Temporal dynamics of woolly mammoth genome erosion prior to extinction.

A number of species have recently recovered from near-extinction. Although these species have avoided the immediate extinction threat, their long-term viability remains precarious due to the potential genetic consequences of population declines, which are poorly understood on a timescale beyond a few generations. Woolly mammoths (Mammuthus primigenius) became isolated on Wrangel Island around 10,000 years ago and persisted for over 200 generations before becoming extinct around 4,000 years ago. To study the evolutionary processes leading up to the mammoths' extinction, we analyzed 21 Siberian woolly mammoth genomes. Our results show that the population recovered quickly from a severe bottleneck and remained demographically stable during the ensuing six millennia. We find that mildly deleterious mutations gradually accumulated, whereas highly deleterious mutations were purged, suggesting ongoing inbreeding depression that lasted for hundreds of generations. The time-lag between demographic and genetic recovery has wide-ranging implications for conservation management of recently bottlenecked populations.

Testosterone histories from tusks reveal woolly mammoth musth episodes.

Hormones in biological media reveal endocrine activity related to development, reproduction, disease and stress on different timescales1. Serum provides immediate circulating concentrations2, whereas various tissues record steroid hormones accumulated over time3,4. Hormones have been studied in keratin, bones and teeth in modern5-8 and ancient contexts9-12; however, the biological significance of such records is subject to ongoing debate10,13-16, and the utility of tooth-associated hormones has not previously been demonstrated. Here we use liquid chromatography with tandem mass spectrometry paired with fine-scale serial sampling to measure steroid hormone concentrations in modern and fossil tusk dentin. An adult male African elephant (Loxodonta africana) tusk shows periodic increases in testosterone that reveal episodes of musth17-19, an annually recurring period of behavioural and physiological changes that enhance mating success20-23. Parallel assessments of a male woolly mammoth (Mammuthus primigenius) tusk show that mammoths also experienced musth. These results set the stage for wide-ranging studies using steroids preserved in dentin to investigate development, reproduction and stress in modern and extinct mammals. Because dentin grows by apposition, resists degradation, and often contains growth lines, teeth have advantages over other tissues that are used as records of endocrine data. Given the low mass of dentin powder required for analytical precision, we anticipate dentin-hormone studies to extend to smaller animals. Thus, in addition to broad applications in zoology and palaeontology, tooth hormone records could support medical, forensic, veterinary and archaeological studies.

Late Quaternary dynamics of Arctic biota from ancient environmental genomics.

During the last glacial-interglacial cycle, Arctic biotas experienced substantial climatic changes, yet the nature, extent and rate of their responses are not fully understood1-8. Here we report a large-scale environmental DNA metagenomic study of ancient plant and mammal communities, analysing 535 permafrost and lake sediment samples from across the Arctic spanning the past 50,000 years. Furthermore, we present 1,541 contemporary plant genome assemblies that were generated as reference sequences. Our study provides several insights into the long-term dynamics of the Arctic biota at the circumpolar and regional scales. Our key findings include: (1) a relatively homogeneous steppe-tundra flora dominated the Arctic during the Last Glacial Maximum, followed by regional divergence of vegetation during the Holocene epoch; (2) certain grazing animals consistently co-occurred in space and time; (3) humans appear to have been a minor factor in driving animal distributions; (4) higher effective precipitation, as well as an increase in the proportion of wetland plants, show negative effects on animal diversity; (5) the persistence of the steppe-tundra vegetation in northern Siberia enabled the late survival of several now-extinct megafauna species, including the woolly mammoth until 3.9 ± 0.2 thousand years ago (ka) and the woolly rhinoceros until 9.8 ± 0.2 ka; and (6) phylogenetic analysis of mammoth environmental DNA reveals a previously unsampled mitochondrial lineage. Our findings highlight the power of ancient environmental metagenomics analyses to advance understanding of population histories and long-term ecological dynamics.

Meta-proteomic analysis of two mammoth's trunks by EVA technology and high-resolution mass spectrometry for an indirect picture of their habitat and the characterization of the collagen type I, alpha-1 and alpha-2 sequence.

The recent paleoproteomic studies, including paleo-metaproteomic analyses, improved our understanding of the dietary of ancient populations, the characterization of past human diseases, the reconstruction of the habitat of ancient species, but also provided new insights into the phylogenetic relationships between extant and extinct species. In this respect, the present work reports the results of the metaproteomic analysis performed on the middle part of a trunk, and on the portion of a trunk tip tissue of two different woolly mammoths some 30,000 years old. In particular, proteins were extracted by applying EVA (Ethylene-Vinyl Acetate studded with hydrophilic and hydrophobic resins) films to the surface of these tissues belonging to two Mammuthus primigenus specimens, discovered in two regions located in the Russian Far East, and then investigated via a shotgun MS-based approach. This approach allowed to obtain two interesting results: (i) an indirect description of the habitat of these two mammoths, and (ii) an improved characterization of the collagen type I, alpha-1 and alpha-2 chains (col1a1 and col1a2). Sequence characterization of the col1a1 and col1a2 highlighted some differences between M. primigenius and other Proboscidea together with the identification of three (two for col1a1, and one for col1a2) potentially diagnostic amino acidic mutations that could be used to reliably distinguish the Mammuthus primigenius with respect to the other two genera of elephantids (i.e., Elephas and Loxodonta), and the extinct American mastodon (i.e., Mammut americanum). The results were validated through the level of deamidation and other diagenetic chemical modifications of the sample peptides, which were used to discriminate the "original" endogenous peptides from contaminant ones. The data have been deposited to the ProteomeXchange with identifier < PXD029558 > .

Meta-proteomic analysis of the Shandrin mammoth by EVA technology and high-resolution mass spectrometry: what is its gut microbiota telling us?

During the last decade, paleoproteomics allowed us to open a direct window into the biological past, improving our understanding of the phylogenetic relationships of extant and extinct species, past human diseases, and reconstruction of the human diet. In particular, meta-proteomic studies, mainly carried out on ancient human dental calculus, provided insights into past oral microbial communities and ancient diets. On the contrary, very few investigations regard the analysis of ancient gut microbiota, which may enable a greater understanding of how microorganisms and their hosts have co-evolved and spread under the influence of changing diet practices and habitat. In this respect, this paper reports the results of the first-ever meta-proteomic analysis carried out on a gut tissue sample some 40,000 years old. Proteins were extracted by applying EVA (ethylene-vinyl acetate) films to the surface of the gut sample of a woolly mammoth (Mammuthus primigenus), discovered in 1972 close to the Shandrin River (Yakutia, Russia), and then investigated via a shotgun MS-based approach. Proteomic and peptidomic analysis allowed in-depth exploration of its meta-proteome composition. The results were validated through the level of deamidation and other diagenetic chemical modifications of the sample peptides, which were used to discriminate the "original" endogenous peptides from contaminant ones. Overall, the results of the meta-proteomic analysis here reported agreeing with the previous paleobotanical studies and with the reconstructed habitat of the Shandrin mammoth and provided insight into its diet. The data have been deposited to the ProteomeXchange with identifier < PXD025518 > .

Fifty thousand years of Arctic vegetation and megafaunal diet.

Although it is generally agreed that the Arctic flora is among the youngest and least diverse on Earth, the processes that shaped it are poorly understood. Here we present 50 thousand years (kyr) of Arctic vegetation history, derived from the first large-scale ancient DNA metabarcoding study of circumpolar plant diversity. For this interval we also explore nematode diversity as a proxy for modelling vegetation cover and soil quality, and diets of herbivorous megafaunal mammals, many of which became extinct around 10 kyr bp (before present). For much of the period investigated, Arctic vegetation consisted of dry steppe-tundra dominated by forbs (non-graminoid herbaceous vascular plants). During the Last Glacial Maximum (25-15 kyr bp), diversity declined markedly, although forbs remained dominant. Much changed after 10 kyr bp, with the appearance of moist tundra dominated by woody plants and graminoids. Our analyses indicate that both graminoids and forbs would have featured in megafaunal diets. As such, our findings question the predominance of a Late Quaternary graminoid-dominated Arctic mammoth steppe.

Tracking the origins of Yakutian horses and the genetic basis for their fast adaptation to subarctic environments.

Yakutia, Sakha Republic, in the Siberian Far East, represents one of the coldest places on Earth, with winter record temperatures dropping below -70 °C. Nevertheless, Yakutian horses survive all year round in the open air due to striking phenotypic adaptations, including compact body conformations, extremely hairy winter coats, and acute seasonal differences in metabolic activities. The evolutionary origins of Yakutian horses and the genetic basis of their adaptations remain, however, contentious. Here, we present the complete genomes of nine present-day Yakutian horses and two ancient specimens dating from the early 19th century and ∼5,200 y ago. By comparing these genomes with the genomes of two Late Pleistocene, 27 domesticated, and three wild Przewalski's horses, we find that contemporary Yakutian horses do not descend from the native horses that populated the region until the mid-Holocene, but were most likely introduced following the migration of the Yakut people a few centuries ago. Thus, they represent one of the fastest cases of adaptation to the extreme temperatures of the Arctic. We find cis-regulatory mutations to have contributed more than nonsynonymous changes to their adaptation, likely due to the comparatively limited standing variation within gene bodies at the time the population was founded. Genes involved in hair development, body size, and metabolic and hormone signaling pathways represent an essential part of the Yakutian horse adaptive genetic toolkit. Finally, we find evidence for convergent evolution with native human populations and woolly mammoths, suggesting that only a few evolutionary strategies are compatible with survival in extremely cold environments.

Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome.

Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics.

Development of a microarray-based method for allergen-specific IgE and IgG4 detection.

BACKGROUND: sIgE and sIgG4 detection is necessary for more accurate and effective type I hypersensitivity diagnostics and the estimation of disease development. Typically, the analyses of these antibodies are performed separately with the help of various specialized systems. The aim of this study was to develop a microarray-based method for the simultaneous quantitative detection of sIgE and sIgG4 to the most common allergens in a single sample. METHODS: A quantitative method for the simultaneous detection of sIgE and sIgG4 was developed based on the technology of hydrogel microchips previously designed at Engelhardt Institute of Molecular Biology, Russian Academy of Sciences (EIMB RAS). The microarray contained gel pads with immobilized allergens and gel pads that allow for the obtaining of sIgE and sIgG4 internal calibration curves for each allergen during the assay. The possibility of the simultaneous detection of sIgE and sIgG4 was developed using the corresponding Cy5 and Cy3 fluorescent dyes. RESULTS: The multiplex immunoassay method using hydrogel microarrays developed in this study allowed the quantitative detection of sIgE and sIgG4 to 31 allergens from different groups in a single assay. A comparison of the microarray with the existing plate-based analogues (i.e., ALLERG-O-LIQ and sIgG4 ELISA) was performed by analysing 152 blood serum samples and by evaluating Pearson correlation coefficients, ROC analysis, and Passing-Bablok linear regression results. CONCLUSION: The implementation of this method in allergy diagnostics will provide the possibility of simultaneously performing primary patient screening and obtaining additional information concerning the severity of the allergies and the choice of an appropriate therapy.

Species-specific responses of Late Quaternary megafauna to climate and humans.

Despite decades of research, the roles of climate and humans in driving the dramatic extinctions of large-bodied mammals during the Late Quaternary period remain contentious. Here we use ancient DNA, species distribution models and the human fossil record to elucidate how climate and humans shaped the demographic history of woolly rhinoceros, woolly mammoth, wild horse, reindeer, bison and musk ox. We show that climate has been a major driver of population change over the past 50,000 years. However, each species responds differently to the effects of climatic shifts, habitat redistribution and human encroachment. Although climate change alone can explain the extinction of some species, such as Eurasian musk ox and woolly rhinoceros, a combination of climatic and anthropogenic effects appears to be responsible for the extinction of others, including Eurasian steppe bison and wild horse. We find no genetic signature or any distinctive range dynamics distinguishing extinct from surviving species, emphasizing the challenges associated with predicting future responses of extant mammals to climate and human-mediated habitat change.

Sequencing the nuclear genome of the extinct woolly mammoth.

In 1994, two independent groups extracted DNA from several Pleistocene epoch mammoths and noted differences among individual specimens. Subsequently, DNA sequences have been published for a number of extinct species. However, such ancient DNA is often fragmented and damaged, and studies to date have typically focused on short mitochondrial sequences, never yielding more than a fraction of a per cent of any nuclear genome. Here we describe 4.17 billion bases (Gb) of sequence from several mammoth specimens, 3.3 billion (80%) of which are from the woolly mammoth (Mammuthus primigenius) genome and thus comprise an extensive set of genome-wide sequence from an extinct species. Our data support earlier reports that elephantid genomes exceed 4 Gb. The estimated divergence rate between mammoth and African elephant is half of that between human and chimpanzee. The observed number of nucleotide differences between two particular mammoths was approximately one-eighth of that between one of them and the African elephant, corresponding to a separation between the mammoths of 1.5-2.0 Myr. The estimated probability that orthologous elephant and mammoth amino acids differ is 0.002, corresponding to about one residue per protein. Differences were discovered between mammoth and African elephant in amino-acid positions that are otherwise invariant over several billion years of combined mammalian evolution. This study shows that nuclear genome sequencing of extinct species can reveal population differences not evident from the fossil record, and perhaps even discover genetic factors that affect extinction.

Ancient DNA analyses exclude humans as the driving force behind late Pleistocene musk ox (Ovibos moschatus) population dynamics.

The causes of the late Pleistocene megafaunal extinctions are poorly understood. Different lines of evidence point to climate change, the arrival of humans, or a combination of these events as the trigger. Although many species went extinct, others, such as caribou and bison, survived to the present. The musk ox has an intermediate story: relatively abundant during the Pleistocene, it is now restricted to Greenland and the Arctic Archipelago. In this study, we use ancient DNA sequences, temporally unbiased summary statistics, and Bayesian analytical techniques to infer musk ox population dynamics throughout the late Pleistocene and Holocene. Our results reveal that musk ox genetic diversity was much higher during the Pleistocene than at present, and has undergone several expansions and contractions over the past 60,000 years. Northeast Siberia was of key importance, as it was the geographic origin of all samples studied and held a large diverse population until local extinction at approximately 45,000 radiocarbon years before present ((14)C YBP). Subsequently, musk ox genetic diversity reincreased at ca. 30,000 (14)C YBP, recontracted at ca. 18,000 (14)C YBP, and finally recovered in the middle Holocene. The arrival of humans into relevant areas of the musk ox range did not affect their mitochondrial diversity, and both musk ox and humans expanded into Greenland concomitantly. Thus, their population dynamics are better explained by a nonanthropogenic cause (for example, environmental change), a hypothesis supported by historic observations on the sensitivity of the species to both climatic warming and fluctuations.

Ancient DNA of the Don-Hares Assumes the Existence of Two Distinct Mitochondrial Clades in Northeast Asia.

Paleoclimatic changes during the Pleistocene-Holocene transition is suggested as a main factor that led to species extinction, including the woolly mammoth (Mammuthus primigenius), Steller's sea cow (Hydrodamalis gigas) and the Don-hare (Lepus tanaiticus). These species inhabited the territory of Eurasia during the Holocene, but eventually went extinct. The Don-hare is an extinct species of the genus Lepus (Leporidae, Lagomorpha), which lived in the Late Pleistocene-Early Holocene in Eastern Europe and Northern Asia. For a long time, the Don-hare was considered a separate species, but at the same time, its species status was disputed, taking into account both morphological data and mitochondrial DNA. In this study, mitochondrial genomes of five Don-hares, whose remains were found on the territory of Northeastern Eurasia were reconstructed. Firstly, we confirm the phylogenetic proximity of the "young" specimens of Don-hare and mountain or white hare, and secondly, that samples older than 39 Kya form a completely distinct mitochondrial clade.

Genomics of adaptive evolution in the woolly mammoth.

Ancient genomes provide a tool to investigate the genetic basis of adaptations in extinct organisms. However, the identification of species-specific fixed genetic variants requires the analysis of genomes from multiple individuals. Moreover, the long-term scale of adaptive evolution coupled with the short-term nature of traditional time series data has made it difficult to assess when different adaptations evolved. Here, we analyze 23 woolly mammoth genomes, including one of the oldest known specimens at 700,000 years old, to identify fixed derived non-synonymous mutations unique to the species and to obtain estimates of when these mutations evolved. We find that at the time of its origin, the woolly mammoth had already acquired a broad spectrum of positively selected genes, including ones associated with hair and skin development, fat storage and metabolism, and immune system function. Our results also suggest that these phenotypes continued to evolve during the last 700,000 years, but through positive selection on different sets of genes. Finally, we also identify additional genes that underwent comparatively recent positive selection, including multiple genes related to skeletal morphology and body size, as well as one gene that may have contributed to the small ear size in Late Quaternary woolly mammoths.

Ancient DNA Reveals Maternal Philopatry of the Northeast Eurasian Brown Bear (Ursus arctos) Population during the Holocene.

Significant palaeoecological and paleoclimatic changes that took place during Late Pleistocene-Early Holocene transition are considered important factors that led to megafauna extinctions. Unlike many other species, the brown bear (Ursus arctos) has survived this geological time. Despite the fact that several mitochondrial DNA clades of brown bears became extinct at the end of the Pleistocene, this species is still widely distributed in Northeast Eurasia. Here, using the ancient DNA analysis of a brown bear individual that inhabited Northeast Asia in the Middle Holocene (3460 ± 40 years BP) and comparative phylogenetic analysis, we show a significant mitochondrial DNA similarity of the studied specimen with modern brown bears inhabiting Yakutia and Chukotka. In this study, we clearly demonstrate the maternal philopatry of the Northeastern Eurasian U. arctos population during the several thousand years of the Holocene.

Out of America: ancient DNA evidence for a new world origin of late quaternary woolly mammoths.

Although the iconic mammoth of the Late Pleistocene, the woolly mammoth (Mammuthus primigenius), has traditionally been regarded as the end point of a single anagenetically evolving lineage, recent paleontological and molecular studies have shown that successive allopatric speciation events must have occurred within Pleistocene Mammuthus in Asia, with subsequent expansion and hybridization between nominal taxa [1, 2]. However, the role of North American mammoth populations in these events has not been adequately explored from an ancient-DNA standpoint. To undertake this task, we analyzed mtDNA from a large data set consisting of mammoth samples from across Holarctica (n = 160) and representing most of radiocarbon time. Our evidence shows that, during the terminal Pleistocene, haplotypes originating in and characteristic of New World populations replaced or succeeded those endemic to Asia and western Beringia. Also, during the Last Glacial Maximum, mammoth populations do not appear to have suffered an overall decline in diversity, despite differing responses on either side of the Bering land bridge. In summary, the "Out-of-America" hypothesis holds that the dispersal of North American woolly mammoths into other parts of Holarctica created major phylogeographic structuring within Mammuthus primigenius populations, shaping the last phase of their evolutionary history before their demise.