Aim: Understanding the phylogeography of a species complex can provide important insights into its evolutionary history. However, phylogeographic inference often faces the dilemma of regionally inadequate sampling. Pungitius sticklebacks are a case in point: although the highest species diversity is found in Northeast Asia, their phylogeography in this region is still poorly understood.

Location: Northeast Asia

Methods: With the aid of whole genome resequencing data, we investigated the phylogeography of Northeast Asian Pungitius sticklebacks, with newly sampled 83 world-wide Pungitius individuals from 11 locations including eight Chinese locations reported to host only P. sinensis.

Results: We discovered that three of these locations hosted populations of P. kaibarae and P. bussei, species new to the fauna of China. Phylogeographic analyses further clarified the sequence and timing of colonization of Northeast Asia by different Pungitius species, shedding new light on their origins and current distribution ranges. Colonization of inland Northeast Asia by Pungitius sticklebacks occurred in multiple waves, and the widespread P. sinensis expanded its range relatively late in the Pleistocene.

Main Conclusions: This study complements our understanding of the phylogeography of Pungitius sticklebacks by extending sampling to cover an area that comprises nearly half of the known distribution area of this genus in Northeast Asia. The discovery of three Pungitus species from China is of particular interest, as translocations to support locally declining populations have occurred under the assumption that all sticklebacks in China – except the endangered P. stenurus – are P. sinensis, and draws attention to local introduction for their conservation.