The age and diversification of the angiosperms re-revisited

? Premise of the study: It has been 8 years since the last comprehensive analysis of divergence times across the angiosperms. Given recent methodological improvements in estimating divergence times, refined understanding of relationships among major angiosperm lineages, and the immense interest in using large angiosperm phylogenies to investigate questions in ecology and comparative biology, new estimates of the ages of the major clades are badly needed. Improved estimations of divergence times will concomitantly improve our understanding of both the evolutionary history of the angiosperms and the patterns and processes that have led to this highly diverse clade. ? Methods: We simultaneously estimated the age of the angiosperms and the divergence times of key angiosperm lineages, using 36 calibration points for 567 taxa and a "relaxed clock" methodology that does not assume any correlation between rates, thus allowing for lineage-specific rate heterogeneity. ? Key results: Based on the analysis for which we set fossils to fit lognormal priors, we obtained an estimated age of the angiosperms of 167-199 Ma and the following age estimates for major angiosperm clades: Mesangiospermae (139-156 Ma); Gunneridae (109-139 Ma); Rosidae (108-121 Ma); Asteridae (101-119 Ma). ? Conclusions: With the exception of the age of the angiosperms themselves, these age estimates are generally younger than other recent molecular estimates and very close to dates inferred from the fossil record. We also provide dates for all major angiosperm clades (including 45 orders and 335 families [208 stem group age only, 127 both stem and crown group ages], sensu APG III). Our analyses provide a new comprehensive source of reference dates for major angiosperm clades that we hope will be of broad utility.     

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.     

Molecular dating and biogeography of the early placental mammal radiation

The timing and phylogenetic hierarchy of early placental mammal divergences was determined based on combined DNA sequence analysis of 18 gene segments (9779 bp) from 64 species. Using rooted and unrooted phylogenies derived from distinct theoretical approaches, strong support for the divergence of four principal clades of eutherian mammals was achieved. Minimum divergence dates of the earliest nodes in the placental mammal phylogeny were estimated with a quartet-based maximum-likelihood method that accommodates rate variation among lineages using conservative fossil calibrations from nine different nodes in the eutherian tree. These minimum estimates resolve the earliest placental mammal divergence nodes at periods between 64 and 104 million years ago, in essentially every case predating the Cretaceous-Tertiary (K-T) boundary. The pattern and timing of these divergences allow a geographic interpretation of the primary branching events in eutherian history, likely originating in the southern supercontinent Gondwanaland coincident with its breakup into Africa and South America 95-105 million years ago. We propose an integrated genomic, paleontological, and biogeographic hypothesis to account for these earliest splits on the placental mammal family tree and address current discrepancies between fossil and molecular evidence.     

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.     

The impact of fossils and taxon sampling on ancient molecular dating analyses

The number and complexity of molecular dating studies has increased over the past decade. Along with a broadening acceptance of their utility has come significant controversy over the methods and models that are appropriate, as well as the accuracy of the estimates yielded by molecular clock analyses. Radically different age estimates have been published for the same divergences from analyses of different datasets with different fossil constraints obtained with different methods, and the underlying explanation for these differences is often unclear. Here we utilize two previously published datasets to examine the effect of fossil calibrations and taxon sampling on the age estimates for two deep eukaryote divergences in an attempt to discern the relative impact of these factors. Penalized likelihood, non-parametric rate smoothing, and Bayesian methods were utilized to generate age estimates for the origin of the Metazoa from a 7-gene dataset and for the divergence of Eukaryotes from a 129-gene dataset. From these analyses, it is clear that the fossil calibrations chosen and the method for applying constraints to these nodes have a large impact on age estimates, while the degree of taxon sampling within a dataset is less important in terms of the resulting age estimates. Concerns and recommendations for addressing these two factors when initiating a dating analysis are discussed.     

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.     

Paleontological evidence to date the tree of life

The role of fossils in dating the tree of life has been misunderstood. Fossils can provide good "minimum" age estimates for branches in the tree, but "maximum" constraints on those ages are poorer. Current debates about which are the "best" fossil dates for calibration move to consideration of the most appropriate constraints on the ages of tree nodes. Because fossil-based dates are constraints, and because molecular evolution is not perfectly clock-like, analysts should use more rather than fewer dates, but there has to be a balance between many genes and few dates versus many dates and few genes. We provide "hard" minimum and "soft" maximum age constraints for 30 divergences among key genome model organisms; these should contribute to better understanding of the dating of the animal tree of life.     

Molecular phylogenetics of the exoneurine allodapine bees reveal an ancient and puzzling dispersal from Africa to Australia

Previous phylogenetic studies of the bee tribe Allodapini suggested a puzzling biogeographic problem: one of the key basal divergences involved separation of the southern African and southern Australian clades at a very early stage in allodapine evolution, but no taxa occur in the Palaearctic or Asian regions that might suggest a Laurasian dispersal route. However, these studies lacked sufficient sequence data and appropriate maximum likelihood partition models to provide reliable phylogenetic estimates and enable alternative biogeographic hypotheses to be distinguished. Using Bayesian and penalized likelihood approaches and an expanded sequence and taxon set we examine phylogenetic relationships between the Australian, African, and Malagasy groups and estimate divergence times for key nodes. We show that divergence of the three basal Australian clades (known as the exoneurines) occurred at least 25 Mya following a single colonization event, and that this group diverged from the African + Madagascan clade at least 30 Mya, but actual divergence dates are likely to be much older than these very conservative limits. The bifurcation order of the exoneurine clades was not resolved and analyses could not rule out the existence of a hard polytomy, suggesting rapid radiation after colonization of Australia. Their divergence involved major transitions in life history traits and these placed constraints on the kinds of social organization that subsequently evolved in each lineage. Early divergence between the African, Malagasy, and Australian clades presents a major puzzle for historical biogeography: node ages are too recent for Gondwanan vicariance hypotheses, but too early for Laurasian dispersal scenarios. We suggest a scenario involving island hopping across the Indian Ocean via a series of now largely submerged elements of the Kergulen Plateau and Broken Ridge provinces, both of which are known to have had subaerial formations during the Cenozoic. [Bayesian; biogeography; dispersal; Gondwana; Kerguelen Plateau; penalized likelihood.].     

Dating primate divergences through an integrated analysis of palaeontological and molecular data

Estimation of divergence times is usually done using either the fossil record or sequence data from modern species. We provide an integrated analysis of palaeontological and molecular data to give estimates of primate divergence times that utilize both sources of information. The number of preserved primate species discovered in the fossil record, along with their geological age distribution, is combined with the number of extant primate species to provide initial estimates of the primate and anthropoid divergence times. This is done by using a stochastic forwards-modeling approach where speciation and fossil preservation and discovery are simulated forward in time. We use the posterior distribution from the fossil analysis as a prior distribution on node ages in a molecular analysis. Sequence data from two genomic regions (CFTR on human chromosome 7 and the CYP7A1 region on chromosome 8) from 15 primate species are used with the birth-death model implemented in mcmctree in PAML to infer the posterior distribution of the ages of 14 nodes in the primate tree. We find that these age estimates are older than previously reported dates for all but one of these nodes. To perform the inference, a new approximate Bayesian computation (ABC) algorithm is introduced, where the structure of the model can be exploited in an ABC-within-Gibbs algorithm to provide a more efficient analysis.     

Ancient dates or accelerated rates? Morphological clocks and the antiquity of placental mammals

Analyses of a comprehensive morphological character matrix of mammals using ‘relaxed’ clock models (which simultaneously estimate topology, divergence dates and evolutionary rates), either alone or in combination with an 8.5 kb nuclear sequence dataset, retrieve implausibly ancient, Late Jurassic–Early Cretaceous estimates for the initial diversification of Placentalia (crown-group Eutheria). These dates are much older than all recent molecular and palaeontological estimates. They are recovered using two very different clock models, and regardless of whether the tree topology is freely estimated or constrained using scaffolds to match the current consensus placental phylogeny. This raises the possibility that divergence dates have been overestimated in previous analyses that have applied such clock models to morphological and total evidence datasets. Enforcing additional age constraints on selected internal divergences results in only a slight reduction of the age of Placentalia. Constraining Placentalia to less than 93.8 Ma, congruent with recent molecular estimates, does not require major changes in morphological or molecular evolutionary rates. Even constraining Placentalia to less than 66 Ma to match the ‘explosive’ palaeontological model results in only a 10- to 20-fold increase in maximum evolutionary rate for morphology, and fivefold for molecules. The large discrepancies between clock- and fossil-based estimates for divergence dates might therefore be attributable to relatively small changes in evolutionary rates through time, although other explanations (such as overly simplistic models of morphological evolution) need to be investigated. Conversely, dates inferred using relaxed clock models (especially with discrete morphological data and MrBayes) should be treated cautiously, as relatively minor deviations in rate patterns can generate large effects on estimated divergence dates.     

Molecular evidence on plant divergence times

Estimation of divergence times from sequence data has become increasingly feasible in recent years. Conflicts between fossil evidence and molecular dates have sparked the development of new methods for inferring divergence times, further encouraging these efforts. In this paper, available methods for estimating divergence times are reviewed, especially those geared toward handling the widespread variation in rates of molecular evolution observed among lineages. The assumptions, strengths, and weaknesses of local clock, Bayesian, and rate smoothing methods are described. The rapidly growing literature applying these methods to key divergence times in plant evolutionary history is also reviewed. These include the crown group ages of green plants, land plants, seed plants, angiosperms, and major subclades of angiosperms. Finally, attempts to infer divergence times are described in the context of two very different temporal settings: recent adaptive radiations and much more ancient biogeographic patterns.     

Molecular distance and divergence time in carnivores and primates

Numerous studies have used indices of genetic distance between species to reconstruct evolutionary relationships and to estimate divergence time. However, the empirical relationship between molecular-based indices of genetic divergence and divergence time based on the fossil record is poorly known. To date, the results of empirical studies conflict and are difficult to compare because they differ widely in their choice of taxa, genetic techniques, or methods for calibrating rates of molecular evolution. We use a single methodology to analyze the relationship of molecular distance and divergence time in 86 taxa (72 carnivores and 14 primates). These taxa have divergence times of 0.01-55 Myr and provide a graded series of phylogenetic divergences such that the shape of the curve relating genetic distance and divergence time is often well defined. The techniques used to obtain genetic distance estimates include one- and two-dimensional protein electrophoresis, DNA hybridization, and microcomplement fixation. Our results suggest that estimates of molecular distance and divergence time are highly correlated. However, rates of molecular evolution are not constant; rather, in general they decline with increasing divergence time in a linear fashion. The rate of decline may differ according to technique and taxa. Moreover, in some cases the variability in evolutionary rates changes with increasing divergence time such that the accuracy of nodes in a phylogenetic tree varies predictably with time.     

Closing the gap between rocks and clocks using total-evidence dating

Total-evidence dating (TED) allows evolutionary biologists to incorporate a wide range of dating information into a unified statistical analysis. One might expect this to improve the agreement between rocks and clocks but this is not necessarily the case. We explore the reasons for such discordance using a mammalian dataset with rich molecular, morphological and fossil information. There is strong conflict in this dataset between morphology and molecules under standard stochastic models. This causes TED to push divergence events back in time when using inadequate models or vague priors, a phenomenon we term ‘deep root attraction’ (DRA). We identify several causes of DRA. Failure to account for diversified sampling results in dramatic DRA, but this can be addressed using existing techniques. Inadequate morphological models also appear to be a major contributor to DRA. The major reason seems to be that current models do not account for dependencies among morphological characters, causing distorted topology and branch length estimates. This is particularly problematic for huge morphological datasets, which may contain large numbers of correlated characters. Finally, diversification and fossil sampling priors that do not incorporate all the available background information can contribute to DRA, but these priors can also be used to compensate for DRA. Specifically, we show that DRA in the mammalian dataset can be addressed by introducing a modest extra penalty for ghost lineages that are unobserved in the fossil record, for instance by assuming rapid diversification, rare extinction or high fossil sampling rate; any of these assumptions produces highly congruent divergence time estimates with a minimal gap between rocks and clocks. Under these conditions, fossils have a stabilizing influence on divergence time estimates and significantly increase the precision of those estimates, which are generally close to the dates suggested by palaeontologists.This article is part of the themed issue ‘Dating species divergences using rocks and clocks’.     

Bees diversified in the age of eudicots

Reliable estimates on the ages of the major bee clades are needed to further understand the evolutionary history of bees and their close association with flowering plants. Divergence times have been estimated for a few groups of bees, but no study has yet provided estimates for all major bee lineages. To date the origin of bees and their major clades, we first perform a phylogenetic analysis of bees including representatives from every extant family, subfamily and almost all tribes, using sequence data from seven genes. We then use this phylogeny to place 14 time calibration points based on information from the fossil record for an uncorrelated relaxed clock divergence time analysis taking into account uncertainties in phylogenetic relationships and the fossil record. We explore the effect of placing a hard upper age bound near the root of the tree and the effect of different topologies on our divergence time estimates. We estimate that crown bees originated approximately 123 Ma (million years ago) (113–132 Ma), concurrently with the origin or diversification of the eudicots, a group comprising 75 per cent of angiosperm species. All of the major bee clades are estimated to have originated during the Middle to Late Cretaceous, which is when angiosperms became the dominant group of land plants.     

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.     

The age of the angiosperms: a molecular timescale without a clock

The age of the angiosperms has long been of interest to botanists and evolutionary biologists. Many early efforts to date the age of the angiosperms and evolutionary divergences within the angiosperm clade using a molecular clock have yielded age estimates that are grossly inconsistent with the fossil record. We investigated the age of angiosperms using Bayesian relaxed clock (BRC) and penalized likelihood (PL) approaches. Both of these methods allow the incorporation of multiple fossil constraints into the optimization procedure. The BRC method allows a range of values for among-lineage rate of substitution, from a nearly clocklike behavior to a condition in which each branch is allowed an optimal substitution rate, and also accounts for variation in molecular evolution across multiple genes. A topology derived from an analysis of genes from all three plant genomes for 71 taxa was used as a backbone. The effects on age estimates of different genes, single-gene versus concatenated datasets, and the inclusion and assumptions of fossils as age constraints were examined. In addition, the influence of prior distributions on estimates of divergence times was also explored. These results indicate that widely divergent age estimates can result from the different methods (198-139 million years ago), different sources of data (275-122 million years ago), and the inclusion of temporal constraints to topologies. Most dates, however, are between 180-140 million years ago, suggesting a Middle Jurassic-Early Cretaceous origin of flowering plants, predating the oldest unequivocal fossil angiosperms by about 45-5 million years. Nonetheless, these dates are consistent with other recent studies that have used methods that relax the assumption of a strict molecular clock and also agree with the hypothesis that the angiosperms may be somewhat older than the fossil record indicates.     

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.     

Major issues in the origins of ray‐finned fish (Actinopterygii) biodiversity

Ray‐finned fishes (Actinopterygii) dominate modern aquatic ecosystems and are represented by over 32000 extant species. The vast majority of living actinopterygians are teleosts; their success is often attributed to a genome duplication event or morphological novelties. The remainder are ‘living fossils’ belonging to a few depauperate lineages with long‐retained ecomorphologies: Polypteriformes (bichirs), Holostei (bowfin and gar) and Chondrostei (paddlefish and sturgeon). Despite over a century of systematic work, the circumstances surrounding the origins of these clades, as well as their basic interrelationships and diagnoses, have been largely mired in uncertainty. Here, I review the systematics and characteristics of these major ray‐finned fish clades, and the early fossil record of Actinopterygii, in order to gauge the sources of doubt. Recent relaxed molecular clock studies have pushed the origins of actinopterygian crown clades to the mid‐late Palaeozoic [Silurian–Carboniferous; 420 to 298 million years ago (Ma)], despite a diagnostic body fossil record extending only to the later Mesozoic (251 to 66 Ma). This disjunct, recently termed the ‘Teleost Gap’ (although it affects all crown lineages), is based partly on calibrations from potential Palaeozoic stem‐taxa and thus has been attributed to poor fossil sampling. Actinopterygian fossils of appropriate ages are usually abundant and well preserved, yet long‐term neglect of this record in both taxonomic and systematic studies has exacerbated the gaps and obscured potential synapomorphies. At the moment, it is possible that later Palaeozoic‐age teleost, holostean, chondrostean and/or polypteriform crown taxa sit unrecognized in museum drawers. However, it is equally likely that the ‘Teleost Gap’ is an artifact of incorrect attributions to extant lineages, overwriting both a post‐Palaeozoic crown actinopterygian radiation and the ecomorphological diversity of stem‐taxa.     

Molecular Clock Calibrations and Metazoan Divergence Dates

It has recently been argued that living metazoans diverged over 800 million years ago, based on evidence from 22 nuclear genes for such a deep divergence between vertebrates and arthropods (Gu 1998). Two ``internal' calibration points were used. However, only one fossil divergence date (the mammal–bird split) was directly used to calibrate the molecular clock. The second calibration point (the primate–rodent split) was based on molecular estimates that were ultimately also calibrated by the same mammal–bird split. However, the first tetrapods that can be assigned with confidence to either the mammal (synapsid) lineage or the bird (diapsid) lineage are approximately 288 million years old, while the first mammals that can be assigned with confidence to either the primate or the rodent lineages are 65 million years old, or 85 million years old if ferungulates are part of the primate lineage and zhelestids are accepted as ferungulate relatives. Recalibration of the protein data using these fossil dates indicates that metazoans diverged between 791 and 528 million years ago, a result broadly consistent with the palaeontological documentation of the ``Cambrian explosion.' The third, ``external' calibration point (the metazoan–fungal divergence) was similarly problematic, since it was based on a controversial molecular study (which in turn used fossil dates including the mammal–bird split); direct use of fossils for this calibration point gives the absurd dating of 455 million years for metazoan divergences. Similar calibration problems affect another recent study (Wang et al. 1999), which proposes divergences for metazoans of 1000 million years or more: recalibrations of their clock again yields much more recent dates, some consistent with a ``Cambrian explosion' scenario. Molecular clock studies have persuasively argued for the imperfection of the fossil record but have rarely acknowledged that their inferences are also directly based on this same record. Received: 26 January 1999 / Accepted: 14 April 1999