Studies On Speciation Of Galerucella Birmanica (Coleoptera Chrysomelidae)

Galerucella birmanica Jacoby (Coleoptera: Chrysomelidae) is a specialist beetle that is widespread in China and is a potential biocontrol agent of Trapa natans Linnaeus, which is an invasive weed in the Northeastern United States and Canada. Relatively few studies exist that have addressed the population genetics of this beetle. This study was conducted to investigate genetic differentiation of G. birmanica across a large part of its range in China by methods of taxology, bioecology, genetics, biochemistry and molecular biology, in order to discuss the relationships between genetic structure and geography distribution, population variation and the extent of speciation in different level. This investigation will provide some essential information for understanding possible local adaptation and provide useful data for the pest control in a large area, and might be helpful for its potential introduction and release in North America. The main contents and conclusions are as follows:1. Morphological data showed that twelve geographical populations of G. birmanica in China were individed into six groups, which showed a remarkable relation between numerical character variations of populations and geographical distribution. It seems appropriate to apply the morphometric method to distinguish the subspecies of G. birmanica. Furthermore, differentiation of pudendum among the six populations also existed.2. The influence of temperature on the development, survival and fecundity of different water chestnut beetle populations collected from Liaoning, Shandong, Yuannan, Guangdong, Shanghai and Jiangsu Provinces at 19, 22, 25, 28, 31℃were presented. The results showed that temperature was the key factor that affected the developmental speed of water chestnut beetle. Baoshan population had the longest developmental period and Taian population had the shortest. The highest suivival rate was above 60% (Qingpu pupulation) and the lowest was under 40%(Taian population). All the population of water chestnut beetle had an ovipositional peak at procreational prophase, but their fecundity was different. The highest fecundity was exceed 200(Qingpu pupulation) and the lowest was under 100(Guangzhou population). In winter, the super-cooling points and freezing point fell with the rise of latitude.3. Hybrid experiment showed that F1 of hybrids were obtained in the hybridization between Yangzhou population and other populations and that F2 of hybrids were obtained in the selfed and backcrosses. On the other hand, the survival rate of F1 and F2 adults was very low, from 0% to 60%. It may be the reflection of differentiation of G. birmanica populations in genetics.4. The results of electrophoretic zymogram showed that different populations had their own distinct zymograms. The clustering analysis by Squared Euclidean distance coefficients of EST isozyme further proved that the differentiation of geo-populations of G. birmanica had high relativity with geography distance.5. Analysis of mtDNA COI, COII and Cyt b gene showed:①Nucleotide frequencies were 0.714, 0.744 and 0.749 for T+A, respectively. A+T content was higher than G+C obviously, which showed a strong A+T bias. This bias was also apparent at each codon positions, with third position codons having the greatest bias (>0.90). All variable sites were transitions or transversions, with no insertions or deletions.②The average transition and transvesion ratio was 6.3, 3.1 and 6.0, respectively. Transition was obviously more than transversion. Transition frequently occurs between T and C and transversion usually ocurrs between T and A.③Each population sample was characterized by a single and high-frequency haplotype, with few haplotypes shared among all five populations. We found a small number of rare haplotypes, typically present in only one or two individuals and representative of a specific population. It seemed that there was significant levels of genetic differentiation among populations.④The analysis of genetic differentiation and gene flow about different geographic populations showed that Fst values among five populations were larger than 0.33 except North and Huazhong population. The overall Fst may be significant because of the isolation of the populations.⑤The phylogenetic tree of haplotype showed that the phylogeny among haplotypes was related with geographical distribution. The haplotypes from North and Huazhong population were clustered into one group, and South, Northeast and Southwest were clustered into the other three goups. But the degree of variation was lower between the North and Huazhong populations.⑥The TCS network showed that haplotypes appear to have originated from the North and Huazhong regions. It was consist with the the origin center of the water chestnut. So, we supposed that the spread of G. birmanica had followed the spread of water chestnut.6. The results about rDNA ITS1 and ITS2 sequences analysis showed:①ITS1 was a reasonable target region for molecular identification of infraspecies; At the same time we found that the differentiation of 6 geo-populations of Galerucella birmanica had high relativity with host and geography distance.②502 bp DNA fragment was amplified from ITS2 gene for 10 geographic populations and there are 23 variable sites including 10 transition, 3 transversion and 11 deletion and insertion. Baoshan population had the most delete and insert sites ant it has status of subspecies. The phylogeny among populations was related with geographical distribution.7. The results of combinated mtDNA and ITS sequence analysis showed: All the five molecular markers are faster gene in molecular evolution, but they have different evolving speed. It is better to study the relationship between genetic differentiation and geographic distribution by combined different molecular markers. The evolution speed showed: ITS1 >ITS2 >Cyt b >COII >COI8. The phylogenetic relationships among Galerucella showed:①Average A+T content of COI gene in Galerucella is 71.8%, which showed a strong A+T bias. The variation ratio of amino acid is 18.3%. Genetic distances among subfamilies (0.169-0.198) were bigger than distances between species(0.001-0.134) of the same genus. Molecular phylogenetic trees suggested that there was remarkable relation between the evolution of Galerucella and itshost plant.②In the 366 bp fragment of mitochondrial 16S rDNA, A+T content was about 78.5%, which showed a strong A + T bias . The variation ratio of amino acid is 37.7%. Genetic distances showed: intra-species