We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

Molecular dating of species divergences has become an important means to add a temporal dimension to the Tree of Life. Increasingly larger datasets encompassing greater taxonomic diversity are becoming available to generate molecular timetrees by using sophisticated methods that model rate variation among lineages. However, the practical application of these methods is challenging because of the exorbitant calculation times required by current methods for contemporary data sizes, the difficulty in correctly modeling the rate heterogeneity in highly diverse taxonomic groups, and the lack of reliable clock calibrations and their uncertainty distributions for most groups of species. Here, we present a method that estimates relative times of divergences for all branching points (nodes) in very large phylogenetic trees without assuming a specific model for lineage rate variation or specifying any clock calibrations. The method (RelTime) performed better than existing methods when applied to very large computer simulated datasets where evolutionary rates were varied extensively among lineages by following autocorrelated and uncorrelated models. On average, RelTime completed calculations 1,000 times faster than the fastest Bayesian method, with even greater speed difference for larger number of sequences. This speed and accuracy will enable molecular dating analysis of very large datasets. Relative time estimates will be useful for determining the relative ordering and spacing of speciation events, identifying lineages with significantly slower or faster evolutionary rates, diagnosing the effect of selected calibrations on absolute divergence times, and estimating absolute times of divergence when highly reliable calibration points are available.bioinformatics | timescales | relaxed clocks T housands of research studies have reported the use of molecular dating techniques in establishing the timing of species divergences (e.g., refs. 1-5). With the availability of fast and cheap genome sequencing, molecular dating is being applied to increasingly larger datasets that span a much greater diversity of species and harbor extensive heterogeneity of evolutionary rates among lineages. This complexity poses many challenges that limit modern scientific investigations from truly leveraging the genome revolution. First, the application of the fastest molecular dating tools available already requires a very large amount of computational time for datasets containing only a few hundred sequences, which are modest for today's standards (6, 7). Second, current approaches require a priori selection of statistical distributions to model the heterogeneity of rates among branches in the evolutionary tree (e.g., autocorrelated versus uncorrelated rates, 8-12). Use of an incorrect statistical distribution is known to introduce significant bias in such analyses (10,(13)(14)(15). With increasingly larger datasets, it is unlikely that the same rate model will fit evolutionarily distant groups in the same large phylogeny, which exacerbates the...

Phylogenomics refers to the inference of historical relationships among species using genome-scale sequence data and to the use of phylogenetic analysis to infer protein function in multigene families. With rapidly decreasing sequencing costs, phylogenomics is becoming synonymous with evolutionary analysis of genome-scale and taxonomically densely sampled data sets. In phylogenetic inference applications, this translates into very large data sets that yield evolutionary and functional inferences with extremely small variances and high statistical confidence (P value). However, reports of highly significant P values are increasing even for contrasting phylogenetic hypotheses depending on the evolutionary model and inference method used, making it difficult to establish true relationships. We argue that the assessment of the robustness of results to biological factors, that may systematically mislead (bias) the outcomes of statistical estimation, will be a key to avoiding incorrect phylogenomic inferences. In fact, there is a need for increased emphasis on the magnitude of differences (effect sizes) in addition to the P values of the statistical test of the null hypothesis. On the other hand, the amount of sequence data available will likely always remain inadequate for some phylogenomic applications, for example, those involving episodic positive selection at individual codon positions and in specific lineages. Again, a focus on effect size and biological relevance, rather than the P value, may be warranted. Here, we present a theoretical overview and discuss practical aspects of the interplay between effect sizes, bias, and P values as it relates to the statistical inference of evolutionary truth in phylogenomics.

Molecular clocks have been used to date the divergence of humans and chimpanzees for nearly four decades. Nonetheless, this date and its confidence interval remain to be firmly established. In an effort to generate a genomic view of the human-chimpanzee divergence, we have analyzed 167 nuclear protein-coding genes and built a reliable confidence interval around the calculated time by applying a multifactor bootstrap-resampling approach. Bayesian and maximum likelihood analyses of neutral DNA substitutions show that the human-chimpanzee divergence is close to 20% of the ape-Old World monkey (OWM) divergence. Therefore, the generally accepted range of 23.8 -35 millions of years ago for the ape-OWM divergence yields a range of 4.98 -7.02 millions of years ago for human-chimpanzee divergence. Thus, the older time estimates for the human-chimpanzee divergence, from molecular and paleontological studies, are unlikely to be correct. For a given the ape-OWM divergence time, the 95% confidence interval of the human-chimpanzee divergence ranges from ؊12% to 19% of the estimated time. Computer simulations suggest that the 95% confidence intervals obtained by using a multifactor bootstrapresampling approach contain the true value with >95% probability, whether deviations from the molecular clock are random or correlated among lineages. Analyses revealed that the use of amino acid sequence differences is not optimal for dating humanchimpanzee divergence and that the inclusion of additional genes is unlikely to narrow the confidence interval significantly. We conclude that tests of hypotheses about the timing of human-chimpanzee divergence demand more precise fossil-based calibrations.Bayesian analysis ͉ molecular clock ͉ hominid ͉ fossil ͉ primate D etermining the age of the most recent common ancestor of humans and their closest African ape relatives has been the subject of scientific inquiry for over a century. This age is important for assessing the evolutionary rate of morphological and molecular changes in humans, assigning key fossils in hominoid phylogeny, and estimating when the common ancestor of all humans lived. The paleontological approach has been hampered by the paucity of fossils of some lineages. The first chimpanzee fossil, for example, was only just reported in 2005 (1). As recently as the mid-1960s (2), the African apes were considered to be distant relatives of the human lineage, but subsequent molecular phylogenetic analyses have shown that the chimpanzee and human are sister species and have led to a revision of the age of their divergence (3-13). Currently, the earliest unequivocal upright hominids at 4.2 millions of years ago (Ma) provide the minimum age for human-chimpanzee divergence (14), and some recently proposed early hominids dated between 5 and slightly more than 6 Ma are thought to provide an estimate close to the actual species divergence (15-19). However, a divergence time of 12.5 Ma for humans and chimpanzees has been entertained recently as well (20), and so the fossil record and its inte...