Extinct Cave Research Articles

Iteratively mapping ancient DNA to reconstruct highly divergent mitochondrial genomes: An evaluation of software, parameters and bait reference

Abstract The ability to obtain ancient DNA (aDNA) from extinct species has greatly improved our ability to study their evolutionary histories. Most aDNA studies focus on mitochondrial DNA for both population‐level and species‐level insights, due to its relatively high abundance and increased obtainability. Although several approaches are available to assemble mitogenomes, high levels of DNA damage and non‐target contaminating DNA make many approaches unsuitable for aDNA. Further complicating matters for most extinct species is the lack of a sufficiently closely related extant relative to act as a reference. Iterative mapping can assemble mitogenomes without using a close reference sequence, and has been applied to aDNA from various extinct species. Despite its widespread use, the accuracy of the reconstructed assemblies is yet to be comprehensively assessed. Here, we investigated the influence of (i) mapping software (Burrows–Wheeler Alignment [BWA], MITObim and NOVOplasty), (ii) parameters and (iii) bait reference phylogenetic distance on assembly accuracy using two simulated datasets: spotted hyena and various mammalian bait references; southern cassowary and various avian bait references, and two empirical datasets: extinct cave hyena; two extinct sloth species. Specifically, we assessed the accuracy of results through three parameters: pairwise distance to a reference conspecific mitogenome, number of incorrectly inserted base pairs and total length of the assembly. We found large discrepancies in the accuracy of reconstructed assemblies using different mapping software, parameters and bait references. Results also varied depending on the dataset, thereby making broad, generalised conclusions difficult. However, the overall most accurate results were obtained with either BWA using a minimum mapping quality of 20 and relaxed mismatch parameters (‐n 0.001 ‐o 2), or MITObim using a mismatch value of 3 or 5. We also show accuracy could be further improved by combining results from multiple bait references. Our study provides information on how divergent mitogenomes are best reconstructed from aDNA. By obtaining the most accurate reconstruction possible, one can be more confident as to the reliability of downstream analyses, and the evolutionary inferences made from them.

The genome and diet of a 35,000-year-old Canis lupus specimen from the Paleolithic painted cave, Chauvet-Pont d'Arc, France.

The Chauvet-Pont-d'Arc Cave (Ardèche, France) contains some of the oldest Paleolithic paintings recorded to date, as well as thousands of bones of the extinct cave bear, and some remains and footprints of other animals. As part of the interdisciplinary research project devoted to this reference cave site, we analyzed a coprolite collected within the deep cave. AMS radiocarbon dating of bone fragments from the coprolite yielded an age of 30,450 ± 550 RC yr. BP (AAR-19656; 36,150-34,000 cal BP), similar to ages assigned to Paleolithic artwork and cave bear remains from the same cave sector. Using high-throughput shotgun DNA sequencing, we demonstrated a high abundance of canid DNA and lesser amounts of DNA from the extinct cave bear. We interpret the sample as feces from a canid that had consumed cave bear tissue. The high amount of canid DNA allowed us to reconstruct a complete canid mitochondrial genome sequence (average coverage: 83×) belonging to a deeply divergent clade of extinct mitochondrial wolf lineages that are most closely related to coeval (~35 ka) Belgian wolves. Analysis of the nuclear genome yielded a similar coverage for the X chromosome (2.4×) and the autosomes (range: 2.3-3.2×), indicating that the Chauvet canid was a female. Comparing the relationship of the nuclear genome of this specimen with that of a variety of canids, we found it more closely related to gray wolves' genomes than to other wild canid or dog genomes, especially wolf genomes from Europe and the Middle East. We conclude that the coprolite is feces from an animal within an extinct wolf lineage. The consumption of cave bear by this wolf likely explains its intrusion into the dark cave sectors and sheds new light on the paleoecology of a major cave site.

Paleodiet and niche partitioning among the easternmost European cave bears based on tooth wear analysis

ABSTRACT Extinct cave bears inhabited a large part of Eurasia in a wide diversity of habitats during the Pleistocene. This study investigates via tooth wear analysis the feeding habits of 66 individuals belonging to three genetically different species located in the Urals and eastern Europe: Ursus rossicus from Kizel cave, U. kanivetz from Secrets cave, and U. ingressus from Shiriaevo 1 cave. For the microwear analysis, the three species were compared with a reference collection of extant ursids. The dietary space of U. kanivetz does not match any of the extant species’ spaces. U. rossicus and U. ingressus tended towards some of the extant species’ measurements without any overlapping. For all three sites, the same microwear pattern was identified for all age groups (juvenile, prime, and old adult), meaning no differences related to ontogeny were found. The extinct species presented a larger number of coarse scratches compared to extant species, pointing to a preference for abrasive food items like dry grass or shrubs typical of the mammoth steppe biome during the cold season. These results indicate a niche partitioning between U. rossicus and U. kanivetz that co-existed in the same area, and in this way they avoiding competition.

The Preliminary Analysis of Cave Lion Cubs Panthera spelaea (Goldfuss, 1810) from the Permafrost of Siberia

A preliminary description is presented of the well-preserved frozen mummies of two cubs of the extinct cave lion Panthera spelaea (finds of 2017–2018, Semyuelyakh River, Yakutia, eastern Siberia, Russia). The fossil lion cubs were found in close proximity, but they do not belong to the same litter, since their radiocarbon ages differ: the female (named ‘Sparta’) was dated to 27,962 ± 109 uncal years BP, and the male (named ‘Boris’) was dated to 43,448 ± 389 uncal years BP. The lion cubs have similar individual ages, 1–2 months. The general tone of the colour of the fur coat of Sparta is greyish to light brown, whereas, in Boris, the fur is generally lighter, greyish yellowish. It is, therefore, possible that light colouration prevailed with age in cave lions and was adaptive for northern snow-covered landscapes. The article discusses the results of computed tomography of cubs of the cave lion, the possible reasons for their death, and the peculiarities of their existence in the Siberian Arctic.

Three-dimensional dental topography and feeding ecology in the extinct cave bear.

The cave bear (Ursus spelaeus s.l.) was an iconic extinct bear that inhabited the Pleistocene of Eurasia. The cause of extinction of this species is unclear and to identify the actual factors, it is crucial to understand its feeding preferences. Here, we quantified the shape descriptor metrics in three-dimensional (3D) models of the upper teeth (P4-M2) of the cave bear to make inferences about its controversial feeding behaviour. We used comparative samples, including representatives of all living bear species with known diets, as a template. Our topographic analyses show that the complexity of upper tooth rows in living bears is more clearly associated with the mechanical properties of the items consumed than with the type of food. Cave bears exhibit intermediate values on topographic metrics compared with the bamboo-feeder giant panda (Ailuropoda melanoleuca) and specialists in hard mast consumption (Ursus arctos and Ursus thibetanus). The crown topography of cave bear upper teeth suggests that they could chew on tough vegetal resources of low quality with high efficiency, a characteristic that no living bear currently displays. Our results align with a climate-driven hypothesis to explain the extinction of cave bear populations during the Late Pleistocene.

Early\xa0Pleistocene 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.

Heavy reliance on plants for Romanian cave bears evidenced by amino acid nitrogen isotope analysis

Heavy reliance on plants is rare in Carnivora and mostly limited to relatively small species in subtropical settings. The feeding behaviors of extinct cave bears living during Pleistocene cold periods at middle latitudes have been intensely studied using various approaches including isotopic analyses of fossil collagen. In contrast to cave bears from all other regions in Europe, some individuals from Romania show exceptionally high δ15N values that might be indicative of meat consumption. Herbivory on plants with high δ15N values cannot be ruled out based on this method, however. Here we apply an approach using the δ15N values of individual amino acids from collagen that offsets the baseline δ15N variation among environments. The analysis yielded strong signals of reliance on plants for Romanian cave bears based on the δ15N values of glutamate and phenylalanine. These results could suggest that the high variability in bulk collagen δ15N values observed among cave bears in Romania reflects niche partitioning but in a general trophic context of herbivory.

Hyena paleogenomes reveal a complex evolutionary history of cross-continental gene flow between spotted and cave hyena.

The genus Crocuta (African spotted and Eurasian cave hyenas) includes several closely related extinct and extant lineages. The relationships among these lineages, however, are contentious. Through the generation of population-level paleogenomes from late Pleistocene Eurasian cave hyena and genomes from modern African spotted hyena, we reveal the cross-continental evolutionary relationships between these enigmatic hyena lineages. We find a deep divergence (~2.5 Ma) between African and Eurasian Crocuta populations, suggesting that ancestral Crocuta left Africa around the same time as early Homo. Moreover, we find discordance between nuclear and mitochondrial phylogenies and evidence for bidirectional gene flow between African and Eurasian Crocuta after the lineages split, which may have complicated prior taxonomic classifications. Last, we find a number of introgressed loci that attained high frequencies within the recipient lineage, suggesting some level of adaptive advantage from admixture.

Diet reconstruction in cave bears from craniodental morphology: past evidences, new results and future directions

ABSTRACTThe diet of the cave bear (Ursus spelaeus) is a controversial topic, as different paleobiological approaches (e.g. dental wear, isotopic biochemistry, skull morphometrics) result in different dietary inferences for the cave bear, ranging from carnivory to pure herbivory. Here, we review the main results obtained from these approaches, with special emphasis on those obtained from the morphometric analyses of the cave bear craniodental skeleton. Then, we compute a between-group Principal Components Analysis from a set of 3D-landmarks digitized on 103 mandibles of living bears and extinct cave bears and using a phylomorphospace approach. Moreover, we also reconstructed the evolutionary trajectory of the cave bear mandible from the hypothetical shape of its inferred ancestor. Our results indicate that the mandible of the cave bear possess specific traits indicative of a highly-herbivorous diet or, at least, more herbivorous than their closest living relative, the brown bear (Ursus arctos). Moreover, we also propose new directions for future research to obtain more detailed inferences on the potential food resources consumed by the cave bear being crucial to understand the ‘life and death’ of this vanished animal.

A three-dimensional analysis of tooth-root morphology in living bears and implications for feeding behaviour in the extinct cave bear

ABSTRACTThe morphology of both crowns and tooth-roots reflects dietary specialisation in mammalian carnivores. In this article, we analyse the tooth-root morphology of maxillary teeth from CT scans of living bears (Ursus arctos, Ursus americanus, Ursus maritimus, Ursus thibetanus, Melursus ursinus, Helarctos malayanus, Tremarctos ornatus and Ailuropoda melanoleuca) in order to make inferences about the diet and feeding behaviour of the extinct cave bear (Ursus spelaeus sensu lato). Specifically, we investigate two major mitochondrial clades of extinct cave bears recognized by previous authors: Ursus ingressus and Ursus spelaeus (U. spelaeus spelaeus, U. spelaeus ladinicus, U. spelaeus eremus). Our results indicate a close association between tooth-root surface area and feeding behaviour in all living bear species. Tooth-root surface area values of cave bears suggest that they relied more on vegetative matter than living brown bears (Ursus arctos) but subtle differences between these species/subspecies could also indicate different feeding strategies among the members of cave bear complex.

Ancient DNA reveals differences in behaviour and sociality between brown bears and extinct cave bears.

Ancient DNA studies have revolutionized the study of extinct species and populations, providing insights on phylogeny, phylogeography, admixture and demographic history. However, inferences on behaviour and sociality have been far less frequent. Here, we investigate the complete mitochondrial genomes of extinct Late Pleistocene cave bears and middle Holocene brown bears that each inhabited multiple geographically proximate caves in northern Spain. In cave bears, we find that, although most caves were occupied simultaneously, each cave almost exclusively contains a unique lineage of closely related haplotypes. This remarkable pattern suggests extreme fidelity to their birth site in cave bears, best described as homing behaviour, and that cave bears formed stable maternal social groups at least for hibernation. In contrast, brown bears do not show any strong association of mitochondrial lineage and cave, suggesting that these two closely related species differed in aspects of their behaviour and sociality. This difference is likely to have contributed to cave bear extinction, which occurred at a time in which competition for caves between bears and humans was likely intense and the ability to rapidly colonize new hibernation sites would have been crucial for the survival of a species so dependent on caves for hibernation as cave bears. Our study demonstrates the potential of ancient DNA to uncover patterns of behaviour and sociality in ancient species and populations, even those that went extinct many tens of thousands of years ago.

Mitogenomics of the Extinct Cave Lion, Panthera spelaea (Goldfuss, 1810), Resolve its Position within the Panthera Cats

The extinct cave lion ( Panthera spelaea ) was an apex predator of the Pleistocene, and one of the largest felid species ever to exist. We report the first mitochondrial genome sequences for this species, derived from two Beringian specimens, one of which has been radiocarbon dated to 29,860 ± 210 14C a BP. Phylogenetic analysis confirms the placement of the cave lion as the sister taxon to populations of the modern lion ( P. leo ). Using newly recovered stem pantherine fossils to calibrate a molecular clock, we estimate that P. spelaea and P. leo diverged about 1.89 million years ago (95% credibility interval: 1.23–2.93 million years), highlighting the likely position of this extinct carnivore as a distinct species.

Dietary ecology of the extinct cave bear (Ursus spelaeus): evidence of omnivory as inferred from dental microwear textures

The diet of the extinct European cave bear, Ursus spelaeus, has widely been debated. Diverging from the extant brown bear (Ursus arctos) approximately 1.2 million years ago, the cave bear is one of the most ubiquitous fossil bears occurring in Europe during the Middle and Late Pleistocene. Early morphological studies suggested that the cave bear was likely specialized on processing tough and/or abrasive foods, while later two-dimensional low-magnification microwear studies suggested that they were omnivorous and may have consumed more bone than U. arctos. Here, we used dental microwear texture analysis (DMTA) to further interpret the diet of the cave bear. Microscopic wear features were assessed and compared to modern ursids, including the cave bears' closest living relative, U. arctos. Results suggest that U. spelaeus consumed a diet with a diversity of textural properties, similar to most other bears and only distinguishable from the hyper-carnivorous polar bear (Ursus maritimus). Further, only U. marit...

‘Neanderthal bone flutes’: simply products of Ice Age spotted hyena scavenging activities on cave bear cubs in European cave bear dens

Punctured extinct cave bear femora were misidentified in southeastern Europe (Hungary/Slovenia) as ‘Palaeolithic bone flutes’ and the ‘oldest Neanderthal instruments’. These are not instruments, nor human made, but products of the most important cave bear scavengers of Europe, hyenas. Late Middle to Late Pleistocene (Mousterian to Gravettian) Ice Age spotted hyenas of Europe occupied mainly cave entrances as dens (communal/cub raising den types), but went deeper for scavenging into cave bear dens, or used in a few cases branches/diagonal shafts (i.e. prey storage den type). In most of those dens, about 20% of adult to 80% of bear cub remains have large carnivore damage. Hyenas left bones in repeating similar tooth mark and crush damage stages, demonstrating a butchering/bone cracking strategy. The femora of subadult cave bears are intermediate in damage patterns, compared to the adult ones, which were fully crushed to pieces. Hyenas produced round–oval puncture marks in cub femora only by the bone-crushing premolar teeth of both upper and lower jaw. The punctures/tooth impact marks are often present on both sides of the shaft of cave bear cub femora and are simply a result of non-breakage of the slightly calcified shaft compacta. All stages of femur puncturing to crushing are demonstrated herein, especially on a large cave bear population from a German cave bear den.

Coprolites as a source of information on the genome and diet of the cave hyena

We performed high-throughput sequencing of DNA from fossilized faeces to evaluate this material as a source of information on the genome and diet of Pleistocene carnivores. We analysed coprolites derived from the extinct cave hyena (Crocuta crocuta spelaea), and sequenced 90 million DNA fragments from two specimens. The DNA reads enabled a reconstruction of the cave hyena mitochondrial genome with up to a 158-fold coverage. This genome, and those sequenced from extant spotted (Crocuta crocuta) and striped (Hyaena hyaena) hyena specimens, allows for the establishment of a robust phylogeny that supports a close relationship between the cave and the spotted hyena. We also demonstrate that high-throughput sequencing yields data for cave hyena multi-copy and single-copy nuclear genes, and that about 50 per cent of the coprolite DNA can be ascribed to this species. Analysing the data for additional species to indicate the cave hyena diet, we retrieved abundant sequences for the red deer (Cervus elaphus), and characterized its mitochondrial genome with up to a 3.8-fold coverage. In conclusion, we have demonstrated the presence of abundant ancient DNA in the coprolites surveyed. Shotgun sequencing of this material yielded a wealth of DNA sequences for a Pleistocene carnivore and allowed unbiased identification of diet.

Low regional diversity of late cave bears mitochondrial DNA at the time of Chauvet Aurignacian paintings

The Chauvet-Pont d'Arc and Deux-Ouvertures caves, located along the Ardèche River (France), contain abundant remains of the extinct cave bear ( Ursus spelaeus). Because they also display a variety of Palaeolithic anthropogenic evidences, such as the earliest charcoal drawings recorded to date (Chauvet-Pont d'Arc), and delicate engravings (Deux-Ouvertures), they offer the opportunity of studying the interaction between animals and human beings during a key period for Pleistocene species extinctions. We characterized cave bear specimens from these two sites by radiocarbon dating, stable isotopes, and mitochondrial DNA analysis. In Chauvet-Pont d'Arc, we obtained radiocarbon ages that ranged between 29,000 and 37,300 years before present (BP). The Deux-Ouvertures cave bear specimens clustered to the bottom of this time frame, returning radiocarbon ages of 27,440–30,220 years BP. Cave bear nitrogen isotope values were all compatible with a vegetarian diet. Mitochondrial DNA analysis, carried out on a highly variable domain of the control region, evidenced only two cave bear haplotypes, including a new haplotype, and a common one which largely predominated. We detected both haplotypes in Chauvet-Pont d'Arc, but only recorded the predominant one in the Deux-Ouvertures Cave. Our data put forward the surprising observation that cave bears inhabited Ardèche over a short period of time, from about 37,000 to 27,400 years BP. They were notably present during the first (Aurignacian) phase of human intrusions in Chauvet-Pont d'Arc, 30,000–32,000 years BP. This points to the possible competition for cave sites, presumably on a seasonal scale considering the cave bear habit for hibernation. During this time period, the small number of haplotypes is at variance with the extensive genetic diversity reported elsewhere for much more ancient specimens.

Isotopic evidence for dietary ecology of cave lion ( Panthera spelaea) in North-Western Europe: Prey choice, competition and implications for extinction

The prey choice of extinct cave lions Panthera spelaea was determined using bone collagen isotopic signatures in the Belgian Ardennes and the Swabian Jura between 40,000 and 25,000 years ago as well as in the Late-glacial of the northwestern Alp foreland and of the Paris Basin. More than 370 specimens of large carnivorous and herbivorous mammals from 25 sites coeval with cave lion were analyzed. The isotopic results point to an individualistic prey choice for cave lions, with some individuals more oriented on reindeer and others on young cave bears. The isotopic signatures and therefore dietary choice of cave lions did not overlap with those of cave hyenas, indicating competitive exclusion between the large predators. The most recent western European cave lions seem to have been consuming mainly reindeer until the local extirpation of this prey species, which coincides chronologically with their own extinction. This restricted prey choice may be involved in the extinction of this large predator in Western Europe.

Deciphering the complete mitochondrial genome and phylogeny of the extinct cave bear in the Paleolithic painted cave of Chauvet

Retrieving a large amount of genetic information from extinct species was demonstrated feasible, but complete mitochondrial genome sequences have only been deciphered for the moa, a bird that became extinct a few hundred years ago, and for Pleistocene species, such as the woolly mammoth and the mastodon, both of which could be studied from animals embedded in permafrost. To enlarge the diversity of mitochondrial genomes available for Pleistocene species, we turned to the cave bear (Ursus spelaeus), whose only remains consist of skeletal elements. We collected bone samples from the Paleolithic painted cave of Chauvet-Pont d'Arc (France), which displays the earliest known human drawings, and contains thousands of bear remains. We selected a cave bear sternebra, radiocarbon dated to 32,000 years before present, from which we generated overlapping DNA fragments assembling into a 16,810-base pair mitochondrial genome. Together with the first mitochondrial genome for the brown bear western lineage, this study provides a statistically secured molecular phylogeny assessing the cave bear as a sister taxon to the brown bear and polar bear clade, with a divergence inferred to 1.6 million years ago. With the first mitochondrial genome for a Pleistocene carnivore to be delivered, our study establishes the Chauvet-Pont d'Arc Cave as a new reservoir for Paleogenetic studies. These molecular data enable establishing the chronology of bear speciation, and provide a helpful resource to rescue for genetic analysis archeological samples initially diagnosed as devoid of amplifiable DNA.

Two Sets of Cave Bear DNA Uncover the Bear Facts

A paper published online this week helps untangle bear phylogeny by presenting the mitochondrial genome of the extinct cave bear, Ursus spelaeus. Another paper, published last July, also reported the complete mitochondrial DNA of the cave bear as well as that of the extinct American short-faced bear, Arctodus simus.

Palaeogenomics

In many ways, palaeogenomics began when the first ancient DNA sequence was reported. This first sequence was derived from a stuffed museum specimen of the quagga, an extinct mammal related to the zebra. Unspecified and unselected DNA was extracted from the quagga specimen, cloned into a bacterial library, and then sequenced. It took another 17 years and the development of PCR before two independent groups successfully sequenced the complete mitochondrial genomes from several extinct moa species. Only 4 years later, using the original approach of cloning nonspecific ancient DNA extract and shotgun sequencing, the first ancient nuclear DNA sequences were determined, this time from the extinct cave bear. Since these early successes, palaeogenomics has rapidly expanded, because of both technological development and increasing interest in ancient DNA research. New methods, developed since the cave bear sequence was reported, have produced nuclear DNA on a megabase scale from two extinct species, the mammoth and the Neanderthal, our closest relative. For both species, low-coverage genome-sequencing projects have been proposed. It is likely that these will be successful, given the rapid technical development in sequencing techniques. This review carefully examines both the promise and the current limitations of palaeogenomic analyses for both mitochondrial and nuclear DNA.