The X chromosome seems to be enriched in genes that cause reproductive isolation between species in the genus Drosophila . In addition, many sibling species including the An. gambiae complex are outcomes of recent speciation processes associated with paracentric inversions involving this chromosome . The X chromosome banding patterns and inversion frequencies studies of Brazilian south and south-east An. cruzii populations, showed three X chromosomal forms (A, B and C), suggesting a process of incipient speciation [9, 10]. The authors observed that the majority of mosquitoes from Juquitiba population had form A, while form B predominated in Cananéia [9, 10]. In the current study, although there are no fixed differences in the cpr gene between Juquitiba and Cananéia, a moderately high F
value was observed. In An. gambiae, cpr is located on the X chromosome. Therefore, if this molecular marker has a similar chromosomal location in An. cruzii, it might be associated with the chromosomal forms described by Ramirez & Dessen [9, 10].
Comparisons of the F
values observed with cpr and timeless in all pair-wise comparisons involving the five populations analysed in the current study and in Rona et al  show only a partial consistence but this is expected. Wang-Sattler et al  demonstrated that the phylogenetic relationships in the An. gambiae complex could vary widely between different genomic regions, thus indicating the mosaic nature of the genome of these species .
As mentioned above the cpr gene in An. gambiae is X-linked, while timeless is autosomal. Assuming these two markers have similar locations in An. cruzii, cpr is expected to be under more efficient selection than the timeless, since in species with X/Y sex determination, as An. cruzii, rare recessive mutations are fully expressed in the heterogametic sex, which could lead to 'faster-X evolution' if a large proportion of mutations are fixed by positive selection . If positive selection is more efficient on the X chromosome, one expects it to harbour less variability than the autosomes . The X chromosome is indeed less variable than the autosomes in non-African populations of Drosophila simulans . Comparing timeless and cpr, the first is more polymorphic than the latter, but the latter shows higher differentiation among the southern populations of An. cruzii.
Since An. cruzii is polymorphic for chromosomal inversions and Ramirez & Dessem [9, 10] found evidence for sibling species carrying different X chromosomal forms, another hypothesis that might explain the differences between the two markers is the suppressed-recombination model of speciation proposed by Coluzzi [33, 38].
Analysis of the molecular polymorphism and genetic differentiation of the timeless gene among Brazilian populations of An. cruzii suggested that the population from Itatiaia (Rio de Janeiro State) is in a process of differentiation and incipient speciation . High F
values between Itatiaia (Rio de Janeiro State) and the other populations from south and south-east Brazil was reported here. In addition, comparison of Itatiaia with other populations revealed some fixed differences and only a few shared polymorphisms. Moreover, the haplotype network shows that Itatiaia is clearly separated in an isolated group (Figure 3). These results, therefore, suggest that this population represents a different species in the An. cruzii complex.
Preliminary evidence was also presented here that raised the possibility of the existence of two different sympatric incipient species in Itatiaia. This is based on the analysis of the genetic differentiation of the cpr gene and a reanalysis of the recently published timeless data . Although a putative heterozygote was found in cpr analysis considering the AG repeats and shared polymorphisms were observed in timeless, high F
values were detected between Itatiaia A and Itatiaia B in these two molecular markers, as well as fixed differences which seem to indicate that these two groups might represent different incipient species. Inspection of the neighbour-joining tree presented in the timeless study  reveals that the individuals classified here as Itatiaia A are clearly isolated in a separated branch. However, the individuals classified as Itatiaia B are mixed with the other individuals from south and south-east populations. In that study, only one putative heterozygote was found (Ita01) carrying alleles of the two Itatiaia groups. Unfortunately this DNA sample was lost and therefore it was not possible to analyse the cpr gene of this individual mosquito. The sample sizes available for the two Itatiaia groups are quite small and further work is clearly needed to determined beyond any doubt that two sympatric incipient sibling species exist in this locality but the results presented here seems to indicate that might be the case.
Analysis of a number of other molecular markers will allow a more precise estimate of the Itatiaia population differentiation and might provide a more complete representation of the divergence history of this species complex.