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We suggest that using a calibration point allowed BEAST to estimate a more realistic clock rate.
The split between the Sardinian clade and its mainland sister Bathysciola zariquieyi was used for estimating the evolutionary rate of cox1, using a calibration point of 33 MY ago as the time of vicariant separation of both lineages (Fig. 1).
By also using a calibration point set at 12 My for the Mus/Rattus split, other studies suggested younger (5.1 to 5.2 Mya: Suzuki et al. [ 18]) or similar (6.8 to 7.8 Mya: Chevret et al. [ 20]; 7.6 ± 1.1 Mya: Veyrunes et al. [ 21]) timing for the initial divergence of subgenera within the genus Mus (inclusive of Nannomys).
On the one hand, the calibration in Zane et al. [ 16] refers to previous reports [ 5, 95] using a calibration point of "perciform radiation" at 60 Mya, which is highly tentative, since we actually don't know what a perciform is [ 96].
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This number is used as a calibration point for molecular clock dating using BEAST.
Thus, 91 Myr was used as a calibration point for dataset A. Secondary calibration (calibrating a node with a date provided by a previous analysis) is a commonly used alternative given the absence of a method for direct calibration (e.g. fossil or geological [ 61- 63]).
For each analysis (row), bold text indicates the node(s) used as a calibration point.
The oldest European anchovy fossil is 10 million years old [ 49], thus this date was used as a calibration point.
Previously estimated divergence time between domestic horse and Somali wild ass of 2.0 Ma was used as a calibration point (Forstén 1992).
The separation of mosquitoes and Drosophila at 227 Ma (95% confidence interval 210 244 Ma), as suggested by a recent study (Wheat and Wahlberg 2013), was used as a calibration point.
However, when the archaeological date for the colonization of the Canary Islands was used as a calibration point in a U6 Bayesian phylogenetic analysis based also on complete sequences, the U6b1a age estimation was 4.8 (2,9-7.1 2,9-7.19].
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