A living bdelloid rotifer from 24,000-year-old Arctic permafrost.

In natural, permanently frozen habitats, some organisms may be preserved for hundreds to tens of thousands of years. For example, stems of Antarctic moss were successfully regrown from an over millennium-old sample covered by ice for about 400 years1. Likewise, whole campion plants were regenerated from seed tissue preserved in relict 32,000-year-old permafrost2, and nematodes were revived from the permafrost of two localities in northeastern Siberia, with source sediments dated over 30,000 years BP3. Bdelloid rotifers, microscopic multicellular animals, are known for their ability to survive extremely low temperatures4. Previous reports suggest survival after six to ten years when frozen between -20° to 0°C4-6. Here, we report the survival of an obligate parthenogenetic bdelloid rotifer, recovered from northeastern Siberian permafrost radiocarbon-dated to ∼24,000 years BP. This constitutes the longest reported case of rotifer survival in a frozen state. We confirmed the finding by identifying rotifer actin gene sequences in a metagenome obtained from the same sample. By morphological and molecular markers, the discovered rotifer belongs to the genus Adineta, and aligns with a contemporary Adineta vaga isolate collected in Belgium. Experiments demonstrated that the ancient rotifer withstands slow cooling and freezing (∼1°C min-1) for at least seven days. We also show that a clonal culture can continuously reproduce in the laboratory by parthenogenesis.

Frozen Zoo: a collection of permafrost samples containing viable protists and their viruses.

BACKGROUND: Permafrost, frozen ground cemented with ice, occupies about a quarter of the Earth's hard surface and reaches up to 1000 metres depth. Due to constant subzero temperatures, permafrost represents a unique record of past epochs, whenever it comes to accumulated methane, oxygen isotope ratio or stored mummies of animals. Permafrost is also a unique environment where cryptobiotic stages of different microorganisms are trapped and stored alive for up to hundreds of thousands of years. Several protist strains and two giant protist viruses isolated from permafrost cores have been already described. NEW INFORMATION: In this paper, we describe a collection of 35 amoeboid protist strains isolated from the samples of Holocene and Pleistocene permanently frozen sediments. These samples are stored at -18°C in the Soil Cryology Lab, Pushchino, Russia and may be used for further studies and isolation attempts. The collection strains are maintained in liquid media and may be available upon request. The paper also presents a dataset which consists of a table describing the samples and their properties (termed "Sampling events") and a table describing the isolated strains (termed "Occurrences"). The dataset is publicly available through the GBIF portal.

Isolates from ancient permafrost help to elucidate species boundaries in Acanthamoeba castellanii complex (Amoebozoa: Discosea).

Acanthamoeba castellanii species complex (genotype T4) comprises of more than ten species with unclear synonymy. Its molecular phylogeny has several conflicts with published morphological data. In this paper, we analyze morphometric traits and temperature preferences in six new strains belonging to A. castellanii complex isolated from Arctic permafrost in the framework of molecular phylogeny. This integrative approach allows us to cross-link genotypic and phenotypic variability and identify species-level boundaries inside the complex. We also analyze previously known and newly found discrepancies between the nuclear and mitochondrial gene-based phylogenies. We hypothesize that one reason for these discrepancies may be the intragenomic polymorphism of ribosomal RNA genes.

Metagenomes from Late Pleistocene Ice Complex Sediments of the Siberian Arctic.

The late Pleistocene Ice Complex (also known as Yedoma) encompasses ice-rich permafrost formed when alluvial and/or aeolian sediments accumulated under cold climatic conditions. Three metagenomes obtained from Yedoma deposits continually frozen for periods up to 60,000 years are reported here.

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.

In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba.

Acanthamoeba species are infected by the largest known DNA viruses. These include icosahedral Mimiviruses, amphora-shaped Pandoraviruses, and Pithovirus sibericum, the latter one isolated from 30,000-y-old permafrost. Mollivirus sibericum, a fourth type of giant virus, was isolated from the same permafrost sample. Its approximately spherical virion (0.6-µm diameter) encloses a 651-kb GC-rich genome encoding 523 proteins of which 64% are ORFans; 16% have their closest homolog in Pandoraviruses and 10% in Acanthamoeba castellanii probably through horizontal gene transfer. The Mollivirus nucleocytoplasmic replication cycle was analyzed using a combination of "omic" approaches that revealed how the virus highjacks its host machinery to actively replicate. Surprisingly, the host's ribosomal proteins are packaged in the virion. Metagenomic analysis of the permafrost sample uncovered the presence of both viruses, yet in very low amount. The fact that two different viruses retain their infectivity in prehistorical permafrost layers should be of concern in a context of global warming. Giant viruses' diversity remains to be fully explored.

Two metagenomes from late pleistocene northeast siberian permafrost.

The present study reports metagenomic shotgun sequencing of microbial communities of two ancient permafrost horizons of the Russian Arctic. Results demonstrate a significant difference in microbial community structure of the analyzed samples in general and microorganisms of the methane cycle in particular.

Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.

The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 µm in diameter and adenine-thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 µm in length and guanine-cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 µm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health.