Peter Andolfatto - US grants

Understanding the process of how new species form (speciation) is central to modern biology. Species are most commonly identified as groups of animals that are reproductively isolated from other such groups. This collaborative project will use two swallowtail butterfly species, Papilio glaucus and P. canadensis, and a discrete zone where hybrids of these two species can be found (a hybrid zone), as a model system to study the genetic basis of reproductive isolation. These two species differ in several ecologically important traits that are linked to their sex chromosomes, yet are still partly capable of forming fertile hybrids. These features allow study of the genetics of reproductive isolation between these species in the context of these ecological differences. The research will produce a genetic map of the Papilio X-chromosome and will identify regions of the X-chromosome that are linked to ecologically important traits. The degree to which natural selection in the hybrid zone prevents between-species exchanges of genetic material that is closely linked to these traits will be tested.

Papilio swallowtails are a common and easily recognizable butterfly in North America, and will serve as a publicly accessible tool for the study of speciation. This work will significantly contribute to genetic resources for butterfly and moth researchers. Insights gained by the proposed research will be broadly applicable to understanding speciation in a wide variety of plant and animal groups. In addition, publicly available software will be developed and made available to facilitate the study of speciation by other researchers.

Central to evolutionary biology is understanding how reproductive isolation between two closely related species develops. This study uses two swallowtail butterfly species, Papilio glaucus and Papilio canadensis, as a system to study the genetic basis of reproductive isolation between newly emerging species. The two species differ in many ecological traits that are linked to their sex chromosomes, yet where their ranges meet they are still capable of forming fertile hybrids. The investigators predict that some genes are able to move through the hybrid zone, while genes linked to species-specific ecologically important traits do not. Implementing both standard and novel population genetic approaches, thirty sex-linked genes will be examined in parental and hybrid populations of P. glaucus and P. canadensis to test the prediction. Using parental populations, the study will also elucidate which sex-linked traits were important in the initial divergence of the two species.

The Papilio hybrid zone is a well-known geographic hotspot for species turnover and hybridization in many plants and animals in North America. Insights gained by the proposed research will improve our understanding of complex ecological systems. This study will significantly contribute to genetic resources for non-model organisms. In addition, publicly available software will be developed that can be applied to many biological species.

DESCRIPTION (provided by applicant): A growing body of evidence supports the view that regulatory evolution - the evolution of where and when a gene is expressed - is the primary genetic mechanism behind the modular organization, functional diversification, and origin of novel traits in higher organisms. Most elements regulating gene expression in eukaryotic genomes reside in noncoding DNA (i.e. DNA that does not encode protein). Recent studies suggest that much of the noncoding portion of the Drosophila melanogaster genome is evolutionarily constrained, implying that these regions are important for an organism

Many toxic animals have bright warning colors to deter predators such as birds and lizards. Often a group of unrelated species mimic one another by sharing a single defensive pattern. For insects such as butterflies that use wing patterns to recognize their own species, especially when they are choosing mates, mimics of different species might be confusing. This group has recently found that the color vision of toxic passion vine or Heliconius butterflies of South and Central America is able to distinguish the Heliconius yellow wing pigments from those of their mimics, whereas the two types of yellow pigment produce colors that are indistinguishable for bird color vision. This project will investigate the differences in color vision between species and sexes of Heliconius butterflies, using molecular, anatomical, biochemical, electrophysiological and next-generation sequencing methods. The resulting data and models of color vision will provide evidence on how color is used for both choosing mates and finding leaves on which to lay eggs and flowers to feed. This study will make predictions about Heliconius color vision that can be tested behaviorally. This research will reveal in unprecedented detail the evolution of color vision within a group of closely-related animals in relation to the signals they encounter in their daily lives. Whereas much is know about sexual differences in animal coloration, virtually nothing is known about sexual differences in color vision. This is a major gap that can now be addressed. Spectral data resulting from this project will be deposited in the Dryad Data Repository http://datadryad.org/ and made freely available to the public. There will be training of a postdoc, graduate and undergraduate students in intracellular recording and anatomical methods, as well as in the analysis of next-generation sequencing data. This research group will recruit undergraduate math and chemistry majors to analyze color reflectance spectra.

Some organisms exhibit traits that seem likely to hinder their survival, such as bright colors or elaborate tails or fins. Counterintuitively, these traits can actually facilitate an organism?s success because they can increase their ability to find mates. In swordtail fish, males have evolved an elongated fin, or ?sword? that helps them attract mates. Females of many swordtail species prefer males with the sword ornament, but females of one species have evolved a disdain of the sword. This project will investigate which genes are responsible for producing the sword ornament. Once the genes responsible for producing a trait are identified, population genetic methods can be used to determine the strength of selection on these genes in different species. This project will also test hypotheses about when and how the sword originated.

Understanding the genetic basis of traits and the strength of selection on these traits is important in a number of biological fields including evolutionary biology and biomedical research. In addition, this project will collaborate with the Princeton Prison Teaching Initiative to bring ongoing research into the classroom and give students hands-on experience with data analysis and interpretation. This grant will also support the development of educational resources for K-12 instructors to help students learn about genetic and evolutionary processes.

? DESCRIPTION (provided by applicant): The goal of this research is to dissect the causative genes and mutations underlying a complex trait, and trace the stepwise process by which the identified causative alleles arose and spread through an isolated population to generate a fixed morphological phenotype. The species Drosophila santomea exhibits a recently evolved, drastic shift in its pigment patterns that represents an optimal model system in which to dissect complex polygenic traits. Our previous work identified the gene underlying one of four major QTL contributing to this trait. Analysis of multiple individuals in the population revealed that causative alleles of this gene arose several times in parallel in the D. santomea population (i.e. a soft sweep). Here, we propose to employ molecular genetic techniques (introgression mapping and transgenic complementation) to identify causative genes and mutations responsible for two additional QTL. Using a combination of molecular and genomic techniques (in situ hybridization, RNA-seq), we will then assess how these loci interact with each other, as well as how they impact the genome-wide profile of expression. Finally, we will survey population variation at these additional causative loci to assess whether a similar soft sweep occurred, and determine whether these genes exhibit signs of positive selection. This study will provide a rare vista of a complex morphological trait that integrates molecular studies of gene function with processes occurring at the population level.

DESCRIPTION (provided by applicant): Despite its place as the centerpiece of evolutionary biology, fundamental questions remain about adaptive evolution that are difficult to address with the current set of examples of adaptations dissected to the molecular level. Instances of parallel evolution allow one to evaluate multiple outcomes of the process of adaptation under a common regime of natural selection. We propose to examine situations in which large assemblages of species experience parallel evolution as a tool to understand the genetic basis of specific adaptations and the dynamics of adaptation more generally. Specifically, we propose to focus will be on the medically important interaction between Na+,K+-ATPases, and their regulatory steroidal-glycosides, in the context of the natural genetic diversity underlying plant-herbivore (and predator-prey) conflicts. Our preliminary work reveals that diverse herbivorous insects specializing on steroidal-glycoside producing plants have repeatedly evolved insensitive versions of Na+,K+-ATPase in a surprisingly predictable manner. In particular, we discovered that when species only have a single copy of the gene, evolution appears to be largely limited to two functionally important amino acid residues. On the other hand, when a species has multiple copies of the gene, we found that these copies diverge in function and evolve tissue-specific expression patterns. Using a highly integrative suite of techniques (RNA and genome sequencing, computational biology, genome engineering as well as biochemical, physiological and behavioral assays), we will generate a large comparative genomics database to identify key amino acid changes underlying insensitivity of Na+,K+-ATPase to steroidal- glycosides and quantify their effects on organism performance. The resulting data and insights will comprise a major contribution to our understanding of adaptation and the origins of organismal complexity. In addition, given the medical importance of the Na+,K+-ATPase/steroid-glycoside interaction, our work will yield insights into the development of drugs to treat a number of Na+,K+-ATPase-associated neurological and physiological disorders in humans, as well as reduce the detrimental side-effects resulting from steroid-glycoside treatment of cardiac arrhythmias and congestive heart failure.

? DESCRIPTION (provided by applicant) The goal of this research is to dissect the causative genes and mutations underlying a rapidly evolving complex trait and to understand the nature of constraints on the evolution of this trait. Such an endeavor requires an experimental system in which drastic changes occur over short evolutionary timeframes in species that can be easily manipulated genetically. The posterior lobe in the Drosophila melanogaster species group is a recently-derived structural feature of male genitalia important in mating. The structure has evolved strikingly different morphology in each of the four species of this group over the past two million years. Our preliminary work has documented the apparent co-option and re-deployment of a highly conserved gene regulatory network (GRN) encoding the larval posterior spiracle in creating novel phenotypic variation in the adult posterior lobe. This finding stimulates us to hypothesize mechanisms that would allow the rapidly evolving lobe to change while leaving the spiracle structure unaltered. Among the members of this GRN is the gene pox neuro (poxn) which we have shown is divergently upregulated in D. simulans relative to D. melanogaster and that over-expression in D. melanogaster leads to enlargement of the lobe as observed in D. simulans. Additionally, we have shown that the upregulation of poxn in D. simulans is caused by sequence divergence in a posterior lobe-specific enhancer contained within the gene. Using a population genetic method to detect natural selection, we have identified a signature of selection that coincides with the posterior lobe enhancer of poxn. Here, we propose to employ genomic (i.e. RNA-seq, population genomics) and molecular genetic techniques (i.e. transgenic expression and complementation assays) to identify other causative genes and mutations responsible for differences in posterior lobe size and shape among species of the D. melanogaster species group (Aims 1 and 2). With a large collection of such molecular changes in hand, we will examine how co-opted nodes of the posterior lobe have diverged in their lobe functions without altering the spiracle structure (Aim 3).