Conclusions: guidelines for optimizing preservation conditions
Standardized protocols are essential for repeatable and reliable biodiversity monitoring, and our results allow to propose guidelines to improve and standardize the preservation of soil samples for eDNA metabarcoding analyses (Fig. 5):
1) If sampling occurs close to lab facilities, or a mobile lab is available, extracting DNA as soon as possible is the best approach. Storing samples a few hours at room temperature does not have major impact on the outcome of analyses, especially if the focus is not on rare MOTUs;
2) If lab facilities are available after a short-time transportation, storing samples in the fridge (0-4°C) for a few days is a safe approach as it does not have a significant impact on community composition, and only moderately affects MOTUs richness. However, this approach can be problematic if the aim is to estimate MOTUs richness, and particularly the occurrence of rare MOTUs. The feasibility of this strategy also depends on the number and volume of samples, and to the possibility of maintaining the cold chain;
3) If monitoring in remote areas, sample desiccation (e.g. using silica gel) and long-term preservation at room temperature is a reasonable approach, and it is particularly convenient when working with a large volume of samples. This approach preserves ecological signal, but can affect the detection of some taxa, particularly among the rarest ones. Therefore, this approach is suboptimal for monitoring programs aiming at detecting rare MOTUs.
An effective application of eDNA metabarcoding to biodiversity monitoring is complex, and protocols of sample preservation are key methodological choices that has to be taken into account when designing a metabarcoding-based monitoring. When working in difficult and remote environments researchers are faced to the trade-offs between a faithful representation of biodiversity, and multiple logistic constraints in the field. Accurate a-priori planning is often the basis of successful monitoring programs and our guidelines can help researchers and practitioners to identify the best approach to sample preservation, depending on the studied taxa and research goals.