Studies On Phylogeny Of Chimonanthus And Domestication Of Chimonanthus Praecox

Wintersweet (Calycanthaceae, Chimonanthus praecox), widely cultivated in many provinces in china, is a Chinese endemic winter-flowering plant which has a long history of cultivation. In this study, cpDNA sequences and AFLP (Amplified Fragment Length Polymorphism) markers were applied to study the domestication of Ch.praecox firstly. Then phylogenetic trees of Chimonanthus were constructed based on ITS and cpDNA sequences. With the combination of morphology data, we finally discussed the intragenic relationships in Chimonanthus and some taxonomic recommendations were given. There are2main conclusions of our research:1. Phylogeny and species defined of ChimonanthusSamples including species of Chimonanthus and Calycanthus floridus were all surveyed by ITS and cpDNA fragments. The molecular phylogenetic tree of ITS revealed that Chimonanthus were divided into two lineages:Ch.praecox and Ch.campanulatus had obvious relationship and the support rate is very high; Ch.zhejiangensis, Ch.salicifolius, Ch.nitens and Ch.grammatus were clustered together. The latter was also divided into two subgroups:Ch.salicifolius and Ch.grammatus had close relationship, Ch.zhejiangensis and Ch.nitens had close relationship. cpDNA sequence supports the relationship of Ch.praecox and Ch.campanulatus. The united data of ITS and cpDNA established Ch.praecox and Ch.campanulatus are sister group and suggest both species come from a common ancestor, the similar characteristics of flowering, flower color, leaf crumple odorless also showed the close relationship in both species.The analysis of relationships among the populations of Ch. praecox shows WBK, WSLJ and WYCH were formed a sub branch; it revealed those geographically close populations maybe from the same ancestor and gene communication more easily than other groups. CWH and WWX formed another sub branch showed some individuals of CWH were introduced from WWX. The other groups showed parallel branches. Regardless of the populations of the cultivated or wild, it need for further study of population genetics.PCoA analysis of the external morphological characters the show that Ch.zhejiangensis and Ch.nitens overlap and difficult to distinguish. Ch.nitens distribute in Hunan, Fujian, Guangxi, Guizhou and other provinces but Zhejiang province hasn’t distributed, Ch.zhejiangensis only distributed in Longquan, zhejiang province. Combined with the features of morphological, molecular and geographical distribute, we induced Ch.zhejiangensis is the most eastern boundary group and only is the kind of ecological type (ecotype) of Ch.nitens rather than independent taxa. Therefore, we supports Ch.zhejiangensis merge into Ch.nitens, as the synonym of Ch.nitens. Ch.zhejiangensis, Ch.nitens and Ch.grammatus had called by a joint name as the Ch.nitens complex; the small variations may be caused by environmental factors. ITS and morphological data of this paper also showed Ch.grammatus had some differences at the molecular level, as well as leaf size, shape and tepals with Ch.zhejiangensis and Ch.nitens and not support merge into Ch.nitens. It needs to be further study by increasing the gene fragment.2. Domestication of Ch. praecox based on cpDNA sequences and AFLP.cpDNA sequence (psbA-trnH, trnS1-G1and rrnS2-G2) and AFLP(EcoRI-CAA (FAM)/MseI-TC,oRI-TTT (FAM)/Mse-AGG andoRI-CAA (FAM)/MceI-TA) were used in13cultivated populations and8wild populations of Ch. praecox.9polymorphic sites classified into9haplotypes were detected in these three fragments for282individuals of21populations. The whole haplotype network is a star-style topology with haplotype A (highest frequency) as a radiating center, deriving from it are the other eight haplotypes (B, C, D, E, F, G, H and I). There were7haplotypes (77.8%of total) in wild populations and only2(12.5%) harbored haplotype diversity, while6haplotypes were in cultivated populations and8(61.5%) harbored haplotype diversity. Results of haplotype network showed that4haplotypes (A, B, C and D) in cultivated populations shared with wild populations,5derived haplotypes is peculiar to populations,4shared haplotypes found in different wild populations. The wild populations has a high haplotype diversity and nucleotide diversity:/ht=0.819, πT=0.0011; AMOVA analysis showed that the gene flow (Nm) was0.52and there was obvious genetic differentiation in the cultivated populations. The genetic variation of cultivated populations was49.19%among populations and50.81%within the populations. In the wild populations, the genetic variation among populations was78.12%and gene flow (Nm) was only0.14. The cpDNA sequence suggested that most genetic variance occurred among populations in wild populations and in cultivated populations the two statistical numbers were similar. Results of haplotype network showed that Ch. praecox might experience multiple origin events.315bands were amplified in282individuals by3pairs of AFLP markers and309bands were polymorphic (98.41%). The highest genetic diversity occurred in CCQ cultivated population (h=0.1518) and the lowest in EMS cultivated population (h=0.0724).Genetic diversity in wild populations were a little higher than cultivated populations (h in cultivated populations were0.2383; in wild populations were0.2834). AMOVA analysis results keep consistent to cpDNA sequence. In all of populations of Ch. praecox, strong differentiation among21populations was found suggested that genetic variance occurred among populations (cpDNA:0.7478and AFLP:0.6187)。Results of PCoA analysis, Neighbor-joining and UPGMA analysis and Structure calculation revealed that all populations by two different genes pools and formed two intraspecific lineages.8of13cultivated populations with WWX and WLA shared the same gene pool,5cultivated populations of CYS,CJXF,CXT,CFZ and CHF the remaining six wild populations shared the same gene pool. cpDNA and AFLP data suggests Ch.praecox may be multiple origins. One origination center of modern cultivated C. praecox is hypothesized to eastern China where Zhejiang LA is; while southwest China is another origination center including Chongqing Wuxi, Sichuan Wanyuan and Guizhou Guiyang. It consistent with the modern cultivation center of Ch. praecox. Cultivated populations of CJXF, CXT and CHF with some endemic haplotypes located in central China. It worthy of further study to know whether there were wild population existences.The study also reveals Ch. praecox is not experienced obvious genetic bottleneck for founder effect, artificial breeding and clonal propagation in the cultivation process insignificant, and the commercial, cultural intercourse make cultivated populations of Ch. praecox maintain higher genetic diversity.Based on the above analysis, this study suggests the important populations of Ch. praecox should pay attention to in situ conservation, the major cultivated populations should carry out the genetic screening to all members and establish a genetic profile in order to conservation and use some special individuals. The specific haplotype in different populations is an important repository to breeding fine varieties and quality gene and provide the basis for the cultivation and breeding of Ch. praecox.