Sarah R Hoy

Alexandre Millon

Steve J Petty

D Philip Whitfield

Xavier Lambin
email: x.lambin@abdn.ac.uk

Food availability and predation

Keywords: Accipiter gentilis, breeding decisions, breeding propensity, clutch size, juvenile survival, life-history trade-offs, northern goshawk, reproductive strategies, Strix aluco, tawny owl

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conditions (e.g. food availability or predation) varies according to its intrinsic attributes (e.g. age, previous allocation of resources towards reproduction).

2. We used 29 years of reproductive data from marked female tawny owls and natural variation in food availability (field vole) and predator abundance (northern goshawk) to quantify the extent to which extrinsic and intrinsic factors interact to influence owl reproductive traits (breeding propensity, clutch size and nest abandonment).

3.

Extrinsic and intrinsic factors appeared to interact to affect breeding propensity (which accounted for 83% of the variation in owl reproductive success). Breeding propensity increased with vole density, although increasing goshawk abundance reduced the strength of this relationship. Owls became slightly more likely to breed as they aged, although this was only apparent for individuals who had fledged chicks the year before.

4.

Owls laid larger clutches when food was more abundant. When owls were breeding in territories less exposed to goshawk predation, 99.5% of all breeding attempts reached the fledging stage. In contrast, the probability of breeding attempts reaching the fledging stage in territories more exposed to goshawk predation depended on the amount of resources an owl had already allocated towards reproduction (averaging 87.7% for owls with clutches of 1-2 eggs compared to 97.5% for owls with clutches of 4-6 eggs).

Introduction

Understanding how different factors influence reproductive decisions is a central issue in ecology and conservation biology, as the number of offspring produced is a key driver of population dynamics [START_REF] Nichols | Estimation of sexspecific survival from capture-recapture data when sex is not always known[END_REF][START_REF] Sedinger | Fidelity and breeding probability related to population density and individual quality in black brent geese Branta bernicla nigricans[END_REF]. The impact of some extrinsic factors on reproductive decisions, such as food availability, are well understood (reviewed in [START_REF] White | The role of food, weather and climate in limiting the abundance of animals[END_REF]. In contrast the impact of others, such as predation risk is more equivocal, even when the same predator and prey species are examined [START_REF] Sergio | Intraguild predation in raptor assemblages: a review[END_REF]. Quantifying the indirect effect of predation risk on prey reproductive decisions under natural conditions is difficult, but merits further investigation as it can theoretically destabilize predator-prey dynamics, under certain circumstances [START_REF] Kenward | Breeding suppression and predator-prey dynamics[END_REF].

Furthermore, despite the influence of food availability and predation risk on reproductive success being extensively studied, the extent to which these two extrinsic factors interact to affect reproductive decisions remains poorly understood (but see [START_REF] Sergio | Spatial refugia and the coexistence of a diurnal raptor with its intraguild owl predator[END_REF].

Food availability is frequently reported to have a positive influence on the proportion of individuals in the population breeding and the number of offspring produced [START_REF] Arcese | Effects of population density and supplemental food on reproduction in song sparrows[END_REF][START_REF] Pietiäinen | Seasonal and individual variation in the production of offspring in the Ural owl, Strix uralensis[END_REF][START_REF] Petty | Ecology of the Tawny Owl Strix Aluco in the Spruce Forests of Northumberland and Argyll[END_REF][START_REF] Millon | Dampening prey cycle overrides the impact of climate change on predator population dynamics: A long-term demographic study on tawny owls[END_REF]. However, breeding individuals and individuals producing more offspring per breeding attempt are often more vulnerable to predation compared to non-breeding individuals [START_REF] Magnhagen | Predation risk as a cost of reproduction[END_REF][START_REF] Hoogland | Selective predation on Utah prairie dogs[END_REF] or those producing fewer offspring [START_REF] Ercit | Egg load decreases mobility and increases predation risk in female black-horned tree crickets (Oecanthus nigricornis)[END_REF]. Consequently, in years when predation risk is high, individuals of long-lived iteroparous species may attempt to minimize their vulnerability to predation by: i) refraining from breeding [START_REF] Spaans | Dark-bellied Brent geese Branta bernicla bernicla forego breeding when arctic foxes Alopex lagopus are present during nest initiation[END_REF]; ii) reducing the number or quality of offspring [START_REF] Doligez | Clutch size reduction as a response to increased nest predation rate in the collared flycatcher[END_REF][START_REF] Zanette | Perceived Predation Risk Reduces the Number of Offspring Songbirds Produce per Year[END_REF]; or iii) abandoning the breeding attempt at an early stage [START_REF] Sergio | Spatial refugia and the coexistence of a diurnal raptor with its intraguild owl predator[END_REF][START_REF] Chakarov | Mesopredator release by an emergent superpredator: a natural experiment of predation in a three level guild[END_REF]. Indeed, experimental studies have shown that individuals respond to variation in predation risk by making facultative decisions to alter their allocation of resources towards reproduction, so as to reduce their own, or their offspring's vulnerability to predators [START_REF] Ghalambor | Fecundity-survival trade-offs and parental risktaking in birds[END_REF][START_REF] Doligez | Clutch size reduction as a response to increased nest predation rate in the collared flycatcher[END_REF][START_REF] Fontaine | Parent birds assess nest predation risk and adjust their reproductive strategies[END_REF][START_REF] Zanette | Perceived Predation Risk Reduces the Number of Offspring Songbirds Produce per Year[END_REF]. However, according to life history theory, such changes in reproductive strategies should arise only when the losses incurred from not breeding, or not completing a breeding attempt, are compensated for by future reproductive success [START_REF] Stearns | The Evolution of Life Histories[END_REF]).

This intrinsic trade-off between current reproductive success and future reproductive potential is thought to be an important factor shaping reproductive decisions [START_REF] Stearns | The Evolution of Life Histories[END_REF].

For many long-lived species, the strength of this trade-off is thought to vary over an individual's lifetime [START_REF] Proaktor | Age-related shapes of the cost of reproduction in vertebrates[END_REF], as both survival-and reproduction-related traits are age-dependant, often declining in later life [START_REF] Nussey | Senescence in natural populations of animals: widespread evidence and its implications for biogerontology[END_REF]. Furthermore, changes in extrinsic conditions can also cause the strength of this intrinsic trade-off to vary, via their influence on survival probabilities and ultimately the individual's future reproductive potential [START_REF] Barbraud | Environmental conditions and breeding experience affect costs of reproduction in Blue Petrels[END_REF][START_REF] Hamel | Maternal characteristics and environment affect the costs of reproduction in female mountain goats[END_REF]. Consequently, an individual's reproductive response to changes in extrinsic conditions is predicted to vary according to their intrinsic attributes, with individuals becoming increasingly committed to their current reproductive attempt as they age, to compensate for the decline in future breeding prospects [START_REF] Clutton-Brock | Reproductive effort and terminal investment in iteroparous animals[END_REF]. However, few studies have examined whether intrinsic and extrinsic factors interact to explain variation in reproductive success (but see [START_REF] Wiklund | The adaptive significance of nest defence by merlin, Falco columbarius, males[END_REF][START_REF] Kontiainen | Aggressive ural owl mothers recruit more offspring[END_REF][START_REF] Rauset | Reproductive patterns result from age-related sensitivity to resources and reproductive costs in a mammalian carnivore[END_REF], despite theory predicting such a link [START_REF] Williams | Natural selection, the cost of reproduction, and a refinement of lack's principle[END_REF][START_REF] Ricklefs | On the evolution of reproductive strategies in birds: Reproductive effort[END_REF].

In this study, we used 29-years of breeding data collected on an intensively monitored population of individually identifiable female tawny owls (Strix aluco) to examine the extent to which owl reproductive decisions varied in relation to two extrinsic factors, natural variation in the abundance of their main prey (field vole, Microtus agrestis; Petty 1999), and their main predator (a diurnal raptor, northern goshawk, Accipiter gentilis; [START_REF] Hoy | Age and sex-selective predation as moderators of the overall impact of predation[END_REF].

In another study site, predation by diurnal raptors was found to account for 73% of natural tawny owl mortality after the fledging stage, when parents are still provisioning food for their young [START_REF] Sunde | Diurnal exposure as a risk sensitive behaviour in tawny owls Strix aluco ?[END_REF] and in our study site predation on adult owls was biased towards breeding females [START_REF] Hoy | Age and sex-selective predation as moderators of the overall impact of predation[END_REF]. It is expected that breeders and parents of larger broods spend more time hunting to provision food for their offspring, which may make these parents more exposed to predation by goshawks. Consequently, in years when predation risk is high, individuals may attempt to minimise their vulnerability to predation by reducing the amount of resources they allocate towards reproduction (breeding less frequently or laying smaller clutches). However, as the seasonal peak in goshawk predation on tawny owls occurs after owls have already initiated breeding attempts [START_REF] Petty | The decline of common kestrels Falco tinnunculus in a forested area of northern England: the role of predation by Northern Goshawks Accipiter gentilis[END_REF], the main response of individuals to variation in predation risk may manifest itself as an increased tendency to abandon breeding attempts at an early stage. Therefore in this study we examined how three different reproductive decisions: i) breeding propensity; ii) clutch size; and iii) whether breeding attempts were completed to the fledging stage varied in relation to fluctuations in food availability and predation risk.

We also investigated whether owl reproductive decisions were related to the following intrinsic attributes, current and previous allocation of resources towards breeding (clutch size and reproductive success the year before, respectively) and the age of the individual, as lifehistory theory predicts an intrinsic trade-off between current and future allocation of resources towards reproduction [START_REF] Williams | Natural selection, the cost of reproduction, and a refinement of lack's principle[END_REF], as survival and reproductive rates are agedependent in tawny owls [START_REF] Millon | Natal conditions alter age-specific reproduction but not survival or senescence in a long-lived bird of prey[END_REF].

Changes in extrinsic conditions are also likely to affect the probability of offspring being recruited into the breeding population, via their effect on juvenile owl survival [START_REF] Sunde | Diurnal exposure as a risk sensitive behaviour in tawny owls Strix aluco ?[END_REF][START_REF] Sunde | Predators control post-fledging mortality in tawny owls, Strix aluco[END_REF][START_REF] Koning | Long-term study on interactions between tawny owls Strix aluco , jackdaws Corvus monedula and northern goshawks Accipiter gentilis[END_REF][START_REF] Millon | Pulsed resources affect the timing of first breeding and lifetime reproductive success of tawny owls[END_REF]. Thus, the influence of extrinsic conditions on juvenile survival should influence the adaptive basis for reproductive decisions, for instance, how beneficial it is to allocate resources towards a reproductive attempt. Consequently, we also examined how juvenile survival varied in relation to temporal fluctuations in food availability and predation risk.

Methods

Study site and owl monitoring

Tawny owl reproduction has been continuously monitored in a 176 km² central section of Kielder Forest (55°13′N, 2°33′W) since 1979, using nest boxes [START_REF] Petty | Value of nest boxes for population studies and conservation of owls in coniferous forests in Britain[END_REF]. Kielder

Forest, mainly planted with Sitka Spruce (Picea sitchensis), lacks natural tree cavities, therefore owls breed almost exclusively in nestboxes [START_REF] Petty | Value of nest boxes for population studies and conservation of owls in coniferous forests in Britain[END_REF]. Each year, all nest boxes were checked for occupancy, to record clutch size, the number of chicks fledging and to ring chicks. Tawny owls do not breed every year after becoming reproductively active and only breed once per year, but can re-lay if the first breeding attempt fails early (during laying or the early incubation period; [START_REF] Petty | Ecology of the Tawny Owl Strix Aluco in the Spruce Forests of Northumberland and Argyll[END_REF]). In such cases, we only included the second breeding attempt, such that each individual contributed only one breeding attempt per year to our analysis. In some cases, the monitoring of a nestbox resulted in owls abandoning their breeding attempts. We therefore excluded all such breeding attempts (N = 51/965) from all our analyses. Breeding females were captured every year using a modified angler's landing net which was placed over the entrance of the nestbox, when their chicks were 1-2 weeks old. The identity of breeding females was established from their metal ring numbers, and any unmarked breeding females (entering the population as immigrants) were ringed upon capture so that they would subsequently be individually identifiable. Tawny owls are highly site faithful, and in our study site >98% remained in the same territory where they first started breeding [START_REF] Petty | Ecology of the Tawny Owl Strix Aluco in the Spruce Forests of Northumberland and Argyll[END_REF]). Therefore we determined the identity of a female occupying a territory when no breeding took place or when the breeding attempt failed prior to trapping in the following way. When the same female was recorded breeding in a territory both before and after the year(s) where no female was caught, we assumed the same individual was involved.

However, when different females were recorded either side of a year(s) when females were not caught, we deemed the identity of the breeder unknown and excluded such breeding attempts from our analyses. A total of 914 breeding attempts took place between 1985 and 2013 where the identity of the female was known, or could be assumed in 89% of cases (N = 813).

Analysis

To determine the extent to which owl breeding decisions were affected by fluctuating extrinsic and intrinsic factors, we examined: i) breeding propensity, ii) clutch size and iii) whether breeding attempts were completed using generalised linear mixed effect models (GLMM) with the appropriate error structure in R version 3.0.3 (R Core Development Team 2014). The identity of the breeding female and the year of a breeding attempt were fitted as random effects to account for individuals breeding in more than one year, and any residual temporal variation in response variables not attributable to the fitted temporal covariates of interest (food availability and predation risk). In all analyses both the additive and 2-way interactive effects of fixed effect covariates were tested. We visually checked for any residual spatial-autocorrelation in all response variables not explained by the covariates included in the selected best models using correlograms [START_REF] Zuur | Mixed Effects Models and Extensions in Ecology with R[END_REF].

We examined causes of variation in breeding propensity by analysing whether an individual bred or did not breed each year after becoming reproductively active, up until its last recorded breeding attempt (fitted as a binary covariate). We examined breeding propensity in this way for the following reasons. We excluded first-time breeding attempts as the breeding propensity of such attempts would necessarily be one and this may bias the results. We did not include the years prior to the first breeding attempt because there is no way to identify a new recruit in a territory before it first bred and it was unknown whether individuals had made a facultative decision not to breed the year(s) before they first bred, or whether they were incapable of breeding regardless of extrinsic conditions. Furthermore, some individuals were only recorded breeding once, thus we had no way of determining whether such individuals were alive and had decided to not to breed in the subsequent year(s) after their only recorded breeding attempt or whether these individuals were dead. When at least one egg was laid in a territory known to be occupied by a particular female, we recorded that as a breeding attempt. Less than 2% (N= 5) of the 268 different females recorded breeding in Kielder Forest were known to have skipped breeding for three or more consecutive years.

Therefore, we assumed an individual was dead if it had not been re-captured in the last 3 years of the study (i.e. after 2010). In this analysis, we excluded all individuals that could not be assumed dead or were known to be alive (i.e. were recorded breeding) in 2013 (N = 40), to remove any bias that unknown non-breeding events occurring in the last few years of the study period could induce.

To determine the extent to which owls adjust the amount of resources they allocate towards reproduction in response to variation in food availability and predation risk, we modelled variation in clutch size. In addition, we examined the decision or capability to continue with a breeding attempt by classifying each breeding attempt as "complete", if at least one chick fledged, or "incomplete" if not (fitted as a binary covariate). These two analyses were based on a different dataset to that used for the breeding propensity analysis, as it contained all breeding attempts by all known individuals (N = 241), including first time-breeders between 1985-2013.

Measures of food availability and predation risk

Field voles are the main year-round prey of tawny owls in Kielder Forest, representing on average 62% of prey brought to the nestbox (N = 1423; Petty 1999). As tawny owls are vole specialists in our study site, variation in the abundance of alternative food sources probably had only a limited impact on owl breeding decisions. Field vole densities were monitored in spring and autumn at 17-21 sites within the owl monitoring area, every year since 1985 (for methods see Lambin, Petty, & MacKinnon 2000). Vole densities in the spring and autumn were positively correlated (r = 0.65, N = 27, P <0.001). The amount of vole prey available in early spring (prior to egg laying) has previously been shown to affect owl reproduction; in years of high food availability more pairs attempted to breed and clutch sizes were larger [START_REF] Petty | Ecology of the Tawny Owl Strix Aluco in the Spruce Forests of Northumberland and Argyll[END_REF][START_REF] Millon | Dampening prey cycle overrides the impact of climate change on predator population dynamics: A long-term demographic study on tawny owls[END_REF]. Therefore, spring vole densities were used as a proxy for owl food availability in all analyses. Field vole densities were asynchronous but spatially structured across Kielder Forest (i.e. travelling waves; [START_REF] Lambin | Spatial asynchrony and periodic travelling waves in cyclic populations of field voles[END_REF]. However, this pattern has changed over time with a gradual loss of spatial structure [START_REF] Bierman | Changes over time in the spatiotemporal dynamics of cyclic populations of field voles (Microtus agrestis L.)[END_REF].

Such changes in prey spatial synchrony may affect how easy it is for owls to predict the amount of food available in their territory, and hence influence their reproductive decisions. Therefore, we also examined the extent to which tawny owl breeding decisions were affected by changes in the spatial synchrony of field vole densities. To do so, we first calculated spatial variation in field vole densities as the coefficient of variation (standard deviation divided by the mean) in spring vole densities between survey sites, each year. However, spatial variation in vole densities may be less important in years when food is abundant, compared to when it is scarce. Therefore, we classified years as either being of low overall food abundance if the averaged spring vole density was below the median value for all years, or high if not. We then included an interaction between spatial variation in vole densities and the categorical covariate of overall vole densities to test this hypothesis.

Northern goshawks (hereafter goshawks) have been continuously monitored since the first breeding attempt in 1973 [START_REF] Petty | Goshawks Accipiter gentilis. The Atlas of Breeding Birds in Northumbria[END_REF]. Each year occupied goshawk homeranges were identified and over the last 40 years the number of occupied home-ranges has increased from one to 25-33. Goshawks are known predators of tawny owls, with breeding female owls being three times more likely to be killed than adult males; predation is also heavily biased towards juveniles [START_REF] Hoy | Age and sex-selective predation as moderators of the overall impact of predation[END_REF]. Goshawk dietary data collected in Kielder Forest suggests that as the breeding population of goshawks increased, the mean number of owls killed each year by goshawks has also increased. An average of 5 [3-8; 95% CI] owls were killed each year when less than 15 goshawk home-ranges were occupied, compared to an average of 159 [141-176; 95% CI] owls killed each year when more than 24 goshawk home-ranges were occupied (see Appendix S1). Consequently, as predation on owls has increased with the abundance of goshawks in the forest, we used the total number of occupied goshawk home-ranges in a 964 km² area of Kielder Forest as a proxy of temporal variation in predation risk. However, as goshawks were monitored over a larger area than tawny owls, we also used an additional proxy of temporal variation in predation risk. Local goshawk abundance was measured as the number of goshawk home-ranges whose nest sites were within 5.8 km (the estimated goshawk foraging distance) of the owl monitoring area, calculated in the same way described in [START_REF] Hoy | Age and sex-selective predation as moderators of the overall impact of predation[END_REF]. Spatial variation in predation risk has also been found to influence reproductive decisions [START_REF] Sergio | Spatial refugia and the coexistence of a diurnal raptor with its intraguild owl predator[END_REF]. Therefore, we investigated the extent to which owl reproductive decisions varied in relation to two spatial proxies of predation risk: (i) distance from an owl's nest to the nearest goshawk nest site; and (ii) the location of an owl's territory in relation to all goshawks nest sites, (i.e. connectivity of an owl territory to all goshawk nest sites). The connectivity measure of predation risk takes into account all goshawk nest sites, but weights the influence each goshawk nest site has on this index of predation risk, according to its distance from the focal owl nest site (for further details and method see Appendix S2). These spatial covariates of predation risk were calculated for each owl territory, every year. Although common buzzards Buteo buteo are abundant in our study site and are known to kill tawny owls [START_REF] Mikkola | Owls killing and killed by other owls and raptors in Europe[END_REF]), we did not include buzzards in any of our analyses of owl predation risk. This was because dietary data showed us that buzzard predation on owls in our study site was negligible (unpublished data). None of the temporal proxies of food availability were significantly correlated with the temporal covariates of predation risk. However, no two proxies of predation risk or two proxies of food availability were included in the same model as they were collinear (see Appendix S3 for all cross correlation coefficients). All temporal and spatial covariates were standardised (had a mean of 0 and a standard error of 1) to enable their effect sizes to be compared.

Intrinsic attributes

When testing the hypothesis that the response of an individual to changes in extrinsic conditions varied according to age, we used the number of years elapsed since the individuals first recorded breeding attempt, because the exact age of 94 breeding females entering the population as adult immigrants was unknown. However, most (89%) female owls had commenced breeding by the time they were 3 years old [START_REF] Millon | Pulsed resources affect the timing of first breeding and lifetime reproductive success of tawny owls[END_REF] and there had been no change in the mean age at first reproduction over the study period, neither for immigrants nor local recruits entering the owl population (unpublished data).

Consequently, the number of years elapsed since an individual's first recorded breeding attempt is closely related to its age, and the length of an individual's breeding lifespan is also highly correlated with actual lifespan (r = 0.91; N = 163). We tested the hypothesis that previous investment in reproduction influenced an individual's current reproductive decisions in relation to changes in predation risk and food availability by fitting a binary covariate reflecting whether a female owl had successfully raised offspring to the fledgling stage the previous year. Lastly, we investigated whether the likelihood of an individual completing a breeding attempt to the fledging stage was related to clutch size, taking clutch size as a proxy for the extent to which an individual had already allocated resources towards the current reproductive attempt. All descriptive statistics are shown with the standard deviation (SD).

Juvenile survival

As recapture data were not available for male owls in all years, our analysis of juvenile owl survival was based on female owls only, ringed as chicks between 1985 and 2012 (N=1,082), with the last recapture of individuals in 2013. The sex of individuals never recaptured as adults or sexed as chicks using DNA was unknown, as juvenile owls cannot be accurately sexed without molecular analyses. However, the sex ratio of chicks born in our study site was even 1:1 (N =312, over 4 years; Appleby et al. 1997). Consequently, we randomly assigned half the number of chicks born each year minus the number known to be female as females, as done in previous analyses [START_REF] Nichols | Estimation of sexspecific survival from capture-recapture data when sex is not always known[END_REF][START_REF] Millon | Pulsed resources affect the timing of first breeding and lifetime reproductive success of tawny owls[END_REF]. The rest of these chicks were assumed to be males and excluded from the analysis. Owls were only recaptured when breeding and owls usually starting to breeding between the ages 1-4 (89% before age 3; [START_REF] Millon | Pulsed resources affect the timing of first breeding and lifetime reproductive success of tawny owls[END_REF]. Recapture probabilities were therefore modelled as time-dependent and age-specific [(1, 2-3, 4+)] as done in [START_REF] Millon | Pulsed resources affect the timing of first breeding and lifetime reproductive success of tawny owls[END_REF]. This analysis was carried out in E-SURGE version 1.9.0 [START_REF] Choquet | Program E-SURGE: A Software Application for Fitting Multievent Models[END_REF]. Goodness-of-fit tests were carried out in U-CARE 2.3.2 [START_REF] Choquet | U-CARE 2.2 User's Manual[END_REF]. In this analysis only, rather than using spring vole densities (measured in March) as the measure of food availability, we used autumn densities of field voles (measured in September-October), as they have previously been shown to be more closely related to changes in juvenile tawny owl survival [START_REF] Millon | Pulsed resources affect the timing of first breeding and lifetime reproductive success of tawny owls[END_REF][START_REF] Millon | Natal conditions alter age-specific reproduction but not survival or senescence in a long-lived bird of prey[END_REF][START_REF] Millon | Dampening prey cycle overrides the impact of climate change on predator population dynamics: A long-term demographic study on tawny owls[END_REF]. Temporal proxies of predation risk were the same as those used in the previous analyses. Spatial proxies of predation risk were calculated as before, but using the natal nestbox, and were modelled as an individual covariate. Model selection in all of the above analyses was based on Akaike's information criterion corrected for small sample size (AICc; [START_REF] Burnham | Model Selection and Multimodel Inference: A Practical Information-Theoretic Approach, 2nd Editio[END_REF].

Results

Breeding propensity

When averaged across years, the probability of a female breeding after becoming reproductively active was 0.78 ± 0.17 (range: 0.21-0.99). Variation in breeding propensity appeared most strongly related to changes in extrinsic conditions (Table 1). In years when local goshawk abundance was relatively low (fewer than 10 home-ranges occupied) breeding propensity increased from an average of 0.33 ± 0.18, when food availability was also low, to an average of 0.95 ± 0.06 in years of high food availability (Fig. 1a). However, in years when goshawk abundance was high the relationship between breeding propensity and food availability was less apparent (Fig. 1a). Breeding propensity also appeared to vary according to intrinsic attributes (proxies of age and previous allocation of resources to reproduction); however the association between breeding propensity and intrinsic attributes was much weaker in comparison to the relationship with extrinsic factors (Fig. 1b; Table 1; Appendix S4). Breeding propensity was estimated to increase slightly as owls aged. However this trend was only observed for individuals who had successfully fledged chicks the year before.

Clutch size

Owl clutch size averaged 2.85 ± 0.82 (range: 1-6; N = 850), with 92.8% of clutches containing 2-4 eggs. The largest clutches were laid in years of high spring vole densities with clutch size increasing from an average of 2.38 [2.28-2.48; 95% CI] in years when vole densities were below 50 voles ha -1 to 2.98 [2.82-3.14; 95% CI] in years when vole densities were above 150 voles ha -1 (Fig. 2). There was no evidence to suggest that variation in clutch size was related to predation risk or female age (Table 2; Appendix S5).

Completing a breeding attempt to the fledging stage

On average, 96% of breeding attempts (N = 813) were completed. Clutch size and connectivity to goshawk nest sites explained the most variation in whether a breeding attempt was completed (Table 3; Appendix S6). Irrespective of clutch size, the percentage of a breeding attempts observed to reach the fledging stage was close to 100% (N = 193/194) for owls breeding in territories not well connected to goshawk nest sites, hence less exposed to predation (i.e. in territories not in close proximity to many goshawks nest sites; Fig. 3).

However, for owls breeding in territories relatively well connected to goshawk nest sites, hence exposed to predation (i.e. in close proximity to several goshawk nest sites in that year) the probability of breeding attempts being completed decreased from 97.5% (N = 39/40 breeding attempts) when owls had clutches containing four or more eggs to 87.7% (N = 57/65 breeding attempts) when clutches contained 1-2 eggs (Fig 3).

Juvenile survival

Juvenile survival averaged 0.18 ± SE 0.02. Autumn vole densities explained the most variation in juvenile survival (slope on logit scale: β = 0.42 ± 0.1; %Deviation = 34.5).

Juvenile survival was estimated to increase with autumn vole densities (Appendix S7

). There was no evidence of a relationship between juvenile owl survival and any proxy of predation risk (Table 4).

Discussion

In this study we examined how reproduction in female tawny owls (breeding propensity, clutch size and nest abandonment) was influenced by both extrinsic (food availability and predation risk) and intrinsic factors (age, previous and current allocation of resources towards reproduction) and any interactions between these factors. Our main findings were as follows: i) breeding propensity was highest in years when food (field vole densities in spring) was abundant and predation risk (goshawk abundance) was low. However, in years when goshawk abundance was relatively high the association between breeding propensity and food availability was less apparent. Breeding propensity also appeared to be related to intrinsic attributes (but to a lesser extent than extrinsic factors), as owls which had successfully fledged chicks the year before were slightly more likely to breed as they aged compared to owls which had not fledged chicks. ii) Clutch size was positively associated with spring vole densities but was unrelated to predation risk or any intrinsic attributes examined.

iii) On average 96% of breeding attempts were completed, however owls with small clutches (1-2 eggs), and breeding in territories more exposed to goshawk predation, were less likely to complete their breeding attempt compared to owls with larger clutches breeding in less exposed territories. iv) Juvenile owl survival was positively correlated with food availability in the autumn but was unrelated to predation risk. Overall, these findings represent rare evidence about how extrinsic and intrinsic factors interact to shape reproductive decisions in a long-lived iteroparous predator.

Breeding propensity

Breeding propensity was closely correlated with food availability (measured as field vole densities in spring) in the early years of the study, when predation risk (goshawk abundance) was relatively low (Fig. 1a). However, as predator abundance increased over the study period, the positive effect of food availability on breeding propensity diminished. These results indicate that breeding propensity is not purely constrained by the amount of food available prior to the breeding season. They also suggest that owls may be capable of assessing changes in predation risk and make facultative decisions about whether to allocate resources to reproduction, as shown for other species [START_REF] Sih | The effects of predators on habitat use, activity and mating behaviour of a semi-aquatic bug[END_REF][START_REF] Candolin | Reproduction under predation risk and the trade-off between current and future reproduction in the threespine stickleback[END_REF][START_REF] Ghalambor | Fecundity-survival trade-offs and parental risktaking in birds[END_REF][START_REF] Zanette | Perceived Predation Risk Reduces the Number of Offspring Songbirds Produce per Year[END_REF]. Unfortunately, we were unable to determine the exact nature of the link between food availability, predation risk and the observed changes in owl reproduction, as our approach was necessarily correlative, given the spatial scale of the processes considered. Therefore, we cannot rule out the possibility that changes other than average vole density in spring or goshawk abundance may have co-occurred to cause the observed variation in breeding propensity. However, we also examined whether changes in the spatial dynamics of food availability and predation risk were related to breeding propensity. Life history theory predicts that individuals should only forgo breeding when the cost of not breeding is compensated for by future reproductive gains [START_REF] Stearns | The Evolution of Life Histories[END_REF]). An analysis of breeding female owl survival in our study site suggests that it was lowest in years when goshawk abundance was relatively high and owl food availability was low (unpublished data). Consequently, we suggest that the higher breeding propensity observed in years when goshawks were abundant and food was scarce could plausibly reflect that these environmental conditions (being adverse for owls for a number of consecutive years towards the end of the study period) have made intermittent breeding a less beneficial strategy, as the cost of not breeding now is less likely to be compensated for in the future.

We also found evidence suggesting that a detectable but relatively small amount of variance in breeding propensity was associated with the age of the female owl and their previous allocation of resources towards reproduction, as breeding propensity increased slightly with age for females which had fledged chicks the previous year. This could indicate that some individuals are inherently of "high quality" and do not face a strong trade-off between current and future investment in reproduction. While, the effect sizes were relatively small in comparison with the strength of the correlations between breeding propensity and extrinsic conditions (food availability and predation risk; Fig. 1), our results demonstrate the dual intrinsic and extrinsic influence on the decision to reproduce.

Clutch size

The strong positive effect of food availability on clutch size is concordant with results from several other studies (Fig. 2; [START_REF] Ballinger | Reproductive strategies: food availability as a source of proximal variation in a lizard[END_REF][START_REF] Crawford | The influence of food availability on breeding success of African penguins Spheniscus demersus at Robben Island, South Africa[END_REF][START_REF] Lehikoinen | The impact of climate and cyclic food abundance on the timing of breeding and brood size in four boreal owl species[END_REF]).

However, we found no evidence of an association between clutch size and any proxy of predation risk. Due to the latitude of our study site, nights are relatively long prior to the breeding season. Hence, there is little overlap in the activity-periods of nocturnal tawny owls and diurnal goshawks, compared to late spring and summer when nights are relatively short.

Furthermore, female goshawks are thought to leave Kielder Forest in winter, returning in February, just prior to owls laying (unpublished data). Therefore, predation risk for owls might potentially be relatively low prior to the breeding season, when female owls are building up the body reserves needed for breeding, which could, in part, explain why we found no evidence of a relationship between clutch size and predation risk.

Completing a breeding attempt to the fledging stage

As predicted by life-history theory, individuals who had allocated more towards reproduction (e.g. by laying larger clutches), were more likely to continue their breeding attempt to the fledging stage, a finding consistent with previous studies (e.g. [START_REF] Delehanty | Effect of clutch size on incubation persistence in male Wilson's Phalaropes (phalaropus tricolor[END_REF].

Predation risk was the only extrinsic predictor of whether breeding attempts reached the fledging stage, with individuals breeding in territories more exposed to predation risk being less likely to complete a breeding attempt (Fig. 3); a result congruent with another study examining the effect of spatial variation in predation risk on reproductive success [START_REF] Sergio | Spatial refugia and the coexistence of a diurnal raptor with its intraguild owl predator[END_REF]. Goshawks start displaying over territories and building nests in late March and April in the UK [START_REF] Kenward | Breeding suppression and predator-prey dynamics[END_REF], hence are likely to become even more conspicuous to owls, after owls have already committed to breeding. Furthermore, predation risk for both adult and fledgling owls increased throughout the breeding season [START_REF] Petty | The decline of common kestrels Falco tinnunculus in a forested area of northern England: the role of predation by Northern Goshawks Accipiter gentilis[END_REF][START_REF] Hoy | Age and sex-selective predation as moderators of the overall impact of predation[END_REF]. Therefore, the tendency of owls not to complete breeding attempts in territories where predation risk is presumably high, is consistent with females (having already commenced breeding), attempting to reduce their own vulnerability to predation as the breeding season progresses. Alternatively, as 23% of breeders which did not complete a breeding attempt were never recaptured in the study site again, the higher failure rates in territories well connected to areas of high goshawk activity could also reflect that some parents in those territories were predated by goshawks and hence were unable to complete the breeding attempt.

Juvenile survival

Our analysis confirmed that juvenile owl survival was positively related to food availability [START_REF] Millon | Pulsed resources affect the timing of first breeding and lifetime reproductive success of tawny owls[END_REF][START_REF] Millon | Natal conditions alter age-specific reproduction but not survival or senescence in a long-lived bird of prey[END_REF][START_REF] Millon | Dampening prey cycle overrides the impact of climate change on predator population dynamics: A long-term demographic study on tawny owls[END_REF]. Estimates of juvenile owl survival were lowest in low vole years (Appendix S7). If mothers were able to predict the food conditions that their offspring would experience they should be less inclined to allocate resources towards reproduction in low vole years, due to the reduced probability of these offspring being recruited into the population. This may in part explain why individuals allocated relatively few resources towards reproduction (i.e. smaller clutch sizes) in years when food was scarce.

Reproductive strategies in relation to changing environmental conditions

A reproductive strategy can be defined as the set of decisions which influence the number of offspring an individual produces. Owl breeding strategies appeared to change in response to extrinsic conditions. Individuals allocated more resources towards reproduction (in terms of breeding propensity and clutch size) in years when food was abundant (Fig. 1 & Fig. 2).

Although we found no evidence to support our prediction that owls would attempt to minimise their vulnerability to predation by breeding less frequently or laying smaller clutches in years when predation risk was high, we did find evidence to suggest that owls responded to changes in predation risk, by making facultative decisions about whether to continue with their breeding attempt. However, the observed increase in incomplete nesting attempts with increasing predation risk also be partly due to parent(s) being killed, hence being unable to complete the breeding attempt, rather than a facultative decision not to continue the attempt. There was no year-to-year collinearity between our temporal covariates of predation risk and food availability. However, when averaged over a larger time scale (5 years) these covariates were correlated, and hence both environmental conditions changed simultaneously in opposite ways, with spring vole densities decreasing and predation risk increasing over the course of the study period. Therefore, we were unable to fully disentangle the effects of food availability and predation risk on owl breeding decisions. As the overall percentage of failed breeding attempts was very low (4% on average), the main reproductive decisions influencing reproductive output were primarily breeding propensity then clutch size. Indeed, the proportion of the population breeding and average clutch size explained 83% and 16% of the total variation in annual reproductive success (measured as the average number of chicks fledged per occupied owl territory) of the tawny owl population respectively. Whereas whether breeding attempts were completed only explained 0.1% of the total variation in reproductive success (see Appendix S8). Consequently, food availability seemed to have a greater impact on breeding propensity than changing predation risk (Fig. 1, Table 1) and be the main extrinsic factor driving variation in reproductive output, thus shaping reproductive strategies in tawny owls. However, the strength of the relationship between reproductive output and food availability weakened as predation risk increased.

As food availability declined (specifically as vole populations switched from high to low amplitude cycles; [START_REF] Cornulier | Europe-wide dampening of population cycles in keystone herbivores[END_REF] and predation risk increased, tawny owls seemed to breed more frequently, but invested less per breeding attempt. By spreading reproductive effort more evenly across years, a 'bet-hedging' reproductive strategy, minimises variation in reproductive success, and can actually increase an individual's fitness in certain situations [START_REF] Slatkin | Hedging one's evolutionary bets[END_REF][START_REF] Starrfelt | Bet-hedging-a triple trade-off between means, variances and correlations[END_REF]. Consequently, given that owl survival was lowest in years when food was scarce and goshawk abundance was high, our results could reflect that owls have switched from an intermittent reproductive strategy of saving resources to invest more in one, or a few reproductive attempts in the future, to a 'bet-hedging' reproductive strategy.

Together our results suggest that extrinsic conditions and intrinsic attributes have a combined and interactive effect on reproductive decisions. Changes in extrinsic conditions, particularly food availability, were the main factors shaping owl reproductive decisions, as the association between intrinsic attributes and owl breeding decisions was relatively weak in comparison.

This could in part be due to environmental variation in this system being relatively high because of the cyclical dynamics of vole populations, and the relatively recent recovery of an apex predator, thus swamping the contribution of intrinsic attributes to reproductive strategies. Although many of our results were in line with previous studies and theoretical predictions, our comprehensive approach highlights the complex nature of how intrinsic and extrinsic trade-offs act in combination to shape tawny owl reproduction. Furthermore, the length of this study has enabled us to provide some empirical evidence, albeit correlative, of long-lived predators altering their life-history strategies in response to changes in multiple interacting environmental factors.

Fig. 1 .

 1 Fig. 1. Variation in the probability of adult female tawny owls breeding in relation to changes

Fig. 3 .

 3 Fig. 3. The mean proportion of tawny owl breeding attempts which were observed to reach

Table 1 .

 1 Parameter estimates and model selection examining how tawny owls breeding 685 propensity varies in relation to fluctuations in predation risk (total goshawk abundance; local 686 goshawk abundance; connectivity of the owls territory to all predator nest sites; distance the 687 owl was nesting from the nearest predator) and food availability (spring vole densities; spatial 688 variation in vole densities across the study site). Breeding propensity was also analysed in 689 relation to whether the individual had successfully bred the previous year and the number of 690 years elapsed since the owl first started breeding (a measure of age). The most parsimonious

	691
	692	model is emboldened.
		Model	np	Estimate	SE	ΔAICc
		1. Null	3			27.99
		2. Total goshawk	4	0.40	0.24	27.37
		3. Local goshawk	4	0.45	0.25	27.08
		4. Connectivity to goshawks	4	-0.03	0.12	29.97
		5. Nearest goshawk	4	0.05	0.10	29.75
		6. Spring voles density	4	1.09	0.26	16.20
		7. Categorical spring vole density (CSV)	6	-0.83	0.56	23.87
		Spatial variation in vole densities (SVVD)		-0.62	0.44
		CSV x SVVD		0.03	0.60
		8. Breeding success previous year (BS)	4	0.34	0.22	27.81
		9. Years since 1 st reproduction (Y1st)	4	0.07	0.03	24.45
		10. Spring voles	5	1.14	0.23	10.84
		+ Local goshawk		0.51	0.18
		11. Spring voles (SV)	6	1.15	0.23	6.29
		+ Local goshawk (LG)		0.14	0.21
		SV x LG		-0.68	0.26
		12. Breeding success previous year	5	0.34	0.23	24.33
		+ Years since 1st reproduction		0.07	0.03
		13. Breeding success previous year	6	-0.30	0.35	21.03
		Years since 1st reproduction		-0.01	0.05
		BS x Y1st		0.14	0.06
		14. Breeding success previous year	9	-0.34	0.35	0
		Years since 1st reproduction		-0.01	0.05
		BS x Y1st		0.13	0.06
		Spring voles		1.17	0.23
		Local goshawk		0.13	0.22
		SV x LG		-0.69	0.26
	693
	694

Table 2 .

 2 Parameter estimates and model selection to determine whether variation in tawny owl investment in reproduction (clutch size) was related to proxies of predation risk (total goshawk abundance; local goshawk abundance; connectivity of the owls territory to all predator nest sites; distance the owl was nesting from the nearest predator), food availability (spring vole densities; spatial variation in vole densities across the study site) and intrinsic attributes (whether the individual had successfully bred the previous year and the number of years since the individuals first breeding attempt). The most parsimonious model is highlighted in bold.

	Model	np	Estimate	SE	ΔAICc
	1. Null	3			17.11
	2. Total goshawk	4	-0.035	0.032	17.99
	3. Local goshawk	4	-0.017	0.033	18.88
	4. Connectivity to goshawk	4	0.007	0.024	19.04
	5. Nearest goshawk	4	-0.007	0.022	19.02
	6. Spring vole density	4	0.125	0.023	0.00
	7. Categorical spring vole density (CSV)	6	-0.130	0.059	6.52
	Spatial variation in vole densities (SVVD)		-0.068	0.036
	CSV x SVVD		-0.020	0.060
	8. Breeding success previous year	4	0.028	0.046	18.75
	9. Years since 1st reproduction	4	0.002	0.006	18.97

Table 4 .

 4 Model selection for annual survival of female tawny owls in their first year of life 714 between 1985 and 2013 in relation to predation risk (total goshawk abundance; local goshawk 715 abundance; connectivity of the owls territory to all predator nest sites; distance the owl was nesting 716 from the nearest predator) and food availability (autumn vole density). Recapture probability was 717 modelled as [a(1,2-3,4+)+t]. The most parsimonious model is emboldened.

	718
	Models

Acknowledgments

We thank B. Sheldon and two anonymous reviewers for all their helpful comments on a previous version of the manuscript. Our thanks also go to M. Davison, B. Little, P. Hotchin, D. Anderson and all other field assistants for their help with data collection and Forest Enterprise, particularly Tom Dearnley and Neville Geddes for facilitating work in Kielder Forest. This work was partly funded by Natural Research Limited and a Natural Environment Research Council studentship NE/J500148/1 to SH and grant NE/F021402/1 to XL. Forest

Research funded all the fieldwork on goshawks, tawny owls and field voles during 1973-1996. In addition, we are grateful to English Nature and the BTO for issuing licences to visit goshawk nest sites.

Data accessibility

All data associated with the study which is not given in the text is available in the Dryad Digital Repository. http://dx.doi.org/10.5061/dryad.6n579.

Table 3. Model estimates and selection for analyses investigating the relationship between the probability of tawny owl breeding attempts being completed to the fledgling stage and proxies of predation risk (total goshawk abundance; local goshawk abundance; connectivity of the owls territory to all predator nest sites; distance the owl was nesting from the nearest predator), food availability (spring vole densities; spatial variation in vole densities across the study site) and attributes intrinsic to the breeder (whether they had successfully bred the previous year and the number of years since their first breeding attempt) and the breeding attempt (clutch size). The most parsimonious model is emboldened.

Model

Supporting Information

The following supporting information is available for this article:

Appendix S1: Estimating the number of tawny owls killed each year by the goshawk population.

Appendix S2: Method used to calculate the connectivity measure of predation risk for each owl territory.


Nicolas Lieury

Nolwenn Drouet-Hoguet

Sandrine Ruette
email: sandrine.ruette@oncfs.gouv.fr

Sébastien Devillard

Michel Albaret

Alexandre Millon

Reproductive senescence in the red fox Rural populations of the red fox Vulpes vulpes show little evidence of reproductive senescence

Keywords: Litter size, Vulpes vulpes, Placental scar count, Embryo count, Reproductive senescence

The ageing theory predicts fast and early senescence for fast-living species. We investigated whether the pattern of senescence of a medium-sized, fast-living and heavily-culled mammal, the red fox (Vulpes vulpes), fits this theoretical prediction. We used cross-sectional data from a large-scale culling experiment of red fox conducted over six years in five study sites located in two regions of France to explore the age-related variation in reproductive output. We used both placental scars and embryos counts from 755 vixens' carcasses aged by the tooth cementum method (age range: 1-10), as proxies for litter size. Mean litter size per vixen was 4.7 ± 1.4. Results from Generalized Additive Mixed Models revealed a significant variation of litter size with age. Litter size peaked at age 4 with 5.0 ± 0.2 placental scars and decreased thereafter by 0.5 cubs per year. Interestingly, we found a different age-specific variation when counting embryos which reached a plateau at age 5-6 (5.5 ± 0.2) and decreased slower than placental scars across older ages, pointing out embryo resorption as a potential physiological mechanism of reproductive senescence in the red fox. Contrary to our expectation, reproductive senescence is weak, occurs late in life and takes place at an age reached by less than 11.7% of the population such that very few females exhibit senescence in these heavily culled populations.

Introduction

Senescence, or ageing is the gradual deterioration of physical condition and cellular functioning, which results in a decline in fitness with age [START_REF] Kirkwood | Why do we age?[END_REF][START_REF] Sharp | Reproductive senescence in a cooperatively breeding mammal[END_REF]. Ageing can be expressed as a reduction in survival probability and/or a deterioration of reproductive efficiency, including decrease in the probability to give birth and reduced litter size. It is now recognized that both reproductive and actuarial senescence are widespread in the wild. Senescence rate greatly vary across individuals [START_REF] Bouwhuis | Individual variation in rates of senescence: natal origin effects and disposable soma in a wild bird population[END_REF], populations [START_REF] Lemaître | Early-late life trade-offs and the evolution of ageing in the wild[END_REF] and species [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF][START_REF] Nussey | Senescence in natural populations of animals: widespread evidence and its implications for biogerontology[END_REF]. Life-history theory provides a framework for predicting the variability of ageing across species. Major life-history traits, such as the age at first reproductive event, reproductive lifespan and number and size of offspring, vary across species, even when bodysize is controlled for [START_REF] Bielby | The fast-slow continuum in mammalian life history: an empirical reevaluation[END_REF][START_REF] Gittleman | Carnivore life history patterns: Allometric, phylogenetic, and ecological associations[END_REF][START_REF] Harvey | Life history variation in Primates[END_REF][START_REF] Read | Life history differences among the eutherian radiations[END_REF][START_REF] Stearns | The influence of size and phylogeny on patterns of covariation among life-history traits in the mammals[END_REF]. Such response led to the concept of "fast-slow continuum" of life-history variations, which categorises species from short-lived and highly reproductive species to long-lived species showing reduced reproductive output [START_REF] Cody | A general theory of clutch size[END_REF][START_REF] Cole | The population consequences of life history phenomena[END_REF][START_REF] Dobzhansky | Evolution in the tropics[END_REF][START_REF] Gaillard | An analysis of demographic tactics in bird and mammals[END_REF][START_REF] Lack | The significance of clutch size[END_REF][START_REF] Promislow | Living fast and dying young: A comparative analysis of life-history variation among mammals[END_REF][START_REF] Read | Life history differences among the eutherian radiations[END_REF][START_REF] Stearns | The influence of size and phylogeny on patterns of covariation among life-history traits in the mammals[END_REF]. As synthesised by [START_REF] Gaillard | Life Histories, Axes of Variation[END_REF], the fast-slow continuum can be interpreted as the range of possible solutions to the trade-off between reproduction and survival. The variation in ageing pattern along the continuum of senescence has been assessed by [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF]. These authors showed that both agespecific mortality and fertility patterns were strongly heterogeneous among vertebrates. Using data from 20 populations of intensively monitored vertebrates, they concluded that ageing is influenced by the species' position on the fast-slow continuum, which sets the principles of a continuum of senescence that predicts fast and early senescence for fast-living species [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF].

The red fox Vulpes vulpes is a medium-sized carnivore, known to have a fast reproductive rate with high productivity and early sexual maturity [START_REF] Englund | Some aspects of reproduction and mortality rates in Swedish foxes (Vulpes vulpes), 1961 -63 and 1966 -69[END_REF][START_REF] Harris | Age-related fertility and productivity in red foxes, Vulpes vulpes, in suburban London[END_REF][START_REF] Harris | Demography of two urban fox (Vulpes vulpes) populations[END_REF][START_REF] Ruette | Reproduction of the red fox Vulpes vulpes in western France: does staining improve estimation of litter size from placental scar counts?[END_REF]. According to the life history theory of ageing, red fox is therefore expected to display an early and fast senescence. To date, the demography of red fox has been mainly studied in anthropogenic contexts, and evidence of senescence in this species is mixed [START_REF] Artois | Reproduction du renard roux (Vulpes vulpes) en France: rythme saisonnier et fécondité des femelles[END_REF][START_REF] Cavallini | Reproduction of the red fox Vulpes vulpes in Central Italy[END_REF][START_REF] Harris | Age-related fertility and productivity in red foxes, Vulpes vulpes, in suburban London[END_REF][START_REF] Harris | Demography of two urban fox (Vulpes vulpes) populations[END_REF][START_REF] Marlow | Demographic characteristics and social organisation of a population of red foxes in a rangeland area in Western Australia[END_REF].

In France, red fox are hunted or even culled when locally classified as a pest species preying upon farmed and game species. Between 2002 and 2011, we conducted a fox culling experiment to measure the impact of removals on fox population dynamics in two rural regions [START_REF] Lieury | Compensatory Immigration Challenges Predator Control: An Experimental Evidence-Based Approach Improves Management[END_REF]. This landscape-scale experiment thus provided a unique opportunity to study the age-specific variation in reproduction. We addressed the variation in reproductive output with age, expecting an early onset of senescence. Recent papers have recommended a better comprehension of heterogeneity among life history traits in the wild, so as to improve the detection of cryptic senescence and its underlying mechanisms [START_REF] Hewison | Phenotypic quality and senescence affect different components of reproductive output in roe deer[END_REF][START_REF] Massot | An integrative study of ageing in a wild population of common lizards[END_REF][START_REF] Nussey | Senescence in natural populations of animals: widespread evidence and its implications for biogerontology[END_REF]. Thus, looking at a single reproductive trait might be misleading regarding senescence. Therefore we analysed two proxies of litter size (counts of placental scars and embryos) which may shed light on the underlying physiology of reproductive senescence.

Material and Methods

Study area and data collection

Data were obtained from culling campaigns performed as part of a large-scale culling experiment of the red fox in two French regions over six years [START_REF] Lieury | Compensatory Immigration Challenges Predator Control: An Experimental Evidence-Based Approach Improves Management[END_REF]. The carcasses of 899 vixens were collected in five distinct rural study areas (average size: 246 ± 53 km²; Fig. 1). All sites were located in the same range latitude: in Brittany (sites A, B and C; ≥10 km apart; 48°10'N, 03°00'W) and Champagne (sites D and E separated by the Seine River; 48°40'N, 04°20'E). Brittany landscape was dominated by bocage mixing farming and arable lands, with little forested area. In contrast, Champagne sites presented open field systems (mostly cereals and vineyard) and a larger forest cover compared to Brittany. The study took place from 2002 to 2011 but was not synchronous across all five sites. Hunting occurred between October and February, and trapping occurred between December and April.

Culling at the den occurred in April. Night shooting occurred only in sites D-E between December and May (see Lieury et al., 2015 for details).

Reproductive parameters

An estimation of the litter size could be made for 755 reproductive females with undamaged uterus among the 899 vixens collected (84%; Table 1). We used the number of embryos and the number of placental scars as two proxies for litter size (respectively on 394 and 361 individuals). When counting embryos only prenatal losses during early-pregnancy stages are considered while with placental scar counts, all losses between implantation and birth are taken into account. For pregnant females (i.e. females which were culled from February to April), embryos were counted. For the others, uteri were collected 12-48 h after the death of the animal, and soaked in water before freezing and stored at -20°C until examination. Uteri horns were opened longitudinally and examined for placental scars [START_REF] Elmeros | Placental scar counts and litter size estimations in ranched red foxes (Vulpes vulpes)[END_REF][START_REF] Lindström | Placental scar in the red fox (Vulpes vulpes L.) revisited[END_REF]. When the evaluation of litter size was questionable, we used a staining method to facilitate the identification of active placental scars [START_REF] Ruette | Reproduction of the red fox Vulpes vulpes in western France: does staining improve estimation of litter size from placental scar counts?[END_REF].

The staining method allows for the identification of atypical scars, i.e. with a singular aspect when compared to others from the same uterus or from other uteri at the same period of examination. However it does not permit the distinction of scars that could have persisted from earlier pregnancies from those that have been due to resorption or abortion [START_REF] Ruette | Reproduction of the red fox Vulpes vulpes in western France: does staining improve estimation of litter size from placental scar counts?[END_REF]. So, we did not estimate resorption rates from atypical placental scars counts.

Age determination and age classes

The age of foxes at death was determined from the carcasses based on the number of annual growth lines visible in the tooth cementum, the date of death and the expected date of birth on April 1 st [START_REF] Ruette | Reproduction of the red fox Vulpes vulpes in western France: does staining improve estimation of litter size from placental scar counts?[END_REF]. Canine teeth, or premolar teeth when canines were unavailable or damaged, were extracted from the lower jaw following Matson's laboratories (Milltown, MT, USA) procedures [START_REF] Harris | Age determination in the red fox (Vulpes vulpes) -an evaluation of technique efficiency as applied to as sample of suburban fixes[END_REF]. Foxes were assigned to age-classes based on their recruitment into the adult population on February 1 st of the year following birth (i.e. at the age of 10 months old). Animals between 10 and 22 months of age were classified as ageclass 1 (yearlings) whereas older ones were classified as age-class 2, 3, and up to 10.

Modelling and data analysis

Although the Poisson distribution has been often applied to the counts of offspring such as litter size, the Gaussian distribution actually fits better such reproductive data that are typically associated with a narrower variance than expected under a Poisson distribution (Devenish-Nelson et al., 2013a;[START_REF] Mcdonald | A Comparison of regression models for small counts[END_REF]. We thus developed a model for age-dependent variation in litter size accounting for both among-sites and among-years variability [START_REF] Artois | Reproduction du renard roux (Vulpes vulpes) en France: rythme saisonnier et fécondité des femelles[END_REF]Devenish-Nelson et al., 2013b;[START_REF] Ruette | Reproduction of the red fox Vulpes vulpes in western France: does staining improve estimation of litter size from placental scar counts?[END_REF] with a Gaussian distribution of error.

We used generalized additive mixed models (GAMM; [START_REF] Wood | Generalized additive models: an introduction with R[END_REF] to explore the relationship between vixen age and litter size without a priori hypothesis on its shape [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF]. Year and geographic area (study sites, 'Site', or region, 'Region') were tested as random factors to account for their potential confounding effects on litter size. Litter size may indeed depend on i) variations in habitat quality among sites or regions, ii) inter-annual variations in climate conditions or resources availability and iii) spatio-temporal variations of population densities between sites or regions.

Finally, we also tested the effect of the type of measure for litter size (i.e. placental scars vs. embryos) by adding a fixed effect 'Type' in the model.

We thus developed a full GAMM for the variations of litter size (LS) as follows: LS = s(Age)×Type + Age|Site + 1|Year,

The bars indicate the addition of a random effect of the 'Year' on the intercept (1) or of the site on the slope (Age). The parameterization s(Age)×Type denotes that the non-linear effect of vixen age was modelled independently for each type of the litter size proxy 'Type'.

Following [START_REF] Zuur | Mixed effects models and extensions in ecology with R[END_REF], we first started from the full random model and evaluated whether the age-specific variation in LS was similar among sites (random parameterisations: Age|Site vs. 1|Site), whether the spatial variation among regions was negligible when compared to the spatial variation among sites (1|Region vs. 1|Site) and whether the random effect of the year (1|Year) was important. According to [START_REF] Zuur | Mixed effects models and extensions in ecology with R[END_REF], parameters were estimated using Restricted Maximum Likelihood (REML) for random effects and Maximum Likelihood (ML) for fixed effects. Model selection was based on the AICc (Akaike Information Criterion corrected from small sample size; [START_REF] Burnham | Model selection and multimodel inference: a practical information-theoric approach[END_REF]. Once the random effects were selected, we performed an AICc-based model selection of fixed effects [START_REF] Zuur | Mixed effects models and extensions in ecology with R[END_REF] to test whether the type of measure affected age-specific variation in LS.

Finally, we estimated the rate of senescence by using least-squares linear regression models fitted through the mean values of each litter size, from the onset of senescence onwards, as predicted by the most parsimonious GAMM. Each point was weighted by the inverse of the variance so as to account for the small number of individuals in the oldest age classes.

All analyses were carried out in R.2.15.1 using packages mgcv and AICcmodavg (R Development Core Team, 2012;[START_REF] Wood | Generalized additive models: an introduction with R[END_REF]. Descriptive statistics of the data were presented as mean ± 1 SD and model estimates as mean ± 1 SE.

Results

Pooled over sites, years and age, litter size averaged 4.9 ± 1.4 when based on embryo counts and 4.5 ± 1.4 from counts of placental scars (see Table 2 for detailed results by age class).

From GAMM, all models including the random effects of the Year, the Site or the Region and the fixed effect of Age and Type had substantial support (ΔAICc < 2, Table 3). We retained the simplest of those models (Table 3). Placental scars count increased up to 5.0 ± 0.2 at the age of 4 (black line and dots in Fig. 2). From the age of 4 onwards, it significantly declined at a rate of senescence of 0.5 ± 0.02 cubs per year (Fig. 2). This pattern was consistent across study areas (random effects '1|Site' retained; Table 3.A), thereby suggesting that senescence pattern is likely to be a generalized process in red fox populations. We found divergence in senescence patterns between the two proxies of litter size (fixed effect s(Age)×Type retained; Table 3.B). Embryo counts peaked at age five but the rate of senescence in embryo counts afterwards was much reduced compared to placental scars (0.1 ± 0.01 cubs per year; Fig. 2). Finally, only a small proportion of females were killed after the age of 4 and 5 (11.7 and 5.6% respectively, median age at death: 2 years, Fig. 2), such that very few females exhibited senescence in these heavily culled populations.

Discussion

We took advantage of a large dataset collected over 10 years from a landscape-scale culling experiment in rural France, to investigate the deterioration in reproductive output with age in the red fox. Contrary to our expectation, our results revealed a weak and late reproductive senescence in this species. The onset of senescence occurred late (four years old) relatively to the age structure of the population (median age at death: two years old). The decline in litter size after four or five years old depending on the proxy used was significant but clearly more pronounced for placental scars count than for embryos count, suggesting increased embryo resorption as a likely physiological mechanism of senescence. This weak and late senescence concerned very few females in the populations (i.e. less than 11.7% of the females in the population reached the age of the onset of senescence) so that the impact of senescence on the dynamics of these heavily culled populations is likely to be negligible.

Limits inherent to post-mortem and cross-sectional data for investigating senescence

Monitoring reproductive performance in red fox is challenging on a large scale, due to its nocturnal, cryptic and elusive behaviour. We used post-mortem examination of carcasses to measure litter size and age. Although these methods may overcome some of the challenges of studying reproduction in free-ranging carnivore populations, we are aware of the inherent weaknesses in their applications. First, we estimated red fox age from cementum annuli lines in teeth. Although the method is widely used in carnivores studies such as red fox [START_REF] Harris | Age determination of badgers (Meles meles) from tooth wear: the need for a pragmatic approach[END_REF] or hyaena [START_REF] Van Horn | Age estimation and dispersal in the spotted hyena (Crocuta crocuta)[END_REF], misclassification has been noted due to some animals that did not develop a cementum line in one year (Grau et al., 1970 on raccoons;King, 1991 on stoats;[START_REF] Matson | Progress in cementum aging of martens and fishers[END_REF]. Deposition of cementum annuli and tooth wear may also vary with diet, season and region [START_REF] Costello | Reliability of the cementum annuli technique for estimating age of black bears in New Mexico[END_REF] on black bears). The method has not been applied on red foxes of known age. Thus we could not rule out some misclassifications although not quantifiable. Working with dead animals, we used placental scars and embryos counts as proxies for litter size. Placental scars counts provide a possible overestimate of litter size, due to embryos resorption, prenatal mortality and stillborn litters [START_REF] Vos | Reproductive performance of the red fox, Vulpes vulpes, in Garmish-Partenkirchen, Germany, 1987-1992[END_REF][START_REF] Elmeros | Placental scar counts and litter size estimations in ranched red foxes (Vulpes vulpes)[END_REF]. Inversely in a certain time postpartum, litter size might be underestimated by placental scars count due to the regeneration of uterine tissues [START_REF] Harris | Age-related fertility and productivity in red foxes, Vulpes vulpes, in suburban London[END_REF][START_REF] Harris | Demography of two urban fox (Vulpes vulpes) populations[END_REF][START_REF] Heydon | Demography of rural foxes (Vulpes vulpes) in relation to cull intensity in three contrasting regions of Britain[END_REF][START_REF] Lindström | Placental scar in the red fox (Vulpes vulpes L.) revisited[END_REF][START_REF] Marlow | Demographic characteristics and social organisation of a population of red foxes in a rangeland area in Western Australia[END_REF][START_REF] Mcilroy | The reproductive performance of female red foxes, Vulpes vulpes, in central-western New South Wales during and after a drought[END_REF][START_REF] Ruette | Reproduction of the red fox Vulpes vulpes in western France: does staining improve estimation of litter size from placental scar counts?[END_REF].

Our approach relies on the use of data from large-scale culling experiments to investigate senescence in five population replicates. Yet, the inference of senescence from life-table studies using cross-sectional data has been questionable for a long time. Indeed, the needs to consider sources of heterogeneity, such as unequal probability of sampling, individual heterogeneity, climate, density or early life conditions, advocate for following individuals throughout their life [START_REF] Gaillard | Senescence in natural populations of mammals: a reanalysis[END_REF][START_REF] Gaillard | An analysis of demographic tactics in bird and mammals[END_REF][START_REF] Nussey | Senescence in natural populations of animals: widespread evidence and its implications for biogerontology[END_REF][START_REF] Reid | Age-specific reproductive performance in red-billed choughs Pyrrhocorax pyrrhocorax: patterns and processes in a natural population[END_REF].

However, as non-selective methods of culling (trapping and hunting) were used, there is no reason to expect bias toward low or high reproductive individuals, since the age of adult's foxes could not be visually assessed. Moreover, we took into account the variability between populations by using samples from two contrasted regions and over several years. Nevertheless, it is important to consider both within (improvement, senescence) and betweenindividuals (selective appearance and disappearance) process in the estimation of patterns of age-dependent reproduction [START_REF] Reid | Age-specific reproductive performance in red-billed choughs Pyrrhocorax pyrrhocorax: patterns and processes in a natural population[END_REF][START_REF] Van De Pol | Age-dependent traits: a new statistical model to separate within and between individual effects[END_REF]. For instance, if individuals with high reproduction have poorer survival, mean reproduction may decline across older age because only individuals that invest little on reproduction survive. Selective disappearance has thus been found to partly mask the age-related changes in reproductive traits in ungulates [START_REF] Nussey | Measuring senescence in wild animal populations: towards a longitudinal approach[END_REF][START_REF] Nussey | The rate of senescence in maternal performance increases with early-life fecundity in red deer[END_REF].

We have no possibility to check for that kind of individual heterogeneity, determined by genetic and/or natal environment conditions. However, we found senescence in both traits i.e. numbers of placental scars and embryos and have no reason to expect a different sampling bias in vixens collected before or after parturition. Moreover, we did not observe a reduction in litter size variance with age expected in case of selective appearance or disappearance process (result not shown).

Besides, cross-sectional data are not systematically biased by individual heterogeneity and earlier studies revealing reproductive senescence from such data had been a posteriori validated by longitudinal data [START_REF] Hanks | Reproduction of elephant, Loxodonta africana, in the Luangwa Valley, Zambia[END_REF]. Hence, we are confident that our approach provides a relatively accurate picture of the age-related pattern in red fox reproduction. However, we call for long-term individual-based time series throughout longitudinal datasets to confirm senescence in free-ranging red fox populations.

Reproductive senescence in the red fox

Age-related reproductive output in red fox has long been discussed, but without reaching unanimous findings regarding senescence. Our results confirmed the increase of litter size with age among the young age-classes with a maximum reached at the age of 4-5 years old (see also [START_REF] Englund | Some aspects of reproduction and mortality rates in Swedish foxes (Vulpes vulpes), 1961 -63 and 1966 -69[END_REF][START_REF] Harris | Age-related fertility and productivity in red foxes, Vulpes vulpes, in suburban London[END_REF][START_REF] Lindström | Food limitation and social regulation in a red fox population[END_REF]. However, the decrease in litter size for older vixens has rarely been evidenced [START_REF] Artois | Reproduction du renard roux (Vulpes vulpes) en France: rythme saisonnier et fécondité des femelles[END_REF][START_REF] Cavallini | Reproduction of the red fox Vulpes vulpes in Central Italy[END_REF][START_REF] Marlow | Demographic characteristics and social organisation of a population of red foxes in a rangeland area in Western Australia[END_REF]. Moreover, litter size estimated from placental scars was even reported to be independent of age in several red foxes populations (France: [START_REF] Artois | Reproduction du renard roux (Vulpes vulpes) en France: rythme saisonnier et fécondité des femelles[END_REF]Central Italy: Cavallini and Santini, 1996;Denmark: Elmeros et al., 2003;and in Western Australia: Marlow et al., 2000). Here we were able to reveal a weak senescence pattern in reproduction in vixens from five to ten years old, and that affects reproduction at a rate of one cub less every two years when considering placental scars. [START_REF] Harris | Age-related fertility and productivity in red foxes, Vulpes vulpes, in suburban London[END_REF] and [START_REF] Harris | Demography of two urban fox (Vulpes vulpes) populations[END_REF] described for the first time reproductive senescence in London urban fox population. In a sample of 192 vixens, litter size significantly decreased in their fifth and sixth breeding season. Our results obtained in rural areas where fox densities are lower, are in accordance with those results. Interestingly in South-East Australia, i.e. in a context of invasion, reproductive parameters peaked in fifth-and sixth-year vixens, but vixens over eight years of age produced as many cubs as first-year breeders did [START_REF] Mcilroy | The reproductive performance of female red foxes, Vulpes vulpes, in central-western New South Wales during and after a drought[END_REF].

Reproductive senescence has been identified in several natural populations of mammals including ungulates, primates and domestic livestock [START_REF] Beehner | The ecology of conception and pregnancy failure in wild baboons[END_REF][START_REF] Ericsson | Age-related reproductive effort and senescence in free-ranging moose, Alces alces[END_REF][START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF][START_REF] Nussey | The rate of senescence in maternal performance increases with early-life fecundity in red deer[END_REF][START_REF] Promislow | Senescence in Natural Populations of Mammals: A Comparative study[END_REF]. To date only little evidence of reproductive senescence exists in carnivores, most of them focusing on long-lived species such as lions [START_REF] Packer | Reproductive success of lions[END_REF], bears [START_REF] Schwartz | Reproductive maturation and senescence in the female brown bear[END_REF]; but see Dugdale et al., 2011 on badgers). Only recently, senescence has been detected in free-ranging American mink, Neovison vison, a short-lived species with early age at first parturition [START_REF] Melero | Density-and age-dependent reproduction partially compensates culling efforts of invasive non-native American mink[END_REF].

The proposal formulated by [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF] that the magnitude of senescence is tightly associated with life history, mainly the slow-fast continuum, has been previously verified in populations of similar traits such as marmots [START_REF] Berger | Agespecific survival in the socially monogamous alpines marmot (Marmota marmota): evidence of senescence[END_REF], meerkats [START_REF] Sharp | Reproductive senescence in a cooperatively breeding mammal[END_REF], ground squirrels [START_REF] Broussard | Senescence and age-related reproduction of female Columbian ground squirrels[END_REF], opossums [START_REF] Austad | Retarded senescence in an insular population of Virginia opossums (Didelphis virginiana)[END_REF], and badgers [START_REF] Dugdale | Age-specific breeding success in a wild mammalian population: selection, constraint, restraint and senescence[END_REF]. Our findings provided evidence of weak reproductive senescence in the fast-living red fox, which occurred late (4-5 years old) relatively to the agestructure of our populations, and, therefore does not fully support the proposal of [START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF]. Furthermore, it concerned only very few females, since only a small proportion of vixens were killed after the age of 4 and 5.

Increasing embryo resorption with age: a physiological mechanism underpinning reproductive senescence?

Interestingly, senescence was more pronounced on placental scars than on the number of embryos. It suggested that gestation failure is the most likely cause of the decline in red fox litter size, rather than a decrease in ovulation rate. Spontaneous embryo resorption is an important issue in obstetrics, but also in livestock breeding and wildlife breeding programs. In wild species, increasing implantation failure with age has been identified in several taxa such as roe deer (Borg, 1970, Hewison andGaillard, 2011). Accordingly, reproductive senescence resulted from a combination uterine defects and reduction in oocyte numbers in elephant [START_REF] Hanks | Reproduction of elephant, Loxodonta africana, in the Luangwa Valley, Zambia[END_REF]. The success of embryo to develop depends on a complex series of cellular and molecular mechanisms associated with hormonal balance [START_REF] Cross | Implantation and the placenta: key pieces of the developmental puzzle[END_REF][START_REF] Finn | The implantation reaction[END_REF].

According to the disposal soma theory of ageing, individuals should invest less effort in the maintenance of somatic tissues for those that invested early in life, based on the best allocation of resources among the various metabolic tasks [START_REF] Kirkwood | Evolution of ageing[END_REF][START_REF] Kirkwood | Evolution of senescence: late survival sacrificed for reproduction[END_REF]. In the case of red fox, the ageing of reproductive tracts (mainly uterus) probably plays an important role in the decrease of red fox litter size with age.

Finally, our study highlighted that reproductive senescence occurs in red fox populations, although being weak and occurring late in life. The consequences of reproductive senescence on red fox population dynamics might be negligible due to the low proportion of females in the population that reached the age at the onset of senescence. In the context of intensive removal through hunting and trapping acting on population densities [START_REF] Lieury | Compensatory Immigration Challenges Predator Control: An Experimental Evidence-Based Approach Improves Management[END_REF], a proper assessment of the effect of the variation in population density and removal pressure over time and populations on the reproductive performance is needed to investigate process such as compensatory reproduction. Lines represent GAMM predictions (plain) and their associated standard error (dashed).

Figure 1 .

 1 Figure 1. Location showing the sites where the landscape-scale culling experiments of red

Figure 2 .

 2 Figure 2. Variation in litter size of the red fox in relation to the age of vixens (in years)

  Fig. 1

Acknowledgements

We are grateful to the regional and local Hunter's associations, and we warmly thank all people involved in field activity. We are grateful to the regional and local Hunters' Associations, especially Y. Desmidt, J.-L. Pilard, P. Hecht, C. Mercuzot, J. Desbrosse, and C. Urbaniak for sustaining the program. We warmly thank F. Drouyer, B. Baudoux, N. Haigron, C. Mangeard, and T. Mendoza for efficient support in the fieldwork, our colleagues working on hares, especially Y. Bray and J. Letty and all local people in charge of hunting, and hunters and trappers who helped in counting and collecting foxes. This study was partially funded by the Regional Hunters' Association of Champagne-Ardenne, and the Hunters' Associations of Aube and Ille-et-Vilaine.

This work was supported by the Regional Hunters' Association of Champagne-Ardenne, and the Hunters' Associations of Aube and Ille-et-Vilaine.

Reproductive senescence in the red fox