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Amphibians are particularly sensitive to habitat loss and fragmentation caused by the intensification and modernization of farming occurring in the second half of the twentieth century in the Mediterranean basin. However, artificial water bodies, associated with traditional husbandry, proved to be important surrogate for amphibian feeding and reproduction. Here, multilocus genotypes were used to investigate the spatial population structure of Lissotriton vulgaris meridionalis and the role of drinking troughs in supporting viable breeding populations within a rural landscape interested by traditional husbandry and agriculture. Our genetic analysis highlighted the conservation value and the potential stepping-stone function of artificial aquatic sites in the dispersal of the species and for the gene flow maintenance. Indeed, populations of drinking troughs show allelic richness and heterozygosity levels comparable to those from natural ponds and there is no great evidence of genetic bottlenecks. A complex system of artificial aquatic sites and few natural wetlands was identified sustaining a well-structured network of demes highly interconnected with themselves and natural aquatic sites. The conservation of the identified genetic clusters may be useful to prevent further population declines and future loss of genetic diversity within the study area characterized by scarce natural wetlands that frequently dried because of agricultural practices and strong seasonality. Site-specific protection measures are needed to contrast the progressive disappearance of drinking troughs observed in the last years in Italy because of the abandonment of traditional farming practices in favour of modern agriculture and intensive farming.

Background The low cost and rapidity of microsatellite analysis have led to the development of several markers for many species. Because in non-invasive genetics it is recommended to genotype individuals using few loci, generally a subset of markers is selected. The choice of different marker panels by different research groups studying the same population can cause problems and bias in data analysis. A priority issue in conservation genetics is the comparability of data produced by different labs with different methods. Here, we compared data from previous and ongoing studies to identify a panel of microsatellite loci efficient for the long-term monitoring of Apennine brown bears ( Ursus arctos marsicanus ), aiming at reducing genotyping uncertainty and allowing reliable individual identifications overtimes. Results We examined all microsatellite markers used up to now and identified 19 candidate loci. We evaluated the efficacy of 13 of the most commonly used loci analyzing 194 DNA samples belonging to 113 distinct bears selected from the Italian national biobank. We compared data from 4 different marker subsets on the basis of genotyping errors, allelic patterns, observed and expected heterozygosity, discriminatory powers, number of mismatching pairs, and probability of identity. The optimal marker set was selected evaluating the low molecular weight, the high discriminatory power, and the low occurrence of genotyping errors of each primer. We calibrated allele calls and verified matches among genotypes obtained in previous studies using the complete set of 13 STRs (Short Tandem Repeats), analyzing six invasive DNA samples from distinct individuals. Differences in allele-sizing between labs were consistent, showing a substantial overlap of the individual genotyping. Conclusions The proposed marker set comprises 11 Ursus specific markers with the addition of cxx20, the canid-locus less prone to genotyping errors, in order to prevent underestimation (maximizing the discriminatory power) and overestimation (minimizing the genotyping errors) of the number of Apennine brown bears. The selected markers allow saving time and costs with the amplification in multiplex of all loci thanks to the same annealing temperature. Our work optimizes the available resources by identifying a shared panel and a uniform methodology capable of improving comparisons between past and future studies.

Determination of parentage provides valuable information for the conservation of wild populations, for instance, by allowing the monitoring of breeding success and inbreeding. Between 1999 and 2002, nine brown bears ( Ursus arctos ) were translocated to augment the remnant population of a few surviving individuals in the Italian Alps, but only part of them reproduced, with a higher inbreeding risk occurrence in the long-time. Currently, in the Alpine population, parentage tests are assessed through the analysis of 15 microsatellite loci (STRs), but the reduction of genetic variability in future generations will need the use of additional informative markers. Single nucleotide polymorphisms (SNPs) have been proven to be useful and reliable in individual identification and family reconstruction; moreover, they can perform well on low-quality samples. In this study, we analysed 51 SNPs to generate a SNP multilocus genotype dataset of 54 Alpine brown bears ( Ursus arctos ) and compared its performance in parentage analysis with the validated STR dataset. We found that SNPs alone are not sufficient to determine parentage relationships, but the combination of SNPs and STRs provided unambiguous parentage assignments. The combined panel also performed better than STRs when true parents were not present in the dataset and, consequently, showed higher values of assignment probabilities.

Sexually Selected Infanticide (SSI) is thought of as a male reproductive strategy in social mammalian species, because females who lose cubs may quickly re-enter oestrus. SSI has rarely been documented in non-social mammals and, in brown bears, SSI has been studied mainly in an eco-ethological perspective. The authors examined the first genetically documented infanticide case which occurred in May 2015 in brown bears in Italy (Trentino, Central-Eastern Alps). The infanticide killed two cubs and their mother. Hair samples were collected from the corpses as well as saliva, through swabs on mother’s wounds, with the aim of identifying the genotype of the perpetrator. The samples were genotyped by PCR amplification of 15 autosomal microsatellite loci, following the protocol routinely used for individual bear identifications within the Interregional Action Plan for Brown Bear Conservation in the Central-Eastern Alps (PACOBACE). Reliable genotypes were obtained from the mother, cubs and putative perpetrator. The genotypes were matched with those populating the PACOBACE database and genealogies were reconstructed. Both mother and perpetrator genotypes were already present in the database. Kinship analyses confirmed mother-cubs relationships and identified the father of the cubs. In this study, for the first time, the authors used the open-source LRmix STUDIO software, designed to analyse human forensic genetic profiles, to solve a case in wildlife. Through LRmix STUDIO, those alleles that do not belong to the victims were isolated and, finally, the perpetrator was identified. This study presents a method that allows, through the application of different models, the genetic identification of the conspecific perpetrator with the highest probability. The identification of the infanticidal male is relevant for the better management and conservation of wild populations with small effective population size (Ne) and low population growth rate, especially in the case of recently established populations in human-dominated landscapes. This procedure will have predictably wide applications, supplying important data in the monitoring of small and isolated populations.

From September 2006 through May 2010, we repeatedly detected an adult male bear (G70) through non-invasive sampling in the Sibillini National Park (SNP; central Apennines, Italy), at the northernmost periphery of the reported Apennine bear range. Notwithstanding sustained sampling effort, we failed to detect bear G70 in SNP after May 2010, but in autumn 2010 it was twice detected, through non-invasive sampling, in the Duchessa Nature Reserve (76 km south of the SNP), revealing its southward travel across the central Apennines. More than one year later (16 January 2012), a male bear was live-captured in the Sirente-Velino Regional Park showing clinical symptoms of Aujeszki's disease. The bear died overnight, and genotyping revealed it to be bear G70. Although the causes of death were not clearly determined, poisoning, shooting and vehicle accident were ruled out, suggesting more subtle mortality factors (e.g., diseases) were responsible. The long distance movements and the fate of this adult male bear indicate that, even though protected and suitable areas are connected across the Apennines to some degree, the expected expansion of the Apennine bear range from the core distribution might be suffering from undisclosed anthropogenic risks of mortality in the peripheral portions of the range.

An assessment of the genetic diversity and structure of a population is essential for designing recovery plans for threatened species. Italy hosts two brown bear populations, Ursus arctos marsicanus (Uam), endemic to the Apennines of central Italy, and Ursus arctos arctos (Uaa), in the Italian Alps. Both populations are endangered and occasionally involved in human–wildlife conflict; thus, detailed management plans have been in place for several decades, including genetic monitoring. Here, we propose a simple cost-effective microsatellite-based protocol for the management of populations with low genetic variation. We sampled 22 Uam and 22 Uaa individuals and analyzed a total of 32 microsatellite loci in order to evaluate their applicability in individual identification. Based on genetic variability estimates, we compared data from four different STR marker sets, to evaluate the optimal settings in long-term monitoring projects. Allelic richness and gene diversity were the highest for the Uaa population, whereas depleted genetic variability was noted for the Uam population, which should be regarded as a conservation priority. Our results identified the most effective STR sets for the estimation of genetic diversity and individual discrimination in Uam (9 loci, PIC 0.45; PID 2.0 × 10−5), and Uaa (12 loci, PIC 0.64; PID 6.9 × 10−11) populations, which can easily be utilized by smaller laboratories to support local governments in regular population monitoring. The method we proposed to select the most variable markers could be adopted for the genetic characterization of other small and isolated populations.

Brown bears in Italy persist in two isolated populations, one in the Alpine and the other in the Apennine mountain range. Both are threatened and elusive. Non-invasive genetics provides a good way to monitor the populations. Microsatellites (STRs) have been the marker of choice for non-invasive genetic monitoring, but due to non-invasive bad quality samples, these analyses were plagued by low amplification rates and genotyping errors. Moreover, to compare microsatellite genotypes, allele calibration is needed between laboratories, leading to difficulties in individual identification. In contrast, SNP genotyping is directly comparable between laboratories, and more sensitive and accurate. Here we test a 96-marker SNP chip developed for the Scandinavian brown bear population on the Italian populations. A subset of these SNPs was found informative and could reliable confirm species, sex and, only in the Alpine population, distinguish individuals. A total of 51 informative SNPs provided better resolution power than 15 STRs, used in the routine monitoring of the Alpine population in Italy. In contrast, only 15 SNPs were found to be informative for the Apennine population, which did not have enough resolution to discriminate individuals and were less informative than 11 STRs. While highly useful in the Alpine population, additional SNP markers must be included to reach the same level of resolution in the Apennine population.

Habitat loss and fragmentation are threatening amphibians by increasing population isolation. However, artificial waterbodies created for livestock may contrast this phenomenon by providing surrogate habitats for amphibians. Here, we performed a genetic study on an amphibian species, Lissotriton vulgaris meridionalis, in a rural area in central Italy where natural wetlands are disappearing and drinking troughs for cattle watering are widespread. Specifically, we tested a panel of microsatellite markers to identify a suitable tool for addressing conservation genetic issues of this species that is undergoing severe local decline. Twelve of the 20 tested loci produced reliable amplifications and were polymorphic. Three distinct units with a low level of gene flow were distinguished and the population genetic structuring overlapped with geographic distribution. Such loci will be useful to assess the genetic diversity of the species across multiscale levels for its management and conservation.

La presente comunicación centra su contenido en una experiencia singular de transformación educativa en la etapa 0-6 y presenta como rasgo distintivo la implicación y el compromiso de los tres agentes que lo han hecho posible: el Ayuntamiento de Vic, a través de su Concejalía de Educación; la de la Universidad de Vic -Universidad Central de Cataluña (UVic-UCC), tanto a través de diversas profesoras de su Facultad de Educación, Traducción, Deportes y Psicología (FETEP) como a través del Centro de Innovación y Formación en Educación (CIFE), centro dedicado a la formación permanente de los maestros y maestras; y finalmente las

Evidence on the health effects of extreme temperatures and air pollution is copious. However few studies focused on their interaction. The aim of this study is to evaluate daily PM10 and ozone as potential effect modifiers of the relationship between temperature and natural mortality in 25 Italian cities. Time-series analysis was run for each city. To evaluate interaction, a tensor product between mean air temperature (lag 0–3) and either PM10 or ozone (both lag 0–5) was defined and temperature estimates were extrapolated at low, medium, and high levels of pollutants. Heat effects were estimated as percent change in mortality for increases in temperature between 75th and 99th percentiles. Results were pooled by geographical area. Differential temperature-mortality risks by air pollutants were found. For PM10, estimates ranged from 3.9% (low PM10) to 14.1% (high PM10) in the North, from 3.6% to 24.4% in the Center, and from 7.5% to 21.6% in the South. Temperature-related mortality was similarly modified by ozone in northern and central Italy, while no effect modification was observed in the South. This study underlines the synergistic effects of heat and air pollution on mortality. Considering the predicted increase in heat waves and stagnation events in the Mediterranean countries such as Italy, it is time to enclose air pollution within public health heat prevention plans.