The Taxonomic Research Of Pollicaria Gravida

Objective:Polllicaria gravida is considered to be a specie of Pollicaria(Pupinidae),there have been no reports on the systematic morphological and molecular biology studies of living specimens in China.This project hopes to examine the shell and morphological structure of living specimens collected from Yuanjiang County,Yunnan Province.Amplify and sequence the mitochondrial gene 16S rRNA and nuclear genes 18S rRNA and 28S rRNA,the 16S rDNA,18S rDNA and 28S rDNA data sets of Polllicaria gravida and related groups were established,calculate the genetic distances between various groups,construct molecular phylogenetic trees based on the maximum likelihood method(ML),the maximum parsimony method(MP)and the Bayesian Inference(BI)for each data set,provide more basis for clarifying the taxonomic status of Polllicaria gravida from morphological and molecular levels.Methods:1.Collect live specimens of Pollicaria gravida in Yuanjiang County,Yunnan Province.2.The shell height,shell width,aperture height and aperture width were measured separately,and use Excel to calculate the average and standard deviation.3.After dehulled the living specimen and washed the mucus on the body surface of the specimen,dissect the specimen under a stereo microscope,observe and recorde its structure.Draw the anatomical illustration of Pollicaria gravida.4.Use the DNA extraction kit to extract the whole genome DNA of Pollicaria gravida,amplify and sequence 16S rRNA,18S rRNA and 28S rRNA genes by PCR.5.The 16S rDNA,18S rDNA and 28S rDNA sequences of other species of the superfamily Cyclophoridae which Pollicaria gravida belongs to were downloaded from the GenBank of the American Center for Bioinformatics(NCBI).Use Clustal X for sequence alignment.Use MEGA X,select the Kimura 2-parameter nucleotide substitution model,calculate the genetic distances of the 16S rRNA,18S rRNA and 28S rRNA genes in the relevant taxa,and analyze the base mutation saturation.6.Use MEGA X,PAUP 4.0a 165 and Mrbayes 3.2,take Bellamya bengalensis and Viviparus georgianus which belong to Viviparidae as the outer group,select the appropriate nucleotide replacement model,construct the phylogenetic tree according to the single gene and combined gene of 16S rRNA,18Sr RNA and 28S rRNA based on maximum likelihood method(ML),maximum parsimony method(MP)and Bayesian Inference(BI)respectively.Results:1.Two field trips to Yuanjiang County,Yunnan Province,collected 37 and 7 live specimens,respectively,a total of 44.2.Gained the average of the shell height,shell width,aperture height and aperture width of the measured specimens,and compare them with the relevant data of the existing research.The results are basically consistent.3.Compared the anatomical structure of Pollicaria gravida with the existing literature of the species of the genu Pollicaria.The descriptions are basically consistent.4.The lengths of 16S rDNA,18S rDNA and 28S rDNA fragments of Pollicaria gravida are 480 bp,317 bp and 609 bp,respectively,by PCR amplification and sequencing.The sequences are 422 bp,366 bp and 773 bp after alignment with the sequence of the superfamily Cyclophoridae and the outer group.5.Analyzed the genetic distances of Pollicaria gravida with other groups of 16S rRNA,18S rRNA and 28S rRNA genes,the results indicate that Pollicaria gravida has a close relationship with the genu Pollicaria.6.Constructed the phylogenetic trees according to the single gene and combined gene of 16S rRNA,18SrRNA and 28S rRNA of the Pollicaria gravida and other groups based on ML,MP and BI,respectively.Conclusions:1.The shell morphology and anatomy of Pollicaria gravida are basically consistent with the records in the existing literature regarding the genu Pollicaria and Pollicaria gravida.2.Pollicaria gravida is closely related to the species of the genu Pollicaria.