This survey presents the first high-throughput characterisation of fungal distribution based on ITS2 Illumina sequencing of uncultured microbiome from a 1500 years old perennial ice deposit in Scărișoara Ice Cave, Romania. Of the total of 1 751 957 ITS2 sequences, 64% corresponded to 182 fungal operational taxonomic units, showing a low diversity, particularly in older ice strata, and a distinct temporal distribution pattern. Ascomycota was the major phylum in all ice samples, dominating the 400 and 1500 years old ice strata deposited during the cold Little Ice Age (LIA) and Dark Ages Cold Period, while Basidiomycota was mostly present in 900-years old ice formed during the Medieval Warm Period (MWP). Chytridiomycota and Mucoromycota phyla were present in recently formed and 400-years old ice, respectively. Among the 80 identified genera, Cryptococcus victoriae, commonly found in glacial habitats, was identified in all strata. A positive correlation between fungal distribution and ice conductivity, Ca, Na and Sr concentrations was observed across the ice block, with pH values trailing climate variations during LIA and MWP, respectively. Our record highlighted the presence of a complex climate and environmental-driven fungal community in perennial ice strata accumulated during the last 1500 years in Scărișoara Ice Cave.

Ice caves constitute the newly investigated frozen and secluded model habitats for evaluating the resilience of ice-entrapped microbiomes in response to climate changes. This survey identified the total and active prokaryotic and eukaryotic communities from millennium-old ice accumulated in Scarisoara cave (Romania) using Illumina shotgun sequencing of the ribosomal RNA (rRNA) and messenger RNA (mRNA)-based functional analysis of the metatranscriptome. Also, the response of active microbiome to heat shock treatment mimicking the environmental shift during ice melting was evaluated at both the taxonomic and metabolic levels. The putatively active microbial community was dominated by bacterial taxa belonging to Proteobacteria and Bacteroidetes, which are highly resilient to thermal variations, while the scarcely present archaea belonging to Methanomicrobia was majorly affected by heat shock. Among eukaryotes, the fungal rRNA community was shared between the resilient Chytridiomycota and Blastocladiomycota, and the more sensitive Ascomycota and Basidiomycota taxa. A complex microeukaryotic community highly represented by Tardigrada and Rotifera (Metazoa), Ciliophora and Cercozoa (Protozoa), and Chlorophyta (Plantae) was evidenced for the first time in this habitat. This community showed a quick reaction to heat shock, followed by a partial recovery after prolonged incubation at 4°C due to possible predation processes on the prokaryotic cluster. Analysis of mRNA differential gene expression revealed the presence of an active microbiome in the perennial ice from the Scarisoara cave and associated molecular mechanisms for coping with temperature variations by the upregulation of genes involved in enzyme recovery, energy storage, carbon and nitrogen regulation, and cell motility. This first report on the active microbiome embedded in perennial ice from caves and its response to temperature stress provided a glimpse into the impact of glaciers melting and the resilience mechanisms in this habitat, contributing to the knowledge on the functional role of active microbes in frozen environments and their response to climatic changes.

This experimental procedure was designed to isolate total the RNA aiming to obtain intact and high-quality integrity (RIN>7) for further next generation sequencing analysis. The protocol was developed to analyse the environmental microbial active community providing a clear overview of the microbial transcriptional activity in a specific time or after a different condition exposition sets, such as the investigation of thermal adaptation after heat-shock. The procedure is specifically designed to extract total-RNA from water samples although it can be used for various environmental samples as done in Schostag et al. and Bang-Andreasen et al. 2019 in soil with similar accuracy results. The total-RNA yield was >1000 ng/mL with a RIN ranging from 7 to 8.5. The final Illumina library preparation yield ranged between 15 and 31 ng/µL. This protocol is a compiled by using the Qiagen RNeasy® PowerSoil® Total RNA Kit with additional steps according the DNeasy Max®Kit (Qiagen),