Analysis of Molecular Variance Inferred from Metric Distances among DNA Haplotypes: Application to Human Mitochondrial DNA Restriction Data

We present here a framework for the study of molecular variation within a single species. Information on DNA haplotype divergence is incorporated into an analysis of variance format, derived from a matrix of squared-distances among all pairs of haplotypes. This analysis of molecular variance (AMOVA) produces estimates of variance components and F-statistic analogs, designated here as phi-statistics, reflecting the correlation of haplotypic diversity at different levels of hierarchical subdivision. The method is flexible enough to accommodate several alternative input matrices, corresponding to different types of molecular data, as well as different types of evolutionary assumptions, without modifying the basic structure of the analysis. The significance of the variance components and phi-statistics is tested using a permutational approach, eliminating the normality assumption that is conventional for analysis of variance but inappropriate for molecular data. Application of AMOVA to human mitochondrial DNA haplotype data shows that population subdivisions are better resolved when some measure of molecular differences among haplotypes is introduced into the analysis. At the intraspecific level, however, the additional information provided by knowing the exact phylogenetic relations among haplotypes or by a nonlinear translation of restriction-site change into nucleotide diversity does not significantly modify the inferred population genetic structure. Monte Carlo studies show that site sampling does not fundamentally affect the significance of the molecular variance components. The AMOVA treatment is easily extended in several different directions and it constitutes a coherent and flexible framework for the statistical analysis of molecular data.     

Assessing the nucleotide diversity of three aphid species by RAPD

A method is presented for the estimation of nucleotide diversity and genetic structure of populations from RAPD (random amplified polymorphic DNA) data. It involves a modification of the technique developed by Lynch and Crease (1990) for the case of restriction sites as survey data. As new elements the method incorporates (i) dominance correction, (ii) values of asexual reproduction of the populations sampled, and (iii) an analytical variance of the number of nucleotide substitutions per site. Sampling was carried out at two geographic scales for three aphid species. At a macrogeographic scale, populations of Rhopalosiphum padi did not show statistical genetic differentiation. Aphis gossypii and Myzus persicae, which were sampled at a microgeographic scale, showed a higher genetic differentiation than R. padi, it being statistically significant in M. persicae. The major sources of sampling variance within- and between-populations were found to be nucleotide (i.e., the number of alleles used as a function of the number of primers used) and population (i.e., sample size) sampling. Extremely low estimates of nucleotide diversity were obtained for the species studied here. This result is consistent with previous reports on genetic diversity for the same or other aphid species which were based on allozyme polymorphism, mitochondrial DNA variation and qualitative analyses of RAPDs.     

Evolutionary Relationship of DNA Sequences in Finite Populations

With the aim of analyzing and interpreting data on DNA polymorphism obtained by DNA sequencing or restriction enzyme technique, a mathematical theory on the expected evolutionary relationship among DNA sequences (nucleons) sampled is developed under the assumption that the evolutionary change of nucleons is determined solely by mutation and random genetic drift. The statistical property of the number of nucleotide differences between randomly chosen nucleons and that of heterozygosity or nucleon diversity is investigated using this theory. These studies indicate that the estimates of the average number of nucleotide differences and nucleon diversity have a large variance, and a large part of this variance is due to stochastic factors. Therefore, increasing sample size does not help reduce the variance significantly. The distribution of sample allele (nucleomorph) frequencies is also studied, and it is shown that a small number of samples are sufficient in order to know the distribution pattern.     

The effect of ancient DNA damage on inferences of demographic histories

The field of ancient DNA (aDNA) is casting new light on many evolutionary questions. However, problems associated with the postmortem instability of DNA may complicate the interpretation of aDNA data. For example, in population genetic studies, the inclusion of damaged DNA may inflate estimates of diversity. In this paper, we examine the effect of DNA damage on population genetic estimates of ancestral population size. We simulate data using standard coalescent simulations that include postmortem damage and show that estimates of effective population sizes are inflated around, or right after, the sampling time of the ancestral DNA sequences. This bias leads to estimates of increasing, and then decreasing, population sizes, as observed in several recently published studies. We reanalyze a recently published data set of DNA sequences from the Bison (Bison bison/Bison priscus) and show that the signal for a change in effective population size in this data set vanishes once the effects of putative damage are removed. Our results suggest that population genetic analyses of aDNA sequences, which do not accurately account for damage, should be interpreted with great caution.     

Is genomic diversity a useful proxy for census population size? Evidence from a species‐rich community of desert lizards

Species abundance data are critical for testing ecological theory, but obtaining accurate empirical estimates for many taxa is challenging. Proxies for species abundance can help researchers circumvent time and cost constraints that are prohibitive for long‐term sampling. Under simple demographic models, genetic diversity is expected to correlate with census size, such that genome‐wide heterozygosity may provide a surrogate measure of species abundance. We tested whether nucleotide diversity is correlated with long‐term estimates of abundance, occupancy and degree of ecological specialization in a diverse lizard community from arid Australia. Using targeted sequence capture, we obtained estimates of genomic diversity from 30 species of lizards, recovering an average of 5,066 loci covering 3.6 Mb of DNA sequence per individual. We compared measures of individual heterozygosity to a metric of habitat specialization to investigate whether ecological preference exerts a measurable effect on genetic diversity. We find that heterozygosity is significantly correlated with species abundance and occupancy, but not habitat specialization. Demonstrating the power of genomic sampling, the correlation between heterozygosity and abundance/occupancy emerged from considering just one or two individuals per species. However, genetic diversity does no better at predicting abundance than a single day of traditional sampling in this community. We conclude that genetic diversity is a useful proxy for regional‐scale species abundance and occupancy, but a large amount of unexplained variation in heterozygosity suggests additional constraints or a failure of ecological sampling to adequately capture variation in true population size.     

Use of RAPD analyses to estimate population genetic parameters in the alfalfa leaf-cutting bee, Megachile rotundata.

RAPD analyses were performed on five geographically isolated populations of Megachile rotundata. We used haploid males of the alfalfa leaf-cutting bee, M. rotundata, to overcome the limitation of the dominance of RAPD markers in the determination of population genetic parameters. Sixteen primers gave rise to 130 polymorphic and 31 monomorphic bands. The unbiased estimators calculated in this study include within- and between-population heterozygosity, nucleotide divergence, and genetic distance. The genetic diversity (H = 0.32-0.35) was found to be about 10 times that of previous estimates (H = 0.033) based on allozyme data. Contrary to the data obtained at the protein level, our results suggest that Hymenoptera do not have a lower level of genetic variability at the DNA level compared with other insect species. Regardless of the different assumptions underlying the calculation of heterozygosity, divergence, and genetic distance, all five populations showed a parallel interrelationship for the three parameters. We conclude that RAPD markers are a convenient tool to estimate population genetic variation in haploid M. rotundata and that with an adequate sample size the technique is applicable to the evaluation of divergence in diploid populations. Key words : Megachile rotundata, RAPD, heterozygosity, genetic distance, nucleotide divergence.     

Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species

High‐throughput DNA sequencing facilitates the analysis of large portions of the genome in nonmodel organisms, ensuring high accuracy of population genetic parameters. However, empirical studies evaluating the appropriate sample size for these kinds of studies are still scarce. In this study, we use double‐digest restriction‐associated DNA sequencing (ddRADseq) to recover thousands of single nucleotide polymorphisms (SNPs) for two physically isolated populations of Amphirrhox longifolia (Violaceae), a nonmodel plant species for which no reference genome is available. We used resampling techniques to construct simulated populations with a random subset of individuals and SNPs to determine how many individuals and biallelic markers should be sampled for accurate estimates of intra‐ and interpopulation genetic diversity. We identified 3646 and 4900 polymorphic SNPs for the two populations of A. longifolia, respectively. Our simulations show that, overall, a sample size greater than eight individuals has little impact on estimates of genetic diversity within A. longifolia populations, when 1000 SNPs or higher are used. Our results also show that even at a very small sample size (i.e. two individuals), accurate estimates of F_ST can be obtained with a large number of SNPs (≥1500). These results highlight the potential of high‐throughput genomic sequencing approaches to address questions related to evolutionary biology in nonmodel organisms. Furthermore, our findings also provide insights into the optimization of sampling strategies in the era of population genomics.     

Variogram analysis of the spatial genetic structure of continuous populations using multilocus microsatellite data

A geostatistical perspective on spatial genetic structure may explain methodological issues of quantifying spatial genetic structure and suggest new approaches to addressing them. We use a variogram approach to (i) derive a spatial partitioning of molecular variance, gene diversity, and genotypic diversity for microsatellite data under the infinite allele model (IAM) and the stepwise mutation model (SMM), (ii) develop a weighting of sampling units to reflect ploidy levels or multiple sampling of genets, and (iii) show how variograms summarize the spatial genetic structure within a population under isolation-by-distance. The methods are illustrated with data from a population of the epiphytic lichen Lobaria pulmonaria, using six microsatellite markers. Variogram-based analysis not only avoids bias due to the underestimation of population variance in the presence of spatial autocorrelation, but also provides estimates of population genetic diversity and the degree and extent of spatial genetic structure accounting for autocorrelation.     

Optimal Sequencing Strategies for Surveying Molecular Genetic Diversity

Two commonly used measures of genetic diversity for intraspecies DNA sequence data are based, respectively, on the number of segregating sites, and on the average number of pairwise nucleotide differences. Expressions are derived for their variance in the presence of intragenic recombination for a panmictic population of fixed size that is at neutral equilibrium at the region sequenced. We show that, in contrast to the slow decrease in variance with increasing sample size, if the recombination rate is nonzero, the asymptotic rate of decrease of variance with increasing sequence length, for fixed sample size, is quite rapid. In particular, it is close to that which would be obtained by sequencing independent chromosome regions. The correlation between measures of diversity from linked regions is also examined. For a given total number of bases sequenced in a particular region, optimal sequencing strategies are derived. These typically involve sequencing relatively few (three to 10) long copies of the region. Under optimal strategies, the variances of the two measures are very similar for most parameter values considered. Results concerning optimal sequencing strategies will be sensitive to gross departures from the underlying assumptions, such as population bottlenecks, selective sweeps, and substantial population substructure.     

Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a polymerase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in order to estimate population genetic diversity and genetic structure parameters, one must assume that dominant (amplified) alleles are identical in state, recessive (unamplified) alleles are identical in state, AFLP fragments segregate according to Mendelian expectations and that the genotypes of an AFLP locus are in Hardy-Weinberg equilibrium (HWE). The HWE assumption is untestable for natural populations using dominant markers. Restriction fragment length polymorphism (RFLP) markers segregate as codominant alleles, and can therefore be used to test the HWE assumption that is critical for analysing AFLP data. This study examined whether the dominant AFLP markers could provide accurate estimates of genetic variability for the Aedes aegypti mosquito populations of Trinidad, West Indies, by comparing genetic structure parameters using AFLP and RFLP markers. For AFLP markers, we tested a total of five primer combinations and scored 137 putative loci. For RFLP, we examined a total of eight mapped markers that provide a broad coverage of mosquito genome. The estimated average heterozygosity with AFLP markers was similar among the populations (0.39), and the observed average heterozygosity with RFLP markers varied from 0.44 to 0.58. The average FST (standardized among-population genetic variance) estimates were 0.033 for AFLP and 0.063 for RFLP markers. The genotypes at several RFLP loci were not in HWE, suggesting that the assumption critical for analysing AFLP data was invalid for some loci of the mosquito populations in Trinidad. Therefore, the results suggest that, compared with dominant molecular markers, codominant DNA markers provide better estimates of population genetic variability, and offer more statistical power for detecting population genetic structure.     

Spineless and overlooked: DNA metabarcoding of autonomous reef monitoring structures reveals intra- and interspecific genetic diversity in Mediterranean invertebrates

The ability to gather genetic information using DNA metabarcoding of bulk samples obtained directly from the environment is crucial to determine biodiversity baselines and understand population dynamics in the marine realm. While DNA metabarcoding is effective in evaluating biodiversity at community level, genetic patterns within species are often concealed in metabarcoding studies and overlooked for marine invertebrates. In the present study, we implement recently developed bioinformatics tools to investigate intraspecific genetic variability for invertebrate taxa in the Mediterranean Sea. Using metabarcoding samples from Autonomous Reef Monitoring Structures (ARMS) deployed in three locations, we present haplotypes and diversity estimates for 145 unique species. While overall genetic diversity was low, we identified several species with high diversity records and potential cryptic lineages. Further, we emphasize the spatial scale of genetic variability, which was observed from locations to individual sampling units (ARMS). We carried out a population genetic analysis of several important yet understudied species, which highlights the current knowledge gap concerning intraspecific genetic patterns for the target taxa in the Mediterranean basin. Our approach considerably enhances biodiversity monitoring of charismatic and understudied Mediterranean species, which can be incorporated into ARMS surveys.     

Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages.

A Burkholderia cepacia population naturally occurring in the rhizosphere of Zea mays was investigated in order to assess the degree of root association and microbial biodiversity at five stages of plant growth. The bacterial strains isolated on semiselective PCAT medium were mostly assigned to the species B. cepacia by an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (16S rDNA) (ARDRA) with the enzyme AluI. Partial 16S rDNA nucleotide sequences of some randomly chosen isolates confirmed the ARDRA results. Throughout the study, B. cepacia was strictly associated with maize roots, ranging from 0.6 to 3.6% of the total cultivable microflora. Biodiversity among 83 B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique with two 10-mer primers. An analysis of RAPD patterns by the analysis of molecular variance method revealed a high level of intraspecific genetic diversity in this B. cepacia population. Moreover, the genetic diversity was related to divergences among maize root samplings, with microbial genetic variability markedly higher in the first stages of plant growth; in other words, the biodiversity of this rhizosphere bacterial population decreased over time.     

The effects of sample size on population genetic diversity estimates in song sparrows Melospiza melodia

To empirically determine the effects of sample size on commonly used measures of average genetic diversity, we genotyped 200 song sparrows Melospiza melodia from two populations, one genetically depauperate (n=100) and the other genetically diverse (n=100), using eight microsatellite loci. These genotypes were used to randomly create 10,000 datasets of differing sizes (5 to 50) for each population to determine what the effects of sample size might be on several estimates of genetic diversity (number of alleles per locus, average observed heterozygosity, and unbiased average expected heterozygosity) in natural populations of conservation concern. We found that at small sample sizes of 5 to 10 individuals, estimates of unbiased heterozygosity outperformed those based on observed heterozygosity or allelic diversity for both low- and high-diversity populations. We also found that when comparing across populations in which different numbers of individuals were sampled, rarefaction provided a useful way to compare estimates of allelic diversity. We recommend that standard errors should be reported for all diversity estimators, especially when sample sizes are small. We also recommend that at least 20 to 30 individuals be sampled in microsatellite studies that assess genetic diversity when working in a population that has an unknown level of diversity. However, research on critically endangered populations (where large sample sizes are impossible or extremely difficult to obtain) should include measures of genetic diversity even if sample sizes are less than ideal. These estimates can be useful in assessing the genetic diversity of the population.     

Analytical framework for identifying and differentiating recent hitchhiking and severe bottleneck effects from multi-locus DNA sequence data

Hitchhiking and severe bottleneck effects have impact on the dynamics of genetic diversity of a population by inducing homogenization at a single locus and at the genome-wide scale, respectively. As a result, identification and differentiation of the signatures of such events from DNA sequence data at a single locus is challenging. This paper develops an analytical framework for identifying and differentiating recent homogenization events at multiple neutral loci in low recombination regions. The dynamics of genetic diversity at a locus after a recent homogenization event is modeled according to the infinite-sites mutation model and the Wright-Fisher model of reproduction with constant population size. In this setting, I derive analytical expressions for the distribution, mean, and variance of the number of polymorphic sites in a random sample of DNA sequences from a locus affected by a recent homogenization event. Based on this framework, three likelihood-ratio based tests are presented for identifying and differentiating recent homogenization events at multiple loci. Lastly, I apply the framework to two data sets. First, I consider human DNA sequences from four non-coding loci on different chromosomes for inferring evolutionary history of modern human populations. The results suggest, in particular, that recent homogenization events at the loci are identifiable when the effective human population size is 50,000 or greater in contrast to 10,000, and the estimates of the recent homogenization events are agree with the "Out of Africa" hypothesis. Second, I use HIV DNA sequences from HIV-1-infected patients to infer the times of HIV seroconversions. The estimates are contrasted with other estimates derived as the mid-time point between the last HIV-negative and first HIV-positive screening tests. The results show that significant discrepancies can exist between the estimates.     

Nuclear gene sequences and DNA variation of Cryptomeria japonica samples from the postglacial period

Genomic DNA was extracted from heartwood blocks of six Cryptomeria japonica individuals that had been buried (in an area now covered by rice fields) for about 3600 years. Attempts were made to determine the sequences of five nuclear genes following polymerase chain reaction amplification, using previously obtained C. japonica expressed sequence tag (EST) information. We detected 15 nucleotide substitutions and four insertion/deletions (indels) in a partial GapC gene sequence among 13 individuals of the buried and an extant population, which allowed us to estimate the extent of DNA variation within the buried populations, and the level of genetic differentiation between the buried population and the extant population growing in a neighbouring area. For the entire haplotypes of the GapC region, pi and theta nucleotide diversity estimates were 0.0063 and 0.0010, respectively, when both populations were included, while corresponding figures for the buried population alone were 0.0009 and 0.0017. Estimates of DNA divergence statistics (dXY = 0.0062, dA = 0.0005, FST = 0.0832 and KST = 0.0935) suggest that differentiation between the two populations was not great. However, permutation tests gave FST and KST values rejecting the null hypothesis (that populations were not differentiated) at the 5% and 1% probability levels, respectively. The significant genetic differentiation between the two populations was mainly caused by differences in haplotype diversity. The significant level of haplotype diversity in the extant population compared to the buried population might be the result of gene flow from neighbouring artificial forests. Alternatively, it is possible that we failed to detect all the DNA variation in the buried population because of clonal growth in the buried population.     

周公河齐口裂腹鱼种群的遗传多样性和遗传结构

为探讨周公河中齐口裂腹鱼(Schizothorax prenanti)的遗传多样性和遗传结构, 沿周公河连续设定6个采样点进行齐口裂腹鱼采集, 在进行基因组DNA提取后以线粒体控制区(D-loop)为分子标记进行种群遗传多样性和遗传结构的评估。结果从63尾齐口裂腹鱼中共检测到32个单倍型, 核苷酸多样性(π)为0.013, 单倍型多样性(h)为0.966。遗传多样性最高的为张家湾种群(PopF, π=0.018, h=1.000), 核苷酸多样性最低的为罗坝种群(PopC, π=0.010, h=0.970), 单倍型多样性最低的为瓦屋山大坝下游种群(PopB, π=0.015, h=0.867)。种群间共享13个单倍型, 多数齐口裂腹鱼种群间的遗传分化水平中等或较低, 仅Pop C和Pop F (F_st=0.195, P<0.01), 种群Pop A 和PopE (F_st=0.158, P<0.01)分化程度较高, 显示各种群间较密切的遗传关系。周公河不同河段齐口裂腹鱼群体的遗传多样性处于较高的水平, 各种群间具有较近的遗传关系。     

A brief note on the resemblance between relatives in the presence of population stratification

Population stratification occurs when a study population is comprised of several sub-populations, and can result in increased false positive findings in genomewide-association studies. Recently published work shows that sub-population-specific positive assortative mating at the genotypic level results in population stratification. We show that if the allele frequency of a single nucleotide polymorphism responsible for a trait varies between sub-populations and there is no dominance variance, then the heritability of the trait increases, primarily due to an increase in the additive genetic variance of the trait.     

DIFFERING LEVELS OF AMONG-POPULATION DIVERGENCE IN THE MITOCHONDRIAL DNA OF PERIODICAL CICADAS RELATED TO HISTORICAL BIOGEOGRAPHY

Mitochondrial DNA (mtDNA) haplotypes were determined for 118 individuals of 13-and 17-year periodical cicadas (genus Magicicada) collected from 16 localities throughout the Midwest and eastern United States. Two distinct mtDNA lineages, identified as A and B, differ by 2.5% based on analysis of fragment patterns and restriction maps. Observed levels of mtDNA diversity within each lineage are low compared to estimates for other taxa. The two lineages are regionally segregated, with the boundary line occurring at a latitude of approximately 33° North. The levels of mtDNA diversity and population genetic structure differ within the two lineages. There is a remarkably low level of mean mtDNA divergence and no genetic structure in lineage A, whereas lineage B exhibits an order of magnitude higher level of mtDNA diversity and significant genetic structure among sampled populations. The low level of mtDNA diversity in cicadas may be attributed to (1) a population bottleneck that most likely occurred during the Pleistocene, (2) recent colonization following the retreat of the glaciers and the expansion of deciduous forests, and/or (3) high among-family reproductive variance (as a consequence of large population size, high fecundity, aggregative behavior of adults, and clumping of eggs). The difference in mtDNA diversity and population genetic structure between the lineages suggests that they experienced different biogeographic histories; we relate this to Pleistocene changes.     

A simulation study of sample size for DNA barcoding

For some groups of organisms, DNA barcoding can provide a useful tool in taxonomy, evolutionary biology, and biodiversity assessment. However, the efficacy of DNA barcoding depends on the degree of sampling per species, because a large enough sample size is needed to provide a reliable estimate of genetic polymorphism and for delimiting species. We used a simulation approach to examine the effects of sample size on four estimators of genetic polymorphism related to DNA barcoding: mismatch distribution, nucleotide diversity, the number of haplotypes, and maximum pairwise distance. Our results showed that mismatch distributions derived from subsamples of ≥20 individuals usually bore a close resemblance to that of the full dataset. Estimates of nucleotide diversity from subsamples of ≥20 individuals tended to be bell‐shaped around that of the full dataset, whereas estimates from smaller subsamples were not. As expected, greater sampling generally led to an increase in the number of haplotypes. We also found that subsamples of ≥20 individuals allowed a good estimate of the maximum pairwise distance of the full dataset, while smaller ones were associated with a high probability of underestimation. Overall, our study confirms the expectation that larger samples are beneficial for the efficacy of DNA barcoding and suggests that a minimum sample size of 20 individuals is needed in practice for each population.     

Comparing RADseq and microsatellites for estimating genetic diversity and relatedness — Implications for brown trout conservation

The conservation and management of endangered species requires information on their genetic diversity, relatedness and population structure. The main genetic markers applied for these questions are microsatellites and single nucleotide polymorphisms (SNPs), the latter of which remain the more resource demanding approach in most cases. Here, we compare the performance of two approaches, SNPs obtained by restriction‐site‐associated DNA sequencing (RADseq) and 16 DNA microsatellite loci, for estimating genetic diversity, relatedness and genetic differentiation of three, small, geographically close wild brown trout (Salmo trutta) populations and a regionally used hatchery strain. The genetic differentiation, quantified as F_ST, was similar when measured using 16 microsatellites and 4,876 SNPs. Based on both marker types, each brown trout population represented a distinct gene pool with a low level of interbreeding. Analysis of SNPs identified half‐ and full‐siblings with a higher probability than the analysis based on microsatellites, and SNPs outperformed microsatellites in estimating individual‐level multilocus heterozygosity. Overall, the results indicated that moderately polymorphic microsatellites and SNPs from RADseq agreed on estimates of population genetic structure in moderately diverged, small populations, but RADseq outperformed microsatellites for applications that required individual‐level genotype information, such as quantifying relatedness and individual‐level heterozygosity. The results can be applied to other small populations with low or moderate levels of genetic diversity.