Assessing calibration uncertainty in molecular dating: the assignment of fossils to alternative calibration points

Although recent methodological advances have allowed the incorporation of rate variation in molecular dating analyses, the calibration procedure, performed mainly through fossils, remains resistant to improvements. One source of uncertainty pertains to the assignment of fossils to specific nodes in a phylogeny, especially when alternative possibilities exist that can be equally justified on morphological grounds. Here we expand on a recently developed fossil cross-validation method to evaluate whether alternative nodal assignments of multiple fossils produce calibration sets that differ in their internal consistency. We use an enlarged Crypteroniaceae-centered phylogeny of Myrtales, six fossils, and 72 combinations of calibration points, termed calibration sets, to identify (i) the fossil assignments that produce the most internally consistent calibration sets and (ii) the mean ages, derived from these calibration sets, for the split of the Southeast Asian Crypteroniaceae from their West Gondwanan sister clade (node X). We found that a correlation exists between s values, devised to measure the consistency among the calibration points of a calibration set (Near and Sanderson, 2004), and nodal distances among calibration points. By ranking all sets according to the percent deviation of s from the regression line with nodal distance, we identified the sets with the highest level of corrected calibration-set consistency. These sets generated lower standard deviations associated with the ages of node X than sets characterized by lower corrected consistency. The three calibration sets with the highest corrected consistencies produced mean age estimates for node X of 79.70, 79.14, and 78.15 My. These timeframes are most compatible with the hypothesis that the Crypteroniaceae stem lineage dispersed from Africa to the Deccan plate as it drifted northward during the Late Cretaceous.     

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.     

Dated Phylogenies of the Sister Genera Macaranga and Mallotus (Euphorbiaceae): Congruence in Historical Biogeographic Patterns?

Molecular phylogenies and estimates of divergence times within the sister genera Macaranga and Mallotus were estimated using Bayesian relaxed clock analyses of two generic data sets, one per genus. Both data sets were based on different molecular markers and largely different samples. Per genus three calibration points were utilised. The basal calibration point (crown node of all taxa used) was taken from literature and used for both taxa. The other three calibrations were based on fossils of which two were used per genus. We compared patterns of dispersal and diversification in Macaranga and Mallotus using ancestral area reconstruction in RASP (S-DIVA option) and contrasted our results with biogeographical and geological records to assess accuracy of inferred age estimates. A check of the fossil calibration point showed that the Japanese fossil, used for dating the divergence of Mallotus, probably had to be attached to a lower node, the stem node of all pioneer species, but even then the divergence time was still younger than the estimated age of the fossil. The African (only used in the Macaranga data set) and New Zealand fossils (used for both genera) seemed reliably placed. Our results are in line with existing geological data and the presence of stepping stones that provided dispersal pathways from Borneo to New Guinea-Australia, from Borneo to mainland Asia and additionally at least once to Africa and Madagascar via land and back to India via Indian Ocean island chains. The two genera show congruence in dispersal patterns, which corroborate divergence time estimates, although the overall mode and tempo of dispersal and diversification differ significantly as shown by distribution patterns of extant species.     

Phylogenetic analysis of pelecaniformes (aves) based on osteological data: implications for waterbird phylogeny and fossil calibration studies

Background

Debate regarding the monophyly and relationships of the avian order Pelecaniformes represents a classic example of discord between morphological and molecular estimates of phylogeny. This lack of consensus hampers interpretation of the group''s fossil record, which has major implications for understanding patterns of character evolution (e.g., the evolution of wing-propelled diving) and temporal diversification (e.g., the origins of modern families). Relationships of the Pelecaniformes were inferred through parsimony analyses of an osteological dataset encompassing 59 taxa and 464 characters. The relationships of the Plotopteridae, an extinct family of wing-propelled divers, and several other fossil pelecaniforms (Limnofregata, Prophaethon, Lithoptila, ?Borvocarbo stoeffelensis) were also assessed. The antiquity of these taxa and their purported status as stem members of extant families makes them valuable for studies of higher-level avian diversification.

Methodology/Principal Findings

Pelecaniform monophyly is not recovered, with Phaethontidae recovered as distantly related to all other pelecaniforms, which are supported as a monophyletic Steganopodes. Some anatomical partitions of the dataset possess different phylogenetic signals, and partitioned analyses reveal that these discrepancies are localized outside of Steganopodes, and primarily due to a few labile taxa. The Plotopteridae are recovered as the sister taxon to Phalacrocoracoidea, and the relationships of other fossil pelecaniforms representing key calibration points are well supported, including Limnofregata (sister taxon to Fregatidae), Prophaethon and Lithoptila (successive sister taxa to Phaethontidae), and ?Borvocarbo stoeffelensis (sister taxon to Phalacrocoracidae). These relationships are invariant when ‘backbone’ constraints based on recent avian phylogenies are imposed.

Conclusions/Significance

Relationships of extant pelecaniforms inferred from morphology are more congruent with molecular phylogenies than previously assumed, though notable conflicts remain. The phylogenetic position of the Plotopteridae implies that wing-propelled diving evolved independently in plotopterids and penguins, representing a remarkable case of convergent evolution. Despite robust support for the placement of fossil taxa representing key calibration points, the successive outgroup relationships of several “stem fossil + crown family” clades are variable and poorly supported across recent studies of avian phylogeny. Thus, the impact these fossils have on inferred patterns of temporal diversification depends heavily on the resolution of deep nodes in avian phylogeny.     

Phylogeny of extant and fossil Juglandaceae inferred from the integration of molecular and morphological data sets

It is widely acknowledged that integrating fossils into data sets of extant taxa is imperative for proper placement of fossils, resolution of relationships, and a better understanding of character evolution. The importance of this process has been further magnified because of the crucial role of fossils in dating divergence times. Outstanding issues remain, including appropriate methods to place fossils in phylogenetic trees, the importance of molecules versus morphology in these analyses, as well as the impact of potentially large amounts of missing data for fossil taxa. In this study we used the angiosperm clade Juglandaceae as a model for investigating methods of integrating fossils into a phylogenetic framework of extant taxa. The clade has a rich fossil record relative to low extant diversity, as well as a robust molecular phylogeny and morphological database for extant taxa. After combining fossil organ genera into composite and terminal taxa, our objectives were to (1) compare multiple methods for the integration of the fossils and extant taxa (including total evidence, molecular scaffolds, and molecular matrix representation with parsimony [MRP]); (2) explore the impact of missing data (incomplete taxa and characters) and the evidence for placing fossils on the topology; (3) simulate the phylogenetic effect of missing data by creating "artificial fossils"; and (4) place fossils and compare the impact of single and multiple fossil constraints in estimating the age of clades. Despite large and variable amounts of missing data, each of the methods provided reasonable placement of both fossils and simulated "artificial fossils" in the phylogeny previously inferred only from extant taxa. Our results clearly show that the amount of missing data in any given taxon is not by itself an operational guideline for excluding fossils from analysis. Three fossil taxa (Cruciptera simsonii, Paleoplatycarya wingii, and Platycarya americana) were placed within crown clades containing living taxa for which relationships previously had been suggested based on morphology, whereas Polyptera manningii, a mosaic taxon with equivocal affinities, was placed firmly as sister to two modern crown clades. The position of Paleooreomunnea stoneana was ambiguous with total evidence but conclusive with DNA scaffolds and MRP. There was less disturbance of relationships among extant taxa using a total evidence approach, and the DNA scaffold approach did not provide improved resolution or internal support for clades compared to total evidence, whereas weighted MRP retained comparable levels of support but lost crown clade resolution. Multiple internal minimum age constraints generally provided reasonable age estimates, but the use of single constraints provided by extinct genera tended to underestimate clade ages.     

Angiosperm divergence times: the effect of genes, codon positions, and time constraints

An understanding of the evolution of modern terrestrial ecosystems requires an understanding of the dynamics associated with angiosperm evolution, including the timing of their origin and diversification into their extraordinary present-day diversity. Molecular estimates of angiosperm age have varied widely, and many substantially predate the Early Cretaceous fossil appearance of the group. In this study, the effect of different genes, codon positions, and chronological constraints on node ages are examined on divergence time estimates across seed plants, with a special focus on angiosperms. Penalized likelihood was used to estimate divergence times on a phylogenetic hypothesis for seed plants derived from Bayesian analysis, with branch lengths estimated with maximum likelihood. The plastid genes atpB, psaA, psbB, and rbcL were used individually and in combination, using first and second, third, and the three codon positions, including and excluding age constraints on 20 nodes derived from a critical examination of the land-plant fossil record. The optimal level of rate smoothing according to each unconstrained and constrained dataset was obtained with penalized likelihood. Tests for a molecular clock revealed significantly unclocklike rates in all datasets. Addition of fossil constraints resulted in even greater departures from constancy. Consistently with significant deviations from a clock, estimated optimal smoothing values were low, but a strict correlation between rate heterogeneity and optimal smoothing value was not found. Age estimates for nodes across the phylogeny varied, sometimes substantially, with gene and codon position. Nevertheless, estimates based on the four concatenated genes are very similar to the mean of the four individual gene estimates. For any given node, unconstrained age estimates are more variable than constrained estimates and are frequently younger than well-substantiated fossil members of the clade. Constrained estimates of ages of clades are older than unconstrained estimates and oldest fossil representatives, sometimes substantially so. Angiosperm age estimates decreased as rate smoothing increased. Whereas the range of unconstrained angiosperm age estimates spans the fossil age of the clade, the range of constrained estimates is narrower (and older) than the earliest angiosperm fossils. Results unambiguously indicate the relevance of constraints in reducing the variability of ages derived from different partitions of the data and diminishing the effect of the smoothing parameter. Constrained optimizations of divergence times and substitution rates across the phylogeny suggest appreciably different evolutionary dynamics for angiosperms and for gymnosperms. Whereas the gymnosperm crown group originated shortly after the origin of seed plants, a long time elapsed before the origin of crown group angiosperms. Although absolute age estimates of angiosperms and angiosperm clades are older than their earliest fossils, the estimated pace of phylogenetic diversification largely agrees with the rapid appearance of angiosperm lineages in stratigraphic sequences.     

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.     

Multiple Miocene Melastomataceae dispersal between Madagascar, Africa and India

Melastomataceae sensu stricto (excluding Memecylaceae) comprise some 3000 species in the neotropics, 1000 in Asia, 240 in Africa, and 230 in Madagascar. Previous family-wide morphological and DNA analyses have shown that the Madagascan species belong to at least three unrelated lineages, which were hypothesized to have arrived by trans-oceanic dispersal. An alternative hypothesis posits that the ancestors of Madagascan, as well as Indian, Melastomataceae arrived from Africa in the Late Cretaceous. This study tests these hypotheses in a Bayesian framework, using three combined sequence datasets analysed under a relaxed clock and simultaneously calibrated with fossils, some not previously used. The new fossil calibration comes from a re-dated possibly Middle or Upper Eocene Brazilian fossil of Melastomeae. Tectonic events were also tentatively used as constraints because of concerns that some of the family's fossils are difficult to assign to nodes in the phylogeny. Regardless of how the data were calibrated, the estimated divergence times of Madagascan and Indian lineages were too young for Cretaceous explanations to hold. This was true even of the oldest ages within the 95% credibility interval around each estimate. Madagascar's Melastomeae appear to have arrived from Africa during the Miocene. Medinilla, with some 70 species in Madagascar and two in Africa, too, arrived during the Miocene, but from Asia. Gravesia, with 100 species in Madagascar and four in east and west Africa, also appears to date to the Miocene, but its monophyly has not been tested. The study afforded an opportunity to compare divergence time estimates obtained earlier with strict clocks and single calibrations, with estimates based on relaxed clocks and different multiple calibrations and taxon sampling.     

The effects of fossil placement and calibration on divergence times and rates: An example from the termites (Insecta: Isoptera)

Among insects, eusocial behavior occurs in termites, ants, some bees and wasps. Isoptera and Hymenoptera convergently share social behavior, and for both taxa its evolution remains poorly understood. While dating analyses provide researchers with the opportunity to date the origin of eusociality, fossil calibration methodology may mislead subsequent ecological interpretations. Using a comprehensive termite dataset, we explored the effect of fossil placement and calibration methodology. A combined molecular and morphological dataset for 42 extant termite lineages was used, and a second dataset including these 42 taxa, plus an additional 39 fossil lineages for which we had only morphological data. MrBayes doublet-model analyses recovered similar topologies, with one minor exception (Stolotermitidae is sister to the Hodotermitidae, s.s., in the 42-taxon analysis but is in a polytomy with Hodotermitidae and (Kalotermitidae + Neoisoptera) in the 81-taxon analysis). Analyses using the r8s program on these topologies were run with either minimum/maximum constraints (analysis a = 42-taxon and analysis c = 81-taxon analyses) or with the fossil taxon ages fixed (ages fixed to be the geological age of the deposit from which they came, analysis b = 81-taxon analysis). Confidence intervals were determined for the resulting ultrametric trees, and for most major clades there was significant overlap between dates recovered for analyses A and C (with exceptions, such as the nodes Neoisoptera, and Euisoptera). With the exception of isopteran and eusiopteran node ages, however, none of the major clade ages overlapped when analysis B is compared with either analysis A or C. Future studies on Dictyoptera should note that the age of Kalotermitidae was underestimated in absence of kalotermitid fossils with fixed ages.     

Effects of taxon sampling on molecular dating for within‐genus divergence events,when deep fossils are used for calibration

A universal method of molecular dating that can be applied to all families and genera regardless of their fossil records, or lack thereof, is highly desirable. A possible method for eudicots is to use a large phylogeny calibrated using deep fossils including tricolpate pollen as a fixed (124 mya) calibration point. This method was used to calculate node ages within three species‐poor disjunct basal eudicot genera, Caulophyllum, Podophyllum and Pachysandra, and sensitivity of these ages to effects such as taxon sampling were then quantified. By deleting from one to three accessions related to each genus in 112 different combinations, a confidence range describing variation due only to taxon sampling was generated. Ranges for Caulophyllum, Podophyllum and Pachysandra were 8.4–10.6, 7.6–20.0, and 17.6–25.0 mya, respectively. However, the confidence ranges calculated using bootstrapping were much wider, at 3–19, 0–32 and 11–32 mya, respectively. Furthermore, deleting 10 adjacent taxa had a large effect in Pachysandra only, indicating that undersampling effects are significant among Buxales. Changes to sampling density in neighboring clades, or to the position of the fixed fossil calibration point had small to negligible effects. Non‐parametric rate smoothing was more sensitive to taxon sampling effects than was penalized likelihood. The wide range for Podophyllum, compared to the other two genera, was probably due to a high degree of rate heterogeneity within this genus. Confidence ranges calculated by this method could be narrowed by sampling more individuals within the genus of interest, and by sequencing multiple DNA regions from all species in the phylogeny.     

Wing Shape of Four New Bee Fossils (Hymenoptera: Anthophila) Provides Insights to Bee Evolution

Bees (Anthophila) are one of the major groups of angiosperm-pollinating insects and accordingly are widely studied in both basic and applied research, for which it is essential to have a clear understanding of their phylogeny, and evolutionary history. Direct evidence of bee evolutionary history has been hindered by a dearth of available fossils needed to determine the timing and tempo of their diversification, as well as episodes of extinction. Here we describe four new compression fossils of bees from three different deposits (Miocene of la Cerdanya, Spain; Oligocene of Céreste, France; and Eocene of the Green River Formation, U.S.A.). We assess the similarity of the forewing shape of the new fossils with extant and fossil taxa using geometric morphometrics analyses. Predictive discriminant analyses show that three fossils share similar forewing shapes with the Apidae [one of uncertain tribal placement and perhaps near Euglossini, one definitive bumble bee (Bombini), and one digger bee (Anthophorini)], while one fossil is more similar to the Andrenidae. The corbiculate fossils are described as Euglossopteryx biesmeijeri De Meulemeester, Michez, & Engel, gen. nov. sp. nov. (type species of Euglossopteryx Dehon & Engel, n. gen.) and Bombus cerdanyensis Dehon, De Meulemeester, & Engel, sp. nov. They provide new information on the distribution and timing of particular corbiculate groups, most notably the extension into North America of possible Eocene-Oligocene cooling-induced extinctions. Protohabropoda pauli De Meulemeester & Michez, gen. nov. sp. nov. (type species of Protohabropoda Dehon & Engel, n. gen.) reinforces previous hypotheses of anthophorine evolution in terms of ecological shifts by the Oligocene from tropical to mesic or xeric habitats. Lastly, a new fossil of the Andreninae, Andrena antoinei Michez & De Meulemeester, sp. nov., further documents the presence of the today widespread genus Andrena Fabricius in the Late Oligocene of France.     

Integrating fossils in a molecular‐based phylogeny and testing them as calibration points for divergence time estimates in Menispermaceae

Abstract The phylogeny of extant Menispermaceae (Ranunculales) is reconstructed based on DNA sequences of two chloroplast genes (rbcL and atpB) from 94 species belonging to 56 genera. Fossilized endocarps represent 34 genera. The positions of these are inferred using 30 morphological characters and the molecular phylogeny as a backbone constraint. Nine of the thirteen nodes that are each dated by a fossil are used as calibration points for the estimates of molecular divergence times. BEAST is used to estimate stem age (121.2 Myr) and crown age (105.4 Myr) for Menispermaceae. This method does not require an input tree topology and can also account for rate heterogeneity among lineages. The sensitivity of these estimates to fossil constraints is then evaluated by a cross‐validation procedure. The estimated origin for Menispermaceae is dated to the mid‐Jurassic if the customary maximum age of 125 Myr for eudicots is not implemented. All constraints when used alone failed to estimate node ages in some parts of the tree. Fossils from the Palaeocene and Eocene impose strict constraints. Likewise, the use of Prototinomiscium as a dating constraint for Menispermaceae appears to be a conservative approach.     

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.     

Giving the early fossil record of sponges a squeeze

Twenty candidate fossils with claim to be the oldest representative of the Phylum Porifera have been re‐analysed. Three criteria are used to assess each candidate: (i) the diagnostic criteria needed to categorize sponges in the fossil record; (ii) the presence, or absence, of such diagnostic features in the putative poriferan fossils; and (iii) the age constraints for the candidate fossils. All three criteria are critical to the correct interpretation of any fossil and its placement within an evolutionary context. Our analysis shows that no Precambrian fossil candidate yet satisfies all three of these criteria to be a reliable sponge fossil. The oldest widely accepted candidate, Mongolian silica hexacts from c. 545 million years ago (Ma), are here shown to be cruciform arsenopyrite crystals. The oldest reliable sponge remains are siliceous spicules from the basal Cambrian (Protohertzina anabarica Zone) Soltanieh Formation, Iran, which are described and analysed here in detail for the first time. Extensive archaeocyathan sponge reefs emerge and radiate as late as the middle of the Fortunian Stage of the Cambrian and demonstrate a gradual assembly of their skeletal structure through this time coincident with the evolution of other metazoan groups. Since the Porifera are basal in the Metazoa, their presence within the late Proterozoic has been widely anticipated. Molecular clock calibration for the earliest Porifera and Metazoa should now be based on the Iranian hexactinellid material dated to c. 535 Ma. The earliest convincing fossil sponge remains appeared at around the time of the Precambrian‐Cambrian boundary, associated with the great radiation events of that interval.     

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.     

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.     

Neither phylogenomic nor palaeontological data support a Palaeogene origin of placental mammals

O''Leary et al. (O''Leary et al. 2013 Science 339, 662–667. (doi:10.1126/science.1229237)) performed a fossil-only dating analysis of mammals, concluding that the ancestor of placentals post-dated the Cretaceous–Palaeogene boundary, contradicting previous palaeontological and molecular studies that placed the ancestor in the Cretaceous. They incorrectly used fossil ages as species divergence times for crown groups, while in fact the former should merely form minimum-age bounds for the latter. Statistical analyses of the fossil record have shown that crown groups are significantly older than the oldest ingroup fossil, so that fossils do not directly reflect the true ages of clades. Here, we analyse a 20 million nucleotide genome-scale alignment in conjunction with a probabilistic interpretation of the fossil ages from O''Leary et al. Our combined analysis of fossils and molecules demonstrates that Placentalia originated in the Cretaceous.     

Broken gears in the avian molecular clock: new phylogenetic analyses support stem galliform status for Gallinuloides wyomingensis and rallid affinities for Amitabha urbsinterdictensis

Galliformes (landfowl) have been the focus of numerous divergence dating studies that seek a refined understanding of the early radiation of living birds. The Eocene fossil birds Amitabha urbsinterdictensis (Bridger Formation) and Gallinuloides wyomingensis (Green River Formation) have been used extensively in studies dealing with the timing of evolution in crown Galliformes. Divergence estimates from studies incorporating these fossils as calibration points suggest that multiple galliform lineages radiated in the Cretaceous and survived the Cretaceous–Tertiary mass extinction. However, the phylogenetic position of both fossils has been disputed, particularly with regard to crown or stem status. In order to resolve this debate, a new study of A. urbsinterdictensis and G. wyomingensis was undertaken. Further preparation and re‐examination of the A. urbsinterdictensis holotype indicates this fossil falls outside both crown and stem Galliformes, and reveals evidence for a relationship with Rallidae (rails). In order to reassess the status of G. wyomingensis, a matrix of 120 morphological characters was constructed by revising and expanding on previous studies. Phylogenetic analyses using this matrix place G. wyomingensis basal to all crown Galliformes. Stem placement of G. wyomingensis is retained and resolution is improved in combined analyses incorporating sequence data from cytochrome b, NADH dehydrogenase subunit 2, mitochondrial control region, 12S rDNA, and nuclear ovomucoid intron G. All evidence indicates that A. urbsinterdictensis and G. wyomingensis are inappropriate internal calibration points for Galliformes and may have contributed to overestimation of divergence event ages. Though stem galliforms existed in the Cretaceous, the divergence of crown lineages in the Cretaceous remains inconclusively demonstrated. Because few galliform fossils have been evaluated phylogenetically, further investigations into the tempo of galliform evolution must await identification of proper fossil calibration points. © The Willi Hennig Society 2009.     

Fossil calibration of molecular divergence infers a moderate mutation rate and recent radiations for pinus

Silent mutation rate estimates for Pinus vary 50-fold, ranging from angiosperm-like to among the slowest reported for plants. These differences either reflect extraordinary genomic processes or inconsistent fossil calibration, and they have important consequences for population and biogeographical inferences. Here we estimate mutation rates from 4 Pinus species that represent the major lineages using 11 nuclear and 4 chloroplast loci. Calibration was tested at the divergence of Pinus subgenera with the oldest leaf fossil from subg. Strobus (Eocene; 45 MYA) or a recently published subg. Strobus wood fossil (Cretaceous; 85 MYA). These calibrations place the origin of Pinus 190-102 MYA and give absolute silent rate estimates of 0.70-1.31x10(-9) and 0.22-0.42x10(-9).site-1.year-1 for the nuclear and chloroplast genomes, respectively. These rates are approximately 4- to 20-fold slower than angiosperms, but unlike many previous estimates, they are more consistent with the high per-generation deleterious mutation rates observed in pines. Chronograms from nuclear and chloroplast genomes show that the divergence of subgenera accounts for about half of the time since Pinus diverged from Picea, with subsequent radiations occurring more recently. By extending the sampling to encompass the phylogenetic diversity of Pinus, we predict that most extant subsections diverged during the Miocene. Moreover, subsect. Australes, Ponderosae, and Contortae, containing over 50 extant species, radiated within a 5 Myr time span starting as recently as 18 MYA. An Eocene divergence of pine subgenera (using leaf fossils) does not conflict with fossil-based estimates of the Pinus-Picea split, but a Cretaceous divergence using wood fossils accommodates Oligocene fossils that may represent modern subsections. Because homoplasy and polarity of character states have not been tested for fossil pine assignments, the choice of fossil and calibration node represents a significant source of uncertainty. Based on several lines of evidence (including agreement with ages inferred using calibrations outside of Pinus), we conclude that the 85 MYA calibration at the divergence of pine subgenera provides a reasonable lower bound and that further refinements in age and mutation rate estimates will require a synthetic examination of pine fossil history.