Taxonomy And DNA Bar-coding Of The Dichaetophora Tribolita And Sinensis Species Groups(Diptear:Drosophilidae)

The genus Dichaetophora(Diptera:Drosophila)is composed of four specie groups,among which the smallest is the Di.sinensis species group established in 2005 by Hu and Toda with four species found in southern China.In this study,numbers of specimens collected in recent years and then preliminarily identified as of the sinensis species group were further examined and identified into morpho-species.These morpho-species were then tested by incorporating data of DNA-barcoding and geographical origin of the specimens.As a result,all the specimens proved to be of Dichaetophora,and were subdivided into two assemblages.The assemblage I is composed of six new species,all showing morphological affinity to the sinensis species group but differs from them in some characters,thus are included in a newly established species group;all species of the assemblage II,including two new ones,proved to be members of the sinensis group.The study can increase our understanding of the geographical distribution and species diversity of Dichaetophora,moreover,it is fundamental to research the evolutionary history of the genus.The results and conclusions of the present study are summarized as the following.1.Assemblage I:initially four morpho-species were recognized:p.K1,sp.K2,sp.K2-like and sp.K3.The first three of these morpho-species were supported in DN A barcoding analysis.Though all the species pertaining to sp.K3 formed a well supported cluster,three more or less diverged lineages,each composing of specimens from one of the Borneo(Malaysia),Java(Indonesia)and Sumatra(Indonesia)islands,respectively,was recognized.This indicating that,there may be cryptic species harboring in sp.K3.Further detailed morphological inspection revealed some morphological divergence among the three lineages.In addition,comparison of barcode sequences among the relevant species revealed that,multiple diagnostic nucleotide sites exist to distinguish each of the three lineage from the other compared lineage/species.Finally,six new species were identified:trilobita sp.n.,heterochroma sp.n.,flatosternata sp.n.,borneoensis sp.n.,javaensis sp.n.and sumatrensis sp.n.,and a new species group-trilobita species group,was established to include these six species.2.Assemblage Ⅱ:two new species,multisetata sp.n.and infundibularis sp.n.,were finally identified.Co-amplification of orthologous mitochondrial COI sequence and nuclear mitochondrial pseudogenes(NUMTs)was found when amplifying the barcode region of the new species multisetata sp.n.using the Folmer primer pair.In this case,inner,conserved PCR primers were found based on available barcodes to determine the orthologous mitochondrial sequence.By comparing the resulting sequences with those of the other species,and the suspected NUMT sequence,including examining presence of indels/point mutations/in-frame stop codons and base composition at the third codon position,the NUMTs were identified.It was also revealed that,two species previously assigned in the Di.agbo species group,cirricauda and neocirricauda,should be moved to the sinensis species group,with hainanensis,which was described as a member of this species group in 2005,treated as junior synomym of cirricauda.The two new species,multisetata sp.n.and infundibularis sp.n.,were described.