On the Importance of Replication and Sample Size in DNA-Based Biodiversity Surveys

  • May 28, 2024

To paraphrase Heraclitus: “No sample can be collected from the same river twice, for it is not the same river, and it is not the same sample.” More than a bit of philosophical musing, this is a guiding principle for those of us working in the domain of DNA-based biodiversity surveys. Each moment in nature is unique, and each sample reflects a fleeting snapshot of this dynamism. Therefore, the importance of replication and ample sample sizes cannot be overstated.


ecoType eDNA soil samplingThe Case for High-Capacity Sampling in Soil Biodiversity

In terrestrial habitats, soil biodiversity is both rich and elusive. The complex matrix of soil hosts a myriad of organisms, from microbes to invertebrates. Large-volume sampling is critical for capturing this diversity. Small samples may miss rare species and fail to represent the full ecological complexity. Studies have shown that larger samples increase the likelihood of detecting a more comprehensive range of taxa, providing a truer picture of the soil ecosystem (Fierer et al., 2012).


CIFCA inDepth eDNA deploymentHigh-Volume Filtered Water Sampling in Marine Environments

Marine biodiversity, like terrestrial biodiversity, is dynamic and complex. Tidal cycles introduce variability, distributing organisms differently throughout the day. High-volume water sampling across the entire tidal cycle is essential to capture this variability. This approach ensures that transient and less abundant species are detected, offering a more accurate reflection of the marine ecosystem. For example, Deiner et al. (2017) demonstrated that comprehensive sampling protocols could reveal a greater diversity of species in aquatic environments.


The Imperative of Replication

Replication is the cornerstone of robust biodiversity surveys. It mitigates the stochastic nature of biological data and accounts for spatial and temporal variability.

Spatial Replication

In terrestrial environments, spatial replication helps account for habitat heterogeneity. Soil properties can vary significantly over short distances, influencing species composition. By sampling multiple locations within a habitat type, we can provide a more accurate assessment of the spatial distribution of biodiversity (Prosser, 2010).

In freshwater and marine environments, spatial replication is equally important. Water bodies are dynamic, with currents and other hydrological features affecting organism distribution. Replicating samples across different locations ensures a more representative understanding of biodiversity (Thomsen et al., 2012). However, because the environment being sampled is itself dynamic, spatial replicates, particularly in marine environments need to be distributed over vastly larger spatial scales.

Seasonal Replication

Biodiversity is not static; it fluctuates with seasons. Many species exhibit seasonal behaviours such as migration, breeding, or dormancy. Sampling at different times of the year captures these temporal variations. For instance, studies in temperate forests have shown significant seasonal shifts in soil microbial communities (Lauber et al., 2013).

In marine environments, seasonal replication can reveal changes in species composition driven by factors such as temperature and nutrient availability. For example, seasonally replicated eDNA surveys in coastal waters have uncovered shifts in fish community structures (Kelly et al., 2018). 

Annual Replication

Long-term monitoring is crucial for detecting trends and changes in biodiversity over time. Annual replication allows researchers to distinguish between natural variability and significant ecological changes. This is particularly important in the context of climate change and human impact. For example, annual monitoring of freshwater streams has provided insights into long-term trends in fish populations (Hughes et al., 2014).

Recently, Applied Genomics completed analysis of 460 marine eDNA samples to achieve the first ever 12-year eDNA time-series for fish communities, revealing both seasonal patterns and annual trends.


In the pursuit of understanding biodiversity, the importance of replication and adequate sample size cannot be overstated. Whether characterising soil communities, marine ecosystems, or any other habitat, these principles ensure that our surveys are robust, reliable, and reflective of true ecological complexity. By embracing these practices, we can better appreciate the richness and dynamism of life on Earth.


  • Deiner, K., Fronhofer, E. A., Mächler, E., Walser, J.-C., & Altermatt, F. (2017). Environmental DNA reveals that rivers are conveyer belts of biodiversity information. Nature Communications, 8, 14087.
  • Fierer, N., Ladau, J., Clemente, J. C., Leff, J. W., Owens, S. M., Pollard, K. S., ... & Knight, R. (2012). Reconstructing the microbial diversity and function of pre-agricultural tallgrass prairie soils in the United States. Science, 336(6087), 1387-1390.
  • Hughes, R. M., Herlihy, A. T., & Wang, L. (2014). The importance of large-scale studies in assessing anthropogenic impacts on streams and rivers. Fisheries, 39(1), 18-28.
  • Kelly, R. P., Shelton, A. O., & Gallego, R. (2018). Understanding PCR processes to draw meaningful conclusions from environmental DNA studies. Scientific Reports, 8(1), 2971.
  • Lauber, C. L., Ramirez, K. S., Aanderud, Z., Lennon, J., & Fierer, N. (2013). Temporal variability in soil microbial communities across land-use types. ISME Journal, 7(8), 1641-1650.
  • Prosser, J. I. (2010). Replicate or lie. Environmental Microbiology, 12(7), 1806-1810.
  • Thomsen, P. F., Kielgast, J., Iversen, L. L., Wiuf, C., Rasmussen, M., Gilbert, M. T. P., ... & Willerslev, E. (2012). Monitoring endangered freshwater biodiversity using environmental DNA. Molecular Ecology, 21(11), 2565-2573.
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