Researchers using cutting-edge environmental DNA (eDNA) technology have uncovered far greater biodiversity in eastern Ontario waterways than traditional monitoring methods detected over more than a decade, demonstrating the transformative potential of DNA-based biomonitoring for freshwater conservation and environmental management.
The study, published in the journal Molecular Ecology, analyzed benthic macroinvertebrates, small aquatic organisms such as insect larvae, crustaceans, and other stream-dwelling species widely regarded as key indicators of freshwater ecosystem health, across 18 streams in the agriculturally dominated South Nation River watershed in eastern Ontario. Environmental DNA researchers found that a single year of DNA metabarcoding identified dramatically more biodiversity than conventional surveys, while also revealing much clearer ecological signals linked to agriculture, water quality, and land use, based on morphology-based data for 10 years gathered within a span of 15 years.
"This study shows that DNA metabarcoding can reveal ecological patterns and biodiversity changes that traditional approaches often miss," said senior author Mehrdad Hajibabaei. "The ability to rapidly and accurately detect species-level changes across freshwater systems could fundamentally improve how we monitor, manage, and protect aquatic ecosystems under increasing environmental stress."
The findings come as freshwater ecosystems worldwide face mounting pressures from agricultural intensification, urban expansion, pollution, and climate change. Agriculture alone occupies more than 37 per cent of Earth's terrestrial surface and is considered one of the leading drivers of biodiversity decline globally.
The South Nation River watershed, covering approximately 3,900 square kilometres in eastern Ontario, provided an ideal test case. The region contains a complex mix of agricultural lands, forests, and expanding urban areas, much of it heavily influenced by extensive subsurface tile drainage systems used to support intensive farming.
To conduct the study, researchers collected benthic macroinvertebrate samples during summer and fall 2023 from streams spanning a gradient from predominantly forested to heavily agricultural landscapes. Using DNA metabarcoding, they extracted and sequenced genetic material from bulk environmental samples, enabling the identification of hundreds of species simultaneously through high-throughput sequencing technologies.
The results revealed striking differences between the DNA-based and traditional monitoring approaches.
DNA metabarcoding detected 282 species across the watershed, with 261 species found exclusively through the DNA approach. Traditional morphology-based methods detected only 22 unique species not recovered by DNA analysis, while just 20 species overlapped between both methods.
At the site level, DNA metabarcoding recovered significantly more species and orders than morphology-based identification. Median species richness per site was 59 species using DNA compared to just 15 using conventional methods.
The DNA analyses also uncovered extensive hidden diversity. Nearly 44 per cent of detected species occurred at only a single site, suggesting many freshwater species may have highly localized distributions that conventional methods fail to capture.
Importantly, the DNA-based approach provided far sharper ecological resolution. Statistical analyses showed that DNA metabarcoding consistently distinguished differences between agricultural, forested, and mixed-use stream systems more clearly than historical morphology datasets collected over many years.
Agricultural streams displayed strong associations with elevated conductivity, turbidity, and altered pH levels, indicators linked to fertilizer runoff, soil disturbance, and intensive land management. Forested streams, by contrast, were associated with higher dissolved oxygen levels and greater biodiversity.
"Even relatively modest changes in community composition can provide important early warning signals of ecosystem stress," the authors wrote. "DNA metabarcoding offers the sensitivity needed to detect these changes before larger ecological degradation becomes apparent."
The study also highlights longstanding limitations of traditional morphology-based biomonitoring. While Ontario's widely used Ontario Benthos Biomonitoring Network protocol has provided valuable stream assessments for years, the method relies heavily on expert taxonomic identification, which can be slow, labour-intensive, and often incapable of reliably distinguishing closely related or immature organisms.
Among nearly 80,000 specimens collected through conventional monitoring from 2008 to 2022, most remained unresolved at the species level. In many survey years, more than 90 per cent of specimens could not be confidently identified to species using morphology alone.
DNA metabarcoding, by contrast, rapidly generated fine-scale biodiversity data across multiple taxonomic levels and successfully identified numerous groups underrepresented or entirely absent in morphology-based records, including several insect and crustacean lineages.
Researchers say the technology could substantially strengthen long-term freshwater monitoring programs, particularly in regions experiencing mixed environmental stressors from agriculture and urbanization.
The study supports growing international momentum toward integrating eDNA approaches into routine environmental assessment programs. Because DNA metabarcoding requires less dependence on specialized taxonomic expertise while offering faster processing and greater reproducibility, it may help agencies scale up monitoring efforts at lower cost and with higher ecological sensitivity.
The authors emphasize that traditional methods still retain value, especially for historical continuity and certain trait-based analyses. However, they argue that DNA metabarcoding should now become a core component of modern freshwater biomonitoring frameworks.
"The future of freshwater biomonitoring will likely combine rapid, scalable DNA-based screening with targeted traditional surveys where needed," the researchers concluded. "This integrated approach offers a more sensitive, timely, and comprehensive understanding of ecological change in freshwater ecosystems facing accelerating human pressures."
With study was led by the Hajibabaei lab at the Centre for Biodiversity Genomics and the Department of Integrative Biology at the University of Guelph with collaborators from AAFC and South Nation Conservation.
"Fine-Scale Ecological Biomonitoring in a Large, Complex Agriculturally Impacted Watershed via eDNA Metabarcoding," was authored by Bráulio S. M. L. Silva, Andrew C. Riley, Emilia Craiovan, Michael Wright, Katherine Watson, David R. Lapen, and Mehrdad Hajibabaei and published in Molecular Ecology. Funding was provided by the New Frontiers in Research Fund, the Illumina Foundation, Environment and Climate Change Canada, and the Canadian Safety and Security Program.
