# Sibling similarity in telomere length in Adélie penguin chicks

Coline Marciau, Sophie Bestley, David Costantini, Olivia Hicks, Mark Hindell, Akiko Kato, Thierry Raclot, Cécile Ribout, Yan Ropert-Coudert, Frédéric Angelier

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Coline Marciau, Sophie Bestley, David Costantini, Olivia Hicks, Mark Hindell, et al.. Sibling similarity in telomere length in Adélie penguin chicks. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology, 2025, 302, pp.111818. 10.1016/j.cbpa.2025.111818. hal-05019497

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# Sibling similarity in telomere length in Adélie penguin chicks

Coline Marciau<sup>a,b,*</sup>, Sophie Bestley<sup>a</sup>, David Costantini<sup>c,d</sup>, Olivia Hicks<sup>b</sup>, Mark Hindell<sup>a</sup>, Akiko Kato<sup>b</sup>, Thierry Raclot<sup>e</sup>, Cécile Ribout<sup>b</sup>, Yan Ropert-Coudert<sup>b</sup>, Frederic Angelier<sup>b</sup>

## ARTICLE INFO

Edited by Michael Hedrick

**Keywords:**

Seabird

Pygoscelis adeliae

Telomere length

Oxidative stress

Early life

Growth

Antarctica

## ABSTRACT

Early life telomere length is thought to influence and predict an individual's fitness. It has been shown to vary significantly in early life compared to adulthood. Investigating the factors influencing telomere length in young individuals is therefore of particular interest, especially as the relative importance of heredity compared to post-natal conditions remains largely uncertain. Adélie penguins are eco-indicators of the Antarctic ecosystem and their population are currently undergoing variable trajectories due to climate change. Here, we conducted a correlative study to investigate how telomere length was influenced by external and internal factors in Adélie penguin chicks. We found that most of the parameters we tested, including sex, body mass, brood size and hatching order as well as parental foraging trip duration, did not significantly influence chick telomere length at 32 days. However, siblings had similar telomere length, suggesting that hereditary factors play a stronger role in determining telomere length at this stage compared to the post-natal environment. In addition, telomere length and oxidative damage did not directly correlate but did interact in a complex way mediated by chick mass. High levels of oxidative damage were associated with longer telomeres in heavy chicks, whereas they were associated with shorter telomeres in light chicks. Although this mass-dependent relationship between telomere length and oxidative damage needs to be confirmed in future studies, it could reflect two different scenarios: (1) short telomeres may mimic the cost of poor nutritional conditions and oxidative damage in light chicks; (2) long telomeres may be maintained despite high oxidative damage in heavy chicks thanks to optimal nutritional conditions.

## 1. Introduction

Early life is a key life-history stage for vertebrates because this period is associated with growth and the ontogeny of multiple organismal systems, that have long-term consequences for survival and reproductive performance later in life (Costantini and Marasco, 2022; Gilbert, 2001; Van De Pol et al., 2006). Developmental conditions depend on both the environment and parental care, which together determine the resources available for growth and development (Uller, 2008). When resources/energy are limited, resources allocated to growth must be traded-off with self-maintenance (Metcalfe and Monaghan, 2001; Stearns, 1992). Theoretical and empirical studies have shown that growth in non-optimal conditions or even compensatory growth (e.g. fast development to catch up a delayed growth) may occur at the

expense of physiological balances with long-lasting consequences for individual life history strategies (Dmitriev, 2011; Metcalfe and Monaghan, 2001; Monaghan and Ozanne, 2018).

In this context, telomere length has drawn increasing attention from evolutionary ecologists (Horn et al., 2010; Monaghan et al., 2022). Telomeres are repeated sequences of non-coding DNA capping the chromosomes and protecting DNA from erosion during cell division and oxidative stress (Blackburn, 2005). Telomere length generally shortens throughout life (Dunshea et al., 2011) and this shortening has been associated with the occurrence of age-related diseases in humans (Blasco, 2005) and with reduced survival in wild vertebrates (Wilbourn et al., 2018). As such, telomere shortening is often viewed not only as a proxy of cellular ageing (Harley et al., 1990), but also as a proxy for remaining lifespan (Tricola et al., 2018; Whittemore et al., 2019), and

<footnote><sup>a</sup> Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, TAS, Australia</footnote>

<footnote><sup>b</sup> Centre d'Etudes Biologiques de Chizé, UMR7372 CNRS-La Rochelle Université, Villiers en Bois, France</footnote>

<footnote><sup>c</sup> Department of Ecological and Biological Sciences, University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy</footnote>

<footnote><sup>d</sup> Unité Physiologie Moléculaire et Adaptation, UMR7221-Muséum National d'Histoire Naturelle-CNRS, 75005 Paris, France</footnote>

<footnote><sup>e</sup> Institut Pluridisciplinaire Hubert Curien, CNRS-UMR7178, Strasbourg, France</footnote>

<footnote>* Corresponding author at: Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, TAS, Australia.
E-mail address: coline.marciau@gmail.com (C. Marciau).</footnote>
telomere shortening is a trait that is considered as a hallmark of ageing in vertebrates (Chakravarti et al., 2021). Indeed, the rate of telomere shortening may vary between individuals of the same species although the extent of this inter-individual variation remains debated (Monaghan, 2010; Bichet et al., 2020; Vedder et al., 2022). A fast rate of telomere shortening has been convincingly linked with poor environmental conditions (Angelier et al., 2018; Chatelain et al., 2020), such as nutritional stress (e.g., Nettle et al., 2014), the occurrence of diseases (e.g., Asghar et al., 2015) and high levels of environmental pollution (e.g., Blévin et al., 2017). Telomere length and the rate of telomere shortening have also been correlated with fitness-related traits such as survival, breeding performance and lifetime reproductive success in several wild bird species (e.g., Angelier et al., 2019; Bauch et al., 2013; Bize et al., 2009; Eastwood et al., 2019; Haussmann et al., 2005; Heidinger et al., 2021).

The developmental life-history stage is thought to be of primary importance when examining the relationship between telomere length, environmental conditions and lifetime reproductive success (Monaghan, 2010). Most telomere shortening seems to occur during this stage and early life telomere dynamic is therefore thought to be a key determinant of adult telomere length, irrespective of adult age (Marasco et al., 2022; Salomons et al., 2009). Intense cell division is one of the primary drivers of telomere attrition, and as a consequence, telomere shortening is much faster in growing individuals than in adults (Hall et al., 2004; Salomons et al., 2009). The energetically demanding growth period is also associated with the production of reactive oxygen species (ROS) through increased metabolic activity (Andreyev et al., 2005; Monaghan and Ozanne, 2018). These ROS will react with other cell compounds leading to oxidative stress and damage to proteins, lipids, and DNA, including telomere sequences (Armstrong and Boonekamp, 2023; Reichert and Stier, 2017). Furthermore, constraining developmental conditions have been shown to induce additional oxidative stress (Costantini et al., 2014; Smith et al., 2016), which in turn accelerates telomere shortening (Alonso-Alvarez et al., 2007). Telomeres are guanine rich regions, and as such are particularly susceptible to oxidation (Haussmann and Marchetto, 2010; von Zglinicki, 2002).

Many studies have found that developmental conditions affect both growth and telomere dynamics, suggesting that growth is intrinsically linked to telomere length in developing individuals. However, several correlative and experimental studies have found that while developmental constraints can accelerate telomere shortening, this faster shortening is not necessarily paralleled by a slower growth or a smaller body size at fledging (e.g. (Badás et al., 2023; Meillère et al., 2015; Nettle et al., 2014; Vedder et al., 2017). Recent studies have even reported that a slower growth or a smaller body size may be associated with longer telomeres in some bird species (e.g., great tits Hsu et al., 2023, house sparrows Ringsby et al., 2015). This suggests that growth and telomere dynamics may co-vary, but in a context-dependent and complex manner (Monaghan and Ozanne, 2018).

According to life history theory, recent studies proposed that a diversion of resources away from telomere maintenance could, under some circumstances, be beneficial if it improves performance in the short term (Vedder et al., 2017; Casagrande and Hau, 2019). When energy is limited, this theory therefore predicts that developing individuals should maintain growth at the expense of their telomeres as growth and body size are key determinants of fledging success and post-fledging survival (Casagrande and Hau, 2019). Although this theory has rarely been tested in growing chicks, it is supported by some empirical studies. For example, chicks that grew faster also showed intense telomere erosion in king penguins (Geiger et al., 2012) and a faster growth appears to be associated with higher oxidative stress and smaller telomeres in this species (Stier et al., 2014). Overall, and to date, the links between growth, oxidative stress, and telomere length remain debated and we currently lack empirical studies focusing on this topic in wild vertebrate species (but see Burraco et al., 2017; Zhang et al., 2023 for example).

To investigate these questions, we conducted a correlative study on

Adélie penguins (*Pygoscelis adeliae*), an Antarctic species that faces increasing variability in environmental conditions around Antarctica (Dugger et al., 2010; Mayewski et al., 2009; Turner et al., 2020). Adélie penguins typically lay two eggs and their chicks grow rapidly for several weeks before fledging (Ainley, 2002). Chicks compete for food and sibling rivalry can have a major impact on growth patterns although both chicks share the same nest and parents (Marciau et al., 2023). Adélie penguin chicks therefore represent an ideal study system to understand not only to what extent post-natal conditions and growth pattern may affect telomere length, but also to test whether the relationship between developmental conditions and telomere length is related to oxidative stress. In this correlative study, we therefore aimed to identify the predictors of telomere length in developing Adélie penguins by sampling at a specific age (32-days old) chicks that had experienced contrasting developmental conditions.

Firstly, we tested whether chicks from the same nest had similar telomere length to determine the extent to which post-natal conditions could attenuate hereditary and maternal effects on telomere length (e.g., Bauch et al., 2022; Becker et al., 2015; Viblanc et al., 2020). Secondly, we investigated the influence of multiple predictors of post-natal conditions on telomere length (i.e., brood size and hatching order, parental foraging effort). Brood size and hatching order may induce nutritional stress while parental ability to breed early and to forage efficiently will determine chick growth with potential consequences for telomere dynamics. According to previous life-history theoretical background (Vedder et al., 2017; Casagrande and Hau, 2019), we might expect growing chicks to maintain their growth at the expense of their telomere length if they are facing poor developmental conditions (e.g. due to large brood size, second-hatched chick, and/or low-quality parents). Finally, we examined whether oxidative stress could explain the link that may exist between telomere length and chick body size. We expected that oxidative stress could be correlated with both telomere length and body size if growth induces oxidative damage to telomeres (Monaghan and Ozanne, 2018).

## 2. Methods

### 2.1. Study site and study species

The study was conducted at Pointe Géologie in Adélie Land, Antarctica (66.7°S, 140.0°E), during the austral summer 2018/2019. Adélie penguins are long lived seabirds that breed exclusively around the Antarctic continent. They gather in large colonies on rocky areas in October and start their breeding cycle. Adélie penguins lay a maximum of two eggs per season. After laying, both partners alternate foraging trips at sea and incubating or brooding periods at the nest. Incubation and chick-rearing last approximatively 35 days and 50 days, respectively. After hatching, the chicks are brooded and guarded by the parents for about 22 days. Then, they gather in creches and are still fed by both parents that forage simultaneously at sea (Ainley, 2002).

### 2.2. Monitoring reproduction

Adélie penguin nests were monitored throughout the season as previously described in Marciau et al. (2023). Briefly, 124 penguins (62 pairs) were individually marked at the beginning of the reproductive season when pairs defend a nest but have not yet laid their eggs (November). After marking, we checked each of the 62 focal nests daily to record laying dates of the two eggs (~mid-November). In December, we then checked each nest for hatching dates of both chicks. A few hours after egg laying, the female leaves the nest to forage at sea while the male incubates the egg. Then, the female relieves the male from its incubation duty and the male leaves the colony to forage at sea. During incubation, the alternate foraging trips last from several days to three weeks. After hatching, the same alternation of parental care at the nest and foraging trips at sea occurs but the foraging trips are much shorter
(1–3 days) as they return to provision chicks. During the chick-rearing period, each nest was visually inspected from a distance every 3 h to monitor the foraging trip durations (hereafter FTD) of both parents. In Adélie penguins, FTD is a relevant proxy of food provisioning and nutritional status of the chicks (Ballard et al., 2010). In our study, 97 chicks hatched in 62 nests of which 27 nests had a single chick (brood size: 1) and 35 nests had two chicks (brood size: 2). When the chicks reached the age of 32 days old, they were captured, weighed (± 10 g), and sampled for blood from the foot vein using a 1 ml heparinised syringe and a 25G needle. Immediately after sampling, the blood was centrifuged (7 min, 5000 rpm). The plasma was then separated from the red blood cells and all the tubes were kept frozen at -20 °C until laboratory analysis (see below). In Adélie penguin chicks, body mass at 32 days old is a reliable proxy of growth because it is highly correlated with body mass gain ($r = 0.89$; $p < 0.001$, Marciau et al., 2023).

## 2.3. Molecular sexing, telomere length and oxidative stress

Molecular sexing was performed at the Centre d'Etudes Biologiques de Chizé (Service d'Analyses Biologiques) by DNA extraction and PCR as previously described in Marciau et al. (2023).

Telomere analyses were conducted following standard quantitative PCR (qPCR) methodology and telomere length was expressed as T/S ratio (Cawthon, 2002; Criscuolo et al., 2009). DNA of each sample was extracted from red blood cells using the Qiagen DNeasy Blood & Tissue Kit. DNA quality was assessed using a spectrophotometer (Nanodrop) and by running gel electrophoresis and all samples were adequate for telomere analyses. Telomere length was then quantified using qPCR and the BioRad SYBR Green Supermix. Telomeric DNA (TEL) and a reference gene (RAG1) were amplified by running 5 plates and all samples were run in triplicates. This gene has previously been used as a single copy gene in several studies and bird species (e.g. Criscuolo et al., 2019; Kärkkäinen et al., 2022), including seabirds (Sebastiano et al., 2023; Carravieri et al., 2023). The RAG1 primers (forward and reverse) used in this study were designed for Adélie penguins. qPCR runs were performed using 5 ng of DNA per reaction and the telomere primers and RAG1 primers were used at a concentration of 800 nM. The efficiencies of the telomere and RAG1 assays were 98.48 % and 102.44 %, respectively (CVs, telomere: 1.2 %, RAG1: 0.8 %). Standard curves to control the amplification efficiency of the reactions were performed by using serial dilutions of DNA from a pooled sample of 32-day old Adélie penguins. The intra- and inter-assay CVs were 0.27 % and 1.05 % for the RAG1 Ct, and 0.31 % and 1.33 % for telomere Ct.

Measurements of two markers of oxidative damage (Reactive Oxygen Metabolites, ROMs; and protein carbonyls) were performed in the laboratory of the Muséum National d'Histoire Naturelle (Unité Physiologie Moléculaire et Adaptation) as described in Marciau et al. (2023). Briefly, ROMs were quantified using the d-ROMs test (Diacron International, Italy) which measures early oxidation compounds. It is expressed as millimolars of H2O2 equivalents. Protein carbonyls were measured using the Protein Carbonyl Colorimetric assay (Cayman Chemical Company, Ann Arbor, MI, USA) based on the protocol described in Levine et al. (1990) and adapted to penguins as in Marciau et al. (2023). To do so, protein carbonyls are first purified, and absorbance is then read at 370 nm. The extinction coefficient of 2,4-dinitrophenylhydrazine (0.022 mmol L21 cm21) is used to calculate protein carbonyl content, which is expressed as nanomoles per milligram of protein. ROMs and protein carbonyls were measured in plasma. The coefficients of variation and the repeatability estimates were as follows: 9.35 % and 0.958 (CI = 0.948, 0.965; $p < 0.001$) for the d-ROMs test; 10.92 % and 0.781 (0.694, 0.840; $p < 0.001$) for the protein carbonyl test.

## 2.4. Statistical analysis

Statistical analyses were performed using R software (ver. 4.2.1, R Core Team, 2023). First, similarity of our variables of interest between

siblings was investigated by running repeatability tests (i.e. intra-class correlation) using the *rptR* package (Stoffel et al., 2017). All chicks were included in these tests. Then, four linear mixed models were performed (using *lme4* package, Bates et al., 2015) to investigate our second and third hypotheses: (i) individuals facing poor developmental conditions (large brood size, second hatched, low parental foraging effort) will have short telomeres and (ii) oxidative damage will be both related to chick mass and telomere length.

In these models, the brood size and hatching order were reduced to one single three-level variable called 'BSHO' (single chick, first-hatched chick, and second-hatched chicks for the nests with two chicks). Parental FTD was calculated as the mean trip duration (in days) during the chick rearing period from hatching to emascipation (i.e., when the chick is left unattended by both parents).

In the first model, we tested how chick body mass 32 days after hatching was related to sex, BSHO and parental FTD. In this species, body mass 32 days after hatching is a reliable proxy of growth (Marciau et al., 2023). There was strong support for including nest as a random effect in all models, which were therefore run as linear mixed models.

In the second model, we examined chick telomere length and tested for the influence of chick sex, BSHO and parental FTD. Finally, in the third and fourth models, we examined telomere length in male and female chick separately to investigate the effect of chick body mass and markers of oxidative damage. We ran one model per sex to avoid collinearity issues because body mass and sex were strongly related. Significance level was set at $p = 0.05$. When interactions were significant, contrast tests were used to compare groups using the *emmeans* package (Lenth, 2022) with degrees of freedom estimated using the Kenward-Roger method.

# 3. Results

## 3.1. Sibling similarity in body mass, telomere length, and oxidative damage levels

We found a strong similarity in telomere length within nests: chicks from the same nest had similar telomere length (Fig. 1A, $R \pm SE = 0.79 \pm 0.07$ $p < 0.001$). In contrast, neither body mass nor oxidative damage markers (ROMs and protein carbonyls) were significantly repeatable within nests, suggesting a low similarity between the chicks of a given nest (Fig. 1B: body mass, $R \pm SE = 0.21 \pm 0.17$, $p = 0.221$; Fig. 1C: ROMs: $R \pm SE = 0.22 \pm 0.16$, $p = 0.092$; Fig. 1D: protein carbonyls: $R \pm SE = 0.25 \pm 0.17$, $p = 0.228$).

## 3.2. Predictors of body mass

Chick body mass was influenced by the sex of the individuals as well as the BSHO factor (Table 1, Fig. 2A). Specifically, male chicks were heavier than females (Table 1, $F_{1,74} = 14.38$, $p < 0.001$). Second hatched chicks were smaller than first hatched chicks from nests with two siblings (contrast: $t = 3.96$, $p < 0.001$) and first hatched chicks were also smaller than chicks from nests with only one chick (contrast: $t = 3.81$, $p < 0.001$).

Chick body mass was also influenced by the paternal foraging trip duration but not the maternal. Specifically, chicks from fathers performing shorter trips were heavier ($coef \pm se = -172.01 \pm 51.58$; Table 1, $F_{1,74} = 11.12$, $p = 0.001$).

## 3.3. Predictors of relative telomere length

When considering all chicks together, telomere length was not significantly influenced by any variable regarding developmental conditions examined in this study (Table 2, Fig. 2B).

Considering male and female chicks separately, telomere length was not directly related to the level of ROMs or protein carbonyls. However, it was influenced by a highly significant interaction between body mass
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Similarity of (A) telomere length, (B) body mass, (C) levels of ROMs, (D) levels of protein carbonyls between siblings at 32 days. C1 and C2 represent the first-hatched and second-hatched chicks of the same nest, respectively. Given R stands for repeatability coefficient.</figcaption></figure>

<caption>**Table 1**</caption>

Influence of sex, BSHO (Brood size and hatching order factor, see methods), parental FTD (foraging trip duration) and their interactions on the body mass of 32 day old Adélie penguin chicks. Terms included in the final best model are notified in the last column. Nest identity was included as a random factor. Estimates with standard errors can be found in the supplementary materials.

<table><thead><tr><th></th><th>NumDF</th><th>DenDF</th><th>F value</th><th>Pr(&gt;F)</th><th>Final model</th></tr></thead><tbody><tr><td>sex</td><td>1</td><td>90</td><td>14.38</td><td>&lt;0.001</td><td>x</td></tr><tr><td>BSHO</td><td>2</td><td>90</td><td>18.24</td><td>&lt;0.001</td><td>x</td></tr><tr><td>Paternal FTD</td><td>1</td><td>90</td><td>11.12</td><td>0.001</td><td>x</td></tr><tr><td>Maternal FTD</td><td>1</td><td>76</td><td>0.45</td><td>0.503</td><td></td></tr><tr><td>sex x BSHO</td><td>2</td><td>76</td><td>2.14</td><td>0.125</td><td></td></tr><tr><td>sex x Paternal FTD</td><td>1</td><td>76</td><td>0.24</td><td>0.625</td><td></td></tr><tr><td>BSHO x Paternal FTD</td><td>2</td><td>76</td><td>1.05</td><td>0.354</td><td></td></tr><tr><td>sex x Maternal FTD</td><td>1</td><td>76</td><td>0.79</td><td>0.378</td><td></td></tr><tr><td>BSHO x Maternal FTD</td><td>2</td><td>76</td><td>0.04</td><td>0.963</td><td></td></tr><tr><td>Sex x BSHO x Paternal FTD</td><td>2</td><td>76</td><td>1.04</td><td>0.359</td><td></td></tr><tr><td>Sex x BSHO x Maternal FTD</td><td>2</td><td>76</td><td>3.58</td><td>0.033</td><td></td></tr></tbody></table>

and ROMs in males, but not in females. A positive relationship between telomere length and level of ROMs was detected in heavy male chicks whereas a negative relationship between telomere length and level of ROMs was detected in light male chicks (Table 3A; Fig. 3A). Thus, in males, telomere length was shortest in small chicks with high ROMs (Fig. 3A). In contrast, these relationships were not evident in female

<figure><img src="image_5.png" /></figure>

chicks (Table 3B; Fig. 3B).

## 4. Discussion

In this correlative study, we investigated the predictors of telomere length in 32-day old Adélie penguin chicks. We found a strong similarity in telomere length between siblings originating from the same nest. Although sex and post-natal conditions (paternal effort, brood size and hatching order) had a significant effect on chick body mass, we found no relationship between chick telomere length and any of these variables. Telomere length was not related to protein carbonyl levels but was related to ROMs in a complex and sex-dependent manner.

### 4.1. Sibling similarity in telomere length

Repeatability tests showed that siblings had a strong similarity in telomere length. This indicates that two individuals originating from the same parents and developing in the same nest have very similar telomere length, as found in other studies (e.g., Atema et al., 2015; Bauch et al., 2022; Westneat et al., 2023). This similarity can result from several factors, including heritability of telomere length, maternal effects, and pre- and post-natal conditions that are shared by the siblings until the time of sampling (Dugdale and Richardson, 2018), although it is difficult to disentangle their relative importance. The lack of similarity in other growth-related variables in siblings (body mass and markers of oxidative damage: ROMs and protein carbonyls) suggests that heritability is likely to play a significant role in determining telomere length in
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Body mass (A) and telomere length (B) of 32-days old chicks in relation to the BSHO category (see methods, 1–1: singleton, 1–2: first hatched and 2–2: second hatched). Female and male are represented in grey and black, respectively. Box-and-whisker plots represent the data: the top and bottom of the boxes represent the top and bottom quartiles, respectively; the line across the box represents the mean. The ends of the whiskers represent the fifth and ninety-fifth percentiles, and dots represent the raw data points.</figcaption></figure>

**Table 2**

Influence of sex, BSHO (Brood size and hatching order factor, see methods), parental FTD (foraging trip duration) and their interactions on telomere length of 32-days old Adélie penguin chicks. Nest identity was included as a random factor. The best model was the null model that did not include any independent variable. Estimates with standard errors can be found in the supplementary materials.

<table><thead><tr><th></th><th>NumDF</th><th>DenDF</th><th>F value</th><th>Pr(&gt;F)</th></tr></thead><tbody><tr><td>sex</td><td>1</td><td>69.18</td><td>0.01</td><td>0.907</td></tr><tr><td>BSHO</td><td>2</td><td>38.69</td><td>1.01</td><td>0.373</td></tr><tr><td>Paternal FTD</td><td>1</td><td>62.83</td><td>2.07</td><td>0.156</td></tr><tr><td>Maternal FTD</td><td>1</td><td>55.60</td><td>0.48</td><td>0.490</td></tr><tr><td>sex x BSHO</td><td>2</td><td>48.21</td><td>0.64</td><td>0.529</td></tr><tr><td>sex x Paternal FTD</td><td>1</td><td>67.16</td><td>2.00</td><td>0.162</td></tr><tr><td>BSHO x Paternal FTD</td><td>2</td><td>40.05</td><td>2.50</td><td>0.095</td></tr><tr><td>sex x Maternal FTD</td><td>1</td><td>67.88</td><td>0.00</td><td>0.971</td></tr><tr><td>BSHO x Maternal FTD</td><td>2</td><td>31.21</td><td>0.70</td><td>0.505</td></tr><tr><td>Sex x BSHO x Paternal FTD</td><td>2</td><td>42.11</td><td>0.56</td><td>0.578</td></tr><tr><td>Sex x BSHO x Maternal FTD</td><td>2</td><td>37.15</td><td>0.13</td><td>0.875</td></tr></tbody></table>

**Table 3**

Relationship between telomere length and the levels of ROMs and protein carbonyls, and body mass in male (A) and female (B) 32-day old Adélie penguin chicks. Nest identity was included as a random factor. Terms included in the final best model are notified in the last column. Estimates with standard errors can be found in the supplementary materials.

<table><thead><tr><th>A</th><th>NumDF</th><th>DenDF</th><th>F value</th><th>Pr (&gt;F)</th><th>Final model</th></tr></thead><tbody><tr><td>Mass</td><td>1</td><td>4.66</td><td>0.06</td><td>0.823</td><td>X</td></tr><tr><td>ROMs</td><td>1</td><td>8.47</td><td>0.97</td><td>0.352</td><td>X</td></tr><tr><td>Protein carbonyls</td><td>1</td><td>43</td><td>0.18</td><td>0.671</td><td></td></tr><tr><td><strong>Mass x ROMs</strong></td><td>1</td><td>5.92</td><td>21.73</td><td><strong>0.004</strong></td><td>X</td></tr><tr><td>Mass x Protein carbonyls</td><td>1</td><td>43</td><td>2.25</td><td>0.141</td><td></td></tr></tbody></table>

<table><thead><tr><th>B</th><th>NumDF</th><th>DenDF</th><th>F value</th><th>Pr (&gt;F)</th><th>Final model</th></tr></thead><tbody><tr><td>Mass</td><td>1</td><td>33.94</td><td>0.55</td><td>0.464</td><td></td></tr><tr><td>ROMs</td><td>1</td><td>13.47</td><td>0.63</td><td>0.441</td><td></td></tr><tr><td>Protein carbonyls</td><td>1</td><td>39.62</td><td>0.28</td><td>0.600</td><td></td></tr><tr><td>Mass x ROMs</td><td>1</td><td>23.64</td><td>0.31</td><td>0.582</td><td></td></tr><tr><td>Mass x Protein carbonyls</td><td>1</td><td>40.93</td><td>0.48</td><td>0.493</td><td></td></tr></tbody></table>

Adélie penguin chicks. To refine this measurement of heritability, futures studies could use an animal model approach (Boonekamp et al., 2022).

Indeed, the low similarity in body mass and oxidative damage markers between two chicks of a given nest suggests that two siblings sharing the same nest and parents can probably have very contrasted post-natal developmental conditions. In Adélie penguins, these differences mainly result from brood competition that affects growth trajectories of the competing siblings with strong effects on body mass and oxidative stress (see also Marciau et al., 2023). Although we did not measure heritability per se, our results support the idea that in Adélie penguins genetic and maternal effects may play a larger role than post-natal developmental conditions in determining offspring telomere length. Our results are therefore consistent with recent studies showing high heritability of telomere length in many vertebrate species (e.g., Asghar et al., 2015; Atema et al., 2015; Bauch et al., 2022; Morosinotto et al., 2022; Sparks et al., 2022; Vedder et al., 2022). However, the importance of heritability in determining telomere length is still highly debated (reviewed in Dugdale and Richardson, 2018) as high heterogeneity in heritability estimates of telomere length has been described in the literature (reviewed in Chik et al., 2022). In this context, the strong similarity in telomere length between Adélie penguin siblings may have been amplified by limited effects of brood competition during the study year, which was characterized by good environmental conditions and a high chick productivity (1.23 chick per nest, see Ropert-Coudert et al., 2018).

From a methodological point of view, we assessed telomere length by qPCR, which is known to measure both interstitial telomeric and terminal telomeric fragments (Foote et al., 2013). Contrary to terminal telomeric fragments, interstitial ones theoretically do not shorten with age and are not affected by environmental conditions (Kärkkäinen et al., 2022). These interstitial telomeric fragments coould thus blur partly our results. Future studies should therefore confirm our results in Adélie penguins with other techniques (e.g., TRF, as in Vedder et al., 2018 for example) that measure terminal telomeric fragments.

## 4.2. Post natal conditions and telomere length: body mass, brood size and hatching order

As expected, we found that brood competition (i.e., hatching order and brood size) and parental foraging effort had a strong effect on body mass of 32-day old chicks, demonstrating that these variables are good proxies for post-natal conditions (see also Marciau et al., 2023).
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 3.** Influence of ROMs and body mass on telomere length of 32 days old male (A and B) and female (C and D) Adelie penguin chick. Surface plots (A: male and C: female) are represented using the predicted values from the models including body mass, the levels of ROMs and their interaction. To assist visualisation, the raw data and the linear regression (red line) between telomere length and the levels of ROMs are presented for male (B) and female (D) chicks that were split into three body mass categories, divided at the lower and upper 30 % percentiles of body mass for each sex. However, body mass was considered as a continuous variable in the models. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
However, contrary to our prediction, these same proxies were unrelated to telomere length. We found a high similarity in telomere length among siblings, suggesting that our technical repeatability in measuring telomere length was appropriate. However, recent studies have emphasized that qPCR have an overall low repeatability that can mask biologically-relevant relationships (Kärkkäinen et al., 2022) and this could explain why we did not find any effect of brood competition on telomere length in our study. The qPCR technique could also explain why we did not find any effect of developmental conditions on telomere length because this technique measures not only terminal telomeric fragments, but also interstitial telomeric fragments that are very long in some species (Foote et al., 2013) and that do not seem to be affected by environmental conditions (Kärkkäinen et al., 2022). Previous studies have reported effects of post-natal conditions, particularly brood competition, on telomere length in several bird species (e.g., Boonekamp et al., 2014; Costanzo et al., 2017; Mizutani et al., 2016; Nettle et al., 2014; Young et al., 2017). However, other studies have found no clear effect of brood competition on telomere length (e.g., Belmaker et al., 2019; Nettle et al., 2016; Vedder et al., 2017; Voillemot et al., 2012), and some studies have reported that post-natal constraints may affect telomere length without a significant effect on body size (e.g., Meillère et al., 2015; Stier et al., 2015). Altogether, these mixed results indicate that the link between post-natal conditions, growth, and telomere length is complex and appears to vary with the environmental context (Badás et al., 2023; Nettle et al., 2016). Importantly, and in combination with other studies, our study suggests that developmental conditions may not always have a strong impact on offspring telomere length.

Recent theoretical studies have proposed the hypothesis that a trade-off between telomere maintenance and growth may occur when energy is limited (Vedder et al., 2017; Casagrande and Hau, 2019; Monaghan and Ozanne, 2018). Accordingly, we expect that chicks should maintain their growth at the expense of their telomere length when facing poor developmental conditions (large brood size, second-hatched chick, and/or low-quality parents). However, we did not find any support for these predictions in our correlative study as most of these post-natal variables were related to body mass (a reliable proxy of growth in Adélie penguin chicks, Marciau et al., 2023), but not to telomere length, at 32 days. Combined with the sibling similarity in telomere length (see previous section), these results suggest that telomere length of Adélie penguin chicks is mainly determined by genetic and pre-natal conditions, rather than post-natal conditions (but see the methodological limits of qPCR described earlier). Similarly, a previous correlative study in common gulls also found that post-natal conditions - influenced by parental age - had no significant effect on telomere length (Sepp et al., 2022). However, previous experimental studies have convincingly shown through cross-fostering designs that post-natal conditions could significantly affect chick telomere length (e.g. Great tits, Salmón et al., 2016; King penguins, Viblanc et al., 2020).

Importantly, heterogeneity in parental quality may explain these discrepancies. Indeed, it may mask a potential negative effect of brood size on offspring telomere length in correlative studies, especially if better parents produce larger broods and are better able to raise them. In addition, the effect of post-natal conditions on telomere length might also vary depending on the harshness of post-natal conditions (Dugdale and Richardson, 2018), and this may explain why post-natal conditions had no detectable effect on telomere length in our study. During our study year, environmental conditions were good, with low chick mortality (1.23 chicks were produced per nest among the 116 nests monitored in total this year), high chick body mass, and short parental foraging trips (see Ropert-Coudert et al., 2018 for comparisons). Importantly, several correlative and experimental studies have reported that post-natal conditions may have a detectable effect on telomere dynamics (i.e. rate of erosion) rather than on telomere length at a given chick age (e.g., Boonekamp et al., 2014; Nettle et al., 2014). Our study therefore supports the idea that the effect of post-natal conditions on telomere length may be blurred by genetic and pre-natal variables,

particularly if post-natal telomere attrition is overall small relative to telomere length at hatching (as would be expected under overall favourable post-natal conditions). Combining correlative and experimental studies is therefore important to fully assess the influence of brood competition on offspring telomere length.

### 4.3. Growth, oxidative stress, and telomere length

Oxidative stress is known to trigger telomere attrition in vitro (Von Zglinicki, 2002) and in vivo (Armstrong and Boonekamp, 2023; Reichert and Stier, 2017). Indeed, reactive oxygen species are expected to induce telomere attrition, particularly due to the guanine rich content of telomeric regions which are particularly sensitive to oxidation (Von Zglinicki, 2002). Previous experimental studies have reported that antioxidant supplementation slows down telomere shortening (e.g., Pineda-Pampliega et al., 2020). However, the link between oxidative stress and telomere attrition is still highly debated (Boonekamp et al., 2017), especially in the context of developing organisms (Armstrong and Boonekamp, 2023). Here, we found no direct and overall positive or negative link between telomere length and two commonly used markers of oxidative damage. In a previous study, we found that post-natal constraints were associated with higher levels of dROMs although this effect was subtle (Marciau et al., 2023). This highlights that the relationship between developmental conditions, oxidative damage, and telomere length is not as straightforward as previously thought. It is also possible that oxidative damage may have accelerated telomere shortening without any detectable effect on telomere length at 32 days after hatching, especially if most of the inter-individual variation in the telomere length is related to genetic or pre-hatching variables (as discussed above). Importantly, we measured oxidative damage when the chicks were 32 days old, but oxidative damage at this age may not be representative of the oxidative damage experienced from hatching to 32 days because (i) newly hatched chicks have an immature antioxidant system and (ii) metabolic demands vary with growth rate which is not constant (Janes, 1997; Starck and Ricklefs, 1998), potentially explaining the lack of relationship between oxidative damage and telomere length.

Although oxidative damage was not related to telomere length overall, we found a complex and significant relationship between the levels of ROMs and telomere length in male chicks (*p*-value = 0.004, Table 3A). Interestingly, this relationship was dependent on the chick body mass with an opposite positive and a negative relationship between telomere length and the levels of ROMs in large and small chicks, respectively. Therefore, telomere length was shortest in small male chicks with high ROMS levels. This complex relationship is only apparent in males and could be related to statistical type 1 error. Therefore, it needs to be confirmed by additional field and experimental studies. Previous studies have found that the link between telomere attrition and body mass may depend on developmental conditions (i.e., brood size, Nettle et al., 2016, Boonekamp et al., 2014) and our study suggests that this may be related to contrasted levels of oxidative damage. A negative effect of oxidative damage on telomere length is expected (Armstrong and Boonekamp, 2023, Reichert and Stier, 2017) and accordingly, higher levels of ROMs were associated with shorter telomere length in small male chicks, which likely suffered from developmental constraints. In contrast, and surprisingly, higher levels of ROMs were associated with longer telomeres in large male chicks. Large chicks have certainly sustained rapid and intense growth, which, in turn, may have induced the production of reactive oxygen species through metabolic activities and replication processes, leading to high levels of ROMs. However, their longer telomeres suggest that these heavier chicks may be able to alleviate the detrimental effects of such oxidative damage on their cellular machinery and their DNA. This could be achieved for example through higher antioxidant capacity.

Recent theoretical studies suggest that growth, oxidative stress, and telomere length are connected in complex ways, mainly because growth is associated with replication processes and increased metabolic activity
that may affect telomere attrition (Casagrande and Hau, 2019; Monaghan and Ozanne, 2018). As a result, the relationship between oxidative damage and telomere length is often less obvious in developing individuals (Armstrong and Boonekamp, 2023). Accordingly, Pineda-Pampliega et al. (2020) found a beneficial effect of antioxidant supplementation on telomere length in white stork chicks (Ciconia ciconia), but they did not find any correlation between the levels of oxidative damage and telomere length. They suggested that proliferating cells of growing chicks experience rapid telomere shortening, which may confound the potential link between telomere length and oxidative stress. In addition, growth-related telomere dynamics can also very between tissues (Demanelis et al., 2020; Burraco et al., 2023) and antioxidant mechanisms can vary across lifetime and development (Martin et al., 2024), leading potentially to such complex relationships between telomere dynamics, growth, and oxidative stress. It remains unclear why such complex relationships between body size, oxidative damage, and telomere length were not found in female chicks, especially since female Adélie penguin chicks may be more sensitive to developmental constraints (Marciau et al., 2023). This may be related to differential parental investment towards female chicks compared to male chicks (Beaulieu et al., 2009; Jennings et al., 2016) but further experimental studies manipulating growth or antioxidant defences in male and female chicks would be required to better understand this sex-dependent relationship.

## 5. Conclusion

Our correlative study provides insight into the ecological predictors of telomere length in the early life of a long-lived seabird species. Supporting the idea that telomere length is heritable, we found a strong similarity in telomere length between siblings and little, if any, effect of post-natal conditions (i.e. brood size, hatching order, parental effort) on telomere length. This similarity in telomere length between siblings was apparent even though siblings showed different growth trajectories, suggesting a strong genetic or pre-natal determination of telomere length in this species - at least during the year of our study, which was characterized by overall favourable environmental conditions. Further correlative and experimental studies should integrate measures of telomere length at different stages during early life to investigate telomere dynamic. Interestingly, we found that telomere length appears to be influenced by oxidative damage (ROMs) but in a complex manner that depends on the body mass and the sex of the chick. Extending such study over several years with contrasted conditions and integrating more parameters such as parental physiological condition and diet analyses would allow to disentangle further growth effect from constraining conditions in chicks oxidative damage and telomere length.

## Funding

The project is part of the research program l'AMMER, supported by the French polar institute (IPEV, Prog 1091) and the WWF-UK. This project was funded by the CNRS and by a research grant allocated by the University of Tasmania (TGRS) to C. Marciau.

## Ethics

All experiments were approved by a French regional ethical committee in animal experimentation (n°84) under the issued permit number APAFIS#2111-2015092414273394 v4. Access to restricted area in the context of the Antarctic Treaty (ASPA) was approved by the French Polar Institute IPEV.

## CRediT authorship contribution statement

Coline Marciau: Writing - original draft, Visualization, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation,

Conceptualization. **Sophie Bestley:** Writing - review & editing, Validation, Supervision, Methodology, Investigation, Funding acquisition.
**David Costantini:** Writing - review & editing, Formal analysis. **Olivia Hicks:** Writing - review & editing, Resources. **Mark Hindell:** Writing - review & editing, Supervision, Funding acquisition. **Akiko Kato:** Writing - review & editing, Funding acquisition. **Thierry Raclot:** Writing - review & editing, Funding acquisition. **Cécile Ribout:** Writing - review & editing, Resources. **Yan Robert-Coudert:** Writing - review & editing, Supervision, Resources, Funding acquisition. **Frederic Angelier:** Writing - review & editing, Validation, Supervision, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare having no competing interests.

## Acknowledgements

We are grateful to Alexandre Baduel and Danuta M. Wisniewska for their valuable assistance in the field. In addition, we thank the "Service d'Analyses Biologiques du CEBC" for their expertise and their technical help in conducting laboratory analyses.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cbpa.2025.111818.

## Data availability

Data will be made available on request.

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