultivars and primers were involved into the analyses. For studying the origin of Csabagyöngye and for proving the parent-progeny relations of Irsai Olivér and Mátrai muskotály, 19 microsatellite markers were applied, while 11 were selected for tracing the origin of Királyleányka. Genetic distances between the varieties were estimated with cluster analysis and demonstrated by dendrogram, proving that the varieties can be discriminated from each other based on the microsatellite allele sizes. Pedigree of Irsai Olivér and Mátrai muskotály has been confirmed by microsatellite allele size results, searching for the parents of Csabagyöngye and Királyleányka is in progress, since the molecular-marker based pedigree does not correspond with the putative origin of these cultivars. Our results excluded progeny-parent relationships in theCsabagyöngye-Bronnerstraube-Muscat ottonel (Ottonel muskotály) and the Királyleányka-Kövérszőlő combinations.

Loss of genetic variability in reintroduced roach (Rutilus rutilus) populations

Genetic variation at five microsatellite loci and in mtDNA was surveyed in reintroduced and ‘control’ populations of roach (Rutilus rutilus) in Sweden. Microsatellite allelic richness and allele size ranges were significantly reduced in reintroduced populations, and mtDNA diversity was nearly significantly reduced in reintroduced populations. These measures of genetic variability were strongly correlated with lake characteristics that influence population size and food availability.

A Bayesian Heterogeneous Analysis of Variance Approach to Inferring Recent Selective Sweeps

The distribution of microsatellite allele sizes in populations aids in understanding the genetic diversity of species and the evolutionary history of recent selective sweeps. We propose a heterogeneous Bayesian analysis of variance model for inferring loci involved in recent selective sweeps by analyzing the distribution of allele sizes at multiple loci in multiple populations. Our model is shown to be consistent with a multilocus test statistic, ln RV, proposed for identifying microsatellite loci involved in recent selective sweeps. Our methodology differs in that it accepts original allele size data rather than summary statistics and allows the incorporation of prior knowledge about allele frequencies using a hierarchical prior distribution consisting of log normal and gamma probability distributions. Interesting features of the model are its ability to simultaneously analyze allele size data for any number of populations and to cope with the presence of any number of selected loci. The utility of the method is illustrated by application to two sets of microsatellite allele size data for a group of West African Anopheles gambiae populations. The results are consistent with the suppressed-recombination model of speciation, and additional candidate loci on chromosomes 2 (079 and 175) and 3 (088) are discovered that escaped former analysis.

Microsatellite allele sizes: a simple test to assess their significance on genetic differentiation.

The mutation process at microsatellite loci typically occurs at high rates and with stepwise changes in allele sizes, features that may introduce bias when using classical measures of population differentiation based on allele identity (e.g., F(ST), Nei's Ds genetic distance). Allele size-based measures of differentiation, assuming a stepwise mutation process [e.g., Slatkin's R(ST), Goldstein et al.'s (deltamu)(2)], may better reflect differentiation at microsatellite loci, but they suffer high sampling variance. The relative efficiency of allele size- vs. allele identity-based statistics depends on the relative contributions of mutations vs. drift to population differentiation. We present a simple test based on a randomization procedure of allele sizes to determine whether stepwise-like mutations contributed to genetic differentiation. This test can be applied to any microsatellite data set designed to assess population differentiation and can be interpreted as testing whether F(ST) = R(ST). Computer simulations show that the test efficiently identifies which of F(ST) or R(ST) estimates has the lowest mean square error. A significant test, implying that R(ST) performs better than F(ST), is obtained when the mutation rate, mu, for a stepwise mutation process is (a) >/= m in an island model (m being the migration rate among populations) or (b) >/= 1/t in the case of isolated populations (t being the number of generations since population divergence). The test also informs on the efficiency of other statistics used in phylogenetical reconstruction [e.g., Ds and (deltamu)(2)], a nonsignificant test meaning that allele identity-based statistics perform better than allele size-based ones. This test can also provide insights into the evolutionary history of populations, revealing, for example, phylogeographic patterns, as illustrated by applying it on three published data sets.

Spatially restricted gene flow and reduced microsatellite polymorphism in the Eurasian otter Lutra lutra in Britain

Although gene flow and population fragmentationwill often have opposed effects on geneticstructure, their actual effects on many elusiveanimal species are unknown. We assessed sucheffects in British populations of the Eurasianotter Lutra lutra by analysis ofgenotypes consisting of 12 microsatellites from618 carcasses representing the period 1982–1998. Spatial patterns of genetic subdivisionand levels of polymorphism in the continuouspopulation in Scotland were estimated. Theseresults were used to infer patterns of geneflow in Scottish otters and assess theinfluence of fragmentation on the geneticstructure of otters in Wales and SW England.The latter showed no higher genetic divergencethan expected given the degree of isolation bydistance found in the Scottish population, andtheir distributions of microsatellite allelesizes provided no evidence for populationbottlenecks. Nonetheless, otters in southernBritain contained significantly lower levels ofmicrosatellite polymorphism than otters inScotland, and the population in the westernpeninsula of SW England was geneticallydistinct. These results suggested that thegenetic structure of the Scottish population isdue more to restricted contemporary gene flowthan to historical fluctuations insubpopulation size, and that the geneticstructure of the southern British populationsis due more to small historical effective sizesthan to recent declines. If spatiallyrestricted gene flow is typical of all Eurasianotter populations then data on dispersal shouldbe taken into account when sitting protectedareas for this species.

Microsatellite evolution in vertebrates: inference from AC dinucleotide repeats.

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.

Microsatellite variation in solitary and social tuco-tucos: molecular properties and population dynamics.

Variation at 15 microsatellite loci was characterized for a population of the solitary Patagonian tuco-tuco (Ctenomys haigi) and a population of group-living colonial tuco-tuco (C. sociabilis), both of which were located in the Limay River Valley of south western Argentina. All loci examined were characterized by uninterrupted di- or trinucleotide repeats in both species; seven of these loci had been isolated from C. haigi and eight had been isolated from C. sociabilis. Across all loci, there was a significant tendency for both number of alleles and heterozygosity to be greater in C. haigi than in C. sociabilis. Cloning and sequencing of multiple PCR products per locus per population revealed no significant biases in allele length, suggesting that this difference in variability was not due to ascertainment bias or to population-wide differences in rates of microsatellite evolution. Instead, differences in microsatellite variation between C. haigi and C. sociabilis were more consistent with current demographic and suspected historical differences between these populations. In particular, interpopulation differences in the distribution of microsatellite allele sizes and allele frequencies suggested that C. sociabilis has been more subject to historical reductions in population size (e.g. population bottlenecks) than has C. haigi. These findings indicate that analyses of microsatellite structure can yield important insights into the population-level phenomena that contribute to diversity at these markers, including differences in population history that continue to influence levels of genetic variability.

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.

MICROSATELLITE EVOLUTION IN VERTEBRATES: INFERENCE FROM AC DINUCLEOTIDE REPEATS

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.Corresponding Editor: B. Bowen

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.