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资助项目
GST, p < 0.01). At the regional level, Chinese and Japanese L. hodgsonii had a similar estimate of genetic diversity (China: Hd = 0.847, HT = 0.869; Japan: Hd = 0.766, HT = 0.867). Populations from China and Japan possess unique sets of haplotypes, and no haplotypes were shared between the regions. Furthermore, both the phyloegenetic and network analyses recovered the haplotypes of China and Japan as two distinct clades. Thus, we suggested the disjunct distribution of L. hodgsonii in China and Japan may present the climatic vicariant relicts of the ancient widely distributed populations. After divergence, this species within each region experienced independent evolutionary process. In China, L. hodgsonii was distributed around the Sichuan Basin. This distribution range can be divided into five regions. They were Jiajin Mountain region, E’mei Mountain region, Yunnan-Guizhou Plateau region, Wushan-Wuling Mountain region and Qinling Mountain region. Twelve haplotypes were indentified within these regions. Each region had its own specific haplotypes, which had different ancestry in the network. We deduced that Chinese L. hodgsonii might survive the LGM in multiple isolated refugia around the Sichuan Basin. In Japan, L. hodgsonii was disjunctively distributed in northern Honshu and Hokkaido. Seven haplotypes were identified within this region. However, the genetic diversity in Honshu (Hd = 0.821) was much higher than that in Hokkaido (Hd = 0.513). And all haplotypes in Hokkaido were derived from Honshu. This haplotype distribution suggested that the northern Honshu could have served as refuge in Japan. Nested clade analysis (NCA) indicated multiple forces including the vicariance and long-distance dispersal affected the disjunctive distribution among populations of L. hodgsonii in Japan.2. The phylogeography of L. tongolensis，Ligularia tongolensis was distributed along the Jinshajiang watershed, Yalongjiang watershed and Wumeng Mountain. In order to deduce the demographic history of this species, we sequenced two chloroplast DNA (cpDNA) intergenic spacers (trnQ-5’rps16, trnL-rpl32) in 140 individuals from 14 populations of three groups (Jinshajiang vs. Yalongjiang vs. Wumeng) within this species range. High levels of haplotype diversity (Hd = 0.814) and total genetic diversity (HT = 0.862) were detected at the species level, based on a total oftwelve haplotypes identified. However, the intrapopulation diversity (HS = 0.349) was low, which led to the high levels of genetic divergence (GST = 0.595, NST = 0.614, FST = 0.597). In consideration of the speciation of L. tongolensis resulting from the uplifts of the Qinghai-Tibetan Plateau (QTP), we thought the present genetic structure of L. tongolensis was shaped by the fragmentation of ancestral populations during the courses of QTP uplifts. This was further supported by the absence of IBD tests (r = –0.291, p = 0.964), which suggest that the differentiation had not occurred in accordance with the isolation by distance model. The genetic differentiation in L. tongolensis appears to be associated with historical events. Meanwhile, H2 and H5, the dominant haplotypes that located on internal nodes and deviated from extinct ancestral haplotype in the network, were detected to be shared between Jinshajiang and Yalongjiang groups. We deduced that ancestral populations of this species might have had a continuous distribution range, which was then fragmented and isolated by the following tectonic events. Finally, the ancestral polymorphism, H2 and H5, were randomly allocated in Jinshajiang watershed and Yalongjiang watershed. Meanwhile, H5 was the dominant haplotype in Jinshajiang watershed; H7 was the domiant haplotype in Yalongjiang watershed and Wumeng Mountain. This haplotype distribution pattern indicated that each group might have served as a refuge for L. tongolensis during the Quaternary Glaciation. Postglacial demographic expansion was supported by unimodal mismatch distribution and star-like phylogenies, with expansion ages of 274 ka B. P. for this 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doichangensis is an endangered plant. In this paper, the megasporogenesis and development of female gametophyte, seed morphological traits and seed germination, seed conservation, micropropagation and acclimatization of this species were studied. Combined with the published results of cytology, molecular genetics and other researches,the mechanisms of extinction, basic biology and technology of germplasm conservation and acclimatization of T. doichangensis were discussed. The main results are summarized as follows:1. Megasporogenesis and development of female gametophyte，Stamens exist under the stigma of T. doichangensis, and the pollen is aborted on the later development stage of pistil, therefore, the pistillate flower in function is hermaphrodite flower in morphology. The ovule is anatropous, bitegmic and crassinucellate. The primary archesporium is hypodermal and single-celled and the sporogenous cell of the nucellus functions directly as a megaspore mother cell which goes meiosis to form a linear tetrad. The chalazal megaspore of the tetrad is functional. The development of embryo sac conforms to the polygonum type. There are six ovules in the ovary of T. doichangensis, and only one develops into a seed in normal fruits. In the process of megasporogenesis and development of female gametophyte, there are several links of abortion, and 93.3% of mature embryo sacs is aborted.2. Morphological characters and germination of seeds，Most of the variation occurred among individual trees within populations in seed morphological traits (length, width and 1000-seed weight) and germination-related indices (germination percentage, germination index and vigor index). In addition, the variation in percentage of well-developed seeds among populations and among individual trees within populations is equal, each accounting for 48%. Each of seed morphological traits has significantly positive correlation with each other (p < 0.01), but they have no significant correlation with percentage of well-developed seeds and germination-related indices. In the same batch of seeds of T. doichangensis, there are light-colored and dark-colored seed coats, and development of light-colored seeds is significantly poorer than that of dark-colored seeds.The sensitivity of seeds to high temperature varys in different stages of seed imbibition. In each stage, heat acclimatization don’t increase germination percentage, germination index and fresh weight of seedlings. If the distilled water is substituted by solution of SA during seed imbibition, seed germination and germination index after heat shock are not significantly different from control, but they are significantly higher than that of other treatments. Moreover, when the seeds are treatmented with SA, the fresh weight of seedlings is significantly higher than that of control and other treatments.3. Seed conservation，Seeds of T. doichangensis belong to orthodox seeds which can tolerate certain level of dehydration. The condition of low temperature and low water content of seeds is conducive to seed conservation.Germination of fresh seeds shows significant variation among populations, howerer, germination of the seeds after storage for one year in room temperature shows no significant variation among populations.High temperature and high relative humidity damages the seeds more severely than high temperature does. In addition, low water content of seeds enable the seeds to be more tolerant to high temperature.The electrical conductivity, dehydrogenase activity and germination percentage have no significant correlation with each other.4. Micropropagation and in vitro conservation，Cotyledonary nodes are a kind of efficient explants. Low salt media are conducive to shoot propagation and root induction.The maximum multiplication rate (20-25 shoots/explant within 4 months) is achieved on quarter-strength Murashige and Skoog (1/4 MS) medium supplemented with 1 mg·L-1 6-benzyladenine (6-BA) and 0.05 mg·L-1 α-naphthaleneacetic acid (NAA).Rooting is promoted by auxins, however, IBA alone or low concentrations of NAA are preferable due to small amount of callus induced. The research has established an efficient protocol for micropropagation of T. doichangensis, and it provides technology support for in vitro conservation of special germplasm of the species.5. Acclimatization，Quercus variabilis, Cyclobalanopsis glaucoides and T. doichangensis belong to the family of Fagaceae, and the natural distribution ranges of the 3 species are decreasing in turn. The research suggests that the ranges of temperature tolerance of the 3 species are decreasing corresponding to their distribution ranges.The high and low semi-lethal temperature of one-year old T. doichangensis is 49.5℃ and -5℃ respectively. It suggests that T. doichangensis has a wide range of basic temperature tolerance. Short-term heat and cold acclimatization cannot expand the range of temperature tolerance. It can be inferred that T. doichangensis may lack induced tolerance to temperature. Under proper conditions, ABA can increase the cold tolerance, and SA can increase the heat tolerance of leaf discs of T. 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