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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. doichangensis.","jscount":"1","jsurl":"/simple-search?field1=all&field=dc.citation.source_filter&advanced=false&query1=%25E4%25BF%259D%25E6%258A%25A4%25E5%25AF%25B9%25E7%25AD%2596&&fq=dc.project.title_filter%3ATrigonobalanus%5C+doichangensis%5C+is%5C+an%5C+endangered%5C+plant.%5C+In%5C+this%5C+paper%2C%5C+the%5C+megasporogenesis%5C+and%5C+development%5C+of%5C+female%5C+gametophyte%2C%5C+seed%5C+morphological%5C+traits%5C+and%5C+seed%5C+germination%2C%5C+seed%5C+conservation%2C%5C+micropropagation%5C+and%5C+acclimatization%5C+of%5C+this%5C+species%5C+were%5C+studied.%5C+Combined%5C+with%5C+the%5C+published%5C+results%5C+of%5C+cytology%2C%5C+molecular%5C+genetics%5C+and%5C+other%5C+researches%2Cthe%5C+mechanisms%5C+of%5C+extinction%2C%5C+basic%5C+biology%5C+and%5C+technology%5C+of%5C+germplasm%5C+conservation%5C+and%5C+acclimatization%5C+of%5C+T.%5C+doichangensis%5C+were%5C+discussed.%5C+The%5C+main%5C+results%5C+are%5C+summarized%5C+as%5C+follows%5C%3A1.%5C+Megasporogenesis%5C+and%5C+development%5C+of%5C+female%5C+gametophyte%EF%BC%8CStamens%5C+exist%5C+under%5C+the%5C+stigma%5C+of%5C+T.%5C+doichangensis%2C%5C+and%5C+the%5C+pollen%5C+is%5C+aborted%5C+on%5C+the%5C+later%5C+development%5C+stage%5C+of%5C+pistil%2C%5C+therefore%2C%5C+the%5C+pistillate%5C+flower%5C+in%5C+function%5C+is%5C+hermaphrodite%5C+flower%5C+in%5C+morphology.%5C+The%5C+ovule%5C+is%5C+anatropous%2C%5C+bitegmic%5C+and%5C+crassinucellate.%5C+The%5C+primary%5C+archesporium%5C+is%5C+hypodermal%5C+and%5C+single%5C-celled%5C+and%5C+the%5C+sporogenous%5C+cell%5C+of%5C+the%5C+nucellus%5C+functions%5C+directly%5C+as%5C+a%5C+megaspore%5C+mother%5C+cell%5C+which%5C+goes%5C+meiosis%5C+to%5C+form%5C+a%5C+linear%5C+tetrad.%5C+The%5C+chalazal%5C+megaspore%5C+of%5C+the%5C+tetrad%5C+is%5C+functional.%5C+The%5C+development%5C+of%5C+embryo%5C+sac%5C+conforms%5C+to%5C+the%5C+polygonum%5C+type.%5C+There%5C+are%5C+six%5C+ovules%5C+in%5C+the%5C+ovary%5C+of%5C+T.%5C+doichangensis%2C%5C+and%5C+only%5C+one%5C+develops%5C+into%5C+a%5C+seed%5C+in%5C+normal%5C+fruits.%5C+In%5C+the%5C+process%5C+of%5C+megasporogenesis%5C+and%5C+development%5C+of%5C+female%5C+gametophyte%2C%5C+there%5C+are%5C+several%5C+links%5C+of%5C+abortion%2C%5C+and%5C+93.3%25%5C+of%5C+mature%5C+embryo%5C+sacs%5C+is%5C+aborted.2.%5C+Morphological%5C+characters%5C+and%5C+germination%5C+of%5C+seeds%EF%BC%8CMost%5C+of%5C+the%5C+variation%5C+occurred%5C+among%5C+individual%5C+trees%5C+within%5C+populations%5C+in%5C+seed%5C+morphological%5C+traits%5C+%5C%28length%2C%5C+width%5C+and%5C+1000%5C-seed%5C+weight%5C%29%5C+and%5C+germination%5C-related%5C+indices%5C+%5C%28germination%5C+percentage%2C%5C+germination%5C+index%5C+and%5C+vigor%5C+index%5C%29.%5C+In%5C+addition%2C%5C+the%5C+variation%5C+in%5C+percentage%5C+of%5C+well%5C-developed%5C+seeds%5C+among%5C+populations%5C+and%5C+among%5C+individual%5C+trees%5C+within%5C+populations%5C+is%5C+equal%2C%5C+each%5C+accounting%5C+for%5C+48%25.%5C+Each%5C+of%5C+seed%5C+morphological%5C+traits%5C+has%5C+significantly%5C+positive%5C+correlation%5C+with%5C+each%5C+other%5C+%5C%28p%5C+%3C%5C+0.01%5C%29%2C%5C+but%5C+they%5C+have%5C+no%5C+significant%5C+correlation%5C+with%5C+percentage%5C+of%5C+well%5C-developed%5C+seeds%5C+and%5C+germination%5C-related%5C+indices.%5C+In%5C+the%5C+same%5C+batch%5C+of%5C+seeds%5C+of%5C+T.%5C+doichangensis%2C%5C+there%5C+are%5C+light%5C-colored%5C+and%5C+dark%5C-colored%5C+seed%5C+coats%2C%5C+and%5C+development%5C+of%5C+light%5C-colored%5C+seeds%5C+is%5C+significantly%5C+poorer%5C+than%5C+that%5C+of%5C+dark%5C-colored%5C+seeds.The%5C+sensitivity%5C+of%5C+seeds%5C+to%5C+high%5C+temperature%5C+varys%5C+in%5C+different%5C+stages%5C+of%5C+seed%5C+imbibition.%5C+In%5C+each%5C+stage%2C%5C+heat%5C+acclimatization%5C+don%E2%80%99t%5C+increase%5C+germination%5C+percentage%2C%5C+germination%5C+index%5C+and%5C+fresh%5C+weight%5C+of%5C+seedlings.%5C+If%5C+the%5C+distilled%5C+water%5C+is%5C+substituted%5C+by%5C+solution%5C+of%5C+SA%5C+during%5C+seed%5C+imbibition%2C%5C+seed%5C+germination%5C+and%5C+germination%5C+index%5C+after%5C+heat%5C+shock%5C+are%5C+not%5C+significantly%5C+different%5C+from%5C+control%2C%5C+but%5C+they%5C+are%5C+significantly%5C+higher%5C+than%5C+that%5C+of%5C+other%5C+treatments.%5C+Moreover%2C%5C+when%5C+the%5C+seeds%5C+are%5C+treatmented%5C+with%5C+SA%2C%5C+the%5C+fresh%5C+weight%5C+of%5C+seedlings%5C+is%5C+significantly%5C+higher%5C+than%5C+that%5C+of%5C+control%5C+and%5C+other%5C+treatments.3.%5C+Seed%5C+conservation%EF%BC%8CSeeds%5C+of%5C+T.%5C+doichangensis%5C+belong%5C+to%5C+orthodox%5C+seeds%5C+which%5C+can%5C+tolerate%5C+certain%5C+level%5C+of%5C+dehydration.%5C+The%5C+condition%5C+of%5C+low%5C+temperature%5C+and%5C+low%5C+water%5C+content%5C+of%5C+seeds%5C+is%5C+conducive%5C+to%5C+seed%5C+conservation.Germination%5C+of%5C+fresh%5C+seeds%5C+shows%5C+significant%5C+variation%5C+among%5C+populations%2C%5C+howerer%2C%5C+germination%5C+of%5C+the%5C+seeds%5C+after%5C+storage%5C+for%5C+one%5C+year%5C+in%5C+room%5C+temperatur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pingbianensis J. L. Huang & X. Z. Liu, is a newly described perennial herb narrowly distributed in South-east Yunnan, China. It belongs to genera Tupistra Ker Gawler(Liliaceae). It usually occurs on outcrops of bare rock, or occasionally as an epiphyte on tree trunks covered with humus and moss. T. pingbianensis is unusual in that it exhibits rarity according to three different ways of measuring rarity, i.e. it has a small geographical range, is a habitat specialist, and always has low abundance where it occurs. Because of this, T. pingbianensis has been listed as an endangered species and catalogued in the Chinese Species Red List. In order to discuss the causes of rarity of T. pingbianensis, the multidisciplinary investigations of the seed and seedling establishment, cytology, breeding system, and population genetic structure of the endangered T. pingbianensis were performed in this thesis. Besides, the corresponding conservation strategies were also proposed according to the above-mentioned. The main results are summarized as follows:1. Biological traits of T. pingbianensis,T. pingbianensis is a perennial herbaceous with a creeping rhizome, thick basal leaves, and an inflorescence that is a terminal spike. Florescence is from November to December, while fruiting occurs between November and December in the next year. Reproduction and spread also occurs clonally via rhizomes, most seeds simply fall from the mother plant and germinate where they land. It occurs on outcrops of bare rock, or occasionally as an epiphyte on tree trunks covered with humus and moss, which are naturally rare habitat. Throughout its small geographical range, T. pingbianensis occurs as discrete, small populations size. 2. Seed germination traits of T. pingbianensis,Seed morphology was observed and effects of substrates soil types, light, sowing depth on germination percentage of the species T. pingbianensis were investigated primarily. The results showed that the average seed size was (1.17±0.02) cm × (0.79±0.01) cm × (0.77±0.01) cm (length × width × thickness), per-hundred-seed-weight was about 35.03±0.12g. Among the three different substrates soil types and sowing depths, seeds of T. pingbianensis germinate best in alkalescence soil and shallow sowing depth (2cm). It could germinate in the both light and dark, but the germination rate can be accelerated by light obviously. Its seed has high germination rate not just in greenhouse, but also in the field. We considered that this is a good strategy to expand its population in the special habit.3. Karyotype evolution status of T. pingbianensis,The karyotype of total eight species in Campylandra, Tupistra and Aspidistra from China were reported. Considering Tupistra has the similar morphological character with Campylandra but resemble Aspidistra in karyotype. The results support the earlier study that Tupistra is a transition between Compylandra and Aspidistra. Besides, our results also showes that the T. pingbianensis and T. fungilliformis has higher karyotype asymmetry than other species in this genera, which means these species have higher karyotype evolution status. 4. Reproduction ecology of T. pingbianensis, The flower phenology, pollinators of T. pingbianensis were documented herein. We also examined the breeding system of T. pingbianensis and seed fitness traits to determine what forms of pollination and mating occur in this naturally rare species, and is there evidence of inbreeding depression in its populations. The results shows that the flowers opened 10-15 days, which suggest stigma and pollen can keep high vitality for a long time (10-15 days). The only pollinators observed on T. pingbianensis flowers were ants (Aphaenogaster smythiesii Forel,Formicidea), springtail (Hypogastrura sp., Hypogastruridae, Collembola) and one species of beetles (Anomala corpulenta Motsch, Rutelidae). These pollinators generally have restricted movement capacities and hence promote geitonogamy or mating between individuals in close proximity within populations. The results of out crossing index (OCI) pollination experiments in our study suggest that T. pingbianensis has an animal-pollinated, mixed selfing and outcrossing breeding systems. However, a pollination experiment also fail to detect significant inbreeding depression upon F1 fruit set, seed weight and germinate rate fitness-traits. Since naturally rare species T. pingbianensis is not seriously genetically impoverished and likely to have adapted to tolerating a high level of inbreeding early in its history. 5. Conservation genetic of T. pingbianensis, The levels and partitioning of genetic diversity were investigated in Tupistra pingbianensis. Here genetic diversity and patterns of genetic variation within and among 11 populations were analyzed using AFLP markers with 97 individuals across its whole geographical range. High levels of genetic variation were revealed both at the species level (P99 = 96.012%; Ht = 0.302) and at the population level (P99 = 51.41%; Hs = 0.224). Strong genetic differentiation among populations was also detected (FST = 0.2961; ⍬Ⅱ= 0.281), which corresponded to results reported for typical animal-pollinated, mixed selfing and outcrossing plant species. Special habitat and its life history traits may play an important role in shaping the genetic diversity and the genetic structure of this species. Based on the special habitat in T. pingbianensis, the most suitable strategy for its conservation is the protection of its habitat. Moreover, given the observed strong genetic differentiation among populations of T. pingbianensis, the preservation of genetic diversity in this species will require the protection of many populations as possible to maintain the current levels of genetic 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a town of Shangri-la County, Diqing Prefecture, was chosen as the main field site for studying the structure and characters of traditional agricultural ecosystem, by using approaches of ethnobotany, cultural anthropology and ecology. Combined with interviewing exercises in Hanpi village, Jiantang Township, this paper also discussed the impact of traditional management on the biocultural diversity. The results showed: Traditional agroecosystem in Shangri-la is an integrated system with three subsystems, which are farming, forest and grazing subsystem. The seasonal shifting grazing activity in Shangri-la, following the natural season change and the recover process of plants, is a sustainable management that protects the local biodiversity. However, along with the decay of shifting grazing tradition recently, the local Tibetans turned to use grass land and forest which is close to villages as the main grazing lands. It increased the pasturing pressure to these areas and caused productivity decreasing and biodiversity. As a symbolic part of Tibetan culture in Shangri-la, the sacred mountain culture has played a significant role in biodiversity conservation by restricting human’s behavior. The Tibetan traditional culture, indigenous knowledge and traditional ecosystem management in Shangri-la has contributed to the biodiversity conservation in this area. However, this research indicated that under the pressure of mainstream culture and market economy, traditional knowledge is vanishing; old crop land races are decreasing; diverse land use management is inclining to be single and seasonal shifting grazing tradition is fading away. The change of diversity to singularity might cause some negative impacts on the local environment and ecosystem. In this paper, advices were also given on how to combine Tibetan traditional knowledge and management experiences into sustainable development of modern agriculture. In this thesis, genetic diversity of Musella lasiocarpa (Franch.) C. Y. Wu ex H. W. Li, a plant endemic to southwest China, was also discussed through the approach of SSR markers. The wild populations of M. lasiocarpa are very rare now due to the habitat fragment and long time human’s disturbance. By conducting broad field investigation, we have found 5 wild populations near the boarder of Yunnan and Sichuan province. Seventeen microsatellite markers were isolated from M. lasiocarpa by using FIASCO method. 8 primers were selected to do the further genetic population structure and genetic diversity analysis. The results showed that genetic diversity of M. lasiocarpa’s wild populations is higher than cultivated populations. The genetic diversity difference between wild and cultivated populations is related to the different reproduction systems. Adopting the way of asexuality reproduction, the genetic basis of cultivated populations become narrow that decrease the genetic diversity. AMOVA analysis showed that 37.19% genetic differentiation is among populations and 62.81% is within population. Genetic differentiation among different populations is related to the limited gene communication. POPGENE analysis indicated that there is very little gene flow among different populations (0.4916), which is the main reason of high genetic differentiation among M. lasiocarpa 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