Mathilde L Tissier
email: mathilde.tissier@hotmail.com

Patrick Bergeron

Dany Garant

Sandrine Zahn

François Criscuolo

Denis Réale

Telomere length positively correlates with pace-of-life in a sex-and cohort-specific way and elongates with age in a wild mammal

Keywords: Aging, life-history strategies, evolutionary trade-offs, reproduction, personality, telomere length

Understanding ageing and the diversity of life histories is a cornerstone in biology. Telomeres, the protecting caps of chromosomes, are thought to be involved in ageing, cancer risks and to modulate lifehistory strategies. They shorten with cell division and age in somatic tissues of most species, possibly limiting lifespan. The resource allocation trade-off hypothesis predicts that short telomeres have thus coevolved with early reproduction, proactive behaviour and reduced lifespan, i.e. a fast Pace-of-Life Syndrome (POLS). Conversely, since short telomeres may also reduce the risks of cancer, the anti-cancer

Published in Molecular Ecology

hypothesis advances that they should be associated with slow POLS. Conclusion on which hypothesis best supports the role of telomeres as mediators of life-history strategies is hampered by a lack of study on wild short-lived vertebrates, apart from birds. Using seven years of data on wild Eastern chipmunks Tamias striatus, we highlighted that telomeres elongate with age and do not limit lifespan in this species.

Furthermore, short telomeres correlated with a slow POLS in a sex-specific way. Females with short telomeres had a delayed age at first breeding and a lower fecundity rate than females with long telomeres, whereas those differences were not recorded in males. Our findings support most predictions adapted from the anti-cancer hypothesis, but none of those made under the resource allocation trade-off hypothesis. Results are in line with an increasing body of evidence suggesting that other evolutionary forces than resource allocation trade-offs shape the diversity of telomere length in adult somatic cells and the relationships between telomere length and life-histories.

Introduction

Biologists are striving to comprehend the physiological and evolutionary mechanisms that underlie the wide range of life-history strategies and the diversity of ageing patterns observed across the tree of life [START_REF] Jones | Diversity of ageing across the tree of life[END_REF][START_REF] Kirkwood | Understanding the Odd Science of Aging[END_REF][START_REF] Ricklefs | The physiology/life-history nexus[END_REF]. According to the life-history theory, individuals trade resources devoted towards current reproduction for those devoted towards maintenance and survival [START_REF] Clutton-Brock | Reproductive Effort and Terminal Investment in Iteroparous Animals[END_REF][START_REF] Kirkwood | Understanding the Odd Science of Aging[END_REF][START_REF] Stearns | The evolution of life histories[END_REF], such that early breeders may age faster than late ones. Still, some species escape that pattern [START_REF] Jones | Diversity of ageing across the tree of life[END_REF]. Yet, the physiological mechanisms underlying ageing remain poorly understood and are still debated [START_REF] Kirkwood | Understanding the Odd Science of Aging[END_REF][START_REF] Lemaître | Ecoevolutionary perspectives of the dynamic relationships linking senescence and cancer[END_REF][START_REF] Ricklefs | The physiology/life-history nexus[END_REF][START_REF] Sahin | Linking functional decline of telomeres, mitochondria and stem cells during ageing[END_REF]. Among diverse interacting molecular markers, telomeres, the protective caps at the end of chromosomes, are widely considered as biomarkers of organismal ageing and longevity [START_REF] Fairlie | Lifelong leukocyte telomere dynamics and survival in a free-living mammal[END_REF][START_REF] Heidinger | Telomere length in early life predicts lifespan[END_REF][START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF][START_REF] Sahin | Linking functional decline of telomeres, mitochondria and stem cells during ageing[END_REF].

One of the current and dominant thinking in life-history is based on the fact that resources are limited, such that allocation trade-offs occur between three key functions: growth, reproduction and selfmaintenance [START_REF] Kirkwood | Understanding the Odd Science of Aging[END_REF][START_REF] Stearns | The evolution of life histories[END_REF] . These functions are associated with physiological costs, such as oxidative stress and accumulation of oxidative damage, or costs associated with telomere maintenance [START_REF] Alonso-Alvarez | Increased susceptibility to oxidative stress as a proximate cost of reproduction[END_REF][START_REF] Metcalfe | Compensation for a bad start: grow now, pay later?[END_REF][START_REF] Monaghan | Oxidative stress as a mediator of life history trade-offs: mechanisms, measurements and interpretation[END_REF]; S. M. [START_REF] Smith | Meta-analysis indicates that oxidative stress is both a constraint on and a cost of growth[END_REF][START_REF] Von Zglinicki | Oxidative stress shortens telomeres[END_REF]. These costs limit the ability of an organism to allocate energy to all three functions and potentially cause ageing, driving life-history strategies [START_REF] Metcalfe | Growth versus lifespan: perspectives from evolutionary ecology[END_REF][START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF][START_REF] Monaghan | Oxidative stress as a mediator of life history trade-offs: mechanisms, measurements and interpretation[END_REF]. In terms of telomeres, the resource allocation trade-offs hypothesis predicts that long telomeric sequences during pre-reproductive life, or at the time of reproduction, are associated with prolonged lifespan [START_REF] Eastwood | Early-life telomere length predicts lifespan and lifetime reproductive success in a wild bird[END_REF][START_REF] Heidinger | Telomere length in early life predicts lifespan[END_REF]. Such long telomeric sequences are also associated to reduced investment into early reproduction, including reduced first litters and delayed reproduction [ [START_REF] Bauch | Telomere length reflects phenotypic quality and costs of reproduction in a long-lived seabird[END_REF][START_REF] Bauer | Chronological and Biological Age Predict Seasonal Reproductive Timing: An Investigation of Clutch Initiation and Telomeres in Birds of Known Age[END_REF][START_REF] Ouyang | Do Hormones, Telomere Lengths, and Oxidative Stress form an Integrated Phenotype? A Case Study in Free-Living Tree Swallows[END_REF], reviewed in [START_REF] Giraudeau | Do Telomeres Influence Pace-of-Life-Strategies in Response to Environmental Conditions Over a Lifetime and Between Generations[END_REF][START_REF] Sudyka | Does Reproduction Shorten Telomeres? Towards Integrating Individual Quality with Life-History Strategies in Telomere Biology[END_REF]; but see [START_REF] Vaillant | Telomere length reflects individual quality in free-living adult king penguins[END_REF]] or to a greater lifetime reproductive success [i.e. LRS [START_REF] Eastwood | Early-life telomere length predicts lifespan and lifetime reproductive success in a wild bird[END_REF][START_REF] Heidinger | Longer telomeres during early life predict higher lifetime reproductive success in females but not males[END_REF]; reviewed in [START_REF] Sudyka | Does Reproduction Shorten Telomeres? Towards Integrating Individual Quality with Life-History Strategies in Telomere Biology[END_REF]]. The Pace-of-Life Syndrome hypothesis (POLS) posits that physiological and behavioural traits (e.g. exploration profile) have co-evolved with life-history traits by mediating the trade-offs between current and future reproduction [START_REF] Montiglio | The pace-of-life syndrome revisited: the role of ecological conditions and natural history on the slow-fast continuum[END_REF][START_REF] Réale | Personality and the emergence of the pace-of-life syndrome concept at the population level[END_REF][START_REF] Ricklefs | The physiology/life-history nexus[END_REF]. Based on differential resource allocation trade-offs at the individual level, the POLS hypothesis proposes that telomeres may mediate individual variation in pace-of-life along a slow-fast continuum [START_REF] Giraudeau | Do Telomeres Influence Pace-of-Life-Strategies in Response to Environmental Conditions Over a Lifetime and Between Generations[END_REF]. Within a population, individuals with long telomeres in their pre-reproductive life are thus expected to display a slower POLS than individuals with short telomeres (Figure 1, [START_REF] Giraudeau | Do Telomeres Influence Pace-of-Life-Strategies in Response to Environmental Conditions Over a Lifetime and Between Generations[END_REF].

Positive relationships between telomere length or telomere dynamics and longevity, fecundity rate or LRS have been confirmed at the intraspecific level [START_REF] Bichet | Telomere length is repeatable, shortens with age and reproductive success, and predicts remaining lifespan in a longlived seabird[END_REF][START_REF] Bize | Telomere dynamics rather than age predict life expectancy in the wild[END_REF][START_REF] Eastwood | Early-life telomere length predicts lifespan and lifetime reproductive success in a wild bird[END_REF][START_REF] Heidinger | Telomere length in early life predicts lifespan[END_REF][START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF][START_REF] Sudyka | Does Reproduction Shorten Telomeres? Towards Integrating Individual Quality with Life-History Strategies in Telomere Biology[END_REF]. However, when considering inter-specific comparisons, the resource allocation trade-offs hypothesis fails to explain the relationship between telomere length and lifespan [START_REF] Haussmann | Telomeres shorten more slowly in long-lived birds and mammals than in short–lived ones[END_REF][START_REF] Risques | All's well that ends well: why large species have short telomeres[END_REF]. While it would predict that telomere length increases with species size and longevity, the reverse pattern is observed: large and long-lived species have shorter telomeres than small and short-lived ones [START_REF] Risques | All's well that ends well: why large species have short telomeres[END_REF][START_REF] Seluanov | Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan[END_REF]. In addition, although telomeres are widely reported to shorten with age, due to cell division or oxidative stress, in birds and humans [START_REF] Bichet | Telomere length is repeatable, shortens with age and reproductive success, and predicts remaining lifespan in a longlived seabird[END_REF][START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF][START_REF] Pauliny | Age-independent telomere length predicts fitness in two bird Species[END_REF], an increasing body of evidence show that this pattern is not universal among vertebrates [START_REF] Remot | Decline in telomere length with increasing age across nonhuman vertebrates: A meta-analysis[END_REF] and even that telomere elongation may occur seasonally or in older individuals in several species [START_REF] Criscuolo | Seasonal variation in telomere dynamics in African striped mice[END_REF]Hoelzl, Smith, et al., 2016;[START_REF] Panasiak | Telomere Dynamics in the Diploid and Triploid Rainbow Trout (Oncorhynchus mykiss) Assessed by Q-FISH Analysis[END_REF][START_REF] Spurgin | Spatio-temporal variation in lifelong telomere dynamics in a long-term ecological study[END_REF]. Furthermore, the negative relationship between age and telomere length is absent in some species [START_REF] Fairlie | Lifelong leukocyte telomere dynamics and survival in a free-living mammal[END_REF][START_REF] Foley | Growing old, yet staying young: The role of telomeres in bats' exceptional longevity[END_REF]Hoelzl, Smith, et al., 2016;[START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF], questioning the generality of this pattern and on the role of telomeres as biomarkers of senescence under the resource allocation trade-offs hypothesis.

An alternative hypothesis has recently emerged, seeking to explain why large and long-lived species have shorter telomeres than small and short-lived ones: the anti-cancer hypothesis [START_REF] Pepke | On the comparative biology of mammalian telomeres: Telomere length co-evolves with body mass, lifespan and cancer risk[END_REF][START_REF] Seluanov | Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan[END_REF][START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF]. This evolutionary hypothesis advances that long lifespans have coevolved with short telomeres [START_REF] Risques | All's well that ends well: why large species have short telomeres[END_REF][START_REF] Seluanov | Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan[END_REF][START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF] because of a trade-off between preventing senescence and avoiding cancer [START_REF] Lemaître | Ecoevolutionary perspectives of the dynamic relationships linking senescence and cancer[END_REF][START_REF] Pepke | On the comparative biology of mammalian telomeres: Telomere length co-evolves with body mass, lifespan and cancer risk[END_REF][START_REF] Risques | All's well that ends well: why large species have short telomeres[END_REF]. Long-lived species do not invest in costly mechanisms to prevent the shortening of telomeres as a mechanism to limit cancer, but pay a senescence cost [START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF]. In contrast, because they have long telomeres, short-lived species have a low probability to live old due to higher risks of developing cancer, and thus follow a "live fast and die young strategy". Shortlived species have thus evolved different types of telomere maintenance compared to large species and do not show replicative senescence as a mechanism to protect from cancer [START_REF] Lemaître | Ecoevolutionary perspectives of the dynamic relationships linking senescence and cancer[END_REF][START_REF] Risques | All's well that ends well: why large species have short telomeres[END_REF][START_REF] Seluanov | Telomerase activity coevolves with body mass not lifespan[END_REF][START_REF] Seluanov | Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan[END_REF]. Hence, they can overcome this senescence-cancer trade-off and have longer telomeres than long-lived species [START_REF] Risques | All's well that ends well: why large species have short telomeres[END_REF][START_REF] Seluanov | Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan[END_REF][START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF]. So far, this anti-cancer hypothesis has only been applied to species comparisons and mostly focuses on mammals [START_REF] Pepke | On the comparative biology of mammalian telomeres: Telomere length co-evolves with body mass, lifespan and cancer risk[END_REF][START_REF] Risques | All's well that ends well: why large species have short telomeres[END_REF]. It has not been used to explain variation of telomere dynamics at the inter-individual level. To do so, the patterns observed must be cleaned from any age effect, since even species that maintain an active telomerase in the adult soma, display telomere shortening with age (François Criscuolo et al., 2018). Still, selective forces and evolutionary mechanisms leading to the negative link between telomere length and lifespan at the inter-species level, should find their origin at the within-species level, and may vary between large and small-bodied species [START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF]. In addition, intra-individual differences in telomere mechanisms should be detectable already early in life (i.e. as they would be associated with short or long-lived individual trajectories) [START_REF] Noguera | Embryonic and postnatal telomere length decrease with ovulation order within clutches[END_REF]. Under high cancer-risks, when applied to the within-species level, the anti-cancer hypothesis predicts opposite relationships between telomere length and POL than the resource allocation trade-offs hypothesis, with long telomeres being associated with a fast pace-of-life (Figure 1). The resource allocation trade-offs and the anti-cancer hypotheses are not mutually exclusive but could translate into different, and opposite, selective pressures on telomere length [START_REF] Gomes | Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination[END_REF][START_REF] Pepke | On the comparative biology of mammalian telomeres: Telomere length co-evolves with body mass, lifespan and cancer risk[END_REF][START_REF] Risques | All's well that ends well: why large species have short telomeres[END_REF][START_REF] Young | The role of telomeres in the mechanisms and evolution of life-history trade-offs and ageing[END_REF].

the study of the greatest diversity of taxa. However, studies on reptiles, amphibians and wild, small and short-lived mammals are particularly rare [START_REF] Pepke | On the comparative biology of mammalian telomeres: Telomere length co-evolves with body mass, lifespan and cancer risk[END_REF][START_REF] Remot | Decline in telomere length with increasing age across nonhuman vertebrates: A meta-analysis[END_REF][START_REF] Wilbourn | The relationship between telomere length and mortality risk in non-model vertebrate systems: a metaanalysis[END_REF] telomere length and life history is best supported by the resource allocation trade-offs hypothesis or the anti-cancer hypothesis in this species. Studies on this population revealed that behavioural traits, such as exploration profile, are an integral part of the POLS as they correlate with several life-history traits; i.e. fast explorers display fast life histories, namely an early age at first reproduction and a high fecundity [START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF][START_REF] Montiglio | Intra-individual variability in fecal cortisol metabolites varies with lifetime exploration and reproductive life history in eastern chipmunks (Tamias striatus)[END_REF]. We thus tested whether telomere length at first breeding opportunity was linked to speed of exploration and age at first breeding (Figure 1B.a & b). We also investigated if telomeres shorten with age, as observed in most vertebrates studied [START_REF] Bichet | Telomere length is repeatable, shortens with age and reproductive success, and predicts remaining lifespan in a longlived seabird[END_REF][START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF][START_REF] Pauliny | Age-independent telomere length predicts fitness in two bird Species[END_REF], or not, as predicted under the anti-cancer hypothesis(Figure 1B.c). We then assessed the presence of a correlation between telomere length and two common measures of the pace-of-life: fecundity rate and lifespan (Figure 1B.d & e). Finally, the "phenotypic quality" hypothesis has received a lot of attention from studies looking at the role of telomeres as mediators of life-history trade-offs [START_REF] Eastwood | Early-life telomere length predicts lifespan and lifetime reproductive success in a wild bird[END_REF][START_REF] Giraudeau | Do Telomeres Influence Pace-of-Life-Strategies in Response to Environmental Conditions Over a Lifetime and Between Generations[END_REF][START_REF] Heidinger | Telomere length in early life predicts lifespan[END_REF], but see [START_REF] Bergeron | Individual quality: tautology or biological reality[END_REF]. This hypothesis derivates from the resource allocation trade-offs hypothesis and states that individuals who inherited long telomeres or benefited from good environmental conditions during key life history stages, such as early life, may be able to invest more energy in all life-history traits. In addition to predictions made under the life-history trade-offs hypothesis, the phenotypic quality hypothesis thus posits that a positive relationship should exist between telomere length in early life and LRS, as individuals with long telomeres are assumed to successfully cope with the costs of reproduction [START_REF] Sudyka | Does Reproduction Shorten Telomeres? Towards Integrating Individual Quality with Life-History Strategies in Telomere Biology[END_REF]. We therefore tested this by looking at the relationship between telomere length and LRS (Fig. 1B.f).

Figure 1. Expected relationships between telomere length (TL) and life histories under the resource allocation trade-offs and anti-cancer hypotheses (A) and detailed predictions on the expected relationships between pre-reproductive TL, life-history and behavioural traits (B). We define pre-reproductive telomere length here as telomere length before reproduction (early life TL measured in juveniles) or at first reproduction (TL at first breeding opportunity). Traits characterizing the pace-of-life (a, b, d, e) and the expected relationship between TL and age (c) or TL and LRS (f) are shown. Empty white symbols and dashed lines are attributed to the resource allocation trade-off hypothesis, filled coloured symbols and solid lines to the anti-cancer hypothesis (slow individuals are in grey and fast individuals are in blue, for references on other figures). The shape of the chipmunk symbols represents the speed of the pace-of-life (slow VS fast). * According to the anti-cancer hypothesis, no significant erosion of telomeres would be expected with age in small-bodied species under high cancer risk, considering that small-bodied species have evolved different telomere maintenance and anti-cancer mechanisms than large species [START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF]. ** Within the framework of the resource allocations trade-offs hypothesis, the phenotypic quality hypothesis posits that individuals with long telomeres cope well with the costs of reproduction and display a greater LRS. Under these hypotheses, LRS will depend on the outcome between fecundity rate and lifespan.

Materials and Methods

Model species, study system and site

We monitored a population of Eastern chipmunks in a deciduous forest of southern Québec, Canada (Mont-Sutton; 45°05′ N, 72°25′ W), on three sites from 2013 to 2019 (Sites 1 and 2 of 7.84 ha each, and site 3 of 4 ha). Eastern chipmunks are omnivorous, food-hoarding Sciurid rodents that display periodic torpor events in winter (Elliott, 1978;Humphries, Thomas, & Speakman, 2002). Each year, from May to September, we conducted daily trapping from 8:00 a.m. until dusk, using Longworth traps (Bergeron, Réale, Humphries, & Garant, 2011a). Upon capture, chipmunks were marked with ear tags and a Trovan PIT tag and we recorded their body mass, sex, reproductive status and determined their age class (juveniles or adults; see supplementary methods). Individuals weighing > 80 g at first capture in May or displaying reproductive signs over the entire reproductive season were considered as adults. We classified as juvenile individuals weighing less than 70 g at first capture and showing no signs of sexual maturity over the entire season, i.e. absence of darkened scrotum for males and undeveloped mammae for females (Bergeron et al., 2011;[START_REF] Tissier | Consumption of red maple in anticipation of beech mast-seeding drives reproduction in eastern chipmunks[END_REF]. Chipmunks weighing between 70 g and 80 g at first capture and that could not be classified as juveniles or adults were considered as NA (Bergeron, Réale, et al., 2011a;[START_REF] Tissier | Consumption of red maple in anticipation of beech mast-seeding drives reproduction in eastern chipmunks[END_REF].

From 2013 to 2019, we captured a total of 819 chipmunks, including 209 adults at their first breeding opportunity (Table S1). Ear tissue punches were collected on each individual for genotyping. We measured relative telomere length (rTL) on all the individuals for which untouched tissue or DNA samples were remaining after genotyping, resulting in 70 individuals with a rTL measurement at first breeding opportunity (27 females and 43 males; see Table S1). Capitalizing on a long-term monitoring of the population, we also had data on behavioural and life-history traits for a portion of these individuals, namely on their exploration profile (n=48), fecundity rate (n=37), LRS (n=35) and minimum lifespan (n=51). Chipmunks live on average 30 months (Bergeron, Réale, et al., 2011a). The maximum recorded lifespan is 7 years in this population (unpublished results). Previous studies showed that chipmunks exclusively reproduce in the summer before and in the spring after a beech mast in autumn in this system (Bergeron, Réale, et al., 2011a;[START_REF] Tissier | Consumption of red maple in anticipation of beech mast-seeding drives reproduction in eastern chipmunks[END_REF]. Individuals cannot reproduce before their first winter.

Therefore, first breeding opportunity either occurs at 7 or 15 months-old for the summer and spring cohorts, respectively (Montiglio, Garant, Bergeron, Dubuc Messier, & Réale, 2014). First breeding opportunity corresponds to the first time for which we can have a pre-reproductive measure on most residents, considering the low recapture rate of juveniles. Note that we make the distinction between first breeding opportunity, which occurs at either 7 or 15 months old, and age at first effective reproduction, which may occur at 22 months old, for individuals that failed to reproduce at their first opportunity [START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF].

Tissue collection and DNA extraction

We collected ear tissue punches upon capture per individual during their first two years of life. Since we usually do not collect DNA samples on individuals older than two years, a sample was specifically collected on a subsample of 3-6-year-old individuals (n=12), among which 7 individuals for which we had a measure of rTL at first breeding opportunity, to investigate changes in telomere length with age (see below). Upon collection, ear tissue punches were transferred to 95% ethanol Eppendorf tubes, transferred from the field to laboratory conditions within 2-4 hours where they were stored at +4°C until extraction, which occurred within two months after collection. Upon extraction, DNA samples were stored at -80°C. DNA extraction was carried out using a modified version of a validated salting-out method (Aljanabi & Martinez, 1997; see supplementary methods). Briefly, after allowing ethanol to evaporate from the tissue sample, saline extraction buffer (2 mM ethylenediaminetetraacetic acid, 10 mM Tris-HCl, 0.4 M NaCl), sodium dodecyl sulphate and proteinase K were added to the sample and incubated overnight at 60 °C.

The following morning, more proteinase K was added; the mixture was vortexed and incubated for 2 additional hours at 60 °C. Once digested, 300 μl of 6 M saline solution was added and the mixture was vortexed and centrifuged for 30 min at 6000 g. We then transferred 600 μl of the aqueous solution into a clean 1.5-ml microtube. The DNA was precipitated by a 1-h incubation at 20 °C with 600 μl isopropanol and pelleted by centrifugation for 20 min at 10 000 g. Pellet was rinsed with 200 μl of 70% ethanol, spun again at 10 000 g for 10 min, and left to air dry overnight. The next day, the pellet was resuspended using 200 μl of distilled water. After having determined the DNA concentration using gel electrophoresis, samples were diluted to a concentration of 5 ng/μl for polymerase chain reaction (PCR) amplification and frozen at -80°C until being processed for genotyping or telomeres measurements.

Genotyping and parental assignment

We conducted microsatellite genotyping using 23 loci previously employed in the study system (Bergeron, Réale, Humphries, & Garant, 2011b;Chambers & Garant, 2010;Vandal, Houle, Archambault, Réale, & Garant, 2020). The detailed genotyping protocol, is described in (Chambers & Garant, 2010;Vandal et al., 2020). The parentage assignment protocol is fully described in (Bergeron, Réale, et al., 2011b). Briefly, we captured weaned juveniles that emerged from their known maternal burrow. We used CERVUS (Kalinowski, Taper, & Marshall, 2007) to corroborate the maternal identity and assigned paternity based on microsatellite analyses (Chambers and Garant 2010). When we had no information on the maternal burrow (i.e. when juveniles were captured during systematic trapping on the grid), we used COLONY (Wang & Santure, 2009) to reconstruct family groups (Bergeron et al., 2011b).

Assessment of relative telomere length (rTL)We used DNA extracted from ear tissue punches collected in spring to measure relative telomere length (rTL, expressed as T/S ratio). Telomeres length is commonly measured on ear tissues in rodents (Hehar & Mychasiuk, 2016;Turbill et al., 2013), and strongly correlates with telomere length in other tissues, including brain tissues such as hippocampus and prefrontal cortex (Hehar & Mychasiuk, 2016) or buccal mucosa (Turbill et al., 2013). In total, we obtained rTL on 204 chipmunks of known age, 74 at their first breeding opportunity (among which 70 for which we had lifehistory data as detailed above), 106 at emergence from the maternal burrow, 12 individuals aged 18-24 months, and 12 individuals aged 3-6 years. There were 20 individuals for which we had two rTL measurements in spring. At their first breeding opportunity, individuals were either 7 months or 15 months of age [START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF]. In both cases, individuals were captured in spring after their first winter. Individuals aged 7 months were about to initiate reproduction at that time whereas individuals aged 15 months already initiated it. To investigate change in telomere length with age, we randomly selected 20 out of 30 individuals for which we had sufficient remaining DNA to measure rTL at two ages in spring, ensuring to have a representative sampling of our population (samples from different years, sexes, and ages; see Table S1). We also measured rTL on juveniles sampled upon emergence from the maternal burrow (i.e. weaning, ~30-45 days of age). The average recapture rate of juveniles after their first winter is 14.5% in our population (9% for males and 20% for females; unpublished data, see also [START_REF] Loew | Sex-biased dispersal in eastern chipmunks, Tamias striatus[END_REF] reporting similar proportions in other chipmunk populations). Combined with the exclusion of 3 individuals that were aged of 1 year-old at the time of the study, this reduced our initial sample of 106 to a subset of 12 juveniles for which we had data on both telomere length and lifespan.

We first assessed the quality of each DNA sample by gel electrophoresis (DNA integrity: samples were conserved only in absence of smears). Using a Nanodrop ND1000 (ThermoFisher Scientific, Waltham, MA, USA) we then measured DNA concentration and purity of each sample looking at the ratios of absorbance at 260nm (ratio 260/280) and at 230nm (ratio 230/260). We fixed exclusion criteria at a minimum concentration of 10ng/µL, and ratios at 1.7-2.0 (260/280) and >1.5 (230/260). Regarding the qPCR reaction, the rTL (T/S ratio) measurement is based on the determination of a number of amplification cycles necessary to detect a lower threshold of fluorescent signal. Cycle number is proportional to the sample telomere length (T), or to the number of copies of a non-variable copy number gene (or control gene S used as a reference). As a reference non-variable copy number (non-VCN) gene, we used a 54 bp portion of the c-myc proto-oncogene, which was tested for non-variability in copy number (i.e. we checked in the genome that there were not 2 copies of the gene in chipmunks), as described for other species [START_REF] Nussey | Measuring telomere length and telomere dynamics in evolutionary biology and ecology[END_REF][START_REF] Smith | Chasing telomeres, not red herrings, in evolutionary ecology[END_REF]. We first select 2 (2-5) non-VCN genes candidate sequences. Then we conducted a pilot study comprising a subset of 10 (10-20) samples (on a range of samples representing both sexes and all our populations used in the final sample set) to allow pairwise correlation of rTLs estimated from the different non-VCN gene. We then conducted rTL measurements by quantitative real-time amplification (Cawthon, 2002) adapted for ear tissues and for Eastern chipmunks.

Forward and reverse telomeric primers were 5'-CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG -3' and 5'-GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT -3', respectively. Primer sequences for the non-VCN gene were 5'-GAG GGC CAA GTT GGA CAG TG -3', and 5' -TTG CGG TTG TTG CTG ATC TG -3', respectively.

Measurements were done on two 384-well plates. We added 10 ng (2 μl) of DNA in each well, together with 8 μl of GoTaq QPCR Mix (Promega, Madison, WI, USA); with an extra 100 µM final concentration of dNTPs; and forward and reverse primers at 5 µM. Cycling conditions included: 1) an initial hold at 95 °C for 2 min; 2) 30 cycles (telomere) of 95 °C for 15 sec, 56 °C for 30 sec, and 72 °C for 30 sec or 40 cycles (non-VCN) of 95 °C for 15 sec, 60 °C for 30 sec and 72°C for 1 min, with fluorescence readings recorded at this step; and 3) a final melt step was set at 60 °C to 95 °C in 0.5 °C increments (see Figure S1). Cycling was performed on an Automated Thermal Cycler, Q real-time PCR instrument (CFX-384, Biorad, Hercules, CA, USA). Each plate contained samples ran in duplicates, a no-template control (sterile water used for dilution of samples), duplicates of 4 individuals and duplicates of a control well of 10 ng of 100 pooled individuals' DNA sample (i.e. repeated on each qPCR and used as the "golden sample", the reference sample with a value of T/S = 1, used to calculate the values of the other points). Calculation of rTL (T/S ratio) was carried-out using the software LinRegPCR (V.12.5) as described in (Pfaffl, 2001).. Efficiencies of qPCR reactions were of 99.9% for the control and 99.8% for telomeres in plate 1 and of 100% for the control and 99.9% for telomeres in Plate 2. Intraclass correlation coefficient for intra-and inter-runs (ICC; based on 12 samples repeated over the two 385-well plates; see (Eisenberg, Nettle, & Verhulst, 2020;Verhulst et al., 2016)) were of 0.976 and 0.992, respectively. Regarding measurements on change in rTL with age, samples were spread over the two plates as follows: we measured the two rTL of a same individual on two different plates for 9 individuals (three on plate 1 and six on plate 2) and on the same plate for 11 individuals (three on plate 1, eight on plate 2), thus preventing that plate effect would influence the results.

Life-history and exploration variables

We considered two measures of fecundity: the average number of offspring per reproductive event as a measure of fecundity rate, and the lifetime number of assigned offspring at weaning as a measure of the lifetime reproductive success (LRS). We calculated the minimum lifespan in months, as the difference between the date of last capture and either the date of first capture (i.e. emergence from the maternal burrow) or the date of latest possible birth for the cohort (i.e. for individuals missed at emergence from the burrow and only captured after their first winter). For instance, given the recorded patterns of reproduction on our study sites since 2012 [START_REF] Tissier | Consumption of red maple in anticipation of beech mast-seeding drives reproduction in eastern chipmunks[END_REF], an unmarked adult first caught in spring 2013 would be assigned to an emergence date in June 2012 (spring cohort). An individual first captured in June 2016 would be assigned to an emergence date in September 2015 (summer cohort). We considered only residents for this analysis and thus excluded 7 individuals based on this criterion, considering that we had data on telomere length at first breeding opportunity and exploration profile for those individuals but no information about their life-history. We also excluded 12 individuals that were still alive and aged one year-old for the same reasons. We considered as "residents" individuals that were present on the sites at their first reproductive opportunity (dispersion rarely occur afterwards; Elliott 1978) and that were caught more than 5 times during their life/over several seasons (to exclude potential dispersers or individuals at the periphery of the sites, for which we would not have accurate life-history data).

We quantified chipmunk behavioural response to a novel environment, as a measure of their exploratory behaviour and their activity, by conducting open-field tests (Gharnit, [START_REF] Tissier | Consumption of red maple in anticipation of beech mast-seeding drives reproduction in eastern chipmunks[END_REF]Montiglio, Garant, Thomas, & Réale, 2010). All the open-field tests were conducted on site. Upon capture, individuals were placed in an opaque tubular entrance for one minute for acclimation before the test.

Then, we gently pushed them inside the open-field arena (80 x 80 x 40 cm, without any refuge and gridded at the base) and closed the entrance with a trapping grid [START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF][START_REF] Réale | Personality and the emergence of the pace-of-life syndrome concept at the population level[END_REF]. Chipmunk behaviour was videotaped for 90 s and we hereafter counted the number of lines crossed during this period (using The Observer XT11 software; Nodus). Individuals were tested twice, allowing us to calculate individual's best linear unbiased predictor (BLUPs) for exploration (Gharnit et al., 2020). We subsequently simulated 1000 series of BLUPs and used the standardized mean individual exploration BLUPs of the 1000 estimates to characterize the exploration profile (from slow/thorough to fast/superficial) of each individual in our analyses (Gharnit et al., 2020).

Data Analyses

Model 1 -Determinants of the rTL at first breeding opportunity

We investigated which variables influenced rTL at first breeding opportunity using a Linear Model (LM).

The rTL was log-transformed and Z-transformed before being included in the models [START_REF] Heidinger | Telomere length in early life predicts lifespan[END_REF]Verhulst, 2020). Considering the reported effects of age and sex, alone or in interaction, on TL [START_REF] Barrett | Sex differences in telomeres and lifespan[END_REF][START_REF] Bauch | Sex-specific telomere length and dynamics in relation to age and reproductive success in Cory's shearwaters[END_REF][START_REF] Heidinger | Longer telomeres during early life predict higher lifetime reproductive success in females but not males[END_REF], we included age at first breeding opportunity (categorical variable), sex and their interaction as fixed effects in our model. Note that age at first breeding opportunity is a life-history stage, which occurs either at 7 or 15 months old, depending on whether individuals were born in the previous spring or summer (Bergeron, Réale, et al., 2011a;[START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF]. Site, exploration score and the age*exploration and sex*exploration interactions were also included as fixed effects. These two-way interactions were reported to affect other life-history traits in this species (Bergeron, Réale, et al., 2011a;[START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF]. Finally, individual body mass was included as a covariate, as a proxy of growth and because telomere length covaries with body mass [START_REF] Pepke | On the comparative biology of mammalian telomeres: Telomere length co-evolves with body mass, lifespan and cancer risk[END_REF].

Models 2, 3 and 4 -Relationship between rTL and life-history traits

We investigated the links between rTL at first breeding opportunity and fecundity (response variable, expressed as the total number of offspring weaned or sired /number of reproductive event) using a LM (model 2), assuming the normality of the residuals, which was confirmed by a Kolmogorov-Smirnov test.

We investigated the relationship between rTL and LRS using a Generalized Linear Model (GLM; model 3) with a Poisson distribution, after verifying the absence of overdispersion. Finally, we used a LM to investigate the relationship between rTL at first breeding opportunity and minimum lifespan. We assessed the normality of the residuals with a Kolmogorov-Smirnov test (LM, model 4). As normality was not met, lifespan was expressed in Log10(months+1) [START_REF] Eastwood | Early-life telomere length predicts lifespan and lifetime reproductive success in a wild bird[END_REF]. We included age at first breeding opportunity (7 or 15 months), average adult body mass (linear term in models 2 and 3 and both the linear and quadratic terms in model 4 to account for the quadratic relationship between body mass and lifespan in our data; unpublished results), sex, site, rTL and the double-way interactions (rTL*sex, rTL*age at first breeding opportunity and sex*age at first breeding opportunity) as fixed effects in the three models. We faced issues of model convergence when including the year of birth as a random effect in two of the three models. We thus included year as a fixed continuous effect in all full models. We also included the minimum lifespan as fixed effect in models 2 to account for the duration of breeding opportunities. Body mass, year, and exploration score (mean individual exploration BLUP) were standardized (Z-transformed) before inclusion in the models.

Models 5, 6 and 7: relationships between age, rTL and telomere dynamic and between rTL at weaning and lifespan

Using Linear Mixed Models (LMMs, model 5), we investigated the effect of age on telomere length. We first tested the relationship between age and rTL in spring at the population level (n=204), with age, sex, site, cohort of birth and sex*cohort of birth as fixed effects, and the year of birth as a random effect to account for measurements on individuals born within the same years. Then, we looked at the relationship between age and rTL at the individual level, using the 20 individuals for which we had repeated spring measurements of telomere length (model 6). We included sex and age as fixed effects and the ID (intercept) and age (slope) as random effects in this model to separate within-from between-subject effects (van de [START_REF] Van De Pol | A simple method for distinguishing within-versus between-subject effects using mixed models[END_REF]. We tested for within-individual repeatability in rTL by estimating the marginal R 2 using a LMM as detailed in [START_REF] Spurgin | Spatio-temporal variation in lifelong telomere dynamics in a long-term ecological study[END_REF]. A recent meta-analysis highlighted that within individual repeatability in telomere length depends on the duration of the study (time between the two measures of telomere length in a same individual; see [START_REF] Kärkkäinen | Within-individual repeatability in telomere length: A meta-analysis in nonmammalian vertebrates[END_REF].

Considering that it varied from 7 to 428 months in our study, we also conducted the same analysis on individuals for which there was <12 months between the two measures of telomere length (n=11). We finally assessed the relationship between early life rTL and lifespan on the subset of 12 juveniles, using a LM (model 7).

Model selection and parsimony

Starting from the full models, we removed one by one the least significant variables, only if their removal reduced the AICc . Both full and reduced models are presented in the online supplementary material (Tables S2-S6). We implemented false discovery rates to correct for multiple comparisons (Pike, 2011)

Results

Correlates of telomere length at first breeding opportunity

We found no effect of age on rTL at first breeding opportunity (t=0.34, p=0.74) but found a significant exploration*age interaction (LM, t=3.42, p=0.001; model 1, Table S2B): rTL increased with speed of exploration, but only for individuals experiencing a first breeding opportunity at 15 months of age (R 2 = 0.64, Figure 2A, Table S2B). We also found an effect of the sex*age interaction, with females that had a first breeding opportunity at 7 months of age showing longer telomeres than females having a first breeding opportunity at 15 months of age (LM, t=2.62, p=0.01), whereas we found no differences in males (Figure 2B, Table S2B).

Figure 2. Determinants of relative Telomere Length (rTL) at first breeding opportunity. In (A) interaction between exploration and age at first breeding opportunity on rTL (Table S2, n=48, sample sizes by age at first breeding, 7 months: n=14 males and 13 females; 15 months: n=14 males and 7 females; sex symbols 380 are solely represented on the figures for informative purposes; no interaction with sex was found). The 381 dashed line represents a non-significant relationship in individuals aged 7 months. In (B) the interaction 382 between sex and age at first breeding opportunity on rTL is shown (means ± SE; Table S2). Females whose 383 first breeding opportunity occurred at 7 months old had significantly longer telomeres than females with a 384 first breeding opportunity at 15 months old, whereas no significant difference was observed in males. The 385 rTL, expressed as T/S ratio, has been LN transformed before conducting the analyses. Z-scores are 386 presented for exploration and rTL. 387

Change in telomere length with age

We found a within-individual repeatability in rTL of R = 0.126 for all 20 chipmunks for which we had to measures of rTL. Repeatability for the subset of individuals sampled at <12 months was R = 0.378. We found a positive relationship between age and rTL at the population level (model 5, LMM, n=204, F 1;190 =37.57, t=6.13, p<0.001, Figure 3A), but no effect of the sex (F 2;169.6 =1.26, p=0.71), cohort of birth (F 2;28.5 =0.56, p=0.85) or their two-way interaction (F 3;169.7 =1.00, p=0.32; Table S3A).

We also found that telomere length of both male and female chipmunks increased with age at the within individual level (model 6, LMM, n=20, t=3.981, p<0.001, Table S3B), although two individuals displayed a slight reduction in rTL with age and one female showed no elongation or shortening (Figure 3B). Our LMM revealed no sex effect on change in telomere length (n= 9 males and 11 females , t=0.42, p=0.67, Table S3B). positive relationship between age and rTL was recorded (R 2 =0.12, t=6.18, p<0.001, Table S3A), with an appearance of long telomeres (i.e. with a T/S ratio > 2.70) in individuals of >7 months of age (these long telomeres were not recorded in juveniles sampled at emergence from the maternal burrow, 0 months, n=106). (B) Repeated measurements of relative telomere length in individual chipmunks sampled twice in their life in spring. Telomeres increased significantly with age (t=3.98, p<0.001, Table S3B). Data are displayed for females (diamonds and black lines) and males (triangles and grey lines) as informative purposes (we found no significant difference between the sexes, Table S3B); plates are also represented by dotted, dashed and solid lines to highlight that elongation was not caused by methodological artefacts.

Telomere length at first breeding opportunity and life-history traits

We found no effect of age or body mass on fecundity rate, expressed as the average number of offspring per reproductive event (model 2, p>0.05; Table S4). However, we found that the sex*rTL interaction had a significant effect on this trait (model 2, t=2.75, FDR-adjusted p-value = 0.02; Table S4): fecundity rate positively covaried with rTL at first breeding opportunity in females, but not in males (Figure 4A, R 2 females =0.33, p=0.001). Regarding LRS, however, we found no significant link between rTL at first breeding opportunity and LRS, nor of the sex*rTL interaction, that were both excluded from the final selected model (model 3, Figure 4B & Table S5). Our model, however, revealed a positive and significant correlation between lifespan and LRS (χ 2 =4.50, p=0.03; Table S5). No effects of the cohort of birth (spring or summer)

were found on either the fecundity rate or the LRS (p>0.05, Table S4 & Table S5).

Figure 4. Relationship between relative Telomere length (rTL) at first breeding opportunity and (A) fecundity rate (average number of offspring per reproductive event) or (B) LRS, in males (triangles) and in females (diamonds). We found a sex*rTL interaction effect on fecundity rate (Table S4) but no effect of rTL or the interaction on LRS (Table S5). The full line represents a significant relationship between rTL and fecundity rate in females (A; Table S4), whereas the dashed lines represent no significant relationships.

Regarding lifespan, we found no significant effect of rTL nor of the age at first breeding opportunity*rTL interaction (model 4, t=0.63 & t=1.43, FDR-adjusted p-values = 0.60 and p= 0.24, respectively; Table S6). We only observed a significant effect of the year of birth on lifespan (t=3.07, FDR-adjusted p = 0.02; Table S6). A supplementary analysis on a subset of 12 juveniles revealed that telomere length at the emergence from the maternal burrow (i.e. weaning) also tended to be negatively associated with longevity (model 7, R 2 =0.33, p=0.05; Figure 5B). (diamonds) are represented for informative purposes. We found no effects of rTL on lifespan and no effects of sex or age at first breeding opportunity on the relationship between rTL and lifespan (Table S6). In (B) early life rTL, measured at emergence from the maternal burrow, is shown with lifespan expressed in months. The dashed line represents a non-significant linear regression. The rTL, expressed as the T/S ratio, is presented as Z scores of LN transformed data.

Discussion

We found that telomeres elongated with age both at the population level and within individuals, although we initially predicted that telomeres length would either shortened with age or would be maintained , under the resource allocation trade-offs and the anti-cancer hypotheses, respectively. Telomere length at first breeding opportunity positively correlated with speed of exploration, age and subsequent fecundity rate (thus with a fast pace-of-life), but did so in a sex-specific or cohort-specific manner. However, we found no correlation between telomere length and LRS or lifespan. Our results, thus, supported three of the five predictions from the anti-cancer hypothesis, but none of the predictions from the resource allocations tradeoffs or the phenotypic quality hypotheses (Figure 1). Our findings suggest that resource allocation trade-offs alone are unlikely to drive ageing and the evolution of life-histories in this species.

Correlates of telomere length at first breeding opportunity

We found that fast and superficial explorers, reported to have a higher fecundity rate than slow and thorough explorers in this population [START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF], also had longer telomeres, but solely in late breeders (i.e. individuals whose first breeding opportunity occurred at 15 months old). This result confirms predictions derived from the anti-cancer hypothesis. It echoes findings from a previous study on wild chipmunks, highlighting that fast explorers showed lower daily energy expenditure levels than did slow explorers, contrary to predictions made under the resource-allocation trade-offs hypothesis (Careau et al., 2015). The absence of relationship between telomere length and exploration profile in early breeders in our study could be explained by a greater variation in their exploration profile or telomere length compared to late breeders, as already reported for exploration in this species [START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF]. This greater variation and the absence of a significant relationship with telomere length could represent different behavioural/life-history strategies that could be genetically fixed in late breeders compared to early ones.

Alternatively, it could reflect a greater plasticity of early breeders compared to late ones. Early-breeding female chipmunks (7 months of age) were shown to be faster explorers than late-breeding ones in the studied population [START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF]. Here, we show that these early-breeding females have longer telomeres than late-breeding females, as predicted by the anti-cancer hypothesis. Because telomeres elongated with age in this population, this result cannot be explained by a simple age difference between females of the two groups (i.e. with older females having shorter telomeres because of a greater erosion with age). This result suggests that, for the same age, females with a first breeding opportunity before 15 months of age had longer telomeres than females with a first breeding opportunity at 15 months of age.

However, given the intrinsic link between cohort of birth and age at first breeding opportunity in this system, early breeding and late-breeding females would never have the same age at the same period of their life cycle (Bergeron et al. 2011[START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF][START_REF] Tissier | Consumption of red maple in anticipation of beech mast-seeding drives reproduction in eastern chipmunks[END_REF]. Comparing their telomere length at the same age would thus be biased by life-cycle stage and season, and therefore by varying intrinsic and environmental factors that could have an influence on their telomeres. Together, our findings support the idea that rTL early in life positively covaries, in a cohort-and sex-specific manner, with speed of exploration and age at first breeding opportunity, two metrics commonly used to map individuals along the slow-fast continuum of the POLS (Araya-Ajoy et al., 2018;[START_REF] Réale | Personality and the emergence of the pace-of-life syndrome concept at the population level[END_REF]. These results also suggest that resource allocation trade-offs alone are unlikely to drive the evolution of life-histories in this system and that other forces, such as cancer risks, are at play (see [START_REF] Cohen | Are trade-offs really the key drivers of ageing and life span?[END_REF][START_REF] Kokko | Towards cancer-aware life-history modelling[END_REF][START_REF] Lemaître | Ecoevolutionary perspectives of the dynamic relationships linking senescence and cancer[END_REF][START_REF] Pepke | On the comparative biology of mammalian telomeres: Telomere length co-evolves with body mass, lifespan and cancer risk[END_REF]).

Telomere elongation with age

Following the anti-cancer hypothesis, telomeres were not predicted to shorten with age, but were not expected to significantly elongate at the within individual and population levels (Figure 3). Most studies conducted so far found that telomeres do shorten with age in humans, captive-bred animals and wild, longlived vertebrates (Bichet et al., 2019;[START_REF] Bize | Telomere dynamics rather than age predict life expectancy in the wild[END_REF][START_REF] Eastwood | Early-life telomere length predicts lifespan and lifetime reproductive success in a wild bird[END_REF][START_REF] Heidinger | Telomere length in early life predicts lifespan[END_REF][START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF][START_REF] Sudyka | Does Reproduction Shorten Telomeres? Towards Integrating Individual Quality with Life-History Strategies in Telomere Biology[END_REF]. This usually translates into shorter telomeres being measured in older than in younger individuals at the population level [START_REF] Haussmann | Telomeres shorten more slowly in long-lived birds and mammals than in short–lived ones[END_REF][START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF][START_REF] Pauliny | Age-independent telomere length predicts fitness in two bird Species[END_REF]. Here, we found the reversed patterns, with a positive relationship between age and rTL (Figure 3A) and a within individual elongation of telomeres with age (Figure 3B). This positive relationship cannot solely be explained by a selective disappearance of individuals with short telomeres (Dantzer & Fletcher, 2015), as an appearance of long telomeres (with a T/S ratio >2.70, which is above the maximum recorded in the 106 juveniles sampled) seem to appear with age in this population (Figure 3A). Within individual repeatability in telomere length was R=0.128 (whole sample) and R=0.378 (subset of individuals sampled at <12 months apart), which is equivalent or above repeatability recorded in other mammal studies using qPCR essays [START_REF] Fairlie | Lifelong leukocyte telomere dynamics and survival in a free-living mammal[END_REF][START_REF] Van Lieshout | Individual variation in early-life telomere length and survival in a wild mammal[END_REF]. However, repeatability within individuals is based on several hypotheses: 1) telomere length on year n is predictive of what will happen in year n+1; 2) telomere length shorten with age and 3) the rate of shortening is similar between individuals of a same cohort. In our case, however, these hypotheses are not met, especially considering that we did not record a no reduction in telomere length with age but a lengthening.

Considering the significant elongation of telomeres recorded at the within individual level combined with the significant increase observed at the population level (n=204), also reported in other species (Hoelzl, Smith, et al., 2016;[START_REF] Spurgin | Spatio-temporal variation in lifelong telomere dynamics in a long-term ecological study[END_REF], telomere elongation with age is unlikely to be due to a methodological artefact of the qPCR method. It is also unlikely to be caused by a simple increase in interstitial telomeric sequences as suggested in previous work [START_REF] Aksenova | At the Beginning of the End and in the Middle of the Beginning: Structure and Maintenance of Telomeric DNA Repeats and Interstitial Telomeric Sequences[END_REF]. One FISH-labelling study conducted in a squirrel species indeed found no specific staining of telomere probes at the interstitial location and highlighted that the occurrence of multiple interstitial nucleolar organizer regions is rare in rodents [START_REF] Fagundes | Multiple interstitial ribosomal sites (NORs) in the Brazilian squirrel Sciurus aestuans ingrami (Rodentia, Sciuridae) with 2n= 40: An overview of Sciurus cytogenetics[END_REF]. However, using the native telomeric restriction fragment assay (TRF) to measure the length of terminal telomeres would allow to confirm these statements. The native TRF assay allows to obtain an absolute telomere length measurement (without interstitial telomeric sequences), unlike the qPCR assay, which only provides an estimated relative telomere length compared to a control gene (S. [START_REF] Smith | Chasing telomeres, not red herrings, in evolutionary ecology[END_REF]. However, the TRF assay is difficult to implement on longitudinal studies of small wild mammals because it requires high concentrations of DNA difficult to obtain with least-invasive approaches (Dantzer et al., 2020;[START_REF] Nussey | Measuring telomere length and telomere dynamics in evolutionary biology and ecology[END_REF][START_REF] Smith | Chasing telomeres, not red herrings, in evolutionary ecology[END_REF].

Few other studies also found a positive relationship between age and telomere length [START_REF] Haussmann | Telomeres shorten more slowly in long-lived birds and mammals than in short–lived ones[END_REF]Hoelzl, Smith, et al., 2016). In edible dormice (Glis glis), young individuals significantly shorten their telomeres with age while old individuals (>6 years old) slightly elongate their telomeres when approaching the end of their life (Hoelzl, Smith, et al., 2016). This pattern has been attributed to the peculiar life-history strategy of this species, that may skip reproduction and hibernate for up to almost a year [START_REF] Hoelzl | Telomere dynamics in free-living edible dormice (Glis glis): the impact of hibernation and food supply[END_REF]Hoelzl, Smith, et al., 2016). Here, we found that telomere elongation occurred in chipmunks of all ages. A within-individual seasonal elongation of telomeres has also been recorded in few rodent species [START_REF] Criscuolo | Seasonal variation in telomere dynamics in African striped mice[END_REF]Hoelzl, Cornils, et al., 2016). In our study, telomere length was always measured in spring. We can, thus, exclude a potential seasonal effect on the elongation pattern observed, which was recorded both in young and in old individuals. However, we cannot exclude that an environmental effect -e.g. pulsed availability of food resources that have a strong impact on behaviour, reproduction and life-history strategies in this system (Bergeron, Réale, Humphries, & Garant, 2011a;[START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF][START_REF] Tissier | Consumption of red maple in anticipation of beech mast-seeding drives reproduction in eastern chipmunks[END_REF] -may partially explain this elongation (Hoelzl, Cornils, et al., 2016). Nonetheless, the observation of telomere elongation with age under controlled conditions with unlimited food resources and using the Q-FISH procedure (see [START_REF] Panasiak | Telomere Dynamics in the Diploid and Triploid Rainbow Trout (Oncorhynchus mykiss) Assessed by Q-FISH Analysis[END_REF], highlights that food alone unlikely explains these patterns of telomere elongation.

Our results, combined with recent literature on telomere dynamics in small [START_REF] Criscuolo | Seasonal variation in telomere dynamics in African striped mice[END_REF][START_REF] Foley | Growing old, yet staying young: The role of telomeres in bats’ exceptional longevity[END_REF]Hoelzl, Smith, et al., 2016) and large mammals (Kirby, Johnson, Alldredge, & Pauli, 2019;Lemaître et al., 2021), as well as in fishes [START_REF] Panasiak | Telomere Dynamics in the Diploid and Triploid Rainbow Trout (Oncorhynchus mykiss) Assessed by Q-FISH Analysis[END_REF] and birds [START_REF] Spurgin | Spatio-temporal variation in lifelong telomere dynamics in a long-term ecological study[END_REF], pinpoint that a much greater variety of telomere dynamics patterns may exist than previously reported, including within-individual telomere elongation, and that these patterns likely correlate with pace of life. Proximal mechanisms explaining telomere elongation in chipmunks will have to be investigated in the future, but we can hypothesize that telomerase activity is involved (S. [START_REF] Smith | Telomerase activity in ecological studies: What are its consequences for individual physiology and is there evidence for effects and trade-offs in wild populations[END_REF]. Telomerase is the enzyme that repairs telomeres (Chan & Blackburn, 2004). It is repressed after growth in the somatic tissues of large and long-lived vertebrates, but not in the somatic tissues of small and short-lived ones (Chan & Blackburn, 2004;[START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF]. Among 18 rodent species, Eastern chipmunks show the highest bodymass specific telomerase activity in their somatic tissues [START_REF] Seluanov | Telomerase activity coevolves with body mass not lifespan[END_REF][START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF]. We initially expected that this high activity of telomerase would prevent telomere erosion with age. However, it could in fact be involved in the significant and consistent telomere elongation that we recorded at the within individual and population levels, as observed, for instance, in sperm of large mammals, including humans, where telomerase is maintained active [START_REF] Kimura | Offspring's Leukocyte Telomere Length, Paternal Age, and Telomere Elongation in Sperm[END_REF][START_REF] Kozik | Increased telomere size in sperm cells of mammals with long terminal (TTAGGG)n arrays[END_REF][START_REF] Ozturk | Telomerase Activity and Telomere Length in Male Germ Cells[END_REF][START_REF] Tian | Evolution of telomere maintenance and tumour suppressor mechanisms across mammals[END_REF]. However, the qPCR assay did not allow us to assess the strength of this elongation, nor to compare the absolute length of telomeres recorded in this population with other studies in rodents, which could be done using the TRF or Q-FISH approaches [START_REF] Kozik | Increased telomere size in sperm cells of mammals with long terminal (TTAGGG)n arrays[END_REF][START_REF] Nussey | Measuring telomere length and telomere dynamics in evolutionary biology and ecology[END_REF]. Future studies could tackle those issues by optimizing DNA extraction from buccal swabs, which offers a promising avenue to obtain sufficient DNA in small mammals with a non-invasive technique [START_REF] Hoelzl | Telomere dynamics in free-living edible dormice (Glis glis): the impact of hibernation and food supply[END_REF][START_REF] Kozik | Increased telomere size in sperm cells of mammals with long terminal (TTAGGG)n arrays[END_REF], while assessing whether telomerase, beyond its impact on telomere length or dynamics, acts as a mediator of life-history strategies [START_REF] Criscuolo | Experimental manipulation of telomere length: does it reveal a corner-stone role for telomerase in the natural variability of individual fitness[END_REF][START_REF] Smith | Telomerase activity in ecological studies: What are its consequences for individual physiology and is there evidence for effects and trade-offs in wild populations[END_REF].

Early-life telomere length and life-history traits

Fecundity rates positively co-varied with telomere length, as predicted by the anti-cancer hypothesis, but did so in a sex-specific manner. This absence of relationship between rTL and fecundity rate in males is not surprising, considering the differences of parental investment between sexes. In chipmunks, males engage in scrambling competitions for mates but do not provide parental care (Bergeron, Réale, Humphries, & Garant, 2011b), whilst females bear the costs of gestation and lactation, which are high in small mammals [START_REF] Speakman | The physiological costs of reproduction in small mammals[END_REF]. Previous studies revealed sex-differences in stress reactivity and behaviour, likely associated to differential investment into reproduction [START_REF] Montiglio | Pulsed resources and the coupling between life-history strategies and exploration patterns in eastern chipmunks (Tamias striatus)[END_REF][START_REF] Montiglio | Intra-individual variability in fecal cortisol metabolites varies with lifetime exploration and reproductive life history in eastern chipmunks (Tamias striatus)[END_REF]. The sex-specific relationship observed could reflect the sexually discordant selection on telomeres reported in other vertebrate species, through sex-specific telomere dynamics or sex-specific relationships between telomere length and life-history traits [START_REF] Barrett | Sex differences in telomeres and lifespan[END_REF][START_REF] Bauch | Sex-specific telomere length and dynamics in relation to age and reproductive success in Cory's shearwaters[END_REF][START_REF] Brown | Causes and consequences of telomere lengthening in a wild vertebrate population[END_REF][START_REF] Heidinger | Longer telomeres during early life predict higher lifetime reproductive success in females but not males[END_REF], or a well-reported sex-specific differences in POLS [START_REF] Tarka | Sex differences in life history, behavior, and physiology along a slow-fast continuum: a meta-analysis[END_REF]. Since fecundity rate is considered as one of the best metrics of individual paceof-life (Araya-Ajoy et al., 2018), this positive relationship between rTL and fecundity rate in females, independently of their cohort of birth, provides another support for the anti-cancer hypothesis. Finally, we found no link between early life telomere length and LRS, neither in males nor in females, as expected under the "phenotypic quality" hypothesis [START_REF] Sudyka | Does Reproduction Shorten Telomeres? Towards Integrating Individual Quality with Life-History Strategies in Telomere Biology[END_REF].

We failed to find any significant relationship between lifespan and rTL in Eastern chipmunks. An absence of correlation between telomere length and lifespan has previously been reported at the between-species level in rodents [START_REF] Seluanov | Telomerase activity coevolves with body mass not lifespan[END_REF]. Nonetheless, the supplementary analysis on a subset of 12 juveniles shows a trend for a negative correlation between telomere length and lifespan . It also highlights that the two juveniles with the shortest telomeres at weaning displayed an above-average lifespan of more than 40 months, i.e. the average lifespan is of ~30 months in this population (Bergeron, Réale, et al., 2011a).

Although anecdotal, this observation goes against the idea of a selective disappearance of individuals with short telomeres, supported by our results on telomere elongation with age. Lifespan, commonly linked to telomere length in birds and humans (Bichet et al., 2019;[START_REF] Eastwood | Early-life telomere length predicts lifespan and lifetime reproductive success in a wild bird[END_REF][START_REF] Heidinger | Telomere length in early life predicts lifespan[END_REF][START_REF] Sahin | Linking functional decline of telomeres, mitochondria and stem cells during ageing[END_REF], is commonly used as a metric of the POLS [START_REF] Giraudeau | Do Telomeres Influence Pace-of-Life-Strategies in Response to Environmental Conditions Over a Lifetime and Between Generations[END_REF][START_REF] Ricklefs | The physiology/life-history nexus[END_REF]. However, it could be a less valuable metric than fecundity rate to assess the pace of life of species with low survival probabilities such as chipmunks, as it displays less inter-individual variation compared to fecundity rate and generation time (Araya-Ajoy et al., 2018). This could potentially explain why we found no significant relationship in adults and solely a trend in juveniles, together with a lack of statistical power. Competing interests: Authors declare no competing interests; Data and materials availability: All data are available as supplementary material.

Figure 3 .

 3 Figure 3. Relationships between relative telomere length (Z scores of LN(T/S ratio)) and age (months since the emergence from the maternal burrow) in Eastern chipmunks. (A) Relative telomere length in spring according to age at the population level (n=204). A

Figure 5 .

 5 Figure 5. Relationship between relative telomere length (rTL) and lifespan. In (A) rTL of adults according to their age at their first breeding opportunity is represented with lifespan expressed in Log 10 (Months+1). Individuals with a first breeding opportunity at 7 months old (fast) are shown in dark blue, whereas individuals first mating at 15 months old (slow) are shown in light grey; both males (triangles) and females

  long-term research project, including data collection, exploration measurements and DNA extraction. D.R. and M.L.T. conceived the theoretical framework of this study. S.Z. and F.C. designed the qPCR measurements, which were performed by S.Z. with help from M.L.T. M.L.T conducted statistical analyses with advices from D.G., D.R. and P.B. M.L.T wrote the manuscript with edits from all other authors. Funding: Financial support was provided by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grants to D.R. (#2013-262747), D.G. (#2016-03638) and P.B. (#2016-04193), and by a Fonds de Recherche du Québec Nature et technologies (FRQNT) team grant #2019-PR-254944 to D.R., D.G. and P.B. The salary of M.L.T. was supported by a PBEEE postdoctoral fellowship from the FRQNT #263036.

2018). Capitalizing on a long-term monitoring of a wild population of Eastern chipmunks (Tamias striatus),

Acknowledgments

We are grateful to V. Lemieux for his help in managing DNA samples and to E. Gharnit for sharing data on exploration profile. We thank the many students and crew members who assisted with field, laboratory work and data collection within the Chipmunk Project. We also thank the Nature Conservancy of Canada and the Réserve Naturelle des Montagnes Vertes (Québec, Canada) for providing access to the chipmunk study sites.

We thank the anonymous reviewers for their helpful comments and feedbacks on a previous version of the manuscript. Animal welfare statement: Animals were captured and handled in compliance with the Canadian Council on Animal Care, under the approval of the Animal Ethics Committees (CIPA) of the Université de Sherbrooke (protocol number: DG2015-01-Université de Sherbrooke), Bishop's University (protocole number: PB2015-02 -Bishop's University) and the Université du Québec à Montréal Ethics Committee (protocol number: DR0317-C1760-0320). Author Contributions: D.G., D.R. and P.B. conceived and supervised the chipmunk

Consequently, we found no support for a positive relationship between rTL and lifespan or LRS, as expected under the phenotypic quality hypothesis. We argue that the concept of phenotypic "quality" should be used and interpreted with caution, especially when fitness is used to both define and assess quality [START_REF] Bergeron | Individual quality: tautology or biological reality[END_REF].

Conclusion

To our knowledge, this study is the first to test the anti-cancer hypothesis at the within-species level, within the framework of the life-history theory. Our results, based on a long-term monitoring of a wild rodent, support several predictions following the anti-cancer hypothesis, but not the resource-allocation trade-offs hypothesis. We also show within-individual elongation of telomeres with age, in individuals from all age classes. Previous studies revealed that Eastern chipmunks show the highest body-mass specific telomerase activity in their somatic tissues. The elongation of telomeres recorded with age in chipmunks could thus be compared to closely related species. Recent literature suggests that patterns of telomere elongation exist in other vertebrate species, especially in non-model and non-avian species. Further investigations are thus needed to disentangle to which extent life histories and telomere dynamics of species are more likely to be driven by resource allocation trade-offs alone or by higher order constraints that depend on species size and taxa.

Supplementary material

Tables S1 to S6 Figures S1 and S2

Captions for external databases S1-S3

External databases S1-S3