Studies On The Molecular Markers And Phylogeny Of The Scarabaeidae Beetles From Northern China

Scarabaeidae，which belongs to Coleoptera Polyphaga Scarabaeoidea, include13subfamilies about1600genus27000species. The phytophagous scarab beetles are majorpests damaging agriculture and forestry, and the coprophagous beetles have an importantsignificance in maintaining ecological balance and protecting the environment.Classification of researches on Scarabidae mainly based on morphological characteristics.The taxonomic status is always controversial, the research that aim at beetles groups innorthern is vital. The paper was based on the research of the best methods of preservationand DNA extraction of specimens, and the mitochondrial16S rRNA and nuclear28S rRNAgenetic molecular markers. It combined with morphological characteristics and used thebioinformatics analysis to explored the taxonomy and phylogenetic relationships betweenthe different subfamilies and tribes of Scarabidae in North of China.(1)In this study, by electrophoresis and PCR amplification of nuclear28S rDNA genesequence results, compared three preservations of anhydrous ethanol,75%ethanol, driedneedle; and three methods for DNA extraction of SDS-proteinase K method, CTABmethod and saturated NaCl method. The results showed that the best preservation methodof Scarabaeidae beetles was anhydrous ethanol preservation,and the optimum extractionmethod was SDS-proteinase K method.(2)The fragment length of mtDNA16S ribosomal RNA and nuclear28S ribosomalDNA of the Scarabaeidae species obtained in our experiments were415bp and531bprespectively. The mean base composition for16S rRNA was39.9％thymine,7.6％cytosine,36.7％adenine,15.8％guanine. The A+T content was76.6%and exhibitedobviously A+T nucleotide composition bias. The mean base composition for28S rDNAwas20.3％thymine,24.4％cytosine,24.5％adenine,30.7％guanine. The G+C content was55.1%and exhibited obviously G and C composition bias of nuclear DNA. (3)The numbers of variable sites and parsimony informative sites for16S rRNA were166and156respectively. The numbers of variable sites and parsimony informative sitesfor28S rDNA were79and66respectively. Both of the two datasets had enough variablesites and parsimony informative sites.(4)The average frequency of transition and transversion were26and42times for16SrRNA gene fragments. Transition mainly occurred mainly between T and A andtransversion occurred between A and G. The frequencies of transversion (10) were higherthan ones of transition (9) for28S rDNA gene fragments. Transition mainly occurredmainly between T and C and transversion occurred between A and T．(5)The genetic distances for16S rRNA of seven subfamilies between11.8％and19.6％. The inter-tribal genetic distances were between6.5％and22.4％and the intra-tribalgenetic distances were from0％to13.2％. The genetic distances for28S rRNA of sevensubfamilies between1.8％and5.6％. The inter-tribal genetic distanceswere between0.4％and7.2％and the intra-tribal genetic distances were from0％to3.5％. The resultsindicated that the inter-tribal relationships were more distant than intra-tribal relationshipsand the inter-genus relationships were more distant than intra-genus.(6)Phylogenetic tree analysis: The NJ, MP phylogenetic trees of the subfamilyMelolonthinae taxon recovered on the basis of16S rRNA and28S rDNA sequencesindicated that, the phylogenetic relationships constructed by molecular phylogeny and theclassification method according to position of scarab belly valve given the similar outcome.The species of the phytophagous taxa—Rutelinae, Cetoniinae、Trichiinae、Dynastinae andMelolonthinae had gathered for a classification of clusters, then Scarabaeinae andAphodiinae formed another cluster. Dynastinae and Rutelinae had closely geneticrelationships, so did Cetoniinae and Trichiinae. Melolonthinae formed a separate branchof the tree that indicated the relationships were far with other relatively.In the analysis of the tribe of categories, Adoretini tribe and Anomalini tribe ofRutelinae had a clear relationship,which was consistent with the morphological taxonomy.Cetoniinae tribe of Cetoniinae were clustered into a higher support rate. Dicranocephalus wallichi and Melolonthinae gethered as one with a low degree of confidence. Melolonthiniclustered into their own tribe respectively with higher boostrap values. But some species ofRhizotrogini were not group into its own tribes. The location of Sericini couldn’t becompletely confirmed in the NJ, MP phylogenetic trees. Aphodiinae gatheredthemselves first with a higher confidence rate, and then gathered with the Scarabaeinae.The use of16S rRNA,28S rRNA sequences as molecular markers seems to be a goodtool for the identification and studies of systematics of Scarabaeidae populations.