Phylogeography, Phylogeny And Genetic Diversity Of Chimonanthus (Calycanthaceae)

Chimonanthus Lindl. is a perennial shrub endemic to China with important ornamental and medicinal values, belongs to the family Calycanthaceae. The main distribution of wild populations is limited in the subtropical hills and valleys of the south of Qinling Mountains, the west of Yunnan-Guizhou Plateau, and the north of the Nanling Mountains regions. The present distributions of it are vulnerable to disturbance. The molecular clock estimates suggest that relict Ch. of the Neogene (late Tertiary) diverged from Calycanthus in the early Pliocene. The current genetic structure and geographical distribution were influenced significantly by the climate of Quaternary glacial.Our present work investigated the genetic structure, phylogeography and phlogeny of Ch. including25populations from China. The analysis was carried out based on nuclear sequence data from the internal transcribed specer (ITS) region of the ribosomal DNA and chloroplast (cp) DNA data from trnL-F, trnS-G, and trnH-psbA intergenic spacer, located in the large single-copy region of the cp genome. Using of the ancient geological, paleoclimatic data and revegetation information of the last glacial period, the inference was made that the possible glacial refuge of Ch. plants in the Quaternary Ice Age. In additional, the genetic structure and genetic diversity were estimated based on inter-simple sequence repeat (ISSR) markers from Ch. praecox and Ch. salicifolius. Combined with results of the cpDNA sequence data, we proposed an appropriate protection strategy for different populations of different species. The main results were listed as follows:1)25populations (264individuals) were analyzed using cpDNA trnL-F、trnS-G、and trnH-psbA intergenic spacer, and56variable sites were detected, which produced40haplotypes. Analysis of cpDNA haplotypes from different populations suggested that two phylogeographical groups existed a double star-like relationship in this genus. Populations from Dabashan, Wushan, Wulingshan Mountains, mainly distributed in the Central China to southwest, had a unique ancestral haplotypes (H3). Areas around Lushan, Tianmushan, Wuyishan, Xuefengshan mountains, mainly distributed in the East China, had another unique ancestral haplotypes (H3).All other private haplotypes from different populations originated from the two ancestral haplotypes. Accoding to the analysis of the molecular genetic variation of different populations, combined with the geological history events, we speculated that the present populations are evolved independently in their respective refuge, i.e. their present distribution areas. Neutrality test and mismatch distribution neither support any significant postglacial population expansion.2) Chose trnL-F genes of chloroplast genome and ITS sequences of nuclear genome, we adopted maximum likelihood, maximum parsimony and neighbor joining method to resolve the systematic problems of6species and1variety of this genus. In the end, we combined both data of trnL-F and ITS to explain the relationships among these species. Ch. praecox and Ch. campanulatus var. guizhouensis diverged from this genus firstly grouped into one branch (BS>81%). And leftover of this genus grouped into another main branch including five species, such as Ch. salicifolius, Ch. grammatus, Ch. nitens, Ch. zhejiangensis, and Ch. campanulatus. Ch. campanulatus var guizhouensis determined previously as a variety of Ch. campanulatus by the flexible morphological and closed geographical characteristics. Inconsistent results with previous showed a far relationship in molecular evidence between them. Our results supported (BS>87%) that Ch. zhejiangensis was treated as a synonyms of Ch. salicifolius.3)According to cpDNA data, haplotype diversity (h=0.90500±0.01200) and nucleotide diversity (π=0.00339±0.00013) of the genus is relatively high. In order to estimate the genetic diversity of populations from two ancestral haplotypes, two species from17populations were chose based on ISSR. For both species, the genetic diversity was low at the species level(Ch.praecox:PPB=32.49%, h=0.0968,I=0.1488; Ch. salicifolius:PPB=34.25%, h=0.1192,I=0.1777), but relatively higher at the species level (Ch. praecox:PPB=86.03%, h=0.1552,I=0.0.2635; Ch. salicifolius:PPB=81.25%, h=0.2401,I=0.3684). The hierarchical AMOVA revealed high levels of among-population genetic differentiation in both species, in line with the gene differentiation coefficient and the limited among-population gene flow (Ch. praecox:ΦST=0.4650, GST=0.3784, Nm=0.8213; Ch. salicifolius:ΦST=0.5912, GST=0.5036, Nm=0.4929). Both neighbor-joining (NJ) cluster analysis and Principal Coordinate Analysis (PCoA) clustered all17populations into two major groups, which corresponded to the two separate species. An isolation-by-distance pattern was revealed in Ch. salicifolius (r=0.703, P=0.048,999permutations), but not in Ch. praecox (r=0.168, P=0.185,999permutations). For the respect of description of the richness of the species genetic diversity, results from cpDNA and ISSR data are consistent. 4) Maternal inheritance of cpDNA and nuclear gene ISSR markers showed a significant differentiation among these populations. In order to protect the genetic diversity of these plants distributed in these refuge areas, we should use in situ conservation strategies. Highlights include the following populations:SMF, BKH, JSJ, LAI, XNS of Ch. praecx; SYC and GFD of Ch. salicifolius; GZG of Ch. zhejiangensis; ZYM and YSK of Ch. nitens; HCO of Ch. grammatus; LQN of Ch. campanulatus. If need an ex situ conservation strategy in emergency, sampling method should be in the light of the characteristics of above-metioned populations, also should include XSZ of Jiangxi Province, ALU and XYT of Guizhou Province, especially.