Using a rate of 0.01 subs/site/Myr, only three of the 22 species had phylogroup divergences occurring within the past 250 kyr, whereas three species had Pliocene (.1.8 Myr) ages (Table 1). By applying a higher, revised rate of 0.075 subs/site/Myr, obtained from an internally-calibrated analysis of amakihi subspecies [22] (see Materials and Methods), we found that the evidence shifted significantly towards support for late Pleistocene divergences. Only three species yielded phylogroup divergences exceeding 250 kyr. Our date estimates do not take into account the uncertainty associated with the imported, revised rate, but if the highest rate implied by the upper 95% credibility limit on the original rate estimate [0.111 subs/site/Myr; 22] is applied to the data, then all of the resulting divergence time estimates fall within the past 250 kyr. Our results should be interpreted cautiously because of the dubious applicability of a single substitution rate to different avian species. Collectively, however, our analyses demonstrate that the published molecular evidence used to challenge the LPO hypothesis is weakened by the application of a revised rate. This does not necessarily signify that glacial cycles provided conditions that were exceptionally suitable for allopatric speciation among birds, and it remains unclear whether the tempo of avian speciation in the Pleistocene was actually elevated in comparison Table 1. Divergence time estimates for conspecific phylogroups from 22 bird species Familya Species Genetic Divergence time distanceb estimatec (Myr) Rate = 0.01 Rate = 0.075 subs/site/ subs/site/ Myr Myr Inappropriate Calibration to preceding geological periods [36,39,40]. Nevertheless, the sensitivity of tests of the LPO to the assumed substitution rate emphasises the importance of selecting an appropriate calibration. This concern also applies to similar studies of late Pleistocene divergences among other organisms [49]. Demographic History of the Bowhead Whale The bowhead whale (Balaena mysticetus) was subjected to intensive commercial exploitation from the 17th to early 20th centuries. During this period, over 90,000 individuals were taken from the Spitsbergen stock alone [50]. This stock remains critically endan- gered, while two of the other four designated stocks are endangered. Bowhead whales are exceptionally long-lived, with a maximum longevity well in excess of 100 years, and appear to reach sexual maturity at around 25 years of age [51]. These observations suggest a long generation time, which presents obvious difficulties for population management and stock recovery. For these reasons, the demographic history of this species is of significant ecological, commercial, and conservational interest. An analysis of modern DNA samples from the mitochondrial control region of 98 individuals found that the most recent common ancestor (MRCA) of modern bowhead whales lived around 267 kyr ago [9]. The estimate was calibrated using a value of 3.4 Myr for the age of the split between bowhead whales (Balaena) and right whales (Eubalaena). This calibration point was informed by the fossil record, which is relatively sparse for balaenids; the oldest fossil that can be confidently assigned to either the Balaena or Eubalaena lineage dates from the Pliocene (5.2 to 2.6 Myr BP). Several lines of evidence, however, suggest a more protracted evolutionary history for the two lineages. First, the Pliocene Balaena fossils appear to be highly derived [52]. Second, there is a conspicuous hiatus between the Pliocene fossils and the most ancient fossil representative of Balaenidae, Morenocetus parvus, which dates from the earliest Miocene around 20–23 Myr BP [52]. Independent support for a deeper split between the two genera was provided by analysis of whole mitochondrial genomes, which produced an age estimate of 17 Myr [53]. The palaeontological uncertainty over the age of the bowhead- right whale split, the possible antiquity of the divergence event, and the external nature of this calibration all argue against its use in studying the recent demographic history of bowhead whales. To investigate the effect of calibration choice, we use ancient DNA sequences to infer an internally-calibrated evolutionary time-scale. We reconstructed the demographic history of bowhead whales using Bayesian skyline plot (BSP) analysis, which generates a plot of the estimated effective population size through time [5] (see Materials and Methods). We found evidence of a population expansion in the late Pleistocene (Figure 2). This pattern emerges for three data configurations (modern only, ancient only, and combined modern and ancient), suggesting that it is not an exclusive artefact of the chronologically heterogeneous sampling of ancient DNA sequences. The age of the MRCA of all individuals was estimated at 153 kyr BP, with a 95% highest posterior density (HPD) of 49.6–294 kyr BP. The estimated timings of the MRCA and population expansion of bowhead whales are more recent than those inferred by Rooney et al. [9]. The estimated substitution rate was 0.159 subs/site/Myr (95% HPD: 0.051–0.272 subs/site/Myr). Although this is lower than other rate estimates obtained in some ancient DNA studies [for lists, see 22,54], it is significantly higher than the substitution rates obtained using fossil-based point calibrations of either 3.4 or 17 Myr for the bowhead-right whale split, which yield estimates of 0.017 subs/site/Myr (95% HPD: 0.009–0.0029 subs/site/Myr) and 0.0034 subs/site/Myr (95% HPD: 0.0019–0.0057 subs/site/ Emberizinae Passerella iliaca Paridae Parulinae Certhiidae Turdidae Vireonidae Paridae Parulinae Turdidae Parulinae Mimidae Paridae Poecile gambeli Wilsonia pusilla Polioptila caerulea Catharus guttatus Vireo gilvus Poecile carolinensis Dendroica petechia Catharus ustulatus Geothlypis trichas Toxostoma redivivum Baeolophus inornatus 0.781 Emberizinae Melospiza melodia Sylviidae Chamaea fasciata Tyrannidae Empidonax traillii Paridae Baeolophus ridgwayi Emberizinae Calcarius lapponicus Vireonidae Vireo solitarius Picidae Picoides dorsalis Emberizinae Spizella breweri Fringillidae Carduelis hornemanni 0.551 5.442 5.188 4.008 3.397 3.228 2.900 2.858 2.377 1.420 1.033 0.824 0.708 0.704 0.614 0.558 0.550 0.532 0.457 0.291 Emberizinae Zonotrichia leucophrys 0.231 aAll taxa are members of Order Passeriformes, with the exception of Picidae (Order Piciformes). bGamma-corrected distance estimated by Weir and Schluter [42]. cDivergence times older than 250 kyr are italicised. doi:10.1371/journal.pone.0001615.t001 2.721 2.594 2.004 1.698 1.614 1.450 1.429 1.189 0.710 0.517 0.412 0.390 0.354 0.352 0.307 0.279 0.275 0.275 0.266 0.228 0.146 0.116 0.363 0.346 0.267 0.226 0.215 0.193 0.191 0.158 0.095 0.069 0.055 0.052 0.047 0.047 0.041 0.037 0.037 0.037 0.035 0.030 0.019 0.015 PLoS ONE | www.plosone.org 3 February 2008 | Volume 3 | Issue 2 | e1615