The multilocus results presented here revealed a high level of differentiation between Florianópolis, Itatiaia A and Itatiaia B indicating that these populations represent indeed different species in the An. cruzii complex. Species as evolutionary lineages are expected to show greater evolutionary independence from one another than are populations within species . Hey & Pinho  discuss the use of two measures of evolutionary independence, gene flow and divergence time, for species diagnosis, which help to differentiate intraspecific differences from interspecific differences. The two measures of evolutionary independence were also correlated with F
estimates. For simplicity, the authors suggested a threshold criterion of gene flow < 1, the separation time >1 (based on IM results) and the F
> 0.35 to species diagnosis. Among the six F
values calculated for the pairwise Florianópolis and Itatiaia (including A and B), only two of them are higher than 0.35 (F
= 0.67 and 0.71). Nevertheless, the mean F
among the six loci (0.38) and both measures of evolutionary independence (gene flow and divergence time) are above the threshold criteria. The comparison made between Itatiaia A and Itatiaia B showed similar results, but the migration parameter in the direction of Itatiaia B shown values higher than the suggested threshold, consistent with the idea of incipient speciation.
Previously, we observed no evidence of association between function (circadian x ribosomal genes) in the divergence levels between the sibling species of Florianópolis and Itaparica . In the current work, Clk and RpS29 showed the highest F
values between Florianópolis and Itatiaia (considering Itatiaia A and B together), while Rp49 and RpS2 showed the highest F
values between Itatiaia A and B. Interestingly, these four genes are located on the chromosome 2 of Anopheles gambiae, albeit the three ribosomal protein genes are on the right arm while Clk is on the left arm. Many authors have postulated that chromosomal inversions could promote speciation since their recombination suppressing effects facilitate the maintenance and accumulation of differences between interbreeding populations [37–39]. Ramirez & Dessem [22, 23], studying the polytene chromosomes of Anopheles cruzii from south and southeast regions of Brazil, revealed the existence of three putative species, which differed mainly in the banding patterns of the X chromosome. Nevertheless, different autosomal inversions seemed to be associated with each form of X chromosome and it is tempting to speculate that some of the markers we used and showed high levels of differentiation between the sibling species are located within or nearby the inversions studied by Ramirez & Dessem [22, 23]. Future in situ hybridization experiments using these markers as probes might confirm if that is the case.
The observation of a putative hybrid  and the varying degrees of differentiation observed in the six loci between Itatiaia A and Itatiaia B, suggest that these two siblings are in a process of incipient speciation. Similar results were found in the Anopheles gambiae complex [40–42] that maintains a genome in a mosaic form, with regions of low and high divergence. These variations among the different regions of the genome in the divergence between the An. cruzii siblings have two main explanations: i) the maintenance of ancestral polymorphisms as these cryptic species have separated recently and ii) differential introgression in different genomic regions between the species reflecting locations where gene flow occurs freely or is restricted by selection.
The two conflicting but no mutually exclusive hypotheses of retention of ancestral polymorphism and introgression between closely related species are often difficult to distinguished. Introgression can sometimes be excluded based on the geographic separation of two species and in this case, the shared polymorphisms can be classified as ancestral . This is perhaps the situation found between the allopatric Florianópolis and Itatiaia (A and B) with no strong indication of migration, in either direction, as suggested by the IM results. On the other hand, the introgression hypothesis is of course more likely if the species are sympatric. This is probably the case of Itatiaia A and Itatiaia B, where the introgression hypothesis seem confirmed by the IM results that revealed nonzero values of migration parameter between the latter species, only in the direction of Itatiaia B.
Asymmetric introgression has been documented in a variety of taxa [44–48], including mosquitoes. For example, Donnelly et al. , comparing haplotype frequencies in allopatric and sympatric populations of An. arabiensis and An. gambiae, found unidirectional introgression, from the former into the latter species, only in sympatric populations. Gomes et al.  found differential introgression in the Culex pipiens complex, in spite of the high levels of genetic differentiation between its forms.
Marsden et al.  found similar results in the An. gambiae complex, between M and S molecular forms, and they argue, as one of the possible explanations, that the asymmetric introgression may be a consequence of the differences in relative abundance of the two taxa (S form–the genetic recipient–was more common than M form–the genetic donor–in all sites assessed), which could result in higher levels of backcrossing between the hybrids and the more abundant species. Our results are also consistent with this explanation as the estimates of population sizes and migration parameters from the IM program suggest that Itatiaia A (the genetic donor) has a smaller estimated population size than Itatiaia B (the genetic recipient).
Marsden et al.  also proposed that the asymmetric introgression can contribute to the maintenance of differentiation between M and S, since the unidirectional movement of nuclear genes from the M into the S form would prevent homogenization of their gene pools because of the conservation of unique polymorphism within the S form and a lack of admixture in M. Indeed, the observed genetic diversity was considerably lower in Itatiaia A than in Itatiaia B.
The estimated divergence time (based on the Da values) between Itatiaia A and Itatiaia B is ~ 0.2 Mya and between Florianópolis and Itatiaia (including A and B) is ~ 0.6 Mya. This indicates an earlier speciation process splitting the populations of Florianópolis and Itatiaia, followed by a more recent separation between the two sympatric species in the latter locality.
The time of divergence of these three sibling species as well as that between the Bahia and the south and southeast populations (~ 2.4 Mya) , points to the importance of Pleistocene environmental changes (glaciations and inter-glaciations periods)  as factors in the diversification of An. cruzii species complex. Since temperature and water are essential for the development of Anopheles spp. immature stages , the hypothesis that Pleistocene environmental changes driving Anopheles diversification seems plausible. Furthermore, in the case of An. cruzii (Kerteszia subgenus), its larval development is associated with water trapped in Bromeliads plants, which are restricted to the rainforest [4, 7]. So, a number of studies show that several malaria vectors, including An. cruzii, have revealed patterns of genetic divergence associated with the Pleistocene environmental changes [26, 51–55].
The process of successive rainforest contractions and expansions was a very important consequence of these environmental modifications possibly favoring the differentiation and speciation of forest obligate species [56–58]. Accordingly, a number of studies of closely related species demonstrated the importance of the Pleistocene climatic changes in shaping the Brazilian Atlantic forest biodiversity [59, 60], since patterns of endemicity in many forest obligate species are concordant in geographic distribution [59, 61–63]. For example, Mata et al. , studying the molecular phylogeny and biogeography of the eastern Tapaculo birds in Brazilian Atlantic forest, corroborated the importance of the Bahia refuge as an avian center of endemism and also concluded that the southeast (Serra da Mantiqueira, where Itatiaia is located) lineage is genetically different from the southern populations.
Turchetto-Zolet et al.  studying the molecular variation patterns in Schizolobium parahyba (Fabaceae), a widespread tree in the Brazilian Atlantic forest, found high levels of genetic diversity in populations from the southeast region and low levels in southern populations. They argue that the low genetic diversity in the south may be the result of a founder effect followed by a range expansion after glacial periods. Lorenz-Lemke et al.  and Palma-Silva et al.  also reported expansion toward the south in Atlantic forest plants. However, in the case of An. cruzii, Florianópolis (south sibling species) showed a higher level of genetic diversity than the Itatiaia (southeast) sympatric sibling species. Therefore, it is likely that there was a persistence of An. cruzii populations in the south during the contraction of the forest, as was proposed for toads , instead of a southern colonization of the Atlantic forest from northern regions.