Permanent genetic resources added to Molecular Ecology Resources Database 1 February 2012 - 31 March 2012.

This article documents the addition of 171 microsatellite marker loci and 27 pairs of single nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bombus pauloensis, Cephalorhynchus heavisidii, Cercospora sojina, Harpyhaliaetus coronatus, Hordeum vulgare, Lachnolaimus maximus, Oceanodroma monteiroi, Puccinia striiformis f. sp. tritici, Rhea americana, Salmo salar, Salmo trutta, Schistocephalus solidus, Sousa plumbea and Tursiops aduncus. These loci were cross-tested on the following species: Aquila heliaca, Bulweria bulwerii, Buteo buteo, Buteo swainsoni, Falco rusticolus, Haliaeetus albicilla, Halobaena caerulea, Hieraaetus fasciatus, Oceanodroma castro, Puccinia graminis f. sp. Tritici, Puccinia triticina, Rhea pennata and Schistocephalus pungitii. This article also documents the addition of 27 sequencing primer pairs for Puffinus baroli and Bulweria bulwerii and cross-testing of these loci in Oceanodroma castro, Pelagodroma marina, Pelecanoides georgicus, Pelecanoides urinatrix, Thalassarche chrysostoma and Thalassarche melanophrys.

Distribution of lesions in red and fallow deer naturally infected with Mycobacterium bovis.

Wild deer have an important role in the epidemiology of bovine tuberculosis (bTB). The aims of this study were (1) to compare the pattern of lesions present in wild red (Cervus elaphus) and fallow (Dama dama) deer that were naturally infected with Mycobacterium bovis, and (2) to use this information to develop a sampling strategy for the isolation of M. bovis from the lymphoid tissues of the head of these animals. Culture of head lymphoid tissues demonstrated that 28 of 95 red deer and 22 of 100 fallow deer sampled were infected with M. bovis. Approximately 30% of each deer population had no gross lesions. Fallow deer were significantly more likely to have thoracic lesions than red deer. Lesions were observed in the retropharyngeal lymph nodes of 64% of the culture-positive red deer and 43% of the culture positive fallow deer. One third of the red deer, but none of the fallow deer, had well-encapsulated abscess lesions. There were no microscopical differences in the lesions in the lymph nodes of the red and fallow deer. Bacteriological culture from both the tonsil and retropharyngeal lymph nodes increased the rate of isolation of M. bovis by 22% over culture of the retropharyngeal lymph nodes alone in both species. These findings indicate that investigation of wild deer for bTB-compatible lesions should include examination of the medial retropharyngeal, left tracheobronchial, mediastinal, mesenteric and ileocaecal lymph nodes. Sampling for bacteriological culture from head lymphoid tissues should be from the tonsil and the medial retropharyngeal lymph nodes. These protocols may prove useful in bTB surveillance and control in regions where wild deer contribute to the circulation of M. bovis.

Population fragmentation leads to isolation by distance but not genetic impoverishment in the philopatric Lesser Kestrel: a comparison with the widespread and sympatric Eurasian Kestrel.

Population fragmentation is a widespread phenomenon usually associated with human activity. As a result of habitat transformation, the philopatric and steppe-specialist Lesser Kestrel Falco naumanni underwent a severe population decline during the last century that increased population fragmentation throughout its breeding range. In contrast, the ubiquitous Eurasian Kestrel Falco tinnunculus did not suffer such adverse effects, its breeding range still remaining rather continuous. Using microsatellites, we tested the effects of population fragmentation on large-scale spatial patterns of genetic differentiation and diversity by comparing these two sympatric and phylogenetically related species. Our results suggest that habitat fragmentation has increased genetic differentiation between Lesser Kestrel populations, following an isolation-by-distance pattern, while the population of Eurasian Kestrels is panmictic. Contrary to expectations, we did not detect significant evidence of reduced genetic variation or increased inbreeding in Lesser Kestrels. Although this study reports genetic differentiation in a species that has potential for long-distance dispersal but philopatry-limited gene flow, large enough effective population sizes and migration may have been sufficient to mitigate genetic depauperation. A serious reduction of genetic diversity in Lesser Kestrels would, therefore, only be expected after severe population bottlenecks following extreme geographic isolation.

Multiple ways to become red: pigment identification in red feathers using spectrometry.

Red hues are a challenge in studies on the evolution of bird coloration, as multiple pigments such as carotenoids, pheomelanin, psittacofulvins, porphyrins, turacin, haemoglobin and even exogenous iron-oxides, may confer red colors. Determining the pigment type is paramount and here we investigate the differences in spectrum reflectance for six pigments resulting in red colorations in feathers of different species, with a focus on discriminating among melanins and carotenoids. Pigment chemical identification was obtained from the literature or using High Performance Liquid Chromatography (HPLC) in our laboratory. We have also derived discriminant formulas for identification of the major known types of pigments based on parameters of the reflectance curves obtained with a portable spectrometer. Our results indicate that the reflectance patterns of coloration perceived as red patches widely differ. The distinction between carotenoid- and melanin-based reflectance curves is relatively straightforward: sigmoid versus straight slope. The spectral reflectance curves of feathers containing red psittacofulvins are sigmoid, whereas iron oxide and porphyrin-containing feathers recall pheomelanins in rendering a straight slope. In the case of turacin-based coloration, the spectral shape is unique. For the pigments with enough number of species sampled (i.e., carotenoids, melanins and psittacofulvins) the differences in reflectance shape are important enough to allow separation of carotenoid and melanin derived colorations based on reflectance curves alone.

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, F(1) 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.

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.

Organization and variation of the mitochondrial control region in two vulture species, Gypaetus barbatus and Neophron percnopterus.

We report the first entire mitochondrial DNA (mtDNA) control region sequences in two endangered vulture species, the bearded vulture (Gypaetus barbatus) and the Egyptian vulture (Neophron percnopterus). Results showed that the general organization of vulture control regions was very similar to other birds, with three distinct domains: a left variable domain (DI), a central conserved one (DII) including the F, E, D, and C boxes, and a right domain (DIII) containing the CSB1 sequence. However, due to the presence of long tandem repeats, vulture control regions differed from other avian control regions both in size and nucleotide composition. The Egyptian vulture control region was found to be the largest sequenced so far (2031 bp), due to the simultaneous presence of repeats in both DI (80 bp) and DIII (77 bp). Low variation was found in vulture control regions, particularly in G. barbatus, as the probable result of populations declines in the last few centuries.

Ecological, morphological and phylogenetic correlates of interspecific variation in plasma carotenoid concentration in birds.

Carotenoids are important as pigments for bright coloration of animals, and as physiologically active compounds with a wide array of health-related benefits. However, the causes of variation in carotenoid acquisition and physiology among species are poorly known. We measured the concentration of carotenoids in the blood of 80 wild bird species differing in diet, body size and the extent of carotenoid-based traits. Preliminary analyses showed that diet significantly explains interspecific variability in plasma carotenoids. However, dietary influences were apparently overridden by phylogenetic relationships among species, which explained most (65%) of this variability. This phylogenetic effect could be due partly to its covariation with diet, but may also be caused by interspecific differences in carotenoid absorption from food to the blood stream, mediated, for example by endothelial carriers or gut parasites. Carotenoid concentrations also decreased with body size (which may be explained by the allometric relationship between ingestion rate and body mass), and correlated positively with the extent of carotenoid-dependent coloration of plumage and bare parts. Therefore, the acquisition of carotenoids from the diet and their use for both health and display functions seem to be constrained by ecological and physiological aspects linked to the phylogeny and size of the species.

Coprophagy: an unusual source of essential carotenoids.

The rare Egyptian vulture (Neophron percnopterus) stands out among the Old World vultures (Family Accipitridae) because of its brightly ornamented head, which is coloured yellow by carotenoid pigments, and its practice of feeding on faeces. Here we show that Egyptian vultures obtain these pigments from the excrement of ungulates. To our knowledge, this is the first demonstration that faeces can be used as a source of carotenoids by a vertebrate.

Captivity diets alter egg yolk lipids of a bird of prey (the American kestrel) and of a galliforme (the red-legged partridge).

The salient feature of the fatty acid profile of kestrel eggs collected in the wild was the very high proportion of arachidonic acid (15.2%+/-0.7% of fatty acid mass, n=5) in the phospholipid fraction of the yolk. Kestrels in captivity fed on day-old chickens produced eggs that differed from those of the wild birds in a number of compositional features: the proportion of linoleic acid was increased in all the lipid fractions; the proportion of arachidonic acid was increased in yolk phospholipid and cholesteryl ester; the proportion of alpha-linolenic acid was decreased in all lipid classes, and that of docosahexaenoic acid was decreased in phospholipid and cholesteryl ester. Partridge eggs from the wild contained linoleic acid as the main polyunsaturate of all the yolk lipid fractions. Captive partridges maintained on a formulated diet very rich in linoleic acid produced eggs with increased levels of linoleic, arachidonic, and n-6 docosapentaenoic acids in the phospholipid fraction; reduced proportions of alpha-linolenic acid were observed in all lipid classes, and the proportion of docosahexaenoic acid was markedly reduced in the phospholipid fraction. Thus, captive breeding of both the kestrel and the partridge increases the n-6/n-3 polyunsaturate ratio of the yolk lipids.

Astaxanthin is the major carotenoid in tissues of white storks (Ciconia ciconia) feeding on introduced crayfish (Procambarus clarkii).

We studied the carotenoid pigments in plasma, skin and body fat of white storks (Ciconia ciconia) from a colony in Spain feeding mainly on the recently introduced red swamp crayfish (Procambarus clarkii). In control colonies, where crayfish was absent, plasma was collected for comparison. Our objective was to determine whether the astaxanthin contained in the crayfish reached the blood, accumulated in fat, and finally was deposited in the red-colored bill and legs. If that was true, the visual cues provided by those tegumentary areas would be altered, with potential behavioral consequences. Plasma carotenoids were directly extracted with acetone, whereas skin and fat samples needed harsher conditions, i.e. grinding, sonication and extraction with diethyl ether. The extracts were analyzed by thin-layer chromatography (TLC) and UV/Vis spectroscopy. In crayfish-eating storks, astaxanthin was confirmed to be the dominant pigment in all analyzed tissues. This red pigment was absorbed unchanged in the gut, and was responsible for the red color of plasma and the abnormal orange pigmentation of the feather-covered skin. It was also present in large quantities in the exposed bill and tarsi, which are typically red-colored in the stork. Control storks with no crayfish in the diet only presented lutein in their plasma.

Genetics, local environment and health as factors influencing plasma carotenoids in wild American kestrels (Falco sparverius).

Carotenoids are important as pigments for bright coloration of animals, and as physiologically active compounds with a wide array of health-related functions. Carotenoid-dependent coloration may have evolved as a signal to conspecifics; however, factors that may limit availability of carotenoids are poorly known. We investigated how the acquisition of carotenoids may be constrained by availability in the environment, diet, genetic make-up and health status of wild American kestrels (Falco sparverius). Plasma concentrations of siblings at the time of fledging showed a high degree of resemblance; however, a cross-fostering experiment revealed that variance was largely explained by nest of rearing, rather than nest of origin, thus indicating a low genetic component. A multivariate analysis of attributes of nestlings (sex, size, plasma proteins, immune function), parental reproduction (laying date, clutch size) and rearing conditions (brood size, size hierarchy, nestling mortality) showed only a small significant effect of leucocyte differentials on carotenoid concentrations of nestlings. A strong environmental effect on plasma carotenoids was demonstrated by levels of adult kestrels being correlated within mated pairs, and having a significant association with the abundance of voles, the primary prey species, per territory.

Diet explains interpopulation variation of plasma carotenoids and skin pigmentation in nestling white storks.

Carotenoids have a dietary origin in birds, but mechanisms by which they are absorbed in the gut, transported in the blood, metabolized at various sites, and deposited in the integument remain poorly understood. Variation in both plasma carotenoid levels and external color may reflect different access to dietary carotenoids or individual physiological differences in the uptake and deposition of carotenoids. We compared total plasma carotenoid concentration in nestling white storks (Ciconia ciconia) from 11 Spanish colonies in two consecutive years. The main food item in one of the colonies was the red swamp crayfish (Procambarus clarkii), a recently introduced species. Storks in the remaining colonies ate a variety of foods but no crayfish. Total plasma carotenoid levels in the colony where crayfish were consumed were about five times higher than in any other colony. These differences were maintained after controlling for the significant interyear variability, as well as for sex, age, and body mass of birds. Skin pigmentation also differed, being intensely orange in storks that consumed crayfish but white (unpigmented) in the remaining individuals. With thin-layer chromatography (TLC) and electronic absorption spectroscopy, astaxanthin was confirmed as the major carotenoid in crayfish as well as in the plasma, skin, and body fat of crayfish-eating storks, whereas lutein was the main carotenoid in plasma samples from the other colonies. These results indicate that a newly available carotenoid in the environment, astaxanthin, can be absorbed in large quantities from the gut and be transported in the blood before deposition in different tissues.

A comparison of spectrophotometry and color charts for evaluating total plasma carotenoids in wild birds.

The study of the role of carotenoids on the physiology and evolutionary ecology of birds demands methods for their quantification in the bloodstream. We compared color-chart scores of plasma hue with the actual concentration of plasma carotenoids obtained by spectrophotometry in 356 wild birds from 26 species. Repeatability of chart scores between three independent observers was high. However, color scores did not correlate with the spectrophotometric results in interspecific analyses. Within species (n = 3), one showed no relationship and two showed weak but significant positive correlations. Hemoglobin, and probably other substances, may mask the color of carotenoids, making the accurate use of color charts difficult. Spectrophotometry should be the method of choice as it permits precise quantifications of total plasma carotenoids and objective comparisons among studies.

Growth of hand-reared American kestrels I. The effect of two different diets and feeding frequency.

Seventy captive-bred American kestrels (Falco sparverius) were hand-reared on diets of either day-old cockerels (Gallus domesticus) (n = 38) or laboratory mice (Mus musculus) (n = 32) at meal frequencies of 4 or 6 times per day. Ad libitum food was provided in all meals. A proximate analysis of the two diets was performed and showed that cockerels when compared to mice contained more crude protein (60.0% versus 42.7% dry matter) and less fat (28.1% versus 46.5% dry matter). Body mass of nestlings was recorded daily until fledging, while lengths of the antebrachium and 9th primary remex were measured at intervals of 2 to 5 days. Data were fitted to the Richards growth model. Cockerel-fed nestlings exhibited growth patterns similar to those found in free-ranging kestrels. Mouse-fed birds however, grew more slowly which could be related to inadequate protein intake. No effect of hand-feeding frequency (4 versus 6 times daily) on growth rates was noted under any of the two diets. Irrespective of the dietary group, female kestrels were significantly heavier at the end of the experiment, as expected from the reversed sexual size dimorphism present in adult American kestrels.

Growth of hand-reared American kestrels. II. Body composition and wingloading of fledglings hand-fed two different diets.

The body composition of young American kestrels (Falco sparverius) hand-fed either a protein-rich diet (day-old cockerels Gallus domesticus) or a fat-rich diet (laboratory mice Mus musculus) was determined one day after fledging. Mouse-fed fledglings (n = 16) had significantly greater fat deposits than cockerel-fed birds (n = 15), while the crude protein content of the carcass was unaffected by the diets. At fledging, mouse-fed birds showed a significantly greater wingloading than cockerel-fed birds. Larger fat reserves (as in mouse-fed birds) might be mobilised in the event of a food shortage and thus these birds would be at an advantage in relation to fledglings with smaller reserves. On the other hand, large fat deposition, which alters wingloading, might impair the flight performance of the fledglings.

Organochlorine and heavy metal contamination in non-viable eggs and its relation to breeding success in a Spanish population of Lesser Kestrels (Falco naumanni).

Residues of organochlorines, polychlorinated biphenyls (PCBs), and heavy metals (mercury, cadmium, lead, copper and zinc) were measured in unhatched eggs of Lesser Kestrels (Falco naumanni) collected in southern Spain in 1988-1991. Although contaminants were detected in all eggs, the amounts were generally below levels known to have negative effects on reproduction. This is consistent with the relatively high hatching rate (about 80%) in the studied population. The nestling mortality was severe, however, apparently due to starvation. It cannot be discounted that pesticides had an indirect effect on the kestrel's breeding success by reducing the populations of prey.