Philip C. J. Donoghue

Best Practices for Justifying Fossil Calibrations

Our ability to correlate biological evolution with climate change, geological evolution, and other historical patterns is essential to understanding the processes that shape biodiversity. Combining data from the fossil record with molecular phylogenetics represents an exciting synthetic approach to this challenge. The first molecular divergence dating analysis (Zuckerkandl and Pauling 1962) was based on a measure of the amino acid differences in the hemoglobin molecule, with replacement rates established (calibrated) using paleontological age estimates from textbooks (e.g., Dodson 1960). Since that time, the amount of molecular sequence data has increased dramatically, affording ever-greater opportunities to apply molecular divergence approaches to fundamental problems in evolutionary biology. To capitalize on these opportunities, increasingly sophisticated divergence dating methods have been, and continue to be, developed. In contrast, comparatively, little attention has been devoted to critically assessing the paleontological and associated geological data used in divergence dating analyses. The lack of rigorous protocols for assigning calibrations based on fossils raises serious questions about the credibility of divergence dating results (e.g., Shaul and Graur 2002; Brochu et al. 2004; Graur and Martin 2004; Hedges and Kumar 2004; Reisz and Muller 2004a, 2004b; Theodor 2004; van Tuinen and Hadly 2004a, 2004b; van Tuinen et al. 2004; Benton and Donoghue 2007; Donoghue and Benton 2007; Parham and Irmis 2008; Ksepka 2009; Benton et al. 2009; Heads 2011). The assertion that incorrect calibrations will negatively influence divergence dating studies is not controversial. Attempts to identify incorrect calibrations through the use of a posteriori methods are available (e.g., Near and Sanderson 2004; Near et al. 2005; Rutschmann et al. 2007; Marshall 2008; Pyron 2010; Dornburg et al. 2011). We do not deny that a posteriori methods are a useful means of evaluating calibrations, but there can be no substitute for a priori assessment of the veracity of paleontological data. Incorrect calibrations, those based upon fossils that are phylogenetically misplaced or assigned incorrect ages, clearly introduce error into an analysis. Consequently, thorough and explicit justification of both phylogenetic and chronologic age assessments is necessary for all fossils used for calibration. Such explicit justifications will help to ensure that divergence dating studies are based on the best available data. Unfortunately, the majority of previously published calibrations lack explicit explanations and justifications of the age and phylogenetic position of the key fossils. In the absence of explicit justifications, it is difficult to distinguish between correct and incorrect calibrations, and it becomes difficult to reevaluate previous claims in light of new data. Paleontology is a dynamic science, with new data and perspectives constantly emerging as a result of new discoveries (see Kimura 2010 for a recent case where the age of the earliest known record of a clade was more than doubled). Calibrations based upon the best available evidence at a given time can become inappropriate as the discovery of new specimens, new phylogenetic analyses, and ongoing stratigraphic and geochronologic revisions refine our understanding of the fossil record. Our primary goals in this paper are to establish the best practices for justifying fossils used for the temporal calibration of molecular phylogenies. Our examples derive mainly, but not exclusively, from the vertebrate fossil record. We hope that our recommendations will lead to more credible calibrations and, as a result, more reliable divergence dates throughout the tree of life. A secondary goal is to help the community (researchers, editors, and reviewers) who might be unfamiliar with fossils to understand and overcome the challenges associated with using paleontological data. In order to accomplish these goals, we present a specimen-based protocol for selecting and documenting relevant fossils and discuss future directions for evaluating and utilizing phylogenetic and temporal data from the fossil record. We likewise encourage biologists relying on nonfossil calibrations for molecular divergence estimates (e.g., ages of island or mountain range formations, continental drift, and biomarkers) to develop their own set of rigorous guidelines so that their calibrations may also be evaluated in a systematic way.

MicroRNAs and the advent of vertebrate morphological complexity

The causal basis of vertebrate complexity has been sought in genome duplication events (GDEs) that occurred during the emergence of vertebrates, but evidence beyond coincidence is wanting. MicroRNAs (miRNAs) have recently been identified as a viable causal factor in increasing organismal complexity through the action of these ≈22-nt noncoding RNAs in regulating gene expression. Because miRNAs are continuously being added to animalian genomes, and, once integrated into a gene regulatory network, are strongly conserved in primary sequence and rarely secondarily lost, their evolutionary history can be accurately reconstructed. Here, using a combination of Northern analyses and genomic searches, we show that 41 miRNA families evolved at the base of Vertebrata, as they are found and/or detected in lamprey, but not in either ascidians or amphioxus (or any other nonchordate taxon). When placed into temporal context, the rate of miRNA acquisition and the extent of phenotypic evolution are anomalously high early in vertebrate history, far outstripping any other episode in chordate evolution. The genomic position of miRNA paralogues in humans, together with gene trees incorporating lamprey orthologues, indicates that although GDEs can account for an increase in the diversity of miRNA family members, which occurred before the last common ancestor of all living vertebrates, GDEs cannot account for the origin of these novel families themselves. We hypothesize that lying behind the origin of vertebrate complexity is the dramatic expansion of the noncoding RNA inventory including miRNAs, rather than an increase in the protein-encoding inventory caused by GDEs.

Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny

The fossil record suggests a rapid radiation of placental mammals following the Cretaceous–Paleogene (K–Pg) mass extinction 65 million years ago (Ma); nevertheless, molecular time estimates, while highly variable, are generally much older. Early molecular studies suffer from inadequate dating methods, reliance on the molecular clock, and simplistic and over-confident interpretations of the fossil record. More recent studies have used Bayesian dating methods that circumvent those issues, but the use of limited data has led to large estimation uncertainties, precluding a decisive conclusion on the timing of mammalian diversifications. Here we use a powerful Bayesian method to analyse 36 nuclear genomes and 274 mitochondrial genomes (20.6 million base pairs), combined with robust but flexible fossil calibrations. Our posterior time estimates suggest that marsupials diverged from eutherians 168–178 Ma, and crown Marsupialia diverged 64–84 Ma. Placentalia diverged 88–90 Ma, and present-day placental orders (except Primates and Xenarthra) originated in a ∼20 Myr window (45–65 Ma) after the K–Pg extinction. Therefore we reject a pre K–Pg model of placental ordinal diversification. We suggest other infamous instances of mismatch between molecular and palaeontological divergence time estimates will be resolved with this same approach.

Conodont affinity and chordate phylogeny.

Current information on the conodonts Clydagnathus windsorensis (Globensky) and Promissum pulchrum Kovács‐ Endrödy, together with the latest interpretations of conodont hard tissues, are reviewed and it is concluded that sufficient evidence exists to justify interpretation of the conodonts on a chordate model. A new phylogenetic analysis is undertaken, consisting of 17 chordate taxa and 103 morphological, physiological and biochemical characters; conodonts are included as a primary taxon. Various experiments with character coding, taxon deletion and the use of constraint trees are carried out. We conclude that conodonts are cladistically more derived than either hagfishes or lampreys because they possess a mineralised dermal skeleton and that they are the most plesiomorphic member of the total group Gnathostomata. We discuss the evolution of the nervous and sensory systems and the skeleton in the context of our optimal phylogenetic tree. There appears to be no simple evolution of free to canal‐enclosed neuromasts; organised neuromasts within canals appear to have arisen at least three times from free neuromasts or neuromasts arranged within grooves. The mineralised vertebrate skeleton first appeared as odontodes of dentine or dentine plus enamel in the paraconodont/euconodont feeding apparatus. Bone appeared later, co‐ordinate with the development of a dermal skeleton, and it appears to have been primitively acellular. Atubular dentine is more primitive than tubular dentine. However, the subsequent distribution of the different types of dentine (e.g. mesodentine, orthodentine), suggests that these tissue types are homoplastic. The topology of relationships and known stratigraphic ranges of taxa in our phylogeny predict the existence of myxinoids and petromyzontids in the Cambrian.

Synchrotron X-ray tomographic microscopy of fossil embryos

Fossilized embryos from the late Neoproterozoic and earliest Phanerozoic have caused much excitement because they preserve the earliest stages of embryology of animals that represent the initial diversification of metazoans. However, the potential of this material has not been fully realized because of reliance on traditional, non-destructive methods that allow analysis of exposed surfaces only, and destructive methods that preserve only a single two-dimensional view of the interior of the specimen. Here, we have applied synchrotron-radiation X-ray tomographic microscopy (SRXTM), obtaining complete three-dimensional recordings at submicrometre resolution. The embryos are preserved by early diagenetic impregnation and encrustation with calcium phosphate, and differences in X-ray attenuation provide information about the distribution of these two diagenetic phases. Three-dimensional visualization of blastomere arrangement and diagenetic cement in cleavage embryos resolves outstanding questions about their nature, including the identity of the columnar blastomeres. The anterior and posterior anatomy of embryos of the bilaterian worm-like Markuelia confirms its position as a scalidophoran, providing new insights into body-plan assembly among constituent phyla. The structure of the developing germ band in another bilaterian, Pseudooides, indicates a unique mode of germ-band development. SRXTM provides a method of non-invasive analysis that rivals the resolution achieved even by destructive methods, probing the very limits of fossilization and providing insight into embryology during the emergence of metazoan phyla.

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.

microRNAs reveal the interrelationships of hagfish, lampreys, and gnathostomes and the nature of the ancestral vertebrate

Hagfish and lampreys are the only living representatives of the jawless vertebrates (agnathans), and compared with jawed vertebrates (gnathostomes), they provide insight into the embryology, genomics, and body plan of the ancestral vertebrate. However, this insight has been obscured by controversy over their interrelationships. Morphological cladistic analyses have identified lampreys and gnathostomes as closest relatives, whereas molecular phylogenetic studies recover a monophyletic Cyclostomata (hagfish and lampreys as closest relatives). Here, we show through deep sequencing of small RNA libraries, coupled with genomic surveys, that Cyclostomata is monophyletic: hagfish and lampreys share 4 unique microRNA families, 15 unique paralogues of more primitive microRNA families, and 22 unique substitutions to the mature gene products. Reanalysis of morphological data reveals that support for cyclostome paraphyly was based largely on incorrect character coding, and a revised dataset is not decisive on the mono- vs. paraphyly of cyclostomes. Furthermore, we show fundamental conservation of microRNA expression patterns among lamprey, hagfish, and gnathostome organs, implying that the role of microRNAs within specific organs is coincident with their appearance within the genome and is conserved through time. Together, these data support the monophyly of cyclostomes and suggest that the last common ancestor of all living vertebrates was a more complex organism than conventionally accepted by comparative morphologists and developmental biologists.

The impact of the representation of fossil calibrations on Bayesian estimation of species divergence times.

Bayesian inference provides a powerful framework for integrating different sources of information (in particular, molecules and fossils) to derive estimates of species divergence times. Indeed, it is currently the only framework that can adequately account for uncertainties in fossil calibrations. We use 2 Bayesian Markov chain Monte Carlo programs, MULTIDIVTIME and MCMCTREE, to analyze 3 empirical datasets to estimate divergence times in amphibians, actinopterygians, and felids. We evaluate the impact of various factors, including the priors on rates and times, fossil calibrations, substitution model, the violation of the molecular clock and the rate-drift model, and the exact and approximate likelihood calculation. Assuming the molecular clock caused seriously biased time estimates when the clock is violated, but 2 different rate-drift models produced similar estimates. The prior on times, which incorporates fossil-calibration information, had the greatest impact on posterior time estimation. In particular, the strategies used by the 2 programs to incorporate minimum- and maximum-age bounds led to very different time priors and were responsible for large differences in posterior time estimates in a previous study. The results highlight the critical importance of fossil calibrations to molecular dating and the need for probabilistic modeling of fossil depositions, preservations, and sampling to provide statistical summaries of information in the fossil record concerning species divergence times.

Origin and evolution of the integumentary skeleton in non-tetrapod vertebrates

Most non‐tetrapod vertebrates develop mineralized extra‐oral elements within the integument. Known collectively as the integumentary skeleton, these elements represent the structurally diverse skin‐bound contribution to the dermal skeleton. In this review we begin by summarizing what is known about the histological diversity of the four main groups of integumentary skeletal tissues: hypermineralized (capping) tissues; dentine; plywood‐like tissues; and bone. For most modern taxa, the integumentary skeleton has undergone widespread reduction and modification often rendering the homology and relationships of these elements confused and uncertain. Fundamentally, however, all integumentary skeletal elements are derived (alone or in combination) from only two types of cell condensations: odontogenic and osteogenic condensations. We review the origin and diversification of the integumentary skeleton in aquatic non‐tetrapods (including stem gnathostomes), focusing on tissues derived from odontogenic (hypermineralized tissues, dentines and elasmodine) and osteogenic (bone tissues) cell condensations. The novelty of our new scenario of integumentary skeletal evolution resides in the demonstration that elasmodine, the main component of elasmoid scales, is odontogenic in origin. Based on available data we propose that elasmodine is a form of lamellar dentine. Given its widespread distribution in non‐tetrapod lineages we further propose that elasmodine is a very ancient tissue in vertebrates and predict that it will be found in ancestral rhombic scales and cosmoid scales.

Phase-contrast X-ray microtomography links Cretaceous seeds with Gnetales and Bennettitales

Over the past 25 years the discovery and study of Cretaceous plant mesofossils has yielded diverse and exquisitely preserved fossil flowers that have revolutionized our knowledge of early angiosperms, but remains of other seed plants in the same mesofossil assemblages have so far received little attention. These fossils, typically only a few millimetres long, have often been charred in natural fires and preserve both three-dimensional morphology and cellular detail. Here we use phase-contrast-enhanced synchrotron-radiation X-ray tomographic microscopy to clarify the structure of small charcoalified gymnosperm seeds from the Early Cretaceous of Portugal and North America. The new information links these seeds to Gnetales (including Erdtmanithecales, a putatively closely related fossil group), and to Bennettitales—important extinct Mesozoic seed plants with cycad-like leaves and flower-like reproductive structures. The results suggest that the distinctive seed architecture of Gnetales, Erdtmanithecales and Bennettitales defines a clade containing these taxa. This has significant consequences for hypotheses of seed plant phylogeny by providing support for key elements of the controversial anthophyte hypothesis, which links angiosperms, Bennettitales and Gnetales.