FINAL REPORT Assessing Pollinator Communities via Environmental DNA (eDNA) Metacommunity Assay SERDP Project RC19-1102 APRIL 2020 Dr. Mark Davis Dr. Lynsey R Harper Dr. Brenda Molano-Flores University of Illinois Urbana-Champaign Mr. Joseph Benito Dr. Matthew L Niemiller The University of Alabama in Huntsville Distribution Statement A Page Intentionally Left Blank 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. Page Intentionally Left Blank Form Approved REPORT DOCUMENTATION PAGE OMB No. 0704-0188 The public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing the burden, to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) 2. REPORT TYPE 3. DATES COVERED (From - To) 06/04/2020 SERDP Final Report 4/15/2019 - 4/21/2020 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Assessing Pollinator Communities via Environmental DNA (eDNA) 19-P-0025 Metacommunity Assay 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Mark Davis, Brenda Molano-Flores, Lynsey Harper RC19-1102 University of Illinois Urbana-Champaign 5e. TASK NUMBER Matthew Niemiller,Joseph Benito The University of Alabama in Huntsville 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION Illinois Natural History Survey, Prairie Research Institute REPORT NUMBER University of Illinois Urbana-Champaign RC19-1102 1816 South Oak St. Champaign, IL 61820 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S) Strategic Environmental Research and Development Program SERDP 4800 Mark Center Drive, Suite 17D03 Alexandria, VA 22350-3605 11. SPONSOR/MONITOR'S REPORT NUMBER(S) RC19-1102 12. DISTRIBUTION/AVAILABILITY STATEMENT DISTRIBUTION STATEMENT A. Approved for public release: distribution unlimited. 13. SUPPLEMENTARY NOTES 14. ABSTRACT Pollinators provide essential ecosystem services and are critical components of resilient ecosystems. Yet, pollinators are suffering staggering global declines leading to increased listing under the Endangered Species Act. This presents challenges for DoD installations. The technical objectives of this project were to: 1) develop and validate broad-spectrum pollinator eDNA metabarcoding primers, 2) test the efficacy of multiple capture methods to obtain Next Generation Sequencing-ready pollinator eDNA from flowers, and 3) validate eDNA sampling and microfluidic metabarcoding methods in controlled and field experiments with mixed plant/pollinator communities. 15. SUBJECT TERMS Biodiversity, Community Ecology, Environmental DNA, Microfluidic Metabarcoding, Pollinator Communities, Species Richness 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON ABSTRACT OF a. REPORT b. ABSTRACT c. THIS PAGE PAGES Mark Davis 19b. TELEPHONE NUMBER (Include area code) 72 UNCLASS UNCLASS UNCLASS UNCLASS 217-300-0980 Standard Form 298 (Rev. 8/98) Prescribed by ANSI Std. Z39.18 Page Intentionally Left Blank TABLE OF CONTENTS Abstract 1 Executive Summary 4 Introduction 4 Objectives 5 Technical Approach 6 Results and Discussion 13 Results 13 Discussion 36 Implications for Future Research and Benefits 45 Implications for Future Research 45 Benefits 46 Literature Cited 47 Appendices 56 i LIST OF TABLES Table Page 1 Heat map depicting results of in vitro primer validation 14 2 Summary of analyses statistically comparing homogeneity of multivariate dispersions and dissimilarity between the communities of different flower species 18 3 Summary of analyses statistically comparing homogeneity of multivariate dispersions and dissimilarity between communities produced by different sample types 24 4 Summary of analyses statistically comparing homogeneity of multivariate dispersions and dissimilarity between the communities produced by different preservation/extraction methods 27 5 Summary of analyses statistically comparing homogeneity of multivariate dispersions and dissimilarity between communities produced by different primer sets 30 6 Summary of analyses statistically comparing homogeneity of multivariate dispersions and dissimilarity between communities produced by two bioinformatics pipelines 34 ii LIST OF FIGURES Figure Page 1 Greenhouse colonies 6 2 Heat map showing the frequency of contamination in metabarcoding process controls 15 3 Heat map showing the frequency of taxa detected in baseline samples 17 4 Heat map showing the frequency of taxa detected in greenhouse and field samples from focal flower species 18 5 Bipartite network showing interactions between invertebrate families and our focal flower species 19 6 Boxplot showing the number of taxa in eDNA samples from different flower species 20 7 NMDS plots of communities from different flower species 22 8 Boxplot showing the number of taxa in eDNA samples taken from different sources of plant material 23 9 NMDS plots of communities produced by different sample types 25 10 Boxplot showing the number of taxa in eDNA samples preserved and extracted using different methods 26 11 NMDS plots of communities produced by different preservation/extraction methods 28 12 Boxplot showing the number of eDNA taxa in eDNA samples detected using different primer sets 29 13 NMDS plots of communities produced by different primer sets 32 14 Boxplot showing the number of taxa in eDNA samples processed using different bioinformatics pipelines 33 15 NMDS plots of communities produced by different bioinformatics pipelines 35 16 Focal flower species morphology 39 17 A carpenter bee exhibiting nectar robbing behavior 40 18 A recommended workflow for pollinator eDNA microfluidic metabarcoding 44 iii LIST OF 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 wash buffer BLAST Basic Local Alignment Search Tool BLCA Bayesian Least Common Ancestor bp base pairs BOLD Barcode of Life Data System oC degrees Celsius COI mitochondrial cytochrome c oxidase subunit 1 locus CRUX Creating Reference libraries Using eXisting tools CTAB cetyl trimethyl ammonium bromide df degrees of freedom DNA deoxyribonucleic acid DoD U.S. Department of Defense eDNA environmental DNA EDTA ethylenediaminetetraacetic acid EMBL European Molecular Biology Laboratory EtOH ethanol (ethyl alcohol) FR Federal Register g gram or gravitation constant GLM generalized linear model hr hour IBCP Illinois Bat Conservation Program INHS Illinois Natural History Survey LCA least common ancestor M million metaBEAT Metabarcoding and Environmental DNA Analysis Tool min minute mL milliliter mtDNA mitochondrial DNA MVDISP multivariate dispersion NaCl sodium chloride iv NCBI National Center for Biotechnology Information nDNA Nuclear DNA ng nanogram NMDS non-metric multidimensional scaling nt nucleotide NGS next-generation sequencing OTU operational taxonomic unit P probability value (p-value) PCI phenol-chloroform-isoamyl PERMANOVA permutational multivariate analysis of variance PCR polymerase chain reaction PRI Prairie Research Institute r correlation coefficient R2 coefficient of determination RNA ribonucleic acid RTE rare, threatened, and endangered sec second SE standard error SERDP Strategic Environmental Research and Development Program TAE Tris-acetate-EDTA TE Tris-EDTA T&E threatened and endangered UIUC University of Illinois at Urbana-Champaign μL microliter μM micromolar UV ultraviolet V volt v version x times (multiple of) χ2 chi-square v KEYWORDS Biodiversity, Community Ecology, Environmental DNA, Microfluidic Metabarcoding, Pollinator Communities, Species Richness vi ACKNOWLEDGEMENTS We are grateful for the Strategic Environmental Research and Development Program for funding this research. We thank Patricia Dickerson, Evelyn Knittle, and Lauren Paddock for technical assistance. We are deeply grateful to Rosalie Metallo, Montgomery Flack, and Clinton Shipley at the University of Illinois at Urbana-Champaign Plant Care facility for technical, logistical, and moral support of our research. We appreciate Dr. Mark R. Band, Dr. Alvaro G. Hernandez, Dr. Christy L. Wright and the staff