Comparative analysis of quantitative variation in CHC profiles of the D. buzzatii species cluster revealed that CHC evolution has been somewhat conserved and associated with the evolutionary divergence of these species. Thus, CHC differentiation among these populations has not evolved so quickly as to erase evidence of phylogenetic affinity suggesting that variation in CHCs in this group of Drosophila can be predicted, to some extent, by species ancestry. Here, a key observation was the degree of CHC chemical conservation between the D. buzzatii and D. mojavensis clusters (Table 2) where most molecular structures, retention times, and carbon chainlengths were conserved, but species-specific CHC amounts varied quantitatively. The D. mojavensis cluster is also part of the mulleri complex, but is endemic to North America [59, 72, 73]. As these species groups are restricted to different continents and diverged ca 10-15 mya [74, 75], CHC biosynthesis and expression have been conserved over a large portion of the D. repleta group phylogeny. The most conserved chemical compounds were 2-methyloctacosane (2-MeC28) and 2-methyltriacontane (2-MeC30). These two compounds are not only shared within and between both clusters but are also found in a variety of other insect species . In retrospect, such conserved CHCs may not be surprising, but few attempts have been made to assess broad-scale variation in CHCs in groups of related species. Thus, CHC evolution in these D. repleta group species has a significant phylogenetic component based on a core group of C29, C31, C33, C35, C37 and C39 hydrocarbons (Additional File 3: Figure S3) with additional species and population-specific variations on this theme.
The multiple functional roles for insect cuticular hydrocarbons has been appreciated for some time . In arthropods with longer chain length CHCs (>20 carbon atoms), effects of desiccation are reduced because longer CHCs have higher melting temperatures [78, 79], consistent with observations that xeric adapted Drosophila species exhibit longer chain length CHCs than mesic species . Although saturated compounds, n-alkanes, provide increased protection against desiccation, branched and unsaturated compounds decrease melting temperatures and can cause increased rates of water loss across insect epicuticles . In Drosophila, alkenes and alkadienes have pheromonal activity in a number of species [14, 81–84]. In experimental populations of D. melanogaster that responded to increased desiccation conditions, CHC differences did evolve, but there were no associated changes in sexual isolation suggesting that CHCs involved in desiccation resistance were different from those used for mate choice . In other insects like paper wasps  and honeybees , branched alkanes and/or alkenes are more easily identified by other individuals than linear alkanes and therefore serve as recognition cues while n-alkanes function primarily to reduce water loss. Given the conservation of CHC compounds in the desert-adapted D. buzzatii and D. mojavensis species groups, significant sexual dimorphism in CHC profiles (Table 3), and the presence of branched and unsaturated molecules in the CHCs of all of these species, we expect that D. buzzatii cluster CHCs serve as both physiological mechanisms to control transcuticular water flux as well as in chemical communication, i.e. mate recognition. Nevertheless, the role of CHCs as pheromones has yet to be confirmed in the D. buzzatii cluster. Preliminary results revealed undetectable pheromonal activity in CHC perfuming experiments with D. seriema and D. buzzatii even though significant amounts of CHCs were transferred between males (Oliveira et. al., unpubl. data). However, we initially chose these species for perfuming studies because of the ability to detect CHC transfers. This result may not be representative of other, more closely related species in the cluster because D. seriema and D. buzzatii were so reproductively divergent (in mate choice trials, Oliveira et. al., unpubl. data) that alterations in CHCs had little effect despite the significant CHC differences between them. Further perfuming trials with all D. buzzatii cluster species are clearly needed.
The detection of positive phylogenetic signal using the three different data sets: (1) D. buzzatii + D. mojavensis cluster; (2) D. buzzatii cluster; and (3) D. buzzatii cluster (without D. serido populations) + D. mojavensis cluster (Table 4, Additional Files 9 and 11, respectively) supports the hypothesis that phylogenetic signal was strong enough to be detected by different methods independent of their assumptions. Moreover, positive phylogenetic signal was observed when just the D. buzzatii cluster species were used supporting that some CHCs were conserved in the cluster. These results were even more robust when the divergent D. serido populations were removed from the analysis. We hypothesize that CVs that were weakly correlated with the phylogeny, mainly CV2, were influenced by CHCs that may be responding to the ambient environment or other forces, i.e. these are traits involved in mate recognition like courtship songs, pheromones, or coloration that should evolve more rapidly due to sexual or stabilizing selection [88–91].
Contrasting results have been reported regarding the presence of phylogenetic signal in studies of character evolution that have implicated CHCs and other volatile compounds in mate and/or species recognition. For example, Jallon and David  concluded that "Hydrocarbon variations do not match the phylogeny" in eight species of the D. melanogaster group. Symonds and Elgar  reported little association between aggregation pheromone composition and phylogenetic relationships in bark beetles since closely related species were as different, if not more so, than more distantly related species. Conversely, Symonds and Wertheim  found that more closely related Drosophila species had more chemically similar aggregation pheromones and concluded that there was a positive relationship between phylogenetic distance and pheromone differentiation. Cuticular hydrocarbons in pine engraver beetles have been used to identify different species and thus have systematic value . Some phylogenetic trends in species-specific CHCs were also reported in Hawaiian swordtail crickets . However, known phylogenetic relationships among 78 ant species in five subfamilies showed "no similarity" to cuticular hydrocarbon differences based on chemical structures . Male courtship songs were homoplasic in the Drosophila willistoni species complex , showed evidence of diversification, character loss, and reversal in the D. repleta group , and converged in green lacewings . In birds, sexually selected traits like male plumage and bower characters exhibited low phylogenetic signal [97, 98], while male songs were more conserved . We suggest that phylogenetic diversification of insect CHCs may be more conservative than courtship songs or avian plumage characteristics because the complex underlying biochemical and physiological machinery required to synthesize and express CHCs in arthropods [9, 100, 101] may be more conserved than in other traits. Thus, similarity in cuticular hydrocarbon profiles among species may represent a phylogenetic constraint due to their mode of production. Certainly, more comparative studies involving mating signals will be necessary to determine whether the presence of phylogenetic signal is a rule or an exception for pheromonal or behavioral traits.
Evolution of the D. buzzatii cluster and CHCs
Attempts to resolve a phylogeny using the mtDNA data  failed to resolve all species into individual evolutionary lineages. Specifically, D. gouveai, D. serido, and D. seriema show substantial geographic variation and considerable phylogenetic incongruence (Additional File 1: Figure S1). Incomplete lineage sorting or hybridization could be responsible, as well as natural selection on mtDNA function . Phylogenetic reconstruction based on the nuclear period (per) gene by Franco et al.  resolved the relationships among D. gouveai, D. borborema and D. seriema (Figure 5). Although per grouped populations of D. serido together, they were not placed as a sister taxa of D. antonietae, as predicted by chromosomal inversion data (Figure 2). Therefore, the position of "D. serido" has yet to be resolved.
The large and very significant intraspecific differences in D. serido CHCs (Figure 3A) does not suggest a gradual model of CHC evolution, but were consistent with previously described differentiation between populations that inhabit northeastern Brazil in the caatinga (e.g. Milagres, Bahia) and those from the east coast of Brazil (e.g. Mucuri, Bahia and Arraial do Cabo, Rio de Janeiro, see Figure 1). The observation that the CHCs of the coastal D. serido population from Macaé, Rio de Janeiro did not match this pattern of differentiation further suggests that this stock was contaminated (see results for details). Here, the scale of intraspecific CHC variation was greater than interspecific variation for the remaining six species, and included multiple CHC components (Figure 4). Genetic divergence between populations of D. serido in these regions includes mtDNA haplotype differentiation , cytological differences, amounts of heterochromatin in metaphase chromosomes , and frequency differences of polymorphic inversions [41, 104]. These observations together with our results showing large intraspecific CHC differences strongly suggest the presence of several more cryptic species in this group.