The alignment of sets of sequences and the construction of phyletic trees: An integrated method

Summary In this paper we argue that the alignment of sets of sequences and the construction of phyletic trees cannot be treated separately. The concept of good alignment is meaningless without reference to a phyletic tree, and the construction of phyletic trees presupposes alignment of the sequences.We propose an integrated method that generates both an alignment of a set of sequences and a phyletic tree. In this method a putative tree is used to align the sequences and the alignment obtained is used to adjust the tree; this process is iterated. As a demonstration we apply the method to the analysis of the evolution of 5S rRNA sequences in prokaryotes.     

The evolution of stramenopiles and alveolates as derived by “substitution rate calibration» of small ribosomal subunit RNA

The substitution rate of the individual positions in an alignment of 750 eukaryotic small ribosomal subunit RNA sequences was estimated. From the resulting rate distribution, an equation was derived that gives a more precise relationship between sequence dissimilarity and evolutionary distance than hitherto available. Trees constructed on the basis of evolutionary distances computed by this new equation for small ribosomal subunit RNA sequences from ciliates, apicomplexans, dinoflagellates, oomycetes, hyphochytriomycetes, bicosoecids, labyrinthuloids, and heterokont algae show a more consistent tree topology than trees constructed in the absence of substitution rate calibration. In particular, they do not suffer from anomalies caused by the presence of extremely long branches.     

When is it safe to use an oversimplified substitution model in tree- making?

The choice of an "optimal" mathematical model for computing evolutionary distances from real sequences is not currently supported by easy-to-use software applicable to large data sets, and an investigator frequently selects one of the simplest models available. Here we study properties of the observed proportion of differences (p- distance) between sequences as an estimator of evolutionary distance for tree-making. We show that p-distances allow for consistent tree- making with any of the popular methods working with evolutionary distances if evolution of sequences obeys a "molecular clock" (more precisely, if it follows a stationary time-reversible Markov model of nucleotide substitution). Next, we show that p-distances seem to be efficient in recovering the correct tree topology under a "molecular clock," but produce "statistically supported" wrong trees when substitutions rates vary among evolutionary lineages. Finally, we outline a practical approach for selecting an "optimal" model of nucleotide substitution in a real data analysis, and obtain a crude estimate of a "prior" distribution of the expected tree branch lengths under the Jukes-Cantor model. We conclude that the use of a model that is obviously oversimplified is inadvisable unless it is justified by a preliminary analysis of the real sequences.      

Anchor-based whole genome phylogeny (ABWGP): a tool for inferring evolutionary relationship among closely related microorganisms [corrected

Phenotypic behavior of a group of organisms can be studied using a range of molecular evolutionary tools that help to determine evolutionary relationships. Traditionally a gene or a set of gene sequences was used for generating phylogenetic trees. Incomplete evolutionary information in few selected genes causes problems in phylogenetic tree construction. Whole genomes are used as remedy. Now, the task is to identify the suitable parameters to extract the hidden information from whole genome sequences that truly represent evolutionary information. In this study we explored a random anchor (a stretch of 100 nucleotides) based approach (ABWGP) for finding distance between any two genomes, and used the distance estimates to compute evolutionary trees. A number of strains and species of Mycobacteria were used for this study. Anchor-derived parameters, such as cumulative normalized score, anchor order and indels were computed in a pair-wise manner, and the scores were used to compute distance/phylogenetic trees. The strength of branching was determined by bootstrap analysis. The terminal branches are clearly discernable using the distance estimates described here. In general, different measures gave similar trees except the trees based on indels. Overall the tree topology reflected the known biology of the organisms. This was also true for different strains of Escherichia coli. A new whole genome-based approach has been described here for studying evolutionary relationships among bacterial strains and species.     

Techniques for determining protein sequence evolution applied to primates

Each amino acid in a protein is considered to be an individual, mutable characteristic of the species from which the protein is extracted. For a branching tree representing the evolutionary history of the known sequences in different species, our computer programs use majority logic and parsimony of mutations to determine the most likely ancestral amino acid for each position of the protein at each node of the tree. The number of mutations necessary between the ancestral and present species is summed for each branch and the entire tree. The programs then move branches to make many different configurations, from which we select the one with the minimum number of mutations as the most likely evolutionary history. We used this method to elucidate primate phylogeny from sequences of fibrinopeptides, carbonic anhydrase, and the hemoglobin beta, delta and alpha chains. All available sequences indicate that the early Pongidae had diverged into two lines before the divergence of an ancestor for the human line alone. We have constructed some probable ancestral sequences at major points during primate evolution and have developed tentative trees showing the order of divergences and evolutionary distances among primate groups. Further questions on primate evolution could be answered in the future by the detemination of the appropriate sequences.     

Anchor-Based Whole Genome Phylogeny (ABWGP): A Tool for Inferring Evolutionary Relationship among Closely Related Microorganims

Phenotypic behavior of a group of organisms can be studied using a range of molecular evolutionary tools that help to determine evolutionary relationships. Traditionally a gene or a set of gene sequences was used for generating phylogenetic trees. Incomplete evolutionary information in few selected genes causes problems in phylogenetic tree construction. Whole genomes are used as remedy. Now, the task is to identify the suitable parameters to extract the hidden information from whole genome sequences that truly represent evolutionary information. In this study we explored a random anchor (a stretch of 100 nucleotides) based approach (ABWGP) for finding distance between any two genomes, and used the distance estimates to compute evolutionary trees. A number of strains and species of Mycobacteria were used for this study. Anchor-derived parameters, such as cumulative normalized score, anchor order and indels were computed in a pair-wise manner, and the scores were used to compute distance/phylogenetic trees. The strength of branching was determined by bootstrap analysis. The terminal branches are clearly discernable using the distance estimates described here. In general, different measures gave similar trees except the trees based on indels. Overall the tree topology reflected the known biology of the organisms. This was also true for different strains of Escherichia coli. A new whole genome-based approach has been described here for studying evolutionary relationships among bacterial strains and species.     

Performance comparison between k-tuple distance and four model-based distances in phylogenetic tree reconstruction

Phylogenetic tree reconstruction requires construction of a multiple sequence alignment (MSA) from sequences. Computationally, it is difficult to achieve an optimal MSA for many sequences. Moreover, even if an optimal MSA is obtained, it may not be the true MSA that reflects the evolutionary history of the underlying sequences. Therefore, errors can be introduced during MSA construction which in turn affects the subsequent phylogenetic tree construction. In order to circumvent this issue, we extend the application of the k-tuple distance to phylogenetic tree reconstruction. The k-tuple distance between two sequences is the sum of the differences in frequency, over all possible tuples of length k, between the sequences and can be estimated without MSAs. It has been traditionally used to build a fast ‘guide tree’ to assist the construction of MSAs. Using the 1470 simulated sets of sequences generated under different evolutionary scenarios, the neighbor-joining trees and BioNJ trees, we compared the performance of the k-tuple distance with four commonly used distance estimators including Jukes–Cantor, Kimura, F84 and Tamura–Nei. These four distance estimators fall into the category of model-based distance estimators, as each of them takes account of a specific substitution model in order to compute the distance between a pair of already aligned sequences. Results show that trees constructed from the k-tuple distance are more accurate than those from other distances most time; when the divergence between underlying sequences is high, the tree accuracy could be twice or higher using the k-tuple distance than other estimators. Furthermore, as the k-tuple distance voids the need for constructing an MSA, it can save tremendous amount of time for phylogenetic tree reconstructions when the data include a large number of sequences.     

An evolutionary tree for invertebrate globin sequences

Summary A phylogenetic tree was constructed from 245 globin amino acid sequences. Of the six plant globins, five represented the Leguminosae and one the Ulmaceae. Among the invertebrate sequences, 7 represented the phylum Annelida, 13 represented Insecta and Crustacea of the phylum Arthropoda, and 6 represented the phylum Mollusca. Of the vertebrate globins, 4 represented the Agnatha and 209 represented the Gnathostomata. A common alignment was achieved for the 245 sequences using the parsimony principle, and a matrix of minimum mutational distances was constructed. The most parsimonious phylogenetic tree, i.e., the one having the lowest number of nucleotide substitutions that cause amino acid replacements, was obtained employing clustering and branch-swapping algorithms. Based on the available fossil record, the earliest split in the ancestral metazoan lineage was placed at 680 million years before present (Myr BP), the origin of vertebrates was placed at 510 Myr BP, and the separation of the Chondrichthyes and the Osteichthyes was placed at 425 Myr BP. Local molecular clock calculations were used to date the branch points on the descending branches of the various lineages within the plant and invertebrate portions of the tree. The tree divided the 245 sequences into five distinct clades that corresponded exactly to the five groups plants, annelids, arthropods, molluscs, and vertebrates. Furthermore, the maximum parsimony tree, in contrast to the unweighted pair group and distance Wagner trees, was consistent with the available fossil record and supported the hypotheses that the primitive hemoglobin of metazoans was monomeric and that the multisubunit extracellular hemoglobins found among the Annelida and the Arthropoda represent independently derived states.     

Evaluation measures of multiple sequence alignments.

Multiple sequence alignments (MSAs) are frequently used in the study of families of protein sequences or DNA/RNA sequences. They are a fundamental tool for the understanding of the structure, functionality and, ultimately, the evolution of proteins. A new algorithm, the Circular Sum (CS) method, is presented for formally evaluating the quality of an MSA. It is based on the use of a solution to the Traveling Salesman Problem, which identifies a circular tour through an evolutionary tree connecting the sequences in a protein family. With this approach, the calculation of an evolutionary tree and the errors that it would introduce can be avoided altogether. The algorithm gives an upper bound, the best score that can possibly be achieved by any MSA for a given set of protein sequences. Alternatively, if presented with a specific MSA, the algorithm provides a formal score for the MSA, which serves as an absolute measure of the quality of the MSA. The CS measure yields a direct connection between an MSA and the associated evolutionary tree. The measure can be used as a tool for evaluating different methods for producing MSAs. A brief example of the last application is provided. Because it weights all evolutionary events on a tree identically, but does not require the reconstruction of a tree, the CS algorithm has advantages over the frequently used sum-of-pairs measures for scoring MSAs, which weight some evolutionary events more strongly than others. Compared to other weighted sum-of-pairs measures, it has the advantage that no evolutionary tree must be constructed, because we can find a circular tour without knowing the tree.     

Distant horizontal gene transfer is rare for multiple families of prokaryotic insertion sequences

Horizontal gene transfer in prokaryotes is rampant on short and intermediate evolutionary time scales. It poses a fundamental problem to our ability to reconstruct the evolutionary tree of life. Is it also frequent over long evolutionary distances? To address this question, we analyzed the evolution of 2,091 insertion sequences from all 20 major families in 438 completely sequenced prokaryotic genomes. Specifically, we mapped insertion sequence occurrence on a 16S rDNA tree of the genomes we analyzed, and we also constructed phylogenetic trees of the insertion sequence transposase coding sequences. We found only 30 cases of likely horizontal transfer among distantly related prokaryotic clades. Most of these horizontal transfer events are ancient. Only seven events are recent. Almost all of these transfer events occur between pairs of human pathogens or commensals. If true also for other, non-mobile DNA, the rarity of distant horizontal transfer increases the odds of reliable phylogenetic inference from sequence data.     

A protein alignment scoring system sensitive at all evolutionary distances

Summary Protein sequence alignments generally are constructed with the aid of a substitution matrix that specifies a score for aligning each pair of amino acids. Assuming a simple random protein model, it can be shown that any such matrix, when used for evaluating variable-length local alignments, is implicitly a log-odds matrix, with a specific probability distribution for amino acid pairs to which it is uniquely tailored. Given a model of protein evolution from which such distributions may be derived, a substitution matrix adapted to detecting relationships at any chosen evolutionary distance can be constructed. Because in a database search it generally is not known a priori what evolutionary distances will characterize the similarities found, it is necessary to employ an appropriate range of matrices in order not to overlook potential homologies. This paper formalizes this concept by defining a scoring system that is sensitive at all detectable evolutionary distances. The statistical behavior of this scoring system is analyzed, and it is shown that for a typical protein database search, estimating the originally unknown evolutionary distance appropriate to each alignment costs slightly over two bits of information, or somewhat less than a factor of five in statistical significance. A much greater cost may be incurred, however, if only a single substitution matrix, corresponding to the wrong evolutionary distance, is employed.     

A measure of the denseness of a phylogenetic network

Summary The concept of phylogenetic denseness bears critically on the accuracy of evolutionary pathways inferred from experimentally sequenced proteins isolated from extant species. In this paper I develop an objective measure,, of denseness to supplement previous intuitive concepts and which permits one to use this concept in comparing the quality of different evolutionary reconstructions. This measure is used to examine several published phylogenetic trees: insulin, a-hemoglobin,-hemoglobin, myoglobin, cytochromec, and the parvalbumin family. The paper emphasizes 1) the importance of denseness in accurately estimating the number of nucleotide replacements which separate homologous sequences when this estimation is made by the method of parsimony, 2) the value of this concept in assessing the quality of those estimates, and 3) the use of this concept as a biologically practical heuristic method for identifying poorly studied regions in a phylogenetic tree, whether or not the tree was obtained by the parsimony method.     

Complete Mitochondrial Genomes and Eutherian Evolution

Recent large-scale nuclear DNA phylogenies have supported unconventional interordinal relationships among modern eutherians as well as divergence dates (100 mya) that substantially predate the first appearance of fossils from modern eutherians near the Cretaceous/Cenozoic (K/T) boundary (65-70 mya). For comparison to the nuclear data, I analyzed 12 complete mitochondrial DNA (mtDNA) protein-coding genes (10,677 bp) from 53 eutherian taxa, using maximum-likelihood methods to estimate model parameters (GTR + I + ) and to optimize topology and branch-length estimates. Although closely resembling the nuclear DNA trees, the mtDNA maximum-likelihood tree is just one of seven statistically indistinguishable ( lnL 1.747) trees, each suggesting different evolutionary relationships. This 53-taxon data set and another including 56 taxa provide no statistically significant support for a monophyletic afrotherian clade. In fact, these mitochondrial DNA sequences fail to support the monophyly of three putative eutherian divisions suggested by the nuclear data (Afrotheria, Laurasiatheria or Euarchontoglires). By comparison to well-supported branches describing relationships among families, those describing interordinal relationships are extremely short and only tenuously supported. Neither these sequences, nor sequences simulated under a known tree, fully resolve any interordinal relationship. Even simulated sequences that are twice as long (22kb) as mtDNA protein-coding genes are too short and too saturated to resolve the deepest and shortest interordinal relationships. Further, the mammalian mtDNA sequences appear to depart significantly from molecular-clock and quartet dating assumptions. Unlike recent nuclear DNA studies, I find that mtDNA genes, by themselves, are inadequate to describe relationships or divergence times at the base of the eutherian tree.     

Efficient biased estimation of evolutionary distances when substitution rates vary across sites

This paper deals with phylogenetic inference when the variability of substitution rates across sites (VRAS) is modeled by a gamma distribution. We show that underestimating VRAS, which results in underestimates for the evolutionary distances between sequences, usually improves the topological accuracy of phylogenetic tree inference by distance-based methods, especially when the molecular clock holds. We propose a method to estimate the gamma shape parameter value which is most suited for tree topology inference, given the sequences at hand. This method is based on the pairwise evolutionary distances between sequences and allows one to reconstruct the phylogeny of a high number of taxa (>1,000). Simulation results show that the topological accuracy is highly improved when using the gamma shape parameter value given by our method, compared with the true (unknown) value which was used to generate the data. Furthermore, when VRAS is high, the topological accuracy of our distance-based method is better than that of a maximum likelihood approach. Finally, a data set of Maoricicada species sequences is analyzed, which confirms the advantage of our method.     

STRUCLA: a WWW meta-server for protein structure comparison and evolutionary classification

MOTIVATION: Evolutionary relationships of proteins have long been derived from the alignment of protein sequences. But from the view of function, most restraints of evolutionary divergence operate at the level of tertiary structure. It has been demonstrated that quantitative measures of dissimilarity in families of structurally similar proteins can be applied to the construction of trees from a comparison of their three-dimensional structures. However, no convenient tool is publicly available to carry out such analyses. RESULTS: We developed STRUCLA (STRUcture CLAssification), a WWW tool for generation of trees based on evolutionary distances inferred from protein structures according to various methods. The server takes as an input a list of PDB files or the initial alignment of protein coordinates provided by the user (for instance exported from SWISS PDB VIEWER). The user specifies the distance cutoff and selects the distance measures. The server returns series of unrooted trees in the NEXUS format and corresponding distance matrices, as well as a consensus tree. The results can be used as an alternative and a complement to a fixed hierarchy of current protein structure databases. It can complement sequence-based phylogenetic analysis in the 'twilight zone of homology', where amino acid sequences are too diverged to provide reliable relationships.     

Reconstruction of Certain Phylogenetic Networks from Their Tree-Average Distances

Trees are commonly utilized to describe the evolutionary history of a collection of biological species, in which case the trees are called phylogenetic trees. Often these are reconstructed from data by making use of distances between extant species corresponding to the leaves of the tree. Because of increased recognition of the possibility of hybridization events, more attention is being given to the use of phylogenetic networks that are not necessarily trees. This paper describes the reconstruction of certain such networks from the tree-average distances between the leaves. For a certain class of phylogenetic networks, a polynomial-time method is presented to reconstruct the network from the tree-average distances. The method is proved to work if there is a single reticulation cycle.     

Median-joining networks for inferring intraspecific phylogenies.

Reconstructing phylogenies from intraspecific data (such as human mitochondrial DNA variation) is often a challenging task because of large sample sizes and small genetic distances between individuals. The resulting multitude of plausible trees is best expressed by a network which displays alternative potential evolutionary paths in the form of cycles. We present a method ("median joining" [MJ]) for constructing networks from recombination-free population data that combines features of Kruskal's algorithm for finding minimum spanning trees by favoring short connections, and Farris's maximum-parsimony (MP) heuristic algorithm, which sequentially adds new vertices called "median vectors", except that our MJ method does not resolve ties. The MJ method is hence closely related to the earlier approach of Foulds, Hendy, and Penny for estimating MP trees but can be adjusted to the level of homoplasy by setting a parameter epsilon. Unlike our earlier reduced median (RM) network method, MJ is applicable to multistate characters (e.g., amino acid sequences). An additional feature is the speed of the implemented algorithm: a sample of 800 worldwide mtDNA hypervariable segment I sequences requires less than 3 h on a Pentium 120 PC. The MJ method is demonstrated on a Tibetan mitochondrial DNA RFLP data set.     

The number of nucleotides required to determine the branching order of three species,with special reference to the human-chimpanzee-gorilla divergence

Summary A mathematical theory for computing the probabilities of various nucleotide configurations among related species is developed, and the probability of obtaining the correct tree (topology) from nucleotide sequence data is evaluated using models of evolutionary trees that are close to the tree of mitochondrial DNAs from human, chimpanzee, gorilla, orangutan, and gibbon. Special attention is given to the number of nucleotides required to resolve the branching order among the three most closely related organisms (human, chimpanzee, and gorilla). If the extent of DNA divergence is close to that obtained by Brown et al. for mitochondrial DNA and if sequence data are available only for the three most closely related organisms, the number of nucleotides (m^*) required to obtain the correct tree with a probability of 95% is about 4700. If sequence data for two outgroup species (orangutan and gibbon) are available, m^* becomes about 2600–2700 when the transformed distance, distance-Wagner, maximum parsimony, or compatibility method is used. In the unweighted pair-group method, m^* is not affected by the availability of data from outgroup species. When these five different tree-making methods, as well as Fitch and Margoliash's method, are applied to the mitochondrial DNA data (1834 bp) obtained by Brown et al. and by Hixson and Brown, they all give the same phylogenetic tree, in which human and chimpanzee are most closely related. However, the trees considered here are gene trees, and to obtain the correct species tree, sequence data for several independent loci must be used.     

The phylogeny of trypsin-related serine proteases and their zymogens. New methods for the investigation of distant evolutionary relationships.

The sequence of all presently known trypsin-related serine proteases and their zymogens of animal and bacterial origin were optimally aligned on the basis of three different scoring schemes for amino acid comparisons. Sequence homology was found to extend into the activation peptides. The gaps resulting from the alignment of the sequences of the active enzymes formed the basis for a new procedure based on position and number of gaps, which allowed the correct topology of the evolutionary relationship of thrombin and the pancreatic enzymes trypsin, chymotrypsin and elastase to be determined. The procedure was applied in an analogous manner to changes in disulfide bridges as well as to a selected set of amino acid positions.Evolutionary distances between proteins were estimated by minimum, base differences as well as according to the stochastic model of evolution. These distances were used successfully to find the best topology of evolutionary relationships. The fact that the branch lengths in evolutionary trees were less affected by the number of sequences considered when evolutionary distances between contemporary sequences were measured in minimum base differences than when measured according to the stochastic model of evolution, suggested in our specific case, that minimum base differences yielded estimates of evolutionary distance closer to reality than the stochastic model of evolution.All these techniques combined yielded the following picture for the evolution of the four protease families. Prothrombin and the zymogens of the pancreatic serine proteases had a common ancestor with tryptic specificity. After the initial divergence, the gene for trypsinogen duplicated. Evidence was found that the duplicated gene underwent drastic changes for a short period of time to become eventually the common ancestor of chymotrypsin and elastase. The phylogenetic tree elaborated for these enzyme families and the methods introduced to determine its topology, should readily allow determination of the attachment site of branches leading to newly sequenced serine proteases, provided their amino acid sequence can be aligned fairly unambiguously. In addition, the consequences of the alignment of the different serine proteases for the relationship of zymogen to enzyme are discussed.