Population estimators or progeny tests: what is the best method to assess null allele frequencies at SSR loci?

Nuclear SSRs are notorious for having relatively high frequencies of null alleles, i.e. alleles that fail to amplify and are thus recessive and undetected in heterozygotes. In this paper, we compare two kinds of approaches for estimating null allele frequencies at seven nuclear microsatellite markers in three French Fagus sylvatica populations: (1) maximum likelihood methods that compare observed and expected homozygote frequencies in the population under the assumption of Hardy-Weinberg equilibrium and (2) direct null allele frequency estimates from progeny where parent genotypes are known. We show that null allele frequencies are high in F. sylvatica (7.0% on average with the population method, 5.1% with the progeny method), and that estimates are consistent between the two approaches, especially when the number of sampled maternal half-sib progeny arrays is large. With null allele frequencies ranging between 5% and 8% on average across loci, population genetic parameters such as genetic differentiation (F _ST) may be mostly unbiased. However, using markers with such average prevalence of null alleles (up to 15% for some loci) can be seriously misleading in fine scale population studies and parentage analysis.     

A method for adjusting allele frequencies in the case of microsatellite allele drop‐out

A new method is proposed to adjust allele frequencies when allelic drop‐out is common. This method assumes Hardy–Weinberg equilibrium (HWE), and treats the problematic alleles as a one‐locus two‐allele system with dominance. By assuming that the homozygote frequency of the ‘recessive’ allele is measured correctly, we can back calculate the allele frequency of the ‘dominant’ allele, and adjust the heterozygote frequency accordingly. The drawback is that multilocus genotypes cannot be constructed and tests that use deviations from Hardy–Weinberg such as tests for bottlenecks become impossible. An example is given where a large homozygote excess (F_IS = 0.44) is adjusted to a reasonable level (F_IS = 0.046). The effect of scoring error was set in relation to sampling error and while F_IS values can be seriously biased, F_ST values are not necessarily so, if scoring error and sample size are both low. As sample size increases, the effect of scoring error increases.     

Population Structure of the Giant Garter Snake, Thamnophis gigas

The giant garter snake, Thamnophis gigas, is a threatened species endemic to California’s Central Valley. We tested the hypothesis that current watershed boundaries have caused genetic differentiation among populations of T. gigas. We sampled 14 populations throughout the current geographic range of T. gigas and amplified 859 bp from the mitochondrial gene ND4 and one nuclear microsatellite locus. DNA sequence variation from the mitochondrial gene indicates there is some genetic structuring of the populations, with high F_ST values and unique haplotypes occurring at high frequency in several populations. We found that clustering populations by watershed boundary results in significant between-region genetic variance for mtDNA. However, analysis of allele frequencies at the microsatellite locus NSU3 reveals very low F_ST values and little between-region variation in allele frequencies. The discordance found between mitochondrial and microsatellite data may be explained by aspects of molecular evolution and/or T. gigas life history characteristics. Differences in effective population size between mitochondrial and nuclear DNA, or male-biased gene flow, result in a lower migration rate of mitochondrial haplotypes relative to nuclear alleles. However, we cannot exclude homoplasy as one explanation for homogeneity found for the single microsatellite locus. The mitochondrial nucleotide sequence data supports conservation practices that identify separate management units for T. gigas.     

Fifteen new polymorphic microsatellite loci for the meadow brown butterfly,Maniola jurtina

We characterized fifteen microsatellite markers for the butterfly Maniola jurtina. For the six studied populations (96 samples) the total number of alleles per locus ranged from 3 to 55 and mean overall expected heterozygosity across all loci was 0.74. In spite of a high frequency of null alleles detected in part of the loci, a recurrent phenomenon in Lepidopteron, the estimation of pairwise F_ST seems rather insensitive to the presence of these null alleles as shown by the high correlation between F_ST calculated after correction for the presence of null alleles and non-corrected F_ST, indicating that the loci may be usable in population genetics, more specifically for the study of populations genetics structure.     

Combining multiple analytical approaches for the identification of population structure and genetic delineation of two subspecies of the endemic Arabian burnet moth Reissita simonyi (Zygaenidae; Lepidoptera)

Habitat fragmentation and landscape topology may influence the genetic structure and connectivity between natural populations. Six microsatellite loci were used to infer the population structure of 35 populations (N = 788) of the alpine Arabian burnet moth Reissita simonyi (Lepidoptera, Zygaenidae) in Yemen and Oman. Due to the patchy distribution of larval food plants, R. simonyi is not continuously distributed throughout the studied area and the two recognized subspecies of this endemic species (Reissita s. simonyi/R. s. yemenicola) are apparently discretely distributed. All microsatellites showed prevalence of null alleles and therefore a thorough investigation of the impact of null alleles on different population genetic parameters (F _ST, inbreeding coefficients, and Population Graph topologies) is given. In general, null alleles reduced genetic covariance and independence of allele frequencies resulting in a more connected genetic topology in Population Graphs and an overestimation of pairwise F _ST values and inbreeding coefficients. Despite the presence of null alleles, Population Graphs also showed a much higher genetic connectivity within subspecies (and lower genetic differentiation (via F _ST)) than between; supporting existing taxonomic distinction. Partial Mantel tests showed that both geographical distance and altitude were highly correlated with the observed distribution of genetic structure within R. simonyi. In conclusion, we identified geographical and altitudinal distances in R. simonyi as well as an intervening desert area to be the main factors for spatial genetic structure in this species and show that the taxonomic division into two subspecies is confirmed by genetic analysis.     

Evidence of recent population bottlenecks and inbreeding in British populations of Bechstein’s bat, Myotis bechsteinii

We investigated the population genetics of seven maternity roosts of Bechstein’s bats widely distributed across the south of England. Across all of the populations sampled, two mitochondrial DNA microsatellite loci were fixed for single haplotypes. Genetic diversity across eight nuclear microsatellite loci was similar in all seven populations, with a mean He of 0.727. However, six of the populations showed substantial homozygote excess, with F _IS estimates greater than zero, indicative of recent inbreeding. Bottleneck tests also implied that six of the populations have experienced recent declines. Genetic differentiation among the populations was low, with a mean intersite F _ST estimate of 0.041. There was no significant isolation by distance using allele frequency-based criteria (F _ST and genetic distances), however, a weak correlation was found using the allele size-based R _ST criterion. Assignment tests were unable to distinguish the seven sampling sites as distinct clusters. Mean intra-roost relatedness (r) was 0.079, indicative of recent inbreeding relative to German populations. All but one of the bats had one or more half or full siblings in its maternity roost. In addition, family relationships of individuals within a colony were significantly commoner than family relationships among four proximal roosts <8 km apart. The results are discussed in the context of conservation requirements for this rare British bat.     

Genetic variation in two populations of the rough‐skinned newt (Taricha granulosa) assessed using novel tetranucleotide microsatellite loci

Seven novel tetranucleotide microsatellite loci were identified from a partial genomic DNA library, enriched for GATA‐motif microsatellites, from the rough‐skinned newt (Taricha granulosa). All loci were polymorphic, and one displayed a high frequency null allele. A related species, T. rivularis, displays strong site fidelity and detectable population structure over small spatial scales, so we assessed genetic variation in two samples of T. granulosa separated by 16 km. Distributions of allele frequencies differ significantly between our two sites, but small values of F_ST and Rho_ST suggest that the populations are linked by a large amount of gene flow.     

Sampling for Microsatellite-Based Population Genetic Studies: 25 to 30 Individuals per Population Is Enough to Accurately Estimate Allele Frequencies

One of the most common questions asked before starting a new population genetic study using microsatellite allele frequencies is “how many individuals do I need to sample from each population?” This question has previously been answered by addressing how many individuals are needed to detect all of the alleles present in a population (i.e. rarefaction based analyses). However, we argue that obtaining accurate allele frequencies and accurate estimates of diversity are much more important than detecting all of the alleles, given that very rare alleles (i.e. new mutations) are not very informative for assessing genetic diversity within a population or genetic structure among populations. Here we present a comparison of allele frequencies, expected heterozygosities and genetic distances between real and simulated populations by randomly subsampling 5–100 individuals from four empirical microsatellite genotype datasets (Formica lugubris, Sciurus vulgaris, Thalassarche melanophris, and Himantopus novaezelandia) to create 100 replicate datasets at each sample size. Despite differences in taxon (two birds, one mammal, one insect), population size, number of loci and polymorphism across loci, the degree of differences between simulated and empirical dataset allele frequencies, expected heterozygosities and pairwise F_ST values were almost identical among the four datasets at each sample size. Variability in allele frequency and expected heterozygosity among replicates decreased with increasing sample size, but these decreases were minimal above sample sizes of 25 to 30. Therefore, there appears to be little benefit in sampling more than 25 to 30 individuals per population for population genetic studies based on microsatellite allele frequencies.     

Genetic variation and population structure in Korean populations of sand dune speciesSalsola komarovi (Chenopodiaceae)

Salsola komarovi lljin is a herbaceous annual native to the sand dunes and beaches of Japan, northern China, Sakhalln and Korea. Starch-gel electrophoresis was conducted on leaves and stems collected from 300 plants in eight Korean populations. The mean number of alleles per locus (A _p=1.51), mean expected heterozygosity (He _p=0.116), and total genetic diversity (H _T=0.279) were comparable with those for species with similar life history and ecological traits. A general conformance of genotype frequencies to Hardy-Weinberg expectations (meanF _IS=−0.030) indicates thatS. komarovi is an outcrossing species. Slightly more than 20% of the genetic variation was found among populations (F _ST=0.204). In addition, significant differences in allele frequency were detected between populations at all 11 polymorphic loci (P<0.001). Nei's genetic identities range from 0.885 to 0.985 with a mean of 0.942. However, indirect estimates of the number of migrant per generation (0.97, calculated fromF _ST and 0.31, calculated from seven private alleles) indicate that the levels of gene flow is low among Korean populations. Although the species maintains a moderate level of genetic variation within populations, the small, isolated natural populations of the species have been severely destructed by human activities, particularly in summer season. If this is true, conservation efforts should be focused on those populations that currently maintain the most genetic diversity (e.g., populations of Cheju Island and coast of the southwestern Korean Peninsula).     

Polymorphic microsatellites in the African freshwater snail,Bulinus forskalii (Gastropoda,Pulmonata)

Eleven microsatellites were isolated in the freshwater snail Bulinus forskalii, intermediate host for the medically important trematode Schistosoma intercalatum. Characterization in 60 snails from three populations of B. forskalii from Cameroon revealed 4 to 18 alleles per locus. Low observed heterozygosity but higher expected heterozygosity, high F_IS estimates, significant departures from Hardy–Weinberg equilibrium and genotypic linkage disequilibria all indicate that B. forskalii is a preferential selfer. High F_ST estimates suggest that effective dispersal is limited and genetic drift is an important determinant of genetic structure. The potential utility of the microsatellite primers in other closely related Bulinus species was explored.     

Microgeographic structure of Anopheles gambiae in western Kenya based on mtDNA and microsatellite loci

The population genetic structure of the Anopheles gambiae in western Kenya was studied using length variation at five microsatellite loci and sequence variation in a 648-nt mtDNA fragment. Mosquitoes were collected from houses in villages spanning up to 50 km distance, The following questions were answered, (i) Are mosquitoes in a house more related genetically to each other than mosquitoes between houses? (ii) What degree of genetic differentiation occurs on these geographical scales? (iii) How consistent are the results obtained with both types of genetic markers? At the house level, no differentiation was detected by F_ST and R_ST, and the band sharing index test revealed no significant associations of alleles across loci. Likewise, indices of kinship based on mtDNA haplotypes in houses were even lower than in the pooled sample. Therefore, the hypothesis that mosquitoes in a house are more related genetically was rejected. At increasing geographical scales, microsatellite allele distributions were similar among all population samples and no subdivision of the gene pool was detected by F_ST or R_ST. Likewise, estimates of haplotype divergence of mtDNA between populations were not higher than the within population estimates, and mtDNA-based F_ST values were not significantly different from zero. That sequence variation in mtDNA provided matching results with microsatellite loci (while high genetic variation was observed in all loci), suggested that this pattern represents the whole genome. The minimum area associated with a deme of A. gambiae in western Kenya is therefore larger than 50 km in diameter.     

Microsatellite DNA Polymorphism in Intensely Enhanced Populations of Sea Trout (Salmo trutta) in the Southern Baltic

Sea trout (Salmo trutta) is an anadromous form of brown trout, a commercially important salmonid species in Europe. Stocking has been used to compensate for the decrease of natural populations and maintenance of fishery and angling catches. Over 1.5 million smolts and 4.5 million alevins are released to Polish coastal rivers each year. Variation at 7 microsatellite loci (Ssa197, Ssa171, Ssa85, Str15, Str73, Str591, and Str543) was used to study genetic polymorphism of spawners returning to 6 rivers. Application of distance method for comparison of pairs of populations based on number of different alleles (F _ST) revealed significant differences between Vistula and Wieprza, and Parseta as well as between Drweca and Wieprza, and Slupia. The level of heterozygosity was similar between most of the studied sea trout populations; considerable differences were found only for Str591. Differences in frequencies of a few alleles between populations were observed. An exact test of sample differentiation based on allele frequencies confirmed lack of significance of differentiation between the 6 pairs of populations (F _ST and R _ST). No admixture was observed in the studied populations. Possible effects of stocking on the genetic polymorphism of the sea trout populations in wild with implications for biotechnology are discussed.     

Moderately and Highly Polymorphic Microsatellites Provide Discordant Estimates of Population Divergence in Sockeye Salmon, Oncorhynchus nerka

Mutation rate can vary widely among microsatellite loci. This variation may cause discordant single-locus and multi-locus estimates of F_ST, the commonly used measure of population divergence. We use 16 microsatellite and five allozyme loci from 14 sockeye salmon populations to address two questions about the affect of mutation rate on estimates of F_ST: (1) does mutation rate influence F_ST estimates from all microsatellites to a similar degree relative to allozymes?; (2) does the influence of mutation rate on F_ST estimates from microsatellites vary with geographic scale in spatially structured populations? For question one we find that discordant estimates of F_ST among microsatellites as well as between the two marker classes are correlated with mean within-population heterozygosity (H_S) and thus are likely due to differences in mutation rate. Highly polymorphic microsatellites (H_S > 0.84) provide significantly lower estimates of F_ST than moderately polymorphic microsatellites and allozymes (H_S < 0.60). Estimates of F_ST from binned allele frequency data and R_ST provide more accurate measures of population divergence for highly polymorphic but not for moderately polymorphic microsatellites. We conclude it is more important to pool loci of like H_S rather than marker class when estimating F_ST. For question two we find the F_ST values for moderately and highly polymorphic loci, while significantly different, are positively correlated for geographically proximate but not geographically distant population pairs. These results are consistent with expectations from the equilibrium approximation of Wright's infinite island model and confirm that the influence of mutation on estimates of F_ST can vary in spatially structured populations presumably because the rate of migration varies inversely with geographic scale.     

Genetic Diversity and Structure of Two Prominent Zebu Cattle Breeds Adapted to the Arid Region of India Inferred from Microsatellite Polymorphism

This study aims to assess the genetic diversity and population structure of two major zebu dairy breeds (Tharparkar and Rathi) adapted to the arid region of Rajasthan state of India. Various variability estimates indicate the existence of sufficient within-breed genetic diversity. Mean estimates of F-statistics are significantly different from zero: F _IS = 0.112 ± 0.029, F _IT = 0.169 ± 0.033, F _ST = 0.065 ± 0.017. The overall positive value of F _IS (0.112) and an F _IT value (0.169) that is more than the F _ST (0.065) indicate departure from random mating. The drift-based estimates reflect a moderate yet significant level of breed differentiation between the Tharparkar and Rathi breeds. The evaluation of an exact test, showing that allele frequencies across all the loci differed significantly, supports the population differentiation. This is paralleled by the outcome of neighbor-joining clustering based on allele-sharing distance measures. The allocation of a high percentage of individuals (95.7%) to their population of origin and correspondence analysis further substantiates the existence of a cohesive genetic structure in both the breeds.     

Application of Microsatellite Markers to Population Genetics Studies of Japanese Flounder Paralichthys olivaceus

We examined population genetic structure by means of microsatellite analysis among 7 Japanese flounder (Paralichthys olivaceus) populations collected from coastal sea areas around Japan. As was expected, all of the 11 microsatellite loci examined were variable in all populations (number of alleles per locus, 15.2–18.2; average of expected heterozygosity, 0.74–0.78). Eleven population pairs in 21 possible pairwise comparisons showed significant genetic heterogeneity associated with allele frequency distributions or fixation index (F _ST ). Modified Cavalli-Sforza chord distance (D _A ) and Nei's standard genetic distance (D _ST ) ranged from 0.051 to 0.090, and from 0.000 to 0.025, respectively. There was evidence that the populations assessed in this study were not drawn from a single panmictic population; however, it appears that Japanese flounder populations around Japan are not well-structured, as an estimate of the fixation index value among the 7 localities was very low (F _ST = 0.0025). Received April 27, 2001; accepted June 27, 2001.     

Population structure of nuclear and mitochondrial DNA variation among humpback whales in the North Pacific

The population structure of variation in a nuclear actin intron and the control region of mitochondrial DNA is described for humpback whales from eight regions in the North Pacific Ocean: central California, Baja Peninsula, nearshore Mexico (Bahia Banderas), offshore Mexico (Socorro Island), southeastern Alaska, central Alaska (Prince Williams Sound), Hawaii and Japan (Ogasawara Islands). Primary mtDNA haplotypes and intron alleles were identified using selected restriction fragment length polymorphisms of target sequences amplified by the polymerase chain reaction (PCR–RFLP). There was little evidence of heterogeneity in the frequencies of mtDNA haplotypes or actin intron alleles due to the year or sex composition of the sample. However, frequencies of four mtDNA haplotypes showed marked regional differences in their distributions (Φ_ST = 0.277; P < 0.001; n = 205 individuals) while the two alleles showed significant, but less marked, regional differences (Φ_ST = 0.033; P < 0.013; n = 400 chromosomes). An hierarchical analysis of variance in frequencies of haplotypes and alleles supported the grouping of six regions into a central and eastern stock with further partitioning of variance among regions within stocks for haplotypes but not for alleles. Based on available genetic and demographic evidence, the southeastern Alaska and central California feeding grounds were selected for additional analyses of nuclear differentiation using allelic variation at four microsatellite loci. All four loci showed significant differences in allele frequencies (overall F_ST = 0.043; P < 0.001; average n = 139 chromosomes per locus), indicating at least partial reproductive isolation between the two regions as well as the segregation of mtDNA lineages. Although the two feeding grounds were not panmictic for nuclear or mitochondrial loci, estimates of long-term migration rates suggested that male-mediated gene flow was several-fold greater than female gene flow. These results include and extend the range and sample size of previously published work, providing additional evidence for the significance of genetic management units within oceanic populations of humpback whales.     

Population genetic structure of the gynodioecious Thymus vulgaris L. (Labiatae) in southern France

In a self-compatible gynodioecious species, the abundance of female plants (which are obligate outcrossers) relative to hermaphrodites (which may self and outcross) may be a critical factor influencing genetic diversity and population structure. In the gynodioecious Thymus vulgaris L., female frequency varies from 5 to 95%, providing a suitable model to examine this issue. In this study, we use allozyme markers to (1) evaluate the relationship between female frequency, genetic diversity and population structure, (2) determine whether females and hermaphrodites vary in heterozygote deficiency and (3) examine whether other factors such as plant density are related to heterozygote deficiency. Twenty three natural populations, with female frequencies ranging from 11 to 92%, were sampled in and around the St-Martin-de-Londres basin in southern France. Based on four polymorphic allozyme loci, we found no significant correlation between female frequency and heterozygote deficiency. A significant (P < 0.05) F_IS value over loci and over populations of 0.11 was detected. The F_IS value per population showed a significant heterozygote deficiency in 11 of the 23 populations. However, no significant difference between female and hermaphrodite F_IS values was found. A significant heterozygote deficiency only occurred in populations of intermediate density. There was little differentiation among populations (F_ST = 0.038) nor among subpopulations within each population. The significant F_IS values are thus mostly due to inbreeding effects. The lack of a correlation between F_IS values and female frequency may be due to outcrossing in hermaphrodites and/or restoration of male fertility which may occur to a greater extent at low female frequency. The similarity of female and hermaphrodite F_IS values indicates that females may occasion high levels of biparental inbreeding.     

Calculations of population differentiation based on GST and D: forget GST but not all of statistics!

G _ST‐values and its relatives (F_ST) belong to the most used parameters to define genetic differences between populations. Originally, they were developed for allozymes with very low number of alleles. Using highly polymorphic microsatellite markers it was often puzzling that G_ST‐values were very low but statistically significant. In their papers, Jost (2008) and Hedrick (2005) explained that G_ST‐values do not show genetic differentiation, and Jost suggested calculating D‐values instead. Theoretical mathematical considerations are often difficult to follow; therefore, we chose an applied approach comparing two artificial populations with different number of alleles at equal frequencies and known genetic divergence. Our results show that even for more than one allele per population G_ST‐values do not calculate population differentiation correctly; in contrast, D‐values do reflect the genetic differentiation indicating that data based on G_ST‐values need to be re‐evaluated. In our approach, statistical evaluations remained similar. We provide information about the impact of different sample sizes on D‐values in relation to number of alleles and genetic divergence.     

Comparing the van Oosterhout and Chybicki‐Burczyk methods of estimating null allele frequencies for inbred populations

In spite of the usefulness of codominant markers in population genetics, the existence of null alleles raises challenging estimation issues in natural populations that are characterized by positive inbreeding coefficients (F > 0). Disregarding the possibility of F > 0 in a population will generally lead to overestimates of null allele frequencies. Conversely, estimates of inbreeding coefficients (F) may be strongly biased upwards (excess homozygotes), in the presence of nontrivial frequencies of null alleles. An algorithm has been presented for the estimation of null allele frequencies in inbred populations (van Oosterhout method), using external estimates of the F‐statistics. The goal of this study is to introduce a modification of this method and to provide a formal comparison with an alternative likelihood‐based method (Chybicki‐Burczyk). Using simulated data, we illustrate the strengths and limitations of these competing methods. Under most circumstances, the likelihood method is preferable, but for highly inbred organisms, a modified van Oosterhout method offers some advantages.     

Empirical Distributions of F ST from Large-Scale Human Polymorphism Data

Studies of the apportionment of human genetic variation have long established that most human variation is within population groups and that the additional variation between population groups is small but greatest when comparing different continental populations. These studies often used Wright’s F _ST that apportions the standardized variance in allele frequencies within and between population groups. Because local adaptations increase population differentiation, high-F _ST may be found at closely linked loci under selection and used to identify genes undergoing directional or heterotic selection. We re-examined these processes using HapMap data. We analyzed 3 million SNPs on 602 samples from eight worldwide populations and a consensus subset of 1 million SNPs found in all populations. We identified four major features of the data: First, a hierarchically F _ST analysis showed that only a paucity (12%) of the total genetic variation is distributed between continental populations and even a lesser genetic variation (1%) is found between intra-continental populations. Second, the global F _ST distribution closely follows an exponential distribution. Third, although the overall F _ST distribution is similarly shaped (inverse J), F _ST distributions varies markedly by allele frequency when divided into non-overlapping groups by allele frequency range. Because the mean allele frequency is a crude indicator of allele age, these distributions mark the time-dependent change in genetic differentiation. Finally, the change in mean-F _ST of these groups is linear in allele frequency. These results suggest that investigating the extremes of the F _ST distribution for each allele frequency group is more efficient for detecting selection. Consequently, we demonstrate that such extreme SNPs are more clustered along the chromosomes than expected from linkage disequilibrium for each allele frequency group. These genomic regions are therefore likely candidates for natural selection.