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.     

Establishing a time-scale for plant evolution

? Plants have utterly transformed the planet, but testing hypotheses of causality requires a reliable time-scale for plant evolution. While clock methods have been extensively developed, less attention has been paid to the correct interpretation and appropriate implementation of fossil data. ? We constructed 17 calibrations, consisting of minimum constraints and soft maximum constraints, for divergences between model representatives of the major land plant lineages. Using a data set of seven plastid genes, we performed a cross-validation analysis to determine the consistency of the calibrations. Six molecular clock analyses were then conducted, one with the original calibrations, and others exploring the impact on divergence estimates of changing maxima at basal nodes, and prior probability densities within calibrations. ? Cross-validation highlighted Tracheophyta and Euphyllophyta calibrations as inconsistent, either because their soft maxima were overly conservative or because of undetected rate variation. Molecular clock analyses yielded estimates ranging from 568-815 million yr before present (Ma) for crown embryophytes and from 175-240 Ma for crown angiosperms. ? We reject both a post-Jurassic origin of angiosperms and a post-Cambrian origin of land plants. Our analyses also suggest that the establishment of the major embryophyte lineages occurred at a much slower tempo than suggested in most previous studies. These conclusions are entirely compatible with current palaeobotanical data, although not necessarily with their interpretation by palaeobotanists.     

Major transitions in the evolution of early land plants: a bryological perspective

Background Molecular phylogeny has resolved the liverworts as the earliest-divergent clade of land plants and mosses as the sister group to hornworts plus tracheophytes, with alternative topologies resolving the hornworts as sister to mosses plus tracheophytes less well supported. The tracheophytes plus fossil plants putatively lacking lignified vascular tissue form the polysporangiophyte clade. Scope This paper reviews phylogenetic, developmental, anatomical, genetic and paleontological data with the aim of reconstructing the succession of events that shaped major land plant lineages. Conclusions Fundamental land plant characters primarily evolved in the bryophyte grade, and hence the key to a better understanding of the early evolution of land plants is in bryophytes. The last common ancestor of land plants was probably a leafless axial gametophyte bearing simple unisporangiate sporophytes. Water-conducting tissue, if present, was restricted to the gametophyte and presumably consisted of perforate cells similar to those in the early-divergent bryophytes Haplomitrium and Takakia. Stomata were a sporophyte innovation with the possible ancestral functions of producing a transpiration-driven flow of water and solutes from the parental gametophyte and facilitating spore separation before release. Stomata in mosses, hornworts and polysporangiophytes are viewed as homologous, and hence these three lineages are collectively referred to as the 'stomatophytes'. An indeterminate sporophyte body (the sporophyte shoot) developing from an apical meristem was the key innovation in polysporangiophytes. Poikilohydry is the ancestral condition in land plants; homoiohydry evolved in the sporophyte of polysporangiophytes. Fungal symbiotic associations ancestral to modern arbuscular mycorrhizas evolved in the gametophytic generation before the separation of major present-living lineages. Hydroids are imperforate water-conducting cells specific to advanced mosses. Xylem vascular cells in polysporangiophytes arose either from perforate cells or de novo. Food-conducting cells were a very early innovation in land plant evolution. The inferences presented here await testing by molecular genetics.     

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.     

Multigene phylogeny of land plants with special reference to bryophytes and the earliest land plants

A widely held view of land plant relationships places liverworts as the first branch of the land plant tree, whereas some molecular analyses and a cladistic study of morphological characters indicate that hornworts are the earliest land plants. To help resolve this conflict, we used parsimony and likelihood methods to analyze a 6, 095-character data set composed of four genes (chloroplast rbcL and small-subunit rDNA from all three plant genomes) from all major land plant lineages. In all analyses, significant support was obtained for the monophyly of vascular plants, lycophytes, ferns (including PSILOTUM: and EQUISETUM:), seed plants, and angiosperms. Relationships among the three bryophyte lineages were unresolved in parsimony analyses in which all positions were included and weighted equally. However, in parsimony and likelihood analyses in which rbcL third-codon-position transitions were either excluded or downweighted (due to apparent saturation), hornworts were placed as sister to all other land plants, with mosses and liverworts jointly forming the second deepest lineage. Decay analyses and Kishino-Hasegawa tests of the third-position-excluded data set showed significant support for the hornwort-basal topology over several alternative topologies, including the commonly cited liverwort-basal topology. Among the four genes used, mitochondrial small-subunit rDNA showed the lowest homoplasy and alone recovered essentially the same topology as the multigene tree. This molecular phylogeny presents new opportunities to assess paleontological evidence and morphological innovations that occurred during the early evolution of terrestrial plants.     

Carbon isotopes support the presence of extensive land floras pre-dating the origin of vascular plants

Multiple lines of evidence indicate that Earth's land masses became green some 2.7 Ga ago, about 1 billion years after the advent of life. About 2.2 billion years later, land plants abruptly appear in the fossil record and diversify marking the onset of ecologically complex terrestrial communities that persist to the present day. Given this long history of land colonization, surprisingly few studies report direct fossil evidence of emergent vegetation prior to the continuous record of life on land that starts in the mid-Silurian (ca. 420–425 Ma ago). Here we compare stable carbon isotope signatures of fossils from seven Ordovician–Silurian (450–420 Ma old) Appalachian biotas with signatures of coeval marine organic matter and with stable carbon isotope values predicted for Ordovician and Silurian liverworts (BRYOCARB model). The comparisons support a terrestrial origin for fossils in six of the biotas analyzed, and indicate that some of the fossils represent bryophyte-grade plants. Our results demonstrate that extensive land floras pre-dated the advent of vascular plants by at least 25 Ma. The Appalachian fossils represent the oldest direct evidence of widespread colonization of continents. These findings provide a new search image for macrofossil assemblages that contain the earliest stages of land plant evolution. We anticipate they will fuel renewed efforts to search for direct fossil evidence to track the origin of land plants and eukaryotic life on continents further back in geologic time.     

The evolution of chloroplast RNA editing

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.     

Assessing the quality of molecular divergence time estimates by fossil calibrations and fossil-based model selection

Estimates of species divergence times using DNA sequence data are playing an increasingly important role in studies of evolution, ecology and biogeography. Most work has centred on obtaining appropriate kinds of data and developing optimal estimation procedures, whereas somewhat less attention has focused on the calibration of divergences using fossils. Case studies with multiple fossil calibration points provide important opportunities to examine the divergence time estimation problem in new ways. We discuss two cross-validation procedures that address different aspects of inference in divergence time estimation. 'Fossil cross-validation' is a procedure used to identify the impact of different individual calibrations on overall estimation. This can identify fossils that have an exceptionally large error effect and may warrant further scrutiny. 'Fossil-based model cross-validation' is an entirely different procedure that uses fossils to identify the optimal model of molecular evolution in the context of rate smoothing or other inference methods. Both procedures were applied to two recent studies: an analysis of monocot angiosperms with eight fossil calibrations and an analysis of placental mammals with nine fossil calibrations. In each case, fossil calibrations could be ranked from most to least influential, and in one of the two studies, the fossils provided decisive evidence about the optimal molecular evolutionary model.     

Introduction and synthesis: Plant phylogeny and the origin of major biomes

Phylogenetic trees based upon DNA sequence data, when calibrated with a dimension of time, allow inference of: (i) the pattern of accumulation of lineages through time; (ii) the time of origin of monophyletic groups; (iii) when lineages arrived in different geographical areas; (iv) the time of origin of biome-specific morphologies. This gives a powerful new view of the history of biomes that in many cases is not provided by the incomplete plant fossil record. Dated plant phylogenies for angiosperm families such as Leguminoaceae (Fabaceae), Melastomataceae sensu stricto, Annonaceae and Rhamnaceae indicate that long-distance, transoceanic dispersal has played an important role in shaping their distributions, and that this can obscure any effect of tectonic history, previously assumed to have been the major cause of their biogeographic patterns. Dispersal from other continents has also been important in the assembly of the Amazonian rainforest flora and the Australian flora. Comparison of dated biogeographic patterns of plants and animals suggests that recent long-distance dispersal might be more prevalent in plants, which has major implications for community assembly and coevolution. Dated plant phylogenies also reveal the role of past environmental changes on the evolution of lineages in species-rich biomes, and show that recent Plio-Pleistocene diversification has contributed substantially to their current species richness. Because of the critical role of fossils and morphological characters in assigning ages to nodes in phylogenetic trees, future studies must include careful morphological consideration of fossils and their extant relatives in a phylogenetic context. Ideal study systems will be based upon DNA sequence data from multiple loci and multiple fossil calibrations. This allows cross-validation both of age estimates from different loci, and from different fossil calibrations. For a more complete view of biome history, future studies should emphasize full taxon sampling in ecologically important groups, and should focus on geographical areas for which few species-level phylogenies are available, such as tropical Africa and Asia. These studies are urgent because understanding the history of biomes can both inform conservation decisions, and help predict the effects of future environmental changes at a time when biodiversity is being impacted on an unprecedented scale.     

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

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

Integrating fossil preservation biases in the selection of calibrations for molecular divergence time estimation

The selection of fossil data to use as calibration age priors in molecular divergence time estimates inherently links neontological methods with paleontological theory. However, few neontological studies have taken into account the possibility of a taphonomic bias in the fossil record when developing approaches to fossil calibration selection. The Sppil-Rongis effect may bias the first appearance of a lineage toward the recent causing most objective calibration selection approaches to erroneously exclude appropriate calibrations or to incorporate multiple calibrations that are too young to accurately represent the divergence times of target lineages. Using turtles as a case study, we develop a Bayesian extension to the fossil selection approach developed by Marshall (2008. A simple method for bracketing absolute divergence times on molecular phylogenies using multiple fossil calibrations points. Am. Nat. 171:726-742) that takes into account this taphonomic bias. Our method has the advantage of identifying calibrations that may bias age estimates to be too recent while incorporating uncertainty in phylogenetic parameter estimates such as tree topology and branch lengths. Additionally, this method is easily adapted to assess the consistency of potential calibrations to any one calibration in the candidate pool.     

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.     

Deep origin and gradual evolution of transporting tissues: Perspectives from across the land plants

The evolution of transporting tissues was an important innovation in terrestrial plants that allowed them to adapt to almost all nonaquatic environments. These tissues consist of water-conducting cells and food-conducting cells and bridge plant–soil and plant–air interfaces over long distances. The largest group of land plants, representing about 95% of all known plant species, is associated with morphologically complex transporting tissue in plants with a range of additional traits. Therefore, this entire clade was named tracheophytes, or vascular plants. However, some nonvascular plants possess conductive tissues that closely resemble vascular tissue in their organization, structure, and function. Recent molecular studies also point to a highly conserved toolbox of molecular regulators for transporting tissues. Here, we reflect on the distinguishing features of conductive and vascular tissues and their evolutionary history. Rather than sudden emergence of complex, vascular tissues, plant transporting tissues likely evolved gradually, building on pre-existing developmental mechanisms and genetic components. Improved knowledge of the intimate structure and developmental regulation of transporting tissues across the entire taxonomic breadth of extant plant lineages, combined with more comprehensive documentation of the fossil record of transporting tissues, is required for a full understanding of the evolutionary trajectory of transporting tissues.

Combining fossil records and recent molecular research provides insights into the origin and evolutionary progress of transporting tissue development in land plants.     

Moss and liverwort xyloglucans contain galacturonic acid and are structurally distinct from the xyloglucans synthesized by hornworts and vascular plants

Xyloglucan is a well-characterized hemicellulosic polysaccharide that is present in the cell walls of all seed-bearing plants. The cell walls of avascular and seedless vascular plants are also believed to contain xyloglucan. However, these xyloglucans have not been structurally characterized. This lack of information is an impediment to understanding changes in xyloglucan structure that occurred during land plant evolution. In this study, xyloglucans were isolated from the walls of avascular (liverworts, mosses, and hornworts) and seedless vascular plants (club and spike mosses and ferns and fern allies). Each xyloglucan was fragmented with a xyloglucan-specific endo-glucanase and the resulting oligosaccharides then structurally characterized using NMR spectroscopy, MALDI-TOF and electrospray mass spectrometry, and glycosyl-linkage and glycosyl residue composition analyses. Our data show that xyloglucan is present in the cell walls of all major divisions of land plants and that these xyloglucans have several common structural motifs. However, these polysaccharides are not identical because specific plant groups synthesize xyloglucans with unique structural motifs. For example, the moss Physcomitrella patens and the liverwort Marchantia polymorpha synthesize XXGGG- and XXGG-type xyloglucans, respectively, with sidechains that contain a beta-D-galactosyluronic acid and a branched xylosyl residue. By contrast, hornworts synthesize XXXG-type xyloglucans that are structurally homologous to the xyloglucans synthesized by many seed-bearing and seedless vascular plants. Our results increase our understanding of the evolution, diversity, and function of structural motifs in land-plant xyloglucans and provide support to the proposal that hornworts are sisters to the vascular plants.     

Dating the Monocot–Dicot Divergence and the Origin of Core Eudicots Using Whole Chloroplast Genomes

We estimated the dates of the monocot–dicot split and the origin of core eudicots using a large chloroplast (cp) genomic dataset. Sixty-one protein-coding genes common to the 12 completely sequenced cp genomes of land plants were concatenated and analyzed. Three reliable split events were used as calibration points and for cross references. Both the method based on the assumption of a constant rate and the Li–Tanimura unequal-rate method were used to estimate divergence times. The phylogenetic analyses indicated that nonsynonymous substitution rates of cp genomes are unequal among tracheophyte lineages. For this reason, the constant-rate method gave overestimates of the monocot–dicot divergence and the age of core eudicots, especially when fast-evolving monocots were included in the analysis. In contrast, the Li–Tanimura method gave estimates consistent with the known evolutionary sequence of seed plant lineages and with known fossil records. Combining estimates calibrated by two known fossil nodes and the Li–Tanimura method, we propose that monocots branched off from dicots 140–150 Myr ago (late Jurassic–early Cretaceous), at least 50 Myr younger than previous estimates based on the molecular clock hypothesis, and that the core eudicots diverged 100–115 Myr ago (Albian–Aptian of the Cretaceous). These estimates indicate that both the monocot–dicot divergence and the core eudicots age are older than their respective fossil records.     

A timeline for terrestrialization: consequences for the carbon cycle in the Palaeozoic

The geochemical carbon cycle is strongly influenced by life on land, principally through the effects of carbon sequestration and the weathering of calcium and magnesium silicates in surface rocks and soils. Knowing the time of origin of land plants and animals and also of key organ systems (e.g. plant vasculature, roots, wood) is crucial to understand the development of the carbon cycle and its effects on other Earth systems. Here, we compare evidence from fossils with calibrated molecular phylogenetic trees (timetrees) of living plants and arthropods. We show that different perspectives conflict in terms of the relative timing of events, the organisms involved and the pattern of diversification of various groups. Focusing on the fossil record, we highlight a number of key biases that underpin some of these conflicts, the most pervasive and far-reaching being the extent and nature of major facies changes in the rock record. These effects probably mask an earlier origin of life on land than is evident from certain classes of fossil data. If correct, this would have major implications in understanding the carbon cycle during the Early Palaeozoic.     

Caveats on the use of fossil calibrations for molecular dating: a comment on Near et al

We reassess a study on a fossil-calibrated molecular clock that provides a new method for evaluating the accuracy of calibration points. We address several pitfalls that molecular systematists should be aware of when calculating rates of molecular evolution based on fossil calibrations. These caveats involve the substantiation and accurate use of geologic dates, the inappropriate use of fixed calibration points, and the explicit and objective phylogenetic placement of fossil taxa. Paleontological data, like molecular data, should be treated with the utmost rigor.