Cryptic Species in the <I>Anopheles</I> (<I>Nyssorhynchus</I>) <I>albitarsis</I> (Diptera: Culicidae) Complex: Incongruence Between Random Amplified Polymorphic DNA-Polymerase Chain Reaction Identification and Analysis of Mitochondrial DNA <I>COI</I> Gene Sequences

Abstract

Random amplified polymorphic DNA (RAPD) diagnostic bands are one tool used to differentiate cryptic mosquito species in the Anopheles albitarsis Complex. Monophyly of four species (A. albitarsis Lynch-Arribálzaga, A. albitarsis B, A. deaneorum Rosa-Freitas, and A. marajoara Galvão & Damasceno) currently identified with the RAPD technique was assessed using sequences of the cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) gene. Maximum parsimony, maximum likelihood, and Bayesian analyses support monophyly for A. albitarsis s.s., A. albitarsis B, and A. deaneorum. Anopheles marajoara, as identified by RAPD banding patterns, was either polyphyletic or paraphyletic in all phylogenetic analyses. The phylogenetic pattern and within-species genetic distances observed in A. marajoara suggest the existence of a previously unidentified species (species E) in northern Brazil and Venezuela. Diagnostic RAPD bands were unable to distinguish between A. marajoara and species E, probably because of the low number of correlated bands used to identify species and weaknesses of the RAPD technique, in particular, violations of the untested assumption of homology of comigrating bands. A. marajoara (even without species E) is paraphyletic with respect to A. deaneorum; if A. deaneorum is a separate species from A. marajoara, then A. marajoara may consist of two or more species in Amazonian Brazil. Based on mtDNA COI sequences, there are at least four phylogenetic species within the Albitarsis Complex: A. albitarsis s.s., A. albitarsis B, A. marajoara, and species E; the species status of A. deaneorum is ambiguous.

The Anopheles albitarsis complex with the recognition of Anopheles oryzalimnetes Wilkerson and Motoki, n. sp. and Anopheles janconnae Wilkerson and Sallum, n. sp. (Diptera: Culicidae)

“…A preferable method of identification is the use of a combination of species-specific primers based on rDNA ITS2 sequence developed by Li and Wilkerson (2005). Lehr et al (2005) and Wilkerson et al (2005) corroborated the monophyly of An. albitarsis when using COI mtDNA sequence data and combined COI, ND4 mtDNA, ITS2, 28S rDNA sequence data, respectively.…”

“…An. albitarsis is found in Argentina, Provinces of Misiones, Corrientes and Buenos Aires; Brazil, states of Santa Catarina, Paraná (PR) and SP (Wilkerson et al 1995c, Lehr et al 2005; and Paraguay, state of Alto Paraná (Wilkerson et al 1995c). …”

“…marajoara can be distinguished from other species of the complex by species specific ITS2 sequence. Lehr et al (2005), using COI mtDNA sequence data, found at least two species under the name An. marajoara among specimens that were first identified by the RAPD-PCR profile and ITS2 sequence.…”

“…albitarsis B (Wilkerson et al 1995c), An. albitarsis E (Lehr et al 2005) and An. albitarsis F (Brochero et al 2007).…”

“…albitarsis B. Recently, Lehr et al (2005), based on the results of phylogenetic analyses of the mitochondrial cytochrome c oxidase subunit I (mtDNA COI) gene, proposed a fifth species in the complex, An. albitarsis E. In contrast, Wilkerson (2005, 2006) observed consistent and diagnostic, but intragenomically variable differences in the ITS2 of the four species of An.…”