Mitochondrial COI and Cytb gene as valid molecular identification marker of sandfly species (Diptera: Psychodidae) in China

•Six species were identified by the morphological features, belonging to Phlebotomus and Sergentomyia (Ph. chinensis s. l., Ph. stantoni, Se. bailyi, Se. iyengari, Se. squamirostris, and Se. squamipleuris).•MtDNA COI and Cytb genes are optimal for DNA barcoding to identify these sandfly species.•Sequence alignment data of mtDNA Cytb suggested that Ph. chinensis s. l. from Sichuan should be Ph. sichuanensis, and the sandfly specimens collected from Henan were Ph. chinensis s. s..•There was obvious genetic divergence in Se. bailyi populations from China. The accurate identification of sandfly species is crucial because some species transmit medically significant diseases, including leishmaniasis, bartonellosis and sandfly fever. However, due to the high similarity of the external morphology in sandfly species, identification can only be performed using internal morphological characteristics after dissection, which is time consuming and requires highly experienced staff. Thus, the introduction of suitable molecular markers may solve these identification problems. This study screened suitable DNA barcodes to identify common sandfly species in China. The phlebotomine sandflies were collected from Sichuan, Henan and Hainan Provinces from 2014 to 2016. The species were identified by the morphological characteristics of the pharyngeal armature and spermatheca. The genomic DNA of sandfly was extracted individually, and mitochondrial DNA (mtDNA) cytochrome C oxidase subunit I (COI) and cytochrome B (Cytb) as well as the 18S subunit of ribosomal DNA (rDNA) were amplified using polymerase chain reaction (PCR). Additionally, intraspecific and interspecific differences (p-distance) were calculated to evaluate the feasibility of the three gene fragments as a DNA barcode. The phylogeny trees of all sandfly species in this study were constructed using neighbor joining (NJ) method. Six species were identified by the morphological features, belonging to Phlebotomus and Sergentomyia, as Ph. chinensis s. l., Ph. stantoni, Se. bailyi, Se. iyengari, Se. squamirostris, and Se. squamipleuris. Analysis based on three gene fragments revealed some degree of intraspecific polymorphism among these sandfly species in China. The largest intraspecific variation occurred in Ph. chinensis s. l. (mtDNA COI, p-distance = 0.042; mtDNA Cytb, p-distance = 0.071), but the 18S rDNA fragment showed a small variation (p-distance = 0.005). The ranges of interspecific p-distances for mtDNA COI and mtD