population genetic processes. For example, the observed genetic variation within a species can be inflated by the presence of ancestral polymorphisms that have been inherited from the parent species [25]. Nevertheless, it is apparent that considerable disparities can result between long-term substitution rates and instantaneous mutation rates, with direct and indirect estimates of the latter frequently yielding high values [26–28]. This disparity can be magnified by saturation at mutational hot spots [29], which can obscure the presence of past polymorphisms and cause long-term rates to be underestimated if inadequate corrections are applied. In a phylogenetic analysis, transient polymorphisms manifest themselves as excess nucleotide changes on the branches near the tips of the tree (Figure 1) [30,31]. They disappear from the population through time, so that the deeper a branch is in the tree, the fewer transient polymorphisms it will carry. When multiple species are compared within a single tree, the majority of nucleotide changes observed along the deepest branches, including the branch between the study species and outgroup, are likely to represent substitutions. Consequently, deeper calibration points will lead to slower observed rates [21,23]. This can lead to considerable overestimates of times to divergence, particularly when slow substitution rates are obtained from interspecific comparisons in a phylogenetic context and then extrapolated to population-level data [21,32]. We believe that the inappropriate use of extraspecific or external calibration points may have misled a substantial number of molecular ecological studies. Here we present three detailed case studies in which the conclusions are significantly altered by using intraspecific, internal calibration points. Results and Discussion Avian Speciation in the Late Pleistocene In Eurasia and North America, the Pleistocene was a time of significant fluctuations in climate and the distribution of habitats. Cyclical glacial advance and retreat produced dramatic changes in local environmental conditions over the past 250 kyr [33], leading Inappropriate Calibration to the view that these changes were conducive to an increased rate of avian speciation, a concept embodied in the ‘‘Late Pleistocene Origins’’ (LPO) hypothesis [for a recent review, see 1]. This hypothesis readily lends itself to testing because it makes specific predictions about divergence times, which can be estimated from molecular data. A study of mitochondrial DNA from avian sister species found that divergences occurred prior to the Pleistocene [34]. On the basis of this evidence the authors suggested that the speciation events were associated with early glacial expansion in the Northern Hemisphere around 2.4 Myr before present (BP). In contrast, an analysis of diverging conspecific populations or ‘phylogroups’, which were interpreted as reflecting incipient speciation, supported a more recent time-scale for avian diversification [35]. The debate has continued in numerous subsequent studies [36–42]. In nearly all of these studies, the authors collected a series of genetic divergence estimates, then divided the distances by a known substitution rate in order to produce an estimate of the time since divergence. The reliability of such estimates depends absolutely on the accuracy of the imported rate. Without exception, the substitution rate used in tests of the LPO hypothesis has been the ‘traditional’ mitochondrial rate of 0.01 substitutions per site per million years (subs/site/Myr), which has long been adopted as a standard in avian molecular studies [43,44]. There are several reasons why the use of this rate may be questionable. First, the rate is not universally applicable among different avian taxa, with considerable rate heterogeneity detected among lineages [45,46]. Second, the rate was calculated in a phylogenetic context, whereas the study of incipient species (and perhaps recently diverged species) is a genealogical issue [32]. Studies of intraspecific data from birds have generally yielded faster substitution rate estimates [23,47], peaking at a rate of 0.95 subs/site/Myr for the mitochondrial hypervariable region 1 in Ade´lie penguins [48]. To investigate the impact of employing these higher rates in investigations of the LPO hypothesis, we re- analysed sets of genetic distances made in published studies. Figure 1. Phylogenetic tree illustrating the impact of using extraspecific and intraspecific calibration points. The tree shows the locations of nucleotide changes (small vertical bars). The nucleotide changes within the study species represent segregating sites, some of which will be fixed in the long term, but most of which will be removed by drift or selection. The changes between the study species and outgroup species represent substitutions. If an estimate of the evolutionary rate is calibrated using an external calibration point, such as the split between the study and outgroup species, then the intraspecific rate will be underestimated. This will lead to an overestimation of times to divergence, including the age of the most recent common ancestor of the study species. doi:10.1371/journal.pone.0001615.g001 PLoS ONE | www.plosone.org 2 February 2008 | Volume 3 | Issue 2 | e1615