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Wildlife must adapt to human presence to survive in the Anthropocene, so it is critical to understand species responses to humans in different contexts. We used camera trapping as a lens to view mammal responses to changes in human activity during the COVID-19 pandemic. Across 163 species sampled in 102 projects around the world, changes in the amount and timing of animal activity varied widely. Under higher human activity, mammals were less active in undeveloped areas but unexpectedly more active in developed areas while exhibiting greater nocturnality. Carnivores were most sensitive, showing the strongest decreases in activity and greatest increases in nocturnality. Wildlife managers must consider how habituation and uneven sensitivity across species may cause fundamental differences in human-wildlife interactions along gradients of human influence., (© 2024. The Author(s).)

Rehabilitation of injured or immature individuals has become an increasingly used conservation and management tool. However, scientific evaluation of rehabilitations is rare, raising concern about post-release welfare as well as the cost-effectiveness of spending scarce financial resources. Over the past 20 years, events of juvenile Eurasian lynx presumably orphaned have been observed in many European lynx populations. To guide the management of orphaned lynx, we documented survival, rehabilitation and fate after the release and evaluated the potential relevance of lynx orphan rehabilitation for population management and conservation implications. Data on 320 orphaned lynx was collected from 1975 to 2022 from 13 countries and nine populations. The majority of orphaned lynx (55%) were taken to rehabilitation centres or other enclosures. A total of 66 orphans were released back to nature. The portion of rehabilitated lynx who survived at least one year after release was 0.66. Release location was the best predictor for their survival. Of the 66 released lynx, ten have reproduced at least once (8 females and 2 males). Conservation implications of rehabilitation programmes include managing genetic diversity in small, isolated populations and reintroducing species to historical habitats. The lynx is a perfect model species as most reintroduced populations in Central Europe show significantly lower observed heterozygosity than most of the autochthonous populations, indicating that reintroduction bottlenecks, isolation and post-release management have long-term consequences on the genetic composition of populations. The release of translocated orphans could be a valuable contribution to Eurasian lynx conservation in Europe. It is recommended to release orphans at the distribution edge or in the frame of reintroduction projects instead of a release in the core area of a population where it is not necessary from a demographic and genetic point of view. Rehabilitation programmes can have conservation implications that extend far beyond individual welfare benefits., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Molinari-Jobin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Integrating data across studies with traditional microsatellite genetic markers requires careful calibration and represents an obstacle for investigation of wide-ranging species where populations require transboundary management. We used the "yardstick" method to compare results published across Europe since 2002 and new wolf (Canis lupus) genetic profiles from the Carpathian Mountains in Central Europe and the Dinaric Mountains in Southeastern Europe, with the latter as our reference population. We compared each population with Dinaric wolves, considering only shared markers (range 4-17). For each population, we calculated standard genetic diversity indices plus calibrated heterozygosity (Hec) and allelic richness (Ac). Hec and Ac in Dinaric (0.704 and 9.394) and Carpathian wolves (0.695 and 7.023) were comparable to those observed in other large and mid-sized European populations, but smaller than those of northeastern Europe. Major discrepancies in marker choices among some studies made comparisons more difficult. However, the yardstick method, including the new measures of Hec and Ac, provided a direct comparison of genetic diversity values among wolf populations and an intuitive interpretation of the results. The yardstick method thus permitted the integration of diverse sources of publicly available microsatellite data for spatiotemporal genetic monitoring of evolutionary potential., (© 2023. Springer Nature Limited.)

COVID-19 lockdowns in early 2020 reduced human mobility, providing an opportunity to disentangle its effects on animals from those of landscape modifications. Using GPS data, we compared movements and road avoidance of 2300 terrestrial mammals (43 species) during the lockdowns to the same period in 2019. Individual responses were variable with no change in average movements or road avoidance behavior, likely due to variable lockdown conditions. However, under strict lockdowns 10-day 95th percentile displacements increased by 73%, suggesting increased landscape permeability. Animals' 1-hour 95th percentile displacements declined by 12% and animals were 36% closer to roads in areas of high human footprint, indicating reduced avoidance during lockdowns. Overall, lockdowns rapidly altered some spatial behaviors, highlighting variable but substantial impacts of human mobility on wildlife worldwide.

Trace element pollution can adversely affect the brains of individuals and thus impact the entire population of apex predators, such as large European carnivores. We assessed exposure to prominent neurotoxicants As, Cd, Hg and Pb by measuring their brain stem levels in brown bears ( n = 114), grey wolves ( n = 8), Eurasian lynx ( n = 3), and golden jackals ( n = 2) sampled in 2015-2022 in Croatia. The highest of the non-essential elements was the Pb level in the bears' brains (median, Q1-Q3; 11.1, 7.13-24.1 μg/kg wet mass), with 4% of animals, all subadults, exceeding the established normal bovine levels (100 μg/kg wet mass). Species-specific differences were noted for Ca, Cd, Cu, Fe, Pb and Se brain levels. Female brown bears had higher As brain levels than males. Cubs and yearlings had lower brain Cd, but higher Zn, while subadults had higher Cu than adult bears. Hepatic As, Cd, Cu and Hg levels were shown to be a moderate proxy for estimating brain levels in bears (r S = 0.30-0.69). Multiple associations of As, Cd, Hg and Pb with essential elements pointed to a possible interaction and disturbance of brain Ca, Cu, Fe, Se and Zn homeostasis. Non-essential element levels in the brains of four studied species were lower than reported earlier for terrestrial meso-carnivores and humans. The age and sex of animals were highlighted as essential factors in interpreting brain element levels in ecotoxicological studies of large carnivores.

Intra- and inter-specific gene flow are natural evolutionary processes. However, human-induced hybridization is a global conservation concern across taxa, and the development of discriminant genetic markers to differentiate among gene flow processes is essential. Wolves (Canis lupus) are affected by hybridization, particularly in southern Europe, where ongoing recolonization of historic ranges is augmenting gene flow among divergent populations. Our aim was to provide diagnostic canid markers focused on the long-divergent Iberian, Italian and Dinaric wolf populations, based on existing genomic resources. We used 158 canid samples to select a panel of highly informative single nucleotide polymorphisms (SNPs) to (i) distinguish wolves in the three regions from domestic dogs (C. l. familiaris) and golden jackals (C. aureus), and (ii) identify their first two hybrid generations. The resulting 192 SNPs correctly identified the five canid groups, all simulated first-generation (F1) hybrids (0.482 ≤ Q i  ≤ 0.512 between their respective parental groups) and all first backcross (BC1) individuals (0.723 ≤ Q i  ≤ 0.827 to parental groups). An assay design and test with invasive and non-invasive canid samples performed successfully for 178 SNPs. By separating natural population admixture from inter-specific hybridization, our reduced panel can help advance evolutionary research, monitoring, and timely conservation management., (© 2022. The Author(s).)

The Balkan Peninsula and the Dinaric Mountains possess extraordinary biodiversity and support one of the largest and most diverse wolf ( Canis lupus ) populations in Europe. Results obtained with diverse genetic markers show west-east substructure, also seen in various other species, despite the absence of obvious barriers to movement. However, the spatial extent of the genetic clusters remains unresolved, and our aim was to combine fine-scale sampling with population and spatial genetic analyses to improve resolution of wolf genetic clusters. We analyzed 16 autosomal microsatellites from 255 wolves sampled in Slovenia, Croatia, Bosnia and Herzegovina (BIH), and Serbia and documented three genetic clusters. These comprised (1) Slovenia and the regions of Gorski kotar and Lika in Croatia, (2) the region of Dalmatia in southern Croatia and BIH, and (3) Serbia. When we mapped the clusters geographically, we observed west-east genetic structure across the study area, together with some specific structure in BIH-Dalmatia. We observed that cluster 1 had a smaller effective population size, consistent with earlier reports of population recovery since the 1980s. Our results provide foundation for future genomic studies that would further resolve the observed west-east population structure and its evolutionary history in wolves and other taxa in the region and identify focal areas for habitat conservation. They also have immediate importance for conservation planning for the wolves in one of the most important parts of the species' European range., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

The global lockdown to mitigate COVID-19 pandemic health risks has altered human interactions with nature. Here, we report immediate impacts of changes in human activities on wildlife and environmental threats during the early lockdown months of 2020, based on 877 qualitative reports and 332 quantitative assessments from 89 different studies. Hundreds of reports of unusual species observations from around the world suggest that animals quickly responded to the reductions in human presence. However, negative effects of lockdown on conservation also emerged, as confinement resulted in some park officials being unable to perform conservation, restoration and enforcement tasks, resulting in local increases in illegal activities such as hunting. Overall, there is a complex mixture of positive and negative effects of the pandemic lockdown on nature, all of which have the potential to lead to cascading responses which in turn impact wildlife and nature conservation. While the net effect of the lockdown will need to be assessed over years as data becomes available and persistent effects emerge, immediate responses were detected across the world. Thus, initial qualitative and quantitative data arising from this serendipitous global quasi-experimental perturbation highlights the dual role that humans play in threatening and protecting species and ecosystems. Pathways to favorably tilt this delicate balance include reducing impacts and increasing conservation effectiveness., Competing Interests: Authors declare no competing interests., (© 2021 Published by Elsevier Ltd.)

Where direct killing is rare and niche overlap low, sympatric carnivores may appear to coexist without conflict. Interference interactions, harassment and injury from larger carnivores may still pose a risk to smaller mesopredators. Foraging theory suggests that animals should adjust their behaviour accordingly to optimise foraging efficiency and overall fitness, trading off harvest rate with costs to fitness. The foraging behaviour of red foxes, Vulpes vulpes, was studied with automated cameras and a repeated measures giving-up density (GUD) experiment where olfactory risk cues were manipulated. In Plitvice Lakes National Park, Croatia, red foxes increased GUDs by 34% and quitting harvest rates by 29% in response to wolf urine. In addition to leaving more food behind, foxes also responded to wolf urine by spending less time visiting food patches each day and altering their behaviour in order to compensate for the increased risk when foraging from patches. Thus, red foxes utilised olfaction to assess risk and experienced foraging costs due to the presence of a cue from gray wolves, Canis lupus. This study identifies behavioural mechanisms which may enable competing predators to coexist, and highlights the potential for additional ecosystem service pathways arising from the behaviour of large carnivores. Given the vulnerability of large carnivores to anthropogenic disturbance, a growing human population and intensifying resource consumption, it becomes increasingly important to understand ecological processes so that land can be managed appropriately.

Tissue element investigations of apex terrestrial mammals are very scarce in Europe. We quantified 16 essential and nonessential elements in the kidney cortex, liver, and muscle tissue of 467 brown bears (Ursus arctos), 125 gray wolves (Canis lupus), one Eurasian lynx (Lynx lynx), and three golden jackals (Canis aureus) from Croatia by inductively coupled plasma mass spectrometry (ICP-MS). Renal cadmium (0.6% of animals) and lead (1%) and hepatic lead (5%) were found in toxicologically relevant levels for mammals only in bears, while the other elements were within normal range. The association of age, sex, season, and region with measured tissue elements in bear and wolf was estimated by multiple regression analyses. Age-related accumulation of cadmium was observed in bears and wolves. Lead tissue content increased with the age of bears but declined in wolves. Female bears and wolves had higher arsenic, iron, and thallium than males in some tissues. Also, cadmium, mercury, copper, zinc, selenium, molybdenum, and uranium were more abundant only in female bears. Male bears had higher potassium, zinc, and magnesium, while male wolves had higher calcium in some tissues compared to female wolves. Seasonal differences were mainly observed for bears' tissues and region-specific differences only in wolves. The bear kidneys had the highest levels of cobalt, copper, molybdenum, cadmium, and lead among the four studied species. The element levels reported for bears and wolves represent baseline values for the Dinaric population.

Background: Babesia spp. and Theileria spp. are important emerging causes of disease in dogs. Alongside these domesticated hosts, there is increasing recognition that these piroplasms can also be found in a range of wild animals with isolated reports describing the presence of these pathogen in foxes (Vulpes vulpes) and captive grey wolves (Canis lupus). The prevalence and impact of these infections in free-ranging populations of canids are unknown. To gain a better insight into the epidemiology and pathogenesis of piroplasm infections in free-ranging grey wolves, pathological and molecular investigations into captive and free-ranging grey wolves in Croatia were performed., Results: The carcasses of 107 free-ranging wolves and one captive wolf were the subjects of post-mortem investigations and sampling for molecular studies. A blood sample from one live captured wolf for telemetric tracking was also used for molecular analysis. PCR amplification targeting the 18S RNA gene revealed that 21 of 108 free-ranging wolves and one captive animal were positive for Theileria/Babesia DNA. Subsequent sequencing of a fragment of the 18S RNA gene revealed that 7/22 animals were positive for Babesia canis while the other amplified sequence were found to be identical with corresponding 18S rDNA sequences of Theileria capreoli isolated from wild deer (15/22). Haematological and cytological analysis revealed the presence of signet-ring shaped or pear-shaped piroplasms in several animals with the overall parasite burden in all positive animals assessed to be very low. Pathological investigation of the captive animal revealed fatal septicemia as a likely outcome of hemolytic anaemia. There was little or no evidence of hemolytic disease consistent with babesiosis in other animals., Conclusion: Importantly, the presence of B. canis in free-ranging grey wolves has not been described before but has been reported in a single fox and domestic dogs only. That B. canis infections cause disease in dogs but have little impact on wolf health possibly suggests that the wolf is the natural and the domestic dog is a secondary host. Surprisingly, the frequent finding of Theileria capreoli in wolves suggests that this Theileria species is not restricted to ungulates (cervids) but commonly infects also this carnivore species. Nevertheless, the potential role that these asymptomatically infected animals may play in the dispersal of these pathogens to susceptible sympatric species such as domesticated dogs requires further investigation.

The European wolf (Canis lupus) is a large carnivore species present in limited areas of Europe with several small populations still being considered as endangered. Wolves can be infected by a wide range of protozoan and metazoan parasites with some of them affecting free-living wolf health condition. On this account, an epidemiological survey was conducted to analyze the actual parasite fauna in Croatian wild wolves. In total, 400 individual faecal samples were collected during field studies on wolf ecology in the years 2002-2011. Parasite stages were identified by the sodium acetate acetic acid formalin (SAF)-technique, carbolfuchsin-stained faecal smears and Giardia/Cryptosporidium coproantigen-ELISAs. A subset of taeniid eggs-positive wolf samples was additionally analyzed by PCR and subsequent sequencing to identify eggs on Echinococcus granulosus/E. multilocularis species level. In total 18 taxa of parasites were here detected. Sarcocystis spp. (19.1%) occurred most frequently in faecal samples, being followed by Capillaria spp. (16%), ancylostomatids (13.1%), Crenosoma vulpis (4.6%), Angiostrongylus vasorum (3.1%), Toxocara canis (2.8%), Hammondia/Neospora spp. (2.6 %), Cystoisospora ohioensis (2.1%), Giardia spp. (2.1%), Cystoisospora canis (1.8%), Cryptosporidium spp. (1.8%), Trichuris vulpis (1.5%), Taenia spp. (1.5%), Diphyllobothrium latum (1.5%), Strongyloides spp. (0.5%), Opisthorchis felineus (0.5%), Toxascaris leonina (0.3%), Mesocestoides litteratus (0.3%) and Alaria alata (0.3%). Some of the here identified parasites represent relevant pathogens for wolves, circulating between these carnivorous definitive hosts and a variety of mammalian intermediate hosts, e. g. Taenia spp. and Sarcocystis spp., while others are considered exclusively pathogenic for canids (e.g. A. vasorum, C. vulpis, T. vulpis, Cystoisospora spp.). This study provides first records on the occurrence of the two relevant anthropozoonotic parasites, Giardia spp. and Cryptosporidium spp., in wild wolves from Croatia., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The ongoing refugee crisis in Europe has seen many countries rush to construct border security fencing to divert or control the flow of people. This follows a trend of border fence construction across Eurasia during the post-9/11 era. This development has gone largely unnoticed by conservation biologists during an era in which, ironically, transboundary cooperation has emerged as a conservation paradigm. These fences represent a major threat to wildlife because they can cause mortality, obstruct access to seasonally important resources, and reduce effective population size. We summarise the extent of the issue and propose concrete mitigation measures.

Controlling for background demographic effects is important for accurately identifying loci that have recently undergone positive selection. To date, the effects of demography have not yet been explicitly considered when identifying loci under selection during dog domestication. To investigate positive selection on the dog lineage early in the domestication, we examined patterns of polymorphism in six canid genomes that were previously used to infer a demographic model of dog domestication. Using an inferred demographic model, we computed false discovery rates (FDR) and identified 349 outlier regions consistent with positive selection at a low FDR. The signals in the top 100 regions were frequently centered on candidate genes related to brain function and behavior, including LHFPL3, CADM2, GRIK3, SH3GL2, MBP, PDE7B, NTAN1, and GLRA1. These regions contained significant enrichments in behavioral ontology categories. The 3rd top hit, CCRN4L, plays a major role in lipid metabolism, that is supported by additional metabolism related candidates revealed in our scan, including SCP2D1 and PDXC1. Comparing our method to an empirical outlier approach that does not directly account for demography, we found only modest overlaps between the two methods, with 60% of empirical outliers having no overlap with our demography-based outlier detection approach. Demography-aware approaches have lower-rates of false discovery. Our top candidates for selection, in addition to expanding the set of neurobehavioral candidate genes, include genes related to lipid metabolism, suggesting a dietary target of selection that was important during the period when proto-dogs hunted and fed alongside hunter-gatherers.

The mountain forest ecosystem of Gorski Kotar is distant from any significant sources of environmental pollution, though recent findings have revealed that this region is among the most intense (137)Cs contaminated area in Croatia. Therefore, the aim of this study was to investigate (137)Cs and (40)K load in three large predator species in the mountain forest ecosystem. Radionuclides mass activities were determined by the gamma-spectrometric method in the muscle tissue of brown bear (47), wolf (7), lynx (1) and golden jackal (2). The highest (137)Cs mass activity was found in lynx (153 Bq kg(-1)), followed by brown bear (132 Bq kg(-1)), wolf (22.2 Bq kg(-1)), and golden jackal (2.48 Bq kg(-1)). Analysis of 63 samples of dietary items suggests that they are not all potentially dominant sources of (137)Cs for wildlife. The most important source of radionuclides for the higher parts of the food-chain from the study area were found to be the mushroom species wood hedgehog (Hydnum repandum), with a transfer factor TF of 5.166, and blueberry (Vaccinium myrtillus) as a plant species (TF=2.096). Food items of animal origin indicated higher mass activity of radionuclides and therefore are possible moderate bioindicators of environmental pollution. The results also revealed that possible unknown wild animal food sources are a caesium source in the study region, and further study is required to illuminate this issue., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this pilot study, we investigated levels of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in the adipose tissues of two free-ranging terrestrial carnivores from Croatia sampled in 2010 and 2011: the brown bear (Ursus arctos; N=32) and the grey wolf (Canis lupus; N=29). Concentrations of ∑OCPs and ΣPCBs ranged from 0.45 to 4.09 ng g(-1) lipid mass (lm) and from 0.93 to 8.52 ng g(-1) lm in brown bear, and from 1.18 to 5.67 ng g(-1) lm and 2.68 to 48.9 ng g(-1) lm in grey wolf adipose tissues, respectively. PCBs were dominant accounting for over 72% of total analyzed persistent organic pollutants (POPs). The sum of six indicator non-dioxin-like PCBs (Σ6IndNDL PCBs) made up 60-93% and 58-85% of the total congener concentrations in brown bears and wolves, respectively. Although the levels of the measured parameters were significantly higher in grey wolves than in bears, the contaminant profiles of the two species were similar, with γ-HCH, HCB, β-HCH and DDE as major OCP contaminants, and PCB-153>PCB-180≈PCB-170>PCB-138 as the dominant congeners. The sum of hexachlorocyclohexanes (ΣHCHs) and 8 toxicologically relevant dioxin-like PCBs (Σ8ToxDL PCBs) was higher in the males than in the females of the brown bear. Concentrations of ΣDDTs, HCB, ΣOCP, ΣPCBs, Σ6IndNDL PCBs, and toxicologically relevant non-dioxin-like PCBs (ΣToxNDL PCBs) were significantly positively correlated with lipid content in the grey wolf. Concentrations of OCPs and PCBs in brown bears and wolves from Croatia were low and normal for large terrestrial mammals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Ecological and environmental heterogeneity can produce genetic differentiation in highly mobile species. Accordingly, local adaptation may be expected across comparatively short distances in the presence of marked environmental gradients. Within the European continent, wolves (Canis lupus) exhibit distinct north-south population differentiation. We investigated more than 67-K single nucleotide polymorphism (SNP) loci for signatures of local adaptation in 59 unrelated wolves from four previously identified population clusters (northcentral Europe n = 32, Carpathian Mountains n = 7, Dinaric-Balkan n = 9, Ukrainian Steppe n = 11). Our analyses combined identification of outlier loci with findings from genome-wide association study of individual genomic profiles and 12 environmental variables. We identified 353 candidate SNP loci. We examined the SNP position and neighboring megabase (1 Mb, one million bases) regions in the dog (C. lupus familiaris) genome for genes potentially under selection, including homologue genes in other vertebrates. These regions included functional genes for, for example, temperature regulation that may indicate local adaptation and genes controlling for functions universally important for wolves, including olfaction, hearing, vision, and cognitive functions. We also observed strong outliers not associated with any of the investigated variables, which could suggest selective pressures associated with other unmeasured environmental variables and/or demographic factors. These patterns are further supported by the examination of spatial distributions of the SNPs associated with universally important traits, which typically show marked differences in allele frequencies among population clusters. Accordingly, parallel selection for features important to all wolves may eclipse local environmental selection and implies long-term separation among population clusters.

The conservation of large carnivores is a formidable challenge for biodiversity conservation. Using a data set on the past and current status of brown bears (Ursus arctos), Eurasian lynx (Lynx lynx), gray wolves (Canis lupus), and wolverines (Gulo gulo) in European countries, we show that roughly one-third of mainland Europe hosts at least one large carnivore species, with stable or increasing abundance in most cases in 21st-century records. The reasons for this overall conservation success include protective legislation, supportive public opinion, and a variety of practices making coexistence between large carnivores and people possible. The European situation reveals that large carnivores and people can share the same landscape., (Copyright © 2014, American Association for the Advancement of Science.)

Free-living game can be an important source of dietary cadmium and lead; the question is whether exposure to these two elements is such that it might cause adverse health effects in the consumers. The aim of this study was to estimate dietary exposure to cadmium, lead, and mercury from free-living big game (fallow deer, roe deer, red deer, wild boar, and brown bear), and to mercury from small game (pheasant and hare), hunted in Croatia from 1990 to 2012. The exposure assessment was based on available literature data and our own measurements of metal levels in the tissues of the game, by taking into account different consumption frequencies (four times a year, once a month and once a week). Exposure was expressed as percentage of (provisional) tolerable weekly intake [(P)TWI] values set by the European Food Safety Authority (EFSA). Consumption of game meat (0.002-0.5 % PTWI) and liver (0.005-6 % PTWI) assumed for the general population (four times a year) does not pose a health risk to consumers from the general population, nor does monthly (0.02-6 % PTWI) and weekly (0.1-24 % PTWI) consumption of game meat. However, because of the high percentage of free-living game liver and kidney samples exceeding the legislative limits for cadmium (2-99 %) and lead (1-82 %), people should keep the consumption of certain game species' offal as low as possible. Children and pregnant and lactating women should avoid eating game offal altogether. Free-living game liver could be an important source of cadmium if consumed on a monthly basis (3-74 % TWI), and if consumed weekly (11-297 % TWI), it could even give rise to toxicological concern.

Hybridization and introgression can impact the evolution of natural populations. Several wild canid species hybridize in nature, sometimes originating new taxa. However, hybridization with free-ranging dogs is threatening the genetic integrity of grey wolf populations (Canis lupus), or even the survival of endangered species (e.g., the Ethiopian wolf C. simensis). Efficient molecular tools to assess hybridization rates are essential in wolf conservation strategies. We evaluated the power of biparental and uniparental markers (39 autosomal and 4 Y-linked microsatellites, a melanistic deletion at the β-defensin CBD103 gene, the hypervariable domain of the mtDNA control-region) to identify the multilocus admixture patterns in wolf x dog hybrids. We used empirical data from 2 hybrid groups with different histories: 30 presumptive natural hybrids from Italy and 73 Czechoslovakian wolfdogs of known hybrid origin, as well as simulated data. We assessed the efficiency of various marker combinations and reference samples in admixture analyses using 69 dogs of different breeds and 99 wolves from Italy, Balkans and Carpathian Mountains. Results confirmed the occurrence of hybrids in Italy, some of them showing anomalous phenotypic traits and exogenous mtDNA or Y-chromosome introgression. Hybridization was mostly attributable to village dogs and not strictly patrilineal. The melanistic β-defensin deletion was found only in Italian dogs and in putative hybrids. The 24 most divergent microsatellites (largest wolf-dog FST values) were equally or more informative than the entire panel of 39 loci. A smaller panel of 12 microsatellites increased risks to identify false admixed individuals. The frequency of F1 and F2 was lower than backcrosses or introgressed individuals, suggesting hybridization already occurred some generations in the past, during early phases of wolf expansion from their historical core areas. Empirical and simulated data indicated the identification of the past generation backcrosses is always uncertain, and a larger number of ancestry-informative markers is needed.

Muscle (n = 111), liver (n = 111), and kidney cortex (n = 101) samples from brown bears (Ursus arctos) were collected in the 2009 and 2010 hunting seasons in Croatia and analysed for selenium (Se), cadmium (Cd), and total mercury (Hg). The aim was to assess the levels of these elements according to age, sex, and season of collection, and to investigate possible Se/Cd and Se/Hg interactions. Median Se concentrations were 0.139 μg/g in muscle, 0.409 μg/g in liver and 1.75 μg/g wet mass in kidney cortex. Median Cd and Hg were 0.0078 and 0.0018 μg/g in muscle, 1.09 and 0.031 μg/g in liver, and 16.5 and 0.206 μg/g wet mass in the renal cortex, respectively. Se/Cd molar ratios were less than 1 in the kidney cortex, and close to or above 1 in liver and muscle, respectively. Toxic Cd and Hg correlated with Se in all of the studied tissues. Sex differences were found for all three elements (except Se in liver), with females having higher tissue concentration than males. Only Cd showed age-dependence. Bear samples collected in fall had higher Se in muscles, and Hg in muscles and liver compared to samples collected in spring. Element concentrations in brown bear tissues were within the range of previously reported studies. Bear meat is considered a rich source of Se, safe for consumption with regard to its Cd and Hg content. According to the molar ratio and correlation results, we assume that Se binding is not the primary detoxification pathway for Cd and Hg in brown bears.

To identify genetic changes underlying dog domestication and reconstruct their early evolutionary history, we generated high-quality genome sequences from three gray wolves, one from each of the three putative centers of dog domestication, two basal dog lineages (Basenji and Dingo) and a golden jackal as an outgroup. Analysis of these sequences supports a demographic model in which dogs and wolves diverged through a dynamic process involving population bottlenecks in both lineages and post-divergence gene flow. In dogs, the domestication bottleneck involved at least a 16-fold reduction in population size, a much more severe bottleneck than estimated previously. A sharp bottleneck in wolves occurred soon after their divergence from dogs, implying that the pool of diversity from which dogs arose was substantially larger than represented by modern wolf populations. We narrow the plausible range for the date of initial dog domestication to an interval spanning 11-16 thousand years ago, predating the rise of agriculture. In light of this finding, we expand upon previous work regarding the increase in copy number of the amylase gene (AMY2B) in dogs, which is believed to have aided digestion of starch in agricultural refuse. We find standing variation for amylase copy number variation in wolves and little or no copy number increase in the Dingo and Husky lineages. In conjunction with the estimated timing of dog origins, these results provide additional support to archaeological finds, suggesting the earliest dogs arose alongside hunter-gathers rather than agriculturists. Regarding the geographic origin of dogs, we find that, surprisingly, none of the extant wolf lineages from putative domestication centers is more closely related to dogs, and, instead, the sampled wolves form a sister monophyletic clade. This result, in combination with dog-wolf admixture during the process of domestication, suggests that a re-evaluation of past hypotheses regarding dog origins is necessary.

European wolves (Canis lupus) show population genetic structure in the absence of geographic barriers, and across relatively short distances for this highly mobile species. Additional information on the location of and divergence between population clusters is required, particularly because wolves are currently recolonizing parts of Europe. We evaluated genetic structure in 177 wolves from 11 countries using over 67K single nucleotide polymorphism (SNP) loci. The results supported previous findings of an isolated Italian population with lower genetic diversity than that observed across other areas of Europe. Wolves from the remaining countries were primarily structured in a north-south axis, with Croatia, Bulgaria, and Greece (Dinaric-Balkan) differentiated from northcentral wolves that included individuals from Finland, Latvia, Belarus, Poland and Russia. Carpathian Mountain wolves in central Europe had genotypes intermediate between those identified in northcentral Europe and the Dinaric-Balkan cluster. Overall, individual genotypes from northcentral Europe suggested high levels of admixture. We observed high diversity within Belarus, with wolves from western and northern Belarus representing the two most differentiated groups within northcentral Europe. Our results support the presence of at least three major clusters (Italy, Carpathians, Dinaric-Balkan) in southern and central Europe. Individuals from Croatia also appeared differentiated from wolves in Greece and Bulgaria. Expansion from glacial refugia, adaptation to local environments, and human-related factors such as landscape fragmentation and frequent killing of wolves in some areas may have contributed to the observed patterns. Our findings can help inform conservation management of these apex predators and the ecosystems of which they are part.

A microwave-assisted digestion method for the determination of cadmium (Cd) and lead (Pb) by inductively coupled plasma mass spectrometry (ICP-MS) was optimised on certified reference material (CRM) (bovine liver, BCR-185R) and wolf liver samples. Different factors influencing digestion efficiency (temperature, time, composition of the digestion mixture, sample mass) were tested. Validation included linearity (up to 200 μg L(-1) for Cd and Pb), detection (0.003 μg L(-1) for Cd and 0.035 μg L(-1) for Pb), and quantification (0.008 μg L(-1) for Cd and 0.081 μg L(-1) for Pb) limits. Good agreement between measured and certified values was achieved in all conditions, with recoveries ranging from 94 % to 111 % for Cd and from 95 % to 105 % for Pb. The precision of the method, expressed as relative standard deviation, was up to 3 % for Cd and 8 % for Pb. The best digestion parameters (260 °C, 30 min, 1 mL HNO3+4 mL H2O, 0.1 g of CRM) based on accuracy and precision were applied on two wolf liver samples to evaluate the need for the predigestion step (freeze-drying) and appropriate mass of the sample. Freeze-drying improved precision and minimising the tissue mass to 0.1 g reduced the matrix effect. Using these optimised digestion conditions, we determined Cd and Pb in 40 wolf livers collected in Croatia, and their medians (0.055 μg g(-1) and 0.107 μg g(-1), respectively) were in the range of previously reported data for the grey wolf.

Root hairs are an important model in studies of cell differentiation and development in higher plants. The function of NADPH oxidase-related reactive oxygen species (ROS) in root hair development has been reported extensively in studies on Arabidopsis. In this study, we investigated the mechanism of the initiation of root hair formation, mediated by the peroxidase-dependent production of the highly reactive hydroxyl radical in barley (Hordeum vulgare L.). The distribution of ROS, including the hydroxyl radical (OH) and superoxide (O(2)(-)) was assessed using hydroxyphenyl fluorescein and nitroblue tetrazolium chloride, respectively, in the roots of wild-type plants and two root-hair mutants: root-hairless (rhl1.a) and with root hair growth blocked at the primordium stage (rhp1.b). Peroxidase-dependent OH accumulation was linked to root hair initiation and growth in plants where root hair formation was at least initiated, whereas OH was not detectable in the epidermis of the root-hairless mutant rhl1.a. O(2)(-) distribution in the roots of rhl1.a and rhp1.b mutants was not impaired and did not influence the root hair phenotype. Peroxidase inhibitor treatments of wild-type roots dramatically reduced the ability of growing roots to form root hairs and thus phenocopied the root-hairless phenotype. Expression of two candidate peroxidase genes, HvPRX45 and HvPRX2, was analyzed and their possible role in root hair-specific production of hydroxyl radicals was discussed. We propose a model of a two-step, coordinated ROS formation process in root hair cells that involves root hair-specific peroxidase(s) and root hair-specific NADPH oxidase necessary for a proper root hair formation in barley., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The aim of the present study was to investigate the prevalence of Trichinella infection in wolves (Canis lupus) in a 17,468 km(2) area in Croatia. Muscle samples were collected from 67 wolves between 1996 and 2007 and analyzed by artificial digestion. Muscle larvae were detected in 21 wolves (31%) and genotyped by multiplex PCR. Trichinella britovi was the predominant species confirmed in 90% (19 wolves) while Trichinella spiralis was detected in 9% (2 wolves). The presence of the so called "domestic"Trichinella species was a surprise since, to date, only T. britovi had been reported in wild animals in this region. The larval burdens in infected animals ranged from 0.3 to 45.9 larvae per gram. The prevalence of infected animals varied by geographic region; infected animals were found in the region of Gorski Kotar (20%) which has very similar environment to the region of Lika, where almost all wolves were found infected. Interestingly, this is the first report of infected wolves in Dalmatia.

Effects of various intrinsic and extrinsic factors on 17 hematologic values from 56 brown bears (Ursus arctos) sampled in Croatia from 1981 to 2005 were evaluated. Differences between female and male bears were detected for number of erythrocytes, sedimentation rate after 30 min, and number of leukocytes and segmented neutrophils. Significant differences between free-living vs. captive and snared vs. not snared bears were detected for the same three parameters: leukocytes, segmented neutrophils, and eosinophils. It was concluded that the physical exertion of bears snared by leg, rather than their free-living status, influenced differences of results among these groups. The obtained mean values are useful reference values for the species.

Sixty seven serum samples collected from 43 European brown bears (Ursus arctos) from Croatia were tested for < or = 31 serum chemistry parameters. Results were grouped and compared by bears origin (method of capture), sex, age, mass, and season sampled. Greatest differences were found between captive and free-living bears, and minor differences were found when sex, age, mass, or season of sampling were compared. Creatine kinase was significantly different among three categories of bears with the highest mean value of 924 IU/l in snare captured free-living bears compared to 67.8 IU/l in captive ones. Results of these tests provide reference values for European brown bears.