Juan J. Negro

Bovine Tuberculosis in Doñana Biosphere Reserve: The Role of Wild Ungulates as Disease Reservoirs in the Last Iberian Lynx Strongholds

Doñana National Park (DNP) in southern Spain is a UNESCO Biosphere Reserve where commercial hunting and wildlife artificial feeding do not take place and traditional cattle husbandry still exists. Herein, we hypothesized that Mycobacterium bovis infection prevalence in wild ungulates will depend on host ecology and that variation in prevalence will reflect variation in the interaction between hosts and environmental risk factors. Cattle bTB reactor rates increased in DNP despite compulsory testing and culling of infected animals. In this study, 124 European wild boar, 95 red deer, and 97 fallow deer were sampled from April 2006 to April 2007 and analyzed for M. bovis infection. Modelling and GIS were used to identify risk factors and intra and inter-species relationships. Infection with M. bovis was confirmed in 65 (52.4%) wild boar, 26 (27.4%) red deer and 18 (18.5%) fallow deer. In the absence of cattle, wild boar M. bovis prevalence reached 92.3% in the northern third of DNP. Wild boar showed more than twice prevalence than that in deer (p<0.001). Modelling revealed that M. bovis prevalence decreased from North to South in wild boar (p<0.001) and red deer (p<0.01), whereas no spatial pattern was evidenced for fallow deer. Infection risk in wild boar was dependent on wild boar M. bovis prevalence in the buffer area containing interacting individuals (p<0.01). The prevalence recorded in this study is among the highest reported in wildlife. Remarkably, this high prevalence occurs in the absence of wildlife artificial feeding, suggesting that a feeding ban alone would have a limited effect on wildlife M. bovis prevalence. In DNP, M. bovis transmission may occur predominantly at the intra-species level due to ecological, behavioural and epidemiological factors. The results of this study allow inferring conclusions on epidemiological bTB risk factors in Mediterranean habitats that are not managed for hunting purposes. Our results support the need to consider wildlife species for the control of bTB in cattle and strongly suggest that bTB may affect animal welfare and conservation.

Sexual Dichromatism in Birds Independent of Diet, Parasites and Androgens

Sexual selection may explain why male animals are typically more colourful than females. Females may choose brightly coloured males for mating because colour is a reliable signal of a male’s genetic resistance to parasites, or that he can bear the cost of the immunosuppressant effects of androgens. Bright yellows, oranges and reds are the product of carotenoid pigments, which are known to have significant health benefits. Therefore, bright colours may be indicative of a bird’s quality because it shows access to a superior diet or superior foraging ability. We maintained populations of American kestrels and loggerhead shrikes in captivity that were largely free of parasites and fed a uniform diet. Male kestrels were more brightly coloured than females in the colour of their ceres, lores and legs, and there were pronounced age- and gender-specific patterns to concentrations of carotenoids in their plasma. Even though shrikes do not show any carotenoid-based colouration, the sexes had pronounced differences in plasma carotenoids. Carotenoids in kestrels were unrelated to androgen levels, but the correlation between carotenoids and plasma proteins suggest colour may be a condition-dependent trait. These results suggest that neglected physiological processes may regulate carotenoids, and hence some colour variation need not be explained by parasites, androgens or diet.

Phylogeography, genetic structure and diversity in the endangered bearded vulture (Gypaetus barbatus, L.) as revealed by mitochondrial DNA

Bearded vulture populations in the Western Palearctic have experienced a severe decline during the last two centuries that has led to the near extinction of the species in Europe. In this study we analyse the sequence variation at the mitochondrial control region throughout the species range to infer its recent evolutionary history and to evaluate the current genetic status of the species. This study became possible through the extensive use of museum specimens to study populations now extinct. Phylogenetic analysis revealed the existence of two divergent mitochondrial lineages, lineage A occurring mainly in Western European populations and lineage B in African, Eastern European and Central Asian populations. The relative frequencies of haplotypes belonging to each lineage in the different populations show a steep East–West clinal distribution with maximal mixture of the two lineages in the Alps and Greece populations. A genealogical signature for population growth was found for lineage B, but not for lineage A; futhermore the Clade B haplotypes in western populations and clade A haplo‐types in eastern populations are recently derived, as revealed by their peripheral location in median‐joining haplotype networks. This phylogeographical pattern suggests allopatric differentiation of the two lineages in separate Mediterranean and African or Asian glacial refugia, followed by range expansion from the latter leading to two secondary contact suture zones in Central Europe and North Africa. High levels of among‐population differentiation were observed, although these were not correlated with geographical distance. Due to the marked genetic structure, extinction of Central European populations in the last century re‐sulted in the loss of a major portion of the genetic diversity of the species. We also found direct evidence for the effect of drift altering the genetic composition of the remnant Pyrenean population after the demographic bottleneck of the last century. Our results argue for the management of the species as a single population, given the apparent ecological exchangeability of extant stocks, and support the ongoing reintroduction of mixed ancestry birds in the Alps and planned reintroductions in Southern Spain.

Population genetics after fragmentation: the case of the endangered Spanish imperial eagle ( Aquila adalberti )

The highly endangered Spanish imperial eagle, Aquila adalberti, has suffered from both population decline and fragmentation during the last century. Here we describe the current genetic status of the population using an extensive sampling of its current distribution range and both mitochondrial control region sequences and nuclear microsatellite markers. Results were evaluated in comparison to those obtained for the Eastern imperial eagle, Aquila heliaca, its nearest extant relative. Mitochondrial haplotype diversity was lower in the Spanish than in the Eastern species whereas microsatellite allelic richness and expected heterozygosity did not differ. Both allelic richness and expected heterozygosity were lower in the small Parque Nacional de Doñana breeding nucleus compared to the remaining nuclei. A signal for a recent genetic bottleneck was not detected in the current Spanish imperial eagle population. We obtained low but significant pairwise FST values that were congruent with a model of isolation by distance. FST and exact tests showed differentiation among the peripheral and small Parque Nacional de Doñana population and the remaining breeding subgroups. The centrally located Montes de Toledo population did not differ from the surrounding Centro, Extremadura and Sierra Morena populations whereas the latter were significantly differentiated. On the other hand, a Bayesian approach identified two groups, Parque Nacional de Doñana and the rest of breeding nuclei. Recent migration rates into and from Parque Nacional de Doñana and the rest of breeding nuclei were detected by assignment methods and estimated as 2.4 and 5.7 individuals per generation, respectively, by a Bayesian approach. We discuss how management strategies should aim at the maintenance of current genetic variability levels and the avoidance of inbreeding depression through the connection of the different nuclei.

Extensive polymorphism and geographical variation at a positively selected MHC class II B gene of the lesser kestrel (Falco naumanni)

Understanding the selective forces that shape genetic variation in natural populations remains a high priority in evolutionary biology. Genes at the major histocompatibility complex (MHC) have become excellent models for the investigation of adaptive variation and natural selection because of their crucial role in fighting off pathogens. Here we present one of the first data sets examining patterns of MHC variation in wild populations of a bird of prey, the lesser kestrel, Falco naumanni. We report extensive polymorphism at the second exon of a putatively functional MHC class II gene, Fana‐DAB*1. Overall, 103 alleles were isolated from 121 individuals sampled from Spain to Kazakhstan. Bayesian inference of diversifying selection suggests that several amino acid sites may have experienced strong positive selection (ω = 4.02 per codon). The analysis also suggests a prominent role of recombination in generating and maintaining MHC diversity (ρ = 4Nc = 0.389 per codon, θ = 0.017 per codon). Both the Fana‐DAB*1 locus and a set of eight polymorphic microsatellite markers revealed an isolation‐by‐distance pattern across the Western Palaearctic (r = 0.67; P = 0.01 and r = 0.50; P = 0.04, respectively). Nonetheless, geographical variation at the MHC contrasts with relatively uniform distributions in the frequencies of microsatellite alleles. In addition, we found lower fixation rates in the MHC than those predicted by genetic drift after controlling for neutral mitochondrial sequences. Our results therefore underscore the role of balancing selection as well as spatial variations in parasite‐mediated selection regimes in shaping MHC diversity when gene flow is limited.

Carotenoids in Eggs and Plasma of Red‐Legged Partridges: Effects of Diet and Reproductive Output

Carotenoids are important dietary constituents in birds. They serve as pigments and play numerous physiological roles in both the laying hen and developing embryo. However, factors determining the absorption of carotenoids and their allocation to different functions are numerous and complex, and causal relationships are generally poorly known. Our objective was to determine the degree to which carotenoid levels in egg yolks and the plasma of hens were influenced by differences in diet and reproductive output in captive red‐legged partridges. Carotenoid concentrations were measured by high performance liquid chromatography in two feeds (high and low carotenoid content) and in yolks and plasma of hens near the start and end of laying. Early in the laying season, plasma and yolk carotenoids varied with diet and were correlated with one another. Late in the season, a dietary effect was evident only for yolks, and there was no relationship between plasma and egg levels of individual hens. However, plasma carotenoids at the end of laying were strongly correlated with the number of eggs that had been laid. Dietary availability, although important, could explain some variation in carotenoid levels in plasma and egg yolks only in the context of reproductive history.

Birds of a Feather: Neanderthal Exploitation of Raptors and Corvids

The hypothesis that Neanderthals exploited birds for the use of their feathers or claws as personal ornaments in symbolic behaviour is revolutionary as it assigns unprecedented cognitive abilities to these hominins. This inference, however, is based on modest faunal samples and thus may not represent a regular or systematic behaviour. Here we address this issue by looking for evidence of such behaviour across a large temporal and geographical framework. Our analyses try to answer four main questions: 1) does a Neanderthal to raptor-corvid connection exist at a large scale, thus avoiding associations that might be regarded as local in space or time?; 2) did Middle (associated with Neanderthals) and Upper Palaeolithic (associated with modern humans) sites contain a greater range of these species than Late Pleistocene paleontological sites?; 3) is there a taphonomic association between Neanderthals and corvids-raptors at Middle Palaeolithic sites on Gibraltar, specifically Gorhams, Vanguard and Ibex Caves? and; 4) was the extraction of wing feathers a local phenomenon exclusive to the Neanderthals at these sites or was it a geographically wider phenomenon?. We compiled a database of 1699 Pleistocene Palearctic sites based on fossil bird sites. We also compiled a taphonomical database from the Middle Palaeolithic assemblages of Gibraltar. We establish a clear, previously unknown and widespread, association between Neanderthals, raptors and corvids. We show that the association involved the direct intervention of Neanderthals on the bones of these birds, which we interpret as evidence of extraction of large flight feathers. The large number of bones, the variety of species processed and the different temporal periods when the behaviour is observed, indicate that this was a systematic, geographically and temporally broad, activity that the Neanderthals undertook. Our results, providing clear evidence that Neanderthal cognitive capacities were comparable to those of Modern Humans, constitute a major advance in the study of human evolution.

Causes of natal dispersal in the lesser kestrel: Inbreeding avoidance or resource competition?

1. Natal dispersal of seven cohorts (1988-94) of the colonial lesser kestrel (Falco naumanni) was studied in southern Spain. We ringed 1852 nestlings and resighted 321 (17.3%) which survived at least the first migration to Africa. Of the returning individuals, 57% settled in their natal colonies and the remaining 43% dispersed to other colonies. Resights fell off markedly with increasing distance from the natal colony; more than 90% of resights were within 30 km of the birthplace. 2. Contrary to most avian studies, dispersal from the natal site was not sex-biased in our population. The proportion of dispersing males (41%, n = 171) did not differ significantly from that of females (45%, n = 150). Excluding philopatric individuals, the median dispersal distance for males (19 km) did not differ from that of females (18.5 km). 3. Siblings (72 twos) dispersed or were philopatric independently of each other. This result provides no support for either a genetic basis of dispersal (which predicts the same strategy for both siblings) or the inbreeding avoidance hypothesis (which predicts greater dispersal in one sex than the other). This latter hypothesis also conflicts with our observation that the presence of the parent of the opposite sex in the natal colony has no effect on whether returning individuals disperse. 4. Generalized linear models (GLMs) showed that the probability of dispersal for an individual increased when the absolute growth of its natal colony decreased and also from beginning to end of the study, coincidental with a general population increase. These results point to a link between colony saturation and dispersal. GLMs also showed that dispersal distances tended to increase with size of the natal colony and higher breeding success. These results are also consistent with intraspecific competition for resources. 5. Some individuals were observed in two different colonies in their year of first return. These observations, and the lack of active inbreeding avoidance, suggest a scenario for the dispersal process: individuals would prefer to settle in their natal colonies. If they cannot secure a nest site or a mate, they will have to decide whether to stay as nonbreeders or to disperse in search of another breeding chance elsewhere. The actual decision to disperse could be triggered by the level of aggression the unmated individuals encounter in their natal colonies.

Population fragmentation leads to spatial and temporal genetic structure in the endangered Spanish imperial eagle

The fragmentation of a population may have important consequences for population genetic diversity and structure due to the effects of genetic drift and reduced gene flow. We studied the genetic consequences of the fragmentation of the Spanish imperial eagle (Aquila adalberti) population into small patches through a temporal analysis. Thirty‐four museum individuals representing the population predating the fragmentation were analysed for a 345‐bp segment of the mitochondrial control region and a set of 10 nuclear microsatellite loci. Data from a previous study on the current population (N = 79) were re‐analysed for this subset of 10 microsatellite markers and results compared to those obtained from the historical sample. Three shared mitochondrial haplotypes were found in both populations, although fluctuations in haplotype frequencies and the occurrence of a fourth haplotype in the historical population resulted in lower current levels of haplotype and nucleotide diversity. However, microsatellite markers revealed undiminished levels of nuclear diversity. No evidence for genetic structure was observed for the historical Spanish imperial eagle population, suggesting that the current pattern of structure is the direct consequence of population fragmentation. Temporal fluctuations in mitochondrial and microsatellite allelic frequencies were found between the historical and the current population as well as for each pairwise comparison between historical and current Centro and historical and current Parque Nacional de Doñana nuclei. Our results indicate an ancestral panmictic situation for the species that management policies should aim to restore. A historical analysis like the one taken here provides the baseline upon which the relative role of recent drift in shaping current genetic patterns in endangered species can be evaluated and this knowledge is used to guide conservation actions.

Hybridization between white‐headed ducks and introduced ruddy ducks in Spain

The ruddy duck, Oxyura jamaicensis, was introduced to Great Britain in the mid‐20th century and has recently spread to other Western European countries. In Spain, ruddy ducks hybridize with the globally endangered white‐headed duck, Oxyura leucocephala. We assessed the effects of hybridization on the Spanish white‐headed ducks, which constitute 25% of the global population of this species, using a panel of eight nuclear intron markers, 10 microsatellite loci, and mtDNA control region sequences. These data allowed parental individuals, F1 hybrids, and the progeny of backcrossing to be reliably distinguished. We show that hybrids between the two species are fertile and produce viable offspring in backcrosses with both parental species. To date, however, we found no extensive introgression of ruddy duck genes into the Spanish white‐headed duck population, probably due to the early implementation of an effective ruddy duck and hybrid control programme. We also show that genetic diversity in the expanding European ruddy duck population, which was founded by just seven individuals, exceeds that of the native Spanish white‐headed duck population, which recently recovered from a severe bottleneck. Unless effective control of ruddy ducks is continued, genetic introgression will compromise the unique behavioural and ecological adaptations of white‐headed ducks and consequently their survival as a genetically and evolutionary distinct species.