Molecular phylogenetics and biogeography of the Neocellia Series of Anopheles mosquitoes in the Oriental Region Katy Morgan a , Samantha M. O’Loughlin a,b , Fong Mun-Yik a,c , Yvonne-Marie Linton d Pradya Somboon e , Sein Min f , Pe Than Htun f , Simone Nambanya g , Indira Weerasinghe h Tho Sochantha i , Anil Prakash j , Catherine Walton a, * a Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK b NERC Centre for Population Biology, Imperial College, London, UK c University of Malaya, Kuala Lumpur, Malaysia d Department of Entomology, The Natural History Museum, London, UK e Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand f Department of Medical Research, Yangon, Myanmar g Centre of Malariology, Parasitology and Entomology, Vientiane, Laos h Institute of Medical Research, Colombo, Sri Lanka i National Centre for Malaria Control, Parasitology and Entomology, Phnom Penh, Cambodia j Regional Medical Research Centre, Dibrugarh, Assam, India article info Article history: Received 29 July 2008 Revised 28 January 2009 Accepted 29 January 2009 Available online 6 February 2009 Keywords: Anopheles Southeast Asia Phylogeography Biogeography Pleistocene climatic change Allopatric speciation Ecological speciation abstract Molecular studies of population divergence and speciation across the Oriental Region are sparse, despite the region’s high biodiversity and extensive Pliocene and Pleistocene environmental change. A molecular phylogenetic study of the Neocellia Series of Anopheles mosquitoes was undertaken to identify patterns of diversification across the Oriental Region and to infer the role of Pleistocene and Pliocene climatic change. A robust phylogeny was constructed using CO2 and ND5 mitochondrial genes and ITS2 and D3 nuclear ribosomal markers. Bayesian analysis of mitochondrial genes was used to date divergence events. The repeated contraction and expansion of forest habitat resulting from Pleistocene climatic fluctuations appears to have had a substantial impact on intraspecific diversification, but has not driven speciation within this group. Primarily early to mid Pliocene speciation was detected within the Annularis Group, whereas speciation within the Maculatus and Jamesii Groups occurred during the mid and late Pliocene. Both allopatric divergence driven by late Pliocene environmental changes and ecological adaptation, involving altitudinal replacement and seasonality, are likely to have influenced speciation in the Macul- atus Group. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Southeast Asia is an important region in terms of global biodi- versity, containing four of the 25 global biodiversity hotspots de- scribed by Myers et al. (2000). These are the island regions of Sundaland, Wallacea and the Philippines, and the mainland region of Indo-Burma. Together these regions contain an estimated 9.7% of the world’s known endemic plant species and 8.3% of the known endemic vertebrate species, the majority of which are concen- trated within tropical forest habitat (Taylor et al., 1999; Myers et al., 2000; Brook et al., 2003). Sri Lanka and the Western Ghats of India represent an additional biodiversity hotspot in the Oriental Region, which shares similar flora and fauna to the Southeast Asian hotspots. Research on Oriental biodiversity has been neglected rel- ative to that of other regions, and consequently little is understood of the processes underlying the generation of diversity across the region (Sodhi et al., 2004). The mosquito genus Anopheles is species rich within the Oriental Region, containing multiple sibling species complexes that occupy a wide variety of ecological niches (Reid, 1968; Collins and Paskewitz, 1996; Foley et al., 2007a). The genus Anopheles is therefore a good model for studying the distribution of biodiversity and the factors influencing population divergence and speciation across the region. Numerous hypotheses have been put forward to explain the species richness of the tropics, a phenomenon that has been dem- onstrated in mosquitoes (Foley et al., 2007a), as well as numerous other taxa (for reviews see Gaston, 2000; Hill and Hill, 2005; Mittlebach et al., 2007). One of the oldest and most debated of these hypotheses is the refuge model, originally proposed by Haffer 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.01.022 * Corresponding author. Address: Michael Smith Building, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Fax: +44 0161 275 5082. E-mail address: catherine.walton@manchester.ac.uk (C. Walton). Molecular Phylogenetics and Evolution 52 (2009) 588–601 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev