The Effect of Inappropriate Calibration: Three Case Studies in Molecular Ecology Simon Y. W. Ho1¤*, Urmas Saarma2,3, Ross Barnett4, James Haile1, Beth Shapiro1 1 Department of Zoology, University of Oxford, Oxford, United Kingdom, 2 Department of Zoology, Institute of Ecology and Earth Sciences, University of Tartu, Tartu, Estonia, 3 Estonian Biocentre, Tartu, Estonia, 4 Department of Biology, University of York, York, United Kingdom Abstract Time-scales estimated from sequence data play an important role in molecular ecology. They can be used to draw correlations between evolutionary and palaeoclimatic events, to measure the tempo of speciation, and to study the demographic history of an endangered species. In all of these studies, it is paramount to have accurate estimates of time- scales and substitution rates. Molecular ecological studies typically focus on intraspecific data that have evolved on genealogical scales, but often these studies inappropriately employ deep fossil calibrations or canonical substitution rates (e.g., 1% per million years for birds and mammals) for calibrating estimates of divergence times. These approaches can yield misleading estimates of molecular time-scales, with significant impacts on subsequent evolutionary and ecological inferences. We illustrate this calibration problem using three case studies: avian speciation in the late Pleistocene, the demographic history of bowhead whales, and the Pleistocene biogeography of brown bears. For each data set, we compare the date estimates that are obtained using internal and external calibration points. In all three cases, the conclusions are significantly altered by the application of revised, internally-calibrated substitution rates. Collectively, the results emphasise the importance of judicious selection of calibrations for analyses of recent evolutionary events. Citation: Ho SYW, Saarma U, Barnett R, Haile J, Shapiro B (2008) The Effect of Inappropriate Calibration: Three Case Studies in Molecular Ecology. PLoS ONE 3(2): e1615. doi:10.1371/journal.pone.0001615 Editor: Peter Bennett, University of Kent, United Kingdom Received October 21, 2007; Accepted January 22, 2008; Published February 20, 2008 Copyright: 2008 Ho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: S.Y.W.H. was supported by the Leverhulme Trust. U.S. was supported by the Estonian Science Foundation (grant 7040). J.H. was supported by the Arts and Humanities Research Board. B.S. was supported by the Royal Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *E-mail: drsimonho@gmail.com ¤ Current address: Centre for Macroecology and Macroevolution, School of Botany and Zoology, Australian National University, Canberra, Australian Capital Territory, Australia Introduction Evolutionary time-scales estimated from molecular data form the foundation for a diverse range of molecular ecological studies, including those of biogeography, speciation, conservation genetics, and population biology. Molecular chronologies allow us to examine correlations between evolutionary events and palaeocli- matic phenomena, such as glaciations and sea level changes [1–3], or biotic factors, including faunal and floral changes and human migration [4–6]. For conservation purposes, we can measure the phylogenetic distinctiveness of a species or the antiquity of a specific population [7,8], while past and present effective population sizes can be inferred in studies of population biology [9,10]. All of these studies depend on accurate estimates of molecular time-scales and substitution rates which, primarily due to methodological limitations, have been made with varying degrees of rigour in the past. Estimating time-scales from genetic data is laden with methodological obstacles. One of the chief difficulties is the selection of an appropriate calibration [11–14], which is necessary for converting measures of genetic divergence into units of absolute or geological time. Calibrating information can be incorporated into an analysis in one of several ways [15], of which the most widely used are: (i) fixing the age of a phylogenetic divergence event on the basis of independent palaeontological, archaeological, or biogeographic data; and (ii) importing a substitution rate obtained from independent data. A third calibration method is the inclusion of heterochronous sequences of known age, such as ancient DNA sequences extracted from radiocarbon-dated samples [16,17]. The fossil record has played a key role in calibrating molecular estimates of evolutionary rates and divergence times in the tree of life [18]. This role has expanded in recent years due to advances in phylogenetic methods [15,19]. Ecological studies, however, often investigate evolutionary processes within species, such as the timing of dispersal, migration, or extinction events [4,20]. These occur on genealogical time-scales rather than the longer phylogenetic time-scales typically associated with fossil calibra- tions. Using deep fossil calibrations in ecological studies can present a methodological problem because different stages of the substitution process are being observed over genealogical and phylogenetic time-scales [21]. When sequences are taken from individuals within a population or species, the differences among them represent segregating sites or polymorphisms, many of which are transient and will be removed by genetic drift or purifying selection [21–24]. In contrast, differences between sequences taken from different species represent past fixations (substitutions). This is, of course, a simplistic portrayal of the situation, as the species boundary is often indistinct and the two scales are directly linked because mutation and substitution are aspects of the same PLoS ONE | www.plosone.org 1 February 2008 | Volume 3 | Issue 2 | e1615