# Cleaning of Phototrophic Biofilms in a Show Cave: The Case of Tesoro Cave, Spain
## 3 Results and Discussion  
### Composition of biofilms in Different Cave Sectors  
#### 3.1.1 Sector T1  
The cave was divided into three sectors with different ecological and environmental characteristics. Sector T1, largely represented by Sala del Lago, was influenced by the artificial lake occupying almost the entire sector, which provided high relative humidity, near saturation. In addition, the connection with the exterior was relevant through the water. This and the lighting produced abundant phototrophic biofilms composed of cyanobacteria, microalgae and bryophytes near the lighting lamps. The biofilms were dominated by the cyanobacterium _Phormidium_ sp. (Figure 2B), the chlorophyte _Myrmecia israelensis_ (Figure 2A) and the rhodophyte _Cyanidium_ sp. It is remarkable that _Cyanidium_ sp. was only found in this sector and not in the biofilms from the rest of the cave. The presence of _Cyanidium_ sp. in Tesoro Cave, which was also observed in Nerja Cave some 50 km away [14], deserves a special mention. The reports on the genus _Cyanidium_, identified by microscopy and morphology and retrieved from non-extremophilic habitats, are very scarce, but not those from extreme and acidophilic environments, where _Cyanidium caldarium_ is well represented [30; 31]. _Cyanidium_ sp. would not correspond to _C. caldarium_ due to its very different habitat and ecology. In fact, _Cyanidium_ strains from caves cluster into a separate monophyletic lineage within the class _Cyanidiophyceae_[32]. The first morphospecies of the genus _Cyanidium_ that did not inhabit acidic ecosystems or volcanic areas were described from samples from caves (_Cyanidium chilense_) and from fissures in coastal rocks, both in Chile [33]. Subsequently, by means of molecular analysis, three species of non-extremophilic aerophytic _Cyanidium_ have been described, two of them _Cyanidium_ sp. Monte Rotaro and _Cyanidium_ sp. Sybil Cave, inhabitants of Italian caves [34; 35], and the third, _Cyanidium_ sp. Atacama, in a cave on the Chilean coast [36]. The sequences of clones related to the genus _Cyanidium_ identified in Tesoro Cave present identities of 95.09%, 94.75% and 92.76% with the strains from Atacama, Monte Rotaro and Sybil Cave, respectively (Figure 3). In the phylogenetic tree, the Tesoro Cave sequences form a robust clade far from previous _Cyanidium_ isolates, suggesting that they could represent a different species of the genus. Figure 2: **(A)** Cells of _Myrmecia israelensis. **(B)** Filaments of _Phormidium_ sp. Scale bar: 10 μm. In many cases, the biofilms also showed abundance of the hypophyte _Eucladium verticillatum_. The cause was the intense illumination by means of incandescent lamps, as shown in Figure 4. _Eucladium verticillatum_ usually live outdoors in very pronounced shaded areas and produce rhizoid buds. However, this hypophyte is common in limestone caves, where it grows at varied ranges of light irradiation [37, 38]. [PERSON] [39] reported the presence of _E. verticillatum_ in Altamira Cave in 1955. We also find it in Encajero Cave, in Quesada, Jaen, with a thick spongy growth giving rise to considerable tufa formations, associated with _Stichococcus bacillaris_[40]. Figure 4: Growth of the hypophyte _Eucladium verticillatum_. **(A)** Sala del Volcan. (**B**) Sala del Aguila. Figure 3: Maximum-likelihood phylogenetic tree based on 165 rRNA gene sequences showing the relationship of clones from phototrophic biofilms collected in Tesoro Cave to isolated species of the genus _Cyanidium_. Bootstrap values (>50%) are expressed as percentages of 1000 replicates. The 165 rRNA gene sequence of _Bangia fuscopurpurea_ was used as the outgroup. Bar: 0.02 substitutions per nucleotide position. In the studied cases, cells of _M. israelensis, Phormidium_ sp. and _E. verticillatum_ formed a dense biofilm. Scanning electron microscopy images of the biofilms showed a dense network of _M. israelensis_ cells (Figure 5A) and filaments of _Phormidium_ sp. (Figure 5B). The cells of _M. israelensis_ and _Phormidium_ sp.