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Background L1 retrotransposons have co-evolved with their mammalian hosts for the entire history of mammals and currently compose ~ 20% of a mammalian genome. B1 retrotransposons are dependent on L1 for retrotransposition and span the evolutionary history of rodents since their radiation. L1s were found to have lost their activity in a group of South American rodents, the Sigmodontinae, and B1 inactivation preceded the extinction of L1 in the same group. Consequently, a basal group of sigmodontines have active L1s but inactive B1s and a derived clade have both inactive L1s and B1s. It has been suggested that B1s became extinct during a long period of L1 quiescence and that L1s subsequently reemerged in the basal group. Results Here we investigate the evolutionary histories of L1 and B1 in the sigmodontine rodents and show that L1 activity continued until after the L1-extinct clade and the basal group diverged. After the split, L1 had a small burst of activity in the former group, followed by extinction. In the basal group, activity was initially low but was followed by a dramatic increase in L1 activity. We found the last wave of B1 retrotransposition was large and probably preceded the split between the two rodent clades. Conclusions Given that L1s had been steadily retrotransposing during the time corresponding to B1 extinction and that the burst of B1 activity preceding B1 extinction was large, we conclude that B1 extinction was not a result of L1 quiescence. Rather, the burst of B1 activity may have contributed to L1 extinction both by competition with L1 and by putting strong selective pressure on the host to control retrotransposition. Electronic supplementary material The online version of this article (10.1186/s13100-019-0164-5) contains supplementary material, which is available to authorized users.

Natural selection acting on synonymous mutations in protein-coding genes influences genome composition and evolution. In viruses, introducing synonymous mutations in genes encoding structural proteins can drastically reduce viral growth, providing a means to generate potent, live-attenuated vaccine candidates. However, an improved understanding of what compositional features are under selection and how combinations of synonymous mutations affect viral growth is needed to predictably attenuate viruses and make them resistant to reversion. We systematically recoded all nonoverlapping genes of the bacteriophage ΦX174 with codons rarely used in its Escherichia coli host. The fitness of recombinant viruses decreases as additional deoptimizing mutations are made to the genome, although not always linearly, and not consistently across genes. Combining deoptimizing mutations may reduce viral fitness more or less than expected from the effect size of the constituent mutations and we point out difficulties in untangling correlated compositional features. We test our model by optimizing the same genes and find that the relationship between codon usage and fitness does not hold for optimization, suggesting that wild-type ΦX174 is at a fitness optimum. This work highlights the need to better understand how selection acts on patterns of synonymous codon usage across the genome and provides a convenient system to investigate the genetic determinants of virulence.

L1s are transposable elements that move by a copy-and-paste mechanism that continuously increases their copy number in the genome, such that each genome has a record of the L1 history in that host lineage. They make up about 20% of the genomes of eutherian mammals and have played a major role in shaping genome evolution. Chiroptera has the lowest average genome size among mammalian orders and the only documented case of L1 extinction affecting an entire mammalian family. Herein, L1 activity and extinction are characterized in all families of the order Chiroptera using a method that enriches for the youngest lineages of L1s in the genome. In addition to the previously reported L1 extinction in Pteropodidae, L1 extinction was documented to occur in Mormoops blainvilli, but this event did not affect all species of Mormoopidae. Further, there was no evidence of concordance between the evolution of L1s and their chiropteran host. There were two L1 lineages present before the divergence of all extant bats. Both lineages are extinct in the Pteropodidae. One or the other L1 lineage is extinct in almost all bat families, but Taphozous melanopogon maintains active members of both. Most intriguingly, some families within the Rhinolophoidea retain one active L1 lineage whereas other families retain the other, creating a deep discontinuity between L1 phylogeny and chiropteran phylogeny. These results indicate that there have been numerous losses of active L1 lineages over the history of chiropteran evolution, but that all chiropteran families except Pteropodidae have retained L1 activity.

Natural selection acting on synonymous mutations in protein-coding genes influences genome composition and evolution. In viruses, introducing synonymous mutations in genes encoding structural proteins can drastically reduce viral growth, providing a means to generate potent, live attenuated vaccine candidates. However, an improved understanding of what compositional features are under selection and how combinations of synonymous mutations affect viral growth is needed to predictably attenuate viruses and make them resistant to reversion. We systematically recoded all non-overlapping genes of the bacteriophage ΦX174 with codons rarely used in itsE. colihost. The fitness of recombinant viruses decreases as additional deoptimizing mutations are made to the genome, although not always linearly, and not consistently across genes. Combining deoptimizing mutations may reduce viral fitness more or less than expected from the effect size of the constituent mutations and we point out difficulties in untangling correlated compositional features. We test our model by optimizing the same genes and find that the relationship between codon usage and fitness does not hold for optimization, suggesting that wild-type ΦX174 is at a fitness optimum. This work highlights the need to better understand how selection acts on patterns of synonymous codon usage across the genome and provides a convenient system to investigate the genetic determinants of virulence.

Here we present a novel protocol for the construction of saturation single-site—and massive multisite—mutant libraries of a bacteriophage. We segmented the ΦX174 genome into 14 nontoxic and nonreplicative fragments compatible with Golden Gate assembly. We next used nicking mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near-comprehensive single-site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned). Libraries possessed greater than 99% of all 11 860 programmed mutations. Golden Gate cloning was then used to assemble the complete ΦX174 mutant genome and generate libraries of infective viruses. This protocol will enable reverse genetics experiments for studying viral evolution and, with some modifications, can be applied for engineering therapeutically relevant bacteriophages with larger genomes.

In microbial communities, viruses compete with each other for host cells to infect. As a consequence of competition for hosts, viruses evolve inhibitory mechanisms to suppress their competitors. One such mechanism is superinfection exclusion, in which a preexisting viral infection prevents a secondary infection. The bacteriophage ΦX174 exhibits a potential superinfection inhibition mechanism (in which secondary infections are either blocked or resisted) known as the reduction effect. In this auto-inhibitory phenomenon, a plasmid containing a fragment of the ΦX174 genome confers resistance to infection among cells that were once permissive to ΦX174. Taking advantage of this plasmid system, we examine the inhibitory properties of the ΦX174 reduction effect on a range of wild ΦX174-like phages. We then assess how closely the reduction effect in the plasmid system mimics natural superinfection inhibition by carrying out phage–phage competitions in continuous culture, and we evaluate whether the overall competitive advantage can be predicted by phage fitness or by a combination of fitness and reduction effect inhibition. Our results show that viral fitness often correctly predicts the winner. However, a phage’s reduction sequence also provides an advantage to the phage in some cases, modulating phage–phage competition and allowing for persistence where competitive exclusion was expected. These findings provide strong evidence for more complex dynamics than were previously thought, in which the reduction effect may inhibit fast-growing viruses, thereby helping to facilitate coexistence.

Here we present a novel protocol for the construction of saturation single-site—and massive multi-site—mutant libraries of a bacteriophage. We segmented the ΦX174 genome into 14 non-toxic and non-replicative fragments compatible with golden gate assembly. We next used nicking mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near-comprehensive single-site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned). Libraries possessed greater than 99% of all 11,860 programmed mutations. Golden Gate cloning was then used to assemble the complete ΦX174 mutant genome and generate libraries of infective viruses. This protocol will enable reverse genetics experiments for studying viral evolution and, with some modifications, can be applied for engineering of therapeutically relevant bacteriophages with larger genomes.

BackgroundL1 retrotransposons have co-evolved with their mammalian hosts for the entire history of mammals and currently make up to 20% of a typical mammalian genome. B1 retrotransposons are dependent on L1 for retrotransposition and span the evolutionary history of rodents since their radiation. L1s were found to have lost their activity in a group of South American rodents, the Sigmodontinae, and B1 inactivation preceded the extinction of L1 in the same group. Consequently, a basal group of sigmodontines have active L1s but inactive B1s and a derived clade have both inactive L1s and B1s. It has been suggested that B1s became extinct during a long period of L1 quiescence and that L1s subsequently reemerged in the basal group.ResultsHere we investigate the evolutionary histories of L1 and B1 in the sigmodontine rodents and show that L1 activity continued until after the split of the L1-extinct clade and the basal group. After the split, L1s had a small burst of activity in the former group, followed by extinction. In the basal group, activity was initially low but was followed by a dramatic increase in L1 activity. We found the last wave of B1s retrotransposition was large and probably preceded the split between the two rodent clades.ConclusionsGiven that L1s had been steadily retrotransposing during the time corresponding to B1 extinction and that the burst of B1 activity preceding B1 extinction was large, we conclude that B1 extinction was not a result of L1 quiescence. Rather, the burst of B1 activity may have contributed to L1 extinction both by competition with L1 and by putting strong selective pressure on the host to control retrotransposition.

Fitness landscapes map genotypes to organismal fitness. Their topography depends on how mutational effects interact–epistasis–and is important for understanding evolutionary processes such as speciation, the rate of adaptation, the advantage of recombination, and predictability versus stochasticity of evolution. The growing amount of empirical data has made it possible to better test landscape models empirically. We argue that this endeavor will benefit from the development and use of meaningful null models against which to compare more complex models. Here we develop statistical and computational methods for fitting fitness data from mutation combinatorial networks to three simple models: additive, multiplicative and stickbreaking. We employ a Bayesian framework for doing model selection. Using simulations, we demonstrate that our methods work and we explore their statistical performance: bias, error, and the power to discriminate among models. We then illustrate our approach and its flexibility by analyzing several previously published datasets. An R-package that implements our methods is available in the CRAN repository under the nameStickbreaker.

[Graphic][1] Paul Joyce was born in 1958 in Butte, Montana, and grew up there. Many of the qualities that defined Paul in life were apparent in childhood. The sixth of 12 children, he was the youngest of the big kids but adopted by the younger ones—he was the bridge in his family and he

The 2014 Ebola virus outbreak in West Africa was the largest in recorded history and resulted in over 11,000 deaths. It is important to develop strategies for treatment and containment to avoid future epidemics of this magnitude. One strategy is to anticipate how the evolution of the virus might compromise treatment efforts. In this presentation I will show results from a molecular modeling study of the Ebola virus glycoprotein. As part of this study we have initiated a watch list of mutations that could potentially reduce the effectiveness of vaccines and antibody therapies. The list was generated by considering every possible mutation of the glycoprotein and including those that disrupt binding to protective antibodies but do not disrupt the protein function. This study emphasizes the need for more experimental structures of Ebola in order to expand the watch list.

Since the recent devastating outbreak of Ebola virus disease in western Africa, there has been significant effort to understand the evolution of the deadly virus that caused the outbreak. There has been a considerable investment in sequencing Ebola virus (EBOV) isolates, and the results paint an important picture of how the virus has spread in western Africa. EBOV evolution cannot be understood outside the context of previous outbreaks, however. We have focused this study on the evolution of the EBOV glycoprotein gene (GP) because one of its products, the spike glycoprotein (GP1,2), is central to the host immune response and because it contains a large amount of the phylogenetic signal for this virus. We inferred the maximum likelihood phylogeny of 96 nonredundant GP gene sequences representing each of the outbreaks since 1976 up to the end of 2014. We tested for positive selection and considered the placement of adaptive amino acid substitutions along the phylogeny and within the protein structure of GP1,2. We conclude that: 1) the common practice of rooting the phylogeny of EBOV between the first known outbreak in 1976 and the next outbreak in 1995 provides a misleading view of EBOV evolution that ignores the fact that there is a non-human EBOV host between outbreaks; 2) the N-terminus of GP1 may be constrained from evolving in response to the host immune system by the highly expressed, secreted glycoprotein, which is encoded by the same region of the GP gene; 3) although the mucin-like domain of GP1 is essential for EBOV in vivo, it evolves rapidly without losing its twin functions: providing O-linked glycosylation sites and a flexible surface.

In relating genotypes to fitness, models of adaptation need to both be computationally tractable and qualitatively match observed data. One reason that tractability is not a trivial problem comes from a combinatoric problem whereby no matter in what order a set of mutations occurs, it must yield the same fitness. We refer to this as the bookkeeping problem. Because of their commutative property, the simple additive and multiplicative models naturally solve the bookkeeping problem. However, the fitness trajectories and epistatic patterns they predict are inconsistent with the patterns commonly observed in experimental evolution. This motivates us to propose a new and equally simple model that we call stickbreaking. Under the stickbreaking model, the intrinsic fitness effects of mutations scale by the distance of the current background to a hypothesized boundary. We use simulations and theoretical analyses to explore the basic properties of the stickbreaking model such as fitness trajectories, the distribution of fitness achieved, and epistasis. Stickbreaking is compared to the additive and multiplicative models. We conclude that the stickbreaking model is qualitatively consistent with several commonly observed patterns of adaptive evolution.

We present a joint experimental/theoretical investigation into the roles of spatial structure and time in the competition between two pathogens for a single host. We suggest a natural mechanism by which competing pathogens can coexist when host evolution and competitive dynamics occur on similar timescales. Our experimental system consisted of a single bacterial host species and two competing bacteriophage strains grown on agar plates, with a serial transfer of samples of the bacteriophage population to fresh host populations after each incubation cycle. The experiments included two incubation times and two transfer protocols that either maintained or disrupted the spatial structure of the viruses at each transfer. The same bacteriophage acted as the dominant competitor under both transfer protocols. A striking difference between the treatments is that the weak competitor was able to persist in the long-incubation experiments but not in the short-incubation experiments. Mathematical and experimental evidence suggest that coexistence is due to the appearance of resistant mutant host cells that provide a transient “spatiotemporal refuge” for the weaker competitor. Our mathematical model is individual based, captures the stochastic spatial dynamics down to the level of individual cells, and helps to explain the differences in behavior under the various experimental conditions.

LINE-1 (L1) retrotransposons are the most abundant type of mammalian retroelement. They have profound effects on genome plasticity and have been proposed to fulfill essential host functions, yet it remains unclear where they lie on the spectrum from parasitism to mutualism. Their ubiquity makes it difficult to determine the extent of their effects on genome evolution and gene expression because of the relative dearth of animal models lacking L1 activity. We have isolated L1 sequences from 11 megabat species by a method that enriches for recently inserted L1s and have done a bioinformatic examination of L1 sequences from a 12th species whose genome was recently shotgun sequenced. An L1 extinction event appears to have occurred at least 24 million years ago (MYA) in an ancestor of the megabats. The ancestor was unusual in having maintained two highly divergent long-term L1 lineages with different levels of activity, which appear, on an evolutionary scale, to have simultaneously lost that activity. These megabat species can serve as new animal models to ask what effect loss of L1 activity has on mammalian genome evolution and gene expression.

In modeling evolutionary genetics, it is often assumed that mutational effects are assigned according to a continuous probability distribution, and multiple distributions have been used with varying degrees of justification. For mutations with beneficial effects, the distribution currently favored is the exponential distribution, in part because it can be justified in terms of extreme value theory, since beneficial mutations should have fitnesses in the extreme right tail of the fitness distribution. While the appeal to extreme value theory seems justified, the exponential distribution is but one of three possible limiting forms for tail distributions, with the other two loosely corresponding to distributions with right-truncated tails and those with heavy tails. We describe a likelihood-ratio framework for analyzing the fitness effects of beneficial mutations, focusing on testing the null hypothesis that the distribution is exponential. We also describe how to account for missing the smallest-effect mutations, which are often difficult to identify experimentally. This technique makes it possible to apply the test to gain-of-function mutations, where the ancestral genotype is unable to grow under the selective conditions. We also describe how to pool data across experiments, since we expect few possible beneficial mutations in any particular experiment.

Epistatic interactions between mutations are widespread. Theoretical investigations have shown that variability in epistatic effects influences fundamental evolutionary processes, yet few empirical studies have identified causes or the extent of this variation. We examined variation in epistatic effects of mutations at two host recognition sites in phiX174 bacteriophage. We calculated epistatic effects from the sum of fitness effects (log scale) of two single mutants and their corresponding double mutant for five combinations of mutations in six conditions. We found that epistatic effects differed in sign, degree, and variability across conditions. The data highlight that even between single mutations at the same two sites the sign and variability of epistatic effects are affected by environment. We discuss these findings in the context of studying the role of epistasis in evolution.

Background Defining factors that contributed to the fixation of a high number of underdominant chromosomal rearrangements is a complex task because not only molecular mechanisms must be considered, but also the uniqueness of natural history attributes of each taxon. Ideally, detailed investigation of the chromosome architecture of an organism and related groups, placed within a phylogenetic context, is required. We used multiple approaches to investigate the dynamics of chromosomal evolution in lineages of bats with considerable karyotypic variation, focusing on the different facets contributing to fixation of the exceptional chromosomal changes in Tonatia saurophila. Integration of empirical data with proposed models of chromosome evolution was performed to understand the probable conditions for Tonatia’s karyotypic evolution. Results The trajectory of reorganization of chromosome blocks since the common ancestor of Glossophaginae and Phyllostominae subfamilies suggests that multiple tandem fusions, as well as disruption and fusions of conserved phyllostomid chromosomes were major drivers of karyotypic reshuffling in Tonatia. Considerable variation in the rates of chromosomal evolution between phyllostomid lineages was observed. Thirty–nine unique fusions and fission events reached fixation in Tonatia over a short period of time, followed by ~12 million years of chromosomal stasis. Physical mapping of repetitive DNA revealed an unusual accumulation of LINE-1 sequences on centromeric regions, probably associated with the chromosomal dynamics of this genus. Conclusions Multiple rearrangements have reached fixation in a wave-like fashion in phyllostomid bats. Different biological features of Tonatia support distinct models of rearrangement fixation, and it is unlikely that the fixations were a result of solely stochastic processes in small ancient populations. Increased recombination rates were probably facilitated by expansion of repetitive DNA, reinforced by aspects of taxon reproduction and ecology.

A cornerstone of evolutionary ecology is that population density affects adaptation: r and K selection is the obvious example. The reverse is also appreciated: adaptation impacts population density. Yet, empirically demonstrating a direct connection between population density and adaptation is challenging. Here, we address both evolution and ecology of population density in models of viral (bacteriophage) chemostats. Chemostats supply nutrients for host cell growth, and the hosts are prey for viral reproduction. Two different chemostat designs have profoundly different consequences for viral evolution. If host and virus are confined to the same chamber, as in a predator-prey system, viral regulation of hosts feeds back to maintain low viral density (measured as infections per cell). Viral adaptation impacts host density but has a small effect on equilibrium viral density. More interesting are chemostats that supply the viral population with hosts from a virus-free refuge. Here, a type of evolutionary succession operates: adaptation at low viral density leads to higher density, but high density then favors competitive ability. Experiments support these models with both phenotypic and molecular data. Parallels to these designs exist in many natural systems, so these experimental systems may yield insights to the evolution and regulation of natural populations.

The observation that LINE-1 transposable elements are enriched on the X in comparison to the autosomes led to the hypothesis that LINE-1s play a role in X chromosome inactivation. If this hypothesis is correct, loss of LINE-1 activity would be expected to result in species extinction or in an alternate pathway of dosage compensation. One such alternative pathway would be to evolve a karyotype that does not require dosage compensation between the sexes. Two of the three extant species of the Ryukyu spiny rat Tokudaia have such a karyotype; both males and females are XO. We asked whether this karyotype arose due to loss of LINE-1 activity and thus the loss of a putative component in the X inactivation pathway. Although XO Tokudaia has no need for dosage compensation, LINE-1s have been recently active in Tokudaia osimensis and show higher density on the lone X than on the autosomes.

A large percentage of the repetitive elements in mammalian genomes are retroelements, which have been moved primarily by LINE-1 retrotransposons and endogenous retroviruses. Although LINE-1 elements have remained active throughout the mammalian radiation, specific groups of endogenous retroviruses generally remain active for comparatively shorter periods of time. Identification of an unusual extinction of LINE-1 activity in a group of South American rodents has opened a window for examination of the interplay in mammalian genomes between these ubiquitous retroelements. In the course of a search for any type of repetitive sequences whose copy numbers have substantially changed in Oryzomys palustris , a species that has lost LINE-1 activity, versus Sigmodon hispidus , a closely related species retaining LINE-1 activity, we have identified an endogenous retrovirus family differentially amplified in these two species. Analysis of three full-length, recently transposed copies, called mysTR elements, revealed gag , pro , and pol coding regions containing stop codons which may have accumulated either before or after retrotransposition. Isolation of related sequences in S. hispidus and the LINE-1 active outgroup species, Peromyscus maniculatus , by PCR of a pro-pol region has allowed determination of copy numbers in each species. Unusually high copy numbers of approximately 10,000 in O. palustris versus 1,000 in S. hispidus and 4,500 in the more distantly related P.maniculatus leave open the question of whether there is a connection between endogenous retrovirus activity and LINE-1 inactivity. Nevertheless, these independent expansions of mysTR represent recent amplifications of this endogenous retrovirus family to unprecedented levels.

Bacteriophage φX174 was evolved on a continuous supply of sensitive hosts for 180 days (∼13,000 phage generations). The average rate of nucleotide substitution was nearly 0.2% (11 substitutions)/20 days, and, surprisingly, substitutions accumulated in a clock-like manner throughout the study, except for a low rate during the first 20 days. Rates of silent and missense substitutions varied over time and among genes. Approximately 40% of the 71 missense changes and 25% of the 58 silent changes have been observed in previous adaptations; the rate of parallel substitution was highest in the early phase of the evolution, but 7% of the later changes had evolved in previous studies of much shorter duration. Several lines of evidence suggest that most of the changes were adaptive, even many of the silent substitutions. The sustained, high rate of adaptive evolution for 180 days defies a model of adaptation to a constant environment. We instead suggest that continuing molecular evolution reflects a potentially indefinite arms race, stemming from high levels of co-infection and the resulting conflict among genomes competing within the same cell.

LINE-1 transposable elements (L1s) are ubiquitous in mammals and are thought to have remained active since before the mammalian radiation. Only one L1 extinction event, in South American rodents in the genus Oryzomys, has been convincingly demonstrated. Here we examine the phylogenetic limits and evolutionary tempo of that extinction event by characterizing L1s in related rodents. Fourteen genera from five tribes within the Sigmodontinae subfamily were examined. Only the Sigmodontini, the most basal tribe in this group, demonstrate recent L1 activity. The Oryzomyini, Akodontini, Phyllotini, and Thomasomyini contain only L1s that appear to have inserted long ago; their L1s lack open reading frames, have mutations at conserved amino acid residues, and show numerous private mutations. They also lack restriction site-defined L1 subfamilies specific to any species, genus or tribe examined, and fail to form monophyletic species, genus or tribal L1 clusters. We determine here that this L1 extinction event occurred roughly 8.8 million years ago, near the divergence of Sigmodon from the remaining Sigmodontinae species. These species appear to be ideal model organisms for studying the impact of L1 inactivity on mammalian genomes.

Short Interspersed Nuclear Elements, or SINEs, retrotranspose despite lacking protein-coding capability. It has been proposed that SINEs utilize enzymes produced in trans by Long Interspersed Nuclear Elements, or LINEs. Strong support for this hypothesis is found in LINE and SINE pairs that share sequence homology; however, LINEs and SINEs in primates and rodents are only linked by an insertion site motif. We have now profiled L1 LINE and B1 SINE activity in 24 rodent species including candidate taxa for the first documented L1 extinction. As expected, there was no evidence for recent activity of B1s in species that also lack L1 activity. However, B1 silencing appears to have preceded L1 extinction, since B1 activity is also lacking in the genus most closely related to those lacking active L1s despite the presence of active L1s in this genus. A second genus with active L1s but inactive B1s was also identified.

The Lyon repeat hypothesis postulates that long interspersed elements (LINEs) play a role in X-chromosome inactivation. Evidence to support this hypothesis includes the observation that the degree of inactivation of autosomes translocated to the X chromosome is correlated with LINE density on that autosome. We examined the distribution of LINEs in the fruit bat Carollia brevicauda, which has an autosomal translocation to the X that occurred at least 7 million years ago. A quantitative analysis of LINE accumulation on multiple metaphase chromosome spreads revealed a significant accumulation on the original X relative to the attached autosome, the homolog of that autosome (Y2), and chromosome 1. Previous replication studies indicate that for the X and attached autosome, only the original X chromosome replicates late in Carollia females, and that the attached autosome replicates in the same timeframe as other autosomes. These data are compatible with the Lyon repeat hypothesis, and the possibility that LINEs act as booster elements for X inactivation remains a viable hypothesis. We address the procedures and limitations of quantitative analysis based on in situ hybridization.

Although L1 sequences are present in the genomes of all placental mammals and marsupials examined to date, their activity was lost in the megabat family, Pteropodidae, ∼24 million years ago. To examine the characteristics of L1s prior to their extinction, we analyzed the evolutionary history of L1s in the genome of a megabat, Pteropus vampyrus, and found a pattern of periodic L1 expansion and quiescence. In contrast to the well-characterized L1s in human and mouse, megabat genomes have accommodated two or more simultaneously active L1 families throughout their evolutionary history, and major peaks of L1 deposition into the genome always involved multiple families. We compared the consensus sequences of the two major megabat L1 families at the time of their extinction to consensus L1s of a variety of mammalian species. Megabat L1s are comparable to the other mammalian L1s in terms of adenosine content and conserved amino acids in the open reading frames (ORFs). However, the intergenic region (IGR) of the reconstructed element from the more active family is dramatically longer than the IGR of well-characterized human and mouse L1s. We synthesized the reconstructed element from this L1 family and tested the ability of its components to support retrotransposition in a tissue culture assay. Both ORFs are capable of supporting retrotransposition, while the IGR is inhibitory to retrotransposition, especially when combined with either of the reconstructed ORFs. We dissected the inhibitory effect of the IGR by testing truncated and shuffled versions and found that length is a key factor, but not the only one affecting inhibition of retrotransposition. Although the IGR is inhibitory to retrotransposition, this inhibition does not account for the extinction of L1s in megabats. Overall, the evolution of the L1 sequence or the quiescence of L1 is unlikely the reason of L1 extinction., Author Summary Most of a typical mammalian genome is occupied by transposable elements, which have played an important role in shaping these genomes, and L1s account for approximately half of this transposable element load. Mammals have evolved several mechanisms to control L1 retrotransposition, and yet L1s remain active in almost all mammalian lineages. However, L1s were found to have gone extinct in the megabat family ∼24 million years ago. We were able to trace megabat L1s to the ancestral L1 families shared by all mammals as well as identify bat-specific L1 families. Unlike most well-characterized mammals which have a single active L1 lineage, multiple L1 lineages have persisted in megabats throughout their evolutionary history. When the L1 extinction occurred in megabats, two active lineages lost their ability to retrotranspose almost simultaneously after a burst of activity. We synthesized the L1 from the most active family at the time of extinction and found a long intergenic spacer between its two protein coding genes. Tissue culture assays of the reconstructed megabat L1 revealed that both genes supported retrotransposition, but that the spacer is inhibitory. Despite the inhibition, this family accounted for 18% of the L1s detected in the megabat genome.

Background Most clinical and natural microbial communities live and evolve in spatially structured environments. When changes in environmental conditions trigger evolutionary responses, spatial structure can impact the types of adaptive response and the extent to which they spread. In particular, localized competition in a spatial landscape can lead to the emergence of a larger number of different adaptive trajectories than would be found in well-mixed populations. Our goal was to determine how two levels of spatial structure affect genomic diversity in a population and how this diversity is manifested spatially. Methodology/Principal Findings We serially transferred bacteriophage populations growing at high temperatures (40°C) on agar plates for 550 generations at two levels of spatial structure. The level of spatial structure was determined by whether the physical locations of the phage subsamples were preserved or disrupted at each passage to fresh bacterial host populations. When spatial structure of the phage populations was preserved, there was significantly greater diversity on a global scale with restricted and patchy distribution. When spatial structure was disrupted with passaging to fresh hosts, beneficial mutants were spread across the entire plate. This resulted in reduced diversity, possibly due to clonal interference as the most fit mutants entered into competition on a global scale. Almost all substitutions present at the end of the adaptation in the populations with disrupted spatial structure were also present in the populations with structure preserved. Conclusions/Significance Our results are consistent with the patchy nature of the spread of adaptive mutants in a spatial landscape. Spatial structure enhances diversity and slows fixation of beneficial mutants. This added diversity could be beneficial in fluctuating environments. We also connect observed substitutions and their effects on fitness to aspects of phage biology, and we provide evidence that some substitutions exclude each other.

Genomes of the closely related bacteriophages ϕX174 and S13 are 5386 bases long and differ at 114 nucleotides, affecting 28 amino acids. Both parental phages were adapted to laboratory culture conditions in replicate lineages and analysed for nucleotide changes that accumulated experimentally. Of the 126 experimental substitutions, 90% encoded amino–acid changes, and 62% of the substitutions occurred in parallel in more than one experimental line. Furthermore, missense changes at 12 of the experimental sites were at residues differing between the parental phages; in ten cases the ϕX174 experimental lineages were convergent with the S13 parent, or vice versa, at both the nucleotide and amino–acid levels. Convergence at a site was even obtained in both directions in three cases. These results point to a limited number of pathways taken during evolution in these viruses, and also raise the possibility that much of the amino–acid variation in the natural evolution of these viruses has been selected.

High temperature inhibits the growth of the wild-type bacteriophage phiX174. Three different point mutations were identified that each recovered growth at high temperature. Two affected the major capsid protein (residues F188 and F242), and one affected the internal scaffolding protein (B114). One of the major capsid mutations (F242) is located in a beta strand that contacts B114 in the procapsid during viral maturation, whereas the other capsid mutation (F188) is involved in subunit interactions at the threefold axis of symmetry. Selective coefficients of these mutations ranged from 13.9 to 3.8 in the inhibitory, hot environment, but all mutations reduced fitness at normal temperature. The selective effect of one of the mutations (F242) was evaluated at high temperature in four different genetic backgrounds and exhibited epistasis of diminishing returns: as log fitness of the background genotype increased from -0.1 to 4.1, the fitness boost provided by the F242 mutation decreased from 3.9 to 0. 8. These results support a model in which viral fitness is bounded by an upper limit and the benefit of a mutation is scaled according to the remaining opportunity for fitness improvement in the genome.

The large number of L1 [long interspersed elements (LINE)-1] sequences found in the genome is due to the insertion of copies of the retrotransposon over evolutionary time. The majority of copies appear to be replicates of a few active, or “master” templates. A continual replacement of master templates over time gives rise to lineages distinguishable by their own unique set of shared-sequence variants. A previous analysis of L1 sequences in deer mice, Peromyscus maniculatus and P. leucopus, revealed two active L1 lineages, marked by different rates of evolution, whose most recent common ancestor predates the expansion of the Peromyscus species. Here we exploit lineage-specific, shared-sequence variants to reveal a paucity of Lineage 2 sequences in at least one species, P. californicus. The dearth of Lineage 2 copies in P. californicus suggests that Lineage 2 may have been unproductive until after the most recent common ancestor of P. californicus and P. maniculatus. We also show that Lineage 1 appears to have a higher rate of evolution in P. maniculatus relative to either P. californicus or P. leucopus. As a phylogenetic tool, L1 lineage-specific variants support a close affinity between P. californicus and P. eremicus relative to the other species examined.

Replicate lineages of the bacteriophage ϕX 174 adapted to growth at high temperature on either of two hosts exhibited high rates of identical, independent substitutions. Typically, a dozen or more substitutions accumulated in the 5.4-kilobase genome during propagation. Across the entire data set of nine lineages, 119 independent substitutions occurred at 68 nucleotide sites. Over half of these substitutions, accounting for one third of the sites, were identical with substitutions in other lineages. Some convergent substitutions were specific to the host used for phage propagation, but others occurred across both hosts. Continued adaptation of an evolved phage at high temperature, but on the other host, led to additional changes that included reversions of previous substitutions. Phylogenetic reconstruction using the complete genome sequence not only failed to recover the correct evolutionary history because of these convergent changes, but the true history was rejected as being a significantly inferior fit to the data. Replicate lineages subjected to similar environmental challenges showed similar rates of substitution and similar rates of fitness improvement across corresponding times of adaptation. Substitution rates and fitness improvements were higher during the initial period of adaptation than during a later period, except when the host was changed.

Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy). No analogous studies have been carried out for protein-protein binding. Here we use a structure-based model of the major coat protein in a simple virus, bacteriophage φX174, to estimate the free energy of folding of a single coat protein and binding of five coat proteins within a pentameric unit. We confirm and extend previous work in finding that most accessible substitutions destabilize both protein folding and protein-protein binding. We compare the pool of accessible substitutions with those observed among the φX174-like wild phage and in experimental evolution with φX174. We find that observed substitutions have smaller effects on stability than expected by chance. An analysis of adaptations at high temperatures suggests that selection favors either substitutions with no effect on stability or those that simultaneously stabilize protein folding and slightly destabilize protein binding. We speculate that these mutations might involve adjusting the rate of capsid assembly. At normal laboratory temperature there is little evidence of directional selection. Finally, we show that cumulative changes in stability are highly variable; sometimes they are well beyond the bounds of single substitution changes and sometimes they are not. The variation leads us to conclude that phenotype selection acts on more than just stability. Instances of larger cumulative stability change (never via a single substitution despite their availability) lead us to conclude that selection views stability at a local, not a global, level.

BackgroundChromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution — some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 – 75 Gb, 12–74 Gb of which are lost from pre-somatic cell lineages at germline – soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown.ResultsHere, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes ofMesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome.ConclusionsOur results suggest that germline genome expansion inM. edaxreflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms.

Observing organisms that evolve in response to strong selection over very short time scales allows the determination of the molecular mechanisms underlying adaptation. Although dissecting these molecular mechanisms is expensive and time-consuming, general patterns can be detected from repeated experiments, illuminating the biological processes involved in evolutionary adaptation. The bacteriophage φX174 was grown for 50 days in replicate chemostats under two culture conditions: Escherichia coli C as host growing at 37°C and Salmonella typhimurium as host growing at 43.5°C. After 50 days, greater than 20 substitutions per chemostat had risen to detectable levels. Of the 97 substitutions, four occurred in all four chemostats, five arose in both culture conditions, eight arose in only the high temperature S. typhimurium chemostats, and seven arose only in the E. coli chemostats. The remaining substitutions were detected only in a single chemostat, however, almost half of these have been seen in other similar experiments. Our findings support previous studies that host recognition and capsid stability are two biological processes that are modified during adaptation to novel hosts and high temperature. Based upon the substitutions shared across both environments, it is apparent that genome replication and packaging are also affected during adaptation to the chemostat environment, rather than to temperature or host per se. This environment is characterized by a large number of phage and very few hosts, leading to competition among phage within the host. We conclude from these results that adaptation to a high density environment selects for changes in genome replication at both protein and DNA sequence levels.

It has been proposed that the most extensively studied mammalian retrotransposons replicate by some form of a master template model. This conclusion has been drawn largely from DNA sequence analysis and is based on phylogenetic tree topology, the presence and ordering of shared variants, the degree of divergence between elements within a subfamily, and the shape of the distribution of pairwise differences between elements. To investigate how robust these parameters are as predictors of the model of transposition, computer simulations of the two most extreme transposition models, the Random Template Model and the Strict Master Model, were carried out. A prototype of a computer simulator for studying retrotransposition is presented. The simulator is a versatile digital workbench that maintains DNA sequence data and allows manipulation of a range of factors including reverse transcriptase and in situ mutation rates, transposition template, and transposition rate. All parameters previously used as predictors of the model of transposition were markedly different for the two extreme models when evaluated using large sample sizes of sequences from experiments simulating up to 15 million years of evolution.

Mys is a retrovirus-like transposable element found throughout the genus Peromyscus. Several mys subfamilies identified on the basis of restriction site variation occur in more than one species. The distribution of these subfamilies is consistent with the accepted species phylogeny, suggesting that mys was present in the ancestor of Peromyscus and has been active through much of the evolution of this genus. Quantitative Southern blot analysis was used to examine the variability of subfamilies in P. leucopus and maniculatus. We found that subfamilies with phylogenetically narrow distributions were more variable in copy number both within and between species than subfamilies with a broader distribution. Taken together, our data suggest that mys has undergone multiple rounds of transposition since the peromyscine radiation, and that five subfamilies have been amplified during the evolution of the leucopus-maniculatus species complex.

Parallelism is important because it reveals how inherently stochastic adaptation is. Even as we come to better understand evolutionary forces, stochasticity limits how well we can predict evolutionary outcomes. Here we sought to quantify parallelism and some of its underlying causes by adapting a bacteriophage (ID11) with nine different first-step mutations, each with eight-fold replication, for 100 passages. This was followed by whole-genome sequencing five isolates from each endpoint. A large amount of variation arose—281 mutational events occurred representing 112 unique mutations. At least 41% of the mutations and 77% of the events were adaptive. Within wells, populations generally experienced complex interference dynamics. The genome locations and counts of mutations were highly uneven: mutations were concentrated in two regulatory elements and three genes and, while 103 of the 112 (92%) of the mutations were observed in ≤4 wells, a few mutations arose many times. 91% of the wells and 81% of the isolates had a mutation in the D-promoter. Parallelism was moderate compared to previous experiments with this system. On average, wells shared 27% of their mutations at the DNA level and 38% when the definition of parallel change is expanded to include the same regulatory feature or residue. About half of the parallelism came from D-promoter mutations. Background had a small but significant effect on parallelism. Similarly, an analyses of epistasis between mutations and their ancestral background was significant, but the result was mostly driven by four individual mutations. A second analysis of epistasis focused on de novo mutations revealed that no isolate ever had more than one D-promoter mutation and that 56 of the 65 isolates lacking a D-promoter mutation had a mutation in genes D and/or E. We assayed time to lysis in four of these mutually exclusive mutations (the two most frequent D-promoter and two in gene D) across four genetic backgrounds. In all cases lysis was delayed. We postulate that because host cells were generally rare (i.e., high multiplicity of infection conditions developed), selection favored phage that delayed lysis to better exploit their current host (i.e., ‘love the one you’re with’). Thus, the vast majority of wells (at least 64 of 68, or 94%) arrived at the same phenotypic solution, but through a variety of genetic changes. We conclude that answering questions about the range of possible adaptive trajectories, parallelism, and the predictability of evolution requires attention to the many biological levels where the process of adaptation plays out.

The 2014 Ebola virus (EBOV) outbreak in West Africa is the largest in recorded history and resulted in over 11,000 deaths. It is essential that strategies for treatment and containment be developed to avoid future epidemics of this magnitude. With the development of vaccines and antibody-based therapies using the envelope glycoprotein (GP) of the 1976 Mayinga strain, one important strategy is to anticipate how the evolution of EBOV might compromise these efforts. In this study we have initiated a watch list of potential antibody escape mutations of EBOV by modeling interactions between GP and the antibody KZ52. The watch list was generated using molecular modeling to estimate stability changes due to mutation. Every possible mutation of GP was considered and the list was generated from those that are predicted to disrupt GP-KZ52 binding but not to disrupt the ability of GP to fold and to form trimers. The resulting watch list contains 34 mutations (one of which has already been seen in humans) at six sites in the GP2 subunit. Should mutations from the watch list appear and spread during an epidemic, it warrants attention as these mutations may reflect an evolutionary response from the virus that could reduce the effectiveness of interventions such as vaccination. However, this watch list is incomplete and emphasizes the need for more experimental structures of EBOV interacting with antibodies in order to expand the watch list to other epitopes. We hope that this work provokes experimental research on evolutionary escape in both Ebola and other viral pathogens.