Design of soil preservation conditions
We did not test full-factorial combinations of different preservation
periods and conditions, and we did not consider freezing, which is
unrealistic when dealing with large numbers and / or volumes of samples,
as is the case for more and more metabarcoding studies. Furthermore,
freezing is generally impossible when sampling remote areas, where
maintaining a cold chain cannot be ensured given the logistical
challenges and, in the best cases, it is replaced by preservation in a
cool box (i.e. 4°C or more).
In the design of treatments, we considered approaches allowing
preservation at different temperatures and for different periods. For
preservation condition 2, we accounted for a certain delay (six hours)
between sampling and extraction, that could correspond to local
transportation from the sampling area to the nearest base station.
Sometimes, even in the case of an in situ extraction, samples
remain at ambient temperature for hours prior to extraction, especially
when monitoring a large area or a tricky ground, but this can have an
impact on the final results (Delavaux et al., 2020). Preservation at 4°C
is among the most frequent approaches to soil preservation (Dickie et
al., 2018). This can be attained through portable refrigerators and
requires the cold chain not to be interrupted at any point during
transportation, which is only possible when lab facilities are
accessible in a relatively short time (Hoffmann et al., 2016; Huerlimann
et al., 2020). However, one of the most attractive characteristics of
eDNA metabarcoding is its capacity to provide biodiversity data for
understudied areas (e.g. tropical and arctic areas; mountain chains).
Preservation conditions 4 and 5 of this study refer to the situations
where samples are located far from lab facilities, in areas for which
traditional biomonitoring is particularly challenging. When sampling
these areas, the time lag between sample collection and subsequent
molecular analysis can be particularly relevant. The use of silica gel
to preserve soil samples is then a good solution (Chase & Hills, 1991;
Guo, Yang, Chen, Li, & Guo, 2018): silica gel allows removing up to
26% of its weight in water in a relatively short time (overnight;
Taberlet, pers. communication), is cost-effective, easy to transport,
and is not an issue for aircraft transportation (no flammable or
dangerous preservatives). Silica gel can be added in situ during
sampling (protocol 4) or within a short time lag, allowing for local
transportation from the sampling area to the nearest facilities
(protocol 5). Afterwards, samples can be stored at room temperature,
protected from sunlight to avoid photo-degradation of DNA.