Gene flow and gene flux shape evolutionary patterns of variation in Drosophila subobscura.

Gene flow (defined as allele exchange between populations) and gene flux (defined as allele exchange during meiosis in heterokaryotypic females) are important factors decreasing genetic differentiation between populations and inversions. Many chromosomal inversions are under strong selection and their role in recombination reduction enhances the maintenance of their genetic distinctness. Here we analyze levels and patterns of nucleotide diversity, selection and demographic history, using 37 individuals of Drosophila subobscura from Mount Parnes (Greece) and Barcelona (Spain). Our sampling focused on two frequent O-chromosome arrangements that differ by two overlapping inversions (OST and O(3+4)), which are differentially adapted to the environment as observed by their opposing latitudinal clines in inversion frequencies. The six analyzed genes (Pif1A, Abi, Sqd, Yrt, Atpα and Fmr1) were selected for their location across the O-chromosome and their implication in thermal adaptation. Despite the extensive gene flux detected outside the inverted region, significant genetic differentiation between both arrangements was found inside it. However, high levels of gene flow were detected for all six genes when comparing the same arrangement among populations. These results suggest that the adaptive value of inversions is maintained, regardless of the lack of genetic differentiation within arrangements from different populations, and thus favors the Local Adaptation hypothesis over the Coadapted Genome hypothesis as the basis of the selection acting on inversions in these populations.

Finding of male-killing Spiroplasma infecting Drosophila melanogaster in Africa implies transatlantic migration of this endosymbiont.

We report the identification of male-killing Spiroplasma in a wild-caught female Drosophila melanogaster from Uganda, the first such infection to be found in this species outside of South America. Among 38 female flies collected from Namulonge, Uganda in April, 2005, one produced a total of 41 female offspring but no males. PCR testing of subsequent generations revealed that females retaining Spiroplasma infection continued to produce a large excess of female progeny, while females that had lost Spiroplasma produced offspring with normal sex ratios. Consistent with earlier work, we find that male-killing and transmission efficiency appear to increase with female age, and we note that males born in sex ratio broods display much lower survivorship than their female siblings. DNA sequence comparisons at three loci suggest that this Spiroplasma strain is closely related to the male-killing strain previously found to infect D. melanogaster in Brazil, although part of one locus appears to show a recombinant history. Implications for the origin and history of male-killing Spiroplasma in D. melanogaster are discussed.

Cross-species comparison of Drosophila male accessory gland protein genes.

Drosophila melanogaster males transfer seminal fluid proteins along with sperm during mating. Among these proteins, ACPs (Accessory gland proteins) from the male's accessory gland induce behavioral, physiological, and life span reduction in mated females and mediate sperm storage and utilization. A previous evolutionary EST screen in D. simulans identified partial cDNAs for 57 new candidate ACPs. Here we report the annotation and confirmation of the corresponding Acp genes in D. melanogaster. Of 57 new candidate Acp genes previously reported in D. melanogaster, 34 conform to our more stringent criteria for encoding putative male accessory gland extracellular proteins, thus bringing the total number of ACPs identified to 52 (34 plus 18 previously identified). This comprehensive set of Acp genes allows us to dissect the patterns of evolutionary change in a suite of proteins from a single male-specific reproductive tissue. We used sequence-based analysis to examine codon bias, gene duplications, and levels of divergence (via dN/dS values and ortholog detection) of the 52 D. melanogaster ACPs in D. simulans, D. yakuba, and D. pseudoobscura. We show that 58% of the 52 D. melanogaster Acp genes are detectable in D. pseudoobscura. Sequence comparisons of ACPs shared and not shared between D. melanogaster and D. pseudoobscura show that there are separate classes undergoing distinctly dissimilar evolutionary dynamics.

Diversity and selection in sorghum: simultaneous analyses using simple sequence repeats.

Although molecular markers and DNA sequence data are now available for many crop species, our ability to identify genetic variation associated with functional or adaptive diversity is still limited. In this study, our aim was to quantify and characterize diversity in a panel of cultivated and wild sorghums (Sorghum bicolor), establish genetic relationships, and, simultaneously, identify selection signals that might be associated with sorghum domestication. We assayed 98 simple sequence repeat (SSR) loci distributed throughout the genome in a panel of 104 accessions comprising 73 landraces (i.e., cultivated lines) and 31 wild sorghums. Evaluation of SSR polymorphisms indicated that landraces retained 86% of the diversity observed in the wild sorghums. The landraces and wilds were moderately differentiated (F st=0.13), but there was little evidence of population differentiation among racial groups of cultivated sorghums (F st=0.06). Neighbor-joining analysis showed that wild sorghums generally formed a distinct group, and about half the landraces tended to cluster by race. Overall, bootstrap support was low, indicating a history of gene flow among the various cultivated types or recent common ancestry. Statistical methods (Ewens-Watterson test for allele excess, lnRH, and F st) for identifying genomic regions with patterns of variation consistent with selection gave significant results for 11 loci (approx. 15% of the SSRs used in the final analysis). Interestingly, seven of these loci mapped in or near genomic regions associated with domestication-related QTLs (i.e., shattering, seed weight, and rhizomatousness). We anticipate that such population genetics-based statistical approaches will be useful for re-evaluating extant SSR data for mining interesting genomic regions from germplasm collections.

Genome-wide variation in the human and fruitfly: a comparison.

Average levels of nucleotide diversity are ten-fold lower in humans than in the fruitfly, Drosophila melanogaster. Despite this difference, apparently as a result of a lower population size, patterns of genomic diversity are strikingly similar in being correlated with local rates of recombination, and influenced by similar interactions between positive natural selection and recombination. Both species also show lower levels of variation on average in non-African compared to African populations, reflecting a similar evolutionary history and perhaps both natural selection and founder effects in new environments.

The evolutionary analysis of "orphans" from the Drosophila genome identifies rapidly diverging and incorrectly annotated genes.

In genome projects of eukaryotic model organisms, a large number of novel genes of unknown function and evolutionary history ("orphans") are being identified. Since many orphans have no known homologs in distant species, it is unclear whether they are restricted to certain taxa or evolve rapidly, either because of a lack of constraints or positive Darwinian selection. Here we use three criteria for the selection of putatively rapidly evolving genes from a single sequence of Drosophila melanogaster. Thirteen candidate genes were chosen from the Adh region on the second chromosome and 1 from the tip of the X chromosome. We succeeded in obtaining sequence from 6 of these in the closely related species D. simulans and D. yakuba. Only 1 of the 6 genes showed a large number of amino acid replacements and in-frame insertions/deletions. A population survey of this gene suggests that its rapid evolution is due to the fixation of many neutral or nearly neutral mutations. Two other genes showed "normal" levels of divergence between species. Four genes had insertions/deletions that destroy the putative reading frame within exons, suggesting that these exons have been incorrectly annotated. The evolutionary analysis of orphan genes in closely related species is useful for the identification of both rapidly evolving and incorrectly annotated genes.

Dynamics of microsatellite divergence under stepwise mutation and proportional slippage/point mutation models.

Recently Kruglyak, Durrett, Schug, and Aquadro showed that microsatellite equilibrium distributions can result from a balance between polymerase slippage and point mutations. Here, we introduce an elaboration of their model that keeps track of all parts of a perfect repeat and a simplification that ignores point mutations. We develop a detailed mathematical theory for these models that exhibits properties of microsatellite distributions, such as positive skewness of allele lengths, that are consistent with data but are inconsistent with the predictions of the stepwise mutation model. We use our theoretical results to analyze the successes and failures of the genetic distances (delta(mu))(2) and D(SW) when used to date four divergences: African vs. non-African human populations, humans vs. chimpanzees, Drosophila melanogaster vs. D. simulans, and sheep vs. cattle. The influence of point mutations explains some of the problems with the last two examples, as does the fact that these genetic distances have large stochastic variance. However, we find that these two features are not enough to explain the problems of dating the human-chimpanzee split. One possible explanation of this phenomenon is that long microsatellites have a mutational bias that favors contractions over expansions.

Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila.

Sequence comparisons of genomes or expressed sequence tags (ESTs) from related organisms provide insight into functional conservation and diversification. We compare the sequences of ESTs from the male accessory gland of Drosophila simulans to their orthologs in its close relative Drosophila melanogaster, and demonstrate rapid divergence of many of these reproductive genes. Nineteen ( approximately 11%) of 176 independent genes identified in the EST screen contain protein-coding regions with an excess of nonsynonymous over synonymous changes, suggesting that their divergence has been accelerated by positive Darwinian selection. Genes that encode putative accessory gland-specific seminal fluid proteins had a significantly elevated level of nonsynonymous substitution relative to nonaccessory gland-specific genes. With the 57 new accessory gland genes reported here, we predict that approximately 90% of the male accessory gland genes have been identified. The evolutionary EST approach applied here to identify putative targets of adaptive evolution is readily applicable to other tissues and organisms.

Microsatellite variation in colonizing and palearctic populations of Drosophila subobscura.

The recent colonization of North America by Drosophila subobscura has provided a great opportunity to analyze a colonization process from the beginning. A comparative study using 10 microsatellite loci was conducted for five European and two North American populations. No genetic differentiation between European populations was detected, indicating that gene flow is high among them and that the microsatellites used in the present work represent neutral markers not subject to differentiation due to selection. Extensive reduction in the number of alleles and a significant decrease in heterozygosity in colonizing populations were detected that could be explained by the founder effect and a subsequent quick but not infinite expansion. Assuming that all alleles present in the colonized area were carried by the sample of colonizers, we estimated that most probably 4-11 individuals expanded in the new area. F(ST) and the chord distance measures reflect the colonization process more accurately, since drift has been the major force in differentiating the Old and New World populations, and thus other measures considering allele size differences, such as Rho(ST) and deltamu2, are less reliable for studying nonequilibrium populations. Finally, our results were consistent with the two-phase microsatellite mutational model, indicating that most alleles are generated by gain or loss of a repeat unit, while some alleles originate by more complex mutations.

Polymorphism in abalone fertilization proteins is consistent with the neutral evolution of the egg's receptor for lysin (VERL) and positive darwinian selection of sperm lysin.

The evolution of species-specific fertilization in free-spawning marine invertebrates is important for reproductive isolation and may contribute to speciation. The biochemistry and evolution of proteins mediating species-specific fertilization have been extensively studied in the abalone (genus Haliotis). The nonenzymatic sperm protein lysin creates a hole in the egg vitelline envelope by species-specifically binding to its egg receptor, VERL. The divergence of lysin is promoted by positive Darwinian selection. In contrast, the evolution of VERL does not depart from neutrality. Here, we cloned a novel nonrepetitive region of VERL and performed an intraspecific polymorphism survey for red (Haliotis rufescens) and pink (Haliotis corrugata) abalones to explore the evolutionary forces affecting VERL. Six statistical tests showed that the evolution of VERL did not depart from neutrality. Interestingly, there was a subdivision in the VERL sequences in the pink abalone and a lack of heterozygous individuals between groups, suggesting that the evolution of assortative mating may be in progress. These results are consistent with a model which posits that egg VERL is neutrally evolving, perhaps due to its repetitive structure, while sperm lysin is subjected to positive Darwinian selection to maintain efficient interaction of the two proteins during sperm competition.

Positive Darwinian selection drives the evolution of several female reproductive proteins in mammals.

Rapid evolution driven by positive Darwinian selection is a recurrent theme in male reproductive protein evolution. In contrast, positive selection has never been demonstrated for female reproductive proteins. Here, we perform phylogeny-based tests on three female mammalian fertilization proteins and demonstrate positive selection promoting their divergence. Two of these female fertilization proteins, the zona pellucida glycoproteins ZP2 and ZP3, are part of the mammalian egg coat. Several sites identified in ZP3 as likely to be under positive selection are located in a region previously demonstrated to be involved in species-specific sperm-egg interaction, suggesting the selective pressure is related to male-female interaction. The results provide long-sought evidence for two evolutionary hypotheses: sperm competition and sexual conflict.

Distribution and abundance of microsatellites in the yeast genome can Be explained by a balance between slippage events and point mutations.

We fit a Markov chain model of microsatellite evolution introduced by Kruglyak et al. to data on all di-, tri-, and tetranucleotide repeats in the yeast genome. Our results suggest that many features of the distribution of abundance and length of microsatellites can be explained by this simple model, which incorporates a competition between slippage events and base pair substitutions, with no need to invoke selection or constraints on the lengths. Our results provide some new information on slippage rates for individual repeat motifs, which suggest that AT-rich trinucleotide repeats have higher slippage rates. As our model predicts, we found that many repeats were adjacent to shorter repeats of the same motif. However, we also found a significant tendency of microsatellites of different motifs to cluster.

High density of long dinucleotide microsatellites in Drosophila subobscura.

We isolated 96 dinucleotide repeats with five or more tandemly repeated units from a subgenomic Drosophila subobscura library. The mean repeat unit length of microsatellite clones in D. subobscura is 15, higher than that observed in other Drosophila species. Population variation was assayed in 32-40 chromosomes from Barcelona, Spain, using 18 randomly chosen microsatellite loci. Positive correlation between measures of variation and perfect repeat length measures (mean size, most common, and longest allele) is consistent with a higher mutation rate in loci with longer repeat units. Levels of microsatellite variation measured as variance in repeat number and heterozygosity in D. subobscura were similar to those of Drosophila pseudoobscura and higher than those of Drosophila melanogaster and Drosophila simulans. Our data suggest that higher levels of microsatellite variation, and possibly density, in D. subobscura compared with D. melanogaster are due to both a higher average effective population and a higher intrinsic slippage rate in the former species.

Microsatellite variation in populations of Drosophila pseudoobscura and Drosophila persimilis.

We have isolated, characterized and mapped 33 dinucleotide, three trinucleotide and one tetranucleotide repeat loci from the four major chromosomes of Drosophila pseudoobscura. Average inferred repeat unit length of the dinucleotide repeats is 12 repeat units, similar to D. melanogaster. Assays of D. pseudoobscura and populations of its sibling species, D. persimilis, using 10 of these loci show extremely high levels of variation compared with similar studies of dinucleotide repeat variation in D. melanogaster populations. The high levels of variation are consistent with an average mutation rate of approximately 10(-6) per locus per generation and an effective population size of D. pseudoobscura approximately four times larger than that of D. melanogaster. Consistent with allozymes and nucleotide sequence polymorphism, the dinucleotide repeat loci reveal minimal structure across four populations of D. pseudoobscura. Finally, our preliminary recombinational mapping of 24 of these microsatellites suggests that the total recombinational genome size may be larger than previously inferred using morphological mutant markers.

Large number of replacement polymorphisms in rapidly evolving genes of Drosophila. Implications for genome-wide surveys of DNA polymorphism.

We present a survey of nucleotide polymorphism of three novel, rapidly evolving genes in populations of Drosophila melanogaster and D. simulans. Levels of silent polymorphism are comparable to other loci, but the number of replacement polymorphisms is higher than that in most other genes surveyed in D. melanogaster and D. simulans. Tests of neutrality fail to reject neutral evolution with one exception. This concerns a gene located in a region of high recombination rate in D. simulans and in a region of low recombination rate in D. melanogaster, due to an inversion. In the latter case it shows a very low number of polymorphisms, presumably due to selective sweeps in the region. Patterns of nucleotide polymorphism suggest that most substitutions are neutral or nearly neutral and that weak (positive and purifying) selection plays a significant role in the evolution of these genes. At all three loci, purifying selection of slightly deleterious replacement mutations appears to be more efficient in D. simulans than in D. melanogaster, presumably due to different effective population sizes. Our analysis suggests that current knowledge about genome-wide patterns of nucleotide polymorphism is far from complete with respect to the types and range of nucleotide substitutions and that further analysis of differences between local populations will be required to understand the forces more completely. We note that rapidly diverging and nearly neutrally evolving genes cannot be expected only in the genome of Drosophila, but are likely to occur in large numbers also in other organisms and that their function and evolution are little understood so far.

DNA sequence variation and the recombinational landscape in Drosophila pseudoobscura: a study of the second chromosome.

The relationship between rates of recombination and DNA sequence polymorphism was analyzed for the second chromosome of Drosophila pseudoobscura. We constructed integrated genetic and physical maps of this chromosome using molecular markers at 10 loci spanning most of its physical length. The total length of the map was 128.2 cM, almost twice that of the homologous chromosome arm (3R) in D. melanogaster. There appears to be very little centromeric suppression of recombination, and rates of recombination are quite uniform across most of the chromosome. Levels of sequence variation (theta(W), based on the number of segregating sites) at seven loci (tropomyosin 1, Rhodopsin 3, Rhodopsin 1, bicoid, Xanthine dehydrogenase, Myosin light chain 1, and ribosomal protein 49) varied from 0.0036 to 0.0167. Generally consistent with earlier studies, the average estimate of theta(W) at total sites is 1.5-fold higher than that in D. melanogaster, while average theta(W) at silent sites is almost 3-fold higher. These estimates of variation were analyzed in the context of a background selection model under the same parameters of mutation rate and selection as have been proposed for D. melanogaster. It is likely that a significant fraction of the higher level of sequence variation in D. pseudoobscura can be explained by differences in regional rates of recombination rather than a larger species-level effective population size. However, the distribution of variation among synonymous, nonsynonymous, and noncoding sites appears to be quite different between the species, making direct comparisons of neutral variation, and hence inferences about effective population size, difficult. Tajima's D statistics for 6 out of the 7 loci surveyed are negative, suggesting that D. pseudoobscura may have experienced a rapid population expansion in the recent past or, alternatively, that slightly deleterious mutations constitute an important component of standing variation in this species.