|其他摘要||Abstract: According to the investigation of geographical distributions, the putative natural hybrid Rhododendron agastum Balf. f. et W. W. Smith and its assumed parents R. delavayi Franch., R. decorum Franch.and R. irroratum Franch are sympatrically distibuted in Yunnan, China. And the four species belongs to three subsection of the Section Ponticum under Subgenus Hymenanthes of Rhododendron. In this paper we study the morphological characters, the palynology, the pollination biology, sequences of the nuclear ribosomal DNA ITS region, the chloroplast DNA trnL-F intron-spacer and RFLP analysis of ITS sequences of them in order to confirm our assumed review of the relationship among them. We also compared these data with an artificial hybrid between R. decorum (♀) × R. delavayi (♂). Based on the results, we conclude that R. agastum is a natural hybrid between a female R. delavayi and a male R. decorum, and R. irroratm is also a natural hybrid between R. decorum and R. delavayi. The main research results are summarized as follows:
1. Geographic distributions
We totally investigated 7 sites of the geographic distributions of four Rhododendron species in Yunnan, China. Two sites including Junzishan in shizong county and Maxiongshan in zhanyi county of eastern Yunnan, and five sites of Dapingdi, Jinzan and Dazhuping in Yangbi county (western slope of cangshan), Shibaoshan in Jianchuan county and Dalicangshan (eastern slope) of the northwestern Yunnan. R. agastum, R. delavayi, R. decorum and R. irroratum are all sympatrically distributed in Junzishan, Maxiongshan, Dapingdi and Jinzhanmaidi. However, Dazhuping possesses R. delavayi, R. irroratum, and R. agastum, without R. decorum. And Shibaoshan possesses R. delavayi, R. decorum and R. agastum without R. irroratum. The results show that the four Rhododendron species are basically sympatrically distributed. R. decorum grows about the lower altitude, R. delavayi distributes more widely than the other three species in Yunnan Province. Except for horizontal distribution, the putative hybrids R. agastum and R. irroratum are occupant of the higher altitude.
We analyzed the morphological characters of the four species specimens which were collected in 2005 and 2006 from the above mentioned seven natural populations in the eastern and northwestern Yunnan and in Kunming Botanic Garden. We compared the qualitative characters and the quantitative characters of the four Rhododendron species, also used PAC analysis. The results show that morphology of R. delavayi and R. decorum are obviously distinct, and morphology of R. agastum and R. irroratum are intermediate between the two parents R. delavayi and R. decorum.
The pollen morphology of R. delavayi, R. decorum, R. agastum and R. irroratum are all pollen tetrad, approaching to sphericity, three apertures. The pollen size of R. delavayi and R. decorum are distinct, R. delavayi is the smallest (37.00-51.60um), but R. decorum is the largest (39.00-54.20um). The pollen size of R. agastum and R. irroratum are similar (41.00-53.00um and 40.40-53.00um), and are intermediate between R. delavayi and R. decorum. Extine of R. delavyi and R. decorum are distinct. R. delavayi is very smooth，but R. decorum have many coarse adorns. And extine of R. agastum and R. irroratum are intermediate between the two parents of R. delavayi and R. decorum.
4. Pollination Biology
We investigated the flowering period, floral visitors and conducted a series of pollination experiments to examine breeding systems and hybrid compatibility in four Rhododendron species in Junzishan. We observed that R. delavayi was flowering from the beginning of March to the end of May, while R. agastum and R. irroratum were flowering from the beginning of March to the beginning of April. After their flowering periods were end, R. decorum began to flower until the end of May. The anthesis of the single flower in four Rhododendron species was about one week. Their pollen vigor and pistil reception are the highest within flowering 3-5 days. The floral visitors belong to Hymenoptera and Diptera Order. Apiscerana Fabricius is the mutual visitor of the four Rhododendron species, but Colletes sp is proprietary visitor for R. agastum and R. irroratum. We found that R. delavayi, R. agastum and R. decorum are self-incompatible except R. irroratum. No breeding barrier was detected in four Rhododendron species by artificial pollination, and the hybrid seeds were fertile. The hybrids between four species showed equivalent fitness as their parents. Based on the above resultd, we suggest that the role of potential interspecific pollination is essential to the hybrid speciation and evolution of Rhododendron species.
The observation of chromosomes revealed that Rhododendron delavayi, R. agastum, R. irroratum and R. decorum are all diploids, x=13, and confirmed that R. agastum is a diploid hybrid speciation. But the result of the meiotic observation is not very good in this study.
6. Molecular Biology
We collected materials of Rhododendron delavayi, R. decorum, R. agastum and R. irroratum from 19 natural populations in different places, and randomly choose 1 to 3 individuals from every population to analysis their nuclear DNA ITS sequences and chloroplast DNA trnL-F sequences. According to the ITS sequences of the four Rhododendron species, three restriction enzyme Bsp68I, Bsh1285I and MvaI were used in the PCR-RFLP analysis. The nuclear DNA ITS sequences and chloroplast DNA trnL-F sequences revealed R. delavayi and R. decorum are clearly distinct, and the cloning ITS sequences of R. agastum and R. irroratum shows that the two hybrid have cloning ITS sequences from R. delavayi and R. decorum, and have another cloning ITS sequences. The trnL-F sequences of R. agastum are all from R. delavayi, but trnL-F sequences of R. irroratum are partly from R. delavayi, partly from R. decorum. PCR-RFLP analysis of ITS sequence reveals that R. agastum is a natural hybrid between R. delavayi and R. decorum and R. irroratum is also a hybrid between R. delavayi and R. decorum. During the course of hybrid evolution, backcrossing may be happened for many times.|