Molecular Phylogeny Of Steganinae And Systematic Taxonomy Of Leucophenga In The Oriental Region

Steganinae is one of the two subfamilies of family Drosophilidae;genus Leucophenga is the largest genus in the Steganinae.Molecular phylogenetic studies of the Steganinae and the Leucophenga are very few.To date,there are only 23 species of the genus Drosophila in the Drosophilidae with complete or incomplete mitochondrial genomes.This study attempts to use mitochondrial genomes as the entry point.High throughput sequencing data of 77 species of 13 genera within the Steganinae,including 54 species of the Leucophenga was obtained from low-coverage whole genome sequencing.These data was used for mitochondrial genome assembly,and the assembled mitochondrial genomes were used for phylogenetic analysis and divergence time estimation.Simultaneously,the basic characteristics of whole mitochondrial genome of one species on behalf of the Steganinae were studied.Besides,1,106 DNA barcoding sequences were used for analyzing and accessing the species delimitation accuracy of the Leucophenga by three methods.Based on the mitochondrial genomes and DNA barcoding sequences,a phylogenetic analysis of the Leucophenga was conducted.Systematic taxonomy of 100 species of the Leucophenga in the Oriental region was studied based on the results obtained from above-mentioned analyses.The main results of this study are summarized as follows:(1)Mitochondrial genomes could be maximum assembled by using the combination of NOVOPlasty and Geneious.(2)One hundred and forty-seven mitochondrial genomes of 143 species of 20 genera of two families were assembled in this study,including 81 ones of 77 species of the Steganinae,63 species(ones)of the subfamily Drosophilinae,and three species(ones)of the family Ephydridae.The species number of assembled mitochondrial genomes in this study was accounted for about one-third of current dipteran species number in Gen Bank.(3)The mitochondrial genome of the Steganinae is typical arthropod mitochondrial genome.The evolutionary rates of protein-coding genes in 155 mitochondrial genomes ofthe Drosophilidae were analyzed and indicated that nad2,atp8,nad5,nad4,nad4 l,and nad6 genes are more suitable molecular genetic markers for phylogenetic analysis,and nad2,nad3,nad6,and Cytb are suitable for delimiting closely-related or cryptic species.(4)The phylogenetic analysis of 158 mitochondrial genomes of 153 species indicated genera Acletoxenus and Luzonimyia might be a more ancient clade.Except for the Leucophenga,the other 12 genera which with relatively insufficient samplings were unapplicable to explain the intergeneric genetic relationships within the Steganinae.Phylogenetic relationships within the Leucophenga and the Drosophilinae were very clear in the case of sufficient species samplings and mitochondrial genomes.These results indicated the divergence and change of habitats of important groups within the Drosophilidae might be affected by the Ice Age.(5)Results of divergence time estimation shown that the initial divergence of the Drosophilidae dated back to 41.06 million years ago(Mya).Both the species radiation of the Steganinae main clade except for the genera Acletoxenus and Luzonimyia(35.24 Mya)and the Drosophilinae(33.86 Mya)are very close to the boundary between the Eocene and Oligocene epochs.Divergence of the main clade of the two main subgenera of the Leucophenga occurs in 24.64 Mya,which is very close to the boundary between the Oligocene and Miocene epochs.Corresponding groups of these the two subgenera were mostly collected from tussocks and tree trunks,respectively.However,the only exception of the latter is the maculata species group,and its divergence time(11.28 Mya)is very close to the boundary between the middle and late Miocene epochs.(6)Arranging the results of used species delimitation methods for 1,106 DNA barcoding sequences of the Leucophenga by decreasing accuracy: ABGD > GMYC(single-threshold model)> b PTP > m PTP.The highest accuracy was only 61.27%.Therefore,it is not recommended to use only these methods and DNA barcoding sequences for delimiting Leucophenga species.(7)Compated with the phylogenetic framework based on only mitochondrial genomes,the phylogenetic framework based on both them and DNA barcoding sequences provided roughly similar topology except for two end nodes.The Leucophenga was divided into three main clades and multiple sub-clades,the monophyly of the ornata species group and the proxima species group was not supported.(8)The phylogenetic results of the Leucophenga were basically adopted and combined with systematic taxonomy in this study.This genus was suggested to be divided as three subgenus: the Leucophenga,the Herbiaccola subgenus nov.,and the Prolixinervissubgenus nov.Two new species groups were suggested to be established for the subgenus Leucophenga: the L.(L.)atrinervis species group nov.and the L.(L.)quadripunctata species group nov.Three new species groups were suggested to be established for the subgenus Herbiaccola: the L.(H.)forcipula species group nov.,the L.(H.)jacobsoni species group nov.,and the L.(H.)varinervis species group nov.(9)Main diagnostic characters,geographical distributions,related figures and other data,and a morphological key were provided for 100 Leucophenga species from the Oriental region in the study of their systematic taxonomy.