2.1. Sampling design
We adopted a replicated two-factorial sampling design wherein sediment
samples were repeatedly sampled at progressive distance from the tourist
path and at progressive distance from the entrance in four Italian show
caves (Fig. 1).
In each cave, we identified four sampling areas at progressive distance
from the cave entrance: Section I to IV, with Section I being the
closest to the entrance and Section IV the furthest. Sections I, II and
III were located in areas open to the public (touristic areas, T), while
Section IV was located in an area closed to the public in the deepest
part of the cave (control area, C). The length of each Section was one
third of the tourist path length. Within each sampling section of the
touristic part, we identified three sampling transects placed at
progressive distance from the tourist path: the High Pressure (HP)
transect at 0-3 m from the tourist path; the Medium Pressure (MP)
transect at 3 to 5 m from the tourist path; and the Low Pressure (LP)
transect at > 5 m from the tourist path. In the control
area (Section IV), three sampling transects were randomly placed to
obtain a representative view of the natural conditions of the cave.
Along each transect, we collected 9 random replicates of alluvial
sediment within 9 squares of 10 cm2. Sediment was
collected on the ground up to 3 cm depth with sterile Falcon® tubes (50
mL). Samples were preserved in a thermal bag until the arrival at the
laboratory, where the 9 replicates for each sub-transect were pooled
together and homogenised. In this way, we obtained a total of 12 samples
(4 transects × 3 sub-transects) for each show cave. We focused on soil
as it proved to be the compartment with the highest microbial diversity
(see Alonso et al., 2019). As seasonal differences have been observed in
the subterranean microbiota (Mammola et al., 2017), samplings were
performed in Summer, between June and September 2020, in order to avoid
possible seasonal variations that could mask the effect of tourism on
the soil microbiota.
Sediment samples were sieved, under sterile conditions, by removing
coarse rock debris. Their physical and chemical properties were
evaluated by measuring pH, concentration of Organic Carbon, total
Nitrogen and the percentage of sand (%Sand), silt (%Silt) and clay
(%Clay) with standard protocols by Regione Piemonte - Laboratorio
Agrochimico - Settore Fitosanitario e Servizi Tecnico-Scientifici.