A phylogenomic approach to microbial evolution

To study the origin and evolution of biochemical pathways in microorganisms, we have developed methods and software for automatic, large-scale reconstructions of phylogenetic relationships. We define the complete set of phylogenetic trees derived from the proteome of an organism as the phylome and introduce the term phylogenetic connection as a concept that describes the relative relationships between taxa in a tree. A query system has been incorporated into the system so as to allow searches for defined categories of trees within the phylome. As a complement, we have developed the pyphy system for visualising the results of complex queries on phylogenetic connections, genomic locations and functional assignments in a graphical format. Our phylogenomics approach, which links phylogenetic information to the flow of biochemical pathways within and among microbial species, has been used to examine more than 8000 phylogenetic trees from seven microbial genomes. The results have revealed a rich web of phylogenetic connections. However, the separation of Bacteria and Archaea into two separate domains remains robust.     

POPE--a tool to aid high-throughput phylogenetic analysis

POPE (Phylogeny, Ortholog and Paralog Extractor) provides an integrated platform for automatic ortholog identification. Intermediate steps can be visualized, modified and analyzed in order to assess and improve the underlying quality of orthology and paralogy assignments. AVAILABILITY: POPE is available for download from the website: http://www.well.ox.ac.uk/~tota/pope.     

Assessment of phylogenomic and orthology approaches for phylogenetic inference

MOTIVATION: Phylogenomics integrates the vast amount of phylogenetic information contained in complete genome sequences, and is rapidly becoming the standard for reliably inferring species phylogenies. There are, however, fundamental differences between the ways in which phylogenomic approaches like gene content, superalignment, superdistance and supertree integrate the phylogenetic information from separate orthologous groups. Furthermore, they all depend on the method by which the orthologous groups are initially determined. Here, we systematically compare these four phylogenomic approaches, in parallel with three approaches for large-scale orthology determination: pairwise orthology, cluster orthology and tree-based orthology. RESULTS: Including various phylogenetic methods, we apply a total of 54 fully automated phylogenomic procedures to the fungi, the eukaryotic clade with the largest number of sequenced genomes, for which we retrieved a golden standard phylogeny from the literature. Phylogenomic trees based on gene content show, relative to the other methods, a bias in the tree topology that parallels convergence in lifestyle among the species compared, indicating convergence in gene content. CONCLUSIONS: Complete genomes are no guarantee for good or even consistent phylogenies. However, the large amounts of data in genomes enable us to carefully select the data most suitable for phylogenomic inference. In terms of performance, the superalignment approach, combined with restrictive orthology, is the most successful in recovering a fungal phylogeny that agrees with current taxonomic views, and allows us to obtain a high-resolution phylogeny. We provide solid support for what has grown to be a common practice in phylogenomics during its advance in recent years. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

The average common substring approach to phylogenomic reconstruction.

We describe a novel method for efficient reconstruction of phylogenetic trees, based on sequences of whole genomes or proteomes, whose lengths may greatly vary. The core of our method is a new measure of pairwise distances between sequences. This measure is based on computing the average lengths of maximum common substrings, which is intrinsically related to information theoretic tools (Kullback-Leibler relative entropy). We present an algorithm for efficiently computing these distances. In principle, the distance of two l long sequences can be calculated in O(l) time. We implemented the algorithm using suffix arrays our implementation is fast enough to enable the construction of the proteome phylogenomic tree for hundreds of species and the genome phylogenomic forest for almost two thousand viruses. An initial analysis of the results exhibits a remarkable agreement with "acceptable phylogenetic and taxonomic truth." To assess our approach, our results were compared to the traditional (single-gene or protein-based) maximum likelihood method. The obtained trees were compared to implementations of a number of alternative approaches, including two that were previously published in the literature, and to the published results of a third approach. Comparing their outcome and running time to ours, using a "traditional" trees and a standard tree comparison method, our algorithm improved upon the "competition" by a substantial margin. The simplicity and speed of our method allows for a whole genome analysis with the greatest scope attempted so far. We describe here five different applications of the method, which not only show the validity of the method, but also suggest a number of novel phylogenetic insights.     

The primary divisions of life: a phylogenomic approach employing composition-heterogeneous methods

The three-domains tree, which depicts eukaryotes and archaebacteria as monophyletic sister groups, is the dominant model for early eukaryotic evolution. By contrast, the ‘eocyte hypothesis’, where eukaryotes are proposed to have originated from within the archaebacteria as sister to the Crenarchaeota (also called the eocytes), has been largely neglected in the literature. We have investigated support for these two competing hypotheses from molecular sequence data using methods that attempt to accommodate the across-site compositional heterogeneity and across-tree compositional and rate matrix heterogeneity that are manifest features of these data. When ribosomal RNA genes were analysed using standard methods that do not adequately model these kinds of heterogeneity, the three-domains tree was supported. However, this support was eroded or lost when composition-heterogeneous models were used, with concomitant increase in support for the eocyte tree for eukaryotic origins. Analysis of combined amino acid sequences from 41 protein-coding genes supported the eocyte tree, whether or not composition-heterogeneous models were used. The possible effects of substitutional saturation of our data were examined using simulation; these results suggested that saturation is delayed by among-site rate variation in the sequences, and that phylogenetic signal for ancient relationships is plausibly present in these data.     

On a functional-morphological approach to phylogenetic reconstruction: A critique

A method of phylogenetic reconstruction as proposed by a number of scientists of the Senckenberg Research Institute is discussed. The method is based on functional-morphological studies, the evolutionary adaptation principle of Bock and Von Wahlert (1965) and so-called model reconstruction. It is argued in this paper that direction of the adaptation process cannot be determined because of lack of knowledge about particular selective forces and that theories of model reconstruction are not open to contradiction in the sense of Popperian falsification. Although it has been claimed that the method provides the only valid directional argument for morphoclines in cladistic studies, it remains unclear how to proceed when morphoclines show contradictory polarities. Moreover, it is doubtful whether polarities of morphoclines can be determined independently of phylogenetic hypotheses, and also whether the use of multistate morphoclines is methodologically valid. By relying on a particular evolutionary theory, i.e. the neo-Darwinian theory, and consequently assigning natural selection as the major agent of directional progress, the Senckenburg method of phylogenetic reconstruction restricts itself to microevolutionary change and, therefore, cannot be used when other hypotheses on the evolutionary process appear to explain the speciation process more plausibly, i.e. hypotheses on macroevolution. Furthermore, it is an unproved statement that evolution always proceeds according to the principle of economy.     

Birds perching on bushes: Networks to visualize conflicting phylogenetic signals during early avian radiation

Hybridization is increasingly seen as an important source of adaptive genetic variation and biotic diversity. Recent phylogenetic studies on the early evolution of birds suggest that the early diversification of neoavian orders perhaps involved a period of extensive hybridization or incomplete lineage sorting. Phylogenetic error, saturation, long-branch attraction, and convergence make it difficult to detect ancient hybridization events and differentiate them from incomplete lineage sorting using sequence data. We used recently published retroposon marker data to visualize the early radiation of Neoaves within a phylogenetic network approach, and found that the most basal neoavian taxa indeed show a complex pattern of reticulated relationships. Moreover, the reticulation levels of different parts of the network are consistent with the insertion pattern of the retroposon elements. The use of network-based analyses on homoplasy-free data shows true conflicting signals and the taxa involved that are not represented in trees.     

Identification of the mammalian Not gene via a phylogenomic approach

Despite the great morphological diversity of early embryos, the underlying mechanisms of gastrulation are known to be broadly conserved in vertebrates. However, a number of genes characterized as fulfilling an essential function in this process in several model organisms display no clear ortholog in mammalian genomes. We have devised an in silico phylogenomic approach, based on exhaustive similarity searches in vertebrate genomes and subsequent bayesian phylogenetic analyses, to identify such missing genes, presumed to be highly divergent. This approach has been used to identify mammalian orthologs of Not, an homeodomain containing gene previously characterized in Xenopus, chick and zebrafish as playing a critical role in the formation of the notochord. This attempt led to the identification of a highly divergent mammalian Not-related gene in the mouse, human and rat. The results from phylogenetic reconstructions, synteny analyses, expression pattern analyses in wild-type and mutant mouse embryos, and overexpression experiments in Xenopus embryos converge to confirm these genes as representatives of the Not family in mammals. The identification of the mammalian Not gene delivers an important component for the understanding of the genetics underlying notochord formation in mammals and its evolution among vertebrates. The phylogenomic method used to retrieve this gene thus provides a tool, which can complement or validate genome annotations in situations when they are weakly supported.     

A streamlined approach to high-throughput proteomics

Proteomics has rapidly become an important tool for life science research, allowing the integrated analysis of global protein expression from a single experiment. To accommodate the complexity and dynamic nature of any proteome, researchers must use a combination of disparate protein biochemistry techniques, often a highly involved and time-consuming process. Whilst highly sophisticated, individual technologies for each step in studying a proteome are available, true high-throughput proteomics that provides a high degree of reproducibility and sensitivity has been difficult to achieve. The development of high-throughput proteomic platforms, encompassing all aspects of proteome analysis and integrated with genomics and bioinformatics technology, therefore represents a crucial step for the advancement of proteomics research. ProteomIQ (Proteome Systems) is the first fully integrated, start-to-finish proteomics platform to enter the market. Sample preparation and tracking, centralized data acquisition and instrument control, and direct interfacing with genomics and bioinformatics databases are combined into a single suite of integrated hardware and software tools, facilitating high reproducibility and rapid turnaround times. This review will highlight some features of ProteomIQ, with particular emphasis on the analysis of proteins separated by 2D polyacrylamide gel electrophoresis.     

A streamlined approach to high-throughput proteomics

Proteomics has rapidly become an important tool for life science research, allowing the integrated analysis of global protein expression from a single experiment. To accommodate the complexity and dynamic nature of any proteome, researchers must use a combination of disparate protein biochemistry techniques, often a highly involved and time-consuming process. Whilst highly sophisticated, individual technologies for each step in studying a proteome are available, true high-throughput proteomics that provides a high degree of reproducibility and sensitivity has been difficult to achieve. The development of high-throughput proteomic platforms, encompassing all aspects of proteome analysis and integrated with genomics and bioinformatics technology, therefore represents a crucial step for the advancement of proteomics research. ProteomIQ? (Proteome Systems) is the first fully integrated, start-to-finish proteomics platform to enter the market. Sample preparation and tracking, centralized data acquisition and instrument control, and direct interfacing with genomics and bioinformatics databases are combined into a single suite of integrated hardware and software tools, facilitating high reproducibility and rapid turnaround times. This review will highlight some features of ProteomIQ, with particular emphasis on the analysis of proteins separated by 2D polyacrylamide gel electrophoresis.     

Lifeact: a versatile marker to visualize F-actin

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.     

Colorimetric approach to high-throughput mutation analysis

High-throughput genomic mutation screening for primary tumors has characteristically been expensive, labor-intensive, and inadequate to detect low levels of mutation in a background of wild-type signal. We present a new, combined PCR and colorimetric approach that is inexpensive, simple, and can detect the presence of 1% mutation in a background of wild-type. We compared manual dideoxy sequencing of p53 for eight lung cancer samples to a novel assay combining a primer extension step and an enzymatic colorimetric step in a 96-well plate with covalently attached oligonucleotide sequences. For every sample, we were able to detect the presence or absence of the specific mutation with a statistically significant difference between the sample optical density (OD) and the background OD, with a sensitivity and specificity of 100%. This assay is straightforward, accurate, inexpensive, and allows for rapid, high-throughput analysis of samples, making it ideal for genomic mutation or polymorphism screening studies in both clinical and research settings.     

The nucleotide-sugar transporter family: a phylogenetic approach

Nucleotide sugar transporters (NST) establish the functional link of membrane transport between the nucleotide sugars synthesized in the cytoplasm and nucleus, and the glycosylation processes that take place in the endoplasmic reticulum (ER) and Golgi apparatus. The aim of the present work was to perform a phylogenetic analysis of 87 bank annotated protein sequences comprising all the NST so far characterized and their homologues retrieved by BLAST searches, as well as the closely related triose-phosphate translocator (TPT) plant family. NST were classified in three comprehensive families by linking them to the available experimental data. This enabled us to point out both the possible ER subcellular targeting of these transporters mediated by the dy-lysine motif and the substrate recognition mechanisms specific to each family as well as an important acceptor site motif, establishing the role of evolution in the functional properties of each NST family.     

Functional proteomics: a promising approach to find novel components of the circadian system

In the postgenome era, the analysis of entire subproteomes in correlation with their function has emerged due to high throughput technologies. Early approaches have been initiated to identify novel components of the circadian system. For example, in the marine dinoflagellate Lingulodinium polyedra, a chronobiological proteome assay was performed, which resulted in the identification of already known circadian expressed proteins as well as novel temporal controlled proteins involved in metabolic pathways. In the green alga Chlamydomonas reinhardtii, two circadian expressed proteins (a protein disulfide isomerase and a tetratricopeptide repeat protein) were identified by functional proteomics. Also, the first hints of temporal control within chloroplast proteins of Arabidopsis thaliana were identified by proteome analysis.     

The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach

The animal sialyltransferases are Golgi type II transmembrane glycosyltransferases. Twenty distinct sialyltransferases have been identified in both human and murine genomes. These enzymes catalyze transfer of sialic acid from CMP-Neu5Ac to the glycan moiety of glycoconjugates. Despite low overall identities, they share four conserved peptide motifs [L (large), S (small), motif III, and motif VS (very small)] that are hallmarks for sialyltransferase identification. We have identified 155 new putative genes in 25 animal species, and we have exploited two lines of evidence: (1) sequence comparisons and (2) exon-intron organization of the genes. An ortholog to the ancestor present before the split of ST6Gal I and II subfamilies was detected in arthropods. An ortholog to the ancestor present before the split of ST6GalNAc III, IV, V, and VI subfamilies was detected in sea urchin. An ortholog to the ancestor present before the split of ST3Gal I and II subfamilies was detected in ciona, and an ortholog to the ancestor of all the ST8Sia was detected in amphioxus. Therefore, single examples of the four families (ST3Gal, ST6Gal, ST6GalNAc, and ST8Sia) have appeared in invertebrates, earlier than previously thought, whereas the four families were all detected in bony fishes, amphibians, birds, and mammals. As previously hypothesized, sequence similarities among sialyltransferases suggest a common genetic origin, by successive duplications of an ancestral gene, followed by divergent evolution. Finally, we propose predictions on these invertebrates sialyltransferase-related activities that have not previously been demonstrated and that will ultimately need to be substantiated by protein expression and enzymatic activity assays.     

The evolution of peafowl and other taxa with ocelli (eyespots): a phylogenomic approach

The most striking feature of peafowl (Pavo) is the males'' elaborate train, which exhibits ocelli (ornamental eyespots) that are under sexual selection. Two additional genera within the Phasianidae (Polyplectron and Argusianus) exhibit ocelli, but the appearance and location of these ornamental eyespots exhibit substantial variation among these genera, raising the question of whether ocelli are homologous. Within Polyplectron, ocelli are ancestral, suggesting ocelli may have evolved even earlier, prior to the divergence among genera. However, it remains unclear whether Pavo, Polyplectron and Argusianus form a monophyletic clade in which ocelli evolved once. We estimated the phylogeny of the ocellated species using sequences from 1966 ultraconserved elements (UCEs) and three mitochondrial regions. The three ocellated genera did form a strongly supported clade, but each ocellated genus was sister to at least one genus without ocelli. Indeed, Polyplectron and Galloperdix, a genus not previously suggested to be related to any ocellated taxon, were sister genera. The close relationship between taxa with and without ocelli suggests multiple gains or losses. Independent gains, possibly reflecting a pre-existing bias for eye-like structures among females and/or the existence of a simple mutational pathway for the origin of ocelli, appears to be the most likely explanation.     

A phylogenetic approach to cultural evolution

There has been a rapid increase in the use of phylogenetic methods to study the evolution of languages and culture. Languages fit a tree model of evolution well, at least in their basic vocabulary, challenging the view that blending, or admixture among neighbouring groups, was predominant in cultural history. Here, we argue that we can use language trees to test hypotheses about not only cultural history and diversification, but also bio-cultural adaptation. Phylogenetic comparative methods take account of the non-independence of cultures (Galton's problem), which can cause spurious statistical associations in comparative analyses. Advances in phylogenetic methods offer new possibilities for the analysis of cultural evolution, including estimating the rate of evolution and the direction of coevolutionary change of traits on the tree. They also enable phylogenetic uncertainty to be incorporated into the analyses, so that one does not have to treat phylogenetic trees as if they were known without error.