"Genetics of hybrid sterility in Drosophila" National Science Foundation (Population Biology, DEB), 6/01/2005-5/31/2008.

"Chromosomal inversions and the persistence of species" National Science Foundation (Population Biology, DEB), 8/15/2003-7/31/2007.

"Genetics of speciation factors in Drosophila mojavensis" National Science Foundation (Population Biology, DEB), 9/01/2002-8/31/2006, collaborative with Bill Etges and Mike Ritchie.

"Identification of misregulated gene products and disrupted genetic pathways in male hybrids of Drosophila pseudoobscura and D. persimilis" National Science Foundation (Population Biology, DEB), 12/01/2000-11/30/2002.

"Genetics of reinforcement in Drosophila," National Science Foundation (Population Biology, DEB), 8/01/2000-6/30/2004.

"Genetics of speciation in Drosophila," National Institutes of Health (NIGMS, R01), 7/01/1998-6/30/2003, collaborative with Jody Hey.
The long term goal of the proposed research is to reveal the evolutionary mechanisms that have given rise to biological species. Speciation is difficult to study, even with pairs of closely related species, because their divergence and isolation from each other limits the degree to which the genetic basis of their divergence can be studied. The proposed work will be on Drosophila pseudoobscura, D. p. bogotana and D. persimilis, three closely related and frequently studied new world species. It is possible to generate fertile hybrids of these species, so one can map and count genes that are responsible for the traits associated with isolation. Three complementary approaches to the study of species differences will be joined in this work. A dense microsattelite map has been developed for these species, and these markers will be use for mapping genes responsible for isolation traits, such as sterility, hybrid inviability, and mater discrimination. (2) At the same time as these studies proceed, the genomic regions that flank the individual microsattelite loci will be the subject of a thorough comparative DNA sequence study of intraspecific and interspecific and interspecific variation. Together these two studies will show which genes are linked to those that contribute to isolation, and it will show the historical pattern of divergence that has occurred for these same regions. Previous research has shown that these species have a history that includes more gene flow for some genomic regions than for others. This means that natural selection, acting against gene flow, has been a critical part of their speciation and divergence. (3) A third study will also be done to measure the amount of introgression, from one species to another, that can occur for each microsatellite locus. These introgression experiments will be done in the laboratory. In contrast to the mapping of isolation traits, the introgression mapping does not rely on investigator identified phenotypes. The introgression mapping will assess the relative fitness of the marked genomic regions, when introgressed into closely related species. Each of these three components draws on similar speciation research done in the past on these three species, though in the proposed work, the mapping will be on a much finer scale with many more markers than has been used previously. The proposed work will also permit entirely novel comparisons of the three types of information which can be brought together to develop a fuller and more integrated picture of how these species came to diverge and the genetic basis of the evolutionary forces that have kept them distinct.

"Sex mediated effects on recombination rates," Sigma Xi to Laurie Stevison, 12/2007.
In meiosis, recombination plays an important role in both merging beneficial alleles and purging deleterious alleles, thus allowing mutation and selection to act in concert to promote higher fitness4,7. Because increased recombination is often the result of genomic instability in stressful environments, it is not necessarily a favorable outcome5. Factors that alter recombination rate are therefore important in understanding how the environment affects genetic diversity.
Several studies have demonstrated that factors such as maternal age1,11, temperature3,8, nutrition6, and recently, number of matings9 can significantly alter recombination rate. This last effect suggests the possibility that males may, through mating, alter recombination rate in females. Because males and females are expected to originate from the same environment in nature, experiments have not been conducted testing physiological responses to environment in males and females separately9. There are several reasons that a significant difference between males and females would be biologically meaningful. First, in systems where males and females have different dispersal patterns, the environmental condition of one sex may differ from another. Second, the physiological response of reduced recombination may occur faster in one sex suggesting increased sensitivity to environment. Finally, because meiosis is not complete at the time of mating9, males may directly alter female recombination rate via seminal proteins2,10.
Here, I propose an experiment to test for sex-specific differences in recombination rate using an experimental cross of males and females of Drosophila simulans with high temperature as the environmental cue to alter recombination rate. The F1 progeny of four crosses will be reared in the maternal environment and females will be backcrossed to males from the maternal environment. To control for differences in development time, both males and females will be crossed 3-5 days post-eclosion. To further control for maternal age, known to have a significant impact on recombination rate, females will only be allowed to lay eggs for 3 days1,11.
I will measure recombination rate using 1000 progeny from each of four backcrosses by genotyping offspring at approximately 10 microsatellite markers evenly spaced along a single chromosome arm (apx. 6 cM apart). Two crosses will serve as controls with an expected increase in recombination rate in high temperature as has been shown previously8. If the physiological response of the females is responsible for the increased recombination, then they will not be significantly different from the high temperature control. If however males induce recombination rate variation in females (perhaps mediated through environmentally induced differences in seminal proteins9), then they will not be significantly different from the high temperature control. Finally, if physiological changes in both sexes are responsible for altered recombination rate, then both males and females will be intermediate of the controls.
Because increased recombination rate can lead to loss of genomic integrity, environmental stress may present a trade-off where adaptation is preferred over the risk of genomic instability4,5. Examination of the stresses that present this trade-off and physiological processes by which organisms alter their recombination rate can lead to understanding of the environmental conditions that may compromise genomic integrity.

"Postcopulatory sexual selection and the evolution of reproductive proteins in Drosophila," Sigma Xi to Audrey Chang, 4/2006.
Sperm competition often occurs in species where females are promiscuous. In response to this form of postcopulatory sexual selection (PSS), males of many species have evolved seminal fluid proteins to ensure post-mating fertilization. If sexual selection drives the evolution of these proteins, then the rate of evolution of male proteins for fertilization success should be greater in species where sperm competition is intense (i.e., when females frequently remate) than in species where sperm competition is minimal (i.e., when females rarely remate). Such a correlation between female promiscuity and reproductive protein evolution has been demonstrated in primates but only for a single gene.
Drosophila are ideal for testing the role of PSS in driving the evolution of male reproductive proteins. First, Drosophila exhibit highly variable mating systems. Second, accessory gland proteins (ACPs), transferred by males to females during mating, evolve rapidly and will likely show the signature of PSS. Studies demonstrate that some ACPs influence female behavior and physiology after mating and may be involved in sperm competition. Third, the genome sequences of 12 Drosophila species are available.
To test for a correlation between PSS intensity and ACP evolution, data will be gathered from the published literature on remating frequency, sperm length, and testis size relative to body size. These variables are indicators of the intensity of sexual selection within species. Classes for the intensity of sexual selection (High, Medium, and Low) will be designated based on these three variables. Three species are chosen to represent each of the classes such that: 1) preference is given to species for which the genome is available and to species closely related to “genome species" and 2) there are differences in the strength of sexual selection between relatives, hence controlling for phylogenetic effects. Species chosen are as follows: High: D. virilis, D. littoralis, D. kanekoi; Medium: D. mojavensis, D. melanogaster, D. americana; Low: D. simulans, D. arizonae, D. obscura.
Gene sequences for 10 ACPs will be obtained from available genomes and direct sequencing. The 10 ACPs will be chosen based on ortholog presence in all 9 species (determined by BLAST searches) and will represent a random subset of the 52 ACPs currently confirmed in D. melanogaster. When direct sequencing is required, primer design will be based on from the available genome of the most closely-related species. dN (nonsynonymous substitution rate), dS (synonymous substitution rate), and the lineage-specific dN/dS ratio (a measure of the rate of protein evolution relative to the mutation rate) will be calculated using PAML for each gene. If PSS drives the rate of evolution of a given ACP, then species in the "High" class should have higher dN/dS values than species in the "Low" class.
Until the advent of whole-genome sequencing, the influence of sexual selection on evolution has primarily been recognized at the morphological level. The abundance of studies on mating systems, the availability of sequenced genomes, and the well-resolved phylogeny of Drosophila make it a fruitful testing ground for the role of sexual selection reproductive protein evolution.

"Consubspecific sperm precedence in Drosophila," Sigma Xi to Darren Burkett, 12/23/1999.
In Drosophila, second male sperm precedence is a documented occurrence among intraspecific matings. In contrast, when a Drosophila female is mated with both a conspecific and heterospecific male, regardless of order, the conspecific sperm fertilize most of the female's eggs (e.g., Price, 1997). The conspecific sperm exhibit traits that increase their transmissibility and viability within the reproductive tract and/or hinder the success of the heterospecific sperm. Such precedence can lead to barriers that cause complete reproductive isolation between species.
Although the phenomenon of conspecific sperm precedence (CSP) is known to exist, it is not known how rapidly it evolves. If it plays a major role in the divergence of species, it should begin to evolve before speciation is complete. I propose to test for con[sub]specific sperm precedence in two subspecies of Drosophila (D.pseudoobscura pseudoobscura & D.p. bogotana).
I will collect males and females eight days prior to mating, and sort according to sex. On the eighth day, some females will be mated first to a consubspecific male followed by a heterosubspecific male, and other females will be mated in the reverse order. I will observe & time all copulations and remove males shortly after copulation. Females will be stored individually and offspring collected as they appear. Once fifty offspring have been collected from each female they will be scored for paternity. The two subspecies will be differentiated from one another using PCR techniques. We have forty highly variable microsatellites that I can use to score paternity. Microsatellites will be amplified by PCR and the products will be scored on agarose gels. Paternity will be assigned to a particular male if offspring bear his allele and not that of the other male.
Although CSP has been identified in numerous taxa, this study will help to establish how early in the speciation process it evolves. Also, if CSP is identified in these taxa, genetic studies of this phenomenon will be possible.

"The evolution of X chromosome inactivation in Drosophila," Sigma Xi to Brian Counterman, 1/2004.
The X chromosome is generally inactivated during male germline differentiation (Lifschytz and Lindsley, 1972; Kelly et al 2002). Surprisingly, little is known about the evolutionary forces driving X chromosome germline inactivation (XI) (Rastelli and Kuroda, 1998). XI may result from the evolution of genes with conflicting fitness effects between males and females (sexual antagonism) (Rogers, Carr and Pomiankowski, 2003). As a result of sexual antagonism, sex specific genes may differentially accumulate on the X chromosome over autosomes (Rice, 1984). An accumulation of female specific genes on the X chromosome may provide the selective pressure for XI since female specific genes may be detrimental during spermatogenesis (Wu and Xu, 2003; Rogers, Carr and Pomiankowski. 2003).
In Drosophila, the X chromosome: (1) has an excess of female specific genes (Parisi et al., 2003; Ranz et al., 2003) (2) is a hot spot for sexual antagonism (Gibson, Chippindale and Rice, 2002) and (3) is inactivated during germline differentiation in males (Cooper, 1951; Lifschytz and Lindsley, 1972). As such, Drosophila provides a unique opportunity for testing the role of sexual antagonism in driving the evolution of XI. If XI were driven by sexual antagonism, female specific genes expressed during spermatogenesis would affect germline development in males. We can test this prediction by examining sperm development in Drosophila lines containing chromosomal duplications from the X chromosome on to autosomes (X-A). Genes on X-A duplications would escape inactivation during spermatogenesis (Lindsley and Zimm, 1992) and thus would make it possible to study the effects of sexual antagonism on XI.
We will make reciprocal crosses between X-A duplication lines of Drosophila and examine sperm development, testis formation and fertility in F1 individuals homozygote for the autosomal duplicate. We will use two controls: (1) X-X duplicates to control for duplication effects and (2) X-A duplicates carrying the Xist gene as a control for chromosomal and gene expression effects (Xist causes chromosomal inactivation wherever it occurs, Jaafar et al., 1989). We will cross-reference phenotypic data with expression data from public databases (Parisi et al., 2003; Ranz et al., 2003). We predict (1) fertile males will carry mostly male specific genes or genes expressed in both sexes, while (2) duplications with an excess of female specific genes will be present in sterile males.
Understanding the evolutionary causes of XI will advance our comprehension of sex chromosome evolution. Sex chromosome evolution lies at the heart of many evolutionary phenomena such as sexual selection, speciation and the origins of sex.

"Testing the faster X theory in Lepidoptera," Sigma Xi to Brian Counterman, 5/2003.
To understand the process of species formation, one must identify the genetics of underlying phenotypes, such as species mating discrimination and hybrid sterility. A disproportionate number of traits involved in speciation map to the X chromosome, resulting in a 'large X effect' (1). This observation is most pronounced in Lepidoptera (butterflies/moths), where an astounding number of behavioral and morphological differences between species map to the X chromosome (2). Some authors suggest the large X effect results from more rapid evolution of X chromosome genes relative to autosomal genes.
I propose to test for faster evolution of X chromosome genes (also known as the 'faster X theory') in host strains of the fall armyworm moth, Spodoptera frugiperda, where there are confirmed large X effects on reproductive isolation (2, 4). To test the faster X theory, I will develop a cDNA library of expressed genes in S. frugiperda. I will determine the chromosomal linkage of these genes and compare the rate of amino acid substitutions on the X vs. autosomes between host strains of S. frugiperda. If X-linked genes have a significantly higher rate of amino acid substitution relative to autosomal-linked genes, I will conclude that X chromosome genes evolve faster.
In S. frugiperda, I predict there is disproportionate accumulation of X-linked speciation phenotypes as a result of faster evolution of the X chromosome. If faster evolution of the X chromosome is found in S. frugiperda, this will be the first compelling direct genetic evidence for the faster X theory. In contrast, results suggesting the X is not evolving faster in S. frugiperda, would promote further research of alternate theories for the purported large X effect as well as studies of the large X effect in other Lepidoptera species. With either result, this study will address recurrent models of molecular population genetics, provide new research aims regarding testing the large X effect, and promote Lepidoptera as a model system for studying the speciation process.

"A test of the balanced inversion model of speciation," Sigma Xi to Loren Henagan, 5/2002.
The study of speciation is the backbone of studying the diversity of life and the origin of life forms. Species are kept from fusing through factors that block gene exchange and introgression, such as hybrid sterility. The genetic basis of hybrid male sterility has been studied extensively in the hybridizing North American species Drosophila pseudoobscura and D. persimilis. These studies found that sterility results from incompatibilities between genes in inverted regions of the X and second chromosome. No hybrid sterility-conferring genes were found outside these inverted regions, and Noor et al (2001) argued that inversions may allow hybrid sterility to persist. Briefly, they said that alleles which confer sterility in uninverted regions would be lost upon hybridization, while those in inverted regions can persist, suggesting that chromosomal inversions may thereby play a special role in speciation (Rieseberg 2001).
I propose to test this hypothesis using genetic studies of Drosophila pseudoobscura bogotana. D. p. bogotana is found in South America, is allopatric to the two other taxa, and does not naturally hybridize with them. If the hypothesis of Noor et al (2001) is correct, then sterility in hybrids of D. p. bogotana and D. persimilis should NOT map exclusively to the inverted regions, as no hybridization has occurred. This will contrast the previous observation that hybrid sterility DOES map exclusively to inverted regions in hybrids of D. pseudoobscura and D. persimilis.
I will cross D. p. bogotana females to D. persimilis males and backcross the resulting fertile female hybrids to D. p. bogotana males to produce backcross progeny (BCbog). I will also cross D. pseudoobscura females to D. persimilis males and backcross the resulting fertile female hybrids to D. pseudoobscura males to produce other backcross progeny (BCps). I will dissect the BCps and BCbog males' testes. The model predicts that a greater fraction of BCbog than BCps males will be sterile. Next, I will use microsatellites to identify the arrangements in the backcross progeny at the sites inverted between the species. I will then determine if the arranged regions are associated with all of the hybrid sterility- i.e., if individuals homozygous for all arrangements from one species are nearly always fertile. The hypothesis predicts that the arranged regions will be associated with virtually all of the hybrid sterility in BCps males but not BCbog males. These experiments may provide a strong experimental confirmation or refutation of Noor et al's inversion hypothesis.

"The genetic basis of courtship song differences between Drosophila pseudoobscura and D. persimilis," Sigma Xi to Marcus Williams, 12/23/1999.
Drosophila males emit species specific sounds from their wings during courtship, commonly called "courtship songs". Previous studies have shown strong associations between courtship song elements and mating success in several Drosophila species. It is now widely accepted that at least two song parameters are key in female mate recognition in Drosophila (e.g., Ewing and Bennet-Clark, 1968; Cowling and Burnet, 1981; Kyriacou and Hall, 1982; Tomaru et al., 1995; Noor and Aquadro, 1998). Studies have also shown that courtship song differentiation evolves faster than sexual isolation and hybrid sterility among species in the Drosophila willistoni group (Gleason and Ritchie, 1998). However, little is known about the genetic basis of differences between species in courtship song characteristics.
For the past year, I have examined the courtship songs of two closely related species, Drosophila pseudoobscura and D. persimilis, and their hybrids. I have already recorded and analyzed the songs of 500 backcross hybrid males bred by crossing Drosophila pseudoobscura and D. persimilis and backcrossing the resulting F1 hybrids to D. pseudoobscura. I have found that two song traits, interpulse interval and intrapulse frequency, seem to differentially affect mating success in the two species. Mating success with D. pseudoobscura females is strongly correlated with short interpulse intervals (p<0.0001) whereas mating success with D. persimilis is strongly correlated with high intrapulse frequency (p=0.0012) among hybrid males.
Now, I will use the DNA from these hybrid males to determine the genetic basis of the song differences between these species. I hypothesize that the courtship song differences between species are based on many changes of small effect on different chromosomes, as suggested by the coarse mapping of Pugh and Ritchie (1996) in Drosophila simulans and D. mauritiana. I will genotype these hybrids using 13 molecular markers, most of them microsatellites, which are located along all five chromosomes of these species. Microsatellites will be amplified by PCR, and some of the products will be scored on 2% agarose gels while others will be genotyped on acrylamide gels. The data from this genotyping will then be assayed using composite interval mapping software (e.g. QTL Cartographer) to determine whether a significant association exists between having a particular genomic region from a species and having courtship song characteristics from that species.
Courtship song differences between Drosophila species have never before been analyzed with this many genetic markers. This study will help to explain the genetic changes associated with an important behavioral character and perhaps even the speciation process in these taxa.

"Local adaptation and the signature of speciation," Sigma Xi to Daniel Ortíz-Barrientos, 12/2001.
Closely related species that co-occur in nature remain distinct by not mating with each other. Natural selection may strengthen the ability to discriminate between conspecific and heterospecific partners if maladapted hybrids are produced [1]. This theory, called reinforcement, predicts that mate discrimination is stronger in females derived from areas where multiple species co-occur than from areas where only one species is present. This prediction, however, seems counterintuitive when applied to species with high rates of gene flow. If alleles that confer mate discrimination spread out of the shared habitat, then discrimination should be equally strong in areas where only one species exists. Nonetheless, cases of reinforcement have been reported in taxa with high rates of gene flow [2].
I hypothesize that alleles conferring mating discrimination and alleles providing local adaptation are often tightly linked. In this case, the effects of genes conferring local adaptation will restrict the diffusion of alleles that allow females to discriminate among partners into other areas. Chromosomal inversions may suppress recombination, thereby providing a simple way to link local adaptation and mate discrimination alleles.
Drosophila pseudoobscura and D. persimilis provide an example of speciation by reinforcement [3] despite the extensive level of gene flow detected among populations of D. pseudoobscura [4]. Intriguingly, a rich inversion polymorphism on one chromosome shows dramatic differences among populations of D. pseudoobscura. Environmental factors and individual fitness are thought to be responsible for this differentiation [5]. Surprisingly, some inversions are only present where the two species co-occur, and thus only present where female species discrimination has shown to be stronger. Furthermore, I have mapped population differences in species discrimination to the chromosome bearing the inversion polymorphism.
I will collect D. pseudoobscura from areas where both species occur and determine their inversion-types. Subsequently, I will assay female mate choice of females in the laboratory for 50 lineages. If mating discrimination and local adaptation alleles are linked through inversions, then (a) females bearing inversion-types that are abundant in areas where only D. pseudoobscura is present will exercise weak species discrimination, while (b) females bearing inversion types that are only present in shared areas of the species will consistently exercise high species discrimination.
These species offer a unique opportunity to understand the relationship between speciation and adaptation within species. Understanding this relationship will allow us to better understand speciation by reinforcement, and to identify the effects of inversions and other chromosomal rearrangements on speciation.

"On the appearance of speciation by reinforcement," Sigma Xi to Daniel Ortíz-Barrientos, 12/21/2000.
Speciation by reinforcement predicts that females of a particular species that occur in the same region with heterospecifics (sympatry) should exhibit higher species mating discrimination than females derived from areas where only one species is present (allopatry)[1]. This difference between sympatry and allopatry results from the strengthening of mating discrimination by natural selection to avoid maladaptive hybridization[2]. Gene flow among discriminating and non-discriminating conspecific populations could obscure the pattern predicted by reinforcement, though, as the alleles conferring higher discrimination may spread across all populations. However, the pattern predicted by reinforcement is commonly observed, even in species which have high levels of gene flow between discriminating and non-discriminating populations. This paradox suggests that some mechanism is preventing the spread of the high discrimination phenotype out of sympatry.
This paradox could be explained by one or both of two following hypothesis: (a) natural selection favors less discriminating females in populations allopatric to other species, perhaps because these females do not needlessly postpone mating[3] and/or (b) mating discrimination differences found among sympatric and allopatric populations are maintained by asymmetrical gene flow among populations, with most migration occurring from allopatric to sympatric populations. The first hypothesis predicts that conspecific mating should occur faster in individuals derived from allopatric populations than those from sympatric populations, thus increasing the number of potential offspring. The second hypothesis predicts that levels of genetic diversity should be higher in populations sympatric to heterospecifics than in allopatric populations. This will result in a significant genetic structure between allopatry and sympatry. I propose to test these hypotheses in the sibling species Drosophila pseudoobscura and Drosophila persimilis, which have been suggested to have speciated by reinforcement[4] in the presence of extensive intraspecific gene flow[5] and minimal genetic structure[6].
I will collect flies from six D. pseudoobscura populations: 3 sympatric to D. persimilis and three allopatric to D. persimilis. I will evaluate the time to copulation in flies derived from these populations using a no-choice mating design to determine whether the higher discrimination exercised by D. pseudoobscura females in sympatry increases mating latency in conspecific pairings. Likewise, I will examine the numbers of alleles and average heterozygosity of 15 microsatellite markers[6] in flies collected in these populations to evaluate the level of structure between sympatric and allopatric populations and whether migration may be asymmetric between these populations.
The results of this investigation will shed light on the historical aspects and mechanisms that might be contributing to the sexual isolation of species and thus, to the origin of species.

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