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.     

Integration of Bayesian molecular clock methods and fossil-based soft bounds reveals early Cenozoic origin of African lacertid lizards

Background  

Although current molecular clock methods offer greater flexibility in modelling evolutionary events, calibration of the clock with dates from the fossil record is still problematic for many groups. Here we implement several new approaches in molecular dating to estimate the evolutionary ages of Lacertidae, an Old World family of lizards with a poor fossil record and uncertain phylogeny. Four different models of rate variation are tested in a new program for Bayesian phylogenetic analysis called TreeTime, based on a combination of mitochondrial and nuclear gene sequences. We incorporate paleontological uncertainty into divergence estimates by expressing multiple calibration dates as a range of probabilistic distributions. We also test the reliability of our proposed calibrations by exploring effects of individual priors on posterior estimates.     

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.     

Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae)

Discussions aimed at resolution of the Tree of Life are most often focused on the interrelationships of major organismal lineages. In this study, we focus on the resolution of some of the most apical branches in the Tree of Life through exploration of the phylogenetic relationships of darters, a species-rich clade of North American freshwater fishes. With a near-complete taxon sampling of close to 250 species, we aim to investigate strategies for efficient multilocus data sampling and the estimation of divergence times using relaxed-clock methods when a clade lacks a fossil record. Our phylogenetic data set comprises a single mitochondrial DNA (mtDNA) gene and two nuclear genes sampled from 245 of the 248 darter species. This dense sampling allows us to determine if a modest amount of nuclear DNA sequence data can resolve relationships among closely related animal species. Darters lack a fossil record to provide age calibration priors in relaxed-clock analyses. Therefore, we use a near-complete species-sampled phylogeny of the perciform clade Centrarchidae, which has a rich fossil record, to assess two distinct strategies of external calibration in relaxed-clock divergence time estimates of darters: using ages inferred from the fossil record and molecular evolutionary rate estimates. Comparison of Bayesian phylogenies inferred from mtDNA and nuclear genes reveals that heterospecific mtDNA is present in approximately 12.5% of all darter species. We identify three patterns of mtDNA introgression in darters: proximal mtDNA transfer, which involves the transfer of mtDNA among extant and sympatric darter species, indeterminate introgression, which involves the transfer of mtDNA from a lineage that cannot be confidently identified because the introgressed haplotypes are not clearly referable to mtDNA haplotypes in any recognized species, and deep introgression, which is characterized by species diversification within a recipient clade subsequent to the transfer of heterospecific mtDNA. The results of our analyses indicate that DNA sequences sampled from single-copy nuclear genes can provide appreciable phylogenetic resolution for closely related animal species. A well-resolved near-complete species-sampled phylogeny of darters was estimated with Bayesian methods using a concatenated mtDNA and nuclear gene data set with all identified heterospecific mtDNA haplotypes treated as missing data. The relaxed-clock analyses resulted in very similar posterior age estimates across the three sampled genes and methods of calibration and therefore offer a viable strategy for estimating divergence times for clades that lack a fossil record. In addition, an informative rank-free clade-based classification of darters that preserves the rich history of nomenclature in the group and provides formal taxonomic communication of darter clades was constructed using the mtDNA and nuclear gene phylogeny. On the whole, the appeal of mtDNA for phylogeny inference among closely related animal species is diminished by the observations of extensive mtDNA introgression and by finding appreciable phylogenetic signal in a modest sampling of nuclear genes in our phylogenetic analyses of darters.     

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.     

Sources of error and confidence intervals in estimating the age of angiosperms from rbcL and 18S rDNA data

Molecular estimates of the age of angiosperms have varied widely, and many greatly predate the Early Cretaceous appearance of angiosperms in the fossil record, but there have been few attempts to assess confidence limits on ages. Experiments with rbcL and 18S data using maximum likelihood suggest that previous angiosperm age estimates were too old because they assumed equal rates across sites-use of a gamma distribution of rates to correct for site-to-site variation gives 10-30 my (million years) younger ages-and relied on herbaceous angiosperm taxa with high rates of molecular evolution. Ages based on first and second codon positions of rbcL are markedly older than those based on third positions, which conflict with the fossil record in being too young, but all examined data partitions of rbcL and 18S depart substantially from a molecular clock. Age estimates are surprisingly insensitive to different views on seed-plant relationships. Randomization schemes were used to quantify confidence intervals due to phylogenetic uncertainty, substitutional noise, and lineage effects (deviations from a molecular clock). Estimates of the age of crown-group angiosperms range from 68 to 281 mya (million years ago), depending on data, tree, and assumptions, with most ～140-190 mya (Early Jurassic-earliest Cretaceous). Approximate 95% confidence intervals on ages are wider for rbcL than 18S, ranging up to 160 my for phylogenetic uncertainty, 90 my for substitutional noise, and 70 my for lineage effects. These intervals overlap the oldest occurrences of angiosperms in the fossil record, as well as some estimates from previous molecular studies.     

Testing the impact of calibration on molecular divergence times using a fossil-rich group: the case of Nothofagus (Fagales)

Although temporal calibration is widely recognized as critical for obtaining accurate divergence-time estimates using molecular dating methods, few studies have evaluated the variation resulting from different calibration strategies. Depending on the information available, researchers have often used primary calibrations from the fossil record or secondary calibrations from previous molecular dating studies. In analyses of flowering plants, primary calibration data can be obtained from macro- and mesofossils (e.g., leaves, flowers, and fruits) or microfossils (e.g., pollen). Fossil data can vary substantially in accuracy and precision, presenting a difficult choice when selecting appropriate calibrations. Here, we test the impact of eight plausible calibration scenarios for Nothofagus (Nothofagaceae, Fagales), a plant genus with a particularly rich and well-studied fossil record. To do so, we reviewed the phylogenetic placement and geochronology of 38 fossil taxa of Nothofagus and other Fagales, and we identified minimum age constraints for up to 18 nodes of the phylogeny of Fagales. Molecular dating analyses were conducted for each scenario using maximum likelihood (RAxML + r8s) and Bayesian (BEAST) approaches on sequence data from six regions of the chloroplast and nuclear genomes. Using either ingroup or outgroup constraints, or both, led to similar age estimates, except near strongly influential calibration nodes. Using "early but risky" fossil constraints in addition to "safe but late" constraints, or using assumptions of vicariance instead of fossil constraints, led to older age estimates. In contrast, using secondary calibration points yielded drastically younger age estimates. This empirical study highlights the critical influence of calibration on molecular dating analyses. Even in a best-case situation, with many thoroughly vetted fossils available, substantial uncertainties can remain in the estimates of divergence times. For example, our estimates for the crown group age of Nothofagus varied from 13 to 113 Ma across our full range of calibration scenarios. We suggest that increased background research should be made at all stages of the calibration process to reduce errors wherever possible, from verifying the geochronological data on the fossils to critical reassessment of their phylogenetic position.     

Investigating phylogenetic relationships of sunfishes and black basses (Actinopterygii: Centrarchidae) using DNA sequences from mitochondrial and nuclear genes

The 32 species of the Centrarchidae are ecologically important components of the diverse fish communities that characterize North American freshwater ecosystems. In spite of a rich history of systematic investigations of centrarchid fishes there is extensive conflict among previous hypotheses that may be due to restricted taxon or character sampling. We present the first phylogenetic analysis of the Centrarchidae that combines DNA sequence data from both the mitochondrial and nuclear genomes and includes all described species. Gene sequence data were collected from a complete mtDNA protein coding gene (NADH subunit 2), a nuclear DNA intron (S7 ribosomal protein intron 1), and a portion of a nuclear DNA protein-coding region (Tmo-4C4). Phylogenetic trees generated from analysis of the three-gene dataset were used to test alternative hypotheses of centrarchid relationships that were gathered from the literature. Four major centrarchid lineages are present in trees generated in maximum parsimony (MP) and Bayesian maximum likelihood analyses (BML). These lineages are Acantharchus pomotis, Micropterus, Lepomis, and a clade containing Ambloplites, Archoplites, Centrarchus, Enneacanthus, and Pomoxis. Phylogenetic trees resulting from MP and BML analyses are highly consistent but differ with regard to the placement of A. pomotis. Significant phylogenetic incongruence between mtDNA and nuclear genes appears to result from different placement of Micropterus treculi, and is not characteristic of relationships in all other parts of the centrarchid phylogeny. Slightly more than half of the 27 previously proposed hypotheses of centrarchid relationships were rejected based on the Shomodaira-Hasegawa test.     

Rapid allopatric speciation in logperch darters (Percidae: Percina)

Theory predicts that clades diversifying via sympatric speciation will exhibit high diversification rates. However, the expected rate of diversification in clades characterized by allopatric speciation is less clear. Previous studies have documented significantly higher speciation rates in freshwater fish clades diversifying via sympatric versus allopatric modes, leading to suggestions that the geographic pattern of speciation can be inferred solely from knowledge of the diversification rate. We tested this prediction using an example from darters, a clade of approximately 200 species of freshwater fishes endemic to eastern North America. A resolved phylogeny was generated using mitochondrial DNA gene sequences for logperches, a monophyletic group of darters composed of 10 recognized species. Divergence times among logperch species were estimated using a fossil calibrated molecular clock in centrarchid fishes, and diversification rates in logperches were estimated using several methods. Speciation events in logperches are recent, extending from 4.20 +/- 1.06 million years ago (mya) to 0.42 +/- 0.22 mya, with most speciation events occurring in the Pleistocene. Diversification rates are high in logperches, at some nodes exceeding rates reported for well-studied adaptive radiations such as Hawaiian silverswords. The geographic pattern of speciation in logperches was investigated by examining the relationship between degree of sympatry and the absolute age of the contrast, with the result that diversification in logperches appears allopatric. The very high diversification rate observed in the logperch phylogeny is more similar to freshwater fish clades thought to represent examples of sympatric speciation than to clades representing allopatric speciation. These results demonstrate that the geographic mode of speciation for a clade cannot be inferred from the diversification rate. The empirical observation of high diversification rates in logperches demonstrates that allopatric speciation can occur rapidly.     

Fossil-calibrated molecular phylogenies reveal that leaf-mining moths radiated millions of years after their host plants

Coevolution has been hypothesized as the main driving force for the remarkable diversity of insect-plant associations. Dating of insect and plant phylogenies allows us to test coevolutionary hypotheses and distinguish between the contemporaneous radiation of interacting lineages vs. insect 'host tracking' of previously diversified plants. Here, we used nuclear DNA to reconstruct a molecular phylogeny for 100 species of Phyllonorycter leaf-mining moths and 36 outgroup taxa. Ages for nodes in the moth phylogeny were estimated using a combination of a penalized likelihood method and a Bayesian approach, which takes into account phylogenetic uncertainty. To convert the relative ages of the moths into dates, we used an absolute calibration point from the fossil record. The age estimates of (a selection of) moth clades were then compared with fossil-based age estimates of their host plants. Our results show that the principal radiation of Phyllonorycter leaf-mining moths occurred well after the main radiation of their host plants and may represent the dominant associational mode in the fossil record.     

Dating the arthropod tree based on large-scale transcriptome data

Molecular sequences do not only allow the reconstruction of phylogenetic relationships among species, but also provide information on the approximate divergence times. Whereas the fossil record dates the origin of most multicellular animal phyla during the Cambrian explosion less than 540 million years ago(mya), molecular clock calculations usually suggest much older dates. Here we used a large multiple sequence alignment derived from Expressed Sequence Tags and genomes comprising 129genes (37,476 amino acid positions) and 117 taxa, including 101 arthropods. We obtained consistent divergence time estimates applying relaxed Bayesian clock models with different priors and multiple calibration points. While the influence of substitution rates, missing data, and model priors were negligible, the clock model had significant effect. A log-normal autocorrelated model was selected on basis of cross-validation. We calculated that arthropods emerged ~600 mya. Onychophorans (velvet worms) and euarthropods split ~590 mya, Pancrustacea and Myriochelata ~560 mya, Myriapoda and Chelicerata ~555 mya, and 'Crustacea' and Hexapoda ~510 mya. Endopterygote insects appeared ~390 mya. These dates are considerably younger than most previous molecular clock estimates and in better agreement with the fossil record. Nevertheless, a Precambrian origin of arthropods and other metazoan phyla is still supported. Our results also demonstrate the applicability of large datasets of random nuclear sequences for approximating the timing of multicellular animal evolution.     

Timing of the evolutionary history of Corallinaceae (Corallinales,Rhodophyta)

The temporal dimension of the most recent Corallinaceae (order Corallinales) phylogeny was presented here, based on first occurrence time estimates from the fossil record. Calibration of the molecular clock of the genetic marker SSU entailed a separation of Corallinales from Hapalidiales in the Albian (Early Cretaceous ~105 mya). Neither the calibration nor the fossil record resolved the succession of appearance of the first three emerging subfamilies: Mastophoroideae, Corallinoideae, and Neogoniolithoideae. The development of the tetra/bisporangial conceptacle roofs by filaments surrounding and interspersed among the sporangial initials was an evolutionary novelty emerging at the Cretaceous–Paleogene boundary (~66 mya). This novelty was shared by the subfamilies Hydrolithoideae, Metagoniolithoideae, and Lithophylloideae, which diverged in the early Paleogene. Subclades within the Metagoniolithoideae and Lithophylloideae diversified in the late Oligocene–middle Miocene (~28–12 mya). The most common reef corallinaceans (Hydrolithon, Porolithon, Harveylithon, “Pneophyllum” conicum, and subclades within Lithophylloideae) appeared in this interval in the Indo‐Australian Archipelago.     

Tempo of hybrid inviability in centrarchid fishes (Teleostei: Centrarchidae)

Hybrid viability decreases with divergence time, a pattern consistent with a so-called speciation clock. However, the actual rate at which this clock ticks is poorly known. Most speciation-clock studies have used genetic divergence as a proxy for time, adopting a molecular clock and often far-distant calibration points to convert genetic distances into age. Because molecular clock assumptions are violated for most genetic datasets and distant calibrations are of questionable utility, the actual rate at which reproductive isolation evolves may be substantially different than current estimates suggest. We provide a robust measure of the tempo at which hybrid viability declines with divergence time in a clade of freshwater fishes (Centrarchidae). This incompatibility clock is distinct from a speciation clock because speciation events in centrarchids appear to be driven largely by prezygotic isolation. Our analyses used divergence times estimated with penalized likelihood applied to a phylogeny derived from seven gene regions and calibrated with six centrarchid fossils. We found that hybrid embryo viability declined at mean rate of 3.13% per million years, slower than in most other taxa investigated to date. Despite measurement error in both molecular estimated ages and hatching success of hybrid crosses, divergence time explained between 73% and 90% of the variation in hybrid viability among nodes. This high correlation is consistent with the gradual accumulation of many genetic incompatibilities of small effect. Hybrid viability declined with the square of time, consistent with an increasing rate of accumulation of incompatibilities between divergent genomes (the snowball effect). However, the quadratic slope is due to a lag phase resulting from heterosis among young species pairs, a phenomenon rarely considered in predictions of hybrid fitness. Finally, we found that reciprocal crosses often show asymmetrical hybrid viabilities. We discuss several alternative explanations for this result including possible deleterious cytonuclear interactions. Speciation-clock studies have been a small cottage industry recently, but there are still novel insights to be gained from analyses of more taxonomic groups. However, between-group comparisons require more careful molecular-clock calibration than has been the norm.     

A new time-scale for ray-finned fish evolution

The Actinopterygii (ray-finned fishes) is the largest and most diverse vertebrate group, but little is agreed about the timing of its early evolution. Estimates using mitochondrial genomic data suggest that the major actinopterygian clades are much older than divergence dates implied by fossils. Here, the timing of the evolutionary origins of these clades is reinvestigated using morphological, and nuclear and mitochondrial genetic data. Results indicate that existing fossil-based estimates of the age of the crown-group Neopterygii, including the teleosts, Lepisosteus (gar) and Amia (bowfin), are at least 40 Myr too young. We present new palaeontological evidence that the neopterygian crown radiation is a Palaeozoic event, and demonstrate that conflicts between molecular and morphological data for the age of the Neopterygii result, in part, from missing fossil data. Although our molecular data also provide an older age estimate for the teleost crown, this range extension remains unsupported by the fossil evidence. Nuclear data from all relevant clades are used to demonstrate that the actinopterygian whole-genome duplication event is teleost-specific. While the date estimate of this event overlaps the probable range of the teleost stem group, a correlation between the genome duplication and the large-scale pattern of actinopterygian phylogeny remains elusive.     

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.     

Calibration choice, rate smoothing, and the pattern of tetrapod diversification according to the long nuclear gene RAG-1

A phylogeny of tetrapods is inferred from nearly complete sequences of the nuclear RAG-1 gene sampled across 88 taxa encompassing all major clades, analyzed via parsimony and Bayesian methods. The phylogeny provides support for Lissamphibia, Theria, Lepidosauria, a turtle-archosaur clade, as well as most traditionally accepted groupings. This tree allows simultaneous molecular clock dating for all tetrapod groups using a set of well-corroborated calibrations. Relaxed clock (PLRS) methods, using the amniote = 315 Mya (million years ago) calibration or a set of consistent calibrations, recovers reasonable divergence dates for most groups. However, the analysis systematically underestimates divergence dates within archosaurs. The bird-crocodile split, robustly documented in the fossil record as being around approximately 245 Mya, is estimated at only approximately 190 Mya, and dates for other divergences within archosaurs are similarly underestimated. Archosaurs, and particulary turtles have slow apparent rates possibly confounding rate modeling, and inclusion of calibrations within archosaurs (despite their high deviances) not only improves divergence estimates within archosaurs, but also across other groups. Notably, the monotreme-therian split ( approximately 210 Mya) matches the fossil record; the squamate radiation ( approximately 190 Mya) is younger than suggested by some recent molecular studies and inconsistent with identification of approximately 220 and approximately 165 Myo (million-year-old) fossils as acrodont iguanians and approximately 95 Myo fossils colubroid snakes; the bird-lizard (reptile) split is considerably older than fossil estimates (< or = 285 Mya); and Sphenodon is a remarkable phylogenetic relic, being the sole survivor of a lineage more than a quarter of a billion years old. Comparison with other molecular clock studies of tetrapod divergences suggests that the common practice of enforcing most calibrations as minima, with a single liberal maximal constraint, will systematically overestimate divergence dates. Similarly, saturation of mitochondrial DNA sequences, and the resultant greater compression of basal branches means that using only external deep calibrations will also lead to inflated age estimates within the focal ingroup.     

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.     

The impact of fossil stratigraphic ranges on tip-calibration,and the accuracy and precision of divergence time estimates

The molecular clock provides the only viable means of establishing realistic evolutionary timescales but it remains unclear how best to calibrate divergence time analyses. Calibrations can be applied to the tips and/or to the nodes of a phylogeny. Tip-calibration is an attractive approach since it allows fossil species to be included alongside extant relatives in molecular clock analyses. However, most fossil species are known from multiple stratigraphical horizons and it remains unclear how such age ranges should be interpreted to codify tip-calibrations. We use simulations and empirical data to explore the impact on precision and accuracy of different approaches to informing tip-calibrations. In particular, we focus on the effect of using tip-calibrations defined using the oldest vs youngest stratigraphic occurrences, the full stratigraphical range, as well as confidence intervals on these data points. The results of our simulations show that using different calibration approaches leads to different divergence-time estimates and demonstrate that concentrating tip-calibrations near the root of the dated phylogeny improves both precision and accuracy of estimated divergence times. Finally, our results indicate that the highest levels of accuracy and precision are achieved when fossil tips are calibrated based on the fossil occurrence from which the morphological data were derived. These trends were corroborated by analysis of an empirical dataset for Ursidae. Overall, we conclude that tip-dating analyses should, in particular, employ tip calibrations close to the root of the tree and they should be calibrated based on the age of the fossil used to inform the morphological data used in Total Evidence Dating.     

Phylogenetics of Cancer crabs (Crustacea: Decapoda: Brachyura).

We used morphological, mitochondrial DNA sequence, paleontological, and biogeographical information to examine the evolutionary history of crabs of the genus Cancer. Phylogenies inferred from adult morphology and DNA sequence of the cytochrome oxidase I (COI) gene were each well resolved and well supported, but differed substantially in topology. Four lines of evidence suggested that the COI data set accurately reflected Cancer phylogeny: (1) in the phylogeny inferred from morphological data, each Atlantic species was sister taxon to an ecologically similar Pacific species, suggesting convergence in morphology; (2) a single trans-Arctic dispersal event, as indicated by the phylogeny inferred from COI, is more parsimonious than two such dispersal events, as inferred from morphology; (3) test and application of a maximum likelihood molecular clock to the COI data yielded estimates of origin and speciation times that fit well with the fossil record; and (4) the tree inferred from the combined COI and morphology data was closely similar to the trees inferred from COI, although notably less well supported by the bootstrap. The phylogeny inferred from maximum likelihood analysis of COI suggested that Cancer originated in the North Pacific in the early Miocene, that the Atlantic species arose from a North Pacific ancestor, and that Cancer crabs invaded the Atlantic from the North Pacific 6-12 mya. This inferred invasion time is notably prior to most estimates of the date of submergence of the Bering Strait and the trans-Arctic interchange, but it agrees with fossil evidence placing at least one Cancer species in the Atlantic about 8 mya.     

To what extent do new fossil discoveries change our understanding of clade evolution? A cautionary tale from burying beetles (Coleoptera: Nicrophorus)

Divergence time estimates derived from phylogenies are crucial to infer historical biogeography and diversification dynamics. Yet, the impact of fossil record incompleteness on macroevolutionary reconstructions remains equivocal. Here, we investigate to what extent gaps in the fossil record can impinge downstream evolutionary inferences in the beetle family Silphidae. Recent discoveries have pushed back the fossil record of this group from the Eocene into the Jurassic. We estimated the divergence times of the family using both its currently understood fossil record and the fossil record known prior to these recent discoveries. All fossil calibrations were informed with different parametric distributions to investigate the weight of priors on posterior age estimates. Based on time‐calibrated trees, we assessed the impact of fossil calibrations on the inference of ancestral ranges and diversification rate dynamics in the genus Nicrophorus. Depending upon the selected sets of fossil constraints, the age discrepancies had a major impact on the macroevolutionary inferences: the biogeographic extrapolations relative to paleogeography are markedly contrasting, and the calculated rates at which species form or go extinct (and when they varied) are strikingly different. We show that soft prior distributions do not necessarily alleviate such shortcomings therefore preventing the inference of reliable macroevolutionary patterns in groups presenting a taphonomic bias in their fossil record.