|摘要||极度濒危植物西畴含笑（Michelia coriacea Chang et B.L.Chen）仅零星分布于中国云南省东南部的石灰岩山坡、路边等地的常绿杂木林中。野外调查发现，西畴含笑现有的种群个体约300—500株，成年植株开花繁茂，但结实率极低，加之当地人群的干扰，生境极度破碎化，种群自然更新困难，有灭绝的危险。本文首次对西畴含笑的种群结构、传粉生物学特性、胚胎发育、遗传多样性等方面进行综合研究，旨在探讨西畴含笑的致濒因子，为制定该物种的长期有效保护策略提供科学依据。
观察发现西畴含笑开花繁茂，但其自然座果率（6.67%）和结实率（0.2%）都极低。人工授粉实验表明，人工授粉能够极大地提高其座果率（F4, 25 = 35.139, P<0.0001）和结实率（F4, 25 = 85.022, P < 0.0001）。对栽培植株和自然居群植株的访花昆虫的种类和行为观察统计显示，甲虫、地蜂（Andrenidae）和熊蜂（Apidae）可能是西畴含笑的有效传粉者，但这些传粉者的访花频率都不高，其中栽培植株的访花频率为0.27次/小时，自然居群植株仅为0.09次/小时。人工异株异花授粉植株的座果率和结实率都高于人工同株异花授粉的植株（P<0.05），因此可以推测出小居群的西畴含笑在其有性生殖过程中极易产生近交衰退。西畴含笑在自然条件下的有效传粉者的不足及小居群内植株可能发生的近交衰退，应是导致其结实率低的重要因素。
采用ISSR分子标记，对西畴含笑2个居群36单株成年个体的遗传多样性水平和遗传结构进行了检测和分析。结果表明，在物种水平上，多态位点百分（PPB）为93%，Nei’s基因多样性指数（H）为0.264，Shannon’s指数信息指数（I）为0.410；在居群水平上，多态位点百分数（PPB）为76.5%，Nei’s基因多样性指数（H）为0.229，Shannon’s指数信息指数（I）为0.353。西畴居群（XC）拥有较高的遗传变异（PPB=88%, H = 0.255, and I = 0.394），而麻栗坡居（MLP）的遗传变异水平较低（PPB=65%, H = 0.203, and I = 0.311）。西畴含笑居群间的遗传分化系数Gst为0.1197，居群间没有明显的遗传分化，这表明在总的遗传多样性中有88.03%的遗传变异存在于居群内；AMOVA分析显示，几乎所有遗传变异存在于居群内（100.00%, p<0.001）。与木兰科其他植物相比西畴含笑维持了较高的遗传多样性。
西畴含笑现存分布区狭窄，生境被破坏严重，动物对种子破坏严重，幼苗和幼树储备严重不足，使种群更新受到严重阻碍，居群呈快速减少趋势；西畴含笑在大小孢子发生及雌雄配子体发育过程中存在严重败育，有效传粉媒介不足，以及近交衰退的发生是导致西畴含笑生殖失败的主要原因。对西畴含笑的保护迫在眉睫。西畴含笑两居群间极低的遗传分化和较高的遗传多样性，这为西畴含笑的保护提供理论基础。对西畴含笑的保护，要采取就地保护和迁地保护相结合的措施：在就地保护的同时采取人工辅助授粉措施，提高结实率，促进种群的自然恢复。科学选择西畴含笑的母株，开展其人工繁育技术的研究，在此基础上开展该物种迁地保护（植物园或其他栽培场所的活体保存，以及种子和离体培养物的种质库保存等）和再引种，实现种群的人工恢复。; Michelia coriacea，the critically endangered tree endemic to Southeast Yunnan province, China, is individually or as a small patches, scattered on the limestone mountain slopes or secondary shrubby woods and on the roadside. Field investigations revealed that the total M. coriacea is only around 300-500 individuals. The species flowered well, but its fruit and seed set were extremely low. In addition, the habitat has been badly degraded and its populations are isolated each other due to the heavily logging and vegetation destruction in past decade. Undoubtedly, M. coriacea is facing a high risk to be extincted. In order to probe into courses of the extremely low fruit/seed set and its endangerment status, the population ecology, biological characters of pollination, embryo development and the genetic diversity were comprehensively studied.
1. Population structure of M. coriacea
The ecological plots were set up in population Xichou (XC) and Malipo (MLP) and the individual number, size class and age structure of M. coriacea in the populations were investigated. The result showed that the DBH-class and age-class distributions of living individuals of M. coriacea were generally multimodal. Only 53 individuals were found in the 6 plots of 2400 m2. In population MLP (Malipo), the total 33 individuals of the species in 3 plots were counted, which included 6 individuals more than 100 year. In population XC (Xichou) 20 individuals of the species in 3 plots were found and all the individuals were less than 40 years. Although the population structure of M. coriacea in population XC and MLP was different, both of the population structures showed the declining trend.
2. Characters of pollination biology in M. coriacea
Field investigations indicated that M. coriacea in the extant populations can normally bear abundant flowers, but fruit set and fertile seed production per fruit was only 6.67% and 0.2% respectively. Hand pollination could effectively enhance the fruit-set (F4,25=35.139, P<0.0001) and seed set per fruit (F4,25=85.022, P<0.0001). Both cultivated and wild M. coriacea had an extremely low frequency of pollinators, and some beetles, a few species of Andrenidae and some Bombus species are likely to be the effective pollinators. The frequency of floral visitors of both cultivated and wild M. coriacea was low, which were 0.27 times•hour-1 and 0.09 times•hour-1, respectively. The fruit set and seed set per fruit from controlled cross-pollination were much higher than that from controlled self-pollination (p<0.05), and thus it is inferred that in small populations of M. coriacea the inbreed depression might occur in the seed production. It appears that low number of effective pollinators and inbreeding depression could be main reasons to cause the low seed set of M. coriacea in the wild population.
3. Embryological habits of M. coriacea
The sexual development of M. coriacea was investigated by using the paraffin section method. The anther of M. coriacea was tetrasporangiate and its anther wall was composed of 5-7 layers cells, and the tapetum belonged to glandular type. In the meiosis of microsporocyte, cytokinesis was modified simultaneous type. The tetrads were tetrahedral, decussate and sometimes were anomalous type. Mature pollen grain was two-celled. The ovule was anatropous, bitegmic, and crassinucellar. The megaspore mother cell undergone meiotic divisions and developed into a linear type of tetrads. The chalazal megaspore of the tetrad was functional. The functional megaspore underwent three successively nuclear divisions to give rise to eight–nucleate embryo sac which was Polygonum Type. The great abortion could be observed in process of megaspore and microspore genesis and the development of gametophytes. The abnormality in the reproductive development of the species could limit the fruit set and seed production.
4. Genetic diversity and genetic structure of M. coriacea
The genetic diversity and differentiation of two populations with total of 36 M. coriacea individuals were detected with inter-simple sequence repeat (ISSR) DNA markers. A relatively high level of genetic diversity was revealed: PPB=93%, H=0.264, I=0.410 at the species level; and PPB=76.5%, H=0.229, I=0.353 at the population level. The coefficient of genetic differentiation between populations (Gst) was 0.1197, and which indicated that the genetic variation between the populations was lower that among the populations. The result was also confirmed by AMOVA analysis, which showed that almost all the variation (100.00%, p<0.001) occurred within populations. The high genetic diversity and low genetic differentiation might mean that recent human over exploitation lead to this species rare and endangered.
In summary, extant populations of M. coriacea are very fragile. The species is facing a high risk of extinction due to its limited individuals within the extremely fragmentized habitat caused by the human disturbance. The abnormal development of stamen and pistil, lack of pollinators, and inbreeding depression may be main causes of low fruit / seed set. Currently the species still present the relatively higher genetic diversity comparing with other species in Magnoliaceae. To strengthen habitat pretection and to ensure all the existing M. coriacea individuals can be well protected, are the priorities for the species conservation. It may be essential that the measure of hand pollination among individuals is used to improve the fruit/seed set. Meanwhile, the actions of Ex-Situ conservations in the botanic gardens and germplasm bank based on the propagation from the sampled M. coriacea individuals are urgently needed. Further more, the population re-enforcement and restoration of M. coriacea by using the propagated saplings, are certainly needed for the long term preservation of the species.|