Time scale of eutherian evolution estimated without assuming a constant rate of molecular evolution

Controversies over the molecular clock hypothesis were reviewed. Since it is evident that the molecular clock does not hold in an exact sense, accounting for evolution of the rate of molecular evolution is a prerequisite when estimating divergence times with molecular sequences. Recently proposed statistical methods that account for this rate variation are overviewed and one of these procedures is applied to the mitochondrial protein sequences and to the nuclear gene sequences from many mammalian species in order to estimate the time scale of eutherian evolution. This Bayesian method not only takes account of the variation of molecular evolutionary rate among lineages and among genes, but it also incorporates fossil evidence via constraints on node times. With denser taxonomic sampling and a more realistic model of molecular evolution, this Bayesian approach is expected to increase the accuracy of divergence time estimates.     

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

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

Timing primate evolution: Lessons from the discordance between molecular and paleontological estimates

The molecular clock has become an increasingly important tool in evolutionary biology and biological anthropology. Nevertheless, a source of contention with respect to this method is the frequent discordance with fossil‐based estimates of divergence times. The primate radiation is a case in point: Numerous studies have dated the major primate nodes (reviewed in Steiper and Young, 1 , 2 ) and there are many instances where molecular and fossil‐based estimates of divergence times differ (Fig. 1). Some investigators have recently focused on phenomena such as stratigraphic dating, the stochastic nature of molecular time estimates, and other sources as potential biases in molecular clock estimates. 3 , 4 In this paper we do not focus on accuracy or statistical error; rather, we argue that discordance is a predictable phenomenon that provides valuable information about the tempo and mode of primate molecular and morphological evolution. Using this perspective, we reexamine the principal theoretical and methodological factors that lead to discordance between molecular and fossil estimates of the origins of taxa and discuss how a better understanding of these factors can help to improve our understanding of primate evolution.     

Bayesian relaxed clock estimation of divergence times in foraminifera

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

A mitogenomic timescale for birds detects variable phylogenetic rates of molecular evolution and refutes the standard molecular clock

Current understanding of the diversification of birds is hindered by their incomplete fossil record and uncertainty in phylogenetic relationships and phylogenetic rates of molecular evolution. Here we performed the first comprehensive analysis of mitogenomic data of 48 vertebrates, including 35 birds, to derive a Bayesian timescale for avian evolution and to estimate rates of DNA evolution. Our approach used multiple fossil time constraints scattered throughout the phylogenetic tree and accounts for uncertainties in time constraints, branch lengths, and heterogeneity of rates of DNA evolution. We estimated that the major vertebrate lineages originated in the Permian; the 95% credible intervals of our estimated ages of the origin of archosaurs (258 MYA), the amniote-amphibian split (356 MYA), and the archosaur-lizard divergence (278 MYA) bracket estimates from the fossil record. The origin of modern orders of birds was estimated to have occurred throughout the Cretaceous beginning about 139 MYA, arguing against a cataclysmic extinction of lineages at the Cretaceous/Tertiary boundary. We identified fossils that are useful as time constraints within vertebrates. Our timescale reveals that rates of molecular evolution vary across genes and among taxa through time, thereby refuting the widely used mitogenomic or cytochrome b molecular clock in birds. Moreover, the 5-Myr divergence time assumed between 2 genera of geese (Branta and Anser) to originally calibrate the standard mitochondrial clock rate of 0.01 substitutions per site per lineage per Myr (s/s/l/Myr) in birds was shown to be underestimated by about 9.5 Myr. Phylogenetic rates in birds vary between 0.0009 and 0.012 s/s/l/Myr, indicating that many phylogenetic splits among avian taxa also have been underestimated and need to be revised. We found no support for the hypothesis that the molecular clock in birds "ticks" according to a constant rate of substitution per unit of mass-specific metabolic energy rather than per unit of time, as recently suggested. Our analysis advances knowledge of rates of DNA evolution across birds and other vertebrates and will, therefore, aid comparative biology studies that seek to infer the origin and timing of major adaptive shifts in vertebrates.     

Testing the molecular clock: molecular and paleontological estimates of divergence times in the Echinoidea (Echinodermata)

The phylogenetic relationships of 46 echinoids, with representatives from 13 of the 14 ordinal-level clades and about 70% of extant families commonly recognized, have been established from 3 genes (3,226 alignable bases) and 119 morphological characters. Morphological and molecular estimates are similar enough to be considered suboptimal estimates of one another, and the combined data provide a tree that, when calibrated against the fossil record, provides paleontological estimates of divergence times and completeness of their fossil record. The order of branching on the cladogram largely agrees with the stratigraphic order of first occurrences and implies that their fossil record is more than 85% complete at family level and at a resolution of 5-Myr time intervals. Molecular estimates of divergence times derived from applying both molecular clock and relaxed molecular clock models are concordant with estimates based on the fossil record in up to 70% of cases, with most concordant results obtained using Sanderson's semiparametric penalized likelihood method and a logarithmic-penalty function. There are 3 regions of the tree where molecular and fossil estimates of divergence time consistently disagree. Comparison with results obtained when molecular divergence dates are estimated from the combined (morphology + gene) tree suggests that errors in phylogenetic reconstruction explain only one of these. In another region the error most likely lies with the paleontological estimates because taxa in this region are demonstrated to have a very poor fossil record. In the third case, morphological and paleontological evidence is much stronger, and the topology for this part of the molecular tree differs from that derived from the combined data. Here the cause of the mismatch is unclear but could be methodological, arising from marked inequality of molecular rates. Overall, the level of agreement reached between these different data and methodological approaches leads us to believe that careful application of likelihood and Bayesian methods to molecular data provides realistic divergence time estimates in the majority of cases (almost 80% in this specific example), thus providing a remarkably well-calibrated phylogeny of a character-rich clade of ubiquitous marine benthic invertebrates.     

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.     

Nuclear rDNA-based molecular clock of the evolution of triatominae (Hemiptera: reduviidae), vectors of Chagas disease

The evolutionary history and times of divergence of triatomine bug lineages are estimated from molecular clocks inferred from nucleotide sequences of the small subunit SSU (18S) and the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA of these reduviids. The 18S rDNA molecular clock rate in Triatominae, and Prosorrhynchan Hemiptera in general, appears to be of 1.8% per 100 million years (my). The ITS-2 molecular clock rate in Triatominae is estimated to be around 0.4-1% per 1 my, indicating that ITS-2 evolves 23-55 times faster than 18S rDNA. Inferred chronological data about the evolution of Triatominae fit well with current hypotheses on their evolutionary histories, but suggest reconsideration of the current taxonomy of North American species complexes.     

Bayesian models of episodic evolution support a late precambrian explosive diversification of the Metazoa

Multicellular animals, or Metazoa, appear in the fossil records between 575 and 509 million years ago (MYA). At odds with paleontological evidence, molecular estimates of basal metazoan divergences have been consistently older than 700 MYA. However, those date estimates were based on the molecular clock hypothesis, which is almost always violated. To relax this hypothesis, we have implemented a Bayesian approach to describe the change of evolutionary rate over time. Analysis of 22 genes from the nuclear and the mitochondrial genomes under the molecular clock assumption produced old date estimates, similar to those from previous studies. However, by allowing rates to vary in time and by taking small species-sampling fractions into account, we obtained much younger estimates, broadly consistent with the fossil records. In particular, the date of protostome-deuterostome divergence was on average 582 +/- 112 MYA. These results were found to be robust to specification of the model of rate change. The clock assumption thus had a dramatic effect on date estimation. However, our results appeared sensitive to the prior model of cladogenesis, although the oldest estimates (791 +/- 246 MYA) were obtained under a suboptimal model. Bayes posterior estimates of evolutionary rates indicated at least one major burst of molecular evolution at the end of the Precambrian when protostomes and deuterostomes diverged. We stress the importance of assumptions about rates on date estimation and suggest that the large discrepancies between the molecular and fossil dates of metazoan divergences might partly be due to biases in molecular date estimation.     

Calibrating molecular estimates of substitution rates and divergence times in birds

Many evolutionary studies of birds rely on the estimation of molecular divergence times and substitution rates. In order to perform such analyses, it is necessary to incorporate some form of calibration information: a known substitution rate, radiometric ages of heterochronous sequences, or inferred ages of lineage splitting events. All three of these techniques have been employed in avian molecular studies, but their usage has not been entirely satisfactory. For example, the 'traditional' avian mitochondrial substitution rate of 2% per million years is frequently adopted without acknowledgement of the associated uncertainty. Similarly, fossil and biogeographic information is almost always converted into an errorless calibration point. In both cases, the resulting estimates of divergence times and substitution rates will be artificially precise, which has a considerable impact on hypothesis testing. In addition, using such a simplistic approach to calibration discards much of the information offered by the fossil record. A number of more sophisticated calibration methods have recently been introduced, culminating in the development of probability distribution-based calibrations. In this article, I discuss the use of this new class of methods and offer guidelines for choosing a calibration technique.     

The origin and diversification of eukaryotes: problems with molecular phylogenetics and molecular clock estimation

Determining the relationships among and divergence times for the major eukaryotic lineages remains one of the most important and controversial outstanding problems in evolutionary biology. The sequencing and phylogenetic analyses of ribosomal RNA (rRNA) genes led to the first nearly comprehensive phylogenies of eukaryotes in the late 1980s, and supported a view where cellular complexity was acquired during the divergence of extant unicellular eukaryote lineages. More recently, however, refinements in analytical methods coupled with the availability of many additional genes for phylogenetic analysis showed that much of the deep structure of early rRNA trees was artefactual. Recent phylogenetic analyses of a multiple genes and the discovery of important molecular and ultrastructural phylogenetic characters have resolved eukaryotic diversity into six major hypothetical groups. Yet relationships among these groups remain poorly understood because of saturation of sequence changes on the billion-year time-scale, possible rapid radiations of major lineages, phylogenetic artefacts and endosymbiotic or lateral gene transfer among eukaryotes. Estimating the divergence dates between the major eukaryote lineages using molecular analyses is even more difficult than phylogenetic estimation. Error in such analyses comes from a myriad of sources including: (i) calibration fossil dates, (ii) the assumed phylogenetic tree, (iii) the nucleotide or amino acid substitution model, (iv) substitution number (branch length) estimates, (v) the model of how rates of evolution change over the tree, (vi) error inherent in the time estimates for a given model and (vii) how multiple gene data are treated. By reanalysing datasets from recently published molecular clock studies, we show that when errors from these various sources are properly accounted for, the confidence intervals on inferred dates can be very large. Furthermore, estimated dates of divergence vary hugely depending on the methods used and their assumptions. Accurate dating of divergence times among the major eukaryote lineages will require a robust tree of eukaryotes, a much richer Proterozoic fossil record of microbial eukaryotes assignable to extant groups for calibration, more sophisticated relaxed molecular clock methods and many more genes sampled from the full diversity of microbial eukaryotes.     

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

Background  

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

Intercontinental and intracontinental biogeography patterns and methods

The study of biogeography has benefited from the exponential increase of DNA sequence data from recent molecular systematic studies, the development of analytical methods in the last decade concerning divergence time estimation and geographic area analyses, and the availability of large-scale distributiofi data of species in many groups of organisms. The underlying principle of divergence time estimation from DNA and protein data is that sequence divergence depends on the product of evolutionary rate and time. With their molecular clock hypothesis, Zuckerkandl and Pauling （1965） separated rates of molecular evolution from time by incorporating fossil evidence. Originally,     

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.     

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.     

Evolutionary diversification of reef corals: a comparison of the molecular and fossil records

Understanding historical patterns of diversity dynamics is of paramount importance for many evolutionary questions. The fossil record has long been the only source of information on patterns of diversification, but the molecular record, derived from time-calibrated phylogenies, is becoming an important additional resource. Both fossil and molecular approaches have shortcomings and biases. These have been well studied for fossil data but much less so for molecular data and empirical comparisons between approaches are lacking. Here, we compare the patterns of diversification derived from fossil and molecular data in scleractinian reef coral species. We also assess the robustness of molecular diversification rates to poor taxon sampling. We find that the temporal pattern of molecular diversification rates is robust to incomplete sampling when rates are calculated per interval. The major obstacle of molecular methods is that rate estimates are distorted because diversification rates can never be negative, whereas the fossil record suffers from incomplete preservation and inconsistent taxonomy. Nevertheless, the molecular pattern of diversification is comparable to the pattern we observe in the fossil record, with the timing of major diversification pulses coinciding in each dataset. For example, both agree that the end-Triassic coral extinction was a catastrophic bottleneck in scleractinian evolution.     

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.     

Exploring the correlations between sequence evolution rate and phenotypic divergence across the Mammalian tree provides insights into adaptive evolution

Sequence evolution behaves in a relatively consistent manner, leading to one of the fundamental paradigms in biology, the existence of a ??molecular clock??. The molecular clock can be distilled to the concept of accumulation of substitutions, through time yielding a stable rate from which we can estimate lineage divergence. Over the last 50?years, evolutionary biologists have obtained an in-depth understanding of this clock??s nuances. It has been fine-tuned by taking into account the vast heterogeneity in rates across lineages and genes, leading to ??relaxed?? molecular clock methods for timetree reconstruction. Sequence rate varies with life history traits including body size, generation time and metabolic rate, and we review recent studies on this topic. However, few studies have explicitly examined correlates between molecular evolution and morphological evolution. The patterns observed across diverse lineages suggest that rates of molecular and morphological evolution are largely decoupled. We discuss how identifying the molecular mechanisms behind rapid functional radiations are central to understanding evolution. The vast functional divergence within mammalian lineages that have relatively ??slow?? sequence evolution refutes the hypotheses that pulses in diversification yielding major phenotypic change are the result of steady accumulation of substitutions. Patterns rather suggest phenotypic divergence is likely caused by regulatory alterations mediated through mechanisms such as insertions/deletions in functional regions. These can rapidly arise and sweep to fixation faster than predicted from a lineage??s sequence neutral substitution rate, enabling species to leapfrog between phenotypic ??islands??. We suggest research directions that could illuminate mechanisms behind the functional diversity we see today.     

Combining fossil and molecular data to date the diversification of New World Primates

Recent methodological advances in molecular dating associated with the growing availability of sequence data have prompted the study of the evolution of New World Anthropoidea in recent years. Motivated by questions regarding historical biogeography or the mode of evolution, these works aimed to obtain a clearer scenario of Platyrrhini origins and diversification. Although some consensus was found, disputed issues, especially those relating to the evolutionary affinities of fossil taxa, remain. The use of fossil taxa for divergence time analysis is traditionally restricted to the provision of calibration priors. However, new analytical approaches have been developed that incorporate fossils as terminals and, thus, directly assign ages to the fossil tips. In this study, we conducted a combined analysis of molecular and morphological data, including fossils, to derive the timescale of New World anthropoids. Differently from previous studies that conducted total‐evidence analysis of molecules and morphology, our approach investigated the morphological clock alone. Our results corroborate the hypothesis that living platyrrhines diversified in the last 20 Ma and that Miocene Patagonian fossils compose an independent evolutionary radiation that diversified in the late Oligocene. When compared to the node ages inferred from the molecular timescale, the inclusion of fossils augmented the precision of the estimates for nodes constrained by the fossil tips. We show that morphological data can be analysed using the same methodological framework applied in relaxed molecular clock studies.