## 3 Results
### Seasonal Changes of Microbial Biodiversity  
#### 3.3.1 Alpha Diversity of Bacterial and Fungal Communities  
To determine significant changes in microbial diversity associated with subsequent seasons a high-throughput sequencing (Illumina technology) of PCR amplicons (of marker  
Figure 5: Concentrations in ppm of selected chemical compounds in the air in the Northern Pergola at the Museum of King John III’s Palace at Wilanow. The data represent averages with standard deviation. * -Indicates significant differences according to the Student’s _t_-test (\(p\leq 0.05\)) when comparing the concentration of selected chemical compounds between winter (blue bars) and summer (yellow bars).  
genes, i.e., regions V3/V4 of 16S rRNA genes for bacteria and ITS1 region for fungi) was applied. Analysis of preliminary reads showed a high diversity of the bacterial communities, with a total of 1,551,839 reads belonging to 15,310 amplicon sequence variants (ASVs), and 697,259 sequences finally analyzed. The number of reads for fungal biodiversity analysis was higher-4,389,046 reads with 2,640,212 finally analyzed sequences that clustered into 1706 ASVs. The numbers of sequences excluded from the samples are shown in Supplementary Table S1. DNA sequencing data of analyzed samples revealed the presence of 177 different bacterial genera (without uncultured or unidentified genera), belonging to 32 different classes, and 118 fungal genera (excluding unidentified genera), belonging to 29 classes (Supplementary Figure S2). Furthermore, the percentage of identified bacterial taxa was higher compared to fungal taxa, which is associated with a still intensively verified and supplemented classification within the fungi kingdom [36; 37].  
We measured alpha diversity using the Chao1, Shannon, and Simpson indices to evaluate differences between the microbial richness and biodiversity of the samples collected in winter and summer seasons (Figure 6). The Chao1 index, used to calculate the microbiological richness, ranged from 100 to 1000 in summer and from 100 to 1600 in winter for bacterial amplicons, and from 80 to 400 in summer and from 120 to 820 in winter for fungal amplicons. Our results also showed that the bacterial communities are more diverse than the fungal consortia (higher Shannon and Simpson indices for bacterial amplicons). In addition, both the diversity and richness of bacteria and fungi show a greater range of indices values for samples taken in winter compared to those analyzed in summer.  
The comparison of alpha diversity in samples collected in summer and winter in a pairwise manner allowed to identify microbiology stable biocenoses. In case of bacterial communities (Figure 6a), these seem to be stable in sites 5 and 8 (with lichen crusts). Moreover, these stable biocenoses had also the highest values of analyzed indices in both seasons. Our results showed that other biocenoses were affected by seasons. Sites  
Figure 6: Alpha diversity of bacterial (**a**) and fungal (**b**) communities. For all analyzed samples, Chao1, Shannon and Simpson indices were calculated. The samples were colored and connected by sampling site, as well as grouped by season as indicated in legend.  
2, 4, and 7 had lower bacterial community richness and diversity in winter comparing with summer, while the opposite effect was observed in the site no 1. Higher bacterial community richness in winter and similar diversity in both seasons was observed in site no 3, while in site no 6 with green biofilms, the richness (Chao1 indices) was stable and diversity (Simpson indices) decreased in winter.  
In the case of fungi, richness (Chao1 indices) increased during winter in all sites except no 6 (Figure 6b). Despite a decrease in richness in that site in winter, as well as the increase of richness in sites no 1 and 8, the mycological diversity (Shannon and Simpson indices) in these samples was stable. The evenness (Simpson indices) of fungal communities in sites 3, 4, and 5 increased in winter, while the opposite effect was observed in sites 2 and 7.