Statistical Studies on Protein Polymorphism in Natural Populations II. Gene Differentiation between Populations

With the aim of testing the validity of the mutation-drift hypothesis, we examined the pattern of genetic differentiation between populations by using data from Drosophila, fishes, reptiles, and mammals. The observed relationship between genetic identity and correlation of heterozygosities of different populations or species was generally in good agreement with the theoretical expectations from the mutation-drift theory, when the variation in mutation rate among loci was taken into account. In some species of Drosophila, however, the correlation was unduly high. The relationship between the mean and variance of genetic distance was also in good agreement with the theoretical prediction in almost all organisms. We noted that both the distribution of heterozygosity within species and the pattern of genetic differentiation between species can be explained by the same set of genetic parameters in each group of organisms. Alternative hypotheses for explaining these observations are discussed.     

Statistical Studies on Protein Polymorphism in Natural Populations. III. Distribution of Allele Frequencies and the Number of Alleles per Locus

With the aim of understanding the mechanism of maintenance of protein polymorphism, we have studied the properties of allele frequency distribution and the number of alleles per locus, using gene-frequency data from a wide range of organisms (mammals, birds, reptiles, amphibians, Drosophila and non-Drosophila invertebrates) in which 20 or more loci with at least 100 genes were sampled. The observed distribution of allele frequencies was U-shaped in all of the 138 populations (mostly species or subspecies) examined and generally agreed with the theoretical distribution expected under the mutation-drift hypothesis, though there was a significant excess of rare alleles (gene frequency, 0 ~ 0.05) in about a quarter of the populations. The agreement between the mutation-drift theory and observed data was quite satisfactory for the numbers of polymorphic (gene frequency, 0.05 ~ 0.95) and monomorphic (0.95 ~ 1.0) alleles.—The observed pattern of allele-frequency distribution was incompatible with the prediction from the overdominance hypothesis. The observed correlations of the numbers of rare alleles, polymorphic alleles and monomorphic alleles with heterozygosity were of the order of magnitude that was expected under the mutation-drift hypothesis. Our results did not support the view that intracistronic recombination is an important source of genetic variation. The total number of alleles per locus was positively correlated with molecular weight in most of the species examined, and the magnitude of the correlation was consistent with the theoretical prediction from mutation-drift hypothesis. The correlation between molecular weight and the number of alleles was generally higher than the correlation between molecular weight and heterozygosity, as expected.     

Is there a pattern of gene differentiation in the Indian populations

Summary Indian populations divided into a number of endogamous groups consisting of different castes, languages, religions, and tribes provide unique opportunities for examining the extent and nature of genetic differentiation at a microevolutionary stage. The genetic relationships between some of these Indian population groups have been examined using electrophoretic data from several biochemical loci in a gene diversity analysis. Does this type of analysis provide any insight into what causes such gene differentiation? What patterns of genetic variation emerge from these empirical findings? Answers are sought by relating the observed heterozygosity, genetic distance, and allied statistics to a mutation-drift hypothesis. The statistics used are: (1) interlocus mean and variance of heterozygosity, (2) mean and variance of genetic distance, and (3) correlation of heterozygosity and gene identity. The observed relationships between these sets of statistics agree well with the ones predicted by the hypothesis that different alleles at protein loci are selectively equivalent and gene frequency change occurs predominantly due to genetic drift     

STATISTICAL ANALYSIS OF HETEROZYGOSITY DATA: INDEPENDENT SAMPLE COMPARISONS

The distribution of mean heterozygosities under an infinite allele model with constant mutation rate was examined through simulation studies. It was found that, although the variance of the distribution decreases with increasing numbers of loci examined as expected, the shape of the distribution may remain skewed or bimodal. The distribution becomes symmetrical for increasing mean heterozygosity levels and numbers of loci. As a result, parametric statistical tests may not be valid for making comparisons among populations or species. Independent sample t-tests were examined in detail to determine the frequency of rejection of the null hypothesis when pairs of samples are drawn from populations with the same mean heterozygosity. Differing numbers of loci and levels of mean heterozygosity were examined. For mean heterozygosity levels above 7.5%, t-tests provide the proper rejection rate, with as few as five loci. When mean heterozygosity is as low as 2.5%, the t-test is conservative even when 40 loci are examined in each population. Independent sample t-tests were then examined for their power to detect true differences between populations as the degree of difference and number of loci vary. Although large differences can be found with high certainty, differences on the order of 5% heterozygosity may require that large numbers of loci (>40) be examined in order to be 80% or more certain of detecting them. In addition, it is emphasized that, for small numbers of loci (<25), the statistical detection of differences of interesting magnitude requires that relatively rare sampling events occur and that much larger differences be observed among the samples than exist for the population means. Two reasons exist for the lack of sensitivity of the test procedures. First, when mean heterozygosity levels are low, the non-normality of the sample means is perhaps most important. Second, even when mean heterozygosity levels are high or when sample sizes are large enough so sample means are approximately normally distributed, the intrinsically high interlocus variance of heterozygosity estimates makes the tests insensitive to the presence of heterozygosity differences that might be biologically meaningful. Finally, the implications of the results of this study are discussed with regard to observed low levels of correlation between heterozygosity and other explanatory variables.     

Allozyme and RFLP Heterozygosities as Correlates of Growth Rate in the Scallop Placopecten Magellanicus: A Test of the Associative Overdominance Hypothesis

Several studies have reported positive correlations between the degree of enzyme heterozygosity and fitness-related traits. Notable among these are the correlations between heterozygosity and growth rate in marine bivalves. Whether the correlation is the result of intrinsic functional differences between enzyme variants at the electrophoretic loci scored or arises from non-random genotypic associations between these loci and others segregating for deleterious recessive genes (the associative overdominance hypothesis) is a matter of continuing debate. A prediction of the associative overdominance hypothesis, not shared by explanations that treat the enzyme loci as causative agents of the correlation, is that the correlation is not specific to the type of genetic marker used. We have tested this prediction by scoring heterozygosity at single locus nuclear restriction fragment length polymorphisms (RFLPs) in a cohort of juvenile scallops (Placopecten magellanicus) in which growth rate was known to be positively correlated with an individual's degree of allozyme heterozygosity. A total of 222 individuals were scored for their genotypes at seven allozyme loci, two nonspecific protein loci of unknown function and eight nuclear RFLPs detected by anonymous cDNA probes. In contrast to the enzyme loci, no correlation was observed between growth rate and the degree of heterozygosity at the DNA markers. Furthermore, there was no relationship between the magnitude of heterozygote deficiency at a locus and its effect on the correlation. The differences observed between the effects of allozyme and RFLP heterozygosity on growth rate provide evidence against the associative overdominance hypothesis, but a strong case against this explanation must await corroboration from similar studies in different species.     

On the Pattern of Polymorphisms at Major Histocompatibility Complex Loci

The pattern of polymorphisms at major histocompatibility complex loci was studied by computer simulations and by DNA sequence analysis. Two types of selection, overdominance plus short-term selection and maternal–fetal incompatibility, were simulated for a gene family with intra- and interlocus gene conversion. Both types of selection were found to be consistent with the observed patterns of polymorphisms. It was also found that the more interlocus conversion occurs, the higher the divergence becomes at both nonsynonymous and synonymous sites. The ratio of nonsynonymous-to-synonymous divergence among alleles decreases as the interlocus conversion rate increases. These results agree with the interpretation that the rate of interlocus conversion is lower in human genes than in genes of other nonprimate mammals. This is because, in the latter, synonymous divergence at the ARS (antigen recognition site) is often higher than that at the non-ARS, whereas in the former, this is not so. Also, the ratio of nonsynonymous to synonymous substitutions at the ARS tends to be higher in human genes than in other mammalian genes. The main difference between overdominance plus short-term selection and maternal–fetal interaction is that the number of alleles and heterozygosity per locus are higher in the latter than in the former under the presumed selection intensities. However, the average divergence among alleles tends to be lower in the latter than in the former under similar conditions. Received: 30 September 1997 / Accepted: 15 December 1997     

Genetic structure of the Sardinian population at the D1S80 VNTR locus and population diversity

We typed the Sardinian population at the D1S80 VNTR locus. Nineteen alleles were detected in a sample of 92 unrelated individuals, allele frequency distribution showing a modal pattern mostly in agreement with other Caucasoid populations. A high degree of heterozygosity (observed value=80.4%) was present. Goodness-of-fit tests demonstrated no departure from Hardy-Weinberg expectations. Data regarding heterozygosity, number of alleles and singletons appeared in accordance with the IAM mutation-drift equilibrium model and showed no evidence of hidden substructuring. Allele 34 exhibited in Sardinians the highest frequency never observed in Caucasians. Nonetheless, the comparison with other European populations did not disclose Sardinian genetic peculiarity. Indeed, measures of genetic divergence among Europeans demonstrated definitely smaller values at the D1S80 locus in comparison with those calculated over a high number of (pre-DNA) polymorphic loci. High mutation rate and selective neutrality typical of VNTRs could account for the observed moderate genetic divergence. Isolation and genetic drift, on the other hand, may have determined certain deviations in allele frequency distribution, as occurred to allele 34 in the Sardinian population.     

Estimation of genetic diversity in Siri cattle from India

Siri cattle, a dual-purpose breed of India is currently showing declining population trend. Siri animals have been developed through natural selection and show high adaptation to wide range of hilly terrain (altitudes 150–2500 m). The present work evaluated the genetic diversity of 23 FAO recommended microsatellite loci in a sample of 50 animals. The allele and genotype frequencies, heterozygosities and gene diversity were estimated. A total of 141 alleles were detected by the 23 microsatellite markers investigated. Microsatellites were highly polymorphic with mean allelic number 6.13 ± 1.63 (ranging from 3–10 per locus). The observed heterozygosity in the population varied from 0.26–0.80 with the mean of 0.53 ± 0.16, indicating substantial genetic variation in this population. Heterozygote deficiency and mutation-drift equilibrium hypothesis were also examined. Population exhibited heterozygote deficit of 22.1%. Population was found to be in mutation-drift equilibrium. Substantial genetic variability verified in Siri cattle despite its reducing population size suggests that this breed has a rich reservoir of genetic diversity. This fact and its marked environmental adaptation reinforce the importance of its preservation as a pure breed, and (or) its use in agricultural exploitation.     

Multilocus Structure of Natural Populations of HORDEUM SPONTANEUM

The association of alleles among different loci was studied in natural populations of Hordeum spontaneum, the evolutionary progenitor of cultivated barley. The variance of the number of heterozygous loci in two randomly chosen gametes affords a useful measure of such association. The behavior of this statistic in several particular models is described. Generally, linkage (gametic phase) disequilibrium tends to increase the variance above the value expected under complete independence. This increase is greatest when disequilibria are such as to maximize the sum of squares of the two-locus gametic frequencies.—When data on several loci per individual are available, the observed variance may be tested for its agreement with that expected under the hypothesis of complete interlocus independence, using the sampling theory of this model. When applied to allozyme data from 26 polymorphic populations of wild barley, this test demonstrated the presence of geographically widespread multilocus organization. On average, the variance was 80% higher than expected under random association. Gametic frequencies for four esterase loci in both of these populations of wild barley and two composite crosses of cultivated barley were analyzed. Most generations of the composites showed less multilocus structure, as measured by the indices of association, than the wild populations.     

Statistical properties of the variation at linked microsatellite loci: implications for the history of human Y chromosomes [published erratum appears in Mol Biol Evol 1997 Mar;14(3):354]

It has recently been suggested that observed levels of variation at microsatellite loci can be used to infer patterns of selection in genomes and to assess demographic history. In order to evaluate the feasibility of these suggestions it is necessary to know something about how levels of variation at microsatellite loci are expected to fluctuate due simply to stochasticity in the processes of mutation and inheritance (genetic sampling). Here we use recently derived properties of the stepwise mutation model to place confidence intervals around the variance in repeat score that is expected at mutation-drift equilibrium and outline a statistical test for whether an observed value differs significantly from expectation. We also develop confidence intervals for the time course of the buildup of variation following a complete elimination of variation, such as might be caused by a selective sweep or an extreme population bottleneck. We apply these methods to the variation observed at human Y-specific microsatellites. Although a number of authors have suggested the possibility of a very recent sweep, our analyses suggest that a sweep or extreme bottleneck is unlikely to have occurred anytime during the last approximately 74,000 years. To generate this result we use a recently estimated mutation rate for microsatellite loci of 5.6 x 10(-4) along with the variation observed at autosomal microsatellite loci to estimate the human effective population size. This estimate is 18,000, implying an effective number of 4,500 Y chromosomes. One important general conclusion to emerge from this study is that in order to reject mutation-drift equilibrium at a set of linked microsatellite loci it is necessary to have an unreasonably large number of loci unless the observed variance is far below that expected at mutation-drift equilibrium.      

No evidence for parallel sympatric speciation in cichlid species of the genus Pseudotropheus from north-western Lake Malawi

To test the hypothesis of parallel speciation by sexual selection, we examined length variation at six microsatellite loci of samples from four sites of four to six putative species belonging to two subgenera of rocky shore mbuna cichlids from Lake Malawi. Almost all fixation indices were significantly different from zero, suggesting that there is presently little or no gene flow among allopatric populations or sympatric species. Analysis of variance indicated that genetic distances among allopatric populations of putative conspecifics were significantly lower than among sympatric populations of heterospecifics. The topology of trees based on distance matrices was also largely consistent with the hypothesis that the putative species are monophyletic and have thus not evolved in parallel in their present locations. If parallel speciation does occur in Malawi cichlids, it may be on a larger spatial scale than investigated in our study.     

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

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

Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE.

Allelic data for the D1S80 locus was obtained by using the PCR and subsequent analysis with a high-resolution, horizontal PAGE technique and silver staining. Compared with RFLP analysis of VNTR loci by Southern blotting, the approach described in this paper offers certain advantages: (1) discrete allele resolution, (2) minimal measurement error, (3) correct genotyping of single-band VNTR patterns, (4) a nonisotopic assay, (5) a permanent record of the electrophoretic separation, and (6) reduced assay time. In a sample of 99 unrelated Caucasians, the D1S80 locus demonstrated a heterozygosity of 80.8% with 37 phenotypes and 16 alleles. The distribution of genotypes is in agreement with expected values according to the Hardy-Weinberg equilibrium. Furthermore, the observed number of alleles and the level of heterozygosity, obtained through the protocol described here, were congruent with each other in accordance with the expectation of a mutation-drift equilibrium model for a single, homogeneous, random-mating population. Therefore, the analysis of D1S80 and similar VNTR loci by amplified fragment length polymorphism (AMP-FLP) may prove useful as models for population genetic issues for VNTR loci analyzed by RFLP typing via Southern blotting.     

Associations between heterozygosity and morphological variance

Recent studies have contrasted the expression of phenotypic traits, such as variance in morphological characters, with levels of genetic variation (heterozygosity) as determined by electrophoretic analysis of protein-coding loci. The theoretical basis for interpreting significant covariation stems in part from Lerner's work on genetic homeostasis, which predicts that within populations increased heterozygosity will produce decreased morphological variance, owing to a buffering effect of heterosis during development. However, the prediction for the relationship between genic heterozygosity and the variance of morphological traits among populations is unclear. To determine if a relationship existed between heterozygosity and morphological variance, we compared estimates of heterozygosity and morphological variance across 15 population samples of the fox sparrow and 17 samples of the pocket gopher. The estimates of morphological variance included coefficients of variation for each character and the variance of individual scores about the population mean in a principal components analysis. Although several recent studies have reported a significant relationship between heterozygosity and morphological variance, we found that the two measures do not covary significantly.     

Testing for a historical population bottleneck in wild Verreaux's sifaka (Propithecus verreauxi verreauxi) using microsatellite data

The degree to which historical human activities negatively impacted past and present lemur species is a long-standing question in primatology. At present, most evidence addressing this issue comes from archaeology, paleontology, and behavioral studies. Genetic data provide another source of evidence. In this study, six microsatellite loci, genotyped on more than 360 wild Verreaux's sifaka, are used in order to test the hypothesis that this population experienced a population bottleneck in the last 2000 years. Excess heterozygosity is compared with the heterozygosity expected under mutation-drift equilibrium in order to test for the genetic signature of a rapid population contraction in the past. The results indicate that the sifaka population did not experience a population bottleneck. Various methodological and conceptual implications of this result are discussed.     

Effect of mass selection on the within-family genetic variance in finite populations

Summary The adequacy of an expression for the withinfamily genetic variance under pure random drift in an additive infinitesimal model was tested via simulation in populations undergoing mass selection. Two hundred or one thousand unlinked loci with two alleles at initial frequencies of 1/2 were considered. The size of the population was 100 (50 males and 50 females). Full-sib matings were carried out for 15 generations with only one male and one female chosen as parents each generation, either randomly or on an individual phenotypic value. In the unselected population, results obtained from 200 replicates were in agreement with predictions. With mass selection, within-family genetic variance was overpredicted by theory from the 12th and 4th generations for the 1,000 and 200 loci cases, respectively. Taking into account the observed change in gene frequencies in the algorithm led to a much better agreement with observed values. Results for the distribution of gene frequencies and the withinlocus genetic covariance are presented. It is concluded that the expression for the within-family genetic variance derived for pure random drift holds well for mass selection within the limits of an additive infinitesimal model.     

EFFECTS OF POPULATION BOTTLENECKS ON GENETIC DIVERSITY AS MEASURED BY ALLOZYME ELECTROPHORESIS

Low levels of allozyme heterozygosity in populations are often attributed to previous population bottlenecks; however, few experiments have examined the relationship between heterozygosity and bottlenecks under natural conditions. The composition and number of founders of 55 experimental populations of the eastern mosquitofish (Gambusia holbrooki), maintained under simulated field conditions, were manipulated to examine the effects of bottlenecks on three components of allozyme diversity. Correlations between observed and expected values of allozyme heterozygosity, proportions of polymorphic loci, and numbers of alleles per locus were 0.423, 0.602, and 0.772, respectively. The numbers of polymorphic loci and of alleles per locus were more sensitive indicators of differences in genetic diversity between the pre-bottleneck and post-bottleneck populations than was multiple-locus heterozygosity. In many populations, single- and multiple-locus heterozygosity actually increased as a result of the founder event. The weak relationship between a population's heterozygosity and the number and composition of its founders resulted from an increase in the variance of heterozygosity due to drift of allele frequencies. There was little evidence that selection influenced the loss of allozyme variation. When it is not possible to estimate heterozygosity at a large number of polymorphic loci, allozyme surveys attempting to detect founder events and other types of bottlenecks should focus on levels of locus polymorphism and allelic diversity rather than on heterozygosity.     

DNA variability and recombination rates at X-linked loci in humans.

We sequenced 11,365 bp from introns of seven X-linked genes in 10 humans, one chimpanzee, and one orangutan to (i) provide an average estimate of nucleotide diversity (pi) in humans, (ii) investigate whether there is variation in pi among loci, (iii) compare ratios of polymorphism to divergence among loci, and (iv) provide a preliminary test of the hypothesis that heterozygosity is positively correlated with the local rate of recombination. The average value for pi was low 0.063%, SE = 0.036%, about one order of magnitude smaller than for Drosophila melanogaster, the species for which the best data are available. Among loci, pi varied by over one order of magnitude. Statistical tests of neutrality based on ratios of polymorphism to divergence or based on the frequency spectrum of variation within humans failed to reject a neutral, equilibrium model. However, there was a positive correlation between heterozygosity and rate of recombination, suggesting that the joint effects of selection and linkage are important in shaping patterns of nucleotide variation in humans.     

Polymorphism and evolution in the constant region of the T-cell receptor beta chain in an advanced teleost fish

Sequence, PCR and Southern data are presented as evidence that, as in mammals, two gene loci encode C regions of the TCR beta chain in the bicolor damselfish, Stegastes partitus. The loci are distinguished by an insertion of ten amino acids in the c-d loop at one locus and by a high interlocus divergence of the third intron and fourth exon sequences. Unlike their mammalian counterparts, the damselfish TCRBC genes encode highly polymorphic regions. None of the eight complete cDNA or four partial genomic DNA sequences presented from a single animal are identical; and three of the four animals examined are heterozygous at both loci, suggesting high heterozygosity in the damselfish population. Coding regions of the eight cDNA clones differ by up to 12% at the DNA level and 23% at the amino acid level. Polymorphism is concentrated primarily in the less evolutionarily conserved regions, suggesting that this variation may be selectively neutral. However, a comparison of the variation between synonymous and non-synonymous sites suggests that at least a portion of the observed variation results from selection. As in mammals, a gradient of sequence homogenization between the two loci is observed. Data presented here suggest that both interlocus homogenization and the sharing of polymorphic segments are likely achieved by partial gene conversion.     

Microsatellite variation in Drosophila melanogaster and Drosophila simulans: a reciprocal test of the ascertainment bias hypothesis

Interspecific comparisons of microsatellite loci have repeatedly shown that the loci are longer and more variable in the species from which they are derived (the focal species) than are homologous loci in other (nonfocal) species. There is debate as to whether this is due to directional evolution or to an ascertainment bias during the cloning and locus selection processes. This study tests these hypotheses by performing a reciprocal study. Eighteen perfect dinucleotide microsatellite loci identified from a Drosophila simulans library screen and 18 previously identified in an identical Drosophila melanogaster library screen were used to survey natural populations of each species. No difference between focal and nonfocal species was observed for mean PCR fragment length. However, heterozygosity and number of alleles were significantly higher in the focal species than in the nonfocal species. The most common allele in the Zimbabwe population of both species was sequenced for 31 of the 36 loci. The length of the longest stretch of perfect repeat units is, on average, longer in the focal species than in the non-focal species. There is a positive correlation between the length of the longest stretch of perfect repeats and heterozygosity. The difference in heterozygosity can thus be explained by a reduction in the length of the longest stretch of perfect repeats in the nonfocal species. Furthermore, flanking-sequence length difference was noted between the two species at 58% of the loci sequenced. These data do not support the predictions of the directional-evolution hypothesis; however, consistent with the ascertainment bias hypothesis, the lower variability in nonfocal species is an artifact of the microsatellite cloning and isolation process. Our results also suggest that the magnitude of ascertainment bias for repeat unit length is a function of the microsatellite size distribution in the genomes of different species.