Review ["Telling the Evolutionary Time : Molecular Clocks and the Fossil Record" edited by Philip C. J. Donoghue and M. Paul Smith]

Determining the temporal scale of biological evolution has traditionally been the preserve of paleontology, with the timing of species originations and major diversifications all being read from the fossil record. However, the ages of the earliest (correctly identified) records will underestimate actual origins due to the incomplete nature of the fossil record and the necessity for lineages to have evolved sufficiently divergent morphologies in order to be distinguished. The possibility of inferring divergence times more accurately has been promoted by the idea that the accumulation of genetic change between modern lineages can be used as a molecular clock (Zuckerkandl and Pauling, 1965). In practice, though, molecular dates have often been so old as to be incongruent even with liberal readings of the fossil record. Prominent examples include inferred diversifications of metazoan phyla hundreds of millions of years before their Cambrian fossil record appearances (e.g., Nei et al., 2001) and a basal split between modern birds (Neoaves) that is almost double the age of their earliest recognizable fossils (e.g., Cooper and Penny, 1997).

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.

Taxonomy and palaeoecology of Quaternary faunas from caves in eastern tropical Queensland : a record of broad-scale environmental change

Analysis of fossils from cave deposits at Mount Etna (eastern-central Queensland) has established that a species-rich rainforest palaeoenvironment existed in that area during the middle Pleistocene. This unexpected finding has implications for several fields (e.g., biogeography/phylogeography of rainforest-adapted taxa, and the impact of climate change on rainforest communities), but it was unknown whether the Mount Etna sites represented a small refugial patch of rainforest or was more widespread. In this study numerous bone deposits in caves in north-east Queensland are analysed to reconstruct the environmental history of the area during the late Quaternary. Study sites are in the Chillagoe/Mitchell Palmer and Broken River/Christmas Creek areas. The cave fossil records in these study areas are compared with dated (middle Pleistocene-Holocene) cave sites in the Mount Etna area. Substantial taxonomic work on the Mount Etna faunas (particularly dasyurid marsupials and murine rodents) is also presented as a prerequisite for meaningful comparison with the study sites further north. Middle Pleistocene sites at Mount Etna contain species indicative of a rainforest palaeoenvironment. Small mammal assemblages in the Mount Etna rainforest sites (>500-280 ka) are unexpectedly diverse and composed almost entirely of new species. Included in the rainforest assemblages are lineages with no extant representatives in rainforest (e.g., Leggadina), one genus previously known only from New Guinea (Abeomelomys), and forms that appear to bridge gaps between related but morphologically-divergent extant taxa ('B-rat' and 'Pseudomys C'). Curiously, some taxa (e.g., Melomys spp.) are notable for their absence from the Mount Etna rainforest sites. After 280 ka the rainforest faunas are replaced by species adapted to open, dry habitats. At that time the extinct ‘rainforest’ dasyurids and rodents are replaced by species that are either extant or recently extant. By the late Pleistocene all ‘rainforest’ and several ‘dry’ taxa are locally or completely extinct, and the small mammal fauna resembles that found in the area today. The faunal/environmental changes recorded in the Mount Etna sites were interpreted by previous workers as the result of shifts in climate during the Pleistocene. Many samples from caves in the Chillagoe/Mitchell-Palmer and Broken River/Christmas Creek areas are held in the Queensland Museum’s collection. These, supplemented with additional samples collected in the field as well as samples supplied by other workers, were systematically and palaeoecologically analysed for the first time. Palaeoecological interpretation of the faunal assemblages in the sites suggests that they encompass a similar array of palaeoenvironments as the Mount Etna sites. ‘Rainforest’ sites at the Broken River are here interpreted as being of similar age to those at Mount Etna, suggesting the possibility of extensive rainforest coverage in eastern tropical Queensland during part of the Pleistocene. Likewise, faunas suggesting open, dry palaeoenvironments are found at Chillagoe, the Broken River and Mount Etna, and may be of similar age. The 'dry' faunal assemblage at Mount Etna (Elephant hole Cave) dates to 205-170 ka. Dating of one of the Chillagoe sites (QML1067) produced a maximum age for the deposit of approximately 200 ka, and the site is interpreted as being close to that age, supporting the interpretation of roughly contemporaneous deposition at Mount Etna and Chillagoe. Finally, study sites interpreted as being of late Pleistocene-Holocene age show faunal similarities to sites of that age near Mount Etna. This study has several important implications for the biogeography and phylogeography of murine rodents, and represents a major advance in the study of the Australian murine fossil record. Likewise the survey of the northern study areas is the first systematic analysis of multiple sites in those areas, and is thus a major contribution to knowledge of tropical Australian faunas during the Quaternary. This analysis suggests that climatic changes during the Pleistocene affected a large area of eastern tropical Queensland in similar ways. Further fieldwork and dating is required to properly analyse the geographical extent and timing of faunal change in eastern tropical Queensland.

Thorough assessment of DNA preservation from fossil bone and sediments excavated from a late Pleistocene–Holocene cave deposit on Kangaroo Island, South Australia

Fossils and sediments preserved in caves are an excellent source of information for investigating impacts of past environmental changes on biodiversity. Until recently studies have relied on morphology-based palaeontological approaches, but recent advances in molecular analytical methods offer excellent potential for extracting a greater array of biological information from these sites. This study presents a thorough assessment of DNA preservation from late Pleistocene–Holocene vertebrate fossils and sediments from Kelly Hill Cave Kangaroo Island, South Australia. Using a combination of extraction techniques and sequencing technologies, ancient DNA was characterised from over 70 bones and 20 sediment samples from 15 stratigraphic layers ranging in age from >20 ka to ∼6.8 ka. A combination of primers targeting marsupial and placental mammals, reptiles and two universal plant primers were used to reveal genetic biodiversity for comparison with the mainland and with the morphological fossil record for Kelly Hill Cave. We demonstrate that Kelly Hill Cave has excellent long-term DNA preservation, back to at least 20 ka. This contrasts with the majority of Australian cave sites thus far explored for ancient DNA preservation, and highlights the great promise Kangaroo Island caves hold for yielding the hitherto-elusive DNA of extinct Australian Pleistocene species.

The fossil calibration database - A new resource for divergence dating

Fossils provide the principal basis for temporal calibrations, which are critical to the accuracy of divergence dating analyses. Translating fossil data into minimum and maximum bounds for calibrations is the most important, and often least appreciated, step of divergence dating. Properly justified calibrations require the synthesis of phylogenetic, paleontological, and geological evidence and can be difficult for non-specialists to formulate. The dynamic nature of the fossil record (e.g., new discoveries, taxonomic revisions, updates of global or local stratigraphy) requires that calibration data be updated continually lest they become obsolete. Here, we announce the Fossil Calibration Database (http://fossilcalibrations.org), a new open-access resource providing vetted fossil calibrations to the scientific community. Calibrations accessioned into this database are based on individual fossil specimens and follow best practices for phylogenetic justification and geochronological constraint. The associated Fossil Calibration Series, a calibration-themed publication series at Palaeontologia Electronica, will serve as one key pipeline for peer-reviewed calibrations to enter the database.

Four mammal fossil calibrations: Balancing competing palaeontological and molecular considerations

With the introduction of relaxed-clock molecular dating methods, the role of fossil calibration has expanded from providing a timescale, to also informing the models for molecular rate variation across the phylogeny. Here I suggest fossil calibration bounds for four mammal clades, Monotremata (platypus and echidnas), Macropodoidea (kangaroos and potoroos), Caviomorpha-Phiomorpha (South American and African hystricognath rodents), and Chiroptera (bats). In each case I consider sources of uncertainty in the fossil record and provide a molecular dating analysis to examine how the suggested calibration priors are further informed by other mammal fossil calibrations and molecular data.

Time-dependent rates of molecular evolution

For over half a century, it has been known that the rate of morphological evolution appears to vary with the time frame of measurement. Rates of microevolutionary change, measured between successive generations, were found to be far higher than rates of macroevolutionary change inferred from the fossil record. More recently, it has been suggested that rates of molecular evolution are also time dependent, with the estimated rate depending on the timescale of measurement. This followed surprising observations that estimates of mutation rates, obtained in studies of pedigrees and laboratory mutation-accumulation lines, exceeded long-term substitution rates by an order of magnitude or more. Although a range of studies have provided evidence for such a pattern, the hypothesis remains relatively contentious. Furthermore, there is ongoing discussion about the factors that can cause molecular rate estimates to be dependent on time. Here we present an overview of our current understanding of time-dependent rates. We provide a summary of the evidence for time-dependent rates in animals, bacteria and viruses. We review the various biological and methodological factors that can cause rates to be time dependent, including the effects of natural selection, calibration errors, model misspecification and other artefacts. We also describe the challenges in calibrating estimates of molecular rates, particularly on the intermediate timescales that are critical for an accurate characterization of time-dependent rates. This has important consequences for the use of molecular-clock methods to estimate timescales of recent evolutionary events.

Molecules, morphology, and ecology indicate a recent, amphibious ancestry for echidnas

The semiaquatic platypus and terrestrial echidnas (spiny anteaters) are the only living egg-laying mammals (monotremes). The fossil record has provided few clues as to their origins and the evolution of their ecological specializations; however, recent reassignment of the Early Cretaceous Teinolophos and Steropodon to the platypus lineage implies that platypuses and echidnas diverged >112.5 million years ago, reinforcing the notion of monotremes as living fossils. This placement is based primarily on characters related to a single feature, the enlarged mandibular canal, which supplies blood vessels and dense electrosensory receptors to the platypus bill. Our reevaluation of the morphological data instead groups platypus and echidnas to the exclusion of Teinolophos and Steropodon and suggests that an enlarged mandibular canal is ancestral for monotremes (partly reversed in echidnas, in association with general mandibular reduction). A multigene evaluation of the echidna–platypus divergence using both a relaxed molecular clock and direct fossil calibrations reveals a recent split of 19–48 million years ago. Platypus-like monotremes (Monotrematum) predate this divergence, indicating that echidnas had aquatically foraging ancestors that reinvaded terrestrial ecosystems. This ecological shift and the associated radiation of echidnas represent a recent expansion of niche space despite potential competition from marsupials. Monotremes might have survived the invasion of marsupials into Australasia by exploiting ecological niches in which marsupials are restricted by their reproductive mode. Morphology, ecology, and molecular biology together indicate that Teinolophos and Steropodon are basal monotremes rather than platypus relatives, and that living monotremes are a relatively recent radiation.

Branch-length estimation bias misleads molecular dating for a vertebrate mitochondrial phylogeny

Despite recent methodological advances in inferring the time-scale of biological evolution from molecular data, the fundamental question of whether our substitution models are sufficiently well specified to accurately estimate branch-lengths has received little attention. I examine this implicit assumption of all molecular dating methods, on a vertebrate mitochondrial protein-coding dataset. Comparison with analyses in which the data are RY-coded (AG → R; CT → Y) suggests that even rates-across-sites maximum likelihood greatly under-compensates for multiple substitutions among the standard (ACGT) NT-coded data, which has been subject to greater phylogenetic signal erosion. Accordingly, the fossil record indicates that branch-lengths inferred from the NT-coded data translate into divergence time overestimates when calibrated from deeper in the tree. Intriguingly, RY-coding led to the opposite result. The underlying NT and RY substitution model misspecifications likely relate respectively to “hidden” rate heterogeneity and changes in substitution processes across the tree, for which I provide simulated examples. Given the magnitude of the inferred molecular dating errors, branch-length estimation biases may partly explain current conflicts with some palaeontological dating estimates.

'Nothing exists for one cause' : putting biology back into evolution

The opening phrase of the title is from Charles Darwin’s notebooks (Schweber 1977). It is a double reminder, firstly that mainstream evolutionary theory is not just about describing nature but is particularly looking for mechanisms or ‘causes’, and secondly, that there will usually be several causes affecting any particular outcome. The second part of the title is our concern at the almost universal rejection of the idea that biological mechanisms are sufficient for macroevolutionary changes, thus rejecting a cornerstone of Darwinian evolutionary theory. Our primary aim here is to consider ways of making it easier to develop and to test hypotheses about evolution. Formalizing hypotheses can help generate tests. In an absolute sense, some of the discussion by scientists about evolution is little better than the lack of reasoning used by those advocating intelligent design. Our discussion here is in a Popperian framework where science is defined by that area of study where it is possible, in principle, to find evidence against hypotheses – they are in principle falsifiable. However, with time, the boundaries of science keep expanding. In the past, some aspects of evolution were outside the current boundaries of falsifiable science, but increasingly new techniques and ideas are expanding the boundaries of science and it is appropriate to re-examine some topics. It often appears that over the last few decades there has been an increasingly strong assumption to look first (and only) for a physical cause. This decision is virtually never formally discussed, just an assumption is made that some physical factor ‘drives’ evolution. It is necessary to examine our assumptions much more carefully. What is meant by physical factors ‘driving’ evolution, or what is an ‘explosive radiation’. Our discussion focuses on two of the six mass extinctions, the fifth being events in the Late Cretaceous, and the sixth starting at least 50,000 years ago (and is ongoing). Cretaceous/Tertiary boundary; the rise of birds and mammals. We have had a long-term interest (Cooper and Penny 1997) in designing tests to help evaluate whether the processes of microevolution are sufficient to explain macroevolution. The real challenge is to formulate hypotheses in a testable way. For example the numbers of lineages of birds and mammals that survive from the Cretaceous to the present is one test. Our first estimate was 22 for birds, and current work is tending to increase this value. This still does not consider lineages that survived into the Tertiary, and then went extinct later. Our initial suggestion was probably too narrow in that it lumped four models from Penny and Phillips (2004) into one model. This reduction is too simplistic in that we need to know about survival and ecological and morphological divergences during the Late Cretaceous, and whether Crown groups of avian or mammalian orders may have existed back into the Cretaceous. More recently (Penny and Phillips 2004) we have formalized hypotheses about dinosaurs and pterosaurs, with the prediction that interactions between mammals (and groundfeeding birds) and dinosaurs would be most likely to affect the smallest dinosaurs, and similarly interactions between birds and pterosaurs would particularly affect the smaller pterosaurs. There is now evidence for both classes of interactions, with the smallest dinosaurs and pterosaurs declining first, as predicted. Thus, testable models are now possible. Mass extinction number six: human impacts. On a broad scale, there is a good correlation between time of human arrival, and increased extinctions (Hurles et al. 2003; Martin 2005; Figure 1). However, it is necessary to distinguish different time scales (Penny 2005) and on a finer scale there are still large numbers of possibilities. In Hurles et al. (2003) we mentioned habitat modification (including the use of Geogenes III July 2006 31 fire), introduced plants and animals (including kiore) in addition to direct predation (the ‘overkill’ hypothesis). We need also to consider prey switching that occurs in early human societies, as evidenced by the results of Wragg (1995) on the middens of different ages on Henderson Island in the Pitcairn group. In addition, the presence of human-wary or humanadapted animals will affect the distribution in the subfossil record. A better understanding of human impacts world-wide, in conjunction with pre-scientific knowledge will make it easier to discuss the issues by removing ‘blame’. While continued spontaneous generation was accepted universally, there was the expectation that animals continued to reappear. New Zealand is one of the very best locations in the world to study many of these issues. Apart from the marine fossil record, some human impact events are extremely recent and the remains less disrupted by time.

Neck length and mean arterial pressure in the sauropod dinosaurs

How blood was able to reach the heads of the long-necked sauropod dinosaurs has long been a matter of debate and several hypotheses have been presented. For example, it has been proposed that sauropods had exceptionally large hearts, multiple ‘normal’ sized hearts spaced at regular intervals up the neck or held their necks horizontal, or that the siphon effect was in operation. By means of an experimental model, we demonstrate that the siphon principle is able to explain how blood was able to adequately perfuse the sauropod brain. The return venous circulation may have been protected from complete collapse by a structure akin to the vertebral venous plexus. We derive an equation relating neck height and mean arterial pressure, which indicates that with a mean arterial pressure similar to that of the giraffe, the maximum safe vertical distance between heart and head would have been about 12 m. A hypothesis is presented that the maximum neck length in the fossil record is due to the siphon height limit. The equation indicates that to migrate over high ground, sauropods would have had to either significantly increase their mean arterial pressure or keep their necks below a certain height dependent on altitude.

Geomolecular dating and the origin of placental mammals

In modern evolutionary divergence analysis the role of geological information extends beyond providing a timescale, to informing molecular rate variation across the tree. Here I consider the implications of this development. I use fossil calibrations to test the accuracy of models of molecular rate evolution for placental mammals, and reveal substantial misspecification associated with life history rate correlates. Adding further calibrations to reduce dating errors at specific nodes unfortunately tends to transfer underlying rate errors to adjacent branches. Thus, tight calibration across the tree is vital to buffer against rate model errors. I argue that this must include allowing maximum bounds to be tight when good fossil records permit, otherwise divergences deep in the tree will tend to be inflated by the interaction of rate errors and asymmetric confidence in minimum and maximum bounds. In the case of placental mammals I sought to reduce the potential for transferring calibration and rate model errors across the tree by focusing on well-supported calibrations with appropriately conservative maximum bounds. The resulting divergence estimates are younger than others published recently, and provide the long-anticipated molecular signature for the placental mammal radiation observed in the fossil record near the 66 Ma Cretaceous–Paleogene extinction event.

Avian diversification patterns across the K-Pg boundary: Influence of calibrations, datasets, and model misspecification

Birds represent the most diverse extant tetrapod clade, with ca. 10,000 extant species, and the timing of the crown avian radiation remains hotly debated. The fossil record supports a primarily Cenozoic radiation of crown birds, whereas molecular divergence dating analyses generally imply that this radiation was well underway during the Cretaceous. Furthermore, substantial differences have been noted between published divergence estimates. These have been variously attributed to clock model, calibration regime, and gene type. One underappreciated phenomenon is that disparity between fossil ages and molecular dates tends to be proportionally greater for shallower nodes in the avian Tree of Life. Here, we explore potential drivers of disparity in avian divergence dates through a set of analyses applying various calibration strategies and coding methods to a mitochondrial genome dataset and an 18-gene nuclear dataset, both sampled across 72 taxa. Our analyses support the occurrence of two deep divergences (i.e., the Palaeognathae/Neognathae split and the Galloanserae/Neoaves split) well within the Cretaceous, followed by a rapid radiation of Neoaves near the K-Pg boundary. However, 95% highest posterior density intervals for most basal divergences in Neoaves cross the boundary, and we emphasize that, barring unreasonably strict prior distributions, distinguishing between a rapid Early Paleocene radiation and a Late Cretaceous radiation may be beyond the resolving power of currently favored divergence dating methods. In contrast to recent observations for placental mammals, constraining all divergences within Neoaves to occur in the Cenozoic does not result in unreasonably high inferred substitution rates. Comparisons of nuclear DNA (nDNA) versus mitochondrial DNA (mtDNA) datasets and NT- versus RY-coded mitochondrial data reveal patterns of disparity that are consistent with substitution model misspecifications that result in tree compression/tree extension artifacts, which may explain some discordance between previous divergence estimates based on different sequence types. Comparisons of fully calibrated and nominally calibrated trees support a correlation between body mass and apparent dating error. Overall, our results are consistent with (but do not require) a Paleogene radiation for most major clades of crown birds.

Identification of alcohol involvement in injury-related hospitalisations using routine data compared to medical record review

Objective: To quantify the extent to which alcohol related injuries are adequately identified in hospitalisation data using ICD-10-AM codes indicative of alcohol involvement. Method: A random sample of 4373 injury-related hospital separations from 1 July 2002 to 30 June 2004 were obtained from a stratified random sample of 50 hospitals across 4 states in Australia. From this sample, cases were identified as involving alcohol if they contained an ICD-10-AM diagnosis or external cause code referring to alcohol, or if the text description extracted from the medical records mentioned alcohol involvement. Results: Overall, identification of alcohol involvement using ICD codes detected 38% of the alcohol-related sample, whilst almost 94% of alcohol-related cases were identified through a search of the text extracted from the medical records. The resultant estimate of alcohol involvement in injury-related hospitalisations in this sample was 10%. Emergency department records were the most likely to identify whether the injury was alcohol-related with almost three-quarters of alcohol-related cases mentioning alcohol in the text abstracted from these records. Conclusions and Implications: The current best estimates of the frequency of hospital admissions where alcohol is involved prior to the injury underestimate the burden by around 62%. This is a substantial underestimate that has major implications for public policy, and highlights the need for further work on improving the quality and completeness of routine administrative data sources for identification of alcohol-related injuries.