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Whole genome-based phylogenetic analysis of free-living microorganisms.

A phylogenetic 'tree of life' has been constructed based on the observed presence and absence of families of protein-encoding genes observed in 11 complete genomes of free-living microorganisms. Past attempts to reconstruct the evolutionary relation-ships of microorganisms have been limited to sets of genes rather than complete genomes. Despite apparent rampant lateral gene transfer among microorganisms, these results indicate a single robust underlying evolutionary history for these organisms. Broadly, the tree produced is very similar to the small subunit rRNA tree although several additional phylogenetic relationships appear to be resolved, including the relationship of Archaeoglobus to the methanogens studied. This result is in contrast to notions that a robust phylogenetic reconstruction of microorganisms is impossible due to their genomes being composed of an incomprehensible amalgam of genes with complicated histories and suggests that this style of genome-wide phylogenetic analysis could become an important method for studying the ancient diversification of life on Earth. Analyses using informational and operational subsets of the genes showed that this 'tree of life' is not dependent on the phylogenetically more consistent informational genes.

Computational Biology↗

Limitations of relative apparent synapomorphy analysis (RASA) for measuring phylogenetic signal.

In this paper we use hypothetical and empirical data matrices to evaluate the ability of relative apparent synapomorphy analysis (RASA) to measure phylogenetic signal, select outgroups, and identify terminals subject to long-branch attraction. In all cases, except for equal character-state frequencies, RASA indicated extraordinarily high levels of phylogenetic information for hypothetical data matrices that are uninformative regarding relationships among the terminals. Yet, regardless of the number of characters or character-state frequencies, RASA failed to detect phylogenetic signal for hypothetical matrices with strong phylogenetic signal. In our empirical example, RASA indicated increasing phylogenetic signal for matrices for which the strict consensus of the most parsimonious trees is increasingly poorly resolved, clades are increasingly poorly supported, and for which many relationships are in conflict with more widely sampled analyses. RASA is an ineffective approach to identify outgroup terminal(s) with the most plesiomorphic character states for the ingroup. Our hypothetical example demonstrated that RASA preferred outgroup terminals with increasing numbers of convergent character states with ingroup terminals, and rejected the outgroup terminal with all plesiomorphic character states. Our empirical example demonstrated that RASA, in all three cases examined, selected an ingroup terminal, rather than an outgroup terminal, as the best outgroup. In no case was one of the two outgroup terminals even close to being considered the optimal outgroup by RASA. RASA is an ineffective means of identifying problematic long-branch terminals. In our hypothetical example, RASA indicated a terminal as being a problematic long-branch terminal in spite of the terminal being on a zero-length branch and having no possibility of undergoing long-branch attraction with another terminal. RASA also failed to identify actual problematic long-branch terminals that did undergo long-branch attraction, but only after following Lyons-Weiler and Hoelzer's (1997) three-step process to identify and remove terminals subject to long-branch attraction. We conclude that RASA should not be used for any of these purposes.

Algorithms↗

Bayesian model adequacy and choice in phylogenetics.

Bayesian inference is becoming a common statistical approach to phylogenetic estimation because, among other reasons, it allows for rapid analysis of large data sets with complex evolutionary models. Conveniently, Bayesian phylogenetic methods use currently available stochastic models of sequence evolution. However, as with other model-based approaches, the results of Bayesian inference are conditional on the assumed model of evolution: inadequate models (models that poorly fit the data) may result in erroneous inferences. In this article, I present a Bayesian phylogenetic method that evaluates the adequacy of evolutionary models using posterior predictive distributions. By evaluating a model's posterior predictive performance, an adequate model can be selected for a Bayesian phylogenetic study. Although I present a single test statistic that assesses the overall (global) performance of a phylogenetic model, a variety of test statistics can be tailored to evaluate specific features (local performance) of evolutionary models to identify sources failure. The method presented here, unlike the likelihood-ratio test and parametric bootstrap, accounts for uncertainty in the phylogeny and model parameters.

Animals↗

Molecular evolution of ependymin and the phylogenetic resolution of early divergences among euteleost fishes.

The rate and pattern of DNA evolution of ependymin, a single-copy gene coding for a highly expressed glycoprotein in the brain matrix of teleost fishes, is characterized and its phylogenetic utility for fish systematics is assessed. DNA sequences were determined from catfish, electric fish, and characiforms and compared with published ependymin sequences from cyprinids, salmon, pike, and herring. Among these groups, ependymin amino acid sequences were highly divergent (up to 60% sequence difference), but had surprisingly similar hydropathy profiles and invariant glycosylation sites, suggesting that functional properties of the proteins are conserved. Comparison of base composition at third codon positions and introns revealed AT-rich introns and GC-rich third codon positions, suggesting that the biased codon usage observed might not be due to mutational bias. Phylogenetic information content of third codon positions was surprisingly high and sufficient to recover the most basal nodes of the tree, in spite of the observation that pairwise distances (at third codon positions) were well above the presumed saturation level. This finding can be explained by the high proportion of phylogenetically informative nonsynonymous changes at third codon positions among these highly divergent proteins. Ependymin DNA sequences have established the first molecular evidence for the monophyly of a group containing salmonids and esociforms. In addition, ependymin suggests a sister group relationship of electric fish (Gymnotiformes) and Characiformes, constituting a significant departure from currently accepted classifications. However, relationships among characiform lineages were not completely resolved by ependymin sequences in spite of seemingly appropriate levels of variation among taxa and considerably low levels of homoplasy in the data (consistency index = 0.7). If the diversification of Characiformes took place in an "explosive" manner, over a relatively short period of time this pattern should also be observed using other phylogenetic markers. Poor conservation of ependymin's primary structure hinders the design of efficient primers for PCR that could be used in wide-ranging fish systematic studies. However, alternative methods like PCR amplification from cDNA used here should provide promising comparative sequence data for the resolution of phylogenetic relationships among other basal lineages of teleost fishes.

Amino Acid Sequence↗

Nuclear-encoded rDNA group I introns: origin and phylogenetic relationships of insertion site lineages in the green algae.

Group I introns are widespread in eukaryotic organelles and nuclear-encoded ribosomal DNAs (rDNAs). The green algae are particularly rich in rDNA group I introns. To better understand the origins and phylogenetic relationships of green algal nuclear-encoded small subunit rDNA group I introns, a secondary structure-based alignment was constructed with available intron sequences and 11 new subgroup ICI and three new subgroup IB3 intron sequences determined from members of the Trebouxiophyceae (common phycobiont components of lichen) and the Ulvophyceae. Phylogenetic analyses using a weighted maximum-parsimony method showed that most group I introns form distinct lineages defined by insertion sites within the SSU rDNA. The comparison of topologies defining the phylogenetic relationships of 12 members of the 1512 group I intron insertion site lineage (position relative to the E. coli SSU rDNA coding region) with that of the host cells (i.e., SSU rDNAs) that contain these introns provided insights into the possible origin, stability, loss, and lateral transfer of ICI group I introns. The phylogenetic data were consistent with a viral origin of the 1512 group I intron in the green algae. This intron appears to have originated, minimally, within the SSU rDNA of the common ancestor of the trebouxiophytes and has subsequently been vertically inherited within this algal lineage with loss of the intron in some taxa. The phylogenetic analyses also suggested that the 1512 intron was laterally transferred among later-diverging trebouxiophytes; these algal taxa may have coexisted in a developing lichen thallus, thus facilitating cell-to-cell contact and the lateral transfer. Comparison of available group I intron sequences from the nuclear-encoded SSU rDNA of phycobiont and mycobiont components of lichens demonstrated that these sequences have independent origins and are not the result of lateral transfer from one component to the other.

Base Sequence↗

The correlation of protein structure and evolution of a protein-coding gene: phylogenetic inference using cytochrome oxidase III.

Discriminating phylogenetic signal from noise in DNA sequence data is a difficult problem in phylogenetic inference at higher systematic levels. For protein-coding genes, noise at synonymous (silent) positions can be filtered by deleting entire codon positions or types of change at a codon position. This method is not appropriate for replacement sites, because changes at each site within a codon may not be independent. This research presents a method using information from protein structure to evaluate variation in replacement sites. Analysis of the correlation of amino acid variation with protein structure identified rapidly evolving codons in the COIII gene. In a series of phylogenetic analyses attempting to recover a known set of vertebrate relationships, downweighting these labile codons produced the most accurate results. Structural correlates of variable and invariant residues identified in this study can be used to increase the accuracy of models used for phylogenetic inference. Viewing amino acid variation within a phylogenetic framework provided insight into residue changes important in the secondary and tertiary structures of the molecule, changes that were correlated between pairs of neighboring residues or between residues in neighboring helices.

Amino Acid Substitution↗

Potentials and limitations of histone repeat sequences for phylogenetic reconstruction of Sophophora.

Simplified DNA sequence acquisition has provided many new data sets that are useful for phylogenetic reconstruction, including single- and multiple-copy nuclear and organellar genes. Although transcribed regions receive much attention, nontranscribed regions have recently been added to the repertoire of sequences suitable for phylogenetic studies, especially for closely related taxa. We evaluated the efficacy of a small portion of the histone repeat for phylogenetic reconstruction among Drosophila species. Histone repeats in invertebrates offer distinct advantages similar to those of widely used ribosomal repeats. First, the units are tandemly repeated and undergo concerted evolution. Second, histone repeats include both highly conserved coding and variable intergenic regions. This composition facilitates application of "universal" primers spanning potentially informative sites. We examined a small region of the histone repeat, including the intergenic spacer segments of coding regions from the divergently transcribed H2A and H2B histone genes. The spacer (about 230 bp) exists as a mosaic with highly conserved functional motifs interspersed with rapidly diverging regions; the former aid in alignment of the spacer. There are no ambiguities in alignment of coding regions. Coding and noncoding regions were analyzed together and separately for phylogenetic information. Parsimony, distance, and maximum-likelihood methods successfully retrieve the corroborated phylogeny for the taxa examined. This study demonstrates the resolving power of a small histone region which may now be added to the growing collection of phylogenetically useful DNA sequences.

Animals↗

An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes.

Elongation factor 1 alpha (EF-1 alpha) is a highly conserved ubiquitous protein involved in translation that has been suggested to have desirable properties for phylogenetic inference. To examine the utility of EF-1 alpha as a phylogenetic marker for eukaryotes, we studied three properties of EF-1 alpha trees: congruency with other phyogenetic markers, the impact of species sampling, and the degree of substitutional saturation occurring between taxa. Our analyses indicate that the EF-1 alpha tree is congruent with some other molecular phylogenies in identifying both the deepest branches and some recent relationships in the eukaryotic line of descent. However, the topology of the intermediate portion of the EF-1 alpha tree, occupied by most of the protist lineages, differs for different phylogenetic methods, and bootstrap values for branches are low. Most problematic in this region is the failure of all phylogenetic methods to resolve the monophyly of two higher-order protistan taxa, the Ciliophora and the Alveolata. JACKMONO analyses indicated that the impact of species sampling on bootstrap support for most internal nodes of the eukaryotic EF-1 alpha tree is extreme. Furthermore, a comparison of observed versus inferred numbers of substitutions indicates that multiple overlapping substitutions have occurred, especially on the branch separating the Eukaryota from the Archaebacteria, suggesting that the rooting of the eukaryotic tree on the diplomonad lineage should be treated with caution. Overall, these results suggest that the phylogenies obtained from EF-1 alpha are congruent with other molecular phylogenies in recovering the monophyly of groups such as the Metazoa, Fungi, Magnoliophyta, and Euglenozoa. However, the interrelationships between these and other protist lineages are not well resolved. This lack of resolution may result from the combined effects of poor taxonomic sampling, relatively few informative positions, large numbers of overlapping substitutions that obscure phylogenetic signal, and lineage-specific rate increases in the EF-1 alpha data set. It is also consistent with the nearly simultaneous diversification of major eukaryotic lineages implied by the "big-bang" hypothesis of eukaryote evolution.

Algorithms↗

Molecular evolution and phylogenetic utility of Wolbachia ftsZ and wsp gene sequences with special reference to the origin of male-killing.

A detailed assessment of the evolution and phylogenetic utility of two genes, ftsZ and wsp, was used to investigate the origin of male-killing Wolbachia, previously isolated from the ladybird Adalia bipunctata and the butterfly Acraea encedon. The analysis included almost all available sequences of B-group Wolbachia and two outgroup taxa and showed that (1) the two gene regions differ in phylogenetic utility, (2) sequence variation is here correlated with phylogenetic information content, (3) both genes show significant rate heterogeneity between lineages, (4) increased substitution rates are associated with homoplasy in the data, (5) wsp sequences of some taxa appear to be subject to positive selection, and (6) only a limited number of clades can be inferred with confidence due to either lack of phylogenetic information or the presence of homoplasy. With respect to the evolution of male-killing, the two genes nevertheless seemed to provide unbiased information. However, they consistently produce contradictory results. Current data therefore do not permit clarification of the origin of this behavior. In addition, A. bipunctata was found to be a host to two recently diverged strains of male-killing Wolbachia that showed increased substitution rates for both genes. Moreover, the wsp gene, which codes for an outer membrane protein, was found to be subject to positive selection in these taxa. These findings were postulated to be the product of high selection pressures due to antagonistic host-symbiont interactions in this ladybird species. In conclusion, our study demonstrates that the results of a detailed phylogenetic analysis, including characterization of the limitations of such an approach, can serve as a valuable basis for an understanding of the evolution of Wolbachia bacteria. Moreover, particular features of gene evolution, such as elevated substitution rates or the presence of positive selection, may provide information about the dynamics of Wolbachia-host associations.

Bacterial Outer Membrane Proteins↗

Mitochondrial DNA sequences and multiple data sets: a phylogenetic study of phytophagous beetles (Chrysomelidae: Ophraella).

This paper presents the phylogenetic infrastructure for an integrated historical and experimental study of host use evolution in the chrysomelid leaf beetle genus Ophraella. We report the collection of sequence data from the 16S ribosomal RNA (446 bp) and the cytochrome oxidase subunit I (420 bp) mitochondrial genes from 12 species of Ophraella and two outgroups. Sequence analysis revealed a strong A + T nucleotide bias, high interspecific COI sequence divergences (up to 21.4%) that greatly exceeded those for 16S (up to 5.9%), high intraspecific COI divergences (up to 3.8%), a dearth of amino acid substitutions in COI, and differing substitution patterns in ribosomal stems and loops. Intraspecific variation in COI haplotypes generally supported the genealogical coherence of Ophraella lineages, while suggesting two cases of paraphyletic species. Separate phylogenetic analyses of 16S and COI data sets yielded largely congruent trees. A combined 16S + COI analysis yielded a single shortest tree under maximum parsimony that was identical to trees provided by successive approximations, neighbor-joining, and maximum-likelihood methods. This topology proved robust to various forms of weighting and most nodes were highly supported (by bootstrap analysis). Separate parsimony analyses of mtDNA and previously collected morphological and electromorphic data sets revealed congruent estimates of all cladistic relationships except those within one clade. Analysis of the pooled data sets in a combined approach additionally provided support for the basal placement of two species from this clade, although the topology for the remaining species was weakly supported and incongruent with the mtDNA tree. Each data set contained significantly structured phylogenetic signal with respect to this clade, and data sets exhibited limited conflict (character incongruence) with each other. The combined data set, however, was found to lack phylogenetic signal. These observations may imply that pooling heterogeneously evolving classes of data obscured the phylogenetic signal in each, a potential limitation of the combined approach.

Algorithms↗

Phylogenetics of flowering plants based on combined analysis of plastid atpB and rbcL gene sequences.

Following (1) the large-scale molecular phylogeny of seed plants based on plastid rbcL gene sequences (published in 1993 by Chase et al., Ann. Missouri Bot. Gard. 80:528-580) and (2) the 18S nuclear phylogeny of flowering plants (published in 1997 by Soltis et al., Ann. Missouri Bot. Gard. 84:1-49), we present a phylogenetic analysis of flowering plants based on a second plastid gene, atpB, analyzed separately and in combination with rbcL sequences for 357 taxa. Despite some discrepancies, the atpB-based phylogenetic trees were highly congruent with those derived from the analysis of rbcL and 18S rDNA, and the combination of atpB and rbcL DNA sequences (comprising approximately 3000 base pairs) produced increased bootstrap support for many major sets of taxa. The angiosperms are divided into two major groups: noneudicots with inaperturate or uniaperturate pollen (monocots plus Laurales, Magnoliales, Piperales, Ceratophyllales, and Amborellaceae-Nymphaeaceae-Illiciaceae) and the eudicots with triaperturate pollen (particularly asterids and rosids). Based on rbcL alone and atpB/rbcL combined, the noneudicots (excluding Ceratophyllum) are monophyletic, whereas in the atpB trees they form a grade. Ceratophyllum is sister to the rest of angiosperms with rbcL alone and in the combined atpB/rbcL analysis, whereas with atpB alone, Amborellaceae, Nymphaeaceae, and Illiciaceae/Schisandraceae form a grade at the base of the angiosperms. The phylogenetic information at each codon position and the different types of substitutions (observed transitions and transversions in the trees vs. pairwise comparisons) were examined; taking into account their respective consistency and retention indices, we demonstrate that third-codon positions and transitions are the most useful characters in these phylogenetic reconstructions. This study further demonstrates that phylogenetic analysis of large matrices is feasible.

Base Sequence↗

Phylogenetic relationships of marine bacteria, mainly members of the family Vibrionaceae, determined on the basis of 16S rRNA sequences.

The phylogenetic relationships of 50 reference strains, mostly marine bacteria which require Na+ for growth, were determined on the basis of 600 16S rRNA nucleotides by using reverse transcriptase sequencing. Strains belonging to 10 genera were included (four genera of the family Vibrionaceae, the genus Aeromonas of the family Aeromonadaceae, and the genera Alteromonas, Marinomonas, Shewanella, Pseudomonas, and Deleya). The sequences were aligned, the similarity values and evolutionary distance values were determined, and a phylogenetic tree was constructed by using the neighbor-joining method. On the basis of our results, the family Vibrionaceae was separated into at least seven groups (genera and families). Vibrio marinus clearly was on a line of descent that was remote from other vibrios. As determined by the similarity and evolutionary distance values, V. marinus is more distantly related to the family Vibrionaceae than the members of the Aeromonadaceae are. Also, Vibrio cholerae strains formed a separate group with Vibrio mimicus at the genus level. Of 30 species of the Vibrionaceae, 17 formed a large phylogenetic cluster. The genus Listonella was found to be a heterogeneous group, and the species were distributed in various subgroups of the Vibrionaceae. The separation of the family Aeromonadaceae from the family Vibrionaceae and the separation of the genera Marinomonas and Shewanella from the genus Alteromonas were confirmed in this phylogenetic study. However, a marine Pseudomonas species, Pseudomonas nautica, was clearly separated from two terrestrial Pseudomonas species. Each group that was separated by the phylogenetic analysis had characteristic 16S rRNA sequence patterns that were common only to species in that group. Therefore, the characteristic sequences described in this paper may be useful for identification purposes.

Base Sequence↗

Phylogenetic and physiological diversity of Arthrobacter strains isolated from unconsolidated subsurface sediments.

Forty strains of Gram-positive, aerobic, heterotrophic bacteria isolated from saturated subsurface lacustrine, paleosol and fluvial sediments at the US Department of Energy's Hanford Site in south central Washington State were characterized by phylogenetic analysis of 16S rRNA gene sequences and by determination of selected morphological, physiological and biochemical traits. Phylogenetic analyses of 16S rDNA sequences from subsurface isolates in the context of similar sequences from previously described bacterial species indicated that 38 of the subsurface strains were most closely related to Arthrobacter: The other two strains appeared to be most closely related to Kocuria. The subsurface isolates fell into seven phylogenetically coherent and distinct clusters, indicating that there was a significant degree of diversity among them. Additional diversity was detected by analysis of cellular fatty acids and physiological traits. The general morphological, physiological and biochemical traits of the subsurface strains were consistent with those of Arthrobacter, Micrococcus and genera recently separated from Micrococcus, such as Kocuria. Some of the subsurface strains were phylogenetically closely related to certain species of Arthrobacter. (16S rDNA sequence similarities >99%). However, most of the subsurface isolates did not cluster with previously established species in phylogenetic analyses of 16S rRNA gene sequences or with hierarchical cluster analysis of cellular fatty acid profiles. Moreover, many of the subsurface isolates that were most closely related to Arthrobacter. also differed from all established species of that genus in several of their specific physiological characteristics. Most of the subsurface isolates, then, are likely to be novel strains or species of Arthrobacter.

Arthrobacter↗

Phylogenetic taxonomy of the family Chlorobiaceae on the basis of 16S rRNA and fmo (Fenna-Matthews-Olson protein) gene sequences.

A new taxonomy of the green sulfur bacteria is proposed, based on phylogenetic relationships determined using the sequences of the independent 16S rRNA and fmo (Fenna-Matthews-Olson protein) genes, and supported by the DNA G + C content and sequence signatures. Comparison of the traditional classification system for these bacteria with their phylogenetic relationship yielded a confusing picture, because properties used for classification (such as cell morphology, photosynthetic pigments and substrate utilization) do not concur with their phylogeny. Using the genetic information available, strains and species assigned to the genera Chlorobium, Pelodictyon and Prosthecochloris are considered, and the following changes are proposed. Pelodictyon luteolum is transferred to the genus Chlorobium as Chlorobium luteolum comb. nov. Pelodictyon clathratiforme and Pelodictyon phaeoclathratiforme are transferred to the genus Chlorobium and combined into one species, Chlorobium clathratiforme comb. nov. The name Pelodictyon will become a synonym of Chlorobium. Strains known as Chlorobium limicola subsp. thiosulfatophilum that have a low DNA G + C content (52-52.5 mol%) are treated as strains of Chlorobium limicola; those with a high DNA G + C content (58.1 mol%) are transferred to Chlorobaculum gen. nov., as Chlorobaculum thiosulfatiphilum sp. nov. Chlorobium tepidum is transferred to Chlorobaculum tepidum comb. nov., and defined as the type species of the genus Chlorobaculum. Strains assigned to Chlorobium phaeobacteroides, but phylogenetically distant from the type strain of this species, are assigned to Chlorobium limicola and to Chlorobaculum limnaeum sp. nov. Strains known as Chlorobium vibrioforme subsp. thiosulfatophilum are transferred to Chlorobaculum parvum sp. nov. Chlorobium chlorovibrioides is transferred to 'Chlorobaculum chlorovibrioides' comb. nov. The type strain of Chlorobium vibrioforme is phylogenetically related to Prosthecochloris, and is therefore transferred to this genus as Prosthecochloris vibrioformis comb. nov. Consequently, the name Chlorobium vibrioforme will become a synonym of Prosthecochloris vibrioformis, and other strains that were assigned to this species are now considered to belong to Chlorobium luteolum, Chlorobium phaeovibrioides and 'Chlorobaculum chlorovibrioides', according to their phylogenetic relatedness.

Bacterial Proteins↗

Phylogenetic analysis of correlation structure in stalk-eyed flies (Diasemopsis, Diopsidae).

Morphological divergence among species may be constrained by the pattern of genetic variances and covariances among traits within species. Assessing the existence of such a relationship in nature requires information on the stability of intraspecific correlation and covariance structure and the correspondence of this structure to the pattern of evolutionary divergence within a lineage. Here, we investigate these issues for nine morphological traits and 15 species of stalk-eyed flies in the genus Diasemopsis. Within-species matrices for these traits were generated from phenotypic data for all the Diasemopsis species and from genetic data for a single Diasemopsis species, D. dubia. The among-species pattern of divergence was assessed by calculating the evolutionary correlations for all pairwise combinations of the morphological traits along the phylogeny of these species. Comparisons of intraspecific matrices reveal significant similarity among all species in the phenotypic correlations matrices but not the covariance matrices. In addition, the differences in correlation structure that do exist among species are not related to their phylogenetic placement or change in the means of the traits. Comparisons of the phenotypic and phylogenetic matrices suggest a strong relationship between the pattern of evolutionary change among species and both the intraspecific correlation structure and the stability of this structure among species. The phenotypic and the phylogenetic matrices are significantly similar, and pairs of traits whose intraspecific correlations are more stable across taxa exhibit stronger coevolution on the phylogeny. These results suggest either the existence of strong constraints on the pattern of evolutionary change or a consistent pattern of correlated selection shaping both the phenotypic and phylogenetic matrices. The genetic correlation structure for D. dubia, however, does not correspond with patterns found in the phenotypic and phylogenetic data. Possible reasons for this disagreement are discussed.

Animals↗

Orthonormal transform to decompose the variance of a life-history trait across a phylogenetic tree.

In recent years, there has been an increased interest in studying the variability of a quantitative life-history trait across a set of species sharing a common phylogeny. However, such studies have suffered from an insufficient development of statistical methods aimed at decomposing the trait variance with respect to the topological structure of the tree. Here we propose a new and generic approach that expresses the topological properties of the phylogenetic tree via an orthonormal basis, which is further used to decompose the trait variance. Such a decomposition provides a structure function, referred to as an "orthogram," which is relevant to characterize in both graphical and statistical aspects the dependence of trait values on the topology of the tree ("phylogenetic dependence"). We also propose four complementary test statistics to be computed from orthogram values that help to diagnose both the intensity and the nature of phylogenetic dependence. The relevance of the method is illustrated by the analysis of three phylogenetic data sets, drawn from the literature and typifying contrasted levels and aspects of phylogenetic dependence. Freely available routines which have been programmed in the R framework are also proposed.

Analysis of Variance↗

Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli.

Repetitive-element PCR (rep-PCR) fingerprinting is a promising molecular typing tool for Escherichia coli, including for discriminating between pathogenic and nonpathogenic clones, but is plagued by irreproducibility. Using the ERIC2 and BOXA1R primers and 15 E. coli strains from the ECOR reference collection (three from each phylogenetic group, as defined by multilocus enzyme electrophoresis [MLEE], including virulence-associated group B2), we rigorously assessed the effect of extremely elevated annealing temperatures on rep-PCR's reproducibility, discriminating power, and ability to reveal MLEE-defined phylogenetic relationships. Modified cycling conditions significantly improved assay reproducibility and discriminating power, allowing fingerprints from different cyclers to be analyzed together with minimal loss of resolution. The correspondence of rep-PCR with MLEE with respect to tree structure and regression analysis of distances was substantially better with modified than with standard cycling conditions. Nonetheless, rep-PCR was only a fair surrogate for MLEE, and when fingerprints from different days were compared, it failed to distinguish between different clones within all-important phylogenetic group B2. These findings indicate that although the performance and phylogenetic fidelity of rep-PCR fingerprinting can be improved substantially with modified assay conditions, even when so improved rep-PCR cannot fully substitute for MLEE as a phylogenetic typing method for pathogenic E. coli.

DNA Fingerprinting↗

Phylogenetic analysis of anaerobic thermophilic bacteria: aid for their reclassification.

Small subunit rDNA sequences were determined for 20 species of the genera Acetogenium, Clostridium, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobium, and Thermobacteroides, 3 non-validly described species, and 5 isolates of anaerobic thermophilic bacteria, providing a basis for a phylogenetic analysis of these organisms. Several species contain a version of the molecule significantly longer than that of Escherichia coli because of the presence of inserts. On the basis of normal evolutionary distances, the phylogenetic tree indicates that all bacteria investigated in this study with a maximum growth temperature above 65 degrees C form a supercluster within the subphylum of gram-positive bacteria that also contains Clostridium thermosaccharolyticum and Clostridium thermoaceticum, which have been previously sequenced. This supercluster appears to be equivalent in its phylogenetic depth to the supercluster of mesophilic clostridia and their nonspore-forming relatives. Several phylogenetically and phenotypically coherent clusters that are defined by sets of signature nucleotides emerge within the supercluster of thermophiles. Clostridium thermobutyricum and Clostridium thermopalmarium are members of Clostridium group I. A phylogenetic tree derived from transversion distances demonstrated the artificial clustering of some organisms with high rDNA G+C moles percent, i.e., Clostridium fervidus and the thermophilic, cellulolytic members of the genus Clostridium. The results of this study can be used as an aid for future taxonomic restructuring of anaerobic sporogenous and asporogenous thermophillic, gram-positive bacteria.

Bacteria, Anaerobic↗