BACKGROUND
understanding the process of ageing and the factors that influence individual’s lifespan is a major focus in evolutionary biology and biomedical sciences.
a growing body of evidence suggests that genome integrity maintenance is essential to guarantee healthy organismal ageing, and that telomeres have an important role in this maintenance process
<cit> .
telomeres are highly conserved non-coding dna repeat sequences that cap the end of linear eukaryotic chromosomes n in vertebrates and n in insects;
<cit> ).
in so doing, they prevent the end of chromosomes of being wrongly recognized as broken or damaged
<cit> .
because dna double-stranded break activates dna damage response pathways and induces cell cycle arrest and apoptosis
<cit> , the presence of ‘healthy’ telomeres is essential to distinguish damaged chromosomes from healthy ones
<cit> .
interestingly, because dna polymerase is unable to copy the very end of chromosomes during replication of normal somatic cells , telomeres are shortening at each cell division until they reach a critical size below which cell replicative senescence is triggered
<cit> .
although telomeres can be restored by the reverse transcriptase enzyme known as telomerase, this enzyme is mainly active in germinal and stem cells
<cit> .
high telomerase activity in somatic cells has been linked to cellular proliferation and cancer
<cit> , and down-regulation of telomerase in somatic tissues is thought to have evolved as a tumor suppressing mechanism
<cit> .
hence, it has been suggested that telomere dynamics has an important influence on organismal ageing
<cit> , and that inter-individual variation in telomere length predicts, at least partially, inter-individual variation in life expectancy
<cit> .
accordingly, telomere length measured early in life or at adulthood has been found to predict subsequent survival, and in turn life expectancy, in organisms as diverse as humans
<cit> , mice
<cit> , lizards
<cit>  and birds
<cit> .
the relative contributions of genetic and environmental factors on the large inter-individual variation in telomere length among individuals of the same age remain elusive, and this advancement could help understanding the nature of the large inter-individual variation in life expectancy
<cit> .
the study of human genetic disorder and of genetically modified organisms has provided abundant evidence of diverse genetic pathways involved in the regulation of telomere length .
recent genome-wide association surveys in yeast
<cit>  and humans
<cit>  have also confirmed that telomere length is a polygenic trait.
yet, how much of the variation of telomere length in a population is transmitted to the next generation  has received little attention despite its importance in our understanding of the response to selection and evolvability of telomere length, and putatively life expectancy
<cit> .
presently, available heritability estimates of telomere length come almost exclusively from comparisons between twins or parent-offspring in humans, with reported heritability estimates ranging between  <dig>  and  <dig>
<cit> .
to our knowledge, only two studies so far have investigated heritability of telomere length in wild animals, showing significant heritability ranging between  <dig>  and  <dig>  in the sand lizard .
besides genetic factors, there is increasing evidence that telomere length is influenced by environmental factors acting during development
<cit>  and by adult lifestyle
<cit> .
the effects of the environment on telomere length can be substantial , and early life conditions are probably particularly important in shaping telomere length due to fast cell divisions during development
<cit> .
environmental factors leading to increased oxidative stress and dna damage can further accelerate telomere erosion
<cit> .
early life conditions have been reported to profoundly influence later life, and thus telomeres have also been suggested as potential effectors linking early life conditions to later organismal ageing
<cit> .
accordingly, a recent longitudinal study on captive zebra finches  has demonstrated that telomere length at 25 days of age was a strong predictor of realized lifespan
<cit> .
how early growth conditions affect telomere length and subsequent survival remains poorly known
<cit>  and warrants further studies, especially in natural populations where organisms are subjected to large environmental variations.
here, we experimentally studied the relative importance of origin  and early growth conditions on telomere length close to the end of the growth period in a natural population of an altricial bird species, the collared flycatcher  .
to do so, we performed a brood size manipulation experiment in the study population: broods hatched on the same day were matched in triplets, and part of the hatchlings were exchanged among broods of each triplet to create one brood reduced by two nestlings, one control brood  and one brood enlarged by two nestlings within each triplet.
we then investigated the effect of the brood size manipulation experiment on nestling body mass, tarsus length and telomere length close to fledging, and estimated the heritability of these three nestling phenotypic traits by comparing siblings reared in different nests .
we split the total variance of each nestling phenotypic trait using linear mixed models where we entered as explanatory variables the brood size manipulation treatment as one fixed factor and the nest of origin and the triplet as two random factors
<cit> .
the ‘triplet’ factor controls for any difference in a group of nests  and the ‘origin’ factor accounts for variation due to genetic and early maternal effects.
heritability  was estimated by using the variance components given by the linear mixed model as: h2 = va / vp, where va represents the additive genetic variance, and vp the total phenotypic variance .
telomere length was quantified using a real-time quantitative pcr developed to measure relative telomere length in birds
<cit> .
RESULTS
before the brood size manipulation, there was no significant difference in the number of two-day-old nestlings between reduced , control  and enlarged broods .
ten days after the manipulation, there were fewer nestlings in reduced compared to control broods and in control compared to enlarged broods , showing that the treatment was efficient, but nestlings were heavier in reduced compared to enlarged broods .
brood size manipulation had no significant effect on nestling tarsus length  and relative telomere length .
significance of fixed effects were tested using wald f-statistics and of random effects using log-likelihood ratio tests .
after controlling for the brood size manipulation, our linear mixed models showed significant additive genetic variance and/or maternal effects  on nestling body mass, tarsus length and relative telomere length .
this translated into heritability estimates of h2 =  <dig>  for body mass, h2 =  <dig>  for tarsus length and of h2 =  <dig>  for relative telomere length .
there was a significant effect of environmental conditions other than growth conditions  on relative telomere length and body mass but not on tarsus length .
because one important source of non-manipulated early environmental conditions associated to the effect of triplet is the hatching date, we computed two additional mixed models where hatching date was entered as a fixed covariable to investigate its influence on nestling body mass and telomere length and, in turn, heritability estimates.
hatching date was significantly negatively related to nestling body mass, as could be predicted , but not to telomere length .
as a consequence, including this covariable slightly increased the heritability estimate of body mass  but did not change the estimate for relative telomere length .
we did not test it for tarsus length because the effect of triplet was not significant for this variable.
vp=va=vt=vr+andh2=va/vp.
nestling sex had no significant influence on 12-day-old nestling body mass , tarsus length  or relative telomere length .
thus, the effect of nestling sex was dropped from the final models.
discussion
variation in telomere length has been demonstrated to predict subsequent survival in numerous organisms
<cit> .
fascinatingly, a recent study in a captive population of zebra finches where telomere length was measured at various time points throughout the life of each individual showed that telomere length in early life  was the strongest predictor of realized lifespan
<cit> .
furthermore, although individuals with long telomeres early in life maintained longer telomeres throughout their life compared to individuals with short telomere early in life, telomere length early in life is a much stronger predictor of realized lifespan when compared to telomere length in adulthood
<cit> .
these findings emphasize the importance of understanding the factors that determine telomere length early in life.
here, we report results from a brood size manipulation experiment testing the relative importance of early growth conditions and genetics and early maternal effects on telomere length measured close to the end of the growth period in nestling collared flycatchers.
in agreement with previous studies in this and other passerine species
<cit> , offspring raised in enlarged broods, and thus facing increased sibling competition and lower access to food, were in significantly poorer condition compared to offspring raised in reduced broods, as reflected by lower body mass.
however, we found no evidence that the brood size manipulation significantly influenced offspring structural body size, as measured by tarsus length, and telomere length.
body mass is known to quickly respond to short time environmental changes; skeletal traits are less sensitive to environmental variation
<cit> .
hence, a significant effect of treatment on fledgling body mass, but not tarsus length, is not surprising.
yet, the lack of significant effect of treatment on telomere length is more surprising, and it emphasizes that telomere length at the end of the growth period might not accurately reflect the past developmental conditions after hatching in this bird species.
the lack of difference in telomere length between experimental treatments however needs to be interpreted with caution for the three following reasons.
first, telomere length was measured using red blood cells present in the blood sample; red blood cells have a turnover of  <dig> to 40 days in birds
<cit> .
thus, we cannot exclude that the effect of early growth conditions on telomere length in red blood cells will become apparent only  <dig> to 25 days after fledging, which takes place around 16 days of age in the collared flycatcher.
second, our mixed models revealed that part of the variation in offspring telomere length and body mass, but not tarsus length, was significantly explained by the effect of nest triplet.
because broods matched in triplets had hatched on the same day and were in most cases located in the same forest plot, this random term is controlling, among other things, for seasonal and spatial effects on telomere length.
hence, the significant nest triplet effect on offspring telomere length suggests that some non-manipulated environmental factors have influenced telomere length, and thereby that telomere length is sensitive to environmental conditions.
here, note that additional analyses pointed out that hatching date was not affecting telomere length, and thus important early environmental variables remain to be identified.
finally,  <dig> was a very good year for breeding and the development of nestling collared flycatchers in our study site, with a nest failure probability  from  <dig> to 50% lower than in the previous years .
thus, replicating this brood size manipulation in years with contrasting environmental conditions is needed to investigate the importance of cohort and treatment effects on variation in offspring telomere length at fledging.
our mixed models showed a significant effect of the nest of origin on offspring body mass, tarsus length and telomere length, which translated into significant heritability estimates for these three traits.
the low but nonetheless significant estimate of heritability of telomere length  in fledgling collared flycatchers contrasts with previously published heritability estimates, which were notably higher
<cit> .
at least three reasons can be evocated to explain discrepancies in telomere length heritability across studies.
first, most heritability estimates reported in previous studies are based on telomere restricted fragment  measurements rather than quantitative pcr measurements.
although the two methods have been demonstrated to produce comparable telomere measurements in humans
<cit> , mice
<cit>  and birds
<cit> , we cannot exclude that lower heritability estimates in flycatchers are rooted in the methodological approach to quantify telomere length
<cit> .
both trf and qpcr measurements present methodological difficulties to accurately estimate telomere length
<cit> , and systematic variation in measurement errors of telomere length between the two methods could hamper comparisons of heritability estimates among studies.
second, maternal effects can inflate sibling resemblances
<cit> , calling for caution when interpreting heritability estimates based on sib-sib comparison  or on parent-offspring regression .
however, the heritability estimates obtained here for body mass  and tarsus length  are consistent with previous estimates obtained in the same study population using a mixed model
<cit> .
therefore, this gives confidence in the low heritability estimate of telomere length found in the present study.
finally, telomere length and its heritability level frequently differ between the sexes
<cit> , and it has been proposed that telomere length is primarily inherited from the heterogametic sex
<cit> , namely fathers in humans
<cit>  and in some reptiles as the sand lizard
<cit>  and mothers in birds
<cit> .
here, we found no difference in telomere length between the sexes in nestling collared flycatchers, but computing sex-specific heritability estimates would be needed to assess whether our estimate could be affected by this inheritance mechanism.
unfortunately, the models did not converge when computing sex-specific estimates because of a limited number of opposite-sex siblings reared in different nests .
additional studies in the wild are required for assessing the relative importance of early growth conditions and genetics and maternal effects on telomere length measured at various ages to better understand the factors shaping the variation in telomere length in natural populations and for testing the links between telomere length in early life and later survival.
strong natural selection on a phenotypic trait can quickly deplete its additive genetic variation and, in turn, its heritability
<cit> .
the lower heritability of telomere length compared to body mass and tarsus length may supports the hypothesis that telomere length is under stronger selection, and in turn is more closely associated with fitness in this natural population.
assessing the shape and strength of natural selection on telomere length is needed to test this hypothesis and get insights on the evolutionary potential of telomere length in the wild.
CONCLUSIONS
this study shows that telomere length of nestlings close to fledging was weakly, but nonetheless significantly, influenced by genetics and/or maternal effects taking place before hatching in a natural populations of birds.
furthermore, although the brood size manipulation experiment was efficient at altering nestling body mass, there was no evidence that this experimental manipulation of nestling early growth conditions affected the length of their telomeres.

 understanding the process of ageing and the factors that influence individual’s lifespan is a major focus in evolutionary biology and biomedical sciences. a growing body of evidence suggests that genome integrity maintenance is essential to guarantee healthy organismal ageing, and that telomeres have an important role in this maintenance process. telomeres are highly conserved non-coding dna repeat sequences that cap the end of linear eukaryotic chromosomes n in vertebrates and n in insects; ). in so doing, they prevent the end of chromosomes of being wrongly recognized as broken or damaged. because dna double-stranded break activates dna damage response pathways and induces cell cycle arrest and apoptosis, the presence of ‘healthy’ telomeres is essential to distinguish damaged chromosomes from healthy ones. interestingly, because dna polymerase is unable to copy the very end of chromosomes during replication of normal somatic cells, telomeres are shortening at each cell division until they reach a critical size below which cell replicative senescence is triggered. although telomeres can be restored by the reverse transcriptase enzyme known as telomerase, this enzyme is mainly active in germinal and stem cells. high telomerase activity in somatic cells has been linked to cellular proliferation and cancer, and down-regulation of telomerase in somatic tissues is thought to have evolved as a tumor suppressing mechanism. hence, it has been suggested that telomere dynamics has an important influence on organismal ageing, and that inter-individual variation in telomere length predicts, at least partially, inter-individual variation in life expectancy. accordingly, telomere length measured early in life or at adulthood has been found to predict subsequent survival, and in turn life expectancy, in organisms as diverse as humans, mice, lizards and birds. the relative contributions of genetic and environmental factors on the large inter-individual variation in telomere length among individuals of the same age remain elusive, and this advancement could help understanding the nature of the large inter-individual variation in life expectancy. the study of human genetic disorder and of genetically modified organisms has provided abundant evidence of diverse genetic pathways involved in the regulation of telomere length. recent genome-wide association surveys in yeast and humans have also confirmed that telomere length is a polygenic trait. yet, how much of the variation of telomere length in a population is transmitted to the next generation has received little attention despite its importance in our understanding of the response to selection and evolvability of telomere length, and putatively life expectancy. presently, available heritability estimates of telomere length come almost exclusively from comparisons between twins or parent-offspring in humans, with reported heritability estimates ranging between and. to our knowledge, only two studies so far have investigated heritability of telomere length in wild animals, showing significant heritability ranging between and in the sand lizard. besides genetic factors, there is increasing evidence that telomere length is influenced by environmental factors acting during development and by adult lifestyle. the effects of the environment on telomere length can be substantial, and early life conditions are probably particularly important in shaping telomere length due to fast cell divisions during development. environmental factors leading to increased oxidative stress and dna damage can further accelerate telomere erosion. early life conditions have been reported to profoundly influence later life, and thus telomeres have also been suggested as potential effectors linking early life conditions to later organismal ageing. accordingly, a recent longitudinal study on captive zebra finches has demonstrated that telomere length at 25 days of age was a strong predictor of realized lifespan. how early growth conditions affect telomere length and subsequent survival remains poorly known and warrants further studies, especially in natural populations where organisms are subjected to large environmental variations. here, we experimentally studied the relative importance of origin and early growth conditions on telomere length close to the end of the growth period in a natural population of an altricial bird species, the collared flycatcher. to do so, we performed a brood size manipulation experiment in the study population: broods hatched on the same day were matched in triplets, and part of the hatchlings were exchanged among broods of each triplet to create one brood reduced by two nestlings, one control brood and one brood enlarged by two nestlings within each triplet. we then investigated the effect of the brood size manipulation experiment on nestling body mass, tarsus length and telomere length close to fledging, and estimated the heritability of these three nestling phenotypic traits by comparing siblings reared in different nests. we split the total variance of each nestling phenotypic trait using linear mixed models where we entered as explanatory variables the brood size manipulation treatment as one fixed factor and the nest of origin and the triplet as two random factors. the ‘triplet’ factor controls for any difference in a group of nests and the ‘origin’ factor accounts for variation due to genetic and early maternal effects. heritability was estimated by using the variance components given by the linear mixed model as: h2 = va / vp, where va represents the additive genetic variance, and vp the total phenotypic variance. telomere length was quantified using a real-time quantitative pcr developed to measure relative telomere length in birds.  before the brood size manipulation, there was no significant difference in the number of two-day-old nestlings between reduced, control and enlarged broods. ten days after the manipulation, there were fewer nestlings in reduced compared to control broods and in control compared to enlarged broods, showing that the treatment was efficient, but nestlings were heavier in reduced compared to enlarged broods. brood size manipulation had no significant effect on nestling tarsus length and relative telomere length. significance of fixed effects were tested using wald f-statistics and of random effects using log-likelihood ratio tests. after controlling for the brood size manipulation, our linear mixed models showed significant additive genetic variance and/or maternal effects on nestling body mass, tarsus length and relative telomere length. this translated into heritability estimates of h2 =  for body mass, h2 =  for tarsus length and of h2 =  for relative telomere length. there was a significant effect of environmental conditions other than growth conditions on relative telomere length and body mass but not on tarsus length. because one important source of non-manipulated early environmental conditions associated to the effect of triplet is the hatching date, we computed two additional mixed models where hatching date was entered as a fixed covariable to investigate its influence on nestling body mass and telomere length and, in turn, heritability estimates. hatching date was significantly negatively related to nestling body mass, as could be predicted, but not to telomere length. as a consequence, including this covariable slightly increased the heritability estimate of body mass but did not change the estimate for relative telomere length. we did not test it for tarsus length because the effect of triplet was not significant for this variable. vp=va=vt=vr+andh2=va/vp. nestling sex had no significant influence on 12-day-old nestling body mass, tarsus length or relative telomere length. thus, the effect of nestling sex was dropped from the final models. discussion variation in telomere length has been demonstrated to predict subsequent survival in numerous organisms. fascinatingly, a recent study in a captive population of zebra finches where telomere length was measured at various time points throughout the life of each individual showed that telomere length in early life was the strongest predictor of realized lifespan. furthermore, although individuals with long telomeres early in life maintained longer telomeres throughout their life compared to individuals with short telomere early in life, telomere length early in life is a much stronger predictor of realized lifespan when compared to telomere length in adulthood. these findings emphasize the importance of understanding the factors that determine telomere length early in life. here, we report results from a brood size manipulation experiment testing the relative importance of early growth conditions and genetics and early maternal effects on telomere length measured close to the end of the growth period in nestling collared flycatchers. in agreement with previous studies in this and other passerine species, offspring raised in enlarged broods, and thus facing increased sibling competition and lower access to food, were in significantly poorer condition compared to offspring raised in reduced broods, as reflected by lower body mass. however, we found no evidence that the brood size manipulation significantly influenced offspring structural body size, as measured by tarsus length, and telomere length. body mass is known to quickly respond to short time environmental changes; skeletal traits are less sensitive to environmental variation. hence, a significant effect of treatment on fledgling body mass, but not tarsus length, is not surprising. yet, the lack of significant effect of treatment on telomere length is more surprising, and it emphasizes that telomere length at the end of the growth period might not accurately reflect the past developmental conditions after hatching in this bird species. the lack of difference in telomere length between experimental treatments however needs to be interpreted with caution for the three following reasons. first, telomere length was measured using red blood cells present in the blood sample; red blood cells have a turnover of to 40 days in birds. thus, we cannot exclude that the effect of early growth conditions on telomere length in red blood cells will become apparent only to 25 days after fledging, which takes place around 16 days of age in the collared flycatcher. second, our mixed models revealed that part of the variation in offspring telomere length and body mass, but not tarsus length, was significantly explained by the effect of nest triplet. because broods matched in triplets had hatched on the same day and were in most cases located in the same forest plot, this random term is controlling, among other things, for seasonal and spatial effects on telomere length. hence, the significant nest triplet effect on offspring telomere length suggests that some non-manipulated environmental factors have influenced telomere length, and thereby that telomere length is sensitive to environmental conditions. here, note that additional analyses pointed out that hatching date was not affecting telomere length, and thus important early environmental variables remain to be identified. finally, was a very good year for breeding and the development of nestling collared flycatchers in our study site, with a nest failure probability from to 50% lower than in the previous years. thus, replicating this brood size manipulation in years with contrasting environmental conditions is needed to investigate the importance of cohort and treatment effects on variation in offspring telomere length at fledging. our mixed models showed a significant effect of the nest of origin on offspring body mass, tarsus length and telomere length, which translated into significant heritability estimates for these three traits. the low but nonetheless significant estimate of heritability of telomere length in fledgling collared flycatchers contrasts with previously published heritability estimates, which were notably higher. at least three reasons can be evocated to explain discrepancies in telomere length heritability across studies. first, most heritability estimates reported in previous studies are based on telomere restricted fragment measurements rather than quantitative pcr measurements. although the two methods have been demonstrated to produce comparable telomere measurements in humans, mice and birds, we cannot exclude that lower heritability estimates in flycatchers are rooted in the methodological approach to quantify telomere length. both trf and qpcr measurements present methodological difficulties to accurately estimate telomere length, and systematic variation in measurement errors of telomere length between the two methods could hamper comparisons of heritability estimates among studies. second, maternal effects can inflate sibling resemblances, calling for caution when interpreting heritability estimates based on sib-sib comparison or on parent-offspring regression. however, the heritability estimates obtained here for body mass and tarsus length are consistent with previous estimates obtained in the same study population using a mixed model. therefore, this gives confidence in the low heritability estimate of telomere length found in the present study. finally, telomere length and its heritability level frequently differ between the sexes, and it has been proposed that telomere length is primarily inherited from the heterogametic sex, namely fathers in humans and in some reptiles as the sand lizard and mothers in birds. here, we found no difference in telomere length between the sexes in nestling collared flycatchers, but computing sex-specific heritability estimates would be needed to assess whether our estimate could be affected by this inheritance mechanism. unfortunately, the models did not converge when computing sex-specific estimates because of a limited number of opposite-sex siblings reared in different nests. additional studies in the wild are required for assessing the relative importance of early growth conditions and genetics and maternal effects on telomere length measured at various ages to better understand the factors shaping the variation in telomere length in natural populations and for testing the links between telomere length in early life and later survival. strong natural selection on a phenotypic trait can quickly deplete its additive genetic variation and, in turn, its heritability. the lower heritability of telomere length compared to body mass and tarsus length may supports the hypothesis that telomere length is under stronger selection, and in turn is more closely associated with fitness in this natural population. assessing the shape and strength of natural selection on telomere length is needed to test this hypothesis and get insights on the evolutionary potential of telomere length in the wild.  this study shows that telomere length of nestlings close to fledging was weakly, but nonetheless significantly, influenced by genetics and/or maternal effects taking place before hatching in a natural populations of birds. furthermore, although the brood size manipulation experiment was efficient at altering nestling body mass, there was no evidence that this experimental manipulation of nestling early growth conditions affected the length of their telomeres. 
the weak heritability of telomere length suggests a close association with fitness in natural populations. although our treatment significantly affected body mass at fledging, we found no evidence that increased sibling competition affected nestling tarsus length and telomere length. however, mixed models showed that brood triplets explained a significant part of the variance in body mass and telomere length, but not tarsus length, emphasizing that unmanipulated early environmental factors influenced telomere length. for comparison, the heritability of nestling body mass and tarsus length was and, respectively, which was in the range of previously published estimates for those two traits in this species. evidence is accumulating that telomere length is a good predictor of life expectancy, especially early in life, thus calling for determining the factors that affect telomere length at this stage. here, we investigated the relative influence of early growth conditions and origin on telomere length of collared flycatchers at fledging. these models also revealed low, but significant, heritability of telomere length. those findings in a wild bird population demonstrate that telomere length at the end of the growth period is weakly, but significantly, determined by genetic and/or maternal factors taking place before hatching. however, we found no evidence that the brood size manipulation experiment, and by extension the early growth conditions, influenced nestling telomere length.
 evidence is accumulating that telomere length is a good predictor of life expectancy, especially early in life, thus calling for determining the factors that affect telomere length at this stage. here, we investigated the relative influence of early growth conditions and origin on telomere length of collared flycatchers at fledging. we experimentally transferred hatchlings among brood triplets to create reduced, control and enlarged broods.  although our treatment significantly affected body mass at fledging, we found no evidence that increased sibling competition affected nestling tarsus length and telomere length. however, mixed models showed that brood triplets explained a significant part of the variance in body mass and telomere length, but not tarsus length, emphasizing that unmanipulated early environmental factors influenced telomere length. these models also revealed low, but significant, heritability of telomere length. for comparison, the heritability of nestling body mass and tarsus length was and, respectively, which was in the range of previously published estimates for those two traits in this species.  those findings in a wild bird population demonstrate that telomere length at the end of the growth period is weakly, but significantly, determined by genetic and/or maternal factors taking place before hatching. however, we found no evidence that the brood size manipulation experiment, and by extension the early growth conditions, influenced nestling telomere length. the weak heritability of telomere length suggests a close association with fitness in natural populations. 