James E. Tarver

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.

Dinosaurs and the Cretaceous Terrestrial Revolution

The observed diversity of dinosaurs reached its highest peak during the mid- and Late Cretaceous, the 50 Myr that preceded their extinction, and yet this explosion of dinosaur diversity may be explained largely by sampling bias. It has long been debated whether dinosaurs were part of the Cretaceous Terrestrial Revolution (KTR), from 125–80 Myr ago, when flowering plants, herbivorous and social insects, squamates, birds and mammals all underwent a rapid expansion. Although an apparent explosion of dinosaur diversity occurred in the mid-Cretaceous, coinciding with the emergence of new groups (e.g. neoceratopsians, ankylosaurid ankylosaurs, hadrosaurids and pachycephalosaurs), results from the first quantitative study of diversification applied to a new supertree of dinosaurs show that this apparent burst in dinosaurian diversity in the last 18 Myr of the Cretaceous is a sampling artefact. Indeed, major diversification shifts occurred largely in the first one-third of the groups history. Despite the appearance of new clades of medium to large herbivores and carnivores later in dinosaur history, these new originations do not correspond to significant diversification shifts. Instead, the overall geometry of the Cretaceous part of the dinosaur tree does not depart from the null hypothesis of an equal rates model of lineage branching. Furthermore, we conclude that dinosaurs did not experience a progressive decline at the end of the Cretaceous, nor was their evolution driven directly by the KTR.

A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome

Although microRNAs (miRNAs) are among the most intensively studied molecules of the past 20 years, determining what is and what is not a miRNA has not been straightforward. Here, we present a uniform system for the annotation and nomenclature of miRNA genes. We show that less than a third of the 1,881 human miRBase entries, and only approximately 16% of the 7,095 metazoan miRBase entries, are robustly supported as miRNA genes. Furthermore, we show that the human repertoire of miRNAs has been shaped by periods of intense miRNA innovation and that mature gene products show a very different tempo and mode of sequence evolution than star products. We establish a new open access database--MirGeneDB ( http://mirgenedb.org )--to catalog this set of miRNAs, which complements the efforts of miRBase but differs from it by annotating the mature versus star products and by imposing an evolutionary hierarchy upon this curated and consistently named repertoire.

Reconstruction of Family-Level Phylogenetic Relationships within Demospongiae (Porifera) Using Nuclear Encoded Housekeeping Genes

Background Demosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges. Methodology/Principal Findings We generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosap, Myxospongiaep, Spongillidap, Haploscleromorphap (the marine haplosclerids) and Democlaviap. We found conflicting results concerning the relationships of Keratosap and Myxospongiaep to the remaining demosponges, but our results strongly supported a clade of Haploscleromorphap+Spongillidap+Democlaviap. In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillidap) are sister to Haploscleromorphap rather than part of Democlaviap. Within Keratosap, we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiaep, Chondrosida and Verongida were monophyletic. A well-supported clade within Democlaviap, Tetractinellidap, composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlaviap. Within Tetractinellidap, we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida. Conclusions/Significance These results, using an independent nuclear gene set, confirmed many hypotheses based on ribosomal and/or mitochondrial genes, and they also identified clades with low statistical support or clades that conflicted with traditional morphological classification. Our results will serve as a basis for future exploration of these outstanding questions using more taxon- and gene-rich datasets.

Evolutionary history of plant microRNAs

microRNAs (miRNAs) are short noncoding regulatory genes that perform important roles in plant development and physiology. With the increasing power of next generation sequencing technologies and the development of bioinformatic tools, there has been a dramatic increase in the number of studies surveying the miRNAomes of plant species, which has led to an explosion in the number of described miRNAs. Unfortunately, very many of these new discoveries have been incompletely annotated and thus fail to discriminate genuine miRNAs from small interfering RNAs (siRNAs), fragments of longer RNAs, and random sequence. We review the published repertoire of plant miRNAs, discriminating those that have been correctly annotated. We use these data to explore prevailing hypotheses on the tempo and mode of miRNA evolution within the plant kingdom.

miRNAs: Small Genes with Big Potential in Metazoan Phylogenetics

microRNAs (miRNAs) are a key component of gene regulatory networks and have been implicated in the regulation of virtually every biological process found in multicellular eukaryotes. What makes them interesting from a phylogenetic perspective is the high conservation of primary sequence between taxa, their accrual in metazoan genomes through evolutionary time, and the rarity of secondary loss in most metazoan taxa. Despite these properties, the use of miRNAs as phylogenetic markers has not yet been discussed within a clear conceptual framework. Here we highlight five properties of miRNAs that underlie their utility in phylogenetics: 1) The processes of miRNA biogenesis enable the identification of novel miRNAs without prior knowledge of sequence; 2) The continuous addition of miRNA families to metazoan genomes through evolutionary time; 3) The low level of secondary gene loss in most metazoan taxa; 4) The low substitution rate in the mature miRNA sequence; and 5) The small probability of convergent evolution of two miRNAs. Phylogenetic analyses using both Bayesian and parsimony methods on a eumetazoan miRNA data set highlight the potential of miRNAs to become an invaluable new tool, especially when used as an additional line of evidence, to resolve previously intractable nodes within the tree of life.

Do miRNAs have a deep evolutionary history

The recent discovery of microRNAs (miRNAs) in unicellular eukaryotes, including miRNAs known previously only from animals or plants, implies that miRNAs have a deep evolutionary history among eukaryotes. This contrasts with the prevailing view that miRNAs evolved convergently in animals and plants. We re‐evaluate the evidence and find that none of the 73 plant and animal miRNAs described from protists meet the required criteria for miRNA annotation and, by implication, animals and plants did not acquire any of their respective miRNA genes from the crown ancestor of eukaryotes. Furthermore, of the 159 novel miRNAs previously identified among the seven species of unicellular protists examined, only 28 from the algae Ectocarpus and Chlamydomonas, meet the criteria for miRNA annotation. Therefore, at present only five groups of eukaryotes are known to possess miRNAs, indicating that miRNAs have evolved independently within eukaryotes through exaptation of their shared inherited RNAi machinery.

Molecular signatures of plastic phenotypes in two eusocial insect species with simple societies

Significance In eusocial insect societies, such as ants and some bees and wasps, phenotypes are highly plastic, generating alternative phenotypes (queens and workers) from the same genome. The greatest plasticity is found in simple insect societies, in which individuals can switch between phenotypes as adults. The genomic, transcriptional, and epigenetic underpinnings of such plasticity are largely unknown. In contrast to the complex societies of the honeybee, we find that simple insect societies lack distinct transcriptional differentiation between phenotypes and coherently patterned DNA methylomes. Instead, alternative phenotypes are largely defined by subtle transcriptional network organization. These traits may facilitate genomic plasticity. These insights and resources will stimulate new approaches and hypotheses that will help to unravel the genomic processes that create phenotypic plasticity. Phenotypic plasticity is important in adaptation and shapes the evolution of organisms. However, we understand little about what aspects of the genome are important in facilitating plasticity. Eusocial insect societies produce plastic phenotypes from the same genome, as reproductives (queens) and nonreproductives (workers). The greatest plasticity is found in the simple eusocial insect societies in which individuals retain the ability to switch between reproductive and nonreproductive phenotypes as adults. We lack comprehensive data on the molecular basis of plastic phenotypes. Here, we sequenced genomes, microRNAs (miRNAs), and multiple transcriptomes and methylomes from individual brains in a wasp (Polistes canadensis) and an ant (Dinoponera quadriceps) that live in simple eusocial societies. In both species, we found few differences between phenotypes at the transcriptional level, with little functional specialization, and no evidence that phenotype-specific gene expression is driven by DNA methylation or miRNAs. Instead, phenotypic differentiation was defined more subtly by nonrandom transcriptional network organization, with roles in these networks for both conserved and taxon-restricted genes. The general lack of highly methylated regions or methylome patterning in both species may be an important mechanism for achieving plasticity among phenotypes during adulthood. These findings define previously unidentified hypotheses on the genomic processes that facilitate plasticity and suggest that the molecular hallmarks of social behavior are likely to differ with the level of social complexity.

Bayesian methods outperform parsimony but at the expense of precision in the estimation of phylogeny from discrete morphological data

Different analytical methods can yield competing interpretations of evolutionary history and, currently, there is no definitive method for phylogenetic reconstruction using morphological data. Parsimony has been the primary method for analysing morphological data, but there has been a resurgence of interest in the likelihood-based Mk-model. Here, we test the performance of the Bayesian implementation of the Mk-model relative to both equal and implied-weight implementations of parsimony. Using simulated morphological data, we demonstrate that the Mk-model outperforms equal-weights parsimony in terms of topological accuracy, and implied-weights performs the most poorly. However, the Mk-model produces phylogenies that have less resolution than parsimony methods. This difference in the accuracy and precision of parsimony and Bayesian approaches to topology estimation needs to be considered when selecting a method for phylogeny reconstruction.

The Interrelationships of Placental Mammals and the Limits of Phylogenetic Inference

Placental mammals comprise three principal clades: Afrotheria (e.g., elephants and tenrecs), Xenarthra (e.g., armadillos and sloths), and Boreoeutheria (all other placental mammals), the relationships among which are the subject of controversy and a touchstone for debate on the limits of phylogenetic inference. Previous analyses have found support for all three hypotheses, leading some to conclude that this phylogenetic problem might be impossible to resolve due to the compounded effects of incomplete lineage sorting (ILS) and a rapid radiation. Here we show, using a genome scale nucleotide data set, microRNAs, and the reanalysis of the three largest previously published amino acid data sets, that the root of Placentalia lies between Atlantogenata and Boreoeutheria. Although we found evidence for ILS in early placental evolution, we are able to reject previous conclusions that the placental root is a hard polytomy that cannot be resolved. Reanalyses of previous data sets recover Atlantogenata + Boreoeutheria and show that contradictory results are a consequence of poorly fitting evolutionary models; instead, when the evolutionary process is better-modeled, all data sets converge on Atlantogenata. Our Bayesian molecular clock analysis estimates that marsupials diverged from placentals 157–170 Ma, crown Placentalia diverged 86–100 Ma, and crown Atlantogenata diverged 84–97 Ma. Our results are compatible with placental diversification being driven by dispersal rather than vicariance mechanisms, postdating early phases in the protracted opening of the Atlantic Ocean.