The Hyrcanus group of Anopheles (Anopheles) in China (Diptera: Culicidae): species discrimination and phylogenetic relationships inferred by ribosomal DNA internal transcribed spacer 2 sequences.

The Hyrcanus group comprises many closely related species with wide distributions in the Oriental and Palaearctic regions. The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA were determined for 12 species in China--An. crawfordi, An. hyrcanus, An. junlianensis, An. kunmingensis, An. kweiyangensis, An. lesteri, An. liangshanensis, An. peditaeniatus, An. pullus, An. sinensis, and two unknown species within the group. The length of the ITS2 ranged from 436 bp in An. hyrcanus to 469 bp in An. crawfordi, with GC contents of 44.9-46.8%. Intraspecific variation was found in three species (An. junlianensis, An. liangshanensis, and An. pullus) at the level of 0.0-0.4%, whereas interspecific differences ranged from 1.6% between An. liangshanensis and An. kunmingensis to 50.8% between An. peditaeniatus and sp. 1. The ITS2 comparisons revealed two unknown species, verified the valid species status for An. kunmingensis, and found An. pullus in China. We agree that An. anthropophagus is a junior synonym of An. lesteri. The validation of An. junlianensis awaits recognition of the molecular identity of the entity identified as An. yatsushiroensis. The ITS2 divergences were used for inferring phylogenetic relationships among 12 species in China. The estimation revealed close relationships among An. liangshanesis, An. kunmingensis, An. kweiyangensis, An. lesteri, and An. sinensis. Our study emphasizes the need for the molecular identity of the species members in integrated studies in systematics, bionomics, and population genetics for the Hyrcanus group.