| Atractylodes DC (Asteraceae) is a small genus endemic to East Asia. Species in the genus are perennial herbs, and the dried rhizome of them (except A. carlinoides) have been used as the famous Traditional Chinese Medicine (TCM)—Atractylodes Macrocephala Rhizoma (common name:"Baizhu" in Chinese and "Byakujutsu" in Japanese) and Atractylodes Lancea Rhizoma (common name:"Cangzhu" in Chinese and "So-jutsu" in Japanese). A. macrocephala is one species in the genus Atractylodes, which has been widely used in traditional herbal remedies in East Asia, and has a long and widespread cultivation history in China. Nowadays, with the standardization of TCM, domestication of medicinal herbs and molecular authentication become more and more important. In this study, we first reconstructed the phylogeny of the genus Atractylodes based on four chloroplast intergenic regions and nuclear ribosomal DNA ITS and18S-ETS data; and then studied the population genetics and domestication process of A. macrocephala through sequencing chloroplast DNA fragments and genotyping eight nuclear microsatellite markers. We studied the resolution capability of different DNA barcodes on Atractylodes, and evaluated the function of them for revealing species boundaries. Futhermore, studies on molecular identification of A. macrocephala germplasm were also conducted based on chloroplast DNA and nrITS.The main findings are as follows:1) Phylogeny of Atractylodes.Through a wide sampling of species of Atractylodes, we obtained five taxa, one subspecies, and one cultivated variety from China, Korea and Japan to represent the genus. Based on four chloroplast intergenic regions(trnL-trnF, ndhJ-trnF, trnD-psbM and psbE-petL) and nuclear ribosomal DNA ITS and18S-ETS, we reconstructed the molecular phylogenetic tree of Atractylodes using maximum parsimony, maximum likelihood and bayesian inference methods. The results showed that A. carlinoides and A. macrocephala were monophyletic based on both data sets. The phylogenetic tree of the genus was better resolved with the nuclear ribosomal markers compared to the chloroplast markers. We attributed this pattern to incomplete lineage sorting of ancestral polymorphism of A. japonica, A. lancea and A. coreana, as well as relatively lower mutation rate of chloroplast genome, thus suggested that nuclear gene markers such as ITS and18S-ETS might be more suitable in resolving phylogenetic relationships of the genus. The nuclear ribosomal phylogeny showed that A. lancea formed two well-supported clades matching the geographic distribution pattern, while A. coreana was not monophyletic and belonged to the northern clade of A. lancea. Samples of A. japonica clustered into one clade and showed a close relationship with the northern clade of A. lancea.’Pingzhu’variety belonged to the clade of A. macrocephala and formed a well-supported clade together.2) Isolation of polymorphic microsatellite markers from the Chinese medicinal herb A. macrocephala.In this study,15polymorphic microsatellite markers were developed from A. macrocephala using the compound microsatellite marker technique. Levels of polymorphism within the15markers were assessed using83individuals from two wild and two cultivated populations in China. The number of alleles per locus ranged from2to20, with an average of9.9alleles. Observed and expected heterozygosities ranged from0.083to1.000and from0.097to0.938, respectively. These markers will be valuable for germplasm classification and identification, as well as for assessing the genetic diversity and spatial genetic structure among wild and cultivated populations of A. macrocephala.3) Population genetics and domestication of A.macrocephala.We used two cpDNA regions (trnL-trnF and psbE-petL) and eight microsatellite markers to investigate genetic diversity and population genetic structure of A. macrocephala. Genetic diversity in cultivated populations was higher than that of wild populations based on SSR makers, which might due to human-mediated population expansion and gene flow of wild germplasm, and rapid declines of wild populations. Moreover, the differentiation in wild populations was larger than that of cultivated populations. For cpDNA data, genetic variance occurred among populations were52.85%in wild populations, while3.12%occurred in cultivated populations; for SSR data, genetic variance occurred among populations were52.85%in wild populations, while4.56%occurred in cultivated populations.3groups were detected for466samples of A. macrocephala based on the results of STRUCTURE and UPGMA analysis of SSR data. The cultivated populations belonged to2groups, which indicated that the cultivated A. macrocephala might be multiple-origin. The major traditional production area—Zhejiang, might originated from central or western China based on our data; while the other one—at the junction regions of Jiangxi, Hunan and Hubei, might be local origin. We concluded that Tiantai, Zhejiang and Sangzhi, Hunan to be the earliest provenance of cultivated A. macrocephala based on the analysis of contemporary gene flow using BAYESASS.4) Genetic evidence for geo-authentic A. macrocephala of Zhejiang Province and special germplasm from wild populations.Cultivated populations from Zhejiang (C6, C7, C16, and C17-introduced from Zhejiang) occurred in the same cluster, which could be the genetic evidence for geo-authentic A. macrocephala from Zhejiang. However, due to habitat deterioration and over-exploitation, the wild germplasm of A. macrocehala have suffered rapid declines. Wild populations with high genetic diversity, especially the population from Qimen, Anhui (W1), may contain special genes that are very important for genetic improvement of cultivated A. macrocephala.5) Research on DNA barcoding of Atractylodes and molecular identification of A. macrocephala germplasm.DNA barcoding is a new molecular technique developed recently in which species identification was performed using DNA sequences from a small fragment of the genome, thus might overcome many defects on traditional classification. In this study,20individuals representing5species (A. carlinoids, A. macrocephala, A. lancea, A. chinensis, A. japonica) and1variant (A. chinensis var. coreand) were used to assess the effectiveness and universality of four barcode makers (rbcL, matK, psbA-trnH, and nrITS) in Atractylodes. Wilcoxon signed rank tests were used to detect the intra-and inter-specific differences, and the results showed that ITS>> psbA-trnH>> matK>>rbcL for inter-specific level and ITS=psbA-trnH>>matK>> rbcL for intra-specific level. Analysis of NJ, UPGMA, MP and ML all showed that single ITS could discriminate all the5species, while failed to identify A. chinensis and A. chinensis var. coreana even increasing sequences on it. Finally, we recommended single ITS to identify the species of Atractylodes considering costs and efficiency. Moreover, we studied the molecolar identify of the representative germplasm of A. macrocephala based on chloroplast DNA(rbcL, matK, psbA-trnH, trnL-trnF and psbE-petL) and nrITS. The results showed that significant differentiation occurred between cultivated populations and wild population from Qimen, Anhui, while no obvious differentiation were detected within cultivated A. macrocephala due to human-mediated gene flow.6) Some taxonomic revisions were proposed for Atractylodes.We proposed to give some revisions within Atractylodes by combing molecular phylogenetic results, DNA barcoding analysis, morphological characters and geographic distribution pattern.(1) Samples of A. lancea from northern China, which formed a monophyletic clade based on combined nuclear ribosomal DNA data, could be treated as A. chinensis (Bunge) Koidz., while A. lancea only included the southern clade;(2) A. coreana was embedded in the northern clade of A. lancea, and might be an ecological type of it, then could be treated as A. chinensis (Bunge) Koidz. var. coreana.(3) Combining molecular data, morphological data and practical application, we recommended the so called’Pingzhu’ to be a variety of A. macrocephal(A. macrocephala’Pingzhu’).(4) A. japonica which is distributed in Northeast China, Japan and Korea, and was sister group to A. chinensis based on our molecular data, was a distinct species with a pattern of allopatric speciation process. |
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