Phylogenetic Relationship Of Some Testudines Species Inferred From Mitochondrial And Nuclear Gene Markers

Mitochondrial 12SrRNA, 16SrRNA, and two nuclear genes (Rag2 and R35) sequence were utilized to analyze the molecular systematic characters of turtles.1) 12SrRNA: Mitochondrial 12SrRNA gene segment sequences of 15 Tortoises individuals were obtained by using PCR amplification and sequencing. After alignment, 433 bp consensus sequences of 12SrRNA were obtained. Among them, 191 variable sites were detected, accounting for 43.2% of total sequence, and 108 were parsim-informative sites. The average contents of A, T, G, and C were 21.46%, 34.42%, 25.85%, and 18.27%, respectively. The content of A+T(55.9%) is bigger than G+C(44.2%). In the 433 nucleotide sites, 34 transitional and 21 transversional sites with 35 indels were found, and the ratio between transition and transversion was 1.67. We downloaded the 12SrRNA sequences of other Tortoise species from NCBI and analyzed the phylogenetic relationships of them together with our own sequences. After alignment, 414 bp consensus sequences of 12SrRNA and 24.0%, respectively. The content of A+T (57.9%) is bigger than G+C (42.1%). 30 transition and 14 transversion in 414 nucleotide sites, and the ratio between transition and transversion were 2.12. Interspecific, intergenus, and interfamily genetic distances were calculated using the Kimura 2-parameter model with the pairwise deletion option. The NJ method was used to perform phylogenetic analysis. The results indicate that Bataguridae is closer to Testudinidae than that of Emydidae to Testudinidae. The results in this paper do not support the idea that the genus Cuora should be divided into two genera, Cuora and Cistoclemmys, and supporting the opinion that Platysternon should be refereed as a genus within Chelydridae.2) 16SrRNA: Mitochondrial DNA 16SrRNA gene segment sequences of 13 turtle individuals were obtained by using PCR amplification and sequencing. We downloaded the 16SrRNA sequences of other 47 turtles from NCBI and analyzed the phylogenetic characters of them together with our own 13 sequences. The genetic information indexes, including base composition,The ratio between transition and transversion (Ts/Tv ratio) and Kimura-2 parameter genetic distance (K-2-P),were calculated. 480bp consensus sequences of 16SrRNA were obtained from these 60 sequences after aligned. 229 variable sites were detected in all turtle individuals, accounting for 47.7% of total sites, with 186 parsim-informative and 80 indels. The average contents of A, T, G, and C were 33.6%, 24.3%, 18.5%, and 23.7%, respectively. The Ts/Tv ratio was 1.95. The K-2-P distances between the seven hinged turtles were 0.004～0.063, with average value 0.03; between 21 genus within Bataguridae were 0.035～0.120, with average 0.088; and between 8 families within Cryptodira (excluding Platysternon megacephalum) were 0.071～0.259, with average 0.169. Topologies of the NJ, MP and ML trees were different. The results indicate that 1) Family Emydidae is the sister taxon to Bataguridae plus Testudinidae, and Bataguridae is closer to Testudinidae than that of Emydidae to Testudinidae, 2) the taxonomica status of Chinemys should be revised, and classified into Genus Mauremys, and that Pyxidea should be classified into Cuora, 3) the results do not support the idea that the genus Cuora should be divided into two genera, i.e., Cuora and Cistoclemmys. Above conclusions are consistent with the results of mtDNA 12SrRNA sequences. However, this study supported the opinion that Platysternon should be elevated to family status (i.e. Platysternidae), contradicting with the study of mtDNA 12SrRNA sequences.3) Nuclear genes: R35 intron segment sequences of 2 turtles and Rag2 gene segment sequences of 6 turtle individuals were obtained by using PCR amplification and sequencing. We downloaded the homologous sequences of other turtles from NCBI and analyzed the phylogenetic characters of them together with our own sequences. The results are as follows: 941bp consensus sequences of R35 were obtained from these sequences after aligned. 620bp consensus sequences of Rag2 were obtained from these sequences after aligned. After aligned, 1561bp consensus sequences of combined R35/Rag2 date set ,505 variable sites were detected in all turtles individuals, accounting for 32.35%of total sites, with 239 parsim-informative and 139 indels. The average contents of A, T, G, and C were 29.5%,28.5%,22.8%and 19.2%, respectively. The Ts/Tv ratio was 1.64. The K-2-P distances between the Cheloniidae and Chelydridae was minimum (0.025); and The K-2-P distances between the Trionychidae and Podocnemididae was maximum (0.182). The phylogenetic trees indicate that 1) Family Emydidae is the sister taxon to Geoemydidae plus Testudinidae, 3) the results strong support that Testudinoidea is the sister taxon to Chelydridae plus Chelonioidea,2) the results also support that Family Chelydridae is the sister taxon to Chelonioidea.