Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani

The complete nucleotide sequence (16,650 bp) of the mitochondrial genome of the salamander Mertensiella luschani (Caudata, Amphibia) was determined. This molecule conforms to the consensus vertebrate mitochondrial gene order. However, it is characterized by a long non-coding intervening sequence with two 124-bp repeats between the tRNA(Thr) and tRNA(Pro) genes. The new sequence data were used to reconstruct a phylogeny of jawed vertebrates. Phylogenetic analyses of all mitochondrial protein-coding genes at the amino acid level recovered a robust vertebrate tree in which lungfishes are the closest living relatives of tetrapods, salamanders and frogs are grouped together to the exclusion of caecilians (the Batrachia hypothesis) in a monophyletic amphibian clade, turtles show diapsid affinities and are placed as sister group of crocodiles+birds, and the marsupials are grouped together with monotremes and basal to placental mammals. The deduced phylogeny was used to characterize the molecular evolution of vertebrate mitochondrial proteins. Amino acid frequencies were analyzed across the main lineages of jawed vertebrates, and leucine and cysteine were found to be the most and least abundant amino acids in mitochondrial proteins, respectively. Patterns of amino acid replacements were conserved among vertebrates. Overall, cartilaginous fishes showed the least variation in amino acid frequencies and replacements. Constancy of rates of evolution among the main lineages of jawed vertebrates was rejected.     

Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny

The relative efficiencies of different protein-coding genes of the mitochondrial genome and different tree-building methods in recovering a known vertebrate phylogeny (two whale species, cow, rat, mouse, opossum, chicken, frog, and three bony fish species) was evaluated. The tree-building methods examined were the neighbor joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML), and both nucleotide sequences and deduced amino acid sequences were analyzed. Generally speaking, amino acid sequences were better than nucleotide sequences in obtaining the true tree (topology) or trees close to the true tree. However, when only first and second codon positions data were used, nucleotide sequences produced reasonably good trees. Among the 13 genes examined, Nd5 produced the true tree in all tree-building methods or algorithms for both amino acid and nucleotide sequence data. Genes Cytb and Nd4 also produced the correct tree in most tree-building algorithms when amino acid sequence data were used. By contrast, Co2, Nd1, and Nd41 showed a poor performance. In general, large genes produced better results, and when the entire set of genes was used, all tree-building methods generated the true tree. In each tree-building method, several distance measures or algorithms were used, but all these distance measures or algorithms produced essentially the same results. The ME method, in which many different topologies are examined, was no better than the NJ method, which generates a single final tree. Similarly, an ML method, in which many topologies are examined, was no better than the ML star decomposition algorithm that generates a single final tree. In ML the best substitution model chosen by using the Akaike information criterion produced no better results than simpler substitution models. These results question the utility of the currently used optimization principles in phylogenetic construction. Relatively simple methods such as the NJ and ML star decomposition algorithms seem to produce as good results as those obtained by more sophisticated methods. The efficiencies of the NJ, ME, MP, and ML methods in obtaining the correct tree were nearly the same when amino acid sequence data were used. The most important factor in constructing reliable phylogenetic trees seems to be the number of amino acids or nucleotides used.      

A New Method of Inference of Ancestral Nucleotide and Amino Acid Sequences

A statistical method was developed for reconstructing the nucleotide or amino acid sequences of extinct ancestors, given the phylogeny and sequences of the extant species. A model of nucleotide or amino acid substitution was employed to analyze data of the present-day sequences, and maximum likelihood estimates of parameters such as branch lengths were used to compare the posterior probabilities of assignments of character states (nucleotides or amino acids) to interior nodes of the tree; the assignment having the highest probability was the best reconstruction at the site. The lysozyme c sequences of six mammals were analyzed by using the likelihood and parsimony methods. The new likelihood-based method was found to be superior to the parsimony method. The probability that the amino acids for all interior nodes at a site reconstructed by the new method are correct was calculated to be 0.91, 0.86, and 0.73 for all, variable, and parsimony-informative sites, respectively, whereas the corresponding probabilities for the parsimony method were 0.84, 0.76, and 0.51, respectively. The probability that an amino acid in an ancestral sequence is correctly reconstructed by the likelihood analysis ranged from 91.3 to 98.7% for the four ancestral sequences.     

The problem of rooting rapid radiations

There are many examples of groups (such as birds, bees, mammals, multicellular animals, and flowering plants) that have undergone a rapid radiation. In such cases, where there is a combination of short internal and long external branches, correctly estimating and rooting phylogenetic trees is known to be a difficult problem. In this simulation study, we tested the performances of different phylogenetic methods at estimating a tree that models a rapid radiation. We found that maximum likelihood, corrected and uncorrected neighbor-joining, and corrected and uncorrected parsimony, all suffer from biases toward specific tree topologies. In addition, we found that using a single-taxon outgroup to root a tree frequently disrupts an otherwise correct ingroup phylogeny. Moreover, for uncorrected parsimony, we found cases where several individual trees (in which the outgroup was placed incorrectly) were selected more frequently than the correct tree. Even for parameter settings where the correct tree was selected most frequently when using extremely long sequences, for sequences of up to 60,000 nucleotides the incorrectly rooted trees were each selected more frequently than the correct tree. For all the cases tested here, tree estimation using a two taxon outgroup was more accurate than when using a single-taxon outgroup. However, the ingroup was most accurately recovered when no outgroup was used.     

Invariable sites models and their use in phylogeny reconstruction

Phylogenetic inference is well known to be problematic if both long and short branches occur together in the underlying tree. With biological data, correcting for this problem may require simultaneous consideration for both substitution biases and rate heterogeneity between lineages and across sequence positions. A particular form of the latter is the presence of invariable sites, which are well known to mislead estimation of genetic divergences. Here we describe a capture-recapture method to estimate the proportion of invariable sites in an alignment of amino acids or nucleotides. We use it to investigate phylogenetic signals in 18S ribosomal DNA sequences from Holometabolus insects. Our results suggest that, as taxa diverged, their 18S rDNA sequences have altered in both their distribution of sites that can vary as well as in their base compositions.     

Protein structural influences in rhodopsin evolution

Incorporating specific structural information can be important for developing a realistic model of evolution for phylogenetic reconstruction of protein-coding genes. We analyzed 62 sequences of vertebrate rhodopsin. The bovine rhodopsin structure was used to label residue sites by surface accessibility, secondary structure, and transmembrane (TM) location. Residue sites with amino acid differences were identified; using maximum parsimony (MP), homoplasious residues were identified. Residues were analyzed for patterns that would indicate correlation of rate with secondary structure, surface accessibility, or position relative to the lipid bilayer. Surface residues, especially those residing in one of the seven TM helices, were significantly correlated with high rates of amino acid substitution. This category of residues, defined solely by protein structural characteristics, potentially defined a class enriched in homoplasious residues. MP analysis using all sites led to a tree with anomalies in the relationships of amphibian, mammalian, bird, and alligator species. Analysis excluding the structurally defined residue class recovered a more accurate phylogeny. A model is presented for including structural influences on rate in phylogenetic inference.     

Inhibitory sequences within the B-domain stabilize circulating factor V in an inactive state

Blood coagulation factor V circulates as a procofactor with little or no procoagulant activity. It is activated to factor Va by thrombin following proteolytic removal of a large central B-domain. Although this reaction is well studied, the mechanism by which bond cleavage and B-domain release facilitate the transition to the active cofactor state has not been defined. Here we show that deletion or substitution of specific B-domain sequences drives the expression of procoagulant function without the need for proteolytic processing. Conversion to the constitutively active cofactor state is related, at least in part, to a cluster of amino acids that is highly basic and well conserved across the vertebrate lineage. Our findings demonstrate that discrete sequences in the B-domain serve to stabilize the inactive procofactor state, with proteolysis primarily functioning to remove these inhibitory constraints. These unexpected results provide new insight into the mechanism of factor V activation.     

Accounting for evolutionary rate variation among sequence sites consistently changes universal phylogenies deduced from rRNA and protein-coding genes.

Phylogenetic analyses of gene and protein sequences have led to two major competing views of the universal phylogeny, the evolutionary tree relating the three kinds of living organisms, Bacteria, Archaea, and Eukarya. In the first scheme, called "the archaebacterial tree, " organisms of the same type are clustered together. In the second scenario, called "the eocyte tree," the archaeal phylum of Crenarchaeota is more closely related to eukaryotes than are other Archaea. A major property of the evolution of functional ribosomal and protein-encoding genes is that the rate of nucleotide and amino acid substitution varies across sequence sites. Here, using distance-based and maximum-likelihood methods, we show that universal phylogenies of ribosomal RNAs and RNA polymerases built by ignoring this variation are biased toward the archaebacterial tree because of attraction between long branches. In contrast, taking among-site rate variability into account gives support for the eocyte tree.     

The phylogenetic relationship of tetrapod, coelacanth, and lungfish revealed by the sequences of forty-four nuclear genes

The origin of tetrapods is a major outstanding issue in vertebrate phylogeny. Each of the three possible principal hypotheses (coelacanth, lungfish, or neither being the sister group of tetrapods) has found support in different sets of data. In an attempt to resolve the controversy, sequences of 44 nuclear genes encoding amino acid residues at 10,404 positions were obtained and analyzed. However, this large set of sequences did not support conclusively one of the three hypotheses. Apparently, the coelacanth, lungfish, and tetrapod lineages diverged within such a short time interval that at this level of analysis, their relationships appear to be an irresolvable trichotomy.     

Overdispersion of the molecular clock: temporal variation of gene-specific substitution rates in Drosophila

Simple models of molecular evolution assume that sequences evolve by a Poisson process in which nucleotide or amino acid substitutions occur as rare independent events. In these models, the expected ratio of the variance to the mean of substitution counts equals 1, and substitution processes with a ratio greater than 1 are called overdispersed. Comparing the genomes of 10 closely related species of Drosophila, we extend earlier evidence for overdispersion in amino acid replacements as well as in four-fold synonymous substitutions. The observed deviation from the Poisson expectation can be described as a linear function of the rate at which substitutions occur on a phylogeny, which implies that deviations from the Poisson expectation arise from gene-specific temporal variation in substitution rates. Amino acid sequences show greater temporal variation in substitution rates than do four-fold synonymous sequences. Our findings provide a general phenomenological framework for understanding overdispersion in the molecular clock. Also, the presence of substantial variation in gene-specific substitution rates has broad implications for work in phylogeny reconstruction and evolutionary rate estimation.     

Limited effects of among-lineage rate variation on the phylogenetic performance of molecular markers

Variation in substitution rates among evolutionary lineages (among-lineage rate variation or ALRV) has been reported to negatively affect the estimation of phylogenies. When the substitution processes underlying ALRV are modeled inadequately, non-sister taxa with similar substitution rates are estimated incorrectly as sister species due to long-branch attraction. Recent advances in modeling site-specific rate variation (heterotachy) have reduced the impacts of ALRV on phylogeny estimation in several empirical and simulated datasets. However, the addition of parameters to the substitution model reduces power to estimate each parameter correctly, which can also lead to incorrect phylogeny estimation. A potential solution to this problem is to identify the levels of ALRV that negatively impact phylogeny estimation such that molecular markers with non-deleterious levels of ALRV can be identified. To this end, we used analyses of empirical and simulated gene datasets to evaluate whether levels of ALRV identified in a mitochondrial genomic dataset for salamanders negatively impacted phylogeny estimation. We simulated data with and without ALRV, holding all other evolutionary parameters constant, and compared the phylogenetic performance of both simulated and empirical datasets. Overall, we found limited, positive effects of ALRV on phylogeny estimation in this dataset, the majority of which resulted from an increase in substitution rate on short branches. We conclude that ALRV does not always negatively impact phylogeny estimation. Therefore, ALRV can likely be disregarded as a criterion for marker selection in comparable phylogenetic studies.     

Maximum likelihood inference of protein phylogeny and the origin of chloroplasts

Summary A maximum likelihood method for inferring protein phylogeny was developed. It is based on a Markov model that takes into account the unequal transition probabilities among pairs of amino acids and does not assume constancy of rate among different lineages. Therefore, this method is expected to be powerful in inferring phylogeny among distantly related proteins, either orthologous or parallogous, where the evolutionary rate may deviate from constancy. Not only amino acid substitutions but also insertion/deletion events during evolution were incorporated into the Markov model. A simple method for estimating a bootstrap probability for the maximum likelihood tree among alternatives without performing a maximum likelihood estimation for each resampled data set was developed. These methods were applied to amino acid sequence data of a photosynthetic membrane protein,psbA, from photosystem II, and the phylogeny of this protein was discussed in relation to the origin of chloroplasts.     

Lactate dehydrogenase A as a highly abundant eye lens protein in platypus (Ornithorhynchus anatinus): upsilon (upsilon)-crystallin

Vertebrate eye lenses mostly contain two abundant types of proteins, the alpha-crystallins and the beta/gamma-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (upsilon)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as epsilon-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/upsilon-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate ldh-a gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.     

Rates of genome evolution and branching order from whole genome analysis

Accurate estimation of any phylogeny is important as a framework for evolutionary analysis of form and function at all levels of organization from sequence to whole organism. Using alignments of nonrepetitive components of opossum, human, mouse, rat, and dog genomes we evaluated two alternative tree topologies for eutherian evolution. We show with very high confidence that there is a basal split between rodents (as represented by the mouse and rat) and a branch joining primates (as represented by humans) and carnivores (as represented by dogs), consistent with some but not the most widely accepted mammalian phylogenies. The result was robust to substitution model choice with equivalent inference returned from a spectrum of models ranging from a general time reversible model, a model that treated nucleotides as either purines and pyrimidines, and variants of these that incorporated rate heterogeneity among sites. By determining this particular branching order we are able to show that the rate of molecular evolution is almost identical in rodent and carnivore lineages and that sequences evolve approximately 11%-14% faster in these lineages than in the primate lineage. In addition by applying the chicken as outgroup the analyses suggested that the rate of evolution in all eutherian lineages is approximately 30% slower than in the opossum lineage. This pattern of relative rates is inconsistent with the hypothesis that generation time is an important determinant of substitution rates and, by implication, mutation rates. Possible factors causing rate differences between the lineages include differences in DNA repair and replication enzymology, and shifts in nucleotide pools. Our analysis demonstrates the importance of using multiple sequences from across the genome to estimate phylogeny and relative evolutionary rate in order to reduce the influence of distorting local effects evident even in relatively long sequences.     

Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes

A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.     

A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation

The shikimate pathway is essential for survival of the apicomplexan parasites Plasmodium falciparum, Toxoplasma gondii and Cryptosporidium parvum. As it is absent in mammals it is a promising therapeutic target. Herein, we describe the genes encoding the shikimate pathway enzymes in T. gondii. The molecular arrangement and phylogeny of the proteins suggests homology with the eukaryotic fungal enzymes, including a pentafunctional AROM. Current rooting of the eukaryotic evolutionary tree infers that the fungi and apicomplexan lineages diverged deeply, suggesting that the arom is an ancient supergene present in early eukaryotes and subsequently lost or replaced in a number of lineages.     

Rates of genomic divergence in humans,chimpanzees and their lice

The rate of DNA mutation and divergence is highly variable across the tree of life. However, the reasons underlying this variation are not well understood. Comparing the rates of genetic changes between hosts and parasite lineages that diverged at the same time is one way to begin to understand differences in genetic mutation and substitution rates. Such studies have indicated that the rate of genetic divergence in parasites is often faster than that of their hosts when comparing single genes. However, the variation in this relative rate of molecular evolution across different genes in the genome is unknown. We compared the rate of DNA sequence divergence between humans, chimpanzees and their ectoparasitic lice for 1534 protein-coding genes across their genomes. The rate of DNA substitution in these orthologous genes was on average 14 times faster for lice than for humans and chimpanzees. In addition, these rates were positively correlated across genes. Because this correlation only occurred for substitutions that changed the amino acid, this pattern is probably produced by similar functional constraints across the same genes in humans, chimpanzees and their ectoparasites.