# 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
Metabarcoding has been applied to characterize microorganisms associated to speleothems (e.g., stalagmites, stalactites, and flowstones). Biomineralization is a naturally occurring process in which microbial activity influences the formation of geological structures like speleothems [70; 71; 72]. Bacteria have been reported as important actors for carbonate mineral nucleation and growth [73]. _Proteobacteria_ and _Actinobacteria_ were found to be the predominant phyla associated with speleothems (Figure 1). _Actinobacteria_ are known to be involved in biomineralization processes in karstic ecosystems [74; 75; 76; 77; 78] and are dominant in rock and sediment samples, while proteobacteria were more abundant in air and water samples, explaining their retrieval in speleothems [61]. The metabarcoding study of bacterial communities associated with the different cave speleothems highlighted specific variations in assemblages that were related to mineralogy and geochemistry [79]. In Lake Cave (Australia), _Cyanobacteria_, _Firmicutes_, and _Planctomycetes_ were less abundant in the stalactites, where a greater proportion of aragonite and less dolomite was reported compared with stalagmites [79]. Recent work focused on the microbial entrapment in stalactites and their possible environmental origin in the Agios Athanasios cave (Greece) showing bacterial transport from the upper ground cave environment through water movement, especially from plant roots, when considering _Rhizobiales_, _Pseudarthiobacter_ (_Micrococcales_) or _Gaiella_ (_Thermoleophilia_), _Pseudomonas_ (_Gammaproteobacteria_), _Ralstonia_ (_Betaproteobacteria_), and _Pantoea_ (_Gammaproteobacteria_) [80]. This study also reported that some bacterial genera retrieved in cave speleothems, such as _Polaromonas_, _Thiopfundum_, or the _Verrucomicobia_ phylum, support the concept that stalactites could be used as proxies of paleoecology to infer paleoclimate variations [80]. In the same cave, another work investigated the diversity of the rarest _Actinobacteria_ families and genera, suggesting that stalactites represent microbial arts, entrapping and preserving DNA signal of such microorganisms at a paleomicrobiological scale [81]. Metabarcoding has documented microorganisms present in soil at the cave floor. Many studies focusing on cave microbial ecology targeted the soil in these caves. _Actinobacteria_ and _Proteobacteria_ dominate metabarcoding dataset (an average of 44% and 24%, respectively) in anthropized and non-anthropized cave soil. For the microeukaryotic community, reads affiliated to _Ascomycata_ were largely preponderant (an average of 67%), followed by _Basiodomycota_ phyla (an average of 14%). However, tourist activity can have an impact on the composition of soil microbial communities by bringing organic matter and dust from the outside on shoes, fallen hair, and flakes of skin [76]. A tourist that became sick during his visit to Castanar de lbor Cave (Spain) raised the organic carbon content of the cave soil from 0.10% to 0.28% where he vomited [82]. Surprisingly, no major difference in structure and composition of soil microbial communities between anthropized and non-anthropized caves was observed at the phyla level (Figure 1). Only the _Firmicutes_ phylum displayed a difference, with 9.7% of sequences in soil of non-anthropized caves compared with 4.1% of sequences in anthropized caves. This shows the necessity to go to lower taxonomic levels to observe possible differences. Currently, there is a crucial lack of knowledge on archaeal diversity in cave floors, whether anthropized or not. Metabarcoding also identified microorganisms associated to arthropods and bats living in caves. These animals can act as biotic vectors facilitating the colonization of microorganisms in karstic environments [5; 66; 83; 26]. They can promote the dispersion of _Bacteria_, _Archaea_, and _Fungi_ by carrying them on their bodies or hosting them in their gut [84; 85; 86].