Enhanced monsoon duration and soil acidification from acid rain are expected to impact the distribution of microbial communities in surface and subsurface environments, although these impacts are poorly understood for most systems. In central China, soluble carbonate bedrock forms extensive karst landscapes. Current predictions are that the amount of monsoonal precipitation and acid rainfall in central China will increase, which is expected to lead to changes in the pH balance of karst ecosystems. To evaluate the role of pH, total organic carbon, and other geochemical parameters (e.g., Ca2+, Mg2+, NH4+, NOx, SO42-) in shaping bacterial communities within a single karst system in central China, samples were collected from the thin surface soils overlying Heshang Cave, cave sediments, and weathered cave passage rocks from the entrance, twilight, and dark zones, as well as from epikarstic drip waters inside the cave. Illumina sequencing of 16S rRNA genes and multivariate statistical analyses revealed that each tested community was distinct and the community variability was significantly correlated with pH, total organic carbon, and potassium concentrations. Specifically, surface soils were dominated by Acidobacteria, Verrucomicrobia and Planctomycetes, and diversity significantly decreased with acidic pH values. Nitrospirae, Gemmatimonadetes, Firmicutes, and Chloroflexi were unique to cave sediments, while Actinobacteria and Proteobacteria dominated weathered rocks and drip waters, respectively. The results reveal important implications regarding the effects of acidification on bacterial communities in karst areas, and on the control of pH in shaping bacterial communities throughout a karst system. Increased water flux into and through karst habitats due to monsoonal precipitation may result in deeper penetration of acidic solutions into karst and shift the bacterial communities inside the cave in the future.

Caves are nutrient-limited and dark subterranean ecosystems. To date, attention has been focused on geological research of caves in China, whilst indigenous microbial diversity has been insufficiently characterized. Here, we report the fungal diversity in the pristine, oligotrophic, karst Heshang Cave, central China, using a culture-dependent method coupled with the analysis of the fungal rRNA-ITS gene sequences. A total of 194 isolates were obtained with six different media from 14 sampling sites of sediments, weathered rocks, and bat guanos. Phylogenetic analysis clustered the 194 sequenced isolates into 33 genera within 15 orders of three phyla, Ascomycota, Basidiomycota, and Zygomycota, indicating a high degree of fungal diversity in the Heshang Cave. Notably, 16 out of the 36 fungal genera were also frequently observed in solution caves around the world and 23 genera were previously found in carbonate cave, indicating potential similarities among fungal communities in cave ecosystems. However, 10 genera in this study were not reported previously in any solution caves, thus expanding our knowledge about fungal diversity in cave ecosystems. Moreover, culturable fungal diversity varied from one habitat to another within the cave, being the highest in sediments, followed by weathered rocks and bat guanos as indicated by α-diversity indexes. At the genus level, Penicillium accounted for 40, 54, and 52% in three habitats of sediments, weathered rocks, and bat guanos, respectively. Trichoderma, Paecilomyces, and Aspergillus accounted for 9, 22, and 37% in the above habitats, correspondingly. Despite of the dominance of Penicillium in all samples, β-diversity index indicated significant differences between each two fungal communities in the three habitats in view of both the composition and abundance. Our study is the first report on fungal communities in a natural pristine solution cave system in central China and sheds light on fungal diversity and functions in cave ecosystems.

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Deciphering of the mycobiome in pristine karst caves has been impeded by constraints of remote locations, inaccessibility to specimens and technical limitations, which greatly restricted in-depth understanding of mycobiomes in subterranean ecosystem. Here, mycobiomes of Heshang Cave in south-western karst region of China were investigated by Illumina HiSeq sequencing of fungal rRNA-ITS1 gene across different habitats. In total 793,502 ITS1 reads and 2,179 OTUs from 778 Mb reads after stringent quality control (Q30) and 453 genera, 72 orders and 19 classes within 6 phyla were detected. Ascomycota (42% OTUs) dominated across the five habitats. Shannon-Wiener index varied from 1.25 to 7.62 and community richness was highest in drip waters, followed by weathered rocks, bat guanos, sediments, and air samples. Mycobiomes displayed specificity to five habitats and more distinct OTUs were found in weathered rocks (12%) and drip waters (9%). In contrast, only 6.60% core OTUs were shared by five habitats. Notably, weathered rocks possessed more indicator groups and were revealed for the first time to be dominated by Sordariomycetes (43%). The community richness of air mycobiomes increased from cave entrance to the innermost part and dominated by the indicator groups of Penicillium mallochii (>30%) and P. herquei (>9%). Our work represents the largest attempt to date to a systematical investigation of oligotrophic solution-cave-associated mycobiomes in China. Our discovery of high diversity of mycobiomes in Heshang Cave also suggests that eukaryotic microorganisms may play a crucial role in subsurface environments.

Microbial lipids in stalagmites are increasingly used to reconstruct the paleoclimate change, necessitating the investigations on microbial communities in dripping waters. A pilot molecular survey was conducted on bacterial communities of dripping waters at two sites (1D and 3D) in Heshang Cave of Hubei province in central China for a period of 2008 to 2013. The samples were subjected to genome DNA extraction, 16S rRNA gene amplification, clone library construction and phylogenetic analysis to explore the seasonal variation of bacterial communities and their association with environmental factors including regional air temperature， precipitation, cave temperature, pH, conductivity and dripping rate of the water samples. Seasonal variations were clearly observed in components and diversities of bacterial communities at both sites. Bacterial community was dominated by Gammaproteobacteria in autumn and winter, whereasDownloaded by [FU Berlin] at 04:42 04 July 2015 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2Betaproteobacteria became dominant in samples collected in summer and spring. Among the environmental factors investigated, regional air temperature was found to have a strong impact on bacterial communities indicated by cluster and redundancy analysis. Moreover the bacterial biodiversity was observed to increase with the temperature rising. Bacteria identified in dripping water were either oligotrophs or able to acquire nutrients from minerals under oligotrophic conditions. They may also be able to induce calcite precipitation in cave systems. Our data shed light on the potential of microbes used as a tool for the reconstruction of paleo-temperature as well as on the ecological functions of bacterial communities in oligotrophic caves.

In general, the constant physicochemical conditions and limited nutrient sources over long periods in the subsurface support a stable ecosystem in karst cave. Previous studies on cave microbial ecology were mostly focused on community composition, diversity, and the relationship with local environmental factors.

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