A glimpse into darkness: Diversity of culturable cyanobacteria, green algae and fungi from subaerial cave biofilms

Caves and hypogean environments provide stable microclimates characterized by uniform temperatures, constant humidity, and low light levels. In such sites, extremely low light irradiance can support the growth of subaerial biofilms (SABs) dominated by unique photosynthetic communities of cyanobacteria accompanied by chlorophytes, heterotrophic bacteria, and fungi. This study aimed to apply a culture-dependent approach to uncover the diversity of cyanobacteria, green algae, and fungi from SABs of a cave in Northern Spain. We isolated a total of 58 cyanobacteria grouped into 21 genera based on their 16S rRNA gene sequences; 24 green algae grouped into 10 genera based on their SSU rRNA gene sequences, and 41 fungi fell into 13 genera according to phylogenies based on the ITS rRNA region between the 16S and 23S rRNA genes (ITS rRNA region). The SABs were dominated by cyanobacteria, which also reflected by high diversity, including calcium-carbonate sheath-producing species such as Geitleria calcarea and Scytonema julianum. Typical cave-inhabiting species such as Chalicogloea cavernicola, Timaviella karstica or Oculatella subterranea were also isolated, alongside potentially new genera and species. Associated green algae were predominantly located closer to the cave entrance and included various lineages of the genera Jenufa and Chromochloris. The fungal community directly derived from the SABs was dominated by decomposers, saprophytes, and phytoparasitic representatives such as Sporobolomyces, Stereum, and Phlebia, with a corresponding set of enzymes that were evaluated for all fungal isolates. The results strongly support the hypothesis that specialized cyanobacterial communities are often located in caves as a result of niche specialization.