Ecological drivers of breeding periodicity in four forest neotropical eagles.

We explore the potential factors that affect clutch initiation in four Neotropical large raptors (Harpy eagle-HE, Crested eagle-CE, Ornate hawk-eagle-OHE, and Black hawk-eagle-BHE) by analyzing 414 clutch events mostly obtained from captive individuals. Differences in how clutch initiation is associated with changes in photoperiod were found between HE and both hawk-eagles, and between CE and BHE. Changes in temperature at the time of clutch initiation only differed between HE and OHE, whereas changes in precipitation varied between BHE and all other species. Principal Component Analysis of these environmental cues showed that ellipses in the dataset of each species overlap, but only ellipses from CE and OHE had the same variation trends. This means that although these species live under similar ecological conditions, they exhibit three different patterns of response to environmental cues. Apparently, these patterns are not associated with phylogenetic relatedness because species belonging to the same clade do not show the same response pattern. Diet diversity analysis revealed that HE has the least varied diet, and CE and OHE the most varied diet. The fact that species who fit the same reproductive timing response to environmental cues show similar diets leads us to hypothesize that breeding in these eagles was most likely shaped by food availability.

Genomic diversity and differentiation between island and mainland populations of white-tailed eagles (Haliaeetus albicilla).

Divergence in the face of high dispersal capabilities is a documented but poorly understood phenomenon. The white-tailed eagle (Haliaeetus albicilla) has a large geographic dispersal capability and should theoretically be able to maintain genetic homogeneity across its dispersal range. However, following analysis of the genomic variation of white-tailed eagles, from both historical and contemporary samples, clear signatures of ancient biogeographic substructure across Europe and the North-East Atlantic is observed. The greatest genomic differentiation was observed between island (Greenland and Iceland) and mainland (Denmark, Norway and Estonia) populations. The two island populations share a common ancestry from a single mainland population, distinct from the other sampled mainland populations, and despite the potential for high connectivity between Iceland and Greenland they are well separated from each other and are characterized by inbreeding and little variation. Temporal differences also highlight a pattern of regional populations persisting despite the potential for admixture. All sampled populations generally showed a decline in effective population size over time, which may have been shaped by four historical events: (1) Isolation of refugia during the last glacial period 110-115,000 years ago, (2) population divergence following the colonization of the deglaciated areas ~10,000 years ago, (3) human population expansion, which led to the settlement in Iceland ~1100 years ago, and (4) human persecution and exposure to toxic pollutants during the last two centuries.

Adaptation of the classical end-point ITS-PCR for the diagnosis of avian trichomonosis to a real-time PCR reveals Bonelli's eagle as a new host for Trichomonas gypaetinii.

Avian trichomonosis is a parasitic disease caused mainly by Trichomonas gallinae and other Trichomonas species. It can be asymptomatic, or it can produce a necrotic lesion in the upper digestive tract and spread to other organs, causing the death of the infected birds. In this study, we aimed to evaluate an adapted real-time PCR method for the diagnosis of different genotypes and species of avian oropharyngeal trichomonads. Fifty-six samples from the oropharynx of Bonelli's eagles (Aquila fasciata) obtained between 2018 and 2019 were analyzed using the real-time PCR and the end-point PCR, both targeting trichomonads ITS, and the results were compared by a coefficient of agreement. All positive samples were sequenced. The analysis showed a higher percentage of detection of real-time PCR ITS compared with end-point PCR ITS (64.3 vs 55.4%), and good agreement value (Kappa = 0.816). Melting temperature value for resulting amplicons of real-time PCR for avian trichomonads was 83.45 ± 0.72 °C. Genotypes A, D, and III were found among the sequences. Moreover, Trichomonas gypaetinii, a common species in scavenger birds, is reported for the first time in Bonelli's eagles.

MHC Reflects Fine-Scale Habitat Structure in White-Tailed Eagles, Haliaeetus albicilla.

The major histocompatibility complex (MHC) genes code for key immune receptors responsible for recognition of intra- and extracellular pathogens (MHC class I and class II, respectively). It was hypothesized that MHC polymorphism can be maintained via fluctuating selection resulting from between-habitat variation in pathogen regimes. We examined associations between MHC class I and class II genes and habitat structure in an apex avian predator, the white-tailed eagle, Haliaeetus albicilla. We genotyped MHC class I and class II genes in ca. 150 white-tailed eagle chicks from nearly 100 nesting territories distributed across 3 distinct populations in Poland. Habitat structure was quantified at the level of foraging territories and directly at the nest sites. We found strong support for associations of habitat traits with diversity and allelic composition at the MHC class II. Forest area within territory and forest productivity were identified as the major habitat predictors of MHC class II polymorphism, whereas other habitat traits (distance to nearest open water, grassland, and water area within territory or understory presence) showed fewer associations with class II alleles. In contrast, there was little support for associations between MHC class I genes and habitat structure. All significant associations were apparent at the within-population level rather than between populations. Our results suggest that extracellular (rather than intracellular) pathogens may exert much stronger selective pressure on the white-tailed eagle. Associations of habitat structure with MHC class II may reflect fluctuating (balancing) selection, which maintains MHC diversity within populations.

Molecular characterization of Trichomonas gypaetinii isolated from the upper alimentary tract of Steller's sea eagles (Haliaeetus pelagicus) and white-tailed sea eagles (Haliaeetus albicilla) in Hokkaido, Japan.

Recent phylogenetic and morphologic studies of Trichomonas spp. suggests that there are more than 3 species that infect the upper alimentary tract of wild birds, which include T. gallinae, T. stableri, and T. gypaetinii. In this study, investigations were conducted on the prevalence of trichomonads in the upper alimentary tract of 12 Steller's sea eagles (Haliaeetus pelagicus) and 18 white-tailed sea eagles (H. albicilla). All birds were rescued from the wild and kept at a rehabilitation facility in Hokkaido, Japan, for variable durations and did not show any symptoms of trichomonosis. The ITS1-5.8SrRNA-ITS2 (ITS) genomic region of Trichomonas spp. was detected from 29 samples by PCR, and flagellates were confirmed from 4 samples by culture. Morphologic observations and measurement recordings were conducted under a light microscope on trophozoites obtained from the cultured isolates. Genomic sequences of the ITS, 18S ribosomal RNA (18S rRNA), Fe-hydrogenase, and RNA polymerase II largest subunit (Rpb1) regions were determined by direct sequencing, and phylogenetic analyses were conducted with previously published sequences of Trichomonas spp. All isolates were concluded as T. gypaetinii based on morphologic and molecular characterizations of the ITS and 18S rRNA genes. This is the first study to isolate T. gypaetinii from Haliaeetus eagles and further provide novel sequences of the Fe-hydrogenase and Rpb1 genes of T. gypaetinii. Both genomic regions also confirmed that T. gypaetinii belong to independent clusters from other Trichomonas spp.

Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis.

The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski's horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme.

Elevated Heterozygosity in Adults Relative to Juveniles Provides Evidence of Viability Selection on Eagles and Falcons.

Viability selection yields adult populations that are more genetically variable than those of juveniles, producing a positive correlation between heterozygosity and survival. Viability selection could be the result of decreased heterozygosity across many loci in inbred individuals and a subsequent decrease in survivorship resulting from the expression of the deleterious alleles. Alternatively, locus-specific differences in genetic variability between adults and juveniles may be driven by forms of balancing selection, including heterozygote advantage, frequency-dependent selection, or selection across temporal and spatial scales. We use a pooled-sequencing approach to compare genome-wide and locus-specific genetic variability between 74 golden eagle (Aquila chrysaetos), 62 imperial eagle (Aquila heliaca), and 69 prairie falcon (Falco mexicanus) juveniles and adults. Although genome-wide genetic variability is comparable between juvenile and adult golden eagles and prairie falcons, imperial eagle adults are significantly more heterozygous than juveniles. This evidence of viability selection may stem from a relatively smaller imperial eagle effective population size and potentially greater genetic load. We additionally identify ~2000 single-nucleotide polymorphisms across the 3 species with extreme differences in heterozygosity between juveniles and adults. Many of these markers are associated with genes implicated in immune function or olfaction. These loci represent potential targets for studies of how heterozygote advantage, frequency-dependent selection, and selection over spatial and temporal scales influence survivorship in avian species. Overall, our genome-wide data extend previous studies that used allozyme or microsatellite markers and indicate that viability selection may be a more common evolutionary phenomenon than often appreciated.

High genetic diversity and low differentiation retained in the European fragmented and declining Greater Spotted Eagle (Clanga clanga) population.

Characterising genetic diversity and structure of populations is essential for effective conservation of threatened species. The Greater Spotted Eagle (Clanga clanga), a large and globally vulnerable raptor, is extinct or in severe decline in most of its previous range in Europe. We assessed whether the remnants of European population are genetically impoverished, and isolated from each other. We evaluated levels of genetic diversity and population structuring by sequencing mitochondrial pseudo-control region and 10 introns from various nuclear genes, and estimated length diversity in 23 microsatellite markers. The European population has expanded since the late Pleistocene, and does not exhibit signs of a recent population bottleneck. The global genetic diversity in Europe was rather similar to that detected in other similar species. Microsatellites suggested shallow but significant differentiation between the four extant populations in Estonia, Poland, Belarus and Russia (Upper Volga region) populations, but introns and mtDNA showed that only the Estonian population differed from the others. Mitochondrial diversity was highest in the northernmost Estonian population, introns suggested lower diversity in Upper Volga, microsatellites indicated equal diversity among populations. A recent bottleneck was detected in Poland, which is consistent with the observed repopulation of the region. We conclude that significant gene flow and high genetic diversity are retained in the fragmented Greater Spotted Eagle populations; there is currently no need for genetic augmentation in Europe.

Allelic diversity and selection at the MHC class I and class II in a bottlenecked bird of prey, the White-tailed Eagle.

BACKGROUND: Genes of the Major Histocompatibility Complex (MHC) are essential for adaptive immune response in vertebrates, as they encode receptors that recognize peptides derived from the processing of intracellular (MHC class I) and extracellular (MHC class II) pathogens. High MHC diversity in natural populations is primarily generated and maintained by pathogen-mediated diversifying and balancing selection. It is, however, debated whether selection at the MHC can counterbalance the effects of drift in bottlenecked populations. The aim of this study was to assess allelic diversity of MHC genes in a recently bottlenecked bird of prey, the White-tailed Eagle Haliaeetus albicilla, as well as to compare mechanisms that shaped the evolution of MHC class I and class II in this species. RESULTS: We showed that significant levels of MHC diversity were retained in the core Central European (Polish) population of White-tailed Eagles. Ten MHC class I and 17 MHC class II alleles were recovered in total and individual birds showed high average MHC diversity (3.80 and 6.48 MHC class I and class II alleles per individual, respectively). Distribution of alleles within individuals provided evidence for the presence of at least three class I and five class II loci the White-tailed Eagle, which suggests recent duplication events. MHC class II showed greater sequence polymorphism than MHC class I and there was much stronger signature of diversifying selection acting on MHC class II than class I. Phylogenetic analysis provided evidence for trans-species similarity of class II, but not class I, sequences, which is likely consistent with stronger balancing selection at MHC class II. CONCLUSIONS: Relatively high MHC diversity retained in the White-tailed Eagles from northern Poland reinforces high conservation value of local eagle populations. At the same time, our study is the first to demonstrate contrasting patterns of allelic diversity and selection at MHC class I and class II in an accipitrid species, supporting the hypothesis that different mechanisms can shape evolutionary trajectories of MHC class I and class II genes.

Genetic determination of migration strategies in large soaring birds: evidence from hybrid eagles.

The relative contributions of genetic and social factors in shaping the living world are a crucial question in ecology. The annual migration of birds to their wintering grounds and back provides significant knowledge in this field of research. Migratory movements are predominantly genetically determined in passerine birds, while in large soaring birds, it is presumed that social (cultural) factors play the largest role. In this study, we show that genetic factors in soaring birds are more important than previously assumed. We used global positioning system (GPS)-telemetry to compare the autumn journeys and wintering ranges of two closely related large raptorial bird species, the greater spotted eagle Clanga clanga and the lesser spotted eagle Clanga pomarina, and hybrids between them. The timing of migration in hybrids was similar to that of one parental species, but the wintering distributions and home range sizes were similar to those of the other. Tracking data were supported by habitat suitability modelling, based on GPS fixes and ring recoveries. These results suggest a strong genetic influence on migration strategy via a trait-dependent dominance effect, although we cannot rule out the contribution of social interactions.

The axon degeneration gene SARM1 is evolutionarily distinct from other TIR domain-containing proteins.

Many forms of neurodegenerative disease are characterized by Wallerian degeneration, an active program of axonal destruction. Recently, the important player which enacts Wallerian degeneration was discovered, the multidomain protein SARM1. Since the SARM1 protein has classically been thought of as an innate immune molecule, its role in Wallerian degeneration has raised questions on the evolutionary forces acting on it. Here, we synthesize a picture of SARM1's evolution through various organisms by examining the molecular and genetic changes of SARM1 and the genes around it. Using proteins that possess domains homologous to SARM1, we established distances and Ka/Ks values through 5671 pairwise species-species comparisons. We demonstrate that SARM1 diverged across species in a pattern similar to other SAM domain-containing proteins. This is surprising, because it was expected that SARM1 would behave more like its TIR domain relatives. Going along with this divorce from TIR, we also noted that SARM1's TIR is under stronger purifying selection than the rest of the TIR domain-containing proteins (remaining highly conserved). In addition, SARM1's synteny analysis reveals that the surrounding gene cluster is highly conserved, functioning as a potential nexus of gene functionality across species. Taken together, SARM1 demonstrates a unique evolutionary pattern, separate from the TIR domain protein family.

Phylogeny and new taxonomy of the Booted Eagles (Accipitriformes: Aquilinae).

We present a phylogeny of all booted eagles (38 extant and one extinct species) based on analysis of published sequences from seven loci. We find molecular support for five major clades within the booted eagles: Nisaetus (10 species), Spizaetus (4 species), Clanga (3 species), Hieraaetus (6 species) and Aquila (11 species), requiring generic changes for 14 taxa. Additionally, we recommend that the Long-crested Eagle (Lophaetus occipitalis) and the Black Eagle (Ictinaetus malaiensis) remain in their monotypic genera, due to their distinctive morphology. We apply the recently resurrected genus Clanga for the spotted eagles (previously Aquila spp.) to resolve the paraphyly of the genus Aquila such that the clade including the Booted Eagle (H. pennatus), Little Eagle (H. morphnoides), Pygmy Eagle (H. weiskei), Ayres's Eagle (H. ayresii) and Wahlberg's Eagle (H. wahlbergi) can remain in the genus Hieraaetus. The Rufous-bellied Eagle should be retained in the genus Lophotriorchis. For consistency in English names, we recommend that the term "hawk-eagles" be used only for the species in the genera Nisaetus and Spizaetus. We suggest following new or modified English names: Cassin's Eagle (Aquila africana), Bonaparte's Eagle (A. spilogaster), Ayres's Eagle (Hieraaetus ayresii), and Black-and-chestnut Hawk-Eagle (Spizaetus isidori).

Nuclear and Mitochondrial DNA Analyses of Golden Eagles (Aquila chrysaetos canadensis) from Three Areas in Western North America; Initial Results and Conservation Implications.

Understanding the genetics of a population is a critical component of developing conservation strategies. We used archived tissue samples from golden eagles (Aquila chrysaetos canadensis) in three geographic regions of western North America to conduct a preliminary study of the genetics of the North American subspecies, and to provide data for United States Fish and Wildlife Service (USFWS) decision-making for golden eagle management. We used a combination of mitochondrial DNA (mtDNA) D-loop sequences and 16 nuclear DNA (nDNA) microsatellite loci to investigate the extent of gene flow among our sampling areas in Idaho, California and Alaska and to determine if we could distinguish birds from the different geographic regions based on their genetic profiles. Our results indicate high genetic diversity, low genetic structure and high connectivity. Nuclear DNA Fst values between Idaho and California were low but significantly different from zero (0.026). Bayesian clustering methods indicated a single population, and we were unable to distinguish summer breeding residents from different regions. Results of the mtDNA AMOVA showed that most of the haplotype variation (97%) was within the geographic populations while 3% variation was partitioned among them. One haplotype was common to all three areas. One region-specific haplotype was detected in California and one in Idaho, but additional sampling is required to determine if these haplotypes are unique to those geographic areas or a sampling artifact. We discuss potential sources of the high gene flow for this species including natal and breeding dispersal, floaters, and changes in migratory behavior as a result of environmental factors such as climate change and habitat alteration. Our preliminary findings can help inform the USFWS in development of golden eagle management strategies and provide a basis for additional research into the complex dynamics of the North American subspecies.

Reduction of Genetic Diversity of the Harpy Eagle in Brazilian Tropical Forests.

Habitat loss and fragmentation intensify the effects of genetic drift and endogamy, reducing genetic variability of populations with serious consequences for wildlife conservation. The Harpy Eagle (Harpia harpyja) is a forest dwelling species that is considered near threatened and suffers from habitat loss in the forests of the Neotropical region. In this study, 72 historical and current samples were assessed using eight autosomal microsatellite markers to investigate the distribution of genetic diversity of the Harpy Eagle of the Amazonian and Atlantic forests in Brazil. The results showed that the genetic diversity of Harpy Eagle decreased in the regions where deforestation is intense in the southern Amazon and Atlantic Forest.

Reference genes for quantitative gene expression studies in multiple avian species.

Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

The genome sequence of a widespread apex predator, the golden eagle (Aquila chrysaetos).

Biologists routinely use molecular markers to identify conservation units, to quantify genetic connectivity, to estimate population sizes, and to identify targets of selection. Many imperiled eagle populations require such efforts and would benefit from enhanced genomic resources. We sequenced, assembled, and annotated the first eagle genome using DNA from a male golden eagle (Aquila chrysaetos) captured in western North America. We constructed genomic libraries that were sequenced using Illumina technology and assembled the high-quality data to a depth of ∼40x coverage. The genome assembly includes 2,552 scaffolds >10 Kb and 415 scaffolds >1.2 Mb. We annotated 16,571 genes that are involved in myriad biological processes, including such disparate traits as beak formation and color vision. We also identified repetitive regions spanning 92 Mb (∼6% of the assembly), including LINES, SINES, LTR-RTs and DNA transposons. The mitochondrial genome encompasses 17,332 bp and is ∼91% identical to the Mountain Hawk-Eagle (Nisaetus nipalensis). Finally, the data reveal that several anonymous microsatellites commonly used for population studies are embedded within protein-coding genes and thus may not have evolved in a neutral fashion. Because the genome sequence includes ∼800,000 novel polymorphisms, markers can now be chosen based on their proximity to functional genes involved in migration, carnivory, and other biological processes.

Did postglacial sea-level changes initiate the evolutionary divergence of a Tasmanian endemic raptor from its mainland relative?

Populations on continental islands are often distinguishable from mainland conspecifics with respect to body size, appearance, behaviour or life history, and this is often congruent with genetic patterns. It is commonly assumed that such differences developed following the complete isolation of populations by sea-level rise following the Last Glacial Maximum (LGM). However, population divergence may predate the LGM, or marine dispersal and colonization of islands may have occurred more recently; in both cases, populations may have also diverged despite ongoing gene flow. Here, we test these alternative hypotheses for the divergence between wedge-tailed eagles from mainland Australia (Aquila audax audax) and the threatened Tasmanian subspecies (Aquila audax fleayi), based on variation at 20 microsatellite loci and mtDNA. Coalescent analyses indicate that population divergence appreciably postdates the severance of terrestrial habitat continuity and occurred without any subsequent gene flow. We infer a recent colonization of Tasmania by marine dispersal and cannot discount founder effects as the cause of differences in body size and life history. We call into question the general assumption of post-LGM marine transgression as the initiator of divergence of terrestrial lineages on continental islands and adjacent mainland, and highlight the range of alternative scenarios that should be considered.

Karyotype reorganization with conserved genomic compartmentalization in dot-shaped microchromosomes in the Japanese mountain hawk-eagle (Nisaetus nipalensis orientalis, Accipitridae).

The karyotype of the Japanese mountain hawk-eagle (Nisaetus nipalensis orientalis) (2n = 66) consists of a large number of medium-sized and small chromosomes but only 4 pairs of dot-shaped microchromosomes, in contrast to the typical avian karyotype with a small number of macrochromosomes and many indistinguishable microchromosomes. To investigate the drastic karyotype reorganization in this species, we performed a molecular cytogenetic characterization employing chromosome in situ hybridization and molecular cloning of centromeric heterochromatin. Cross-species chromosome painting with chicken chromosome-specific probes 1-9 and Z and a paint pool of 20 microchromosome pairs revealed that the N. n. orientalis karyotype differs from chicken by at least 13 fissions of macrochromosomes and 15 fusions between microchromosomes and between micro- and macrochromosomes. A novel family of satellite DNA sequences (NNO-ApaI) was isolated, consisting of a GC-rich 173-bp repeated sequence element. The NNO-ApaI sequence was localized to the C-positive centromeric heterochromatin of 4 pairs of microchromosomes, which evolved concertedly by homogenization between the microchromosomes. These results suggest that the 4 pairs of dot-shaped microchromosomes have retained their genomic compartmentalization from other middle-sized and small chromosomes.

Molecular identification of traces from the White-tailed Sea Eagle.

PURPOSE: Over the preceeding decades, after periods of dramatic decline and extinction in many parts of Europe, the White-tailed Sea Eagle (Haliaeetus albicilla) has re-colonized traditional breeding areas. However, this large apex predator remains threatened, not only by the bioaccumulation of environmental pollutants, but also by targeted poisoning and poaching. In connection with a forensic case, a novel PCR assay was developed for the sensitive and specific detection of sea eagle DNA traces in questioned samples of unknown origin. METHODS: The assay amplifies a fragment of the popular phylogenetic marker gene cytochrome b. Primers were designed to bind sites with relatively high variability between homologous sequences from H. albicilla and other related European birds of prey. RESULTS: Assay sensitivity was sufficient for single cell analysis. Specificity was tested in vitro and the primers did not cross-detect DNA from humans, chicken and the following raptors: Common Buzzard (Buteo buteo), Northern Goshawk (Accipiter gentilis), Red Kite (Milvus milvus) and Black Kite (Milvus migrans). Applicability for the analysis of poor quality samples was demonstrated with extracts from field-collected small molted down feathers that did not contain detectable amounts of sea eagle nuclear DNA. Amplicons of the expected size were generated, purified and sequenced. Sequence data were subjected to Basic Local Alignment Search Tool analysis and affiliated with cytochrome b from H. albicilla. CONCLUSIONS: The novel PCR primers allowed for the correct assignment of traces from H. albicilla, even in mixed samples and in cases with limited and degraded biological material.

Non-invasive sex identification of the white-bellied sea eagle (Haliaeetus leucogaster) through genetic analysis of feathers.

The white-bellied sea eagle, Haliaeetus leucogaster, displays reversed sexual size dimorphism and is monomorphic for adult plumage coloration. Early attempts to identify sex in sexually monomorphic birds were based on morphological or chromosomal characters, but since avian W-specific DNA sequences were identified, PCR amplification has become commonly used for molecular sexing. We used a PCR test employing primers that amplify two homologous fragments of both the CHD-W gene, unique to females, and the CHD-Z gene, occurring in both sexes. This test was applied to five individuals of H. leucogaster from the Malacca Zoo and to male and female domestic chickens, Gallus domesticus, for comparison. All individuals were sexed successfully with high reproducibility. We conclude that this PCR-based test with feathers as the DNA source is a reliable sexing method for H. leucogaster. This sexing technique is objective and non-invasive and could be used to test sex ratio theories, as well as to help improve conservation and management actions for captive breeding program of this species in Malaysia.