Molecular-based divergence dating methods, or molecular clocks, are the primary neontological tool for estimating the temporal origins of clades. While the appropriate use of vertebrate fossils as external clock calibrations has stimulated heated discussions in the paleontological community, less attention has been given to the quality and implementation of other calibration types. In lieu of appropriate fossils, many studies rely on alternative sources of age constraints based on geological events, substitution rates and heterochronous sampling, as well as dates secondarily derived from previous analyses. To illustrate the breadth and frequency of calibration types currently employed, we conducted a literature survey of over articles published from to Current patterns in calibration practices were disproportionate to the number of discussions on their proper use, particularly regarding plants and secondarily derived dates, which are both relatively neglected in methodological evaluations. Based on our survey, we provide a comprehensive overview of the latest approaches in clock calibration, and outline strengths and weaknesses associated with each.
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Sorry, that molecular dating calibrations more than word!
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Gheerbrant E. Flynn J. A radiation of arboreal basal eutherian mammals beginning in the Late Cretaceous of India. Janke A. Mammalian evolution may not be strictly bifurcating. Time scale of eutherian evolution estimated without assuming a constant rate of molecular evolution. Genes Genet. Huelsenbeck J. The fossilized birth-death process for coherant calibration of divergence time estimates.
Accounting for calibration uncertainty in phylogenetic estimation of evolutionary divergence times. Accuracy of rate estimation using relaxed-clock models with a critical focus on the early metazoan radiation.
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May 26, Molecular-based divergence dating methods, or molecular clocks, are the primary neontological tool for estimating the temporal origins of clades. While the appropriate use of vertebrate fossils as external clock calibrations has stimulated heated discussions in the paleontological community, less attention has been given to the quality and implementation of other calibration ojasjobz.com by: Jan 07, Consequently, node-based calibrations will continue to play an important role in molecular dating. As we have demonstrated, establishing accurate constraints should not rely on a posteriori methods, and so node-based calibrations established using a priori methods will remain especially significant for groups for whom the molecular clock is the Cited by: This may result in poorly informative calibrations and an alternative, time-consuming approach would be to conduct as many molecular dating analyses as there are positions of a fossil on the phylogeny (Rutschmann et al., ). Finally, in many cases, only an intuitive assignment to extant taxa, similar to using an overall similarity criterion Cited by:
The impact of fossils and taxon sampling on ancient molecular dating analyses. Spiny Norman in the Garden of Eden? Dispersal and early biogeography of Placentalia. Bielby J. Fritz S. O'Dell J. Orme C. Safi K. Sechrest W. Boakes E. Carbone C. Connolly C. Cutts M. Foster J. Greyner R. Habib M.
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Marivaux L. Adaci M. Bensalah M. Rodrigues H. Hautier L. Mahboubi M. Mebrouk F. Tabuce R. Vianey-Liaud M. Laurin M. Fossils, molecules, divergence times, and the origin of lissamphibians. Linked cts of nonmarine Cretaceous-Tertiary boundary events. Carnegie Mus. Meredith R. Janecka J.
The molecular clock is a figurative term for a technique that uses the mutation rate of biomolecules to deduce the time in prehistory when two or more life forms ojasjobz.com biomolecular data used for such calculations are usually nucleotide sequences for DNA, RNA, or amino acid sequences for ojasjobz.com benchmarks for determining the mutation rate are often fossil or archaeological dates. Molecular-based divergence dating methods, or molecular clocks, are the primary neontological tool for estimating the temporal origins of clades. While the appropriate use of vertebrate fossils as external clock calibrations has stimulated heated discussions in the paleontological community, less attention has been given to the quality and Cited by: Bayesian dating, Caenophidia, cross-validation, fossil calibrations, Hydrophiinae, molecular clock, nucleotide saturation, snakes Ideally, molecular clock calibrations are obtained from accurately dated fossils that can be assigned to nodes with high phylogenetic precision (Graur and Martin ), but reality is generally far from this ideal.
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O'Brien S. Madsen O. Scally M. Douady C. Stanhope M. Resolution of the early placental mammal radiation using Bayesian phylogenetics. Nabholz B. Glemin S. Galtier N.
Strong variations of mitochondrial mutation rate across mammals-the longevity hypothesis. Gebo D. Dagosto M. Tafforeau P. Beard K. The oldest known primate skeleton and early haplorhine evolution. Nature : 60 - Mammalian phylogeny: Shaking the tree.
Nowack M. Smith A. Simpson C. Zwickl D. A simple method for estimating informative node age priors for the fossil calibration of molecular divergence time analyses. PLoS One 8 : e O'Leary M. Bloch J. Gaudin T. Giallombardo A. Giannini N. Goldberg S. Kraatz B. Luo Z. Perini F. Randall Z.
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Pagel M. Meade A. Bayesian analysis of correlated evolution of discrete characters by reversible-jump Markov chain Monte Carlo. Parham J. Calway T. Head J. Holroyd P. Imris R. Joyce W. Smith N. Tarver J. Angielczyk K. Greenwood J. Hipsley C. Jacobs L. Makovicky P. Smith K.
Theodor J. Warnock R. Best practices for justifying fossil calibrations. Branch-length estimation bias misleads molecular dating for a vertebrate mitochondrial phylogeny.
Gene : - Bennett T. Molecules, morphology, and ecology indicate a recent, amphibious ancestry for echidnas. Four mammal fossil calibrations: Balancing competing palaeontological and molecular considerations. Electronica 17 : 1. Pyron R. Divergence time estimation using fossils as terminal taxa and the origins of Lissamphibia. Rannala B. Bayesian estimation of species divergence times from multiple loci using multiple calibrations.
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Schulmeister S. Murray D. Rasnitsyn A. A total-evidence approach to dating with fossils, applied to the early radiation of the Hymenoptera. Sanderson M. A nonparametric approach to estimating divergence times in the absence of rate constancy.
Sauquet H. A practical guide to molecular dating. Comptes Rendus Palevol. Scotland R. Olmstead R. Bennett J. Phylogeny reconstruction: The role of morphology.
Simons E. Ryan T. Attia Y. Additional remains of Wadilemur elegansa primitive stem galagid from the late Eocene of Egypt. Seymour K. Habersetzer J. Gunnell G. Primitive Early Eocene bat from Wyoming and the evolution of flight and echolocation.
Simpson G. The principles of classification and a classification of mammals. The adequacy of morphology for reconstructing the early history of placental mammals. Cleven G. Waddell V. Amrine H. Endemic African mammals shake the phylogenetic tree. Nature : 61 - The historical biogeography of Mammalia. Emerling C. Park J. Macroevolutionary dynamics and historical biogeography of primate diversification inferred from a species supermatrix. Plos Biol. Science : b.
Eizrik E. Placental mammal diversification and the Cretaceous-Tertiary boundary. Swofford D. Sunderland : Sinauer Associates.
Carbon Dating: Calibration
For example, underestimating the true rate of hidden substitution results in tree compression: However, if the rate of hidden substitutions were to be overestimated, the reverse would be true.
These effects are further complicated by the calibration placement. For example, if only deep splits are calibrated, then recent nodes will be biased to be younger under tree extension and older under tree compression. Slowly evolving genes, as are typical for nuclear DNA, are less prone to such saturation effects; however, nuclear DNA data are not completely immune to these issues; problems of saturation also can emerge for slowly evolving nuclear loci if deeper divergences are being investigated.
Molecular dating calibrations
More importantly, although the effects of saturation have been documented for estimating divergence times Brandley et al. Indeed, recent papers using calibrated molecular clocks to date divergences among advanced snake clades highlight the extent of controversy about the placements of certain fossils Wuster et al. In part, this controversy exists because of the relatively poor nature of the snake fossil record.
Well preserved and relatively complete caenophidian fossils date back no further than the Miocene Rage and often belong to extant genera RageSzyndlar and RageSzyndlar and Ragethus are of little value as calibration points for most studies. Earlier caenophidian fossils mostly comprise isolated vertebrae, the taxonomic affinities of which have been strongly debated McDowellRage Perhaps, the most controversial calibrations concern the origin of caenophidian snakes themselves, which has been assigned dates of 38 myr Sanders and LeeKelly et al.
As such, very different dates have been used to calibrate the caenophidian molecular clock Nagy et al. Details of fossils tested using three approaches for evaluating candidate fossil calibrations. Notes: Constraints are given as absolute values millions of years before present and the corresponding lognormal mean, standard deviation, and zero offset of the calibration prior used in BEAST analyses. Phylogenetic placement of nodes is shown on Figure 1. In this paper, we use advanced snakes as a test case to compare three previously published methods for evaluating fossil calibrations: the single-fossil cross-validation method of Near et al.
Briefly, the single-fossil cross-validation approach Near et al. The approach of Marshall aims to identify candidate calibrations with the best fossil coverage and then tests whether these fossils are potential outliers.
Was molecular dating calibrations consider
Finally, the Bayesian multicalibration approach evaluates one or more alternative calibrations in a set by comparing the Bayesian prior and posterior probabilities PPs at fossil-calibrated nodes Sanders and Lee We explicitly evaluate the effects of using sequence data with different rates of molecular evolution on the best fossils identified by each method using the same mitochondrial and nuclear sequence data set each with identical taxon sampling for each method.
In addition, we evaluate whether saturation effects can be ameliorated by 1 removing the third codon position of the mitochondrial coding regions and 2 analyzing a combined nuclear and mitochondrial data set. Our study focused on testing alternative placements or ages of controversial fossil calibrations as is typical for groups with poor fossil records ; however, our approach is relevant for any situation where numerous candidate fossil calibrations exist.
Colubroid classification is in flux Vidal et al. We use the traditional colubroid classification as comprising viperids, elapids, and colubrids, including colubrid subfamilies recently elevated to higher taxonomic ranks McDowellRageLawson et al.
We specifically selected fossil calibrations that often have been used to date recent caenophidian divergences Nagy et al.
Details of the fossil calibrations evaluated are given in Table 1. The mitochondrial data comprised 16S rRNA bpNADH dehydrogenase subunit 4 ND4 bpand cytochrome b bpand the nuclear data comprised the oocyte maturation factor gene c- mos - bp and the recombination activating gene 1 RAG bp. Sequences were edited in SeqMan Lasergene v. Following alignment, coding region sequences were translated into amino acid sequences in MacClade v. No premature stop codons were observed, so we are confident that the mitochondrial sequences obtained were mitochondrial in origin, and that the nuclear genes were not nonfunctional nuclear copies pseudogenes.
In order to evaluate saturation in each of the mitochondrial codon positions, we also constructed saturation plots for the first, second, and third codon positions of the ND4 and cytochrome b genes.
The best-fit models of molecular evolution for the nuclear and mitochondrial data sets were selected based on Akaike Information Criteria AIC implemented in ModelTest 3. We evaluated alternative partitioning strategies using a modified version of the Akaike information criterion for small sample sizes AIC c and Bayesian information criterion BIC McGuire et al.
AIC c and BIC values incorporate a penalty for increasing the number of parameters in the model, thus potentially avoiding problems with model overparameterization.
Caveats on the use of fossil calibrations for molecular dating: a reply to Parham and Irmis. Am. Nat. , - (doi/). Mar 15, A limiting factor in many molecular dating studies is shortage of reliable calibrations. Current methods for choosing calibrations (e.g. cross-validation) treat them as either correct or incorrect, whereas calibrations probably lie on a continuum from highly accurate to very poor. Recently, a total-evidence or (hereafter) tip-dating method to use fossil calibrations for molecular clock analyses has been pro-posed by Pyron and Ronquist et al. This approach requires morphological data to simultaneously infer the placement of the fossil in the phylogeny and to calibrate the tree. Tip dating.
Bayesian analyses four incrementally heated chains run for 2, generations sampled every th generation with all substitution parameters and rates allowed to vary across partitions were conducted in MrBayes Ronquist and Huelsenbeck and used to evaluate combinations of character partition and evolutionary model.
However, MrBayes returned unrealistic estimates of alpha for the nDNA1 gamma distribution of rate heterogeneity Bayesian relaxed molecular clocks, which assume rates of molecular evolution are uncorrelated but lognormally distributed among lineages Drummond et al.
Yule and birth-death models performed similarly in all preliminary analyses, so the birth-death model Gernhard with a uniform prior was used to model cladogenesis for all final analyses. ESS values greater than are generally regarded as being sufficient to obtain a reliable posterior distribution Drummond et al.
ESS values typically were much larger than for most parameters in each analysis. Bayesian chronograms from BEAST analyses with tree roots constrained to 97 myr indicating the position of 10 candidate fossil-calibrated nodes root and nodes evaluated in this study. The second difference in our approach was that, rather than using the cross-validations to exclude specific fossils, we used them in a more exploratory fashion to evaluate the alternative placements of three fossils as calibrations for their respective stem nodes 4, 6, and 8 and crown nodes 5, 7, and 9 clades Table 1.
We also evaluated three different pairs referred to as calibration sets of fossil dates for two nodes, the most recent common ancestor MRCA of Caenophidia Fig. Each alternative set of fossil dates for nodes 2 and 3 Table 1 : Sets A, B, C was evaluated by conducting a separate iteration of the cross-validation exercise i. The molecular and fossil dates for the other eight single-fossil-calibrated nodes were the same for the three calibration sets.
Preliminary analyses revealed that the shallower calibrations Fig. BEAST runs for single-fossil cross-validations were conducted as follows: nDNA-4, generations sampled every generations, mtDNA-5, generations sampled every generations, and mtDNA3rdExcl, generations sampled every generations.
The approach of Marshall involves generating an ultrametric tree that is uncalibrated with respect to the fossil record and then mapping all candidate fossil calibrations onto the tree to determine which of the calibrated lineages has the best temporal fossil coverage.
Specifically, the method aims to identify the lineage, for which the oldest fossil for that lineage sits proportionally closest to the node of its MRCA true time of origin and therefore has the best temporal coverage. Marshall emphasizes two assumptions of the method: 1 the proportional branch lengths of the ultrametric tree are accurate and 2 fossilization is random: However, the method also assumes that fossils are accurately dated and assigned correctly to their respective lineages see below for further discussion.
The first and arguably most important step in the approach of Marshall is to generate a reliable ultrametric phylogeny that is uncalibrated with respect to the fossil record using an appropriate relaxed clock algorithm. Given that obtaining accurate proportional branch lengths of the ultrametric tree is critical to the success of this method, we generated a number of ultrametric trees using different approaches and compared the results.
As such, we followed the approach of Marshall and obtained ultrametric trees using r8s Sanderson The lizard taxa were pruned from the optimal ML and Bayesian trees and the resulting rooted trees used to obtain ultrametric trees in r8s, again fixing the root age to an arbitrary value of We used semiparametric penalized likelihood PL Sanderson and optimal smoothing parameters identified from the cross-validation procedure in r8s as follows: MrBayes tree-smoothing parameter of with log penalty function and ML tree-smoothing parameter of with additive penalty function.
Given that Smith et al. We tested for possible fossil outliers by comparing the distribution of ESF i values to a uniform distribution using the Kolmorgorov-Smirnov test, on the assumption that ESF i values for fossil outliers lie outside a uniform distribution Marshall One limitation of this approach is that it is most effective if there is just one outlier Marshallp. We were testing the alternative stem and crown placements of three fossils. As such, the ESF i values for the crown placements that inevitably will be larger than the ESF i values for their stem placements might potentially cluster together, thereby making it impossible to identify them as outliers.
In order to address this issue, we modified the approach of Marshall to test the alternative placements of these fossils see Results section for details. We used the method of Sanders and Lee to evaluate three alternative dates for two nodes with controversial fossil calibrations in a Bayesian multicalibration framework.
In our case, the single-fossil cross-validations identified plausible congruent calibration sets comprising six fossil-calibrated nodes that included nodes 2 and 3 but could not distinguish between the different possible ages assigned to these two nodes Table 1 - Sets A, Band C. In addition, the ESF i values for the same six fossil-calibrated nodes indicated that none were outliers. However, ESF i values cannot be used to evaluate alternative dates for the same node because the oldest date will inevitably have the highest empirical coverage, even if that date is not correct.
Moreover, ESF i values from different ultrametric trees identified different fossils as having the highest empirical coverage see below for details. We evaluated the alternative ages for nodes 2 and 3 using three sets of BEAST multicalibration analyses that incorporated the four congruent calibrations and the Set A, B, and C node 2 and 3 calibration ages in turn. For each analysis, we compared the prior and posterior distributions of all six fossil-calibrated nodes, with the expectation that the node 2 and 3 calibration set most consistent with the other four fossil-dated nodes would return posterior distributions for all six calibrated nodes that were similar to their prior constraints Sanders and Lee We also conducted a fourth set of analyses using the four congruent fossils with no constraints on nodes 2 and 3 Set D and compared the unconstrained and constrained node 2 and 3 age estimates.
MCMC runs comprised 4, generations for the nuclear data and 10, generations for both mitochondrial data sets. In each case, MCMC runs were sampled every generations. Given that certain combinations of priors can interact to generate unexpected effective joint priors, we also performed an analysis for each calibration set without data empty alignments to ensure that the effective priors were similar to the original priors.
We assessed how informative the data were by comparing the effective priors with posteriors obtained using data Drummond et al. These analyses indicated that the effective priors were similar to the original priors and the posteriors obtained from the data departed from the priors indicating informative data. The final nDNA alignment had characters of which were variable and were parsimony informative, whereas the mtDNA alignment had characters of which were variable and were parsimony informative, and the mtDNA3rdExcl had characters of which were variable and were parsimony informative.
Saturation plots revealed an abundance of hidden substitutions in all three codon positions of the mitochondrial data set Fig. Corrected genetic distances were calculated using the estimated best-fit models of sequence evolution obtained from AIC criterion in ModelTest.
Note the different axis scales for the nuclear and mitochondrial data sets. Note the different x -axis scales for b, c, and d. In all cases, the results of single-fossil cross-validations using mean and median age estimates from BEAST were highly consistent, so we present only the results from the mean age estimates. Nuclear DNA cross-validations produced similar results for each calibration set, with values indicating that four fossils consistently produced older molecular divergence estimates for other candidate fossil-calibrated nodes, whereas the other six fossils produced younger divergence estimates; however, the relative magnitude of these tendencies differed between calibration sets Fig.
SS values ranked the four node calibrations that consistently produced older molecular divergence estimates for other FAs as the most incongruent fossils Fig. Sequential removal of fossil calibrations from most to least divergent, as ranked by SS values Fig.
At this point, s values for se ts B and C were small and subsequent removal of fossils did not markedly decrease s values Fig. Starting s values for set A were much larger than for sets B and C and did not drop to low values until the fifth fossil calibration node 2 was removed and then remained low Fig.
Histogram of the mean differences and standard errors between fossil and estimated MAs for each of three sets of 10 single-fossil-calibrated nodes from a nuclear DNA; b mitochondrial DNA; and c mitochondrial DNA with third codon position removed. FAs for 8 of the 10 candidate nodes were identical for each set, differing only for nodes 2 and 3 see Fig.
FAs used as constraints are given in Table 1. For a single node xthe FA at node x was used as a single calibration prior. MA estimates were obtained for the nine other candidate nodes, for which FAs were available. SS values for each candidate fossil calibration node when used as the single calibration prior in each of the three calibration sets for a nuclear DNA; b mitochondrial DNA; and c mitochondrial DNA with third codon position removed.
Effect of sequentially removing candidate fossil calibrated nodes on sthe average squared deviation of D i values for the remaining fossil calibrations in each set. Fossils were removed based on highest to lowest SS values calculated from all 10 fossil-calibrated nodes. Removal order shown on the x -axis of the first four most incongruent fossils was identical for each calibration set but then differed between sets. Most notably, the four fossil calibrations nodes 4, 5, 7, and 9 that returned much older nuclear DNA values for FAs at other candidate calibration nodes either produced younger or only slightly older estimates of FAs for mtDNA Fig.
In addition, the tendency for nodes 6 and 8 to produce younger MAs for fossil dates at other nodes was more extreme for the mitochondrial than nuclear data, and this was true for both mitochondrial data sets Fig.
Share your molecular dating calibrations consider, that
By contrast, node 1 produced older ages at other nodes for both mtDNA data sets, whereas this node produced younger dates for nuclear DNA. Given these differences, it is not surprising that mitochondrial SS values ranked fossils differently than nuclear SS values Fig. In addition, values for the younger set A calibrations at nodes 2 and 3 did not follow the same pattern as for sets B and C Fig.
Sets B andC had highest SS values at nodes 6 and 8; however, removing these nodes only slightly decreased s values, which did not decline sharply until subsequent removals of the third and fourth ranked fossils and then remained low Fig. Interestingly, node 1 was the most incongruent fossil for the younger set A calibrations for the entire mtDNA data set and s values dropped sharply when it was removed. Subsequent removal of the three next most incongruent fossils did not produce further decreases in sbut s decreased with the removal of the fifth and subsequent fossils Fig.
By contrast, node 8 was the most incongruent fossil for all three calibration sets for the mtDNA data set with third codon position excluded, and s values did not drop sharply until the first two most incongruent nodes were excluded in each case Fig.
The four ultrametric trees obtained from the nDNA data set differed in their proportional branch lengths, resulting in differing ESF i values for the candidate fossil calibrations Table 2. Nonetheless, the four highest ESF i values in decreasing order for the ML and MrBayes ultrametric trees were for nodes 9, 7, 5, and 4 Table 2the same nodes identified as least congruent by the cross-validation analyses.
Lack of resolution in the ML and Bayesian nDNA trees resulted in nodes 4 and 5 forming a polytomy: As such, it was not possible to evaluate the alternative placements of this fossil calibration as the ESF i values for the stem and crown placement were identical.
Dec 10, Molecular dating has been transformed by relaxed clock methods that allow the rate of molecular evolution to vary across the phylogeny (Sanderson ; Thorne and Kishino ; and see Welch and Bromham ).Single point calibrations have in turn been superseded by multiple sets of calibration bounds or ojasjobz.com by:
Moreover, issues regarding the taxonomic affinities of these fossils Table 1; and Supplementary material suggest that it is not possible to accurately place them on the phylogeny despite their use to date caenophidian divergences in previous studies: Guicking et al.
As such, we excluded them from the outlier analysis. ESF i for candidate fossil calibrations for nuclear and mitochondrial data sets calculated using proportional branch lengths obtained from uncalibrated ultrametric trees produced using different methods. The remaining ultrametric trees were produced in r8s using the optimal ML and Bayesian MrBayes phylogenies.
Uncalibrated ultrametric trees were obtained by fixing the root to an arbitrary value of and using PL with the logarithmic log or additive add penalty function and the optimal smoothing parameter obtained from cross-validation shown in the column heading.
Nodes and corresponding ESF values highlighted in bold for each ultrametric tree indicate the fossil with the highest empirical coverage after removing fossils identified as outliers i. See text for more details. Nodes 7 and 9 were the shallower crown placements of the two candidate fossil calibrations, for which the alternative deeper stem placements also were evaluated. Obviously, the candidate fossils cannot correctly be assigned to both the stem and crown nodes so, prior to testing whether the distributions of ESF i values conformed to uniform distributions, we removed the ESF i values for the corresponding stem placements of each fossil nodes 6 and 8.
These inconsistent results highlight the sensitivity of this approach to differences in proportional branch lengths obtained from ultrametric trees obtained using different methods see below for further discussion. Given that the weight of evidence suggested that crown placement of the Naja fossil was an outlier, we removed the ESF i values for node 9 and reinserted the ESF i values for the corresponding stem placement of the fossil node 8.
The resulting distributions of ESF i values for the MrBayes and ML ultrametric trees also were rejected as belonging to uniform distributions, suggesting that the crown placement of the putative Laticauda fossil at node 7 also is an outlier. The resulting distributions of ESF i values were not rejected as belonging to uniform distributions.
In terms of the MrBayes tree, the inclusion of ESF i values for both potential outliers nodes 7 and 9 may have resulted in the artifact mentioned by Marshallwhereby the larger ESF i values of outliers group together making it impossible to distinguish the resultant distribution from a uniform distribution thereby failing to identify node 9 as an outlier.
In order to explore this possibility, we removed the ESF i for node 7 and retained the ESF i of the corresponding stem placement at node 6. The resulting distribution of ESF i values did not conform to a uniform distribution, supporting node 9 as an outlier. Overestimation of shorter branches has recently been demonstrated for Bayesian approaches Schwartz and Muellerand the smaller difference between ESF i values for nodes 9 and 7 for the Bayesian than ML trees may reflect overestimation of short branches in the crown Naja clade by MrBayes.
However, the distributions of ESF i values conformed to uniformity for all three mitochondrial ultrametric trees ML and MrBayesand this result was true for distributions including just one potential crown node outlier and the corresponding stem placement of the other fossil : Thus, no outliers were identified. There were consistent differences in the plausible sets of congruent fossil calibrations identified from the cross-validations from nuclear and mitochondrial DNA, and the fossil outliers identified from nuclear but not mitochondrial data based on ESF i values.
These differences are almost certainly due to the effects of nucleotide saturation for mtDNA see Discussion section. As such, we conducted the multicalibration analyses using the six fossil-calibrated nodes selected by the nuclear data.
By contrast, age estimates for nodes 2 and 3 differed considerably between calibration sets, in part reflecting the influence of their calibration priors but also reflecting inconsistencies between these priors and the other four fossil calibrations Fig. Moreover, age estimates for nodes 2 and 3 tended to converge on ages estimated by set D Fig. This tendency was most pronounced for node 2, for which the set A age estimate was far more similar to the set D estimate than to the set A calibration prior.
Indeed, the set A prior and posterior distributions barely overlapped Fig. Similarly, the set B estimated age for node 2 also was closer to the set D estimate than to the set B calibration prior, with the set B maximum age estimate 70 million years younger than its calibration prior Fig.
The node 3 age nDNA estimates were more similar to their respective calibration priors, but again, posterior distributions diverged from priors towards the unconstrained set D age estimate. The set A estimated mean age was slightly older than its calibration prior, but posterior and prior distributions were identical, whereas the set C age estimate also was identical to the mean and minimum bounds of the calibration prior Fig.
Each calibration set comprised four calibration priors that were identical among sets tree root, nodes 1, 6, and 8 and two priors that differed among sets nodes 2 and 3. Lognormal calibration priors are shown as wider shaded bars with the lognormal mean shown as a black square on the bar. MA estimates for nuclear black barsmitochondrial white barsand mitochondrial DNA with third codon position removed gray bars are shown in pairs for each calibration set at each node. At nodes 2 and 3, the respective calibration prior is shown immediately below the corresponding nuclear and mitochondrial age estimates.
Calibration priors at the other four nodes are shown below all four sets of MA estimates. Prior and posterior distributions are shown on a diagrammatic chronogram depicting the backbone of the phylogeny; however, this chronogram does not represent the results of any specific analysis.
Mitochondrial age estimates were invariably older for the shallower nodes 3, 6, and 8 than their respective calibration priors and, with one exception, also for the corresponding nDNA age estimates. By contrast, mitochondrial node 1 age estimates for all calibration sets were similar to the calibration prior and to nuclear DNA age estimates, and this was true for both mitochondrial data sets Fig. Nonetheless, the tendency for mtDNA to return older age estimates at shallow nodes and the tree root was much pronounced when the third codon positions were excluded, with mtDNA3rdExcl age estimates for nodes 6 and 8 age intermediate to the nDNA and mtDNA age estimates and tending to converge on mean nDNA age estimates for node 3 and the tree root Fig.
Although mitochondrial age estimates for node 2 from the entire data set showed the same tendency as nuclear ages to converge on the unconstrained set D age estimates irrespective of the calibration prior use this was not the case for mtDNA with third codon positions excluded Fig.
Indeed, with the exception of set C, the node 2 mtDNA3rdExcl age estimates tended to converge on the calibration prior resulting in age estimates that were younger than the corresponding nDNA estimates, and this was also true for the node 3 set A age estimate Fig.
Increasing awareness of the importance of identifying reliable fossils to calibrate molecular clocks has resulted in the development of several methods for evaluating and employing fossil calibrations reviewed by Ho and Phillips Each approach has advantages and limitations, as we demonstrate by comparing three different approaches with particular emphasis on the impact of nucleotide saturation on the fossils selected.
The cross-validation method Near et al. Nonetheless, the method has been used in several recent studies Near and SandersonNoonan and Chippindale bRutschmann et al. By contrast, the use of ESF aims to identify one fossil with the best empirical coverage Marshall ; however, accurate results are highly dependant on meeting the assumptions of the method see below.
Unlike the cross-validation approach, ESFs have only been used in one previous study Davis et al. This study obtained an ultrametric tree in r8s using PL with log penalty function following the advice of Marshallbased on empirical evidence that PL using the log penalty function produces the most reliable ultrametric trees Smith et al.
However, Davis et al. Our study demonstrated that ultrametric trees generated from ML and Bayesian nDNA phylogenies using the log penalty function were incongruent in terms of the magnitude and order of the ESFs Table 2 and the fossil outliers identified. By contrast, results from the ML ultrametric tree using the additive penalty function were more similar to those obtained for the MrBayes tree. At the very least, these results suggest that the findings of Smith et al. These conflicting results highlight a major limitation of ESFs, which is the reliance on accurate proportional branch lengths which we do not know, or the entire dating process would be considerably easier.
The final step of Marshall's approach uses the lineage with the highest coverage to calibrate the tree and estimate divergences. However, we were evaluating several controversial FAs for this node Table 1 and, by default, the highest coverage will be assigned to the oldest fossil so ESFs cannot be used for this task.
The third method we evaluated, which uses a Bayesian framework to evaluate several candidate fossils in a multicalibration framework Sanders and Leeis ideally suited for the task. However, one limitation of this method is that at least some of the candidate calibrations are assumed to be reliable, with just one or two calibrations being evaluated. In addition, multiple calibrations can interact with each other to generate different effective priors; However, the extent of this effect can be evaluated explicitly Drummond et al.
Nonetheless, one limitation of our study was that the calibrations for nodes 2 and 3 were evaluated in pairs based on their previous use in other studies and, as such, the best combination may not have been included in our analyses. Rutschmann et al. However, this approach will be subject to the same saturation effects demonstrated in our study and, as such, the effects of using rapidly and slowly evolving gene regions or codon positions for evaluating the internal consistency of calibrations will need to be considered.
There is a growing consensus that the advantages of using multiple independent fossil calibrations significantly outweigh any disadvantages Ho and Phillips Moreover, the use of multiple calibrations allows the explicit modeling of rate variation among lineages.
For the molecular dating calibrations necessary words... super
The limitations of using just one calibration in BEAST analyses for modeling rate variation are highlighted in the chronogram from the mitochondrial data set with third codon positions removed: The two basal branches extending from the tree root on the BEAST chronogram were massively stretched and the remaining internal branches overly compressed Fig.
The addition of multiple calibrations ameliorated this effect Fig. Although the mtDNA3rdExcl ultrametric tree generated in r8s did not suffer from similarly stretched basal branches results not shownthe approach of Marshall ultimately relies on just one calibration to date the phylogeny, and our analyses demonstrated the highly variable results that could be obtained using different methods to generate the ultrametric tree Table 2.
Moreover, although this approach might be realistic for groups with exceptionally good fossil records provided that the hurdle of obtaining a reliable ultrametric tree can be overcomeon its own, it is likely to produce highly misleading results in the majority of cases where the fossil record is less than ideal.
The differences in the plausible sets of congruent fossil calibrations identified from the cross-validations from nuclear and mitochondrial DNA, as well as fossil outliers identified from nuclear but not mitochondrial data based on ESF i values, can be entirely accounted for by saturation effects. The saturation plots revealed strong mitochondrial saturation in the data set Fig. The saturation effects on tree topology, and corresponding age estimates of fossil-calibrated nodes, are clearly evident in Figure 1.
Compared with the nuclear chronogram Fig. In terms of the cross-validations, the three sets of nuclear cross-validations identified the same four shallow fossil-calibrated nodes 4, 5, 7, and 9 as least congruent with the six other candidate calibrations tested.
These nodes also had the highest ESF i values Table 2with nodes 7 and 9 being identified as outliers by three of the four nuclear DNA ultrametric trees. By contrast, mitochondrial cross-validations identified nodes 6 and 8 as least congruent for sets B and C and also set A when the third codon positions were removed.
Thus, for two fossils Naja and Laticaudanuclear DNA favored stem placement nodes 8 and 8whereas mtDNA favored crown placement nodes 7 and 9directly as the result of saturation effects. Specifically, if a crown group is constrained with the same fossil calibration as its respective stem group, the placement of a fossil at the shallower crown node will return older estimates at other nodes than stem placement, irrespective of data type. However, because mitochondrial distances were artificially shortened due to compression of internal branches resulting from nucleotide saturationthe tendency for crown placement to produce much older age estimates for other fossil-calibrated nodes, which was so strongly apparent for nuclear DNA, disappeared for mtDNA: Instead, stem placement resulted in younger age estimates at deeper fossil-calibrated nodes.
Similarly, the compressed internal branches for mtDNA resulted in smaller differences between the larger ESF i values; thus, ESF i distributions did not deviate from uniformity with the result that fossil outliers were not identified.
Evaluating these results in terms of the actual fossils Table 1 and Supplementary material A further suggests that misleading results were obtained from the mitochondrial data due to the effects of saturation.
The effects of mitochondrial saturation are also evident in many studies estimating divergence times in snakes.