Biodiversity Data Journal :
Data Paper (Biosciences)
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Corresponding author: Stas Malavin (stas.malavin@issp.psn.ru)
Academic editor: Anna Maria Fiore-Donno
Received: 28 Feb 2020 | Accepted: 03 Jul 2020 | Published: 10 Jul 2020
© 2020 Stas Malavin, Lyubov Shmakova, Jean-Michel Claverie, Elizaveta Rivkina
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Malavin S, Shmakova L, Claverie J-M, Rivkina E (2020) Frozen Zoo: a collection of permafrost samples containing viable protists and their viruses. Biodiversity Data Journal 8: e51586. https://doi.org/10.3897/BDJ.8.e51586
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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.
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.
Permafrost, microbiology collection, protists, giant viruses
Permafrost, or perennially frozen ground, is the ground that remains below zero degrees Celsius for two or more consecutive years (
The sediments, from which living microorganisms have been successfully isolated, date back to a million years BP. These strains are of great scientific interest for several reasons. First, this is a remarkable case of the organism's hardiness, far exceeding the traditional view on how long an organism can survive, even in the suspended stage of the life cycle. Although subzero temperatures down to −50°C are not incompatible with certain metabolic reactions in bacteria (
Amoeboid protists, a polyphyletic group of eukaryotic, mostly unicellular, microorganisms with inconstant cell shape, are an important component of all soil ecosystems (e.g.
The data on the taxonomic composition of amoeboid protists in the Arctic and Antarctic are scarce compared to the better-studied temperate areas.
The finding of live protists in the permafrost layers up to hundreds of thousands years old significantly expands our view on the survival capabilities of eukaryotes and raises questions about the mechanisms that make this survival possible. Despite intensive research during the last 40 years, our understanding of these mechanisms in unicellular organisms is still far from completion
An interesting peculiarity about the described samples is the isolation of two new giant double-stranded DNA viruses from one of them (
The samples were collected in the field and stored constantly frozen during all periods of transportation and processing. In the lab, a part of each sample was used to isolate protist strains. The remaining part has never been melted and is stored at −18°C. The isolation was done in sterile conditions. Revived protists were cloned and are maintained as bacterised or axenic cultures on plastic or agar with liquid overlay.
Drilling was performed using a mobile drilling rig (core-drilling machine) UKB-12/25 (V.V. Vorovsky Machine-Building Plant, Moscow, Russia) operated without flushing and blowing (Fig.
Outcrops are natural exposures of permafrost sediments formed at sea and river banks. The advantage of sampling from the outcrop wall is the possibility of visual inspection and description of the whole sediment layer, including preserved soils (Fig.
Buried terminal nesting chambers of ground squirrel (Urocitellus sp.) burrows (Fig.
During the development of the permafrost microbiological sampling technique, several tests for contamination of the core interior were established at different phases of sampling and storage.
Later,
Based on the negative results obtained for bacteria and fluorescent beads, i.e. particles around 2 μm in diameter or less, we consider that protist cysts, which are at least five times larger, cannot move inside the frozen ground and thus could not have penetrated the sediments much later than they were deposited. In the same way, the contamination of the inner part of the samples during sampling and laboratory processing is highly unlikely.
The samples were obtained from three areas in High Eurasian Arctic, i.e. the Gydan Peninsula, the Bykovskiy Peninsula and the Kolyma Lowland (Fig.
Sampling event id | Parent event id | Year of collection | Locality | Locality link | Latitude | Longitude | Depth below surface, m |
D-01/01-2.2 | D-01/01 | 2001 | Bykovskiy Peninsula | geonames.org/2025770 | 71.783284 | 129.3611761 | 2.16 |
D-07/03-5.0 | D-07/03 | 2003 | Bykovskiy Peninsula | geonames.org/2025770 | 71.775537 | 129.330297 | 5 |
D-05/13-2.5 | D-05/13 | 2013 | Ngarka-Khortiyakha River flood land, 800 m from the mouth, 100 m from the bank | geonames.org/1497773 | 71.463875 | 76.992927 | 2.5 |
D-05/13-5.0 | D-05/13 | 2013 | Ngarka-Khortiyakha River flood land, 800 m from the mouth, 100 m from the bank | geonames.org/1497773 | 71.463875 | 76.992927 | 5 |
D-05/13-6.0 | D-05/13 | 2013 | Ngarka-Khortiyakha River flood land, 800 m from the mouth, 100 m from the bank | geonames.org/1497773 | 71.463875 | 76.992927 | 6 |
D-04/13-2.5 | D-04/13 | 2013 | Southeast of the Yayne-Vonga Bay, low terrace separated from the sea by laida | geonames.org/1545199 | 72.348887 | 78.546807 | 2.5 |
D-04/13-3.5 | D-04/13 | 2013 | Southeast of the Yayne-Vonga Bay, low terrace separated from the sea by laida | geonames.org/1545199 | 72.348887 | 78.546807 | 3.5 |
D-01/13-2.0 | D-01/13 | 2013 | West of Lake Tirebyato, 10 m from the terrace cliff | geonames.org/1544900 | 72.350733 | 75.118445 | 2 |
D-01/13-4.0 | D-01/13 | 2013 | West of Lake Tirebyato, 10 m from the terrace cliff | geonames.org/1544900 | 72.350733 | 75.118445 | 4 |
D-01/13-8.0 | D-01/13 | 2013 | West of Lake Tirebyato, 10 m from the terrace cliff | geonames.org/1544900 | 72.350733 | 75.118445 | 8 |
D-03/13-1.0 | D-03/13 | 2013 | Southeast of the Yayne-Vonga Bay, laida rear welt | geonames.org/1545199 | 71.429709 | 72.991683 | 1 |
D-07/13-2.0 | D-07/13 | 2013 | Mongocheyakha River Mouth | geonames.org/1498452 | N/D | N/D | 2 |
D-03/15-3.5 | D-03/15 | 2015 | Alazeya River | geonames.org/2127297 | 69.3388694 | 154.9969472 | 3.5 |
D-03/15-14.2 | D-03/15 | 2015 | Alazeya River | geonames.org/2127297 | 69.3388694 | 154.9969472 | 14.2 |
P-1084T | P-1084 | 2000 | Kolyma River, Stanchikovskiy Yar | geonames.org/12123736 | 68.370155 | 161.415553 | N/A |
P-1086AT2 | P-1086 | 2000 | Kolyma River, Stanchikovskiy Yar | geonames.org/12123736 | 68.370155 | 161.415553 | N/A |
P-318-08-69a | P-318-08 | 2008 | Kolyma River, Duvannyy Yar | geonames.org/12123735 | 68.628232 | 159.194842 | N/A |
C-02/19-1 | C-02/19 | 2019 | Kolyma River, Duvannyy Yar | geonames.org/12123735 | 68.635026 | 159.07798 | N/A |
B-34/19 | B-34/19 | 2019 | Kolyma River, Duvannyy Yar | geonames.org/12123735 | 68.630072 | 159.153383 | N/A |
We isolated protists from permafrost samples using enrichment cultivation. Specifically, three portions of ca. 1 cm3 from the inner part of each frozen sample were placed into 90-mm Petri dishes filled with 10 ml autoclaved mineral Prescott and James (PJ) medium (
Preliminarily, we identified isolated strains to the lowest possible level using keys and diagrams as in
Strains of the described collection. LM—Light microscopy; TEM—Transmitted electron microscopy; SEM—scanning electron microscopy; M—Molecular phylogeny; Kyr—thousands of years.
Strain | Identification | Identification basis | Sample | Location | Geological epoch | Estimated age, Kyr | Description reference | GenBank accession number (SSU) |
Amoebozoa | ||||||||
Discosea | ||||||||
SCL-am7 | Acanthamoeba sp. | LM | D-01/01-2.2 | Bykovskiy Pen. | Late Pleistocene | 12–28 | ||
SCL-am8 | Acanthamoeba sp. | LM, M | P-1086AT2 | Kolyma Lowland | Late Pleistocene | 34–37 |
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MK124583 |
SCL-am9 | Acanthamoeba sp. | LM, M | D-07/03-5.0 | Bykovskiy Pen. | Late Pleistocene | 28–48 | MK124584 | |
SCL-14-2 | Acanthamoeba sp. | LM, M | D-05/13-5.0 | Gydan Pen. | Holocene | MK124585 | ||
SCL-14-3 | Acanthamoeba sp. | LM, M | D-04/13-3.5 | Gydan Pen. | Late Pleistocene | ~30 | MK124586 | |
SCL-14-9 | Acanthamoeba sp. | LM, M | D-05/13-6.0 | Gydan Pen. | Late Pleistocene | MK124587 | ||
SCL-14-12 | Acanthamoeba sp. | LM, M | P-1084T | Kolyma Lowland | Late Pleistocene | 34–37 | MK124588 | |
SCL-19-2 | Acanthamoeba sp. | LM | C-02/19-1 | Kolyma Lowland | Late Pleistocene | 42–43 | ||
SCL-16-1 | Cochliopodium sp. | LM | D-03/15-3.5 | Kolyma Lowland | Late Pleistocene | |||
SCL-16-3 | Vannella sp. | LM | D-03/15-3.5 | Kolyma Lowland | Late Pleistocene | |||
SCL-15-5 | Amoebozoa indet. | LM | D-03/15-14.2 | Kolyma Lowland | Middle Pleistocene | 600–1000 | ||
SCL-14-10 | Amoebozoa indet. | LM | D-05/13-2.5 | Gydan Pen. | Holocene | |||
SCL-19-3 | Amoebozoa indet. | LM | C-02/19-1 | Kolyma Lowland | Late Pleistocene | 42–43 | ||
Evosea: Variosea | ||||||||
SCL-flam1 | Flamella pleistocenica Shmakova et al., 2016 | LM, TEM, M | P-318-08-69a | Kolyma Lowland | Late Pleistocene | 42–43 |
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KP208180 |
SCL-flam2 | Flamella beringiania Shmakova et al., 2016 | LM, TEM, M | P-1084T | Kolyma Lowland | Late Pleistocene | 34–37 | KP219428 | |
SCL-flam3 | Flamella beringiania Shmakova et al., 2016 | LM, TEM, M | D-04/13-3.5 | Gydan Pen. | Late Pleistocene | ~30 | KP219429 | |
SCL-flam4 | Flamella beringiania Shmakova et al., 2016 | LM, TEM, M | D-05/13-5.0 | Gydan Pen. | Holocene | KP219430 | ||
SCL-flam5 | Flamella pleistocenica Shmakova et al., 2016 | LM, TEM, M | D-05/13-2.5 | Gydan Pen. | Holocene | KP219431 | ||
SCL-flam6 | Flamella beringiania Shmakova et al., 2016 | LM, TEM, M | D-01/13-4.0 | Gydan Pen. | Holocene | KP219432 | ||
SCL-flam9 | Flamella sp. | LM | D-03/15-3.5 | Kolyma Lowland | Late Pleistocene | |||
SCL-19-1 | Flamella sp. | LM | C-02/19-1 | Kolyma Lowland | Late Pleistocene | 42–43 | ||
SCL-19-8 | Flamella sp. | LM | B-34/19 | Kolyma Lowland | Late Pleistocene | 42–43 | ||
SCL-14-8 | Filamoeba sp. | LM | D-01/13-8.0 | Gydan Pen. | Late Pleistocene | 15–17 | ||
SCL-Parc | Phalansterium arcticum Shmakova et al., 2018 | LM, TEM, M | D-01/13-2.0 | Gydan Pen. | Holocene | 8.6 |
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KX844828 |
SCL-14-1 | BNFA | LM | D-05/13-2.5 | Gydan Pen. | Holocene | |||
SCL-14-4 | BNFA | LM | D-05/13-6.0 | Gydan Pen. | Late Pleistocene | |||
SCL-14-6 | BNFA | LM | D-03/13-1.0 | Gydan Pen. | Holocene? | |||
SCL-14-7 | BNFA | LM | D-07/13-2.0 | Gydan Pen. | Holocene? | |||
SCL-14-11 | BNFA | LM | D-04/13-2.5 | Gydan Pen. | Late Pleistocene | |||
SCL-16-4 | BNFA | LM | D-03/15-3.5 | Kolyma Lowland | Late Pleistocene | |||
SCL-16-5 | BNFA | LM | D-03/15-3.5 | Kolyma Lowland | Late Pleistocene | |||
Heterolobosea | ||||||||
SCL-16-2 | Heterolobosea indet. | LM | D-03/15-3.5 | Kolyma Lowland | Late Pleistocene | |||
SCL-16-8 | Heterolobosea indet. | LM | D-03/15-3.5 | Kolyma Lowland | Late Pleistocene | |||
SCL-16-9 | Heterolobosea indet. | LM | D-03/15-3.5 | Kolyma Lowland | Late Pleistocene | |||
Rhizaria | ||||||||
SCL-16-6 | Lecythium sp. | LM | D-03/15-3.5 | Kolyma Lowland | Late Pleistocene | |||
Viruses | ||||||||
Pithovirus sibericum | TEM, SEM, M | P-1084T | Kolyma Lowland | Late Pleistocene | 34–37 |
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NC023423 | |
Mollivirus sibericum | TEM, SEM, M | P-1084T | Kolyma Lowland | Late Pleistocene | 34–37 |
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NC027867 |
For the isolation of viruses, 400 mg of the sample were resuspended in 6 ml of PJ. Each 3 ml were supplemented with 300 μl of Amphotericin B (Fungizone) at 250 μg/ml. A volume of 1.65 ml of this solution was left overnight under stirring at 4°C. After decantation, the supernatant was centrifuged at 800×g for 5 min. Acanthamoeba castellanii strain Neff (ATCC 30010™) culture adapted to Fungizone was inoculated with 100 μl supernatant and with the pellet resuspended in 50 μ buffer (Tris 20 mM, CaCl 21 mM, pH 7.4). Acanthamoeba cells were cultured at 32°C in microplates with 1 ml of PPYG medium (
When the cell lysis was completed, cultures were centrifuged for 5 min at 500×g to remove cellular debris and virus particles were pelleted by a 30-min centrifugation at 3,000×g. The pellet was then washed twice in PBS and centrifuged at 5,000×g for 15 min on a discontinuous sucrose gradient (30%/40%/50%/60% wt/vol). Purified particles were studied by scanning electron microscopy. Genomic DNA was recovered from 1.8 × 1010 purified particles and sequenced in paired-end flow cell on the Illumina MiSeq system using 151 base read chemistry. Viruses were identified and described based on their genome sequences, SEM of the virion morphology and TEM of the virion factory morphology (
Age of the isolated strains. The specific property of the described collection is that its strains are of ancient, mostly Pleistocene, origin and represent a part of a disappeared ecosystem. Due to the small number of cells in the frozen sediments, it is not possible to date these cells directly, thus an indirect method is needed. In the case of syncryogenetic formation, when freezing of the sediments occurs together with their deposition, all particles, including bacterial and fungal spores and protist cysts, become frozen at approximately the same time. If frozen deposits have not melted, which may be inferred from cryotexture, distribution of methane or other signatures, no particles of bacterial size or larger could have penetrated from the surface. Thus, one could assume the age of the cells trapped in permafrost to be roughly the same as the age of the permafrost itself or, in other words, that the found cells originate from the time of the last sediment freezing. This time may be determined by radiocarbon (14C) dating of carbon-containing remnants and substances produced by the biota before sedimentation-freezing occurred.
Gydan Peninsula. Based on radiocarbon and optically-stimulated luminescence (OSL) dating, sediments associated with massive ice formations in the Gydan Peninsula are considered to be of the Late Pleistocene estuarine-alluvial origin (
The borehole D-04/13 was drilled on a low sea terrace and, below the thin cover layer, penetrates the Late Pleistocene sediments. At 4 m, these sediments were radiocarbon-dated to 34300 ± 1200 years BP (
Sediments penetrated by the borehole D-05/13 split into two benches, with a border located between 5 and 6 m. The upper bench is considered to be of the Holocene origin, while the lower formed during the Late Pleistocene (
Bykovskiy Peninsula. The Bykovskiy Peninsula harbours the most studied Late Pleistocene deposits in Siberia, called the Yedoma suite (
Kolyma Lowland. In this area, the Late Pleistocene Yedoma suite is also widely distributed. Samples C-02/19-1, B-34/19 and P-318-08-69a were taken from the Duvannyy Yar exposure on the Kolyma River, in its lowermost part (5–12 m above the river level). The sampled sediments were silty and sandy loams with numerous inclusions of roots and branches of shrubs which correspond to the allochthonous peat layer dated 42 to 43 thousand years (
The GBIF dataset "Amoeboid protists isolated from ancient Siberian permafrost" (
Usage of strains from the collection: The protist strains from the collection are freely available for non-commercial use upon request to Pushchino Scientific Center for Biological Research RAS. The distribution of strains used in the ongoing research projects will be discussed on an individual basis. The purpose of the strain usage must be stated explicitly and may be made public. Strains may not be passed to a third person without the official permission of the rights holder. The collection must be clearly referenced as the source of the strain while in public use.
Sampling events (i.e. boreholes, wall sampling, borrow samples) with linked occurrences (i.e. clonal cultures isolated from the samples obtained)
Column label | Column description |
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id | Sample id |
dynamicProperties | Sample type in Darwin Core json format |
eventID | Same as "id" |
parentEventID | Parent event— usually a borehole |
samplingProtocol | Sampling protocol |
eventDate | Date (year) of sampling |
fieldNumber | Field tag of the sample |
eventRemarks | Sample description made by a collector |
locationID | A link to a location of sampling at geonames.org |
higherGeography | Higher geography of the sampling site |
continent | Always "Asia" |
country | Always "Russia" |
countryCode | Country code |
locality | Sampling locality |
minimumDepthInMetres | The beginning depth of the core (if applicable) |
maximumDepthInMetres | The ending depth of the core (if applicable) |
decimalLatitude | Decimal latitude |
decimalLongitude | Decimal longitude |
geodeticDatum | Geodetic datum (always EPSG:4326) |
georeferencedBy | Collector |
georeferencedDate | Same as eventDate |
earliestEpochOrLowestSeries | Earliest estimated epoch of deposit formation |
latestEpochOrHighestSeries | Latest estimated epoch of deposit formation |
lithostratigraphicTerms | Lithostratigraphic terms |
Characteristics of the isolated strains
Column label | Column description |
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id | Sample ID |
type | Always "Collection" |
language | Always "en" |
rightsHolder | The rights holder, always "Pushchino Scientific Center for Biological Research RAS" |
accessRights | Always "not-for-profit use only" |
collectionCode | Always "SCL" |
ownerInstitutionCode | Always "PSCBR" |
basisOfRecord | Always"MaterialSample" |
occurrenceID | Strain ID |
catalogNumber | The same |
disposition | "in collection" or "missing" |
associatedReferences | Publications where the strain was described |
associatedSequences | Publicly available sequences of the strain |
organismScope | Always "clonal culture" |
organismRemarks | Strain maintainance |
eventID | Sample ID |
eventDate | Collection date (year) |
identifiedBy | By whom the strains was identified |
identificationReferences | Resources used for identification |
typeStatus | Type status of the strain |
scientificName | Nearest possible identification following the GBIF taxonomy |
scientificName | Always "Protozoa" (GBIF taxonomy) |
taxonRank | Rank of the nearest identified taxon |
nomenclaturalCode | Always "ICZN" |
Radiocarbon (14C) ages of the dated samples.
Column label | Column description |
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id | Sample ID |
measurementType | Always "Age" |
measurementValue | Age value |
measurementAccuracy | 1 standard deviation |
measurementUnit | Always "years BP" |
measurementMethod | Always "Radiocarbon, AMS" |
measurementRemarks | Measurement remarks |
We are obliged to all persons participating the field sampling. We are thankful to A.M. Fiorre-Donno, G. Torruella and A. Kudryavtsev for their valuable comments and suggestions and to I. Dick for the language corrections. The study was supported by the Russian Foundation for Basic Research grants nos. 17-54-150003 and 18-04-00824 and the Russian Federal Target Program no. 0191-2019-0044.