Genomic plasticity and rapid host switching can promote the evolution of generalism : a case study in the zoonotic pathogen Campylobacter

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/I02464X/1, the Medical Research Council (MRC) grants MR/M501608/1 and MR/L015080/1, and the Wellcome Trust grant 088786/C/09/Z. GM was supported by a NISCHR Health Research Fellowship (HF-14–13).Peer reviewedPublisher PD

The role of clonal interference across genetic backgrounds and environments

The study of adaptation in microorganisms has led to a significant expansion in knowledge at many biological levels, ranging from biochemistry and genetics, to ecology and demography. Experimental evolution, in particular, has been invaluable at elucidating how complex the adaptive dynamics in microbial populations can be. One of the most fundamental characteristics of these dynamics is the distribution of beneficial mutations driving the adaptive process. How often do microorganisms acquire these mutations? And what are their expected effects? These questions have been at the heart of evolutionary biology from the very beginning, and the studies that have tackled these difficult issues have been tremendously enlightening about adaptive processes. However, the increasing awareness of the complexity of the environment where microorganisms live requires constant development of new approaches to answer these fundamental questions about their evolution. Large population sizes lead to increased levels of clonal interference, and thus to a deviation from the expected outcome in classical regimes of periodic selection. Genetic variation within an evolving population, which is now easily detected by sequencing technologies, can create complex interactions between phenotypes. Environments with antagonistic biotic interactions, pose very different selective pressures from the ones experienced when a species grows alone. All these factors influence adaptation in microorganisms and, importantly, drive the pathogenicity traits that create severe clinical and epidemiological problems

The role of the gut microbiota in the subsistence of antibiotic resistance

"Antibiotic resistance is one of the major contemporary threats to global health. Studies on evolutionary biology, molecular biology and genetics have revealed that many phenomena contribute for the subsistence of resistant bacteria. The environment has been shown to be a key factor, capable of altering fitness costs and the epistasis patterns between resistance determinants. Still, few studies have ventured to assess the costs of antibiotic resistance in natural environments, and such studies are centered on pathogens. It is now known that commensal bacteria can act as reservoirs of resistance, and that resistant commensals can evolve to express pathogenicity and share resistance genes with pathogens. Here, we explore how selection acts on resistant, commensal E. coli in the mouse gut. (...)

Herbicide cycling has diverse effects on evolution of resistance in Chlamydomonas reinhardtii

Cycling pesticides has been proposed as a means of retarding the evolution of resistance, but its efficacy has rarely been empirically tested. We evolved populations of Chlamydomonas reinhardtii in the presence of three herbicides: atrazine, glyphosate and carbetamide. Populations were exposed to a weekly, biweekly and triweekly cycling between all three pairwise combinations of herbicides and continuously to each of the three herbicides. We explored the impacts of herbicide cycling on the rate of resistance evolution, the level of resistance selected, the cost of resistance and the degree of generality (cross-resistance) observed. Herbicide cycling resulted in a diversity of outcomes: preventing evolution of resistance for some combinations of herbicides, having no impacts for others and increasing rates of resistance evolution in some instances. Weekly cycling of atrazine and carbetamide resulted in selection of a generalist population. This population had a higher level of resistance, and this generalist resistance was associated with a cost. The level of resistance selected did not vary amongst other regimes. Costs of resistance were generally highest when cycling was more frequent. Our data suggest that the effects of herbicide cycling on the evolution of resistance may be more complex and less favourable than generally assumed

Effects of sex and competition on evolutionary survival of Chlamydomonas reinhardtii populations in deteriorating environments

Ongoing global change has made understanding the factors that affect adaptation and survival of populations in the context of changing environments a central problem in evolutionary biology. Special focus has been given to the probability of survival through genetic adaptation to lethal environments; a process termed evolutionary rescue. Many studies of this process, both theoretical and empirical, have been carried out over the last two decades. As a result, we now understand how a number of factors may affect the probability of population survival. However, two factors that are known to affect evolutionary responses, mode of reproduction and interspecific interaction, have received limited attention. The main aim of my work was to investigate whether and how mode of reproduction and negative interspecies interactions (competition) affect the probability of evolutionary rescue. To achieve this goal, I set up a series of selection experiments, by propagating populations of unicellular alga Chlamydomonas reinhardtii in various stressful conditions, and monitored their survival and fitness. To investigate the effect of sex in these experiments, I manipulated mode of reproduction, by constructing the experimental populations allowed to reproduce either only sexually or asexually or both. To investigate the effect of competition, I manipulated the presence of the competitor(s) in the experimental populations, by cultivating them either in presence or absence of the competitor. I first tested the effect of rate of environmental deterioration and mode of reproduction on extinction dynamics and evolutionary rescue of the experimental populations. I found positive correlation between the rate of extinctions and the rate of environmental deterioration. The experiment revealed an interaction between mode of reproduction and the rate of deterioration, manifested through significantly reduced extinction rate of sexual populations relative to asexual populations in environment deteriorating at intermediate rate. I then investigated the effect of sex and competition on the probability of evolutionary rescue, by propagating the experimental populations in environment deteriorating in a simple way (the change comprising a single abiotic factor) and complex way (the change of both abiotic and biotic factors). I found the negative effect of competition on the probability of evolutionary rescue, and beneficial effect of sex in both types of environmental deterioration, reflected in higher number of rescued populations relative to asexual group. I then tested whether phylogenetic relatedness between a competitor and the focal species and the extent of their ecological similarity affect the likelihood of evolutionary rescue, by subjecting the experimental populations to the presence of 10 different competitors, isolated from two different types of habitats, and each being positioned on a different branch of the phylogenetic tree of Chlamydomonas genus. The probability of evolutionary rescue was contingent on the identity of a competitor species, but the results showed no significant effects of phylogenetic relatedness and ecological similarity. Finally, I investigated which experimental factors could potentially select for the long-term maintenance of sex, by subjecting the experimental populations to different types of selective environments (directional and fluctuating change of abiotic factors, the presence of the competitor) and monitoring the frequency of sex over the course of time. No selective environment significantly increased the rate of sex in the experimental populations. In contrast, I found reduction in frequency of sex in the populations subjected to fluctuating environmental change. My results demonstrate that both mode of reproduction and competition affect the probability of evolutionary rescue, which is generally positively affected by sex and negatively affected by competition. However, these general effects may be altered by other factors, namely mode of environmental change and the identity of the competitor species

Adaptation to high ethanol reveals complex evolutionary pathways

Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts

Adaptation from standing genetic variation and from mutation

Dissertation presented to obtain the Ph.D degree in Evolutionary BiologyUnderstanding the genetic basis of adaptation is crucial to explain the emergence and maintenance of the multitude of life forms we find on Earth today. Perhaps even more importantly, gaining knowledge about how fast organisms can cope with environmental changes may prove crucial in a world being altered at increasing speed due to the human actions. The study of adaptive evolution may therefore have major implications (and applications) in Agriculture, Conservation of endangered species and even Human Health. Natural selection has long been appreciated as one of the predominant evolutionary mechanisms and it enjoys a solid theoretical framework regarding its requirements, its effects and its limitations. Empirically, however, it has proved quite challenging to study. In wild populations natural selection is particularly difficult to characterize and measure since in these settings other evolutionary mechanisms (such as genetic drift or gene flow) often occur simultaneously. In addition to this, the different evolutionary mechanisms may vary greatly in time and in space with respect to their relative influences on the evolutionary dynamics of populations.(...

The impact of genetic interactions in antibiotic resistance

Tese de doutoramento, Biologia (Genética), Universidade de Lisboa, Faculdade de Ciências, 2012Genetic interactions, both between genetic material and between this and the surrounding environment of a given individual are important factors for understanding the process of evolution of natural populations. The study of this interactions as well as their integration in evolutionary terms may have several applications such as, for example, understanding the observed prevalence of antibiotic resistance in natural populations. In this thesis the patterns of genetic interactions between multiple-resistances to antibiotics were explored. In particular, epistasis and genotype-by-environment interactions operating among antibiotic resistances were studied. To measure levels of epistasis occurring between multiple-antibiotic-resistances in the complete absence of antibiotics, mutants resistant to three antibiotics commonly used in clinic were first generated: nalidixic acid, rifampicin and streptomycin. With these mutants double resistant mutants were created. By measuring the costs of each mutation individually and jointly the type of epistasis operating between different sets of resistance mutations was determined. The cost of double resistance was majorly lower than expected, revealing the presence of positive epistasis between mutations conferring resistance to the studied antibiotics. The same patterns were observed for interactions between a set of resistance mutations and conjugative plasmids with multiple-resistance factors. However, in this case, extreme cases of positive epistasis coined sign epistasis were observed for a large fraction of combinations. This type of interactions was also observed in the previous study yet less frequently. Naturally occurring bacteria are often faced with a multitude of environments. The action of environmental changes in the effects of antibiotic resistance mutations was studied for a group of mutations resistant to four antibiotics in three environments differing in the CHAPTER I viii number of comprised environmental stresses. This study revealed, for most cases, a strong pattern of interactions between the different genotypes and the environment.As interacções genéticas, quer entre material genético quer entre este e o ambiente que rodeia um determinado organismo, são factores importantes para entender o processo de evolução de populações naturais. O estudo destas interacções bem como a sua integração em termos evolutivos pode ter diversas aplicações tais como, por exemplo, a compreensão da prevalência da resistência a antibióticos observada em populações naturais. Nesta tese exploraram-se os padrões de interacções genéticas entre múltiplas resistências a antibióticos nomeadamente, epistasia e as interacções genótipo-ambiente que ocorrem em resistências a antibióticos. Para medir os níveis de epistasia entre múltiplas resistências a antibióticos na ausência dos mesmos, foram construídos mutantes resistentes a três antibióticos vulgarmente usados em clínica: ácido nalidíxico, rifampicina e estreptomicina. Com estes mutantes foram gerados duplos mutantes. Medindo os custos de cada uma das mutações individualmente e em conjunto determinou-se o tipo de epistasia a operar entre diferentes conjuntos de mutações de resistência. O custo da dupla resistência foi maioritariamente menor do que o esperado, revelando a presença de epistasia positiva entre mutações que conferem resistência aos antibióticos estudados. Os mesmos padrões foram observados para interacções entre um conjunto de mutações que conferem resistência e plasmídeos conjugativos com múltiplos factores de resistência. No entanto, neste caso, foram observados numa larga fracção de combinações casos extremos de epistasia positiva denominados epistasia de sinal, situação também observada anteriormente mas em menor frequência. Na natureza, as bactérias enfrentam frequentemente uma multiplicidade de ambientes. A acção das alterações ambientais nos efeitos das mutações que conferem resistência a antibióticos foi CHAPTER I x estudada num grupo de mutações resistentes a quatro antibióticos em três ambientes que diferem no número de factores ambientais prejudiciais comportados por cada um. Este estudo revelou, para a maioria dos casos, um forte padrão de interacções entre os vários genótipos e o ambiente.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/40162/2007