# Diversity and Seasonal Dynamics of Airborne Fungi in Nerja Cave, Spain
## 2 Materials and Methods
### Aerobiology  
Two sampling campaigns were carried out on 9 July 2016 and 12 January 2017. These two campaigns corresponded to the stronger ventilation (winter, UAF-mode ventilation) and lower ventilation (summer, DAF-mode ventilation) periods, previously described by L\(\ddot{\text{i}}\)n\(\acute{\text{a}}\)n et al. [13]. In each sampling, a total of 10 control sites were selected inside and one outside the cave. The sites sampled were: Touristic Galleries (Entrance Hall, Nativity Hall, Cascade Hall, Ghosts Hall, Cataclysm Hall), High Galleries (Kitchen Hall, Heracles Hall, Immersity Hall), and New Galleries (Lance Hall and Mountain Hall), in addition to the exterior air (Figure 1).  
Samplings were performed with a Duo SAS (Surface Air System) Super 360 system (International pBL, Milan, Italy). This equipment is a type of suction impact collector that was widely used in aerobiological studies [8; 10]. It allows the detection of a great diversity of cultivable fungi by filtering a preselected volume of air through two heads provided with a series of orifices, which impact upon Petri dishes.  
At each sampling site, samples were taken in duplicate. The volume of filtered air was 100 L in 35 s. This volume was established because at higher volumes the number of spores was too high for an accurate count [8]. The culture medium used to promote fungal growth was dichloran rose Bengal-chloramphenicol-agar (DRBCA, Merck, Darmstadt, Germany).  
Figure 1: Cave map and location of the aerobiology sampling points and microclimatic stations. 1: Cave Exterior, 2: Entrance Hall, 3: Nativity Hall, 4: Cascade Hall, 5: Ghosts Hall, 6: Cataclysm Hall, 7: Kitchen Hall, 8: Heracles Hall, 9: Immensity Hall, 10: Lance Hall, 11: Mountain Hall. PC: Pintada Cave, GC: non accessible cave from surface, WS: weather station, E1: tourist entrance, E2 and E3: sinkholes. Location of PC, GC, and WS is approximate.  
The Petri dishes were incubated at 25 \({}^{\circ}\)C and after 5 days counted. After counting, the fungi were isolated, cultured and the DNA extracted.  
The methodology was thoroughly described by [PERSON] et al. [19] and [PERSON] et al. [30]. DNA was extracted from the mycelia of each fungal strain. Mycelia were scraped from the plates and transferred to a 1.5 mL Eppendorf tube containing 500 \(\upmu\)l lysing buffer and 200 \(\upmu\)l glass beads. The mixture was shaken in a cell disrupter (Fast Prep-24, Solon, OH, USA) at full speed for 3 min.  
Fungal internal transcribed spacer (ITS) regions, including ITS1, 5.8S rDNA and ITS2, were amplified using the primers ITS1 (5\({}^{\prime}\)-TCCGTAGGTGAAACCTGCGG-3\({}^{\prime}\)) and ITS4 (5\({}^{\prime}\)-TCCCTCCGTATTGTATATGC-3\({}^{\prime}\)). Amplification protocol comprised one cycle of denaturation (94 \({}^{\circ}\)C for 2 min), followed by 35 cycles of denaturation (94 \({}^{\circ}\)C for 1 min), and a terminal elongation step (72 \({}^{\circ}\)C for 5 min).  
PCR products were analyzed by Macrogen Inc. (Amsterdam, The Netherlands) and sequenced using the same primer set. For phylogenetic identification of fungal strains, the sequences were compared using BLASTn algorithm with nonredundant databases of sequences deposited at the National Center for Biotechnology Information ([[http://www.ncbi.nlm.nih.gov/](http://www.ncbi.nlm.nih.gov/)]([http://www.ncbi.nlm.nih.gov/](http://www.ncbi.nlm.nih.gov/))). The sequences are available from the GenBank database under accession numbers: MZ374012-MZ374055 and MZ375763-MZ375863.