Morphological and Genetic Sex Identification of White-Tailed Eagle

J Ornithol (2007) 148:435–442 DOI 10.1007/s10336-007-0156-y ORIGINAL ARTICLE Morphological and genetic sex identification of white-tailed eagle Haliaeetus albicilla nestlings Bjo¨rn Helander Æ Frank Hailer Æ Carles Vila` Received: 8 December 2006 / Revised: 8 May 2007 / Accepted: 10 May 2007 / Published online: 26 June 2007 Ó Dt. Ornithologen-Gesellschaft e.V. 2007 Abstract Identifying the sex of bird nestlings is relevant to Keywords Haliaeetus albicilla Á Molecular sexing Á studies of behaviour and ecology and is often a central issue Morphological sexing Á Sexual dimorphism Á Raptors in the management of endangered or captive populations. The white-tailed eagle Haliaeetus albicilla is a formerly threatened Eurasian raptor which is closely monitored in Introduction many countries due to its high exposure to environmental pollutants in the food chain. The aim of this study was to Sex identification can provide researchers with important evaluate the reliability of sex identification methods for information regarding the ecology and behaviour of bird white-tailed eagle nestlings based on morphological mea- species (e.g. Ellegren and Sheldon 1997) and also yields surements that can be recorded at the nest by a single person valuable insights into their conservation and management and with minimum disturbance. The sex of each bird was (Morris and Doak 2002; Sutherland 2002). A knowledge of independently determined using molecular (genetic) meth- the sex of individuals is relevant, for example, for studies ods. One measure of tarsus width allowed the correct iden- of demography, sex ratios, population viability analyses tification of sex for 96% of the nestlings from southern and (Gilpin and Soule´ 1986), territorial behaviour, life history central Sweden. However, we found that the criteria for sex or species management through the translocation of nes- identification were not directly applicable to the population tlings. Despite the lack of external sexual organs, the sex of in Swedish Lapland, where nestlings are typically thinner, adult individuals of many bird species can be ascertained probably due to a limited food supply. These results show by their plumage (colouration or form). The sex of juve- that sexing in the field of white-tailed eagle nestlings can be niles, however, is generally hard to discern. Many raptor feasible with high accuracy based on a limited number of species exhibit sexual size dimorphism even at the nestling measurements. However, the criteria employed to separate or subadult stages (e.g. Bortolotti 1984a, b, c; Masterov sexes may have to be adjusted for each population. 2000; Shephard et al. 2004), potentially enabling sex identification based on morphometry. However, if the traits used for sex discrimination also show geographical varia- tion (see, for example, Bent 1961; Salomonsen 1979; Merz Communicated by M. Wink. and Merz 2004; Nebel 2006), their accuracy may vary from population to population. Moreover, problems can arise B. Helander (&) when the degree of dimorphism varies or when individuals Department of Contaminant Research, are sampled at different times during their nestling period Swedish Museum of Natural History, Box 50007, 104 05 Stockholm, Sweden (Bortolotti 1984b; Setiawan et al. 2004). We present here e-mail: [email protected] an assessment of the accuracy of one such sex identification method in two populations of white-tailed eagle Ha- F. Hailer Á C. Vila` liaeetus albicilla. Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, White-tailed eagles are protected and classified as vul- Norbyva¨gen 18d, 752 36 Uppsala, Sweden nerable or threatened over most of their distribution range. 123 436 J Ornithol (2007) 148:435–442 Although ringing of nestlings is taking place in many year 2000 the number of territorial pairs was estimated at countries, there is a mandate to reduce disturbance at nest 250 in the southern and central areas and 50 in Lapland sites as much as possible. Since nests are often built in tree (Helander 2003a). These two populations have likely been tops, simple and quick methods are preferable to make the demographically independent from other populations in handling of nestlings on the tree more comfortable and northern Europe during the past decades (Helander 2003b) reduce both risk and stress to the nestlings. This precludes and are genetically differentiated from each other (Ceder- the development of multivariate methods for sex determi- berg et al. 2003; Hailer et al. 2006). The present study nation (Shephard et al. 2004). A method to sex white-tailed includes samples from throughout the species range in eagles nestlings based on two tarsal measurements has Lapland and from a major part of its range in southern and been suggested (Helander 1981). This approach takes into central Sweden (nests south of latitude 61°N). account the relatively larger size of the tarsus in females than in males and is applicable to field work conditions Nestling measurement and sampling with only one person handling the eagle nestlings in the treetop. Nevertheless, no formal assessment of the value of Nest trees were climbed when nestlings were expected to this method to identify the sex of the birds has been carried be between 4 and 8 weeks old. Before taking measure- out. An accurate assessment of the validity of this method ments, each nestling was assigned an estimated age based will require that it be compared to an independent and on its stage of development and feathering (Heinroth and more precise approach. Heinroth 1967; Helander 1981, 1982). Nestlings were Molecular genetic methods offer the possibility for sexing ringed lying flat on their belly with the feet stretched of birds based on the fact that females are heterogametic backwards and held with one hand. Before the rings were (carry two different sex chromosomes, ZW), whereas males attached, a calliper was used to measure the thickness of are homogametic (with two Z chromosomes, ZZ). Several the tarsus to the nearest 0.5 mm at the thinnest point (lo- methods based on the polymerase chain reaction (PCR) have cated approximately mid-way between the toes and the been suggested (Fridolfsson and Ellegren 1999; Griffiths joint of the heel; tars1). At the same location, the thickest et al. 1998). One commonly applied method is based on the tarsus diameter was also measured (tars2), at an approxi- PCR amplification of an intron within the CHD1 gene, which mately right angle to the first measurement. With the is present on both sex chromosomes, using primers located in nestling still lying on its belly after ringing, a metal-coil conserved exonic regions flanking the intron. If the amplified ruler was used to measure the length of the folded wing to intron shows a pronounced length difference between the Z the nearest 5 mm from the carpal joint to the tip of the and W chromosome gene copies, males and females can be longest primary (wing). The nestling was then put into a discriminated on the basis of a simple agarose gel electro- plastic micromesh bag and weighed on a 10 kg Pesola phoresis. The product of a PCR amplification will then spring balance (Pesola AG, Baar, Switzerland). Weight produce two bands in females and just one in males. was recorded to the nearest 0.05 kg and was then corrected In the present study, we evaluate the reliability of sex for the weight of the bag and for crop contents (weight). identification methods for white-tailed eagle nestlings The estimated volume of the crop was used as a proxy for based on morphological measurements. We also assess if the weight of its contents (Helander 1981). morphology-based methods to identify sex in one popula- Blood (0.5 ml) was sampled from the brachial vein tion also can be used in other populations with different using sterile techniques and was buffered in 1 ml EDTA/ ecological conditions. SSC and kept frozen until treatment in the laboratory. From broods containing more than one chick in the southern population, the larger chick was often chosen for sampling Methods (potentially implying a bias towards the – larger – females; see Discussion). In the northern (Lapland) population, nests Study period and populations commonly contained only one chick (Helander 2003a), but when broods of two occurred, both nestlings were gener- The 211 samples used for this study were collected be- ally sampled to increase the number of samples from this tween 1999 and 2003 across the range of the white-tailed smaller population. eagle (Haliaeetus albicilla) in Sweden. Its present distribution comprises two separate areas: a population along the Molecular sex identification Baltic coast and also extends into freshwaters in central and southern Sweden, and an inland population in Lapland DNA was extracted from the blood samples using a stan- (northern Sweden), mainly north of latitude 66°N. In the dard proteinase K and phenol-chloroform procedure 123 J Ornithol (2007) 148:435–442 437 (Sambrook et al. 1989). To identify the sex of each nest- Previous studies have shown that food resources are ling, we used the primers 2550F and 2718R (Fridolfsson limited for the eagles in Lapland and, probably as a result, and Ellegren 1999) to amplify an intron within the CHD1 nestlings tend to be thinner during the investigated nestling genes on the Z and W chromosomes. For the PCR ampli- period (Helander 1983). To avoid potential confounding fication, we used approximately 10 ng of genomic DNA, effects of the different habitat characteristics, data analysis 3 pmol of each primer, 2 nmol of each dNTP, 0.3 U of was performed treating the two populations separately: HotStar Taq (Qiagen, Hilden, Germany) and 1· of HotStar methods for nestling sex identification were developed Taq reaction buffer (Qiagen) containing Tris–Cl, KCl, based on the population in southern and central Sweden.

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