Abstract. Cryptic species and lineages characterize Anopheles nuneztovari s.l. Gabaldón, an important malaria vector in South America. We investigated the phylogeographic structure across the range of this species with cytochrome oxidase subunit I ( COI ) mitochondrial DNA sequences to estimate the number of clades and levels of divergence. Bayesian and maximum-likelihood phylogenetic analyses detected four groups distributed in two major monophyletic clades (I and II). Samples from the Amazon Basin were clustered in clade I, as were subclades II-A and II-B, whereas those from Bolivia/Colombia/ Venezuela were restricted to one basal subclade (II-C). These data, together with a statistical parsimony network, confirm results of previous studies that An. nuneztovari is a species complex consisting of at least two cryptic taxa, one occurring in Colombia and Venezuela and the another occurring in the Amazon Basin. These data also suggest that additional incipient species may exist in the Amazon Basin. Divergence time and expansion tests suggested that these groups separated and expanded in the Pleistocene Epoch. In addition, the COI sequences clearly separated An. nuneztovari s.l. from the closely related species An. dunhami Causey, and three new records are reported for An. dunhami in Amazonian Brazil. These findings are relevant for vector control programs in areas where both species occur. Our analyses support dynamic geologic and landscape changes in northern South America, and infer particularly active divergence during the Pleistocene Epoch for New World anophelines.* Address correspondence to Vera Margarete Scarpassa, Instituto Nacional de Pesquisas da Amazônia, Avenida André Araujo, no. 2936, Bairro Aleixo, Manaus, CEP 69060-001, Amazonas, Brazil. E-mail: vera@inpa.gov.br 858 SCARPASSA AND CONN field-collected specimens or crossing experiments between suspected taxa, distinct modes and rates of evolu tion of the various molecular markers used, and a recent temporal framework for evolutionary processes within An. nuneztovari s.l. 30 CURRENT TAXONOMIC SITUATION Anopheles nuneztovari was originally described from San Carlos, Cojedes State, in western Venezuela, on the basis of on male genitalia. 31 Rozeboom and Gabaldón 32 described An. goeldii from morphology of eggs, larvae, females, and males collected in Belterra (Fortlandia), on the Tapajós River, in Pará State, Brazil. ( Figure 1 ).We investigated the phylogeographic structure of An. nuneztovari s.l. from 17 localities in northern South America (Amazonian Brazil, Bolivia, Colombia, Suriname, and Venezuela) to estimate the number of clades and levels of divergence by using an 873-basepair fragment of the mtDNA COI gene. Within Amazonian Brazil, we included 11 localities, 5 of which have not been studied (MA, PI, BR, CS, CR; Figure 1 ), to address three questions. 1) How many lineages exist within Table 1
MATERIALS AND METHODSMosquito collection and identification. Samples of An. nuneztovari s.l. were collected from 11 sites in Amazonian Brazil (MA, NM, B...