| Summary: | Ancient trees are famous for their life spans of hundreds or even thousands of years. These trees are rare, a testament to history and are important for scientific research. <i>Platycladus orientalis</i>, with the longest life span and a beautiful trunk, has become the most widely planted tree species and is believed to be sacred in China. Extensive declines in habitat area and quality pose the greatest threats to the loss of genetic diversity of ancient <i>P. orientalis</i> trees in the middle reaches of the Yellow River. Strengthening the protection of <i>P. orientalis</i> genetic resources is of great significance for the long-term development of reasonable conservation and breeding strategies. To better understand the genetic diversity and population structure of <i>P. orientalis</i>, we successfully analyzed four polymorphic chloroplast simple sequence repeat (cpSSR) loci and applied them to diversity and population structure analyses of 202 individuals from 13 populations in the middle reaches of the Yellow River. Based on the cpSSR data, 16 alleles were detected across 202 individuals, and a moderate level of genetic diversity was inferred from the genetic diversity parameters (<i>H</i> = 0.367 and <i>A</i><sub>R</sub> = 1.964). The mean pairwise genetic differentiation coefficient (<i>F</i>st) between populations was 0.153, indicating relatively high genetic population differentiations. Analysis of molecular variance (AMOVA) showed that only 8% of the variation occurred among populations. Structure analysis divided the 13 <i>P. orientalis</i> populations into two groups with no significant geographic population structure, which was consistent with the unweighted pair group method with arithmetic mean (UPGMA) and Mantel test results. These results may indicate that transplanting and cultivation by ancient human activities are the main factors responsible for the revealed pattern of genetic differentiation of ancient <i>P. orientalis</i> populations. Our research is of great significance for the future establishment of protection schemes and scientific breeding of <i>P. orientalis</i>.
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