Developing Phylogenomic-Based Diagnostic Tools for Species Identification Across Diptera

The following is a proposal for AFRI Foundational and Applied Sciences Program FY 2019; Program Area: Crosscutting Programs; Program Area Priority: Tactical Sciences for Agricultural Biosecurity (code A1181). This project will evaluate cutting-edge phylogenomic approaches (https://doi.org/10.1111/1755-0998.12783) for their utility in a diagnostic context and create laboratory workflows and analytical tools for cost-effective and rapid use of such diagnostic markers. By focusing on plant and animal pests in the economically damaging dipteran families Tephritidae and Calliphoridae, the proposed research will demonstrate the utility, applicability, and universality of molecular diagnostic markers at multiple phylogenetic scales (i.e. Dipterawide down to species complexes and population-level assessments of widespread species). The proposed research has several expected outcomes, in both basic and applied settings, including: the most data rich systematic datasets generated for both tephritids and calliphorids, which will be used to generate phylogenomic hypotheses to reevaluate historically debated relationships within these groups (e.g. subgeneric relationships within tephritids and species-level relationships within calliphorids); robust population-level datasets to understand diversity of species complexes and range-wide population structure; development and demonstration of userfriendly molecular diagnostic tools for species identification and geographic source determination; creation of bioinformatic tools to facilitate rapid and user-friendly analysis of data generated with such diagnostic markers. This project is an international collaboration involving two senior collaborators within USDA-ARS, and several collaborating investigators from other domestic and international institutions, including regulatory agencies and diagnostic labs. Specific objectives: I. Generate Highly-Multiplexed Amplicon-based Phylogenomic (HiMAP) data across multiple families of Diptera I-A. Utilize comparative genomics of existing resources to identify and characterize phylogenetically informative markers at variable phylogenetic scales, focusing on Tephritidae and Calliphoridae I-B. Generate phylogenomic datasets for Tephritidae I-C. Generate phylogenomic datasets for Calliphoridae I-D. Generate complementary COI datasets to act as a baseline for systematics/diagnostics II. Demonstrate the utility of these diagnostic markers for resolving systematics, performing species delimitation/identification, and evaluating population genomic patterns II-A. Phylogenomic analyses for systematic inference of Tephritidae II-B. Phylogenomic analyses for systematic inference of Calliphoridae II-C. Using genomic data for species delimitation/identification in species complexes of Tephritidae II-D. Species delimitation and population genomics of widespread pests in Calliphoridae III. Evaluate and demonstrate the utility, applicability, and universality of these diagnostic markers at multiple phylogenetic scales III-A. Evaluate the efficacy of diagnostic markers at broad (Diptera) and fine (population-level) phylogenetic scales III-B. Develop wet-lab techniques for rapid and cost-effective sequencing of diagnostic markers III-C. Generate diagnostic data from contemporary specimens of interest (intercepted specimens) III-D. Develop bioinformatic tools to facilitate the analysis of diagnostic marker data