The spatial heterogeneity of soil and the microbial communities therein, however, does not only persist on the microscale, but certainly also on a centimeter, meter, field, or ecosystem scale \cite{Becker_2006,Wolfe_2006,Franklin_2003}. Sampling “the same soil” a few meters apart or at different depths in the soil profile might result in individual samples with varying biogeochemical properties such as pH, water saturation, soil texture, and also plant root distribution. Thus, the assumption that replicates are similar can certainly introduce bias into soil microbiome interpretation. For example, a recent study showed that the intraplot spatial variability in microbial communities was pronounced throughout a season even though 10-15 cores were randomly sampled in individual subplots and pooled \cite{Carini_2020}⁠.  Similar shifts in bacterial and fungal communities were observed in these subplots, suggesting that there were distinct and consistent differences between these commonly termed “replicate” soil samples. Pooling of samples, individual extractions of DNA/RNA and/or amplification reactions made from a single DNA template is usually considered to minimize the effect of soil heterogeneity on a larger scale but still differences prevailed in the study. In another example, chemical soil properties as well as microbial biomass and communities were observed to exhibit high levels of spatial variation across 49 samples in a 6 \(\times\) 6 m forest plot \cite{_tursov__2016}⁠. The authors concluded that “forest topsoil should be considered a spatially heterogeneous environment in which the mean estimates of ecosystem-level processes and microbial community composition may confound the existence of highly specific microenvironments ”. These considerations point to the fact that existing intraplot variability has to be critically discussed and acknowledged in any study on soil microbiomes and their potential environmental drivers. Otherwise, drawing of macroecological and generalistic conclusions from soil samples taken and pooled with large distance from each other yield speculative information at best \cite{Zhang_2020}. The critical importance of adequate sample replication is further discussed in section XYZ.

Temporal considerations and ... 'Relic' DNA obscures biological patterns

When designing an experiment, one must not only consider the spatial scales at which microorganisms live and interact, but as well the temporal scale at which sampling should occur to capture dynamics of interest.
In soil, studies that include a temporal effect often observed changes in microbial communities from short time scales such as days and weeks to longer time periods such as season and years (REFS).
A critical parameter when investigating temporal scales is the rate of community change detectable with amplicon sequencing which represents a snapshot of microbial prevalence at a given moment. Given that estimation of microbial turnover in soil range from days to years (REFs), it is difficult to assess the best temporal sampling strategy a priori
In a forest soil....the temporal dynamics most likely differ from a highly-used agricultural soil, processes in the rhizosphere including community assembly are expected to occur at higher rates than in bulk soil (Kuzyakov?).