Species Boundary Delimitation And Molecular Detection Of The Genus Fruhstorferiola In China(Orthoptera:Acrididae:Melanoplinae)

The genus Fruhstorferiola Willemse,1922,belongs to Orthoptera,Acridoidea,Catantopidae,Melaninae.The purpose of this study is to enrich the data of molecular systematics of the genus Fruhstorferiola,and to explore the monophylity of the genus Fruhstorferiola and the independence of some morphological species in the genus Fruhstorferiola.In this study,72 mitochondrial genomes of 7 species of the genus Fruhstorferiola Willemse,1922 were obtained by high-throughput sequencing combined with cloning and sequencing.And the main characteristics of the mitochondrial genomes of Fruhstorferia were summarized.Based on the data of 20 species of mitochondria from GenBank,the phylogeny of the genus Fruhstorferia was studied by various methods(ML,BI,etc.).Based on the mitochondrial gene data of 72 species,NJ clustering analysis,genetic distance calculation,haplotype network construction and other methods were used to study the species demarcation of the genus Fruhstorferia.The success rate of bar code molecular identification was tested by the mitochondrial COX1 gene data of 72 species and the accumulated COX1 gene data of the genus Fruhstorferia,The main results are as follows:1.Mitochondrial genome characterization of 7 species of genus Fruhstorferiola.The mitochondrial genome size of 7 species of genus Fruhstorferiola ranged from 15679bp to 15727bp.The gene composition is the same as that of Drosophila yakubay and the gene rearrangement is the translocation of trnK and trnD genes compared with Drosophila yakuba.With the prominent A+T content,the content of A+T base in the whole mitochondrial genome is more than 75%.All of the 13 protein coding genes were standard ATN.Most of the termination codons are complete TAA or TAG,only the termination codon of COXI gene is incomplete T.21 transport RNA secondary structures are the usual clover-leaf structure,only tmSAGN secondary structure lacks a DHU arm,which does not constitute the usual clover-leaf structure.G-U mismatch is the main type of mismatch.Mitochondrial genome characterization of 7 species of genus Fruhstorferiola was consistent with the reportedof Acridoidea.2.The phylogenetic analysis of genus Fruhstorferiola base on 13PCGs and 2rRNA gene sequence.The phylogenetic trees obtained in this studyhas the same topological structure on the higher order element,which are consistent with the morphological classification system.At the same time,all of the phylogenetic trees support the monophylicity of the genus Fruhstorferiola.In the genus Fruhstorferiola,most of the system trees show that F.tonkinensis is located at the root position;some species in the F.viridifemorata group are interlaced with some species in the F.huangshanensis group,so it is difficult to distinguish them.3.The species boundary delimitation analysis of genus Fruhstorferiola base on 13PCGs and 2rRNA gene sequence.(1)The phylogeny of F.sibynecerca and F.cerinitibia.Morphologically,there was no significant difference between F.sibynecerca and F.cerinitibia.The phylogenetic tree and the haplotype network showed that,F.cerinitibia and F.sibynecerca were mixed with each other,which could not form a independent monophyletic branch.Genetic distance analysis showed that the genetic distance between the two species almost overlapped.In conclusion,it can be concluded that the two species are synonymous.(2)The phylogeny of F.huayinensis and F.kulinga.Morphologically,the body length of F.huayinensisand F.kulingawas significantly shorter than that of other species in same genus.In most of the results of molecular data analysis in this study,there are relatively independent branches between the F.huayinensis and the F.kulinga.But in partial phylogenetic tree and haplotype network,they are not monophyletic to each other.The sampling range of the two species in this study was insufficient.The phylogenetic analysis of the two species needs more information to further analyze.(3)The phylogeny of F.viridifemorata and F.huangshanensis group.The results of most phylogenetic trees show that the monophyletic branches formed by the F.huangshanensis group fall in the branches of the F.viridifemorata,but there are obvious differences in morphological characteristics between them.Genetic distance analysis showed that there was a very small genetic distance between F.viridifemorata and F.huangshanensis group,even a different degree of overlap between the intraspecific and interspecific genetic distances;in almost all the haplotype network maps of gene sequences,F.viridifemorata and F.huangshanensis group could not form a single line of each other.At present,the mitochondrial genome data can not fully confirm whether there is incomplete pedigree separation between F.viridifemorata and F.huangshanensis group.In the future,more data such as nuclear genes are needed to verify the conjecture.(4)The success of rate detection from Molecular identification barcode.The fuzzy identification and wrong identification sequences mainly appeared between F.sibynecerca and F.cerinitibia,F.huayinensis and F.kulinga,F.viridifemorata and F.huangshanensis group.Based on the data of phylogeny and genetic distance,it is speculated that the main reason for the low accuracy is incomplete pedigree selection and classification.