| Objective:1.Based on the 16 S rRNA gene,a phylogenetic tree of freshwater crabs which distributed in karst region of south-west China was established,and the relationship between the geographic region of China's freshwater crabs and the southwest karst landform was discussed.2.To construct a geographic information system(GIS)for freshwater crabs in the southwestern karst region for all the 640 samples of 31 genus,129 species collected in eight provinces and cities in the southwest,aiming to find out the inner relationship between the distribution patterns and the species diversity of freshwater crabs and karst topography that possibly exist.3.The mitochondrial genomes of 7 species of freshwater crabs from 7 genus of karst landforms in south-west China were obtained,and phylogenetic analysis and comparative mitogenomics was carried out,trying to clarify the molecular phylogenetic relationship of the freshwater crabs in the southwestern China.Deducing the divergence time of the genus and species of freshwater crabs in the region.Methods:1.Based on the preliminary study on the morphological classification and molecular kinship of the freshwater crabs in the karst area of south-west China,the selection of samples was based on the ?species? of freshwater crabs,and the specific collection locations of the specimens were determined based on the karst geomorphological features.The research team went to the proposed location to collect specimens according to the project plan,and collected the data from the species resource library,then selected the freshwater crabs collected from the southwestern karst area over the years to be used in this research.The selected specimens were subjected to morphological identification,and the latitude and longitude of the specimen collection site were recorded to distinguish whether they were in karst landforms.2.Extract the DNA of the sample and perform amplification and measurement of 16 S rRNA gene.Search the NCBI database and download the sequence of 16 S rRNA gene belonging to the eight provinces and cities in southwest China.The phylogenetic analysis based on the 16 S rRNA gene was established by combining the sequence measured in this experiment and the sequences download from NCBI database.3.Based on the distribution data of karst space in south-west China provided by the Karst Science Data Center(www.karstdata.cn),the geographic information system(GIS)is used to construct the distribution map of freshwater crabs in the eight provinces and cities in southwestern China.4.Obtain the mitochondrial genome of 7 species of freshwater crabs from 7 genus in southwestern China,analyze their genome composition and gene arrangement order,analyze their tRNA structure and codon usage,and combine 13 protein-coding genes of other Brachyura species to conduct phylogenetic analysis and divergence time estimation.Conclusion:1.There are 2 families,48 geuns and 324 species of freshwater crabs that distributed in China,and 85.42% of genus and 60.49% of species are concentrated in the eight provinces and cities in the southwest of China.2.The phylogenetic analysis based on the 16 S rRNA gene showed that basically all freshwater crabs formed their branches in genus,and the species that distributed in Yunnan Province formed an independent clade,and most of them were unique in Yunnan Province.3.For the phylogenetic tree that constructed based on 16 S rRNA gene,the phenomenon of disappearance of gene interval occurs,especially the interspecies classification of the species under Longpotamon is not ideal.4.By constructing a geographic information system for freshwater crabs in the eight provinces and city of southwest China,it can be found that under each province/city,there is no significant correlation between the distribution patterns and species diversity of freshwater crabs and karst landforms.5.Climate,latitude and karst topography all play an important role in the formation,survival and differentiation of freshwater crabs.Among them,climate and latitude determine whether freshwater crabs have a suitable environment for survival,while karst topography plays an important role in the species differentiation.6.The length of the mitochondrial genome of 7 species of freshwater crabs from 7 genus under Potamidae in Southwest China's karst region is between 19236 bp(Aparapotamon similium)to 16547 bp(Sinolapotamon patellifer).All species contained 37 genes and a control region as typical mitochondrial genome of metazoan,and the nucleotide composition showed significant AT bias.7.Among the seven species that sequenced in this study,structurally unusual tRNA genes(tRNA-Ile and tRNA-Met)appeared in the mitochondrial genome of Neilupotamon xinganense and Bottapotamon lingchuanense.8.Compared with the Brachyuran ground pattern and the gene order of other freshwater crabs,a new genetic arrangement was found the first time in Neilupotamon xinganense and Bottapotamon lingchuanense.In this case,the rearrangement of two protein-coding genes,including the COX1 gene,occurs,which is rare in Brachyura species.9.The ML tree and BI tree based on the 13 protein-coding genes of mitochondrial genome of Brachyura species showed similar topologies with high confidence,and the analysis results were consistent with the current mainstream classification system.10.The divergence time estimation based on 13 protein-coding genes shows that the differentiation time of freshwater crabs was 113.3008 Ma,and the differentiation of Gecarcinucoidea and Potamidea was 74.8013 Ma.11.The differentiation time of Potamiscus motuoense coincided with the formation time of Qinghai-Tibet Plateau and the Yunnan-Guizhou Plateau,suggesting that Potamiscus motuoense and Aparapotamon similium may come from a common ancestor,and the geographical isolation caused by the development of the Hengduan Mountains contributed to the differentiation of the species.12.The differentiation time of the seven species distributed under the karst topography in southwestern China is basically earlier than that of the freshwater crab species in the other regions,indicating that the freshwater crabs distributed in the karst region have a more ancient history and may be the ancestors of some freshwater crabs in mainland China.In the southwestern karst region of China,especially the karst region in the Yunnan-Guizhou Plateau may be the birthplace of some freshwater crabs in mainland China. |
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