Optimizing eDNA Replication for Standardized Application in Lotic Systems in Aotearoa, New Zealand

National standardization of ecological sampling protocols between different agencies in Aotearoa New Zealand has historically been difficult to attain, creating challenges for combining datasets for national scale analyses. The introduction of new methods for biological monitoring, such as environmental DNA (eDNA), presents an opportunity to standardize aquatic sampling protocols prior to widespread adoption. The objective of this study was to optimize eDNA sample replication for the consistent characterization of freshwater fish and macroinvertebrate communities in flowing waters, and ultimately, to inform the development of robust national monitoring standards. A comparison of field replication and extraction methods (pooling of preservation buffer) was also trialed as part of this high replication (n = 16) eDNA study to assess any potential benefits in measuring species richness and reducing processing costs alongside replication optimisation. This involved two ‘syringe’ sampling methods (‘standard’ and ‘boosted’, eight each) conducted across 54 riverine sites throughout the country. No significant difference was found for species richness between the standardized (eight replicates) or boosted (16 replicates composited to eight) eDNA methods for fish and macroinvertebrates. Results indicated that six replicates were needed to consistently detect 89.5% of fish species likely to be present using field-based syringe eDNA sampling and preservation. However, an altitudinal species richness effect was observed for fish. For macroinvertebrates, six replicates were required to identify 86% of taxa identified to the NEMS (National Environmental Monitoring Standards) level used for the Macroinvertebrate Community Index (MCI: usually genera) while eight replicates were required to detect 89% of NEMS taxa. For fish and macroinvertebrate biodiversity, this study suggests that six replicates are a reasonable trade-off between effective community characterization and cost in New Zealand lotic systems.