Comparison research and phylogenetic implications of mitochondrial control regions in four soft-shelled turtles of Trionychia (Reptilia, Testudinata)

The mitochondrial control regions (CRs) and flanking sequences of Pelodiscus sinensis, Apalone ferox, Palea steindachneri and Carettochelys insculpta were obtained using Long-PCR with gene-specific primers. The CR lengths of the four species were 1843 bp, 1356 bp, 1725 bp, and 969 bp. The base composition percentages of A+T were 60.5%, 60.7%, 65.7%, 64.7%, respectively. Combined with CR sequences of other three soft-shelled turtles published in GenBank (Pelodiscus sinensis, Korea, AY962573; Dogania subplana, AF366350; Lissemys punctata, EF050073), we compared the CR structures and identified three functional domains (TAS, CD and CSB) in which conserved sequence blocks (TAS, CSB-F, CSB-1, CSB-2 and CSB-3) were also successfully identified according to their sequence similarities to those of other turtles. The variable numbers of tandem repeats (VNTRs 1) with 50-52 bp motif were identified at 5'-end of CR among the five soft-shelled turtles P. sinensis (China), P. sinensis (Korea), A. ferox, P. steindachneri, D. subplana. The copy number of the VNTRs varied from 5 to 15. VNTRs 2 with 2-11 bp motif were identified in the 3'- end of CR among all of the six soft-shelled turtles with variable number of motifs from 4 to 29. Moreover, VNTRs 3 with 6 bp motif were identified between CSB-1 and CSB-2 of CR both in P. sinensis (China) and P. sinensis (Korea), in which the number of motifs varied from 19 to 29. The types and distribution of VNTRs of the six soft-shelled turtles were also discussed. With Alligator mississippiensis as an outgroup, combined with the CR sequences (excluding VNTRs) of other five turtles which were published in GenBank, the molecular phylogenetic trees were constructed using PAUP 4.0b10 and MrBayes ver. 3.0. The results strongly supported the monophyly of Carretochelyidae and Carettochelyidae as sister group to an assemblage of Cryptodira. Our research suggested that the earliest phylogenetic tree splits into three separated basal branches; the Pelomedusidira (Pelomedusa subrufa), the Carettochelyidae (C. insculpta), and an assemblage of Cryptodira and the C. insculpta that might be a representation of distinctive suborder.