Molecular variation, systematics and distribution of the Anopheles fluviatilis complex in southern Asia B. CHEN 1,2 , R. K. BUTLIN 1,3 , P. M. PEDRO 1 , X. Z. WANG 4 and R. E. HARBACH 5 1 School of Biology, The University of Leeds, Leeds, U.K., 2 College of Plant Protection, South-west Agricultural University, Chongqing, China, 3 Animal and Plant Sciences, The University of Sheffield, Sheffield, U.K., 4 Yunnan Institute of Parasitic Diseases, Yunnan, China and 5 Department of Entomology, The Natural History Museum, London, U.K. Abstract. Species of the Anopheles fluviatilis James and Anopheles minimus com- plexes (Diptera: Culicidae) are difficult to distinguish morphologically. Members of the two complexes have been confused and, consequently, their distributions and roles in malaria transmission are uncertain. We identified numerous mosquitoes from China, Thailand, Vietnam, India, Nepal, Pakistan and Iran by single-strand conformation polymorphism (SSCP) and/or sequencing, and ana- lysed the variation in the 28S D3 region of ribosomal DNA for members of the Minimus Subgroup and also the internal transcribed spacer region 2 (ITS2) for members of the An. fluviatilis complex. The D3 region is highly conserved between taxa and therefore could serve as a standard for molecular diagnosis of the subgroup members. D3 sequence, bionomics and malaria transmission data provide further evidence that An. fluviatilis S in India is conspecific with An. minimus C in South-east Asia. An. fluviatilis T has three ITS2 haplotypes (designated T1, T2 and Y) and its distribution in India, Nepal, Pakistan and Iran is confirmed. An. fluviatilis U is well defined on cytogenetic grounds in Uttar Pradesh, India, but is very close to An. fluviatilis T and the two species may hybridize in some regions. Variant ITS2 sequences suggest the possible existence of two additional taxa within the An. fluviatilis complex, one in Iran and another in India, provisionally designated An. fluviatilis forms V and X, respectively. The distributions of members of the An. fluviatilis and An. minimus complexes in south-central Asia are summarized. Key words. Anopheles fluviatilis, Anopheles minimus, distribution, molecular variation, ribosomal DNA, systematics, southern Asia. Introduction Anopheles fluviatilis James was described from the Central Provinces and Jeypore Hill Tracts of India (James, 1902). Rao (1984) suggested that differences in population densi- ties, feeding preferences and Plasmodium infection rates indicated the presence of two biological forms of this spe- cies. Cytotaxonomic studies subsequently revealed the pre- sence of three cryptic species within An. fluviatilis in India, designated species S, T and U, which are distinguished by fixed inversions in polytene chromosome arm 2 (Subbarao et al., 1994). In the absence of chromosomal data, Manonmani et al. (2001) referred to two of these species from localities in Orissa State in east-central India as spe- cies X and Y based on differences in the internal tran- scribed spacer region 2 (ITS2) of rDNA. Subsequent correlation with chromosome banding revealed that 94% of species X were species S and 90% of species Y were species T (Manonmani et al., 2003). Naddaf et al. (2002, 2003) examined the ITS2 sequence in Iranian specimens and found they were identical (100% similarity) to that of species T (as species Y) in India. Recently, Singh et al. Correspondence: Dr Ralph E. Harbach, Department of Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD, U.K. Fax: þ 44 (0)20 7942 5229; e-mail: r.harbach@nhm.ac.uk Medical and Veterinary Entomology (2006) 20, 33–43 # 2006 The Authors Journal compilation # 2006 The Royal Entomological Society 33