Molecular adaptation of Ammonia Monooxygenase during independent pH specialization in Thaumarchaeota

Acknowledgements. This work was funded by Natural Environment Research Council Fellowship NE/J019151/1 and by institutional funding from within the University of Aberdeen.

Directed evolution of a thermostable esterase

We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14°C increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.

A test of alternative models of diversification in tropical rainforests: Ecological gradients vs. rainforest refugia

Comparison of mitochondrial and morphological divergence in eight populations of a widespread leaf-litter skink is used to determine the relative importance of geographic isolation and natural selection in generating phenotypic diversity in the Wet Tropics Rainforest region of Australia. The populations occur in two geographically isolated regions, and within each region, in two different habitats (closed rainforest and tall open forest) that span a well characterized ecological gradient. Morphological differences among ancient geographic isolates (separated for several million years, judging by their mitochondrial DNA sequence divergence) were slight, but morphological and life history differences among habitats were large and occurred despite moderate to high levels of mitochondrial gene flow. A field experiment identified avian predation as one potential agent of natural selection. These results indicate that natural selection operating across ecological gradients can be more important than geographic isolation in similar habitats in generating phenotypic diversity. In addition, our results indicate that selection is sufficiently strong to overcome the homogenizing effects of gene flow, a necessary first step toward speciation in continuously distributed populations. Because ecological gradients may be a source of evolutionary novelty, and perhaps new species, their conservation warrants greater attention. This is particularly true in tropical regions, where most reserves do not include ecological gradients and transitional habitats.

Plasmodium falciparum antigenic diversity: Evidence of clonal population structure

Plasmodium falciparum, the agent of malignant malaria, is one of mankind’s most severe scourges. Efforts to develop preventive vaccines or remedial drugs are handicapped by the parasite’s rapid evolution of drug resistance and protective antigens. We examine 25 DNA sequences of the gene coding for the highly polymorphic antigenic circumsporozoite protein. We observe total absence of silent nucleotide variation in the two nonrepeated regions of the gene. We propose that this absence reflects a recent origin (within several thousand years) of the world populations of P. falciparum from a single individual; the amino acid polymorphisms observed in these nonrepeat regions would result from strong natural selection. Analysis of these polymorphisms indicates that: (i) the incidence of recombination events does not increase with nucleotide distance; (ii) the strength of linkage disequilibrium between nucleotides is also independent of distance; and (iii) haplotypes in the two nonrepeat regions are correlated with one another, but not with the central repeat region they span. We propose two hypotheses: (i) variation in the highly polymorphic central repeat region arises by mitotic intragenic recombination, and (ii) the population structure of P. falciparum is clonal—a state of affairs that persists in spite of the necessary stage of physiological sexuality that the parasite must sustain in the mosquito vector to complete its life cycle.

The onset and extent of genomic instability in sporadic colorectal tumor progression

Cancer cell genomes contain alterations beyond known etiologic events, but their total number has been unknown at even the order of magnitude level. By sampling colorectal premalignant polyp and carcinoma cell genomes through use of the technique inter-(simple sequence repeat) PCR, we have found genomic alterations to be considerably more abundant than expected, with the mean number of genomic events per carcinoma cell totaling approximately 11,000. Colonic polyps early in the tumor progression pathway showed similar numbers of events. These results indicate that, as with certain hereditary cancer syndromes, genomic destabilization is an early step in sporadic tumor development. Together these results support the model of genomic instability being a cause rather than an effect of malignancy, facilitating vastly accelerated somatic cell evolution, with the observed orderly steps of the colon cancer progression pathway reflecting the consequences of natural selection.

Origins and antiquity of X-linked triallelic color vision systems in New World monkeys

It is known that the squirrel monkey, marmoset, and other related New World (NW) monkeys possess three high-frequency alleles at the single X-linked photopigment locus, and that the spectral sensitivity peaks of these alleles are within those delimited by the human red and green pigment genes. The three alleles in the squirrel monkey and marmoset have been sequenced previously. In this study, the three alleles were found and sequenced in the saki monkey, capuchin, and tamarin. Although the capuchin and tamarin belong to the same family as the squirrel monkey and marmoset, the saki monkey belongs to a different family and is one of the species that is most divergent from the squirrel monkey and marmoset, suggesting the presence of the triallelic system in many NW monkeys. The nucleotide sequences of these alleles from the five species studied indicate that gene conversion occurs frequently and has partially or completely homogenized intronic and exonic regions of the alleles in each species, making it appear that a triallelic system arose independently in each of the five species studied. Nevertheless, a detailed analysis suggests that the triallelic system arose only once in the NW monkey lineage, from a middle wavelength (green) opsin gene, and that the amino acid differences at functionally critical sites among alleles have been maintained by natural selection in NW monkeys for >20 million years. Moreover, the two X-linked opsin genes of howler monkeys (a NW monkey genus) were evidently derived from the incorporation of a middle (green) and a long wavelength (red) allele into one chromosome; these two genes together with the (autosomal) blue opsin gene would immediately enable even a male monkey to have trichromatic vision.

Working memory constrains human cooperation in the Prisoner’s Dilemma

Many problems in human society reflect the inability of selfish parties to cooperate. The “Iterated Prisoner’s Dilemma” has been used widely as a model for the evolution of cooperation in societies. Axelrod’s computer tournaments and the extensive simulations of evolution by Nowak and Sigmund and others have shown that natural selection can favor cooperative strategies in the Prisoner’s Dilemma. Rigorous empirical tests, however, lag behind the progress made by theorists. Clear predictions differ depending on the players’ capacity to remember previous rounds of the game. To test whether humans use the kind of cooperative strategies predicted, we asked students to play the iterated Prisoner’s Dilemma game either continuously or interrupted after each round by a secondary memory task (i.e., playing the game “Memory”) that constrained the students’ working-memory capacity. When playing without interruption, most students used “Pavlovian” strategies, as predicted, for greater memory capacity, and the rest used “generous tit-for-tat” strategies. The proportion of generous tit-for-tat strategies increased when games of Memory interfered with the subjects’ working memory, as predicted. Students who continued to use complex Pavlovian strategies were less successful in the Memory game, but more successful in the Prisoner’s Dilemma, which indicates a trade-off in memory capacity for the two tasks. Our results suggest that the set of strategies predicted by game theorists approximates human reality.

Evolutionary theory predicts late-life mortality plateaus

Most demographic data indicate a roughly exponential increase in adult mortality with age, a phenomenon that has been explained in terms of a decline in the force of natural selection acting on age-specific mortality. Scattered demographic findings suggest the existence of a late-life mortality plateau in both humans and dipteran insects, seemingly at odds with both prior data and evolutionary theory. Extensions to the evolutionary theory of aging are developed which indicate that such late-life mortality plateaus are to be expected when enough late-life data are collected. This expanded theory predicts late-life mortality plateaus, with both antagonistic pleiotropy and mutation accumulation as driving population genetic mechanisms.

Functional and physiological consequences of genetic variation at phosphoglucose isomerase: Heat shock protein expression is related to enzyme genotype in a montane beetle

Allele frequency variation at the phosphoglucose isomerase (PGI) locus in Californian populations of the beetle Chrysomela aeneicollis suggests that PGI may be undergoing natural selection. We quantified (i) apparent Michaelis-Menten constant (Km) of fructose 6-phosphate at different temperatures and (ii) thermal stability for three common PGI genotypes (1–1, 1–4, and 4–4). We also measured air temperature (Ta) and beetle body temperature (Tb) in three montane drainages in the Sierra Nevada, California. Finally, we measured 70-kDa heat shock protein (Hsp70) expression in field-collected and laboratory-acclimated beetles. We found that PGI allele 1 predominated in the northernmost drainage, Rock Creek (RC), which was also significantly cooler than the southernmost drainage, Big Pine Creek (BPC), where PGI allele 4 predominated. Allele frequencies and air temperatures were intermediate in the middle drainage, Bishop Creek (BC). Differences among genotypes in Km (1–1 > 1–4 > 4–4) and thermal stability (4–4 > 1–4 > 1–1) followed a pattern consistent with temperature adaptation. In nature, Tb was closely related to Ta. Hsp70 expression in adult beetles decreased with elevation and differed among drainages (BPC > BC > RC). After laboratory acclimation (8 days, 20°C day, 4°C night) and heat shock (4 h, 28–36°C), Hsp70 expression was greater for RC than BPC beetles. In RC, field-collected beetles homozygous for PGI 1–1 had higher Hsp70 levels than heterozygotes or a 4–4 homozygote. These results reveal functional and physiological differences among PGI genotypes, which suggest that montane populations of this beetle are locally adapted to temperature.

Truncated hexa-octahedral magnetite crystals in ALH84001: Presumptive biosignatures

McKay et al. [(1996) Science 273, 924–930] suggested that carbonate globules in the meteorite ALH84001 contained the fossil remains of Martian microbes. We have characterized a subpopulation of magnetite (Fe3O4) crystals present in abundance within the Fe-rich rims of these carbonate globules. We find these Martian magnetites to be both chemically and physically identical to terrestrial, biogenically precipitated, intracellular magnetites produced by magnetotactic bacteria strain MV-1. Specifically, both magnetite populations are single-domain and chemically pure, and exhibit a unique crystal habit we describe as truncated hexa-octahedral. There are no known reports of inorganic processes to explain the observation of truncated hexa-octahedral magnetites in a terrestrial sample. In bacteria strain MV-1 their presence is therefore likely a product of Natural Selection. Unless there is an unknown and unexplained inorganic process on Mars that is conspicuously absent on the Earth and forms truncated hexa-octahedral magnetites, we suggest that these magnetite crystals in the Martian meteorite ALH84001 were likely produced by a biogenic process. As such, these crystals are interpreted as Martian magnetofossils and constitute evidence of the oldest life yet found.

Declines of biomes and biotas and the future of evolution

Although panel discussants disagreed whether the biodiversity crisis constitutes a mass extinction event, all agreed that current extinction rates are 50–500 times background and are increasing and that the consequences for the future evolution of life are serious. In response to the on-going rapid decline of biomes and homogenization of biotas, the panelists predicted changes in species geographic ranges, genetic risks of extinction, genetic assimilation, natural selection, mutation rates, the shortening of food chains, the increase in nutrient-enriched niches permitting the ascendancy of microbes, and the differential survival of ecological generalists. Rates of evolutionary processes will change in different groups, and speciation in the larger vertebrates is essentially over. Action taken over the next few decades will determine how impoverished the biosphere will be in 1,000 years when many species will suffer reduced evolvability and require interventionist genetic and ecological management. Whether the biota will continue to provide the dependable ecological services humans take for granted is less clear. The discussants offered recommendations, including two of paramount importance (concerning human populations and education), seven identifying specific scientific activities to better equip us for stewardship of the processes of evolution, and one suggesting that such stewardship is now our responsibility. The ultimate test of evolutionary biology as a science is not whether it solves the riddles of the past but rather whether it enables us to manage the future of the biosphere. Our inability to make clearer predictions about the future of evolution has serious consequences for both biodiversity and humanity.

Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions

The evolution of novelty in tightly integrated biological systems, such as hormones and their receptors, seems to challenge the theory of natural selection: it has not been clear how a new function for any one part (such as a ligand) can be selected for unless the other members of the system (e.g., a receptor) are already present. Here I show—based on identification and phylogenetic analysis of steroid receptors in basal vertebrates and reconstruction of the sequences and functional attributes of ancestral proteins—that the first steroid receptor was an estrogen receptor, followed by a progesterone receptor. Genome mapping and phylogenetic analyses indicate that the full complement of mammalian steroid receptors evolved from these ancient receptors by two large-scale genome expansions, one before the advent of jawed vertebrates and one after. Specific regulation of physiological processes by androgens and corticoids are relatively recent innovations that emerged after these duplications. These findings support a model of ligand exploitation in which the terminal ligand in a biosynthetic pathway is the first for which a receptor evolves; selection for this hormone also selects for the synthesis of intermediates despite the absence of receptors, and duplicated receptors then evolve affinity for these substances. In this way, novel hormone-receptor pairs are created, and an integrated system of increasing complexity elaborated. This model suggests that ligands for some “orphan” receptors may be found among intermediates in the synthesis of ligands for phylogenetically related receptors.

A power law for cells

Darwin observed that multiple, lowly organized, rudimentary, or exaggerated structures show increased relative variability. However, the cellular basis for these laws has never been investigated. Some animals, such as the nematode Caenorhabditis elegans, are famous for having organs that possess the same number of cells in all individuals, a property known as eutely. But for most multicellular creatures, the extent of cell number variability is unknown. Here we estimate variability in organ cell number for a variety of animals, plants, slime moulds, and volvocine algae. We find that the mean and variance in cell number obey a power law with an exponent of 2, comparable to Taylor's law in ecological processes. Relative cell number variability, as measured by the coefficient of variation, differs widely across taxa and tissues, but is generally independent of mean cell number among homologous tissues of closely related species. We show that the power law for cell number variability can be explained by stochastic branching process models based on the properties of cell lineages. We also identify taxa in which the precision of developmental control appears to have evolved. We propose that the scale independence of relative cell number variability is maintained by natural selection.

Evolution of genome–phenome diversity under environmental stress

The genomic era revolutionized evolutionary biology. The enigma of genotypic-phenotypic diversity and biodiversity evolution of genes, genomes, phenomes, and biomes, reviewed here, was central in the research program of the Institute of Evolution, University of Haifa, since 1975. We explored the following questions. (i) How much of the genomic and phenomic diversity in nature is adaptive and processed by natural selection? (ii) What is the origin and evolution of adaptation and speciation processes under spatiotemporal variables and stressful macrogeographic and microgeographic environments? We advanced ecological genetics into ecological genomics and analyzed globally ecological, demographic, and life history variables in 1,200 diverse species across life, thousands of populations, and tens of thousands of individuals tested mostly for allozyme and partly for DNA diversity. Likewise, we tested thermal, chemical, climatic, and biotic stresses in several model organisms. Recently, we introduced genetic maps and quantitative trait loci to elucidate the genetic basis of adaptation and speciation. The genome–phenome holistic model was deciphered by the global regressive, progressive, and convergent evolution of subterranean mammals. Our results indicate abundant genotypic and phenotypic diversity in nature. The organization and evolution of molecular and organismal diversity in nature at global, regional, and local scales are nonrandom and structured; display regularities across life; and are positively correlated with, and partly predictable by, abiotic and biotic environmental heterogeneity and stress. Biodiversity evolution, even in small isolated populations, is primarily driven by natural selection, including diversifying, balancing, cyclical, and purifying selective regimes, interacting with, but ultimately overriding, the effects of mutation, migration, and stochasticity.

Directionality principles in thermodynamics and evolution

Directionality in populations of replicating organisms can be parametrized in terms of a statistical concept: evolutionary entropy. This parameter, a measure of the variability in the age of reproducing individuals in a population, is isometric with the macroscopic variable body size. Evolutionary trends in entropy due to mutation and natural selection fall into patterns modulated by ecological and demographic constraints, which are delineated as follows: (i) density-dependent conditions (a unidirectional increase in evolutionary entropy), and (ii) density-independent conditions, (a) slow exponential growth (an increase in entropy); (b) rapid exponential growth, low degree of iteroparity (a decrease in entropy); and (c) rapid exponential growth, high degree of iteroparity (random, nondirectional change in entropy). Directionality in aggregates of inanimate matter can be parametrized in terms of the statistical concept, thermodynamic entropy, a measure of disorder. Directional trends in entropy in aggregates of matter fall into patterns determined by the nature of the adiabatic constraints, which are characterized as follows: (i) irreversible processes (an increase in thermodynamic entropy) and (ii) reversible processes (a constant value for entropy). This article analyzes the relation between the concepts that underlie the directionality principles in evolutionary biology and physical systems. For models of cellular populations, an analytic relation is derived between generation time, the average length of the cell cycle, and temperature. This correspondence between generation time, an evolutionary parameter, and temperature, a thermodynamic variable, is exploited to show that the increase in evolutionary entropy that characterizes population processes under density-dependent conditions represents a nonequilibrium analogue of the second law of thermodynamics.