| Summary: | Divergence-time estimation critically improves the understanding of biogeography processes underlying the distribution of species, especially when fossil data is not available. We hypothesise that the Asian black-spined toad, <i>Duttaphrynus melanostictus</i>, expanded into the Eastern Indomalaya following the Quaternary glaciations with the subsequent colonisation of new landscapes during the Last Glacial Maximum. Divergence dating inferred from 364 sequences of mitochondrial <i>tRNAGly ND3</i> supported the emergence of a common ancestor to the three <i>D. melanostictus</i> clades around 1.85 (±0.77) Ma, matching with the Lower to Mid-Pleistocene transition. <i>Duttaphrynus melanostictus</i> then dispersed into Southeast Asia from the central Indo-Pacific and became isolated in the Southern Sundaic and Wallacea regions 1.43 (±0.10) Ma through vicariance as a result of sea level oscillations. The clade on the Southeast Asian mainland then colonised the peninsula from Myanmar to Vietnam and expanded towards Southeastern China at the end of the Mid-Pleistocene Revolution 0.84 (±0.32) Ma. Population dynamics further highlight an expansion of the Southeast Asian mainland population towards Taiwan, the Northeastern edge of the species’ range after the last interglacial, and during the emergence of the Holocene human settlements around 7000 BP. Thus, the current divergence of <i>D. melanostictus</i> into three segregated clades was mostly shaped by Quaternary glaciations, followed by natural dispersion events over land bridges and accelerated by anthropogenic activities.
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