New methods employing multilocus genotypes to select or exclude populations as origins of individuals

A new method for assigning individuals of unknown origin to populations, based on the genetic distance between individuals and populations, was compared to two existing methods based on the likelihood of multilocus genotypes. The distribution of the assignment criterion (genetic distance or genotype likelihood) for individuals of a given population was used to define the probability that an individual belongs to the population. Using this definition, it becomes possible to exclude a population as the origin of an individual, a useful extension of the currently available assignment methods. Using simulated data based on the coalescent process, the different methods were evaluated, varying the time of divergence of populations, the mutation model, the sample size, and the number of loci. Likelihood-based methods (especially the Bayesian method) always performed better than distance methods. Other things being equal, genetic markers were always more efficient when evolving under the infinite allele model than under the stepwise mutation model, even for equal values of the differentiation parameter F(st). Using the Bayesian method, a 100% correct assignment rate can be achieved by scoring ca. 10 microsatellite loci (H approximately 0.6) on 30-50 individuals from each of 10 populations when the F(st) is near 0.1.     

Global population genetic structure and male-mediated gene flow in the green sea turtle (Chelonia mydas): analysis of microsatellite loci

We assessed the degree of population subdivision among global populations of green sea turtles, Chelonia mydas, using four microsatellite loci. Previously, a single-copy nuclear DNA study indicated significant male-mediated gene flow among populations alternately fixed for different mitochondrial DNA haplotypes and that genetic divergence between populations in the Atlantic and Pacific Oceans was more common than subdivisions among populations within ocean basins. Even so, overall levels of variation at single-copy loci were low and inferences were limited. Here, the markedly more variable microsatellite loci confirm the presence of male-mediated gene flow among populations within ocean basins. This analysis generally confirms the genetic divergence between the Atlantic and Pacific. As with the previous study, phylogenetic analyses of genetic distances based on the microsatellite loci indicate a close genetic relationship among eastern Atlantic and Indian Ocean populations. Unlike the single-copy study, however, the results here cannot be attributed to an artifact of general low variability and likely represent recent or ongoing migration between ocean basins. Sequence analyses of regions flanking the microsatellite repeat reveal considerable amounts of cryptic variation and homoplasy and significantly aid in our understanding of population connectivity. Assessment of the allele frequency distributions indicates that at least some of the loci may not be evolving by the stepwise mutation model.     

Biased mutations and microsatellite variation

Mutation bias is one of the forces that may constrain the variation at microsatellite loci. Here, we study the dynamics of population statistics and the genetic distance between two populations under multiple stepwise mutations with linear bias and random drift. Expressions are derived for these statistics as functions of time, as well as at mutation-drift equilibrium. Applying these expressions to published data on humans and chimpanzees, the regression coefficient of mutation bias on allele size was estimated to be at least between - 0.0064 and -0.013. The assumption of mutational bias produces larger estimates of divergence times than are obtained in its absence; in particular, the time of split between African and non-African human populations is estimated to be between 183,000 and 222,000 years, assuming one-step mutations and no selection. With multistep mutations, the divergence time is estimated to be lower.      

Dissimilarity of individual microsatellite profiles under different mutation models: Empirical approach

Microsatellites (simple sequence repeats, SSRs) still remain popular molecular markers for studying neutral genetic variation. Two alternative models outline how new microsatellite alleles evolve. Infinite alleles model (IAM) assumes that all possible alleles are equally likely to result from a mutation, while stepwise mutation model (SMM) describes microsatellite evolution as stepwise adding or subtracting single repeat units. Genetic relationships between individuals can be analyzed in higher precision when assuming the SMM scenario with allele size differences as a proxy of genetic distance. If population structure is not predetermined in advance, an empirical data analysis usually includes (a) estimating proximity between individual SSR profiles with a selected dissimilarity measure and (b) determining putative genetic structure of a given set of individuals using methods of clustering and/or ordination for the obtained dissimilarity matrix. We developed new dissimilarity indices between SSR profiles of haploid, diploid, or polyploid organisms assuming different mutation models and compared the performance of these indices for determining genetic structure with population data and with simulations. More specifically, we compared SMM with a constant or variable mutation rate at different SSR loci to IAM using data from natural populations of a freshwater bryozoan Cristatella mucedo (diploid), wheat leaf rust Puccinia triticina (dikaryon), and wheat powdery mildew Blumeria graminis (monokaryon). We show that inferences about population genetic structure are sensitive to the assumed mutation model. With simulations, we found that Bruvo's distance performs generally poorly, while the new metrics are capturing the differences in the genetic structure of the populations.     

Constraints on Allele Size at Microsatellite Loci: Implications for Genetic Differentiation

Microsatellites are promising genetic markers for studying the demographic structure and phylogenetic history of populations. We present theoretical arguments indicating that the usefulness of microsatellite data for these purposes may be limited to a short time perspective and to relatively small populations. The evolution of selectively neutral markers is governed by the interaction of mutation and random genetic drift. Mutation pressure has the inherent tendency to shift different populations to the same distribution of alleles. Hence, mutation pressure is a homogenizing force, and population divergence is caused by random genetic drift. In case of allozymes or sequence data, the diversifying effect of drift is typically orders of magnitude larger than the homogenizing effect of mutation pressure. By a simple model, we demonstrate that the situation may be different for microsatellites where mutation rates are high and the range of alleles is limited. With the help of computer simulations, we investigate to what extent genetic distance measures applied to microsatellite data can nevertheless yield useful estimators for phylogenetic relationships or demographic parameters. We show that predictions based on microsatellite data are quite reliable in small populations, but that already in moderately sized populations the danger of misinterpretation is substantial.     

Microsatellite variation among divergent populations of stalk-eyed flies, genus Cyrtodiopsis

Microsatellite primers are often developed in one species and used to assess neutral variability in related species. Such analyses may be confounded by ascertainment bias (i.e. a decline in amplification success and allelic variability with increasing genetic distance from the source of the microsatellites). In addition, other factors, such as the size of the microsatellite, whether it consists of perfect or interrupted tandem repeats, and whether it is autosomal or X-linked, can affect variation. To test the relative importance of these factors on microsatellite variation, we examine patterns of amplification and allelic diversity in 52 microsatellite loci amplified from five individuals in each of six populations of Cyrtodiopsis stalk-eyed flies that range from 2.2 % to 11.2% mitochondrial DNA sequence divergence from the population used for microsatellite development. We find that amplification success and most measures of allelic diversity declined with genetic distance from the source population, in some cases an order of magnitude faster than in birds or mammals. The median and range of the repeat array length did not decline with genetic distance. In addition, for loci on the X chromosome, we find evidence of lower observed heterozygosity compared with loci on autosomes. The differences in variability between X-linked and autosomal loci are not adequately explained by differences in effective population sizes of the chromosomes. We suggest, instead, that periodic selection events associated with X-chromosome meiotic drive, which is present in many of these populations, reduces X-linked variation.     

Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Rapid genetic divergence in postglacial populations of threespine stickleback (Gasterosteus aculeatus): the role of habitat type, drainage and geographical proximity

The goal of this study was to evaluate the role of common ancestry, and of geographical or reproductive isolation, in genetic divergence in populations of threespine sticklebacks (Gasterosteus aculeatus). Using seven DNA microsatellite loci we compared the effects of habitat type, drainage system and geographical proximity on genetic distance among 16 populations situated in an area in Schleswig-Holstein (Germany) that became deglaciated approximately 12 000 years ago. Stickleback population structure correlated only weakly with drainage system, whereas the primary divergence was among habitat types. Phylogenetic analysis revealed that lake (n = 7) and river (n = 5) populations formed two distinct clades (Cavalli-Sforza's and Edwards' chord distance, 82-100% bootstrap support) at approximately equal genetic distances to a third clade, comprising putative estuarine (n = 4) ancestors. Allele frequencies in lake and river populations represented different subsets of the genetically more diverse estuarine populations. In nested amovas approximately twice the genetic variance was distributed among lake vs. river vs. estuarine populations as compared with the combined effects of drainage system and geographical distance. Limited gene flow between habitat types must have been established after postglacial colonization, suggesting ecological hybrid inferiority or behavioural mating barriers between ecotypes. Within estuarine and lake populations, population differentiation followed an isolation-by-distance model. Given the high observed heterozygosities within the 16 study populations (HO = 0.65-0.87), the mean divergence between lake and river population pairs (FST = 0.18 +/- 0.007) would be reached after 300-6000 generations in a stepwise mutation model, depending on the size of N(e). This demonstrates both the utility of hypervariable microsatellites for detecting recent population divergences and the danger of operating at temporal or spatial scales which are beyond their resolution.     

Can clone size serve as a proxy for clone age?An exploration using microsatellite divergence in Populus tremuloides

In long‐lived clonal plants, the overall size of a clone is often used to estimate clone age. The size of a clone, however, might be largely determined by physical or biotic interactions, obscuring the relationship between clone size and age. Here, we use the accumulation of mutations at 14 microsatellite loci to estimate clone age in trembling aspen (Populus tremuloides) from southwestern Canada. We show that the observed patterns of genetic divergence are consistent with a model of increasing ramet population size, allowing us to use pairwise genetic divergence as an estimator of clone age. In the populations studied, clone size did not exhibit a significant relationship with microsatellite divergence, indicating that clone size is not a good proxy for clone age. In P. tremuloides, the per‐locus per‐year neutral somatic mutation rate across 14 microsatellite loci was estimated to lie between 6 × 10^?7 (lower bound) and 4 × 10^?5 (upper bound).     

Independent evolutionary origins of landlocked alewife populations and rapid parallel evolution of phenotypic traits

Alewife, Alosa pseudoharengus, populations occur in two discrete life-history variants, an anadromous form and a landlocked (freshwater resident) form. Landlocked populations display a consistent pattern of life-history divergence from anadromous populations, including earlier age at maturity, smaller adult body size, and reduced fecundity. In Connecticut (USA), dams constructed on coastal streams separate anadromous spawning runs from lake-resident landlocked populations. Here, we used sequence data from the mtDNA control region and allele frequency data from five microsatellite loci to ask whether coastal Connecticut landlocked alewife populations are independently evolved from anadromous populations or whether they share a common freshwater ancestor. We then used microsatellite data to estimate the timing of the divergence between anadromous and landlocked populations. Finally, we examined anadromous and landlocked populations for divergence in foraging morphology and used divergence time estimates to calculate the rate of evolution for foraging traits. Our results indicate that landlocked populations have evolved multiple times independently. Tests of population divergence and estimates of gene flow show that landlocked populations are genetically isolated, whereas anadromous populations exchange genes. These results support a 'phylogenetic raceme' model of landlocked alewife divergence, with anadromous populations forming an ancestral core from which landlocked populations independently diverged. Divergence time estimates suggest that landlocked populations diverged from a common anadromous ancestor no longer than 5000 years ago and perhaps as recently as 300 years ago, depending on the microsatellite mutation rate assumed. Examination of foraging traits reveals landlocked populations to have significantly narrower gapes and smaller gill raker spacings than anadromous populations, suggesting that they are adapted to foraging on smaller prey items. Estimates of evolutionary rates (in haldanes) indicate rapid evolution of foraging traits, possibly in response to changes in available resources.     

Influence of mutational and sampling factors on the estimation of demographic parameters in a "continuous" population under isolation by distance

In numerous species, individual dispersal is restricted in space so that "continuous" populations evolve under isolation by distance. A method based on individual genotypes assuming a lattice population model was recently developed to estimate the product Dsigma2, where D is the population density and sigma2 is the average squared parent-offspring distance. We evaluated the influence on this method of both mutation rate and mutation model, with a particular reference to microsatellite markers, as well as that of the spatial scale of sampling. Moreover, we developed and tested a nonparametric bootstrap procedure allowing the construction of confidence intervals for the estimation of Dsigma2. These two objectives prompted us to develop a computer simulation algorithm based on the coalescent theory giving individual genotypes for a continuous population under isolation by distance. Our results show that the characteristics of mutational processes at microsatellite loci, namely the allele size homoplasy generated by stepwise mutations, constraints on allele size, and change of slippage rate with repeat number, have little influence on the estimation of Dsigma2. In contrast, a high genetic diversity (approximately 0.7-0.8), as is commonly observed for microsatellite markers, substantially increases the precision of the estimation. However, very high levels of genetic diversity (>0.85) were found to bias the estimation. We also show that statistics taking into account allele size differences give unreliable estimations (i.e., high variance of Dsigma2 estimation) even under a strict stepwise mutation model. Finally, although we show that this method is reasonably robust with respect to the sampling scale, sampling individuals at a local geographical scale gives more precise estimations of Dsigma2.     

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.     

Genetic divergence disclosing a rapid prehistorical dispersion of native americans in central and South america

An accurate estimate of the divergence time between Native Americans is important for understanding the initial entry and early dispersion of human beings in the New World. Current methods for estimating the genetic divergence time of populations could seriously depart from a linear relationship with the true divergence for multiple populations of a different population size and significant population expansion. Here, to address this problem, we propose a novel measure to estimate the genetic divergence time of populations. Computer simulation revealed that the new measure maintained an excellent linear correlation with the population divergence time in complicated multi-population scenarios with population expansion. Utilizing the new measure and microsatellite data of 21 Native American populations, we investigated the genetic divergences of the Native American populations. The results indicated that genetic divergences between North American populations are greater than that between Central and South American populations. None of the divergences, however, were large enough to constitute convincing evidence supporting the two-wave or multi-wave migration model for the initial entry of human beings into America. The genetic affinity of the Native American populations was further explored using Neighbor-Net and the genetic divergences suggested that these populations could be categorized into four genetic groups living in four different ecologic zones. The divergence of the population groups suggests that the early dispersion of human beings in America was a multi-step procedure. Further, the divergences suggest the rapid dispersion of Native Americans in Central and South Americas after a long standstill period in North America.     

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.     

Evolutionary and statistical properties of three genetic distances

Many genetic distances have been developed to summarize allele frequency differences between populations. I review the evolutionary and statistical properties of three popular genetic distances: DS, DA, and theta;, using computer simulation of two simple evolutionary histories: an isolation model of population divergence and an equilibrium migration model. The effect of effective population size, mutation rate, and mutation mechanism upon the parametric value between pairs of populations in these models explored, and the unique properties of each distance are described. The effect of these evolutionary parameters on study design is also investigated and similar results are found for each genetic distance in each model of evolution: large sample sizes are warranted when populations are relatively genetically similar; and loci with more alleles produce better estimates of genetic distance.     

Spatial and population genetic structure of microsatellites in white pine

We evaluated the population genetic structure of seven microsatellite loci for old growth and second growth populations of eastern white pine (Pinus strobus). From each population, located within Hartwick Pines State Park, Grayling, Michigan, USA, 120-122 contiguous trees were sampled for genetic analysis. Within each population, genetic diversity was high and inbreeding low. When comparing these populations, there is a significant, but small (less than 1%), genetic divergence between populations. Spatial distance between populations or timber harvest at the second growth site were reasonable explanations for the observed minor differences in allele frequencies between populations. Spatial autocorrelation analysis suggested that, for the old growth population, weak positive structuring at 15 m fits the isolation by distance model for a neighbourhood size of about 100 individuals. In comparison, genotypes were randomly distributed in the second growth population. Thus, logging may have decreased spatial structuring at the second growth site, suggesting that management practices may be used to alter natural spatial patterns. In addition, the amount of autocorrelation in the old growth population appears to be lower for some of the microsatellites, suggesting higher numbers of rare alleles and that higher mutation rates may have directly affected spatial statistics by reducing structure.     

Microsatellite Variation in Two Populations of Free-Ranging Yellow Baboons (Papio hamadryas cynocephalus)

We investigated genetic variation at six microsatellite (simple sequence repeat) loci in yellow baboons (Papio hamadryas cynocephalus) at two localities: the Tana River Primate Reserve in eastern Kenya and Mikumi National Park, central Tanzania. The six loci (D1S158, D2S144, D4S243, D5S1466, D16S508, and D17S804) were all originally cloned from and characterized in the human genome. These microsatellites are polymorphic in both baboon populations, with the average heterozygosity across loci equal to 0.731 in the Tana River sample and 0.787 in the Mikumi sample. The genetic differentiation between the two populations is substantial. Kolmogornov–Smirnov tests indicate that five of the six loci are significantly different in allele frequencies in the two populations. The mean F _ST across loci is 0.069, and Shriver's measure of genetic distance, which was developed for microsatellite loci (Shriver et al., 1995), is 0.255. This genetic distance is larger than corresponding distances among human populations residing in different continents. We conclude that (a) the arrays of alleles present at these six microsatellite loci in two geographically separated populations of yellow baboons are quite similar, but (b) the two populations exhibit significant differences in allele frequencies. This study illustrates the potential value of human microsatellite loci for analyses of population genetic structure in baboons and suggests that this approach will be useful in studies of other Old World monkeys.     

Genetic evidence of recent population contraction in the southernmost population of giant pandas

Anthropogenic habitat loss and fragmentation have been implicated in the endangerment and extinction of many species. Here we assess genetic variation and demographic history in the southernmost population of giant pandas (Ailuropoda melanoleuca) that continues to be threatened by habitat degradation and fragmentation, using noninvasive genetic sampling, mitochondrial control region sequence and 12 microsatellite loci. Compared to other giant panda populations, this population has medium-level genetic diversity based on the measure of both mitochondrial and nuclear markers. Mitochondrial DNA-based demographic analyses revealed that no historical population expansion or contraction has occurred, indicating a relatively stable population size. However, a Bayesian-coalescent method based on the observed allele distribution and allele frequencies of microsatellite clearly did detect, quantify and date a recent decrease in population size. Overall, the results indicate that a population contraction in the order of 95-96% has taken place over the last 910-999 years and is most likely due to anthropogenic habitat loss. These findings highlight the need for a greater focus on habitat protection and restoration for the long-term survival of this giant panda population.     

Comparison of genetic diversity at microsatellite loci and quantitative traits in hatchery populations of Japanese flounder Paralichthys olivaceus

Relationships of genetic diversity at microsatellite loci and quantitative traits were examined in hatchery-produced populations of Japanese flounder using a relatively straightforward experiment. Five hatchery populations produced by wild-caught and domesticated broodstocks were used to examine the effects of different levels (one to three generations) of domestication on the genetic characteristics of hatchery populations. Allelic richness at seven microsatellite loci in all hatchery populations was lower than that in a wild population. Genetic variation measured by allelic richness and heterozygosity tended to decrease with an increase in generations of domestication. In addition, the degree of genetic differentiation from a wild population increased with an increase in generations of domestication. Significant differences in three morphometric traits (dorsal and anal fin ray counts and vertebral counts) and three physiological traits (high temperature, salinity and formalin tolerance) were observed among the hatchery populations. The degree of phenotypic difference among populations was larger in morphometric traits than in physiological traits. The divergence pattern of some quantitative traits was similar to that observed at microsatellite loci, suggesting that domestication causes the decrease of genetic variation and the increase of genetic differentiation for some quantitative traits concomitantly with those for microsatellite loci. Significant positive correlation was observed between F _ST and the degree of phenotypic difference in the three morphometric traits and formalin tolerance, indicating that genetic variation at microsatellite loci predicts the degree of phenotypic divergence in some quantitative traits.     

THE INFLUENCE OF SELF-FERTILIZATION AND POPULATION DYNAMICS ON THE GENETIC STRUCTURE OF SUBDIVIDED POPULATIONS: A CASE STUDY USING MICROSATELLITE MARKERS IN THE FRESHWATER SNAIL BULINUS TRUNCATUS

The distribution of neutral genetic variability within and among sets of populations results from the combined actions of genetic drift, migration, extinction and recolonization processes, mutation, and the mating system. We here analyzed these factors in 38 populations of the hermaphroditic snail Bulinus truncatus. The sampling area covered a large part of the species range. The variability was analyzed using four polymorphic microsatellite loci. A very large number of alleles (up to 55) was found at the level of the whole study. Observed heterozygote deficiencies within populations are consistent with very high selfing rates, generally above 0.80, in all populations. These should depress the variability within populations, because of low effective size, genetic hitchhiking, and background selection, whatever the model of mutation assumed. However, that some populations exhibit much more variability than others suggests that historical demographic processes (e.g., population size variation, bottlenecks, or founding events) may play a significant role. A hierarchical analysis of the distribution of the variability across populations indicates a strong pattern of isolation by distance, whatever the geographical scale considered. Our analysis also illustrates how the mutation rate may affect population differentiation, as different mutation rates result in different levels of homoplasy at microsatellite loci. The effects of both genetic drift and gene flow vary with the temporal and spatial scales considered in B. truncatus populations.