Genetic differentiation between sandfly populations of Phlebotomus chinensis and Phlebotomus sichuanensis (Diptera: Psychodidae) in China inferred by microsatellites

Phlebotomus chinensis is a primary vector of visceral leishmaniasis; it occurs in various biotopes with a large geographical distribution, ranging from Yangtze River to northeast China. Phlebotomus sichuanensis, a species closely related to P. chinensis in high altitude regions, has a long term disputation on its taxonomic status. Both species occur in the current epidemic regions and are responsible for the transmission of leishmaniasis. Population genetic analysis will help to understand the population structure and infer the relationship for morphologically indistinguishable cryptic species. In this study, microsatellite markers were used for studying the genetic differentiation between P. chinensis and P. sichuanensis. Sandflies were collected in 6 representative localities in China in 2005-2009. Ten microsatellite loci were used to estimate population genetic diversity. The intra-population genetic diversity, genetic differentiation and effective population size were estimated. All 10 microsatellite loci were highly polymorphic across populations, with high allelic richness and heterozygosity. Hardy-Weinberg disequilibrium was found in 23 out of 60 (38.33%) comparisons associated with heterozygote deficits, which was likely caused by the presence of null allele and the Wahlund effect. Bayesian clustering analysis revealed three clusters. The cluster I included almost all specimens in the sample SCD collected at high altitude habitats in Sichuan. The other two clusters were shared by the remaining 5 populations, SCJ in Sichuan, GSZ in Gansu, SXL and SXX in Shaanxi and HNS in Henan. The diversity among these 5 populations was low (FST = -0.003-0.090) and no isolation by distance was detected. AMOVA analysis suggested that the variations were largely derived from individuals within populations and among individuals. Consistently, the analysis of ribosomal DNA second internal transcribed spacer (ITS2) sequence uncovered three types of variants, which corresponded with the three gene pools revealed by microsatellites. The data suggested that the SCD population carried a distinct gene pool, which was differentiated from the other populations. The high altitude ecological habitats, distinctive ITS2 and herein divergence inferred by microsatellite loci support the species status of P. sichuanensis. The P. chinensis populations did not have a significant divergence from each another.