Nest-Building Effort is Not An Indicator of Male Quality In Bonelli’s Eagles, Aquila fasciata

Raptors often use a variety of materials to build their nests (natural, such as branches, but also non-natural objects), presumably due to their insulating properties, their suitability to advertise occupancy of the nest, and to decrease pathogen and parasite loads. The amount of branches used in a nest is an indicator of parental quality and is often associated with increased breeding success. However, in raptors where both sexes collaborate in nest construction, it is unclear whether the effort expended by males (taking the amount of material carried to the nest as the potential predictor) could constitute an honest signal of parental quality to female conspecics. We examined data on sex, type of material brought to the nest, breeding experience, timing, and nest-building investment prior to egg-laying from 32 identiable Bonelli’s Eagles (Aquila fasciata) during the pre-laying period to investigate the relative contribution of the sexes to the amount of nest material gathered. We asked: (1) whether the nest-building investment of males could provide information to the females about their quality; and (2) whether the amount of material delivered to the nest by the male was related to breeding success. Despite the considerable investment of males in nest-building during the pre-laying period, our results indicate that this effort is not a consistent indicator of male quality to the female. Therefore, male nest-building behaviour and investment by Bonelli’s Eagles cannot be considered as an extended expression of their phenotype (an extended phenotypic signal). Nest-building behaviour by males in the early and late stages of nest-building, and the fact that males were not signicantly more active builders, are discussed in the contexts of signaling nest occupancy to conspecics and competitors, the decrease of parasite loads, and the strengthening of the pair-bond during the pre-laying period.


Introduction
Many animals use a wide variety of materials, designs, nest-site, and building techniques to build nests for shelter and reproduction 1,2,3 . Nests are essential structures for reproduction in some mammals and the majority of birds, and provide shelter and protection from inclement weather and predators for the eggs and nestlings 4,5 . Unlike animals that do not build their own nests, instead using the abandoned nests of heterospeci cs to lay eggs and rear their young 6,7 , many species expend considerable time and energy in the construction of one or multiple elaborate nests for breeding 8,9 , with varying degrees of differential parental investment (i.e., exclusively maternal, paternal, or biparental; see 10 . Although there is currently no consensus on the adaptive signi cance of building multiple nests and its effects on breeding success 11 , one of the hypotheses that has attracted more interest among researchers has been that they act to attract mates 12,13,14,15,16 . This hypothesis postulates that individuals could signal their current status, or the quality of their territory, through the construction of multiple nests 17,18,19 . In addition, in those species where the elaboration of the nest falls to both sexes, it could be a sexually selective behaviour that allows each sex to collect reliable information on the quality of its mate 20 .
Avian nests are often concealed and camou aged 2 , but many species build prominent nests or use conspicuous materials for nest ornamentation 21 . Previous studies have suggested that birds and mammals select materials for nest-building based on their thermal or structural properties 22 , which provide important bene ts for the eggs and young 23,24 . For example, the use of feathers, fresh fragments of aromatic plants, or even cigarette butts as nest materials can play a key role reducing the adverse effects of pathogenic bacteria and parasites on eggshells 25 and nestlings 26,27 , so improving the growth and condition of chicks at edging 28 to increase parental breeding success 29,30 . These direct bene ts alone often make it bene cial to choose mates most able to build well-constructed nests 31,32,4 .
At the same time, the type of nest material can provide indirect bene ts for builder birds. For example, during bird courtship displays, the degree of nest decoration and the speed and/or e ciency of gathering and transporting nest material may have additional or complementary functions, such as signaling: the extended phenotype of mate quality 33,34,35 ; genetic quality 14 ; nest-site occupancy; social status to potential intruders 21 ; breeding experience 36,37 and the willingness to attract partners and to invest in reproduction 20,38 . The nest-building experience of a mate may be an important factor in uencing how and where nests are built in order to improve breeding success, because their previous experience enables them to optimize the nest characteristics to their particular requirements, or possibly because experience is a surrogate of quality 39 . Previous experience can in uence decisions relating to the choice of nestmaterial 36, 40 . In addition, experience in nest-building plays an important role in decision making in future building endeavors, because a bird will have had practice in how to use different materials from its environment to build a nest or to increase the speed of construction 41 . Therefore, if we assume that nestbuilding ability is not wholly genetically determined 42,37 , older and more experienced males could gather more nest-building material than younger, less experienced males which would construct simpler nests than older birds. This behaviour could encourage females mated to more active males to make greater parental investments compared to females mated to less active males.
Previous research has shown that male displays of nest-building ability act as inter-sexual signals to attract females, mainly by the addition of green materials, owers, feathers, and even stones to the nest 17,43,30 . Males contribute to nest-building in many different avian species. Because nest-building is a costly activity in terms of both time and energy, and has tness consequences, it would pay females to encourage male nest-building behaviour 5 . In this way, the number and size of items gathered for a nest could provide females with information on male condition or willingness to invest in reproduction 20 . To date, two hypotheses have been postulated to explain why females increase their parental effort when caring for the offspring of attractive males: (1) the partner-compensation hypothesis (PCH) postulates that females mated to attractive males elevate their own level of care to achieve increased reproductive success 44,45 and (2) the differential allocation hypothesis (DAH) postulates that females mated to more attractive males are willing to contribute greater levels of parental investment compared with females mated to less attractive males 46,33 .
While investigations into extended phenotypic signals have been principally carried out on a variety of passerine species 47 , this topic has not yet been explored in other avian groups, such as raptors. Previous studies documenting the supply of nest-material to cliff nests by several raptor species have focused on: the functional aspects of decreasing ectoparasite loads in nests 29,48 ; indicators of nest-site selection criteria 49 social dominance; territory quality; means of signaling nest occupancy 21 ; and indicators of the evolutionary load of past tree-nesting behaviour 50 . Although the parental behaviour of both sexes of Bonelli's Eagle (Aquila fasciata) during breeding has been recently studied 51 , the role of nest-building behaviour has been generally poorly explored in raptors. Bonelli's Eagles build multiple large nests which they use alternately between years 29 . Bonelli's Eagle nests consist of a large basal structure of hard materials (sticks) and a nest cup lined with the green branches of aromatic trees and shrubs, and other materials such as fresh and dead grasses 52 . Both sexes build the nests throughout the 3-4 month period before egg-laying, although females may build while males gather nest-material 52 . This raises the possibility that nest-building per se can be used as a signal of male quality by females, if male nestbuilding behaviour and stick supply are consistent traits of a given male. An experimental approach is essential to disentangle whether stick supply to the nest serves as a signal or is an inherent indicator of mate quality. Our research is also of wider interest, because studies on the functionality of nest-building behaviour in biparental species are relatively uncommon compared to those on species where nestbuilding falls exclusively on either males or females 47 .
The objectives of our study were: (1) to describe the nest-building behaviour of Bonelli's Eagles during the pre-laying period; (2) to assess a possible relationship between a male's nest-building effort and his experience (quality); and (3) to investigate a possible relationship between male traits and reproductive success in this species. We hypothesized that male investment in nest-building (taken as the number of branches carried to the nest) could act as an honest signal informing female conspeci cs of the parental quality of their mates. If this were the case, females would bene t from choosing mates with good nestbuilding abilities. We therefore predicted correlations between males which made a high contribution to nest-building and their experience and increased fecundity.

The species
Our model species, the Bonelli's Eagle, is a large-sized eagle which inhabits mountainous areas across the Palearctic, Indo-Malayan and, marginally, the Afro-tropical regions 53 . It is a long-lived, sexually dimorphic species, and is monogamous and territorial. It builds its own nests using materials gathered by both sexes before egg-laying 54 . Bonelli's Eagles build large, open, perennial nests mainly on cliffs and sometimes in trees 55 , with platforms composed of tree and shrub sticks and branches that can be reused for several years, or even decades, as new materials are added every breeding season 29,54 . The existence of alternative nests is often associated with competitive exclusion and ectoparasite deterrence 56 .
Their clutch size ranges from one to three (less than 1% of clutches having three eggs 57 ). Their diet is generalist, based principally on the European Wild Rabbit (Oryctolagus cuniculus) complemented with medium-sized birds such as pigeons (Columba spp., Red-legged Partridges (Alectoris rufa) and corvids, as well as Ocellated Lizards (Timon lepidus) 54 . Regarding its reproductive biology, there is detailed information on the copulatory behaviour of territorial pairs at nest-sites prior to egg-laying 58 , and the parental investment of both sexes during breeding 51 . This last study revealed a marked division in parental duties in Bonelli's Eagles: females invested more effort in incubation, nest attendance, chickfeeding and nest-building, while males contributed more to the provision of food.
Bonelli's Eagle is considered to be a agship conservation species 59,60 . It typically inhabits semiarid and arid landscapes with sparse woodland, and is declining in numbers and range throughout the

Field Work
Our study population consisted of 16 Bonelli's Eagle pairs, nesting on cliffs at altitudes of up to 900 m.
During 2010-2016, pairs were monitored from October to one week after egg-laying (between January-February). Pairs were observed once a week. On each observation day, we recorded the nest construction activity during the daylight hours from 06:00-18:00, the shortest observation period being 4 hours (interrupted due to adverse weather) and the longest being 12 hours. In total, 4,131 hours of nest monitoring were carried out using 20-60× telescopes, from points overlooking the territories at a distance of about 600-800 m from the nest. This distance did not appear to alarm the birds or affect their behaviour 58 .
Following 66 , nest visits were recorded during each observation period, regardless of whether males or females arrived with or without nest-material. At each nest visit, we recorded for each sex: (1) the type of material brought to the nest, whether fresh material (mainly green branches used for building the nest structure and decoration, and lining the interior of the nest) or hard material (mainly withered branches and sticks used for building the nest structure); (2) the date and time of material supply to the nest; (3) the number of branches or sticks supplied/individual/day (nest-building effort); (4) the return of a previously identi ed individual to a speci c nest; and (5) the sex of an individual.

Individual Identi cation
Capture-recapture methods involving identi cation based on photographs is recognized as a reliable method to monitor wildlife populations and assess ecological aspects such as population size and structure, survival, site-delity, occupancy, lifetime reproductive success and other variables 67,68,69,70,71 .
Following previous studies on vultures 72  For analytical purposes, the monitoring season was divided into 16 weeks during the pre-laying period (weeks 1-16, counting backwards from the moment of egg-laying). Results were then aggregated into four, month-long time-periods (a discrete quantitative variable, range: 1-4; October-January) to assess the monthly distribution of the types of material gathered for nest-building (hard or fresh) by males and females. The pre-laying period spanned the time between the last pre-dispersal ights of the young Bonelli's Eagles of the previous clutch 76, 77 , the beginning of courtship and nest-building in mid-October, up to the moment of egg-laying 58 . We determined the onset of laying by direct observation (i.e., onset of incubation and changeovers). Laying dates were recorded with a maximum error of ± 1 day.
Using our individual identi cations, the experience of each known individual in a territory was determined by the number of consecutive years spent in the same territory, provided that the individual concerned was seen to be installed in the territory when rst observed 69 . In addition, the monitored territories were visited at least four times post-laying to record the number of edglings and to gather data on productivity. Nestlings observed at ≥ 50 days old were assumed to have edged successfully 78,79 .

Data analysis
All statistical analyses were performed with R 4.0.4 80 . We tested six hypotheses in total. For hypotheses 1-5, we rst applied a generalized linear mixed model (GLMM) to assess the factors determining the amount of material (number of branches) carried to the nest by parents and to explore whether the investment of males in nest-building could act as an honest signal informing female conspeci cs on their parental quality, ghting ability and social dominance. To test these hypotheses, we quanti ed the daily rates of nest-material supply as the response variable under a negative binomial distribution. We included a bird's sex and experience and the week as possible predictors of nest-building investment. Hypothesis 1 postulated that males invested more effort than females in nest-building. We then explored the possible differences between the sexes in relation to the type of material delivered to the nest (hard or fresh). Hypotheses 2 and 3 postulated that males gather higher proportions of either hard or fresh materials, respectively, than females. Hypothesis 4 postulated that male nest-building investment depends on male experience, expressed as the number of years that a male has bred in the same territory (as the predictive variable). Hypothesis 5 postulated that the addition of material by males increased throughout the prelaying period. To account for possible correlation effects between the factors in the data, male/female identity was included as a random factor.
Hypothesis 6 explored whether male nest-building investment affected breeding productivity, because females may increase their parental effort by preferentially investing in the offspring of attractive males. We performed a second GLMM using the breeding quality index (hereafter, BQI) as a response factor under a normal distribution. BQI was de ned as an individual's ability to produce offspring compared with the average success of others in the same year. BQI was calculated as the difference between the number of eaglets edged for a particular individual/territory and the average number of eaglets in the monitored territories in the same year 81 . In this analysis, male nest-building investment was considered as a predictive factor determining productivity. Male identity was again considered as a random factor. Mean values are reported with their con dence intervals. Statistical signi cance was set at P < 0.05.

Results
The materials used for nest construction comprised principally hard and fresh branches, principally of Aleppo Pine, with a small number of shrub species (Table 1). During the pre-laying period, males (mean: 2.00; 95% CI: 1.42-2.82) were more active than females (mean: 1.64; 95% CI: 1.17-2.30) in construction activity, although the difference was not signi cant ( Table 2). The supply of material to the nest began 16 weeks prior to egg-laying. In both sexes, there were two peaks in construction activity: the rst, between 112-78 days prior to egg-laying (October and early November); and the second, between 35 days prior to egg-laying and clutch completion (end December and January). In contrast, the supply of nest-material was very low between 77-36 days prior to egg-laying (Fig. 2).  During the pre-laying period, the amount of hard and fresh material gathered showed a temporal variation in both males and females ( Fig. 3a and   During the day, Bonelli's Eagles showed two peaks of material gathering: between 09:00-12:00 h; and later in the day (16:00 h). In addition, both sexes showed similar nest-building activity (Fig. 4).
The nest-building effort of males during pre-laying was not signi cantly related either to their level of breeding experience at the territory, or to the week ( Table 2). In addition, there were no signi cant relationships between the amount of material carried to the nest by males and their reproductive performance (Table 2).

Discussion
Our observations showed that Bonelli's Eagles selected their nest-site and began bringing hard and fresh material up to four months prior to egg-laying 52 . The onset of eagle visits to the nest, in October, and the start of nest-building using larger quantities of ne and coarse dry branches, might be related to the need to create a larger nest structure, including size, thickness, mass, and cup volume, which can in uence the nest's thermal properties 23,22 . This buffers the impact of adverse environmental conditions on the development of embryos and nestlings 82 and signals nest occupancy by increasing the visibility of the nest-site to conspeci cs and competitors (e.g., Golden Eagles, Aquila chrysaetos), even at large distances 83,21 .
Our results showed temporal changes in the types of material supplied to the nest: the supply of hard material occurred in the early visits to the nest and during the start of nest-building, whereas the supply of green material tended to increase as the laying date approached ( Fig. 3a and b). Previous studies have shown that green material brought to the nest can regulate the nest temperature and may help to decrease ectoparasite and pathogen loads 84,85,50 and improve breeding success 29 . Although fresh nestmaterial was gathered from vegetation rich in resins, including nine plant species, the most abundant plant delivered to the nests was greenery from pines (Table 1). Pines are characterized by a high level of aromatic compounds, highly repellent to insects 29,28 .
Previous studies have highlighted the important pair-bond existing between territorial Bonelli's Eagles throughout the year 86,87,88 . During the pre-laying period, while copulation activity occurred principally in the evening, with a progressive increase from the afternoon onwards 58 , both sexes showed the strongest nest-building activity during the morning, with a strong peak between 09:00-11:00 h, showing that birds worked together on nest construction activities (Fig. 4).
Behavioural studies exploring the function of nest-building behaviour in biparental species are scarce 47 .
In raptors, both partners invest in nest construction 89,66,90 . Thus, if male investment in nest construction is a consistent trait of any particular male, it is likely that females will use male nest-building effort to assess male parental quality and that the amount of branches and sticks that males deliver to the nest would become a sexually selected trait 20,33 . Previous studies describing biparental care in Bonelli's Eagles have shown a sex-biased specialization in parental duties 51,91 . Females invested signi cantly more effort than males in gathering nest-material during the breeding season 51 . In contrast, our ndings show that during the pre-laying period, the construction activities of males were important, and although the males provided slightly more material they were not more active builders than the females. In fact, our model does not demonstrate that sex is important in explaining the amount of material supplied to nests. Therefore, neither sex appears to use the nest-building process as a signal of their mate's quality. Our results, therefore, do not lend weight to Hypothesis 1. Neither were any inter-sexual differences observed with regard to the type of nest-material gathered (hard and fresh). This nding is consistent with the results of 66 , who found that in Pyrenean Bearded Vultures (Gypaetus barbatus), there were no observed inter-sexual differences concerning the amount supplied of either of the two common nest-materials used (branches and wool). Therefore, our results do not support Hypotheses 2 or 3.
However, breeding experience could be a decisive factor determining the investment effort of males 83,92 .
If the nest-building abilities of males are not wholly genetically determined, breeding experience could in uence decisions relating to nest material choice 36,40 and the amount of material gathered for nest construction 37 . In this way, younger eagles, with less experience of nest construction, should supply lower amounts of sticks and branches than older, more experienced eagles 37 . However, our model did not demonstrate any importance of this factor in explaining the effort of nest-building by males, thus rejecting Hypothesis 4.
In some bird species where both partners put similar effort into nest construction, the pair may reduce their effort as the laying date approaches 93,94 . In other taxa, the parents carry material to the nest throughout the entire breeding season (until after the chicks edge 9 ). In Bearded Vultures, for example, males invest more effort in nest-building than females, showing a peak of construction activity between 4-2 weeks prior to egg-laying 66 . These studies showed a progressive increase in material delivered to the nest throughout the pre-laying period. However, our results indicated a bimodal response of males to nestbuilding (Figure 3a), rejecting Hypothesis 4. This pattern could be explained by the ful lment of two adaptive functions: (1) signaling occupancy of the nest against conspeci cs and competitors (e.g., Golden Eagles) at the beginning of courtship; and (2) accumulation of the maximum amount of green material as the laying date approaches, with the aim of reducing the parasite and pathogen loads in order to maximize the viability of the eggs and the chicks.
Previous researches have shown that males may signal their condition, health, or parental quality to mates by building large or elaborate nests or by intense nest-building activity 20,33,47 . At rst glance, one might expect that male investment in nest-building could act as an honest signal informing females about their parental quality. Male nest-building behaviour could therefore be considered as a sexually selected trait: females might pair with males which are active builders, regardless of their construction ability (good or bad), and females would adjust their reproductive investment based on the nest-building investment exhibited by their mates. Nevertheless, our analysis did not nd a relationship between male nest-building investment and consequent reproductive performance. In this respect, our results are not consistent with the DAH 33 , thus rejecting our sixth hypothesis.

Conclusions
Our results indicate that male investment in nest-building does not appear to serve as a signal of male quality and, therefore, does not act as an honest signal to indicate their parental quality to their mates. Male nest-material provisioning rates therefore do not have a positive impact on reproductive success.
However, given that nest-building imposes high costs in both time and energy 1 , high levels of male nestbuilding could help females reduce their energy expenditure and help them to maintain optimal physical condition for reproduction 95 . This, in turn, supports the idea that a balanced distribution of nest-building activity, and of parental care in general, is bene cial to the breeding effort of a pair 93 .

Declarations Ethic statements
Bird-photographing procedures, camara trapping and monitoring of Bonelli´s Eagles were conducted under permits and following the protocols approved by the competent Regional Government of Region of Murcia (Resolución AUF/2020/0107). All the work was conducted in accordance with relevant national and international guidelines, and conforms to all legal requirements in compliance with the Ethical Principles in Animal Research.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information les).