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  1. Abstract Background

    Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination between fossil and living microorganisms in ancient cryogenic environments. DNA repair protocols were combined with high throughput sequencing (HTS) of separate iDNA and eDNA fraction to reconstruct metagenome-assembled genomes (MAGs) from ancient microbial DNA entrapped in Siberian coastal permafrost.

    Results

    Despite the severe DNA damage in ancient permafrost, the coupling of DNA repair and HTS resulted in a total of 52 MAGs from sediments across a chronosequence (26–120 kyr). These MAGs were compared with those derived from the same samples but without utilizing DNA repair protocols. The MAGs from the youngest stratum showed minimal DNA damage and thus likely originated from viable, active microbial species. Many MAGs from the older and deeper sediment appear related to past aerobic microbial populations that had died upon freezing. MAGs from anaerobic lineages, includingAsgardarchaea, however exhibited minimal DNA damage and likely represent extant living microorganisms that have become adapted to the cryogenic and anoxic environments. The integration of aspartic acid racemization modeling and metaproteomics further constrained the metabolic status of the living microbial populations. Collectively, combining DNA repair protocols with HTS unveiled the adaptive strategies of microbes to long-term survivability in ancient permafrost.

    Conclusions

    Our results indicated that coupling of DNA repair protocols with simultaneous sequencing of iDNA and eDNA fractions enabled the assembly of MAGs from past and living microorganisms in ancient permafrost. The genomic reconstruction from the past and extant microbial populations expanded our understanding about the microbial successions and biogeochemical alterations from the past paleoenvironment to the present-day frozen state. Furthermore, we provided genomic insights into long-term survival mechanisms of microorganisms under cryogenic conditions through geological time. The combined strategies in this study can be extrapolated to examine other ancient non-permafrost environments and constrain the search for past and extant extraterrestrial life in permafrost and ice deposits on Mars.

     
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  2. Ice-rich permafrost in the circum-Arctic and sub-Arctic (hereafter pan-Arctic), such as late Pleistocene Yedoma, are especially prone to degradation due to climate change or human activity. When Yedoma deposits thaw, large amounts of frozen organic matter and biogeochemically relevant elements return into current biogeochemical cycles. This mobilization of elements has local and global implications: increased thaw in thermokarst or thermal erosion settings enhances greenhouse gas fluxes from permafrost regions. In addition, this ice-rich ground is of special concern for infrastructure stability as the terrain surface settles along with thawing. Finally, understanding the distribution of the Yedoma domain area provides a window into the Pleistocene past and allows reconstruction of Ice Age environmental conditions and past mammoth-steppe landscapes. Therefore, a detailed assessment of the current pan-Arctic Yedoma coverage is of importance to estimate its potential contribution to permafrost-climate feedbacks, assess infrastructure vulnerabilities, and understand past environmental and permafrost dynamics. Building on previous mapping efforts, the objective of this paper is to compile the first digital pan-Arctic Yedoma map and spatial database of Yedoma coverage. Therefore, we 1) synthesized, analyzed, and digitized geological and stratigraphical maps allowing identification of Yedoma occurrence at all available scales, and 2) compiled field data and expert knowledge for creating Yedoma map confidence classes. We used GIS-techniques to vectorize maps and harmonize site information based on expert knowledge. We included a range of attributes for Yedoma areas based on lithological and stratigraphic information from the source maps and assigned three different confidence levels of the presence of Yedoma (confirmed, likely, or uncertain). Using a spatial buffer of 20 km around mapped Yedoma occurrences, we derived an extent of the Yedoma domain. Our result is a vector-based map of the current pan-Arctic Yedoma domain that covers approximately 2,587,000 km 2 , whereas Yedoma deposits are found within 480,000 km 2 of this region. We estimate that 35% of the total Yedoma area today is located in the tundra zone, and 65% in the taiga zone. With this Yedoma mapping, we outlined the substantial spatial extent of late Pleistocene Yedoma deposits and created a unique pan-Arctic dataset including confidence estimates. 
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  3. ABSTRACT 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. 
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  4. ABSTRACT Here, we report the draft genome sequence of Microbacterium sp. strain Gd 4-13, isolated from late Pleistocene permafrost of marine origin located on the Gydanskiy Peninsula. Genome sequence analysis was performed to understand strain survivability mechanisms under permafrost conditions and to expand biotechnology applications. 
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  5. ABSTRACT The prevalence of microbial life in permafrost up to several million years (Ma) old has been well documented. However, the long-term survivability, evolution, and metabolic activity of the entombed microbes over this time span remain underexplored. We integrated aspartic acid (Asp) racemization assays with metagenomic sequencing to characterize the microbial activity, phylogenetic diversity, and metabolic functions of indigenous microbial communities across a ∼0.01- to 1.1-Ma chronosequence of continuously frozen permafrost from northeastern Siberia. Although Asp in the older bulk sediments (0.8 to 1.1 Ma) underwent severe racemization relative to that in the youngest sediment (∼0.01 Ma), the much lower d -Asp/ l -Asp ratio (0.05 to 0.14) in the separated cells from all samples suggested that indigenous microbial communities were viable and metabolically active in ancient permafrost up to 1.1 Ma. The microbial community in the youngest sediment was the most diverse and was dominated by the phyla Actinobacteria and Proteobacteria . In contrast, microbial diversity decreased dramatically in the older sediments, and anaerobic, spore-forming bacteria within Firmicutes became overwhelmingly dominant. In addition to the enrichment of sporulation-related genes, functional genes involved in anaerobic metabolic pathways such as fermentation, sulfate reduction, and methanogenesis were more abundant in the older sediments. Taken together, the predominance of spore-forming bacteria and associated anaerobic metabolism in the older sediments suggest that a subset of the original indigenous microbial community entrapped in the permafrost survived burial over geological time. IMPORTANCE Understanding the long-term survivability and associated metabolic traits of microorganisms in ancient permafrost frozen millions of years ago provides a unique window into the burial and preservation processes experienced in general by subsurface microorganisms in sedimentary deposits because of permafrost’s hydrological isolation and exceptional DNA preservation. We employed aspartic acid racemization modeling and metagenomics to determine which microbial communities were metabolically active in the 1.1-Ma permafrost from northeastern Siberia. The simultaneous sequencing of extracellular and intracellular genomic DNA provided insight into the metabolic potential distinguishing extinct from extant microorganisms under frozen conditions over this time interval. This in-depth metagenomic sequencing advances our understanding of the microbial diversity and metabolic functions of extant microbiomes from early Pleistocene permafrost. Therefore, these findings extend our knowledge of the survivability of microbes in permafrost from 33,000 years to 1.1 Ma. 
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  6. The gas shows in the permafrost zone represent a hazard for exploration, form the surface features, and are improperly estimated in the global methane budget. They contain methane of either surficial or deep-Earth origin accumulated earlier in the form of gas or gas hydrates in lithological traps in permafrost. From these traps, it rises through conduits, which have tectonic origin or are associated with permafrost degradation. We report methane fluxes from 20-m to 30-m deep boreholes, which are the artificial conduits for gas from permafrost in Siberia. The dynamics of degassing the traps was studied using static chambers, and compared to the concentration of methane in permafrost as analyzed by the headspace method and gas chromatography. More than 53 g of CH4 could be released to the atmosphere at rates exceeding 9 g of CH4 m−2 s−1 from a trap in epigenetic permafrost disconnected from traditional geological sources over a period from a few hours to several days. The amount of methane released from a borehole exceeded the amount of the gas that was enclosed in large volumes of permafrost within a diameter up to 5 meters around the borehole. Such gas shows could be by mistake assumed as permanent gas seeps, which leads to the overestimation of the role of permafrost in global warming. 
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  7. A genomic reconstruction belonging to the genus Methanosarcina was assembled from metagenomic data from a methane-producing enrichment of Antarctic permafrost. This is the first methanogen genome reported from permafrost of the Dry Valleys and can help shed light on future climate-affected methane dynamics. 
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  8. A comparative analysis of the metagenomes from two 30 000-year-old permafrost samples, one of lake-alluvial origin and the other from late Pleistocene Ice Complex sediments, revealed significant differences within microbial communities. The late Pleistocene Ice Complex sediments (which have been characterized by the absence of methane with lower values of redox potential and Fe2+ content) showed a low abundance of methanogenic archaea and enzymes from both the carbon and nitrogen cycles, but a higher abundance of enzymes associated with the sulfur cycle. The metagenomic and geochemical analyses described in the paper provide evidence that the formation of the sampled late Pleistocene Ice Complex sediments likely took place under much more aerobic conditions than lake-alluvial sediments. 
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