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Lemon verbena infusions are widely appreciated due to their agreeable lemony flavor and medicinal properties. In this study, the antioxidant potential, phenolic profile, and free amino acid profile of lemon verbena infusions from different commercial brands were studied. Characterization by UHPLC-QTOF-HRMS allowed the identification of 34 phenolics. The free amino acid profile (by RP-HPLC-FLD) was assessed for the first time, allowing the quantification of 16 amino acids. Furthermore, the infusions showed high antioxidant activity by different assays (ferric reducing antioxidant power, DPPH• scavenging, and oxygen radical absorbance capacity assays), which in turn were significantly correlated with total phenolics and total flavonoid contents. Notwithstanding, phenylalanine seemed to have also an impact on the antioxidant activity of the infusions, with significant correlations found. Finally, significant differences were found in all the evaluated parameters for one of the four commercial brands herein studied, which was possibly related to the different geographical origins of this sample. Overall, these lemon verbena infusions proved to be rich in a huge variety of bioactive compounds that can provide therapeutic potential.

[Introduction] Francisella tularensis is a highly infectious bacterium that causes the zoonotic disease tularemia. The development of genotyping methods, especially those based on whole-genome sequencing (WGS), has recently increased the knowledge on the epidemiology of this disease. However, due to the difficulties associated with the growth and isolation of this fastidious pathogen in culture, the availability of strains and subsequently WGS data is still limited., [Methods] To surpass these constraints, we aimed to implement a culture-free approach to capture and sequence F. tularensis genomes directly from complex samples. Biological samples obtained from 50 common voles and 13 Iberian hares collected in Spain were confirmed as positive for F. tularensis subsp. holarctica and subjected to a WGS target capture and enrichment protocol, using RNA oligonucleotide baits designed to cover F. tularensis genomic diversity., [Results] We obtained full genome sequences of F. tularensis from 13 animals (20.6%), two of which had mixed infections with distinct genotypes, and achieved a higher success rate when compared with culture-dependent WGS (only successful for two animals). The new genomes belonged to different clades commonly identified in Europe (B.49, B.51 and B.262) and subclades. Despite being phylogenetically closely related to other genomes from Spain, the detected clusters were often found in other countries. A comprehensive phylogenetic analysis, integrating 599 F. tularensis subsp. holarctica genomes, showed that most (sub)clades are found in both humans and animals and that closely related strains are found in different, and often geographically distant, countries., [Discussion] Overall, we show that the implemented culture-free WGS methodology yields timely, complete and high-quality genomic data of F. tularensis, being a highly valuable approach to promote and potentiate the genomic surveillance of F. tularensis and ultimately increase the knowledge on the genomics, ecology and epidemiology of this highly infectious pathogen.

Thanks to the Pan-European Common Birds Monitoring Scheme and Breeding Bird Survey, the temporal changes in abundance of the Red-legged Partridge are known in most countries of its area of distribution. However, these are based on abundance indices and no strong inference is possible about potential causes of local trends. Robust estimates of abundance or densities at national scale are still lacking. Information from hunting bags cannot be used owing to the inclusion of released Red-legged Partridges in those numbers. Even in the absence of released individuals in hunting bags, these estimates cannot be used owing to a lack of knowledge of variations in reporting behaviour in Europe and potential age-specific mortality. Numbers at the local scale are obtained using similar (although not equal) methods across countries based on line/point transect methods. Given that some capture-recapture studies have been carried out from time to time in most of the distribution range, their results could help to estimate the demographic parameters required for management. Finally, given that these common methods are applied with, possibly, some avoidable differences, a more integrative strategy of methodological design and spatial sampling may make it possible to extrapolate estimates from regional to national/continental scale, and to provide assessments of abundance as well as spatial and temporal variation in Red-legged Partridge population size. This global approach may increase the current value of monitoring to all stakeholders interested in Red-legged Partridges.

Measures of performance in wild ungulates must consider density dependent and stochastic factors such as parasitic rates (as natural enemies in intimae relation with hosts) along with the impact of population management. In order to standardize parasite counts to be useful ecological indicators, it is essential to test host and parasite responses to the abovementioned. In the Iberian Peninsula, the parasitic nematode Elaphostrongylus cervi and its host the red deer (Cervus elaphus) provide an excellent practical scenario to illustrate how parasitic rates should be interpreted under different management contexts. Our study focuses on two distinct management scenarios, one deer population (LM) with supplemental feeding and one population (QM) with almost no human intervention, aside from hunting. We aim to assess how management (supplemental feeding) modulates the relationships of density dependent, stochastic and individual factors, and E. cervi faecal larvae counts in the red deer, so as with other physiological traits (body condition and spleen weight - as a proxy to immunological status). Overall, we have shown the decisive role that density and precipitation can have in a Mediterranean environment, with different trends in the two studied areas (e.g. negative correlation between E. cervi and red deer density in supplemented population, in opposition to un-supplemented). The crucial role that precipitation combined with density can have on deer dynamics, along with the artificial feeding as buffer of some traits, reinforces the need for an adaptive management, and the demand to manage each population considering the surrounding environment. Management strategies selected in each site can establish populations dynamics and health and should be closely monitored in a long-term context, as it occurs in our study.

Intra-annual wood density fluctuations are valuable indicators of the role of the local climate and provenance origins on tree growth. These intra-ring wood features were the most informative about local adaptation and phenotypic plasticity ofPinus halepensisMill. in a provenance trial. This highlights the importance of taking provenance into account when selecting forest reproductive material to mitigate the effects of climate change. Context: It is essential to disentangle the influence of climate from the effect of provenance on inter- and intra-annual wood traits to identify populations’ responses to climate changes. This will be even more relevant in the drought-prone Mediterranean region, where species have high genetic differentiation among populations. Aims: We investigated the effects of provenance and climate driving Pinus halepensis Mill. wood traits, as imprinted phenotypic adaptations to the Mediterranean climate. Methods: Inter- and intra-annual wood trait variation (ring-, early-, and latewood width, wood density) was studied by microdensitometry in 8 provenances from Spain, France, Greece, and Italy in a common-garden experiment. We related wood traits and density fluctuations with inter-annual climate variations at the trial site and the provenance origin. Results: Provenances showed distinct responses to climate regarding tree growth, wood density, and intra-annual fluctuations. Density fluctuations in latewood were more frequent than in earlywood. Climate at the provenance origin was a more important force for density fluctuations than climatic variations at the planting site, with different trends between coastal and inland provenances, and higher frequency of latewood fluctuations in provenances from arid and semiarid sites. Conclusion: Differences in P. halepensis wood density fluctuations confirmed the imprinted origin provenance control in response to climate variations and indicated a relation with local adaptation of these xylem anatomical

Gold nanoparticles (AuNPs) are interesting for the design of new cancer theranostic tools, mainly due to their biocompatibility, easy molecular vectorization, and good biological half-life. Herein, we report a gold nanoparticle platform as a bimodal imaging probe, capable of coordinating Gd3+ for Magnetic Resonance Imaging (MRI) and 67Ga3+ for Single Photon Emission Computed Tomography (SPECT) imaging. Our AuNPs carry a bombesin analogue with affinity towards the gastrin releasing peptide receptor (GRPr), overexpressed in a variety of human cancer cells, namely PC3 prostate cancer cells. The potential of these multimodal imaging nanoconstructs was thoroughly investigated by the assessment of their magnetic properties, in vitro cellular uptake, biodistribution, and radiosensitisation assays. The relaxometric properties predict a potential T1- and T2- MRI application. The promising in vitro cellular uptake of 67Ga/Gd-based bombesin containing particles was confirmed through biodistribution studies in tumor bearing mice, indicating their integrity and ability to target the GRPr. Radiosensitization studies revealed the therapeutic potential of the nanoparticles. Moreover, the DOTA chelating unit moiety versatility gives a high theranostic potential through the coordination of other therapeutically interesting radiometals. Altogether, our nanoparticles are interesting nanomaterial for theranostic application and as bimodal T1- and T2- MRI / SPECT imaging probes.

Wild ungulates have expanded their geographical range across Europe and Portugal is no exception. Despite the known benefits associated with the increase of these populations (e.g. increased prey for wild carnivores), the negative impacts also need to be taken into account (e.g. damages in agriculture and forestry, ungulate–vehicle collisions). Additionally, their role as reservoirs of zoonotic agents has gained scientific relevance due to the potential human health risks, impact on livestock and food safety. In northeast Portugal, Montesinho Natural Park, three species of ungulates occur in sympatry, the wild boar Sus scrofa, the red deer Cervus elaphus and the roe deer Capreolus capreolus. Considering their close association with humans and livestock, it is essential to understand their role as reservoirs of infectious diseases, namely as vectors for parasitic infections. In order to achieve this, 112 fresh faecal samples were collected to assess, by means of coprological analyses, their parasite diversity, prevalence and mean intensity. In total, 88 (78.60%, ±69.81–85.76) samples were infected with at least one parasite species. Parasite prevalence was different among the three species, with the red deer showing higher prevalence values (83.6%), then the wild boar (80.2%) and the roe deer (46.7%). The results have revealed that these species carry parasites that not only represent a health problem for domestic ruminants and domestic pigs (e.g. Muellerius sp., Trichostrongylidae, Strongylidae, Metastrongylus sp., Moniezia benedeni, Eimeria spp. and Cystoisospora sp.) but they can also pose a potential public health risk (e.g. Balantidium coli). The implementation of surveillance programs must include regular monitoring protocols of wild ungulates.

Throughout recent years, ungulates have experienced significant increases in numbers and geographic range sizes in Europe, becoming locally overabundant populations in some regions. Changes in legislation regarding poaching, abandonment of land and re‐naturalisation of habitats, and decreasing numbers of hunters, among other things, have led to alarming scenarios in wild ungulate biology. Although ungulates bring some financial benefits for ecosystems and society through tourism and hunting, the problems associated with populations that are no longer controlled can outweigh the advantages. Damage to forestry and agriculture, ungulate‐vehicle collisions, and diseases are among the most concerning problems related to ungulate overabundance. To deal with these problems and to decide on the best management strategy to apply, it is essential to have tools available to monitor populations with an integrative approach based on ecological change indicators, and to assess population and ecosystem status. Furthermore, in a globalised world, people’s opinions matter, and sociological studies regarding human perception of wild mammals must take place in order to allow proper management, including consideration of people’s expectations as well as animal and ecosystem needs. Successful and unsuccessful management strategies have already been attempted, and the knowledge of consequences over time enables an adaptive approach. Management of ungulate populations is a complex subject, and each case should be studied, analysing the cost_performance balance of measures to be taken, and ensuring ongoing financial means to carry out and continue with successful ecosystem management strategies. Multidisciplinary teams should be built, including biologists, veterinarians, stakeholders, sociologists, and others, to deal with the management of European wild ungulate populations.

Reliability of population size and density estimates is one of the most contentious issues when evaluating the conservation status of species. Non-invasive DNA monitoring, combined with spatially explicit capture-recapture approaches (SCR), is recurrently presented as a reliable procedure to achieve accurate, precise and feasible estimates. However, conservation practitioners frequently face the dilemma of which molecular marker type would perform better for their objectives. Here, using as case study the eastern subpopulation of the endangered Cantabrian brown bear, and the same dataset of non-invasive samples, we evaluated which molecular marker perform better (microsatellites vs. SNPs), and whether the selection of the marker could impact on estimates. Our results show similar posterior estimates for bear density and for the scale parameter, resulting in consistent population estimates, around 50 bears, regardless the molecular marker type selected, microsatellites: 48.4 (95%BCI = 33.8–67.3) or SNPs: 52.7 (95%BCI = 36.0–74.8). Using our dataset, we provide evidence for the importance of running several replicates for both microsatellites and SNPs when genotyping non-invasive samples, and how for the same number of molecular markers, microsatellites were statistically more powerful. The positive trend observed in small large carnivore populations requires the adaptation of monitoring approaches. The quality of indexes commonly used, such as the minimum annual counts of females with cubs of the year in our study case, may be more difficult to maintain over time as populations increase in size and range. We therefore recommend the implementation of a regular monitoring based on non-invasive DNA monitoring and SCR approaches.

Viruses are ubiquitous in nature; however, very few have been identified in the Leporid species. In the fall of 2018, an outbreak of myxomatosis in Iberian hares (Lepus granatensis) was reported in Spain and a novel recombinant myxoma virus strain (MYXV-Tol) was identified. To investigate variability within the recombinant region of the MYXV-Tol and identify any potential viral coinfections, samples (ear, eyelid or vaginal) of Iberian hares were collected from Spain and analyzed. The presence of the recombinant region of the MYXV-Tol was confirmed in six out of eleven samples analyzed. Additionally, a polyomavirus (family Polyomaviridae), representing a putative new species, and anelloviruses (family Anelloviridae) belonging to two putative species were identified, some as coinfection with the recombinant MYXV-Tol. The two polyomavirus genomes were identified in two hares and share >99% genome-wide identity. Based on the analysis of their large T-antigen, the new polyomavirus clusters in a distant clade from other mammals sharing <64% amino acid identity. A total of 14 anelloviruses were identified, which share 63–99% genome-wide identity. Overall, our results show a coinfection of different DNA viruses in the studied samples and raise awareness regarding the extensive unsampled diversity of viruses in hares.

To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR). The current knowledge of the molecular determinants and mechanistic details of the DDR owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of DSBs at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNA-templated processes and the specific contribution of the chromatin environment. In this review, we present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR.

The human dimension of wildlife is a subject of increasing interest, especially considering the potential impact of people's perceptions on decision-making concerning wildlife management and thus on species' distribution and abundance. This is particularly important for species that inhabit human-dominated landscapes, where conflicts are likely to arise. These conflicts typically emerge between different human collectives when their interests collide and thus an assessment of their perceptions is valuable. Throughout Europe, ungulates have expanded, and Portugal is no exception. The expansion of red deer (Cervus elaphus) comes with benefits (e.g. hunting opportunities), but also with costs (e.g. vehicle collision or damage to crops), that can shape people's perceptions of these populations. To assess perceptions of red deer populations in continental Portugal, we developed a questionnaire survey with three interest groups: general public, farmers and hunters (total n = 1532). Our results show that perceptions about red deer were generally positive with a high acknowledgement of deer benefits, which we link to a broad level of sympathy towards this species. In addition, farmers showed a higher concern with crop damage caused by deer than non-farmers. Nonetheless, a general lack of knowledge regarding wildlife was present, particularly among younger people, who are potentially more apart and disconnected from nature. Now is the time to implement preventive and mitigation measures – e.g. fencing, reduction of population density – which can be a small contribution to address this problem. The focus should be put on people's acceptance of the required strategies to manage deer populations (which can also include unpopular practices, such as hunting). For that, an evaluation of people's attitudes towards deer populations and awareness of associated problems is essential. People's opinions, as well as the success of monitoring and management strategies, should be evaluated

Anthropogenic hybridization is one of the greatest threats to global biodiversity. It incites human-mediated gene flow between non-native/exotic and native taxa, which can have irreversible effects on native species or locally adapted populations, eventually leading to extinction. The red deer, Cervus elaphus, is a game species that, due to its extraordinary economic value, has been introduced in several regions throughout Europe. However, the consequences of those introductions on native populations, namely on their genetic background, have been poorly addressed. This study is focused on the Iberian Peninsula and aims to: (i) assess the extent of anthropogenic hybridization/introgression of introduced red deer into the native Iberian populations; (ii) evaluate the impact of red deer management regimes on the observed hybridization/introgression patterns; and (iii) assess how hybridization/introgression influence the current genetic diversity of native Iberian populations. A set of 11 microsatellites and a 329 bases pair fragment of the mitochondrial D-loop gene were used to estimate nuclear admixture and mitochondrial introgression in 1,132 individuals sampled across 46 red deer populations throughout Iberia. A Bayesian approach implemented in the STRUCTURE program was employed to investigate the proportion of admixture between native populations and non-native red deer. Results showed that 17% of individuals presented signs of non-native recent ancestors and 10.1% had non-native mitochondrial haplotypes, reaching an overall hybridization/introgression rate of 23%. Non-native or hybrid individuals were found throughout 40 Iberian red deer populations, and the percentages per population varied between 3.3 and 75.0%, independently of the management regime. Mitochondrial introgression was observed across 15 Iberian red deer populations, being more frequent in free-ranging individuals (16.2%) than in fenced populations (9.2%) but was completely absent from public-owned

The red deer (Cervus elaphus) is a widespread wild ungulate in Europe that has suffered strong anthropogenic impacts over their distribution during the last centuries, but also at the present time, due its economic importance as a game species. Here we focus on the evolutionary history of the red deer in Iberia, one of the three main southern refugial areas for temperate species in Europe, and addressed the hypothesis of a cryptic refugia at higher latitudes during the Last Glacial Maximum (LGM). A total of 911 individuals were sampled, genotyped for 34 microsatellites specifically developed for red deer and sequenced for a fragment of 670 bp of the mitochondrial (mtDNA) D-loop. The results were combined with published mtDNA sequences, and integrated with species distribution models and historical European paleo-distribution data, in order to further examine the alternative glacial refugial models and the influence of cryptic refugia on European postglacial colonization history. Clear genetic differentiation between Iberian and European contemporary populations was observed at nuclear and mtDNA levels, despite the mtDNA haplotypes central to the phylogenetic network are present across western Europe (including Iberia) suggesting a panmictic population in the past. Species distribution models, fossil records and genetic data support a timing of divergence between Iberian and European populations that overlap with the LGM. A notable population structure was also found within the Iberian Peninsula, although several populations displayed high levels of admixture as a consequence of recent red deer translocations. Five D-loop sub-lineages were found in Iberia that belong to the Western European mtDNA lineage, while there were four main clusters based on analysis of nuclear markers. Regarding glacial refugial models, our findings provide detailed support for the hypothesis that red deer may have persisted in cryptic northern refugia in western Europe during the LGM, most

Myxomatosis is a lethal disease in wild European and domestic rabbits (Oryctolagus cuniculus), which is caused by a Myxoma virus (MYXV) infection—a leporipoxvirus that is found naturally in some Sylvilagus rabbit species in South America and California. The introduction of MYXV into feral European rabbit populations of Australia and Europe, in the early 1950s, demonstrated the best-documented field example of host–virus coevolution, following a cross-species transmission. Recently, a new cross-species jump of MYXV has been suggested in both Great Britain and Spain, where European brown hares (Lepus europaeus) and Iberian hares (Lepus granatensis) were found dead with lesions consistent with those observed in myxomatosis. To investigate the possibility of a new cross-species transmission event by MYXV, tissue samples collected from a wild Iberian hare found dead in Spain (Toledo region) were analyzed and deep sequenced. Our results reported a new MYXV isolate (MYXV Toledo) in the tissues of this species. The genome of this new virus was found to encode three disruptive genes (M009L, M036L, and M152R) and a novel ~2.8 kb recombinant region, which resulted from an insertion of four novel poxviral genes towards the 3’ end of the negative strand of its genome. From the open reading frames inserted into the MYXV Toledo virus, a new orthologue of a poxvirus host range gene family member was identified, which was related to the MYXV gene M064R. Overall, we confirmed the identity of a new MYXV isolate in Iberian hares, which, we hypothesized, was able to more effectively counteract the host defenses in hares and start an infectious process in this new host.

Constraints in the characterization of microbiota community that circulates in the host have limited the extent of co-infection studies in natural populations. In this study, we used a metaproteomics approach to characterize the mandibular lymph nodes microbiota of wild boar (Sus scrofa) naturally exposed to an increasing trend of Mycobacterium tuberculosis complex (MTC) infection. Our results showed a reduction in microbiota diversity and changes in the composition, structure and functionality of the microbiota community associated with an increase in tuberculosis prevalence, from 45% in 2002/06 to 83% in 2009/12. These temporal changes were accompanied by an increase in the relative abundance of Babesia, Theileria and Pestivirus genera and a decrease in the Ascogregarina and Chlorella. A positive association was also evidenced between the prevalence of tuberculosis and the presence of microbial proteins responsible for carbohydrate transport and metabolism. Our findings suggest MTC-host-microbiota interactions at the population level, which may occur in order to ensure sufficient metabolic resources for MTC survival, growth and transmission. We strongly recommend the use of metaproteomics when studying microbiota communities in wildlife populations, for which traditional diagnostic techniques are limited and in which new organisms with a pathogenic potential for domestic animals and humans may appear.

As a top predator, the Iberian wolf (Canis lupus signatus) plays a major role shaping interactions within food webs. Due to its conservation status in Portugal, i.e. endangered, it is important to understand the role of parasites in this population, since they can be a limiting factor for the population fitness and trophic interactions and, ultimately, their survival. From November 2017 to August 2018, 33 fresh faecal samples were collected in several transects distributed throughout Montesinho Natural Park. Samples were analysed by means of four coprological techniques. A total of three helminth parasites (Ancylostoma spp., Uncinaria spp. and Eucoleus aerophilus) were identified based on size and morphology. The overall prevalence was low (15.5%), being Ancylostoma spp. the most prevalent parasite. The three parasites found are of major concern, once they are pathogenic to humans and other wild and domestic animals. We suggest surveillance programs that include both parasite and wildlife monitoring. To our knowledge, this is the first coprological study performed with this Iberian wolf population.

Active orthoses (AOs) are becoming relevant for user-oriented training in gait rehabilitation. This implies efficient responses of AO¿s low-level controllers with short time modeling for medical applications. This thesis investigates, in an innovative way, the performance of Feedback-Error Learning (FEL) control to time-effectively adapt the AOs¿ responses to user-oriented trajectories and changes in the dynamics due to the interaction with the user. FEL control comprises a feedback PID controller and a neural network feedforward controller to promptly learn the inverse dynamics of two AOs. It was carried out experiments with able-bodied subjects walking on a treadmill and considering external disturbances to user-AO interaction. Results showed that the FEL control effectively tracked the user-oriented trajectory with position errors between 5% to 7%, and with a mean delay lower than 25 ms. Compared to a single PID control, the FEL control decreased by 16.5% and 90.7% the position error and delay, respectively. Moreover, the feedforward controller was able to learn the inverse dynamics of the two AOs and adapt to variations in the user-oriented trajectories, such as speed and angular range, while the feedback controller compensated for random disturbances. FEL demonstrated to be an efficient low-level controller for controlling AOs during gait rehabilitation.

Double-walled oxide nanotube structures are interesting for a wide range of applications, from photocatalysis to drug delivery. In this work, a progressive oxidation method to fabricate double-walled nanotube structures is reported in detail. The approach is based on the electrodeposition of metallic iron nanowires, in porous alumina templates, followed by a selective chemical etching, nanoscale Kirkendall effect, a fast oxidation and out-diffusion of the metallic core structure during thermal annealing. To validate the formation mechanism of such core-shell structure, chemical composition and atomic structure were assessed. The resulting hematite nanotubes have a high degree of uniformity, along several microns, and a nanoscopic double-walled structure.

This study analysed the distribution and abundance of both polygastric (asexual) and gonophores of the calycophoran siphonophores Muggiaea atlantica and Muggiaea kochii in the Mondego estuary (Portugal), as well as the influence of local environment and prey availability on the observed patterns. Samples were collected in three different zones of the estuary, from December 2013 to November 2015. In general, both species nectophores and total gonophores followed a similar trend, with higher abundances during spring-summer periods and increased densities in the downstream areas of the estuary, where the marine influence is stronger. Inter-annual differences occurred mainly for M. atlantica nectophores, which showed higher abundances in 2015, particularly in the upstream areas. Both species displayed a shift in the annual peak abundance, from bimodal in 2014 to unimodal in 2015. Patterns observed were not only strongly related with an increase of the marine influence in the estuary, but also with the presence of zooplanktonic prey, such as cirripede cypris, calanoid nauplius, Poecilostomatoida, and copepodites of Cyclopoida. This study represents a step towards a better knowledge of the biology and ecology of siphonophores

[Aim]: The distribution of hares (Lepus spp.) in southern Europe was markedly different at the Last Glacial Maximum. Historical hybridization during range replacements led to high frequencies of mtDNA introgression from Lepus timidus into populations of three other species in northern Iberian Peninsula, even though L. timidus subsequently disappeared from the region. We use palaeontological records to provide new insights on the biogeographic history of these range replacements, combining ancient DNA and ecological niche modelling. [Location]: Southern France. [Taxon]: Hares (genus Lepus). [Methods]: DNA was extracted from hare bones collected at Neolithic sites in southern France (5.5–7.5 thousand years before present; kyr bp) with uncertain species assignment. One mtDNA fragment was sequenced and 27 SNPs from 11 nuclear genes with species-diagnostic information were genotyped. Distributions of L. granatensis, L. europaeus, and L. timidus were modelled using spatial and bioclimatic predictors, and the favourability function. Each model was transferred to 6 kyr bp and favourabilities were combined to determine the species with the highest environmental favourability in each sampled locality. [Results]: Ancient DNA analysis showed that the screened specimens belong to L. granatensis, a species presently confined to the Iberian Peninsula, but carried mtDNA haplotypes from L. timidus. Niche models show that L. granatensis achieved highest favourability in southern France 6 kyr bp reinforcing the molecular evidences. [Main conclusions]: L. granatensis replaced L. timidus populations from Iberia to southern France, being present in this region 5.5 kyr bp. Subsequent westwards invasion of L. europaeus likely replaced these L. granatensis populations towards northern Iberia, establishing current ranges. Despite successive species replacements, introgressed mtDNA haplotypes from L. timidus mark the distribution of the species in northern Iberian Peninsula before it disappea

Managers have, over the years, searched methods with which to monitor wildlife populations that will have the best cost-performance balance for each case scenario. Monitoring techniques are of particular importance when attempting to attain either population density estimates or species richness values, since they enable management decisions to be made. It is, therefore, imperative to assess the capability of the methods employed to detect a wide range of species as a means to evaluate their cost-efficiency. Seventeen mammal species on Mediterranean farmland were sampled using three monitoring methods: camera trapping, spotlight counts and indirect indices based on track and faeces counts. The method able to detect the greatest number of species with the best cost-performance balance was the indirect index. There were no significant differences among seasons when applying the same sampling methodology. Studies analysing methods with which to monitor wildlife populations are in great demand in Europe, where attempts are gradually being made to standardise protocols, reduce field effort and costs, and maximise data potential and data gathering over the years. Our manuscript contributes towards finding a cost-effective method and a standardised field protocol to be applied in Mediterranean habitats.

Identifying the ecological and anthropogenic processes that affect wildlife physiology, and that may operate as chronic stressors, is of prime importance to implementing appropriate management and conservation strategies. Although advances have been made in understanding the physiological ecology of wild ungulates, little is known of how multiple intrinsic and ecological factors work, either independently or synergistically, to modulate their stress responses. By using faecal glucocorticoid metabolites (FGM) as indicators of stress, a set of environmental and human determinants affecting the stress physiology of wild red deer (Cervus elaphus) was examined in the Mediterranean ecosystems of south-western Europe, where this species is subjected to contrasting weather regimes and hunting management systems. Variation-partitioning techniques were also used to estimate the comparative influence of factors related to an individual's intrinsic characteristics, environmental conditions and management practices in shaping physiological stress levels. Our results showed that factors related to hunting management were the main drivers of FGM variation in red deer, followed by those related to the environmental conditions and individuals’ traits, and their effects were closely associated to spatio-temporal variability. Holding massive hunting events involving the use of hounds, as well as high population densities, were related to more long-term stress levels in the populations studied. Evidence was also found that supplementary feeding practices may mitigate the negative effects of reduced food availability in overabundant deer populations. Weather conditions were also significant factors explaining variation in stress levels; accumulated rainfall and an increase in ambient temperatures during the coldest months were associated with a decrease in stress hormone levels. No differences in hormonal concentrations were found between males and females, but higher levels of hormone

Porphyrins are pigments produced in most animal cells during the synthesis of heme, but their importance for external coloration is unclear. Owls (Order Strigiformes) are among the few animals that accumulate porphyrins in the integument, where it could serve as a means of signaling. Here we hypothesized that the porphyrin content of feathers may depend on body condition and breeding site quality in Eurasian eagle owl (Bubo bubo) fledglings and, thus, constitute amplifiers of the quality of the area where they are born. Using high-performance liquid chromatography, we found 2 porphyrins (protoporphyrin IX and coproporphyrin III) in the body feathers of 19 eagle owl fledglings from 7 breeding territories. Coproporphyrin III, but not protoporphyrin IX feather concentration, was positively associated with the body mass of fledglings and with the quality of the breeding sites where they were reared with respect to food quality and availability. As coproporphyrin III is produced under oxidative stress, we suggest that good breeding sites may lead to fledglings in good condition. This, in turn, may make fledglings induce a certain level of free radical and coproporphyrin III production to signal to conspecifics their site-mediated capacity to cope with oxidative stress. This is the first time that porphyrin content in the integument has been found to be related to individual quality, opening a new scenario for studying evolution of animal coloration.

Speciation is a process proceeding from weak to complete reproductive isolation. In this continuum, naturally hybridizing taxa provide a promising avenue for revealing the genetic changes associated with the incipient stages of speciation. To identify such changes between two subspecies of rabbits that display partial reproductive isolation, we studied patterns of allele frequency change across their hybrid zone using whole-genome sequencing. To connect levels and patterns of genetic differentiation with phenotypic manifestations of subfertility in hybrid rabbits, we further investigated patterns of gene expression in testis. Geographic cline analysis revealed 253 regions characterized by steep changes in allele frequency across their natural region of contact. This catalog of regions is likely to be enriched for loci implicated in reproductive barriers and yielded several insights into the evolution of hybrid dysfunction in rabbits: (i) incomplete reproductive isolation is likely governed by the effects of many loci, (ii) protein–protein interaction analysis suggest that genes within these loci interact more than expected by chance, (iii) regulatory variation is likely the primary driver of incompatibilities, and (iv) large chromosomal rearrangements appear not to be a major mechanism underlying incompatibilities or promoting isolation in the face of gene flow. We detected extensive misregulation of gene expression in testis of hybrid males, but not a statistical overrepresentation of differentially expressed genes in candidate regions. Our results also did not support an X chromosome-wide disruption of expression as observed in mice and cats, suggesting variation in the mechanistic basis of hybrid male reduced fertility among mammals.

Tuberculosis (TB) affects a wide range of host species worldwide. Understanding host-pathogen co-evolution remains a global challenge owing to complex interactions among host genetic factors, pathogen traits and environmental conditions. We used an endemic wild boar population that had undergone a huge increase in Mycobacterium bovis infection prevalence, from 45% in 2002/06 to 83% in 2009/12, to understand the effects of host genetics on host TB outcomes and disease dynamics. Host genomic variation was characterized using a high-density single nucleotide polymorphism (SNP) array, while host TB phenotype was assessed using both gross pathology and mycobacterial culture. Two complementary genome-wide association (GWAS) analyses were conducted: (i) infected-uninfected; and (ii) 2002/06-2009/12. The SNPs with the highest allelic frequency differences between time-periods and TB outcomes were identified and validated in a large dataset. In addition, we quantified the expression levels of some of their closest genes. These analyses highlighted various SNPs (i.e. rs81465339, rs81394585, rs81423166) and some of the closest genes (i.e. LOC102164072, BDNF/NT-3, NTRK2, CDH8, IGSF21) as candidates for host genetic susceptibility. In addition to TB-driven selection, our findings outline the putative role of demographic events in shaping genomic variation in natural populations and how population crashes and drift may impact host genetic susceptibility to TB over time.

Monitoring changes in the nutritional status of wild populations is crucial to understanding how species respond to natural or human-mediated variations in food availability and quality. It is a fundamental step for decision-making in wildlife management. Many studies on nutritional ecology still tend to rely on the use of single indicators. Nonetheless, nutrition has multiple interdependent facets that should be combined to better understand how organisms interact with their environment through feeding. Here, data from a large-scale cross-sectional survey conducted between 2010 and 2013 were used to examine the influence of environmental conditions and management practices on the variation of faecal nitrogen content (FN, index of dietary quality) and kidney fat index (KFI, indicator of nutritional condition) in red deer Cervus elaphus in the Mediterranean ecosystems of Iberia. Variations in the nutritional indicators in relation to sex, age, faecal fibre contents and faecal counts of lungworm larvae were also analysed. Our results showed that FN levels were statistically and positively correlated with vegetation primary productivity (P = 0.003). In contrast, faecal fibre fractions were significantly and negatively correlated with FN concentrations (P = 0.024 for hemicellulose, P < 0.0001 for cellulose and lignin). Moreover, a negative, marginally significant relationship was found between FN and the average altitude of the study areas (P = 0.055). The KFI was statistically higher in females than in males (P < 0.0001) and increased with increasing FN levels (P = 0.01). Fat stores were also statistically and positively correlated with vegetation productivity (P = 0.001) and with the amount of supplementary food provided (P = 0.04). The effect of high-quality diets on deer kidney fat deposits was gradually lower as the population density increased (P = 0.02). This result was more marked in non-food-supplemented populations. Both FN and KFI were statistically higher du

The most characteristic feature of domestic animals is their change in behavior associated with selection for tameness. Here we show, using high-resolution brain magnetic resonance imaging in wild and domestic rabbits, that domestication reduced amygdala volume and enlarged medial prefrontal cortex volume, supporting that areas driving fear have lost volume while areas modulating negative affect have gained volume during domestication. In contrast to the localized gray matter alterations, white matter anisotropy was reduced in the corona radiata, corpus callosum, and the subcortical white matter. This suggests a compromised white matter structural integrity in projection and association fibers affecting both afferent and efferent neural flow, consistent with reduced neural processing. We propose that compared with their wild ancestors, domestic rabbits are less fearful and have an attenuated flight response because of these changes in brain architecture.

Separation techniques hyphenated with high-resolution mass spectrometry have been a true revolution in analytical separation techniques. Such instruments not only provide unmatched resolution, but they also allow measuring the peaks accurate masses that permit identifying monoisotopic formulae. However, data files can be large, with a major contribution from background noise and background ions. Such unnecessary contribution to the overall signal can hide important features as well as decrease the accuracy of the centroid determination, especially with minor features. Thus, noise and baseline correction can be a valuable pre-processing step. The methodology that is described here, unlike any other approach, is used to correct the original dataset with the MS scans recorded as profiles spectrum. Using urine metabolic studies as examples, we demonstrate that this thorough correction reduces the data complexity by more than 90%. Such correction not only permits an improved visualisation of secondary peaks in the chromatographic domain, but it also facilitates the complete assignment of each MS scan which is invaluable to detect possible comigration/coeluting species.

The dwarf phenotype characterizes the smallest of rabbit breeds and is governed largely by the effects of a single dwarfing allele with an incompletely dominant effect on growth. Dwarf rabbits typically weigh under 1 kg and have altered craniofacial morphology. The dwarf allele is recessive lethal and dwarf homozygotes die within a few days of birth. The dwarf phenotype is expressed in heterozygous individuals and rabbits from dwarf breeds homozygous for the wild-type allele are normal, although smaller when compared to other breeds. Here, we show that the dwarf allele constitutes a ~ 12.1 kb deletion overlapping the promoter region and first three exons of the HMGA2 gene leading to inactivation of this gene. HMGA2 has been frequently associated with variation in body size across species. Homozygotes for null alleles are viable in mice but not in rabbits and probably not in humans. RNA-sequencing analysis of rabbit embryos showed that very few genes (4–29 genes) were differentially expressed among the three HMGA2/dwarf genotypes, suggesting that dwarfism and inviability in rabbits are caused by modest changes in gene expression. Our results show that HMGA2 is critical for normal expression of IGF2BP2, which encodes an RNA-binding protein. Finally, we report a catalog of regions of elevated genetic differentiation between dwarf and normal-size rabbits, including LCORL-NCAPG, STC2, HOXD cluster, and IGF2BP2. Levels and patterns of genetic diversity at the LCORL-NCAPG locus further suggest that small size in dwarf breeds was enhanced by crosses with wild rabbits. Overall, our results imply that small size in dwarf rabbits results from a large effect, loss-of-function (LOF) mutation in HMGA2 combined with polygenic selection.

1. Protected areas are one of the most common strategies for wildlife conservation world-wide. However, their effectiveness is rarely evaluated. In Europe, after the outbreak of bovine spongiform encephalopathy, a restrictive sanitary regulation (EC 1774/2002) prohibited the abandonment of dead livestock in extensive farming (extensive livestock) in the field, which led to negative consequences for scavengers. As an attempt to mitigate this negative impact, a new regulation was approved (EC 142/2011) to allow farmers to leave extensive livestock carcasses in the so-called `Protection areas for the feeding of necrophagous species of European interest' (PAFs). 2. Our general aims were to quantify (i) the proportion of breeding distribution of targeted scavenger species overlapping PAFs; (ii) the extensive livestock carrion biomass available inside PAFs; (iii) the proportion of breeding distribution of non-targeted scavenger species falling within PAFs; (iv) the overlap between the home range of vultures and PAFs, as well as the extent to which vultures move through different administrative units; and (v) the savings in greenhouse gas (GHG) emissions in relation to the pre-PAF scenario. 3. After assessing the status of PAF implementation in every region of peninsular Spain, we analysed the large-scale spatial information of extensive livestock carrion availability and scavenger breeding distribution, movement data of GPS-tracked vultures, and the annual GHG emissions associated with the transport of livestock carcasses. 4. Most regions established PAFs in their territories, although design criteria were variable. The breeding distribution of targeted species was better represented within PAFs than that of non-targeted species. The extensive livestock carrion biomass potentially available for scavengers within PAFs represented 34·9% of the annual extensive livestock biomass generated in peninsular Spain. The overlap between the home range of GPS-marked vulture populatio

Comparative landscape genetics studies can provide key information to implement cost-effective conservation measures favouring a broad set of taxa. These studies are scarce, particularly in Mediterranean areas, which include diverse but threatened biological communities. Here, we focus on Mediterranean wetlands in central Iberia and perform a multi-level, comparative study of two endemic pond-breeding amphibians, a salamander (Pleurodeles waltl) and a toad (Pelobates cultripes). We genotyped 411 salamanders from 20 populations and 306 toads from 16 populations at 18 and 16 microsatellite loci, respectively, and identified major factors associated with population connectivity through the analysis of three sets of variables potentially affecting gene flow at increasingly finer levels of spatial resolution. Topographic, land use/cover, and remotely sensed vegetation/moisture indices were used to derive optimized resistance surfaces for the two species. We found contrasting patterns of genetic structure, with stronger, finer scale genetic differentiation in Pleurodeles waltl, and notable differences in the role of fine-scale patterns of heterogeneity in vegetation cover and water content in shaping patterns of regional genetic structure in the two species. Overall, our results suggest a positive role of structural heterogeneity in population connectivity in pond-breeding amphibians, with habitat patches of Mediterranean scrubland and open oak woodlands (“dehesas”) facilitating gene flow. Our study highlights the usefulness of remotely sensed continuous variables of land cover, vegetation and water content (e.g., NDVI, NDMI) in conservation-oriented studies aimed at identifying major drivers of population connectivity.

In the literature on territorial innovation systems there is a constant tension between the effect of endogenous and exogenous factors and their relative importance. In order to address this tension several approaches have sought to conceptualize the relational and multi-scalar dimension of innovation and to explain how external knowledge and economic flows in conjunction with regional contexts combine to produce regional growth, stagnation or decline. This paper will contribute to this debate, while also highlighting that even at the local level there is substantial heterogeneity between firms. It will also shed light on the role of intermediary organizations that can help address information and power asymmetries between multinational corporations and less competitive local firms. Empirically it will draw on the case study of the Portuguese moulds industry, which has successfully adapted to shifts in the global economy by drawing on local resources while also remaining integrated into global value chains.

Glacial refugia protected and promoted biodiversity during the Pleistocene, not only at a broader scale, but also for many endemics that contracted and expanded their ranges within refugial areas. Understanding the evolutionary history of refugial endemics is especially important in the case of endangered species to recognize the origins of their genetic structure and thus produce better informed conservation practices. The Iberian Peninsula is an important European glacial refugium, rich in endemics of conservation concern, including small mammals, such as the Cabrera vole (Microtus cabrerae). This near-threatened rodent is characterized by an unusual suite of genetic, life history and ecological traits, being restricted to isolated geographic nuclei in fast-disappearing Mediterranean subhumid herbaceous habitats. To reconstruct the evolutionary history of the Cabrera vole, we studied sequence variation at mitochondrial, autosomal and sex-linked loci, using invasive and noninvasive samples. Despite low overall mitochondrial and nuclear nucleotide diversities, we observed two main well-supported mitochondrial lineages, west and east. Phylogeographic modelling in the context of the Cabrera vole's detailed fossil record supports a demographic scenario of isolation of two populations during the Last Glacial Maximum from a single focus in the southern part of the Iberian Peninsula. In addition, our data suggest subsequent divergence within the east, and secondary contact and introgression of the expanding western population, during the late Holocene. This work emphasizes that refugial endemics may have a phylogeographic history as rich as that of more widespread species, and conservation of such endemics includes the preservation of that genetic legacy.

Recent advances in the cost-efficiency of sequencing technologies enabled the combined DNA- and RNA-sequencing of human individuals at the population-scale, making genome-wide investigations of the inter-individual genetic impact on gene expression viable. Employing mRNA-sequencing data from the Geuvadis Project and genome sequencing data from the 1000 Genomes Project we show that the computational analysis of DNA sequences around splice sites and poly-A signals is able to explain several observations in the phenotype data. In contrast to widespread assessments of statistically significant associations between DNA polymorphisms and quantitative traits, we developed a computational tool to pinpoint the molecular mechanisms by which genetic markers drive variation in RNA-processing, cataloguing and classifying alleles that change the affinity of core RNA elements to their recognizing factors. The in silico models we employ further suggest RNA editing can moonlight as a splicing-modulator, albeit less frequently than genomic sequence diversity. Beyond existing annotations, we demonstrate that the ultra-high resolution of RNA-Seq combined from 462 individuals also provides evidence for thousands of bona fide novel elements of RNA processing—alternative splice sites, introns and cleavage sites—which are often rare and lowly expressed but in other characteristics similar to their annotated counterparts.

Environmental changes are driving rapid geographic shifts of suitable environmental conditions for species. These might survive by tracking those shifts, however successful responses will depend on the spatial distribution of suitable habitats (current and future) and on their connectivity. Most herptiles (i.e., amphibians and reptiles) have low dispersal abilities, and therefore herptiles are among the most vulnerable groups to environmental changes. Here we assessed the vulnerability of herptile species to future climate and land use changes in fragmented landscapes. We developed and tested a methodological approach combining the strengths of Species Distribution Models (SDMs) and of functional connectivity analysis. First, using SDMs we forecasted current and future distributions of potential suitable areas as well as range dynamics for four herptile species in Portugal. SDM forecasts for 2050 were obtained under two contrasting emission scenarios, translated into moderate (low-emissions scenario) or large (high-emissions scenario) changes in climate and land use conditions. Then, we calculated and analysed functional connectivity from areas projected to lose environmental suitability towards areas keeping suitable conditions. Landscape matrix resistance and barrier effects of the national motorway network were incorporated as the main sources of fragmentation. Potential suitable area was projected to decrease under future conditions for most test species, with the high-emissions scenario amplifying the losses or gains. Spatiotemporal patterns of connectivity between potentially suitable areas signalled the most important locations for maintaining linkages and migration corridors, as well as potential conflicts due to overlaps with the current motorway network. By integrating SDM projections with functional connectivity analysis, we were able to assess and map the vulnerability of distinct herptile species to isolation or extinction under environmental change sce

Various algorithms have been developed to improve the quantity and quality of information that can be extracted from complex datasets obtained using hyphenated mass spectrometric techniques. While different approaches are possible, the key step often consists in arranging the data into a large series of profiles known as extracted ion profiles. Those profiles, similar to mono-dimensional separation profiles, are then processed to detect potential chromatographic peaks. This allows extracting from the dataset a large number of peaks that are characteristics of the compounds that have been separated. However, with mass spectrometry (MS) detection, the response is usually a complex signal whose pattern depends on the analyte, the MS instrument and the ionization method. When converted to ionic profiles, a single separated analyte will have multiple images at different m/z range. In this manuscript we present a hierarchical agglomerative clustering algorithm to group profiles with very similar feature. Each group aims to contain all profiles that are due to the transport and monitoring of a single analyte. Clustering results are then used to generate a 2 dimensional representation, called clusters plot, which allows an in-depth analysis of the MS dataset including the visualization of poorly separated compounds even when their intensity differs by more than two orders of magnitude. The usefulness of this new approach has been validated with data from capillary electrophoresis time of flight mass spectrometry hyphenated via an electrospray ionization. Using a mixture of 17 low molecular endogenous compounds it was verified that ionic profiles belonging to each compounds were correctly clustered even with very low degree of separation (R below 0.03). The approach was also validated using a urine sample. While with the total ion profile 15 peaks could be distinguished, 70 clusters were obtained allowing a much thorough analysis. In this particular example, the total comput

Post processing of dataset obtained using separation techniques hyphenated with mass spectrometry can allow automation of the recovery of target information, improve the visualization of the data or allow for mathematical separations of convoluted data. Despite the growing popularity and proven successes, those techniques are still only used by few. This is mainly due to the supposed difficulty to open hyphenated dataset, as well as use or develop efficient chemometrics tools. However, due to the work of the Human Proteome Organization (HUPO), the IUPAC and others, there is now MS file formats that are vendor-neutral and can easily be open using various language such as Java, Python, Matlab or R. In the past decades, using those formats, various tools (commercially or freely available) have been designed to help in the post-processing of hyphenated datasets. For proteomic applications, the best known is probably XCMS. However, each of these software is designed for a particular purpose and will not fit all purposes. In the context of our work, we developed a Matlab toolbox that allows openning of MS files in the mzML format and perform various calculations. The aim of this manuscript is to present this toolbox, called Finnee, as well as some key functions. While it is necessary to own a working Matlab license, the toolbox can be used without any programing skills. Every function is protected with the BSD license allowing anyone to study the source code, modify and share it, as long as its copyright notices and the license's disclaimers of warranty are maintained. The source code is available and will be maintain in the GitHub repository (https://github.com/glerny/finnee). Updates and tutorials will be posted in https://finneeblog.wordpress.com/.

[EN]: The European rabbit, Oryctolagus cuniculus, has long been an important model for immunological surveys and, thus, its immunoglobulin genes have been, to some extent, well studied. Regarding the immunoglobulins heavy chain genes, particularities have been revealed that set the European rabbit apart from all studied mammals. Among these are the preferential usage of one VH gene in VDJ rearrangements, the existence of trans-species polymorphism in the IGHV locus, the unusually large genetic distances between VH allelic lineages and the fact that the antibody repertoire is diversified in this species only after birth. Additionally, the European rabbit has a single IgG, contrarily to most mammals that have multiple IgG subclasses, and 13 IgA subclasses, being the most complex IgA system of all studied mammals. Despite all these surprising facts study of other Leporids IGH genes are limited. Since the 1950’s the European rabbit populations have been decimated by two viral fatal diseases, Myxomatosis and Rabbit haemorragic disease, the second closely related to the European brown hare syndrome that has been causing population declines in hares populations, another leporid. Given this context, the study of leporids immune system genes is of utmost importance. In this thesis we aimed at extending knowledge of immunoglobulin genes, both variable and constant regions of the Heavy chain, to other Leporids../.., [PO]: O coelho-bravo, Oryctolagus cuniculus, tem sido um importante modelo para a investigação na área da imunologia e, como tal, os genes que sintetizam as imunoglobulinas têm sido bastante estudados. Nos genes envolvidos na produção da cadeia pesada das imunoglobulinas foram descobertas várias características que distinguem o coelho dos restantes mamíferos. Entre elas podemos destacar o uso preferencial de apenas um gene VH nos rearranjos dos genes VDJ, a existência de polimorfismos trans-específicos no locus IGHV, as distâncias genéticas elevadas entre linhagens alélicas dos genes VH e ainda o facto de a diversificação dos anticorpos ocorrer apenas nas semanas após o nascimento. Para além destas características, o coelho-bravo apresenta ainda outras peculariedades como ter uma só IgG, quando a maioria dos mamíferos tem várias subclasses de IgG, e 13 subclasses de IgA, tendo o mais complexo sistema de IgA de todos os mamíferos estudados. Apesar de se conhecerem estas particularidades nas imunoglobulinas do coelho-bravo, os genes IGH de outros Leporídeos têm sido pouco estudados. Desde a década de 1950 as populações de coelho-bravo têm sido dizimadas por duas doenças virais que apresentam mortalidades muito elevadas, a Mixomatose e a Doença Hemorrágica do Coelho. O vírus que causa a Doença Hemorrágica do Coelho pertence à mesma família do vírus que causa a Síndrome da lebre-comum, doença que tem causado declínios em populações de lebres (Lepus sp.), outro Leporídeo. Neste contexto, o estudo dos genes do sistema imunitário dos Leporídeos assume particular importância. Assim, o objectivo desta tese é desenvolver o conhecimento dos genes das imunoglobulinas, das regiões variáveis e constante da cadeia pesada, para outros Leporídeos../..

The reduction of game and fish populations has increased investment in management practices. Hunting and fishing managers use several tools to maximize harvest. Managers need to know the impact their management has on wild populations. This issue is especially important to improve management efficacy and biodiversity conservation. We used questionnaires and field bird surveys in 48 hunting estates to assess whether red-legged partridge Alectoris rufa young/ adult ratio and summer abundance were related to the intensity of management (provision of supplementary food and water, predator control and releases of farm-bred partridges), harvest intensity or habitat in Central Spain. We hypothesized that partridge abundance would be higher where management practices were applied more intensively. Variation in young/ adult ratio among estates was best explained by habitat, year and some management practices. Density of feeders and water points had a positive relationship with this ratio, while the density of partridges released and magpies controlled were negatively related to it. The variables with greatest relative importance were feeders, releases and year. Variations in postbreeding red-legged partridge abundance among estates were best explained by habitat, year, the same management variables that influenced young/adult ratio, and harvest intensity. Harvest intensity was negatively related to partridge abundance. The other management variables had the same type of relationship with abundance as with young/adult ratio, except magpie control. Variables with greatest relative importance were habitat, feeders, water points, releases and harvest intensity. Our study suggests that management had an overall important effect on post-breeding partridge abundance. However, this effect varied among tools, as some had the desired effect (increase in partridge abundance), whereas others did not or even had a negative relationship (such as release of farm-reared birds) and can be th

[EN]: About 21 thousands years ago the Iberian Peninsula was very cold and most of Europe was even covered by snow! This was at the end of the Pleistocene, the geologic epoch from the Quaternary period that predated the current, the Holocene. And it was during this epoch that most of the planet suffered many glacial events and that’s why the Quaternary is also known as the glacial period. The modern glacial period, because previously there were another four! One of the major consequences of these climatic oscillations was changes in the geographic distribution and genetic variability of species, especially those that inhabited regions most affected by glaciations. But why are we talking about Quaternary, Pleistocene, Iberian Peninsula, Europe and snow? Because we will be talking about hares!, [PT]: Há cerca de 21 mil anos a Península Ibérica estava muito gelada e grande parte da Europa estava mesmo coberta de neve! Estávamos no final do Pleistoceno, a época geológica do período Quaternário que antecedeu a actual, o Holoceno. E foi durante esta época que uma grande parte do planeta viveu muitos eventos glaciares e é por isso que o Quaternário é também conhecido como o período glaciar. Período glaciar moderno, porque houve outros quatro! Uma das grandes consequências destas oscilações climáticas foi a alteração da distribuição geográfica e variabilidade genética das espécies, principalmente daquelas que viviam nas regiões mais afectadas pelas glaciações. Mas porque estamos a falar em Quaternário, Pleistoceno, Península Ibérica, Europa e neve? Porque vamos falar de lebres!, [ES]: Hace cerca de 21 mil años la Península Ibérica estaba helada y gran parte de Europa estaba también cubierta de nieve. Estábamos al final del Pleistoceno, la época geológica del Cuaternario que antecedió la actual, el Holoceno. Fue durante esta época cuando una gran parte del planeta vivió numerosos eventos glaciares y por eso es que también se conoce al Cuaternario como el periodo glacial. ¡Periodo glacial moderno, porque hubo otros cuatro! Una de las grandes consecuencias de estas oscilaciones climáticas fue la alteración de la distribución geográfica y variabilidad genética de las especies, principalmente de aquellas que vivían en las regiones más afectadas por las glaciaciones. Pero, ¿por qué estamos hablando del Cuaternario, Pleistoceno, Península Ibérica, Europa y nieve? ¡Porque vamos a hablar de liebres!

[Aim]: A striking case of mitochondrial DNA (mtDNA) introgression occurs in the Iberian Peninsula: the mtDNA of Lepus timidus, a species that inhabited this territory during the Last Glacial Maximum (LGM), is almost fixed in the Iberian range of Lepus europaeus, is very frequent in the northern half of Iberia within Lepus granatensis, and is fixed in Lepus castroviejoi. Here, we used a transdisciplinary approach to understand the ecological drivers of the distribution of this introgressed mtDNA in the Iberian hare species. [Location]: Europe, with an emphasis on the Iberian Peninsula. [Methods]: First, we modelled the climatic niche of L. timidus to predict the favourability for the species during the LGM. Second, we gathered data regarding L. timidus mtDNA introgression for 1137 individuals (139 populations) from the Iberian Peninsula. We tested whether the climatic favourability for L. timidus during the LGM reflects the frequency of the introgressed mtDNA in the carrier hare populations. [Results]: The climatic favourability for the LGM is positively related to the presence of individuals with L. timidus mtDNA, and discriminates better than by chance between individuals with and without this lineage. At the population level, the favourability values relate to the observed frequencies of mtDNA introgression and discriminate between populations with and without L. timidus mtDNA. [Main conclusions]: The results suggest that the geographical distribution of L. timidus mtDNA introgression is linked to L. timidus distribution in Iberia during the LGM. Our study helps to improve our understanding of the role of climate in the reticulated evolutionary history of hares, and is a first step towards understanding the ecological impact of mtDNA introgression on Iberian hares.

Wild and domestic ruminants are susceptible to Bluetongue virus (BTV) infection. Three BTV serotypes (BTV-4, BTV-1 and BTV-8) have been detected in Spain in the last decade. Even though control strategies have been applied to livestock, BTV circulation has been frequently detected in wild ruminant populations in Spain. The aim of the present study is to assess the role for wild ruminants in maintaining BTV after the vaccination programs in livestock in mainland Spain. A total of 931 out 1,914 (48.6%) serum samples, collected from eight different wild ruminant species between 2006 and 2011, were BTV positive by ELISA. In order to detect specific antibodies against BTV-1, BTV-4 and BTV-8, positive sera were also tested by serumneutralisation test (SNT). From the ELISA positive samples that could be tested by SNT (687 out of 931), 292 (42.5%) showed neutralising antibodies against one or two BTV serotypes. For each BTV seroptype, the number of outbreaks in livestock (11,857 outbreaks in total) was modelled with pure autoregressive models and the resulting smoothed values, representing the predicted number of BTV outbreaks in livestock at municipality level, were positively correlated with BTV persistence in wild species. The strength of this relationship significantly decreased as red deer (Cervus elaphus) population abundance increased. In addition, BTV RNA was detected by real time RT-PCR in 32 out of 311 (10.3%) spleen samples from seropositive animals. Although BT outbreaks in livestock have decreased substantially after vaccination campaigns, our results indicated that wild ruminants have been exposed to BTV in territories where outbreaks in domestic animals occurred. The detection of BTV RNA and spatial association between BT outbreaks in livestock and BTV rates in red deer are consistent with the hypothesis of virus circulation and BTV maintenance within Iberian wild ruminant populations.

The population dynamics of wild ungulates, particularly wild boar (Sus scrofa), are modulated by biotic (e.g. predation) and abiotic (environmental) determinants. Despite the evident potential interference of predation in the environmental patterns of wild boar population abundance, studies including both predation and abiotic factors are scarce. Here, using spatially explicit predictive models, we investigated the effects of habitat features on the relative abundance of wild boar populations and how the abundance of boars is related to frequency of Iberian wolf (Canis lupus signatus; hereafter, wolf) in the area. Wild boar relative abundance was determined by hunting bag statistics, including hunting effort-related variables (in order to avoid problems derived from modeling rates) as covariates, while wolf attacks to livestock were considered as a proxy of wolf frequency in the drive. After modeling, variation partitioning procedures were used to determine the relative importance of each factor and their overlaid effects. Our results showed that wild boar and wolf relative abundances are associated. According to previous knowledge on the wild boar ecology, we found that the species abundance is positively related to the percentage of surface occupied by mature forest and heather providing high food diversity and refuge, but these environmental variables achieved a low explanatory capacity in the models in relation to wolf frequency. The holistic approach followed in this study was attended to open new perspectives for thinking on the wolf-livestock conflict and to adequate wild boar management strategies taking into account hunting interests and natural processes.

The common midwife toad (Alytes obstetricans), widely distributed in the northern half of the Iberian Peninsula and part of Western Europe, is currently subdivided into four subspecies: A. o. obstetricans, A. o. boscai, A. o. pertinax and A. o. almogavarii. However, the delimitation of these subspecies and their ranges are still under discussion because strong discordances have been found between morphological and molecular data, and especially among different genetic markers. Here, we screen a set of novel microsatellite loci and mtDNA sequences of A. obstetricans populations representative of all currently recognized subspecies to investigate the correspondence between genetic groupings inferred from clustering analysis of microsatellite genotypes and the described subspecies and test whether patterns of mtDNA variation are concordant with those genetic clusters. Our results confirm previous expectations of extremely high intraspecific diversity in A. obstetricans in Iberia. Analyses of microsatellite and mtDNA data were concordant in recovering five well-defined groups, of which three correspond to previously defined subspecies, while the two additional clusters correspond to populations of subspecies A. o. boscai separated by the Douro River. Our results suggest the occurrence of two distinct genetic units within A. o. boscai that likely result from a long independent evolutionary history, thus deserving special attention from a conservation point of view.

Mediterranean painted frogs (genus Discoglossus Otth, 1837) are distributed across western Europe, North Africa and some Mediterranean islands. Previous studies have focused on their phylogenetic relationships, but the taxonomic position of the Iberian taxa (D. galganoi and D. jeanneae) is still under debate. By using microsatellites, patterns and rates of admixture can be quantified. We report the characterisation of eighteen novel polymorphic microsatellite loci in Iberian painted frogs. These loci were also tested in all other species of Discoglossus and in the recently rediscovered and highly endangered relative Latonia nigriventer. Two to eleven loci amplified in these species, and the number of polymorphic loci ranged from zero (in Latonia) to eight (in D. scovazzi). The new markers will be useful in addressing questions related to the evolutionary history, population structure, and conservation of Iberian Discoglossus. They also have potential for use in the North African species D. scovazzi and D. pictus, the latter of which is an invasive species rapidly expanding its range in southeast France and northeast Spain.

Salmonellosis is one of the most common and widely distributed foodborne diseases. The emergence of Salmonella strains that are resistant to a variety of antimicrobials is a serious global public health concern. Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) is one of these emerging epidemic multidrug resistant strains. Here we collate information from the diverse and comprehensive range of experiments on Salmonella proteomes that have been published. We then present a new study of the proteome of the quinolone-resistant Se20 strain (phage type DT104B), recovered after ciprofloxacin treatment and compared it to the proteome of reference strain SL1344. A total of 186 and 219 protein spots were recovered from Se20 and SL1344 protein extracts, respectively, after two-dimensional gel electrophoresis. The signatures of 94% of the protein spots were successfully identified through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). Three antimicrobial resistance related proteins, whose genes were previously detected by polymerase chain reaction (PCR), were identified in the clinical strain. The presence of these proteins, dihydropteroate synthase type-2 (sul2 gene), aminoglycoside resistance protein A (strA gene) and aminoglycoside 6'-N-acetyltransferase type Ib-cr4 (aac(6')-Ib-cr4 gene), was confirmed in the DT104B clinical strain. The aac(6')-Ib-cr4 gene is responsible for plasmid-mediated aminoglycoside and quinolone resistance. This is a preliminary analysis of the proteome of these two S. Typhimurium strains and further work is being developed to better understand how antimicrobial resistance is developing in this pathogen.

Ready-to-eat baby-leaf vegetables market has been growing and offering to consumers convenient, healthy and appealing products, which may contain interesting bioactive compounds. In this work, the composition and the evolution of the phenolic compounds from different baby-leaf vegetables during refrigerated storage was studied. The phenolic compounds were extracted using pressurized liquid extraction (PLE) and the phenolic profile of each sample was analyzed and quantified by using LC-MS and LC-DAD methods, respectively, at the beginning and at the end of a 10-day storage period. The baby-leaf vegetables studied included green lettuce, ruby red lettuce, swiss chard, spinach, pea shoots, watercress, garden cress, mizuna, red mustard, wild rocket and spearmint samples and a total of 203 phenolic compounds were tentatively identified and quantified. The main naturally phenolic compounds identified correspond to glycosylated flavonoids, with exception of green lettuce and spearmint leaves which had a higher content of hydroxycinnamic acids. Quantification of the main compounds showed a 10-fold higher content of total phenolic content of ruby red lettuce (483mgg-1) in relation to the other samples, being the lowest values found in the garden cress (12.8mgg-1) and wild rocket leaves (8.1mgg-1). The total phenolic content only showed a significant change (p<0.05) after storage in the green lettuce (+17.5%), mizuna (+7.8%), red mustard (-23.7%) and spearmint (-13.8%) leaves. Within the different classes of phenolic compounds monitored, the flavonols showed more stable contents than the hydroxycinnamic and hydroxybenzoic acids, although the behavior of each compound varied strongly among samples. © 2014.

In separation techniques hyphenated to mass spectrometry (MS) the bulk from the separation step is continuously flowing into the mass spectrometer where the compounds, arriving at each separation time, are ionized and further separated based on their m/. z ratio. An MS detector is recognized as being a universal detector, although it can also be a very selective instrument. In spite of these advantages, classical two dimensional representations from these hyphenated systems, such as those based on the base peak of electropherogram/chromatogram or on the total ion of electropherogram/chromatogram, usually hide a large number of features that if correctly assessed will show the presence of co-migrating species and/or the low abundant ones. The uses of peak picking algorithms to detect and measure as many peaks as possible from a dataset allow extracting much more information. However, a single migrating compound usually produces a multiplicity of ions, making difficult to differentiate peaks generated by the same compound from other peaks due e.g., to closely co-migrating/eluting species. In this work, a new representation is proposed and its usefulness demonstrated with experimental data from capillary electrophoresis-hyphenated to a time of flight mass spectrometer via an electrospray interface. This representation, called centergram, is obtained after using a peak picking methodology that detects electrophoretic peaks of single ions and measure their positions. The centergram is the histogram (i.e. the count of the number of observations that fall into each one of the intervals, known as bins, as determined by the user) of the measured positions. The intensity of the bars in this histogram will indicate the amount of peaks in the whole dataset whose centers are within each interval. As a compound that has been separated and has entered the MS instrument will produce multiple images at the same position along the m/. z dimension, the centergram will exhibit a series