Population Genomics of Sub-Saharan Drosophila melanogaster: African Diversity and Non-African Admixture

Drosophila melanogaster has played a pivotal role in the development of modern population genetics. However, many basic questions regarding the demographic and adaptive history of this species remain unresolved. We report the genome sequencing of 139 wild-derived strains of D. melanogaster, representing 22 population samples from the sub-Saharan ancestral range of this species, along with one European population. Most genomes were sequenced above 25X depth from haploid embryos. Results indicated a pervasive influence of non-African admixture in many African populations, motivating the development and application of a novel admixture detection method. Admixture proportions varied among populations, with greater admixture in urban locations. Admixture levels also varied across the genome, with localized peaks and valleys suggestive of a non-neutral introgression process. Genomes from the same location differed starkly in ancestry, suggesting that isolation mechanisms may exist within African populations. After removing putatively admixed genomic segments, the greatest genetic diversity was observed in southern Africa (e.g. Zambia), while diversity in other populations was largely consistent with a geographic expansion from this potentially ancestral region. The European population showed different levels of diversity reduction on each chromosome arm, and some African populations displayed chromosome arm-specific diversity reductions. Inversions in the European sample were associated with strong elevations in diversity across chromosome arms. Genomic scans were conducted to identify loci that may represent targets of positive selection within an African population, between African populations, and between European and African populations. A disproportionate number of candidate selective sweep regions were located near genes with varied roles in gene regulation. Outliers for Europe-Africa F_ST were found to be enriched in genomic regions of locally elevated cosmopolitan admixture, possibly reflecting a role for some of these loci in driving the introgression of non-African alleles into African populations.     

Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster

Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-β-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.     

Surprising Differences in the Variability of Y Chromosomes in African and Cosmopolitan Populations of Drosophila melanogaster

The nonrecombining Drosophila melanogaster Y chromosome is heterochromatic and has few genes. Despite these limitations, there remains ample opportunity for natural selection to act on the genes that are vital for male fertility and on Y factors that modulate gene expression elsewhere in the genome. Y chromosomes of many organisms have low levels of nucleotide variability, but a formal survey of D. melanogaster Y chromosome variation had yet to be performed. Here we surveyed Y-linked variation in six populations of D. melanogaster spread across the globe. We find surprisingly low levels of variability in African relative to Cosmopolitan (i.e., non-African) populations. While the low levels of Cosmopolitan Y chromosome polymorphism can be explained by the demographic histories of these populations, the staggeringly low polymorphism of African Y chromosomes cannot be explained by demographic history. An explanation that is entirely consistent with the data is that the Y chromosomes of Zimbabwe and Uganda populations have experienced recent selective sweeps. Interestingly, the Zimbabwe and Uganda Y chromosomes differ: in Zimbabwe, a European Y chromosome appears to have swept through the population.     

Different forces drive the evolution of the Acp26Aa and Acp26Ab accessory gland genes in the Drosophila melanogaster species complex.

The Acp26Aa and Acp26Ab genes that code for male accessory gland proteins are tandemly arranged in the species of the Drosophila melanogaster complex. An approximately 1.6-kb region encompassing both genes has been sequenced in 10, 24, and 18 lines from Spain, Ivory Coast, and Malawi, respectively; the previously studied 10 lines from North Carolina have also been included in the analyses. A total of 110 nucleotide and 4 length polymorphisms were detected. Silent variation for the whole Acp26A region was slightly higher in African than in non-African populations, while for both genes nonsynonymous variation was similar in all populations studied. Based on Fst estimates no major genetic differentiation was detected between East and West Africa, while in general non-African populations were strongly differentiated from both African populations. Comparison of polymorphism and divergence at synonymous and nonsynonymous sites revealed that directional selection acting on amino acid replacement changes has driven the evolution of the Acp26Aa protein in the last 2.5 myr.     

Selective Pressures on <Emphasis Type="Italic">Drosophila</Emphasis> Chemosensory Receptor Genes

The evolution and patterns of selection of genes encoding 10 Drosophila odorant receptors (Or) and the sex pheromone receptor Gr68a were investigated by comparing orthologous sequences across five to eight ecologically diverse species of Drosophila. Using maximum likelihood estimates of dN/dS ratios we show that all 11 genes sampled are under purifying selection, indicating functional constraint. Four of these genes (Or33c, Or42a, Or85e, and Gr68a) may be under positive selection, and if so, there is good evidence that 12 specific amino acid sites may be under positive selection. All of these sites are predicted to be located either in loop regions or just inside membrane spanning regions, and interestingly one of the two sites in Gr68a is in a similar position to a previously described polymorphism in Gr5a that causes a shift in sensitivity to its ligand trehalose. For three Ors, possible evidence for positive selection was detected along a lineage. These include Or22a in the lineage leading to D. mauritiana and Or22b in the lineage leading to D. simulans. This is of interest in light of previous data showing a change in ligand response profile for these species in the sensory neuron (ab3A) which expresses both Or22a and Or22b in D. melanogaster. In summary, while the main chemosensory function and/or structural integrity of these 10 Or genes and Gr68a are evolutionarily preserved, positive selection appears to be acting on some of these genes, at specific sites and along certain lineages, and provides testable hypotheses for further functional experimentation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. David Pollock]     

Natural variation at the Drosophila melanogaster Or22 odorant receptor locus is associated with changes in olfactory behaviour

In insects, many critical olfactory behaviours are mediated by the large odorant receptor (Or) gene family, which determines the response properties of different classes of olfactory receptor neurons (ORNs). While ORN responses are generally conserved within and between Drosophila species, variant alleles of the D. melanogaster Or22 locus have previously been shown to alter the response profile of an ORN class called ab3A. These alleles show potential clinal variation, suggesting that selection is acting at this locus. Here, we investigated if the changes seen in ab3A responses lead to changes in olfactory-related behaviours. We show that variation at the Or22 locus and in the ab3A neurons are not fully compensated for by other ORNs and lead to overall changes in antennal odorant detection. We further show that this correlates with differences in odorant preference behaviour and with differences in oviposition site preference, with flies that have the chimaeric short allele strongly preferring to oviposit on banana. These findings indicate that variation at the Or22 locus leads to changes in olfactory-driven behaviours, and add support to the idea that the ab3A neurons are of especial importance to the ecology of Drosophila flies.     

Odorant receptor (Or) genes: polymorphism and divergence in the D. melanogaster and D. pseudoobscura lineages

Background

In insects, like in most invertebrates, olfaction is the principal sensory modality, which provides animals with essential information for survival and reproduction. Odorant receptors are involved in this response, mediating interactions between an individual and its environment, as well as between individuals of the same or different species. The adaptive importance of odorant receptors renders them good candidates for having their variation shaped by natural selection.

Methodology/Principal Findings

We analyzed nucleotide variation in a subset of eight Or genes located on the 3L chromosomal arm of Drosophila melanogaster in a derived population of this species and also in a population of Drosophila pseudoobscura. Some heterogeneity in the silent polymorphism to divergence ratio was detected in the D. melanogaster/D. simulans comparison, with a single gene (Or67b) contributing ∼37% to the test statistic. However, no other signals of a very recent selective event were detected at this gene. In contrast, at the speciation timescale, the MK test uncovered the footprint of positive selection driving the evolution of two of the encoded proteins in both D. melanogaster —OR65c and OR67a —and D. pseudoobscura —OR65b1 and OR67c.

Conclusions

The powerful polymorphism/divergence approach provided evidence for adaptive evolution at a rather high proportion of the Or genes studied after relatively recent speciation events. It did not provide, however, clear evidence for very recent selective events in either D. melanogaster or D. pseudoobscura.     

Drosophila: sentinels of environmental toxicants

Synthetic insecticides have been used intensively for the past50 years in many parts of the world. Insect populations, bothtarget and nontarget, have responded by evolving resistance.One of the nontarget insects is Drosophila melanogaster, whichis well-suited for genetic analysis and has been particularlywell-studied in both laboratory and field populations. Resistanceto several insecticides, including two for which significantresistance in field populations has not been found, has beengenerated in susceptible laboratory strains following mutagenesis,allowing comprehensive study of the resistance genes. Fieldpopulations of D. melanogaster have evolved resistance to many,but not all, insecticides in use today. Both the genetic andbiochemical mechanisms that underlie resistance in this insectare similar to those in other insects. Therefore, D. melanogastercan be a sentinel organism for long-term release of toxicantsinto the environment. While it remains useful for genetic analysisof resistance, a better understanding of the movement and populationstructures of this insect will be a prerequisite for its sentinelutilization at specific locales.     

Or47b-neurons promote male-mating success in Drosophila

Drosophila performs elaborate well-defined rituals of courtship, which involve several types of sensory inputs. Here, we report that Or47b-neurons promote male-mating success. Males with Or47b-neurons silenced/ablated exhibit reduced copulation frequency and increased copulation latency. Copulation latency of Or47b-manipulated flies increased proportionately with size of the assay arena, whereas in controls it remained unchanged. While competing for mates, Or47b-ablated males are outperformed by intact controls. These results suggest the role of Or47b-neurons in promoting male-mating success.     

Isolation, Properties and a Possible Function of a Water-Soluble Chlorophyll a/b-Protein from Brussels Sprouts

A water-soluble Chl a/b-protein (CP673) was isolated and purifiedfrom Brussels sprouts (Brassica oleracea L. var. gemmifera DC).The protein had a molecular mass of 78 kDa and an isoelectricpoint of 4.7, consisted of three or four subunits of 22 kDaand was extremely heat-stable. Although CP673 contained aboutone Chl a per protein, the blue and red absorption bands ofChl a that consisted of three or four Chl a forms with differentabsorption maxima suggested that there are several differentmodes or sites of binding for Chl a. Chl a/b ratio of largerthan 10 also indicated that Chl b is present only in a smallfraction of CP673. The heterogeneity of CP673 in terms of compositionand binding of Chl suggests that Chl is not an intrinsic componentof the Chl-protein. Homology search showed that the N-terminalamino acid sequence of CP673 is highly homologous with thatof a 22 kDa protein that accumulates in water-stressed leavesof two Brassicaceae plants, rapeseed and radish, but not withthose of the light-harvesting Chl a/b-proteins of photosynthesis.A possible function of the water-soluble Chl-protein was discussed. (Received September 17, 1996; Accepted November 18, 1996)     

Absence of short period interspersion of repetitive and non-repetitive sequences in the DNA of Drosophila melanogaster

A sensitive search has been made in Drosophila melanogaster DNA for short repetitive sequences interspersed with single copy sequences. Five kinds of measurements all yield the conclusion that there are few short repetitive sequences in this genome: 1) Comparison of the kinetics of reassociation of short (360 nucleotide) and long (1,830 nucleotide) fragments of DNA; 2) reassociation kinetics of long fragments (2,200 nucleotide) with an excess of short (390 short nucleotide) fragments; 3) measurement of the size of S1 nuclease resistant reassociated repeated sequences; 4) measurement of the hyperchromicity of reassociated repetitive fragments as a function of length; 5) direct assay by kinetics of reassociation of the amount of single copy sequence present on 1,200 nucleotide long fragments which also contain repetitive sequences.     

Similar Levels of X-linked and Autosomal Nucleotide Variation in African and non-African populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

Levels of molecular diversity in Drosophila have repeatedly been shown to be higher in ancestral, African populations than in derived, non-African populations. This pattern holds for both coding and noncoding regions for a variety of molecular markers including single nucleotide polymorphisms and microsatellites. Comparisons of X-linked and autosomal diversity have yielded results largely dependent on population of origin.     

Evolution of Olfactory Receptors Tuned to Mustard Oils in Herbivorous Drosophilidae

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.     

Preparation of Chlorophyll a, Chlorophyll b and Bacteriochlorophyll a by Column Chromatography with DEAE-Sepharose CL-6B and Sepharose CL-6B

Chlorophylls extracted from spinach leaves were made free fromcarotenoids, pheophytins, chlorophyllides and plastoquinonesby column chromatography with DEAE-Sepharose CL-6B. Chlorophylla and b were separated by column chromatography with SepharoseCL-6B. By a combined use of DEAE-Sepharose CL-6B and SepharoseCL-6B, pure chlorophyll a and b were prepared from the leavesin a short time. Bacteriochlorophyll a_p extracted from a photosyntheticbacterium Chromatium vinosum was made free from carotenoids,bacteriopheophytin, ubiquinone and lipids by column chromatographywith Sepharose CL-6B. (Received April 19, 1983; Accepted June 16, 1983)     

Reproduction rates and secondary production of three species of the rotifer genus Synchaeta in the estuarine Potomac River

Rotifers are a relatively well-studied component of lacustrinesystems but their role is only poorly understood in estuaries.Three species of the genus Synchaeta—S. baltica, S. triophthalmaand S.cecilia—dominate the cold-water assemblage of rotifersin Chesapeake Bay. Laboratory experiments were conducted todetermine the temperature dependence of egg development time(EDT) for each species; EDT varied over an approximate rangeof 90–9 h as temperature (T) varied from 2 to 22°C.The EDT/temperature relationships could be closely fitted bya simple polynomial equation of the form log(EDT) = a + b(logT) + c(log T)² for each species. Natural populations of thesethree rotifers were sampled during a cruise in the Potomac River(7–11 March 1983). Estimates of specific reproductiverates (b) were calculated based on the previously defined EDT/temperaturerelationship and the observed ratio of eggs/rotifers for eachspecies. The two most abundant species, S.triophthalma and S.cecilia,showed a clear dependence of b on the observed chlorophyll aconcentrations. Maximum reproductive rates ({small tilde}0.015h^–1) were attained only at relatively high phytoplanktondensities within a bloom of Heterocapsa triquetra where thechlorophyll a concentrations exceeded 10 µg l^–1.Estimates of secondary production suggest that Synchaeta spp.may contribute to the trophic flow of carbon in this systemwith a significance at least similar to that of the planktoniccopepods.     

Sequence and evolution of the Drosophila pseudoobscura glycerol-3-phosphate dehydrogenase locus

The Gpdh genomic region has been cloned and sequenced in Drosophila pseudoobscura. A total of 6.8 kb of sequence was obtained, encompassing all eight exons of the gene. The exons have been aligned with the sequence from D. melanogaster, and the rates of synonymous and nonsynonymous substitution have been compared to those of other genes sequenced in these two species. Gpdh has the lowest rate of nonsynonymous substitution yet seen in genes sequenced in both D. pseudoobscura and D. melanogaster. No insertion/deletion events were observed, and the overall architecture of the gene (i.e., intron sites, etc.) is conserved. An interesting amino acid reversal was noted between the D. melanogaster Fast allele and the D. pseudoobscura gene.     

Non-African populations of Drosophila melanogaster have a unique origin

Drosophila melanogaster is widely used as a model in DNA variation studies. Patterns of polymorphism have, however, been affected by the history of this species, which is thought to have recently spread out of Africa to the rest of the world. We analyzed DNA sequence variation in 11 populations, including four continental African and seven non-African samples (including Madagascar), at four independent X-linked loci. Variation patterns at all four loci followed neutral expectations in all African populations, but departed from it in all non-African ones due to a marked haplotype dimorphism at three out of four loci. We also found that all non-African populations show the same major haplotypes, though in various frequencies. A parsimonious explanation for these observations is that all non-African populations are derived from a single ancestral population having undergone a substantial reduction of polymorphism, probably through a bottleneck. Less likely alternatives involve either selection at all four loci simultaneously (including balancing selection at three of them), or admixture between two divergent populations. Small but significant structure was observed among African populations, and there were indications of differentiation across Eurasia for non-African ones. Since population history may result in non-equilibrium variation patterns, our study confirms that the search for footprints of selection in the D. melanogaster genome must include a sufficient understanding of its history.     

Effect of Sodium Dodecyl Sulfate on Structure and Spectroscopic Characteristics of Water-Soluble Chlorophyll Protein Complex Isolated from Stems of Lepidium virginicum

The relationship between structure and spectroscopic characteristicsof the watersoluble chlorophyll protein complex isolated fromstems of Lepidium virginicum (CP663S) was studied. Additionof 0.08% SDS induced a red shift of the 663 nm absorption maximum.At the same time, under excitation at 435 nm, the maximum offluorescence emission shifted from 672 nm to 675 nm and thefluorescence yield increased. When CP663S was excited at 480nm, the 660 nm emission band of chlorophyll b became more prominent.Fluorescence lifetime of emission from chlorophyll a increasedon addition of SDS. The energy transfer from chlorophyll b tochlorophyll a was decreased by the SDS addition, as judged bythe fluorescence spectra and lifetime measurement. Symmetricalpositive and negative peaks of the circular dichroism (CD) spectrumaround 669 nm, which indicate the interaction between chlorophylla molecules at short distances, disappeared after addition ofSDS. These SDS-induced changes of spectroscopic characteristicsoccurred in similar SDS concentration ranges and were reversible.SDS polyacrylamide gel electrophoresis cleaved CP663S into subunits.Chlorophyll molecules moved with protein moieties. Glutaraldehydetreatment suppressed the effects of SDS on absorption, fluorescenceand CD characteristics. We conclude that chlorophyll moleculesin CP663S are in the hydrophobic region of the protein and theinteraction between chlorophyll a molecules occurs at shortdistances. Changes of spectroscopic characteristics are a resultof cleavage of CP663S. ¹Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received November 22, 1982; Accepted May 31, 1983)     

Population genomic analysis uncovers African and European admixture in Drosophila melanogaster populations from the south‐eastern United States and Caribbean Islands

Drosophila melanogaster is postulated to have colonized North America in the past several 100 years in two waves. Flies from Europe colonized the east coast United States while flies from Africa inhabited the Caribbean, which if true, make the south‐east US and Caribbean Islands a secondary contact zone for African and European D. melanogaster. This scenario has been proposed based on phenotypes and limited genetic data. In our study, we have sequenced individual whole genomes of flies from populations in the south‐east US and Caribbean Islands and examined these populations in conjunction with population sequences from the west coast US, Africa, and Europe. We find that west coast US populations are closely related to the European population, likely reflecting a rapid westward expansion upon first settlements into North America. We also find genomic evidence of African and European admixture in south‐east US and Caribbean populations, with a clinal pattern of decreasing proportions of African ancestry with higher latitude. Our genomic analysis of D. melanogaster populations from the south‐east US and Caribbean Islands provides more evidence for the Caribbean Islands as the source of previously reported novel African alleles found in other east coast US populations. We also find the border between the south‐east US and the Caribbean island to be the admixture hot zone where distinctly African‐like Caribbean flies become genomically more similar to European‐like south‐east US flies. Our findings have important implications for previous studies examining the generation of east coast US clines via selection.     

Transposable elements in individual genotypes of Drosophila simulans

Transposable elements are abundant, dynamic components of the genome that affect organismal phenotypes and fitness. In Drosophila melanogaster, they have increased in abundance as the species spread out of Africa, and different populations differ in their transposable element content. However, very little is currently known about how transposable elements differ between individual genotypes, and how that relates to the population dynamics of transposable elements overall. The sister species of D. melanogaster, D. simulans, has also recently become cosmopolitan, and panels of inbred genotypes exist from cosmopolitan and African flies. Therefore, we can determine whether the differences in colonizing populations are repeated in D. simulans, what the dynamics of transposable elements are in individual genotypes, and how that compares to wild flies. After estimating copy number in cosmopolitan and African D. simulans, I find that transposable element load is higher in flies from cosmopolitan populations. In addition, transposable element load varies considerably between populations, between genotypes, but not overall between wild and inbred lines. Certain genotypes either contain active transposable elements or are more permissive of transposition and accumulate copies of particular transposable elements. Overall, it is important to quantify genotype‐specific transposable element dynamics as well as population averages to understand the dynamics of transposable element accumulation over time.