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Gene families underlie genetic innovation and phenotypic diversification. However, our understanding of the early genomic and functional evolution of tandemly arranged gene families remains incomplete as paralog sequence similarity hinders their accurate characterization. The Drosophila melanogaster-specific gene family Sdic is tandemly repeated and impacts sperm competition. We scrutinized Sdic in 20 geographically diverse populations using reference-quality genome assemblies, read-depth methodologies, and qPCR, finding that ∼90% of the individuals harbor 3-7 copies as well as evidence of population differentiation. In strains with reliable gene annotations, copy number variation (CNV) and differential transposable element insertions distinguish one structurally distinct version of the Sdic region per strain. All 31 annotated copies featured protein-coding potential and, based on the protein variant encoded, were categorized into 13 paratypes differing in their 3' ends, with 3-5 paratypes coexisting in any strain examined. Despite widespread gene conversion, the only copy present in all strains has functionally diverged at both coding and regulatory levels under positive selection. Contrary to artificial tandem duplications of the Sdic region that resulted in increased male expression, CNV in cosmopolitan strains did not correlate with expression levels, likely as a result of differential genome modifier composition. Duplicating the region did not enhance sperm competitiveness, suggesting a fitness cost at high expression levels or a plateau effect. Beyond facilitating a minimally optimal expression level, Sdic CNV acts as a catalyst of protein and regulatory diversity, showcasing a possible evolutionary path recently formed tandem multigene families can follow toward long-term consolidation in eukaryotic genomes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The animal chemosensory system is involved in essential biological processes, most of them mediated by proteins encoded in multigene families. These multigene families have been fundamental for the adaptation to new environments, significantly contributing to phenotypic variation. This adaptive potential contrasts, however, with the lack of studies at their upstream regions, especially taking into account the evidence linking their transcriptional changes to certain phenotypic effects. Here, we explicitly characterize the contribution of the upstream sequences of the major chemosensory gene families to rapid adaptive processes. For that, we analyze the genome sequences of 158 lines from a population of Drosophila melanogaster that recently colonized North America, and integrate functional and transcriptional data available for this species. We find that both, strong negative and strong positive selection, shape transcriptional evolution at the genome-wide level. The chemosensory upstream regions, however, exhibit a distinctive adaptive landscape, including multiple mutations of small beneficial effect and a reduced number of cis-regulatory elements. Together, our results suggest that the promiscuous and partially redundant transcription and function of the chemosensory genes provide evolutionarily opportunities for rapid adaptive episodes through weak polygenic selection., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Animal olfactory systems have a critical role for the survival and reproduction of individuals. In insects, the odorant-binding proteins (OBPs) are encoded by a moderately sized gene family, and mediate the first steps of the olfactory processing. Most OBPs are organized in clusters of a few paralogs, which are conserved over time. Currently, the biological mechanism explaining the close physical proximity among OBPs is not yet established. Here, we conducted a comprehensive study aiming to gain insights into the mechanisms underlying the OBP genomic organization. We found that the OBP clusters are embedded within large conserved arrangements. These organizations also include other non-OBP genes, which often encode proteins integral to plasma membrane. Moreover, the conservation degree of such large clusters is related to the following: 1) the promoter architecture of the confined genes, 2) a characteristic transcriptional environment, and 3) the chromatin conformation of the chromosomal region. Our results suggest that chromatin domains may restrict the location of OBP genes to regions having the appropriate transcriptional environment, leading to the OBP cluster structure. However, the appropriate transcriptional environment for OBP and the other neighbor genes is not dominated by reduced levels of expression noise. Indeed, the stochastic fluctuations in the OBP transcript abundance may have a critical role in the combinatorial nature of the olfactory coding process.

The IT-insulin/target of rapamycin (TOR)-signal transduction pathway is a relatively well-characterized pathway that plays a central role in fundamental biological processes. Network-level analyses of DNA divergence in Drosophila and vertebrates have revealed a clear gradient in the levels of purifying selection along this pathway, with the downstream genes being the most constrained. Remarkably, this feature does not result from factors known to affect selective constraint such as gene expression, codon bias, protein length, and connectivity. The present work aims to establish whether the selective constraint gradient detected along the IT pathway at the between-species level can also be observed at a shorter time scale. With this purpose, we have surveyed DNA polymorphism in Drosophila melanogaster and divergence from D. simulans along the IT pathway. Our network-level analysis shows that DNA polymorphism exhibits the same polarity in the strength of purifying selection as previously detected at the divergence level. This equivalent feature detected both within species and between closely and distantly related species points to the action of a general mechanism, whose action is neither organism specific nor evolutionary time dependent. The detected polarity would be, therefore, intrinsic to the IT pathway architecture and function.

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.

Nucleotide variation at the genomic region encompassing the odorant-binding protein genes OS-E and OS-F (OS region) was surveyed in two populations of Drosophila simulans, one from Europe and the other from Africa. We found that the European population shows an atypical and large haplotype structure, which extends throughout the approximately 5-kb surveyed genomic region. This structure is depicted by two major haplotype groups segregating at intermediate frequency in the sample, one haplogroup with nearly no variation, and the other at levels more typical for this species. This pattern of variation was incompatible with neutral predictions for a population at a stationary equilibrium. Nevertheless, neutrality tests contrasting polymorphism and divergence data fail to detect any departure from the standard neutral model in this species, whereas they confirm the non-neutral behavior previously observed at the OS-E gene in D. melanogaster. Although positive Darwinian selection may have been responsible for the observed unusual nucleotide variation structure, coalescent simulation results do not allow rejecting the hypothesis that the pattern was generated by a recent bottleneck in the history of European populations of D. simulans.

The Olfactory Specific-E and -F genes (OS-E and OS-F) belong to the odorant-binding protein gene family, which includes the general odorant-binding proteins and the pheromone-binding proteins. In Drosophila melanogaster, these genes are arranged in tandem in a genomic region near the centromere of chromosome arm 3R. We examined the pattern of DNA sequence variation in an approximately 7-kb genomic region encompassing the two OS genes in four species of the melanogaster subgroup of Drosophila and in a population sample of D. melanogaster. We found that both the OS-E and the OS-F gene are present in all surveyed species. Nucleotide divergence estimates would support that the two genes are functional, although they diverge in their functional constraint. The pattern of nucleotide variation in D. melanogaster also differed between genes. Variation in the OS-E gene region exhibited an unusual and distinctive pattern: (i) a relatively high number of fixed amino acid replacements in the encoded protein and (ii) a peak of nucleotide polymorphism around the OS-E gene. These results are unlikely under the neutral model and suggest the action of natural selection in the evolution of the two odorant-binding protein genes.

An approximately 1.3-kb region including the rp49 gene plus its 5' and 3' flanking regions was sequenced in 24 lines of Drosophila simulans (10 from Spain and 14 from Mozambique). Fifty-four nucleotide and 8 length polymorphisms were detected. All nucleotide polymorphisms were silent: 52 in noncoding regions and 2 at synonymous sites in the coding region. Estimated silent nucleotide diversity was similar in both populations (pi = 0.016, for the total sample). Nucleotide variation revealed an unusual haplotype structure showing a subset of 11 sequences with a single polymorphism. This haplotype was present at intermediate frequencies in both the European and the African samples. The presence of such a major haplotype in a highly recombining region is incompatible with the neutral equilibrium model. This haplotype structure in both a derived and a putatively ancestral population can be most parsimoniously explained by positive selection. As the rate of recombination in the rp49 region is high, the target of selection should be close to or within the region studied.

Nucleotide variation at the ribosomal protein 49 (rp49) gene region has been studied in 75 lines of Drosophila subobscura belonging to four chromosomal arrangements (Ost, O3+4, O3+4+8, and O3+4+23). The location of the rp49 gene region within the inversion loop differs among heterokaryotypes: it is very close to one of the breakpoints in heterozygotes involving Ost chromosomes, while it is in a more central position in all other heterokaryotypes. The distribution of nucleotide polymorphism in the different arrangements is consistent with a monophyletic origin of the inversions. The data also provide evidence that gene conversion and possibly double crossover are involved in shuffling nucleotide variation among gene arrangements. The analyses reveal that the level of genetic exchange is higher when the region is located in a more central position of the inverted fragment than when it is close to the breakpoints. The pairwise difference distributions as well as the negative values of Tajima's and Fu and Li's statistics further support the hypothesis that nucleotide variation within chromosomal arrangements still reflects expansion after the origin of the inversions. Under the expansion model, we have estimated the time of origin of the studied inversions.

DNA sequence variation studies report the transfer of small segments of DNA among different sequences caused by gene conversion events. Here, we provide an algorithm to detect gene conversion tracts and a statistical model to estimate the number and the length distribution of conversion tracts for population DNA sequence data. Two length distributions are defined in the model: (1) that of the observed tract lengths and (2) that of the true tract lengths. If the latter follows a geometric distribution, the relationship between both distributions depends on two basic parameters: psi, which measures the probability of detecting a converted site, and phi, the parameter of the geometric distribution, from which the average true tract length, 1/(1-phi), can be estimated. Expressions are provided for estimating phi by the method of the moments and that of the maximum likelihood. The robustness of the model is examined by computer simulation. The present methods have been applied to the published rp49 sequences of Drosophila subobscura. Maximum likelihood estimate of phi for this data set is 0.9918, which represents an average conversion tract length of 122 bp. Only a small percentage of extant conversion events is detected.