AbstractApplying BEAST v1.10.4, we constructed a Bayesian Inference tree comprising 322 taxa, primarily representing Paleoptera (Odonata and Ephemeroptera; Pterygota), Zygentoma and Archaeognatha (Apterygota; paraphyly), and Neoptera (Plecoptera; Pterygota), based on a 2685 bp sequence dataset. Our analyses revealed that robust dating required the incorporation of both Quaternary and pre‐Quaternary dates. To achieve this, our dating incorporated a 1.55 Ma (Quaternary) geological event (the formation of the Ryukyu Islands) and a set of chronologically well‐founded fossil dates, spanning from up to 400 Ma (Devonian) for the stem Archaeognatha, 320 Ma (Carboniferous) for the crown of Paleoptera, 300 Ma (Carboniferous) for the crown Ephemeroptera, and 280 Ma (Permian) for the crown Odonata, down to 1.76 Ma (Quaternary) for Calopteryx japonica, encompassing a total of 22 calibration points (events: 6, fossils: 16; Quaternary: 7, pre‐Quaternary: 15). The resulting dated tree aligns with previous research, albeit with some dates being overestimated. This overestimation was mainly due to the lack of Quaternary calibration and the exclusive dependence on pre‐Quaternary calibration, though the application of maximum age constraints also played a role. Our minimum age dating demonstrates that the molecular clock did not uniformly progress, rendering rate dating an inapplicable approach. We observed that the base substitution rate is time‐dependent, with an exponential increase evident from around 20 Ma (Miocene) to the present time, exceeding an order of magnitude. The extensive radiation and speciation of Insecta and Paleoptera, potentially resulting from the severe climatic changes associated with the Quaternary, including the commencement of glacial and interglacial cycles, may have significantly contributed to this increase in base substitution rates. Additionally, we identified a potential peak in base substitution rates during the Carboniferous period, around 320 million years ago, possibly corresponding to the Late Paleozoic Ice Age.
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