Ruben Limiñana
email: ruben.lm@gmail.com

Beatriz E Arroyo

Martín Surroca

Vicente Urios

Abilio Reig-Ferrer

Influence of habitat on nest location and reproductive output of Montagu"s Harriers breeding in natural vegetation

Keywords: Circus pygargus, colonial species, conservation, natural vegetation, Spain Paare und die mit mehr Nachbarn hatten einen niedrigeren Bruterfolg die Wiesenweihe, nicht zu einer Veränderung der Verbreitung führen. Auch können Gebiete mit gutem Habitat trotzdem nicht besiedelt werden, da soziale Faktoren wie die Präsenz von Artgenossen eine wichtige Rolle

We examined distribution and breeding success of semi-colonial Montagu"s Harriers in relation to habitat in Castellon province (Eastern Spain). Breeding areas used by harriers at 1 km 2 scale were characterized by having intermediate percentages of scrub cover, their nesting habitat, and also had intermediate coverage of herbaceous crops and nonirrigated orchards. Out of all habitat variables considered, only percentage of herbaceous crops within 500 metres from individual nests had a positive and significant effect on breeding output of the species, suggesting that this habitat may be efficiently used by harriers to forage. Breeding output was also related to laying date and number of breeding neighbours within 500 metres around nests, with pairs laying later and having a higher number of breeding neighbours showing lower fledged brood sizes.

Number of neighbours (but not laying date) was positively related to scrub cover within 500 metres and to cover of herbaceous crops within 2000 metres. Conservation actions for Montagu"s Harrier in the study area should be aimed to preserve areas of scrub with nearby presence of herbaceous crops or natural grasslands. However, habitat improvement for semi-colonial species such as Montagu"s Harrier may not result in a change of species distribution area, and good habitat areas may remain unoccupied, as social factors like presence of conspecifics play an important role in breeding area selection for these species.

INTRODUCTION

Declines of many bird populations have been linked to a reduction or degradation of their preferred habitats [START_REF] Browne | Habitat availability and use by Turtle Doves Streptopelia turtur between 1965 and 1995: an analysis of Common Birds Census data[END_REF][START_REF] Fernández | Bird-habitat relationship for the declining Pampas meadowlark populations in the southern Pampas grasslands[END_REF][START_REF] Julliard | Common birds facing global changes: what makes a species at risk?[END_REF][START_REF] Robinson | The importance of arable habitat for farmland birds in grassland landscapes[END_REF]. Both nesting and foraging habitats may play an important role in limiting bird population numbers or distribution (e.g. [START_REF] Newton | Population limitation in birds[END_REF]). Thus, conservation of bird species is frequently based on protection of their habitats, except in those cases when direct intervention is necessary (e.g. rescue of nestlings of a given species, supplementary food campaigns, captive breeding and release of birds in small populations, etc.; [START_REF] Cade | Management of threatened bird species: evaluation of the hands-on approach[END_REF][START_REF] Oro | Testing the Goodness of Supplementary Feeding to Enhance Population Viability in an Endangered Vulture[END_REF]. For example, protected areas for birds (such as the Special Protection Areas -or SPAs designated under the EC Birds Directive) usually consider financial incentives for sustainable management of the land.

Like many birds, raptors are usually highly selective with respect to their habitats, especially regarding the availability of suitable nesting areas, although foraging habitats may also have an important effect at the time of choosing a site during the breeding season [START_REF] Newton | Population limitation in birds[END_REF]. Breeding habitat (which include both nesting and foraging habitats) may limit species productivity or distribution (e.g. [START_REF] Benton | Linking agricultural practice to insect and bird populations: a historical study over three decades[END_REF][START_REF] Soh | High sensitivity of montane bird communities to habitat disturbance in Peninsular Malaysia[END_REF][START_REF] Suárez | Nesting habitat selection by booted eagles Hieraaetus pennatus and implications for management[END_REF]. In these cases, increasing availability or suitability of preferred habitats (e.g. restoring nesting habitats or increasing the availability of foraging habitats) may potentially lead to increasing population sizes [START_REF] Carrete | Predicting the implications of conservation management: a territorial occupancy model of Bonelli"s eagle in Murcia, Spain[END_REF][START_REF] Hiraldo | A demographic model for a population of the endangered lesser kestrel is southern Spain[END_REF]. Understanding the strength of the relationships between habitat and species distribution or breeding success may be important to manage protected areas and to predict how changes in habitat may influence population dynamics, and thus contribute to the development of successful conservation programmes [START_REF] López-López | Modelling breeding habitat preferences of Bonelli"s eagle (Hieraaetus fasciatus) in relation to topography, disturbance, climate and land use at different spatial scales[END_REF], 2007;[START_REF] Suárez | Nesting habitat selection by booted eagles Hieraaetus pennatus and implications for management[END_REF]Tapia et al. 2004;[START_REF] Arroyo | Hunting habitat selection by hen harriers on moorland: implications for conservation[END_REF]).

However, the majority of raptor studies have been carried out on territorial species (but see [START_REF] García-Ripollés | Modelling nesting habitat preferences of Eurasian Griffon vulture Gyps fulvus in eastern Iberian Peninsula[END_REF][START_REF] Poirazidis | Modelling nesting habitat as a conservation tool for the Eurasian black vulture (Aegypius monachus) in Dadia Nature Reserve, northeastern Greece[END_REF][START_REF] Sergio | Adaptive selection of foraging and nesting habitat by black kites (Milvus migrans) and its implications for conservation: a multi-scale approach[END_REF], for semicolonial and colonial raptor species). Colonial or semi-colonial species may be atypical, because habitat selection may play a relatively smaller role in breeding spatial distribution for these species (e.g. [START_REF] Cornulier | Composants de la distribution spatiale d´un prédateur : effets respectifs de l´habitat, des ressources alimentaires et des interactions comportementales[END_REF][START_REF] Cornulier | Assessing the influence of environmental heterogeneity on bird spacing patterns: a case study with two raptors[END_REF], whereas factors like conspecific attraction may be more important [START_REF] Cornulier | Composants de la distribution spatiale d´un prédateur : effets respectifs de l´habitat, des ressources alimentaires et des interactions comportementales[END_REF];

Sergio and Penteriani 2005, but see also [START_REF] Sergio | Sequential settlement and site dependence in a migratory raptor[END_REF]. For these species, increasing the availability of preferred habitats might be inefficient to ensure the occupancy of given areas (see e.g. [START_REF] Reed | Behavioural constraints and conservation biology: conspecific attraction and recruitment[END_REF].

The Montagu"s Harrier (Circus pygargus) is a semi-colonial ground-nesting Palaearctic raptor [START_REF] Cramp | The birds of the Western Paleartic[END_REF]. The species is considered vulnerable in France and the Iberian Peninsula [START_REF] Blanco | The use of conspecific reproductive success for breeding patch selection in terrestrial migratory species[END_REF][START_REF] Salamolard | Le Busard cendré[END_REF][START_REF] Snprcn | Livro vermelho dos vertebrados de Portugal[END_REF], which are the strongholds of its western European populations. In Western Europe, this species mainly builds nests within cereal crops [START_REF] Arroyo | Conservation of the Montagu"s Harrier (Circus pygargus) in agricultural areas[END_REF], but some populations nest in natural vegetation [START_REF] Cramp | The birds of the Western Paleartic[END_REF]. The importance of protecting populations breeding in natural vegetation has been highlighted [START_REF] Arroyo | Conservation of the Montagu"s Harrier (Circus pygargus) in agricultural areas[END_REF]Limiñana et al. 2006a), but most recent conservation measures have been directed towards populations breeding in agricultural habitats. One population nesting in natural vegetation is located in inland Castellon province in eastern Spain (Limiñana et al. 2006a). This population has increased exponentially from three pairs in early 80s to nearly 150 pairs in 2007, although population growth has slowed down since 2002 (Limiñana et al. 2006a;[START_REF] Soutullo | Density-dependent regulation of population size in colonial breeders: Allee and buffer effects in the migratory Montagu"s Harrier[END_REF]. Harrier breeding sites in Castellon face an uncertain future, as the area is currently subject to social and commercial developments, such as the recent building of an airport. A better understanding of the relationship between breeding habitat availability, harrier distribution and breeding performance would enable more effective conservation of Montagu"s Harrier in this area.

In this paper, we first examine the relationship between habitat and nest occurrence, to assess the habitats preferred for breeding within the study area. Secondly, we examine the relationship between habitat, timing of breeding, number of neighbours and breeding output. We discuss the importance of habitat in explaining breeding distribution or success, and its implications for conservation in this semi-colonial species.

METHODS

Study site and species

The study was carried out in Castellon province (Eastern Spain, Fig. 1) where the species breeds in the inland corridors between the mountain ranges. Montagu"s Harriers first bred in the study area in early 1980s. The only nesting habitat used in this area by the species is Mediterranean scrub, dominated by Kermes Oak (Quercus coccifera).

Other vegetation types in the region are non-irrigated crops (including cereal fields and orchards), as well as pine plantations. More details on the study area can be found in Limiñana et al. (2006aLimiñana et al. ( , 2006b) ) and [START_REF] Soutullo | Density-dependent regulation of population size in colonial breeders: Allee and buffer effects in the migratory Montagu"s Harrier[END_REF]. The main prey types for this Montagu"s Harrier population are passerines and insects (especially orthopterans and small coleopterans), with other prey such as small mammals and lizards being taken less frequently [START_REF] Limiñana | Uso del habitat y dieta del aguilucho cenizo, C. pygargus, en el interior de la provincia de Castellón[END_REF]).

This study is based on data from 2005-2007, when positions of located nests were recorded using a GPS. The area searched to locate Montagu"s Harrier nests was ca.

1050 Km 2 (Fig. 1); field effort to locate the nests and monitor breeding performance was kept constant through the study period. Population size in the study area was stable between 2001 and 2005 at ca. 100 pairs (see Limiñana et al. 2006a and[START_REF] Soutullo | Density-dependent regulation of population size in colonial breeders: Allee and buffer effects in the migratory Montagu"s Harrier[END_REF]. In 2006 and 2007 it increased to 129 and 145 breeding pairs, respectively. In 2005, a total of 80 Montagu"s Harrier nests were located and positions of 76 of them were recorded using a GPS. In 2006, 96 nests were located and positions of 85 of them were recorded. In 2007, there were 101 located nests and positions of 86 were recorded.

Breeding data (number of eggs and nestlings) on these nests were recorded during nests visits. All pairs in 2005 which nests were not located were inside known colonies, so the fact that we are not including them in the analyses of harrier nesting occurrence is not likely to strongly affect the results (as they were all in grid cells that were otherwise occupied). Some nests in 2006 and 2007 appeared in new areas for which we did not have accurate habitat data (see below), but study of aerial photographs confirmed that these areas were similar to those included in these analyses in terms of habitat composition.

Habitat variables

Habitat composition in the study area was determined using the 1:10 000 Land Use map of the Comunidad Valenciana which is, in turn, based on recent aerial photographs (taken at the time period of the study). We had access to aerial photographs (7x5 km)

and to the Land Use map corresponding to the areas where Montagu´s Harriers nested up to 2005. We imposed a 1 km 2 UTM grid over the study area and calculated the proportion of each habitat type within each of the grid cells using a Geographic

Information System (ArcView 3.2). We also evaluated (using the GIS) whether each grid cell was occupied or not by breeding harriers, and how many nests were located within each one. This scale has been extensively used in other studies of harrier occupancy [START_REF] Arroyo | Conservation of the Montagu"s Harrier (Circus pygargus) in agricultural areas[END_REF][START_REF] Arroyo | Habitat use and range management on priority areas for hen harriers: 2004 report[END_REF]Tapia et al. 2004). To evaluate breeding performance in relation to breeding habitat, we calculated habitat composition around each nest at two different radii (500 metres and 2000 metres) using the GIS. The area of a circle with 500 metres radius corresponds roughly to the area covered by a 1 km 2 grid cell, and also corresponds roughly to the area that a female uses for hunting around the nest, at least in Mediterranean areas [START_REF] García | Food-niche differentiation in sympatric Hen and Montagu"s harriers[END_REF]. We also used the 2000 metres radius since it includes a large part of the male core home range, also according to studies in southern Europe [START_REF] Arroyo | Estudio de la ecología poblacional del aguilucho cenizo (Circus pygargus) en Extremadura[END_REF][START_REF] Cornulier | Composants de la distribution spatiale d´un prédateur : effets respectifs de l´habitat, des ressources alimentaires et des interactions comportementales[END_REF]. By using both radii, we can be sure that we are accounting for both nesting and foraging habitat in our analyses on the effect of breeding habitat on reproductive performance.

Overall, habitat variables calculated (both for circles and grid cells) were the following: percentage of scrub, percentage of forest (mainly pine plantations), percentage of herbaceous crops (mainly cereal crops) and percentage of non-irrigated orchards (mainly almond and olive). Across the study area, scrub covered 47% of the surface, orchards covered 38%, herbaceous crops 8% and forest 6%.

Statistical analyses

Hierarchical partitioning (HP) was used to identify the most likely habitat variables explaining the occurrence of Montagu"s Harrier nests in the grid encompassing the study area [START_REF] Chevan | Hierarchical partitioning[END_REF]. Hierarchical partitioning computes all of the possible hierarchical models that can be developed with a set of independent predictive variables; this is to say that if U, V and W are variables, HP computes single-order (U, V, W), second order (UV, UW, VW) and higher-order (UVW) models and tests whether the addition of a given variable produces an improvement in goodness of fit. For each independent variable, their explanatory power is segregated into the independent effect "I" and the effects caused jointly with other variables "J" (MacNally 2000). This analysis was conducted in R (R Development Core Team 2009) with the "hier.part" package (Walsh and MacNally 2003), using logistic regression and log-likelihood as goodnessof-fit measure. As suggested by [START_REF] Macnally | Multiple regression and inference in ecology and conservation biology: further comments on identifying important predictor variables[END_REF], significance of the individual contribution of each variable included in the analysis was evaluated by a randomization procedure based on 999 randomizations. Grid cell occupancy was defined as 1 = occupied (if at least one nest was present in the cell in at least one year), and 0 = unoccupied (if no nests were known in the cell). Only those cells for which we had information on at least 75% of its surface for habitat variables were used for the analyses. The initial model included percentage of each habitat variable: scrub, forest, herbaceous crops and orchards, as well as their quadratic terms. Quadratic terms were included since other studies of harriers have shown that percentage cover of certain habitats may be optimal at intermediate levels [START_REF] Arroyo | Habitat use and range management on priority areas for hen harriers: 2004 report[END_REF].

To test whether frequency of use (the number of years that a given cell had been occupied during the study) varied in relation to habitat, we used categorical modelling, with the procedure CATMOD within SAS 9.1 (SAS Institute 1999).

Secondly, we evaluated the relationship between breeding habitat and breeding output using Generalized Linear Mixed Models (GLMM). Laying date usually influences breeding output in raptors, with pairs laying later having a lower productivity (e.g. [START_REF] Newton | Seasonal trend in the breeding performance of Sparrowhawks[END_REF][START_REF] Pietiäinen | Seasonal and individual variation in the production of offspring in the ural owl Strix uralensis[END_REF]. It is thus important to control for this variable when analysing factors influencing breeding output. We calculated laying date by backdating from nestling age, assuming an incubation period of 30 days [START_REF] Cramp | The birds of the Western Paleartic[END_REF]. Nestling age was estimated from length of the eighth primary wing feather following [START_REF] Arroyo | Breeding ecology and nest dispersion of Montagu's harrier Circus pygargus in central Spain[END_REF]. Laying date was not known for nests that failed, since most of them failed during the incubation period (65% of total nest losses in the period [2005][2006][2007] or before the first visit in the nestling stage (so chick age could not be assessed). To analyse breeding output in relation to habitat, we performed two separate (and complementary) analyses. We first evaluated whether breeding success (production of at least one fledged young from a nest) depended on habitat around the nest (at either 500 or 2000 m). This binomial response variable (coded as 1 = successful nest, 0 = unsuccessful nest) was modelled using a binomial error distribution and a logit link function, with year included as a random variable. Secondly, we evaluated whether fledged brood size (number of fledglings per successful pair, modelled using a Poisson error distribution and a log link function) depended on habitat within 500 or 2000 m, when controlling for laying date, also including year as a random variable. In semicolonial species, local density (i.e. number of breeding neighbours) may also influence breeding output because of density-dependent competition within colonies [START_REF] Arroyo | Breeding ecology and nest dispersion of Montagu's harrier Circus pygargus in central Spain[END_REF]. Hence, we also calculated number of breeding neighbours within 500 m from each nest to include this variable in the models as a covariate. We used 500 m because this approximates the maximum distance between nests of the same semi-colony [START_REF] Arroyo | Breeding ecology and nest dispersion of Montagu's harrier Circus pygargus in central Spain[END_REF][START_REF] Cornulier | Assessing the influence of environmental heterogeneity on bird spacing patterns: a case study with two raptors[END_REF][START_REF] Limiñana | Biología reproductiva del aguilucho cenizo (Circus pygargus) en el interior de la provincia de Castellón[END_REF]). Thus, initial models included laying date (for fledged brood size only), number of breeding neighbours, and the habitat variables that had a significant contribution to harrier occurrence in the HP analysis (proportion of scrub and its quadratic term, and quadratic terms of herbaceous crops and orchards, see Results). Backward-forward selection and AIC comparisons were used to identify the final models (those with lower AIC values).

Because of semi-coloniality, there could be spatial correlation in results according to the position of nests (i.e., there may be lower variance within than between colonies). In fact, the variable "colony" was almost significant in models of breeding output with only colony as explanatory variable. Hence, we also used the variable "colony" as a random variable in the GLMM, to explain breeding output in relation to habitat (although in final models, this random variable was not significant in any analyses). As specified above, semi-colonies were defined based on distances between nests, with distances between closest semi-colonies being larger than 1000 m (and thus, much larger than distances between nests within the same semi-colony).

Finally, we evaluated whether laying date or number of neighbours (assuming a normal error distribution and using an identity link function) of individual nests varied according to habitat around them. All the analyses related to breeding output were carried out using SAS 9.1.

RESULTS

Breeding occurrence and habitat

The habitat variables that best explained the occurrence of Montagu"s Harrier nests in the study area were the percentage of scrub and its quadratic term, which had each an independent contribution of more than 20% (Table 1). In addition, there was also a significant influence of the quadratic terms of percentage cover of herbaceous crops and non-irrigated orchards (Table 1). The quadratic term for scrub showed a preference for areas not entirely covered by this habitat, but with an intermediate degree of habitat diversity, as also illustrated by the significant contribution of the quadratic terms of farmland variables. Overall, cells used in at least one year had a higher proportion of scrub than non-occupied cells, and a lower proportion of both herbaceous crops and non-irrigated orchards (Fig. 2).

Additionally, the frequency of use (the probability that cells were used only one year, two or three of the years of the study, or not at all) varied quadratically with scrub cover (χ 2 3 = 18.14, p = 0.0004 for scrub cover, χ 2 3 = 16.15, p = 0.001 for scrub cover squared), and with orchard cover (χ 2 3 = 7.69, p = 0.053 for orchard cover, χ 2 3 = 9.66, p = 0.022 for orchard cover squared). It was not significantly related to any other habitat variable (all p > 0.20). Grid cells occupied all three years were more likely to have between 40 and 80% of scrub cover (Fig. 3a), and between 0.1 and 60% of orchards (Fig. 3b).

Breeding parameters and habitat

We found no relationship between breeding success and either number of neighbours or any habitat variable within 500 m or 2000 m (all p > 0.2).

When considering habitat within 500 m, fledged brood size was significantly related to laying date, number of neighbours and proportion of scrub and herbaceous crops (F 1,138 = 18.54, p < 0.0001 for laying date; F 1,78 = 2.87, p = 0.095 for number of neighbours;

F 1,33 = 3.24, p = 0.081 for scrub cover; F 1,53 = 5.15, p = 0.027 for herbaceous crops cover squared). Fledged brood size decreased with laying date, number of neighbours and scrub cover, and increased with higher availability of herbaceous crops (parameter estimates: -0.01 0.002; -0.015 0.009; -0.003 0.002 and 0.0005 0.0002 respectively).

When considering habitat within 2000 m, the only significant variables explaining fledged brood size were laying date and number of neighbours (F 1,150 = 21.78, p < 0.0001 for laying date; F 1,51 = 15.92, p = 0.0002 for number of neighbours) (parameter estimates: -0.01 0.002; and -0.03 0.01 respectively). No habitat variable was retained in the final model.

Number of breeding neighbours was significantly higher for nests placed in areas with higher scrub cover within 500 m (F 1,245 = 17.17, p < 0.0001), or with intermediate scrub cover and relatively high herbaceous crops cover within 2000m (F 1,242 = 41.43, p < 0.0001 for scrub cover, F 1,242 = 46.11 p < 0.0001 for scrub cover squared, F 1,242 = 6.43, p = 0.009 for herbaceous crops cover, Fig. 4).

No significant relationship between laying date and any habitat variable at either at 500 or 2000 m was found (all p > 0.2).

DISCUSSION

Our results show that distribution of Montagu"s Harrier nests within the occupied area in inland Castellon province is influenced by habitat, as found in other populations of the species or other raptors (see e.g. Tapia et al. 2004;[START_REF] Arroyo | Hunting habitat selection by hen harriers on moorland: implications for conservation[END_REF][START_REF] López-Iborra | Modelling the distribution of short-toed eagle (Circaetus gallicus) in semi-arid Mediterranean landscapes: identifying important explanatory variables and their implications for its conservation[END_REF]. Probability of occurrence of harrier nests was significantly related to the availability of scrub, their nesting habitat. This relationship was not linear, but quadratic: probability and regularity of occurrence in a 1 km 2 area was greatest at intermediate levels of scrub cover. Also, the areas used by harriers for breeding had intermediate values of both herbaceous crops and non-irrigated orchards at the 1 km 2 scale. A quadratic relationship between nesting habitat and probability of occupancy was also found for Hen Harriers Circus cyaneus in Scotland. These birds use heather Calluna spp. as nesting habitat, but forages primarily over a mosaic of heather and grassland, so they favour areas with both foraging and nesting habitat to breed [START_REF] Arroyo | Habitat use and range management on priority areas for hen harriers: 2004 report[END_REF]. Similar results were also found in Galicia, NW Spain, where Montagu"s Harriers also breed in natural vegetation. There, plots occupied by harriers had a greater extent of scrub (nesting habitat) than unoccupied squares, but also a higher degree of pastureland (their preferred foraging habitat; Tapia et al. 2004) [START_REF] Martínez | Hábitat de caza y nidificación del aguilucho cenizo Circus pygargus en el Parque Natural de La Mata-Torrevieja (Alicante, SE de España): Efectos de la estructura de la vegetación y de la densidad de presas[END_REF][START_REF] Guixé | Caracterització de les àrees de nidificació de l'esparver cendrós a la plana de Lleida[END_REF][START_REF] Arroyo | Estudio de la ecología poblacional del aguilucho cenizo (Circus pygargus) en Extremadura[END_REF]. Also, Montagu"s Harriers in the study area have been observed hunting in open areas with herbaceous vegetation (e.g. grasslands and cereal crops; [START_REF] Limiñana | Uso del habitat y dieta del aguilucho cenizo, C. pygargus, en el interior de la provincia de Castellón[END_REF].

This result suggests that areas with intermediate scrub cover and nearby presence of orchards or herbaceous crops are optimal, but that areas with too much or too little scrub cover are suboptimal for harrier breeding, due to a lack of foraging and nesting habitat, respectively. We could thus expect that breeding output would be higher in areas with intermediate scrub and crop/orchard cover, where harriers probably assure their nesting and foraging needs in a more profitable way.

We found no effect of any of the habitat variables considered on harrier breeding success, and similarly habitat within 2000 m of the nest had not a significant effect on fledged brood size. However, fledged brood size was positively related to percentage cover of herbaceous crops within 500 m (and, concordantly, almost significantly negatively related to the percentage cover of scrub within 500 m). It is noteworthy that only herbaceous crops, not non-irrigated orchards, had a significant effect on breeding output, suggesting that this habitat type may be better for foraging harriers, possibly because prey are more easily captured in this habitat than in orchards [START_REF] Martínez | Hábitat de caza y nidificación del aguilucho cenizo Circus pygargus en el Parque Natural de La Mata-Torrevieja (Alicante, SE de España): Efectos de la estructura de la vegetación y de la densidad de presas[END_REF].

Overall, fledged brood size was mostly influenced by laying date and number of neighbours, both of which had a negative effect on breeding output of the species. Pairs laying later had a lower fledged brood size, a pattern common in many raptor species that may be related to individual quality (e.g. [START_REF] Newton | Seasonal trend in the breeding performance of Sparrowhawks[END_REF]. However, there was no relationship between laying date and habitat, which suggests that optimal breeding sites (in terms of habitat) are not necessarily occupied before less optimal sites.

This may indicate that breeding site occupancy in the species may be better explained by the ideal free model rather than by the ideal despotic distribution [START_REF] Fretwell | On territorial behaviour and other factors influencing habitat distribution in birds, theoretical development[END_REF]. In any case, as we are only using data from three years, explanations of harrier settlement and distribution in the study area derived from these models should be taken with caution (see also [START_REF] Soutullo | Density-dependent regulation of population size in colonial breeders: Allee and buffer effects in the migratory Montagu"s Harrier[END_REF].

Fledged brood size was also related to number of breeding neighbours, with a higher number of nearby breeding neighbours resulting in lower fledged brood sizes. This may reflect local competition for food or other resources, or a higher amount of time spent in conspecific interactions, in larger or denser nest clusters [START_REF] Mougeot | Breeding biology of the Red kite Milvus milvus in Corsica[END_REF]. On the other hand, predation may also play a role in explaining this pattern, as a higher density of harriers breeding in the same scrub patch could attract more the attention of predators, which may result in lower fledged brood sizes in these areas.

However, predation rate of Montagu"s Harrier nests in the study area is very low (e.g. a maximum of 12 nests out of 80 located nest were predated in 2005, and a maximum of 7 out of 96 nests were predated in 2006), and no effect of either habitat or number of neighbours was found on nesting success, which suggests that the effect observed refers to partial brood reduction or clutch size differences. Interestingly, number of breeding neighbours was related to habitat, being highest in areas with high scrub cover within 500 m or with intermediate cover of scrub and also a high percentage of herbaceous crops cover within 2000 m. This suggests that the best habitat conditions to host large colonies are areas where scrub is very abundant at a lower scale (ca. 500 m), but intermixed with foraging areas (herbaceous crops) at a larger scale (ca. 2000 m).

Our results thus suggest that habitat influences nest distribution in this species.

However, in semi-colonial species, nest or colony location may be strongly influenced by factors like presence of conspecifics and their breeding success (Boulinier and Danchin 1997;[START_REF] Sergio | Public information and territory establishment in a loosely colonial raptor[END_REF], which may have an even stronger effect than habitat quality [START_REF] Arroyo | Conservation of the Montagu"s Harrier (Circus pygargus) in agricultural areas[END_REF]Cornulier and Bretagnolle 2005). Quality of the nesting patch (e.g. size of the scrub patch, availability of places to locate the nest or availability of nearby good feeding areas) appears to be the factor that determines the number of pairs in it (number of neighbours, thus colony size), and density within nesting patches (i.e. local competition by interference) may determine when to settle in a new patch rather than in an existing colony [START_REF] Soutullo | Density-dependent regulation of population size in colonial breeders: Allee and buffer effects in the migratory Montagu"s Harrier[END_REF]. Location of new colonies might be chosen at random among patches of similar quality, or determined by other factors, such as distance to occupied patches [START_REF] Hanski | Metapopulation Ecology[END_REF].

This means that, for this species, it may be difficult to predict the impact of small scale habitat changes on population size or distribution at a larger scale. Improvement of breeding habitat for colonial or semi-colonial species such as Montagu"s Harrier may result in higher nest numbers at the colony or semi-colony level (and even this may be cupped up because of interspecific competition), but it may not necessarily result in the creation of new colonies (or the occupancy of areas previously unoccupied). In these cases, it may be important to evaluate which other factors (e.g. past occurrence or productivity of breeding birds in that area, distance to other occupied sites, etc.) may influence the distribution and probability of occurrence [START_REF] Arroyo | Conservation of the Montagu"s Harrier (Circus pygargus) in agricultural areas[END_REF]. If protected/managed areas with good habitat are unoccupied, it may be possible to consider whether some of those parameters may be manipulated for management purposes (for example, by artificially increasing the productivity in certain areas through hacking, or by using decoys to attract them there, as has been done with the Montagu´s Harrier [START_REF] Pomarol | Releasing Montagu"s Harrier (Circus pygargus) by the method of hacking[END_REF] or other semi-colonial species such as osprey (Thibault et al. 1995).

On the other hand, our results also suggest that habitat (in particular, availability of foraging areas nearby the nest) influences breeding output. Similar results were found in Hen Harriers [START_REF] Amar | Influence of habitat on breeding performance of Hen Harriers in Orkney[END_REF]. This suggests that habitat management in areas that are occupied regularly may have an impact, increasing local density (as seen above), as well as the productivity of pairs breeding there (and thus, potentially, the likelihood of that area being occupied in subsequent years). In our study area, this habitat seems to be the herbaceous crops, being an open habitat where Montagu"s Harriers can easily catch its prey (e.g. [START_REF] Martínez | Hábitat de caza y nidificación del aguilucho cenizo Circus pygargus en el Parque Natural de La Mata-Torrevieja (Alicante, SE de España): Efectos de la estructura de la vegetación y de la densidad de presas[END_REF]. In that respect, it is important to note this habitat type is one of the most restricted in the study area (only 8% of the surface of the study area).

Thus, an increase in its availability may enhance the suitability of the area for breeding Montagu´s Harriers. However, this should ideally not be done at the expense of scrub availability, because the latter is important for harriers to choose an area to locate their nests, and important to hold high local densities (which may enhance the suitability of the area due to conspecific attraction). In the study area, the major land-use change observed in recent years is the abandonment of traditional farming practices and nonintensive agriculture for new intensive agricultural practices, mainly the transformation of herbaceous crop fields into more lucrative irrigated orchards. As well as being less well-suited to Montagu"s Harrier hunting, the latter habitat may hold a lower density of prey, due to a decrease in prey habitat suitability (especially for ground-nesting passerines) and an increased use of pesticides. Hence, for species conservation in the study area, it would be useful to develop measures to encourage farmers to stop the transformation of herbaceous crops into orchards, or even encourage them to create new herbaceous crops.

In conclusion, the results of this study suggest that conservation of semi-colonial species, like Montagu"s Harrier, should not be solely based on increasing availability of nesting habitat (or on protecting only these habitats), without taking into account social factors [START_REF] Reed | Behavioural constraints and conservation biology: conspecific attraction and recruitment[END_REF] and the importance of foraging habitats [START_REF] Sergio | Adaptive selection of foraging and nesting habitat by black kites (Milvus migrans) and its implications for conservation: a multi-scale approach[END_REF][START_REF] Amar | Influence of habitat on breeding performance of Hen Harriers in Orkney[END_REF][START_REF] Arroyo | Hunting habitat selection by hen harriers on moorland: implications for conservation[END_REF]. Habitat improvement may result in higher local densities and breeding success, but local actions aimed at preserving or enhancing nesting habitat in irregularly occupied areas for semi-colonial species may result in an inefficient investment of available conservation resources for these species. Indeed, habitat manipulation may be inefficient for changing species distribution (and thus, potentially, total breeding numbers at a regional scale). Thus, having a complete framework for a target species, including breeding habitat (both nesting and foraging), social factors and relationships between populations of the species would improve the effectiveness of conservation effort and investment. Also, protecting several core areas may be efficient for conservation of semi-colonial species [START_REF] Sergio | Adaptive selection of foraging and nesting habitat by black kites (Milvus migrans) and its implications for conservation: a multi-scale approach[END_REF][START_REF] Poirazidis | Modelling nesting habitat as a conservation tool for the Eurasian black vulture (Aegypius monachus) in Dadia Nature Reserve, northeastern Greece[END_REF]

Fig. 2 .

 2 Fig. 2. Mean ( SE) percentage of different habitats in 1 km 2 grid cells occupied (n = 53) and unoccupied (n = 985) by breeding harriers in the study area.

Fig. 3 .

 3 Fig. 3. Mean ( SE) frequency of occupancy (i.e., 0, 1, 2 or 3 years) of 1 km 2 grid cells in the period 2005 -2007, according to scrub coverage (above) and orchard cover (below).

Fig. 4 .

 4 Fig. 4. Mean ( SE) number of neighbours within 500 m in relation to scrub cover within 500 m of the nest (above) or within scrub cover and herbaceous crops within 2000 m of the nest (below).

  . Our results thus indicate that Montagu"s Harriers in Castellon prefer areas with heterogeneous land uses, where both scrub (nesting habitat) and farmland occur, and suggest that harriers probably use these non-irrigated orchards and herbaceous crops for foraging in the study area. Indeed, Montagu"s Harriers in other parts of Spain often hunt in open areas with herbaceous vegetation, including grasslands and arable fields, as well as open orchards

Table 1 .

 1 , especially if such areas are regularly occupied, hold a large number of breeding pairs or are more productive. Tapia L, Domínguez J, Rodríguez L (2004) Modeling habitat use and distribution of Hen harriers (Circus cyaneus) and Montagu's harrier (Circus pygargus) in a mountainous area in Galicia, northwestern Spain. J Raptor Res 38:133-140. Wilson MW, Irwin S, Norriss DW, Newton SF, Collins K, Kelly TC, O"Halloran J (2009) The importance of pre-thicket conifer plantations for nesting Hen Harriers Circus cyaneus in Ireland. Ibis 151:332-343.TABLES Results of hierarchical partitioning analysis performed to assess the importance of habitat on the occurrence of Montagu"s Harrier nests in 1 km 2 scale in inland Castellon province (E Spain). I and J represent the independent and joint contribution of each habitat variable respectively. %I is the percentage of the total I accounted for each habitat variable. z-score is the randomization test for the independent contributions of each habitat variable calculated from 999 randomizations. *p<0.05. Location of the study area (grey polygon) and overall distribution of nests.

	Thibault JC, Bretagnolle V, Dominici JM (1995) Recovery of a resident population of
	osprey on Corsica. J Raptor Res 29:204-207
	Walsh C, MacNally R (2003) Hierarchical partitioning. R Project for Statistical
	Computing. http://cran.r-project.org/

ACKNOWLEDGEMENTS

We are thankful to Aeropuerto de Castellon for support and funding. A part of the data presented in this work comes from the monitoring and conservation programme for Montagu"s Harrier in Castellon, and have been provided by D.G. de Gestión del Medio Natural (Consellería de Medio Ambiente, Agua, Urbanismo y Vivienda, Generalitat Valenciana). We are particularly thankful to Juan Jiménez and also to Sara Ferreras and Teresa de Chiclana for helping in the fieldwork. Finally, we are indebted to C.

Trierweiler, Pascual López-López, Mark Wilson and two anonymous referees, whose comments improved a previous version of the manuscript. First author had a grant from the Generalitat Valenciana. This paper was part of his PhD thesis at the Universidad de Alicante. All the research done for this study complies with the current laws of Spain.


Rita Hargitai
email: harrita@freemail.hu

Rafael Mateo

János Török

Shell thickness and pore density in relation to shell colouration, female characteristics, and environmental factors in the collared flycatcher Ficedula albicollis

Keywords: biliverdin, eggshell pigmentation, eggshell porosity, eggshell structure, eggshell thickness, Ficedula albicollis Zusammenfassung Schalendicke und Porendichte in Bezug zu Schalenfärbung, Weibchenmerkmale und Umweltfaktoren beim Halsbandschnäpper Ficedula albicollis Die Struktur der Eischale von Vögeln kann wichtige Folgen für das Wachstum und die Entwicklung der Embryonen haben, doch über die Faktoren, die für Variation in den Eigenschaften der Eischale bei . Wir haben analysiert, ob die Eischalenfarbe von der Schalenstruktur oder dem Pigmentgehalt beeinflusst wird und ob Weibchenmerkmale, Legedatum, lokale Brutpaardichte und Gelegegröße die Schalendicke und Porendichte beeinflussen. Wir fanden, dass blaugrüne und ultraviolette Farben der Eischale nicht mit der Schalendicke, Porendichte und Eigröße zusammenhingen. Eier mit einer höheren Biliverdinkonzentration zeigten eine geringere UV-Reflexion und stärkere Reflexion im blaugrünen Bereich des Spektrums. Wir fanden, dass Weibchen in besserem Ernährungszustand, angezeigt durch ihre höhere Körpermasse korrigiert für Tarsuslänge, Eier mit dickerer Schale legten. Es ist möglich, dass Weibchen in besserer Kondition mehr Zeit haben, kalziumreiche Nahrung zu suchen, und daher Eier mit stärkerer Schale produzieren. Die keiner signifikanten Beziehung zu der

HAL is

Introduction

Avian eggs have hard eggshells, which are composed of 95% calcite, one of the crystalline forms of calcium carbonate, and 3.5% proteins, proteoglycans and glycoproteins [START_REF] Nys | Avian eggshell mineralization[END_REF]. The main function of the shell is to provide mechanical protection for the developing embryo [START_REF] Ar | The avian egg: mass and strength[END_REF]). In addition, embryos use the shell as a source of calcium for the ossification of the developing skeleton [START_REF] Packard | Comparative aspects of calcium metabolism in embryonic reptiles and birds[END_REF]. During egg laying, female birds require large amounts of calcium-rich material, such as mollusc shells, woodlice and millipedes [START_REF] Bureš | Sources and timing of calcium intake during reproduction in flycatchers[END_REF], as their normal food usually does not contain sufficient calcium for eggshell formation [START_REF] Graveland | Arthropods and seeds are not sufficient as calcium sources for shell formation and skeletal growth in passerines[END_REF][START_REF] Graveland | Timing of the calcium intake and effect of calcium deficiency on behaviour and egg laying in captive Great tits Parus major[END_REF][START_REF] Bureš | Sources and timing of calcium intake during reproduction in flycatchers[END_REF]. A shortage of calcium during egg formation could result in thinnershelled eggs, and thus poor hatchability due to excessive water loss or shell breakage (Graveland et al. 1994, Eeva and[START_REF] Eeva | Egg shell quality, clutch size and hatching success of the great tit (Parus major) and the pied flycatcher (Ficedula hyoleuca) in an air pollution gradient[END_REF].

To provide the necessary communication between the developing embryo and the external environment, the shell is perforated by microscopic pores [START_REF] Ar | Pores in avian eggshells: gas conductance, gas exchange and embryonic growth rate[END_REF]. Exchange of respiratory gases and water vapour is affected by the porosity of the shell and the partial pressure gradients of the gases across the shell [START_REF] Ar | The avian egg: water vapor conductance, shell thickness, and functional pore area[END_REF][START_REF] Paganelli | The physics of gas exchange across the avian eggshell[END_REF][START_REF] Rahn | Role of diffusion in gas exchange of the avian egg[END_REF]. In addition, the rate of water loss through the eggshell is negatively affected by eggshell thickness, as it lengthens the pathway for water vapour [START_REF] Ar | Pores in avian eggshells: gas conductance, gas exchange and embryonic growth rate[END_REF]. The oxygen availability and thus the growth rate of the embryo may be limited by the pore number of the shell [START_REF] Burton | A comparison of the effects of eggshell porosity on the respiration and growth of domestic fowl, duck and turkey embryos[END_REF]Tullett 1983, Zimmermann and[START_REF] Zimmermann | Egg size, eggshell porosity, and incubation period in the marine bird family Alcidae[END_REF].

In many bird species, the cuticle and the calcified layer of the eggshell are pigmented, often with complex patterns. Blue-green eggshell colouration results from the pigment biliverdin, which typically permeates the whole eggshell, while red-brown spots or patches are the result of protoporphyrin [START_REF] Kennedy | A survey of avian eggshell pigments[END_REF]Vevers 1976, Mikšík et al. 1996). Avian eggshell colouration has traditionally been interpreted as a response to selection pressures imposed by nest predators and brood parasites, although other functions, e.g. filtering solar radiation, strengthening the eggshell, or serving as a sexually selected signal of female quality, have also been proposed [START_REF] Underwood | Adaptive significance of egg coloration[END_REF][START_REF] Moreno | Avian egg colour and sexual selection: does eggshell pigmentation reflect female condition and genetic quality?[END_REF][START_REF] Gosler | Why are birds' eggs speckled?[END_REF][START_REF] Higham | Speckled eggs: water-loss and incubation behavior in the great tit Parus major[END_REF][START_REF] Kilner | The evolution of egg colour and patterning in birds[END_REF]). In addition to human-visible light, most passerines can also detect near ultraviolet (UV-A) light (Cuthill et al. 2000, Ödeen and[START_REF] Ödeen | Complex distribution of avian color vision systems revealed by sequencing the SWS1 opsin from total DNA[END_REF], thus, UV reflection may also be an important aspect of egg colouration [START_REF] Cherry | Egg colour matching in an African cucko, as revealed by ultraviolet-visible reflectance spectrophotometry[END_REF]Bennett 2001, Avilés et al. 2006).

Ultraviolet colour is produced by light scattering and it may provide information on the nanostructure of the tissue [START_REF] Prum | Two-dimensional Fourier analysis of the spongy medullary keratin of structurally coloured feather barbs[END_REF][START_REF] Prum | Structural colouration of avian skin: convergent evolution of coherently scattering dermal collagen arrays[END_REF][START_REF] Shawkey | Nanostructure predicts intraspecific variation in ultraviolet-blue plumage colour[END_REF]. The basis of eggshell UV-colour has not been determined yet; it could be affected by the structure of the calcified layer and/or by the pigment content of the shell.

In this study, we explored potential causes of variation in eggshell colour (blue-green and UV chroma) and structure (shell thickness and porosity) in a wild passerine, the collared flycatcher (Ficedula albicollis). In a previous study, we have shown that the average blue-green colour of a clutch was unrelated to female morphological and serological characteristics and increased with laying date in the collared flycatcher [START_REF] Hargitai | Eggshell colouration in relation to female condition, male attractiveness and egg quality in the collared flycatcher (Ficedula albicollis)[END_REF]). Here, one of our aims was to determine if the blue-green and UV reflectances of the eggshell are affected by the pigment concentration and the structure of the shell. We predicted that biliverdin pigment concentration in the eggshell would be positively related to blue-green chroma, as demonstrated in earlier studies [START_REF] Moreno | Experimental evidence that egg color indicates female condition at egg laying in a songbird[END_REF][START_REF] López-Rull | Egg pigmentation reflects female and egg quality in the spotless straling Sturnus unicolor[END_REF]. In contrast, as biliverdin absorb light in the UV part of the spectrum [START_REF] Falchuk | A role for biliverdin IX alpha in dorsal axis development of Xenopus laevis embryos[END_REF], we expected that eggs with more pigmentation would show less reflectance in the UV region, which hypothesis has not been tested before to our knowledge. Also, we expected that eggs with thicker shells and less pore channels would contain more pigments and thus be more colourful. Alternatively, biliverdin pigment may strengthen the shell similarly to protoporphyrin [START_REF] Gosler | Why are birds' eggs speckled?[END_REF][START_REF] Jagannath | Eggshell pigmentation indicates pesticide contamination[END_REF][START_REF] Sanz | Eggshell pigmentatioin pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF], therefore, thinner-shelled eggs with a larger number of pores may show more intense blue-green colour. Until now, the relationship between blue-green eggshell colour and shell thickness has been tested only in a few species, where no associations were detected [START_REF] Jagannath | Eggshell pigmentation indicates pesticide contamination[END_REF][START_REF] López-Rull | Egg pigmentation reflects female and egg quality in the spotless straling Sturnus unicolor[END_REF], Hargitai et al. 2010).

Although eggshell thickness and porosity may have important consequences for embryonic growth and development, relatively little is known about the factors responsible for variation in these eggshell characteristics of wild birds. We tested if eggshell thickness and shell porosity are influenced by morphological and serological characteristics of the mother, such as body mass, body size, and stress level, and by local breeding density, laying date, and clutch size. As females seek dietary calcium sources during egg formation [START_REF] Bureš | Sources and timing of calcium intake during reproduction in flycatchers[END_REF], we expected that females in better nutritional condition, indicated by their higher mass controlled for tarsus length, would lay eggs with stronger shells, as they may be better foragers of calciumrich materials too. In additon, we hypothesized that females with a higher level of physiological stress would produce eggs with thinner shells. As far as we are aware, these hypotheses have not been tested before for wild passerines.

Breeding density may be related to eggshell thickness through affecting the level of food competition between females [START_REF] Alatalo | Density-dependence in breeding success of the pied flycatcher (Ficedula hypoleuca)[END_REF][START_REF] Török | Density dependence in reproduction of the collared flycatcher (Ficedula albicollis) at high population levels[END_REF][START_REF] Hargitai | Egg composition in relation to social environment and maternal physiological condition in the collared flycatcher[END_REF], and thus having a negative effect on shell thickness, however, this hypothesis has not been tested before for wild songbirds. Alternatively, high density areas may be of higher quality with more resources [START_REF] Doliguez | Availability and use of public information and conspecific density for settlement decisions in the collared flycatcher[END_REF], where individuals of better quality may be able to settle. In this case, we may expect females to produce eggs with stronger shells in an area of higher density. Nevertheless, we suppose that this hypothesis is less likely in our case, as forest soil, vegetation structure and age of oak trees were homogenous in the study area. We also expected that eggshells of eggs laid later in the breeding season may be thinner as late-breeding females may be lower-quality individuals (Garamszegi et al. 2004a, Verhulst and[START_REF] Verhulst | The timing of birds' breeding season: a review of experiments that manipulated timing of breeding[END_REF], which may not be able to acquire sufficient amount of calcium to produce strong-shelled eggs. Eggshell thickness may also vary during the breeding season due to seasonal variation in the availability of calcium-rich resources. Seasonal decline in eggshell thickness has been found in a study of the blue tit (Cyanistes caeruleus; [START_REF] Dauwe | The effect of heavy metal exposure on egg size, eggshell thickness and the number of spermatozoa in blue tit Parus caeruleus eggs[END_REF]), but other studies found no significant variation in eggshell thickness with laying date [START_REF] Eeva | Egg shell quality, clutch size and hatching success of the great tit (Parus major) and the pied flycatcher (Ficedula hyoleuca) in an air pollution gradient[END_REF][START_REF] Mänd | Increased eggshell porosity in replacement clutches of the black-headed gull Larus ridibundus[END_REF], Massaro and Davis 2004).

The seasonal variation in eggshell thickness and pore density could also be explained by some adaptive hypotheses. Later breeding has been shown to result in lower reproductive success in small insectivorous passerines as a result of declining food availability [START_REF] Norris | Seasonal variation in the reproductive success of Blue Tits: an experimental study[END_REF][START_REF] Wiggins | Seasonal decline in collared flycatcher Ficedula albicollis reproductive success: an experimental approach[END_REF]. In several bird species, there is a seasonal decline in incubation length [START_REF] Eichholz | Factors affecting duration of incubation in Black Brant[END_REF][START_REF] Hipfner | Seasonal declines in incubation periods of Brünnich's Guillemots Uria lomvia: testing proximate causes[END_REF], Massaro et al. 2004), which could reflect adjustments by late-laying parents to improve the survival prospects of their offspring. [START_REF] Hipfner | Seasonal declines in incubation periods of Brünnich's Guillemots Uria lomvia: testing proximate causes[END_REF] hypothesized that later-laid eggs might have higher porosity, which may allow embryos to develop more rapidly as a consequence of a higher gas exchange rate. In addition, later-breeding females may produce eggs with thinner shells as an adaptation to allow embryos to develop more rapidly and emerge faster during hatching (Massaro and Davis 2004). So far, these hypotheses have only been tested for non-passerine birds [START_REF] Mänd | Increased eggshell porosity in replacement clutches of the black-headed gull Larus ridibundus[END_REF], Massaro and Davis 2004[START_REF] Boersma | Magellanic Penguin eggshell pores: does number matter?[END_REF], although accelerated embryonic development of later-laid clutches would be beneficial for insectivorous songbirds too.

Females of small passerines have to consume enough calcium each day for egg formation during the laying period (Graveland andBerends 1997, Pahl et al. 1997), so we might not expect a decline in shell thickness with increasing clutch size, or there may even be a positive relationship between clutch size and shell thickness as a result of the effect of female quality [START_REF] Eeva | Egg shell quality, clutch size and hatching success of the great tit (Parus major) and the pied flycatcher (Ficedula hyoleuca) in an air pollution gradient[END_REF]. However, females laying more eggs might reduce pore number per egg so that nest humidity remains constant in clutches of different egg numbers. Also, we expected that egg size would be positively related to shell thickness, but negatively to pore density in line with previous findings [START_REF] Tullett | Determinants of avian eggshell porosity[END_REF][START_REF] Rahn | Shell mass, thickness and density of avian eggs derived from the tables of Schonwetter[END_REF][START_REF] Eeva | Egg shell quality, clutch size and hatching success of the great tit (Parus major) and the pied flycatcher (Ficedula hyoleuca) in an air pollution gradient[END_REF]. Moreover, eggs with thicker shells may have more pores so as to compensate for the greater length of each pore channel.

Methods

Study species and field procedures

The study was carried out on a population of collared flycatchers breeding in a sessile oak (Quercus petraea) dominated woodland in the Pilis Mountains (47° 43′N, 19° 01′E), Hungary, in 2005. The study area was a nestbox plot of approximately 17.2 ha, where altogether 98 collared flycatcher pairs bred in the given year. The woodland has clayed brown forest soil on volcanic stones (pH = 5-6). The collared flycatcher is a long-distance migratory, insectivorous passerine. This species is predominantly monogamous (Garamszegi et al. 2004b[START_REF] Herényi | Lifetime offspring production in relation to attractiveness, breeding lifespan and mating status in male collared flycatchers[END_REF], and females usually lay five to seven eggs of one clutch per breeding season [START_REF] Török | Unpredictable food supply modifies costs of reproduction and hampers individual optimization[END_REF]). Collared flycatcher eggs appear light blue-green in the human visible spectrum, and have a peak of reflectance at 490 nm at the visible spectrum, and at 320 nm at UV wavelengths [START_REF] Hargitai | Eggshell colouration in relation to female condition, male attractiveness and egg quality in the collared flycatcher (Ficedula albicollis)[END_REF]. Females were captured in the nestbox during incubation, weighed with a Pesola spring balance (to the nearest 0.1 g), and the length of the tarsus was measured with a calliper (to the nearest 0.1 mm).

Eggs were marked according to laying order with a waterproof marker, and two eggs (third and fourth) per clutch were collected on the day of laying from 24 clutches. For ethical reasons and due to legal restrictions, no whole clutches were collected in this study. Egg length and breadth were measured with a calliper (to the nearest 0.1 mm). Egg size was estimated from Hoyt's formula [START_REF] Hoyt | Practical methods of estimating volume and fresh weight of bird eggs[END_REF]; size = length × breadth 2 × 0.51).

Female collared flycatchers defend a small breeding territory around their nestbox against conspecific females, particularly during the nest-building and egg-laying periods [START_REF] Král | Intrasexual aggression of female collared flycatchers (Ficedula albicollis): competition for male parental care?[END_REF] similarly to their sibling species, the pied flycatcher (Ficedula hypoleuca; [START_REF] Slagsvold | Female-female aggression explains polyterritoriality in male pied flycatchers[END_REF][START_REF] Rätti | Characteristics and level of aggression by female pied flycatchers at different distances from the nest hole[END_REF]. However, the foraging areas of flycatchers often overlap with those of neighbouring pairs [START_REF] Von Haartman | Territory in the pied flycatcher Muscicapa hypoleuca[END_REF]. Previously, it has been shown that egg yolk mass was negatively related to the number of neighbouring pairs in the collared flycatcher [START_REF] Hargitai | Egg composition in relation to social environment and maternal physiological condition in the collared flycatcher[END_REF], which could indicate that high local breeding density could limit food availability in this species. We calculated a local breeding density index by summing up the number of breeding collared flycatcher pairs in neighbouring (i.e. adjacent) nestboxes that started egg laying until the laying of the fifth egg of the clutch in question. As nestboxes were placed in a grid system, each nestbox, except for those located at the edge of the study plot, had eight nestboxes adjacent to it. Distance between nestboxes was approximately 30-50 m.

Serological analysis

A blood sample was taken from the female's brachial vein during incubation. A drop of blood was smeared on a microscope slide, air dried, fixed in absolute methanol, and later stained with Giemsa's solution. The proportion of different types of leucocytes was assessed by examining a total of 50 leucocytes under ×1000 magnification with oil immersion. An index of the relative abundance of heterophil granulocytes to lymphocytes (H/L ratio) was used to assess the stress level of the females [START_REF] Gross | Evaluation of the heterophil/lymphocyte ratio as a measure of stress in chickens[END_REF]Siegel 1983, Maxwell 1993).

Egg colour measurement

Egg colour was measured on the day of laying using a portable battery-driven USB2000 spectrometer with a Mini DT deuterium-halogen light source (Ocean Optics Europe, Duiven, The Netherlands). The reflectance spectrum was determined between 320 and 700 nm at 0.38 nm intervals. Inclusion of ultraviolet (UV) is necessary as most bird species are sensitive to near UV light [START_REF] Cuthill | Ultraviolet vision in birds[END_REF]. Eggs were placed with their side in the hole of a brick-formed aluminium reflection probe holder (RPH-1, Ocean Optics Europe), so that eggs completely filled the hole. A bifurcated 400-μm fibre optic probe was used, and the sampling optic was held perpendicular and at a distance of 3 mm from the eggshell surface. Three measurements were taken from each egg by slightly changing the position of the egg in the hole. Reference calibration was performed with a WS-1 white standard after every second egg. Mean reflectance was calculated by averaging the three measurements. The spectral curves were generated by using the OOIBase32 software with a 3,500 ms integration time. The reflectance spectrum was double peaked with a prominent peak around 400-580 nm (blue-green), and around 320 nm (UV). Blue-green chroma (R 400-580 /R 320-700 ) and UV chroma (R 320-400 /R 320-700 ) were calculated as the proportion of the reflectance occurring in the given part of the spectrum, corresponding to purity or saturation. Variations in eggshell colour variables were higher among eggs than within eggs (blue-green chroma: r = 0.90, F 47,96 = 28.10, P < 0.001, UV chroma: r = 0.85, F 47,96 = 17.69, P < 0.001; [START_REF] Lessells | Unrepeatable repeatabilities: a common mistake[END_REF].

Eggshell thickness and pore density Pieces of eggshell were taken from the equatorial region of two eggs (third and fourth) per clutch. Shell thickness was measured with a Mitutoyo Digimatic Micrometer (395-271) with a ball-point end at a precision of 0.001 mm at four places of the shell. Variation in eggshell thickness was greater among eggs than within eggs (r = 0.68, F 46,139 = 9.48, P < 0.001), indicating that these measurements were repeatable [START_REF] Lessells | Unrepeatable repeatabilities: a common mistake[END_REF]. We used the mean of the four measurements as an average eggshell thickness of an egg.

Organic shell membranes were removed by immersing shells in household bleach (5.4% aquous sodium hypochlorite) for 1-2 min. Eggshells were then rinsed in tap water, drained and allowed to dry. The inner surface of shell fragments was painted with 1% aqueous Evans blue.

Capillary forces filled the pore channels rendering them visible as fine blue dots on the external surface of the shell. A paper mat with an exposed area of 25 mm 2 was placed over the shell.

Pores were counted in two such areas using a stereomicroscope with a magnification of 40x. Six shell samples were too small, and only one area was used for pore density measurement. Variation in pore density was higher among eggs than within eggs (r = 0.41, F 40,41 = 2.41, P = 0.003), showing that these measurements were repeatable [START_REF] Lessells | Unrepeatable repeatabilities: a common mistake[END_REF].

Biliverdin analysis

Biliverdin analysis was based on the method described by [START_REF] Mateo | Determination of porphyrins and biliverdin in bile and excreta of birds by a single liquid chromatography-ultraviolet detection analysis[END_REF] with some modifications. Biliverdin was extracted from 7-19 mg eggshell pieces from the equatorial region of one egg (fourth) per clutch by adding 315 μl acetonitrile and 263 μl HCl 3N. After 15 min of the addition of the reagents, the tubes were capped and subsequently vortexed for 15 s and sonicated for 10 s in an Ultrasons-H. Samples were centrifuged for 10 min at 12000 rpm and the supernatant, containing the pigments, was transferred to glass vials for later high pressure liquid chromatography (HPLC) analysis. Extraction was repeated twice by adding 400 μl acetonitrile to the samples. The acetonitrile phases were combined and HPLC analysis was conducted with an Agilent 1100 system with a Waters Spherisorb 5 μm ODS2 (4.6 mm × 100 mm) column at a 1.5 ml/min flow rate. The initial mobile phase composition was ammoniumacetate (1 M, pH: 5.16) 75% and methanol 25% for 2 min after sample injection, and then a gradient was applied with a linear change in 8 min to 95% methanol and 5% ammonium acetate, followed by 2 min at these conditions. At this moment, the phase composition returned to the initial conditions in 3 min, and was then stabilized for another 3 min before the next sample injection. The column was maintained at 70˚ C and the detection was performed at 400 nm wavelength. The quantification of samples was performed using calibration curves of biliverdin standard added to white eggshells of domestic hen [START_REF] Moreno | Experimental evidence that egg color indicates female condition at egg laying in a songbird[END_REF].

Concentration of biliverdin was expressed as nmol/g of dry weight of eggshell.

Statistical analyses

Data were analysed with Pearson's correlation and general linear models. Separate analyses were performed with female characteristics (body mass, tarsus length, and H/L ratio; Table 1) and environmental factors (laying date and local breeding density; Table 1) as the independent variables with pore density and shell thickness as the dependent variable. Eggshell thickness, pore density, egg size, and biliverdin concentration were entered together as explanatory variables in separate models with blue-green chroma and UV chroma as the dependent variable.

In the models involving biliverdin concentration, data of the fourth egg of each clutch were used; in all other cases, the mean values of the third and fourth eggs per clutch were entered into the models. In all models, a stepwise analysis based on backward deletion procedure was employed, removing nonsignificant (P > 0.05) effects from the model one by one in decreasing order of P. To avoid nonsignificance due to overparameterization, we re-entered nonsignificant effects to the final model one by one, and present these F-and P-values. We also report standardized regression coefficients (beta) as effect size estimates and the 95% confidence intervals around those measures to facilitate the interpretation of the results [START_REF] Nakagawa | Effect size, confidence interval and statistical significance: a practical guide for biologists[END_REF]. Log-transformed biliverdin concentration and H/L ratio were used in order to obtain normal distribution of data. Analyses were performed by STATISTICA, version 5.5 (StatSoft Inc.).

Results

Our results revealed that blue-green eggshell colour was unrelated to shell thickness, pore density and egg size (Table 2; for egg size see also [START_REF] Hargitai | Eggshell colouration in relation to female condition, male attractiveness and egg quality in the collared flycatcher (Ficedula albicollis)[END_REF]), but showed a positive relationship with biliverdin concentration in the shell (Table 2; Fig. 1). We also tested if UV reflection of the shell was influenced by shell structure or biliverdin pigment concentration.

Similarly, we found that shell thickness, pore density and egg size had no significant associations with UV chroma (Table 2), but there was a negative correlation between biliverdin concentration and UV chroma (Table 2; Fig. 2). Thus, eggs with higher concentration of biliverdin pigment showed lower UV reflection and higher reflection in the blue-green part of the spectrum. Moreover, we found no significant correlation between the biliverdin concentration of the eggshell and shell thickness (r = -0.01, n = 23, P = 0.96) or pore density (r = -0.15, n = 23, P = 0.49).

Concerning female morphological and serological characteristics, female H/L ratio and tarsus length did not influence shell thickness (Table 3), but we found that females of higher body mass laid eggs with thicker shells (Table 3; Fig. 3). We also tested the effect of female condition by using female mass as an independent variable and tarsus length as a covariate; the effect of female condition was also significant (F 1,18 = 4.69, P = 0.044, beta = 0.45, Cnf. Lmt. = 0.01 and 0.89). Female body mass, tarsus length and H/L ratio had no significant relationships with shell porosity (Table 3). Moreover, we analysed if environmental factors influenced the thickness and pore density of the eggshell, but neither laying date nor local breeding density was related to eggshell characteristics (Table 3). We found that eggshell thickness was unrelated to clutch size (r = -0.07, n = 23, P = 0.74). However, pore density showed a negative relationship with the number of eggs within a clutch (r = -0.57, n = 22, P = 0.005), that is, eggs in clutches of 7 eggs had less pores than eggs in clutches of 5 eggs. As clutch size declined with laying date (F 1,21 = 6.18, P = 0.021, beta = -0.48, Cnf. Lmt. = -0.88 and -0.08), we examined whether the negative relationship between pore density and clutch size could be accounted for the confounding effect of laying date. However, we found that the effect of laying date on pore density was nonsignificant, while the effect of clutch size remained significant (laying date: F 1,19 = 0.41, P = 0.53, beta = -0.14, Cnf. Lmt. = -0.58 and 0.31; clutch size: F 1,19 = 9.10, P = 0.007, beta = -0.64, Cnf. Lmt. = -1.09 and -0.20).

Our results showed that eggshell thickness was unrelated to shell porosity (r = -0.05, n = 23, P = 0.83), but there was a trend for larger eggs to have thicker shells, although it was nonsignificant (r = 0.36, n = 24, P = 0.081). However, we detected no significant association between egg size and the porosity of the eggshell (r = 0.15, n = 23, P = 0.51).

Discussion

Our study showed that eggshell biliverdin concentration was positively related to blue-green chroma, in line with the findings of earlier studies [START_REF] Moreno | Experimental evidence that egg color indicates female condition at egg laying in a songbird[END_REF][START_REF] López-Rull | Egg pigmentation reflects female and egg quality in the spotless straling Sturnus unicolor[END_REF]).

In addition, eggshells containing higher concentration of biliverdin pigment showed less intense UV reflection. However, neither shell thickness nor shell porosity was related to eggshell colouration or the biliverdin pigment concentration of the shell. In concert with our results, blue-green eggshell colouration was unrelated to shell thickness in a study of the sparrowhawk (Accipiter nisus; [START_REF] Jagannath | Eggshell pigmentation indicates pesticide contamination[END_REF], the spotless starling (Sturnus unicolor; [START_REF] López-Rull | Egg pigmentation reflects female and egg quality in the spotless straling Sturnus unicolor[END_REF], the cuckoo (Cuculus canorus; [START_REF] Hargitai | Eggshell characteristics and yolk composition in the common cuckoo Cuculus canorus: are they adapted to brood parasitism?[END_REF]) and the great reed warbler (Acrocephalus arundinaceus; [START_REF] Hargitai | Eggshell characteristics and yolk composition in the common cuckoo Cuculus canorus: are they adapted to brood parasitism?[END_REF]. In several studies, the brown-red eggshell pigment protoporphyrin was shown to be associated to shell thickness [START_REF] Gosler | Why are birds' eggs speckled?[END_REF][START_REF] Jagannath | Eggshell pigmentation indicates pesticide contamination[END_REF][START_REF] Sanz | Eggshell pigmentatioin pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF], suggesting that protoporphyrin might have a structural function in strengthening the eggshell [START_REF] Solomon | Egg and eggshell quality[END_REF][START_REF] Gosler | Why are birds' eggs speckled?[END_REF].

However, despite the similar molecular structure of the two pigment types [START_REF] Lang | A review of eggshell pigmentation[END_REF]), it appears that biliverdin pigmentation of the eggshell does not have such a function.

Our results suggest that UV reflectance of the shell is generated by the structure of the calcified layer, as biliverdin pigmentation decreased UV chroma. This hypothesis is also supported by Sieffermann et al. (2006), who found that white eggs also reflected light in the UV range. Egg pigments appear to decrease the intensity of UV reflectance by absorbing light in these wavelengths. However, we could not detect any associations between UV chroma and shell thickness or pore density, suggesting that nanostructural characteristics of the shell and shell pigmentation may be responsible for the UV reflectance, and shell thickness and porosity do not influence it.

Besides proteins and lipids, laying birds need to consume large quantities of calcium-rich material in order to possess sufficient calcium for egg formation [START_REF] Simkiss | Calcium in reproductive physiology[END_REF]. Acquiring a sufficient amount of calcium is time-consuming even in a calcium-rich environment, thus, there may exist a trade-off between feeding time and time spent searching for calcium [START_REF] Graveland | Timing of the calcium intake and effect of calcium deficiency on behaviour and egg laying in captive Great tits Parus major[END_REF]. We found that females in better nutritional condition produced eggs with thicker shells, although body size and stress level showed no significant relationships with shell thickness. It is possible that females in better condition have more time available for searching calcium-rich food items and thus could produce eggs with stronger shells. Moreover, these birds may be better foragers of both nutrients and calcium, as egg size, which is also related to female condition [START_REF] Hargitai | Effects of environmental conditions and parental quality on the inter-and intraclutch egg size variation in the collared flycatcher (Ficedula albicollis)[END_REF], and shell thickness showed a weak, almost significant positive relationship, which might be significant with a larger sample size. The positive relationship between shell thickness and egg size has also been shown in two other songbirds, the great tit (Parus major) and the pied flycatcher [START_REF] Eeva | Egg shell quality, clutch size and hatching success of the great tit (Parus major) and the pied flycatcher (Ficedula hyoleuca) in an air pollution gradient[END_REF]. In the peregrine falcon (Falco peregrinus peregrinus), it was also reported that heavier females laid clutches of larger eggs with thicker shells, although in other falcon species this relationship was not significant [START_REF] Castilla | Determinants of eggshell strength in endangered raptors[END_REF]. Experimental studies also found that calcium supplemented birds laid thicker-shelled and also larger eggs, probably as the decrease in searching time for calcium might incur a higher intake of nutrients or a lower energy expenditure [START_REF] Tilgar | Effect of calcium availability and habitat quality on reproduction in pied flycatcher Ficedula hypoleuca and great tit Parus major[END_REF][START_REF] Mänd | Does supplementary calcium reduce the cost of reproduction in the pied flycatcher Ficedula hypoleuca?[END_REF].

Contrary to our expectation, we found no evidence in collared flycatchers that eggshell thickness was related to laying date or local breeding density. This suggests that the availability of calcium-rich materials does not vary significantly during the laying period, and it is not affected by local competition for calcium-rich materials. However, the laying date interval of the analysed clutches (Table 1) might have been too short to detect such differences (see also later), therefore, we must treat our results with caution. Nevertheless, our results are in concordance with previous findings. Eggshell thickness did not vary with laying date in great tits [START_REF] Eeva | Egg shell quality, clutch size and hatching success of the great tit (Parus major) and the pied flycatcher (Ficedula hyoleuca) in an air pollution gradient[END_REF], pied flycatchers [START_REF] Eeva | Egg shell quality, clutch size and hatching success of the great tit (Parus major) and the pied flycatcher (Ficedula hyoleuca) in an air pollution gradient[END_REF], black-headed gulls (Larus ridibundus; Mänd 1996) and yellow-eyed penguins either (Megadyptes antipodes; Massaro and Davis 2004).

Apart from the effect of physiological constraints, eggshell structure may also vary seasonally due to the strategic adjustment of the female. Since later laying has often been associated with lower reproductive success as a result of declining food availability during the period of parental care [START_REF] Norris | Seasonal variation in the reproductive success of Blue Tits: an experimental study[END_REF][START_REF] Wiggins | Seasonal decline in collared flycatcher Ficedula albicollis reproductive success: an experimental approach[END_REF], there may be a reproductive advantage for females breeding later in the season to reduce incubation time. It has been shown in numerous studies that total pore area of the eggshell affects water vapour conductance, and both of them are positively related to the growth rate of embryos and hatchability of eggs [START_REF] Burton | A comparison of the effects of eggshell porosity on the respiration and growth of domestic fowl, duck and turkey embryos[END_REF][START_REF] Ar | Pores in avian eggshells: gas conductance, gas exchange and embryonic growth rate[END_REF][START_REF] Soliman | Relationship between shell porosity, shell thickness, egg weight loss, and embryonic development in Japanese quail eggs[END_REF][START_REF] Massaro | Differences in egg size, shell thickness, pore density, pore diameter and water vapour conductance between first and second eggs of Snares Penguins Eudyptes robustus and their influence on hatching asynchrony[END_REF][START_REF] Zimmermann | Egg size, eggshell porosity, and incubation period in the marine bird family Alcidae[END_REF]). In the yellow-eyed penguin, Massaro and Davis (2004) detected an increase in pore density with laying date which may allow embryos to develop more rapidly than earlier laid eggs. Until now, this hypothesis has not been tested for small passerines, although accelerated embryonic development of later-laid clutches would be beneficial for insectivorous songbirds too. However, we could not detect a seasonal variation in eggshell pore density in the collared flycatcher. It is possible that late-breeding collared flycatcher females reduce incubation length by starting incubation before all eggs within a clutch are laid, thereby producing hatching asynchrony, which may benefit the first hatched nestlings [START_REF] Slagsvold | Asynchronous versus synchronous hatching in birds: experiments with the pied flycatcher[END_REF][START_REF] Szegedi | The effect of environmental conditions on hatching asynchrony in the collared flycatcher[END_REF]. Alternatively, the laying date interval of the analysed clutches (11 days; Table 1) might have been too short to detect such differences. The total laying date interval of collared flycatchers breeding in our study plot in 2005 was 19 days, and we do not have data of clutches at the end of the breeding season and of clutches of repeat laying, where we may expect the largest increase in pore density. This could limit the interpretation of our result. However, in accordance with our result, eggshell pore density did not differ between eggs of early and late clutches in the black-headed gull [START_REF] Mänd | Increased eggshell porosity in replacement clutches of the black-headed gull Larus ridibundus[END_REF] and the Magellanic penguin either (Spheniscus magellanicus; Boersma and Rebstock 2009).

Songbirds do not store calcium for egg formation in the skeleton, they must collect calcium on a daily basis during the laying period [START_REF] Graveland | Arthropods and seeds are not sufficient as calcium sources for shell formation and skeletal growth in passerines[END_REF][START_REF] Graveland | Timing of the calcium intake and effect of calcium deficiency on behaviour and egg laying in captive Great tits Parus major[END_REF][START_REF] Pahl | Songbirds do not create long-term stores of calcium in their legs prior to laying: results from the high-resolution radiography[END_REF]. Therefore, we might not expect a decline in shell thickness with increasing clutch size as calcium is not utilized from a limited store, and indeed we found no relationship between number of eggs within a clutch and shell thickness. However, we found that eggs in clutches of more eggs had lower pore density than eggs in clutches of fewer eggs.

Porosity of the shell and the gas pressure gradient betwen the internal environment and the microenvironment of the nest affect gas conductance [START_REF] Ar | The avian egg: water vapor conductance, shell thickness, and functional pore area[END_REF]. If the rate of water loss and nest humidity are to remain constant in clutches of different egg numbers, then pore density should be reduced in proportion to the increase in egg number.

To sum up, our data showed that the blue-green and UV chroma of the eggshell were related to shell biliverdin concentration, as predicted. We found that females in better nutritional condition produced thicker-shelled eggs, although shell thickness was not related to the body size and stress level of the female. In contrast to our expectation, the structure of collared flycatcher eggs was not associated with laying date and local breeding density. However, we found that eggs in larger clutches had lower pore density than eggs in smaller clutches. Further research on eggshell thickness and porosity may provide a better understanding of the factors that influence variation in eggshell characteristics, and their effects on incubation period and hatching success of wild birds. Table 2 Results of separate GLMs with blue-green chroma and UV chroma as dependent variables, and eggshell thickness, pore density, egg size and biliverdin concentration as independent variables. Final models (from stepwise deletion procedure) with significant effects are marked in bold. We present the effects of nonsignificant variables after re-entering them to the final model. Effect size estimates (beta) and their 95% confidence intervals (Conf. Lmt.) are also reported.

Table 3 Results of separate GLMs with eggshell thickness and pore density as dependent variables, and environmental factors (laying date and local breeding density) and female traits (tarsus length, body mass and H/L ratio) as independent variables. Final models (from stepwise deletion procedure) with significant effects are marked in bold. We present the effects of nonsignificant variables after re-entering them to the final model. Effect size estimates (beta)

and their 95% confidence intervals (Conf. Lmt.) are also reported.

Fig. 1 Fig. 2 Fig. 3

 123 Fig.1Relationship between eggshell blue-green chroma (R 400-580 /R 320-700 ) and eggshell

Table 1 n

 1 Minimum Maximum Mean SD

	Clutch size	23	5	7	6.3	0.8
	Laying date (1= 30 April)	24	1	11	4.8	2.3
	Local breeding density (number of
	occupied neighbour nests)	24	1	6	3.4	1.1
	Tarsus length (cm)	23	1.68	1.88	1.80 0.06
	Body mass (g)	23	13.9	17.7	15.3 0.94
	H/L ratio (%)	21	0.06	1.63	0.45 0.37

Table 2

 2

			Blue-green chroma			UV-chroma
						Cnf. Lmt.					Cnf. Lmt.
		F	df	P	Beta	(lower,upper)	F	df	P	Beta	(lower, upper)
	Eggshell thickness 0.23 1, 20	0.64	-0.09 (-0.51, 0.32)	0.23 1, 20	0.64	0.10 (-0.32, 0.52)
	Pore density	0.02 1, 20	0.90	0.03 (-0.39, 0.45)	0.01 1, 20	0.98 -0.01 (-0.43, 0.42)
	Egg size	1.23 1, 20	0.28	-0.22 (-0.63, 0.19)	0.59 1, 20	0.45	0.16 (-0.27, 0.58)
	Biliverdin
	concentration	5.61 1, 21	0.027	0.46 (0.06, 0.86)	4.60 1, 21	0.044 -0.42 (-0.83, -0.01)

Table 1

 1 Sample sizes (n), minimum, maximum, mean and standard deviation (SD) values of the independent variables analysed in this paper.

Acknowledgements

We are grateful to R. Főző, L.Z. Garamszegi, G. Hegyi, M. Herényi, M. Laczi, G. Nagy, B. Rosivall, B. Szigeti and E. Szöllősi for assistance during fieldwork. We are indebted to P. Camarero, M. Martínez-Haro and J. Moreno for suggestions and help in the laboratory analyses. We thank two anonymous referees for their constructive comments. This study was supported by the Hungarian Scientific Research Fund (OTKA, grants no. T49650 and K75618), the Hungarian Ministry of Education (FKFP 0021/2002), the Eötvös Loránd University, and the Pilis Park Forestry. The Hungarian Ministry of Environment and Water provided permissions for the collection of collared flycatcher eggs for this study (permission no. 15951/2005). This research received support from the SYNTHESYS Project (http://www.synthesys.info/), which is financed by European Community Research Infrastructure Action under the FP6 "Structuring the European Research Area" Programme. The study complied with the current laws of Hungary.