The Molecular Phylogenetic Research Of Ruidocollaris And Sinochlora

The two genera of Ruidocollaris and Sinochlora belong to the subfamily Phaneropterinae (Insecta:Orthoptera:Tettigoniidae). Due to their extremely sensitivity to environmental changes. species within both genera can act as indicator species, which have special significance for environmental protection and biodiversity conservation. Both genera are rich in Oriental Region in China, with some species endemic to Hengduan Mountain and eastern Tibetan Plateau. The research of Ruidocollaris and Sinochlora genus for Phaneropterinae and taxonomic studying is very important.Since early 19th centuries, Foreign entomologists have started research regarding Phaneropterinae and published many monographs books. In China, however, there is still limited research on phylogenetic relationships of the subfamily Phaneropterinae as a whole or at lower taxonomic levels. Our present study analyzed, for the first time, the phylogenetic relationships of two genera Ruidocollaris and Sinochlora, which enriched our knowledge on Phaneropterinae and promoted further research on this subfamily.Using a combination of Conventional PCR, Touch-down PCR, and Gradient PCR, we obtained partial mitochondrial genes and nuclear genes sequences in 65 individuals from 5 genera in Ruidocollaris and Sinochlora as well as outgroup taxa. To analyze the phylogenetic relationships between the two genera and those within genera, the phylogenetic tree was reconstructed using Bayesian approach. The main results and conclusions are shown below:1. The ethanol soaked specimen and the dry specimen was preserved for many years, but they can be use to extract DNA and amplified specific gene. However, temperature should be focused on during the extracting, and physical damage should be avoided, for these facetors will lead to DNA degradation.2. The six mitochondrial genes(COX1, COX2, COX3, ND1,16SRNA, and 12SRNA) and three nuclear genes (H3, ITS1, and ITS2) were sequenced. The Ruidocollaris and Sinochlora genus mitochondrial genes and nucleargenes are 3750bp and 1050bp respectively.3. The third codon positions of mitochondrial protein-coding genes achieved mutational saturation. The mitochondrial genes were A+T-biased, with an A+T content of 60%, whereas the nuclear genes biased toward G+C, with a G+C content of 55%. With the exception of nuclear genes in Sinochlora, the genetic distance among species is generally high. 4. The phylogenetic tree inferred from combined genes was more robust than that based on single genes. The Ruidocollaris and Sinochlora genus are separated with the outside genus, thus supporting the monophyly of the two genera.5. The phylogenetic relationship within Ruidocollaris is (((R. convexipennis tibentensis)+(R. apennis+R. convexipennis))+((R. obscura)+(R. latilobalis+(R. trunctolobata+R. rubescens)))). Within Sinochlora, the relationship is (S. libetensis+((S. sinensis+(S. trapezialis +S. stylosa))+((S.longifissa+S. sp)+(S. hainnanensis+(S. retrolateralis+S. paraminor+ S.aequalis))).6. Within each of the two genera, species in Tibet formed a clade and diverged first from others. This might indicate that geographic barriers arisen from the uplift of Tibetan Plateau resulted in isolation and independent evolution of species.7. Many species in Sinochlora formed a multifurcative branch, revealing the breakout of speciation. S. szechwanenis, a widely distributed species, did not form a monophyletic clade, indicating gene introgression played an important role in speciation and evolution.