The Characterization of Microbiome and Interactions on Weathered Rocks in a Subsurface Karst Cave, Central China

Abstract

Karst caves are a natural oligotrophic subsurface biosphere widely distributed in southern China. Despite the progress in bacterial and fungal diversity, the knowledge about interactions between bacteria, fungi, and minerals is still limited in caves. Hence, for the first time, we investigated the interaction between bacteria and fungi living on weathered rocks in the Heshang Cave via high-throughput sequencing of 16S rRNA and ITS1 genes, and co-occurrence analysis. The mineral compositions of weathered rocks were analyzed by X-ray diffraction. Bacterial communities were dominated by Actinobacteria (33.68%), followed by Alphaproteobacteria (8.78%), and Planctomycetia (8.73%). In contrast, fungal communities were dominated by Sordariomycetes (21.08%) and Dothideomycetes (14.06%). Mineral substrata, particularly phosphorus-bearing minerals, significantly impacted bacterial (hydroxyapatite) and fungal (fluorapatite) communities as indicated by the redundancy analysis. In comparison with fungi, the development of bacterial communities was more controlled by the environmental selection indicated by the overwhelming contribution of deterministic processes. Co-occurrence network analysis showed that all nodes were positively linked, indicating ubiquitous cooperation within bacterial groups and fungal groups, as well as between bacteria and fungi under oligotrophic conditions in the subsurface biosphere. In total, 19 bacterial ASVs and 34 fungal OTUs were identified as keystone taxa, suggesting the fundamental role of fungi in maintaining the microbial ecosystem on weathered rocks. Ascomycota was most dominant in keystone taxa, accounting for 26.42%, followed by Actinobacteria in bacteria (24.53%). Collectively, our results confirmed the highly diverse bacterial and fungal communities on weathered rocks, and their close cooperation to sustain the subsurface ecosystem. Phosphorus-bearing minerals were of significance in shaping epipetreous bacterial and fungal communities. These observations provide new knowledge about microbial interactions between bacteria, fungi, and minerals in the subterranean biosphere.

Keywords: co-occurrence network; community assembly; karst caves; microbial interaction; phosphate-bearing minerals; subsurface biosphere.

FIGURE 1

Alpha-diversity indices and non-metric multidimensional scaling (NMDS) analysis of bacterial (A,B,E) and fungal (C,D,F) communities of weathered rocks in the Heshang Cave, Hubei province, China. PZ: photic zone; TZ: twilight zone; AZ: aphotic zone. The mark with * indicates significant differences between the two groups (P < 0.05).

FIGURE 2

Bacterial (A,B) and fungal community (C,D) compositions of the top 15 classes (A,C) and genera (B,D) of weathered rocks in the Heshang Cave. Linear discriminant analysis (LDA) effect size taxonomic histogram with LDA = 4 of bacterial (E) and LDA = 3 of fungal (F) communities of the weathered rocks in the Heshang Cave, Hubei province, China. PZ: photic zone, TZ: twilight zone, AZ: aphotic zone. C and G after the sampling zone abbreviation indicate classes and genera of microbial taxonomy in the corresponding sampling location.

FIGURE 3

Redundancy analysis of mineral compositions and bacterial communities (A) and fungal communities (B) of the weathered rocks in the Heshang Cave, China. Mineral phases significantly impacting microbial communities were marked with one (with P < 0.05) or two blue stars (P < 0.01), respectively.

FIGURE 4

Phylogenetic and null model analyses show the assembly processes of bacterial (A) and fungal communities (B) in the photic zone (PZ), twilight zone (TZ), and aphotic zone (AZ) tested by βNTI values. The contribution of each ecological process to microbial community assembly in different zones in the Heshang Cave (C). PZ_B, TZ_B, and AZ_B indicate the ecological processes responsible for bacterial community assembly in the photic zone, twilight zone, and aphotic zone, respectively, whereas those PZ_F, TZ_F, and AZ_F denote fungal community assembly.

FIGURE 5

Co-occurrence networks of bacterial and fungal communities of weathered rocks in the Heshang Cave colored by bacteria and fungi (A) and by modules (B). The proportion of ASVs/OTUs based on sampling zonation in each module (C). The proportion of bacterial ASVs and fungal OTUs in each module (D). The within-module connectivity (Zi)–among-module connectivity (Pi) plot shows distribution of ASVs/OTUs according to their module-based topological roles in the co-occurrence networks of bacterial and fungal communities (E). Each dot represents an ASV in the dataset of bacteria (pink) or an OTU of fungi (green). PZ: photic zone, TZ: twilight zone, AZ: aphotic zone.