Microsatellites and the Genetics of Highly Selfing Populations in the Freshwater Snail Bulinus Truncatus

Hermaphrodite tropical freshwater snails provide a good opportunity to study the effects of mating system and genetic drift on population genetic structure because they are self-fertile and they occupy transient patchily distributed habitats (ponds). Up to now the lack of detectable allozyme polymorphism prevented any intrapopulation studies. In this paper, we examine the consequences of selfing and bottlenecks on genetic polymorphism using microsatellite markers in 14 natural populations (under a hierarchical sampling design) of the hermaphrodite freshwater snail Bulinus truncatus. These population genetics data allowed us to discuss the currently available mutation models for microsatellite sequences. Microsatellite markers revealed an unexpectedly high levels of genetic variation with <=41 alleles for one locus and gene diversity of 0.20-0.75 among populations. The values of any estimator of F(is) indicate high selfing rates in all populations. Linkage disequilibria observed at all loci for some populations may also indicate high levels of inbreeding. The large extent of genetic differentiation measured by F(st), R(st) or by a test for homogeneity between genic distributions is explained by both selfing and bottlenecks. Despite a limited gene flow, migration events could be detected when comparing different populations within ponds.     

Population genetic structure of wild Phaseolus lunatus (Fabaceae), with special reference to population sizes

To set up an in situ conservation strategy for Phaseolus lunatus, we analyzed the genetic structure of 29 populations in the Central Valley of Costa Rica. Using 22 enzyme loci, we quantified the proportion of polymorphic loci (P(p)), the mean number of alleles per locus (A), and the mean effective number of alleles per locus (A(e)), which equaled to 10.32%, 1.10, and 1.05, respectively. The total heterozygosity (H(T)), the intrapopulation genetic diversity (H(S)), and the interpopulation genetic diversity (D(ST)) were 0.193, 0.082, and 0.111, respectively. The genotypic composition of the analyzed populations showed a deviation from the Hardy-Weinberg proportions (F(IT) = 0.932). This disequilibrium was due to either genetic differentiation between populations (F(ST) = 0.497) or nonrandom mating within populations (F(IS) = 0.866). From the level of genetic differentiation between populations and the private alleles frequencies estimates, gene flow was calculated: Nm(W) = 0.398 and Nm(S) = 0.023, respectively. The results suggested that wild Lima bean maintains most of its isozyme variation among populations. Significant positive correlation was observed between population size and P(p), A, and H(o) (observed heterozygosity), whereas no correlation was observed with the average fixation index of population (F). The loss of genetic variability in populations was attributed to inbreeding and the bottleneck effects that characterized the target populations. In situ conservation and management procedures for wild Lima bean are discussed.     

Simultaneous estimation of all the parameters of a stepwise mutation model.

Minisatellite and microsatellite are short tandemly repetitive sequences dispersed in eukaryotic genomes, many of which are highly polymorphic due to copy number variation of the repeats. Because mutation changes copy numbers of the repeat sequences in a generalized stepwise fashion, stepwise mutation models are widely used for studying the dynamics of these loci. We propose a minimum chi-square (MCS) method for simultaneous estimation of all the parameters in a stepwise mutation model and the ancestral allelic type of a sample. The MCS estimator requires knowing the mean number of alleles of a certain size in a sample, which can be estimated using Monte Carlo samples generated by a coalescent algorithm. The method is applied to samples of seven (CA)n repeat loci from eight human populations and one chimpanzee population. The estimated values of parameters suggest that there is a general tendency for microsatellite alleles to expand in size, because (1) each mutation has a slight tendency to cause size increase and (2) the mean size increase is larger than the mean size decrease for a mutation. Our estimates also suggest that most of these CA-repeat loci evolve according to multistep mutation models rather than single-step mutation models. We also introduced several quantities for measuring the quality of the estimation of ancestral allelic type, and it appears that the majority of the estimated ancestral allelic types are reasonably accurate. Implications of our analysis and potential extensions of the method are discussed.SINCE the discovery that a large number of loci with tandemly repeated sequences in human and many eukaryote species are highly polymorphic because of copy number variation of the repeats in different individuals (Jeffreys 1985; Litt and Luty 1989; Weber and May 1989), allele size data from such loci are rapidly becoming the dominant source of genetic markers for genome mapping, forensic testing, and population studies. Loci with repeat sequences longer than 5 bp are generally referred to as minisatellite or variable number tandem repeat loci, and those with repeat sequences between 2 to 5 bp are referred to as microsatellite or short tandem repeat loci (Tautz 1993). Because mutations change the copy number of such loci in a stepwise fashion, rapid accumulation of population samples from minisatellite and microsatellite loci has resurrected the interest of the stepwise mutation model (SMM), which was popular in the 1970s.     

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.     

Variation after a selective sweep in a subdivided population

The effect of genetic hitchhiking on neutral variation is analyzed in subdivided populations with differentiated demes. After fixation of a favorable mutation, the consequences on particular subpopulations can be radically different. In the subpopulation where the mutation first appeared by mutation, variation at linked neutral loci is expected to be reduced, as predicted by the classical theory. However, the effect in the other subpopulations, where the mutation is introduced by migration, can be the opposite. This effect depends on the level of genetic differentiation of the subpopulations, the selective advantage of the mutation, the recombination frequency, and the population size, as stated by analytical derivations and computer simulations. The characteristic outcomes of the effect are three. First, the genomic region of reduced variation around the selected locus is smaller than that predicted in a panmictic population. Second, for more distant neutral loci, the amount of variation increases over the level they had before the hitchhiking event. Third, for these loci, the spectrum of gene frequencies is dominated by an excess of alleles at intermediate frequencies when compared with the neutral theory. At these loci, hitchhiking works like a system that takes variation from the between-subpopulation component and introduces it into the subpopulations. The mechanism can also operate in other systems in which the genetic variation is distributed in clusters with limited exchange of variation, such as chromosome arrangements or genomic regions closely linked to targets of balancing selection.     

GENETIC VARIABILITY OF GELIDIUM CANARIENSIS (RHODOPHYTA) DETERMINED BY ISOZYME ELECTROPHORESIS1

The populations of Gelidium canariensis (Grunow) Seoane-Camba from the Canary Islands were analyzed for genetic variability by isozyme electrophoresis in 1989 and 1990. Each population was divided into sporophytic and gametophytic subpopulations. Twenty-three to 27 putative alleles corresponding to 22 gene loci were analyzed. Sev-enteen loci were monomorphic in all six subpopulations, and five were polymorphic in at least one subpopulation. Significant deviations from Hardy-Weinberg equilibrium were found. The amount of genetic variability (percentage of polymorphic loci, mean number of alleles per locus, and average gene diversity) of haploid subpopulations was lower than that of diploid subpopulations. No correlation between genetic distance and geographical distance was found. Low genetic differentiation between sporophytic and gametophytic subpopulations of the same locality was obsewed in two populations. The low genetic diversity and genetic differentiation suggest that the genetic structure of the populations of G. canariensis from the Canary Islands is due to a combination of founder effects and the predominance of asexual reproduction. Initial differences in gene frequencies may have persisted because of insufficient time to reach a higher level of differentiation.     

Genetic diversity within and between natural populations of Rattus norvegicus

The levels of gene diversity for 17 polymorphic loci in natural populations of wild rats were examined for three separate locations in North and South America. The level of gene diversity in the total sample for the RT1.A locus, the dominant class I histocompatibility locus in the major histocompatibility (RT1) complex of the rat, was 0.807. The degree of gene diversity for nonalloantigenic loci scattered throughout the rat genome was 0.215, a level comparable to, if not slightly higher than, that for other mammalian species. The large and consistent levels of diversity for individuals within each population suggest that significant deviations from random mating have occurred within each group. Conclusions from analyzing genetic distance and the index of genetic differentiation between the three populations are consistent with these populations' geographic isolation and small effective population size. Assuming that the separation of the North and South American groups has existed for approximately 300 years, the effective size of these populations is estimated to be approximately 1,500 individuals. Apparent differences in the distribution of the number and frequency of alleles in the major histocompatibility complexes of mice and rats and the level of genetic differentiation among separate rat populations may be due to the effects of genetic drift in small populations.     

Minisatellite mutational processes reduce Fst estimates

We have used a new method for binning minisatellite alleles (semi-automated allele aggregation) and report the extent of population diversity detectable by eleven minisatellite loci in 2,689 individuals from 19 human populations distributed widely throughout the world. Whereas population relationships are consistent with those found in other studies, our estimate of genetic differentiation (F(st)) between populations is less than 8%, which is lower than comparative estimates of between 10%-15% obtained by using other sources of polymorphism data. We infer that mutational processes are involved in reducing F(st) estimates from minisatellite data because, first, the lowest F(st) estimates are found at loci showing autocorrelated frequencies among alleles of similar size and, second, F(st) declines with heterozygosity but by more than predicted assuming simple models of mutation. These conclusions are consistent with the view that minisatellites are subject to selective or mutational constraints in addition to those expected under simple step-wise mutation models.     

Analysis of linkage disequilibrium in an island model

Linkage disequilibria for two loci in a finite island model were parameterized. The total linkage disequilibrium was decomposed into three components, gametic, demic, and population, for which corresponding unbiased estimators were established. Other statistics encountered provided measures of differentiation corresponding to the hierarchical structure of the ecological model. Under the assumption of linkage equilibrium, the variances and covariances of these estimators and statistics were formulated in terms of descent measures, functions of gene frequencies, and the numbers of individuals, demes, and populations sampled. The functions of gene frequencies fall into two classes, one representing the differentiation of genes at each locus, and the other representing the association of genes between the loci. For a neutral model with extinction, migration, and linkage, transition equations were derived for the descent measures which also take into account deme size and numbers of dems within the population. With the addition of unequal mutation rates for a finite number of alleles at each locus, the transition equations were solved for the descent measures in the equilibrium state. This permitted the exact numerical evaluation of the effects of the sampling and ecological dimensions and of extinction, migration, and mutation rates in any parameter range. Some numerical results were presented for the effects of linkage, extinction, migration, and sampling on the variances of various measures of linkage disequilibrium and genetic differentiation. Also, some results were compared with the approximate numerical results of Ohta which agreed fairly well in the parameter ranges she considered, but not so well in other ranges.     

Selection of high heterozygosity popcorn varieties in Brazil based on SSR markers

We analyzed genetic structure and diversity among eight populations of popcorn, using SSR loci as genetic markers. Our objectives were to select SSR loci that could be used to estimate genetic diversity within popcorn populations, and to analyze the genetic structure of promising populations with high levels of heterozygosity that could be used in breeding programs. Fifty-seven alleles (3.7 alleles per locus) were detected; the highest effective number of alleles (4.21) and the highest gene diversity (0.763) were found for the Umc2226 locus. A very high level of population differentiation was found (F(ST) = 0.3664), with F(ST) for each locus ranging from 0.1029 (Umc1664) to 0.6010 (Umc2350). This analysis allowed us to identify SSR loci with high levels of heterozygosity and heterozygous varieties, which could be selected for production of inbred lines and for developing new cultivars.     

Large scale population structure and gene flow in the planktonic developing periwinkle, Littorina striata, in Macaronesia (Mollusca: Gastropoda)

Allozymes were used to investigate the genetic structure of 42 populations of the planktonic developing, Macaronesian periwinkle Littorina striata, throughout its entire geographic range (Azores, Madeira, Canary Islands and Cape Verde Islands). This periwinkle is presumed to have a high dispersal and gene flow potential, because it has a planktonic development. It is therefore expected to show little population genetic differentiation. Indeed, based on Wright's hierarchical F-statistics, no significant genetic differentiation could be detected among populations, at any of the specified hierarchical levels (i.e. population, island, and archipelago). Nevertheless, the Cape Verde Islands seemed genetically more diverse (highest mean number of alleles per locus). The number of loci revealing a significant genetic heterogeneity increased with increasing distance between populations, while private alleles based gene flow (Nm) estimates also revealed a tendency towards a geographical pattern. The distribution of rare and private alleles, might account for these observations which suggested some geographical effect. Because of the low frequency at which these alleles occur, their influence on the genetic population structure is negligible, and not picked up by F-statistics.     

A vectorized method of importance sampling with applications to models of mutation and migration

An importance-sampling method is presented for computing the likelihood of the configuration of population genetic data under general assumptions about population history and transitions among states. The configuration of the data is the number of chromosomes sampled that are in each of a finite set of states. Transitions among states are governed by a Markov chain with transition probabilities dependent on one or more parameters. The method assumes that the joint distribution of coalescence times of the underlying gene genealogy is independent of the genetic state of each lineage. Given a set of coalescence times, the probability that a pair of lineages is chosen to coalesce in each replicate is proportional to the contribution that the coalescence event makes to the probability of the data. This method can be applied to gene genealogies generated by the neutral coalescent process and to genealogies generated by other processes, such as a linear birth-death process which provides a good approximation to the dynamics of low-frequency alleles. Two applications are described. In the first, the fit of allele frequencies at two microsatellite loci sampled in a Sardinian population to the one-step mutation model is tested. The one-step model is rejected for one locus but not for the other. The second application is to low-frequency alleles in a geographically subdivided population. The geographic location is the allelic state, and the alleles are assumed to be sufficiently rare that their dynamics can be approximated by a linear birth-death process in which the birth and death rates are independent of geographic location. The analysis of eight low-frequency allozyme alleles found in the glaucous-winged gull, Larus glaucescens, illustrates how geographically restricted dispersal can be detected.     

The effect of linkage and population size on inbreeding depression due to mutational load.

Using a stochastic model of a finite population in which there is mutation to partially recessive detrimental alleles at many loci, we study the effects of population size and linkage between the loci on the population mean fitness and inbreeding depression values. Although linkage between the selected loci decreases the amount of inbreeding depression, neither population size nor recombination rate have strong effects on these quantities, unless extremely small values are assumed. We also investigate how partial linkage between the loci that determine fitness affects the invasion of populations by alleles at a modifier locus that controls the selfing rate. In most of the cases studied, the direction of selection on modifiers was consistent with that found in our previous deterministic calculations. However, there was some evidence that linkage between the modifier locus and the selected loci makes outcrossing less likely to evolve; more losses of alleles promoting outcrossing occurred in runs with linkage than in runs with free recombination. We also studied the fate of neutral alleles introduced into populations carrying detrimental mutations. The times to loss of neutral alleles introduced at low frequency were shorter than those predicted for alleles in the absence of selected loci, taking into account the reduction of the effective population size due to inbreeding. Previous studies have been confined to outbreeding populations, and to alleles at frequencies close to one-half, and have found an effect in the opposite direction. It therefore appears that associations between neutral and selected loci may produce effects that differ according to the initial frequencies of the neutral alleles.     

Effect of mutation on genetic differentiation among nonequilibrium populations

The usefulness of GST and similar measures of genetic differentiation has been questioned repeatedly because of their dependence on the amount of heterozygosity within populations, creating problems when comparing degrees of divergence at loci with different mutation rates. Although the effect of mutation on GST is expected to be small in the early phases of divergence, it is unclear for how long after separation from a common ancestral population that GST is largely unaffected by mutation and by the resulting effect on heterozygosity. We address this question through analysis of the recursion equations for gene identity under the infinite allele model of mutation, and derive conditions describing when the effect of mutation on GST can be ignored under mutation-migration-drift equilibrium conditions and during the preceding transition phase. An important result is that during the transition phase GST is not only affected by mutation, but also by the heterozygosity in the base population from which the subpopulations diverged. The effect of mutation on GST is significant from the very start of the divergence process when initial heterozygosity is low, whereas GST is only weakly affected by mutation in the early phases of differentiation when initial heterozygosity is high. Thus, differentiation following a severe bottleneck is strongly dependent on mutation. The standardized measure of differentiation, G'ST, suggested by Hedrick (2005), may be helpful when comparing amounts of divergence at loci with different mutation rates under steady-state conditions, provided that migration is very low. In many other situations the use of G'ST might be misleading, however, and its application should be exercised with caution.     

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.     

小毛茛居群的遗传分化及其与空间隔离的相关性

采用聚丙烯酰胺凝胶电泳技术,对分布于华中地区的11个小毛茛（Ranunculus ternatus）居群的遗传分化进行了检测。对8个酶系统17个酶位点上的分析结果表明,该种各居群 的各项遗传多样性指标处于一个相对较低的水平：多态位点比率（P）为0～53.0%,平均每位点等位基因数（A）为1～1.647,平均预期杂合度（H_e）和观察杂合度（H_o）分别为0～0.108和0～0.102。居群间遗传一致度甚高（I＝0.9754～0.9991）。根据Nei′s遗传距离所作出的聚类分析表明,豫南信阳地区3个居群与湖北省武汉地区8 个居群之间关系较远。而在武汉地区,长江以北的居群及长江以南的部分居群分别相聚在一 起。用GPS定位方法得到居群间空间距离并据此聚类,结果显示了该种的遗传分化与地理因 素 的相关性,并推测出长江的隔离作用加强了两岸居群间的遗传分化。同时发现一个生于独特 生境的居群在表型和遗传结构上都已与其他邻近居群有了很大分异,由于该居群在所检测的 酶位点上均无特有等位基因出现,作者认为不宜将其作为新种或新变种处理。     

Likelihood analysis of ongoing gene flow and historical association

Abstract.— We develop a Monte Carlo-based likelihood method for estimating migration rates and population divergence times from data at unlinked loci at which mutation rates are sufficiently low that, in the recent past, the effects of mutation can be ignored. The method is applicable to restriction fragment length polymorphisms (RFLPs) and single nucleotide polymorphisms (SNPs) sampled from a subdivided population. The method produces joint maximum-likelihood estimates of the migration rate and the time of population divergence, both scaled by population size, and provides a framework in which to test either for no ongoing gene flow or for population divergence in the distant past. We show the method performs well and provides reasonably accurate estimates of parameters even when the assumptions under which those estimates are obtained are not completely satisfied. Furthermore, we show that, provided that the number of polymorphic loci is sufficiently large, there is some power to distinguish between ongoing gene flow and historical association as causes of genetic similarity between pairs of populations.     

Genetic diversity and differentiation of guanaco populations from Argentina inferred from microsatellite data

Genotype data from 14 microsatellite markers were used to assess the genetic diversity and differentiation of four guanaco populations from Argentine Patagonia. These animals were recently captured in the wild and maintained in semi-captivity for fibre production. Considerable genetic diversity in these populations was suggested by the finding of a total of 162 alleles, an average mean number of alleles per locus ranging from 6.50 to 8.19, and H(e) values ranging from 0.66 to 0.74. Assessment of population differentiation showed moderate but significant values of F(ST)=0.071 (P=0.000) and R(ST)=0.083 (P=0.000). An amova test showed that the genetic variation among populations was 5.6% while within populations it was 94.4%. A number of 6.6 migrants per generation may support these results. Unambiguous individual assignment to original populations was obtained for the Pilcaniyeu, Las Heras and La Esperanza populations. The erroneous assignment of 18.75% Rio Mayo individuals to the Las Heras population can be explained by the low genetic differentiation found between these two populations. Thirty-nine of 56 loci per population combinations were in Hardy--Weinberg disequilibrium because of guanaco heterozygote deficiency, which may be explained by population subdivision. The high level of genetic diversity of the guanacos analysed here indicates that the Patagonian guanaco constitutes an important genetic resource for conservation or economic utilization programmes.     

The effect of recurrent floods on genetic composition of marble trout populations

A changing global climate can threaten the diversity of species and ecosystems. We explore the consequences of catastrophic disturbances in determining the evolutionary and demographic histories of secluded marble trout populations in Slovenian streams subjected to weather extremes, in particular recurrent flash floods and debris flows causing massive mortalities. Using microsatellite data, a pattern of extreme genetic differentiation was found among populations (global F(ST) of 0.716), which exceeds the highest values reported in freshwater fish. All locations showed low levels of genetic diversity as evidenced by low heterozygosities and a mean of only 2 alleles per locus, with few or no rare alleles. Many loci showed a discontinuous allele distribution, with missing alleles across the allele size range, suggestive of a population contraction. Accordingly, bottleneck episodes were inferred for all samples with a reduction in population size of 3-4 orders of magnitude. The reduced level of genetic diversity observed in all populations implies a strong impact of genetic drift, and suggests that along with limited gene flow, genetic differentiation might have been exacerbated by recurrent mortalities likely caused by flash flood and debris flows. Due to its low evolutionary potential the species might fail to cope with an intensification and altered frequency of flash flood events predicted to occur with climate change.     

The apportionment of dinucleotide repeat diversity in Native Americans and Europeans: a new approach to measuring gene identity reveals asymmetric patterns of divergence

The purpose of this paper is to assess the extent of gene identity and differentiation at 33 dinucleotide repeat loci (377 total alleles) within and among three European and three Native American populations. In order to do this, we show that a maximum-likelihood method proposed for phylogenetic trees (Cavalli-Sforza and Piazza 1975) can be used to estimate gene identity (Nei 1987) with respect to any hierarchical structure. This method allows gene differentiation to be evaluated with respect to any internal node of a hierarchy. It also allows a generalization of F- and G-statistics to situations with unequal expected levels of differentiation. Our principal finding is that levels of genetic differentiation are unique to specific populations and levels of nesting. The populations of European origin show very little internal differentiation; moreover, their continental average is close to the total population defined by the aggregate of Europeans and Native Americans. By contrast, the Native American populations show moderate levels of internal differentiation, and a great distance between their continental average and the total. The results of analyses of subsets of loci that were selected to have high gene diversities in either Europeans or Native Americans closely parallel those from the total set of loci. This suggests that the principal results are unlikely to be caused by a European ascertainment bias in locus selection. In summary, our findings demonstrate that partitions of gene diversity into within- and between-populations components are heavily biased by the populations analyzed and the models fitted. Optimistically, however, more information is available to analyze population history and evolution by quantifying, as we have done, the uniqueness of patterns of differentiation.