Bayesian relaxed clock estimation of divergence times in foraminifera

Accurate and precise estimation of divergence times during the Neo-Proterozoic is necessary to understand the speciation dynamic of early Eukaryotes. However such deep divergences are difficult to date, as the molecular clock is seriously violated. Recent improvements in Bayesian molecular dating techniques allow the relaxation of the molecular clock hypothesis as well as incorporation of multiple and flexible fossil calibrations. Divergence times can then be estimated even when the evolutionary rate varies among lineages and even when the fossil calibrations involve substantial uncertainties. In this paper, we used a Bayesian method to estimate divergence times in Foraminifera, a group of unicellular eukaryotes, known for their excellent fossil record but also for the high evolutionary rates of their genomes. Based on multigene data we reconstructed the phylogeny of Foraminifera and dated their origin and the major radiation events. Our estimates suggest that Foraminifera emerged during the Cryogenian (650-920 Ma, Neo-Proterozoic), with a mean time around 770 Ma, about 220 Myr before the first appearance of reliable foraminiferal fossils in sediments (545 Ma). Most dates are in agreement with the fossil record, but in general our results suggest earlier origins of foraminiferal orders. We found that the posterior time estimates were robust to specifications of the prior. Our results highlight inter-species variations of evolutionary rates in Foraminifera. Their effect was partially overcome by using the partitioned Bayesian analysis to accommodate rate heterogeneity among data partitions and using the relaxed molecular clock to account for changing evolutionary rates. However, more coding genes appear necessary to obtain more precise estimates of divergence times and to resolve the conflicts between fossil and molecular date estimates.     

Bayesian estimation of species divergence times under a molecular clock using multiple fossil calibrations with soft bounds

We implement a Bayesian Markov chain Monte Carlo algorithm for estimating species divergence times that uses heterogeneous data from multiple gene loci and accommodates multiple fossil calibration nodes. A birth-death process with species sampling is used to specify a prior for divergence times, which allows easy assessment of the effects of that prior on posterior time estimates. We propose a new approach for specifying calibration points on the phylogeny, which allows the use of arbitrary and flexible statistical distributions to describe uncertainties in fossil dates. In particular, we use soft bounds, so that the probability that the true divergence time is outside the bounds is small but nonzero. A strict molecular clock is assumed in the current implementation, although this assumption may be relaxed. We apply our new algorithm to two data sets concerning divergences of several primate species, to examine the effects of the substitution model and of the prior for divergence times on Bayesian time estimation. We also conduct computer simulation to examine the differences between soft and hard bounds. We demonstrate that divergence time estimation is intrinsically hampered by uncertainties in fossil calibrations, and the error in Bayesian time estimates will not go to zero with increased amounts of sequence data. Our analyses of both real and simulated data demonstrate potentially large differences between divergence time estimates obtained using soft versus hard bounds and a general superiority of soft bounds. Our main findings are as follows. (1) When the fossils are consistent with each other and with the molecular data, and the posterior time estimates are well within the prior bounds, soft and hard bounds produce similar results. (2) When the fossils are in conflict with each other or with the molecules, soft and hard bounds behave very differently; soft bounds allow sequence data to correct poor calibrations, while poor hard bounds are impossible to overcome by any amount of data. (3) Soft bounds eliminate the need for "safe" but unrealistically high upper bounds, which may bias posterior time estimates. (4) Soft bounds allow more reliable assessment of estimation errors, while hard bounds generate misleadingly high precisions when fossils and molecules are in conflict.     

一个适于最大似然法估计物种分化年代的进化速率平滑近似方法

众所周知 ,物种分化年代的估计对分子钟 (进化速率恒定 )假定很敏感。另一方面 ,在远缘物种 (例如哺乳纲不同目的动物 )的比较中 ,分子钟几乎总是不成立的。这样在估计分化时间时考虑不同进化区系的速率差异至为重要。最大似然法可以很自然地考虑这种速率差异 ,并且可以同时分析多个基因位点的资料以及同时利用多重化石校正数据。以前提出的似然法需要研究者将进化树的树枝按速率分组 ,本文提出一个近似方法以使这个过程自动化。本方法综合了以前的似然法、贝斯法及近似速率平滑法的一些特征。此外 ,还对算法加以改进 ,以适应综合数据分析时某些基因在某些物种中缺乏资料的情形。应用新提出的方法来分析马达加斯加的倭狐猴的分化年代 ,并与以前的似然法及贝斯法的分析进行了比较     

Integrating fossil preservation biases in the selection of calibrations for molecular divergence time estimation

The selection of fossil data to use as calibration age priors in molecular divergence time estimates inherently links neontological methods with paleontological theory. However, few neontological studies have taken into account the possibility of a taphonomic bias in the fossil record when developing approaches to fossil calibration selection. The Sppil-Rongis effect may bias the first appearance of a lineage toward the recent causing most objective calibration selection approaches to erroneously exclude appropriate calibrations or to incorporate multiple calibrations that are too young to accurately represent the divergence times of target lineages. Using turtles as a case study, we develop a Bayesian extension to the fossil selection approach developed by Marshall (2008. A simple method for bracketing absolute divergence times on molecular phylogenies using multiple fossil calibrations points. Am. Nat. 171:726-742) that takes into account this taphonomic bias. Our method has the advantage of identifying calibrations that may bias age estimates to be too recent while incorporating uncertainty in phylogenetic parameter estimates such as tree topology and branch lengths. Additionally, this method is easily adapted to assess the consistency of potential calibrations to any one calibration in the candidate pool.     

Are pollen fossils useful for calibrating relaxed molecular clock dating of phylogenies? A comparative study using Myrtaceae

The identification and application of reliable fossil calibrations represents a key component of many molecular studies of evolutionary timescales. In studies of plants, most paleontological calibrations are associated with macrofossils. However, the pollen record can also inform age calibrations if fossils matching extant pollen groups are found. Recent work has shown that pollen of the myrtle family, Myrtaceae, can be classified into a number of morphological groups that are synapomorphic with molecular groups. By assembling a data matrix of pollen morphological characters from extant and fossil Myrtaceae, we were able to measure the fit of 26 pollen fossils to a molecular phylogenetic tree using parsimony optimisation of characters. We identified eight Myrtaceidites fossils as appropriate for calibration based on the most parsimonious placements of these fossils on the tree. These fossils were used to inform age constraints in a Bayesian phylogenetic analysis of a sequence alignment comprising two sequences from the chloroplast genome (matK and ndhF) and one nuclear locus (ITS), sampled from 106 taxa representing 80 genera. Three additional analyses were calibrated by placing pollen fossils using geographic and morphological information (eight calibrations), macrofossils (five calibrations), and macrofossils and pollen fossils in combination (12 calibrations). The addition of new fossil pollen calibrations led to older crown ages than have previously been found for tribes such as Eucalypteae and Myrteae. Estimates of rate variation among lineages were affected by the choice of calibrations, suggesting that the use of multiple calibrations can improve estimates of rate heterogeneity among lineages. This study illustrates the potential of including pollen-based calibrations in molecular studies of divergence times.     

A Simple Method for Estimating Informative Node Age Priors for the Fossil Calibration of Molecular Divergence Time Analyses

Molecular divergence time analyses often rely on the age of fossil lineages to calibrate node age estimates. Most divergence time analyses are now performed in a Bayesian framework, where fossil calibrations are incorporated as parametric prior probabilities on node ages. It is widely accepted that an ideal parameterization of such node age prior probabilities should be based on a comprehensive analysis of the fossil record of the clade of interest, but there is currently no generally applicable approach for calculating such informative priors. We provide here a simple and easily implemented method that employs fossil data to estimate the likely amount of missing history prior to the oldest fossil occurrence of a clade, which can be used to fit an informative parametric prior probability distribution on a node age. Specifically, our method uses the extant diversity and the stratigraphic distribution of fossil lineages confidently assigned to a clade to fit a branching model of lineage diversification. Conditioning this on a simple model of fossil preservation, we estimate the likely amount of missing history prior to the oldest fossil occurrence of a clade. The likelihood surface of missing history can then be translated into a parametric prior probability distribution on the age of the clade of interest. We show that the method performs well with simulated fossil distribution data, but that the likelihood surface of missing history can at times be too complex for the distribution-fitting algorithm employed by our software tool. An empirical example of the application of our method is performed to estimate echinoid node ages. A simulation-based sensitivity analysis using the echinoid data set shows that node age prior distributions estimated under poor preservation rates are significantly less informative than those estimated under high preservation rates.     

Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny

The fossil record suggests a rapid radiation of placental mammals following the Cretaceous-Paleogene (K-Pg) mass extinction 65 million years ago (Ma); nevertheless, molecular time estimates, while highly variable, are generally much older. Early molecular studies suffer from inadequate dating methods, reliance on the molecular clock, and simplistic and over-confident interpretations of the fossil record. More recent studies have used Bayesian dating methods that circumvent those issues, but the use of limited data has led to large estimation uncertainties, precluding a decisive conclusion on the timing of mammalian diversifications. Here we use a powerful Bayesian method to analyse 36 nuclear genomes and 274 mitochondrial genomes (20.6 million base pairs), combined with robust but flexible fossil calibrations. Our posterior time estimates suggest that marsupials diverged from eutherians 168-178 Ma, and crown Marsupialia diverged 64-84 Ma. Placentalia diverged 88-90 Ma, and present-day placental orders (except Primates and Xenarthra) originated in a ～20 Myr window (45-65 Ma) after the K-Pg extinction. Therefore we reject a pre K-Pg model of placental ordinal diversification. We suggest other infamous instances of mismatch between molecular and palaeontological divergence time estimates will be resolved with this same approach.     

Performance of a divergence time estimation method under a probabilistic model of rate evolution

Rates of molecular evolution vary over time and, hence, among lineages. In contrast, widely used methods for estimating divergence times from molecular sequence data assume constancy of rates. Therefore, methods for estimation of divergence times that incorporate rate variation are attractive. Improvements on a previously proposed Bayesian technique for divergence time estimation are described. New parameterization more effectively captures the phylogenetic structure of rate evolution on a tree. Fossil information and other evidence can now be included in Bayesian analyses in the form of constraints on divergence times. Simulation results demonstrate that the accuracy of divergence time estimation is substantially enhanced when constraints are included.     

Assessing concordance of fossil calibration points in molecular clock studies: an example using turtles

Although still controversial, estimation of divergence times using molecular data has emerged as a powerful tool to examine the tempo and mode of evolutionary change. Two primary obstacles in improving the accuracy of molecular dating are heterogeneity in DNA substitution rates and accuracy of the fossil record as calibration points. Recent methodological advances have provided powerful methods that estimate relative divergence times in the face of heterogeneity of nucleotide substitution rates among lineages. However, relatively little attention has focused on the accuracy of fossil calibration points that allow one to translate relative divergence times into absolute time. We present a new cross-validation method that identifies inconsistent fossils when multiple fossil calibrations are available for a clade and apply our method to a molecular phylogeny of living turtles with fossil calibration times for 17 of the 22 internal nodes in the tree. Our cross-validation procedure identified seven inconsistent fossils. Using the consistent fossils as calibration points, we found that despite their overall antiquity as a lineage, the most species-rich clades of turtles diversified well within the Cenozoic. Many of the truly ancient lineages of turtles are currently represented by a few, often endangered species that deserve high priority as conservation targets.     

Divergence time and evolutionary rate estimation with multilocus data

Bayesian methods for estimating evolutionary divergence times are extended to multigene data sets, and a technique is described for detecting correlated changes in evolutionary rates among genes. Simulations are employed to explore the effect of multigene data on divergence time estimation, and the methodology is illustrated with a previously published data set representing diverse plant taxa. The fact that evolutionary rates and times are confounded when sequence data are compared is emphasized and the importance of fossil information for disentangling rates and times is stressed.     

Divergence Times and the Evolutionary Radiation of New World Monkeys (Platyrrhini,Primates): An Analysis of Fossil and Molecular Data

The estimation of phylogenetic relationships and divergence times among a group of organisms is a fundamental first step toward understanding its biological diversification. The time of the most recent or last common ancestor (LCA) of extant platyrrhines is one of the most controversial among scholars of primate evolution. Here we use two molecular based approaches to date the initial divergence of the platyrrhine clade, Bayesian estimations under a relaxed-clock model and substitution rate plus generation time and body size, employing the fossil record and genome datasets. We also explore the robustness of our estimations with respect to changes in topology, fossil constraints and substitution rate, and discuss the implications of our findings for understanding the platyrrhine radiation. Our results suggest that fossil constraints, topology and substitution rate have an important influence on our divergence time estimates. Bayesian estimates using conservative but realistic fossil constraints suggest that the LCA of extant platyrrhines existed at ca. 29 Ma, with the 95% confidence limit for the node ranging from 27–31 Ma. The LCA of extant platyrrhine monkeys based on substitution rate corrected by generation time and body size was established between 21–29 Ma. The estimates based on the two approaches used in this study recalibrate the ages of the major platyrrhine clades and corroborate the hypothesis that they constitute very old lineages. These results can help reconcile several controversial points concerning the affinities of key early Miocene fossils that have arisen among paleontologists and molecular systematists. However, they cannot resolve the controversy of whether these fossil species truly belong to the extant lineages or to a stem platyrrhine clade. That question can only be resolved by morphology. Finally, we show that the use of different approaches and well supported fossil information gives a more robust divergence time estimate of a clade.     

Calibration uncertainty in molecular dating analyses: there is no substitute for the prior evaluation of time priors

Calibration is the rate-determining step in every molecular clock analysis and, hence, considerable effort has been expended in the development of approaches to distinguish good from bad calibrations. These can be categorized into a priori evaluation of the intrinsic fossil evidence, and a posteriori evaluation of congruence through cross-validation. We contrasted these competing approaches and explored the impact of different interpretations of the fossil evidence upon Bayesian divergence time estimation. The results demonstrate that a posteriori approaches can lead to the selection of erroneous calibrations. Bayesian posterior estimates are also shown to be extremely sensitive to the probabilistic interpretation of temporal constraints. Furthermore, the effective time priors implemented within an analysis differ for individual calibrations when employed alone and in differing combination with others. This compromises the implicit assumption of all calibration consistency methods, that the impact of an individual calibration is the same when used alone or in unison with others. Thus, the most effective means of establishing the quality of fossil-based calibrations is through a priori evaluation of the intrinsic palaeontological, stratigraphic, geochronological and phylogenetic data. However, effort expended in establishing calibrations will not be rewarded unless they are implemented faithfully in divergence time analyses.     

Comparison of likelihood and Bayesian methods for estimating divergence times using multiple gene Loci and calibration points, with application to a radiation of cute-looking mouse lemur species

Divergence time and substitution rate are seriously confounded in phylogenetic analysis, making it difficult to estimate divergence times when the molecular clock (rate constancy among lineages) is violated. This problem can be alleviated to some extent by analyzing multiple gene loci simultaneously and by using multiple calibration points. While different genes may have different patterns of evolutionary rate change, they share the same divergence times. Indeed, the fact that each gene may violate the molecular clock differently leads to the advantage of simultaneous analysis of multiple loci. Multiple calibration points provide the means for characterizing the local evolutionary rates on the phylogeny. In this paper, we extend previous likelihood models of local molecular clock for estimating species divergence times to accommodate multiple calibration points and multiple genes. Heterogeneity among different genes in evolutionary rate and in substitution process is accounted for by the models. We apply the likelihood models to analyze two mitochondrial protein-coding genes, cytochrome oxidase II and cytochrome b, to estimate divergence times of Malagasy mouse lemurs and related outgroups. The likelihood method is compared with the Bayes method of Thorne et al. (1998, Mol. Biol. Evol. 15:1647-1657), which uses a probabilistic model to describe the change in evolutionary rate over time and uses the Markov chain Monte Carlo procedure to derive the posterior distribution of rates and times. Our likelihood implementation has the drawbacks of failing to accommodate uncertainties in fossil calibrations and of requiring the researcher to classify branches on the tree into different rate groups. Both problems are avoided in the Bayes method. Despite the differences in the two methods, however, data partitions and model assumptions had the greatest impact on date estimation. The three codon positions have very different substitution rates and evolutionary dynamics, and assumptions in the substitution model affect date estimation in both likelihood and Bayes analyses. The results demonstrate that the separate analysis is unreliable, with dates variable among codon positions and between methods, and that the combined analysis is much more reliable. When the three codon positions were analyzed simultaneously under the most realistic models using all available calibration information, the two methods produced similar results. The divergence of the mouse lemurs is dated to be around 7-10 million years ago, indicating a surprisingly early species radiation for such a morphologically uniform group of primates.     

Comparative historical biogeography of Plateumaris leaf beetles (Coleoptera: Chrysomelidae) in Japan: interplay between fossil and molecular data

Aim In an attempt to use molecular and fossil data interactively in historical biogeography, we studied the phylogeography of five Plateumaris leaf beetles in Japan using mitochondrial cytochrome oxidase subunit I (COI) sequence data to explore interspecific differences in phylogeographical patterns and estimate the timings of colonization and geographical differentiation. Location A total of 461 beetles from five species on Hokkaido, Honshu and Kyushu islands of Japan were analysed with 117 beetles from three conspecies and two congeners from the mainland (Russia, including Sakhalin; Korea; Mongolia; Belgium; France; hereafter, the continent). Methods Using the sequence data from a 750‐bp portion of the COI gene, we studied the phylogeny of COI haplotypes, intraspecific population differentiation using analysis of molecular variance and the Mantel test, and intraspecific phylogeography using nested clade analysis. In addition, divergence times between the continental and Japanese lineages, as well as among the various Japanese lineages, were estimated using a Bayesian approach with node constraints based on fossil records of extant species. Results Three widely distributed species showed different degrees of geographical differentiation corresponding to their different colonization history in Japan. Bayesian estimates of divergence time revealed that one of two endemic species, which originated before the late Pliocene, attained intraspecific differentiation through the Pliocene and Pleistocene, whereas another endemic species has been confined in one locality, and three non‐endemic species colonized Japan after the mid‐Pleistocene. Main conclusions Molecular analyses of an insect group with relatively abundant fossil data can contribute greatly to the understanding of diverse biogeographical histories of related species in a region. Bayesian estimates of divergence time could be used to assess the variable evolutionary rates of the COI gene, and may be applied to other related insect species.     

Exploring uncertainty in the calibration of the molecular clock

Calibration is a critical step in every molecular clock analysis but it has been the least considered. Bayesian approaches to divergence time estimation make it possible to incorporate the uncertainty in the degree to which fossil evidence approximates the true time of divergence. We explored the impact of different approaches in expressing this relationship, using arthropod phylogeny as an example for which we established novel calibrations. We demonstrate that the parameters distinguishing calibration densities have a major impact upon the prior and posterior of the divergence times, and it is critically important that users evaluate the joint prior distribution of divergence times used by their dating programmes. We illustrate a procedure for deriving calibration densities in Bayesian divergence dating through the use of soft maximum constraints.     

Bayesian analysis of combined chloroplast loci, using multiple calibrations, supports the recent arrival of Melastomataceae in Africa and Madagascar

A new biogeographic scenario for Melastomataceae (Morley and Dick, American Journal of Botany 90(11) pp. 1638-1645, 2003) accepts an ndhF-based phylogeny for the family by Renner et al. (American Journal of Botany 88(7): 1290-1300, 2001), but rejects those authors' divergence time estimates. Morley and Dick concluded that Gondwanan vicariance, rather than the more recent long dispersal proposed by Renner et al. explains the presence of the family in Africa and Madagascar. To assess the strength of this conclusion, a Bayesian analysis was conducted on three times the amount of sequence data used before (ndhF, rbcL, rpl16; 3100 base pairs [bp], excluding all gaps). The Bayesian approach to divergence time estimation does not rely on a strict molecular clock and employs multiple simultaneous minimal or maximal bounds on node ages. Reliance on northern mid-latitude fossils of Melastomataceae for calibrations was avoided or reduced by using alternative fossil and tectonic calibrations, including all those suggested by Morley and Dick. Results reaffirm the relatively recent spread of melastome lineages among the southern continents and refute the breakup of Gondwana as a plausible explanation for the presence of Dissochaeteae/Sonerileae in Madagascar and Africa and the presence of Melastomeae in Africa and Southeast Asia. Melastomeae appear to have reached Africa around 17-15 million years (my) ago, while Dissochaeteae and Sonerileae apparently reached Madagascar at 17-15 and 20-18 my ago. I also explored the effects of constraining Melastomeae to minimally 76 my old (to have reached Africa by island hopping as postulated by Morley and Dick). This resulted in an estimate for their arrival in Africa of 35 my ago and for Dissochaeteae and Sonerileae in Madagascar of 28 and 33 my ago, still implying long-distance dispersal. The Bayesian 95% credibility ranges around these dates, however, are large. Regardless of the increasing sophistication of molecular estimates of divergence time, Gondwanan scenarios will remain untestable as long as biases in the fossil record can justifiably be invoked to explain away the absence of fossils.     

Fossil calibrations and molecular divergence time estimates in centrarchid fishes (Teleostei: Centrarchidae)

Molecular clock methods allow biologists to estimate divergence times, which in turn play an important role in comparative studies of many evolutionary processes. It is well known that molecular age estimates can be biased by heterogeneity in rates of molecular evolution, but less attention has been paid to the issue of potentially erroneous fossil calibrations. In this study we estimate the timing of diversification in Centrarchidae, an endemic major lineage of the diverse North American freshwater fish fauna, through a new approach to fossil calibration and molecular evolutionary model selection. Given a completely resolved multi-gene molecular phylogeny and a set of multiple fossil-inferred age estimates, we tested for potentially erroneous fossil calibrations using a recently developed fossil cross-validation. We also used fossil information to guide the selection of the optimal molecular evolutionary model with a new fossil jackknife method in a fossil-based model cross-validation. The centrarchid phylogeny resulted from a mixed-model Bayesian strategy that included 14 separate data partitions sampled from three mtDNA and four nuclear genes. Ten of the 31 interspecific nodes in the centrarchid phylogeny were assigned a minimal age estimate from the centrarchid fossil record. Our analyses identified four fossil dates that were inconsistent with the other fossils, and we removed them from the molecular dating analysis. Using fossil-based model cross-validation to determine the optimal smoothing value in penalized likelihood analysis, and six mutually consistent fossil calibrations, the age of the most recent common ancestor of Centrarchidae was 33.59 million years ago (mya). Penalized likelihood analyses of individual data partitions all converged on a very similar age estimate for this node, indicating that rate heterogeneity among data partitions is not confounding our analyses. These results place the origin of the centrarchid radiation at a time of major faunal turnover as the fossil record indicates that the most diverse lineages of the North American freshwater fish fauna originated at the Eocene-Oligocene boundary, approximately 34 mya. This time coincided with major global climate change from warm to cool temperatures and a signature of elevated lineage extinction and origination in the fossil record across the tree of life. Our analyses demonstrate the utility of fossil cross-validation to critically assess individual fossil calibration points, providing the ability to discriminate between consistent and inconsistent fossil age estimates that are used for calibrating molecular phylogenies.     

Speciational history of Australian grass finches (Poephila) inferred from thirty gene trees

Multilocus genealogical approaches are still uncommon in phylogeography and historical demography, fields which have been dominated by microsatellite markers and mitochondrial DNA, particularly for vertebrates. Using 30 newly developed anonymous nuclear loci, we estimated population divergence times and ancestral population sizes of three closely related species of Australian grass finches (Poephila) distributed across two barriers in northern Australia. We verified that substitution rates were generally constant both among lineages and among loci, and that intralocus recombination was uncommon in our dataset, thereby satisfying two assumptions of our multilocus analysis. The reconstructed gene trees exhibited all three possible tree topologies and displayed considerable variation in coalescent times, yet this information provided the raw data for maximum likelihood and Bayesian estimation of population divergence times and ancestral population sizes. Estimates of these parameters were in close agreement with each other regardless of statistical approach and our Bayesian estimates were robust to prior assumptions. Our results suggest that black-throated finches (Poephila cincta) diverged from long-tailed finches (P. acuticauda and P. hecki) across the Carpentarian Barrier in northeastern Australia around 0.6 million years ago (mya), and that P. acuticauda diverged from P. hecki across the Kimberley Plateau-Arnhem Land Barrier in northwestern Australia approximately 0.3 mya. Bayesian 95% credibility intervals around these estimates strongly support Pleistocene timing for both speciation events, despite the fact that many gene divergences across the Carpentarian region clearly predated the Pleistocene. Estimates of ancestral effective population sizes for the basal ancestor and long-tailed finch ancestor were large (about 521,000 and about 384,000, respectively). Although the errors around the population size parameter estimates are considerable, they are the first for birds taking into account multiple sources of variance.     

A Bayesian evaluation of human mitochondrial substitution rates

Accurate estimates of mitochondrial substitution rates are central to molecular studies of human evolution, but meaningful comparisons of published studies are problematic because of the wide range of methodologies and data sets employed. These differences are nowhere more pronounced than among rates estimated from phylogenies, genealogies, and pedigrees. By using a data set comprising mitochondrial genomes from 177 humans, we estimate substitution rates for various data partitions by using Bayesian phylogenetic analysis with a relaxed molecular clock. We compare the effect of multiple internal calibrations with the customary human-chimpanzee split. The analyses reveal wide variation among estimated substitution rates and divergence times made with different partitions and calibrations, with evidence of substitutional saturation, natural selection, and significant rate heterogeneity among lineages and among sites. Collectively, the results support dates for migration out of Africa and the common mitochondrial ancestor of humans that are considerably more recent than most previous estimates. Our results also demonstrate that human mitochondrial genomes exhibit a number of molecular evolutionary complexities that necessitate the use of sophisticated analytical models for genetic analyses.