A molecular phylogeny of Anopheles annulipes (Diptera: Culicidae) sensu lato: The most species-rich anopheline complex

The Australasian Annulipes Complex is the most species-rich among Anopheles mosquitoes, with at least 15 sibling species suspected. Members of this complex are the most likely vectors of malaria in the past in southern Australia and are involved in the spread of myxomatosis among rabbits. In this, the first comprehensive molecular study of the Annulipes Complex, 23 ITS2 rDNA variants were detected from collections throughout Australia and Papua New Guinea, including diagnostic variants for the previously identified An. annulipes species A–G. Specimens of each ITS2 variant were sequenced for portions of the mitochondrial COI, COII and nuclear EF-1α genes. Partitioned Bayesian and Maximum Parsimony analyses confirmed the monophyly of the Annulipes Complex and revealed at least 17 clades that we designate species A–Q. These species belong to two major clades, one in the north and one mainly in the south, suggesting that climate was a driver of species radiation. We found that 65% (11) of the 17 sibling species recorded here had unique COI sequences, suggesting that DNA barcoding will be useful for diagnosing species within the Annulipes Complex. A comparison of the taxa revealed morphological characters that may be diagnostic for some species. Our results substantially increase the size of the subgenus Cellia in Australasia, and will assist species-level studies of the Annulipes Complex.