INTRODUCTION
Environmental DNA (eDNA) can be defined as the mixture of complex, often
degraded, DNA that organisms leave behind in their environment (i.e.
soil, water, sediments, etc.). By studying short,
taxonomically-informative DNA fragments obtained from eDNA samples, it
is possible to identify the associated taxa and therefore to survey
biodiversity. Coined as “eDNA metabarcoding”, this approach has
revolutionized several branches of ecology and environmental sciences
during the last decade, by providing relatively quick, non-invasive, and
standardized assessments of present or past biodiversity of animals,
plants and microorganisms (Taberlet, Bonin, Zinger, & Coissac, 2018).
Metabarcoding is particularly valuable for monitoring biodiversity over
large geographical or taxonomic scales (De Vargas et al., 2015;
Delgado-Baquerizo et al., 2018; Zinger et al., 2019b). Furthermore, it
gives access to biodiversity components that are elusive to conventional
survey methods. For instance, it allows the rapid assessment of
microbial soil biodiversity, which is extremely complex, time-consuming
and imperfect when using direct observations, culturing techniques or
microscopy (Giovannoni, Britschgi, Moyer, & Field, 1990; Ward, Weller,
& Bateson, 1990).
Metabarcoding relies on a succession of several steps: 1) sampling; 2)
preservation of the collected material until lab processing; 3) DNA
extraction; 4) PCR amplification of a selected genomic region; 5)
high-throughput sequencing of amplicons; and 6) analysis of sequences
using bioinformatics and statistical tools (Zinger, Bonin, et al.,
2019a). Each step is critical to obtain robust taxonomic inventories and
diversity estimates, and an increasing number of studies have assessed
how methodological choices across the different steps could influence
the conclusions of a study (Calderón‐Sanou, Münkemüller, Boyer, Zinger,
& Thuiller, 2020; Cantera et al., 2019; Chen & Ficetola, 2020; Nichols
et al., 2018; Taberlet et al., 2018). So far, despite this growing body
of literature, little attention has been accorded to the effect of
different preservation conditions of the collected environmental
material before lab processing (i.e. step 2). We thus know neither under
which conditions the collected material should be stored, nor how long
it can be stored to avoid biases in taxonomic inventories.
While more is known for water samples (see e.g. Kumar, Eble, & Gaither,
2020; Majaneva et al., 2018), in the case of soil biodiversity research,
methodological analyses on the effects of sample preservation are
largely dismissed probably because the majority of metabarcoding studies
have so far been performed in temperate areas where access to lab
facilities is often easy (Hoffmann, Schubert, & Calvignac-Spencer,
2016; Huerlimann et al., 2020). In such cases, sample preservation is
sometimes not necessary at all, or at least not over long periods of
time. However, one great promise of metabarcoding is its potential for
providing biodiversity data for remote areas, where biodiversity
monitoring is essential but difficult. When sampling in remote or
inaccessible areas (e.g. tropical and arctic areas; mountain chains),
samples are rarely collected nearby lab facilities and an immediatein situ DNA extraction is generally not possible due to logistic
constraints (but see Zinger, Taberlet, et al., 2019b for a notable
exception). More generally, with the ever-increasing number of samples
analyzed during a typical metabarcoding study, sample preservation is
more and more indispensable, and the time lag between sample collection
and subsequent molecular processing makes it particularly relevant to
understand the impact of sample preservation, and to identify
preservation strategies that do not bias the conclusions of studies.
In an optimal metabarcoding study, communities recovered from preserved
samples should ideally be identical to those retrieved if samples had
been processed immediately after sampling. However, inappropriate
preservation conditions can cause both DNA degradation and the
proliferation of certain taxonomic groups, with respect to others,
before DNA extraction (Cardona et al., 2012; Orchard, Standish, Nicol,
Dickie, & Ryan, 2017). This can in turn affect taxa detection and also
the relative contributions of different taxonomic groups to the overall
biodiversity. A recent review suggested that the majority of eDNA
metabarcoding studies do not provide accurate information about sample
treatment before processing (Dickie et al., 2018). Almost half of the
studies do not report how samples were stored and conserved, and 30% of
them store samples at 0-4°C, and thus at a temperature where many
bacteria and fungi continue to be active and potentially affecting the
whole sample. About 15% of the studies stored samples in a range of
5-35°C, which is considered as a poor practice, and only 10 % stored
them below 0°C (Dickie et al., 2018).
So far, the consequences of preservation practices and the resulting
deviations from immediate processing and analyses have rarely been
studied quantitatively. Yet, Lauber, Zhou, Gordon, Knight, & Fierer
(2010) tested the effect of storing samples from soil, human gut and
skin at different temperatures and did not detect any significant effect
on bacterial communities, while Orchard et al. (2017) found that storage
time and temperature can affect colonization by arbuscular mycorrhizal
fungi, with subsequent impacts on the reconstruction of communities.
Differences between these studies may be due to their different
protocols. However, they also focused on different taxonomic groups,
which may react differently to storage period and temperature. Other
studies use desiccation for conserving plant and animal tissues for
subsequent genomic studies (e.g. Chase & Hills, 1991), which has proven
efficient and convenient. Although not widely used for metabarcoding
samples, desiccation is another attractive option, and has a potential
for being largely implemented in soil sample preservation. A clear
understanding of the effect of different preservation methods,
especially across various groups of taxa, is thus pivotal for a robust
application of eDNA metabarcoding to biodiversity monitoring in general,
and that of remote areas in particular.
Here, using eDNA metabarcoding of different taxonomic groups in soil
systems, we tested: (i) how preservation methods influence overall
richness estimates and what the role of rarely observed taxa is; (ii)
how preservation methods influence identified community structure and
its turn-over between different habitats; and (iii) what the best
practices are under limited laboratory access. More specifically, we
first selected three soil preservation methods (room temperature, 4°C,
desiccation by addition of silica gel) because they are commonly used in
the literature (room temperature and 4°C) or because they are easy to
implement in the field (desiccation and room temperature). Then, we
assessed the impact of these preservation methods applied to different
durations in order to mimick logistic constraints, and compared the
communities obtained with those observed in ideal conditions, i.e. when
eDNA is extracted immediately after sampling (within less than one
hour). We examined bacterial, fungal and eukaryotic communities to cover
a broad taxonomic range, since different taxa can be differentially
affected by sample preservation conditions (Cardona et al., 2012;
Orchard et al., 2017).