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Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life 1,2 . Here we report on how an engineered minimal cell 3,4 contends with the forces of evolution compared with the Mycoplasma mycoides non-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations in ftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology 5,6 . Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells 2,7-9 ., (© 2023. The Author(s).)

Most aspects of the molecular biology of cells involve tightly coordinated intermolecular interactions requiring specific recognition at the nucleotide and/or amino acid levels. This has led to long-standing interest in the degree to which constraints on interacting molecules result in conserved vs. accelerated rates of sequence evolution, with arguments commonly being made that molecular coevolution can proceed at rates exceeding the neutral expectation. Here, a fairly general model is introduced to evaluate the degree to which the rate of evolution at functionally interacting sites is influenced by effective population sizes ( N e ), mutation rates, strength of selection, and the magnitude of recombination between sites. This theory is of particular relevance to matters associated with interactions between organelle- and nuclear-encoded proteins, as the two genomic environments often exhibit dramatic differences in the power of mutation and drift. Although genes within low N e environments can drive the rate of evolution of partner genes experiencing higher N e , rates exceeding the neutral expectation require that the former also have an elevated mutation rate. Testable predictions, some counterintuitive, are presented on how patterns of coevolutionary rates should depend on the relative intensities of drift, selection, and mutation.

It has previously been shown that, conditional on its fixation, the time to fixation of a semi-dominant deleterious autosomal mutation in a randomly mating population is the same as that of an advantageous mutation. This result implies that deleterious mutations could generate selective sweep-like effects. Although their fixation probabilities greatly differ, the much larger input of deleterious relative to beneficial mutations suggests that this phenomenon could be important. We here examine how the fixation of mildly deleterious mutations affects levels and patterns of polymorphism at linked sites-both in the presence and absence of interference amongst deleterious mutations-and how this class of sites may contribute to divergence between-populations and species. We find that, while deleterious fixations are unlikely to represent a significant proportion of outliers in polymorphism-based genomic scans within populations, minor shifts in the frequencies of deleterious mutations can influence the proportions of private variants and the value of FST after a recent population split. As sites subject to deleterious mutations are necessarily found in functional genomic regions, interpretations in terms of recurrent positive selection may require reconsideration., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A majority of cellular proteins function as part of multimeric complexes of two or more subunits. Multimer formation requires interactions between protein surfaces that lead to closed structures, such as dimers and tetramers. If proteins interact in an open-ended way, uncontrolled growth of fibrils can occur, which is likely to be detrimental in most cases. We present a statistical physics model that allows aggregation of proteins as either closed dimers or open fibrils of all lengths. We use pairwise amino-acid contact energies to calculate the energies of interacting protein surfaces. The probabilities of all possible aggregate configurations can be calculated for any given sequence of surface amino acids. We link the statistical physics model to a population genetics model that describes the evolution of the surface residues. When proteins evolve neutrally, without selection for or against multimer formation, we find that a majority of proteins remain as monomers at moderate concentrations, but strong dimer-forming or fibril-forming sequences are also possible. If selection is applied in favor of dimers or in favor of fibrils, then it is easy to select either dimer-forming or fibril-forming sequences. It is also possible to select for oriented fibrils with protein subunits all aligned in the same direction. We measure the propensities of amino acids to occur at interfaces relative to noninteracting surfaces and show that the propensities in our model are strongly correlated with those that have been measured in real protein structures. We also show that there are significant differences between amino acid frequencies at isologous and heterologous interfaces in our model, and we observe that similar effects occur in real protein structures.

The evolution of mitochondrial genomes and their population-genetic environment among unicellular eukaryotes are understudied. Ciliate mitochondrial genomes exhibit a unique combination of characteristics, including a linear organization and the presence of multiple genes with no known function or detectable homologs in other eukaryotes. Here we study the variation of ciliate mitochondrial genomes both within and across 13 highly diverged Paramecium species, including multiple species from the P. aurelia species complex, with four outgroup species: P. caudatum, P. multimicronucleatum, and two strains that may represent novel related species. We observe extraordinary conservation of gene order and protein-coding content in Paramecium mitochondria across species. In contrast, significant differences are observed in tRNA content and copy number, which is highly conserved in species belonging to the P. aurelia complex but variable among and even within the other Paramecium species. There is an increase in GC content from ∼20% to ∼40% on the branch leading to the P. aurelia complex. Patterns of polymorphism in population-genomic data and mutation-accumulation experiments suggest that the increase in GC content is primarily due to changes in the mutation spectra in the P. aurelia species. Finally, we find no evidence of recombination in Paramecium mitochondria and find that the mitochondrial genome appears to experience either similar or stronger efficacy of purifying selection than the nucleus., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

A recent article reassessing the Neutral Theory of Molecular Evolution claims that it is no longer as important as is widely believed. The authors argue that "the neutral theory was supported by unreliable theoretical and empirical evidence from the beginning, and that in light of modern, genome-scale data, we can firmly reject its universality." Claiming that "the neutral theory has been overwhelmingly rejected," they propose instead that natural selection is the major force shaping both between-species divergence and within-species variation. Although this is probably a minority view, it is important to evaluate such claims carefully in the context of current knowledge, as inaccuracies can sometimes morph into an accepted narrative for those not familiar with the underlying science. We here critically examine and ultimately reject Kern and Hahn's arguments and assessment, and instead propose that it is now abundantly clear that the foundational ideas presented five decades ago by Kimura and Ohta are indeed correct., (© 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.)

How genetic variation is generated and maintained remains a central question in evolutionary biology. When presented with a complex environment, microbes can take advantage of genetic variation to exploit new niches. Here we present a massively parallel experiment where WT and repair-deficient ( ∆mutL ) Escherichia coli populations have evolved over 3 y in a spatially heterogeneous and nutritionally complex environment. Metagenomic sequencing revealed that these initially isogenic populations evolved and maintained stable subpopulation structure in just 10 mL of medium for up to 10,000 generations, consisting of up to five major haplotypes with many minor haplotypes. We characterized the genomic, transcriptomic, exometabolomic, and phenotypic differences between clonal isolates, revealing subpopulation structure driven primarily by spatial segregation followed by differential utilization of nutrients. In addition to genes regulating the import and catabolism of nutrients, major polymorphisms of note included insertion elements transposing into fimE (regulator of the type I fimbriae) and upstream of hns (global regulator of environmental-change and stress-response genes), both known to regulate biofilm formation. Interestingly, these genes have also been identified as critical to colonization in uropathogenic E. coli infections. Our findings illustrate the complexity that can arise and persist even in small cultures, raising the possibility that infections may often be promoted by an evolving and complex pathogen population., Competing Interests: The authors declare no conflict of interest.

Mutations contribute to genetic variation in all living systems. Thus, precise estimates of mutation rates and spectra across a diversity of organisms are required for a full comprehension of evolution. Here, a mutation-accumulation (MA) assay was carried out on the endosymbiotic bacterium Teredinibacter turnerae. After ∼3,025 generations, base-pair substitutions (BPSs) and insertion-deletion (indel) events were characterized by whole-genome sequencing analysis of 47 independent MA lines, yielding a BPS rate of 1.14 × 10-9 per site per generation and indel rate of 1.55 × 10-10 events per site per generation, which are among the highest within free-living and facultative intracellular bacteria. As in other endosymbionts, a significant bias of BPSs toward A/T and an excess of deletion mutations over insertion mutations are observed for these MA lines. However, even with a deletion bias, the genome remains relatively large (∼5.2 Mb) for an endosymbiotic bacterium. The estimate of the effective population size (Ne) in T. turnerae is quite high and comparable to free-living bacteria (∼4.5 × 107), suggesting that the heavy bottlenecking associated with many endosymbiotic relationships is not prevalent during the life of this endosymbiont. The efficiency of selection scales with increasing Ne and such strong selection may have been operating against the deletion bias, preventing genome erosion. The observed mutation rate in this endosymbiont is of the same order of magnitude of those with similar Ne, consistent with the idea that population size is a primary determinant of mutation-rate evolution within endosymbionts, and that not all endosymbionts have low Ne.

One of the long-standing mysteries of evolutionary genomics is the source of the wide phylogenetic diversity in genome nucleotide composition (G + C versus A + T), which must be a consequence of interspecific differences in mutation bias, the efficiency of selection for different nucleotides or a combination of the two. We demonstrate that although genomic G + C composition is strongly driven by mutation bias, it is also substantially modified by direct selection and/or as a by-product of biased gene conversion. Moreover, G + C composition at fourfold redundant sites is consistently elevated above the neutral expectation-more so than for any other class of sites.

Using data from 83 isolates from a single population, the population genomics of the microcrustacean Daphnia pulex are described and compared to current knowledge for the only other well-studied invertebrate, Drosophila melanogaster These two species are quite similar with respect to effective population sizes and mutation rates, although some features of recombination appear to be different, with linkage disequilibrium being elevated at short ([Formula: see text] bp) distances in D. melanogaster and at long distances in D. pulex The study population adheres closely to the expectations under Hardy-Weinberg equilibrium, and reflects a past population history of no more than a twofold range of variation in effective population size. Fourfold redundant silent sites and a restricted region of intronic sites appear to evolve in a nearly neutral fashion, providing a powerful tool for population genetic analyses. Amino acid replacement sites are predominantly under strong purifying selection, as are a large fraction of sites in UTRs and intergenic regions, but the majority of SNPs at such sites that rise to frequencies [Formula: see text] appear to evolve in a nearly neutral fashion. All forms of genomic sites (including replacement sites within codons, and intergenic and UTR regions) appear to be experiencing an [Formula: see text] higher level of selection scaled to the power of drift in D. melanogaster , but this may in part be a consequence of recent demographic changes. These results establish D. pulex as an excellent system for future work on the evolutionary genomics of natural populations., (Copyright © 2017 by the Genetics Society of America.)

Mutation is the ultimate source of all genetic variation and is, therefore, central to evolutionary change. Previous work on Paramecium tetraurelia found an unusually low germline base-substitution mutation rate in this ciliate. Here, we tested the generality of this result among ciliates using Tetrahymena thermophila. We sequenced the genomes of 10 lines of T. thermophila that had each undergone approximately 1,000 generations of mutation accumulation (MA). We applied an existing mutation-calling pipeline and developed a new probabilistic mutation detection approach that directly models the design of an MA experiment and accommodates the noise introduced by mismapped reads. Our probabilistic mutation-calling method provides a straightforward way of estimating the number of sites at which a mutation could have been called if one was present, providing the denominator for our mutation rate calculations. From these methods, we find that T. thermophila has a germline base-substitution mutation rate of 7.61 × 10 - 12 per-site, per cell division, which is consistent with the low base-substitution mutation rate in P. tetraurelia. Over the course of the evolution experiment, genomic exclusion lines derived from the MA lines experienced a fitness decline that cannot be accounted for by germline base-substitution mutations alone, suggesting that other genetic or epigenetic factors must be involved. Because selection can only operate to reduce mutation rates based upon the "visible" mutational load, asexual reproduction with a transcriptionally silent germline may allow ciliates to evolve extremely low germline mutation rates., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Although the human germline mutation rate is higher than that in any other well-studied species, the rate is not exceptional once the effective genome size and effective population size are taken into consideration. Human somatic mutation rates are substantially elevated above those in the germline, but this is also seen in other species. What is exceptional about humans is the recent detachment from the challenges of the natural environment and the ability to modify phenotypic traits in ways that mitigate the fitness effects of mutations, e.g., precision and personalized medicine. This results in a relaxation of selection against mildly deleterious mutations, including those magnifying the mutation rate itself. The long-term consequence of such effects is an expected genetic deterioration in the baseline human condition, potentially measurable on the timescale of a few generations in westernized societies, and because the brain is a particularly large mutational target, this is of particular concern. Ultimately, the price will have to be covered by further investment in various forms of medical intervention. Resolving the uncertainties of the magnitude and timescale of these effects will require the establishment of stable, standardized, multigenerational measurement procedures for various human traits., (Copyright © 2016 by the Genetics Society of America.)

An enduring mystery of evolutionary genomics concerns the mechanisms responsible for lineage-specific expansions of genome size in eukaryotes, especially in multicellular species. One idea is that all excess DNA is mutationally hazardous, but weakly enough so that genome-size expansion passively emerges in species experiencing relatively low efficiency of selection owing to small effective population sizes. Another idea is that substantial gene additions were impossible without the energetic boost provided by the colonizing mitochondrion in the eukaryotic lineage. Contrary to this latter view, analysis of cellular energetics and genomics data from a wide variety of species indicates that, relative to the lifetime ATP requirements of a cell, the costs of a gene at the DNA, RNA, and protein levels decline with cell volume in both bacteria and eukaryotes. Moreover, these costs are usually sufficiently large to be perceived by natural selection in bacterial populations, but not in eukaryotes experiencing high levels of random genetic drift. Thus, for scaling reasons that are not yet understood, by virtue of their large size alone, eukaryotic cells are subject to a broader set of opportunities for the colonization of novel genes manifesting weakly advantageous or even transiently disadvantageous phenotypic effects. These results indicate that the origin of the mitochondrion was not a prerequisite for genome-size expansion.

Many cellular functions depend on highly specific intermolecular interactions, for example transcription factors and their DNA binding sites, microRNAs and their RNA binding sites, the interfaces between heterodimeric protein molecules, the stems in RNA molecules, and kinases and their response regulators in signal-transduction systems. Despite the need for complementarity between interacting partners, such pairwise systems seem to be capable of high levels of evolutionary divergence, even when subject to strong selection. Such behavior is a consequence of the diminishing advantages of increasing binding affinity between partners, the multiplicity of evolutionary pathways between selectively equivalent alternatives, and the stochastic nature of evolutionary processes. Because mutation pressure toward reduced affinity conflicts with selective pressure for greater interaction, situations can arise in which the expected distribution of the degree of matching between interacting partners is bimodal, even in the face of constant selection. Although biomolecules with larger numbers of interacting partners are subject to increased levels of evolutionary conservation, their more numerous partners need not converge on a single sequence motif or be increasingly constrained in more complex systems. These results suggest that most phylogenetic differences in the sequences of binding interfaces are not the result of adaptive fine tuning but a simple consequence of random genetic drift.

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.

Despite much theoretical work, the molecular-genetic causes and evolutionary consequences of asexuality remain largely undetermined. Asexual animal species are rare, evolutionarily short-lived, and thought to suffer mutational meltdown as a result of lack of recombination. Whole-genome analysis of 11 sexual and 11 asexual genotypes of Daphnia pulex indicates that current asexual lineages are in fact very young, exhibit no signs of purifying selection against accumulating mutations, and have extremely high rates of gene conversion and deletion. The reconstruction of chromosomal haplotypes in regions containing SNP markers associated with asexuality (chromosomes VIII and IX) indicates that introgression from a sister species, Daphnia pulicaria, underlies the origin of the asexual phenotype. Silent-site divergence of the shared chromosomal haplotypes of asexuals indicates that the spread of asexuality is as recent as 1,250 y, although the origin of the meiosis-suppressing element or elements could be substantially older. In addition, using previous estimates of the gene conversion rate from Daphnia mutation accumulation lines, we are able to age each asexual lineage. Although asexual lineages originate from wide crosses that introduce elevated individual heterozygosities on clone foundation, they also appear to be constrained by the inbreeding-like effect of loss of heterozygosity that accrues as gene conversion and hemizygous deletion expose preexisting recessive deleterious alleles of asexuals, limiting their evolutionary longevity. Our study implies that the buildup of newly introduced deleterious mutations (i.e., Muller's ratchet) may not be the dominant force imperiling nonrecombining populations of D. pulex, as previously proposed.

One of the most striking features of proteins is their common assembly into multimeric structures, usually homomers with even numbers of subunits all derived from the same genetic locus. However, although substantial structural variation for orthologous proteins exists within and among major phylogenetic lineages, in striking contrast to patterns of gene structure and genome organization, there appears to be no correlation between the level of protein structural complexity and organismal complexity. In addition, there is no evidence that protein architectural differences are driven by lineage-specific differences in selective pressures. Here, it is suggested that variation in the multimeric states of proteins can readily arise from stochastic transitions resulting from the joint processes of mutation and random genetic drift, even in the face of constant directional selection for one particular protein architecture across all lineages. Under the proposed hypothesis, on a long evolutionary timescale, the numbers of transitions from monomers to dimers should approximate the numbers in the opposite direction and similarly for transitions between higher-order structures.

It has been argued that the evolution of plant genome size is principally unidirectional and increasing owing to the varied action of whole-genome duplications (WGDs) and mobile element proliferation. However, extreme genome size reductions have been reported in the angiosperm family tree. Here we report the sequence of the 82-megabase genome of the carnivorous bladderwort plant Utricularia gibba. Despite its tiny size, the U. gibba genome accommodates a typical number of genes for a plant, with the main difference from other plant genomes arising from a drastic reduction in non-genic DNA. Unexpectedly, we identified at least three rounds of WGD in U. gibba since common ancestry with tomato (Solanum) and grape (Vitis). The compressed architecture of the U. gibba genome indicates that a small fraction of intergenic DNA, with few or no active retrotransposons, is sufficient to regulate and integrate all the processes required for the development and reproduction of a complex organism.

Enormous phylogenetic variation exists in the number and sizes of introns in protein-coding genes. Although some consideration has been given to the underlying role of the population-genetic environment in defining such patterns, the influence of the intracellular environment remains virtually unexplored. Drawing from observations on interactions between co-transcriptional processes involved in splicing and mRNA 3'-end formation, a mechanistic model is proposed for splice-site recognition that challenges the commonly accepted intron- and exon-definition models. Under the suggested model, splicing factors that outcompete 3'-end processing factors for access to intronic binding sites concurrently favor the recruitment of 3'-end processing factors at the pre-mRNA tail. This hypothesis sheds new light on observations such as the intron-mediated enhancement of gene expression and the negative correlation between intron length and levels of gene expression., (© 2013 WILEY Periodicals, Inc.)

Although observations from biochemistry and cell biology seemingly illustrate hundreds of examples of exquisite molecular adaptations, the fact that experimental manipulation can often result in improvements in cellular infrastructure raises the question as to what ultimately limits the level of molecular perfection achievable by natural selection. Here, it is argued that random genetic drift can impose a strong barrier to the advancement of molecular refinements by adaptive processes. Moreover, although substantial improvements in fitness may sometimes be accomplished via the emergence of novel cellular features that improve on previously established mechanisms, such advances are expected to often be transient, with overall fitness eventually returning to the level before incorporation of the genetic novelty. As a consequence of such changes, increased molecular/cellular complexity can arise by Darwinian processes, while yielding no long-term increase in adaptation and imposing increased energetic and mutational costs.

Although the mechanisms by which complex cellular features evolve constitute one of the great unsolved problems of evolutionary biology, it is clear that the emergence of new protein-protein interactions, often accompanied by the diversification of duplicate genes, is involved. Using information on the levels of protein multimerization in major phylogenetic groups as a guide to the patterns that must be explained and relying on results from population-genetic theory to define the relative plausibility of alternative evolutionary pathways, a framework for understanding the evolution of dimers is developed. The resultant theory demonstrates that the likelihoods of alternative pathways for the emergence of protein complexes depend strongly on the effective population size. Nonetheless, it is equally clear that further advancements in this area will require comparative studies on the fitness consequences of alternative monomeric and dimeric proteins.

The boundaries between prokaryotes, unicellular eukaryotes and multicellular eukaryotes are accompanied by orders-of-magnitude reductions in effective population size, with concurrent amplifications of the effects of random genetic drift and mutation. The resultant decline in the efficiency of selection seems to be sufficient to influence a wide range of attributes at the genomic level in a non-adaptive manner. A key remaining question concerns the extent to which variation in the power of random genetic drift is capable of influencing phylogenetic diversity at the subcellular and cellular levels. Should this be the case, population size would have to be considered as a potential determinant of the mechanistic pathways underlying long-term phenotypic evolution. Here we demonstrate a phylogenetically broad inverse relation between the power of drift and the structural integrity of protein subunits. This leads to the hypothesis that the accumulation of mildly deleterious mutations in populations of small size induces secondary selection for protein-protein interactions that stabilize key gene functions. By this means, the complex protein architectures and interactions essential to the genesis of phenotypic diversity may initially emerge by non-adaptive mechanisms.

Despite substantial attention from theoreticians, the evolutionary mechanisms that drive intra- and interspecific variation in the mutation rate remain unclear. It has often been argued that mutation rates associated with the major replicative polymerases have been driven down to their physiological limits, defined as the point at which further enhancement in replication fidelity incurs a cost in terms of reproductive output, but no evidence in support of this argument has emerged for cellular organisms. Here, it is suggested that the lower barrier to mutation rate evolution may ultimately be defined not by molecular limitations but by the power of random genetic drift. As the mutation rate is reduced to a very low level, a point will eventually be reached at which the small advantage of any further reduction is overwhelmed by the power of drift. This hypothesis is consistent with a number of observations, including the inverse relationship between the per-site mutation rate and genome size in microbes, the negative scaling between the per-site mutation rate and effective population size in eukaryotes, and the elevated error rates associated with less frequently deployed polymerases and repair pathways.

Understanding the mechanisms of evolution requires information on the rate of appearance of new mutations and their effects at the molecular and phenotypic levels. Although procuring such data has been technically challenging, high-throughput genome sequencing is rapidly expanding knowledge in this area. With information on spontaneous mutations now available in a variety of organisms, general patterns have emerged for the scaling of mutation rate with genome size and for the likely mechanisms that drive this pattern. Support is presented for the hypothesis that natural selection pushes mutation rates down to a lower limit set by the power of random genetic drift rather than by intrinsic physiological limitations, and that this has resulted in reduced levels of replication, transcription, and translation fidelity in eukaryotes relative to prokaryotes., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A central problem in evolutionary theory concerns the mechanisms by which adaptations requiring multiple mutations emerge in natural populations. We develop a series of expressions that clarify the scaling of the time to establishment of complex adaptations with population size, mutation rate, magnitude of the selective disadvantage of intermediate-state alleles, and the complexity of the adaptation. In general, even in the face of deleterious intermediate steps, the time to establishment is minimized in populations with very large size. Under a broad range of conditions, the time to establishment also scales by no more than the square of the mutation rate, regardless of the number of sites contributing to the adaptive change, demonstrating that the emergence of complex adaptations is only weakly constrained by the independent acquisition of mutations at the underlying sites. Mutator alleles with deleterious side effects have only moderate effects on the rate of adaptation in large populations but can cause a quantum decrease in the time to establishment of some adaptive alleles in small populations, although probably not at a high enough rate to offset the increased deleterious mutation load. Transient hypermutability, whereby a subset of gamete-producing cells mutate at an elevated rate in a nonheritable manner, may also elevate the rate of adaptation, although the effect is modest and appears to result from a simple increase in the rate of transitions between intermediate states rather than from the saltational production of doublet mutations. Taken together, these results illustrate the plausibility of the relatively rapid emergence of specific complex adaptations by conventional population genetic mechanisms and provide insight into the relative incidences of various paths of allelic adaptation in organisms with different population genetic features.

Photoreactivation, one of the first DNA repair pathways to evolve, is the direct reversal of premutagenic lesions caused by ultraviolet (UV) irradiation, catalyzed by photolyases in a light-dependent, single-enzyme reaction. It has been experimentally shown that photoreactivation prevents UV mutagenesis in a broad range of species. In the absence of photoreactivation, UV-induced photolesions are repaired by the more complex and much less efficient nucleotide excision repair pathway. Despite their obvious beneficial effects, several lineages, including placental mammals, lost photolyase genes during evolution. In this study, we ask why photolyase genes have been lost in those lineages and discuss the significance of these losses in the context of the evolution of the genomic mutation rates. We first perform an extensive phylogenomic analysis of the photolyase/cryptochrome family, to assess what species lack each kind of photolyase gene. Then, we estimate the ratio of nonsynonymous to synonymous substitution rates in several groups of photolyase genes, as a proxy of the strength of purifying natural selection, and we ask whether less evolutionarily constrained photolyase genes are more likely lost. We also review functional data and compare the efficiency of different kinds of photolyases. We find that eukaryotic photolyases are, on average, less evolutionarily constrained than eubacterial ones and that the strength of natural selection is correlated with the affinity of photolyases for their substrates. We propose that the loss of photolyase genes in eukaryotic species may be due to weak natural selection and may result in a deleterious increase of their genomic mutation rates. In contrast, the loss of photolyase genes in prokaryotes may not cause an increase in the mutation rate and be neutral in most cases.

The Sm family of proteins is closely associated with RNA metabolism throughout all life. These proteins form homomorphic and heteromorphic rings consisting of six or seven subunits with a characteristic central pore, the presence of which is critical for binding U-rich regions of single-stranded RNA. Eubacteria and Archaea typically carry one or two forms of Sm proteins and assemble one homomorphic ring per Sm protein. Eukaryotes typically carry 16 or more Sm proteins that assemble to form heteromorphic rings which lie at the center of a number of critical RNA-associated small nuclear ribonucleoproteins (snRNPs). High Sm protein diversity and heteromorphic Sm rings are features stretching back to the origin of eukaryotes; very deep phylogenetic divisions among existing Sm proteins indicate simultaneous evolution across essentially all existing eukaryotic life. Two basic forms of heteromorphic Sm rings are found in eukaryotes. Fixed Sm rings are highly stable and static and are assembled around an RNA cofactor. Flexible Sm rings also stabilize and chaperone RNA but assemble in the absence of an RNA substrate and, more significantly, associate with and dissociate from RNA substrates more freely than fixed rings. This suggests that the conformation of flexible Sm rings might be modified in some specific manner to facilitate association and dissociation with RNA. Diversification of eukaryotic Sm proteins may have been initiated by gene transfers and/or genome clashes that accompanied the origin of the eukaryotic cell itself, with further diversification driven by a greater need for steric specificity within increasingly complex snRNPs.

Although the matter has been subject to considerable theoretical study, there are numerous open questions regarding the mechanisms driving the mutation rate in various phylogenetic lineages. Most notably, empirical evidence indicates that mutation rates are elevated in multicellular species relative to unicellular eukaryotes and prokaryotes, even on a per-cell division basis, despite the need for the avoidance of somatic damage and the accumulation of germline mutations. Here it is suggested that multicellularity discourages selection against weak mutator alleles for reasons associated with both the cellular and the population-genetic environments, thereby magnifying the vulnerability to somatic mutations (cancer) and increasing the risk of extinction from the accumulation of germline mutations. Moreover, contrary to common belief, a cost of fidelity need not be invoked to explain the lower bound to observed mutation rates, which instead may simply be set by the inability of selection to advance very weakly advantageous antimutator alleles in finite populations.

The nuclear genomes of multicellular animals and plants contain large amounts of noncoding DNA, the disadvantages of which can be too weak to be effectively countered by selection in lineages with reduced effective population sizes. In contrast, the organelle genomes of these two lineages evolved to opposite ends of the spectrum of genomic complexity, despite similar effective population sizes. This pattern and other puzzling aspects of organelle evolution appear to be consequences of differences in organelle mutation rates. These observations provide support for the hypothesis that the fundamental features of genome evolution are largely defined by the relative power of two nonadaptive forces: random genetic drift and mutation pressure.

Most of the phenotypic diversity that we perceive in the natural world is directly attributable to the peculiar structure of the eukaryotic gene, which harbors numerous embellishments relative to the situation in prokaryotes. The most profound changes include introns that must be spliced out of precursor mRNAs, transcribed but untranslated leader and trailer sequences (untranslated regions), modular regulatory elements that drive patterns of gene expression, and expansive intergenic regions that harbor additional diffuse control mechanisms. Explaining the origins of these features is difficult because they each impose an intrinsic disadvantage by increasing the genic mutation rate to defective alleles. To address these issues, a general hypothesis for the emergence of eukaryotic gene structure is provided here. Extensive information on absolute population sizes, recombination rates, and mutation rates strongly supports the view that eukaryotes have reduced genetic effective population sizes relative to prokaryotes, with especially extreme reductions being the rule in multicellular lineages. The resultant increase in the power of random genetic drift appears to be sufficient to overwhelm the weak mutational disadvantages associated with most novel aspects of the eukaryotic gene, supporting the idea that most such changes are simple outcomes of semi-neutral processes rather than direct products of natural selection. However, by establishing an essentially permanent change in the population-genetic environment permissive to the genome-wide repatterning of gene structure, the eukaryotic condition also promoted a reliable resource from which natural selection could secondarily build novel forms of organismal complexity. Under this hypothesis, arguments based on molecular, cellular, and/or physiological constraints are insufficient to explain the disparities in gene, genomic, and phenotypic complexity between prokaryotes and eukaryotes.

A recent paper in this journal has challenged the idea that complex adaptive features of proteins can be explained by known molecular, genetic, and evolutionary mechanisms. It is shown here that the conclusions of this prior work are an artifact of unwarranted biological assumptions, inappropriate mathematical modeling, and faulty logic. Numerous simple pathways exist by which adaptive multi-residue functions can evolve on time scales of a million years (or much less) in populations of only moderate size. Thus, the classical evolutionary trajectory of descent with modification is adequate to explain the diversification of protein functions.

Unlike the situation in prokaryotes, most eukaryotic messenger RNAs contain a moderately long 5' untranslated region (UTR). Such leader sequences impose a burden on eukaryotic genes by providing substrate for the mutational origin of premature translation-initiation codons, which generally result in defective proteins. To gain an insight into the expansion of 5' UTRs in eukaryotic genomes, we present a simple null model in which the evolution of transcription-initiation sites is entirely driven by the stochastic mutational flux of core-promoter sequences and premature translation-initiation codons. This model yields results consistent with a variety of heretofore disconnected observations, including the form of length distributions of 5' UTRs, the relatively low variance in UTR features among distantly related eukaryotes, the universal reliance on relatively simple core-promoter sequences, and the elevated density of introns in the 5' UTR. We suggest that the reduced effective population sizes of most eukaryotes impose a population-genetic environment conducive to the movement of core promoters to random positions, subject to the constraint imposed by the upstream accumulation of premature translation-initiation codons. If this hypothesis is correct, then selection for gene-specific regulatory features need not be invoked to explain either the origin of lengthy eukaryotic 5' UTRs or the 1,000-fold range of 5'-UTR lengths among genes within species. Nevertheless, once permanently established, expanded 5' UTRs may have provided a novel substrate for the evolution of mechanisms for posttranscriptional regulation of eukaryotic gene expression. These results provide a further example of how an increase in the power of random genetic drift can passively promote the evolution of forms of gene architecture that ultimately facilitate the evolution of organismal complexity.

Asexual taxa are short-lived, suggesting that transitions to asexuality represent evolutionary dead-ends. However, with high rates of clonal origin and coexistence of asexuals and sexuals via selective asymmetries, asexuality may persist in the long term as a result of a dynamic equilibrium between clonal origin and extinction. Few such systems have been studied in detail. Here, we investigate the evolutionary history of asexual lineages of Daphnia pulex, which are derived from sexual relatives via the inheritance of a dominant female-limited meiosis-suppressing locus and inhabit ponds throughout northeastern North America (NA). Our extensive sampling and subsequent phylogenetic analysis using mitochondrial sequence data reveals a young and genetically diverse asexual assemblage, reflecting high rates of clonal origin due to the contagious nature of asexuality. Yet, asexuality is restricted to two phylogroups (B and C) with historical and/or present associations with northeastern NA and is absent from a northwestern phylogroup (A), supporting a recent northeastern origin of asexuality in this species. Furthermore, macrogeographic patterns of genetic variability indicate that phylogroups B and C recolonized northeastern NA from opposite directions, yet their presently overlapping geographic distributions are similarly divided into an eastern asexual and a western sexual region. We attribute these patterns to a recent contagious spread of asexuality from a northeastern source. If environment-mediated selective asymmetries play no significant role in determining the outcome of competitive interactions between sexuals and asexuals, regions of contact may be setting the stage for continued asexual conquests.

Gene duplication is widely regarded as the predominant mechanism by which genes with new functions and associated phenotypic novelties arise. However, the mutational events and population-genetic mechanisms that lead to the short-term preservation of duplicate genes are not necessarily the same as those exhibited by well-established paralogs en route to the origin of new beneficial features. Thus, although recent genome-wide analyses have revealed striking patterns of protein-sequence divergence among the members of surviving paralogous gene families, the mechanisms responsible for the historical development of these patterns remain unclear.

Homopolymeric nucleotide runs, also called mononucleotide microsatellites, are a ubiquitous, dominant, and mutagenic feature of eukaryotic genomes. A clear understanding of the forces that shape patterns of homopolymer evolution, however, is lacking. We provide a focused investigation of the abundance, chromosomal distribution, and mutation spectra of the four strand-specific homopolymer types (A, T, G, C) >or=8 bp in the genome of Caenorhabditis elegans. A and T homopolymers vastly outnumber G and C HPs, and the run-length distributions of A and T homopolymers differ significantly from G and C homopolymers. A scanning window analysis of homopolymer chromosomal distribution reveals distinct clusters of homopolymer density in autosome arms that are regions of high recombination in C. elegans. Dramatic biases are detected among closely spaced homopolymers; for instance, we observe 994 A homopolymers immediately followed by a T homopolymer (5' to 3') and only 8 instances of T homopolymers directly followed by an A homopolymer. Empirical homopolymer mutation assays in a set of C. elegans mutation-accumulation lines reveal an approximately 20-fold higher mutation rate for G and C homopolymers compared to A and T homopolymers. Nuclear A and T homopolymers are also found to mutate approximately 100-fold more slowly than mitochondrial A and T homopolymers. This integrative approach yields a total nuclear genome-wide homopolymer mutation rate estimate of approximately 1.6 mutations per genome per generation.

The significance of gene duplication in provisioning raw materials for the evolution of genomic diversity is widely recognized, but the early evolutionary dynamics of duplicate genes remain obscure. To elucidate the structural characteristics of newly arisen gene duplicates at infancy and their subsequent evolutionary properties, we analyzed gene pairs with < or =10% divergence at synonymous sites within the genome of Caenorhabditis elegans. Structural heterogeneity between duplicate copies is present very early in their evolutionary history and is maintained over longer evolutionary timescales, suggesting that duplications across gene boundaries in conjunction with shuffling events have at least as much potential to contribute to long-term evolution as do fully redundant (complete) duplicates. The median duplication span of 1.4 kb falls short of the average gene length in C. elegans (2.5 kb), suggesting that partial gene duplications are frequent. Most gene duplicates reside close to the parent copy at inception, often as tandem inverted loci, and appear to disperse in the genome as they age, as a result of reduced survivorship of duplicates located in proximity to the ancestral copy. We propose that illegitimate recombination events leading to inverted duplications play a disproportionately large role in gene duplication within this genome in comparison with other mechanisms.

Complete genomic sequences from diverse phylogenetic lineages reveal notable increases in genome complexity from prokaryotes to multicellular eukaryotes. The changes include gradual increases in gene number, resulting from the retention of duplicate genes, and more abrupt increases in the abundance of spliceosomal introns and mobile genetic elements. We argue that many of these modifications emerged passively in response to the long-term population-size reductions that accompanied increases in organism size. According to this model, much of the restructuring of eukaryotic genomes was initiated by nonadaptive processes, and this in turn provided novel substrates for the secondary evolution of phenotypic complexity by natural selection. The enormous long-term effective population sizes of prokaryotes may impose a substantial barrier to the evolution of complex genomes and morphologies.

The helix-hairpin-helix (HhH) superfamily of base excision repair DNA glycosylases is composed of multiple phylogenetically diverse enzymes that are capable of excising varying spectra of oxidatively and methyl-damaged bases. Although these DNA repair glycosylases have been widely studied through genetic, biochemical, and biophysical approaches, the evolutionary relationships of different HhH homologs and the extent to which they are conserved across phylogeny remain enigmatic. We provide an evolutionary framework for this pervasive and versatile superfamily of DNA glycosylases. Six HhH gene families (named AlkA: alkyladenine glycosylase; MpgII: N-methylpurine glycosylase II; MutY/Mig: A/G-specific adenine glycosylase/mismatch glycosylase; Nth: endonuclease III; OggI: 8-oxoguanine glycosylase I; and OggII: 8-oxoguanine glycosylase II) are identified through phylogenetic analysis of 234 homologs found in 94 genomes (16 archaea, 64 bacteria, and 14 eukaryotes). The number of homologs in each gene family varies from 117 in the Nth family (nearly every genome surveyed harbors at least one Nth homolog) to only five in the divergent OggII family (all from archaeal genomes). Sequences from all three domains of life are included in four of the six gene families, suggesting that the HhH superfamily diversified very early in evolution. The phylogeny provides evidence for multiple lineage-specific gene duplication events, most of which involve eukaryotic homologs in the Nth and AlkA gene families. We observe extensive variation in the number of HhH superfamily glycosylase genes present in different genomes, possibly reflecting major differences among species in the mechanisms and pathways by which damaged bases are repaired and/or disparities in the basic rates and spectra of mutation experienced by different genomes.

The mechanisms responsible for the proliferation and subsequent stabilization of introns within the eukaryotic lineage have remained elusive. In the early stages of eukaryotic evolution, most introns may have been mildly deleterious at the time of insertion, but enough of them eventually acquired integral roles in transcript processing that few eukaryotic species can any longer survive without them. We suggest that the proliferation of spliceosomal introns was facilitated by the evolution of nonsense-mediated decay, an ancient and (in many cases) intron-dependent mechanism for eliminating aberrant mRNA molecules resulting from errors in transcription and splicing and from mutations at the DNA level. The spatial distribution of introns, as revealed by whole-genome analysis, is consistent with expectations for a model in which maximum protective coverage of a gene stochastically evolves over time.

Although gene duplication has generally been viewed as a necessary source of material for the origin of evolutionary novelties, the rates of origin, loss, and preservation of gene duplicates are not well understood. Applying steady-state demographic techniques to the age distributions of duplicate genes censused in seven completely sequenced genomes, we estimate the average rate of duplication of a eukaryotic gene to be on the order of 0.01/ gene/million years, which is of the same order of magnitude as the mutation rate per nucleotide site. However, the average half-life of duplicate genes is relatively small, on the order of 4.0 million years. Significant interspecific variation in these rates appears to be responsible for differences in species-specific genome sizes that arise as a consequence of a quasi-equilibrium birth-death process. Most duplicated genes experience a brief period of relaxed selection early in their history and a minority exhibit the signature of directional selection, but those that survive more than a few million years eventually experience strong purifying selection. Thus, although most theoretical work on the gene-duplication process has focused on issues related to adaptive evolution, the origin of a new function appears to be a very rare fate for a duplicate gene. A more significant role of the duplication process may be the generation of microchromosomal rearrangements through reciprocal silencing of alternative copies, which can lead to the passive origin of post-zygotic reproductive barriers in descendant lineages of incipient species.

Although the widespread proliferation of introns in eukaryotic protein-coding genes remains one of the most poorly understood aspects of genomic architecture, major advances have emerged recently from large-scale genome sequencing projects and functional analyses of mRNA-processing events. Evidence supports the idea that spliceosomal introns were not only present in the stem eukaryote but diverged into at least two distinct classes very early in eukaryotic evolution. Some rough estimates of intron turnover rates are provided, and a testable hypothesis for the origin of new introns is proposed. In light of recent findings on the molecular natural history of splicing, various aspects of the phylogenetic and physical distributions of introns can now be interpreted in a theoretical framework that jointly considers the population-genetic roles of mutation, random genetic drift, and natural selection.

Understanding the utility and limitations of molecular markers for predicting the evolutionary potential of natural populations is important for both evolutionary and conservation genetics. To address this issue, the distribution of genetic variation for quantitative traits and molecular markers is estimated within and among 14 permanent lake populations of Daphnia pulicaria representing two regional groups from Oregon. Estimates of population subdivision for molecular and quantitative traits are concordant, with QST generally exceeding GST. There is no evidence that microsatellites loci are less informative about subdivision for quantitative traits than are allozyme loci. Character-specific comparison of QST and GST support divergent selection pressures among populations for the majority of life-history traits in both coast and mountain regions. The level of within-population variation for molecular markers is uninformative as to the genetic variation maintained for quantitative traits. In D. pulicaria, regional differences in the frequency of sex may contribute to variation in the maintenance of expressed within-population quantitative-genetic variation without substantially impacting diversity at the genic level. These data are compared to an identical dataset for 17 populations of the temporary-pond species, D. pulex.

This paper describes software created to search for and analyze pairs of duplicate genes within a genome. The process is based on a program that uses aligned amino acid sequences to generate a corresponding alignment of the underlying nucleotide sequences and perform a codon by codon comparison of the nucleotides. Observed numbers of nucleotide substitutions can be used to make inferences about the ages of gene duplication events and the effects of natural selection acting on duplicate genes.

Gene duplication has generally been viewed as a necessary source of material for the origin of evolutionary novelties, but it is unclear how often gene duplicates arise and how frequently they evolve new functions. Observations from the genomic databases for several eukaryotic species suggest that duplicate genes arise at a very high rate, on average 0.01 per gene per million years. Most duplicated genes experience a brief period of relaxed selection early in their history, with a moderate fraction of them evolving in an effectively neutral manner during this period. However, the vast majority of gene duplicates are silenced within a few million years, with the few survivors subsequently experiencing strong purifying selection. Although duplicate genes may only rarely evolve new functions, the stochastic silencing of such genes may play a significant role in the passive origin of new species.

The accumulation of deleterious mutations is thought to be a major factor preventing the long-term persistence of obligately asexual lineages relative to their sexual ancestors. This phenomenon is also of potential relevance to sexual species that harbor asexually propagating organelle genomes. A comparative study of the transfer RNA genes in animal mitochondrial and nuclear genomes demonstrates that the former accumulate nucleotide substitutions much more rapidly than do the latter, and several lines of evidence are consistent with the idea that the excess substitutions are mildly deleterious. First, the average binding stability between complementary strands in the stems of mitochondrial tRNAs is less than half that in nuclear tRNAs. Second, most loop sizes in the mitochondrial tRNAs have experienced a net reduction in size over evolutionary time, and they are nearly 50 times more variable in the mitochondrial than in the nuclear genome. Third, although nearly 20% of the nucleotides in nuclear tRNA genes (particularly those involved in tertiary interactions) are invariant across all animal taxa and all tRNA species, there are no invariant sites in the mitochondrial tRNAs. These observations, as well as results from recent laboratory experiments, are consistent with the hypothesis that nonrecombining organelle genomes are subject to gradual loss of fitness due to the cumulative chance fixation of mildly deleterious mutations.