Mating dynamics of a sperm-limited drosophilid, Zaprionus indianus.

When males have large sperm, they may become sperm limited and mating dynamics may be affected. One such species is Zaprionus indianus, a drosophilid that is an introduced pest species in the Americas. We examined aspects of mating behavior in Z. indianus to determine the senses necessary for mating and measure female and male remating habits. We found that vision is necessary for successful copulation, but wings, which produce courtship song, are not needed. Males need their foretarsi to successfully copulate and although the foretarsi may be needed for chemoreception, their role in hanging on to the female during copulation may be more important for successful mating. Females that mate once run out of sperm in approximately five days, although mating a second time greatly increases offspring production. Females do not seem to exert pre-mating choice among males with respect to mating with a familiar versus a novel male. Males are not capable of mating continuously and fail to produce offspring in many copulations. Overall, females of this species benefit from polyandry, providing an opportunity to study sexual selection in females. In addition, the dynamics of male competition for fertilizing eggs needs to be studied.

Phenology of Drosophila species across a temperate growing season and implications for behavior.

Drosophila community composition is complex in temperate regions with different abundance of flies and species across the growing season. Monitoring Drosophila populations provides insights into the phenology of both native and invasive species. Over a single growing season, we collected Drosophila at regular intervals and determined the number of individuals of the nine species we found in Kansas, USA. Species varied in their presence and abundance through the growing season with peak diversity occurring after the highest seasonal temperatures. We developed models for the abundance of the most common species, Drosophila melanogaster, D. simulans, D. algonquin, and the recent invasive species, D. suzukii. These models revealed that temperature played the largest role in abundance of each species across the season. For the two most commonly studied species, D. melanogaster and D. simulans, the best models indicate shifted thermal optima compared to laboratory studies, implying that fluctuating temperature may play a greater role in the physiology and ecology of these insects than indicated by laboratory studies, and should be considered in global climate change studies.

Quantitative Genetic Mapping and Genome Assembly in the Lesser Wax Moth Achroia grisella.

Specific characteristics of the male Achroia grisella acoustic mating signal determine a male's attractiveness toward females. These features are genetically variable in populations, and mapping experiments have been used to identify loci contributing to song variation, and understand the evolutionary forces acting on this important sexual trait. Here we built on this foundation and carried out QTL (Quantitative Trait Locus) mapping using >1,000 recombinant individuals, genotyping this large cohort at thousands of sequence-based markers covering the entire collection of 30 A. grisella chromosomes. This dense marker set, coupled with our development of an annotated, draft genome of A. grisella, allowed us to link >3,000 genome scaffolds, >10,000 predicted genes, and close to 275Mb of genome sequence to chromosomes. Our QTL mapping confirmed a fraction of the QTL identified in a previous study, and additionally revealed novel loci. Collectively, QTL explained only small fractions of the phenotypic variance, suggesting many more causative factors remain below the detection threshold of our study. A surprising, and ultimately challenging feature of our study was the low level of intrachromosomal recombination present in our mapping population. This led to difficulty ordering markers along linkage groups, necessitating a chromosome-by-chromosome mapping approach, rather than true interval mapping, and precluded confident ordering/orienting of scaffolds along each chromosome. Nonetheless, our study increased the genomic resources available for the A. grisella system. Enabled by ever more powerful technologies, future investigators will be able to leverage our data to provide more detailed genetic dissection of male song variation in A. grisella.

Assessing the use of wing ornamentation and visual display in female choice sexual selection.

Conspicuous sexual dimorphism is often ascribed to sexual selection. When the differences between the sexes are ornamental, this is thought to indicate a role for female choice. In spotted winged Drosophila species courtship, a male positioned in front of a female waves his wings, which have a patch of melanization on the exterior margin. In this study, we examine both female preference for wing spots and the role of vision in mating success in three species of the suzukii group: Drosophila biarmipes, D. suzukii, and D. subpulchrella. To assess female preference for wing spot, we removed the spot with a novel, non-invasive method, and competed spotless males with males with two spots. Phenotype did not affect mating success in any species. To eliminate the potential effect of competitive behavior on male mating success, we also ran a no-choice analysis. Mating frequency and timing was not different between phenotypes within these species. The effect of vision on mating success was assessed by comparing mating success of spotted males between light and dark conditions, both for frequency of mating, as well as timing of multiple courtship parameters. Species varied in the extent that lack of vision negatively affected mating success. Though vision is important for mating success, the spot itself may not be providing the signal that females use to make mating decisions.

Costs of cold acclimation on survival and reproductive behavior in Drosophila melanogaster.

Fitness is determined by the ability of an organism to both survive and reproduce; however, the mechanisms that lead to increased survival may not have the same effect on reproductive success. We used nineteen natural Drosophila melanogaster genotypes from the Drosophila Genetic Reference Panel to determine if adaptive plasticity following short-term acclimation through rapid cold-hardening (RCH) affects mating behavior and mating success. We confirmed that exposure to the acclimation temperature is beneficial to survival following cold stress; however, we found that this same acclimation temperature exposure led to less efficient male courtship and a significant decrease in the likelihood of mating. Cold tolerance and the capacity to respond plastically to cold stress were not correlated with mating behavior following acclimation, suggesting that the genetic control of the physiological effects of the cold temperature exposure likely differ between survival and behavioral responses. We also tested whether the exposure of males to the acclimation temperature influenced courtship song. This exposure again significantly increased courtship duration; however, courtship song was unchanged. These results illustrate costs of short-term acclimation on survival and reproductive components of fitness and demonstrate the pronounced effect that short-term thermal environment shifts can have on reproductive success.

Development of a Genomic Resource and Quantitative Trait Loci Mapping of Male Calling Traits in the Lesser Wax Moth, Achroia grisella.

In the study of sexual selection among insects, the Lesser Waxmoth, Achroia grisella (Lepidoptera: Pyralidae), has been one of the more intensively studied species over the past 20 years. Studies have focused on how the male calling song functions in pair formation and on the quantitative genetics of male song characters and female preference for the song. Recent QTL studies have attempted to elucidate the genetic architecture of male song and female preference traits using AFLP markers. We continued these QTL studies using SNP markers derived from an EST library that allowed us to measure both DNA sequence variation and map loci with respect to the lepidopteran genome. We report that the level of sequence variation within A. grisella is typical among other Lepidoptera that have been examined, and that comparison with the Bombyx mori genome shows that macrosynteny is conserved. Our QTL map shows that a QTL for a male song trait, pulse-pair rate, is situated on the Z chromosome, a prediction for sexually selected traits in Lepidoptera. Our findings will be useful for future studies of genetic architecture of this model species and may help identify the genetics associated with the evolution of its novel acoustic communication.

Reaction norm variants for male calling song in populations of Achroia grisella (Lepidoptera: Pyralidae): toward a resolution of the lek paradox.

Significant additive genetic variance often occurs for male advertisement traits in spite of the directional selection imposed by female choice, a problem generally known in evolutionary biology as the lek paradox. One hypothesis, which has limited support from recent studies, for the resolution of this paradox is the role of genotype x environment interaction in which no one genotype exhibits the superior performance in all environments--a crossover of reaction norms. However, these studies have not characterized the actual variation of reaction norms present in natural populations, and the extent to which crossover maintains genetic variance remains unknown. Here, we present a study of genotype x environment interaction for the male calling song in populations of Achroia grisella (Lepidoptera: Pyralidae; lesser waxmoth). We report significant variance among reaction norms for male calling song in two North American populations of A. grisella as measured along temperature, food availability, and density gradients, and there is a relatively high incidence of crossover of the temperature reaction norms. This range of reaction norm variants and their crossover may reflect the co-occurrence of plastic and canalized genotypes, and we argue that the different responses of these variants along environmental gradients may contribute toward the maintenance of genetic variance for male song.

Quantitative trait loci for cuticular hydrocarbons associated with sexual isolation between Drosophila simulans and D. sechellia.

The identification of genes with large effects on sexual isolation and speciation is an important link between classic evolutionary genetics and molecular biology. Few genes that affect sexual isolation and speciation have been identified, perhaps because many traits influencing sexual isolation are complex behaviors. Cuticular hydrocarbons (CHs) of species of the Drosophila melanogaster group play a large role in sexual isolation by functioning as contact pheromones influencing mate recognition. Some of the genes that play key roles in determining species-specific CHs have been identified. We have performed separate quantitative trait locus (QTL) analyses of 7-tricosene (7-T) and 7,11-heptacosadiene (7,11-HD), the two major female CHs differing between D. simulans and D. sechellia. We find that approximately 40% of the phenotypic variance in each CH is associated with two to four chromosomal regions. A region on the right arm of chromosome 3 contains QTL that affect both traits, but other QTL are in distinct chromosomal regions. Epistatic interactions were detected between two pairs of QTL for 7,11-HD such that if either were homozygous for the D. simulans allele, the fly was similar to D. simulans in phenotype, with a low level of 7,11-HD. We discuss the location of these regions with regard to candidate genes for CH production, including those for desaturases.

Mutations and natural genetic variation in the courtship song of Drosophila.

All Drosophila species have a courtship repertoire by which a male stimulates a female to mate with him. In many species, males vibrate their wings to produce courtship song, an element of courtship that plays an important role in female choice. Each species has a unique courtship song, with the major differences among species songs being in timing and/or structure. Analysis of genetic mutations has revealed 17 genes that affect courtship song in Drosophila melanogaster. Most of the genes were first identified as affecting another trait and were subsequently shown to affect song. Quantitative genetic studies have demonstrated a polygenetic additive genetic architecture for many song traits. Few candidate genes, identified through the classical genetic approach, coincide with the regions implicated as affecting natural variation. With many new tools in genetic analysis and the multiple Drosophila genome projects currently underway, the ability to relate mutational and quantitative analyses will improve.

Do quantitative trait loci (QTL) for a courtship song difference between Drosophila simulans and D. sechellia coincide with candidate genes and intraspecific QTL?

The genetic architecture of traits influencing sexual isolation can give insight into the evolution of reproductive isolation and hence speciation. Here we report a quantitative trait loci (QTL) analysis of the difference in mean interpulse interval (IPI), an important component of the male courtship song, between Drosophila simulans and D. sechellia. Using a backcross analysis, we find six QTL that explain a total of 40.7% of the phenotypic variance. Three candidate genes are located in the intervals bounded by two of the QTL and there are no significant QTL on the X chromosome. The values of mean IPI for hybrid individuals imply the presence of dominant alleles or epistasis. Because unisexual hybrid sterility prevents an F(2) analysis, we cannot distinguish dominant from additive genetic effects at the scale of QTL. A comparison with a study of QTL for intraspecific variation in D. melanogaster shows that, for these strains, the QTL we have identified for interspecific variation cannot be those that contribute to intraspecific variation. We find that the QTL have bidirectional effects, which indicates that the genetic architecture is compatible with divergence due to genetic drift, although other possibilities are discussed.

Analysis of a shift in codon usage in Drosophila.

In order to gain further insight into a shift in codon usage first observed in Drosophila willistoni we have analyzed seven genes in six species in the lineage leading to D. willistoni. This lineage contains the willistoni and saltans species groups. Sequences were obtained from GenBank or newly sequenced for this study. All species studied showed significant difference in codon usage compared to D. melanogaster for about one third of all amino acids. Within the willistoni/saltans lineage, codon usage is homogeneous, indicating that the shift in codon usage occurred prior to the diversification of extant species in this lineage which we estimate to date to about 20 million years ago. Thus the shift is old and has been stable. We also examined introns from these genes and the G/C composition at four-fold degenerate sites in an effort to detect a change in mutation bias. There is little or no evidence for a difference in mutation bias compared to D. melanogaster. We also considered whether relaxed selection (possibly due to reduced population sizes) or reduced recombination (due to numerous naturally occurring inversions) could account for the shift and concluded these factors alone are insufficient to explain the patterns observed. A change in the relative abundance of isoaccepting tRNAs is one of the few explanations that can account for the observations. Particularly intriguing is the fact that the greatest changes in codon usage have occurred for amino acids with two-fold C/T ending codons for which it is known that posttranscriptional modification occurs in tRNAs from a G in the wobble position to Queuosine that changes optimal binding from C to a slight preference for U. However, we do not argue that this shift was adaptive in nature, rather it may be an example of a "frozen accident."

A MOLECULAR PHYLOGENY OF THE DROSOPHILA WILLISTONI GROUP: CONFLICTS BETWEEN SPECIES CONCEPTS?

The six sibling species of the Neotropical Drosophila willistoni group have a long history in studies of evolutionary biology, yet to date only one molecular study, which used allozymes, has been published on the phylogeny of the group. Here we present a phylogeny of the siblings based on the sequences of two nuclear genes, period (per) and Alcohol dehydrogenase (Adh), as well as the mitochondrial gene Cytochrome oxidase I (COI). Taken individually, only per has a strong phylogenetic signal supporting a well-resolved phylogeny of the group, and this phylogeny is different from that obtained using allozymes. The COI dataset by itself produces trees that disagree with per, and neither that data nor the Adh data have a strong phylogenetic signal, as indicated by low bootstrap values for all analyses. Combining the Adh and COI datasets results in the same tree as per alone. Combining all three genes results in the same topology, which is strongly supported. Two problematic taxa, D. pavlovskiana and a "Carmody strain," which were identified as potentially separate species based on reproductive isolation, clearly cluster in the phylogenetic analyses within D. paulistorum and D. equinoxialis, respectively. Thus, there appears to be a conflict between the biological species concept and the phylogenetic species concept.

EVOLUTION OF COURTSHIP SONG AND REPRODUCTIVE ISOLATION IN THE DROSOPHILA WILLISTONI SPECIES COMPLEX: DO SEXUAL SIGNALS DIVERGE THE MOST QUICKLY?

Reproductive isolation increases with genetic distance between species. Although sexual selection may drive divergence of sexual signals and traits, causing rapid evolution of sexual isolation, quantitative data supporting this idea are rare. We examine the rates of divergence of a species-specific courtship signal, sexual isolation, and postmating isolation in the Drosophila willistoni group. Both types of isolation increase with genetic distance and postmating isolation is the most strongly correlated with genetic divergence, suggesting this has the least variable divergence rate. Song divergence is not correlated with genetic divergence. Homoplasy in song pattern results in poorly resolved phylogenies that are different from molecular phylogenies. Song evolves more quickly than sexual isolation, which evolves more quickly than postmating isolation.

MITOCHONDRIAL DNA PHYLOGENIES FOR THE DROSOPHILA OBSCURA GROUP.

Species belonging to the obscura group of the genus Drosophila have long held a central position in evolutionary studies, especially in experimental population genetics. Despite the considerable amount of accumulated knowledge, many of the phylogenetic relationships of the species in the group remain unclear. Here we present DNA sequence data for the mitochondrial gene cytochrome oxidase I (COI) for 13 species native to both the Old and New Worlds. We combine these data with seven other mitochondrial gene sequences from previous studies, for a total of over 3 kb per species. Strongly supported conclusions include: (1) the two North American subgroups, pseudoobscura and affinis, are each monophyletic; and (2) among Eurasian species two unambiguous clades are identified, one containing D. tristis, D. ambigua, and D. obscura and the other containing D. guanche, D. subobscura, and D. madeirensis. Constructing firm hypotheses connecting these four major clades is problematic with all datasets. Major ambiguities are the number of invasions giving rise to the North American obscura species and the relationships among the Eurasian species. The inadequacy of the mtDNA data to resolve these ambiguities does not reside in lack of changes; the transversions-only parsimony tree has 283 informative characters. Rather, the problems are likely intrinsic to the history of the group: while radiating in temperate Eurasia, North America was colonized once or twice, followed by one or two radiations in the New World.