EXECUTIVE SUMMARY Assessing Pollinator Communities via Environmental DNA (eDNA) Metacommunity Assay SERDP Project RC19-1102 APRIL 2020 Dr. Mark Davis Dr. Lynsey Harper Dr. Brenda Molano-Flores University of Illinois Urbana-Champaign Mr. Joseph Benito Dr. Matthew Niemiller The University of Alabama in Huntsville Distribution Statement A This report was prepared under contract to the Department of Defense Strategic Environmental Research and Development Program (SERDP). The publication of this report does not indicate endorsement by the Department of Defense, nor should the contents be construed as reflecting the official policy or position of the Department of Defense. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the Department of Defense. EXECUTIVE SUMMARY Project: RC19-1102 TABLE OF CONTENTS Page 1.0 INTRODUCTION ..................................................................................................................1 2.0 OBJECTIVES .........................................................................................................................2 3.0 TECHNOLOGY DESCRIPTION ..........................................................................................2 3.1 GREENHOUSE EXPERIMENT...................................................................................2 3.2 FIELD EXPERIMENT ..................................................................................................3 3.3 DNA EXTRACTION, QUANTIFICATION, AND PLATING ....................................4 3.4 PRIMER SELECTION ..................................................................................................5 3.5 IN SILICO PRIMER VALIDATION ............................................................................5 3.6 IN VITRO PRIMER VALIDATION ............................................................................5 3.7 MICROFLUIDIC METABARCODING.......................................................................6 3.8 METABEAT PIPELINE ...............................................................................................6 3.9 ANACAPA TOOLKIT PIPELINE................................................................................7 4.0 PERFORMANCE ASSESSMENT ........................................................................................8 4.1 PRIMER DEVELOPMENT AND VALIDATION .......................................................8 4.2 CONTAMINATION ......................................................................................................9 4.3 GREENHOUSE RESULTS.........................................................................................12 4.4 FIELD RESULTS ........................................................................................................12 4.5 DIVERSITY ACROSS FLOWER SPECIES ..............................................................16 4.6 DIVERSITY ACROSS SAMPLE TYPES ..................................................................19 4.7 DIVERSITY ACROSS PRESERVATION/EXTRACTION METHOD ....................22 4.8 DIVERSITY ACROSS PRIMER SETS ......................................................................25 4.9 DIVERSITY ACROSS BIOINFORMATICS PIPELINES ........................................29 5.0 IMPLEMENTATION ISSUES ............................................................................................32 5.1 BENEFITS ...................................................................................................................33 6.0 REFERENCES .....................................................................................................................33 APPENDIX A FLOWER SPECIES THAT COMPRISED THE GREENHOUSE COMMUNITY .................................................................................................................. A-1 APPENDIX B DETAILS OF THE PRIMER PANEL SELECTED FOR POLLINATOR EDNA MICROFLUIDIC METABARCODING. ....................................B-1 APPENDIX C RESULTS OF IN SILICO PRIMER TESTING FOR EACH PRIMER PAIR CONSIDERED .............................................................................C-1 i LIST OF FIGURES Page Figure 1. The Team Established a Mixed Species Colony of Plants to Ensure a Constant Supply of Pollen and Nectar (A). A Subset of Plants Was then Moved to an Isolated Greenhouse Room Where a Colony of Common Eastern Bumblebees Was Established (B). ................................................... 3 Figure 2. Heat Map Showing Frequency of Contaminants Detected in Metabarcoding Process Controls. ............................................................................... 11 Figure 3. Heat Map Showing the Frequency of Taxa Detected in Baseline Samples Collected from Flower Species Before They Were Introduced to the Greenhouse Containing Common Eastern Bumblebee (B. impatiens). ...................... 13 Figure 4. Heat Map Showing the Frequency of Taxa Detected in Greenhouse and Field Samples Collected from the Four Focal Flower Species. ........................................... 14 Figure 5. Bipartite Network Showing Interactions Between Invertebrate Families and the Four Focal Flower Species. ............................................................. 15 Figure 6. Boxplot Showing the Number of Taxa Detected in eDNA Samples from Different Flower Species. ................................................................................... 16 Figure 7. NMDS Plots of Communities (Jaccard dissimilarity) from Different Flower Species (Colored Points/Ellipses). .................................................................. 18 Figure 8. Boxplot Showing the Number of Taxa Detected in eDNA Samples Taken from Different Sources of Plant Material. ....................................................... 19 Figure 9. NMDS Plots of Communities (Jaccard dissimilarity) Produced by Different Sample Types (Colored Points/Ellipses). .................................................... 21 Figure 10. Boxplot Showing the Number of Taxa Detected in eDNA Samples that Were Preserved and Extracted Using Different Methods. ................................... 22 Figure 11. NMDS Plots of Communities (Jaccard Dissimilarity) Produced by Different Preservation/Extraction Methods (Colored Points/Ellipse). ....................................... 24 Figure 12. Boxplot Showing the Number of Taxa Detected in eDNA Samples Using Different Primer Sets. ....................................................................................... 25 Figure 13. NMDS Plots of Communities (Jaccard Dissimilarity) Produced by Different Primer Sets (Colored Points/Ellipse). .......................................................... 28 Figure 14. Boxplot Showing the Number of Taxa Detected in eDNA Samples Processed using Different Bioinformatics Pipelines. .................................................. 29 Figure 15. NMDS Plots of Communities (Jaccard Dissimilarity) Produced by Different Bioinformatics Pipelines (Colored Points/Ellipse). .................................................... 31 LIST OF TABLES Page Table 1. The Subset of Primers with Approximately Appropriate Annealing Temperature and Fragment Length Were Tested with 11 Locally Occurring Bee Species Using PCR and Verified via Gel Electrophoresis. .................. 10 ii Table 2. Summary of Analyses Statistically Comparing Homogeneity of MVDISP Between the Communities of Different Flower Species (ANOVA) and Variation in Community Composition of eDNA Samples from Different Flower Species (PERMANOVA). ........................................ 17 Table 3. Summary of Analyses Statistically Comparing Homogeneity of MVDISP between the Communities Produced by Different Sample Types (ANOVA) and Variation in Community Composition of eDNA Samples Sourced from Different Material (PERMANOVA). ..................................... 20 Table 4. Summary of Analyses Statistically Comparing Homogeneity of MVDISP Between the Communities Produced by Different Preservation/Extraction Methods (ANOVA) and Variation in Community Composition of eDNA Samples Preserved and Extracted Using Different Protocols (PERMANOVA). ......................................................................... 23 Table 5. Summary of Analyses Statistically Comparing Homogeneity of MVDISP Between the Communities Produced by Different Primer Sets (ANOVA) and Variation in Community Composition of eDNA Samples When Amplified with Different Primer Sets (PERMANOVA). ................... 26 Table 6. Summary of Analyses Statistically Comparing Homogeneity of MVDISP Between the Communities Produced by Different Bioinformatics Pipelines (ANOVA) and Variation in Community Composition of eDNA Samples Processed Using Anacapa or MetaBEAT (PERMANOVA). ........................ 30 Table 7. Greenhouse Community Flower Species ................................................................... A-1 Table 8. Primer Panel Details ................................................................................................... B-1 Table 9. Results of in Silico Primer Testing ............................................................................. C-1 iii ACRONYMS AND ABBREVIATIONS 16s mitochondrial 16s ribosomal RNA locus 18s nuclear ribosomal RNA locus 28S nuclear ribosomal RNA locus AE Qiagen elution buffer AL Qiagen lysis buffer ANOVA analysis of variance ASV amplicon sequence variant ATL Qiagen tissue lysis buffer AW1 Qiagen wash buffer AW2 Qiagen