Epigenetic clocks, sex markers and age-class diagnostics in three harvested large mammals.

The development of epigenetic clocks, or the DNA methylation-based inference of age, is an emerging tool for ageing in free ranging populations. In this study, we developed epigenetic clocks for three species of large mammals that are the focus of extensive management throughout their range in North America: white-tailed deer, black bear and mountain goat. We quantified differential DNA methylation patterns at over 30,000 cytosine-guanine sites (CpGs) from tissue samples of all three species (black bear n = 49; white-tailed deer n = 47; mountain goat n = 45). We used a penalized regression model (elastic net) to build explanatory (black bear r = .95; white-tailed deer r = .99; mountain goat r = .97) and robust (black bear Median Absolute Error or MAE = 1.33; white-tailed deer MAE = 0.29; mountain goat MAE = 0.61) models of age or clocks. We also characterized individual CpG sites within each species that demonstrated clear differences in methylation levels between age classes and sex, which can be used to develop a suite of accessible diagnostic markers. This tool has the potential to contribute to wildlife monitoring by providing easily obtainable representations of age structure in managed populations.

Mid-holocene Brown Bear (Ursus arctos) from the Bolshoy Lyakhovsky Island (New Siberian Islands).

A morphological description is provided for a unique find of a frozen mummified subfossil brown bear (Ursus arctos L., 1758), found for the first time ever. The find is a well-preserved bear carcass of approximately 3500 years in age. Results of computed tomography and DNA testing are discussed.

Assembling mitogenome of Himalayan Black Bear (U. t. laniger) from low depth reads and its application in drawing phylogenetic inferences.

The complete mitogenome of Himalayan black bear (Ursus thibetanus laniger) from Indian Himalayan region was assembled following the modified approach of mitochondrial baiting and mapping using the next-generation sequencing reads. The complete mitogenome was of 16,556 bp long, consisted of 37 genes that contained 13 protein-coding genes, 22 tRNAs, 2 rRNAs and 1 control region. The complete base composition was 31.33% A, 15.24% G, 25.45%C, and 27.98%T and gene arrangement was similar to the other sub-species of Asiatic black bear. The relative synonymous codon usage analysis revealed the maximum abundance of Isoleucine, Tyrosine, Leucine and Threonine. The assembled mitogenome of U. t. laniger exhibited 99% similarity with the mitogenomes of Himalayan black bear available from Nepal and Tibetan Plateau-Himalaya region. The findings of the present study has proven low depth sequencing data, adequate and highly efficient in rapid recovering the mitochondrial genome by overcoming the conventional strategies of obtaining long-range PCR and subsequently drawing phylogenetic inferences.

Diet drives convergent evolution of gut microbiomes in bamboo-eating species.

Gut microbiota plays a critical role in host physiology and health. The coevolution between the host and its gut microbes facilitates animal adaptation to its specific ecological niche. Multiple factors such as host diet and phylogeny modulate the structure and function of gut microbiota. However, the relative contribution of each factor in shaping the structure of gut microbiota remains unclear. The giant (Ailuropoda melanoleuca) and red (Ailurus styani) pandas belong to different families of order Carnivora. They have evolved as obligate bamboo-feeders and can be used as a model system for studying the gut microbiome convergent evolution. Here, we compare the structure and function of gut microbiota of the two pandas with their carnivorous relatives using 16S rRNA and metagenome sequencing. We found that both panda species share more similarities in their gut microbiota structure with each other than each species shares with its carnivorous relatives. This indicates that the specialized herbivorous diet rather than host phylogeny is the dominant driver of gut microbiome convergence within Arctoidea. Metagenomic analysis revealed that the symbiotic gut microbiota of both pandas possesses a high level of starch and sucrose metabolism and vitamin B12 biosynthesis. These findings suggest a diet-driven convergence of gut microbiomes and provide new insight into host-microbiota coevolution of these endangered species.

Genetics of the Andean bear (Tremarctos ornatus; Ursidae, Carnivora) in Ecuador: when the Andean Cordilleras are not an Obstacle.

One of the top carnivores in the Andean mountains is the Andean bear (Tremarctos ornatus, Ursidae), the only bear in South America. This is a flagship and key umbrella species in Ecuador because its conservation has a positive impact on the conservation of many other species in the Andes. But to preserve, first one must know the genetic characteristics of a species, among other things. For this, we analyzed six mitochondrial genes and seven nuclear DNA microsatellites of 108 Andean bear specimens sampled throughout Ecuador. We adopted three strategies for analyzing the data: by Province, by Region (north vs south), and by Cordillera. Four main results were obtained. First, the mitochondrial genetic diversity levels were elevated, but there were no differences in genetic diversity by Province or by Cordillera. By Regions, southern Ecuador had higher genetic diversity levels than to northern Ecuador. The genetic diversity for the microsatellites was only medium for the Andean bear at this country. Second, there was clear and significant evidence of female population expansions, for the overall sample, by Province, Region, and Cordillera. This population expansion was determined to have occurred in the time interval of 30,000-20,000 years ago (YA), during the last phase of the Pleistocene. We detected a population decrease to have occurred more recently, within the last 5000 years. It continued until about 300-200 YA when a population increase was again detected. Third, there were, practically, no phylogeographic pattern nor genetic differentiation among Andean bear populations in Ecuador by Province or by Cordillera for either mitochondrial or microsatellite markers. There was a little more genetic differentiation between northern and southern areas. Fourth, there was no trace of significant spatial genetic structure for the Andean bear in Ecuador in agreement with the genetic differentiation analyses. This shows that the Andean Cordilleras in this country did not present an obstacle to the dispersion of this species. Therefore, all of the Andean bear specimens in Ecuador should be treated as a unique Management Unit (MU) for conservation purposes, differently to that determined for other countries as Colombia.

Biomechanical simulations reveal a trade-off between adaptation to glacial climate and dietary niche versatility in European cave bears.

The cave bear is one of the best known extinct large mammals that inhabited Europe during the "Ice Age," becoming extinct ≈24,000 years ago along with other members of the Pleistocene megafauna. Long-standing hypotheses speculate that many cave bears died during their long hibernation periods, which were necessary to overcome the severe and prolonged winters of the Last Glacial. Here, we investigate how long hibernation periods in cave bears would have directly affected their feeding biomechanics using CT-based biomechanical simulations of skulls of cave and extant bears. Our results demonstrate that although large paranasal sinuses were necessary for, and consistent with, long hibernation periods, trade-offs in sinus-associated cranial biomechanical traits restricted cave bears to feed exclusively on low energetic vegetal resources during the predormancy period. This biomechanical trade-off constitutes a new key factor to mechanistically explain the demise of this dominant Pleistocene megafaunal species as a direct consequence of climate cooling.

Using bear rub data and spatial capture-recapture models to estimate trend in a brown bear population.

Trends in population abundance can be challenging to quantify during range expansion and contraction, when there is spatial variation in trend, or the conservation area is large. We used genetic detection data from natural bear rubbing sites and spatial capture-recapture (SCR) modeling to estimate local density and population growth rates in a grizzly bear population in northwestern Montana, USA. We visited bear rubs to collect hair in 2004, 2009-2012 (3,579-4,802 rubs) and detected 249-355 individual bears each year. We estimated the finite annual population rate of change 2004-2012 was 1.043 (95% CI = 1.017-1.069). Population density shifted from being concentrated in the north in 2004 to a more even distribution across the ecosystem by 2012. Our genetic detection sampling approach coupled with SCR modeling allowed us to estimate spatially variable growth rates of an expanding grizzly bear population and provided insight into how those patterns developed. The ability of SCR to utilize unstructured data and produce spatially explicit maps that indicate where population change is occurring promises to facilitate the monitoring of difficult-to-study species across large spatial areas.

High genetic diversity and distinct ancient lineage of Asiatic black bears revealed by non-invasive surveys in the Annapurna Conservation Area, Nepal.

Asiatic black bears (Ursus thibetanus) have a widespread distribution in mountain landscapes, and are considered vulnerable globally, but are low-priority species for conservation in Nepal. Habitat fragmentation, illegal hunting, and human-bear conflict are the major threats to Asiatic black bears across their global range. Having an adequate level of genetic variation in a population helps with adapting to rapidly changing environments, and thus is important for the long-term health of bear populations. Accordingly, we conducted non-invasive surveys of bear populations in the Annapurna Conservation Area (ACA) to elucidate genetic diversity, genetic structure, and the phylogenetic relationship of Asiatic black bears from this region of Nepal to other subspecies. To assess levels of genetic diversity and population genetic structure, we genotyped eight microsatellite loci using 147 samples, identifying 60 individuals in an area of approximately 525 km2. We found that the Asiatic black bear population in the ACA has maintained high levels of genetic diversity (HE = 0.76) as compared to other bear populations from range countries. We did not detect a signature of population substructure among sampling localities and this suggests that animals are moving freely across the landscape within the ACA. We also detected a moderate population size that may increase with the availability of suitable habitat in the ACA, so bear-related conflict should be addressed to ensure the long-term viability of this expanding bear populations. Primers specific to bears were designed to amplify a 675 bp fragment of the mitochondrial control region from the collected samples. Three haplotypes were observed from the entire conservation area. The complete mitochondrial genome (16,771 bp), the first obtained from wild populations of the Himalayan black bear (U. t. laniger), was also sequenced to resolve the phylogenetic relationships of closely related subspecies of Asiatic black bears. The resulting phylogeny indicated that Himalayan black bear populations in Nepal are evolutionary distinct from other known subspecies of Asiatic black bears.

A cerebellar substrate for cognition evolved multiple times independently in mammals.

Given that complex behavior evolved multiple times independently in different lineages, a crucial question is whether these independent evolutionary events coincided with modifications to common neural systems. To test this question in mammals, we investigate the lateral cerebellum, a neurobiological system that is novel to mammals, and is associated with higher cognitive functions. We map the evolutionary diversification of the mammalian cerebellum and find that relative volumetric changes of the lateral cerebellar hemispheres (independent of cerebellar size) are correlated with measures of domain-general cognition in primates, and are characterized by a combination of parallel and convergent shifts towards similar levels of expansion in distantly related mammalian lineages. Results suggest that multiple independent evolutionary occurrences of increased behavioral complexity in mammals may at least partly be explained by selection on a common neural system, the cerebellum, which may have been subject to multiple independent neurodevelopmental remodeling events during mammalian evolution.

Comparison of Methods for Molecular Species Delimitation Across a Range of Speciation Scenarios.

Species are fundamental units in biological research and can be defined on the basis of various operational criteria. There has been growing use of molecular approaches for species delimitation. Among the most widely used methods, the generalized mixed Yule-coalescent (GMYC) and Poisson tree processes (PTP) were designed for the analysis of single-locus data but are often applied to concatenations of multilocus data. In contrast, the Bayesian multispecies coalescent approach in the software Bayesian Phylogenetics and Phylogeography (BPP) explicitly models the evolution of multilocus data. In this study, we compare the performance of GMYC, PTP, and BPP using synthetic data generated by simulation under various speciation scenarios. We show that in the absence of gene flow, the main factor influencing the performance of these methods is the ratio of population size to divergence time, while number of loci and sample size per species have smaller effects. Given appropriate priors and correct guide trees, BPP shows lower rates of species overestimation and underestimation, and is generally robust to various potential confounding factors except high levels of gene flow. The single-threshold GMYC and the best strategy that we identified in PTP generally perform well for scenarios involving more than a single putative species when gene flow is absent, but PTP outperforms GMYC when fewer species are involved. Both methods are more sensitive than BPP to the effects of gene flow and potential confounding factors. Case studies of bears and bees further validate some of the findings from our simulation study, and reveal the importance of using an informed starting point for molecular species delimitation. Our results highlight the key factors affecting the performance of molecular species delimitation, with potential benefits for using these methods within an integrative taxonomic framework.

Evolutionary history of enigmatic bears in the Tibetan Plateau-Himalaya region and the identity of the yeti.

Although anecdotally associated with local bears (Ursus arctos and U. thibetanus), the exact identity of 'hominid'-like creatures important to folklore and mythology in the Tibetan Plateau-Himalaya region is still surrounded by mystery. Recently, two purported yeti samples from the Himalayas showed genetic affinity with an ancient polar bear, suggesting they may be from previously unrecognized, possibly hybrid, bear species, but this preliminary finding has been under question. We conducted a comprehensive genetic survey of field-collected and museum specimens to explore their identity and ultimately infer the evolutionary history of bears in the region. Phylogenetic analyses of mitochondrial DNA sequences determined clade affinities of the purported yeti samples in this study, strongly supporting the biological basis of the yeti legend to be local, extant bears. Complete mitochondrial genomes were assembled for Himalayan brown bear (U. a. isabellinus) and black bear (U. t. laniger) for the first time. Our results demonstrate that the Himalayan brown bear is one of the first-branching clades within the brown bear lineage, while Tibetan brown bears diverged much later. The estimated times of divergence of the Tibetan Plateau and Himalayan bear lineages overlap with Middle to Late Pleistocene glaciation events, suggesting that extant bears in the region are likely descendants of populations that survived in local refugia during the Pleistocene glaciations.

A basal ursine bear (Protarctos abstrusus) from the Pliocene High Arctic reveals Eurasian affinities and a diet rich in fermentable sugars.

The skeletal remains of a small bear (Protarctos abstrusus) were collected at the Beaver Pond fossil site in the High Arctic (Ellesmere I., Nunavut). This mid-Pliocene deposit has also yielded 12 other mammals and the remains of a boreal-forest community. Phylogenetic analysis reveals this bear to be basal to modern bears. It appears to represent an immigration event from Asia, leaving no living North American descendants. The dentition shows only modest specialization for herbivory, consistent with its basal position within Ursinae. However, the appearance of dental caries suggest a diet high in fermentable-carbohydrates. Fossil plants remains, including diverse berries, suggests that, like modern northern black bears, P. abstrusus may have exploited a high-sugar diet in the fall to promote fat accumulation and facilitate hibernation. A tendency toward a sugar-rich diet appears to have arisen early in Ursinae, and may have played a role in allowing ursine lineages to occupy cold habitats.

Phylogenetic Conflict in Bears Identified by Automated Discovery of Transposable Element Insertions in Low-Coverage Genomes.

Phylogenetic reconstruction from transposable elements (TEs) offers an additional perspective to study evolutionary processes. However, detecting phylogenetically informative TE insertions requires tedious experimental work, limiting the power of phylogenetic inference. Here, we analyzed the genomes of seven bear species using high-throughput sequencing data to detect thousands of TE insertions. The newly developed pipeline for TE detection called TeddyPi (TE detection and discovery for Phylogenetic Inference) identified 150,513 high-quality TE insertions in the genomes of ursine and tremarctine bears. By integrating different TE insertion callers and using a stringent filtering approach, the TeddyPi pipeline produced highly reliable TE insertion calls, which were confirmed by extensive in vitro validation experiments. Analysis of single nucleotide substitutions in the flanking regions of the TEs shows that these substitutions correlate with the phylogenetic signal from the TE insertions. Our phylogenomic analyses show that TEs are a major driver of genomic variation in bears and enabled phylogenetic reconstruction of a well-resolved species tree, despite strong signals for incomplete lineage sorting and introgression. The analyses show that the Asiatic black, sun, and sloth bear form a monophyletic clade, in which phylogenetic incongruence originates from incomplete lineage sorting. TeddyPi is open source and can be adapted to various TE and structural variation callers. The pipeline makes it possible to confidently extract thousands of TE insertions even from low-coverage genomes (∼10×) of nonmodel organisms. This opens new possibilities for biologists to study phylogenies and evolutionary processes as well as rates and patterns of (retro-)transposition and structural variation.

Assessing SNP genotyping of noninvasively collected wildlife samples using microfluidic arrays.

Noninvasively collected samples are a common source of DNA in wildlife genetic studies. Currently, single nucleotide polymorphism (SNP) genotyping using microfluidic arrays is emerging as an easy-to-use and cost-effective methodology. Here we assessed the performance of microfluidic SNP arrays in genotyping noninvasive samples from grey wolves, European wildcats and brown bears, and we compared results with traditional microsatellite genotyping. We successfully SNP-genotyped 87%, 80% and 97% of the wolf, cat and bear samples, respectively. Genotype recovery was higher based on SNPs, while both marker types identified the same individuals and provided almost identical estimates of pairwise differentiation. We found that samples for which all SNP loci were scored had no disagreements across the three replicates (except one locus in a wolf sample). Thus, we argue that call rate (amplification success) can be used as a proxy for genotype quality, allowing the reduction of replication effort when call rate is high. Furthermore, we used cycle threshold values of real-time PCR to guide the choice of protocols for SNP amplification. Finally, we provide general guidelines for successful SNP genotyping of degraded DNA using microfluidic technology.

The effect of body size evolution and ecology on encephalization in cave bears and extant relatives.

BACKGROUND: The evolution of larger brain volumes relative to body size in Mammalia is the subject of an extensive amount of research. Early on palaeontologists were interested in the brain of cave bears, Ursus spelaeus, and described its morphology and size. However, until now, it was not possible to compare the absolute or relative brain size in a phylogenetic context due to the lack of an established phylogeny, comparative material, and phylogenetic comparative methods. In recent years, many tools for comparing traits within phylogenies were developed and the phylogenetic position of cave bears was resolved based on nuclear as well as mtDNA. RESULTS: Cave bears exhibit significantly lower encephalization compared to their contemporary relatives and intraspecific brain mass variation remained rather small. Encephalization was correlated with the combined dormancy-diet score. Body size evolution was a main driver in the degree of encephalization in cave bears as it increased in a much higher pace than brain size. In Ursus spelaeus, brain and body size increase over time albeit differently paced. This rate pattern is different in the highest encephalized bear species within the dataset, Ursus malayanus. The brain size in this species increased while body size heavily decreased compared to its ancestral stage. CONCLUSIONS: Early on in the evolution of cave bears encephalization decreased making it one of the least encephalized bear species compared to extant and extinct members of Ursidae. The results give reason to suspect that as herbivorous animals, cave bears might have exhibited a physiological buffer strategy to survive the strong seasonality of their environment. Thus, brain size was probably affected by the negative trade-off with adipose tissue as well as diet. The decrease of relative brain size in the herbivorous Ursus spelaeus is the result of a considerable increase in body size possibly in combination with environmental conditions forcing them to rest during winters.

Comparative genomics reveals convergent evolution between the bamboo-eating giant and red pandas.

Phenotypic convergence between distantly related taxa often mirrors adaptation to similar selective pressures and may be driven by genetic convergence. The giant panda (Ailuropoda melanoleuca) and red panda (Ailurus fulgens) belong to different families in the order Carnivora, but both have evolved a specialized bamboo diet and adaptive pseudothumb, representing a classic model of convergent evolution. However, the genetic bases of these morphological and physiological convergences remain unknown. Through de novo sequencing the red panda genome and improving the giant panda genome assembly with added data, we identified genomic signatures of convergent evolution. Limb development genes DYNC2H1 and PCNT have undergone adaptive convergence and may be important candidate genes for pseudothumb development. As evolutionary responses to a bamboo diet, adaptive convergence has occurred in genes involved in the digestion and utilization of bamboo nutrients such as essential amino acids, fatty acids, and vitamins. Similarly, the umami taste receptor gene TAS1R1 has been pseudogenized in both pandas. These findings offer insights into genetic convergence mechanisms underlying phenotypic convergence and adaptation to a specialized bamboo diet.

Retreat and extinction of the Late Pleistocene cave bear (Ursus spelaeus sensu lato).

The cave bear (Ursus spelaeus sensu lato) is a typical representative of Pleistocene megafauna which became extinct at the end of the Last Glacial. Detailed knowledge of cave bear extinction could explain this spectacular ecological transformation. The paper provides a report on the youngest remains of the cave bear dated to 20,930 ± 140 14C years before present (BP). Ancient DNA analyses proved its affiliation to the Ursus ingressus haplotype. Using this record and 205 other dates, we determined, following eight approaches, the extinction time of this mammal at 26,100-24,300 cal. years BP. The time is only slightly earlier, i.e. 27,000-26,100 cal. years BP, when young dates without associated collagen data are excluded. The demise of cave bear falls within the coldest phase of the last glacial period, Greenland Stadial 3. This finding and the significant decrease in the cave bear records with cooling indicate that the drastic climatic changes were responsible for its extinction. Climate deterioration lowered vegetation productivity, on which the cave bear strongly depended as a strict herbivore. The distribution of the last cave bear records in Europe suggests that this animal was vanishing by fragmentation into subpopulations occupying small habitats. One of them was the Kraków-Czestochowa Upland in Poland, where we discovered the latest record of the cave bear and also two other, younger than 25,000 14C years BP. The relatively long survival of this bear in karst regions may result from suitable microclimate and continuous access to water provided by deep aquifers, indicating a refugial role of such regions in the Pleistocene for many species.

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.