Ancient genomic studies have extensively explored human-microbial interactions, yet research on non-human animals remains limited. In this study, we analyzed ancient microbial DNA from 483 mammoth remains spanning over 1 million years, including 440 newly sequenced and unpublished samples from a 1.1-million-year-old steppe mammoth. Using metagenomic screening, contaminant filtering, damage pattern analysis, and phylogenetic inference, we identified 310 microbes associated with different mammoth tissues. While most microbes were environmental or post-mortem colonizers, we recovered genomic evidence of six host-associated microbial clades spanning Actinobacillus, Pasteurella, Streptococcus, and Erysipelothrix. Some of these clades contained putative virulence factors, including a Pasteurella-related bacterium that had previously been linked to the deaths of African elephants. Notably, we reconstructed partial genomes of Erysipelothrix from the oldest mammoth sample, representing the oldest authenticated host-associated microbial DNA to date. This work demonstrates the potential of obtaining ancient animal microbiomes, which can inform further paleoecological and evolutionary research.

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.

Temporal genomic data hold great potential for studying evolutionary processes such as speciation. However, sampling across speciation events would, in many cases, require genomic time series that stretch well back intothe Early Pleistocene subepoch. Although theoretical models suggest that DNA should survive on this timescale(1), the oldest genomic data recovered so far are from a horse specimen dated to 780-560 thousand years ago(2). Here we report the recovery of genome-wide data from three mammoth specimens dating to the Early and Middle Pleistocene subepochs, two of which are more than one million years old. We find that two distinct mammoth lineages were present in eastern Siberia during the Early Pleistocene. One of these lineages gave rise to the woolly mammoth and the other represents a previously unrecognized lineage that was ancestral to the first mammoths to colonize North America. Our analyses reveal that the Columbian mammoth of North America traces its ancestry to a Middle Pleistocene hybridization between these two lineages, with roughly equal admixture proportions. Finally, we show that the majority of protein-coding changes associated with cold adaptation in woolly mammoths were already present one million years ago. These findings highlight the potential of deep-time palaeogenomics to expand our understanding of speciation and long-term adaptive evolution.

A pioneer comprehensive study of several diminutive last-generation woolly mammoth teeth (M3) found on the coast of the East Siberian Sea between the mouths of the Alazeya and Malaya Kuropatoch'ya rivers was conducted. Two teeth belonged to one individual. These teeth have a similar lamellar frequency and enamel thickness as teeth of Mammuthus primigenius Blumenbach. The molar crowns from the lower Alazeya region are similar in size to those of the small Late Pleistocene-Holocene mammoths from Wrangel Island. However, the number of plates (17-19, excluding talons) is much lower than that in the teeth of typical Late Pleistocene M. primigenius (23-25). The age data of the examined teeth are beyond the limits of the C-14 dating method (>45 000 years BP). Nevertheless, palaeobotanical data allow correlation of the enclosing sediments with the warm Kazantsevo Interglacial (Eemian, MIS 5e) and reconstruction of the average annual temperature, which was warmer than present-day temperatures. These conditions are confirmed by the delta O-18 isotopes from the structurally bound carbonate in tooth enamel. The ancient landscape was wetter and more forested than modern landscapes. The diminution of M3 size and loss of posterior plates were a result of the overall decrease in body size, likely in response to landscape change and narrowing of resource space. Mammoths from the lower Alazeya region demonstrate a stage of significant size reduction, although the dwarfing was not finalized. Their teeth are the oldest amongst the small teeth found in west Beringia.

The grey wolf (Canis lupus Linnaeus, 1758) is one of the most widespread large carnivores on Earth, and occurs throughout the Arctic. Although wolf diet is well studied, we have scant information from high Arctic areas. Global warming is expected to increase the importance of predation for ecosystem regulation in Arctic environments. To improve our ability to manage Arctic ecosystems under environmental change, we therefore need knowledge about Arctic predator diets. Prey remains in 54 wolf scats collected at three sites in the high Arctic region surrounding the Hall Basin (Judge Daly Promontory, Ellesmere Island, Canada, and Washington Land and Hall Land, both in northwestern Greenland) pointed to a dietary importance of arctic hare (Lepus arcticus Ross, 1819; 55% frequency of occurrence) and muskoxen (Ovibos moschatus (Zimmermann, 1780); 39% frequency of occurrence), although we observed diet variation among the sites. A literature compilation suggested that arctic wolves (Canis lupus arctos Pocock, 1935) preferentially feed on caribou (Rangifer tarandus (Linnaeus, 1758)) and muskoxen, but can sustain themselves on arctic hares and Greenland collared lemmings (Dicrostonyx groenlandicus (Traill, 1823)) in areas with limited or no ungulate populations. We suggest that climate change may alter the dynamics among wolves, arctic hare, muskoxen, and caribou, and we encourage further studies evaluating how climate change influences predator-prey interactions in high Arctic environments.

Species worldwide are subject to contractions in both abundance and geographical range, and their persistence in a changing environment may thus depend on the ability to survive in small and fragmented populations. Despite the urgent need to understand how extinction works, our knowledge of pre-extinction genetic processes is limited because techniques allowing population and conservation genomics to be studied in wild threatened populations have become available only recently. In this thesis, I used the last surviving population of the woolly mammoth (Mammuthus primigenius) as a model for studying pre-extinction population dynamics. I used ancient DNA as a tool to study microevolutionary processes in real time, analysing genetic changes in response to environmental shifts at the end of the last Ice Age and exploring impacts of genetic drift and inbreeding as woolly mammoths became isolated on Wrangel Island and survived for 6000 years at small population size. Using mitochondrial genomes, I found evidence of a founder effect that decreased the maternal diversity to a single lineage at the time when mammoths became trapped on Wrangel Island (~10,500 years ago). Moreover, a two- to three-fold higher mitochondrial mutation rate in Holocene and a fixed, potentially detrimental mutation in the ATP6 gene encoding for one of the key enzymes of the oxidative phosphorylation pathway, is consistent with the hypothesis that selection is less effective in removing deleterious mutations in small populations. A loss of diversity was also observed in an immunity gene that belongs to the major histocompatibility complex (MHC), even though the MHC is considered to be under balancing selection. Low-coverage genomic data was analysed in order to estimate endogenous DNA content and molecular sex of the mammoth samples. The observation of a male bias (69%) in the sex ratio led to the conclusion that male mammoths were more likely to die in a way that ensured good preservation. Another potential way of getting information about life history strategies of extinct species, which was explored here, is by measuring testosterone levels in mammoth hair shafts in connection with molecular sex inference. Finally, given that previous estimates have suggested a very small Holocene effective population size on Wrangel Island and thus that the population may have been too small to avoid genome erosion, four mammoths were sequenced to a high coverage in order to look for genomic consequences of small population size. When compared to mammoths from the Pleistocene mainland population, Wrangel Island mammoths had lower levels of genome-wide diversity and had a higher proportion of their genomes allocated in runs of homozygosity, which are large fragments completely depleted of diversity. Importantly, genome erosion appears to have accelerated in the last ten generations before the extinction, resulting in the last known woolly mammoth having almost 40% of its genome without any genetic diversity. Overall, these results highlight how genetic drift and inbreeding triggered genomic deterioration in the last surviving woolly mammoth population. Although Wrangel Island was a refugium, where mammoths survived for thousands of years after the last Ice Age, and the causal factors of the final extinction are not yet clear, isolation and small population size without any possibility of new gene flow may have contributed to reduced fitness, and thus to extinction. 

Testosterone is a key regulator in vertebrate development, physiology and behaviour. Whereas technology allows extraction of a wealth of genetic information from extant as well as extinct species, complementary information on steroid hormone levels may add a social, sexual and environmental context. Hair shafts have been previously used to sequence DNA from >50000 C-14 years old Siberian woolly mammoths (Mammuthus primigenius). Hair-testing has also been used to measure endogenous steroids in multiple extant species. Here we use small quantities of woolly mammoth hair samples to measure testosterone, and a genomics-based approach to determine sex, in permafrost-preserved mammoths dated to c. 10000-60000 C-14 years. Our validated method opens up exciting opportunities to measure multiple steroids in keratinized tissues from extinct populations of mammals. This may be specifically applied to investigating life histories, including the extinct Quaternary megafauna populations whose remains are preserved in the permafrost throughout the northern hemisphere.

While present-day taxa are valuable proxies for understanding the biology of extinct species, it is also crucial to examine physical remains in order to obtain a more comprehensive view of their behavior, social structure, and life histories [1, 2]. For example, information on demographic parameters such as age distribution and sex ratios in fossil assemblages can be used to accurately infer socioecological patterns (e.g., [3]). Here we use genomic data to determine the sex of 98 woolly mammoth (Mammuthus primigenius) specimens in order to infer social and behavioral patterns in the last 60,000 years of the species' existence. We report a significant excess of males among the identified samples (69% versus 31%; p < 0.0002). We argue that this male bias among mammoth remains is best explained by males more often being caught in natural traps that favor preservation. Wehypothesize that this is a consequence of social structure in proboscideans, which is characterized by matriarchal hierarchy and sex segregation. Without the experience associated with living in a matriarchal family group, or a bachelor group with an experienced bull, young or solitary males may have been more prone to die in natural traps where good preservation is more likely.

The onset of the Holocene was associated with a global temperature increase, which led to a rise in sea levels and isolation of the last surviving population of woolly mammoths on Wrangel Island. Understanding what happened with the population's genetic diversity at the time of the isolation and during the ensuing 6000 years can help clarify the effects of bottlenecks and subsequent limited population sizes in species approaching extinction. Previous genetic studies have highlighted questions about how the Holocene Wrangel population was established and how the isolation event affected genetic diversity. Here, we generated high-quality mitogenomes from 21 radiocarbon-dated woolly mammoths to compare the ancestral large and genetically diverse Late Pleistocene Siberian population and the small Holocene Wrangel population. Our results indicate that mitogenome diversity was reduced to one single haplotype at the time of the isolation, and thus that the Holocene Wrangel Island population was established by a single maternal lineage. Moreover, we show that the ensuing small effective population size coincided with fixation of a nonsynonymous mutation, and a comparative analysis of mutation rates suggests that the evolutionary rate was accelerated in the Holocene population. These results suggest that isolation on Wrangel Island led to an increase in the frequency of deleterious genetic variation, and thus are consistent with the hypothesis that strong genetic drift in small populations leads to purifying selection being less effective in removing deleterious mutations.