Genetic diversity of Dinaric brown bears (Ursus arctos) in Croatia with implications for bear conservation in Europe

Brown bears have lost most of their range on the European continent. The remaining western populations are small, isolated and highly endangered. The Dinaric-Pindos brown bear population is the western-most stable population and the fourth largest in Europe. It has been recognized as a potential source for recolonization of populations whose survival is at risk. Indeed, several translocations of Dinaric bears to Italy, Austria and France have recently been made. Despite the importance of the Dinaric bear population, its genetic status remains poorly understood. Using tissue samples from 156 hunted or accidentally killed Dinaric bears in Croatia, this study analysed genetic diversity at 12 microsatellite loci, as well as population structure and past reductions in size. In addition, a subset of 59 samples was used to assess diversity of the mitochondrial DNA control region. The results indicate that Dinaric bears have high nuclear genetic diversity, as compared to other extant brown bear populations, despite genetic evidence of a bottleneck caused by past persecutions. However, haplotype diversity was low, probably as a result of male-biased dispersal and female philopatry. Not surprisingly, no evidence of population sub-structure was found using nuclear markers, as the bear habitat has remained continuous and the highway network has been built only recently. Management should focus on maintaining habitat connectivity and keeping the effective population size as large as possible. In addition, when removing individuals, care should be taken not to further deplete the population of rare haplotypes. A coordinated transboundary management of the entire Dinaric-Pindos brown bear population should be a priority for its long-term conservation.     

Maintenance of high levels of allelic variation in spite of a severe bottleneck in population size: the brown bear (Ursus arctos) in the Western Carpathians

Genetic variation in an isolated brown bear population of the Western Carpathians was studied by electrophoretic analysis of 51 presumptive allozyme loci. In spite of a severe population bottleneck at the beginning of the 1930s (40 survivors), average heterozygosity (H=5.3%) was within the range commonly found in mammals and the mean number of alleles per polymorphic locus (A_p=3, over five polymorphic loci) was very high. Possible reasons for the maintenance of high allelic variation are discussed.     

Integrating sign surveys and telemetry data for estimating brown bear (Ursus arctos) density in the Romanian Carpathians

Accurate population size estimates are important information for sustainable wildlife management. The Romanian Carpathians harbor the largest brown bear (Ursus arctos) population in Europe, yet current management relies on estimates of density that lack statistical oversight and ignore uncertainty deriving from track surveys. In this study, we investigate an alternative approach to estimate brown bear density using sign surveys along transects within a novel integration of occupancy models and home range methods. We performed repeated surveys along 2‐km segments of forest roads during three distinct seasons: spring 2011, fall‐winter 2011, and spring 2012, within three game management units and a Natura 2000 site. We estimated bears abundances along transects using the number of unique tracks observed per survey occasion via N‐mixture hierarchical models, which account for imperfect detection. To obtain brown bear densities, we combined these abundances with the effective sampling area of the transects, that is, estimated as a function of the median (± bootstrapped SE) of the core home range (5.58 ± 1.08 km²) based on telemetry data from 17 bears tracked for 1‐month periods overlapping our surveys windows. Our analyses yielded average brown bear densities (and 95% confidence intervals) for the three seasons of: 11.5 (7.8–15.3), 11.3 (7.4–15.2), and 12.4 (8.6–16.3) individuals/100 km². Across game management units, mean densities ranged between 7.5 and 14.8 individuals/100 km². Our method incorporates multiple sources of uncertainty (e.g., effective sampling area, imperfect detection) to estimate brown bear density, but the inference fundamentally relies on unmarked individuals only. While useful as a temporary approach to monitor brown bears, we urge implementing DNA capture–recapture methods regionally to inform brown bear management and recommend increasing resources for GPS collars to improve estimates of effective sampling area.     

Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition

We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature.     

Detailed model of shelter areas for the Cantabrian brown bear

We propose a methodology to determine critical areas for brown bear (Ursus arctos) in the Cantabrian Mountains (Spain), considered as shelter areas in which there are concentrations of winter dens and groups of resting sites. We assessed a function which enabled a precise spatial distribution model to be constructed. This function helps to explain the present division of the range of the bears into two isolated populations and to identify areas for protection with the aim of increasing their probabilities of survival or connection.

This has been done using a multivariate analysis and by applying the results to variables modelled on a geographic information system. We took a location containing 161 bear dens and resting sites. These positions have been characterised by modelling variables in a grid of 50-m cells derived from the topography (digital model of elevations, orientations, slopes, altitude variability in an area around each location). The variables include those that quantify distance weighting according to difficulties associated with access to different disturbance elements (infrastructures or buildings), variables that quantify distances to refuges (forests, scrubs, and rocky areas) and their shape (perimeter, thickness, eccentricity or shape of the forest and type of rocky areas). We constructed a logistic regression model with this data that locates and highlights shelter areas with an 86% average rate of precision. This result reveals that the eastern population has shelter areas around the present range, indicating a possible expansion area; however, there is a gap measuring close to 30 km between both populations that is almost entirely lacking in shelters and that contains important infrastructures. Increasing refuge conditions in the gap to connect shelter areas will make it possible to recover the area as a corridor.     

Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa

Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample.     

Assessment of a brown midrib (BMR) mutant gene on the nutritive value of sudangrass using in vitro and in vivo techniques

The brown midrib (BMR) gene has been reported to reduce the lignin concentration in plants, which contributed to increased fiber digestion in ruminants. Three studies were completed to compare the digestibility of a BMR mutant of sudangrass (sorghum bicolor subsp. Drummondii) versus a non-BMR (‘Piper’) variety when included in diets fed to sheep (Study 1), to complete a rumen in vitro assessment of sheep and lactating cow diets (Study 2), and to compare digestibility when included in the diet fed to lactating dairy cows (Study 3). Four wether sheep were used in a 2 × 2 Latin square experiment (Study 1) with total fecal collection to determine total tract apparent digestibility of pelleted Piper (P) and BMR (P-BMR) sudangrass hays. Forage pellets consisted of either P-BMR or P hay with added urea to meet the maintenance crude protein (CP) requirement of the sheep. Digestibility of organic matter (OM; P<0.01), dry matter (DM; P<0.01), acid detergent fiber (ADF; P<0.05), and neutral detergent fiber (aNDFom; P<0.07) was higher for P-BMR than P sudangrass. In vitro rumen digestibility of aNDFom using cattle rumen fluid was higher at 24 (P<0.01), 48 (P<0.01) and 72 h (P<0.01) of fermentation for P-BMR versus P (Study 2). Four lactating Holstein dairy cows (251 ± 30 days in milk) and fitted with ruminal and duodenal cannulae were used in a 4 × 4 Latin square experiment. Total mixed rations (TMR) contained 180 g/kg DM shredded sudangrass hay and 180 g/kg sliced alfalfa hay, but the proportion of P to P-BMR sudangrass varied as 100:0, 66:34, 34:66, or 0:100. Yields of milk and milk protein were highest at the 66:34 level (Quadratic: P=0.06 and 0.07, respectively), but composition of milk fat, protein and lactose, as well as DM intake, did not differ (Study 3), probably because forestomach and total tract apparent digestion of aNDFom and OM did not differ due to sudangrass source.     

Biogeography of Hysterangiales (Phallomycetidae, Basidiomycota)

To understand the biogeography of truffle-like fungi, DNA sequences were analysed from representative taxa of Hysterangiales. Multigene phylogenies and the results of ancestral area reconstructions are consistent with the hypothesis of an Australian, or eastern Gondwanan, origin of Hysterangiales with subsequent range expansions to the Northern Hemisphere. However, neither Northern Hemisphere nor Southern Hemisphere taxa formed a monophyletic group, which is in conflict with a strictly vicariant scenario. Therefore, the occurrence and importance of long-distance dispersal could not be rejected. Although a pre-Gondwanan origin of Hysterangiales remains as a possibility, this hypothesis requires that Hysterangiales exist prior to the origin of the currently recognized ectomycorrhizal plants, as well as the arrival of mycophagous animals in Australia. This also requires that a basal paraphyletic assemblage represents parallel evolution of the ectomycorrhizal symbiosis, or that Hysterangiales was mycorrhizal with members of the extinct flora of Gondwana. Regardless, models for both ancient and more recent origins of Hysterangiales are consistent with truffle-like fungi being capable of transoceanic dispersal.     

Functional macronutritional generalism in a large omnivore,the brown bear

We combine a recently developed framework for describing dietary generalism with compositional data analysis to examine patterns of omnivory in a large widely distributed mammal. Using the brown bear (Ursus arctos) as a model species, we collected and analyzed data from the literature to estimate the proportions of macronutrients (protein, carbohydrate, and lipid) in the diets of bear populations. Across their range, bears consumed a diversity of foods that resulted in annual population diets that varied in macronutrient proportions, suggesting a wide fundamental macronutrient niche. The variance matrix of pairwise macronutrient log‐ratios indicated that the most variable macronutrient among diets was carbohydrate, while protein and lipid were more proportional or codependent (i.e., relatively more constant log‐ratios). Populations that consumed anthropogenic foods, such agricultural crops and supplementary feed (e.g., corn), had a higher geometric mean proportion of carbohydrate, and lower proportion of protein, in annual diets. Seasonally, mean diets were lower in protein and higher in carbohydrate, during autumn compared to spring. Populations with anthropogenic subsidies, however, had higher mean proportions of carbohydrate and lower protein, across seasons compared to populations with natural diets. Proportions of macronutrients similar to those selected in experiments by captive brown bears, and which optimized primarily fat mass gain, were observed among hyperphagic prehibernation autumn diets. However, the majority of these were from populations consuming anthropogenic foods, while diets of natural populations were more variable and typically higher in protein. Some anthropogenic diets were close to the proportions selected by captive bears during summer. Our results suggest that omnivory in brown bears is a functional adaptation enabling them to occupy a diverse range of habitats and tolerate variation in the nutritional composition and availability of food resources. Furthermore, we show that populations consuming human‐sourced foods have different dietary macronutrient proportions relative to populations with natural diets.     

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.     

Tetranucleotide microsatellite loci from the black bear (Ursus americanus)

We describe primers and polymerase chain reaction conditions to amplify 21 tetranucleotide microsatellite DNA loci in black bears (Ursus americanus). We tested primers using individuals from two populations, one each in Georgia and Florida. Among individuals from Georgia (n = 29), primer pairs yielded an average of 2.9 alleles (range, one to four) and an average observed heterozygosity (H_O) of 0.50 (range, 0.00 to 0.79). Among individuals from Florida (n = 19), primer pairs yielded an average of 5.7 alleles (range, one to 14) and an H_O of 0.55 (range, 0.00 to 1.00). A comparison of previously developed markers with individuals from Georgia suggests that bear populations in Georgia and Florida have reduced allelic diversity relative to other populations.     

Monitoring the effective population size of a brown bear (Ursus arctos) population using new single-sample approaches

The effective population size (N(e) ) could be the ideal parameter for monitoring populations of conservation concern as it conveniently summarizes both the evolutionary potential of the population and its sensitivity to genetic stochasticity. However, tracing its change through time is difficult in natural populations. We applied four new methods for estimating N(e) from a single sample of genotypes to trace temporal change in N(e) for bears in the Northern Dinaric Mountains. We genotyped 510 bears using 20 microsatellite loci and determined their age. The samples were organized into cohorts with regard to the year when the animals were born and yearly samples with age categories for every year when they were alive. We used the Estimator by Parentage Assignment (EPA) to directly estimate both N(e) and generation interval for each yearly sample. For cohorts, we estimated the effective number of breeders (N(b) ) using linkage disequilibrium, sibship assignment and approximate Bayesian computation methods and extrapolated these estimates to N(e) using the generation interval. The N(e) estimate by EPA is 276 (183-350 95% CI), meeting the inbreeding-avoidance criterion of N(e) > 50 but short of the long-term minimum viable population goal of N(e) > 500. The results obtained by the other methods are highly consistent with this result, and all indicate a rapid increase in N(e) probably in the late 1990s and early 2000s. The new single-sample approaches to the estimation of N(e) provide efficient means for including N(e) in monitoring frameworks and will be of great importance for future management and conservation.     

The Tanaka Line shaped the phylogeographic pattern of the cotton tree (Bombax ceiba) in southwest China

The Tanaka Line is considered to be an important phytogeographic boundary in southwest China, especially in Yunnan province. This Line has been deemed to separate East Asia's Sino-Himalayan and Sino-Japanese floras. However, it is not clear whether there is a special phylogeographic pattern for plants occurring across the Tanaka Line. To better understand the role of the Tanaka Line in shaping genetic structure of plant species occurring either side of this line, we employed Bombax ceiba, an economically and ecologically important tree species with a distribution across the Tanaka Line, as a proxy to study whether or not the Tanaka Line acts as a boundary to gene flow. We scanned and analyzed genetic variation at three chloroplast DNA fragments (psbB-psbF, trnL-rpl32 and psbI-psbK) within and among 17 natural populations (201 individuals). We identified eight chloroplast haplotypes (A-H) in total. Geographically, seven haplotypes were found southwest of the Tanaka Line, but only two haplotypes (B and H) were located northeast of the Tanaka Line. Meanwhile, both mismatch distribution analysis and environmental niche modeling (ENM) analysis suggested that multiple glacial refugia were maintained in the southwest range of B. ceiba during the last glacial maximum and that northeastern populations underwent strenuous retreatment during the Quaternary climatic oscillations. The present study highlights the importance of historical climate change and topographical circumstances in shaping population structure across the Tanaka Line.