Theoretical foundation of the balanced minimum evolution method of phylogenetic inference and its relationship to weighted least-squares tree fitting

Due to its speed, the distance approach remains the best hope for building phylogenies on very large sets of taxa. Recently (R. Desper and O. Gascuel, J. Comp. Biol. 9:687-705, 2002), we introduced a new "balanced" minimum evolution (BME) principle, based on a branch length estimation scheme of Y. Pauplin (J. Mol. Evol. 51:41-47, 2000). Initial simulations suggested that FASTME, our program implementing the BME principle, was more accurate than or equivalent to all other distance methods we tested, with running time significantly faster than Neighbor-Joining (NJ). This article further explores the properties of the BME principle, and it explains and illustrates its impressive topological accuracy. We prove that the BME principle is a special case of the weighted least-squares approach, with biologically meaningful variances of the distance estimates. We show that the BME principle is statistically consistent. We demonstrate that FASTME only produces trees with positive branch lengths, a feature that separates this approach from NJ (and related methods) that may produce trees with branches with biologically meaningless negative lengths. Finally, we consider a large simulated data set, with 5,000 100-taxon trees generated by the Aldous beta-splitting distribution encompassing a range of distributions from Yule-Harding to uniform, and using a covarion-like model of sequence evolution. FASTME produces trees faster than NJ, and much faster than WEIGHBOR and the weighted least-squares implementation of PAUP*. Moreover, FASTME trees are consistently more accurate at all settings, ranging from Yule-Harding to uniform distributions, and all ranges of maximum pairwise divergence and departure from molecular clock. Interestingly, the covarion parameter has little effect on the tree quality for any of the algorithms. FASTME is freely available on the web.     

Statistical properties of molecular tree construction methods under the neutral mutation model

Summary The statistical properties of three molecular tree construction methods—the unweighted pair-group arithmetic average clustering (UPG), Farris, and modified Farris methods—are examined under the neutral mutation model of evolution. The methods are compared for accuracy in construction of the topology and estimation of the branch lengths, using statistics of these two aspects. The distribution of the statistic concerning topological construction is shown to be as important as its mean and variance for the comparison.Of the three methods, the UPG method constructs the tree topology with the least variation. The modified Farris method, however, gives the best performance when the two aspects are considered simultaneously. It is also shown that a topology based on two genes is much more accurate than that based on one gene.There is a tendency to accept published molecular trees, but uncritical acceptance may lead one to spurious conclusions. It should always be kept in mind that a tree is a statistical result that is affected strongly by the stochastic error of nucleotide substitution and the error intrinsic to the tree construction method itself.     

Secondary structure model for the ITS-2 precursor rRNA of strongyloid nematodes of equids: implications for phylogenetic inference

In order to maximise the positional homology in the primary sequence alignment of the second internal transcribed spacer for 30 species of equine strongyloid nematodes, the secondary structures of the precursor ribosomal RNA were predicted using an approach combining an energy minimisation method and comparative sequence analysis. The results indicated that a common secondary structure model of the second internal transcribed spacer of these nematodes was maintained, despite significant interspecific differences (2–56%) in primary sequences. The secondary structure model was then used to refine the primary second internal transcribed spacer sequence alignment. The “manual” and “structure” alignments were both subjected to phylogenetic analysis using three different tree-building methods to compare the effect of using different sequence alignments on phylogenetic inference. The topologies of the phylogenetic trees inferred from the manual second internal transcribed spacer alignment were usually different to those derived from the structure second internal transcribed spacer alignment. The results suggested that the positional homology in the second internal transcribed spacer primary sequence alignment was maximised when the secondary structure model was taken into consideration.     

Studying the evolutionary relationships and phylogenetic trees of 21 groups of tRNA sequences based on complex networks

To find out the evolutionary relationships among different tRNA sequences of 21 amino acids, 22 networks are constructed. One is constructed from whole tRNAs, and the other 21 networks are constructed from the tRNAs which carry the same amino acids. A new method is proposed such that the alignment scores of any two amino acids groups are determined by the average degree and the average clustering coefficient of their networks. The anticodon feature of isolated tRNA and the phylogenetic trees of 21 group networks are discussed. We find that some isolated tRNA sequences in 21 networks still connect with other tRNAs outside their group, which reflects the fact that those tRNAs might evolve by intercrossing among these 21 groups. We also find that most anticodons among the same cluster are only one base different in the same sites when S ≥ 70, and they stay in the same rank in the ladder of evolutionary relationships. Those observations seem to agree on that some tRNAs might mutate from the same ancestor sequences based on point mutation mechanisms.     

Independent gene evolution in the potato actin gene family demonstrated by phylogenetic procedures for resolving gene conversions and the phylogeny of angiosperm actin genes

Summary Nine different actin DNA sequences were isolated from the common potato,Solanum tuberosum, and the nucleotide sequence of five actin loci and of two allelic variants are presented. Unlike the wide variation in intron position among animal actin genes, the potato actin genes have three introns situated in the same positions as reported for all other angiosperm actin genes. Using a novel combination of analytical procedures (G-test and compatibility analysis), we could not find evidence of frequent large or small nonreciprocal exchanges of genetic material between the sequenced loci, although there were a few candidates. Resolution of such gene conversion events and the quantification of independence of gene evolution in multigene families is critical to the inference of phylogenetic relationships. Comparison with actin genes in other angiosperm species suggests that the actin multigene family can be divided into a number of subfamilies, evolved by descent rather than gene conversion, which are of possible functional origin, with one major subfamily diversification occurring before the divergence of monocots and dicots. The silent rate of nucleotide substitution was estimated to be similar to that suggested for a number of other plant nuclear genes, whereas the replacement rate was extremely slow, suggestive of selective constraints.     

Evolutionary aspects of the synuclein super-family and sub-families based on large-scale phylogenetic and group-discrimination analysis

Over the last decade, many genetic studies have suggested that the synucleins, which are small, natively unfolded proteins, are closely related to Parkinson’s disease and cancer. Less is known about the molecular basis of this role. A comprehensive analysis of the evolutionary path of the synuclein protein family may reveal the relationship between evolutionarily conserved residues and protein function or structure. The phylogeny of 252 unique synuclein sequences from 73 organisms suggests that gamma-synuclein is the common ancestor of alpha- and beta-synuclein. Although all three sub-families remain highly conserved, especially at the N-terminal, nearly 15% of the residues in each sub family clearly diverged during evolution, providing crucial guidance for investigations of the different properties of the members of the superfamily. His50 is found to be an alpha-specific conserved residue (91%) and, based on mutagenesis, evolutionarily developed a secondary copper binding site in the alpha synuclein family. Surprisingly, this site is located between two well-known polymorphisms of alpha-synuclein, E46K and A53T, which are linked to early-onset Parkinson’s disease, suggesting that the mutation-induced impairment of copper binding could be a mechanism responsible for alpha-synuclein aggregation.     

The role of parsimony,outgroup analysis,and theory of evolution in phylogenetic systematics

It is argued that both the principle of parsimony and the theory of evolution, especially that of natural selection, are essential analytical tools in phylogenetic systematics, whereas the widely used outgroup analysis is completely useless and may even be misleading. In any systematic analysis, two types of patterns of characters and character states must be discriminated which are referred to as completely and incompletely resolved. In the former, all known species are presented in which the characters and their states studied occur, whereas in the latter this is not the case. Dependent on its structure, a pattern of characters and their states may be explained by either a unique or by various conflicting, equally most parsimonious hypotheses of relationships. The so-called permutation method is introduced which facilitates finding the conflicting, equally most parsimonious hypotheses of relationships. The utility of the principle of parsimony is limited by the uncertainty as to whether its application in systematics must refer to the minimum number of steps needed to explain a pattern of characterts and their states most parsimoniously or to the minimum number of evolutionary events assumed to have caused these steps. Although these numbers may differ, the former is usually preferred for simplicity. The types of outgroup analysis are shown to exist which are termed parsimony analysis based on test samples and cladistic type of outgroup analysis. Essentially, the former is used for analysing incompletely resolved patterns of characters and their states, the latter for analysing completely resolved ones. Both types are shown to be completely useless for rejecting even one of various conflicting, equally most parsimonious hypotheses of relationships. According to contemporary knowledge, this task can be accomplished only by employing the theory of evolution (including the theory of natural selection). But even then, many phylogenetic-systematic problems will remain unsolved. In such cases, arbitrary algorithms like those offered by phenetics can at best offer pseudosolutions to open problems. Despite its limitations, phylogenetic systematics is superior to any kind of aphylogenetic systematics (transformed cladistics included) in approaching a (not: the) “general reference system” of organisms.     

Characterization of two rice genes for nuclear-encoded chloroplast ribosomal protein L12 and phylogenetic analysis of the acquisition of transit peptides and gene duplication

 We have identified two genes coding for chloroplast ribosomal protein L12 encoded in the nuclear genome of rice (Oryza sativa). These genes were designated rpl12-1 and rpl12-2 (rpl12, ribosomal protein L12). Northern analysis with specific probes revealed that both genes are transcribed. The expression of each gene seems to have a different regulatory machinery. It is also suggested that the expression of rpl12-1 is controlled in an organ-specific manner. The deduced amino-acid sequences of the mature peptide parts are more conserved than those of the transit peptide parts in both monocotyledonous and dicotyledonous plants. A phylogenetic tree was constructed using the nucleotide sequences of the transit peptide region of the rpl12s of reported plant. The tree includes estimates of when the transit peptides were acquired, and when the genes were duplicated, in the course of evolution. According to our hypothesis, the nuclear-translocated chloroplast ribosomal protein L12 gene obtained its transit peptide after the divergence of monocots and dicots, then gene duplications occurred independently in monocots and dicots, and subsequently rice and rye branched apart. Received: 4 October 1997 / Accepted: 5 January 1998     

Evolution of RNA polymerases and branching patterns of the three major groups of archaebacteria

Summary The amino acid sequences of the largest subunits of the RNA polymerases I, II, and III from eukaryotes were compared with those of archaebacterial and eubacterial homologs, and their evolutionary relationships were analyzed in detail by a recently developed tree-making method, the likelihood method of protein phylogeny, as well as by the neighbor-joining method and the parsimony method, together with bootstrap analyses. It was shown that the best tree topologies predicted by the first two methods are identical, whereas the last one predicts a distinct tree. The maximum likelihood tree revealed that, after the separation from archaebacteria, the three eukaryotic RNA polymerases diverged from an ancestral precursor in the eukaryotic lineage. This result is contrasted with the published result showing multiple origins for the three eukaryotic polymerases. It was shown that eukaryotic RNA polymerase I evolved much more rapidly than RNA polymerases II and III: The N-terminal half of RNA polymerase I shows an extraordinarily high evolutionary rate, possibly due to relaxed functional constraints. In contrast the evolutionary rate of archaebacterial RNA polymerase is remarkably limited. In addition, including the second largest subunit of the RNA polymerase, a detailed analysis for the branching pattern of the three major groups of archaebacteria was carried out by the maximum likelihood method. It was shown that the three major groups of archaebacteria are likely to form a single cluster; that is, archaebacteria are likely to be monophyletic as originally proposed by Woese and his colleagues.     

Statistical uncertainty and its propagation in the analysis of quantitative polymerase chain reaction data: Comparison of methods

Most methods for analyzing real-time quantitative polymerase chain reaction (qPCR) data for single experiments estimate the hypothetical cycle 0 signal y₀ by first estimating the quantification cycle (C_q) and amplification efficiency (E) from least-squares fits of fluorescence intensity data for cycles near the onset of the growth phase. The resulting y₀ values are statistically equivalent to the corresponding C_q if and only if E is taken to be error free. But uncertainty in E usually dominates the total uncertainty in y₀, making the latter much degraded in precision compared with C_q. Bias in E can be an even greater source of error in y₀. So-called mechanistic models achieve higher precision in estimating y₀ by tacitly assuming E = 2 in the baseline region and so are subject to this bias error. When used in calibration, the mechanistic y₀ is statistically comparable to C_q from the other methods. When a signal threshold y_q is used to define C_q, best estimation precision is obtained by setting y_q near the maximum signal in the range of fitted cycles, in conflict with common practice in the y₀ estimation algorithms.     

Purification,characterization, and molecular cloning of chitinases from the stomach of the threeline grunt Parapristipoma trilineatum

Two chitinase isozymes, PtChiA and PtChiB, were purified from the stomach of the threeline grunt, Parapristipoma trilineatum. The molecular masses of PtChiA and PtChiB were estimated to be 50 and 60 kDa by SDS-PAGE, respectively. Both chitinases were stable at pH 3.0–6.0 (acidic) and showed the optimum pH toward both short and long substrates in the acidic region (pH 2.5–5.0). PtChiA and PtChiB preferentially degraded the second and third glycosidic bonds from the non-reducing end of N-acetylchitooligosaccharides, respectively. PtChiA and PtChiB exhibited wide substrate specificities toward crystalline chitin. Moreover, 2 cDNAs encoding PtChiA and PtChiB, PtChi-1 and PtChi-2, respectively, were cloned. The deduced amino acid sequences of both chitinase cDNAs comprised N-terminal signal peptides, glycoside hydrolase 18 catalytic domains, linker regions, and C-terminal chitin-binding domains. Phylogenetic tree analysis of vertebrate chitinases revealed that fish stomach chitinases including PtChi-1 and PtChi-2 form unique chitinase groups, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), which differ from the acidic mammalian chitinase (AMCase) group. The present results suggest that fish have a chitin-degrading enzymatic system in which 2 different chitinases, AFCase-1 and AFCase-2, with different degradation patterns are expressed in the stomach.     

Complete nucleotide sequences of seven eubacterial genes coding for the elongation factor Tu: functional,structural and phylogenetic evaluations

The nucleotide sequences of cloned genes coding for the elongation factor Tu of seven eubacteria have been determined. These genes were fiom Anacystis nidulans, Bacillus subtilis, Bacteroides fragilis, Deinonema spec., Pseudomonas cepacia, Shewanella putrefaciens and Streptococcus oralis. The primary structures of the genes were compared to the available sequences of prokaryotic elongation factors Tu and eukaryotic elongation factors 1 alpha. A conservation profile was determined for homologous amino acid residues. Sites of known or putative functions are usually located at highly conserved positions or within highly conserved sequence stretches. The aligned 24 amino acid sequences were used as basis for a phylogenetic analysis. The phylogenetic tree corroborates the kingdom as well as phylum concept deduced from 16S rRNA data.Abbreviations EF-Tu elongation factor Tu - GDP guanosine 5-diphosphate - GTP guanosine 5-triphosphate; tuf gene, gene coding for elongation factor Tu     

Comparison of the accuracies of several phylogenetic methods using protein and DNA sequences

A biologically realistic method was used to simulate evolutionary trees. The method uses a real DNA coding sequence as the starting point, simulates mutation according to the mutational spectrum of Escherichia coli-including base substitutions, insertions, and deletions-and separates the processes of mutation and selection. Trees of 8, 16, 32, and 64 taxa were simulated with average branch lengths of 50, 100, 150, 200, and 250 changes per branch. The resulting sequences were aligned with ClustalX, and trees were estimated by Neighbor Joining, Parsimony, Maximum Likelihood, and Bayesian methods from both DNA sequences and the corresponding protein sequences. The estimated trees were compared with the true trees, and both topological and branch length accuracies were scored. Over the variety of conditions tested, Bayesian trees estimated from DNA sequences that had been aligned according to the alignment of the corresponding protein sequences were the most accurate, followed by Maximum Likelihood trees estimated from DNA sequences and Parsimony trees estimated from protein sequences.     

Using AFLP markers and the Geneland program for the inference of population genetic structure

The use of dominant markers such as amplified fragment length polymorphism (AFLP) for population genetics analyses is often impeded by the lack of appropriate computer programs and rarely motivated by objective considerations. The point of the present note is twofold: (i) we describe how the computer program Geneland designed to infer population structure has been adapted to deal with dominant markers; and (ii) we use Geneland for numerical comparison of dominant and codominant markers to perform clustering. AFLP markers lead to less accurate results than bi-allelic codominant markers such as single nucleotide polymorphisms (SNP) markers but this difference becomes negligible for data sets of common size (number of individuals n≥100, number of markers L≥200). The latest Geneland version (3.2.1) handling dominant markers is freely available as an R package with a fully clickable graphical interface. Installation instructions and documentation can be found on http://www2.imm.dtu.dk/~gigu/Geneland.     

Generic revision of Cypricercinae McKenzie, 1971 (Crustacea,Ostracoda), with the description of three new genera and one new species and a phylogenetic analysis of the subfamily

The Cypricercinae are one of the most speciose subfamilies of non-marine ostracods, with more than 170 described species, mostly from the tropics. Although the identity of the subfamily as such is clear, because of the presence of unifying characters such as the Triebel’s loop in the attachment of the caudal ramus, the supra-specific taxonomy of this group has long been confused because of lack of good generic and tribal characters. Here, the generic characters of the Cypricercinae are revised. Eleven genera are retained in this subfamily, including three new genera: Bradleytriebella n. gen., Nealecypris n. gen. and Pseudostrandesia n. gen. Tanycypris siamensis n. sp. is described from Thailand. In addition, five species [Bradleystrandesia fuscata (Jurine, 1820), Bradleytriebella tuberculata (Hartmann, 1964), Nealecypris obtusa (Klie, 1933), Pseudostrandesia striatoreticulata (Klie, 1932), Spirocypris horrida (Sars, 1926)] are redescribed. A key to the genera is given. We propose three tribes: the nominal tribe Cypricercini McKenzie, 1971, as well as two new tribes, Bradleystrandesiini n. trib. and Nealecypridini n. trib. To evaluate the systematic relationships within this subfamily, phylogenetic analyses, based on morphological characters of valves and soft parts, were conducted. The Neighbour Joining (NJ) tree strongly supports the classification into three independent tribes, whereas the Maximum Parsimony (MP) tree shows that Bradleystrandesiini n. trib is actually a subgroup of the Cypricercini. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: Luigi Naselli-Flores