FINAL REPORT Development of an Environmental Metagenetics Approach for Monitoring Aquatic Biodiversity SERDP Project RC-2240 JULY 2017 Michael E. Pfrender University of Notre Dame 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 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 this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this 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) 07-07-2017 SERDP Final Report 09/25/2012 – 09/25/17 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Development of an Environmental Metagenetics Approach for Monitoring Aquatic 5b. GRANT NUMBER Biodiversity 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Deiner, Kristy: Jerde, Christopher; Evans, Nathan; Lamberti, Gary; Li, Yiyuan; Olds, 12 RC01-012 / RC02240 Brett; Renshaw, Mark; Lodge, David; Pfrender, Michael E. 5e. TASK NUMBER 1-5 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION University of Notre Dame REPORT Not re Dame, Indiana, USA 46556 NUMBER RC-2240 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) DoD Strategic Environmental SERDP Research and Development Program (SERDP) 11. SPONSOR/MONITOR’S REPORT Alexandria,AND ADDRESS(ES) VA 22350-3605 NUMBER(S) RC-2240 12. DISTRIBUTION / AVAILABILITY STATEMENT Distribution A: unlimited public release 13. SUPPLEMENTARY NOTES 14. ABSTRACT Our overall hypothesis is that genetic-based approaches are more accurate than existing methods in estimating species richness because they are more sensitive in the detection of rare species, whether invasive, threatened, or endangered. Our project was divided into five tasks. Task 1: Develop aquatic environmental DNA (eDNA) metagenetic assays for fishes and amphibians using ultrasequencing and validated for selected species with quantitative real time PCR (qPCR). Task 2: Test the assays on artificial aquatic mesocosm-scale assemblages of known species richness and diversity. Task 3: Test the assays in natural environments whose species richness has been thoroughly evaluated by traditional methods. Task 4: Apply the assays to natural environments with unknown species richness at different spatial scales, for comparison with traditional methods for estimating species richness. Task 5: Apply and compare alternative models for estimating species richness and biodiversity across a variety of aquatic systems. To accomplish these objectives, we developed and applied a new metagenetic toolbox of PCR primers based on multiple mitochondrial loci for the detection via sequencing of freshwater fish and amphibian species richness. By applying these tools to experimental and natural systems we demonstrated that eDNA methods entail much less sampling effort than traditional methods while providing more sensitive estimates of species presence. Therefore, eDNA yielded higher and likely more accurate estimates of species richness than traditional methods. 15. SUBJECT TERMS Metagenetics, metagenomics, metabarcoding, ultrasequencing, eDNA, species detection, species richness, aquatic biodiversity 16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. 19a. NAME OF OF ABSTRACT NUMBER RESPONSIBLE PERSON OF PAGES Michael Pfrender a. REPORT b. ABSTRACT c. THIS PAGE 103 19b. TELEPHONE NUMBER (include area code) UNCLASS UNCLASS UNCLASS UNCLASS (574)631-0591 Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18 Page Intentionally Left Blank Table of Contents ABSTRACT ................................................................................................................................................... 1 OBJECTIVES ................................................................................................................................................ 1 TECHNICAL APPROACH .............................................................................................................................. 1 RESULTS ..................................................................................................................................................... 1 BENEFITS .................................................................................................................................................... 2 OBJECTIVES ................................................................................................................................................ 3 BACKGROUND ........................................................................................................................................... 4 TASK 1: DEVELOP AQUATIC ENVIRONMENTAL DNA METAGENETIC ASSAYS ....................... 5 METHODS ................................................................................................................................................... 5 Marker choice and primer design .................................................................................................... 5 eDNA sample collection and extraction ........................................................................................... 5 Library preparation ............................................................................................................................ 7 Contamination and error controls .................................................................................................... 8 Bioinformatics pipeline ...................................................................................................................... 8 Reference database generation ...................................................................................................... 10 RESULTS AND DISCUSSION ...................................................................................................................... 10 Marker choice and primer design .................................................................................................. 10 eDNA sample collection and extraction ......................................................................................... 11 Library preparation .......................................................................................................................... 13 Contamination and error controls .................................................................................................. 13 Bioinformatics pipeline .................................................................................................................... 14 Reference database generation ...................................................................................................... 14 TASK 2: ARTIFICIAL AQUATIC MESOCOSOM ................................................................................. 15 METHODS ................................................................................................................................................. 15 RESULTS AND DISCUSSION ...................................................................................................................... 15 TASK 3: TESTS IN NATURAL ENVIRONMENTS WITH KNOWN SPECIES RICHNESS ............... 16 METHODS ................................................................................................................................................. 16 Juday Creek, St. Joseph County, Indiana, USA ........................................................................... 16 Lawler pond, Fort Custer, Michigan Army National Guard, Michigan, USA ........................ 21 RESULTS AND DISCUSSION ...................................................................................................................... 25 Juday Creek, St. Joseph County, Indiana, USA ........................................................................... 25 Lawler pond, Fort Custer, Michigan Army National Guard, Michigan, USA ........................ 27 TASK 4: TESTS IN NATURAL ENVIRONMENTS WITH UNKNOWN SPECIES RICHNESS .......... 29 METHODS ................................................................................................................................................. 29 Eagle