The Complete Mitochondrial Genome Analysis Of Four Clemmys Complex Turtles (Testudines: Geoemydidae) With Their Molecular Phylogenetic Relationships

The Clemmys complex in Geoemydidae is composed of Mauremys, Annamemys, Chinemys, Ocadia and Sacalia genera. Up to now, researches based on morphological and different molecular data, have not obtained wide and consistent conclusion to that whether the four genera (Mauremys, Annamemys, Chinemys, Ocadia) should be put into an expanded Mauremys genus, and to the phylogenetic position of Sacalia in Testudinata and the phylogenetic relationships with its related genera. In this paper, the complete mitochondrial genomes (mtDNA) of four species (Ocadia sinensis, Mauremys japonica, Sacalia bealei and Sacalia quadriocellata) were determined with the universal PCR, LA-PCR and sequencing technology, their genome characters were analyzed, and their phylogenetic relationships were discussed based on the complete mtDNA data.The mtDNA size of O. sinensis, M. japonica, S. bealei and S. quadriocellata were 16461 bp,16443 bp,16561 bp and 16551 bp, respectively, all included 13 protein-coding genes, 22 tRNA, 2 rRNA, a highly variable control region (CR) and an initiation site for transcription and replication (OL). The location of genes and base composition in the mtDNA are identical to that of typical vertebrates, indicating the high conservation in turle mitochondrial genome evolution. An extra insertion in ND3 gene were found in O. sinensis, M. japonica and S. quadriocellata of this study (205, 172 and 174 site, respectively), but not in S. bealei. The fact that some cases are unique suggests that frameshifting over these sites does not have a regulatory role.Combined with the mtDNA sequences published previously, the CR of five representative turtles (M. mutica, M. japonica, A. annamensis, O. sinensis and C. reevesii) of Mauremys complex, three functional domains (TAS, CD and CSB) and their conservation sequence boxes (TAS, CSB-F, CSB-1, CSB-2 and CSB-3) in CR were identified successfully, and their variable number tandem repeats (VNTR) were analyzed primarily. At the 3'end of CR, 6 motif types of VNTRs of five species were recognized. TTATATTA may stand for the VNTR motif of ancestor species of these five turtles, other motifs may have undergone changes from this motif. According to this, we can infer their relationships among these species: M. mutica and A. annamensis are closer, they originated earlier than other three species; there are closer relationships among C. reevesii, O. sinensis and M. japonica; and C. reevesii originated earlier than O. sinensis and M. japonica. In the analysis of mtDNA CR of two valid species in Sacalia, we found that the CR structure in two species of Sacalia (S. bealei and S. quadriocellata) were highly conserved, however, distant TA motif repeats ((TA)5, (TA) 5, (TA)10) in S. quadriocellata CR were not detected in S. bealei. Such distant motifs may be as a character to distinguish the two species.Phylogenetic relationships were reconstructed with Maximum Parsimony (MP), Maximum likelihood (ML) and Bayesian inferences (BI) methods based on 14,016 bp complete mtDNA data set from 27 turtle taxa. All the results confidently support the expanded Mauremys genus including genera of Mauremys, Annamemys, Ocadia and Chinemys, as well the sister relationship between Sacalia and Cyclemys (Cyclemys atripons as the representative). Using"relaxed"molecular clock models, we estimated divergences time among turtle lineages with fossil evidence based on above phylogenetic framework. Dating analysis revealed that divergences among the most diverse families of extant cryptodirans occurred from recent million years ago, showing their late interspecies divergences relatively.