The Pattern of Mammalian Evolution and the Relative Rate of Molecular Evolution

The rates of nucleotide substitution at four genes in four orders of eutherian mammals are compared in relative rate tests using marsupial orthologs for reference. There is no evidence of systematic variation in evolutionary rate among the orders. The sequences are used to reconstruct the phylogeny of the orders using maximum likelihood, parsimony and compatibility methods. A branching order of rodent then ungulate then primate and lagomorph is overwhelmingly indicated. The nodes of the nucleotide based cladograms are widely separated in relation to the total lengths of the branches. The assumption of a star phylogeny that underlies Kimura's test for molecular evolutionary rate variation is shown to be invalid for eutherian mammals. Excess variance in nucleotide or amino acid differences between mammalian orders, above that predicted by neutral theory is explained better by variation in divergence time than by variation in evolutionary rate.     

Detecting substitution-rate heterogeneity among regions of a nucleotide sequence

Likelihood-ratio statistics are proposed to test for heterogeneity in nucleotide substitution rate among regions of a DNA sequence. The tests examine three-sequence phylogenies, and two specific tests are proposed: a test to detect rate heterogeneity among genic regions within a sequence, over all evolutionary lineages; and a test to detect rate heterogeneity among regions in a specific evolutionary lineage. Simulations examine the ability of tests to detect a single region that varies in nucleotide substitution rate relative to the remainder of the sequence. A 50-bp region with a fivefold substitution-rate increase can be detected > or = 90% of the time when it is found in all three lineages of the phylogeny, and a 50-bp region of fivefold rate increase can be detected with approximately 70% power when it is found in only one evolutionary lineage. Simulation also examines the effect of transition- and transversion-rate differences. The tests are applied to published DNA sequences. While the tests are powerful, significant results can be difficult to interpret biologically.      

Structure and evolution of opossum,guinea pig,and porcupine cytochrome b genes

Summary We have sequenced the mitochondrial cytochrome b gene from the guinea pig, the African porcupine, and a South American opossum. A phylogenetic analysis, which includes 22 eutherian and four other vertebrate cytochrome b sequences, indicates that the guinea pig and the porcupine constitute a natural clade (Hystricomorpha) that is not a sister group to the clade of mice and rats (Myomorpha). Therefore, the hypothesis that the Rodentia is paraphyletic receives additional support. The artiodactyls, the perissodactyls, and the cetaceans form a group that is separated from the primates and the rodents. The 26 sequences are used to study the structure/function relationships in cytochrome b, whose function is electron transport. Most of the amino acid residues involved in the two reaction centers are well conserved in evolution. The four histidines that are believed to ligate the two hemes are invariant among the 26 sequences, but their nearby residues are not well conserved in evolution. The eight transmembrane domains represent some of the most divergent regions in the cytochrome b sequence. The rate of nonsynonymous substitution is considerably faster in the human and elephant lineages than in other eutherian lineages; the faster rate might be due to coevolution between cytochrome b and cytochrome c. Offprint requests to: W.-H. Li     

Different rates of substitution may produce different phylogenies of the eutherian mammals

Summary In an attempt to resolve some points of branching order in the phylogeny of the eutherian mammals, a phylogenetic analysis of 26 nuclear and 6 mitochondrial genes was undertaken using a maximum likelihood method on a constant rate stochastic model of molecular evolution. Seventeen of the nuclear genes gave a primates/artiodactyls grouping highest support whereas three of the mitochondrial genes found a rodents/artiodactyls grouping to be best supported. The primates/rodents grouping was never the best supported. On the assumption that rodents are indeed an outgroup to primates and artiodactyls and that the latter taxa diverged 70 million years ago, an estimation was made, for each gene, of the time of divergence of the rodent lineage. In most cases such estimates were beyond the limits set by present interpretations of the paleontological record as were many estimates of the divergence time of mouse and rat. These results suggest that, although there is locus variation, the divergent position of the rodent lineage may be an artifact of an elevated rate of nucleotide substitution in this order.     

Reconciling extreme branch length differences: decoupling time and rate through the evolutionary history of filmy ferns

The rate of molecular evolution is not constant across the Tree of Life. Characterizing rate discrepancies and evaluating the relative roles of time and rate along branches through the past are both critical to a full understanding of evolutionary history. In this study, we explore the interactions of time and rate in filmy ferns (Hymenophyllaceae), a lineage with extreme branch length differences between the two major clades. We test for the presence of significant rate discrepancies within and between these clades, and we separate time and rate across the filmy fern phylogeny to simultaneously yield an evolutionary time scale of filmy fern diversification and reconstructions of ancestral rates of molecular evolution. Our results indicate that the branch length disparity observed between the major lineages of filmy ferns is indeed due to a significant difference in molecular evolutionary rate. The estimation of divergence times reveals that the timing of crown group diversification was not concurrent for the two lineages, and the reconstruction of ancestral rates of molecular evolution points to a substantial rate deceleration in one of the clades. Further analysis suggests that this may be due to a genome-wide deceleration in the rate of nucleotide substitution.     

Divergence of conserved non-coding sequences: rate estimates and relative rate tests

In many eukaryotic genomes only a small fraction of the DNA codes for proteins, but the non-protein coding DNA harbors important genetic elements directing the development and the physiology of the organisms, like promoters, enhancers, insulators, and micro-RNA genes. The molecular evolution of these genetic elements is difficult to study because their functional significance is hard to deduce from sequence information alone. Here we propose an approach to the study of the rate of evolution of functional non-coding sequences at a macro-evolutionary scale. We identify functionally important non-coding sequences as Conserved Non-Coding Nucleotide (CNCN) sequences from the comparison of two outgroup species. The CNCN sequences so identified are then compared to their homologous sequences in a pair of ingroup species, and we monitor the degree of modification these sequences suffered in the two ingroup lineages. We propose a method to test for rate differences in the modification of CNCN sequences among the two ingroup lineages, as well as a method to estimate their rate of modification. We apply this method to the full sequences of the HoxA clusters from six gnathostome species: a shark, Heterodontus francisci; a basal ray finned fish, Polypterus senegalus; the amphibian, Xenopus tropicalis; as well as three mammalian species, human, rat and mouse. The results show that the evolutionary rate of CNCN sequences is not distinguishable among the three mammalian lineages, while the Xenopus lineage has a significantly increased rate of evolution. Furthermore the estimates of the rate parameters suggest that in the stem lineage of mammals the rate of CNCN sequence evolution was more than twice the rate observed within the placental amniotes clade, suggesting a high rate of evolution of cis-regulatory elements during the origin of amniotes and mammals. We conclude that the proposed methods can be used for testing hypotheses about the rate and pattern of evolution of putative cis-regulatory elements.     

Time scale of eutherian evolution estimated without assuming a constant rate of molecular evolution

Controversies over the molecular clock hypothesis were reviewed. Since it is evident that the molecular clock does not hold in an exact sense, accounting for evolution of the rate of molecular evolution is a prerequisite when estimating divergence times with molecular sequences. Recently proposed statistical methods that account for this rate variation are overviewed and one of these procedures is applied to the mitochondrial protein sequences and to the nuclear gene sequences from many mammalian species in order to estimate the time scale of eutherian evolution. This Bayesian method not only takes account of the variation of molecular evolutionary rate among lineages and among genes, but it also incorporates fossil evidence via constraints on node times. With denser taxonomic sampling and a more realistic model of molecular evolution, this Bayesian approach is expected to increase the accuracy of divergence time estimates.     

Rates of nucleotide substitution in Cornaceae (Cornales)-Pattern of variation and underlying causal factors

Identifying causes of genetic divergence is a central goal in evolutionary biology. Although rates of nucleotide substitution vary among taxa and among genes, the causes of this variation tend to be poorly understood. In the present study, we examined the rate and pattern of molecular evolution for five DNA regions over a phylogeny of Cornus, the single genus of Cornaceae. To identify evolutionary mechanisms underlying the molecular variation, we employed Bayesian methods to estimate divergence times and to infer how absolute rates of synonymous and nonsynonymous substitutions and their ratios change over time. We found that the rates vary among genes, lineages, and through time, and differences in mutation rates, selection type and intensity, and possibly genetic drift all contributed to the variation of substitution rates observed among the major lineages of Cornus. We applied independent contrast analysis to explore whether speciation rates are linked to rates of molecular evolution. The results showed no relationships for individual genes, but suggested a possible localized link between species richness and rate of nonsynonymous nucleotide substitution for the combined cpDNA regions. Furthermore, we detected a positive correlation between rates of molecular evolution and morphological change in Cornus. This was particularly pronounced in the dwarf dogwood lineage, in which genome-wide acceleration in both molecular and morphological evolution has likely occurred.     

Reconstruction of ancestral nucleotide sequences and estimation of substitution frequencies in a star phylogeny

Maximum likelihood phylogeny reconstruction methods are widely used in uncovering and assessing the evolutionary history and relationships of natural systems. However, several simplifying assumptions commonly made in this analysis limit the explanatory power of the results obtained. We present an algorithm that performs the phylogenetic analysis without making the common assumptions for sequence data from at least three leaf nodes in a star phylogeny. In particular, the underlying nucleotide substitution model does not have to be reversible and may include neighbor-dependent processes like the CpG methylation deamination process (CpG-effect). The base composition of the sequences at the external nodes and the one of the ancestral sequence may be different from each other and they do not have to be stationary state distributions of the corresponding substitution model. The algorithm is able to reconstruct the ancestral base composition and accurately estimate substitution frequencies in the branches of the star phylogeny. Extensive tests on simulated data validate the very favorable performance of the algorithm. As an application we present the analysis of aligned genomic sequences from human, mouse, and dog. Different substitution pattern can be observed in the three lineages.     

Estimating the rate of evolution of the rate of molecular evolution

A simple model for the evolution of the rate of molecular evolution is presented. With a Bayesian approach, this model can serve as the basis for estimating dates of important evolutionary events even in the absence of the assumption of constant rates among evolutionary lineages. The method can be used in conjunction with any of the widely used models for nucleotide substitution or amino acid replacement. It is illustrated by analyzing a data set of rbcL protein sequences.      

Relative rates of nuclear DNA evolution in human and Old World monkey lineages

Rates of substitution were compared between humans and Old World monkeys for sequences in or adjacent to 19 genes. The comparison of 21,299 sites in noncoding regions indicates that the substitution rate is approximately 43% greater in the Old World monkey lineage. However, 83% of the compared sites are in the region of the beta-globin gene family. Outside this region there is no consistent pattern of rate difference between the two lineages. Comparison of the coding regions of 16 genomically dispersed genes, involving 1,592 synonymous sites and 5,275 nonsynonymous sites, showed a faster rate of substitution in the human lineage at the nonsynonymous sites of the prion gene, but otherwise no evidence of rate difference between the two lineages. It is concluded that rate differences between these two lineages may be specific to certain regions of the genome rather than being a general phenomenon. This conclusion needs to be confirmed by comparison of a larger number of genomically dispersed sequences. It is, however, consistent with the results of DNA-DNA hybridization experiments, which show no difference in evolutionary rate between the two lineages.      

The molecular clock ticks regularly in muroid rodents and hamsters

Summary Extensive DNA sequence data are used to compare the rates of nucleotide substitution in the mouse, rat, and hamster lineages. A relative rate test using hamster sequences as references shows that the rates of synonymous and nonsynonymous substitution in the mouse and rat lineages are nearly equal and a test using human sequences as references shows that the rates in the mouse, rat, and hamster lineages are also nearly equal. Under the assumptions that the guinea pig lineage and the myomorph (mouse, rat, and hamster) lineage diverged 70–100 million years (Myr) ago and that the rate of nucleotide substitution has been constant in all these lineages since their divergence, the date of the mouse-rat split is estimated to be between 20 and 29 Myr ago, which is considerably older than the date ( 12 Myr) suggested by available rodent fossils and considerably younger than the date ( 35 Myr) suggested by Wilson and colleagues. The murid-hamster split is estimated to be 1.6 times older than the mouse-rat split.     

Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution

An excess of nonsynonymous substitutions over synonymous ones is an important indicator of positive selection at the molecular level. A lineage that underwent Darwinian selection may have a nonsynonymous/synonymous rate ratio (dN/dS) that is different from those of other lineages or greater than one. In this paper, several codon-based likelihood models that allow for variable dN/dS ratios among lineages were developed. They were then used to construct likelihood ratio tests to examine whether the dN/dS ratio is variable among evolutionary lineages, whether the ratio for a few lineages of interest is different from the background ratio for other lineages in the phylogeny, and whether the dN/dS ratio for the lineages of interest is greater than one. The tests were applied to the lysozyme genes of 24 primate species. The dN/dS ratios were found to differ significantly among lineages, indicating that the evolution of primate lysozymes is episodic, which is incompatible with the neutral theory. Maximum- likelihood estimates of parameters suggested that about nine nonsynonymous and zero synonymous nucleotide substitutions occurred in the lineage leading to hominoids, and the dN/dS ratio for that lineage is significantly greater than one. The corresponding estimates for the lineage ancestral to colobine monkeys were nine and one, and the dN/dS ratio for the lineage is not significantly greater than one, although it is significantly higher than the background ratio. The likelihood analysis thus confirmed most, but not all, conclusions Messier and Stewart reached using reconstructed ancestral sequences to estimate synonymous and nonsynonymous rates for different lineages.      

Intraspecific phylogeny and lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes

Listeria monocytogenes is a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years. In order to test hypotheses regarding L. monocytogenes lineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based on prfA virulence gene cluster sequences from 113 L. monocytogenes isolates. The results of the multigene phylogenetic analyses confirm that L. monocytogenes comprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested. However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment. Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the three L. monocytogenes lineages may represent distinct species within the framework of the cohesion species concept.     

Duplication and adaptive evolution of the COR15 genes within the highly cold-tolerant Draba lineage (Brassicaceae)

Plants have evolved diverse adaptive mechanisms that enable them to tolerate abiotic stresses, to varying degrees, and such stresses may have strongly influenced evolutionary changes at levels ranging from molecular to morphological. Previous studies on these phenomena have focused on the adaptive evolution of stress-related orthologous genes in specific lineages. However, heterogenetic evolution of the paralogous genes following duplication has only been examined in a very limited number of stress-response gene families. The COR15 gene encodes a low molecular weight protein that plays an important role in protecting plants from cold stresses. Although two different copies of this gene have been found in the model species, Arabidopsis thaliana, evolutionary patterns of this small gene family in plants have not been previously explored. In this study, we cloned COR15-like sequences and performed evolutionary analyses of these sequences (including those previously reported) in the highly cold-tolerant Draba lineage and related lineages of Brassicaceae. Our phylogenetic analyses indicate that all COR15-like sequences clustered into four clades that corresponded well to the morphological lineages. Gene conversions were found to have probably occurred before/during the divergence of Brassica and Draba lineage. However, repeated, independent duplications of this gene have occurred in different lineages of Brassicaceae. Further comparisons of all sequences suggest that there have been significant inter-lineage differences in evolutionary rates between the duplicated and original genes. We assessed the likelihood that the differences between two well-supported gene subfamilies that appear to have originated from a single duplication, COR15a and COR15b, within the Draba lineage have been driven by adaptive evolution. Comparisons of their non-synonymous/synonymous substitution ratios and rates of predicted amino acid changes indicate that these two gene groups are evolving under different selective pressures and may be functionally divergent. This functional divergence was confirmed by comparing site-specific shifts in evolution indexes of the two groups of predicted proteins. The evidence of differential selection and possible functional divergence suggests that the duplication may be of adaptive significance, with possible implications for the explosive diversification of the Draba lineage during the cooling Quaternary stages and the following worldwide colonization of arid alpine and artic regions.     

Molecular Phylogeny of Conjugating Green Algae (Zygnemophyceae,Streptophyta) Inferred from SSU rDNA Sequence Comparisons

Nuclear-encoded SSU rDNA sequences have been obtained from 64 strains of conjugating green algae (Zygnemophyceae, Streptophyta, Viridiplantae). Molecular phylogenetic analyses of 90 SSU rDNA sequences of Viridiplantae (inciuding 78 from the Zygnemophyceae) were performed using complex evolutionary models and maximum likelihood, distance, and maximum parsimony methods. The significance of the results was tested by bootstrap analyses, deletion of long-branch taxa, relative rate tests, and Kishino-Hasegawa tests with user-defined trees. All results support the monophyly of the class Zygnemophyceae and of the order Desmidiales. The second order, Zygnematales, forms a series of early-branching clades in paraphyletic succession, with the two traditional families Mesotaeniaceae and Zygnemataceae not recovered as lineages. Instead, a long-branch Spirogyra/Sirogonium clade and the later-diverging Netrium and Roya clades represent independent clades. Within the order Desmidiales, the families Gonatozygaceae and Closteriaceae are monophyletic, whereas the Peniaceae (represented only by Penium margaritaceum) and the Desmidiaceae represent a single weakly supported lineage. Within the Desmidiaceae short internal branches and varying rates of sequence evolution among taxa reduce the phylogenetic resolution significantly. The SSU rDNA-based phylogeny is largely congruent with a published analysis of the rbcL phylogeny of the Zygnemophyceae (McCourt et al. 2000) and is also in general agreement with classification schemes based on cell wall ultrastructure. The extended taxon sampling at the subgenus level provides solid evidence that many genera in the Zygnemophyceae are not monophyletic and that the genus concept in the group needs to be revised.     

Purifying selection and positive selection on the myxovirus resistance gene in mammals and chickens

The myxovirus resistance gene (Mx) expresses antiviral activity in many species, e.g. mouse, human and chicken. It is not clear if the antiviral activity of Mx has evolved in these species to inhibit a set of species-specific pathogens, nor what factors drive Mx evolution in different animal lineages. Therefore, it is important to determine the evolutionary pattern of Mx and positively selected sites which affect the antiviral activity of the Mx gene in mammals and birds. We used sequence comparisons among species to detect positively selected sites by conducting phylogenetic analysis. The two-ratio model was significantly better than the one-ratio model in four species (mouse, rat, chicken and duck, p<0.05). Although selection pressure varied among different lineages, Mx had strong purifying selection in mammals and positive selection in chicken and duck lineages. Relative rate test revealed that Mx evolved faster in chickens than in ducks (Tajima's relative rate test, chi(2)=7.17, p<0.01). In the further analysis using a branch-site model A test, 8 sites were positively selected in the chicken lineage while no positive selection signals were observed for any site in the other lineages. The branch-site model A test had a omega value of 4.374 for the chicken lineage (2Deltal=14.20, d.f.=1, p<0.001). Comparisons of all currently available Mx mRNA sequences showed that these predicted positively selected sites had been fixed in the chicken lineage, suggesting that the chicken Mx gene evolved within the species to resist newly challenging environments. There is an increased selection constraint leading to mammals, while positive selection has acted on the chicken Mx.