A Study On The Classification And Segregation Diversity Of Chinese Genus

The genus Itara(Orthoptera:Gryllidea)was established by Francis Walker in 1869 with Itara sericea Walker,1869 as the type species.Up to now,in total 57 species within 10 subgenera have been reported worldwide and mostly are distributed in Asia-tropical areas.Among them,eight species of Itara recorded from China are located in southern region in our country.The external morphological characteristics of the individuals of genus Itara are very similar,and the definite identification of the known species is mainly based on male genitalia features.Although we have conducted systematical studies on classification to make a clear understanding of the taxonomy of most Chinese species in this genus,there are still many changes on male's genitalia in some individuals remaining indistinct or can not be identified.For that reason,we could not confirm whether they possess an intraspecific relationship or an interspecific relationship.Previous studies on Itara mostly adopted morphological methods but few molecular markers were used on them.Therefore,relevant research on this group was carried out with the help of molecular biology methods in this study,we hope to provide some necessary supplements to morphological taxonomy and make clearer on the relationship of these species.Here,DNA barcode of COI and 16S rDNA gene markers were used to identity and explore cryptic diversity of Itara species based on distance-based method and tree method(Neighbor-joining method and Bayesian method).Based on the Neighbor-joining method and Bayesian method,we conducted molecular phylogenetic analysis combined sequences of 16S rDNA-COI-COII and 16S rDNA-CO?-CO?-Cytb to reveal the interspecific phylogenetic relationship of Chinese Itara speices.Furthermore,some morphometric methods were carried out to analyze the multifarious phenotypes of male's genitalia of individuals and inter-individuals.The rules of variation of phenotypic diversity were carefully summarized by genetic distance and phylogenetic relationships among species.Then,we also eagger to reveal some causes of these morphological diversities.The main results are as follows:(1)In this study,a total of 316 male individuals of Itara species were collected from Yunnan,Guangdong,Hainan,Shenzhen,etc.And all samples were classified as 8 species by genitalia morphological characteristics.(2)Seven species of Itara of China were classificated and determinated by DNA barcoding COI and 16SrDNA gene sequences,and the cryptic diversity was explored.I.vietnamensis,I.communi and Ldicrana were classified as Itara vietnamensis group.I.minor,I.denudate,I.aperta and I.sonabilis were proved different species,which consistented with the morphological classification.The molecular experiment result indicated that there is no cryptic species was found in the study.(3)We also compared the efficiency of DNA barcode 16S rDNA and COI gene sequence.The results showed that both of them could distinguish Itara speicies,and COI gene is superior to the 16SrDNA in recognition efficiency.(4)Based on the Neighbor-joining method(NJ)and Bayesian method,we conducted molecular phylogenetic analysis by using gene markers of COI,16S rDNA,16S rDNA-COI-COII and 16S rDNA-COI-COII-Cytb to reveal interspecific phylogenetic relationship of the seven species of Itara.The results showed that two trees are basically presenting same topology.Based on the principle of two methods,the genetic relationship in the seven species was shown by the Bayesian tree with high confidence:(I.sonabilis +(I.Apert a +(Itara vietnamensis group(I.vietnamensis,I.communi,I.dicrana)+(I.denudate + I.minor)))).(5)The geometric morphometric methods were used to analyze overall shapes and changed types of epiphallus(viewed laterally),the primary morphological identification features of the three species(I.vietnamens,I.communis and I.dicrana)in Itara vietnamensis group.The results showed significant differences presented in the size and the shape of epiphallus(lateral view)of those three species.The genetic diversity of K2P(Kimura's 2-parameter)genetic distances was calculated by using 16S rDNA-COI-COII-Cytb gene haplotype sequence,and the molecular system was studied based on Neighbor-joining method(NJ)and Bayesian method.The results indicated that there was no difference in genetic distance in them and did not show closer relationship in same species or same geographical population.We found that male genitalia features showed a significant difference while mitochondrial DNA are difficult to distinguish.Therefore,we speculated the situation could be due to the positive selection on nuclear gene,but there is no associated gene in mitochondria.And the morphological variation occurred mainly because some mutations emerged in nuclear genes which would control the formation of genitalia morphology.However,both the causes of short period of variation and the absence of reproductive isolation will not contribute to enough variation in mitochondrial gene which could express some difference when comparing their sequences.But we did not conduct any research on the cause and mechanism of this situation.Therefore,the cause of the morphology diversity of Itara vietnamensis group's three species remains to be further research.