MOLECULAR BIOLOGY/GENOMICS Ribosomal DNA ITS2 Sequences Differentiate Six Species in the Anopheles crucians Complex (Diptera: Culicidae)

Anopheles cruciansWiedemann(sensu lato)was investigated for thepresenceof cryptic species using rDNA ITS2 sequences. This complex of species presently contains the named species An. crucians, An. bradleyi King, and An. georgianus King. Adult female mosquitoes were collected at 28 sites in Alabama, Florida, Georgia, North Carolina, Mississippi, and Louisiana, resulting in 245 progeny broods. Species were identiÞed using preliminarymorphological characters, and the internal transcribed spacer two (ITS2) was ampliÞed from all broods. The result was Þve distinct sizes of ampliÞcation product, and based on morphological characters, one of the size classes was suspected to consist of two species. All six putative species were then sequenced: Þve directly, and the sixth, because of extreme intragenomic (each individual with many variants) size variability, cloned. The ITS2 sequences were markedly distinct for all six species. Species designations and ITS2 sequence lengths (base pairs in parentheses) were A (461), B (1,000 ), C (204), D (293), E (195), and An. bradleyi (208). Species B showed both large intraspeciÞc and intragenomic sequence variability and is distinguished by having the longest ITS2 found so far in anAnopheles. Based on these data, we found that all species could be identiÞed with polymerase chain reaction (PCR) using a mixture of four primers in a single reaction. Members of this complex were often found in sympatry, with the adults of Þve species collected at a single site in central Florida.