The overall hypothesis of this project 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. This work was divided into five tasks. Task 1: Develop aquatic environmental DNA (eDNA) metagenetic assays for fishes and amphibians using traditional polymerase chain reaction (PCR) and ultrasequencing, 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.

The research team developed and applied a new metagenetic toolbox of PCR primers for multiple mitochondrial loci for the detection via sequencing of freshwater fish and amphibian species richness. Details for each task follow.

Task 1: To develop markers the research team used three in silico approaches of literature searches, primer design software, and visual searches of alignments from publicly available sequences. Using experiments, they tested alternative methods for field (DNA sample collection) and laboratory (extraction, library preparation, controls) protocols to increase capture of Edna, reduce contamination, and achieve room temperature preservation of eDNA. They gathered fish and amphibian specimens from other investigators, museums, and their own collections to create a reference database. They assessed different bioinformatics pipelines with respect to the goal of identifying rare species.

Task 2: The research team conducted a replicated mesocosm experiment to test the precision of the metagenetic approach for measuring known fish and amphibian species richness; different treatments contained different relative and absolute abundances of the same eight fish species and one amphibian species.

Task 3: The research team conducted side-by-side sampling with both eDNA and traditional methods in Juday Creek, an Indiana stream with a fish community that has been sampled for many years, and Lawler Pond at Ft. Custer Training Center, Michigan.

Task 4: The research team conducted eDNA sampling in Eagle Creek at Ft. Custer, and in streams inmultiple watersheds at Camp Pendleton, California.

Task 5: The research team used the bias-corrected Chao II estimator on eDNA incidence data to estimate fish species richness in the mesocosm study, Juday Creek, and Lawler Pond.

Using eDNA assays in experimental mesocosms the research detected 100% of stocked species. In a lake and a stream community, using eDNA they detected 100% of the species that were detected by intensive sampling with traditional means (traps, nets, electrofishing), plus additional species not captured. Using Chao estimators, species richness estimates based on eDNA exceeded those based on traditional sampling. Details for each task follow.

Task 1: The research team identified six primer pairs that are effective for fishes and amphibians. They identified new protocols for DNA sample collection (e.g., use of cellulose nitrate filers, CI extraction), and laboratory protocols including various controls to identify contamination, and discovered that Longmire’s preservative provides room temperature preservation for at least two weeks. They created a reference database of sequences for a total of 47 species, including targetspecies, species used in the experiments, and species known to occur at the field sites. Finally, they developed a new bioinformatics pipeline to analyze metagenetic data.

Task 2: In the mesocosm experiment, the metagenetic methods detected all fish and amphibian species in all treatments and all replicates.

Task 3: In Juday Creek, eDNA detected all 12 species caught with traditional methods. eDNA detected four additional species known from the region but never before detected in Juday Creek; these species may be present in the sampled reaches or upstream. From the Ft. Custer pond, eDNA detected all 10 fish species detected with traditional methods plus as many as 11 additional species (depending on the level of bioinformatics stringency the research team applied).

Task 4: In Eagle Creek, the research team detected a total of 23 fish species with eDNA, with species number increasing downstream. In Camp Pendleton, they detected 27 native species and 10 exotic species, all previously known from the base. They additionally detected the federally protected Tidewater Goby and the state listed Arroyo chub and Arroyo toad at locations previously known for their occurrence. They did not detect the federally listed Southern Steelhead.

Task 5: Under plausible bioinformatics stringency, species richness estimated using the Chao II estimator from eDNA exceeded the number of species detected with eDNA (and exceeded by even more the number of species detected with traditional methods).

The research projects in tasks 1-5 built upon each other. Collectively, these projects 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.