The Research Of Mitogenomic Phylogeny In Mauremys Sensu Lato And The Adaptive Evolution Of Mitochondrial ND5 Gene In Turtles

Species of Mauremys sensu lato have a very wide distribution all over the Eurasia. The interspecific phylogeny of Mauremys sensu lato has always been a research hot spot due to the special distribution. Previous molecular research on this group was based on several mt genes or nuclear genes and the conclusions by different molecular markers were controversial. The phylogeography and the historical speciation of this genus are still poorly understood.Several unique properties of mitochondrial DNA(mt DNA), including its high copy number, high mutation rate, lack of recombination and maternal inheritance, have made it an ideal molecule marker for studies in phylogeny and origins of species. The mt DNA is generally assumed to evolve under neutral or nearly-neutral selection due to its high mutation rate. However, with the deepening study in cell energy metabolism, the significant biological function of the mt DNA protein products has been verified in aerobic respiration, which suggested the variation of mt DNA is under the regulation of selection pressure. The research also showed that mt DNA mutations were closely associated with many diseases.Mitogenomic structure of turtles is relatively conserved and only a few of them have the double control regions and the displacement of protein-coding gene. The evolutionary dynamics and the selection pressure mechanism of these special mt DNA still need further explore.In this study, the complete mt DNA of M. leprosa, M. caspica, M. rivulata and M. mutica was determined. Phylogenetic trees of Mauremys sensu lato were reconstructed based on Maximum-likelihood(ML) and Bayesian inference(BI) using mt genome data. Interspecific divergence times were estimated by fossil calibration. And the ancestral area were reconstructed combining the related geographic events. Combined with the species of Testudinidae, Geoemydidae and Trionychidae, the mt evolution pattern and its adaptive mechanism were explored based on q PCR and bioinformatic analyses.The main results and conclusions are as follows:1. Phylogenetic results of Mauremys sensu lato suggested that the four old genera(Mauremys, Annamemys, Chinemys, and Ocadia) which divided by morphological feature were paraphyletic and should be merged into the genus(Mauremys sensu lato). Ancestral area reconstruction and divergence time estimation suggested Southeast Asia may be the area of origin for the common ancestral species of this genus and genetic drift may have played a decisive role in species divergence due to the isolated event of a glacial age. And M. japonica may have been speciated due to the creation of the island of Japan. The detection of extensive gene flow suggested no vicariance occurred between Asia and Southeast Asia. Inconsistent results between gene flow assessment and phylogenetic analysis revealed the hybrid origin of M. mutica(Southeast Asian).2. Up to March 2016, 115 complete mt DNA of turtles had been released involved 11 families, 61 species. Most of them have a conservative mitogenomic structure and only a few turtles have the double control regions and the displacement of protein-coding gene. High variation in the mt DNA of P. megacephalum has made it an ideal research material here. Compared with the mt DNA of M. reevesii which has the typical characteristics of vertebrates, the impact of gene expression by the transfer of ND5 gene and the multiplication of the control region were investigated using Real-time Quantitative PCR(q PCR). Combined with the species of Testudinidae, Geoemydidae and Trionychidae, the selection pressure of the high variable mt DNA was estimated and the protein tertiary structure of mt ND5 subunit of P. megacephalum and M. reevesii were predicted respectively to explore the influence on energy metabolism by the mutation of base sequence. The results suggested the highly variable mt genome of turtles may has its adaptive significance associated with energy metabolism. And new transcriptional pattern were arosen by ND5 gene transfer and multiple control regions, i.e., mitochondria with double-CR has three transcription start sites and initiated to transcribe three pre-RNA in different length. The up-regulation of ND5 expression were achieved by the shortening in the primary transcript. High non-synonymous nucleotide substitutions rate may result in the increase of transmembrane area of ND5 subunit in order to enhance the ability of ATP synthesis and provide more energy for cells.Here, the phylogeny and historical speciation of Mauremys sensu lato were investigated based on mt genome. We also assessed the interspecific gene flow level using five unlinked microsatellites loci to further explore the interspecific hybrid degree. Combined with the species of Testudinidae, Geoemydidae and Trionychidae, the mt evolution pattern and its adaptive mechanism were explored here based on the analyses of its highly mutated mt ND5 gene.