| Batrachium bungei is a perennial aquatic herb, which belongs to the family Ranunculaceae. It is a self-compatible species, which can reproduce sexually both by selfed and out-crossed seeds and vegetatively through rhizomes or a detached unit. In China, B. bungei has been reported from several provinces, e.g. Liaoning, Hebei, Zhejiang, Sichuan, Qinghai, Yunnan, Shanxi, Jiangsu, Gansu provinces, and usually forms dense beds of vegetation. However, in recent years, as a consequence of human activity and habitat loss, the distribution range of this species has been reduced greatly. In some places this species is nearly extinct. The phenomenon of habitat fragmentation is the obvious character for B. bungei’s distribution. The current distribution range is mainly confined to the Hengduan Mountains region. The morphology of this species often varies with the habitat, on account of different degrees of genetic differentiation and environmental modifications. Due to its north-south trending ridges and rivers and complex topography, the Hengduan Mountains have been generally considered as a centre of active speciation and is one of the global biodiversity ’hotspots’. In this region most studies were about the animals and terrestrial plants and there is few published data on researching aquatic herbaceous plant. B. bungei appears to be an ideal candidate for researching. In this thesis, we studied the reproductive character and population genetic differentiation of B. bungei in the Hengduan Mountains.1. The floral quantitative characters among populations of B. bungei have been studied. Results show that positive correlations between pollen size and pistil length provide advantaged condition for sexual reproduction. Pollens can pass through styles with enough energy because of this positive correlation. This result doesn’t show a trade-off relationship between pollen size and pollen number or between pollen number and ovule number, nor does between pollen number and ovule number. There is a positive correlation between pollen-ovule ratio and ovule size. In the sexual allocation investment study, we found that pollen volume per flower correlates positively with pollen number per flower and pollen size respectively. We consider that the allocation mode is related to the nutrition condition. As to the female function, the ovule volume per flower correlates positively with ovule volume per flower but no correlation with ovule number. Ovule size has a lager variation range than ovule number. This indicates that ovule size has more plasticity characters than ovule number in changeful environment. The steady character of ovule number can guarantee the success of sexual reproduction for B. bungei.2. The author studied seed germination characters of the 8 populations of B. bungei under the same germination condition but by 4 different storage methods which were room temperature and dry, cold and dry, cold and wet, cold and below water. Results show that the average seed germination rate is 28.78%by the cold and below water storage method. Comparing to other aquatic plants, this rate is higher than Sagittaria pygmaea, S. trifolia and S. potamogetifolia, but lower than Ranalisma rostratum, S. guyanensis and S. trifolia var. trifolia. The four storage methods have no great effect on the final germination rates of the 8 populations of B. bungei, but we can see that comparing to the room temperature and dry storage method, the cold storage methods (including cold and dry, cold and wet, cold and below water) can improve seed germination rate obviously, especially the cold and wet, cold and below water methods. While, the case stated above doesn’t suit for TSQ02 and CJK populations. Results can tell us that water has become the very important factor for the seed germination of B. bungei during the long suffering circumstance and evolution course.3. Genetic variation and clonal diversity of 8 natural populations of B. bungei were investigated using inter-simple sequence repeats (ISSR) markers. A total of 155 different genotypes or clones were identified among the 442 samples by using 10 effective ISSR primers. Based on ISSR data analyses, the proportion of distinguishable genotypes ranged from 0.14 to 0.7 (mean PD=0.37) for B. bungei populations. The values of D ranged from 0.27 to 0.97 and the values of E ranged from 0.11 to 0.89 (mean D=0.73, E=0.64). For the whole species, the values of D, PD and E were 0.98,0.35 and 0.95, respectively. Results also show that the PPB value within each population ranged from 2.7%to 16.0%, the value of h was from 0.0023 to 0.0506 and the value of I was from 0.0054 to 0.0758. At the species level, the percentage of polymorphic band (PPB), the genetic diversity (h) and the Shannon index (I) were 69.3%,0.1649 and 0.2703, respectively. Analysis of molecular variance (AMOVA) shows that a large proportion of genetic variation (85.3%) resided among populations, while only a small proportion (14.7%) resided within populations. In the present study although the mean value of the genotypic diversity (mean PD: 0.37 and mean D:0.73) detected in B. bungei was similar to the estimated values for herbaceous clonal plant species (mean PD:0.44 and mean D:0.74), the within-population genotypic diversity was shown to be a relative high level. The results may imply that when seedling recruitment continues to take place after establishment of a population, a pattern of many small genets is expected. In contrast, populations in which there is no further seedling recruitment after an initial colonization event exhibit a rapid loss of genets, resulting in a few large genets. This species is self-compatible and often with a tendency towards cleistogamy. Due to the lack of effective pollinators within populations of B. bungei in the Hengduan Mountains region, the sexual recombination by outcrossing may not happen frequently. In addition, a number of remnant genets in each population could also be contributed to the high levels of clonal diversity found in this study. The generally low levels of genetic diversity in B. bungei could be explained by its reproductive strategies. It is likely that selfing and clonal growth might be possible causes that contributed to the low levels of genetic diversity found in populations of B. bungei. Furthermore, founder events during postglacial recolonizations and bottlenecks may also contribute to the low levels of genetic diversity within populations of B. bungei. The geographic isolation of B. bungei populations would limit the gene flow, which will result in high genetic differentiation among populations. The results of Mantel test analysis show that there is no correlation between the genetic distance and geographical distance.4. The spatial structure of genetic variation of 191 individuals from 6 populations of B. bungei was investigated using ISSR markers followed by spatial autocorrelation analysis. These samples were subject to spatial autocorrelation analysis in two approaches of equal gene frequency and equal distance interval, respectively. Ten ISSR primers were used and polymorphic bands were selected to calculate Moran’s I spatial autocorrelation coefficient for each population. The results reveal that a spatial structure of genetic variation existed in SC population of B. bungei (with a positive autocorrelation within 2ed distance class). In contrast, the other 5 populations (SW, SQ, TSQ01, TSQ02 and LTCX) are found to be randomly distributed. The different spatial distribution patterns may be attributed to the environment differences. For example, the flowing water can be the most influencing factor because it can bring the detached individuals of B. bungei to other place and settle down to become a new clone which may be a different genet campared to surrounding individuals. |
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