Ribosomal DNA sequence markers differentiate Two species of the Anopheles maculatus (Diptera: Culicidae) complex in the Philippines

The Anopheles maculatus Theobald complex includes important vectors of malaria. Based on chromosomal and morphological evidence, two species in this complex occur in the Philippines. Because separation of these species, An. dispar Rattanarithikul & Harbach and An. greeni Rattanarithikul & Harbach, is problematic due to the difficulty or unreliability of the identification methods currently available, we sought a molecular technique for identifying these two species. We sequenced two regions of nuclear ribosomal DNA; the second internal transcribed spacer (ITS2) and the third domain (D3) of the 28S gene, from An. maculatus sensu late (s.l.) collected throughout the Philippines. Two sequence groups were identified that corresponded morphologically to An. dispar and An. greeni. Four percent of the 318-320 bp ITS2 and 2.5% of the 367 bp D3 differed between the two species. No evidence of intraspecific variation in sequences was found. From the sequence data, we developed a more reliable and easier method for identifying An. dispar and An. green., based on a HaeII restriction fragment-length polymorphism in a polymerase chain reaction amplified fragment of ITS2. This method will facilitate future vector studies, which will be necessary, as previous data collected on An. maculatus s.l. in the Philippines is unreliable given the multispecies nature of this taxon.