The Determination Of The Mitochondrial Genome Of The Genus Chrysalis Based On High-throughput Sequencing And The Study Of Molecular Systematics

With the revolution of polymerase chain reaction,molecular maker has been widely used in taxonomic identification,species delimitation and phylogenetic reconstruction.Complete mitogenome has been considered as an efficient and useful marker.More and more complete mitogenomes are available and have been widely applied in the phylogenetic analyses owing to the development of high-throughput sequencing technology.The insect mitochondrial genome consists of 37 genes and a control region.However,one should be very cautious to choose suitable dataset and nucleotide substitution model for phylogeny construction in different scale.Besides,being used as a molecular marker for phylogeny,the mitogenome is also widely used in biogeography,accompanied with fossil records on the clade node,to speculate on the origin and differentiation time of the species,and combined with geological events on the time axis.The genus Chorthippus is the most speciose genus in Gomphocerinae.There are more than 200 species in the world,mainly distributed in Eurasia,a few species in Africa and only one species in North America.In China,it is mainly distributed in the north of the Yangtze River.In 2012,Zheng Zhemin proposed that the subgenus Megaulacobothrus should be upgraded as an independent genus.Thus,the classification scheme of subgenus composition in China is the same as in the international one.There are three subgenera in the genus Chorthippus:Altichorthippus,Chorthippus and Glyptobothrus.Although the genus has been studied based on morphological and molecular(short molecular markers)characters,its origin and evolution process are still inconclusive.In this study,15 mitogenomes of Chorthippus were obtained by using next-generation sequencing and the structure variation among species were analyzed.Depending on the different mitogenome datasets,the phylogenetic relationship of the genus Chorthippus was inferred using Bayesian inference and Maximum likelihood methods.Finally,the dating tree of the genus Chorthippus was constructed based on the mitogenome dataset and fossil calibration.The aims of this study are to explore effective and useful molecular markers for phylogenetic inference,and infer dating tree to determine Chorthippus divergence time and its potential colonization route.The conclusions of this paper mainly include three parts:1.Fifteen mitogenomes were obtained efficiently and quickly using the next-generation sequencing.The results showed that the gene order and orientation within the Chorthippus mitogenome are identical with those described in Acrididae family.The total length of the mitogenome ranges from 15,602 bp to 15,620 bp with the composition of 13 protein-coding genes(PCGs),two rRNA genes(rrnL and rrnS)and 22 tRNA genes.There is no gene order arrangement between species.The length of overlapping bases between genes were different.In the comparison of nucleotide compositional skew,the datasets showed A bias and C bias in the whole mitogenome,rRNAs(rrnL and rrnS),and AT-rich region(Dloop region);and A bias and G bias in all protein-coding genes(PCGs)and tRNAs,respectively.The length of the protein-coding gene is relatively conservative.The results of relative synonymous codon usage indicated that it preferred to use A/T than G/C in the third site of the codon.2.The phylogenetic analysis showed no significant difference in tree topologies based on different data sets,except for tRNAs.Although the combined data sets(PCGs+rRNAs+tRNAs)are considered to have the most comprehensive phylogenetic information,it is not suitable for phylogenetic studies of all subfamilies.Therefore,the data sets should be selected carefully depending on the taxon-level.The results of phylogeny showed that the genus Ch.orthippus is not a monophyletic group.Except for rRNAs,all data sets support monophyly of the subgenus Chorthippus,while the species of the other two subgenera Altichorthippus and Glyptobothrus are nested within each other and grouped into three main clades:the first clade formed by(C.hsiai +(C.albonemus + C.genheensis),the second clade formed by(C.fallax ?(C.atridorsus ?C.biguttulus))and the last one(C.intermedius ?(C.louguanensis+(C.huchengensis ?C.hammarstroemi))).Our results proposed to merge subgenera Altichorthippus into Glyptobothrus as one independent genus.3.The divergence time of the genus Chorthippus was probably in the late Miocene.The common ancestor of the subgenus Chorthippus originated in the late Miocene.The subgenus Chorthippus diverged recently.The results inferred that there probably was a refuge for this subgenus in the Quaternary glacial period;after the climate became warm,it spread widely.At last,we need more sampling to confirm the results in this study.The attempt of nuclear marker with more variable sites to solve the phylogenetic problem between genera Altichorthippus and Glyptobothrus will be applied in the next step.