4. DISCUSSION
The conversion of caves into touristic attractions has major impacts on
subterranean ecosystems (Fernandez-Cortes et al., 2011; Mulec, 2014;
Pulido-Bosh et al., 1997), and causes large-scale indirect anthropogenic
disturbances, such as land use change (Jiménez-Sánchez et al., 2008) and
climate change (Mammola et al., 2019). Yet, studies show inconsistent
responses of the microbial communities to human disturbance, pointing
out either a positive (Mammola et al., 2017; Marques et al., 2016),
negative (Alonso et al., 2019; Shapiro & Pringle, 2010) or variable
effect (Bercea et al., 2019; Mulec & Oarga-Mulec, 2012). These
controversial outcomes are likely attributed to differences in the
examined groups and to the environmental variations across different
caves (Pfendler et al., 2018, 2019). Examining human-induced changes in
subterranean microbial communities across different caves is often
hampered by the great differences in local species composition, which is
strictly influenced by geographic and geological features such as cave
size, morphology, and water dynamics (Saiz-Jimenez, 2012). To overcome
these limitations, we simultaneously analysed the response of the three
main microbial groups inhabiting caves, namely Fungi, Bacteria and
Archaea, combining the data collected across multiple show caves. By
relating both species richness and community composition in the tourist
parts to those observed in control areas, we could depict the
human-induced effects on microbial communities, without confounding
factors due to local environmental features. By adopting a metacommunity
framework, we disentangled the effect of visitors on subterranean
microbial communities from the habitat filter and the dispersal
limitation for the first time.
Our results pointed out an effect of visitors on the diversity and
composition of microbial communities in show cave sediments, but with
different outcomes depending on the examined microbial group. When
separately analysing the response of the three groups, our results
pointed out that human disturbance does not affect the species richness
of the prokaryotic component, as demonstrated by the absence of a
significant response of the models fitted on the ASV-ratios. However, we
clearly showed that visitors influence the composition of microbial
communities of Bacteria and Archaea by causing a turnover of species as
demonstrated by the decline of β-replacement at increasing distance from
the tourist path. These results are in accordance with the outcomes of a
meta-analysis testing the response of microbial communities to
disturbance, which showed only a weak response in terms of species
richness but the effects were evident on the community composition
(Jurburg et al., 2021). The observed species turnover on the tourist
path could be ascribed to the replacement of resident species by
propagules of microorganisms vehiculated by visitors’ shoes and clothes,
which are spread around while visiting the cave (Mulec, 2014;
Saiz-Jimenez, 2012; Zhelyazkova et al., 2020), similarly to what
observed by other authors (Alonso et al. 2019; Dong et al., 2020; Ikner
et al. 2007).
The response of Fungi showed a U-shaped response to the human
disturbance. The ASV-ratio in samples close to the path was similar to
those found in control sites, but decreased significantly at
intermediate distance. This outcome suggests that visitors increase
fungal species richness either directly —by vehiculating propagules of
novel species (Jurado et al., 2021; Mammola et al., 2017; Taylor et al.,
2013)— or indirectly —by introducing high amounts of organic matter,
beyond the lampenflora biomass proliferating in the visited paths due to
the lighting system, that favour more competitive species (Jurado et
al., 2010; Marquez et al., 2016; Addesso et al., 2020). However, in
sampling sites located deeper in the cave, the pattern is reversed, with
increasing ASV-ratio at increasing distance from the tourist path. We
can hypothesize that fungal communities at the cave entrance are
composed of different species, which can cope well with the new
conditions induced by visitors with respect to the assemblages in the
deepest parts. On the contrary, fungal communities in the control area
are composed of resident species that are expected to be less resilient
to human disturbance.
Variation of fungal communities at different cave depths is supported by
the observed decline in species replacement at increasing distance from
the cave entrance. Based on these results, the composition of species
assemblages of Fungi is driven by dispersal, as demonstrated by their
higher turnover at the entrance of the cave with respect to the deepest
parts. This is in agreement with previous studies demonstrating that
Fungi from the external environment can be vehiculated in caves by air
circulation (Docampo et al., 2011; Ogórek et al., 2014, 2016).
Environmental filtering plays a significant role in determining the
assemblages of Fungi and, to a lesser extent, of Archaea. Both groups
showed a decline of their ASV-ratio at increasing dissimilarity of
sediment composition from the control areas, together with an increase
in species replacement for Fungi. Given that the distance from the
tourist path and from the cave entrance did not influence sediment
composition, this response can be specifically ascribed to the filter
exerted by the environment. By examining the contribution of each
physical and chemical parameter to the overall variation of sediment
composition, we could identify Nitrogen concentration and, to a lesser
extent, pH and the % of clay as the most important variables. We can
therefore hypothesize that these parameters filter species based on
their ecological requirements. This is in agreement with literature
data, which pointed out a key role of substrate as a driver of species
richness and composition of cave assemblages of both Fungi (Cailhol et
al., 2020; Kukla et al., 2018) and Archaea (Marques et al., 2017).
Overall, by adopting a metacommunity framework, we could provide new
perspectives on the dynamics and patterns of microbial communities under
human disturbance. Specifically, our results highlighted that
anthropogenic pressure affects all microbial communities but with
different effects. Similarly, the three examined groups show
differential responses to environmental filtering and dispersal pointing
out that a proper understanding of the underlying selective mechanisms
require a comprehensive and multi-taxonomic approach.
Funding: this work
was realized within the framework of the PRIN SHOWCAVE “A
multidisciplinary research project to study, classify and mitigate the
environmental impact in tourist caves” - code 2017HTXT2R (PI: Marco
Isaia), funded by the Italian Ministry of Education, University and
Research. The SHOWCAVE project is endorsed by AGTI (Associazione Grotte
Turistiche Italiane) and the SSI (Società Speleologica Italiana). The
grant of EP is co-financed by the PON “Research and Innovation”
Programme (Axis IV “Education and Research for recovery” – Action
IV.6 “Research contracts on Green themes”).
Conflicts of interest: the authors declare no conflicts of
interest.
Acknowledgments: we are grateful to Benedetta Baroni for her
help during the field work. We also thank Cooperativa Alto Corsaglia
(Grotta di Bossea), Mondolè touristic office (Grotta del Caudano),
Vittorio Verole and Mario Verole (Grotta del Vento), and to the
president, Dr. Francescantonio D’Orilia, and the scientific director,
Prof. Mariana Amato, of MIdA Foundation (Grotte di Pertosa-Auletta) for
endorsing our research and for their logistic support during the
sampling activities.