Michael Robert Lynch - US grants

animal population genetics; gene mutation; bacterial genetics; biochemical evolution; natural selections; animal breeding; genetic models; computer simulation; genetic strain; fertility; inbreeding; microorganism growth; microorganism culture; Escherichia coli; Branchiopoda;

Intellectual Merit. This project is focused on one of biology's deepest mysteries - the evolutionary causes and consequences of recombination. The investigative team consists of cell biologists, ecologists, parasitologists, quantitative geneticists, genomicists, and mathematicians. The study organism, the planktonic microcrustacean Daphnia pulex, provides an exceptional array of opportunities for recombination research that is unavailable with any other system: a wide range of recombination intensities among natural populations, the presence of multiple sexual and asexual lineages, a powerful set of genomic tools, well understood ecology, ease of experimental manipulation, and a "living-fossil" record that can be resurrected from lake sediments. Specific goals include: 1) characterization of the genetic changes associated with the transition from meiotic to mitotic progeny production; 2) evaluation of whether the mutation rate (including the activity of mobile-genetic elements) is affected by meiosis; 3) a test of the hypothesis that mutation load accumulates in the absence of recombination; 4) evaluation of the extent to which recombination modifies the range of variation upon which natural selection acts; and 5) a test of the hypothesis that host-parasite evolution drives the evolution of recombination and sex. These studies will be informed by an integrated research program involving high-throughput sequencing, microarray analysis, and quantitative-genetic surveys. Guided by the empirical results, mathematical models will also be developed for understanding the evolutionary fates of genomic features of asexual organisms. Finally, the results of this study will be integrated into an emerging evolutionary framework suggesting that many aspects of the genomic architecture of multicellular organisms arose passively in response to mildly deleterious mutation accumulation in populations with small effective sizes.
Broader Impacts. The potential impacts of this project on science, society, and education are numerous. First, an undergraduate program will help instill an interdisciplinary philosophy while broadening career choices for students from multiple institutions, with a particular focus on minority recruitment. Second, the research program will be tightly integrated with the newly founded Daphnia Genomics Consortium, an international group of scientists from across the life sciences (http://daphnia.cgb.indiana.edu/). This will firmly establish D. pulex as a premier model system for studies in ecological and evolutionary genomics. Third, the research has significant applied implications in the areas of parasite-resistance evolution, clonal propagation, and genetic engineering.

Transposable elements (TEs) are regions of DNA that have the capacity to move around the genome in which they are found. Despite their longtime characterization as "junk", TEs have become recognized as important and ubiquitous components of eukaryotic genomes. As a result, there is great interest in what factors influence the dynamics of TE movement. Recombination among host genomes during sexual reproduction may have an especially interesting role in TE proliferation because it simultaneously provides a mechanism by which TEs may easily be gained (e.g. due to insertions when DNA molecules break and exchange material), and an opportunity for TEs to be lost via independent assortment of chromosomes lacking new insertions. Using Daphnia pulex as a model, we can examine the role of recombination in TE proliferation during both sexual and asexual reproduction.

Understanding TE movement is important because TEs introduce a significant source of genetic variation upon which natural selection can act. In some cases, TEs insert into genes and disrupt normal function leading to disease. Alternatively, TEs can insert into or near genes, providing novel or regulatory function. Understanding how recombination influences TE dynamics is a key part of understanding their overall role in the genome.

Intellectual Merit:
Because all aspects of biodiversity ultimately derive from DNA-level resources, a mechanistic theory of evolution must start at the genomic level. This work focuses on the hypothesis that many aspects of genome evolution are driven by nonadaptive forces (random genetic drift, mutation, and recombination) rather than natural selection responding to external ecological forces. Using mathematical/computational analysis, the research will exploit the enormous set of resources available from whole-genome sequencing projects, from viruses to organelles to bacteria and multicellular species. Because evolution is a population-level process, priority will be on developing a general theory for genomic evolution consistent with well-established principles of population genetics, while spanning the fields of computational biology and informatics, molecular and cellular biology, and systems biology.
The overall goal is to help transform the descriptive field of comparative genomics into a more explanatory field of evolutionary genomics. Under the proposed hypothesis, many of the complex genomic features that are the hallmark of multicellular species emerged largely as a consequence of a substantial reduction in the efficiency of selection, whereas the streamlined genomes of microbial species result from highly efficient selection opposing the accumulation of mutationally hazardous excess DNA. At the very least, the theory to be developed provides a null (nonadaptive) hypothesis for interpreting the evolution of genome complexity.

Broader Impact:
Through the integration of these various subprojects, the overall goal of the proposed research is to help transform the descriptive field of comparative genomics into a more explanatory field of evolutionary genomics, as well as to help develop the intellectual infrastructure that will ultimately be necessary for the emergence of a field of evolutionary cell biology. In addition, the research project will be intertwined with a set of educational goals, including: 1) the establishment of an annual set of projects associated with a genome evolution class that services students from both the life sciences and informatics; 2) the recruitment of mathematically inclined biology undergraduates into a two- to three-year research program; and 3) the development of an integrative ?training-grant?-like environment among the grant participants.

Intellectual Merit.
The evolutionary causes and consequences of sexual vs. asexual reproduction remain among the deepest mysteries in evolutionary genetics. Although the mechanisms responsible for the suppression of meiosis, which involve a loss of homologous recombination and segregation, appear to be widely available phylogenetically, the molecular basis for the conversion of meiotic haploid gamete production to mitotic diploid egg production remains unknown. The project will address this issue using the microcrustacean Daphnia pulex to elucidate the molecular and cell biological mechanisms underlying the transition to parthenogenetic egg production. The principal investigator's previous research has identified a key gene whose modification appears to be necessary to convert meiosis to mitosis, and as well as identifying chromosomal regions containing two to three others. To perform functional studies of these genes, it will be necessary to develop stable transgenic lines of D. pulex containing reporter gene constructs of the putative meiosis suppressor. If successful, the project will lead to an enhanced understanding of the mechanisms underlying the molecular basis of meiosis and meiosis suppression, which would in turn have considerable implications for applied breeding programs.

Broader Impact.
The establishment of reliable transgenic methods will open up the Daphnia system to inquiry of a wide array of additional questions in molecular, cellular, and developmental biology, thereby greatly broadening the investigative capacity of this model system beyond the already substantial research community embodied in the Daphnia Genomics Consortium. Educational and training efforts will be focused on the career development of a postdoctoral researcher as well as on exposing undergraduate researchers to the full spectrum of scientific inquiry, from the development and implementation of new techniques to critical aspects of research design, hypothesis testing, and publication. Indiana University supports a number of summer programs for high-school and undergraduate minority students, and the principal investigator will continue to sponsor such students.

Intellectual Merit. With the ability to completely characterize the genomes of closely related species and individuals within species, it is now possible to elucidate the mechanisms by which evolution proceeds at the molecular level, via both the promotion of adaptations within species and the establishment of new species. This project involves a comparative survey of the genome sequences of the complete set of cryptic species of the Paramecium aurelia assemblage of ciliated protozoans. Just prior to the radiation of this complex, the common ancestor experienced a complete doubling of the nuclear genome, and preliminary evidence suggests that silencing of alternative redundant gene copies in sister lineages has led to map changes that may operate as effective reproductive isolating barriers. The relatively young age of the complex, combined with its large number of constituent species and relatively simple genomic architecture, provides a powerful and unprecedented resource for understanding the roles that gene duplication plays in the generation of biodiversity. By establishing the complete history of all ancestral gene copies over a finely dissected phylogeny, the patterns of preservation vs. demise of various functional classes of duplicate genes will be evaluated. The analyses will also reveal the temporal patterns of gene loss that eventually lead to the acquisition of new equilibrium genomic states in the descendant taxa, as well as clarify the extent to which gene resurrections occur. With the inclusion of information on gene expression, several key hypotheses on the evolution of duplicate genes will be tested. Lending an exceptional level of power to the analyses is the availability of information on the rate and complete molecular spectrum of mutations for two aurelia species. This provides a formal basis for deciphering the forces of evolution operating on duplicate genes by providing a null model for the fates of genes in the absence of selection (e.g., positive selection for preservation or active promotion of gene loss by mutational degradation). As the first study of this sort in a natural assemblage of unicellular eukaryotes, this project has the potential to greatly expand our understanding the mechanisms of genome evolution, providing a complement to the much richer set of observations on multicellular species.

Broader Impacts. The data generated by this study will serve as a critical and permanent resource for the Paramecium genetics community, while also providing the first detailed data on the dynamics of duplicate genes on an evolutionarily interpretable time scale. The data will be organized into the existing ParameciumDB web-based data system, allowing users to readily query the entire species assemblage for the status and evolutionary history of the full set of paralogous genes back to the ancestor of the P. aurelia complex. In addition, the database will be integrated into a community-level effort at incorporating ciliates in classroom research. Finally, the project will support the training of a graduate student and a postdoctoral fellow, with a goal of establishing them as leaders in the re-emerging field of Paramecium evolutionary genetics.

DESCRIPTION (provided by applicant): The goal of this project is to obtain high-quality sequence for the genomes of ~150 isolates of the microcrustacean Daphnia pulex, as well as lower-coverage data for ~5000 additional geographically distributed genotypes for population-genetic analysis. The study species is a major model system employed in the research of a large international consortium of life scientists. As the assayed genotypes will be maintained indefinitely in a clonal fashion, the resultant data set will serve as a permanent resource for the research community. Innovative features of the project include the direct sequencing and assembly of haplotypes via whole-genome amplification of isolated sperm, and the application of maximum-likelihood methods for estimating patterns of within- and among-individual variation and linkage disequilibrium. The availability of direct estimates of the rate and molecular spectra of de novo mutations in all major lineages to be studied provides a level of power for the interpretation of molecular population-genetic data that has not been possible in prior work, e.g., estimation of the power of genetic drift in each study population. In addition to providing a community resource, which will leverage additional work from numerous other labs, the sampling scheme for this survey is designed to generate results bearing on several long-standing problems in evolutionary genetics. First, the study species harbors a large number of permanently asexual lineages, resulting from an unusual system of sex-limited meiosis suppression that promotes the recurrent production of novel asexual clones via backcrossing of males to the sexual species. Analyses of asexual lineages with a range of ages will provide an unprecedented opportunity to evaluate the genome-wide causes and consequences of the loss of recombination, and such analyses are further enhanced by the presence of two chromosomes that never recombine, even via male transmission. Second, D. pulex harbors substantial numbers of novel introns. Collection of hundreds of neo-introns, analysis of their molecular features, and elucidation of their genealogical distributions will set the stage for future functional work on the mechanisms of intron origin, one of the great mysteries in evolutionary genomics. Third, one extensive lineage of D. pulex has undergone a prolonged population bottleneck, and comparison of this to ~50 other populations will cover essentially the full range of effective population sizes known in metazoan species, providing a unique genome- wide analysis of the consequences of variation in the strength of random genetic drift. Finally, as only about half of the genotypes to be sequenced are capable of male production, comparative analyses will provide insight into the mechanisms of sex determination in this system, clarifying the proposed existence of a nonrecombining mating-system chromosome, and evaluating the consequences of such a genetic environment on an otherwise freely recombining genetic background.

This award is designated as a Global Venture Fund Award and is being co-funded by NSF's Office of International Science and Engineering. A species' ability to withstand sudden changes in the environment is limited by internal and external factors. In this project, researchers from the US and India, using zebrafish as a model, join forces to identify the specific mechanisms by which populations undergo rapid shifts in behavior. This in an integrated project as the research team will conduct an ecological and genomic survey of Indian fish in the wild, and then transport fish to laboratories in the US for detailed tests of sensory ability. In addition, new statistical methods will be developed to predict the impact of genetic, physiological, and ecological mechanisms on the ability of zebrafish to adapt to sudden change. The results are expected to yield insight into the relative importance of genomic, physiological, and habitat constraints. Statistical and software tools to predict the impact of pollution and climate change on the survival of key organisms will be disseminated broadly to other researchers via the internet. The research will also increase basic knowledge on the behavior and ecology of zebrafish, a model organism used commonly in biomedical research. The collaborative team includes a third-grade teacher who will use internet videoconferencing to add an international component to classroom exercises based on this research.