Author's personal copy A molecular phylogeny of mosquitoes in the Anopheles barbirostris Subgroup reveals cryptic species: Implications for identification of disease vectors Claudia Paredes-Esquivel a , Martin J. Donnelly a , Ralph E. Harbach b , Harold Townson a, * a Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK b Department of Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK article info Article history: Received 26 June 2008 Revised 13 October 2008 Accepted 15 October 2008 Available online 1 November 2008 Keywords: Anopheles barbirostris Anopheles campestris Bayesian analysis Phylogeny mtDNA COI rRNA ITS2 Southeast Asia Thailand Indonesia Vector identification abstract The Barbirostris Subgroup of the genus Anopheles includes six mosquito species that are almost identical in adult morphology, but differ in their roles in the transmission of malaria and filariasis within Southeast Asia. The lack of robust, diagnostic morphological characters in adults has contributed to extensive mis- identification of the species. Mosquitoes were collected from localities in Thailand and Indonesia, with an emphasis on specimens identified in the field as An. barbirostris and An. campestris. A 754 bp COI mito- chondrial gene fragment was sequenced from 136 specimens and the rDNA ITS2 region (c.1600– 1800 bp) from 51 specimens. Phylogenetic analyzes based on Bayesian methods, distance measures and Maximum-parsimony produced five clades (I–V) that are congruent between the nuclear and mito- chondrial data sets. Based on adult female morphology, it is deduced that three of these clades, I–III, are members of the Barbirostris Complex whereas Clade V is An. campestris. The identity of Clade IV is as yet unknown. Using a haplotype network analysis, Clade III was found to have a star-like genealogy, suggest- ing population expansion. There were no shared haplotypes between clades. In Afrotropical anopheline mosquitoes, speciation has been linked to the expansion of human populations and the development of agriculture. We postulate that the radiation of species within the Barbirostris Subgroup in Southeast Asia may similarly be linked to human population expansion and the agrarian revolution. The develop- ment of a propensity for feeding on the blood of humans in some species of the Subgroup would have led to the transmission of malaria protozoa and filarial nematodes. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Speciation in the Anopheles gambiae species complex, which in- cludes the principal vectors of malaria in Africa, is postulated to have been driven by human impact on the environment subse- quent to the Neolithic revolution, associated in turn with signifi- cant changes in climate and the move from a forest to savannah environment (Coluzzi et al., 1985, 2002). Studies based on micro- satellite loci and the ND5 region of the mitochondrial genome in An. gambiae and An. arabiensis led to the proposal that populations in East Africa had undergone recent range expansion rather than being at mutation-drift equilibrium (Donnelly et al., 2001). These authors suggested that the expansion could have been contempo- raneous with the agricultural revolution in sub-Saharan Africa. There have been fewer such studies of malaria vectors in Southeast Asia but increasingly there is evidence of extensive speciation with most nominal species being found to comprise species complexes (Harbach, 2004; Phuc et al., 2003; Walton et al., 2000). The ques- tion remains, to what extent has this speciation been driven by anthropogenic factors linked to the expansion of human popula- tions in the region? The Barbirostris Subgroup of the genus Anopheles (Reid, 1968; Harbach, 2004) includes six formally described species (Reid, 1962) of almost identical morphology: An. barbirostris, An. campes- tris, An. donaldi, An. franciscoi, An. hodgkini and An. pollicaris. They differ markedly in feeding preferences and hence their importance as vectors of pathogens that cause disease in humans. The precise identification of these species requires the examination of adults with associated larval and pupal exuviae (Harrison and Scanlon, 1975) but morphological examination of reared specimens is te- dious and time-consuming, and hence is not widely used in field studies. Field-collected adult female mosquitoes may lose scales that are crucial to accurate identification and may easily be misidentified. As a consequence, the distributions and vectorial roles of species of the Barbirostris Subgroup are poorly known (Apiwathnasorn et al., 2002; Harrison and Scanlon, 1975; Limrat et al., 2001). Anopheles campestris appears to be highly anthropophagic but it is often confused in the field with the more zoophagic An. barbiros- tris (Reid, 1962). The pattern of wing scales used to distinguish these species (Reid, 1962) has been considered ‘‘not valid” in 1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2008.10.011 * Corresponding author. Fax: +44 151 705 3369. E-mail address: htownson@liverpool.ac.uk (H. Townson). Molecular Phylogenetics and Evolution 50 (2009) 141–151 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev