# Contributions of DNA Sequencing Technologies to the Integrative Monitoring of Karstic Caves
## 4 NGS Taxonomic Assessment of Cave Microorganisms by Metabarcoding
### Metabarcoding-Based Knowledge of Caves  
At a global cave scale, data obtained across anthropized and non-anthropized caves indicate that the microbial community is largely dominated by _Proteobacteria_ and _Actinobacteria_ among _Bacteria_, _Thaumarchaeota_ and _Euryarchaeota_ among _Archaea_, and _Ascomycota_ when considering _Fungi_ among microeukaryotic communities. However, many differences exist in microbial diversity and structure considering the contrasted compartments present in caves (i.e., water, air, speleothems, sediments, etc. ), which are detailed below. Metabarcoding has been useful in documenting microorganisms in cave water. Water infiltration through carbonate rocks is an important process in cave functioning, as it can cause CO\({}_{2}\)-mediated chemical dissolution of limestone, resulting in the formation of karstic caves, and plays a role in the microbial colonization of caves after their formation (for example, via rhizosphere channels going through the epikarst). Water has been studied in both non-anthropized and anthropized caves where a high prevalence of _Proteobacteria_ was reported (Figure 1) [54; 55], among which the _Gammaproteobacteria_ class was overwhelmingly dominant [56; 57]. Generally, water samples from karstic caves present highly diverse microorganisms [57; 58], which could be positively correlated with the water content of the epikarst, as it promotes microbial transportation, as found below farmland [59] and forest [60]. Bacterial co-occurrence networks suggested richer and more complex interactions, in terms of number of nodes, edges, clustering coefficient, and average degree, in cave water compared with mineral substrates (i.e., sediments and rocks) [61]. However, cave water studies are still scarce and lack the consideration of Archaea and microeukaryotes. Metabarcoding has identified airborne microorganisms in caves. Microorganisms may enter in caves and karst ecosystems following air currents. Air flow transporting organic particles and gaseous organics as condensation [5; 62] forms clouds of water microparticles (hydroaerosols) that support adhesion of microorganisms on walls [63; 64]. _Bacteroides_, _Firmicutes_, and _Ascomycota_ were significantly enriched in air samples compared with other compartments in anthropized and non-anthropized caves [61; 65]. Cave fungal dynamics were investigated by [PERSON] et al. (2019), and co-occurrence networks metrics showed that the bacterial community in air samples was less connected than those in cave sediments and water. Being mainly transported as spores, _Fungi_ in air samples were extensively described in comparison with _Bacteria_ and _Archaea_, which are more likely to enter caves as cells adhering to dust particles [64; 66]. Investigations into airborne _Fungi_ in caves showed that the quantity of fungal spores decreased with the distance from the entrance of the cave [25; 67] and the numbers of human visitors [68]. Yet, anthropized caves present a more diversified fungal microbiota, with a greater proportion of _Basidiomycota_ (+11%) and _Mucoromycota_ (+19%) than in non-anthropized caves (Figure 1) [69]. Figure 1: Composition of bacterial, archaeal, and eukaryotic communities based on metabarcoding and metagenomic data. The size of the circles represents the mean relative abundance of each lineage across the caves studied. Only lineages with relative proportion \(>\) 0.5% are shown. Sequences were retrieved in the results sections or supplementary data of research articles or in sequence databases (e.g., RDP classifier, Greengenes, 119 SILVA, 123 SILVA, 123.1 SILVA, UNITE 7.0, etc.). These sequences were affiliated according to the best hit obtained by BLASTn against Nucleotide collection (nr/nt) and the relative abundance of phyla was calculated. Bubble plots were generated using R (v.4.0.2) software. ‘No data’ is indicated when data are not available. See Supplementary Table S1 for further information on the experimental conditions that are included in the comparison.