Table 2. Bayesian age estimates for nodes labelled in Figure 3 Node Clade Age estimate (kyr) External calibration Internal calibration Mean 95% HPD Mean 95% HPD A B C D E F G H I J K L M N I II IIa IIb III IIIb IIIc IV IIa and IIb IIb and IIc IIIa and IIIb IIIa; modern only 415 415 72 208 319 112 525 374 362 380 260 458 198–646 209–659 17–149 55–398 115–548 50–197 307–793 89–741 67–735 168–620 147–394 167–755 49–323 74 94 16 43 51 58 35 32 65 86 75 31 111 45–109 62–133 10–26 19–73 26–85 48–72 79–148 11–75 9–74 36–110 67–108 40–130 7–59 IV; modern US only 164 All brown bears 1,159 745–1,622 211 143–295 doi:10.1371/journal.pone.0001615.t002 Concluding Remarks Our three case studies demonstrate that the findings of molecular ecological studies can be altered significantly by the choice of calibration. Our results show that divergence time estimates can change by an order of magnitude when relatively recent, internal calibration points are used (Table 3). Deep calibration points, particularly those based on the fossil record, lead to inferred evolutionary scenarios that are drastically different from those implied by internal, intraspecific calibrations. The large disparity between estimates obtained by internal and external calibration also highlights the importance of judicious selection of calibrations for recent evolutionary events, even if one does not subscribe to the hypothesis of time-dependent rate estimates [68,69]. Intraspecific calibrations can be obtained in one of several ways. As illustrated in the bowhead whale and brown bear examples above, the radiocarbon ages of ancient samples can be used as Table 3. Summary of estimates made using internal and external calibrations for the three data sets presented in this study Evolutionary event Age estimate (kyr) Common ancestor of bowhead whales External calibration = 3.4 Myr External calibration = 17 Myr Common ancestor of brown bears 1,159 Mean divergence time of conspecific avian phylogroups aRange of 22 divergence time estimates doi:10.1371/journal.pone.0001615.t003 External calibration Internal calibration Mean 95% HPD Mean 95% HPD 1,140 4,123 878 497–1,880 2,071–6,386 745–1,622 116–2,721a 154 211 117 50–295 143–295 15–363a Inappropriate Calibration calibrations on the tips of the tree. Alternatively, internal calibrations can be obtained from biogeography [70,71], although such calibrations can be difficult to interpret and specify correctly [12,68]. The fossil record will rarely be able to offer intraspecific calibrations unless substantial diagnostic variation exists within a species, for example among subspecies or conspecific populations. Frequently, however, intraspecific calibration data are unavailable for many data sets, leaving the less desirable option of importing a substitution rate estimated from another (preferably related) species. A considerable disadvantage of intraspecific calibrations is that they will often produce date estimates with a larger degree of uncertainty than those made using external calibrations. There are two reasons for this: (i) external calibrations are often placed at the root of the tree; and (ii) the removal of outgroup species reduces the information in the data set. Moreover, molecular date estimates can come with substantial uncertainty, so that it can be difficult to draw strong conclusions from molecular date estimates if the associated estimation error is taken into account. Therefore, a reduction in precision appears to be the consequence of using intraspecific calibrations, but the improvement in accuracy should come as a worthwhile recompense. Materials and Methods Avian Speciation in the Late Pleistocene In order to test the hypothesis of late Pleistocene origins for birds, we obtained a set of pairwise genetic distances estimated from avian mitochondrial cytochrome b by Weir and Schluter [42]. Of these genetic distances, we restrict our re-analysis to the 22 measured between conspecific phylogroups from species with a geographic range extending above 40uN, the approximate boundary of Pleistocene glaciation in North America [72] (Table 1). To convert the distance estimates to time durations, two different substitution rates were applied. The first rate was 0.01 subs/site/Myr, which was used in the original study and corresponds to the canonical 1% rate in birds. The second rate was 0.075 subs/site/Myr, based on an analysis of cytochrome b sequences from two subspecies of amakihi from Hawaii and Maui [22]; this is one of the few internally-calibrated rate estimates available for avian cytochrome b. Demographic History of the Bowhead Whale To investigate the demographic history of modern bowhead whale populations, we assembled a data set comprising both ancient and modern DNA sequences. Ancient DNA (aDNA) sequences (ages from ranging from 30 years to 51 kyr) from the mitochondrial control region of 99 bowhead whales were obtained from the study by Borge et al. [73]. Modern DNA sequences for 68 haplotypes were obtained from the study by Rooney et al. [9]. Demographic history was inferred from these data using Bayesian skyline plot [BSP; 5] analyses, performed using BEAST 1.4 [74] with the substitution model selected by comparison of Akaike Information Criterion values. Three BSP analyses were per- formed: (i) aDNA sequences only; (ii) modern DNA sequences only; and (iii) ancient and modern DNA sequences combined. For all three analyses, 10 group sizes were used for the BSP. Posterior distributions of parameters were investigated using Markov chain Monte Carlo (MCMC) analysis, with samples from the posterior drawn every 10,000 steps over a total of 50,000,000 steps, with the first 10% discarded as burn-in. In the two analyses using aDNA sequences, time-scales were calibrated using the radiocarbon dates of the 99 ancient samples. The analysis of modern DNA sequences was calibrated by specifying a normally-distributed prior on the substitution rate (mean 0.149 subs/site/Myr, standard deviation 0.052 subs/site/ PLoS ONE | www.plosone.org 6 February 2008 | Volume 3 | Issue 2 | e1615