Analysis On Phylogenetic Relationships Of The Representative Species Of Melolonthinae Based On Three Gene Sequences

Melolonthinae (Coleoptera; Searabaeoidea:Scarabaeidae) is one of the largest and most diverse subfamilies in the family Scarabaeidae, with approximately750genera and nearly11,000species worldwide. The larvae of some species from Melolonthinae are generally phytophagous, damaging a wide variety of crops, pastures, and turf. Therefore, studies on Melolonthinae speices have the important value of application to prevention and control pests.However, the classification of Melolonthinae groups has been always controversial. Molecular biotechnology is an essential method of complement to morphological studies. The relevant research on the molecular phylogeny of subfamily Melolonthinae started late and had very limited reports. The studies on the taxonomy and systematics of Melolonthinae based on the gene sequences have an important significance.In this study, the mitchondrial COI,16S rRNA and nuclear28S rDNA gene fragments of some tribes and species from Melolonthinae were amplified by PCR technique and were sequenced used as molecular markers. The nucleotide composition, the genetic distances, the ratios of si/sv and other parameters were calculated using software MEGA4.0ect. The molecular phylogenetic trees were reconstructed based on CO I,16S rRNA and nuclear28S rDNA gene by software PAUP4.0. The phylogenetic relationships within and between tribes and genera were analyzed and the application of these sequences to Melolonthinae was explored.The results showed as follows:(1) The fragment length of mtDNA cytochrome oxidase subunit I,16S ribosomal RNA (16S rRNA) and nuclear28S ribosomal DNA (28S rDNA) of the subfamily Melolonthinae species obtained in our experiments were739bp,505bp and556bp respectively. The mean base composition for CO I was31.6%adenine,36.5%thymine,14.2%guanine,17.7%cytosine. The mean base composition for16S rRNA was33.9%adenine,39.0%thymine,17.7%guanine,9.3%cytosine. The mean base composition for28S D3-D6region was24.7%adenine,20.3%thymine,30.7%guanine,24.3%cytosine. The aligned data exhibited strong A+T nucleotide bias in the mitchondrial DNA and G+C nucleotide bias in the nuclear ribosomal DNA by an estimate with the help of software MEGA4.0. (2)The numbers of variable sites and parsimony informative sites for CO I genefragments produced the aligments of739positions were318and242respectively. Thenumbers of vairable sites and parsimony informative sites for16S rRNA produced thealigments of505positions were208and166respectively. The numbers of variable sitesand parsimony informative sites for28S rDNA produced the aligments of556positionswere53and36respectively. The vairable sites occured on third codon sites with thehighest frequencies for the three gene sequences.(3)The average frequency of transition (TS) and transversion (TV) were67and64times for CO I gene fragments, with TS and TV substitution mainly occurred between Cand T and between A and T, The average frequency of transition (TS) and transversion (TV)were26and42times for16S rRNA gene fragments, with TS and TV substitution mainlyoccurred between A and G and between A and T. The average frequency of transition (TS)and transversion (TV) were7and6times for28S rDNA gene fragments，with TS and TVsubstitution mainly occurred between C and T, A and T, C and G. The substitution dataindicated that the sequences of nuclear28S rDNA were more conservative than themitchondiral CO I and16S rRNA. The multiple substitutions were detected by analysisusing sotfware DAMBE and reached no saturation,(4)The genetic distances (TS+TV) of intra-tirbe of Hopliini，Sericini, Melolonthinifor CO I sequences by K-2-parameter were13.5%,10.7%^19.9%respectively and thegenetic distances of inter-tribe were from20.4%to22,5%.The intra-tirbal geneticdistances for16S rRNA of Hopliini, Sericini, Melolonthini were between9.1%and12.6%and the inter-tribal genetic distances were from13.9%to18.8%. The intra-tribal geneticdistances for28S rDNA of Hopliini, Sericini，Melolonthini were between2.2%and2.8%and the inter-tribal genetic distances were from0.8%to2.0%. The results indicated thatthe inter-tirbal relationships were more distant than intra-tribal relationships and theinter-genus relationships were more distant than intra-genus. In addition，species ofHolotrichia have the obvious diversity and the representive species between Trematodesand Brahmina were sister species.(5)The NJ，MP, ML phylogenetic trees of the subfamily Melolonthinae taxonrecovered on the basis of CO I，16S rRNA and28S rDNA sequences give the similarapproximately the phylogenetic relationships. There are three clades in the most molecular trees that the species of Melolonthini，Sericini, Hopliini clustered into their their own triberespectively with higher boostrap values. The ifrst was comprised of a large well-supportedgroup of Melolonthini including the genera of Holotrichia, AmphimaUon，Trematodes,Brahmina, Hoplosternus and Polyphylla. The second clade of Melolonthinae wascomprised of the representative species from the genus Serica, Maladera, Microserica tothe tribe Sericini. The third clade was comprised of species from the tirbe Hopliini. Themolecular trees also indicated that CO I，16S rRNA and28S rDNA had high resolution ininter-genus and intra-genus of Melolonthinae such as the genus Hoplia, Apogonia,Eriesthis, Serica, Microserica^Maladera and Onthophagus. Almost every genus membersgrouped ifrst into their own linages. However, the species of Holotrichia did not group intoits own genus，showing that the monophyly of the genus Holotrichia still was argued, andthis result was consistent with Coca-Abia’s. Therefore, the conclusion inferred from thisstudy is that CO I,16S rRNA and28S rDNA are the effective molecular markers inanalysis on the phylogeny of Melolonthinae taxon.