Molecular Phylogenetic Analysis On Representative Species Of The Subfamily Scarabaeinae Based On Gene Sequences

The dung beetles (Scarabaeinae) include appromaxitely5000species and exhibit a diverse great diversity of morphology and nesting behaviors. The many amounts of beetles of the subfamily have the important significance in the protection of habitant and ecology because of breeding on the excrement of animal and human. There have been contradictory on the classification and phylogeny of Scarabaeinae group based on morphology. In this study, the mitochondrial DNA CO I,16S rRNA,12S S rRNA genes and nuclear28S rRNA gene of the subfamily Scarabaeinae were obtained by PCR technique and were analyzed by the software DNAstar, DAMBE, MEGA5.0, PAUP*4b10, combining with morphological and behavioral characters. The genetic diversity and phylogenetic relationships of Scarabaeinae taxon were discussed. The results are as follows:1. The3’region of cytochrome oxidase I (CO I) was amplified using primers Pat and Jerry and exhibited the adequately variable information, suggesting that this region can be used as DNA barcoding for analysis on classification and phylogeny of the subfamily Scarabaeinae.2. Read lengths for CO I gene were all about731bp long. Of those, the conserved sites(C) were446, variable sites (V) were285and parsimony informative sites (Pi) were253of the matrix Scarabaeinae species. The T+A content was77.7%and exhibited obviously A+T nucleotide composition bias. The frequencies of transition (39) were lower than ones of transversion(56). Transition mainly occurred mainly between C and T and transversion occurred between A and T. The genetic distance of representative species was13.5%between the genus Onthophagus and Caccobius and was16.7%between the genus Onthophagus and Catharsius. 3. The sequences16S rRNA gene obtained were all about440bp long. Of those, the conserved sites were267, variable sites were170and parsimony informative sites were152. The T+A content was76.1%and also exhibited obviously A+T nucleotide composition bias. The frequencies of transversion (34) were higher than ones of transition (15). Transition mainly occurred mainly between A and G and transversion occurred between A and T. The number of multiple substitutions major occurred on third codon base. The mean16S rRNA divergence was8.3%between species Onthophagus and Caccobius and was13.1%between the species Onthophagus and Catharsius. The results were similar with CO Ⅰ gene.4. The sequences of12S rRNA gene were all about398bp long. Of those, the conserved sites were284, the variable sites were114and the parsimony informative sites were100. The T+A content was77.7%and also exhibited obviously mitochondrial nucleotide composition bias. The frequencies of transversion (21) were higher than ones of transition (16). Transition mainly occurred mainly between T and C and transversion occurred between A and T. The mean divergence of12S rRNA sequences was8.0%between species Onthophagus and Caccobius and was15.5%between the species Onthophagus and Sisyphus.5. The28S rRNA gene fragment were all about589bp (C=489; V=59; Pi=41). The G+C content was53.9%and also exhibited obviously G and C composition bias of nuclear DNA. The results were consistent with the base composition bias in other kinds of insects. The frequencies of transversion (9) were higher than ones of transition (8). Transition mainly occurred mainly between T and C and transversion occurred between A and T. The mean28rRNA divergence was1.4%between species Onthophagus and Caccobius and was3.7%between the species Onthophagus and Catharsius. The numbers of divergence and substitution in the mitochondrial DNA were few than nuclear DNA, demonstrating that the sequences of nuclear DNA were conservative. 6. The molecular trees reconstructed based on three genes revealed that the genera of Onthophagus and Caccobius species clustered into one clade and showed two genera probably were paraphyly. Almost all individuals of the species from the genera Copris, Sisyphus and Catharsius clustered firstly in their own species lineages, then all species clustered in their own genus respectively, indicating that the relationships of intra-species were closer than that of inter-species and the intra-generic relationships was closer than the inter-generic relationships. The species between Onthophagus lenzi and Onthophagus fodiens was sister species based on analysis on CO I,16S rRNA,28S rRNA. The species Onthophagus gibbulus and Onthophagus marginalis nigrimargo was sister species based on analysis on12S rRNA sequences, which were approximately consistent with morphological characters.The use of CO I,16S rRNA,12S rRNA,28S rRNA sequences as molecular markers seems to be a good tool for the identification and studies on systematics of Scarabaeinae group.