Nuclear and chloroplast DNA phylogeography reveal two refuge areas with asymmetrical gene flow in a temperate walnut tree from East Asia.

• Recently, there has been a debate about whether the temperate forests of East Asia merged or fragmented during glacial periods in the Pleistocene. Here, we tested these two opposing views through phylogeographical studies of the temperate-deciduous walnut tree, Juglans mandshurica (Juglandaceae) in northern and northeastern China, as well as Japan and Korea. • We assessed the genetic structure of 33 natural populations using 10 nuclear microsatellite loci and seven chloroplast DNA (cpDNA) fragments. • The cpDNA data showed the complete fixation of two different haplotype lineages in northeastern vs northern populations. This pronounced phylogeographic break was also indicated by nuclear microsatellite data, but there were disparities regarding individual populations. Among those populations fixed for haplotype A (the northeastern group), three were clustered in the northern group and four showed evidence of mixed ancestry based on microsatellite data. • Our results support the hypothesis that two independent refugia were maintained across the range of J. mandshurica in the north of China during the last glacial maximum, contrary to the inference that all temperate forests migrated to the south (25-30°N). The discordance between the patterns revealed by cpDNA and microsatellite data indicate that asymmetrical gene flow has occurred between the two refugia.