Natural-trap ursid mortality and the Kurtén Response

Ursid mortality data have long been used to evaluate associations between cave-bear remains (Ursus deningeri and U. spelaeus) and hominin (Homo sp.) remains. Typically, such ursid assemblages produce mortality patterns that indicate that juvenile and old bears died during hibernation, a pattern that is used to suggest that humans and bears occupied the same caves at different times. However, a different kind of mortality pattern can also be used to suggest human influence on cave bears, particularly under circumstances when bears and humans compete for habitat. In particular, data from Lawson Cave and Jerry Long Cave, Missouri indicate that young-adult North American black bears (Ursus americanus) are prone to capture in natural-trap caves. Similar faunal data from Sima de los Huesos in Spain, where cave-bear and hominin remains are found in the same deposit, might also suggest that the bears died from falling into a natural trap. It is concluded that mortality analysis of ursid remains from caves is a useful tool with which to evaluate accumulation histories of cave deposits and relations between humans, artifacts, and cave-bear remains. In particular, ursid mortality data are relevant to the Kurtén Response, a hypothesis reiterated in the recent literature that implicates human encroachment on ursid habitat (e.g., cave den sites) as a potential cause in cave-bear extinction.     

Hominid exploitation of the environment and cave bear populations. The case of Ursus spelaeus Rosenmüller-Heinroth in Amutxate cave (Aralar, Navarra-Spain)

Cave bears (Ursus deningeri and U. spelaeus) and hominids (Homo heidelbergensis, H. neanderthalensis, and H. sapiens) were potential competitors for environmental resources (subterranean and open air). Here, we examined the age at death of cave bear (Ursus spelaeus Rosenmüller-Heinroth) specimens from Amutxate cave in order to shed light on the effect of resource sharing between cave bears and hominids. After studying dental wear of the deciduous and permanent dentitions, the ontogenetic development of mandibles, and incremental layers of cement (annuli), we defined five age groups differentiated by marked development and size gaps. Our findings indicate that after hibernating, bears abandoned the den, thereby leaving the subterranean environment (caves) free for temporary hominid occupation-this would explain the subtle traces of hominid presence in many dens. However, a simple calculation based on age at death of subadult and adult cave bear specimens in Amutxate cave, extrapolated to the whole cave area, showed that the area surrounding this cave hosted bears for at least 9,000 years. This length of habitation, quite similar to the time-span derived from amino acid racemization and electron spin resonance, indicates that bear populations in the Amutxate cave constituted a serious constraint for hominid exploitation of the environment.     

Ancient DNA analysis reveals divergence of the cave bear, Ursus spelaeus, and brown bear, Ursus arctos, lineages

The cave bear, Ursus spelaeus, represents one of the most frequently found paleontological remains from the Pleistocene in Europe. The species has always been confined to Europe and was contemporary with the brown bear, Ursus arctos. Relationships between the cave bear and the two lineages of brown bears defined in Europe, as well as the origins of the two species, remain controversial, mainly due to the wide morphological diversity of the fossil remains, which makes interpretation difficult [1, 2]. Sequence analysis of ancient DNA is a useful tool for resolving such problems because it provides an independent source of data [3]. We previously amplified a short DNA fragment of the mitochondrial DNA control region (mt control region) of a 40,000-year-old Ursus spelaeus sample [4]. In this paper, we describe the DNA analysis of two mtDNA regions, the control region and the cytochrome b gene. Control region sequences were obtained from ten samples of cave bears ranging from 130,000 to 20,000 years BP, and one particularly well-conserved sample gave a complete cyt b sequence. Our data demonstrate that cave bears split largely before the lineages of brown bears around 1.2 million years ago. Given its abundance, its wide distribution in space and time, and its large morphological diversity, the cave bear is a promising model for direct observation of the evolution of sequences throughout time, extinction periods, and the differentiation of populations shaped by climatic fluctuations during the Pleistocene.     

Ancient DNA evidence for the loss of a highly divergent brown bear clade during historical times

The genetic diversity of present-day brown bears (Ursus arctos) has been extensively studied over the years and appears to be geographically structured into five main clades. The question of the past diversity of the species has been recently addressed by ancient DNA studies that concluded to a relative genetic stability over the last 35,000 years. However, the post-last glacial maximum genetic diversity of the species still remains poorly documented, notably in the Old World. Here, we analyse Atlas brown bears, which became extinct during the Holocene period. A divergent brown bear mitochondrial DNA lineage not present in any of the previously studied modern or ancient bear samples was uncovered, suggesting that the diversity of U. arctos was larger in the past than it is now. Specifically, a significant portion (with respect to sequence divergence) of the intraspecific diversity of the brown bear was lost with the extinction of the Atlas brown bear after the Pleistocene/Holocene transition.     

The first detection of Babesia species DNA from Japanese black bears (Ursus thibetanus japonicus) in Japan

In this study, we tried to detect protozoan blood parasites from the liver or blood of 156 Japanese black bears (Ursus thibetanus japonicus) in Iwate Prefecture of Japan by polymerase chain reaction. Two amplicons (approximately 540 bp and 480 bp) were detected by amplification for V4 hyper-variable regions of the 18S rRNA gene. Approximately 540-bp products were obtained in 119 samples (76.3%) and were considered to be DNA of Hepatozoon ursi. Approximately 480-bp products were obtained in 22 samples (14.1%) and were considered to be DNA of Babesia species. The nucleotide sequences (1635 bp) of the 18S rRNA gene of Babesia sp. were very similar (99.3%) to those (AY190123, AY190124) of Babesia sp. detected previously from Ixodes ovatus. Phylogenetic analysis showed that Babesia sp. detected in this study closely related to Babesia sp. derived from raccoons in Japan and the U.S.A. This is the first report of Babesia species detected from Japanese black bears.     

A unique population of cave bears (Carnivora: Ursidae) from the Middle Pleistocene of Kents Cavern,England, based on dental morphometrics

The ‘breccia’ stratum from Kents (we follow local tradition in using the form ‘Kents’, without an apostrophe) Cavern, England, has been well known for its rich yield of cave-bear material since excavations began in the mid-19th century. Recent work has established that the bears are of latest MIS 12 or earliest MIS 11 age. A life table based on a collection of 67 molariform teeth is consistent with the use of the cave as a hibernaculum. Univariate and morphological assessment of the teeth shows an unusual range of primitive and more derived characters. Multivariate morphometric analysis of cave-bear teeth from the site demonstrates that these animals, while currently assignable to Ursus deningeri sensu lato, are nevertheless morphologically distinct and not simply late deningeri on a hypothetical chronospecific continuum.     

Fossil avian eggshell preserves ancient DNA

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.     

Comparing the performance of three ancient DNA extraction methods for high‐throughput sequencing

The DNA molecules that can be extracted from archaeological and palaeontological remains are often degraded and massively contaminated with environmental microbial material. This reduces the efficacy of shotgun approaches for sequencing ancient genomes, despite the decreasing sequencing costs of high‐throughput sequencing (HTS). Improving the recovery of endogenous molecules from the DNA extraction and purification steps could, thus, help advance the characterization of ancient genomes. Here, we apply the three most commonly used DNA extraction methods to five ancient bone samples spanning a ~30 thousand year temporal range and originating from a diversity of environments, from South America to Alaska. We show that methods based on the purification of DNA fragments using silica columns are more advantageous than in solution methods and increase not only the total amount of DNA molecules retrieved but also the relative importance of endogenous DNA fragments and their molecular diversity. Therefore, these methods provide a cost‐effective solution for downstream applications, including DNA sequencing on HTS platforms.     

Mitochondrial DNA haplogrouping of the brown bear,Ursus arctos (Carnivora: Ursidae) in Asia,based on a newly developed APLP analysis

Sequence analyses of the complete brown bear, Ursus arctos, mitochondrial DNA (mtDNA) genome have detected scattered single nucleotide polymorphisms (SNPs) that define distinct mtDNA haplogroups in phylogeographical studies. The degraded DNA in historical samples, such as stuffed or excavated specimens, however, is often not suitable for sequence analyses. To address this problem, we developed an amplified product length polymorphism (APLP) analysis for mtDNA‐haplogrouping U. arctos specimens by detecting haplogroup‐specific SNPs. We verified the validity and utility of this method by analysing up to 170‐year‐old skin samples from U. arctos specimens collected widely across continental Eurasia. We detected some of the same haplogroups as those occurring in eastern Hokkaido (Japan) and eastern Alaska in continental Eurasia (the Altai and the Caucasus). Our results show that U. arctos in eastern Hokkaido and eastern Alaska descended from a common ancestor in continental Eurasia, and suggest that U. arctos occupied several refugia in southern Asia during the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 627–635.     

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

ABSTRACT

The 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.     

COMPARATIVE ANALYSIS OF METHODS FOR THE PURIFICATION OF DNA FROM LOW-BIOMASS SAMPLES BASED ON TOTAL YIELD AND CONSERVED MICROBIAL DIVERSITY

Despite advances in the specificity and sensitivity of molecular biological technologies, the efficient recovery of DNA from low-biomass samples remains extremely challenging. Optimal methods to purify biomolecules from such environments should (1) achieve the greatest total yield and (2) reflect the true microbial diversity of the sample. These attributes were assessed from five DNA purification regimes: a standard-manual procedure, MoBio Ultraclean and Promega Wizard kits, and an automated Axcyte AutoLyser method with and without bead-beating agitation. A homogenous mixture of known concentrations of nine distinct bacterial lineages isolated from low-biomass environments was prepared and suitable aliquots of subsamples were processed in parallel. DNA products from each of these methods were then subjected to polymerase chain reaction (PCR), quantitative PCR and 16S rRNA clone-library analysis. The Axcyte AutoLyser outperformed all other purification regimes examined. This automated method consistently both yielded the highest concentration of PCR-amplifiable DNA, and reported species composition most consistent with the starting solution.

PRACTICAL APPLICATIONS

This communication carefully examines the effectiveness of common DNA purification regimes as well as an automated method. Comparative analyses convincingly demonstrate that the different methods not only result in different recovery of genomic DNA, but more importantly, different estimations of microbial diversity in the sample. This report will hopefully inspire investigators from various industries (pharmaceutical, ecological, medical, semiconductor, etc.) who find themselves in the initial phases of large-scale studies to devote a significant effort into optimizing sample extraction protocols to achieve the most accurate information.     

The status of ribosomal RNA genes during nuclear DNA reversion in flax

Heritable changes in plant weight and nuclear DNA content may be induced in certain varieties of flax by different fertilizer environments. The large stable form (L) has 16% more nuclear DNA that the small stable form (S). In subsequent generations in particular environments the nuclear DNA difference disappears, although the plant weight differences remain, while in other environments both DNA and plant weight differences are maintained. The large stable form has 56-70% more rRNA genes than the small stable form. The majority of this difference in rDNA is maintained in types where the nuclear difference has reverted. It is suggested that the expression of the flax genome may be susceptible to a reduction in the multiplicity of rRNA genes which limits the availability of protein synthetic machinery needed for growth.     

Simplified Method for Dissolved DNA Determination in Aquatic Environments

A method was developed for the determination of dissolved DNA in aquatic environments. The method is based upon the concentration of dissolved DNA by ethanol precipitation of 0.2-μm-pore-size filtered water. The DNA in concentrated extracts was quantified by the fluorescence of Hoechst 33258-DNA complexes. Fluorescence not attributable to DNA was corrected for by DNase I digestion of the extracts and averaged 25% of the total fluorescence for all samples. The effectiveness of the procedure for concentrating dissolved DNA was demonstrated by the efficient (>90%) recovery of internal standards. Concentrations of dissolved DNA from a variety of marine and freshwater environments ranged from 0.2 to 44 μg/liter, with the highest values being obtained for estuarine and river environments. The method is simple, specific for DNA, and more sensitive than previously described methods for the determination of extracellular DNA.     

Impacts of sampling location within a faeces on DNA quality in two carnivore species

We investigated the influence of sampling location within a faeces on DNA quality by sampling from both the outside and inside of 25 brown bear (Ursus arctos) scats and the side and the tip of 30 grey wolf (Canis lupus) scats. The outside of the bear scat and side of the wolf scat had significantly lower nuclear DNA microsatellite allelic dropout error rates (U. arctos: P = 0.017; C. lupus: P = 0.025) and significantly higher finalized genotyping success rates (U. arctos: P = 0.017; C. lupus: P = 0.012) than the tip and inside of the scat. A review of the faecal DNA literature indicated that <45% of studies report the sampling location within a faeces indicating that this methodological consideration is currently underappreciated. Based on our results, we recommend sampling from the side of canid scats and the outside portion of ursid scats to obtain higher quality DNA samples. The sampling location within a faeces should be carefully considered and reported as it can directly influence laboratory costs and efficiency, as well as the ability to obtain reliable genotypes.     

Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.     

Ancient DNA studies

Now that the hype surrounding Jurassic Park has settled down and we have become relatively used to dramatic headlines announcing the recovery of DNA from exotic fossilized remains, scientists working on ancient DNA are beginning to reflect on the long-term prospects and implications of the subject.¹ The science of ancient DNA has grown exponentially since its birth only ten years ago, and despite serious technical difficulties, it promises to become a revolutionary research tool in anthropology and molecular evolution. The use of bone DNA typing in particular has already yielded useful insights into Polynesian prehistory as well as spectacular applications in the forensic identification of skeletal remains.     

The early life social environment and DNA methylation

Although epidemiological data provides evidence that there is an interaction between genetics (nature) and the social and physical environments (nurture) in human development; the main open question remains the mechanism. The pattern of distribution of methyl groups in DNA is different from cell-type to cell type and is conferring cell specific identity on DNA during cellular differentiation and organogenesis. This is an innate and highly programmed process. However, recent data suggests that DNA methylation is not only involved in cellular differentiation but that it is also involved in modulation of genome function in response to signals from the physical, biological and social environments. We propose that modulation of DNA methylation in response to environmental cues early in life serves as a mechanism of life-long genome "adaptation" that molecularly embeds the early experiences of a child ("nurture") in the genome ("nature"). There is an emerging line of data supporting this hypothesis in rodents, non-human primates and humans that will be reviewed here. However, several critical questions remain including the identification of mechanisms that transmit the signals from the social environment to the DNA methylation/demethylation enzymes.     

Nouvelles recherches dans le bassin Plio-Pléistocène d’Anagni (Latium méridional, Italie)

The Anagni intra-apennine basin is especially rich in palaeontological and archaeological sites, spanning between middle Villafranchian and upper Pleistocene. The Italian Institute of Human Palaeontology has carried out researches in this region in the last 30 years. At Costa San Giacomo, middle Villafranchian yellow sands contain Mastodon arvernensis and Elephas meridionalis. At Colle Marino, a travertine layer and lime-clay contains mode 1 lithic industry, below any volcanic layers whose lower limit is 700 Ky old. Excavations have been restarted at Fontana Ranuccio, a 458 Ky Acheulian site with rich mammalian fauna (Elephas antiquus, Ursus deningeri, Bos primigenius, Dama clactoniana, Equus mosbachensis), a remarkable bone industry and four human teeth. Drillings, for about 40 m, have identified Matuyama-Brunhes limit 23 m under the main volcanic pyroclastite, in a thick limnic clay layer.     

First report of Taenia arctos (Cestoda: Taeniidae) from grizzly (Ursus arctos horribilis) and black bears (Ursus americanus) in North America

The cestode Taenia arctos was found at necropsy in the small intestine of a grizzly (Ursus arctos horribilis) and a black bear (Ursus americanus) from Kananaskis Country in southwestern Alberta, Canada. The autolysis of the tapeworm specimens precluded detailed morphological characterization of the parasites but molecular analysis based on mitochondrial DNA cytochrome c oxidase subunit 1 gene confirmed their identity as T. arctos. This is the first report of T. arctos from definitive hosts in North America. Its detection in Canadian grizzly and black bears further supports the Holarctic distribution of this tapeworm species and its specificity for ursids as final hosts. Previously, T. arctos was unambiguously described at its adult stage in brown bears (Ursus arctos arctos) from Finland, and as larval stages in Eurasian elk (Alces alces) from Finland and moose (Alces americanus) from Alaska, USA. Given the morphological similarity between T. arctos and other Taenia species, the present study underlines the potential for misidentification of tapeworm taxa in previous parasitological reports from bears and moose across North America. The biogeographical history of both definitive and intermediate hosts in the Holarctic suggests an ancient interaction between U. arctos, Alces spp., and T. arctos, and a relatively recent host-switching event in U. americanus.