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Due to genetic variation in the ancestor of two populations or two species, the divergence time for DNA sequences from two populations is variable along the genome. Within genomic segments all bases will share the same divergence—because they share a most recent common ancestor—when no recombination event has occurred to split them apart. The size of these segments of constant divergence depends on the recombination rate, but also on the speciation time, the effective population size of the ancestral population, as well as demographic effects and selection. Thus, inference of these parameters may be possible if we can decode the divergence times along a genomic alignment. Here, we present a new hidden Markov model that infers the changing divergence (coalescence) times along the genome alignment using a coalescent framework, in order to estimate the speciation time, the recombination rate, and the ancestral effective population size. The model is efficient enough to allow inference on whole-genome data sets. We first investigate the power and consistency of the model with coalescent simulations and then apply it to the whole-genome sequences of the two orangutan sub-species, Bornean (P. p. pygmaeus) and Sumatran (P. p. abelii) orangutans from the Orangutan Genome Project. We estimate the speciation time between the two sub-species to be thousand years ago and the effective population size of the ancestral orangutan species to be , consistent with recent results based on smaller data sets. We also report a negative correlation between chromosome size and ancestral effective population size, which we interpret as a signature of recombination increasing the efficacy of selection., Author Summary We present a hidden Markov model that uses variation in coalescence times between two distantly related populations, or closely related species, to infer population genetics parameters in ancestral population or species. The model infers the divergence times in segments along the alignment. Using coalescent simulations, we show that the model accurately estimates the divergence time between the two populations and the effective population size of the ancestral population. We apply the model to the recently sequenced orangutan sub-species and estimate their divergence time and the effective population size of their ancestor population.

The authors propose that short repeat sequences may play an important role in causing the pervasive clustering of mutations across diverse genomes from prokaryotes to humans., The genome-sequencing gold rush has facilitated the use of comparative genomics to uncover patterns of genome evolution, although their causal mechanisms remain elusive. One such trend, ubiquitous to prokarya and eukarya, is the association of insertion/deletion mutations (indels) with increases in the nucleotide substitution rate extending over hundreds of base pairs. The prevailing hypothesis is that indels are themselves mutagenic agents. Here, we employ population genomics data from Escherichia coli, Saccharomyces paradoxus, and Drosophila to provide evidence suggesting that it is not the indels per se but the sequence in which indels occur that causes the accumulation of nucleotide substitutions. We found that about two-thirds of indels are closely associated with repeat sequences and that repeat sequence abundance could be used to identify regions of elevated sequence diversity, independently of indels. Moreover, the mutational signature of indel-proximal nucleotide substitutions matches that of error-prone DNA polymerases. We propose that repeat sequences promote an increased probability of replication fork arrest, causing the persistent recruitment of error-prone DNA polymerases to specific sequence regions over evolutionary time scales. Experimental measures of the mutation rates of engineered DNA sequences and analyses of experimentally obtained collections of spontaneous mutations provide molecular evidence supporting our hypothesis. This study uncovers a new role for repeat sequences in genome evolution and provides an explanation of how fine-scale sequence contextual effects influence mutation rates and thereby evolution., Author Summary An intriguing observation made during the comparison of genomes is that insertion and deletion mutations (indels) cluster together with nucleotide substitutions. Two (not mutually exclusive) hypotheses have been proposed to explain this phenomenon. The first postulates that an indel mutation causes an increase in the likelihood of the surrounding sequence incurring nucleotide substitutions, while the second claims that the region of DNA in which such a cluster is located is more likely to sustain both indels and substitutions. Here, we present evidence suggesting that the region of DNA, and not the indel, is associated with the accumulation of clusters of mutations over evolutionary time scales. We find that repeat sequences are closely associated with a large proportion of indels and that the abundance of repeat sequences is linked with regions of increased nucleotide diversity. By analysing molecular data and measuring the mutation rates of genes engineered to contain repeats, we find that the mutation rate can be manipulated by the insertion of long repeat sequences. On the basis of these results, we propose a model in which repeat sequences are prone to cause stalling of the high-fidelity DNA polymerase, leading to the recruitment of error-prone repair polymerases which then replicate the surrounding sequence with a higher-than-average error rate.

In a series of publications, I and my colleagues have developed hypotheses for how the evolution of various aspects of genome architecture is expected to proceed under conditions in which the forces of random genetic drift and mutation predominate (e.g., [1]–[15]). These models, collectively referred to below as the mutational-hazard (hereafter, MH) hypothesis, are sometimes represented as neutral models [16], [17], but this is not correct, as the key component of each model is the deleterious mutational consequence of excess DNA. The MH hypothesis is, however, a nonadaptational model, in that it yields expectations on the structure of genomes without invoking external selective forces. It is likely that some aspects of these models will need to be changed as more is learned about the molecular consequences of various aspects of gene structure and the nature of mutation. Such modifications will not alter the need for baseline null hypotheses in attempts to defend adaptive explanations for variation in genomic architecture [9]. Nevertheless, any theory that strives to provide a unifying explanation for diverse sets of genomic observations must be scrutinized extensively from a variety of angles and interpreted in the context of well-established molecular and population-genetic processes. Although I will argue that a recent challenge to the MH hypothesis by Whitney and Garland ([18]; hereafter, WG) contains numerous problems, this exchange may help clarify more broadly misunderstood issues.

Ecologists have increasingly come to understand that evolutionary change on short time-scales can alter ecological dynamics (and vice-versa), and this idea is being incorporated into community ecology research programs. Previous research has suggested that the size and topology of the gene network underlying a quantitative trait should constrain or facilitate adaptation and thereby alter population dynamics. Here, I consider a scenario in which two species with different genetic architectures compete and evolve in fluctuating environments. An important trade-off emerges between adaptive accuracy and adaptive speed, driven by the size of the gene network underlying the ecologically-critical trait and the rate of environmental change. Smaller, scale-free networks confer a competitive advantage in rapidly-changing environments, but larger networks permit increased adaptive accuracy when environmental change is sufficiently slow to allow a species time to adapt. As the differences in network characteristics increase, the time-to-resolution of competition decreases. These results augment and refine previous conclusions about the ecological implications of the genetic architecture of quantitative traits, emphasizing a role of adaptive accuracy. Along with previous work, in particular that considering the role of gene network connectivity, these results provide a set of expectations for what we may observe as the field of ecological genomics develops.

Comparative genomic analyses of primates offer considerable potential to define and understand the processes that mold, shape, and transform the human genome. However, primate taxonomy is both complex and controversial, with marginal unifying consensus of the evolutionary hierarchy of extant primate species. Here we provide new genomic sequence (∼8 Mb) from 186 primates representing 61 (∼90%) of the described genera, and we include outgroup species from Dermoptera, Scandentia, and Lagomorpha. The resultant phylogeny is exceptionally robust and illuminates events in primate evolution from ancient to recent, clarifying numerous taxonomic controversies and providing new data on human evolution. Ongoing speciation, reticulate evolution, ancient relic lineages, unequal rates of evolution, and disparate distributions of insertions/deletions among the reconstructed primate lineages are uncovered. Our resolution of the primate phylogeny provides an essential evolutionary framework with far-reaching applications including: human selection and adaptation, global emergence of zoonotic diseases, mammalian comparative genomics, primate taxonomy, and conservation of endangered species., Author Summary Advances in human biomedicine, including those focused on changes in genes triggered or disrupted in development, resistance/susceptibility to infectious disease, cancers, mechanisms of recombination, and genome plasticity, cannot be adequately interpreted in the absence of a precise evolutionary context or hierarchy. However, little is known about the genomes of other primate species, a situation exacerbated by a paucity of nuclear molecular sequence data necessary to resolve the complexities of primate divergence over time. We overcome this deficiency by sequencing 54 nuclear gene regions from DNA samples representing ∼90% of the diversity present in living primates. We conduct a phylogenetic analysis to determine the origin, evolution, patterns of speciation, and unique features in genome divergence among primate lineages. The resultant phylogenetic tree is remarkably robust and unambiguously resolves many long-standing issues in primate taxonomy. Our data provide a strong foundation for illuminating those genomic differences that are uniquely human and provide new insights on the breadth and richness of gene evolution across all primate lineages.

In the quest to reconstruct the Tree of Life, researchers have increasingly turned to phylogenomics, the inference of phylogenetic relationships using genome-scale data (Box 1). Mesmerized by the sustained increase in sequencing throughput, many phylogeneticists entertained the hope that the incongruence frequently observed in studies using single or a few genes [1] would come to an end with the generation of large multigene datasets. Yet, as so often happens, reality has turned out to be far more complex, as three recent large-scale analyses, one published in PLoS Biology [2]–[4], make clear. The studies, which deal with the early diversification of animals, produced highly incongruent (Box 2) findings despite the use of considerable sequence data (see Figure 1). Clearly, merely adding more sequences is not enough to resolve the inconsistencies.

Recent research has provided mechanistic insight into the important contributions of the gut microbiota to vertebrate biology, but questions remain about the evolutionary processes that have shaped this symbiosis. In the present study, we showed in experiments with gnotobiotic mice that the evolution of Lactobacillus reuteri with rodents resulted in the emergence of host specialization. To identify genomic events marking adaptations to the murine host, we compared the genome of the rodent isolate L. reuteri 100-23 with that of the human isolate L. reuteri F275, and we identified hundreds of genes that were specific to each strain. In order to differentiate true host-specific genome content from strain-level differences, comparative genome hybridizations were performed to query 57 L. reuteri strains originating from six different vertebrate hosts in combination with genome sequence comparisons of nine strains encompassing five phylogenetic lineages of the species. This approach revealed that rodent strains, although showing a high degree of genomic plasticity, possessed a specific genome inventory that was rare or absent in strains from other vertebrate hosts. The distinct genome content of L. reuteri lineages reflected the niche characteristics in the gastrointestinal tracts of their respective hosts, and inactivation of seven out of eight representative rodent-specific genes in L. reuteri 100-23 resulted in impaired ecological performance in the gut of mice. The comparative genomic analyses suggested fundamentally different trends of genome evolution in rodent and human L. reuteri populations, with the former possessing a large and adaptable pan-genome while the latter being subjected to a process of reductive evolution. In conclusion, this study provided experimental evidence and a molecular basis for the evolution of host specificity in a vertebrate gut symbiont, and it identified genomic events that have shaped this process., Author Summary The gastrointestinal microbiota of vertebrates is important for nutrient utilization, resistance against pathogens, and immune maturation of its host, but little is known about the evolutionary relationships between vertebrates and individual bacterial members of these communities. Here we provide robust evidence that the evolution of the gut symbiont Lactobacillus reuteri with vertebrates resulted in the emergence of host specialization. Genomic approaches using a combination of genome sequence comparisons and microarray analysis were used to identify the host-specific genome content in rodent and human strains and the evolutionary events that resulted in host adaptation. The study revealed divergent patterns of genome evolution in rodent and human lineages and a distinct genome inventory in host-restricted sub-populations of L. reuteri that reflected the niche characteristics in the gut of their particular vertebrate hosts. The ecological significance of representative rodent-specific genes was demonstrated in gnotobiotic mice. In conclusion, this work provided evidence that the vertebrate gut symbiont Lactobacillus reuteri, despite the likelihood of horizontal transmission, has remained stably associated with related groups of vertebrate hosts over evolutionary time and has evolved a lifestyle specialized to these host animals.

The modification of DNA by methylation is an important epigenetic mechanism that affects the spatial and temporal regulation of gene expression. Methylation patterns have been described in many contexts within and across a range of species. However, the extent to which changes in methylation might underlie inter-species differences in gene regulation, in particular between humans and other primates, has not yet been studied. To this end, we studied DNA methylation patterns in livers, hearts, and kidneys from multiple humans and chimpanzees, using tissue samples for which genome-wide gene expression data were also available. Using the multi-species gene expression and methylation data for 7,723 genes, we were able to study the role of promoter DNA methylation in the evolution of gene regulation across tissues and species. We found that inter-tissue methylation patterns are often conserved between humans and chimpanzees. However, we also found a large number of gene expression differences between species that might be explained, at least in part, by corresponding differences in methylation levels. In particular, we estimate that, in the tissues we studied, inter-species differences in promoter methylation might underlie as much as 12%–18% of differences in gene expression levels between humans and chimpanzees., Author Summary It has long been hypothesized that changes in gene regulation have played an important role in primate evolution. However, despite the wealth of comparative gene expression data, there are still only few studies that focus on the mechanisms underlying inter-primate differences in gene regulation. In particular, we know relatively little about the degree to which changes in epigenetic profiles might explain differences in gene expression levels between primates. To this end, we studied DNA methylation and gene expression levels in livers, hearts, and kidneys from multiple humans and chimpanzees. Using these comparative data, we were able to study the evolution of gene regulation in the context of conservation of or changes in DNA methylation profiles across tissues and species. We found that inter-tissue methylation patterns are often conserved between humans and chimpanzees. In addition, we also found a large number of gene expression differences between species, which might be explained, at least in part, by corresponding differences in methylation levels. We estimate that, in the tissues we studied, inter-species differences in methylation levels might underlie as much as 12%–18% of differences in gene expression levels between humans and chimpanzees.

BackgroundBeavers are one of the largest and ecologically most distinct rodent species. Little is known about their evolution and even their closest phylogenetic relatives have not yet been identified with certainty. Similarly, little is known about the timing of divergence events within the genus Castor.Methodology/principal findingsWe sequenced complete mitochondrial genomes from both extant beaver species and used these sequences to place beavers in the phylogenetic tree of rodents and date their divergence from other rodents as well as the divergence events within the genus Castor. Our analyses support the phylogenetic position of beavers as a sister lineage to the scaly tailed squirrel Anomalurus within the mouse related clade. Molecular dating places the divergence time of the lineages leading to beavers and Anomalurus as early as around 54 million years ago (mya). The living beaver species, Castor canadensis from North America and Castor fiber from Eurasia, although similar in appearance, appear to have diverged from a common ancestor more than seven mya. This result is consistent with the hypothesis that a migration of Castor from Eurasia to North America as early as 7.5 mya could have initiated their speciation. We date the common ancestor of the extant Eurasian beaver relict populations to around 210,000 years ago, much earlier than previously thought. Finally, the substitution rate of Castor mitochondrial DNA is considerably lower than that of other rodents. We found evidence that this is correlated with the longer life span of beavers compared to other rodents.Conclusions/significanceA phylogenetic analysis of mitochondrial genome sequences suggests a sister-group relationship between Castor and Anomalurus, and allows molecular dating of species divergence in congruence with paleontological data. The implementation of a relaxed molecular clock enabled us to estimate mitochondrial substitution rates and to evaluate the effect of life history traits on it.

Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens. Furthermore, our study highlights the remarkable evolvability of T4SSs and their effector proteins, explaining their broad application in bacterial interactions with the environment., Author Summary Adaptive radiation is the rapid origination of an array of species by the divergent colonization of disparate ecological niches. In the case of pathogenic bacteria, radiations can lead to the emergence of novel human pathogens. Being divergently adapted to a range of different mammalian hosts, including humans as reservoir or incidental hosts, the genus Bartonella represents a suitable model to study genomic mechanisms underpinning divergent adaptation of pathogens. Here we show that two distinct lineages of Bartonella have radiated in parallel, resulting in two arrays of evolutionary distinct species adapted to overlapping sets of mammalian hosts. Such parallelisms display excellent models to reveal insights into the genetic mechanisms underlying these independent evolutionary processes. Our genome-wide analysis identifies a striking evolutionary parallelism in a horizontally-acquired protein secretion system in the two lineages. The parallel evolutionary trajectory of this system in the two lineages is characterized by the convergent origination of a wide array of adaptive functions dedicated to the cellular interaction within the mammalian hosts. The parallel evolution of the two radiating lineages on the ecological as well as on the molecular level suggests that the horizontal acquisition and the functional diversification of the secretion system display an evolutionary key innovation underlying adaptive evolution.

Here we investigate the correlations between coding sequence substitutions as a function of their separation along the protein sequence. We consider both substitutions between the reference genomes of several Drosophilids as well as polymorphisms in a population sample of Zimbabwean Drosophila melanogaster. We find that amino acid substitutions are “clustered” along the protein sequence, that is, the frequency of additional substitutions is strongly enhanced within ≈10 residues of a first such substitution. No such clustering is observed for synonymous substitutions, supporting a “correlation length” associated with selection on proteins as the causative mechanism. Clustering is stronger between substitutions that arose in the same lineage than it is between substitutions that arose in different lineages. We consider several possible origins of clustering, concluding that epistasis (interactions between amino acids within a protein that affect function) and positional heterogeneity in the strength of purifying selection are primarily responsible. The role of epistasis is directly supported by the tendency of nearby substitutions that arose on the same lineage to preserve the total charge of the residues within the correlation length and by the preferential cosegregation of neighboring derived alleles in our population sample. We interpret the observed length scale of clustering as a statistical reflection of the functional locality (or modularity) of proteins: amino acids that are near each other on the protein backbone are more likely to contribute to, and collaborate toward, a common subfunction., Author Summary Genes are templates for proteins, yet evolutionary studies of genes and proteins often bear little resemblance. Analyses of gene evolution typically treat each codon independently, quantifying gene evolution by summing over the constituent codons. In contrast, studies of protein evolution generally incorporate protein structure and interactions between amino acids explicitly. We investigate correlations in the evolution of codons as a function of their distance from each other along the protein coding sequence. This approach is motivated by the expectation that codons near each other in sequence often encode amino acids belonging to the same functional unit. Consequently, these amino acids are more likely to interact and/or experience similar selective regimes, introducing correlation between the evolution of the underlying codons. We find codon evolution in Drosophilids to be correlated over a characteristic length scale of ≈10 codons. Specifically, the presence of a non-synonymous substitution substantially increases the probability of further such substitutions nearby, particularly within that lineage. Further analysis suggests both functional interactions between amino acids and correlation in the strength of selection contribute to this effect. These findings are relevant for understanding the relative importance of different modes of selection, and particularly the role of epistasis, in gene and protein evolution.

The cancer stem cell (CSC) concept is a highly debated topic in cancer research. While experimental evidence in favor of the cancer stem cell theory is apparently abundant, the results are often criticized as being difficult to interpret. An important reason for this is that most experimental data that support this model rely on transplantation studies. In this study we use a novel cellular Potts model to elucidate the dynamics of established malignancies that are driven by a small subset of CSCs. Our results demonstrate that epigenetic mutations that occur during mitosis display highly altered dynamics in CSC-driven malignancies compared to a classical, non-hierarchical model of growth. In particular, the heterogeneity observed in CSC-driven tumors is considerably higher. We speculate that this feature could be used in combination with epigenetic (methylation) sequencing studies of human malignancies to prove or refute the CSC hypothesis in established tumors without the need for transplantation. Moreover our tumor growth simulations indicate that CSC-driven tumors display evolutionary features that can be considered beneficial during tumor progression. Besides an increased heterogeneity they also exhibit properties that allow the escape of clones from local fitness peaks. This leads to more aggressive phenotypes in the long run and makes the neoplasm more adaptable to stringent selective forces such as cancer treatment. Indeed when therapy is applied the clone landscape of the regrown tumor is more aggressive with respect to the primary tumor, whereas the classical model demonstrated similar patterns before and after therapy. Understanding these often counter-intuitive fundamental properties of (non-)hierarchically organized malignancies is a crucial step in validating the CSC concept as well as providing insight into the therapeutical consequences of this model., Author Summary Cancer is in essence a genetic disease that leads to uncontrolled cell proliferation, invasion and metastasis. The cancer stem cell (CSC) hypothesis states that tumors are not just a mass of uniform malignant cells but they are hierarchically organized, like normal tissues. At the top of such a hierarchy are cancer stem cells that fuel tumor growth in the long run, whereas the majority of other cells are able to divide only a few times. The experiments that support the CSC hypothesis are often criticized as being difficult to interpret. A novel approach to test the CSC paradigm is to integrate mathematical modeling with DNA variation data that carry the phylogenetic history of cells. We have developed a model that simulates the occurrence of such changes under both the CSC hypothesis and the classical, purely stochastic scenario. We found that although a CSC-driven tumor has a smaller number of tumorigenic cells, it triggers more malignant properties such as invasive growth, heterogeneity and evolutionary escape from peaks in the fitness landscape. These properties, that are unique to the CSC model, are enhanced even further when a treatment is applied to the tumor.

BackgroundHepatitis E virus (HEV) is an enterically transmitted hepatropic virus. It segregates as four genotypes. All genotypes infect humans while only genotypes 3 and 4 also infect several animal species. It has been suggested that hepatitis E is zoonotic, but no study has analyzed the evolutionary history of HEV. We present here an analysis of the evolutionary history of HEV.Methods and findingsThe times to the most recent common ancestors for all four genotypes of HEV were calculated using BEAST to conduct a Bayesian analysis of HEV. The population dynamics for genotypes 1, 3 and 4 were analyzed using skyline plots. Bayesian analysis showed that the most recent common ancestor for modern HEV existed between 536 and 1344 years ago. The progenitor of HEV appears to have given rise to anthropotropic and enzootic forms of HEV, which evolved into genotypes 1 and 2 and genotypes 3 and 4, respectively. Population dynamics suggest that genotypes 1, 3 and 4 experienced a population expansion during the 20(th) century. Genotype 1 has increased in infected population size ∼30-35 years ago. Genotype 3 and 4 have experienced an increase in population size starting late in the 19(th) century until ca.1940-45, with genotype 3 having undergone additional rapid expansion until ca.1960. The effective population size for both genotype 3 and 4 rapidly declined to pre-expansion levels starting in ca.1990. Genotype 4 was further examined as Chinese and Japanese sequences, which exhibited different population dynamics, suggesting that this genotype experienced different evolutionary history in these two countries.ConclusionsHEV appears to have evolved through a series of steps, in which the ancestors of HEV may have adapted to a succession of animal hosts leading to humans. Analysis of the population dynamics of HEV suggests a substantial temporal variation in the rate of transmission among HEV genotypes in different geographic regions late in the 20(th) Century.

While the majority of multiexonic human genes show some evidence of alternative splicing, it is unclear what fraction of observed splice forms is functionally relevant. In this study, we examine the extent of alternative splicing in human cells using deep RNA sequencing and de novo identification of splice junctions. We demonstrate the existence of a large class of low abundance isoforms, encompassing approximately 150,000 previously unannotated splice junctions in our data. Newly-identified splice sites show little evidence of evolutionary conservation, suggesting that the majority are due to erroneous splice site choice. We show that sequence motifs involved in the recognition of exons are enriched in the vicinity of unconserved splice sites. We estimate that the average intron has a splicing error rate of approximately 0.7% and show that introns in highly expressed genes are spliced more accurately, likely due to their shorter length. These results implicate noisy splicing as an important property of genome evolution., Author Summary Most human genes are split into pieces, such that the protein-coding parts (exons) are separated in the genome by large tracts of non-coding DNA (introns) that must be transcribed and spliced out to create a functional transcript. Variation in splicing reactions can create multiple transcripts from the same gene, yet the function for many of these alternative transcripts is unknown. In this study, we show that many of these transcripts are due to splicing errors which are not preserved over evolutionary time. We estimate that the error rate in the splicing of an intron is about 0.7% and demonstrate that there are two major types of splicing error: errors in the recognition of exons and errors in the precise choice of splice site. These results raise the possibility that variation in levels of alternative splicing across species may in part be to variation in splicing error rate.

BACKGROUND: To utilize the power of high-throughput sequencers, target enrichment methods have been developed. The majority of these require reagents and equipment that are only available from commercial vendors and are not suitable for the targets that are a few kilobases in length. METHODOLOGY/PRINCIPAL FINDINGS: We describe a novel and economical method in which custom made long-range PCR products are used to capture complete human mitochondrial genomes from complex DNA mixtures. We use the method to capture 46 complete mitochondrial genomes in parallel and we sequence them on a single lane of an Illumina GA(II) instrument. CONCLUSIONS/SIGNIFICANCE: This method is economical and simple and particularly suitable for targets that can be amplified by PCR and do not contain highly repetitive sequences such as mtDNA. It has applications in population genetics and forensics, as well as studies of ancient DNA.

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs.

Gene expression responds to changes in conditions but also stochastically among individuals. In budding yeast, both expression responsiveness across conditions (“plasticity”) and cell-to-cell variation (“noise”) have been quantified for thousands of genes and found to correlate across genes. It has been argued therefore that noise and plasticity may be strongly coupled and mechanistically linked. This is consistent with some theoretical ideas, but a strong coupling between noise and plasticity also has the potential to introduce cost–benefit conflicts during evolution. For example, if high plasticity is beneficial (genes need to respond to the environment), but noise is detrimental (fluctuations are harmful), then strong coupling should be disfavored. Here, evidence is presented that cost–benefit conflicts do occur and that they constrain the evolution of gene expression and promoter usage. In contrast to recent assertions, coupling between noise and plasticity is not a general property, but one associated with particular mechanisms of transcription initiation. Further, promoter architectures associated with coupling are avoided when noise is most likely to be detrimental, and noise and plasticity are largely independent traits for core cellular components. In contrast, when genes are duplicated noise–plasticity coupling increases, consistent with reduced detrimental affects of expression variation. Noise–plasticity coupling is, therefore, an evolvable trait that may constrain the emergence of highly responsive gene expression and be selected against during evolution. Further, the global quantitative data in yeast suggest that one mechanism that relieves the constraints imposed by noise–plasticity coupling is gene duplication, providing an example of how duplication can facilitate escape from adaptive conflicts., Author Summary Gene expression needs to respond to changes in conditions, but also varies stochastically among cells in a homogenous environment. It has been argued that these two levels of expression variation may be coupled, relating to the same underlying molecular mechanisms. However, such a strong coupling between expression “plasticity” and expression “noise” may introduce cost–benefit conflicts during evolution. For example, if plasticity is beneficial, but noise is detrimental, then coupling will be disfavored. In this work, evidence is presented that such cost–benefit conflicts do occur and that they constrain the evolution of gene expression in yeast. In contrast to recent conclusions, it is shown that noise–plasticity coupling is not a general result, but rather one associated with particular mechanisms of transcription initiation. Promoter architectures associated with coupling are avoided when noise is detrimental, and noise and plasticity are not coupled for core cellular components. Noise–plasticity coupling is therefore not a general property of gene expression, but an evolvable trait that may constrain the evolution of gene expression and be selected against during evolution. Further, gene duplication may facilitate escape from the adaptive conflict imposed by coupling.

The frequent dispensability of duplicated genes in budding yeast is heralded as a hallmark of genetic robustness contributed by genetic redundancy. However, theoretical predictions suggest such backup by redundancy is evolutionarily unstable, and the extent of genetic robustness contributed from redundancy remains controversial. It is anticipated that, to achieve mutual buffering, the duplicated paralogs must at least share some functional overlap. However, counter-intuitively, several recent studies reported little functional redundancy between these buffering duplicates. The large yeast genetic interactions released recently allowed us to address these issues on a genome-wide scale. We herein characterized the synthetic genetic interactions for ∼500 pairs of yeast duplicated genes originated from either whole-genome duplication (WGD) or small-scale duplication (SSD) events. We established that functional redundancy between duplicates is a pre-requisite and thus is highly predictive of their backup capacity. This observation was particularly pronounced with the use of a newly introduced metric in scoring functional overlap between paralogs on the basis of gene ontology annotations. Even though mutual buffering was observed to be prevalent among duplicated genes, we showed that the observed backup capacity is largely an evolutionarily transient state. The loss of backup capacity generally follows a neutral mode, with the buffering strength decreasing in proportion to divergence time, and the vast majority of the paralogs have already lost their backup capacity. These observations validated previous theoretic predictions about instability of genetic redundancy. However, departing from the general neutral mode, intriguingly, our analysis revealed the presence of natural selection in stabilizing functional overlap between SSD pairs. These selected pairs, both WGD and SSD, tend to have decelerated functional evolution, have higher propensities of co-clustering into the same protein complexes, and share common interacting partners. Our study revealed the general principles for the long-term retention of genetic redundancy., Author Summary Eukaryotic cells show remarkable robustness against external perturbations, which has been thought to be attributed, at least in part, to the extensive gene duplication events in eukaryotic genomes. By duplication, genes are likely to gain redundant copies for backup purposes, however, this notion contradicts the population genetic theory that genetic redundancy is evolutionarily unstable. In this study, we used yeast as a model organism to delineate the evolutionary trajectory of genetic robustness by gene duplication, utilizing the comprehensively characterized synthetic genetic interaction data in the yeast genome. We showed that the evolution of genetic robustness by duplication follows a neutral mode, with the loss of backup capacity proportional to the divergence time. However, natural selection was also acting on a few pairs to maintain their long-term backup capacity; and these pairs are slowly evolving, are co-clustered in the same protein complexes, and tend to interact with the similar partners. This study unravels the general principles underlying the evolution of the cellular robustness arising from genetic redundancy.

Natural killer (NK) cells serve essential functions in immunity and reproduction. Diversifying these functions within individuals and populations are rapidly-evolving interactions between highly polymorphic major histocompatibility complex (MHC) class I ligands and variable NK cell receptors. Specific to simian primates is the family of Killer cell Immunoglobulin-like Receptors (KIR), which recognize MHC class I and associate with a range of human diseases. Because KIR have considerable species-specificity and are lacking from common animal models, we performed extensive comparison of the systems of KIR and MHC class I interaction in humans and chimpanzees. Although of similar complexity, they differ in genomic organization, gene content, and diversification mechanisms, mainly because of human-specific specialization in the KIR that recognizes the C1 and C2 epitopes of MHC-B and -C. Humans uniquely focused KIR recognition on MHC-C, while losing C1-bearing MHC-B. Reversing this trend, C1-bearing HLA-B46 was recently driven to unprecedented high frequency in Southeast Asia. Chimpanzees have a variety of ancient, avid, and predominantly inhibitory receptors, whereas human receptors are fewer, recently evolved, and combine avid inhibitory receptors with attenuated activating receptors. These differences accompany human-specific evolution of the A and B haplotypes that are under balancing selection and differentially function in defense and reproduction. Our study shows how the qualitative differences that distinguish the human and chimpanzee systems of KIR and MHC class I predominantly derive from adaptations on the human line in response to selective pressures placed on human NK cells by the competing needs of defense and reproduction., Author Summary Natural killer (NK) cells are versatile lymphocytes that make essential contributions to immune defense and placental reproduction. Essential to NK cell development, diversification and function are variable families of surface receptors that recognize equally variable determinants of polymorphic major histocompatibility complex (MHC) class I molecules, better known as the tissue types matched in clinical organ transplantation. These ligand-receptor interactions evolve rapidly, exhibiting much species specificity and convergent evolution. Consequently, mice represent a poor model, because their receptors are so disparate from the independently evolved human counterparts that are restricted to simian primates. To identify unique and shared aspects of human NK cell biology, we have defined the genomics, population biology, and immunology of variable chimpanzee NK cell receptors and ligands to a level permitting accurate, informed comparison with the well-characterized human system. In both receptors and ligands there are dramatic, qualitative differences between humans and chimpanzees. We show these differences arose during human evolution from the last common human–chimpanzee ancestor, while the chimpanzee system remained relatively stable. That two so closely related species exhibit major differences in NK cell receptors and ligands testifies to the strong and varying selection imposed by the different demands and competing needs of defense and reproduction.

The human genome is characterised by many runs of homozygous genotypes, where identical haplotypes were inherited from each parent. The length of each run is determined partly by the number of generations since the common ancestor: offspring of cousin marriages have long runs of homozygosity (ROH), while the numerous shorter tracts relate to shared ancestry tens and hundreds of generations ago. Human populations have experienced a wide range of demographic histories and hold diverse cultural attitudes to consanguinity. In a global population dataset, genome-wide analysis of long and shorter ROH allows categorisation of the mainly indigenous populations sampled here into four major groups in which the majority of the population are inferred to have: (a) recent parental relatedness (south and west Asians); (b) shared parental ancestry arising hundreds to thousands of years ago through long term isolation and restricted effective population size (N-e), but little recent inbreeding (Oceanians); (c) both ancient and recent parental relatedness (Native Americans); and (d) only the background level of shared ancestry relating to continental N-e (predominantly urban Europeans and East Asians; lowest of all in sub-Saharan African agriculturalists), and the occasional cryptically inbred individual. Moreover, individuals can be positioned along axes representing this demographic historic space. Long runs of homozygosity are therefore a globally widespread and under-appreciated characteristic of our genomes, which record past consanguinity and population isolation and provide a distinctive record of the demographic history of an individual's ancestors. Individual ROH measures will also allow quantification of the disease risk arising from polygenic recessive effects.

Two bursts of gene gains occurred on the mammalian X chromosome contribute to an age-dependent chromosomal distribution of male-biased genes., Mammalian X chromosomes evolved under various mechanisms including sexual antagonism, the faster-X process, and meiotic sex chromosome inactivation (MSCI). These forces may contribute to nonrandom chromosomal distribution of sex-biased genes. In order to understand the evolution of gene content on the X chromosome and autosome under these forces, we dated human and mouse protein-coding genes and miRNA genes on the vertebrate phylogenetic tree. We found that the X chromosome recently acquired a burst of young male-biased genes, which is consistent with fixation of recessive male-beneficial alleles by sexual antagonism. For genes originating earlier, however, this pattern diminishes and finally reverses with an overrepresentation of the oldest male-biased genes on autosomes. MSCI contributes to this dynamic since it silences X-linked old genes but not X-linked young genes. This demasculinization process seems to be associated with feminization of the X chromosome with more X-linked old genes expressed in ovaries. Moreover, we detected another burst of gene originations after the split of eutherian mammals and opossum, and these genes were quickly incorporated into transcriptional networks of multiple tissues. Preexisting X-linked genes also show significantly higher protein-level evolution during this period compared to autosomal genes, suggesting positive selection accompanied the early evolution of mammalian X chromosomes. These two findings cast new light on the evolutionary history of the mammalian X chromosome in terms of gene gain, sequence, and expressional evolution., Author Summary Some evolutionary theories predict that the X chromosome will be enriched for genes with male functions. However, recent studies showed there had been gene traffic in which autosomal male-biased genes were retroposed from X-linked parental genes. A question remains about whether this pattern also holds for all types of new genes. Herein, using comparative genomic analysis, we dated all human and mouse genes to the vertebrate phylogenetic tree. We found that the X chromosome evolved with two bursts of gene origination events. The recent burst includes mainly male-biased genes in contrast to older X-linked genes that are often female-biased in expression. Meiotic sex chromosome inactivation contributes to this dynamic since it silences the older but not the younger X-linked genes. The older burst was after the split of eutherian mammals and the marsupial opossum, and the genes from this burst were quickly incorporated into transcriptional networks of multiple tissues, especially in the brain. The transcriptional expansion, together with the rapid protein evolution of the preexisting old X-linked genes, suggests that positive selection was acting in the early evolution of the mammalian X chromosome. These two lines of findings revealed extensive gene evolution in the mammalian X chromosome.

Haploinsufficiency, wherein a single functional copy of a gene is insufficient to maintain normal function, is a major cause of dominant disease. Human disease studies have identified several hundred haploinsufficient (HI) genes. We have compiled a map of 1,079 haplosufficient (HS) genes by systematic identification of genes unambiguously and repeatedly compromised by copy number variation among 8,458 apparently healthy individuals and contrasted the genomic, evolutionary, functional, and network properties between these HS genes and known HI genes. We found that HI genes are typically longer and have more conserved coding sequences and promoters than HS genes. HI genes exhibit higher levels of expression during early development and greater tissue specificity. Moreover, within a probabilistic human functional interaction network HI genes have more interaction partners and greater network proximity to other known HI genes. We built a predictive model on the basis of these differences and annotated 12,443 genes with their predicted probability of being haploinsufficient. We validated these predictions of haploinsufficiency by demonstrating that genes with a high predicted probability of exhibiting haploinsufficiency are enriched among genes implicated in human dominant diseases and among genes causing abnormal phenotypes in heterozygous knockout mice. We have transformed these gene-based haploinsufficiency predictions into haploinsufficiency scores for genic deletions, which we demonstrate to better discriminate between pathogenic and benign deletions than consideration of the deletion size or numbers of genes deleted. These robust predictions of haploinsufficiency support clinical interpretation of novel loss-of-function variants and prioritization of variants and genes for follow-up studies., Author Summary Humans, like most complex organisms, have two copies of most genes in their genome, one from the mother and one from the father. This redundancy provides a back-up copy for most genes, should one copy be lost through mutation. For a minority of genes, one functional copy is not enough to sustain normal human function, and mutations causing the loss of function of one of the copies of such genes are a major cause of childhood developmental diseases. Over the past 20 years medical geneticists have identified over 300 such genes, but it is not known how many of the 22,000 genes in our genome may also be sensitive to gene loss. By comparing these ∼300 genes known to be sensitive to gene loss with over 1,000 genes where loss of a single copy does not result in disease, we have identified some key evolutionary and functional similarities between genes sensitive to loss of a single copy. We have used these similarities to predict for most genes in the genome, whether loss of a single copy is likely to result in disease. These predictions will help in the interpretation of mutations seen in patients.

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite.

The X chromosome often plays a central role in hybrid male sterility between species, but it is unclear if this reflects underlying regulatory incompatibilities. Here we combine phenotypic data with genome-wide expression data to directly associate aberrant expression patterns with hybrid male sterility between two species of mice. We used a reciprocal cross in which F1 males are sterile in one direction and fertile in the other direction, allowing us to associate expression differences with sterility rather than with other hybrid phenotypes. We found evidence of extensive over-expression of the X chromosome during spermatogenesis in sterile but not in fertile F1 hybrid males. Over-expression was most pronounced in genes that are normally expressed after meiosis, consistent with an X chromosome-wide disruption of expression during the later stages of spermatogenesis. This pattern was not a simple consequence of faster evolutionary divergence on the X chromosome, because X-linked expression was highly conserved between the two species. Thus, transcriptional regulation of the X chromosome during spermatogenesis appears particularly sensitive to evolutionary divergence between species. Overall, these data provide evidence for an underlying regulatory basis to reproductive isolation in house mice and underscore the importance of transcriptional regulation of the X chromosome to the evolution of hybrid male sterility., Author Summary The X chromosome plays an important role in the development of reproductive isolation between species, but the basis for this has remained unclear. One possible explanation is that sperm development is sensitive to disruption of X-linked gene regulation. In mice, evidence linking abnormal gene expression on the X chromosome with reproductive isolation has been lacking until now. Here we use experimental crosses within and between species of mice and genome-wide expression data to identify aberrant expression patterns associated with hybrid male sterility. We observed chromosome-wide over-expression of the X chromosome during spermatogenesis in sterile hybrid males and developmentally localized this breakdown to an apparent disruption of X-inactivation. Collectively, these results highlight the importance of gene regulation to the evolution of reproductive isolation and support the hypothesis that improper expression of the X chromosome during spermatogenesis is an important mechanism contributing to the rapid evolution of hybrid male sterility.

Chromosomal location has a significant effect on the evolutionary dynamics of genes involved in sexual dimorphism, impacting both the pattern of sex-specific gene expression and the rate of duplication and protein evolution for these genes. For nearly all non-model organisms, however, knowledge of chromosomal gene content is minimal and difficult to obtain on a genomic scale. In this study, we utilized Comparative Genomic Hybridization (CGH), using probes designed from EST sequence, to identify genes located on the X chromosome of four species in the stalk-eyed fly genus Teleopsis. Analysis of log2 ratio values of female-to-male hybridization intensities from the CGH microarrays for over 3,400 genes reveals a strongly bimodal distribution that clearly differentiates autosomal from X-linked genes for all four species. Genotyping of 33 and linkage mapping of 28 of these genes in Teleopsis dalmanni indicate the CGH results correctly identified chromosomal location in all cases. Syntenic comparison with Drosophila indicates that 90% of the X-linked genes in Teleopsis are homologous to genes located on chromosome 2L in Drosophila melanogaster, suggesting the formation of a nearly complete neo-X chromosome from Muller element B in the dipteran lineage leading to Teleopsis. Analysis of gene movement both relative to Drosophila and within Teleopsis indicates that gene movement is significantly associated with 1) rates of protein evolution, 2) the pattern of gene duplication, and 3) the evolution of eyespan sexual dimorphism. Overall, this study reveals that diopsids are a critical group for understanding the evolution of sex chromosomes within Diptera. In addition, we demonstrate that CGH is a useful technique for identifying chromosomal sex-linkage and should be applicable to other organisms with EST or partial genomic information., Author Summary The distribution and organization of genes on chromosomes vary widely among animals. Chromosomes can change in number and size, as well as gene composition, over short evolutionary time scales. Furthermore, chromosome location can influence how genes are expressed in various tissues and how they evolve. The sex chromosomes, in particular, have a dynamic impact on gene movement, expression, and evolution. Uncovering the chromosomal location of genes has traditionally been difficult for non-model organism species. In this study, we assess sex chromosome linkage using a new method that hybridizes DNA from males and females to probes representing over 3,400 genes in stalk-eyed flies. This technique identifies 533 genes (15%) that are located on the X chromosome with the remaining genes located on two autosomes. Comparison of these genes with their location in Drosophila indicates that the X chromosome in stalk-eyed flies is nearly completely homologous to the autosome 2L in D. melanogaster. This result reveals the formation of a neo-X chromosome in the lineage leading to stalk-eyed flies and indicates that stalk-eyed flies provide a valuable new system to study the origin and evolution of sex chromosomes.

The functional structure of all biologically active molecules is dependent on intra- and inter-molecular interactions. This is especially evident for RNA molecules whose functionality, maturation, and regulation require formation of correct secondary structure through encoded base-pairing interactions. Unfortunately, intra- and inter-molecular base-pairing information is lacking for most RNAs. Here, we marry classical nuclease-based structure mapping techniques with high-throughput sequencing technology to interrogate all base-paired RNA in Arabidopsis thaliana and identify ∼200 new small (sm)RNA–producing substrates of RNA–DEPENDENT RNA POLYMERASE6. Our comprehensive analysis of paired RNAs reveals conserved functionality within introns and both 5′ and 3′ untranslated regions (UTRs) of mRNAs, as well as a novel population of functional RNAs, many of which are the precursors of smRNAs. Finally, we identify intra-molecular base-pairing interactions to produce a genome-wide collection of RNA secondary structure models. Although our methodology reveals the pairing status of RNA molecules in the absence of cellular proteins, previous studies have demonstrated that structural information obtained for RNAs in solution accurately reflects their structure in ribonucleoprotein complexes. Furthermore, our identification of RNA–DEPENDENT RNA POLYMERASE6 substrates and conserved functional RNA domains within introns and both 5′ and 3′ untranslated regions (UTRs) of mRNAs using this approach strongly suggests that RNA molecules are correctly folded into their secondary structure in solution. Overall, our findings highlight the importance of base-paired RNAs in eukaryotes and present an approach that should be widely applicable for the analysis of this key structural feature of RNA., Author Summary At the heart of RNA functionality, maturation, and regulation is the formation of intricate secondary structures that are dependent on specific nucleotide base-pairing interactions encoded within their sequences. These interactions can either be within (intra-molecular) or between (inter-molecular (heteroduplex)) RNA molecules. Although it is clear that secondary structure is abundantly important for the functionality and regulation of RNAs, comprehensive base-pairing interaction data are completely lacking for the majority of these molecules. To address this, we have developed a new approach for studying the base-pairing interactions of RNA molecules by marrying classical nuclease-based structure mapping techniques with high-throughput sequencing technology. We have used this approach to identify known and novel substrates of the base-paired RNA producing enzyme RNA–DEPENDENT RNA POLYMERASE6, reveal conserved functionality within introns and both 5′ and 3′ untranslated regions (UTRs) of mRNAs, uncover a novel population of functional RNAs, and produce a genome-wide collection of RNA secondary structure models by identifying the base-pairing interactions within each RNA molecule. Our findings demonstrate that our methodology should be widely applicable for the identification and analysis of base-paired RNAs in all biological organisms.

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy., Author Summary Leaf-cutter ants form massive subterranean colonies containing millions of workers that harvest hundreds of kilograms of leaves each year. They use these leaves to grow a mutualistic fungus that serves as the colony's primary food source. By farming fungus in specialized garden chambers, these dominant Neotropical herbivores facilitate rapid large-scale plant biomass conversion. Our understanding of this degradation process, and the responsible microbial community, is limited. In this study, we track the degradation of plant polymers in leaf-cutter ant fungus gardens and characterize the microbial community potentially mediating this process. We show that cellulose and hemicelluloses are degraded in the fungus gardens and that a previously unknown microbial community containing a diversity of bacteria is present. Metagenomic analysis of this community's genetic content revealed many genes predicted to encode enzymes capable of degrading plant cell walls. The ability of leaf-cutter ants to maintain an external microbial community with high plant biomass-degrading capacity likely represents a key step in the establishment of these ants as widespread, dominant insect herbivores in the Neotropics. This system is an important model for understanding how microbial communities degrade plant biomass in natural systems and has direct relevancy for bioenergy, given recent interest in cellulosic biofuels.

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome., A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest., Author Summary In contrast to the compact sequence of viruses and bacteria, determining the complete genome sequence of complex vertebrate genomes can be a daunting task. With the advent of “next-generation” sequencing platforms, it is now possible to rapidly sequence and assemble a vertebrate genome, especially for species for which genomic resources—genetic maps and markers—are currently available. We used a combination of two next-generation sequencing platforms, Roche 454 and Illumina GAII, and unique assembly tools to sequence the genome of the agriculturally important turkey, Meleagris gallopavo. Our draft assembly comprises approximately 1.1 gigabases of which 917 megabytes are assigned to specific chromosomes. Comparisons of the turkey genome sequence with those of the chicken, Gallus gallus, and the zebra finch, Taeniopygia guttata, provide insights into the evolution of the avian lineage. This genome sequence will facilitate discovery of agriculturally important genetic variants.

Mutation is the engine that drives evolution and adaptation forward in that it generates the variation on which natural selection acts. Mutation is a random process that nevertheless occurs according to certain biases. Elucidating mutational biases and the way they vary across species and within genomes is crucial to understanding evolution and adaptation. Here we demonstrate that clonal pathogens that evolve under severely relaxed selection are uniquely suitable for studying mutational biases in bacteria. We estimate mutational patterns using sequence datasets from five such clonal pathogens belonging to four diverse bacterial clades that span most of the range of genomic nucleotide content. We demonstrate that across different types of sites and in all four clades mutation is consistently biased towards AT. This is true even in clades that have high genomic GC content. In all studied cases the mutational bias towards AT is primarily due to the high rate of C/G to T/A transitions. These results suggest that bacterial mutational biases are far less variable than previously thought. They further demonstrate that variation in nucleotide content cannot stem entirely from variation in mutational biases and that natural selection and/or a natural selection-like process such as biased gene conversion strongly affect nucleotide content., Author Summary Natural selection sorts through the variability generated by mutation and biases evolution toward fitter outcomes. However, because mutation is itself not entirely random it can also bias the direction of evolution independently of selection. For instance, it is often assumed that the extreme variation observed in nucleotide content among bacteria (from ∼20% to ∼80% GC) is predominantly driven by extreme differences in mutational biases towards or away from GC. Here, we show that bacterial lineages that recently developed clonal, pathogenic lifestyles evolve under weak selection and that polymorphisms in these bacteria can be used as a fair proxy for mutational spectra. We analyze large sequence datasets from five clonal pathogens in four diverse bacterial clades spanning most of the range of genomic nucleotide content. We find that, surprisingly, mutation is AT-biased in every case to a very similar degree and in each case it is dominated by transitions from C/G to T/A. This demonstrates that mutational biases are far les variable than previously assumed and that variation in bacterial nucleotide content is not due entirely to mutational biases. Rather natural selection or a selection like process such as biased gene conversion strongly affect nucleotide content in bacteria.

The genomic GC-content of bacteria varies dramatically, from less than 20% to more than 70%. This variation is generally ascribed to differences in the pattern of mutation between bacteria. Here we test this hypothesis by examining patterns of synonymous polymorphism using datasets from 149 bacterial species. We find a large excess of synonymous GC→AT mutations over AT→GC mutations segregating in all but the most AT-rich bacteria, across a broad range of phylogenetically diverse species. We show that the excess of GC→AT mutations is inconsistent with mutation bias, since it would imply that most GC-rich bacteria are declining in GC-content; such a pattern would be unsustainable. We also show that the patterns are probably not due to translational selection or biased gene conversion, because optimal codons tend to be AT-rich, and the excess of GC→AT SNPs is observed in datasets with no evidence of recombination. We therefore conclude that there is selection to increase synonymous GC-content in many species. Since synonymous GC-content is highly correlated to genomic GC-content, we further conclude that there is selection on genomic base composition in many bacteria., Author Summary Shortly after it was proved that DNA was the genetic material it became apparent that organisms, and in particular bacteria, use the four letters of the genetic code to very different extents; the use of G and C varies from less than 20% in some species, to more than 70% in others. This variation in the use of G and C is usually attributed to differences in the pattern of mutation between species. Here we test this hypothesis, and show that, on the contrary, there seems to be pervasive selection on the base composition of the bacterial genome, particularly in GC-rich species. This suggests that many, if not all, sites may be subject to natural selection in many bacteria. Unfortunately, the reason why some bacteria should be selected for high GC-content remains unclear.

Positive selection is known to occur when the environment that an organism inhabits is suddenly altered, as is the case across recent human history. Genome-wide association studies (GWASs) have successfully illuminated disease-associated variation. However, whether human evolution is heading towards or away from disease susceptibility in general remains an open question. The genetic-basis of common complex disease may partially be caused by positive selection events, which simultaneously increased fitness and susceptibility to disease. We analyze seven diseases studied by the Wellcome Trust Case Control Consortium to compare evidence for selection at every locus associated with disease. We take a large set of the most strongly associated SNPs in each GWA study in order to capture more hidden associations at the cost of introducing false positives into our analysis. We then search for signs of positive selection in this inclusive set of SNPs. There are striking differences between the seven studied diseases. We find alleles increasing susceptibility to Type 1 Diabetes (T1D), Rheumatoid Arthritis (RA), and Crohn's Disease (CD) underwent recent positive selection. There is more selection in alleles increasing, rather than decreasing, susceptibility to T1D. In the 80 SNPs most associated with T1D (p-value

BackgroundAn ancient cyanobacterial incorporation into a eukaryotic organism led to the evolution of plastids (chloroplasts) and subsequently to the origin of the plant kingdom. The underlying mechanism and the identities of the partners in this monophyletic event remain elusive.Methodology/principal findingsTo shed light on this evolutionary process, we sequenced the genome of a cyanobacterium residing extracellularly in an endosymbiosis with a plant, the water-fern Azolla filiculoides Lam. This symbiosis was selected as it has characters which make it unique among extant cyanobacterial plant symbioses: the cyanobacterium lacks autonomous growth and is vertically transmitted between plant generations. Our results reveal features of evolutionary significance. The genome is in an eroding state, evidenced by a large proportion of pseudogenes (31.2%) and a high frequency of transposable elements (approximately 600) scattered throughout the genome. Pseudogenization is found in genes such as the replication initiator dnaA and DNA repair genes, considered essential to free-living cyanobacteria. For some functional categories of genes pseudogenes are more prevalent than functional genes. Loss of function is apparent even within the 'core' gene categories of bacteria, such as genes involved in glycolysis and nutrient uptake. In contrast, serving as a critical source of nitrogen for the host, genes related to metabolic processes such as cell differentiation and nitrogen-fixation are well preserved.Conclusions/significanceThis is the first finding of genome degradation in a plant symbiont and phenotypically complex cyanobacterium and one of only a few extracellular endosymbionts described showing signs of reductive genome evolution. Our findings suggest an ongoing selective streamlining of this cyanobacterial genome which has resulted in an organism devoted to nitrogen fixation and devoid of autonomous growth. The cyanobacterial symbiont of Azolla can thus be considered at the initial phase of a transition from free-living organism to a nitrogen-fixing plant entity, a transition process which may mimic what drove the evolution of chloroplasts from a cyanobacterial ancestor.

Genome-wide studies of post-transcriptional mRNA regulation in model organisms indicate a “post-transcriptional RNA regulon” model, in which a set of functionally related genes is regulated by mRNA–binding RNAs or proteins. One well-studied post-transcriptional regulon by Puf3p functions in mitochondrial biogenesis in budding yeast. The evolution of the Puf3p regulon remains unclear because previous studies have shown functional divergence of Puf3p regulon targets among yeast, fruit fly, and humans. By analyzing evolutionary patterns of Puf3p and its targeted genes in forty-two sequenced fungi, we demonstrated that, although the Puf3p regulon is conserved among all of the studied fungi, the dedicated regulation of mitochondrial biogenesis by Puf3p emerged only in the Saccharomycotina clade. Moreover, the evolution of the Puf3p regulon was coupled with evolution of codon usage bias in down-regulating expression of genes that function in mitochondria in yeast species after genome duplication. Our results provide a scenario for how evolution like a tinker exploits pre-existing materials of a conserved post-transcriptional regulon to regulate gene expression for novel functional roles., Author Summary It is well known that the evolution of gene expression can account for significant phenotypic diversity among species. Gene expression is regulated at various levels. Many studies have demonstrated gene expression changes caused by mutations in transcription. Post-transcriptional regulation is also crucial for splicing, translation, localization, and degeneration of mRNAs in eukaryotes and, thus, is important in determining the abundance of gene expression. Changes in post-transcriptional regulons are also important in the evolution of gene expression. In this study we investigated the evolution of a particular post-transcriptional regulon, Puf3p, in fungal species. Our results illustrated an important evolutionary mode for evolution of post-transcriptional regulon, i.e., pre-existing materials of conserved post-transcriptional regulons can be recruited to regulate gene expression for novel functional roles.

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We "shotgun" sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300x average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20x while heteroplasmic variants required >200x coverage. Several Sanger "misses" were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.

Background: Nonrandom distribution of rearrangements is a common feature of eukaryotic chromosomes that is not well understood in terms of genome organization and evolution. In the major African malaria vector Anopheles gambiae, polymorphic inversions are highly nonuniformly distributed among five chromosomal arms and are associated with epidemiologically important adaptations. However, it is not clear whether the genomic content of the chromosomal arms is associated with inversion polymorphism and fixation rates. Methodology/Principal Findings: To better understand the evolutionary dynamics of chromosomal inversions, we created a physical map for an Asian malaria mosquito, Anopheles stephensi, and compared it with the genome of An. gambiae. We also developed and deployed novel Bayesian statistical models to analyze genome landscapes in individual chromosomal arms An. gambiae. Here, we demonstrate that, despite the paucity of inversion polymorphisms on the X chromosome, this chromosome has the fastest rate of inversion fixation and the highest density of transposable elements, simple DNA repeats, and GC content. The highly polymorphic and rapidly evolving autosomal 2R arm had overrepresentation of genes involved in cellular response to stress supporting the role of natural selection in maintaining adaptive polymorphic inversions. In addition, the 2R arm had the highest density of regions involved in segmental duplications that clustered in the breakpoint-rich zone of the arm. In contrast, the slower evolving 2L, 3R, and 3L, arms were enriched with matrixattachment regions that potentially contribute to chromosome stability in the cell nucleus. Conclusions/Significance: These results highlight fundamental differences in evolutionary dynamics of the sex chromosome and autosomes and revealed the strong association between characteristics of the genome landscape and rates of chromosomal evolution. We conclude that a unique combination of various classes of genes and repetitive DNA in each arm, rather than a single type of repetitive element, is likely responsible for arm-specific rates of rearrangements. Published version

The understanding of selective constraints affecting genes is a major issue in biology. It is well established that gene expression level is a major determinant of the rate of protein evolution, but the reasons for this relationship remain highly debated. Here we demonstrate that gene expression is also a major determinant of the evolution of gene dosage: the rate of gene losses after whole genome duplications in the Paramecium lineage is negatively correlated to the level of gene expression, and this relationship is not a byproduct of other factors known to affect the fate of gene duplicates. This indicates that changes in gene dosage are generally more deleterious for highly expressed genes. This rule also holds for other taxa: in yeast, we find a clear relationship between gene expression level and the fitness impact of reduction in gene dosage. To explain these observations, we propose a model based on the fact that the optimal expression level of a gene corresponds to a trade-off between the benefit and cost of its expression. This COSTEX model predicts that selective pressure against mutations changing gene expression level or affecting the encoded protein should on average be stronger in highly expressed genes and hence that both the frequency of gene loss and the rate of protein evolution should correlate negatively with gene expression. Thus, the COSTEX model provides a simple and common explanation for the general relationship observed between the level of gene expression and the different facets of gene evolution., Author Summary The analysis of gene evolution is a powerful approach to recognize the genetic features that contribute to the fitness of organisms. It was shown previously that selective constraints on protein sequences increase with expression level. This observation was surprising because there is a priori no reason why lowly expressed genes should be less important than highly expressed genes for the proper function of an organism. Here we show that selective pressure on the evolution of gene dosage, which is another important aspect of gene evolution, is also directly dependent on gene expression level. To explain these observations, we propose a model based on the fact that gene expression is a costly process (notably protein synthesis), so that there is an optimal expression level for each gene corresponding to a trade-off between the benefit and the cost of its expression. This model predicts that selective pressure on gene expression level or on the encoded protein should on average be stronger in highly expressed genes, providing a simple and common explanation for the general relationship observed between gene expression and the different facets of gene evolution.

The equine-associated obligate pathogen Burkholderia mallei was developed by reductive evolution involving a substantial portion of the genome from Burkholderia pseudomallei, a free-living opportunistic pathogen. With its short history of divergence (∼3.5 myr), B. mallei provides an excellent resource to study the early steps in bacterial genome reductive evolution in the host. By examining 20 genomes of B. mallei and B. pseudomallei, we found that stepwise massive expansion of IS (insertion sequence) elements ISBma1, ISBma2, and IS407A occurred during the evolution of B. mallei. Each element proliferated through the sites where its target selection preference was met. Then, ISBma1 and ISBma2 contributed to the further spread of IS407A by providing secondary insertion sites. This spread increased genomic deletions and rearrangements, which were predominantly mediated by IS407A. There were also nucleotide-level disruptions in a large number of genes. However, no significant signs of erosion were yet noted in these genes. Intriguingly, all these genomic modifications did not seriously alter the gene expression patterns inherited from B. pseudomallei. This efficient and elaborate genomic transition was enabled largely through the formation of the highly flexible IS-blended genome and the guidance by selective forces in the host. The detailed IS intervention, unveiled for the first time in this study, may represent the key component of a general mechanism for early bacterial evolution in the host., Author Summary It has been known for some time that bacteria undergo genome-reduction when they transition from a free-living state to a constantly host-restricted state. High levels of IS element expansion were also found in these bacteria, and the IS elements were suggested to play a role in genome reductive evolution. Here we provide evidence for stepwise IS actions as the exclusive mechanism that mediates bacterial genomic changes during the early stage of constant host-bacterial association, by unveiling the processes that resulted in the development of B. mallei genome. We show the details of the multi-level interplay of IS elements, which facilitate the wide spread of the IS copies, and the overall mechanics in genome reduction and rearrangement. These processes appeared to operate as chain reactions mediating elaborate genomic transition, without seriously affecting the original gene expression patterns. The absence of differential gene expression in the resulting genome suggests that changes in transcriptional regulation that are often observed in other old bacterial genomes may take place subsequent to the IS-mediated steps, along with gradual nucleotide-level changes.

Much of the eukaryotic genome is known to be mobile, largely due to the movement of transposons and other parasitic elements. Recent work in plants and Drosophila suggests that mobility is also a feature of many nontransposon genes and gene families. Indeed, analysis of the Arabidopsis genome suggested that as many as half of all genes had moved to unlinked positions since Arabidopsis diverged from papaya roughly 72 million years ago, and that these mobile genes tend to fall into distinct gene families. However, the mechanism by which single gene transposition occurred was not deduced. By comparing two closely related species, Arabidopsis thaliana and Arabidopsis lyrata, we sought to determine the nature of gene transposition in Arabidopsis. We found that certain categories of genes are much more likely to have transposed than others, and that many of these transposed genes are flanked by direct repeat sequence that was homologous to sequence within the orthologous target site in A. lyrata and which was predominantly genic in identity. We suggest that intrachromosomal recombination between tandemly duplicated sequences, and subsequent insertion of the circular product, is the predominant mechanism of gene transposition., Author Summary Repetitive DNA, such as satellite repeats and transposons, is ubiquitous throughout the genome. Such repeats have been associated with DNA loss, circle formation, and gene transposition in plants and Drosophila. In this work we suggest that, in plants, one mechanism of gene mobility is intrachromosomal recombination via tandem repeats. In addition, we have demonstrated that the classes of genes that tend to form tandem duplications are more likely to have transposed than other gene classes. We conclude that tandem duplications may particularly facilitate gene excision and may also provide targets for gene insertion.

Background: Genetic ancestry is known to impact outcomes of genotype-phenotype studies that are designed to identify risk for common diseases in human populations. Failure to control for population stratification due to genetic ancestry can significantly confound results of disease association studies. Moreover, ancestry is a critical factor in assessing lifetime risk of disease, and can play an important role in optimizing treatment. As modern medicine moves towards using personal genetic information for clinical applications, it is important to determine genetic ancestry in an accurate, cost-effective and efficient manner. Self-identified race is a common method used to track and control for population stratification; however, social constructs of race are not necessarily informative for genetic applications. The use of ancestry informative markers (AIMs) is a more accurate method for determining genetic ancestry for the purposes of population stratification. Methodology/Principal Findings: Here we introduce a novel panel of 36 microsatellite (MSAT) AIMs that determines continental admixture proportions. This panel, which we have named Continental Ancestry Informative Markers or CoAIMs, consists of MSAT AIMs that were chosen based upon their measure of genetic variance (Fst), allele frequencies and their suitability for efficient genotyping. Genotype analysis using CoAIMs along with a Bayesian clustering method (STRUCTURE) is able to discern continental origins including Europe/Middle East (Caucasians), East Asia, Africa, Native America, and Oceania. In addition to determining continental ancestry for individuals without significant admixture, we applied CoAIMs to ascertain admixture proportions of individuals of self declared race. Conclusion/Significance: CoAIMs can be used to efficiently and effectively determine continental admixture proportions in a sample set. The CoAIMs panel is a valuable resource for genetic researchers performing case-control genetic association studies, as it can control for the confounding effects of population stratification. The MSAT-based approach used here has potential for broad applicability as a cost effective tool toward determining admixture proportions.

An association between sex-specific fitness and gene expression in the fruit fly provides an estimate of number, identity and function of sexually antagonistic genes in this species., When selective pressures differ between males and females, the genes experiencing these conflicting evolutionary forces are said to be sexually antagonistic. Although the phenotypic effect of these genes has been documented in both wild and laboratory populations, their identity, number, and location remains unknown. Here, by combining data on sex-specific fitness and genome-wide transcript abundance in a quantitative genetic framework, we identified a group of candidate genes experiencing sexually antagonistic selection in the adult, which correspond to 8% of Drosophila melanogaster genes. As predicted, the X chromosome is enriched for these genes, but surprisingly they represent only a small proportion of the total number of sex-biased transcripts, indicating that the latter is a poor predictor of sexual antagonism. Furthermore, the majority of genes whose expression profiles showed a significant relationship with either male or female adult fitness are also sexually antagonistic. These results provide a first insight into the genetic basis of intralocus sexual conflict and indicate that genetic variation for fitness is dominated and maintained by sexual antagonism, potentially neutralizing any indirect genetic benefits of sexual selection., Author Summary Males and females of many species are different: many of these differences are thought to have evolved because the sexes often have needs and strategies that do not coincide. For example, in fruit-flies, females may do best by concentrating their efforts in acquiring resources to be able to lay more eggs, while males would benefit most from increasing their mating and fertilization success. Such differences generate a sexual “conflict of interests”, and since as a general rule each behavioural, morphological or physiological characteristic is regulated by the same set of genes in the two sexes, this conflict takes place ultimately at the genetic level. In our study, we combined data on the reproductive success of different lines of fruit-flies with their gene expression profiles. We show that a large proportion of genes that contribute to male fertilization success are detrimental for female fecundity, and vice-versa. These results indicate that an optimal genotype for both sexes does not exist: many genes maintain different variants because they have opposite effects in males and females, perhaps helping to explain how genetic diversity is maintained in the face of selection.

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of diseases. Since the first isolation of C. pneumoniae TWAR in 1965, all human isolates have been essentially clonal, providing little evolutionary insight. To address this gap, we investigated the genetic diversity of 30 isolates from diverse geographical locations, from both human and animal origin (amphibian, reptilian, equine and marsupial). Based on the level of variation that we observed at 23 discreet gene loci, it was clearly evident that the animal isolates were more diverse than the isolates of human origin. Furthermore, we show that C. pneumoniae isolates could be grouped into five major genotypes, A-E, with A, B, D and E genotypes linked by geographical location, whereas genotype C was found across multiple continents. Our evidence strongly supports two separate animal-to-human cross species transfer events in the evolutionary history of this pathogen. The C. pneumoniae human genotype identified in the USA, Canada, Taiwan, Iran, Japan, Korea and Australia (non-Indigenous) most likely originated from a single amphibian or reptilian lineage, which appears to have been previously geographically widespread. We identified a separate human lineage present in two Australian Indigenous isolates (independent geographical locations). This lineage is distinct and is present in Australian amphibians as well as a range of Australian marsupials., Author Summary Chlamydia pneumoniae is an intracellular bacterial pathogen with an extremely diverse host range (humans, amphibians, reptiles and marsupials). We selected 23 target genes in order to investigate genetic diversity: seven of these had been lost or gained by C. pneumoniae, a further six were conserved, four were polymorphic (defined by greater than 20 SNPs per 1 kbp; in this study), and six were truncated or length polymorphic in one strain or the other. Our research highlights that C. pneumoniae animal isolates are much more genetically diverse than C. pneumoniae human isolates, and have crossed the host barrier to humans on at least two occasions. Our study provides new insights into the evolution of this complex pathogen.

Assessing the contribution of promoters and coding sequences to gene evolution is an important step toward discovering the major genetic determinants of human evolution. Many specific examples have revealed the evolutionary importance of cis-regulatory regions. However, the relative contribution of regulatory and coding regions to the evolutionary process and whether systemic factors differentially influence their evolution remains unclear. To address these questions, we carried out an analysis at the genome scale to identify signatures of positive selection in human proximal promoters. Next, we examined whether genes with positively selected promoters (Prom+ genes) show systemic differences with respect to a set of genes with positively selected protein-coding regions (Cod+ genes). We found that the number of genes in each set was not significantly different (8.1% and 8.5%, respectively). Furthermore, a functional analysis showed that, in both cases, positive selection affects almost all biological processes and only a few genes of each group are located in enriched categories, indicating that promoters and coding regions are not evolutionarily specialized with respect to gene function. On the other hand, we show that the topology of the human protein network has a different influence on the molecular evolution of proximal promoters and coding regions. Notably, Prom+ genes have an unexpectedly high centrality when compared with a reference distribution (P = 0.008, for Eigenvalue centrality). Moreover, the frequency of Prom+ genes increases from the periphery to the center of the protein network (P = 0.02, for the logistic regression coefficient). This means that gene centrality does not constrain the evolution of proximal promoters, unlike the case with coding regions, and further indicates that the evolution of proximal promoters is more efficient in the center of the protein network than in the periphery. These results show that proximal promoters have had a systemic contribution to human evolution by increasing the participation of central genes in the evolutionary process.

selection pressure on a gene, revealed from its evolutionary history, is determined by the role played by the gene, i.e., its biological function. The known conservation of a gene’s sequence coupled with the knowledge of the timing/dating of evolutionary events provides clues about the gene’s function. If a gene is preserved in all species with high sequence similarity and there are only a few duplication events along its evolutionary history, we have high confidence that its orthologs have the same function in different species. On the other hand, a large number of duplications and/or deletions along a gene’s evolutionary history could indicate neofunctionalization and/or nonorthologous gene displacement [3], and consequently, orthologs in different genomes may have different functions. These facts highlight the significance of functionoriented ortholog identification. In this article, we will review the general procedures to identify orthologs and make ortholog groups. We will focus on the functional analyses of orthologs, review previous work to assess functional consistency of orthologs, and make suggestions to construct better ortholog groups. Lastly, because orthologs can only be identified when the whole gene inventories from all the involved species are examined, the distribution of identified orthologs among species is an immediate result of looking into the composition of ortholog groups. Composition of ortholog groups, which bears important information for downstream research and applications, will also be briefly discussed.

Background The role of long non-coding RNAs (lncRNAs) in controlling gene expression has garnered increased interest in recent years. Sequencing projects, such as Fantom3 for mouse and H-InvDB for human, have generated abundant data on transcribed components of mammalian cells, the majority of which appear not to be protein-coding. However, much of the non-protein-coding transcriptome could merely be a consequence of ‘transcription noise’. It is therefore essential to use bioinformatic approaches to identify the likely functional candidates in a high throughput manner. Principal Findings We derived a scheme for classifying and annotating likely functional lncRNAs in mammals. Using the available experimental full-length cDNA data sets for human and mouse, we identified 78 lncRNAs that are either syntenically conserved between human and mouse, or that originate from the same protein-coding genes. Of these, 11 have significant sequence homology. We found that these lncRNAs exhibit: (i) patterns of codon substitution typical of non-coding transcripts; (ii) preservation of sequences in distant mammals such as dog and cow, (iii) significant sequence conservation relative to their corresponding flanking regions (in 50% cases, flanking regions do not have homology at all; and in the remaining, the degree of conservation is significantly less); (iv) existence mostly as single-exon forms (8/11); and, (v) presence of conserved and stable secondary structure motifs within them. We further identified orthologous protein-coding genes that are contributing to the pool of lncRNAs; of which, genes implicated in carcinogenesis are significantly over-represented. Conclusion Our comparative mammalian genomics approach coupled with evolutionary analysis identified a small population of conserved long non-protein-coding RNAs (lncRNAs) that are potentially functional across Mammalia. Additionally, our analysis indicates that amongst the orthologous protein-coding genes that produce lncRNAs, those implicated in cancer pathogenesis are significantly over-represented, suggesting that these lncRNAs could play an important role in cancer pathomechanisms.

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.

It is often claimed that genes affecting health in old age, such as cardiovascular and Alzheimer diseases, are beyond the reach of natural selection. We show in a simulation study based on known genetic (apolipoprotein E) and non-genetic risk factors (gender, diet, smoking, alcohol, exercise) that, because there is a statistical distribution of ages at which these genes exert their influence on morbidity and mortality, the effects of selection are in fact non-negligible. A gradual increase with each generation of the epsilon2 and epsilon3 alleles of the gene at the expense of the epsilon4 allele was predicted from the model. The epsilon2 allele frequency was found to increase slightly more rapidly than that for epsilon3, although there was no statistically significant difference between the two. Our result may explain the recent evolutionary history of the epsilon 2, 3 and 4 alleles of the apolipoprotein E gene and has wider relevance for genes affecting human longevity.

Dosage sensitivity is an important evolutionary force which impacts on gene dispensability and duplicability. The newly available data on human copy-number variation (CNV) allow an analysis of the most recent and ongoing evolution. Provided that heterozygous gene deletions and duplications actually change gene dosage, we expect to observe negative selection against CNVs encompassing dosage sensitive genes. In this study, we make use of several sources of population genetic data to identify selection on structural variations of dosage sensitive genes. We show that CNVs can directly affect expression levels of contained genes. We find that genes encoding members of protein complexes exhibit limited expression variation and overlap significantly with a manually derived set of dosage sensitive genes. We show that complexes and other dosage sensitive genes are underrepresented in CNV regions, with a particular bias against frequent variations and duplications. These results suggest that dosage sensitivity is a significant force of negative selection on regions of copy-number variation.

Background Aphids are one of the most important insect taxa in terms of ecology, evolutionary biology, genetics and genomics, and interactions with endosymbionts. Additionally, many aphids are serious pest species of agricultural and horticultural plants. Recent genetic and genomic research has expanded molecular resources for many aphid species, including the whole genome sequencing of the pea aphid, Acrythosiphon pisum. However, the invasive soybean aphid, Aphis glycines, lacks in any significant molecular resources. Methodology/Principal Findings Two next-generation sequencing technologies (Roche-454 and Illumina GA-II) were used in a combined approach to develop both transcriptomic and genomic resources, including expressed genes and molecular markers. Over 278 million bp were sequenced among the two methods, resulting in 19,293 transcripts and 56,688 genomic sequences. From this data set, 635 SNPs and 1,382 microsatellite markers were identified. For each sequencing method, different soybean aphid biotypes were used which revealed potential biotype specific markers. In addition, we uncovered 39,822 bp of sequence that were related to the obligatory endosymbiont, Buchnera aphidicola, as well as sequences that suggest the presence of Hamiltonella defensa, a facultative endosymbiont. Conclusions and Significance Molecular resources for an invasive, non-model aphid species were generated. Additionally, the power of next-generation sequencing to uncover endosymbionts was demonstrated. The resources presented here will complement ongoing molecular studies within the Aphididae, including the pea aphid whole genome, lead to better understanding of aphid adaptation and evolution, and help provide novel targets for soybean aphid control.

BackgroundThe genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels.Methodology/principal findingsWe designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource).Conclusions/significanceOur comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology.