Divergent patterns of telomere shortening in tropical compared to temperate stonechats

Abstract Telomeres have emerged as important biomarkers of health and senescence as they predict chances of survival in various species. Tropical birds live in more benign environments with lower extrinsic mortality and higher juvenile and adult survival than temperate birds. Therefore, telomere biology may play a more important role in tropical compared to temperate birds. We measured mean telomere length of male stonechats (Saxicola spp.) at four age classes from tropical African and temperate European breeding regions. Tropical and temperate stonechats had similarly long telomeres as nestlings. However, while in tropical stonechats pre‐breeding first‐years had longer telomeres than nestlings, in temperate stonechats pre‐breeding first‐years had shorter telomeres than nestlings. During their first breeding season, telomere length was again similar between tropical and temperate stonechats. These patterns may indicate differential survival of high‐quality juveniles in tropical environments. Alternatively, more favorable environmental conditions, that is, extended parental care, may enable tropical juveniles to minimize telomere shortening. As suggested by previous studies, our results imply that variation in life history and life span may be reflected in different patterns of telomere shortening rather than telomere length. Our data provide first evidence that distinct selective pressures in tropical and temperate environments may be reflected in diverging patterns of telomere loss in birds.


| INTRODUC TI ON
Variation in life histories is thought to result from differential allocation of limited resources to competing life history traits. Such tradeoffs and the resulting optimal resource allocation may vary with environmental conditions (Stearns, 1992). For example, tropical environments have favored a slow pace of life, that is, reduced fecundity but increased life span, in many vertebrates (Ricklefs & Wikelski, 2002). This is especially well studied in birds where tropical species produce fewer, but higher quality offspring (Jetz, Sekercioglu, & Böhning-Gaese, 2008;Martin, 2015), have lower basal metabolic rates (Tieleman et al., 2009;Wiersma, Muñoz-Garcia, Walker, & Williams, 2007) and live longer (Møller, 2007;Peach, Hanmer, & Oatley, 2001) than temperate species. Therefore, a comparison between tropical and temperate species may reveal physiological constraints that may limit the evolution of alternative combinations of life history traits (Ricklefs & Wikelski, 2002).
An important candidate mechanism with respect to physiological constraints of growth, reproduction and survival are telomeres (Haussmann & Marchetto, 2010). Telomeres are noncoding DNAprotein caps at the end of eukaryotic chromosomes that protect genomic integrity, but shorten during cell division and potentially when exposed to oxidative stress (Boonekamp, Bauch, Mulder, & Verhulst, 2017;Reichert & Stier, 2017;Zglinicki, 2002). Critically, short telomeres eventually lead to cell senescence or death (Blackburn, 2000(Blackburn, , 2005, and the accumulation of cells with short telomeres may be one of the factors that causes aging and senescence in vertebrates (López-Otín, Blasco, Partridge, Serrano, & Kroemer, 2013).
Furthermore, an increasing number of studies in birds show that individuals with longer telomeres or little telomere attrition have better survival prospects than individuals with short telomeres or high levels of telomere attrition (reviewed in Wilbourn et al., 2018).
This has been especially well studied in zebra finches (Taeniopygia guttata), for which it has been shown that long telomeres in early life are associated with increased survival and a long life span (Heidinger et al., 2012). In addition, studies in a variety of species show that telomere dynamics are sensitive to environmental influences such as variations in food availability (Spurgin et al., 2018), parasitic diseases (Asghar et al., 2015), and exposure to stress (Hau et al., 2015).
Fewer studies have compared telomere length between taxa that vary in their life histories and life span. In mammals, a comparative study found that short-lived, small species have longer telomeres and higher telomerase expression than long-lived, large species (Gomes et al., 2011). In a study on rodents, no relationship between maximum lifespan and telomere length was detected (Seluanov et al., 2007). In birds, absolute telomere length does not seem to relate to variation in lifespan between species; however, longer-lived avian species seem to have lower rates of telomere shortening than shorter-lived species (Dantzer & Fletcher, 2015;Haussmann et al., 2003;Sudyka, Arct, Drobniak, Gustafsson, & Cichoan, 2016;Tricola et al., 2018). This relationship between rate of telomere loss and maximum lifespan in birds may be caused by variation between species in how well telomeres are maintained throughout their lifespan.
In addition, it may reflect selective disappearance of low-quality individuals with short telomeres. In longer-lived species, that experience lower levels of extrinsic mortality, individual condition, and thus telomere dynamics, may play a greater role as determinants of mortality (Kirkwood & Austad, 2000). Therefore, selective disappearance of individuals with short telomeres may be more apparent in long-lived species (Tricola et al., 2018).
Tropical species live in less seasonal environments with lower levels of adult extrinsic mortality than temperate ones (Brown, 2014). Consequently, tropical songbirds have higher survival probabilities than temperate birds Muñoz, Kéry, Martins, & Ferraz, 2018). Therefore, stronger selective disappearance of individuals with short telomeres is expected in tropical compared to temperate birds. However, mortality rates are age-specific, and therefore, the strength of selective disappearance may vary with age. In birds, mortality is usually highest during the first year of life, especially directly after fledging (Cox, Thompson, Cox, & Faaborg, 2014;Naef-Daenzer & Grüebler, 2016). As predicted by life history theory (McNamara, Barta, Wikelski, & Houston, 2008), juvenile survival is in general higher in tropical compared to temperate birds (Lloyd, Martin, & Roskaft, 2016;Remes & Matysiokova, 2016). Tropical parents take care of their fewer fledglings for considerably longer than temperate birds and may thereby be able to lower extrinsic mortality in juveniles (Styrsky, Brawn, & Robinson, 2005). We, therefore, hypothesize that differential survival of highquality fledglings should be more apparent in tropical compared to temperate birds. Assuming that telomeres are bioindicators of somatic state and individual quality we expect that in tropical birds, individuals with short telomeres disappear faster from a population than in temperate birds both during the critical first year of life and later as adults.
In addition, there is good evidence that tropical species invest more into self-maintenance, but are less fecund than temperate species. For example, tropical species exhibit stronger sickness behavior after infection during the breeding season than temperate species (Owen-Ashley, Hasselquist, Raberg, & Wingfield, 2008). Furthermore, in tropical species, reproductive workload is reduced as they lay smaller clutches, thereby caring for fewer young and expending less energy than temperate birds (Nilsson, 2002;Tieleman et al., 2006). Thus, they may reduce high levels of oxidative stress (Noguera, 2017) and potential telomere loss associated with breeding (Reichert et al., 2014). In addition, tropical songbirds seem to have lower post-natal metabolic rates and slower, more sustained growth despite similar nestling times than temperate birds (Martin, 2015;Ton & Martin, 2016). These slower growth trajectories in combination with increased parental care per offspring may favor lower levels of telomere attrition during early life in tropical birds, which in turn may be important determinants of their longer life spans (Monaghan & Ozanne, 2018). Thus, longer-lived tropical species, which invest more in growth and self-maintenance than in fecundity, are expected to show longer telomeres as nestlings or a slower rate of telomere loss than short-lived temperate species. To determine how life history variation has shaped variation in telomeres, comparisons between the same or closely related species in different environments are necessary.

| ME THODS
Blood samples were collected from two phylogenetically closely related stonechat species (Table 1)  African stonechats are residents, European populations vary in migratory strategy from short-distance migrants to residents (Table 1).
Samples from some of these birds were also used to study baseline and stress-induced corticosterone concentrations of tropical and temperate stonechats during the breeding season (Apfelbeck, Helm, et al., 2017). All samples were analyzed with telomere restriction fragment analysis (TRF) and we restricted our analysis to males to optimize sample sizes across species and age classes.

| Capture methods
Male stonechats were caught between 07:00 hr and 18:00 hr with baited clap net traps but also in some cases additionally lured by a mounted decoy and playback. Traps were observed continuously and upon capture birds were immediately removed from the traps.
Nestlings were bled during the last third of the nestling stage (i.e., between day 8 and 14 post-hatch). Pre-breeding first-year males were caught 3-6 months (see Table 1) after their likely hatching dates, after completing post-juvenile moult, but before their first breeding season. Breeding first-year males were caught during their first breeding season before their first post-nuptial moult. All males were measured (weight, tarsus, and wing length), checked for moult, ringed with a numbered aluminum ring and a combination of three color rings and were then released back into their territories. We determined the age of all individuals caught as either in their first year or as adults (≥2 years) based on feather moult pattern of the wings (Flinks, 1994). Nestlings and fledglings were ringed with a numbered aluminum ring only.

| Blood sampling
Blood samples (~120 µl) were taken within 3 min of capture by venipuncture of the wing vein or (less often) with an insulin sy-  (Nussey et al., 2014;, and thus, preferentially samples should be treated similarly across groups. Although in our study storage method varied across samples, we are confident that this did not influence our results as DNA integrity was checked for each sample by standard gel electrophoresis (Kimura et al., 2010). Samples that showed signs of degradation were not included in the TRF assay. Upon return from the field, samples were stored at ~2°C whenever possible. During periods of transportation, samples were stored at room temperature.

| Sex determination of nestling and fledgling samples
Molecular sexing was carried out by amplification of the chromodomain-helicase-DNA binding (CHD) genes in 10 µl PCR reactions following the standard procedure described in Fridolfsson and Ellegren (1999) and Griffiths, Double, Orr, and Dawson (1998). Invitrogen, CA). The background was fixed as the nadir of the low-MW region on the gel (<1 kb). Samples were distributed among six gels and mixed by population and age class. One stonechat sample was run three times on each gel to determine intra-and inter-gel coefficients of variation, which were 4.86% and 7.54%, respectively.

| Statistical analysis
Data were analyzed within the R environment (R version 3.2.2; R Core Team, 2016) and the packages arm (Gelman & Su, 2018), JAGS (Plummer, 2003), and runjags (Denwood, 2016). Linear models were used to determine whether variation in mean telomere length was related to breeding region (tropical, temperate), age class or the interaction between breeding region and age class. We tested whether tropical stonechats had longer telomeres than temperate stonechats and whether differences between taxa declined with age by comparing tropical and temperate male stonechats in different age classes (nestling, first year pre-breeding, first year breeding, ≥2 years). We applied linear models using tropical nestlings as reference level. Body mass was included as a covariate in the initial model, but dropped in the final model as it did not detectably explain variance in the data. Because samples from tropical stonechats covered the widest range of age classes, we ran a separate linear model on tropical stonechats including the additional factor level "fledglings." We chose a Bayesian approach to draw inferences from the models. Bayesian statistics estimate probability distributions of the parameters in the model (i.e., posterior distributions) given the data and prior knowledge about the distribution of the data (specified as

| RE SULTS
Overall, temperate and tropical male stonechats showed similar mean telomere lengths ( Note. The second column shows the estimated difference from the intercept. In this case the reference level was "tropical nestlings." The third column shows the mean estimates for each factor level, which were calculated from column 2. As the reference level was "tropical nestlings," for temperate birds the estimated difference has to be added to the estimate obtained for tropical birds for each age class. When 0 (zero) is not included in the credible intervals there is an effect of this parameter on the dependent variable (shown in bold). than nestlings (negative difference from intercept, Table 2, Figure 2).
Analysis of tropical stonechats across the whole range of age classes revealed that average telomere length was similar in nestlings and fledglings of tropical stonechats (

| D ISCUSS I ON
Our data provide first evidence that distinct selective pressures in tropical and temperate environments may be reflected in diverging patterns of telomere loss between age classes. Similar to other studies (e.g., Spurgin et al., 2018), we find that mean telomere length decreased fastest during the first year of life in temperate stonechats.
In contrast, in tropical stonechats mean telomere length increased initially from nestlings to fledglings to first year pre-breeding before dropping in first year breeding males. This suggests that tropical compared to temperate stonechats may either experience lower levels of telomere loss and/or more pronounced differential survival of individuals with long telomeres during their first year of life.
In tropical birds, the post-fledging period has emerged as a critical period during reproduction that may have a considerable impact on the fitness of tropical birds. Tropical birds experience high levels of nest predation (Martin, 1992), but high levels of adult survival.
Therefore, tropical parents may raise small clutches in favor of extended offspring care (Russell, Yom-Tov, & Geffen, 2004;Tarwater, Ricklefs, Maddox, & Brawn, 2011), which has been shown to increase fledgling survival (Grüebler & Naef-Daenzer, 2010). Tropical stonechats lay on average smaller clutches (3) than temperate stonechats (5) and care for fledglings for several weeks after fledging, often allowing juveniles to remain on their territories (Dittami & Gwinner, 1985;Scheuerlein, Van't Hof, & Gwinner, 2001). In addition, especially under high predation pressure, stonechats often skip a second clutch in favor of their fledglings (Scheuerlein et al., 2001). In addition, as environmental conditions and parental care during growth can influence telomere loss and maintenance (e.g., Costanzo et al., 2017), extended parental care may create more favorable conditions for tropical fledglings and juveniles that may allow them to maintain their telomeres better than temperate ones. A number of recent studies in temperate songbirds during the nestling period have shown that the rearing environment has an influence on telomere attrition rates in early life (e.g., Salmon et al., 2016;Soler et al., 2017). For example, growing up in large broods, high begging effort and low food availability hasten telomere loss in nestlings (Costanzo et al., 2017;Nettle et al., 2017;Young et al., 2017 , 2014;Salmon et al., 2017). The effect of extended parental care in tropical birds on telomere dynamics in fledglings should be further investigated, for example through brood size manipulation experiments.
In contrast to telomere length in juveniles, telomere length at the end of the nestling stage did not differ between temperate and tropical stonechats despite smaller clutches in tropical stonechats, which favor increased feeding rates per offspring (Martin, 2015).
As ground-nesting birds in open habitat, high nest predation rates may favor fast growth and consequently similar telomere loss during development in tropical and temperate environments. In tropical stonechats, frequent presence of predators reduced the growth rate of nestlings (Scheuerlein & Gwinner, 2006), highlighting that lower growth rates in stonechats may not represent a mechanism for (supposedly) slower aging of tropical stonechats. However, direct comparisons of growth rates in nestlings of tropical and temperate stonechats are necessary to further clarify this issue.
Selective disappearance of individuals with short telomeres during the first year of life also seems to take place in temperate stonechats, albeit later than in tropical stonechats, potentially during the first migration and overwinter period. Migration or the winter period are especially challenging for inexperienced, subordinate juvenile birds and accordingly mortality during migration and winter is higher for juveniles than for adults (Ekman, 1984;Rotics et al., 2016). Only the highest quality individuals with the longest telomeres may be able to survive, which may lead to longer telomeres in first year breeding males compared to males caught before their first winter (first year pre-breeding). Thus, mean telomere length across age classes may indicate when selective disappearance of low-quality individuals with short telomeres is most likely to occur.
In contrast to our predictions, tropical and temperate stonechats had telomeres of similar length during their first breeding season and as adults. Reproduction, especially parental care, is energetically costly (Nilsson, 2002), can lead to oxidative DNA damage (Noguera, 2017) and potentially increased telomere loss (Heidinger et al., 2012;Reichert et al., 2014

| CON CLUS IONS
To the best of our knowledge, this is one of the first studies to compare mean telomere length across several age classes in closely related species that breed in tropical and temperate environments and differ in their pace of life. As indicated by previous interspecific studies, our results suggest that variation in life history and life span may be reflected in different patterns of telomere loss between species rather than absolute telomere length. Our data reveal that mean telomere length across age classes may indicate during which life-cycle phases individuals with short telomeres, and thus of potentially low quality, are most likely to disappear from a population. These patterns closely fit with expectations from life history theory and match variation in parental behavior and juvenile mortality between tropical and temperate birds.

ACK N OWLED G M ENTS
We want to thank John O`Halloran for organizing the fieldwork in Ireland, and Guillermo López, Musa Makomba, Isaac (Monduli), Julius (Pare), Jack Kiiru, and Ann Gathitu for help with fieldwork. We are also grateful to Pat Monaghan for advice and access to laboratory space. We further thank the National Museums of Kenya (NMK) and the Tanzania Wildlife Research Institute (TAWIRI) for providing support for fieldwork. We also want to thank Dominic Cram and an anonymous reviewer for valuable comments on an earlier version of this manuscript.

E TH I C A L A PPROVA L
All experimental procedures were approved by the governmental authorities of the respective countries (Germany: Regional

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R ' S CO NTR I B UTI O N
BA and BH conceived of the study. BA, HF, JCI, KGM, PS collected the field data. BA and ZS conducted the molecular laboratory work.
WB, KG, and MH supported and supervised the laboratory work.
BA carried out the statistical analysis and drafted the manuscript.
MH and BH helped drafting the manuscript. All authors gave final approval for publication.

DATA ACCE SS I B I LIT Y
The dataset supporting this article has been uploaded as Supporting