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.     

Genetic diversity of endangered brown bear (Ursus arctos) populations at the crossroads of Europe, Asia and Africa

Aim  Middle East brown bears ( Ursus arctos syriacus Hemprich and Ehrenberg, 1828) are presently on the edge of extinction. However, little is known of their genetic diversity. This study investigates that question as well as that of Middle East brown bear relationships to surrounding populations of the species.
Location  Middle East region of south-western Asia.
Methods  We performed DNA analyses on 27 brown bear individuals. Twenty ancient bone samples (Late Pleistocene to 20th century) from natural populations and seven present-day samples obtained from captive individuals were analysed.
Results  Phylogenetic analyses of the mitochondrial sequences obtained from seven ancient specimens identify three distinct maternal clades, all unrelated to one recently described from North Africa. Brown bears from Iran exhibit striking diversity (three individuals, three haplotypes) and form a unique clade that cannot be linked to any extant one. Individuals from Syria belong to the Holarctic clade now observed in Eastern Europe, Turkey, Japan and North America. Specimens from Lebanon surprisingly appear as tightly linked to the clade of brown bears now in Western Europe. Moreover, we show that U. a. syriacus in captivity still harbour haplotypes closely linked to those found in ancient individuals.
Main conclusion  This study brings important new information on the genetic diversity of brown bear populations at the crossroads of Europe, Asia and Africa. It reveals a high level of diversity in Middle East brown bears and extends the historical distribution of the Western European clade to the East. Our analyses also suggest the value of a specific breeding programme for captive populations.     

First DNA sequences from Asian cave bear fossils reveal deep divergences and complex phylogeographic patterns

Until recently, cave bears were believed to have only inhabited Europe. However, recent morphological evidence suggests that cave bears' geographic range extended as far east as Transbaikalia, Eastern Siberia. These Asian cave bears were morphologically distinct from European cave bears. However, how they related to European lineages remains unclear, stressing the need to assess the phylogenetic and phylogeographic relationship between Asian cave bears and their European relatives. In this work, we address this issue using a 227 base-pair fragment of the mitochondrial control region obtained from nine fossil bone samples from eight sites from the Urals, Caucasus, Altai Mountains, Ukraine and Yana River region in Eastern Siberia. Results of the phylogenetic analyses indicate that (i) the cave bear from the Yana River is most closely related to cave bears from the Caucasus region; (ii) the Caucasus/Yana group of bears is genetically very distinct from both European cave bears and brown bears, suggesting that these bears could represent an independent species; and (iii) the Western European cave bear lineage reached at least temporarily to the Altai Mountains, 7000 km east of their known centre of distribution. These results suggest that the diversity of cave bears was greater than previously believed, and that they could survive in a much wider range of ecological conditions than previously assumed. They also agree with recent studies on other extinct and extant species, such as wolves, hyenas and steppe bison, which have also revealed higher genetic and ecological diversity in Pleistocene populations than previously known.     

Mitogenetic structure of brown bears (Ursus arctos L.) in northeastern Europe and a new time frame for the formation of European brown bear lineages

We estimated the phylogenetic relationships of brown bear maternal haplotypes from countries of northeastern Europe (Estonia, Finland and European Russia), using sequences of mitochondrial DNA (mtDNA) control region of 231 bears. Twenty-five mtDNA haplotypes were identified. The brown bear population in northeastern Europe can be divided into three haplogroups: one with bears from all three countries, one with bears from Finland and Russia, and the third composed almost exclusively of bears from European Russia. Four haplotypes from Finland and European Russia matched exactly with haplotypes from Slovakia, suggesting the significance of the current territory of Slovakia in ancient demographic processes of brown bears. Based on the results of this study and those from the recent literature, we hypothesize that the West Carpathian Mountains have served either as one of the northernmost refuge areas or as an important movement corridor for brown bears of the Eastern lineage towards northern Europe during or after the last ice age. Bayesian analyses were performed to investigate the temporal framework of brown bear lineages in Europe. The molecular clock was calibrated using Beringian brown bear sequences derived from radiocarbon-dated ancient samples, and the estimated mutation rate was 29.8% (13.3%-47.6%) per million years. The whole European population and Western and Eastern lineages formed about 175,000, 70,000 and 25,000 years before present, respectively. Our approach to estimating the time frame of brown bear evolution demonstrates the importance of using an appropriate mutation rate, and this has implications for other studies of Pleistocene populations.     

Evidence for reproductive isolation between cave bear populations

The European cave bear (Ursus spelaeus), which became extinct around 15,000 years ago, had several morphologically different forms. Most conspicuous of these were small Alpine cave bears found at elevations of 1,600 to 2,800 m. Whereas some paleontologists have considered these bears a distinct form, or even a distinct species, others have disputed this. By a combination of morphological and genetic methods, we have analyzed a population of small cave bears from Ramesch Cave (2,000 m altitude) and one of larger cave bears from Gamssulzen Cave (1,300 m), situated approximately 10 km apart in the Austrian Alps (Figure 1A). We find no evidence of mitochondrial gene flow between these caves during the 15,000 years when they were both occupied by cave bears, although mitochondrial DNA sequences identical to those from Gamssulzen Cave could be recovered from a site located about 200 km to the south in Croatia. We also find no evidence that the morphology of the bears in the two caves changed to become more similar over time. We suggest that the two cave bear forms may have represented two reproductively isolated subspecies or species.     

Ancient DNA analyses reveal high mitochondrial DNA sequence diversity and parallel morphological evolution of late pleistocene cave bears

Cave bears (Ursus spelaeus) existed in Europe and western Asiauntil the end of the last glaciation some 10,000 years ago.To investigate the genetic diversity, population history, andrelationship among different cave bear populations, we havedetermined mitochondrial DNA sequences from 12 cave bears thatrange in age from about 26,500 to at least 49,000 years andoriginate from nine caves. The samples include one individualfrom the type specimen population, as well as two small-sizedhigh-Alpine bears. The results show that about 49,000 yearsago, the mtDNA diversity among cave bears was about 1.8-foldlower than the current species-wide diversity of brown bears(Ursus arctos). However, the current brown bear mtDNA gene poolconsists of three clades, and cave bear mtDNA diversity is similarto the diversity observed within each of these clades. The resultsalso show that geographically separated populations of the high-Alpinecave bear form were polyphyletic with respect to their mtDNA.This suggests that small size may have been an ancestral traitin cave bears and that large size evolved at least twice independently.     

Recent history of the brown bear in the Maghreb

Bones of the brown bear (Ursus arctos, mammalia, Carnivora) found in a cave of the Akouker massif (Djurdjura, Algeria) have been dated according to the 14C method as belonging to the historical times (420-600 A.D.). The bone and teeth measurements correspond to a small-sized animal, the smallest ever found in the Maghreb. A review of fossil bears in the Quaternary faunas of North Africa clearly shows that the area of distribution, which was initially wide, shrank at the end of the Upper Pleistocene. The bears had temporarily taken to mountainous areas difficult to access. The bone remains discovered up to now prove that the brown bear was represented by individuals or populations showing a large diversity of size.     

Geographic and genetic boundaries of brown bear (Ursus arctos) population in the Caucasus

The taxonomic status of brown bears in the Caucasus remains unclear. Several morphs or subspecies have been identified from the morphological (craniological) data, but the status of each of these subspecies has never been verified by molecular genetic methods. We analysed mitochondrial DNA sequences (control region) to reveal phylogenetic relationships and infer divergence time between brown bear subpopulations in the Caucasus. We estimated migration and gene flow from both mitochondrial DNA and microsatellite allele frequencies, and identified possible barriers to gene flow among the subpopulations. Our suggestion is that all Caucasian bears belong to the nominal subspecies of Ursus arctos. Our results revealed two genetically and geographically distinct maternal haplogroups: one from the Lesser Caucasus and the other one from the Greater Caucasus. The genetic divergence between these haplogroups dates as far back as the beginning of human colonization of the Caucasus. Our analysis of the least‐cost distances between the subpopulations suggests humans as a major barrier to gene flow. The low genetic differentiation inferred from microsatellite allele frequencies indicates that gene flow between the two populations in the Caucasus is maintained through the movements of male brown bears. The Likhi Ridge that connects the Greater and Lesser Caucasus mountains is the most likely corridor for this migration.     

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.     

Microevolution of the mitochondrial DNA control region in the Japanese brown bear (Ursus arctos) population.

We investigated nucleotide sequences of the mitochondrial DNA control region to describe natural genetic variations and to assess the relationships between subpopulations of the brown bear Ursus arctos on Hokkaido Island, Japan. Using the polymerase chain reaction product-direct sequencing technique, partial sequences (about 930 bases) of the control region were determined for 56 brown bears sampled throughout Hokkaido Island. A sequence alignment revealed that the brown bear control region included a variable sequence on the 5' side and a repetitive region on the 3' side. Phylogenetic trees reconstructed from the 5' variable region (696-702 bases) exhibited 17 haplotypes, which were clustered into three groups (Clusters A, B, and C). The distribution of each group did not overlap with those of the others, and the three different areas were located in separate mountainous forests of Hokkaido Island. Furthermore, most of the phylogenetically close haplotypes within each group were distributed geographically close to each other. In addition, the 3' repetitive region (arrays of 10 bases) exhibited a much faster mutation rate than the 5' variable region, resulting in heteroplasmy. Such mitochondrial DNA divergence in each group could have occurred after the brown bears migrated from the continent to Hokkaido and became fixed in the different areas.     

Genetic turnovers and northern survival during the last glacial maximum in European brown bears

The current phylogeographic pattern of European brown bears (Ursus arctos) has commonly been explained by postglacial recolonization out of geographically distinct refugia in southern Europe, a pattern well in accordance with the expansion/contraction model. Studies of ancient DNA from brown bear remains have questioned this pattern, but have failed to explain the glacial distribution of mitochondrial brown bear clades and their subsequent expansion across the European continent. We here present 136 new mitochondrial sequences generated from 346 remains from Europe, ranging in age between the Late Pleistocene and historical times. The genetic data show a high Late Pleistocene diversity across the continent and challenge the strict confinement of bears to traditional southern refugia during the last glacial maximum (LGM). The mitochondrial data further suggest a genetic turnover just before this time, as well as a steep demographic decline starting in the mid‐Holocene. Levels of stable nitrogen isotopes from the remains confirm a previously proposed shift toward increasing herbivory around the LGM in Europe. Overall, these results suggest that in addition to climate, anthropogenic impact and inter‐specific competition may have had more important effects on the brown bear's ecology, demography, and genetic structure than previously thought.     

Phylogeography and mitochondrial diversity of extirpated brown bear (Ursus arctos) populations in the contiguous United States and Mexico

The fossil record indicates that the brown bear (Ursus arctos) colonized North America from Asia over 50 000 years ago. The species historically occupied the western United States and northern Mexico but has been extirpated from over 99% of this range in the last two centuries. To evaluate colonization hypotheses, subspecific classifications, and historical patterns and levels of genetic diversity in this region, we sequenced 229 nucleotides of the mitochondrial DNA control region in 108 museum specimens. The work was set in a global context by synthesizing all previous brown bear control region sequences from around the world. In mid-latitude North America a single moderately diverse clade is observed, represented by 23 haplotypes with up to 3.5% divergence. Only eight of 23 haplotypes (35%) are observed in the extensively sampled extant populations suggesting a substantial loss of genetic variability. The restriction of all haplotypes from mid-latitude North America to a single clade suggests that this region was founded by bears with a similar maternal ancestry. However, the levels and distributions of diversity also suggest that the colonizing population was not a small founder event, and that expansion occurred long enough ago for local mutations to accrue. Our data are consistent with recent genetic evidence that brown bears were south of the ice prior to the last glacial maximum. There is no support for previous subspecies designations, although bears of the southwestern United States may have had a distinctive, but recent, pattern of ancestry.     

Determination of the evolutionary mode of Austrian alpine cave bears by uranium series dating

Middle and Late Pleistocene sediments in many caves in Central and South Europe contain large numbers of bones and teeth of the cave bear (Ursus spelaeus). The cave bear differs in many characteristics from the recent brown bear and shows a rapid evolution especially in the changes of the teeth due to adaptation to pure herbivorous nutrition. The shifts of the morphotype frequencies of the fourth premolar from the upper jaw were used as a measure of the evolution. The uranium series method is the only suitable tool for the absolute age determination of the fossil bones with ages beyond the time range accessible to the radiocarbon method. By applying this method to the Herdengel cave profile the evolutionary rate of the cave bears was determined. Uranium series data from the fossil bones were partly verified by an independent carbonate speleothem age. For both, bone layers and carbonate formation found in stratigraphic relation, the determined ages correspond to a normal time sequence. According to the relatively fine time scale obtained by absolute dating, the evolutionary mode of the cave bears was determined as gradual. The main novelty of this study is the dating of the successive layers of the Herdengel cave and the determination of evolutionary stages of the cave bear in them.     

New data on the phylogeography and genetic diversity of the brown bear Ursus arctos Linnaeus, 1758 of Northeastern Eurasia (mtDNA control region polymorphism analysis)

An analysis of polymorphism of the fragment of the control region of mitochondrial DNA of 53 tissue samples of the brown bear Ursus arctos from several regions of the eastern part of Russia was carried out. It was found that most of the described haplotypes belong to cluster 3a, the most common in Eurasia, and do not form regionally specific haplogroups. However, among the bears from Western and Eastern Siberia, as well as the island of Kunashir, three haplotypes were identified, which are close to the haplogroup typical of Eastern Hokkaido bears. The assumption was made of the existence in Siberia and the Far East of one or more Pleistocene refugia.     

Phylogeography and pleistocene evolution in the North American black bear

To determine the extent of phylogeographic structuring in North American black bear (Ursus americanus) populations, we examined mitochondrial DNA sequences (n = 118) and restriction fragment length polymorphism profiles (n = 258) in individuals from 16 localities. Among the bears examined, 19 lineages falling into two highly divergent clades were identified. The clades differ at 5.0% of nucleotide positions, a distance consistent with an origin 1.8 MYA, and have different but overlapping geographical distributions. Areas of clade cooccurrence show that eastern and western populations are currently mixing, but regional differences in lineage distribution suggest that mixing has begun only recently. The long-term population history of black bears appears to be characterized predominantly by long-term regional isolation followed by recent contact and hybridization. Congruence between the pattern of diversity observed in black bears and patterns of forest refuge formation during the Pleistocene supports earlier speculation that Pleistocene forest fragmentations underlie a common pattern in the phylogeography of North American forest taxa.      

Sequencing mtDNA of the cave bearUrsus spelaeus from the Bavarian Alps is feasible by nested and touchdown PCR

Ancient DNA from bones of the extinctUrsus spelaeus Rosamueller et Hainroth, 1794 found in the Bavarian Alps has been amplified by PCR. Two out of five samples yielded a distinct band of 135 bp originating from the mtDNA control region. A combination of nested and touchdown PCR supported the amplification. Analysis of the nucleotide sequences revealed four transitions compared to the French cave bear sequence, the only cave bear data known so far (Genbank database X80259, AF121779). The consensus distant matrix tree clustered the two cave bears next to the brown bearUrsus arctos Linnaeus, 1758.     

Genetic variability of brown bear (<Emphasis Type="Italic">Ursus arctos</Emphasis> L., 1758)

We perform an analysis of the diversity of the control region fragment (570 bp) and ten nuclear microsatellite loci for 36 specimens of the brown bear (Ursus arctos) from several regions of the Caucasus. Four mitochondrial haplogroups that significantly differ from each other are revealed. An assumption of reiterative brown bear colonization of the Caucasus is made. Microsatellite data analysis shows a medium level of heterozygosity and clear differentiation between Caucasian and European bears combined with a slight distinction between the Caucasian regions.     

Stable isotopes data (delta13C, delta15N) from the cave bear (Ursus spelaeus): a new approach to its palaeoenvironment and dormancy

Palaeoclimatic data that can be extracted from the isotopic signatures of delta13C and delta15N, which are found in fossil bone collagen, should be analysed according to the specific metabolism of each species. Although Ursus spelaeus is an extinct species, its metabolism is assimilated to current, closely related species of bear. In this study, bone collagen isotopic signatures (delta13C and delta15N) of cave bears from Late Pleistocene Alpine sites were compared to those that have already been documented. The delta13C signature did not seem to follow a systematic trend according to climatic conditions, probably as a consequence of the high variability present in the values of C3 plants, which were the basis of feeding. On the contrary, the delta15N signature displayed higher values in sites corresponding to colder periods in which the delta15N signature appeared to be dominated by the physiology of dormancy. Then, due to the reuse of urea in synthesizing amino acids, the delta15N signature systematically increased along with dormancy duration. This was related to the length of winter and, in turn, depended on climate.