Neotropical anophelines have shown an extraordinary diversity and complexity due, in part, to the capability of dipterans to adapt to and utilize a broad variety of ecological niches . The subgenus Nyssorhynchus provides an example of extensive morphological and genetic variation within taxa, and several studies have focused on the processes driving this differentiation. Some hypotheses are related to climatic changes that occurred in different epochs, provoking drastic modifications to the habitats of different organisms [2, 3]. However, this remains controversial because of the paucity of available evidence-based data [4–6]. Furthermore, factors affecting speciation and population differentiation, such as ecology, behaviour and genetics, evolve at different rates and are not necessarily congruent .
Some of these taxa, estimated to be 10% of all anophelines, are directly responsible for malaria psite transmission and their accurate identification is necessary for the implementation of effective control strategies. Understanding the current distribution of species, investigating past or recent demographic events (population growth or contraction), gene flow, as well as human interventions (exploitation of new ecological niches, introduction of non endemic species, among others), can provide powerful tools and valuable predictors for the management of pathogens transmitted by anophelines.
Anopheles triannulatus s.l., subgenus Nyssorhynchus,  was first described from adult females in central Brazil and subsequently reported in Central America (Costa Rica, Nicaragua and Panama), in the majority of South American countries [9, 10], and recently in some Caribbean islands . This species has been previously described under different names (syn.bachmanni Petrocchi, syn. chagasi Galvao, syn. cuyabensis Neiva and Pinto, syn. davisi Paterson and Shannon, syn. perezi Shannon and Del Ponte). Morphological variation was later considered intraspecific and attributed to adaptation to different habitats [9, 12–18]. However, recent investigations based on morphological characters of the male genitalia and immature stages led to the designation of a new species, Anopheles halophylus, and elevated the status from polymorphic species to complex, (i.e. the Anopheles triannulatus complex) [18–20].
Genetic distance analysis of allozymes and RAPD detected a third species, An. triannulatus C, and showed that An. halophylus and An. triannulatus species C formed a reciprocally monophyletic group . Apart from this preliminary finding, very little is known about the phylogenetic relationships of members of the An. triannulatus complex, except for the results of analysis of sequences of cpr and timeless genes , which confirmed previous findings and suggested that An. halophylus and An. triannulatus C are in the process of incipient speciation.
Seasonal population density and behavioral differences have also been reported within the complex, for example, potential species-specific preferences for different larval habitats of species that occur in sympatry, e.g., An. halophylus and An. triannulatus species C [19, 22].
An. triannulatus s.l. has been incriminated in human malaria transmission in different regions of Brazil [23–26]), and probably Peru and Venezuela [27, 28], although the role of each species within the complex remains unknown. However, zoophilic and exophilic behaviour has been much commonly reported in Brazil [29–32].
The study of sibling species is aggravated by the difficulty of identification based exclusively on morphological characters when key traits, especially in adult females, may exhibit great phenotypic plasticity . Nowadays, DNA sequences are an essential tool for delineating and identifying species, as well as for gathering information about the genetic variation within species complexes. The mitochondrial gene cytochrome oxidase subunit 1 (COI) is usually informative in phylogenetic reconstruction and geographic variability [34–36]. To represent the nuclear genome the single-copy white gene was chosen [37, 38]. The ribosomal DNA (r-DNA) internal transcribed spacer 2 (ITS2) was used because it plays an important role in distinguishing cryptic anopheline species [39, 40].
The major objective of this study was to reconstruct and clarify the evolutionary relationships based on mtDNA and nuclear sequences, to illustrate the demographic history at the population level, and to provide information on the distribution of An. triannulatus s.l. in several countries across its range. The information obtained would be useful to distinguish the species and help to focus scarce vector control resources on species involved in malaria transmission.