Fungi in perennial ice from Scărișoara Ice Cave (Romania)

Abstract

Screening of 1,000-years old ice layers from the perennial ice block of Scărișoara Ice Cave (NW Romania) revealed the presence of fungal communities. Using culture-dependent methods and molecular techniques based on DGGE fingerprinting of 18S rRNA gene fragments and sequencing, we identified 50 cultured and 14 uncultured fungi in presently-forming, 400 and 900 years old ice layers, corresponding to 28 distinct operational taxonomic units (OTUs). The dominant ice-contained fungal OTUs were related to Ascomycota, Basidiomycota and Cryptomycota phyla. Representatives of Mucoromycota and Chytridiomycota were also isolated from recent and 400 years old ice samples. The cryophilic Mrakia stokesii was the most abundant fungal species found in the cave ice samples of all prospected ages, alongside other cryophilic fungi also identified in various glacial environments. Ice deposits formed during the Little Ice Age (dated between AD 1,250 and 1,850) appeared to have a higher fungal diversity than the ice layer formed during the Medieval Warm Period (prior to AD 1,250). A more complex fungal community adapted to low temperatures was obtained from all analyzed ice layers when cultivated at 4 °C as compared to 15 °C, suggesting the dominance of cold-adapted fungi in this glacial habitat. The fungal distribution in the analyzed cave ice layers revealed the presence of unique OTUs in different aged-formed ice deposits, as a first hint for putative further identification of fungal biomarkers for climate variations in this icy habitat. This is the first report on fungi from a rock-hosted cave ice block.

Figure 1

DGGE profile of 18S rRNA gene amplicons from Scărișoara ice samples. Fungal 18S rRNA gene fragments were amplified from 1-S, 1-L, 400-O, 900-O and 900-I (A) environmental ice samples, and (B) the corresponding enrichment cultures obtained in T1, T2, LB and LBG media at 4 °C and 15 °C, and analyzed by DGGE as previously described. The number on each sample amplicon corresponds to the fungal sequence (Table 1).

Figure 2

Distribution of fungal OTUs in cave ice chronosequence. VENN diagram indicates the number of distinct and shared fungal OTUs in Scărişoara ice samples 1-S, 1-L, 400-O, 900-O and 900-I. The GenBank closest match and distribution (presence/absence: +/−) of 7 fungal strains common to different cave ice layers are indicated in the table.

Figure 3

Phylogenetic tree of fungi from ice samples collected from Scărişoara Ice Cave and from the corresponding enrichment cultures, based on 18S rDNA. The cave ice 18S rRNA gene sequences and closest fungal match (Table 1) were used for the phylogenetic tree construction, using Glomus mosseae [NG017178] as outgroup for tree rooting.