Detection and characterization of ancient fungi entrapped in glacial ice

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Abstract
Glacial ice is a unique matrix that is capable of protecting microorganisms for long-term preservation. The organisms entrapped in glacial ice can provide information on evolutionary processes and ancient biodiversity. The major objective of this study was to detect and characterize ancient fungi in glacial ice and determine their viability. We developed a protocol using rigorous decontamination procedures, conventional cultural methods, and molecular techniques (polymerase chain reaction amplification of fungal ribosomal DNA internal transcribed spacers, followed by sequence analyses) to examine the ancient fungi entrapped in glacial ice. Fifteen glacial ice subcores, ranging in age from several hundred to 140 000 yr, from two locations in Greenland were used in this study. One hundred ninety fungal isolates were obtained using conventional cultural methods. In this paper we report the morphological and molecular characteristics of eight isolates, including two Penicillium spp., three Cladosporium spp., one Ulocladium sp., one basidiomycete (Pleurotus sp.), and one unknown ascomycete. In addition, ten fungal sequences were amplified and sequenced directly from ice meltwater. One of them was identified as a basidiomycete. The other nine were classified as ascomycetes, of which three sequences had very low similarities with contemporary sequences in Gen-Bank. The results from this study may facilitate the future use of ancient fungi in studies of fungal ecology, distribution, epidemiology, phylogeny, and evolution.