Microbial diversity and proxy species for human impact in Italian karst caves

Microbial community composition in cave sediments

The ITS1 dataset generated 5,793,980 raw sequence reads, resulting in 5,458,895 gene quality-filtered reads, ranging from 1252 up to 540,803 per sample. After singletons and rare taxa (< 5 reads) removal (1108 out of 10,595 ASVs total), a total of 9176 high-quality ASVs were obtained (Table S1). A total of 5,453,881 raw reads were generated from 16S rDNA dataset and accounted for a total of 4,806,902, which were grouped into 31,878 ASVs (out of a total of 65,037 ASVs) after quality filtering, with sequencing depths between samples ranging from 2066 to 265,442 reads .

Subsequently, the total 16S dataset was splitted by grouping the bacterial (31,015 ASVs) and archaeal (863 ASVs) ASVs separately for the downstream analyses (Table S2; Table S3). The 18S dataset was processed through a specific evaluation, extracting and analyzing only the sequences related to the microalgal component. A total of 97 ASVs were mapped for a total of 10,084 quality filtered reads, spread over 4 different groups (Alveolata, Chloroplastida, Chromista and Stramenopiles), using the Class taxonomic rank as the threshold for taxonomic identification as a consequence of the weak accuracy in the assignment towards higher ranks (Table S4).

Considering the total composition of microbial communities in cave sediments as a unique dataset (Fig. 1A), fungi represented the most abundant group (51.4%), followed by bacteria (46.6%), while archaea (1.8%) and micro-algae (0.2%) constituted minor fractions.

When analyzing the assemblage composition of each cave individually (Fig. 1B), Bossea cave was characterized by an almost equal presence of fungi (50.22%) and bacteria (49.16%). Caudano and Vento caves were dominated by fungi (57.17% and 61.46%, respectively), while the bacterial community accounted for 42.37 and 37.22% of the total assemblage, respectively. Conversely, Costacalda and the Pertosa-Auletta caves showed a higher bacterial presence (66.66% and 52.70%, respectively) compared to fungi (32.05 and 41.48%, respectively). Concerning the archaea domain, Pertosa-Auletta cave was the most representative with 5.74% of the total microbial assemblage, followed by Costacalda (1.27%), Vento (0.85%), Bossea (0.52%) and Caudano (0.41%) caves. The algal component was scarcely represented along all caves, with a relative abundance ranging from 0.47 for Vento cave to 0.01% for Costacalda cave.

Fungal community composition

In all caves, the dominant phylum was Ascomycota (Fig. 2A) (ranging from 60% in Costacalda to 76.25% in Pertosa Auletta), followed by Basidiomycota and Mortierellomycota. A broad different distribution of fungal taxa pattern emerged between the four show -caves and the wild cave (Fig. 2A,B,C,D). Notably, the genus Mortierella (Fig. 2D) was particularly abundant in the Costacalda wild cave (up to 31.8%), while in the show- caves did not exceed 10% (from 2.31% in Caudano up to 9.30% in Vento). The opposite was observed for the genus Candida (class Saccharomycetes), which was more abundant in the tourist caves (ranging from 13.36% in Caudano to 37.32% in Vento). Also, the genus Dipodascus was mainly present in the anthropized settings, even if at much lower extent compared to Candida, ranging from 0.14% in Pertosa-Auletta to 4% in Bossea. The genus Archaeorhizomyces (class Archaeorhizomycetes) was reported in show- caves only, albeit with scarce relative abundance (e.g. 0.01% Bossea up to 2.70% Vento). A number of cave-specific genera in the class Sordariomycetes have been recorded: for instance, Chaetomium (7.50%) in Caudano, Sarocladium (6.4%) in Pertosa-Auletta, Cephalotrichum (6.30%) and Rodentomyces (5.60%) in Costacalda ; differently, the genus Humicola (average abundance 1.39%) was found in all caves (Fig. 2). The class Eurotiomycetes was mainly represented by the genera Aspergillus (ranging from 0.01% in Costacalda to 6.77% in Pertosa-Auletta) and Penicillium (ranging from 0.17% in Vento to 6.45% in Pertosa-Auletta), while the genus Gymnoascus was most abundant in Costacalda and Bossea caves, accounting for 3.72% and 1.50% respectively. Likewise, the genera Tetracladium and Pseudogymnoascus (not shown in the Top 15) were the dominant members of Leotiomycetes throughout the dataset, up to1.96% and 1.12% in Costacalda , respectively (Fig. 2D). Agaricomycetes class was particularly abundant in show-caves (Caudano 12.6%, Pertosa-Auletta 8.25%, Bossea 4.44%, Vento 4.30%, Costacalda 1.2%), with the genus Bjerkandera being the only one reported among the top 15 (Fig. 2D). Yet, despite a considerable proportion of classes Tremellomycetes (ranging from 1.41% at Pertosa-Auletta to 12.7% at Bossea) and Dothideomycetes (ranging from 1.64% at Pertosa-Auletta to 7.50% at Caudano) among caves, they were mainly represented by two genera, i.e. Apiotrichum (Trichosporonaceae) and Phoma (Didymellaceae). Apiotrichum was spotted across all sites investigated, while Phoma appeared more prevalent among tourist caves (Vento: 3.45%, Caudano: 1.51%, Bossea: 1.10%, Pertosa-Auletta: 0.07%, Costacalda: 0.004%). Some other fungal classes showed different abundance patterns between tourist and wild cave, such as: Malasseziomycetes, (over 1% in show-caves vs. 0.15% of Costacalda), Microbotryomycetes (Bossea: 0.93%, Vento: 0.3%, Caudano: 0.16%, Pertosa-Auletta: 0.13%, vs. Costacalda: < 0.001%) and Geminibasidiomycetes (Costacalda: 1.52%, vs. all tourist caves: < 0.2%). Finally, lichenized fungi class of Lecanoromycetes was especially reported in Bossea (0.85%) and Vento (0.52%) caves.

Bacterial community composition

A different bacterial distribution was found in each cave (Fig. 3A–D). For instance, the phylum Pseudomonadota dominated throughout the dataset, with remarkable presence of its main classes (Gamma, Alpha, Beta and Delta-Proteobacteria) and by different genera reported in the top 15 (Fig. 3). The class Gamma-Proteobacteria was mainly represented by the genus Pseudomonas, particularly abundant in tourist caves (ranging from 11.1% in Bossea to 17.78% in Pertosa-Auletta, vs. Costacalda 2.95%). Conversely, the genera Sphingomonas (class Alpha-Proteobacteria, 2.46%), Lysobacter (class Gamma-Proteobacteria, 2.34%) and Polaromonas (class Beta-Proteobacteria, 1.8%) were prevalent in the pristine site, while Hyphomicrobium genus (class Alpha-Proteobacteria) was largely spread across all habitats (average of 0.6%). Three Acidobacteria sub-groups, i.e. Gp6 (mean 6.0%; highest in Bossea: 8.2%), Gp17 (mean 2. 0%), Gp16 (mean 1.74%), Gp4 (mean 1.35%) and the two main Actinobacteriota genera, i.e. Gaiella (with 0.78% in Pertosa-Auletta up to 4.51% in Vento caves) and Arthrobacter (ranging from 0.24% in Vento up to 2% in Pertosa-Auletta caves) showed a broad distribution across all caves. Yet, among Actinobacteriota, the genus Bifidobacterium was recorded only in show-caves (between 0.3% in Caudano and 2.5% in Pertosa-Bossea caves). However, similar abundance trends across all surveyed sites were also reported for the family Planctomycetaceae (ranging from 2.7% in Vento up to 4.27% in Bossea caves) and the genus Nitrospira (between 0.7% for Caudano and 1.75% of Costacalda caves). Other remarkable compositional spectra were represented by Bacillota with classes Clostridia (ranging from 0.48% in Caudano up to 1.62% in Bossea, vs. 0.07% in Costacalda) and Bacilli (from 3.15% in Caudano up to 7.66% in Pertosa-Auletta and 1.81% for Costacalda caves) with genus Lactobacillus (1.64% mean in show-caves, vs. 0.007% in Costacalda) all typically found in anthropized settings. Conversely, the Flavobacterium genus (Phylum Bacteroidota, 2.47% in Costacalda) was more than 8 times higher in the natural one than in show -caves.

Archaeal community composition

Archaea showed 2 main compositional trends describing the archaeome throughout the investigated caves (Fig. 4A,B,C,D). The first group is recurrent in all investigated caves and includes Thaumarchaeota and Euryarchaeota as the most abundant phyla, with the genera Nitrososphaera (ranging from 21.38% in Bossea to 41.35% in Pertosa-Auletta caves), Nitrosopumilus (between 14.23% and 41.76% in Bossea e Pertosa-Auletta) and Methanomassiliicoccus (ranging from 3% in Pertosa-Auletta to 21.35% in Costacalda). The same distribution across all caves was observed for taxa in the phylum Woesearchaeota, even if far less represented. The second group included taxa more frequent in show -caves as for the genera Acidianus (Phylum Chrenarcheota, class Thermoprotei, ranging from 2.62% Bossea to 11.34% in Pertosa-Auletta), Ignicoccus (average in show-caves 2.04%) and Thermocladium (4.14% highest value in Pertosa-Auletta); the genera Methanothermobacter and Methanobacterium (Phylum Euryarcheota, class Methanobacteria) were also found mainly in show -caves, the first with an average of 2.33% and the second with a frequency ranging between 0.55% and 3.9%. Other less abundant genera such as Methanosphaera and Thermococcus could be included in this second group.

Micro-algal community composition

Algae is by definition a polyphyletic group; therefore, the micro-algal assemblage (Fig. 5A,B) was derived by clustering members belonging to different kingdoms in order to investigate the micro-photosynthetic component. The compositional scenario was characterized by the Class Chrysophyceae (phylum Ochrophyta) dominating in most of the caves, in particular in Costacalda (81.5%) and Pertosa-Auletta (39.76%) caves. Vento cave was dominated by Cryptophyceae (35.3%) members of the phylum Cryptophyta. Thereafter, community composition became patchy as no other widespread classes were found across the dataset. For instance, Dinophyceae showed a high abundance in the Caudano cave (25.86%), but it dropped in Vento (12.1%), Pertosa-Auletta and Costacalda (11.57%) caves; on the other hand, Trebouxiophyceae showed a decreasing trend along Caudano (20.10%) del Vento (17.37%), Bossea (11.47%) and Pertosa-Auletta (7.25%) caves. The class Diatomea was particularly present in Bossea cave instead (24.48%).

Shared and unique taxa

Venn diagrams (Fig. 6) displayed the ASVs that typically characterized each cave (Unique ASVs) and those that are representative of the community core, shared throughout the dataset. Regarding the fungal community, the core was scarcely populated (200 unique ASVs), accounting for only 2.2% of the total composition. The ASVs shared between all caves were represented by 54 genera; in particular Mortierella (13%) and Candida (10%). ollowed by Penicillium (5.4%), Humicola (3.8%), Cephalotrichum (3.1%), Geomyces (2.3%), Pseudogymnoascus (1.5%) and Aspergillus (1.5%) genera commonly found in cave environments. When considering the unique ASVs, Bossea cave had the highest number of unique ASVs (1281) representing 14% of the total composition, spread over 178 fungal genera. The other caves, showed a conspicuous number of unique ASVs, in particular the Caudano cave (1144 ASVs, 12.5% of the total), displayed the highest number of genera (192) identified among the typical ASVs, followed by Vento cave showed 1,124 unique ASVs (12.2%) and Pertosa-Auletta cave characterized by 999 unique ASVs (10.9%) with 171 identified genera. The wild Costacalda cave counted 102 unique ASVs (1.1%), with only 30 genera among the site-specific ASVs reported.

The bacterial community (Fig. 6) gravitated around a robust core of 4,674 ASVs (15.1%) and 283 genera, among which Acidobacteria Gp6 (15.8%), Gaiella (6%) and Nitrospira (1.9%) were the most abundant. However, the entire cluster of subgroups-Acidobacteria accounted for 37% of the shared ASVs. On the other hand, there were few unique ASVs for each cave, with Pertosa-Auletta cave showing 2,292 ASVs (7.4%), followed by Bossea cave (1,870 ASVs, 6%), Vento cave (1,260 ASVs, 4.1%), Caudano cave (742 ASVs, 2.4%) and Costacalda cave (262 ASVs, 0.8%). Moreover, some peculiar genera were uniquely reported for the different caves, such as Legionella (3.3%) in Bossea, Gemmatimonas (4.5%) in Caudano, Lactobacillus (9.5%) Desulfomicrobium (3.8%) and Clostridium sensu stricto (3.6%) in Pertosa-Auletta.

The distribution of ASVs belonging to the archaeal component among caves (Fig. 6) was sharply defined. Indeed, while the core group (79 ASVs, 9.1%) and unique ASVs of Bossea (20 ASVs, 2.3%), Vento (15 ASVs, 1.7%), Costacalda (9 ASVs, 1%) and Caudano (3 ASVs, 0.3%) caves were represented by a low number of ASVs, the Pertosa-Auletta cave was characterized by 355 unique ASVs (41%), taxonomically labeled by Acidianus (33%) and Thermoplasma (14%) genera. However, the genera of archaeal methanogens constituted a solid cluster either across the common core (24.3%) and unique ASVs for each cave.

The distribution of taxa detected for the phototrophic assemblage (Fig. 6) was extremely polarized between caves. One pole was represented by Costacalda Cave for which no unique ASVs were recorded. The other one was the cave of Pertosa-Auletta which accounted for 28 unique ASVs (28.9%), mostly belonging to the Chlorophyceae (39%), Chrysophyceae (32%) and Diatomea (18%) classes.

Human related microbial taxa

Due to the high abundance of some common human-related microbial taxa found in the show -caves investigated (i.e. Malasseziomycetes class and Candida genus for fungi, Lactobacillus and Bifidobacterium genera for bacteria), we decided to explore the occurrence of less abundant guilds too as potential proxies for direct human impact.

Throughout the fungal dataset (Fig. 7A) tourist caves were especially characterized by an higher significant presence of dermatophytes with the genus Trichosporon (Bossea 0.26%, Vento 0.084%, Caudano 0.0054% and Pertosa-Auletta 0.0001%, vs. 0% in Costacalda; p < 0.05) and Cutaneotrichosporon on average 0.05% for the show-caves, vs. 0% in Costacalda (p < 0.05). The genus Arthroderma, here recorded in all caves analyzed, is instead a geophilic dermatophyte mostly occurring in soil, but also frequent in cave environments, and it is rarely associated with human and animal cutaneous infections¹⁹. Moreover, Malassezia spp., here mainly found in human impacted caves (Bossea 0.07%, Caudano 1.02%, Pertosa-Auletta 1.10% and Vento 1.30%, vs. Costacalda 0.15%; p > 0.05), currently exist as a commensal fungi of the mammalian skin, and associated with atopic dermatitis in inflammatory skin disorders only.

Similarly, for bacterial assemblage (Fig. 7B), many widespread human tissues-inhabiting members of Bacilli class characterized the tourist caves, in contrast to Costacalda wild cave, where their relative abundance never exceeded the 0.007%; in particular Staphylococcus (ranging from 0.082% in Pertosa-Auletta to 0.48% in Bossea; p < 0.05), Streptococcus (from 0.061% in Pertosa-Auletta to 0.47% in Bossea; p < 0.01), Lactococcus (as a mean of 0.21% in the show -caves, vs. 0.003% in Costacalda; p < 0.05) and Lac tobacillus (with 1.7% on average in show -caves, vs. 0.007% in Costacalda; p < 0.05) showed a higher significant occurrence within show-caves.

Furthermore, even the Clostridium genus (from 0.5% in Caudano to 1.6% in Bossea, vs. 0.07% in Costacalda; p > 0.05) seemed to exhibit a higher occurrence across the anthropized settings.

Source: Ecology - nature.com