Stanislav V. Gubin

Regeneration of whole fertile plants from 30,000-y-old fruit tissue buried in Siberian permafrost

Whole, fertile plants of Silene stenophylla Ledeb. (Caryophyllaceae) have been uniquely regenerated from maternal, immature fruit tissue of Late Pleistocene age using in vitro tissue culture and clonal micropropagation. The fruits were excavated in northeastern Siberia from fossil squirrel burrows buried at a depth of 38 m in undisturbed and never thawed Late Pleistocene permafrost sediments with a temperature of −7 °C. Accelerator mass spectrometry (AMS) radiocarbon dating showed fruits to be 31,800 ± 300 y old. The total γ-radiation dose accumulated by the fruits during this time was calculated as 0.07 kGy; this is the maximal reported dose after which tissues remain viable and seeds still germinate. Regenerated plants were brought to flowering and fruiting and they set viable seeds. At present, plants of S. stenophylla are the most ancient, viable, multicellular, living organisms. Morphophysiological studies comparing regenerated and extant plants obtained from modern seeds of the same species in the same region revealed that they were distinct phenotypes of S. stenophylla. The first generation cultivated from seeds obtained from regenerated plants progressed through all developmental stages and had the same morphological features as parent plants. The investigation showed high cryoresistance of plant placental tissue in permafrost. This natural cryopreservation of plant tissue over many thousands of years demonstrates a role for permafrost as a depository for an ancient gene pool, i.e., preexisting life, which hypothetically has long since vanished from the earths surface, a potential source of ancient germplasm, and a laboratory for the study of rates of microevolution.

Viable Protozoa in Late Pleistocene and Holocene Permafrost Sediments

It has been established that viable prokaryotic and eukaryotic organisms may remain viable for a long time under natural conditions at constant low temperatures. For example, viable anaerobic and aerobic bacteria [1–4], cyanobacteria, unicellular green algae [5, 6], yeast [7], and mycelial fungi [8] have been found in permafrost sediments dated to several hundred million to several thousand million years ago. In addition, spores of mosses and seeds of higher plants capable of germination after long-term cryopreservation have been found [9]. The study of the communities of viable paleoorgan-isms offers the unique possibility to make substantial progress in understanding the preservation of life in the cryosphere during geologically significant periods of time and analyzing cryoanabiosis and psychrophily, as well as the ecological and evolutionary characteristics of organisms that lived in previous geological epochs. Obviously, these studies would be incomplete without studying various lower eukaryotes belonging to Protozoa ; however, there were no reliable data on the presence of viable protozoa in permafrost layers until this study. On the other hand, various forms of cryptobiosis [10] are known to be widespread among modern representatives of most macrotaxa of protozoa [11, 12]. The purpose of this study was to find viable forms of protozoa in syngenetically frozen late Pleistocene and Holocene sediments and isolate these organisms. The age of the biota in syncryogenic layers corresponds to the age of the sediments forming these layers, which can be reliably dated by the radiocarbon method in the case of late Pleistocene sediments. The possibility to determine the age of the biota determined the choice of late Pleistocene syncryogenic sediments in the eastern sector of the Arctic region, including the Kolyma Lowland and the Laptev Sea coast (the Byk-ovskii Peninsula and Cape Chukochii) as the object of the study. We studied late Pleistocene sediments from a 28 000-to 35000-year-old glacial complex and the soils and material of fossil rodent burrows that were buried in it in the Stanchikovskii Yar outcrop on the Malyi Anyui River. The glacial complex (cryopedolith) was represented by powdered loam containing sparse thin roots of grassy plants, fine plant detritus, and humus compounds; the loam was transformed by processes involved in synlithogenic soil formation. Its ice content was as large as 40–80% due to thick ice veins and texture-forming ice. The buried soils were represented by peat and a profile of humus–peat gley soil, and the material of fossil burrows, by …

Viability of higher plant seeds of late pleistocene age from permafrost deposits as determined by in vitro culturing.

Studies of loess–ice deposits in coastal lowland regions of northern Yakutia revealed that bulk permafrost rocks, chronologically attributed to the late Pleistocene epoch, contained many buried burrows of rodents. These fossil burrows were found to contain large paleological ecology material, including fruitage and seeds of plants. In some burrows, the number of items of this material reached 600000–800000. Most frequently, these were burrows of a species of ground squirrel of the subgenus Urocitellus [5].

Paleosol complex in the uppermost Mikhailovian Horizon (Viséan, Lower Carboniferous) in the southern flank of the Moscow Syneclise

The beds composing an unconformity at the boundary of the Mikhailovian and Venevian horizons in the Polotnyanyi Zavod quarry (Kaluga Region) are investigated. A detailed study has shown that, at the base of black “rhizoid” limestone, a pedocomplex of two paleosols of different genesis is formed. From below upwards, rocks of subaerial (Paleosol 1 of the rendzina type), subaerial–subaquatic (Paleosol 2 on bog marl), and palustrine genesis replace each other. Both paleosols display a horizontal structure of profiles; the presence of the root systems, imprints of plants, and traces of animal’s activity; the presence of soil new formations (micritic calcite with a light isotopic composition of C, Fe-concretions ). In paleosols, some geochemical indices (PWI, CIA, CIA-K), the ratios Ba/Sr, Rb/Sr, and concentration of Ga increase. The soil complex was formed under conditions of a warm subhumid climate. In the structure and lateral scale of distribution, the object investigated is comparable to the Everglades marsh landscape of Florida (United States).

DNA analysis of a 30,000-year-old Urocitellus glacialis from northeastern Siberia reveals phylogenetic relationships between ancient and present-day arctic ground squirrels

In contrast to the abundant fossil record of arctic ground squirrels, Urocitellus parryii, from eastern Beringia, only a limited number of fossils is known from its western part. In 1946, unnamed GULAG prisoners discovered a nest with three mummified carcasses of arctic ground squirrels in the permafrost sediments of the El’ga river, Yakutia, Russia, that were later attributed to a new species, Citellus (Urocitellus) glacialis Vinogr. To verify this assignment and to explore phylogenetic relationships between ancient and present-day arctic ground squirrels, we performed 14C dating and ancient DNA analyses of one of the El’ga mummies and four contemporaneous fossils from Duvanny Yar, northeastern Yakutia. Phylogenetic reconstructions, based on complete cytochrome b gene sequences of five Late Pleistocene arctic ground squirrels and those of modern U. parryii from 21 locations across western Beringia, provided no support for earlier proposals that ancient arctic ground squirrels from Siberia constitute a distinct species. In fact, we observed genetic continuity of the glacialis mitochondrial DNA lineage in modern U. parryii of the Kamchatka peninsula. When viewed in a broader geographic perspective, our findings provide new insights into the genetic history of U. parryii in Late Pleistocene Beringia.

Reply to Oxelman et al.: On the taxonomic status of the plants regenerated from 30,000-y-old fruit tissue buried in Siberian permafrost

We are grateful to Oxelman et al. (1) for their interest in the problem of taxonomic identification of plants regenerated from the fruit tissues of Late Pleistocene age and plants grown from the seeds of extant species growing in the Kolyma Lowland (2). The authors’ arguments regarding the determination of plant species are rather convincing. Indeed, our regenerated plants, as well as extant examples, show a high degree of similarity with plants of the Silene linnaeana Czerepanov (Lychnis sibirica L.) group, especially concerning the shape of inflorescences and calyces. At the same time, contemporary examples of the Silene genus found in Kolyma Lowland in the area of excavated burrows were identified as Silene stenophylla according to Tolmachev et al. (3). Moreover, the number of styles in our regenerated plants, as well as in extant ones, was found to be preferentially three (rarely 4 to 5), and the number of teeth in the dehiscing capsule was twice as many as the number of styles, which was typical for S. stenophylla, whereas the S. linnaeana group was indicated to have five styles (4). The areas of distribution S. stenophylla and some species of the S. linnaeana group overlap in Kolyma Lowland (3), which is in accordance with the fact of simultaneous discovery of their seeds in fossil burrows (5).