Landscape features influencing gene flow and connectivity of an endangered passerine.

Dispersal of individuals and gene flow are crucial aspects to maintain genetic diversity and viability of populations, especially in the case of threatened species. Landscape composition and structure may facilitate or limit individual movement within and among populations. We used a landscape genetics approach to assess the connectivity patterns of the threatened Dupont's lark (Chersophilus duponti subsp. duponti), considering their genetic patterns and the landscape features associated with its gene flow in Spain. We analysed the genetic relatedness based on 11 species-specific polymorphic microsatellites on 416 Dupont's lark individuals sampled across peninsular Spain between 2017 and 2019, covering most of the European distribution of the species. To assess the relationship between the landscape composition and the species gene flow, we estimated genetic distance at the individual level (Dps). Next, we built a set of environmental surfaces from two time periods (years 1990 and 2018), based on factors such as land use and topography, influencing individuals' movement. We then obtained resistance surfaces from an optimization process on landscape variables. Landscape genetics analyses were done for single and composite surface models for each year separately. Our findings from both time points show that scatter or mosaic-structured vegetation composed by low agricultural and tree cover and high presence of sclerophyllous shrubs favoured Dupont's lark dispersal, while dense and continuous tree cover, as well as areas of intensive agriculture, were limiting factors. Our results suggest the importance of steppe habitat patches for the species' establishment and dispersal. In addition, our results provide key information to develop conservation measures, including conserving and restoring steppe habitats as scattered and/or mosaic-structured vegetation that could warrant the connectivity and persistence of Dupont's lark populations.

Habitat type modulates sharp body mass oscillations in cyclic common vole populations.

Cyclic rodent populations exhibit pronounced changes in body mass associated with the population cycle phase, long-known as Chitty effect. Although Chitty effect is a common epiphenomenon in both America and Europe, there is still incomplete evidence about the generality of these patterns across the entire range of most species. Moreover, despite decades of research, the underlying factors driving Chitty effect remains poorly understood. Here, we examined the influence of intrinsic and extrinsic factors that may underlie observed patterns in vole size variation in the Iberian common vole Microtus arvalis asturianus. We weighed and measured 2816 adult voles that were captured during 6 trapping periods. Vole numbers and body mass showed strong period- and phase-related variation both in females and males, demonstrating marked Chitty effect in the studied population. Body mass of adult males correlated with body length, evidencing that heavier males are also structurally larger. Statistical models showed that probability of occurrence of large-sized vole (> 37 g) was significantly more likely in reproductive males, during increase and peak phases, and it was modulated by habitat, with crop fields and field margins between crops showing an increased likelihood. We suggest an effect of the habitat on vole body mass mediated by predation.

Genetic footprints of a rapid and large-scale range expansion: the case of cyclic common vole in Spain.

In the Anthropocene, many species are rapidly shifting their ranges in response to human-driven habitat modifications. Studying patterns and genetic signatures of range shifts helps to understand how species cope with environmental disturbances and predict future shifts in the face of global environmental change. We investigated the genetic signature of a contemporary wide-range expansion observed in the Iberian common vole Microtus arvalis asturianus shortly after a colonization event. We used mtDNA and microsatellite data to investigate patterns of genetic diversity, structure, demography, and gene flow across 57 localities covering the historical range of the species and the newly colonized area. The results showed a genetic footprint more compatible with a true range expansion (i.e. the colonization of previously unoccupied areas), than with a model of "colonization from within" (i.e. local expansions from small, unnoticed populations). Genetic diversity measures indicated that the source population was likely located at the NE of the historical range, with a declining gradient of genetic diversity towards the more recently invaded areas. At the expansion front, we observed the greatest gene flow and smallest pairwise differences between nearby localities. Both natural landscape features (rivers) and recent anthropogenic barriers (roads, railways) explained a large proportion of genetic variance among populations and had a significant impact on the colonization pathways used by voles.

Wild micromammal host spectrum of zoonotic eukaryotic parasites in Spain. Occurrence and genetic characterisation.

Micromammals have historically been recognized as highly contentious species in terms of the maintenance and transmission of zoonotic pathogens to humans. Limited information is currently available on the epidemiology and potential public health significance of intestinal eukaryotes in wild micromammals. We examined 490 faecal samples, grouped into 155 pools, obtained from 11 micromammal species captured in 11 Spanish provinces for the presence of DNA from Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Blastocystis sp. The presence of Leishmania spp. was investigated in individual spleen samples. All micromammal species investigated harboured infections by at least one eukaryotic parasite, except Apodemus flavicollis, Myodes glareolus, Sorex coronatus and Sciurus vulgaris, but the sample size for these host species was very low. Cryptosporidium spp. was the most prevalent species found (3.7%, 95% confidence interval [CI]: 2.2-5.7), followed by G. duodenalis (2.8%, 95% CI: 1.6-4.6) and E. bieneusi (2.6%, 95% CI: 1.4-4.3). All pooled faecal samples tested negative for Blastocystis sp. Leishmania infantum was identified in 0.41% (95% CI: 0.05-1.46) of the 490 individual spleen samples analysed. Sequence analyses allowed the identification of Cryptosporidium andersoni (5.9%), C. ditrichi (11.7%), C. muris (5.9%), C. parvum (5.9%), C. tyzzeri (5.9%), rat genotypes CR97 (5.9%) and W19 (5.9%), vole genotypes V (11.7%) and VII (5.9%) and Cryptosproridium spp. (35.3%) within Cryptosporidium (n = 17). Known genotypes C (66.7%) and Peru11 (25.0%) and a novel genotype (named MouseSpEb1, 8.3%) were detected within E. bieneusi (n = 12). None of the G. duodenalis-positive samples could be genotyped at the assemblage level. Molecular data indicate that wild micromammals were primarily infected by rodent-adapted species/genotypes of eukaryotic pathogens and thereby have a limited role as a source of human infections. The presence of ruminant-adapted species C. andersoni along with finding C. parvum is indicative of an overlap between domestic/peri-domestic and sylvatic transmission cycles of these agents.

Weasel exposure to the anticoagulant rodenticide bromadiolone in agrarian landscapes of southwestern Europe.

Bromadiolone is an anticoagulant rodenticide (AR) commonly used as a plant protection product (PPP) against rodent pests in agricultural lands. ARs can be transferred trophically to predators/scavengers when they consume intoxicated live or dead rodents. ARs exposure in weasels Mustela nivalis, small mustelids specialized on rodent predation, is poorly known in southern Europe. Moreover, in this species there is no information on bioaccumulation of AR diastereomers e.g., cis- and trans-bromadiolone. Trans-bromadiolone is more persistent in the rodent liver and thus, is expected to have a greater probability of trophic transfer to predators. Here, we report on bromadiolone occurrence, total concentrations and diastereomers proportions (trans- and cis-bromadiolone) in weasels from Castilla y León (north-western Spain) collected in 2010-2017, where bromadiolone was irregularly applied to control outbreaks of common voles Microtus arvalis mainly with cereal grain bait distributed by the regional government. We also tested variables possibly associated with bromadiolone occurrence and concentration, such as individual features (e.g., sex), spatio-temporal variables (e.g., year), and exposure risk (e.g., vole outbreaks). Overall bromadiolone occurrence in weasels was 22% (n = 32, arithmetic mean of concentration of bromadiolone positives = 0.072 mg/kg). An individual showed signs of bromadiolone intoxication (i.e., evidence of macroscopic hemorrhages or hyperaemia and hepatic bromadiolone concentration > 0.1 mg/kg). All the exposed weasels (n = 7) showed only trans-bromadiolone diastereomer in liver, whilst a single analyzed bait from those applied in Castilla y León contained trans- and cis-bromadiolone at 65/35%. Bromadiolone occurrence and concentration in weasels varied yearly. Occurrence was higher in 2012 (100% of weasels), when bromadiolone was widely distributed, compared to 2016-2017 (2016: 20%; 2017: 8.33%) when bromadiolone was exceptionally permitted. The highest concentrations happened in 2014 and 2017, both years with vole outbreaks. Our findings indicate that specialist rodent predators could be exposed to bromadiolone in areas and periods with bromadiolone treatments against vole outbreaks.

FREQUENT LEPTOSPIRA SPP. DETECTION BUT ABSENCE OF TULA ORTHOHANTAVIRUS IN MICROTUS SPP. VOLES, NORTHWESTERN SPAIN.

The common vole (Microtus arvalis) is a major agricultural pest in Europe and is a reservoir for several zoonotic agents, such as Leptospira spp. and Tula orthohantavirus (TULV). However, little is known about the occurrence of those pathogens in voles from Spain, where the species has largely expanded its distribution range in the past decades, causing agricultural pests and zoonotic diseases. For a molecular survey, 580 common voles and six Lusitanian pine voles (Microtus lusitanicus) were collected in 26 localities from four provinces of northwestern Spain. We assessed the presence of Leptospira spp. DNA in kidney tissue by PCR targeting the lipL32 gene, detecting a prevalence of 7.9% (95% confidence interval, 5.9-10.4) for common voles and of 33.3% (95% confidence interval, 4.3-77.7) for Lusitanian pine voles. We identified Leptospira kirschneri in 24 animals and Leptospira borgpetersenii in two animals, using secY gene-specific PCR. We analyzed environmental and demographic factors (such as age class, weight, and sex) and population dynamics data for their potential effect on the Leptospira spp. prevalence in those voles. The Leptospira spp. DNA detection rate in common voles increased significantly with maximum air temperature, vole weight, and amount of accumulated rainfall during the 90 d before capture and within the peak phase of the population cycle. We assessed the presence of TULV in lung tissue of 389 voles by reverse-transcription PCR, with no positive results. The absence of TULV might be explained by the evolutionary isolation of the common vole in Spain. The detection of two Leptospira genomospecies underlines the necessity for further typing efforts to understand the epidemiology of leptospiral infection in the common vole and the potential risk for human health in Spain.

Lack of detectable genetic isolation in the cyclic rodent Microtus arvalis despite large landscape fragmentation owing to transportation infrastructures.

Although roads are widely seen as dispersal barriers, their genetic consequences for animals that experience large fluctuations in population density are poorly documented. We developed a spatially paired experimental design to assess the genetic impacts of roads on cyclic voles (Microtus arvalis) during a high-density phase in North-Western Spain. We compared genetic patterns from 15 paired plots bisected by three different barrier types, using linear mixed models and computing effect sizes to assess the importance of each type, and the influence of road features like width or the age of the infrastructure. Evidence of effects by roads on genetic diversity and differentiation were lacking. We speculate that the recurrent (each 3-5 generations) episodes of massive dispersal associated with population density peaks can homogenize populations and mitigate the possible genetic impact of landscape fragmentation by roads. This study highlights the importance of developing spatially replicated experimental designs that allow us to consider the large natural spatial variation in genetic parameters. More generally, these results contribute to our understanding of the not well explored effects of habitat fragmentation on dispersal in species showing "boom-bust" dynamics.

Investigating the Role of Micromammals in the Ecology of Coxiella burnetii in Spain.

Coxiella burnetii, the causal agent of human Q fever and animal Coxiellosis, is a zoonotic infectious bacterium with a complex ecology that results from its ability to replicate in multiple (in)vertebrate host species. Spain notifies the highest number of Q fever cases to the ECDC annually and wildlife plays a relevant role in C. burnetii ecology in the country. However, the whole picture of C. burnetii hosts is incomplete, so this study seeks to better understand the role of micromammals in C. burnetii ecology in the country. Spleen samples from 816 micromammals of 10 species and 130 vaginal swabs from Microtus arvalis were analysed by qPCR to detect C. burnetii infection and shedding, respectively. The 9.7% of the spleen samples were qPCR positive. The highest infection prevalence (10.8%) was found in Microtus arvalis, in which C. burnetii DNA was also detected in 1 of the 130 vaginal swabs (0.8%) analysed. Positive samples were also found in Apodemus sylvaticus (8.7%), Crocidura russula (7.7%) and Rattus rattus (6.4%). Positive samples were genotyped by coupling PCR with reverse line blotting and a genotype II+ strain was identified for the first time in one of the positive samples from M. arvalis, whereas only partial results could be obtained for the rest of the samples. Acute Q fever was diagnosed in one of the researchers that participated in the study, and it was presumably linked to M. arvalis handling. The results of the study are consistent with previous findings suggesting that micromammals can be infected by C. burnetii. Our findings additionally suggest that micromammals may be potential sources to trace back the origin of human Q fever and animal Coxiellosis cases in Europe.

Linked-read sequencing enables haplotype-resolved resequencing at population scale.

The feasibility to sequence entire genomes of virtually any organism provides unprecedented insights into the evolutionary history of populations and species. Nevertheless, many population genomic inferences - including the quantification and dating of admixture, introgression and demographic events, and inference of selective sweeps - are still limited by the lack of high-quality haplotype information. The newest generation of sequencing technology now promises significant progress. To establish the feasibility of haplotype-resolved genome resequencing at population scale, we investigated properties of linked-read sequencing data of songbirds of the genus Oenanthe across a range of sequencing depths. Our results based on the comparison of downsampled (25×, 20×, 15×, 10×, 7×, and 5×) with high-coverage data (46-68×) of seven bird genomes mapped to a reference suggest that phasing contiguities and accuracies adequate for most population genomic analyses can be reached already with moderate sequencing effort. At 15× coverage, phased haplotypes span about 90% of the genome assembly, with 50% and 90% of phased sequences located in phase blocks longer than 1.25-4.6 Mb (N50) and 0.27-0.72 Mb (N90). Phasing accuracy reaches beyond 99% starting from 15× coverage. Higher coverages yielded higher contiguities (up to about 7 Mb/1 Mb [N50/N90] at 25× coverage), but only marginally improved phasing accuracy. Phase block contiguity improved with input DNA molecule length; thus, higher-quality DNA may help keeping sequencing costs at bay. In conclusion, even for organisms with gigabase-sized genomes like birds, linked-read sequencing at moderate depth opens an affordable avenue towards haplotype-resolved genome resequencing at population scale.

Blood concentrations of p,p'-DDE and PCBs in harriers breeding in Spain and Kazakhstan.

Organochlorine compounds (OC) are of interest in current biomonitoring studies because of their well-known persistence, accumulation capacity and the adverse effects they caused in the past. P,p'-DDE has been shown to cause severe reproductive failures and population declines in birds of prey. However, there are knowledge gaps regarding OC exposure for some species (e.g. harriers), the historical record and the broader picture. The main goal was to evaluate exposure to p,p'-DDE and PCBs in two raptor species: Montagu's and pallid harriers (Circus pygargus and Circus macrourus), and to investigate if birds from different breeding areas and wintering grounds differ in pollutant levels. For this purpose, we collected blood of adult and nestling Montagu's and pallid harriers breeding in the natural steppes of Kazakhstan, and adult and nestling Montagu's harriers breeding in agricultural and natural habitats of Spain, in 2007-2008. We determined the blood concentrations of p,p'-DDE and PCBs. Adult harriers generally showed higher concentrations of p,p'-DDE and PCBs than nestlings, probably because they had more time for a progressive accumulation of these compounds due to a higher intake than excretion rate. The p,p'-DDE concentrations in adults were equivalent in all the studied areas. The ratio p,p'-DDE/PCB 153 was higher in adults than in nestlings, suggesting that a portion of the p,p'-DDE in adult harriers may have come from p,p'-DDT applied in the past in the wintering areas. Overall, the concentrations of p,p'-DDE and ∑PCBs reported were generally low and below any demonstrated threshold of harm.

Pollutants and diet influence carotenoid levels and integument coloration in nestlings of an endangered raptor.

Carotenoid-based traits or ornaments, such as yellow-red integuments (feathers, beaks, legs or eye-rings) displayed by birds, play key roles in social communication by reliably advertising an individual's quality or health. In some species, these traits are displayed not only by adults but also by nestlings, and function in parent-offspring communication or sibling competition by advertising an individual's physical or physiological condition. Pollutants such as organochlorine compounds (OCs) could have disruptive effects on the coloration of these traits, thereby interfering with communication processes. Such effects have been reported in adult birds, but are still largely unknown for nestlings. Here we investigated associations between polychlorinated biphenyl (PCB) and dichlorodiphenyltrichloroethane (DDT) blood-levels, circulating carotenoid levels and the yellow-orange coloration of the cere and tarsi of wild Black Harrier Circus maurus nestlings, a scarce raptor endemic to southern Africa. As carotenoid pigments must be acquired through the diet, we also tested for an effect of dietary composition. The orangeness-purity of cere and tarsi coloration positively correlated with circulating carotenoid levels, and increased with both nestling age and the proportion of birds consumed in the diet. Circulating carotenoid levels and the orangeness-purity of colored integuments were unrelated to blood PCB levels, although the brightness of integuments (i.e. lack of pigmentation) increased with PCB levels. Nestlings with more DDT had lower levels of circulating carotenoids and reduced carotenoid-based coloration (i.e. higher hue and lower saturation, reflecting a yellow rather than orange and less intense color, respectively). Together, our results are consistent with the hypothesis that OC contaminants, in particular DDT, may disrupt carotenoid-based signaling in exposed nestlings.

Changes in behaviour and faecal glucocorticoid levels in response to increased human activities during weekends in the pin-tailed sandgrouse.

Human recreational activities are becoming increasingly widespread and frequent, a fact that may potentially exacerbate their effects on wildlife. These human-related disturbances on animals may induce behavioural and physiological changes that can ultimately affect their fitness, showing a similar anti-predator response that against natural predator or other threats. Here, we combine the use of behavioural and physiological approaches to assess the potential effect of winter human activities on a threatened farmland bird in Europe, the pin-tailed sandgrouse (Pterocles alchata). We compared before, during and after weekend variations in human activity rates, pin-tailed sandgrouse behaviour (flocking and flying behaviour, interspecific association in mixed flocks and habitat use) and faecal glucocorticoid metabolite concentrations. Human disturbances, in particular those associated with hunting activities, peaked during weekends. Sandgrouse showed significant behavioural changes (increased sandgrouse-only flock sizes, increased proportion of birds flying and changes in habitat use) during weekends and higher faecal glucocorticoid metabolite concentrations after the weekends compared with during or before weekends. Therefore, physiological stress levels could be modulated by behavioural adjustments such as increased flock sizes and changes in habitat use that may allow sandgrouse to cope with increased human disturbance rates during weekends. Nevertheless, temporal and spatial organization of hunting days among groups of estates might be good strategies to buffer these potential adverse effects on wintering pin-tailed sandgrouse and other steppe species of conservation concern, while preserving a socio-economically important activity such as hunting.

Sex-dependent differences in avian malaria prevalence and consequences of infections on nestling growth and adult condition in the Tawny pipit, Anthus campestris.

BACKGROUND: Parasites play pivotal roles in host population dynamics and can have strong ecological impacts on hosts. Knowledge of the effects of parasites on hosts is often limited by the general observation of a fraction of individuals (mostly adults) within a population. The aim of this study was to assess the prevalence of malaria parasites in adult (≥ 1 year old) and nestling (7-11 day old) Tawny pipits Anthus campestris, to evaluate the influence of the host sex on parasite prevalence in both groups of age, and explore the association between infections and body condition (adults) and growth (nestlings). METHODS: Two hundred Tawny pipits (105 adults and 95 nestlings) from one Spanish population were screened for avian malaria parasites (Haemoproteus and Plasmodium) using the polymerase chain reaction (PCR)-based methods. Body condition (body mass against a linear measure of size) was measured in adults and growth rate (daily mass gain) was calculated for nestlings. RESULTS: The overall prevalence of infection was 46%. Sixteen different mitochondrial cytochrome b haplotypes of Plasmodium spp. and one Haemoproteus spp. haplotype were found. Malaria parasites were equally prevalent in nestlings and adults (45 and 46%, respectively). Males were more likely to be infected by parasites than females, and this sex-bias parasitism was evident in both adults and nestlings. Furthermore, a lower daily mass gain during nestling growth in males than in females following infections were found, whereas the effect of infections on body condition of adults was detrimental for females but not for males. CONCLUSIONS: Age-specific differences in physiological trade-offs and ecological factors, such as nest predation would explain, at least in part, the observed host sex and age-related patterns in Tawny pipits.

Detection of vancomycin-resistant Enterococcus faecalis ST6-vanB2 and E. faecium ST915-vanA in faecal samples of wild Rattus rattus in Spain.

The detection of vancomycin-resistant-enterococci (VRE) among wild animals represents a worrisome public health concern. The objectives of the study were to determine the possible presence of VRE in faecal samples of wild small mammals in Spain, to characterize the vancomycin resistance mechanisms and genetic lineages of recovered isolates and to know the diversity of enterococcal species in these animals. A total of 155 faecal samples from small mammals were inoculated in Slanetz-Bartley agar supplemented or not with vancomycin (Van-SB/SB plates). The antimicrobial susceptibility profile to 12 antimicrobials and the presence of 20 antimicrobial resistance genes was analyzed. The structure of Tn1546 and the presence of gelE, cylA, asa, esp and hyl genes was studied. Multilocus-sequence-typing (MLST) technique was also performed. VRE isolates were recovered in Van-SB plates in 11 samples. Two samples contained vanB2-positive E. faecalis isolates of lineage ST6, which showed a multiresistance phenotype and harboured the virulence genes gelE and asa. One sample contained a vancomycin-resistant E. faecium isolate of the new lineage ST915, with the vanA gene included into Tn1546 (truncated with IS1542 and IS1216 elements). The vanB2 and vanA isolates were obtained from Rattus rattus. The remaining eight VRE-positive samples contained species with intrinsic vancomycin-resistance mechanisms: E. casseliflavus (n=5) and E. gallinarum (n=3). One hundred and forty-seven vancomycin-susceptible-enterococcal isolates were obtained in SB plates, and E. faecalis and E. faecium were the most frequent detected species. This is the first report of vanB2-containing enterococci in wild animals.

Niche-habitat mechanisms and biotic interactions explain the coexistence and abundance of congeneric sandgrouse species.

Ascertaining which niche processes allow coexistence between closely related species is of special interest in ecology. We quantified variations in the environmental niches and densities of two congeneric species, the pin-tailed and the black-bellied sandgrouse (Pterocles alchata and Pterocles orientalis) in allopatry and sympatry under similar abiotic, habitat and dispersal contexts to understand their coexistence. Using principal component analysis, we defined environmental gradients (niche dimensions) including abiotic, habitat and anthropogenic variables, and calculated niche breadth, position and overlap of both species in sympatry and allopatry. Additionally, sandgrouse density was modelled as a function of the niche dimensions and the density of the other species. We found evidence that each species occupies distinct environmental niches in sympatry and in allopatry. The black-bellied sandgrouse exploits a broader range of environmental conditions (wider niche breadth) while the pin-tailed sandgrouse reaches high densities where conditions seem to match its optimum. In sympatry, both species shift their niches to intermediate positions, indicating the importance of abiotic factors in setting coexistence areas. Environmental conditions determine regional densities of pin-tailed sandgrouse whereas biotic interactions explain the density of the black-bellied sandgrouse in areas with abiotic conditions similarly conducive for both species. Highly suitable areas for the pin-tailed sandgrouse fall beyond the upper thermal limit of the black-bellied sandgrouse, leading to niche segregation and low densities for the latter. Finally, local niche shift and expansion plus possible heterospecific aggregation allow the pin-tailed sandgrouse to thrive in a priori less favourable environments. This work provides insight into how different mechanisms allow species coexistence and how species densities vary in sympatry compared to allopatry as a result of environmental filtering and biotic interactions.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene in wild small mammals in Spain.

OBJECTIVES: To determine the rate of Staphylococcus aureus faecal carriage in 101 wild small mammals in Spain and to characterize the isolates obtained. METHODS: Faecal samples were seeded on mannitol salt agar and ORSAB plates. The presence of the resistance genes mecA, mecC and blaZ and the new blaZ allotype associated with staphylococcal cassette chromosome mec (SCCmec) XI (blaZ-SCCmecXI) was studied by PCR. S. aureus isolates were characterized by spa typing, agr typing and multilocus sequence typing. The presence of immune evasion cluster (IEC) genes and virulence genes was analysed by PCR. RESULTS: S. aureus was detected in 13/101 studied faecal samples and one isolate per positive sample was further studied. Two S. aureus isolates were methicillin-resistant S. aureus (MRSA) (recovered from wood mice, Apodemus sylvaticus) and 11 were methicillin-susceptible S. aureus (MSSA). Both MRSA isolates harboured the mecC gene and the novel blaZ-SCCmecXI, were typed as spa-t1535/agrIII/ST1945(CC130)/SCCmecXI (where ST stands for sequence type and CC stands for clonal complex), carried the exfoliative toxin etd2 gene and were IEC type E. Eight different spa types were identified among the 11 MSSA isolates (five new) and six different sequence types were identified (two new). All MSSA strains were susceptible to the antibiotics tested except one blaZ-positive penicillin-resistant isolate (spa-t120/agrII/ST15). MSSA isolates were ascribed to the CCs (number of strains) CC5 (1), CC1956 (4) and singleton (6). Nine of 11 MSSA isolates carried the cna virulence gene. Only one MSSA isolate carried IEC genes (type C). CONCLUSIONS: This is the first report of MRSA carrying mecC in faecal samples of wild small mammals in Spain. These resistant isolates carried genes of the IEC system, unusual in S. aureus from animals. Wild small mammals could be a reservoir of the mecC gene with important implications for public health.

Local genetic structure on breeding grounds of a long-distance migrant passerine: the bluethroat (Luscinia svecica) in Spain.

Breeding site fidelity can be determined by environmental features, which depending on their heterogeneous distribution may shape the genetic landscape of a population. We used 10 microsatellite loci to study the genetic variation of 83 bluethroats (Luscinia svecica azuricollis) across 14 localities within the Spanish breeding population and assess the relative influence of different habitat characteristics (physiography and vegetation) on genetic differentiation. Based on the genetic variation of this population, we identified 3 geographically consistent genetic clusters that on average showed a higher genetic differentiation than among other north European populations, even those belonging to different subspecies. The inferred genetic clusters occurred in geographic areas that significantly differed in elevation. The highest genetic differentiation was observed between sites at different mountain ranges, as well as between the highest altitude sites in the northeastern locale, whereas vegetation type did not explain a significant percentage of genetic variation. The lack of correlation between geographic and genetic distances suggests that this pattern of genetic structure cannot be explained as a consequence of isolation by distance. Finally, we discuss the importance of preserving areas encompassing high environmental and genetic variation as a means of preserving evolutionary processes and adaptive potential.

Demographic history, genetic structure and gene flow in a steppe-associated raptor species.

BACKGROUND: Environmental preferences and past climatic changes may determine the length of time during which a species range has contracted or expanded from refugia, thereby influencing levels of genetic diversification. Connectivity among populations of steppe-associated taxa might have been maximal during the long glacial periods, and interrupted only during the shorter interglacial phases, potentially resulting in low levels of genetic differentiation among populations. We investigated this hypothesis by exploring patterns of genetic diversity, past demography and gene flow in a raptor species characteristic of steppes, the Montagu's harrier (Circus pygargus), using mitochondrial DNA data from 13 breeding populations and two wintering populations. RESULTS: Consistent with our hypothesis, Montagu's harrier has relatively low genetic variation at the mitochondrial DNA. The highest levels of genetic diversity were found in coastal Spain, France and central Asia. These areas, which were open landscapes during the Holocene, may have acted as refugia when most of the European continent was covered by forests. We found significant genetic differentiation between two population groups, at the SW and NE parts of the species' range. Two events of past population growth were detected, and occurred ca. 7500-5500 and ca. 3500-1000 years BP in the SW and NE part of the range respectively. These events were likely associated with vegetation shifts caused by climate and human-induced changes during the Holocene. CONCLUSIONS: The relative genetic homogeneity observed across populations of this steppe raptor may be explained by a short isolation time, relatively recent population expansions and a relaxed philopatry. We highlight the importance of considering the consequence of isolation and colonization processes in order to better understand the evolutionary history of steppe species.

Age and breeding effort as sources of individual variability in oxidative stress markers in a bird species.

Oxidative stress is the imbalance between the production of pro-oxidant substances and the level of antioxidant defenses, which leads to oxidative damage. It has been proposed that senescence is the result of accumulated oxidative damage throughout life. In birds, the sources of individual variability in oxidative stress are still poorly understood. Among these sources, age, as related to senescence, should be particularly relevant. Furthermore, recent findings suggest that breeding effort may also deeply influence susceptibility to oxidative stress. However, there is still no evidence of a link between breeding effort and oxidative damage in any vertebrate. Here we analyzed 288 captive red-legged partridges (Alectoris rufa) across a wide age range (i.e., 1-8 yr old), thus including potentially senescent birds. In spite of limitations due to the cross-sectional approach, results revealed that old birds produced less offspring and endured higher levels of oxidized glutathione and peroxidized lipids in erythrocytes than did middle-aged individuals. Old birds also showed higher plasma total antioxidant status and uric acid levels than did younger birds, but lower amounts of circulating carotenoids. Furthermore, hatching success was negatively correlated to lipid peroxidation in females but not in males, supporting the hypothesis that breeding effort promotes oxidative damage.

Testosterone-mediated trade-offs in the old age: a new approach to the immunocompetence handicap and carotenoid-based sexual signalling.

The immunocompetence handicap hypothesis proposes that testosterone mediates a trade-off between sexual signalling and immunocompetence in males. Such a trade-off could favour the reliability of sexual signals on the basis that testosterone required for signal expression also promotes immunosuppression. However, the immunosuppressive activity of testosterone has not been convincingly demonstrated. We propose that the optimal solution to the testosterone-mediated trade-off should change with age, explaining ambiguous results in the past. Testosterone and ageing would promote two simultaneous immunosuppressive challenges unaffordable for low-quality males. Oxidative stress, as intimately related to ageing and immunosenescence, could contribute to enhance signal reliability. In this context, traits coloured by carotenoids (yellow-red traits) could play a crucial role due to the immunostimulatory and antioxidant properties of these pigments. Here, old and middle-aged male red-legged partridges were treated with testosterone or manipulated as controls. In the presence of high-testosterone levels, middle-aged males increased both circulating carotenoid levels and colour expression, whereas their cell-mediated immunity was not significantly altered. However, in old males, neither circulating carotenoids nor sexual signalling increased when treated with testosterone, but immunosuppression was detected. The link between testosterone and carotenoids could favour the reliability of sexual signals throughout the life.