Inappropriate Calibration Figure 3. Maximum clade credibility tree from Bayesian analysis of mitochondrial control region sequences from 56 modern and 51 ancient brown bear samples, with a time scale calibrated using the radiocarbon dates of the ancient sequences. Major clades and their geographic localities are given. Posterior probabilities are given for the nodes A-N, which are referred to in the text and in Table 2. doi:10.1371/journal.pone.0001615.g003 internally-calibrated date estimate suggests an MRCA during the previous (Weichselian) glacial period, but the 95% HPD of the externally-calibrated estimate encompasses several glacial and interglacial periods, prohibiting a detailed correlation with environmental change. The evolutionary time-frame of the Eastern European clade, which makes up part of clade IIIa, is less clear. It is interesting that there is no distinct subdivision between European and Alaskan brown bears in clade IIIa, which could be interpreted as evidence for recent expansion into Alaska and Europe from a single source population, although further sampling of late Pleistocene brown bears in Europe and Asia will be required to test this hypothesis. Brown bears are believed to have established the first populations in North America around 50–75 kyr BP, during the early and middle parts of the last (Wisconsinan; equivalent to the Weichselian in Europe) glaciation [66,67]. Age estimates for clades II, III, and IV (Table 2) therefore support previous claims that the modern lineages were established prior to the initial colonization of North America [57,61], most probably during the previous interglacial/glacial transition. Conversely, the MRCA of bears from the Alaskan ABC islands (clade IIa, node C) existed around 16 kyr BP (95% HPD: 10–26 kyr BP), consistent with the timing of the most recent glacial/interglacial transition. The age of the MRCA of polar bears (node D) is estimated at 43 kyr BP (95% HPD 19–73 kyr BP), and the divergence between polar bears and ABC islands bears (node F) is estimated to have occurred about 58 kyr BP (95% HPD: 48–72 kyr BP). Despite the internal calibration with a multitude of dated tips, the 95% HPD intervals for these estimates are wide, indicating only that polar bears diverged from other brown bears some time after the warmest part of the Wisconsinan glacial period, but lacking the power to identify any specific environmental event or geographic location. This example illustrates the impact of inappropriate calibration on inferences made from molecular data. It also demonstrates that it can be difficult to correlate demographic data inferred from phylogenies with large-scale environmental fluctuations, due to the considerable statistical uncertainty that is often associated with intraspecific estimates of divergence times. It is possible to reduce this uncertainty by increasing sample size and alignment length, but the most powerful approach might involve the use of molecular data in conjunction with alternative methods, such as radiocarbon dating to find terminal occurrences. PLoS ONE | www.plosone.org 5 February 2008 | Volume 3 | Issue 2 | e1615