Molecular phylogeny of Anopheles hyrcanus group members based on ITS2 rDNA

Abstract

Background: The Anopheles hyrcanus group includes 25 species, and is widely distributed in the Oriental and Palaearctic regions. Several species within this group are vectors of malaria, lymphatic filariasis and Japanese encephalitis. It is difficult or impossible to identify cryptic species based on their morphological characteristics, with some closely related species of the Hyrcanus Group have similar adult morphological characteristics. Thus, their molecular identification has been an important complementary method to traditional morphological taxonomy.

Methods: We used 461 ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) sequences relating to 19 species to reconstruct the molecular phylogeny of the Hyrcanus Group across its range. In addition, we compared the performance of rDNA ITS2 to that of mitochondrial DNA (mtDNA) cytochrome c oxidase subunit 1 gene (cox1) to assess the genetic divergence of Hyrcanus Group sibling species.

Results: Based on Kimura's 2-parameter (K2P) distance model, the average conspecific ITS2 divergence was 0.003, whereas sequence divergence between species averaged 0.480. Average ITS2 sequence divergences were almost 160 times higher among the Hyrcanus Group members than within each species. Two sets of sibling species, An. lesteri Baisas & Hu, 1936 and An. paraliae Sandosham, 1959; and An. sinensis Wiedemann, 1828, An. belenrae Rueda, 2005, and An. kleini Rueda, 2005, were resolved by ITS2. Each of these species was represented as an independent lineage in the phylogenetic tree. Results suggest that An. pseudopictus Grassi, 1899 and An. hyrcanus (Pallas, 1771) are most likely a single species. We uncovered two new ITS2 lineages that require further study before resolving their true taxonomic status, and designed a diagnostic polymerase chain reaction (PCR) assay to distinguish five morphologically similar species.

Conclusions: Nuclear and mitochondrial genes generally provided consistent results for subgroup division. Compared to cox1, ITS2 is a more reliable tool for studying phylogenetic relationships among closely related mosquito taxa. Based on species-specific differences in ITS2 sequences, the multiplex PCR assay developed here can be used to improve the efficiency of vector identification. Thus, this research will promote the progress of malaria vector surveillance in both epidemic and non-epidemic areas of South and East Asia.

Keywords: Anopheles; DNA barcoding; Malaria.

Fig. 1

Neighbour-joining phylogenetic tree of the Hyrcanus Group based on ITS2 sequences from GenBank and our original data. Bootstrap values (1000 replicates) of neighbour-joining analyses are shown above/below the main lineages. Lineage designation is indicated on the right. The geometric shapes correspond to different subgroups of the Hyrcanus Group, according to the classification of Harbach [1], Lesteri Subgroup; Nigerrimus Subgroup; unclassified species. Bars represent 0.04 substitutions per site. Anopheles lindesayi and An. claviger were used as the outgroup taxa

Fig. 2

Intra- and interspecific ITS2 divergences in 19 Hyrcanus Group members determined using Kimura’s 2-parameter distance. The barcoding gap ranged 0.014–0.081. Y-axis, genetic divergence; x-axis, Hyrcanus Group members

Fig. 3

Polymerase chain reaction (PCR) products of a multiplex PCR assay. Lane M: PCR marker; Lanes 1 and 2: An. peditaeniatus; Lanes 3 and 4: An. hyrcanus; Lanes 5 and 6: An. lesteri; Lanes 7 and 8: An. pullus; Lanes 9 and 10: An. sinensis; Lanes 11 and 12: negative controls

Fig. 4

ITS2 and cox1 sequence divergences in the Hyrcanus Group. The minimum interspecific (intergroup) divergence is plotted against the maximum intraspecific divergence. ITS2 has more dots above the diagonal line in the top left-hand corner than cox1, indicating low intraspecific and high interspecific divergence