Europe‐wide biogeographical patterns in the diet of an ecologically and epidemiologically important mesopredator,the red fox Vulpes vulpes: a quantitative review

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Wolf population genetics in Europe: a systematic review,meta‐analysis and suggestions for conservation and management

The grey wolf (Canis lupus) is an iconic large carnivore that has increasingly been recognized as an apex predator with intrinsic value and a keystone species. However, wolves have also long represented a primary source of human–carnivore conflict, which has led to long‐term persecution of wolves, resulting in a significant decrease in their numbers, genetic diversity and gene flow between populations. For more effective protection and management of wolf populations in Europe, robust scientific evidence is crucial. This review serves as an analytical summary of the main findings from wolf population genetic studies in Europe, covering major studies from the ‘pre‐genomic era’ and the first insights of the ‘genomics era’. We analyse, summarize and discuss findings derived from analyses of three compartments of the mammalian genome with different inheritance modes: maternal (mitochondrial DNA), paternal (Y chromosome) and biparental [autosomal microsatellites and single nucleotide polymorphisms (SNPs)]. To describe large‐scale trends and patterns of genetic variation in European wolf populations, we conducted a meta‐analysis based on the results of previous microsatellite studies and also included new data, covering all 19 European countries for which wolf genetic information is available: Norway, Sweden, Finland, Estonia, Latvia, Lithuania, Poland, Czech Republic, Slovakia, Germany, Belarus, Russia, Italy, Croatia, Bulgaria, Bosnia and Herzegovina, Greece, Spain and Portugal. We compared different indices of genetic diversity in wolf populations and found a significant spatial trend in heterozygosity across Europe from south‐west (lowest genetic diversity) to north‐east (highest). The range of spatial autocorrelation calculated on the basis of three characteristics of genetic diversity was 650?850 km, suggesting that the genetic diversity of a given wolf population can be influenced by populations up to 850 km away. As an important outcome of this synthesis, we discuss the most pressing issues threatening wolf populations in Europe, highlight important gaps in current knowledge, suggest solutions to overcome these limitations, and provide recommendations for science‐based wolf conservation and management at regional and Europe‐wide scales.     

Assessing the Importance of Organic Matrix Materials in Biofilm Chemical Reactivity: Insights from Proton and Cadmium Adsorption onto the Commercially Available Biopolymer Alginate

Biofilms coat the exterior of most water-exposed interfaces, from the surfaces of sediments and rocks to the interior walls of fluid transport systems and even medical and dental apparatus. Composed of a diverse assemblage of microbial species growing in a matrix of extracellular polymeric substances (EPS), biofilms are well-known for their ability to sorb metals and nucleate mineral phases. In this study, purified alginate, a major polysaccharide component of some algal and bacterial EPS, was studied to ascertain its chemical reactivity towards dissolved cadmium and protons, and thus better constrain its role in overall EPS reactivity. FTIR analysis and compositional constraints based on known molecular structure indicate that alginate’s geochemical behaviour is dominated by a single carboxyl functional group. Correspondingly, potentiometric titration data were best fit using a single functional group acidity constant (pK_a) and site concentration of 3.98 ± 0.01 and 1.728 ± 0.02 mol/kg, respectively, which are in agreement with typical carboxyl acidity (pK_a 3–6) and carboxyl functional group concentration based on alginate polymer composition. The logarithm of the Cd-carboxyl complexation constant (log K) was determined to be ?0.52 ± 0.22, lower than carboxyl-Cd stability constants reported from independent studies of isolated microbes. Together, these results place important constraints on organic matrix contributions to overall biofilm reactivity.     

Regional and local patterns of ectomycorrhizal fungal diversity and community structure along an altitudinal gradient in the Hyrcanian forests of northern Iran

? Altitudinal gradients strongly affect the diversity of plants and animals, yet little is known about the altitudinal effects on the distribution of microorganisms, including ectomycorrhizal fungi. ? By combining morphological and molecular identification methods, we addressed the relative effects of altitude, temperature, precipitation, host community and soil nutrient concentrations on species richness and community composition of ectomycorrhizal fungi in one of the last remaining temperate old-growth forests in Eurasia. ? Molecular analyses revealed 367 species of ectomycorrhizal fungi along three altitudinal transects. Species richness declined monotonically with increasing altitude. Host species and altitude were the main drivers of the ectomycorrhizal fungal community composition at both the local and regional scales. The mean annual temperature and precipitation were strongly correlated with altitude and accounted for the observed patterns of richness and community. ? The decline of ectomycorrhizal fungal richness with increasing altitude is consistent with the general altitudinal richness patterns of macroorganisms. Low environmental energy reduces the competitive ability of rare species and thus has a negative effect on the richness of ectomycorrhizal fungi. Because of multicollinearity with altitude, the direct effects of climatic variables and their seasonality warrant further investigation at the regional and continental scales.     

Construction of a medium-density horse gene map

A medium-density map of the horse genome (Equus caballus) was constructed using genes evenly distributed over the human genome. Three hundred and twenty-three exonic primer pairs were used to screen the INRA and the CHORI-241 equine BAC libraries by polymerase chain reaction and by filter hybridization respectively. Two hundred and thirty-seven BACs containing equine gene orthologues, confirmed by sequencing, were isolated. The BACs were localized to horse chromosomes by fluorescent in situ hybridization (FISH). Overall, 165 genes were assigned to the equine genomic map by radiation hybrid (RH) (using an equine RH(5000) panel) and/or by FISH mapping. A comparison of localizations of 713 genes mapped on the horse genome and on the human genome revealed 59 homologous segments and 131 conserved segments. Two of these homologies (ECA27/HSA8 and ECA12p/HSA11p) had not been previously identified. An enhanced resolution of conserved and rearranged chromosomal segments presented in this study provides clarification of chromosome evolution history.     

Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen

Different culture systems were evaluated for their ability to support attachment and proliferation of the somatic cells obtained from ovine semen. Ejaculates (n = 14) were collected from eight rams representing three breeds, Dorper, Suffolk and Hampshire. All samples were processed immediately and somatic cells were obtained from 11 of the 14 ejaculates. These cells had classic epithelial morphology and expressed cytokeratin, indicating they were of epithelial origin. Cells from four rams with the greatest growth rates were used for subsequent studies. Cells were cultured in four different media for 5 days and total numbers of attached cells vs. total numbers of seeded cells were counted and compared each day. Four media were evaluated: (1) a supplemented medium composed of DMEM/F12, 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 30 μg/ml bovine pituitary extract, 5 μg/ml insulin, 10 ng/ml cholera toxin, and 50 μg/ml gentamycin; (2) sheep fetal fibroblast (SFF)-conditioned medium; (3) swiss 3T3 fibroblast-conditioned medium; and (4) basic medium composed of DMEM/F12, 10% FBS, and 50 μg/ml gentamycin. Cell proliferation was greater in the supplemented medium, SFF-conditioned medium, and 3T3 fibroblast-conditioned medium compared to the basic medium by day 2 of culture (p < 0.05, n = 24), and greater in supplemented medium compared to the SFF-conditioned medium and 3T3 fibroblast-conditioned medium by day 4 of culture (p < 0.05, n = 24). Three different surfaces: (1) Matrigel basement membrane matrix-coated plastic; (2) collagen I-coated plastic; and (3) uncoated plastic were evaluated for their ability to support proliferation and attachment of the cells obtained from semen. Cell proliferation was greater when cells were cultured on the Matrigel-coated compared to the collagen I-coated and uncoated plastic by day 2 of culture (p < 0.05, n = 16). Cell attachment was greater when cells were plated on the Matrigel-coated and collagen I-coated plastic compared to the uncoated plastic (p < 0.05, n = 16). These studies describe an effective system for the culture and proliferation of epithelial cells obtained from ovine semen samples. The system may increase the likelihood of obtaining cells from frozen semen, which could be used for cloning to recover animals of genetic value in which semen is the only material that is available.     

The molecular dissection of mtDNA haplogroup H confirms that the Franco-Cantabrian glacial refuge was a major source for the European gene pool

Complete sequencing of 62 mitochondrial DNAs (mtDNAs) belonging (or very closely related) to haplogroup H revealed that this mtDNA haplogroup--by far the most common in Europe--is subdivided into numerous subhaplogroups, with at least 15 of them (H1-H15) identifiable by characteristic mutations. All the haplogroup H mtDNAs found in 5,743 subjects from 43 populations were then screened for diagnostic markers of subhaplogroups H1 and H3. This survey showed that both subhaplogroups display frequency peaks, centered in Iberia and surrounding areas, with distributions declining toward the northeast and southeast--a pattern extremely similar to that previously reported for mtDNA haplogroup V. Furthermore, the coalescence ages of H1 and H3 (~11,000 years) are close to that previously reported for V. These findings have major implications for the origin of Europeans, since they attest that the Franco-Cantabrian refuge area was indeed the source of late-glacial expansions of hunter-gatherers that repopulated much of Central and Northern Europe from ~15,000 years ago. This has also some implications for disease studies. For instance, the high occurrence of H1 and H3 in Iberia led us to re-evaluate the haplogroup distribution in 50 Spanish families affected by nonsyndromic sensorineural deafness due to the A1555G mutation. The survey revealed that the previously reported excess of H among these families is caused entirely by H3 and is due to a major, probably nonrecent, founder event.     

Modelling the Influence of Major Baltic Inflows on Near-Bottom Conditions at the Entrance of the Gulf of Finland

A coupled hydrodynamic-biogeochemical model was implemented in order to estimate the effects of Major Baltic Inflows on the near-bottom hydrophysical and biogeochemical conditions in the northern Baltic Proper and the western Gulf of Finland during the period 1991–2009. We compared results of a realistic reference run to the results of an experimental run where Major Baltic Inflows were suppressed. Further to the expected overall decrease in bottom salinity, this modelling experiment confirms that in the absence of strong saltwater inflows the deep areas of the Baltic Proper would become more anoxic, while in the shallower areas (western Gulf of Finland) near-bottom average conditions improve. Our experiment revealed that typical estuarine circulation results in the sporadic emergence of short-lasting events of near-bottom anoxia in the western Gulf of Finland due to transport of water masses from the Baltic Proper. Extrapolating our results beyond the modelled period, we speculate that the further deepening of the halocline in the Baltic Proper is likely to prevent inflows of anoxic water to the Gulf of Finland and in the longer term would lead to improvement in near-bottom conditions in the Baltic Proper. Our results reaffirm the importance of accurate representation of salinity dynamics in coupled Baltic Sea models serving as a basis for credible hindcast and future projection simulations of biogeochemical conditions.     

Copy Number Variation in the Horse Genome

We constructed a 400K WG tiling oligoarray for the horse and applied it for the discovery of copy number variations (CNVs) in 38 normal horses of 16 diverse breeds, and the Przewalski horse. Probes on the array represented 18,763 autosomal and X-linked genes, and intergenic, sub-telomeric and chrY sequences. We identified 258 CNV regions (CNVRs) across all autosomes, chrX and chrUn, but not in chrY. CNVs comprised 1.3% of the horse genome with chr12 being most enriched. American Miniature horses had the highest and American Quarter Horses the lowest number of CNVs in relation to Thoroughbred reference. The Przewalski horse was similar to native ponies and draft breeds. The majority of CNVRs involved genes, while 20% were located in intergenic regions. Similar to previous studies in horses and other mammals, molecular functions of CNV-associated genes were predominantly in sensory perception, immunity and reproduction. The findings were integrated with previous studies to generate a composite genome-wide dataset of 1476 CNVRs. Of these, 301 CNVRs were shared between studies, while 1174 were novel and require further validation. Integrated data revealed that to date, 41 out of over 400 breeds of the domestic horse have been analyzed for CNVs, of which 11 new breeds were added in this study. Finally, the composite CNV dataset was applied in a pilot study for the discovery of CNVs in 6 horses with XY disorders of sexual development. A homozygous deletion involving AKR1C gene cluster in chr29 in two affected horses was considered possibly causative because of the known role of AKR1C genes in testicular androgen synthesis and sexual development. While the findings improve and integrate the knowledge of CNVs in horses, they also show that for effective discovery of variants of biomedical importance, more breeds and individuals need to be analyzed using comparable methodological approaches.     

An Invasive Vector of Zoonotic Disease Sustained by Anthropogenic Resources: The Raccoon Dog in Northern Europe

The raccoon dog (Nyctereutes procyonoides) is an introduced species in Europe with a continually expanding range. Since the species is capable of affecting local ecosystems and is a vector for a number of severe zoonotic diseases, it is important to understand its food habits. Raccoon dog diet was studied in Estonia by examining the contents of 223 stomach samples collected during the coldest period of the year, August to March, in 2010–2012. The most frequently consumed food categories were anthropogenic plants (e.g. cereals, fruits; FO = 56.1%) and carrion (e.g. carcasses of artiodactyls and carnivores; FO = 48.4%). Carrion was also the only food category that was consumed significantly more frequently by raccoon dogs exhibiting symptoms of sarcoptic mange than by uninfected animals. Small mammals, which represent intermediate hosts for the zoonotic tapeworm Echinococcus multilocularis, were more commonly recorded in samples also containing anthropogenic plants than expected by chance. Comparison of raccoon dog and red fox (Vulpes vulpes) diet in Estonia revealed higher overlap than found elsewhere in Europe, with ‘carrion’ and ‘anthropogenic plants’ making up the bulk of both species’ diet; however, raccoon dogs were more omnivorous than red foxes. Our results suggest that while the use of most food categories reflects the phenology of natural food sources, ‘anthropogenic plants’ and ‘carrion’ provide an essential resource for raccoon dogs during the coldest period of the year, with the latter resource especially important for individuals infected with sarcoptic mange. Since both of these food categories and small mammals are often found at supplementary feeding sites for wild boar (Sus scrofa), this game management practice may facilitate high densities of mesocarnivores and promote the spread of some severe zoonotic diseases, including alveolar echinococcosis, trichinellosis, rabies and sarcoptic mange.