| Phellodendron amurense Rupr. (Rutaceae) is a tertiary paleotropical flora relict plant, endangered medicinal plant and important woody plant in China, which has important scientific, medicinal and economic values. The wild populations of P. amurense have declined gradually and are difficult to recover due to defeorestation and excessive harvesting. It is very urgent to protect the species diversity of P. amurense.This study was committed to research on survival status, age structure, genetic diversity, genetic structure, species evolution and the populations dynamic history of P. amurense, as well as to establish a method to trace the crude herbs of Phellodendri Cortex, based on sampling and survey over the entire distribution area of P. amurense. We aimed to provide a theoretical basis and technological methods for biodiversity conservation of P. amurense.We carried out the survey of the survive status of wild populations of P. amurense. We found that the distribution area of P. amurense is about 2300 hectares, which has declined 19.3% comparing with documentary records and it mainly distributed in Changbai mountain, Zhangguangcai mountain and the southern of Xiaoxiang’an mountain in China at present. The consumption of resource was mainly by medicine use. The wild populations showed a trend of decline that the young individuals was 15.2% only and the adult individuals accounted for 68.5%. P. amurense could be divided into three types based on the morphological character by inhabitant in the distribution.By using ISSR (inter-simple sequence repeat) molecular marker, we assessed the genetic diversity within and among 17 wild populations of P. amurense. The results revealed a low level of genetic diversity between and among populations. At species level, the effective number of alleles per locus (Na) was 2.0000, the effective number of alleles per locus (Ne) was 1.4271, Nei’s gene diversity (H) was 0.2620, and Shannon’s information index (I) was 0.4078, respectively. At population level, the four genetic indexs were lower than those of species level. The coefficient of genetic differentiation (Gst) was 0.2731 indicated that the 27.31 percentage of genetic variation only exist among populations. The gene flow among populations (Nm) was very low as 1.3308. UPGMA cluster analysis showed that there was no correlation between genetic variance and habitat differences. Each distributed area should get equal attention when we take measures to protect P. amurense.Phylogeographical analyses were carried out to study the species differentied and populations dynamic history. The results revealed that significant genetic differentiation and lineage geographical structure (Nst> Gst, P< 0.05) exist in P. amurense and P. chinense, as well as each populations of P. amurense. The haplotype analysis showed P. amurense owned the original haplotype of Phellodendron, with all haplotypes of P. chinense evolved from P. amurense. The area of southern of Changbai mountain and Yanshan were likely the refuge for P. amurense in the Quaternary Ice Age.We selected ITS and psbA-trnH sequence as DNA barcoding candidate genes for distinguishing Phellodendron species. It showed that psbA-trnH and YTS+psbA-trnH sequence were more efficacy than ITS sequences in identification of Phellodendron amurense species from other Phellodendron species. Moreover, three SNP sites could trace the three original plants of the crude Phellodendri Cortex accurately, which were consists of 173rd bp of ITS sequence,206th bp and 397th bp of psbA-trnH sequence. By simulating sampling, we found that at least 1-2 samples from 12 populations are required to represent of the genetic diversity of P. amurense. |
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