Experimental evolution, loss-of-function mutations, and "the first rule of adaptive evolution"

Adaptive evolution can cause a species to gain, lose, or modify a function; therefore, it is of basic interest to determine whether any of these modes dominates the evolutionary process under particular circumstances. Because mutation occurs at the molecular level, it is necessary to examine the molecular changes produced by the underlying mutation in order to assess whether a given adaptation is best considered as a gain, loss, or modification of function. Although that was once impossible, the advance of molecular biology in the past half century has made it feasible. In this paper, I review molecular changes underlying some adaptations, with a particular emphasis on evolutionary experiments with microbes conducted over the past four decades. I show that by far the most common adaptive changes seen in those examples are due to the loss or modification of a pre-existing molecular function, and I discuss the possible reasons for the prominence of such mutations.     

Population genetics, pleiotropy, and the preferential fixation of mutations during adaptive evolution

Ongoing debate centers on whether certain types of mutations are fixed preferentially during adaptive evolution. Although there has been much discussion, no quantitative framework currently exists to test for these biases. Here, we describe a method for distinguishing between the two processes that likely account for biased rates of substitution: variation in mutation rates and variation in the probability that a mutation becomes fixed once it arises. We then use this approach to examine the type and magnitude of these biases during evolutionary transitions across multiple scales: those involving repeated origins of individual traits (flower color change), and transitions involving broad suites of traits (morphological and physiological trait evolution in plants and animals). We show that fixation biases can be strong at both levels of comparison, likely due to differences in the magnitude of deleterious pleiotropy associated with alternative mutation categories. However, we also show that the scale at which these comparisons are made greatly influences the results, as broad comparisons that simultaneously analyze multiple traits obscure heterogeneity in the direction and magnitude of these biases. We conclude that preferential fixation of mutations likely is common in nature, but should be studied on a trait-by-trait basis.     

Compensatory mutations, antibiotic resistance and the population genetics of adaptive evolution in bacteria

In the absence of the selecting drugs, chromosomal mutations for resistance to antibiotics and other chemotheraputic agents commonly engender a cost in the fitness of microorganisms. Recent in vivo and in vitro experimental studies of the adaptation to these "costs of resistance" in Escherichia coli, HIV, and Salmonella typhimurium found that evolution in the absence of these drugs commonly results in the ascent of mutations that ameliorate these costs, rather than higher-fitness, drug-sensitive revertants. To ascertain the conditions under which this compensatory evolution, rather than reversion, will occur, we did computer simulations, in vitro experiments, and DNA sequencing studies with low-fitness rpsL (streptomycin-resistant) mutants of E. coli with and without mutations that compensate for the fitness costs of these ribosomal protein mutations. The results of our investigation support the hypothesis that in these experiments, the ascent of intermediate-fitness compensatory mutants, rather than high-fitness revertants, can be attributed to higher rates of compensatory mutations relative to that of reversion and to the numerical bottlenecks associated with serial passage. We argue that these bottlenecks are intrinsic to the population dynamics of parasitic and commensal microbes and discuss the implications of these results to the problem of drug resistance and adaptive evolution in parasitic and commmensal microorganisms in general.     

Parallel evolution of adaptive mutations in Plasmodium falciparum mitochondrial DNA during atovaquone-proguanil treatment

Here we provide direct evidence that two adaptive nucleotide changes in the same codon (268) of the cytochrome b gene (pfcytb) each occurred repeatedly in independent Plasmodium falciparum lineages exposed to the antimalarial drug atovaquone-proguanil (AP). We analyzed the history of 7 AP resistance alleles from clinical isolates by sequencing the mitochondrial (mt) genome that encodes the pfcytb gene and found that a distinct mt haplotype was associated with each AP resistance allele. By comparing mt sequences and microsatellite genotypes of the isolates both before treatment initiation and at the day of failure for each uncured patient, we observed that the AP resistance alleles occurred and spread within the patients. These data demonstrate that identical AP resistance alleles have multiple independent origins and provide an example of parallel evolution driven by drug treatment selection in P. falciparum.     

Scalable method to determine mutations that occur during adaptive evolution of Escherichia coli

Denaturing HPLC was used to determine mutations occurring during the adaptive evolution of Escherichia coli K-12. The strains were evolved over 700 generations on glycerol as the sole carbon source from a sub-optimal to an optimal growth rate. The mutations detected by direct sequencing of amplicons of the glycerol-phosphate regulon repressor (glpR) gene were a synonymous substitution Val20Val in two separately evolved strains. Non-synonymous substitutions, Val119Gly and Gly179Trp, were also observed in each of the two strains. This procedure can be scaled to determine genome-scale sequence variations that have occurred during adaptive evolution.     

Predicting adaptive evolution

Phylogenetic trees reconstruct past evolution and can provide evidence of past evolutionary pressure on genes and on individual codons. In addition to tracing past evolutionary events, molecular phylogenetics might also be used to predict future evolution. Our ability to verify adaptive hypotheses using phylogenetics has broad implications for vaccine design, genomics and structural biology.     

Microbial adaptive evolution

Bacterial species can adapt to significant changes in their environment by mutation followed by selection, a phenomenon known as “adaptive evolution.” With the development of bioinformatics and genetic engineering, research on adaptive evolution has progressed rapidly, as have applications of the process. In this review, we summarize various mechanisms of bacterial adaptive evolution, the technologies used for studying it, and successful applications of the method in research and industry. We particularly highlight the contributions of Dr. L. O. Ingram. Microbial adaptive evolution has significant impact on our society not only from its industrial applications, but also in the evolution, emergence, and control of various pathogens.     

Predictive adaptive responses and human evolution

The importance of a single genotype being able to produce different phenotypes in different environments (phenotypic plasticity) is widely recognized in evolutionary theory and its adaptive significance is clear. In most cases, the developing organism responds to an environmental cue by producing a selectively and immediately appropriate phenotype. One subset of phenotypic responses to environmental stimuli, however, does not necessarily provide an immediate selective advantage. Rather, these kinds of responses, which we call 'predictive adaptive responses' (PARs), act primarily to improve fitness at a later stage of development. We argue that PARs have had an important role in human evolution, and that their recognition and interpretation has major significance for public health.     

Development and evolution of adaptive polyphenisms

Phenotypic plasticity is the primitive character state for most if not all traits. Insofar as developmental and physiological processes obey the laws of chemistry and physics, they will be sensitive to such environmental variables as temperature, nutrient supply, ionic environment, and the availability of various macro- and micronutrients. Depending on the effect this phenotypic plasticity has on fitness, evolution may proceed to select either for mechanisms that buffer or canalize the phenotype against relevant environmental variation or for a modified plastic response in which some ranges of the phenotypic variation are adaptive to particular environments. Phenotypic plasticity can be continuous, in which case it is called a reaction norm, or discontinuous, in which case it is called a polyphenism. Although the morphological discontinuity of some polyphenisms is produced by discrete developmental switches, most polyphenisms are due to discontinuities in the environment that induce only portions of what is in reality a continuous reaction norm. In insect polyphenisms, the environmental variable that induces the alternative phenotype is a token stimulus that serves as a predictor of, but is not itself, the environment to which the polyphenism is an adaptation. In all cases studied so far, the environmental stimulus alters the endocrine mechanism of metamorphosis by altering either the pattern of hormone secretion or the pattern of hormone sensitivity in different tissues. Such changes in the patterns of endocrine interactions result in the execution of alternative developmental pathways. The spatial and temporal compartmentalization of endocrine interactions has produced a developmental mechanism that enables substantial localized changes in morphology that remain well integrated into the structure and function of the organism.     

Genome evolution: recombination speeds up adaptive evolution

Mutual interference among linked genetic sites subject to selection may reduce the level of adaptation. A recent study detected this effect using data on protein sequence evolution and codon usage in Drosophila.     

Fluctuation domains in adaptive evolution

We derive an expression for the variation between parallel trajectories in phenotypic evolution, extending the well known result that predicts the mean evolutionary path in adaptive dynamics or quantitative genetics. We show how this expression gives rise to the notion of fluctuation domains-parts of the fitness landscape where the rate of evolution is very predictable (due to fluctuation dissipation) and parts where it is highly variable (due to fluctuation enhancement). These fluctuation domains are determined by the curvature of the fitness landscape. Regions of the fitness landscape with positive curvature, such as adaptive valleys or branching points, experience enhancement. Regions with negative curvature, such as adaptive peaks, experience dissipation. We explore these dynamics in the ecological scenarios of implicit and explicit competition for a limiting resource.     

The evolution and adaptive significance of heterostyly

The origin and adaptive significance of heterostylous breeding systems have fascinated evolutionary biologists since Darwin's early work on the subject. Models of the evolution of heterostyly differ in the emphasis given to different selective forces and in the sequence in which the physiological and morphological components of the polymorphism are thought to arise. Recent field studies of the population biology of heterostylous plants provide support for Darwin's hypothesis that the style-stamen polymorphism promotes disassortative pollination among the floral morphs.     

A new insight into arable weed adaptive evolution: mutations endowing herbicide resistance also affect germination dynamics and seedling emergence

Background and Aims

Selective pressures exerted by agriculture on populations of arable weeds foster the evolution of adaptive traits. Germination and emergence dynamics and herbicide resistance are key adaptive traits. Herbicide resistance alleles can have pleiotropic effects on a weed''s life cycle. This study investigated the pleiotropic effects of three acetyl-coenzyme A carboxylase (ACCase) alleles endowing herbicide resistance on the seed-to-plant part of the life cycle of the grass weed Alopecurus myosuroides.

Methods

In each of two series of experiments, A. myosuroides populations with homogenized genetic backgrounds and segregating for Leu1781, Asn2041 or Gly2078 ACCase mutations which arose independently were used to compare germination dynamics, survival in the soil and seedling pre-emergence growth among seeds containing wild-type, heterozygous and homozygous mutant ACCase embryos.

Key Results

Asn2041 ACCase caused no significant effects. Gly2078 ACCase major effects were a co-dominant acceleration in seed germination (1·25- and 1·10-fold decrease in the time to reach 50 % germination (T₅₀) for homozygous and heterozygous mutant embryos, respectively). Segregation distortion against homozygous mutant embryos or a co-dominant increase in fatal germination was observed in one series of experiments. Leu1781 ACCase major effects were a co-dominant delay in seed germination (1·41- and 1·22-fold increase in T₅₀ for homozygous and heterozygous mutant embryos, respectively) associated with a substantial co-dominant decrease in fatal germination.

Conclusions

Under current agricultural systems, plants carrying Leu1781 or Gly2078 ACCase have a fitness advantage conferred by herbicide resistance that is enhanced or counterbalanced, respectively, by direct pleiotropic effects on the plant phenology. Pleiotropic effects associated with mutations endowing herbicide resistance undoubtedly play a significant role in the evolutionary dynamics of herbicide resistance in weed populations. Mutant ACCase alleles should also prove useful to investigate the role played by seed storage lipids in the control of seed dormancy and germination.     

Mutational reversions during adaptive protein evolution

Adaptation is often regarded as the sequential fixation of individually, intrinsically beneficial mutations. Contrary to this expectation, we find a surprisingly large number of evolutionary trajectories on which natural selection first favors a mutation, then favors its removal, and later still favors its ultimate restoration during the course of antibiotic resistance evolution. The existence of reversion trajectories implies that natural selection may not follow the most parsimonious path separating two alleles, even during adaptation. Altogether, this discovery highlights the unusual and potentially circuitous routes natural selection can follow during adaptation.     

The early adaptive evolution of calmodulin

Interaction between gene duplication and natural selection in molecular evolution was investigated utilizing a phylogenetic tree constructed by the parsimony procedure from amino acid sequences of 50 calmodulin- family protein members. The 50 sequences, belonging to seven protein lineages related by gene duplication (calmodulin itself, troponin-C, alkali and regulatory light chains of myosin, parvalbumin, intestinal calcium-binding protein, and glial S-100 phenylalanine-rich protein), came from a wide range of eukaryotic taxa and yielded a denser tree (more branch points within each lineage) than in earlier studies. Evidence obtained from the reconstructed pattern of base substitutions and deletions in these ancestral loci suggests that, during the early history of the family, selection acted as a transforming force on expressed genes among the duplicates to encode molecular sites with new or modified functions. In later stages of descent, however, selection was a conserving force that preserved the structures of many coadapted functional sites. Each branch of the family was found to have a unique average tempo of evolutionary change, apparently regulated through functional constraints. Proteins whose functions dictate multiple interaction with several other macromolecules evolved more slowly than those which display fewer protein-protein and protein-ion interactions, e.g., calmodulin and next troponin-C evolved at the slowest average rates, whereas parvalbumin evolved at the fastest. The history of all lineages, however, appears to be characterized by rapid rates of evolutionary change in earlier periods, followed by slower rates in more recent periods. A particularly sharp contrast between such fast and slow rates is found in the evolution of calmodulin, whose rate of change in earlier eukaryotes was manyfold faster than the average rate over the past 1 billion years. In fact, the amino acid replacements in the nascent calmodulin lineage occurred at residue positions that in extant metazoans are largely invariable, lending further support to the Darwinian hypothesis that natural selection is both a creative and a conserving force in molecular evolution.      

The evolution of adaptive immune systems

A clonally diverse anticipatory repertoire in which each lymphocyte bears a unique antigen receptor is the central feature of the adaptive immune system that evolved in our vertebrate ancestors. The survival advantage gained through adding this type of adaptive immune system to a pre-existing innate immune system led to the evolution of alternative ways for lymphocytes to generate diverse antigen receptors for use in recognizing and repelling pathogen invaders. All jawed vertebrates assemble their antigen-receptor genes through recombinatorial rearrangement of different immunoglobulin or T cell receptor gene segments. The surviving jawless vertebrates, lampreys and hagfish, instead solved the receptor diversification problem by the recombinatorial assembly of leucine-rich-repeat genetic modules to encode variable lymphocyte receptors. The convergent evolution of these remarkably different adaptive immune systems involved innovative genetic modification of innate-immune-system components.