The results presented here confirm the high level of differentiation between the Itaparica and Florianópolis sibling species of the An. cruzii complex [5, 6].
Less differentiation might have been expected in the three genes that code for the highly conserved ribosomal proteins (Rp49, RpS29 and RpS2) than in loci possibly involved in the control of mating rhythms (timeless, Clock and cycle) [7, 8]. The latter three genes are potentially important in maintaining temporal reproductive isolation between closely related species, and might be involved in the speciation process in some insects. In fact, Rona et al.  showed very high differentiation between Itaparica and the more southern Brazilian populations, including Florianópolis, using the timeless gene as a molecular marker.
However, very high F
values were detected in all loci between these two sibling species and they were even higher for Rp49, RpS29 and RpS2 (0.8854, 0.8865 and 0.8502, respectively for the whole fragment) than for timeless, Clock and cycle (0.8150, 0.7088 and 0.5806, respectively for the whole fragment). Mazzoni et al.  found similar results in a multilocus analysis between two sand fly vectors of leishmaniasis.
No indication of migration was found in either direction in the multiple IM simulations, which was consistent with the very high differentiation values for all loci. Itaparica also presented lower levels of variability than those from Florianópolis, possibly indicating a smaller population size. This is confirmed by IM results, which also indicated a smaller effective population size for Itaparica. The estimated difference in population sizes seems coherent, since the southern An. cruzii sibling species found in Florianópolis is distributed throughout most of the southern and southeastern Brazilian Atlantic Forest (from Santa Catarina to Espírito Santo State) while the northeastern sibling species found in Itaparica seems to occur only in a more restricted region .
The multilocus results corroborate previous data [5, 6] indicating that these populations represent two different species in the An. cruzii complex. This was also confirmed by NJ trees, which show that Florianópolis and Itaparica are clearly separated in two isolated groups, except perhaps in the case of cycle which suggests persistence of ancestral polymorphisms in Florianópolis. However, this gene fragment presents a very small number of variable sites in the Itaparica sample.
The estimated divergence time from 1.1 to 3.6 Mya, based on the IM results, corresponds to the end of the Pliocene and beginning of the Pleistocene . Significant climate changes, including the onset of heavy Northern Hemisphere glaciation, around 2.75 Mya, occurred at the end of the Pliocene . A very important consequence of this cooling was an extensive increase in aridification, which lead to fragmentation of forests, including the Brazilian Atlantic Forest [18, 19]. Interestingly, Carnaval et al.  discussed the hypothesis of refugia for neotropical species occurring in the Atlantic Forest. Itaparica is located in an area proposed to be a large central refugium in the Brazilian Atlantic Forest and another refugium is proposed in the southern and southeastern Brazil. Climate changes have been proposed to explain the differentiation among many groups such as fruit flies , insect vectors  as well as many forest-obligate species [20, 23–25]. Since An. cruzii is endemic to the Atlantic Forest, it seems likely that differentiation between its populations might have occurred due to forest fragmentation, which might have split a single ancestral species into two or more isolated groups.