Genetic drift promotes and recombination hinders speciation on holey fitness landscapes.

Dobzhansky and Muller proposed a general mechanism through which microevolution, the substitution of alleles within populations, can cause the evolution of reproductive isolation between populations and, therefore, macroevolution. As allopatric populations diverge, many combinations of alleles differing between them have not been tested by natural selection and may thus be incompatible. Such genetic incompatibilities often cause low fitness in hybrids between species. Furthermore, the number of incompatibilities grows with the genetic distance between diverging populations. However, what determines the rate and pattern of accumulation of incompatibilities remains unclear. We investigate this question by simulating evolution on holey fitness landscapes on which genetic incompatibilities can be identified unambiguously. We find that genetic incompatibilities accumulate more slowly among genetically robust populations and identify two determinants of the accumulation rate: recombination rate and population size. In large populations with abundant genetic variation, recombination selects for increased genetic robustness and, consequently, incompatibilities accumulate more slowly. In small populations, genetic drift interferes with this process and promotes the accumulation of genetic incompatibilities. Our results suggest a novel mechanism by which genetic drift promotes and recombination hinders speciation.

Structural analysis of MALAT1 long noncoding RNA in cells and in evolution.

Although not canonically polyadenylated, the long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is stabilized by a highly conserved 76-nt triple helix structure on its 3' end. The entire MALAT1 transcript is over 8000 nt long in humans. The strongest structural conservation signal in MALAT1 (as measured by covariation of base pairs) is in the triple helix structure. Primary sequence analysis of covariation alone does not reveal the degree of structural conservation of the entire full-length transcript, however. Furthermore, RNA structure is often context dependent; RNA binding proteins that are differentially expressed in different cell types may alter structure. We investigate here the in-cell and cell-free structures of the full-length human and green monkey (Chlorocebus sabaeus) MALAT1 transcripts in multiple tissue-derived cell lines using SHAPE chemical probing. Our data reveal levels of uniform structural conservation in different cell lines, in cells and cell-free, and even between species, despite significant differences in primary sequence. The uniformity of the structural conservation across the entire transcript suggests that, despite seeing covariation signals only in the triple helix junction of the lncRNA, the rest of the transcript's structure is remarkably conserved, at least in primates and across multiple cell types and conditions.

Precision patient education using a "flipped classroom" approach.

OBJECTIVES: To improve patient education delivered over telemedicine by using a "flipped classroom"-inspired approach. METHODS: A "flipped classroom" is an education strategy used to engage active learning by sending students home with lecture material and reserving classroom time for collaborative learning. To adapt this approach for use in radiation oncology patient education, three pieces of written education material were created: introduction to radiation oncology, treatment planning scan, and treatment delivery. An automated system was created to deliver precisely timed emails at three time points ahead of appointments. Appointment time was then used for collaborative learning with our staff. As a primary endpoint, email engagement metrics were tracked via the automated system. Secondarily, enrolled patients were surveyed to assess level of understanding (before vs. after intervention), anxiety (before vs. after intervention), and satisfaction. Additionally, email delivery timing, clarity, relevance, and patient support were evaluated. Data analyses test the impact of active learning against our existing education approaches. RESULTS: Overall, 77.1% of the emails were opened, and of those, patients accessed 72.2% of the education material. Patients re-read the education material 4.6 times on average. Active learning increased patient understanding regarding the purpose of the treatment planning scan (p = 0.031) and increased patient understanding of what to expect during daily radiation treatments (p = 0.0078). Patients reported reduced anxiety (p = 0.031) and high scores for satisfaction, timing, clarity, relevance, and overall support. CONCLUSIONS: Patient engagement with the education material was high, and they continued to access it many times. Active learning enhances patient comprehension of complex treatment information leading to decreased anxiety. Furthermore, this technique can be incorporated into existing telemedicine with basic technology.

Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants.

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. While bat, camel, and human DPP4 support MERS-CoV infection, several DPP4 orthologs, including mouse, ferret, hamster, and guinea pig DPP4, do not. Previous work revealed that glycosylation of mouse DPP4 plays a role in blocking MERS-CoV infection. Here, we tested whether glycosylation also acts as a determinant of permissivity for ferret, hamster, and guinea pig DPP4. We found that, while glycosylation plays an important role in these orthologs, additional sequence and structural determinants impact their ability to act as functional receptors for MERS-CoV. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and better inform our understanding of virus-receptor interactions associated with disease emergence and host susceptibility.IMPORTANCE MERS-CoV is a recently emerged zoonotic virus that is still circulating in the human population with an ∼35% mortality rate. With no available vaccines or therapeutics, the study of MERS-CoV pathogenesis is crucial for its control and prevention. However, in vivo studies are limited because MERS-CoV cannot infect wild-type mice due to incompatibilities between the virus spike and the mouse host cell receptor, mouse DPP4 (mDPP4). Specifically, mDPP4 has a nonconserved glycosylation site that acts as a barrier to MERS-CoV infection. Thus, one mouse model strategy has been to modify the mouse genome to remove this glycosylation site. Here, we investigated whether glycosylation acts as a barrier to infection for other nonpermissive small-animal species, namely, ferret, guinea pig, and hamster. Understanding the virus-receptor interactions for these DPP4 orthologs will help in the development of additional animal models while also revealing species-specific differences impacting MERS-CoV host range.

The emergence of performance trade-offs during local adaptation: insights from experimental evolution.

Environmental heterogeneity is considered a general explanation for phenotypic diversification, particularly when heterogeneity causes populations to diverge via local adaptation. Performance trade-offs, such as those stemming from antagonistic pleiotropy, are thought to contribute to the maintenance of diversity in this scenario. Specifically, alleles that promote adaptation in one environment are expected to promote maladaptation in alternative environments. Contrary to this expectation, however, alleles that underlie locally adaptive traits often fail to exhibit fitness costs in alternative environments. Here, we attempt to explain this paradox by reviewing the results of experimental evolution studies, including a new one of our own, that examined the evolution of trade-offs during adaptation to homogeneous versus heterogeneous environments. We propose that when pleiotropic effects vary, whether or not trade-offs emerge among diverging populations will depend critically on ecology. For example, adaptation to a locally homogeneous environment is more likely to occur by alleles that are antagonistically pleiotropic than adaptation to a locally heterogeneous environment, simply because selection is blind to costs associated with environments that are not experienced locally. Our literature review confirmed the resulting prediction that performance trade-offs were more likely to evolve during selection in homogeneous than heterogeneous environments. The nature of the environmental heterogeneity (spatial versus temporal) and the length of the experiment also contributed in predictable ways to the likelihood that performance trade-offs evolved.

Evolutionary rescue and the coexistence of generalist and specialist competitors: an experimental test.

Competition for resources is thought to play a critical role in both the origins and maintenance of biodiversity. Although numerous laboratory evolution experiments have confirmed that competition can be a key driver of adaptive diversification, few have demonstrated its role in the maintenance of the resulting diversity. We investigate the conditions that favour the origin and maintenance of alternative generalist and specialist resource-use phenotypes within the same population. Previously, we confirmed that competition for hosts among φ6 bacteriophage in a mixed novel (non-permissive) and ancestral (permissive) host microcosm triggered the evolution of a generalist phenotype capable of infecting both hosts. However, because the newly evolved generalists tended to competitively exclude the ancestral specialists, coexistence between the two phenotypes was rare. Here, we show that reducing the relative abundance of the novel host slowed the increase in frequency of the generalist phenotype, allowing sufficient time for the specialist to further adapt to the ancestral host. This adaptation resulted in 'evolutionary rescue' of the specialists, preventing their competitive exclusion by the generalists. Thus, our results suggest that competition promotes both the origin and maintenance of biodiversity when it is strong enough to favour a novel resource-use phenotype, but weak enough to allow adaptation of both the novel and ancestral phenotypes to their respective niches.

Comparison of SIV and HIV-1 genomic RNA structures reveals impact of sequence evolution on conserved and non-conserved structural motifs.

RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1(NL4-3). One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1(NL4-3) also occur at the 5' polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve.

Architecture and secondary structure of an entire HIV-1 RNA genome.

Single-stranded RNA viruses encompass broad classes of infectious agents and cause the common cold, cancer, AIDS and other serious health threats. Viral replication is regulated at many levels, including the use of conserved genomic RNA structures. Most potential regulatory elements in viral RNA genomes are uncharacterized. Here we report the structure of an entire HIV-1 genome at single nucleotide resolution using SHAPE, a high-throughput RNA analysis technology. The genome encodes protein structure at two levels. In addition to the correspondence between RNA and protein primary sequences, a correlation exists between high levels of RNA structure and sequences that encode inter-domain loops in HIV proteins. This correlation suggests that RNA structure modulates ribosome elongation to promote native protein folding. Some simple genome elements previously shown to be important, including the ribosomal gag-pol frameshift stem-loop, are components of larger RNA motifs. We also identify organizational principles for unstructured RNA regions, including splice site acceptors and hypervariable regions. These results emphasize that the HIV-1 genome and, potentially, many coding RNAs are punctuated by previously unrecognized regulatory motifs and that extensive RNA structure constitutes an important component of the genetic code.

Competition and the origins of novelty: experimental evolution of niche-width expansion in a virus.

Competition for resources has long been viewed as a key agent of divergent selection. Theory holds that populations facing severe intraspecific competition will tend to use a wider range of resources, possibly even using entirely novel resources that are less in demand. Yet, there have been few experimental tests of these ideas. Using the bacterial virus (bacteriophage) 6 as a model system, we examined whether competition for host resources promotes the evolution of novel resource use. In the laboratory, 6 exhibits a narrow host range but readily produces mutants capable of infecting novel bacterial hosts. Here, we show that when 6 populations were subjected to intense intraspecific competition for their standard laboratory host, they rapidly evolved new generalist morphs that infect novel hosts. Our results therefore suggest that competition for host resources may drive the evolution of host range expansion in viruses. More generally, our findings demonstrate that intraspecific resource competition can indeed promote the evolution of novel resource-use phenotypes.

Empirical Evidence That Complexity Limits Horizontal Gene Transfer.

Horizontal gene transfer (HGT) is a major contributor to bacterial genome evolution, generating phenotypic diversity, driving the expansion of protein families, and facilitating the evolution of new phenotypes, new metabolic pathways, and new species. Comparative studies of gene gain in bacteria suggest that the frequency with which individual genes successfully undergo HGT varies considerably and may be associated with the number of protein-protein interactions in which the gene participates, that is, its connectivity. Two nonexclusive hypotheses have emerged to explain why transferability should decrease with connectivity: the complexity hypothesis (Jain R, Rivera MC, Lake JA. 1999. Horizontal gene transfer among genomes: the complexity hypothesis. Proc Natl Acad Sci U S A. 96:3801-3806.) and the balance hypothesis (Papp B, Pál C, Hurst LD. 2003. Dosage sensitivity and the evolution of gene families in yeast. Nature 424:194-197.). These hypotheses predict that the functional costs of HGT arise from a failure of divergent homologs to make normal protein-protein interactions or from gene misexpression, respectively. Here we describe genome-wide assessments of these hypotheses in which we used 74 existing prokaryotic whole genome shotgun libraries to estimate rates of horizontal transfer of genes from taxonomically diverse prokaryotic donors into Escherichia coli. We show that 1) transferability declines as connectivity increases, 2) transferability declines as the divergence between donor and recipient orthologs increases, and that 3) the magnitude of this negative effect of divergence on transferability increases with connectivity. These effects are particularly robust among the translational proteins, which span the widest range of connectivities. Whereas the complexity hypothesis explains all three of these observations, the balance hypothesis explains only the first one.

Sexual reproduction selects for robustness and negative epistasis in artificial gene networks.

The mutational deterministic hypothesis for the origin and maintenance of sexual reproduction posits that sex enhances the ability of natural selection to purge deleterious mutations after recombination brings them together into single genomes. This explanation requires negative epistasis, a type of genetic interaction where mutations are more harmful in combination than expected from their separate effects. The conceptual appeal of the mutational deterministic hypothesis has been offset by our inability to identify the mechanistic and evolutionary bases of negative epistasis. Here we show that negative epistasis can evolve as a consequence of sexual reproduction itself. Using an artificial gene network model, we find that recombination between gene networks imposes selection for genetic robustness, and that negative epistasis evolves as a by-product of this selection. Our results suggest that sexual reproduction selects for conditions that favour its own maintenance, a case of evolution forging its own path.

Major coexisting human immunodeficiency virus type 1 env gene subpopulations in the peripheral blood are produced by cells with similar turnover rates and show little evidence of genetic compartmentalization.

A distinctive feature of chronic human immunodeficiency virus type 1 (HIV-1) infection is the presence of multiple coexisting genetic variants, or subpopulations, that comprise the HIV-1 population detected in the peripheral blood. Analysis of HIV-1 RNA decay dynamics during the initiation of highly active antiretroviral therapy (HAART) has been a valuable tool for modeling the life span of infected cells that produce the bulk HIV-1 population. However, different HIV-1 target cells may have different turnover rates, and it is not clear whether the bulk HIV-1 RNA decay rate actually represents a composite of the decay rates of viral subpopulations compartmentalized in different cellular subsets with different life spans. Using heteroduplex tracking assays targeting the highly variable V3 or V4-V5 regions of the HIV-1 env gene in eight subjects, we found that all detectable coexisting HIV-1 variants in the peripheral blood generally decayed at similar rates during the initiation of HAART, suggesting that all of the variants were produced by cells with similar life spans. Furthermore, single genome amplification and coreceptor phenotyping revealed that in two subjects coexisting HIV-1 variants with distinct CXCR4 or CCR5 coreceptor phenotypes decayed with similar rates. Also, in nine additional subjects, recombination and a lack of genetic compartmentalization between X4 and R5 variants were observed, suggesting an overlap in host cell range. Our results suggest that the HIV-1 env subpopulations detectable in the peripheral blood are produced by cells with similar life spans and are not genetically isolated within particular cell types.

Genetic architecture and the evolution of sex.

Theoretical investigations of the advantages of sex have tended to treat the genetic architecture of organisms as static and have not considered that genetic architecture might coevolve with reproductive mode. As a result, some potential advantages of sex may have been missed. Using a gene network model, we recently showed that recombination imposes selection for robustness to mutation and that negative epistasis can evolve as a by-product of this selection. These results motivated a detailed exploration of the mutational deterministic hypothesis, a hypothesis in which the advantage of sex depends critically on epistasis. We found that sexual populations do evolve higher mean fitness and lower genetic load than asexual populations at equilibrium, and, under moderate stabilizing selection and large population size, these equilibrium sexual populations resist invasion by asexuals. However, we found no evidence that these long- and short-term advantages to sex were explained by the negative epistasis that evolved in our experiments. The long-term advantage of sex was that sexual populations evolved a lower deleterious mutation rate, but this property was not sufficient to account for the ability of sexual populations to resist invasion by asexuals. The ability to resist asexual invasion was acquired simultaneously with an increase in recombinational robustness that minimized the cost of sex. These observations provide the first direct evidence that sexual reproduction does indeed select for conditions that favor its own maintenance. Furthermore, our results highlight the importance of considering a dynamic view of the genetic architecture to understand the evolution of sex and recombination.

Compensatory evolution in RNA secondary structures increases substitution rate variation among sites.

There is growing evidence that interactions between biological molecules (e.g., RNA-RNA, protein-protein, RNA-protein) place limits on the rate and trajectory of molecular evolution. Here, by extending Kimura's model of compensatory evolution at interacting sites, we show that the ratio of transition to transversion substitutions (kappa) at interacting sites should be equal to the square of the ratio at independent sites. Because transition mutations generally occur at a higher rate than transversions, the model predicts that kappa should be higher at interacting sites than at independent sites. We tested this prediction in 10 RNA secondary structures by comparing phylogenetically derived estimates of kappa in paired sites within stems (kappa(p)) and unpaired sites within loops (kappa(u)). Eight of the 10 structures showed an excellent match to the quantitative predictions of the model, and 9 of the 10 structures matched the qualitative prediction kappa(p) > kappa(u). Only the Rev response element from the human immunovirus (HIV) genome showed the reverse pattern, with kappa(p) < kappa(u). Although a variety of evolutionary forces could produce quantitative deviations from the model predictions, the reversal in magnitude of kappa(p) and kappa(u) could be achieved only by violating the model assumption that the underlying transition (or transversion) mutation rates were identical in paired and unpaired regions of the molecule. We explore the ability of the APOBEC3 enzymes, host defense mechanisms against retroviruses, which induce transition mutations preferentially in single-stranded regions of the HIV genome, to explain this exception to the rule. Taken as a whole, our findings suggest that kappa may have utility as a simple diagnostic to evaluate proposed secondary structures.

Hotter is better and broader: thermal sensitivity of fitness in a population of bacteriophages.

Hotter is better is a hypothesis of thermal adaptation that posits that the rate-depressing effects of low temperature on biochemical reactions cannot be overcome by physiological plasticity or genetic adaptation. If so, then genotypes or populations adapted to warmer temperatures will have higher maximum growth rates than those adapted to low temperatures. Here we test hotter is better by measuring thermal reaction norms for intrinsic rate of population growth among an intraspecific collection of bacteriophages recently isolated from nature. Consistent with hotter is better, we find that phage genotypes with higher optimal temperatures have higher maximum growth rates. Unexpectedly, we also found that hotter is broader, meaning that the phages with the highest optimal temperatures also have the greatest temperature ranges. We found that the temperature sensitivity of fitness for phages is similar to that for insects.

The genetic basis of thermal reaction norm evolution in lab and natural phage populations.

Two major goals of laboratory evolution experiments are to integrate from genotype to phenotype to fitness, and to understand the genetic basis of adaptation in natural populations. Here we demonstrate that both goals are possible by re-examining the outcome of a previous laboratory evolution experiment in which the bacteriophage G4 was adapted to high temperatures. We quantified the evolutionary changes in the thermal reaction norms--the curves that describe the effect of temperature on the growth rate of the phages--and decomposed the changes into modes of biological interest. Our analysis indicated that changes in optimal temperature accounted for almost half of the evolutionary changes in thermal reaction norm shape, and made the largest contribution toward adaptation at high temperatures. Genome sequencing allowed us to associate reaction norm shape changes with particular nucleotide mutations, and several of the identified mutations were found to be polymorphic in natural populations. Growth rate measures of natural phage that differed at a site that contributed substantially to adaptation in the lab indicated that this mutation also underlies thermal reaction norm shape variation in nature. In combination, our results suggest that laboratory evolution experiments may successfully predict the genetic bases of evolutionary responses to temperature in nature. The implications of this work for viral evolution arise from the fact that shifts in the thermal optimum are characterized by tradeoffs in performance between high and low temperatures. Optimum shifts, if characteristic of viral adaptation to novel temperatures, would ensure the success of vaccine development strategies that adapt viruses to low temperatures in an attempt to reduce virulence at higher (body) temperatures.

Recombination drives the evolution of mutational robustness.

Recombination can impose fitness costs as beneficial parental combinations of alleles are broken apart, a phenomenon known as recombination load. Computational models suggest that populations may evolve a reduced recombination load by reducing either the likelihood of recombination events (bring interacting loci in physical proximity) or the strength of interactions between loci (make loci more independent of one another). We review evidence for each of these possibilities and their consequences for the genotype-fitness relationship. In particular, we expect that reducing interaction strengths between loci will lead to genomes that are also robust to mutational perturbations, but reducing recombination rates alone will not. We note that both mechanisms most likely played a role in the evolution of extant populations, and that both can result in the frequently-observed pattern of physical linkage between interacting loci.

Population structure promotes the evolution of costly sex in artificial gene networks.

We build on previous observations that Hill-Robertson interference generates an advantage of sex that, in structured populations, can be large enough to explain the evolutionary maintenance of costly sex. We employed a gene network model that explicitly incorporates interactions between genes. Mutations in the gene networks have variable effects that depend on the genetic background in which they appear. Consequently, our simulations include two costs of sex-recombination and migration loads-that were missing from previous studies of the evolution of costly sex. Our results suggest a critical role for population structure that lies in its ability to align the long- and short-term advantages of sex. We show that the addition of population structure favored the evolution of sex by disproportionately decreasing the equilibrium mean fitness of asexual populations, primarily by increasing the strength of Muller's Ratchet. Population structure also increased the ability of the short-term advantage of sex to counter the primary limit to the evolution of sex in the gene network model-recombination load. On the other hand, highly structured populations experienced migration load in the form of Dobzhansky-Muller incompatibilities, decreasing the effective rate of migration between demes and, consequently, accelerating the accumulation of drift load in the sexual populations.

High frequency of mutations that expand the host range of an RNA virus.

The ability of a virus population to colonize a novel host is predicted to depend on the equilibrium frequency of potential colonists (i.e., genotypes capable of infecting the novel host) in the source population. In this study, we investigated the determinants of the equilibrium frequency of potential colonists in the RNA bacteriophage 6. We isolated 40 spontaneous mutants capable of infecting a novel Pseudomonas syringae host and sequenced their host attachment genes to identify the responsible mutations. We observed 16 different mutations in the host attachment gene and used a new statistical approach to estimate that 39 additional mutations were missed by our screen. Phenotypic and fitness assays confirmed that the proximate mechanism underlying host range expansion was an increase in the ability to attach to the novel host and that acquisition of this ability most often imposed a cost for growth rate on two standard hosts. Considered in a population genetic framework, our data suggest that host range mutations should exist in phage populations at an equilibrium frequency (3 x 10(-4)) that exceeds the phage mutation rate by more than two orders of magnitude. Thus, colonization of novel hosts is unlikely to be limited by an inability to produce appropriate mutations.

Experimental estimate of the abundance and effects of nearly neutral mutations in the RNA virus phi 6.

Although the frequency and effects of neutral and nearly neutral mutations are critical to evolutionary patterns and processes governed by genetic drift, the small effects of such mutations make them difficult to study empirically. Here we present the results of a mutation-accumulation experiment designed to assess the frequencies of deleterious mutations with undetectable effects. We promoted the accumulation of spontaneous mutations by subjecting independent lineages of the RNA virus 6 to repeated population bottlenecks of a single individual. We measured fitness following every bottleneck to obtain a complete picture of the timing and effects of the accumulated mutations with detectable effects and sequenced complete genomes to determine the number of mutations that were undetected by the fitness assays. To estimate the effects of the undetected mutations, we implemented a likelihood model developed for quantitative trait locus (QTL) data (Otto and Jones 2000) to estimate the number and effects of the undetected mutations from the measured number and effects of the detected mutations. Using this method we estimated a deleterious mutation rate of U = 0.03 and a gamma effects distribution with mean s=0.093 and coefficient of variation = 0.204. Although our estimates of U and s fall within the range of recent mutation rate and effect estimates in eukaryotes, the fraction of mutations with detectable effects on laboratory fitness (39%) appears to be far higher in 6 than in eukaryotes.