Andrea Erhardt Scope 13-104B Water Resources Research Institute Annual Base Program 2021-2026

Urbanization degrades streams by altering hydrology1,2, eroding stream channels2,3, impairing water quality4,5, and reducing biodiversity of aquatic organisms4,6–8. Because of these issues, biotic communities in urban streams are frequently characterized by low biodiversity and high pollution tolerance9,10. Stream restoration can improve stream health, but restoration approaches such as natural channel design (NCD) are costly and labor-intensive. Riparian reforestation may present a relatively low-investment opportunity providing some water quality improvement11, in addition to other ecosystem service benefits 12 13. However, previous research suggests that the effects of riparian reforestation on stream health are highly context-dependent, with some studies documenting improved aquatic diversity in reforested reaches14,15 and others concluding that reforestation had no effect16,17. Thus, while riparian reforestation is a relatively simple restoration activity with wide applicability and broad possible ecosystem service benefits, the effects of riparian reforestation on in-stream water quality and stream-dwelling macroinvertebrates remain unclear. Lexington’s urban streams have a troubled history, influenced in part by aging storm and sanitary sewer infrastructure and resulting outflows during high flow events18. Several stream sites throughout the city have been restored using riparian reforestation as part of the Reforest the Bluegrass program, or other conservation programs, a major goal of which was water quality improvement19. Previous unpublished research by the current investigators in reforested stream reaches in Lexington, KY demonstrated consistent water quality improvement from upstream to downstream sampling locations in several sampled sites for some chemical constituents (e.g., nitrate), but no consistent patterns with respect to macroinvertebrate communities sampled using leaf packs. The proposed project will support continued water quality analysis and conventional macroinvertebrate surveys, but will elevate and expand the scope of this analysis by adding an environmental DNA (eDNA)-based metabarcoding protocol. Since its initial use in macroorganism studies in 200820, eDNA has become firmly established as a reliable method capable of characterizing organism presence21–23, and perhaps abundance24–26, and is transforming the manner in which ecological studies are conducted27. The recent addition of metabarcoding to eDNA enables taxonomic composition analysis of entire communities through the identification of millions of DNA fragments/per sample, providing a powerful survey approach that is rapidly emerging as a cost-effective method for biodiversity studies28–32. Aquatic invertebrates are among the most studied organism groups to date using this technique, providing a plethora of molecular tools, databased sequences, and protocols 28,29,31,33,34. Numerous studies have detected anthropogenically induced alterations in aquatic organism communities using this technique32,35–38, often when traditional methods failed to detect these differences32,35.