No evidence of sexual selection in a repetition of Bateman's classic study of Drosophila melanogaster.

We are unique in reporting a repetition of Bateman [Bateman AJ (1948) Heredity (Edinb) 2:349-368] using his methods of parentage assignment, which linked sex differences in variance of reproductive success and variance in number of mates in small populations of Drosophila melanogaster. Using offspring phenotypes, we inferred who mated with whom and assigned offspring to parents. Like Bateman, we cultured adults expressing dramatic phenotypes, so that each adult was heterozygous-dominant at its unique marker locus but had only wild-type alleles at all other subjects' marker loci. Assuming no viability effects of parental markers on offspring, the frequencies of parental phenotypes in offspring follow mendelian expectations: one-quarter will be double-mutants who inherit the dominant gene from each parent, the offspring from which Bateman counted the number of mates per breeder; half of the offspring must be single mutants inheriting the dominant gene of one parent and the wild-type allele of the other parent; and one-quarter would inherit neither of their parent's marker mutations. Here we show that inviability of double-mutant offspring biased inferences of mate number and number of offspring on which rest inferences of sex differences in fitness variances. Bateman's method overestimated subjects with zero mates, underestimated subjects with one or more mates, and produced systematically biased estimates of offspring number by sex. Bateman's methodology mismeasured fitness variances that are the key variables of sexual selection.

Evolution of genes and genomes on the Drosophila phylogeny.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

Sexual isolation between North American and Bogota strains of Drosophila pseudoobscura.

Sexual isolation, the reduced ability of organisms of different species to successfully mate, is one of the reproductive barriers that prevent gene flow between different taxa. Various species-specific signals during courtship are attributed to the sexual isolation between them. Drosophila pseudoobscura has been widely used to study the behavioral and genetic mechanisms underlying selection for sexual isolation, as a model system for speciation. D. pseudoobscura and its sibling species, D. persmilis, live together in many locations but are reproductively isolated from one another. North American geographic strains of D. pseudoobscura from the American West mate at random. Several decades ago, D. pseudoobscura was collected in the vicinity of Bogota, Colombia, and later named the subspecies D. pseudoobscura bogotana. Nearly 5,000 matings were observed in this study. We analyzed mating behavior and cuticular hydrocarbon profiles as well as courtship within and between North American and Bogota strains of D. pseudoobscura. Here we report for the first time that Bogota strains of D. pseudoobscura do show statistically significant sexual isolation from North American strains. In addition, there are quantitative variations in cuticular hydrocarbons as well as in courtship behavior between Bogota and North American strains, and females of both North American and Bogota strains show strong preference for North American strain males having high mating propensities, suggesting that the Bogota strains are at an early stage that could lead to a separate species.

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps.

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.

The Transcriptional Repressor BS69 is a Conserved Target of the E1A Proteins from Several Human Adenovirus Species.

Early region 1A (E1A) is the first viral protein produced upon human adenovirus (HAdV) infection. This multifunctional protein transcriptionally activates other HAdV early genes and reprograms gene expression in host cells to support productive infection. E1A functions by interacting with key cellular regulatory proteins through short linear motifs (SLiMs). In this study, the molecular determinants of interaction between E1A and BS69, a cellular repressor that negatively regulates E1A transactivation, were systematically defined by mutagenesis experiments. We found that a minimal sequence comprised of MPNLVPEV, which contains a conserved PXLXP motif and spans residues 112⁻119 in HAdV-C5 E1A, was necessary and sufficient in binding to the myeloid, Nervy, and DEAF-1 (MYND) domain of BS69. Our study also identified residues P113 and L115 as critical for this interaction. Furthermore, the HAdV-C5 and -A12 E1A proteins from species C and A bound BS69, but those of HAdV-B3, -E4, -D9, -F40, and -G52 from species B, E, D, F, and G, respectively, did not. In addition, BS69 functioned as a repressor of E1A-mediated transactivation, but only for HAdV-C5 and HAdV-A12 E1A. Thus, the PXLXP motif present in a subset of HAdV E1A proteins confers interaction with BS69, which serves as a negative regulator of E1A mediated transcriptional activation.

Mechanisms of genetic exchange within the chromosomal inversions of Drosophila pseudoobscura.

We have used the inversion system of Drosophila pseudoobscura to investigate how genetic flux occurs among the gene arrangements. The patterns of nucleotide polymorphism at seven loci were used to infer gene conversion events between pairs of different gene arrangements. We estimate that the average gene conversion tract length is 205 bp and that the average conversion rate is 3.4 x 10(-6), which is 2 orders of magnitude greater than the mutation rate. We did not detect gene conversion events between all combinations of gene arrangements even though there was sufficient nucleotide variation for detection and sufficient opportunity for exchanges to occur. Genetic flux across the inverted chromosome resulted in higher levels of differentiation within 0.1 Mb of inversion breakpoints, but a slightly lower level of differentiation in central inverted regions. No gene conversion events were detected within 17 kb of an inversion breakpoint suggesting that the formation of double-strand breaks is reduced near rearrangement breakpoints in heterozygotes. At least one case where selection rather than proximity to an inversion breakpoint is responsible for reduction in polymorphism was identified.

Mutual interest between the sexes and reproductive success in Drosophila pseudoobscura.

The pre-mating behavior of female Drosophila pseudoobscura has been considered passive and "coy" relative to more active, "ardent," and indiscriminate male behavior. To test whether this long-held view-the "received wisdom" about mating behavior in Drosophila-is really true we carried out observations on how often D. pseudoobscura females approached males prior to courtship and copulation. By including only virgin females and males in the experiments, we eliminated the possibility that males are "coy" due to sperm limitation and females flexibly "coy" due to male manipulations that may affect the duration of remating inhibition. We observed the movements of females and males in vials during the first five minutes of exposure to one another. Video records revealed females went toward males as frequently as males toward females; we inferred that females were as interested in males as males in females. The total number of offspring emerging as adults correlated significantly with mutual, precourtship interest of both males and females in their vial-mates and latency to copulation. Thus, we hypothesize that females in nature approach males, perhaps actively soliciting male courtship simply by remaining close to them.

Indiscriminate females and choosy males: within- and between-species variation in Drosophila.

The classic view of choosy, passive females and indiscriminate, competitive males gained theoretical foundations with parental investment theory. When females invest more in offspring than males, parental investment theory says that selection operates so that females discriminate among males for mates (i.e., females are choosy and passive) and males are indiscriminate (i.e., males are profligate and competitive). Here we report tests of predictions using Drosophila pseudoobscura and D. melanogaster, with typical asymmetry in gamete sizes (females > males), and in D. hydei with far less asymmetry in gamete size. Experimental observations revealed that the labels "choosy, passive females" and "profligate, indiscriminate males" did not capture the variation within and between species in premating behavior. In each of the species some females were as active in approaching males (or more so) than males in approaching females, and some males were as discriminating (or more so) than females. In pairs focal males and females responded differently to opposite-sex than to same-sex conspecifics. Drosophila hydei were less sex-role stereotyped than the other two species consistent with parental investment theory. However, D. pseudoobscura females approached males more often than did D. melanogaster females, and male D. hydei approached females as often as males of the other two species, both results inconsistent with parental investment theory. Male D. pseudoobscura and D. hydei were more likely to approach males in same-sex pairs than male D. melanogaster, inconsistent with parental investment theory.

Mendel's law reveals fatal flaws in Bateman's 1948 study of mating and fitness.

Bateman's experimental study of Drosophila melanogaster produced conclusions that are now part of the bedrock premises of modern sexual selection. Today it is the most cited experimental study in sexual selection, and famous as the first experimental demonstration of sex differences in the relationship between number of mates and relative reproductive success. We repeated the experimental methodology of the original to evaluate its reliability. The results indicate that Bateman's methodology of visible mutations to assign parentage and reproductive success to subject adults is significantly biased. When combined in offspring, the mutations decrease offspring survival, so that counts of mate number and reproductive success are mismeasured. Bateman's method overestimates the number of subjects with no mates and underestimates the number with one or more mates for both sexes. Here we discuss why Bateman's paper is important and present additional analyses of data from our monogamy trials. Monogamy trials can inform inferences about the force of sexual selection in populations because in monogamy trials male-male competition and female choice are absent. Monogamy trials also would have provided Bateman with an a priori test of the fit of his data to Mendel's laws, an unstated, but vital assumption of his methodology for assigning parentage from which he inferred the number of mates per individual subject and their reproductive success. Even under enforced monogamous mating, offspring frequencies of double mutant, single mutant and no mutant offspring were significantly different from Mendelian expectations proving that Bateman's method was inappropriate for answering the questions he posed. Double mutant offspring (those with a mutation from each parent) suffered significant inviability as did single mutant offspring whenever they inherited their mother's marker but the wild-type allele at their father's marker locus. These inviability effects produced two important inaccuracies in Bateman's results and conclusions. (1) Some matings that actually occurred were invisible and (2) reproductive success of some mothers was under-estimated. Both observations show that Bateman's conclusions about sex differences in number of mates and reproductive success were unwarranted, based on biased observations. We speculate about why Bateman's classic study remained without replication for so long, and we discuss why repetition almost 60 years after the original is still timely, necessary and critical to the scientific enterprise. We highlight overlooked alternative hypotheses to urge that modern tests of Bateman's conclusions go beyond confirmatory studies to test alternative hypotheses to explain the relationship between mate number and reproductive success.

Preference status does not indicate intrinsic quality differences in Drosophila pseudoobscura.

The good genes hypothesis states that choosers prefer individuals of high intrinsic quality to individuals of lower intrinsic quality. Variation in longevity is thought to reflect, in part, intrinsic quality differences of individuals in addition to the costs of mating and reproduction. Here we report longevity variation of Drosophila pseudoobscura, a species in which previous experiments have demonstrated that individual mate preferences (pre-touching mate assessments) of females and males are associated with enhanced numbers of eclosed (adult) offspring and higher egg-to-adult survival (offspring viability). Using mate assessment arenas and protocols similar to those in a previous experiment that demonstrated fitness benefits to breeders and their offspring of mating with individuals they preferred, we tested the following predictions: (i) preferred discriminatees live longer than non-preferred discriminatees; (ii) males live longer than females; and (iii) virgins live longer than mated individuals. The experiment yielded 938 individuals for longevity analysis. Sex and mating status affected longevity: males lived longer than females, virgin females lived longer than mated females, but there were no differences in longevity for mated and virgin males. Non-preferred discriminatees of both sexes survived as long as preferred discriminatees, a result inconsistent with the prediction of the good genes hypothesis for mate preferences. To our knowledge, this is the first demonstration of the cost of reproduction for females in D. pseudoobscura and of longevity variation of preferred and non-preferred discriminatees.

Experimental constraints on mate preferences in Drosophila pseudoobscura decrease offspring viability and fitness of mated pairs.

Using Drosophila pseudoobscura, we tested the hypothesis that social constraints on the free expression of mate preferences, by both females and males, decrease offspring viability and reproductive success of mating pairs. Mate preference arenas eliminated intrasexual combat and intersexual coercion. The time female and male choosers spent in arena tests near either of two opposite-sex individuals measured the preferences of choosers. We placed choosers in breeding trials with their preferred or nonpreferred discriminatee when they met the minimum criteria for showing the same preference in two consecutive tests. There was no statistically significant difference in the frequency of female and male choosers meeting minimal preference criteria. There was a significant difference between female and male choosers for offspring viability, with female choice having the greater effect, but there was not a significant difference in the overall reproductive success of male and female choosers. There were significant differences in fitness between matings to preferred and nonpreferred partners. Female and male choosers paired with their nonpreferred discriminatees had offspring of significantly lower viability, as predicted by the constraints hypothesis. Reproductive success, our measure of overall fitness, was greater when males or females mated with the partner they preferred rather than the one they did not prefer.

The hypothesis of reproductive compensation and its assumptions about mate preferences and offspring viability.

The Compensation Hypothesis says that parents and prospective parents attempt to make up for lowered offspring viability by increasing reproductive effort to produce healthy, competitive offspring and by increasing investment in less viable, but still-living progeny (parental effects). The hypothesis assumes that offspring viability is lower when individuals are constrained (often through sexual conflict) to breed with individuals they do not prefer. We review results of experimental tests of the offspring-viability assumption in Tanzanian cockroaches, fruit flies, pipefish, wild mallards, and feral house mice. Experimental constraints on mating preferences lowered offspring viability in each of the studies. Females breeding under constraints laid more eggs or gave birth to more young than females breeding without or with fewer constraints on their mating preferences, and males mating under constraints on their mate preferences ejaculated more sperm than males mating without constraints. The number of eggs laid or offspring born was higher when female choosers were experimentally constrained to reproduce with males they did not prefer. Constrained females may increase fecundity to enhance the probability that they produce adult offspring with rarer phenotypes with survival benefits against offspring generation pathogens. Similarly, ejaculation of more sperm when males are paired with females they do not prefer may be a mechanism that provides more variable sperm haplotypes for prospective mothers or that may provide nutritional benefits to mothers and zygotes.

Sexual isolation between sympatric and allopatric populations of Drosophila pseudoobscura and D. persimilis.

According to reinforcement theory, sexual isolation between species in sympatry is strengthened by natural selection against maladaptive hybrids. Drosophila pseudoobscura and D. persimilis from four locations where these species are sympatric, and from three locations where only D. pseudoobscura has been found, were utilized in studies of sexual isolation. Multiple-choice observation chambers were used to record matings between sympatric and allopatric strains of the two species. There was a wide variation in sexual isolation between the two species in the four localities we studied. The average isolation index for sympatric strains of the species was not significantly different from the average index for allopatric strains. There were no meaningful differences between the isolation indices in sympatric and allopatric strains of the species. The failure to find a relationship is likely the result of gene flow among populations within the two species.

VIABILITIES OF AMYLASE GENOTYPES IN DROSOPHILA PSEUDOOBSCURA ON STARCH AND MALTOSE FOOD.

In order to study the relationship between selection on an enzyme and the environment, viability was measured for genotypes at the α-amylase (Amy) locus of D. pseudoobscura on food containing either starch as the primary source of carbohydrates or, as a control, maltose, the breakdown product of starch by amylase. These conditions were chosen to reveal possible differences among these genotypes in their abilities to survive on starch food. Counted numbers of larvae were cultured on food with low or high levels of starch, or with maltose, at both 20°C and 25°C. Only under the most stressful environmental conditions, on food with a low starch concentration at 25°C, did the genotypes differ significantly in viability. Four population cages maintained on food with either maltose or starch as carbohydrate sources were set up to assay differences in overall fitness among the Amy genotypes. No selective differences were observed over 10 generations, and we infer that components of fitness other than viability also differed little among the Amy genotypes on the two types of food. Thus our efforts to manipulate the environmental levels of starch, the substrate of amylase, led to measurable selective differences only under very stressful conditions.

Evolutionary genomics of inversions in Drosophila pseudoobscura: evidence for epistasis.

Drosophila pseudoobscura harbors a rich polymorphism for paracentric inversions on the third chromosome, and the clines in the inversion frequencies across the southwestern United States indicate that strong natural selection operates on them. Isogenic inversion strains were made from isofemale lines collected from four localities, and eight molecular markers were mapped on the third chromosome. Nucleotide diversity was measured for these loci and formed the basis of an evolutionary genomic analysis. The loci were differentiated among inversions. The inversions did not show significant differences among populations, however, likely the result of extensive gene flow among populations. Some loci had significant reductions in nucleotide diversity within inversions compared with interspecies divergence, suggesting that these loci are near inversion breakpoints or are near targets of directional selection. Linkage disequilibrium (LD) levels tended to decrease with distance between loci, indicating that some genetic exchange occurs among gene arrangements despite the presence of inversions. In some cases, however, adjacent genes had low levels of interlocus LD and loosely linked genes had high levels of interlocus LD, suggesting strong epistatic selection. Our results support the hypothesis that the inversions of D. pseudoobscura have emerged as suppressors of recombination to maintain positive epistatic relationships among loci within gene arrangements that developed as the species adapted to a heterogeneous environment.