A molecular specimen bank for contemporary and future study captures landscape-scale biodiversity baselines before Klamath River dam removal.

Global restoration and conservation of freshwater biodiversity are represented in practice by works such as the Klamath River Renewal Project (KRRP), the largest dam removal and river restoration in the United States, which has reconnected 640 river kilometers. With dam removals, many biological outcomes remain understudied due to a lack of pre-impact data and complex ecosystem recovery timeframes. To avoid this, we created the KRRP molecular library, an environmental specimen bank, for long-term curation of environmental nucleic acids collected from the restoration project. We used these initial samples, environmental DNA metabarcoding, and generalized linear mixed-effects models to evaluate patterns of pre-dam removal fish richness and diversity. Demonstrating the suitability to resolve biological differences, the baseline shows that tributary and mainstem streams had greater native fish diversity and 2.3-10.7 times greater native fish species richness than reservoirs. These and future sampling efforts should, at a minimum, allow tracking of fish community response to ecosystem restoration. Anticipating the acceleration of omics innovation, we preserved samples for long-term storage and identified requisite phases for sustained function and adaptation of the molecular library: securing a physical storage facility for genetic material, establishing a governance structure, and confirming support for archive management.