Viable Nematodes from Late Pleistocene Permafrost of the Kolyma River Lowland.

We have obtained the first data demonstrating the capability of multicellular organisms for longterm cryobiosis in permafrost deposits of the Arctic. The viable soil nematodes Panagrolaimus aff. detritophagus (Rhabditida) and Plectus aff. parvus (Plectida) were isolated from the samples of Pleistocene permafrost deposits of the Kolyma River Lowland. The duration of natural cryopreservation of the nematodes corresponds to the age of the deposits, 30 000-40 000 years.

Morphology, biology and phylogeny of Phalansterium arcticum sp. n. (Amoebozoa, Variosea), isolated from ancient Arctic permafrost.

A new species, Phalansterium arcticum sp. n., was isolated from an 8580-year-old Arctic permafrost layer. This organism typically lives as a sedentary uniflagellated cell enclosed in a thin flexible mucilaginous sheath, but can form naked swimming cells and amoeboid cells with eruptive pseudopodia accompanied with the formation of short, filopodia-like projections. In an SSU rDNA phylogenetic tree, it robustly groups with other species of this genus. Along with a description of the species, we also add new details to the description of the cell division of Phalansterium and the feeding process in this organism.

Viable Species of Flamella (Amoebozoa: Variosea) Isolated from Ancient Arctic Permafrost Sediments.

Six viable strains of amoebae belonging to the genus Flamella (Amoebozoa, Variosea) were isolated from permafrost sediments sampled in the Russian Arctic region. Two of them are from late Pleistocene permafrost in North-East Siberia, and four--from Holocene and late Pleistocene in North-West Siberia. Light- and electron-microscopic study and molecular phylogeny show that these isolates represent two new species belonging to the genus Flamella. Both species are cyst-forming. This is a remarkable case of high resistance of protozoan cysts, allowing them to survive and recover an amoebae population after a very long, geologically significant period of rest; a "snapshot" of evolution in time. This study directly shows for the first time that amoeba cysts can be conserved not only for years and decades but for many thousand years and then recover, contributing to the formation of an active microbial community. We propose to name the new species as Flamella pleistocenica n.sp. and Flamella beringiania n.sp. Phylogenetic analysis shows that the genus Flamella is a robust and potentially species-rich group of Variosea.

Ciliates from ancient permafrost: Assessment of cold resistance of the resting cysts.

There is evidence that resting cysts of soil ciliates and numerous taxa of other protists can survive in permafrost for thousands of years at subzero temperatures; however, our knowledge about mechanisms of long term cryobiosis remains incomplete. In order to better understand the means by which ancient cysts survive, we investigated resistance to cyclical supercooling stress of resting cysts of the soil ciliate Colpoda steinii (Colpodida, Ciliophora). Three clonal strains were used for comparison, isolated from Siberian tundra soil, ancient Holocene (5-7,000 y) and late Pleistocene (32-35,000 y) permafrost sediments. To determine the viability of the ancient and contemporary ciliate cysts we improved and validated a cultivation-independent method of vital fluorescent staining with a combination of two nucleic acid binding dyes, acridine orange and propidium iodide. The viability of Colpoda steinii cysts during low-temperature experiments was measured using both the proposed vital fluorescent staining method and standard germination test. Our results indicate that the dual-fluorescence technique is a more accurate, rapid, and efficient method for estimating cyst viability. We found that cysts of ancient ciliates display lower tolerance to the impact of cyclical cold compared to cysts of contemporary ciliates from Siberian permafrost affected soils.