BackgroundAnopheles nuneztovari sensu lato comprises cryptic species in northern South America, and the Brazilian populations encompass distinct genetic lineages within the Brazilian Amazon region. This study investigated, based on two molecular markers, whether these lineages might actually deserve species status.MethodsSpecimens were collected in five localities of the Brazilian Amazon, including Manaus, Careiro Castanho and Autazes, in the State of Amazonas; Tucuruí, in the State of Pará; and Abacate da Pedreira, in the State of Amapá, and analysed for the COI gene (Barcode region) and 12 microsatellite loci. Phylogenetic analyses were performed using the maximum likelihood (ML) approach. Intra and inter samples genetic diversity were estimated using population genetics analyses, and the genetic groups were identified by means of the ML, Bayesian and factorial correspondence analyses and the Bayesian analysis of population structure.ResultsThe Barcode region dataset (N = 103) generated 27 haplotypes. The haplotype network suggested three lineages. The ML tree retrieved five monophyletic groups. Group I clustered all specimens from Manaus and Careiro Castanho, the majority of Autazes and a few from Abacate da Pedreira. Group II clustered most of the specimens from Abacate da Pedreira and a few from Autazes and Tucuruí. Group III clustered only specimens from Tucuruí (lineage III), strongly supported (97 %). Groups IV and V clustered specimens of A. nuneztovaris.s. and A. dunhami, strongly (98 %) and weakly (70 %) supported, respectively. In the second phylogenetic analysis, the sequences from GenBank, identified as A. goeldii, clustered to groups I and II, but not to group III. Genetic distances (Kimura-2 parameters) among the groups ranged from 1.60 % (between I and II) to 2.32 % (between I and III). Microsatellite data revealed very high intra-population genetic variability. Genetic distances showed the highest and significant values (P = 0.005) between Tucuruí and all the other samples, and between Abacate da Pedreira and all the other samples. Genetic distances, Bayesian (Structure and BAPS) analyses and FCA suggested three distinct biological groups, supporting the barcode region results.ConclusionsThe two markers revealed three genetic lineages for A. nuneztovaris.l. in the Brazilian Amazon region. Lineages I and II may represent genetically distinct groups or species within A. goeldii. Lineage III may represent a new species, distinct from the A. goeldii group, and may be the most ancestral in the Brazilian Amazon. They may have differences in Plasmodium susceptibility and should therefore be investigated further.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1217-6) contains supplementary material, which is available to authorized users.

Lutzomyia umbratilis is the main vector of Leishmania guyanensis in the Brazilian Amazon and in neighboring countries. Previous biological and molecular investigations have revealed significant differences between L. umbratilis populations from the central Brazilian Amazon region. Here, a phylogeographic survey of L. umbratilis populations collected from nine localities in the Brazilian Amazon was conducted using two mitochondrial genes. Statistical analyses focused on population genetics, phylogenetic relationships and species delimitations. COI genetic diversity was very high, whereas Cytb diversity was moderate. COI genealogical haplotypes, population structure and phylogenetic analyses identified a deep genetic differentiation and three main genetic groups. Cytb showed a shallower genetic structure, two main haplogroups and poorly resolved phylogenetic trees. These findings, allied to absence of isolation by distance, support the hypothesis that the Amazon and Negro Rivers and interfluves are the main evolutionary forces driving L. umbratilis diversification. The main three genetic groups observed represent three evolutionary lineages, possibly species. The first lineage occurs north of the Amazon River and east of Negro River, where Le. guyanensis transmission is intense, implying that L. umbratilis is an important vector there. The second lineage is in the interfluve between north of Amazon River and west of Negro River, an area reported to be free of Le. guyanensis transmission. The third lineage, first recorded in this study, is in the interfluve between south of Amazonas River and west of Madeira River, and its involvement in the transmission of this parasite remains to be elucidated.

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ABSTRACT. Anopheles nuneztovari sensu lato consists of cryptic species and genetic lineages, one of which is an important human malaria vector in the northern part of South America. Population structure and evolutionary genetics studies may help in the definition and delimitation of the species and lineages within this species complex, which is relevant information for organizations involved in malaria control efforts. In this study, 10 new microsatellite markers were isolated from 2 repeat-enriched genomic libraries of A. nuneztovari s.l. and were characterized in 37-48 mosquitoes of this species. All loci were highly polymorphic and encompassed 5-25 alleles per locus. The observed (H O ) and expected (H E ) heterozygosities ranged from 0.354 to 0.866 and from 0.613 to 0.932, respectively. Six of the 10 new loci showed significant deviations from the HardyWeinberg equilibrium, and no linkage disequilibrium was detected. The loci described in this study were more polymorphic than the 18 previously characterized loci and appear to be promising markers for use in investigating the fine-scale population genetic structure and the boundaries of the cryptic species and lineages within the A. nuneztovari complex.

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