The effects of sex on extinction dynamics of Chlamydomonas reinhardtii depend on the rate of environmental change.

The continued existence of sex, despite many the costs it entails, still lacks an adequate explanation, as previous studies demonstrated that the effects of sex are environment-dependent: sex enhances the rate of adaptation in changing environments, but the benefits level off in benign conditions. To the best of our knowledge, the potential impact of different patterns of environmental change on the magnitude of these benefits received less attention in theoretical studies. In this paper, we begin to explore this issue by examining the effect of the rate of environmental deterioration (negatively correlated with population survival rate), on the benefits of sex. To investigate the interplay of sex and the rate of environmental deterioration, we carried out a long-term selection experiment with a unicellular alga (Chlamydomonas reinhardtii), by manipulating mode of reproduction (asexual, facultative or obligate sexual) and the rate of environmental deterioration (an increase of salt concentration). We monitored both the population size and extinction dynamics. The results revealed that the relative advantage of sex increased at the intermediate rate and plateaued at the highest rate of environmental deterioration. Obligate sexual populations had the slowest extinction rate under the intermediate rate of environmental deterioration, while facultative sexuality was favoured under the high rate-treatment. To the best of our knowledge, our study is the first to demonstrate that the interplay of sex and the rate of environmental deterioration affects the probability of survival, which indicates that mode of reproduction may be an important determinant of survival of the anthropogenic-induced environmental change.

The effect of heterospecific and conspecific competition on inter-individual differences in tungara frog tadpole (Engystomops pustulosus) behavior.

Repeated social interactions with conspecifics and/or heterospecifics during early development may drive the differentiation of behavior among individuals. Competition is a major form of social interaction and its impacts can depend on whether interactions occur between conspecifics or heterospecifics and the directionality of a response could be specific to the ecological context that they are measured in. To test this, we reared tungara frog tadpoles (Engystomops pustulosus) either in isolation, with a conspecific tadpole or with an aggressive heterospecific tadpole, the whistling frog tadpole (Leptodactylus fuscus). In each treatment, we measured the body size and distance focal E. pustulosus tadpoles swam in familiar, novel and predator risk contexts six times during development. We used univariate and multivariate hierarchical mixed effect models to investigate the effect of treatment on mean behavior, variance among and within individuals, behavioral repeatability and covariance among individuals in their behavior between contexts. There was a strong effect of competition on behavior, with different population and individual level responses across social treatments. Within a familiar context, the variance in the distance swam within individuals decreased under conspecific competition but heterospecific competition caused more variance in the average distance swam among individuals. Behavioral responses were also context specific as conspecific competition caused an increase in the distance swam within individuals in novel and predator risk contexts. The results highlight that the impact of competition on among and within individual variance in behavior is dependent on both competitor species identity and context.

The distribution of fitness effects of spontaneous mutations in Chlamydomonas reinhardtii inferred using frequency changes under experimental evolution.

The distribution of fitness effects (DFE) for new mutations is fundamental for many aspects of population and quantitative genetics. In this study, we have inferred the DFE in the single-celled alga Chlamydomonas reinhardtii by estimating changes in the frequencies of 254 spontaneous mutations under experimental evolution and equating the frequency changes of linked mutations with their selection coefficients. We generated seven populations of recombinant haplotypes by crossing seven independently derived mutation accumulation lines carrying an average of 36 mutations in the haploid state to a mutation-free strain of the same genotype. We then allowed the populations to evolve under natural selection in the laboratory by serial transfer in liquid culture. We observed substantial and repeatable changes in the frequencies of many groups of linked mutations, and, surprisingly, as many mutations were observed to increase as decrease in frequency. Mutation frequencies were highly repeatable among replicates, suggesting that selection was the cause of the observed allele frequency changes. We developed a Bayesian Monte Carlo Markov Chain method to infer the DFE. This computes the likelihood of the observed distribution of changes of frequency, and obtains the posterior distribution of the selective effects of individual mutations, while assuming a two-sided gamma distribution of effects. We infer that the DFE is a highly leptokurtic distribution, and that approximately equal proportions of mutations have positive and negative effects on fitness. This result is consistent with what we have observed in previous work on a different C. reinhardtii strain, and suggests that a high fraction of new spontaneously arisen mutations are advantageous in a simple laboratory environment.

De Novo Mutation Rate Variation and Its Determinants in Chlamydomonas.

De novo mutations are central for evolution, since they provide the raw material for natural selection by regenerating genetic variation. However, studying de novo mutations is challenging and is generally restricted to model species, so we have a limited understanding of the evolution of the mutation rate and spectrum between closely related species. Here, we present a mutation accumulation (MA) experiment to study de novo mutation in the unicellular green alga Chlamydomonas incerta and perform comparative analyses with its closest known relative, Chlamydomonas reinhardtii. Using whole-genome sequencing data, we estimate that the median single nucleotide mutation (SNM) rate in C. incerta is µ = 7.6 × 10-10, and is highly variable between MA lines, ranging from µ = 0.35 × 10-10 to µ = 131.7 × 10-10. The SNM rate is strongly positively correlated with the mutation rate for insertions and deletions between lines (r > 0.97). We infer that the genomic factors associated with variation in the mutation rate are similar to those in C. reinhardtii, allowing for cross-prediction between species. Among these genomic factors, sequence context and complexity are more important than GC content. With the exception of a remarkably high C→T bias, the SNM spectrum differs markedly between the two Chlamydomonas species. Our results suggest that similar genomic and biological characteristics may result in a similar mutation rate in the two species, whereas the SNM spectrum has more freedom to diverge.

The effects of individual nonheritable variation on fitness estimation and coexistence.

Demographic theory and data have emphasized that nonheritable variation in individual frailty enables selection within cohorts, affecting the dynamics of a population while being invisible to its evolution. Here, we include the component of individual variation in longevity or viability which is nonheritable in simple bacterial growth models and explore its ecological and evolutionary impacts. First, we find that this variation produces consistent trends in longevity differences between bacterial genotypes when measured across stress gradients. Given that direct measurements of longevity are inevitably biased due to the presence of this variation and ongoing selection, we propose the use of the trend itself for obtaining more exact inferences of genotypic fitness. Second, we show how species or strain coexistence can be enabled by nonheritable variation in longevity or viability. These general conclusions are likely to extend beyond bacterial systems.

Sex increases the probability of evolutionary rescue in the presence of a competitor.

The explanation for the continued existence of sex, despite its many costs, remains one of the major challenges of evolutionary biology. Previous experimental studies have demonstrated that sex increases the rate of adaptation in novel environments relative to asexual reproduction. Whereas these studies have investigated the impact of sex on adaptation to stressful abiotic environments, the potential for biotic interactions to influence this advantage of sex has been largely ignored. Species rarely exist in isolation in natural conditions, so the impact of sex on adaptation to a stressful abiotic environment may be altered by the interactions between coexisting species. To investigate the interplay of sex and competition on adaptation to deteriorating conditions, we allowed populations of the unicellular alga (Chlamydomonas reinhardtii) to evolve in an environment to which they were initially poorly adapted. We manipulated both their mode of reproduction and the presence of a competitor, and monitored population size and proportion of evolutionary rescue events for each mode of reproduction. The results indicate that sex may be the beneficial strategy in the presence of the competitor. Sexual populations had highest probability of evolutionary rescue irrespective of the presence of the competitor. The overall advantage of sex was also manifested through higher level of adaptedness of survived sexual populations relative to asexual populations. Since competitive interactions are commonplace in nature, one of the explanations for the maintenance of sex by natural selection may be the increased rate of adaptation of sexual populations both in the presence and absence of competitors.

Patterns of population structure and complex haplotype sharing among field isolates of the green alga Chlamydomonas reinhardtii.

The nature of population structure in microbial eukaryotes has long been debated. Competing models have argued that microbial species are either ubiquitous, with high dispersal and low rates of speciation, or that for many species gene flow between populations is limited, resulting in evolutionary histories similar to those of macroorganisms. However, population genomic approaches have seldom been applied to this question. Here, we analyse whole-genome resequencing data for all 36 confirmed field isolates of the green alga Chlamydomonas reinhardtii. At a continental scale, we report evidence for putative allopatric divergence, between both North American and Japanese isolates, and two highly differentiated lineages within N. America. Conversely, at a local scale within the most densely sampled lineage, we find little evidence for either spatial or temporal structure. Taken together with evidence for ongoing admixture between the two N. American lineages, this lack of structure supports a role for substantial dispersal in C. reinhardtii and implies that between-lineage differentiation may be maintained by reproductive isolation and/or local adaptation. Our results therefore support a role for allopatric divergence in microbial eukaryotes, while also indicating that species may be ubiquitous at local scales. Despite the high genetic diversity observed within the most well-sampled lineage, we find that pairs of isolates share on average ~9% of their genomes in long haplotypes, even when isolates were sampled decades apart and from different locations. This proportion is several orders of magnitude higher than the Wright-Fisher expectation, raising many further questions concerning the evolutionary genetics of C. reinhardtii and microbial eukaryotes generally.

Inferring the distribution of fitness effects of spontaneous mutations in Chlamydomonas reinhardtii.

Spontaneous mutations are the source of new genetic variation and are thus central to the evolutionary process. In molecular evolution and quantitative genetics, the nature of genetic variation depends critically on the distribution of effects of mutations on fitness and other quantitative traits. Spontaneous mutation accumulation (MA) experiments have been the principal approach for investigating the overall rate of occurrence and cumulative effect of mutations but have not allowed the phenotypic effects of individual mutations to be studied directly. Here, we crossed MA lines of the green alga Chlamydomonas reinhardtii with its unmutated ancestral strain to create haploid recombinant lines, each carrying an average of 50% of the accumulated mutations in a large number of combinations. With the aid of the genome sequences of the MA lines, we inferred the genotypes of the mutations, assayed their growth rate as a measure of fitness, and inferred the distribution of fitness effects (DFE) using a Bayesian mixture model. We infer that the DFE is highly leptokurtic (L-shaped). Of mutations with absolute fitness effects exceeding 1%, about one-sixth increase fitness in the laboratory environment. The inferred distribution of effects for deleterious mutations is consistent with a strong role for nearly neutral evolution. Specifically, such a distribution predicts that nucleotide variation and genetic variation for quantitative traits will be insensitive to change in the effective population size.

Hook innovation boosts foraging efficiency in tool-using crows.

The New Caledonian crow is the only non-human animal known to craft hooked tools in the wild, but the ecological benefit of these relatively complex tools remains unknown. Here, we show that crows acquire food several times faster when using hooked rather than non-hooked tools, regardless of tool material, prey type and extraction context. This implies that small changes to tool shape can strongly affect energy-intake rates, highlighting a powerful driver for technological advancement.

Using Biological Insight and Pragmatism When Thinking about Pseudoreplication.

Pseudoreplication is controversial across experimental biology. Researchers in the same field can disagree on whether a given study suffers from pseudoreplication and on to what extent any pseudoreplication undermines the value of a study. A recent survey indicated that concerns about pseudoreplication can strongly impact peer review of manuscripts submitted for publication. Here we explore controversies around pseudoreplication, identify issues requiring resolution, and in each case offer a resolution. We emphasise that having non-independence in data points and pseudoreplicating are not the same thing. Researchers should be able to demonstrate that in a given experiment they have minimised and controlled the risk of non-independence weakening their study. If they do that to the satisfaction of others, they have avoided pseudoreplication.

Fitness change in relation to mutation number in spontaneous mutation accumulation lines of Chlamydomonas reinhardtii.

Although all genetic variation ultimately stems from mutations, their properties are difficult to study directly. Here, we used multiple mutation accumulation (MA) lines derived from five genetic backgrounds of the green algae Chlamydomonas reinhardtii that have been previously subjected to whole genome sequencing to investigate the relationship between the number of spontaneous mutations and change in fitness from a nonevolved ancestor. MA lines were on average less fit than their ancestors and we detected a significantly negative correlation between the change in fitness and the total number of accumulated mutations in the genome. Likewise, the number of mutations located within coding regions significantly and negatively impacted MA line fitness. We used the fitness data to parameterize a maximum likelihood model to estimate discrete categories of mutational effects, and found that models containing one to two mutational effect categories (one neutral and one deleterious category) fitted the data best. However, the best-fitting mutational effects models were highly dependent on the genetic background of the ancestral strain.

Evolutionary consequences of multidriver environmental change in an aquatic primary producer.

Climate change is altering aquatic environments in a complex way, and simultaneous shifts in many properties will drive evolutionary responses in primary producers at the base of both freshwater and marine ecosystems. So far, evolutionary studies have shown how changes in environmental drivers, either alone or in pairs, affect the evolution of growth and other traits in primary producers. Here, we evolve a primary producer in 96 unique environments with different combinations of between one and eight environmental drivers to understand how evolutionary responses to environmental change depend on the identity and number of drivers. Even in multidriver environments, only a few dominant drivers explain most of the evolutionary changes in population growth rates. Most populations converge on the same growth rate by the end of the evolution experiment. However, populations adapt more when these dominant drivers occur in the presence of other drivers. This is due to an increase in the intensity of selection in environments with more drivers, which are more likely to include dominant drivers. Concurrently, many of the trait changes that occur during the initial short-term response to both single and multidriver environmental change revert after about 450 generations of evolution. In future aquatic environments, populations will encounter differing combinations of drivers and intensities of selection, which will alter the adaptive potential of primary producers. Accurately gauging the intensity of selection on key primary producers will help in predicting population size and trait evolution at the base of aquatic food webs.

Statistical model specification and power: recommendations on the use of test-qualified pooling in analysis of experimental data.

A common approach to the analysis of experimental data across much of the biological sciences is test-qualified pooling. Here non-significant terms are dropped from a statistical model, effectively pooling the variation associated with each removed term with the error term used to test hypotheses (or estimate effect sizes). This pooling is only carried out if statistical testing on the basis of applying that data to a previous more complicated model provides motivation for this model simplification; hence the pooling is test-qualified. In pooling, the researcher increases the degrees of freedom of the error term with the aim of increasing statistical power to test their hypotheses of interest. Despite this approach being widely adopted and explicitly recommended by some of the most widely cited statistical textbooks aimed at biologists, here we argue that (except in highly specialized circumstances that we can identify) the hoped-for improvement in statistical power will be small or non-existent, and there is likely to be much reduced reliability of the statistical procedures through deviation of type I error rates from nominal levels. We thus call for greatly reduced use of test-qualified pooling across experimental biology, more careful justification of any use that continues, and a different philosophy for initial selection of statistical models in the light of this change in procedure.

The effect of sex on the repeatability of evolution in different environments.

The adaptive function of sex has been extensively studied, while less consideration has been given to the potential downstream consequences of sex on evolution. Here, we investigate one such potential consequence, the effect of sex on the repeatability of evolution. By affecting the repeatability of evolution, sex could have important implications for biodiversity, and for our ability to make predictions about the outcome of environmental change. We allowed asexual and sexual populations of Chlamydomonas reinhardtii to evolve in novel environments and monitored both their change in fitness and variance in fitness after evolution. Sex affected the repeatability of evolution by changing the importance of the effect of selection, chance, and ancestral constraints on the outcome of the evolutionary process. In particular, the effects of sex were highly dependent on the initial genetic composition of the population and on the environment. Given the lack of a consistent effect of sex on repeatability across the environments used here, further studies to dissect in more detail the underlying reasons for these differences as well as studies in additional environments are required if we are to have a general understanding of the effects of sex on the repeatability of evolution.

Caging and Uncaging Genetics.

It is important for biology to understand if observations made in highly reductionist laboratory settings generalise to harsh and noisy natural environments in which genetic variation is sorted to produce adaptation. But what do we learn by studying, in the laboratory, a genetically diverse population that mirrors the wild? What is the best design for studying genetic variation? When should we consider it at all? The right experimental approach depends on what you want to know.

Cathelicidins Have Direct Antiviral Activity against Respiratory Syncytial Virus In Vitro and Protective Function In Vivo in Mice and Humans.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infection in infants, causing significant morbidity and mortality. No vaccine or specific, effective treatment is currently available. A more complete understanding of the key components of effective host response to RSV and novel preventative and therapeutic interventions are urgently required. Cathelicidins are host defense peptides, expressed in the inflamed lung, with key microbicidal and modulatory roles in innate host defense against infection. In this article, we demonstrate that the human cathelicidin LL-37 mediates an antiviral effect on RSV by inducing direct damage to the viral envelope, disrupting viral particles and decreasing virus binding to, and infection of, human epithelial cells in vitro. In addition, exogenously applied LL-37 is protective against RSV-mediated disease in vivo, in a murine model of pulmonary RSV infection, demonstrating maximal efficacy when applied concomitantly with virus. Furthermore, endogenous murine cathelicidin, induced by infection, has a fundamental role in protection against disease in vivo postinfection with RSV. Finally, higher nasal levels of LL-37 are associated with protection in a healthy human adult RSV infection model. These data lead us to propose that cathelicidins are a key, nonredundant component of host defense against pulmonary infection with RSV, functioning as a first point of contact antiviral shield and having additional later-phase roles in minimizing the severity of disease outcome. Consequently, cathelicidins represent an inducible target for preventative strategies against RSV infection and may inform the design of novel therapeutic analogs for use in established infection.

Direct Estimate of the Spontaneous Mutation Rate Uncovers the Effects of Drift and Recombination in the Chlamydomonas reinhardtii Plastid Genome.

Plastids perform crucial cellular functions, including photosynthesis, across a wide variety of eukaryotes. Since endosymbiosis, plastids have maintained independent genomes that now display a wide diversity of gene content, genome structure, gene regulation mechanisms, and transmission modes. The evolution of plastid genomes depends on an input of de novo mutation, but our knowledge of mutation in the plastid is limited to indirect inference from patterns of DNA divergence between species. Here, we use a mutation accumulation experiment, where selection acting on mutations is rendered ineffective, combined with whole-plastid genome sequencing to directly characterize de novo mutation in Chlamydomonas reinhardtii. We show that the mutation rates of the plastid and nuclear genomes are similar, but that the base spectra of mutations differ significantly. We integrate our measure of the mutation rate with a population genomic data set of 20 individuals, and show that the plastid genome is subject to substantially stronger genetic drift than the nuclear genome. We also show that high levels of linkage disequilibrium in the plastid genome are not due to restricted recombination, but are instead a consequence of increased genetic drift. One likely explanation for increased drift in the plastid genome is that there are stronger effects of genetic hitchhiking. The presence of recombination in the plastid is consistent with laboratory studies in C. reinhardtii and demonstrates that although the plastid genome is thought to be uniparentally inherited, it recombines in nature at a rate similar to the nuclear genome.

Extensive de novo mutation rate variation between individuals and across the genome of Chlamydomonas reinhardtii.

Describing the process of spontaneous mutation is fundamental for understanding the genetic basis of disease, the threat posed by declining population size in conservation biology, and much of evolutionary biology. Directly studying spontaneous mutation has been difficult, however, because new mutations are rare. Mutation accumulation (MA) experiments overcome this by allowing mutations to build up over many generations in the near absence of natural selection. Here, we sequenced the genomes of 85 MA lines derived from six genetically diverse strains of the green alga Chlamydomonas reinhardtii. We identified 6843 new mutations, more than any other study of spontaneous mutation. We observed sevenfold variation in the mutation rate among strains and that mutator genotypes arose, increasing the mutation rate approximately eightfold in some replicates. We also found evidence for fine-scale heterogeneity in the mutation rate, with certain sequence motifs mutating at much higher rates, and clusters of multiple mutations occurring at closely linked sites. There was little evidence, however, for mutation rate heterogeneity between chromosomes or over large genomic regions of 200 kbp. We generated a predictive model of the mutability of sites based on their genomic properties, including local GC content, gene expression level, and local sequence context. Our model accurately predicted the average mutation rate and natural levels of genetic diversity of sites across the genome. Notably, trinucleotides vary 17-fold in rate between the most and least mutable sites. Our results uncover a rich heterogeneity in the process of spontaneous mutation both among individuals and across the genome.

Experimental adaptation to marine conditions by a freshwater alga.

The marine-freshwater boundary has been suggested as one of the most difficult to cross for organisms. Salt is a major ecological factor and provides an unequalled range of ecological opportunity because marine habitats are much more extensive than freshwater habitats, and because salt strongly affects the structure of microbial communities. We exposed experimental populations of the freshwater alga Chlamydomonas reinhardtii to steadily increasing concentrations of salt. About 98% of the lines went extinct. The ones that survived now thrive in growth medium with 36 g⋅L(-1) NaCl, and in seawater. Our results indicate that adaptation to marine conditions proceeded first through genetic assimilation of an inducible response to relatively low salt concentrations that was present in the ancestors, and subsequently by the evolution of an enhanced inducible response to high salt concentrations. These changes appear to have evolved through reversible and irreversible modifications, respectively. The evolution of marine from freshwater lineages is an example that clearly indicates the possibility of studying certain aspects of major ecological transitions in the laboratory.

PrfA regulation offsets the cost of Listeria virulence outside the host.

Virulence traits are essential for pathogen fitness, but whether they affect microbial performance in the environment, where they are not needed, remains experimentally unconfirmed. We investigated this question with the facultative pathogen Listeria monocytogenes and its PrfA virulence regulon. PrfA-regulated genes are activated intracellularly (PrfA 'ON') but shut down outside the host (PrfA 'OFF'). Using a mutant PrfA regulator locked ON (PrfA*) and thus causing PrfA-controlled genes to be constitutively activated, we show that virulence gene expression significantly impairs the listerial growth rate (µ) and maximum growth (A) in rich medium. Deletion analysis of the PrfA regulon and complementation of a L. monocytogenes mutant lacking all PrfA-regulated genes with PrfA* indicated that the growth reduction was specifically due to the unneeded virulence determinants and not to pleiotropic regulatory effects of PrfA ON. No PrfA*-associated fitness disadvantage was observed in infected eukaryotic cells, where PrfA-regulated virulence gene expression is critical for survival. Microcosm experiments demonstrated that the constitutively virulent state strongly impaired L. monocytogenes performance in soil, the natural habitat of these bacteria. Our findings provide empirical proof that virulence carries a significant cost to the pathogen. They also experimentally substantiate the assumed, although not proven, key role of virulence gene regulation systems in suppressing the cost of bacterial virulence outside the host.