New technologies to assess genotype-phenotype relationships

The accelerating pace of the discovery of genes has far surpassed our capabilities to understand their biological function--in other words, the phenotypes they engender. We need efficient and comprehensive large-scale phenotyping technologies. This presents a difficult challenge because phenotypes are numerous and diverse, and they can be observed and annotated at the molecular, cellular and organismal level. New technologies and approaches will therefore be required. Here, I describe recent efforts to develop new and efficient technologies for assessing cellular phenotypes.     

Inference of genotype-phenotype relationships in the antigenic evolution of human influenza A (H3N2) viruses

Distinguishing mutations that determine an organism's phenotype from (near-) neutral 'hitchhikers' is a fundamental challenge in genome research, and is relevant for numerous medical and biotechnological applications. For human influenza viruses, recognizing changes in the antigenic phenotype and a strains' capability to evade pre-existing host immunity is important for the production of efficient vaccines. We have developed a method for inferring 'antigenic trees' for the major viral surface protein hemagglutinin. In the antigenic tree, antigenic weights are assigned to all tree branches, which allows us to resolve the antigenic impact of the associated amino acid changes. Our technique predicted antigenic distances with comparable accuracy to antigenic cartography. Additionally, it identified both known and novel sites, and amino acid changes with antigenic impact in the evolution of influenza A (H3N2) viruses from 1968 to 2003. The technique can also be applied for inference of 'phenotype trees' and genotype-phenotype relationships from other types of pairwise phenotype distances.     

Continental drift, paleoclimatology, and the evolution and biogeography of birds

An analysis is presented of the influence that late Mesozoic and Tertiary paleogeography and paleoclimatology may have had on the evolution and biogeography of birds. Many intercontinental connections, especially in the Southern Hemisphere, persisted until the late Cretaceous and/or early Tertiary. Moreover, climates at these times were warmer and more equable than in the late Tertiary, and birds could breed in and disperse through high latitudes. It is concluded that a number of avian orders and families had their origin in Gondwanaland and predrift configurations of the continents were major determinants of their biogeography. Penguins, ratites, galliforms, and suboscines among others are the best examples. Tropical-subtropical Eurasia was probably the centre of origin for the oscines, and primitive stocks entered the New World mostly through Beringia and mostly prior to the Miocene (but also via a North Atlantic land connection prior to the early Eocene). Continental drift and paleoclimatology have clearly influenced the evolution and biogeography of birds, and future advances in the systematics of the higher taxa will undoubtedly provide further confirmation of this.     

The effects of genetic drift in experimental evolution

Experimental evolution is characterized by exponential or logistic growth and periodic population bottlenecks. The fate of a rare beneficial mutation in these systems is influenced both by the bottleneck effect and by genetic drift. This paper explores the effects of incorporating genetic drift into models of fixation probability in populations with periodic bottlenecks. To model the inherent stochasticity during the growth phase in these populations, we assume a Poisson distribution of offspring. An analytical solution is developed to calculate the fixation probability and a computer simulation is used to verify the results. We find that the fixation rate of a favourable mutant is significantly lower when genetic drift is considered; fixation probability is reduced by over 25% for realistic experimental protocols. Our method is valid for both weak and strong selection; since very large selection coefficients have been reported in the experimental literature, this is an important improvement over previous results.     

Mother-infant bonding and the evolution of mammalian social relationships

A wide variety of maternal, social and sexual bonding strategies have been described across mammalian species, including humans. Many of the neural and hormonal mechanisms that underpin the formation and maintenance of these bonds demonstrate a considerable degree of evolutionary conservation across a representative range of these species. However, there is also a considerable degree of diversity in both the way these mechanisms are activated and in the behavioural responses that result. In the majority of small-brained mammals (including rodents), the formation of a maternal or partner preference bond requires individual recognition by olfactory cues, activation of neural mechanisms concerned with social reward by these cues and gender-specific hormonal priming for behavioural output. With the evolutionary increase of neocortex seen in monkeys and apes, there has been a corresponding increase in the complexity of social relationships and bonding strategies together with a significant redundancy in hormonal priming for motivated behaviour. Olfactory recognition and olfactory inputs to areas of the brain concerned with social reward are downregulated and recognition is based on integration of multimodal sensory cues requiring an expanded neocortex, particularly the association cortex. This emancipation from olfactory and hormonal determinants of bonding has been succeeded by the increased importance of social learning that is necessitated by living in a complex social world and, especially in humans, a world that is dominated by cultural inheritance.     

Rank and relationships in the evolution of spoken language

If evolutionary benefits associated with language were predominantly referential, as many theorists assume, then there must have been a preparatory stage in which an 'appetite' for information, not evident in the other primates, developed. To date, no such stage has been demonstrated. The problem dissipates, however, if it is assumed that language emerged from a function more nearly shared with other primates. An obvious candidate is displaying. Here I argue that performative functions associated with oral sound-making provided initial pressures for vocal communication by promoting rank and relationships. These benefits, I suggest, facilitated conflict avoidance and resolution, collaboration, and reciprocal sharing of needed resources. By valuing the performative applications of language, which continue in modern humans, one can more easily derive speech from the social-vocal behaviours of non-human primates, providing greater continuity in accounts of linguistic evolution     

Directed enzyme evolution via small and effective neutral drift libraries

Small libraries for directed evolution can be obtained by neutral drifts that maintain the protein's original function, yielding highly polymorphic, stable and evolvable variants. We describe methods for preparing such libraries, using serum paraoxonase (PON1). An optimized GFP variant fused to PON1 reported levels of soluble, functional enzyme, enabling selection by flow cytometry and identification of enzyme variants exhibiting improved specific and total activities toward several substrates, including toxic organophosphates.     

Trends, stasis, and drift in the evolution of nematode vulva development

BACKGROUND: A surprising amount of developmental variation has been observed for otherwise highly conserved features, a phenomenon known as developmental system drift. Either stochastic processes (e.g., drift and absence of selection-independent constraints) or deterministic processes (e.g., selection or constraints) could be the predominate mechanism for the evolution of such variation. We tested whether evolutionary patterns of change were unbiased or biased, as predicted by the stochastic or deterministic hypotheses, respectively. As a model, we used the nematode vulva, a highly conserved, essential organ, the development of which has been intensively studied in the model systems Caenorhabditis elegans and Pristionchus pacificus. RESULTS: For 51 rhabditid species, we analyzed more than 40 characteristics of vulva development, including cell fates, fate induction, cell competence, division patterns, morphogenesis, and related aspects of gonad development. We then defined individual characters and plotted their evolution on a phylogeny inferred for 65 species from three nuclear gene sequences. This taxon-dense phylogeny provides for the first time a highly resolved picture of rhabditid evolution and allows the reconstruction of the number and directionality of changes in the vulva development characters. We found an astonishing amount of variation and an even larger number of evolutionary changes, suggesting a high degree of homoplasy (convergences and reversals). Surprisingly, only two characters showed unbiased evolution. Evolution of all other characters was biased. CONCLUSIONS: We propose that developmental evolution is primarily governed by selection and/or selection-independent constraints, not stochastic processes such as drift in unconstrained phenotypic space.     

Group selection, individual selection, and the evolution of genetic drift.

In a subdivided population, genetic drift affects variation between groups, and thus it can have an important effect on the outcome of evolution (Wright, 1978). The rate of genetic drift is determined, in part, by the behaviour of population members. This paper presents three mathematical models in which behavioural traits that affect the rate of genetic drift are allowed to coevolve with traits that are under selection at the group and individual levels. The results show that if group selection is strong relative to individual selection, then behavioural traits that enhance the rate of genetic drift will tend to increase in frequency. The strength of this effect depends, in part, on the way in which vacant sites are colonized.     

The evolution of sex: empirical insights into the roles of epistasis and drift

Despite many years of theoretical and experimental work, the explanation for why sex is so common as a reproductive strategy continues to resist understanding. Recent empirical work has addressed key questions in this field, especially regarding rates of mutation accumulation in sexual and asexual organisms, and the roles of negative epistasis and drift as sources of adaptive constraint in asexually reproducing organisms. At the same time, new ideas about the evolution of sexual recombination are being tested, including intriguing suggestions of an important interplay between sex and genetic architecture, which indicate that sex and recombination could have affected their own evolution.     

Genetic defects in fatty acid beta-oxidation and acyl-CoA dehydrogenases. Molecular pathogenesis and genotype-phenotype relationships.

Mitochondrial fatty acid oxidation deficiencies are due to genetic defects in enzymes of fatty acid beta-oxidation and transport proteins. Genetic defects have been identified in most of the genes where nearly all types of sequence variations (mutation types) have been associated with disease. In this paper, we will discuss the effects of the various types of sequence variations encountered and review current knowledge regarding the genotype-phenotype relationship, especially in patients with acyl-CoA dehydrogenase deficiencies where sufficient material exists for a meaningful discussion. Because mis-sense sequence variations are prevalent in these diseases, we will discuss the implications of these types of sequence variations on the processing and folding of mis-sense variant proteins. As the prevalent mis-sense variant K304E MCAD protein has been studied intensively, the investigations on biogenesis, stability and kinetic properties for this variant enzyme will be discussed in detail and used as a paradigm for the study of other mis-sense variant proteins. We conclude that the total effect of mis-sense sequence variations may comprise an invariable--sequence variation specific--effect on the catalytic parameters and a conditional effect, which is dependent on cellular, physiological and genetic factors other than the sequence variation itself.     

High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative genomic hybridization

We have used array comparative genomic hybridization to map DNA copy-number changes in 94 patients with cri du chat syndrome who had been carefully evaluated for the presence of the characteristic cry, speech delay, facial dysmorphology, and level of mental retardation (MR). Most subjects had simple deletions involving 5p (67 terminal and 12 interstitial). Genotype-phenotype correlations localized the region associated with the cry to 1.5 Mb in distal 5p15.31, between bacterial artificial chromosomes (BACs) containing markers D5S2054 and D5S676; speech delay to 3.2 Mb in 5p15.32-15.33, between BACs containing D5S417 and D5S635; and the region associated with facial dysmorphology to 2.4 Mb in 5p15.2-15.31, between BACs containing D5S208 and D5S2887. These results overlap and refine those reported in previous publications. MR depended approximately on the 5p deletion size and location, but there were many cases in which the retardation was disproportionately severe, given the 5p deletion. All 15 of these cases, approximately two-thirds of the severely retarded patients, were found to have copy-number aberrations in addition to the 5p deletion. Restriction of consideration to patients with only 5p deletions clarified the effect of such deletions and suggested the presence of three regions, MRI-III, with differing effect on retardation. Deletions including MRI, a 1.2-Mb region overlapping the previously defined cri du chat critical region but not including MRII and MRIII, produced a moderate level of retardation. Deletions restricted to MRII, located just proximal to MRI, produced a milder level of retardation, whereas deletions restricted to the still-more proximal MRIII produced no discernible phenotype. However, MR increased as deletions that included MRI extended progressively into MRII and MRIII, and MR became profound when all three regions were deleted.     

Sex ratio polymorphism: The impact of mutation and drift on evolution

This paper addresses the question, which sex ratio will evolve in a population that is subject to mutation and drift. The problem is analyzed using a simulation model as well as analytical methods. A detailed simulation model for the evolution of a population's allele distribution shows that for the sex ratio game a wide spectrum of different population states may evolve from on the one hand a monomorphic state with one predominant allele and with all other alleles suppressed by the forces of selection, to on the other hand a polymorphism determined by recurrent mutations. Which of these states will evolve depends on the population size, the mating system and the rate of mutations. For the sex ratio game the evolutionary stable strategy (ESS), as defined by evolutionary game theory, can only predict the population sex ratio but not the underlying stable population state. A comparison of different approaches to the problem shows that false predictions of the stable population states might result from two simplifying assumptions that are fairly common in evolutionary biology: a) it is assumed that mutations are rare events and there is never more than one mutant gene present in a population at any one time; b) a deterministic relationship is assumed between the fitness assigned to an individual's strategy and the individual's contribution to the gene pool of future generations.     

Cytogenetic evolution in the Triticinae: Homoeologous relationships

The subtribe Triticinae consists of five genera, namely Aegilops, Agropyron, Haynaldia, Secale and Triticum, all with x=7 as the basic number and possibly derived from a common ancestor (PP). The homoeologous relationships known to exist between individual chromosomes from different genomes of hexaploid wheat, are being extended to other members of the subtribe. The available work in the area of homoeologous relationships in the subtribe Triticinae has been reviewed in detail. Homoeologous relationships of one chromosome of each of the four species of Aegilops i.e. Ae. comosa, Ae. caudata, Ae. umbellulata and Ae. bicornis are now known. Similarly, in the genus Agrophyron, relationships of six chromosomes of A. clongatum (2n=70), two chromosomes of A. clongatum (2n=14) and of two chromosomes of A. intermedium are known. The genus Secale, for which the author's own work is also available, has been most extensively studied and relationships of at least five chromosomes of S. cereale are now known. No other species of Secale has been used in similar studies. In the genus Haynaldia, no chromosome could be shown to have any relationship with a wheat chromosome, perhaps due to low erossability with Triticum aestivum. The above work has been discussed and reviewed.     

Phylogenetic relationships of Neotropical honeycreepers and the evolution of feeding morphology

Phylogenetic relationships among Neotropical honeycreepers were studied by using cytochrome b sequence data. Although honeycreepers were once placed in their own family (Coerebidae), these species did not form a monophyletic group in any of the phylogenies in this study. Thus, our results indicate that nectar-feeding evolved independently multiple times among these birds. The Neotropical honeycreepers are best considered disparate members of a larger radiation of tanagers and finches, in which bill size and shape, and associated feeding behaviors have changed frequently to fill a variety of niches. Our phylogenies also provide specific taxonomic recommendations on the placement of each honeycreeper genus. The use of the taxon Coerebidae is no longer warranted given the well-supported relationships between different honeycreeper species and a variety of tanagers and finches.     

Protein evolution: structure-function relationships of the oncogene beta-catenin in the evolution of multicellular animals

Beta-catenin functions as a cytoskeletal linker protein in cadherin-mediated adhesion and as a signal mediator in wnt-signal transduction pathways. We use a novel integrative approach, combining evolutionary, genomic, and three-dimensional structural data to analyze and trace the structural and functional evolution of beta-catenin genes. This approach also enabled us to examine the effects of gene duplication on the structure and function of beta-catenin genes in Drosophila, C. elegans, and vertebrates. By sampling a large number of different taxa, we identified both ancestral and derived motifs and residues within the different regions of the beta-catenin proteins. Projecting amino acid substitutions onto the three- dimensional structure established for mouse beta-catenin, we identified specific domains that exhibit loss and gain of selective constraints during beta catenin evolution. Structural changes, changes in the amino acid substitution rate, and the appearance of novel functional domains in beta-catenin can be mapped to specific branches on the metazoan tree. Together, our analyses suggest that a single, beta-catenin gene fulfilled both adhesion and signaling functions in the last common ancestor of metazoans some 700 million years ago. In addition, gene duplications facilitated the evolution of beta-catenins with novel functions and allowed the evolution of multiple, single-function proteins (cell adhesion or wnt-signaling) from the ancestral, dual-function protein. Integrative methods such as those we have applied here, utilizing the 'natural experiments' present in animal diversity, can be employed to identify novel and shared functional motifs and residues in virtually any protein among the proteomes of model systems and humans.