Common walnut (Juglans regia L.) is an economically important hardwood tree species cultivated worldwide for its high quality wood and edible nuts. It is generally accepted that after the last glaciation J. regia survived and grew in almost completely isolated stands in Asia, and that ancient humans dispersed walnuts across Asia and into new habitats via trade and cultural expansion. The history of common walnut in China is a matter of debate, however. We estimated the genetic diversity and spatial genetic structure of 31 walnut populations sampled across its Chinese range using 22 microsatellite markers (13 neutral and 9 non-neutral). Using historical data and population genetic analysis, including approximate Bayesian analysis (ABC), we reconstructed the demographic history of J. regia in China. The genetic data indicated the likely presence of J. regia in glacial refugia in the Xinjiang province (Northwest China), Northeastern China (Beijing, Shandong, and Changbai Mountains), Central China (Qinling and Baishan Mountains and Xi’an), and Southwestern China (Tibet, Yunnan, Guizhou, and Sichuan provinces). Based on DIY-ABC analysis, we identified three ancient lineages of J. regia in China. Two lineages (subpopulation A and subpopulation B+C) diverged about 2.79 Mya, while Southwestern China, and Qinling and Baishan Mountains lineages diverged during the Quaternary glaciations (about 1.13 Mya). Remnants of these once-distinct genetic clusters of J. regia may warrant ecological management if they are to be retained as in situ resources. A population size expansion in Northeastern China was detected in the last five centuries. The present distribution of walnut in China resulted from the combined effects of expansion/contraction from multiple refugia after the Last Glacial Maximum and later human exploitation.

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The common walnut (Juglans regia L.) and iron walnut (J. sigillata Dode) are well-known economically important species cultivated for their edible nuts, high-quality wood, and medicinal properties and display a sympatric distribution in southwestern China. However, detailed research on the genetic diversity and introgression of these two closely related walnut species, especially in southwestern China, are lacking. In this study, we analyzed a total of 506 individuals from 28 populations of J. regia and J. sigillata using 25 EST-SSR markers to determine if their gene introgression was related to sympatric distribution. In addition, we compared the genetic diversity estimates between them. Our results indicated that all J. regia populations possess slightly higher genetic diversity than J. sigillata populations. The Geostatistical IDW technique (HO, PPL, NA and PrA) revealed that northern Yunnan and Guizhou provinces had high genetic diversity for J. regia while the northwestern Yunnan province had high genetic diversity for J. sigillata. AMOVA analysis revealed that significant genetic variation was mainly distributed within population as 73% in J. regia and 76% in J. sigillata. The genetic differentiation (FST) was 0.307 between the two walnut species (p < 0.0001), which was higher than FST values within populations (J. regia FST = 0.265 and J. sigillata FST = 0.236). However, the STRUCTURE analysis of the J. regia and J. sigillata populations revealed two genetic clusters in which gene introgression exists, therefore, the boundary of separation between these two walnut species is not clear. Moreover, these results were validated by NJ and UPGMA analysis with additional conformation from the PCoA. Based on the SSR data, our results indicate that J. sigillata is an ecotype of J. regia. Taken together, these results reveal novel information on population genetics and provide specific geographical regions containing high genetic diversity of the Juglans species sampled, which will assist in future conservation management.