Discovery of recessive effect of human polymerase δ proofreading deficiency through mutational analysis of POLD1-mutated normal and cancer cells.

Constitutional heterozygous pathogenic variants in the exonuclease domain of POLE and POLD1, which affect the proofreading activity of the corresponding polymerases, cause a cancer predisposition syndrome characterized by increased risk of gastrointestinal polyposis, colorectal cancer, endometrial cancer and other tumor types. The generally accepted explanation for the connection between the disruption of the proofreading activity of polymerases epsilon and delta and cancer development is through an increase in the somatic mutation rate. Here we studied an extended family with multiple members heterozygous for the pathogenic POLD1 variant c.1421T>C p.(Leu474Pro), which segregates with the polyposis and cancer phenotypes. Through the analysis of mutational patterns of patient-derived fibroblasts colonies and de novo mutations obtained by parent-offspring comparisons, we concluded that heterozygous POLD1 L474P just subtly increases the somatic and germline mutation burden. In contrast, tumors developed in individuals with a heterozygous mutation in the exonuclease domain of POLD1, including L474P, have an extremely high mutation rate (>100 mut/Mb) associated with signature SBS10d. We solved this contradiction through the observation that tumorigenesis involves somatic inactivation of the wildtype POLD1 allele. These results imply that exonuclease deficiency of polymerase delta has a recessive effect on mutation rate.

Complex fitness landscape shapes variation in a hyperpolymorphic species.

It is natural to assume that patterns of genetic variation in hyperpolymorphic species can reveal large-scale properties of the fitness landscape that are hard to detect by studying species with ordinary levels of genetic variation. Here, we study such patterns in a fungus Schizophyllum commune, the most polymorphic species known. Throughout the genome, short-range linkage disequilibrium (LD) caused by attraction of minor alleles is higher between pairs of nonsynonymous than of synonymous variants. This effect is especially pronounced for pairs of sites that are located within the same gene, especially if a large fraction of the gene is covered by haploblocks, genome segments where the gene pool consists of two highly divergent haplotypes, which is a signature of balancing selection. Haploblocks are usually shorter than 1000 nucleotides, and collectively cover about 10% of the S. commune genome. LD tends to be substantially higher for pairs of nonsynonymous variants encoding amino acids that interact within the protein. There is a substantial correlation between LDs at the same pairs of nonsynonymous mutations in the USA and the Russian populations. These patterns indicate that selection in S. commune involves positive epistasis due to compensatory interactions between nonsynonymous alleles. When less polymorphic species are studied, analogous patterns can be detected only through interspecific comparisons.

Changes to DNA known as mutations may alter how the proteins and other components of a cell work, and thus play an important role in allowing living things to evolve new traits and abilities over many generations. Whether a mutation is beneficial or harmful may differ depending on the genetic background of the individual ­ that is, depending on other mutations present in other positions within the same gene ­ due to a phenomenon called epistasis. Epistasis is known to affect how various species accumulate differences in their DNA compared to each other over time. For example, a mutation that is rare in humans and known to cause disease may be widespread in other primates because its negative effect is canceled out by another mutation that is standard for these species but absent in humans. However, it remains unclear whether epistasis plays a significant part in shaping genetic differences between individuals of the same species. A type of fungus known as Schizophyllum commune lives on rotting wood and is found across the world. It is one of the most genetically diverse species currently known, so there is a higher chance of pairs of compensatory mutations occurring and persisting for a long time in S. commune than in most other species, providing a unique opportunity to study epistasis. Here, Stolyarova et al. studied two distinct populations of S. commune, one from the USA and one from Russia. The team found that ­ unlike in humans, flies and other less genetically diverse species ­ epistasis maintains combinations of mutations in S. commune that individually would be harmful to the fungus but together compensate for each other. For example, pairs of mutations affecting specific molecules known as amino acids ­ the building blocks of proteins ­ that physically interact with each other tended to be found together in the same individuals. One potential downside of having pairs of compensatory mutations in the genome is that when the organism reproduces, the process of making sex cells may split up these pairs so that harmful mutations are inherited without their partner mutations. Thus, epistasis may have helped shape the way S. commune and other genetically diverse species have evolved.

Characteristics and possible mechanisms of formation of microinversions distinguishing human and chimpanzee genomes.

Genomic inversions come in various sizes. While long inversions are relatively easy to identify by aligning high-quality genome sequences, unambiguous identification of microinversions is more problematic. Here, using a set of extra stringent criteria to distinguish microinversions from other mutational events, we describe microinversions that occurred after the divergence of humans and chimpanzees. In total, we found 59 definite microinversions that range from 17 to 33 nucleotides in length. In majority of them, human genome sequences matched exactly the reverse-complemented chimpanzee genome sequences, implying that the inverted DNA segment was copied precisely. All these microinversions were flanked by perfect or nearly perfect inverted repeats pointing to their key role in their formation. Template switching at inverted repeats during DNA replication was previously discussed as a possible mechanism for the microinversion formation. However, many of definite microinversions found by us cannot be easily explained via template switching owing to the combination of the short length and imperfect nature of their flanking inverted repeats. We propose a novel, alternative mechanism that involves repair of a double-stranded break within the inverting segment via microhomology-mediated break-induced replication, which can consistently explain all definite microinversion events.

Two linked loci under mutation-selection balance and Muller's ratchet.

We report complete analysis of a deterministic model of deleterious mutations and negative selection against them at two haploid loci without recombination. As long as mutation is a weaker force than selection, mutant alleles remain rare at the only stable equilibrium, and otherwise, a variety of dynamics are possible. If the mutation-free genotype is absent, generally the only stable equilibrium is the one that corresponds to fixation of the mutant allele at the locus where it is less deleterious. This suggests that fixation of a deleterious allele that follows a click of the Muller's ratchet is governed by natural selection, instead of random drift.

Genomic signatures of recombination in a natural population of the bdelloid rotifer Adineta vaga.

Sexual reproduction is almost ubiquitous among extant eukaryotes. As most asexual lineages are short-lived, abandoning sex is commonly regarded as an evolutionary dead end. Still, putative anciently asexual lineages challenge this view. One of the most striking examples are bdelloid rotifers, microscopic freshwater invertebrates believed to have completely abandoned sexual reproduction tens of Myr ago. Here, we compare whole genomes of 11 wild-caught individuals of the bdelloid rotifer Adineta vaga and present evidence that some patterns in its genetic variation are incompatible with strict clonality and lack of genetic exchange. These patterns include genotype proportions close to Hardy-Weinberg expectations within loci, lack of linkage disequilibrium between distant loci, incongruent haplotype phylogenies across the genome, and evidence for hybridization between divergent lineages. Analysis of triallelic sites independently corroborates these findings. Our results provide evidence for interindividual genetic exchange and recombination in A. vaga, a species previously thought to be anciently asexual.

Rapid Accumulation of Mutations in Growing Mycelia of a Hypervariable Fungus Schizophyllum commune.

The basidiomycete Schizophyllum commune has the highest level of genetic polymorphism known among living organisms. In a previous study, it was also found to have a moderately high per-generation mutation rate of 2×10-8, likely contributing to its high polymorphism. However, this rate has been measured only in an experiment on Petri dishes, and it is unclear how it translates to natural populations. Here, we used an experimental design that measures the rate of accumulation of de novo mutations in a linearly growing mycelium. We show that S. commune accumulates mutations at a rate of 1.24×10-7 substitutions per nucleotide per meter of growth, or ∼2.04×10-11 per nucleotide per cell division. In contrast to what has been observed in a number of species with extensive vegetative growth, this rate does not decline in the course of propagation of a mycelium. As a result, even a moderate per-cell-division mutation rate in S. commune can translate into a very high per-generation mutation rate when the number of cell divisions between consecutive meiosis is large.

Genetics of Adaptation of the Ascomycetous Fungus Podospora anserina to Submerged Cultivation.

Podospora anserina is a model ascomycetous fungus which shows pronounced phenotypic senescence when grown on solid medium but possesses unlimited lifespan under submerged cultivation. In order to study the genetic aspects of adaptation of P. anserina to submerged cultivation, we initiated a long-term evolution experiment. In the course of the first 4 years of the experiment, 125 single-nucleotide substitutions and 23 short indels were fixed in eight independently evolving populations. Six proteins that affect fungal growth and development evolved in more than one population; in particular, in the G-protein alpha subunit FadA, new alleles fixed in seven out of eight experimental populations, and these fixations affected just four amino acid sites, which is an unprecedented level of parallelism in experimental evolution. Parallel evolution at the level of genes and pathways, an excess of nonsense and missense substitutions, and an elevated conservation of proteins and their sites where the changes occurred suggest that many of the observed fixations were adaptive and driven by positive selection.

Architecture of Parallel Adaptation in Ten Lacustrine Threespine Stickleback Populations from the White Sea Area.

Adaptation of threespine stickleback to freshwater involves parallel recruitment of freshwater alleles in clusters of closely linked sites, or divergence islands (DIs). However, it remains unclear to what extent the DIs and the alleles that constitute them coincide between populations that underwent adaptation to freshwater independently. We examine threespine sticklebacks from ten freshwater lakes that emerged 500-1500 years ago in the White Sea basin, with the emphasis on repeatability of genomic patterns of adaptation among the lake populations and the role of local recombination rate in the distribution and structure of DIs. The 65 detected DIs are clustered in the genome, forming 12 aggregations, and this clustering cannot be explained by the variation of the recombination rate. Only 21 of the DIs are present in all the freshwater populations, likely being indispensable for successful colonization of freshwater environment by the ancestral marine population. Within most DIs, the same set of single nucleotide polymorphisms (SNPs) distinguish marine and freshwater haplotypes in all the lake populations; however, in some DIs, freshwater alleles differ between populations, suggesting that they could have been established by recruitment of different haplotypes in different populations.

Bursts of amino acid replacements in protein evolution.

Evolution can occur both gradually and through alternating episodes of stasis and rapid changes. However, the prevalence and magnitude of fluctuations of the rate of evolution remain obscure. Detecting a rapid burst of changes requires a detailed record of past evolution, so that events that occurred within a short time interval can be identified. Here, we use the phylogenies of the Baikal Lake amphipods and of Catarrhini, which contain very short internal edges which make this task feasible. We detect six salient bursts of evolution of individual proteins during such short time periods, each involving between six and 38 amino acid substitutions. These bursts were extremely unlikely to have occurred neutrally, and were apparently caused by positive selection. On average, in the course of a time interval required for one synonymous substitution per site, a protein undergoes a strong burst of rapid evolution with probability at least approximately 0.01.

Prevalence of loss-of-function alleles does not correlate with lifetime fecundity and other life-history traits in metazoans.

BACKGROUND: Natural selection is possible only because all species produce more offsprings than what is needed to maintain the population. Still, the lifetime number of offspring varies widely across species. One may expect natural selection to be stronger in high-fecundity species. Alternatively, natural selection could be stronger in species where a female invests more into an individual offspring. This issue needed to be addressed empirically. RESULTS: We analyzed the prevalence of loss-of-function alleles in 35 metazoan species and have found that the strength of negative selection does not correlate with lifetime fecundity or other life-history traits. CONCLUSIONS: Higher random mortality in high-fecundity species may negate the effect of increased opportunity for selection. Perhaps, invariance of the strength of negative selection across a wide variety of species emerges because natural selection optimized the life history in each of them, leading to the strongest possible competition. REVIEWERS: This article was reviewed by Nicolas Galtier and I. King Jordan.

Through Sex, Nature Is Telling Us Something Important.

Theoretically, a variety of mechanisms can make amphimixis advantageous due to reshuffling of offspring genotypes. Recently, it has been shown experimentally that some of these mechanisms can indeed work in artificial populations. However, we still do not know which of them, if any, are relevant in nature, and the available indirect estimates seem to suggest that neither negative nor positive selection in natural populations is strong enough to provide evolutionary protection for obligate amphimixis. Thus, progress in understanding the evolution of amphimixis will depend on direct measurements of the strength of natural selection.

Are Nonsense Alleles of Drosophila melanogaster Genes under Any Selection?

A gene which carries a bona fide loss-of-function mutation effectively becomes a functionless pseudogene, free from selective constraint. However, there is a number of molecular mechanisms that may lead to at least a partial preservation of the function of genes carrying even drastic alleles. We performed a direct measurement of the strength of negative selection acting on nonsense alleles of protein-coding genes in the Zambian population of Drosophila melanogaster. Within those exons that carry nonsense mutations, negative selection, assayed by the ratio of missense over synonymous nucleotide diversity levels, appears to be absent, consistent with total loss of function. In other exons of nonsense alleles, negative selection was deeply relaxed but likely not completely absent, and the per site number of missense alleles declined significantly with the distance from the premature stop codon. This pattern may be due to alternative splicing which preserves function of some isoforms of nonsense alleles of genes.

Negative selection in humans and fruit flies involves synergistic epistasis.

Negative selection against deleterious alleles produced by mutation influences within-population variation as the most pervasive form of natural selection. However, it is not known whether deleterious alleles affect fitness independently, so that cumulative fitness loss depends exponentially on the number of deleterious alleles, or synergistically, so that each additional deleterious allele results in a larger decrease in relative fitness. Negative selection with synergistic epistasis should produce negative linkage disequilibrium between deleterious alleles and, therefore, an underdispersed distribution of the number of deleterious alleles in the genome. Indeed, we detected underdispersion of the number of rare loss-of-function alleles in eight independent data sets from human and fly populations. Thus, selection against rare protein-disrupting alleles is characterized by synergistic epistasis, which may explain how human and fly populations persist despite high genomic mutation rates.

High-quality genome assembly of Capsella bursa-pastoris reveals asymmetry of regulatory elements at early stages of polyploid genome evolution.

Polyploidization and subsequent sub- and neofunctionalization of duplicated genes represent a major mechanism of plant genome evolution. Capsella bursa-pastoris, a widespread ruderal plant, is a recent allotetraploid and, thus, is an ideal model organism for studying early changes following polyploidization. We constructed a high-quality assembly of C. bursa-pastoris genome and a transcriptome atlas covering a broad sample of organs and developmental stages (available online at http://travadb.org/browse/Species=Cbp). We demonstrate that expression of homeologs is mostly symmetric between subgenomes, and identify a set of homeolog pairs with discordant expression. Comparison of promoters within such pairs revealed emerging asymmetry of regulatory elements. Among them there are multiple binding sites for transcription factors controlling the regulation of photosynthesis and plant development by light (PIF3, HY5) and cold stress response (CBF). These results suggest that polyploidization in C. bursa-pastoris enhanced its plasticity of response to light and temperature, and allowed substantial expansion of its distribution range.

Transcriptome-based phylogeny of endemic Lake Baikal amphipod species flock: fast speciation accompanied by frequent episodes of positive selection.

Endemic species flocks inhabiting ancient lakes, oceanic islands and other long-lived isolated habitats are often interpreted as adaptive radiations. Yet molecular evidence for directional selection during species flocks radiation is scarce. Using partial transcriptomes of 64 species of Lake Baikal (Siberia, Russia) endemic amphipods and two nonendemic outgroups, we report a revised phylogeny of this species flock and analyse evidence for positive selection within the endemic lineages. We confirm two independent invasions of amphipods into Baikal and demonstrate that several morphological features of Baikal amphipods, such as body armour and reduction in appendages and sensory organs, evolved in several lineages in parallel. Radiation of Baikal amphipods has been characterized by short phylogenetic branches and frequent episodes of positive selection which tended to be more frequent in the early phase of the second invasion of amphipods into Baikal when the most intensive diversification occurred. Notably, signatures of positive selection are frequent in genes encoding mitochondrial membrane proteins with electron transfer chain and ATP synthesis functionality. In particular, subunits of both the membrane and substrate-level ATP synthases show evidence of positive selection in the plankton species Macrohectopus branickii, possibly indicating adaptation to active plankton lifestyle and to survival under conditions of low temperature and high hydrostatic pressures known to affect membranes functioning. Other functional categories represented among genes likely to be under positive selection include Ca-binding muscle-related proteins, possibly indicating adaptation to Ca-deficient low mineralization Baikal waters.

Local fitness landscape of the green fluorescent protein.

Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.

Extraordinary Genetic Diversity in a Wood Decay Mushroom.

Populations of different species vary in the amounts of genetic diversity they possess. Nucleotide diversity π, the fraction of nucleotides that are different between two randomly chosen genotypes, has been known to range in eukaryotes between 0.0001 in Lynx lynx and 0.16 in Caenorhabditis brenneri. Here, we report the results of a comparative analysis of 24 haploid genotypes (12 from the United States and 12 from European Russia) of a split-gill fungus Schizophyllum commune. The diversity at synonymous sites is 0.20 in the American population of S. commune and 0.13 in the Russian population. This exceptionally high level of nucleotide diversity also leads to extreme amino acid diversity of protein-coding genes. Using whole-genome resequencing of 2 parental and 17 offspring haploid genotypes, we estimate that the mutation rate in S. commune is high, at 2.0 × 10(-8) (95% CI: 1.1 × 10(-8) to 4.1 × 10(-8)) per nucleotide per generation. Therefore, the high diversity of S. commune is primarily determined by its elevated mutation rate, although high effective population size likely also plays a role. Small genome size, ease of cultivation and completion of the life cycle in the laboratory, free-living haploid life stages and exceptionally high variability of S. commune make it a promising model organism for population, quantitative, and evolutionary genetics.

Comparative genome analysis of Pseudogymnoascus spp. reveals primarily clonal evolution with small genome fragments exchanged between lineages.

BACKGROUND: Pseudogymnoascus spp. is a wide group of fungi lineages in the family Pseudorotiaceae including an aggressive pathogen of bats P. destructans. Although several lineages of P. spp. were shown to produce ascospores in culture, the vast majority of P. spp. demonstrates no evidence of sexual reproduction. P. spp. can tolerate a wide range of different temperatures and salinities and can survive even in permafrost layer. Adaptability of P. spp. to different environments is accompanied by extremely variable morphology and physiology. RESULTS: We sequenced genotypes of 14 strains of P. spp., 5 of which were extracted from permafrost, 1 from a cryopeg, a layer of unfrozen ground in permafrost, and 8 from temperate surface environments. All sequenced genotypes are haploid. Nucleotide diversity among these genomes is very high, with a typical evolutionary distance at synonymous sites dS ≈ 0.5, suggesting that the last common ancestor of these strains lived >50 Mya. The strains extracted from permafrost do not form a separate clade. Instead, each permafrost strain has close relatives from temperate environments. We observed a strictly clonal population structure with no conflicting topologies for ~99% of genome sequences. However, there is a number of short (~100-10,000 nt) genomic segments with the total length of 67.6 Kb which possess phylogenetic patterns strikingly different from the rest of the genome. The most remarkable case is a MAT-locus, which has 2 distinct alleles interspersed along the whole-genome phylogenetic tree. CONCLUSIONS: Predominantly clonal structure of genome sequences is consistent with the observations that sexual reproduction is rare in P. spp. Small number of regions with noncanonical phylogenies seem to arise due to some recombination events between derived lineages of P. spp., with MAT-locus being transferred on multiple occasions. All sequenced strains have heterothallic configuration of MAT-locus.

Fast evolution from precast bricks: genomics of young freshwater populations of threespine stickleback Gasterosteus aculeatus.

Adaptation is driven by natural selection; however, many adaptations are caused by weak selection acting over large timescales, complicating its study. Therefore, it is rarely possible to study selection comprehensively in natural environments. The threespine stickleback (Gasterosteus aculeatus) is a well-studied model organism with a short generation time, small genome size, and many genetic and genomic tools available. Within this originally marine species, populations have recurrently adapted to freshwater all over its range. This evolution involved extensive parallelism: pre-existing alleles that adapt sticklebacks to freshwater habitats, but are also present at low frequencies in marine populations, have been recruited repeatedly. While a number of genomic regions responsible for this adaptation have been identified, the details of selection remain poorly understood. Using whole-genome resequencing, we compare pooled genomic samples from marine and freshwater populations of the White Sea basin, and identify 19 short genomic regions that are highly divergent between them, including three known inversions. 17 of these regions overlap protein-coding genes, including a number of genes with predicted functions that are relevant for adaptation to the freshwater environment. We then analyze four additional independently derived young freshwater populations of known ages, two natural and two artificially established, and use the observed shifts of allelic frequencies to estimate the strength of positive selection. Adaptation turns out to be quite rapid, indicating strong selection acting simultaneously at multiple regions of the genome, with selection coefficients of up to 0.27. High divergence between marine and freshwater genotypes, lack of reduction in polymorphism in regions responsible for adaptation, and high frequencies of freshwater alleles observed even in young freshwater populations are all consistent with rapid assembly of G. aculeatus freshwater genotypes from pre-existing genomic regions of adaptive variation, with strong selection that favors this assembly acting simultaneously at multiple loci.