The Effect of Inappropriate Calibration: Three Case Studies in Molecular Ecology

Abstract

Time-scales estimated from sequence data play an important role in molecular ecology. They can be used to draw correlations between evolutionary and palaeoclimatic events, to measure the tempo of speciation, and to study the demographic history of an endangered species. In all of these studies, it is paramount to have accurate estimates of time-scales and substitution rates. Molecular ecological studies typically focus on intraspecific data that have evolved on genealogical scales, but often these studies inappropriately employ deep fossil calibrations or canonical substitution rates (e.g., 1% per million years for birds and mammals) for calibrating estimates of divergence times. These approaches can yield misleading estimates of molecular time-scales, with significant impacts on subsequent evolutionary and ecological inferences. We illustrate this calibration problem using three case studies: avian speciation in the late Pleistocene, the demographic history of bowhead whales, and the Pleistocene biogeography of brown bears. For each data set, we compare the date estimates that are obtained using internal and external calibration points. In all three cases, the conclusions are significantly altered by the application of revised, internally-calibrated substitution rates. Collectively, the results emphasise the importance of judicious selection of calibrations for analyses of recent evolutionary events.

Comparing Evolutionary Patterns and Variability in the Mitochondrial Control Region and Cytochrome b in Three Species of Baleen Whales

“…Using a control region rate of 5.4% per Myr brings Ne estimates calculated from the control region into excellent agreement with those calculated from multiple nuclear introns in gray whales (Alter et al 2007). In addition, this rate falls within the 95% highest posterior density rate estimates for bowhead whales based on Bayesian analysis of ancient and modern DNA samples (Ho et al 2007(Ho et al , 2008. However, without an equivalent nuclear dataset for the other whale species, we cannot yet generalize about the reliability of control region rates calculated using a cytochrome b clock across all baleen whales.…”

The origins of the enigmatic Falkland Islands wolf

“…The accuracy of molecular divergence date estimates is directly related to the quality of calibration data, and their proximity to the date of interest. In this regard, the use of deep fossil calibration points has been shown to be inappropriate for intra-specific and recent inter-specific divergences 18,19 , such as those within Dusicyon. The radiocarbon dates associated with the ancient sequences of D. avus provide much closer calibration points to the late Pleistocene events under consideration than the early canid fossils used in Slater et al 7 , and importantly do not suffer from the same uncertainty over taxonomic identification and phylogenetic position.…”

“…S2). The combined morphological and molecular analysis recovered one most parsimonious tree of 3782.017 steps information at the tip of the tree to facilitate molecular dating 19 ( Supplementary Fig. S5).…”

“…To examine the problems caused by the temporal dependency of molecular dates, we performed molecular dating analyses using either internal (radiocarbon), or external (fossil), dates. The advantages of using internal calibrations to study recent evolutionary events are well established 18,19 , and given a divergence event thought to be late Pleistocene (natural dispersal) or Holocene (human dispersal), the internal radiocarbon dates for D. avus appear far more appropriate than external fossil dates of 4-32 Ma (with largely unknown error margins) and uncertain taxonomic/ phylogenetic position.…”

“…However, even if this date estimate was approximately accurate, the colonization event could have been considerably older or younger than this depending on the demographic history, any subsequent lineage extinctions 16 , and the relationship between the FIW and unsampled mainland relatives 17 . Furthermore, the use of external fossil calibrations to date recent events is known to be problematic due to the temporal dependency of molecular rates 18,19 . Either way, while the estimated 330 ka date and wide error margins (70-640 ka) clearly predates human arrival, it provided little evidence as to how the FIW might have colonized the islands.…”