Diversity and Seasonal Dynamics of Airborne Fungi in Nerja Cave, Spain

: Nerja Cave, Southern Spain, was revealed as an important biodiversity reservoir from which several novel species of Aspergillus were described. We carried out an aerobiological study in Nerja Cave to assess the origin of airborne fungi. This study quantiﬁed the fungi present in the air of ten representative halls covering the three sectors comprising the cave: Touristic Galleries, High Galleries, and New Galleries. Microclimatological monitoring allowed us to understand the dynamic of airborne fungi in two seasons of the year (winter and summer), corresponding to the strongest and the lowest cave ventilation, and to validate the inﬂuence that the transport of airborne fungi from outside may have on the cave itself. The data show that cold air enters in winter, as conﬁrmed by the abundant presence of Aspergillus and Penicillium spores inside and outside the cave. In summer, the abundance of some fungi in the air of Nerja Cave, which are not detected outside, indicates a stagnation or low ventilation, and therefore, the concentration of fungal spores is maxima. The high occurrence of Cladosporium outside the cave and the scarce abundance inside support the cave stagnation in this season.


Introduction
One of the most important topics in the conservation of caves with Paleolithic paintings is the assessment of the microbial communities inhabiting speleothems, rocks, water pools and air. A wide diversity of approaches on cave fungi can be found in the literature [1][2][3][4][5][6] and aerobiological studies were introduced as a management tool to control airborne fungi in caves with paintings [7][8][9][10].
The microbiology of Nerja Cave, Southern Spain, was studied regarding the impact of lighting on the development of phototrophic communities [20][21][22][23][24], but scarcely from an aerobiological point of view. A preliminary study carried out by Del Rosal et al. [25] was focused on the survey of pathogenic bacteria and the genus Aspergillus, very abundant in the cave air in summer. Further studies by Docampo et al. [26,27] using nonviable Hirst-type volumetric pollen traps located near the entrance and at the end of the visited area found that Aspergillus/Penicillium were the most abundant spore types, representing 50% of the total, followed by Cladosporium. However, the morphology of the spores, studied by microscopy, does not allow distinguishing between Aspergillus and Penicillium nor the identification of species.
The main objective of this aerobiological study was to assess the presence of airborne fungi in the cave to elucidate its origin. The identification of fungi was carried out by molecular methods. The study quantified the fungi present in the air from 10 representative halls covering the three sectors composing the cave: Touristic Galleries, High Galleries, and New Galleries. A sampling point outside the cave was used as a control of environmental fungi. This monitoring allowed to know the dynamic of airborne fungi in two seasons of the year (winter and summer), corresponding to the strongest and the lowest cave ventilation, and to validate the influence that the transport of airborne fungi from outside may have on the cave itself.

Site Description
Nerja Cave is one of the biggest and most important show caves in Spain, with a volume of about 300,000 m 3 , almost 5 km of passages, and more than 400,000 visitors annually. It is also one of the most significant prehistoric sites in Europe, with more than 500 artistic representations. About a third of the cave, the Touristic Galleries, is open to the public since 1960. The other two sections, named High Galleries and New Galleries, only can be accessed by researchers.
The climate of the area is coastal Mediterranean, with an average precipitation slightly lower than 500 mm yr −1 . The external air average temperature ranges from about 13 • C to 26 • C (January and August, respectively), with an average annual value of 18.8 • C [28].
The cave is developed within fissured and karstified Triassic dolomitic marbles more than 500 m thick at the unsaturated zone of the Sierra Almijara carbonate massif. A significant portion of the dolomitic marbles is devoid of vegetation. The soil cover is scarce, with a thickness lower than 0.15 m, and the natural vegetation consists mainly of shrubs and pine trees. However, in the areas nearest to the touristic entrance, there are several gardens with many ornamental plants [29], and thus, the vegetation cover is much more developed over the Touristic Galleries than over the nonvisited sector.
The cavity develops between 123 and 191 m a.s.l. According to the outside orography, rock thickness above the cave varies from 4 to 50 m in the area open to tourism, and exceeds 80 m in the nonvisited area. The cavity has three natural entrances: an entrance with a semicircular form and about 12 m 2 of surface (E1 in Figure 1), which is used for the access and exit of visitors and two subcircular sinkholes (E2 and E3 in Figure 1).
Nerja Cave is connected to at least two other nearby cavities, Pintada Cave (PC)-a small cavity with a natural entrance located to 250 m a.s.l.-and another great and nonaccessible cave from the surface (GC), found recently with indirect methods [13]. Although there is no speleological connection between these three caves, they are effectively linked for air circulation patterns from the surface.
Four ventilation regimes (winter, summer, spring, and autumn) and two ventilation modes with opposite airflows direction (named DAF-and UAF-modes) were defined in Nerja Cave karstic system during the annual cycle [13]. In all the cases, the natural ventilation of the system is determined by variations in the differences of density between the external and internal air.
The 222 Rn concentration of cave air was registered every 30 min using a Radim 5WP radon monitor (GT-Analytic KG). The detectable activity is between 80 Bq/m 3 and 150 KBq/m 3 (for one-hour measurements with a statistical error ± 20%) ( Table 1).
The environmental data of the external air were provided by the instrumental equipment placed in the weather station (WS in Figure 1) located a few meters from Nerja Cave entrances.

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 Liñán 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, Immensity 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 pBI, 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, Darm-
The 222 Rn concentration of cave air was registered every 30 min using a Radim 5WP radon monitor (GT-Analytic KG). The detectable activity is between 80 Bq/m 3 and 150 KBq/m 3 (for one-hour measurements with a statistical error ± 20%) ( Table 1).
The environmental data of the external air were provided by the instrumental equipment placed in the weather station (WS in Figure 1) located a few meters from Nerja Cave entrances.

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 Liñán 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, Immensity 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 pBI, 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, Darmstad, Germany).

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The Petri dishes were incubated at 25 • C and after 5 days counted. After counting, the fungi were isolated, cultured and the DNA extracted.
The methodology was thoroughly described by Sanchez-Moral et al. [19] and Dominguez-Moñino 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 µl lysing buffer and 200 µl 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 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ). Amplification protocol comprised one cycle of denaturation (94 • C for 2 min), followed by 35 cycles of denaturation (94 • C for 1 min), and a terminal elongation step (72 • 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/). The sequences are available from the GenBank database under accession numbers: MZ374012-MZ374055 and MZ375763-MZ375863. Table 1   Inside the cave, the air temperature in H-4 hall ranged between 18.15 and 20.08 • C (m= 19.21 • C) and the relative humidity ranged between 54% and 98% (m = 85%). The air CO 2 content ranged from 489 to 1483 ppmv, with a mean value of 797 ppmv.

Microclimatology
In the H-11 hall, the air temperature ranged between 19.75 and 19.92 • C (m = 19.86 • C) so the difference between the maximum and minimum temperatures was lower than 0.2 • C ( Table 1). The air CO 2 content ranged from 499 to 986 ppmv, with a mean value of 633 ppmv. The air relative humidity was 100% during all the studied period.
In H-8 intermediate values of air temperature (m = 19.40 • C) and relative humidity (m = 95%) were recorded. In this case, the air CO 2 content ranged from 512 to 1170 ppmv, with a mean value of 699 ppmv. Therefore: (1) the air of the tourist sector is colder, less wet, and has a higher CO 2 content than in that of the nonvisited galleries of Nerja Cave, (2) the temperature and relative humidity variability is noticeably reduced in the nonvisited sector, located below greater thickness of rock and at greater distances from the cave entrances (E1, E2 and E3 in Figure 1), and (3) the variation coefficients (v in Table 1) in the external media are higher than in the cave, except for air CO 2 . Figure 2 shows the temporal evolution of the main environmental parameters relative to the external air and cave air during 2016 and 2017. In summer, the temperature of the external air (T ext ) is higher than that of the cave air (T int ), and the latter is denser than the exterior (Figure 2A,B). CO 2 concentrations in the Touristic Galleries ( Figure 2C) reach maximum values, coinciding with a lower index of natural ventilation (daily average values of 222 Rn over the 400 Bq/m 3 , Figure 2A) and a greater number of visitors ( Figure 2B).
Air relative humidity is high in the cave, with values ranging between 95 and 100% ( Figure 2D). When the external temperature markedly exceeds the cave temperature (∆T > 5.0 • C) the DAF-mode ventilation is activated (yellow orange bars in Figure 2). The cave air, denser than the external air, is blown out by the lowest entrances, and atmospheric air is sucked in by the highest entrances. During the day corresponding to the summer campaign (9 July 2016), ∆T was 7.53 • C (daily average value).
In winter, the external air is generally colder and denser than the cave air, although exceptionally short-term periods exist in which T ext > T int (Figure 2 A,B). CO 2 concentrations inside the Touristic Galleries are minimal and much more similar to those of the external atmosphere ( Figure 2C), given the more effective natural ventilation (daily average values of 222 Rn of 80 Bq/m 3 ) and lower number of visitors.
Cave air relative humidity is minimum in the Touristic Galleries and shows the same temporal evolution than the external air ( Figure 2D). When T ext < T int , the external air, denser and colder than the internal air, easily enters Nerja Cave through the lowest entrances, moves through the Touristic Galleries, arrives at the High and New Galleries (the same day or 1-2 days later), and finally leaves the cave through Pintada Cave. During the day corresponding to the winter campaign (12 January 2017), the daily average value of ∆T was −5.30 • C. Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 19

Aerobiology
Two aerobiological samplings were performed to cover the aerodynamic periods in Nerja Cave (Figure 3): high ventilation, corresponding to the winter station (12 January 2017), and very low ventilation produced in the summer station (9 July 2016). 2017), and very low ventilation produced in the summer station (9 July 2016).
The concentration of fungal spores outside was relatively high in summer and near the half in winter. In general, the concentration of fungal spores in all the galleries was considerably lower in the summer period with respect to the higher ventilation period, as shown in Tables 2 and 3. Inside the cave, the fungal patterns were different depending on the galleries trail.
Two behaviors were distinguished: in the Touristic Galleries, fungal spore abundances increased between 2-and 5-times in winter, while the figures were greater (from 7-to 133-times in the High and New Galleries).
Remarkable were the low concentrations in the Lance Hall (Table 3), while in the Kitchen and Heracles halls, they were very high. Obviously these abundances should be related with definite sources and/or the aerodynamic of the halls. The lower ventilation period (summer) is characterized by the abundant presence of spores of three genera, Parengyodontium, Aspergillus, and Penicillium. Other less abundant genera were Cladosporium, Alternaria, Aureobasidium, Bipolaris, and Amphichorda (Table 2). In total, 16 different species were retrieved inside the cave and 13 outside.
Parengyodontium album was only limited to the visited Touristic Galleries: Entrance, Cascade, and Ghosts Halls, where reached abundances between 70 and 90%. However, Aspergillus versicolor, also abundant, was distributed across the three galleries: Touristic (Nativity and Ghosts Halls), High (Kitchen and Immensity Halls), and New (Mountain Hall).
Other Aspergillus species was A. spelaeus, only identified in the Kitchen Hall. Six species of Penicillium were collected in the Touristic and High Galleries, from which the abundances of P. flavigenum, P. sumatrense, P. pancosmium, and P. nodositatum were above 10% in any of the halls and those of P. citreosulfuratum and P. steckii below 10%. Interestingly, P. flavigenum was the unique fungus in Heracles Hall and A. versicolor in Mountain Hall.
Outside, none of the most abundant fungi inside the cave could be identified, and only Cladosporium cladosporioides (43.06% outside and 5.0% inside) and Aureobasidium The concentration of fungal spores outside was relatively high in summer and near the half in winter. In general, the concentration of fungal spores in all the galleries was considerably lower in the summer period with respect to the higher ventilation period, as shown in Tables 2 and 3. Inside the cave, the fungal patterns were different depending on the galleries trail.
Two behaviors were distinguished: in the Touristic Galleries, fungal spore abundances increased between 2-and 5-times in winter, while the figures were greater (from 7-to 133-times in the High and New Galleries).
Remarkable were the low concentrations in the Lance Hall (Table 3), while in the Kitchen and Heracles halls, they were very high. Obviously these abundances should be related with definite sources and/or the aerodynamic of the halls.

Summer Campaign, 9 July 2016
The lower ventilation period (summer) is characterized by the abundant presence of spores of three genera, Parengyodontium, Aspergillus, and Penicillium. Other less abundant genera were Cladosporium, Alternaria, Aureobasidium, Bipolaris, and Amphichorda (Table 2). In total, 16 different species were retrieved inside the cave and 13 outside.
Parengyodontium album was only limited to the visited Touristic Galleries: Entrance, Cascade, and Ghosts Halls, where reached abundances between 70 and 90%. However, Aspergillus versicolor, also abundant, was distributed across the three galleries: Touristic (Nativity and Ghosts Halls), High (Kitchen and Immensity Halls), and New (Mountain Hall).
Other Aspergillus species was A. spelaeus, only identified in the Kitchen Hall. Six species of Penicillium were collected in the Touristic and High Galleries, from which the abundances of P. flavigenum, P. sumatrense, P. pancosmium, and P. nodositatum were above 10% in any of the halls and those of P. citreosulfuratum and P. steckii below 10%. Interestingly, P. flavigenum was the unique fungus in Heracles Hall and A. versicolor in Mountain Hall.
Outside, none of the most abundant fungi inside the cave could be identified, and only Cladosporium cladosporioides (43.06% outside and 5.0% inside) and Aureobasidium pullulans (5.56% outside and 5.0%) were represented in the Ghosts Hall, and Alternaria tenuissima in the Nativity Hall (6.67%).

Winter Campaign, 12 January 2017
The higher ventilation period (winter) showed a great number of different species inside the cave (25), but less diversity outside (8) ( Table 3). Seventeen out of 25 species attained abundance above 10% in any of the halls ( Table 3). The sampling was dominated by species of Penicillium (9), Aspergillus (9), and Cladosporium (2). Single species of Arthrinium, Aureobasidium, Botrytis, Cutaneotrichosporon, Scopulariopsis, and Talaromyces were also identified. The major diversity of fungal species was found in the halls of the Touristic Galleries (7 to 10 species), middle in the New Galleries (4 to 6 species), and low in the High Galleries (3 and 4 species).
Two fungi, A. spelaeus and P. flavigenum were isolated from eight halls, A. ustus from four, and Aspergillus pulvericola and P. glabrum from 3 out of 10 halls. Aspergillus was not found in Cascade Hall, nor Penicillium in Lance Hall.
Two halls, Kitchen and Heracles, showed huge spore concentrations (2170 and 1330 CFU m −3 , respectively). P. flavigenum and A. spelaeus dominated in both cases, in addition to A. baeticus in Kitchen and A. ustus in Heracles halls.
In the sampling outside the cave, 6 out of 8 fungal species were also retrieved inside, and only Alternaria tenuissima and Monocillium indicum were not present in the cave air. However, A. tenuissima was also recovered inside and outside the cave in summer.

Discussion
The microclimatological study in Nerja Cave shows two well defined ventilation patterns corresponding to summer (low ventilation) and winter (high ventilation). The type of ventilation regime is closely related with the concentration of airborne fungi in the cave. In fact, the higher number of spores was observed in summer due to air stagnation or low ventilation, whereas in winter, ventilation helped to remove and/or dilute spore concentrations. Similar phenomena were described in other caves [7,19]. Figure 3 shows the distribution of the most abundant fungi across the different galleries and seasons. Several patterns can be observed, namely the practical absence of Basidiomycota. This pattern is opposite to that found in caves from Northern Spain, where the input of wood-rotting Basiomycota spores related to Quercus forests predominates in the ventilation period (winter) [18]. In Nerja Cave's surroundings, forests and dead trees are not prevalent.
Another well-defined pattern is the higher diversity of Ascomycota in the air in winter with relatively medium spore abundances, whereas in the summer period, the abundances of a few Ascomycota (Aspergillus versicolor, Parengyodontium album, Penicillium flavigenum) were remarkable, but they were not retrieved outside the cave.
The presence of Ascomycota in the two seasons, irrespective of the high or low ventilation period, could be associated with the phototrophic biofilms. Dominguez-Moñino et al. [30] related the abundance of Parengyodontium, Aspergillus, and Penicillium in the air of Tesoro Cave (Rincon de la Victoria, Malaga) at some 40 km from Nerja, with the abundant biofilms of cyanobacteria and algae originated by the artificial lighting used for allowing the visits. Nerja Cave houses also abundant phototrophic biofilms near the entrance, just in the Touristic Galleries. A similar association between phototrophs and Ascomycota was reported by other authors [31,32].
The senescence and lysis of old phototrophic biofilms can provide nutrients for the fungi and lead to the abundance of airborne spores in the halls close to the cave entrance. Jurado et al. [24] reported complex trophic interactions in Nerja Cave between the components of the biofilms, including processes of predation of bacteria and amoebas, and the occurrence of nematodes, fungi, and other Eukaryota.
The higher diversity and relative abundance of Ascomycota spores in winter are consistent with the entry of air according to the ventilation mode of Nerja Cave during this period of the annual cycle. During the winter campaign, the difference ∆T is negative. The colder and denser external air enters Nerja Cave through the lower entrances (E1, E2 and E3 in Figure 1) and moves from the tourist to nontourist sector (UAF-mode). So, the strong ventilation during this period contributes to the entry of atmospheric air inside the cave, to its rapid exit through the Pintada Cave, and thus, to the rapid renovation of the air cave. This is exemplified by the abundant occurrence of Cladosporium cladosporioides, Aspergillus spelaeus, and Penicillium flavigenum in the halls of the Touristic Galleries and outside.
Parengyodontium album (syn. Engyodontium album, Beauveria alba, Tritirachium album) [33], identified only in summer, was limited to the air of the visited area (Touristic Galleries), which could correspond to the presence of insects in spring and summer, since the fungus is a parasite of arthropods, and also to the presence of phototrophic biofilms in these galleries. Novakova et al. [34] found this species only in the air of the cave, but not in the sediments.
Jurado et al. [35] and Leplat et al. [36] reviewed the entomopathogenic fungi related to cultural heritage sites and associated Pa. album with the presence of arthropods. This was also reported in Lascaux Cave [37]. Data on the role of fungi in caves suggested that the species related to arthropods contribute to their mortality and favor the dispersal of spores. Jurado et al. [35] recommended the control of arthropod populations as a method to reduce fungal contamination and colonization of substrates, sediments, and rocks [35].
The spores of Pa. album were also widely distributed in the air of Castañar de Ibor, Ardales, Gruta de las Maravillas, and Tesoro caves [30,38,39]. In addition, Pa. album is common in other subterranean environments [40][41][42][43] and wall paintings, and in this last case associated to arthropods [44][45][46]. A review on cave fauna reported the presence of many different classes of arthropods in Nerja, including endemisms [47]. The abundant presence of spores of Pa. album and other entomopathogenic fungi in the air in Ardales and Tesoro caves [30] emphasizes the likely role of arthropods as a vector of these fungi.
The genus Aspergillus is one of the most abundant in the air of Nerja Cave. Aspergillus showed greater diversity in winter, with the species A. versicolor, A. spelaeus, A. baeticus, A. ustus, A. pulvericola, A. welwitschiae, A. oryzae, and A. ochraceus, while in summer were only recovered A. versicolor and A. spelaeus.
The abundance of Aspergillus was shown in previous publications, although the species could not be identified due to the protocol used [26,27]. Novakova et al. [32] isolated 72 fungi from sediments and air in Nerja, among them 18 different species of Aspergillus, from which two were only present in the air and three in both sediments and air. This suggests that an important source of Ascomycota in the cave, the sediments, could not be disregarded. We isolated seven species of Aspergillus from the cave air, and the most abundant species present in the two seasonal samplings were A. versicolor, A. spelaeus, A. ustus, and A. baeticus. The first two showed a marked seasonality, so while A. versicolor was abundant in summer and scarce in winter, A. spelaeus showed the inverse trend, being highly represented in almost all the halls in winter and occasionally in summer (8.7% in Kitchen Hall).
Aspergillus versicolor, relatively abundant outside in winter, was only found in the deepest Mountain Hall. Thus, according to the ventilation mode of the winter period (UAF-mode), their relationship with the external atmosphere is discarded. On the contrary, A. versicolor is not detected outside the cave in summer, but its concentration is high inside the cave. The highest frequency of A. versicolor in summer, unlike other species of the genus Aspergillus, points to a relationship with the increasing of nutrient sources, i.e., phototrophic biofilms or bat droppings, and could be explained by the ventilation mode of Nerja Cave during this period of the year (DAF-mode). The colder and denser Nerja Cave air is removed through the lower entrances (E1, E2 and E3 in Figure 1) and external air is sucked in through the Pintada Cave and other nonaccessible entrances located in the Almijara range. This suction effect causes the air previously stored in the PC and the GC to reach Nerja Cave. Thus, the source of these fungi would not be the external atmosphere but the cave air previously stored in PC, GC, and other possible subterranean voids connected to Nerja Cave and located in higher altitude.
This also applies to A. spelaeus, found in 2011 with some frequency in the sediments of Nerja Cave. The study of the isolated strains established the bases for the description of these strains as a new species [57]. A subsequent visit in 2012 allowed the isolation of new strains [32,57]. As far as we know, no other records on this Aspergillus were published.
Another new species of Aspergillus isolated in Nerja was A. baeticus. This was described in 2012 [58], and the type strain was isolated from sediments from Gruta de las Maravillas (Aracena, Huelva) along with other strains from Tesoro Cave. In addition, this fungus was isolated from air, cave sediments, and a dead spider (Agraecina cristiani) in Movile Cave, as well as from a bat cadaver in Demänovska Peace Cave [59], and from cave sediments in Nerja Cave [32].
The new species Aspergillus europaeus was isolated in 2011 from soils near Nerja and Movile caves, although no isolates were obtained directly from the caves [57]. We isolated this fungus in the air of the Entrance Hall in winter, suggesting an origin from the soil outside the cave and their transport inside, according to the UAF-mode ventilation [28].
Less abundant Aspergillus species identified in the air of Nerja Cave were also recorded in different caves such as A. ochraceus [55,[60][61][62]. No cave record was found for A. pulvericola and A. welwitschiae.
The genus Penicillium is widely distributed in caves, and in Nerja, it was recorded in the two seasonal samplings in almost all the halls; although generally with little abundance in summer, not so in winter. In summer, six species of Penicillium were recorded in all the halls except in the Lance, while in winter, a total of 10 species were recovered in all the halls but not in the Lance. This is the same trend recorded for Aspergillus; very abundant in winter, and scarce in summer.
The most abundant Penicillium species was P. flavigenum, both in summer and winter, although with marked differences in their total abundances and presence in the halls (Tables 2 and 3), and P. glabrum, which was only identified in winter. P. flavigenum and P. glabrum are relatively common in caves [8,49,50,55,[63][64][65][66]. Madsen et al. [67] found that P. glabrum was among the three most frequent species of fungi present inside Danish houses.
The presence of spores of Penicillium, in autumn and winter, in the air of Tesoro Cave, was important [30], as it suggests that this genus is abundant in the air of the caves of Malaga province in periods when ventilation is greater.
Talaromyces brunneus was isolated in winter from Ghosts and Cataclysm halls. The basionym is Penicillium brunneum. The references to this fungus in the literature are scarce, only to the original isolation from miller rice from Thailand [68], the production of anthraquinones [69] and the cellulolytic activity of a strain isolated from a cave [70].
In Nerja Cave, the spores of the genus Cladosporium were detected in the two seasonal samplings, both outside and inside the cave. In summer, C. cladosporioides accounted for 43.1% of all spores outdoor, but only 5.3% in the Ghosts Hall; C. lebrasiae was only found in the Entrance Hall, with 6.5% abundance, denoting clearly the existence of a low ventilation period. However, in winter, the situation changed drastically, since the concentration of C. cladosporioides spores outside was 30.2%, but its abundance increased in the different halls, such as the Entrance Hall (33.3%) and in Nativity Hall (42.2%), as correspondent with a higher ventilation period. In addition, C. sphaerospermum was represented in Nativity (4.6%), Ghosts (5.3%), Cataclysm (12.1%), Immensity (3.7%), and Mountain (1.9%) halls.
Garcia-Anton et al. [17] (2014) proved that in Altamira Cave Cladosporium spores had a clear external origin, as the data obtained for Nerja Cave also suggest that the presence of these fungi in the cave was due to transport from outside through air currents. It also stands out for its abundance in the air of Nerja Cave when the ventilation in the cave is greater (winter) compared to its practical absence in summer, with less or little ventilation. Likewise abundant were the species of the genus Cladosporium in the seasonal samplings (autumn and winter) in Ardales Cave [30].
Other remarkable fungal species were Aureobasidium pullulans, Botrytis cinerea, Arthrinium arundinis, Alternaria tenuissima, Bipolaris cynodontis, all plant pathogens, but also isolated from caves [5,32,50,61,62,74,75]. The origin inside the cave may be due to the vegetation around the cavity. In addition, other noticeable fungi were Amphichorda felina, Cutaneotrichosporon mucoides, and Scopulariopsis brevicaulis. Amphichorda is one of the most ubiquitous entomopathogenic fungal genera. The species A. felina (syn. Beauveria felina, Isaria felina) appeared only in the Kitchen Hall with 8.7% abundance, but it deserves to be noticed for their ecology, since is a fungus colonizing animal feces in Spanish caves [76] and isolated from other caves [77,78]. Scopulariopsis brevicaulis is often recorded in indoor environments [79] and isolated from bat skin [80] as well as Cutaneotrichosporon mucoides (syn. Trichosporon mucoides), the later common in caves populated by bats [81,82]. Scopulariopsis brevicaulis was previously isolated from the air and sediments of Nerja Cave [32].
The abundance of Cladosporium cladosporioides spores in the air outside the cave is notable. Species of the genus Cladosporium are very common outdoor, at any time of the year, and also inside buildings, along with Alternaria, Aspergillus, and Penicillium. Outdoor, according to Gómez de Ana et al. [83], (2006), Cladosporium and Penicillium are very common in the four seasons of the year, but Penicillium was more abundant in winter and Aspergillus in summer. In Nerja Cave, both Penicillium and Aspergillus showed outdoor higher abundances in winter than in summer, as well as a high diversity in the cave air. However, A. versicolor stood out for their abundance in the cave in summer. This showed the influence that the outside air can have on the dispersal of the spores of these fungi inside the cave in winter and the influence of the air previously stored in other caves connected to the ventilation of Nerja Cave in summer.

Conclusions
The studies in Nerja and other Andalusian caves were essential for the description of new species of the genus Aspergillus [57,58], and specifically for A. spelaeus, which type strain was isolated from Nerja [94]. This suggests that these caves are reservoirs of new species of fungi as well as bacteria [95,96], and their study contributes to increasing the knowledge of biodiversity on the planet.
Considering the results, the influence that the external air has on Nerja Cave during the winter is demonstrated. Cold air enters in winter, and this is confirmed by the abundant presence of Aspergillus and Penicillium spores inside the cave, without ruling out that the reservoirs of these fungi were already installed in the cave itself (sediments, phototrophic biofilms) and that the increased air flow carries away the spores. In summer, the abundance of fungal spores in Nerja Cave, which are not detected outside, as in the case of A. versicolor, suggests a different source for these microorganisms, linked to the nutrient sources and other subterranean voids and caves near Nerja Cave. Other seasonal events, such as the presence of entomopathogenic fungal spores in summer, were also reflected in the air of the cave.
The data reported in this work are in agreement with the findings of Docampo et al. [26,27]. These authors reported that spores of Aspergillus and Penicillium were, by far, the most abundant in the air of the cave and were retrieved throughout the year, followed by Cladosporium. The influence of the ventilation is clearly shown by the high abundances of Cladosporium spores outside the cave both in summer and winter. The high occurrence in the halls near the entrance in winter is related to ventilation, and the practical absence inside the cave in summer is due to low ventilation or stagnation period.
The conditions of access and visit to a cave have an impact due to the mobilization of air and dispersal of aerial microorganisms. However, this is minimized in the summer season, when there is a greater influx of visitors, as shown by the low concentration of spores found inside. However, the high abundance of Aspergillus and Penicillium spores in the Kitchen and Heracles halls in winter requires a more detailed study to understand their origin and nature and to be able to reduce the concentrations of spores, which are much larger than in other neighboring halls (Cataclysm and Immensity).