Marcel van Tuinen

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.

Genetic Response to Climatic Change: Insights from Ancient DNA and Phylochronology

Understanding how climatic change impacts biological diversity is critical to conservation. Yet despite demonstrated effects of climatic perturbation on geographic ranges and population persistence, surprisingly little is known of the genetic response of species. Even less is known over ecologically long time scales pertinent to understanding the interplay between microevolution and environmental change. Here, we present a study of population variation by directly tracking genetic change and population size in two geographically widespread mammal species (Microtus montanus and Thomomys talpoides) during late-Holocene climatic change. We use ancient DNA to compare two independent estimates of population size (ecological and genetic) and corroborate our results with gene diversity and serial coalescent simulations. Our data and analyses indicate that, with population size decreasing at times of climatic change, some species will exhibit declining gene diversity as expected from simple population genetic models, whereas others will not. While our results could be consistent with selection, independent lines of evidence implicate differences in gene flow, which depends on the life history strategy of species.

Calibration of galliform molecular clocks using multiple fossils and genetic partitions.

For more than a century, members of the traditional avian order Galliformes (i.e., pheasants, partridges, junglefowl, and relatives) have been among the most intensively studied birds, but still a comprehensive timeframe for their evolutionary history is lacking. Thanks to a number of recent cladistic interpretations for several galliform fossils, candidates now exist that can potentially be used as accurate internal calibrations for molecular clocks. Here, we describe a molecular timescale for Galliformes based on cytochrome b and ND2 using nine mostly internal fossil-based anchorpoints. Beyond application of calibrations spanning the entire evolutionary history of Galliformes, care was taken to investigate the effects of calibration choice, substitution saturation, and rate heterogeneity among lineages on divergence time estimation. Results show broad consistency in time estimation with five out of the nine total calibrations. Our divergence time estimates, based on these anchorpoints, indicate that the early history of Galliformes took place in the Cretaceous, including the origin of the basal-most megapode and perhaps cracid lineages, but that the remaining morphological diversification likely started in the earliest Tertiary. The multi-calibration/multi-genetic partition approach used here highlights the importance of understanding the genetic saturation, variation, and rate constancy spectra for the accurate calculation of divergence times by use of molecular clocks.

The shifting baseline of northern fur seal ecology in the northeast Pacific Ocean

Historical data provide a baseline against which to judge the significance of recent ecological shifts and guide conservation strategies, especially for species decimated by pre-20th century harvesting. Northern fur seals (NFS; Callorhinus ursinus) are a common pinniped species in archaeological sites from southern California to the Aleutian Islands, yet today they breed almost exclusively on offshore islands at high latitudes. Harvest profiles from archaeological sites contain many unweaned pups, confirming the presence of temperate-latitude breeding colonies in California, the Pacific Northwest, and the eastern Aleutian Islands. Isotopic results suggest that prehistoric NFS fed offshore across their entire range, that California populations were distinct from populations to the north, and that populations breeding at temperate latitudes in the past used a different reproductive strategy than modern populations. The extinction of temperate-latitude breeding populations was asynchronous geographically. In southern California, the Pacific Northwest, and the eastern Aleutians, NFS remained abundant in the archaeological record up to the historical period ≈200 years B.P.; thus their regional collapse is plausibly attributed to historical hunting or some other anthropogenic ecosystem disturbance. In contrast, NFS populations in central and northern California collapsed at ≈800 years B.P., long before European contact. The relative roles of human hunting versus climatic factors in explaining this ecological shift are unclear, as more paleoclimate information is needed from the coastal zone.

ANCIENT DNA EVIDENCE OF PROLONGED POPULATION PERSISTENCE WITH NEGLIGIBLE GENETIC DIVERSITY IN AN ENDEMIC TUCO-TUCO (CTENOMYS SOCIABILIS)

Abstract We traced a population of Ctenomys sociabilis, a highly endemic South American tuco-tuco, through 1,000 years to assess its response to climatic change and recent human disturbance. Samples were obtained from a late-Holocene raptor roost in Parque Nacional Nahuel Huapi, Argentina, which produced a diverse and abundant rodent fauna, with >10 genera extending from the present to 950 ± 50 years ago (CAMS-45936). The site (Estancia Nahuel Huapi locality 1) was located near the center of the present geographic range of C. sociabilis, which occurs throughout 8 of 9 stratigraphic levels in the site. To examine genetic structure through time, we extracted ancient DNA from 16 teeth at those levels and from 1 modern tooth at the surface for a total of 17 specimens. Cytochrome-b sequences from ancient and modern specimens were compared with a modern tuco-tuco sequence from the extant local population. Our results show that of those 17 specimens, all but 1 had identical sequences. Further, these sequences were identical to a representative of the modern population. Thus, that population has remained genetically identical for at least 1,000 years in the face of climatic change, human disturbance, and proximity of other tuco-tuco species (C. haigi, C. maulinus) with adjacent geographic distributions. Our findings indicate that a population bottleneck contributing to low genetic diversity of C. sociabilis occurred before 1,000 years ago and that late-Holocene climatic change occurred without a corresponding impact on the genetic diversity of this species.

Error in Estimation of Rate and Time Inferred from the Early Amniote Fossil Record and Avian Molecular Clocks

The best reconstructions of the history of life will use both molecular time estimates and fossil data. Errors in molecular rate estimation typically are unaccounted for and no attempts have been made to quantify this uncertainty comprehensively. Here, focus is primarily on fossil calibration error because this error is least well understood and nearly universally disregarded. Our quantification of errors in the synapsid–diapsid calibration illustrates that although some error can derive from geological dating of sedimentary rocks, the absence of good stem fossils makes phylogenetic error the most critical. We therefore propose the use of calibration ages that are based on the first undisputed synapsid and diapsid. This approach yields minimum age estimates and standard errors of 306.1 ± 8.5 MYR for the divergence leading to birds and mammals. Because this upper bound overlaps with the recent use of 310 MYR, we do not support the notion that several metazoan divergence times are significantly overestimated because of serious miscalibration (sensuLee 1999). However, the propagation of relevant errors reduces the statistical significance of the pre-K–T boundary diversification of many bird lineages despite retaining similar point time estimates. Our results demand renewed investigation into suitable loci and fossil calibrations for constructing evolutionary timescales.

Extracting time from phylogenies: Positive interplay between fossil and genetic data

Abstract Temporal information on mammalian evolution allows testing of hypotheses about the mode of speciation and extinction, comparison of rates of evolution across taxa, and correlation of cladogenesis with important geological processes. Important insights can be made from combining data from fossils and molecules (DNA sequences), and relevant methods are expanding. When considering these methods, careful consideration should be given to features affecting time estimation (e.g., accuracy of tree construction, branch length estimation, taxonomic and genomic sampling, and sources of genetic and calibration errors). We report on the available methods that aid in evolutionary time estimation from molecular data and corresponding fossils. We recommend several steps to improve this process. First, we recommend using appropriate DNA substitution models to help correct DNA distance estimates and construct trees with more reliable branch lengths. Second, we recommend using multiple fossil calibration dates where possible. Third, in general, we recommend a conservative approach to time estimation by reporting inherent errors associated with genetic distances, calibration selection and application. In conclusion, temporal data derived from molecular clocks should be regarded in light of their dependence on paleontological information and their synergy with fossil data rather than competing with paleontological information. Thus, despite the varied sources of error, we encourage the extraction of time from molecular data with careful examination of potential biases.

THE EFFECT OF EXTERNAL AND INTERNAL FOSSIL CALIBRATIONS ON THE AVIAN EVOLUTIONARY TIMESCALE

Abstract Molecular clocks can provide insights into the evolutionary timescale of groups with unusually biased or fragmentary fossil records, such as birds. In those cases, it is advantageous to establish internal anchor points—molecular time estimates—using the best external fossil calibrations. In turn, those anchor points can be used as calibrations for more detailed time estimation within the group under study. This method also avoids the inherent problems in drawing conclusions about the evolution of a group based on data tied to the poor fossil record of that same group. The galliform-anseriform divergence (∼90 million years ago) is an example of such an ideal anchor point for molecular clock analyses in birds.

A New Specimen of the Fossil Palaeognath Lithornis from the Lower Eocene of Denmark

Abstract “Buy preparations?! [but] we have not enough money left to buy gunpowder”—comment of the British Prime Minister William Pitt during the Napoleonic Wars (recorded by Flower, 1898). The original holotype of Lithornis vulturinus was purchased by the British government in 1799 as part of a collection to “be maintained in its integrity to serve the education of the citizens”. Palaeognathous birds (Aves, Palaeognathae) are uncontroversially the most basal clade among modern birds (Neornithes), having been defined for more than 100 years on the basis of their palatal morphology. However, because many fossil specimens that have been described to date lack detailed skull material (especially in association with postcrania), aspects of the early evolutionary history of these birds remain unclear, and their relationships on the basis of anatomical characters are as yet unresolved. In this paper we present a new and exceptionally well-preserved specimen of the Lower Eocene fossil palaeognath Lithornis that has a remarkable three-dimensionally preserved and complete skull. New anatomical information provided by this Danish fossil leads us to suggest that a number of cranial characters previously considered diagnostic for ratites may in fact be primitive among palaeognaths. The presence of members of Lithornithidae in the Lower Eocene (earliest Tertiary) is consistent with the hypothesis that basal divergences within Palaeognathae occurred at an earlier geological time, perhaps prior to the Cretaceous–Tertiary (K–T) boundary, as has been proposed based on evidence from much less well-preserved fossil material.

Synthesizing and databasing fossil calibrations: Divergence dating and beyond

Divergence dating studies, which combine temporal data from the fossil record with branch length data from molecular phylogenetic trees, represent a rapidly expanding approach to understanding the history of life. National Evolutionary Synthesis Center hosted the first Fossil Calibrations Working Group (3–6 March, 2011, Durham, NC, USA), bringing together palaeontologists, molecular evolutionists and bioinformatics experts to present perspectives from disciplines that generate, model and use fossil calibration data. Presentations and discussions focused on channels for interdisciplinary collaboration, best practices for justifying, reporting and using fossil calibrations and roadblocks to synthesis of palaeontological and molecular data. Bioinformatics solutions were proposed, with the primary objective being a new database for vetted fossil calibrations with linkages to existing resources, targeted for a 2012 launch.