A new view of territoriality in large eagles: the territory pre-exists regardless of their occupants.

1. The knowledge about territoriality and space use by predators is a topic of great interest in the study of animal behavior and conservation biology. Examining the plasticity of territory limits, the topology and how territories can be modi�ed depending on their owners is important to deepen into the behavior of territorial species. 2. Here, we analyzed the variations and tested the similarity of the home-range of individuals from the same territory using the data provided by 51 Bonelli's eagles from 22 different territories, tagged with GPS/GSM dataloggers in eastern Spain. 3. We analyzed the percentage of overlap of the annual home-range considering the 95% kernel density estimator between individuals of the same territory. Also, we analyzed the variation in the home range size according to three different kernel levels (95%, 75% and 50%) and the annual eccentricity of each individual home-range as a complementary metric to study the topology of territories across years. We also analyzed the changes in territory size and topology after the replacement of territory owners either by a single individual or by the entire pair. 4. Our results show that


Introduction
The study of spacing patterns and territoriality is essential for many aspects of animal biology.There are several criteria for de ning territoriality in animals.From a behavioral point of view, there are different forms of territoriality; one is area-defended territoriality, with aggression and warnings at territory boundaries (Maher and Lott, 1995).Originally this idea of area-defended territoriality was de ned as the defense of territory by one male against other males of the same species (Kaufmann, 1983).Some authors de ned territoriality as involving some xed spatial area (Brown, 1975;Kaufmann, 1983).Other authors state that territories need not be xed but are defended areas that could change over time and across space and be mobile (Wilson, 1975).Another behavioral criterion is site-speci c dominance, de ning territory as the home-range of an aggressive and dominant animal against intruders (Emlen, 1957).This de nition supports the idea of territory exclusion and the existence of overlapping areas between neighboring territories with hostile owners (Wittenberger, 1981).From an ecological point of view, they de ne territoriality as the exclusive occupation and use of a de ned area (Maher and Lott, 1995).In addition, territories may vary due to population density, food quantity and quality, habitat characteristics, and the individuals inhabiting them (Maher and Lott, 2000).
The description and use of the territories by different species of vertebrates have been described since the beginning of zoology and animal behavior studies.Most of these studies have been based on large mammals (e.g.Lorimer, 2010; Kalan et al., 2016), large predators (e.g.Cruz et al., 2021) and large raptors (e.g.Newton, 1979;Ferrer and Bison, 2003;Blas and Hiraldo, 2010;Bosch et al., 2010;Hernández-Matías et al., 2010).
Territoriality of raptors has been studied in different research.We nd studies that analyze the behavior and use of space in reproductive cycles (e.g.López-López et al., 2016a), feeding (e.g.Barton and Houston, 1994;Costantini et al., 2005), territorial occupation (e.g.Martinez et al., 2008) and habitat selection (e.g.Tanferna et al., 2013;Barrientos and Arroyo, 2014).However, the physiognomy and determination that territories exist as a singular element by themselves have not been studied to date (Fryxell, and Lundberg, 1997;Adams, 2001;López-Sepulcre and Kokko, 2005).The Bonelli's eagle (Aquila fasciata) is a strongly territorial raptor that defends its territory in pairs, exhibiting a typical behavior in which males and females y together for cooperative hunting.This species shows a large worldwide distribution across Europe, north of Africa, and southern Asia.In Europe, it occupies Mediterranean habitats with Mediterranean evergreen forest and an abrupt orography.The species is classi ed as "Near Threatened" (NT) in Europe (BirdLife International, 2015) and "Vulnerable" (VU) in Spain (SEO/Bird-Life, 2021).
The objective of the present work is to test whether the existence of stable and de ned territories is independent of their owners in the case of Bonelli's eagles.To this end, we use telemetry (GPS/GSM) data information to examine the plasticity of territory limits, the topology and how territories can be modi ed depending on their owners.Here, to this end, we evaluate the space use of different individuals within the same territory on eagles' ranging behavior across years.If the territory is xed independently of its occupants, no differences in annual home-range size and the territory eccentricity would be expected between individuals that occupy each territory during consecutive years (i.e., the null hypothesis).In contrast, each individual of one territory may show different space use patterns and thus annual home-range size and territory eccentricity should be different between individuals that inhabit the same territory across time (i.e., the alternative hypothesis).

Study area
The study area is located in eastern Spain including Albacete, Alicante, Castellón, Cuenca and Valencia provinces.The area covers approximately 10000 km 2 with an average altitude ranging between sea level and 1500 m a.s.l.The climate is Mediterranean with an average annual temperature that varies between 8°C in the interior mountains and 17°C in the coastal areas.The dominant landscape is composed of Mediterranean scrublands, oak forests (Quercus faginea, Quercus suber) and Mediterranean evergreen forests (Quercus ilex, Pinus halepensis and Pinus nigra).

Tracking
Adult and subadult Bonelli's eagles were trapped in their breeding territories and tracked between 2015 and 2021.In some cases, individuals were trapped and tagged in the same territory after the death or disappearance of one or both occupants (Table S1, see Supplementary Material).All individuals were tagged with GPS/GSM solar energy dataloggers manufactured by e-obs GmbH (Munich, Germany) and Ornitela (Vilnius, Lithuania) using a backpack con guration employing a Te on tubular harness designed to ensure that the harness fell off at the end of the tag's life.The weight of the transmitters was 48 and 50 g, respectively, and represented 1.66 to 2.86% (mean = 2.25%, SD = 0.38%) of the body mass of eagles, below the 3% threshold established to avoid negative effects on behavior (Kenward, 2001;García et al. 2021).The duty cycle of the transmitters was programmed to record a GPS location at ve-minute intervals (López-López et al., 2021), from 1 h before sunrise to 1 h after sunset, year-round during the study period (2015-2021).Transmitters' data was retrieved, stored and managed through the Movebank online repository (http://www.movebank.org/).

Ethics statement
Trapping and marking activities were authorized and conducted under permissions issued by regional authorities (Conselleria de Agricultura, Medio Ambiente, Cambio climático y Desarrollo Rural, Generalitat Valenciana, Spain) and all efforts were made to minimize handling time to avoid any suffering to eagles.

Home-range and space use analysis
We used kernel density methods (KDE) (Worton, 1989) to calculate home-range size and evaluate space use.We computed different levels of kernel isopleths as follows to allow comparison with similar studies: the 95% kernel isopleth (K95%) was considered as the total area of the home-range (Samuel et al., 1985); the 75% kernel isopleth (K75%) was considered as the intermediate area of active use, which includes the feeding and resting areas; and the 50% kernel isopleth (K50%) is the core area of the homerange, where the nest is usually found (Kie et al., 2010).We obtained these kernel levels from the next day after tagging to the end day of data transmission (e.g.animal's death, end of transmission) using the "reproducible home-range" (rhr) R package for statistical computing (Signer and Balkenhol, 2015; R Core Team, 2020).
Secondly, we computed the eccentricity of each territory as the distance from the arithmetic center of all the annual K95% locations (i.e., the centroid of the polygon) of each individual to the nest location within each territory.The rgeos and raster R packages were used to calculate the eccentricity (Bivand and Rundel, 2020;Hijmans, 2020).

Data analysis
We analyzed the annual territorial overlap between the different individuals that occupied the same territory across years.To this end, we calculated the percentage of annual overlap between the total home-ranges sizes (K95%) of individuals from the same territory using the raster R package (Hijmans, 2020).Furthermore, we use a Generalized Linear Mixed Model (GLMM; Zuur et al., 2009) to analyze the variation of annual home-range size and the eccentricity within each territory, considering "Territory" as a random factor.The variation is examined in relation to three xed factors, "Year", "Sex" and animal "ID".Annual home-range sizes according to the three different spatial estimators (K95%, K75% and K50%) and the annual eccentricity was logarithmically transformed and was used as the response variable.The R package used for the analysis was lme4 (Bates et al., 2015).We also computed the correlation between the home range area according to the K95% and the eccentricity to check the relationship between both parameters and the morphology of the territories.
We further described in detail the turnover events in which a territory owner was replaced by another individual who was subsequently tagged.In these cases, we computed the percentage of overlap between home ranges of those individuals (previous and new ones).

Results
Overall, we obtained 4.791.080GPS locations (mean = 101098.42;SD = 81578.23;range = 1016-257640 locations) of the 51 eagles that were tracked in this study.A total of 51 territorial adult and subadult Bonelli's eagles, including 26 males and 25 females, were trapped in 22 different territories (Figure S1 in Supp.Mat.).

Correlation between eccentricity and territory size
We found a signi cant positive correlation between home-range size and eccentricity (R = 0.4; p < 0.001; Fig. 2)".Therefore, the higher the home-range size, the higher the eccentricity.

Turnover events
There were seven territories with replacements in their owners, with eight replacements in total: 1) the A territory had one simple male replacement (Abel died in September 2016 by unknown causes and was replaced by Adan, which was tagged in January 2017).The annual overlap percentage of these individuals in consecutive years was Abel 2016 -Adan 2017 = 89.2%(Fig. 3A; Table S3  range on the old D territory (Dino -Dora pair) were between 50.7-77.5(Fig. 3C; Table S6 and Table S25 in Supp.Mat.); 4) the F territory had one simple female replacement (Flora died in December 2016 by electrocution at a power line and was replaced by Fauna, tagged in May 2017).The annual overlap percentage between these individuals was Flora 2016 -Fauna 2017 = 93.0%(Fig. 3D; Table S8 in Supp.Mat.); 5) the territory H had one simple male replacement (Helios died in January 2017 by electrocution at a power line and was replaced by Haeckel, tagged in April 2017 ).The annual overlap percentage between these males was Helios 2016 -Haeckel 2017 = 95.1% (Fig. 3E; Table S10 in Supp.Mat.); 6) the P territory had one simple male replacement (Popper died in October 2019 by collision with a power line and was replaced by Pino, tagged in June 2020).The annual overlap percentage between these individuals was Popper 2019 -Pino 2020 = 97.2% (Fig. 3F; Table S18 in Supp.Mat.); 7) the S territory had one simple female replacement (Sabina died in February 2020 by collision with a fence and was replaced by Salvia, tagged in June 2020).The annual overlap percentage between these females was Sabina 2019 -Salvia 2020 = 99.3% (Fig. 3G; Table S20 in Supp.Mat.).

Discussion
Territoriality is one of the most decisive aspects in ecology and behavior of animals, humans included, with important evolutionary implications.Hence, the study of territoriality in long-lived vertebrates might be a good proxy for this purpose.In this study we propose a new view in spatial ecology of large vertebrates by which territories exist by themselves and are independent of the individuals occupying them.To the best of our knowledge, this had not been taken into account in studies of territoriality to date.To this end, we have taken advantage of a large sample size either in terms of number of locations (4791080 GPS locations), number of territories occupied by different individuals (22 territories), and in terms of study duration (seven years).

Annual territorial overlap between individuals within the same territory
Our results show that the area and delimitation of territories remain the same over the years regardless of the individuals inhabiting them, with a high mean percentage of overlap between individuals occupying the same territory (  Fernández et al., 2009).The higher percentage recorded in this study is probably due to the larger sample size (n = 51 individuals) and the higher precision of the raw data (GPS/GSM telemetry versus combined Argos/GPS telemetry or radio-tracking).Therefore, our results show the delity and similarity between individuals occupying the same territory and the de nition of territorial limits do not change over time.
Interestingly, our results also show that space use could also be oppressed by neighboring territorial pairs.Our overlap analysis shows that the shape of the territory is also maintained in addition to its extension.Moreover, the topology of the territory is not similar between neighboring pairs; whereas some are elongated, others are rounded; others are more irregular, adapting to the physiognomy of the terrain and maintaining these limits and this differentiated shape across individuals and time.
The invariability of the territories is also supported by the statistical results of the GLMM in the sense that the limits of the total home-range size (K95%) do not vary signi cantly either over time or by the different occupants.In fact, both the extension of active use areas (i.e.feeding and resting; K75%) and the core areas of the territory (K50%) are invariant between years and between individuals within the same territory.Smaller home-range size of females (K95%, K75% and K50%) is explained by incubation tasks during the breeding season (López-López et al., 2021).

Territory eccentricity
The invariance in eccentricity within each territory corroborates the stability of territory physiognomy.
Eccentricity has been previously studied in Bonelli's eagles between breeding and non-breeding periods (Bosch et al., 2010), nding a signi cant eccentricity value for breeding areas in relation to the global home range.In other species such as the booted eagle (Aquila pennata), eccentricity has been also studied without nding differences between sexes and between breeding and non-breeding seasons (López-López et al., 2016a).
The location of the nest in the different topologies of the territories can also vary, either centrally or eccentrically.Despite these differences, the distance between the centroid and the nest remains constant among the different individuals occupying each territory over time.These results suggest that territory use is similar for different individuals regardless of nest location and the maintenance of territories independent of time and owners is once again rea rmed.

Simple double replacement cases
To further demonstrate our hypothesis, we now discuss the cases of territories where single or double substitution occurred.The members of the pairs occupying the territories in most cases of replacement had died or disappeared and the new member maintained the same territory as the previous one.How does the fact that both members of the pair disappear at the same time affect the physiognomy and boundaries of the territory?
Our hypothesis on the invariability of the territory is shown again.For example, in the rst case of a double turnover, despite the disappearance of the pair, the neighbors did not occupy a part of the territory by extending the boundary of their territory in that direction.Instead, they maintained their boundaries with the same con guration and eccentricity.In a second case, two neighboring pairs did not extend their territorial boundaries into the territory of the pair that had disappeared.The pair that did so inhabited practically all of the new territory, maintaining their original territory with pre-existing boundaries in the occupied territory and did not extend the area of the occupied territory nor its shape.
This could happen because the territory of the missing pair was surrounded by marked pairs except on one side, which overlooks an orange grove and urban territories that were not occupied by any Bonelli's eagle pair.In successive years, this pair that occupied two territories, gradually reduced its extension but maintained the core area of both territories, and the neighbors did not occupy part of the abandoned territory.In the case of simple replacements, our results show that the new member of the pair adapts the limits of its home-range to the previous one, also similar to the other member of the pair (the nonsubstituted one).
To date, it was thought that territory boundaries were maintained by pressure from neighbors (Fryxell, and Lundberg, 1997;Adams, 2001; López-Sepulcre and Kokko, 2005) and disappeared when the defending pair died, dis guring the boundaries and shape of the territory, resulting in the eventual occupation by neighboring pairs.The boundaries of the territories of the pair inhabiting the empty territory would also be dis gured.

Evolutionary implications
The turnover events recorded in this study by means of accurate GPS/GSM telemetry have made possible to assess the outcome of individual turnover from established territories in long-lived vertebrates.To the best of our knowledge, this is the rst time the pre-existence of territories with their entity, regardless of the individuals that occupy them, has been supported by eld information.
In many animal strategies, the evolution of territoriality re ects the balance between bene t and cost (Ord, 2021).Species whose males use territories to monopolize access to females appear to incur higher costs than those that defend only food resources (Adams, 2001;Ord, 2021).On the other hand, Wilson (1975) considered that territory could change in time and space, that it may not be a xed space.On the contrary, other argued that territories are xed spaces (Brown, 1975;Kauffmann, 1983).We consider that the existence of stable territories regardless of the individuals occupying them may represent an evolutionary advantage that we could divide into the following points: 1) territoriality increases population stability and oating individuals form a buffer against uctuations (López-Sepulcre and Kokko, 2005); 2) the age and the quality of a territory (measured as reproductive success) are correlated (Ferrer and Bisson, 2003).We, therefore, distinguish a constant struggle in the oating population to reach the best territories, usually replacing agonistically a member of the pair or, in the case of a natural loss, by occupying the vacant in the territory as soon as possible.In fact, our eld observations show that replacement after a vacancy takes place usually in less than few weeks.In contrast, the formation of new territories by tracked animals has not been recorded throughout the study period.In this continuous struggle, the best specimens settle in the best territories, which are the ones that favor the continuity of the population.The main evolutionary advantage is that no energy is wasted in founding, exploiting and defending new territories with limited chances of success.We have proposed this territorial strategy for Bonelli's eagles, it remains to be seen which other animal groups or species are also partially or adapted to this strategy.

Implications for conservation
The assessment of the independent pre-existence of territories is of great value for the conservation of territorial species.Hence, the identi cation of territories regardless of the state of occupancy and its preservation in an adequate state of conservation will facilitate their occupation when an eventual recovery of the species could take place.Finally, this study highlights the role of modern telemetry techniques to get a better understanding of the exact delimitation of territories.This tool makes possible to act with the greatest precision, for example, in the delimitation of protected areas or for taking management actions for endangered species.

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Table 1
Generalized Linear Mixed Model (GLMMs) results.Signi cant variables are highlighted in bold.
in Supp.Mat.); 2) the B territory had two replacements, one simple female replacement (Berta died in July 2015 by a collision with a power line and was replaced by Boira, tagged in November 2015).The overlap percentage of this year was Berta 2015 -Boira 2015 = 66.50%.Also one double turnover (Boira and Blas drowned in an irrigation pond in June 2016 (López-López et al., 2016b) and were replaced by individuals from the oating population, Boj and Bruma in 2017, which were tagged at the same time in April 2017).The annual percentage of overlap of home-ranges between 2016 and 2017 (period in which the complete replacement occurred) were: Boira 2016 -Bruma 2017 = 89.7%;Boira2016-Boj 2017 = 84.0%;Blas2016-Bruma 2017 = 92.2%;andBlas2016 -Boj 2017 = 84.3%(Fig.3B;TableS4of Supp.Mat.); 3) the D territory had a double replacement (Dino and Dora were poisoned at the same time in March 2018 (López-López and Urios, 2018) and Adan and Aura (neighbors of the A territory) occupied both territories (territories A and D) only two days after the death of the pair Dino-Dora.The annual overlap percentages of the A pair's home-ranges compared with the year before the death of the D pair (2017) were between 69.3-92.6%.The annual overlap percentages of the new territory emplacement of the A pair's home Bosch et al., 2010)s means that the territory remains constant regardless of whether it is used by one pair, by a new pair or by a pair formed by a previous and a new individual.Our result of the overlap between different occupants of a territory has never been studied before, neither in Bonelli's eagle nor in other vertebrate species.Instead of that, there are previous studies that measured the inter-annual delity by each individual or overlap between sexes.The overlap percentage of the occupants of a territory recorded in our study is higher in comparison to these previous studies in Bonelli's eagle of individual overlap percentage recorded by GPS (30.3%, n = 7, Pérez-García et al., 2013; 76.8%, n = 8, Martínez-Miranzo et al., 2016), but lower than the percentage of overlap between sexes recorded by radio-tracking (98.6%, n = 10,Bosch et al., 2010).The overlap percentage is also higher than Watson et al., 2014)dividual inter-annual delity overlap described in other raptor species such as the Golden eagle (Aquila chrysaetos) (60%, n = 8, radio-tracking, Marzluff et al., 1997; 70% (99% kernel), n = 17, GPS,Watson et al., 2014), and Spanish imperial eagle (Aquila adalberti) (75%, n = 8, radio-tracking,