Phylogenetic Network of Mitochondrial COI Gene Sequences Distinguishes 10 Taxa Within the Neotropical Albitarsis Group (Diptera: Culicidae), Confirming the Separate Species Status of Anopheles albitarsis H (Diptera: Culicidae) and Revealing a Novel Lineage, Anopheles albitarsis J.

The Neotropical Albitarsis Group is a complex assemblage of essentially isomorphic species which currently comprises eight recognized species-five formally described (Anopheles albitarsis Lynch-Arribalzaga, An. deaneorum Rosa-Freitas, An. janconnae Wilkerson and Sallum, An. marajoara Galvao and Damasceno, An. oryzalimnetes Wilkerson and Motoki) and three molecularly assigned (An. albitarsis F, G & I)-and one mitochondrial lineage (An. albitarsis H). To further explore species recognition within this important group, 658 base pairs of the mitochondrial DNA cytochrome oxidase subunit I (COI) were analyzed from 988 specimens from South America. We conducted statistical parsimony network analysis, generated estimates of haplotype, nucleotide, genetic differentiation, divergence time, and tested the effect of isolation by distance (IBD). Ten clusters were identified, which confirmed the validity of the eight previously determined species, and confirmed the specific status of the previous mitochondrial lineage An. albitarsis H. High levels of diversity were highlighted in two samples from Pará (= An. albitarsis J), which needs further exploration through additional sampling, but which may indicate another cryptic species. The highest intra-specific nucleotide diversity was observed in An. deaneorum, and the lowest in An. marajoara. Significant correlation between genetic and geographical distance was observed only in An. oryzalimnetes and An. albitarsis F. Divergence time within the Albitarsis Group was estimated at 0.58-2.25 Mya, during the Pleistocene. The COI barcode region was considered an effective marker for species recognition within the Albitarsis Group and a network approach was an analytical method to discriminate among species of this group.

Morphometric comparisons of the scanning electron micrographs of the eggs of Anopheles (Nyssorhynchus) darlingi Root (Diptera: Culicidae).

Anopheles darlingi Root is the principal vector of Plasmodium in Brazil, but its biological variability is not well known. Morphometric analyses of scanning electron microscopy images of the eggs of An. darlingi were conducted using individuals collected in nine states of Brazil (Acre, Amapá, Espírito Santo, Pará, Paraná, Rio de Janeiro, Rondônia, São Paulo, and Tocantins). Ten attributes of the eggs (seven continuous variables and three discrete variables) were respectively measured or counted and analyzed to determine if populations from different geographical regions or biomes could be distinguished. Univariate analysis showed that the eggs from Espírito Santo were the narrowest whereas representatives from Tocantins populations had the smallest floats. Results of multivariate analyses of continuous variables showed that the first principal component (PC1), mainly represented by all four float attributes, helped to differentiate populations. The second principal component (PC2) comprised roughly the length and width of the egg. PC1 of discrete variables corresponded to the number of ribs on the float whereas PC2 was approximately equivalent to the number of discs on the micropyle. Based on those variables (continuous and discrete separately), multivariate discriminant analysis indicated that eggs from individuals collected in Tocantins were distinct from the other populations. Among sampled localities, the one from the state of Tocantins was situated within the Cerrado biome whereas the locality from São Paulo state was at the border of Cerrado, within a transition zone of the Atlantic Forest biome. Generally, the climate in the Cerrado biome was more arid than in areas of the Amazon and Atlantic Forest biomes, and the temperature had the highest range. Coincidentally, based on morphometric data, cluster analysis distinguished the population from Cerrado, Tocantins from all other populations. Results of multiple regression analysis of the variables showed no correlation between egg variables and latitude or climatic variables. We concluded that eggs were polymorphic and that some morphological patterns were regional. Although no environmental influence on the egg attributes was unequivocally detected, a potential association cannot be entirely discarded. Consequently, we hypothesize that morphological traits of the immature stages, especially from the eggs, convey evolutionary information regarding to this species.

New records of Anopheles homunculus in central and Serra do Mar biodiversity corridors of the Atlantic Forest, Brazil.

Two new records of Anopheles homunculus in the eastern part of the Atlantic Forest are reported. This species was found for the first time in Barra do Ouro district, Maquiné municipality, Rio Grande do Sul state, located in the southern limit of the Atlantic Forest. The 2nd new record was in the Serra Bonita Reserve, Camacan municipality, southeast Bahia state. These records extend the geographical distribution of An. homunculus, suggesting that the species may be widely distributed in coastal areas of the Atlantic Forest. It is hypothesized that the disjunct distribution of the species may be caused by inadequate sampling, and also difficulties in species identification based only on female external characteristics. Species identification was based on morphological characters of the male, larva, and pupa, and corroborated by DNA sequence analyses, employing data from both 2nd internal transcribed spacer of nuclear ribosomal DNA and of mitochondrial cytochrome c oxidase subunit I.

Resurrection of two species from synonymy of Anopheles (Nyssorhynchus) strodei Root, and characterization of a distinct morphological form from the Strodei Complex (Diptera: Culicidae).

Anopheles albertoi Unti and Anopheles arthuri Unti are revived from the synonymy with Anopheles strodei Root, and a distinct morphological form (designated in this study as Anopheles CP Form) from the Strodei Complex of Anopheles (Nyssorhynchus) is characterized. The male genitalia of An. arthuri and An. albertoi are described and illustrated for the first time. An. strodei, An. arthuri, and An. albertoi were first distinguished based on scanning electron microphotos of the eggs, and then each egg type was associated with diagnostic characters of the male genitalia. Identification of Anopheles CP Form was based on morphological characters of the male genitalia, characterized and illustrated in this study. Molecular phylogenetic analysis was most clear when an outgroup was not included, in which case using the nuclear white gene, or the white gene in combination with the mitochondrial cytochrome c oxidase subunit I (COI) gene, clearly separated these four taxa. When Anopheles quadrimaculatus Say and Anopheles stephensi Liston were included as an outgroup, combined white and COI data resolved An. strodei and An. albertoi, whereas An. arthuri was not well resolved. The single sequence of Anopheles CP Form was recovered well separated from other groups in all analyses.

Intraespecific variation on the aedeagus of Anopheles oswaldoi (Peryassú) (Diptera: Culicidae).

Anopheles (Nyssorhynchus) oswaldoi (Peryassú) comprises a species complex in South America. Specimens from two localities in east Mata Atlântica were characterized both at the morphological and molecular level as An. oswaldoi s.s. Intraspecific variation of the shape of the apex of the aedeagus of the male genitalia of specimens of Anopheles (Nyssorhynchus) oswaldoi s.s. from Vale do Ribeira, Mata Atlântica, São Paulo State, Brazil, was observed. Distinctive aedeagus of the specimens from Vale do Ribeira, Mata Atlântica, were evaluated, illustrated and compared to that of An. oswaldoi s.s.

Intraespecific variation on the aedeagus of Anopheles oswaldoi (Peryassú) (Diptera: Culicidae) / Variação intraespecífica no edeago de Anopheles oswaldoi (Peryassú) (Diptera: Culicidae)

Anopheles (Nyssorhynchus) oswaldoi (Peryassú) comprises a species complex in South America. Specimens from two localities in east Mata Atlântica were characterized both at the morphological and molecular level as An. oswaldoi s.s. Intraspecific variation of the shape of the apex of the aedeagus of the male genitalia of specimens of Anopheles (Nyssorhynchus) oswaldoi s.s. from Vale do Ribeira, Mata Atlântica, São Paulo State, Brazil, was observed. Distinctive aedeagus of the specimens from Vale do Ribeira, Mata Atlântica, were evaluated, illustrated and compared to that of An. oswaldoi s.s.

Anopheles (Nyssorhynchus) oswaldoi (Peryassú) compreende complexo de espécies crípticas na América do Sul. Espécimes de duas localidades situadas no leste da Mata Atlântica foram empregados para caracterizar morfologica e molecularmente An. oswaldoi s.s. Foram observadas e avaliadas variações na forma do ápice do edeago da genitália masculina de espécimes de Anopheles (Nyssorhynchus) oswaldoi s.s. do Vale do Ribeira, Mata Atlântica, estado de São Paulo, e nas sequências do segundo espaçador interno transcrito (ITS2). Os espécimes com edeagos distintos apresentaram seqüências idênticas de ITS2. Os tipos distintos de edeago encontrados nos exemplares do Vale do Ribeira, Mata Atlântica, foram ilustrados.