The Validity Of Three Subspecies Of Platysternon Megacephalum And Evolutionary Pattern Of Its Mitochondrial Duplicated Control Regions

Platysternon megacephalum is the sole living specie of Platysternidae with3subspecies, P.m.megacephalum, P.m.shiui, P.m.peguens, based on morphylogicalcharacters and geological distribution. However, these morphylogical characters areontogenetic, containing implicit errors. The potential hybridization among specieswhich share an overlapping distribution could lead to amibigious morphylogicalcharacters. It is nessecery to discuss the validity of three subspecies ofP.megacephalum at molecular level.The validity of subspecies was explored using combined data of nu DNA andmt DNA from10speciemens of P. megacephalum by PCR method. Throughcomparative of mt DNA, a long tandem repeat (130bp) was recognized only at the5’end of TAS region of P.m.peguense, followed by a poly-C stretch. Only one motifcould be found as TATTA at VNTRs in mt DNA of P.m.peguense, while twocomplex motifs, TGTTA and GTTGTTATATAATATAT, were recognized both atother two subspecies. These motif heterogeneities suggested that P.m.peguensedistinct with other two subspecies siginificantly. Then we calculated the genenticdistances and reconstructed phylogenetic trees using nu DNA and mt DNArespectively. Then the genetic distances calculated using nu DNA and mt DNAwere0.009and0.093between P.m.peguense and other two subspecies, which hasbeen up to species level. Furthermore, the phylogenetic trees indicated thatP.m.peguense distinct with other two subspecies significantly. Therefore, werecommend to rise the divergence subspecies P.m.peguense up to a species with thename Platysternon peguense.The largest non-coding region of mitochondrial genome, containing lotsessential elements involving in replication and transcription of mt DNA, wasnamed control region. Increasing cases of duplicated control regions have beendocumented in a wide range of species, although only one in most metazoan’s mt DNA.It has been documented previously by us that the mt DNA ofP.megacephalum has a unique mt genome of6genes rearrangement and highsimilar duplicated control regions. Almost duplicated CRs have high similarity ofsequences which are proposed as concerted evolution, while some otherevolutionary patterns of duplicated control reigons in some animals’ mt DNA. Itremains unkonwn that how the duplicated control regions evolve in the mt DNA ofP.megacephalum.In this research,22specimens were collected through the distribution rangesof this turtle. The complete sequences of duplicated CRs were amplified usinglong-PCR and nested PCR method. Then the evolution pattern of CRs was exploredat micro-evolution level combined with phylogenetic and recombination test.The phylogenetic trees reconstructed using different method,Neighbor-Joining (NJ), Maximum Likelihood (ML) and Bayesian Inference (BI)shared nearly consensus topology structure. CRⅠalways cluster together with theirorthology copies from different individuals rather than with their counterpart, CRⅡfrom the same individuals. These results indicated that the duplicated CRs of mtDNA from P.megacephalum evolved independently instead of concertedly.In addition, a remarkable individuals, T2, griped our attention. The CRⅠof T2got together with clade B, but the CRⅡgather with clade C. The individual T2hasbeen proved as a hybridization of two subspecies using nu DNA. Thus, wehypothesize that the duplication control regions might have originated fromheterology recombination of mitochondrial genomes.Based on recombination test using RDP software, the break points wereidentified occurring around420bp and1080bp at the mt DNA of T2. AReplication Forks Barrier (RFB) found in TAS region of mt DNA of Aceroswaldeni might intrige recombinantion. A homologous RFB was identified aroundthe first breakpoint at420bp. Moreover, a variable number of tandem repeats(VNTRs) have significant heterogeneities at both length and motif at differentsubspecies downstream of1080bp in CRⅠ. These heterogeneities might behelpful in recombination. Meanwhile, a special sequence, located at the3’ end ofCR Ⅱ, have been identified as homology with Muts gene in mt DNA ofOctocorallia. This sequence was inferred to take action of a mispairing repair for CRⅡ and flanking tRNA genes. These recombination related elements are theexactly reason for duplicated CRs maintain function with high sequence similarityduring evolution period.This is the first research on the validity of supspecies of Platysternonmegacephalum using mt DNA combined with nu DNA. The subspeicies P.peguense distincted with other two subspecies significantly based on the geneticdistances and phylogenetic trees. Therefore, we recommend rise P. peguense to aspecies with a name Platysternon peguense. Then, the evolutionary pattern ofduplicated control region of mt DNA of this turtle were discussed at a microevolution level using phylogenetic methods combined with recombination test. Theresults suggested the duplicated control regions of mt DNA of P. megacephalumoriginated from heterology recombination and evolved independently.