【作者】 权秋梅；

【导师】 黎云祥； 吴卫；

【作者基本信息】 四川农业大学 ， 药用植物学， 2011， 博士

【摘要】 本文通过定位观测、显微观察、室内测定等研究方法,以三个生境中的巫山淫羊藿(Epimedium wushanense)种群为研究对象,对巫山淫羊藿的传粉生态学进行了研究,其主要结果如下：1.巫山淫羊藿花期为3月初到4月中下旬,种群开花一般要历时22～27d；单株花期约12～17d；单花花期一般为3～4d。三个种群中个体水平的开花振幅呈单峰型曲线,均具有较高的开花同步指数,表现出一种集中开花的模式。座果数与开花高峰期、终花期、花期持续时间和花蕾数均呈极为显著的正相关关系,但座果数与始花期无相关关系。开花物候指数均值以及变异系数分析表明：三个种群中花期持续时间、单花持续时间、终花期、开花高峰期和座果数差异极其显著；总花数、开花振幅和结实率差异显著；开花同步指数和始花期无显著差异性。三个种群中除同步性指数外,其余指数的变异系数均有差异。此外巫山淫羊藿结实率受到距长短的影响。因而巫山淫羊藿的开花物候和生殖特征受到微环境影响,同时生殖特征还受到花部特征距的影响。2.巫山淫羊藿存在三种花部特征：即雌蕊长度等于或者短于雄蕊(PESS)、雌蕊长于雄蕊(PLS)、雌蕊长于雄蕊并发生偏离雄蕊的弯曲生长(SAA),三种异位方式分别在种群中占54%、23.5%和22.5%。其中具有PLS和SAA特征的巫山淫羊藿结实率无差异,但是两者的结实率均显著高于PESS。巫山淫羊藿属于雄蕊先熟,两性器官同时具有可授性重叠期为1d。开花时花粉活力在40%～90%左右,能维持约3～4d；其柱头可授时间可以持续4～5d。由于两性器官的空间异位,可授性为雄蕊先熟,花序为无限花序,因此巫山淫羊藿昆虫自下而上的访花方式和雄蕊先熟可以降低由于同株异花授粉而造成的自交败育。巫山淫羊藿杂交指数为5,花粉-胚珠比等于7102.34±365.77,结合人工套袋和授粉实验结果可以确定巫山淫羊藿的繁育系统属于专性异交为主,传粉过程需要传粉者。由于巫山淫羊藿的繁育系统为自交不亲和,因此在花部特征中PLS和SAA有利于促进和提高巫山淫羊藿的生殖成功。野外套袋实验表明巫山淫羊藿自然传粉的结实率要显著低于人工异花授粉。因而花部综合特征强烈的影响着传粉者,从而影响着巫山淫羊藿的生殖成功。3.在巫山淫羊藿具有12种访花者,分别隶属于膜翅目和双翅目,其中有两种熊蜂为巫山淫羊藿有效传粉者,分别以觅食花粉和盗取花蜜为主要回报物。访花者的来访频率最高的时间是一天中的13：00-14：00。巫山淫羊藿在不同开花天数的花蜜量随着开花时间增加而上升,其中开花第二天花蜜分泌量显著增加。套袋的花蜜体积日变化呈上升趋势,在13：30以后要显著高于13：30以前的花蜜体积。由于受到传粉者觅食影响,未套袋的花蜜体积日变化呈下降趋势,传粉者觅食时间主要集中在13：00-15：30。花蜜含糖量在一天内的变化呈单峰趋势,在13：30时花蜜的糖含量达到最大值。因而巫山淫羊藿的花蜜分泌和糖量的变化是通过影响有效传粉者的取食行为,从而影响该植物的生殖成功。诱物实验表明传粉者的主要诱物为产生花蜜的距,缺失距会影响传粉者的访问,显著影响巫山淫羊藿的生殖成功。4.对不同生境中巫山淫羊藿结实特性比较研究表明：(1)不同花部特征的植株在三个种群中繁殖投资差异不大,如：三个生境中的每株分枝数、每株花序数和每株开花数差异均不显著。(2)不同花部特征在三个生境中每株结实数、每株结实率、每花序结实数和每花序结实率存在显著差异,其中PLS和SAA要显著大于PESS。(3)在不同生境中,三种花部特征的单株分枝数和单株花序数均无显著差异。由于种群1开花数量相对较少,因而在结实方面均值均要小于种群2和种群3。(4)不同花部特征的果实饱满种子数、败育数和败育率均具有显著差异,但在不同生境间均无显著的差异。说明种子数更多地受到花部特征的影响。(5)在PLS和SAA花部特征的种子长和宽均要显著大于PESS,但种子数无显著差异。表明不同花部特征对巫山淫羊藿的种子质量存在着显著影响。总之,巫山淫羊藿的结实特性受到生境和花部特征的双重影响,其中花型比生境对巫山淫羊藿结实特性的影响更为明显。5.开花的位置效应对巫山淫羊藿的生殖成功具有显著影响,其中基部和中部的果实形态参数、种子形态参数和种子数均要显著高于顶部；在败育率中,基部和中部的败育较少,而顶部的种子败育率最高,因而顶部的果实重量要显著低于基部和中部。在开花时间效应对巫山淫羊藿的影响中,果实形态参数和种子数为早期和中期的大于晚期。在开花时间中种子宽度无显著差异,长度略有差异。种子败育率在晚期较高,而在早期和中期较低,因此早期和中期的果实重量要显著高于晚期。因此开花的位置和时间对巫山淫羊藿生殖成功具有强烈的影响。当我们采摘巫山淫羊藿来进行实验或者果实留种时,应采摘早期和中期,基部和中部的果实,这样会使效果更好。6.巫山淫羊藿在有性繁殖期间克隆片段密度对其各个形态特征的影响显著。从回归分析的R2指数来看,巫山淫羊藿的克隆片段密度增大对三个生长时期的制约作用是由弱至强,有性繁殖期间随克隆片段密度的增加其资源投资趋向于有性繁殖；非参数检验表明：巫山淫羊藿在有性繁殖期间明显增加其叶数、叶宽等来获取更多的资源用于提高其繁殖能力,以取得生存。7.培养基内蔗糖、硝酸钙和硼酸在一定浓度范围内对巫山淫羊藿的花粉萌发和花粉管生长起促进的作用,但超过一定浓度时会有不同程度的抑制作用；镁和钾对花粉萌发及花粉管生长影响不均显著。虽然在正交实验中培养基组分间没有明显的交互作用,但蔗糖、硝酸钙和硼酸对巫山淫羊藿花粉萌发的影响极其显著。巫山淫羊藿最适花粉液体培养基为20%蔗糖+10mg/LH3BO3+20mg/L Ca(NO3)·4H2O;在pH值为5.0、20℃和600LX光照时巫山淫羊藿花粉萌发和花粉管生长最好。8.在比较野生和栽培的巫山淫羊藿淫羊藿苷的含量中,发现其叶片含量相对较高,但二者各个部分均不能达到用药标准。试验还发现,栽培和野生的巫山淫羊藿中叶片黄酮含量都能达到用药标准,其余的部位均不能达到用药标准。由于栽培环境的相对光照强度提高,栽培的巫山淫羊藿均存在矮化现象,但有效成份增加,而每株分枝数和叶数却比野生的淫羊藿和巫山淫羊藿多,提高了栽培巫山淫羊藿的产量。由于环境改变,栽培中巫山淫羊藿的单花花期、单株花期和种群花期均要比野生种群的持续时间要长,而野生的开花振幅要比栽培巫山淫羊藿大。栽培巫山淫羊藿种群表现出集中开花的模式,开花振幅曲线为单峰,相对开花强度也比较低,这与野生种群一致。而栽培的巫山淫羊藿可能会因为缺失有效传粉者而造成有性生殖的困难。更多还原

【Abstract】 To study the pollination biology of Epimedium wushanense, a species endemic to China, different methods such as field and microscopic observations and laboratory measurement were adopted, the results were as follow:1. The flowering span of the population, individual, inflorescence and single flowering was approximately 22～27,12～17 and 3～4d, respectively. Flowering amplitudes curves were a single peak and marked synchrony, and the E. wushanense exhibited a "mass-flowering" pattern with similar at the three populations. Correlation analysis between phenology index and number of the fuirt set indicated that number of the fuirt set had a positive correlation to the peak flowering date, last flowering date, number of buds, duration per plant respectively, and had not a correlation to first flowering date. Comparisons of means and CV of flowering phenology suggested that means of flowering phenology indexes were very significant difference among duration per plant, duration per flower, end date, peak flowering date and fruit numbers, and significant among total flowers, flowering amplitude, fruit setting, but on significant between synchrony and onset. The CV of flowering phenology was remarkably different in phenology indexes, except Synchrony. The fuirt set was also affected by the spur of E. wushanense. The conclusion was that the flowering phenology and reproductive features are affected by micro-environment, and the fuirt set was also affected by the spur of E. wushanense.2. Three distinct kinds of flowers were observed in this species:(1) about 54% of the flowers had pistils that were of equal length or shorter than the stamens (PESS); (2) in 23.5% of the flowers, the pistils were longer than the stamens (PLS), and (3) in 22.5% of the flowers, the styles were positioned axially away from the anthers (SAA). No significant difference in fruit setting rate was observed between PLS and SAA flowers, but PESS flowers set less fruits than the other two. During flowering, the pollen vigor of E. wushanense was 40%～90% and could be maintained for 4～5days. Spatial ectopia, protogyny and indefinite inflorescence were observed to be the characteristics of most reproductive organs of E. wushanense, and the bottom-up pathway of flower visiting by visitors could therefore reduce the possibility of self-cross abortion which caused by cross pollination in one plant. The out-crossing index and pollen-ovule ratio (P/O) was 4 and 3959.51±386.83, respectively. A breeding experiment also indicated that the species was self-incompatible, and revealed that the presence of PLS and SAA could increase outcrossing and fruit set. A field experiment showed that open-pollinated flowers of E. wushanense produced significantly less fruits than an artificial xenogamy experiment. The results therefore showed that floral morphology could significantly affect fruit set in E. wushanense.3. Nine species of hymenoptera and diptera were recorded as visitors, three of them were effective visitors. The principal visitors and effective pollinators were. Bombus spl and Bombus sp2., and the rewards were pollen and nectar. The highest visiting frequency in a day was observed at 13:00～14:00. There was a positive correlation between the nectar volume and the flowering day of E. wushanense, and the nectar volume significant increased at second flowering day in bag flowers. Daily variation of nectar volume of E. wushanense gradually increased, and the nectar volume of the time of after 13:30 was significant higher than the time before 13:30. Because of pollinators, the nectar volume gradually reduced during a day, and the pollinators had more behavior between 13:00 and 15:30. Daily variation of sugar content of nectar of E. wushanense was a single peak, and the reach to top value at 13:30. which reveal that variation of sugar and pollinator, and the nectar secretion andvariation of sugar could affect the reproductive success by pollinators. The attractant experiments indicated that the fruit set of flowers whose spurs were removed was significantly lower than that of flowers whose stamens were removed, suggesting that the spurs of the flowers could strongly increase the pollinator visitation and fruit set.4. The survey of the fruit-setting characteristics of E. wushanense revealed in the three habitats indicated:(1) the resource and reproduction of floral characteristics had no difference among the three habitats, eg. No significant differences of branches per plant, inflorescences number per plant and flowers per plant were observed among the three habitats. (2) Fruit-setting rate per plant Fruit-setting rate per plant Fruit setting per inflorescence fruit setting rate per inflorescence of PLS and SAA were remarkably higher than the PESS. (3) The branches per plant and inflorescences number per plant of three floral characteristics had no difference in three habitats. The flowers per plant and fruit set of population 1 was less than the other populations. (4) The full seed per fruit, percentage of seeds abortion per fruit and number of seeds abortion per fruit were significant different in three floral characteristics, but among the population, which revealed the seeds per fruit had been affected more by floral characteristics. (5) The three floral characteristics had no difference in seeds per fruit, but the PLS and SAA were remarkably longer than the PESS in the length and wide of seed. This indicated the quality of seed have been strongly affected by floral characteristics. The result suggest that the seed setting characteristics of E. wushanense were affected by its flower types and habitats and more bv the latter.5. The significant effects of space on reproductive success of E. wushanense. The fruit morphological parameters of base and middle were higher than the top, the percentage of seeds abortion at base and middle were higher than top. The parameters of fruit and seed of fruit in the early and middle stage of flowering were bigger and much more than the last stage of flowering. In three flowering stages, no significantly was measured in width of seed, but the length of seed. The percentage of seeds abortion at the last stage of flowering were higher than the early and middle stage of flowering, which suggested the weight of fruit in the early and middle stage of flowering were remarkably weightier than the last stage of flowering. So, the of space and flowering time significant effected on reproductive success of E. wushanense. And in order to make work more effective,we should pick the fruits of E. wushanense at the early or middle stage of flowering, and the base position of the inflorescences to do experiment or for medicine.6. Based on the results of regression, the intensity of density dependence effect was enhanced, and more resources were allocated to sexual rep roduction with the density of clone fragments increased. The results of nonparametric test revealed that the leaf number and leaf breadth of clone were gradually increased to gain more resource to enhance the rep roduction capacity. 7.The pollen germination and pollen tube growth of E. wushanense would be enhanced when the concentration of borate, calcium nitrate and sucrose of the culture medium were within a certain range. If the concentrations were too high or low, the pollen germination and pollen tube growth would be inhibited. The effect of magnesium and kalium on pollen germination and pollen tube growth were insignificant. Sucrose, Ca(NO3)2·4H2O and H3BO3 had significant effect on pollen germination, but the interactions among medium components were insignificant. The optimum culture medium and environment conditions which support the germination and growth were 20% sucrose+10 mg/L H3BO3+20 mg/L Ca(NO3)2·4H2O, pH 5.0,20℃and 600 LX light intensity respectively.8. All parts of the icariin contents in wild and cultivated E. wushanense cannot meet the standard, which might be related to harvest seasons. Both of the flavonoids contents in wild and cultivated leaves of E. wushanense can meet the standard, while other parts can not. Because of the relative light intensity increased in cultivated, the effective composition content of two kinds of Epimedium increased. The dwarf phenomenon was occurred in cultivated E. wushanense, but the number of branches per plant in Cultivated were more than wild, the output of E. wushanense increased. Because of environment changed, the flowering span of the population, individual, inflorescence and single flowering in cultivated were longer than the wild population, and the flowering amplitudes of wild were higher than the cultivated. Flowering amplitudes curve of was a single peak and marked synchrony, and the E. wushanense exhibits a "mass-flowering" pattern with similar at wild population. And no pollinators would lead to the less reproductive success in cultivated E. wushanense.更多还原