Studies On Domestication&Phylogeography Of Scrophularia Ningpoensis Hemsley And Phylogeny Of Eastern Asian Scrophidaria

Scrophularia ningpoensis Hemsley, known as one of the famous Traditional Chinese Medicine (TCM)--"Zhebawei", used in the Chinese Materia Medica (CMM) belonging to the family Scrophulariaceae, has a long history of widespread use in China. Root of this medicinal herb is used to treat inflammation, laryngitis, tonsillitis, abscesses of carbuncles, constipation and this species is widely cultivated in Zhejiang, Sichuan, Hubei Provinces and so on. Nowadays, with the standardization of TCM, domestication of medicinal herbs and molecular authentication become more and more important. In this study, firstly, we used the ISSR (Inter-Simple Sequence Repeats) molecular markers to detect the genetic diversity and population genetic structure in cultivated S. ningpoensis. Further more, one pair of SCAR (Sequence Characterized Amplified Region) primers was developed to identify S. ningpoensis originated from Zhejiang Province. Secondly, cpDNA sequences and AFLP (Amplified Fragment Length Polymorphism) markers were applied to study the domestication of S. ningpoensis. Thirdly, phylogenetic trees of East-Asian Scrophularia were constructed based on ITS and cpDNA sequences. Finally, the Phylogeography of S. ningpoensis species complex was studied by cpDNA sequences and AFLP molecular markers. There are4main conclusions of our research:1. Genetic diversity and population genetic structure of cultivated S. ningpoensis and the development of SCAR markersTwelve ISSR universal primers were applied in8cultivated population and2wild populations of S. ningpoensis, which revealed that two wild populations (TM1W, TWW) harbored higher polymorphic percentage (48.39%,35.48%) than cultivated populations (9.68%-20.97%). In cultivated populations, the polymorphic percentage of population YC and PA were the lowest. AMOVA analysis suggested that genetic variance occurred among populations is76.67%and that occurred within population is23.33%, which resulted from differentiation in populations. UPGMA dendrogram for ten populations of S. ningpoensis based on Nei's genetic distance revealed that cultivated populations grouped in one clade, within which populations from Pan'an County were closer than others.Based on ISSR analysis, primer UBC874provided an approximately1500bp band unique to populations originated from Zhejiang Province. After gel purified, cloned and sequenced, this DNA fragment turned out to be1306bp. A pair of22bp SCAR primers (CC874u and CC874d) was designed for the amplification of this DNA fragment. All samples from different regions were amplified by SCAR primers CC874u and CC874d and PCR products show that a single band about1000bp was only in accessions originated from Zhejiang Province, which proved that CC874u and CC874d are useful for identifying S. ningpoensis originated from Zhejiang Province.2. Domestication of S. ningpoensiscpDNA (psbA-trbH and trnL-F) and AFLP molecular markers were used in14cultivated populations and14wild populations of S. ningpoensis.27polymorphic sites classified into22haplotypes were detected by cpDNA. There were only4haplotypes in cultivated popoulations and every population only harbored single haplotype:the haplotype diversity is0.486and nucleotide diversity is0.002; whereas21haplotypes were in wild populations:haplotype diversity is0.919, nucleotide diversity is0.003. Results of haplotype network showed that four haplotypes in cultivated populations shared with many wild populations were located in different clades, which suggested that S. ningpoensis might experience multiple origin events and LSW wild population in Jiangxi Province probably was involved in the origin of cultivated S. ningpoensis in Zhejiang Province.Two hundreds eighty nine bands were amplified in315individuals by6pairs of AFLP markers and261bands were polymorphic (90.31%). Genetic diversity in wild populations were higher than cultivated populations (h in cultivated populations were from0.0076to0.0875; in wild populations were from0.0791to0.1614). As to the percentage of polymorphic bands, in wild populations were from22.15%to50.87%, in the cultivated were from3.11%to27.68%. Moreover, the differentiation in cultivated populations was larger than which in the wild:in cultivated populations, genetic variance occurred among populations were76.97%, genetic variance occurred in populations were23.03%; in the wild, those two statistical numbers were39.60%and60.40%respectively.Results of PCoA,Neighbor-Joining and UPGMA analysis were consistent, which showed that all cultivated populations grouped together with two wild populations: HNW and LSW, located in Hunan and Jiangxi Provinces respectively. This clarified that the origin of cultivated S. ningpoensis were Hunan and Jiangxi Province, precisely, the HNW and LSW wild populations. That point contrary to current belief that Zhejiang Province was involved in the origin of cultivated S. ningpoensis. Moreover, cultivated populations from Zhejiang (PAC, XJC, GZC—introduced from Zhejiang) occurred in same cluster represented genetic identity, which can be the genetic evidence for geo-authentic S. ningpoensis from Zhejiang. Due to habitat deterioration and over exploitation, the wild genetic resources of S. ningpoensis have suffered rapid declines. But some native populations (LSW, JHW etc.) have high genetic diversity may contain special genes that are very important for the plant's growth and use.3. Phylogeny of East Asian ScrophulariaSamples including21species of Scrophularia in China mainland,5species in Japan,3species in South Korea and7species in America-Europe were all surveyed by ITS and cpDNA fragments (trnQ-rps16, psbA-trnH, trnL-F), combined with sequences of15North American species which were download from Genebank. The molecular phylogenetic tree revealed that Scrophularia in East Asian were divided into four lineages:S. ningpoensis species complex including S. ningpoensis, S. buergeriana, S. kakudensis and S. yoshimurae; diploid clade with Sect. Tomiphyllum; the rest Scrophularia species distributed in China; South Korea-Japan-North America clade. S. ningpoensis, S. buergeriana, S. kakudensis and S. yoshimurae form a monophyletic clade with high bootstrap support and shared with very similar morphological characters can be treated as S. ningpoensis species complex. Our study also threw light on the origin of East Asian Scrophularia:Species in South Korea and Japan might be derived from North America; Species in China might be from Europe-Central Asian and then evolved into two lineages. Based on relaxed clock model in BEAST analysis, time dating was estimated:four lineages in East Asian formed at52.12mya; S. ningpoensis species complex rose at26mya in the Miocene epoch; other Scrophularia species in China were evolved at41.25mya in the Eocene epoch, the same time as diploid clade; South Korea and Japan groups were migrated from North America at10.7-7.25mya in the Pliocene epoch. 4. Phylogeography of S. ningpoensis species complexSamples of S. ningpoensis species complex comprises28populations of S. ningpoensis,7populations of S. buergeriana,6populations of S. kakudensis and one population of S. yoshimurae. cpDNA fragments (psbA-trbH and trn"L-F) combined AFLP molecular markers were used for all the42populations.Thirty four polymorphic sites and27haplotypes were detected in538individuals by cpDNA sequences. Two haplotypes were share by S. ningpoensis and S. buergeriana; one haplotype was share by S. buergeriana and S. kakudensis; S. yoshimurae only contained one haplotype which was share with S. ningpoensis. The haplotype shared by S. ningpoensis and S. buergeriana (population in Lingyuan, Liaoning) located in the center of the haplotype network. By Coalescence Theory, the haplotype in the center of the network can be the candidate of ancestors. Considered with chromosome numbers in Scrophularia (S. buergeriana:2n=30; S. ningpoensis:2n=90; S. kakudensis:2n=36), we indicated that LYW population of S. buergeriana might be the ancestor of S. ningpoensis species complex. Two wild populations LSW (Jiangxi Province) and TM2W (Mt. Tianmu) harbored five and six cpDNA haplotypes respectively, and contain S10and S6rare haplotypes. So we indicated the glacial refugia for S. ningpoensis were Jiangxi and Mt. Tianmu.Results of AFLP analysis showed the Nei's genetic diversity in S. ningpoensis species complex were:S. ningpoensis was0.2202, S. buergeriana was0.1543, S, kakudensis was0.1512and S. yoshimurae was0.0685. Hierarchical analysis of molecular variance revealed that genetic variance occurred among species were32.62%; genetic variances occurred among populations in species were38.10%and that within populations were29.28%. Results of PCoA, Neighbor-Joining and STRUCTURE analysis all clarified the differentiation among three species:S. ningpoensis, S. buergeriana and kakudensis. So we confirmed the taxonomic status of these three species. But results from cpDNA sequences suggested that these three species must from the same ancestor. S. ningpoensis species complex might rise in early Miocene epoch when Weather changed from dry to warm and humid. At that time,s. buergeriana (wild population in Liaoning, China--LYW) considered as diploid migrated towards east to form S. kakudensis, and migrated towards south to form S. ningpoensis and S. yoshimurae.S. yoshimurae and S. ningpoensis grouped together and came from the same gene pool. So these two species might be synonym and we suggested that S. yoshimurae can be treated as a subspecies of S. ningpoensis Hemsley.