Application of fingermark enhancement techniques on Clydesdale Bank and Royal Bank of Scotland £10 and £5 polymer banknotes in a pseudo-operational trial.

The aim of this study was to apply the two most effective enhancement sequences for latent fingermarks on Clydesdale Bank and Royal Bank of Scotland polymer banknotes (£10 and £5), as determined in a previous study, to a pseudo-operational trial. The two enhancement sequences established as being the most effective for these types of notes were superglue fuming using PolycyanoUV followed by black magnetic powder, and black powder suspension. Both enhancement sequences included a fluorescence examination before enhancement, and after-treatment using white light, followed by Infrared light. The study conducted by Joannidis et al. was carried out in a controlled laboratory environment where all variables, i.e., the position and age of each fingermark, were known and controlled. However, these conditions do not accurately mirror those of polymer notes seized as part of a criminal investigation. The two most effective enhancement sequences were therefore tested in a pseudo-operational trial to determine whether they would be as effective when applied to banknotes that mimicked those seized in an investigation. To mimic these conditions 102 banknotes from each bank (each containing a mixture of circulated and uncirculated banknotes) were left out in the laboratory for four weeks for the laboratory staff to randomly handle. The results from this pseudo operational trial confirmed the outcomes of the previous study. Superglue fuming (using PolyCyano UV) followed by black magnetic powder was found to be effective in enhancing fingermarks on both Clydesdale Bank and Royal Bank of Scotland polymer banknotes (£10 and £5). This was closely followed in effectiveness by powder suspension which, although it gave slightly poorer results than superglue followed by black magnetic powder, was also effective at enhancing ridge detail. This study also confirmed that Infrared light (730-800 nm), with the addition of an 815 nm filter for notes processed using superglue and black magnetic powder,aided in the reduction of background pattern interference when photographing any ridge detail.

Lockdown labs: Pivoting to remote learning in forensic science higher education.

Forensic Science training and education is reliant on the application of knowledge to casework scenarios and the development of key practical skills that provide a platform for career development in the field. The COVID-19 pandemic introduced a number of challenges to effectively deliver practical content online and remotely, whilst still meeting intended learning outcomes, accreditation requirements, and attaining a high level of student engagement and experience. The MSc Forensic Science programme featured in this study is a one-year degree programme with a strong emphasis on the practical elements of forensic science, and a diverse international student cohort. Therefore, the restrictions associated with the pandemic made it very difficult not only to plan the delivery of material but also to adapt the content itself for effective online and remote learning. By focusing on the intended learning outcomes, a number of innovative teaching practices were developed to successfully transition from face-to-face teaching to online and remote delivery. A range of online and practical resources were developed, including a laboratory home kit, demonstration videos, online practical technique simulations (produced by Learning Science), data analysis tasks, and interactive workshops and activities, all designed to consolidate student learning and build confidence, in preparation for such a time that on campus practical teaching could resume. The initial feedback received from these activities from both staff and students was extremely positive and the transition from classroom to online teaching was a success, as reflected in student attainment and later student feedback. Students reported that they had a better understanding of what was expected of them, including knowledge of protocols and techniques, and felt much more confident moving into the next stage of their learning development. Even though the practical laboratory sessions were the most significantly affected by the restrictions associated with the pandemic, and resulted in reduced interaction for the students, this was counteracted by virtual sessions and workshops, which gave students the opportunity to engage with each other and communicate their thoughts and opinions, ultimately building key presentation and group working skills. This case study will detail the pivot to remote learning, as well as critically evaluating the feedback from students and discussing the changes that are likely to be retained as longer-term teaching practices, versus those that were a necessary temporary addition or adjustment in response to the pandemic.

Developments in forensic DNA analysis.

The analysis of DNA from biological evidence recovered in the course of criminal investigations can provide very powerful evidence when a recovered profile matches one found on a DNA database or generated from a suspect. However, when no profile match is found, when the amount of DNA in a sample is too low, or the DNA too degraded to be analysed, traditional STR profiling may be of limited value. The rapidly expanding field of forensic genetics has introduced various novel methodologies that enable the analysis of challenging forensic samples, and that can generate intelligence about the donor of a biological sample. This article reviews some of the most important recent advances in the field, including the application of massively parallel sequencing to the analysis of STRs and other marker types, advancements in DNA mixture interpretation, particularly the use of probabilistic genotyping methods, the profiling of different RNA types for the identification of body fluids, the interrogation of SNP markers for predicting forensically relevant phenotypes, epigenetics and the analysis of DNA methylation to determine tissue type and estimate age, and the emerging field of forensic genetic genealogy. A key challenge will be for researchers to consider carefully how these innovations can be implemented into forensic practice to ensure their potential benefits are maximised.

Population genetics and forensic utility of 23 autosomal PowerPlex Fusion 6C STR loci in the Kuwaiti population.

This study evaluates the forensic utility of 23 autosomal short tandem repeat markers in 400 samples from the Kuwaiti population, of which four markers (D10S1248, D22S1045, D2S441 and SE33) are reported for the first time for Kuwait. All the markers were shown to exhibit no deviation from Hardy-Weinberg equilibrium, nor any linkage disequilibrium between and within loci, indicating that these loci are inherited independently, and their allele frequencies can be used to estimate match probabilities in the Kuwaiti population. The low combined match probability of 7.37 × 10-30 and the high paternity indices generated by these loci demonstrate the usefulness of the PowerPlex Fusion 6C kit for human identification in this population, as well as to strengthen the power of paternity testing. Off-ladder alleles were seen at several loci, and these were identified by examining their underlying nucleotide sequences. Principal component analysis (PCA) and STRUCTURE showed no genetic structure within the Kuwaiti population. However, PCA revealed a correlation between geographic and genetic distance. Finally, phylogenetic trees demonstrated a close relationship between Kuwaitis and Middle Easterners at a global level, and a recent common ancestry for Kuwait with its northern neighbours of Iraq and Iran, at a regional level.

Determination of the most effective enhancement process for latent fingermarks on Clydesdale Bank and Royal Bank of Scotland £5 and £10 polymer banknotes.

Fingermarks are commonly found at crime scenes and can be used to link an individual to an object and/or place. One common evidence type regularly encountered in the course of a criminal investigation is banknotes, and the recovery of fingermarks from these notes can give an indication of who has handled them. This study was carried out in order to determine the most effective sequential processing techniques for recovering latent fingermarks on the new £5 and £10 Clydesdale Bank and Royal Bank of Scotland polymer banknotes. No previous studies have been published on the recovery of latent fingermarks from these types of polymer notes; therefore, this work provides valuable insight into the challenges associated with these notes. Initial experimentation was done in order to determine the best light source to be used in combination with each sequential process tested. From this, infrared (730-800nm) and ultraviolet (350-380nm) light were chosen for use in the main study. Black iron oxide powder suspension and black magnetic powder were two of the enhancement treatments tested, both of which are recommended 'Category A' processes in the Fingermark Visualisation Manual produced by the Home Office. Superglue fuming - using PolyCyano UV-which is a Category C process was also used, as well as the recently developed infrared fluorescent powder, fpNATURAL®2. Three fingermark donors were selected for this study-one good, one medium and one poor-and each donor deposited a 10-mark depletion series onto both sides of each type of note. Superglue fuming (using PolyCyano UV) followed by black magnetic powder was found to be the most effective sequential process for enhancement of fingermarks on all note types tested. Infrared (730-800nm) light with an 815nm filter was the most effective light source for enhancing ridge detail for this enhancement sequence. This process is now being implemented for use with these note types in Scotland.

Population genetics of 30 insertion/deletion polymorphisms in the Kuwaiti population.

This study evaluates the forensic utility of the 30 insertion and deletion (indel) markers contained in the Qiagen Investigator® DIPplex kit in the Kuwaiti population (n = 150). All but one of the 30 markers were shown to conform to the expectations of the Hardy-Weinberg Equilibrium. Linkage disequilibrium tests showed no statistically significant deviation from independence. The high combined power of discrimination (CPD > 99.999%) and low combined match probability (CMP) of 2.736 × 10-13 provide a satisfactory level of discrimination, allowing the DIPplex loci to be used as forensic markers for individual identification in Kuwait. The paternity indices indicate the usefulness of the DIPplex kit as a supplementary typing system for challenging paternity cases in Kuwait.

Evaluating the effect of body fluid mixture on the relative expression ratio of blood-specific RNA markers.

The estimation of the time elapsed since a biological stain was deposited at a crime scene can provide crucial information to a forensic investigation, indicating either when a crime was committed, or whether the biological evidence was deposited at the time of a known crime event. This would enable the investigators to limit the number of suspects and to assess alibis. The relative expression ratios (RERs) of body fluid-specific RNA markers are promising molecular tools for indicating the age of biological stains. However, the nature of some forensic samples found at crime scenes could be challenging, as they frequently occur in a mixture of different body fluid types. The research presented here has utilised reverse transcription quantitative PCR (RT-qPCR) to explore the impact of bloodstains being present in mixtures with other body fluids (saliva or semen) on the resulting RERs of blood-specific markers. The expression level of three blood-specific markers (HBA, HBB and miR16) along with two reference genes (18S and U6) were analysed across multiple ageing time points in pure and mixed bloodstains. For some markers, no significant differences were found when comparing RERs in pure and mixed bloodstains, however some RERs were altered in mixed stains. This indicates that the presence of body fluid mixtures may have a significant effect on the RERs of some blood-specific markers. This should therefore be considered when selecting markers for estimating the age of stains, particularly when multiple body fluids are thought to be present.

Identifying blood-specific age-related DNA methylation markers on the Illumina MethylationEPIC® BeadChip.

The past decade has seen rapid development in DNA methylation (DNAm) microarrays, including the Illumina HumanMethylation27 and HumanMethylation450 (450K) chips, which have played an essential role in identifying and evaluating age-related (AR) DNAm markers in different tissues. Recently, a new array, the Illumina MethylationEPIC (EPIC) was introduced, with nearly double the number of probes as the 450K (∼850,000 probes). In this study, we test these newly added probes for age association using a large cohort of 754 DNAm profiles from blood samples assayed on the EPIC BeadChip, for individuals aged 0-88 years old. 52 AR CpG sites (Spearman's abs(rho) >0.6 and P-value <10-83) were identified, 21 of which were novel sites and mapped to 18 genes, nine of which (LHFPL4, SLC12A8, EGFEM1P, GPR158, TAL1, KIAA1755, LOC730668, DUSP16, and FAM65C) have never previously been reported to be associated with age. The data were subsequently split into a 527-sample training set and a 227-sample testing set to build and validate two age prediction models using elastic net regression and multivariate regression. Elastic net regression selected 425 CpG markers with a mean absolute deviation (MAD) of 2.6 years based on the testing set. To build a multivariate linear regression model, AR CpG sites with R2 > 0.5 at FDR < 0.05 were input into stepwise regression to select the best subset for age prediction. The resulting six CpG markers were linearly modelled with age and explained 81% of age-correlated variation in DNAm levels. Age estimation accuracy using bootstrap analysis was 4.5 years, with 95% confidence intervals of 4.56 to 4.57 years based on the testing set. These results suggest that EPIC BeadChip probes for age estimation fall within the range of probes found on the previous Illumina HumanMethylation platforms in terms of their age-prediction ability.

Estimating time since deposition using quantification of RNA degradation in body fluid-specific markers.

The first appearance of ribonucleic acid (RNA) in forensic science research was in 1984 in the study of post-mortem tissues. Since then, many studies have explored the role of gene expression and its potential applications in forensic science. The two main RNA molecules that have been subject to increasing interest in the forensic science community are messenger RNA (mRNA) and microRNA (miRNA). Identification of body fluid type and estimating the time since deposition can be of immense value to criminal investigations. Determining the time since deposition or age of a biological stain can help to indicate either when a crime happened, or whether the biological evidence was deposited before/after a known crime event, in order for samples to be excluded. The research presented here has used reverse transcription quantitative PCR to examine the relative expression ratio (RER) in two types of body fluid-specific markers (saliva and semen), to develop a method to estimate the age of biological stains. mRNA and miRNA markers specific to saliva and semen, along with three reference genes were selected. Biological samples from 20 participants were stored in a dark dry place at room temperature to simulate natural ageing. A series of desired ageing points were set and total RNA was extracted when samples reached each desired point. The degradation behaviour of each RNA marker was analysed, showing that they exhibited unique degradation profiles across a one-year storage interval for saliva and semen samples, where miRNAs and the U6 reference gene were shown to have high stability. The RERs exhibit a non-linear relationship with body fluid stain age and can be considered as a potential method for body fluid stain age estimation, hence the time since deposition.

Variation in the Intensity of Selection on Codon Bias over Time Causes Contrasting Patterns of Base Composition Evolution in Drosophila.

Four-fold degenerate coding sites form a major component of the genome, and are often used to make inferences about selection and demography, so that understanding their evolution is important. Despite previous efforts, many questions regarding the causes of base composition changes at these sites in Drosophila remain unanswered. To shed further light on this issue, we obtained a new whole-genome polymorphism data set from D. simulans. We analyzed samples from the putatively ancestral range of D. simulans, as well as an existing polymorphism data set from an African population of D. melanogaster. By using D. yakuba as an outgroup, we found clear evidence for selection on 4-fold sites along both lineages over a substantial period, with the intensity of selection increasing with GC content. Based on an explicit model of base composition evolution, we suggest that the observed AT-biased substitution pattern in both lineages is probably due to an ancestral reduction in selection intensity, and is unlikely to be the result of an increase in mutational bias towards AT alone. By using two polymorphism-based methods for estimating selection coefficients over different timescales, we show that the selection intensity on codon usage has been rather stable in D. simulans in the recent past, but the long-term estimates in D. melanogaster are much higher than the short-term ones, indicating a continuing decline in selection intensity, to such an extent that the short-term estimates suggest that selection is only active in the most GC-rich parts of the genome. Finally, we provide evidence for complex evolutionary patterns in the putatively neutral short introns, which cannot be explained by the standard GC-biased gene conversion model. These results reveal a dynamic picture of base composition evolution.

The Discovery, Distribution, and Evolution of Viruses Associated with Drosophila melanogaster.

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont--which is known to be protective against virus infections in Drosophila--we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host-virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.

Strain-specific and pooled genome sequences for populations of Drosophila melanogaster from three continents.

To contribute to our general understanding of the evolutionary forces that shape variation in genome sequences in nature, we have sequenced genomes from 50 isofemale lines and six pooled samples from populations of Drosophila melanogaster on three continents. Analysis of raw and reference-mapped reads indicates the quality of these genomic sequence data is very high. Comparison of the predicted and experimentally-determined Wolbachia infection status of these samples suggests that strain or sample swaps are unlikely to have occurred in the generation of these data. Genome sequences are freely available in the European Nucleotide Archive under accession ERP009059. Isofemale lines can be obtained from the Drosophila Species Stock Center.

The relation between recombination rate and patterns of molecular evolution and variation in Drosophila melanogaster.

Genetic recombination associated with sexual reproduction increases the efficiency of natural selection by reducing the strength of Hill-Robertson interference. Such interference can be caused either by selective sweeps of positively selected alleles or by background selection (BGS) against deleterious mutations. Its consequences can be studied by comparing patterns of molecular evolution and variation in genomic regions with different rates of crossing over. We carried out a comprehensive study of the benefits of recombination in Drosophila melanogaster, both by contrasting five independent genomic regions that lack crossing over with the rest of the genome and by comparing regions with different rates of crossing over, using data on DNA sequence polymorphisms from an African population that is geographically close to the putatively ancestral population for the species, and on sequence divergence from a related species. We observed reductions in sequence diversity in noncrossover (NC) regions that are inconsistent with the effects of hard selective sweeps in the absence of recombination. Overall, the observed patterns suggest that the recombination rate experienced by a gene is positively related to an increase in the efficiency of both positive and purifying selection. The results are consistent with a BGS model with interference among selected sites in NC regions, and joint effects of BGS, selective sweeps, and a past population expansion on variability in regions of the genome that experience crossing over. In such crossover regions, the X chromosome exhibits a higher rate of adaptive protein sequence evolution than the autosomes, implying a Faster-X effect.

Estimation of the spontaneous mutation rate per nucleotide site in a Drosophila melanogaster full-sib family.

We employed deep genome sequencing of two parents and 12 of their offspring to estimate the mutation rate per site per generation in a full-sib family of Drosophila melanogaster recently sampled from a natural population. Sites that were homozygous for the same allele in the parents and heterozygous in one or more offspring were categorized as candidate mutations and subjected to detailed analysis. In 1.23 × 10(9) callable sites from 12 individuals, we confirmed six single nucleotide mutations. We estimated the false negative rate in the experiment by generating synthetic mutations using the empirical distributions of numbers of nonreference bases at heterozygous sites in the offspring. The proportion of synthetic mutations at callable sites that we failed to detect was <1%, implying that the false negative rate was extremely low. Our estimate of the point mutation rate is 2.8 × 10(-9) (95% confidence interval = 1.0 × 10(-9) - 6.1 × 10(-9)) per site per generation, which is at the low end of the range of previous estimates, and suggests an effective population size for the species of ∼1.4 × 10(6). At one site, point mutations were present in two individuals, indicating that there had been a premeiotic mutation cluster, although surprisingly one individual had a G→A transition and the other a G→T transversion, possibly associated with error-prone mismatch repair. We also detected three short deletion mutations and no insertions, giving a deletion mutation rate of 1.2 × 10(-9) (95% confidence interval = 0.7 × 10(-9) - 11 × 10(-9)).

Codon usage bias and effective population sizes on the X chromosome versus the autosomes in Drosophila melanogaster.

Codon usage bias (CUB) in Drosophila is higher for X-linked genes than for autosomal genes. One possible explanation is that the higher effective recombination rate for genes on the X chromosome compared with the autosomes reduces their susceptibility to Hill-Robertson effects, and thus enhances the efficacy of selection on codon usage. The genome sequence of D. melanogaster was used to test this hypothesis. Contrary to expectation, it was found that, after correcting for the effective recombination rate, CUB remained higher on the X than on the autosomes. In contrast, an analysis of polymorphism data from a Rwandan population showed that mean nucleotide site diversity at 4-fold degenerate sites for genes on the X is approximately three-quarters of the autosomal value after correcting for the effective recombination rate, compared with approximate equality before correction. In addition, these data show that selection for preferred versus unpreferred synonymous variants is stronger on the X than the autosomes, which accounts for the higher CUB of genes on the X chromosome. This difference in the strength of selection does not appear to reflect the effects of dominance of mutations affecting codon usage, differences in gene expression levels between X and autosomes, or differences in mutational bias. Its cause therefore remains unexplained. The stronger selection on CUB on the X chromosome leads to a lower rate of synonymous site divergence compared with the autosomes; this will cause a stronger upward bias for X than A in estimates of the proportion of nonsynonymous mutations fixed by positive selection, for methods based on the McDonald-Kreitman test.

Molecular evolution in nonrecombining regions of the Drosophila melanogaster genome.

We study the evolutionary effects of reduced recombination on the Drosophila melanogaster genome, analyzing more than 200 new genes that lack crossing-over and employing a novel orthology search among species of the melanogaster subgroup. These genes are located in the heterochromatin of chromosomes other than the dot (fourth) chromosome. Noncrossover regions of the genome all exhibited an elevated level of evolutionary divergence from D. yakuba at nonsynonymous sites, lower codon usage bias, lower GC content in coding and noncoding regions, and longer introns. Levels of gene expression are similar for genes in regions with and without crossing-over, which rules out the possibility that the reduced level of adaptation that we detect is caused by relaxed selection due to lower levels of gene expression in the heterochromatin. The patterns observed are consistent with a reduction in the efficacy of selection in all regions of the genome of D. melanogaster that lack crossing-over, as a result of the effects of enhanced Hill-Robertson interference. However, we also detected differences among nonrecombining locations: The X chromosome seems to exhibit the weakest effects, whereas the fourth chromosome and the heterochromatic genes on the autosomes located most proximal to the centromere showed the largest effects. However, signatures of selection on both nonsynonymous mutations and on codon usage persist in all heterochromatic regions.

Genetic diversity, population structure and Wolbachia infection status in a worldwide sample of Drosophila melanogaster and D. simulans populations.

Drosophila melanogaster and its close relatives have been extremely important model species in the development of population genetic models that serve to explain patterns of diversity in natural populations, a major goal of evolutionary biology. A detailed picture of the evolutionary history of these species is beginning to emerge, as the relative importance of forces including demographic changes and natural selection is established. A continuing aim is to characterise levels of genetic diversity in a large number of populations of these species, covering a wide geographic area. We have used collections from five previously un-sampled wild populations of D. melanogaster and two of D. simulans, across three continents. We estimated levels of genetic diversity within, and divergence between, these populations, and looked for evidence of genetic structure both between ancestral and derived populations, and amongst derived populations. We also investigated the prevalence of infection with the bacterial endosymbiont Wolbachia. We found that D. melanogaster populations from Sub-Saharan Africa are the most diverse, and that divergence is highest between these and non-Sub-Saharan populations. There is strong evidence for structuring of populations between Sub-Saharan Africa and the rest of the world, and some evidence for weak structure amongst derived populations. Populations from Sub-Saharan Africa also differ in the prevalence of Wolbachia infection, with very low levels of infection compared to populations from the rest of the world.

Ancestral polymorphisms in Drosophila pseudoobscura and Drosophila miranda.

Ancestral polymorphisms are defined as variants that arose by mutation prior to the speciation event that generated the species in which they segregate. Their presence may complicate the interpretation of molecular data and lead to incorrect phylogenetic inferences. They may also be used to identify regions of the genome that are under balancing selection. It is thus important to take into account the contribution of ancestral polymorphisms to variability within species and divergence between species. Here, we extend and improve a method for estimation of the proportion of ancestral polymorphisms within a species, and apply it to a dataset of 33 X-linked and 34 autosomal protein-coding genes for which sequence polymorphism data are available in both Drosophila pseudoobscura and Drosophila miranda, using Drosophila affinis as an outgroup. We show that a substantial proportion of both X-linked and autosomal synonymous variants in these two species are ancestral, and that a small number of additional genes with unusually high sequence diversity seem to have an excess of ancestral polymorphisms, suggestive of balancing selection.

Determinants of synonymous and nonsynonymous variability in three species of Drosophila.

We estimated the intensity of selection on preferred codons in Drosophila pseudoobscura and D. miranda at X-linked and autosomal loci, using a published data set on sequence variability at 67 loci, by means of an improved method that takes account of demographic effects. We found evidence for stronger selection at X-linked loci, consistent with their higher levels of codon usage bias. The estimates of the strength of selection and mutational bias in favor of unpreferred codons were similar to those found in other species, after taking into account the fact that D. pseudoobscura showed evidence for a recent expansion in population size. We examined correlates of synonymous and nonsynonymous diversity in these species and found no evidence for effects of recurrent selective sweeps on nonsynonymous mutations, which is probably because this set of genes have much higher than average levels of selective constraints. There was evidence for correlated effects of levels of selective constraints on protein sequences and on codon usage, as expected under models of selection for translational accuracy. Our analysis of a published data set on D. melanogaster provided evidence for the effects of selective sweeps of nonsynonymous mutations on linked synonymous diversity, but only in the subset of loci that experienced the highest rates of nonsynonymous substitutions (about one-quarter of the total) and not at more slowly evolving loci. Our correlational analysis of this data set suggested that both selective constraints on protein sequences and recurrent selective sweeps affect the overall level of codon usage.

Estimating the parameters of selection on nonsynonymous mutations in Drosophila pseudoobscura and D. miranda.

We present the results of surveys of diversity in sets of >40 X-linked and autosomal loci in samples from natural populations of Drosophila miranda and D. pseudoobscura, together with their sequence divergence from D. affinis. Mean silent site diversity in D. miranda is approximately one-quarter of that in D. pseudoobscura; mean X-linked silent diversity is about three-quarters of that for the autosomes in both species. Estimates of the distribution of selection coefficients against heterozygous, deleterious nonsynonymous mutations from two different methods suggest a wide distribution, with coefficients of variation greater than one, and with the average segregating amino acid mutation being subject to only very weak selection. Only a small fraction of new amino acid mutations behave as effectively neutral, however. A large fraction of amino acid differences between D. pseudoobscura and D. affinis appear to have been fixed by positive natural selection, using three different methods of estimation; estimates between D. miranda and D. affinis are more equivocal. Sources of bias in the estimates, especially those arising from selection on synonymous mutations and from the choice of genes, are discussed and corrections for these applied. Overall, the results show that both purifying selection and positive selection on nonsynonymous mutations are pervasive.