The evolutionary processes governing the divergence of animal genitalia are mostly unknown and constitute one of the most intriguing pieces of a mayor puzzle that is speciation [1-5]. In many arthropod groups male genitalia evolves at particularly high rates and this special feature constitutes the mechanistic basis of its use as a specific diagnostic trait [6,7]. Notwithstanding the taxonomic importance of genitalic morphology, intraspecific studies addressing the causes and consequences of intraspecific morphological variation are scarce. Such studies, using methods successfully applied in evolutionary biology, offer the opportunity to gain new insights into the evolutionary processes and forces involved in genitalic evolution .
Three main hypotheses have been proposed to explain the evolution of genital morphology: the lock and key, the pleiotropy and the sexual selection hypotheses. The lock and key hypothesis  states that male genitalia evolve as a species-specific trait in order to properly fit in female genitalic organs. This theory predicts a canalized development of male genitalia and low levels of phenotypic and genotypic variation, since genitalic traits are expected to be under strong stabilizing selection [3,9].
The pleiotropy hypothesis assumes that genital variation is largely neutral. Since genital and non-genital morphological traits are implicitly genetically correlated, changes of allele frequencies at loci pleiotropically affecting general morphology and genitalia may lead to rapid and arbitrary evolution of genitalic traits [2,10,11]. The sexual selection hypothesis states that morphological differences in male genitalia are related to variation in fertilization success and that morphological divergence is driven by sexual selection .
The study of the evolution of male genitalia may be even more complicated not only because it may be influenced by both natural and sexual selection, but also because it's phenotypic expression might be influenced by environmental factors  as occurs for other morphological traits. Thus, the joint study of intraspecific variation and interspecific divergence may provide a useful approach for the understanding of the underlying genetic architecture of genital traits and the evolutionary processes involved [13-15]. In this sense, it has been suggested that differences in genital morphology between closely related species would be largely polygenic . Such claim is based on the single study comparing the morphology of male posterior lobe in two closely related species of Drosophila and their hybrids . Therefore, it is clear that more studies are necessary to further support this affirmation and to determine whether simple genetic differences can account for the evolution of fast evolving and complex structures such as male genitalia.
The aedeagus, which is the intromittent organ of male genitalia , is considered the main diagnostic trait for species recognition in the Drosophila repleta group . To this group belong the South American D. buzzatii and D. koepferae [19,20], which are morphologically undistinguishable except for conspicuous differences in male genitalia. These species are reproductively isolated by partial ecological isolation , sexual isolation and post mating barriers . Both species can breed and feed on the necrotic tissues of several cacti species [23,24], however they exhibit a certain degree of niche separation; D. buzzatii is mainly adapted to breed on decaying tunas (genus Opuntia), while D. koepferae prefers the necrotic stems of columnar cacti of the genera Cereus and Trichocereus . Though sexual isolation between these species is strong, behavioral barriers can be forced in the laboratory, since D. buzzatii males can inseminate D. koepferae females and female hybrid offspring can be successfully backcrossed to D. buzzatii males . Furthermore, recent population genetic studies have provided indirect evidence of past or recent gene flow between these species [26,27].
The knowledge of the ecology of these cactophilic Drosophila [21,24,28,29], coupled with the possibility to produce interspecific hybrids in the laboratory and the potential for natural hybridization, makes this pair of species into an excellent model for speciation studies, particularly those addressing the genetic and ecological basis of morphological change associated to interspecific divergence.
In this work we investigate the sources of phenotypic variation, genetic and environmental, by examining genitalic size and shape in flies of several isofemale lines of both species and interspecific hybrids raised in two different species of host cacti.