Topological Estimation Biases with Covarion Evolution

Covarion processes allow changes in evolutionary rates at sites along the branches of a phylogenetic tree. Covarion-like evolution is increasingly recognized as an important mode of protein evolution. Several recent reports suggest that maximum likelihood estimation employing covarion models may support different optimal topologies than estimation using standard rates-across-sites (RAS) models. However, it remains to be demonstrated that ignoring covarion evolution will generally result in topological misestimation. In this study we performed analytical and theoretical studies of limiting distances under the covarion model and four-taxon tree simulations to investigate the extent to which the covarion process impacts on phylogenetic estimation. In particular, we assessed the limits of an RAS model-based maximum likelihood method to recover the phylogenies when the sequence data were simulated under the covarion processes. We find that, when ignored, covarion processes can induce systematic errors in phylogeny reconstruction. Surprisingly, when sequences are evolved under a covarion process but an RAS model is used for estimation, we find that a long branch repel bias occurs.     

A mixed branch length model of heterotachy improves phylogenetic accuracy

Evolutionary relationships are typically inferred from molecular sequence data using a statistical model of the evolutionary process. When the model accurately reflects the underlying process, probabilistic phylogenetic methods recover the correct relationships with high accuracy. There is ample evidence, however, that models commonly used today do not adequately reflect real-world evolutionary dynamics. Virtually all contemporary models assume that relatively fast-evolving sites are fast across the entire tree, whereas slower sites always evolve at relatively slower rates. Many molecular sequences, however, exhibit site-specific changes in evolutionary rates, called "heterotachy." Here we examine the accuracy of 2 phylogenetic methods for incorporating heterotachy, the mixed branch length model--which incorporates site-specific rate changes by summing likelihoods over multiple sets of branch lengths on the same tree--and the covarion model, which uses a hidden Markov process to allow sites to switch between variable and invariable as they evolve. Under a variety of simple heterogeneous simulation conditions, the mixed model was dramatically more accurate than homotachous models, which were subject to topological biases as well as biases in branch length estimates. When data were simulated with strong versions of the types of heterotachy observed in real molecular sequences, the mixed branch length model was more accurate than homotachous techniques. Analyses of empirical data sets confirmed that the mixed branch length model can improve phylogenetic accuracy under conditions that cause homotachous models to fail. In contrast, the covarion model did not improve phylogenetic accuracy compared with homotachous models and was sometimes substantially less accurate. We conclude that a mixed branch length approach, although not the solution to all phylogenetic errors, is a valuable strategy for improving the accuracy of inferred trees.     

Testing for covarion-like evolution in protein sequences

The covarion hypothesis of molecular evolution proposes that selective pressures on an amino acid or nucleotide site change through time, thus causing changes of evolutionary rate along the edges of a phylogenetic tree. Several kinds of Markov models for the covarion process have been proposed. One model, proposed by Huelsenbeck (2002), has 2 substitution rate classes: the substitution process at a site can switch between a single variable rate, drawn from a discrete gamma distribution, and a zero invariable rate. A second model, suggested by Galtier (2001), assumes rate switches among an arbitrary number of rate classes but switching to and from the invariable rate class is not allowed. The latter model allows for some sites that do not participate in the rate-switching process. Here we propose a general covarion model that combines features of both models, allowing evolutionary rates not only to switch between variable and invariable classes but also to switch among different rates when they are in a variable state. We have implemented all 3 covarion models in a maximum likelihood framework for amino acid sequences and tested them on 23 protein data sets. We found significant likelihood increases for all data sets for the 3 models, compared with a model that does not allow site-specific rate switches along the tree. Furthermore, we found that the general model fit the data better than the simpler covarion models in the majority of the cases, highlighting the complexity in modeling the covarion process. The general covarion model can be used for comparing tree topologies, molecular dating studies, and the investigation of protein adaptation.     

Covarion structure in plastid genome evolution: a new statistical test

Covarion models of molecular evolution allow the rate of evolution of a site to vary through time. There are few simple and effective tests for covarion evolution, and consequently, little is known about the presence of covarion processes in molecular evolution. We describe two new tests for covarion evolution and demonstrate with simulations that they perform well under a wide range of conditions. A survey of covarion evolution in sequenced plastid genomes found evidence of covarion drift in at least 26 out of 57 genes. Covarion evolution is most evident in first and second codon positions of the plastid genes, and there is no evidence of covarion evolution in third codon positions. Therefore, the significant covarion tests are likely due to changes in the selective constraints of amino acids. The frequency of covarion evolution within the plastid genome suggests that covarion processes of evolution were important in generating the observed patterns of sequence variation among plastid genomes.     

Evolutionary processes among sylvatic dengue type 2 viruses

Sylvatic dengue viruses (DENV) are transmitted in an enzootic cycle between nonhuman primates and arboreal Aedes mosquitoes in Southeast Asia and West Africa. Although previous analyses have revealed the evolutionary processes among endemic (human) DENV, little is known about viral evolution in the sylvatic cycle. Through an analysis of 14 complete coding regions of sylvatic Dengue type 2 virus sampled over a 33-year period, we show that both the rate of evolutionary change and the pattern of natural selection are similar among endemic and sylvatic DENV, although the latter have a uniquely high frequency of positive selection in the NS4B protein gene. Our findings support a recent cross-species transmission event and suggest the possibility of future DENV reemergence from the sylvatic cycle.     

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.     

Heterotachy processes in rhodophyte-derived secondhand plastid genes: Implications for addressing the origin and evolution of dinoflagellate plastids

Serial transfer of plastids from one eukaryotic host to another is the key process involved in evolution of secondhand plastids. Such transfers drastically change the environment of the plastids and hence the selection regimes, presumably leading to changes over time in the characteristics of plastid gene evolution and to misleading phylogenetic inferences. About half of the dinoflagellate protists species are photosynthetic and unique in harboring a diversity of plastids acquired from a wide range of eukaryotic algae. They are therefore ideal for studying evolutionary processes of plastids gained through secondary and tertiary endosymbioses. In the light of these processes, we have evaluated the origin of 2 types of dinoflagellate plastids, containing the peridinin or 19'-hexanoyloxyfucoxanthin (19'-HNOF) pigments, by inferring the phylogeny using "covarion" evolutionary models allowing the pattern of among-site rate variation to change over time. Our investigations of genes from secondary and tertiary plastids derived from the rhodophyte plastid lineage clearly reveal "heterotachy" processes characterized as stationary covarion substitution patterns and changes in proportion of variable sites across sequences. Failure to accommodate covarion-like substitution patterns can have strong effects on the plastid tree topology. Importantly, multigene analyses performed with probabilistic methods using among-site rate and covarion models of evolution conflict with proposed single origin of the peridinin- and 19'-HNOF-containing plastids, suggesting that analysis of secondhand plastids can be hampered by convergence in the evolutionary signature of the plastid DNA sequences. Another type of sequence convergence was detected at protein level involving the psaA gene. Excluding the psaA sequence from a concatenated protein alignment grouped the peridinin plastid with haptophytes, congruent with all DNA trees. Altogether, taking account of complex processes involved in the evolution of dinoflagellate plastid sequences (both at the DNA and amino acid level), we demonstrate the difficulty of excluding independent, tertiary origin for both the peridinin and 19'-HNOF plastids involving engulfment of haptophyte-like algae. In addition, the refined topologies suggest the red algal order, Porphyridales, as the endosymbiont ancestor of the secondary plastids in cryptophytes, haptophytes, and heterokonts.     

Testing for differences in rates-across-sites distributions in phylogenetic subtrees

It has long been recognized that the rates of molecular evolution vary amongst sites in proteins. The usual model for rate heterogeneity assumes independent rate variation according to a rate distribution. In such models the rate at a site, although random, is assumed fixed throughout the evolutionary tree. Recent work by several groups has suggested that rates at sites often vary across subtrees of the larger tree as well as across sites. This phenomenon is not captured by most phylogenetic models but instead is more similar to the covarion model of Fitch and coworkers. In this article we present methods that can be useful in detecting whether different rates occur in two different subtrees of the larger tree and where these differences occur. Parametric bootstrapping and orthogonal regression methodologies are used to test for rate differences and to make statements about the general differences in the rates at sites. Confidence intervals based on the conditional distributions of rates at sites are then used to detect where the rate differences occur. Such methods will be helpful in studying the phylogenetic, structural, and functional bases of changes in evolutionary rates at sites, a phenomenon that has important consequences for deep phylogenetic inference.     

Evolution of endemic and sylvatic lineages of dengue virus

Recent disease outbreaks have raised awareness of tropical pathogens, especially mosquito-borne viruses. Dengue virus (DENV) is a widely studied mammalian pathogen transmitted by various species of mosquito in the genus Aedes, especially Aedes aegypti and Aedes albopictus. The prevailing view of the research community is that endemic viral lineages that cause epidemics of DENV in humans have emerged over time from sylvatic viral lineages, which persist in wild, non-human primates. These notions have been examined by researchers through phylogenetic analyses of the envelope gene (E) from the four serotypes of DENV (serotypes DENV-1 to DENV-4). In these previous reports, researchers used visual inspection of a phylogeny in order to assert that sylvatic lineages lead to endemic clades. In making this assertion, these researchers also reasserted the model of periodic sylvatic to endemic disease outbreaks. Since that study, there has been a significant increase in data both in terms of metadata (e.g., place and host of isolation) and genetic sequences of DENV. Here, we re-examine the model of sylvatic to endemic shifts in viral lineages through a phylogenetic tree search and character evolution study of metadata on the tree. We built a dataset of nucleotide sequences for 188 isolates of DENV that have metadata on sylvatic or endemic sampling along with three orthologous sequences from West Nile virus as the outgroup for the phylogenetic analysis. In contrast to previous research, we find that there are several shifts from endemic to sylvatic lineages as well as sylvatic to endemic lineages, indicating a much more dynamic model of evolution. We propose that a model that allows oscillation between sylvatic and endemic hosts better captures the dynamics of DENV transmission.     

The identifiability of tree topology for phylogenetic models, including covarion and mixture models.

For a model of molecular evolution to be useful for phylogenetic inference, the topology of evolutionary trees must be identifiable. That is, from a joint distribution the model predicts, it must be possible to recover the tree parameter. We establish tree identifiability for a number of phylogenetic models, including a covarion model and a variety of mixture models with a limited number of classes. The proof is based on the introduction of a more general model, allowing more states at internal nodes of the tree than at leaves, and the study of the algebraic variety formed by the joint distributions to which it gives rise. Tree identifiability is first established for this general model through the use of certain phylogenetic invariants.     

A covarion-based method for detecting molecular adaptation: application to the evolution of primate mitochondrial genomes

A new method for detecting site-specific variation of evolutionary rate (the so-called covarion process) from protein sequence data is proposed. It involves comparing the maximum-likelihood estimates of the replacement rate of an amino acid site in distinct subtrees of a large tree. This approach allows detection of covarion at the gene or the amino acid levels. The method is applied to mammalian-mitochondrial-protein sequences. Significant covarion-like evolution is found in the (simian) primate lineage: some amino acid positions are fast-evolving (i.e. unconstrained) in non-primate mammals but slow-evolving (i.e. highly constrained) in primates, and some show the opposite pattern. Our results indicate that the mitochondrial genome of primates reached a new peak of the adaptive landscape through positive selection.     

Testing the covarion hypothesis of molecular evolution

The covarion hypothesis of molecular evolution states that the fixation of mutations may alter the probability that any given position will fix the next change. Tests of this hypothesis using the divergence of real sequences are compromised because models of rate variation among sites (e.g., the gamma version of the one-parameter equation) predict sequence divergence values similar to those for the covarion process. This study therefore focuses on the extent to which the varied and unvaried codons of two well-diverged taxa are the same, because fewer are expected by the covarion hypothesis than by the gamma model. The data for these tests are the protein sequences of Cu, Zn superoxide dismutase (SOD) for mammals and plants. Simulation analyses show that the covarion hypothesis makes better predictions about the frequencies of varied and unhit positions in common between these two taxa than does the gamma version of the one-parameter model. Furthermore, the analysis of SOD tertiary structure demonstrates that mammal and plant variabilities are distributed differently on the protein. These results support the conclusions that the variable and invariable codons of mammal and plant SODs are different and that the covarion model explains the evolution of this protein better than the gamma version of the one-parameter process. Unlike other models, the covarion hypothesis accounts for rate fluctuations among positions over time, which is an important parameter of molecular evolution.      

A Molecular Genetic Timescale for the Diversification of Autotrophic Stramenopiles (Ochrophyta): Substantive Underestimation of Putative Fossil Ages

Background

Stramenopiles constitute a large and diverse eukaryotic clade that is currently poorly characterized from both phylogenetic and temporal perspectives at deeper taxonomic levels. To better understand this group, and in particular the photosynthetic stramenopiles (Ochrophyta), we analyzed sequence data from 135 taxa representing most major lineages. Our analytical approach utilized several recently developed methods that more realistically model the temporal evolutionary process.

Methodology/Principal Findings

Phylogenetic reconstruction employed a Bayesian joint rate- and pattern-heterogeneity model to reconstruct the evolutionary history of these taxa. Inferred phylogenetic resolution was generally high at all taxonomic levels, sister-class relationships in particular receiving good statistical support. A signal for heterotachy was detected in clustered portions of the tree, although this does not seem to have had a major influence on topological inference. Divergence time estimates, assuming a lognormally-distributed relaxed molecular clock while accommodating topological uncertainty, were broadly congruent over alternative temporal prior distributions. These data suggest that Ochrophyta originated near the Proterozoic-Phanerozoic boundary, diverging from their sister-taxon Oomycota. The evolution of the major ochrophyte lineages appears to have proceeded gradually thereafter, with most lineages coming into existence by ∼200 million years ago.

Conclusions/Significance

The evolutionary timescale of the autotrophic stramenopiles reconstructed here is generally older than previously inferred from molecular clocks. However, this more ancient timescale nevertheless casts serious doubt on the taxonomic validity of putative xanthophyte/phaeophyte fossils from the Proterozoic, which predate by as much as a half billion years or more the age suggested by our molecular genetic data. If these fossils truly represent crown stramenopile lineages, then this would imply that molecular rate evolution in this group proceeds in a fashion that is fundamentally incompatible with the relaxed molecular clock model employed here. A more likely scenario is that there is considerable convergent morphological evolution within Heterokonta, and that these fossils have been taxonomically misdiagnosed.     

The Identifiability of Covarion Models in Phylogenetics

Covarion models of character evolution describe inhomogeneities in substitution processes through time. In phylogenetics, such models are used to describe changing functional constraints or selection regimes during the evolution of biological sequences. In this work the identifiability of such models for generic parameters on a known phylogenetic tree is established, provided the number of covarion classes does not exceed the size of the observable state space. `Generic parameters' as used here means all parameters except possibly those in a set of measure zero within the parameter space. Combined with earlier results, this implies both the tree and generic numerical parameters are identifiable if the number of classes is strictly smaller than the number of observable states.     

Microevolution of Dengue viruses circulating among primary school children in Kamphaeng Phet, Thailand

To determine the extent and structure of genetic variation in dengue viruses (DENV) on a restricted spatial and temporal scale, we sequenced the E (envelope) genes of DENV-1, -2, and -3 isolates collected in 2001 from children enrolled in a prospective school-based study in Kamphaeng Phet, Thailand, and diagnosed with dengue disease. Our analysis revealed substantial viral genetic variation in both time and space, with multiple viral lineages circulating within individual schools, suggesting the frequent gene flow of DENV into this microenvironment. More-detailed analyses of DENV-2 samples revealed strong clustering of viral isolates within individual schools and evidence of more-frequent viral gene flow among schools closely related in space. Conversely, we observed little evolutionary change in those viral isolates sampled over multiple time points within individual schools, indicating a low rate of mutation fixation. These results suggest that frequent viral migration into Kamphaeng Phet, coupled with population (school) subdivision, shapes the genetic diversity of DENV on a local scale, more so than in situ evolution within school catchment areas.     

Chronology of Deep Nodes in the Neotropical Primate Phylogeny: Insights from Mitochondrial Genomes

The evolution of Neotropical Primates (NP) is permeated by factors associated with the pattern of diversification and the biogeography of the major lineages. These questions can be better understood by providing a robust estimate of the chronological scenario of NP evolution, a reason why molecular dating methods have been widely applied. One aspect of especial interest is the timing of diversification of the major NP lineages (pitheciids, atelids and cebids), which may have resulted from rapid episodes of adaptive radiation, a question that requires NP divergence time estimates with accurate statistical certainty. In this study, we evaluated the primate timescale focused on the age of nodes of NP radiation. We investigated the performance of complete primate mitochondrial genomes as traditional molecular markers of primate evolution and further including original mitochondrial data from the endangered muriqui, Brachyteles arachnoides (Accession No. {"type":"entrez-nucleotide","attrs":{"text":"JX262672","term_id":"403488686","term_text":"JX262672"}}JX262672). Comparisons of the age estimates at NP nodes based on mitochondrial genomes with those obtained from a nuclear supermatrix showed similar degrees of uncertainty. Further molecular data and more informative calibration priors are required for a more precise understanding of the early NP diversification.     

Tree imbalance causes a bias in phylogenetic estimation of evolutionary timescales using heterochronous sequences

Phylogenetic estimation of evolutionary timescales has become routine in biology, forming the basis of a wide range of evolutionary and ecological studies. However, there are various sources of bias that can affect these estimates. We investigated whether tree imbalance, a property that is commonly observed in phylogenetic trees, can lead to reduced accuracy or precision of phylogenetic timescale estimates. We analysed simulated data sets with calibrations at internal nodes and at the tips, taking into consideration different calibration schemes and levels of tree imbalance. We also investigated the effect of tree imbalance on two empirical data sets: mitogenomes from primates and serial samples of the African swine fever virus. In analyses calibrated using dated, heterochronous tips, we found that tree imbalance had a detrimental impact on precision and produced a bias in which the overall timescale was underestimated. A pronounced effect was observed in analyses with shallow calibrations. The greatest decreases in accuracy usually occurred in the age estimates for medium and deep nodes of the tree. In contrast, analyses calibrated at internal nodes did not display a reduction in estimation accuracy or precision due to tree imbalance. Our results suggest that molecular‐clock analyses can be improved by increasing taxon sampling, with the specific aims of including deeper calibrations, breaking up long branches and reducing tree imbalance.     

PROCOV: maximum likelihood estimation of protein phylogeny under covarion models and site-specific covarion pattern analysis

Background  

The covarion hypothesis of molecular evolution holds that selective pressures on a given amino acid or nucleotide site are dependent on the identity of other sites in the molecule that change throughout time, resulting in changes of evolutionary rates of sites along the branches of a phylogenetic tree. At the sequence level, covarion-like evolution at a site manifests as conservation of nucleotide or amino acid states among some homologs where the states are not conserved in other homologs (or groups of homologs). Covarion-like evolution has been shown to relate to changes in functions at sites in different clades, and, if ignored, can adversely affect the accuracy of phylogenetic inference.     

Analyses of evolutionary dynamics in viruses are hindered by a time-dependent bias in rate estimates

Time-scales of viral evolution and emergence have been studied widely, but are often poorly understood. Molecular analyses of viral evolutionary time-scales generally rely on estimates of rates of nucleotide substitution, which vary by several orders of magnitude depending on the timeframe of measurement. We analysed data from all major groups of viruses and found a strong negative relationship between estimates of nucleotide substitution rate and evolutionary timescale. Strikingly, this relationship was upheld both within and among diverse groups of viruses. A detailed case study of primate lentiviruses revealed that the combined effects of sequence saturation and purifying selection can explain this time-dependent pattern of rate variation. Therefore, our analyses show that studies of evolutionary time-scales in viruses require a reconsideration of substitution rates as a dynamic, rather than as a static, feature of molecular evolution. Improved modelling of viral evolutionary rates has the potential to change our understanding of virus origins.