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Abstract Background Drosophila Frequenin (Frq), the homolog of the mammalian Neuronal Calcium Sensor-1 (NCS-1), is a high affinity calcium-binding protein with ubiquitous expression in the nervous system. This protein has an important role in the regulation of neurotransmitter release per synapse, axonal growth and bouton formation. In D. melanogaster, Frequenin is encoded by two genes (frq1 and frq2), a very unexpected feature in the Frq/NCS-1 subfamily. These genes are located in tandem in the same genomic region, and their products are 95% identical in their amino acid sequence, clearly indicating their recent origin by gene duplication. Here, we have investigated the factors involved in this unusual feature by examining the molecular evolution of the two frq genes in Drosophila and the evolutionary dynamics of NCS family in a large set of bilaterian species. Results Surprisingly, we have found no amino acid replacements fixed across the twelve Drosophila species surveyed. In contrast, synonymous substitutions have been prevalent in the evolution of the coding region of frq1 and frq2, indicating the presence of strong functional constraints following gene duplication. Despite that, we have detected that significant evolutionary rate acceleration had occurred in Frq1 in early times from the duplication, in which positive selection (likely promoting functional diversification) had probably an important role. The analysis of sequence conservation and DNA topology at the non-coding regions of both genes has allowed the identification of DNA regions candidates to be cis-regulatory elements. The results reveal a possible mechanism of regulatory diversification between frq1 and frq2. Conclusions The presence of two Frequenins in Drosophila and the rapid accumulation of amino acid substitutions after gene duplication are very unusual features in the evolution of the Frq/NCS-1 subfamily. Here we show that the action of positive selection in concordance with some extent of regulatory diversification might explain these findings. Selected amino acid substitutions in Frq1 likely contributed to the functional divergence between the two duplicates, which, in turn, should have diverged in their regulation by Ecdysone-induced early genes.

Abstract Background The Drosophila Odorant-Binding Protein (Obp) genes constitute a multigene family with moderate gene number variation across species. The OS-E and OS-F genes are the two phylogenetically closest members of this family in the D. melanogaster genome. In this species, these genes are arranged in the same genomic cluster and likely arose by tandem gene duplication, the major mechanism proposed for the origin of new members in this olfactory-system family. Results We have analyzed the genomic cluster encompassing OS-E and OS-F genes (Obp83 genomic region) to determine the role of the functional divergence and molecular adaptation on the Obp family size evolution. We compared nucleotide and amino acid variation across 18 Drosophila and 4 mosquito species applying a phylogenetic-based maximum likelihood approach complemented with information of the OBP three-dimensional structure and function. We show that, in spite the OS-E and OS-F genes are currently subject to similar and strong selective constraints, they likely underwent divergent evolution. Positive selection was likely involved in the functional diversification of new copies in the early stages after the gene duplication event; moreover, it might have shaped nucleotide variation of the OS-E gene concomitantly with the loss of functionally related members. Besides, molecular adaptation likely affecting the functional OBP conformational changes was supported by the analysis of the evolution of physicochemical properties of the OS-E protein and the location of the putative positive selected amino acids on the OBP three-dimensional structure. Conclusion Our results support that positive selection was likely involved in the functional differentiation of new copies of the OBP multigene family in the early stages after their birth by gene duplication; likewise, it might shape variation of some members of the family concomitantly with the loss of functionally related genes. Thus, the stochastic gene gain/loss process coupled with the impact of natural selection would influence the observed OBP family size.