Population genomics of the muskox' resilience in the near absence of genetic variation.

Genomic studies of species threatened by extinction are providing crucial information about evolutionary mechanisms and genetic consequences of population declines and bottlenecks. However, to understand how species avoid the extinction vortex, insights can be drawn by studying species that thrive despite past declines. Here, we studied the population genomics of the muskox (Ovibos moschatus), an Ice Age relict that was at the brink of extinction for thousands of years at the end of the Pleistocene yet appears to be thriving today. We analysed 108 whole genomes, including present-day individuals representing the current native range of both muskox subspecies, the white-faced and the barren-ground muskox (O. moschatus wardi and O. moschatus moschatus) and a ~21,000-year-old ancient individual from Siberia. We found that the muskox' demographic history was profoundly shaped by past climate changes and post-glacial re-colonizations. In particular, the white-faced muskox has the lowest genome-wide heterozygosity recorded in an ungulate. Yet, there is no evidence of inbreeding depression in native muskox populations. We hypothesize that this can be explained by the effect of long-term gradual population declines that allowed for purging of strongly deleterious mutations. This study provides insights into how species with a history of population bottlenecks, small population sizes and low genetic diversity survive against all odds.

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.

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.

Population dynamics and demographic history of Eurasian collared lemmings.

BACKGROUND: Ancient DNA studies suggest that Late Pleistocene climatic changes had a significant effect on population dynamics in Arctic species. The Eurasian collared lemming (Dicrostonyx torquatus) is a keystone species in the Arctic ecosystem. Earlier studies have indicated that past climatic fluctuations were important drivers of past population dynamics in this species. RESULTS: Here, we analysed 59 ancient and 54 modern mitogenomes from across Eurasia, along with one modern nuclear genome. Our results suggest population growth and genetic diversification during the early Late Pleistocene, implying that collared lemmings may have experienced a genetic bottleneck during the warm Eemian interglacial. Furthermore, we find multiple temporally structured mitogenome clades during the Late Pleistocene, consistent with earlier results suggesting a dynamic late glacial population history. Finally, we identify a population in northeastern Siberia that maintained genetic diversity and a constant population size at the end of the Pleistocene, suggesting suitable conditions for collared lemmings in this region during the increasing temperatures associated with the onset of the Holocene. CONCLUSIONS: This study highlights an influence of past warming, in particular the Eemian interglacial, on the evolutionary history of the collared lemming, along with spatiotemporal population structuring throughout the Late Pleistocene.

Evolutionary consequences of genomic deletions and insertions in the woolly mammoth genome.

Woolly mammoths had a set of adaptations that enabled them to thrive in the Arctic environment. Many mammoth-specific single nucleotide polymorphisms (SNPs) responsible for unique mammoth traits have been previously identified from ancient genomes. However, a multitude of other genetic variants likely contributed to woolly mammoth evolution. In this study, we sequenced two woolly mammoth genomes and combined these with previously sequenced mammoth and elephant genomes to conduct a survey of mammoth-specific deletions and indels. We find that deletions are highly enriched in non-coding regions, suggesting selection against structural variants that affect protein sequences. Nonetheless, at least 87 woolly mammoth genes contain deletions or indels that modify the coding sequence, including genes involved in skeletal morphology and hair growth. These results suggest that deletions and indels contributed to the unique phenotypic adaptations of the woolly mammoth, and were potentially critical to surviving in its natural environment.

Development and Optimization of a Silica Column-Based Extraction Protocol for Ancient DNA.

Rapid and cost-effective retrieval of endogenous DNA from ancient specimens remains a limiting factor in palaeogenomic research. Many methods have been developed to increase ancient DNA yield, but modifications to existing protocols are often based on personal experience rather than systematic testing. Here, we present a new silica column-based extraction protocol, where optimizations were tested in controlled experiments. Using relatively well-preserved permafrost samples, we tested the efficiency of pretreatment of bone and tooth powder with a bleach wash and a predigestion step. We also tested the recovery efficiency of MinElute and QIAquick columns, as well as Vivaspin columns with two molecular weight cut-off values. Finally, we tested the effect of uracil-treatment with two different USER enzyme concentrations. We find that neither bleach wash combined with a predigestion step, nor predigestion by itself, significantly increased sequencing efficiency. Initial results, however, suggest that MinElute columns are more efficient for ancient DNA extractions than QIAquick columns, whereas different molecular weight cut-off values in centrifugal concentrator columns did not have an effect. Uracil treatments are effective at removing DNA damage even at concentrations of 0.15 U/µL (as compared to 0.3 U/µL) of ancient DNA extracts.

Modeling Eastern Russian High Arctic Geese (Anser fabalis, A. albifrons) during moult and brood rearing in the 'New Digital Arctic'.

Many polar species and habitats are now affected by man-made global climate change and underlying infrastructure. These anthropogenic forces have resulted in clear implications and many significant changes in the arctic, leading to the emergence of new climate, habitats and other issues including digital online infrastructure representing a 'New Artic'. Arctic grazers, like Eastern Russian migratory populations of Tundra Bean Goose Anser fabalis and Greater White-fronted Goose A. albifrons, are representative examples and they are affected along the entire flyway in East Asia, namely China, Japan and Korea. Here we present the best publicly-available long-term (24 years) digitized geographic information system (GIS) data for the breeding study area (East Yakutia and Chukotka) and its habitats with ISO-compliant metadata. Further, we used seven publicly available compiled Open Access GIS predictor layers to predict the distribution for these two species within the tundra habitats. Using BIG DATA we are able to improve on the ecological niche prediction inference for both species by focusing for the first time specifically on biological relevant population cohorts: post-breeding moulting non-breeders, as well as post-breeding parent birds with broods. To assure inference with certainty, we assessed it with 4 lines of evidence including alternative best-available open access field data from GBIF.org as well as occurrence data compiled from the literature. Despite incomplete data, we found a good model accuracy in support of our evidence for a robust inference of the species distributions. Our predictions indicate a strong publicly best-available relative index of occurrence (RIO). These results are based on the quantified ecological niche showing more realistic gradual occurrence patterns but which are not fully in agreement with the current strictly applied parsimonious flyway and species delineations. While our predictions are to be improved further, e.g. when synergetic data are made freely available, here we offer within data caveats the first open access model platform for fine-tuning and future predictions for this otherwise poorly represented region in times of a rapid changing industrialized 'New Arctic' with global repercussions.

Ancient and modern genomes unravel the evolutionary history of the rhinoceros family.

Only five species of the once-diverse Rhinocerotidae remain, making the reconstruction of their evolutionary history a challenge to biologists since Darwin. We sequenced genomes from five rhinoceros species (three extinct and two living), which we compared to existing data from the remaining three living species and a range of outgroups. We identify an early divergence between extant African and Eurasian lineages, resolving a key debate regarding the phylogeny of extant rhinoceroses. This early Miocene (∼16 million years ago [mya]) split post-dates the land bridge formation between the Afro-Arabian and Eurasian landmasses. Our analyses also show that while rhinoceros genomes in general exhibit low levels of genome-wide diversity, heterozygosity is lowest and inbreeding is highest in the modern species. These results suggest that while low genetic diversity is a long-term feature of the family, it has been particularly exacerbated recently, likely reflecting recent anthropogenic-driven population declines.

Ancient horse genomes reveal the timing and extent of dispersals across the Bering Land Bridge.

The Bering Land Bridge (BLB) last connected Eurasia and North America during the Late Pleistocene. Although the BLB would have enabled transfers of terrestrial biota in both directions, it also acted as an ecological filter whose permeability varied considerably over time. Here we explore the possible impacts of this ecological corridor on genetic diversity within, and connectivity among, populations of a once wide-ranging group, the caballine horses (Equus spp.). Using a panel of 187 mitochondrial and eight nuclear genomes recovered from present-day and extinct caballine horses sampled across the Holarctic, we found that Eurasian horse populations initially diverged from those in North America, their ancestral continent, around 1.0-0.8 million years ago. Subsequent to this split our mitochondrial DNA analysis identified two bidirectional long-range dispersals across the BLB ~875-625 and ~200-50 thousand years ago, during the Middle and Late Pleistocene. Whole genome analysis indicated low levels of gene flow between North American and Eurasian horse populations, which probably occurred as a result of these inferred dispersals. Nonetheless, mitochondrial and nuclear diversity of caballine horse populations retained strong phylogeographical structuring. Our results suggest that barriers to gene flow, currently unidentified but possibly related to habitat distribution across Beringia or ongoing evolutionary divergence, played an important role in shaping the early genetic history of caballine horses, including the ancestors of living horses within Equus ferus.

Combining modern tracking data and historical records improves understanding of the summer habitats of the Eastern Lesser White-fronted Goose Anser erythropus.

The Lesser White-fronted Goose (Anser erythropus), smallest of the "gray" geese, is listed as Vulnerable on the IUCN Red List and protected in all range states. There are three populations, with the least studied being the Eastern population, shared between Russia and China. The extreme remoteness of breeding enclaves makes them largely inaccessible to researchers. As a substitute for visitation, remotely tracking birds from wintering grounds allows exploration of their summer range. Over a period of three years, and using highly accurate GPS tracking devices, eleven individuals of A. erythropus were tracked from the key wintering site of China, to summering, and staging sites in northeastern Russia. Data obtained from that tracking, bolstered by ground survey and literature records, were used to model the summer distribution of A. erythropus. Although earlier literature describes a patchy summer range, the model suggests a contiguous summer habitat range is possible, although observations to date cannot confirm A. erythropus is present throughout the modeled range. The most suitable habitats are located along the coasts of the Laptev Sea, primarily the Lena Delta, in the Yana-Kolyma Lowland, and smaller lowlands of Chukotka with narrow riparian extensions upstream along major rivers such as the Lena, Indigirka, and Kolyma. The probability of A. erythropus presence is related to areas with altitude less than 500 m with abundant wetlands, especially riparian habitat, and a climate with precipitation of the warmest quarter around 55 mm and mean temperature around 14°C during June-August. Human disturbance also affects site suitability, with a gradual decrease in species presence starting around 160 km from human settlements. Remote tracking of animal species can bridge the knowledge gap required for robust estimation of species distribution patterns in remote areas. Better knowledge of species' distribution is important in understanding the large-scale ecological consequences of rapid global change and establishing conservation management strategies.

Pre-extinction Demographic Stability and Genomic Signatures of Adaptation in the Woolly Rhinoceros.

Ancient DNA has significantly improved our understanding of the evolution and population history of extinct megafauna. However, few studies have used complete ancient genomes to examine species responses to climate change prior to extinction. The woolly rhinoceros (Coelodonta antiquitatis) was a cold-adapted megaherbivore widely distributed across northern Eurasia during the Late Pleistocene and became extinct approximately 14 thousand years before present (ka BP). While humans and climate change have been proposed as potential causes of extinction [1-3], knowledge is limited on how the woolly rhinoceros was impacted by human arrival and climatic fluctuations [2]. Here, we use one complete nuclear genome and 14 mitogenomes to investigate the demographic history of woolly rhinoceros leading up to its extinction. Unlike other northern megafauna, the effective population size of woolly rhinoceros likely increased at 29.7 ka BP and subsequently remained stable until close to the species' extinction. Analysis of the nuclear genome from a ∼18.5-ka-old specimen did not indicate any increased inbreeding or reduced genetic diversity, suggesting that the population size remained steady for more than 13 ka following the arrival of humans [4]. The population contraction leading to extinction of the woolly rhinoceros may have thus been sudden and mostly driven by rapid warming in the Bølling-Allerød interstadial. Furthermore, we identify woolly rhinoceros-specific adaptations to arctic climate, similar to those of the woolly mammoth. This study highlights how species respond differently to climatic fluctuations and further illustrates the potential of palaeogenomics to study the evolutionary history of extinct species.

Early Pleistocene origin and extensive intra-species diversity of the extinct cave lion.

The cave lion is an extinct felid that was widespread across the Holarctic throughout the Late Pleistocene. Its closest extant relative is the lion (Panthera leo), but the timing of the divergence between these two taxa, as well as their taxonomic ranking are contentious. In this study we analyse 31 mitochondrial genome sequences from cave lion individuals that, through a combination of 14C and genetic tip dating, are estimated to be from dates extending well into the mid-Pleistocene. We identified two deeply diverged and well-supported reciprocally monophyletic mitogenome clades in the cave lion, and an additional third distinct lineage represented by a single individual. One of these clades was restricted to Beringia while the other was prevalent across western Eurasia. These observed clade distributions are in line with previous observations that Beringian and European cave lions were morphologically distinct. The divergence dates for these lineages are estimated to be far older than those between extant lions subspecies. By combining our radiocarbon tip-dates with a split time prior that takes into account the most up-to-date fossil stem calibrations, we estimated the mitochondrial DNA divergence between cave lions and lions to be 1.85 Million ya (95% 0.52- 2.91 Mya). Taken together, these results support previous hypotheses that cave lions existed as at least two subspecies during the Pleistocene, and that lions and cave lions were distinct species.

Genome-Based Sexing Provides Clues about Behavior and Social Structure in the Woolly Mammoth.

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. We hypothesize 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.

Mitogenome evolution in the last surviving woolly mammoth population reveals neutral and functional consequences of small population size.

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.

Changes in variation at the MHC class II DQA locus during the final demise of the woolly mammoth.

According to the nearly-neutral theory of evolution, the relative strengths of selection and drift shift in favour of drift at small population sizes. Numerous studies have analysed the effect of bottlenecks and small population sizes on genetic diversity in the MHC, which plays a central role in pathogen recognition and immune defense and is thus considered a model example for the study of adaptive evolution. However, to understand changes in genetic diversity at loci under selection, it is necessary to compare the genetic diversity of a population before and after the bottleneck. In this study, we analyse three fragments of the MHC DQA gene in woolly mammoth samples radiocarbon dated to before and after a well-documented bottleneck that took place about ten thousand years ago. Our results indicate a decrease in observed heterozygosity and number of alleles, suggesting that genetic drift had an impact on the variation on MHC. Based on coalescent simulations, we found no evidence of balancing selection maintaining MHC diversity during the Holocene. However, strong trans-species polymorphism among mammoths and elephants points to historical effects of balancing selection on the woolly mammoth lineage.

Synchronous genetic turnovers across Western Eurasia in Late Pleistocene collared lemmings.

Recent palaeogenetic studies indicate a highly dynamic history in collared lemmings (Dicrostonyx spp.), with several demographical changes linked to climatic fluctuations that took place during the last glaciation. At the western range margin of D. torquatus, these changes were characterized by a series of local extinctions and recolonizations. However, it is unclear whether this pattern represents a local phenomenon, possibly driven by ecological edge effects, or a global phenomenon that took place across large geographical scales. To address this, we explored the palaeogenetic history of the collared lemming using a next-generation sequencing approach for pooled mitochondrial DNA amplicons. Sequences were obtained from over 300 fossil remains sampled across Eurasia and two sites in North America. We identified five mitochondrial lineages of D. torquatus that succeeded each other through time across Europe and western Russia, indicating a history of repeated population extinctions and recolonizations, most likely from eastern Russia, during the last 50 000 years. The observation of repeated extinctions across such a vast geographical range indicates large-scale changes in the steppe-tundra environment in western Eurasia during the last glaciation. All Holocene samples, from across the species' entire range, belonged to only one of the five mitochondrial lineages. Thus, extant D. torquatus populations only harbour a small fraction of the total genetic diversity that existed across different stages of the Late Pleistocene. In North American samples, haplotypes belonging to both D. groenlandicus and D. richardsoni were recovered from a Late Pleistocene site in south-western Canada. This suggests that D. groenlandicus had a more southern and D. richardsoni a more northern glacial distribution than previously thought. This study provides significant insights into the population dynamics of a small mammal at a large geographical scale and reveals a rather complex demographical history, which could have had bottom-up effects in the Late Pleistocene steppe-tundra ecosystem.

Complete genomes reveal signatures of demographic and genetic declines in the woolly mammoth.

The processes leading up to species extinctions are typically characterized by prolonged declines in population size and geographic distribution, followed by a phase in which populations are very small and may be subject to intrinsic threats, including loss of genetic diversity and inbreeding. However, whether such genetic factors have had an impact on species prior to their extinction is unclear; examining this would require a detailed reconstruction of a species' demographic history as well as changes in genome-wide diversity leading up to its extinction. Here, we present high-quality complete genome sequences from two woolly mammoths (Mammuthus primigenius). The first mammoth was sequenced at 17.1-fold coverage and dates to ∼4,300 years before present, representing one of the last surviving individuals on Wrangel Island. The second mammoth, sequenced at 11.2-fold coverage, was obtained from an ∼44,800-year-old specimen from the Late Pleistocene population in northeastern Siberia. The demographic trajectories inferred from the two genomes are qualitatively similar and reveal a population bottleneck during the Middle or Early Pleistocene, and a more recent severe decline in the ancestors of the Wrangel mammoth at the end of the last glaciation. A comparison of the two genomes shows that the Wrangel mammoth has a 20% reduction in heterozygosity as well as a 28-fold increase in the fraction of the genome that comprises runs of homozygosity. We conclude that the population on Wrangel Island, which was the last surviving woolly mammoth population, was subject to reduced genetic diversity shortly before it became extinct.

Holarctic genetic structure and range dynamics in the woolly mammoth.

Ancient DNA analyses have provided enhanced resolution of population histories in many Pleistocene taxa. However, most studies are spatially restricted, making inference of species-level biogeographic histories difficult. Here, we analyse mitochondrial DNA (mtDNA) variation in the woolly mammoth from across its Holarctic range to reconstruct its history over the last 200 thousand years (kyr). We identify a previously undocumented major mtDNA lineage in Europe, which was replaced by another major mtDNA lineage 32-34 kyr before present (BP). Coalescent simulations provide support for demographic expansions at approximately 121 kyr BP, suggesting that the previous interglacial was an important driver for demography and intraspecific genetic divergence. Furthermore, our results suggest an expansion into Eurasia from America around 66 kyr BP, coinciding with the first exposure of the Bering Land Bridge during the Late Pleistocene. Bayesian inference indicates Late Pleistocene demographic stability until 20-15 kyr BP, when a severe population size decline occurred.

Microsatellite genotyping reveals end-Pleistocene decline in mammoth autosomal genetic variation.

The last glaciation was a dynamic period with strong impact on the demography of many species and populations. In recent years, mitochondrial DNA sequences retrieved from radiocarbon-dated remains have provided novel insights into the history of Late Pleistocene populations. However, genotyping of loci from the nuclear genome may provide enhanced resolution of population-level changes. Here, we use four autosomal microsatellite DNA markers to investigate the demographic history of woolly mammoths (Mammuthus primigenius) in north-eastern Siberia from before 60 000 years ago up until the species' final disappearance c.4000 years ago. We identified two genetic groups, implying a marked temporal genetic differentiation between samples with radiocarbon ages older than 12 thousand radiocarbon years before present (ka) and those younger than 9ka. Simulation-based analysis indicates that this dramatic change in genetic composition, which included a decrease in individual heterozygosity of approximately 30%, was due to a multifold reduction in effective population size. A corresponding reduction in genetic variation was also detected in the mitochondrial DNA, where about 65% of the diversity was lost. We observed no further loss in genetic variation during the Holocene, which suggests a rapid final extinction event.