Figure 2. Bayesian skyline plots showing the recent demo- graphic history of bowhead whales, estimated using phyloge- netic analysis of three alignments of the mitochondrial control region: (a) combined alignment of 68 modern haplotypes and 99 radiocarbon-dated, ancient DNA sequences; (b) modern sequences only; and (c) ancient sequences only. All three plots are drawn to the vertical and horizontal scales. doi:10.1371/journal.pone.0001615.g002 Myr), respectively. By using these two rate estimates to specify a prior on the substitution rate in analyses of the modern bowhead whale sequences, the age of the MRCA of all individuals is estimated to be 1.1 Myr BP (95% HPD: 0.497–1.88 Myr BP) and 4.12 Myr BP (95% HPD: 2.07–6.39 Myr BP), respectively. These are substantially older than the estimate of 150 kyr obtained in the BSP analyses calibrated by radiocarbon dates. The results suggest that previous estimates of the evolutionary time-scale of bowhead whales have been misled by an inappro- priate fossil calibration, the effect of which has been to produce overestimates of times to coalescence. Our re-analysis suggests that population expansion in bowhead whales occurred relatively recently and that short time periods may suffice for the generation of appreciable levels of genetic diversity. This has important consequences for studies of conservation genetics, many of which have relied on substitution rates obtained by adopting relatively deep calibration points. Pleistocene Biogeography of the Brown Bear The last few years have seen a proliferation of large data sets that sample individuals from a population over thousands to tens of thousands of generations. These data enable the direct testing of hypotheses concerning the relationship between past and present Inappropriate Calibration distributions of species and populations [20], and the impact of environmental events on the phylogeography of a species [4,55]. In these studies, it is evident that without accurate estimates of substitution rates, there is a risk of misidentifying causal environmental factors for inferred demographic changes. Brown bears (Ursus arctos) present an interesting illustration of this point. Modern brown bear populations are distributed throughout Europe, Asia, and North America, and exhibit significant maternally-linked phylogeographic structure across this range [20,56–58] (see tree topology in Figure 3). The modern distribution of brown bears is believed to be the consequence of post-glacial expansion from local refuges, and to have remained relatively stable since this expansion [3,57,59–61]; however, a more recent study of ancient DNA found a more complex phylogeographic history for Western European brown bears [62]. Although this work did not entirely exclude the classic refuge scenario, it insinuated that past refugium theories might be oversimplifications [62]. Nevertheless, ancient DNA studies have revealed that the modern European distribution of brown bear lineages differs from that prior to the last glacial maximum [55], whereas in North America four distinct periods of constant population structure could be identified over the last 50 kyr, interposed by brief periods of rapid demographic change [20]. These findings suggest that brown bears in both Europe and North America experienced frequent local extinctions and range expansions during the climatically volatile late Pleistocene. Saarma et al. [3] investigated the possibility that the present distribution of bears in northern Europe is the result of post-glacial expansion from a refuge in the West Carpathian mountains. Using a substitution rate estimated from radiocarbon-dated North American brown bears [20], they estimated that the MRCA of the Eastern lineage (node I, Figure 3) existed 24 kyr BP (95% HPD: 6–50 kyr BP), with dates of 67 kyr BP (95% HPD: 20– 131 kyr BP) for the Western lineage (node A) and 174 kyr BP (95% HPD: 61–314 kyr BP) for all European bears (node N). The estimates came with wide 95% HPD intervals, but were consistent with recent expansions of the Eastern and Western lineages and a mid-late Pleistocene MRCA for all brown bears. These relatively recent estimates for the origin of the European brown bear lineages stand in contrast to the more ancient estimates of Hofreiter et al. [63], which were calibrated using the split between brown bears and their sister species (cave bears; Ursus speleaus) at 1.2–1.7 Myr [64,65]. This calibration put the ages of the MRCAs for the two European lineages of brown bears during the mid-early Pleistocene: 640 kyr BP (95% CI: 290–1,390 kyr BP) for the Eastern lineage and 350 kyr BP (95% CI: 150–790 kyr BP) for the Western lineage. The disparity between the two sets of estimates illustrates the difference between internal and external calibration, but the estimates are difficult to compare directly because they were made using different approaches. To investigate this further, we performed a Bayesian phylogenetic analysis of an alignment of 107 brown bear sequences using two separate modes of calibration: (i) internal calibration using 31 radiocarbon-dated ancient DNA samples; and (ii) importation of a rate estimated using the external cave-brown bear split. The inferred tree topology was identical for both analyses (Figure 3). Internal calibration produced a rate estimate of 0.390 subs/site/Myr (95% HPD: 0.264–0.526 subs/site/Myr), whereas external calibration yielded a slower rate of 0.061 subs/site/Myr (95% HPD: 0.045–0.075 subs/site/Myr), with correspondingly older MRCA age estimates (Table 2). Both of our age estimates for the MRCA of the Western lineage (clade I) were similar to those made by previous studies [3,20]. The PLoS ONE | www.plosone.org 4 February 2008 | Volume 3 | Issue 2 | e1615