Fabia U. Battistuzzi

Estimating divergence times in large molecular phylogenies

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.

Evolution of Modern Birds Revealed by Mitogenomics: Timing the Radiation and Origin of Major Orders

Mitochondrial (mt) genes and genomes are among the major sources of data for evolutionary studies in birds. This places mitogenomic studies in birds at the core of intense debates in avian evolutionary biology. Indeed, complete mt genomes are actively been used to unveil the phylogenetic relationships among major orders, whereas single genes (e.g., cytochrome c oxidase I [COX1]) are considered standard for species identification and defining species boundaries (DNA barcoding). In this investigation, we study the time of origin and evolutionary relationships among Neoaves orders using complete mt genomes. First, we were able to solve polytomies previously observed at the deep nodes of the Neoaves phylogeny by analyzing 80 mt genomes, including 17 new sequences reported in this investigation. As an example, we found evidence indicating that columbiforms and charadriforms are sister groups. Overall, our analyses indicate that by improving the taxonomic sampling, complete mt genomes can solve the evolutionary relationships among major bird groups. Second, we used our phylogenetic hypotheses to estimate the time of origin of major avian orders as a way to test if their diversification took place prior to the Cretaceous/Tertiary (K/T) boundary. Such timetrees were estimated using several molecular dating approaches and conservative calibration points. Whereas we found time estimates slightly younger than those reported by others, most of the major orders originated prior to the K/T boundary. Finally, we used our timetrees to estimate the rate of evolution of each mt gene. We found great variation on the mutation rates among mt genes and within different bird groups. COX1 was the gene with less variation among Neoaves orders and the one with the least amount of rate heterogeneity across lineages. Such findings support the choice of COX 1 among mt genes as target for developing DNA barcoding approaches in birds.

A Major Clade of Prokaryotes with Ancient Adaptations to Life on Land

Evolutionary trees of prokaryotes usually define the known classes and phyla but less often agree on the relationships among those groups. This has been attributed to the effects of horizontal gene transfer, biases in sequence change, and large evolutionary distances. Furthermore, higher level clades of prokaryote phyla rarely are supported by information from ecology and cell biology. Nonetheless, common patterns are beginning to emerge as larger numbers of species are analyzed with sophisticated methods. Here, we show how combined evidence from phylogenetic, cytological, and environmental data support the existence of an evolutionary group that appears to have had a common ancestor on land early in Earths history and includes two-thirds of known prokaryote species. Members of this terrestrial clade (Terrabacteria), which includes Cyanobacteria, the gram-positive phyla (Actinobacteria and Firmicutes), and two phyla with cell walls that differ structurally from typical gram-positive and gram-negative phyla (Chloroflexi and Deinococcus-Thermus), possess important adaptations such as resistance to environmental hazards (e.g., desiccation, ultraviolet radiation, and high salinity) and oxygenic photosynthesis. Moreover, the unique properties of the cell wall in gram-positive taxa, which likely evolved in response to terrestrial conditions, have contributed toward pathogenicity in many species. These results now leave open the possibility that terrestrial adaptations may have played a larger role in prokaryote evolution than currently understood.

Statistics and Truth in Phylogenomics

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.

Performance of Relaxed-Clock Methods in Estimating Evolutionary Divergence Times and Their Credibility Intervals

The rapid expansion of sequence data and the development of statistical approaches that embrace varying evolutionary rates among lineages have encouraged many more investigators to use DNA and protein data to time species divergences. Here, we report results from a systematic evaluation, by means of computer simulation, of the performance of two frequently used relaxed-clock methods for estimating these times and their credibility intervals (CrIs). These relaxed-clock methods allow rates to vary in a phylogeny randomly over lineages (e.g., BEAST software) and in autocorrelated fashion (e.g., MultiDivTime software). We applied these methods for analyzing sequence data sets simulated using naturally derived parameters (evolutionary rates, sequence lengths, and base substitution patterns) and assuming that clock calibrations are known without error. We find that the estimated times are, on average, close to the true times as long as the assumed model of lineage rate changes matches the actual model. The 95% CrIs also contain the true time for >or=95% of the simulated data sets. However, the use of incorrect lineage rate model reduces this frequency to 83%, indicating that the relaxed-clock methods are not robust to the violation of underlying lineage rate model. Because these rate models are rarely known a priori and are difficult to detect empirically, we suggest building composite CrIs using CrIs produced from MultiDivTime and BEAST analysis. These composite CrIs are found to contain the true time for >or=97% data sets. Our analyses also verify the usefulness of the common practice of interpreting the congruence of times inferred from different methods as a reflection of the accuracy of time estimates. Overall, our results show that simple strategies can be used to enhance our ability to estimate times and their CrIs when using the relaxed-clock methods.

Timing the origin of human malarias: the lemur puzzle

BackgroundTiming the origin of human malarias has been a focus of great interest. Previous studies on the mitochondrial genome concluded that Plasmodium in primates, including those parasitic to humans, radiated relatively recently during a process where host switches were common. Those investigations, however, assumed constant rate of evolution and tightly bound (fixed) calibration points based on host fossils or host distribution. We investigate the effect of such assumptions using different molecular dating methods. We include parasites from Lemuroidea since their distribution provides an external validation to time estimates allowing us to disregard scenarios that cannot explain their introduction in Madagascar.ResultsWe reject the assumption that the Plasmodium mitochondrial genome, as a unit or each gene separately, evolves at a constant rate. Our analyses show that Lemuroidea parasites are a monophyletic group that shares a common ancestor with all Catarrhini malarias except those related to P. falciparum. However, we found no evidence that this group of parasites branched with their hosts early in the evolution of primates. We applied relaxed clock methods and different calibrations points to explore the origin of primate malarias including those found in African apes. We showed that previous studies likely underestimated the origin of malarial parasites in primates.ConclusionsThe use of fossils from the host as absolute calibration and the assumption of a strict clock likely underestimate time when performing molecular dating analyses on malarial parasites. Indeed, by exploring different calibration points, we found that the time for the radiation of primate parasites may have taken place in the Eocene, a time consistent with the radiation of African anthropoids. The radiation of the four human parasite lineages was part of such events. The time frame estimated in this investigation, together with our phylogenetic analyses, made plausible a scenario where gorillas and humans acquired malaria from a Pan lineage.

More reliable estimates of divergence times in Pan using complete mtDNA sequences and accounting for population structure

Here, we report the sequencing and analysis of eight complete mitochondrial genomes of chimpanzees (Pan troglodytes) from each of the three established subspecies (P. t. troglodytes, P. t. schweinfurthii and P. t. verus) and the proposed fourth subspecies (P. t. ellioti). Our population genetic analyses are consistent with neutral patterns of evolution that have been shaped by demography. The high levels of mtDNA diversity in western chimpanzees are unlike those seen at nuclear loci, which may reflect a demographic history of greater female to male effective population sizes possibly owing to the characteristics of the founding population. By using relaxed-clock methods, we have inferred a timetree of chimpanzee species and subspecies. The absolute divergence times vary based on the methods and calibration used, but relative divergence times show extensive uniformity. Overall, mtDNA produces consistently older times than those known from nuclear markers, a discrepancy that is reduced significantly by explicitly accounting for chimpanzee population structures in time estimation. Assuming the human–chimpanzee split to be between 7 and 5 Ma, chimpanzee time estimates are 2.1–1.5, 1.1–0.76 and 0.25–0.18 Ma for the chimpanzee/bonobo, western/(eastern + central) and eastern/central chimpanzee divergences, respectively.

Molecular evidence for an Asian origin of monitor lizards followed by Tertiary dispersals to Africa and Australasia

Monitor lizards are emblematic reptiles that are widely distributed in the Old World. Although relatively well studied in vertebrate research, their biogeographic history is still controversial. We constructed a molecular dataset for 54 anguimorph species, including representatives of all families with detailed sampling of the Varanidae (38 species). Our results are consistent with an Asian origin of the Varanidae followed by a dispersal to Africa 41 (49–33) Ma, possibly via an Iranian route. Another major event was the dispersal of monitors to Australia in the Late Eocene–Oligocene 32 (39–26) Ma. This divergence estimate adds to the suggestion that Australia was colonized by several squamate lineages prior to the collision of the Australian plate with the Asian plate starting 25 Ma.

Fast and Slow Implementations of Relaxed-Clock Methods Show Similar Patterns of Accuracy in Estimating Divergence Times

Phylogenetic analyses are using increasingly larger data sets for estimating divergence times. With this increase in data sizes, the computation time required is becoming a bottleneck in evolutionary investigations. Our recent study of two relaxed-clock programs (BEAST and MultiDivTime [MDT]) showed their usefulness in time estimation; however, they place a significant computational time burden on biologists even for moderately small data sets. Here, we report speed and accuracy of another relaxed-clock program (MCMCTree, MC2T). We find it to be much faster than both MDT and BEAST while producing comparable time estimates. These results will encourage the analysis of larger data sets as well as the evaluation of the robustness of estimated times to changes in the model of evolutionary rates and clock calibrations.

A Protocol for Diagnosing the Effect of Calibration Priors on Posterior Time Estimates: A Case Study for the Cambrian Explosion of Animal Phyla

We present a procedure to test the effect of calibration priors on estimated times, which applies a recently developed calibration-free approach (RelTime) method that produces relative divergence times for all nodes in the tree. We illustrate this protocol by applying it to a timetree of metazoan diversification (Erwin DH, Laflamme M, Tweedt SM, Sperling EA, Pisani D, Peterson KJ. 2011. The Cambrian conundrum: early divergence and later ecological success in the early history of animals. Science 334:1091-1097.), which placed the divergence of animal phyla close to the time of the Cambrian explosion inferred from the fossil record. These analyses revealed that the two maximum-only calibration priors in the pre-Cambrian are the primary determinants of the young divergence times among animal phyla in this study. In fact, these two maximum-only calibrations produce divergence times that severely violate minimum boundaries of almost all of the other 22 calibration constraints. The use of these 22 calibrations produces dates for metazoan divergences that are hundreds of millions of years earlier in the Proterozoic. Our results encourage the use of calibration-free approaches to identify most influential calibration constraints and to evaluate their impact in order to achieve biologically robust interpretations.