Variation in mutation, recombination, and transposition rates in Drosophila melanogaster and Drosophila simulans.

The rates of mutation, recombination, and transposition are core parameters in models of evolution. They impact genetic diversity, responses to ongoing selection, and levels of genetic load. However, even for key evolutionary model species such as Drosophila melanogaster and Drosophila simulans, few estimates of these parameters are available, and we have little idea of how rates vary between individuals, sexes, or populations. Knowledge of this variation is fundamental for parameterizing models of genome evolution. Here, we provide direct estimates of mutation, recombination, and transposition rates and their variation in a West African and a European population of D. melanogaster and a European population of D. simulans Across 89 flies, we observe 58 single-nucleotide mutations, 286 crossovers, and 89 transposable element (TE) insertions. Compared to the European D. melanogaster, we find the West African population has a lower mutation rate (1.67 × 10-9 site-1 gen-1 vs. 4.86 × 10-9 site-1 gen-1) and a lower transposition rate (8.99 × 10-5 copy-1 gen-1 vs. 23.36 × 10-5 copy-1 gen-1), but a higher recombination rate (3.44 cM/Mb vs. 2.06 cM/Mb). The European D. simulans population has a similar mutation rate to European D. melanogaster, but a significantly higher recombination rate and a lower, but not significantly different, transposition rate. Overall, we find paternal-derived mutations are more frequent than maternal ones in both species. Our study quantifies the variation in rates of mutation, recombination, and transposition among different populations and sexes, and our direct estimates of these parameters in D. melanogaster and D. simulans will benefit future studies in population and evolutionary genetics.

Rates and spectra of de novo structural mutations in Chlamydomonas reinhardtii.

Genetic variation originates from several types of spontaneous mutation, including single-nucleotide substitutions, short insertions and deletions (indels), and larger structural changes. Structural mutations (SMs) drive genome evolution and are thought to play major roles in evolutionary adaptation, speciation, and genetic disease, including cancers. Sequencing of mutation accumulation (MA) lines has provided estimates of rates and spectra of single-nucleotide and indel mutations in many species, yet the rate of new SMs is largely unknown. Here, we use long-read sequencing to determine the full mutation spectrum in MA lines derived from two strains (CC-1952 and CC-2931) of the green alga Chlamydomonas reinhardtii The SM rate is highly variable between strains and between MA lines, and SMs represent a substantial proportion of all mutations in both strains (CC-1952 6%; CC-2931 12%). The SM spectra differ considerably between the two strains, with almost all inversions and translocations occurring in CC-2931 MA lines. This variation is associated with heterogeneity in the number and type of active transposable elements (TEs), which comprise major proportions of SMs in both strains (CC-1952 22%; CC-2931 38%). In CC-2931, a Crypton and a previously undescribed type of DNA element have caused 71% of chromosomal rearrangements, whereas in CC-1952, a Dualen LINE is associated with 87% of duplications. Other SMs, notably large duplications in CC-2931, are likely products of various double-strand break repair pathways. Our results show that diverse types of SMs occur at substantial rates, and support prominent roles for SMs and TEs in evolution.

The distribution of fitness effects of spontaneous mutations in Chlamydomonas reinhardtii inferred using frequency changes under experimental evolution.

The distribution of fitness effects (DFE) for new mutations is fundamental for many aspects of population and quantitative genetics. In this study, we have inferred the DFE in the single-celled alga Chlamydomonas reinhardtii by estimating changes in the frequencies of 254 spontaneous mutations under experimental evolution and equating the frequency changes of linked mutations with their selection coefficients. We generated seven populations of recombinant haplotypes by crossing seven independently derived mutation accumulation lines carrying an average of 36 mutations in the haploid state to a mutation-free strain of the same genotype. We then allowed the populations to evolve under natural selection in the laboratory by serial transfer in liquid culture. We observed substantial and repeatable changes in the frequencies of many groups of linked mutations, and, surprisingly, as many mutations were observed to increase as decrease in frequency. Mutation frequencies were highly repeatable among replicates, suggesting that selection was the cause of the observed allele frequency changes. We developed a Bayesian Monte Carlo Markov Chain method to infer the DFE. This computes the likelihood of the observed distribution of changes of frequency, and obtains the posterior distribution of the selective effects of individual mutations, while assuming a two-sided gamma distribution of effects. We infer that the DFE is a highly leptokurtic distribution, and that approximately equal proportions of mutations have positive and negative effects on fitness. This result is consistent with what we have observed in previous work on a different C. reinhardtii strain, and suggests that a high fraction of new spontaneously arisen mutations are advantageous in a simple laboratory environment.

Recommendations for improving statistical inference in population genomics.

The field of population genomics has grown rapidly in response to the recent advent of affordable, large-scale sequencing technologies. As opposed to the situation during the majority of the 20th century, in which the development of theoretical and statistical population genetic insights outpaced the generation of data to which they could be applied, genomic data are now being produced at a far greater rate than they can be meaningfully analyzed and interpreted. With this wealth of data has come a tendency to focus on fitting specific (and often rather idiosyncratic) models to data, at the expense of a careful exploration of the range of possible underlying evolutionary processes. For example, the approach of directly investigating models of adaptive evolution in each newly sequenced population or species often neglects the fact that a thorough characterization of ubiquitous nonadaptive processes is a prerequisite for accurate inference. We here describe the perils of these tendencies, present our consensus views on current best practices in population genomic data analysis, and highlight areas of statistical inference and theory that are in need of further attention. Thereby, we argue for the importance of defining a biologically relevant baseline model tuned to the details of each new analysis, of skepticism and scrutiny in interpreting model fitting results, and of carefully defining addressable hypotheses and underlying uncertainties.

De Novo Mutation Rate Variation and Its Determinants in Chlamydomonas.

De novo mutations are central for evolution, since they provide the raw material for natural selection by regenerating genetic variation. However, studying de novo mutations is challenging and is generally restricted to model species, so we have a limited understanding of the evolution of the mutation rate and spectrum between closely related species. Here, we present a mutation accumulation (MA) experiment to study de novo mutation in the unicellular green alga Chlamydomonas incerta and perform comparative analyses with its closest known relative, Chlamydomonas reinhardtii. Using whole-genome sequencing data, we estimate that the median single nucleotide mutation (SNM) rate in C. incerta is µ = 7.6 × 10-10, and is highly variable between MA lines, ranging from µ = 0.35 × 10-10 to µ = 131.7 × 10-10. The SNM rate is strongly positively correlated with the mutation rate for insertions and deletions between lines (r > 0.97). We infer that the genomic factors associated with variation in the mutation rate are similar to those in C. reinhardtii, allowing for cross-prediction between species. Among these genomic factors, sequence context and complexity are more important than GC content. With the exception of a remarkably high C→T bias, the SNM spectrum differs markedly between the two Chlamydomonas species. Our results suggest that similar genomic and biological characteristics may result in a similar mutation rate in the two species, whereas the SNM spectrum has more freedom to diverge.

Comparative genomics of Chlamydomonas.

Despite its role as a reference organism in the plant sciences, the green alga Chlamydomonas reinhardtii entirely lacks genomic resources from closely related species. We present highly contiguous and well-annotated genome assemblies for three unicellular C. reinhardtii relatives: Chlamydomonas incerta, Chlamydomonas schloesseri, and the more distantly related Edaphochlamys debaryana. The three Chlamydomonas genomes are highly syntenous with similar gene contents, although the 129.2 Mb C. incerta and 130.2 Mb C. schloesseri assemblies are more repeat-rich than the 111.1 Mb C. reinhardtii genome. We identify the major centromeric repeat in C. reinhardtii as a LINE transposable element homologous to Zepp (the centromeric repeat in Coccomyxa subellipsoidea) and infer that centromere locations and structure are likely conserved in C. incerta and C. schloesseri. We report extensive rearrangements, but limited gene turnover, between the minus mating type loci of these Chlamydomonas species. We produce an eight-species core-Reinhardtinia whole-genome alignment, which we use to identify several hundred false positive and missing genes in the C. reinhardtii annotation and >260,000 evolutionarily conserved elements in the C. reinhardtii genome. In summary, these resources will enable comparative genomics analyses for C. reinhardtii, significantly extending the analytical toolkit for this emerging model system.

Inbred lab mice are not isogenic: genetic variation within inbred strains used to infer the mutation rate per nucleotide site.

For over a century, inbred mice have been used in many areas of genetics research to gain insight into the genetic variation underlying traits of interest. The generalizability of any genetic research study in inbred mice is dependent upon all individual mice being genetically identical, which in turn is dependent on the breeding designs of companies that supply inbred mice to researchers. Here, we compare whole-genome sequences from individuals of four commonly used inbred strains that were procured from either the colony nucleus or from a production colony (which can be as many as ten generations removed from the nucleus) of a large commercial breeder, in order to investigate the extent and nature of genetic variation within and between individuals. We found that individuals within strains are not isogenic, and there are differences in the levels of genetic variation that are explained by differences in the genetic distance from the colony nucleus. In addition, we employ a novel approach to mutation rate estimation based on the observed genetic variation and the expected site frequency spectrum at equilibrium, given a fully inbred breeding design. We find that it provides a reasonable per nucleotide mutation rate estimate when mice come from the colony nucleus (~7.9 × 10-9 in C3H/HeN), but substantially inflated estimates when mice come from production colonies.

Patterns of population structure and complex haplotype sharing among field isolates of the green alga Chlamydomonas reinhardtii.

The nature of population structure in microbial eukaryotes has long been debated. Competing models have argued that microbial species are either ubiquitous, with high dispersal and low rates of speciation, or that for many species gene flow between populations is limited, resulting in evolutionary histories similar to those of macroorganisms. However, population genomic approaches have seldom been applied to this question. Here, we analyse whole-genome resequencing data for all 36 confirmed field isolates of the green alga Chlamydomonas reinhardtii. At a continental scale, we report evidence for putative allopatric divergence, between both North American and Japanese isolates, and two highly differentiated lineages within N. America. Conversely, at a local scale within the most densely sampled lineage, we find little evidence for either spatial or temporal structure. Taken together with evidence for ongoing admixture between the two N. American lineages, this lack of structure supports a role for substantial dispersal in C. reinhardtii and implies that between-lineage differentiation may be maintained by reproductive isolation and/or local adaptation. Our results therefore support a role for allopatric divergence in microbial eukaryotes, while also indicating that species may be ubiquitous at local scales. Despite the high genetic diversity observed within the most well-sampled lineage, we find that pairs of isolates share on average ~9% of their genomes in long haplotypes, even when isolates were sampled decades apart and from different locations. This proportion is several orders of magnitude higher than the Wright-Fisher expectation, raising many further questions concerning the evolutionary genetics of C. reinhardtii and microbial eukaryotes generally.

Inferring the distribution of fitness effects of spontaneous mutations in Chlamydomonas reinhardtii.

Spontaneous mutations are the source of new genetic variation and are thus central to the evolutionary process. In molecular evolution and quantitative genetics, the nature of genetic variation depends critically on the distribution of effects of mutations on fitness and other quantitative traits. Spontaneous mutation accumulation (MA) experiments have been the principal approach for investigating the overall rate of occurrence and cumulative effect of mutations but have not allowed the phenotypic effects of individual mutations to be studied directly. Here, we crossed MA lines of the green alga Chlamydomonas reinhardtii with its unmutated ancestral strain to create haploid recombinant lines, each carrying an average of 50% of the accumulated mutations in a large number of combinations. With the aid of the genome sequences of the MA lines, we inferred the genotypes of the mutations, assayed their growth rate as a measure of fitness, and inferred the distribution of fitness effects (DFE) using a Bayesian mixture model. We infer that the DFE is highly leptokurtic (L-shaped). Of mutations with absolute fitness effects exceeding 1%, about one-sixth increase fitness in the laboratory environment. The inferred distribution of effects for deleterious mutations is consistent with a strong role for nearly neutral evolution. Specifically, such a distribution predicts that nucleotide variation and genetic variation for quantitative traits will be insensitive to change in the effective population size.

Understanding the Factors That Shape Patterns of Nucleotide Diversity in the House Mouse Genome.

A major goal of population genetics has been to determine the extent by which selection at linked sites influences patterns of neutral nucleotide diversity in the genome. Multiple lines of evidence suggest that diversity is influenced by both positive and negative selection. For example, in many species there are troughs in diversity surrounding functional genomic elements, consistent with the action of either background selection (BGS) or selective sweeps. In this study, we investigated the causes of the diversity troughs that are observed in the wild house mouse genome. Using the unfolded site frequency spectrum, we estimated the strength and frequencies of deleterious and advantageous mutations occurring in different functional elements in the genome. We then used these estimates to parameterize forward-in-time simulations of chromosomes, using realistic distributions of functional elements and recombination rate variation in order to determine whether selection at linked sites can explain the observed patterns of nucleotide diversity. The simulations suggest that BGS alone cannot explain the dips in diversity around either exons or conserved noncoding elements. A combination of BGS and selective sweeps produces deeper dips in diversity than BGS alone, but the inferred parameters of selection cannot fully explain the patterns observed in the genome. Our results provide evidence of sweeps shaping patterns of nucleotide diversity across the mouse genome and also suggest that infrequent, strongly advantageous mutations play an important role in this. The limitations of using the unfolded site frequency spectrum for inferring the frequency and effects of advantageous mutations are discussed.

Inferring the Probability of the Derived vs. the Ancestral Allelic State at a Polymorphic Site.

It is known that the allele ancestral to the variation at a polymorphic site cannot be assigned with certainty, and that the most frequently used method to assign the ancestral state-maximum parsimony-is prone to misinference. Estimates of counts of sites that have a certain number of copies of the derived allele in a sample (the unfolded site frequency spectrum, uSFS) made by parsimony are therefore also biased. We previously developed a maximum likelihood method to estimate the uSFS for a focal species using information from two outgroups while assuming simple models of nucleotide substitution. Here, we extend this approach to allow multiple outgroups (implemented for three outgroups), potentially any phylogenetic tree topology, and more complex models of nucleotide substitution. We find, however, that two outgroups and the Kimura two-parameter model are adequate for uSFS inference in most cases. We show that using parsimony to infer the ancestral state at a specific site seriously breaks down in two situations. The first is where the outgroups provide no information about the ancestral state of variation in the focal species. In this case, nucleotide variation will be underestimated if such sites are excluded. The second is where the minor allele in the focal species agrees with the allelic state of the outgroups. In this situation, parsimony tends to overestimate the probability of the major allele being derived, because it fails to account for the fact that sites with a high frequency of the derived allele tend to be rare. We present a method that corrects this deficiency and is capable of providing nearly unbiased estimates of ancestral state probabilities on a site-by-site basis and the uSFS.

Fitness decline under osmotic stress in Caenorhabditis elegans populations subjected to spontaneous mutation accumulation at varying population sizes.

The consequences of mutations for population fitness depends on their individual selection coefficients and the effective population size. An earlier study of Caenorhabditis elegans spontaneous mutation accumulation lines evolved for 409 generations at three population sizes found that Ne   = 1 populations declined significantly in fitness whereas the fitness of larger populations (Ne   = 5, 50) was indistinguishable from the ancestral control under benign conditions. To test if larger MA populations harbor a load of cryptic deleterious mutations that are obscured under benign laboratory conditions, we measured fitness under osmotic stress via exposure to hypersaline conditions. The fitness of Ne   = 1 lines exhibited a further decline under osmotic stress compared to benign conditions. However, the fitness of larger populations remained indistinguishable from that of the ancestral control. The average effects of deleterious mutations in Ne   = 1 lines were estimated to be 22% for productivity and 14% for survivorship, exceeding values previously detected under benign conditions. Our results suggest that fitness decline is due to large effect mutations that are rapidly removed via selection even in small populations, with implications for conservation practices. Genetic stochasticity may not be as potent and immediate a threat to the persistence of small populations as other demographic and environmental stochastic factors.

Detecting positive selection in the genome.

Population geneticists have long sought to understand the contribution of natural selection to molecular evolution. A variety of approaches have been proposed that use population genetics theory to quantify the rate and strength of positive selection acting in a species' genome. In this review we discuss methods that use patterns of between-species nucleotide divergence and within-species diversity to estimate positive selection parameters from population genomic data. We also discuss recently proposed methods to detect positive selection from a population's haplotype structure. The application of these tests has resulted in the detection of pervasive adaptive molecular evolution in multiple species.

Fitness change in relation to mutation number in spontaneous mutation accumulation lines of Chlamydomonas reinhardtii.

Although all genetic variation ultimately stems from mutations, their properties are difficult to study directly. Here, we used multiple mutation accumulation (MA) lines derived from five genetic backgrounds of the green algae Chlamydomonas reinhardtii that have been previously subjected to whole genome sequencing to investigate the relationship between the number of spontaneous mutations and change in fitness from a nonevolved ancestor. MA lines were on average less fit than their ancestors and we detected a significantly negative correlation between the change in fitness and the total number of accumulated mutations in the genome. Likewise, the number of mutations located within coding regions significantly and negatively impacted MA line fitness. We used the fitness data to parameterize a maximum likelihood model to estimate discrete categories of mutational effects, and found that models containing one to two mutational effect categories (one neutral and one deleterious category) fitted the data best. However, the best-fitting mutational effects models were highly dependent on the genetic background of the ancestral strain.

The Recombination Landscape in Wild House Mice Inferred Using Population Genomic Data.

Characterizing variation in the rate of recombination across the genome is important for understanding several evolutionary processes. Previous analysis of the recombination landscape in laboratory mice has revealed that the different subspecies have different suites of recombination hotspots. It is unknown, however, whether hotspots identified in laboratory strains reflect the hotspot diversity of natural populations or whether broad-scale variation in the rate of recombination is conserved between subspecies. In this study, we constructed fine-scale recombination rate maps for a natural population of the Eastern house mouse, Mus musculus castaneus We performed simulations to assess the accuracy of recombination rate inference in the presence of phase errors, and we used a novel approach to quantify phase error. The spatial distribution of recombination events is strongly positively correlated between our castaneus map, and a map constructed using inbred lines derived predominantly from M. m. domesticus Recombination hotspots in wild castaneus show little overlap, however, with the locations of double-strand breaks in wild-derived house mouse strains. Finally, we also find that genetic diversity in M. m. castaneus is positively correlated with the rate of recombination, consistent with pervasive natural selection operating in the genome. Our study suggests that recombination rate variation is conserved at broad scales between house mouse subspecies, but it is not strongly conserved at fine scales.

Mitochondrial Mutation Rate, Spectrum and Heteroplasmy in Caenorhabditis elegans Spontaneous Mutation Accumulation Lines of Differing Population Size.

Mitochondrial genomes of metazoans, given their elevated rates of evolution, have served as pivotal markers for phylogeographic studies and recent phylogenetic events. In order to determine the dynamics of spontaneous mitochondrial mutations in small populations in the absence and presence of selection, we evolved mutation accumulation (MA) lines of Caenorhabditis elegans in parallel over 409 consecutive generations at three varying population sizes of N = 1, 10, and 100 hermaphrodites. The N =1 populations should have a minimal influence of natural selection to provide the spontaneous mutation rate and the expected rate of neutral evolution, whereas larger population sizes should experience increasing intensity of selection. New mutations were identified by Illumina paired-end sequencing of 86 mtDNA genomes across 35 experimental lines and compared with published genomes of natural isolates. The spontaneous mitochondrial mutation rate was estimated at 1.05 × 10-7/site/generation. A strong G/C→A/T mutational bias was observed in both the MA lines and the natural isolates. This suggests that the low G + C content at synonymous sites is the product of mutation bias rather than selection as previously proposed. The mitochondrial effective population size per worm generation was estimated to be 62. Although it was previously concluded that heteroplasmy was rare in C. elegans, the vast majority of mutations in this study were heteroplasmic despite an experimental regime exceeding 400 generations. The frequencies of frameshift and nonsynonymous mutations were negatively correlated with population size, which suggests their deleterious effects on fitness and a potent role for selection in their eradication.

Global population divergence and admixture of the brown rat (Rattus norvegicus).

Native to China and Mongolia, the brown rat (Rattus norvegicus) now enjoys a worldwide distribution. While black rats and the house mouse tracked the regional development of human agricultural settlements, brown rats did not appear in Europe until the 1500s, suggesting their range expansion was a response to relatively recent increases in global trade. We inferred the global phylogeography of brown rats using 32 k SNPs, and detected 13 evolutionary clusters within five expansion routes. One cluster arose following a southward expansion into Southeast Asia. Three additional clusters arose from two independent eastward expansions: one expansion from Russia to the Aleutian Archipelago, and a second to western North America. Westward expansion resulted in the colonization of Europe from which subsequent rapid colonization of Africa, the Americas and Australasia occurred, and multiple evolutionary clusters were detected. An astonishing degree of fine-grained clustering between and within sampling sites underscored the extent to which urban heterogeneity shaped genetic structure of commensal rodents. Surprisingly, few individuals were recent migrants, suggesting that recruitment into established populations is limited. Understanding the global population structure of R. norvegicus offers novel perspectives on the forces driving the spread of zoonotic disease, and aids in development of rat eradication programmes.

Inferring the Frequency Spectrum of Derived Variants to Quantify Adaptive Molecular Evolution in Protein-Coding Genes of Drosophila melanogaster.

Many approaches for inferring adaptive molecular evolution analyze the unfolded site frequency spectrum (SFS), a vector of counts of sites with different numbers of copies of derived alleles in a sample of alleles from a population. Accurate inference of the high-copy-number elements of the SFS is difficult, however, because of misassignment of alleles as derived vs. ancestral. This is a known problem with parsimony using outgroup species. Here we show that the problem is particularly serious if there is variation in the substitution rate among sites brought about by variation in selective constraint levels. We present a new method for inferring the SFS using one or two outgroups that attempts to overcome the problem of misassignment. We show that two outgroups are required for accurate estimation of the SFS if there is substantial variation in selective constraints, which is expected to be the case for nonsynonymous sites in protein-coding genes. We apply the method to estimate unfolded SFSs for synonymous and nonsynonymous sites in a population of Drosophila melanogaster from phase 2 of the Drosophila Population Genomics Project. We use the unfolded spectra to estimate the frequency and strength of advantageous and deleterious mutations and estimate that ∼50% of amino acid substitutions are positively selected but that <0.5% of new amino acid mutations are beneficial, with a scaled selection strength of Nes ≈ 12.

Direct Estimate of the Spontaneous Mutation Rate Uncovers the Effects of Drift and Recombination in the Chlamydomonas reinhardtii Plastid Genome.

Plastids perform crucial cellular functions, including photosynthesis, across a wide variety of eukaryotes. Since endosymbiosis, plastids have maintained independent genomes that now display a wide diversity of gene content, genome structure, gene regulation mechanisms, and transmission modes. The evolution of plastid genomes depends on an input of de novo mutation, but our knowledge of mutation in the plastid is limited to indirect inference from patterns of DNA divergence between species. Here, we use a mutation accumulation experiment, where selection acting on mutations is rendered ineffective, combined with whole-plastid genome sequencing to directly characterize de novo mutation in Chlamydomonas reinhardtii. We show that the mutation rates of the plastid and nuclear genomes are similar, but that the base spectra of mutations differ significantly. We integrate our measure of the mutation rate with a population genomic data set of 20 individuals, and show that the plastid genome is subject to substantially stronger genetic drift than the nuclear genome. We also show that high levels of linkage disequilibrium in the plastid genome are not due to restricted recombination, but are instead a consequence of increased genetic drift. One likely explanation for increased drift in the plastid genome is that there are stronger effects of genetic hitchhiking. The presence of recombination in the plastid is consistent with laboratory studies in C. reinhardtii and demonstrates that although the plastid genome is thought to be uniparentally inherited, it recombines in nature at a rate similar to the nuclear genome.